Carbohydrate Chemistry Volume 15 Part II
A Specialist Periodical Report
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Carbohydrate Chemistry Volume 15 Part II
A Specialist Periodical Report
Carbohydrate Chemistry Volume 15 Part II Macromolecules A Review of the Literature Published during 1981 Senior Reporter J. F. Kennedy, University of Birmingham Reporters
D. P. Atkins, University of Birmingham
1. M. Morrison, Hannah Research Institute, Ayr C. M. Sturgeon, University of Edinburgh R. J. Sturgeon, Heriot- Watt University, Edinburgh C. A. White, University of Birmingham
The Royal Society of Chemistry Burlington House, London W I V OBN
ISBN 0-85186-152-0 ISSN 0576-7172
Copyright @ 1986 The Royal Society of Chemistry All Rights Reserved No part of this book may be reproduced or transmitted in any form or by any means graphic, electronic, including photocopying, recording, taping, or information storage and retrieval systems - without written permission from The Royal Society of Chemistry
-
Printed in Great Britain by Whitstable Litho Ltd., Whitstable, Kent
Preface This Report, t h e f i f t e e n t h o f t h i s T i t l e , is, l i k e its predecessors, a c o m p r e h e n s i v e summary of p u b l i c a t i o n s , b o t h p r i m a r y and s e c o n d a r y , p r e s e n t e d i n a c o n c i s e and r e a d a b l e form. In s p i t e o f t h e s u p p o r t i v e comments we h a v e r e c e i v e d for t h i s T i t l e from r e a d e r s i n v a r i o u s p a r t s o f t h e w o r l d , r e f e r e n c e must be made t o t h e 1985 d e c i s i o n o f t h e Royal S o c i e t y o f C h e m i s t r y t o cease p u b l i c a t i o n o f ' C a r b o h y d r a t e C h e m i s t r y ' P a r t I1 i n i t s p r e s e n t form.
T h i s d e c i s i o n h a s been b a s e d on
i n s u f f i c i e n t income t o s u p p o r t t h e h i g h c o s t o f p r o d u c t i o n .
Such a c e s s a t i o n
was h i n t e d a t i n t h e p r e f a c e t o Volume 14 a s b e i n g a p o s s i b i l i t y .
It is
r e g r e t t a b l e n o t o n l y s i n c e t h e c a r b o h y d r a t e macromolecular c h e m i s t r y may no l o n g e r b e a v a i l a b l e i n s u c h a n a c c e s s i b l e form b u t a l s o s i n c e so much e f f o r t h a s b e e n p u t i n t o t h e change t o t h e p r o d u c t i o n o f camera-ready c o p y d e s p i t e t h e problems a s s o c i a t e d w i t h t h e c o m p l e x i t i e s o f c a r b o h y d r a t e names and s t r u c t u r e s and a b b r e v i a t e d forms t h e r e o f . I n t h e p r o d u c t i o n o f t h i s volume I h a v e been s u p p o r t e d o n c e a g a i n by c o l l e a g u e s who have u n s t i n t i n g l y c o n t r i b u t e d t o many volumes o f t h i s T i t l e and by Drs C . A. White and D. P. A t k i n s who j o i n e d i n t h e p r o d u c t o n o f t h i s volume t o e a s e t h e work-load.
I a g a i n v e r y g r a t e f u l l y acknowledge t h e c o n s i d e r a b l e
e f f o r t p u t i n by e v e r y r e p o r t e r , and t h e h e l p a n d a d v i c e t h e y have g i v e n . F i n a l l y , t h e e d i t o r i a l and o t h e r a s s i s t a n c e p r o v i d e d by Mrs R.H.
Pape,
A s s i s t a n t E d i t o r , Books, a t t h e Royal S o c i e t y o f C h e m i s t r y i n t h e p r o d u c t i o n o f t h i s Report i s much a p p r e c i a t e d . A p r i l 1986
JOHN F. KENNEDY
Contents Chapter 1
Introduction By J . F. Kennedy
1
Chapter 2
G e n e r a l Methods By R. J. S t u r g e o n
3
1
Gas-Li q u i d Chromatography
3
2 Column and Ion-exchange Chromatography
5
3 T h i n - l a y e r Chromatography
5
4 H i g h - p r e s s u r e L i q u i d Chromatography
6
5 Electrophoresis
9
6 A n a l y t i c a l Methods
10
S t r u c t u r a l Methods N. M. R. S p e c t r o s c o p y Mass S p e c t r o m e t r y M i s c e l l a n e o u s Methods
13 13 14 15
P l a n t a n d Algal P o l y s a c c h a r i d e s
21
1
Introduction
21
2
Starch
21
3 Fruct a n s
31
4 Ce 11u l o s e
32
5 H e m i c e l l u l oses
39
6 Pectins
48
7 Gums a n d M u c i l a g e s
53
8 Algal Polysaacharides
56
7
Chapter 3
By I. M. M o r r i s o n
Microbial P o l y s a c c h a r i d e s By C. A. Whibe
70
1
Teichoic Acids
70
2
Peptidoglycans
77
Chapter 4
viii
Carbohydrate Chemistry 3
Lipopolysaccharides
89
4
Capsular Polysaccharides
107
5
E x t r a c e l l u l a r and I n t r a c e l l u l a r P o l y s a c c h a r i d e s
119
6
Miscellaneous B a c t e r i a l Polysaccharides
136
7
Fungal Polysaccharides D - G l ucan s D-Ma nn a n s Chitin Miscellaneous Cell-wall Polysaccharides
141 145 150 157 158
C l y c o p r o t e i n s , G l y c o p e p t i d e s , P r o t e o g l y c a n s , and Animal P o l y s a c c h a r i d e s By R . J. S t u r g e o n
168
1
Microbial Glycoproteins Fungal G l y c o p r o t e i n s Viral Glycoproteins
168 168 169
2
Plant Glycoproteins
178
Chapter 5
3 Lectins
179
4
Fibronectin
193
5
Collagen
197
6
Glycogen
202
7
Glycosaminoglycans and Proteoglycans Analysis O c c u r r e n c e , I s o l a t i o n , and S t r u c t u r e Biosynthesis Pa t h o l o g y
203 20 3 205 21 6 219
8 Cell and T i s s u e G l y c o p r o t e i n s
222
9
Cell-surface Glycoproteins
234
10
G l y c o p r Q t e i n Hormones
249
11
Milk G l y c o p r o t e i n s
253
12
Serum G l y c o p r o t e i n s
25 8
13
Immunoglobulins
269
14
Erythrocyte Glycoproteins
284
15
S a l i v a r y a n d Mucous G l y c o p r o t e i n s
292
16
U r i n a r y G l y c o p r o t e i n s , G l y c o p e p t i d e s , and 01 i g o s a c c h a r i d e s
298
17
Avian C l y c o p r o t e i n s
30 1
18 M i s c e l l a n e o u s C l y c o p r o t e i n s a n d C h i t i n
307
Contents
Chapter
19 Analysis of Glycoproteins
310
20 Biosynthesis of Glycoprotelns
314 347
6 Enzymes By J. F. Kennedy 1
Introduction General Aspects and Nomenclature Methods of Assay Kinetics Mechanisms o f Action Applications Enzyme Immobilization
2
fi-D-2-Acetamido-2-deoxygalactosidases, B-D-2-Acetamido-2-deoxyglucosidases, 6-D-2-Acetamido-2-deoxyhexosidases
3 a-L-Arabinofuranosidases
347 347 347 348 348 348 349 and
and a-L-Arabinopyranosidases
350 376
4 B-D-Fructofuranosidases
378
5 D-Fructose-1.6-bisphosphatase
383
6 B-D- and a-L-Fucosidases
384
7
aa-
and 8-D-Galactosidases and D-Galactolipid-oriented and B-D-Galactosidases
390
8
a-
and B-D-Glucosidases
414
9 B-D-Glucuronidases
441
10 a-L-Iduronidases
445
11
446
a-
and B-D-Uannosidases
12 Ne ur ami n id a ses ( Sia 1 ida ses 1
450
13 6-D-Xylosidases
455
14
e8-D-2-Ace tamido-2-deoxygluc anases
456
15 Agarases
457
16 Alginases and Alginate Lyases
458
17 a4mylases
458
18 6-Amylases
477
19 Amylo-l,6-D-glucosidases
481
20 Cellulases
482
21
497
Chitinases
22 Dextranases 23
( 1-3 1-aCD-Glucanases
498 499
Carbohydrate Chemistry
X
500
24 endo-( 1-3)-~-D-Glucanases 25
exo- ( 1 -
-
502
4) B-D-Gl ucanases
-
503
endo-(1-6)-B-D-Glucanases
506
28 D-Glucanases (Miscellaneous)
507
26 endo-( 1-4)-~-D-Glucanases 27
29
G 1ucoamy 1a se s
509
30
Glycanases (Miscellaneous)
513
31
Heparin Hydrolases
514
32
Hyaluronidases
515
33
Inulinases
518
34
Isoamylases
35
Laminaranases
519
36
Ly so2 yme s
519
37
Oligo-1 ,6-D-glucosidases
528
3a
Pectate, Pectin, and Poly-D-galacturonate
39
Poly-D-galacturonases
40
exo-Poly-D-galacturonate
41
Pullulanases
534
42
Sucrose-a-D-glucohydrolase
535
Lyases
529 531
Lyases
534
43 aa- and B B-Trehalases
536
44
endo- ( 1*4) -8-D-Xyl -
537
45
Xylanases (Miscellaneous)
539
46
Carbohydrate Isomerases D-Xylose Isomerases (D-Glucose Isomerase) L-Ribose Isomerases
54 1 54 1 544
47
Carbohydrate Oxidases D-Glucose Oxidases L-Galactonolactone Oxidases L-Gulonolac tone Oxidases Cellobiose Oxidases L-Fructose Oxidases D-Galactose Oxidases
545 545 54 a 549 549 549 54 9
anases
48 Carbohydrate T r an sferases
550
49
L-Iduronic Acid 2-Sulphate Sulphatases
555
50
Arylsulphatases
556
xi
Contents 558
51
2-Acetamido-2-deoxy-D-glucose
52
M i s c e l l a n e o u s Enzymes Dextransucrase Syn t h a s e s Lya se s De h y d r o g e n a s e s Levansucrase Pectinesterases
558 558 560 562 562 563 564
G l y c o l i p i d s and G a n g l i o s i d e s By I. M. M o r r i s o n
578
1
Introduction
578
2
A n a l y t i c a l and G e n e r a l Methods
578
3
Gangliosides
579
4
Animal G l y c o l i p i d s
592
5
Plant Glycolipids
605
6
Microb i a 1 G l y c o l i p i d s
609
Chemical S y n t h e s i s and M o d i f i c a t i o n o f O l i g o s a c c h a r i d e s , P o l y s a c c h a r i d e s , G l y c o p r o t e i n s , Enzymes, and G l y c o l i p i d s By C. M. S t u r g e o n
61 8
Chapter 7
Chapter 8
6-Sulphate Sulphates
1 S y n t h e s i s of P o l y s a c c h a r i d e s , O l i g o s a c c h a r i d e s , G l y c o p r o t e i n s , G l y c o p e p t i d e s , and G l y c o l i p i d s Polysacchar i d e s Oligosaccharides Glycoproteins G 1y c o p e p t i d e s Glycolipids 2
3
M o d i f i c a t i o n o f P o l y s a c c h a r i d e s and O l i g o s a c c h a r i d e s a n d Uses of M o d i f i e d P o l y s a c c h a r i d e s and O l i g o s a c c h a r i d e s Introduction Agarose Amylose Cellulose Chitin Chitosan Cycloamyloses De x t r a n s D-Glucan s G 1ycosami n o g l y c a n s D-Mannans M i s c e l l a n e o u s P o l y s a c c h a r i d e s , O l i g o s a c c h a r i d e s , and G 1y c o l i p i d s Starch M o d i f i c a t i o n of G l y c o p r o t e i n s and Uses of Modified G 1y c o p r o t e i n s Albumins Antibodies Phytohaemagglutinins Transferrin
61 8 61 8 61 9 630 63 1 632 633 633 6 36 675 676 67 9 680 68 1 683 685 685 685 686 687 687 688 688 68 9 689
Carbohydrate Chemistry
xii Miscellaneous Glycoproteins Immobilized D e r i v a t i v e s of Glycoproteins Immobilized C e l l s
4 Modification of Enzymes and Uses of Modified Enzymes Acid Phosphatase B-D-Fucosidase B-D-Galactosidase Glucoamylase D-Glucose Isomerase Hya lur onid a s e Lysozyme Miscellaneous Glycoenzymes Immobilized Derivatives of Enzymes
Author Index
689 690 71 4 718 71 8 718 71 8 718 718 718 71 8 719 719 772
Abbreviations
The f o l l o w i n g a b b r e v i a t i o n s have been u s e d : C h e m i c a l s a n d Chemical Groups AD P adenosine 5 I-diphosphate AMP adenosine 5'-phosphate ATP adenosine 5'-triphosphate CDP c y t i d i n e 5'-diphosphate CM P c y t i d i n e 5'-phosphate DEAE N,N-diethylaminoethyl DMF E,N-dimethylformamide DM SO dimethyl sulphoxide DNA deoxyribonucleic acid GDP guanosine 5'-phosphate RNA ribonucleic acid THF tetrahydrofuran RIS trimethylsilyl UDP u r i d i n e 5'-diphosphate P h y s i c o c h e m i c a l Methods c.d. c i r c u l a r d i c h r o is m d.s.c. d i f f e r e n t i a l scanning calorimetry e.s.r. e l e c t r o n s p i n resonance gas-liquid chromatography-mass spectrometry g c -m. s g.1.c. gas-liquid chromatography g.p.c. g e l permeation chromatography h.p.1.c. high-performance l i q u i d chromatography i.r. infrared m.s. mass s p e c t r o m e t r y n u c l e a r magnetic resonance n .m.r. 0.r .d. optical rotatory dispersion t.1.c. thin-layer chromatography U.V. ultraviolet
..
.
1
Introduction BY J. F. KENNEDY
The o b j e c t i v e s o f t h e P a r t I 1 R e p o r t h a v e r e m a i n e d i n t h e a r e a o f p r o v i d i n g a summary o f t h e l i t e r a t u r e o f c a r b o h y d r a t e - c o n t a i n i n g and c a r b o h y d r a t e - d i r e c t e d macromolecules,
but s i n c e t h e i n c e p t i o n of
C a r b o h y d r a t e Chemistry’ f o u r t e e n volumes and t h e r e f o r e f o u r t e e n y e a r s ago,
t h e coverage has n e c e s s a r i l y changed.
T h i s change has o f t e n
b e e n n o t h i n g more t h a n k e e p i n g up t o d a t e w i t h i m p r o v e m e n t s i n t h e s c i e n t i f i c u n d e r s t a n d i n g o f t h e m a c r o m o l e c u l e s concerned.
However,
s i n c e w h a t h a v e come t o be c a l l e d b i o t e c h n o l o g i c a l p r o c e s s e s the l i f e aspects o f
molecules
-
-
i.e.
h a v e become a d r i v i n g f o r c e i n
t o d a y ’ s s c i e n t i f i c t h i n k i n g , s o i n t h i s T i t l e we h a v e c o n t i n u e d t o c i t e a p p l i c a t i o n s of
t h e m a c r o m o l e c u l e s as w e l l as t o r e p o r t on t h e
b a s i c science. The i n t r o d u c t i o n t o
Volume 14 P a r t
comprehensive and s e t t h e scene f o r Report.
111 w a s n e c e s s a r i l y
subsequent
volumes o f t h i s
I n i t , t h e f o l l o w i n g a s p e c t s were covered:
1.
Scope and C o v e r a g e o f t h e R e p o r t ,
2.
O r g a n i z a t i o n , N o m e n c l a t u r e , a n d Use o f t h e R e p o r t ,
3.
S i g n i f i c a n t Advances i n M a c r o m o l e c u l a r C a r b o h y d r a t e
4.
C o n c l u s i o n s and R e a d e r s h i p ,
Chemistry,
and r e a d e r s o f t h i s volume a r e advised t o c o n s u l t t h e p r e v i o u s one t o d e r i v e maximum b e n e f i t . March
1982 saw
Carbohydrate Research
-
the
e a r l y on i n i t s l i f e t i m e , important carbohydrate
publication
of
a n o t a b l e landmark.
the
100th volume
This publication,
of
very
became a p r i m a r y p u b l i c a t i o n c h a n n e l f o r work,
particularly
academic,
and i t i s
p l e a s i n g t o see t h a t t h e j o u r n a l l o o k s e a s i l y s e t f o r a n o t h e r one h u n d r e d volumes.
The s i s t e r j o u r n a l ,
p u b l i s h e s a r t i c l e s more i n t h e f i e l d s research,
Carbohydrate of
Polymers,
which
a p p l i c a t i o n and i n d u s t r i a l
h a s a l o n g way t o go t o c a t c h t h i s u p ,
because i t i s a
2
Carbohydrate Chemistry
much y o u n g e r j o u r n a l .
N e v e r t h e l e s s t h e r e i s scope f o r b o t h j o u r n a l s
t o c o n t i n u e s u c c e s s f u l l y w i t h t h e c o n t i n u a l s i g n i f i c a n t advances i n b o t h a c a d e m i c and i n d u s t r i a l c a r b o h y d r a t e c h e m i s t r y . A s p e c i a l i s s u e o f C a r b o h y d r a t e R e s e a r c h 2 was p u b l i s h e d t o
honour
Professor
Sumio
Umezawa
and
his
work
on
antibiotics,
p a r t i c u l a r l y i n the f i e l d o f sugar-containing antibiotics. Re f e r e n c e s 1.
Kennedy,
J.F.
(Macromolecules), Reports), 2.
i n ed.
‘Carbohydrate
Chemistry’,
Kennedy
(Specialist
J.F.
The R o y a l S o c i e t y o f C h e m i s t r y ,
p.
1.
T.
Tsuchiya,
Carbohydr.
e., 1982,
109,
London,
1.
Part
I1
Periodical
1983,
Vol.
14,
2
General Methods BY R. J. STURGEON 1 G a s - L i q u i d Chromatoqraphy
An i m p r o v e d g.1.c.
method f o r t h e simultaneous d e t e r m i n a t i o n o f
a l d i t o l a c e t a t e s o f n e u t r a l and amino-sugars
has been r e p o r t e d . ’
S h o r t r e t e n t i o n t i m e s and b a s e l i n e s e p a r a t i o n b e t w e e n Q - g l u c o s e a n d a-galactose
were
recorded.
The
method
has
been
used
for
the
a n a l y s i s o f s i a l o g l y c o p r o t e i n s f r o m bone. A c h i r a l p o l y s i l o x a n e s t a t i o n a r y phase has been used i n t h e
9.1. c.
analysis o f mixtures o f neutral
s e p a r a t i o n of 3-g-methylg.1.c.
and a m i n o - s u g a r s . 2
Complete
t h e a l d i t o l a c e t a t e s o f t h e n e u t r a l sugars,
including
and 4 - ~ - m e t h y l - ~ - g l u c i t o l s , was a c c o m p l i s h e d .
Capillary
s e p a r a t i o n o f monosaccharides as t h e i r a l d i t o l a c e t a t e s has
been r e p ~ r t e d . ~
2-Amino-2-deoxy-Q-galactose
and 2-amino-2-deoxy-~-glucose,
a f t e r r e d u c t i o n w i t h sodium b o r o h y d r i d e t o t h e c o r r e s p o n d i n g amino a l c o h o l s , and c o n v e r s i o n t o t h e t r i f l u o r o a c e t y l d e r i v a t i v e s , have b e e n s e p a r a t e d b y g.1.c. flame thermionic
and d e t e c t e d u s i n g a n i t r o g e n - s p e c i f i c
detector.4
The m e t h o d may
be a p p l i e d t o
the
d e t e r m i n a t i o n o f amino-sugars i n glycoconjugates. The q u a n t i t a t i v e g.1.c.
a n a l y s i s o f sucrose i n t h e presence o f
t h e o x i m e s o f Q - g l u c o s e and Q - f r u c t o s e has been r e p o r t e d u s i n g a b u f f e r e d o x i m a t i o n reagent.5
No h y d r o l y s i s o f t h e s u c r o s e t a k e s
p l a c e d u r i n g t h e o x i m a t i o n procedure,
n o r do t h e r e a g e n t s a f f e c t t h e
subsequent s i l y l a t i o n o f t h e d i s a c c h a r i d e . l - M e t h y l - i m i d a z o l e has been used as a s o l v e n t and a c a t a l y s t
f o r t h e p r e p a r a t i o n o f a l d o n o n i t r i l e a c e t a t e s o f aldoses.6 methods have been m o d i f i e d f o r t h e a n a l y s i s o f g l y c o s e s , sugars,
and S m i t h d e g r a d a t i o n p r o d u c t s by u s i n g g.1.c.
of t h e i r aldononitrile acetate
derivative^.^
Existing amino-
determination
M o n o s a c c h a r i d e s h a v e been c o n v e r t e d t o p r o d u c e one d e r i v a t i v e f o r each aldose,
as d e t e c t e d by g.l.c.,
the formation o f aldoximes,
i n a sequence w h i c h i n v o l v e s
t h e i r r e d u c t i o n w i t h borane t o t h e
4
Carbohydrate Chemistry
corresponding aminopolyols,
l-
and s u b s e q u e n t c o n v e r s i o n t o t h e
e t hox y c a r bo n y 1-0t r i m e t hy 1s i1y 1 d e r iv a t iv e s
.
diastereomers
formation
upon d e r i v a t i z a t i o n .
The
K e t o s es pr o d uc e t w o
o f side
and
decomposition products i s n e g l i g i b l e . The m o n o s a c c h a r i d e c o m p o s i t i o n o f d e t e r m i n e d b y g.1.c.-m.s.
of
g l y c o c o n j u g a t e s has been
the g - t r i m e t h y l s i l y l
ethers
after
l i b e r a t i o n o f t h e component s u g a r s , E - d s a c e t y l a t i o n , and d e a m i n a t i o n o f amino-sugars
and n e u r a m i n i c acid.9
6-Deoxyhexoses,
hexose, a n d
p e n t o s e s , d e r i v e d f r o m c a r d i a c g l y c o s i d e s , have been e s t i m a t e d by g.1.c.-m.s. may
also
of be
their g-trimethylsilyl
c a p i l l a r y columns.12 convenient
r e s o l u t i o n g.1.c.
Monosaccharides
These d e r i v a t i v e s have been f o u n d
alternative
quantitation o f
ethers.lO,ll
as 2 - t r i m e t h y l s i l y l a l d i t o l s on f u s e d - s i l i c a
separated
to
the
g-acetate
acid hydrolysates
of
to offer
derivatives
for
polysaccharides.
a
the High
s e p a r a t i o n s o f a l d i t o l a c e t a t e s on f u s e d - s i l i c a
w a l l - c o a t e d open t u b u l a r col u mns ha ve been achieved.13 A d i f f e r e n t i a l g.1.c. i n carbohydrate procedure,
me t h od f o r d e t e r m i n a t i o n o f u r o n i c a c i d s
mixtures
has
be en
deve10ped.l~
I n a two-step
n e u t r a l s u g a r s a r e f i r s t d e t e r m i n e d by g.1.c.
a l d o n o n i t r i l e acetates,
of their
b e f o r e t h e u r o n i c a c i d s i n t u r n a r e reduced
and c o n v e r t e d t o t h e same d e r i v a t i v e s .
The r e l a t i v e p r o p o r t i o n s o f
Q - m a n n u r o n i c a c i d and C - g u l u r o n i c a c i d s i n a l g i n i c a c i d have been d e t e r m i n e d i n a g.1.c.
me t h od . 1 5
After lowering the viscosity o f
t h e a l g i n i c a c i d by l i m i t e d a c i d h y d r o l y s i s ,
c a r b o x y l groups a r e
f i r s t e s t e r i f i e d b y r e a c t i o n w i t h l-ethyl-3-{3-(dimethylamino)propyll-carbodi-imide,
t h e n r e d u c e d w i t h so dium b o r o h y d r i d e , end t h e
r e s u l t i n g m i x t u r e o f hexosans i s c o n v e r t e d by a c i d h y d r o l y s i s t o monosaccharides.
The m o n o s a c c h a r i d e s i n t u r n a r e r e d u c e d w i t h s o d i u m
b o r o h y d r i d e t o h e x i t o l s w h i c h can be a n a l y s e d as t h e b u t a n e b o r o n i c esters. New c h i r a l s t a t i o n a r y p h a s e s f o r t h e g.1.c. enantiomers
of
monosaccharides
amines, have
amino-alcohols,
been
described.l6
separation o f the hydroxy-acids,
The
separation
and
af
m ono s a c c h a r id e e n a n t i o m e r s as t h e 0 - t r i f l u o r a a c e t a t e a is a c h i e v e d on a s t a t i o n a r y phase by a t t a c h m e n t o f ~ - v a l y i - S - a - p h e n y l s t a t i o n a r y phase.
ethylamide t o the p o l y s i l o x a n e U r i n a r y p o l y o l s have been e s t i m a t e d by g.1.c. of
t h e i r acetates.17
I n t e r f e r e n c e i n t h e chromatographic
the
functionalized cyanoethyl
side
chains
of
p a t t e r n s due
t o m o n o s a c c h a r i d e s was o v e r c o m e b y f o r m i n g m e t h y l o x i m e - a c e t a t e d e r i v a t i v e s o f r e d u c i n g sugars.
2: General Methods
5
2 Column a n d I o n - e x c h a n g e C h r o m a t o g r a p h y
Macroporous m i c r o s p h e r i c a l c e l l u l o s e h a s been used as a s t a t i o n a r y p h a s e i n t h e c h r o m a t o g r a p h y o f w a t e r - s o l u b l e h i g h and low m o l e c u l a r weight carbohydrates.18 D i s t r i b u t i o n c o e f f i c i e n t s of t h e polymer molecules depend on t h e hydrodynamic d i a m e t e r s o f t h e polymer molecules. The h y d r o d y n a m i c b e h a v i o u r o f r e d u c e d g l y c o p o l y p e p t i d e s h a s b e e n s t u d i e , d by g e l f i l t r a t i o n i n g u a n i d i n e h y d r o c h l o r i d e i n c o n j u n c t i o n w i t h h.p.1.c.” Even t h o u g h c a r b o h y d r a t e - r i c h g l y c o p e p t i d e s may o c c a s i o n a l l y y i e l d a n u n d e r e s t i m a t e d m o l e c u l a r w e i g h t value, t h e method a p p e a r s t o be u s e f u l f o r t h e r a p i d e s t i m a t i o n of molecular weights of simple polypeptides. The s e p a r a t i o n o f 2-amino-2-deoxy-Q-glucose from 2-amino-2deoxy-n-galactose i n t h e r o u t i n e amino-acid a n a l y s i s of p l a n t glycop r o t e i n s h a s b e e n i m p r o v e d by t h e u s e o f l o n g e r c o l u m n s o n t h e a m i n o - a c i d a n a l y s e r . 2 0 An i m p r o v e d c h r o m a t o g r a p h i c s e p a r a t i o n o f s u g a r s and s u g a r a l c o h o l s on c a t i o n - e x c h a n g e r e s i n s (Ca2+) i s reported.21 The a d d i t i o n o f s m a l l a m o u n t s o f t r i e t h y l a m i n e t o the eluant catalyses the mutarotation of reducing sugars, resulting i n r e d u c e d p e a k w i d t h s w i t h o u t a f f e c t i n g t h e e l u t i o n times. T h e s e p a r a t i o n of s t r o n g l y basic ion-exchange r e s i n s f o r t h e s e p a r a t i o n o f a l d i t o l s f r o m m o n o s a c c h a r i d e s has p r e v i o u s l y b e e n r e p o r t e d a s a method f o r t h e e s t i m a t i o n o f t h e d e g r e e o f p o l y m e r i z a t i o n o f n e u t r a l o l i g o - and p o l y - s a c c h a r i d e s . I n t h i s p r o c e d u r e , t h e a l d i t o l s were r e p o r t e d n o t t o b i n d t o t h e r e s i n , a n d were e s t i m a t e d a f t e r I t h a s now b e e n s h o w n t h a t i n a n u m b e r o f d e r i v a t i z a t i o n by g.1.c. cases a l d i t o l s do b i n d t o t h e r e s i n s , b u t t h e i r e l u t i o n from t h e r e s i n i s i n f l u e n c e d by m e t h a n o l , ammonium a c e t a t e , a n d ammonium forrnate.22 Cation-exchange r e s i n s i n t h e s i l v e r form r e t a i n o l i g o saccharides t o a g r e a t e r e x t e n t t h a n t h e calcium forms o f t h e r e s i n s , r e s u l t i n g i n a g r e a t e r number o f o l i g o s a c c h a r i d e s b e i n g s e p a r a t e d . 23
3 Thin-layer Chromatography
A c o n t i n u o u s - f l o w t.1.c. s y s t e m h a s b e e n d e v e l o p e d f o r t h e i d e n t i f i c a t i o n a n d m e a s u r e m e n t o f u r i n a r y c a r b o h y d r a t e s i m p r e g n a t e d on p a p e r . 24 M u r a m i c a c i d , 2-am i n o - 2 - d e o x y -Q-gl u c o s e , 2-am i n o - 2 - d e o x y Q-galactose,and t h e i r corresponding aminodeoxyalditols have been s e p a r a t e d b y t w o - d i m e n s i o n a l t.1.c. as t h e i r 2 , 4 - d i n i t r o p h e n y l
Carbohydrate Chemistry
6
derivative^.'^
Some o f
the
factors
influencing the derivatization
and s e p a r a t i o n o f t h e s e s u g a r s a r e r e p o r t e d .
4 H i q h - p r e s s u r e L i q u i d Chromatography A r e v i e w o f t h e d i f f e r e n t t y p e s o f s i l i c a and m o b i l e phases t h a t a r e
u s e d i n t h e h.p.1.c.
of
s u g a r s has been published.26
column p r e p a r a t i o n , pre-column d e r i v a t i z a t i o n ,
Methods o f
and d e t e c t i o n methods
are discussed. C h r o m a t o g r a p h y on b o r o n i c a c i d s i l i c a r e d u c e s t h e s e p a r a t i o n t i m e of
diol-containing
compared
to
less
p o l y m e t h a c r y l i c acid.27 the
separation
glycoproteins.
of
s u b s t a n c e s w i t h o u t loss o f
r i g i d
polymers
such
as
r e s o l u t i o n when cellulose
substances
Proteoglycans
such
as
catecholamines
from bovine nasal,
and
bovine a r t i c u l a r
and r a t c h o n d r o s a r c o m a c a r t i l a g e h a v e been a n a l y s e d by h.p.1.c. silica-based
and
T h i s s t a t i o n a r y p h a s e h a s been p r o p o s e d f o r
m a t e r i a l bonded w i t h an a m i d e phase.28
on a
The b i o c h e m i c a l
i n t e g r i t y o f t h e proteoglycans i s r e t a i n e d d u r i n g t h i s procedure. High-performance obtain
data
for
size-exclusion
hydrodynamic
c h r o m a t o g r a p h y h a s been u s e d t o
molecular
radii
of
dextrans.29
G l y c o p r o t e i n hormones and g l y c o p r o t e i n a l l e r g e n s f r o m p l a n t and a n i m a l o r i g i n h a v e been a n a l y s e d by s i z e - e x c l u s i o n c h r o m a t o g r a p h y o n t h e TSK s e r i e s o f m o d i f i e l d s i l i c a s u n d e r h.p.1.c.
condition^.^^
S i l i c a g e l c h e m i c a l l y m o d i f i e d w i t h aminopropyl groups, s i l i c a g e l w i t h 1,4-diaminobutane effective
i n the
h.p.l.~.~l
The
separation o f method
has
n e u t r a l o l i g o s a c c h a r i d e s by
been
used
p u r i f i c a t i o n o f neutral oligosaccharides b r a n c h i n g and d e r i v e d f r o m
or
p r e s e n t i n t h e m o b i l e phase, i s successfully
with d i f f e r e n t
dolichol-linked
in
the
degrees o f
oligosaccharide
intermediates. O l i g o s a c c h a r i d e s (up t o d.p. hydrolysis of
s t a r c h have
12) o b t a i n e d f r o m a c i d and e n z y m i c
been s e p a r a t e d by
reversed-phase
h. p. 1. c. 32 A method f o r t h e r a p i d s e p a r a t i o n o f a n i o n i c o l i g o s a c c a r i d e s has been d e v e l o p e d u s i n g an anion-exchange s u p e r i o r speed,
column which o f f e r s
r e s o l u t i o n , and y i e l d when c o m p a r e d w i t h o t h e r i o n -
exchange m e t h o d s o r h i g h - v o l t a g e
p a p e r e l e c t r o p h ~ r e s i s . ~B~a s e l i n e
r e s o l u t i o n o f anionic oligosaccharides which d i f f e r i n t h e i r net negative charge i s achieved,
r e g a r d l e s s o f whether t h e charge i s
i m p a r t e d by n e u r a m i n o s y l o r p h o s p h a t e m o i e t i e s .
7
2: General Methods A
h.p.1.c.
procedure
for
the
separation
and i s o l a t i o n o f
h a s been d e v e l o p e d u s i n g an i s o c r a t i c
neuraminosyl-oligosaccharides
s y s t e m t o g i v e b a s e l i n e s e p a r a t i o n o f n e u r a m i n i c a c i d , 3’neuraminosyl-lactose.34
A complex m i x t u r e o f
a n d 6‘-
oligosaccharides
o b t a i n e d b y a l k a l i n e b o r o h y d r i d e d e g r a d a t i o n o f human,
bronchial
mucous
on
g l y c o p r o t e i n s has
been s e p a r a t e d
by
h.p.1.c.
bonded
p r i m a r y a m i n e p a c k i n g u s i n g a l i n e a r g r a d i e n t s o l v e n t system.35
The
s e p a r a t i o n o f i s o m e r i c o l i g o s a c c h a r i d e s i s achieved, suggesting t h a t t h e t o t a l number o f h y d r o x y l g r o u p s , as w e l l as t h e i r c o n f i g u r a t i o n , d e t e r m i n e s t h e r e t e n t i o n t i m e o f each o l i g o s a c c h a r i d e . A m e t h o d h a s been d e v e l o p e d f o r s e p a r a t i o n o f r e d u c e d ,
o l i g o s a c c h a r i d e s b y h . p . l . ~ . ~ ~p - G l u c i t o l oligosaccharides
containing
b e t w e e n one
neutral
and r e d u c e d p - g l u c o and t w e n t y
Q-glucosyl
r e s i d u e s have been s e p a r a t e d . The m e t h o d h a s a l s o b e e n a p p l i e d t o t h e analysis
and p r e p a r a t i v e
isolation o f
glycoprotein-derived
o l i g o s a c c h a r i d e s o b t a i n e d by e n z y m i c r e l e a s e by e n d o g l y c o s i d a s e s o r by c h e m i c a l r e l e a s e by h y d r a z i n o l y s i s .
Subnanomolar q u a n t i t i e s o f
o l i g o s a c c h a r i d e s may be d e t e c t e d when s o d i u m b o r o t r i t i d e i s u s e d i n t h e r e d u c t i o n step. The u r i n a r y e x c r e t i o n o f i s o m e r i c c h o n d r o i t i n s u l p h a t e s h a s been m o n i t o r e d by h.p.1.c.
o f t h e unsaturated disaccharides produced
by d i g e s t i o n w i t h c h o n d r o i t i n s u l p h a t e lyases.37 v a r i a t i o n i n t h e c o m p o s i t i o n o f t h e h.p.1.c.
A systematic
m o b i l e p h a s e was u s e d
for the selection o f the optimal conditions for separation. Endo-B-p-2-acetamido-2-deoxy-P-glucanase
a c t i v i t y h a s been a s s a y e d
using the dansyl derivative o f the L-asparaginyl oligosaccharide
(11-
Mane)5-(Q-GlceNAc)2-L-Asn
the
as
the
substrate.38
Analysis
of
product, dansyl 2-acetamido-2-deoxy-~-glucosyl-~-asparagine, has method.
The l o w e r l i m i t
nmol o f dansyl glycopeptides.
Reducing sugars
b e e n a c h i e v e d b y a r e v e r s e - p h a s e h.p.1.c. of
d e t e c t i o n i s 0.1
h a v e been r e a c t e d w i t h d a n s y l h y d r a z i n e p r i o r t o t h e i r s e p a r a t i o n by F l u o r e s c e n t d e t e c t i o n p r o v i d e s a s e n s i t i v i t y o f 10 p m o l
h.p.l.c.39
p e r i n j e c t i o n f o r t h e monosaccharide components o f g l y c o p r o t e i n s . Numerous a l k y l mannosides,
and a r y l P - g l u c o s i d e s ,
Q-glucosiduronic acids,and
d e r i v a t i v e s have been s e p a r a t e d by h . p . l . ~ . ~ ’ h.p.1.c.
been d e s c r i b e d . 4 1
sides.42
Q-
A rapid isocratic
method f o r t h e e s t i m a t i o n o f n e u r a m i n i c a c i d i n serum has
u s i n g 0.003M h.p.1.c.
Q-galactosides,
t h e i r a c e t y l and b e n z o y l
Separation i s achieved on a cation-exchange r e s i n
s u l p h u r i c a c i d as t h e m o b i l e phase.
Reversed-phase
h a s b e e n u s e d i n t h e s e p a r a t i o n o f some m e t h y l g l y c o -
I n t h e aldohexose series,
the pyranosides e l u t e before
8
Carbohydrate Chemistry
furanosides,
but i n the aldopentose series the furanosides e l u t e
first. Mono- a n d d i - s a c c h a r i d e s separated
by
h.p.1.c.
on
and p o l y h y d r i c a l c o h o l s have been
silica
columns
e t h ~ l e n e p e n t a m i n e . ~ The ~ method,
modified with
tetra-
which o f f e r s a h i g h degree o f
r e s o l u t i o n , r e q u i r e s low o p e r a t i n g temperatures. T r y p t i c glycopeptides from t o g r a p h e d u s i n g reverse-phase a hydrophilic for the
p a i r i n g agent,
the analysis of enzymic
reduced,
digestion
chondroitin sulphate,
v i r a l s o u r c e s have been chroma-
ion-pair
p a r t i t i o n chromatography w i t h
phosphoric acid.44
An h.p.1.c.
method
unsaturated disaccharides derived from
and
sodium
borohydride
dermatan sulphate,
h e p a r i n h a s been d e s c r i b e d . 4 5
reduction of
heparan s u l p h a t e , and
The m e t h o d a v o i d s t h e p o s s i b i l i t y o f
o b t a i n i n g anomeric forms o f t h e unsaturated disaccharides. An i m p r o v e d s e p a r a t i o n o f m o n o s a c c h a r i d e s by h.p.1.c. achieved using porous m i c r o p a r t i c l e
Fourteen oligosaccharylpyrophosphoryl dolichols, L-asparagine-linked separated
by
precursors o f
the
o l i g o s a c c h a r i d e s o f g l y c o p r o t e i n s , have been
h.p.1.c.
on
p r o d u c t s were shown t o enzyme-catalysed
h a s been
c a r b o h y d r a t e columns.46
silica
Some
retain their
reactions.
t h e i r b o r a t e c o m p l e x e s by h.p.
activity
of as
the
resolved
substrates
in
M o n o s a c c h a r i d e s h a v e been s e p a r a t e d as a n i o n - e x c h a n g e c h r ~ m a t o g r a p h y . ~The ~
s u g a r s a r e d e t e c t e d f l u o r i m e t r i c a l l y a f t e r s e p a r a t i o n w i t h 2-cyanoacetamide.
Monosaccharide
residues of
glycoproteins,
after
c o n v e r s i o n t o 2 - a m i n o p y r i d y l d e r i v a t i v e s , h a v e been p u r i f i e d by i o n exchange c h r o m a t o g r a p h y and t h e n s e p a r a t e d by h.p.1.c. phase columns.49
on reversed
A l t h o u g h i t was d i f f i c u l t t o d e t e c t mono- a n d d i -
s a c c h a r i d e d e r i v a t i v e s because o f i n t e r f e r e n c e f r o m 2 - a m i n o p y r i d i n e and o t h e r f l u o r e s c e n t m a t e r i a l s ,
higher oligosaccharides
can be
separated. I m p r o v e m e n t s i n a d e t e c t o r f o r l i q u i d c h r o m a t o g r a p h y b a s e d on o p t i c a l a c t i v i t y o f t h e components a l l o w
100 n g o f mono-
or di-
s a c c h a r i d e t o be d e t e ~ t e d . ~ ' S i m u l t a n e o u s d e t e r m i n a t i o n o f s i x c a r b o h y d r a t e c o m p o n e n t s o f human u r i n e ,
which i s i n j e c t e d a f t e r
d e i o n i z a t i o n , h a s been a c c o m p l i s h e d .
I m p r o v e m e n t s h a v e been made t o
the sensitivity o f refractive-index
d e t e c t o r s by e l i m i n a t i n g n o i s e
c a u s e d by p o o r t e m p e r a t u r e c o n t r o l o f t h e d e t e c t o r a n d e l u a n t . 5 1 The u s e o f 2 - c y a n o a c e t a m i d e i n p o s t - c o l u m n d e t e c t i o n o f a l d o s e s has
been
evaluated.52
The
condensation reactions obtained
h e a t i n g carbohydrates w i t h 3-methyl-2-benzothiazolinone alkali
containing
2-methoxyethanol
have
been
by
hydrazone i n described.53
2: General Methods
9
Monosaccharide
aldoses
and
ketoses
and
disaccharides
chromogens w h i c h can be measured a t 390 nm.
t h e method f o r p o s t - c o l u m n d e t e c t i o n o f c a r b o h y d r a t e s h.p.1.c.
produce
The p o t e n t i a l i n u s i n g s e p a r a t e d by
i s discussed.
5 Electrophoresis The a c c u r a c y o f m o l e c u l a r w e i g h t e s t i m a t e s f o r g l y c o p r o t e i n s h a s been e v a l u a t e d
by
SDS-pore
gradient
electrophoresis
of
glyco-
p r o t e i n s o f known m o l e c u l a r w e i g h t and known a m i n o - a c i d
and
c a r b o h y d r a t e c o m p ~ s i t i o n . ~A ~ c o m p a r i s o n was made o f T r i s - g l y c i n e and Tris-borate-e.d.t.a. by 2 - m e r c a p t o e t h a n o l . formation of
b u f f e r s y s t e m s w i t h and w i t h o u t
reduction
A l t h o u g h b o t h t h e g r a d i e n t g e l system and t h e
b o r a t e complexes w i t h t h e i n d i v i d u a l c a r b o h y d r a t e
m o i e t i e s of the g l y c o p r o t e i n c o n t r i b u t e t o t h e p r e c i s i o n o f t h e molecular weight estimate,
i t i s t h e thermodynamic and h y d r o d y n a m i c
p r o p e r t i e s o f t h e i n d i v i d u a l g l y c o p r o t e i n which determine
its
m o b i l i t y on g e l e l e c t r o p h o r e s i s . L e c t i n s have been us ed f o r transferred separation
to
on
the
d e t e c t i o n of
n i t r o c e l l u l o s e sheets polyacrylamide
asialoglycopeptides
have
gels.55
been
after
glycoproteins
electrophoretic
Fluorescein-derivatized
resolved
by
polyacrylamide
gel
e l e c t r o p h ~ r e s i s . ~When ~ t h e method i s used i n c o n j u n c t i o n w i t h s p e c i f i c exoglycosidase d i g e s t i o n o f the glycopeptides, correlation
i s
observed
between
the
relative
a linear
electrophoretic
m o b i l i t y and t h e number o f m o n o s a c c h a r i d e r e s i d u e s removed by t h e enzymes. a-Glucuronic
a c i d and I - i d u r o n i c
a c i d may be s e p a r a t e d by
e l e c t r o p h o r e s i s on T i t a n I11 c e l l u l o s e a c e t a t e p l a t e s . 5 7
The method
has been a p p l i e d t o t h e a n a l y s i s o f u r o n i c a c i d s i n c h o n d r o i t i n 4and 6 - s u l p h a t e s
and dermatan s u l p h a t e .
A n a l y t i c a l i s o t a c h o p h o r e s i s has been used i n t h e e s t i m a t i o n o f n e u r a m i n i c acid.58
Under t h e c o n d i t i o n s o f assay n e u r a m i n i c a c i d
a p p e a r s as a t r o u g h b e t w e e n t w o buffer.
U.V.
i m p u r i t y components o f t h e
The t r o u g h l e n g t h i s p r o p o r t i o n a l
t o t h e amount
of
n e u r a m i n i c a c i d i n j e c t e d i n t o t h e system. E l e c t r o p h o r e t i c and c h r o m a t o g r a p h i c methods f o r s e p a r a t i n g c a r b o h y d r a t e s have been r e v i e w e d i n t h e l a t e s t e d i t i o n o f a t r e a t i s e on t h e c h e m i s t r y and b i o c h e m i s t r y o f
carbohydrate^.^'
Carbohydrate Chemistry
10 6 A n a l y t i c a l Methods Modifications o f
the
Park-Johnson
ferricyanide
submicromethod
for
t h e assay o f r e d u c i n g g r o u p s i n c a r b o h y d r a t e s have been r e p o r t e d . 6 0 O x a l i c a c i d i s used as a s o l v e n t f o r f e r r i c f e r r o c y a n i d e i n p l a c e o f sodium d o d e c y l s u l p h a t e , w h i c h g i v e s t u r b i d s o l u t i o n s . When a l d o s e s
are determined i o d o m e t r i c a l l y
magnesium o x i d e ,
f o r m a t i o n o f i o d a t e ions.61
i n t h e presence o f
t a k e s p l a c e w i t h t h e minimum
no o v e r - o x i d a t i o n
C o l o r i m e t r i c analyses o f carbohydrates
have been c a r r i e d o u t on a l i q u i d s c i n t i l l a t i o n c o u n t e r o v e r a w i d e r r a n g e o f c o n c e n t r a t i o n s t h a n can be measured w i t h a c o n v e n t i o n a l l i g h t spectrophotometer.62 hydroxybenzoic
The u s e o f a l k a l i n e s o l u t i o n s o f 4 -
acid hydrazide for the determination o f reducing
s u g a r s i n e x t r a c t s o f p l a n t t i s s u e s has been e v a l u a t e d . 6 3 w i t h a s e n s i t i v i t y range o f
An e n z y m i c m i c r o a s s a y f o r l a c t o s e , 12.5-500
n m o l p e r sample, h a s been developed.64
upon t h e h y d r o l y s i s o f l a c t o s e t o 4 - g l u c o s e
The method i s based
and 4 - g a l a c t o s e
by
0-4-
g a l a c t o s i d a s e f o l l o w e d b y t h e c o l o r i m e t r i c a s s a y o f t h e f r e e Qglucose u s i n g t h e Q-glucose oxidase procedure. A s i m p l e d e t e c t i o n method f o r 9 - g a l a c t o s e phosphate i n blood-impregnated screening
test
for
and Q - g a l a c t o s e
1-
f i l t e r p a p e r has been d e v e l o p e d as a
galactosaemia.65
When
Q-galactose
( o r Q-
g a l a c t o s e 1-phosphate a f t e r t r e a t m e n t w i t h phosphatase) i s o x i d i z e d by a - g a l a c t o s e d e h y d r o g e n a s e ,
t h e r e s u l t i n g NADH may be m e a s u r e d
I n a s i m i l a r approach t h e m i c r o d e t e r m i n a t i o n o f
fluorimetrically.
Q - g a l a c t o s e and 1 - g a l a c t o s e 1 - p h o s p h a t e i n d r i e d b l o o d s p o t s has been achieved.66 galactose determined phosphate
Q-Galactose i s determined f l u o r i m e t r i c a l l y
dehydrogenase using ester
the with
whilst
same
the
Q-galactose
procedure
alkaline
after
phosphatase.
with
1-phosphate
4i s
hydrolysis
of
I n Nelson’s
Somogyi
method f o r t h e d e t e r m i n a t i o n o f r e d u c i n g s u g a r s , r e a g e n t has been r e p l a c e d by t h e F o l i n - C i o c a l t e u
the
t h e arsenomolybdate p h e n o l reagent.67
The method compares f a v o u r a b l y w i t h t h e o r i g i n a l method i n s t a b i l i t y o f c o l o u r and r e p r o d u c i b i l i t y . a n a l y t i c a l element,
A new t y p e o f m u l t i l a y e r f i l m
consisting o f a spreading layer,
a blocking
l a y e r , an e n z y m e l a y e r ( c o m p o s e d o f a - g l u c o s e o x i d a s e ,
peroxidase
and
dye),
and
a
transparent
layer,
d e t e r m i n a t i o n o f 1-glucose i n blood.68 i n d r o p p e d on t h e f i l m ,
has
been d e v e l o p e d
for
the
A f t e r a spot o f whole b l o o d
the a-glucose concentration i s determined
without further manipulations. S u l p h y d r y l r e a g e n t s such as c y s t e i n e and g l u t a t h i o n e i n t e r -
11
2: General Methods fere
with
the
P-glucose
oxidase
assay
for
Q-glucose.69
This
i n t e r f e r e n c e c a n be e l i m i n a t e d by u s e o f N - e t h y l m a l e i m i d e .
Q-
G l u c o s e has been e s t i m a t e d e n z y m i c a l l y
using a commercial analyser
a f t e r e l u t i o n f r o m b l o o d s p o t t e d on t o
filter
paper.70
An oxygen-
s t a b i l i z e d enzyme e l e c t r o d e has been d e s i g n e d f o r t h e a n a l y s i s o f Qg l u c o s e i n f e r m e n t a t i o n broths.71 I n a new e n z y m a t i c method f o r t h e d e t e r m i n a t i o n o f [)-glucose human s e r a ,
t h e h y d r o g e n p e r o x i d e p r o d u c e d by Q - g l u c o s e o x i d a s e
measured f r o m
in
is
t h e change i n absorbance due t o o x i d a t i o n o f NAD(P)H
i n t h e presence o f catalase,
a l d e h y d e dehydrogenase, and a h i g h
c o n c e n t r a t i o n o f ethanol.72 S e v e r a l a d v a n t a g e s o f t h i s m e t h o d , a s c o m p a r e d w i t h o t h e r Qg l u c o s e o x i d a s e methods c o u p l e d w i t h l e u c o dyes, estimation
o f Q-glucose
hexokinase-Q-glucose
are recorded.
i n haemolysed b l o o d samples
The
using the
6-phosphate dehydrogenase method has been
described.73 Two
new
systems
have
been
developed
measurement o f pH i n s m a l l volumes.74
for
the d i f f e r e n t i a l
The a p p a r a t u s i s c a p a b l e o f
f o l l o w i n g t h e pH change caused by h e x o k i n a s e - c a t a l y s e d Q - g l u c o s e a n d ATP, 18 m M .
r e a c t i o n of
when t h e s u g a r c o n c e n t r a t i o n i s i n t h e r a n g e 1-
The s y s t e m can be used t o m o n i t o r [ ) - g l u c o s e c o n c e n t r a t i o n s i n
biological fluids.
Optimal
p r o p e r t i e s o f D-glucose been s t u d i e d
i n order
d e t e r m i n a t i ~ n . ~The ~
reaction conditions
and k i n e t i c
dehydrogenase f r o m B a c i l l u s m e q a t e r i u m have to
develop a method
Em
value
of
the
for
serum q - g l u c o s e
enzyme f o r
Q-glucose
i n f l u e n c e d by t h e pH o f t h e medium and by i o n i c s t r e n g t h . conditions end-point
f o r t h e use o f R-glucose assay
i s
Suitable
dehydrogenase i n r a t e assay and
methods were i d e n t i f i e d .
a-Glucose
concentrations
d e t e r m i n e d i n serum u s i n g t h e s e methods show good c o r r e l a t i o n w i t h t h o s e o b t a i n e d w i t h t h e h e x o k i n a s e method.
n-Glucose
has been c o v a l e n t l y a t t a c h e d t o n y l o n t u b i n g . 7 6
dehydrogenase
The a c t i v i t y and pH
o p t i m u m o f t h e bound enzyme depends on t h e t r a n s i t i o n m e t a l s u s e d as s p a c e r s b e t w e e n t h e enzyme and t h e s u p p o r t . A
fluorimetric
r e l e a s e d from
method f o r t h e measurement
glycoproteins
by a - L - f u c o s i d a s e
o f a-l-fucopyranose has been r e p o r t e d . 7 7
The I - f u c o s e i s o x i d i z e d w i t h I - f u c o s e d e h y d r o g e n a s e a n d NAD, a n d t h e NADH p r o d u c e d i s used i n c o n j u n c t i o n w i t h d i a p h o r a s e t o c o n v e r t the nonfluorescent
dye r e s a z u r i n t o t h e h i g h l y f l u o r e s c e n t
product
resorufin. I-Fucose
has been measured r a d i o m e t r i c a l l y
u s i n g a method
c a p a b l e o f m e a s u r i n g q u a n t i t i e s o f t h e s u g a r a s l o w a s 25 p m 0 1 . ~ ~
Carbohydrate Chemistry
12
The a s s a y c o u p l e s I - f u c o s e d e h y d r o g e n a s e t o I = - g l u t a m a t e d e h y d r o genase and L - g l u t a m a t e d e c a r b o x y l a s e t o y i e l d 14C02 w h i c h i s d e r i v e d f r o m {l-14C)-a-ketoglutarate.
L-Fucose
dehydrogenase has been used
p r e v i o u s l y i n f l u o r e s c e n c e assays o f t h e sugar, limited
when
the
sample
to
be
assayed c o n t a i n s
fluorescent
substances,
commonly
glycopeptides
and membrane
preparations
on c o lu m n s h a v i n g a r e l a t i v e l y
b u t t h e method i s
observed
endogenous
i n
hydrolysed
a t low operating pressures
l e n g t h y l i f e and h i g h l i n e a r sample
capacity. The p h l o r o g l u c i n o l m e t h o d f o r t h e e s t i m a t i o n o f Q - x y l o s e i n u r i n e has been a s s e s s e d w i t h r e s p e c t t o t h e i n t e r f e r e n c e f r o m l o w l e v e l s o f ~-glucose.'g A micromethod f o r t h e d e t e r m i n a t i o n o f f r e e o r g l y c o p r o t e i n bound n e u r a m i n i c a c i d on t h e b a s i s o f o x i d a t i o n w i t h p e r i o d i c a c i d and
reaction
with
thiobarbiturate
has
been
A
described.80
o f the t h i o b a r b i t u r i c acid technique for the d e t e r m i n a t i o n o f n e u r a m i n i c a c i d ha s been developed.81 Elimination modification
o f i n t e r f e r e n c e caused by 2 - d e o x y - P - r i b o s e
without
loss o f n e u r a m i n i c a c i d ,
extraction
of
the
chromogen.
i s
The
and s e v e r a l o t h e r sugars, achieved
by
neuraminic
pH-dependent
acid
in
level
s i a l o g l y c o c o n j u g a t e s ha s been measured i n a new e n z y m i c method a f t e r r e l e a s e f r o m g l y c o p r o t e i n s w i t h n eu rami n i da se.82
Neuraminic a c i d i s
cleaved t o release pyruvate using N-acetylneuraminic followed peroxide
by o x i d a t i o n o f which
pyruvate oxidase t o
i s measured c o l o r i m e t r i c a l l y
chlorophenol-4-am
i n o - a n t i p y r i n e m e t ho d.
a c i d aldolase,
produce
by t h e
hydrogen
peroxidase-4-
C o r r e l a t i o n o f r e s u 1t s
w i t h t h o s e o b t a i n e d u s i n g a c h e m i c a l m e t h o d o f a n a l y s i s was reported. Free neuraminic a c i d s p l i t from red-blood-cell receptors t o N e w c a s t l e d i s e a s e v i r u s has been d e t e r m i n e d d u r i n g s i m u l t a n e o u s e l u t i o n and h a e m o l y s i s o f t h e c e l l s . 8 3 The p r o c e d u r e depends on t h e presence o f neuramin id ase a c t i v i t y on t h e preadsorbed v i r i o n s . F l u o r e s c a m i n e 14 -p h e ny 1s p ir o ( f u r a n - 2 - ( 3H ),1' -( 3 ' H ) furan)-3,3'-dione}
- i so b e n z o -
may be a p p l i e d t o t h e q u a n t i t a t i v e a n d q u a l i -
t a t i v e a n a l y s i s o f amino-sugars.84
The m e t h o d h a s b e e n a p p l i e d t o
of acid hydrolysis of chitin. Methods have been d e v e l o p e d f o r t h e i d e n t i f i c a t i o n o f t h e g l y c o s i d i c
the
analysis
of
the
product
l i n k a g e s b e t w e e n amino-sugars
a nd I - a s p a r a g i n e ,
C - s e r i n e , and
C,-
t h r e o n i n e i n g l y c o p r ~ t e i n s . ~A~f t e r a l k a l i n e e l i m i n a t i o n i n t h e p r e s e n c e o f s o d iu m b o r o t r i t i d e , sugar
components
separation
o f
of the
a c i d hydrolysis,and
0-glycosyl dansyl
linkages
are
hexosaminitols
dansylation, identified by
the
after
thin-layer
13
2: General Methods electrophoresis
.
The 1i n k age co m po un d 4! -( 2 -ace t a m ido -2 -deo x y -S-E
glycosidic
linkage
region of
glycoproteins
by
-
L-
g l u c o p y r a n o s y l ) hydrogen I - a s p a r a g i n a t e i s i s o l a t e d from t h e
partial acid
h y d r o l y s i s and i d e n t i f i e d as i t s d a n s y l d e r i v a t i v e . The m a n n i t o l l e v e l s i n serum samples f r o m dogs a f t e r m a n n i t o l i n f u s i o n have been measured u s i n g t h e s p e c t r o p h o t o m e t r i c measurement o f t h e i n i t i a l r a t e o f NADH f o r m a t i o n when t h e sugar i s o x i d i z e d by a b a c t e r i a l m a n n i t o l dehydrogenase p r e p a r a t i o n . 8 6
7 S t r u c t u r a l Methods A comprehensive
review
o f p h y s i c a l methods used f o r s t r u c t u r a l
a n a l y s i s o f c a r b o h y d r a t e s has been p u b l i s h e d . 5 9 high-resonance
n.m.r.
spectroscopy,
spectroscopy, and X - r a y Spectroscopy.-A
N.m.r.
m.s.,
The methods i n c l u d e
polarimetry,
U.V.
and i.r.
diffraction. r e v i e w d e a l i n g w i t h 13C n.m.r.
spectroscopy
o f p o l y s a c c h a r i d e s has been p ~ b l i s h e d . ~ ' The p r i m a r y s t r u c t u r e s o f p o l y s a c c h a r i d e s and t h e i r d e r i v a t i v e s have
been
investigated
c r o s s p o l a r i z a t i o n n.m.r.
using
magic-angle
13C
spectroscopy.88
spinning-
Although the r e s o l u t i o n o f
t h e i n d i v i d u a l c a r b o n r e s o n a n c e s was n o t p e r f e c t when x a n t h a n gum and i t s o c t y l a m i d e d e r i v a t i v e w e r e u s e d , considerably
the technique provided
more d e t a i l t h a n t h e c o r r e s p o n d i n g 13C n.m.r.
i n an e q u i v a l e n t p e r i o d o f t i m e .
A 1 3 C n.m.r.
studies
a n a l y s i s method has
been d e s c r i b e d f o r t h e i d e n t i f i c a t i o n o f c a r b o h y d r a t e r e s i d u e s i n
n-
g a l a c t o s i d e s a n d Q - g l u c o ~ i d e s . ~A ~ s s i g n m e n t s c a n be made t o t h e a n o m e r i c c o n f i g u r a t i o n and t o t h e r i n g s i z e o f t h e s u g a r s . of
13C
s p e c t r o s c o p y i s a p r a c t i c a l method f o r f o l l o w i n g t h e k i n e t i c s
N.m.r.
enzymic
digestion
of
individual
carbohydrate
residues
of
g l y c o p e p t i d e s and f o r d e t e r m i n i n g t h e s t r u c t u r e o f t h e p r o d u c t s o f p a r t i a l d i g e ~ t i o n . ~ ' I n a s t u d y o f t h e h y d r o l y s i s o f hen o v a l b u m i n g l y c o p e p t i d e s by a-n-mannosidase,
t h e method showed t h a t some a-n-
mannopyranosyl-(1 + 3)-P-mannopyranosyl faster
r a t e than the corresponding ( 1
The r e s o l u t i o n - e n h a n c e d
*
500 MHz 'H
oligo-(a-mannopyranosyl)-l-asparagines are
chemical-shift
mainly
n.m.r.
spectra o f three
N4-
can be i n t e r p r e t e d i n t e r m s o f
t h e complete p r i m a r y s t r u c t u r e s o f these structures
linkages are cleaved a t a
6 ) linkages.
compound^.^^
b a s e d upon, i n t e r p r e t a t i o n o f
The p r o p o s e d the
sets o f
v a l u e s o f H - 1 s and H-2s o f c o n s t i t u t i n g P-mannopyran-
14
Carbohydrate Chemistry
o s y l residues.
The d a t a a r e s e n s i t i v e t o t h e t y p e and c o n f i g u r a t i o n
of t h e g l y c o s i d i c l i n k a g e o f t h e Q - m a n n o p y r a n o s y l r e s i d u e and t o t h e p o s i t i o n o f t h a t su ga r i n t h e c h a i n . M as s S p e c t r o m e t r y , -
Carbohydrates,
have
using
been
measured
a
mass
a f t e r s e p a r a t i o n by h.p.l.c., detector.92
The
solvent
i s
evaporated a f t e r n e b u l i z a t i o n i n a heated column producing f i n e l y d i v i d e d s o l u t e p a r t i c l e s w h i c h p a s s t h r o u g h a l i g h t beam.
Light
s c a t t e r e d f r o m t h e p a r t i c l e s i s d e t e c t e d by a p h o t o m u l t i p l i e r and a m p l i f i e d on a c h a r t r e c o r d e r . with refractive-index
The mass d e t e c t o r ,
detection,
when c o m p a r e d
showed a t e n - f o l d
increase i n
s e n s i t i v i t y , i m p r o v e d s t a b i l i t y , a n d an a b i l i t y t o d e t e c t p r o d u c t s s e p a r a t e d by g r a d i e n t e l u t i o n . By c a r e f u l s e l e c t i o n o f d e r i v a t i v e s a n d a n a l y s i s c o n d i t i o n s , m.s.
has
been
applied
dodecasaccharide
to
derived
a n a l y s i s b y m.s.
of
oligosaccharides
has
methylated aldoses
the
from
a
microscale
some s y n t h e t i c f u l l y been
have
reported.94
been
sequencing
g l y ~ o l i p i d . ’ ~ The
separated
of
a
structural
methylated I-rhamno-
A
number
of
partially
by
g.1.c.
on
capillary
columns as t h e i r t r i m e t h y l s i l y l a t e d d i e t h y l d i t h i o a c e t a l d e r i v a tives.”
S i n g l e peaks f o r each d e r i v a t i v e a r e o b t a i n e d and m.s.
u s e d i n t h e i d e n t i f i c a t i o n o f peaks.
is
Thirteen methyl ethers o f
m e t h y l N-acetyl-N-methyl-a-Q-neuraminate
m e t h y l g l y c o s i d e have been
N-
i d e n t i f i e d b y g.1.c.-m.s.
a f t e r p a r t i a l methylation o f methyl
acetyl-u-g-neuraminate
methyl g l y ~ o s i d e . ’ ~ Chemical-ionization
(methane) m.s. of
partially
has been used t o enhance t h e s e n s i t i v i t y o f d e t e c t i o n methylated
alditol
acetates
of
neutral
and
a m i n d - s ~ g a r s . ~The ~ mass s p e c t r a o f 5 2 p a r t i a l l y m e t h y l a t e d a n d acetylated methyl
glycosides
of
a-galactose,
a-mannose,
Q-glucose
and 2-ace t a m id o - 2 - d eo xy - Q - g l u cose have been d e t e r m i n e d . 98
A scheme
f o r t h e r a p i d d e t e r m i n a t i o n o f t h e p o s i t i o n o f t h e m e t h y l and a c e t y l r e s i d u e s is proposed. Field-desorption
m.s.
has
been
used
to
analyse
s a c c h a r i d e s c o n t a i n i n g f i v e t o f o u r t e e n he xose u n i t s , derivatization.”
The
molecular
weight
o f the oligosaccharide
be d e t e r m i n e d by means o f t h e a bu nd an t q u a s i - m o l e c u l a r t y p e MNa+,
MH’,
MNa22+,and
MNa33+.
structural elucidation of
x
i o n s of
can the
The me t h od has t h e p o t e n t i a l f o r
oligosaccharides
p r o d u c t s u p t o a r a n g e o f 3.5
oligo-
without p r i o r
lo3
and t h e i r
mass u n i t s .
reaction
Neuraminic a c i d
i s o l a t e d f r o m e r y t h r o c y t e g h o s t s h a s b e e n e s t i m a t e d by m e t h a n e chemical-ionization
m.s.
of
the 2 - t r i m e t h y l s i l y l
methyl glycoside
15
2: General Methods
-
u s in g p h e n y 1 2 - a c e t am ido - 2 d e o xy -( 3,4,6- tr i-0- t r i m e t h y 1s i 1y 1)-a-Q g l u c o p y r a n o s i d e as i n t e r n a l s t a n d a r d . l o O sugar
can
be
detected
i n
the
-
Nanogram l e v e l s o f t h e
presence o f
other
contaminating
substances. The a n o m e r i c c o n f i g u r a t i o n o f a 2 - a c e t a m i d o - 2 - d e o x y - Q glucopyranosyl residue linked t o
a
secondary
h y d r o x y l group
a n o t h e r s u g a r r e s i d u e h a s b e e n e s t a b l i s h e d f r o m g.1.c.-m.s.
of
data
o b t a i n e d when s u c h a d i s a c c h a r i d e is s u c c e s s f u l l y t r e a t e d w i t h periodate,
s o d i u m b o r o h y d r i d e , and a c e t i c anhydride.”’
Miscellaneous
Methods.-A
modification
o f
the
Hakomori
m e t h y l a t i o n r e a g e n t has been r e p o r t e d , u s i n g p o t a s s i u m h y d r i d e i n place o f
sodium
hydride.lo2
The
preparation o f
the
potassium
m e t h y l s u l p h e n y l m e t h i d e i s f a s t e r a n d may b e p e r f o r m e d a t a m b i e n t temperature.
Significantly
fewer
impurities
i n
the
reaction
p r o d u c t s a r e a l s o observed. I n t e r n a l 8-2-substituted
N-glycolylneuraminic
m e t h y l a t e d s a c c h a r i d e s undergo d e - N - a c y l a t i o n f u l l y methylated N-glycolylneuraminic
acid residues o f
whereas t h e t e r m i n a l
a c i d r e s i d u e s do n o t u n d e r t h e
conditions o f m e t h a n o l y s i ~ . ~ ~ T h~i s, ~d i~f f~e r e n c e i n t h e s t a b i l i t y o f N - a c y l g r o u p s h a s been o b s e r v e d i n p o l y s i a l o s y l c h a i n s c o m p r i s i n g either N-glycolylneuraminic
a c i d or N-acetylneuraminic acid.
On m e t h y l a t i o n and s u b s e q u e n t s a p o n i f i c a t i o n ,
a-a-glucopyranosyluronic galactopyranosyluronic
.
acid)-Q-xylose
acid)-a-xylose
2-2-(4-g-methyland
4-g-(a-Q-
g i v e f o u r and t w o
methyl
g l y c o s ides, r e s p e c t ive l y lo5 I n v e s t i g a t i o n o f t h e i r s t r u c t u r e s u s i n g t h e H a k o m o r i m e t h y l a t i o n p r o c e d u r e showed t h a t t h e 4 - 2 - s u b s t i t u t e d uronic acid residues afford 6-elimination products i n addition t o the permethylated derivatives. u n s u b s t i t u t e d 4’-OH
The a l d o b i o u r o n i c a c i d w i t h a n
g r o u p gave t h e p r o d u c t s o f d i r e c t m e t h y l a t i o n .
The h y d r a z i n o l y s i s - n i t r o u s
acid deamination o f glycopeptides
leads t o the s p e c i f i c cleavage o f 2-acetamido-2-deoxy-~-glucosyl l i n k a g e s . lo6 The 2,5-anhy d r o -P-mannose-con t a i n i n g
o l i g o s a c c h a r i des
thus o b t a i n e d a r e r e d u c e d w i t h sodium b o r o h y d r i d e , m e t h y l a t e d , and a n a l y s e d by
g.1.c.-m.s.
P a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s have been s e p a r a t e d by g.1.c.
on g l a s s c a p i l l a r y columns.107
column and t h e
application to
s a c c h a r i d e s are r e p o r t e d .
Some c h a r a c t e r i s t i c s o f t h e
structural analysis of
poly-
Methylation techniques i n the s t r u c t u r a l
a n a l y s i s o f g l y c o p r o t e i n s and g l y c o l i p i d s h a v e been r e v i e w e d . l o 8 Kinetic
data
for
eleven
common
methyl glucopyranosidea
i n u n b u f f e r e d s o d i u m p e r i o d a t e h a v e b e e n reported:”
The r e s u l t s
16
Carbohydrate Chemistry
i n d i c a t e t h e e x t e n t o f v a r i a t i o n s i n r e a c t i v i t y t h a t can a r i s e f r o m differences i n configuration, i l l u s t r a t e the r o l e
o v e r a l l reaction rates. branches
on
the
e s p e c i a l l y a t t h e anomeric centre,
intermediate
0.f
The s i t e o f s u b s t i t u t i o n o f p e r i p h e r a l
core
Q-mannosyl
residues
carbohydrate u n i t s o f glycoproteins
of
complex-type
have been s t u d i e d by S m i t h
By u s i n g g l y c o p e p t i d e s o f known s t r u c t u r e
degradation.’”
i s s h o w n t h a t t h e s i t e o f a t t a c h m e n t o f t h e (1
( 1 ) it 4)-linked, t h i r d
+
b r a n c h t o t h e Q-mannose c o r e i n t r i - and t e t r a - a n t e n n a r y may be i d e n t i f i e d . used
for
and
hemiacetals i n determining
the
cleavage
of
the
structures
i s the concentration o f a c i d
The c r i t i c a l f a c t o r
periodate-oxidized
product.
The
r e s u l t s support t h e concept t h a t i n g l y c o p r o t e i n s from most sources the
(1 + 4 ) - l i n k e d
branch i s attached t o
the
(1
+
3)-linked
mannosyl r e s i d u e o f t h e core p o r t i o n i n t h e complex-type The
possible
conformations
analysed using semi-empirical neuraminic
acid
may
hydroxymethyl group of i s
consistent
with
preponderantly i n the
m e t h y l acarboxyl
’H- a n d 13C-n.m.r. C.d.
and B - a - g l y c o s i d e s
of
a r o u n d 220 nm a r i s e group
and a r e
p o s i t i v e f o r B-linked
acid
i n
been
different
the
terminal
The p r e s e n t m o d e l
spectroscopic
data,
but
s p e c t r a have been r e c o r d e d f o r
neuraminic acid.’l2
from
negative
have
I n solution,
two
orientation of
t h e g l y c e r o l s i d e chain.
d i f f e r s from e a r l i e r models. effects
neuraminic
p o t e n t i a l f u n c t i o n s . 11’
exist
conformations which d i f f e r
of
Q-
structures.
t h e ,” for
+
H*
The C o t t o n
transition of
a-q-linked
glycosides
the and
glycosides.
The r e s u l t s o f X - r a y d i f f r a c t i o n a n a l y s i s o f t h e c r y s t a l l i n e c o n f o r m a t i o n o f homo- and r e g u l a r h e t e r o - Q - g l u c a n
c h a i n s have been
used t o e s t a b l i s h t h e h y p o t h e s i s t h a t t h e g l y c o s i d i c l i n k a g e t y p e i s t h e d e t e r m i n a n t o f p o l y s a c c h a r i d e c o n f o r m a t i o n . 11’
I n t h i s respect,
p o l y s a c c h a r i d e s a r e more l i k e s y n t h e t i c p o l y m e r s t h a n p r o t e i n s o r n u c l e o t i d e s . I n t h e l a t t e r i t is v a r i a t i o n s i n t h e s u b s t i t u e n t s w h i c h are responsible f o r the conformational diversity.
The c o n t i n u i n g
c o m p i l a t i o n o f c r y s t a l s t r u c t u r e s o f carbohydrates,
n u c l e o s i d e s , and
n u c l e o t i d e s has been p u b l i s h e d . An i m p r o v e d m e t h o d f o r
the determination o f
the molecular
w e i g h t s o f o l i g o s a c c a r i d e s by a c o m b i n a t i o n o f p a p e r e l e c t r o p h o r e s i s and
h.p.1.c.
reported.l15
of
their
2-aminopyridyl
derivatives
has
been
The e l e c t r o p h o r e t i c m o b i l i t i e s o f t h e d e r i v a t i v e s a r e
i n d e p e n d e n t o f l i n k a g e p o s i t i o n s , a n o m e r i c c o n f i g u r a t i o n s , and t h e p r e s e n c e o f a c e t a m i d o groups, 0-Q-Glucans,
but
not
up t o m o l e c u l a r w e i g h t s o f 1.7 x their
oligo-
or
lo3.
mono-saccharide
17
2: General Methods
R - B - D - G ~ ~ ~ N A-t~ 2)-a-D-Manp-(l - ( ~ -m
R-D-GlcpNAc-(1 -
-
+
6)-B-D-Manp=
3 4 1
-f
4)-a-D-Manp =
2
+
1 6-D-G1 c ~ N A c =
( 1 ) R = o t h e r glycosyl r e s i d u e s
+
R
18
Carbohydrate Chemistry
degradation products, interact with Congo Red.116 Radial diffusion of 6-Q-glucanases into a substrate-bearing gel slab has been used a s a sensitive method for assay o f the enzymes, by measuring the clearing zone formed by enzyme action on the substrate. The use of glycosyltransferases in assessing oligosaccharide structure has been reviewed."' Re fe rences 1 2 3 4 5 6 7 8 9 10 11 12
13 14 15 16 17 18 19 20 21 22 23 24
T.Anastassiades, R.Wzic, and O.Puzic, J.Chra~togr., 1981, 225, 309. C.Green, V.M.Doctor, G.Holzer, and J.Or0, J.Chranatogr., 1981, 207, 268. J.Klok, E.H.Nieberg van Velzen, J.W.Leeuw, and P.A.Schenck, J.Chromatogr., 1981, 207, 273. T.Shinohara, J.Chranatogr., 1981, 207, 262. K.J.S&affler and P.G.More1 du Boil, J.ChrmtOgr., 1981, 207, 221. C.C.Chen and G.D.M&innis, Carbohydr.Res., 1981, 90, 127. S.H.Turner and R.Cherniak, QrMydr.Res., 1981, 95, 137. H.Frank, H.J.C.das Neveqand E.Bayer, J.Chranatoqr., 1981, 207, 2l3. I.Mononen, Qrbohydr.Res., 1981, 88, 39. and W.Kubelka, J.Chranatoqr. , 1981, 210, 291. B.Kow, J.Jureni-, J.Jurenitsch, B.Kapp, LGabler-Kolacsek,and W.Kubel)ca, J.Chromatogr., 1981, 210, 337 AXW.inl.dbury, D.J.Halliday, and D.G.Medcalf, J.Chranatogr., 1981, 213, 146. R.Oshirm, A.Yoshikawa, and K.Kumanotani, J.Chranatogr., 1981, 213, 141. J.L&rfeld, Anal.BiOdlgn., 1981, 115, 410. H.S .Prihar, B.K. Bugashetti,ad D.S .Feingold, Anal .Biochm., 1981, 114,294. W.A.Ktjnig, I.Benecke,and S.Sievers, J.Chranatoqr., 1981, 217, 71. J.N.Mount and M.F.Laker, J.Chranatogr., 1981, 226, 191. Y.A.Eltekov, N.M.Strakhova, J.K&lal, J.Pegka, and J.Stamberg, J.pOlym.Sci., PolymSymp., 1980,68, 247. N.Ui, J.Chranatogr., 1981, 215, 289. I.E.P. Taylor, P h y t m h d s t r y , 1981, 20, 2769. L.A.T. Verhaar and B.F.M. Kuster, J.Chranatogr. , 1981, 210, 279. M.Tanaka, Carbohydr.Res., 1981, 88, 1. H.D.Smbel1 and KM.Bmbst, J.ChranatOgr., 1981, 212, 51. J.R.Alonso-Fendndez, M.D. Bbveda, C .Parrado, J.Peih, and J.M.Fraga - ., J.Chranatogr., 1981; 217, 357. M.J.Talieri, N.Kilic,and J.S.Thampson, J.Chrarratogr., 1981 206, 353. L.A.T.Verfiaar and B.F.M.Kuster, J.Chranatosr., 1981, 220, 3U. KGlad, SDhlson, Uansson, M.O.Mansson, and IGMosbach, J.Chromatogr., 1981, 200, 254. EXSchwartz, J.Stevens, and D.E.Scfimidt , Anal.Biod'lem., 1981, 112, 170. M.E.Hhr?l and P.G.Squire, J.Chranatogr., 1981, 210, 443. D.H.Calam and J.Davidson, J.ChramtOgr., 1981, 218, 581. S.J.Turm, Andl.Biochem., 1981, 118, 278. N.W.H.Cheetham, P.SirimaMe,and W.R.Day, J.Chra~togr., 1981, 207, 439. J.U.Baenziger and M.NaWicz, Anal.Biochem., 1981, 112, 357. M.Le Bergh, P.Koppn,and D.H.van den Eijnden, Carbohydr.Res., 1981, 94, 225. RBoersm, GLamblin, P.Degad, ad PJXowsel, CarbohydrJIes., 1981, 94, C7. c7. S.J.Mellis and J.U.Baenziger, Anal.Biod'Iem., 1981, 114,276. G.J.L.Lee and H.TieckelmaM, J.Chrarratogr., 1981, 222, 23. H.Iwase, T.Morinaga, Y.T.Li,and S.C.Li, Anal.Biochan., 1981, 113,93. W.F.Alpfels, Anal.Biochem., 1981, 114, 153. K.Yoshida, K.Kotsubo, and H.ShigaMtsu, J.Chranatogr., 1981, 208, 104. H.K.B.Silver, KAKrim, M.J.Gray, and F.A.Salinas, J.Chromatogr., 1981, 224, 381. ~
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
2: General Methods
42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79
80 81 82 83 84 85 86 87 88 89 90 91 92
19
N.W.H.Cheetham and P.Sirixnanne, J.Chrana r., 1981, 208, 100. D.L.Hendrix, R.E.Lee, J.G.Ba~t,and H.J%J.Chr-Gr., 1981, 210, 45. M. C.Kenp, W.L.Hollaway , R.L.Prestidge, J.C.Bennett, and R.W.Canpans, ., 1981, 4, 587. J.Li id ChrG.J.L?Lee, D.W.LzJ.W. Pav,and H.Tieckelmann, JShramtoqr., 1981, 212, 65. M.T.Yang, L.P.Milligan,and G.W.Mathisan, J.Chrana r., 1981, 209, 316. 1981, 110, G.B.Wells, S.J.Turco, BAHanson,and R.L.Lester ,?nal.BiochemT 397. S.Honda, M.Takahashi, K.Kakehi,and S.Gann0, Anal.Biochan., 1981, 113,130. S.Hase, T.Ikenaka, and Y.Matsushh, J.Biochem. (Tokyo), 1981, 90, 407. J.C.Ku0 and E.S.Yeung, J.Chranatogr., 1981, 223, 321. S.I.M.Johncodc and P.J.Wagstaffe, Analyst, 1980, 581. S.Honda, M.Takahasi, Y.Nishimura, K.Kakehi, and S.Gann0, Anal.Biochem., 1981, 118, 162. S.Honda, Y.Nishimura, H.Chiba,and K.Kakehi, Anal.Chim.Acta, 1981, 131,293. J.F.Poduslo, AMl.BioChem., 1981, 114,131. W.F.GLass, R.C.Briggs, and L.S.Hnilica, Anal.Biochem., 1981, 115,219. R.D.Poretz and G.Pieczenik, Anal.Biochem., 1981, 115, 170. I.Miymto and S.Nagase, Anal.Biochem., 1981, 115,308. E.Weiland, W.Thorn, and F.Bl&r, J.Chranatogr., 1981, 214, 156. M.I.Horowitz, in 'Carbohydrates: Chemistry and Biochemistry', 2nd Edition, ed. W.Pigman and D.Horton, A m d d c Press, New York, 1980, Vol.lB, p.1445. M.Porro, S.Viti, G.Antoni, and P.Neri, Anal.Biochem., 1981, 118, 301. H.S.Isbel1 and H.L.Frush, Carbohydr.Res., 1981, 92, 131. I.M.Morri-n and R.E.Brice, Carbhydr.Res., 1981, 98, 237. M.J.Kozio1, Anal.Chim.Acta, 1981, 128,195. D .P.Kotler, A.R.Tierney, and N.S .Rosenweig, A M 1.Bi&em., 1981, 110, 393. H.Misuma ,H.Wada,M.Kawakami ,H.Ninomiya, and T.Shohmori ,Clin.Chim.Acta, 1981, 111, 27. Y.Fujimura, S.Ishii, M.Kawamura, and H.Naruse, Anal.Biochem., 1981, 117,187. C.Hatanaka and Y.Kobara, Agric.Biol.Chgn., 1980, 44, 2943. ROhkubo, S.Kamei, M.Yamanaka, F.Arai, M.Kitajima,and RKondo, Clin.Chem., 1981, 27, 1287. N.Haugaard, J.Cutler, and M.R.Ruggieri, Anal.Biochem., 1981, 116, 341. R.Taylor and C.Pennodr, Clin.Chan., 1981, 27, 1624. S.O.Enfors, Eslzyme Microb.Te&nol., 1981, 3, 29. F.Hehz and T.W.Beushausen, J.Clin.Chen.Clin.Biochem., 1981, 19, 977. H.Sdlebusch, M. Sarger, E.Munz, A.C.Kessler, and W.Zwz, J.Clin.Chem.Clin. Biochan., 1980, 18, 885. RMosca, G.Gossi, M.Luzzana, L.R.Bernardi, W.S.Friauf, R.L.Berger, H.P.Hopkins, a d V.Carey, Anal.Biochem., 1981, 112, 287. M.Sugiura, S.Hayakawa, Y.It0, and K.Hirano, Chem.Pharm.Bull., 1981, 2,146 E.Biss&, A.Scholer,and D.J.von der Schmitt, J.App.Biochan., 1981, 2, 176. M.A.Cchenford, J.C.Urbammki,and J.A. Dain, Anal.Biochem., 1981, 112, 76. D.S.Grove and G.S.Serif, Aml.Biochem., 1981, 111, 122. J.P.Straub, Clin.Chem., 1981, 27, 198. R.Kattermann and R.Krieger, J.Clin.Chan.Clin.Biochen., 1981, 19, 31. J.Roboz, M.Suttajit,and J.G.B&esi, Anal.Biochem., 1981, 110, 380. K.Sugahara, K.Sughto, O.Nanura, and T.Usui, ClhChhActa, 1981, 108,493. B.Rivetz,M.A.Lipkind,E.Shichmanter, and E.Bogin,Experientia, 1980, 36, 370. A.C.Chen and R.T.Mayer,J.Chrawitogr., 1981, 207, 445. D.C.Farwel1 and A.S.Dian, Aml.Biochem., 1981, 113, 423. C.H.Blomquist, B.D.Snyder, and W.G.Neihaus, J.Clin.Chem.ClinBiochem., 1981, 19, 139. P.A.J. Garin, Adv.Carbohydr.Chem.Biochem., 1981, 38, 13. L.D.Hal1 and M.Yalpani, Carbhydr.Res., 1981, 91, Q. R.C.Beier, B.P.Mundy, and G.A.Strobe1, Can.J.Chem., 1981, 58, 2800. E.Berman and A.Allerhand, J.Biol.Chem., 1981, 256, 6657. H. van Halbeek, L.Dorland, GA.Veldink, J.F.G.Vliegenthar t, J.C.M ichalski, J.Montreui1, G.Strecker, and W.E.Hull, FEBS Lett., 1980, 2,65. R.MacRae and J.Dick, J.Qlranakgr., 1951, 210, 138.
m,
20
93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 ll5 116 117
Carbohydrate Chemistry
M.E.Brek, G.C . H a n s s a n , K.-A.Karlsm, H .&if ler, W .Pimlott,and B.E.Samuelsson, FEES Lett., 1981, 124,299. V.KovbEik, P.KodE, and A.LiplAk, Carb0hydr.Res. ,1981, 98, 242. S.Honda, M.Nagata,and K.Kakehi, J.ChraMtogr., 1981, 209, 299. C.Bnnrier, Y.Leroy, J.MontreUi1, B.Fournet, and J.P.Kamerliq, J.Qlrcmatogr. , 1981, 210, 487. R.A.Laine, Anal.Biochan., 1981, 116, 383. B.Fournet, G.Strecker, Y.Leroy, and J.Montreui1, AnaLBiochem., 1981,,&I 489. M.Linscheid, J . D a n g o ~ , A.L.Burlingam, A.Dell,and C.E.Ballou, Proc.Natl .Acad.Sci .USA, 1981, 78, 1471. J.Ashraf , DAButterfield, J.Jarnef elf and RALaine, J.Lipid Res., 1980,2& 1137. S.R.Sarfati, Chrbohydr. Res., 1981, 94, 236. L.R.Phillips and B.A.Fraser, Carbohydr.Res., 1981, 90, 149. S.Inoue and G.Matsumura, FEE!S Lett., 1980, 12, 33. S.I~~W? and M.Iwasaki, Biochen.Biophys.Res.Carm., 1980, 2, 162. K.Shimizu, Carbohydr.Res., 1981, 92, 65. G.Strecka, A.PieraeCretel, B.Fournet, G.Spik, and J.Montreui1, 17. Anal.Bi&an., 1981, I&, N.Shihya, J.chramatogr., 1981, 208, 96. H.Rauvala, J . F h , T.Krusius, J.Karkkainen,and J.Jarnefelt, Adv.Carbohyr.ch&.Bicchan., 1981, 38, 389: K.M.Aalmo and T.J. Painter, Carbohydr.Res., 1981, 89, 73. T.Krusius and J.Finne, Carbohydr.Res., 1981, 90, 203. K.Veluraja and V.S.R.Rao, Biochim.Biophys.Acta, 1980, 630, 442. H.Ogum and K.Furuhata,'Tetrahedron Lett., 1981, 22, 4265. R.H.Marchessault and Y.Deslandes, Chrbohydr.Polymers, 1981, I, 31. G.A.Jeffrey and M.Sundaralingan, Adv.Carbohydr.Chem.Bi&en., 1981I 38, 417. S.Hase, T.Ikenaka,and Y.Matsushima, J.Bio&an.(Tokyo), 1981, 90, 127'5. P.J.Wocd, Carbohydr.Res., 1981, 94, C19. T.A.Beyer, J.E.Sadler, J.I.Rearick, J.C.Paulson, and R.L.Hil1, Adv.Enzp1 .Related Areas Mol .Biol , 1981, 52, 23.
.
3
Plant and Algal Polysaccharides BY I. M. MORRISON 1 Introduction
volume of the comprehensive treatise on the biochemistry of plants has been devoted to the structure and function of carbohydrates.' The proceedings of a symposium on the mechanism of saccharide polymerization and depolymerization have been published with several articles relevant to this chapter.' The state of macromolecular chemistry at the present time, with particular reference to the pulp and paper industry, has been r e ~ i e w e d . ~ A
2 Starch
The structure of the hydrated amylose-iodine complex has been determined by combined methods of X-ray diffraction and stereochemical packing analysis and shown to be an orthorhombic 13.60, = 23.42,and 2 (the fibre unit cell with dimensions of 2 Two amylose chains pass through the unit cell repeat) = 8. and iodide ions were present as an almost linear chain in the centre of the six-fold, left-handed amylose helix. Eight molecules of hydration per unit cell were located in good hydrogen-bonding positions between the amylose helices. The crystal structure of lJh amylose has been refined by similar methods to show another orthorhombic unit cell of dimensions ,a = 13.65, b = 23.70,and 2 = 8.05i.5 The chain conformation is a left-handed, six-fold helix with 0-6 in the position Bauche to both 0-5 and C-4. The water molecules are located inside the helical channel of the amylose and in the interstitial spaces, forming an intensive hydrogen-bonded network. Starch has been degraded by plasma and thermolysis in the ionization chamber of a mass spectrometer, and the s u g h and oligosaccharides formed were characterized by direct chemical ionization mass spectrometry.6 The technique, especially with negative ions, extends mass spectrometry to polymers. The molecular-size distribution of modified corn starch has
22
Carbohydrate Chemistry
been determined by size-exclusion chromatography. The high performance liquid chromatography of malto-oligosaccharides has been reported. The degree of mechanical damage imparted to starch granules during flour milling has been correlated with various absorbancies in the near -infrared reflectance spectrum. The wavelengths of importance corresponded to overtones and combinations of vibration frequencies due to free and hydrogen-bonded hydroxy groups in the starch. A resonance Raman spectroscopic study has been made of the blue Similar spectra were colouring of iodine/iodide in amylose.l o obtained regardless of the KI and I 2 concentrations, degree of polymerization, and excitation wavelengths, but the relative intensities of the lines changed. It was concluded that the basic unit changed from 162- to I 2- through I s2- with decreasing KI concentration. A simple straight-line relationship has been obtained between average chain length of linear amyloglucans and extent of iodine staining." The wavelength of maximal optical absorbance, the maximal absorbance,and the iodine-binding capacity per chain are all linearly related to the chain length. The sorption of water and water-soluble alcohols by starch granules in aqueous suspension has been studied,and potato and corn starches absorb 33 and 28% water, respectively, at pH 7.'* The method was not suitable for cationic starches. Alcohols were also absorbed in large amounts. The kinetics of adsorption of 2,3-dialdehydostarch can be expressed by an exponential equation uniform heterogeneous which is applicable for energetically cellulose surfaces.13 The activation energy did not depend on the initial concentration of starch in solution but increased linearly with the increased amount of starch adsorbed. Some physicochemical properties of heat-moisture treatment of starches have been determined. The water-binding capacity and enzyme susceptibility increased, while the swelling power decreased. The starches with the highest moisture content before heating had the lowest swelling powers. The same treatments had adverse effects on the functional properties and baking potential o f the starches.15 An enzymatic and an acidic hydrolysis procedure have been compared f o r the analysis of starch in feeds and digesta.16 Comparable results were obtained for purified starches and feeds with high starch contents. Only in treated materials like digesta
23
3: Plant and Algal Polysaccharides
with more than 17% cellulose did problems arise due to hydrolysis of cellulose by the acidic treatment. The enzymes do not need to be highly purified to be used in starch determination even in the presence of cellulose and hemicelluloses. A series of n.m.r. spectra of whole seeds of various types have been obtained by using cross-polarization magic-angle spinning techniques. Selected signals provided a means of comparing the protein content relative to the starch content within a group of seed varieties. The multi-branched nature of amylose samples from several plant sources has been revealed by methods for estimating reducing and non-reducing residues. The isoamylase of a Pseudomonas partially split the branch linkage in potato amylose. The concurrent action of pullulanase from Aerobacter and @-amylase from sweet potato hydrolysed the amylose completely. An enzymatic method of determining the A and B chains in amylopectin has led to a ratio of 1 : 1 , not 2:l as previously suggested. 2o Partial debranching with pullulanase gave results A chains are consistent with earlier suggestions that predominantly and selectively removed by this enzyme. The changes in physicochemical properties of starches isolated from maize, barley,and triticale grains which had been allowed to sprout for various lengths of time have been determined.2’ The water-binding capacity initially decreased but then increased with increasing time whereas swelling power decreased and solubilities and enzyme susceptibilities both increased with time, A procedure adapted for the rapid analysis of total starch and amylose has been used on a series of 37 barley genotypes.22 The amylose content was not related to either starch content or grain yield. Investigations of the starch granules of wheat throughout their development have suggested that there are two distinct populations of granules which are initiated at separate stages of kernel d e v e l ~ p m e n t . ~This ~ theory was supported by results using a Coulter counter as well as light and electron microscopy and chemical analysis. In studies on the characterization of legume starches, the fine structures of amylose and amylopectin have been determined by the use of pullulanase, 6-amylase, and glucoamylase .24 The results indicated tbat there were some 1+6 substituted a-D-glucopyranosyl residues in amylose. Similar studies were carried out on
’
24
Carbohydrate Chemktry
acid-treated starches.25 A fast hydrolysis of amorphous and gel phases occurred with a slow hydrolysis of crystalline regions. The results were consistent with the structure being a cluster type In attempts to separate the starch and protein fractions of legumes, the starch fraction was found to contain between 7.7 and 20.1% protein while the protein fraction contained 0.0-4.6% starch.2 6 During germination of dry beans at 22OC, the total starch, amylose, and amylopectin contents all declined over the first 6-day period when the amy1ose:amylopectin ratio also declined.27 The solubility and swelling characteristics of the starch from the Great Northern bean have been found to be both temperature and pH dependent. 2 8 The solubility was highest at pH 6.0. Oxidation increased the solubility as a function of temperature whereas acetylation decreased the solubility. Acetylation and oxidation both reduced the swelling while addition of free fatty acids reduced the viscosity and raised the gelatinization temperature. The gelatinization temperature of the starch from the winged bean was 60-70°C and it exhibited single-stage swelling and low s ~ l u b i l i t y . ~It ~ was very soluble in DMSO and had an amylose content of 38.5%. The gelatinized starch was more susceptible to a-amylase and glucoamylase than the native starch. The morphological and physicochemical properties of starch from mature green and ripe bananas have been deter~nined.~' The granules were irregularly shaped with spheroid and elongated forms predominating. Other parameters were similar to those of the starch from mung beans. The starch from mature, unripe breadfruit has been isolated and characterized by the use of pullulanase and The @-amylase followed by gel-permeat ion chromatography. granules were 10-20 pm in size with an amylose content of 18.2% and 8-amylolysis limit of 58%. The debranched amylopectin had chains of 15 and 38-45 residues,respectively, in the ratio 4.4:l. The relationships between contents of dry matter, starch, total fermentables, and specific gravity in cassava storage roots have = a been determined.32 A curvilinear relationship of the form + 5 was found to exist between dry matter and starch content and a similar one for specific gravity and starch. The native starch from fermented cocoa beans has been isolated by solvent extraction and flocculation of non-starch material at pH 2.7.33 The egg-shaped granules had a mean size of 4 . 4 pm, an
-
'
x-'
3: Plant and Algal Polysaccharides
25
amylose content of 30.4%, a gelatinization temperature of 52.5in 1M KOH. 68°C and a limiting viscosity number of 166.2 ml l'g Kuzu starch contains 21% amylose and 124 mg kg-' p h o ~ p h o r u s . ~ ~ The B-amylolysis limits of the amylose and amylopectin were 76 and 57, respectively, with 20.5 being the average chain length of the amylopectin. The granules were of the Ca crystalline type. A non-aqueous procedure, using glycerol and 3-chloro-1,2-propanediol, has been developed for the isolation from maize of starch granules with associated metabolite^.^^ Electron microscopy failed to demonstrate the presence of the amyloplast's membrane but it may be dried onto the granule surface. Soluble metabolites were found which may derive from the stroma of amyloplasts. The distribution of starch components of maize (Zea mays) and their properties have been investigated by gel filtration after debranching and by exhaustive hydrolysis of the granules with glucoamylase .36 Based on the results, the relationship between genotypes and properties of starch components was discussed. For gene produced example, introduction of the amylose-extender ( E ) amylopectins of longer average chain lengths. The starch and phytoglycogen fractions from normal and sugary (E) maize have been compared, and negligible differences were observed between 2 inbred lines and 2 9~ conversions of the lines studied.37 The phytoglycogen was shown to be distributed in a soluble fraction and a particulate fraction which also contained some amylose. These observations are consistent with a pattern of conversion of starch into phytoglycogen in endosperm. The starch of wild rice (Zizania aquatica) has been isolated and is a very small granule (2-7 urn) of angular and polygonal shape.38 It has an A-type pattern on X-ray diffraction but an amylose content of only 2%. Other properties were compared with those of wheat starch. Normal- and waxy-type starches have been isolated from two types of Amaranthus hypochondriacus and found to be round granules ( 1 urn diameter) with similar X-ray diffraction diagrams to other A-types. 39 The amylose contents were 1 4 and O%, respectively,while the amylopectins were similar to those from rice and maize starches. The elephant yam (Amorphophallus campanulatus) has yielded a starch which exhibited single-stage swelling and moderate solubility in water but low resistance to solubilization in
26
Carbohydrate Chemistry
DMS0.40 The amylose content was 24.5%. The amylolytic susceptibility of the native starch was low but that of the gelatinized starch was high. Oxidation of malto-oligosaccharides has yielded the series of oligomers with aldonolactones at the reducing end position and these give diamides with alkylenediamines in 30-755 yield.41 The effect of these derivatives on the enzymatic activity of potato phosphorylase was determined. The digestion of hydroxypropyl wheat starches by porcine pancreatic a-amylase has been examined.42 The digestibility of the of gelatinized starches decreased with increasing degree substitution, and the action pattern favoured a non-repetitive attack whereas the digestibility of the native starches increased with extent of modification. The hydrolysates from the above digestion of starches with degrees of substitution from 0-0.17 were separated into total digest, oligo- and polysaccharide fractions and further analysed .43 Increasing the degree of substitution caused a reduction in the reducing value, an increase in the average degree of polymerization, and a greater proportion of oligosaccharides in the hydrolysate. The oligosaccharide fraction had double the hydroxypropyl content of the undigested starch. The oligosaccharide fraction was further analysed by h.p. 1.c. on a u-BondapakR carbohydrate column .44 The proportion of oligosaccharides with three and four ;-glucose residues increased and those with five !-glucose residues decreased as the degree of substitution increased. The changes in molecular weight of amylose and amylopectin during thermal dispersion in water have been followed by paper Grain disaggregation and mo lecular chromatography.4 5 disintegration of amylopectin proceeded more rapidly than in amylose. The effect of sodium hydroxide was more marked on amylose than amylopectin. Aqueous suspensions of starch at neutral pH values were heated by microwave energy in sealed tubes.46 The colour of the hydrolysates darkened with increasing time and pressure. The total acidity increased and oligosaccharides in the range monomer to octamer were determined. The amounts of glyceraldehyde, dihydroxyacetone, and 2-hydroxyl,+propanedial induced in maize starch by Y-radiation have been determined by a g.c. procedure,and the influence of several .parameters such as dose and temperature has been in~estigated.~’
27
3: Plant and Algal Polysaccharides
This is the first report of 2-hydroxy-1,3-propanedial from irradiated starch. The influence of the polysaccharide structure on the oxidation process with Ce4+, Mn3+, and V 5 + ions has been studied,and it was confirmed that the rates were highest for compounds containing a-glycol groups.48 The nature of the functional groups and the conformation of the macromolecular unit exert an influence on the oxidation rate. An enzymatic procedure has been developed to measure starch in cereal product^.^' The precision of the test is 2 1.5% and is applicable to a wide range of products. The amylases from pearl millet have an action pattern similar to those of other cereal a-amylases but are able to hydrolyse wheat starch more readily than millet starch.50 The immature wheat endosperm aleurone with seed coat and embryo detached has produced considerably less a-amylase activity than immature whole or de-embryonated wheat kernels. The isoenzyme composition of the incubated endosperm aleurone was unique suggesting that the seed coat may contain factors required for normal a-amylase isoenzyme synthesis. An enzymatic assay for the determination of a-amylase in serum has been developed which employs maltoheptaose as substrate and a coupled enzymatic indicator system.52 H.p.1.c. was used to establish the action pattern of hydrolysis of the maltoheptaose. The stoichiometric equations for the action of a-amylase alone and a-amylase and a-glucosidase together were derived. The multiple-attack model for the kinetics of hog pancreatic a-amylase action has been a n a l y ~ e d . ~The ~ model explains the dependence of ,V and 2 on the degree of polymerization of substrates. The distribution in the degree of polymerization of the hydrolysis products has been used to calculate the number of unitary movements of the enzyme during the single enzymesubstrate meeting. A mixture of 6-amylase and pullulanase from Bacillus cereus has been used to produce maltose from a variety of starches which had previously been liquefied by the a-amylase of Bacillus ~ u b t i l i s . ~ ~ The maximum yield was 87-88% obtained at pH 6.0-8.0, 55"C,and 30h hydrolysis of a 5% solution. Similar results were obtained if acidic conditions were used for the liquefaction. Two inbred lines of rye (Secale cereale), whose kernels had only 1-3% of the 6-amylase activity of normal lines, have been
'
28
Carbohydrate Chemistry
investigated using an anti-wheat B-amylase immune serum which cross-reacted with the rye enzyme.55 The results indicated that the reduced activity was due neither to the presence of an inhibitor nor to the production of inactive enzymes. An inducible cell-bound glucoamylase, which degrades cyclodextrins, has been isolated from a Flavobacterium species. 56 The final degradation product in all cases was ;-glucose with small amounts of maltose. Amylopectin and glycogen were very poor substrates. The enzymes of starch metabolism in the developing grains of a high-lysine barley mutant have been determir~ed.~’The activity of ADP-E-g1ucose:starch synthetase was lower while those of UDP-Eglucose-pyrophosphorylase and starch phosphorylase were higher than in normal barley. The structural changes in starch molecules during malting of barley have been investigated by comparing a normal malt where 14% of the starch had been lost with an over-modified malt where 68% of the starch had been lost.58 In the normal malt, 14% of the large granules were eroded but the small granules remained intact whereas 38% of the large granules were eroded in the over-modified malt with a marked reduction in the small granules. Malting resulted in an apparent increase in the amylose content. There was no apparent difference in the amylopectin structure between the two malts. A reduction in light intensity during starch synthesis in developing wheat grains has reduced the starch synthetase I activity but not the starch phosphorylase activity. 59 The low starch production was not due to an insufficient supply of assimilates. [ 35S]-Methionine has been incorporated into B-amylase from germinating rice seeds.60 The specific radioactivity of the enzyme derived from scutellum was significantly greater than that from the endosperm. These results indicate that, at the onset of germination, 6-amylase is synthesized in the scutellum and only at a later stage is an inactive, latent form of the enzyme activated in the endosperm. This latter form becomes dominant during the later stages of germination. The role of the recessive amylose-extender ( E ) allele in the biosynthesis of maize starch has been investigated.61 Multiple forms of starch synthetase were observed, some of them appearing at the same time as the rapid increase in amylose content. The
29
3: Plant and Algal Polysaccharides
presence of modified amylopectin synthesized by the enzymes which were affected by the ae allele was suggested. Debranching of these amylopectins followed by analysis of the products was attempted to confirm this theory, but the results were inconclusive. Starch granule-bound D-glucan synthetase has been isolated from cotton leaves.63 On further separation, amylopectin was the only glycosidically linked with protein and this had E-glucan Q-glucan synthetase activity. The amylose component was free from covalently bound protein. On germination, both in the dark and light, a DMSO-soluble P-glucan is synthesized in the cotyledons of lupins .64 Chemical analysis indicated that it was starch-like but its chromatographic behaviour differed from that of amylopectin. The concentrations of starch synthetase and debranching enzyme from developing seeds of Pisum sativum have been mea~ured.'~ Primed starch synthetase activity increased from day 8 to day 14 after anthesis but then decreased by 50% at 26 days,whereas citrate-stimulated starch synthetase activity was highest at 10 days after anthesis thereafter falling to a low value. Debranching activity increased from day 8 to day 18 after anthesis. The changes in starch synthetase and debranching activity resulted from changes in the concentration of a few enzyme forms and not from the appearance of different enzyme forms. The @-amylase activities of some soybean varieties are times lower than others yet they have similar starch metabolism. It was therefore concluded that starch metabolism was independent of the 8-amylase activity. The starch contents of plantains and bananas, ripened under similar conditions, have been determined with a content of 1% in ripe bananas and 9% in ripe plantain^.'^ These values fell to zero and 3%,respectively,in over-ripe material. Phosphorylase I1 from potato tubers has been purified and characterized.68 No separation of primed from unprimed activity was achieved. The molecular weight was 9.6 x l o 4 and the native enzyme was a dimer. The reaction product was a protein-bound P-glucan which possessed long ( 1+4)-a-E-glucan chains. 6 9 Since protein was also present in amylose from potato starch grains, it is suggested that these findings may be further evidence of a from a precursor common biosynthetic pathway for (1+4)-a-D-glucans protein. The rate of starch breakdown in isolated chloroplasts from
'*
''
Carbohydrate Chemistry
30
spinach is similar to that found in whole leaves. These rates have been measured both in the dark and in light and compared with the rates of starch synthesis in the light.’l’ Illumination has little inhibitory effect on breakdown at high phosphate levels but 67% inhibition was observed at low phosphate levels. Since C02 evolution is prevented by illumination, the oxidative pentose phosphate pathway must have been inhibited. The balance between accumulation and breakdown is very sensitive to the rate at which carbon is withdrawn from the chloroplast in exchange for inorganic phosphate. The unusually high solution torque developed by the starch from the Hinoat variety of oats when a hot paste is cooled to 75-80°C has been abolished in the presence of acetic acid at pH 3.0.’12 Salts at 0.1M concentration reduced the torque value as did increasing sucrose concentrations. The gelatinization of wheat starch as modified by xanthan, guar and cellulose gums has been investigated and each gum hastens the onset of initial paste viscosity.’13 The reaction seemed to be a strong association with the amylose component since the B-amylolysis limit of the amylose is reduced. The starch-xanthan gum exhibited a pseudoplastic behaviour whereas the starch-guar gum resisted shearing at low shear rates. The syneresis of curdlan gel has been repressed by the addition of starch before heating but not by other sugars.74 The role of starch in this process was discussed. A number of starch esters of herbicides have been prepared from activated pregelatinized starch and evaluated as slow release agents.’15 With sterile soil a picloram ester was phytotoxic for 48 days. Unmodified starch esters were hydrolysed at a slower rate than gelatinized starch esters. A Flavobacterium from tap water was able to grow on tap water but the addition of starch greatly enhanced its growth.76 X-Ray and neutron-diffraction studies on hexa-amylose hexahydrate crystal forms A and B have given an indication of the chain-like and circular arrangement of hydrogen bonds within the molecules. ’17 In the circles, homodromic and antidromic orientations of the hydrogen bonds are observed, with the homodromic circles being more stable by 8% per hydrogen bond. The changes in electronic charges on H and 0 atoms are greater in homodromic circles and the dipole moments are only ~2.30 in the chain-like and antidromic homodromic circles but 6-8D in
-
3: Plant and Algal Polysaccharides
31
arrangements. -X-Ray analysis of the hexa-amylose-3-nitroaniline ( 1 : l ) hexahydrate complex has shown that the hexa-amylose molecules are stacked along the z-axis in a head-to-tail fashion to form a channel-type structure .78 A stereo-drawing of the packing feature of the complex with its associated water molecules was shown. Similar studies have been conducted on the hexa-amylose-benzaldehyde ( 1 : l ) hexahydrate complex and a similar packing was observed with the hexa-amylose molecule being tilted at 11.5" against the channel axis." The benzene ring is inserted into the hexa-amylose ring from the secondary hydroxy side while the carbonyl group is in Van der Waals contact with the primary hydroxy side of the next hexa-amylose molecule. A correlation between the penetration depth of the C atoms of a variety of phenols in the cavity of hexa-amyloseand the changes in chemical shifts has been investigated.80 The their 1 3 C n.m.r. formation and molecular dynamics of cycloamylose inclusion complexes with phenylalanine have been investigated by 1 3 C n.m.r. spectroscopy.8 The influence of the cavity size was determined and the cyclohepta-amylose-phenylalanine system was most strongly coupled. The cyclohexa-amylose coupling was shallow and loose while that of the octa-amylose was deep and loose. The induced c.d. and U.V. spectra ofhepta-amylose-disubstituted benzene systems has been studied with respect to their molecular structures.82 The dipole moment was correlated with rotational intensity and the energy-level transition calculated by molecular orbital methods. The use of a hexa-amylose mobile phase in the separation of mono-substi tuted phenols by t .I. c. has been reported .83 The llf values depended on both the structural features of the phenolic compounds and the concentration of hexa-amylose in the mobile phase. A selective attack of dichlorocarbene at the 4-position of phenolate ion was achieved with hexa-amylose as catalyst.84 The mechanism was studied by lH and 13C n.m.r. spectroscopy.
3 Fructans The has and
crystalline conformation of inulin [(2+1)-B-E-fructofurananl been determined by conformational analysis coupled with g-ray electron- diffraction data.85 The only two models possible
32
Carbohydrate Chemistry
correspond to 5-fold helices, one left-handed and the other right-handed. Steric interactions of the substituents and the exanomeric effect were believed to be responsible for the observed conformational differences. An invertase and an inulase from germinating garlic (Allium sativum) bulbs have been separated and purified.86 The invertase had no effect on inulin whereas the inulase, as well as hydrolysing inulin, could hydrolyse sucrose and raffinose. The enzymes had similar molecular weights (7.6 x l o 4 ) . In the early stages of development the activities increased in paralle1,but at later stages the invertase activity was higher. A pathway for fructan trisaccharide synthesis from sucrose has been reported.87 Two non-reducing hexasaccharides ( 1 ) and (2) have been isolated from the roots of Asparagus officinalis and their structures confirmed by the usual methods.88 A novel tetrasaccharide ( 3 ) has been isolated from Chinese milk vetch honey.89 It is probably synthesised 2 erlose which is produced from sucrose in nectar.
4 Cellulose A history of the study of cellulose structure has been published which also considered how this knowledge has been extended to synthetic polymers. A hydrate of cellulose I1 has been prepared and its crystal structure determined.91 The unit cell contains disaccharide sections of two chains,and an antiparallel arrangement of adjacent chains was assumed. The chains are stacked in the same way as in cellulose I1 with the water molecules between the stacks,but it was not possible to position the water molecules in a regular arrangement. The treatment of cotton and rayon with ethylenediamine resulted in the transformation of cellulose IV to cellulose I11 as determined by X-ray analy~is.’~ Thus ethylenediamine-methanol can affect lattice modifications. The transformation of cellulose I into cellulose I11 in Valonia macrophysa, preformed in ethylenediamine, has maintained the external appearance of the microfibrils as shown by electron microscopy. 93 This conversion must have involved a fracturing and fibrillation of the initial crystals. ‘ H n.m.r. spectroscopy has shown that both ramie and cotton of native have a unique phase structure characteristic
33
3: Plant and Algal Polysaccharides
B-~-Fruf-(2+l)-~-~-Fruf-(2-tl)-~-~-Fruf-(
-
1 .f
2 B-D-Fruf -
(1)
B-E-Fruf- (2+1)-B-E-Fruf-(2+6)-a-D-Glcp- (1+2)-B-!-Fruf -
1
.f
B-D-Fruf-(Z+l (2)
2 )-$-E-Fruf
34
Carbohydrate Chemiitry
cellul0se.9~ The non-crystalline component did not exhibit micro-Brownian segmental motion even in the swollen state. The phase structure was distinctly different from that of regenerated cellulose. An n.m.r. spectroscopic study of the interactions between cadoxen and cellulose has been carried out using signals from 'H, 13C, and 113Cd.95 A well recorded 13C spectrum was obtained which could be assigned by comparison with the spectra of simpler sugars. The 13Cd and 13C results were not consistent with the formation of chelate-alcoholate complexes with the C-2 and C-3 hydroxy groups. Hydrogen bonding is probably the dominant interaction. It is further suggested that the metal ion serves the dual purpose of holding two amino groups in a favourable orientation to hydrogen bond with a pair of equatorial hydroxy groups on the cellulose. The solubilization of cellulose and other plant structural carbohydrates has been achieved in a mixture of 4-methylmorpholine g-oxide and DMSO.' The separation and purification of homogeneous polgsaccharide fractions could then be made. Hydroxymethylcellulose has been prepared and characterized by i.r. and n.m.r. s p e c t r o s ~ o p y . ~To ~ achieve dissolution of cellulose, a high degree of molecular substitution was required, but once solution was achieved the molecular substitution could be lowered, the actual value of precipitation depending on the solvent. A rapid separation of cello-oligosaccharides (degree of polymerization 2-9) has been obtained on a cation-exchange resin in 60 min with water as eluant.98 The cellulose content of raw and cooked vegetables has been determined by extraction procedures using detergent solutions.9 9 The U.V. spectra of extracts of hydrocellulose with the hydroxides of Li', Na', K ' , Ca2+, Sr2+,and Ba2+ were identical."' The curves of yellowing absorption coefficients against loss in weight of hydrocellulose in boiling solutions of these alkalis were linear, the slopes giving apparent molar absorption coefficients in the order Na+= K+>Li+>Ba2+>Sr2+>Ca2+.The divalent cations reduced the yield of chromogen from the 4-deoxy-~-glycero-2,3-hexodiulose released from reducing end groups of the hydrocellulose. In the same systems, greater weight losses and production of acidity were obtained with the alkalineearth cations than alkali-metal cations when the OH- ion
35
3: Plant and Algal Polysaccharides
concentration was 0.02M while the reverse was found at 1.OM. l o ' The results were explained in terms of a hypothesis that divalent cations exercise their catalytic effects through enhanced ionization of the ;-glucose moiety at the reducing terminus of cellulose chains. The selectivity (viscosity at a given Kappa number) of softwood Kraft pulp has been shown to increase dramatically when soaked with S02-H20 in the presence of diethylenetriaminepentamethylene phosphonic acid before oxygen bleaching in the presence of magnesium sulphate. O 2 Addition of manganese(VI1) sulphate to soaked pulps led to a severe loss in selectivity while to dry pulps a slight increase was obtained. Addition of the phosphonic acid to manganese(VI1)-treated pulps caused a large increase in selectivity. Aqueous sulphur dioxide treatment of cellulose pulp of and cotton linters dramatically reduced the degree polymerization of the cellulose but did not affect its enzymatic digestibility or decrease its crystallinity as previously reported. 1 0 3 The acetyl content of cellulose acetate and a variety of woods has been determined by aminolysis with pyrrolidine followed by g.c. of the resultant I-acetylpyrrolidine. O 4 The distribution of the sulphate ester groups in the natural cellulose sulphate of Buccinum undatum has been determined by 1 3 C n.m.r.; the results were compared with those from a randomly The relative substituted synthetic cellulose sulphate.' O5 simplicity of the natural cellulose sulphate spectrum indicated that this polymer consisted of blocks of disulphated (1+4)-B-;-glucan chains separated by blocks of unsulphated chains. (1+4)-~-Q-glucan A variety of sulphoalkylcelluloses have been synthesized in NaOH solutions at 65°C. l o 6 Increasing temperature and NaOH concentrations increased the sulphoalkylation as well as the yield of water- and alkali-soluble fractions. The relative oxyanion formation at hydroxy groups of q-glucosides and cellulose has been reviewed O7 Native cellulose and pulps from a variety of hardwoods have been fractionated by the nitric acid rnethod.'O8 A regression analysis showed a direct correlation between the age of the tree and the degree of polymerization of the cellulose. The values were a useful indicator of the papermaking potential of the wood. Cellulose powders obtained by mechanical disintegration have
'
'
.'
36
Carbohydrate Chemistry
larger average particle sizes and broader size distributions than those obtained by hydrolytic degradation.l o g Microcrystallization of bleached sulphate pulp from beechwood and reed increased the crystallinity index from 66 to 71% and 56 to 61%, respectively, as determined by X-ray analysis, but decreased the yield from 86 to 75% and 74 to 67%, respectively.'" The average length of the microcrystalline pulp fibres was 55 um compared with 220 urn for the control fibres. By the use of polarizing microscopy, the microfibrils in the spherulites formed in the pellicle of bacterial cellulose have been shown to be radially or tangentially orientated. Some of the results indicated that the initial stage of nucleation may be a function of intermediate lipid-carbohydrate compounds as in the lyotropic liquid crystal of the soap micelle. When virulent strains of Agrobacterium tumefaciens bind to carrot cell walls, fibrils develop which surround the bacteria and anchor them to the cell surface.'12 The fibrils are composed of cellulose and are synthesized by the bacteria since they are produced on dead as well as living carrot cells. Within this entrapped mass, the bacteria are still able to multiply and form large clusters. On pyrolysis of cellulose under various conditions, 82 compounds have been identified as volatile product^."^ The maximum yield in a nitrogen atmosphere was achieved at 600-650°C while in air a 50-200°C lower temperature was required. The volatile products were qualitatively similar to those found in cigarette smoke. Combined q.c. gas-phase thermal fragmentation has been developed for the separation and identification of pyrolysis The compounds were products from bark polysaccharides.' identified by their fragmentation pattern which was useful in demonstrating the thermal stability of the parent compounds. Various polymer initiating systems, especially redox systems involving cerium(II1) and iron(I1) salts,had little effect on the thermal properties of cotton cellulose.' l 5 The cellulose from Acetobacter xylinum has been used for visualizing the action of the cellulase enzyme system from Trichoderma reesei by high-resolution electron microscopy.' l 6 The enzymes initially bind to the cellulose ribbon, but within 10 min the ribbon is split along its longitudinal axis into bundles of microfibrils which are thinned until they are dissolved,which takes about 30 min. Separation of the various enzyme components
'''
'
3: Plant and Algal Polysaccharides
37
produced less dramatic changes confirming the synergistic mode of action of these enzymes. The cellulase from T. viride has been immobilized on SepharoseR 4B.l17 The immobilized enzyme had a similar Km to the free enzyme, the activity increased up to 50°C and did not decrease after 4 cycles of batch use and recovery. Treatment of finely divided Pinus densiflora with the cellulase from T. viride at 40°C and pH 4.0 resulted in hydrolysis of 80-90% of the carbohydrate present .I18 A series of mono- and oligo-saccharides were obtained from bagasse and pith by cellulase hydrolysis."' The presence of pentoses and their oligosaccharides in hydrolysates of alkali-treated bagasse showed that hemicelluloses had not been completely removed by this treatment. The action of the cellulase from Penicillium funiculosum on a variety of cellulosic substrates has been investigated by scanning electron microscopy and X-ray diffraction analysis. 120 The differences were interpreted in terms of the a-cellulose content, surface area, degree of crystallinity, and crystallite dimensions of the substrate. The kinetics of hydrolysis of ball-milled cotton linters by eight cellulase complexes from Trichoderma, Geotrichum, and Asperuillus species have been studied under steady-state and nonThe steady-state rate of D-glucose steady-state conditions. 12' formation was proportional to the endo-(1-+4)-B-D-glucanase content. A mechanism was proposed in which the enzyme which first attacks the insoluble substrate is this m - P - g l u c a n a s e . This contradicts the accepted view for a prehydrolytic C1 enzyme. The C 1 role is filled by the endo-g-glucanase. Partial acid hydrolysis has been investigated as a pretreatment to enhance the enzymatic hydrolysis of cellulosic materials. 2 2 Up to 100% of the potential E-glucose could be obtained,and this was due to removal of hemicellulose, reduction in the degree of polymerization, and changes in the crystal structure of the cellulose. The purification of a cellulase isoenzyme from kidney bean abscission zones has been carried out by affinity chromatography. 1 2 3 Antibodies raised against this cellulase (PI = 9.5) did not react with the cellulase (PI = 4.5) obtained from 2,bE-treated abscission zones. examined, only 3 1 were Of 60 species of soil fungi
Carbohydrate Chemktry
38
cellulolytic.1 2 4 The- cellulolytic activity of 25 species of pyrenomycetous fungi has been determined with 18 being able to utilize cellulose. 125 A Bacteroides species, of human faecal origin, was able to degrade isolated cell walls of peanuts.' 2 6 When autospores of Oocystis solitaria were treated with Congo Red or Calcofluor White a more diffuse fibrillar material was obtained [which could be chains of (1+4)-B-D-glucanl rather than the discrete microfibrils of untreated cells. 2 7 Using intact cotton fibres, it has been shown by pulse-chase experiments that (1+3)-8-~-glucans (callose) have a high rate of turnover and are likely to be intermediates in cellulose biosynthesis at the stage of secondary cell-wall formation. 28 Protoplasts from cultured soybean (Glycine max) cells have been used to study polysaccharide synthesis during the early stages of cell-wall regeneration. 29 Label from !-glucose was rapidly incorporated into cellulose but neither UDP- nor GDP-P-glucose was rapidly incorporated into (1+3)-B-~-glucans, Calcofluor White ST can increase the rate of ;-glucose polymerization into the cellulose of Acetobacter xylinum.' 30 At a concentration of >O.lmM, the rate was four times the control but the assembly of crystalline cellulose I microfibrils was disrupted. It is concluded that polymerization and crystallization are cell-directed, coupled processes and that the rate of crystallization determines the rate of polymerization. Coupling must be maintained for biogenesis of cellulose I. Addition of coumarin (1 g kg-') to Phaseolus vulgaris leaves inhibited the synthesis of cellulose I but stimulated the synthesis of a-cel1ulose.l3' A persisting cellulosic wall has been demonstrated by light microscopy in microspore mother cells during microsporogenesis in Allium tuberosum and Cyclamen persicum. 1 3 2 Cellulose metabolism by the flagellate Trichonympha, isolated from a termite, has been studied.'33 The protozoon, in culture, harboured no endosymbiotic bacteria and produced acetate, CO,, and H, from cellulose. The cellulolytic activity is therefore an inherent property. A new method of determining the extent of binding of an ionic dye to a polyelectrolyte in solution has used the fact that dyes can adsorb to a cellulose dialysis membrane.'34 The permeability of cellulosic walls to macromolecules may
'
'
'
39
3: Plant and Algal Polysaccharides
limit the ability of enzymes to alter the properties of these walls. 135 Since proteins of molecular weight 6 x lo4 can permeate a substantial portion of the wall, previous results may have underestimated this property. The fermentation of cellulose by Acetivibrio cellulolyticus, Methanosarcina barkeri, and a Desulphovibrio species has been culture allowed a rapid studied. 3 6 Only a three-component conversion to C02 and CH4 and utilized 85% of the cellulose present. This value was increased to 90% by increasing the concentrations of M. barkeri.
5 Hemicelluloses
A number of sugar residues were found in hydrolysates of the water-soluble hemicelluloses of rice brans. 3 7 Although L-arabinose was the major sugar residue, there was no direct evidence for the presence of an L-arabinan. The intracellular site of the synthesis of 4-arabinose-containing cell-wall precursors in suspension-cultured tobacco cells has been determined as the Golgi bodies with transport being vesicles in the low-density fraction.1 3 8 A D-galactan has been isolated from the leaves of Aloe barbadensis by hot-water extraction and separation from contaminating pectin, ;-gluco-q-mannan, and 4-arabinan. 1 3 ’ The (l+4)-linked ;-galactan had one branch point at 0-6 for every 26 residueq and the molecular weight of the acetylated polysaccharide was 3.7 x 104. The formation of monodispersed polysaccharides on Smith degradation of the acidic k-arabino-;-galactan from rapeseed (Brassica campestris) strongly suggests a molecular structure composed of regularly repeated subunits. 1 4 * The molecular weight of the subunit was 1.2 x lo4 and the resistance of the polysaccharide to subsequent Smith degradations suggested that the main chain was a (1*3)-?-galactan. An 4-arabino-2-galactan-protein has been released by sonication of a crude cell organelle fraction from hypocotyl tissue of bean seedlings (Phaseolus vulj?aris) and suspension cultured cells. 1 4 ’ The macromolecule (mol. wt. 1.4 x l o 5 ) contained 90% carbohydrate and 10% protein with the major sugar residues being ;-galactose, L-arabinose,and uronic acids and the major amino-acids present being k-hydroxyproline, &-serine,and L-alanine. The carbohydrate
’
-
Carbohydrate Chemistry
40
was linked the first two amino-acids and also to 4-threonine with the carbohydrate portion of the linkage regions containing residues of L_-arabinose,q-galactose , 8-glucose,and k-rhamnose .14' The L-hydroxyproline glycosides were different from those in nonextractable cell-wall protein but have similarities with those in the wall proteins of the algae. The changes in the 5-arabino-q-galactan-proteins of the carrot have been studied during germination and growth.143 An extracellular k-arabino-8-galactan-protein has been obtained from suspension-cultured tobacco cells. 44 The complex (mol. wt. 2.24 x l o 5 ) contained 5.5% protein and the sugar moiety was a A B-q-glucopyranosyluronic typical L-arabino-(1+3)-8-galactan. acid residue was present at the non-reducing terminus attached to 0-6 of a P-galactopyranosyl residue. Auxin-induced changes in the sugar composition, intrinsic weight distribution of matrix viscosity, and molecular polysaccharides in the epicotyl cell wall of Vigna angularis have been determined. 1 4 5 The greatest change was the reduction in 4-arabino-q-galactan content, but other changes in pectin and E-xylo-g-glucan content were also observed. The linkage regions in cell walls of rice coleoptiles have been characterized as involving 0 _ 3 - ~ - g a l a c t o p y r a n o s y l - ~ - s e r i n e and N4 - ( 2-ac etam i do 2- d eoxy- B -E- g1ucopyran0s y1 ) hydrogen LThe linkage region in the water-soluble asparaginate. 1 4 6 4-arabino-E-galactan-protein complex from wheat endosperm has been characterized as g4 B ga1acto p yrano s y1 4 -trans hyd ro xy proline. 14' Methylation analysis of the glycopeptide linkage region from cell walls of Chlamydomonas reinhardii has revealed that g-glycosidically linked g-galactofuranosyl residues are present. 1 48 Bacteroides thetaiotaomicron, a polgsaccharide degrading bacterium from the human colon,can use larch L-arabino-;-galactan as its carbon source with yields similar to those obtained on p-glucose. 149 The crystal structure of a regenerated form of (1+3)-a-D-glucan has been determined by X-ray diffraction analysis and stereochemical model refinement as an orthorhombic unit cell. 50 The chain conformation is almost completely extended and is very close to a 2/1 helix even though the dimer residue is the crystallographic repeat unit. The vacuum c.d. spectrum of (1+6)-~-~-glucan has a single
-
c
-
---
-
-
-
41
3: Plant and Algal Polysaccharides
positive band near 177 nm.151 As gels formed, a negative band at 190 nm appeared followed by a blue shift in both bands with ageing. The specificity of the interaction of direct dyes with polysaccharides has been studied by changes in solubility and in the fluorescence and absorption spectra of the dyes.’52 The strongest interactions were shown by polysaccharides with (l+4)-linked-B-Q-glucopyranosyl residues, but contiguous (1+3)-8-P-glucans exhibited some complex formation. Dyes differed in their affinity. The direct dye, Calcofluor, has been used in a fluorimetric assay for (1+3)(1+4)-B-D-glucans in beer, wort, malt, and barley. The B-E-glucan content of developing and germinating kernels of several varieties of barley has been determined 5 4 The E-glucan content varied considerably but did not seem to be related to the B-g-glucanase activity of the variety. A g-glucan is present in the cotyledons of Mirabilis jalapa seeds.155 Methylation, periodate oxidation, and graded and enzymatic hydrolysis studies have indicated 34 (l+4)- and 3 ( l + 3 ) linkages for every 38 ;-glucose residues. The branch point appears at 0-2. In some places, at least three (1+3)-linked P-glucopyranosyl residues are in sequence. Both a and 8-glycosidic linkages are present. The hemicellulose fraction accounted for 47% of the pollen tube wall of Camellia japonica and comprised essentially c-glucose The polysaccharide had a ( 1 +3)-B-;-glucan chain with residues. 15‘ a degree of polymerization of 21. The pollen tube wall of C. sasanqua, C. sinensis, Tulipa gesneriana,and Lilium longiflorum also consisted mostly of ;-glucose residues,and the hemicellulose fractions were essentially pure D - g l u c a n ~ . ’ ~The ~ deposition of (1+3)-8-;-glucan during megagametogenesis in two species of Oenothera is partly in continuum with the wall and partly in the degenerating cytoplasm. The addition of B-g-glucanase before the use of u-amylase has been used to aid filtration in the measurement of fibre contents by detergent procedures. 59 The fact that fungal g-glucans will stimulate soybeans to accumulate phytoalexins has prompted an investigation into the (1+3)-B-~-glucanases and B-P-glucosidases of soybean and their hydrolytic patterns.16’ The B-9-glucosylase I enzyme has been purified. Several lines of evidence suggest that the
.’
’
’
42
Carbohydrate Chemisrry
(1+3)-8-;-glucanase and 8-P-glucosidase activities exhibited by I preparations are catalysed by the same enzyme. B-g-glucosylase Treatment of the 2-glucan elicitor from the walls of Phytophthora megasperma by E-glucosylase I results in extensive hydrolysis and loss of 94% of its activity.lbl It is unlikely that some other enzyme(s) may be involved in the overall reaction. The endo- and exo-8-P-glucanases from Zea mays have been partially purified. l b 2 The endo-8-q-glucanase (mol. wt. 2.6 x lo4> showed a marked preference for substrates with mixed linkages,and it probably initiated the solubilization of wall g-glucan. The exo-8-E-glucanase (mol. wt. 6.0 x lo4) was required for extensive hydrolysis in vitro. During coleoptile development, the autolytic activity of the cell wall increased dramatically but did not parallel the growth potential of the tissue. Conditions which induce a transmembrane electrical potential, positive with respect to the inside of the membrane vesicles, have resulted in a 4-12-fold stimulation of the activity of membraneassociated 8-q-glucan synthetases in a preparation from developing cotton (Gossypium hirsutum) fibres.1 6 3 The products were mainly (1+3)-8-;-glucans but some increase in (1-+4)-B-D-glucan content was also found. No (1+4)-a-q-glucan was formed. The products of 8-q-glucan synthetases have been characterized using specific -9-glucan hydrolases. 6 4 The B-Q-glucan synthesis by particulate enzymes from suspension-cultured endosperm cell walls from ryegrass has been studied. 6 5 The 8-E-glucan synthetase activity of the fungus Saprolemia monoica has been assayed and a product is observed which contains The both 1 +3- and 1 -+blinked B-g-glucopyranosyl residues. 6 6 presence of Mg2+ ions affects the structure of the product. In the absence of Mg2+, only a (1-+3)-8-P-glucan is formed, but as the substrate concentration is reduced and Mg2+ ion concentration is increased the proportion of lj3-linked residues in the product is reduced and a pure ( 1-+4)-B-Q-glucan is formed. High molecular weight E-glucans have been isolated from mycelial walls of Fusarium oxysporum.' 6 7 The P-glucans contained both 1+3- and 1-+6-linked residues in contrast to the E-glucans from Colletotrichum lindemuthianum, which contained 1+3- and 1-+4-linkedresidues. However, the two types of 9-glucans had very similar effects in eliciting browning and phytoalexin production in green-bean cotyledons. An exo-(l+3)-B-;-glucanase derived from Sclerotinia libertiana
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3: Plant and Algal Polysaccharides
has
induced
growth
43
of Avena sativa coleoptiles and degraded the No endo-( 1+4)or endo-( 1+3)-B-pglucanase activity could be detected. The exo-(1+3)-8-;-glucanase from a Basidiomycete did not induce growth. The metabolism of a e-glucan from the cactus Opuntia bigelovii has been studied in a desert en~ir0nment.l~’The D-glucan content increased slightly during a 9-week drought period but then decreased during the irrigation period. The ;-glucan is probably storage material. The g-xylo-;-glucan from the walls of etiolated mung beans has been studied by acidic and enzymatic hydrolyses, the latter by a preparation from Aspergillus oryzae. 170 Cellobiose, cellotriose, and cellotetraose were obtained from the acid hydrolysate while isoprimeverose ( 4 ) and the pentasaccharide ( 5 ) were characterized from the enzymic hydrolysate. Similar studies using the cellulase from Trichoderma viride produced three types of oligosaccharide, hepta-, nona-, and deca-saccharides, and their structures were partially characterized. 1 7 ’ The structure of a cell-wall polysaccharide isolated from cotyledons of tora bean has been established by methylation analysis, Smith degradation, and enzymatic fragmentation. 1 7 2 The main features were those of a E-galacto-E-xylo-D-glucan where a (1+4)-8-P-glucan main chain is substituted at 0-6 by and 8-8-galactopyranosyl-E-xylopyranosyl residues. D-xylopyranosyl A further feature was the presence of (1+5)-a-L-arabinan side chains (degree of polymerization = 8 ) at some 0-3 positions of the main chain. A Q-xylo-E-glucan has been isolated from the cell walls of potato (Solanum tuberosum), and the methylation analysis and fragmentation results confirmed the usual structure. 73 Some of the g-xylose residues carried further substitution at 0-2 with L-arabinofuranosyl and 9-galactopyranosyl residues. The biosynthesis of the P-xylo-P-glucan in cultured soybean cells has been studied. 7 4 Two types were obtained from cultured cell walls (mol. wts. 1.8 x l o 5 and 6 x l o 4 ) and one from the culture medium, the molecular weight of which shifted from 6 x l o 4 to 3 x l o 4 during the progress of cultivation. The compositions were very similar and two kinds of oligosaccharide repeating units were present, namely a heptasaccharide (Q-xy1ose:D-glucose 3:4) and a nonasaccharide (?-galactose:&-fucose:;-xylose:Q-glucose (1:1:3:4)). Incubation of a particulate fraction from soybean ( 1 + 3 ) (1+4)-8-p-glucans. -
44
Carbohydrate Chemistry
3: Plant and Algal Polysaccharides
45
cells with UDP-14C-Q-glucose or UDP-I C-E-xylose produced a A. oryzae enzymes gave a labelled polymer.175 Digestion with labelled disaccharide characterized as isoprimeverose (4) by the usual methods. Since isoprimeverose is the smallest structural unit of D-xylo-q-glucan, the polymer can be synthesized from these nucleotide sugars. The results have been used as the basis of an 76 They have also assay for Q-xylo-P-glucan:q-xylosyltransferase.l been used to suggest that this transferase can be distinguished from other polysaccharide synthetase activities. The metabolism of polysaccharides by pea stem segments treated with and without auxin has been studied by a centrifugation technique.’ 7 7 Auxin enhanced the levels of e-xylose and ;-glucose in ethanol-insoluble polysaccharide, and these sugars were in a Q-xylo-;-glucan. Similar studies using ethene showed that this treatment lowered the amount of Q-xylo-9-glucan in ethanol-insoluble polysaccharides. 78 The vacuum u.v.-c.d. spectra of guar, tara, and carob p-galacto-E-mannans, which had D-galactose contents of 3 9 , 25,and 198, respectively,have been re~0rded.l~’A positive band at 169 nm and a negative band at 149 nm were observed whose relative intensities increased systematically with decreasing ;-galactose content for solid films. Some of the differences were attributed to conformational restriction of ;-galactose residues by chain packing with a consequent increase in the c.d. intensities from these residues and a cancellation of q-mannan backbone contributions. High-performance size-exclusion chromatography of guar gum has indicated that the molecular weight was >2.0 x 1O6.l8’ The seeds of AstraRalus sinicus contain a polysaccharide which ratio of 1:2.3 and a molecular weight has a p-galactose:p-mannose of 1.6 x 104.181 The results of methylation and Smith degradation analyses indicated that the P-galactopyranosyl residues were attached to 0-6 of the main chain but that both (1+3)-and (1+4)-linkages were present in the main c-mannan chain. Treatment of guar gum with an a-E-galactosidase has produced a polymer in which 75% of the E-galactose residues have been removed, but there was no reduction in viscosity.182 Further treatment did reduce the viscosity and led to D-mannan-type precipitates. The interaction of guar gum with xanthan gum is increased by removal of the Q-galactose residues. The chemical structure of the 2-galacto-q-mannan component of
46
Carbohydrate Chemistry
Trypanosoma cruzi has been determined by 13C n.m.r. spectroscopy and confirmed that B-IJ-galactofuranosyl residues were linked at the non-reducing terminus to 0-3 of P-mannose residues.’83 E-Galacto-g-mannans are the major storage reserve carbohydrate in the embryo of fenugreek but they are not mobilized during the first 24 h of germination. 84 A pure D-gluco-g-mannan has been recovered from the hot-water extract from Aloe barbadensis .185 The methylated polysaccharide (mol. wt. 1.5 x l o 4 ) contained residues of 2,3,4,6-tetra-, 2,3,6-tri-, and 2,3-di-g-methyl-P-mannose and 2,3,6-tri-g-methylD-glucose in the ratios 1.3:18.3:1.2:1.0. ;-Mannose was observed in hydrolysates of the holocellulose of Panicum coloratum leaves.186 The material containing this sugar residue was particularly resistant to extraction from the a-cellulose fraction. The conditions for the preparation of a rigid and homogeneous gel from Konjac mannan have been p ~ b 1 i s h e d . l ~ ~ Fractionation of cell-wall preparations from grass leaves has shown that the less dense fractions had a higher ;-xylan content as well as a higher content of lignin and acetyl residues. 88 Fully methylated P-xylan-type oligosaccharides have been analysed by mass spectrometry and the fragmentation patterns compared.189 Within the series of di- and tri-saccharides studied, the criteria proposed permitted reliable determinations of any of the theoretically possible branching points. The structure of the water-soluble 4-arabino-Q-xylan from delignified bark of Cinnamomum zeylanicum has been established.”’ The main (1+4)-B-;-xylan chain is substituted both at 0-2 and 0-3 with b-arabinofuranosyl and 3-C+a-~-xylopyranosyl-~-arabinofuranosyl groups. The isolation, purification,and partial characterization of a g-glucurono-L-arabino-;-xylan, a previously unobserved component of the primary cell wall of dicotyledonous plants, have been described.lgl It represents 5% of the wall and has the usual structural features of this type of hemicellulose. A procedure has been developed for the fractionation of the non-starchy polysaccharides of wheat bran.’ 9 2 Determination of the activity of Ca2+ ions bound to carboxy groups in the E-xylan from white willow has shown that 4-~-methyl-~-glucopyranosyluronic acid groups are concentrated in blocks where at least every second a-Q-xylopyranosyl residue
’
3: Plant and Algal Polysaccharides
47
contains a single uronic acid residue linked by a ( 1 + 2 ) bond.lg3 These acidic blocks alternate with blocks of unsubstituted D-xylose units which are three times as long. The hemicellulose contents of leaves, nodes,and internodes of wheat straw were similar.lg4 Ozone and sulphur dioxide at 70°C for 72 h solubilized virtually all the wheat hemicelluloses. 9 5 Results were also presented on the digestion of the hemicelluloses by rumen micro-organisms. Similar studies with rumen organisms have been carried out on NaOH-treated straw. l g 6 The hemicellulose content of cotton straw is reduced by 50% on treatment with ozone. l g 7 Auxin was found to have no effect on the structure of the L-arabino-q-xylans isolated from Avena coleoptiles. 9 8 The pentosans of pearl millet (Pennisetum americanum) have been separated into four fractions and each was found to contain varying amounts of seven sugar residues.lg9 Each fraction also contained protein. A mechanism for the oxidative gelation of the water-soluble pentosan of wheat flour has been proposed that suggests an attack by addition of a protein radical to the activated double bond of a ferulic acid residue.200 The substrate-binding site of the endo-(1-+4)-8-~-xylanase from the yeast Cryptococcus albidus has been investigated using a series of (1+4)-8-;-xylan oligosaccharides which were labelled with tritium at the reducing end.201 The substrate binding site is composed of four subsites with the catalytic groups located in the centre. As a consequence of an assymetric distribution of negative values of affinity around the binding site, the enzyme displays a strong preference to attack near the reducing end of the substrate. The mechanism of action was found to involve pathways other than a simple hydrolytic cleavage.202 Some IJ-xylosyl transfer occurred to increase the molecular size of the substrate and the transfer came from the non-reducing end of the molecule. All features of the degradation of oligosaccharides by this E-xylanase were consistent with the lysozyme-type reaction mechanism Particulate enzyme preparations obtained from homogenates of differentiated xylem cells from sycamore trees have catalysed the synthesis of a D_-xylan from UDP-q-xylose. 203 The polysaccharide had the expected structure. Various properties of the enzyme and its requirements were determined. The specific activity of the
.
48
Carbohydrate Chemistry
enzyme system increased markedly as cells differentiated from the vascular cambium to the xylem.204 The increase was closely correlated with the enhanced deposition of D-xylan occurring during the formation of secondary thickening. The control of hemicellulose synthesis during differentiation of vascular tissue in bean callus and hypocotyl has been investigated and the E-xylan synthetase induction correlated with the induction of phenylalanine ammonia-lyase and with lignin synthesis.*05 A cell-wall degrading agent has been isolated from Chlorella 206 and characterized as a (1+4)-8-Q-xylanase. Residual carbohydrates have been removed almost completely from milled wood lignin preparations of birchwood by treating with NaOH-dioxane. 2 0 7 Treatment with HC1-dioxane did not decrease the carbohydrate content. Studies using i.r. and n.m.r. spectroscopy indicated that a benzyl ester type of linkage existed between lignin and 'the carbohydrates. The lignin-carbohydrate complex from the milled wood lignin fraction of Pinus densiflora contained three sub-fractions on gel filtration.2 0 8 Two of the fractions were homogeneous and their compositions were determined. Methylation analysis showed that the carbohydrate moiety exhibited multiple branching with the major backbone being composed of (1+4)-p-mannan chains. A water-soluble lignin-carbohydrate complex has been released from jute (Corcharus capsularis) fibre by reduction with borohydride. * 0 9 Analysis of the pxylans from the original and borohydride-treated fibres indicated that 34% of the acidic side chains of the g-xylan were linked to lignin by ester bonds. Lignin-carbohydrate complexes have been released from wheat straw by prolonged hot-water extraction.2 The bound sugars were identified as g-glucose, 4-arabinose, q-xylose,and ;-galactose residues with uronic acids being absent. The higher molecular weight fractions were richer in !-glucose residues with the lower molecular weight ones being richer in E-xylose and 4-arabinose residues.
6 Pectins
A
high-performance gel-permeation chromatographic method has been developed to determine the molecular weight distribution of pectins.211 High-methoxy, low-methoxy,and amidated pectins can be analysed within 15 min.
3: Plant and Algal Polysaccharides
49
An investigation of oligo- and poly-galacturonic acids has been carried out by potentiometry and c.d.212 It was shown that no Ca2+ ion is ever fixed in excess of stoichiometry. The c.d. spectra, in spectroscopic data, suggested that, in agreement with 1 3 C n.m.r. dilute solutions, the polymer adopts two conformations. The gelation of pectin under conditions of low water activity has been investigated by c.d., competitive inhibition, and mechanical properties. 2 1 3 The optimum degree of esterification for pectin gelation under conditions of low water activity is 70%. The reduction in gel strength at lower degrees of esterification is not merely due to increased charge density but also results from the loss of a significant contribution which the ester group makes to the stability of the interchain junctions. Gel permeation chromatography, c.d., and viscosity measurements showed that appreciable aggregations of aqueous solutions of pectin occur at low concentrations in pectins of both low and high ester content . 2 1 The aggregation is independent of divalent cations. The Arrhenius slopes were greater at lower temperatures and they increase with conditions such as pH, ionic strength, concentration, and degree of esterification that are known to favour interchain association of pectin. The c.d. spectra at various pH values and in the presence of additives indicated that two modes of interchain association contribute to the network formation in calcium pectate gels.215 The first is by interchain chelation of Ca2+ ions and the second is by non-ionic interchain associations analogous to those in pectin gels with low water activity. The contribution of the latter type increases on lowering the pH, as the Ca2+ ion binding capacity is diminished and interchain electrostatic repulsions are minimized. The mechanical properties and thermal stability of a gel are a function of the proportion of each type. A relationship has been sought between the ability of various ions to inhibit growth and their ability to cause gelation of isolated pectins, their binding affinity for isolated cell walls, and their binding affinity for purified pectin.216 A good correspondence was found with the second factor. Pectic gel formation was not involved in cation-induced growth inhibition. The previously determined activity coefficients of Ca2+ ion in oligo-g-galacturonates were plotted and extrapolated to infinity.217 In molecular disperse solutions the binding of Ca2+ ions was electrostatic, whereas in aggregated molecules a chelate
Carbohydrate Chemistry
50
intermolecular binding also took place. The non-reducing terminal uronic acid residues bind Ca2+ ions less firmly than to the inner units of the chain. Lemon pectin and pectic acids have been hydrolysed into acid-soluble and acid-insoluble products and separated by gel filtration.*18 The latter product had a molecular weight of 3.03.2 x l o 4 whereas the former could be separated into two components (mol. wts. 2.2 -2.5 x l o 4 and < 6 x l o 3 ) . The larger of these two components contained only (1+4)-a-Q-galacturonan chains while the smaller contained residues of E-galacturonic acid and L_-rhamnose as well as other neutral sugars. Cell-wall material has been isolated from cabbage (Brassica oleracea) and methylated.21 Hydrolysis of the product and analysis of the fragments revealed some of the structural features many of which were derived from the pectin present. Two pectic fractions, comprising 52% of the cell wall, have been isolated from potatoes. 2 2 0 Both fractions contained a range of molecular types differing in composition. The more easily extracted fraction had a high 4-arabinose:E-galactose ratio as well as a high D-galacturonic acid content. The more easily extractable fraction was probably held by Ca2+ ion bonds. An endo-poly-2-galacturonanase from Aspergillus japonica has been used for the isolation and characterization of cell-wall pectic Methylation analysis of the four substances from potato tubers. fractions obtained by gel filtration was used to elucidate their structure. The two highest molecular size fragments had very similar compositions while the intermediate fraction had a very complex structure including P-galacturonic acid residues which were branched at C-3. Sequential extractions of Rosa glauca cell walls showed that two different types of acidic polysaccharide were present . 2 2 2 One was extracted with chelating agents while the other remained in close association with the a-cellulose fraction. A structurally complex pectic polysaccharide, k-rhamno-Pgalacturonan I, has been isolated from the primary cell wall of suspension-cultured sycamore cells. 223 The polysaccharide (mol. wt. 2 x l o 5 ) has the usual pectin backbone to which is attached a wide variety of side chains containing &-arabinosyl and/or P-galactosyl residues. Tomato pectin esterase has been used to prepare a high molecular weight pectin from citrus.224 The product from the free
’”
51
3: Plant and Algal Polysaccharides
ca.
enzyme had a degree of esterification of 2% whereas an immobilized enzyme gave a value of z . 1 9 8 . The molecular weight was lowered by less than 5% by the enzymatic treatments. The steric course of the methyl esterification reaction of a has been studied using 5-adenosylmethionine and an p-galacturonan enzyme preparation from Phaseolus a ~ r e u s .The ~ ~methyl ~ group of the methionine derivative was chiral, possessing 'H, 2H, and 3H substituents,and it was shown that transfer of the methyl group to the oxygen of the carboxy group proceeds with inversion of the configuration of the methyl group. mg g-l) was found in a wide variety of pectin Silica ( 1 . 4 - 2 . 3 samples. 226 Pectic substances were the major components of cell-wall material from pea flour and concentrates. 227 Detergent methods were shown to underestimate the pectin contents in a range of dietary fibres.228 For example, 50% of orange pectin was recovered in the acid-detergent residue and 38% in the neutral-detergent residue. Many of the physical properties of the dietary fibre from apples were shown to be due to the high pectin content.229 The interactions of bile acids and their conjugates with pectins of different degrees of esterification in the pH range 3.5-7.3 have spectroscopy, but no molecular been investigated by 'H n.m.r. interactions were apparent .23 O The pH-dependent interaction between pea cell-wall polymers possibly involved in wall deposition and growth has been investigated and there occurs, at neutral pH values, a pH- and time-dependent binding of soluble pectin, in the walls, to a heat-labile protein component of the wall.231 The reaction is also observed in water extracts of walls. The reaction is inhibited at low pH values and by Ca2+ ions and is lectin-like in nature. An elicitor of phytoalexin accumulation has been solubilized from cell walls of soybeans.232 The active material contains residues of E-galacturonic acid, L-rhamnose,and E-xylose and is probably pectic in origin. Similar elicitors could be prepared from suspension-cultured tobacco, sycamore, and wheat cells. A pectic polysaccharide, solubilized by the action of a fungal endo-(1+4)-a-~-galacturonanase from sycamore cell walls, possesses activity similar to the proteinase-inhibitor inducing factor isolated from tomato leaves.2 3 3 The synthesis and accumulation of proteinase inhibitor I in excised tomato leaves can be induced with oligosaccharides obtained by endo-(1+4)-a-D-galacturonanase -
52
Carbohydrate Chemistry
action of a pectic polysaccharide from tomato leaves .234 These oligosaccharides released by enzymic action at a wound or site of infection may play a hormone-like role in regulating plant defence responses in unwounded tissue some distance from the site of release. Pectic multienzyme preparations have been separated by h.p.1.c. using both isocratic and linear-gradient conditions.235 Three D_-galacturonanases have been isolated from carrots, two from root tissue and one from cell-suspension cultures.236 All three enzymes were of similar size (mol. wt. 4.8 x l o 4 > and were exo-enzymes. The only major differences were in the binding properties of the enzymes to cell-wall material. Pectic enzymes have been extracted from haustoria of Cassytha filiformis growing on Nerium indicum and Hibiscus rosa-sinensis and both strains contained exo-(1+4)-a-~-galacturonanase and exo-pectin methylgalacturonanase activity. 2 3 7 The two strains differed markedly in the production of lyase enzymes: the Nerium strain produced endo-pectic acid lyase while the Hibiscus strain produced endo-pectin lyase. 4 A g-galacturonanase (mol. wt. 3.6 x 10 ) has been isolated from Sodium citrus fruit infected with Penicillium italicum. 238 P-galacturonan was a better substrate than citrus pectin. The P-galacturonanase activity in seed cavity tissue from harvested cucumber fruit increases 20-fold after the fruit produces a transient burst of ethene during maturation.239 A single pectic enzyme, g-galacturonanase, is produced by Rhizoctonia solani in culture and during infection of cotton seedlings.240 The enzyme (mol. wt. 4.2 x l o 4 > has a pH optimum of 5.2.
The P-galacturonanase from Rhizopus stolonifer has been shown to be an elicitor of casbene synthetase activity in castor bean seedlings.241 The enzyme is a glycoprotein (mol. wt. 3.2 x lo4) and contains 20% carbohydrate. The principal sugar residues present are E-mannose (92%) and 2-amino-2-deoxy-~-glucose (8%) and the enzyme is an e n d o - h y d r ~ l a s e . ~ ~Other ~ features of the enzyme were reported. The active site of the endo-q-galacturonanase from tomato has been studied using oligo-2-galacturonic acids and their aldehydo derivatives.243 The relationship of the substrate chain length to the initial and maximum reaction rates was determined. The topology of the active site was suggested from the results. The
3: Plant and Algal Polysaccharides
53
;-galacturonanase of ripe tomatoes has been extracted in two isoenzyme forms, I and II.244 The enzymes have different properties although their polypeptides are similar. Green fruit contains a factor which can convert form I1 into form 1,and the amount of this factor increases during ripening. The characteristics of the pectin methylesterase from potato have been determined and the role of the enzyme in commercial dehydrated mashed-potato production has been examined. 2 4 5 Ca2+ ions. but not Mg2+ ions, retard solubilization. Texture measurements suggested a firming effect which could be attributed to Ca2+ ion release during pre-cooking and stabilization of Ca2+ ion bridges during cooling. The changes in pectinesterase activity and other pectin changes in mango varieties during storage and ripening have been investigated.246 In a study of the microbiology of wetwood, the importance of Clostridia species in the degradation of pectin has been assessed.247 High levels of pectinolytic enzymes were produced in culture filtrates of Phoma exigua and Graphium penicillioides isolated from decaying seeds of Phaseolus aureus and Cyamopsis tetragonalobus,respectively.248 The susceptibility of strawberry varieties to breakdown in sulphate liquor by the m-g-galacturonanase from Zygomycete spoilage fungi has been in~estigated.~~' Fruit waste containing de-esterified pectin as a thickener has been assessed for its potential use in canned products.250
7 Gums and Mucilages The carbohydrate gum from bael (AeRle marmelos) seeds has been resolved into four glycoprotein fractions. 2 5 1 One of these contained residues of :-galactose, !-glucose, L-arabinose, and &-rhamnose in the molar ratios 6:2:8:3. The linkages amongst these residues were determined as well as the anomeric configuration of the glycosyl residues. The structure of the linkage region was established as Q3-4-arabinopyranosyl-4-threonine. A histological and a histochemical comparison of the mucilages on the root tips of several grasses has been made,and it was concluded that there were two types.252 One is a gelatinous material originating from the root cap and the other is a firm uniformly thick mucilage overlying the columnar epidermal cells.
54
Carbohydrate Chemistry
Reactions of the outer layer and cell wall indicated that carboxy groups were present and these were absent from the inner mucilage. A mucous polysaccharide has been isolated from the bulbs of Narcissus t a ~ e t t a . ~It~ was ~ homogeneous and contained g-mannose and g-glucose residues in the ratio 5:l with a molecular weight of 1.19 x lo’. The g-acetyl content was 22.79,and these groups were present on C - 6 and on C-2 and C-6 of most p-mannose residues. The main chain is a (1+4)-B-g-hexopyran. The glycoprotein from neem (Azadirachta indica) gum (mol. wt. 1.3 x lo4) contained 50% carbohydrate.254 The sugar residues present were Q-mannose, 2-acetamido-2-deoxy-D-glucose, p-galactose, 4-arabinose, 4-fucose, q-xylose,and g-glucose in the ratios 14:13:5:3:1:1:1. The linkage between the carbohydrate and the protein was an y 4 - (2-acetamido-2-deoxy-8-D-glucopyranosyl) hydrogen L-asparaginate bond. Another glycoprotein from the same This material contained source contained only 19% ~arbohydrate.~’~ the same sugar residues in the ratio 4:3:2:3:2:1:1. The linkage region was the same. The mucilage of Opuntia ficus-indica and the partially degraded mucilage have been investigated by methylation analysis and periodate oxidation.256 The results show that the mucilage is composed of l,4-substituted a-D-galactopyranosyluronic acid and 1,2-substituted B-L-rhamnopyranosyl residues to which short chains of 1,6-substituted B-P-galactopyranosyl residues were attached at 0-4 of all of the &-rhamnopyranosyl residues. Most of the 9-galactosyl residues carry branches at 0-3 while some are branched at 0-4. Partial acid hydrolysis of this mucilage has e its polymeryielded ~ - ~ --~ - g a l a c t o p y r a n o s y l - ( 1 ’ 6 ) - ~ - g a l a c t o sand homologous trimer as well as fourteen oligosaccharides that contain L-arabinose residues and most of which have D-xylosyl residues at the non-reducing terminus.257 The oligosaccharides ( 6 ) and (7) were the most abundant, Similar studies have been reported on the mucilage of 0. aurantiaca with similar results.258 The gum exudates from some Prosopis species have been analysed by the Smith degradation procedure. 259 The degraded gums have chain was molecular weights of 6 x lo3. A simple (1+3)-!-galactan obtained after only two degradations showing that the skeleton was similar to those of Acacia gums. Autohydrolysis of the gum exudate from the Spondias dulcis tree gave a gum containing !-galactose, ;-arabinose,and D-galacturonic acid in the ratio 3:3:1.260 Three neutral and three acidic
3: Plant and Algal Polysaccharides
55
@-P-Xylp-(1+5)-a-&-AraL-(l-+5)-L-Araf (7)
Carbohydrate Chemistry
56
oligosaccharides were obtained and their structures characterized to suggest a sequence for the repeating unit. Chromium(V1) trioxide oxidation was used to determine the anomeric configuration of the sugar residues. 8 Algal Polysaccharides
Based on chemical shifts and C-H coupling constants from model molecules, the structural conformation of aqueous agarose solutions and gels has been determined from the 1 3 C n.m.r. spectra. 2 6 1 Similar studies on the agaroses of Pterocladia species and Gracilaria secundata have been reported and all signals in the spectra were assigned to various ;-galactose derivatives and agaro-oligosaccharides.26 The chemical composition and rheological properties of the agar isolated from Gelidium purpurascens before and after alkaline treatment have been reported .263 The D_-xylose, ;-glucose, and p-glucuronic acid residues in the agar were removed on alkaline treatment together with 86% of the protein. Sequential extraction of the alga accounted for low yields of agar as losses incurred on precipitation with ethanol. A method has been presented for the determination of the backbone conformations of polymer chains conforming to a given helical type.264 The method is illustrated by its application to agarose. The high resolution 13C n.m.r. spectra of slightly depolymerized alginates have been interpreted.265 The sequence of the monomer units k-guluronate (G) and p-mannuronate (M) markedly influences the chemical shifts. Some of the individual carbon resonances were resolved into four lines,which was evidence for a dependence on the identities of the units immediately before and after them in the polymer chain. The relative intensities of the signals permitted a rapid computation of the monomeric composition (M/G ratio), the composition of end units (M/G ratio), and the monomeric sequence in terms of a complete set of four diad and eight triad frequencies. Any region with a strictly alternating M and G sequence was very short. The cation-specific vacuum ultraviolet c.d. spectra of alginate solutions, gels, and solid films have been recorded.266 A band at 185 nm has been assigned to carboxy groups while those at 169 and 149 nm were assigned to changes in the polymer backbone.
*
-
3: Plant and Algal Polysaccharides
57
1-Carrageenan has shown substantial helix formation in the presence of Li', Na', and Me4N+ ions as measured by optical rotation, but the polymer does not gel. 2 6 7 Under identical Cs+,and NHq' ions. conditions, the polymer does gel with K+, Rb', Initially the individual The gel formation is in three stages. coils form double helices,then the double helices form domains, which finally form aggregates around the heavier Group I ions. A detailed assignment of the 13C chemical shifts of K and I-carrageenan in their Na' and K + forms has been given.268 Evidence for the conformational transition induced by temperature variation in the absence of any gel formation of K-carrageenan is also presented. The evidence is based on both n.m.r. and optical rotation experiments. Measurements of rigidity moduli and 39K and 23Na n.m.r. spectra have been used to investigate the roles played by Ca2+, K',and Na+ ions in 1-carrageenan gels. 2 6 9 Differences in binding were reflected in the rheological properties of the gels. Potassium 1-carrageenate gels have been prepared and studied by photon nm.270 The molecular and correlation spectroscopy at 633 vibrational motions of the gels were interpreted in terms of their elastic properties. Below the sol-gel transition temperature, oscillatory correlation functions were found with the frequency remaining constant for two weeks. Measurements of the shear modulus for Ca2+, K+, and Na+ 1-carrageenate gels as a function of biopolymer concentration and temperature showed that the Ca 2 + salt appears to possess a random glass-like structure whereas the K + and Na+ forms show a pseudocrystalline structure.271 The differences were tentatively attributed to differences in the solubility of the salt forms which, through controlling the gelation temperature, moderated the kinetics of the gelation process. The optical rotation and the conductivity of K-carrageenans in aqueous solutions have been investigated as functions of temperature in the presence of various electrolytes . 2 7 2 The activity coefficients of Na+ and K + ions have been determined and correlated with the conformation. The K + activity coefficient under ordered conformation is in agreement with a mechanism involving dimerization. The use of the carrageenan from the red alga Eucheuma striatum has been investigated as a possible substitute for agar in bacteriological tests. 2 7 3
58
Carbohydrate Chemistry
Neocarratetraose 4-2-monosulphate B-hydrolase has been isolated from cell-free extracts of Pseudomonas carrageenovora.2 7 4 The hydrolysis products are neocarrabiose and neocarrabiose 4-2-sulphate. The pathway for the sequential degradation of the tetrasaccharide was proposed. A-Carrageenan has been shown to have a hypotensive effect in the rat that was dependent on platelet stimulation.275 Also in rats, intraperitoneal injection of ellagic acid and carrageenan The reduced paw oedema induced by carrageenan itself.276 relationship between the anticoagulant activity and the pro- and anti-inflammatory mechanism in A-carrageenan-induced inflammation has been investigated.277 The gelation behaviour of solutions of chitosan on reaction with acid anhydrides has been studied, and the effects of the time to onset of gelation and of varying the nature and concentration of the anhydride, the chitosan concentration, the temperature,and the nature of the cosolvent were all examined.278 Gelation occurred due to the decreased solubility of the polymer molecules brought on by g-acylation,and all other parameters influenced the rate of N-acylation. The syneresis of the gels was examined. The degree of N-acylation required to initiate gelation depended on the molecular weight of the acyl anhydride and the concentrations of c h i t ~ s a n . ~The ~ ~ energy of activation of N-acylation was determined for tj-acetylation and N-hexanoylation. A mechanism was proposed for the gelation process. An i.r. spectroscopic method has been used to determine the amide content of highly deacylated chitosans.280 A plot of the ratio of absorbance at the amide band ( 1 6 5 5 cm-1) to the C-H stretch frequency (2867 cm-l) against the degree of deacetylation was linear in the deacetylation range 90-100%. The degree of N-acetylation of chitosan was rapidly determined by periodate oxidation and i.r. spectroscopic methods. 281 The i.r. methods gave higher results but were thought to be more reliable. A new method of chitin determination, based on deacetylation and g.1.c. analysis of the liberated acetic acid,has been applied to a variety of chitosans.282 The results were in good agreement with other methods even though the rate of deacetylation of some chitosans is 10 times that of others. A highly crystalline form of a-chitin has been found in the grasping spines of the marine worm Sagitta and been examined by electron microscopy and electron diffraction.283
3: Plant and Algal Polysaccharides
59
The chitin from carapace of Penaeus japonicus has been purified by a proteolytic enzyme from Pseudomonas malt~philia.'~~No deacetylation occurred and the protein content was reduced to virtually zero. The molecular weight of the enzymically prepared chitin was higher than that obtained by alkaline treatment. Polyelectrolytic complexes have been prepared from chitosan and sodium carboxymethylcellulose. 2 8 5 None were soluble in aprotic solvents but the series prepared at high pH values were more soluble in hot formic acid than those prepared at low pH values. The former were also different from the latter in the density of carboxy sites.286 A 'H n.m.r. method has been used to investigate the binding of methyl di-tj-acetyl-8-chitobioside to wheat-germ agglutinin. 2 8 7 The linewidth broadening of the methoxy protons allowed a calculation of kinetic association constants to be obtained. The bovine serum chitinase has been characterized as a true (l+4)-8-D-2-acetamido-2-deoxy-glucanase without any s - 8 - q - 2 acetamido-2-deoxy-glucosidase activity. 288 The purification and properties of the enzyme were reported. The chitin synthesizing system of insect tissue has been reported.289 A study on the adsorption of heavy-metal ions by chitin phosphate and chitosan phosphate has been made F g o The chemical composition of the porphyran from Porphyra columbina was in good agreement with data for similar polysaccharides. 29 One fraction had an optical rotation similar to that of the porphyran from P. capensis while the optical rotation of another fraction was similar to that of the phycocolloid of P. umbilicalis. The molar composition of the latter was D-galactose:6-~-methyl-D-galactose:3,6-anhydro-~ga1actose:sulphate in the ratio 1.00:~0.57:0.35:0.65. Several spectra could be assigned to various signals in the 'H n.m.r. anomeric protons. A biophotoreactor, using immobilized cells of Porphyridium cruentum in a polyurethane 'mousse', has been used to produce a capsular polysaccharide sulphate which is secreted into solution.29 Sulphated polysaccharides have been isolated from Caulerpa species and their function in wound healing has been assessed.293 Fucoidanase activities have been observed in fractions from the hepatopancreas of abalone. 2 9 4 The primary product from one enzyme
'
Carbohydrate Chemistry
60
was Ti-fucose while another enzyme gave a series of neutral oligosaccharides. Laminarin, L-fucans, and alginic acid have all been isolated from Dictyopteris p l a g i o ~ r a m m a . The ~ ~ ~ laminarin contained chains terminating in a !-glucose residue and those terminating in a The &-fucans were present in Q-mannitol residue in the ratio 3 : l . a variety of extracts and each had a slightly different ratio of ~-fucose:~-xylose:~-galactose:~-mannose:~-glucuronic acid:sulphate. The structural features elucidated were similar to those fromother L-fucans. Polysaccharide material from Eucheuma spinosum appears to consist of two different components containing low and high sulphate contents. 2 9 6 The major component had the higher sulphate content while the molecular sizes of the two polysaccharides were d i fferent A series of acidic oligosaccharides (8) (20) has been obtained by graded acid hydrolysis of the methylated acidic polysaccharide associated with the coccoliths of the alga Emiliania huxleyi: the above structures were characterized. 2 9 7 The results along with methylation data from the native, carboxyreduced, and desulphated carboxy-reduced polysaccharides have been used to give a proposed structure for this complex polymer.298 Glycogen accumulation in vegetative cells of the blue-green alga Anabaena is a light-dependent process.299 The amount of glycogen produced was high during sporulation and this increased with the onset of maturation of the spores. The amino-acids L-methionine, k-tyrosine, b-glycine, and k-histidine gave the greatest enhancement of glycogen accumulation in supplemented cultures although all the amino-acids examined had a positive effect.3 0 0 The storage D-glucans in and isoenzyme distribution of Cyanidium caldarium have been in~estigated.~” The observations propose this organism to be a primitive Rhodophytan which is an algal-bridge linking the prokaryotic blue-green and the red algae. It seems to be a true transitional organism between the nucleated and anucleated algae and is unlikely to be an endosymbiotic association of more than two cells as speculated elsewhere. Two major arsenical constituents of the brown kelp Ecklonia radiata have been isolated and characterized as
.
-
2-hydroxy-~-sulphopropyl-5-deoxy-5-(dimethylarsenoso)-furanoside
and 2,3-dihydroxypropyl-5-deoxy-5(dimethylarsenoso)-furanoside
.302
3: Plant and Algal Polysaccharides
61
a-E-GalpA-(1-+6)-a-D-Manp-(l+3)-E-Man (8)
E-GalpA- ( 1 -4)- p G a l p - (14 -Man ) (9)
~-GalpA-(1+4)-~-GalpA-(1-+2/6)-Manp-(1+3)-Man
(10)
g-GalpA- ( 1 +4 ) -g-GalpA- ( 1 +3 ) -E-Xyl (1 1 )
q-GalpA-( 1+2)-L=-Manp6Me-( 1+4)-!-GalpA-(
1+2)-&-Rha -
(12)
g-GalpA-( 1+2)-Manp-( l+&)-E-GalpA-( (13)
1+2)-L-Rha
62
Carbohydrate Chemistry
g-GalpA( 1 +2 ) -Manp- ( 1 +4) -E-GalA (16)
g-GalpA- ( 1 +2) -4-Manp6Me- ( 1 +4) -E-GalpA- ( 1 +2 ) -L_-ManGMe (17)
~-GalpA-(1+2)-~-Manp6Me-(1+4)-g-GalpA-(l-+2)-Man
(18)
3: Plant and Algal Polysaccharides
63
References 1
2 3 4 5 6 7 8
9 10 11
12 13
14 15 16 17
18
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
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.-
I.
-
64
50 51
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E.Perciva1, M.A.Rahman, and H.Weige1, Ph tochemistr 1981, 20, 1579. H.K.Tong, K.H.Lee, and H.A.Wong, Carbohyir. Res., 1;81, 88, %8. A.M.J.Fichtinger-Schepman, J.P.Kamerling, C.Versluis, and J.F.G.Vliegenthart, Carbohydr. Res., 1980, 86, 215. A.M.J.Fichtinger-Schepman, J.P.Kamerling, C.Versluis, and J.F.G.Vliegenthart, Carbohydr. Res., 1981, 93, 105. T.A.Sanna and S.Kanta, 2. ALlRem. Mikrobiol., 1979, l9, 571. T.A.Sarma and S.Kanta, Z. Ablgem. Mikrobiol., 1980, 20, 653. J.Seckbach and J.F.Frederick, Microbios, 1980, 29, 135. J.S.Edmonds and K.A.Francesconi, Nature (London), 1981, 28q, 602.
298 299 300 30 1 302
1980,
2,
323.
29
1979,
100, 455.
1980,
3, 132-
87.
Agric. Chem. Soc. Jpn.,
,
1979,
1,
2,
Micro bial Polysaccharides BY C.A. WHITE 1
T e i c h o i c Acids
A review h a s s u m m a r i z e d t h e b i o s y n t h e s i s , a s s e m b l y , a n d l o c a t i o n o f t e i c h o i c and t e i c h u r o n i c acids, which i n c l u d e s t h e b i o s y n t h e s i s o f l i p o t e i c h o i c a c i d s and t h e i r involvement i n t e i c h o i c a c i d biosytheses.’ M e m b r a n e s f r o m B a c i l l u s s u b t i l i s W23 s y n t h e s i z e d a l i p i d precursor of the linkage unit that attaches teichoic acid t o the cell wall I t c o n t a i n e d glycerophosphoryl-2-acetamido-2-deoxy-~g l u c o s e , l i n k e d t h r o u g h a n a c i d - l a b i l e bond t o a l f p i d . Glycerol-3-phosphate c y t i d y l y l t r a n s f e r a s e , Q-ribitol-5phosphate cytidylyltransferase, and p o l y ( r i b i t o 1 phosphate) s y n t h e t a s e a c t i v i t i e s have been measured i n c u l t u r e s o f B a c i l l u s s u b ti l i s W23 a s t h e y became p h o s p h a t e - s t a r v e d e i t h e r i n b a t c h c u l t u r e o r d u r i n g changeover from p o t a s s i u m l i m i t a t i o n t o p h o s p h a t e l i m i t a t i o n i n a ~ h e m o s t a t . ~The r e s u l t s i n d i c a t e d t h a t r e p r e s s i o n o f s y n t h e s i s o f a l l t h r e e enzymes o c c u r r e d a t t h e o n s e t o f p h o s p h a t e s t a r v a t i o n a n d t h a t t h i s was a c c o m p a n i e d by i n h i b i t i o n o r i n a c t i v a t i o n o f glycerol-3-phosphate c y t i d y l y l t r a n s f e r a s e and poly(ribito1 phosphate) synthetase. T h e s e r e s u l t s show t h a t t h e i n i t i a l response t o phosphate s t a r v a t i o n i n v o l v e s more than Syntheses of both i n h i b i t i o n o f o n e enzyme as p r e v i o u s l y proposed. linkage u n i t and p o l y ( r i b i t o 1 phosphate) are i n h i b i t e d independently. P r o t o p l a s t s o f B a c i l l u s s u b t i l i s W23 r e a d i l y s y n t h e s i z e d r i b i t o l t e i c h o i c acid from n u c l e o t i d e p r e c u r s o r s i n t h e s u r r o u n d i n g medium. W i t h C D P - r i b i t o l t h e y made p o l y ( r i b i t o 1 p h o s p h a t e ) , p r e s u m a b l y a t t a c h e d t o l i p o t e i c h o i c a c i d ~ a r r i e r :w~h e n C D P - g l y c e r o l were a l s o p r e s e n t , a 1 0 - f o l d a n d UDP-2-acetamido-2-deoxy-Q-glucose increase i n the r a t e of polymer s y n t h e s i s occurred, and t h e product c o n t a i n e d both t h e main c h a i n and t h e l i n k a g e u n i t . S y n t h e s i s was i n h i b i t e d by t r y p s i n o r 4 - c h l o r o m e r c u r i b e n z e n e s u l p h o n a t e i n t h e
.’
4: Microbial Polysaccharides
71
and i t was concluded t h a t i t occurred a t t h e o u t e r s u r f a c e During s y n t h e s i s , which was a l s o achieved r e a d i l y by whole c e l l s a f t e r a b r i e f p e r i o d o f w a l l l y s i s , t h e CMP p o r t i o n of t h e n u c l e o t i d e p r e c u r s o r s d i d n o t pass through t h e membrane. No evidence was o b t a i n e d f o r a t r a n s p h o s p h o r y l a t i o n mechanism f o r t h e I t was s u g g e s t e d t h a t r e a c t i o n w i t h translocation process. exogenous s u b s t r a t e s was due t o t e m p o r a r y e x p o s u r e of a p r o t e i n component of t h e enzyme complex a t t h e o u t e r s u r f a c e of t h e membrane d u r i n g t h e normal b i o s y n t h e t i c cycle. T o l u e n i t e d c e l l s of B a c i l l u s s u b t i l i s W23 s y n t h e s i z e d t h e t e i c h o i c a c i d , p o l y ( r i b i t o 1 phosphate), from exogenous precursor^.^ The s y n t h e s i s was dependent on concomitant s y n t h e s i s of t h e l i n k a g e u n i t t h a t j o i n s t e i c h o i c a c i d t o p e p t i d o g l y c a n . Under c o n d i t i o n s t h a t reduced c e l l a u t o l y t i c a c t i v i t y , a l a r g e p r o p o r t i o n of t h e t e i c h o i c a c i d became l i n k e d t o t h e c e l l w a l l , i n d e p e n d e n t l y o f p e p t i d o g l y c a n s y n t h e s i s . The s p e c i f i c a c t i v i t y of t h e s y s t e m was more t h a n 30 t i m e s t h a t o f i s o l a t e d membranes, s o t h a t a c t i v i t y c o u l d be measured r e a d i l y i n t h e c e l l s from 2 m l of an e x p o n e n t i a l c u l t u r e of b a c t e r i a . The i n f l u e n c e o f t h e l e c t i n s c o n c a n a v a l i n A a n d phytohaemagglutinin A and t h e p o l y e l e c t r o l y t e s d e x t r a n - s u l p h a t e ( a s i t s s o d i u m s a l t ) and d i e t h y l a m i n o e t h y l - d e x t r a n ( a s i t s hydrochlori d e ) o n b a c t e r i a l t r a n s f o r m a t i o n s i n B a c i l l u s s u b t i l i s h a s been s t u d i e d 6 i n o r d e r t o d i s c r i m i n a t e t h e t a r g e t o f l e c t i n s and p o l y e l e c t r o l y t e s i n competent c e l l s . B a c i l l u s s u b t i l i s var. n i g e r WM was grown i n continuous c u l t u r e under p h o s p h a t e - l i m i t e d and under m a g n e s i u m - l i m i t e d c o n d i t i o n s . Whole c e l l s , c e l l w a l l s , a n d t h e i s o l a t e d w a l l p o l y m e r s p e p t i d o g l y c a n , t e i c h o i c a c i d , and t e i c h u r o n i c a c i d were analysed by Curie-point p y r o l y s i s mass ~ p e c t r o m e t r y . ~ C h a r a c t e r i s t i c i o n peaks f o r t h e w a l l p o l y m e r s were e s t a b l i s h e d and f a c i l i t a t e d t h e i n t e r p r e t a t i o n o f the mass pyrograms of w a l l s and whole c e l l s . The mass pyrograms o f magnesium-limited c e l l s showed t h e c h a r a c t e r i s t i c peaks f o r p r o t e i n , peptidoglycan, and t e i c h o i c acid. A t u n i c a m y c i n - l i k e a n t i b i o t i c 2 4 0 1 0 a t a c o n c e n t r a t i o n of 1 pg/ml s e l e c t i v e l y inhibited the v i v o s y n t h e s i s of g l y c e r o l t e i c h o i c a c i d o f c e l l w a l l s i n B a c i l l u s c e r e u s AHU 1030.8 I n c u b a t i o n of membranes o f t h i s s t a i n w i t h 2-acetamido-Z-deoxy-Qglucopyranosyl p y r o p h o s p h o r y l u n d e c a p r e n o l and UDP-Z-acetamido-2deoxy-a-mannase l e d t o f o r m a t i o n o f a g l y c o l i p i d having a s a c c h a r i d e moiety i d e n t i c a l w i t h t h e c e l l - w a l l t e i c h o i c a c i d l i n k a g e u n i t (1). medium,
o f t h e membrane.
Carbohydrate Chemistry
72
The membranes a l s o c a t a l y s e d t r a n s f e r o f g l y c e r o l p h o s p h a t e u n i t s The from CDP-glycerol t o t h i s disaccharide-linked l i p i d . b i o s y n t h e s i s o f t h e cell-wall g l y c e r o l t e i c h o i c acid i n t h i s s t r a i n seemed t o involve t h e disaccharide-linked l i p i d as an i n t e r m e d i a t e .
8-Q-Man~NAc-(1+4)-Q-GlcNAc (1)
The p r e s e n c e o f a c t i v e , or o f o n l y r e v e r s i b l y i n a c t i v a t e d , enzyme s y s t e m s f o r t h e s y n t h e s i s and i n c o r p o r a t i o n o f t e i c h o i c acid h a s b e e n d e t e r m i n e d 9 by w i t h d r a w i n g s a m p l e s o f b a c t e r i a f r o m a p h o s p h a t e - p u l s e d c h e m o s t a t c u l t u r e i n t o p h o s p h a t e - r i c h medium t h a t contained chloramphenicol. The i n c r e a s e d c o n t e n t o f w a l l t e i c h o i c a c i d f o l l o w i n g i n c u b a t i o n o f t h e s e s a m p l e s was d e t e r m i n e d by chemical analysis o f the recovered bacteria. Q - A l a n y l - l i p o t e i c h o i c a c i d from L a c t o b a c i l l u s casei c o n t a i n s a p o l y ( g l y c e r o 1 p h o s p h a t e ) m o i e t y t h a t i s s e l e c t i v e l y a c y l a t e d w i t h 0a l a n i n e ester residues." To c h a r a c t e r i z e f u r t h e r t h e m e c h a n i s m o f Q - a l a n i n e s u b s t i t u t i o n , i n t e r m e d i a t e s were s o u g h t t h a t p a r t i c i p a t e i n t h e assembly o f t h i s l i p o t e i c h o i c acid. From t h e i n c o r p o r a t i o n system u t i l i z i n g either t o l u e n e - t r e a t e d c e l l s or a combination o f membrane f r a g m e n t s a n d s u p e r n a t a n t f r a c t i o n , a series o f membranec o m p o u n d s was f o u n d . The a s s a y a s s o c i a t e d Q-{14C)alanyl-lipophilic o f t h e s e compounds depended on t h e i r e x t r a c t a b i l i t y i n t o m o n o p h a s i c c h l o r o f o r m -met h a n o 1-water (0.8 :3.2 :1 . 0 , vo l / v o l / v o 1) a n d s u b s e q u e n t p a r t i t i o n i n g i n t o chloroform. Four l i n e s o f evidence suggested t h a t t h e Q - a l a n y l - l i p o p h i l i c compounds are i n t e r m e d i a t e s i n t h e s y n t h e s i s o f Q - a l a n y l - l i p o t e i c h o i c acid. First, p a r t i a l d e g r a d a t i o n o f t h e p o l y ( g l y c e r o 1 p h o s p h a t e ) m o i e t y o f Q - a l a n y l - l i p o t e i c h o i c a c i d by phosphodiesterase II/phosphatase from Aspergillus n i g e r generated a series o f P - a l a n y l - l i p o p h i l i c compounds similar t o t h o s e e x t r a c t e d from t o l u e n e - t r e a t e d c e l l s d u r i n g t h e i n c o r p o r a t i o n o f Q - a l a n i n e . Second, e n z y m a t i c d e g r a d a t i o n o f t h e Q - a l a n y l - l i p o p h i l i c compounds by t h e a b o v e p r o c e d u r e g a v e Q - a l a n y l - g l y c e r o l , t h e same d e g r a d a t i o n p r o d u c t o b t a i n e d from a - a l a n y l - l i p o t e i c h o i c acid. Third, the i n c o r p o r a t i o n o f 9 - a l a n i n e i n t o t h e s e c o m p o u n d s r e q u i r e d t h e same components as t h e i n c o r p o r a t i o n o f Q - a l a n i n e i n t o membraneFourth, t h e phosphate-induced l o s s a s s o c i a t e d Q - l i p o t e i c h o i c acid. o f g- { C 1 a 1a n i n e - 1a be1 1e d 1i p o p h i 1i c c o m p o u n d s c o u 1d be c o r r e 1a t e d with t h e stimulation of phosphatidylglycerol synthesis i n the p r e s e n c e o f e x c e s s p h o s p h a t e . T h e s e e x p e r i m e n t s were i n t e r p r e t e d t o
73
4: Microbial Polysaccharides indicate that
the Q-alanyl-lipophilic
compounds w e r e Q - a l a n y l -
l i p o t e i c h o i c a c i d w i t h s h o r t p o l y m e r c h a i n s and were m o s t l i k e l y i n t e r m e d i a t e s i n t h e assembly o f t h e completed polymer,
a-alanyl-
l i p o t e i c h o i c acid. Teichuronic a c i d i s o l a t e d from the c e l l w a l l s o f Micrococcus
-----luteus
has
been
spectroscopy."
examined
by
natural-abundance
n.m.r.
13C
P r o t o n - d e c o u p l e d a n d p r o t o n - c o u p l e d s p e c t r a were
o b t a i n e d f o r n a t i v e t e i c h u r o n i c a c i d and a l s o a f t e r t h e t e i c h u r o n i c a c i d h a d been o x i d i z e d w i t h p e r i o d a t e and r e d u c e d w i t h b o r o h y d r i d e . The s p e c t r a a r e c o n s i s t e n t w i t h t h e s t r u c t u r e ( 2 ) .
Teichuronic a c i d
s y n t h e s i z e d i n v i t r o f r o m s u i t a b l e s u b s t r a t e s by t h e p a r t i c u l a t e enzyme f r a c t i o n o b t a i n e d f r o m M i c r o c o c c u s l u t e u s y i e l d e d a 13C n.m.r.
spectrum which i s i n d i s t i n g u i s h a b l e from t h a t o f the n a t i v e
teichuronic
acid,
indicating a
teichuronic a c i d synthesized
in
structural identity
o f
the
v i t r o w i t h t h a t i s o l a t e d from c e l l
walls.
+4)-B-Q-Man~ANAc-(l+6)-ct-Q-Glc~-(l+ (2) The
membrane-bound
enzymes
of
Micrococcus varians
which
p a r t i c i p a t e i n t h e syntheses o f t h e t e i c h o i c a c i d main c h a i n and linkage
unit
have
been
solubilized
f r a c t i o n a t e d by s u c r o s e d e n s i t y - g r a d i e n t f r a c t i o n s were obtained:
with
Triton
X-100
centrifugation.12
a heavy f r a c t i o n ,
and
Two m a i n
c o n t a i n i n g enzymes
e f f e c t i n g s y n t h e s i s o f t h e main chain attached t o t h e l i n k a g e u n i t , w h i c h was a s s o c i a t e d w i t h o n l y a s m a l l amount o f l i p i d ,
and a l i g h t
f r a c t i o n , w h i c h was r i c h i n p r e n y l p h o s p h a t e a n d c a t a l y s e d o n l y linkage-unit
synthesis.
The s e p a r a t i o n b y d e n s i t y w a s n o t b a s e d
e n t i r e l y on p o l y p e p t i d e c h a i n l e n g t h , as some o f t h e s h o r t e s t c h a i n s a p p e a r e d i n t h e d e n s e r f r a c t i o n s a n d some r e l a t i v e l y h i g h - m o l e c u l a r weight peptides occurred i n the l i g h t e s t fraction.
High a c t i v i t y
f o r l i n k a g e - u n i t s y n t h e s i s was o b s e r v e d i n a f r a c t i o n c o n t a i n i n g o n l y a few p e p t i d e s .
A d d i t i o n o f f i c a p r e n y l p h o s p h a t e t o t h e enzyme
p r e p a r a t i o n s h a d no s t i m u l a t o r y e f f e c t .
I t was c o n c l u d e d t h a t t h e
enzymes f o r m a i n - c h a i n and l i n k a g e - u n i t
s y n t h e s e s f o r m one o r m o r e
f a i r l y t i g h t l y a s s o c i a t e d c o m p l e x e s and t h a t p o l y p r e n y l p h o s p h a t e i s an i n t e g r a l ,
f i r m l y bound component o f t h e complex i n w h i c h t h e
linkage u n i t i s synthesized. The m a i n c h a i n o f t e i c h o i c a c i d s c a n be a s s e m b l e d i n c e l l - f r e e membrane p r e p a r a t i o n s by t h e t r a n s f e r o f r e s i d u e s f r o m t h e
Carbohydrate Chemistry
74 appropriate nucleotide precursors t o a m p h i p h i l i c molecule, c e l l wall,
an i n c o m p l e t e l y c h a r a c t e r i z e d
lipoteichoic acid carrier.13
However,
linkage u n i t which i s synthesized independently. that,
i n the
the main chain i s attached t o peptidoglycan through a
i n these c e l l - f r e e
linkage units
are
systems,
also
able
to
It i s believed
l i p i d intermediates carrying
accept
residues
n u c l e o t i d e p r e c u r s o r s t o b u i l d up t h e m a i n c h a i n .
directly
from
I t was shown t h a t
t h e m a i n c h a i n a t t a c h e d t o l i p o t e i c h o i c a c i d c a r r i e r was t r a n s f e r r e d from l i p o t e i c h o i c acid c a r r i e r t o l i p i d s containing the linkage unit,
Thus,
i n t h e s e systems,
t h e r e a p p e a r t o be t w o r o u t e s t o t h e
biosynthesis o f teichoic acid/linkage-unit
complexes,
one by d i r e c t
a s s e m b l y o f t h e m a i n c h a i n o n l i n k a g e - u n i t l i p i d s a n d t h e o t h e r by t r a n s f e r o f the preassembled main chain from l i p o t e i c h o i c a c i d carrier t o the linkage unit.
I t was a l s o s h o w n t h a t l i n k a g e - u n i t
l i p i d s f r o m d i f f e r e n t o r g a n i s m s w e r e i n t e r c h a n g e a b l e and t h a t t h e s e w e r e u s e d f o r p o l y m e r s y n t h e s i s by B a c i l l u s s u b t i l i s 3610,
i n which
t h e t e i c h o i c a c i d i s a p o l y ( g l y c e r o 1 phosphate). The i n c r e a s i n g u s e o f S t a p h y l o c o c c u s a u r e u s r i b i t o l t e i c h o i c acid,
which possesses a 2-acetamido-2-deoxy-B-Q-glucopyranosyl
residue,
i n s e r o l o g i c a l d i a g n o s i s e s p e c i a l l y i n b a c t e r e m i a and
osteoarticular antigen.14
infections
I n the
course
prompted the of
the
a g g l u t i n i n immobilized on U l t r o g e l , acetamido-2-deoxy-Q-glucopyranosyl
separate
ribitol
This study
was
teichoic
purification of this
purification
a c i d from
the
wheat-germ
which has a f f i n i t y residues, other
was
for
2-
employed
to
contaminating antigens.
concerned w i t h the abnormal a f f i n i t y
acetamido-2-deoxy-B-~-glucosy~ r i b i t o l
S t a p h y l o c o c c u s a u r e u s f o r t h e wheat-germ
teichoic
of
t h e 2-
acid
o f
a g g l u t i n i n i m m o b i l i z e d on
U l t r o g e l w h i c h i s dependent on t h e i o n i c f o r c e s and t h e n a t u r e o f the contaminants. Native substitution w i t h the p-alanine ester o f lipoteichoic acids a f f e c t s t h e i r immunological properties, divalent cations,
the capacity to bind
and l i p o t e i c h o i c a c i d c a r r i e r a c t i v i t y . 1 5
i n f l u e n c e o f the Q-alanine e s t e r on a n t i - a u t o l y t i c
The
a c t i v i t y was
t e s t e d , u s i n g e x t r a c e l l u l a r a u t o l y s i n f r o m S t a p h y l o c o c c u s a u r e u s and n i n e l i p o t e i c h o i c acids w i t h a l a n i n e /phosphorus
molar ratios o f
b e t w e e n 0.23
h i g h e s t w i t h Q-
a n d 0.71.
The i n h i b i t o r y a c t i v i t y ,
a 1 a n i n e - f r e e 1i p o t e ic h o i c increasing alanine content, g r e a t e r t h a n 0.6. not
inhibitory,
a c id, e x p o n e n t ia 11y
Correspondingly, i n
de c r ea s e d
w it h
approaching zero a t s u b s t i t u t i o n s of
contrast
d i p o l a r i o n i c p h o s p h o l i p i d s were to
negatively
charged
ones.
75
4: Microbial Polysaccharides
G l y c o s y l a t i o n o f l i p o t e i c h o i c a c i d up t o a n e x t e n t o f 0.5 d i d n o t depress i n h i b i t o r y a c t i v i t y , was c o m p a r a t i v e l y s m a l l . various sources,
and e v e n a t a d e g r e e o f 0.8
the effect
On c o m p a r i s o n o f l i p o t e i c h o i c a c i d s f r o m
d i f f e r e n c e s i n l i p i d s t r u c t u r e s and c h a i n l e n g t h s
were w i t h o u t e f f e c t .
The i n h i b i t o r y a c t i v i t y d r a s t i c a l l y d e c r e a s e d
when t h e g l y c o l i p i d c a r r i e d a s i n g l e g l y c e r o p h o s p h a t e r e s i d u e or t h e
(3).
h y d r o p h i l i c c h a i n had t h e u n u s u a l s t r u c t u r e were o b t a i n e d w i t h t h e more complex
The same r e s u l t s
system o f
autolysis o f
I t was s u g g e s t e d t h a t t h e a n t i -
Staphylococcus a u r e u s c e l l s .
a u t o l y t i c a c t i v i t y o f l i p o t e i c h o i c a c i d r e s i d u e s i n a sequence o f glycerophosphate
units
and
that
the
negative
charges
a p p r o p r i a t e l y spaced p h o s p h o d i e s t e r groups p l a y a c r u c i a l r o l e . g-alanine
of The
e s t e r e f f e c t was d i s c u s s e d w i t h r e s p e c t t o t h e p u t a t i v e
vivo regulation
in
o f a u t o l y s i s by l i p o t e i c h o i c a c i d .
+6)-a-Q-Gale-(1+6)-a-g-Gale-O-CH2
I
a-g-Gale-0-CH
l
o
CH 2-0 -P+
OH (3) Streptococcus mutans
Ingbritt
was
grown i n a chemostat
d e s t i n e d d i l u t i o n r a t e s i n e i t h e r 0.5% ! - f r u c t o s e and a t d e s t i n e d pH v a l u e s i n 0.5% f r u c t o s e . a f f e c t e d by t h e c a r b o h y d r a t e s o u r c e , l o w e s t y i e l d b e i n g a t pH 5.5
at
o r 0.5% p - g l u c i t o l
The y i e l d o f c e l l s was
as w e l l as by t h e pH,
i n 0.5% p - f r u c t o s e . 1 6
with the
!-Fructose-grown
c e l l s showed g r e a t e r s u s c e p t i b i l i t y t o l y s i s by a m u r a m i d a s e t h a n t h e corresponding g-glucose-grown
cells,
b u t t h e r e w e r e no m a r k e d
differences i n the l y t i c s u s c e p t i b i l i t i e s o f the corresponding c e l l w a l l preparations or i n the serological r e a c t i v i t i e s o f w a l l lysates w i t h antiserum t o Streptococcus
mutans
Ingbritt.
The
greatest
amounts o f c e l l u l a r l i p o t e i c h o i c a c i d were o b t a i n e d a t h i g h d i l u t i o n r a t e s i n b o t h Q - f r u c t o s e and Q - g l u c i t o l , values
i n
!-fructose.
The
greatest
a s w e l l a s a t h i g h pH
amounts
of
l i p o t e i c h o i c a c i d were f o u n d a t l o w d i l u t i o n r a t e s ,
extracellular as e s t i m a t e d by
r o c k e t i m m u n o e l e c t r o p h o r e s i s and a l s o by h a e m a g g l u t i n a t i o n .
Three
m a j o r e x t r a c e l l u l a r p r o t e i n components w e r e s e p a r a t e d by s o d i u m dodecyl sulphate-polyacrylamide of
g e l e l e c t r o p h o r e s i s , and t h e e f f e c t s
g r o w t h c o n d i t i o n s on t h e s e components were d e t e r m i n e d .
Results
76
Carbohydrate Chemistry
f o r batch-grown c u l t u r e s showed t h a t t h e r e was g e n o t y p i c v a r i a t i o n The i n t h e s u s c e p t i b i l i t y o f c e l l s t o l y s i s by a m u r a m i d a s e . e n h a n c e m e n t o f l i p o t e i c h o i c a c i d p r o d u c t i o n o f p - f r u c t o s e a n d ag l u c i t o l i n b a t c h c u l t u r e s was n o t i d e n t i c a l i n r e p r e s e n t a t i v e n o r was t h e e f f e c t o f s t r a i n s o f S t r e p t o c o c c u s m u t a n s s e r o t y p e 2, p-fructose found uniformly i n r e p r e s e n t a t i v e s t r a i n s of t h e d i f f e r e n t Streptococcus mutans serotypes. T h e p r o b l e m s c a u s e d by t h e n o n - s p e c i f i c b i n d i n g o f l i p o t e i c h o i c a c i d and glucan-binding p r o t e i n s o f Streptococcus mutans t o immunosorbent columns prepared from cyanogen bromide-activated S e p h a r o s e 48 h a v e b e e n d e s c r i b e d . 1 7 H u m a n p o l y m o r p h o n u c l e a r l e u k o c y t e s were s h o w n t o p o s s e s s s p e c i f i c binding sites f o r l i p o t e i c h o i c acid.18 L i p o t e i c h o i c acid b i n d i n g was r e v e r s i b l e a n d t i m e a n d t e m p e r a t u r e d e p e n d e n t . Scatchard p l o t analysis revealed an apparently single population o f 6.6 x l o 6 l i p o t e i c h o i c acid b i n d i n g s i t e s p e r human p o l y m o r p h o n u c l e a r l e u k o c y t e s w i t h a d i s s o c i a t i o n c o n s t a n t o f 5.6 ~ J M . Attachment of an a v i r u l e n t unencapsulated, M-negative of group A s t r e p t o c o c c i t o human p o l y m o r p h o n u c l e a r l e u k o c y t e s was i n h i b i t e d by l i p o t e i c h o i c a c i d b u t n o t by o t h e r b a c t e r i a l s o m a t i c a n t i g e n s tested. O c c u p a t i o n o f 30% o f t h e l i p o t e i c h o i c a c i d b i n d i n g s i t e s r e s u l t e d i n g r e a t e r t h a n 70% i n h i b i t i o n o f s t r e p t o c o c c a l a t t a c h m e n t o f human p o l y m o r p h o n u c l e a r l e u k o c y t e s . In contrast, lipoteichoic acid failed t o block attachment o f Escherichia c o l i o r antibodycoated streptococci, indicating that binding sites f o r E s c h e r i c h i a c o l i and t h e Fc p o r t i o n o f immunoglobulin G are d i s t i n c t from t h o s e f o r l i p o t e i c h o i c a c i d . Immunofluorescent studies demonstrated t h a t l i p o t e i c h o i c acid uniformly bound t o p o l y m o r p h o n u c l e a r l e u k o c y t e m e m b r a n e s f o r a s l o n g a s 2 h a t 37'C. C r o s s - l i n k i n g o f human p o l y m o r p h o n u c l e a r l e u k o c y t e s - b o u n d l i p o t e i c h o i c acid r e s u l t e d i n r a p i d capping o f l i p o t e i c h o i c r e c e p t o r sites. The r e s u l t s s u g g e s t t h a t l i p o t e i c h o i c a c i d i s a m o n o v a l e n t l i g a n d i n t e r a c t i n g w i t h m o b i l e r e c e p t o r s i n t h e p l a s m a membrane o f human p o l y m o r p h o n u c l e a r l e u k o c y t e s . 'H a n d 1 3 C n.m.r. has been shown t o d i s t i n g u i s h e a s i l y b e t w e e n c o v a l e n t l y and i o n i c a l l y a s s o c i a t e d a-alanine i n l i p o t e i c h o i c a c i d s , w i t h 13C n.m.r. showing regular 1,3-linkages in the polyglycerophosphate chain and a s s i g n a b l e resonances f o r t h o s e s u b u n i t s c a r r y i n g P - a l a n i n e and s u g a r r e s i d u e s . 1 9
4: Microbial Polysaccharides Peptidoglycans
2 The
77
immunological a c t i v i t i e s o f
b a c t e r i a l p e p t i d o g l y c a n s have
been r e v i e w e d and t h e s t r u c t u r e s o f p a r t i c u l a r p e p t i d o g l y c a n s and groups o f p e p t i d o g l y c a n s r e l a t e d t o a c t i v i t y . 2 0 Pep t i d o g l y c a n monomer (a-GlceNAc-Mur NAc-L-A l a - Q -i s o g l u t am i n e
-meso-diaminopimelic
acid-E-Ala-Q-Ala),
was i n c u b a t e d w i t h s l i c e s o f
d i s a c c h a r i d e and p e n t a p e p t i d e p o r t i o n s , mouse l i v e r ,
kidney,or
blood cells,
p l a s m a , a n d serum.21
unchanged a f t e r
-
l a b e l l e d w i t h 14C i n both the
s p l e e n as w e l l as w i t h mouse a n d human b l o o d , P e p t i d o g l y c a n monomer was i s o l a t e d
i n c u b a t i o n s w i t h mouse o r g a n s
and b l o o d c e l l s .
H o w e v e r , u p o n i n c u b a t i o n w i t h mouse o r human b l o o d , 1 0 - 5 0 % o f t h e p e p t i d o g l y c a n monomer u n d e r w e n t h y d r o l y s i s t o t h e c o r r e s p o n d i n g disaccharide
and p e n t a p e p t i d e .
serum
than
more
metabolized:
90% o f
the
After
i n c u b a t i o n s w i t h plasma
{14C)peptidoglycan
monomer
and was
a b o u t 5 0 % o f t h e a d m i n i s t e r e d r a d i o a c t i v e d o s e was
r e c o v e r e d i n t h e d i s a c c h a r i d e u n i t and a b o u t 35% i n t h e p e n t a p e p t i d e
I t was s u g g e s t e d t h a t i n b l o o d , p l a s m a , a n d
part.
man an N - a c e t y l m u r a m o y l - l - a l a n i n e
s e r u m o f mouse a n d
amidase e x i s t s
which s p l i t s the
a m i d e bond b e t w e e n t h e l a c t y l c a r b o x y l g r o u p o f t h e m u r a m y l r e s i d u e and t h e amino
mo l e c u l e
.
An
group o f
the peptide moiety
isotope-dilution
micro-analysis
using
procedure
for
quantitative N-terminal
3H- l a b e 1l e d 1- f l u o r o - 2 , 4 - d i n i t r o ben zene and a
m i x t u r e o f f r e e and a c e t y l a t e d l 4 C - 1 a b e l l e d s t a n d a r d s h a s been
described.22
The
derivatives
chromatography.
p r i m a r i l y intended f o r the estimation o f c e l l - w a l l peptidoglycan,
a m i n o a c i d s as i n t e r n a l
2,4-dinitrophenyl
a r e s e p a r a t e d by p o l y a m i d e t h i n - l a y e r
peptides.
i n the peptidoglycan
crosslinkage
Although
i n bacterial
t h e method i s c a p a b l e o f e x t e n s i o n t o o t h e r
When t e s t e d o n l y s o z y m e ,
t h e p r o c e d u r e gave r e s u l t s w h i c h
w e r e i n good a g r e e m e n t w i t h a c c e p t e d v a l u e s . The
conformational
energies
of
complexes
copolymers of 2-acetamido-2-deoxy-~-glucose w i t h hen e g g - w h i t e
of
alternating
and 2 - a c e t y l m u r a m i c a c i d
l y s o z y m e h a v e b e e n computed.23
This involved a
complete search o f t h e c o n f o r m a t i o n a l space a t t h e a c t i v e s i t e o f t h e enzyme a v a i l a b l e t o t h e s e s u b s t r a t e s and m i n i m i z a t i o n o f t h e conformational energies of ho m opo 1y m e r
( a -G 1ceNA c 16 ,
t h e n o n c o v a l e n t complexes.
G ~ c ~ N A cb )i n~d s p r e f e r e n t i a l l y on t h e l e f t s i d e o f cleft,
i n v o l v i n g r e s i d u e s s u c h as &-Arg-45,
The a l t e r n a t i n g c o p o l y m e r
As w i t h t h e
t h e hex as a c c h a r ide (Q -G lceNA c -M u r NAc ) 2I-Asn-46,
(Q-GlceNAc-MurNAc)j,
(g -
the active-site and i - T h r - 4 7 .
however,
binds with
78
Carbohydrate Chemistry
i t s F-site r e s i d u e p r e f e r e n t i a l l y on t h e r i g h t s i d e o f t h e a c t i v e s i t e c l e f t , i n v o l v i n g r e s i d u e s s u c h a s L-Phe-34 a n d L-Arg-114. The l a c t i c a c i d s i d e c h a i n p r e v e n t s good b i n d i n g t o t h e F s i t e on t h e l e f t side. This r e s u l t can e x p l a i n t h e higher rate of c a t a l y s i s f o r t h e cell-wall s u b s t r a t e ( t h e a l t e r n a t i n g co-polymer). The r e l a t i v e a f f i n i t i e s o f t h e d i s a c c h a r i d e Q-GlcQNAc-MurNAc f o r a l l s e q u e n t i a l p a i r s o f s i t e s A-F ( i n c l u d i n g E a n d F s i t e s o n b o t h s i d e s o f t h e It is found t h a t t h e highest a f f i n i t y o f c l e f t ) are determined. t h i s d i s a c c h a r i d e i s f o r s i t e s C a n d D a n d r i g h t - s i d e s i t e s E a n d F. The e n e r g y o f t h e r e c e n t l y d e t e r m i n e d X-ray c r y s t a l l o g r a p h i c s t r u c t u r e o f MurNAc-Q-GlceNAc-MurNAc b o u n d t o t h e B, C, a n d D s i t e s o f hen egg-white lysozyme has been minimized and found t o l e a d a conformation q u i t e s i m i l a r t o one which has been predicted p r e v i o u s l y f o r t h e t r i s a c c h a r i d e ( Q - G ~ c ~ N A c ) ~ .T h e D r i n g i s u n d i s t o r t e d and binds c l o s e t o t h e s u r f a c e o f t h e a c t i v e - s i t e c l e f t . T h e s t r u c t u r e c a n b e e x t e n d e d i n t o s i t e s E a n d F by a d d i t i o n o f t w o g-GlceNAc r e s i d u e s , b u t o n l y o n t h e l e f t s i d e o f t h e a c t i v e - s i t e cleft. T h i s i n d i c a t e s t h a t p o l y m e r s b o u n d w i t h t h e i r D-site r e s i d u e s near t h e s u r f a c e o f t h e c l e f t must bind t o sites E and F on t h e l e f t s i d e of t h e c l e f t , as a l s o predicted previously. A series o f seven d i f f e r e n t UDP-N-acetylmuramyl peptide p r e c u r s o r s o f b a c t e r i a l c e l l - w a l l p e p t i d o g l y c a n h a s b e e n e x a m i n e d by r e v e r s e - p h a s e h i g h - p r e s s u r e l i q u i d c h r ~ m a t o g r a p h y . ~M~i x t u r e s o f t h e s e c o m p o u n d s were s u c c e s s f u l l y a n d r a p i d l y a n a l y s e d by u s i n g Waters U B o n d a p a k C 1 8 c o l u m n a s a s t a t i o n a r y p h a s e a n d i s o c r a t i c e l u t i o n s w i t h 0.05M a m m o n i u m p h o s p h a t e o r f o r m a t e b u f f e r s o f a p p r o p r i a t e pH. A c c u r a t e q u a n t i t a t i o n c o u l d a l s o be r e a d i l y a c h i e v e d by r e v e r s e - p h a s e h i g h - p r e s s u r e l i q u i d c h r o m a t o g r a p h y . A l l t h e s e t e c h n i q u e s are e x t r e m e l y u s e f u l f o r t h e p u r i f i c a t i o n o f these compounds and f o r a wide r a n g e o f b i o c h e m i c a l s t u d i e s c o n c e r n i n g t h e cytoplasmic s t e p s of t h e b i o s y n t h e s i s of peptidoglycan. T h e s o l i d - s t a t e c o n f o r m a t i o n a l a n a l y s i s o f Ac-Q-Ala-a-AlaOH.H20, c a r r i e d o u t by i n f r a r e d a b s o r p t i o n a n d X - r a y d i f f r a c t i o n , has i n d i c a t e d t h a t t h e molecules are not extended i n a r e g u l a r c o n f o r m a t i o n , b u t r a t h e r t h a t t h e y a r e p a r t i a l l y f o l d e d , 2 5 t h e +,$* torsional angles of the carboxyl-terminal reside i n p a r t i c u l a r being i n t h e r e g i o n o f t h e l e f t - h a n d e d a - h e l i x o f t h e R a m a c h a n d r a n map. The a c e t y l a m i n o a n d p e p t i d e g r o u p s are found i n t h e u s u a l t r a n s conformation, the latter e x h i b i t i n g a deviation from r i g i d p l a n a r i t y . Only i n t e r m o l e c u l a r hydrogen bonds o c c u r i n t h e c r y s t a l state. The s o l u t i o n c o n f o r m a t i o n a l a n a l y s i s , p e r f o r m e d by i n f r a r e d
4: Microbial Polysaccharides
79
a b s o r p t i o n a n d c.d., h a s r e v e a l e d t h a t t h e amount o f i n t r a m o l e c u l a r N-H. .O=C h y d r o g e n - b o n d e d f o l d e d f o r m s , i f a n y , s h o u l d be extremely small, even i n deuteriochloroform at high dilution. In water, solvated, unordered s p e c i e s l a r g e l y predominate. Incubation o f growing Bacillus s u b t i l i s with p e n i c i l l i n G led t o the s e c r e t i o n of a peptidoglycan-related polymer and a nonglycanb o u n d p e n t a p e p t i d e i n t o t h e c u l t u r e medium.26 T h e s e c r e t e d p o l y m e r was i s o l a t e d a n d c h a r a c t e r i z e d a s a l i n e a r c e l l - w a l l g l y c a n s t r a n d s u b s t i t u t e d p r e d o m i n a n t l y by u n c r o s s - l i n k e d p e n t a p e p t i d e s i d e c h a i n s . P o l y m e r f o r m a t i o n a n d s e c r e t i o n were m o s t l i k e l y t h e r e s u l t of continued s y n t h e s i s and elongation o t nascent glycan s t r a n d s i n t h e a b s e n c e o f s u b s e q u e n t p r o c e s s i n g by p e p t i d o g l y c a n t r a n s p e p t i d a s e o f Q - a 1a n i n e c a r b o x y p e p t i d a s e e n z y m e s . The nonglycan-bound L-Ala-n-iso-Glu-meso-diaminopimelic acid-P-Ala-D-Ala pentapeptide w a s p r o b a b l y f o r m e d by a n t j - a c e t y l m u r a m o y l - l - a l a n i n e amidase a c t i v e on t h e peptide side c h a i n s of t h e uncross-linked polymer. The u n c r o s s - l i n k e d p e p t i d o g l y c a n p o l y m e r was s h o w n t o be a g o o d s u b t r a t e for penicillin-sensitive P-alanine carboxypeptidase p u r i f i e d from mem b r a n e s o f B a c i l l u s s u b t i l i s , B a c i l l u s s t e a r o t h e r m o p h i l u s , a n d Escherichia c o l i . 9 - A l a n i n e r e l e a s e was n o t , h o w e v e r , c o u p l e d t o the c r o s s - l i n k i n g o f peptide side chains, suggesting t h a t t h e s e e n z y m e s do n o t f u n c t i o n a s p e p t i d o g l y c a n t r a n s p e p t i d a s e i n v i v o . No t r a n s p e p t i d a s e o r Q - a l a n i n e c a r b o x y p e p t i d a s e a c t i v i t y was d e t e c t e d i n m i x t u r e s o f h i gh-m o l e c u l a r-w e i g h t p e n i c i 11i n - b i n d i n g p r o t e i n s f r o m ga3L&s s u b t i l i s , Bacillus stearothermophilus, or Staphylococcus aureus. Possible reasons for the inability t o d e m o n s t r a t e these a c t i v i t i e s are discussed. I n a d d i t i o n , a n Nacetylmuramoyl-i-alanine amidase a c t i v i t y which c o p u r i f i e s w i t h penicillin-binding proteins with Bacillus s u b t i l i s , Staphylococcus a u r e u s , a n d E s c h e r i c h i a c o l i was p a r t i a l l y c h a r a c t e r i z e d . C e l l - w a l l p o l y m e r s were m e a s u r e d b o t h i n t h e c e l l s a n d i n t h e c e l l - f r e e medium o f s a m p l e s f r o m s t e a d y - s t a t e c h e m o s t a t c u l t u r e s o f B a c i l l u s s u b t i l i s , growing a t v a r i o u s rates under magnesium or phosphate limitation.” The p r e s e n c e o f b o t h p e p t i d o g l y c a n a n d a n i o n i c wall polymers i n t h e c u l t u r e s u p e r n a t a n t showed t h e occurrence of wall turnover i n t h e s e c u l t u r e s . Variable p r o p o r t i o n s o f t h e t o t a l p e p t i d o g l y c a n p r e s e n t i n t h e c u l t u r e s a m p l e s were f o u n d o u t s i d e t h e cells i n d u p l i c a t e c u l t u r e s , i n d i c a t i n g t h a t t h e rate of peptidoglycan turnover is variable i n Bacillus subtilis. Besides p e p t i d o g l y c a n , a n i o n i c w a l l p o l y m e r s were d e t e c t e d i n t h e c u l t u r e supernatant, teichoic acid i n magnesium-limited cultures, and
Carbohydrate Chemistry
80 teichuronic acid i n phosphate-limited cultures. the
ratio
between
concentrations
was
than i n the walls.
the
peptidoglycan
significantly
lower
and
I n s e v e r a l samples,
the
anionic-polymer
i n the extracellular
fluid
T h i s d i v e r g e n c y was a t t r i b u t e d t o t h e o c c u r r e n c e
o f d i r e c t secretion o f anionic polymers a f t e r t h e i r synthesis. An enzyme w h i c h c a t a l y s e s t h e h y d r o l y s i s o f a c e t a m i d o g r o u p s o f 2-ace t am ido -2-deox y - g - g l u c o p y r anosy 1 r e s i d u e s i n c e l l - w a 11 pep t id o g l y c a n was f o u n d i n t h e s u p e r n a t a n t and 20,0009
pellet fractions o f
Autolysis of the l a t t e r fraction resulted i n
B a c i l l u s cereus.28
s o l u b i l i z a t i o n and a c t i v a t i o n o f t h e d e a c e t y l a s e . bacteria, of
2-amino-2-deoxy-P-glucopyranosyl
i n t h e deacetylase. able
from
the
basis
behaviour.
Among v a r i o u s
s t r a i n s o f B a c i l l u s cereus which c o n t a i n h i g h p r o p o r t i o n s residues are
particularly
2-acetamido-2-deoxy-~-glucose-6-phosphate
of
rich
The p e p t i d o g l y c a n d e a c e t y l a s e i s d i s t i n g u i s h -
their
cellular
d i s t r i b u t i o n and
deacetylase
on
chromatographic
The r a t e o f r e a c t i o n o f t h e d e a c e t y l a s e w i t h (P-GlceNAc-
MurNAcI3 i s l e s s t h a n 1/100 o f t h a t w i t h p e p t i d o g l y c a n , w h i l e t h e enzyme i s i n a c t i v e t o w a r d s (Q-GlceNAc-Mur N A ~ ) ~ - t j - G l c e N A c - M u r N A c , and mo n o m e r ic 2 - a c e t a m id o - 2 - d e o x y -g
- g 1u c o s e
d e r iv a t iv e s
.
The enzyme
also deacetylates p a r t i a l l y g-hydroxyethylated chitin,
for half-
m a x i m u m a c t i v i t i e s w e r e f o u n d t o b e 0 . 2 9 a n d 6.9 mg p e r m l ( 0 . 1 7 a n d 20mM w i t h r e s p e c t t o 2 - a c e t a m i d o - 2 - d e o x y - ~ - g l u c o p y r a n o s y l respectively.
residues),
The o c c u r r e n c e o f t h i s enzyme a c c o u n t s f o r t h e f o r m -
a t i o n o f c e l l - w a l l peptidoglycan containing non-acetylated
2-amino-
2-deoxy-~-glucopyranosyl residues. The c e l l w a l l s i s o l a t e d f r o m a x e n i c a l l y g r o w n l e p r o s y - d e r i v e d c o r y n e b a c t e r i a were s u b m i t t e d t o v a r i o u s c h e m i c a l and e n z y m a t i c degradations.
The g l y c a n s t r a n d s o f t h e w a l l p e p t i d o g l y c a n w e r e
e s s e n t i a l l y composed o f 2-acetamido-2-deoxy-~-glucopyranosyl-~acetylmuramic
acid disaccharide
units.29
S m a l l amounts
of
2-
acetamido-2-deoxy-~-glucopyranosyl-~-glycolylmuramic a c i d (less than 10%) were a l s o d e t e c t e d .
The m u r a m i c a c i d r e s i d u e s o f a d j a c e n t
g l y c a n s t r a n d s a r e s u b s t i t u t e d by a m i d a t e d t e t r a p e p t i d e u n i t s w h i c h , i n turn,
are c r o s s - l i n k e d through d i r e c t l i n k a g e s extending between
the C-terminal diaminopimelic
2-alanine acid
r e s i d u e o f one t e t r a p e p t i d e a n d t h e meso-
residue
of
another
tetrapeptide.
Such
a
s t r u c t u r e i s very s i m i l a r t o t h a t o f the w a l l peptidoglycan found i n the
taxonomically
related
micro-organisms
of
Corynebacteriurn,
Mycobacterium, and N o r c a r d i a groups. Two
different
cell-wall
peptidoglycan synthetase
c a r r i e d by p e n i c i l l i n - b i n d i n g
systems a r e
p r o t e i n s 1 A a n d 1B p u r i f i e d f r o m
4: Microbial Polysaccharides
-E--s-c h e r i c h i a
-
81
c o l i . Both s y s t e m s c o n s i s t of two enzyme a c t i v i t i e s c a r r y i n g o u t s u c c e s s i v e r e a c t i o n s of peptidoglycan s y n t h e s i s from t h e 1 i p i d - 1i n k e d p r e c u r s o r 2 - ace t a m i do - 2 - d e o x y -B - g l u c o p y r a n o s y 1-!acetylmuramyl-(pentapeptide)-diphosphate-undecaprenol, namely t h o s e o f p e p t i d o g l y c a n t r a n s g l y c o s y l a s e a n d B-lactam a n t i b i o t i c - s e n s i t i v e transpeptidase. The a c t i v i t i e s o f t h e two enzyme s y s t e m s d i f f e r i n o p t i m a l c o n d i t i o n s a n d s e n s i t i v i t i e s o f B-lactam a n t i b i o t i c s . The p r o p e r t i e s of the p u r i f i e d p e n i c i l l i n - b i n d i n g p r o t e i n 1 A have been r e p o r t e d . 30 A p u r i f i e d p r e p a r a t i o n o f N-acetylmuramoyl-L-alanine a m i d a s e , a m u r e i n h y d r o l a s e f r o m E s c h e r i c h i a c o l i , was f o u n d t o l o s e i t s a c t i v i t y during incubation i n the presence of b a c t e r i a l phospholipid suspension^.^' W h e t h e r i t was c o - d i s p e r s e d w i t h t h e p h o s p h o l i p i d s o r a d d e d t o s o n i c a t e d p h o s p h o l i p i d s u s p e n s i o n , t h e e n z y m e was i n h i b i t e d (or i n a c t i v a t e d ) from t h e f i r s t m i n u t e s o f i n c u b a t i o n a t 37OC. As t h e p h o s p h a t i d y l g l y c e r o l / c a r d i o l i p i n r a t i o o f t h e p h o s p h o l i p i d s u s p e n s i o n was i n c r e a s e d ( a l l o t h e r t h i n g s b e i n g e q u a l ) , a f u r t h e r d e c r e a s e o f a m i d a s e a c t i v i t y was o b s e r v e d . T h e h i g h e s t l o s s e s o f a c t i v i t y were f o u n d a f t e r c o - d i s p e r s i o n o f t h e enzyme and t h e s u b s t r a t e t o g e t h e r w i t h t h e p h o s p h o l i p i d s , t h e r e s u l t i n g suspension being formed of l a r g e r multilayered vesicles, a s r e v e a l e d by e l e c t r o n m i c r o s c o p y . I n t h e s e c o n d i t i o n s , t h e e f f e c t o n e n z y m e a c t i v i t y w a s o n l y p a r t i a l l y a c c o u n t e d f o r by t h e p r o p o r t i o n o f t h e e n z y m e t h a t was e n t r a p p e d i n t h e v e s i c l e s . T h e entrapment c a p a c i t y of t h e enzyme ( u s i n g a 35S-labelled enzyme p r e p a r a t i o n ) a n d o f t h e s u b s t r a t e ( 3 H - l a b e l l e d ) by t h e m u l t i l a m e l l a r p h o s p h o l i p i d i c vesicles d i d n o t s i g n i f i c a n t l y change a s a f u n c t i o n o f t h e i r r e l a t i v e c o n t e n t o f p h o s p h a t i d y l g l y c e r o l and c a r d i o l i p i n . T h e m e c h a n i s m by w h i c h a m i n o a c i d s t a r v a t i o n o f E s c h e r i c h i a c o l i induces r e s i s t a n c e against t h e l y t i c and b a c t e r i c i d a l e f f e c t s o f p e n i c i l l i n has been studied.32 Starvation of Escherichia coli s t r a i n W7 o f t h e a m i n o a c i d s I - l y s i n e o r I - m e t h i o n i n e r e s u l t e d i n t h e r a p i d development of r e s i s t a n c e t o a u t o l y t i c cell-wall d e g r a d a t i o n , w h i c h may be e f f e c t i v e l y t r i g g e r e d i n g r o w i n g b a c t e r i a by a n u m b e r o f c h e m i c a l o r p h y s i c a l t r e a t m e n t s . T h e m e c h a n i s m o f t h i s e f f e c t i n t h e amino acid-starved cells involved t h e production of a murein r e l a t i v e l y r e s i s t a n t t o the hydrolytic a c t i o n of crude m u r e i n h y d r o l a s e e x t r a c t s prepared from n o r m a l l y g r o w i n g E s c h e r i c h i a c o l i . R e s i s t a n c e t o t h e a u t o l y s i n s was n o t d u e t o t h e c o v a l e n t l y linked lipoprotein. Resistance t o murein hydrolase developed most r a p i d y a n d most e x c l u s i v e l y i n t h e p o r t i o n of c e l l wall s y n t h e s i z e d
82
Carbohydrate Chemistry
a f t e r the onset of
amino a c i d s t a r v a t i o n .
I-lysine
Lysozyme d i g e s t s o f t h e
m u r e i n s y n t h e s i z e d d u r i n g t h e f i r s t 10 m i n o f
autolysin-resistant
starvation yielded
( i n addition t o the characteristic
degradation products) a high-molecular-weight
m a t e r i a l t h a t was
absent from the lysozyme d i g e s t s o f c o n t r o l c e l l - w a l l
preparations.
I t has been p r o p o s e d t h a t i n h i b i t i o n o f p r o t e i n s y n t h e s i s c a u s e s a r a p i d m o d i f i c a t i o n o f murein s t r u c t u r e a t the c e l l - w a l l
g r o w t h zone
i n s u c h a manner t h a t a t t a c h m e n t o f m u r e i n h y d r o l a s e m o l e c u l e s i s inhibited.
The m e c h a n i s m may i n v o l v e some a s p e c t s o f t h e r e l a x e d
c o n t r o l system since p r o t e c t i o n a g a i n s t p e n i c i l l i n - i n d u c e d l y s i s d e v e l o p e d much s l o w e r
i n amino
acid-starved
relaxed controlled
c e l l s t h a n i n i s o g e n i c s t r i n g e n t l y c o n t r o l l e d (*A+)
(=A)
Physiological concentrations
of
bacteria.
guanosine 5’-diphosphate
i n h i b i t e d t h e s y n t h e s i s o f l i p i d i n t e r m e d i a t e s and p e p t i d o g l y c a n c a t a l y s e d by coli.33
a p a r t i c u l a t e enzyme p r e p a r a t i o n f r o m
concentration i n the
Escherichia
The i n h i b i t i o n o f t h e s e r e a c t i o n s was d e p e n d e n t o n t h e of
assay.
guanosine The
5’-diphosphate
degree o f
3’-diphosphate
inhibition of
and
MgC12
lipid-intermediate
s y n t h e s i s d e c r e a s e d a s t h e m o l a r r a t i o o f MgC12 : g u a n o s i n e 5 ’ diphosphate 3’-diphosphate
was i n c r e a s e d ,
a n d no i n h i b i t i o n was
o b s e r v e d a b o v e a MgC12 : g u a n o s i n e 5 ’ - d i s p h o s p h a t e ratio
o f 2.5.
i n h i b i t i o n by significant
3’-diphosphate
The s y n t h e s i s o f p e p t i d o g l y c a n was m o r e s e n s i t i v e t o guanosine 5’-disphosphate
inhibition
occurred
under
3’-diphosphate,
conditions
where
and lipid
i n t e r m e d i a t e s y n t h e s i s was u n a f f e c t e d ( i . e .
a t MgC12 : g u a n o s i n e 5’-
diphosphate 3’-diphosphate
o r more).
other
nucleotides
did
r a t i o s o f 2.5
not
inhibit
the
A
synthesis
variety o f of
l i p i d
i n t e r m e d i a t e s and p e p t i d o g l y c a n . Azureomycin B (10 pg/ml), a z u r e a nov.
sp.,
a new a n t i b i o t i c f r o m P s e u d o n o c a r d i a
caused t h e a c c u m u l a t i o n o f
i n h i b i t i o n o f p e p t i d o g l y c a n s y n t h e s i s i n an particulate fraction {3H)pentapeptide
l i p i d i n t e r m e d i a t e and
2
v i t r o system u s i n g a
f r o m B a c i l l u s meqaterium KM
a n d c o l d UDP-Q-GlcgNAc
p e n t a p e p t i d e and U D P - { 3 H ) - Q - G l c ~ N A c as
substrate^.^^
c o n c e n t r a t i o n s o f a zu re o mycin B ( o v e r 100ug/ml), a c c u m u l a t i o n was a l s o i n h i b i t e d . E s c h e r i c h i a c o l i Y-10 p e n t a p e p t i d e were
UDP-M urNAcA t higher
lipid-intermediate
When p a r t i c u l a t e f r a c t i o n f r o m
and UDP-{14C}-p-Glc~NAc
used,
with
o r c o l d UDP-MurNAc-
accumulation o f
and c o l d UDP-MurNAc-
l i p i d i n t e r m e d i a t e and
i n h i b i t i o n o f p e p t i d o g l y c a n s y n t h e s i s were a l s o observed.
This
i n d i c a t e d t h a t t h e p r i m a r y t a r g e t o f azureomycin B i s t h e t r a n s f e r o f t h e d i s a c c h a r i d e p e p t i d e u n i t (a-GlcgNAc-M urNAc-pen t a p e p t i d e
1
4: Microbial Polysaccharides
83
from lipid-bound p r e c u r s o r t o acceptor. ---m e s o - L a n t h i o n i n e was i d e n t i f i e d i n t h e h y d r o l y s a t e o f -_----------F u s o b a c t e r i u m --------n u c l e a t u m p e p t i d o g l y c a n by g . c . m . s . and t h e d i s t r i b u t i o n o f t h i s unusual sulphur-containing dibasic amino acid among p e p t i d o g l y c a n s of v a r i o u s s p e c i e s a n d s t r a i n s of g e n u s F u s o b a c t e r i u m i n v e s t i g a t e d . 35 The p e p t i do g l y c a n s o f F u s o b a c t e r i u rn b a c t e r i a c o u l d be s u b d i v i d e d a c c o r d i n g t o c o n s t i t u e n t d i b a s i c a m i n o acid into: a lanthionine type, a diaminopimelic acid type,and a mixed type. The a m i n o a c i d s i n t h e p e p t i d o g l y c a n o f F u s o b a c t e r i u m n u c l e a t u m F e v 1 h a v e b e e n r e p o r t e d t o be Q - g l u t a m i c a c i d , m e s o - l a n t h i o n i n e , a n d p - ( 4 2 % ) a n d L _ - a l a n i n e (58%).36 A b o u t 7 0 % o f t h e l a n t h i o n i n e r e s i d u e s were n o t s u s c e p t i b l e t o d i n i t r o p h e n y l a t i o n , e v i d e n t l y because they a r e involved i n cross-linkages. Consequently, lysozyme d i g e s t i o n o f t h e p e p t i d o g l y c a n y i e l d e d 20 t o 2 5 % u n c r o s s - l i n k e d A chemical analysis o f d i s a c c h a r i d e t r i - and t e t r a - p e p t i d e s . i s o l a t e d g l y c o p e p t i d e s i n d i c a t e d t h a t the s t r u c t u r e of t h e b u i l d i n g b l o c k o f t h i s p e p t i d o g l y ca n i s 2 - a c e t am i d o - 2 - d e o x y -p - g l u c o s e -tja c e t y l m u r a m i c acid-L-alanine-p-glut am i c a c i d - m e s o - l a n t h i o n i n e ( -p a l a n i n e 1. E v i d e n c e h a s b e e n p r e s e n t e d t o s u p p o r t t h e c l a s s i f i c a t i o n o f t h e F u s o b a c t e r i u m n u c l e a t u m F e v 1 p e p t i d o g l y c a n a s a new A16, d i r e c t l y c r o s s - l i n ked, m e s o - l a n t h i o nine-co n t a i n i n g pep t i d 0 g l y can. The d e n s i t y p r o p e r t i e s o f b a c t e r i a l c e l l walls, when c e n t r i f u g e d under c o n d i t i o n s o f low o s m o l a l i t y , s u p p o r t t h e e v i d e n c e f o r an open s t r u c t u r e f o r c e l l walls from Micrococcus l y s o d e i k t i c u s , B a c i l l u s lichenifgymlp, C a c t o b a c i l l u s fermenturn, and P r o p r i o n i b a c t e r i u m t h e o n i i .37 The C - m y c o s i d i c g l y c o p e p t i d o l i p i d t y p i n g a n t i g e n s f r o m a l l serovars i n the t4ycgbacterium "'ig~/Mycobacterium intracellulare/Mycobacterium s c r o f u l a c e u m c o m p l e x h a v e b e e n e x a m i n e d t o varying extents.38 D e t a i l e d a n a l y s i s o f t h o s e from s e r o v a r s 8, 9 , 1 6 , a n d 25 s h o w t h a t t h e a n t i g e n s c o n s i s t o f s h o r t a c e t y l a t e d o l i g o s a c c h a r i d e s l i n k e d t o a common f a t t y acyl-peptidyl-g-(3,4-di-gmethyl-I=-rhamnose) c o r e (4). The o l i g o s a c c h a r i d e u n i t s , i n a f o r m s u i t a b l e f o r c h e m i c a l s t u d i e s , were l i b e r a t e d a s o l i g o s a c c h a r i d e a l d i t o l s on t r e a t m e n t o f the g l y c o p e p t i d o l i p i d s w i t h a l k a l i n e borohydride solution. The a l d i t o l i n t h e reduced o l i g o s a c c h a r i d e s f r o m a l l s o u r c e s was 6 - d e o x y - l - t a l i t o l . M o r e o v e r I - r h a m n o s e was a l s o a l w a y s p r e s e n t , i n d i c a t i n g t h a t a b a s a l d i s a c c h a r i d e , L-rhamnosyl-6deoxy-l-talosyl, is always l i n k e d t o t h e a l l o - t h r e o n i n e i n t h e acylpeptide. I n a d d i t i o n t h e o l i g o s a c c h a r i d e s from the
Carbohydrate Chemistry
84
g l y c o p e p t i d o l i p i d s of each s e r o v a r were d i s t i n g u i s h e d by t h e i r own i n d i v i d u a l i s t i c sugars: 3-g-methyl-~-glucose i n serovar 8 2,3-diO-methyl-l-fucose i n s e r o v a r 9 , 2-2-methyl-&-fucose i n s e r o v a r 25, 4-g-methyl-_L-rhamnose i n t h e o l i g o s a c c h a r i d e from o n e of t h e two g l y c o p e p t i d o l i p i d s i n s e r o v a r 16, and a p p a r e n t l y a n o t h e r I-rhamnose substituent i n the other oligosaccharide. The g l y c o p e p t i d o l i p i d a n t i g e n s i n t h e i r s t r u c t u r a l p r i n c i p a l s , c e l l u l a r l o c a t i o n , and physiological r o l e bear a s t r i k i n g miniscular resemblance to c e l l w a l l c o m p o n e n t s o f o t h e r b a c t e r i a s u c h a s O - a n t i g e n i c and R a n t i g e n i c 1i po p o l y sa ccha r i de s. {Fatty A c y l } - ~ - P h e - ~ - a T h r - ~ - A l a - ~ - A l a n i n o l - O - ~ - R h a ~ 3 , 4 M e 2
1
T
{ O l i g o s a c c h a r i de } -0-Ac (4)
The s p e c i f i c o l i g o s a c c h a r i d e u n i t s o f t h e C - m y c i s i d i c g l y c o p e p t i d o l i p i d a n t i g e n s from s e r o v a r i e t i e s i n t h e Mycobacterium -avium/Mycobacterium L n t r a c e l l u l G / M y c o b a c t e r i u m scrofulaceum complex were l i b e r a t e d a s o l i g o s a c c h a r i d e a l d i t o l s by t r e a t m e n t of t h e glycopeptidolipids w i t h a l k a l i n e borohydride. The c o m p l e t e s t r u c t u r e s o f t h e o l i g o s a c c h a r i d e a l d i t o l s have been d e r i v e d f r o m t h e 'H n . m . r . spectra or those of t h e i r permethylated (or p e r t r i d e u t e r i o m e t h y l a t e d ) d e r i v a t i v e s , t h e m a s s s p e c t r a of t h e m e t h y l a t e d d e r i v a t i v e s , and from m e t h y l a t i o n f r a g m e n t a t i o n analysis.39 P e r i o d a t e o x i d a t i o n was a l s o used t o c o n f i r m t h e p o s i t i o n o f t h e l i n k b e t w e e n t h e u l t i m a t e and p e n u l t i m a t e s u g a r s . S t r u c t u r e s (5)-(7) w e r e p r o p o s e d ( w i t h some e x t r a p o l a t i o n o f e n a n t i o m e r i c c o n f i g u r a t i o n s where e v i d e n c e f o r a s s i g n m e n t i s not y e t c o m p l e t e ) f o r t h e o l i g o s a c c h a r i d e a l d i t o l s from s e r o v a r s 8 , 9, and 25, r e s p e c t i ve 1y 6 -Deox y -I - t a 1i t o 1 ( 6-de ox y -_h - t a 1ose i n t h e o r i g i na 1 glycopeptidolipid) invariably occupies the reducing terminus. LRhamnose i s i n v a r i a b l y t h e p e n u l t i m a t e s u g a r , and t h e l i n k between I-rhamnose and 6-deoxy-L-talose i s i n v a r i a b l y (1+2). Moreover, t h e r e s u l t s p o i n t t o t h e o u t e r o n e o r two a p p e n d a g e s f o r t h e p r o v i s i o n of individually distinctive features required for antigen speci f i c it y The s u r f a c e p r o p e r t i e s o f c e l l s o f M y c o b a c t e r i u m B C G , Mycobac ----t e r i u m p h 1e i , My co b a c t e r i u m smgm a t i s, an d My c o b a c t e r i u m
.
.
4: Microbial Polysaccharides
85
-m-i c r o t i
have been shown t o be i d e n t i c a l ,
medium,
t h e age o f t h e c e l l s ,
various
v i g o r o u s t r e a t r n e n t ~ . ~ ' The n e g a t i v e s u r f a c e c h a r g e f o r
i r r e s p e c t i v e o f the growth
and t h e c o l o n i a l m o r p h o l o g y , and a f t e r
a l l these species a r i s e s from the phosphate groups o f
cells of
phosphodiester
linkages
between
the
a r a b i n o g a l a c t a n of the b a s i c c e l l - w a l l
peptidoglycan
and
the
s t r u c t u r e , w h i c h i s common t o
a l l species o f Mycobacteria. The p e p t i d o g l y c a n o f N e i s s e r i a g o n o r r h o e a g h a s b e e n f o u n d t o contain g-acetyl
groups,
by
use
of
the
hydroxamate
formation
and f o u n d t o be o n l y p a r t l y s e n s i t i v e t o l y ~ o z y m e . ~ ~
reaction,
B-~-Glc~3Me-(1+3)-a-~-Rhae-(1+2)-6-deoxy-~-Talitol 0
6
'\/C
/\
Me
CO,
a-L-Fuce2,3Me2-[
1 + 4 ) - u - L - F u c ~ 2 , 3 M e ~ - [ 1+3)-a-L-Rhae-[
1+2)-
6-deoxy-k-tali to (6)
Low c o n c e n t r a t i o n s o f B - l a c t a m
a n t i b i o t i c s caused an i n c r e a s e d
uptake o f r a d i o a c t i v e 2-amino-2-deoxy-Q-glucose dodecyl
sulphate-insoluble
gonorrhoeae.42
T h e r e was
peptidoglycan of no a p p r e c i a b l e
i n t o the sodium
growing
Neisseria
change i n t h e
[small)
amount o f sodium d o d e c y l s u l p h a t e - s o l u b l e p o l y m e r p r e s e n t i n t h e cultures.
The sodium d o d e c y l s u l p h a t e - i n s o l u b l e
product i n control
c e l l s was o n l y p a r t i a l l y d i s s o l v e d by e g g - w h i t e
lysozyme (about
40%), b u t c o u l d a l l be r e l e a s e d by t h e C h a l a r o p s i s B muramidase. c e l l s exposed t o B - l a c t a m s
s u s c e p t i b l e t o l y s o z y m e i n c r e a s e d t o 60%. C h a l a r o p s i s B d i g e s t s by t h i n - l a y e r contained
disaccharide-peptide
Examination o f
the
c h r o m a t o g r a p h y showed t h a t t h e y
monomers
a c e t y l a t i o n and b i s - d i s a c c h a r i d e - p e p t i d e a c e t y l g r o u p s , o r w i t h none.
I n
the proportion o f labelled peptidoglycan
with
and
without
d i m e r s w i t h one o r t w o
g0-
B-Lactam a n t i b i o t i c s caused a decrease
i n t h e d e g r e e o f 2 - a c e t y l a t i o n b u t d i d n o t g r e a t l y a f f e c t t h e amount
86
Carbohydrate Chemistry
of peptidoglycan cross-linking.
They a l s o e n l a r g e d t h e b a c t e r i a and
c o n s e r v e d a n d t h i c k e n e d t h e s e p t a t h a t c o u l d be o b s e r v e d i n t h i n s e c t i o n s by e l e c t r o n m i c r o s c o p y . r e s u l t s and
e f f e c t s o f B-lactams
The r e l a t i o n s h i p b e t w e e n t h e s e
2
on
v i t r o synthesis o f peptido-
g l y c a n by e t h e r - t r e a t e d N e i s s e r i a g o n o r r h o e a e has been d i s c u s s e d .
-m i r a b i l i s
The m u r e i n o f
i s r e p o r t e d t o be u n i q u e w i t h
respect t o the g-acetylation
o f p a r t o f i t s N-acetylmuramic a c i d
residues.43
synchronized
Working
with
r a d i o a c t i v e 2-acetam ido-2-deoxy-Q-glucose
c e l l s and p r o v i d i n g w i t h t h e medium,
the
m u r e i n was p u l s e - l a b e l l e d i n v i v o f o r 1 0 m i n p e r i o d s a t d i f f e r e n t times of the c e l l cycle.
A f t e r i s o l a t i o n o f m u r e i n and e n z y m a t i c
cleavage w i t h endo-N,g-diacetylmuramidase d i s t r i b u t i o n of radio-activity
from Chalaropsis,
the
between u n c r o s s l i n k e d m u r e i n s u b u n i t s
(monomers) and t h e p e p t i d e - c r o s s l i n k e d
I t was
d i m e r s was a n a l y s e d .
found t h a t t h e m u r e i n s y n t h e s i z e d a t d i f f e r e n t t i m e s d u r i n g t h e c e l l c y c l e d i d n o t show s i g n i f i c a n t v a r i a t i o n r e g a r d i n g t h e d e g r e e o f c r o s s l i n k a g e and t h e d e g r e e o f g - a c e t y l a t i o n .
of g-acetylation sevenfold
of
l o w e r than t h a t o f murein from
asynchronous c u l t u r e s . non-g-acetylated
A pulse-chase
chase,
generation later, became e v i d e n t .
degree
continuously
labelled
experiment revealed t h a t only
p a r t o f w h i c h became c - a c e t y l a t e d s u b s e q u e n t l y .
S i n c e a c e r t a i n amount o f the
the
m u r e i n s u b u n i t s were i n c o r p o r a t e d i n t o t h e g r o w i n g
murein sacculus, during
However,
p u l s e - l a b e l l e d m u r e i n was f o u n d t o be a b o u t
which
newly
incorporated subunits
reappeared
i n
the
sacculus
was
lost
about
one
l i m i t e d murein turnover during the c e l l cycle
The d e g r e e o f c r o s s l i n k a g e o f b o t h t h e g - a c e t y l a t e d
as w e l l as t h e n o n - ) - a c e t y l a t e d d u r i n g subsequent growth.
newly
synthesized murein increased
The l a b e l l e d p e p t i d e - c r o s s l i n k e d
dimers
w e r e c l e a v e d by an e n d o p e p t i d a s e i s o l a t e d P r o t e u s m i r a b i l i s .
The
d i s t r ib u t i o n o f r a d i o a c t i v i t y be t w een t h e r e s u 1t i n g no n - 2 - ace t y 1a t e d and 2 - a c e t y l a t e d a c e t y l a t e d dimer
monomers
n o n - g - a c e t y 1a t e d s u b u n it subsequent b i o s y n t h e s i s .
m ono-)-ace subunits,
indicated that
most
fraction carried radioactivity
t y l at e d dimer
w h i ch
w as
of
the
mono-2-
exclusively
n o n - 2 - a c e t y 1a t e d
Since o n l y a s m a l l p o r t i o n o f t h e l a b e l l e d f r a c t i o n c a r r i e d r a d i o a c t iv i t y
i n
represented
an i n t e r m e d i a t e
i n the g-acetylation
both
i t was
and o n l y a t s p e c i f i c t i m e s o f m u r e i n b i o s y n t h e s i s ,
concluded t h a t t h i s p o r t i o n o f t h e l a b e l l e d mono-)-acetylated fraction
i n the during
dimer
process.
A d e f i n e d sequence o f s t e p s o f m u r e i n b i o s y n t h e s i s has been p r o p o s e d as a r e s u l t o f t h e r e s u l t s d e scrib e d . A n o v e l mur e i n b u i l d i n g b l o c k ,
2-ace tam i d o - 2 - d e o x y - Q - g 1 ycosy 1-
a7
4: Microbial Polysaccharides
-N - a c e t y l m u r a m o y l - d i p e p t i . d e , P r ot e u s m i r a b i l i s ,
formed d u r i n g t h e c e l l - d i v i s i o n c y c l e o f
has been r e p o r t e d . 4 4
g l u c o s e r e s i d u e was f o u n d t o c o n t a i n ,
The 2 - a c e t a m i do-2-deoxy-gi n some i n s t a n c e s , 2 - a c e t y l
groups. The Pseudomonas a e r u g i n o s a o u t e r membrane was i s o l a t e d w i t h attached
peptidoglycan
and
ethylenediaminetetraacetate, major
outer
membrane
fractioned
and lysozyme.
proteins
with
X-100,
I are noncovalently
H2, a n d
F,
Triton
The d a t a s u g g e s t e d t h a t
associated with the p e p t i d ~ g l y c a n . ~ ’ The u n i q u e p r e s e n c e o f
a polyamine covalently
p e p t i d o g l y c a n h a s b e e n r e p ~ r t e d . ~ The ~ , ~p o~l y a m i n e was f o u n d t o e x i s t a s a c o m p o n e n t o f c e l l - w a l l Selenomonas
ruminantium,
a
strictly
linked to a cadaverine
peptidoglycan o f
anaerobic
bacterium.
{ l 4 C } C a d a v e r i n e added t o t h e g r o w t h medium was i n c o r p o r a t e d i n t o t h e cells,
a n d a b o u t 70% o f t h e t o t a l r a d i o a c t i v i t y i n c o r p o r a t e d was
found i n the peptidoglycan fraction.
When t h e { 1 4 C ) c a d a v e r i n e -
l a b e l l e d p e p t i d o g l y c a n p r e p a r a t i o n was a c i d h y d r o l y s e d , 14C
counts were recovered as cadaverine.
l a b e l l e d p e p t i d o g l y c a n p r e p a r a t i o n was t h r e e s m a l l f r a g m e n t s w h i c h were ninhydrin reaction. was composed o f
a l l o f the
{14C)cadaverine-
digested w i t h lysozyme i n t o
r a d i o a c t i v e and were p o s i t i v e i n
One m a j o r s p o t ,
QL-alanine,
The
a compound o f t h e f r a g m e n t s ,
Q-glutamic
acid,
diaminopimelic acid,
c a d a v e r i n e , m u r a m i c a c i d , and 2 - a m i n o - 2 - d e o x y - Q - g l u c o s e . two
amino
groups
peptidoglycan,
of
cadaverine
was
a n d t h e o t h e r was f r e e .
covalently
One o f t h e
linked
to
the
The c h e m i c a l c o m p o s i t i o n o f
t h e p e p t i d o g l y c a n p r e p a r a t i o n o f t h i s s t r a i n was d e t e r m i n e d t o be a s f o l 1ow s:
-
I-a 1a n i n e -Q-
a 1a n i ne-Q- g l u t am ic a c i d - mes o d i am in o p im e 1ic
acid-cadaverine-muramic (1.O:l
.O:l.O:l
.0:1.1:0.9:1
acid-2-amino-2-deoxy-~-glucose
.O).
T h e { I 4 C ) c a da ve r i n e - l a b e 1 l e d p e p t ido g l y c a n was d e g r a d e d w i t h t h e l y t i c enzymes p r e p a r e d f r o m S t r e p t o m y c e s a l b u s G i n t o t h r e e s m a l l f r a g m e n t s i n c l u d i n g a m a j o r f r a g m e n t (band A compound).48 B a n d A c o m p o u n d was c o m p o s e d o f I - a l a n i n e , d i a m i n o p i m e l i c acid,
Q - g l u t a m i c a c i d , meso-
Q-alanine, and c a d a v e r i n e i n t h e m o l a r r a t i o
0.98:1.0:1.0:0.98:0.97.
Diaminopimelic
acid,
&-alanine, and
c a d a v e r i n e w e r e N - t e r m i n a l r e s i d u e s i n b a n d A compound.
{14Clcadaverine-labelled acid hydrolysis,
band A
compound was
t w o p e p t i d e f r a g m e n t s were o b t a i n e d .
c o n s i s t e d o f d i a m i n o p i m e l i c a c i d and Q - a l a n i n e , was t h e N - t e r m i n a l of I-alanine,
subjected
amino a c i d ,
When t h e to
partial
One o f them
diaminopimelic acid
and t h e o t h e r f r a g m e n t was composed
Cj-glutamic acid, and cadaverine, o f which I - a l a n i n e
88
Carbohydrate Chemistry
and c a d a v e r i n e w e r e N - t e r m i n a l . p e p t i d e s t r u c t u r e of
I t was c o n c l u d e d t h a t t h e p r i m a r y
b a n d A compound i s L - a l a n y l - E - g l u t a m o y l - m e s o -
d i a m i n o p i m e l y l - g - a l a n i n e and t h a t c a d a v e r i n e l i n k s c o v a l e n t l y t o t h e g-glutamic a c i d residue. S t r e p t o c o c c u s m u t a n s BHT was g r o w n i n T o d d - H e w i t t d i a l y s a t e medium c o n t a i n i n g 2-acetamido-2-deoxy-E-{
1 4 C ) g l u c o s e f o r 6 t o 11
generations.
A f t e r t r e a t m e n t w i t h c o l d and h o t t r i c h l o r o a c e t i c a c i d
and t r y p s i n ,
5 2 t o 65% o f t h e r a d i o a c t i v i t y r e m a i n e d p r e s e n t i n
insoluble peptidoglycan-containing residues.
Hen e g g - w h i t e l y s o z y m e
or rnutanolysin t r e a t m e n t of t h e peptidoglycan residues r e s u l t e d i n t h e r e l e a s e o f 80 a n d 97%, r e s p e c t i v e l y , o f t h e 1 4 C l a b e l t o t h e supernatant f r a ~ t i o n . ~ ’ H y d r o c h l o r i c a c i d h y d r o l y s a t e s o f such supernatants present
in
showed t h a t insoluble
compounds t h a t
essentially
peptidoglycan
a l l of
the
fractions
c o m i g r a t e d on p a p e r c h r o m a t o g r a p h y
deoxy-!-glucose
( 6 0 % ) o r m u r a m i c a c i d (30%).
radioactivity
was
present
i n
w i t h 2-amino-2-
Treatment o f whole
c e l l s w i t h l o w and h i g h c o n c e n t r a t i o n s o f lysozyme a l o n e r e s u l t e d i n l o s s e s o f 45 and 70% o f t h e i n s o l u b l e p e p t i d o g l y c a n ,
respectively,
y e t r e l e a s e o f d e o x y r i b o n u c l e i c a c i d f r o m c e l l s was n o t d e t e c t e d . Sequential addition inorganic
salts
of
after
appropriate
concentrations
lysozyme treatment
l i b e r a t i o n o f deoxyribonucleic acid.
of
selected
did result i n the
Deoxyribonucleic acid release
was c o r r e l a t e d w i t h a f u r t h e r r e l e a s e o f p e p t i d o g l y c a n f r o m t h e insoluble fraction.
However, t h e t o t a l amount o f p e p t i d o g l y c a n l o s t
e f f e c t e d by t h e l o w c o n c e n t r a t i o n o f l y s o z y m e and NaSCN ( l y s i s ) was s i g n i f i c a n t l y l e s s t h a n t h e amount o f p e p t i d o g l y c a n h y d r o l y s e d by h i g h c o n c e n t r a t i o n s o f l y s o z y m e a l o n e (no l y s i s ) ,
which suggested
t h a t t h e o v e r a l l amount o f p e p t i d o g l y c a n l o s t d i d n o t c o r r e l a t e w e l l with cellular lysis.
The t o t a l a m o u n t o f i n s o l u b l e p e p t i d o g l y c a n
l o s t a t the highest s a l t concentrations greater
t e s t e d was f o u n d t o b e
t h a n c o u l d b e a c c o u n t e d f o r by l y s o z y m e - s e n s i t i v e
o f the peptidoglycan, obtained
suggested
possibly implicating autolysins. that
topologically localized,
hydrolysis
of
linkages
The r e s u l t s
peptidoglycan
bonds
in
b u t s t r a t e g i c a l l y i m p o r t a n t s i t e s was a
more s i g n i f i c a n t f a c t o r i n t h e sequence t h a t r e s u l t s i n loss o f cellular
integrity (lysis).
A p e p t i d o g l y c a n l a y e r o f Treponema p a l l i d u m k a z a n was i s o l a t e d
by
solubilization of
whole
cells
with
1% w a r m
sodium
dodecyl
s u l p h a t e and s u b s e q u e n t d i g e s t i o n o f an i n s o l u b l e r e s i d u e w i t h proteases.
E l e c t r o n m i c r o s c o p y r e v e a l e d t h a t t h e p e p t i d o g l y c a n was
i s o l a t e d as a s i n g l e - l a y e r e d s a c c u l u s o f l e s s t h a n 5 nm t h i c k n e s s ,
89
4: Microbial Polysaccharides f r e e d f r o m a x i a l f i l a m e n t s and an e n v e l o p e sheath.50
An i s o l a t e d
p e p t i d o g l y c a n f r a c t i o n was m a i n l y c o m p o s e d o f 2 - a m i n o - 2 - d e o x y - Q glucose,
muramic acid,
alanine,
g l y c i n e i n molar r a t i o s of
0-glutamic
acid,
L-orthithine,
and
Amino-
0.65:0.68:1.63:1.00:0.75:1.03.
and c a r b o x y l - t e r m i n a l a m i n o a c i d a n a l y s e s s u g g e s t e d t h e i n v o l v e m e n t
o f a t l e a s t a p a r t o f the g l y c i n e r e s i d u e i n c r o s s - l i n k i n g between t h e a m i n o g r o u p o f i - o r n i t h i n e r e s i d u e a t one s t r a n d o f t h e s t e m peptide
subunit
neighbouring
and
the
strand.
carboxyl
The
group
treponemal
of
alanine
peptidoglycan
o f
the
lacked the
i m m u n o a d j u v a n t a c t i v i t y b o t h t o s t i m u l a t e a n t i b o d y p r o d u c t i o n and t o induce delayed-type
hypersensitivity
the properties necessary t o splenocytes
and g u i n e a - p i g
against
stimulate
peritoneal
ovalbumin,
as w e l l as
guinea-pig
macrophages,
and
mouse
unlike the c e l l
w a l l s or p e p t i d o g l y c a n s ( g r o u p A t y p e o f S c h l e i f e r and K e n d l e r s ’ classification) the
i s o l a t e d from
mammal.
However,
many b a c t e r i a l s p e c i e s p a r a s i t i c t o
the
peptidoglycan
activated
the
human
complement s y s t e m t h r o u g h t h e a l t e r n a t i v e pathway, as w e l l as t h e c l a s s i c a l one, rabbit
blood
and caused a l i b e r a t i o n o f 5 - h y d r o x y t r y p t a m i n e platelets
i n
a
similar
manner
to
the
i n
cell-wall
p e p t i d o g l y c a n s o f b o t h g r o u p A and B t y p e s .
Lipopolysaccharides
3
The c h e m i s t r y and b i o l o g i c a l s i g n i f i c a n c e o f 3-deoxy-g-manno-2o c t u l o s o n i c a c i d have been r e v i e w e d Y 5 l elucidation
and
polysaccharides, and
enzymology
i t s
location
chemical
of
an
and
including i t s structure linkages
i n
b a c t e r i a l
s y n t h e s i s and m o n o s a c c h a r i d e c h e m i s t r y , inhibitor
to
metabolism.
Bacterial
l i p o p o l y s a c c h a r i d e and i t s l i p i d A component has been r e v i e w e d briefly.52
C o n f i r m a t i o n o f t h e s t r u c t u r e o f 3-deoxy-P-manno-2-
o c t u l o s o n i c a c i d by X - r a y t h e r e p o r t of
t h e X-ray
c r y s t a l l o g r a p h y h a s been b r o u g h t n e a r e r by s t r u c t u r e o f m e t h y l ( m e t h y l 4,5,7,6-tetra-g-
a c e t y 1-3-deoxy-a-Q-manno-2-octulopyranosid I o n a t e (8).53 The
l i p o p o l y s a c c h a r i d e
f r o m
A c t i n o b a c L l l u s
a c t i n o m y c e t e m c o m i t a n s s t r a i n s Y4 and N27 was i s o l a t e d by t h e p h e n o l water procedure.
Morphologically,
and branched f i l a m e n t s weight.54
Chemical
lipopolysaccharide carbohydrate,
of
which
analysis both
the molecule consisted o f ribbon
c o m p r i s e d 3% o f of
strains
the
the cellular
isolated
showed
them
l i p i d , 3-deoxy-Q-manno-2-octulosonic
and to
consist
acid,
dry
purified of
heptose,
90
Carbohydrate Chemistry
amino-deoxyhexose, and p h o s p h a t e .
The m a j o r f a t t y a c i d s o f t h e l i p i d
A m o i e t y were m y r i s t i c and B - h y d r o x y m y r i s t i c a c i d s . f uco se,
p- g a l a c t o se,
p - g l u c o se,
h e p t o se,
I-Rhamnose,
2-amino-2-deoxy-Q-gluco
Ise,
a n d 2 -am i n o -2-deox y -Q-ga 1a c t o se c o m p r i s e d t h e mono sa c c h a r ide pa r t i o n o f the lipopolysaccharide.
Biological-activity
both lipopolysaccharide molecules t o
be a c t i v e
r e a c t i o n a n d i n v i t r o 45Ca bone r e s o r p t i o n ,
studies revealed
i n t h e Schwartzman
a s w e l l as i n macrophage
a c t i v a t i o n a n d l e t h a l i t y and i n p l a t e l e t a g g r e g a t i o n .
The c o r e o l i g o s a c c h a r i d e o f Aeromonas h y d r o p h i l a (Chemotype
111) l i p o p o l y s a c c h a r i d e has
been
i n ~ e s t i g a t e d . ~ The ~
i n v o l v e d t h e use o f m e t h y l a t i o n a n a l y s i s , trioxide,
p a r t i a l hydrolysis with acid,
periodate oxidation,
degradation,
and t a g g i n g o f t h e r e d u c i n g end group.
unusual
having
i n
constituent.
3 - a c e t am id o - 3 , 6 - d i
Structure
studies
o x i d a t i o n and chromium
(9) was
deoxy-L- g l ucose
proposed
for
Smith
The c o r e i s the
as
a
core
o l i g osaccharide. a -Q - G 1 c g
1
I
6 dd-B-L-GlcgNAc-( -
1+3) -a-e-Galg-(
1+3)-a-LQ-Hepp(
1+2) -a-LQ-Hepe
4
I
1 a -Q - G 1 c e
LQ -H e p e
=
L-g 1y c e r o -Q - m a nno - h e p t o p y r a no s e (91
The sugar c o m p o s i t i o n o f t h e O - a n t i g e n i c lipopolysaccharides i s o l a t e d f r o m Group F (once c l a s s i f i e d a s Aeromonas) v i b r i o s was
91
4: Microbial Polysaccharides
analysed. 56 3-Deoxy-Q-manno-2-oct uloso n i c a c i d was t o t a l l y absent from t h e l i p o p o l y s a c c h a r i d e s . As common component s u g a r s , p g l u c o s e , a - g a l a c t o s e , i - q l y c e r o - Q - m a n n o - h e p t o s e , and 2-amino-2d e o x y - P - g l u c o s e w e r e p r e s e n t . The G r o u p F v i b r i o s e x a m i n e d w e r e f o u n d t o be d i v i d e d i n t o t w o g r o u p s , d e s i g n a t e d t e n t a t i v e l y a s g r o u p s 1 a n d 11, o n t h e b a s i s o f t h e p a t t e r n of t h e s u g a r composition of t h e i r l i p o p o l y s a c c h a r i d e s . As a d d i t i o n a l s u g a r c o m po ne n t s , 2 - am i no - 2 d e ox y -Q - m a n no s e , 2 - am i no - 2,6 - d i d e ox y -Q - g 1u co s e, and t w o u n i d e n t i f i e d amino s u g a r s were p r e s e n t i n group I , while Irhamnose, Z - a m i n o - 2 - d e o x y - Q - g a l a c t o s e , an u n i d e n t i f i e d amino sugar, and a r e l a t i v e l y h i g h c o n t e n t o f a - g l y c e r o - Q - m a n n o - h e p t o s e were found i n g r o u p 11. The main f a t t y a c i d s p r e s e n t i n l i p o p o l y s a c c h a r i d e s from B a c t e r o i d e s f r a g i l i s NCTC 9343 were i d e n t i f i e d a s 13-methylt e t r a d e c a n o i c , B-3-h~d r ox ypent ade c a n o i c , p-3- hy drox y hex ade canoi c, p3 - h y d r o x y - 1 5 - m e t h y l - h e x a d e c a n o i c , and 0 - 3 - h y d r o x y h e p t a d e c a n o i c acids.57 Of t h e s e 1 3 - m e t h y l - t e t r a d e c a n o i c a c i d i s e x c l u s i v e l y e s t e r b o u n d , and 3 - h y d r o x y - 1 5 - m e t h y l - h e x a d e c a n o i c acid i s exclusively i n v o l v e d i n amide l i n k a g e . The o t h e r 3-hydroxy f a t t y a c i d s a r e b o t h e s t e r and amide b o u n d . A l l 3-hydroxy f a t t y a c i d s p o s s e s s t h e c o n f i g u r a t i o n , and t h e 3 - h y d r o x y l group o f e s t e r - l i n k e d 3-hydroxy f a t t y a c i d s i s n o t s u b s t i t u t e d . L i p o p o l y s a c c h a r i d e s of r e l a t e d Bacteroides species (Bacteroides thetaiotaomicron, Bacteroides o v a t u s , B a c t e r o i d e s d i s t a s o n i s , a n d B a c t e r o i d e s v u l g a t u s 1 showed a f a t t y - a c i d spectrum w i t h s i m i l a r and d i s t i n c t f e a t u r e s compared t o t h a t o f Bacteroides f r a g i l i s lipopolysaccharides. Pur i f i e d 1i p o po 1y s a cch a r i de e x t r a c t e d w i t h pheno 1-water from smooth B r u c e l l a a b o r t u s was h y d r o l y s e d w i t h 1% a c e t i c a c i d a t 1 0 0 ° C . 5 8 The d e g r a d e d p o l y s a c c h a r i d e r e l e a s e d gave r e a c t i o n s o f i d e n t i t y w i t h t h e n a t i v e p o l y s a c c h a r i d e hapten i n phenol-water- o r t r i c h l o r o a c e t i c a c i d - e x t r a c t e d e n d o t o x i n preparations o f Brucella a b o r t u s w i t h t h e p o l y s a c c h a r i d e e x t r a c t e d by t r i c h l o r o a c e t i c a c i d from rough B r u c e l l a m e l i t e n s i s s t r a i n 8115. The l a t t e r p o l y s a c c h a r i d e was p r e s e n t i n t h e s o l u b l e c y t o p l a s m i c f r a c t i o n , b u t n o t i n t h e membrane f r a c t i o n , of d i s r u p t e d 8115 c e l l s . I t could n o t be e x t r a c t e d from t h r e e rough m u t a n t s o f B r u c e l l a a b o r t u s o r from -------B r u c e l l a ----canis or Brucella o v i s cells. B o t h the degraded p o l y s a c c h a r i de and na t i ve p o l y saccha r i de hap t e n shared de t e rm i nan t s p r e s e n t o n s m o o t h l i p o p o l y s a c c h a r i d e and m i s s i n g from t h e rough B r ucella g e l i t e n s i s polysaccharide. S u g a r s found i n p u r i f i e d l i p o p o l y s a c c h a r i d e , t h e n a t i v e h a p t e n , and degraded p o l y s a c c h a r i d e
-
a-
92
Carbohydrate Chemistry
a - -~ - G l c ~ N A c - ( 1 + 2 ) - a-- ~ - G l c ~ - ( l + 2 ) --a - ~ - G a l ~ - ( l + 3 ) - a - ~ - G l c ~ - ( l + 6
I
1
a-Q-Gale (10)
a-~-Gal~-(1+2)-a-~-Gal~-(l+2)-a-~-Glc~-(l+3)-a-~-Glc~-(l+ 3
I
1
B-P-GlcQ (11)
i 1
a-Q-Gale (12)
a - -~ - G l c ~ - ( 1 + 2 ) - a - ~ - G l c ~ - ( l + 2 ) - a - ~ - G a l ~ - ( l + 3 ) - a - ~ - G l c ~ ( l + 3
I
1 a-9-GlceNAc (13)
a-Q-Gal~-(1+2)-a-~-Gal~-(l+2)-a-q-Glc~-(l+3)-a-~-Glc~-(l+ -
i 1
B-P-Galg (14)
~-~-Glc~NAc-(1+6)-a-~-Glc~-(l+2)-a-g-Glc~-(l+3)-a-~-Glc~-(l+ 6
I
1
a-P -Ga le
93
4: Microbial Polysaccharides
-
-
i n c l u d e d Q-manno s e , Q g l u c o s e , 2 -am ino -2,6 - d i de ox y -Q g l u c o s e , 2 -
-
am ino-2-deox y -tj- g l u c o se,
a n d 3 - d e o x y -Q m a n n 0 - 2 - 0 c t u l o so n i c a c i d.
The B r u c e l l a m e l i t e n s i s p o l y s a c c h a r i d e c o n t a i n e d o n l y a t r a c e amount o f 2-amino-2,6-dideoxy-Ba c i d d e t e c t a b l e by
g l uco se a n d n o 3-deoxy-B- m a n n o - o c t u l o s o n i c
t h e t h i o b a r b i t u r a t e assay.
Sera
from
some
r a b b i t s i m m u n i z e d w i t h p u r e s m o o t h l i p o p o l y s a c c h a r i d e a n d some, not
all,
cows
infected
with
field strain of
but
Brucella abortus
recognized the determinants missing from the Brucella melitensis polysaccharide.
A subclass-specific enzyme-linked
immunoassay
showed t h a t most o f t h e a n t i b o d y i n s e r a f r o m i n f e c t e d cows w h i c h b i n d s t o smooth l i p o p o l y s a c c h a r i d e and t o t h e n a t i v e h a p t e n i s o f t h e immunoglobulin G 1 subclass. The c h e m i c a l s t r u c t u r e o f t h e 0 - s p e c i f i c
----------Citrobacter
036 l i p o p o l y s a c c h a r i d e has
methylation analysis s p e ~ t r o m e t r y . ~T~h i s
using gas-liquid polysaccharide
polysaccharide o f
been e s t a b l i s h e d
chromatography i s
a
linear
by
and mass
homopolymer
composed o f ( 1+2) - 1 i n k e d 4-deox y-6 - Q - a r a b i n o - hexopy r a n o s y 1 r e s i d u e s . cores
from
E n t e r o b a c t e r a ce ae 1ipo po 1y s a c c h a r i de s h a v e be e n i n ve s t iga t e d,
The
structures
for
the
us in g
s p e c i f i c d e g r a d a t i o n a n d 'H methods.60
hexose
n.m.r.
regions
of
studies as the p r i n c i p a l
Complete s t r u c t u r e s f o r these r e g i o n s i n t h e Salmonella,
t h e E s c h e r i c h i a c o l i R1,
R2,
R3,
R4,
and E s c h e r i c i a c o l i K12 and
E s c h e r i c h i a c o l i B c o r e s a r e p r o p o s e d ((10)-(16), r e s p e c t i v e l y ) .
The
apparent s i m i l a r i t i e s between these s t r u c t u r e s i n d i c a t e a c l o s e relationship.
They a r e a l l composed o f f i v e h e x o s e r e s i d u e s . t h e E s c h e r i c h i a c o l i R2 c o r e s d i f f e r
The S a l m o n e l l a a n d
o n l y i n t h e n a t u r e o f one
h e x o s e r e s i d u e a n d t h e E s c h e r i c h i a c o l i R I , R2, R4 a n d E h e r i c h i a
c o l i K12 c o r e s a l l c o n t a i n c o l i core. There a r e o n l y a n d t h e R4 c o r e s .
t h e disaccharide u n i t of
The S a l m o n e l l a a n d t h e R3 c o r e ,
t h e same P-Glc-Q-Gal-Q-Glc
the Escherichia
minor s t r u c t u r a l d i f f e r e n c e s between R 1 trisaccharide unit.
finally,
contain
Some c o m p l e m e n t a r y
i n f o r m a t i o n o n t h e s t r u c t u r e o f t h e h e p t o s e r e g i o n has a l s o been obtained.
T h i s r e g i o n i s more u n i f o r m , a n d i t w o u l d n o t be s u r p r i s i n g
ifa l l c o m p l e t e h e p t o s e r e g i o n s i n S a l m o n e l l a , A r i z o n a ,
Citrobacter,
E s c h e r i c h i a c o l i , S h i g e l l a , K l e b s i e l l a , Enterobacter,and p r o v e d t o h a v e e s s e n t i a l l y t h e same s t r u c t u r e ( 1 7 ) .
Serratia
One r e s u l t o f
94
Carbohydrate Chemistry
t
N
u I
0
n Y
n I
b N t U
I
0
0 Y I
N
n
z
m t
I
U
I
N
u
Y
N
I
u N r 0 z
I
e
0 0
a
I
2
N
I
u a .t a-
e-N
4
0 1 1 All
I
N
a
I
I
n
M
t
4 v
I
a-al
2 X
b-4
I
n u
2 I nil
A1I
A1I I
a I
n
M
t
4 u
I
a 4
U
w-4
I 0 1 1 I
a
a
I
n
0 .
M 4
L)
w
4 u I
al m
4
U
I nit I
a
n I
N
t
4
0
v
Q Z
I
?
4
U
I 0 1 1 I
0 .
.-I
a c
al
N-4
4
U I
nit
a I
o
95
4: Microbial Polysaccharides this
study6'
was
the
finding
that
a l l
&-glycero-!-manno-
h e p t o p y r a n o s y l r e s i d u e s seem t o be u - l i n k e d . The h e p t o s e r e g i o n o f t h e l i p o p o l y s a c c h a r i d e o f E s c h e r i c h i a The Q - g l u c o s y l r e s i d u e l i n k e d t o c o l i K12 CR34 h a s been s t u d i e d . 6 1 t h e h e p t o s e I 1 w a s f o u n d t o b e s u b s t i t u t e d by a P - g a l a c t o s y l g r o u p a n d t h e l i n e a r c h a i n o f t h e c o r e p o l y s a c c h a r i d e h a s t w o (1+3) heptoses.
linked
The h e p t o s e I 1 i s s u b s t i t u t e d by a l a t e r a l (1+7) l i n k e d
h e p t o s e I11 and t h e h e p t o s e I i s l i n k e d i n (1+5) t o 2-deoxy-Q-mannoo c t u l o s o n i c acid.
h e p t o s e I,
The t h r e e s u g a r s o f t h e l i n e a r c h a i n ,
h e p t o s e 11, and Q-glucose, a r e s u b s t i t u t e d by p h o s p h a t e ,
pyrophosphate,
o r p y r o p h o s p h o r y l e t h a n o l a m i n e g r o u p s l i n k e d t o C-4 h y d r o x y l g r o u p s . i n some p o l y s a c c h a r i d e c h a i n s , one o r t w o s u b s t i t u t i n g
However,
g r o u p s may b e a b s e n t , w h i c h
may e x p l a i n t h e h e t e r o g e n e i t y i n t h e
l e n g t h o f the core polysaccharide chains. Structural studies o f
lipopolysaccharides o f Escherichia c o l i
K 1 2 have d e m o n s t r a t e d t h a t s m o o t h s t r a i n s p r o d u c e l i p o p o l y s a c c h a r i d e
w i t h c o n s i d e r a b l e h e t e r o g e n e i t y w i t h r e s p e c t t o t h e l e n g t h o f 0a n t i g e n i c c h a i n s w h e n "P
n.m.r.
methods
average
only
p r o v i d e d an
heterogeneity.62
was u s e d , a l t h o u g h t r a d i t i o n a l composition
over
S t a n d a r d i s o l a t i o n p r o c e d u r e s were
a
range
of
shown t o f a i l
t o e x t r a c t some 30% o f t h e t o t a l l i p o p o l y s a c c h a r i d e p r e s e n t i n t h e cells,
t h e s i g n i f i c a n c e o f w h i c h was d i s c u s s e d i n r e l a t i o n t o o u t e r
membrane s t r u c t u r e . When l i p o p o l y s a c c h a r i d e s f r o m E s c h e r i c h i a c o l i 8 w e r e s o n i c a t e d together w i t h pure spin-labelled phospholipids without the a d d i t i o n o f unlabelled phospholipids,
e x t e n s i v e l i n e b r o a d e n i n g was o b s e r v e d
due t o t h e c l o s e p r o x i m i t y o f s p i n - l a b e l l e d m o l e c u l e s t o e a c h o t h e r , a result suggesting that spin-labelled phospholipids existed i n s e g r e g a t e d domains c o n t a i n i n g few Such
mixed
bilayers
were
l i p o p o l y s a c c h a r i d e molecules.63
incubated
under
various
conditions,
i n c l u d i n g t h e a d d i t i o n o f N a C l a n d MgC12 t o t h e m e d i u m a n d t h e i n c o r p o r a t i o n o f t h e m a j o r outer-mem b r a n e p r o t e i n , bilayer,
porin,
i n t o the
a n d t h e i n t e r m i x i n g o f t h e d o m a i n s w a s f o l l o w e d by t h e
decrease i n l i n e width.
The d i f f u s i o n o f t h e l a b e l l e d p h o s p h o l i p i d s
i n t o l i p o p o l y s a c c h a r i d e d o m a i n s was h a r d l y d e t e c t a b l e when t h e m i x e d b i 1ay e r co n t a ine d s p i n - l a be 1l e d p h osp h o l ip i ds a n d 1ipo po 1y s a c c h a r i de i n approximately equimolar was o b s e r v e d when a 1 7 - f o l d present,
ratios.
Although progressive d i f f u s i o n
m o l a r e x c e s s o f l i p o p o l y s a c c h a r i d e was
i t i s v e r y slow even under t h e o p t i m a l c o n d i t i o n s ,
r e q u i r i n g s e v e r a l days f o r a n e a r l y complete mixing. s e r i e s o f experiments,
usually
I n another
s p i n - l a b e l l e d p h o s p h o l i p i d s were d i l u t e d w i t h
96
Carbohydrate Chemistry
a 1 0 0 - f o l d e x c e s s of u n l a b e l l e d p h o s p h o l i p i d s and t h e n mixed w i t h lipopolysaccharides. I n t h e s e e x p e r i m e n t s , t h e f l u i d i t y of t h e d o m a i n s c o n t a i n i n g s p i n - l a b e l l e d p h o s p h o l i p i d s was shown t o be i d e n t i c a l , e v e n a f t e r 3 d a y s of i n c u b a t i o n , w i t h t h e f l u i d i t y of b i l a y e r s containing only phospholipids, i n c o n t r a s t t o t h e e x p e c t a t i o n of t h e d i m i n i s h e d f l u i d i t y i f p h o s p h o l i p i d m o l e c u l e s became f i n e l y i n t e r s p e r s e d w i t h l i p o p o l y s a c c h a r i d e m o l e c u l e s . These two d i f f e r e n t l i n e s o f a p p r o a c h t h e r e f o r e s u p p o r t e d t h e i d e a t h a t p h o s p h o l i p i d (and most probably l i p o p o l y s a c c h a r i d e ) domains i n m i x e d b i l a y e r s t e n d t o be r a t h e r s t a b l e and p e r s i s t f o r l o n g p e r i o d s o f time. The n a t u r a l a f f i n i t y of v a r i o u s b a c t e r i a l g l y c o p e p t i d e s and l i p o p o l y s a c c h a r i d e s f o r mammalian c e l l membranes has been e s t i m a t e d q u a n t i t a t i v e l y by c o m p a r i s o n w i t h t h e a d s o r p t i o n of l i p o p o l y s a c c h a r i d e from E s c h e r i c h i a c o l i N C T C 8623 t o e r y t h r o c y t e s , t h y m o c y t e s , bone-marrow c e l l s , s p l e e n c e l l s , p e r i t o n e a l lymphocytes, and macro phage^.^^ I m m u n o p o t e n t i a t i n g a c t i v i t y was e s t i m a t e d b y m e a s u r i n g t h e a b i l i t y of t h e b a c t e r i a l f r a c t i o n s t o s t i m u l a t e a h u m o r a l r e s p o n s e t o o v a l b u m i n i n H A M / l C R m i c e . When t h e a f f i n i t y f o r mammalian c e l l membranes was compared w i t h t h e s t i m u l a t i o n of t h e a n t i b o d y r e s p o n s e , i t was found t h a t a n e g a t i v e c o r r e l a t i o n f o r P 0 . 0 0 0 5 ) and a p o s i t i v e p e r i t o n e a l m a c r o p h a g e s ( g s = -0.94, c o r r e l a t i o n f o r p e r i t o n e a l l y m p h o c y t e s ( z s = + 0 . 9 7 , P 0 . 0 0 0 5 ) and s p l e e n c e l l s (rs = +0.76, 0.005) e x i s t e d . A heptose-deficient l i p o p o l y s a c c h a r i d e s t r a i n of E c h e r i c h i a c o l i 0 8 , s t r a i n F151, was f o u n d t o r e l e a s e p o r t i o n s of i t s o u t e r membrane when c e l l s w e r e e x p o s e d t o l O m M c i t r a t e b u f f e r ( p H 2.75) f o r 30 m i n and s u b s e q u e n t l y e x p o s e d t o l O O m M t r i s ( h y d r o x y m e t h y 1 ) The o u t e r - m e m b r a n e c o m p o n e n t aminomethane b u f f e r (pH 8 . 0 p 5 r e l e a s e d was f o u n d t o be composed of p r o t e i n , l i p o p o l y s a c c h a r i d e , phospholipid (card i o l i p i n , phospha t i d y l e t h a n o lam ine, and p h o s p h a t i d y l g l y c e r o l ) , and a l k a l i n e p h o s p h a t a s e . T h e outer-membrane component was r e l e a s e d from t h e c e l l envelope i n t h e a b s e n c e of c e l l l y s i s , a s no n - g l u c o s e - 6 - p h o s p h a t e dehydrogenase a c t i v i t y or s u c c i n a t e dehydrogenase a c t i v i t y was d e t e c t e d . M o r p h o l o g i c a l l y , t h e o u t e r - membrane component appeared t o c o n s i s t of l a m i n a r f r a g m e n t s and v e s i c l e s which had an a s s o c i a t e d a l k a l i n e p h o s p h a t a s e a c t i v i t y . I n t h e c h r o m a t i n or" s p l e e n c e l l s of m i c e and r a t s i m m u n i z e d w i t h l i p o p o l y s a c c h a r i d e s f r o m E s c h e r i c h i a c o l i , a new s p e c i e s , and a n t i g e n n o n s p e c i f i c f r a c t i o n , o f n o n - h i s t o n e c h r o m a t i n p r o t e i n s has been d e s c r i b e d . 6 6 The p o s s i b l e r o l e of t h i s f r a c t i o n i n t h e
97
4: Microbial Polysaccharides
r e g u l a t o r y p r o c e s s o f gene a c t i v a t i o n d u r i n g t h e immune r e s p o n s e as e x p r e s s e d by
the synthesis of
t h e IgM c l a s s o f
antibodies
was
discussed. E x p e r i m e n t a l e v i d e n c e h a s been p r e s e n t e d w h i c h i n d i c a t e s t h a t
1,2-dihydro-l-hydroxy-6-methyl-2-(propanesulphonyl)-thieno(3,2-~-~ (1,2,3)-diazaborine
(18),
a heterocyclic,
boron-containing
sub-
s t a n c e , p r e v e n t s b a c t e r i a l p r o l i f e r a t i o n by i n h i b i t i n g l i p o p o l y s a c c h a r i d e b i o ~ y n t h e s i s . ~T h~e r e w a s a r e d u c t i o n i n Q - g a l a c t o s e i n c o r p o r a t i o n i n t o the lipopolysaccharide o f whole c e l l s , n e w l y f o r m e d l i p o p o l y s a c c h a r i d e c h a i n s appear t h e p r e s e n c e o f t h e d i a z a b o r i n e compound, t r o n microscopy.
and no
i n whole b a c t e r i a i n
as d e m o n s t r a t e d by e l e c -
The 3 - d e o x y - ~ - m a n n o - 2 - o c t u l o s o n i c
acid metabolism
seemed t o be t h e t a r g e t o f t h e d i a z a b o r i n e compound as L - a r a b i n o s e 5 - p h o s p h a t e i n c o r p o r a t i o n i n t o m a c r o m o l e c u l a r m a t e r i a l was a f f e c t e d .
OH
The p u r i f i e d l i p o p o l y s a c c h a r i d e e n d o x i n - l i k e c o m p o n e n t f r o m L i s t e r i a monocytogenes has been shown t o a c t as b o t h an i n v i t r o mitogen
for
lymphocytes
and
i n
in
vivo
adjuvant
i n
antibody
production. A
novel peptidoglycan-associated
l i p o p r o t e i n was
found i n t h e
c e l l e n v e l o p e o f P r o t e u s ~ i r a b i l i s . T~h ~i s p r o t e i n was s h o w n t o h a v e an a p p a r e n t polyacrylamide gel,
molecular of
18,000.
weight,
i n sodium dodecyl
sulphate
The p r o t e i n was p r e s e n t i n t h e c e l l
envelope i n a form very c l o s e l y , but n o t c o v a l e n t l y , associated w i t h t h e p e p t i d o g l y c a n l a y e r a n d was r e c o v e r e d p r e d o m i n a n t l y f r o m t h e o u t e r membrane a f t e r s e p a r a t i o n o f t h e c e l l e n v e l o p e s . 1-14C)P a l m i t i c a c i d and I 2 - 3 H I g l y c e r o l were i n c o r p o r a t e d i n t o t h e p r o t e i n . A
fatty-acid-containing
polypeptide
(lipopolypeptide)
by d i g e s t i o n o f t h e p u r i f i e d p e p t i d o g l y c a n - a s s o c i a t e d
was
isolated
lipoprotein
It w i t h t r y p s i n i n t h e p r e s e n c e o f 0.05% s o d i u m d o d e c y l s u l p h a t e . was c o m p o s e d o f 3 1 a m i n o a c i d r e s i d u e s , an u n i d e n t i f i e d compound {XI,
and ca. 3 f a t t y - a c i d
residue^.^' A
l i p o - o l i g o p e p t i d e was a l s o
98
Carbohydrate Chemistry
isolated after
further
t h e absence o f composed o f
4
compound { X I ,
digestion of
sodium amino
a n d ca.
dodecyl
lipopolypeptide with t r y p s i n i n
sulphate.
acid residues
3 fatty-acid
Lipo-oligopeptide
(L-Asx,
2 I-Ser,
residues.
was
L-Lys),
The C - t e r m i n a l
a
amino
a c i d s o f l i p o p o l y p e p t i d e and l i p m l i g o p e p t i d e were d e t e r m i n e d as
I-
arginine
N-terminus
o f
lipopolypeptide,
or
and
L-lysine,
respectively.
peptidoglycan-associated
lipoprotein,
The
l i p o a l i g o p e p t i d e c o u l d n o t be i d e n t i f i e d by c o n v e n t i o n a l N - t e r m i n a l a n a l y s i s , i n d i c a t i n g t h a t t h e N - t e r m i n u s i s p r o b a b l y masked.
The
a m i n o a c i d s e q u e n c e o f t h e l i p w l i g o p e p t i d e d e r i v e d f r o m t h e Nterminal
--Proteus
region o f
the peptidoglycan-associated
lipoprotein o f
m i r a b i l i s was d e t e r m i n e d t o be { X I - k - S e r - I = - S e r - L - A s n - i -
L Y S . ~ The ~ u n i d e n t i f i e d compound { X I p r e s e n t a t t h e N - t e r m i n u s was i d e n t i f i e d a s g l y c e r y l - l - c y s t e i n e {S-(p r o p a n e -2’,3’- d i o 1) -3- t h i o - 2 amino-propanoic
acid).
lipopolypeptide,
The p a r t i a l a m i n o a c i d s e q u e n c e o f t h e
w h i c h c o n t a i n e d t h e l i p o a l i g o p e p t i d e a t i t s N-
t e r m i n a l p a r t , w a s a l s o d e t e r m i n e d , m a i n l y by Edman d e g r a d a t i o n . The s t r u c t u r e o f t h e N - t e r m i n a l p a r t o f p e p t i d o g l y c a n - a s s o c i a t e d lipoprotein
was d e t e r m i n e d t o
be
(3 f a t t y
acids
glyceryl-L-Cys-I-
-
Ser-L-Ser-L-Asn-I-Lys-L-Asn-L-Asn-l-Asp-I-Asp-~-Glu-I-Thr-l-Asp-L-Thr-L-
Ser.....}.
These p r o p e r t i e s , e x c e p t f o r m o l e c u l a r w e i g h t a n d n o n -
covalent association w i t h the peptidoglycan, t h o s e o f Braun’s l i p o p r o t e i n . Braun’s
showed r e s e m b l a n c e t o
However, t h e p r o t e i n was d i s t i n c t f r o m
l i p o p r o t e i n i n r e g a r d t o amino a c i d c o m p o s i t i o n .
A similar
p e p t i d o g l y c a n - a s s o c i a t e d l i p o p r o t e i n was p r e s e n t w i d e l y i n t h e c e l l envelopes o f various Gram-negative contains
about
twelve
times
as
l i p o p r o t e i n as E s c h e r i c h i a c o l i . associated lipoprotein o f
bacteria. much
Proteus m i r a b i l i s
peptidoglycan-associated
Antiserum against peptidoglycan-
-m i r a b i l i s
was c r o s s - r e a c t i v e
a g a i n s t p e p t i do g l y c a n - a s s o c i a t e d l i p o p r o t e i n o f
E s c h e r ic h ia c o 1i,
b u t n o t a g a i n s t Braun’s l i p o p r o t e i n o f E s c h e r i c h i a c o l i . The
stimulation
of
incorporation o f
{3HIg-galactose
into
membrane g l y c o c o n j u g a t e s , m e a s u r e d i n a p r e c i p i t a t i o n t e s t ,
was u s e d
as a c r i t e r i o n f o r a c t i v a t i o n o f
I n this
assay,
bone-marrow
p u r i f i e d bacterial lipopolysaccharide,
cells.72
lipoprotein,
and
m u r e i n monomer and d i m e r f r a g m e n t s a l l a c t i v a t e d r a t bone-marrow cells i n vitro. t i m e course,
The r e s p o n s e was dose d e p e n d e n t ,
a n d was n o t s e r u m d e p e n d e n t .
followed a defined
c-Acetylated murein dimer
f r a g m e n t s f r o m P r o t e u s m i r a b i l i s wtere much l e s s a c t i v e t h a n t h e i r
u n s u b s t it u t e d c o u n t e r p a r t s , i n d i c a t i n g a s t r u c t u r a l spe c i f ic i t y f o r
murein activation.
Removal o f a d h e r e n t a n d phagocy t i z i n g c e l l s f r o m
4: Microbial Polysaccharides
99
t h e marrow suspensions d i d n o t a l t e r t h e s e r e s u l t s . The l a b e l l e d , a c t i v a t e d c e l l s c o n s t i t u t e d a d i s t i n c t p o p u l a t i o n of buoyant d e n s i t y 1 . 0 6 4 t o 1 . 0 6 9 g/cm3 when c e n t r i f u g e d o n a c o n t i n u o u s g r a d i e n t of P e r c o l l . Enrichment of t h e t a r g e t - c e l l p o p u l a t i o n was achieved by a c o m b i n a t i o n o f a d h e r e n t - c e l l r e m o v a l and d i s c o n t i n u o u s d e n s i t y g r a d i e n t c e n t r i f u g a t i o n t o remove g r a n u l o c y t e s and e r y t h r o p o i e t i c cells. I t was c o n c l u d e d t h a t a p o p u l a t i o n o f m y e l o p o i e t i c p r e c u r s o r s could be a c t i v a t e d by d i r e c t c o n t a c t w i t h b a c t e r i a l c e l l w a l l c o n s t i t u e n t s . The s t i m u l a t i o n o f Q - g a l a c t o s e i n c o r p o r a t i o n was n o t coupled t o a c t i v e d e o x y r i b o n u c l e i c a c i d s y n t h e s i s i n t h e marrow cells. T h u s , t h e a c t i v a t i o n was i n t e r p r e t e d a s an i n d u c t i o n o f d i f f e r e n t i a t i o n r a t h e r than a m i t o t i c e v e n t . To o b t a i n i n f o r m a t i o n about t h e n a t u r e of t h e immunogens i n t h e r i b o s o m a l v a c c i n e ( f r a c t i o n 11) o f Pseudomonas a e r u g i n o s a , t h e s p e c i f i c i t y of r a b b i t a n t i b o d i e s t o F r a c t i o n I 1 has been ~ t u d i e d . ’ ~ Crossed i m m u n o e l e c t r o p h o r e s i s d e m o n s t r a t e d t h e p r e s e n c e of a n t i b o d i e s which p r e c i p i t a t e d w i t h ribosomal a n t i g e n s , b u t n o t w i t h lipopolysaccharide. B y means of an e n z y m e - l i n k e d irnmunosorbent a s s a y , a n t i b o d i e s t o l i p o p o l y s a c c h a r i d e were d e t e c t e d i n a n t i b o d i e s t o f r a c t i o n 11. A n t i b o d i e s t o f r a c t i o n I1 c o u l d p r o t e c t mice a g a i n s t a l e t h a l c h a l l e n g e w i t h Pseudomonas a e r u g i n o s a . Absorption experiments demonstrated t h a t t h e p r o t e c t i v e a b i l i t y of a n t i b o d i e s t o f r a c t i o n I1 was due t o cell-envelope a n t i g e n s , whereas a n t i b o d i e s t o ribosomal antigens d i d n o t contribute t o the protection. A n t i b o d i e s t o l i p o p o l y s a c c h a r i d e c o u l d be d e t e c t e d i n mice 1 week a f t e r a s i n g l e v a c c i n a t i o n w i t h f r a c t i o n 11. I t was concluded t h a t t h e p r o t e c t i v e a c t i v i t y of f r a c t i o n I1 was due, a t l e a s t i n p a r t , t o t h e p r e s e n c e of l i p o p o l y s a c c h a r i d e i n t h e r i b o s o m a l v a c c i n e . Treatment o f f r a c t i o n I1 w i t h r i b o n u c l e a s e decreased t h e p r o t e c t i v e a c t i v i t y of t h e r i b o s o m a l v a c c i n e . A d d i t i o n of s y n t h e t i c polyadenylic acid-polyuridylic acid restor ed t h e p ro t e c t i v e a c t i v i t y o f r i b o n u c l e a s e - t r e a t e d f r a c t i o n 11, i n d i c a t i n g t h a t R N A i n t h e r i b o s o m a l v a c c i n e m i g h t a c t a s an a d j u v a n t o r a c a r r i e r i n t h e p r e s e n t a t i o n of t h e c o n t a m i n a t i n g c e l l - e n v e l o p e a n t i g e n s . The p r o t e c t i v e a c t i v i t y and t h e t o x i c i t y of f r a c t i o n I1 were compared w i t h t h e p r o t e c t i v e a c t i v i t y and t h e t o x i c i t y of f r a c t i o n I , which co n t a i ne d c e 1l-en ve 1ope co m P O ne n t s , i n c 1u d i n g 1ipopol y s a c c h a r i de , and o f p u r i f i e d l i p o p o l y s a c c h a r i d e . The r e s u l t s i n d i c a t e d t h a t p r o t e c t i o n by t h e ribosomal vaccine was a s s o c i a t e d w i t h a s l i g h t l y h i g h e r t o x i c i t y than was p r o t e c t i o n by f r a c t i o n I , whereas p u r i f i e d l i p o p o l y s a c c h a r i d e was t h e most t o x i c vaccine.
100
Carbohydrate Chemistry
A k a l i n e t r e a t m e n t o f P s e u d o m o n a s gg~g~ifiisg t y p e 5 l i p o p o l y s a c c h a r i d e r e s u l t e d i n r e d u c e d t o x i c i t y a s m e a s u r e d by b o t h t h e L i m u l u s amoebocyte assay and t h e r a b b i t p y r o g e n i c i t y test.74 Chemical a n a l y s i s o f t h e d e a c y l a t e d l i p o p o l y s a c c h a r i d e r e v e a l e d t h a t e s t e r - l i n k e d f a t t y a c i d s were r e m o v e d w h i l e t h e a m i d e - l i n k e d f a t t y acids remained i n t a c t . The n e u t r a l and amino s u g a r c o m p o s i t i o n s f o r n a t i v e l i p o p 0 l y s a c c h a r i de a n d d e a c y 1a t e d l i p o p o l y s a c c h a r i d e were identical within experimental error. Antigenic determinants f o r c o m p l e m e n t - d e p e n d e n t h u m a n o p s o n i c a n t i b o d y were r e t a i n e d u n d e r these d e a c y l a t i o n c o n d i t i o n s . To e n h a n c e i t s i m m u n o g e n i c i t y , d e a c y l a t e d l i p o p o l y s a c c h a r i d e was c o v a l e n t l y c o u p l e d t o P s e u d o m o n a s p i l i and t h e 1,4-diaminobutyl d e r i v a t i v e s o f Pseudomonas exotoxin A and t e t a n u s t o x o i d . Q u a n t i t a t i v e amino s u g a r a n a l y s e s r e v e a l e d t h a t 2.6 a n d 3 . 2 m o l o f d e a c y l a t e d l i p o p o l y s a c c h a r i d e s were c o v a l e n t l y bound t o a m i n o b u t y l Pseudomonas e x o t o x i n A and a m i n o b u t y l t e t a n u s toxoid, r e s p e c t i v e l y . Gel-electrophoresis data i n d i c a t e d a t least 1 mol of d e a c y l a t e d l i p o p o l y s a c c h a r i d e c o v a l e n t l y bound p e r p i l u s subunit protein. I n i t i a l immunologic data i n d i c a t e d that antibody a g a i n s t d e a c y l a t e d 1 i p o p o l y s a c c h a r i . d e c o u l d be i n d u c e d w h e n t h e deacylated l i p o p o l y s a c c h a r i d e i s c o v a l e n t l y attached t o p r o t e i n carriers. The p o l y s a c c h a r i d e m o i e t y was i s o l a t e d by m i l d a c i d h y d r o l y s i s f r o m t h e slime g l y c o p r o t e i n o f P s e u d o m o n a s a e r u g i n o s a s t r a i n BI. After gel f i l t r a t i o n , the polysaccharide obtained from the c a r b o h y d r a t e p e a k f r a c t i o n s was f o u n d t o b e l i p i d - a n d p r o t e i n free.75 A n a l y s e s i n d i c a t e d t h a t t h e p o l y s a c c h a r i d e c o n t a i n e d t h e c a r b o h y d r a t e components o f the p a r e n t g l y c o l i p o p r o t e i n . Molecular s i z e o f t h e p o l y s a c c h a r i d e was e s t i m a t e d by g e l f i l t r a t i o n a s 70,000 t o 10,000. The p o l y s a c c h a r i d e showed no i n d i c a t i o n s o f t o x i c i t y i n mice a t d o s e s f a r i n e x c e s s o f t h e l e t h a l d o s e f o r t h e p a t e n t g l y c o l i p o p r o t e i n , n o r d i d t h e mice d e v e l o p t h e l e u k o p e n i a t h a t characteristically follows intraperitoneal injection of glycolipoprotein. The p o l y s a c c h a r i d e acted a s a n i n h i b i t o r o f i n d i re c t haem a g g l u t i n a t i o n o f g l y c o 1i po p r o t e i n- c o a t e d e r t h r o cy t e s i n t h e p r e s e n c e o f a n t i - g l y c o l i p o p r o t e i n s e r u m . H o w e v e r , i t was n o t a n t i g e n i c i t s e l f i n rabbits. Coupled w i t h methylated bovine serum albumin, t h e p o l y s a c c h a r i d e c o n t i n u e d t o lack t h e l e u k o p e n i c and t o x i c p r o p e r t i e s o f the p a r e n t g l y c o l i p o p r o t e i n , but the coupled p o l y s a c c h a r i d e was c a p a b l e o f s t i m u l a t i n g i n d i r e c t h a e m a g g l u t i n a t i n g antibody a g a i n s t both the polysaccharide and the g l y c o l i p o p r o t e i n coating erythrocytes. Moreover, t h e antibody t o the coupled
101
4: Microbial Polysaccharides
p o l y s a c c h a r i d e p r o t e c t e d m i c e a g a i n s t c h a l l e n g e w i t h l e t h a l doses o f v i a b l e Pseudomonas a e r u g i n o s a w i t h t h e same e f f e c t i v e n e s s a s a n t i g l y c o l i p o p r o t e i n serum. The i d e n t i f i c a t i o n o f t h e new a m i n o s u g a r
d i d e o x y - Q - g l u c o p r a n u r o n i c a c i d (19)
2,3-diacetamido-2,3-
a s a c o n s t i t u e n t o f t h e 0-
s p e c i f i c p o l y s a c c h a r i d e s o f Pseudomonas a e r u g i n o s a s t r a i n 1 7 0 0 1 4 h a s been b r o u g h t a b o u t by use o f 1 3 C n.m.r.
AcHN
a n d c h e m i c a l methods.76
AcHN
OH
The p r o c e d u r e u s e d f o r t h e i s o l a t i o n o f from
strains
o f
Pseudomonas
the lipopolysaccharide
aeruginosa appears
t o
c o n f l i c t i n g r e s u l t s i n t h e i n t e r p r e t a t i o n o f 31P n.m.r. The
use
of
the
Westphal
lipopolysaccharide with
extraction
procedure7'
to
gives
a h i g h e r phosphorus content
o b t a i n e d by u s e d o f t h e B ~ v i pnr o~c e ~d u r e .
lead
spectra. a
than that
The h i g h e r l e v e l s o f
n.m.r.
p h o s p h o r u s c o n t e n t h a v e b e e n a t t r i b u t e d 7 7 b y u s e o f "P
to
the presence o f t r i p h o s p h a t e residues, probably i n t h e l i p i d A and inner-core
regions.
The
presence
of
triphosphate residues i n
-P-s-e u d o m o n a s a e r u g i n o s a i s i n c o n t r a s t t o
results obtained
for
S a l m o n e l l a s p e c i e s and E s c h e r i c h i a c o l i . Among t h e a e r o b i c p s e u d o m o n a d s ,
the closely related species
Pseudomonas d i m i n u t a and Pseudomonas v e s i c u l a r i s f o r m subgroup,
the
inclusion of
which i n
becoming i n c r e a s i n g l y suspect.
the
an i s o l a t e d
genus Pseudomonas
i s
Chemotaxonomic grounds f o r t h e
s e p a r a t e s t a t u s o f t h e s e s p e c i e s i n c l u d e t h e p o s s e s s i o n by t h e i r type
strains
o f
a
unique
range
o f
polar
l i p i d s
and
l i p o p o l y s a c c h a r i d e s i n w h i c h l i p i d A i s based o n 2,3-diamino-2,3d i de ox y -Q- g l uco se r a t h e r t h a n 2- am ino -2 -de ox y -Q - g l
UCG se
.
The 1i po
-
p o l y s a c c h a r i d e f r o m Pseudomonas d i m i n u t a NCTC 8545 h a s a n u m b e r o f o t h e r unusual f e a t u r e s i n c l u d i n g t h e presence o f Q-threo-2-pentu l o s e ( i - x y l u l o s e ) .79 B a c t e r i o p h a g e 12P caused l y s i s o f not
other
plant
Specificity of
pathogenic
or
Pseudomonas p h a s e o l i c o l a b u t
saprophytic
pseudonomads.80
t h e b a c t e r i o p h a g e may depend o n r e c o g n i t i o n o f some
Carbohydrate Chemistry
102 unique
feature
of
polysaccharides.
the
Pseudomonas
phaseolicola
cell-surface
Exopolysaccharide and l i p o p o l y s a c c h a r i d e from
----------P s e u d o m o n a s e ----------haseolicola
were
more e f f i c i e n t a t
inhibiting
bacteriophage attachment than were i d e n t i c a l l y prepared e x t r a c t s from
five
other
phaseolicola
pseudomonad s p e c i e s .
w i t h
Mutants
bacteriophage
of
Pseudomonas
r e s i s t a n c e
produced
e x o p o l y s a c c h a r i d e and l i p o p o l y s a c c h a r i d e d i f f e r e n t i n amount and composition from those o f the parental strain. the
e x o p o l y s a c c h a r i d e and
P-s-e u d o m o n a s e h aseolicola -
Thus i n some manner
lipopolysaccharide
structures
o f
are d i s t i n c t from those o f other p l a n t
p a t h o g e n i c and s a p r o p h y t i c s p e c i e s .
The d i f f e r e n c e may p r e v e n t
r e c o g n i t i o n a n d i n i t i a t i o n o f r e s i s t a n c e e v e n t s when Pseudomonas phaseolicola challenges i t s susceptible host plant, The l i p o p o l y s a c c h a r i d e o f
bean.
R h o d o m i c r o b i u m v a n n i e l i i A T C C 17100
h a s been s t u d i e d a n d f o u n d t o c o n t a i n a - g l u c o s e a n d Q-mannose ( m o l a r r a t i o 2:l)
3-2-
w i t h t r a c e amounts o f p - g l u c o s e and p - x y l o s e and
m e t h y l - Q - x y l o s e . 81
2-Ace t a m i d o - 2 - d e o x y - Q - g 1 ucose,
3-deoxy-~-manno-2-octulosonic
u r o n i c acids, and
a c i d were a l s o found
t o be m a j o r
co m po ne n t s t o g e t h e r w it h 6-hy d r ox y p a 1m i t ic a c i d (ex c l u s ive 1y am ide bound),
6-hydroxymyristic
bound).
H e p t o s e s a n d p h o s p h a t e w e r e f o u n d t o be a b s e n t .
A f r a c t i o n (ca.
acid, and m y r i s t i c a c i d ( m a i n l y e s t e r The l i p i d
4 0 % o f t h e m a t e r i a l d r y w e i g h t ) l i b e r a t e d by m i l d
a c i d h y d r o l y s i s c o n t a i n s Q-mannose ( i n t h e n o n r e d u c i n g t e r m i n a l p o s i t i o n s ) i n a d d i t i o n t o 2-amino-2-deoxy-~-glucose
a n d an unknown
n i n h y d r i n - p o s i t i v e compound, b u t t h e p r e s e n c e o f a-mannose a s a t r u e c o n s t i t u e n t o f l i p i d A (as opposed t o i t b e l o n g i n g t o an a d d i t i o n a l , c o n t a m i n a t i n g l i p i d a s may be t h e c a s e i n l i p o p o l y s a c c h a r i d e s f r o m
---Chromatium
vinosum
I___-
determined. f r a c t io n a r e
and T h i o c a p s a r o s e o p e r s i c i n a ) has y e t
The m a i n c o n s t i t u e n t s o f
p - g l uc o s e ,
acid, and u r o n i c a c i d s .
a -m a nno s e ,
I t should
the
to
be
degraded p o l y s a c c h a r i d e
3 -de o x y -Q - m a n n o - 2 - o c t u l o s o n i c be
noted that
uronic acids
co n t r i b u t e ne ga t ive c h a r g e s i n a p h o s p h a t e - f r e e 1i p o po 1y sa c c h a r ide Interestingly,
9-xylose
and 3 - 2 - m e t h y l - a - x y l o s e
were
.
enriched
r e l a t i v e t o t h e o t h e r s u g a r s when R h o d o m i c r o b i u m v a n n i e l i i A T C C 1 7 1 0 0 was g r o w n f o r s e v e r a l p a s s a g e s i n t h e same R8AH medium. There
is
a
common
structure
(core
l i p o p o l y s a c c h a r i d e s o f R h o d o s p i r i l l u m tenue.”
region)
i n
the
I t i s composed o f a
b r a n c h e d t r i s a c c h a r i d e o f L - g l y c e r o - p - m a n n o - h e p t o s e ( a n d o f 3-deox y ~-manno-2-octulosonic acid),
a s r e v e a l e d by m e t h y l a t i o n a n a l y s e s o f
degraded polysaccharides o f f o u r d i f f e r e n t R h o d o s p i r i l l u m tenue strains.
The s t r u c t u r e i s s i m i l a r o r m i g h t e v e n be i d e n t i c a l t o t h e
103
4: Microbial Polysaccharides i n n e r core o f e n t e r o b a c t e r i a l 0 antigens.
I n addition,
each o f t h e
four R h o d o s p i r i l l u m tenue lipopolysaccharides contains a s t r a i n s p e c if ic r e g io n t h a t c o n s is t s o f h e p t o s e ( s ) ( & -9 1y c e r o - Q - m a n n o h e p t o s e o r a-glycero-p-manno-heptose o r b o t h ) o r hexoses. There i s a p a r t i a l s u b s t i t u t i o n o f t h e core r e g i o n and t h e s t r a i n - s p e c i f i c r e g i o n by p h o s p h o r u s , The
showing m i c r o h e t e r o geneit y .
lipopolysaccharide
from
polymixin-resistant pmrA
the
m u t a n t s o f S a l m o n e l l a t y p h i m u r i u m has been c h a r a c t e r i z e d a n d f o u n d t o be a n u n u s u a l t y p e o f p h o s p h o l i p i d ( 2 0 ) b u i l t up o f a c e n t r a l d i s a c c h a r i de o f t w o 2-am ino -2-deox y g l uco se r e s i d ue s. 83 Pho sp h a t e
-n-
e s t e r s a r e bound i n t h e 4-
and l - p o s i t i o n s
r e s p e c t i v e l y , t o g i v e t h e p h o s p h o l i p i d backbone.
o f r e s i d u e s I 1 a n d I, The l i p i d c h a r a c t e r
i s g i v e n by s u b s t i t u t i o n o f t h e b a c k b o n e w i t h 7 m o l o f l o n g - c h a i n f a t t y acids:
4 mol o f 3-hydroxymyristic
nonhydroxylated f a t t y acids,
2. 1
a c i d and 3 m o l o f
m o l each o f l a u r i c ,
myristic,and
p a l m i t i c acids, r e s p e c t i v e l y , w i t h s m a l l amounts o f 2 - h y d r o x y m y r i t i c acid.
The e x a c t p o s i t i o n s o f t h e s e g r o u p s h a v e o n l y p a r t l y b e e n
identified.52,83
The backbone c o n t a i n s t w o a m i n o g r o u p s s u b s t i t u t e d
by 3 - h y d r o x y m y r i s t i c h y d r o x y l groups,
a c i d and,
besides t h e two phosphate-substituted
another f o u r h y d r o x y l groups o f which p o s i t i o n 3 o f
r e s i d u e I 1 forms t h e l i n k t o t h e s p e c i f i c p o l y s a c c h a r i d e v i a 3-
deoxy-9-manno-2-octulosonic ester-bound backbone,
acid.
3-hydroxymyristic
There i s evidence t h a t
the
two
acids are linked d i r e c t l y t o the
s u b s t i t u t i n g t w o o f t h e t h r e e a v a i l a b l e h y d r o x y l groups.
The n o r m a l f a t t y a c i d s ,
i n c o n t r a s t , a p p e a r t o be l i n k e d a t t h e 3 -
hydroxy groups o f t h e 3 - h y d r o x y m y r i s t i c a c i d residues:
m y r i s t i c and
2- hy d r ox ym y r i s t ic a c i d s s u b s t it u t e t h e e s t e r - b o un d 3- hy d r ox ym y r i s t ic
acid, w h i l e p r e l i m i n a r y acids acid.
to
evidence i n d i c a t e s
be e s t e r - l i n k e d
to
t h e l a u r i c and m y r i s t i c
t h e t w o amide-bound
3-hydroxymyristic
I n t h i s way l i p i d A i s c h a r a c t e r i z e d by a h i g h c o n t e n t o f a
r a t h e r unusual s t r u c t u r e o f long-chain hydroxymyristate.
Thus o n l y
the
s u b s t i t u t e t h e backbone d i r e c t l y , linkage,
fatty-acid
e s t e r s o f 3-
3-hydroxylated
acids would
t w o i n amide and t w o i n e s t e r
w h i l e t h e n o n - h y d r o x y l a t e d a c i d s (and 2 - h y d r o x y m y r i s t i c
a c i d ) w o u l d be e s t e r - l i n k e d t o t h e 3 - h y d r o x y m y r i s t i c
a c i d groups.
M u t a t i o n s i n gene r f a H o f S a l m o n e l l a t y p h i m u r i u m a t 8 4 u n i t s o n t h e l i n k a g e map make l i p o p o l y s a c c h a r i d e o f c h e m o t y p e s Ra, a n d R c . ~ F~ - F a c t o r
e x p r e s s i o n i n RfaH'
f o l l o w i n g p r o p e r t i e s when c o m p a r e d w i t h RfaH'
Flac,
Rb2,
Rb3,
s t r a i n s was r e d u c e d i n t h e
number o f phage f 2 i n f e c t i v e c e n t r e s ,
strains:
transfer o f
l y s i s by a n d p r o p a g a t i o n
o f phages f 2 and M13, p r o p o r t i o n o f c e l l s w i t h v i s i b l e F - p i l i ,
and
Carbohydrate Chemistry
1 04
where F
= -C=O
I
( 2 m o l e s p e r 3 hydroxyl groups)
HC-O(R )
I
(7H2) 10 CH 3
and R = l a u r i c a c i d ( 1 m o l e ) m y r i s t i c a c i d ( 1 mole) p a l m i t i c a c i d ( 1 mole) 2-hydroxymyristic a c i d ( t r a c e )
(201
105
4: Microbial Polysaccharides
f o r m a t i o n o f m a t i n g a g g r e g a t e s w i t h F- c e l l s . Inhibition o f m u l t i p l i c a t i o n o f Br60, a f e m a l e - s p e c i f i c phage, was n o t r e d u c e d i n Rfah' F l a c s t r a i n s . P l a s m i d t r a n s f e r f r o m RfaH' s t r a i n s was r e d u c e d u n a f f e c t e d f o r I n c g r o u p s B, Ia, L, N,
f o r I n c g r o u p s F I , F I I , a n d T, P,and W ,
a n d i n c r e a s e d f o r I n c g r o u p M when c o m p a r e d w i t h p l a s m i d
t r a n s f e r f r o m RfaH'
strains.
R e d u c e d F - f a c t o r f u n c t i o n i n RfaH'
s t r a i n s was n o t due t o d e f e c t i v e l i p o p o l y s a c c h a r i d e s i n c e s t r a i n s with
mutations
rfa
i n other
genes
were
unaffected i n plasmid
Gene r f a H a p p e a r s t o be h o m o l o g o u s w i t h gene s f r B i n
transfer.
E s c h e r i c h i a c o l i K-12,
which maps a t t h e same l o c a t i o n ,
influences
F-factor function,
and a f f e c t s s y n t h e s i s o f l i p o p o l y s a c c h a r i d e .
gene
s f r B has
product
an t i t e r m i na t o r The
.
of
been p r o p o s e d t o
mobility
of
membrane
lipopolysaccharide,
i s
essential
components,
for
particularly
biogenesis
membrane and i s a p r i m a r y e v e n t i n phage i n f e c t i o n . f l u i d dynamic p r o p e r t i e s o f
the outer
The
be t r a n s c r i p t i o n
of
the outer To d e f i n e t h e
membrane a s
related to
f u n c t i o n more a c c u r a t e l y t h e c a p a b i l i t y t o measure l a t e r a l d i f f u s i o n coefficients
vivo
pf
r h o d a m i n a t e d G30 l i p o p o l y s a c c h a r i d e f u s e d
i n t o S a l m o n e l l a t y p h i m u r i u m GPOA f i l a m e n t o u s b a c t e r i a h a s been developed.85 ( f 1uo r e s c e nce
The
method
used
r e d i s t r ib u t io n a f t e r
extends
the
FRAP
p h o t obl e a c h i n g) t o
procedure
b a c t e r i a and
the r e s u l t s demonstrate r a p i d d i f f u s i o n o f lipopolysaccharide
(2.0
2 0.9) A
cm2s-l)
x
=
f a m i l y o f mutants o f Salmonella tyhimurium w i t h a l t e r e d
lipopolysaccharide to
(D
over micrometre distances.
freeze-thaw
c o r e c h a i n l e n g t h s were assessed f o r s e n s i t i v i t y
and o t h e r
stresses.86
Deep r o u g h s t r a i n s w i t h
decreased c h a i n l e n g t h i n t h e l i p o p o l y s a c c h a r i d e c o r e were more s u s c e p t i b l e t o novobiocin, l a u r y l sulphate
p o l y m y x i n B,
d u r i n g growth,
to
bacitracin,
and sodium
ethylenediaminetetracetic
acid
and sodium l a u r y l s u l p h a t e i n r e s t i n g suspension, and t o s l o w and r a p i d freeze-thaw
i n w a t e r and s a l i n e ,
more outer-membrane Variations
i n
the
dramatically affect neomycin,
lipopolysaccharide the s e n s i t i v i t y of
chain
length
did not
the s t r a i n s t o t e t r a c y c l i n e ,
o r NaCl i n g r o w t h c o n d i t i o n s o r t h e degree o f f r e e z e - t h a w -
induced cytoplasmic-membrane strains
and t h e s e s t r a i n s e x h i b i t e d
damage t h a n t h e w i l d t y p e o r l e s s r o u g h s t r a i n s .
incorporated
damage.
larger
The d e e p e r r o u g h i s o g e n i c
quantities
o f
less-stable
l i p o p o l y s a c c h a r i d e and l e s s p r o t e i n i n t o t h e o u t e r membrane t h a n d i d t h e w i l d t y p e o r l e s s rough mutants, a f f e c t e d outer-membrane
i n d i c a t i n g t h a t the mutations
synthesis o r organization or
both.
106
Carbohydrate Chemistry
Nikaido’s
model of
t h e r o l e o f l i p o p o l y s a c c h a r i d e and p r o t e i n i n
determining the resistance o f Gram-negative b a c t e r i a o f lowm o l e c u l a r - w e i g h t h y d r o p h o b i c a n t i b i o t i c s was d i s c u s s e d i n r e l a t i o n t o t h e s t r e s s of freeze-thaw. The s p e c i f i c a n t i - l i p o p o l y s a c c h a r i d e s e r u m a n t i b o d y r e s p o n s e i n BALB/c
mice, b e f o r e and a f t e r an o r a l b o o s t e r w i t h d i f f e r e n t non-
pathogenic s t r a i n s o f Salmonella typhimurium, ELISA t e c h n i q u e . 8 7
was m e a s u r e d b y a n
Serum r e s p o n s e t o l i p o p o l y s a c c h a r i d e was
found
t o depend on t h e l e n g t h o f t h e l i p o p o l y s a c c h a r i d e p o l y s a c c h a r i d e moiety i n rough mutants.
T h e M206 s m o o t h m u t a n t i n d u c e d a l e s s
marked a n t i - l i p o p o l y s a c c h a r i d e response.
The i m m u n o g e n i c i t y o f t h e
p o l y s a c c h a r i d i c c o r e a p p e a r s t o be m o d i f i e d by t h e 0 a n t i g e n .
The
s p e c i f i c r e s p o n s e i n t h e g u t o n l y becomes marked f o l l o w i n g a f t e r b o o s t e r o f Ra, Rc,and The 2 - h a p t e n i c reported
to
contain
However, 2 - h a p t e n products of
M206 m u t a n t s . p o l y s a c c h a r i d e o f S a l m o n e l l a m o n t e v i d e o h a s been glyceraldehyde
partial hydrolysis o f
separate experiments perchloric
acid
at
i t s
preparatlons from a
terminus.
lipopolysaccharide,
gave { 3 H ) g l y c e r o l
a n d {3H)NaBH4.
reducing
galE mutant contained
Further
which
upon t r e a t m e n t study
of
i n
with
the g-hapten
r e d u c i n g t e r m i n u s s u g g e s t e d t h a t i t was a c t u a l l y P-mannose.88 The c e l l - w a l l
polymers o f the thermophilic cyanobacterium
S y n e c h o c o c c u s PCC6716 h a v e b e e n i s o l a t e d a n d a n a l y s e d , t h e m a j o r compounds o f t h e t h e r m o p h i l i c s t r a i n b e i n g s i m i l a r b u t n o t i d e n t i c a l t o those o f the mesophilic strains.89
The l i p o p o l y s a c c h a r i d e was
f o u n d t o c o n t a i n B - h y d r o x y p a l m i t i c a c i d as t h e o n l y h y d r o x y l a t e d f a t t y acid together
with myristic,
p a l m i t i c , and p a l m i t o l e i c a c i d s .
The c a r b o h y d r a t e s p r e s e n t w e r e Q - g l u c o s e , I - r h a m n o s e , 2 - a m i n o - 2 deoxy-a-glucose,and w i t h traces of
an u n i d e n t i f i e d n i n h y d r i n - p o s i t i v e component
Q-mannose,
Q-galactose,
H e p t o s e s and 3 - d e o x y - a - m a n n o - 2 - o c t u l o s o n i c
L-fucose,
and P - x y l o s e .
a c i d w e r e f o u n d n o t t o be
present. The
ability
of
various
bacterial
l i p o p o l y s a c c h a r i d e s and
mycoplasmal l i p o p o l y s a c c h a r i d e s ( l i p o g l y c a n s ) t o i n d u c e macrophagemediated t u m o u r - c e l l was d e t e r m i n e d . 9 0
a-x a n t u m less
o r A c h o l e p l a s m a q r a n u l a r u m had no a c t i v i t y o r
activity
0128:812,
k i l l i n g and L i m u l u s a m e b o c y t e l y s a t e c l o t t i n g L i p o g l y c a n s f r o m t h e mycoplasma Acholeplasma
than
lipopolysaccharides
from
lo4
to
Escherichia
lo5 coli
E s c h e r i c h i a c o l i K235, o r S a l m o n e l l a m i n n e s o t a R595 i n
c a u s i n g L i m u l u s l y s a t e c l o t t i n g and t u m o u r - c e l l
k i l l i n g by
p e r i t o n e a l macrophages f r o m n o r m a l or b a c i l l u s C a l m e t t e - G u e r i n -
4: Microbial Polysaccharides
107
i n f e c t e d mice. Previous s t u d i e s have s h o w n t h a t t h e l i p i d A p o r t i o n o f b a c t e r i a l l i p o p o l y s a c c h a r i d e i s r e s p o n s i b l e f o r t h e e f f e c t s on macrophage-mediated tumour-cell k i l l i n g and L i m u l u s l y s a t e c l o t t i n g . The k n o w n d i f f e r e n c e s i n t h e l i p i d s t r u c t u r e s o f b a c t e r i a l l i p o p o l y s a c c h a r i d e s and mycoplasmal lipopolysaccharides ( l i p o g l y c a n s ) may account f o r t h e noted d i f f e r e n c e s i n t h e b i o l o g i c p o t e n c i e s observed.
4
Capsular Polysaccharfdes
The i n t e r a c t i o n o f l e c t i n s and l e c t i n - l i k e s u b s t a n c e s w i t h b a c t e r i a h a s been r e v i e w e d 9 ' and t h e i n t e r a c t i o n d i s c u s s e d a s a means o f d i s t i n g u i s h i n g between m i c r o b i a l s p e c i e s , i d e n t i f y i n g t h e p r e s e n c e of a p a r t i c u l a r s u g a r r e s i d u e and f r a c t i o n a t i n g t h e po 1y s a c c h a r i des from b a c t e r i a . 91 S o l u b l e a n t i g e n s were o b t a i n e d by e x t r a c t i n g f i v e s e r o t y p e s t r a i n s o f C l o s t r i d i u m p e r f r i n g e n s t y p e A w i t h w a t e r a t 100°C.92 The t y p e - s p e c i f i c p o l y s a c c h a r i d e s w e r e p r e c i p i t a t e d w i t h e t h a n o l , and t h e common a n t i g e n s were recovered from t h e e t h a n o l s u p e r n a t a n t s b y c o n c e n t r a t i o n , d i a l y s i s , and l y o p h i l i z a t i o n . Refluxing the w a t e r - e x t r a c t e d c e l l r e s i d u e s w i t h 1 %a c e t i c a c i d f o l l o w e d b y c o n c e n t r a t i o n , d i a l y s i s , and l y p o p h i l i z a t i o n gave a d d i t i o n a l common antigen fractions. A comprehensive, s i d e - b y - s i d e comparison of t h e antigen fractions, the ethanol precipitate, the ethanol supernatant, and t h e a c e t i c a c i d s u p e r n a t a n t , r e v e a l e d t h a t common a n t i g e n s were r e c o v e r e d i n a l l t h r e e f r a c t i o n s and t h a t t h r e e d i s t i n c t e n t i t i e s w e r e r e s p o n s i b l e f o r t h e f o r m a t i o n o f t h e o b s e r v e d common immunoprecipitin l i n e s . Whereas many f r a c t i o n s possessed a l l t h r e e i m m u n o p r e c i p i t i n l i n e s , o t h e r s c o n t a i n e d o n l y one o r t w o . The s e r o l o g i c a l h o m o l o g y observed between t h e v a r i o u s a n t i g e n f r a c t i o n s was a p p a r e n t l y a consequence of 2 - a c e t a m i d o - 2 - d e o x y - ~ - g l u c o s e and 2acetamido-2-deoxy-P-mannose c o n t a i n i n g polymers. The common a n t i g e n s were presumably a s s o c i a t e d w i t h t h e c e l l envelope and may be t h e t y p e o f m a r k e r s s o u g h t p r e v i o u s l y b y o t h e r s f o r t h e serological identification of C l o s t r i d i u m perfringens. The bacterium formerly named Fusobacterium p o l y s a c c h a r o l y t i c u m and now r e c l a s s i f i e d a s a C l o s t r i d i u m s p e c i e s h a s been s h o w n t o produce s p o r e s and have a c e l l - w a l l s t r u c t u r e o f t h e Gram-positive type d e s i t e i t s appearance a s Gram-negative when s t a i n e d . 9 3 The c a p s u l a r p o l y s a c c h a r i d e s f r o m C r y t o c o c c u s n e o f o r m a n s
108
Carbohydrate Chemistry
s e r o t y p e A and from a p o s s i b l e , mutant s t r a i n t h e r e o f have been s t r u c t u r a l l y a n a l y ~ e d . ~T h~e l a t t e r m a t e r i a l i s s i m i l a r t o t h e c a p s u l a r a n t i g e n o f C r y t o c o c c u s n e o f o r m a n s s e r o t y p e D. Epidemiological and immunological evidence i n d i c a t e s t h a t t h e K 1 c a p s u l a r p o l y s a c c h a r i d e c o n f e r s the p r o p e r t y o f v i r u l e n c e on Escherichia coli. One a p p r o a c h t o s t u d y i n g w h e t h e r t h e K 1 a n t i g e n is s u f f i c i e n t t o confer virulence or i f other Escherichia c o l i s t r u c t u r e s are necessary i s t o i s o l a t e t h e K 1 genes f o r g e n e t i c and biochemical analysis. R e c o m b i n a n t DNA m e t h o d o l o g y p r o v i d e d a p o w e r f u l t o o l f o r s u c h an a p p r o a c h . The m o l e c u l a r c l o n i n g o f t h e E s c h e r i c h i a c o l i K 1 a n t i g e n genes h a s been reported.95 The c l o n e d K 1 g e n e s s y n t h e s i z e d a c a p s u l e i n Escherjchja c o l i K12 i n d i s t i n g u i s h a b l e c h e m i c a l l y and i m m u n o l o g i c a l l y from t h a t o f w i l d type K 1 strains. T h e c a p s u l a r p o l y s a c c h a r i d e was i s o l a t e d f r o m E s c h e r i c h i a c o l i 010:K5:H4: i t c o u l d n o t be o b t a i n e d f r o m a n u n c a p s u l a t e d ( K 5 - ) m u t a n t . I t c o n t a i n s 2 - a c e tam i do - 2 - d e o x y - Q - g l u c o se a n d E - g l u c u r o n i c a c i d i n a m o l a r r a t i o o f 1:1.96 A c i d h y d r o l y s i s o f t h e a c i d i c p o l y s a c c h a r i d e a s w e l l a s S m i t h d e g r a d a t i o n a n d d e g r a d a t i o n by deamination of the carboxyl-reduced p o l y s a c c h a r i d e s u g g e s t e d t h a t t h e p o l y s a c c h a r i d e is composed o f a d i s a c c h a r i d e r e p e a t i n g u n i t . T h e d a t a o b t a i n e d by m e t h y l a t i o n a n a l y s i s a n d n.m.r. s p e c t r o s c o p y indicated t h a t the repeating sequence of the capsular polysaccharide i s similar t o t h a t o f desulpho-heparin w i t h the s t r u c t u r e (21). + 4 ) -B-Q-GlcQA-( 1+4) - a - Q - G l C Q N A C - (
1+
(21) T h e l i n k a g e p a t t e r n o f t h e K6 a n t i g e n was i n v e s t i g a t e d u s i n g m a t e r i a l f r o m t h e u r i n a r y p a t h o g e n E s c h e r i c h i a c o l i LP 1092.97 The p o l y s a c c h a r i d e c o n s i s t s o f 9 - r i b o s e a n d 3-deoxy-Q-manno-2Colorimetric procedures, Smith o c t u l o s o n a t e i n a r a t i o o f 2:l. d e g r a d a t i o n , m e t h y l a t i o n a n a l y s i s , a n d n.m.r. s p e c t r o s c o p y were a p p l i e d t o t h e whole polysaccharide and t o a t r i s a c c h a r i d e r e p e a t i n g u n i t o b t a i n e d by m i l d - a c i d - c a t a l y s e d h y d r o l y s i s . T o g e t h e r , t h e d a t a were c o m p a t i b l e o n l y w i t h a b r a n c h e d - c h a i n s t r u c t u r e ( 2 2 ) . A method has been developed t o s e p a r a t e t h e c e l l e n v e l o p e o f e n c a p s u l a t e d ( t y p e b) Haemophilus i n f l u e n z a e i n t o i t s o u t e r - and inner-membrane components w i t h p r o c e d u r e s t h a t a v o i d e d two p r o b l e m s encountered i n f r a c t i o n a t i o n o f t h i s envelope: (i) t h e tendency o f t h e o u t e r and i n n e r membranes t o h y b r i d i z e and ( i i ) t h e tendency o f
4: Microbial Polysaccharides
109
the apparently
f r a g i l e i n n e r membrane t o f r a g m e n t i n t o d i f f i c u l t l y
sedimentable
units.98
Log
radioactively labelled, press.
phase
cells,
whose
lipids
were
were l y s e d by passage t h r o u g h a F r e n c h
The l y s a t e was a p p l i e d t o a d i s c o n t i n u o u s s u c r o s e g r a d i e n t ,
a n d e n v e l o p e - r i c h m a t e r i a l was c o l l e c t e d b y c e n t r i f u g a t i o n o n t o a c u s h i o n o f dense s u c r o s e under c a r e f u l l y c o n t r o l l e d c o n d i t i o n s . This
material
centrifugation
was i n
a
then
further
sucrose
fractionated
gradient
to
f r a c t i o n s w h i c h were p a r t i a l l y c h a r a c t e r i z e d . density,
by
yield
isopycnic
four
membrane
On t h e b a s i s o f t h e i r
radioactivity,
bouyant
composition,and
content o f phospholipid, protein, lipopolysaccharide,
and s u c c i n i c dehydrogenase, follows:
fraction 1
inner-membrane vesicles
these f r a c t i o n s
outer-membrane
were
entrapped
inner
f r a c t i o n o f i n n e r membrane,
p o o r f r a c t i o n o f i n n e r membrane.
polypeptide i d e n t i f i e d as
vesicles with very l i t t l e
c o n t a m i n a t i o n (<4%), f r a c t i o n 2
containing
protein-rich
-
ultrastructure,
-
membrane,
outer-membrane fraction
3
-
a
and f r a c t i o n 4 - a p r o t e i n -
F r a c t i o n s 3 and 4 c o n t a i n e d a b o u t
25% outer-mem b r ane c o n t a m i n a t i o n .
i 1
6-P-R ibf where KDO = 3 - d e o x y - B - ~ - m a n n o - 2 - o c t u l o p y r a n o s o n i c a c i d
(22) The s t r u c t u r e o f
t h e Haemophilus influenzae type e capsular
p o l y s a c c h a r i d e h a s been d e t e r m i n e d by a c o m b i n a t i o n o f c h e m i c a l and s p e c t r o s c o p i c methods.’’
The s t r u c t u r e o f t h e r e p e a t i n g u n i t o f t h e
p o l y m e r was f o u n d t o be l i n e a r ( 2 3 ) .
+3)-B-~-Glc~NAc-(1+4)-8-~-ManpANAc-(l+ (23) The s t r u c t u r e s o f t w o c a p s u l a r p o l y s a c c h a r i d e s e l a b o r a t e d by H a e m o e----hilus ----respectively, n.m.r.
influenzae type ---------_
e,
strains
have been i n v e s t i g a t e d ,
NCTC
8 4 5 5 and 8 4 7 2 ,
m e t h y l a t i o n a n a l y s i s and
s p e c t r o m e t r y b e i n g t h e p r i n c i p a l m e t h o d s used.100
concluded t h a t
the polysaccharides
h a v i n g t h e s t r u c t u r e (24).
a r e composed o f
It i s
repeating units
I n t h e p o l y s a c c h a r i d e from
s t r a i n NCTC
Carbohydrate Chemistry
110
8472, a l l o f t h e r e p e a t i n g u n i t s c o n t a i n t h e 8 - Q - f r u c t o f u r a n o s y l group.
The p o l y s a c c h a r i d e f r o m s t r a i n NCTC 8455,
only traces o f a-fructose,
however,
contains
c o r r e s p o n d i n g t o a p p r o x i m a t e l y one g r o u p
p e r 100 r e p e a t i n g u n i t s .
+3)-B-Q-GlceNAc-(+4)-B-B-ManpANAc-(
1+
3
I
2 B-Q-Fruf (24) T h e c a p s u l a r p o l y s a c c h a r i d e f r o m K l e b s i e l l a K4 c o n t a i n s t h e t e t r a s a c c h a r i d e r e p e a t i n g sequence (2 5 ). t h e measurement of
'H
n. m . r .
s p e c t r o s c o p y and
o p t i c a l r o t a t i o n were used t o e s t a b l i s h t h e
a n o m e r i c l i n k a g e s i n t h e p o l y s a c c h a r i d e and i n t h e o l i g o s a c c h a r i d e s The r e p e a t i n g u n i t a l s o c o n t a i n s
d e r i v e d by p a r t i a l h y d r o l y s i s . l o 1 one g - a c e t y l
group.
+ 3 ) - a - p -- G l c e - (
1+2) -a-e-GlceA-
( 1 + 3 ) - a - Q - M a n ~ - ( 1+3) -B-Q-Glcp-(
1+
(25)
A
glycanase
---------Klebsiella
activity
associated
bacteriophage
g l u c o p y r a n o s y l - ( 1+3)-4,6-0-(
No.6
with
catalyses
the
particles o f
cleavage
o f
0-13-Q-
l-carboxyethy1idene)-B-Q-mannopyranose
linkages i n K l e b s i e l l a serotype 6 capsular polysaccharide.lo2
O f 74
h e t e r o l o g o u s K l e b s i e l l a p o l y s a c c h a r i d e s and t w o d e r i v a t i v e s o f type 6
glycan,
only
the
t y p e 1 and
type 57
a d d i t i o n a l l y d e g r a d e d by t h e phage 6 enzyme.
polymers
the
were
The r e p e a t i n g u n i t s i n
t h e t h r e e s u b s t r a t e s h a v e a 1 ~ + 3 ~ , l e c ~ + ~ - l i n kc he adi n Q - g l u c o - or Q-galacto-pyranosyl
residue
i n
common
(which
constitutes
the
r e d u c i n g end a f t e r g l y c a n a s e a c t i o n ) , and a c a r b o x y l group o n t h e next hexopyranosyl residue.
O f t h e 72 p o l y s a c c h a r i d e s n o t a f f e c t e d
b y t h e v i r a l e n z y m e , a t l e a s t t h e t y p e 11 a n d t y p e 2 1 g l y c a n s a l s o c o n t a i n t h e same h o m o l o g y o f p r i m a r y s t r u c t u r e .
This indicates t h a t
t h e conformation a t t h e glycanase r e c o g n i t i o n s i t e a l s o c o n s t i t u t e s an i m p o r t a n t f e a t u r e o f t h e s u b s t r a t e s .
A B - -Q - g a l a c t o s i d a s e a s s o c i a t e d w i t h b a c t e r i o p h a g e 4 1 8 h a s b e e n
used t o depolymerize t h e capsular
polysaccharide o f Klebsiella
s e r o t y p e K18 i n t o t h e h e x a s a c c h a r i d e c o r r e s p o n d i n g t o one r e p e a t i n g u n i t o f the polymer.lo3
The n.m.r.
s p e c t r a o f t h e polymer and o f
4: Microbial Polysaccharides
111
t h i s o l i g o s a c c h a r i d e a r e comparable,
leading t o the conclusion t h a t
the
the
conformations
i n
solution
of
repeating
unit
of
these
substances a r e s i m i l a r . The s t r u c t u r e o f t h e c a p s u l a r p o l y s a c c h a r i d e o f K l e b s i e l l a K26 has
been
determined
by
oxidation,
p a r t i a l hydrolysis,
periodate N.m.r.
the
techniques
of
methylation,
and f 3 - e l i m i n a t i 0 n . l ~ ~
s p e c t r o s c o p y ( I H and 13C) was u s e d t o e s t a b l i s h t h e n a t u r e o f
t h e anomeric the
using
different
l i n k a g e s and t o i d e n t i f y o l i g o s a c c h a r i d e s o b t a i n e d by degradative
t e c h n i q u e s employed.
The p o l y s a c c h a r i d e
i s comprised o f r e p e a t i n g u n i t s o f t h e heptasaccharide ( 2 6 ) .
1 CL -Q -G1 C e
6
I
1
B -p -
CQ
i 1
B-Q-Gale
The
capsular
(Enterobacter Q-galactose,
polysaccharide
aerogenes) a-glucose,
was
found t o
I-rhamnose,
from
Klebsiella
type
73
c o n t a i n e q u i m o l a r amounts o f
and 0 - g l u c u r o n i c
h y d r o l y s i s o f t h e p o l y s a c c h a r i d e gave one a l d o b i o -
acid.lo5
Acid
and a l d o t r i o -
u r o n i c a c i d , w h o s e s t r u c t u r e s w e r e e s t a b l i s h e d by a c i d h y d r o l y s i s a n d by m e t h y l a t i o n a n a l y s i s .
The a n o m e r i c c o n f i g u r a t i o n s o f t h e
d i f f e r e n t sugar r e s i d u e s were d e t e r m i n e d from s p e c i f i c r o t a t i o n s o f t h e p o l y s a c c h a r i d e and t h e a l d o b i o - a n d a l d o t r i o - u r o n i c a c i d s , and also
by
oxidation
of
the
native
p o l y s a c c h a r i d e w i t h chromium t r i o x i d e . polysaccharide
provided information
and
the
carboxyl-reduced
Methylation a n a l y s i s o f the about
the
linkages
of
the
112
Carbohydrate Chemistry
different
sugar
structure
(27)
residues. was
Based on a l l o f
assigned
t o
the
these r e s u l t s ,
repeating unit
of
the the
polysaccharide. The v a c c i n a t i n g p o w e r o f a p o l y s a c c h a r i d e p r e p a r a t i o n f r o m K l e b s i e l l a pneumoniae t y p e I has been r e p o r t e d . l o 6
The e s s e n t i a l
p a r t p l a y e d by t h i s e x t r a c t i n t h e v a c c i n a t i n g c a p a c i t i e s o f t h e r i b o s o m a l p r e p a r a t i o n s f r o m t h e same b a c t e r i u m i s d e m o n s t r a t e d .
+3)-f3-~-Rha~-(1+4)-8-~-Galg-(l+4)-B-g-Glcg-(l+ 3
I
1
6 - q -G 1CQA (27)
Two p e a k s w e r e o b t a i n e d b y c a e s i u m c h l o r i d e d e n s i t y - g r a d i e n t ultracentrifugation
o f
K l e b s i e l l a pneumoniae
ribosomal
Peak I c o n t a i n e d c a p s u l a r p o l y s a c c h a r i d e ,
preparations.lo7
l i p o p o l y s a c c h a r i d e , p r o t e i n , a n d l e s s t h a n 0.5% r i b o n u c l e i c a c i d . Peak I 1 c o n s i s t e d m a i n l y o f r i b o n u c l e i c a c i d , w i t h l o w a m o u n t s o f p r o t e i n and c a p s u l a r p o l y s a c c h a r i d e . polysaccharide content,
Expressed as
capsular
t h e 50% p r o t e c t i v e dose o f peak I and o f
n o n - f r a c t i o n a t e d r i b o s o m a l p r e p a r a t i o n s was n e a r l y c o n s t a n t ( 2 . 6 1.2
ng,
respectively).
ribonucleic acid, ribonucleic
Since
peak
and
I c o n t a i n e d l e s s t h a n 0.5%
these r e s u l t s provide evidence t h a t ribosomal
acid i s not
required for
protection of
m i c e by
K l e b s i e l l a pneumoniae c a p s u l a r p o l y s a c c h a r i d e , w h i c h c o n t a m i n a t e s ribosomal preparations. An a n t i t u m o u r
c a r b o x y m e t h y l a t e d (1+3)-f3-;-glucan
was
found t o
h a v e a p o t e n t a b i l i t y t o cause g e l a t i o n o f t h e a m o e b o c y t e l y s a t e o f h o r s e s h o e c r a b ( L i m u l u s ) a t c o n c e n t r a t i o n s as l o w as
mg/ml.lo8
The g e l a t i o n o f t h e l y s a t e a n d t h e a c t i v a t i o n o f t h e p r o c l o t t i n g enzyme i n t h e l y s a t e caused by t h i s P - g l u c a n w e r e u n i q u e i n t h a t these reactions occurred a t concentrations ranging from mg/ml.
The o p t i m u m c o n c e n t r a t i o n was
c o n c e n t r a t i o n s above 10'' pattern
was
also
polysaccharides.
mg/ml
to
no g e l a t i o n o c c u r r e d .
observed i n
common w i t h
to
mg/ml, other
and a t
This gelation antitumour
The mechanism o f t h e g e l a t i o n c a u s e d by g - g l u c a n
was r e v e a l e d t o b e d i s t i n c t l y d i f f e r e n t f r o m t h a t w o r k i n g i n t h e g e l a t i o n c a u s e d by e n d o t o x i n s . The p r o c l o t t i n g e n z y m e , o r f a c t o r 8, i s n o t s e n s i t i v e t o t h e
113
4: Microbial Polysaccharides c a r b o x y m e t h y l a t e d (1+3)-B-Q-glucan
and t h e c o a g u l a t i o n pathway i n
Limulus
by
I _
amoebocytes
component
i s
(tentatively
activated
sensitization G)
named f a c t o r
by
of
a
third
the p-glucan,
which
m e d i a t e s t h e a c t i v a t i o n o f t h e p r o c l o t t i n g enzyme t o i t s a c t i v e form . l o 9 Serum s a m p l e s w e r e c o l l e c t e d f r o m 1 2 0 h e a l t h y a d u l t v o l u n t e e r s (105 Caucasians and 15 Negros) b e f o r e and a f t e r i m m u n i z a t i o n w i t h
meningococcal
polysaccharide
group
B
vaccine.
Antibodies
m e n i n g o c o c c a l g r o u p B were measured and s e r a w e r e t y p e d f o r Gm and Km(1)
a1lotypes.l''
between t h e Km(1)
A significant
to
several
a s s o c i a t i o n was f o u n d
a l l o t y p e and immune r e s p o n s e t o m e n i n g o c o c c a l
group B i n Caucasians. C o l o m i n i c a c i d and m e n i n g o c o c c a l g r o u p B p o l y s a c c h a r i d e ,
both
homopolymers o f 5-acetamido-3,5-deoxy-n-glycero-a-~-
(2+8)-linked
qalacto-2-nonulosonic
acid,
a r e made w a t e r - i n s o l u b l e
either
by
r e a c t i o n w i t h a c a r b o d i - i m i d e i n a q u e o u s s o l u t i o n a t pH 4.75 o r b y t r e a t m e n t w i t h 48% a q u e o u s h y d r o f l u o r i c a c i d a t O ° C f o r 48 h. 1 . r . s p e c t r a o f t h e p r o d u c t s show a m a j o r b a n d n e a r 1 7 5 0 c m - l , with ester
formation.
consistent
T h i s band v i r t u a l l y d i s a p p e a r e d a f t e r
a l k a l i treatment.'"
mild,
E s t e r i f i c a t i o n a l s o o c c u r r e d by i n c u b a t i n g t h e
native polysaccharides
b e l o w pH 6.0,
and t h e i r i.r.
s p e c t r a showed
t h a t t h e d e g r e e o f e s t e r i f i c a t i o n i n c r e a s e s a s t h e pH i s l o w e r e d . The r e l a t i v e l y l o w water-soluble
molecular
molecular ester formation. shows
that
weight
o f these p a r t i a l l y e s t e r i f i e d ,
polymers i s consistent w i t h i n t r a - rather than i n t e r Counter-current
esterification
immunoprecipitation.
13C
colominic acid provides
of
N.m.r.
strong
E.
9% i s
spectroscopy evidence
for
Meningococcal serogroup C polysaccharide,
intramolecular
fully
to
abolish
esterified between
an a d j o i n i n g r e s i d u e .
a (2+9)-linked
homopolymer
- d id e o x y -9 -9 1y c e r o - a-D_ - -9a 1a c t o - 2 -no n u 1o s o n ic
containing g-acetyl However,
of
cross-linking
t h e c a r b o x y l g r o u p o f one r e s i d u e a n d HO-9 o f o f 5 -a c e t a m id o - 3,5
immunoelectrophoresis
sufficient
g r o u p s a t C-7
and/or
e s t e r i f i c a t i o n after
upon g - d e a c e t y l a t i o n o f
C-8,
does n o t
carbodi-imide
the polysaccharide,
a c id
undergo
treatment.
esterification
occurs. Hydrolysis
E s c he r i c hia c o l i
of
the
meningococcal
groups
A,
B, a n d
C
and
K 9 2 p o l y s a c c h a r i d e s b y 60% a q u e o u s h y d r o f l u o r i c
a c i d l i b e r a t e d v a r i o u s 1 , 2 - d i a ~ y l g l y c e r o l s . ~ These ~~ were e x t r a c t e d w i t h chloroform,
trimethylsilylated,
1,2-diacylglycerols polysaccharide,
and a n a l y s e d by g.c.m.s.
were t h e major components i s o l a t e d .
80 t o
90% d i p a l m i t o y l
glycerol
and
10
Two
I n each to
20%
Carbohydrate Chemistry
114 d i s t e a r o y l g l y c e r o l were i d e n t i f i e d .
or mixed
No m o n o a c y l g l y c e r o l s
d i a c y l g l y c e r o l s were noted. The p r e s e n c e o f t h e h y d r o p h o b i c e n d c a u s e s t h e p o l y s a c c h a r i d e s t o a g g r e g a t e i n a m i c e l l a r f o r m and may b e t h e e n t i t y by w h i c h t h e p o l y s a c c h a r i d e r e m a i n s a t t a c h e d t o t h e outer
membrane
of
the
bacterium, g i v i n g
rise
to
the
structure
r e c o g n i z e d as a c a p s u l e . The
currently
-meningitidis
United States-licensed
group
C NeissefAg
v a c c i n e , composed o f t h e g - a c e t y l - p o s i t i v e
polysaccharide,
capsular
i s p o o r l y i m m u n o g e n i c and does n o t a f f o r d p r o t e c t i o n
f r o m d i s e a s e t o i n f a n t s and young c h i l d r e n . meningitidis 2-acetyl-negative t i t e r s i n adults irnmunogenicity
Group C
t h a n does t h e g - a c e t y l - p o s i t i v e
of
these
Neisseria
polysaccharide vaccine induces higher
vaccines
was
compared
vaccine. i n
The
2-year-old
~ h i 1 d r e n . l ' ~ R e a c t i o n s were m i n i m a l and d i d n o t d i f f e r b e t w e e n t h e two vaccines. greater
The p o s t v a c c i n a t i o n g e o m e t r i c mean t i t e r was t w o f o l d
i n the g-acetyl-negative
antibody per ml). b o t h groups.
group
(1.58
age
0.73
pg o f
F u r t h e r s t u d y r e g a r d i n g i m m u n o g e n i c i t y o f and t h e
anamnestic response t o t h e g - a c e t y l - n e g a t i v e the
versus
The r a t e s o f d e c l i n e i n t i t e r w e r e s i m i l a r i n
group
( ~ 1 8months)
at
v a c c i n e is w a r r a n t e d i n
highest
r i s k
for
invasive
meningococcal disease. Phase-contrast
and f l u o r e s c e n c e m i c r o s c o p y o b s e r v a t i o n s showed
t h a t pea s y m b i o n t R h i z o b i u m l e g u m i n o s a r u m a d s o r b e d t o p e a - r o o t hairs,
b u t non-symbiont
extent.l14
r h i z o b i a l s t r a i n s o n l y adsorbed t o a s m a l l
1 4 C - L a b e l l e d c e l l s were u s e d t o assay t h e number o f
r h i z o b i a l c e l l s adsorbed t o a pea r o o t . lipopolysaccharides obtained
from
Capsular p o l y s a c c h a r i d e s o r R h i z o b i g m h?gghu~osarum
s p e c i f i c a l l y i n h i b i t e d t h e a d s o r p t i o n o f 14C-Rhizobium l e g u m i n o s a r u m c e l l s t o a pea r o o t and s p e c i f i c a l l y adsorbed t o p e a - r o o t h a i r s . Also,
they
reacted specifically
with pea-seed
r e s u l t s suggest t h a t capsular polysaccharides
lectins.
These
or lipopolysaccharides
p l a y an i m p o r t a n t r o l e i n h o s t - s p e c i f i c a d s o r p t i o n .
The i n t e r a c t i o n
b e t w e e n t h e p o l y s a c c h a r i d e s and pea l e c t i n s c o u l d be t h e key t o determining h o s t s p e c i f i c i t y i n t h e i n f e c t i o n process o f Rhizobiumpea symbiosis. The
ultrastructural
locations
p o l y s a c c h a r i d e and t h e t y p e - s p e c i f i c
of
the
group-specific
polysaccharides Ia,
I b , 11, and I11 o f group B s t r e p t o c o c c i ( S t r e p t o c o c c u s a g a l a c t i a e ) were s t u d i e d
on i s o l a t e d w a l l s by d i r e c t i m m u n o f e r r i t i n t e c h n i q u e . ' l 5
The t y p e s
o f p o l y s a c c h a r i d e s w e r e l o c a t e d e x c l u s i v e l y on t h e o u t e r s i d e o f t h e w a l l on w h i c h t h e y f o r m e d a d i s t i n c t c a p s u l e .
Except f o r s t r a i n
115
4: Microbial Polysaccharides
58/59 ( t y p e I a ) t h e t h i c k n e s s o f t h e c a p s u l e was c h a r a c t e r i s t i c o f each s t r a i n i n v e s t i g a t e d . I n a l l strains the type-specific f e r r i t i n l a b e l l i n g was c o n f i n e d t o t h e o u t e r s u r f a c e o f t h e c a p s u l e . The g r o u p - s p e c i f i c p o l y s a c c h a r i d e c o u l d be d e m o n s t r a t e d o n t h e o u t e r s u r f a c e i n s t r a i n s 59/59
( t y p e I b ) a n d 8/66
m o s t o f t h e w a l l s o f s t r a i n 58/59
(group B v a r i a n t ) and on
(type Ia).
The f a i l u r e t o d e t e c t
t h i s a n t i g e n o n t h e o u t e r s i d e o f t h e w a l l s o f s t r a i n s 60/59 ( t y p e 11) and 13/63
( t y p e 1 1 1 ) a n d some w a l l s o f s t r a i n 58/59
due t o t h e t h i c k n e s s o f t h e t y p e o f The
release
o f
serotype
was p r o b a b l y
polysaccharide capsule. I11
group
B
streptococcal
p o l y s a c c h a r i d e s i n t o t h e s u p e r n a t a n t f l u i d was e x a m i n e d u n d e r a v a r i e t y of
physiological
low-molecular-weight
conditions.'16
throughout e x p o n e n t i a l growth, the stationary
phase o f
Release o f
I11 a n t i g e n s
type
b o t h high- and
was f a i r l y
constant
b u t i n c r e a s e d m a r k e d l y upon e n t e r i n g
growth.
I n c r e a s e d a - g l u c o s e and d e c r e a s e d
phosphate c o n c e n t r a t i o n s b o t h caused a l a r g e i n c r e a s e i n r e l e a s e o f antigens.
I n h i b i t i o n o f protein synthesis i n exponentially growing
c e l l s by c h l o r a m p h e n i c o l (10 p g / m l )
caused a c o n d i t i o n o f unbalanced
g r o w t h i n w h i c h a n t i g e n r e l e a s e was i n c r e a s e d g r e a t l y o v e r c o n t r o l values.
S t r a i n v a r i a b i l i t y i n a n t i g e n r e l e a s e was a l s o o b s e r v e d .
S t r a i n s w h i c h a r e known t o be h i g h n e u r a m i n i d a s e p r o d u c e r s r e l e a s e d elevated l e v e l s o f both lowa n t i ge ns.
and h i g h - m o l e c u l a r - w e i g h t
t y p e I11
No n- ne uram in i dase-p r o d u c i n g s t r a i n s r e l e a s e d co n s i d e r a b l y
l e s s high-molecular-weigh t antigen molecular-weight producers.
b u t s i m i l a r l e v e l s o f t h e low-
a n t i g e n compared w i t h t h e
high
neuraminidase
S t r a i n D136C, a t y p e 1 1 1 n o n - n e u r a m i n i d a s e p r o d u c e r ,
released n e g l i g i b l e q u a n t i t i e s o f the high-molecular-weight i n the supernatant f l u i d .
antigen
These r e s u l t s i n d i c a t e t h a t b o t h t h e
p h y s i o l o g i c a l e n v i r o n m e n t a n d t h e t y p e I11 s t r a i n a r e i m p o r t a n t i n determining the quantity o f type-specific
antigen released i n t o the
culture fluid. I m m u n i z a t i o n o f r a b b i t s w i t h g r o u p B t y p e I11 S t r e p t o c o c c u s organisms induces two d i s t i n c t populations o f antibodies with a specificity
for
antigen of
t h e s e organisms.'"
determinants of
the native capsular polysaccharide Some o f
the
s t r u c t u r a l and
conformational features o f the two determinants responsible f o r the f o r m a t i o n o f t h e s e a n t i b o d i e s were e l u c i d a t e d by 13C
n.m.r.
and
s e r o l o g i c a l s t u d i e s o n t h e n a t i v e t y p e 111 p o l y s a c c h a r i d e a n d some of i t s s t r u c t u r a l l y m o d i f i e d analogues. determinant
The s p e c i f i c i t y o f t h e
corresponding t o the major population o f antibodies is
depende n t o n t h e p r e se n c e o f 5 - a c e t am ido -3,5
- d i d e o x y -Q - g l y c e r o -a -
116
Carbohydrate Chemistry
qalacto-2-nonulosonic
a c i d r e s i d u e s on t h e n a t i v e t y p e 111 a n t i g e n ,
and,although
these residues are not an i n t e g r a l part o f
determinant,
they
c a r box y l g r o u p s o f 2-nonulosonic
exert
conformational
control
over
the
it.
The
- d i deox y -D-gl y c e r o - Q - g a l a c t o -
t h e 5-ace t am ido -3,5
a c i d r e s i d u e s a r e an i m p o r t a n t f a c t o r i n t h i s c o n t r o l
mechanism, w h i c h c o u l d p o s s i b l y i n v o l v e i n t r a m o l e c u l a r h y d r o g e n bo n d i n g
.
-
The t e r m ina 1 5 -ace t a m ido 3,5 - d i de o x y -Q -g 1y c e r o
2-nonulosonic penultimate
acid residues control
B-Q-galactopyranose
backbone o f t h e n a t i v e a n t i g e n . i s c r i t i c a l t o the determinant
- p -g a 1a c t o -
the orientation of
residues
with
respect
The o r i e n t a t i o n o f
the
to
the
these residues
because t h e d e t e r m i n a n t i s p r o b a b l y
s m a l l a n d i s l o c a t e d p r e c i s e l y a t t h e j u n c t i o n o f t h e same
B-Q-
galactopyranose r e s i d u e s w i t h t h e backbone o f t h e n a t i v e t y p e I11 antigen.
The d e t e r m i n a n t
corresponding t o the other population o f
antibodies i s n o t 5-acetamido-3,5-dideoxy-P-glycero-a-galacto-2n o n u l s o n i c a c i d dependent.
This determinant i s located on the
backbone o f t h e n a t i v e a n t i g e n i n t h e v i c i n i t y o f determinant branches.
but
on
the
opposite
I n t h i s position,
side
to
the
the other
oligosaccharide
i t s c o n f o r m a t i o n i s u n a f f e c t e d e v e n by
t h e removal o f t h e o l i g o s a c c h a r i d e branches from t h e n a t i v e antigen.
I t has been d e m o n s t r a t e d t h a t mouse I g G 3 a n t i b o d i e s a r e h i g h l y p r o t e c t i v e against i n f e c t i o n w i t h Streptococcus pneumoniae.ll8 The s t r u c t u r e o f t h e c a p s u l a r p o l y s a c c h a r i d e f r o m S t r e p t o c o c c u s pneumoniae
type
4
has
been
i n v e ~ t i g a t e d . " ~
spectroscopy, m e t h y l a t i o n analysis,
p r i n c i p a l methods o f s t r u c t u r a l i n v e s t i g a t i o n , the p o l y s a c c h a r i d e i s composed o f
Using
n.m.r.
and Smith degradation as t h e i t i s concluded t h a t
tetrasaccharide repeating units
h a v i n g t h e s t r u c t u r e (26). + 4 ) - B -Q-ManpNA c - ( 1+3 ) -a-C. -FuceNA c - ( 1+3)
-
a-Q-GaleNAc-( 1+4) - a - Q - G a l p - ( 1+ 2
3
\I /"\
Me
C02H
The c h e m i c a l c o m p o s i t i o n a n d i m m u n o c h e m i c a l c h a r a c t e r i z a t i o n o f t h e f o ur c r o s s - r e a c t i o n pneumo co c c a l c a p s u l a r p o l y s a c c h a r i d e s w i t h i n g r o u p 9 ( t y p e s 9N,
9A,
9L,
and 9V) h a v e been i n v e s t i g a t e d . l Z 0
Their
s e r o l o g i c a l r e a c t i o n s w e r e s t u d i e d by u s i n g u n a b s o r b e d a n t i s e r a
117
4: Microbial Polvsaccharides
p r e p a r e d by i m m u n i z i n g r a b b i t s w i t h p n e u m o c o c c i o f e a c h o f t h e f o u r group 9 capsular polysaccharide types. most extensive cross-reactions polysaccharides. r e m o v e d 63,
96,
A b s o r p t i o n w i t h t y p e 9N, 9L, o r 9V p o l y s a c c h a r i d e o r 87%, r e s p e c t i v e l y , o f t h e h e t e r o l o g o u s - a n t i b o d i e s
f r o m t h e t y p e 9A a n t i s e r u m . contained n-glucose, acid.
Type 9A a n t i s e r u m showed t h e w i t h the four group 9
A l l f o u r of t h e group 9 polysaccharides and Q - -glucuronic
2-acetamido-2-deoxy-Q-mannose,
I n addition,
t y p e s 9N a n d 9 L h a d 2 - a c e t a m i d o - 2 - d e o x y - Q -
g l u c o s e , a n d t y p e s 9A, 9L,and
9V c o n t a i n e d Q - g a l a c t o s e .
Reduction
o f t h e u r o n i c a c i d r e s i d u e s o f t h e t y p e 9 p o l y s a c c h a r i d e s removed most
of
their
reactivities,
homologous
and
much
of
their
i n d i c a t i n g an i m p o r t a n t r o l e f o r
component o f t h e i r a n t i g e n i c i t y . p r e p a r a t i o n s had comparable p r o t e i n and n u c l e i c a c i d .
heterologous
the uronic acid
The f o u r g r o u p 9 p o l y s a c c h a r i d e
m o l e c u l a r s i z e s and o n l y t r a c e s o f F u r t h e r s t u d i e s t o e v a l u a t e t h e most
p r o t e c t i v e t y p e among t h e g r o u p 9 s t r a i n s t o be i n c l u d e d i n t h e c u r r e n t p n e u m o c o c c a l v a c c i n e h a v e been d i s c u s s e d . Mild acid hydrolysis o f Pneumococcus t y p e I X (S
1x1
the
capsular
polysaccharide
of
l i b e r a t e d a number o f o l i g o s a c c h a r i d e s
w h i c h w e r e s e p a r a t e d i n t o b a s i c , a c i d i c , and n e u t r a l f r a c t i o n s u s i n g ion-exchange separated
column chromatography.121
into i t s
electrophoresis.
constituent
Each f r a c t i o n was f u r t h e r
oligosaccharides
using high-voltage
The i n d i v i d u a l o l i g o s a c c h a r i d e s were s u b j e c t e d t o
methylation studies.
When u r o n i c a c i d was p r e s e n t
the
methylated
o l i g o m e r was r e d u c e d a n d t h e m e t h y l s u g a r s i n i t w e r e c h a r a c t e r i z e d . Based on t h e s e s t u d i e s s t r u c t u r e s were a s s i g n e d t o t h e o l i g o m e r s . The
five
oligosaccharides characterized
support the revised
s t r u c t u r e assigned t o t h e nonasaccharide r e p e a t i n g u n i t o f polysaccharide
(29)
obtained
by
periodate
oxidation
degradation linked t o methylation analysis.122
type
IX
and S m i t h
Chromium t r i o x i d e
o x i d a t i o n i n d i c a t e d t h a t a l l g l y c o s i d i c l i n k a g e s were o f t h e a-Qtype.
The c a p s u l a r p o l y s a c c h a r i d e f r o m S t r e p t o c o c c u s p n e u m o n i a e t y p e 12F i s composed o f a - g l u c o s y l ,
Q-galactosyl,
2-acetamido-2-deoxy-Q-
g a l a c t o s y l , 2-acetamido-2,6-dideoxy-~-galactosyl,
and 2-acetamido-2-
118
Carbohydrate Chemistry
deoxy-Q --mannopyranosyluronic
acid
residues
i n the
proportions
2:l:l:l.123 The m a i n s t r u c t u r a l e v i d e n c e was a d d u c e d f r o m n.m.r. spectroscopy, methylation analysis, and s p e c i f i c d e g r a d a t i o n s
whereby i t c o u l d be c o n c l u d e d t h a t t h e p o l y s a c c h a r i d e i s composed o f hexasaccharide r e p e a t i n g u n i t s h a v i n g t h e s t r u c t u r e (30). The t y p e X X X I I I S t r e p t o c o c c u s p n e u m o n i a e capsular polysaccharide contains mol),
n-glucose
(pneumococcal)
(2 mol),
Q-galactose
(1
p - g l u c u r o n i c a c i d (1 mol), and 2 - a c e t a m i d o - 2 - d e o x y - ~ - m a n n o s e (1
m o l ) .i24
Structural
i n v e s t i g a t i o n s by m e t h y l a t i o n a n a l y s i s ,
s p e c i f i c degradations,
a n d n.m.r.
spectroscopy
showed t h a t
the
r e p e a t i n g u n i t was a l i n e a r p e n t a s a c c h a r i d e ( 3 1 ) .
+4) -a-L-FuceNAc-(
1+4) -B-g-ManpANAc-( 1+3) -B-Q-GaleNAc-( -
+
3
3
I
I 1
1
a -p - G a l e
a-g-Glce-( 1+2) -a-Q-Glce (30)
+3) -a
-g -Ga lp- ( 1+3 -B B -g-Glce-
-a -MangNA c - ( 1+4)-a -Q -Glee- ( 1+4 ) ( 1+4) -a -g-GlceA- ( 1+
(31) The
specific
c a p s u l a r p o l y s a c c h a r i d e o f t y p e 45 S t r e p t o c o c c u s
pneumoniae was i s o l a t e d i n p u r e f o r m by c h e m i c a l and c h r o m a t o g r a p h i c m e t h o d s a n d f o u n d t o be a h i g h - m o l e c u l a r - w e i g h t ,
glycosidically
l i n k e d p o l y m e r o f a h e x a s a c c h a r i d e r e p e a t i n g u n i t composed o f galactose ( 2 mol),
C-rhamnose
(1 rnol),
(1 m o l ) , 2 - a c e t a m i d o - 2 - d e o x y - g - g a l a c t o s e deoxy-l-fucose
1-
2-acetamido-2-deoxy-~-glucose (1 r n o l ) ,
2-acetamido-2-
(1 mol), and p h o s p h a t e ( 1 m o l ) .12’
The s p e c i f i c c a p s u l a r
p o l y s a c c h a r i d e of
t y p e 46 S t r e p t o c o c c u s
p n e u m o n i a e ( A m e r i c a n t y p e 7 3 ) was i s o l a t e d i n p u r e f o r m and shown t o be a h i g h - m o l e c u l a r - w e i g h t , o f a-galactose
g l y c o s i d i c a l l y l i n k e d p o l y m e r composed
( 2 mol), 2-acetamido-2-deoxy-Q-glucose
(1 m o l ) ,
2-
a c e t a m i d o - 2 - d e o x y - ~ - g a l a c t o s e ( 1 rnol), a n d 2 - a c e t a m i d o - 2 - d e o x y - h -f u c o s e (1 mo1).126 The s p e c i f i c c a p s u l a r p o l y s a c c h a r i d e p r o d u c e d by S t r e p t o c o c c u s p n e u m o n i a e t y p e 9V ( A m e r i c a n t y p e 6 8 ) i s c o m p o s e d o f Q - g l u c u r o n i c a c i d (1 p a r t ) , (1 p a r t ) ,
P-galactose
Q-glucose
Methylation analysis,
(1 p a r t ) ,
(2 parts),
2-acetamido-2-deoxy-~-mannose
and g - a c e t y l
periodate oxidation,
(1.6
parts).l*’
optical rotation,
n.m.r.
4: Microbial Polysaccharides spectroscopy,
119
and p a r t i a l h y d r o l y s i s showed t h a t t h e p o l y s a c c h a r i d e
is an u n b r a n c h e d h i g h - m o l e c u l a r - w e i g h t
l i n e a r polymer of a p a r t i a l l y pentasaccharide repeating u n i t having the s t r u c t u r e
O-acetylated (32).
Extracellular and Intracellular Polysaccharides
5
An e x t r a c e l l u l a r ,
a c i d i c p o l y s a c c h a r i d e has been i s o l a t e d f r o m
t h e c u l t u r e medium o f a spontaneous c e l l u l o s e - n e g a t i v e s t r a i n o f A c e t o b a c t e r xylium.128
C h e m i c a l a n a l y s i s shows t h a t t h e p o l y m e r i s
composed o f
g-mannose,
i n a
n-glucose,
molar
presence
ratio
of
approximating
cellobiose
L-rhamnose, and a - g l u r u r o n i c 3:l:l:l.
units
as
No
evidence
structural
parts
acid
for
the
i n
the
p o l y s a c c h a r i d e h a s been found. An o r g a n i s m p r o d u c i n g e x t r a c e l l u l a r p o l y s a c c h a r i d e was i s o l a t e d from
s o i l and
Stanier.129
i d e n t i f i e d as Aeromonas h y d r o p h i l a (Chester)
The
e f f e c t s o f medium
components
and c u l t u r a l
c o n d i t i o n s on p r o d u c t i o n o f t h e polysaccharide were studied.
The
o p t i m a l c o n c e n t r a t i o n s o f c a r b o n and n i t r o g e n s o u r c e s w e r e 5% and 0.3%,
respectively,
for production o f
o p t i m a l i n i t i a l pH was 7 t o 9.
o b t a i n e d a t 4 t o 8 days f e r m e n t a t i o n . c a r b o n source, i n the
7:3
P-fructose,
From s u c r o s e a n d r a f f i n o s e as
and 4:6, r e s p e c t i v e l y . g-mannose,
p o l y s a c c h a r i d e was p r o d u c e d . c o n t a i n P-galactose, 5:4:2.
The
t h e o r g a n i s m p r o d u c e d l e v a n and a c i d i c p o l y s a c c h a r i d e
ratio of
galactose,
the polysaccharide.
The maximum p o l y s a c c h a r i d e y i e l d was
From Q-glucose,
a-
m a l t o s e , and l a c t o s e m a i n l y a c i d i c
The a c i d i c p o l y s a c c h a r i d e was f o u n d t o
a-mannose, and a - g l u c u r o n i c
acid i n a ratio o f
The a c i d i c p o l y s a c c h a r i d e s o b t a i n e d f r o m s u c r o s e a n d l a c t o s e
seemed t o be t h e same p o l y s a c c h a r i d e . C u r d l a n g e l s h r i n k s even i n w a t e r and causes s y n e r e s i s .
The
s y n e r e s i s is r e p r e s s e d a l m o s t c o m p l e t e l y by t h e a d d i t i o n o f s t a r c h t o curdlan before heating,
b u t n o t r e p r e s s e d by t h e a d d i t i o n o f
d i f f e r e n t s u g a r compounds e v e n o f 10% c o n c e n t r a t i o n .
The r o l e o f
s t a r c h i n r e p r e s s i n g s y n e r e s i s h a s been d i s c u s s e d . 1 3 0 During growth i n l i q u i d culture containing a single c e l l u l o s i c
120
or
Carbohydrate Chemistry non-cellulosic
carbon
source,
a
newly
isolated
strain
A s p e r g i l l u s f u m i g a t u s r e l e a s e d c e l l u l a s e s i n t o t h e medium. amounts produced depended on t h e n i t r o g e n s o u r c e , concentration of Extracellular incubation for
the
carbon source,
cellulolytic activity 6 0 d w i t h 1% ( w / v )
n i t r o g e n source,and
of The
t h e t y p e and
pH, a n d t e m p e r a t u r e . 1 3 1
was s t i l l i n c r e a s i n g a f t e r
CFll cellulose,
a s t a r t i n g pH o f 7.
(NH4)2S04
as
The a c t i v i t i e s o f t h e new
i s o l a t e were compared w i t h t h o s e o f A s p e r q i l l u s f u m i g a t u s I M I 143864 a n d T r i c h o d e r r n a r e e s i QM6a ( A T C C 1 3 6 3 1 1 , a n d i t was s h o w n t o b e a good p r o d u c e r o f B - Q - g l u c o s i d a s e . The a b i l i t y o f B a c t e r o i d e s t h e t a i o t a o m i c r o n ,
an o b l i g a t e a n a e r -
obe f r o m human c o l o n i c m i c r o f l o r a , t o grow i n a c a r b o h y d r a t e - l i m i t e d c o n t i n u o u s c u l t u r e a t g e n e r a t i o n t i m e s r a n g i n g f r o m 3.5 d i v i s i o n was i n v e s t i g a t e d . 1 3 *
glucose, 2-acetAmido-2-deoxy-Q-glucose, amino-2-deoxy-~-glucose.
t o 28 h p e r
F o u r c a r b o h y d r a t e s were t e s t e d :
A t a g e n e r a t i o n t i m e o f 3.5
h per d i v i s -
i o n , t h e g r o w t h y i e l d s f o r b a c t e r i a g r o w i n g on !-glucose,
amido-2-deoxy-~-glucose,
and g - g l u c u r o n i c
a c i d w e r e 77,
2-acet-
68, and 50 g
o f c e l l s (dry weight) per mol of substrate, r e s p e c t i v e l y . y i e l d s a t 28 h p e r d i v i s i o n w e r e 6 1 , respectively.
Q-
E - g l u c u r o n i c acid, and 2-
5 2 , a n d 37 g / m o l
When 2 - a m i n o - 2 - d e o x y - ~ - g l u c o s e
was
Growth
o f substrate,
the carbohydrate
s o u r c e , a s t a b l e p o p u l a t i o n o f b a c t e r i a was a t t a i n a b l e o n l y a t g e n e r a t i o n t i m e s l o n g e r t h a n 12 h per d i v i s i o n . Growth y i e l d s a t 15 and
32
h
per
respectively.
division
were
T h e r e was
no s i g n i f i c a n t
11 a n d
33
g/mol
variation
generation t i m e s i n t h e s p e c i f i c a c t i v i t i e s of
of
substrate,
with increasing
selected glycolipid
- - g l u c o s i d a s e s and e n z y m e s , o f d i s a c c h a r i d e s s u c h a s a- a n d 6 - Q galactosidases, contrast,
o r o f t h e p o l y s a c c h a r i d a s e c h o n d r o i t i n AC l y a s e .
t h e p a t t e r n of
A t a generation t i m e of
propionate
w i t h a trace o f propionate.
concentrations
were
higher
as t h o s e f r o m ! - g l u c o s e
fermentation.
However,
The p r o d u c t s w e r e t h e same
when 2 - a c e t a m i d o - 2 -
was f e r m e n t e d , t h e c o n c e n t r a t i o n o f a c e t a t e was much
h i g h e r a t a l l g e n e r a t i o n t i m e s t h a n when ! - g l u c o s e source.
was t h e c a r b o n
When Q - g l u c u r o n i c a c i d was t h e c a r b o n s o u r c e ,
the main fermentation product, succinate
were
A t 28 h p e r
and s u c c i n a t e
c o n c e n t r a t i o n s w e r e l o w e r t h a n a t 3.5 h p e r d i v i s i o n . f r o m t h e f e r m e n t a t i o n o f 2 - a c e t a m i d o - 2 - d e o x y - p --g l u c o s e deoxy-!-glucose
3.5
t h e m a i n p r o d u c t s f r o m t h e f e r m e n t a t i o n o f !-glucose
w e r e a c e t a t e and s u c c i n a t e , division,
By
fermentation products varied w i t h both the
g e n e r a t i o n t i m e and t h e c a r b o n s o u r c e . h per division,
-!-
detected.
a c e t a t e was
and o n l y t r a c e s o f p r o p i o n a t e and
Another
characteristic
of
the
type
4: Microbial Polysaccharides
121
t h a t v a r i e d w i t h t h e g r o w t h r a t e was t h e a b i l i t y of B a c t e r o i d e s
-t-h--e-t a i o t a o m i c r o n t o p r o d u c e t h e i n d u c i b l e enzyme a - Q - g l u c o s i d a s e
when e x p o s e d t o m a l t o s e . The a b i l i t y o f t h e o r g a n i s m t o p r o d u c e t h i s enzyme d e c l i n e d w i t h i n c r e a s i n g g e n e r a t i o n times. The crude e x t r a c e l l u l a r c e l l u l a s e of C l o s t r i d i u m thermocellum L Q R I ( v i r g i n s t r a i n ) was v e r y a c t i v e and s o l u b i l i z e d m i c r o c r y s t a l l i n e c e l l u l o s e a t one-half t h e r a t e observed f o r t h e e x t r a c e l l u l a r c e l l u l a s e o f T r i c h o d e r m a r e e s i QM9414 ( m u t a n t s t r a i n ) .133 C l o s t r i d i u m t h e r m o c e l l u m c e l l u l a s e a c t i v i t y d i f f e r e d c o n s i d e r a b l y from t h a t o f T r i c h o d e r m a r e e s i a s f o l l o w s : higher endo --6-Q-91 ucanase/--B-Q-gl ucanase a c t i v i t y r a t i o , absence o f e x t r a c e l l u l a r c e l l o b i a s e o r B-a-xylosidase a c t i v i t y , long-chain o l i g o s a c c h a r i d e s i n s t e a d of s h o r t - c h a i n o l i g o s a c c h a r i d e s a s i n i t i a l (15 m i n ) h y d r o l y t i c p r o d u c t s o n m i c r o c r y s t a l l i n e c e l l u l o s e , mainly c e l l o b i o s e o r n-xylobiose a s long-term (24 h ) h y d r o l y s i s p r o d u c t s o f A v i c e l and M N 3 0 0 o r Q - x y l a n , and h i g h a c t i v i t y and s t a b i l i t y a t 6 0 t o 70%. Under optimized r e a c t i o n c o n d i t i o n s , t h e k i n e t i c p r o p e r t i e s (,Vmax0.4 p m o l / m i n p e r mg o f p r o t e i n , e n e r g y o f a c t i v a t i o n 33 k J , t e m p e r a t u r e c o e f f i c i e n t 1.8) o f C l o s t r i d i u m thermocellum c e l l u l o s e s o l u b i l i z i n g a c t i v i t y were c o m p a r a b l e t o t h o s e r e p o r t e d f o r T r i c h o d e r m a r e e s e i , e x c e p t t h a t t h e dyed A v i c e l c o n c e n t r a t i o n a t The c e l l u l o s e half-maximal v e l o c i t y was twofold h i g h e r (182 pM). s o l u b i l i z i n g a c t i v i t y of the two crude c e l l u l a s e s differed c o n s i d e r a b l y i n r e s p o n s e t o v a r i o u s enzyme i n h i b i t o r s . Most n o t a b l y , A g 2 + and Hg2+ e f f e c t i v e l y i n h i b i t e d C l o s t r i d i u m thermocellum b u t n o t Trichoderma r e e s e i c e l l u l a s e a t (20 pM, whereas Ca2+, big2+, and Mn2+ i n h i b i t e d Trichoderma r e e s i b u t n o t C l o s t r i d i u m thermocellum c e l l u l a s e a t >10 m M . B o t h enzymes were i n h i b i t e d by C u 2 + (>29mM), Z n 2 + (>l.OmM), and e t h y l e n e g l y c o l - b i s ( B - a m i n o e t h y l e t h e r ) - l l , N - t e t r a - a c e t i c a c i d ( > l o mM). T r i c h o d e r m a r e e s e i b u t n o t C l o s t r i d i u m t h e r m o c e l l u m c e l l u l o s e - s o l u b i l i z i n g a c t i v i t y was 20% i n h i b i t e d by Q - g l u c o s e ( 7 3 m M ) and c e l l o b i o s e ( 2 9 m M ) . Both c e l l u l a s e s p r e f e r e n t i a l l y cleaved t h e i n t e r n a l g l y c o s i d i c bonds of cello-oligosaccharides. The o v e r a l l r a t e s o f c e l l o - o l i g o s a c c h a r i d e d e g r a d a t i o n were higher f o r Trichoderma r e e s e i than f o r C l o s t r i d i u m -t-h--e r m o c e l l u m c e l l u l a s e , e x c e p t t h a t t h e r a t e s of c o n v e r s i o n o f c e l l o h e x a o s e t o c e l l o t r i o s e were e q u i v a l e n t . The f o r m a t i o n and e x c r e t i o n o f a c e t y l m a l t o s e and a c e t y l a t e d m a l t o d e x t r i n s a f t e r accumulation o f maltose i n E s c h e r i c h i a c o l i have been s t u d i e d . 1 3 4 As a c e t y l m a l t o s e was n o t a s u b s t r a t e f o r t h e m a l t o s e t r a n s p o r t s y s t e m i t was p r o p o s e d t h a t t h e a c e t y l a t i o n
Carbohydrate Chemistry
122
p r o c e s s was an e f f e c t i v e d e t o x i f i c a t i o n mechanism. When E s c h e r i c h i a c o l i s t r a i n CP78 (&+) w a s s t a r v e d f o r ii s o l e u c i n e by t h e a d d i t i o n o f L - v a l i n e , t h e amount o f Q - g l u c o s e i n p o l y m e r i c f o r m i n t h e c e l l s i n c r e a s e d m a r k e d l y compared t o t h a t o f t h e c o n t r o l cells.135
I n contrast,
(*-I.
s t r a i n CP79
increase of
t h i s phenomenon was n o t s e e n i n
The i n c r e a s e i n CP78 was shown t o be due t o t h e
glycogen.
These r e s u l t s i n d i c a t e t h a t
=’
s y n t h e s i s was a u g m e n t e d u n d e r s t r i n g e n t c o n t r o l . using other isogenic pairs o f o t h e r amino acids. valyl-tRNA
strains starved for
When t h e c u l t i v a t i o n t e m p e r a t u r e o f s t r a i n s
and 1 0 8 6 0 2 (&-)
1 0 8 6 0 1 (*+)
&+ and
glycogen
T h i s was c o n f i r m e d
possessing temperature-sensitive
s y n t h e t a s e was s h i f t e d f r o m 3OoC t o 4OoC,
L-
no d i f f e r e n c e
was o b s e r v e d i n t h e r e s p o n s e o f g l y c o g e n s y n t h e s i s b e t w e e n t h e t w o strains.
These
necessary f o r
results
the
guanosine 5’-diphosphate through
indicate
augmentation o f
stimulation
o f
that
3,-diphosphate the activity
i n v o l v e d i n glycogen synthesis.
protein
synthesis
was
glycogen s y n t h e s i s and t h a t did not exert i t s effect
of
pre-existing
enzyme(s)
These c o n c l u s i o n s w e r e s u p p o r t e d by
t h e r e s u l t s o f experiments u s i n g chloramphenicol and r i f a m p i c i n . The r a t e s o f Q - g l u c o s e u t i l i z a t i o n o f CP78 and CP79 w e r e d e c r e a s e d t o n e a r l y t h e same e x t e n t by I - v a l i n e a d d i t i o n .
This suggests t h a t
t h e r e g u l a t i o n s i t e o f glycogen s y n t h e s i s under s t r i n g e n t c o n t r o l resides i n a
step
after
the
transport
of
P-glucose
by
the
p h o p h o t r a n s f e r a s e system. ADP-Q-glucose
pyrophosphorylase from E s c h e r i c h i a c o l i B mutant
CL1136-504 h a s been p u r i f i e d t o h o m o g e n e i t y . t e t r a m e r o f 200,000 subunits.136 enzymes
of
The n a t i v e enzyme i s a
d a l t o n s composed o f f o u r i d e n t i c a l 50,000
Mr
The s t r u c t u r a l g e n e s f o r t h e g l y c o g e n b i o s y n t h e t i c Escherichia coli,
glycogen synthase,
ADP-Q-glucose
p y r o p h o s p h o r y l a s e , a n d b r a n c h i n g enzyme w e r e c l o n e d f r o m c h r o m o s o m a l DNA b a c t e r i a l p l a s m i d , A
pBR322. 137
b a c t e r i a l a g g l u t i n i n was e x t r a c t e d f r o m g r o u n d c o r n ( W I
h y b r i d 64A x W117) seed w i t h p h o s p h a t e - b u f f e r e d s a l i n e (pH 6.0) p r e c i p i t a t e d w i t h (NH4)2S04 a t 7 0 % s a t u r a t i o n .
and
The a c t i v i t i e s o f
t h i s a g g l u t i n i n a g a i n s t 22 s t r a i n s o f E r w i n i a s t e w a r t i i (agent o f b a c t e r i a l w i l t o f c o r n ) t h a t v a r i e d i n v i r u l e n c e were determined.138 Specific agglutination (agglutination t i t e r per m i l l i g r a m o f p r o t e i n p e r m i l l i l i t e r ) values were c o r r e l a t e d n e g a t i v e l y w i t h v i r u l e n c e ratings.
S t r a i n s w i t h h i g h s p e c i f i c a g g J u t i n a t i o n values (15 o r
h i g h e r ) were a v i r u l e n t o r w e a k l y v i r u l e n t ,
s t r a i n s with low s p e c i f i c
a g g l u t i n a t i o n values (10 o r l o w e r ) were h i g h l y v i r u l e n t ,
with two
123
4: Microbial Polysaccharides exceptions.
A
virulent
s t r a i n produced butyrous
c o l o n i e s and
r e l e a s e d o n l y s m a l l amounts o f e x t r a c e l l u l a r p o l y s a c c h a r i d e i n t o t h e medium,
and t h e
fluidal
colonies
cells and
l a c k e d capsules. released
V i r u l e n t s t r a i n s produced
large
amounts
of
extracellular
p o l y s a c c h a r i d e p r o d u c e d by each s t r a i n ( a s d e t e r m i n e d by i n c r e a s e i n viscosity
of
contrast
lipopolysaccharide compositions
strains.
t h e medium) and t h e s p e c i f i c a g g l u t i n a t i o n When c e l l s o f
six
were
fluidal strains
value.
similar were
I n
i n
a l l
washed by
r e p e a t e d l y c e n t i f u g i n g and r e s u s p e n d i n g them i n b u f f e r , t h e y were a g g l u t i n a t e d more s t r o n g l y by c o r n a g g l u t i n i n t h a n were unwashed cells.
When
agglutination
avirulent
cells
were
washed,
their
values d i d not increase s i g n i f i c a n t l y .
c e l l u l a r polysaccharide-deficient
specific
Eight extra-
mutants o f E r w i n i a s t e w a r t i i ,
s e l e c t e d f o r r e s i s t a n c e t o t h e capsule-dependent
b a c t e r i o p h a g e K9,
h a d l o w e r v i r u l e n c e by h i g h e r s p e c i f i c a g g l u t i n a t i o n t h a n d i d t h e i r of
extracellular
p o l y s a c c h a r i d e appears t o be e s s e n t i a l f o r v i r u l e n c e .
corresponding
wild-type
parents.
Production
Extracellular
may p r e v e n t a g g l u t i n a t i o n o f b a c t e r i a i n t h e h o s t , t h u s a l l o w i n g their multiplication, The
dextran
mesenteroides described.13'
produced
obtained
from
Branch
points
by
a
new
strain
contaminated at
0-3
and
of
case 0-2
Leuconostoc
juice
has
been
2:l)
(ratio
were
d e t e r m i n e d by m e t h y l a t i o n w h i l s t t h e o x i d i z e d d e x t r a n was f o u n d t o liberate,
on p a r t i a l a c i d h y d r o l y s i s ,
- - g 1u c o s e ,
a c id ) Q
-
i c a c i d ) ( 1+6)
-0-Q -
6-g-(a-Q-glucopyranosyluronic
glucopyranosy1)-(1+3)-Q-glucose,and acid)-Q-glucose
2-g-(a-a-glucopyranosyluronic
0 - ( a -Q - g 1u c o p y r a n o s y 1u r o n
( w h i c h may h a v e b e e n d e r i v e d f r o m t h e t r i o u r o n i c
a c i d 1. R a b b i t a n t i - d e x t r a n 81355 s e r a p r e p a r e d by i n j e c t i n g r a b b i t s w i t h L e u c o n o s t o c m e s e n t e r o i d e s NRRL 8 1 3 5 5 w e r e s e p a r a t e d o n a S e p h a d e x G75 c o l u m n i n t o t w o f r a c t i o n s , o n e b i n d i n g a n d t h e o t h e r not
binding to
the
column.140
Oligosaccharide
inhibition
of
p r e c i p i t a t i o n o f t h e t w o f r a c t i o n s w i t h d e x t r a n 81355 i n d i c a t e d t h a t b o t h f r a c t i o n s had (1+3)-a-Q-specificity.
However, a n t i b o d i e s i n
t h e n o n - b i n d i n g f r a c t i o n were shown t o be d i r e c t e d a g a i n s t
g l u c o p y r a n o s y l - ( 1 + 3 ) - a - e - g-l u c o p y r a n o s y l - ( l + 6 ) - g l u c o s e ,
g-a-Q-
w h i l s t those
i n t h e b i n d i n g f r a c t i o n were d i r e c t e d a g a i n s t g - a - a - g l u c o p y r a n o s y l -
(1+6)-a-~-glucopyranosyl-(l+3)-~-glucose. (1+6)-a-P-Specific by
anti-dextran
a n t i b o d y was
i n j e c t i n g N4 d e x t r a n - c o n c a n a v a l i n
interactions o f f i v e synthetic linear
A
raised i n rabbits
conjugate,
and
the
dextrans w i t h r a b b i t anti-N4
124
Carbohydrate Chemistry
d e x t r a n were s t u d i e d . l 4 l antibody
The a b i l i t y o f Q - g l u c a n s t o p r e c i p a t e
depended on t h e i r a v e r a g e m o l e c u l a r
t h e same
conditions.
solubility
of
the
This
p h e n o m e n o n was
antigen-antibody
i n h i b i t i o n assay i n d i c a t e d t h a t specific
antibody
samples with
weight,
h i g h e r m o l e c u l a r w e i g h t p r e c i p i t a t i n g more a n t i b o d y ,
n i t r o g e n under
shown t o
complex.
be due
to
Oligosaccharide
t h e maximum s i z e o f t h e (l+6)-a-Q-
combining s i t e corresponded t o
isomaltose.
The
p r e c i p i t a t e d a n t i b o d y c l a s s was shown t o be I g G i m m u n o g l o b u l i n , a n d it
was
mostly
directed
to
linear
non-terminal
a-Q-glucosidic
D e t e r m i n a t i o n o f a n t i b o d y n i t r o g e n and Q-glucan i n t h e
linkages.
precipitates numbers o f
indicated that
e-glucose
the
ratio of
r e s i d u e s was
antibody
molecule t o
1:16 i n t h e e x t r e m e a n t i b o d y
excess r e g i o n . The
concentration
viscosity
(0)
(GI
and
shear-rate
has been s t u d i e d f o r
(y)
dependence
p o l y s a c c h a r i d e s o l u t i o n s ( i n c l u d i n g dextran), and t h e s t r i k i n g g e n e r a l i t i e s a r e observed:142 dilute-to
concentrated-solution
concentration 10
c,* =:
(nsp v a r i e s a t
c1*4
for
viscosity
and
frequency
v i s c o s i t y are c l o s e l y superimposable, p l o t s of
n/n0
a g a i n s t y/yOe1 ( w h e r e
o0
a critical
and a t
shear-rate
dependence
at
s p e c i f i c v i s c o s i t y (nsp)
dilute solutions,
(ii) the
following
(i) the t r a n s i t i o n from
behaviour occurs
4 / { n } , when z e r o - s h e a r
concentrated solutions),
of
a wide range o f random-coil
of
c3*3 f o r
dependence
dynamic
of
(oscillatory)
(iii) double l o g a r i t h m i c i s zero-shear
viscosity,
and
yoe1 i s t h e shear r a t e a t w h i c h y = yo/lO) a r e e s s e n t i a l l y i d e n t i c a l f o r a l l c o n c e n t r a t e d s o l u t i o n s s t u d i e d , and t h u s t h e t w o p a r a m e t e r s
no
and yoe1 c o m p l e t e l y d e f i n e t h e v i s c o s i t y a t a l l shear r a t e s o f
p r a c t i c a l importance. Five strains of
Microccus,
accumulated a,a-trehalose glucose.143
a,a-Trehalose
representing three species,
when g r o w n i n t h e p r e s e n c e o f
a-
was n o t f o u n d i n t w o S t a p h y l o c o c c u s
species or i n Planococcus c i t r e u s . S u d a n o p h i l i c and i o d i n e - s t a i n i n g c y t o p l a s m i c g r a n u l e s were i s o l a t e d a t various developmental stages during growth o f Nocardia asteroides (strain
55)
and g l y c o g e n g r a n u l e s ,
and
i d e n t i f i e d as p o l y - b e t a - h y d r o x y b u t y r a t e
r e ~ p e c t i v e 1 y . l ~O~u r i n g g r o w t h i n n u t r i e n t
b r o t h c o n t a i n i n g 1% D - g l u c o s e , hydroxybutyrate respectively,
and
maximal accumulation o f
glycogen
granules,
o f i t s dry weight,was
up
t o
10
poly-betaand
20%,
obtained i n the filamentous
c e l l s a t 16 h j u s t p r i o r t o the onset o f c e l l fragmentation during t h e s t a t i o n a r y phase.
The d e c r e a s e o f t h e c y t o p l a s m i c g r a n u l e s was
125
4: Microbial Polysaccharides concomitant
with
fragmentation
of
the
cells
to
rod-like
and
s p h e r i c a l c e l l s , s u g g e s t i n g t h a t t h e p o l y m e r s may s e r v e a s c a r b o n and energy
source
during
macrophogenesis.
detected i n t h e t h r e e c e l l forms.
Both g r a n u l e s were
A t higher concentrations o f
a-
g l u c o s e ( 4 % ) more g l y c o g e n g r a n u l e s ( 9 t i m e s ) a c c u m u l a t e d t h a n p o l y b e t a - h y d r o x y b u t y r a t e ( 4 t i m e s ) , a n d g l y c o g e n h y d r o l y s i s was a l s o faster
than
that
of
suggesting
poly-beta-hydroxybutyrate,
preferences o f glycogen over poly-beta-hydroxybutyrate c a r b o n s o u r c e under t h i s g r o w t h c o n d i t i o n . of
granules
were s i g n i f i c a n t l y
concentration
( l o % ) , and
as e n e r g y and
Growth and b i o s y n t h e s i s
r e d u c e d by
very
h i g h !-glucose
the usual pleomorphic developmental stages
were r e d u c e d t o a d i m o r p h i c l i f e c y c l e .
Thus,
b i o s y n t h e s i s of b o t h
g r a n u l e s and morphogenesis a r e under c a t a b o l i t e r e p r e s s i o n .
Both
p o l y m e r s were a l s o f o u n d i n N o c a r d i a b r a s i l i e n s i s and N o c a r d i a otitidis-caviarurn,
indicating that
cytoplasmic accumulation o f
m u l t i p l e g r a n u l e s i s common i n t h e genus. The m e t h o d u s e d t o i s o l a t e a h i g h - m o l e c u l a r - w e i g h t
immunogenic
p o l y s a c c h a r i d e f r o m Pseudomonas a e r u g i n o s a i m m u n o t y p e 1 ( I T - 1 )
was
m o d i f i e d t o p e r m i t t h e i s o l a t i o n o f a s i m i l a r polysaccharide from Pseudomonas a e r u g i n o s a IT-2.145
T h i s a n t i g e n was composed p r i m a r i l y
o f c a r b o h y d r a t e , had a complex monosaccharide c o m p o s i t i o n ,
including
sugars n o t found i n t h e lipopolysaccharide,
a n d was n o n p y r o g e n i c i n
r a b b i t s a n d n o n t o x i c i n m i c e a t h i g h doses.
This m a t e r i a l protected
mice from
challenges
w i t h l i v e homologous
cells.
Pseudomonas
a e r u g i n o s a I T - 2 p o l y s a c c h a r i d e gave a l i n e o f i d e n t i t y w i t h t h e 0 side
chain
of
the
lipopolysaccharide,
polysaccharide i n molecular ability
to
immunize
mice
weight,
but
differed
from
chemical composition,
actively.
Lipopolysaccharide
this and from
Pseudomonas a e r u g i n o s a I T - 2 c o n t a i n e d an i m m u n o l o g i c a l d e t e r m i n a n t n o t found
o n Pseudomonas a e r u g i n o s a I T - 2 p o l y s a c c h a r i d e ,
d e t e c t e d due t o i t s s t a b i l i t y d u r i n g t r e a t m e n t Thus,
a
high-molecular-weight
polysaccharide
Pseudomonas a e r u q i n o s a I T - 2 p o l y s a c c h a r i d e ,
w h i c h was
with dilute alkali. antigen
from
w h i c h was s e r o l o g i c a l l y
i d e n t i c a l t o t h e l i p o p o l y s a c c h a r i d e 0 s i d e c h a i n b u t was c h e m i c a l l y and p h y s i c a l l y d i s t i n c t ,
was r e c o v e r e d .
Also,
l i k e Pseudomonas
a e r u q i n o s a I T - 1 s t r a i n s , Pseudomonas a e r u g i n o s a I T - 2 c o n t a i n s an alkali-stable
i m m u n o d e t e r m i n a n t on t h e l i p o p o l y s a c c h a r i d e t h a t may
represent a c o r e - l i k e antigen. Spontaneous a l g i n a t e - p r o d u c i n g
( m ~ v] a r i a n t s
f r o m s t r a i n s o f Pseudomonas f l u o r e s e n c e s ,
-P-s-e u d o m o n a s
were i s o l a t e d
Pseudomonas p u t i d a , and
mendocina a t a frequency o f 1 i n
lo8
by s e l e c t i n g f o r
126
Carbohydrate Chemistry
c a r b e n i c i l l i n resistance.146
The i n f r a r e d s p e c t r u m o f t h e b a c t e r i a l
e x o p o l y s a c c h a r i d e was t y p i c a l o f
an a c e t y l a t e d a l g i n a t e s i m i l a r
to
t h a t p r e v i o u s l y d e s c r i b e d i n Azotobacter v i n e l a n d i i and i n mucoid v a r i a n t s o f Pseudomonas a e r u g i n o s a . i s o l a t e d f r o m Pseudomonas s t u t z e r i ,
-P-s-e u d o m o n a s
testostergnL,
Mucoid v a r i a n t s
Pseudgmmas
a c i d o v o r a n s , Pseudomonas c e p a c i a , o r
were
not
Pseudomonas p s e u d o a l c a l i g e n e s ,
GLgLn~tg, P s e u d o m o n a s
Pseudomonas m a l t o p h i l i a .
G r a m - n e g a t i v e h y d r o g e n b a c t e r i u m Pseudomonas h y d r o g e n o v o r a was found
t o
excrete
an
anthrone-H2S04
p 0 1 y s a c c h a r i d e . l ~ ~A b o u t
12 g / l i t r e
of
p o s i t i v e
viscous
the polysaccharide
was
p r o d u c e d a u t o t r o p h i c a l l y o n gaseous h y d r o g e n a t t h e s t a t i o n a r y p h a s e of
growth.
Biosynthesis of
the
nitrogen-deficient condition. 39.29%,
H
6.23%,
polysaccharide
49.67%,
0
occurred under
I t s e l e m e n t a r y c o m p o s i t i o n was C 0.21%,
N
p o l y s a c c h a r i d e c o n t a i n e d !-galactose,
and
Q-glucose,
rhamnose as i t s m a i n components.
ash
4.6%.
The
Q-mannose, and
L-
The p o l y s a c c h a r i d e h a d a n t i -
t o b a c c o m o s a i c v i r u s and a n t i - t u m o u r a c t i v i t i e s . The p o s s i b i l i t y o f s e p a r a t i n g p y r u v u l a t e d p o l y s a c c h a r i d e s i n t o pyruvate-rich
and p y r u v a t e - p o o r
u s i n g an a f f i n i t y matrix.148 strand types exists.
f r a c t i o n s has been d e m o n s t r a t e d
T h u s i n some p o l y m e r s , a m i x t u r e o f
The a f f i n i t y m a t r i x was p r e p a r e d by c o u p l i n g
a n t i b o d i e s t o a R h i z o b i u m p o l y s a c c h a r i d e t o Sepharose g e l .
Elution
was a c c o m p l i s h e d by t h e a d d i t i o n o f p y r u v a t e t o t h e e l u t i n g b u f f e r . N o n - p y r u v y l a t e d p o l y s a c c h a r i d e s were n o t a d s o r b e d . Rhizobia
are
Gram-negative
n o d u l a t i n g t h e r o o t s of
bacteria
leguminous p l a n t s ,
e x t r a c e l l u l a r polysaccharide-deficient
normally
capable
of
and t h e f a i l u r e o f f i v e
mutants t o nodulate suggested
that the polysaccharide i s required f o r t h i s nodulation.
I t has
b e e n r e p o r t e d t h a t among a l a r g e r s a m p l e o f m u t a n t s w i t h a l t e r e d polysaccharide production,
i s o l a t e d from two species
( i n c l u d i n g one o f t h e o r i g i n a l s e t p l u s 34 new o n e s ) ,
o f Rhizobium production of
t h e e x t r a c e l l u l a r p o l y s a c c h a r i d e does n o t c o r r e l a t e w i t h a b i l i t y t o n o d u l a t e a p p r o p r i a t e hosts.149
Therefore,
although involvement o f a
m i n o r component c a n n o t be r u l e d o u t , t h e r e i s no e v i d e n c e t h a t t h e whole p o l y s a c c h a r i d e i s r e q u i r e d f o r n o d u l a t i o n . I m m u n o e l e c t r o n m i c r o s c o p y was c o m b i n e d w i t h p a r t i a l characterization o f i s o l a t e d exopolysaccharide t o study binding o f s o y b e a n l e c t i n by R h i z o b i u m j a p o n i c u m s t r a i n USDA 138.150
Lectin-
b i n d i n g a c t i v i t y r e s i d e d i n two forms o f exopolysaccharide produced d u r i n g growth:
an a p p a r e n t l y
very
f o r m and a l o w e r - m o l e c u l a r - w e i g h t
high-molecular-weight d i f f u s i b l e form.
capsular
A t low-speed
4: Microbial Polysaccharides
127
c e n t r i f u g a t i o n , t h e c a p s u l a r form cosedimented w i t h c e l l s t o form a v i s c o u s , w h i t e , c e l l - g e l c o m p l e x which was n o t d i f f u s i b l e i n 1% a g a r , and t h e d i f f u s i b l e form remained i n t h e c e l l - f r e e s u p e r n a t a n t . E l e c t r o n - m i c r o s c o p i c o b s e r v a t i o n of t h e c e l l - g e l c o m p l e x a f t e r l a b e l l i n g w i t h soybean l e c t i n - f e r r i t i n c o n j u g a t e r e v e a l e d t h a t c a p s u l a r p o l y s a c c h a r i d e s , f r e q u e n t l y a t t a c h e d t o o n e end o f t h e c e l l s , w e r e r e c e p t o r s f o r l e c t i n . The o u t e r membrane of t h e c e l l bound no l e c t i n . Various p r e p a r a t i o n s of e x o p o l y s a c c h a r i d e i s o l a t e d from t h e c u l t u r e s u p e r n a t a n t w e r e t e s t e d f o r l e c t i n b i n d i n g , i n t e r a c t i o n w i t h homologous s o m a t i c a n t i g e n , and t h e p r e s e n c e of 3deoxy-a-manno-2-octulosonic a c i d and w e r e c h r o m a t o g r a p h e d i n S e p h a r o s e 48 and 6 8 g e l b e d s . L e c t i n b i n d i n g was r e s t r i c t e d t o a p o l y s a c c h a r i d e component designated a s l e c t i n - b i n d i n g polysaccharide. T h i s polysaccharide, as present i n the cell-free c u l t u r e s u p e r n a t a n t , was a d i f f u s i b l e a c i d i c p o l y s a c c h a r i d e devoid of 3 - d e o x y - ~ - m a n n o - 2 - o c t u l o s o n i c a c i d , w i t h m o l e c u l a r weight of 2 x l o 6 t o 5 x l o 6 . I t was c o n c l u d e d t h a t s o y b e a n l e c t i n - b i n d i n g component o f Rhizobium japonicum i s an e x t r a c e l l u l a r p o l y s a c c h a r i d e and not a l i p o p o l y s a c c h a r i d e and t h a t t h e d i f f u s i b l e l e c t i n - b i n d i n g p o l y s a c c h a r i d e probably d i f f e r s from t h e very h i g h - m o l e c u l a r - w e i g h t l e c t i n - b i n d i n g p o l y s a c c h a r i d e of t h e loose c a p s u l e ( s l i m e ) only i n t h e d e g r e e of p o l y m e r i z a t i o n . The e x t r a c e l l u l a r p o l y s a c c h a r i d e o f R h i z o b i u m m e l i l o t i 201 c o n s i s t s of two a c i d i c p o l y s a c c h a r i d e s , APS-I and APS-I1 .151 APS-I is composed o f D-glucose, D-mannose,and Q - g l u c u r o n i c a c i d i n a molar w h e r e a s APS-I1 i s composed of E - g l u c o s e , r a t i o of 3:3:2, g a l a c t o s e , a - m a n n o s e , a n d p y r u v i c a c i d i n a m o l a r r a t i o of 4:3:2:1. APS-I1 w a s s e p a r a t e d f r o m t h e e x t r a c e l l u l a r p o l y s a c c h a r i d e p r e p a r a t i o n b y h y d r o l y s i n g APS-I t o i t s o c t a s a c c h a r i d e r e p e a t i n g u n i t w i t h a s p e c i f i c enzyme. APS-I and APS-I1 were a l s o s e p a r a t e d by t r e a t m e n t w i t h c e t y l p y r i d i n i u m c h l o r i d e and by p a p e r e l e c t r o p h o r e s i s of t h e d e p y r u v y l a t e d p o l y s a c c h a r i d e . An i m p r o v e d , g l y c o s y l - s e q u e n c i n g method h a s been u s e d f o r e l u c i d a t i n g t h e s t r u c t u r e of t h e a c i d i c p o l y s a c c h a r i d e s e c r e t e d b y R h i z o b i u m -------m e l i l o t i s t r a i n 1021.152 T h e p o l y s a c c h a r i d e was --------methylated, t h e e t h e r p a r t i a l l y hydrolysed, t h e p r o d u c t s were r e d u c e d , and t h e a l d i t o l s e t h y l a t e d . The r e s u l t i n g m i x t u r e of p e r a l k y l a t e d o l i g o s a c c h a r i d e - a l d i t o l s was a n a l y s e d by h.p.1.c. m.s. C h e m i c a l - i o n i z a t i o n mass s p e c t r a were o b t a i n e d a t 2 s i n t e r v a l s , a s t h e v a r i o u s , p e r a l k y l a t e d oligosaccharide-alditols were e l u t e d from t h e h.p.1.c. column. P e r a l k y l a t e d mono-, d i - , t r i - , and t e t r a -
e-
128
carbohydrate Chemistry
s a c c h a r i d e - a l d i t o l s c o u l d be r e a d i l y d e t e c t e d , and a t l e a s t p a r t i a l l y i d e n t i f i e d , by t h e i r M + 1 i o n s , i n conjunction w i t h o t h e r c h a r a c t e r i s t i c i o n s p r e s e n t i n t h e i r c h e m i c a l - i o n i z a t i o n mass s p e c t r a . T h e p e r a l k y l a t e d d i - and t r i - s a c c h a r i d e - a l d i t o l s w e r e f u r t h e r a n a l y s e d b y g.c. m.s. i n t h e e l e c t r o n - i m p a c t mode. The s t r u c t u r e of t h e a c i d i c p o l y s a c c h a r i d e s e c r e t e d by Rhizobium -------m e l i l o t i s t r a i n 1021 i s t h e same a s t h a t of t h e p r e v i o u s l y c h a r a c t e r i z e d p o l y s a c c h a r i d e s e c r e t e d b y Rhizobium m e l i l o t i s t r a i n U-27.
The s t r u c t u r e of an e x t r a c e l l u l a r , a c i d i c p o l y s a c c h a r i d e from R h i z o b i u m m e l i l o t i I F 0 1 3 3 3 6 was s t u d i e d b y a method i n v o l v i n g of successive fragmentation w i t h s p e c i f i c B-e-glycanases F l a v o b a c t e r i u m M64.153 The p o l y s a c c h a r i d e i s composed of r e p e a t i n g u n i t s of t h e o c t a s a c c h a r i d e ( 3 3 ) . An a c i d i c c o m p o n e n t was i d e n t i f i e d a s q-riburonic acid. F i v e c u l t u r e s of Rhizobium m e l i l o t i (57017, 202, 204, 207, 209) and o n e o f R h i z o b i u m t r i f o l i i ( 5 6 0 ) p r o d u c e d w a t e r - s o l u b l e p o l y s a c c h a r i d e s c o n t a i n i n g P-glucose, Q - g a l a c t o s e , a n d p y r u v i c a c i d i n a m o l a r r a t i o of 7 : l : l and some s u c c i n i c and a c e t i c a c i d s . 1 5 4 These were i d e n t i f i e d a s s u c c i n o g l y c a n - l i k e p o l y s a c c h a r i d e s on t h e b a s i s of t h e i r components, m e t h y l a t i o n a n a l y s i s , a n d f r a g m e n t a t i o n w i t h two s p e c i f i c B - Q - g l y c a n a s e s . One c u l t u r e o f R h i z o b i u m m e l i l o t i ( I F 0 13336) produced w a t e r - s o l u b l e p o l y s a c c h a r i d e c o n t a i n i n g ! - g l u c o s e , Q - g a l a c t o s e , 0 - g l u c u r o n i c a c i d , a n d a c e t i c a c i d i n a molar r a t i o o f 5:1:1:2 and an u n i d e n t i f i e d component. Two c u l t u r e s of Rhitobium -------m e l i l o t i (201, 206) produced w a t e r - s o l u b l e p o l y s a c c h a r i d e s c o n t a i n i n g g - g l u c o s e , ; - g a l a c t o s e , g-mannose, and g - g l u c u r o n i c a c i d i n a m o l a r r a t i o o f 4:2:3:1 and 4:1:2:1, r e s p e c t i v e l y , and some p y r u v i c a c i d . Rhizobium t r i f o l i i I F 0 13337 and Rhizobium japonicum I F 0 13338 p r o d u c e d w a t e r - s o l u b l e p o l y s a c c h a r i d e s c o n t a i n i n g Q g l u c o s e , Q - g l a c t a c t o s e , Q - g l u c u r o n i c a c i d , p y r u v i c a c i d , and a c e t i c Two i s o l a t e s f r o m t h e s t o c k a c i d i n a m o l a r r a t i o o f 6:1:1:2:1. c u l t u r e of R h i z o b i u m t r i f o l i i 560 p r o d u c e d l a r g e a m o u n t s o f t h e water-insoluble polysaccharide curdlan. T h i s is t h e f i r s t report i n Rhizobium of t h e o c c u r r e n c e of c u r d l a n and of s p o n t a n e o u s m u t a t i o n s i n a b i l i t y t o produce s u c c i n o g l y c a n - l i k e p o l y s a c c h a r i d e and c u r d l a n . Carbohydrate m u t a n t s were i s o l a t e d from Rhizobium m e l i l o t i L530 u s i n g t h e t r a n s l o c a t a b l e d r u g - r e s i s t a n c e e l e m e n t Tn5.155 Enzyme a s s a y s w i t h c e l l - f r e e e x t r a c t s of f o u r m u t a n t s showed t h a t t h e y lacked p-mannitol dehydrogenase, ; - r i b o s e k i n a s e , ;-xylose isomerase, and !-fructose k i n a s e , r e s p e c t i v e l y . An I - a r a b i n o s e mutant was a l s o
129
4: Microbial Polysaccharides 4
4 U
b
rl
lu
(3
I
4'
m I
n
M 4
+
4
v
.--I (3
I
4' m I
i
4
u
I
4' m I
I
4' m
I
I
2
4
(3 I
4'
m I
n
+
U
4
u
(3
I
4' 0
n
+
U
4 v
I
4'
u
130
Carbohydrate Chemistry
isolated.
U p t a k e s t u d i e s showed t h a t t h e Q - r i b o s e ,
a - x y l o s e , and
p-
f r u c t o s e m u t a n t s s t i l l u t i l i z e d t h e s u g a r s on w h i c h t h e y were u n a b l e t o grow,
p o s s i b l y i n d i c a t i n g t h a t Rhizobium m e l i l o t i possesses
a l t e r n a t i v e m e t a b o l i c r o u t e s w h i c h do n o t r e s u l t i n g r o w t h . mutants were a b l e t o n o d u l a t e a l f a l f a k i n a s e m u t a n t was u n a b l e t o f i x
A l l the
plants, but the n-fructose
n i t r o g e n and t h e L - a r a b i n o s e m u t a n t
showed e i t h e r l a t e or no n i t r o g e n - f i x i n g a b i l i t y . The fulvum,
ADP-!-glucose
pyrophosphorylases
R h o d o s p i r i l l u m molischianum,
from
Rhodospirillum
and R h o d o s p i r i l l u m t e n u e w e r e
p a r t i a l l y p u r i f i e d , and t h e i r k i n e t i c p r o p e r t i e s were studied.156 The e n z y m e f r o m t h e t h r e e o r g a n i s m s was f o u n d t o b e a c t i v a t e d b y p y r u v a t e and t h u s was s i m i l a r
t o t h e R h o d o s p i r i l l u m r u b r u m enzyme
t h a t h a d been p r e v i o u S l y s t u d i e d . fulvum,
The enzymes f r o m R h o d o s p i r i l l u m
R h o d o s p i r i l l u m molischianum,
a l s o a c t i v a t e d by oxamate,
and R h o d o s p i r i l l u m t e n u e were
an a n a l o g o f p y r u v a t e .
Other a-keto
a c i d s , a - k e t o b u t y r a t e and h y d r o x y p y r u v a t e , a c t i v a t e d t o a s m a l l e r extent.
The p r e s e n c e o f p y r u v a t e i n c r e a s e d t h e a p p a r e n t a f f i n i t y
f o r adenosine 5’-triphosphate
a n d M g C 1 2 f o r a l l t h r e e enzymes.
R h o d o s p i r i l l u m m o l i s c h i a n u m enzyme h a s v e r y i n h i b i t i o n by adenosine 5’-monophosphate, phosphate.
However,
pyrophosphorylase monophosphate, ADP.
Rhodospirillum
i s very
sensitive
to
The
l i t t l e sensitivity tenue
to
or inorganic
ADP,
ADP-Q-glucose
i n h i b i t i o n by a d e n o s i n e 5’-
and t h e R h o d o s p i r i l l u m f u l v u m enzyme i s i n h i b i t e d by
Increasing pyruvate concentration reversed the i n h i b i t i o n
o r ADP.
c a u s e d by a d e n o s i n e 5’-monophosphate t h e g l y c o s y l donor f o r s y n t h e s i s of
vivo glycogen pyruvate
and,
synthesis i n
the
Rhodospirillum tenue,
i s
S i n c e ADP-Q-glucose
is
glycogen, i t i s p o s s i b l e t h a t
in
regulated
case
of
by
the
concentration
Rhodozejrillum
fulvum
of and
by t h e r a t i o o f p y r u v a t e c o n c e n t r a t i o n t o
i n h i b i t o r concentration. ADP-Q-glucose
synthetase from
Rhodospirillum tenue
the photosynthetic bacterium
has been p u r i f i e d g r e a t e r
than
95%.
m o l e c u l a r w e i g h t o f t h e e n z y m e i s a p p r o x i m a t e l y 215,000, s u b u n i t m o l e c u l a r w e i g h t o f a b o u t 51,000. composed o f
The enzyme a p p e a r s t o be
f o u r s i m i l a r ifn o t i d e n t i c a l s u b u n i t s . 1 5 7
amino a c i d composition o f
The
with a
t h e enzyme i s s i m i l a r
E s c h e r i c h i a c o l i and S a l m o n e l l a t y p h i m u r i u m ,
Although the t o that
o f
no a p p a r e n t h o m o l o g y
has been o b s e r v e d between t h e i r N - t e r m i n a l amino a c i d sequences. Antisera
prepared against
the
Rhodospirillzm tenue
p a r t i a l l y i n h i b i t the activities o f other photosynthetic bacteria.
ADP-a-glucose
enzyme
synthetases
can from
131
4: Microbial Polysaccharides Q-Mannitol, component
of
extracellular
not
previously
reported
as
an
intracellular
i t has been f o u n d as an end p r o d u c t o f a n a e r o b i c c a r b o h y d r a t e m e t a b o l i s m , bacteria,
although
accumulated w i t h i n s t r a i n s of
a l l 10 s t a p h y l o c o c c a l species t e s t e d
a f t e r a e r o b i c i n c u b a t i o n o f washed c e l l s u s p e n s i o n s i n p h o s p h a t e buffered
1%! - g l u c o s e
f o r 2 h.lS8
b e f o r e and a f t e r i n c u b a t i o n ,
Phenol e x t r a c t s of the c e l l s ,
were a n a l y s e d f o r e - m a n n i t o l c o n t e n t by
p e r i o d a t e u t i l i z a t i o n and p a p e r c h r o m a t o g r a p h y phosphate
content,
with
and f o r q - m a n n i t o l -
Q-mannitol l-phosphate
Staphylococcus aureus Towler,
the
content
of
dehydrogenase.
Q-mannitol
In
increased
f r o m a 0 h v a l u e o f <2.3 t o 1 6 u m o l / g
(dry weight) after incubation,
and t h e l e v e l o f P - m a n n i t o l - p h o s p h a t e
increased from a 0 h value o f
1 t o 8 umol/g.
the per-g-acetyl
The i d e n t i f i c a t i o n o f p - m a n n i t o l was c o n f i r m e d a s e s t e r by g a s - l i q u i d c h r o m a t o g r a p h y and as t h e p e r -
O - m e t h y l e t h e r by mass s p e c t r o m e t r y . A l s o t e s t e d were 5 a d d i t i o n a l --S t a ~ h y-------l o c o c c u s ----__ aureus s t r a i n s and 32 coagulase-negative staphylococcal strains.
A l l s t r a i n s accumulated p-mannito1,even
t h o s e s t r a i n s t h a t c o u l d n o t u t i l i z e exogenous e - m a n n i t o l d u r i n g aerobic growth,
u s u a l l y i n t h e r a n g e 4 t o 25 umol/g.
Furthermore,
t h r e e s t r a i n s accumulated very h i g h g-mannitol levels.
Bacteria
from
found t o
several other
genera were
tested,
accumulate low t o moderate l e v e l s o f incubation conditions.
Q-mannitol
under s i m i l a r
The m e t a b o l i c c o n v e r s i o n o f Q - g l u c o s e t o
i n t r a c e l l u l a r Q-mannitol, common f e a t u r e o f
a n d some w e r e
probably v i a Q-mannitol l-phosphate,
s t a p h y l o c o c c i and a l s o o c c u r s
i s a
i n some o t h e r
bacteria. Soluble extracellular supernatant OMZ
of
a n t i g e n s were p r e p a r e d f r o m t h e c u l t u r e
exponential
growing
9 by a combination o f
cells
Streptococcus
of
sanguis
ammonium s u l p h a t e p r e c i p i t a t i o n a n d
Soluble c e l l - w a l l c h r o m a t o g r a p h y o n a B i o - G e l P 6 column.159 a n t i g e n s w e r e o b t a i n e d f r o m t h e b a c t e r i a l p e l l e t by e x t r a c t i o n w i t h 1 M
phosphate b u f f e r
(pH 6).
---S t r e e -------t o c o c c u s --s a n q--uis serotypes, dextran,
Antisera
against
whole
and S t r e p t o c o c c g L mutans o f
cells of different
10% t r i c h l o r a c e t i c e x t r a c t s o f b a c t e r i a l c e l l w a l l s ,
extracellular
injecting the Extracellular
a n t i g e n s , and w a l l a n t i g e n s w e r e p r e p a r e d by
different antigens
representative strain Lactobacillus salivarius,
antigens
and w a l l of
several
antigens
Bratthall's
times
were
seven
s e r o l o g i c a l groups,
and A c t i n o m y c e s v i s c o s u s .
various agglutinin t i t e r s against heat-killed cells, g e n e r a l l y h i g h e r w i t h homologous c e l l s .
i n rabbits.
prepared from a A l l s e r a showed and t i t e r s w e r e
The c o m p a r i s o n o f t h e
132
Carbohydrate Chemistry
different
antigens,
using
agar-gel
diffusion
and
immuno-
e l e c t r o p h o r e s i s , s h o w e d t h e p r e s e n c e o f e x t r a c e l l u l a r common a n t i g e n s i n
both
extracellular
antigens
and
wall
antigens
between
the
different strains.
Absorption o f
anti-extracellular
antigens sera
with w a l l antigens
and a n t i - w a l l
antigens sera with e x t r a c e l l u l a r
a n t i g e n s s h o w e d t h e e x i s t e n c e o f a s p e c i f i c a n t i g e n common t o a l l b a c t e r i a i n each f r a c t i o n .
Enzymatic treatment o f the antigen
before immunodiffusion demonstrated t h e p r o t e i n n a t u r e o f
the two
a n t i g e n s p r e s e n t i n e x t r a c e l l u l a r a n t i g e n s and w a l l a n t i g e n s . The c o v a l e n t l y b o u n d c a r b o h y d r a t e a n t i g e n e x t r a c t e d f r o m t h e c e l l w a l l o f group B Streptococcus
t y p e l b i s common t o t y p e 1
s e r o t y p e s .160 Two s i a l i c a c i d - c o n t a i n i n g
type
I11 group B s t r e p t o c o c c a l
a n t i g e n s w e r e o b t a i n e d f r o m a s u p e r n a t a n t g r o w t h medium, p u r i f i e d by anion exchange or g e l f i l t r a t i o n , reactivity.161
Quantitation
and f o u n d t o be f r e e o f g r o u p B of
the
high-molecular-weight
e x t r a c e l l u l a r t y p e I11 a n t i g e n i n d i c a t e d t h a t a p p r o x i m a t e l y 2 0 - f o l d m o r e a n t i g e n was r e c o v e r a b l e f r o m t h e g r o w n m e d i u m t h a n c o u l d b e o b t a i n e d by n e u t r a l b u f f e r e x t r a c t i o n o f whole c e l l s . S t a r v e d c e l l s o f S t r e p t o c o c c u s l a c t i s ML3 ( g r o w n p r e v i o u s l y on Q - g a l a c t o s e , l a c t o s e , or m a l t o s e ) a c c u m u l a t e d m e t h y l l - t h i o - B - k g a l a c t o p y r a n o s i d e by t h e 1 a c t o s e : p h o s p h o t r a n s f e r a s e than
98% o f
accumulated
sugar
was
d e r i v a t i v e m e t h y l 1- t h i o - B - g - g a l a c t o p y r a phosphotransferase-system
present
as
system.
a n o s i d e - 6 - p h o s p h a t e . 162
s u g a r (!-glucose,
More
a phosphorylate
Q-mannose,
When
2-deoxy-Q-
g l u c o s e , o r l a c t o s e ) was a d d e d t o t h e m e d i u m s i m u l t a n e o u s l y w i t h methyl l-thio-B-e-galactopyranoside, excluded from
the cells.
!-Galactose
the B-e-galactoside
was
enhanced t h e a c c u m u l a t i o n o f
m e t h y l l-thio-B-Q-galactopyranoside-6-phosphate.
!-Glucose,
p-
o r m a l t o s e p l u s I - a r g i n i n e , when a d d e d t o a s u s p e n s i o n o f m e t h y l l-thio-~-~-galactopyranoside-6-phosphate-loaded mannose,
lactose,
c e l l s o f Streptococcus intracellular solute.
l a c t i s ML3,
e l i c i t e d rapid expulsion of
The m a t e r i a l r e c o v e r e d i n t h e m e d i u m was
e x c l u s i v e l y f r e e methyl l-thio-B-P-galactopyranoside. P-galactoside sugar,
Expulsion o f
r e q u i r e d b o t h e n t r y and m e t a b o l i s m o f an a p p r o p r i a t e
and i n t r a c e l l u l a r d e p h o s p h o r y l a t i o n o f m e t h y l 1-thio-B-Q-
galactopyranoside-6-phosphate galactopyranoside.
preceded e f f l u x o f m e t h y l l-thio-B-Q-
The r a t e o f d e p h o s p h o r y l a t i o n o f
B-Q-galactopyranoside-6-phosphate
methyl l-thio-
by p e r m e a b i l i z e d c e l l s
i n c r e a s e d t w o t o t h r e e f o l d by a d e n o s i n e 5 ' - t r i p h o s p h a t e s t r o n g l y i n h i b i t e d by f l u o r i d e .
was
b u t was
S t r e p t o c o c c u s l a c t i s ML3 ( D G r )
was
4: Microbial Polysaccharides
133
d e r i v e d f r o m S t r e p t o c o c c u s l a c t i s ML3 b y p o s i t i v e s e l e c t i o n f o r r e s i s t a n c e t o 2 - d e o x y - Q - g l u c o s e and was d e f e c t i v e i n t h e enzyme I I M a ncomponent of t h e Q-g1ucose:phosphotransferace system. Neither 0- - g l u c o s e nor Q-mannose excluded methyl 1-thio-B-P-galactopyranoside from c e l l s of S t r e p t o c o c c u s l a c t i s ML3 (DGr), and t h e s e two s u g a r s f a i l e d t o e l i c i t methyl 1-thio-B-1-galactopyranoside e x p u l s i o n from p r e l o a d e d c e l l s of t h e mutant s t r a i n . A c c u m u l a t i o n of m e t h y l 1t h i o - B - Q-- g a l a c t o p y r a n o s i d e - 6 - p h o s p h a t e b y S t r e p t o c o c c u s l a c t i s ML3 c a n be r e g u l a t e d b y t w o i n d e p e n d e n t m e c h a n i s m s whose a c t i v i t i e s promote e x c l u s i o n or e x p u l s i o n of e - g a l a c t o s i d e from t h e c e l l . Antibodies d i r e c t e d a g a i n s t S t r e p t o c o c c u s mutans GS-5 i n t r a c e l l u l a r i n v e r t a s e and Q - g l u c o s y l t r a n s f e r a s e f r a c t i o n s c a p a b l e of s y n t h e s i z i n g p r i m a r i l y w a t e r - s o l u b l e or i n s o l u b l e p-glucans were u s e d t o l o c a l i z e u l t r a s t r u c t u r a l l y t h e e n z y m e s b y means o f t h e This u n l a b e l l e d a n t i b o d y p e r o x i d a s e - a n t i p e r o x i d a s e method.163 immunocytochemical procedure r e v e a l e d t h a t t h e i n t r a c e l l u l a r i n v e r t a s e was a s s o c i a t e d p r i m a r i l y w i t h t h e c y t o p l a s m i c membrane of the cariogenic organism. T h e Q - g l u c o s y l - t r a n s f e r a s e complex r e s p o n s i b l e f o r i n s o l u b l e Q - g l u c a n s y n t h e s i s was l o c a l i z e d a s aggregates attached to the c e l l surface or extracellular p o l y s a c c h a r i d e s of s t r a i n G S - 5 . I n c o n t r a s t , t h e Q-glucosylt r a n s f e r a s e a c t i v i t y s y n t h e s i z i n g p r i m a r i l y w a t e r - s o l u b l e p-glucans was d i s t r i b u t e d u n i f o r m l y o v e r t h e c e l l s u r f a c e o r i n a s s o c i a t i o n w i t h extracellular polysaccharides. The r e s u l t s w e r e d i s c u s s e d r e l a t i v e t o t h e s u c r o s e - m e t a b o l i z i n g a b i l i t y of S t r e p t o c o c c u s mutans. The i s o l a t i o n of t h e d i s a g g r e g a t e d w a t e r - i n s o l u b l e Q-glucans y n t h e s i z i n g p - g l u c o s y l t r a n s f e r a s e s f r o m t h e c u l t u r e f l u i d of S t r e p t o c o c c u s mutans s t r a i n 8-13 ( s e r o t y p e cJ) has been described.164 A d h e s i v e w a t e r - i n s o l u b l e Q - g l u c a n s w e r e shown t o be s y n t h e s i z e d through an o v e r a l l r e a c t i o n by two p r o t e i n components. The Q - g l u c o s y l t r a n s f e r a s e f r o m S t r e p t o c o c c u s m u t a n s 6715 h a s been s e p a r a t e d i n t o t h r e e enzymic f r a c t i o n s t h a t d i f f e r i n t h e i r b i n d i n g t o d e x t r a n and i n t h e i r s y n t h e s i s of d e x t r a n f r o m s ~ c r 0 s e . l ~One ~ enzymic f r a c t i o n ( A F F - I ) does not b i n d t o i n s o l u b l e d e x t r a n , and i t p r o d u c e s an i n s o l u b l e e - g l u c a n . F r a c t i o n A F F - I I U was e l u t e d from a d e x t r a n a f f i n i t y column b y e i t h e r d e x t r a n o r u r e a , whereas f r a c t i o n AFF-IID was e l u t e d only by d e x t r a n . Both of t h e s e f r a c t i o n s produce i n s o l u b l e Q - g l u c a n s from s u c r o s e . S p e c i f i c i n h i b i t i o n by p e r i o d a t e - o x i d i z e d d e x t r a n s of t h e s y n t h e s i s o f a - Q - g l u c a n by S t r e p t o c o c c u s mutans p - g l u c o s y l -
134
Carbohydrate Chemistry
t r a n s f e r a s e prompted a s e a r c h f o r s t r u c t u r a l l y r e l a t e d i n h i b i t o r s t h a t m i g h t be e f f e c t i v e a s a n t i c a r i e s a g e n t . 1 6 6 Clinical dextran u n i t s were d e r i v a t i v e s i n which from 5 t o 5 0 % of t h e !-glucose o x i d i z e d a c t e d a s p o t e n t and s p e c i f i c e n z y m e - i n h i b i t o r s , a s d i d 10%o x i d i z e d d e r i v a t i v e s of d e x t r a n f r a c t i o n s r a n g i n g i n m o l e c u l a r w e i g h t f r o m lo4 t o 2 x l o 6 . W i t h i n t h e s e l i m i t s , d i f f e r e n c e s i n o x i d a t i o n o r m o l e c u l a r weight d i d n o t s i g n i f i c a n t l y a f f e c t t h e high I n contrast, periodate i n h i b i t o r y potency of t h e d e r i v a t i v e s . o x i d a t i o n o f (1+6)-, ( 1 + 3 ) - , a n d ( 1 + 4 ) - l i n k e d o l i g o s a c c h a r i d e s u n i t s and of s u c r o s e and s t r u c t u r a l l y c o n t a i n i n g <-15 a-Q-glucose related trisaccharides y i e l d e d d e r i v a t i v e s t h a t were poor inhibitors. Enzymic h y d r o l y s i s of o x i d i z e d d e x t r a n s caused a l o s s o f t h e i r i n h i b i t o r y power and i n d i c a t e d t h a t , t o a c t a s s p e c i f i c i n h i b i t o r s , o x i d i z e d m o l e c u l e s m u s t c o n t a i n a t l e a s t 1 6 t o 20 Q glucosyl residues. The s i m i l a r , m i n i m u m s i z e r e q u i r e d i n o r d e r t h a t unoxidized o l i g o s a c c h a r i d e s may a c t a s e f f i c i e n t a c c e p t o r s i n t h e ag l uco s y 1t r a n s f e r a s e r e a c t i o n s ugge s t s t h a t t h e i n h i b i t o r y p o t e n c i e s o f o x i d i z e d d e r i v a t i v e s may r e f l e c t t h e i r r e l a t i v e a b i l i t i e s t o b i n d a t t h e a c c e p t o r s i t e of t h e enzyme. Manganese s t i m u l a t e d t h e u t i l i z a t i o n o f Q - g l u c o s e by S t r e p t o c o c c u s mutans, S t r e p t o c o c c u s s a n g u i s , S t r e p t o c o c c u s m i t i o r , and S t r e p t o c o c c u s 1 n i 1 l e r i . l ~ ~S t r e p t o c o c c u s m u t a n s s e r o t y p e C s t r a i n s formed l a r g e r amounts o f intracellular-polysaccharide l a c t i c a c i d and produced a l o w e r t e r m i n a l pH i n p-glucose b r o t h when grown i n t h e p r e s e n c e o f 0.5 m M m a n g a n e s e i o n . Manganese i o n s t i m u l a t e d t h e u t i l i z a t i o n o f P-glucose by r e s t i n g c e l l s u s p e n s i o n s of each of t h e f o u r o r a l s t r e p t o c o c c a l s p e c i e s examined. Fluoride i n h i b i t e d t h e u t i l i z a t i o n o f Q - g l u c o s e by t h e s t r e p t o c o c c i , and m a n g a n e s e i o n , b u t n o t c a l c i u m o r magnesium i o n s , was f o u n d t o c o u n t e r a c t t h e i n h i b i t o r y e f f e c t of f l u o r i d e . S t r e p t o c o c c u s s a n g u i s i s o l a t e d f r o m human d e n t a l p l a q u e h a s been shown t o a d h e r e t o smooth s u r f a c e s ( g l a s s ) , a n d t h i s i n t e r a c t i o n i s a f f e c t e d by S t r e p t o c o c c u s mutans p-gluco s y l t r a n s f e r a s e . 168 The c e l l - w a l l component o f S t r e p t o c o c c u s s a l i v a r i u s HB,which mediates i n the coaggregation w i t h V e i l l o n e l l a a l c a l e s c e n s V1, has been i d e n t i f i e d . 16’ A c o m p a r i s o n o f t h e u s e o f t h e q u a t e r n a r y ammonium s a l t s cetyltrimethylammonium b r o m i d e ( C T A B ) a n d t h e c o m m e r c i a l m i x t u r e C e t a v l o n f o r t h e i s o l a t i o n o f x a n t h a n gum from f e r m e n t a t i o n s o f c a m p e s t r i s i n d i c a t e d t h a t t h e f o r m e r was t h e more -Xanthomonas --e f f i c i e n t c o m p l e x a t i n g agent.170 Although i n both c a s e s more t h a n
4: Microbial Polysaccharides
135
t h e s t o i c h i o m e t r i c r e q u i r e m e n t was n e c e s s a r y t o a c h i e v e q u a n t i t a t i v e r e c o v e r y o f t h e p o l y s a c c h a r i d e , CTAB l e f t o n l y 1.7% m a t e r i a l i n t h e s u p e r n a t a n t f r o m t h e p r e c i p i t a t i o n o f x a n t h a n gum c o m p a r e d t o 1 5 % l e f t by C e t a v l o n , w h i c h was i n t h e a g r e e m e n t w i t h t h e v i e w t h a t t h e e f f i c i e n c y o f q u a t e r n a r y ammonium s a l t s i n c r e a s e s w i t h i n c r e a s e d p a r a f f i n chain length.
An a s s e s s m e n t o f t h e u s e o f C e t a v l o n f o r t h e
i s o l a t i o n o f x a n t h a n gum i n a r e c y c l e p r o c e d u r e showed t h a t an 11.5% l o s s o f p r e c i p i t a n t per cycle occurred. xanthan
gum
was
dispersion of
precipitated
i t s
quaternary
as
the
ammonium
Concentration o f the 2-propanol
I n t h e procedure,
p u r i f i e d K+ complex
i n
salt
the
from
a
2-propanol.
wash p e r m i t t e d r e c o v e r y o f t h e
q u a t e r n a r y ammonium s a l t . The c o m p l e x a t i o n r e a c t i o n o f CTAB w i t h x a n t h a n h a s been s t u d i e d u s i n g 1 4 C - l a b e l l e d CTAB.171
I t was f o u n d t h a t l a r g e p r o p o r t i o n s o f
p r e c i p i t a n t were p r e s e n t i n s o l u t i o n d u r i n g t h e r e a c t i o n b u t a t t h e end-point solution.
o n l y 3.2% o f t h e r e s i d u a l p r e c i p i t a n t r e m a i n e d i n
M u t a g e n e s i s o f Xanthomonas S m p e s t r i s y i e l d e d t w o m a j o r c l a s s e s of
mutant,
different
both having cell-surface f r o m t h e w i l d type.172
polysaccharides fundamentally
The w i l d - t y p e
b a c t e r i u m produced
c o p i o u s amounts o f e x t r a c e l l u l a r s l i m e p o l y s a c c h a r i d e c o n t a i n i n g glucose,
B-mannose, a n d P - g l u c u r o n i c
a c i d i n a r a t i o o f 2:2:1.
e-
Non-
m u c o i d m u t a n t s produced t r a c e amounts o f e x o p o l y s a c c h a r i d e i d e n t i c a l t o the wild-type
product.
Crenated mutants produced m a t e r i a l with
an u n u s u a l c o m p o s i t i o n c o n t a i n i n g s u g a r s n o r m a l l y f o u n d i n t h e lipopolysaccharide.
Analysis
of
lipopolysaccharide fractions
t h e s e s t r a i n s showed t h a t t h e w i l d - t y p e contained predominantly Q-glucose.
from
polysaccharide fraction
Polysaccharides from the two
c l a s s e s o f m u t a n t b a c t e r i a w e r e s i m i l a r and c o n t a i n e d I - r h a m n o s e ,
!-
g a l a c t o s e , a n d s m a l l e r a m o u n t s o f ;-glucose. The r o l e o f
lipid-linked
exopolysaccharide freely
sugars
i n the biosynthesis o f
an
l i b e r a t e d i n t o t h e c u l t u r e medium h a s b e e n
d e m o n s t r a t e d f o r t h e f i r s t t i m e u s i n g Xanthomonas c a m p e s t r i s s t r a i n NRRLB-1459. 173 I t h a s been shown,
u s i n g EDTA-treated
c e l l s and d i f f e r e n t
combinations o f t h e adequate { 14C) l a b e l l e d donors,
t h a t phosphoenol
p y r u v a t e p r o v i d e s t h e a c e t a l r e s i d u e s and t h a t t h e t r a n s f e r
occurs
on t h e t e r m i n a l Q-mannose r e s i d u e o f t h e p e n t a s a c c h a r i d e - p h o s p h a t e phosphate-lipid,
a s j u d g e d b y t h e s o l u b i l i t y p r o p e r t i e s a n d DEAE-
c e l l u l o s e c o l u m n c h r o m a t o g r a p h y o f t h e compounds f o r m e d as w e l l as by
the
behaviour
of
t h e s u b s t a n c e s l i b e r a t e d by
m i l d a c i d and
136
Carbohydrate Chemistry
alkaline
treatments
and
Smith
degradation.174
The
subsequent
p o l y m e r i z a t i o n p r o c e s s l e a d s t o p y r u v u l a t e d x a n t h a n gum.
Bact e r i a l Po 1y saccha r i de s
M i s c e 11aneo us
6
C a l c o f l u o r W h i t e ST,
a s t i l b e n e d e r i v a t i v e used c o m m e r c i a l l y a s
an o p t i c a l b r i g h t e n e r f o r c e l l u l o s e , polymerization i n t o cellulose
increased t h e r a t e o f Q-glucose
by r e s t i n g c e l l s o f t h e G r a m - n e g a t i v e
b a c t e r i u m A c e t o b a c t e r ~ y 1 i n u m . l ~T ~h i s b a c t e r i u m n o r m a l l y p r o d u c e d a
ribbon
of
cellulose
that
i s
a
composite
I n c o n c e n t r a t i o n s above 0.1
microfibrils.
t h e assembly o f
of
crystalline
Calcofluor disrupts
mM,
c r y s t a l l i n e c e l l u l o s e I m i c r o f i b r i l s and t h e i r
i n t e g r a t i o n i n t o a c o m p o s i t e r i b b o n by s t o i c h i o m e t r i c b i n d i n g t o glucose residues o f newly polymerized q-glucan conditions,
t h e r a t e o f p-glucose
e-
Under t h e s e
p o l y m e r i z a t i o n i n c r e a s e s up t o 4
w h e r e a s o x y g e n u p t a k e i n c r e a s e s o n l y 10 t o
times the control rate, 15%.
chains.
These o b s e r v e d e f f e c t s
are
readily reversible.
If
free
C a l c o f l u o r i s washed away o r d e p l e t e d b e l o w t h e t h r e s h o l d v a l u e o f b i n d i n g t o c e l l u l o s e as p o l y m e r i z a t i o n continues,
ribbon production I t was c o n c l u d e d t h a t cell-directed, coupled
a n d t h e n o r m a l r a t e o f p o l y m e r i z a t i o n resume. p o l y m e r i z a t i o n and c r y s t a l l i z a t i o n a r e
p r o c e s s e s and t h a t t h e r a t e o f c r y s t a l l i z a t i o n d e t e r m i n e s
I t was sugge'sted t h a t
o f polymerization.
the rate
coupling must
be
m a i n t a i n e d f o r b i o g e n e s i s o f c r y s t a l l i n e c e l l u l o s e I. D u r i n g g r o w t h o f t w o s t r a i n s o f human c o l o n i c B a c t e r o i d e s , B a c t e r o i d e s o v a t u s ( V P I 0038-1) 51),
on L-arabino-e-xylans
was P - x y l o b i o s e , The
low
and B a c t e r o i d e s e g g e r t h i i
several oligomers,
w e r e r e l e a s e d i n t o t h e medium.176
- tem p e r a t u r e-
E s c h e r i c h i a c o l i 15T'R1
depe nde n t ce 11- d i v i s i on- de f e c t ive
were s t a i n e d w i t h a p e r i o d i c a c i d - S c h i f f microscopy,
m u ta n t
d i s p l a y s a wide range o f c e l l l e n g t h s ( 2 t o
500um) i n l a t e l o g a r i t h m i c p h a s e c u l t u r e s a t 24'C. l i g h t
(VPI B8-
the smallest o f which
When t h e s e c e l l s
technique and viewed under
polysaccharide accumulations appeared as
d i s c r e t e l y s t a i n e d areas a t t h e poles, and a t s i t e s throughout t h e cytoplasm of
elongated cells.177
r e l a t i o n s h i p between the
s i z e of
A s t a t i s t i c a l analysis o f the
stained areas
and c e l l
size
i n d i c a t e d t h a t t h e t o t a l amount o f p o l y s a c c h a r i d e i n c r e a s e d w i t h increasing c e l l length. of
T h i s o c c u r r e d b o t h by a n i n c r e a s e i n s i z e
e x i s t i n g p o l y s a c c h a r i d e - s t a i n e d a r e a s a n d by t h e c r e a t i o n o f new
areas.
Interestingly,
over a wide range o f c e l l size,
the r a t i o o f
137
4: Microbial Polysaccharides
c e l l v o l u m e o c c u p i e d by p o l y s a c c h a r i d e t o t o t a l c e l l v o l u m e r e m a i n e d n e a r l y c o n s t a n t w i t h a mean o f a b o u t 0.16.
These d a t a s u g g e s t t h e
e x i s t e n c e o f a h o m e o s t a t i c mechanism f o r r e g u l a t i n g p o l y s a c c h a r i d e concentration preparations
during
elongation.
specifically
stained
Electron for
microscopy
polysaccharide
o f
revealed
c l u s t e r s o f granules w i t h a s i m i l a r d i s t r i b u t i o n p a t t e r n t o t h a t o f t h e dense a r e a s s e e n by p h a s e - c o n t r a s t
micro~copy.'~~ The g r a n u l e s
were s u s c e p t i b l e t o a-amylase d i g e s t i o n , and c h e m i c a l a n a l y s i s o f t h e e x t r a c t e d and p u r i f i e d p o l y s a c c h a r i d e showed t h a t of
poly-P-glucose,
including
glycogen.
c o n t a i n e d a b o u t t w i c e as much poly-;-glucose
24OC
A t
i t consisted R1 c e l l s
the
and f o u r t i m e s as much
g l y c o g e n as a t 3 7 ' ~ . S t u d i e s were c a r r i e d o u t and
sugar
ColB2Fdr
linkages
to
t o e l u c i d a t e t h e n a t u r e of
F-like
(compatibility
conjugative p i l i
phosphate
encoded by
the
g r o u p F I I ) and EDP208 ( c o m p a t i b i l i t y g r o u p
FV) p l a s m i d s . 17' Both types o f p i l i ,
when i n t h e i n t a c t u n d i s s o c i a t e d s t a t e ,
w e r e f o u n d t o c o n t a i n a p p r o x i m a t e l y 3 rnol o f p h o s p h a t e a n d 3 m o l o f sugar per mol o f p i l i n .
However, f u r t h e r p u r i f i c a t i o n o f t h e t w o
t y p e s o f p i l i n by g e l - f i l t r a t i o n
chromatography
i n t h e presence o f
s o d i u m d o d e c y l s u l p h a t e r e m o v e d a l l o f t h e c a r b o h y d r a t e f r o m EDP208 p i l i n a n d a p p r o x i m a t e l y 6 5 % o f t h e c a r b o h y d r a t e f r o m Colt32 p i l i n . Approximately
0.8
to
remained associated chromatography,
1.0
mol of
with
Q-glucose
ColB2 p i l i n
per
after
mol of
protein
gel-filtration
b u t i t was n o t p o s s i b l e t o d e t e r m i n e w h e t h e r t h i s
was c o v a l e n t l y l i n k e d t o t h e p i l i n sodium dodecyl s u l p h a t e - r e s i s t a n t
or t i g h t l y associated i n a
manner.
Sodium d o d e c y l s u l p h a t e -
g e l c h r o m a t o g r a p h y d i d n o t r e m o v e p h o s p h a t e f r o m e i t h e r Colt32 o r EDP208 p i l i n s .
31P N . m . r .
associated phosphate
i s
spectroscopy indicated t h a t the p i l i n involved i n
a
phosphodiester
Acetone p r e c i p i t a t i o n o f chloroform-methanol
linkage.
extraction of
the
p u r i f i e d p i l i n m a t e r i a l r e d u c e d t h e p h o s p h a t e a s s o c i a t e d w i t h EDP208 p i l i n t o l e s s t h a n 0.04
m o l e c u l e p e r p i l i n monomer.
u n d e r t h e same c o n d i t i o n s , p i l i n monomer.
r e t a i n e d a p p r o x i m a t e l y 0.5
Co1B2 p i l i n , phosphate p e r
The e x t r a c t e d p h o s p h a t e - c o n t a i n i n g m o i e t i e s w e r e
i d e n t i f i e d as p h o s p h a t i d y l g l y c e r o l and p h o s p h a t i d y l e t h a n o l a m i n e by t h i n - l a y e r chromatography.
S i o c e t h e 31P n.m.r.
spectra for both
C o l B 2 a n d EDP208 w e r e i d e n t i c a l a n d n o s i g n a l o t h e r t h a n t h a t o f a p h o s p h o d i e s t e r was d e t e c t e d i n t h e C o l a 2 s p e c t r u m , t h e p h o s p h a t e r e m a i n i n g w i t h t h e C01B2 p i l i n a f t e r c h l o r o f o r m - m e t h a n o l i s m o s t l i k e l y due t o a t i g h t l y bound n o n c o v a l e n t r e s i d u e .
extraction
138
Carbohydrate Chemistry
When p - g l u c o s e i s t h e c a r b o n s o u r c e , g l y c o g e n was f o u n d t o a c c u m u l a t e i n p a r t i a l ( N H 4 + ) o r t o t a l ( N H 4 + and r e q u i r e d a m i n o acids) nitrogen starvation i n Escherichia c o l i s t r a i n s that are r e l A + o r relA-.18' However, glycogen accumulated only i n t h e r e l A + s t r a i n when r e q u i r e d a m i n o a c i d s w e r e d e p l e t e d o r I - i s o l e u c i n e s t a r v a t i o n was induced by L-valine a d d i t i o n . When g l y c e r o l i s t h e carbon s o u r c e , glycogen a c c u m u l a t e s i n both r e l A + and r e l A - s t r a i n s a f t e r I - v a l i n e a d d i t i o n . I t was concluded t h a t t h e relA gene i s not r e q u i r e d f o r glycogen a c c u m u l a t i o n when s y n t h e s i s of a l l n i t r o g e n c o n t a i n i n g compounds of t h e c e l l i s l i m i t e d o r a b o l i s h e d and t h a t , a l t h o u g h t h e relA gene i s needed f o r glycogen t o a c c u m u l a t e i n amino a c i d s t a r v a t i o n , t h i s r e q u i r e m e n t can b e r e p l a c e d b y a high c e l l u l a r c o n c e n t r a t i o n of 3',5'-cyclic AMP. M a l t o s e and l a c t o s e t r a n s p o r t s y s t e m s h a v e been u s e d t o i n v e s t i g a t e t h e a c t i o n o f p r o c a i n e on i n s e r t i o n and a c t i v i t y o f membrane p r o t e i n s and t r a n s l o c a t i o n of e x p o r t e d p r o t e i n s i n E s c h e r i c h i a c o l i . 1 8 1 P r o c a i n e m i l d l y i n h i b i t e d growth on l a c t o s e . The l e v e l o f i n h i b i t i o n was c o n s i s t e n t w i t h t h e s m a l l r e d u c t i o n o b s e r v e d i n a c t i v e and f a c i l i t a t e d t r a n s p o r t f u n c t i o n s of t h e permease. However, p r o c a i n e c a u s e d a s e v e r e r e d u c t i o n of g r o w t h r a t e on m a l t o s e , a s w e l l a s an i n h i b i t i o n of i n d u c t i o n o f m a l t o s e I n both c o n s t i t u t i v e and i n d u c i b l e s t r a i n s , t h e regulon a c t i v i t i e s . s y n t h e s i s of b o t h m a l t o s e t r a n s p o r t a c t i v i t y ( m a l B o p e r o n ) and a m y l o g l y c o s i d a s e a c t i v i t y (malA operon) was i n h i b i t e d . Coordinate i n h i b i t i o n o f s o l u b l e and membrane p r o d u c t s was n o t o b s e r v e d w i t h the operon. B-g-Galactosidase s y n t h e s i s proceeded normally d u r i n g growth on p r o c a i n e , whereas t h e a p p e a r a n c e of new t r a n s p o r t a c t i v i t y was r e d u c e d . R e g a r d l e s s of c a r b o n s o u r c e , p r o c a i n e s p e c i f i c a l l y i n h i b i t e d t h e a p p e a r a n c e o f ompF p r o t e i n i n t h e membrane f r a c t i o n . E s c h e r i c h i a c o l i i s c a p a b l e of g r o w i n g on L - f u c o s e o r Ir h a m n o s e a s a s o l e s o u r c e o f c a r b o n and e n e r g y . When grown u n d e r a n a e r o b i c c o n d i t i o n s on e i t h e r s u g a r , a n i c o t i n a m i d e a d e n i n e dinucleotide-linked L-lactaldehyde reductase a c t i v i t y i s induced. The f u n c t i o n i n g of t h i s enzyme r e s u l t s i n t h e r e g e n e r a t i o n o f o x i d i z e d n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e . C o n d i t i o n s of i n d u c t i o n o f t h e enzyme a c t i v i t y w e r e s t u d i e d and w e r e f o u n d t o d i s p l a y d i f f e r e n t c h a r a c t e r i s t i c s on each sugar.182 I n t h e I-rhamnose-grown c e l l s , t h e i n c r e a s e i n enzyme a c t i v i t y d e t e c t e d u n d e r i n d u c i n g c o n d i t i o n s was a c c o m p a n i e d b y t h e s y n t h e s i s o f t h e r e d u c t a s e , a s measured b y t h e appearance i n t h e e x t r a c t s of a p r o t e i n t h a t r e a c t s
4: Microbial Polysaccharides
139
with propanediol oxidoreductase antibodies. I n c o n t r a s t , i n If u c o s e - g r o w n c e l l s , t h e l e v e l o f t h e r e d u c t a s e a s m e a s u r e d by e n z y m e a n t i b o d y - r e a c t i n g m a t e r i a l was h i g h u n d e r n o n i n d u c i n g a n d i n d u c i n g c o n d i t i o n s . Thus, the i n c r e a s e i n enzyme a c t i v i t y detected i n g o i n g from noninducing t o i n d u c i n g c o n d i t i o n s i n L-fucose-grown c e l l s d i d not depend on t h e appearance of the s p e c i f i c p r o t e i n b u t on t h e activation of the reductase already present i n the cells i n an i n a c t i v e form. The r e d u c t a s e o f b o t h homologous s y s t e m s s h o u l d c o n s e q u e n t l y be r e g u l a t e d by d i f f e r e n t c o n t r o l m e c h a n i s m s . A h i g h - Q - x y l u l o s e m i x t u r e ( p - x y l o s e : p - x y l u l o s e = 3 3 : 6 7 ) was p r e p a r e d from t h e c o l d e t h a n o l e x t r a c t o f p r e i s o m e r i z e d p-xylose Fusarium oxysporum f. sp. s o l u t i o n ( Q - x y 1 o s e : Q - x y l u l o s e = 77:23). l i n i a n d A s p e r g i l l u s n i g e r were d e m o n s t r a t e d t o p r e f e r e n t i a l l y u t i l i z e Q-xylose i n t h e m i x t u r e of p-xylose and g-xylulose.'83 C h r o m a t o g r a p h i c a l l y p u r e g - x y l u l o s e was t h u s o b t a i n e d i n 90% y i e l d . A h i g h - Q - x y l u l o s e m i x t u r e was a l s o i n c u b a t e d w i t h R h o d o t o r u l a t o r u l o i d e s , Klebsiella pneumoniae, Candida u t i l i s , o r Mucor r o u x i . When b o r a t e 1 - X y l o s e a n d g - x y l u l o s e were s i m u l t a n e o u s l y c o n s u m e d . w a s a d d e d t o t h e m i x t u r e Q - x y l u l o s e - b o r a t e c o m p l e x was f o r m e d , a n d i t c o u l d be u s e d t o p r o t e c t g - x y l u l o s e f r o m b e i n g u t i l i z e d . The s u l p h a t e d h e t e r o s a c c h a r i d e f r o m t h e c e l l - w a l l g l y c o p r o t e i n o f H a l o b a c t e r i u m h a l o b i u m has b e e n s h o w n t o c o n t a i n a r e p e t i t i v e s t r u c t u r e w h i c h a r i s e s f r o m a s u l p h a t e d l i p i d - l i n k e d p r e c u r s o r v& a biosynthetic pathway different from that known for g l y c o s a m i n o g l y c a n s . '84 A regular array of exhibiting hexagonal periodicity with a c e n t r e - t o - c e n t r e s p a c i n g o f a b o u t 6 nm was f o u n d o n t h e w a l l s u r f a c e of freeze-etched Lactobacillus buchneri. T h e r e g u l a r a r r a y was c o m p o s e d o f a s u b u n i t p r o t e i n w i t h a m o l e c u l a r w e i g h t o f a b o u t 55000 a s d e t e r m i n e d by s o d i u m d o d e c y l s u l p h a t e - p o l y a c r y l a m i d e g e l e l e c t r o p h ~ r e s i s . ' ~ S~ u b u n i t s i s o l a t e d f r o m t h e w a l l by e x t r a c t i o n w i t h g u a n i d i n e h y d r o c h l o r i d e reassem b l e d i n t o t h e o r i g i n a l r e g u l a r a r r a y a f t e r d i a l y s i s a g a i n s t d i s t i l l e d water. The s u b u n i t s c o u l d r e a t t a c h t o wall f r a g m e n t s from which most o f t h e t e i c h o i c a c i d had been e x t r a c t e d w i t h cold t r i c h l o r o a c e t i c acid, b u t n o t t o wall f r a g m e n t s from which most o f t h e n e u t r a l p o l y s a c c h a r i d e had been r e m o v e d by t r e a t m e n t w i t h h o t f o r m a m i d e . H e t e r o l o g o u s r e a t t a c h m e n t o f t h e s u b u n i t s t o o k p l a c e on t o L a c t o b a c i l l u s casei subsp. casei w a l l f r a g m e n t s w h i c h had been p a r t i a l l y h y d r o l y s e d u n d e r m i l d a c i d conditions. These o b s e r v a t i o n s suggest that t h e s u b u n i t p r o t e i n binds t o a n e u t r a l polysaccharide moiety i n t h e underlying wall
140
Carbohydrate Chemistry
l a y e r b u t n o t t o p e p t i d o g l y c a n or t e i c h o i c acid. T h e f e r m e n t a t i o n o f c e l l u l o s e by m o n o c u l t u r e s o f A c e t i v i b r i o -------c e l l u l o l y ----ticus and cocultures of Acetivibrio cellulolyticusM e t h a n o s a r c i n a b a r k e r i , A c e t i v i b r z cellulolyticus-Desulfovibrio s p . , a n d A c e t i v i b r i o c e l l u l o ~ y t i c u ~ - M e t h a n o s a r c i n ab a r k e r i D e s u l f o v i b r i o s p . was s t u d i e d . 1 8 6 The m o n o c u l t u r e p r o d u c e d e t h a n o l , a c e t a t e , H2,and C02. M o r e a c e t a t e a n d l e s s e t h a n o l was f o r m e d b y t h e c o c u l t u r e s t h a n b y t h e m o n o c u l t u r e . A c e t a t e was u t i l i z e d b y Methanosarcina barkeri i n coculture with Acetivibrio c e l l u l o l y t i c u s a f t e r a l a g p e r i o d , w h e r e a s e t h a n o l was m e t a b o l i z e d by t h e s u l p h a t e r e d u c e r o n l y u n d e r c o n d i t i o n s o f l o w H 2 p a r t i a l p r e s s u r e , k.when c o c u l t u r e d w i t h A c e t i v i b r i o cellulolyticus-Methanosarcina b a r k e r i o r when g r o w n t o g e t h e r w i t h t h e m e t h a n o g e n . Only the t h r e e - c o m p o n e n t c u l t u r e c a r r i e d o u t t h e r a p i d conversion o f c e l l u l o s e t o C02 and methane. F u r t h e r m o r e , t h i s c u l t u r e h y d r o l y s e d t h e most c e l l u l o s e 85% o f t h a t i n i t i a l l y p r e s e n t . T h i s a m o u n t was i n c r e a s e d t o 90% by i n c r e a s i n g t h e population of MethanosarcLC2 b a r k e r i i n t h e t r i c u l t u r e . M e t h a n e p r o d u c t i o n was a l s o i n c r e a s e d , a n d a q u i c k e r f e r m e n t a t i o n r a t e was a c h i e v e d . 2-Amino-2-deoxy h e x o s e m e t a b o l i s m i n r e l a t i o n t o t h e s p h e r u l e w a l l s y n t h e s i s i n P h y s a r u m p o l y c e p h a l u ~ was s t u d i e d b y t h e i n c o r p o r a t i o n of labelled s u g a r i n t o the wall and i n t e r m e d i a r y compounds i n the b i o s y n t h e s i s o f wall polysaccharide.18' The i n c o r p o r a t i o n of 2-amino-2-deoxy-Q-{ 1 4 C ) g a l a c t o s e i n t o t h e wall m a t e r i a l o c c u r r e d a f t e r a l a g p e r i o d o f a b o u t 10 h i n a n i n d u c t i o n medium. N u c l e o t i d e s and s u g a r phosphates i n t h e a c i d - s o l u b l e f r a c t i o n o f s p h e r u l a t i n g p l a s m o d i a were a n a l y s e d by c o l u m n The p r i m a r y l a b e l l e d c h r o m a t o g r a p h y o f Dowex 1-X8 ( f o r m a t e ) . products formed i n the spherulating plasmodia a f t e r incubation with 2 - a m i n o - 2 - d e o x y -Q-{ 1 4 C ) g a l a c t o s e were 2 - a m i n o - 2 - d e o x y - P - g a l a c t o s e 1p h o s p h a t e , UDP-2-amino-2-deoxy-g-galactose, 2-amino-2-deoxy-hexose 6-phosphate, a n d UDP-2-amino-2-deoxy-hexose. S p h e r u l a t i o n was i n s e n s i t i v e t o p o l y o x i n D , w h i l e i t was c o m p l e t e l y b l o c k e d by cycloheximide. T h e a c t i v i t y o f g - g a l a c t o k i n a s e a n d g- - g a l a c t o s e - l phosphate u r i d y l t r a n s f e r a s e increased 4.5-fold during the spherulation. F o u r l y s o z y m e - s e n s i t i v e m u t a n t s were i s o l a t e d a f t e r n i t r o s o g u a n i d i n e mutagenesis o f a lysozyme-insensitive s t r a i n of S t a p h y l o c o c c u s aureus.188 One m u t a n t was s u f f i c i e n t l y e f f e c t i v e f o r t h e i s o l a t i o n o f macromolecules, such as p l a s m i d deoxy-ribonucleic acids, from a c e l l after lysozyme-induced c e l l l y s i s .
4: Microbial Polysaccharides
141
Fungal Polysaccharides
7
The g e n e t i c m a n i p u l a t i o n o f i n d u s t r i a l y e a s t s h a s b e e n r e v i e w e d w i t h p a r t i c u l a r e m p h a s i s o n e t h a n o l p r o d u c t i o n and t o l e r a n c e , genetics
of
brewer's
carbohydrates,
yeast
flocculation,
strains,
metabolism
of
the wort
t r a n s f o r m a t i o n , and p r o t o p l a s t
f u s i o n . 189 The
i n t e r a c t i o n o f l e c t i n s and l e c t i n - l i k e s u b s t a n c e s w i t h
f u n g i f o r i d e n t i f i c a t i o n o f p a r t i c u l a r sugar residues or f u n g a l species o r t h e s e p a r a t i o n o f p o l y s a c c h a r i d e s from f u n g a l c e l l w a l l s h a s b e e n r e v i e w e d .'l The
inhibitory
influence
c r o t o n a l d e h y d e was
of
the
higher
concentration
of
f o l l o w e d d u r i n g t h e b a t c h and l o n g - t e r m
c o n t i n u o u s f e r m e n t a t i o n o f Candida u t i l i s g r o w i n g on s y n t h e t i c M o s t c r o t o n a l d e h y d e .was r e m o v e d f r o m t h e m e d i u m b y ethan01.l~' b i o t r a n s f o r mat i o n . C r o t o n a l d e h y d e i n h i b i t s t h e growth, lengthens t h e l a g phase,and decreases t h e b i o m a s s y i e l d and t h e c o n t e n t o f c r u d e p r o t e i n s i n t h e biomass.
The y e a s t C a n d i d a u t i l i s i s c a p a b l e
o f g r o w i n g on media c o n t a i n i n g very h i g h c o n c e n t r a t i o n s o f i n h i b i t o r i n the in-flow transport
during continuous c u l t i v a t i o n .
oscillations
observed f o r
of
the
content
of
Uncharacteristic
crotonaldehyde
were
w h i c h a c i d i c g r o u p s on t h e c e l l membrane a r e p r o b a b l y
responsible.
A s e n s i t i v e method which i s s u i t a b l e f o r
measuring
v e r y l o w c o n c e n t r a t i o n o f c r o t o n a l d e h y d e i n aqueous s o l u t i o n s has been d e s c r i b e d .
C r o t o n a l d e h y d e a c t s as an u n c o m p e t i t i v e i n h i b i t o r
w i t h s l i g h t mixed type o f i n h i b i t i o n .
An e q u a t i o n d e s c r i b i n g t h e
k i n e t i c s o f i n h i b i t i o n h a s been d e r i v e d . The a d h e r e n c e o f C a n d i d a a l b i c a n s t o a c r y l i c h a s been m e a s u r e d
-in vitro
a f t e r g r o w t h o f t h e yeast t o s t a t i o n a r y phase i n d e f i n e d
medium c o n t a i n i n g g - g l u c o s e ,
sucrose,
m a l t o s e as t h e c a r b o n source.191
g-galactose,
I n e a c h case,
e-fructose,
or
y e a s t a d h e r e n c e was
p r o p o r t i o n a l t o t h e c o n c e n t r a t i o n o f s u g a r i n t h e g r o w t h medium, b u t e q u i m o l a r c o n c e n t r a t i o n s o f d i f f e r e n t s u g a r s p r o m o t e d adherence t o d i f f e r e n t extents.
I n v i t r o a d h e r e n c e was f u r t h e r
i n c r e a s e d by t h e
a d d i t i o n o f d i v a l e n t c a t i o n s t o a s s a y m i x t u r e s b u t was i n h i b i t e d when s a l i v a - t r e a t e d a c r y l i c s t r i p s w e r e u s e d o r when y e a s t s w e r e s u s p e n d e d i n m i x e d s a l i v a d u r i n g t h e assay.
The r a t e o f s p h e r o p l a s t
f o r m a t i o n o f y e a s t s g r o w n i n m e d i a c o n t a i n i n g a 500 m M c o n c e n t r a t i o n o f t h e d i f f e r e n t s u g a r s c o r r e l a t e d w e l l w i t h t h e r e l a t i v e adherence of
the
cells
to
acrylic.
0-Galactose-grown
yeasts
r e s i s t a n t t o s p h e r o p l a s t f o r m a t i o n w i t h Zymolyase-500
were
more
and m o s t
142
Carbohydrate Chemistry w h e r e a s Q - fr u c t o s e - g r o w n o r g a n i s m s were
a d h e r en t t o a c r y l i c ,
least
r e s i s t a n t t o s p h e r o p l a s t f o r m a t i o n and l e a s t a d h e r e n t t o a c r y l i c . These r e s u l t s i n d i c a t e t h a t when g r o w n t o s t a t i o n a r y p h a s e i n m e d i a containing high concentrations o f
c e r t a i n sugars,
u n d e r g o e s a change i n c e l l - s u r f a c e
composition which f a c i l i t a t e s i t s
adherence
to
acrylic
surfaces.
Electron
Candida
microscopy
albicans
of
yeasts
h a r v e s t e d f r o m such m e d i a r e v e a l e d t h e p r e s e n c e o f an a d d i t i o n a l s u r f a c e l a y e r w h i c h may be r e s p o n s i b l e f o r t h i s e n h a n c e d a d h e r e n c e . The new
yeast
s p e c i e s tianein~as~gma n g d i n i g y i h a s b e e n
d e s c r i b e d t o accommodate members o f t h e genus H a n s e n i a s p o r a t h a t a r e unable
to
assimilate
D-glucono-1,4-lactone
and
isolated
from
s t r o m a t a l t i s s u e o f b l a c k k n o t s ( D i b o t r y o n morbosum) o f c h o k e c h e r r y , Prunus
virginiana.192
resemblance,
The
newly
described
taxon
showed
much
by o t h e r c r i t e r i a , t o H a n s e n i a s p o r a v i n e a e van d e r W a l t
e t Tscheuschner
and H a n s e n i a s p o r a o s m o p h i l a ( N i e h a u s ) P h a f f ,
Miller,
and S h i f r i n e . I n
strain
IGC
4052
of
the
amylolytic
yeast
Lipom.yces
kononenkoae g r o w i n g i n s t a r c h - l i n k e d c h e m o s t a t c u l t u r e s t h e c r i t i c a l d i l u t i o n r a t e was r e d u c e d t o a b o u t h a l f o f i t s t h e o r e t i c a l v a l u e due t o severe c a t a b o l i t e r e p r e s s i o n o f amylase f o r m a t i o n w h i l e i t s value i n a repression-resistant The e n z y m e - y i e l d
m u t a n t was n e a r i t s t h e o r e t i c a l value.193
c o e f f i c i e n t s and t h e s p e c . i f i c
production rates o f
a-amylase and g l u c o a m y l a s e passed t h r o u g h maxima a t i n t e r m e d i a t e dilution rates. d e t e r m i n e d by
The s h a p e s o f t h e r e s p e c t i v e c u r v e s w e r e p a r t l y
c a t a b o l i t e repression (parent
s t r a i n ) or
i t s absence
( m u t a n t s t r a i n ) w h i l e i n d u c t i o n d i d n o t seem t o p l a y a r o l e .
An
a d d i t i o n a l g r o w t h - l i n k e d r e g u l a t o r y mechanism seemed t o b e i n v o l v e d . The u s e o f
continuous
c u l t u r e as
compared
with
batch
culture,
i n c r e a s e d t h e maximum b i o m a s s p r o d u c t i v i t y b y a f a c t o r o f 2.2 m u t a n t s t r a i n and b y a f a c t o r o f 1.4
i n the
i n the parent strain.
The a b i l i t y o f n o n - p a t h o g e n i c M y c o b a c t e r i u m s p e c i e s t o grow on Q-galactose
as
the
source
of
carbon has
been
studied.194
The
o u t l i n e o f t h e c a t a b o l i c p a t h w a y h a s been p r e s e n t e d . Glycogen p h o s p h o r y l a s e
from
macroplasmodia o f
p o l y c e p h a l u m was p u r i f i e d 7 6 - f o l d t o h o m o g e n e i t y . 1 9 5
Physarum The n a t i v e
enzyme m i g r a t e d as a s i n g l e p r o t e i n band on a n a l y t i c a l d i s c g e l electrophoresis
coinciding w i t h phosphorylase a c t i v i t y .
After
r e d u c t i o n i n t h e p r e s e n c e o f s o d i u m d o d e c y l s u l p h a t e one p r o t e i n b a n d was d e t e c t a b l e w h i c h c o r r e s p o n d e d t o a n
Mr
o f 93000.
The
m o l e c u l a r w e i g h t o f t h e n a t i v e enzyme d e t e r m i n e d b y g e l s i e v i n g o r gradient-polyacrylamide
gel electrophoresis
was
1 7 2 0 0 0 a n d 186000,
143
4: Microbial Polysaccharides respectively.
The
enzyme
p h o s p h a t e a n d l e s s t h a n 0.1 subunit.
contained about
1 m o l p y r i d o x a l 5'-
m o l c o v a l e n t l y bound p h o s p h a t e p e r
mol
The a m i n o a c i d c o m p o s i t i o n o f t h e enzyme was d e t e r m i n e d .
I n t h e d i r e c t i o n o f p h o s p h o r o l y s i s t h e k i n e t i c d a t a were d e t e r m i n e d by i n i t i a l v e l o c i t y s t u d i e s , mechanism.
assuming a r a p i d e q u i l i b r i u m random
a - G l u c o s e l - p h o s p h a t e and GDP-a-glucose
were c o m p e t i t i v e
i n h i b i t o r s t o w a r d p h o s p h a t e and n o n - c o m p e t i t i v e t o g l y c o g e n . weak a c t i v a t o r o f t h e enzyme, i n h i b i t i o n completely.
a
P h y s a r u m p h o s p h o r y l a s e was c o m p a r e d w i t h
phosphorylases from other kinetic properties.
AMP,
counteracted t h e P-glucose-l-phosphate s o u r c e s on t h e b a s i s o f
c h e m i c a l and
No e v i d e n c e f o r t h e p r e s e n c e o f p h o s p h o r y l a t e d
f o r m s was f o u n d . A s p e c i m e n o f a r h i n o s p o r i d i a l n a s a l p o l y p o f I n d i a n o r i g i n was used t o study
the chemistry
and development
of
the
wall of
Rhinosporidium seeberi using fluorescein isothiocyanate-labelled lectins.196 The s c r e e n i n g o f t w e n t y y e a s t s t r a i n s f o r
ethanol productivity
a t h i g h o s m o t i c pressure a t temperatures r a n g i n g from h a s been d e s c r i b e d . l g 7
5OoC Bx cane m o l a s s e s w e r e p e r f o r m e d . productivity a t a non-inhibitory Most
strains
The e f f e c t
o f t e m p e r a t u r e on
e t h a n o l l e v e l i s weakly pronounced.
fermented poorly
at
Schizosaccharomyces pombe and one Saccharomyces cerevisiae,performed molasses.
32OC t o 45OC
Shake f l a s k f e r m e n t a t i o n s o f 3OoC, 4OoC, a n d
5 O o C Bx
molasses
but
commercial baker's
two
yeast,
well at a l l concentrations o f
I n an e x t e n d e d s t u d y w i t h S c h i z o s a c c h a r o m y c e s pombe (CBS
352) a n d S a c c h a r o m y c e s c e r e v i s i a e (SJAB,
fresh yeast)
simulating a
c o n t i n u o u s r u n , i t was shown t h a t S c h i z o s a c c h a r o m y c e s pombe was l e s s s e n s i t i v e t o h i g h DS t h a n S a c c h a r o m y c e s c e r e v i s i a e .
A t 25% DS t h e
p r o d u c t i v i t y o f S c h i z o s a c c h a r o m y c e s pombe i s a l m o s t t w i c e t h a t o f Saccharomyces c e r e v i s i a e . A p l a s m i d has been c o n s t r u c t e d t h a t a l l o w s t h e s e l e c t i o n
v i v o of
in
gene f u s i o n s b e t w e e n t h e E s c h e r i c h i a c o l i B - g - g a l a c t o s i d a s e
gene a n d t h e y e a s t
( S a c c h a r o m y c e s c e r e v i s a e I E 3 gene.lg8
y e a s t DNA f r a g m e n t
containing the a
A
large
3 gene was p l a c e d u p s t r e a m o f
an a m i n o - t e r m i n a l l y d e l e t e d v e r s i o n o f t h e l a c 2 gene.
The p l a s m i d
v e h i c l e c o n t a i n s sequences t h a t a l l o w s e l e c t i o n a n d m a i n t e n a n c e o f t h e p l a s m i d i n b o t h y e a r s and E s c h e r i c h i a c o l i . S e l e c t i o n f o r Lac' i n E s c h e r i c h i a c o l i y i e l d e d numerous d e l e t i o n s t h a t f u s e d t h e l a c 2 gene t o t h e f i 3
gene and f l a n k i n g y e a s t s e q u e n c e s , t o t h e b a c t e r i a l
tetracycline-resistance
gene f r o m t h e p a r e n t p l a s m i d pBR322,
t h e y e a s t 2-pm p l a s m i d DNA.
and t o
Some o f t h e s e f u s i o n p l a s m i d s p r o d u c e d
Carbohydrate Chemistry
144 B-Q-galactosidase fusions t o the expression of
activity
USA3
when i n t r o d u c e d i n t o y e a s t .
One o f
the
gene i t s e l f has been shown t o p l a c e t h e
6-P-galactosidase
a c t i v i t y under
uracil regulation i n
yeast. The
chemical modification o f
ribosomal subunit
of
yeast
185 r i b o s o m a l R N A
Saccharomyces c e r e v i s i a e
i n the
with
40s
dimethyl
s u l p h a t e h a s been r e ~ 0 r t e d . l ~ ' The d a t a w e r e u s e d t o t e s t t h e m o d e l o f t h e s e c o n d a r y s t r u c t u r e o f e u k a r y o t i c 18s R N A . Commercial baker's yeast (Saccharomyces c e r e v i s i a e ) has been used t o
study
the
conversion
presence of g-xylose.
of
Q-xylulose
an i n c r e a s e i n y e a s t - c e l l d e n s i t y . x y l u l o s e f e r m e n t a t i o n was 35'C, e t h a n o l as t h e
ethanol
i n
the
The r a t e o f t e m p e r a t u r e f o r Q-
a n d t h e o p t i m a l pH r a n g e was 4 t o 6.
The f e r m e n t a t i o n o f Q - x y l u l o s e by y e a s t of
to
The r a t e o f e t h a n o l p r o d u c t i o n i n c r e a s e d w i t h
g l y c e r o l were a l s o produced.200
resulted i n the production
S m a l l amounts o f x y l i t o l and
major product.
The p r o d u c t i o n o f
xylitol
was
i n f l u e n c e d b y pH a s w e l l a s t e m p e r a t u r e .
H i g h pH v a l u e s a n d l o w
temperatures
The
enhanced
fermentation
x y l i t o l production.
decreased
concentrations o f
4%
when
o r more.
x y l u l o s e t o e t h a n o l was density.
the
rate
production of The s l o w
of
Q-xylulose
ethanol yielded
conversion r a t e o f
Q-
i n c r e a s e d by i n c r e a s i n g t h e y e a s t - c e l l
The o v e r a l l p r o d u c t i o n o f e t h a n o l f r o m Q - x y l u l o s e by y e a s t
c e l l u n d e r o p t i m a l c o n d i t i o n s was 90% o f t h e t h e o r e t i c a l y i e l d . Q-Glucose,
Q - g a l a c t o s e , and Q - f r u c t o s e u p t a k e h a s been s t u d i e d
d u r i n g t h e g e r m i n a t i o n o f Streptomyces a n t i b i o t i c u s spores. spores had o n l y t h e Q-glucose uptake system, s o u r c e o f t h e s p o r u l a t i o n medium. i n t h e presence o f Q-galactose energy
source,
detected.*O1
neither
Dormant
whatever t h e carbon
Even when s p o r u l a t i o n t o o k p l a c e
o r Q - f r u c t o s e as t h e s o l e c a r b o n and
Q-galactose
nor
Q-fructose
uptake
was
D u r i n g g e r m i n a t i o n t h e uptake systems f o r these two
s u g a r s w e r e i n d u c i b l e by t h e a p p r o p r i a t e s u g a r .
The i n d u c t i o n t i m e
was l e s s when t h e g e r m i n a t i o n p r o c e s s was more advanced.
Synthesis
o f a l l u p t a k e s y s t e m s was i m m e d i a t e l y b l o c k e d b y t h e a d d i t i o n o f i n h i b i t o r s o f p r o t e i n or RNA synthesis. e x p o n e n t i a l phase o f
Vegetative mycelium i n t h e
g r o w t h showed a s i m i l a r
uptake.
Q-Glucose
fructose
uptake systems.
pattern of
caused r e p r e s s i o n o f t h e Q-galactose The
three
sugar
uptake systems
s t r o n g l y i n h i b i t e d by p o t a s s i u m c y a n i d e , 2 , 4 - d i n i t r o p h e n o l , chloromercuribenzoate,
sugar and
p-
were
and 4-
and showed o p t i m u m t e m p e r a t u r e s f o r u p t a k e a t
4 O o C ( Q - g l u c o s e ) and 45OC ( Q - g a l a c t o s e and Q - f r u c t o s e ) .
G r o w t h w i t h i n t h e pH r a n g e 2 t o 8 o f a s t r a i n o f t h e y e a s t
4: Microbial Polysaccharides
T-o r u l o p s i s
145
p i n t o l o p e s i i was t e s t e d i n m e d i a c o n t a i n i n g
glucose,
P-fructose,
various
O f t h e sugars t e s t e d ,
s u g a r s as c a r b o n and e n e r g y s o u r c e s .
a-
only
and 1-mannose s u p p o r t e d g r o w t h o f t h e yeast.202
I n media c o n t a i n i n g t h o s e s u g a r s ,
t h e o r g a n i s m grew o v e r t h e e n t i r e
pH r a n g e t e s t e d . Microscopic
examination of
h o r s e caecum c o n t e n t s r e v e a l e d
v e g e t a t i v e g r o w t h o f p h y c o m y c e t e f u n g i on p a r t i c l e s o f d i g e s t a ,
and
u n i f l a g e l l a t e d c e l l s s i m i l a r t o f u n g a l zoospores i n t h e l i q u i d phase.203
Three
morphologically
distinct
isolates of
strictly
a n a e r o b i c p h y c o m y c e t e f u n g i were o b t a i n e d f r o m t h e caecum c o n t e n t s and c u l t u r e s i n v i t r o .
Two o f t h e i s o l a t e s w e r e a b l e t o u t i l i z e a
wide range o f p l a n t carbohydrates f o r growth, Q - x y l a n and p a r t i c u l a t e s t a r c h ,
including a-cellulose,
and e x t e n s i v e l y
digested water-
insoluble plant tissues. F u n g i and y e a s t s have been c u l t i v a t e d on m e d i a p r e p a r e d f r o m c a r r a g e e n a n f r o m Eucheuma s t r i a t u m ( S c h m i t ~ ) . ~ ' ~The c u l t u r e s were f o u n d t o compare w e l l w i t h t h o s e grown on agar media.
P-Glucans.
-ambisexualis basis for
-
Chemical analyses o f the hyphal w a l l o f Achlya R a p e r h a v e b e e n p e r f o r m e d i n an a t t e m p t t o p r o v i d e a
understanding the
W a l l s were i s o l a t e d , solubility.
periodate oxidation.
l
l
t h e i r n e u t r a l s u g a r c o n t e n t and t y p e
The d e g r e e o f b r a n c h i n g was e s t i m a t e d b y The o n l y
Three disaccharides,
cellobiose, linkages.
a
The r e s u l t i n g h y d r o l y s a t e s were a n a l y s e d by paper and chromatography f o r
o f glycosidic linkages. glucose.
architecture o f the ~
and f r a c t i o n a t e d by d i f f e r e n t i a l
The w a l l and i t s f r a c t i o n s were t h e n h y d r o l y s e d by a c i d
o r enzymes. gas-liquid
molecular
purified,
were d e t e c t e d ,
monosaccharide laminaribiose,
t h u s i n d i c a t i n g (1+3)-,
A c i d - s o l u b l e (1+3)-B-Q-glucan
detected
was
gentiobiose, (1+6)-,and
Qand
(1+4)-
w i t h s i n g l e (1+6)-linked
r e s i d u e s r e p r e s e n t e d 40% o f t h e w a l l . A l k a l i - s o l u b l e Q-glucan, a l i n e a r p o l y m e r o f (1+3)- and ( 1 + 4 ) - l i n k a g e s w i t h o c c a s i o n a l (1+6)s i d e chains,
r e p r e s e n t e d 7% o f t h e w a l l .
by s o l u b i l i t y and X - r a y d i f f r a c t i o n , The
insoluble
residuum
(6%)
Cellulose,
as d e t e r m i n e d
r e p r e s e n t e d 21% o f t h e w a l l .
which
remained
after
these
s o l u b i l i z a t i o n s had a l i n k a g e p a t t e r n s i m i l a r t o t h e a l k a l i - s o l u b l e fraction.
An i n s o l u b l e component c o n s i s t i n g o f 2-amino-2-deoxy-a-
g l u c o s e r e p r e s e n t e d 3% o f t h e w a l l . w a l l and on h y d r o l y s i s gave including hydroxy-i-proline.
P r o t e i n c o m p r i s e d 10% o f t h e
t h e f u l l spectrum o f amino a c i d s S m a l l amounts o f p h o s p h o r u s - w e r e
.
~
Carbohydrate Chemistry
146 detected
.
The
surface
configuration
of
t h e h y p h a l w a l l s and v a r i o u s
f r a c t i o n s o f t h e w a l l o f A c h l y a a m b i s e x u a l i s Raper were a n a l y s e d b y electron
microscopy
F r a c t i o n a t i o n was
using
carbon-platinum
carried out
with
replicas.206
b o t h enzymes
and
chemical
L a m i n a r i n a s e w i t h o r w i t h o u t p r o t e a s e and a c i d w i t h
solvents.
a l k a l i r e m o v e d t h e a c i d - and a l k a l i - s o l u b l e u n d e r l y i n g p a t t e r n of
microfibrils.
P - g l u c a n s , r e v e a l i n g an
The c o m b i n a t i o n o f c e l l u l a s e ,
l a m i n a r i n a s e , and p r o t e a s e e s s e n t i a l l y d i s s o l v e d t h e hyphae.
The
c e l l u l o s e s o l v e n t cadoxen r e m o v e d t h e m i c r o f i b r i l l a r p a t t e r n w h i c h was e x p o s e d f o l l o w i n g
a c i d and a l k a l i t r e a t m e n t .
f r a c t i o n i s amorphous,
t h e a l k a l i - s o l u b l e and t h e i n s o l u b l e r e s i d u u m
is f a i n t l y m i c r o f i b r i l l a r , strongly microfibrillar.
The a c i d - s o l u b l e
and t h e c e l l u l o s e I1 p r e p a r a t i o n i s
B o t h t h e c e l l u l o s e I and t h e c h i t i n l i k e
fractions are uniformly m i c r o f i b r i l l a r . c o n s i s t s o f an o u t e r m a t r i x o f
(1+3)-
Morphologically,
a n d (1+6)-B-P-glucans
the w a l l covering
an i n n e r c e l l u l o s i c p r o t e i n c o r e . The
polymorphic
fungus
Aureobasidium
pullulans
has
been
f r a c t i o n a t e d i n t o m y c e l i u m and s i n g l e c e l l s a t t h r e e p o i n t s i n t h e growth cycle approximately corresponding t o early, e x p o n e n t i a l phase.
m i d d l e , and l a t e
The a b i l i t y o f c e l l s t o d i v e r t a s s i m i l a t e d
a-
glucose t o form the e x t r a c e l l u l a r polysaccharide p u l l u l a n varies throughout
the
polysaccharide, it.207 Thus,
cycle.
The
spores
are
the
major
source
of
a l t h o u g h t h e hyphae a r e a l s o capable o f p r o d u c i n g
t h e commencement o f p u l l u l a n e x c r e t i o n ,
w h i c h h a s been
a s s o c i a t e d w i t h b l a s t o s p o r e p r o d u c t i o n i n t h e e a r l y phases o f t h e growth cycle, with,
probably arises through
some s t i m u l a t i o n c o n c u r r e n t
though not n e c e s s a r i l y i d e n t i c a l to,
the i n i t i a t i o n o f spore
formation. Lamellar Asperqillus
single niger
degradation as
crystals
have
of
been
an approach
the
a-e-glucan
subJected to
to
nigeran
controlled
from
enzymic
understanding polysaccharide
organization i n these crystals.208
A n a l y s i s o f b o t h r e a c t a n t and
p r o d u c t s by v a r i o u s methods a r g u e s t h a t a s i t u a t i o n e x i s t s where t h e c r y s t a l s a r e i n a c c e s s i b l e a t 2OoC b u t become i n c r e a s i n g l y a c c e s s i b l e as t h e t e m p e r a t u r e a p p r o a c h e s t h a t o f s o l u t i o n f o r t h e c r y s t a l s i n water.
I t was c o n c l u d e d t h a t
chain folds
are
inaccessible t o
e n z y m e s a t 20°C a n d become i n c r e a s i n g l y a c c e s s i b l e a s t h e m e l t i n g t e m p e r a t u r e i s approached.
T h i s c o u l d be due t o i n c r e a s e d m o b i l i t y
o f t h e s u r f a c e c h a i n s as t e m p e r a t u r e i s r a i s e d ,
which produces l a r g e
d y n a m i c l o o p s a n d makes enzyme d e g r a d a t i o n p o s s i b l e .
The g e n e r a l
147
4: Microbial Polysaccharides conclusion
was t h a t
nigeran single-crystal
surfaces are disordered
and i n c r e a s i n g l y m o b i l e c l o s e t o t h e i r d i s s o l u t i o n t e m p e r a t u r e i n water. Antitumour isolated
activities
from
(kikurage,
the
of
two
f r u i t i n g body
an e d i b l e
mushroom),
(1+6)-branched of
(1+3)-B-Q-glucans,
Auricularia
and o t h e r
auricula-judae
branched p o l y s a c c h a r i d e s
or (1+3)-a-Q-
c o n t a i n i n g a backbone c h a i n o f (1+3)-a-!-glucosidic mannosidic
linkages
{and
their
corresponding
(1+3)-a-glycans,
d e r i v e d by m i l d , S m i t h d e g r a d a t i o n ) have been compared.209 these polysaccharides, a water-soluble, (Q-glucan
I)
inhibitory
a c t i v i t y against
mice.
of
Auricularia
auricula-judE
i m p l a n t e d Sarcoma
The a l k a l i - i n s o l u b l e
branches attached,
hydroxy
h a v i n g numerous
periodate oxidation,
and m i l d , a c i d h y d r o l y s i s , t h e r e s u l t i n g ,
attached
at
0-6
having covalently linked poly-
of
the
(1+3)-linked
residues, exhibited potent antitumour a c t i v i t y . ions using the n-glucan-polyalcohol the polyhydroxy
a-glucosyl
Further investigat-
i n d i c a t e d t h a t t h e attachment of
g r o u p s t o t h e (1+3)-B-n-glucan
t h e antitumour potency o f t h e p-glucan.
b a c k b o n e may enhance
On t h e o t h e r hand,
partial
i n t r o d u c t i o n o f c a r b o x y m e t h y l g r o u p s i n t o Q - g l u c a n I1 (d.s. 0.86),
which a l t e r e d the i n s o l u b i l i t y property,
the antitumour
in
II),
showed e s s e n t i a l l y no
was m o d i f i e d by c o n t r o l l e d ,
degraded Q-glucan,
groups
potent,
(g-glucan
When t h e l a t t e r B - g l u c a n ,
borohydride reduction, water-soluble,
exhibited
180 s o l i d t u m o u r
b r a n c h e d (1+3)-B-!-glucan
a m a j o r c o n s t i t u e n t o f t h e f r u i t i n g body, inhibitory activity.
Among
branched (1+3)-B-Q-glucan
activity.
0.47-
f a i l e d t o enhance
The i n t e r r e l a t i o n b e t w e e n t h e a n t i t u m o u r
a c t i v i t y a n d t h e s t r u c t u r e o f t h e b r a n c h e d (1+3)-B-g-glucan d i s c u s s e d , on t h e b a s i s o f m e t h y l a t i o n and 1 3 C n.m.r.
h a s been
studies o f the
p e r i o d a t e - m o d i f i e d Q-glucans. Cultured blastospores o f
Candida a l b i c a n s e x h i b i t
cytoplasmic
g r a n u l a r i t y which o b l i t e r a t e s t h e i d e n t i f i c a t i o n o f i t s organelles. Abundant
glycogen
particles,
identified
by
light
and
electron
m i c r o s c o p y , c o n t r i b u t e d t o t h i s phenomenon t o a l a r g e e x t e n t . * 1 ° The i n h i b i t o r y e f f e c t o f p a p u l a c a n d i n B and a c u l e a c i n A on t h e (1+3)-B-E-glucan
synthase
f r o m G e o t r i c h u m l a c t i s h a s been shown t o
be s p e c i f i c and t o o c c u r b o t h p r o d u c t i o n of
cell-free
in
v i t r o and i n v i v o , l e a d i n g t o t h e
extracts
with
a partially
inactive
s y n t h a s e .*I1 Studies of
c a r b o n r e q u i r e m e n t s f o r s p o r u l a t i o n by Nadsonia
f u l v e s c e n s showed an u n e x p e c t e d a p p e a r a n c e o f of
an exogenous c a r b o n s o u r c e ,
s p o r e s i n t h e absence
when t h e c e l l s w e r e g r o w n on e t h a n o l
148
Carbohydrate Chemistry
p r i o r t o i n d u c t i o n of
sporulation.212
Q-Glucose-grown
c e l l s taken
f r o m e i t h e r t h e l o g a r i t h m i c o r s t a t i o n a r y phase r e q u i r e d a carbon source
(Q-glucose,
or glycerol) for
ethanol,
sporulation.
An
i n h i b i t o r y e f f e c t by a c e t a t e upon s p o r u l a t i o n was d e m o n s t r a t e d ,
and
appeared t o be due t o a r e p r e s s i o n mechanism. The e f f e c t
o f medium components and c u l t u r a l c o n d i t i o n s h a s
been s t u d i e d on t h e p r o d u c t i o n o f p o l y s a c c h a r i d e ( p e n d u l a n ) and g r o w t h o f m y c e l i a i n P o r o d i s c u l u s p e n d u l u s JTS-3009.213 P-mannose,
p-fructose,
cellobiose,
sucrose,
s t a r c h e s w e r e f o u n d t o be s u i t a b l e c a r b o n s o u r c e s . p r o d u c e d and m y c e l i a grew
g-Glucose,
a-mannito1,and
w e l l on ammonium,
various
P e n d u l a n was
nitrate,
and o r g a n i c
nitrogen.
The o p t i m a l c o n c e n t r a t i o n o f n i t r o g e n was 0.03% f o r
production
of
a d d i t i o n of production
pendulan
suitable and
and
mycelium
for
0.04%
amounts
of
growth
yeast
growth.
The
of
extract
mycelia. aided
The
pendulan
o p t i m a l temperature
for
p e n d u l a n p r o d u c t i o n was a b o u t 29OC a n d f o r m y c e l i u m g r o w t h 25 t o The o p t i m a l pH f o r p e n d u l a n p r o d u c t i o n was b e t w e e n pH 6 a n d 8
3OoC.
a n d f o r m y c e l i u m g r o w t h b e t w e e n 5 a n d 6.5.
The s e e d c u l t u r e was
s t a b l e a f t e r s t o r a g e a t 5OC o r a t -2OOC w i t h t h e a d d i t i o n o f 20 t o 3 0 % (v/v) g l y c e r i n . The maximum y i e l d o f p e n d u l a n and m y c e l i a was o b t a i n e d a f t e r 9 0 t o 120 h r o f f e r m e n t a t i o n .
Mouse m a c r o p h a g e s w e r e t r e a t e d w i t h 42 d i f f e r e n t g l y c a n s vitro. size,
-
Macrophages were s t i m u l a t e d 5'-nucleotidase
a c t i v i t y , and t h e
d e ~ x y - ~ - { ~ ~ C ) g l u c ob ys esome glucan.214 chitin).
Not
a l l
stimulatory
incorporation o f
insoluble
insoluble
Laminaran,
pattern similar
as j u d g e d by m o r p h o l o g y ,
glycans
glycans, were
2-amino-2-
e . ~ .y e a s t
stimulatory
w i t h a m o n o s a c c h a r i d e c o n t e n t and b o n d i n g
t o those o f
by c r o s s - l i n k i n g
yeast
Q-glucan,
c o u l d be r e n d e r e d
and i n s o l u b i l i z a t i o n .
T h e r e was a
p r e s u m a b l y p r o d u c i n g complement of
yeast
to
Preincubation o f sera with yeast,
c o n v e r t complement f a c t o r C 3 . effect
p-
(w
c l e a r c o r r e l a t i o n b e t w e e n s t i m u l a t o r y e f f e c t and t h e a b i l i t y
stimulatory
in
cell
cleavage products, g-glucan.
It
was
potentiated the suggested
that
e n d o c y t o s i s o f g l y c a n s w i t h subsequent i n t r a c e l l u l a r t r i g g e r i n g o f a complement
reaction
i s
the
underlying
mechanism
of
glycan
stimulation. I n Saccharomyces c e r e v i s i a e ,
Neurospora crassa,
Aspergillus
n i d u l a n s , and C o p r i n u s c i n e r e u s most o f t h e a l k a l i - i n s o l u b l e 8-/(1+6)-B-Q-glucan
of
the
(1+3)-B-
w a l l c a n be e x t r a c t e d w i t h d i m e t h y l
s ~ l p h o x i d e . ~ The ~ ~same f r a c t i o n ,
and i n Saccaromyces c e r e v i s i a e a
small
be
additional
fraction,
can
extracted
by
a
destructive
149
4: Microbial Polysaccharides procedure fraction
involving of
40% s o d i u m
the Q-glucan
which
hydroxide a t
100°C.
resists
treatment
this
The
small
becomes
s o l u b l e a f t e r a subsequent t r e a t m e n t w i t h n i t r o u s acid, i n d i c a t i n g that i t i s covalently linked t o c h i t i n i n the wall. Q/(l+6)-6-g-glucan
appears
to
be
held
I n contrast,
in
n e a r l y a l l t h e (1+3)-6-
S c h i r o p h y l l u m commune and A g a r i c u s b i s p o r u s ,
insoluble
by
linkage
to
c h i t in. When c e l l s o f S a c c h a r o m y c e s c e r e v i s i a e g r o w i n g e x p o n e n t i a l l y on Q - g l u c o s e as s o l e c a r b o n s o u r c e were
washed and t r a n s f e r r e d t o
b u f f e r e d y e a s t n i t r o g e n b a s e c o n t a i n i n g 100 m M a c e t a t e , t h e y w e r e u n a b l e t o resume g r o w t h f o r s e v e r a l days,whereas a few h o u r s t o grow ( s l o w l y ) on e t h a n o l , pyruvate.216
After the c e l l transfer,
they adapted w i t h i n
a n d w i t h i n 1 2 h t o grow on o x y g e n c o n s u m p t i o n a n d ATP
c o n c e n t r a t i o n d e c r e a s e d r a p i d l y b u t r e c o v e r e d w i t h i n a f e w h o u r s on ethanol,
more s l o w l y on p y r u v a t e ,
and o n l y a f t e r 70 h on a c e t a t e .
When t h e a c e t a t e c u l t u r e h a d l o s t a l l d e t e c t a b l e ATP,
the viable
c e l l t i t r e s l o w l y d e c r e a s e d u n t i l a f t e r 70 h enough c e l l h a d a d a p t e d
t o resume g r o w t h . 15 m M ) ,
A t lower acetate concentrations (optimally 5 t o
ATP d e c r e a s e d l e s s ,
transfer
from P-glucose
and g r o w t h r e s u m e d w i t h i n 1 day.
medium t o b u f f e r
After
p l u s a carbon source,
cells
s p o r u l a t e d e q u a l l y w e l l a t e t h a n o l c o n c e n t r a t i o n s f r o m 20 t o 150 M a n d a t pH 5.5
o r 7.0.
carbon source,
s p o r u l a t i o n was o p t i m a l a t c o n c e n t r a t i o n s b e t w e e n 30
With dihydroxyacetone,
a n d 5 0 m M a n d a b o u t e q u a l a t pH 5.5
a n d 7.0.
transfer
buffer
from
Q-glucose
s p o r u l a t e d a t pH 5.5 mM a c e t a t e
of
o r more.
medium
optimally
to
c e l l s n o t adapted t o gluconeogenesis, acetic
I n contrast, plus
acetate,
after cells
s p o r u l a t i o n showed a b r o a d e r o p t i m u m The r e s u l t s i n d i c a t e d t h a t , i n
a c e t a t e c o n c e n t r a t i o n a b o u t 50 mM.
ATP;
uncharged
a c e t a t e b u t n o t w i t h 50
w i t h 15 m M
A t pH 7.0
another
high concentrations o f neutral
a c i d m o l e c u l e s caused c o m p l e t e c o n s u m p t i o n o f i n t r a c e l l u l a r
consequently t h e c e l l s could n o t adapt t o gluconeogenesis f o r a
long time. Q-Glucan
synthase
Saccharomyces c e r e v i s i a e
activity was
in
partially
b r o k e n i n t h e p r e s e n c e o f sucrose.217
cell-free
extracts
s t a b i l i z e d when c e l l s
of were
Under t h e s e c o n d i t i o n s a
s i g n i f i c a n t amount o f enzyme a c t i v i t y r e m a i n e d i n t h e s u p e r n a t a n t , a f t e r high-speed c e n t r i f u g a t i o n .
When t h i s s u p e r n a t a n t f r a c t i o n was
incubated
microfibrils
with
UDP-Q-glucose,
M i c r o f i b r i l s were i n s o l u b l e i n w a t e r ,
in alkali.
were
synthesized.
e t h a n o l , and a c i d ,
and s o l u b l e
Under t h e e l e c t r o n m i c r o s c o p e t h e y a p p e a r e d more
u n i f o r m w i t h a n a v e r a g e l e n g t h o f a b o u t 0.5
pm.
or less
Alkali-insoluble
150
Carbohydrate Chemistry
r e s i d u e appeared i n t h e fo rm o f d e n se ly packed l o n g e r m i c r o f i b r i l s . After
acidification
of
alkali-solubilized
e-glucan,
shorter
m i c r o f i b r i l s were r e p r e c i p i t a t e d .
M i c r o f i b r i l s were d i g e s t e d by
e2-1,3-B-Q-glucanase.
I n t h e l a t t e r case g- gluc os e
both
m-and
was t h e o n l y p r o d u c t i n d i c a t i n g t h a t t h e m i c r o f i b r i l s c o n s i s t o f 1,3-B-Q-glucan The
w i t h no d e t e c t a b l e b r a n c h e s .
production
of
amyloglucosidase
by
those
strains
Saccharomyces d i a s t a t i c u s which a r e a b l e t o h y d r o l y s e s t a r c h d e x t r i n has been e x p l o i t e d t o d e v i s e a r a p i d method f o r
o f
or the
r e c o g n i t i o n o f such s t r a i n s . 2 1 8 The t r i p l e h e l i x o f a p o l y s a c c h a r i d e S c h i z o p h y l l u m s m m u n e (schizophyllan) o f g mol-'
viscosity-average
m o l e c u l a r w e i g h t 4.8
lo5
x
( i n w a t e r ) was m e l t e d a t d e n a t u r a t i o n t e m p e r a t u r e s b e t w e e n 5
a n d 6OoC i n w a t e r - d m s o
mixtures.
denaturation temperature
values
The
solutions
for
w e r e s t u d i e d by
different
v i s c o m e t r y and
u l t r a c e n t r i f ~ g a t i o n . ~ As ~ ~ denaturation temperature
was i n c r e a s e d ,
t h e i n t r i n s i c v i s c o s i t y o f t h e m i x t u r e c o n t a i n i n g 12.76% ( b y w e i g h t ) of
w a t e r d e c r e a s e d s h a r p l y and,
a t a b o u t 5OoC, i t a p p r o a c h e d t h e
value expected f o r
t h e s i n g l e c h a i n o f s c h i z o p h y l l a n i n p u r e dmso.
Schlieren patterns
of
t h e sample s o l v e n t d e n a t u r e d a t t e m p e r a t u r e s
b e t w e e n 25 and 45OC i n t h e same m i x e d s o l v e n t showed t h e p r e s e n c e o f two s o l u t e species.
The f a s t - s e d i m e n t i n g s p e c i e s d o m i n a t i n g a t
d e n a t u r a t i o n t e m p e r a t u r e s o f 25OC a l m o s t d i s a p p e a r e d a t d e n a t u r a t i o n temperature mixture,
of
45OC.
From s e d i m e n t a t i o n - c o e f f i c i e n t
p u r e water, and dmso t h e f a s t -
data
and s l o w - s e d i m e n t i n g
for
the
species
c o u l d be i d e n t i f i e d w i t h t h e t r i p l e h e l i x and t h e s i n g l e c h a i n o f schizophyllan, that,
respectively.
i n water-dmso
These f i n d i n g s
mixtures
schizophyllan t r i p l e helix
led t o the
c o n t a i n i n g -13% o f
melts i n t o s i n g l e chains
conclusion water,
the
i n all-or-none
f a s h i o n w i t h i n c r e a s i n g temperature.
g-Mannans. - Cell-wall -A s p-e r g----i l l u s ---f u m i g ---atus polysaccharides
components were
comprised
found of
of
mycelia
to
contain
and
E-galacto-Q-mannans
c o p r e c i p i t a t e d w i t h s m a l l proportions o f a g-glucan, i d e n t i f i e d as g l y c o g e n . 2 2 0 mannans
of
which
tentatively
The f i n e s t r u c t u r e s o f t h e g - g a l a c t o - g -
v a r i e d as a f u n c t i o n o f
mycelium,
conidia
alkali-soluble
the c e l l type.
I n a 5-day-old
t h e p o l y s a c c h a r i d e c o n s i s t e d o f a m a i n c h a i n o f (1+6)-
l i n k e d a-;-mannopyranosyl mannopyranosyl
r e s i d u e s s u b s t i t u t e d a t 0 - 2 b y 1 t o 3 a-g-
residues
that
are
(1+2)-interlinked.
Galactofuranosyl residues are (1+6)-linked
B-p-
t o t h e g-mannan c o r e ,
4: Microbial Polysaccharides
151
b e i n g components o f s i d e chains o f average l e n g t h o f
G.
6 residues,
which are (1+5)-interlinked. The 1 0 - d a y - o l d m y c e l i u m h a d a s i m i l a r R-galacto-g-mannan, b u t t h e p r o p o r t i o n o f g l y c o g e n was s m a l l e r . C o n i d i a c o n t a i n p o l y s a c c h a r i d e s o f d i f f e r e n t s t r u c t u r e , a s shown by t h e 1 3 C n.m.r. s p e c t r u m a n d by m e t h y l a t i o n a n a l y s i s . Side chains *composed o f a s i n g l e f 3 - Q - g a l a c t o f u r a n o s y l
g r o u p l i n k e d (1+6) t o
adjacent P-mannopyranosyl residues were i d e n t i f i e d w i t h a m i n o r p r o p o r t i o n o f 6 - 0 - s u b s t i t u t e d p - g a 1a c t o f u r a n o s y 1 r e s i d u e s .
Also
p r e s e n t were nonreducing g - g a l a c t o p y r a n o s y l g r o u p s and 2-amino-2deoxy-glycosyl
residues.
The Q - g l u c a n c o m p o n e n t was n o t g l y c o g e n .
C o n i d i a l w a l l s have much l e s s p r o t e i n t h a n m y c e l i a l w a l l s . P r e d o m i n a n t amino a c i d s i n t h e l a t t e r were & - a s p a r t i c and & - g l u t a m i c acids,
C-tyrosine,
I=-alanine,
and g l y c i n e .
P a l m it i c ,
stearic,
oleic,
a n d l i n o l e i c a c i d s were p r e s e n t i n t h e m y c e l i a l and c o n d i a l w a l l s ; l i n o l e i c a c i d was p r e s e n t i n m i n o r a m o u n t s i n t h e m y c e l i a l w a l l ,
but
was a m a j o r component o f t h e l i p i d f r a c t i o n f r o m w h o l e c e l l s . The p o s s i b i l i t y t h a t d i s s o l v e d g - m a n n a n p o l y s a c c h a r i d e f r o m Candida
may
have
an i n h i b i t o r y
influence on
the
host
defence
mechanisms a s s o c i a t e d w i t h n e u t r o p h i l s and t h e r e b y c o n t r i b u t e t o t h e p a t h o g e n e s i s o f c a n d i d o s e s h a s been i n v e s t i g a t e d . 2 2 1 A model f o r t h i s h y p o t h e s i s was c r e a t e d by u s i n g B-mannan yeast
derived from
baker’s
(Saccharomyces c e r e v i s i a e ) a n d Q-mannan i s o l a t e d f r o m t h e
serum o f a p a t i e n t w i t h c h r o n i c mucocutaneous candidosis.
I t was
o b s e r v e d t h a t t h e s e p-mannans i n h i b i t e d t h e r e s p i r a t o r y b u r s t and r e l e a s e o f m y e l o p e r o x i d a s e s t i m u l a t e d by p h a g o c y t o s i s o f s e r u m o p s o n i z e d zymosan
fi
vitro.
9-Mannan d i d n o t i n h i b i t r e l e a s e o f
t h r e e o t h e r l y s o s o m a l enzymes. of
myeloperoxidase
property of
was
t h e enzyme,
The s e l e c t i v e i n h i b i t i o n o f r e l e a s e
attributable to
a
carbohydrate-binding
w i t h t h e g-mannan c a u s i n g c o - s e d i m e n t a t i o n
o f t h e enzyme w i t h t h e c e l l u l a r f r a c t i o n . V a r i o u s u l t r a s t r u c t u r a l m e t h o d s have been a p p l i e d t o s t u d y t h e l o c a l i z a t i o n of
c h e m i c a l and/or
albicans blastospore c e l l
for the detection of
a n t i g e n i c components o f Candida They c o n c e r n e d :
PATAg r e a c t i o n
polysaccharides on u l t r a t h i n sections
associated w i t h enzymatic
d i g e s t i o n s or
polysaccharide extraction;
t h e i n d i r e c t i m m u n o f e r r i t i n method on i n t a c t c e l l s ; immunoperoxidase method on u l t r a t h i n s e c t i o n o f embedding medium;
the indirect
p a t i e n t s and e x p e r i m e n t a l sera. the
previously
described
the indirect
water-soluble
immunofluorescence t e s t ,
using
The c y t o c h e m i c a l r e s u l t s c o n f i r m e d eight- layer
immunocytochemical methods,
also
i n
organization. agreement
with
The this
Carbohydrate Chemistry
152 stratification, layer
located
important
showed i n a d d i t i o n t o e x t e r n a l s t r u c t u r e s t h a t t h e near
the
plasmalemma
a n t i g e n i c area.
must
be
considered
The Q-mannans r e s p o n s i b l e f o r
as
an
antigenic
d i f f e r e n c e s b e t w e e n s t r a i n s o f C a n d i d a a l b i c a n s and namely t h o s e supporting the
serotype A a c t i v i t y
were
shown t o
be, a t
least,
d i s t r i b u t e d among two o f t h e d e s c r i b e d p e r i p h e r a l l a y e r s . The
cell-wall
C a n d i d a sp.
mutant
of
M-7002, a n d i t s
d i f f e r e n c e i n g-content.
a hydrocarbon-assimilating
yeast,
w i l d t y p e have shown a s i g n i f i c a n t
Each Q-mannan was i s o l a t e d f r o m t h e m u t a n t
and t h e w i l d - t y p e c e l l s by f r a c t i o n a t i o n w i t h C e t a v l o n and c o p p e r reagent.223
Both
phosphate.
Q-mannans
p r o t e i n (18% i n weight low
contain
Q-mannose,
bases) whereas t h e w i l d - t y p e
protein content
(5.1%) w i t h
a h i g h amount
S t r u c t u r a l a n a l y s e s by e n z y m a t i c
(>go%).
Q - g l u c o s e , and
The m u t a n t Q - m a n n a n h a s a r e l a t i v e l y h i g h c o n t e n t o f Q-mannan has a
of
carbohydrate
and c h e m i c a l m e t h o d s
showed t h a t b o t h Q-mannans h a d a ( 1 + 6 ) - l i n k e d a - a - m a n n o s i d i c bone s u b s t i t u t e d a t 0-2 by s i d e c h a i n s o f v a r y i n g l e n t h .
back
The s i d e
c h a i n s o f t h e m u t a n t Q-mannan were shown t o c o n s i s t o f s i n g l e Q mannose
units
and
disaccharide
predominantly (1+2)-linked,
units
whose
while the wild-type
additional side chains o f disaccharides. chains had (1+3)-linkages mannan.
linkages
were
p-mannan had t w o
These a d d i t i o n a l s i d e
w h i c h were s c a r c e l y f o u n d i n t h e m u t a n t Q-
B - E l i m i n a t i o n r e a c t i o n d e m o n s t r a t e d t h a t t h e p-mannans a l s o
contain k-mannosyl oligosaccharides l i n k e d t o p r o t e i n through glycosidic
linkage.
0-
The c h e m i c a l p r o p e r t i e s o f B-mannan o f t h e
Candida m u t a n t i n d i c a t e s t h a t t h e m u t a t i o n m i g h t o c c u r n o t o n l y i n the side-chain s t r u c t u r e but also i n the (1+6)-linked
a-Q-mannan
back bone. The s t r u c t u r e s o f t h e proteomannans f r o m t h e c e l l - w a l l
------Candida
sp.
and
immunochemical
i t s
parent
methods.
strain
Alkaline
were
further
titration
phosphate i s p r e s e n t i n t h e form o f d i e s t e r ,
mutant
s t u d i e d by
showed
and m i l d - a c i d
that
the
treatment
r e l e a s e d Q-mannose f r o m b o t h o f t h e Q - m a n n a n ~ . * ~ I~n t h e w i l d - t y p e Q-mannan, t h e p h o s p h o r y l a t e d s i d e - c h a i n o l i g o s a c c h a r i d e s g a v e 8 5 % s i d e c h a i n h a v i n g ( 1 + 3 ) - a - P- - l i n k a g e
i n h i b i t i o n and a P-mannopentaose i n
the
nonreducing
homologous
terminal
precipitin
also
reaction.
gave
40% i n h i b i t i o n
This
immunodominant s i d e c h a i n o f t h e Candida s p .
indicated wild-type
i n
the
that
the
B-mannan
i s
t h e p h o s p h o r y l a t e d p-mannopentaose w h i c h has an ( 1 + 3 ) - a - Q - l i n k e d nonreducing terminal.
I n the
r e a c t i v i t y i n the cross-reaction
mutant system,
a significant
between t h e mutant a n t i s e r u m and
153
4: Microbial Polysaccharides Kloeckera
brevis
p-mannan
t h e m u t a n t a-mannan
indicates that
oligosaccharide from Kloeckera brevis, (1+2)-linked
a-Q-mannotriose.
immunodeterminant o f
which has phosphorylated
However, no d e f i n i t e r e s u l t s were On t h e b a s i s o f t h e r e s u l t s
obtained i n the i n h i b i t i o n studies. from chemical,
the
may be a s t r u c t u r a l a n a l o g u e o f t h e s i d e - c h a i n
e n z y m a t i c a l , and i m m u n o l o g i c a l e x p e r i m e n t s ,
the side-
c h a i n s t r u c t u r e and m a c r o m o l e c u l a r m o d e l s o f C a n d i d a sp.
wild-type
and m u t a n t mannans w e r e p r o p o s e d . The Q - g a l a c t o - e - m a n n a n
f r o m C o n i d i o b o l u s c o r o n a t u s h a s been
s u b j e c t e d t o m e t h y l a t i o n s t u d i e s and f o u n d t o c o n t a i n (1+2)-, and (1+6)-linked C-2,
C-3,
(1+3)-,
p-mannosyl r e s i d u e s w i t h evidence o f branching a t
a n d C-6 o f t h e a - m a n n o s y l r e s i d u e s . 2 2 5
Q - M a n n o s e was
f o u n d a l s o t o be a t e r m i n a l n o n r e d u c i n g r e s i d u e . was p r e s e n t a s ( 1 + 3 ) - l i n k e d
The Q - g a l a c t o s e
residues,whilst
h a l f the Q-galactose
represented t e r m i n a l nonreducing residues.
The p o l y s a c c h a r i d e
appears t o have s i m i l a r s t r u c t u r a l f e a t u r e s t o those p r e s e n t i n y e a s t s and f i l a m e n t o u s f u n g i . An a l k a l i - s o l u b l e p o l y s a c c h a r i d e , been
isolated
Methylation, that
S-Iawe
from
the
mycelia
periodate oxidation,
of
d e s i g n a t e d as S-Iawe, Epidermophyton
has
floccosum.
and a c e t o l y s i s s t u d i e s s u g g e s t e d
i s composed o f
(1+6)-g-a-g-mannopyranosyl-(l+6)-g-{a-~m a n n o p y r a n o s y 1-( 1+2)1 - g - a - g - m a n n o p y r a n o s y 1 r e p e a t i n g u n i t s . 2 2 6 C o n d e n s a t i o n o f 2,3,4,6-tetra-g-acetyl-a-P-mannopyrannopyranosyl bromide w i t h m e t h y l 3-g-benzyl-4,6-g-benzylidene-u-~-mannopyranoside i n t h e p r e s e n c e o f m e r c u r i c c y a n i d e gave,
i n 70% y i e l d ,
m e t h y l 3-2-benzyl-
4,6-~-benzylidene-2-~-(2,3,4,6-tetra-~-acetyl-~-~-mannopyranosyl)a-P-mannopyranoside. disaccharide
Condensation of
the
debenzylidenated
w i t h 2,3,4,6-tetra-~-acetyl-a-P-mannopyrannopyranosyl
bromide
afforded the corresponding trisaccharide repeating u n i t . F i e l d - d e s o r p t i o n mass s p e c t r o m e t r y h a s b e e n u s e d t o a n a l y s e carbohydrate
polymers
derivatization. oligosacchcride
with
5 to
I n a l l examples, could
be
1 4 hexose u n i t s
the
determined
without
molecular weight
by
means
of
the
prior of
the
abundant
q u a s i m o l e c u l a r i o n s o f t h e t y p e MNa+, MH+, MNa22+, a n d MNa33+.227 Fragmentation a t extents.
deoxy-a-glucitol, MNa+ a t m/z m/z
530,
1519.5.
glycosidic
linkages
was
observed
in
various
The r e d u c e d o l i g o s a c c h a r i d e ( P - m a n n o ~ e ) ~ - 2 - a c e t a m i d o - 2 o b t a i n e d f r o m IgM,
1 5 4 4 , MH+ a t m/z
gave q u a s i m o l e c u l a r - i o n s i g n a l s
1 5 2 2 , MNa22+ a t m/z 784,and
a l l corresponding t o
i t s assumed m o l e c u l a r
MNaj3+ a t weight
of
M y c o b a c t e r i a l methyl-Q-mannose p o l y s a c c h a r i d e s w i t h t h e
g e n e r a l s t r u c t u r e ( ~ - m a n n o s e-) x - ( 3 - ~ - m e t h y l - Q - r n a n n o s e ) y - 3 - ~ - m e t h y l
-
Carbohydrate Chemistry
154 methyl-a-mannoside
were a l s o s u c c e s s f u l l y a n a l y s e d .
;-Mannose-(3-g-
m e t h y l - Q - m a n n o ~ e ) ~ ~ - 3 - ~ - m e t h yml e t h y l - g - m a n n o s i d e , homologue, a t m/z
the largest
gave t h e e x p e c t e d s i g n a l o f t h e q u a s i m o l e c u l a r
2506.
i o n MNa+
The l a r g e r p o l y s a c c h a r i d e s w e r e a n a l y s e d b y u s i n g a
KRATOS M S - 5 0 mass s p e c t r o m e t e r w i t h a h i g h - f i e l d m a g n e t e n a b l i n g f u l l s e n s i t i v i t y t o be m a i n t a i n e d up t o 3 0 0 0 a t o m i c mass u n i t s . P o l y s a c c h a r i d e s up t o m/z
1978 w e r e a n a l y s e d by u s i n g a KRATOS MS-9
mass s p e c t r o m e t e r o p e r a t e d a t 4 k V . which
becomes
spectrometry
at
a
serious
low
The s i g n a l - t o - n o i s e
problem
i n
ratio,
field-desorption
accelerating voltages,
and t h e
low
mass
instrument
s e n s i t i v i t y w e r e i m p r o v e d c o n s i d e r a b l y by u s e o f a m e t h o d o f a d d i n g scans w i t h l o w t o t a l i o n c u r r e n t s o b t a i n e d o v e r a l o n g e r d e s o r p t i o n time.
I n t h i s way, c o m p l e t e sequence i n f o r m a t i o n was o b t a i n e d on 3-
O-methyl-g-mannose
polysaccharides up t o E-mannose-(3-g-methyl-Q-
m a n n o ~ e ) ~ - 3 - g - r n e t h y lm e t h y l - e - m a n n o s i d e Ana 1y s i s o f a p r e s u med methyl-a-mannose
(MNa'
9- m annose (3-0met h y 1- Q -
at
m/z
m annose 1 6 -
1802).
3-0-met hy 1
gave a s p e c t r u m c o n s i s t e n t o n l y w i t h t h e s t r u c t u r e
( Q - m a n n o ~ e ) ~ - ( 3 - ~ - m e t h y l - ~ - m a n n o s e ) ~ - 3 - g - m e t1h ym e t h y 1- Q - m a n n o s i d e , r e v e a l i n g t h e e x i s t e n c e o f a 3-g-methyl-a-mannose
homologue w i t h 2
u n m e t h y l a t e d Q-mannoses a t t h e n o n r e d u c i n g end o f t h e c h a i n . S a c c h a r o m y c e s c e r e v i s i a e w i l d - t y p e and m u t a n t c e l l s a f f e c t e d i n t h e s t r u c t u r e o f Q-mannan o u t e r c h a i n w e r e shown t o p o s s e s s one
major
size,
dolichol-phosphate-phosphate-bound
monosaccharide
composition,
and
in
vivo
oligosaccharide. pattern
obtained
The upon
a c e t o l y s i s and p a p e r c h r o m a t o g r a p h y o f t h e o l i g o s a c c h a r i d e w e r e t h e same
for
a l l
strains
and
for
i s o l a t e d f r o m animal tissues.228 that the dolichol-diphosphate
the
main
derivative occurring
c o n t a i n i n g 2-acetamido-2-deoxy-2-glucose mols),
and Q - g l u c o s e ( 3 m o l s ) ,
protein glycosylation.
The
corresponding
compound
Evidence i s presented i n d i c a t i n g (2 mols),
vivo, e-mannose
and
(9
i s the intermediate involved i n yeast transfer
of
the oligosaccharide t o
p r o t e i n was f o l l o w e d i n v i v o b y t h e e x c i s i o n o f t h e Q - g l u c o s e a n d a t t h e m o s t one a-mannose r e s i d u e . trimmed saccharide moiety.
-
(i.e.
B-Mannoses w e r e t h e n added t o t h e
No d i f f e r e n c e b e t w e e n t h e f i r s t s t a g e s
e x c i s i o n o f monosaccharides) o f t h e p r o c e s s i n g o f t h e p r o t e i n -
bound o l i g o s a c c h a r i d e s by w i l d - t y p e However,
a n d m u t a n t c e l l s was f o u n d .
m u t a n t s c a r r y i n g t h e mnn 1 m u t a t i o n ,
d e v o i d o f t e r m i n a l (1+3)-linked-a-Q-mannosyl
w h i c h a r e known t o b e r e s i d u e s i n t h e Q-
mannan o u t e r c h a i n a n d i n n e r c o r e ,
were f o u n d n o t t o add such Q-
mannose
Q-glucose-free
residues
oligosaccharide.
to
the
already
protein-bound
155
4: Microbial Polysaccharides
The
Q-mannan
of
baker's
yeast (a wild-type s t r a i n o f prepared by means of cetyltrimethylammonium bromide, was i n v e s t i g a t e d f o r its immunochemical properties.229 Upon t r e a t m e n t w i t h 1 0 m M H C 1 a t 1 0 0 ° C f o r 6 0 m i n , t h i s Q - m a n n a n g a v e a m o d i f i e d Q - m a n n a n , Qmannobiose, and Q-mannose. By t h e a c t i o n o f 1 0 0 m M NaOH a t r o o m t e m p e r a t u r e f o r 18 h , a n o t h e r m o d i f i e d Q - m a n n a n was o b t a i n e d t o g e t h e r w i t h a - m a n n o t e t r a o s e , p-m a n n o t r i o s e , n - m a n n o b i o se, a n d a mannose. I n both a c i d - and a l k a l i - d e g r a d a t i o n p r o d u c t s , m a n n o b i o s e was a c o m m o n m a j o r o l i g o s a c c h a r i d e c o m p o n e n t . The r e s u l t s o f m e t h y l a t i o n a n a l y s e s i n d i c a t e d t h a t p-mannobiose i n t h e f o r m e r d e g r a d a t i o n p r o d u c t c o n s i s t e d s o l e l y o f ( 1+3) -a-Q-li n k e d r e s i d u e s , w h e r e a s t h e l a t t e r o n e c o n t a i n e d ( 1 + 2 ) - a n d (1+3)-a-Ql i n k e d r e s i d u e s i n a m o l a r r a t i o o f 1:B:l.O. The f i n d i n g s o f q u a n t i t a t i v e p r e c i p i t i n assay of t h e p a r e n t Q-mannan a n d t h e t w o m o d i f i e d Q-mannans a g a i n s t t h e homologous a n t i - S a c c h a r o m y c e s c e r e v i s i a e serum revealed t h a t t h e amounts o f p r e c i p i t a t e d antibody by e a c h m o d i f i e d p - m a n n a n were n e a r l y i d e n t i c a l , a n d were s i g n i f i c a n t l y smaller t h a n t h a t o f t h e p a r e n t a-mannan. Because t h e a n t i bo dy-p re c i p i t a t i n g a c t i v i t i e s o f b o t h m o d i f i e d g-m a n n a n s were e x a c t l y i d e n t i c a l t o t h a t o f t h e Q - m a n n a n p r e p a r e d by t h e F e h l i n g s o l u t i o n method, i t i s e v i d e n t t h a t t h e (1+3)-linked a-Q-mannobiosyl residues dominate a large p a r t of the serological a c t i v i t y of t h e p a r e n t i n t a c t Q - m a n n a n , a n d t h a t t h e p - m a n n a n p r e p a r e d by t h e F e h l i n g s o l u t i o n method corresponds t o a degradation product l a c k i n g t h e s e i m m uno d o m i n a n t p-manno b i o s y 1 r e s i d u e s . C a r b o x y p e p t i d a s e Y, a v a c u o l a r e n z y m e f r o m S a c c h a r o m y c e s c e r e v i s i a e , was d i g e s t e d w i t h e n d o - B - ~ - 2 - a c e t a m i d o - 2 - d e o x y - ~ - g l u c o sidase H t o release t h e f o u r o l i g o s a c c h a r i d e c h a i n s t h a t a r e l i n k e d t o L-asparagine i n t h e glycoprotein. T h e o l i g o s a c c h a r i d e s were f r a c t i o n a t e d i n t o a n e u t r a l and a c i d i c component, and t h e l a t t e r p r o v e d t o b e p h ~ s p h o r y l a t e d . ~ ~ F' r o m i t s g e l - f i l t r a t i o n p a t t e r n , t h e n e u t r a l f r a c t i o n was s h o w n t o b e a m i x t u r e o f a t l e a s t f o u r h o m o l o g u e s , t h e s m a l l e s t o f w h i c h h a d a 'H n . m . r . s p e c t r u m a l m o s t i d e n t i c a l t o t h a t g i v e n by a n IgM o l i g o s a c c h a r i d e w i t h e i g h t Q mannoses and one 2-acetamido-2-deoxy-~-glucose. The y e a s t o l i g o s a c c h a r i d e h a s o n e a d d i t i o n a l a - p - m a n n o s y l r e s i d u e i n a (1+3)o r (1+6)-linkage, whereas t h e l a r g e r homologues a p p e a r t o have two, three, a n d f o u r more Q-mannose r e s i d u e s . One p h o s p h o r y l a t e d o l i g o s a c c h a r i d e w i t h a - m a n n o s e / p h o s p h a t e r a t i o o f 12.5 was r e d u c e d w i t h NaB3H4 a n d t h e n s u b j e c t e d t o m i l d - a c i d h y d r o l y s i s . This
--------S a c c h a r o m y--c e s ---------cerevisiae),
n-
156
Carbohydrate Chemistry
r e l e a s e d Q-mannose and g-mannobiose t h a t were g l y c o s i d i c a l l y l i n k e d t o t h e p h o s p h a t e group, whereas c o m p l e t e a c i d h y d r o l y s i s y i e l d e d Qmannose 6-phosphate. The recovered oligosaccharide phosphomonoester,
w h i c h c o n t a i n e d 11 o r 1 2 p-mannose
d i g e s t e d e x h a u s t i v e l y w i t h a-a-mannosidase,
residues,
r e a c t i o n was t r e a t e d w i t h a l k a l i n e p h o s p h a t a s e ,
which
yielded
r a d i o a c t ive (Q-m - a n no s e ) 3 - 2 - a c e t a m i d o - 2 - d e o x y - Q-- g l uc it 01.
r e s u l t s sug ge s t e d t h a t
was
and t h e p r o d u c t o f t h i s
-
These
t h e a-Q -m a n no s ida se r e s i s t a nt ph o sp h o r y 1a t e d (341, i n w h i c h s o m e o f
oligosaccharide has the s t r u c t u r e
the
phosphate groups a r e s u b s t i t u t e d w i t h Q-mannobiose i n s t e a d o f mannose.
A
second
mannose/phosphate
phosphorylated oligosaccharide
r a t i o o f 6.5
with
a
2-
e-
probably contains two phosphodiester
b u t i t s s t r u c t u r e has n o t been i n v e s t i g a t e d i n d e t a i l .
groups,
a-Q-Mane( l + ? ) -a +-Mane-
( 1+6) -B -Q-Mang- ( 1+4) -g-ClcNA
cH2
6
1
a -p -M a n p P
I
where +P = o r t h o p h o s p h a t e d i e s t e r l i n k a g e (34) Cell walls o f flocculent strains
(0006) a n d n o n - f l o c c u l e n t
s t r a i n s ( 0 0 1 9 ) o f Saccharomyces uvarum ( C a r l s b e r g e n s i s ) ,
grown i n
d i f f e r e n t m e d i a and t a k e n i n b o t h g r o w t h and s t a t i o n a r y phases,
were
t r e a t e d w i t h w a t e r a n d w i t h 2 % (w/v)
This
treatment yielded four fractions (FI,
potassium hydroxide.
FII,
F I I I , a n d FR).231
The
f r a c t i o n s F I i s o l a t e d f r o m t h e f l o c c u l e n t c e l l w a l l s c o n t a i n e d more 1-mannose
and l e s s p r o t e i n t h a n t h e c o r r e s p o n d i n g f r a c t i o n s F I
i s o l a t e d from
the
non-flocculent
c o m p o s i t i o n was a l s o d i f f e r e n t A radioactive-labelling
c e l l walls.
The
amino
acid
between t h e t w o t y p e s o f f r a c t i o n s .
technique r e v e a l e d t h a t t h e F I and t h e w a l l s
f r o m f l o c c u l e n t c e l l s b o u n d o n a v e r a g e t w o t o t h r e e t i m e s a s much 45Ca a s d i d t h e F I a n d t h e w a l l s f r o m n o n - f l o c c u l e n t
yeast.
The
s u b s t i t u t i o n o f c a r b o x y l g r o u p s i n F I and w a l l s w i t h g l y c i n e m e t h y l e s t e r l e d t o a g r e a t d r o p o f t h e 45Ca b i n d i n g c a p a c i t y , s u g g e s t i n g that
carboxyl
flocculation
groups o f process.
the c e l l walls are involved i n the However,
f l o c c u l a t i o n seems t o
be a
p h e n o m e n o n m o r e c o m p l e x t h a t t h e s i m p l e f o r m a t i o n o f Ca2+ b r i d g e , t h e i n v o l v e m e n t o f l e c t i n - l i k e components e a s i l y removed f r o m t h e
4: Microbial Polysaccharides c e l l walls,
157
s h o u l d n o t be r e j e c t e d .
C h i t i n . - The s t r u c t u r e o f p o l y ( 2 - a m i n o - 2 - d e o x y - B - g a l a c t o s e )
purified
f r o m t h e c u l t u r e f l u i d o f A s p e r g i l l u s p a r a s i t i c u s AHU 7 1 6 5 was studied.
P a r t i a l acid hydrolysis o f t h i s polysaccharide,in
w h i c h 55
t o 65% o f t h e m o n o s a c c h a r i d e r e s i d u e s w e r e t j - u n s u b s t i t u t e d , h e x a m e r ) i n a good y i e l d . 2 3 2
From t h e d a t a on a n a l y s e s o f
p o l y s a c c h a r i d e and i t s o l i g o s a c c h a r i d e s by g e l f i l t r a t i o n , oxidation,
gave a
oligosaccharides (dimer t o
s e r i e s of 2-amino-2-deoxy-Q-galactose
m e t h y l a t i o n a n a l y s i s , and 'H n.m.r.
measurement, t h e p o l y -
s a c c h a r i d e was c h a r a c t e r i z e d a s a l i n e a r c h a i n o f ( 1 + 4 ) - l i n k e d
amino-2-deoxy-a-~-galactosyl r e s i d u e s . 2-deoxy-q-galactosyl
the
periodate 2-
The t j - u n s u b s t i t u t e d 2 - a m i n o -
r e s i d u e s a r e p r o b a b l y d i s t r i b u t e d i n a random
f a s h i o n over t h e polysaccharide chain. The u p t a k e o f 2 - a m i n o - 2 - d e o x y - ~ - g l u c o s e
by t h e s t r o n g l y h a l o -
t o l e r a n t D e b a r y o m y c e s h a n s e n i i a n d t h e n o n - t o l e r a n t Saccharomyces
---------cerevisiae
has
been
studied
to
determine
respiration
and
fermentation rates for these y e a s t ~ . ~ ~ ~ A n t i s e r a s p e c i f i c f o r p u r i f i e d c e l l w a l l s o f F u s a r i u m s o l a n i f. sp.
piSi a n d p h a s e o l i a n d o f s h r i m p - s h e l l c h i t o s a n w e r e u t i l i z e d as
i m m u n o c h e m i c a l p r o b e s t o d e t e r m i n e t h e l o c a t i o n o f f u n g a l components i n
the
pea-Fusarium
inoculation,
interaction.234
Within
15
minutes
after
f u n g a l c e l l - w a l l components appeared t o e n t e r t h e p l a n t
c e l l a n d t o a c c u m u l a t e i n s i d e t h e p l a n t c e l l w a l l as f u n g a l g r o w t h o n t h e p l a n t t i s s u e was i n h i b i t e d . c h i t o s a n and
a l l
components
The a c c u m u l a t i o n p a t t e r n s o f
c o n t a i n i n g 2-amino-2-deoxy-hexose
p o l y m e r s r e s e m b l e d t h o s e o f t h e f u n g a l w a l l components.
Chitosan i s
p r e s e n t on,
the fungal
spore.
and r e l e a s e d f r o m ,
W i t h i n 15 minutes a f t e r a p p l y i n g { 3H)chitosan t o t h e s u r f a c e
of t h e p l a n t t i s s u e , plant
the outer surface o f
cytoplasm
nucleus.
t h e l a b e l was r e a d i l y d e t e c t a b l e w i t h i n t h e
and c o n s p i c u o u s l y
I t was p r o p o s e d t h a t
detectable
within
the potential for
the
plant
transport of
c h i t o s a n between t h e s p o r e s o f F u s a r i u m s o l a n i and pea c e l l s , addition to
i t s potential to
disease-resistance
responses,
inhibit
fungal
suggests
in
g r o w t h and e l i c i t
chitosan has a
major
regulatory r o l e i n t h i s host-parasite interaction. The
vivo differential rates
of
chitin-plus-chitosan
b i o s y n t h e s i s i n Mucor racernosus w e r e d e t e r m i n e d u n d e r a v a r i e t y o f conditions, chitosan
l e a d i n g t o yeast c e l l or m y c e l i a l morphology. was
determined
as
hot
Chitin-
sodium hydroxide-insoluble
r a d i o a c t i v i t y d e r i v e d from 2-acetamido-2-deoxy-P-(
l-3H)glucose
in
158
Carbohydrate Chemistry
the medium. Control experiments demonstrated that the labelled material possessed t h e properties of chitin-plus-chitosan. The r e s u l t s i n d i c a t e d t h a t Mucor y e a s t s h a v e a r e l a t i v e l y low d i f f e r e n t i a l r a t e of chitin-plus-chitosan s y n t h e s i s and t h a t m y c e l i a l c e l l s have a t h r e e f o l d - e l e v a t e d d i f f e r e n t i a l r a t e . 2 3 5 Treatment of aerobic cells with exogenous N6,g2-dibutyryl c y c l i c a d e n o s i n e 3’,5’-monophosphate, an a g e n t which i n d u c e s y e a s t - c e l l morphology, a l s o r e s u l t e d i n a lowered rate of chitin-plus-chitosan synthesis. Control experiments eliminated the possibility t h a t the o b s e r v e d r a t e c h a n g e s were d u e t o c h a n g e s i n e n d o g e n o u s p o o l s i z e , or uptake of exogenous 2-acetamido-2-deoxy-n-( l-3H)glucose, a l t e r a t i o n s i n growth rate. T h e r e f o r e , t h e c h a n g e s were s e e m i n g l y l i n k e d t o m o r p h o g e n e s i s . These r e s u l t s s t r e n g t h e n t h e i d e a t h a t c y c l i c a d e n o s i n e 3’,5’-monophosphate p l a y s an i m p o r t a n t r o l e i n d i m o r p h i s m i n Mucor. I n a d d i t i o n , p u l s e - c h a s e e x p e r i m e n t s s u g g e s t e d t h a t c o n s i d e r a b l e m o d i f i c a t i o n of newly s y n t h e s i z e d c h i t i n p l u s c h i t o s a n i n both y e a s t c e l l s and m y c e l i a o c c u r s i n vivo. The c o m p o n e n t s a n d s t r u c t u r e o f t h e c e l l w a l l o f R h i z o p u s --delemar h a v e b e e n i n v e s t i g a t e d u s i n g p u r i f i e d l y t i c e n z y m e s , p r o t e a s e a n d c h i t o s a n a s e f r o m B a c i l l u s R-4, a n d c h i t i n a s e I1 f r o m Streptomyces o r i e n t a l i s . When t h e s e e n z y m e s were u s e d i n d i v i d u a l l y t h e y o n l y p a r t i a l l y l y s e d t h e c e l l w a l l , b u t when a l l o w e d t o r e a c t on t h e c e l l wall t o g e t h e r a c o m p l e t e l y s i s was a c h i e v e d b y cooperative action.236 T h e s e modes o f a c t i o n o n t h e c e l l w a l l and t h e chemical and m o r p h o l o g i c a l d a t a s u g g e s t e d t h a t t h e cell-wall s t r u c t u r e was d i f f e r e n t i n R h i r o p u s d e l e m a r o f Z y g o m y c e t e s f r o m filamentous f u n g i of Euascomycetes and t h a t its wall s t r u c t u r e might be c o m p o s e d m a i n l y o f c h i t i n f i b r e s c e m e n t e d by c h i t o s a n a n d p r o t e i n or p e p t i d e s s c a t t e r e d i n a m o s a i c manner. D u r i n g c u l t u r e o f R h o d o t o r u l a g l u t i n i s t h e f a l l i n pH f r o m 4.5 t o 2 stimulated the synthesis of chitin. Alternatively keeping the pH a t 4.5 c a u s e d t h e c h i t i n c o n t e n t t o f a l l l a r g e l y . 2 3 7 How-ever, t h e r e was n o p r o p o r t i o n a l v a l u e b e t w e e n t h e c h i t i n - c o n t e n t f a l l a n d t h a t o f t o t a l 2-amino-2-deoxy-hexoses.
Miscellaneous Cell-wall Polysaccharides. - D u r i n g t h e d e v e l o p m e n t o f a c e l l a g g r e g a t e of D i c t y o s t e l i u m d i s c o i d e u m i n t o a f r u i t i n g body, an a n t i g e n i c acid mucopolysaccharide is s y n t h e s i z e d o n l y i n t h e p r e s p o r e c e l l s o f a c e l l mass. T h e s u b c e l l u l a r d i s t r i b u t i o n s o f UDP-Q-ga1actose:polysacchar i d e t r a n s f e r a s e a n d U D P - Q - g l u c o s e pyrophosphorylase involved i n b i o s y n t h e s i s of mucopolysaccharide
159
4: Microbial Polysaccharides h a v e been d e t e r m i n e d . 2 3 8
The t r a n s f e r a s e was s p e c i f i c a l l y l o c a l i z e d
i n the smaller vesicles w i t h l i g h t e r density than the prespores p e c i f i c v a c u o l e s i d e n t i f i a b l e by e l e c t r o n m i c r o s c o p y .
I n contrast
t o t h e enzyme,
t h e a n t i g e n i c m u c o p o l y s a c c h a r i d e was e x c l u s i v e l y
localized
the
transferase,
i n
prespore-specific
vacuoles.
Unlike
soluble fraction.
The s u c r o s e g r a d i e n t p r o f i l e s o f t h e t r a n s f e r a s e
a c t i v i t y i n t h e 5000 x g s u p e r n a t a n t gave t w o m a i n p e a k s . profiles
the
U D P - Q - g l u c o s e p y r o p h o s p h o r y l a s e was c o n f i n e d t o t h e
were
c o m p a r e d among s t a n d i n g
culminating c e l l
mass,
reflected the state o f
the
and
difference i n the
biosynthesis
of
When t h e
migrating slugs
and
profiles closely
t h e a c i d mucopolysaccharide
i n each d e v e l o p m e n t a l s t a g e . An
L- - f u c o - e - g a l a c t a n
and a P-manno-k-fuco-e-galactan
i s o l a t e d f r o m t h e f r u i t b o d i e s o f Ganodermg a p p l a n a t u m , a c o l u m n o f c o n c a n a v a l i n A - S e p h a r o s e 4B.239
were
by e m p l o y i n g
The f o r m e r c o n s i s t s
predominantly o f a main chain o f (1+6)-linked a-k-galactopyranose residues,
G. 3 0 % o f w h i c h a r e s u b s t i t u t e d a t 0-2 by s i n g l e - u n i t a-
&-fucopyranosyl backbone o f
side chains.
(1+6)-linked
The
latter
consists
a-g-galactopyranosyl
mainly
E.
residues,
of
a
50% o f
w h i c h a r e s u b s t i t u t e d w i t h e i t h e r a 3-g-a-g-mannopyranosyl-a-kfucopyranosyl or a-l-fucopyranosyl s u b s t i t u t e d by g - a c e t y l
groups.
s i d e chains.
Both are p a r t i a l l y
These s t r u c t u r a l a t t r i b u t i o n s w e r e
s u p p o r t e d by r e s u l t s o f m e t h y l a t i o n a n a l y s i s , p o l y h y d r i c compounds, The
and by 'H
structures
of
four
p a r t i a l acid-hydrolysate from
a n d 1 3 C n.m.r.
g.1.c.
analysis o f
spectroscopy.
oligosaccharides,
isolated
from
a
o f the glycuronan protuberic acid i s o l a t e d
Kobayasia nipponica,
a n d b y 'H
and 1 3 C n.m.r.
h a v e been e x a m i n e d by c h e m i c a l a n a l y s i s ,
spectroscopy.
They were i d e n t i f i e d as
( B - Q - g l u c o p y r a n o s y l u r o n i c acid)-(1+4)-!-glucuronic i d o p y r a n o s y l u r o n i c acid)-(1+4)-~-glucuronic a c i d ,
0-
a c i d , g-(a-ig-(B-e-gluco-
p y r a n o s y 1u r o n ic a c id ) -( 1+4)-g- ( a - k - i d o p y r ano s y l u r o n i c a c i d )- ( 1+4) -pglucuronic acid,
a n d g-(B-g-glucopyranosyluronic
acid)-(1+4)-O-(B-;-
g l u c o p y r a n o s y l u r o n i c acid)-(1+4)-~-(a-C-idopyranosyluronic ( 1 + 4 ) - ~ - g l u c u r o n i c acid.240
The
acid-resistant
acid)-
portion
of
p r o t u b e r i c a c i d had p r o p e r t i e s s i m i l a r t o those o f t h e o r i g i n a l protuberic
acid.
structural
sequence
These of
trisaccharide repeating u n i t The
major
results
the
linear
derived
from
the
polysaccharide
isolated
from
the
(35).
water-soluble and zone
that
i s
s c l e r o t i a o f Omphalia lapidescence, on D E A E - c e l l u l o s e
suggested
protuberic acid
and p u r i f i e d by c h r o m a t o g r a p h y
electrophoresis,
i s a heteroglycan
160
Carbohydrate Chemistry
(c
+ 56'
1,
water),
which
i s
composed o f
!-glucose,
2-
acetamido-2-deoxy-Q-glucose, r a t i o s 1.0:1.95:2.0.241 degradation,
and Q - g l u c u r o n i c a c i d i n t h e m o l a r r e s u l t s o f periodate oxidation, Smith
The
a n d m e t h y l a t i o n a n a l y s i s showed t h a t t h e p o l y s a c c h a r i d e
h a s a h i g h l y b r a n c h e d s t r u c t u r e i n v o l v i n g 1,3-, 1,3,6-lin ked
9- g l u c o p y r a n o s y l ,
glucopyranosyl, residues.
and 1,3?-
1,4-,
1,6-,
and
1,3,4- 1i n k e d 2 - a c e t a m ido - 2 - d e ox y
and 1 , 4 - l i n k e d
-a -
p-glucopyranosyluronic a c i d
The n o n r e d u c i n g t e r m i n a l p o s i t i o n s a r e o c c u p i e d by t h e
t h r e e component sugars.
This branched glycosaminoglycan i s t h e
f i r s t t o be f o u n d i n t h e f u n g i . +4) -B-Q-GlceA-( -
1+4) -a-$-IdoeA-(
1+4) -B-g-GlcpA-(
+
( 35)
Antigens soluble i n hot trichloracetic
a c i d from
eighteen
s t r a i n s o f P i t y r o s p o r u m o v a l e were s t u d i e d u s i n g r a b b i t a n t i s e r u m t o A m e r i c a n T y p e C u l t u r e C o l l e c t i o n s t r a i n s 2 4 0 2 7 , 12078, a n d 1 4 5 2 1 . These s t r a i n s w e r e f o u n d t o s h a r e t w o a n t i g e n s ,
both o f which were
p r e s e n t i n c e l l s o f P i t y r o s p o r u m o r b i c u l a r e and one o f w h i c h was present i n c e l l s o f Pityrosporum pachydermatis,
P i t y r o s p o r u m canus,
C a n d i d a a l b i c a n s , and R h o d o t o r u l a r ~ b r u m . ~ ~ ~ The c h e m i c a l p r o p e r t i e s o f t h e h o t w a t e r - s o l u b l e
and d i l u t e
a l k a l i - s o l u b l e polysaccharides prepared from the f r u i t bodies o f the mushroom, Five
P s a l l i o t a c a m p e s t r i s ( L i n n ) Fr.,
purified
polysaccharides
were
contained 2-amino-2-deoxy-pglucose
have been s t u d i e d . 2 4 3
obtained,
three
and p - g l u c o s e
of
which
( w i t h p-mannose
a l s o p r e s e n t i n one p o l y s a c c h a r i d e ) w h i l s t t h e r e m a i n i n g t w o w e r e glucans.
n-
From s p e c i f i c r o t a t i o n s t u d i e s a l l t h e p o l y s a c c h a r i d e s
a p p e a r e d t o be c o m p o s e d o f B - Q - g l y c o s i d i c
linkages expect t h e
mannose-containing
was
polysaccharide,
which
composed o f
8-
a-Q-
g l y co s i d i c l i n k a g e s.
I-Fuco-Q-galacto-Q-mannan
was o b t a i n e d f r o m
R o d o t o r u l a g l u t i n i s K-24 by a l k a l i n e e x t r a c t i o n , and a l c o h o l p r e c i p i t a t i o n . of
L-fucose,
1:2.5:2,
and o c c u p i e d a b o u t
successive Smith degradation, degraded,
walls o f
The p u r i f i e d p o l y s a c c h a r i d e was composed
Q - g a l a c t o s e , and Q-mannose
periodate oxidation,
the
pronase digestion,
20% o f
1-mannose
the
i n the molar cell
wall.244
ratio of I n the
r e s i d u e s were r e s i s t a n t t o
whereas most o f Q-galactose r e s i d u e s were
r e l e a s i n g a - t h r e i t o l and L-arabinose.
I t appeared t h a t
B-
mannose r e s i d u e s c o n s i s t e d o f a backbone o f t h e p o l y s a c c h a r i d e , a n d a l l o r p a r t o f t h e P - g a l a c t o s e r e s i d u e s m i g h t be o f a f u r a n o s e t y p e .
161
4: Microbial Polysaccharides By p a r t i a l a c i d h y d r o l y s i s o f
t h e L-fuco-Q-galacto-Q-mannan,
three
kinds o f
oligosaccharide
c o n t a i n i n g L-fucose
obtained
(36-38).
methylation analysis revealed that
The
polysaccharide consisted o f which were
branched a t
galactose residues.
(1+3)-linked
the
Q-mannose r e s i d u e s ,
position with
C-2
L-fucose
were the
a l l of and
p-
Most o f t h e I - f u c o s e r e s i d u e s e x i s t e d i n t h e
nonreducing end of t h e s i d e chain. c o n t a i n e d a s (1+2)-
and L-galactose
Q-Galactose r e s i d u e s were m o s t l y
and ( 1 + 6 ) - l i n k a g e s
i n t h e s i d e chain.
a i d o f e l e c t r o n microscopy t h e l o c a l i z a t i o n of and I-fuco-Q-galacto-Q-mannan
With t h e
t h e p-gluco-p-mannan
i n the c e l l wall of
Rhodutoruh
g l u t i n i s a n d f o r m a t i o n o f s p h e r o p l a s t o f l i v i n g y e a s t c e l l s h a s been
.
de s c r i be d 245 !-Gall-(
1+6) -!-Gall ( 36)
L-Fucp-( 1+2) -Q-Galf-(
1+2) -!-Gal:-(
1+6) -!-Gal1
(37)
E v i d e n c e o f t h e presence o f p o l y s a c c h a r i d e p o l y m e r s h a s been s h o w n i n S t r e p t o m y c e s sp. methods
for
t h e f i r s t time.246
revealed the occurrence of
Cytochemical
polysaccharide
s p o r u l a t i n g hyphae o f S t r e p t o m y c e s v i r i d o c h r o m o g e n e s .
granules i n Onset o f t h e
sporogenesis c o i n c i d e d w i t h t h e appearance o f t h e granules,
which
r e a c h e d a maximum number d u r i n g t h e e a r l y s t a g e s o f m a t u r a t i o n .
The
l a t e r s t a g e s o f m a t u r a t i o n showed a d e c r e a s e o f t h e s e g r a n u l e s ,
and
i n m a t u r e s p o r e s no g r a n u l e s w e r e o b s e r v e d . The l o c a t i o n s o f 2 - a c e t y l
groups i n t h e a c i d i c p o l y s a c c h a r i d e s
A C a n d BC i s o l a t e d f r o m t h e f r u i t b o d i e s o f T r e m e l l a f u c i f o r m i s BERK
h a v e b e e n d e t e r m i n e d by
employing an improved procedure.
The
r e s u l t s indicated t h a t the Q-acetyl
g r o u p s i n A C a n d BC a r e l o c a t e d
at
6 of
p o s i t i o n s 4,6
and b o t h 4
and
a part
of
t h e Q-mannose
r e s i d u e s , a n d a t p o s i t i o n s 2,4 a n d b o t h 2 a n d 4 o f a p a r t o f t h e g l u c u r o n i c a c i d residues.247
Polysaccharides
B-
A C a n d BC d i f f e r
s i g n i f i c a n t l y i n t h a t t h e amount o f t h e 2 - a c e t y l groups l i n k e d t o the
Q-mannose
r e s i d u e s i n BC w a s h i g h e r t h a n t h a t i n AC.
i m p r o v e d p r o c e d u r e s h o u l d be w i d e l y a p p l i c a b l e f o r
l o c a t i o n s o f 2 - a c e t y l groups on a c i d i c polysaccharides. (References begin o v e r l e a f )
The
determining the
Carbohydrate Chemistry
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14,
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164 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99
100 101
102 103 104 105 106
107 108 109
110 111 112
113 114
115 116
117 118
119 120 121 122
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m,
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Carbohydrate Chemistry
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.
5
Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides BY R. J. STURGEON 1 Microbial Glycoproteins
-
Fungal Glycoproteins. The e f f e c t s o f g l y c o s y l a t i o n o n t h e t h e r m a l and p r o t e o l y t i c s t a b i l i t y o f catalase from A s p e r g i l l u s n i g e r D e g l y c o s y l a t i o n o f t h e enzyme r e s u l t s i n a n
have been reported.’
increased s u s c e p t i b i l i t y t o p r o t e o l y s i s and a s m a l l decrease i n t herm 0 s t a b i 1it y
.
V i r u l e n t a n d n o n v i r u l e n t s p e c i e s o f C r y p t o c o c c u s when g r o w n i n c u l t u r e w i t h maximum c a p s u l e p r o d u c t i o n e l a b o r a t e a n e x t r a c e l l u l a r glycoprotein.2
The g l y c o p r o t e i n f r o m
a c e t y l groups and more P-mannosyl-
;.
neoformans contains
and - u r o n i c
a c i d residues
0-
than
does t h e c o r r e s p o n d i n g g l y c o p r o t e i n f r o m t h e n o n - p a t h o g e n i c s p e c i e s , w h i c h does n o t c o n t a i n g - a c e t y l
groups.
Changes i n t h e p l a s m a membrane g l y c o p r o t e i n p a t t e r n d u r i n g c e l l development
i n D i c t y o s t e l i u m d i s c o i d e u m have
been
de~cribed.~
Regulation o f these g l y c o p r o t e i n p a t t e r n s i n v o l v e s t a r v a t i o n pulses of CAMP a n d c e l l - c e l l c o n t a c t .
lo4,
3.1
x 104,and
by a f f i n i t y of
1.
2.8
x
lo4)
T h r e e g l y c o p r o t e i n s (mol.
wts.
chromatography o n i m m o b i l i z e d d i s c o i d i n - 1 ,
d i s c o i d e u ~ . ~T h e s e
3.3
x
1. d i s c o i d e u m
have been i s o l a t e d f r o m
endogenous g l y c o p r o t e i n s
the lectin are
potent
i n h i b i t o r s o f t h e haemagglutination a c t i v i t y o f t h e l e c t i n , and they appear t o have an aggregation-enhancing e f f e c t o n d i f f e r e n t i a t e d cells. thermolabile antigen on the surface o f
A
baker’s
yeast
( S a c c h a r o m y c e s c e r e v i s i a e ) h a s b e e n p u r i f i e d by i m m u n o a d s o r p t i o n c h r ~ m a t o g r a p h y . ~The s u g a r m o i e t y i s c o m p o s e d o n r e s i d u e s o f g a l a c t o s e , Q-mannose, 2 - a c e t am ido - 2 - d e o x y -p- g l u c o se, a n d
w h i l s t t h e p r o t e i n m o i e t y i s composed o f B-amino a c i d s , w h i c h ( G l y , L-Glu, content. that
of
I-Ser,and
p-
I- f u c o s e , four o f
I - A s p ) c o m p r i s e 70% o f t h e a m i n o a c i d
The a n t i g e n i c d e t e r m i n a n t o f t h i s a n t i g e n i s d i s t i n c t f r o m cell-wall
p-mannan.
Cell-wall
p-mannoprotein
o f
169
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
-S a c c h a r o m y c e s
kluyveri
c e l l s has
short
a t t a c h e d t o L-serone o r I-threonine, probably
connected
to
I=-asparagine
d i a c e t y l c h i t o b i o s y l residues.6
oligosaccharide chains
and l o n g p o l y s a c c h a r i d e c h a i n s residues
through
EN’-
The i m m u n o d o m i n a n t s t r u c t u r e o f t h e
which i s found i n b o t h Q - m a n n o p r o t e i n i s c o n t a i n e d i n t h e u n i t (I), the
0-
and N - g l y c o s i d i c c h a i n s of t h e m o l e c ~ l e . ~
1+3) -a-Q-Manp( 1+
B-Q-Mane-( 1+2) -a-Q-Manp-(
6
I 1 a -Q -Mane
(1)
Viral Clycoproteins.
-
The
following topics related t o the
c h e m i s t r y and b i o c h e m i s t r y o f v i r a l c a r b o h y d r a t e s have r e c e n t l y b e e n reviewed:
glycosylation o f
carbohydrates o f v i r a l envelopes,
v i r a l envelopes,
c o m b i n a t i o n s o f h a e m a g g l u t i n i n s and n e u r a m i n i d a s e
sub-types o f i n f l u e n z a A viruses,
l o and t h e s y n t h e s i s a n d a s s e m b l y
o f v e s i c u l a r s t o m a t i t i s v i r u s and S i n d b i s v i r u s g l y c o p r o t e i n s . ” The b i n d i n g o f human i m m u n o g l o b u l i n t o r e t r o v i r u s e s h a s b e e n reported.12
Inhibition studies indicate that
this
binding i s
d i r e c t e d t o the oligosaccharide chains o f the v i r a l glycoproteins and i s n o n - s p e c i f i c I n animals
i n character.
i n f e c t e d by
bovine
leukaemia
virus,
cytotoxic
antibodies t o the envelope g l y c o p r o t e i n are d i r e c t e d against t h e carbohydrate
moiety.13
Immune s e r a
prepared i n r a b b i t s using
p u r i f i e d e n v e l o p e g l y c o p r o t e i n a s immunogen a r e d i r e c t e d a g a i n s t determinants o f
t h e p o l y p e p t i d e backbone o f
the antigen.
carbohydrate moiety o f two bovine leukaemia v i r u s an i m p o r t a n t a n t i g e n i c determinant,
The
glycoproteins i s
s i n c e a r a p i d loss o f a n t i g e n i c
a c t i v i t y o c c u r s a f t e r t r e a t m e n t with g l y c o s i d a ~ e s . ’ ~ The m a j o r v i r u s - s p e c i f i c
p r o t e i n released from c e l l s i n f e c t e d
w i t h f e l i n e l e u k a e m i a v i r u s i s a g l y c o p r o t e i n ( m o l . w t . 4.0 x lo4) which c a r r i e s gene p r o d u c t a n t i g e n i c d e t e r m i n a n t s . l5 Replication-defective Friend murine leukaemia virus p a r t i c l e s c o n t a i n i n g decreased l e v e l s of described.16
The GIX
antigen,
e n v e l o p e g l y c o p r o t e i n have been
which i s expressed o n t h e s u r f a c e o f
t h y m o c y t e s o f c e r t a i n mouse s t r a i n s , the
major
envelope
Comparison o f
glycoprotein
i s an a n t i g e n i c d e t e r m i n a n t o f o f
murine
leukaemia
virus.17
the glycoprotein precursors from two c l o s e l y r e l a t e d
Carbohydrate Chemistry
170 murine
leukaemia
viruses,
d e t e r m i n a n t f o r GIX,
one
of
which
carries
s u g g e s t s t h a t t h e GIX-
the antigenic
v i r u s c o d e s f o r an e x t r a
g l y c o s y l a t i o n s i t e i n t h e e n v e l o p e r e g i o n o f t h e genome, r e l a t i v e t o t h e p r o t e i n f r o m t h e GIX + virus. The gag g l y c o p r o t e i n ( r n o l . w t . 8.0
x
lo4)
of
Rauscher murine leukaemia
g l y c o s y l a t e d carbohydrate chains.18 h a l f o f t h e p r o t e i n a t I-Asn-177 i s found i n a fragment
(rnol.
virus
contains
two
E-
One o c c u r s i n t h e C - t e r m i n a l
o f t h e p 3 0 sequence, 2.3
wt.
lo4)
x
and t h e o t h e r
l o c a t e d i n t h e N-
terminal region. Glycopeptides obtained a f t e r extensive p r o t e o l y t i c digestion o f
the
env
precursors o f ecotropic,
leukaemia
v i r u s e s have
x e n o t r o p i c , and d u a l - t r o p i c
b e e n ~ 0 m p a r e d . l ~T h e
murine
precursor
g l y c o p r o t e i n s o f d u a l - t r o p i c v i r u s e s showed t h e u n u s u a l p r o p e r t y o f m i g r a t i n g t o the c e l l surface without undergoing the normal o l i g o s a c c h a r i d e p r o c e s s i n g and p r o t e o l y t i c c l e a v a g e e v e n t s t h a t a r e observed
for
ecotropic
and
xenotropic
s u b c e l 1u l a r
glycoproteins.
The
glycoproteins
following
murine
leukaemia
distribution of
the
transformation
viral
2:~- r e l a t e d o f
murine
e r y t h r o l e u k a e m i a c e l l s by F r i e n d l e u k a e m i a v i r u s has been s t u d i e d by s u b c e 11u l a r f r a c t i o na t i o n t e c h n i q ue s a n d p u l s e - ch a se ex p e r i m en t s. 2o A
r a p i d a s s o c i a t i o n o f a g l y c o p r o t e i n (rnol.
wt.
5.5
x
lo4)
with the
n u c l e a r membrane a n d e n d o p l a s m i c r e t i c u l u m i s p r o p o s e d w i t h l i t t l e redistribution a f t e r t h i s i n i t i a l association.
Antibodies which
p r e c i p i t a t e p u r i f i e d v i r u s envelope g l y c o p r o t e i n antigens o f Simian sarcoma-associated
virus
and F r i e n d leukaemia
virus
d e t e c t e d i n t h e m a j o r i t y o f n o r m a l human s e r a . * l of
Friend
virus-induced
erythroleukaemia
by
have
been
Immunoprevention vaccination
with
p u r i f i e d v i r a l e n v e l o p e g l y c o p r o t e i n h a s been r e p o r t e d . 2 2 The s t r u c t u r e s o f t h e o l i g o s a c c h a r i d e s
o f the
glycoprotein
e n c o d e d by e a r l y r e g i o n E3 o f a d e n o v i r u s 2 have been e ~ t a b l i s h e d . ~ ~ I n f e c t i o n by t h i s v i r u s d o e s n o t a f f e c t t h e u s u a l c e l l u l a r h i g h Q m a n n o s e g l y c o s y l a t i o n p a t h w a y , and, d e s p i t e b e i n g v i r u s - c o d e d , g l y c o p r o t e i n is
glycosylated i n the
mammalian c e l l - c o d e d g l y c o p r o t e i n .
same
manner d s
a
the
typical
m-RNAs e n c o d e d i n e a r l y r e g i o n 3
o f t h e a d e n o v i r u s t y p e 2 genome h a v e b e e n p u r i f i e d , a n d
proteins
e n c o d e d by d i f f e r e n t mRNAs h a v e b e e n i d e n t i f i e d i n a c e l l - f r e e The p r i m a r y a m i n o a c i d s t r u c t u r e o f
p r o t e i n - s y n t h e s i z i n g s y s t e m .24 a
glycoprotein contains a
hydrophobic signal
sequence,
two
presumptive g l y c o s y l a t i o n sites,and a hydrophobic r e g i o n c l o s e t o t h e C-terminus. The e a r l y r e g i o n E3 o f a d e n o v i r u s t y p e 7 e n c o d e s a glycoprotein
(mol.
wt.
2.8
x
lo4)
and a
protein that are
non-
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
171
e s s e n t i a l f o r growth of t h e v i r u s i n culture.25 These m o l e c u l e s may be a n a l o g u e s o f t h e g l y c o p r o t e i n ( m o l . w t . 2.5 x l o 4 > a n d p r o t e i n c o d e d by E 3 o f a d e n o v i r u s t y p e 2 . H e p a t i t i s B s u r f a c e a n t i g e n c o n t a i n s a p r o t e i n (rnol. w t . 2.5 x l o 4 ) a n d a g l y c o p r o t e i n ( m o l . w t . 3.0 x l o 4 ) o f i d e n t i c a l a m i n o a c i d I t i s p r o p o s e d t h a t t h e t w o a n t i g e n c o m p o n e n t s may composition.26 d i f f e r only i n t h e presence of carbohydrate i n t h e glycoprotein and Some p h y s i c o c h e m i c a l t h a t t h e y a r e i n f a c t t h e same g e n e p r o d u c t . and immunological properties o f a glycopeptide obtained a f t e r p r o t e l y t i c cleavage of h e p a t i t i s B s u r f a c e a n t i g e n have been r e p o r t e d .27 A method f o r determining s p e c i f i c r e a c t i v i t y t o herpes simplex v i r u s t y p e s 1 a n d 2 has been d e v e l o p e d , u s i n g human s e r a t o i m m uno p r e c i p i t a t e s p e c i f i c g l y c o p r o t e i n a n t i g e n s f r o m v i rus - i n f e c t e d c e l l e x t r a c t s . 2 8 The v i r a l g l y c o p r o t e i n s p r e c i p i t a t e d from t h e s e e x t r a c t s w e r e t h e n a n a l y s e d by S D S - p o l y a c r y l a m i d e g e l electrophoresis. Mono c l o n a l a n t i bo d i e s d i r e c t e d a g a i n s t h e r p e s s i m p l e x v i r u s g l y c o p r o t e i n s have been u s e d t o p r o t e c t mice a g , a i n s t a c u t e v i r u s induced neurological disease.29 Evidence t h a t t h e virus-induced g l y c o p r o t e i n gC e x p r e s s e s t y p e - s p e c i f i c a n t i g e n i c d e t e r m i n a n t s , w h e r e a s g l y c o p r o t e i n gD e x p r e s s e s t y p e - c o m m o n d e t e r m i n a n t s , i s reported. By u s i n g a t e m p e r a t u r e - s e n s i t i v e m u t a n t o f h e r p e s s i m p l e x v i r u s t y p e 1, a n d i n h i b i t o r s o f g l y c o s y l a t i o n , s p e c i f i c g l y c o p r o t e i n s o f t h e v i r u s h a v e b e e n s h o w n t o be i n v o l v e d i n T c e l l - m e d i a t e d c y t o l y s i s o f v i r u s - i n f e c t e d cells.30 Two-dimensional gel e l e c t r o p h o r e s i s h a s been used t o i d e n t i f y p o l y p e p t i d e s and g l y c o p r o t e i n s o f h e r p e s s i m p l e x v i r u s t y p e 1.31 Pulse chase e x p e r i m e n t s and t r e a t m e n t o f i n f e c t e d cells w i t h neuraminidase s u g g e s t s t h a t t h r e e g l y c o p r o t e i n s c o n t a i n neuraminic acid and t h a t s y n t h e s i s o f t w o o f t h e t h r e e o c c u r s by a t l e a s t t e n d i s c r e t e s t e p s . The h e r p e s s i m p l e x v i r u s t y p e l - s p e c i f i c g l y c o p r o t e i n has been p u r i f i e d by a f f i n i t y c h r o m a t o g r a p h y o n i m m o b i l i z e d s o y a b e a n agglutinin and Helix pomatia lectin.32 The b i n d i n g o f t h e glycoprotein t o these l e c t i n s indicates the presence of a terminal 2-ace t am i d o - 2 - d e o x y-or -p -ga 1a c t 0 s y 1 r e s i d u e i n a n o l i go sa c c h a r i de , a finding not previously reported f o r glycoproteins of enveloped viruses. Two m o n o c l o n a l a n t i b o d y p r e p a r a t i o n s w h i c h a r e h e r p e s s i m p l e x v i r u s t y p e 2 - s p e c i f i c have been i s o l a t e d . 3 3 One o f t h e a n t i b o d i e s
172
Carbohydrate Chemistry
precipitated
a
glycoprotein with
g l y c o p r o t e i n E of Drecipitated a
the
v i r u s whereas
characteristics similar to
the other antibody preparation
glycoprotein not
previously
described.
This
g l y c o p r o t e i n i s t e n t a t i v e l y d e s i g n a t e d g l y c o p r o t e i n F. E n e r g y d e p l e t i o n o f v i r a l c e l l s c a u s e d by t h e a d d i t i o n t o t h e c u l t u r e medium o f
carbonyl
cyanide
formation o f N,N’-diacetylchitobiosyl the pool size o f affects the
p-glucosyl
pool
size
of
(CCCP),
3-chlorophenylhydrazone
an uncoupler o f o x i d a t i v e phosphorylation,
does n o t a f f e c t
pyrophosphoryl dolichol,
phosphoryl dolichol,
the
or
and o n l y s l i g h t l y
GDP-Q-manno~e.~~ Using t h i s
system,
i n f l u e n z a v i r u s h a e m a g g l u t i n i n c a n be g l y c o s y l a t e d i n c h i c k e m b r y o fibroblasts
o r a nona-a-mannosyl
e i t h e r a penta-a-mannosyl
derivative.
The
construction o f
a
recombinant
c o n s i s t i n g o f an SV40 v e c t o r and a c l o n e d f u l l - l e n g t h
viral
genome
DNA c o d i n g f o r
t h e h a e m a g g l u t i n i n s u r f a c e g l y c o p r o t e i n o f i n f l u e n z a v i r u s h a s been reported.35 produced
a
Infection of glycoprotein
primate cells similar
i n
with
the hybrid
molecular
size
virus
to
the
haemagglutinin o f influenza virus. Some virus,
b i o c h e m i c a l and b i o l o g i c a l p r o p e r t i e s o f i n f l u e n z a C
a s w e l l a s some m o r p h o l o g i c a l a s p e c t s o f t h e o r g a n i z a t i o n o f
t h e g l y c o p r o t e i n s o n t h e v i r a l e n v e l o p e a n d t h e p l a s m a membranes o f infected cells,
have been r e p o r t e d . 3 6
The c a r b o h y d r a t e p o r t i o n o n
t h e h a e m a g g l u t i n i n o f i n f l u e n z a v i r u s does n o t a p p e a r t o be o f m a j o r importance i n d e f i n i n g the a n t i g e n i c i t y o f the h a e m a g g l ~ t i n i n . ~ ~ Although t h e a b i l i t y o f u n t r e a t e d and g l y c o s i d a s e - t r e a t e d v i r u s i n h i b i t
the
binding
haemagglutinin i s almost
o f
antibodies
directed
indistinguishable,
against 50% o f
release o f
carbohydrate from i n t a c t v i r u s p a r t i c l e s s i g n i f i c a n t l y haemaggluti n at i n g
activity.
The
haem a g g l u t i n i n
to the the
affected membrane
g l y c o p r o t e i n o f i n f l u e n z a v i r u s i s composed o f a t r i p l e - s t r a n d e d c o i l e d c o i l o f a - h e l i c e s e x t e n d i n g 76A f r o m t h e membrane, g l o b u l i n r e g i o n o f a n t i p a r a l l e l B-sheet,
which
and a
contains the
r e c e p t o r - b i n d i n g s i t e and the v a r i a b l e a n t i g e n i c determinants, i s p o s i t i o n e d on t o p of l i k e topology,
t h i s stem.38
Each s u b u n i t h a s a n u n u s u a l l o o p -
s t a r t i n g a t t h e membrane, e x t e n d i n g l 3 5 A d i s t a l l y ,
a n d f o l d i n g b a c k t o e n t e r t h e membrane.
Four antigenic s i t e s on t h e
t h r e e - d i m e n s i o n a l s t r u c t u r e have been i d e n t i f i e d . 3 9
A t l e a s t one
a m i n o a c i d s u b s t i t u t i o n i n e a c h s i t e s e e m s t o be r e q u i r e d f o r t h e p r o d u c t i o n o f new e p i d e m i c s t r a i n s . Glycosylated t r y p t i c peptides o f
the haemagglutinin o f
i n f l u e n z a A v i r u s have been s e p a r a t e d u s i n g a c o m b i n a t i o n o f i o n -
173
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides exchange
chromatography
and g e l
f i l t r a t i ~ n . ~ ' Seven d i f f e r e n t
g l y c o s y l a t e d t r y p t i c p e p t i d e classes were o b t a i n e d from t h e HA1 p o l y p e p t i d e , w h i c h i s d e r i v e d f r o m t h e N - t e r m i n a l segment o f t h e h a e m a g g l u t i n i n , a n d o n l y one g l y c o s y l a t e d p e p t i d e f r o m t h e C - t e r m i n a l p o r t i o n (HA2).
Three t y p e s o f c a r b o h y d r a t e c h a i n s ,
complex,
high
a-
mannose, a n d h y b r i d t y p e , a r e p r e s e n t i n t h e H A 1 p o l y p e p t i d e whereas HA2 c o n t a i n s
the
oligosaccharide
complex-type
side
chains
oligosaccharide
of
the
major
chains.
glycoprotein
The
HA1
of
i n f l u e n z a v i r u s h a e m a g g l u t i n i n have been e x a m i n e d f o r a n t i g e n i c activity
using a
qualitative
solid-phase
and
radioimmunoassay.41
quantitative
differences
o l i g o s a c c h a r i d e s i d e c h a i n s were d e t e c t e d ,
the
exception o f
terminus.42 complex
a
highly
c h a i n has been e l u c i d a t e d ,
aggregated
r e g i o n near
A single glycosylated I-asparagine
oligosaccharide
these
the antigenically active
The amino a c i d sequence o f a Hong
side chains a r e cross-reactive.
Kong i n f l u e n z a h a e m a g g l u t i n i n l i g h t (HA2) with
Although
between
structure
was
the
C-
residue bearing a
noted.
The
amino
acid
sequence and o l i g o s a c c h a r i d e d i s t r i b u t i o n f o r t h e h a e m a g g l u t i n i n f r o m t h e e a r l y Hong Kong i n f l u e n z a v i r u s A/Aichi/2/68(X-31) reported.43 variant
Although the
was
found
to
oligosaccharide
be
identical
h a e r n a g g l u t i n i n f r o m A/Memphis/72, specific I-asparaginyl the individual sequence
residues,
carbohydrate
analysis
shows
the
to
that
i n
the
t h e monosaccharide compositions o f
u n i t s were
different.
existence o f
structural
b e t w e e n t h e Hav 7 a n d t h e Hong K o n g (H3) Two o f
heavy
haemagglutinin from
the
found
w i t h sugar u n i t s a t t a c h e d t o s i x
i n f l u e n z a virus.44 chain o f
have been
distribution i n this
Amino
acid
relationships
haemagglutinins of
the six oligosaccharide the
u n i t s on t h e
Hong K o n g
variant
A/Memphis/102/72
have been shown t o be a n t i g e n i c a l l y r e l a t e d t o t h e
h o s t antigen.45
They
attached t o
C-Asn-8
c o n t a i n t h e N-acetyl-lactosamine-type and &-Asn-22.
residues
The a n t i g e n i c s t r u c t u r e o f
i n f l u e n z a v i r u s h a e m a g g l u t i n i n has been d e f i n e d u s i n g h y b r i d o r n a a n t i b o d i e s . 46 The
uncleaved
(HNO)
and
the
cleaved
(HN)
forms
h a e r n a g g l u t i n i n n e u r a m i n i d a s e o f New c a s t l e d i s e a s e v i r u s , Ulster,
the
strain
have been s u b j e c t e d t o e l e c t r o p h o r e s i s u n d e r r e d u c i n g and
non-reducing
conditions.47
The c o n v e r s i o n o f o n e f o r m t o t h e o t h e r
i n v o l v e s t h e r e l e a s e by p r o t e o l y s i s o f a g l y c o p e p t i d e (mol. x lo3).
of
wt.
8.0
T h e i s o e l e c t r i c p o i n t s h a v e b e e n d e t e r m i n e d a n d t h e c.d.
spectra analysed f o r both p o s t - t r a n s l a t i o n a l and uncleaved Newcastle
disease
virus
p r o t e o l y t i c a l l y cleaved
g l y ~ o p r o t e i n s . ~ I~t i s
Carbohydrate Chemistry
174
d e m o n s t r a t e d t h a t c l e a v a g e i s p a r a l l e l e d by a c o n f o r m a t i o n a l c h a n g e of t h e g l y coprot e i n s The d i s s o c i a t i o n a n d r e c o n s t i t u t i o n o f t h e S e m l i k i F o r e s t v i r u s membrane u s i n g n o n - i o n i c d e t e r g e n t have been s t u d i e d . 4 9 Different mechanisms of r e c o n s t i t u t i o n give rise t o symmetric and a s y m m e t r i c vesicles. Glycoprotein E l of the virus i n g - 3 - i n f e c t e d cells is g l y c o s y l a t e d a n d i n s e r t e d i n t o the e n d o p l a s m i c r e t i c u l u m membrane w i t h o u t s i m u l t a n e o u s s y n t h e s i s o f t h e e n v e l o p e p r o t e i n ( m o l . w t. 6.2 x lo4), w h i c h i s a p r e c u r s o r o f t w o o t h e r e n v e l o p e g l y c ~ p r o t e i n s . ~ ' A r e v e r s i b l e defect i n t h e g l y c o s y l a t i o n of t h e membrane p r o t e i n s o f S e m l i k i F o r e s t v i r u s G-1 m u t a n t h a s b e e n d e s c r i b e d . 5 1 At a r e s t r i c t i v e t e m p e r a t u r e t h e v i r a l membrane g l y c o p r o t e i n s a r e a r r e s t e d i n t h e rough e n d o p l a s m i c r e t i c u l u m , but a r e t r a n s p o r t e d t o t h e Golgi complex once t h e c u l t u r e s are s h i f t e d t o a p e r m i s s i v e temperature. An e f f i c i e n t m e t h o d f o r t h e i s o l a t i o n o f l a r g e q u a n t i t i e s o f S e n d a i v i r u s F - g l y c o p r o t e i n by r e d u c t i o n o f t h e i n t a c t v i r u s p a r t i c l e s w i t h d i t h i o t h r e i t o l and s o l u b i l i z a t i o n o f t h e r e d u c e d v i r u s w i t h n o n - i o n i c d e t e r g e n t has b e e n r e p o r t e d . 5 2 Two m e m b r a n e g l y c o p r o t e i n s ( H A N A p r o t e i n a n d F - p r o t e i n ) o f HVJ ( S e n d a i ) v i r u s c o n t a i n r e s i d u e s o f I - f u c o s e , Q - g a l a c t o s e , Q-mannose, a n d 2-ace tam i d o - 2 - d e o x y - & - g l u c o s e , b u t no 2 - a c e t a m i d o - 2 - d e o x y - Q galactose or neuraminic acid.53 Both g l y c o p r o t e i n s c o n t a i n o l i g o s a c c h a r i d e c h a i n s o f t h e h i g h g-mannose t y p e a n d o f t h e complex type. T h e com p l e x - t y p e o l i go s a c c h a r i des c o n t a i n 2 - a c e t a m i d o - 2 deox y - 3 -&a -1- f uco s y 1 - 4 - 2 4 -Q-ga 1a c t o s y 1-Q-g1 uco s y 1 g r o u p s i n t h e i r outer- chain moieties. P a r a m y x o v i r u s f u s i o n (F) p r o t e i n s a r e a c t i v e l y i n v o l v e d i n t h e i n d u c t i o n o f membrane fusion.54 The F p r o t e i n o f S e n d a i v i r u s i s a c t i v a t e d by p r o t e o l y t i c c l e a v a g e t o y i e l d t w o d i s u l p h i d e - l i n k e d p o l y p e p t i d e s (F1 a n d F2). C o n f o r m a t i o n s of t h e i n a c t i v e uncleaved p r e c u r s o r g l y c o p r o t e i n and t h e a c t i v e c l e a v e d form have been D i r e c t e v i d e n c e was o b t a i n e d f o r t h e p r e s e n c e o f compared. hydrophobic binding sites o n t h e active form of t h e F p r o t e i n , but n o t o n t h e i n a c t i v e form. Grow t h - d e p e n d e n t c h a n g e s i n a - m a n n o s y l o l i g o s a c c h a r i d e s d e r i v e d from a s i n g l e s p e c i e s o f membrane g l y c o p r o t e i n have been s t u d i e d i n a v i r a l system.55 E l a n d E 2 g l y c o p r o t e i n s o f S i n d b i s v i r u s e x p r e s s d i f f e r e n t r e l a t i v e q u a n t i t i e s o f f o u r o l i g o s a c c h a r i d e s. Metabolically labelled Sindbis virion and cell-associated v i r a l g l y c o p e p t i d e s h a v e b e e n a n a l y s e d by a c o m b i n a t i o n o f g e l f i l t r a t i o n and specific glycosidase digestion.56 T h e g e n e r a l m o d e l o f I-
.
5: Glycoproteins, Glycopeptides, Proteoglycans, m d Animal Polysaccharides asparaginyl oligosaccharide
p r o c e s s i n g f o r t h e n e u t r a l and a c i d i c -
t y p e s t r u c t u r e s i n normal and l e c t i n - r e s i s t a n t cells
was
confirmed.
175
The
presence o f
Chinese hamster o v a r y
unusual
small
neutral
s t r u c t u r e s i n t h e m a t u r e v i r a l g l y c o p r o t e i n f r o m t h e c e l l l i n e s was a l s o noted. The r o l e o f c a r b o h y d r a t e i n t h e m o r p h o g e n e s i s o f v e s i c u l a r s t o m a t i t i s v i r u s has been s t u d i e d u s i n g t u n i c a m y c i n t o i n h i b i t g l y c o ~ y l a t i o n . ~I ~t i s c o n f i r m e d t h a t d i f f e r e n t g l y c o p r o t e i n s h a v e d i f f e r e n t carbohydrate requirements f o r Simple
mutational
requirement.
changes
Micelles
i n
a
formed
spontaneously p a r t i t i o n i n t o vesicles.58
plasma-mem b r a n e i n s e r t i o n .
protein by
can
also
glycoproteins
affect
of
this
this virus
sonicated phosphatidyl-choline
The h y d r o p h o b i c t a i l f r a g m e n t o f t h e g l y c o p r o t e i n i s
r e s i s t a n t t o p r o t e o l y s i s when t h e g l y c o p r o t e i n is i n s e r t e d i n t o t h e vesicle bilayer.
The i n t r a c e l l u l a r l o c a t i o n o f a n i n t e g r a l membrane
g l y c o p r o t e i n o f v e s i c u l a r s t o m a t i t i s v i r u s h a s been e ~ a m i n e d . ~ ’ I t s p a s s a g e t o t h e c e l l s u r f a c e has been s y n c h r o n i z e d a n d t h u s s t a g e s i n t h e i n t r a c e l l u l a r p a t h w a y t h a t i t f o l l o w s w e r e mapped.
Glycoprotein
o f v e s i c u l a r s t o m a t i t i s v i r u s has b e e n i s o l a t e d a n d i t s b i n d i n g t o c e l l s u r f a c e s studied.60
The s t r u c t u r e s o f t h e o l i g o s a c c h a r i d e s o f
the v i r u s grown i n d i f f e r e n t teratocarcinoma c e l l l i n e s a r e n o t identical
for
sensitivity glycosidases.61 stomatitis sy n t h e s i z i n g
a l l to
cell
lines,
digestion Baby-hamster
virus
and
by
as a
evidenced mixture
by
o f
differences i n endo-
and
KO-
kidney c e l l s i n f e c t e d with v e s i c u l a r
starved
of
Q-glucose
are
capable
o f
-m an no sy 1a t e d 1ip i d -1i n ke d o 1igo sa c c h a r i de s b u t ca nno t
e f f i c i e n t l y c o m p l e t e t h e a s s e m b l y o f Q-Glce3-Q-Maneg-Q-G1cpNAc2pyrophosphoryl dolichol,
r e s u l t i n g i n an a b n o r m a l l y g l y c o s y l a t e d G
p r o t e in .62 Polyoma
virus
requires
oligosaccharide receptors f o r
specific
cell-surface
infection of
sialo-
3T6 c e l l s . 6 3
A
t r i s a c c h a r i d e s e q u e n c e (2) c a n s e r v e a s a s p e c i f i c c e l l - s u r f a c e receptor
both for
polyma
virus-mediated
haemagglutination and f o r
polyoma v i r u s i n f e c t i o n o f h o s t c e l l s . a-Neue5Ac-( 2 + 3 ) - B - Q - G a l p (
1+4) -8-GalpNAc
(2) The a c t i o n o f
toyocamycin on t h e biosynthesis o f
v i r a l
glycoproteins i n c e l l s chronically infected w i t h a murine r e t r o v i r u s i s i n r e d u c i n g t h e content o f envelope g l y c o p r o t e i n (mol.
wt.
7.0 x
176
Carbohydrate Chemistry
104).64
However, t h e
precursor
m o l e c u l a r w e i g h t o f 8.5
x
of
this
glycoprotein,
having a
i s synthesized n o r m a l l y and i s
lo4,
processed i n t o i t s f i n a l products, which accumulate i n t h e c e l l s . from
The p u r i f i e d m a j o r e n v e l o p e g l y c o p r o t e i n ( m o l . w t . 8.5 x l o 4 ) avian m y e l o b l a s t o s i s v i r u s contains 45%carbohydrate i n c l u d i n g
25 % 2 - a c e t am ido - 2 distributed backbone.
- d e ox y -Q - g l u co s e .65
between
seven
to
nine
One s t r u c t u r a l m o d e l ,
p r o p e r t i e s of
the
0 1igo sa c c h a r ide c h a in s a r e
points
on
the
polypeptide
which r e c o n c i l e s t h e hydrodynamic
glycoprotein with
nearly
spherical
architecture
o b s e r v e d by e l e c t r o n m i c r o s c o p y , r e q u i r e s t h e o r g a n i z a t i o n o f t h e p o l y p e p t i d e c h a i n and a p p r o x i m a t e l y h a l f o f t h e c a r b o h y d r a t e i n t o a g l o b u l a r form.
The r e m a i n i n g c o v a l e n t l y l i n k e d o l i g o s a c c h a r i d e s
would extend outwardly
fcom
the globular s t r u c t u r e as randomly
o r i e n t e d chains. I n
order
bunyavirus, c o n t r o l of
to
characterize
Belmont
virus
as
a
possible
e v i d e n c e has been p r e s e n t e d o f s i m i l a r t r a n s l a t i o n a l t h e p r o t e i n s a n d g l y c o p r o t e i n s s p e c i f i e d by
the two
v i r u s e s i n mammalian cells.'' Cauliflower
mosaic
virus
capsid
f 1uo r e s c e i n -de r i va t i z e d co n c a n a v a l i n A ,
glycoproteinaceous nature of The
synthesis,
polypeptide t h us
binds
to
demo n s t r a t i n g t h e
the r n ~ l e c u l e . ' ~
glycosylation, and i d e n t i f i c a t i o n
p r o t e i n s i n d u c e d by E p s t e i n - B a r r
of
v i r u s have b e e n r e p o r t e d . 6 8
s p o n t a n e o u s a n d i n d u c e d s y n t h e s e s of
the
early The
virus antigens i n Raji
c e l l s immobilized on surfaces coated w i t h anti-lymphocy t e g l o b u l i n have been s t u d i e d . "
A m a j o r g l y c o p r o t e i n ( g p 350/220)
B a r r v i r u s h a s been d e t e c t e d by i m m u n o f l u o r e s c e n c e
o f Epstein-
with monoclonal
a n t i b o d i e s o n b o t h t h e p l a s m a membrane a n d i n t h e c y t o p l a s m . 7 0 F o u r s t r u c t u r a l p o l y p e p t i d e s o f H a z a r a v i r u s h a v e been r e s o l v e d
.
by so d i um do de c y 1 s u l p h a t e p o 1y a c r y 1am ide g e l e l e c t r o p h o r e s i s T h r e e g l y c o p r o t e i n s ( m o l . w t s . 8.4 x l o 3 , 4.5 x lo3, a n d 3.0 x l o 3 ) are associated with
the
v i r i o n envelope, and a
nonglycosylated
polypeptide i s associated w i t h t h e nucleocapsid. The GIX
a n t i g e n o f m u r i n e r e t r o v i r u s has b e e n a n a l y s e d u s i n g
m o n o c l o n a l a n t i b ~ d i e s , ' ~r e a c t i v i t y w i t h w h i c h a p p e a r s t o r e q u i r e a stable
configurational
change i n t h e
coinciding w i t h glycosylation,
precursor
protein
rather than d i r e c t p a r t i c i p a t i o n of
carbohydrate i n the antigenic site.
Thus l a t e r e n z y m i c r e m o v a l o f
c a r b o h y d r a t e c h a i n s does n o t a l t e r r e a c t i v i t y w i t h t h e a n t i b o d i e s . Two m e a s l e s v i r u s g l y c o p r o t e i n s ,
a haemagglutinin and a f u s i o n
p r o t e i n , have been i n c o r p o r a t e d i n t o a r t i f i c i a l l i p i d b i l a y e r s . 7 3
177
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides The
resultant
'virosomes'
haemagglutinating
had
activity,
visible
and
should
'spikes'
and
provide a
s t u d y i n g immune responses t o m e a s l e s v i r u s ,
possessed
reagent
for
independent o f the
immunosuppressive e f f e c t s o f t h e whole v i r u s . M o n o s p e c i f i c a n t i b o d i e s have been p r e p a r e d t o each o f e n v e l o p e p a r a m y x o v i r u s g l y c o p r o t e i n s (HN a n d F ) . 7 4
two
Antibodies t o
t h e HN g l y c o p r o t e i n i n h i b i t e d haem a g g l u t i n a t i n g a n d neuram i n i d a s e activities.
A n t i - F a n t i b o d i e s i n h i b i t e d haemolysis.
The c o m p l e t e n u c l e o t i d e s e q u e n c e o f t h e g l y c o p r o t e i n g e n e i n r a b i e s v i r u s has been d e t e r m i n e d . 7 5 to
be!
made o f
several
T h i s has a l l o w e d a p r e d i c t i o n
features o f
the glycoprotein,
from
the
deduced a m i n o a c i d sequence. The e f f e c t s o f c e l l t r a n s f o r m a t i o n o n t h e g l y c o s y l a t i o n a n d p r o c e s s i n g o f P r a g u e C Rous s a r c o m a v i r u s e n v e l o p e p o l y p e p t i d e s have been r e p o r t e d . 7 6 the
The m a j o r d e t e c t a b l e d i f f e r e n c e i s a n i n c r e a s e i n
r e l a t i v e amount
o f
larger acidic-type
containing residues o f neuraminic acid, 2-deoxy-~-glucose,
oligosaccharides
a-galactose, and 2-acetamido-
compared w i t h t h e n e u t r a l - t y p e o l i g o s a c c h a r i d e s .
G l y c o p e p t i d e s d e r i v e d from
a g l y c o p r o t e i n (mol.
been e x a m i n e d a f t e r s e q u e n t i a l
glycosidase
wt.
8.5
digestions
x lo4) h a v e followed
by
g e l f i l t r a t i ~ n . ~The ~ major h y b r i d species has an oligo-Q-mannosyl c o r e o f f i v e Q-mannosyl- and t w o 2-acetamido-2-deoxy-Q-glucosyl residues together
with
substitution
residues w i t h an a c i d i c s i d e a c e t y l n e u r a mi n i c a c i d ,
of
one
of
c h a i n composed o f
the
p-mannosyl
residues o f
N-
Q - g a l a c t o p y r a n o s e , a n d 2-acetam i d o -2-deox y-Q-
glucopyranose. Immunological s t u d i e s on t h e r e l a t i o n s h i p o f t h e major envelope g l y c o p r o t e i n o f HEL-12
v i r u s and t h e analogous g l y c o p r o t e i n s o f
S i m i a n sarcoma-Simian a s s o c i a t e d v i r u s have
been reported.78
A
a n d baboon endogenous v i r u s
glycopeptide
(mol.
w t .
2.0
x
lo5)
a s s o c i a t e d w i t h t h e t r a n s f o r m a t i o n o f S i m i a n sarcoma v i r u s h a s been
.
iden t i f i e d 79 A g l y c o p r o t e i n s p e c i f i e d by s p l e e n f o c u s - f o r m i n g v i r u s i n t h r e e
c e l l l i n e s v a r i e s i n m o l e c u l a r s i z e and p e p t i d e c o m p o s i t i o n b u t retains
both x e n o t r o p i c and e c t o t r o p i c murine leukaemia v i r u s
antigenicity.80
Both q u a l i t a t i v e and q u a n t i t a t i v e d i f f e r e n c e s i n
the post-translational
processing o f t h e envelope g l y c o p r o t e i n s o f
p o l y c y t h a e m i a - and a n a e m i a - i n d u c i n g s t r a i n s o f s p l e e n f o c u s - f o r m i n g v i r u s have been r e p o r t e d . 8 1 The i n v i t r o t r a n s l a t i o n o f U n k u n i e m i v i r u s - s p e c i f i c RNA h a s led to
the identification o f a non-structural
p r o t e i n and a
Carbohydrate Chemistry
178 p r e c u r s o r t o membrane g l y co p r o t e i n s . 82 Vaccinia
wt.
(mol.
virus-induced haemagglutinin i s a single glycoprotein
lo4>
x
8.5
v i r us - i n d u c e d
w h i c h a p p e a r s a t t h e p l a s m a membrane a s a
function. 83
l-lin k e d
A 1 t h ough
predominate i n the molecule,
o 1i g o s a c c h a r i de s
a b o u t 25% o f t h e o l i g o s a c c h a r i d e c h a i n s
which a r e g - g l y c o s i d i c a l l y l i n k e d t o t h e p r o t e i n a r e r e s p o n s i b l e f o r the
biological
activity
of
the
molecule.
Glycoproteins
and
p h o s p h o p r o t e i n s have been i d e n t i f i e d a s s t r u c t u r a l e l e m e n t s o f t h i s
.
v i r u s B4 wt.
Immunogenic g l y c o p r o t e i n s (mol. 1.18
x l o 5 ) i s o l a t e d from
varicella-zoster
lo4,
x 104,and
9.8
vaccine
strains
of
v i r u s have been i d e n t i f i e d . 8 5
I n r e c o n s t i t u t i o n experiments E l and E 2 o f
x
6.5
l a b o r a t o r y and
u s i n g t h e envelope
western equine e n c e p h a l i t i s virus,
glycoproteins
the haemolytic
a c t i v i t y i s associated w i t h the E l glycoprotein which a c t s as a haemaggl u t i n i n . 86
Plant Glycoproteins
2 A
l a r g e number o f l e g u m e - s e e d
glyc~proteins.~’ A glycoproteins functional
high
and t h e i r
significance
p o l y p e p t i d e s have been i d e n t i f i e d a s
of
degree
corresponding
binding lectins
between
might
i n m a i n t a i n i n g l a r g e aggregates
have of
seed some
protein
i n compact i n s o l u b l e f o r m . Twelve N - g l y c o s y l a t e d p o l y p e p t i d e s have been i d e n t i f i e d i n glyoxysomal
membranes
p o l y a c r y lam i d e w t s . 9.0
x
of
castor
electrophoresis
lo4,
7.1
x
lo4,
A glycoprotein,
5.6 x
beans.88
correspond
lo4,
4.7
to
Major
bands o n SDS-
g l yco p r o t e i n s o f m 01.
x 1 0 4 , a n d 4.3
x
lo4.
involved w i t h the control of i n t e r c e l l u l a r
r e c o g n i t i o n and o f p o l l e n i n c o m p a t i b i l i t y has been i s o l a t e d f r o m stigmas o f glycoprotein,
S-allele
g e n o t y p e S2S2
of
Brassica oleracea.89
w h i c h i s n o t p r e c i p i t a t e d by c o n c a n a v a l i n A,
r e s i d u e s o f L-arabinose,
!-galactose,
Evidence t h a t l i p i d - l i n k e d a s e q u e n t i a l manner f r o m
!-glucose,
The
contains
a n d P-mannose.
o l i g o s a c c h a r i d e s can be a s s e m b l e d i n
UDP - 2 - a c e t a m i d o -2-deox y - B - g l uco se a n d GDP -
P-mannose by membrane p r e p a r a t i o n s f r o m d e v e l o p i n g s o y b e a n c o y l e d o n s has
been p r e s e n t e d . ”
Tissue
slices
synthesize products which
resemble t h e completed 7 s storage g l y c o p r o t e i n s . Concanavalin
A
has
been shown
to
bind to
the
endoplasmic
r e t i c u l u m and t h e s t a r c h g r a i n s u r f a c e o f r o o t s t a t o c y t e s ( L e p i d i u m s a t i v u m L9).91
of
cress
The r e c e p t o r f o r t h e l e c t i n a p p e a r s t o be a
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
179
high-molecular- weight glycoprotein which is r e s i s t a n t t o s o l u b i l i t a t i o n a f t e r g l u t a r a l d e h y d e f i x a t i o n of t h e cells. Two s o l u b l e g l y c o p r o t e i n c o m p o n e n t s f r o m r y e g r a s s ( L o l i u m p e r e n n e ) have been i s o l a t e d a n d p a r t i a l l y c h a r a ~ t e r i z e d . ~ ’ They a r e c h e m i c a l l y and i m m u n o l o g i c a l l y d i s t i n c t . One o f t h e g l y c o p r o t e i n s i s t h e m a j o r a l l e r g e n o f r y e - g r a s s p o l l e n and b i n d s s p e c i f i c a l l y t o i m m u n o g l o b u l i n E o f s e n s i t i z e d human s u b j e c t s . 9 3 Immunological c r o s s - r e a c t i v i t y t h a t i s d u e , a t l e a s t i n p a r t , t o common a n t i g e n i c carbohydrate components i s reported. Two t y p e s o f p r o t e i n g l y c o s y l a t i o n i n c e l l - f r e e p r e p a r a t i o n s o f d e v e l o p i n g c o t y l e d o n s o f P h a s e o l u s v u l g a r i s have been p o ~ t u l a t e d . ~ ~ I n a n i n v i t r o s y s t e m , t h e a t t a c h m e n t o f 2-acetamido-2-deoxy-Qglucosyl r e s i d u e s t o phytohaemagglutinin and phaseolin does n o t involve lipid-linked intermediates w h i l s t the glycosylation of a class of p r o t e i n s of heterogeneous molecular weights involves the l i p i d pathway. A q u e o u s e x t r a c t s o f l e a v e s , r o o t s , a n d stems o f g r e e n b e a n , a n d l e a v e s o f mung b e a n , s o y b e a n , a n d l i m a b e a n , c a u s e a g g l u t i n a t i o n o f c e l l s o f a s a p r o p h y t i c b a c t e r i u m P s e u d o m o n a s p ~ t i d a . P~u ~r i f i e d p r e p a r a t i o n s of t h e a g g l u t i n i n are composed of g l y c o p r o t e i n s , containing k-arabinose, e-galactose, and e-galacturonic a c i d as t h e p r e d o m i n a n t c a r b o h y d r a t e c o m p o n e n t s . The p r o p e r t i e s o f l a c c a s e , i s o l a t e d from S c h i n u s m o l l e trees, i n c l u d i n g m o l e c u l a r w e i g h t , amino a c i d and c a r b o h y d r a t e c o m p o s i t i o n , have been d e ~ c r i b e d . ’ ~ The presence o r absence o f an inducing hormonal glycoprotein i n t h e g r o w t h medium o f Volvox l a r t e r i d e t e r m i n e s w h e t h e r t h e algaegrow sexually or asexually.97 The s i t e and time o f f o r m a t i o n o f t h i s g l y c o prot e i n h a v e been est a b l i shed.
3
Lectins
Review a r t i c l e s d e a l i n g w i t h l e c t i n s i n c l u d e a s t u d y of l e c t i n s i n The endogenous b i o l o g i c a l f u n c t i o n s o f l e c t i n s higher plants.98 have b e e n r e ~ i e w e d . ’ ~ E x a m p l e s f r o m p l a n t s , c e l l u l a r slime moulds, The a n d a n i m a l t i s s u e s are u s e d i n t h e a n a l y s i s of t h i s problem. c a r b o h y d r a t e b i n d i n g s p e c i f i c i t y and c e l l - membrane i n t e r a c t i o n o f Vicia s a t i v a l e c t i n have b e e n r e p 0 r t e d . l ” Affinity chromatography f o r t h e p u r i f i c a t i o n o f l e c t i n s h a s been described.lo1 Stable, high c a p a c i t y a f f i n i t y adsorbents used i n t h e i s o l a t i o n o f l e c t i n s have b e e n p r e p a r e d by d e r i v a t i z a t i o n o f e p o x y - a g a r o s e w i t h v a r i o u s
--
Carbohydrate Chemistry
180 carbohydrates .Io2 Isoelectric
focusing
i n polyacrylarnide
gels
containing
immobilized sugars r e s u l t s i n a s h i f t i n the p o s i t i o n o f p r o t e i n bands c a p a b l e o f
i n t e r a c t i o n w i t h these ligands.lo3
These a f f i n i t y
g e l s have been u s e d t o d e m o n s t r a t e i n t e r a c t i o n s w i t h l e c t i n s . s e n s i t i v e assay
A
has been d e v e l o p e d f o r m e a s u r i n g t h e b i n d i n g o f
l e c t i n s t o g l y c o c o n j u g a t e s i n w e l l s of p o l y v i n y l c h l o r i d e ~ 1 a t e s . l ' ~ A chromophore-labelled
Q - g a l a c t o - Q - m a n n a n h a s b e e n p r e p a r e d by
c o u p l i n g a m o n o c h l o r o t r i a z i n y l dye t o guaran.lo5
The p r o d u c t h a s
been u s e d f o r t h e q u a n t i t a t i v e e s t i m a t i o n o f l e c t i n s h a v i n g a f f i n i t y f o r Q - g a l a c t o s y l o r 2-acetam ido-2-deox y - Q - g a l a c t o s y l r e s i d u e s . The f o l l o w i n g l e c t i n s h a v e b e e n p u r i f i e d a n d some o f t h e i r p h y s i c a l a n d c h e m i c a l c h a r a c t e r i s t i c s r e c o r d e d ( T a b l e 1) . l o 6 - l 1 4 I o d i n a t e d l e c t i n s have
been u s e d f o r
the determination o f the
d e g r e e of d e g l y c o s y l a t i o n o f h i g h n - m a n n o s y l g l y c o p r o t e i n s f o l l o w i n g digesti o n w i t h e
- B -2-ace tam i d o -2-deox y - E - g l ucanase H. '15 I n t e r m o l e c u l a r f o r c e s i n l e c t i n- c a r boh y d r a t e in t e r a c t i o n h a v e
been analysed on features.'l6
the
basis o f
their
s t r u c t u r e and chemical
The r o l e o f w a t e r , a s w e l l a s t h a t o f o t h e r p h y s i c o -
c h e m i c a l p a r a m e t e r s i n f l u e n c i n g t h e f o r m a t i o n o f such c o m p l e x e s i n aqueous m e d i a ,
i s a l s o discussed.
A model d e p i c t i n g t h e importance
o f hydrogen bonding and charge-transfer
i n t e r a c t i o n s as t h e main
s o u r c e s o f complex s t a b i l i t y i n t h e a s s o c i a t i o n between l e c t i n s and carbohydrates i s proposed. By p e r f o r m i n g e l e c t r o p h o r e s i s p e r p e n d i c u l a r t o a s t a t i o n a r y pH gradient i n polyacrylamide gels containing a s p e c i f i c l i g a n d e i t h e r covalently f i x e d o r entrapped i n the gel matrix, i t i s possible t o measure d i s s o c i a t i o n c o n s t a n t s o f l e c t i n s a n d t h e i r pH dependence i n a pH r a n g e . l l 7
The m e t h o d h a s b e e n u s e d t o s t u d y t h e l e c t i n s f r o m
R i c i n u s communis a n d L e n s c u l i n a r i s . Aqueous e x t r a c t s f r o m l e a v e s , l e a v e s o f mung bean, saprophytic
r o o t s , a n d s t e m s o f g r e e n bean a n d
soybean,and l i m a bean a g g l u t i n a t e c e l l s o f a putida."
Purified
p r e p a r a t i o n s c o n t a i n b o t h c a r b o h y d r a t e and p r o t e i n ,
r e q u i r e Mg2+ f o r
activity,
bacteria,
Pseudomonas
and a r e h e a t s t a b l e .
pathogenic bacteria
No a g g l u t i n a t i o n o f
A s u r v e y o f t w e n t y - t w o commonly i n g e s t e d f r u i t s , seeds has i n d i c a t e d the presence, interact cells.l19
with
other
plant
or nonpseudomonad s a p r o p h y t e s was o b s e r v e d . i n many,
vegetables, and
o f l e c t i n s w h i c h can
c o m p o n e n t s o f human s a l i v a a n d S t r e p t o c o c c u s
I n t e r a c t i o n s o f soybean and p e a n u t l e c t i n s w i t h
t h a t n o d u l a t e soybean,
peanut, o r
mutans
rhizobia
b o t h p l a n t s have b e e n studied."'
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
0
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182
Carbohydrate Chemisrry
No r e l a t i o n s h i p b e t w e e n t h e a b i l i t y o f p e a n u t o r soybean r h i z o b i a t o n o d u l a t e t h e r e c i p r o c a l h o s t p l a n t and t h e i r a b i l i t y
to bind to
the
l e c t i n o f t h a t p l a n t c o u l d be d e m o n s t r a t e d . The seeds o f P i s u m s a t i v u m ,
-V.
Canavalia ensiformis,
-s a t i v a , a n d R i c i n u s c o m m u n i s c o n t a i n p r o t e i n s ,
Vicia
faba,
termed l e c t i n
binders, which a r e associated w i t h t h e i r r e s p e c t i v e lectins.lZ1
The
p e a l e c t i n b i n d e r i s a t e t r a m e r i c g l y c o p r o t e i n composed o f i d e n t i c a l
w t. 5.1
s u b u n i t s (mol.
x
lo4),
and i t s i n t e r a c t i o n w i t h t h e l e c t i n i s
a b o l i s h e d by l o w pH o r by g - g l u c o s e . w t .
p r o t e i n (mol.
3.5
x
lo4)
The c o n c a n a v a l i n A b i n d e r i s a
c o n t a i n i n g no
covalently
bound
carbohydrate.
A s t u d y o f t h e t i s s u e s o f s o y b e a n a n d j a c k bean s e e d s
demonstrates
t h a t t h e l e c t i n o f a p a r t i c u l a r s p e c i e s may e x h i b i t a
very
narrow
range
glycoconjugates.’**
o f
s p e c i f i c i t y
towards
endogenous
This suggests t h a t n o t o n l y i s t h e r e one m a j o r
l e c t i n i n the cotyledons o f a given plant
b u t a l s o one s o l u b l e
g l y c o p r o t e i n i n t h e c o t y l e d o n s t h a t t h e l e c t i n can r e c o g n i z e .
The
s t r i k i n g s p e c i f i c i t y shown b e t w e e n h o m o l o g o u s l e c t i n a n d r e c e p t o r i s n o t m a i n t a i n e d when t h e l e c t i n i s u s e d t o . i s o l a t e g l y c o p r o t e i n s f r o m t h e same t i s s u e i n o t h e r p l a n t s p e c i e s .
G 1 y co p e p t ide s a n d o l igo s a c c h a r id e s de r ive d f r o m proteins
have
been
different lectins.lZ3
used
g l ycoaspa r a g i ne s ,
define
the
1-g l y co s y 1
specificity
of
twelve
Lectins considered i d e n t i c a l i n terms of
monosaccharide s p e c i f i c i t y d i f f e r e n c e s i n more
to
possess an a b i l i t y
complex
structures.
to recognize fine
The
observation
that
g l yco pe p t i d e s, a n d g l yco p r o t e i ns p o s s e s s a h i g h e r
a f f i n i t y f o r l e c t i n s t h a n t h e r e l a t e d o l i g o s a c c h a r i d e s has been e x p l a i n e d by t h e f a c t t h a t t h e g l y c a n - a m i n o a c i d l i n k a g e l e a d s t o s t r u c t u r e s more r i g i d t h a n t h o s e o f t h e o l i g o s a c c h a r i d e s themselves. Many o f
t h e l e c t i n s u s e d i n t h i s s t u d y seem t o p o s s e s s i n
o r near
t h e i r carbohydrate-binding s i t e a hydrophobic area such as t h a t d e s c r i b e d f o r c o n c a n a v a l i n A,
a n d t h i s c o u l d be t h e c a u s e o f n o n -
s p e c i f i c hydrophobic i n t e r a c t i o n s o f glycopeptides
Fluorescence b i nd i ng o f
between t h e l e c t i n s and r e s i d u e s
o r g l y c o p r o t e i n s , a s d e s c r i b e d f o r c o n c a n a v a l i n A. p o l a r i z a t i o n has been u s e d t o i n v e s t i g a t e t h e
4-m e t h y 1um be 11if e r y 1 - 6 -Q -ga 1a c t o p y r a n 0 s i de
p r e c at o r i u s a g g l ut i n i n
. 24
to
A b r us
L e c t i n a c t i v i t y has been d e t e c t e d i n seeds o f t w e n t y A c a c i a species o f A u s t r a l i a n origin.125
Fourteen of
the
species
had been
r e p o r t e d by o t h e r w o r k e r s t o c o n t a i n no l e c t i n . P e a n u t l e c t i n has been p u r i f i e d by a d s o r p t i o n o n g l u t a r a l d e h y d e cross-linked
h e m a t i c d e s i a l y l a t e d a n d r e t i c u l a t e d human s t r o m a . l Z 6
183
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides The
lectin
(mol.
1.25
wt.
lo5),
x
although
homogeneous on
e l e c t r o p h o r e s i s , y i e l d s e i g h t m a j o r bands o n i s o e l e c t r i c f o c u s i n g . The s c r e e n i n g o f p e a n u t ( A r a c h i s ) a n d i t s w i l d r e l a t i v e s f o r m u l t i p l e m o l e c u l a r f o r m s o f p e a n u t l e c t i n and f o r g e n o t y p e s d e v o i d of
the
b e e n r e ~ 0 r t e d . l ~A ~ p e a n u t
l e c t i n has
lectin-negative
p h e n o t y p e was d e t e c t e d i n f o u r g e n o t y p e s (<0.1% o f t h o s e e x a m i n e d ) . L e c t i n preparations from lectin-containing into a
s m a l l number
profiles.
of
d e f i n e d and
Association constants
genotypes were r e s o l v e d
clearly
related isolectin
and t h e r m o d y n a m i c
t h e b i n d i n g o f s u g a r s t o p e a n u t a g g l u t i n by s p e c t r o s c o p y h a v e been r e p o r t e d . 1 2 8
parameters
U.V.
for
difference
The b i n d i n g k i n e t i c s o f m e t h y l
a- a n d m e t h y l B - P - g a l a c t o p y r a n o s i d e s t o t h e l e c t i n h a v e been s t u d i e d b y 1 3 C n.m.r.
spectroscopy employing methyl Q-galactopyranosides
s p e c i a l l y e n r i c h e d i n 1 3 C a t C-l.129 presented.
A two-step b i n d i n g model i s
Peanut a g g l u t i n i n e x h i b i t s
cryoinsolubility,
which
i s
p a r t i a l l y r e v e r s i b l e and t o t a l l y i n h i b i t e d i n t h e p r e s e n c e o f Qgalactosides.130
The e f f i c a c y
cryoinsolubility
i s
of
related to
the
their
s u g a r s as affinity
inhibitors of
for
the
lectin.
Charge-charge i n t e r a c t i o n s are o f l i t t l e importance, b u t hydrogen bonds
and/or
van
der
Waals
interactions
are
most
responsible for the formation o f cryoprecipitates.
probably
The e f f e c t s o f
c h e m i c a l m o d i f i c a t i o n o f t h e c o n f o r m a t i o n and b i o l o g i c a l a c t i v i t y o f p e a n u t a g g l u t i n h a v e been r e ~ 0 r d e d . l ~When ~ f r e e amino groups a r e modified
with
succinic
benzenesulphonic acid, capacity,
anhydride
and
l-isothiocyanate-4-
the derivatives r e t a i n t h e i r sugar-binding
although the
agglutinating
activity
with
neuraminidase-
t r e a t e d e r y t h r o c y t e s and v a r i o u s tumour c e l l s i s reduced. tyrosine residues
are
modified f i r s t l y
t h e n w i t h 4-aminophenyl-a-Q-91ucopyranoside aminobenzyll-a-Q-neuraminic
activities
are
not
acid,
markedly
When
I-
w i t h t e t r a n i t r o m e t h a n e and and w i t h 2-(4-
t h e a g g l u t i n a t i n g and m i t o g e n i c
altered.
The
i n f l u e n c e of
these
m o d i f i c a t i o n s on t h e c o n f o r m a t i o n o f t h e l e c t i n was e x a m i n e d by c.d. s p e c t r a l studies. The
d i s t r i b u t i o n
-simplicifolia
o f
the
I i s o l e c t i n s (A4,
seeds o f d i f f e r e n t
trees,
f i v e
tetrameric
A3B, A 2 B 2 # AB3,
Bandeiraea
B4> i s o l a t e d from
h a s been a n a 1 ~ s e d . l ~The ~ amount o f e a c h
i s o l e c t i n v a r i e s f r o m seed t o seed, each seed d i s p l a y i n g a u n i q u e distribution of the five isolectins. intact
s imp 1i c i f o 1i a -B. --
cleavage
by
an endo-
It i s postulated that the
I B4 i s o l e c t i n u n d e r goes p r o t e o l y t i c
or =-protease
t o
form
f i r s t t h e AB3
i s o l e c t i n , w h i c h i n t u r n i s t h e n p r o c e s s e d f u r t h e r t o y i e l d t h e A2B2
184
Carbohydrate Chemistry
isolectin,
and so o n u n t i l A4
i s produced.
4-Methylumbelliferyl
g l y c o s i d e s have been used i n b i n d i n g s t u d i e s w i t h t h e l e c t i n s A4,
BSI-B4,
and BS I 1
from
d i a l y s i s,
Equilibrium
Bandeiraea ( G r i f f o n i a )
fluorescent
BSI-
simplicif01ia.l~~
enhancement,
quantitative
p r e c i p i t a t i o n , and h a p t e n i n h i b i t i o n t e c h n i q u e s have been used t o investigate
the
simplicifolia
binding
characteristics
of
four
of
the
2.
I i s 0 1 e c t i n s . l ~ ~One o f t h e m o s t u n u s u a l f e a t u r e s i s
that,although
different,
b o t h t h e A a n d B s u b u n i t s h a v e t h e same
f o r a-Q-galactosyl residues. Although both s u b u n i t s a l s o b i n d 2 - a c e t a m i d o - 2 - d e o x y - ~ - g a l a c t o s y l r e s i d u e s , t h e A s u b u n i t has a n
affinity
association constant
f o r t h e amino sugar more t h a n 1 0 0 0 - f o l d g r e a t e r
t h a n the B s u b u n i t . Affinity
chromatography
i m m o b i l i z e d 4-aminophenyl means of
equations
of
divalent
B-P-glucopyranoside
concanavalin
A
on
h a s been a n a l y s e d by
based o n t h e s i m p l e s t m o d e l ,
i n which t h e l e c t i n
has e q u i v a l e n t a n d i n d e p e n d e n t b i n d i n g s i t e s a n d one l e c t i n m o l e c u l e b i n d s o n l y o n e i m m o b i l i z e d l i g a n d a t a time.135 P o l y a c r y 1am i d e
e l e c t rophore s i s o f
gra d i ent
r e v e a l s the presence o f
various minor
co n c a n a V a l i n A
bands w h i c h a r e n o t
detected
by p o l y a c r y l a m i d e d i s c e l e c t r o p h ~ r e s i s . ~The ~ ~ m i n o r components a r e suggested t o the
combined
s iev i ng
.
be d i f f e r e n t m o l e c u l a r s p e c i e s s e p a r a t i n g by v i r t u e o f effect
of
electrophoretic
mobility
Over w i d e r a n g e s o f t e m p e r a t u r e a n d pH, of only subunits
and m o l e c u l a r
concanavalin A consists
d i m e r s a n d t e t r a m e r ~ . ' ~ ~The l a r g e f r a c t i o n i n commercial preparations
o f hydrolysed
causes s i g n i f i c a n t
populations
o f d i m e r i c species t h a t a s s o c i a t e o n l y weakly or n o t a t a l l .
The
e f f e c t o f t h e b i n d i n g o f saccharide l i g a n d s on t h e r e v e r s i b l e dimert e t r a m e r e q u i l i b r i u m o f t h e l e c t i n has b e e n s t u d i e d by h i g h - s p e e d s e d i m e n t a t i o n e q ~ i 1 i b r i u m . l ~C~o n t r a r y
to
e a r l i e r p u b l i s h e d work,
s a c c h a r i d e b i n d i n g does n o t a p p e a r t o a f f e c t e q u i l i b r i u m o f t h e n a t i v e c o n c a n a v a l i n A, irreversible
behavio ur
i n
preparations
proportions o f hydrolysed subunits.
the
dimer-tetramer
a l t h o u g h i t does i n t r o d u c e containing
sign1f icant
The d i m e r - t e t r a m e r e q u i l i b r i u m
i s l i n k e d t o t h e weak b i n d i n g o f c a l c i u m a t a s i t e p r o b a b l y i n t h e dimer-dimer
interface.
interaction
of
Studies
concanavalin
A
have
with
g l u c o py r a no s y 1- a - e - m a n n o p y r a n o s i de ga l a c t o p y r a n 0 s y l - a - D = - m a n n o p y m a 1 t o ~ i d e . l ~T h ~e disaccharides
i s
existence
proposed.
,
been
Some o f
two the
the
2-2-a-P-
4 - n i t r o p h e n y 1- 2 - 2 - a - Q -
r a n 0 s i de, of
reported of
4-nitrophenyl and
4-ni trophenyl
b i n d i n g modes specificity
for
the
requirements
185
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
f o r t h e i n t e r a c t i o n o f concanavalin A w i t h t h e non-reducing g l y c o s y l group are characterized, Temperature-jump
and a r e s u m m a r i z e d i n Scheme 1.
relaxation studies using 4-methylumbelliferyl
a-Q-mannopyranoside as a f l u o r e s c e n c e i n d i c a t o r l i g a n d have been used i n an e x a m i n a t i o n o f
the
binding kinetics o f
m e t h y l a-Q-
m a n n o p y r a n o s i d e t o c o n c a n a v a l i n A. 140
Scheme 1 An
i.r.-attenuated
total-reflectance
s p e c t r o s c o p i c method has
been u s e d t o s t u d y t h e i n t e r a c t i o n o f m o n o l a y e r s o f c o n c a n a v a l i n A The e f f e c t s o f f i l m
w i t h mono- and p o l y - s a c c h a r i d e s . 1 4 1 pH,
urea,
Mn2+, a n d Ca2+ o n t h e b i n d i n g o f d e x t r a n ,
pressure,
m e t h y l a-Q-
mannopyranoside, and Q - g a l a c t o s e t o t h e l e c t i n w e r e s t u d i e d . thermodynamic parameters o f t h e Mn2+-binding i n the
concanavalin A
dimer
c a l o r i m e t r i c technique.142
state
have
been
The
reaction with
determined
The r e s u l t s i n d i c a t e t h a t
by
a
the free
e n e r g y c h a n g e f o r t h e p r o c e s s i s d o m i n a t e d by a p o s i t i v e e n t r o p y change
and
that
independent. the
binding of
process
i s
effects.
the
two
i d e n t i c a l S1 s i t e s
of
the
dimer
ar,e
C a l o r i m e t r i c s t u d i e s have a l s o been used t o examine saccharides
induced
by
Kinetic
activation of
both
and
to
concanavalin
favourable
equilibrium
Ca2+ i n d i c a t e t h a t
The
A.143
enthalpic
studies
of
and
binding entropic
concanavalin
ion binding at
A
S2 produces a
general ordering o f the ligands a t t h i s s i t e which i n t u r n orders those side-chain residues involved i n saccharide r e ~ o g n i t i 0 n . l ~ ~ The c a r b o h y d r a t e - b i n d i n g
activity
v a r i o u s l e v e l s o f Ca2+ a n d Mn2+
of
concanavalin
has
A containing
b e e n r e ~ 0 r t e d . l ~T h~e
p r e c i p i t a t i o n a c t i v i t y o f t h e l e c t i n w i t h g l y c o g e n as w e l l as t h e binding of affected
lectin to
4-nitrophenyl
a-p-mannopyranoside
by t h e n u m b e r o f b o u n d C a 2 + - i o n s .
are strongly
The k i n e t i c s o f t h e
i n t e r a c t i o n s o f Ca2+ w i t h c o n c a n a v a l i n A i n t h e p r e s e n c e o f C o 2 + , Mn2+, and/or of
Zn2+ h a v e been f o l l o w e d by m e a s u r e m e n t o f t h e q u e n c h i n g
fluorescence of
4-methylumbelliferyl
bound t o ~ r 0 t e i n . l ~Some ~
observations
a-a-mannopyranoside on
metal-ion
when
requirements
a n d s u g a r b i n d i n g by B a n d e i r a e a s i z p l i c i f o l i a I l e c t i n a r e a l s o reported. The c o - o p e r a t i v e
binding properties o f concanavalin A with
186
Carbohydrate Chemistry
g l y c o c o n j u g a t e s a r e dependent o n t h e p r e s e n c e o f h y d r o p h o b i c b i n d i n g sites.14'
The d e g r e e o f c o - o p e r a t i v i t y
can be s u b s t a n t i a l l y a l t e r e d
by c o n f o r m a t i o n a l changes o f t h e l i g a n d . Irradiation of
concanavalin A
tetrameric
with
a high-pressure
me r c u ry lamp i n t h e presence o f c h l o r a c e t a m i d e r e s u l t s i n t h e l e c t i n showing a monomeric m o l e c u l a r w e i g h t w i t h r e t e n t i o n o f b i n d i n g capacity.148 Fluorescein-concanavalin
conjugates
A
are
capable
of
d i s t i n g u i s h i n g b e t w e e n n o r m a l and m a l i g n a n t human ~ e 1 l s . l ~ ' The f o u r
-V i c i a faba
l e c t i n s c o n c a n a v a l i n A,
agglutinin,
Lens c u l i n a r i s a g g l u t i n i n ,
and P i s u m s a t i v u m
be i d e n t i c a l i n t e r m s o f s p e c i f i c i t y
agglutinin,
considered t o
or a-;-glucose,
f o r a-;-mannose
p o s s e s s t h e a b i l i t y t o r e c o g n i z e f i n e d i f f e r e n c e s i n more c o m p l e x carbohydrate structures.150 A
lectin
from
the
a g g l u t i n a t i n g sheep haemolysis
of
seeds
and o t h e r
rabbit
of
Croton
tiglium
erythrocytes
erythrocyte^.'^^
i s
as w e l l
These e f f e c t s
capable as
of
inducing
are i n h i b i t e d
by sheep e r y t h r o c y t e g l y c o p e p t i d e s . D a t u r a s t r a m o n i u m l e c t i n has been p u r i f i e d on c o l u m n s o f diacetylchitobiose
i m m o b i l i z e d on a g a r o s e . l o 6
37% c a r b o h y d r a t e ,
sugar,
of
which I-arabinose
and a h i g h c o n t e n t o f
hydroxy-C-proline i d e n t i c a l with,
residues. the
lectin
i s t h e most
C-cysteine,
glycine,
potato.152
predominant I - s e r i n e , and but not
Although they
chito-oligosaccharides,
N,"-
contains
This l e c t i n i s s i m i l a r to,
l e c t i n from
similar specificities for
The
have
the Datura l e c t i n
shows i t s g r e a t e s t a f f i n i t y f o r g l y c o p e p t i d e s and t h e p o t a t o l e c t i n
for chito-oligosaccharides.
I n both lectins,
the hydroxy-l-proline
residues are substituted w i t h 6-i-arabinofuranosides,
and t h e r e a r e
L-serine residues i n the glycosylated region that are substituted with
a-4-galactopyranosyl
residues.
The
structure
of
the
glycosylated r e g i o n o f the l e c t i n s i s very s i m i l a r t o t h a t o f the hydroxy-&-proline-rich
glycopeptides of plant c e l l walls,
and t h e s e
l e c t i n s c o u l d be p r e c u r s o r s o f s u c h m a t e r i a l . Metal-chelate
affinity
chromatography
p u r i f i c a t i o n o f the Dolichos b i f l o r u s
has been used i n t h e
s e e d 1 e ~ t i n . l ~E q ~u i l i b r i u m
d i a l y s i s s t u d i e s show t h a t t h e l e c t i n h a s t w o c o m b i n i n g s i t e s p e r ma l e c u l e f o r me t h y 1 2-ace t am i d o -2-deoxy - a - g a l a c t 0 s i d e
. 54
Subunit I
i s p r i m a r i l y r e s p o n s i b l e f o r t h e c a r b o h y d r a t e - b i n d i n g p r o p e r t i e s of the lectin.
Monoclonal antibodies s p e c i f i c f o r subunit I of
the
D o l i c h o s b i f l o r u s l e c t i n combine w i t h t h e C - t e r m i n a l p o r t i o n o f t h e subunit
a n d may
be i n t e r a c t i n g w i t h
the
active
site.155
This
187
5: Glycoproteins, Glycopeptides, Proteoglytans, and Animal Polysaccharides
a n t i b o d y does n o t r e a c t w i t h a n o t h e r l e c t i n - l i k e p r o t e i n f r o m t h e s t e m s and l e a v e s o f t h e p l a n t . 1 5 6
The m o n o c l o n a l a n t i b o d y i s o f
i m p o r t a n c e i n t h a t i t can d i s t i n g u i s h s u b u n i t I f r o m s u b u n i t I 1 o f t h e seed l e c t i n . one a n o t h e r
A l t h o u g h t h e s e t w o s u b u n i t s appear t o d i f f e r f r o m
only at
their
C-terminal
I 1 i s not.157
whereas s u b u n i t
Atomic
ends,
subunit
I i s active
absorption spectrophotometry
h a s e s t a b l i s h e d t h e p r e s e n c e o f Ca2+, Mg2+, Mn2+, Zn2+, a n d Cu2+ i n native golichos biflorus subsequent constant
addition of
1 e ~ t i n . l ~ The ~ e f f e c t s o f r e m o v a l and
different
o f the l e c t i n for
m e t a l i o n s on t h e a s s o c i a t i o n are
2-acetamido-2-deoxy-q-galactose
reported. The l e c t i n o f E r y t h r i n a c o r a l l o d e n d r o n s e e d s i s s i m i l a r t o soybean l e c t i n ,
being a glycoprotein
(mol.
1.1 x
wt.
lo5) and
2-acetamido-2-deoxy-qbinding t o 2-amino-2-deoxy-Q-galactose, g a l a c t o s e , a- and B - p - g a l a c t o s i d e s , a n d g - g a l a c t o s e . 1 5 9
-
l e c t i n i s o l a t e d from
A
the roots o f
soybean ( G l y c i n e
!ax)
s e e d l i n g s i s s i m i l a r b u t n o t i d e n t i c a l t o t h e c o r r e s p o n d i n g seed The l e c t i n i s a s s o c i a t e d w i t h t h e o u t e r s u r f a c e s o f t h e
lectin.16'
r o o t and i s c o n c e n t r a t e d i n t h e segments o f t h e r o o t a t w h i c h r o o t h a i r and e a r l y secondary r o o t s a r e observed. and
s o l u b l e soybean l e c t i n , i s of
From e x a m i n a t i o n o f
&
g e n o t y p e s o f G l y c i n e max f o r m e m b r a n e - b o u n d a n d b u f f e r i t seems u n l i k e l y t h a t t h e membrane l e c t i n
c y t o p l a s m i c origin.161
5gJa)
(slycine
I n contrast
l e c t i n
has
erythroagglutinating activity
been
after
t o o t h e r r e p o r t s soybean shown
complete
t o
r e t a i n
removal o f
i t s
Mn2+ f r o m
t h e w h o l e molecule.162 The l e n t i l
subunit
(Lens
s t r u c t u r e and c o m p l e t e a m i n o a c i d s e q u e n c e
cglinaris)
l e c t i n have
been
determined.163
of A
c o m p a r i s o n b e t w e e n t h e s e c o n d a r y s t r u c t u r e o f c o n c a n a v a l i n A and t h e p r o b a b l e s e c o n d a r y s t r u c t u r e o f l e n t i l l e c t i n a s p r e d i c t e d by t w o different
methods
indicates
that
the
folding
of
these
two
p o l y p e p t i d e s has been w e l l c o n s e r v e d d u r i n g e v o l u t i o n . I n a re-examination specifities
of
of
the h i g h - a f f i n i t y
pea and l e n t i l l e c t i n s ,
the
carbohydrate-binding ability
ofvarious
g l y c o p e p t i d e s t o b i n d t o c o l u m n s o f t h e i m m o b i l i z e d l e c t i n s was studied.164
I-Fucose
r e s i d u e s a r e i n d e e d an i m p o r t a n t
determinant
i n t i g h t b i n d i n g t o pea and l e n t i l l e c t i n b u t n o t t o c o n c a n a v a l i n A. The l e c t i n f r o m M a c l u r a p o m i f e r a , a f t e r p u r i f i c a t i o n by i o n exchange and a f f i n i t y
c h r o m a t o g r a p h y , has been r e s o l v e d i n t o f i v e
s t r u c t u r a l l y r e l a t e d p r o t e i n components o f s i m i l a r h a e m a g g l u t i n a t i n g and c a r b o h y d r a t e - b i n d i n g
activity.165
P u r i f i c a t i o n o f the l e c t i n
Carbohydrate Chemistry
188
h a s a l s o been a c h i e v e d by a d s o r p t i o n o n t o p o l y l e u c y l h o g A+H b l o o d f o l l o w e d by e l u t i o n w i t h m e l i b i o s e o r 2 - a c e t a m i d o -
group substances,
2-deoxy-;-galactose.166 104,and 1.2
Three s u b u n i t s (mol.
wt.
1.45
interaction i s important combining
site
of
lo4,
x
x lo4) are present i n the i n t a c t lectin.
1.35
f o r b i n d i n g o f t h e l e c t i n t o sugars.
the
lectin
appears
to
be
x
Hydrophobic
as
large
The as
a
disaccharide. The i n t e r a c t i o n o f been s t u d i e d . 1 6 7 g r o u p s a t t h e C-2,
I w i t h c a r b o h y d r a t e has
mistletoe lectin
I n h i b i t i o n data suggest
that
unmodified hydroxyl
C-3, a n d C-4 p o s i t i o n s o f t h e Q - g a l a c t o p y r a n o s y l
r i n g are essential f o r binding t o the active s i t e o f the l e c t i n . The ligands
association with
the
c h a r a n t i a have of
protein
constants
for
the
P-galactose-specific
been d e t e r m i n e d t h r o u g h
fluorescence.168
binding
of
lectin
from
the
Analysis
of
a
series
light-induced the
iodide
quenching quenching
s u g g e s t s t h a t t h e r e is a s l i g h t i n c r e a s e i n t h e a c c e s s i b i l i t y o f t r y p t o p h a n r e s i d u e s o f t h e l e c t i n on b i n d i n g l a c t o s e . the binding characteristics of sugar
by
fluorescence
i.
of
Momordica
L-
Studies o f
charantia l e c t i n t o i t s specific
spectroscopy
using 4-methylumbelliferyl
f3-g-
g a l a c t o p y r a n o s i d e show t h e q u e n c h i n g o f t h e s u g a r u p o n b i n d i n g t o the
lectin.169
The
binding i s carbohydrate-specific
and i s
i n h i b i t e d by l a c t o s e . Considerable
homology
i n structures
and b i o l o g i c a l a c t i v i t i e s
has been r e p o r t e d f o r 62 c u l t i v a r s o f P h a s e o l u s v u l g a r i s , basis
of
isolectin
A
patterns.’”
number
of
on t h e
cultivars
are
s u f f i c i e n t l y d i f f e r e n t t o w a r r a n t s u b c l a s s i f i c a t i o n and f u r t h e r characterization. Inclusion o f diets
of
rats
nitrogen.171
p u r e l e c t i n s f r o m t h e seeds o f
increases
both
faecal
and
p.
urinary
vulqaris i n losses
of
The a n i m a l s d e v e l o p a n t i b o d i e s o f l o w a v i d i t y f o r t h e
dietary lectins.
L e c t i n t o x i c i t y i s t e n t a t i v e l y suggested t o r e s u l t
f r o m t h e combined e f f e c t s o f i n t e r f e r e n c e w i t h n o r m a l i n t e s t i n a l d i g e s t i o n and/or
absorption o f
p r o t e i n through
damaged e n t e r o c y t e s
and o f s y s t e m i c r e s p o n s e s o f t h e r a t t o t h e i n t e r n a l i s e d l e c t i n . The
subunit
compositions
of
individual
tetrameric
p h y t o h a e m a g g l u t i n i n i s o l e c t i n s f r o m P h a s e o l u s v u l g a r i s have been e x a m i n e d by i s o e l e c t r i c
focusing
and sodium
dodecylsulphate
e l e c t r o p h ~ r e s i s . ~The ~ ~ p r o p o r t i o n o f s u b u n i t s r e s u l t i n g f r o m each i s o l e c t i n was d e t e r m i n e d f o r a d i r e c t
physical conformation o f the
i s o l e c t i n subunit structures. Early
events
i n
the
synthesis
of
polypeptides
of
Ricinis
5: Glycoproteins, Glycopeptides, Proteoglycans, .and Animal Polysaccharides
--communis
189
a g g l u t i n i n t y p e 1 have been r e ~ 0 r t e d . l ~C~o - t r a n s l a t i o n a l
t r a n s l o c a t i o n a c r o s s t h e e n d o p l a s m i c r e t i c u l u m membrane i s a c c o m p a n i e d by p r o t e o l y s i s and, w h e r e a p p r o p r i a t e , core glycosylation. The
agglutinins
from
Solanum
tuberosum
and wheat-germ
a g g l u t i n i n i n t e r a c t w i t h k e r a t a n s u l p h a t e and c h i t i n ~ u 1 p h a t e . l ~ ~ D i f f e r e n c e s i n t h e r e a c t i v i t y between t h e polysaccharides and t h e two
l e c t i n s are
reported.
Chemical m o d i f i c a t i o n
of
amino
or
ca r bo x y 1 gro up s , I - a r g i n i ne , I - m e t h io n i ne, and L - h i s t id i ne r e s i due s does n o t a l t e r s i g n i f i c a n t l y t h e h a e m a g g l u t i n a t i n g a c t i v i t y o f p o t a t o 1 e ~ t i n . l ~I - ~ Tyrosine involved i n the activity,
and I - t r y p t o p h a n findings
residues are closely
which are
similar
to
those
r e p o r t e d f o r o t h e r p r o t e i n s t h a t b i n d o l i g o m e r s o f 2-acetamido-2de ox y -p- g l uco s e. The dependence o f t h e Sophora j a p o n i c a l e c t i n o n b i v a l e n t m e t a l i o n s a s w e l l a s o n t h e pH r a n g e f o r o p t i m a l h a e m a g g l u t i n a t i o n a n d precipitation
i n the
presence
o f
bivalent
e ~ t a b 1 i s h e d . l ~F~u r t h e r c h a r a c t e r i z a t i o n o f
c a t i o n s h a s been
the
combining s i t e o f
t h i s l e c t i n i s reported.
-Ulex
europaeus h a e m a g g l u t i n i n I1 ( C y t i s u s - t y p e
inhibited affinity
by
di-N-acetylchitobiose,
chromatography.l'l
g l uco s e
.
anti-H(O))
i s
been p u r i f i e d
by
sugar w i t h s m a l l e r
@-xylose, !-galactose,
Ribitol
has
The l e c t i n i s a g l y c o p r o t e i n i n w h i c h
Q-mannose i s t h e p r e d o m i n a n t glucose,
and
amounts o f
Q-
L-fucose, and 2-amino-2-deoxy-Q-
t e i c h o i c a c i d o f Staphylococcus
aureus, w h i c h
contains
t e r m i n a 1 2 - ace t am i d o - 2 - d e o x y-B - E - g l uco sy 1 r e s i d u e s , po s s e s s e s a n a b n o r m a l a f f i n i t y f o r i m m o b i l i z e d wheat-germ a g g l u t i n i n i n b i n d i n g a t h i g h i o n i c strength.177 Interactions
of
monomeric
amino
sugars
with
wheat-germ
~pectroscopy.'~~ a g g l u t i n i n h a v e b e e n s t u d i e d b y 'H a n d 19F n.m.r. -N - A c e t y l n e u r a m i n i c a c i d a n d 2 - a c e t a m i d o - 2 - d e o x y - ~ - g l u c o s e have a common b i n d i n g s i t e o n t h e l e c t i n .
D e u t e r i u m n.m.r.
resonance has
been used t o d e l i n e a t e t h e m o l e c u l a r d y n a m i c s o f s u g a r s bound t o 1 e ~ t i n s . l ~2H~ r e l a x a t i o n t i m e s and r o t a t i o n a l for
2- { 2H 1 -ace tam i d o -2-deox y-a-
3 - { 2H 1 - g l uco se
absence o f wheat-germ a g g l u t i n i n were s t u d i e d . a p p e a r s t o have n e g l i g i b l e m o t i o n a l freedom on binding.
correlation
times
i n t h e p r e se nce and The p y r a n o s e r i n g
r e l a t i v e t o the protein
The a c e t a m i d o s i d e c h a i n i s a l s o i m m o b i l i z e d i n t h e
b i n d i n g s i t e , t h e o n l y m o t i o n a v a i l a b l e b e i n g r o t a t i o n o f t h e C2H3 group about i t s t h r e e f o l d axis.
Carbohydrate Chemistry
190
T h e e l u t i o n p r o f i l e s o f v a r i o u s g l y c o p e p t i d e s m o d i f i e d by glycosidase treatment, Smith degradation, acetolysis, and h y d r a z i n o l y s i s show t h a t t h e s t r u c t u r e (3) i s e s s e n t i a l f o r t h e b i n d i n g o f g l y c o p e p t i de s t o immo b i l i z e d w h e a t - g e rm a g g l “ t i n i n 8o Both t h e N,”-diacetylchitobiose m o i e t y and t h e B-Z-acetamido-2d e o x y - a - g l u c o s y l r e s i d u e l i n k e d t o C-4 o f t h e B - l i n k e d Q - m a n n o s y l r e s i d u e c o n t r i b u t e t o t h e i n t e r a c t i o n of t h e g l y c o p e p t i d e w i t h t h e lectin. The s u b s t i t u t i o n a t C-6 o f t h e i n n e r m o s t B-2-acetamido-Zd e o x y - Q - g l u c o s y l r e s i d u e by a n a - I = - f u c o s y l r e s i d u e o r a t C-6 o f t h e B - l i n k e d g - m a n n o s y l r e s i d u e by a n o t h e r g - m a n n o s e r e s i d u e r e d u c e s t h e a f f i n i t y of g l y c o p e p t i d e s f o r the column. In contrast to results f r o m o t h e r w o r k e r s , no i n t e r a c t i o n o f g l y c o p e p t i d e s c o n t a i n i n g 3a c e t y l n e u r a m i n i c acid r e s i d u e s was o b s e r v e d w i t h t h e l e c t i n . Some p r o p e r t i e s o f V i c i a g r a m i n e a l e c t i n p u r i f i e d by a f f i n i t y chromatography have been d e s c r i b e d , a n d t h e b i n d i n g of t h e l e c t i n t o v a r i o u s e r y t h r o c y t e s has been c h a r a c t e r i z e d . l a 1 The c o m p l e t e amino acid s e q u e n c e o f t h e a - s u b u n i t o f V i c i a s-a-t i v a l e c t i n h a s b e e n e s t a b l i s h e d a n d c o m p a r e d w i t h t h e k n o w n s e q u e n c e s o f l e c t i n s f r o m P i s u m s a t i v u m , L e n s c u l i n a r i s , 1. f a b a , a n d C a n a v a 1i a e n s i f o r m is ( co n ca n a v a l i n A );’ 82 A p a r t i a l l y p u r i f i e d h a e m a g g l u t i n i n a s s o c i a t e d w i t h c e l l walls from t h e h y p o c o t y l s o f Vigna r a d i a t a e x h i b i t s b o t h h a e m a g g l u t i n a t i n g activity and a - Q - g a l a c t o s i d a s e activity.183 It is suggested that, s i m i l a r t o t h e s e e d l e c t i n o f t h e same p l a n t , b o t h a c t i v i t i e s m i g h t The n a t i v e m o l e c u l e c o u l d be p r e s e n t i n t h e same m o l e c u l e . d i s s o c i a t e i n t o smaller u n i t s e x h i b i t i n g either of t h e two a c t i v i t i es
.
B-Q-GlcpNAc-( 1+4)-B-Q-Mang-(
1+4) -B-g-GlceNAc-( 1+4) -
B -Q -G1 ceNA c- 1 -A -Asn
A tetrameric Q-galactose-binding
p r o t e i n o f mung b e a n ( V i g n a t h a t d i s p l a y s b o t h h a e m a g g l u t i n i n a c t i v i t y a n d aa - g a l a c t o s i d a s e a c t i v i t y c a n be r e v e r s i b l y d i s s o c i a t e d i n t o a l o w m o 1e c u l a r- w e i gh t m o no m e r i c f o r m w h i c h po s s e s se s o n l y e n z ym i c activity.184 T h e p o s s i b i l i t y o f ffi v i v o c h a n g e s i n s u b u n i t e q u i l i b r i a , when c o m b i n e d w i t h t h e a c c o m p a n y i n g a l t e r a t i o n s i n activity, is suggested a s a possible physiological role of phy t o h a e m a g g l u t i n i n s .
_-----radiata)
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
191
Abrin A , p u r i f i e d from t h e s e e d s of Abrus p r e c a t o r i u s , h a s p h y s i c a l and b i o l o g i c a l p r o p e r t i e s s i m i l a r t o the b e t t e r known a b r i n C.185 P o s s i b l e s t r u c t u r a l r e l a t i o n s h i p s between t h e t w o g l y c o p r o t e i n s a n d a r e l a t e d a g g l u t i n i n from t h e same s o u r c e w e r e examined. A l t h o u g h c o v a l e n t l y a t t a c h e d c a r b o h y d r a t e i s a r e l a t i v e l y minor p a r t of t h e o v e r a l l s t r u c t u r e of r i c i n , o x i d a t i o n w i t h s o d i u m p e r i o d a t e r e s u l t s i n t h e d e s t r u c t i o n of _p-mannosyl r e s i d u e s and l o s s of t o x i c i t y o n t h e HeLa c e l l , w h i l e A-chain and 5 - c h a i n ( c e l l binding) a c t i v i t i e s a r e n o t s i g n i f i c a n t l y altered.186 A hybrid molecule c o n s i s t i n g of t h e 6 chain o f r i c i n l i n k e d t o i n s u l i n v i a a d i s u l p h i d e b o n d b i n d s t o t a r g e t c e l l s and m a i n t a i n s se ve r a l i n s u l i n - l i ke b i o l o g i c a l a c t i v i t i e s .l S e v e r a l human and m u r i n e tumour c e l l l i n e s undergo e x t e n s i v e a g g r e g a t i o n i n t h e p r e s e n c e of f e t u i n and a s i a l o f e t u i n . 1 8 8 This a c t i v i t y can be i n h i b i t e d by l a c t o s e and t o a l e s s e r e x t e n t b y g a l a c t o s e , 2- am i no-2-deox y -a- g a 1 a c t o s e , an d 2 - a c e t am i d o - 2 - de ox y -Q galactose. The i m p l i c a t i o n s o f t h e e x i s t e n c e o f a c a r b o h y d r a t e b i n d i n g p r o t e i n ( s ) o n t h e s u r f a c e o f m a l i g n a n t c e l l s o n t h e i r fi v i v o behaviour a r e d i s c u s s e d . Clq Serum a m y l o i d P c o m p o n e n t ( 9 . 5 s a l - g l y c o p r o t e i n ) , ( s u b c o m p o n e n t of t h e C 1 component o f complement), and C - r e a c t i v e p r o t e i n have l e c t i n - l i k e com b i n i n g s i t e s f o r c e r t a i n Q - g a 1 a ~ t a n s . l ~ ~ Two new a d d i t i o n a l c o m b i n i n g s i t e s o f t h e C - r e a c t i v e p r o t e i n have been d e s c r i b e d . F o e t a l - c a l f s k e l e t a l muscle c o n t a i n s t w o l e c t i n s having b i n d i n g a c t i v i t i e s i n h i b i t e d b y l a c t o s e and a s e p a r a t e h a e m a g g l u t i n a t i n g a c t i v i t y which i s n o t i n h i b i t e d by 1 a c t 0 s e . l ~ ~ The b i n d i n g of a s i a l o o r o s o m u c o i d t o t h e i s o l a t e d r a b b i t h e p a t i c l e c t i n a p p e a r s t o be a s a t u r a b l e and r e v e r s i b l e p r o c e s s a s s h o w n by s t e a d y - s t a t e and k i n e t i c a n a 1 y ~ i s . l ~The ~ minimal m o l e c u l a r w e i g h t (1.04 x lo5) of the intact rat hepatic receptor for a s i a l o g l y c o p r o t e i ns has been measured d i r e c t l y i n plasma rnembranes.lg2 The v a l u e o b t a i n e d i s s h o w n t o be s i g n i f i c a n t l y a f f e c t e d by the presence of d e t e r g e n t employed i n s o l u b i l i z a t i o n and is0 1a t i o n pro ce d u r e s. P r o t e i n h a e m a g g l u t i n i n s have been d e t e c t e d i n h o m o g e n a t e s o f e a c h o f t h e s i x c o x a l d e p r e s s o r m u s c l e s i n t h e l e g of t h e co c kr o a ch 93 The q ua n t i t a t i ve a b i 1i t y of so m e gl y co sa m i no g 1y ca n s t o i n h i b i t these haemagglutinins c o r r e l a t e s w i t h t h e muscles' i n n e r v a t i o n by i d e n t i f i e d motor neurons. The complete amino a c i d sequence of chicken h e p a t i c l e c t i n has
a-
.
192
Carbohydrate Chemistry
been d 0 ~ u m e n t e d . l ~The ~ B-Q-galactoside-specific
l e c t i n present i n
e m b r y o n i c - c h i c k s k e l e t a l m u s c l e does n o t appear t o be i n v o l v e d i n myotube f o r m a t i o n
2
from chick-embryo
k i d n e y b i n d s s t r o n g l y t o asialoglycoconjugates.196
~ i t r 0 . l ~ '
A
developmentally
I t s a f f i n i t y towards 2-amino-2-deoxy-~-glucosyl, and 2-amino-2-deoxy-Q-mannosyl
galactosyl,
regulated l e c t i n
2-amino-2-deoxy-g-
residues i s low.
The
i n h i b i t o r y e f f e c t o f t h e amino s u g a r s i n h a e m a g g l u t i n a t i o n assays i s n o t due t o s i m p l e e l e c t r o s t a t i c i n t e r a ~ t i 0 n . l ~A ~ n e u r a m i n i c a c i d b i n d i n g l e c t i n i s o l a t e d from
r otunda cauda has
t h e horseshoe crab Carcinoscorpius
been p u r i f i e d by
affinity
chromatography
on
i m m o b i l i z e d f e t ~ i n . ' ~The ~ l e c t i n , which i s a g l y c o p r o t e i n (mol.
wt.
lo5)
4.2 x
lo4),
and c o n t a i n s s u b u n i t s (mol.
wts.
2.7
i s antigenically unrelated t o the other
binding lectin, Limulin
limulin,
binds
x
lo4
a n d 2.8 x
neuraminic acid-
from t h e horseshoe c r a b L i m u l u s polyphemus.
specifically
to
glycolylneuraminic acid residue
gangliosides
. 199
bearing
1-
an
The b i n d i n g r e q u i r e s a f r e e
c a r b o x y l g r o u p and a f r e e h y d r o x y l g r o u p a t C-4 o f t h e n e u r a m i n i c acid but the side chain o f the N-glycolylneuraminic required.
Wheat-germ
gangliosides acetamido
agglutinin
binds
b e a r i n g an N - a c e t y l n e u r a m i n i c
group
but
not
the
acid i s not
preferentially acid
c a r b o x y l group
i s
to The
residue.
involved i n the
binding. L e c t i n s o f d i f f e r i n g s p e c i f i c i t i e s have been d e t e c t e d i n t h e crop, midgut, and haemolymph o f t h e i n s e c t Rhodinus p r o l i x u s . 2 0 0 These a r e s p e c i f i c f o r 2 - a c e t a m i d o - 2 - d e o x y - t J - m a n n o s e , 2-acetamido-2deoxy-Q-galactose,
a n d a-
Receptors for
and B-!-galactose.
the
l e c t i n s were d e t e c t e d i n e p i m a s t i g o t e f o r m s o f Trypanosoma c r u z i ,
a
p r o t o z o a n p a r a s i t e o f t h e i n s e c t a n d o f humans. A
possible
----Sarcophaga described.201 smaller
mechanism o f
peregrina
on
induction o f the
injury
of
the
body
insect wall
lectin of has
been
The l a r g e r s u b u n i t o f t h e l e c t i n i s c o n v e r t e d t o t h e
subunit
by
proteolysis,
i n a process essential
for
c o n s t r u c t i n g a c t i v e l e c t i n h a v i n g a f f i n i t y f o r !-galactose. The sponge H a l i c h o n d r i a p a n i c e a c o n t a i n s a l e c t i n t h a t h a s been i s o l a t e d and p u r i f i e d . 2 0 2
F r o m t h e same s p o n g e s p e c i e s , b a c t e r i a
have been i s o l a t e d and i d e n t i f i e d as Pseudomonas i n s o l i t a .
The
l e c t i n , d e t e c t e d on t h e s u r f a c e o f m u c o i d c e l l s f r o m H.panicea,
may
be i n v o l v e d i n a s y m b i o t i c r e l a t i o n s h i p b e t w e e n t h e sponge a n d t h e bacterium. A
mycobacterial haemagglutinin,
o f ~ y x o c o c c u sx a n t h u s ,
a development-specific
lectin
h a s b e e n p ~ r i f i e d . ~ ' ~W, h ~i l e~ ~s i m p l e
193
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
sugars f a i l t o i n h i b i t t h e h a e m a g g l u t i n a t i n g a c t i v i t y o f the l e c t i n , a g l y c o p e p t i d e c o n t a i n i n g t h e t r i s a c c h a r i d e ( 4 ) , common t o many glycoproteins, i s a potent i n h i b i t o r . Two l e c t i n s have
been p u r i f i e d f r o m
A g a r i c u s edulis.’05
Both
a r e c a p a b l e o f a g g l u t i n a t i n g e r y t h r o c y t e s a n d i m m u n o g l o b u l i n s A, and M ,
but
no
monosaccharide
was
capable o f
G,
inhibiting the
interaction. NeupSAc-( 2 + 3 ) - B - Q - G a l p ( 1+3I-!-GalNAc
A chitin-binding
h a e m a g g l u t i n i n h a s been i s o l a t e d f r o m c u l t u r e
C o n id io b o l u s
of
f i1t r a t e s
chromatography
Haemagglutination
i s s t r o n g l y i n h i b i t e d by c h i t o - o l i g o s a c c h a r i d e s . protease, lectin,
p r o d u c e d by
g.
lampranges,
n it y a f fi
f o 11ow in g
lamerrnags
on f o r m a l i n i z e d e r y t h r o c y tes.*06 acts,
A pronase-like
i n company
with the
on erythrocytes t o accelerate the a c t i v i t y of the l e ~ t i n . ~ ’ ’
The l e c t i n h a s c h i t i n - b i n d i n g
p r o p e r t i e s s i m i l a r t o t h o s e o f wheat
germ a g g l u t i n i n . Purpurin,
t h e l e c t i n from
o f seven t e t r a m e r i c forms, The i s o l e c t i n s ,
D i c t y o s t e l i u m purpureum,
i s made up
assembled from f o u r d i s t i n c t subunits.208
although related,
can be f u n c t i o n a l l y d i s c r i m i n a t e d
o n t h e b a s i s o f t h e i r r e l a t i v e a f f i n i t i e s f o r columns d e r i v a t i z e d w i t h complementary s a c c h a r i d e s . Oleic
acid
concentrations d ioleolyl
and
dioleolyl
agglutinate
phosphatidic
rabbit
phospha t i d y 1 c h o l i n e
and r a t i s not
acid
a t
erythrocytes,
low while
haem a g g l ~ t i n a t i n g . ~ ~ ’
E r y t h r o c y t e a g g l u t i n a t i o n by l i p i d s i s i n d i s t i n g u i s h a b l e i n some respects
from
that
haemagglutination
caused
reactions
by
lectins,
exhibit
cell
i n
particular
specificity
and
both are
i n h i b i t e d by c e r t a i n g l y c o p r o t e i n s .
4 A review
Fibronectin
d e a l i n g w i t h c u r r e n t c o n c e p t s c o n c e r n i n g t h e s t r u c t u r e and
f u n c t i o n o f f i b r o n e c t i n has been p u b l i s h e d . 2 1 0 R a t p l a s m a f i b r o n e c t i n h a s been i s o l a t e d and c h a r a c t e r i z e d and
m o n o s p e c i f i c a n t i b o d i e s p r e p a r e d t o it.211
The a n t i b o d i e s o n l y
w e a k l y c r o s s - r e a c t w i t h p l a s m a f i b r o n e c t i n s o f c h i c k e n , horse, and
Carbohydrate Chemistry
194 human.
A
wt.
second g l y c o p r o t e i n (mol.
collagen-binding
activity,
was
Immunologically pure f i b r o n e c t i n cannot p l a s m a by o n e - s t a g e
affinity
x lo4),
7.0
isolated
from
also having rat
plasma.
be p r e p a r e d f r o m
human
chromatography on i m m o b i l i z e d g e l a t i n
o r i m m o b i l i z e d f i b r o n e c t i n due t o n o n - s p e c i f i c a b s o r p t i o n o f o t h e r p r o t e i n s o n t h e support.212
P u r i f i e d f i b r o n e c t i n can be o b t a i n e d
u s i n g a s e c o n d s e p a r a t i o n by g e l p e r m e a t i o n c h r o m a t o g r a p h y . Although
human
amniotic
f l u i d
fibronectin
and
plasma
f i b r o n e c t i n a r e i m m u n o l o g i c a l l y i n d i s t i n g u i s h a b l e and a r e e q u a l l y a c t i v e promoters of c e l l attachment,
they d i f f e r i n carbohydrate
composi t i o n . 2 1 3 solid-phase
A
radioimmunoassay
has been d e v e l o p e d f o r t h e
d e t e r m i n a t i o n o f f i b r o n e c t i n l e v e l s i n plasma.214
An i m m u n o l o g i c a l
a s s a y has been u s e d t o m e a s u r e t h e u p t a k e o f endogenous a n d exogenous fibronectin
by c e l l s i n c u l t u r e . 2 1 5
f i b r o n e c t i n from
The
t h e medium i s i n f l u e n c e d by
cellular
uptake
of
c e l l shape a n d by t h e
presence o f collagen. C u l t u r e d human-mammary n o r m a l t i s s u e s and p r i m a r y fibronectin.*16
epithelial carcinomas
c e l l s derived from
produce
The p r e s e n c e o f c e l l - a s s o c i a t e d
both
large quantities
of
f i b r o n e c t i n as w e l l
a s t h e s y n t h e s i s a n d s e c r e t i o n o f f i b r o n e c t i n i n t o t h e medium by these
c e l l s a n d by
studied
fibroblasts
.
structural
A
comparison
derived from of
these t i s s u e s were
fibronectins
from
t r a n s f o r m e d h a m s t e r c e l l l i n e s has been r e p o r t e d . 2 1 7 major s i t e s of types o f cells,
normal
and
Although
the
p h o s p h o r y l a t i o n i n f i b r o n e c t i n a r e t h e same i n b o t h f i b r o n e c t i n f r o m t r a n s f o r m e d c e l l s a p p e a r s t o be
p h o s p h o r y l a t e d t o a much h i g h e r e x t e n t t h a n t h a t f r o m n o r m a l c e l l s . Human g e r m - c e l l t u m o u r s p r o d u c e f i b r o n e c t i n r e s e m b l i n g i t s a m n i o t i c fluid
variant,
but
differ
from
f ib r o ne c t i n s have been s ugge s t e d t o
plasma
fibronectin.218
Such
be p o t e n t ia 1 o n co de v e l o pm e n t a 1
ma r k e rs. The s p e c i f i c c a l c i u m - d e p e n d e n t serum a m y l o i d P component
for
b i n d i n g r e a c t i v i t y f o r human
f i b r o n e c t i n and a C4- binding
protein
i s reported.219 Human p e r i p h e r a l b l o o d m o n o c y t e s p o s s e s s a t r y p s i n - s e n s i t i v e p l a s m a membrane r e c e p t o r f o r s u r f a c e - b o u n d
fibronectin.220
Binding
o f monocytes t o f i b r o n e c t i n l e a d s t o enhanced f u n c t i o n a l e x p r e s s i o n of
their
plasma-membrane
receptors
for
the
Fc
portion
i m m u n o g l o b u l i n G a n d f o r t h e t h i r d component o f c o m p l e m e n t .
of
It i s
proposed t h a t plasma f i b r o n e c t i n a c t s as a c i r c u l a t i n g probe f o r
195
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
f i b r i n o r f o r e x p o s e d o r a l t e r e d c o l l a g e n a t s i t e s o f t i s s u e damage. Treatment o f f i b r o n e c t i n w i t h e i t h e r m e t h y l m e r c u r i c bromide o r iodoacetamide
as
sulphydryl
blocking
agents
protects
the
g l y c o p r o t e i n a g a i n s t p r e c i p i t a t i o n a l l o s s e s d u r i n g p r e p a r a t i o n . 221 A
combination
o f
electron
microscopy,
c.d.
i.r.
and
spectroscopy, a n d s c a n n i n g m i c r o c a l o r i m e t r y has been u s e d i n a s t u d y o f t h e s t r u c t u r a l o r g a n i z a t i o n o f human p l a s m a f i b r o n e c t i n . 2 2 2 Four
functionally
fibronectin
have
distinct
domains
i n
hamster
plasma
been i s o l a t e d and c h a r a c t e r i z e d f o l l o w i n g
p r o t e o l y t i c d i g e s t i o n o f t h e g l y ~ o p r o t e i n . ~I n~t a ~c t fibronectin i s considered t o contain four carbohydrate chains per subunit, b e i n g a t t a c h e d t o a d o m a i n o f m o l . w t . 4.0 o f mol.
wt.
1.4
lo4
x
three
and one t o a domain
lo5.
x
H e a t d e n a t u r a t i o n o f human p l a s m a f i b r o n e c t i n p r o c e e d s t h r o u g h a t l e a s t three stages, o b s e r v e d a t 6 8 , 82,and
w i t h endothermal denaturing t r a n s i t i o n s 119°C.224
A three-domain s t r u c t u r e f o r t h e
g l y c o p r o t e i n i s p r o p o s e d i n w h i c h t h e domain w h i c h u n f o l d s a t 68' associated
gelatin
with 82'
unfolding at immunological
b i n d i n g and
appears t o
activity.
The
cell
binding,
while
be a s s o c i a t e d w i t h m u c h o f 119'
domain
has h e p a r i n
a c t i v i t y a s w e l l a s some i m m u n o l o g i c a l a c t i v i t y . b i n d i n g domain o f f i b r o n e c t i n i s immunogenic,
i s
that the
binding
The g e l a t i n -
and a n t i s e r a a g a i n s t
t h i s domain r e c o g n i z e c e l l u l a r f i b r o n e c t i n g e l a t i n - b i n d i n g
sites.225
I n h i b i t i o n o f g e l a t i n b i n d i n g b u t n o t c e l l s p r e a d i n g by a n t i - g e l a t i n binding
d o m a i n Fab'
fragments
confirms
the hypothesis
that
f i b r o n e c t i n has separate s i t e s m e d i a t i n g these a c t i v i t i e s . on d i g e s t i o n w i t h c a t h e p s i n D, r e l e a s e s t w o
Plasma f i b r i n o g e n ,
p o o l s o f l o n g - c h a i n p o l y p e p t i d e s o f d i f f e r e n t m o l e c u l a r w e i g h t s and w i t h graded a f f i n i t y
having
weaker
fibronectin;
f o r i m m o b i l i z e d heparin.226
affinity
contains
the
The f r a c t i o n
N-terminal
region
t h a t having the stronger a f f i n i t y consists o f
d i s u l p h i d e - l i n k e d peptide chains o f related structure.
different
o f two
l e n g t h and s h a r i n g a
The h i g h e r - m o l e c u l a r - w e i g h t p o l y p e p t i d e c o n t a i n s
an a d d i t i o n a l t r a n s a m i d a s e - s e n s i t i v e s i t e .
Human p l a s m a f i b r o n e c t i n
h a s b e e n t r e a t e d w i t h ~ h y m o t r y p s i n . ~C~h a ~racterization o f the i s o l a t e d f r a g m e n t s h a s shown t h a t t h e o r d e r o f t h e f u n c t i o n a l domains from t h e N-terminus i s s t a p h y l o c o c c a l binding, l i n k i n g and b i n d i n g s i t e s , area, and h e p a r i n - b i n d i n g Horse serum
gelatin-binding site,
f i b r i n cross-
c e l l attachment
site.
fibronectin
h a s been r e d u c e d a n d a l k y l a t e d t o
p r o d u c e a m o d i f i e d g l y c o p r o t e i n w h i c h no l o n g e r b i n d s t o g e l a t i n ,
196
Carbohydrate Chemistry
b u t s t i l l p r o v i d e s a s u b s t r a t e f o r myoblast attachment.228 was
to
obtained
fibronectin
demonstrate
wt.
(mol.
x
6 .0
chymotryptic
a
fragment
lo4)
was u n a b l e t o p r o m o t e c e l l
a t t a c h m e n t u n l e s s f i b r o n e c t i n was p r e s e n t i n t h e medium, that
the gelatin-binding
fragment
f i b r o ne c t i n- f i b r o ne c t i n b i n d i n g. f i b r o n e c t i n from affinity
also
chromatography
The a c t i n - b i n d i n g
of
suggesting
spe c i f i c a c t i n - b i n d i n g s i t e i n
A
fragments
r e l e a s e d from
wt.
a fragment (mol.
s i t e i s close to,
collagen-binding
site.
2.7
but not i d e n t i c a l with, The
binding o f
human
the
plasma
microtitre
The b i n d i n g i s
b u t n o t by h e p a r i n o r b o v i n e
i n h i b i t e d by b o t h a c t i n a n d g e l a t i n , n o t take
by
x l o 4 ) was i s o l a t e d .
f i b r o n e c t i n t o a c t i n has been s t u d i e d u s i n g a c t i n - c o a t e d
not
After
fibronectin
w e l l s i n e n z y m e - l i n k e d immunosorbent assays.230 serum a l b u m i n .
of
contains a s i t e involved i n
c h i c k e n f i b r o b l a s t s h a s been i d e n t i f i e d . 2 2 9
l i m i t e d proteolysis, reported
Evidence
that
The b i n d i n g o f c o l l a g e n a n d a c t i n t o f i b r o n e c t i n may
place simultaneously.
interfere
with
cell
Since the
attachment,
binding of
the
cell
collagen
does
binding s i t e of
f i b r o n e c t i n i s probably d i f f e r e n t from t h e a c t i n - b i n d i n g
site.
S t r u c t u r a l d i f f e r e n c e s be t w een p l asm a s a n d f i b r o b l a s t c e l l u l a r fibronectins
have
regions.231
One s t r u c t u r a l d i f f e r e n c e o c c u r s n e a r t h e g e l a t i n -
been i d e n t i f i e d i n a t
least
three
different
b i n d i n g s i t e and t h e o t h e r s a r e n e a r h e p a r i n - b i n d i n g s i t e s . A
number
of
cell
types
(HeLa,
Ehrlich
ascites,
and
t r a n s f o r m e d r a t k i d n e y ) show a l o w r a t e o f C a 2 + u p t a k e ,
viral
which i s
stimulated a f t e r incubation with f i b r ~ n e c t i n . ~ ~ ~ The i n t e r a c t i o n o f i s o l a t e d p l a s m a f i b r o n e c t i n w i t h s t i m u l a t e d a n d n o n s t i m u l a t e d human p l a t e l e t s h a s
been ~ h a r a c t e r i z e d . ~ The ~~
s p e c i f i c b i n d i n g o f a l a r g e number o f
fibronectin molecules to
throm b i n - s t i m u l a t e d p l a t e l e t s suggests t h a t
f i b r o n e c t i n may p l a y a
r o l e i n modulating p l a t e l e t
a g g r e g a t i o n i n d u c e d by
adhesion and/or
t h i s stimulus. The i n c o r p o r a t i o n o f C - p r o l i n e and g l y c i n e i n t o f i b r o n e c t i n and c o l l a g e n by c u l t u r e s o f s k i n f i b r o b l a s t s o b t a i n e d f r o m p a t i e n t s w i t h insulin-dependent
diabetes
mellitus
has
been r e p o r t e d . 2 3 4
Changes
i n t h e r a t i o o f f i b r o n e c t i n t o c o l l a g e n between s k i n f i b r o b l a s t s o f n o r m a l and d i a b e t i c s u b j e c t s d u r i n g The XIII,
covalent binding of
h a s been e x a m i n e d i n a s y s t e m
o n macroporous agarose, presence o f beads i s
in
v i t r o a g i n g were not ed.
various s t r u c t u r a l
p r o t e i n s by
i n which p r o t e i n s ,
factor
immobilized
a r e i n c u b a t e d w i t h l a b e l l e d p r o t e i n s i n the
factor X I I I a ,
determined.235
and t h e r a d i o a c t i v i t y Coupling occurred
of
t h e urea-washed
between
fibrin
and
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
197
f i b r o n e c t i n and b e t w e e n m y o s i n and f i b r o n e c t i n . The a m i n o a c i d sequence i n b o v i n e f i b r o n e c t i n w h i c h c o n t a i n s L - g l u t a m i n e r e s i d u e s labelled with
1, 4 - { 1 4 C ) - p u t r e s c i n e
determined.236
--glutamine This L
by
factor
X I 1 1
has
been
residue i s located a t position 3
from t h e N-terminus o f f i b r o n e c t i n . F i b r o n e c t i n and p r o c o l l a g e n a r e r e l e a s e d f r o m t h e m a t r i x o f h u m a n f i b r o b l a s t s by t h r o m b i n . 2 3 7
Collagenase alone i s unable t o
b r i n g about t h e release o f f i b r o n e c t i n .
The r e l e a s e o f t h e s e t w o
g l y c o p r o t e i n s by t h r o m b i n may be i n v o l v e d i n wound h e a l i n g i n v i v o . Hum a n
f i b r one c t in
p l asm a
at
physio logic a l
concentrations
s t i m u l a t e s t h e l e v e l o f t o t a l p l a t e l e t a d h e s ion and t h e i r on s u r f a c e s c o a t e d w i t h f i b r i l l a r collagen.238
spreading
The s t i m u l a t i n g
e f f e c t o f f i b r o n e c t i n i s due t o i t s i n t e r a c t i o n w i t h c o l l a g e n s i n c e f r e e f i b r o n e c t i n does n o t i n f l u e n c e t h e a d h e s i o n
o r spreading o f
platelets. Plasma f i b r o n e c t i n mediates c o l l a g e n t y p e I11
the
b i n d i n g o.f s o l u b l e n a t i v e
macro phage^.^^^
to trypsinized
The a s s o c i a t i o n
i s a u g m e n t e d by h e p a r i n o r h e p a r a n s u l p h a t e , b u t h y a l u r o n i c a c i d reverses the effect. F i b r o n e c t i n h a s been shown t o b i n d t o a g g r e g a t i n g c o l l a g e n fibres.240
Since fibronectin i n h i b i t s the
i t
fibrillogenesis
may
regulate
the
size
rate of
of
collagen
collagen
fibres.
C a r t i l a g e p r o t eo g l y c a n s i n h i b i t fi bro nectin-me d i a t e d adhesion t o collagen.241
The p r e s e n c e o f f i b r o n e c t i n i n c o l l a g e n f i b r i l s o f
human f i b r o b l a s t s has been shown, u s i n g i m m u n o l o g i c a l t e c h n i q u e s . 2 4 2
A g e l a t i n - b i n d i n g p r o t e i n (mol.
wt.
7.0
x
lo4),
which i s q u i t e
d i s t i n c t f r o m f i b r o n e c t i n , i s s y n t h e s i z e d by n o r m a l a n d m a l i g n a n t adherent
cells.243
The g l y c o p r o t e i n seems n o t t o
be a f r a g m e n t
of
f i b r o n e c t i n a n d shows no i m m u n o l o g i c a l c r o s s r e a c t i v i t y w i t h i t .
5
The
Collagen
biochemical
properties
i n c l u d i n g collagen, connective-tissue-
of
basement
have been reviewed.244
r e s e a r c h symposium
membrane
components,
The p r o c e e d i n g s o f a
include chapters dealing w i t h
r e c e n t advances i n methods f o r i n v e s t i g a t i n g t h e m o l e c u l a r s t r u c t u r e of
collagens,
purification collagen.245 c o l l a g e n .246
c h r o m a t o g r a p h i c and e l e c t r o p h o r e t i c of
collagen, A symposium
and
tissue-specific
has been d e v o t e d t o
methods
for
the
differences
i n
the biology
of
Carbohydrate Chemistry I n a novel s t r u c t u r a l model f o r c o l l a g e n , i t i s proposed t h a t t h e r e a r e no i n t e r - c h a i n h y d r o g e n b o n d s a n d v a n d e r Waals' contacts.247 I n s t e a d , a t r i p l e - h e l i c a l s t r u c t u r e i s s t a b i l i z e d by water m o l e c u l e s , w h i c h f o r m i n t e r c h a i n b r i d g e s t h r o u g h h y d r o g e n b o n d s w i t h carbonyl groups. Collagen has been s e l e c t i v e l y s o l u b i l i z e d from m i x t u r e s o f c o l l a g e n and e l a s t i n u s i n g phenol, acetic acid, and water mixtures,248 and a l s o u s i n g c y c l e s i n v o l v i n g pepsin, p a n c r e a t i c e l a s t a s e , and d i t h i ot h r e i P r e p a r a t i o n s f r o m norm a 1 h a m s t e r a n d b a b o o n l u n g s c o n t a i n p r i n c i p a l l y t y p e s I a n d I11 c o l l a g e n . A c h r o m a t o g r a p h i c p r o c e d u r e c o n s i s t i n g of g e l p e r m e a t i o n a n d a n i o n - e x c h a n g e c h r o m a t o g r a p h y f o r p u r i f y i n g n a t i v e t y p e s I , 11, a n d I11 c o l l a g e n s f r o m a v a r i e t y o f s o u r c e s h a s b e e n r e p o r t e d . 2 5 0 The n a t i v e c o l l a g e n p r e p a r e d by t h i s m e t h o d i s m o r e s t a b l e t h a n t h a t p r e p a r e d by s a l t f r a c t i o n a t i o n , a s r e f l e c t e d by t h e h i g h e r a n d s h a r p e r thermal t r a n s i t i o n t e m p e r a t u r e . Radioimmunoassay p r o c e d u r e s f o r 7s c o l l a g e n a n d l a m i n i n h a v e been u s e d t o a n a l y s e t h e s e p r o t e i n s i n s e r u m , c e l l c u l t u r e s , a n d t i s s u e s .251 T h e r e l a t i v e a m o u n t s o f t y p e s I a n d I11 c o l l a g e n s h a v e b e e n e s t i m a t e d a f t e r e x t r a c t i o n of non-collagenous p r o t e i n from r a b b i t The m e t h o d h a s b e e n lung t i s s u e i n sodium dodecyl sulphate.252 a p p l i e d t o s m a l l t i s s u e s a m p l e s s u c h a s w o u l d b e o b t a i n e d by l u n g biopsy. The s k i n c o l l a g e n s f r o m t h e l a m p r e y , E n t o s p h e n u s j a p o n i c u s , a n d t h e g r e a t b l u e s h a r k , P r i o n a c e g l a u c a , c o n t a i n t w o d i s t i n c t acomponents and are classified a s t y p e I-like collagen.253 The h e t e r o g e n e i t y of cyanogen bromide-cleavage p e p t i d e s o f h u m a n c o l l a g e n s , t y p e s I , 11, I I 1 , a n d V , h a v e b e e n d e m o n s t r a t e d by t w o-dim e n s i o n a l p o l y a c r y lam i d e ge 1 e l e c t r o p h o r e s i s. 25 The amino a c i d s e q u e n c e o f t h e o l i g o s a c c h a r i d e a t t a c h m e n t s i t e w i t h i n t h e proa-2 chain of chick type I collagen has been I n a d d i t i o n 0 - m a n n o s e- r i c h g l y c o p e p t i d e s o b t a i n e d i d e n t i f i e d.255 from b o t h p r o a - 1 ( I ) a n d proa-2 c a r b o x y l p r o p e p t i d e s have been i s o l a t e d and characterized. Evidence f o r a s t r u c t u r a l mutation o f procollagen type I i n a p a t i e n t w i t h E h l e r s - D a n l o s s y n d r o m e , t y p e VII, has b e e n The f i b r o b l a s t s o f t h e p a t i e n t s y n t h e s i z e b o t h a n reported.256 abnormal proa-2 chain and a normal proa chain. Human t y p e I1 c o l l a g e n d i f f e r s f r o m t y p e I c o l l a g e n i n m o l e c u l a r p a c k i n g , a s d e m o n s t r a t e d by X - r a y d i f f r a c t i o n s t u d i e s . 2 5 7
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
199
U n d e r p h y s i o l o g i c a l c o n d i t i o n s t h e t y p e I1 c o l l a g e n i s s h o w n t o c o n t a i n 5 0 - 1 0 0 % m o r e water t h a n t h e t y p e I c o l l a g e n . The c o n t e n t o f g l y c o s y l a t e d h y d r o x y - L - l y s i n e r e s i d u e s i s t h o u g h t t o be t h e v a r i a b l e m o s t l i k e l y t o be m o d u l a t i n g f i b r i l l a r h y d r a t i o n . C o l l a g e n s s y n t h e s i z e d by c u l t u r e d b o v i n e c o r n e a l e n d o t h e l i a l cells have been characterized.258 T y p e I11 c o l l a g e n i s t h e m a j o r component both d e p o s i t e d i n t h e e x t r a c e l l u l a r m a t r i x and secreted i n t o t h e media. B a s e m e n t m e m b r a n e c o l l a g e n s , t y p e I V a n d V, a r e a l s o found i n each compartment. The a 2 ( v ) c h a i n o f human c o l l a g e n when c l e a v e d w i t h c y a n o g e n b r o m i d e c o n t a i n s t e n p e p t i d e s , a c c o u n t i n g f o r 956 a m i n o a c i d residues.259 Possible homologies between t h e a2(v) peptides and p e p t i d e s d e r i v e d f r o m o t h e r c o l l a g e n c h a i n s were n o t e d . A n t i b o d i e s have been p r e p a r e d t o c o l l a g e n a s e - r e s i s t a n t t e r m i n a l Competition radioimmunoassay r e g i o n s of pro-type I V collagen.260 e x p e r i m e n t s show r e c o g n i t i o n o f t h e a n t i b o d i e s f o r r e d u c e d a n d a l k y l a t e d 7s c o l l a g e n , s u p p o r t i n g t h e p o s s i b i l i t y t h a t t h i s c o l l a g e n i s a c r o s s l i n k e d domain o f t y p e I V c o l l a g e n . A high-molecular-weight c o l l a g e n o u s p r o t e i n has been i s o l a t e d , a f t e r l i m i t e d p e p s i n d i g e s t i o n , from t h e e n d o m e t r i a l v i l l i o f b o v i n e placenta.261 I t r e s e m b l e s t h e p r o t e i n i s o l a t e d from human a o r t a s i n c o n t a i n i n g t h r e e polypeptide c h a i n s w i t h similar amino acid com po s i t io n 262 The p o l y p e p t i de cha i n s s h ow c h a ra c t e r i s t i cs t y p i c a l o f basement membrane c o l l a g e n s , h a v i n g a high c o n t e n t o f hydroxy-kl y s i n e , w h i c h i s a l m o s t a l l g l y c o s y l a t e d . The o c c u r r e n c e of s i m i l a r a m o u n t s o f L - c y s t e i n e , L-ty r o s i n e , a n d 2 - a m i n o - 2 - d e o x y - Q - g l u c o s e i s not observed i n o t h e r collagen types. An u n u s u a l c o l l a g e n f r a c t i o n h a s b e e n i s o l a t e d a n d c h a r a c t e r i z e d from bovine h y a l i n e c a r t i l a g e and i n t e r v e r t e b r a l disc.263 Evidence s u p p o r t s a model c o n t a i n i n g t h r e e i d e n t i c a l c h a i n s ( m o l . w t . 3 . 3 x l o 4 > l i n k e d by i n t e r c h a i n d i s u l p h i d e b o n d s t o f o r m a s h o r t t r i p l e-he1 i ca 1 p s e u d o a-si z e d com po n e n t The c o l l a g e n o u s d o m a i n o f b o v i n e g l o m e r u l a r b a s e m e n t m e m b r a n e h a s been i s o l a t e d i n s o l u b l e form and shown t o c o n t a i n t h e previously designated polypeptide XIV.264 The amino a c i d a n d carbohydrate compositions and cyanogen bromide p a t t e r n s i n d i c a t e t h a t polypeptide X I V has a s t r u c t u r e similar to t h a t of C chain i s o l a t e d f r o m o t h e r v a s c u l a r t i s s u e s a n d t h a t i t may r e p r e s e n t a m a j o r s t r u c t u r a l segment o f t h e e n t i r e c o l l a g e n o u s domain. The c o l l a g e n o u s domain o f r a b b i t r e n a l t u b u l a r basement membrane i s c o m p o s e d o f a m u l t i p l i c i t y o f c o m p o n e n t s r a n g i n g f r o m m o l . wts. 3 . 4
.
.
200
Carbohydrate Chemistry
lo4
t o 1 0 6 . 265 As a s e l f - a s s e m b l y
system,
renature as t r i p l e - s t r a n d e d
guinea-pig
d e t e r m i n e d by t h e r a t i o s o f t h e a - c h a i n s The c o l l a g e n s f r o m
-e l e g a n s
collagen chains tend t o
m o l e c u l e s whose
chain compositions are
i n the mixture.266
the c u t i c l e o f
t h e nematode C a e n o r h a b d i t i s
h a v e b e e n s e p a r a t e d by m o l e c u l a r - s i e v e c h r o m a t o g r a p h y a n d
shown t o c o n t a i n no h y d r o x y - l - l y s i n e The
collagen
-c--e l l u l o s a e
isolated
from
residues.267 the
platyhelminth
I,-pr o 1i n e .26
ve r t e b r a t e co 11age n ex ce p t t ha t it co n t a i n s no h y d r ox y An e x t r a - e m b r o n i c and
rat
Cysticerus
c o n t a i n s an amino a c i d c o m p o s i t i o n c h a r a c t e r i s t i c o f
contains
base membrane,
collagen
together
R e i c h e r t ' s membrane, with
four
wt.
g l y c o p r ~ t e i n s . ~One ~ ~ o f t h e g l y c o p r o t e i n s (mol. unrelated t o laminin,
o f mouse
non-collagenous
w h i l s t t h e o t h e r t h r e e (mol.
5.0
wts.
x lo4) i s 4.15
x
lo5,
2.45 x lo5, a n d 1.70 x lo5, r e s p e c t i v e l y ) s h a r e some i m m u n o c h e m i c a l ch a r a c t e r i s t ics o f 1am i n i n. A 7 s c o l l a g e n h a s been i d e n t i f i e d a s a c r o s s l i n k i n g domain o f
mouse t u m o u r m a t r i x t y p e I V c o l l a g e n . 2 7 0 Transformed e p i t h e l i a l b o v i n e l e n s c e l l s s y n t h e s i z e and s e c r e t e i n t h e c u l t u r e medium o n l y type
I V
collagen.271
Like
the
interstitial
molecule i s a disulphide-bonded collagenous and a non-collagenous
procollagen,
the
glycoprotein containing both a domain.
The absence o f m a t u r a t i o n o f c o l l a g e n c r o s s - l i n k s
i n fish skin
has been r e p o r t e d . 2 7 2 A c l o s e s i m i l a r i t y e x i s t s between o c t o p u s s k i n c o l l a g e n and
c a l f s k i n t y p e I collagen.273
B o t h c o l l a g e n s r e s e m b l e each o t h e r ,
n o t o n l y i n c h a i n c o m p o s i t i o n b u t a l s o i n chemical composition. The m o l e c u l a r o r g a n i z a t i o n of t h e c o l l a g e n o u s components o f t h e i n t e s t i n a l basement membrane o f A s c a r i s suum has been i n v e s t i g a t e d by c h a r a c t e r i z a t i o n o f t h e i r p h y s i c a l p r o p e r t i e s i n b o t h t h e n a t i v e and denatured states.274
The c o l l a g e n o u s domain c o n s i s t s m a i n l y o f
a component composed o f t w o e s s e n t i a l l y i d e n t i c a l t r i p l e - h e l i c a l s u b u n i t s j o i n e d e n d t o end by d i s u l p h i d e b o n d s w i t h e a c h s u b u n i t being cross-linked
by
disulphide
bonds b e t w e e n a l l
constituent
chains. A
study
of
collagen-sulphated
glycosaminoglycuronan
i n t e r a c t i o n s i n d e v e l o p i n g r a t t a i l t e n d o n has been r e p o r t e d i n 75
t e r m s o f f ib r i11oge ne s i s and f ib r e mat u r a t i on.
Bovine corneal e n d o t h e l i a l c e l l s synthesize predominantly type I11 collagen, V
i n culture
w i t h l e s s e r amounts o f t y p e s I a n d
and a p p a r e n t l y l i t t l e i f any o f t y p e I
V
.
~
~
~
,
~
~
~
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
201
M3A, a d e r i v a t i v e o f the c l o n a l s k e l e t a l m u s c l e myoblast l i n e secretes i n t o t h e c u l t u r e medium a n a b n o r m a l f o r m of c o l l a g e n ( m o l . w t . 5.6 x l o 4 ) a n d a g r e a t l y r e d u c e d a m o u n t o f h i g h - m o l e c u l a r w e i g h t c o l l a g e n a-chains.278 I n a d d i t i o n M 3 A does n o t secrete u n s u l p h a t e d c h o n d r o i t i n w h i l e L6 d o e s . Comparison of the behaviour o f 1e u c i n e am i n o p e p t i d a s e a n d ca r bo x y p e p t i d a s e - m o d i f i e d c o 11 a g e n s suggests that the carboxyl telopeptide has a major r o l e i n the grow t h s t a g e s o f s e l f -assem b l y 2 7 9 M a t r i x - f r e e cells f r o m c h i c k embryo t e n d o n s s y n t h e s i z e pro-y c o l l a g e n c h a i n s which are either not triple-helical o r are i n an u n s t a b l e t r i p l e-he1 i c a l c o n f o r m a t i on.28o These c h a i n s c o n t a i n l e s s h y d r o x y - & - p r o l i n e t h a n t h e t r i p l e - h e l i c a l p r o c o l l a g e n s e c r e t e d by t y e cells. Their content o f glycosylated hydroxy-L-lysine is, h o w e v e r , g r e a t e r t h a n t h a t o f t h e p r o c o l l a g e n s e c r e t e d by t h e c e l l s . The b i o s y n t h e s i s a n d p r o c e s s i n g o f t y p e V p r o c o l l a g e n s i n s e v e r a l c h i c k t i s s u e s p r o d u c e p r o c o l l a g e n s ( p r ~ a l V ) (~p r, o a 2 V I 2 , a n d (proalV)3.281 A comparative b i o s y n t h e t i c s t u d y o f these g l y c o p r o t e i n s by c h i c k t e n d o n f i b r o b l a s t s a n d by h a m s t e r l u n g c e l l c u l t u r e s h a s b e e n r e po r t e d . 82 The N - g l y c o s y l a t i o n o f I - l y s i n e a n d h y d r o x y - l - l y s i n e r e s i d u e s i n t y p e I c o l l a g e n from s t r e p t o z o t o c i n - d i a b e t i c r a t s has been c o n f i r m e d , a n d t h e s t a b i l i t y o f t h e c o m p l e x h a s b e e n s h o w n t o be d u e t o a n Amadori rearrangement.283 This type of glycosylation does not represent an acceleration of the normal maturation process i n collagen involving the reducible cross-links. Post-translational modifications i n the biosynthesis of type I V c o l l a g e n by a h u m a n t u m o u r c e l l l i n e h a v e b e e n s t u d i e d . 2 8 4 The d i f f e r i n g e x t e n t s o f m o d i f i c a t i o n s s h o w n by t h i s c e l l l i n e a n d n o r m a l h u m a n s k i n f i b r o b l a s t s a r e e x p l a i n e d by d i f f e r e n c e s i n t h e a c t i v i t i e s o f L-lysy l h y d r o x y l a s e and hydroxy-k-lysyl-g1 ucosy 1 t r a n s f e r a s e s between t h e two types. Collagen hydroxylases and glycosyltransferases o f cultured human-foetal l u n g f i b r o b l a s t s m i g h t n o t be c o - o r d i n a t e l y regulated.285 Regardless of hydroxylation events, glycosylation of t h e p e p t i d e might be l i m i t e d t o a s p e c i f i c f r a c t i o n of t h e hydroxyI-lysine residues during the post-translational modification of collagen. L6,
.
Carbohydrate Chemistry
202 Glycogen
6
A review d e a l i n g w i t h t h e comparative biochemistry o f s t a r c h and g l y c o g e n has been p u b l i s h e d . 2 8 6
A h i g h l y b r a n c h e d , p o l y d i s p e r s e , h i g h - m o l e c u l a r - w e i gh t g l yco gen from
rat
liver
has
been i s o l a t e d
using centrifugation,
gentle
h e a t i n g , a n d g e l c h r ~ m a t o g r a p h y . ~U~s ~ ing a d u l t fasted rats, t h i s g l y c o g e n was shown t o water-ethanol
be b e t t e r t h a n h i g h - m o l e c u l a r - w e i g h t
extracted
glycogen
for
the
binding of
cold
glycogen
m e t a b o l i z i n g e nz ymes. The
simultaneous
demonstration o f
glycogen and p r o t e i n i n
c a r d i a c t i s s ue g l y co some s has been a c h i e ve d h i s t o chem i c a l l y . 2 8 8 An a b n o r m a l u r i n a r y o l i g o s a c c h a r i d e i n patients with
glycogen storage
p a t t e r n h a s been o b s e r v e d
disease type
III.289 No
ch a r a c t e r i z a t i o n o f t h e o l i g o sa c c h a r i de s was a t t e m p t e d . S t r u c t u r a l aspects o f the c a t a l y t i c and r e g u l a t o r y f u n c t i o n o f g l y c o g e n p h o s p h o r y l a s e have been reviewed.290
The r e g u l a t i o n o f
g l y c o g e n p h o s p h o r y l a s e a n d g l y c o g e n s y n t h a s e by a d r e n a l i n i n S o l e u s
m us c l e in ph o sp h o r y 1as e k i na se - de f ic i ent m ice h a s be e n r e pa r t e d
.
D a t a have been p r o d u c e d w h i c h a r e n o t c o n s i s t e n t w i t h t h e t h a t i n s u l i n a c t i v a t e s g l y c o g e n s y n t h a s e by
91
view
producing an i n h i b i t o r
o f 3’5’ c y c l i c a d e n o s i n e m o n o p h o s p h a t e - d e p e n d e n t
protein
kinase i n
Nor do t h e y s u p p o r t t h e h y p o t h e s i s t h a t
r a t s k e l e t a l muscle.292
i n s u l i n a c t s by d e c r e a s i n g t h e a c t i v i t y o f a n i n h i b i t o r o f a m u l t i s u b s t r a t e p h o s p h o p r o t e i n k i nase. Bovine
heart
glycogen
with
heart.293
combination o f
The
a
synthase
phosphorylated
glycogen
has
synthase
the
been
kinase
specifically isolated
e n z y m e a n d 3’5’
from
c y c l i c AMP-
dependent p r o t e i n k i n a s e l e a d s t o s p e c i f i c p h o s p h o r y l a t i o n i n t h r e e enzyme s i t e s .
Each f o r m o f t h e g l y c o g e n s y n t h a s e , p h o s p h o r y l a t e d i n
s p e c i f i c enzyme s i t e s , A
protein
has b e e n i s o l a t e d a n d s t u d i e d k i n e t i c a l l y .
kinase
from
phosphorylate t h e 8-subunit
rabbit
reticulocytes,
able
i f not
and glycogen synthase k i n a s e from r a b b i t s k e l e t a l muscle are,
.
i d e n t i c a l , c l o s e l y r e 1a t e d p r o t e i n s 2 9 4 Yea s t 1,4 -a -Q - g l u c o s i da s e
, hom o l o go u s
against isolated r a t hepatocytes
have
a 1bum i n , a n d
I gG r a is e d
been c r o s s - l i n k e d
glutaraldehyde t o produce a stable, a c t i v e enryme-polymer (mol.
wt.
1.0
x 106).295
t o
o f e u k a r y o t i c i n i t i a t i o n f a c t o r 2 (IF2),
using
complex
After intravenous i n j e c t i o n i n t o rats,
the
complex
i s p r e f e r e n t i a l l y associated w i t h the Kupffer c e l l s o f
liver.
The p r o c e d u r e h a s b e e n u s e d a s a m o d e l f o r e n z y m e t h e r a p y
the
203
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
f o r t h e p o s s i b l e l o w e r i n g o f h e p a t o c y t e g l y c o g e n c o n t e n t i n t y p e I1 glycogenesis
(Pompe’s
Glycosaminoglycans and P r o t e o g l y c a n s
7
-
Analysis. of
free
disease).
A semi-quantitative micromethod f o r the determination
glycosaminoglycans
i n
serum
has
been
developed as
a
s c r e e n i n g m e t h o d f o r m u c o p o l y s a c c h a r i do s i s . ~ ’ ~ G l y c o s a m i n o g l y c a n s a r e adsorbed i n DEAE-cellusose paper b e f o r e s t a i n i n g w i t h A l c i a n B l u e a n d m e a s u r e m e n t o f t h e o p t i c a l d e n s i t y o f t h e r e s u l t i n g dye complex. G l y c o s a m i n o g l y c a n s have been i d e n t i f i e d and measured a f t e r a s e l e c t i v e and stepwise
sequence
of
treatments w i t h hyaluronidase,
c h o n d r o i t i n s u l p h a t e l y a s e A C a n d ABC, nitrous procedure
To the
determine
remaining
the
@-B-&-galactanase,
result of
each
and
degradative
glycosaminoglycans are subjected t o
cellulose acetate electrophoresis. H y a l u r o n i c a c i d and t h e i s o m e r i c c h o n d r o i t i n s u l p h a t e s have been s i m u l t a n e o u s l y d e t e r m i n e d a f t e r h y d r o l y s i s w i t h c h o n d r o i t i n a s e ABC
( E C 4.2.2.4.).298
( d e t e c t i o n l i m i t 0.02
The m e t h o d i s r e p r o d u c i b l e a n d s e n s i t i v e p m o l d i s a c c h a r i d e 1.
The s p e c i f i c i t y o f a r a d i o i m m u n o a s s a y m e t h o d f o r m e a s u r i n g h u m a n a r t i c u l a r c a r t i l a g i n o u s p r o t e o g l y c a n s h a s been reported.”’ T h e a n t i b o d i e s u s e d show a s p e c i e s s p e c i f i c i t y s i n c e t h e r e i s n o c r o s s - r e a c t i o n w i t h t h e c o r r e s p o n d i n g r a t o r dog p r o t e o g l y c a n s . A n t i g e n i c s i t e s a p p e a r t o be l o c a t e d i n t h e p r o t e i n r e g i o n a n d n o t i n t h e glycosaminoglycan r e g i o n o f t h e molecule. C o m p l e x e s f o r m e d b e t w e e n serum p o l y - a n i o n s a n d c e t y l p y r i d i n i u m c h l o r i d e h a v e been q u a n t i f i e d by l a s e r n e p h e l ~ r n e t r y . ~ ~ ’Measurement o f t h e c o m p l e x e s i n serum b e f o r e a n d a f t e r d i g e s t i o n w i t h s p e c i f i c enzymes e n a b l e s t h e m e a s u r e m e n t o f h y a l u r o n i c a c i d a n d c h o n d r c d t i n s u l p h a t e i n s m a l l v o l u m e s o f serum. c h o n d r o i t i n 4-
and 6 - s u l p h a t e s
The
uronic acid levels i n
and d e r m a t a n s u l p h a t e have been
determined a f t e r electrophoresis o f the l i b e r a t e d uronic acids on T i t a n 111 c e l l u l o s e a c e t a t e An h.p.1.c.
plate^.^"
m e t h o d has
been d e s c r i b e d f o r
the analysis o f
r e d u c e d u n s a t u r a t e d d i s a c c h a r i d e s d e r i v e d f r o m e n z y m i c d i g e s t i o n and sodium borohydride r e d u c t i o n o f c h o n d r o i t i n sulphates, sulphate,
heparan sulphate,and heparin.302
possibility
of
obtaining
anomeric
dermatan
The m e t h o d a v o i d s t h e
forms
of
unsaturated
204
Carbohydrate Chemistry
d i sa ccha r i d e s
.
A modification of measure
the
an amino
sulphaminohexose
s u g a r a n a l y s i s h a s been u s e d t o content
of
heparin
and
heparan
~ u l p h a t e . ~ 'P ~o l y m e r - b o u n d N - s u l p h a t e d 2-amino-2-deoxyhexoses s p e c i f i c a l l y d e a m i n a t e d t o y i e l d f r e e 2,5-anhydro-g-mannose, t h e n measured u s i n g t h e 3 - m e t h y l - 2 - b e n z o t h i a z o l i n o n e The culture
degradation of may
be
m o n i t o r i n g the
proteoglycan
and
release of
soluble
reagent.
collagen
measured simultaneously
are
which i s cells
i n
i n a p l a t e assay
by
by
r a d i o a c t i v e degradation
products
from a d i s h c o a t e d w i t h a g e l c o n t a i n i n g 3 H - l a b e l l e d p r o t e o g l y c a n and 1 4 C - l a b e l l e d c o l l a g e n . 3 0 4
S y n o v i a l c e l l s a r e a b l e t o degrade
both substrates, owing t o the release o f a proteoglycan-degrading n e u t r a l p r o t e i n a s e and o f co lla g e n a se . A
monodimensional c e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s
capable of r e s o l v i n g k e r a t a n sulphate, sulphate,
heparin,
t h e b a s i s of chloride acid,
c h o n d r o i t i n 4-sulphate,and
The e l e c t r o p h o r e t i c b e h a v i o u r
desulphated chondroitin,
sulphate w i t h two d i f f e r e n t buffer concentration, examined.306
A
is
dermatan
h y a l u r o n i c a c i d , on
t h e i r d i f f e r e n t i a l m i g r a t i o n i n H4-e.d.t.a.
so 1u t ions. 305
system
heparan sulphate,
and l i t h i u m o f h y a luronic
heparan sulphate, and c h o n d r o i t i n
b u f f e r s under v a r y i n g c o n d i t i o n s o f
electrophoresis
two-buffer
t i m e , and v o l t a g e has been
monodirectional
electrophoresis
t e c h n i q u e , w h i c h o f f e r s some o f t h e a d v a n t a g e s o f r e s o l u t i o n s e e n w i t h two-dimensional
methods
y e t r e t a i n s t h e a d v a n t a g e s o f band
comparison and q u a n t i t a t i v e dimensional electrophoresis, Intact
chick
flexible.308
limb
analysis
characteristic
of
one-
h a s been p r o p o s e d .
bud
Restrictions
proteoglycan
on
chain
monomer
flexibility
i s
occur
highly i n
the
n e i g h bo u r h o o d o f t h e l i n k a g e b e t w e e n p r o t e i n a n d p o l y s a c c h a r i d e c h a i n s where h i g h l o c a l c h a i n c o n c e n t r a t i o n s cause c o l l i s i o n s and entanglements.
Proteoglycans
from
bovine
nasal,
bovine a r t i c u l a r ,
a n d r a t c h o n d r o s a r c o m a c a r t i l a g e have been a n a l y s e d by h.p.1.c.
or a
s i l i c a - b a s e d m a t e r i a l bonded w i t h a n amide phase.307 Cation binding t o
m u l t i - c h a i n and s i n g l e - c h a i n
g l y c a n p e p t i d e s has been s t u d i e d b y 23Na+ n.m. r. Hyperlipidemic
rabbit
serum
impairs
the
glycosamino-
s p e c t r o s c o p y .309 precipitation
of
g l y c o samino g l y c u r o n a n s f r o m t i s s u e - c u l t u r e medium by c e t y l p y r i d i nium chloride.310 of
samples
derivatives
The i n t e r f e r i n g compounds c a n be r e m o v e d by e x t r a c t i o n i n l i p i d solvents. thereof
on low-density
have
Various
g l y c o s a m i n o g l y c a n s and
been s u b j e c t e d t o a f f i n i t y
l i p o p r o t e i n - s u b s t i t u t e d agarose.311
chromatography By u s e o f
a
205
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
v a r i e t y o f m o d i f i c a t i o n and degradation procedures, t h e chemical c h a r a c t e r i s t i c s t h a t maximize the a f f i n i t y o f a glycan f o r the l i p o p r o t e i n have been assessed.
The i n t e r a c t i o n b e t w e e n t h e t w o
c l a s s e s o f m a c r o m o l e c u l e s i s dependent o n t h e p r e s e n c e o f s u l p h a t e groups and i n c r e a s e s w i t h charge d e n s i t y .
The i n t e r a c t i o n o f serum
l i p o p r o t e i n s and a proteoglycan from
bovine aorta
reported.312
has
been
P o s i t i v e charges o n t h e p r o t e i n moiety o f low-densi t y
l i p o p r o t e i n s are r e q u i r e d f o r the i n t e r a c t i o n , as are presence o f the
protein
core
.
p r o t eo g l y ca n
and
the
glycosaminoglycan
chains
o f
the
P u r i f i e d p r o t e o g l y c a n s f r o m human a r t i c u l a r c a r t i l a g e have b e e n used
i n
the
development
of
a
radioimmunoassay
procedure.313
C o n d i t i o n s f o r l a b e l l i n g and subsequently p u r i f y i n g t h e p r o t e o g l y c a n are described,
Occurrence,
and o p t i m a l c o n d i t i o n s f o r t h e assay a r e d e t a i l e d .
I s o l a t i o n , and S t r u c t u r e .
-
The
f r a c t i o n a t i o n and
c h a r a c t e r i z a t i o n o f p r o t e o g l y cans i s o l a t e d from chondro c y t e c e l l cultures
have
been d e s c r i b e d . 3 1 4
The
proteoglycans
were
compared
with the proteoglycans extracted from bovine nasal o r t r a c h e a l cartilage.
P r o t e o g l y c a n s h a v e been p r e p a r e d f r o m human f e m o r a l - h e a d
a r t i c u l a r cartilage.315 scattering,and proteoglycan
As a r e s u l t o f g e l c h r o m a t o g r a p h y ,
ultracentrifugal studies, molecule
i n cartilage
light
a p h y s i c a l model o f t h e
has
been
proposed.
Three
p r o t e o g l y c a n f r a c t i o n s p r o d u c e d by human-em b r y o l u n g f i b r o b l a s t s h a v e b e e n i s o l a t e d a n d ~ h a r a c t e r i z e d . ~T h~e~ f r a c t i o n s c o n t a i n w t . 4.0 x l o 4 ) , c h o n d r o i t i n
m a i n l y heparan s u l p h a t e c h a i n s (mol. s u l p h a t e c h a i n s (mol. (mol.
2.5
wt.
x
lo4),
lo4),
w t. 4.0 x
respectively.
and dermatan sulphate chains
Glycosaminoglycans i s o l a t e d from
t r y p t i c d i g e s t s o f a t r i a o f t h e human h e a r t have been i d e n t i f i e d a s containing hyaluronic acid, sulphates,
heparan sulphate,
and dermatan sulphate.317
human m e n i s c u s a p p e a r t o be o f t w o types.318 a t
high
buoyant
density
on
caesium
c h o n d r o i t i n 4- and 6 -
The p r o t e o g l y c a n s o f a d u l t Preparations i s o l a t e d
chloride
density-gradient
c e n t r i f u g a t i o n c o n t a i n molecules o f l a r g e hydrodynamic s i z e t h a t a r e composed o f p r o t e i n ,
chondroitin sulphate,
k e r a t a n sulphate, and
neuraminic acid
possess
aggregate
comparable Preparations
and
with from
de rma t a n s u l pha t e,
those low
o f
subunit
and
hy a l i ne- c a r t i l a g e
buoyant
density,
which
are
co n t a i n sm a l l e r- m o l e c u l a r- w e i g h t
w h i c h do n o t i n t e r a c t w i t h h y a l u r o n i c a c i d .
structures
p r o t e o g l y cans. enriched i n p r o t e o g l y cans
The g l y c o s a m i n o g l y c a n
c o m p o s i t i o n s o f c a n i n e m e n i s c i have been reported.319
Although the
Carbohydrate Chemistry
206
c o m p o s i t i o n i s t h e same i n d i f f e r e n t r e g i o n s o f t h e m e n i s c i , t o t a l amounts vary baboons
considerably.
( P a ~ i gp a p i o )
electrophoretic
Articular
contains
mobility
on
a
the
c a r t i l a g e o f young
proteoglycan
large-porosity
gels
c h a r a c t e r i z e d as h a v i n g a high p r o t e i n content.320
of
high
and
i s
Proteoglycans
f r o m p i g a o r t a have b e e n e x t r a c t e d s e q u e n t i a l l y w i t h i n o r g a n i c s a l t s o l u t i o n s under a s s o c i a t i v e and d i s s o c i a t i v e c o n d i t i o n s , fractionation
by
f o l l o w e d by
g e l permeation chromatography i n order
t o
c h a r a c t e r i z e and compare t h e i r c h e m i c a l p r o p e r t i e s and h y d r o d y n a m i c sizes.321 fraction
Low-buoyant-density proteoglycans found i n the l i n k from, a v i a n c a r t i l a g e c o n t a i n some o f t h e a n t i g e n i c However, t h e r e i s
d e t e r m i n a n t s f o u n d o n t h e p r o t e o g l y c a n monomer.322
a t l e a s t one monomer d e t e r m i n a n t w h i c h i s n o t p r e s e n t i n t h e l i n k fraction. Proteoglycan
subunits
of
bovine
nasal
cartilage
contain
phosphate e s t e r groups t h a t a r e s e n s i t i v e t o a c i d phosphatase and are considered t o molecule.323
be a r e g u l a r c o n s t i t u e n t o f
the proteoglycan
S t r u c t u r a l studies on the E-xylosyl-L-serine
linkage
i n p r o t e o g l y c a n s o f human, p o r c i n e , a n d s h a r k c a r t i l a g e s h a v e b e e n reported.324
The f i n d i n g t h a t human a n d p o r c i n e p r o t e o g l y c a n s have
t h e same a m i n o a c i d sequence i n t h e l i n k a g e r e g i o n a s t h a t r e p o r t e d p r e v i o u s l y f o r b o v i n e p r o t e o g l y c a n s u g g e s t s t h a t t h e r e i s a common s t r u c t u r a l f e a t u r e i n t h e core p r o t e i n s o f these mammalian c a r t i l a g e p r o t eo g l y cans. Z o n a l - r a t e c e n t r i f u g a t i o n i n s u c r o s e g r a d i e n t s h a s been u s e d t o study i n t e r a c t i o n s i n proteoglycan aggregation.325
The l i n k p r o t e i n
was shown t o i n t e r a c t w i t h t h e i s o l a t e d h y a l u r o n i c a c i d - b i n d i n g r e g i o n o f t h e p r o t e o g l y c a n monomer. isolated hyaluronic
acid,
the
Since i t also i n t e r a c t s with binding o f
proteoglycans
hyaluronate i n the presence o f l i n k p r o t e i n s i s t r i f u n c t i o n a l .
to The
b i n d i n g p r o p e r t i e s o f c a r t i l a g e l i n k p r o t e i n and t h e hyaluronatebinding region of
cartilage
proteoglycan t o
hyaluronic acid
i m m o b i l i z e d o n a g a r o s e h a v e been c o m p a r e d a n d shown t o h a v e s i m i l a r properties.326
A
gross physical characterization o f the hyaluronic
acid-binding r e g i o n o f proteoglycan from p i g l a r y n g e a l c a r t i l a g e has been a c h i e v e d
using
densitometric
and
small-angle
neutron
s t u d i e s . 327 P l a t e l e t f a c t o r 4 i s a basic t e t r a m e r i c p r o t e i n which i n t e r a c t s w i t h sulphated polymers.328
The t r a n s f e r o f t h i s f a c t o r f r o m i t s
p r o t e o g l y c a n c a r r i e r t o n a t u r a l and s y n t h e t i c p o l y m e r s h a s been reported.
The r e s u l t s o f e l e c t r i c
b i r e f r i n g e n c e s t u d i e s show
that
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
207
c h o n d r o i t i n 4 - s u l p h a t e m o l e c u l e s have a very high d e g r e e o f m o b i l i t y
.
w i t h i n t h e p r o t eo g l y c a n str u c t u r e 329 A novel low-molecular-weight
chondroitin sulphate proteoglycan ( m o l . w t . 7.6 x l o 4 ) h a s b e e n i s o l a t e d f r o m b o v i n e n a s a l c a r t i l a g e , and i n d i f f e r e n t l a y e r s o f bovine h i p a r t i c u l a r cartilage.330 The p r o t e o g l y c a n c o n t a i n s two o r t h r e e c h o n d r o i t i n s u l p h a t e c h a i n s a n d some o l i g o s a c c h a r i d e s , and h i g h c o n t e n t s o f L - l e u c i n e and I - c y s t e i n e as compared w i t h o t h e r proteoglycans. The i n h i b i t o r o f Clq, a s u b u n i t o f t h e f i r s t component o f complement, has been i d e n t i f i e d a s a c h o n d r o i t i n 4-sulphate proteoglycan.331 Unlike the major species o f c a r t i l a g e p r o t e o g l y c a n , the serum p r o t e o g l y c a n does n o t form a complex w i t h h y a l u r o n i c acid. Rat b r a i n c o n t a i n s a s i n g l e p o l y d i s p e r s e m a c r o m o l e c u l e i n w h i c h c h o n d r o i t i n s u l p h a t e c h a i n s and o l i g o s a c c h a r i d e s are both covalently l i n k e d t o a common p r o t e i n c o r e . 3 3 2 The E a n d 2 - g l y c o s i d i c a l l y l i n k e d o l i g o s a c c h a r i d e s of t h e g l y c o p r o t e i n a p p e a r t o be r a n d o m l y The b r a i n p r o t e o g l y c a n appears t o d i s t r i b u t e d i n the proteoglycan. be c a p a b l e o f a t l e a s t a l i m i t e d d e g r e e o f i n t e r a c t i o n w i t h h y a l u r o n i c a c i d t o p r o d u c e l a r g e r a g g r e g a t e s . Some b o v i n e a o r t i c c h o n d r o i t i n s u l p h a t e- a n d d e rma t a n s u l p h a t e- co n t a i n i n g p r o t e o g l y c a n s have been i s o l a t e d and c h e m i c a l l y characterized.333 Disaccharides l i b e r a t e d from c h o n d r o i t i n s u l p h a t e s a n d d e r m a t a n s u l p h a t e by d i g e s t i o n w i t h c h o n d r o i t i n l y a s e A C o r c h o n d r o i t i n l y a s e ABC a r e c o m p l e t e l y s e p a r a t e d o n c e l l u l o s e a c e t a t e p l a t e s by e l e c t r o p h o r e s i s i n barium acetate or c a l c i u m acetate.334 G l y c o s a m i n o g l y c a n s have b e e n i s o l a t e d f r o m t h e f e m u r s o f o e s t r o g e n t r e a t e d male J a p a n e s e Chondroitin 4-sulphate and keratan s u l p h a t e were f o u n d i n t h e f e m u r s , b u t o n l y t h e k e r a t a n s u l p h a t e c o r r e l a t e s w i t h m e d u l l a r y bone p r o d u c t i o n . A proteoglycan isolated f r o m a s c i t e s f l u i d p r o d u c e d by a r a t y o l k - s a c t u m o u r h a s b e e n partially characterized as a chondroitin sulphate p r ~ t e o g l y c a n . ~ ~ ~ De rma t a n s u l p h a t e p r o t e o g l y c a n s h a v e b e e n i s o l a t e d a n d cha r a c t e r i z e d ’ proteoglycans, d i f f e r i n g with respect t o f r o m b o v i n e ~ c l e r a . ~ ~Two the nature o f t h e i r p r o t e i n cores and the co-polymeric s t r u c t u r e of Isopycnic centrifugation studies t h e i r s i d e c h a i n s , were i s o l a t e d . u s i n g caesium c h l o r i d e and caesium s u l p h a t e have been r e p o r t e d o n t h e s e p r o t e o g l y c a n s . 338 B o v i n e s c l e r a c o n t a i n s t w o p r o t e o d e rm a t a n sulphates, the l a r g e r exhibiting self-association i n both gel c h r o m a t o g r a p h y and l i g h t - s c a t t e r i n g e x p e r i m e n t s , and t h e smaller It is showing aggregation only i n l i g h t - s c a t t e r i n g experiments.339 p r o p o s e d t h a t t h e a g g r e g a t i o n i s s o l e l y o r p a r t l y m e d i a t e d by
208
Carbohydrate Chemistry
interaction forms
of
between dermatan s u l p h a t e s i d e
self-association,
e d .
protein-protein interactions,
& v
chains,
although other
polysaccharide-protein
three or four
C a l f s k i n p r o t e o d e r m a t a n s u l p h a t e i s composed o f
wt.
chains o f dermatan s u l p h a t e (mol.
wt.
t o a core p r o t e i n (mol. A
proteodermatan sulphate
5.6 x
lo4)
from
rat
1.7 x l o 4 ) c o v a l e n t l y l i n k e d 2-glycosidic
wt.
3.6
via
46% p r o t e i n w h i c h i s bound t o d e r m a t a n s u l p h a t e Rat
t a i l tendon has
linkages.340
s k i n has been i s o l a t e d and
~ h a r a c t e r i z e d . ~ T h~e ~ p r o t e o g l y c a n ( m o l . linkage.
and
h a v e n o t been e x c l u d e d .
been s t a i n e d
x
lo4)
contains
an 2 - g l y c o s i d i c
with
a
cationic
p h t h a l o c y a n i n dye, C u p r o m e r o n i c B l u e , t o d e m o n s t r a t e t h e l o c a t i o n o f A dermatan s u l p h a t e - r i c h
p r o t e o g l y c a n by e l e c t r o n m i c r o s c o p y . 3 4 2 proteoglycan
i s
distributed
orthogonal array,
about
the
collagen
the transverse elements o f
fibrils
which are
i n
an
located
a l m o s t e x c l u s i v e l y a t t h e d b a n d i n t h e gap zone. Several different proteoglycan species, a Q-glucuronic acidr i c h and an L - i d u r o n i c with two different human
skin
acid-rich
fibroblast
cultures.343
dermatan p o l y s u l p h a t e p e p t i d e s notochord,
proteodermatan sulphate,
proteoheparan sulphates,
Three
I, 11, and
different
a l l contain I-serine,
x y l o s e , a n d Q - g a l a ~ t o s e . ~T~h e~ a - x y l o s y l - g - s e r y l
of
linkage
Rwas
I c o n t a i n s an 2 - g l y c o s y l
d e t e c t e d i n p o l y p e p t i d e 111. P o l y p e p t i d e
l i n k a g e between 2-acetamido-2-deoxy-P-galactose on SephadexR G-50
types
111, i s o l a t e d f r o m h a g f i s h
h a g f i s h skin, and s h a r k s k i n ,
Chromatography
together
have been i s o l a t e d f r o m
and L - s e r i n e .
results i n optimal isolation
o f a h e p a r i n component t h a t i s h i g h l y a c t i v e as m e a s u r e d by s e v e r a l assay
methods.345
highly
The b i n d i n g o f
co-operative,
factors.346
An
exothermic,
examination
of
Methylene Blue t o heparin i s and
stabilized
fundamental
by
entropic
conditions
for
h y d r o p h o b i c - i n t e r a c t i o n c h r o m a t o g r a p h y o f h e p a r i n on h y d r o p h o b i c g e l s has been r e p o r t e d . 3 4 7 applied,
flow
rate,
and t e m p e r a t u r e ,
Column d i m e n s i o n s , amount o f h e p a r i n
e l e c t r o l y t e a n d a c i d i t y o f t h e e l u t i o n medium,
i n f l u e n c e t h e d i s t r i b u t i o n o f h e p a r i n among t h e
f r a c t i o n s s e p a r a t e d on t h e g e l . A d i r e c t m e t h o d h a s been d e v e l o p e d f o r t h e d e t e r m i n a t i o n o f t h e dissociation constant
b e t w e e n h e p a r i n and b o v i n e s e r u m a l b u m i n ,
by
u s i n g f l u o r e s c e n t l y l a b e l l e d h e p a r i n and m o n i t o r i n g t h e f l u o r e s c e n c e i n t e n s i t y change i n d u c e d by t h e p r o t e i n . 3 4 8 Gel f o r m a t i o n o f thymus lymphocytes, c e l l s w i t h h e p a r i n has been observed.349 only,
source o f h e p a r i n i s t h e mast
spleen
or bone marrow
Since the major,
cell,
i f not
and s i n c e h e p a r i n i s
209
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
r e l e a s e d a l o n g w i t h h i s t a m i n e d u r i n g c e r t a i n t y p e s o f a l l e r g i c and inflammatory reactions, i t i s suggested t h a t heparin promotes the a c c u m u l a t i o n o f l e u c o c y t e s i n t h e ex t r a v a s c u l a r space. Heparin
and
chondroitin
glycosaminoglycans which
6-sulphate
a t p H 6.0
are
the
l i p o p r o t e i n f r o m aqueous s o l u t i o n s a n d i n t h e p r e s e n c e of However,
chondroitin 6-sulphate
immobilized low-density same pH.
The
only
p r e c i p i t a t e low-densi t y
i s the only
one t h a t
Ca2+.350 binds
to
l i p o p r o t e i n i n t h e p r e s e n c e o f Ca2+ a t t h e
binding o f
Ca2+ a n d M g 2 + i o n s t o h e p a r i n i n t h e
p r e s e n c e o f added u n i v a l e n t s a l t s has been s t u d i e d u s i n g a dye s p e c t r o s c o p i c method.351 The i n t e r a c t i o n o f M g 2 + w i t h h e p a r i n a p p e a r s t o be i n d e p e n d e n t o f t h e n a t u r e o f t h e c h a r g e d g r o u p s o n t h e polyanion,
b u t Ca2+ b i n d i n g i s c o n s i d e r a b l y s t r o n g e r t h a n Mg2+
b i n d i n g and i s i n excess o f t h e o r e t i c a l p r e d i c t i o n s , s u g g e s t i n g a l o c a l i z e d o r s p e c i f i c i n t e r a c t i o n with heparin.
A selective binding
o f Zn2+ i o n s t o h e p a r i n r a t h e r t h a n t o o t h e r g l y c o s a m i n o g l y c a n s has been observed.352
This observation suggests that,
between
the
negatively
charged
carboxy
g l y c o Sam ino g l y c a n s a n d Z n 2 + c a t i o n s c a n n o t b in d in g
.
Some
chemical,
disaccharides
enzymic,
derived from
d e g r a d a t i o n f o l l o w e d by reported.353 from
which a
Heparin,
and
beef
sodium yields
groups
physical
properties by
c l ea ve s t h e 2-&
conditions
of
of
the
heparin,
been p a r t i a l l y
before for
and a f t e r
sulpham i d a s e ,
to
characterized.354
acid
hydrolysis
that
s u l p h o - l - i d o py r a no s i d i c 1i n ka ge s. 355
substrates
prior
l a r g e r - molecular-w eight fragments,
p u r i f i e d r a d i o - l a b e l l e d d i - s a c c h a r i d e s (5-13) evaluated
o f
b o r o t r i t i d e r e d u c t i o n have been
t e t r a s a c c h a r i d e has
under
the
nitrous acid
a f t e r c a r b o x y l r e d u c t i o n w i t h sodium b o r o t r i t i d e ,
degraded
of
ex p l a i n t h e o b s e r ve d
lung heparin
P a r t i a l tj-desulphation
de g r a da t i ve deam i n a t i o n ,
contrary to a
s i m p l e e l e c t r o s t a t i c in t e r a c t i o n s
p r e v i o u s l y p r o p o s e d hypo t h e s i s,
tj-acetylation
h a s been
selectively The r e s u l t i n g
were c h a r a c t e r i z e d and
o r tj-sulphation,
as
ace t y l c o e n z y m e A:2-am i n o - 2 - d e o x y - a - p -
g l u c o s i de tj- a c e t y 1t r a n s f e r a se , 2 - ace t am i d o -2-deox y-a -Q-g1 uco s i da se,
-
o r 2- a c e t am ido 2 - de ox y f ib r o b l a s t s
.
Tritiated di-
-n-
gl uco s e 6- s u l pha t e s u l pha t a s e in h um a n s k i n
and t e t r a - s a c c h a r i d e s ,
v a r i o u s r e p e a t sequences o f heparin,
evaluated as substrates for the estimation o f present i n s k i n fibroblasts, t y p e A syndrome.356
representative o f the
have been p r e p a r e d and sulphamidase a c t i v i t y
and f o r the d e t e c t i o n o f S a n - f i l l i p 0
Carbohydrate Chemistry
210
A s i m p l e m e t h o d f o r f r a c t i o n a t i n g h e p a r i n uses a n t i t h r o m b i n I 1 1 w h i c h i s n o n c o v a l e n t l y b o u n d t o i m m o b i l i z e d c o n c a n a v a l i n A.357
Active antithrombin i s e l u t e d from the immobilized l e c t i n using m e t h y l a-9-glucoside.
An e l e c t r o p h o r e t i c m e t h o d u s i n g a g a r o s e beads
has been d e v e l o p e d f o r t h e d e t e r m i n a t i o n o f h e p a r i n f r a c t i o n s h a v i n g a h i g h a f f i n i t y f o r a n t i t h r o m b i n III.358 A high-molecular-weight
heparin fraction that
includes
components w i t h a s much a s f i v e t o t e n t i m e s t h e a c t i v i t i e s o f t h e o r i g i n a l h e t e r o g e n e o u s p r e p a r a t i o n s has been i s o l a t e d f r o m lung.359
beef
The f r a c t i o n was c h a r a c t e r i z e d w i t h r e s p e c t t o i t s m o l e c -
u l a r w e i g h t (3.5
lo4),
x
c o m p o s i t i o n , and b i n d i n g t o a n t i t h r o m b i n
HOQoJQ HO NH2
*
'
(5)
R 1 = R2 = H
(6)
R1 = SO3-,
(7)
R1 = H,
OR2
NH2
R2 = H
R2 = SO3-
(8) R1 = R 2 = SO3-
R2
(9) (10)
R 1 = R2 =
(11)
R1
(12)
R 1 = R2 = S0-j-
R1 = SO3-,
= H,
H R2 = H
R2 = S O 3 -
CH20H
H NHAc
111.
OH
Hog rnucosal h e p a r i n , a f t e r p a r t i a l N - d e s u l p h a t i o n ,
co nj uga t e d t o f 1uo r e s c e i ny 1t h io ca r bam oy 1 g r o up s. 3 6 0 h e p a r i n was s e p a r a t e d i n t o l o w - a f f i n i t y w i t h respect
t o a n t i t h r o m b i n 111.
has been
The f 1uo r e s c e n t
and h i g h - a f f i n i t y
Substitution of
fractions
the heparin
m o l e c u l e s h a d no e f f e c t o n t h e b i o l o g i c a l i n t e r a c t i o n w i t h a n t i t h r o m b i n 111.
Evidence f o r a 3 - s - s u b s t i t u t e d
glucosyl residue i n the antithrombin-binding
T h i s s u l p h a t e d amino s u g a r i s a u n i q u e component
been r e p o r t e d . 3 6 1 o f high-affinity
2-amino-2-deoxy-Q-
sequence o f h e p a r i n has
heparin,
antithrom bin-binding
located a t a specific position i n the
sequence o f t h e m o l e c u l e .
Confirmation o f the
e x i s t e n c e o f t h i s component has been o b t a i n e d f r o m 1 3 C n.m.r. r o s c o p i c studies.362
spect-
A h i g h l y a c t i v e and a r e l a t i v e l y i n a c t i v e f o r m
o f w h a l e h e p a r i n have been s u b j e c t e d s e p a r a t e l y t o e n z y m i c d e g r a d a t ion.363
The
disaccharide
2-acetamido-2-deoxy-3-O-(4-deoxy-L-threo-
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
21 1
p y r a n o s y l u r o n i c acid)-!-glucose was f o r m e d e x c l u s i v e l y f r o m t h e h i g h l y a c t i v e f o r m a n d may a r i s e f r o m a n e s s e n t i a l b i n d i n g s e c t i o n f o r a n t i t h r o m b i n 111. Binding constants f o r t h e binding of high-affinity heparin t o a n t i t h r o m b i n 111 a t d i f f e r e n t i o n i c s t r e n g t h s h a v e b e e n d e t e r m i n e d by f l u o r e s c e n c e t i t r a t i o n s a n d e s t i m a t e d f r o m d i s s o c i a t i o n c u r v e s o f t h e h e p a r i n - a n t i t h r o m b i n I11 c o m p l e x . 3 6 4 A thermodynamic e v a l u a t i o n o f t h e d i s s o c i a t i o n o f t h e complex s u g g e s t s t h a t m a x i m a l l y f i v e t o six charged groups are involved i n t h e interaction. The b i n d i n g o f a n t i t h r o m b i n I11 t o h e p a r i n f r a c t i o n s w i t h d i f f e r e n t m o l e c u l a r Binding c o n s t a n t s and s t o i c h i o m e t r i e s w e i g h t s has b e e n s t u d i e d . 3 6 5 f o r t h e b i n d i n g o f t h e s e f r a c t i o n s t o a n t i t h r o m b i n I11 a r e p r e s e n t e d and c o r r e l a t e d with t h e a n t i c o a g u l a n t activities of the f r a c t i o n s . P o r c i n e i n t e s t i n a l h e p a r i n has been p a r t i a l l y degraded t o p r o d u c e a n o c t a s a c c h a r i d e w i t h h i g h a f f i n i t y f o r a n t i t h r o m b i n III!66 T h e o c t a s a c c h a r i de e x h i b i t e d m o r e p o t e n t i n a c t i v a t i o n a c t i v i t i e s f o r t h r o m b i n , f a c t o r Xa, a n d p l a s m a c o a g u l a t i o n t h a n t h e n a t i v e h e p a r i n . T h e h i g h - a f f i n i t y b i n d i n g o f h e p a r i n t o a n t i t h r o m b i n I11 r e q u i r e s t h e p r e s e n c e o f t w o c o n s e c u t i v e t j - s u l p h a t e d 2-am i n o - 2 - d e o x y - p glucosyl residues i n s p e c i f i c positions of the antithrombin-binding sequence.367 Loss o f e i t h e r o n e o f t h e s e N - s u l p h a t e g r o u p s , w i t h o r without tj-acetylation, r e s u l t s i n a d i s t i n c t and appreciable decrease i n binding a f f i n i t y and i n anticoagulant a c t i v i t y . P o r c i n e h e p a r i n h a s b e e n c l e a v e d r a n d o m l y by c h e m i c a l t e c h n i q u e s t o p r o d u c e h e x a saccha r i de s, o c t a s a ccha r i des, de ca sa ccha r i d e s, a n d f r a g m e n t s c o n t a i n i n g 1 4 a n d 16 r e s i d u e s t h a t a r e a b l e t o c o m p l e x with protease inhibitors.368 As a r e s u l t o f s t u d i e s o n t h e a v i d i t y of t h e s e f r a c t i o n s f o r p r o t e a s e i n h i b i t o r s , t h e i r b i n d i n g t o a n t i thrombin, a n d t h e c a t a l y s i s of t h e F a c t o r Xa-antithrom b i n i n t e r a c t ion, i t i s proposed that h e p a r i n p o s s e s s e s m u l t i p l e discrete s t r u c t u r a l d o m a i n s t h a t m o d u l a t e d i f f e r e n t f u n c t i o n s o f a n t i t h r o m b i n 111. T h e c h e m i c a l c o m p o s i t i o n a n d 1 3 C n.m.r. s p e c t r a o f h e p a r i n o c t a s a c c h a r i d e s h a v i n g h i g h a f f i n i t y f o r a n t i t h r o m b i n 111 a n d h i g h a n t i ( f a c t o r X a ) a c t i v i t y , a n d p r e p a r e d by t h r e e i n d e p e n d e n t a p p r o a c h e s , have been s t u d i e d and compared w i t h t h o s e of t h e corresponding i n a c t i v e species.369 Combined w i t h c h e m i c a l d a t a , t h e s p e c t r a of t h e active oligosaccharides and of their fragmentation products afforded information on composition and sequence. The e f f e c t s o f h i g h - a c t i v i t y h e p a r i n f r a c t i o n s of d i f f e r i n g m o l e c u l a r w e i g h t o n a n t i t h r o m b i n I11 i n h i b i t i o n o f e a c h o f t h e s e r i n e p r o t e a s e s known t o o c c u r i n t h e i n t r i n s i c pathway of t h e
212
Carbohydrate Chemistry
c o a g u l a t i o n cascade have been r e p o r t e d . 3 7 0 I n h i b i t i o n o f thrombin, F a c t o r IXa, a n d F a c t o r X I a showed s i m i l a r i t i e s i n t h e dependence o n molecular
weight
of
heparin,
and was
found
to
decreasing molecular weight o f the polysaccharide. F a c t o r Xa,
Factor XIIa,and
decrease
with
Inactivation o f
k a l l i k r e i n was l e s s dependent o n t h e s i z e
o f t h e p o l y sa ccha r i de. Low-molecular-weight
h e p a r i n o f l o w a n t i c o a g u l a n t a c t i v i t y and
h i g h - m o l e c u l a r - w e i g h t h e p a r i n o f c o r r e s p o n d i n g h i g h a c t i v i t y have been s u b j e c t e d t o N - d e s u l p h a t i o n
and t h e r e s u l t i n g amino groups r e -
a c y l a t e d w i t h d a n s y l c h l o r i d e o r r h o d a m i n e B i s ~ t h i o c y a n a t e . ~The ~ ~ results o f binding studies o f the modified heparins t o antithrombin I11 a r e c o n s i s t e n t w i t h t h e p r o p o s a l t h a t a s i n g l e h i g h - m o l e c u l a r weight high-activity
h e p a r i n o c c u p i e s t w o s i t e s when i t b i n d s t o
a n t i t h r o m b i n I11 w h e r e a s l o w - m o l e c u l a r - w e i g h t
low-activity
heparin
binds t o t h e two s i t e s s e p a r a t e l y . P r o t e o h e p a r a n s u l p h a t e p r e p a r e d from
r a t l i v e r p l a s m a membranes
i n h i b i t s t h e g r o w t h o f AH-130 a s c i t e s h e p a t o m a c e l l s . 3 7 2
Heparan
sulphate i s l e s s active. A has
non-collagen
been
occurring
protein,
identified rat
as
basement
a
i n a s s o c i a t i o n w i t h heparan sulphate, normal
constitutent
mem b r a n e . 3 7 3
The
a
naturally
material
of
resembles
p r o t e o g l y c a n s r e c e n t l y i s o l a t e d f r o m t h e EHS sarcoma a n d f r o m b o v i n e g l o m e r u l a r basement membrane. Heparan
sulphate
has
been
i d e n t i f i e d
as
the
major
g l y co s a m i n o g l y can i n b o v i n e r e t i n a l c a p i l l a r y b a s e m e n t m em brane.374 The b i n d i n g o f g l y c o s a m i n o g l y c a n s t o f i b r o n e c t i n a t a d h e s i o n s i t e s o f m u r i n e f i b r o b l a s t s has been r e p o r t e d . 3 7 5 s u l ph a t e p r o t eo g l y ca n s , co n t a in i n g h i gh 1y
l-s u l ph a t e d
Only heparan s e q ue nce s o n
t h e h e p a r a n s u l p h a t e , m e d i a t e s u b s t r a t u m a d h e s i o n t o t h e c e l l s by co-ordinate b i n d i n g t o
f i b r o n e c t i n o n t h e c e l l surface and serum
f i b r o ne c t i n. S e l f - a s s o c i a t i on
be t w een
various
hepa r a n s u l p h a t e s p e c i e s a n d
o l i g o s a c c h a r i d e f r a g m e n t s t h e r e o f h a v e b e e n s t u d i e d by a f f i n i t y
.
ch rom a t o g r a p h y 376 ,377
Se gm e n t s co m p r i s i n g bo t h
L - i d u r o na t e
and
g-
g l u c u r o n a t e may s e r v e a s c o n t a c t z o n e s f o r t h e s e l f - a s s o c i a t i o n , and t h e s t r e n g t h o f b i n d i n g i s dependent on c o o p e r a t i v e i n t e r a c t i o n s b e t w e e n a number o f s u c h zones.
However, v a r i o u s h e p a r a n s u l p h a t e
s u b f r a c t i o n s f r o m t r a n s f o r m e d c e l l s h a v e no a f f i n i t y f o r a g a r o s e g e l s s u b s t i t u t e d w i t h t h e c o r r e s p o n d i n g h e p a r a n s u l p h a t e species.378 T h e l a c k o f a s s o c i a t i o n o f t h e h e p a r a n s u l p h a t e may be c a u s e d by m i n o r changes i n t h e s e q u e n t i a l a r r a n g e m e n t s o f L - i d u r o n a t e -
and
0-
213
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides g l u c u r o n a t e - b e a r i n g r e p e a t i n g segments o f t h e p o l y s a c c h a r i d e . Heparan s u l p h a t e - c l e a v i n g o f a n a s c i t e s h e p a t o m a AH66, sulphate
at
the
cell
enzyme h a s been u s e d t o t r e a t c e l l s f o r which t h e occurrence o f heparan
surface
of
the
plasma
membrane has
been
e s t a b l i s h e d .379 Two p o o l s o f h e p a r a n s u l p h a t e p r o t e o g l y c a n s w i t h d i f f e r e n t b u o y a n t d e n s i t i e s h a v e been s e l e c t i v e l y s o l u b i l i z e d f r o m r a t l i v e r plasma
membranes
detergent.380
by
successive
incubations
with
heparin
and
The d e t e r g e n t - e x t r a c t e d h e p a r a n s u l p h a t e r e p r e s e n t s a
p r o t e o g l y c a n s p e c i e s t h a t has i t s c o r e p r o t e i n i n t h e l i p i d b i l a y e r o f t h e p l a s m a membrane. high
The p r e s e n c e o f h e p a r a n s u l p h a t e h a v i n g a
metabolic activity
has
been d e m o n s t r a t e d i n i s o l a t e d r a t
i n t e s t i n a l e p i t h e l i a l cells.381
A h i g h degree o f s u l p h a t i o n o f i t s
c h a i n s was d e t e c t e d . synthesis.382
H e p a r a n s u l p h a t e i s a p o t e n t i n h i b i t o r o f DNA
The i n h i b i t i o n i s n o t s i m p l y r e l a t e d t o g r o s s c h a r g e
density. B o v i n e v i t r e o u s body h y a l u r o n i c a c i d h a s been p u r i f i e d , t h e use o f
p r o t e o l y t i c enzymes,
by i o n - e x c h a n g e
without
~hromatography.~~~
1251 - L a b e l l e d
h y a l u r o n a t e- b i n d i n g
prepared.384
T h e i r a d s o r p t i o n on an i m m o b i l i z e d h y a l u r o n a t e g e l i n
p r o t e i n s f r o m c a r t i l a g e h a v e been
t h e p r e s e n c e o f f r e e c o m p e t i n g h y a l u r o n i c a c i d has been used as an assay f o r f r e e h y a l u r o n i c a c i d . o f 'H n.m.r. s p e c t r a i n {2H6}-dimethylsulphoxide dodecyltrimethylammonium s a l t s of c h o n d r o i t i n s u l p h a t e s and h y a l u r o n a t e o r s o d i u m s a l t s o f o l i g o m e r s f r o m h y a l u r o n a t e show u n a m b i g u o u s NH s i g n a l s . 3 8 5 some NH,
A s e c o n d a r y s t r u c t u r e i n h y a l u r o n a t e and
chondroitin sulphates, was o b s e r v e d .
i n v o l v i n g a hydrogen-bonded
homologous s e r i e s o f
oligosaccharides
t e s t i c u l a r h y a l ~ r o n i d a s e . ~C.d. ~~ showed t h a t t h e c.d.
acetamido
Sodium h y a l u r o n a t e has been c l e a v e d i n t o an by
the
a n a l y s i s of
action of
bovine
the oligosaccharides
s p e c t r u m o f h y a l u r o n a t e i n aqueous s o l u t i o n a t
n e u t r a l pH d o e s n o t r e f l e c t t o a n y s u b s t a n t i a l d e g r e e a p o l y m e r c o n f o r m a t i o n which r e q u i r e s c o o p e r a t i v e i n t e r a c t i o n between s e v e r a l repeating residues f o r stabilization. The s e q u e n t i a l h y d r o l y s i s o f r o o s t e r comb h y a l u r o n i c a c i d by e-glucuronidase
and f3-Q-2-acetarnido-2-deoxy-glycosidase
B-
has f a i l e d
t o d e m o n s t r a t e t h e p r e s e n c e o f any b r a n c h i n g i n t h e p o l y s a c c h a r i d e chain.387 sugars.
No e v i d e n c e
was
found for
the presence o f
neutral
The t e m p e r a t u r e dependence o f b i n d i n g h y a l u r o n a t e o l i g o m e r s
t o b o v i n e n a s a l p r o t e o g l y c a n has been reported.388 Hyalurononectin,
a human b r a i n g l y c o p r o t e i n c a p a b l e o f b i n d i n g
Carbohydrate Chemistry
214 t o hyaluronic acid, by
hyaluronic
hyaluronidase
has been i s o l a t e d . 3 8 9
acid
and
by
the
but
not
by
either
The b i n d i n g i s i n h i b i t e d
products
hydrolysis
by
glycosaminoglycans
of
or
monosaccharides. The
interactions of
s u l p h a t e d p o l y s a c c h a r i d e s s u c h as
s u l p h a t e and c h i t i n s u l p h a t e
w i t h Solanum t u b e r o s u m
wheat germ a g g l u t i n i n have been r e p o r t e d . 3 9 0
keratan
a g g l u t i n i n and
The p r e s e n c e o f t h e
sulphate groups i n these polysaccharides d i d n o t i n t e r f e r e w i t h t h e i r s p e c i f i c i n t e r a c t i o n s with the l e c t i n s . The d e g r a d a t i o n o f k e r a t a n s u l p h a t e h a s b e e n a c h i e v e d by B - 2 -
For
a c e t a m i d o - 2 - d e o x y - ~ - g l u c o s i d a s e s A a n d 8 (EC 3.2.1.52).391 e n z y m ic a c t i v i t y a s u b s t r a t e w i t h exposed n o n- s ulphat ed 2-deoxy-g-glucosyl
residues a t
t h e non-reducing
end o f
2-acetamidothe polymer
i s required. Keratan
-----Pseudo m o nas glucose
sulphate
=.
has
been
degraded
by
an
extract
of
a
t o g i v e 2 - a c e t a m ido -2-deox y-3-g-Q -ga l a c t o s y 1-B-0-
6 - s ~ l p h a t e . ~ ' ~T h i s
disaccharide
after
reduction with
s o d i u m b o r o t r i t i d e can be u s e d as a s u b s t r a t e f o r t h e measurement o f 2-acetamido-2-deoxy-q-glucose
6-sulphate
sulphatase,
w h i l s t the
d e s u l p h a t e d r e d u c e d d i s a c c h a r i d e c a n be u s e d as a s u b s t r a t e f o r t h e measurement
of
(1+3)-2-acetamido-2-deoxy-B-~-glucosidase.
keratan sulphate from hydrazinolysis,
bovine cornea,
deamination
and
d e g r a d e d by
sodium
releases two t e t r a s a c c h a r i d e f r a c t i o n s , which
(14)
has
been e s t a b l i s h e d . 3 9 3
borohydride
reduction,
t h e s t r u c t u r e o f one o f
80th
r e s i d u e s bear k e r a t a n s u l p h a t e ch a in s.
Peptido-
desulphation,
terminal
rj-mannosyl
A s t r u c t u r e i n w h i c h an
I-
a-Q-Manp-(l+6)-B-q-Mane-(l+?)-g-GlcENAc-L-Asn -
3
I 1 a-E-Manp (14) f u c o s y l r e s i d u e and an N " - d i a c e t y l c h i t o b i o s y l
2-acetamido-2-deoxy-~-glucosyl has
also
been
proposed.
sequence r e p l a c e t h e
residue attached t o the p r o t e i n chain Studies
on t h e
p o l y d i s p e r s i t y and
h e t e r o g e n e i t y o f p r o t e o k e r a t a n s u l p h a t e f r o m c a l f and p o r c i n e c o r n e a have
been
reported.394
The
linkage
region
of
bovine
corneal
p r o t e o k e r a t a n s u l p h a t e i s b r a n c e d a t a Q-mannose r e s i d u e (15).395 The s t r u c t u r a l c h a r a c t e r i s t i c s show t h a t t h e r e i s a g r e a t s i m i l a r i t y
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
m
I
n
n
I
hl 4 .-I
v
1
hl
u
9
E?
d
n
In 4
v
hl
n
v
hl I
0
n
I
a - m
v
u
n CI
v
I
u
I
c311
M
0
-
215
Carbohydrate Chemistry
216
E-
b e t w e e n t h e l i n k a g e r e g i o n o f t h e p r o t e o k e r a t a n s u l p h a t e and t h e g l y c o s i d i c a l l y l i n k e d L - a s p a r a g i n e m o i e t y o f many g l y c o p r o t e i n s .
--
Biosynthesis. ectodermal effects
H y a l u r o n i c a c i d s y n t h e s i s by t h e
ridge of
of
chick
phase
of
l i m b bud has
growth,
c e l l
apical
been r e p o r t e d . 3 9 6
density
and
also
The serum
c o n c e n t r a t i o n a r e i m p o r t a n t c o n t r i b u t o r s f o r t h e i n c o r p o r a t i o n o f 2amino-2-deoxy-9-{
3H1 - g l u c o s e
t i c smooth m u s c l e c e l l s . 3 9 7
i n t o g l y c o s a m i n o g l y c a n s by r a b b i t a o r The s y n t h e s i s o f s u l p h a t e d g l y c o s a m i n o -
in
glycans i n r a t l i v e r explants prepared from regenerating tissue, which the f a c t o r s of
c e l l n e c r o s i s and c o n s e c u t i v e i n f l a m m a t i o n
a s s o c i a t e d w i t h most models o f e x p e r i m e n t a l h e p a t i c f i b r o s i s a r e shows a d e p r e s s i o n by a b o u t 50% o f t h e i n c o r p o r a t i o n o f 2-
excluded,
amino-Z-deoxy-Q-{ 14C) - g l u c o s e i n t o t o t a l
glyco~aminoglycan.~~~
R a t l i v e r p a r e n c h y m a l c e l l s have been e v a l u a t e d f o r
their
a b i l i t y t o s y n t h e s i z e and a c c u m u l a t e h e p a r a n s u l p h a t e as t h e m a j o r component and l o w - s u l p h a t e d c h o n d r o i t i n s u l p h a t e , chondroitin sulphate
dermatan sulphate,
and h y a l u r o n i c a c i d as t h e
minor
ones.399
G l y c o s a m i n o g l y c a n s a r e s y n t h e s i z e d by r a t g l o m e r u l i a n d t r a n s p o r t e d t o g l o m e r u l a r basement membranes. product synthesized, chondroitin
sulphates
are
also
produced.400
The
transfer
of
f a c t o r 4, a b a s i c t e t r a m e r i c p r o t e i n c o m p l e x e d w i t h a
platelet series of studied
Heparan s u l p h a t e i s t h e major
a l t h o u g h s m a l l e r amounts o f h y a l u r o n i c a c i d and
glycosaminoglycans,
by
derivative
measuring of
changes
platelet
factor
t o h e p a r i n has been d e t e c t e d and i n
the
anisotropy
4.401
The
f a c t o r 4 - p r o t e o g l y c a n complex f r o m t h e p l a t e l e t the t r a n s f e r o f the p r o t e i n t o heparin.
of
release
in
the of
dansyl
platelet
vivo results i n
voderate quantities o f
d e r m a t a n s u l p h a t e o r h e p a r a n s u l p h a t e do n o t p r e v e n t t h e t r a n s f e r . The e f f e c t o f c y c l o h e x i m i d e on t h e s y n t h e s i s o f p r o t e o g l y c a n s by c u l t u r e d c h o n d r o c y t e s f r o m t h e Swarm r a t c h o n d r o s a r c o m a h a s been reported.402
The c u l t u r e d c h o n d r o c y t e s c o n t a i n a l a r g e p o o l o f c o r e
protein available for
the addition o f
chondroitin sulphate
chains.
I n t h e absence o f e n t r y o f n e w l y s y n t h e s i z e d c o r e p r o t e i n i n t o t h i s pool following
cycloheximide treatment,
the remaining core p r o t e i n
molecules are processed t o completed proteoglycans w i t h n e a r l y f i r s t order
exponential
synthesis o f
kinetics.
The
biochemical pathways
for
the
c h o n d r o i t i n s u l p h a t e w e r e n o t d r a s t i c a l l y a f f e c t e d by
cycloheximide.
I n h i b i t i o n o f p r o t e i n s y n t h e s i s by c y c l o h e x i m i d e
reduces t h e i n c o r p o r a t i o n of { 35S)-sulphate i n t o heparan s u l p h a t e t o
217
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides about
5% o f
untreated hepatocytes.
4-Nitrophenyl f3-Q-xyloside
p a r t i a l l y reverses the i n h i b i t o r y effect.403 chains
not
bound t o
core p r o t e i n were
Free heparan sulphate
synthesized under
these
conditions. The p a t t e r n o f g l y c o s a m i n o g l y c a n s y n t h e s i s b y s m o o t h - m u s c l e c e l l s f r o m p i g a o r t i c media has been studied.404 s y n t h e s i z e d by media c e l l s e x h i b i t d i f f e r e n t
Proteoglycans
glycosaminoglycan
d i s t r i b u t i o n patterns according t o t h e i r l o c a l i z a t i o n . Cultured c e l l s from red dermal tissue,
containing a large
p r o p o r t i o n o f c e l l s from the h a i r f o l l i c l e s . , produce h y a l u r o n i c acid, dermatan sulphate, sulphate.405
The
c h o n d r o i t i n 4-sulphate,
patterns
of
synthesis
h e p a r i n and heparan
and s u l p h a t i o n
of
the
g l y c o s a m i n o g l y c a n s changed w i t h c o n t i n u e d t i m e i n c u l t u r e . Proteoglycan
synthesis
hydrocortisone-suppressed reported.406
i n
rabbit
normal
articular
and
chronically
cartilage
has
been
The r e p o r t c o n f i r m s t h a t h y d r o c o r t i s o n e c a n e f f e c t
p o o l s i z e s , and a l s o t h e l e v e l o f
UDP-2-acetamido-2-deoxy-p-hexose
a-galactosyltransferases, b u t n o t a - x y l o s y l t r a n s f e r a s e i n v o l v e d i n the synthesis of linkage region.
the chondroitin sulphate carbohydrate-protein
The m a j o r e f f e c t i s t h e d e p r e s s i o n o f p r o t e o g l y c a n
core-protein synthesis.
No n e t change i n t h e p r o t e o g l y c a n s t r u c t u r e
was o b s e r v e d . Insulin
and
r a t-derived,
multiplication-stimulating
somatomedin-like
activity
stimulates
s y n t h e s i s i n r a t c h o n d r o s a r c o m a c h o n d r o c y t e s .407
polypeptide proteoglycan
The s t r u c t u r e s o f
t h e p r o t e o g l y c a n s s y n t h e s i z e d i n response t o t h e hormones compared w i t h t h o s e s y n t h e s i z e d i n t h e medium a l o n e a r e s i m i l a r , chondroitin sulphate chains, hormones,
increase
i n
but the
synthesized i n the presence o f
molecular
weight
by
25-30%.408
The
d i s t r i b u t i o n o f g l y c o s a m i n o g l y c a n s y n t h e s i s by r a t g l o m e r u l i i n v i v o and
in
v i t r o has been reported.409
A d m i n i s t r a t i o n o f a s i n g l e dose o f
2-amino-2-deoxy-g-galactose
t o r a t s causes time-dependent b i p h a s i c changes g l y c o s a m i n o g l y c a n s y n t h e s i s i n l i v e r .410 A rapidly
o f t o t a l occurring
i n h i b i t i o n i s f o l l o w e d by a s i g n i f i c a n t l y enhanced p r o d u c t i o n o f { 35Sl-labelled
glycosaminoglycans i n l a t e r
stages.
Undersulphated
f3-e-xy 10s i d e g l y c o s a m i n o g l y c a n s a r e s e c r e t e d f r o m c h i c k - e m b r y o chondrocytes
i n the
presence o f
the
i o n o p h o r e monensin.411
S t i m u l a t e d p e r i p h e r a l b l o o d monocytes c o n t a i n a f a c t o r c s ) which affects synovial cells i n culture,
depressing markedly collagen
synthesis w h i l e enhancing glycosaminoglycan production.412
The
218
Carbohydrate Chemistry
i n v o 1 vem e n t o f s u c h b i o l o g i c a 11y a c t i v e m o n o c y t e - d e r i ve d s u b s t a n c e (s ) i n c o n n e c t i ve-tiss u e me t a b o l i sm o f i n f 1am ma t o r y l e s i o n s i s discussed. The assembly of proteoglycan a g g r e g a t e s i n c u l t u r e s o f c h o n d r o c y t e s from b o v i n e t r a c h e a l c a r t i l a g e has been studied.413 P r o t e o g l y c a n monomers and h y a l u r o n i c acid a r e e x p o r t e d s e p a r a t e l y The s i z e o f t h e a g g r e g a t e s i n c r e a s e s and combined e x t r a c e l l u l a r l y . g r a d u a l l y w i t h time as t h e p r o p o r t i o n o f monomers bound t o Immunochemical a n a l y s i s of t h e hyaluronic acid increases. proteoglycans h a s i d e n t i f i e d c r o s s - r e a c t i v i t y of molecules i s o l a t e d from d i f f e r e n t species.414 The i n t r a c e l l u l a r p r e c u r s o r p o o l of c o r e p r o t e i n i n t h i s c h o n d r o c y t e system h a s been i d e n t i f i e d and p a r t i a l l y c h a r a c t e r i z e d .415 A s t u d y o f a c c e p t o r s a n d primers f o r c h o n d r o i t i n s u l p h a t e - c h a i n b i o s y n t h e s i s by m i c r o s o m a l p r e p a r a t i o n s f r o m c h i c k - e m b r y o e p i p h y s e a l Incorporation of sugars into c a r t i l a g e has been reported.416 g l y c o s a m i n o g l y c a n s has b e e n u s e d t o o b t a i n i n f o r m a t i o n o n t h e e n t i r e proteoglycan s t r u c t u r e a t t h e s i t e o f glycosaminoglycan synthesis. A s o l u b l e e n z y m e f r o m q u a i l o v i d u c t i n c o r p o r a t e s s u l p h a t e a t 06 o f t h e no n- r e d u c i n g 2 - a c e tam i d o - 2 - d e o x y - ~ - g a l a c t o s y 1 4 - s u l p h a t e r e a c t i o n c a t a l y s e d by t h i s r e s i d u e o f c h o n d r o i t i n ~ u l p h a t e . ~ ~ The ’ e n z y m e i s d i f f e r e n t f r o m t h e s u l p h a t i o n m e d i a t e d by o t h e r k n o w n s u l p h o t r a n s f e r a s e s. The f o r m a t i o n o f C-idopyranosyluronic acid during t h e s y n t h e s i s o f d e r m a t a n s u l p h a t e h a s been s t u d i e d i n human f i b r o b l a s t c u l t u r e s a n d m i c r o s o m e s o f t h e same c e l l s . 4 1 8 E p i m e r i z a t i o n o f ag l ucopy r a n 0 s y l u r o n i c a c i d t o L - i dopy r a n 0 s y l u r o n i c a c i d r e s i d u e s during the biosynthesis o f dermatan sulphate involves an abstraction o f t h e C-5 h y d r o g e n o f t h e u r o n o s y l r e s i d u e . S u b s t r a t e s f o r 2- ace tam i d o - 2 - d e o x y-Q-gl uco s y 1t ra n s f e r a se , w h i c h acts i n conjunction with Q-glucuronosyl transferase i n the b i o s y n t h e s i s o f h e p a r i n , h a v e b e e n p r e p a r e d f r o m a l o w s u l p h a t e d , aglucuronic acid-rich heparan sulphate fraction.419 U s i n g these o l i g o s a c c h a r i d e s , t h e products o f t h e r e a c t i o n have been c h a r a c t e r i z e d a n d some o f t h e k i n e t i c parameters o f t h e enzyme have been determined. S o m e o f t h e o l i g o s a c c h a r i d e s were s h o w n t o s u b s t i t u t e f o r t h e endogenous s u b s t r a t e s and s e r v e as p r i m e r s i n po 1y s a c c h a r i de c h a i n e l o n g a t i o n . T h e s e c r e t i o n o f h e p a r a n s u l p h a t e by c u l t u r e d r a t h e p a t o c y t e s i s i n c r e a s e d i n t h e p r e s e n c e of t h e f l a v e n o i d (+) ~ a t e c h i n . ~ ” T h i s i n c r e a s e i s d u e t o a new s p e c i e s o f h e p a r a n s u l p h a t e l a c k i n g t h e
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
219
c a r b o h y d r a t e - p r o t e i n l i n k a g e b e t w e e n I)-xylose and I - s e r i n e i n t h e no rm a1 h e p a r a n s u l p h a t e p r o t e o g l y c a n . E x t r a c e l l u l a r h y a l u r o n i c acid i s t h e major glycosaminoglycan s y n t h e s i z e d by t h e e p i d e r m i s w h e n i n t a c t s k i n s l i c e s a r e m a i n t a i n e d i n organ culture.421 I n t h e a b s e n c e o f t h e dermis t h i s s y n t h e s i s i s considerably reduced, and the synthesis of sulphated g l yco s a m i n o g l y c a n s , main1y hepa r a n s u l p h a t e , i s u n a f f e c t e d . T h e f o r m a t i o n o f b o t h N- a n d g - s u l p h a t e g r o u p s ( s u l p h a m i d o a n d ester s u l p h a t e groups, r e s p e c t i v e l y ) o f t h e 2-amino-2-deoxy-eg l u c o s y l r e s i d u e s i n p o r c i n e a o r t a h a s b e e n d e m o n s t r a t e d by t h e s t u d y o f s u l p h a t e i n c o r p o r a t i o n from l a b e l l e d 3’-phosphoadenyl sulphate t o heparan sulphate of microsomal f ractions.422 The p r e f e r e n t i a l N-sulphation is obtained f o r incorporation o f labelled precursor over a short period, with P-sulphation occurring on previously 3-sulphated heparan sulphate. The s t i m u l a t i o n o f h y a l u r o n i c a c i d s y n t h e s i s i n mouse s k i n i n r e s p o n s e t o e s t r o g e n t r e a t m e n t i s mediated through e s t r o g e n r e c e p t o r s and i n v o l v e s t h e i n d u c t i o n o f hy a 1 u r o n i c a c i d s y n t h e t a s e . 4 2 3 G l y c o Sam i no g l y c a n s e c r e t i o n from perfused monolayer c u l t u r e s o f r a b b i t ear c h o n d r o c y t e s has been r e p o r t e d . 4 2 4 C o l c h i c i n e has been shown t o a f f e c t t h e l e v e l of glycosaminoglycan synthesis and transport. A short-term incubation system, developed f o r the study of glycosaminoglycan synthesis during t h e early stages o f medullary bone f o r m a t i o n i n J a p a n e s e q u a i l i n t h e p r e s e n c e o f e s t r o g e n , h a s i d e n t i f i e d k e r a t a n s u l p h a t e and c h o n d r o i t i n ~ u l p h a t e . ~ ’ ~
-
The p o l y d i s p e r s i t y p r e s e n t i n a g g r e g a t i n g p r o t e o g l y c a n s n e w l y s y n t h e s i z e d by c h o n d r o c y t e s f r o m r a t chondrosarcoma has been a t t r i b u t e d t o t h e amount of c h o n d r o i t i n sulphate/core p r o t e i n and n o t t o any d e t e c t a b l e d i f f e r e n c e s i n t h e s i z e of t h e c o r e protein.426,427 Glycosaminoglycans i n u r i n e from p a t i e n t s representing t h e major d i f f e r e n t mucopolysaccharidoses have b e e n s e p a r a t e d a n d m e a s u r e d by u s e o f a p r o c e d u r e r e q u i r i n g s m a l l volumes of urine.428 Approximately h a l f t h e p a t i e n t s e x c r e t e d small amounts of heparin. T h e k e r a t a n s u l p h a t e i n samples f r o m M o r q u i o ’ s disease p a t i e n t s m i g r a t e d d i f f e r e n t l y from a u t h e n t i c k e r a t a n s u l p h a t e u n l e s s d i g e s t e d w i t h c h o n d r o i t i n s u l p h a t e l y a s e ABC. Total sulphated glycosaminoglycan l e v e l s i n commercially available preparations of hyaluronic acid and i n the urine of normal individuals and the synovial f l u i d o f individuals w i t h rheumatoid a r t h r i t i s and o s t e o a r t h r i t i s have been measured Pathology.
220
Carbohydrate Chemistry
s p e c t r o p h o t o m e t r i c a l l y as t h e i r A l c i a n b l u e Lowe’s syndrome r e s u l t s i n an u n d e r s u l p h a t i o n o f c h o n d r o i t i n ~ u l p h a t e s . ~ ~ ’ T h i s i s now s h o w n t o be a c o n s e q u e n c e o f a l o w e r l e v e l o f a c t i v e sulphate
(adenosine 3’-phosphate
syndrome
fibroblasts,
caused
pyrophosphatase a c t i v i t y
5’-phosphosulphate) by
i n
an e l e v a t i o n o f
degrading
adenosine
Lowe’s
nucleotide
3’-phosphate
5’-
phosphosulphate. A
low
urinary
e x c r e t i o n o f heparan sulphate i n t h r e e p a t i e n t s
w i t h Lowe’s s y n d r o m e has b e e n o b s e r v e d , a l t h o u g h u r i n a r y e x c r e t i o n o f total
i s o m e r s shows no
g l y c o s a m i n o g l y c a n and c h o n d r o i t i n s u l p h a t e
significant
differences
b e t w e e n Lowe’s
syndrome s u b j e c t s
a n d age-
m a t c h e d c o n t r o l s . 431 A new D,
disease,
tentatively
has been r e c o r d e d . 4 3 2
designated Sanfilippo
It i s
f e a t u r e s o f t h e S a n f i l i p p o syndrome,
c h a r a c t e r i z e d by
disease type the
clinical
excessive e x c r e t i o n o f heparan
s u l p h a t e , a n d t h e i n a b i l i t y t o r e l e a s e i n o r g a n i c s u l p h a t e f r o m 2a c e t a m i d o - 2 - d e o x y - a - g 1 uco sy 1 6 - s u l p h a t e sulphate-derived oligosaccharides.
residues i n heparan
However, t h e r e q u i r e m e n t s f o r
e n z y m a t ic h y d r o 1y s i s o f 2 - a c e t am ido - 2-deox y - g - g 1 uco sy 1 6 - s u l p h a t e linkages are d i f f e r e n t f o r heparan sulphate and k e r a t a n sulphate, when c o m p a r e d w i t h t h o s e p o l y s a c c h a r i d e s f r o m
ordinary
Sanfilippo
syndrome p a t i e n t s . Glycosaminoglycan ne p h r o t i c
s y n drom e has
excretion
i n
urine
been i n ves t i g a t e d.433
from
patients
with
p r ot e i n-asso c i a t e d
A
l o w - c h a r g e f o r m o f c h o n d r o i t i n s u l p h a t e w h i c h i s n o t e x c r e t e d by n o r m a l s u b j e c t s was i d e n t i f i e d . C h o n d r o i t i n 6-sulphate w i t h low sulphate content i s e x c r e t e d i n t h e u r i n e o f p a t i e n t s w i t h an u n u s u a l f o r m o f s p o n d y l o e p i p h y s e a l d y ~ p l a s i a . ~ The ~ ~s e r a o f t h e s e p a t i e n t s show a l o w a c t i v i t y o f 3’phosphoadenos i ne 5’-phosphos u l phat e sulphotransferase, w h i l e the sulphate
i s
a much
urinary
better acceptor
chond r o it in
s u l p h a te
undersulphated chondroit i n of
sulphate
than
standard
c h o n d r o i t i n s u l p h a t e o n i n c u b a t i o n w it h 3 ’-p h o s p h o a d e no s i n e 5 ’pho sphos u l p h a t e a n d norm a1 s u l p h o t r a n s f e r a s e s . P r e c i p i t a t i o n w i t h c e t y l p y r i d i n i u m c h l o r i d e f o l l o w e d by u r o n i c
acid
assay
has
been
used
to
study
urinary
glycosaminoglycan
e x c r e t i o n i n normal subjects and calcium-stone
formers.435
No
s i g n i f i c a n t d i f f e r e n c e i n d a i l y l e v e l s o f g l y c o s a m i n o g l y c a n s was detected. P r o t e o g l y c a n s p r e s e n t i n norm a 1 and a t h e r o s c l e r o t i c human a o r t a s have been i s o l a t e d a n d p a r t i a l l y ~ h a r a c t e r i z e d . ~ ~ ~
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
22 1
The g l y c o s a m i n o g l y c a n c o m p o s i t i o n o f s a m p l e s o f l i v e r f r o m p a t i e n t s w i t h a l c o h o l i c c i r r h o s i s has been compared w i t h t h a t o f no rmal l i v e r . 4 3 7 I n c r e a s e d l e v e l s o f h y d r o x y - l - p r o 1 i n e , h y a l u r o n i c a c i d , h e p a r a n s u l p h a t e , c h o n d r o i t i n 4- a n d 6 - s u l p h a t e s , a n d d e r m a t a n s u l p h a t e were d e t e c t e d i n t h e c i r r h o t i c l i v e r s a m p l e s . Age-related changes i n hydrodynamic s i z e of the proteoglycan monomers o f human c a r t i l a g e a n d o f t h e s p e c i f i c { 3 5 S ) - l a b e l l i n g o f t h e i r chondroi t i n s u l p h a t e and k e r a t a n s u l p h a t e c h a i n s have been de s c r i be d. 4 3 I n o r d e r t o e x c l u d e de g e n e r a t i o n ( 0s t e o a r t h r o t i c ) processes that i n t e r f e r e w i t h age-specific changes, c a r t i l a g e of the p r o c e s s u s x y p h o i d was i n v e s t i g a t e d . A u t o l o g o u s h u m a n k n e e a n d s h o u l d e r c a r t i l a g e show a g e - r e l a t e d changes: t h e p r o t e o g l y c a n c o n t e n t decreases i n c o n t e n t a n d p r o t e o g l y c a n s u b u n i t s decrease i n s i z e . 4 3 9 In contrast, the p r o p o r t i o n s of keratan s u l p h a t e t o c h o n d r o i t i n s u l p h a t e , p r o t e i n t o glycosaminoglycan, and c h o n d r o i t i n 6 - s u l p h a t e t o c h o n d r o i t i n 4s u l p h a t e a l l i n c r e a s e w i t h age. The s t r u c t u r e s o f t h e p r o t e o g l y c a n s o f a r t i c u l a r c a r t i l a g e s o f b o v i n e f o e t u s e s a t v a r i o u s s t a g e s of development have been compared During f o e t a l l i f e , with those of the c a l f and a d u l t a r t i c u l a r c a r t i l a g e proteoglycans are rich i n c h o n d r o i t i n s u l p h a t e a n d c o n t a i n a t a l l a g e s some k e r a t a n s u l p h a t e a n d 2 - g l y c o s i d i c a l l y linked oligosaccharides i n both a keratan-rich region and i n the remainder o f the polysaccharide attachment region. An a n a l y s i s o f c h a n g e s o f s u l p h a t e d g l y c o s a m i n o g l y c a n s a n d related glycosidases during f o e t a l development shows t h a t c h o n d r o i t i n 6 - s u l p h a t e i n c r e a s e s i n c o n c e n t r a t i o n up t o f i f t y d a y s o f f o e t a l d e v e l o p m e n t , and t h e n decreases p r o g r e s s i v e l y u n t i l i t s complete disappearance i n a d u l t tissues.441 The l e v e l s o f h e p a r a t i n s u l p h a t e a n d d e r m a t a n s u l p h a t e do n o t c h a n g e d u r i n g t h i s p e r i o d . The m a j o r p r o t e o g l y c a n s i n t h e a r t i c u l a r c a r t i l a g e o f f o e t a l , calf, and a d u l t a n i m a l s d i f f e r i n t h e c o n t e n t , t y p e s o f s t r u c t u r e o f the chondroitin sulphate, keratan sulphate, and oligosaccharide constituents.442 These changes i n s t r u c t u r e r e f l e c t t h e g r o s s agerelated changes i n t h e chemical composition of t h e t i s s u e . Proteoglycans of t h e nucleus pulposus and annulus f i b r o s u s o f normal a n d d e g e n e r a t e i n v e r t e b r a l d i s c s have been examined.443 A l a r g e p r o p o r t i o n o f t h e proteoglycans found i n d i s c undergo a degree of d e g r a d a t i o n i n t h e h y a l u r o n a t e - b i n d i n g r e g i o n i n d e g e n e r a t e tissue. Glycosaminoglycans o f c u l t u r e d human-foetal melanocytes have
222 been
Carbohydrate Chemistry compared with
cells.444
those
p r o d u c e d by
c u l t u r e d human melanoma
By m a n i p u l a t i n g c e l l s u r f a c e s w i t h enzymes s p e c i f i c f o r
c l e a v i n g glycosaminoglycans from c e l l s , functional
i t i s p o s s i b l e t o change t h e impairment
r e l a t i o n s h i p s such a s adhesion and adhesion
among b o n e - m a r r o w
fibroblast-like
fi
cells cultured
vitro,
mature
l e u c o c y t e s and b l a s t s f r o m n o r m a l marrow and p e r i p h e r a l b l o o d o f leukaem i a p a t i e n t s . 4 4 5 The a b s e n c e o f p r o t e o g l y c a n c o r e p r o t e i n i n c a r t i l a g e f r o m omd/cmd
( c a r t i l a g e m a t r i x d e f i c i e n c y ) mouse446 a n d i n c a r t i l a g e
m u t a n t n a n ~ m - e l i ah ~a s~ ~been r e p o r t e d .
Cell and T i s s u e Glycoproteins
8
Glycoproteins from a d u l t - r a t
b r a i n s y n a p t i c v e s i c l e s h a v e been
f r a c t i o n a t e d by s e q u e n t i a l a f f i n i t y
chromatography i n t h e
presence
o f sodium d o d e c y l s u l p h a t e o n f o u r d i f f e r e n t i m m o b i l i z e d l e ~ t i n s . ~ ~ Nine f r a c t i o n s c o n t a i n i n g o n l y g l y c o p r o t e i n s and d i f f e r i n g i n t h e i r t e r m i n a l s u g a r s were com p o s i t i o n
and
s e p a r a t e d and a n a l y s e d f o r t h e i r c a r b o h y d r a t e
e l e c t rophor et i c
profiles.
considerable
A
h e t e r o g e n e i t y o f t h e g l y c o p r o t e i n p o p u l a t i o n was o b s e r v e d w h i c h c a n n o t be e x p l a i n e d s o l e l y by t h e m i c r o h e t e r o g e n e i t y o f t h e
glycans
o f the synaptic vesicle glycoproteins. A g r o u p of
s t r u c t u r a l l y r e l a t e d g l y c o p r o t e i n s f r o m human b r a i n
and f i b r o b l a s t s ,
which account f o r a l l t h e Thy-1
species cross-
r e a c t i v e antigenic a c t i v i t y present i n these tissues, purified.449
has
been
The i s o l a t e d p r o d u c t s a r e c l o s e l y
r e l a t e d t o each
o t h e r a n d c o u l d be t h e p r o d u c t o f a s i n g l e gene.
T h e y s h o w many
properties antigen.
i n common w i t h
p u r i f i e d forms o f
Human b r a i n g l y c o p r o t e i n s d e p l e t e d of Thy-1
been used f o r
monoclonal antibody
i n t e r a c t s with a determinant (cerebral,
the
production.450
rodent
Thy-1
a n t i g e n have The a n t i b o d y
p r e s e n t o n a l l human b r a i n s u b r e g i o n s
c o r t i c a l grey matter,
white matter,
caudate,
thalmus,
d e n t a t e n u c l e u s , putamen,and c e r e b e l l a r c o r t e x ) b u t i s a b s e n t f r o m a wide range o f o t h e r t i s s u e s (heart,
liver,
spleen).
The d e t e r m i n a n t
i s c o n s e r v e d i n mammalian e v o l u t i o n , a s t h e b r a i n s o f r a t a n d dog have amounts e q u a l t o t h a t f o u n d i n human b r a i n . Some b i o c h e m i c a l c h a r a c t e r i s t i c s , compositions,
examined and compared w i t h s i m i l a r
.
m o us e 45 1
i n c l u d i n g the carbohydrate
o f b r a i n T h y - 1 g l y c o p r o t e i n o f dog a n d man h a v e been c h a r a c t e r i s t i c s i n r a t and
223
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides A new
caprine n e u r o c e r v i c a l storage disease i n r e l a t e d Nubian
g o a t s h a s been c h a r a c t e r i z e d by t h e p r e s e n c e i n b r a i n t i s s u e o f t h e t r i s a c c h a r i d e O-B-g-mannopy r a n 0 sy 1-( 1+4) - 2 - a c e t a m i d o - 2 - d e o x y - Q--
(B-E-
g l u c o p y r a n o s y 1- ( 1 + 4 ) - 2 - a c e t a m id o - 2 - d e o x y - g - g l u c o s e
m a n n o s y l c h i t o b i o s e ) .452 The a c c u m u l a t i o n o f t h e t r i s a c c h a r i d e s u g g e s t s t h e p o s s i b i l i t y o f a g e n e t i c d e f e c t i n B-D=-mannosidase i n the
catabolic
pathway
for
d e f i c i e n c y o f B+-mannosidase
N-linked
complex
glycopeptides.
A
a c t i v i t y was d e m o n s t r a t e d i n a number
o f t i s s u e s f r o m t h e a f f e c t e d goats.453 The l e v e l s o f N - a c e t y l n e u r a m i n i c
a c i d i n w a t e r - s o l u b l e and
membrane f r a c t i o n s o f v a r i o u s r a t b r a i n c o r t e x t i s s u e s have been
.
m ea s u r e d 45 D e v e l o p m e n t a l changes i n t h e b i o s y n t h e s i s a n d c o n c e n t r a t i o n o f i n d i v i d u a l i-f u c o s y l - a n d !-ace t y l n e u r a m i n o s y l - c o n t a i n i n g glycoproteins
associated w i t h
s y n a p t i c membranes and s y n a p t i c
j u n c t i o n s have been r e p o r t e d . 4 5 5 The i n c o r p o r a t i o n o f L - f u c o s e
i n t o s y n a p t i c m i t o c h o n d r i a 1 and
p l a s m a-m em b r a n e g l y c o p r o t e i n s h a s b e e n r e c o r de d .456 mitochondria
s i t u have a g r e a t e r c a p a c i t y
Sy n a p t o som a 1
for the incorporation
o f &-fucose i n t o p r o t e i n t h a n f r e e mitochondria. S y n a p t i c membranes from
sheep b r a i n c o r t e x
incorporate
i n o r g a n i c s u l p h a t e i n t o a range o f g l y c o p r o t e i n s (mol.
lo4
-
1.6
wts.
1.6
x
1 0 ~ 1 . ~ 5 ~
E v i d e n c e h a s been p r e s e n t e d f o r
t h e i n c o r p o r a t i o n o f C-fucose
a n d t h e i d e n t i f i c a t i o n o f I - f u c o s y l g l y c o p r o t e i n s i n sheep c o r t e x sy na p t o som e s .458 The c a r b o h y d r a t e s t r u c t u r e o f g l y c o p r o t e i n s a s s o c i a t e d w i t h r a t c e n t r a l - n e r vo u s - s y s t em my e l i n i s de v e l a pme n t a l l y Although
r e g u l ated.459
t h e p r e c i s e a s s o c i a t i o n o f t h e s e membrane c o m p o n e n t s w i t h
t h e axolemma,
t h e o l i g o d e n d r o g l i a l p l a s m a membrane, o r m y e l i n has n o t
been d e t e r m i n e d ,
t h e o b s e r v e d changes t h a t o c c u r d u r i n g development
s u p p o r t a p r o b a b l e f u n c t i o n a l r o l e f o r many o f them i n t h e m e c h a n i s m o f m y e l i n o g e n e s i s. F l u o r e s c e n t - l a b e l l e d l e c t i n s have been u s e d t o examine t h e g l y c o p r o t e i n compo s i t i o n o f
sub fraction^.^^^
The
r a b b i t p e r i p h e r a l - n e r vo u s - s y s t em my e l i n
g l y c o p r o t e i n composition o f two major
f r a c t i o n s i s more complex t h a n p r e v i o u s l y r e p o r t e d . The s e q u e n c e o f e v e n t s i n t h e d e s t r u c t i o n o f m y e l i n p r o t e i n s and g l y c o p r o t e i n s i n experimental demyelination o f the r a b b i t o p t i c n e r v e h a s been r e p o r t e d . 461 The
presence
of
myelin-associated
glycoprotein
i n the
224
Carbohydrate Chemistry
p e r i p h e r a l nervous system of rats has been demonstrated.462 The presence of t h i s g l y c o p r o t e i n i n t h e periaxonal region o f both peripheral and c e n t r a l m y e l i n s h e a t h s i s c o n s i s t e n t w i t h a similar i n v o l v e m e n t o f the g l y c o p r o t e i n i n axon-sheath c e l l i n t e r a c t i o n s i n t h e p e r i p h e r a l n e r v o u s system a n d t h e c e n t r a l n e r v o u s s y s t e m . Rat brain myelin contains several major glycoproteins i n addition t o t h o s e ( m o l . w t . 1 . 2 x l o 5 a n d 2.7 x l o 4 ) a l r e a d y ~ h a r a c t e r i z e d . ~ ~ ~ F o u r a d d i t i o n a l m a j o r g l y c o p r o t e i n s a n d many m i n o r g l y c o p r o t e i n s a r e r e v e a l e d a f t e r t r e a t m e n t o f i s o l a t e d m y e l i n membrane w i t h n e u r a m i n i dase b e f o r e ox i d a t i o n w i t h g a l a c t o s e ox i da se a n d subsequent r e d u c t i o n w i t h sodium b o r o t r i t i d e . A glycoprotein s o l u b l e i n o r g a n i c s o l v e n t s i s o l a t e d from r a t m y e l i n has b e e n p a r t i a l l y purified and characterized as a sulphated I-fucosyl g 1y c o p ro t e i n .464 The m a j o r g l y c o p r o t e i n (PO) o f c h i c k s c i a t i c - n e r v e m y e l i n h a s been p u r i f i e d and p a r t i a l l y characterized.465 The amino a c i d c o m p o s i t i o n i n d i c a t e s a d e f i n i t e s p e c i e s d i f f e r e n c e when compared w i t h mammalian PO g l y c o p r o t e i n s . Dopamine B-hydroxylase ( E C 1.14.2.1) i s a tetrameric g l y c o p r o t e i n w i t h s u b u n i t s o f m o l . w t . 7.5 x 104.466 I t i s p r e s e n t i n catecholamine s t o r a g e v e s i c l e s o f both a d r e n a l m e d u l l a and sympathetic nerve terminals. The p r e s e n c e o f n e u r a m i n i c a c i d residues on oligosaccharide chains g r e a t l y increases t h e resistance of t h e e n z y m e t o t r y p t i c p r o t e o l y s i s . An a g e - r e l a t e d i n c r e a s e o f n o n - e n z y m a t i c g l y c o s y l a t i o n i n It appears to normal bovine l e n s c r y s t a l l i n s has been observed.467 be a g e n e r a l c h a r a c t e r i s t i c o f t h e a g e i n g p r o c e s s . Bovine thyro g l o b u l i n has been s u b j e c t e d t o s e q u e n t i a l t r e a t m e n t w i t h glycohydrolases i n order t o define t h e components o f the oligosaccharide chain which are important i n binding the g l y c o p r o t e i n t o bovine t h y r o i d membranes.468 Preparations of a s i a l o g a l a c t o t h y r o g l o b u l i n e x h i b i t t h e best b i n d i n g , s u g g e s t i n g t h a t exposed 2-acetamido-2-deoxy-~-glucose r e s i d u e s o n t h e B c a r b o h y d r a t e chain of t h e hormone are i m p o r t a n t i n t h e i n t e r a c t i o n w i t h t h e t h y r o i d membranes. i - T y r o s i n e r e s i d u e s p l a y an i m p o r t a n t r o l e i n The t h y r o g l o b u l i n i n t e r a c t i o n s w i t h t h y r o i d membranes.469 attachment of carbohydrate t o thyroglobulin involves an intermediate (0-G 1 ce3 -p - M a n e g -p- G 1 ceNA c2- py r o pho s p h o r y 1d o 1 i c h o 1) a n d i s f o l 1ow e d r a p i d l y by t h e i n i t i a t i o n o f m o d i f y i n g r e a c t i o n s i n c l u d i n g t h e The r e m o v a l of Q - g l u c o s y l - a n d some Q-mannosyl r e s i d u e s . 4 7 0 i n h i b i t i o n o f i n c o r p o r a t i o n o f c a r b o h y d r a t e i n t o t h y r o g l o b u l i n by
n-
225
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides t u n i carny c i n has been r e p o r t e d .471 Parathyroid secretory protein c o n t a i n i n g 18% c a r b o y d r a t e )
(mol.
wt.
6.7
has been p u r i f i e d f r o m
x
lo4
and
extracts of
b o v i n e p a r a t h y r o i d glands.472 p r o t e i n I, a g l y c o p r o t e i n ( m o l .
Secretory unknown
biological activity,
c h a r a c t e r i z e d from ca r bo h y d r a t e
.
been
wt.
7.0
x 104) o f
isolated
and
partially
b o v i n e p a r a t h y r o i d glands.473
The ! - t e r m i n a l ACTH/LPH
has
fragment
precursor,
i s
a
of
porcine
major
I t c o n t a i n s 2.6%
pro-opiomelanocortin,
product
of
the
m a t u r a t i o n and
i s
g l y ~ o s y l a t e d . ~The ~ ~ a m i n o a c i d sequence i s h o m o l o g o u s t o s i m i l a r p e p t i d e s i n ox a n d man, L --Thr-45
w i t h two s i t e s o f g l y c o s y l a t i o n probably a t
and I - A s n - 6 5 .
Some
of
the
relationships
between
protein
synthesis,
g l y c o s y l a t i o n , cleavage, and s e c r e t i o n o f pro-ACTH-endorphin
i n mouse
p i t u i t a r y tumour c e l l s h a v e been d e s c r i b e d . 4 7 5 B-D-Glucuronidase gland.476
Two
has
been i s o l a t e d f r o m
oligosaccharide
r e d u c t i v e a 1ka 1i n e h y d r o 1y s i s,
alditols
rat preputial
(16-17),
released
by
have be e n c h a r a c t e r i zed.
P-Man3-a-{ ( a-Q-Mane-Q-GlcQNAc )-Q-Mang) -8 -Q-GlcgNA c-8 -Q-GlcNA c - 0 1 ( 16)
Q -M an^^ -4 -a {Q -M a ng2-a -Q -M an e} -B -Q - G 1CQ NA c - 8 -Q - G 1c NA c- 01 (17) Glycoproteins and pepsinogen a r e a s s o c i a t e d i n t h e secretory granules
of
fundic
epithelial
cells
isolated
from
guinea-pig
stomach.477 I n c o r p o r a t i o n o f monosaccharides i n t o r a t l i v e r f e r r i t i n can o c c u r n o n - e n z y m i c a l l y u n d e r p h y s i o l o g i c a l c o n d i t i o n s o f pH a n d i o n i c strength,
i n a process which i s dependent o n t h e c o n c e n t r a t i o n o f
monosaccharides, temperature.478
the
concentration
of
ferritin,
time,
and
Varying degrees o f g l y c o s y l a t i o n m i g h t account f o r
t h e occurrence o f i s o f e r r i t i n s . The s t r u c t u r e a n d d y n a m i c b e h a v i o u r o f t h e o l i g o s a c c h a r i d e s i d e c h a i n o f b o v i n e p a n c r e a t i c r i b o n u c l e a s e B have b e e n s t u d i e d b y 13C n.m .r.
spectroscopy.479
The c o n c l u s i o n s a b o u t t h e p r i m a r y s t r u c t u r e
of t h e c a r b o h y d r a t e s i d e c h a i n s a r e c o n s i s t e n t w i t h r e s u l t s b a s e d o n c h e m i c a l methods. Chem i c a l a n a l y s e s ,
t o g e t h e r w i t h h i s t o c h em i c a l a s s e s s m e n t s ,
226
Carbohydrate Chemistry
have been o b t a i n e d f o r specimens of adenocarcinoma o f t h e colon and histologically normal c o l o n i c epithelium.480 In epithelial g l y c o p r o t e i n s o f normal t i s s u e , t h e m a j o r i t y of t h e n e u r a m i n i c a c i d r e s i d u e s c o n t a i n a s i d e - c h a i n g - a c y l s u b s t i t u e n t a t C - 8 , whereas s i d e - c h a i n s u b s t i t u t i o n o f t h e n e u r a m i n i c a c i d s of t u m o u r Differences i n the glycoproteins i s markedly reduced. s u s c e p t i b i l i t y t o n e u r a m i n i d a s e t r e a t m e n t were a l s o noted between t h e two s o u r c e s of g l y c o p r o t e i n s . Human c o l o n t u m o u r a n t i g e n w h i c h i s r e c o g n i z e d b y l e u c o c y t e adherence i n h i b i t i o n assay i s u n r e l a t e d t o c a r c i n o e m b r y o n i c a n t i g e n (CEA).481 The f o r m e r a n t i g e n , b u t n o t C E A , b i n d s s p e c i f i c a l l y t o immobilized human B2-microglobulin a n t i b o d i e s . B l o o d - g r o u p d e t e r m i n a n t s h a v e b e e n f o u n d on f i v e C E A p r e p a r a t i o n s . 4 8 2 One of t h e p r e p a r a t i o n s h a s an A 1 d e t e r m i n a n t , a n o t h e r h a s a B d e t e r m i n a n t , and a l l h a v e H , L e a , Leb,and MN b l o o d group d e t e r m i n a n t s . The f i n d i n g s a r e c o n s i s t e n t w i t h t h e i d e a t h a t incomplete o r unexpected glycosylation p a t t e r n s occur i n g l y c o p r o t e i n s produced by tumour c e l l s . Since antibodies d i r e c t e d a g a i n s t blood-group s u b s t a n c e s c r o s s - r e a c t w i t h c a r b o h y d r a t e d e t e r m i n a n t s on C E A , c l i n i c a l d e t e r m i n a t i o n s of C E A o r anti-CEA l e v e l s i n serum may be a d v e r s e l y a f f e c t e d . Concurrent measurements o f c a r c i n o e m b r y o n i c a n t i g e n , !-glucose phosphate isomerase, yg l u t a m y l t r a n s f e r a s e , and l a c t a t e dehydrogenase i n m a l i g n a n t , normal a d u l t , and f e t a l colon t i s s u e s g i v e r e s u l t s t h a t a r e c o n s i s t e n t w i t h e a r l i e r o b s e r v a t i o n s t h a t t h e a c t i v i t i e s of t h e s e m a r k e r s a r e i n c r e a s e d s i g n i f i c a n t l y i n t h e serum of p a t i e n t s w i t h m e t a s t a t i c colon cancer.483 Carcinoembryonic a n t i g e n - r e l a t e d a n t i g e n s have been p u r i f i e d and c h a r a c t e r i z e d i n normal a d u l t f a e c e s . 4 8 4 Glycoproteins metabolically l a b e l l e d w i t h 2-amino-Z-deoxy-g{ 3H}-glucose and i s o l a t e d from human r e n a l cancer and normal kidney e p i t h e l i a l c e l l c u l t u r e s h a v e been c o m p a r e d by t w o - d i m e n s i o n a l p o l y a c r y l a m i d e g e l e l e c t r o p h ~ r e s i s . ~Although ~~ o v e r a l l p r o f i l e s of g l y c o p r o t e i n s from tumours and normal c e l l s were e x t r e m e l y s i m i l a r , s e v e r a l s i g n i f i c a n t d i f f e r e n c e s were observed t h a t were c o n s i s t e n t even among a l l o g e n e i c comparisons. A g l y c o p r o t e i n i n h i b i t o r (mol. w t . 1 . 3 3 x l o 4 ) of c a l c i u m o x a l a t e m o n o h y d r a t e c r y s t a l g r o w t h h a s been i s o l a t e d f r o m human kidney t i s s u e c u l t u r e medium.486 T h i s g l y c o p r o t e i n c o n t a i n s 39.4% c a r b o h y d r a t e , c o n s i s t i n g of r e s i d u e s of I=-fucose, Q - g l u c o s e , Eg a l a c t o s e , and neuraminic a c i d . Studies a t the ultrastructural level indicate that the
227
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
g l y c o p r o t e i n GP-2 i s b o t h a c e l l m e m b r a n e a n d a b a s e m e n t m e m b r a n e p r o t e i n i n mouse k i d n e y c e l l s . 4 8 7 L - F u c o s e c a n be i n c o r p o r a t e d i n t o a s e r i e s o f l o w - m o l e c u l a r w e i g h t amino a c i d I - f u c o s i d e s i n normal r a t kidney cells.488 The m o n o s a c c h a r i d e sequence a n d p o s i t i o n o f t h e l i n k a g e o f one o f t h e fucosides
(18) and t h e anomeric c o n f i g u r a t i o n s o f
linkages o f three other L-fucosides
(19-21)
the
I-
carbohydrate
h a v e been i d e n t i f i e d . 4 8 9
T e n t a t i v e evidence i s given t o suggest t h a t these unusual glycosides a r e d e r i v e d f r o m an i - f u c o p r o t e i n .
a -L-Fucp-l+L-Thr (19) a --FucpI 1+I-Se r (20) B - Q - G l c e ( 1+)- a - & - F u c p - l + i - T h r (21) A p a r t i c u l a t e f o l a t e - b i n d i n g p r o t e i n f r o m human p l a c e n t a h a s
been is o l a t e d a n d c h a r a c t e r i z e d.490
I m m u n o f l uo r e s c e n t s t u d i e s u s i n g
antibody d i r e c t e d against t h i s g l y c o p r o t e i n demonstrate t h a t an i m m u n o l o g i c a l l y s i m i l a r p r o t e i n i s p r e s e n t o n t h e p l a s m a membrane o f human e r y t h r o c y t e s ,
s u g g e s t i n g t h a t i t may f u n c t i o n a s a f o l a t e
receptor. Some p h y s i c o c h e m i c a l
properties o f
an a c i d i c g l y c o p r o t e i n
s e c r e t e d by r a t e p i d i d y m a l c e l l s h a v e b e e n ~ h a r a c t e r i z e d . ~ ’ ~The g-galactose,
2-
unexpectedly,
Q-
From amino a c i d sequence s t u d i e s o f g l y c o s y l a t e d component
C3
c a r b o h y d r a t e m o i e t y c o n t a i n s r e s i d u e s o f Q-mannose, a c e t a m i do-2-deox y-1- g l ucose,
neuram in i c a c i d , and,
g l uco se. of
r a t p r o s t a t i c b i n d i n g p r o t e i n , a g l y c o p e p t i d e c o n t a i n i n g 77 amino
acids
has
been c h a r a c t e r i z e d . 4 9 2
An o l i g o s a c c h a r i d e c h a i n i s
a t t a c h e d t o t h e p e p t i d e by a n N - g l y c o s i d i c b o n d t o I - A s n - 1 7 . Bovine c a r t i l a g e m a t r i x g l y c o p r o t e i n (mol. co n t a i n s
three
s u b u n i t s.4 9 3
G l y c o p e p t i de s
wt.
1.48
isolated
x
lo5)
after
p r o t e o l y t i c d i g e s t i o n a r e heterogeneous o n g e l chromatography and c o n t a i n Q-manno s y l a n d 2 - a c e t a m i d o - 2 - d e o x y - a - g l uco s y l r e s i d u e s ,
228
Carbohydrate Chemistry
p r o b a b l y d e r i v e d from o l i g o s a c c h a r i d e s o f the high-Q-mannose t y p e b o u n d t o p r o t e i n by 2 - g l y c o s i d i c l i n k a g e s . Tw o n o n - c o l l a g e n o us i n s o l u b l e s t r u c t u r a l g l y c o p r o t e i n f r a c t i o n s from u n c a l c i f i e d canine-puppy r i b c a r t i l a g e a r e both a s s o c i a t e d w i t h lipid.494 T h e c o m p l e x e s f o r m e d a r e s t a b l e e n o u g h t o be o f p o s s i b l e s i g n i f i c a n c e i n t h e metabolism of connective t i s s u e s . An i n t r i n s i c g l y c o p r o t e i n ( m o l . w t . 5.3 x l o 4 ) o f t h e s a r c o p l a s m i c r e t i c u l u m of r a b b i t s k e l e t a l m u s c l e has been separated f r o m t h e Ca2+ + Mg2+ a d e n o s i n e t r i p h ~ s p h a t a s e . ~ ' ~T h e g l y c o p r o t e i n c o n t a i n s 48% n o n p o l a r a m i n o a c i d s i n a d d i t i o n t o t w o g l y c a n c h a i n s , each c o n t a i n i n g n i n e Q-mannosyl a n d t w o 2 - a c e t a m i d o - Z - d e o x y - Qg l uco s y 1 r e s i d ue s A s u l p h a t e d g l y c o p r o t e i n , e n t a c t i n ( m o l . w t . 1.58 x l o 5 ) , h a s b e e n i s o l a t e d f r o m a n e x t r a c e l l u l a r b a s e m e n t mem b r a n e - l i k e m a t r i x e l a b o r a t e d i n c e l l c u l t u r e by a m o u s e e n d o d e r m a l c e l l l i n e . 4 9 6 I m m u n o e l e c t r o n m i c r o s c o p i c s t u d i e s show t h a t t h e m o u s e k i d n e y antigen is predominantly localized a t the surface o f e p i t h e l i a l c e l l s o f t u b u l e s a n d g l o m e r u l i a d j a c e n t t o t h e b a s e m e n t membrane. Laminin, p r e s e n t i n t h e l a m i n a l u c i d a o f t h e basement membrane, i s c l e a v e d by t h r o m b i n a n d p l a ~ m i n . ~ ' F~r a g m e n t s c l e a v e d by t h e enzymes have been assessed f o r t h e i r a b i l i t y t o m e d i a t e b i n d i n g o f e p i t h e l i a l c e l l s t o t y p e IV c o l l a g e n . P r o t e o l y s i s o f laminin from human p l a c e n t a l and r e n a l b a s e m e n t membranes has been a c h i e v e d w i t h The b i n d i n g o f l a m i n i n t o g l y c o s a m i n o g l y c a n s pepsin.498 (aggregating and non-aggregating subsets o f heparan sulphates and dermatan s u l p h a t e s a s well a s h e p a r i n , c h o n d r o i t i n s u l p h a t e s , and h y a l u r o n i c a c i d ) h a s b e e n s t u d i e d by a f f i n i t y c h r ~ m a t o g r a p h y . ~ ~ ' The b i n d i n g i s s p e c i f i c a n d o c c u r s o n a s i n g l e b i n d i n g s i t e o f laminin. S t u d i e s o n t h e b i o s y n t h e s i s o f l a m i n i n by m u r i n e p a r i e t a l endoderm cells have been r e p ~ r t e d . ~ " Evidence is given f o r the p r e s e n c e o f !-linked oligosaccharide side chains on a l l four p o l y p e p t i d e com po n e n t s o f t h i s g l y c o p r o t e i n . Two e x t r a c e l l u l a r m a t r i x g l y c o p r o t e i n s , s i m i l a r t o l a m i n i n , h a v e been i s o l a t e d from c u l t u r e s o f a mouse e n d o d e r m a l c e l l l i n e . 5 0 1 A s t r u c t u r a l g l y c o p r o t e i n from bovine ligamentum muchae exhibits a dual amine oxidase activity.502 If exposed t o copper i o n s t h e g l y c o p r o t e i n i s c a p a b l e o f f o r m i n g f i b r i l s a b o u t 11 nm i n diameter a n d o f t h e same s i z e a s t h e m i c r o f i b r i l s a s s o c i a t e d m a i n l y with e l a s t i c f i b r e s i n both embryonic and mature t i s s u e s . M o d i f i c a t i o n s i n the l e v e l s of glycosylamines and c r o s s l i n k s observed i n glomerular basement membranes i n s t r e p t o z o t o c i n d i a b e t i c
.
229
5: Glycoproteins, Glycopeptides, Proteoglycans, dnd Animal Polysaccharides
r a t s have been reported.503 Enhanced g - g l y c o s y l a t i o n o f in t e r m o l e c u l a r c r o s s l i n k s a n d i n c r e a s e d f o r m a t i o n o f g l y co s y l am ine c o u l d a f f e c t t h e p h y s i o c o c h e m i c a l p r o p e r t i e s o f t h e membrane. A
glycoprotein
endothelial
(mol.
wt.
1.9
c e l l s i n culture.504
x
lo5)
i s s e c r e t e d by
Immunological
aortic
and s t r u c t u r a l
studies i n d i c a t e t h a t the glycoprotein i s e i t h e r i d e n t i c a l t o o r c l o s e l y r e l a t e d t o thrombospondin,
which i s contained i n p l a t e l e t
granules and r e l e a s e d i n response t o thrombin-induced aggregation. A n t i b o d i e s t o human s e r u m a m y l o i d P b i n d i m m u n o s p e c i f i c a l l y t o the p e r i p h e r a l m i c r o f i b r i l l a r m a n t l e o f e l a s t i c f i b r e s i n s k i n and blood vessels o f normal adults.505 A s i a l o g l y c o p r o t e i n f r a c t i o n f r o m h e r r i n g eggs c o n t a i n s b o t h
a 1 k a l i - l a b i l e a n d a 1k a l i - s t a b l e c a r bohy d r a t e u n i t s .506
Each p e p t i de
c h a i n has an average o f f o u r o l i g o s a c c h a r i d e chains 2 - g l y c o s i d i c a l l y linked r e s i due s A
via
.
2-acetamido-2-deox
probable
structure
y-a- g a l a c t o s y l o f
the
glycan
and
L- t h r e o n i n e
portion
o f
a
s i a l o g l y c o p e p t i d e f r a c t i o n i s o l a t e d f r o m t h e s k i n o f t h e f i s h Labeo r o h i t a h a s been s u g g e s t e d f r o m m e t h y l a t i o n a n a l y s i s and e n z y m i c a n a 1y si s da t a. O7 The p r o t e i n c o m p o s i t i o n o f i s o l a t e d human p l a t e l e t a - g r a n u l e s has
been cha r a ~ t e r i z e d . ~ ' ~C r o s s e d a f fi n it y
i m m u n o e l e c tr o p h o r e s i s
u s i n g l e c t i n s i d e n t i f i e d a t l e a s t seven g l y c o p r o t e i n s , a p p e a r e d t o be s i a l o g l y c o p r o t e i n s . o r i g i n of
The a - g r a n u l e
s i x o f which
component i s t h e
t h e i r enhanced h a e m a g g l u t i n a t i o n a c t i v i t y . 5 0 9
insufficiency
of
the platelet-bound
An
l e c t i n may be t h e cause o f t h e
i n a b i l i t y o f gray p l a t e l e t s t o aggregate n o r m a l l y i n response t o thrombin. T h r o m b o s p o n d i n i s one o f t h e g l y c o p r o t e i n s r e l e a s e d f r o m human A hydrodynamic model o f
b l o o d p l a t e l e t s i n r e s p o n s e t o thrombin.510 thrombospondin i s proposed from molecular weight,
physical
s o l u t i o n measurements o f
p a r t i a l s p e c i f i c volume,
sedimentation coefficient,
and i n t r i n s i c v i s c o s i t y . P l a t e l e t g l y c o p r o t e i n s have been shown t o be u s e f u l p h e n o t y p i c markers
for
cells
o f m e g a k a r y o c y t e lineage.511
Immunofluorescent
s t a i n i n g w i t h an a n t i s e r u m t o h i g h l y p u r i f i e d p l a t e l e t g l y c o p r o t e i n s e x c l u s i v e l y l a b e l s human m e g a k a r y o c y t e s , p l a t e l e t s , a n d a p o p u l a t i o n o f s m a l l human bone-marrow m o n o n u c l e a r c e l l s . Differences
b e t w e e n human l e u c o c y t e a n d f i b r o b l a s t i n t e r f e r o n s
have been reviewed.512 dealing
w i t h
the
A book on i n t e r f e r o n i n c l u d e s a chapter
p u r i f i c a t i o n
and
characterization
of
230
Carbohydrate Chemistry
.
in t e r f e r o n s 5 1 3 Human f i b r o b l a s t
i n t e r f e r o n h a s been p u r i f i e d
chromatography,514
as
well
as
by
S e p h a r ~ s e ~ .E ~ v i d~ e~n c e f o r t h e e x i s t e n c e o f dimer
(mol.wt.
reported. chelate
A
x
4.0
lo4>
and a f f i n i t y
on
Blue
t h i s i n t e r f e r o n as a
a s w e l l a s a monomer
com b i n a t i o n o f h y d r o p h o b i c
chromatography,
by z i n c - c h e l a t e
chromatography (2.0
lo4)
x
chromatography,
chromatography
i s
copper on
Blue
Sepharose* h a s been used i n t h e p u r i f i c a t i o n o f human l y m p h o b l a s t o i d i n t e r f e r o n .516 Size
characteristics
of
human
leucocyte
interferon
r e d u c i n g an d no n- r e d u c i n g co n d i t i o n s ha ve been de t e r m ine d
. l7
under
M e t a b o l i c c a r b o h y d r a t e l a b e l l i n g i n t h e mouse system shows t h a t n o t o n l y m i t o g e n - a c t i v a t e d T a n d B l y m p h o c y t e s , b u t some o f t h e i r subpopulations
can be
d i s t i n g u i s h e d by
display s p e c i f i c sets o f glycoproteins, metabolic
specific glycoprotein
Human l y m pho cy t e s u b p o p u l a t i o n s a 1 so
l a b e l l i n g p a t t e r n s .518 ,519 by
their
carbohydrate
which a r e e a s i l y d e t e c t a b l e
l a b e l l i n g
and
electrophoretic
techniques.520 The b i n d i n g p a t t e r n o f h o r s e r a d i s h p e r o x i d a s e - c o n j u g a t e d p e a n u t l e c t i n o n s e c t i o n s o f l y m p h o i d t i s s u e f r o m d i f f e r e n t s o u r c e s has been
investigated.521
dependent,
The
binding,
which
i s
highly
species-
i s g r e a t e s t t o lymphocytes i n t h y m i c c o r t e x and g e r m i n a l
c e n t r e s i n man, guinea pig,and
mouse, a n d s h e e p .
Lymphoid t i s s u e from hamster,
r a b b i t d i d n o t r e a c t , w h i l s t i n r a t o n l y weak b i n d i n g
t o c e l l s i n t h e t h y m i c c o r t e x and g e r m i n a l c e n t r e s was d e t e c t e d . The o x i d a t i o n o f human p e r i p h e r a l m o n o n u c l e a r c e l l s w i t h sodium periodate r e s u l t s i n lymphocyte activation.522 of
the
oxidant
i s
accompanied
by
the
The m i t o g e n i c e f f e c t
oxidation
of
membrane-
a s s o c i a t e d neuraminosy 1, ! - g a l a c t o s y 1, and I - f u c o s y l r e s i d u e s . The by
r e c e p t o r - m e d i a t e d p i no cy t o sis o f
Q-mannosyl
and
macrophages has been reviewed.523 L-fucosyl
o r P-mannosyl
by macrophages and
uptake o f
g l y copro t e i n s
2-acetam i d o - 2 - d e o x y - q - g l
ucosyl
terminated
r e s i dues
by
Glycoconj ugates b e a r i n g t e r m i n a l
residues are
r e c o g n i z e d and i n t e x n a l i z e d
t h e same r e c e p t o r t h a t m e d i a t e s p l a s m a c l e a r a n c e lysosomal
glycosidases.524
The macrophage
receptor
h a s a b r o a d s p e c i f i c i t y and i s a b l e t o b i n d v a r i o u s g l y c o c o n j u g a t e s . I n human f i b r o b l a s t s , c e l l - s u r face
receptors
i s
t h e r e c o g n i t i o n o f l y s o s o m a l enzymes by mediated
by
g-manno s y l
6-pho spha t e
residues l o c a t e d on oligosaccharide
c h a i n s o n t h e s e enzymes.525
B o t h c a t h e p s i n D and B-hexosaminidase
c o n t a i n t h e phosphate groups
w h i c h a r e d i e s t e r - l i n ke d t o 2-a ce t a m i do-2-de ox y
-a- g l uco s y l
residues.
I
23 1
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides Developmental
changes i n t h e
g l y c o p r o t e i n s s y n t h e s i z e d by
n o r m a l a n d d y s t r o p h i c s a t e l l i t e mouse c e l l s g r o w n i n c u l t u r e h a v e been
observed.526
glycoproteins
Similar,
appear
to
be
but
m u l t i n uc 1e a t e d m y o l u b e s , b u t
not
identical,
synthesized no t
by
I-fucosylated
exclusively
by
normal
dup 1ic a t i n g m ono n uc 1e a t e d
s a t e l l i t e c e l l s o r by o t h e r n o n - m y o g e n i c c e l l s f r o m b o t h s o u r c e s . Changes i n g l y c o p r o t e i n s (mol.
wts.
1.0
x
lo5
a n d 9.0
x lo4,
r e s p e c t i v e l y ) i n m a l i g n a n t t r a n s f o r m a t i o n have been shown t o
be
d i r e c t l y r e l a t e d t o t h e u p t a k e o f P - g l u c o s e by t h e s e c e l l s . 5 2 7 The
purification
and
partial
structures
of
the
major
g l y c o p e p t i d e s i s o l a t e d f r o m c u l t u r e d human melanoma c e l l s a n d f r o m t h e c u l t u r e m e d i a have been r e p o r t e d . 5 2 8 from
The g l y c o p e p t i d e s o b t a i n e d
t h e h i g h l y t u m o r i g e n i c human melanoma c e l l s a r e m a r k e d l y
d i f f e r e n t f r o m t h o s e o b t a i n e d f r o m n o n t u m o r i g e n i c human f e t a l u v e a l me1ano cy t e s . The p l a s m i n o g e n a c t i v a t o r s e c r e t e d by human melanoma c e l l s i n c u l t u r e i s very s i m i l a r to, activator
found
i n
normal
o r i d e n t i c a l with, tissue,
~ r o k i n a s e . ~ ~ The ’ g l y c o p r o t e i n (rnol. i m m o b i l i z e d c o n c a n a v a l i n A,
wt.
exists
but 7.2
as a
the
i s x
plasminogen
different
lo4),
from
p u r i f i e d using
dimer.
Receptors f o r
D o l i c h o s b i f l o r u s a g g l u t i n i n on e m b r y o n a l c a r c i n o m a c e l l s have been i s o l a t e d a n d s h o w n t o be g l y c o p r o t e i n s ( m o l .
wt.
The l e c t i n does n o t b i n d t o d i f f e r e n t i a t e d c e l l s . the content o f neuraminic acid of
x 1
>7.0
0 ~ 1 . ~ ~
Differences i n
low cancer c e l l s ,
high cancer
c e l l s , a n d n o r m a l mouse l u n g c o u n t e r p a r t s have been observed.531 Normal l u n g c e l l s c o n t a i n h i g h e r neuraminic a c i d l e v e l s per c e l l i n c o m p a r i s o n w i t h t h e t r a n s f o r m e d ones. Three
t y p e s o f mononucleosomes i s o l a t e d f r o m
Ehrlich ascites
n u c l e i have been c h a r a c t e r i z e d . 5 3 2 Specific glycoproteins are a s s o c i a t e d w i t h t w o of t h e m o n o n u c l e o s o m e t y p e s w i t h t h e p r i n c i p a l g l y c o p r o t e i n h a v i n g a n apparent mol. w t . A carbohydrate-rich,
lo4)
water-soluble
o f 1.3
x
lo5.
g l y c o p r o t e i n (mol.
wt.
4.5
x
has been i s o l a t e d f r o m d e l i p i d a t e d l u n g l a v a g e f l u i d f r o m a
patient
with pulmonary
alveolar proteinosis.533
The v e r y
high
c a r b o h y d r a t e c o n t e n t i s made up of r e s i d u e s o f n e u r a m i n i c a c i d ( 2 4 ) ,
-
2 ace t a m ido -2 -de o x y -p - g l uco se
ga 1a c t o se ( 23 1 , 2- ace tam ido - 2 -de o x y --!
( 6 1 , g - g a l a c t o s e ( 1 9 1 , p - m a n n o s e (41, I - f u c o s e (11, a n d ! - g l u c o s e (11, p e r m o l e c u l e o f g l y c o p r o t e i n . U n l i k e a number o f c o l l a g e n -
r e l a t e d g l y c o p r o t e i n s w h i c h have been i s o l a t e d f r o m i n s o l u b l e l u n g c o n t e n t s i n pulmonary
proteinosis,
this
g l y c o p r o t e i n does
co n t a i n hy d r oxy-&- p r o l i n e o r hy d r ox y - k - l y s i n e .
not
Carbohydrate Chemistry
232 c v)
I
Q I
. + I nil
u
I nit I
I
m
m I
n
U
t
4 U
I
9 m
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I L=yI
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m I
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t
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A m
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4
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t
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m
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b
C c m c z
b
cv t
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w
oll
-
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cv3
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5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
233
A g l y c o p r o t e i n (mol. w t . 6 . 2 x l o 4 ) , i s o l a t e d f r o m t h e l u n g l a v a g e m a t e r i a l of p a t i e n t s w i t h a l v e o l a r p r o t e i n o s i s , c o n t a i n s 72 r e s i d u e s of g l y c i n e and 5 r e s i d u e s o f hydroxy-I=-proline per m o l e c u l e , i n a d d i t i o n t o Q-mannose, a - g a l a c t o s e , I - f u c o s e , 2acetamido-2-deoxy-Q-glucose, and n e u r a m i n i c a c i d i n t h e m o l a r r a t i o o f 5:4:1:7:3.534 P a r t i a l amino a c i d sequence a n a l y s i s of p e p t i d e s d e r i v e d from t h e d i g e s t i o n o f t h e g l y c o p r o t e i n i n d i c a t e s t h e p r e s e n c e of a l t e r n a t i n g c o l l a g e n o u s and non-collagenous r e g i o n s i n t h e same p o l y p e p t i d e chain. A human a l v e o l a r g l y c o p r o t e i n r e l e a s e s two major g l y c o p e p t i d e f r a c t i o n s on p r o t e ~ l y s i s . ~From ~ ~ s t r u c t u r a l a n a l y t i c a l studies t h e t e n t a t i v e s t r u c t u r e s (22-23) have been proposed. Human a l - a n t i c h y m o t r y p s i n h a s been p u r i f i e d from human p l e u r a l f l u i d a n d from human serum.536 The g l y c o p r o t e i n (mol. w t . 5.8 x l o 3 ) i s o l a t e d from t h e two s o u r c e s h a s t h e same c h e m i c a l composition. A g l y c o p r o t e i n (mol. w t . 3.6 x l o 3 ) w i t h s i m i l a r p r o p e r t i e s t o c o l l a g e n has been i s o l a t e d from t h e l u n g l a v a g e of normal rabbit.537 B 1ood - gro up - spe c i f i c g l yco pro t e i n s o bt a i n e d from ova r i a n- cy st f l u i d s o f A1 a n d A 2 p e r s o n s h a v e b e e n s u b m i t t e d t o a l k a l i n e bo ro h y d r i de e l i m i n a t i on, de -! ace t y 1a t i on, pa r t i a 1 a c i d h y dro 1y s i s , and s u b s e q u e n t l y r e - i - a ~ e t y l a t i o n . ~The ~ ~ t r i s a c c h a r i d e s (24-26) w e r e c h a r a c t e r i z e d f r o m b o t h t h e A1 a n d A 2 m a t e r i a l s . O l i g o s a c c h a r i d e ( 2 6 ) h a s n o t p r e v i o u s l y been d e t e c t e d i n human g l yco pro t e i n s .
a-Q-GaleWc-( 1 + 3 ) - @ 3 - G a l p (1+3) -Q-GlcNAc (24)
-
a -Q Ga lgNA c- ( 1+3 ) -6 -Q - Ga le- ( 1+4) -Q - G 1 c NA c
(25) a-p-GaleNAc-( 1+3) -B-p-Galp( 1+3)-GalNAc-ol 26)
L u b r i c a t i n g g l y c o p r o t e i n 1 h a s been p r e p a r e d from b o v i n e synovial fluid.539 E l e c t r o n - m i c r o s c o p i c s t u d i e s show t h a t t h e g l y c o p r o t e i n i s an a s y m m e t r i c molecule. The m o l e c u l a r model t h a t best f i t s t h e m o l e c u l a r dimensions (222 nm l o n g and 1-2 nm d i a m e t e r ) i s t h a t o f a p a r t i a l l y extended f l e x i b l e rod. Human a m n i o t i c f l u i d c o n t a i n s an i r r e v e r s i b l e t i s s u e i n h i b i t o r
Carbohydrate Chemistry
234 of
c ~ l l a g e n a s e . ~A~l t h~o u g h t h i s g l y c o p r o t e i n i s c l o s e l y a s s o c i a t e d
with
it
fibronectin,
does
not
cross-react
with
antibodies
to
f ib r o ne c t in.
The m i c r o h e t e r o g e n e i t y o f purified
rat
the d i f f e r e n t
a-fetoprotein
variants
carbohydrate chains o f
has
been s t u d i e d . 5 4 1
The
methodology used i n c l u d e s t h e r e l e a s e o f c a r b o h y d r a t e c h a i n s f r o m t h e g l y c o p r o t e i n by h y d r a z i n o l y s i s ,
-N - a c e t y l a t i o n labelled
with
{14C)-acetic
oligosaccharides
the l a b e l l i n g of
anhydride,
on
g l y c a n s by r e -
the f r a c t i o n a t i o n of
immobilized
concanavalin
c h a r a c t e r i z a t i o n o f t h e l a b e l l e d g l y c a n s by t.1.c. has been i s o l a t e d f r o m human a s c i t e s f l u i d . 5 4 2 (27-29)
were r e l e a s e d from
a-Fetoprotein
t h e p o l y p e p t i d e c h a i n by h y d r a z i n o l y s i s .
accumulated duodenal f l u i d , (mol.
wts.
x
5.4
lo4,
contains
1.02
the
and
Oligosaccharides
Human e n t e r o k i n a s e ( e n t e r o p e p t i d a s e EC 3.4.21.91,
subunit
the
A,
p u r i f i e d from
contains three glycosylated subunits x 105,and
1.4
active-site
x 10
1.5 4 3
i-serine
The s m a l l e s t
residue, and
the
o l i g o s a c c h a r i d e c h a i n s o f t h i s u n i t a p p e a r t o be b J - g l y c o s i d i c a l l y linked. The
NAO glycohydrolase
from
Bungarus
fasciatus
(banded k r a i t )
venom h a s b e e n p u r i f i e d t o e l e c t r o p h o r e t i c enzyme
i s a
glycoprotein
(mol.
wt.
1.2
The
lo5)
x
containing
two
s u b u n i t s.
-
Ce 11 s ur Pa ce G 1y Mp r o tei ns
9
with
Two r e c e n t l y p u b l i s h e d t r e a t i s e s h a v e i n c l u d e d c h a p t e r s d e a l i n g c a r b o h y d r a t e s and c e l l membranes,545 and w i t h t h e s t r u c t u r e s
a n d f u n c t i o n s o f t h e c a r b o h y d r a t e m o i e t i e s o f membrane g l y c o p r o t e i n s a n d g l y ~ o l i p i d s . ~R~e v~i e w s T-cell of
recognition,547
n o r m a l and cancerous
importance
of
membrane
and
dealing with glycosyltransferases
cells,548
glycoconjugates the
and
a n d w i t h g l y c o c o n j u g a t e s o f s u r f a c e membranes
multiple
i n
have
been p u b l i s h e d .
the organization
roles
played
co n s t i t ue n t s in m a 1ig na n t t r a n s f o r m a t i o n s
by
of
these
the
The cell
membrane
ha ve bee n r e v i ew e d .549
A
d e t a i l e d account o f h i s t o c h e m i c a l methods f o r c h a r a c t e r i z i n g complex cell-surface
glycoconjugates
by
l i g h t
microscopy
has
been
published. 550 Electron
microscopy
has
been
used
to
map
the
l o c i
of
i m m uno ch em ic a l a c t i ve s i t e s o n i n d i v i d u a l g l y cop r o t e i n m o l e c u l e s.551
This i n v o l v e s t h e complexing o f a g r o u p - s p e c i f i c macromolecule,
such
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
4
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236
Carbohydrate Chemistry
a s a l e c t i n o r an a n t i b o d y , w i t h an a s y m m e t r i c g l y c o p r o t e i n f o l l o w e d by t h e d i r e c t v i s u a l i z a t i o n o f t h e c o m p l e x i n t h e e l e c t r o n m i c r o s co pe A procedure f o r d e t e r m i n i n g membrane p u r i t y of i s o l a t e d c e l l s h a s been r e p ~ r t e d . ” ~ I n t a c t c e l l s a r e l a b e l l e d w i t h p e r o x i d a s e c o n j u g a t e d wheat-germ a g g l u t i n i n . A f t e r d i s r u p t i o n o f t h e i n t a c t l a b e l l e d c e l l s , p l a s m a membrane p u r i f i c a t i o n can be m o n i t o r e d by u l t r a s t r u c t u r a l h i s t o c h e m i c a l ex am i n a t i on o f t h e pe rox i d a s e conjugated l e c t i n . H i s t o c h e m i c a l f i x a t i o n o f c e l l g l y c o p r o t e i n s using p e r i o d a t e oxidation, followed by complexing w i t h L - l y s i n e and p a r a f o r m a l d e h y d e , h a s p r e v i o u s l y been a s s u m e d t o c r o s s l i n k s p e c i f i c a l l y p l a s m a r e s u l t s of an e v a l u a t i o n u s i n g membrane g l y c ~ p r o t e i n s . ’ ~ The ~ Novikoff r a t a s c i t e s h e p a t o c e l l u l a r carcinoma c e l l s suggest a com p l e x i n t e r a c t i o n b e t w e e n p e r i o d a t e - o x i d i z e d g l y c o p r o t e i n s a n d po 1ym e r i c com p l ex e s o f form a 1 de h y de and & - l y si ne C e l l - s u r f a c e g l y c o p r o t e i n s , b u t not g l y c o l i p i d s , from e r y t h r o c y t e s , c o l o n i c t u m o u r c e l l s , and s k i n f i b r o b l a s t s , b i n d t o i m m o b i l i z e d R i c i n u s communis a g g l u t i n i n . 5 5 4 The l a c k of b i n d i n g of t h e s o l u b i l i z e d g l y c o l i p i d s t o t h e i m m o b i l i z e d l e c t i n c o u l d be due t o t h e f a c t t h a t t h e i r m o n o v a l e n c y r e s u l t s i n t o o low a n a f f i n i t y f o r t h e l e c t i n t o c a u s e r e t e n t i o n on t h e column. A p p a r e n t m i n o r c h a n g e s i n c e l l - p r e p a r a t i o n m e t h o d s have been found t o r e s u l t i n major a l t e r a t i o n s i n c e l l b e h a v i o u r , r e f l e c t i n g d i f f e re nce s i n c e l l - s u r f a ce pro pe r t i e s of r a t h e pa t o cy t e s 555 P l a s m a membrane g l y c o p r o t e i n s from r a t h e p a t o c y t e s t h a t had been exposed t o s u b e n d o t h e l i a l s p a c e s of D i s s e have been s e l e c t i v e l y l a b e l l e d w i t h Q - g a l a c t o s e ox i d a s e - s o d i um b o r o t r i t i i d e . Approximately f o r t y gly c o p r o t e i n s were r e s o l v e d by two-dimensional e l e c t ro ph o r e si s. D i f f e r e n t c e l l - s u r f a c e g l y c o p r o t e i n s seem t o be i n v o l v e d i n t h e i n i t i a l r e a c t i o n s of c e l l - c e l l a n d c e l l - c o l l a g e n a d h e s i o n of r a t hepa tocy t e s . 5 5 7 G l y c o p e p t i d e s o b t a i n e d by pronase d i g e s t i o n o f r a t l i v e r plasma m e m b r a n e s have been f r a c t i o n a t e d by a f f i n i t y c h r o m a t o g r a p h y on i m m o b i l i z e d c o n c a n a v a l i n A.558 The primary s t r u c t u r e (29) of a b i a n t e n n a r y glycan from one of t h e f r a c t i o n s has been i d e n t i f i e d by h i g h - r e s o l u t i o n ‘H n.m . r . s p e c t r o s c o p y and m e t h y l a t i o n a n a l y s i s . T h e k i n e t i c behaviour of t r y p s i n i n non-covalent e l e c t r o s t a t i c i n t e r a c t i o n w i t h a s i a l o g l y c o p e p t i d e f r a c t i o n i s o l a t e d from major hepatoma c e l l s u r f a c e s has been reported.’”
.
.
.
5: Glycoproteins, Glycopeptides, Proteeglycans, and Animal Polysaccharides
237
Immunological evidence f o r t h e transmembrane nature o f t h e rat l i v e r r e c e p t o r f o r a s i a l o g l y c o p r o t e i n s has been presented.560 The r e c e p t o r i s exposed on t h e cytoplasmic, as well as t h e e x t e r n a l , s u r f a c e s o f t h e h e p a t o c y t e p l a s m a membrane, a n d d e t e r m i n a n t s o n e a c h s u r f a c e a r e a n t i g e n i c a l l y d i s t i n g u i s h a b l e . The b i o s y n t h e s i s a n d i n s e r t i o n of the r e c e p t o r i n t o endoplasmic reticulum membranes have been s t u d i e d u s i n g a n t i b o d y m o n o s p e c i f i c f o r t h e b i n d i n g protein.561 The b i n d i n g p r o t e i n i s e x c l u s i v e l y s y n t h e s i z e d o n t h e m e m b r a n e - b o u n d r i b o s o m e s and s p a n s t h e m i c r o s o m a l membrane, p r o b a b l y e x p o s i n g t h e C-terminal segment on the cytoplasmic surface and the N-terminal segment containing carbohydrate m o i e t i e s on t h e luminal surface. D e m o n s t r a t i n g a new r o u t e o f c l e a r a n c e o f g l y c o p r o t e i n s f r o m r a t p l a s m a , a b i n d i n g p r o t e i n s p e c i f i c f o r p - m a n n o s y l a n d / o r 2acetamido-2-deoxy-~-glucosyl r e s i d u e s has been i s o l a t e d from rat liver.562 This protein is analogous t o the binding protein s p e c i f i c f o r a s i a l o g l y c o p r o t e i n s , which m e d i a t e s t h e o r i g i n a l r o u t e of clearance, i n its predominant l o c a l i z a t i o n i n t h e l i v e r and i n its r eq u i r eme n t o f c a l c i um b i n d i n g. T h e a s i a l o g l y c o p r o t e i n r e c e p t o r , r e q u i r i n g Ca2+ f o r l i g a n d b i n d i n g , h a s been i d e n t i f i e d o n a c o n t i n u o u s human h e p a t o m a c e l l l i n e , Hep G2.563 There a r e a p p r o x i m a t e l y 150,000 l i g a n d m o l e c u l e s bound p e r c e l l a t 4OC. D i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y h a s been used t o examine t h e t h e rm a 1 d e n a t u r a t i 0 n o f r a b b i t h e p a t i c 9 - g a l a c t o s y l - b i n d i n g Ca2+ a n d l i g a n d b i n d i n g i n f l u e n c e t h e d e n a t u r a t i o n protein.564 process. H u m a n a s i a l o t r a n s f e r r i n s , t y p e s 1 , 2, a n d 3 , a n d r a b b i t a s i a l o t r a n s f e r r i n exhibit unequal binding with p u r i f i e d rat l i v e r p l a s m a membranes.565 The a s i a l o g l y c o p r o t e i n r e c e p t o r o n c u l t u r e d rat h e p a t o c y t e s mediates t h e t o x i c e f f e c t s o f a c o n j u g a t e of a s i a l o f e t u i n and fragment A of d i p h t h e r i a toxin.566 A s i a l o g l y c o p r o te i n s b u t n o t t h e corresponding s i a l o gly coprot e i n s are able t o block t h e a c t i o n o f t h e conjugate. v i v o h e p a t i c u p t a k e by t h e p a r e n c h y m a l Evidence f o r the cells of a Q-galactosyl-terminated glycoprotein (asialofetuin) i n f i s h (Salmo a l p i n u s ) has been r e p o r t e d . 5 6 7 The l e c t i n - d e p e n d e n t r e c o g n i t i o n o f d e s i a l y l a t e d e r y t h r o c y t e s by K u p f e r c e l l s h a s b e e n d i s c u s s e d .568 The s p e c i f i c i t y and k i n e t i c s o f endocy t o s i s o f g l y c o p e p t i d e s a n d g l y c o p r o t e i n s by i s o l a t e d r a t r e t i c u l o e n d o t h e 1 i a l c e l l p r e p a r a t i o n s have been examined.569 The c e l l r e c e p t o r i s h i g h l y
238
Carbohydrate Chemistry
selective
i n i t s
recognition of
subsequently internalized,
oligosaccharides
which
are
and t h e minimum s t r u c t u r e (30) r e q u i r e d
f o r r e c o g n i t i o n and e n d o c y t o s i s i s proposed. R - ( 1+6)-a-P-Mane-(
1+6)-B-B-Man~-( 1+4) -B-Q-GlceNAc-(
1+4)
-
8 -Q-GlceNAc-i-Asn
3
I 1 a -Q -Mane (30)
R = a-Q-Mane o r B-Q-GlCQNAC
The c e l l - s u r f a c e
glycoconjugates of
and o f
four
use o f
1251-substituted
and
by
the
ricin-resistant use o f
baby-hamster
kidney c e l l s
v a r i a n t s h a v e b e e n i n v e s t i g a t e d by t h e
r i c i n which binds t o 1-galactosyl
l e c t i n s which
bind t o
a c e tam ido - 2 -deox y -Q- g l uco sy 1 r e s id ue s .5 70 number o f l e c t i n a n d / o r r i c i n
of
the
2-
r i c i n r e c e p t o r s were found between t h e c e l l
resistance
mo d i f i c a t i o n s
and
M a j o r d i f f e r e n c e s in t h e
surface o f wild-type c e l l s and t h a t o f r i c i n - r e s i s t a n t The
residues,
neuraminosyl
o f
variant
concentration
cells of
i s
variants.
concomitant
certain
to
g l y c o c o nj u g a t e
structures which are accessible t o the sugar-binding proteins. Monkey k i d n e y c e l l s i n f e c t e d w i t h Yaba t u m o u r pox v i r u s e x h i b i t p l a s m a membrane a l t e r a t i o n s when t r e a t e d w i t h b o t h f l u o r e s c e i n l a b e l l e d a n d u n l a b e l l e d c o n c a n a v a l i n A.571 A c o m b i n a t i o n o f i m m u n o l o g i c a l and b i o c h e m i c a l methods have
been u s e d t o i d e n t i f y s u r f a c e membrane c o m p o n e n t s i n v o l v e d i n . c e l l substratum
adhesion.572
Highly p u r i f i e d adhesion-related
wt.
c o n s i s t i n g o f i n t e g r a l membrane g l y c o p r o t e i n s ( m o l .
materials 1.4
x
lo5)
t h a t a r e exposed t o the e x t r a c e l l u l a r environment were i s o l a t e d . Their r o l e i n the process o f cell-substratum adhesion i s n o t y e t un de r s t o o d. Mutant Chinese-hamster ovary c e l l s s e l e c t e d f o r r e s i s t a n c e t o a h a v e a l t e r e d Q-
c o n j u g a t e o f r i c i n and o-(6-phospho)-P-mannopentaose mannose 6 - p h o s p h a t e r e c e p t o r s . 5 7 3
These m u t a n t s e x h i b i t r e d u c e d
u p t a k e and a l t e r e d b i n d i n g o f exogenously added a c i d h y d r o l a s e . mutants
secrete
glucuronidase, and
two
L-f
to
six
times
more
ucosidase than the
a-Q-mannosidase, parent
e l e v a t e d s e c r e t i o n i s n o t due t o a l t e r a t i o n o f p h o s p h a t e r e c o g n i t i o n m a r k e r o n t h e enzymes,
cells.574
The
B-QThis
t h e Q-mannose 6 -
b u t appears t o r e s u l t
f r o m a l t e r a t i o n s i n t h e P-mannose 6 - p h o s p h a t e r e c e p t o r . A r e v i e w has been p r e s e n t e d o n t h e c e l l - s u r f a c e
properties o f
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides the
glycoconjugate
patterns o f
normal,
239
d i f f e r e n t i a t i n g , and
n e o p l a s t i c p a n c r e a t i c a c i n a r c e l l s .5 75 S o l u b i l i z a t i o n w i t h sodium d o d e c y l s u l p h a t e and subsequent e l e c t r o p h o r e t i c f r a c t i o n a t i o n has d e m o n s t r a t e d t h e h e t e r o g e n e i t y o f p r o t e i n s and g l y c o p r o t e i n s i s o l a t e d from
i n t h e i n t e s t i n a l b r u s h b o r d e r membrane
tadpole,
G l y c o p r o t e i n bands t h a t
y o u n g a n d a d u l t Rana c a t e s b e i a n a . 5 7 6 co-migrate
with
maltase,
glucoamylase, a n d
a l k a l i n e p h o s p h a t a s e a c t i v i t y have been i d e n t i f i e d . Sucrase-isomaltase,
an i n t e g r a l membrane g l y c o p r c t e i n t h a t
a p p e a r s i n i n t e s t i n a l m i c r o v i l l u s membranes d u r i n g d i f f e r e n t i a t i o n of intestinal epithelial cells,
has been p u r i f i e d o n i m m o b i l i z e d
m o n o c l o n a l a n t i b o d y - p r o t e i n A columns.577
The m a j o r p a r t o f
the
carbohydrate associated w i t h the g l y c o p r o t e i n i s i n t h e form o f complex o l i g o s a c c h a r i d e s l i n k e d t o A - a s p a r a g i n e . Lectin
binding t o
sealed and d i s r u p t e d d i s c
membranes o f
v e r t e b r a t e r e t i n a l r o d c e l l s supports t h e view t h a t r h o d o p s i n i s a transmem brane p r o t e i n w i t h i t s o l i g o s a c c h a r i d e
components o r i e n t e d
toward t h e i n t e r i o r o f the discs.578
I n d i r e c t evidence favouring
the
a
transmembrane
disposition o f
r o d outer-segment
disc
g l y c o p r o t e i n i s a l s o considered. A human s p e r m a t o z o a 1 a n t i g e n ,
acid,
amino sugars, a n d hexose,
containing residues o f neuraminic
has been p u r i f i e d . 5 7 9
The a n t i g e n
r e a c t s w i t h i m m u n o g l o b u l i n G and i m m u n o g l o b u l i n M a n t i b o d i e s a n d w i t h both types o f antisera. The m a j o r g l y c o p r o t e i n o n t h e p l a s m a membrane o f r a t t e s t i c u l a r
wt.
x
1.1
spermatozoa
(mol.
spermatozoa
pass through
lo5)
has
been i s o l a t e d . 5 8 0
the epididymis,
this
As
glycoprotein
d i s a p p e a r s a n d i s r e p l a c e d by a n o t h e r g l y c o p r o t e i n ( m o l . w t . 3.2 x lo4), which i s a m a j o r component o f e p i d i d y m a l s e c r e t i o n s . A g l y c o p r o t e i n (mol.
ga l a c t o s e
-
,
Q - m a nno s e
,
wt.
1.6
x
lo5)
containing residues o f
e-
2 - a c e t a m ido - 2 - d e o x y - E - g l uco se , a n d 2 -
a c e t am i do 2 -de ox y -9- g a l a c t o se has bee n ide n t i f i e d a s a c e l l - s u r f a ce r e c e p t o r o f b o a r spermatozoa f o r c o n c a n a v a l i n A.581 E n z y m i c r e m o v a l of c e l l - s u r f a c e unmasking and s t i m u l a t i o n o f
n e u r a m i n i c a c i d l e a d s t o an
gonado t r o p h i n - r e c e p t o r
p l a s m a membranes of b o v i n e c o r p u s l u t e u m .582 go na do t r o p h i n - r e c e p t o r
activity i n
A r e l a t i o n s h i p between
i n t e r a c t i o n and g l yco p r o t e i n-bo un d ne uram i n i c
a c i d has been d e m o n s t r a t e d . The sodium c h a n n e l s a x i t o x i n - b i n d i n g s a r c o l emma c o n t a i n s r e s i d u e s of 9-manno se, 2-deox y - Q - g a l a c t o s e ,
component o f r a t h i n d - l i m b
s- g a l a c t o se , 2-ace tam ido-
2-acetam i d o -2-deoxy-e-gl
ucose, and n e u r a m i n i c
Carbohydrate Chemistry acid.583
The t o x i n - b i n d i n g
s i t e i s s p a t i a l l y separated from b i n d i n g
s i t e s f o r l e c t i n s s u c h a s c o n c a n a v a l i n A,
wheat-germ
a g g l u t i n i n , and
R i c i n u s communis. The b i n d i n g s i t e s f o r agglutinin, have
been i d e n t i f i e d . 5 8 4
different 5.0
x
wheat-germ
agglutinin,
R i c i n u s communis
a n d c o n c a n a v a l i n A o n mouse n e u r o b l a s t o m a c e l l membranes glycopeptides,
lo4,
but four
major cell-surface
Concanavalin most
A
binds
w i t h molecular
to
over
weights
twenty
greater
than
of these g l y c o p e p t i d e s were i d e n t i f i e d a s t h e
receptors f o r the lectin.
D e t e r g e n t s have been u s e d t o s o l u b i l i z e t r a n s f e r r i n - t r a n s f e r r i n r e c e p t o r c o m p l e x e s from
r a b b i t r e t i c u l o c y t e membrane.585
Estimates
o f t h e m o l e c u l a r w e i g h t o f t h e t r a n s f e r r i n r e c e p t o r depend o n t h e conditions o f electrophoresis. p a r t i a l l y modified,
I t is s u g g e s t e d t h a t t h e r e c e p t o r i s
p e r h a p s by g l y c o s y l a t i o n .
A s o l u b l e h a e m a g g l u t i n a t i o n a c t i v i t y has b e e n o b t a i n e d f r o m
t e r a t o c a r c i n o m a stem c e l l s . 5 8 6
This haemagglutinin i s s e n s i t i v e t o
t r y p s i n a n d i s i n h i b i t e d by s p e c i f i c c a r b o h y d r a t e s s u c h a s [)-mannans and I - f u c a n s ,
indicating that i t i s a lectin.
The c a r b o h y d r a t e -
and
erythrocyte-binding
s p e c i f i c i t i e s o f t h e s o l u b i l i z e d l e c t i n a r e very
similar to
the cell-surface
that of
o n i n t a c t stem c e l l s , The
effect
of
carbohydrate-binding
component
s u g g e s t i n g i d e n t i t y o f t h e two components. dexamethazone
on
cell-surface
glycoprotein
a c c u m u l a t i o n i n HeLa c e l l s i s r e l a t e d t o i n c r e a s e d s u g a r - l i n k e d do 1i c h o l s y n t h e s i s . The
major
87
plasma
membrane
sialoglycoprotein
a s c i t e s mammary a d e n o c a r c i n o m a
of
13762 r a t
c e l l s i s released from
non-
x e n o t r a n s p l a n t a b l e a n d x e n o t r a n s p l a n t a b l e s u b l i n e s by p r o t e o l y t i c cleavage.588
The s i a l o g l y c o p r o t e i n p r o b a b l y
binds to
t h e membrane
v i a a hydrophobic p o r t i o n o f the p o l y p e p t i d e which remains w i t h t h e
membrane o n r e l e a s e . E v i d e n c e h a s been p r e s e n t e d f o r t h e i n v i v o a s s o c i a t i o n o f t w o c e l l g l y c o p r o t e i n s o f mammary a s c i t e s t u m o u r studies
on
the
redistribution
Redistribution of presence
of
concanavalin A
peanut
o f
cells,
fluorescent
based upon lectins.589
r e c e p t o r s was o b s e r v e d i n t h e
agglutinin, but
a g g l u t i n i n r e c e p t o r s was o b s e r v e d o n l y
redistribution of
peanut
upon t r e a t m e n t o f t h e c e l l s
w i t h c o n c a n a v a l i n A. A pea l e c t i n r e c e p t o r
f r o m 6C3HED m u r i n e a s c i t e s t u m o u r c e l l s
has been d e g r a d e d by t r y p s i n t o r e l e a s e a g l y c o p e p t i d e ( m o l . x
1 0 ~ ) .T h~ is ~ h i g~h - m o l e c u l a r - w e i g h t
wt.
g l y c o p e p t i d e i s absent
non-specific protease digests o f viable cells.
2.0 from
Sequential l e c t i n
24 1
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
a f f i n i t y chromatography o f detergent e x t r a c t s o f c e l l s has p e r m i t t e d the probable i d e n t i f i c a t i o n of the native glycoprotein. C e l l - f r e e m a t r i x m a t e r i a l f r o m human f i b r o b l a s t contains a cell-surface
glycoprotein
wt.
(mol.
lo5)
x
1.4
cultures which
seems t o be c l o s e l y a s s o c i a t e d w i t h t h e p e r i c e l l u l a r f i b r ~ n e c t i n . ~ ~ ~ Like fibronectin,
t h i s g l y c o p r o t e i n can f o r m a n e x t e n s i v e d i s u l p h i d e
crosslinked structure,
wt.
(mol.
2.5
covalent
x l
bonds
p o s s i b l y w i t h a s e c o n d g l y c o p r o t e i n component
~ The) p o. s s ~ i b i l i~ ty th ~a t c o v a l e n t and/or
~ bind
a l l
three
glycoproteins
i n
a
non-
matrix
was
discussed. Rat
Thy-1
antigen,
a glycoprotein o f
111 amino a c i d r e s i d u e s ,
i s a m a j o r membrane m o l e c u l e o f t h y m o c y t e s and b r a i n . 5 9 3 amino
acid
reported.
sequence o f
rat
An
sequence and
apparent
brain
i m m u n o g l o b u l i n was o b s e r v e d .
Thy-1
The f u l l
glycoprotein
structural
Evidence
for
has
been
homology
with
an evolutionary
r e l a t i o n s h i p b e t w e e n Thy-1 a n t i g e n and i m m u n o g l o b u l i n s has been p r e s e n t e d and a h y p o t h e s i s f o r t h e f u n c t i o n a l s i g n i f i c a n c e o f t h e Thy-1 a n t i g e n proposed.594 either
The a n t i g e n e x i s t s i n t w o a l l e l i c f o r m s ,
o r Thy-1.2.595
Thy-1.1
The T h y - 1 . 2
antigen has
been
i d e n t i f i e d by m o n o c l o n a l - a n t i b o d y t e c h n i q u e s a n d shown t o be l o c a t e d on t h e T h y - 1 g l y c o p r o t e i n . Metabolic
carbohydrate
labelling
for
the
analysis
of
g l y c o p r o t e i n s f r o m a c t i v a t e d human l y m p h o c y t e s has been u s e d i n a s t u d y of
the responding c e l l population.596
Numerous g l y c o p r o t e i n s ,
i s o l a t e d f r o m lymp h o cyte s u b s e t s a f t e r s t i m u l a t i o n w i t h m i t o g e n and alloantigen,
have been d e t e c t e d .
moieties of
h i gh-m o l e c u l a r - w e i g h t
lymphocytes
of
T
and
B origins
Differences i n the carbohydrate gly coproteins
have
been
monoclonal a n t i b o d i e s w i t h a n t i - I and a n t i - i
from
human
demonstrated
using
s p e c i f i ~ i t i e s . ~The ~ ~
c h i c k e n a n t i - ( bo v i ne a 2 - m a c r o g l o b u l i n ) a n t i bo d i e s t o
b in d i ng o f
N a m a l v a l y m p h o b l a s t o i d c e l l s has been d e m ~ n s t r a t e d . ~ ’ ~The p o s s i b l e r o l e o f t h e g l y c o p r o t e i n o n t h e c e l l s i s discussed. Treatment
of
c u l t u r e d human l y m p h o c y t e s w i t h
tunicamycin
r e s u l t s i n a decrease o f i n c o r p o r a t i o n o f c a r b o h y d r a t e i n t o p r o t e i n , together
with a
decrease i n the
hormone t o t h e c e l l s . 5 9 9 i n receptor-binding at
surface
functions
human
T
x
inhibit
Antibodies
cytolytic
lymphocytes.600
lo5
i n s u l i n and g r o w t h
t h e decreases a r e m o s t l y
c a p a c i t y and n o t a f f i n i t y .
glycoproteins
of
binding o f
F o r t h e hormones,
A
but
not
monoclonal
directed
suppressor antibody,
m o l e c u l a r w e i g h t f o r m o f T200 ( t h e m a j o r
specific for
t h e 2.2
cell-surface
glycoprotein on lymphoid cells),
has been u s e d t o show
242
Carbohydrate Chemistry
t h a t t h e g l y c o p r o t e i n i s expressed o n l y on B c e l l s and a subset o f bone-marrow
c e l l s w h i c h i n c l u d e s B c e l l precursors.601
A comparison o f
b i n d i n g s i t e s f o r wheat-germ
agglutinin on R a j i
l y m p h o b l a s t o i d c e l l s a n d t h e i r i s o l a t e d n u c l e i a n d p l a s m a membranes i n d i c a t e s s i m i l a r i t i e s i n t h e l e c t i n r e c e p t o r s on t h e o u t e r surface o f lymphoblastoid c e l l s and the c e l l nuclei.602
D i f f e r e n c e s were
o b t a i n e d w i t h i s o l a t e d membranes, a n d t h e s e may be due t o i n v e r s i o n of
t h e membrane v e s i c l e s o r t o t h e i r d e c r e a s e d r i g i d i t y as c o m p a r e d
with the i n t a c t cell. bo r o h y d r ide r e d u c t i on, a n d a c i d h y d r o 1y s i s
P e r i oda t e ox ida t i on,
o f human p e r i p h e r a l lymphocytes, b u t n o t mouse s p l e n o c y t e s o r c a l f l y m p h node c e l l s ,
release
glycerol,
propan-1 ,Z-diol,
carbon analogue o f n e u r a m i n i c acid.603 a r i s e from residues.
Q-galactosyl,
and t h e s e v e n
These f r a g m e n t s p r o b a b l y
I-fucosyl,
and N - a c e t y l n e u r a m i n o s y l
A subpopulation o f chicken B lymphocytes t h a t reacts w i t h
t h e I - f u c o s e - s p e c i f i c l e c t i n f r o m L o t u s t e t r a g o n o l o b u s has been identified.604
The d i s t r i b u t i o n o f t h e l e c t i n - r e a c t i v e
w i t h t h e age o f t h e c h i c k e n .
c e l l s varies
D i f f e r e n t i a l glycosylation o f murine B
c e l l a n d s p l e e n a d h e r e n t c e l l t A a n t i g e n s h a s been r e p o r t e d . 6 0 5 Three p r e d o m i n a n t g l y c o p r o t e i n s have been i s o l a t e d f r o m r a t t h y m o c y t e p l a s m a membranes.606
Two o f t h e g l y c o p r o t e i n s h a v e a
ca r b o h y d r a t e com po s i t i on c h a r a c t e r i s t ic o f The o t h e r
glycoprotein,
carbohydrate similarities to erythrocytes.
units
1-g l yco sy l a t e d
containing about
per
100
glycophorin,
amino
acids,
the major
p r o t e i ns.
twenty 2-glycosylated shows
structural
sialoglycoprotein
o f human
The m o n o c l o n a l a n t i b o d i e s a n t i - T 1 a n d a n t i - T 3 b o t h
r e a c t w i t h a l l human p e r i p h e r a l t h y m u s - d e r i v e d l y m p h o c y t e s a n d w i t h 1 0 % of
t h y m o ~ y t e s . ~Each, ~ ~ however,
surface structures, g l y c o p r o t e i n (mol. glycoprotein
wt.
(mol.
g l y c o p r o t e i n (mol.
recognizes
wt.
6.9
x
wt. 1.0
lo4>
1.9 x
x
lo5>
as
a
receptor
for
cell-
being a
and t h a t o f t h e a n t i - T 3 b e i n g a
lo4>.
A
human
cell-surface
t h a t i s s e l e c t i v e l y e x p r e s s e d by
p r o l i f e r a t i n g human l e u k a e m i c t h y m u s - d e r i v e d identified
different
with the target antigen o f anti-T1
serum
c e l l s has
transferrin.608
been This
g l y c o p r o t e i n c o n t a i n s b o t h complex and h i g h g-manno-oligo s a c c h a r i d e s linked
to
L-asparagine.60g
Glycosylation
i s
not
an
absolute
requirement f o r the receptor t o a c t as an acceptor f o r f a t t y acid, nor f o r transport t o the c e l l surface. Two Q a - 1 a n t i g e n s f r o m m i c e s p l e n o c y t e s have been i s o l a t e d f r o m both b i o s y n t h e t i c a l l y l a b e l l e d c e l l s and from surface i o d i n a t e d cells.610
These g l y c o p r o t e i n s have been shown t o
be d i s t i n c t f r o m
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
243
TL a n t i g e n s .
Primary s t r u c t u r e s of t h e t r a n s p l a n t a t i o n a n t i g e n o f t h e murine m a j o r h i s t o com p a t i b i l i t y c o m p l e x h a ve b e e n r e v i ewe d . 6 1 Partially purified, papain-solubilized mouse l i v e r H-2a h i s t o c o m p a t i b i l i t y a n t i g e n s h a v e b e e n p u r i f i e d f u r t h e r by r e v e r s e p h a s e h.p. 1.c. u n d e r a c i d c o n d i t i o n s , y i e l d i n g B 2 - m i c r o g l o b u l i n a n d H-2a h e a v y c h a i n s . 6 1 2 An a m i n o a c i d s e q u e n c e o f t h e f i r s t 98 a m i n o acid residues a t the N-terminus of the murine major h i s t o c o m p a t i b i l i t y a n t i g e n 6 1 3 a n d o f 98 r e s i d u e s o f t h e m u r i n e h i s t o c o m p a t i b i l i t y a n t i g e n H-2Kd h a s b e e n d e t e r m i n e d . 6 1 4 Comparison of t h e s e q u e n c e s w i t h H-2Kb a n d H-2Db a n t i g e n s r e v e a l s e x t e n s i v e localized differences. Comparison o f t h e primary s t r u c t u r e s o f m u r i n e H-2 a n t i g e n s a n d h u m a n HLA m o l e c u l e s s u g g e s t s a n e v o l u t i o n a r y o r i g i n o f m a j o r h i s t o c o m p a t i b i l i t y products.615 Data p r e s e n t e d o n t h e amino acid sequence o f a n intramembranous hydrophobic segment of t h e H-2Kb m u r i n e h i s t o c o m p a t i b i l i t y a n t i g e n b l 6 a n d o f t h e C t e r m i n a l h y d r o p h o b i c r e g i o n 617 h a v e p r o v i d e d t h e c o m p l e t e p r i m a r y s t r u c t u r e of the glycoprotein. Evidence f o r the existence o f three carbohydrate prosthetic g r o u p s o n m o u s e h i s t o c o m p a t i b i l i t y a n t i g e n s H-2kd a n d H-2Db h a s b e e n r e p o r t e d . l8 P u r i f i e d HLA-A2 a n t i g e n f r o m l y m p h o b l a s t o i d c e l l m e m b r a n e s which h a s been f l u o r e s c e n t l y l a b e l l e d s p e c i f i c a l l y i n i t s C - t e r m i n a l r e g i o n i n t e r a c t s w i t h l y m p h o b l a s t o i d c y t o s k e l e t a l p r o t e i n s when r e c o m b i n e d i n ~ i t r o T. h ~i s ~s y ~s t e m a l l o w s s t u d y o f t h e i n t e r a c t i o n s o f a m e m b r a n e p r o t e i n w i t h c y t o s k e l e t a l e l e m e n t s , a n d may be u s e d t o explore the structural basis and regulation of such interactions. A number o f unusual p r o p e r t i e s o f t h e i n v a r i a n t ( I i ) c h a i n o f t h e m u r i n e Ia a n t i g e n s has been described.620 While a l l of t h e Ia polypeptide chains p o s s e s s N-linked o l i g o s a c c h a r i d e u n i t s , they a l s o show t h a t t h e i n v a r i a n t c h a i n d i f f e r s f r o m t h e p o l y m o r p h i c c h a i n s i n C e l l - s u r f a c e HLAboth t h e number and t y p e o f c a r b o h y d r a t e u n i t s . DR, HLA-ABC a n t i g e n s , a n d g l y c o p h o r i n a r e a l l e x p r e s s e d a t d i f f e r e n t stages during erythroid di fferentiation.621 I n t h e b i o s y n t h e s i s o f HLA a n t i g e n s i n t w o l y m p h o b l a s t o i d c e l l l i n e s , Oaudi a n d Raji, t h e a n t i g e n heavy c h a i n s i n Daudi cells are s y n t h e s i z e d normally, but,although core g l y c o s y l a t i o n t a k e s place, t e r m i n a l g l y c o s y l a t i o n d o e s not.622 The b i o s y n t h e s i s o f t h e h e a v y c h a i n s o f mouse l y m p h o b l a s t o i d h i s t o c o m p a t i b i l i t y a n t i g e n s a n d o f i m m u n o g l o b u l i n M i n v o l v e s t h e same i n t r a c e l l u l a r p a t h w a y a s secretory proteins.623 These p o l y p e p t i d e s p a s s through t h e Golgi
H-2
244
Carbohydrate Chemistry
subsi te,
d e f i n e d by monensin,
and a c q u i r e t e r m i n a l
2
residues d i s t a l to t h i s site.
neuraminic a c i d
v i t r o t r a n s l a t i o n studies on the
b i o s y n t h e s i s o f HLR-DR a n t i g e n s i n d i c a t e t h a t t h e a - a n d $ - c h a i n s a r e s y n t h e s i z e d a s p r e c u r s o r s w i t h s i g n a l sequences,
i n common w i t h
many o t h e r membranes and s e c r e t e d g l y c ~ p r o t e i n s . ~I n~ ~ t h e presence o f a h e t e r o l o g o u s m i c r o s o m a l system, t h e s i g n a l p e p t i d e i s c l e a v e d and ' c o r e '
o l i g o s a c c h a r i d e u n i t s a r e added.625
The e v e n t s i n v o l v e d
i n t h e b i o s y n t h e s i s a n d m a t u r a t i o n o f t h e a n t i g e n s i n v i v o have a l s o been d e s c r i b e d . The
histocompatibility
(H-Y)
Y
antigen
i s
a
minor
h i s t o c o m p a t i b i l i t y antigen detected on the c e l l surface from the h e t erogam e t i c sexes o f species.626
b i r d s , am p h i bian, and i n v e r t e b r a t e
mamm a1 s,
The s e r o l o g i c a l d e t e r m i n a n t o f
the antigen resides i n
t h e carbohydrate p o r t i o n o f the glycoprotein. Three
cell-surface
antigens,
two
g l y c o p r o t e i n s and a p r o t e i n ,
s t r u c t u r a l l y r e l a t e d t o t h e m a j o r human h i s t o c o m p a t i b i l i t y a n t i g e n s h a v e b e e n c h a r a c t e r i z e d f r o m t h e l e u k a e m i c T c e l l l i n e MOLT-4.627 One a n t i g e n i s a g l y c o p r o t e i n (mol.
w t . 4.9 x l o 4 ) and i s a s s o c i a t e d The o t h e r g l y c o p r o t e i n (mol. w t . 4.3 x l o 4 )
w i t h B2-microglobulin. i s also
associated w i t h
B2-microglobulin
d i s t i n c t from t h e h i g h e r - w e i g h t
but
glycoprotein.
i s
serologically
The p r e s e n t s t a t e o f
knowledge o f the primary s t r u c t u r e o f the murine H-2 a l l o a n t i g e n s has been r e v i e w e d . 6 2 8
A speculative model o f the a n t i g e n on the
membrane i s p r e s e n t e d , and i n t e r p r e t a t i o n s o f t h e r e s u l t s o f p r i m a r y s t r u c t u r a l comparisons a r e used t o probe p o s s i b l e answers t o t h e questions
of
the
generation
of
polymorphism
and
genetic
r e l at i o nships. Wax-bean
agglutinin
mimics
the
activities
of
insulin
by
m e d i a t i n g a n t i l i p o l y s i s and t h e o x i d a t i o n o f Q - g l u ~ o s e . ~I ~t ~does not
appear
receptor,
to
compete w i t h
but
exerts
i t s
the
binding of
insulin-like
the
hormone
action
iia
glycoproteins which are d i s t i n c t from insulin-binding
to
i t s
membrane sites.
The
p o s s i b i l i t y that a glycoprotein effector molecule i s an i n t e g r a l p a r t o f t h e h o r m o n a l m e d i a t e d s y s t e m and t h a t t h e l e c t i n - m e d i a t e d o x i d a t i o n of
Q-glucose and a n t i l i p o l y s i s
may
be t r i g g e r e d
via
d i f f e r e n t g l y c o p r o t e i n r e c e p t o r s i s discussed. The p r e v i o u s l y o b s e r v e d i n h i b i t i o n by c o n c a n a v a l i n A o f i n s u l i n b i n d i n g t o i n t a c t r a t f a t c e l l s may be due t o t h e i n t e r a c t i o n o f t h e l e c t i n w i t h a carbohydrate-containing moiety on the d i s t i n c t from,
but capable o f modifying,
p r e v i o u s l y postulated.630
c e l l membrane
the i n s u l i n r e c e p t o r as
I n s u l i n r e c e p t o r s f r o m human p l a c e n t a and
245
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides cultured IM-9
lymphocytes display
similar
specificites for
twelve
i m m o b i l i z e d l e ~ t i n s . Based ~ ~ ~ on b i n d i n g s t u d i e s w i t h l e c t i n s t h e ca rbohy d r a t e m o i e t y o f t h e r e c e p t o r c o n t a i n s 2-ace tam ido-2-deox y-pglucosyl,
a-Q-mannosyl,
and $ + - g a l a c t o s y l
residues.
The i n c r e a s e
i n a f f i n i t y o f the i n s u l i n receptor f o r i n s u l i n a f t e r i t s desorption from l e c t i n s may be due t o t h e r e m o v a l o f an a s s o c i a t e d i n h i b i t o r . Tunicamycin,
which i n h i b i t s N-linked oligosaccharide chain
a d d i t i o n t o nascent polypeptides, i n s u l i n receptor receptors.632
interrupts glycosylation o f the
adipocytes
giving rise to inactive
The i n a c t i v e a g l y c o i n s u l i n r e c e p t o r a c c u m u l a t e s p o s t -
translationally re-enters
i n 3T3-Ll
d u r i n g c h r o n i c t r e a t m e n t w i t h t u n i c a m y c i n and t h e n
t h e g l y c o s y l a t i o n pathway when t h e i n h i b i t o r i s removed,
g i v i n g r i s e t o a f u n c t i o n a l i n s u l i n receptor. A
wt.
1.6
guinea-pig x
lo5)
sensitivity
p e r i t o n e a l macrophage s u r f a c e g l y c o p r o t e i n (rnol.
i s u n i q u e among m a j o r s u r f a c e c o m p o n e n t s i n i t s
to
m i l d t r y p s i n treatment.633
Neuraminic a c i d
r e s i d u e ( s ) w e r e l o c a t e d s o l e l y i n one o f t h e f r a g m e n t s (rnol.
wt.
8.5
x 104).
Heterogeneity
i n s u r f a c e g l y c o p r o t e i n s o f mouse p e r i t o n e a l
macrophage p o p u l a t i o n s has been observed.634 m a c r o p h a g e s w i t h t h e c u l t u r e medium o f fluid,
a new
expressed.
surface
glycoprotein
Upon a c t i v a t i o n o f
the
c e l l - f r e e tumour a s c i t e s
(rnol.
wt.
x
1.35
10')
is
The p o s s i b i l i t y t h a t t h i s m o l e c u l e i s m e r e l y a v a r i a t i o n
o f a pre-existing protein d i f f e r i n g i n i t s extent o f glycosylation,
o r even o n l y a h i g h e r r a t e o f s y n t h e s i s o f a n o r m a l l y m i n o r s u r f a c e g l y c o p r o t e i n , was n o t excluded. Two p r e v i o u s l y unknown macrophage-speci f i c a n t i g e n s (rnol.
wts.
x l o 4 a n d 1.1 x l o 5 ) h a v e b e e n i s o l a t e d by u s e o f a c o m b i n a t i o n o f c e l l h y b r i d i z a t i o n and r e m o v a l o f p r e v i o u s l y r e c o g n i z e d a n t i g e n s
3.2
by immunoadsor be n t s .635 On t h e b a s i s o f a g g l u t i n a t i o n s t u d i e s w i t h a number o f l e c t i n s , guinea-pig
phagocytic
v e s i c l e s have
been shown
r e c e p t o r s o n t h e i r cy t o p 1 asm i c s i d e . 636
Q - m a nno s y 1,
2-deox y -Q- g a l a c t o s y l , deoxy-D - g l ucosy 1, and
N, " - d i a c e
to
Q - G a l a c t o s y 1,
D- g l uco sy 1,
have l e c t i n . 2-acetam i d o -
2 - a c e tam i do-2-
t y l c h i t o b i o s y 1 residues are probably
present on the receptors. L e c t in- l i k e
r e c e p t o rs c a p a b l e o f
b i n d ing Staphylococcus a l b us
h a v e been d e m o n s t r a t e d i n t h e m e m b r a n e s o f p h a g o c y t e s i n c l u d i n g macrophages,
n e u t r o p h i l s , and e o s i n o p h i l s f r o m
v a r i o u s s o u r c e s and
species.637 These r e c e p t o r s a r e l i k e l y t o c o n t r i b u t e t o adherence and p h a g o c y t o s i s i n t h e non-immune a n i m a l .
bacterial
246
Carbohydrate Chemistry
Four independent monoclonal a n t i b o d i e s p r e c i p i t a t e a m u r i n e c e l l - s u r f a c e g l y c o p r o t e i n w h i c h has been i d e n t i f i e d a s a p o l y m o r p h i c d i f f e r e n t i a t i o n a n t i g e n o f m u r i n e mesenchymal c e l l s . 6 3 8 Treatment
of
melanoma
cells with
tunicamycin selectively
inh ib i t s t h e sh e d d i n g o f m e l ano m a- asso c i a t e d g l yco p r o t e i ns w it h o u t a f f e c t i n g t h e i r c e l l - s u r f a c e e x p r e s s i o n .639 K-562 c e l l s ,
o r i g i n a t i n g from the p l e u r a l e f f u s i o n o f a c h r o n i c
my e l o ge no u s l e u k a e m i a pa t i e n t , (erythroglycan, surface
mol.
wt.
e x p r e s s e s a n N - l i n ke d o l i go s a c c h a r i d e
lo3 -
x
7.0
g y ~ o p r o t e i n . ~T h~ e~ e a r l y
biosynthesis
are
the
by
specific cell-
of
erythroglycan
the transferrin-type
maturation o f the oligosaccharide
so
protein structure
that
expressed o n s p e c i f i c glycoproteins. foetal
lo4>,
stages
same a s t h o s e o f
o l i g o ~ a c c h a r i d e . ~H~o w ~ ever, influenced
1.1 x
i s
erythroglycan i s only
The K - 5 6 2 c e l l s e x p r e s s t h e
t y p e (L) a n t i g e n o n d i f f e r e n t g l y c o p r o t e i n s f r o m t h o s e o f
e r y t h r o c y t e s .642 Carbohydrate m o i e t i e s o f r e c e p t o r s f o r immunoglobulin E on r a t b a s o p h i l i c leukaemia c e l l s and r a t mast located i n the
binding s i t e o f
the
c e l l s are not
receptor.643
directly
However,
r e c e p t o r c a r b o h y d r a t e may p l a y a p a r t i n t r a n s p o r t o f
the
the
receptor
t o t h e p l a s m a membrane o r i n i t s o r i e n t a t i o n t h e r e a f t e r .
Cell-
i m m u n o g l o b u l i n E have been i s o l a t e d f r o m r a t
surface receptors f o r
basophilic leukaemia cells.644 The e f f e c t o f t u n i c a m y c i n o n t h e m o l e c u l a r p r o p e r t i e s o f t h e r e c e p t o r have been e v a l u a t e d . The u s u a l diffuse
r e c e p t o r b a n d (rnol.
so d i um
do de c y l s u l p h a t e
r e p l a c e d by one (rnol. i n d i ca t e
that
wt.
wt.
4.5
lo4
x
p o l y a c r y l a m ide
3.8
x lo4),
-
6.2
gel
lo4)
observed on
A number o f l i n e s o f e v i d e n c e
l o w e r - m o l e c u l a r- w e i g h t
this
x
electrophoresis i s protein
represents
ca r b o h y d r a t e-de f ic i e n t r e c e p t o r . Characteristic
g lycoprot e in
prof i l e s
and
carbohydrate
s t r u c t u r e s a r e expressed on d i f f e r e n t l e u k a e m i c c e l l l i n e s blocked at
different
s t a g e s of
stages of
m y e l o i d c e l l d i f f e r e n t i a t i ~ n . ~D ~i f ~f e r e n t
g r a n u l o c y t e d i f f e r e n t i a t i o n can be i d e n t i f i e d by s p e c i f i c
cell-surface
structures.
A murine cell-surface
g l y c o p r o t e i n (rnol.
wt.
8.0
x
lo4)
been i d e n t i f i e d a n d s t u d i e d by u s e o f m o n o c l o n a l a n t i b o d i e s . 6 4 6 d e t e r m i n a n t s o f t h i s m a j o r plasma-membrane by
the antibodies,
are allospecific,
constituent,
differentiation
cells.
The
recognized
and t h e expression o f
glycoprotein i s specific t o certain types o f
has
the
During
r e c e p t o r s f o r c o n c a n a v a l i n A have been shown t o
be
d i s t r i b u t e d i n patches over t h e e n t i r e cell-surface neuroblastoma
247
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides c e l l s . 647
The i m p o r t a n c e o f t h e p l a t e l e t - i s o l a t i o n t e c h n i q u e t o p l a t e l e t r e c o v e r y and p l a t e l e t - membrane i n t e g r i t y h a s b e e n s t r e s s e d . 6 4 8 The
loss o f p l a t e l e t - m e m b r a n e g l y c o p r o t e i n s d u r i n g w a s h i n g p r o c e d u r e s i s p r o b a b l y t h e r e s u l t o f membrane loss f r o m p l a t e l e t s a n d i s n o t due t o the i s o l a t i o n o f a selected p l a t e l e t subpopulation.
Membrane
g l y c o p r o t e i n s h a v e been i s o l a t e d f r o m human p l a t e l e t s a f t e r a f f i n i t y chromatography on i m m o b i l i z e d wheat-germ
a g g l u t i n i n ,649 a n d by h i g h -
voltage free-flow e l e c t r o p h o r e ~ i s . ~ ~ ~ Monoclonal antibodies
have
been
q u a n t i t a t i o n o f the platelet-membrane
used
i s o l a t i o n and
i n
glycoprotein deficient
i n
Glanzmann’s
t h r ~ m b a s t h e n i a . ~ The ~ ~ membrane g l y c o p r o t e i n i s a
complex o f
t w o p l a t e l e t g l y c o p r o t e i n s d e s i g n a t e d I I b and I I I a .
Membrane g l y c o p r o t e i n s o f p l a t e l e t s f r o m n o r m a l and Glanzmann’s thrombasthenic subjects are i d e n t i c a l i n terms o f i s o e l e c t r i c p o i n t s and m o l e c u l a r w e i g h t s ,
and d i f f e r o n l y i n t h e amount o f c e r t a i n
g l y c ~ p r o t e i n s . ~The ~ ~ molecular with
this
disease
i s
due
defect i n p l a t e l e t s from p a t i e n t s
t o
a
deficiency
of
two
membrane
g l y c ~ p r o t e i n s . ~ The ~ ~p o s s i b i l i t y o f a d d i t i o n a l membrane d e f e c t s i n thrombasthenic
platelets
g l y c o p r o t e i n (mol. membranes
from
may
be r e l a t e d t o a n o t h e r s u r f a c e
9.3 x lo4). The g l y c o p r o t e i n s o f p l a t e l e t n o r m a l p a t i e n t s and t h o s e w i t h Glanzmann’s wt.
or
t h r o m b a s t h e n i a have been compared u s i n g c a r b o h y d r a t e - s p e c i f i c protein-specific
l a b e l l i n g techniques
and h i g h - r e s o l u t i o n
d i m e n s i o n a l g e l e 1 e ~ t r o p h o r e s i . s . ~I ~n~ Glanzmann’s
two-
thrombasthenia
t h e absence o r r e d u c t i o n o f t w o m a j o r membrane g l y c o p r o t e i n s i s observed, w h i l e a number o f o t h e r g l y c o p r o t e i n s c o n t a i n i n c r e a s e d l e v e l s o f neuraminic acid. The e x i s t e n c e
i n human p l a t e l e t p l a s m a
m a c r o m o l e c u l a r component (mol.
wt.
>4.0
x
lo6)
membranes o f
c o m p o s i t i o n of a p h o s p h o g l y c o p r o t e i n has been confirmed.655 p h o s p h o g l y c o p r o t e i n i n h i b i t s t h e ADP, thrombin-induced plasma-membrane
a
with the chemical
epinephrine,
The
collagen, and
p l a t e l e t r e a c t i o n w i t h c o n c o m i t a n t changes of
the
structure.
A n a l y s i s of t h e g l y c o p r o t e i n s and p r o t e i n s o f p l a t e l e t s o f f o u r Bernard-Soulier possess
patients
a specific
lesion.656
has
established that
and c h a r a c t e r i s t i c
these
platelets
membrane-glycoprotein
A comparison w i t h t h e r e s u l t s obtained f o r
platelets after
treatment
control
w i t h n e u r a m i n i d a s e has shown t h a t
d e c r e a s e d n e u r a m i n i c a c i d c o n t e n t c a n n o t a l o n e a c c o u n t for observed Bernard-Soulier s u r f a c e a l t e r a t i o n s .
a
the
248
Carbohydrate Chemistry R e c e p t o r s f o r b o v i n e von W i l l e b r a n d f a c t o r have been i d e n t i f i e d
o n w h o l e human p l a t e l e t s , The k i n e t i c s o f
a s w e l l a s o n p l a t e l e t membranes.657
formation o f factor IXa-factor
V I I I complex o n
t h e s u r f a c e o f human p l a t e l e t s h a v e been r e p o r t e d . 6 5 8 T h r o m b i n - i n d u c e d p l a t e l e t a g g r e g a t i o n h a s been i n h i b i t e d by d e r i v a t i v e o f wheat-germ
agglutinin.659
the affinity-chromatographic
used f o r
membranes o f
a g l y c o p r o t e i n (mol.
a
The i m m o b i l i z e d l e c t i n was i s o l a t i o n from 7.4
w t .
i n h i b i t i n g p l a t e l e t a g g r e g a t i o n by t h r o m b i n .
lo4)
x
platelet
capable of
T h i s g l y c o p r o t e i n may
be a p h y s i o l o g i c r e c e p t o r o f t h r o m b i n i n human p l a t e l e t s a n d d i f f e r s the r i s t o c e t i n o r r i s t o c e t i n - v o n
from
Willebrand factor
Platelet
p l a s m a membranes r e t a i n f u n c t i o n a l
following
c e l l l y s i s a n d membrane i s o l a t i o n . 6 6 0
receptor.
aggregation
sites
Isolated platelet
plasma-mem b r a n e g l y c o p r o t e i n s h a v e a g r e a t e r a f f i n i t y f o r t h r o m b i n activated platelets
than control
An i m m u n o l o g i c a l
platelets.
p r o c e d u r e has been u s e d t o e x p l o r e t h e c o n c e p t t h a t a r e d i s t r i b u t i o n of
membrane
secretion.661 agglutinin
receptors
has
may
be
involved
non-agglutinating
A
been
prepared
i n
human
derivative o f
that
precipitates an antibody t o the lectin.
binds
to
platelet
wheat-germ
platelets
and
Pla'telets treated with this
i n a c t i v e d e r i v a t i v e r e l e a s e 5 - h y d r o x y t r y p t a m i n e when e x p o s e d t o bivalent antibody
.
(but
not
monovalent)
fragments
of
the
lectin
A s i a l o g l y c o p r o t e i n I b and a s i a l o g l y c o c a l i c i n have been i s o l a t e d from
the
membranes a n d
from
the
supernatant,
n e u r a m i n i d a s e - t r e a t e d human p l a t e l e t s . 6 6 2
respectively,
of
The t w o g l y c o p r o t e i n s
have been shown t o be c l o s e l y r e l a t e d ,
and evidence s u p p o r t i n g the
idea
from
that
glycocalicin
i s
derived
the
membrane-bound
glycoprotein i s reported. has
been
d e m o n s t r a t e d a t t h e e x t e r n a l s u r f a c e o f human p l a t e l e t s . 6 6 3
The
presence
of
ectosialyltransferase
activity
The
m a j o r endogenous a c c e p t o r i s t h e plasma-mem b r a n e g l y c o p r o t e i n G P I I b . Platelet-membrane
g l y c o p r o t e i n s I I b a n d I I I a h a v e been i s o l a t e d a n d
p u r i f i e d f r o m h u m a n p l a t e l e t mem b r a n e . 6 6 4
The g l y c o p r o t e i n s a r e
a n t i g e n i c a l l y d i f f e r e n t a n d s t r u c t u r a l l y d i s t i n c t g l y c o p r o t e i ns, w h i c h i n t h e n a t i v e s t a t e may f o r m a m a c r o m o l e c u l a r c o m p l e x w i t h a role
i n
mediating
platelet-platelet
c o n s t i t u e n t s o f human p l a t e l e t membranes
interactions.
The
major
G P I I b a n d G P I I I a have been
p u r i f i e d , and the
generation o f monospecific a n t i s e r a t o these a n t i ge n i ca 11y d i f f e r e n t a n d s t r u c t ur a 11y d i s t in c t g l y co p r o t e i n s h a s been d e ~ c r i b e d . ~ M ~ o~r p, h ~o l ~o g~ i c a l e v i d e n c e
demonstrating
249
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides macromolecular complex
formation o f these glycoproteins i n t h e
p l a t e l e t membrane a f t e r t h r o m b i n s t i m u l a t i o n i s a l s o r e p o r t e d . P l a t e l e t g l y c o p r o t e i n I11 and a - a c t i n i n have
been
d i f f e r e n t i a t e d on the basis o f the a b i l i t y o f the former to bind to immobi 1i t e d whea t-germ
a g g l u t i n i n. 6 6 7
F u r t h e r e v i d e n c e h a s been s h o w n t o s u p p o r t t h e h y p o t h e s i s t h a t y o u n g r a b b i t p l a t e l e t s a r e h e m o s t a t i c a l l y m o s t e f f e c t i v e when t h e y a r e r e l e a s e d f r o m bone marrow and t h a t t h e y undergo s i g n i f i c a n t changes i n
the
circulation as
decreased size,
they
age.668
This i s
shown
s y m m e t r i c a l l o s s o f membrane g l y c o p r o t e i n s ,
by and
decreased h e m o s t a t i c e f f e c t i v e n e s s o f the p l a t e l e t s .
wt.
1.1 x 10')
carrying binding
s i t e s f o r c o n c a n a v a l i n A and soybean a g g l u t i n i n
has been p u r i f i e d
A
m a j o r g l y c o p r o t e i n (mol.
f r o m m o s q u i t o p l a s m a membranes.669
The p r e s e n c e o f N - g l y c o s i d i c a l l y
l i n k e d o l i g o s a c c h a r i d e c h a i n s composed o f r e s i d u e s o f e-mannose a n d
2-acetamido-2-deoxy-Q-glucose
(in
the
molar
r a t i o 9:2)
g l y c o s i d i c a l l y l i n k e d 2-acetamido-2-deoxy-q-galacto
and
0-
s y l r e s i d u e s was
e s t a b l i s h ed. Concanavalin A binds t o a surface-membrane
receptor o f the
i n s e c t t r y p a n o s o m a t i d C r i t h i d i a f a s ~ i c u l a t a . ~ ~ The ' receptor
wt.
1.4
x
lo4>
(mol.
may be i d e n t i c a l t o a p r e v i o u s l y r e p o r t e d Q - m a n n a n
from t h a t source. The
in
v i v o synthesis,
membrane i n s e r t i o n , a n d g l y c o s y l a t i o n o f
a m u l t i s u b u n i t i n t e g r a l membrane p r o t e i n o f t h e T o r p e d o e l e c t r o p l a x acetylcholine
r e c e p t o r have
been studied.671
Each o f
s u b u n i t s i s synthesized as an i n d i v i d u a l polypeptide,
the
four
and a l l f o u r
p o l y p e p t i d e chains are independently i n t e g r a t e d i n t o dog pancreas m i c r o s o m e s as transmembrane p r o t e i n s . appears t o
The mechanism o f i n t e g r a t i o n
i n v o l v e a c o t r a n s l a t i o n a l process analogous t o t h a t
d e s c r i b e d f o r v i r a l membrane g l y c o p r o t e i n s .
10
G l y m p r o t e i n Hormones
The s t r u c t u r e a n d f u n c t i o n o f g l y c o p r o t e i n h o r m o n e s , 6 7 2 a n d t h e r e g u l a t i o n o f g l y c o p e p t i d e hormone s y n t h e s i s i n c e l l culture,673 A book d e a l i n g w i t h t h e e v o l u t i o n o f p r o t e i n have been reviewed. s t r u c t u r e and 74
ho rmo ne s
.
function
includes a
chapter
on
glycoprotein
Four g o n a d o t r o p h i n c o m p o n e n t s have been p u r i f i e d f r o m t h e b a s i c p r o t e i n f r a c t i o n o f b u l l f r o g p i t u i t a r y glands.675
The a m i n o a c i d
250
Carbohydrate Chemistry
c o m p o s i t i o n o f t h e four components i s a p p r e c i a b l y d i f f e r e n t f r o m t h a t o f mammalian l u t e i n i z i n g hormone. The b i n d i n g s i t e s o f human g o n a d o t r o p h i n i n t h e i n t r a c e l l u l a r organelles of compared
bovine corpora
with
l u t e a have been c h a r a c t e r i z e d and
plasma-membrane
sites.676
The
synthesis o f
g l y c o p r o t e i n hormone a - s u b u n i t and p l a c e n t a l a l k a l i n e phosphatase has been f o l l o w e d i n t h e p r e s e n c e o f t u n i c a m y c i n , 2-deoxy-P-arabinohexose, and s o d i u m b ~ t y r a t e . ~ ” The g l y c o s y l a t i o n i n h i b i t o r s r e d u c e d t h e b u t y r a t e - s t i m u l a t e d s y n t h e s i s o f t h e s u b u n i t and a l k a l i n e phosphatase t o a greater e x t e n t than t h e i r b a s a l l e v e l s o f synthesis. P r e g n a n t - m a r e s e r u m g o n a d o t r o p h i n d i s s o c i a t e s a t a c i d pH w i t h a p a r a l l e l l o s s o f f o l l i c l e - s t i m u l a t i n g hormone and l u t e i n i z i n g hormone a c t i v i t i e s , s u p p o r t i n g t h e h y p o t h e s i s t h a t t h e same molecular e n t i t y bears the two binding s i t e s for the receptors o f
.
f o 11ic l e -s t im u l a t in g h o r m o n e a n d l u t e i n iz i n g h o r mone 678
Mo l e c u l a r
w e i g h t s t u d i e s o f t h e g o n a d o t r o p h i n i n d i c a t e a v a l u e o f 4.5 o p p o s e d t o p r e v i o u s l y r e p o r t e d v a l u e s o f (5.3-6.4)
x
lo4
x
as
lo4.
A book d e a l i n g w i t h t h e c h e m i s t r y o f a n d h o m o l o g y b e t w e e n human c h o r i o n i c g o n a d o t r o p h i n and p i t u i t a r y l u t e i n i z i n g h o r m o n e h a s been published.679
The
current
understanding
of
immunological
s p e c i f i c i t y o f t h e B - s u b u n i t o f HCG i s e l u c i d a t e d . The
human s e r u m h o r m o n e s
by
immobilized
lutrophin
wheat-germ
agglutinin,
f o l l i t r o p h i n and t h y r o t r o p h i n . 6 8 0 produced
i s
suitable
radioimmunoassay o f immunoassay
and t h e
B-subunit
of
a r e a d s o r b e d by i m m o b i l i z e d c o n c a n a v a l i n A and
choriogonadotrophin
f o r
as
a
matrix
t h e s e hormones. human
which
also
The h o r m o n e - f r e e for A
chorionic
adsorbs
serum thus
standards
i n
highly specific
the
enzyme
gonadotrophin has
been
e s t a b l i s h e d . 681 C u l t u r e d human c h o r i o c a r c i n o m a c e l l s s y n t h e s i z e h i g h 0 - m a n n o s y l
o l i g o s a c c h a r i d e - c o n t a i n i n g f o r m s o f t h e a - s u b u n i t ( m o l . w t . 1.5 x
l o 4 and lo4) o f and
1.8 x
lo4>
and B - s u b u n i t f o r m s (mol.
human c h o r i o n i c g o n a d o t r o p h i n . 6 8 2
B-subunits
of
the
hormone
wt.
1.8 x
involves
the
The f r e e a - s u b u n i t
a n d 2.4 x
formation
accumulation o f incompletely processed forms o f intracellularly.
lo4
The s e c r e t i o n o f t h e aand
these subunits
produced by t h e c e l l s i s a
l a r g e r p o l y p e p t i d e and has a d i f f e r e n t c a r b o h y d r a t e c o m p o s i t i o n t h a n the
corresponding
a-subunit
of
the
placental
human
chorionic
gonadotrophin. A one-step
procedure f o r
the
i s o l a t i o n o f human c h o r i o n i c
gonadotrophin i n m i l l i g r a m amounts, u s i n g s e l e c t i v e s t e a d y - s t a t e
25 1
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides s t a c k i n g o n p o l y a c r y l a m i d e gel,
has been r e p o r t e d . 6 8 3
The p r o d u c t i o n o f human c h o r i o n i c g o n a d o t r o p h i n s u b u n i t s by h o r m o n e - p r o d u c i n g human c a n c e r c e l l s g r o w i n g i n n u d e m i c e i s s i m i l a r but n o t i d e n t i c a l t o t h a t observed i n culture.684
There a p p e a r s t o
be a s h i f t t o w a r d more c o m p l e t e hormone p r o d u c t i o n a n d a d e c r e a s e i n free
s u b u n i t p r o d u c t i o n a s compared t o t h e p a t t e r n i n c u l t u r e d
cells.
A e r o b i c t u m o u r - a s s o c i a t e d b a c t e r i a have been i s o l a t e d a n d
i d e n t i f i e d from m a l i g n a n t t i s s u e s from patients.685 the
i s o l a t e s were
capable o f
A l l b u t one o f
producing t h e B-subunit
o f human
c h o r i o n i c gonadotrophin. D i f f e r e n c e s i n t h e m o l e c u l a r w e i g h t s o f t h e a - s u b u n i t o f human chorionic
gonadotrophin e x c r e t e d i n the
c e l l s have been r e p o r t e d . 6 8 6
u r i n e and s e c r e t e d
by HeLa
The s u b u n i t s e c r e t e d by t h e t u m o u r
c e l l s h a s a h i g h e r m o l e c u l a r w e i g h t a n d may be d u e t o a n i n c r e a s e i n t h e neuraminic a c i d content o f the protein. H um a n cho r i o n i c
to
i t s receptor
go na d o t ro p h i n has
i n r a t testes.687
bee n co v a l e n t l y
c r o s s l i n ke d
Analysis o f the crosslinked
c o m p l e x e s i n d i c a t e s t h a t t h e m e m b r a n e r e c e p t o r may c o n s i s t o f a dimer o f two binding subunits which binds t o the a-subunit o f the hormone. An
assessment
has
b e e n made
o f
the
effects o f
various
m o d i f i c a t i o n s o f t h e t e r m i n a l Q - g a l a c t o s y l r e s i d u e s o f human a s i a l o choriogonado trophin.688
The p r e p a r a t i o n o f t h e d e r i v a t i v e s and t h e
i n f l u e n c e o f d e r i v a t i r a t i o n on t h e i r r a t e s o f plasma clearance, u p t a k e by t h e l i v e r , described.
i m m u n o - r e a c t i v i t y , and t a r g e t t i s s u e b i n d i n g a r e
The r o l e o f c a r b o h y d r a t e i n t h e s u b u n i t i n t e r a c t i o n s a n d
i n t h e b i n d i n g t o t h e r e c e p t o r h a s been a s s e s s e d by d e g l y c o s y l a t i o n of
i n d i v i d u a l a- and B - s u b u n i t s o f human c h o r i o n i c gonado t r ~ p h i n . ~ ~
Removal o f a b o u t 9 0 % and 8 0 % o f t h e c a r b o h y d r a t e f r o m t h e a - and Bs u b u n i t s , r e s p e c t i v e l y , had no e f f e c t o n t h e i m m u n o l o g i c a l a c t i v i t i e s , o n t h e apparent m o l e c u l a r weights, o r on t h e a b i l i t y o f t h e s u b u n i t s
-
F 1uo r e s c e n t l a b e l l e d a-sub u n i t s o f hum an
t o unde r g o r e a s s o c i a t i on. chorionic
gonadotrophin recombine
n o r m a l l y w i t h n a t i v e B-subunits.
L a b e l l i n g p r o c e d u r e s appear t o compromise t h e a b i l i t y o f t h e Bs u b u n i t s t o recombine.690 fluorescent
Since the method o f i n t r o d u c i n g the
label involves p r i o r periodate oxidation o f
the i n t a c t
i t i s p o s s i b l e t h a t p r i m a r y amino g r o u p s o f t h e p r o t e i n compete w i t h added f l u o r e s c e n t amines f o r t h e g e n e r a t e d aldehyde groups t o produce c o v a l e n t c r o s s l i n k i n g between subunits.
hormone,
The p r e s e n c e o f t h e B-subuni t o f human c h o r i o n i c g o n a d o t r o p h i n on
various
tumours
i s
reported to
be
rare on
the
basis
of
252
Carbohydrate Chemistry v a r i o u s t u m o u r s w i t h a n t i - B -HCG
i m m u n o h i s t o chem i c a l s t a i n i n g o f antibodies.691 I-Methionine
residues
of
the
a- a n d B - s u b u n i t s
l u t r o p h i n and b o v i n e t h y r o t r o p h i n have w i t h i o d o a c e t i c acid.692
of
been s p e c i f i c a l l y
bovine
alkylated
The m o d i f i e d r e s i d u e s w e r e i d e n t i f i e d a n d
t h e effects of m o d i f i c a t i o n on recombination with the unmodified c o u n t e r p a r t s u b u n i t , r e c e p t o r - b i n d i n g a c t i v i t y , and c o n f o r m a t i o n w e r e assessed.
Terminal 2-sulphated
deoxy-Q-glucose
residues o f 2-acetamido-2-
h a v e b e e n i d e n t i f i e d i n b o v i n e l u t r ~ p h i n . Human ~ ~ ~
p i t u i t a r y l u t r o p h i n i s a t l e a s t p a r t i a l l y sulphated, i t s placental counterpart,
i n contrast to
LCG.
Evidence f o r t h e amino a c i d sequence o f t h e a - s u b u n i t s o f o v i n e f o l l i c l e - s t i m u l at i n g hormone
a n d 1u t e i n i z i n g h o r m o n e
has
been
presented.694 S i m i l a r s t u d i e s o f t h e amino a c i d sequence o f t h e B - s u b u n i t o f o v i n e f o l l i c l e - s t i m u l a t i n g h o r m o n e have shown a r e m a r k a b l e d e g r e e o f p r e s e r v a t i o n o f s t r u c t u r a l i n f o r m a t i o n i n t h e B - s u b u n i t o f t h i s and o t h e r hormones.695
Two f o r m s o f e q u i n e f o l l i c l e - s t i m u l a t i n g hormone
have b e e n i s o l a t e d . 6 9 6
A h i g h l y s p e c i f i c homologous r a d i o r e c e p t o r
assay f o r t h e hormone i s described. follicle-stimulating
covalently attached to hormone.697
A
An enzyme immunoassay f o r human
h o r m o n e h a s been d e v e l o p e d u s i n g t h e hormone 8-glucose
oxidase and antiserum
radioimmunoassay
to
the
p r o c e d u r e f o r human t h y r o i d -
s t i m u l a t i n g h o r m o n e h a s b e e n i m p r o v e d by r e d u c i n g t h e h e t e r o g e n e i t y o f t h e 1251-human
t h y r o i d - s t i m u l a t i n g hormone t r a c e r . 6 9 8
The c e l l u l a r p r o c e s s i n g , a s s e m b l y , a n d r e l e a s e o f s u b u n i t s i n
-
-
t h y r o id s t im ul a t in g h o r m one b i o sy n t h e s i s h a ve a- and B - s u b u n i t s
are
initially
bee n s t u d ie d.
99 The
s y n t h e s i z e d i n e q u i v a l e n t amounts.
A subsequent excess o f a-subunits
i s observed,
r e s u l t i n g from the
It i s proposed t h a t t h i s n e t production and s e c r e t i o n o f the subunits
i n t r a c e l l u l a r d e g r a d a t i o n o f B-subunits. imbalance i n the
originates a t the level of post-translational
degradation rather
than a t the t r a n s l a t i o n a l process level. The b i o s y n t h e s i s o f c a l c i t o n i n , w t. of
3.5 a
x
lo3),
newly
a 32-amino
a c i d hormone (mol.
i n v o l v e s the g l y c o s y l a t i o n and p r o t e o l y t i c cleavage
synthesized precursor.700
e x t e n s i v e co- a n d p o s t - t r a n s l a t i o n a l
The
precursor
undergoes
p r o c e s s i n g t o t h e s m a l l e r non-
gl y co sy 1a t e d h orm one. Sim i l a r
i - g l u c o sy l - c o n t a i n i n g
o l i g o s a c c h a r i de
lipids
are
s y n t h e s i z e d by t h y r o i d r o u g h m i c r o s o m e s a n d by t h y r o i d c e l l s . 7 0 1 The t r a n s f e r o f Q - g l u c o s e t o t h e s e o l i g o s a c c h a r i d e l i p i d s i s
253
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides d e p r e s s e d b y t h e a d d i t i o n o f GDP-a-mannose. k i n e t i c experiments,
From t h e r e s u l t s o f
i t seems u n l i k e l y t h a t t h e s o l e p a r t i c i p a t i o n
o f newly synthesized oligosaccharide l i p i d s r e s u l t s i n the extensive transfer
o f p-glucose
f r o m UDP-a-glucose
t o t h y r o i d endogenous
p r o t e i n s .702 Two l a r g e g l y c o p r o t e i n f r a g m e n t s r e l a t e d t o t h e N - t e r m i n a l p a r t o f t h e adrenocorticotrophin-8-lipoprotein
p r e c u r s o r a r e t h e end
products of t h e maturation process i n t h e r a t pars intermedia.703 A n o v e l human p i t u i t a r y g l y c o p e p t i d e composed o f 39 a m i n o a c i d
r e s i d u e s a n d one o l i g o s a c c h a r i d e c h a i n h a s been i s o l a t e d f r o m w h o l e pituitaries.704
T h i s g l y c o p e p t i d e e x h i b i t s masked s e q u e n c e h o m o l o g y
t o p i g posterior p i t u i t a r y glycopeptide, g l y c o p e p t i d e s i s o l a t e d f r o m ox,
and t o w h o l e p i t u i t a r y
sheep, a n d p i g .
The i s o l a t i o n and p u r i f i c a t i o n o f a human g l y c o p e p t i d e r e p r e s e n t i n g t h e m a j o r i m m u n o r e a c t i v e f o r m o f t h e p i t u i t a r y N-
It
t e r m i n a l segment o f p r o - o p i o m e l a n o c o r t i n h a v e been r e p o r t e d . 7 0 5 b e a r s an 2 - g l y c o s y l a t i o n
s i t e a t L-Thr-45
and an N - g l y c o s i d i c s i t e
a t L-Asn-65. Human a n d
rhesus-monkey
immunocytochemically
p i t u i t a r i e s have
with antibodies against
been s t a i n e d
the B-subunits
of
p i t u i t a r y g l y c o p r o t e i n h o r m o n e s .706
11
M i l k Glycoproteins
The s t r u c t u r e s ( 3 1 ) o f a h o m o l o g o u s s e r i e s o f o l i g o s a c c h a r i d e s f r o m t h e m i l k o f t h e tamar w a l l a b y (Macropus e u g e n i i ) have been e l u c i d a t e d m a i n l y by 13C n.m.r.
Interactions
of
spectroscopy.707
substrates
g a l a c t o s y l t r a n s f e r a s e have spectroscopy.708
The
and
been
conversion
of
a-lactalbumin
measured
by
with
p-
difference
native a-lactalbumin
to
a
c o n f o r m a t i o n I or D and a l s o t h e c o n v e r s i o n o f It o D have been s t u d i e d by
U.V.
d i f f e r e n c e s p e c t r o s c o p y a n d c.d.
spectroscopy.709
The d a t a h a v e been u s e d t o c a l c u l a t e s t a n d a r d f r e e - e n e r g y
changes
f o r each of t h e t r a n s i t i o n s .
Laser photo-chemically
n u c l e a r p o l a r i z a t i o n n.m.r.
studies f o r f i v e a-lactalbumins from
different
induced dynamic
a n i m a l species c o n f i r m a h i g h degree o f homology f r o m
Carbohydrate Chemistry
254
species t o species with only minor differences i n chemical-shift e n v i r o n m e n t b e t w e e n them.710
A t i g h t l y bound i m p u r i t y f r o m m i l k h a s
a pronounced e f f e c t on t h e &-tryptophan fluorescence o f goat a1a c t a 1b um in. 71
'
The b i n d i n g o f o n e Ca2+ i o n t o b o v i n e a - l a c t a l b u m i n c a u s e s a c o n f o r m a t i o n a l change r e f l e c t e d i n a d e c r e a s e of t h e k - t r y p t o p h a n f l u o r e s c e n c e and a s p e c t r a l s h i f t towards s h o r t e r wavelengths.712 The
s i t e
of
crosslinking
on
a-lactalbumin
Q-
f o r
g a l a c t o s y l t r a n s f e r a s e i n t h e l a c t o s e s y n t h e t a s e complex has been s t u d i e d using t h e mutual e x c l u s i v i t y o f a c e t y l a t i o n and a m i d a t i o n w i t h b i s ( i m i d o e s t e r s ) .713 s i t u a t e d 6.1
4
t o 7.3
-
The r e s u l t s i n d i c a t e t h a t C - l y s i n e - 1 0 8
from
is
an a m i n o g r o u p o n g a l a c t o s y l t r a n s f e r a s e
i n t h e c r o s s l i n k e d complex. Electrophoretic examination (Bibos)
javanicus
has l e d t o
electrophoretically distinct
of
milk
B-lactoglobulins,
Bali cattle three
d e s i g n a t e d E,
Bas new
F, a n d
D e l e t i o n s o r s u b s t i t u t i o n s o f s p e c i f i c amino a c i d s i n the
G.714
p r o t e i n are responsible for the different of
from
the identification of
these
variants.
A
new
electrophoretic properties
a-lactalbumin
variant
was
also
iden t i f i e d. 715 G l y c o p e p t i d e s h a v e been p r e p a r e d f r o m b o v i n e c o l o s t r a l K - c a s e i n a f t e r t r e a t m e n t w i t h cyanogen b r o m i d e a n d pro tease^.^^^ A sequence o f t h i r t e e n amino a c i d r e s i d u e s o n one o f established.
t h e g l y c o p e p t i d e s was
Three o f f o u r L - t h r e o n i n e r e s i d u e s i n t h i s sequence
a r e t h o u g h t t o be t h e s i t e s o f a t t a c h m e n t o f t h r e e p o l y s a c c h a r i d e chains i n t h e casein.
Glycopeptides released from bovine colostrum
K - c a s e i n g l y c o p e p t i d e o b t a i n e d i m m e d i a t e l y a f t e r c a l v i n g c o n t a i n one a d d i t i o n a l p r o s t h e t i c sugar group l i n k e d t o L-threonine i n s t e a d o f o n l y one,
a n d up t o t e n i n t h e c a s e o f b o v i n e ( n o r m a l ) a n d h u m a n
.
ca s e i no g l y co pe p t i de s, r e s pe c t iv e l y 71 been
isolated
from
bovine
Th e t e t r a sa c c ha r i de ( 3 2 ) has
colostrum
K-casein
after
alkaline
One n e u t r a l bo r o h y d r i d e t r e a t m e n t o f c a s e i no g l y c o p e p t i de.718 o l i g o s a c c h a r i d e a l d i t o 1 (33) a n d t h r e e a c i d i c o l i g o s a c c h s a c c h a r i de a l d i t o l s (34-36) taken s i x
have b e e n i d e n t i f i e d f r o m b o v i n e c o l o s t r u m K - c a s e i n
hours a f t e r parturition.719
O l i g o s a c c h a r i d e s (33-34)
-
-
are
ch a r a c t e r is t ic o 1igo sa c ch a r ide s h a v i n g 2 ace t a m ido 2 -de ox y - 3 -2-0 Q galactosyl-g-glucosyl been r e p o r t e d t o
groups a t the non-reducing end occur
i n
normal
K-casein.
-
a n d have n o t
The o t h e r
two
o l i g o s a c c h a r i d e s (35-36) have a l r e a d y been i d e n t i f i e d i n K - c a s e i n from
normal
bovine
milk.
H c h a r a c t e r i z e d i n a 500 M H t '
Oligosaccharide n.m.r.
study.720
(34)
has also
been
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
255
The l o c a l i z a t i o n of g l y c o s y l a t e d K - c a s e i n i n i s o l a t e d b o v i n e c a s e i n m i c e l l e s has been s t u d i e d a t the u l t r a - s t r u c t u r a l l e v e l u s i n g g o l d g r a n u l e s l a b e l l e d w i t h R i c i n u s cgmmunis l e c t i n , w h i c h s p e c i f i c a l l y i n t e r a c t s w i t h 8 - Q - g a l a c t o s y l residues.721 No evidence was o b t a i n e d f o r t h e p r e s e n c e of g l y c o s y l a t e d K - c a s e i n o n t h e s u r f a c e o f g l u t a r a l d e h y d e - f i x e d m i c e l l e s w h e t h e r o r n o t t h e y had The g l y c o p r o t e i n was m a i n l y been t r e a t e d w i t h n e u r a m i n i d a s e . l o c a t e d i n t h e b r i d g i n g n e t w o r k i n t e r c o n n e c t i n g t h e m i c e l l e s , and appeared t o be l o o s e l y a s s o c i a t e d w i t h t h e m i c e l l e s . B-Q-GlceNAc-( 1+3) -g-Gale-( 1+3) -g-GalNAc-ol 6
I 2 a -Ne
ue5 Ac
(32)
B-Q-Galp-( 1+4) -B-Q-GlCeNAC-( 1+6) -Q-GalNAc-01 3
I 1
6 -Q-Galg 3
I 2
R
a -Ne ue5 Ac- ( 2+3 -8
(33)
R = H
(34)
R = a-Neue5Ac
-9 -Gale-
( 1+3)
-a -Gal NA c- 01 6
I 2 R
(35)
R = H
(36)
R = a-Neup5Ac
The i n t e r a c t i o n between bovine K-casein and 6 - l a c t o g l o b u l i n h a s been examined.722 The o l i g o s a c c h a r i d e p o r t i o n o f the g l y c o p r o t e i n i n f l u e n c e s complex formation, and Ca2+ i o n s have an i n h i b i t o r y e f f e c t o n the interaction.
256
Carbohydrate Chemistry S u b c e l l u l a r f r a c t iona t i o n o f o v i ne m am m a r y - g l a n d m em b r a ne s
i n d i c a t es
that
2 - a c e t am id o - 2 - d e o x y -Q- g a l a c t o s y 1 : p o l y p e p t i de
t r a n s f e r a se a n d Q - g a l a c t o s y 1 : 2 - ace t a m i d o - 2 - d e o x y-a-D=-galactosy 1 transferase,
which a r e i n v o l v e d i n t h e assembly o f disaccharide
u n i t s o f x-casein,
a r e l o c a l i z e d c h i e f l y i n G o l g i membranes.723
n e u r a m i n o s y l t r a n s f e r a s e a l s o o c c u r s i n t h e same f r a c t i o n ,
A
probably
s u g g e s t i n g a p o s t - t r a n s l a t i o n a l g l y c o sy l a t i o n p r o c e s s o f x - c a s e i n occurring simultaneously w i t h
lo4)
from
rat
the transport
A c a s e i n component
the Golgi region.
m i l k has
(C-2
been p u r i f i e d
.
by
of
these molecules
casein,
wt.
mol.
ion-exchange
to
3.4
and
x
gel
f i1t r a t i on ch rom a t o g r a phy 7 2 4
only
sugar
detected
i n
2- Ace tam i d o -2-deox y - p - g l uco se w as t h e this glycoprotein. Alkali-labile
o l i g o s a c c h a r i d e s l i b e r a t e d f r o m t h e g l y c o p e p t i d e s o f human m i l k f a t g l o b u l e membrane have been c h a r a c t e r i z e d and compared w i t h t h e corresponding o l i g o s a c c h a r i d e s i s o l a t e d from bovine m i l k f a t g l o b u l e The f o r m a t i o n o f l i p i d - l i n k e d s u g a r s i n t o e x p l a n t s o f
membranes.725
d e v e l o p i n g r a b b i t mammary g l a n d ,
stimulated t o differentiate i n
c u l t u r e w i t h hormones, a n d t h e a p p e a r a n c e o f n o v e l g l y c o p r o t e i n s , whose
synthesis
described.726
i s
dependent
on
this
pathway,
have
I n c u b a t i o n o f a membrane p r e p a r a t i o n from
been
lactating
bo v i ne mam m a r y t i s s u e w i t h UDP - 2 - ace t a m i d o -2-deox y-n-gl ucose and GDP-Q-mannose linked
r e s u l t s i n t h e s y n t h e s i s o f an e n t i r e range o f l i p i d -
saccharides
that
differ
from
one
another
by
one
m o n o s a c c h a r i d e u n i t.727 They a p p e a r t o be p r e c u r s o r - p r o d u c t t y p e o f i n t erm e d i a t e s
for
glycoproteins.
Two i s o m e r i c s t r u c t u r e s
-
the
g l y c o s y l a t i o n o f I - a s p a r a g i ne- l i n k e d for
t h e l i p i d - l i n k e d hexa-
a n d h ep t a sa ccha r i de s a r e p r o PO se d. 7 2 The e l u c i d a t i o n o f t h e s t r u c t u r e s o f t w o c a r b o h y d r a t e u n i t s (37-38),
1-glycosidically
lactotransferrin,
de s c r ibe d
.
l i n k e d t o an &-asparagine
u s i n g 5 0 0 MHz 'H
n.m.r.
residue o f
spectroscopy
has been
S t r u c t u r a l r e l a t e d n e s s b e t w e e n human l a c t o t r a n s f e r r i n a'nd
human
c e r u l o p l a s m i n has been e ~ t a b l i s h e d . ~ ~ The ' comparison o f amino a c i d sequences o f t w o c e r u l o p l a s m i n f r a g m e n t s c o r r e s p o n d i n g t o a t o t a l o f 5 6 4 a m i n o a c i d r e s i d u e s a n d 70% o f t h e l a c t o t r a n s f e r r i n m o l e c u l e (445 r e s i d u e s ) i n d i c a t e s t e n h o m o l o g o u s a r e a s i n c l u d i n g 1 3 3 a m i n o acids.
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
c v)
Q
I J I
t
4
I
4
u I
4'
cn I
.j
t
4
u I
0 Q
z al
4
u I
4' cn
II
U
t
n n
r.03
4
9
M
u
a
m w - 4 m w - - 4 m h l - 4 m I ZE I
4'
m I
4' ?
n
n
4
4
4l
4'
+
M
I
M
t
I
n
n
4 v
u
+
N
I
4' I
n
hl
-4 U
I
a
II
a a
I
n
+
hl
-4
$ m
5
4'
a
v
I
7'
M
u
257
258
Carbohydrate Chemistry 12
Serum G l y c o p r o t e i n s
R a t a 2 - r n a c r o g l o b u l i n h a s been p u r i f i e d f r o m s e r a by s e q u e n t i a l use o f
dextran
sulphate,
ion-exchange
chromatography, and g e l -
p e r m e a t i o n ~ h r o m a t o g r a p h y . ~An ~ ~a n t i s e r u m p r e p a r e d a g a i n s t t h e g l y c o p r o t e i n was u s e d t o a s s a y t h e c o n c e n t r a t i o n o f a 2 - m a c r o g l o b u l i n i n t h e s e r a of n o r m a l r a t s and i n t h e s e r a o f r a t s undergoing an i n f l a m m a t o r y response. Evidence for which
i s
t h e e x i s t e n c e o f t w o t y p e s o f c o m p l e x e s , one o f
covalent
and
the
m a c r o g l o b u l i n and p r o t e a s e s ,
other
of
which
i s
not
has been reported.732
between
a2-
The c o n v e r s i o n
of t h e non-covalent t o t h e covalent form r e q u i r e s t h e presence o f u n b l o c k e d amino groups. Soybean t r y p s i n i n h i b i t o r h a s been u s e d t o a s s e s s t h e d e g r e e o f i n a c c e s s i b i l i t y of porcine t r y p s i n w i t h i n t h e a2-macroglobulint r y p s i n complex.733
Proteinase-binding
on t h e a 2 - m a c r o g l o b u l i n do
not
appear
arranged.
to
surface.
be i d e n t i c a l ,
Evidence
for
the
s i t e s h a v e been i d e n t i f i e d
The t w o s u b u n i t s o f t h i s p r o t e i n
or they
cleavage
are not
of
a
macroglobulin during proteinase-complex discussed.734
thiol
symmetrically
i n a2-
ester
f o r m a t i o n has been
The n a t i v e human g l y c o p r o t e i n c o n t a i n s one r e a c t i v e
l a b i l e t h i o l e s t e r i n each o f i t s four i d e n t i c a l subunits.735
The
c y s t i n e r e s i d u e c o n s t i t u t i n g one p a r t o f t h e t h i o l e s t e r s t r u c t u r e i s l o c a t e d i n an i d e n t i c a l sequence t o c o m p l e m e n t c o m p o n e n t C3. of
a2-macroglobulin
that
f o u n d i n human
Trypsin-induced a c t i v a t i o n o f t h i o l esters
generates
a short-lived
intermediate that
can
r e a c t r a p i d l y t o i n c o r p o r a t e n o t o n l y methylamine or p u t r e s c i n e b u t also
proteins
interaction of
lacking trypsin
protease with
activity.736
this
protease
A
model
inhibitor
for has
the been
p r o p o s e d i n w h i c h t h e i n h i b i t o r can b i n d t o t h e enzyme i n t h r e e d i s t i n c t modes. 737 Human
a2-macroglobulin,
after
derivatization
m o n o d a n s y l c a d a v e r i n e by t r a n s g l u t a m i n a s e - c a t a l y s e d
with
incorporation,
contains two y-glutamyl residues per conjugated molecule,
a n d shows
n o d i f f e r e n c e s i n t r y p s i n b i n d i n g o r m e t h y l a m i n e i n a c t i v a t i o n when compared w i t h t h e n a t i v e a2-macroglobulin.738 methylamine
treatment,
the
a2-macroglobulin
A f t e r t r y p s i n or conjugate
e f f e c t i v e l y r e c o g n i z e d by i t s c e l l u l a r r e c e p t o r . binding o f t r y p s i n t o a2-macroglobulin g r o u p on t h e p r o t e a s e ,
The
i s
not
covalent
o c c u r s by a p r i m a r y a m i n o
a n d i s i n h i b i t e d b y p r i m a r y a m i n e s when
p r e s e n t d u r i n g complex f o r m a t i o n . 7 3 9
Covalent i n c o r p o r a t i o n o f t h e
259
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
same p r i m a r y a m i n e s d u r i n g t r y p s i n b i n d i n g t o a 2 - m a c r o g l o b u l i n indicates transient activation o f the methylamine-reactive s i t e during complexation. The c o v a l e n t l i n k a g e o f n o n - p r o t e o l y t i c proteins t o a2-macroglobulin
d u r i n g i t s r e a c t i o n w i t h p r o t e a s e s has
been f u r t h e r c h a r a c t e r i z e d . 7 4 0 The a m i n o a c i d s e q u e n c e l o c a t i o n o f a p u t a t i v e t r a n s g l u t a m i n a s e c r o s s l i n k i n g s i t e i n human a 2 - m a c r o g l o b u l i n h a s b e e n i d e n t i f ied.741 The a m i n o a c i d s e q u e n c e o f a 3 5 - r e s i d u e i n t e r n a l s t r e t c h o f h u m a n a2-macroglobulin, one
site
cleaved
established.742
w h i c h c o n t a i n s t w o s i t e s c l e a v e d by e l a s t a s e and by t r y p s i n , plasmin, and t h r o m b i n , has been T r y p s i n cleaves a I - L y s - i - L e u bond i n a2-macro-
g l o b u l i n , w h i l e S t a p h y l o c o c c u s a u r e u s V8 p r o t e i n a s e c l e a v e s a n Glu-Gly
I-
bond .743
al-Acid
g l y c o p r o t e i n h a s been m e a s u r e d b y l a s e r n e p h e l o m e t r y i n
a q u i c k and p r e c i s e m e t h o d f o r
d e t e c t i o n and f o l l o w - u p
i n f e c t i o n s i n newborn infants.744 o f r a t al-acid
of
bacterial
The c o m p l e t e n u c l e o t i d e s e q u e n c e
g l y c o p r o t e i n m e s s e n g e r RNA h a s b e e n d e s c r i b e d . 7 4 5
The i n f e r r e d a m i n o a c i d sequence h a s b e e n c o m p a r e d t o t h e p r e v i o u s l y reported
sequence
focusing
has
of
human a l - a c i d
glycoprotein.
been used t o r e v e a l t h e t w o
p o p u l a t i o n s s e p a r a t e d by a f f i n i t y
Isoelectric
al-acid
glycoprotein
chromatography on i m m o b i l i z e d
c o n c a n a v a l i n A and d e r i v e d from w h o l e n o r m a l serum and s e r a f r o m patients
u n d e r g o i n g an
significant
difference
i s
acute
i n f lammatory
observed
response.746
A
between p a t t e r n s o b t a i n e d f r o m
n o r m a l and i n f l a m m a t o r y s e r a . al-Acid
glycoprotein
produces
immunoaffinoelectrophoresis dimension,
thus
with
implying a
three
peaks
concanavalin
degree
of
A
on i n
crossed
the
heterogeneity
first i n
the
These carbohydrate p o r t i o n o f t h i s g l y ~ o p r o t e i n . ~ ~ ~ v a r i a t i o n s have been s t u d i e d under c o n d i t i o n s a s s o c i a t e d w i t h a l t e r a t i o n s i n serum concentration
of
female
sex
hormones.
A
new
type
o f
m i c r o h e t e r o g e n e i t y w i t h r e g a r d t o t h e p o s i t i o n o f a t t a c h m e n t o f an I - f u c o s y l r e s i d u e o n al-acid
g l y c o p r o t e i n h a s been o b s e r v e d f r o m t h e
i m p r o v e d r e s o l v i n g p o w e r a n d e n h a n c e d s e n s i t i v i t y o f 5 0 0 MHz l H n.m.r.
spectroscopy.748 al-Acid
glycoprotein
and
i t s
deglycosylated
derivatives,
p r o d u c e d by e n z y m a t i c c l e a v a g e of n e u r a m i n o s y l , B - g a l a c t o s y l , mannosyl, t o
and 2 - a c e t a m i d o - 2 - d e o x y - ~ - g l u c o s y l r e s i d u e s ,
regulate
immune responses
p a t h o l o g i c a l conditions.749
i n
various
Q-
may f u n c t i o n
physiological
and
The p r e c u r s o r f o r m s o f p l a s m a p r o t e i n s
s y n t h e s i z e d i n human l i v e r h a v e been i n v e s t i g a t e d . 7 5 0
By c o m p a r i n g
260
Carbohydrate Chemistry
t h e chromatographic behaviour of t h i r t e e n d i f f e r e n t plasma p r o t e i n s i s o l a t e d from e i t h e r l i v e r o r blood, f i v e i n t r a c e l l u l a r precursor p r o t e i n s can be i d e n t i f i e d .
Two o f t h e m , t r a n s f e r r i n a n d a l - a c i d
g l y c o p r o t e i n , were p u r i f i e d and c h a r a c t e r i z e d by c o m p a r i n g t h e i r s t r u c t u r e s w i t h those of t h e corresponding plasma forms. forms o f b o t h g l y c o p r o t e i n s l a c k e d n e u r a m i n i c acid, two l i v e r forms
of
al-acid
The l i v e r
and i n a d d i t i o n
g l y c o p r o t e i n are described as h a v i n g
l o w e r n e u t r a l and a m i n o s u g a r c o n t e n t . Rat a - l - a n t i t r y p s i n
has been c h a r a c t e r i z e d w i t h r e s p e c t t o i t s
a m i n o a c i d and c a r b o h y d r a t e c o m p o s i t i o n . 7 5 1
Some o f
i t s chemical
p r o p e r t i e s h a v e b e e n c o m p a r e d t o t h o s e o f human a - l - a n t i t r y p s i n . A n t i b o d i e s p r e p a r e d t o t h e r a t g l y c o p r o t e i n h a v e b e e n s h o w n t o be monospecific.
a-l-Antitrypsin
h a s been f r a c t i o n a t e d on i m m o b i l i z e d
concanavalin A i n t o t w o components which d i f f e r i n t h e degree o f branching
of
their
oligosaccharide
side
chains.752
One
form
c o n t a i n s t h r e e b i a n t e n n a r y s i d e c h a i n s and t h e o t h e r c o n t a i n s t w o b i a n t e n n a r y and one t r i a n t e n n a r y s i d e c h a i n . s t r u c t u r e s have been t e r m e d i s o f o r m s ,
These a l t e r n a t i v e
a t e r m used t o d i f f e r e n t i a t e
them from v a r i a n t s o f g e n e t i c o r p o s t - s e c r e t o r y o r i g i n . A l a t e n t t r y p s i n i n h i b i t o r i s r e l e a s e d f r o m d e n a t u r e d human s e r u m p r o t e i n s b y d i g e s t i o n w i t h t h e r m ~ l y s i n . I~m~m~u n o l o g i c a l c r o s s - r e a t i o n i d e n t i f i e d t h i s i n h i b i t o r as a complex between t h e i n h i b i t o r y a c t i v e p a r t o f t h e g l y c o p r o t e i n and i m m u n o g l o b u l i n G. al-Protease
i n h i b i t o r f r o m r a t serum e x i s t s i n f i v e
These m u l t i p l e f o r m s laser
nephelometric
p r o c e d u r e 7 5 5 and
i m m u n o a ~ s a yh~a v~e~ b e e n d e v e l o p e d f o r The t h e r m a l d e n a t u r a t i o n o f h e p a r i n and a l s o e.d.t.a.757
by
a
sandwich
enzyme
m e a s u r i n g a n t i t h r o m b i n 111.
a n t i t h r o m b i n 111
anions
by
A m o d i f i e d end-
s e q u e n t i a l a d d i t i o n o f n e u r a m i n i c a c i d t o e a c h one. point
forms.754
are derived from three o r i g i n a l forms
such as
i s
s t a b i l i z e d by
phosphate,
sulphate, and
The e f f e c t s o f d i t h i o t h r e i t o l o n t h e m o d u l a t i o n o f
a n t i t h r o m b i n I11 a c t i v i t y by s u l p h a t e d p o l y s a c c h a r i d e s have been reported.758
K i n e t i c analysis o f the heparin-enhanced plasmin-
a n t i t h r o m b i n 111 r e a c t i o n s u p p o r t s t h e t h e o r y t h a t h e p a r i n m u s t b i n d t o
the
enzyme
reaction.759
t o
accelerate
the
plasmin-antithrombin
The b i n d i n g o f h i g h - a f f i n i t y
111
heparin t o antithrombin
I 1 1 h a s been i n v e s t i g a t e d f o l l o w i n g c h a r a c t e r i z a t i o n o f t h e p r o t e i n fluorescence
enhancement
of
I-tryptophan
residues
a n t i t h r o m b i n i n t h e presence and absence o f heparin.760 flow
i n
the
Stopped-
k i n e t i c s t u d i e s o f t h e b i n d i n g i n t e r a c t i o n have a l s o been
reported.761
26 1
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
I n a study o f t h e plasma o f p a t i e n t s c o n g e n i t a l l y d e f i c i e n t i n
a n t i t h r o m b i n 111, o n l y t w o o f f o u r e l e c t r o p h o r e t i c a l l y s e p a r a t e d f o r m s i d e n t i f i e d i n n o r m a l p a t i e n t s ’ p l a s m a c o u l d be detected.762 Isoelectric-focusing
profiles
of
human
antithrombin
demonstrated heterogeneity i n l y o p h i l i z e d s a l t - f r e e
have
111
p r o d u c t s and i n
aged p r o d u c t s .763 A s i n g l e p r o t e o l y t i c c l e a v a g e s i t e i n human a n t i t h r o m b i n h a s
been i d e n t i f i e d between L-Arg-385 w i t h human t h r o m b i n . 7 6 4 -
and I - S e r - 3 8 6
following digestion
T h i s i s a t a p o s i t i o n homologous t o t h a t
observed f o r t h e bovine glycoprotein. The s t r u c t u r e a n d f u n c t i o n o f f a c t o r V I I I c o m p l e x h a v e b e e n r e v i e w e d .765 Factor
VIII-related
f luoroimmunoassay.766
anti-factor with
antigen
The a s s a y ,
has
been
measured
using
a
which uses a f l u o r e s c e n t - l a b e l l e d
V I I I a n t i b o d y as t h e s i n g l e m a j o r r e a g e n t , c o r r e l a t e s
an e x i s t i n g method.
The
molecular
forms
of
factor
V I I I
c o a g u l a n t a n t i g e n i n c l i n i c a l c o n c e n t r a t e s o f f a c t o r I X have been measured u s i n g 1 2 5 1 - l a b e l l e d
factor VIII:C
antibodies.767
Factor
c l o t t i n g a n t i g e n has been measured w i t h t w o d i f f e r e n t
V I I I
i m m u n o r a d i o m e t r i c assays,
one u t i l i z i n g t w o a c q u i r e d a n t i b o d i e s and
one a h a e m o p h i l i c a n t i b o d y . 7 6 8
The r e s u l t s c o n f i r m t h e d i v i s i o n o f
b o t h m o d e r a t e and m i l d h a e m o p h i l i a A i n t o t h r e e sub-groups. h a e m o p h i l i a A,
I n
there i s a quantitative reduction rather than a
q u a l i t a t i v e defect i n the p r o t e i n responsible f o r the f a c t o r V I I I clotting activity. A r a d i o r e c e p t o r assay
factor
VIII/von
f o r q u a n t i t a t i n g p l a s m a l e v e l s o f human
Willebrand’s
factor
has
r e c e p t o r s i t e s o n human p l a t e l e t s . 7 6 9
been
developed
using
The p l a s m a g l y c o p r o t e i n
c o n c e n t r a t i o n s c o r r e l a t e w i t h l e v e l s m e a s u r e d by t h e r i s t o c e t i n i n d u c e d p l a t e l e t - a g g r e g a t i o n method. Aluminium hydroxide but not barium chloride absorption o f factor
VIII p r o c o a g u l a n t
antigen
i s
associated
with
variable
r e c o v e r i e s o f t h e antigen.770 Immobilized p u r i f i c a t i o n of
11, V I I I ,
phospholipid
a number o f
vesicles
have
been
coagulation factors,
used
in
the
including factors
I X , a n d X.771
F i b r i l s u s p e n s i o n s o f t y p e s I,
II,and
111 c o l l a g e n a r e a l l
c a p a b l e o f a d s o r b i n g r i s t o c e t i n c o f a c t o r a c t i v i t y and f a c t o r V I I I r e l a t e d a n t i g e n a c t i v i t y f r o m n o r m a l plasma.772
The b i n d i n g o f
factor VIII/von
W i l l e b r a n d f a c t o r h a s b e e n d e t e c t e d by f o l l o w i n g t h e
adsorption
the
of
labelled
antigen
to
collagen
films.773
In
262
Carbohydrate Chemistry
c o n t r a s t t o r e p o r t s f r o m o t h e r workers, b i n d i n g t o bovine tendon f i b r e s was a l s o d e t e c t e d . The a p p l i c a t i o n o f a f f i n i t y - c h r o m a t o g r a p h i c t e c h n i q u e s f o r s e p a r a t i o n s and s t u d i e s o n molecular i n t e r a c t i o n s of t h e components of t h e blood-coagulation system has been reviewed.774 An e l e c t r o p h o r e t i c sys tern , employing l a r ge-pore polyacry lam i d e g e l s i n n e u t r a l b u f f e r i n t h e absence of s o d i u m d o d e c y l s u l p h a t e , has been used t o f r a c t i o n a t e s i n g l e s p e c i e s o f f a c t o r V I I I m u l t i m e r s under c o n d i t i o n s where b i o l o g i c a l p r o p e r t i e s of f a c t o r V I I I a r e p r e s e r v e d . 7 7 5 F a c t o r V I I I : C , f a c t o r VIII:RCof, and f a c t o r VII1:Ag a c t i v i t i e s a r e e x t r a c t a b l e from t h e g e l s a f t e r e l e c t r o p h o r e s i s and can be a s c r i b e d t o i n d i v i d u a l f a c t o r VIII m u l t i m e r s . Human a n t i - h a e m o p h i l i c f a c t o r (mol. w t . 1.16 x l o 5 ) , c o n t a i n i n g n o d e t e c t a b l e von Willebrand f a c t o r , has been prepared from normal human p l a s m a . 7 7 6 P a r t i a l r e d u c t i o n w i t h d i t h i o t h r e i t o l e x p o s e s c r i t i c a l s u l p h y d r y l g r o u p s , which when a l k y l a t e d m a i n t a i n t h e a n t i - h a e m o p h i l i c f a c t o r w i t h o u t i n a c t i v a t i n g procoagulant a c t i v i t y . F u r t h e r p u r i f i c a t i o n of t h e a n t i - h a e m o p h i l i c f a c t o r was a c h i e v e d a f t e r c o n v e r s i o n of p r o t e i n - 2 - p y r i d y l mixed d i s u l p h i d e c o n j u g a t e s f o l l o w e d b y a f f i n i t y chromatography o n t h i o p r ~ p y l - a g a r o s e . ~ ~ ~ F r a c t i o n a t i o n of p a r t i a l l y p u r i f i e d b o v i n e f a c t o r V I I I o n c a l c i u m c i t r a t e c o l u m n s p r o d u c e s a m a t e r i a l which c o n t a i n s h i g h l e v e l s of f a c t o r VIII procoagulant a c t i v i t y and f a c t o r V I I I - r e l a t e d a n t i g e n b u t d o e s n o t a g g r e g a t e human p l a t e l e t s . 7 7 8 The a p p a r e n t molecular weight of t h e procoagulant f a c t o r is s i g n i f i c a n t l y lower t h a n t h a t of t h e f o r m s of f a c t o r VIII which c o n t a i n p l a t e l e t a g g r e g a t i n g a c t i v i t y , and i t e x h i b i t s a h i g h e r m o b i l i t y o n electrophoresis. Bovine f a c t o r VIII h a s been p r e p a r e d f r e e from p l a t e l e t a g g r e g a t i n g a c t i v i t y . 7 7 9 The p r e p a r a t i o n migrated a s a t r i p l e t on sodium dodecylsulphate-polyacrylamide gel electrophoresis w i t h a p p a r e n t m o l . wts. 9 . 3 x L O 4 , 8.8 x l o 4 , and 8.5 x l o 4 . B o t h p l a s m i n and t h r o m b i n d e s t r o y a p r o t e i n ( m o l . w t . 8.5 x l o 4 ) , p r e s e n t i n h i g h l y p u r i f i e d f a c t o r VIII/von Willebrand f a c t o r , a t a r a t e t h a t p a r a l l e l s t h e loss of f a c t o r V I I I C . 7 8 0 T h i s protein may be n e c e s s a r y f o r f u l l e x p r e s s i o n o f f a c t o r V I I I C and may be d e f e c t i v e i n c a s e s o f haemophilia. The l e v e l s o f f a c t o r V I I I c o a g u l a n t a n t i g e n i n n o r m a l , t h r o m b i n - t r e a t e d , and haemophilic plasma have been a n a l y ~ e d . The ~~~ a n t i g e n d e t e c t e d i n normal plasma i s n o t c o v a l e n t l y l i n k e d t o f a c t o r V I I I / v o n Willebrand f a c t o r m u l t i m e r s and is a b s e n t i n plasma from a
263
5: Glycoproteins, Glycopeptides, ProteoRlycans, and Animal Polysaccharides
number o f h a e m o p h i l i a p a t i e n t s . Thrombin-induced p r o t e o l y s i s o f t h e a n t i g e n has been o b s e r v e d i n n o r m a l plasma. Thrombin i s more e f f e c t i v e
i n potentiating factor
V I I I
p r o c o a g u l a n t a c t i v i t y when t h e a m o u n t o f t h r o m b i n r e l a t i v e t o t h e concentration
of
stoichiometric
rather
activation,
the
factor than
VIII/von
Willebrand
catalytic.782
I n
factor
contrast
the i n a c t i v a t i o n o f thrombin-activated
factor
to
i s i t s
VIII/von
W i l l e b r a n d f a c t o r i s n o t a p r o t e o l y t i c p r o c e s s , and a c t i v a t i o n o f t h i s factor i s a prerequisite for i t s inactivation. F u r t h e r e v i d e n c e o f t h e f o r m a t i o n o f s t a b l e immune complexes b e t w e e n human f a c t o r V I I I a n d i t s h o m o l o g o u s a n t i b o d i e s h a s b e e n published.783
Some e v i d e n c e i s a l s o p r o v i d e d w h i c h s u p p o r t s t h e
view t h a t f a c t o r V I I I c o n s i s t s o f two d i s s i m i l a r subunits which a r e noncovalently linked. Heterogeneity o f molecular size o f factor
i n von Willebrand’s
factor
disease has
VIII/von
Willebrand
been d e m o n s t r a t e d by
a
c o m b i n a t i o n o f SDS p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s and c r o s s e d -
i m m u n o e l e c t r ~ p h o r e s i s . ~ T~h~e
factor
g l y c o p r o t e i n c o n s i s t s o f a s e r i e s of i n m o l e c u l a r w e i g h t f r o m 2.4
x
lo6
VIII/von
Willebrand
multimeric structures ranging t o g r e a t e r t h a n 1 0 x 106.785
Differences i n the s p e c i f i c a c t i v i t y o f the polymeric forms are a f u n c t i o n n o t o n l y of
t h e i r s i z e but also o f quaternary conformation
t h a t may b e d i c t a t e d by d i s u l p h i d e b o n d a r r a n g e m e n t s . A
technique
relationship
has
been
between f a c t o r
developed
i n
VIII-related
order
t o
study
the
a n t i g e n ( V I I I R A g ) and
The V I I I C p a r t o f t h e f a c t o r V I I I c l o t t i n g a n t i g e n (VIIICAg).786 complex i s pre-incubated w i t h 1251-anti-VIIICAg prior to immunoelectrophoresis. Autoradiography o f the immunoprecipitin l i n e produced w i t h a n t i - V I I I R A g i n d i c a t e d t h e presence o r absence o f VIIICAg ( V I I I C )
a s s o c i a t e d w i t h VIIIRAg.
Human f a c t o r
V I I I procoagulant a c t i v i t y i n t e r a c t s w i t h c e r t a i n
phospholipid^.^^^
F u r t h e r e v i d e n c e h a s shown t h a t t h i s a c t i v i t y and
the
factor
VIII/von
Willebrand
factor
V a r i a n t forms of p r o c o a g u l a n t - l i k e f a c t o r
are
separate
entities.
V I I I i n haemophiliacs have
b e e n r e p o r t e d .788 The a c t i v a t i o n o f blood-coagulation
factor
X
t h e i n t r i n s i c pathway i n t h e
s y s t e m i n v o l v e s p h o s p h o l i p i d and f a c t o r
VIII.789
The s t i m u l a t i n g e f f e c t o f p h o s p h o l i p i d i n f a c t o r X a c t i v a t i o n i s m a i n l y due
t o
an e f f e c t
stimulatory effect increase i n the
of
ymax
factor
on t h e V I I I
Km f o r
factor
X,
while
the
i s e x p l a i n e d by i t s 2 0 0 , 0 0 0 - f o l d
o f f a c t o r Xa f o r m a t i o n .
264
Carbohydrate Chemistry
A metal-ion-catalysed conformational change a f f e c t i n g f a c t o r X i s a p r e r e q u i s i t e f o r g l y c o p r o t e i n - l e c t i n p r e c i p i t i n i n t e r a c t i o n . 79b
When Ca2+ o r Mn2+, b u t n o t Mg2+, i s p r e s e n t , s p e c i f i c s u g a r s o n t h e c a r b o h y d r a t e c h a i n s o f f a c t o r X1 b e c o m e a c c e s s i b l e t o w h e a t - g e r m agglutinin. Whether t h i s c a t i o n - g l y c o p r o t e i n i n t e r a c t i o n t a k e s p l a c e t h r o u g h a d i r e c t i n t e r a c t i o n between c a r b o h y d r a t e m o i e t i e s and c a t i o n or by a d i r e c t i n t e r a c t i o n b e t w e e n p r o t e i n m o i e t y a n d c a t i o n , o r b o t h , i s n o t known. A three-step method h a s been developed f o r t h e d e t e r m i n a t i o n o f f a c t o r VII u s i n g a c h r o m o g e n i c s u b s t r a t e . 7 9 1 A p r e p a r a t i v e scheme f o r t h e i s o l a t i o n o f m u l t i p l e c o m p o n e n t s o f complement from one l a r g e p o o l o f human p l a s m a i n which t h e p u r i f i e d p r o d u c t s are c h a r a c t e r i z e d f u n c t i o n a l l y , physicochemically, a n d i m m u n o c h e m i c a 1l y h a s b e e n d e s c r i b e d . 792 The p u r i f i e d s u b c o m p o n e n t C l q o f t h e f i r s t c o m p o n e n t o f m o u s e complement c o n t a i n s h y d r o x y - i - p r o l i n e , h y d r o x y - k - l y s i n e , g l y c i n e , and ~ a r b o h y d r a t e . ” ~ T h e i n t e r a c t i o n o f i s o l a t e d h u m a n c o m p l e m e n t c o m p o n e n t s Clr a n d Cls w i t h c e l l - b o u n d a n d f l u i d - p h a s e C l q h a s b e e n r e p o r t e d . 7 9 4 The b i n d i n g of Clq t o t h e F c p o r t i o n of an a n t i b o d y i n f l u e n c e s i t s a c t i v i t y t w o a r d s Clr a n d Cls a n d i t s a b i l i t y t o a c t i v a t e t h e s e C 1 components. B i n d i n g o f Clr a n d C l s t o C l q i n t h e f l u i d p h a s e a p p e a r s t o change or f i x t h e C l q c o n f o r m a t i o n so t h a t Clq is t h e n able t o bind only t o t h o s e binding sites which can t r i g g e r t h e internal activation. The i n i t i a l r e a c t i o n o f b i n d i n g o f h u m a n c o m p l e m e n t C 3 a n d C 4 t o s u r f a c e s i n v o l v e s similar mechanisms f o r both p r o t e i n s , w i t h t h e b i n d i n g b e i n g by m e a n s o f a c o v a l e n t b o n d ( e s t e r o r a m i d e ) o f w h i c h t h e c a r b o x y l g r o u p i s d o n a t e d by C 3 o r C4.795 Haemolytically active C 3 a n d C4 u n d e r g o p e p t i d e - b o n d c l e a v a g e i n t h e a - c h a i n when incubated under denaturing conditions. I n e a c h case o n l y a s i n g l e p e p t i d e bond i s s p l i t . T h e n o n c o v a l e n t l y a s s o c i a t e d a- a n d B - s u b u n i t s o f t h e e i g h t h component o f human complement ( C 8 ) r e c o m b i n e i n s o l u t i o n t o f o r m n a t i v e C8 w i t h c o n c o m i t a n t a p p e a r a n c e o f C 8 h a e m o l y t i c a c t i v i t y . 7 9 6 S t r u c t u r a l domains which mediate s p e c i f i c i n t e r a c t i o n o f C 8 with its n a t i v e c y t o l y t i c complex are l o c a t e d i n t h e B-subunit. Direct c h e m i c a l e v i d e n c e s h o w s t h a t n o n - e n z y m a t i c g l y c o s y l a t i o n of haemoglobin i n v o l v e s the i n i t i a l f o r m a t i o n o f a r e v e r s i b l e a l d i m i n e ( S c h i f f b a s e ) p r e c u r s o r which s l o w l y r e a r r a n g e s t o a stable k e t o a m i n e by t h e A m a d o r i r e a r r a n g e m e n t . 7 9 7 An e s t i m a t e o f t h e
265
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
d i s t r i b u t i o n o f l a b i l e a l d i m i n e and s t a b l e k e t o a m i n e i n e r y t h r o c y t e s f r o m n o r m a l and d i a b e t i c s o u r c e s i s o b t a i n e d f r o m t h e r a t e c o n s t a n t s f o r t h e p r o d u c t i o n o f t h e two forms. Unless s t r i c t l y standardized conditions are a p p l i e d t o the measurement o f n o n - e n z y m i c a l l y g l y c o s y l a t e d serum p r o t e i n s u s i n g t h e t h i o b a r b i t u r i c a c i d a s s a y , e r r o n e o u s r e s u l t s may b e o b t a i n e d . 7 9 8 The
accuracy
of
the
modifications t o
method has
assay
been
i m p r o v e d by
conditions.
a
number
The c o n d i t i o n s f o r
of the
t h i o b a r b i t u r i c a c i d a s s a y f o r n o n - e n z y m i c g l y c o s y l a t i o n o f human serum a l b u m i n have been examined, and t h o s e o f o p t i m a l s e n s i t i v i t y and r e p r o d u c i b i l i t y a r e r e p o r t e d . 7 9 9 A
colorimetric
haemoglobin, carbohydrate,
based
method on
for
the
the
acid
glycosylated reaction
of
i s c l a i m e d t o be more r e l i a b l e a n d more s e n s i t i v e t h a n
t h e t h i o b a r b i t u r i c a c i d method.800 carbohydrate
estimation of
phenol-sulphuric
content
of
I n a d i f f e r e n t approach,
the
g l y c o s y l a t e d h a e m o g l o b i n i s d e t e r m i n e d by
measuring t h e r e l e a s e o f formaldehyde f o l l o w i n g p e r i o d a t e o x i d a t i o n o f t h e carbohydrate moieties.801
The f o r m a l d e h y d e i s e s t i m a t e d a s
t h e f l u o r e s c e n t c o n d e n s a t i o n p r o d u c t f o r m e d w i t h a c e t y l a c e t o n e and ammonia.
An
improved colorimetric
assay
for
glycosylated
h a e m o g l o b i n depends o n t h e c o n v e r s i o n o f t h e p - g l u c o s e m o i e t y t o 5h y d r o x y m e t h y l f u r f u r a l d e h y d e by o x a l i c a c i d , estimation
of
t h i o b a r b i t u r i c acid.802 a f f e c t e d by
with
2-
The u t i l i t y o f g l y c o s y l a t e d - h a e m o g l o b i n
measurement as an i n d e x o f adversely
f o l l o w e d by c o l o r i m e t r i c
5-hydroxymethylfurfuraldehyde
the
chronic control i n diabetes
interference
from
a
labile
can be
glycosylated
f r a c t i o n t h a t changes r a p i d l y w i t h b l o o d Q - g l u c o s e c o n c e n t r a t i o n . 8 0 3 T h i s f r a c t i o n may b e m e a s u r e d b y h.p.1.c.
or by
electrophoresis.
I n t e r f e r e n c e by Q - g l u c o s e i n t h e c o l o r i m e t r i c e s t i m a t i o n o f g l y c o s y l a t e d p l a s m a p r o t e i n h a s b e e n e l i m i n a t e d by p r e c i p i t a t i o n o f t h e p r o t e i n s w i t h t r i c h l o r a c e t i c acid.804 G l y c o s y l a t e d m i n o r c o m p o n e n t s o f human f e t a l h a e m o g l o b i n h a v e been s e p a r a t e d by ion-exchange
chromatography,
functionally
Sources o f v a r i a t i o n i n t h e i o n -
characterized.805
exchange column
chromatographic
identified,
determination
of
and
glycosylated
h a e m o g l o b i n (HbA) h a v e b e e n d e s c r i b e d . 8 0 6 A
simple,
commercially
a v a i l a b l e method f o r separating
g l y c o s y l a t e d haemoglobin e l e c t r o p h o r e t i c a l l y been
assessed.807
The
method
correlated
on a g a r - g e l well
chromatographic technique. 8-Glucose a l s o b i n d s c o v a l e n t l y t o €-amino
with
f i l m s has a
column
groups o f I - l y s i n e
Carbohydrate Chemistry
266 o f human a p o l i p o p r o t e i n s . 8 0 8
The l e v e l o f g l y c o s y l a t e d a p o p r o t e i n
B o f l o w - d e n s i t y l i p o p r o t e i n s i s i n c r e a s e d i n serum f r o m d i a b e t i c patients. Nonenzymatic g l y c o s y l a t i o n o f p r o t e i n s i n d i a b e t i c s extends beyond h a e m o g l o b i n t o t h e p r o t e i n s o f t h e e r y t h r o c y t e membrane, probably affects
and
o t h e r p r o t e i n s t h a t have s l o w t u r n o v e r a n d a r e
exposed t o h i g h c o n c e n t r a t i o n o f ~ - g l u c o ~ e . ~ ~ ~ Human p l a s m a c o n t a i n s a f a c t o r , factor,
terminal autorosette i n h i b i t i o n
w h i c h can i n h i b i t t h e a b i l i t y o f m u r i n e l y m p h o c y t e s t o b i n d
a u t o l o g o u s e r y t h r o c y tes.810
The p u r i f i e d f a c t o r h a s been i d e n t i f i e d
w t . 8.0 x l o 4 ) , b e i n g a l a r g e r - m o l e c u l a r f r o m w h i c h h i s t i d i n e - r i c h g l y c o p r o t e i n and,
a s a g l y c o p r o t e i n (mol. weight
precursor
possibly,
p l a s m i n o g e n - b i n d i n g p r o t e i n a r e d e r i v e d by p r o t e o l y s i s
during purification. H i s t i d i n e - r i c h g l y c o p r o t e i n f r o m r a b b i t serum b i n d s Cu2+, Zn2+, Hg2+, Cd2+, N i 2 + , consistent
a n d Co2+, w i t h h i g h a f f i n i t y ,
with
t h i s protein having a
vitro, which i s
role i n transport
or
h o m e o s t a s i s o f m e t a l s i n serum.811 Human B 2 - m i c r o g l o b u l i n glycoprotein.812 complex
(mol.
forms
After injection wt,
x
5.5
lo4
-
in
vivo
the
lo4),
x
6.7
with
complexes
serum
rat
with
but
serum
contains a
i;
vitro
i n c u b a t i o n t h e B 2 - m i c r o g l o b u l i n f o r m s a d d i t i o n a l c o m p l e x e s (rnol. 2.0
wt.
lo5). A
com pa r i so n
of
serum
a n a l y s i s by e n z y m e - l i n k e d
pregnancy- s p e c i f i c
B, - g l y c o p r o t e i n
i m m u n o s o r b e n t assay a n d r a d i o i m m u n o a s s a y
has g i v e n good c o r r e l a t i o n between t h e t w o methods.813 A g l y c o p r o t e i n (mol. w t . 5.0 x l o 4 > a s s o c i a t e d w i t h m a l i g n a n c y h a s been i s o l a t e d f r o m human s e r a from c a n c e r p a t i e n t s . 8 1 4 Reaction
of t h i s glycoprotein w i t h various antisera
directed against
normal
serum g l y c o p r o t e i n s h a s e s t a b l i s h e d t h a t i t i s n o t one o f t h e m a j o r ac u t e-phase r e a c t a n t g l y co p r o t e i n s a s s o c i a t e d w it h m a 1 ig n a n cy a n d can be d i s t i n g u i s h e d f r o m c a r c i n o e m b r y o n i c a n t i g e n and a - f e t o p r o t e i n by i t s m o l e c u l a r w e i g h t and c h r o m a t o g r a p h i c b e h a v i o u r . The i n f l u e n c e o f t e m p e r a t u r e ,
pH,
and v a r i o u s enzymes o n t h e
i n t e rme d i a t e s t r u c t u r e s f o r m e d i n t h e t r a n s f o r m a t i o n o f t o f i b r i n h a s been r e p o r t e d . 8 1 5 rod-like
intermediates are
a g g r e g a t i o n p r o c e s s when Babroxobin
(an enzyme
Light-scattering formed a t
fibrinogen
preparation
the
from
the
venom
of
the
beginning
i s treated with
for
the
B o t h r o p s a t r o x m a j o e n s i s l , and C o n t o r t r i x
f i b r i n o gen
s t u d i e s show t h a t
venom
of
of
the
thrombin, the
snake
(an enzyme p r e p a r a t i o n
copperhead snake Ancistrodon c o n t o r t r i x
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
.
261
contortrix) E x t e n s i v e i n a c t i v a t i o n o f t h e i r o n - b i n d i n g c a p a c i t y o f human t r a n s f e r r i n o c c u r s when a p p r o x i m a t e l y f o u r & - t y r o s i n e r e s i d u e s a r e o x i d i z e d w i t h sodium periodate.816 The c o r r e l a t i o n o f d e s t r u c t i o n of t h i s amino a c i d w i t h t h e l o s s o f i r o n - b i n d i n g a c t i v i t y s u g g e s t s t h a t t h e & - t y r o s i n e r e s i d u e s are t h o s e t h a t f u n c t i o n a s l i g a n d s t o metal i o n s bound t o the p r o t e i n . Human s e r u m t h y r o x i n e - b i n d i n g g l o b u l i n h a s b e e n a f f i n i t y l a b e l l e d w i t h N - b r om o a c e t y 1-I- t h y r o x i n e . &-Met h i o n i n e was i d e n t i f i e d as t h e major residue labelled. M i c r o h e t e r o g e n e i t y of a - f e t o p r o t e i n i n human s e r u m has been A practical and d e m o n s t r a t e d by l e c t i n a f f i n o e l e c t r o p h o r e s i s . 8 1 8 e c o n o m i c enzyme immunoassay f o r human a - f e t o p r o t e i n h a s been developed and its performance evaluated.819 Some s t u d i e s o n b i n d i n g a n d i n t e r n a l i z a t i o n o f a s i a l o g l y c o p r o t e i n s by i s o l a t e d r a t h e pa t o cy t e s h a v e b e e n r e p o r t e d .820 The c o nf orm a t i o n s o f t h e N-gl y c o s y l a t e d o l i g o sa c c h a r i d e c h a i n s o f a glycopeptide derived from f e t u i n h a v e b e e n e x a m i n e d by hydrogen-exchange techniques.821 Eight acetamido hydrogens are p r e s e n t , o ri gina t i n g from f i v e 2-acetamido-2-deoxy-eglucosyl r e s i d u e s a n d from three N - a c e t y l n e u r a m i n i c a c i d r e s i d u e s , t o g e t h e r w i t h one hydrogen from t h e oligosaccharide-peptide linkage. Seven of t h e hydrogens exchange a t a rate i n d i c a t i n g free c o n t a c t w i t h s o l v e n t , b u t t h e r e m a i n i n g t w o hydrogens exchange a t a s i g n i f i c a n t l y s l o w e r rate, i m p l y i n g t h e i r i n v o l v e m e n t i n i n t r a m o l e c u l a r hydrogen bonds. The t o t a l carbohydrate content and t h e monosaccharide c o m p o s i t i o n of p l a s m a a p o l i p o p r o t e i n s i s o l a t e d f r o m n o r m a l a n d 2am i no -2 - d e ox y -Q - ga 1a c t o se - t rea t e d r a t s h a ve be e n r e c o r de d. 8 2 2 Radioimmunoassay t e c h n i q u e s have been used t o e v a l u a t e t h e contribution of the carbohydrate moiety t o the immunological r e a c t i v i t y o f human serum l o w - d e n s i t y l i p o p r o t e i n . 8 2 3 Neuraminic a c i d a n d p-mannose, t h e t e r m i n a l r e s i d u e s o f low-density l i p o p r o t e i n g l y c o p e p t i d e s I a n d 11, a r e i m p l i c a t e d i n t h e a n t i g e n i c s i t e ( s ) . The i n t e r a c t i o n s o f f i b r i n o g e n and a s i a l o f i b r i n o g e n w i t h concanavalin A and the blood-clotting properties of the complexes have been described.824 Two o f t h e f o u r p o s s i b l e b i n d i n g s i t e s o n t h e fibrinogen molecule are not a c c e s s i b l e t o concanavalin A tetramers. Asialofibrinogen and its concanavalin A complexes c o a g u l a t e twice a s f a s t a s t h o s e o f f i b r i n o g e n . The t r a n s l a t e d p r o d u c t s o f r a t l i v e r m e s s e n g e r RNA, t o t a l p o l y s o m e s , a n d r o u g h
Carbohydrate Chemistry
268
microsomes i n c l u d e g l y c o s y l a t e d s u b u n i t s o f fibrinogen.825 Both t h e BB- a n d t h e y - c h a i n s a r e g l y c o s y l a t e d I-asparagine moieties and t h e Aa-chains are n o t g l y c o s y l a t e d . The y - c h a i n i s g l y c o s y l a t e d a s a n e a r l y c o t r a n s l a t i o n a l e v e n t , w h i l e t h e BB-chain i s g l y c o s y l a t e d a t o r s l i g h t l y a f t e r t h e t e r m i n a t i o n of p o l y p e p t i d e s y n t h e s i s o f t h a t chain. The p r e s e n c e o f o l i g o s a c c h a r i d e c h a i n s o n I - a s p a r a g i n e - 2 8 8 o f human p l a s m i n o g e n d e c r e a s e d the b i n d i n g o f f r a g m e n t s c o n t a i n i n g t h e h i g h - a f f i n i t y L - l y s i n e - b i n d i n g s i t e ( r e s i d u e s 79-337 o r 7 9 - 3 5 3 ) t o both a2-antiplasmin and fibrin.826 A technique f o r the i d e n t i f i c a t i o n o f glycoprotein antigens i n immune c o m p l e x e s i s o l a t e d from sera of p a t i e n t s w i t h B u r k i t t ' s lymphoma and n a s o p h a r y n g e a l c a r c i n o m a h a s been reported.827 Two c l o s e l y r e l a t e d g l y c o p r o t e i n s were i d e n t i f i e d i n 80% o f t h e s e r a e x a m i n e d f r o m p a t i e n t s w i t h t h o s e t w o d i s e a s e s , b u t were n o t p r e s e n t i n p a t i e n t s w i t h a v a r i e t y of u n r e l a t e d tumours o r i n sera of heal t h y i n d i v i d u a l s . The c o m p a r a t i v e a n a l y s i s o f t h e l e c t i n - b i n d i n g p r o p e r t i e s o f p l a s m a g l y c o p r o t e i n s f r o m c o n t r o l a n d c y s t i c - f i b r o s i s p a t i e n t s has been reported.828 The r e s u l t s o f a comparison o f plasma g l y c o p r o t e i n s f r o m n o rm a 1 c o n t r o l s , c y s t i c - f i b r o s i s p a t i e n t s, a n d c y s t i c - f i b r o s i s h e t e r o z y g o t e s do n o t s u p p o r t t h e h y p o t h e s i s o f a g e n e r a l i z e d d e f e c t i n g l y c o p r o t e i n metabolism a s the b a s i c defect i n c y s t i c fibrosis.829 However, t h e p o s s i b i l i t y o f a m o r e l i m i t e d d e f e c t i n t h e metabolism of c e r t a i n o t h e r t y p e s o f glycoproteins, possibly those involved i n t h e processes o f exocrine excretion, is considered. T r a n s c o r t i n , t h e m a j o r human p l a s m a g l u c o c o r t i c o i d , h a s been i s o l a t e d a n d p a r t i a l l y c h a r a ~ t e r i z e d . ~ ~ 'S o m e o f i t s phy si c o chem i c a 1 pa ram e t e r s, pa r t i c u l a r l y i t s m i c r o h e t e r o g e n e i t y a n d i t s propensity f o r aggregation, are described. Isopiestic data indicate that fish antifreeze glycoproteins b i n d s i g n i f i c a n t l y m o r e water t h a n h a e m o g l o b i n o r c y t o c h r o m e c , o b s e r v a t i o n s w h i c h a r e c o n s i s t e n t w i t h n.m.r. spectroscopic s t u d i e s 831 An i m p r o v e d p r o c e d u r e f o r t h e d e t e r m i n a t i o n o f a n t i f r e e z e g l y c o p r o t e i n a c t i v i t y u s i n g a f r e e z i n g - p o i n t o s m o m e t e r has b e e n described.832 The s e n s i t i v i t y o f t h e m e t h o d h a s a l l o w e d t h e identification of a hitherto overlooked antifreeze a c t i v i t y i n the A t l a n t i c cod (Gadus morhua).
.
269
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides I m m urn g l o b u l i n s
13 A
r a p i d method f o r simultaneously
complexes o f numerous sera
for
the
a n t i g e n h a s been developed.833
s c r e e n i n g t h e immune
p r e s e n c e o f an
unsuspected
An a n t i b o d y - e n z y m e r e a g e n t c a n
i d e n t i f y f r e e x - c a s e i n when no a n t i b o d y i s p r e s e n t a n d a l s o x - c a s e i n i f i t i s bound i n a n immune complex a b s o r b e d o n t o R a j i c e l l s .
level of sensitivity
allowed the demonstration o f
The
the development
and subsequent d i s a p p e a r a n c e o f x - c a s e i n immune complexes i n t h e sera o f e i t h e r immunoglobulin A-deficient o r m i l k - a l l e r g i c p a t i e n t s who h a v e i n g e s t e d a s m a l l amount o f m i l k . I s o e l e c t r i c f o c u s i n g o f a n t i b o d i e s i n agarose g e l s has been r e p o r t e d u s i n g i n e x p e n s i ve l a b o r a t o r y - s y n t h e s i z e d am p h o l y t e s . 8 3 4 The r e s o l v i n g p o w e r o f
these and commercially a v a i l a b l e ampholytes
a r e compared, a n d t h e a d v a n t a g e s o f s u b s t i t u t i n g a g a r o s e g e l s b o n d e d t o p l a s t i c f i l m s f o r polyacrylamide g e l s a r e discussed.
Soluble
immune c o m p l e x e s h a v e been s t u d i e d u s i n g a p r o c e d u r e o f b i n d i n g t o i m m o b i l i z e d p r o t e i n A f o l l o w e d by i s o e l e c t r i c f o c u s i n g t o d e s o r b a n d separate
the
immune
complexes.835
By
this
approach
IgG-type
i m m u n o g l o b u l i n s a n d p u t a t i v e a n t i g e n s can be d e t e c t e d i n b i o l o g i c a l
f l u i d f r o m human specimens. Differences i n binding o f r a t immunoglobulin G sub-classes (IgG1,
IgG2,
IgGZb,
reported.836
a n d I g G 2 c ) t o s t a p h y l o c o c c a l p r o t e i n A have been
A p r o c e d u r e f o r t h e i s o l a t i o n o f p u r e I g G l and I g G 2 c
f r o m n o r m a l r a t s e r u m was d e v e l o p e d .
Immunoglobulins associated
w i t h human p l a t e l e t s b i n d t o i m m o b i l i z e d p r o t e i n A, o f the platelets.837 thrombocytopenic
with r e t e n t i o n
T h i s t e c h n i q u e may be o f v a l u e i n t h e s t u d y o f patients
where
greatly
increased
amounts
o f
a s s o c i a t e d I g G a r e found. The
covalent
attachment
of
and
immunoglobulin G
F(aby)2
f r a g m e n t s t o l i p o s o m e s i s a c h i e v e d by t h e p r i o r p e r i o d a t e o x i d a t i o n o f g l y c o s p h i n g o l i p i d s i n liposomes t o produce aldehyde groups o n t h e vesicle surface,
f o l l o w e d by
protein coupling
a m i n a t i ~ n . ~ U n~d ~ e r t h e c o n d i t i o n s used, external glycolipid, Encapsulated g l y c e r o l v e s i c l e s and r e t a ine d
.
but not internal glycolipid, l-phosphate
encapsulated
The F ( a b ’ I 2
by
fragment
i s
not
goat
i s oxidized.
oxidized
carboxyfluorescein i s o f
reductive
a large proportion o f the within
the
substantially
i m m u n o g l o b u l i n G h a s been
co v a l e n t l y c o u p l e d t o pho s p h a t i d y l e t h a n o l a m i n e w i t h s u b s e q u e n t
Carbohydrate Chemistry a s s o c i a t i o n of t h i s complex w i t h liposomes.839 B i n d i n g assays w i t h antigen-coated Staphylococcus aureus c e l l s a r e used t o e v a l u a t e t h e i m m u n o l o g i c a l l y s p e c i f i c r e c o g n i t i o n and t h e membrane s t a b i l i t y o f t h e liposomes.
Comparative k i n e t i c s t u d i e s o f
ligand dissociation
and D20-induced f l u o r e s c e n t enhancement have been p e r f o r m e d w i t h b o t h h e t e r o g e n e o u s and homogeneous a n t i f l u o r e s c e i n y l i m m u n o g l o b u l i n G antibodies.840
The a n t i - f l u o r e s c e i n
antibody active s i t e contains
b o t h s o l v e n t - a c c e s s i b l e and r e l a t i v e l y - i n a c c e s s i b l e components,
with
t h e b i n d i n g o f l i g a n d i n v o l v i n g b o t h exchangeable hydrogen atoms and other
as
yet
unresolved
interactions.
The
mechanism
of
020
fluorescence enhancement i s discussed i n t e r m s o f i t s c o m p l e x i t y i n v o l v i n g h e t e r o g e n e o u s r a t e mechanisms. Several b i o l o g i c a l effector F c r e g i o n of variant
f u n c t i o n s m e d i a t e d by s i t e s on t h e
human i m m u n o g l o b u l i n G 1 h a v e b e e n s t u d i e d i n t w o
i m m u n o g l o b u l i n G1K
monoclonal
proteins
which
contain
d e l e t i o n s corresponding t o t h e e n t i r e hinge r e g i o n o f t h e heavy chains.841
The f u n c t i o n a l l a c k o f p o t e n c y o f t h e s e i m m u n o g l o b u l i n s
i s r e l a t e d t o t h e c l o s e a s s o c i a t i o n between Fab and Fc r e g i o n s i n t h e s e m o l e c u l e s and t h e l i m i t e d degree o f s e g m e n t a l f l e x i b i l i t y p e r m i t t e d i n t h e absence o f t h e h i n g e r e g i o n .
A major r o l e f o r t h e
C-y2 d o m a i n i n m e d i a t i n g e f f e c t o r f u n c t i o n s i n n o r m a l i m m u n o g l o b u l i n G 1 i s proposed. A model system,
b a s e d on t h e d e s i a l y l a t i o n o f i m m u n o g l o b u l i n G
and g e n e r a t i n g one o f t h e s e r o l o g i c a l changes n o r m a l l y a s s o c i a t e d w i t h t h e p a t h o l o g i c a l c o n d i t i o n o f r h e u m a t o i d a r t h r i t i s , has been developed.842
Rabbit asialo-immunoglobulin
i s immunogenic i n autologous hosts.
G
t e n d s t o a g g r e g a t e and
The n e u r a m i n i c a c i d c o n t e n t o f
r h e u m a t o i d f a c t o r i s o l a t e d f r o m t h e serum o f a r h e u m a t o i d p a t i e n t and i d e n t i f i e d as i m m u n o g l o b u l i n G and i m m u n o g l o b u l i n M i s a l s o lower than t h a t o f the corresponding normal immunoglobulins. I m m o b i l i z e d p e p s i n has been used f o r t h e l i m i t e d c l e a v a g e o f human i m m u n o g l o b u l i n G.843
The F ( a b ’ ) 2 - r i c h
f r a c t i o n was
isolated
f o r i n t r a v e n o u s use. Proximity
relationships
within
the
Fc
segment
of
rabbit
i m m u n o g l o b u l i n G h a v e been a n a l y s e d b y r e s o n a n c e - e n e r g y t r a n s f e r . 8 4 4 The
principle
forces
maintaining
the
integrity
of
the
native
f u n c t i o n a l Fc fragment are t h e strong noncovalent i n t e r a c t i o n s of t h e CH3 d o m a i n s a n d t h e s i n g l e i n t e r - h e a v y - c h a i n
d i s u l p h i d e bond.
T h e c a r b o h y d r a t e s t r u c t u r e s ( 3 9 , 40) a n d t h e c o m p l e t e a m i n o a c i d s e q u e n c e o f a human X - t y p e Sm,
have been determined.845
immunoglobulin l i g h t chain,
One g - g l y c o s i d i c a l l y
protein
l i n k e d c h a i n (40)
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
o u
LL I JI I
Q : I 011
z
a
4
a I
1311
m I
h
U
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4
u
AJ
m m - 4
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I 1311
m
r
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m
a
n
n
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UJ 4
u I
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r
I 1311 I
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0
ct
z al
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4
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o
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z
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27 1
272
Carbohydrate Chemistry
i s attached t o I-Ser-21.
T h i s a p p e a r s t o be t h e f i r s t r e p o r t o f a
tetrasaccharide containing two neuraminic a c i d residues i n the l i g h t c h a i n o f an i m m u n o g l o b u l i n . O l i g o s a c c h a r i d e s o f s i m i l a r s t r u c t u r e o c c u r i n human X - t y p e m o n o c l o n a l i m m u n o g l o b u l i n l i g h t c h a i n s .846
~-N~UQ~AC-(~+~)-B-Q-G~~Q-(~+~)-~-G~~QNAC-L-S~~ 6
I 2 o - Ne u ~ 5 cA
(40) A human m o n o c l o n a l i m m u n o g l o b u l i n G 1 p o s s e s s e s t w o s p e c i e s o f
l i g h t c h a i n ( m o l . w t s . 2.3 x l o 5 a n d 2.8 x lo5, r e ~ p e c t i v e l y ) . ~ ~ ’ The d i f f e r e n c e i n mass is due t o t h e p r e s e n c e o f a c a r b o h y d r a t e m o i e t y on t h e 2.8 K-chains.
x lo4 species, attached w i t h i n the J region o f the
Not a l l of
t h e K-chains
differences i n the primary
are glycosylated,
e v e n t h o u g h no
s t r u c t u r e between the
two
L-chains
s p e c i e s c o u l d be f o u n d . mouse myeloma c e l l l i n e p r o d u c e s an i m m u n o g l o b u l i n G2b w i t h
A
two carbohydrate attachment sites.848 also
occur
i n
two
variant
cell
Alterations i n glycosylation
lines.
The
complete
s t r u c t u r e o f the constant r e g i o n o f L-chains homogeneous
Balilea
rabbit
antibody
primary
derived from
specific
for
type
a I1
pneumococcal p o l y s a c c h a r i d e h a s been e ~ t a b l i s h e d , ~ a ~ s’ h a s t h e a m i n o a c i d s e q u e n c e o f an e u g l o b u l i n - l i k e
X Bence-Jones p r o t e i n NIG-
58. 850 Antibodies
have
been r a i s e d t o
glycolipids,and polysaccharides.
numerous
glycoproteins,
Those a n t i b o d i e s k n o w n t o i n t e r a c t
w i t h carbohydrate d e t e r m i n a n t s on t h e a n t i g e n molecule a r e l i s t e d i n T a b l e 11. Further studies of
the
inhibition of
the monoclonal a n t i -
Ma(group 1) a n t i b o d y i n q u a n t i t a t i v e p r e c i p i t i n a s s a y s h a v e been reported.852 o f
a
I t i s confirmed t h a t the antibody binds t o a portion
B-~-galactopyranosyl-(1+4)-~-B-~-2-acetamido-2-deoxy-
glucopyranosyl-(1+6)-~-~-~-galactopyranosylunit oligosaccharide
structure
i n
a
specific
conformational requirements indicate
that
of
conformation. the
an The
determinant
is
accepted i n t o the antibody-com bining s i t e along a hydrophobic portion of the structure
which includes t h e regions about
C6-Cl’-
;-Galactan
(Continued o v e r l e a f )
Glc~-(1+6)-a-~-Glc~-(1+3)-~-Glc B-(l+6)-!-galactopyranan
1+6)-g-Glc,
a-p-Glce-(
1+3)-a-!-Glce-(
m e t h y l a-g-ManQ
O e x t r a n 81355
Polysaccharides D e x t r a n 8512
!-Gal,
a-g-
p-GlcNAc,
g l ycopro te i n R e t r o v i r u s envelope g l y c o p r o t e i n
Monoclonal
Polyclonal
Monoclonal
Polyclonal
Po l y c l o n a l
I n t a c t oligosaccharide chain
Bovine leukaemia envelope v i r a l
856
855
854
21
12,
13
853
852
Mono c l o n a 1
Ma ( g r o u p 1)
Polyclonal
851
Ref.
Monoclonal
6-g - G a l e - ( 1+4)- 8-9-G lceNA c - ( 1+6 ) - 8-g - G a l e
;-Mane
a-g -Mane
Neue5Ac, a,B-Q-Galg, -
Antibody type
Glucoamy l a s e
a-g-Mane,
on a n t i g e n
g l y c o l i p i d or polysaccharide antigens
Determinant(s)
Antibodies i n t e r a c t i n g with glycoprotein,
Glycoproteins H i s t o c o m p a t i b i l i t y a n t i g e n (H-2K)
Antigen
Table I1
E
E.
3
w
N 4
8
s5
c,
5
h
k 2-
"2 g
0
s
2
Q.
-8
B=.
s
%-
Q
"
a
a-L -F u c~
1
I
3
1 a-L-F u c ~
I
a-~-Fuc~-(1+2)-B-~-Gale-(1+3)-B-~-Glc~NAc-~-~-Gal~-(l~4)-~-Glc 4
SSEA-1
B-g-Gale-(1+4)-g-Glc~NAc, a - L-- F u c e - ( l + Z ) B-q-Galp-(1+4)-q-GlcpNAc B-g-Galp-(1+4)-B-g-Glc~NAc
a - L-- F u c p - ( 1 + 4 ) - g - G l c e " c
Glycosphingolipids
See
Lewisb blood group
below
D e t e r r n i n a n t ( s ) on a n t i g e n
Lewis2 blood group
Glycolipids
Antigen
T a b l e I1 (Continued)
Monoclonal
Monoclonal
Polyclonal
Monoclonal
Antibody t y p e
860
859
858
857
Ref.
P
4
N
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
275
C3’-05”-C6” a s r e p r e s e n t e d i n S c h e m e 2. I t is a s s u m e d t h a t 03’ is i n t r a m o l e c u l a r l y h y d r o g e n b o n d e d t o 05’’ a n d t h a t t h e o t h e r h y d r o x y l groups o f t h e determinant remain engaged in bonding with solvent water. I
I I
combining site .hydrophobic
edge
D i s p l a y s h o w i n g t h a t t h e c o n f o r m a t i o n o f t h e I Ma a n t i genic determinant possesses a region along C6Cl’-C3’-05’’-C6’’ w h i c h c a n b e e x p e c t e d t o be compatible with a hydrophobic region f o r t h e combining site Scheme 2 R a b b i t a n t i - d e x t r a n €31355 h a s b e e n s e p a r a t e d i n t o t w o Gfractions.855 Antibodies of a non-binding f r a c t i o n t o Sephadex 75 are d i r e c t e d a g a i n s t ~-a-Q-glucopyranosyl-(l+6)-~-a-~glucopyranosyl-(1+3)-~-glucosyl r e s i d u e s . The a f f i n i t i e s of t w o m o n o c l o n a l anti-!-galactan immunoglobulin Fab’ fragments have been measured with a linear and a highly b r a n c h e d Q - g a l a ~ t a n . ~A~f f~i n i t y d a t a s h o w t h a t t h e i m m u n o g l o b u l i n s c a n bind i n t e r c a t e n a r i l y t o a (1+6)-B-Q-galactopyranan b u t n o t t o a ( 1+3)-B-~-galactopyranan. F o u r m o n o c l o n a l a n t i b o d i e s p r o d u c e d by hybridomas o b t a i n e d from a mouse immunized with a human a d e n o c a r c i n o m a c e l l l i n e a r e d i r e c t e d a g a i n s t t h e Leb a n t i g e n o f t h e T h e i r s p e c i f i c i t i e s w e re h u m a n Lewis b l o o d - g r o u p s y s t e m . 8 5 7 e s t a b l i s h e d by b i n d i n g s t u d i e s u s i n g p u r i f i e d Leb - a c t i v e g l y c o l i p i d s a n d by h a p t e n i n h i b i t i o n s t u d i e s w i t h o l i g o s a c c h a r i d e s o b t a i n e d f r o m h u m a n milk. T h e a n t i g e n s p e c i f i c i t i e s o f d i f f e r e n t a n t i - L e w i s z sera h a v e b e e n e x a m i n e d by i m m u n o a d s o r p t i o n s t u d i e s u s i n g a d s o r b e n t s w i t h well d e f i n e d c a r b o h y d r a t e u n i t s c o v a l e n t l y b o u n d t o a n i n o r g a n i c
276
Carbohydrate Chemistry
matrix.858 I n c o n t r a s t t o t h o s e of normal a n t i - L e a and a n t i - L e i s e r a , t h e a n t i b o d y - b i n d i n g s i t e o f L e r a n t i b o d i e s was f o u n d t o b e c o n s i d e r a b l y smaller , c o m p r i s i n g t h e s t r u c t u r e a - & - F u c e - ( 1 + 4 ) - Q G lceNAc Two monoclonal a n t i c a r b o h y d r a t e a n t i b o d i e s d i r e c t e d t o w a r d t h e c a r b o h y d r a t e m o i e t y o f g l y c o s p h i n g o l i p i d s c o n t a i n i n g a lacto-!O n e is s p e c i f i c f o r g l y c o s y l T y p e I1 c h a i n h a v e b e e n p r e p a r e d . 8 5 9 t h e T y p e I1 c h a i n H d e t e r m i n a n t ( 4 1 ) a n d t h e o t h e r is d i r e c t e d t o t h e n o n - r e d u c i n g t e r m i n a l 2 - a c e t a m i d o - 2 - d e o x y -4-0-13- Q - g a l a c t o s y l - Q glucosyl disaccharide.
.
a-~-Fucp-(1+2)-B-Q-Gal~-(l+4)-~-Q-GlcpNAc-l+R (41) R = r e s t o f o l i g o s a c c h a r i d e chain A monoclonal antibody r e a c t i n g with e a r l y mouse embryos and m u r i n e e m b r y o n a l c a r c i n o m a cells d e f i n e s t h e s t a g e - s p e c i f i c e m b r y o n i c a n t i g e n ( S S E A - 1 ) a n d is s p e c i f i c f o r t y p e 2 b l o o d - g r o u p antigens.860 T h e c o m b i n i n g s i t e o f t h e a n t i b o d y is c o m p l e m e n t a r y t o t h e s e q u e n c e (42). The a n t i g e n has b e e n i d e n t i f i e d as a complex g l y c o l i p i d .861 ,862 A cold agglutinin resembling anti-I antibodies has been i s o l a t e d from a p a t i e n t s u f f e r i n g from i m m u n ~ b l a s t o m a . ~ ~ ~ H i g h - t i t r e a n t i s e r a s p e c i f i c f o r Lewisa, b, a n d d d e t e r m i n a n t s h a v e b e e n o b t a i n e d by t h e i m m u n i z a t i o n o f r a b b i t s w i t h a r t i f i c i a l a n t i g e n s p r e p a r e d by c o u p l i n g c h e m i c a l l y s y n t h e s i z e d h a p t e n s t o These r e a g e n t s h a v e b e e n u s e d i n t h e bovine serum albumin.864 immunohistochemical analysis of t h e s e antigens i n t h e mucosa of t h e human s t o m a c h a n d d u o d e n u m o f H g r o u p i n d i v i d u a l s .
B-Q-Gale-(1+4)-B-g-Glc~NAc-(l+6)R 3
I 1
-F u c p (42) R = remaining oligosaccharide chain
a-
Monoclonal antibodies, which bind t o an a n t i g e n p r e s e n t i n highly purified carcinoembryonic antigen (CEA) from various tumours, T h e y are c o n s i s t e n t with t h e c o n c e p t t h a t h a v e been isolated.865
277
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides different
immunological
distinguished
by
forms
appropriate
i m munlogical heterogeneity of
many
conventional
of
CEA
antisera. CEA
anti-CEA
exist
which
How e v e r ,
and t h e
sera,
the
in
can
be
of
the
view
polyspecific nature o f possibility
that
these
monoclonal sera are n o t recognizing a unique antigen n o t r e l a t e d t o CEA c a n n o t be excluded. Evaluation
of
a
baboon
antiserum
to
CEA
has
given
similar
r e s u l t s t o a c o m m e r c i a l l y a v a i l a b l e g o a t anti-CEA.866 S p e c i f i c antibody t o a - f e t o p r o t e i n has been prepared t o bind s p e c i f i c a l l y t o a-fetoprotein-producing Human antibodies t o f a c t o r
VIII h a v e b e e n i s o l a t e d f r o m n i n e
Subsequent i m munological c h a r a c t e r i z a t i o n s
h a e m o p h i l i c plasmas.868
revealing restricted heterogeneity i n both light-chain heavy-chain
sub-class
antigen
been
assays,
has
and f o u n d
tumour c e l l s .867
have been described.
measured
with
two
Factor
t y p e and
VIII c l o t t i n g
i m munoradiometric
different
one u t i l i z i n g t w o acquired antibodies and one a haemophilic
a n t i b o d y .768 Monoclonal antibodies i s o l a t e d and t h e i r
to
von
Willebrand’s
with
reactivity
factor
have
been
porcine and human antigens
reported.869 Monoclonal
antibodies
against
human a c i d
a-glucosidase
have
b e e n ~ r e p a r e d . ~ ” T h e a n t i b o d i e s w e r e u s e d t o show t h a t t w o m a j o r c o m p o n e n t s of
the
purified
enzyme
c o n t a i n a t l e a s t one a n t i g e n i c
component i n common. An e n z y m e i m munoassay f o r a u t o a n t i b o d i e s t o t h y r o g l o b u l i n i n h u m a n s e r u m h a s b e e n developed.871 comparison
with
an
indirect
E v a l u a t i o n o f t h e technique by
haemagglutination test
for
anti-
t h y r o g l o b u l i n a n t i b o d i e s gave a c o r r e l a t i o n c o e f f i c i e n t o f 0.85. Sera
from
patients
with
Graves’
disease
contain
antibodies
w h i c h i n h i b i t t h e b i n d i n g o f t h y r o t r o p h i n t o t h y r o i d membranes.872 Immunoglobulin
i s o l a t e d from
G,
t h e sera,
inhibits the binding o f
t h e hormone by b i n d i n g d i r e c t l y t o t h e hormone r e c e p t o r . Antibodies used i n an enzyme immunoassay f o r human c h o r i o n i c gonadotrophin ( h C G ) after
affinity
have been i s o l a t e d i n a h i g h l y
chromatography
on
colums
of
purified form i m mobilized
t r i c o s a p e p t i d e o f t h e hC G Rabbit
transferrin
methionine sulphone
has
been
modified
hydrazide either into
by
introduction
i t s neuraminic
of
acid
r e s i d u e s or i n t o b o t h n e u r a m i n i c a c i d and Q - g a l a c t o s y l residues.873 Rabbits i m munized with t h e modified t r a n s f e r r i n produced antibodies which r e a c t e d w i t h t h e n a t i v e antigen.
278
Carbohydrate Chemistry Mouse i m m u n o g l o b u l i n
G 3 antibodies t o t h e phosphocholine
d e t e r m i n a n t of p n e u m o c o c c a l C c a r b o h y d r a t e a n d t o t y p e 3 pneu m o c o c c a l c a r b o h y d r a t e a r e h i g h l y p r o t e c t i v e against experimental type 3 pneumococcal infection. 874 that
antibodies
of
the
against bacterial infections. ag a inst
A n t ib o d i e s
glucosyltransferase
from
ability to inhibit the The
cross-reactivity
This i s one o f t h e f i r s t r e p o r t s
immunoglobulin D=
G3
protect
- 1 y s y 1: D= -
embryos have been t e s t e d f o r t h e i r
e n r y me r e a c t i o n w i t h the
can
- g a l a ct 0 sy l - hy d r o x y -
chick
of
sub-class
v a r i o u s substrates.875
glucosyltransferase
between
different
species i s low. Monoclonal r a t immunoglobulin G2b
antibody,
a g a i n s t t h e Thy 1.2 a n t i g e n , h a s b e e n c o v a l e n t l y Addition
of
lactose
saturates
the
r i c i n and i n h i b i t s t h e t o x i n f r o m
P_-galactosyl-containing
which i s d i r e c t e d bound t o
Q-galactosyl- binding
sites
b i n d i n g and k i l l i n g c e l l s & a
receptors.
Ricin-monoclonal
h y b r i d s o f t h i s t y p e combine a h i g h degree o f c e l l - t y p e
on the
antibody selectivity
and may have p h a r m a c o l o g i c a l use as a n t i t u m o u r r e a g e n t s . H i gh l y
sp ec i fi c
antibodies
dire c ted
against
c e l l - s u r f ace
i m m u n o g l o b u l i n s on n o r m a l o r n e o p l a s t i c murine B l y m p h o c y t e s have been c o v a l e n t l y molecules
coupled t o
maintain
t o x i c properties.
both
the
their
A
chain
of
r i ~ i n . T~h e~ h~y b r i d
antigen-binding
capacity
and
their
Minute amounts o f t h e conjugates are e f f e c t i v e i n
k i l l i n g target cells i n vitro. The
8aB5-9
antibody
Lab,
deficient
in
cloned hybridoma c e l l l i n e produces a
d i r e c t e d against a major platelet-surface platelets
monoclonal antibody,
of
patients
with
monoclonal
glycoprotein
t h r o m b a ~ t h e n i a . ~ T~h i~s
which i s d i r e c t e d against a platelet-mem brane
complex consisting o f glycoproteins I I b and I I I a ,
has been used t o
enumerate t h e number o f t h e s e complexes o n n o r m a l p l a t e l e t s and t o indicate the
v i r t u a l absence
of the
complex on t h e s u r f a c e
of
thrombasthenic p l a t e l e t s . Four independent
monoclonal antibodies that
recognize
an
m urine cell-surface glycoprotein have been used t o c h a r a c t e r i z e t h e c o m p l e x i t y o f p r o t e i n polymorphism.879
alloantigenic s t r u c t u r e on a
E a c h o f t h e a n t i b o d i e s a p p e a r s t o r e a c t w i t h t h e same o r a c l o s e l y r e l a t e d e p i t o p e on t h e g l y c o p r o t e i n . The
localization
components i n t h e
i m munochemically a n t i s e r a. 880
of
chitosan
pea-Fusarium with
and
other
fungal
cell-wall
i n t e r a c t i o n have been studied
anti-chitosan
and a n t i - f u n g a l
cell-wall
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
279
Monoclonal antibodies specific f o r subunit 1 o f t h e l e c t i n from
--
Dolichos biflorus combine with t h e C -ter minal portion o f t h e s u b u n i t a n d may b e i n t e r a c t i n g w i t h t h e a c t i v e s i t e o r w i t h a d e t e r m i n a n t This antibody, c o n f o r m a t i o n a l l y i n d e p e n d e n t o f t h e a c t i v e site.155
w h i c h d o e s n o t r e a c t w i t h a n o t h e r l e c t i n - l i k e p r o t e i n f r o m t h e stems a n d l e a v e s o f t h e plant,156 c a n d i s t i n g u i s h s u b u n i t 1 f r o m s u b u n i t 2 o f t h e s e e d l e c t i n . 157 The binding o f human immunoglobulin t o t y p e C retroviruses has b e e n a n a l y s e d by r a d i o i m m u n o a s s a y . 1 2 B i n d i n g is d i r e c t e d t o t h e o l i g o s a c c h a r i d e c h a i n s o f t h e v i r a l g l y c o p r o t e i n s a n d is n o n specific in character. Cytotoxic antibodies present in bovine leukaemia virus-infected a n i m a l s are mostly directed a g a i n s t t h e c a r b o h y d r a t e p a r t O P t h e v i r a l e n v e l o p e g 1 y ~ o p r o t e i n . l ~I m m u n e s e r a p r e p a r e d f r o m p u r i f i e d e n v e l o p e g l y c o p r o t e i n s are d i r e c t e d a g a i n s t d e t e r m i n a n t s o f t h e polypeptide backbone of t h e antigen. The majority o f , i f n o t all, n o r ma1 h u m a n s e r a c o n t a i n n a t u r a l l y o c c u r r i n g h e t e r o p h i l a n t i b o d i e s t h a t react with t h e c a r b o h y d r a t e moieties of r e t r o v i r u s envelope glycoproteins.21 T h e s e a n t i v i r a l a n t i b o d i e s are n o t d e t e c t a b l e i n c o r d sera b u t show a n a g e - d e p e n d e n t p e a k i n e a r l y c h i l d h o o d a n d s u b s e q u e n t l y p e r s i s t l i f e -lo n g Monoclonal antibodies d i r e c t e d against herpes simplex virus g l y c o p r o t e i n s h a v e b e e n u s e d t o p r o t e c t mice a g a i n s t a c u t e v i r u s induced neurological disease.29 Virus glycoprotein g c expresses t y p e - s p e c i f i c a n t i g e n i c d e t e r m i n a n t s , w h e r e a s g l y c o p r o t e i n gD e x p r e s s e s t y p e - c o m mon d e t e r m i n a n t s . Monoclonal antibodies t o h e r p e s simplex virus t y p e 2 have been prepared and found t o precipitate t w o d i f f e r e n t v i r a l g l y c o p r o t e i n s . 33 The major glycoprotein (Gp 350/220) o f Epstein-Barr virus h a s b e e n d e t e c t e d b o t h o n t h e p l a s m a m e m b r a n e a n d i n t h e c y t o p l a s m by i m m u n o f l u o r e s c e n c e w i t h a n t i b o d i e s .’O T w o m o n o c l o n a l a n t i b o d i e s r e a c t i n g w i t h A K R l e u k a e m i c cells r e c o g n i z e c l o s e l y r e l a t e d , if n o t i d e n t i c a l , a n t i g e n i c d e t e r m i n a n t s o n t h e Glx g l y ~ o p r o t e i n . ’ ~ Monospecific antibodies t o e a c h of t w o envelope glycoproteins The of paramyxovirus h a v e been p r e p a r e d and characterized.74 e f f e c t s of t h e s e a n t i b o d i e s o n t h e i n f e c t i o u s p r o c e s s a n d t h e o t h e r biological a c t i v i t i e s of t h e virions and of t h e i s o l a t e d glycoproteins are discussed. Amino a c i d s e q u e n c e d i f f e r e n c e s i n t h e V r e g i o n o f murine antibodies t o a(l+3)-P-glucans (dextrans) have been correlated with
.
Carbohydrate Chemistry
280 the
expression o f
cross-reactive
and i n d i v i d u a l i d i o t y p e s ,
an analysis o f t w e l v e d i f f e r e n t d e x t r a n antibodies.881 r e a g e n t s can r e c o g n i z e t w o amino a c i d d i f f e r e n c e s w i t h segments
of
variable
I n anti-dextran
regions.
antibodies,
r e a c t i v e i d i o t y p e s i n v o l v e V-region determinants, idotype determinants secretory The
correlate
immunoglobulin A
hinge
region
variation.
has been i s o l a t e d f r o m
containing
four
cross-
whereas i n d i v i d u a l
D -segment
with
through Idiotype V and D
human
Pure
milk.882
Q-glycosidically
linked
o l i g o s a c c h a r i d e s i s r e l e a s e d en b l o c a f t e r p r o t e o l y t i c d i g e s t i o n . The s t r u c t u r e s ( 4 3 - 4 6 ) to
I=-serine
of t h e s e oligosaccharides,
which are l i n k e d
or I - t h r e o n i n e residues, have been i d e n t i f i e d .
oligosaccharides are oligosaccharides
more complex
linked
to
I-serine
These
and heterogeneous t h a n t h e residues
of
the
hinge
region
f r o m myeloma s e r a i m m u n o g l o b u l i n A. A
partially
characterized
immunoglobulin glycopeptides
has
A
(47-50)
been
glycopeptide
shown
t o
be
from
a
human
mixture
of
milk four
f o l l o w i n g h y d r a t i n o l y s i s and nitrous acid
d e a m i n a t i o n o f t h e g l y c o p e p t i d e . 883 The
membrane
receptor
for
immunoglobulin
A,
isolated
from
r a b b i t l i v e r a n d mammary g l a n d , i s s t r u c t u r a l l y r e l a t e d t o s e c r e t o r y component.884 groups
of
The
membrane s e c r e t o r y
amphiphilic
molecules
component which
is composed o f t w o
are
synthesized
transmem b r a n e p r e c u r s o r s and i n s e r t e d i n t h e c e l l - s u r f a c e
as
membrane
where t h e y a c t as r e c e p t o r s f o r p o l y m e r i c i m m u n o g l o b u l i n A .
R1-(2+3)-B-g-Galp-(1+3)-g-GalNAc 6
I 1
(44)
R 1 = H,
(45)
R 1 = H , R 2 = B-Q-Gale-(1+4)-B-a-Glce"c
(46)
R1 = a-Neup5Ac,
Spectroscopic properties MOPC-315
immunoglobulin
b e t w e e n c.d.
R2
R 1 = R2 = H
(43)
A
of
R2 = @ - g - G l C e N A C R2 = H
light-chain
derivatives o f
h a v e been reported.885
A
m urine
comparison
spectra o f t h e t h r e e f r e e and hapten-bound light-chain
derivatives demonstrates the existence o f conformational transitions i n t h e s e p r o t e i n s i n d u c e d by h a p t e n b i n d i n g . B i o s y n t h e t i c and
fi
v i t r o t r a n s l a t i o n studies have demonstrated
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
4
2 L
I J I
I
M
t
4 U
I
2 m
N
t
4 U
I
0
rI QII
a
h
+
'N 4 U
I
0 4 Z
?
4
U
I 011
m I
n
U
t
4
v
I
al
4
m u
I ail
I
m n I v)
t
N U
I
0
Q:
In
9 a,
z U
m n I
U
t
4
v
I
nl m u
4
4
-
4
4
m u r Y 1 3 :
4
n n n n
I
u u u u
CYl I
m
I 4
13:
F Q o \ O
u e e m
28 1
282
Carbohydrate Chemistry
t h a t membrane-bound and secreted i m m u n o g l o b u l i n A m o l e c u l e s c o n t a i n d i f f e r e n t a - p o l y p e p t i d e c h a i n s t h a t a r e e n c o d e d by d i f f e r e n t a-
m R N A ’s . -
V a r i a n t s h a v e b e e n i s o l a t e d f r o m t h e 3558 B a l b / c m o u s e m y e l o m a (immunoglobulin A,X: anti-a-(1+3)-dextran) with altered g l y c o s y l a t i o n of its heavy c h a i n and decreased r e a c t i v i t y w i t h d e ~ t r a n . T~ h~e ~c a r b o h y d r a t e m u t a n t i m m u n o g l o b u l i n c o n t a i n s m o r e neuraminic acid than does t h e carbohydrate of t h e wild-type immunoglobulin. However, t h e mutant immunoglobulin a p p e a r s t o have t h e same r e a c t i v i t y p a t t e r n w i t h o l i g o s a c c h a r i d e s a s d o e s t h e w i l d t y p e immunoglobulin, and t h u s t h e s i z e o f t h e antibody-combining s i t e s may b e s i m i l a r . By m e a s u r i n g t h e a f f i n i t i e s a t v a r i o u s t e m p e r a t u r e s b e t w e e n a n t i - e - g a l a c t a n i m m u n o g l o b u l i n A 5539 a n d a n t i d e x t r a n i m m u n o g l o b u l i n A W3129, a n d t h e i r r e s p e c t i v e l i g a n d s , t h e e n t h a l p i e s a n d e n t r o p i e s The i n t e r p r e t a t i o n o f t h e of binding have been determined.888 r e s u l t s i s i n a g r e e m e n t w i t h p r e v i o u s w o r k s h o w i n g t h a t 5539 h a s a g r o o v e - t y p e c o m b i n i n g r e g i o n a n d W3129 h a s a c a v i t y - t y p e r e g i o n . The i n t e r a c t i o n of s a c c h a r i d e h a p t e n s w i t h three homogeneous i m m u n o g l o b u l i n A mouse myeloma p r o t e i n s which h a v e s p e c i f i c i t y f o r s a c c h a r i d e s h a s b e e n i n v e s t i g a t e d i n a 270 MHz n . m . r . study.889 D i f f e r e n c e s p e c t r a show t h a t c o m p a r a t i v e l y few p r o t e i n r e s o n a n c e s are p e r t u r b e d on b i n d i n g h a p t e n , s u g g e s t i n g t h a t a c c o m p a n y i n g c o n f o r m a t i o n a l changes are l i m i t e d t o t h e combining s i t e . The primary s t r u c t u r e of t h e I-asparagine-563-linked carbohydrate chain of an immunoglobulin M from a p a t i e n t w i t h W a l d e n s t r o m ’ s m a c r o g l o b u l i n e m i a h a s b e e n r e i n v e s t i g a t e d u s i n g 500 MHz n . m . r . s p e c t r o s c o p y , a n d a new s t r u c t u r e (51) h a s b e e n N o e v i d e n c e was o b t a i n e d t o s u p p o r t a n e a r l i e r proposed.890 claim891 t h a t o n l y o n e a m i n o s u g a r r e s i d u e i s p r e s e n t i n a n u n u s u a l core structure. I n t h e p r e s e n t w o r k t h e n.m.r. spectrum shows a l l t h e s p e c t r a l features c h a r a c t e r i s t i c of an N”-diacetylchitobiosyl u n i t w i t h h e t e r o g e n e i t y e x i s t i n g i n both t h e c a r b o h y d r a t e and peptide moieties. G l y c o s y l a t i o n is n o t r e q u i r e d f o r membrane l o c a l i z a t i o n or s e c r e t i o n o f i m m u n o g l o b u l i n M i n a mouse B c e l l lymphoma.892 However, g l y c o s y l a t i o n d o e s c o n f e r i n c r e a s e d i n t r a c e l l u l a r s t a b i l i t y and r e s i s t a n c e t o p r o t e o l y t i c d i g e s t i o n . The m o l e c u l a r w e i g h t o f t h e h i g h g-mannose c o r e o l i g o s a c c h a r i d e o f i m m u n o g l o b u l i n M h a s been m e a s u r e d by f i e l d - d e s o r p t i o n ~ I . s . ~ ’ ~ High-affinity monoclonal immunoglobulin M antibodies d i r e c t e d
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
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towards e p i t o p e s on h e p a t i t i s B s u r f a c e a n t i g e n have been used i n t h e i m m u n o d i a g n o s i s o f h e p a t i t i s B.894 A P-mannose-rich g l y c o p e p t i d e (52) from a human p a t h o l o g i c a l immunoglobulin M c o n t a i n s o n l y Q-mannosyl and 2-acetamido-2-deoxy-Pg l u c o s y l r e s i d u e s ( m o l a r r a t i o 10:2).895 The r e c o v e r y o f o n e m o l e c u l e o f t h i s g l y c o p e p t i d e per m o l e c u l e of heavy c h a i n a n d t h e d e t e r m i n a t i o n of t h e amino acid s e q u e n c e l e d t o the l o c a t i o n o f t h e g l y c o p e p t i d e o n L-Asn-402 o f t h e Fc p o r t i o n o f t h e h e a v y c h a i n p o f t h e immunoglobulin. The a m i n o a c i d s e q u e n c e a n d l o c a t i o n o f t h r e e g l y c o p e p t i d e s i n t h e Fc r e g i o n o f t h e y c h a i n o f h u m a n i m m u n o g l o b u l i n I g D h a v e b e e n These g l y c o p e p t i d e s h a v e o l i g o s a c c h a r i d e c h a i n s identified.896 l i n k e d t j - g l y c o s i d i c a l l y t o I - A s n - 6 8 , L-Asn-159, a n d I - A s n - 2 1 0 . A t e n t a t i v e a m i n o a c i d s e q u e n c e o f t h e JH r e g i o n , t h e C y l domain, and the hinge r e g i o n of t h e y heavy c h a i n of t h e immunoglobulin h a s been published.897 The h i n g e r e g i o n o f t h e y c h a i n has an u n u s u a l s t r u c t u r e , i n which f o u r or f i v e o l i g o s a c c h a r i d e s c o n t a i n i n g 2-amino-2-deoxy-!-galactose residues a r e a t t a c h e d a t t h e N - t e r m i n a l h a l f o f t h e c h a i n , w h e r e a s t h e Ct e r m i n a l h a l f lacks c a r b o h y d r a t e , is d i s s i m i l a r i n sequence,and has a high charge. A h y b r i d mouse c e l l l i n e (61-8) secretes i m m u n o g l o b u l i n D which i s h e a v i l y g l y c o s y l a t e d , a s i n d i c a t e d by t h e f a c t t h a t t h e u n g l y c o s y l a t e d a c h a i n h a s a m o l e c u l a r w e i g h t o f 4.4 x l o 4 a s c o m p a r e d t o t h e v a l u e o f 6.1 x l o 4 f o r t h e m a j o r s p e c i e s o f g l y c o s y l a t e d y chain.898 A l k a l i - e x t r a c t e d h u m a n e r y t h r o c y t e g h o s t m e m b r a n e s f r o m Rho(D)p o s i t i v e d o n o r s c o m p l e x w i t h human i m m u n e a n t i - R h o ( D ) i m m u n o g l o b u l i n D.899 T h e a n t i b o d y was s h o w n t o b i n d t o b a n d 3 g l y c o p r o t e i n s o f t h e e r y t h r o c y t e membrane.
14
Erythrocyte Glycoproteins
A review dealing with t h e red-cell membrane and its c y t o s k e l e t o n has been publishedgo0 A new a p p r o a c h f o r t h e i s o l a t i o n o f l e c t i n r e c e p t o r s w i t h o u t t h e use of detergents involves t h e mechanical shearing of cells bound t o l e c t i n - c o a t e d macroporous a g a r o s e beads, whereupon t h e r e c e p t o r s r e m a i n a t t a c h e d t o t h e b e a d s and are r e l e a s e d s p e c i f i c a l l y by i n h i b i t o r y s u g a r s . 9 0 1 I n t h i s way a g l y c o p r o t e i n r e l e a s e d f r o m
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
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n e u r a m i n i d a s e - t r e a t e d human e r y t h r o c y t e s a f t e r b i n d i n g t o immobilized peanut a g g l u t i n i n or t o immobilized soybean a g g l u t i n i n was c h a r a c t e r i z e d a s a s i a l o g l y c o p h o r i n . B l o o d - g r o u p H s i t e s o f h u m a n 0 c e l l s h a v e b e e n c o n v e r t e d in v i t r o i n t o g r o u p A s i t e s by t r a n s f e r o f 2 - a c e t a m i d o - 2 - d e o x y - g - { 14c}g a l a c t o s y l r e s i d u e s u s i n g t h e b l o o d - g r o u p A g e n e - d e p e n d e n t a-Q-2a c e t a m i d o - 2 - d e o x y - g a l a c t o s y l t r a n s f e r a s e f r o m h u m a n A1 p l a s m a . 9 0 2 The m a j o r i t y o f t h e r a d i o a c t i v i t y i s t i g h t l y bound t o t h e m e m b r a n e s a n d i s o n l y r e l e a s e d by p r o t e o l y s i s , i m p l y i n g t h a t t h e b u l k o f b l o o d g r o u p H d e t e r m i n a n t s a r e bound t o g l y c o p r o t e i n m a t e r i a l . T h e human e r y t h r o c y t e r e c e p t o r f o r t h e I - f u c o s e - s p e c i f i c l e c t i n p r o d u c e d by S t r e p t o m y c e s s p e c i e s b i n d s t o human e r y t h r o c y t e s b e a r i n g ABO d e t e r m i n a n t s. ’03 I n a h a e m a g g 1u t i n a t i o n - i n h i b i t i o n a s s a y p o l y ( g l y c o s y 1 ) - c e r a m i d e was t h e b e s t i n h i b i t o r w i t h a g l y c o p r o t e i n f r a c t i o n showing only s l i g h t i n h i b i t o r y a c t i v i t y . The d i s t r i b u t i o n o f A a n d B d e t e r m i n a n t s i n t h e p o l y g l y c o s y l p e p t i d e s o f human r e d c e l l s o f b l o o d g r o u p AB h a s b e e n s t u d i e d by i s o l a t i n g b l o o d - g r o u p AB ABH-active c o m p o n e n t s f r o m d e l i p i d a t e d human b l o o d - g r o u p e r y t h r o c y t e mernbranes.’O4 The A and B a n t i g e n i c sites a r e l o c a t e d i n d i f f e r e n t p o l y g l y c o s y l c h a i n s . A p p r o x i m a t e l y h a l f o f t h e bloodg r o u p ABH-active g l y c o p e p t i d e s c a r r y b l o o d - g r o u p A d e t e r m i n a n t s and h a l f c a r r y blood-group 8 d e t e r m i n a n t s . Human e r y t h r o c y t e s h a v e b e e n l a b e l l e d a t t h e c e l l - s u r f a c e a g a l a c t o s y 1 a n d 2 - a c e t a m i d o - 2 - d e o x y - g a l a ct o sy 1 r e si d u e s w i t h s o d i u m b ~ r o t r i t i d e . ~ ’ A~ s i g n i f i c a n t d e c r e a s e i n t h e n u m b e r o f r e s i d u e s d u r i n g a g e i n g o f t h e e r y t h r o c y t e s was d e t e c t e d . Two h e r e d i t a r y p l a t e l e t d i s o r d e r s , Glanzmann’s t h r o m b a s t h e n i a a n d B e r n a r d - S o u l i e r s y n d r o m e , a r e c h a r a c t e r i z e d by d e f e c t s i n v o l v i n g a d h e s i o n a n d a g g r e g a t i o n a n d a r e a s s o c i a t e d w i t h d i f f e r e n t membrane g l y c o p r o t e i n d e f i c i e n ~ i e s . ~ ’ N~ o g l y c o p r o t e i n d e f e c t s h a v e b e e n o b s e r v e d on a n a l y s i s o f t h e e r y t h r o c y t e membrane g l y c o p r o t e i n s of p a t i e n t s o f both d i s o r d e r s . Immunochemical evidence f o r t h e e x i s t e n c e of hybrid s i a l o g l y c o p r o t e i n s o f human e r y t h r o c y t e s h a s b e e n r e p o r t e d . ” ’ U s i n g human e r y t h r o c y t e m e m b r a n e s a s a m o d e l s y s t e m , t h r e e related procedures, based on t h e s p e c i f i c i n t e r a c t i o n of l e c t i n s w i t h membrane c a r b o h y d r a t e , h a v e b e e n d e v e l o p e d f o r t h e i s o l a t 5 o n o f i n s i d e - o u t v e s i c l e s from h e t e r o g e n e o u s p o p u l a t i o n s o f membrane fragments.908 The t e c h n i q u e s s h o u l d p r o v e a p p l i c a b l e t o o t h e r b i o l o g i c a l m e m b r a n e s where i t i s n o t p o s s i b l e t o i n d u c e c o n t r o l l e d s e a l i n g o f membrane f r a g m e n t s i n a d e f i n e d o r i e n t a t i o n a s i s t h e
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case f o r e r y t h r o c y t e membranes. Glycophorin A h a s been r e c o n s t i t u t e d i n small l i p i d v e s i c l e s ( 2 5 0 - 300 I! i n d i a m e t e r ) by u s i n g c h o l a t e d e t e r g e n t s o l u b i l i z a t i o n f o l l o w e d by r a p i d r e m o v a l o f c h o l a t e o n a m o l e c u l a r - s i e v e c o l u m n . 9 0 9 Incorporation is i n a transbilayer fashion, with a high concentration of the molecules orientated with t h e carbohydrateInteraction of the c o n t a i n i n g N-terminus t o t h e v e s i c l e e x t e r i o r . p r o t e i n w i t h t h e h y d r o p h o b i c p o r t i o n o f t h e b i l a y e r c a n be s h o w n by 1~ n.m.r. spectroscopy. The t r a n s l a t i o n a l m o b i l i t y o f g l y c o p h o r i n i n b i l a y e r membranes o f dimyristoylphosphatidylcholine h a s b e e n reported.’1° Evidence f o r m u l t i p l e components of blood-group A and B a n t i g e n s i n human e r y t h r o c y t e m e m b r a n e s - s u g g e s t i v e o f d i f f e r e n t c a r b o h y d r a t e s t r u c t u r e s - h a s been reported.911 An L- - a s p a r a g i n e - l i n k e d o l i g o s a c c h a r i d e c h a i n ( 5 3 ) h a s b e e n i s o l a t e d f r o m a t r y p t i c f r a g m e n t o f h u m a n g l y c o p h o r i n A.912 S t r u c t u r a l similarities between t h i s o l i g o s a c c h a r i d e and t h e s u g a r c h a i n s of immunoglobulin A are e v i d e n t . The b l o o d - g r o u p M a n d N s p e c i f i c d e t e r m i n a n t s o f human e r y t h r o c y t e g l y c o p h o r i n A h a v e t h r e e i d e n t i c a l c a r b o h y d r a t e m o i e t i e s ( 5 4 ) p e r ~ e p t i d e . T~h e~ M~ o r N determinant is t h e N-terminal amino a c i d and a neuraminic a c i d r e s i d u e ( s ) . The a u t h o r s a r g u e t h a t t h e r e i s no c h e m i c a l b a s i s f o r a s s e r t i o n s i n t h e l i t e r a t u r e that M and N a n t i g e n s d i f f e r i n t h e i r oligosaccharide s t r u c t u r e or that t h e N antigen is b i o s y n t h e t i c a l l y t r a n s f o r m e d t o t h e M a n t i g e n by s i a l y l a t i o n . a-Neu~5Ac-(2+3)-B-~-Gal~-(l+3)-~-~-Gal~NAc-l-~-~-Ser/~-Thr 6
I
2 a - Ne u p 5 A c (54) The g - g l y c o s i d i c a l l y l i n k e d o l i g o s a c c h a r i d e c h a i n s o f g l y c o p h o r i n from bovine e r y t h r o c y t e membranes h a v e been i s o l a t e d as r e d u c e d o l i g o s a c c h a r i d e s by a l k a l i n e b o r o h y d r i d e t r e a t m e n t , a n d p u r i f i e d by i o n - e x c h a n g e c h r o m a t o g r a p h y f o l l o w e d by g e l The s t r u c t u r e s o f t h r e e o l i g o s a c c h a r i d e s (55-571, a filtration.914 ~-Gal~-(1+3)-g-Gal~-(l+4)-g-Glc~NAc-(1+3)Q-Gal~-(1+3)-g-GalNAc-o1 (551
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
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penta-,
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of
sequence
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porcine
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.
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of
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acid
erythrocyte
and
carbohydrate
glycoproteins
structural
have
been
variants
of
major
reported.918
The
M M ( M i 1 t e n b e r g e r 111) g l y c o p r o t e i n a p p e a r s t o be a p r o d u c t o f t h e M gene and c o n t a i n s g - g l y c o s i d i c a l l y l i n k e d o l i g o s a c c h a r i d e c h a i n s partially
i d e n t i f i e d by t h e s t r u c t u r e (58). The
o f t h e M-N
locus,
i n t h e N gene.
M 9 i s an a l l o m o r p h
p r o b a b l y e v o l v e d f r o m a s i n g l e base s u b s t i t u t i o n
The r e s u l t i n g s i n g l e a m i n o a c i d s u b s t i t u t i o n a f f e c t s
the post-translational
glycosylation o f neighbouring i-serine
o r I-
threonine resides. The a m i n o a c i d s e q u e n c e o f a N - t e r m i n a l t r y p t i c g l y c o p e p t i d e of the
blood-group
MQ-specific
major
human
s i a l o g l y c o p r o t e i n has been reported.919
erythrocyte
membrane
The a m i n o a c i d s e q u e n c e a n d
t h e s i t e s o f g l y c o s y l a t i o n o f t h e t w e l v e a m i n o a c i d s a t t h e Nterminus
of
blood-group
Ms-specific
major
s i a l o g l y c o p r o t e i n have been established.920 represents
an
evolutionary
link
erythrocyte
The
between blood-group
variant M-
Mc
a n d N-
s p e c i f i c glycoproteins.
Neu~5Ac-Q-Gal~-(1+3)-Q-Gal~NAc-l+~-Thr
I P - G lCQNAC
(58) T h e h u m a n l e u k a e m i a c e l l l i n e K562 s y n t h e s i z e s g l y c o p h o r i n A.921
An i s o l a t e d m e s s e n g e r RNA f r a c t i o n f r o m t h e c e l l s h a s b e e n
used i n a r a b b i t r e t i c u l o c y t e c e l l - f r e e system f o r t h e s y n t h e s i s o f the glycoprotein.
The p r e s e n c e o f m e m b r a n e s i s r e q u i r e d f o r
N-
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
4
0
Q
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4 - u
f4
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1311
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Z
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u
4 u I
m n I
v)
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4
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ZE I
CIII
d I
hl
t
4
u I
0 4 Z
2
4
I
1311
m t
4
a
&I
d
oll
m
291
2 92
Carbohydrate Chemistry
g l y c o s y l a t i o n and 2 - g l y c o s y l a t i o n o f t h e a p o p r o t e i n . Despite t h e i r heterogeneous appearance a f t e r sodium dodecyl sulphate
gel electrophoresis,
glycoprotein o f
band 3 ( t h e
human e r y t h r o c y t e s )
a r e homogeneous p o l y p e p t i d e s . 9 2 2
and i t s
major
transmembrane
p r o t e o l y t i c fragments
The a p p a r e n t h e t e r o g e n e i t y o f b a n d
3 and i t s C - t e r m i n a l r e g i o n may r e f l e c t v a r i a b i l i t y o f g l y c o s y l a t i o n on s o d i u m d o d e c y l s u l p h a t e b i n d i n g .
The c h a r a c t e r i s t i c d i f f u s i o n o f
band 3 g l y c o p r o t e i n as s e e n by S D S - p o l y a c r y l a m i d e g e l e l c t r o p h o r e s i s has
been a t t r i b u t e d t o
heterogeneity
o l i g o s a c c h a r i d e chains.923 further been
of
o l i g o s a c c h a r i d e s o r i g i n a t i n g from
established.924
glycoprotein
The
i s similar
the
molecular
The s t r u c t u r e s ( 5 3 , mechanism
to
that
of
59,
size of
60) o f t h r e e
band 3 g l y c o p r o t e i n have
of
biogenesis
of
cotranslationally
g l y c o p r o t e i n s l i k e v e s i c u l a r s t o m a t i t i s v i r u s G,
band
3
inserted
a n t i g e n , and
HLA-A
g l y c ~ p h o r i n . ~ T h~e ~m a j o r s i a l o g l y c o p r o t e i n s o f r a t e r y t h r o c y t e membrane h a v e been p u r i f i e d by h o t p h e n o l p a r t i t i o n i n g f o l l o w e d by cation-exchange
chromatography.926
Further
chromatographic
r e s o l u t i o n has y i e l d e d a s i n g l e p u r i f i e d g l y c o p r o t e i n (mol. x
lo4)
and a
accounting
mixture of
for
at
least
higher- molecular- weight 23% o f
the
wt.
1.9
glycoproteins
rat-erythrocyte-membrane
neuraminic acid.
15
S a l i v a r y and Mucous G l y c o p r o t e i n s
The f o l l o w i n g a s p e c t s o f t h i s a r e a h a v e been r e v i e w e d :
gastric
m u c o s a l p r o t e c t i o n and g a s t r i c g l y c o p r o t e i n s ,927 n e u r a m i n i c a c i d and mucous
rheology,928
and t h e
neutral oligosaccharides cystic-fibrosis A
i s o l a t i o n and
characterization
of
f r o m human b r o n c h i a l g l y c o p r o t e i n s o f
patients.929
gel-filtration
method has
been
developed
t o
isolate
s i m u l t a n e o u s l y and q u a n t i t a t e s p e c i f i c a l l y r a d i o l a b e l l e d mucous g l y c o p r o t e i n f r o m m u l t i p l e s a m p l e s o f c u l t u r e media.930 Mucin,
i s o l a t e d from a p a t i e n t w i t h c y s t i c f i b r o s i s ,
consists
o f an e x t e n s i v e l y g l y c o s y l a t e d c o r e p r o t e i n w h i c h i s l i n k e d t o a t l e a s t two other proteins.931
D i r e c t evidence f o r the r o l e o f the
m u c i n s i n t h e r h e o l o g i c a l b e h a v i o u r o f s p u t u m h a s been s u m m a r i z e d . Human p a r o t i d I - p r o l i n e - r i c h
g l y c o p r o t e i n (mol.
wt.
3.6
x
lo4)
contains a biantennary oligosaccharide structure s i m i l a r t o that d e s c r i b e d f o r s e v e r a l serum c ~ l y c o p r o t e i n s . ~ ~ ~ M i l d a c i d h y d r o l y s i s has
been used t o
cleave
a
sulphated
293
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
m o n o s a c c h a r i d e f r o m s a l i v a r y m u c i n o f t h e s t u m p t a i l monkey (Macaca a r ~ t o i d e s ) . T~ h~e ~m o n o s a c c h a r i d e was p u r i f i e d b y i o n - e x c h a n g e chromatography
and
identified
sulphate. The i n c o r p o r a t i o n o f { 14C)-mannose
into
as
2-amino-2-deoxy-Q-glucose
L-{ 3 H ) - l e u c i n e
proteins
4-
and 2-acetamido-2-deoxy-P-
and g l y c o p r o t e i n s
submandibular, and s u b l i n g u a l g l a n d s o f
the
of
the
mouse
parotid, has
been
compared. 934 novel
A
neuraminic
acid,
4-g-acetyl-9-g-lactyl-N-
a c e t y l n e u r a m i n i c a c i d , h a s b e e n i d e n t i f i e d as a c o n s t i t u e n t o f h o r s e submandibular- gland
g l y c ~ p r o t e i n s . ~The ~ ~ structure
has
been
e s t a b l i s h e d b y g.1.c.-m.s. A
fluorimetric
porcine
method f o r
submaxillary
mucine
measuring a-h-fucose by
(A')
released from
a-I=-fucosidase
has
been
developed.936 A l k a l i n e borohydride r e d u c t i v e cleavage o f hog submaxillary glycoproteins from three immunologically results
i n
the
release
of
a
series
determined phenotypes
of
neutral
and
acidic
o l i g o s a ~ c h a r i d e - a l d i t o l s . ~A~ l~ l h a v e b e e n i d e n t i f i e d a s p a r t i a l structures
r e p r e s e n t i n g t h e p o s s i b l e c o m p l e t e and b i o s y n t h e t i c a l l y
i n c o m p l e t e s t a g e s o f t h e c h a i n o f t h e p e n t a s a c c h a r i d e (611,
present
in
The
the
glycoprotein
with
blood-group
glycolylneuraminic acid residues,
A activity.
when p r e s e n t ,
2-
are only linked t o
2 - a c e t a m i d o - 2 - d e o x y - Q - g a l a c t it o 1 r e s i d u e s a n d n o t t o P - g a l a c t o s y l r e s i d u e s , as h a d b e e n p r e v i o u s l y r e p o r t e d .
a-Q-Gal~NAc-(1+3)-B-Q-Galp-(1+3)-~-GalNAc-o1 2
6
1
2
I
I
a-i-Fucp
a-N e up5A c
(61) A 2-acetamido- 2- de oxy- 9- gluc osyltr a nsf e r a s e
which a c t s on mucin
s u b s t r a t e s has been d e t e c t e d i n c a n i n e s u b m a x i l l a r y glands.938 Using mucin
o r b e n z y 1 2 - a c e t a m i d o - 2 - d e o x y -3-g-B-P-galactosyl-a-Pt h e t r a n s f e r o f a 2-acetamido-2-deoxy-9-
g a l a c t o s i d e as acceptors,
g l u c o s y l r e s i d u e t o e i t h e r 0-4 o r 0 - 6 o f t h e 2 - a c e t a m i d o - 2 - d e o x y - P galactosyl residue o f the acceptor
occurs.
Detailed substrate
s p e c i f i c i t y of
t h e 2-acetamido-2-deoxy-~-glucosyltransferase
t e s t e d with a
variety
of
when
p o t e n t i a l g l y c o p r o t e i n and s y n t h e t i c
Carbohydrate Chemistry
2 94 disaccharide
a c c e p t o r s has been reported.939
Porcine l i v e r
m i c r o s o m e s c a n i n t r o d u c e r e s i d u e s o f n e u r a m i n i c a c i d f r o m CMPneuraminic a c i d i n t o porcine submaxillary asialo-afuco-mucin, a gg a l a c t o s y l o v i n e s u b m a x i l l a r y a s i a l o m u c i n a n d g a n g l i o s i d e GH1.940
No e v i d e n c e was o b t a i n e d t o i n d i c a t e any t r a n s f e r o f n e u r a m i n i c a c i d t o 2-acetamido-2-deoxy-D-galactosyl residues, and t h e enzyme t r a n s f e r s n e u r a m i n i c a c i d s o l e l y b y f o r m a t i o n o f a n a(2+3)glycosidic linkage t o e-galactosyl
residues.
Rat submaxillary
mucous g l y c o p r o t e i n has been p u r i f i e d a f t e r aqueous e x t r a c t i o n o f t h e g l a n d s f o l l o w e d by r e c y c l i n g g e l - f i l t r a t i o n
~hromatography.~~~
The c h e m i c a l c o m p o s i t i o n o f t h i s m u c i n r e s e m b l e s t h a t o f t h e human c o u n t e r p a r t and i s d i s s i m i l a r t o t h e c o m p o s i t i o n o f r a t s u b l i n g u a l glycoprotein. Chemical
properties of
and a f f i n i t y
of
lectins
for
human
b r o n c h i a l mucous g l y c o p r o t e i n s h a v e been compared.942 The a c t i o n s o f
m e r c a p t o e t h a n o l on s o l u b l e mucous g l y c o p r o t e i n s
o b t a i n e d f r o m b o t h c y s t i c - f i b r o t i c and c h r o n i c - b r o n c h i t i c sputum h a v e been c ~ m p a r e d . ~ The ~ ~ ,e ~ f f e~ c ~t o f t h e r e d u c i n g a g e n t u n d e r n o n - r e d u c i n g c o n d i t i o n s o n t h e g l y c o p r o t e i n s c a n n o t be e x p l a i n e d solely
by
the
disulphide
bond-breaking
action,
but
appears
i n v o l v e t h e e x i s t e n c e o f a mercaptoethanol-inducible
to
mucolytic
s y s t e m w h i c h v a r i e s f r o m one s p u t u m t o a n o t h e r . O l i g o s a c c h a r i d e s o b t a i n e d b y a l k a l i n e d e g r a d a t i o n o f human b r o n c h i a l mucous exchange,
glycoproteins
have been f r a c t i o n a t e d
gel-permeation chromatography,
by
ion
and h . p . l . ~ . ’ ~ ~
S u l p h a t e d g l y c o p r o t e i n s f r o m human g a s t r i c j u i c e h a v e b e e n i s o l a t e d a f t e r a f f i n i t y c h r o m a t o g r a p h y on c o l u m n s o f i m m o b i l i z e d l y ~ i n e . Two ~ ~ m ~ ajor glycoprotein f r a c t i o n s d i f f e r i n g i n t h e i r
LO-
s u l p h a t e a n d n e u r a m i n i c a c i d c o n t e n t were i s o l a t e d . The
nature
of
the
non-covalent
interactions
g l y c o p r o t e i n m o l e c u l e s o f human and p i g g a s t r i c mucous s t u d i e d by mechanical spectroscopy,
between has
been
u s i n g s t o r a g e and l o s s m o d u l i t o
.
c h a r a c t e r ize, r e s p e c t i v e ly, so l i d - 1ik e a n d l i q u i d - 1ik e be h a v i o u r 947 Oligosaccharides having blood-group
Ii a c t i v i t y
have been
i s o l a t e d f r o m sheep g a s t r i c g l y c o p r o t e i n s w h i c h h a d been e n r i c h e d
f o r t h e s e b l o o d - g r o u p a c t i v i t i e s b y a f f i n i t y c h r o m a t o g r a p h y o n an a n t i - I a d s o r b e n t column.948
The f r a c t i o n s o f s m a l l e s t m o l e c u l a r
w e i g h t w i t h b o t h I and i a c t i v i t i e s a r e m i x t u r e s o f h e x a - a n d o c t a saccharides.
From s t u d i e s on t h e hexa- t o o c t a - s a c c h a r i d e
a s t r u c t u r a l model (62) i s proposed which c o n s i s t s o f region,
(b)
a b a c k b o n e r e g i o n h a v i n g (1+3)-
fractions
(2)
a cor,e
and ( 1 + 6 ) - l i n k e d
2-
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides al
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Carbohydrate Chemistry
acetamido-2-deoxy-4-~-B-Q-galactosyl-~-glucosyl b r a n c h e s w i t h I a c t i v i t i e s and l i n e a r r e p e a t i n g (l+3)-linked 2-acetamido-2-deoxy-4-O - 8 - ~ - g a l a c t o s y l - ~ - g l u c o s e u n i t s w i t h i a c t i v i t i e s , a n d (c) a p e r i p h e r a l r e g i o n w i t h b l o o d - g r o u p i s o t y p e a c t i v i t i e s . 949 A p r o t e i n ( m o l . w t . 7.0 x l o 4 ) i s l i n k e d t o p i g g a s t r i c m u c o u s g l y c o p r o t e i n by d i s u l p h i d e b r i d g e s . 9 5 0 On d i s s o c i a t i o n o f t h e g l y c o p r o t e i n i n t o s u b u n i t s by r e d u c t i o n o r p r o t e o l y s i s , t h e p r o t e i n is e i t h e r r e l e a s e d i n t h e reduced form or l o s t on p r o t e o l y t i c digestion.951 P i g g a s t r i c a n d s m a l l - i n t e s t i n a l mucous g l y c o p r o t e i n s a r e c l e a v e d i n t o f o u r s u b u n i t s by p r o t e o l y t i c enzymes.952 A model f o r t h e g a s t r i c mucous g l y c o p r o t e i n (mol. w t . 2 x l o 6 ) , w h e r e on a v e r a g e f o u r s u b u n i t s a r e e a c h j o i n e d t o a p r o t e i n ( m o l . w t . 7.0 x l o 4 ) by d i s u l p h i d e b i n d i n g , i s p r o p o s e d . The i n t e s t i n a l g l y c o p r o t e i n probably h a s a similar o v e r a l l s t r u c t u r e . Rat g a s t r i c m u c o s a l c e l l s a r e c a p a b l e o f t h e s y n t h e s i s and s e c r e t i o n o f a t l e a s t t w o f a m i l i e s o f mucous g l y c o p r o t e i n s o f w i d e l y d i f f e r e n t m o l e c u l a r w e i g h t s a n d r h e o l o g i c a l p r o p e r t i e s .953 Oligosaccharide chains bearing t h e Forssman a n t i g e n i c d e t e r m i n a n t are a s s o c i a t e d w i t h g l y c o p r o t e i n s of t h e dog g a s t r i c mucous.954 Enzymic and immunological evidence i n d i c a t e s t h a t t h e s e g l y c o p r o t e i n s , l i k e F o r s s m a n g l y c o s p h i n g o l i p i d s , c o n t a i n t h e 2a c e t a mido-2-deoxy-3 -O-(2-acetamido-2-deoxy-a-~ - g a l a c t o sy l ) - Q g a l a c t o s e s t r u c t u r e , which d e t e r m i n e s t h e immunological s p e c i f i c i t y of the Forssman a n t i g e n . The v i s c o s i t y a n d g e l - f o r m i n g p o t e n t i a l o f p i g s m a l l - i n t e s t i n a l mucous i n c r e a s e w i t h p r o g r e s s i v e r e m o v a l o f n o n - c o v a l e n t l y bound protein during isolation.955 T h i s g l y c o p r o t e i n ( m o l . w t . 1.7 x l o 6 ) e x h i b i t s A a n d H b l o o d - g r o u p a c t i v i t y a n d c o n t a i n s r e s i d u e s o f If u c o s e , 2 - m a n n o s e , Q - g a l a c t o s e , 2 - a c e t a m i d o - 2 - d e o x y - Q - g l u c o s e , 2acetamido-2-deoxy-Q-galactose, a n d n e u r a m i n i c a c i d . The u n d e g r a d e d g l y c o p r o t e i n c o n t a i n s i n t e r c h a i n d i s u l p h i d e b r i d g e s and is d i s s o c i a t e d by p r o t e o l y s i s o r r e d u c t i o n i n t o s u b u n i t s t h a t a r e d i f f e r e n t i n s i z e and s t r u c t u r e from p i g g a s t r i c mucous gly~oprotein.9~6 Rat c o l o n i c mucous g l y c o p r o t e i n s h a v e b e e n s o l u b i l i z e d i n t h e The absence of p r o t e o l y t i c enzymes or d e n a t u r i n g solvents.957 presence of s e v e r a l high-molecular-weight components, varying i n t h e i r c a r b o h y d r a t e and s u l p h a t e c o n t e n t s , blood-group a n t i g e n i c i t y , a n d n e t c h a r g e , was o b s e r v e d . Evidence f o r the e x i s t e n c e o f two immunochemically d i s t i n c t m u c i n s i s o l a t e d a f t e r p r o t e o l y t i c d i g e s t i o n o f human c o l o n i c m u c i n
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
I
n
M
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I
4 v
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M
t
4 v
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4
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rill
297
298
Carbohydrate Chemistry I t i s n o t c l e a r whether these two mucin a s i n g l e mucin molecule or from t w o
h a s been reported.958 fragments o r i g i n a t e d i f f e r e n t mucins.
from
G l y c o p r o t e i n s f r o m t h e p e r i o v u l a t o r y phase mucins o f t h e b o n n e t monkey,
Macaca
enzymes.959
radiata,
have
been
The p a r t i a l s t r u c t u r e s
degraded
(63-64)
with
proteolytic
o f two oligosaccharide
c h a i n s were i d e n t i f i e d .
Urinary G l y c o p r o t e i n s , Oligosaccharides
16
Some
biochemical
sialidoses,
findings
Glycopeptides,and
concerning
four
different
types
of
on t h e b a s i s o f t h e s p e c i f i c a - n e u r a m i n i d a s e d e f i c i e n c y
and t h e n a t u r e o f t h e s t o r a g e m a t e r i a l , h a v e been r e v i e w e d . 9 6 0 From t h e knowledge o f t h e numerous s t r u c t u r e s o f g l y c o p r o t e i n g l y c a n s , i t i s p o s s i b l e t o d e m o n s t r a t e t h a t t h e o l i g o s a c c h a r i d e s and g l y c o a s p . a r a g i n e s a c c u m u l a t i n g i n t i s s u e s and u r i n e s o f p a t i e n t s w i t h diseases c h a r a c t e r i z e d by o r i g i n a t e from
a
lack
i n lysosomal glycosidases,
g l y c o p r o t e i n g l y c a n s i n c o m p l e t e l y c a t a b ~ l i z e d . ~A ~ ~
scheme f o r t h e n o r m a l a n d p a t h o l o g i c a l c a t a b o l i s m o f g l y c o p r o t e i n s h a s been p r o p o s e d . Three s i a l o g l y c o p r o t e i n s (mol. x
5.3
lo3,
urine.962
of
r e s p e c t i v e l y ) have
been
w t s . 7.79 X lo4, 3.7 x 104,and isolated
from
normal
human
Alkaline borohydride degradation o f the sialoglycoprotein
lowest molecular
weight yielded two s i a l y l a t e d oligosaccharides
w h i c h were p a r t i a l l y c h a r a c t e r i z e d . Patients with tubular or e x c r e t e an a l - m i c r o g l o b u l i n
mixed glomerular-tubular
proteinuria
w h i c h has been shown t o have t h r e e
g l y c o s i d i c a l l y l i n k e d carbohydrate chains o f
l-
structural similarity
t o t h o s e d e r i v e d f r o m many o t h e r s e r u m g l y c o p r ~ t e i n s . ~ ~ ~ A g l y c o p r o t e i n ( m o l . w t . 2.0 x lo4) w h i c h c o m p l e t e l y i n h i b i t s t r y p s i n a t a 1:l m o l a r r a t i o h a s b e e n i s o l a t e d f r m human u r i n e . 9 6 4
I t i s generated from a precursor molecule which i n t u r n i s d e r i v e d from
plasma
inter-a-trypsin
i n h i b i t o r (mol. w t . Kunitz-type
3.0
domains.965
x
inhibitor.
lo4) The
An a c i d - r e s i s t a n t
trypsin
f r o m human u r i n e i s c o m p o s e d o f t w o C-terminal
domain i s r e s p o n s i b l e f o r
a n t i t r y p t i c a c t i v i t y b u t no i n h i b i t o r y a c t i v i t y h a s b e e n d e s i g n a t e d t o t h e o t h e r domain.
B o t h t h e N - t e r m i n a l e x t e n s i o n p e p t i d e and t h e
l a t t e r domain are l i n k e d g - g l y c o s i d i c a l l y respectively.
The 0 - g l y c o s y l
and N - g l y c o s i d i c a l l y ,
residues are attached t o I-serine-10
299
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides w h i l e t h e N--glycosyl
residues are t o I-asparagine-24
on t h e f i r s t
K u n i t z - t y p e domain. 966 G l y c o p e p t i d e s have been i s o l a t e d f r o m p r o t e o l y t i c d i g e s t s o f human T a m m - H o r s f a l l g l y c o p r o t e i n a n d i t s a s i a l o d e r i v a t i v e . 9 6 7 glycoprotein
contains
at
least
five
I-asparagine
The
residues
s u b s t i t u t e d by complex c a r b o h y d r a t e m o i e t i e s ,
t h r e e b e i n g o f one
type,
and t w o c o n t a i n i n g
r e l a t i v e l y r i c h i n ;-galactosyl
residues,
l e s s g - g a l a c t o s e b u t more n e u r a m i n i c a c i d . The
structures
(65-67)
of
three oligo-Q-mannoside-type
h_-
a s p a r a g i n e s i s o l a t e d f r o m t h e u r i n e o f a p a t i e n t w i t h Gaucher's disease
have
been
elucidated
using
MHz
500
'H
n.m.r.
s p e c t r o s c o p y .968 A
reduction or
absence o f
degrading lysosomal amidase
activity of
the glycoprotein
l-aspartamido-$-2-acetamido-2-deoxy-~-
g l u c o s e a m i d o h y d r o l a s e (EC 3.5.1.26)
i s t h e b a s i c enzymic d e f e c t i n
a s p a r t y l g l y c o ~ a m i n u r i a . ~U~r ~i n a r y s i a l y g l y c o c o n j u g a t e s h a v e b e e n s t u d i e d i n a number o f p a t i e n t s w i t h t h i s i n h e r i t e d d e f i c i e n c y . levels
of
the
main
metabolite,
i n v a r i o u s n e u r a l and e x t r a n e u r a l t i s s u e s o f a s p a r t y l g l y c o s a m i n u r i a have been measured
asparaginyl-Q-glucose, a patient suffering by
The
2-acetamido-2-deoxy-l-$-~-
from
g . l . ~ . ~ ~ OC o r r e l a t i o n o f
the
results
with
the
clinical
m a n i f e s t a t i o n s o f t h e d i s e a s e d i d n o t r e v e a l any d i r e c t r e l a t i o n s h i p between t h e amount of
2-acetamido-2-deoxy-l-f3-l=-asparaginyl-~-
g l u c o s e s t o r e d and t h e degree o f o r g a n d y s f u n c t i o n . Six
mono-
and
simultaneously Improvements
in
components a l l o w
di-saccharides
by
h.p.1.c.
a
detector
have
been
determined
o f d e i o n i z e d human urine.971 based on o p t i c a l a c t i v i t y o f t h e
t h e d e t e c t i o n o f 100 n g o f s u g a r .
R1-(1+3)-$-Q-Man~-(1+4)-$-~-G1c~NAc-(1~4)-$-~-G1c~NAc-1+~-Asn 6
I 1 R2-(1+3)-a-g-Mang 6
I 1 R3 (65)
R 1 = R2 = R3 = H R2 = R 3 = H
(66)
R1 = a-Q-Mane,
(67)
R1 = R 2 = R 3 = a-Q-Mane
3 00
Carbohydrate Chemistry The s t r u c t u r e o f
an o l i g o s a c c h a r i d e ( 6 8 ) c o n t a i n i n g n i n e Q -
m a n n o s y l r e s i d u e s and i s o l a t e d f r o m p a t i e n t s w i t h m a n n o s i d o s i s has been s i m i l a r l y a s s i g n e d . 9 7 2
Excessive g i n g i v a l hyperplasia with
s t o r a g e o f Q - m a n n o s e - r i c h o l i g o s a c c h a r i d e s a p p e a r s t o be a u n i q u e feature
present i n a p a t i e n t e x h i b i t i n g mannosidosis.973
c h a r a c t e r i s t i c 0-manno-oligosaccharides
Eight
each t e r m i n a t e d a t
r e d u c i n g end w i t h a 2 - a c e t a m i d o - 2 - d e o x y - ~ - g l u c o s y l r e s i d u e i s o l a t e d from the patients' which
a trisaccharide
urines.
the were
I n contrast t o the urine i n
i s predominant,
tetrasaccharides
and
p e n t a s a c c h a r i d e s a r e more abundant i n g i n g i v a . The u n i q u e s t r u c t u r e ( 6 9 ) o f a t r i s a c c h a r i d e f r o m a p a t i e n t w i t h m a n n o s i d o s i s h a s b e e n d e d u c e d b y 13C n.m.r. terms o f sugar composition,
r i n g forms,
spectroscopy i n
anomeric configurations,
sequence, and i n t e r - r e s i d u e l i n k a g e p o s i t i o n s . 9 7 4 I n a c a s e o f f e l i n e m a n n o s i d o s i s , a m a r k e d d e f i c i e n c y o f a-D-
mannosidase i n b r a i n ,
kidney,and
l i v e r t i s s u e i s accompanied by h i g h
A a_concentrations o f n e u t r a l oligosaccharides i n t h e urine.975 m a n n o s y l - r i c h h e x a s a c c h a r i d e was f o u n d t o be t h e p r e d o m i n a n t
oligosaccharide. A s e t o f procedures f o r s c r e e n i n g o f p a t i e n t s '
urine t o detect
o l i g o s a c c h a r i d e - s t o r a g e d i s e a s e s h a s been d e s c r i b e d . 9 7 6 patients
with
U r i n e s from
mucolipidosis
aspartylglycosaminuria,
I, m a n n o s i d o s i s , f u c o s i d o s i s , and t y p e I g l y c o g e n - s t o r a g e d i s e a s e can be
d i s t i n g u i s h e d b y t.1.c.
B-Q-Galactosidase d e f i c i e n c y i n p a t i e n t s
c a n be d e t e c t e d by e x a m i n a t i o n o f t h e u r i n e u s i n g a c o m b i n a t i o n o f
a-P-Mane-(1+2)-a-a-Mang-(l+2)-a-Q-Man~ 1
I 3 B-B-Manp(l+4)-g-GlcNAc
6
I 1 a-Q-Mane-(1+2)-a-g-Man~-(l+3)-a-p-Manp 6
I 1 a-P-Man~-(1+2)-a-Q-Mang
(68)
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides ion-exchange
c h r o m a t o g r a p h y a n d t.1.c.
301
Excess s i a l y l o l i g o s a c c h a r i d e
e x c r e t i o n i s d e t e c t e d by g e l f i l t r a t i o n f o l l o w e d by e s t i m a t i o n of neuraminic acid. O l i g o s a c c h a r i d e p a t t e r n s o b t a i n e d by g e l f i l t r a t i o n o f u r i n e o f GMl
gangliosidosis
gangliosidosis
(Type
(Type
1) p a t i e n t s d i f f e r
2)
p a t i e n t s . 977
from
The
those
amount
of
of
GM1
total
o l i g o s a c c h a r i d e excreted i n t h e u r i n e of
Type 2 p a t i e n t s i s 10-20%
o f t h a t i n t h e u r i n e o f Type 1 p a t i e n t s .
Oligosaccharides having
t h e s t r u c t u r e s (70-84) o c c u r i n t h e u r i n e o f Type 1 p a t i e n t s .
The
same o l i g o s a c c h a r i d e s w i t h t h e e x c e p t i o n o f ( 7 6 , 7 7 , 7 9 , 8 4 ) a r e e x c r e t e d i n t h e u r i n e s o f Type 2 p a t i e n t s .
a-g-ManQ-(1+3)-B-;-ManE-(l+4)-g-GlcNAc (69)
Four p o s i t i o n a l isomers o f neuraminosyl o l i g o s a c c h a r i d e s have been i s o l a t e d from
t h e u r i n e of
a patient
with sialidosis
with
p a r t i a l d e f i c i e n c y o f B - ~ - g a l a c t o ~ i d a s e . T~h ~e i~r s t r u c t u r e s a r e identical to
oligosaccharides
(70-71)
neuraminosyl or (2+6)-a-neuraminosyl
bearing either
(2+3)-a-
t e r m i n a l residues.
Two s i b l i n g s w i t h n e u r o n a l c e r o i d l i p o f u c o s i n o s i s e x c r e t e d t h e b l o o d - g r o u p A t r i s a c c h a r i d e , 3-g-(2-acetamido-2-deoxy-a-;-galactopy r a n o s y 1) -2-2- (a-C-f u c o p y r a n o s y 1)-;-ga
17
.
l a c t o s e , i n t h e u r i n e 979
Avian Glycoproteins
Absorption spectra,
c.d.
spectra, and sedimentation-equilibrium
v a l u e s have been r e p o r t e d f o r
complexes
of
l u t e i n with
egg
o v a l b u m i n .980 Ovalbumin has been s u b f r a c t i o n a t e d on i m m o b i l i z e d c o n c a n a v a l i n A.981
A
d i s t i n c t i v e carbohydrate
fractions
was n o t e d .
used
demonstrate
t o
H i g h - f i e l d 'H
composition n.m.r.
heterogeneity
i n each
of
four
spectroscopy has been o f
chick
ovalbumin
g l y c ~ p e p t i d e s . ~An~ ~u n a m b i g u o u s a s s i g n m e n t o f a l l C1-H a n d Q m a n n o s e C2-H r e s o n a n c e s was made, e n a b l i n g t h e s t r u c t u r e s o f t h e g l y c o p e p t i d e s t o be assigned.983 The c o m p l e t e a m i n o a c i d s e q u e n c e o f
385 r e s i d u e s ,
has been determined.984
postsynthetic modification: terminus,
hen o v a l b u m i n ,
comprising
Ovalbumin has f o u r s i t e s o f
in addition t o
the
a c e t y l a t e d N-
t h e c a r b o h y d r a t e m o i e t y i s l o c a t e d a t I-Asn-292,
and t h e
3 02
Carbohydrate Chemistry
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Carbohydrate Chemistry
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5: Glycoproteins, Glycopeptides, Proteoglycaris, and Animal Polysaccharides t w o p h o s p h o r y l a t e d L - s e r i n e s a r e a t r e s i d u e s 6 8 a n d 344. Two
glycopeptides
have
been i s o l a t e d f r o m
chicken ~ v a l b u m i n . ~ ’ B ~ oth h i g h Q-mannose-type
pepsin-treated and h y b r i d - t y p e
o l i g o s a c c h a r i d e s w e r e r e l e a s e d by an a l m o n d e m u l s i n g l y c o p e p t i d a s e . N a t u r a l - a b u n d a n c e 1 3 C n.m.r.
s p e c t r o s c o p y h a s been u s e d t o show
t h a t some a - Q - m a n n o p y r a n o s y l - ( 1 + 3 ) - Q - m a n n o p y r a n o s y l ovalbumin
glycopeptides
are
cleaved
much
l i n k a g e s i n hen faster
than
the
c o r r e s p o n d i n g ( 1 + 6 ) - l i n k a g e s b y j a c k - b e a n a - P - m a n n o ~ i d a s e . ~ I~n~ addition the
technique
was
used t o
examine
the
structures o f
p a r t i a l l y cleaved species produced d u r i n g t h e a c t i o n o f glycosidases on o l i g o s a c c h a r i d e s ,
glycopeptides,and glycoproteins.
Ovotransferrin,
i n a d d i t i o n t o human s e r u m t r a n s f e r r i n ,
i s
o x i d i z e d by s o d i u m p e r i o d a t e w i t h a r e s u l t i n g d e s t r u c t i o n o f I=t y r o s i n e r e s i d u e s and a loss o f i r o n - b i n d i n g a c t i v i t y . The
structural
determination o f
one
of
the
glycopeptides
o b t a i n e d by p r o t e o l y s i s o f t u r t l e - d o v e ovomucoid has r e v e a l e d t h e p r e s e n c e o f a t e r m i n a l t r i s a c c h a r i d e (851,
a s p e c i f i c blood-group
P1
a n t i g e n i c d e t e r m i n a n t p r e v i o u s l y d e s c r i b e d f o r human e r y t h r o c y t e P1 antigen.9e7 The (20%),
secondary
structures of
B-structure
(46%), a n d
chicken ovomucoid c o n t a i n a - h e l i x
random
coil
s t r u c t u r e p r e d i c t i o n s f o r chicken egg-white p~blished.~”
and d i s c u s s i o n i n c l u d e s c o n f o r m a t i o n a l
characteristics o f the glycoprotein,
18
Secondary-
ovomucoid have been
Some o f t h e e a r l i e r p r e d i c t i o n s o n s t r u c t u r e a r e
i n c o r p o r a t e d i n t h i s model, reactive site,
(18%).988
t h e secondary s t r u c t u r e a t t h e
a n d t h e s t r u c t u r a l homology among t h r e e domains.
M i s c e l l a n e o u s G l y c o p r o t e i n s and C h i t i n
The s t r u c t u r e s o f f i f t e e n o l i g o s a c c h a r i d e s ( 8 6 - 1 0 0 ) i s o l a t e d f r o m
B-Q-Gale-(1+3)-Q-GlcNAc (86) B-Q-Gale-(1+3)-Q-GalNAc (87)
3 08
Carbohydrate Chemistry B-Q-GalQ-( 1+4) -g-GlcNAc 3
I
1 a-C-Fucg
(88) R - ( 1+2) -B -Q-Gale-( 1+4) -Q-Glc
3
I 1 a-L-Fucp
(89) R = H (90) R = a-L-Fuce
1+-4) -B-Q-GlcpNAc-( 1+3) + - G a l B-Q-Galp-( 92) R1-(
1+2) -a-Q-Mane 1
I 6 R 2 - ( 1+2) -a-Q-Manp-(
1+3) -B +-Mane-(
1+4) -B-Q-GlcNAc
4
I 1 B-Q-GlCQNAC A = a-Neup5Ac-(2+6)-B-Q-Gale-(
B = B-Q-GlCeNAC C = B-Q-Galg-( 1+4) -B-P-GlcpNAc
1-41
-B-Q-GlceNAc
309
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides newborn meconium have been i d e n t i f i e d . ’ ”
I t i s proposed t h a t
e n d o g l y c a n a s e s a r e i n v o l v e d i n t h e p r o d u c t i o n o f t h e s e compounds from glycoproteins. Q - G a l a c t a n s have been i s o l a t e d f r o m t h e albumen g l a n d s f r o m s n a i l s o f t h e g e n u s B i ~ m p h a l a r i a . ~T h~e~ r e s u l t s o f m e t h y l a t i o n a n a l y s i s and p e r i o d a t e o x i d a t i o n d a t a i n d i c a t e t h e p r e s e n c e o f a multibranched
structure
c o n t a i n i n g [1+3)-
Polysaccharides containing b o t h
9-
and (1+6)-linkages.
and l = - g a l a c t o s y l r e s i d u e s have
been i s o l a t e d f r o m t h e a l b u m e n g l a n d s o f f o u r s p e c i e s o f A n t i s e r a t o t h e f o u r g a l a c t a n s showed v a r i o u s degrees o f c r o s s reactivity,
indicating structural differences, ascribable i n part t o
d e t e r m i n a n t s i n v o l v i n g g a l a c t o s e p h o s p h a t e , and p r o b a b l y a l s o t o t h e A B-
l i n k a g e and p o s i t i o n o f I - g a l a c t o s y l r e s i d u e s i n t h e m o l e c u l e . Q-galactopyranan,
--Pomacea
i s o l a t e d from t h e albumen gland o f t h e s n a i l
l i n e a t a , c o n t a i n s 3,4-Q-(l-carboxyethylidene)
groups, as
s h o w n by t h e l i b e r a t i o n o f p y r u v i c a c i d o n a c i d h y d r o l y s i s a n d b y m e t h y l a t i o n data.993
The s t r u c t u r e o f t h e 5-membered a c e t a l g r o u p s spectroscopy.
o f p y r u v i c a c i d was s t u d i e d by 1 3 C n.m.r.
The r o l e o f g l y c o s y l a t i o n i n t h e s e c r e t i o n o f v a r i o u s a v i a n and m a m m a l i a n p r o t e i n s s y n t h e s i z e d i n Xenopus l a e v i s o o c y t e s u n d e r t h e d i r e c t i o n of
heterologous
messenger RNA h a s been r e p o r t e d . 9 9 4
The
presence o f 3 - g l y c o s y l chains on t h e p r o t e i n s d i d n o t appear t o facilitate
secretion.
O l i g o s a c c h a r i d e s w i t h a new t y p e o f c o r e
s t r u c t u r e ( 1 0 1 ) h a v e been i s o l a t e d f r o m t r o u t egg glycoprotein.’’5 The o c c u r r e n c e o f N - g l y c o l y l n e u r a m i n i c a c i d l i n k e d (2-3) i n t e r n a l 2-acetamido-2-deoxy-Q-galactosyl
r e s i d u e has
t o the not
been
d e s c r i b e d i n o t h e r g l y c o p r o t e i n s or g l y c o l i p i d s . Neug5G 2
I 3
8-~-Gal~NAc-(1+4)-B-~-Gal~NAc-(l+4)-8-p-Gal~NAc-(l+3)8-g-Galp-(1+3)-GalNAc-ol (101) B o v i n e h y p o t h a l m i c mRNA t r a n s l a t e d i n a r e t i c u l o c y t e l y s a t e s y s t e m y i e l d s a common p r e c u r s o r [ m o l .
wt.
2.1
x
lo4)
by m i c r o s o m a l
membranes f r o m dog p a n ~ r e a s . ~ ’ ~T h i s p r e c u r s o r b i n d s t o i m m o b i l i z e d c o n c a n a v a l i n A and i s s e n s i t i v e t o t r e a t m e n t w i t h a-p-mannosidase. Purified variant-specific
g l y c o p r o t e i n a n t i g e n s o f Trypanosoma
310
Carbohydrate Chemistry
brucei exist
i n
solution
as
dimers
and o c c a s i o n a l l y as h i g h e r
T.
Two v a r i a n t s u r f a c e g l y c o p r o t e i n s o f b r u c e i have o 1 igo m e r s .99’ been shown t o h a v e a c o n s e r v e d C - t e r m i n a l a m i n o a c i d sequence.998 The g l y c o s y l a t i o n o f t h e m a j o r v a r i a b l e s u r f a c e c o a t g l y c o p r o t e i n o f
-T. b r u c e i
i s i n h i b i t e d by t u n i c a m y ~ i n . N ~ -~L i~n k e d g l y c o s y l a t i o n
occurs subsequent t o s y n t h e s i s o f t h e p r o t e i n . V a r i a n t a n t i g e n s o f Trypanosoma c o n g o l e n s e h a v e b e e n p u r i f i e d by l e c t i n a f f i n i t y c h r o m a t o g r a p h y . loo0 A
molecular- weight
glycopeptides
of
the
analysis of protozoan
the
major
p o l y p e p t i d e s and
Toxoplasma g o n d i i
has
been
r e p o r t e d . lool A s u l p h a t e d g l y c o p r o t e i n (mol.
wt.
1.85 x
lo5)
i s synthesized
i n t h e m u l t i c e l l u l a r organism Velvox c a r t e r i o n l y d u r i n g t h e l i m i t e d p e r i o d o f embryogenesis.1002 function o f t h i s cell-surface
E v i d e n c e f o r an e m b r y o n i c c o n t r o l glycoprotein i s provided.
S m a l l a m o u n t s o f c h i t i n i n a r t h r o p o d c u t i c l e s c a n be m e a s u r e d b y e s t i m a t i n g t h e d e r i v a t i z e d s o l u b l e c h i t o ~ a n . The ~ ~ ~m e~ t h o d i s n o t a f f e c t e d by t h e p r e s e n c e o f n o n - c h i t i n o u s c u t i c u l a r components. C o v a l e n t l y bound c h i t i n - p r o t e i n complexes o f s e v e r a l s p e c i e s o f marine
invertebrates
deproteinization,
a l l
the
have
been
samples
isolated.1004
contained
small
After
amounts
of
r e s i d u a l amino a c i d s w i t h marked s p e c i e s v a r i a t i o n i n t h e i r i d e n t i t y and c o n t e n t . A c o m p a r a b l e s e r i e s o f c h i t i n s d e r i v e d f r o m b l u e , r e d , stone, and h o r s e s h o e c r a b s by t r e a t m e n t w i t h m i l d a c i d ,
H 4 e.d.t.a.,
and
a l k a l i h a v e b e e n i s 0 1 a t e d . l ~ ~The ~ polysaccharides comprise a family
of
closely related
molecular weight,
products
with
variable
solubility,
o p t i c a l r o t a t i o n , and a c e t y l v a l u e s , w h i c h a r e a
f u n c t i o n o f b o t h t h e s p e c i e s f r o m w h i c h t h e y o r i g i n a t e d and t h e i r method o f p r e p a r a t i o n . I n c r e a s e d r a t e s o f h y d r o l y s i s by c h i t i n a s e and l y s o z y m e o f c h i t i n and c h i t o s a n a f t e r o x i d a t i o n w i t h s o d i u m m e t a p e r i o d a t e h a v e been o b s e r v e d . l o o 6 19
The
Analysis o f Glycoproteins
structure,
carbohydrate
elucidation of
structures,
and f u n c t i o n s
of
r e s i d u e s on g l y c o p r o t e i n s have been r e v i e w e d . l o o 7
M e t h o d s f o r a n a l y s i n g c o m p l e x m i x t u r e s o f a n t i g e n s and g l y c o p r o t e i n s have been r e v i e w e d . l o o 8 S i a l o g l y c o p r o t e i n s h a v e been f r a c t i o n a t e d o n an i m m o b i l i z e d
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
31 1
neuraminic acid-binding l e c t i n from the horseshoe crab Carcinoscorpius rotunda ~auda.~"' The r e s o l u t i o n o f t h e i s o e n z y m e s o f a l k a l i n e p h o s p h a t a s e was a c h i e v e d . Polyacrylamide g e l e l e c t r o p h o r e s i s has been used t o r e s o l v e f l u o r e s c e i n - d e r i v a t i z e d asialoglycopeptides.lolo Specific e x o g l y c o s i d a s e d i g e s t i o n of t h e g l y c o p e p t i d e s g e n e r a t e s a series o f d e g r a d a t i o n p r o d u c t s t h a t are r e s o l v e d . A method f o r m o n i t o r i n g t h e r e m o v a l o f poly-e-mannosyl c h a i n s f r o m g l y c o p r o t e i n s by e n d o - 2 - a c e t a m i d o - 2 - d e o x y - Q - g l y c a n a s e H has been described.l15 A f t e r p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f t h e g l y c o p r o t e i n s and t h e i r e n z y m i c a l l y degraded p r o d u c t s , t h e g e l s are overlaid with iodinated lectins. The d e g r e e t o which these l e c t i n s b i n d i s t h e n m e a s u r e d by a u t o r a d i o g r a p h y . A peptide:!-glycanase h a s been p u r i f i e d from almond e m u l s i n and its s p e c i f i c i t y examined w i t h glycopeptides prepared from ovalbumin Both high g-mannose and complex and immunoglobulin M.l0l1 g l y c o p e p t i d e s are h y d r o l y s e d a t t h e B-I=-aspartylglycosylamine linkage. A method f o r t h e f l u o r e s c e n t l a b e l l i n g o f s i a l o g l y c o p r o t e i n s i n v o l v e s o x i d a t i o n w i t h s o d i u m p e r i o d a t e f o l l o w e d by c o n d e n s a t i o n w i t h f l u o r e s c e i n a m i n e o r m o n o d a n s y l - 1 , 2 - d i a m i n o e t h a n e f o l l o w e d by s t a b i l i z a t i o n o f t h e S c h i f f ' s b a s e s by r e d u c t i v e a m i n a t i ~ n . ~ ~ ' L e c t i n s have been used f o r t h e d e t e c t i o n of g l y c o p r o t e i n s transferred t o nitrocellulose sheets, a f t e r t h e i r electrophoretic s e p a r a t i o n on p o l y a c r y l a m i d e g e l s . l0l2 O l i g o s a c c h a r i d e s d e r i v e d f r o m g l y c o p r o t e i n s by e n z y m i c r e l e a s e w i t h e n d o g l y c o s i d a s e s or chemical r e l e a s e by h y d r a z i n o l y s i s h a v e b e e n r e d u c e d by s o d i u m b o r o t r i t i d e a n d s e p a r a t e d by h . p . l . c . 1 ° 1 3 T r e a t m e n t of g l y c o p r o t e i n s w i t h trifluoromethanesulphonic acid a t room t e m p e r a t u r e r e s u l t s i n t h e r a p i d c l e a v a g e o f p e r i p h e r a l s u g a r s , a s l o w loss o f I - s e r i n e a n d I - t h r e o n i n e - l i n k e d 2 - a c e t a m i d o 2-deoxy-p-galactosyl r e s i d u e s , and r e t e n t i o n o f 2 - g l y c o s i d i c a l l y l i n k e d 2-acetamido-2-deoxy-~-glucosyl residues.1°14 The r e a g e n t is safer t o u s e t h a n e i t h e r hydrogen f l u o r i d e or its p y r i d i n e complex. Glycoproteins have been i o d i n a t e d using t h e solid-phase o x i d i z i n g a g e n t 1,3,4,6-tetrachloro-3a,6a-diphenylglycouril ( i o d ~ g e n ) . ' ~ ' ~ The method c o m p a r e s f a v o u r a b l y w i t h t h e s o l i d - p h a s e l a c t o p e r o x i d a s e and c h l o r a m i n e T methods. A method f o r d e t e r m i n a t i o n of p 1 volumes o f g l y c o p r o t e i n u s i n g s p o t a n a l y s i s on c e l l u l o s e acetate l a y e r s i s described.1°16 W i t h pg l u c o s e o x i d a s e and f l u o r e s c e i n - l a b e l l e d c o n c a n a v a l i n A s e n s i t i v i t y
Carbohydrate Chemistry
312 o f 10 n g i s r e a c h e d .
This l e v e l of d e t e c t i o n i s lowered t o 1 ng o f
Q - g l u c o s e o x i d a s e when t h e r e a g e n t s a r e u s e d i n c o m b i n a t i o n w i t h horse-radish
peroxidase.
. .r . s p e c t r o s c o p ic
3C n m
da t a f o r 3 -0- ( 2-ace t a m ido - 2 - de o x y -a-p
-
galactopyranosy1)-N-acetyl-L-serine, 3-0-(2-acetamid0-2-deoxy-a-D- g a l a c t o p y r a n o s y l l - N - a c e t y l - i - t h r e o n i n e , 3-g-a9B-Q-galactopyranosylL_- - s e r i n e, a n d 3 -g-a,B-g - g a 1a c t o p y r a n o s y 1-L- - t h r e o n in e h a v e , b e e n recorded.1°17
These m o d e l compounds r e p r e s e n t common c a r b o h y d r a t e -
p r o t e i n l i n k a g e r e g i o n s of
many g l y c o p r o t e i n s .
The s t r u c t u r e s o f t h e o l i g o s a c c h a r i d e c o m p o n e n t s o f a number o f g l y c o p e p t i d e s have been e s t a b l i s h e d f o l l o w i n g h y d r a z i n o l y s i s - n i t r o u s a c i d deamination,
w h i c h l e a d s t o t h e s p e c i f i c c l e a v a g e o f 2-amino-2-
deoxy-g-glucosyl
l i n k a g e s .883
T h e d e r i v e d 2,5-anhydro-Q-mannose-
c o n t a i n i n g o l i g o s a c c h a r i d e s a r e r e d u c e d w i t h s o d i u m b o r o h y d r i d e and a n a l y s e d by g.1.c.-m.s.
o f t h e i r methylated ethers.
The f l e x i b i l i t y o f b i acetyl-lactosamine
and t r i - a n t e n n a r y
N-
glycans o f the
t y p e has been s t u d i e d by s p i n - l a b e l l i n g
the
n e u r a m i n i c a c i d r e s i d u e s i n g l y c o p e p t i d e s o f known s t r u c t u r e . l o l 8 A
detailed
analysis
of
the
360
MHz 'H
n.m.r.
spectral
p a r a m e t e r s f o r t h e a n o m e r i c a n d C2-H r e s o n a n c e s o f a l a r g e number o f g l y c o p e p t i d e s and o l i g o s a c c h a r i d e s of
known s t r u c t u r e r e v e a l s a
g e n e r a l method f o r t h e d e t e r m i n a t i o n of t h e p r i m a r y s t r u c t u r e o f g l y c o p e p t i d e s f o r m o s t c u r r e n t l y known c l a s s e s o f s t r u c t u r e s . 1 0 1 9 two-dimensional
d i s p l a y formed by p l o t t i n g q-mannosyl
C1-H
A
against
C2-H c h e m i c a l s h i f t s d e m o n s t r a t e s t h a t t h e s e p a i r s o f v a l u e s a r e s e n s i t i v e t o long-range
p e r t u r b a t i o n by r e m o t e s u b s t i t u t i o n by
hexoses as w e l l as t o d i r e c t s u b s t i t u t i o n forty-one
o f these chemical-shift
characterize unique s t r u c t u r a l microenvironments. s e q u e n c e and b r a n c h i n g p a t t e r n f o r
A total of
effects.
c l u s t e r s have been d e f i n e d w h i c h On t h i s b a s i s t h e
most s t r u c t u r e s
can be d e r i v e d .
The h y d r o d y n a m i c b e h a v i o u r o f r e d u c e d g l y c o p o l y p e p t i d e s h a s been
studied
conjunction
by
gel
with
filtration
h.p.l.c.lo2'
in
guanidine
Although
hydrochloride
i n
carbohydrate-rich
g l y c o p o l y p e p t i d e s may o c c a s i o n a l l y y i e l d u n d e r e s t i m a t e d v a l u e s o f molecular weights,
t h e method i s s u i t a b l e f o r t h e r a p i d e s t i m a t i o n
o f m o l e c u l a r w e i g h t s o f s i m p l e p o l y p e p t i d e s and g l y c o p o l y p e p t i d e s . Neuraminic immunodiffusion glycoprotein.1021
acid but
residues not
are
involved i n
the
the r a d i a l immunodiffusion
electro-
of
al-acid
D i f f e r e n t i a l d e t e r m i n a t i o n o f t h e g l y c o p r o t e i n by
t h e t w o i m m u n o l o g i c a l methods o f f e r s a method f o r t h e e s t i m a t i o n o f t h e d e g r e e o f s i a l y l a t i o n o f t h i s and o t h e r s e r u m g l y c o p r o t e i n s .
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
313
T h e s t r u c t u r e s o f s i a l o - o l i g o s a c c h a r i d e s a s d e t e r m i n e d b y 'H n.m.r. s p e c t r o s c o p y t o g e t h e r w i t h m e t h y l a t i o n a n a l y s i s h a v e b e e n shown t o vary w i t h t h o s e d e r i v e d from p e r i o d a t e o x i d a t i o n d a t a alone.1022 E x a m i n a t i o n o f a c i d i c t e t r a s a c c h a r i d e s o b t a i n e d f r o m A', ,'H a n d A - H - h o g s u b m a x i l l a r y m u c i n i n d i c a t e s t h a t n e u r a m i n i c a c i d r e s i d u e s a r e n o t r e l e a s e d f r o m o l i g o s a c c h a r i d e s when p e r i o d a t e o x i d a t i o n i s c a r r i e d o u t a t pH 4.5. The a n i o n i c p r o p e r t i e s o f t h e n e u r a m i n i c a c i d r e s i d u e s were u s e d t o s e p a r a t e t h e p e r i o d a t e o x i d a t i o n p r o d u c t s , t h e r e b y g i v i n g i n f o r m a t i o n on t h e l o c a t i o n of n e u r a m i n i c a c i d on t h e o l i g o s a c c h a r i d e c h a i n . The monosaccharide components of g l y c o p r o t e i n s have been reacted w i t h d a n s y l h y d r a z i n e and the r e s u l t i n g d e r i v a t i v e s s e p a r a t e d b y h . p . l . ~ . l ' ~ ~F l u o r e s c e n t d e t e c t i o n p r o v i d e s a s e n s i t i v i t y o f 10 p m o l per i n j e c t i o n . P a r t i a l l y methylated 2-methyl 2-acetyl methyl glycosides o b t a i n e d by m e t h a n o l y s i s a n d a c e t y l a t i o n o f g l y c o p r o t e i n s h a v e b e e n i d e n t i f i e d by g.1.c.-m.s. analysis.1024 A scheme f o r t h e r a p i d d e t e r m i n a t i o n o f t h e p o s i t i o n o f t h e m e t h y l and a c e t y l r e s i d u e s is proposed. T h e m e t h o d was a p p l i e d t o s i a l o g l y c o p e p t i d e s i s o l a t e d from al-acid glycoprotein. Methylation techniques used i n t h e s t r u c t u r a l a n a l y s i s of g l y c o p r o t e i n s and g l y c o l i p i d s have been reviewed.1025 A m o d i f i c a t i o n o f a g.1.c. m e t h o d a l l o w i n g s i m u l t a n e o u s s e p a r a t i o n o f n e u t r a l and amino s u g a r a l d i t o l acetates has been described.1026 The m e t h o d h a s b e e n a p p l i e d t o t h e m o n i t o r i n g o f t h e f r a c t i o n a t i o n o f complex mixtures of g l y c o p r o t e i n s and glycosaminoglycans. A r a p i d i s o c r a t i c h.p.1.c. method f o r t h e e s t i m a t i o n o f n e u r a m i n i c acid i n serum h a s been described.lo2' S e p a r a t i o n is a c h i e v e d on a c a t i o n - e x c h a n g e r e s i n u s i n g a 0.006N s u l p h u r i c a c i d m o b i l e p h a s e . Reviews d e a l i n g w i t h g l y c o s y l t r a n s f e r a s e s and their u s e i n a s s e s s i n g o l i g o s a c c h a r i d e s t r u c t u r e and s t r u c t u r e - f u n c t i o n r e l a t io n s h i p s h a v e b e e n p u b 1i s h e d l o28 9 02' Lysosomal enzymes contain e-mannosyl 6-phosphate m o i e t i e s which mediate t h e i r t r a n s l o c a t i o n t o lysosomes.1030 A new a s s a y f o r a-Q2-acetamido-2-deoxy-glucosyl p h o s p h o t r a n s f e r a s e using a-methyl-emannoside as a c c e p t o r has been r e p o r t e d . A c o u p l e d e n z y m e a s s a y f o r 2 - a c e t a m i d o - 2 - d e o x y - ~ - g l u c o s e : UDPa - g a l a c t o s e a - g a l a c t o s y l t r a n s f e r a s e h a s been developed t h a t a l l o w s t h e enzyme t o be assayed s p e c t r o p h o t o m e t r i c a l l y and i n d e n a t u r i n g p o l y a c r y l a m i d e gels.1031 I n a sequence o f l i n k e d enzyme r e a c t i o n s t h e l i b e r a t e d UDP i s s u c c e s s i v e l y c o n v e r t e d t o UTP, U D P - a - g l u c o s e ,
.
Carbohydrate Chemistry
314
and f i n a l l y UDP-a-glucuronic a c i d w i t h t h e s u b s e q u e n t p r o d u c t i o n o f NADH. N A D H may t h e n be e s t i m a t e d s p e c t r o p h o t o m e t r i c a l l y o r d e t e c t e d i n polyacrylamide g e l s fluorimetrically.
20
B i o s y n t h e s i s o f Glycoproteins
A r e v i e w of t h e transmembrane a s s e m b l y of membrane and s e c r e t o r y
g l y c o p r o t e i n s h a s b e e n p ~ b 1 i s h e d . l ~ ~ B’ l o o d - g r o u p a n t i g e n s a n d t h e enzymes i n v o l v e d i n t h e i r s y n t h e s i s h a v e been reviewed.1033 Exposure of rat hepatoma t i s s u e c u l t u r e cells t o dexamethasone r e s u l t s i n t h e a p p e a r a n c e o f a new g l y c o p r o t e i n ( g p 3 5 - 5 0 , m o l . w t . 3.5 x l o 4 - 5.0 x l o 4 ) a n d i n c r e a s e d s y n t h e s i s o f a n o t h e r g l y c o p r o t e i n ( m o l . w t . 5.0 x 1 0 ~ 1 . ~ T’h e~ f~o r m e r g l y c o p r o t e i n i s e x p r e s s e d i n n o r m a l l i v e r , whereas t h e l a t t e r i s e x p r e s s e d i n h e p a t o m a c e l l s a n d i s r e g u l a t e d d i f f e r e n t l y by s t e r o i d h o r m o n e s . T r e a t m e n t o f human k i d n e y t u m o u r c e l l s w i t h b u t y r a t e i n c r e a s e s markedly t h e synthesis and sulphation of tumour-cell u n t r e a t e d c u l t u r e d cells release most o f g 1 y ~ o p r o t e i n s . l ~Whereas ~~ t h e i r s u l p h a t e d g l y c o p r o t e i n s i n t o t h e medium, b u t y r a t e - t r e a t e d cells p r e f e r e n t i a l l y accumulate t h e s e products i n t o t h e cell layer. The s y n t h e s i s and a c c u m u l a t i o n of Q - m a n n o s e - c o n t a i n i n g g l y c o p e p t i d e s i n human f i b r o b l a s t c e l l s h a v e been studied.1036 R e s u l t s are c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t m u l t i p l e pathways f o r L - a s p a r a g i n e - l i n k e d g l y c o p r o t e i n b i o s y n t h e s i s a r e p o s s i b l e . The i n c o r p o r a t i o n of Q-mannose, Q-glucose, and 2-acetamido-2-deoxy-Qg l u c o s e i n t o endogenous membrane p r o t e i n s i n calf p i t u i t a r y has been r e p o r t e d . lo3’ Glycosylation of proteins i n the protozoan Crithidia ----------fasciculata does not involve glucosylated lipid-bound oligosaccharides a s intermediates.1038 An o l i g o s a c c h a r i d e c o n t a i n i n g s e v e n P-mannosyl and t w o 2-acetamido-2-deoxy-~-glucosyl residues is t r a n s f e r r e d t o protein before undergoing processing. Carbohydrate-depleted or c h e m i c a l l y d e n a t u r e d g l y c o p r o t e i n s or s y n t h e t i c C-Asn-X-&-Ser/L-Thr-containing p e p t i d e s have been g l y c o s y l a t e d u s i n g hen o v i d u c t membranes as t h e enzyme source.1039 T r i p e p t i d e s may b e g l y c o s y l a t e d b u t t h e l e v e l o f g l y c o s y l a t i o n The i n c r e a s e s as t h e l e n g t h o f t h e p e p t i d e c h a i n i n c r e a s e s . p e p t i d e s were e x a m i n e d b y c . d . i n a q u e o u s l i p i d m i x t u r e s , w h e n i t was o b s e r v e d t h a t t h e p r e s e n c e o f s e c o n d a r y s t r u c t u r e i n t h e l i p i d state promotes the level of glycosylation. The s y n t h e s i s and
315
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides processing
of
I=-asparagine-linked
reviewed.lo40
The
assembly
of
o l i g o s a c c h a r i d e s have been
the
oligosaccharide through i t s transfer
lipid-linked
precursor
t o p r o t e i n and i t s c o n v e r s i o n
t o t h e d i v e r s e a r r a y o f f i n a l products i s discussed.
Glycoprotein
b i o s y n t h e s i s i n mouse L c e l l s i n v o l v e s l i p i d - l i n k e d s a c c h a r i d e s i n the synthesis o f L-asparagine-linked
glycoproteins.1041
The
f o r m a t i o n o f h i g h Q - m a n n o s y l o l i g o s a c c h a r i d e bound t o p r o t e i n can occur
independently o f
higher
lipid-linked
oligosaccharide
synthesis. D o l i c h o l phosphate appears
t o c o n t r o l t h e r a t e of
g l y c o s y l a t i o n d u r i n g t h e development o f sea-urchin
protein
embryos.1042
C o m p a c t i n , an i n h i b i t o r o f p o l y i s o p r e n o i d b i o s y n t h e s i s ,
inhibits A
d o l i c h o l p h o s p h a t e s y n t h e s i s and t h u s g l y c o s y l a t i o n o f p r o t e i n .
second e f f e c t o f t h e i n h i b i t o r i s t h a t a s i g n i f i c a n t f r a c t i o n o f t h e o l i g o s a c c h a r i d e chains synthesized i n t h e presence o f t h e i n h i b i t o r a n d t r a n s f e r r e d t o p r o t e i n a p p e a r t o b e a l t e r e d so t h a t t h e y a r e more n e g a t i v e l y charged.
B o t h de novo s y n t h e s i s o f d o l i c h o l
w e l l as i t s phosphorylation
as
may p l a y a n i m p o r t a n t r o l e i n t h e
i n c r e a s e i n g l y c o p r o t e i n s y n t h e s i s d u r i n g embryonic development o f t h e sea urchin.1043
The p o s s i b l e r o l e o f d o l i c h o l k i n a s e i n t h e
a c t i v a t i o n o f s t o r e d d o l i c h o l , and p e r h a p s t h e pathway f o r i t s
---novo
biosynthesis
i n this
and o t h e r
b i o l o g i c a l systems,
& is
discussed. Turpentine-induced
inflammation
in
rats
causes
increased
s y n t h e s i s o f g l y c o s y l a t e d d o l i c h o l phosphate d e r i v a t i v e s , which a r e intermediates glycoproteins,
i n
the
biosynthesis
of
I-asparagine-linked
and a l s o i n c r e a s e d l e v e l s o f a CTP-dependent
dolichol
k i n a s e . 1044 Immature chick oviduct responsible for
membranes c o n t a i n a t r a n s f e r a s e
transfer o f oligosaccharide pyrophosphoryl d o l i c h o l
t o p r o t e i n acceptors.1045
The enzyme i s s t i m u l a t e d b y e s t r o g e n
treatment. The
metabolism of
lipid-linked
B-mannose
intermediates i n
g l y c o p r o t e i n s y n t h e s i s d u r i n g l i v e r r e g e n e r a t i o n h a s been s t u d i e d i n rat
liver
micro some^.^^^^
i n c o r p o r a t i o n of
p-{
F o l l o w i n g p a r t i a l hepatectomy,
the
1 4 ~ 1 - m a n n o s e f r o m GDP-Q-{ 1 4 ~ 1 - m a n n o s e i n t o
d o l i c h y l p h o s p h o r y l e-mannose a n d p r o t e i n d e c r e a s e s a s c o m p a r e d w i t h controls.
E v i d e n c e i s g i v e n s u g g e s t i n g t h a t t h e i n c o r p o r a t i o n o f Q-
mannose i n t o g l y c o p r o t e i n i n r e g e n e r a t i n g r a t l i v e r may b e r e g u l a t e d a t t h e s t e p of
oligosaccharide transfer.
U s i n g t h e Thy-1'
m u t a n t mouse l y m p h o m a c e l l s o f t h e c l a s s E
316
Carbohydrate Chemistry
c o m p l e m e n t a t i o n g r o u p w h i c h l a c k GDP-Q-mannose : d o l i c h o l phosphoryl mannosyltransferase and which are t h e r e f o r e unable t o i n t e r c o n v e r t GDP-Q-mannose a n d g - m a n n o s y l p h o s p h o r y l d o l i c h o l , f u r t h e r e v i d e n c e h a s been p r o v i d e d t h a t f i v e o f t h e n i n e e-mannosyl r e s i d u e s which are added t o the growing l i p i d - l i n k e d o l i g o s a c c h a r i d e s a r e d o n a t e d d i r e c t l y by G D P - Q - m a n n o s e . l o 4 ’ The r e m a i n i n g f o u r r e s i d u e s a r i s e from Q-mannosyl p h o s p h o r y l d o l i c h o l . The r e a c t i o n s i n v o l v e d i n t h e a d d i t i o n o f g - g l u c o s y l r e s i d u e s t o t h e o l i g o s a c c h a r i d e - l i p i d i n t e r m e d i a t e s by t h y r o i d m i c r o s o m e s have been studied.1048 The p r o p e r t i e s o f t h e enzymes i n v o l v e d i n t h e t r a n s f e r o f !&glucose f r o m i t s p h o s p h o r y l d o l i c h o l d e r i v a t i v e t o endogenous a c c e p t o r s are d e s c r i b e d and t h e p r o d u c t s are c h a r a c t e r i z e d i n terms o f t h e i r s t r u c t u r e a n d c a p a c i t y t o s e r v e a s d o n o r s i n t h e glycosylation of proteins. The b i o s y n t h e s i s and p a r t i a l c h a r a c t e r i z a t i o n of a P-glucose-containing i n s e c t l i p i d - l i n k e d o l i g o s a c c h a r i d e have been reported.lo4’ Its p r o p e r t i e s are c l o s e l y r e l a t e d t o t h e g-glucosylated d o l i c h y l oligosaccharide obtained from mammalian s y s t e m s . A m p h o m y c i n i n h i b i t s t h e t r a n s f e r o f Q - m a n n o s e , g - g l u c o s e , a n d 2acetamido-2-deoxy-!-glucose l-phosphate from their r e s p e c t i v e n u c l e o t i d e d e r i v a t i v e s t o d o l i c h o l p h o s p h a t e by m e m b r a n e p r e p a r a t i o n s f r o m c a l f b r a i n membranes.1050 O t h e r g-mannosyl-, g l u c o s y l - , 2-acetamido-2-deoxy-Q-glucosyl-transferases associated w i t h t h e same p r e p a r a t i o n a r e n o t a f f e c t e d by t h e a n t i b i o t i c . UDP-2-Deoxy-Q-arabino-hexose (UDP-2-deoxy-~-glucose) i n h i b i t s t h e formation o f P-glucosylphosphoryl d o l i c h o l i n chick-embryo cell m e m b r a n e s , b u t h a s n o e f f e c t o n t h e f o r m a t i o n o f N”d i a c e t y l c h i t o b i o s y l p y r o p h o s p h o r y l d 0 1 i c h o l . l ~ ~G~D P - 2 - D e o x y - P ------arabino-hexose inhibits the formation of both the lipid i n t e r m e d i a t e s by c o m p e t i t i o n w i t h p h y s i o l o g i c a l n u c l e o t i d e s u g a r s f o r d o l i c h o l phosphate. The i n f l u e n c e o f v a r i o u s a r y l p h o s p h a t e s and p h o s p h o n a t e s on t h e s y n t h e s i s o f g l y c o s y l p h o s p h o r y l d o l i c h o l , o l i g o s a c c h a r i d e s , and g l y c o p r o t e i n s by r a t l i v e r m i c r o s o m a l f r a c t i o n s h a s b e e n investigated.1052 Phenyl phosphate d i r e c t l y e f f e c t s t h e s y n t h e s i s o f a-mannosyl p h o s p h o r y l d o l i c h o 1 , a n d t h e i n h i b i t i o n o f l i p i d - l i n k e d o l i g o s a c c h a r i d e s a n d g l y c o p r o t e i n s may b e a c o n s e q u e n c e o f t h e effect. The a n t i b i o t i c t s u s h i m y c i n i n h i b i t s t h e f o r m a t i o n o f Qg l u c o s y l - , a-mannosyl-,and 2-acetamido-2-deoxy-~-glucosyl p h o s p h o r y l dolichol, but allows the incorporation of sugars i n t o lipid-linked
a-
317
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides oligosaccharides aorta.1053
i n a particulate
enzyme
The m a j o r o l i g o s a c c h a r i d e
preparation
from
pig
f o r m e d f r o m GDP-D-mannose
in
t h e p r e s e n c e o f t h e a n t i b i o t i c i s (Q-Man~),-(g-GlceNAc)~. T u n i c a m y c i n h a s l i t t l e e f f e c t on t h e p r o t e i n s i n t h e e p i d e r m i s when p i g s k i n s l i c e s a r e m a i n t a i n e d i n o r g a n c u l t u r e ,
although the
synthesis o f s p e c i f i c epidermal glycoconjugates i s affected.1054 The g l y c o s y l a t i o n o f p a r t i c u l a t e g l y c o p r o t e i n s i s d e c r e a s e d , that o f soluble glycoproteins i s hardly affected.
whereas
Inhibition of
s u l p h a t e d g l y c o s a m i n o g l y c a n s b u t n o t o f h y a l u r o n i c a c i d was a l s o noted. The p e p t i d e a n t i b i o t i c t r i d e c a p t i n s t i m u l a t e s t h e i n c o r p o r a t i o n o f g-mannose f r o m GDP-;-(
14C)-mannose
glucose i n t o l i p i d - l i n k e d p i g aorta.1055
and ;-glucose
f r o m UDP-!-{
3H}-
m o n o s a c c h a r i d e s by enzyme f r a c t i o n s f r o m
The a n t i b i o t i c a l s o s t i m u l a t e s t h e i n c o r p o r a t i o n o f
sugars i n t o l i p i d - l i n k e d oligosaccharides. E p i t h e l i a l c e l l s of
the r a t
mannosyl phosphoryl d o l i c h o l dolicho1,which tissues.1056
small intestine synthesize
9-
and o l i g o s a c c h a r y l p y r o p h o s p h o r y l
are s i m i l a r t o those found i n a v a r i e t y o f other The
importance o f
m o n i t o r i n g and c o n t r o l l i n g t h e
d e g r a d a t i o n o f n u c l e o t i d e s u g a r s when g l y c o s y l t r a n s f e r a s e a c t i v i t i e s a r e b e i n g compared i n d i f f e r e n t i a t i n g c e l l s i s d e m o n s t r a t e d . Incubation o f cell-free s a l i n a ) w i t h GDP-g-mannose
e x t r a c t s of
the brine shrimp (Artemia
o r UDP-!-glucose
results i n the formation
o f t h e c o r r e s p o n d i n g d o l i c h y l d e r i v a t i v e s o f t h e sugars.1057
The
enzymic a c t i v i t i e s were d e t e c t e d e a r l y i n t h e development o f t h e e n c y s t e d A r t e m i a embryos. S t a r v a t i o n o f !-glucose
alters lipid-linked oligosaccharide
b i o s y n t h e s i s i n Chinese h a m s t e r o v a r y cells.1058
The c e l l s q u i c k l y
c e a s e s y n t h e s iz i n g t h e (Q- G I c e ) 3(D, - M ane)9-(P-GlceNAc)2 i n s t e a d make p r e d o m i n a n t l y
the
(p-Ma~),(g-GlceNAc)~
s p e c i e s and
species, w h i c h
i s g l u c o s y l a t e d and t r a n s f e r r e d t o p r o t e i n w h e r e i t i s s u b s e q u e n t l y processed,
t h u s d e m o n s t r a t i n g t h e use of
an a l t e r n a t e g l y c o s y l a t i o n
pathway. An o l i g o s a c c h a r y l p y r o p h o s p h o r y l l i p i d f r o m p o r c i n e l i v e r i s composed o f a t e t r a s a c c h a r i d e w i t h e q u a l q u a n t i t i e s o f g-mannose and 2-acetamido-2-deoxy -B-glucose. Q - M a n n o s y l t r a n s f e r a s e I 1 has been p u r i f i e d f r o m r a b b i t l i v e r microsomes.1060
The enzyme c a t a l y s e s
direct transfer
mannose t o o l i g o s a c c h a r y l - p y r o p h o s p h o r y l f o r m a t i o n of
from
GDP-P-
l i p i d s with the resulting
ct(1+3)-~-mannosyl-~-mannose l i n k a g e s .
A number o f c e l l l i n e s o f r i c i n - r e s i s t a n t
baby- h a m s t e r k i d n e y
318
Carbohydrate Chemistry
c e l l s h a v e been a s s a y e d f o r g r o s s c a r b o h y d r a t e c o m p o s i t i o n of c e l l u l a r g l y c o p r o t e i n s , f o r g l y c o s i d a s e and g l y c o s y l t r a n s f e r a s e a c t i v i t y . l o 6 1 None of t h e changes i n g l y c o s y l - t r a n s f e r r e a c t i o n s i n t h e s e l i n e s i s due t o e n h a n c e d g l y c o s i d a s e or s u g a r n u c l e o t i d a s e a c t i v i t i e s i n t h e mutant c e l l s . L a c t o s e s y n t h e t a s e A p r o t e i n from human serum h a s been p u r i f i e d and c h a r a c t e r i z e d 1062 The r e mar kab l e i m muno l o g i c a l d i s s i m i l a r i t y between bovine serum g a l a c t o s y l t r a n s f e r a s e and t h i s enzyme may be r e l a t e d t o major d i f f e r e n c e s i n t h e c a r b o h y d r a t e m o i e t i e s of t h e two enzymes. The b i o s y n t h e s i s of e r y t h r o g l y c a n - l i k e p r o d u c t s b y i n c u b a t i o n of a microsomal f r a c t i o n d e r i v e d from c h r o n i c myelogenous leukaemiad e r i v e d c e l Is w i t h UDP -2-ace t a m i d o -2-deoxy - 9 4 6-3H} - g l u c o s e and UDPQ-{ 6 - 3 H } - g a l a c t o s e h a s been d e s c r i b e d . 1 0 6 3 The h i g h - m o l e c u l a r w e i g h t p r o d u c t s o f t h e t r a n s f e r a s e - c a t a l y s e d r e a c t i o n s were p a r t i a l l y c h a r a c t e r i z e d a f t e r enzymic d e g r a d a t i o n a s a d i - , t r i - , and unidentified oligo-saccharide. The p r e s e n c e of g l y c o s y l t r a n s f e r a s e s on c h r o m a t i n a c c e p t o r s i n monkey l i v e r n u c l e a r membranes h a s b e e n d e m 0 n ~ t r a t e d . l ' ~ ~The e v e n t u a l r o l e of these enzymes i n t h e g l y c o s y l a t i o n of n o n - h i s t o n e proteins is discussed. An a - k - m a n n o s e : B 1 , 2 - ( 2 - a c e t a m i d o - 2 - d e o x y - Q - g l u c o s y l ) t r a n s f e r a s e h a s been i s o l a t e d and p u r i f i e d f r o m p o r c i n e t r a c h e a l mucosa.1065 The enzyme f o r m s 8 1 , 2 bonds b e t w e e n 2 - a c e t a m i d o - 2 d e o x y - Q - g l u c o s e and t e r m i n a l b r a n c h e d p - m a n n o s y l r e s i d u e s o f g l y c o p r o t e i n s and g l y c o p e p t i d e s . I o d i n e i s r e a d i l y i n c o r p o r a t e d f r o m i o d i n e c h l o r i d e i n t o 2g a l a c t o s y l t r a n s f e r a s e from bovine m i l k w i t h t o t a l loss of e n z y m a t i c activity.1066 S u b s t r a t e s and a - l a c t a l b u m i n p r o t e c t a g a i n s t i n a c t i v a t i o n and t h e only amino a c i d modified i s L - t y r o s i n e . An a-Q-galactosyltransferase a c t i v i t y i n E h r l i c h a s c i t e s tumour c e l l s h a s been c h a r a c t e r i z e d . l o 6 ' T h e enzyme h a s b e e n shown t o t r a n s f e r Q - g a l a c t o s y l r e s i d u e s f r o m U D P - Q - g a l a c t o s e t o yacetyl-lactosamine i n t h e s y n t h e s i s of t h e unique t r i s a c c h a r i d e ( 9 9 ) . The e n d o g e n o u s l o c a l i z a t i o n o f U D P - g a l a c t o s e : a s i a l o m u c i n galactosyltransferase a c t i v i t y i n r a t l i v e r endoplasmic reticulum and Golgi a p p a r a t u s has been reported.1068 P a r t i a l l y p u r i f i e d U D P - g a l a c t o s y l t r a n s f e r a s e from bovine m i l k h a s b e e n u s e d t o s y n t h e s i z e m i l l i m o l a r a m o u n t s o f 4-0-B-ggalactopyranosyl-!-glucose, 2-acetamido-2-deoxy-4-~-8-Q-galactosylp- - g l u c o s y l - B - h e x a n o l a m i n e , a n d ~ - 8 - Q - g a l a c t o s y l - ( l + 4 ) - ~ - 8 - 2 -
.
319
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
acetamido-2-deoxy-~-glucosyl-(l~4)-2-acetamido-2-deoxy-~-~glucose.1069 13C-Enriched Q - g a l a c t o p y r a n o s y l m o i e t i e s o f o l i g o s a c c h a r i d e were a l s o s y n t h e s i z e d . Peptides containing a t r i - & - p r o l y l
t h e same
sequence c a r b o x y l t o an
t h r e o n i n e r e s i d u e c a n be 2 - g l y c o s y l a t e d
&-
by crude e x t r a c t s o f p o r c i n e
s u b m a x i l l a r y g l a n d s c o n t a i n i n g UDP-2-acetamido-2-deoxy-Q-galactose p o l y p e p t i d e 2-acetamido-2-deoxy-~-galactose
t r a n s f erase. l o 7 0
:
Using
i t i s revealed t h a t I-threonine
eleven synthetic peptide substrates,
c a n n o t be g l y c o s y l a t e d w i t h o u t a c a r b o x y l t r i - l - p r o l y l
sequence
and
w i t h o u t b l o c k i n g o f t h e N - t e r m i n a l group o f t h e L-threonine. One f o r m o f 2 - a - C - f u c o s y l t r a n s f e r a s e a n d o n e f o r m o f 3 - a - & f u c o s y l t r a n s f e r a s e h a v e b e e n d e t e c t e d i n h u m a n serum.1071 m i l k c o n t a i n s t h r e e f o r m s o f 3-a-L-fucosyltransferase
Human
and one f o r m
o f 4-a-&-fucosyltransferase. Three a - L - f u c o s y l t r a n s f e r a s e single
enzyme.
a c t i v i t i e s have been p u r i f i e d f r o m
The t h r e e a c t i v i t i e s a p p e a r t o b e c o n t a i n e d i n a
human milk.1072
Evidence
for
two
distinct
3-a-L-fucosyltransferase
a c t i v i t i e s i n h u m a n s a l i v a h a s b e e n r e ~ 0 r t e d . l ' ~ B~ o t h e n z y m e s appear t o c a t a l y s e t h e t r a n s f e r o f I - f u c o s e t o 2 - 3 of 2-acetamido-2deoxy-6-Q-glucosyl
residues o f
blood-group
glycoproteins,
t h e t r a n s f e r a s e dependent on t h e e x p r e s s i o n o f t h e
but only
gene h a s t h e
c a p a c i t y t o t r a n s f e r C-fucose t o 0 - 3 o f g - g l u c o s y l r e s i d u e s . E v i d e n c e f o r t h e e x i s t e n c e o f t h e p o s t u l a t e d UDP-2-acetamido-2: g l y c o p r o t e i n 2-acetamido-2-deoxy-~-glucose
deoxy-e-glucose
phosphotransferase involved
the
i n
has
been
e ~ t a b 1 i s h e d . l ' ~ ~T h i s
phosphorylation
o f
the
high
enzyme
1-
i s
e-mannose
o l i g o s a c c h a r i d e u n i t s o f l y s o s o m a l enzymes. The D - m a n n o s y l 6 - p h o s p h a t e on
lysosomal
m o i e t i e s a c t as r e c o g n i t i o n m a r k e r s The
sequential action o f
marker
i s
synthesized
UDP-2-acetamido-2-deoxy-~-glucose
enzyme 2 - a c e t a m i d o - 2 - d e o x y - ~ - g l u c o s e
Q-2-acetamido-2-deoxyglucosyl
l-phosphotransferase
phosphodiesterase.
by
the
: lysosomal
and an a-
I t is p r o p o s e d
t h a t t h e t r a n s f e r a s e c a t a l y s e s t h e i n i t i a l d e t e r m i n i n g s t e p by which newly synthesized acid hydrolases are distinguished from other newly synthesized
glycoproteins
and
thus
are
eventually
targeted to
lysosomes. Glycosylated derivatives o f substrates
for
the
lysozyme have been used as
determination
o f
the
s p e c i f i c i t y
o f
s i a l y l t r a n s f e r a ~ e s . ~C~h~a ~ r a c t e r i s t i c d i f f e r e n c e s between v a r i o u s s i a l y l t r a n s f e r a s e s were d e m o n s t r a t e d . Fetal-calf
liver,
embryonic-chicken
brain,
human p l a c e n t a , a n d
320
Carbohydrate Chemistry
s e v e r a l o t h e r t i s s u e s c o n t a i n C M P - t j - a c e t y l n e u r a m i n y l : 6-ggalactosyl-(l+ 4~-2-acetamido-2-deoxy-~-glucosyl-a-(2~3)-sialylt r a n ~ f e r a s e . " ~ ~T h i s s i a l y l t r a n s f e r a s e a c t i v i t y a p p e a r s t o be due t o an enzyme w h i c h i s d i s t i n c t from a l l o t h e r known s i a l y l transferases. E l e v a t e d s i a l y l t r a n s f e r a s e a c t i v i t i e s have been d e t e c t e d i n t h e i n t e s t i n a l l y m p h of c o l c h i c i n e - t r e a t e d r a t s . 1077 Enterectomy does not p r e v e n t t h e r i s e of serum s i a l y l t r a n s f e r a s e , s u g g e s t i n g t h a t t h e i n t e s t i n e i s not t h e m a j o r s o u r c e of t h e serum enzyme. C o l c h i c i n e has an e f f e c t o n g l y c o p r o t e i n s y n t h e s i s i n r a t l i v e r G o l g i membranes b y a f f e c t i n g g l y c o ~ y l t r a n s f e r a s e s . ~A~l t~h~o u g h b o t h s i a l y l - and g a l a c t o s y l - t r a n s f e r a s e s a r e i n h i b i t e d , t h e i r s e n s i t i v i t y t o t h e drug d i f f e r s . ( R e f e r e n c e s begin o p p o s i t e )
5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
32 1
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5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides
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m.
fatyo,
Enzymes BY J. F. KENNEDY AND D. P. ATKINS
1
Introduction
--
G e n e r a l A s p e c t s and N o m e n c l a t u r e .
V o l u m e 1 o f ‘Enzymes’ c o v e r s
t h e d e v e l o p m e n t o f enzymology f r o m i t s b e g i n n i n g s i n t h e e i g h t e e n t h century,
r e p r e s e n t e d by t h e s t u d i e s o n d i g e s t i o n by Reaumur a n d
Spallanzani,
t o t h e modern o r g a n i c s y n t h e s i s o f r i b o n u c l e a s e by
M e r r i f ie1d.l
The p a p e r s s e l e c t e d emphasize t h e c h e m i c a l n a t u r e and
mode o f a c t i o n o f e n z y m e s r a t h e r t h a n t h e f i e l d o f i n t e r m e d i a r y metabolism.
More
than
twenty
classic
papers
appear
translation for the f i r s t time,
i n c l u d i n g the work of
P e r s o z on enzyme p u r i f i c a t i o n ,
Schwann on p e p s i n ,
here
i n
Payen and
Berzelius
on
c a t a l y s i s , L i e b i g on t h e n a t u r e o f f e r m e n t a t i o n , Buchner o n c e l l free
fermentation,
F i s c h e r on s t e r e o s p e c i f i c enzyme r e a c t i o n s ,
and
S o r e n s e n on pH. The
publication
I n t e r n a t i o n a l Union o f
by
the
Nomenclature
Biochemistry
(NC-IUB)
Committee
‘Enzyme
Recommendations 1978’ has seen a f u r t h e r s u p p l e m e n t .
of
the
Nomenclature, ‘Supplement
2:
C o r r e c t i o n s and A d d i t i o n s ’ i s t h e second l i s t i n g o f amendments t o ‘Enzyme
N o m e n c l a t u r e 1978’ (Academic P r e s s ,
s u p p l e m e n t was p u b l i s h e d i n Eur.J.Biochem., M e t h o d s o f Assay.
-- ‘M e t h o d s
continues t h e coverage
of
p r e s e n t e d i n Volume 70,
New York, 1980,
1979); t h e f i r s t
104, 1.2
i n E n z y m o l o g y ’ , V o l u m e 73, P a r t B,
general P a r t A.3
immunochemical techniques The p a p e r s i l l u s t r a t e t h e
i n g e n u i t y c h a r a c t e r i s t i c o f w o r k e r s who h a v e a d a p t e d t h e a n t i g e n antibody
reaction to
develop
a variety
of
assays which
are
a p p l i c a b l e t o numerous b i o c h e m i c a l and c l i n i c a l p r o b l e m s . The P r o c e e d i n g s o f t h e 4 t h I n t e r n a t i o n a l Symposium, The N e t h e r l a n d s ,
22-26 June,
1981,
and
t h e s t a t u s o f a f f i n i t y chromatography, increasing importance of a f f i n i t y techniques
b i omedical/diagnos t i c a p p l i c a t i o n s .
Veldhoven,
g i v e a s u r v e y o f new d e v e l o p m e n t s i l l u s t r a t i n g the i n i n d u s t r i a l and
Carbohydrate Chemistry
I n Volume 74 o f ‘Methods i n Enzymology’ immunochemical t e c h n i q u e s a r e d e s ~ r i b e d . ~O n e s e c t i o n o f t h i s v o l u m e i s p a r t i c u l a r l y concerned w i t h t h e u s e o f a n t i b o d i e s t o s t u d y enzymes. The l a t e s t v o l u m e i n t h e s e r i e s ‘ A d v a n c e s i n Enzymology a n d R e l a t e d Areas o f M o l e c u l a r B i o l o g y ’ c o n t a i n s a c h a p t e r w h i c h describes t h e use of g l y c o s y l t r a n s f e r a s e s i n t h e assessment o f oligosaccharide structure.6 Kinetics. -- A v e r s a t i l e BASIC p r o g r a m h a s b e e n d e s c r i b e d f o r Direct c u r v e - f i t t i n g i s u n c o n t e s t e d a s t h e ‘Analyzing K i n e t i c method o f c h o i c e f o r e v a l u a t i n g k i n e t i c e x p e r i m e n t s . I f carried o u t by c o m p u t e r , t h e n u m e r i c a l s o l u t i o n o f s u c h p r o b l e m s i s s u p e r i o r t o customary g r a p h i c a l p r o c e d u r e s i n any r e p s e c t . However, most p r o g r a m s a v a i l a b l e t o d a t e c a n n o t b e e x e c u t e d by s m a l l s y s t e m s . T h e a u t h o r s describe a BASIC program f o r d e s k - t o p m i c r o c o m p u t e r s t h a t p e r f o r m s l i n e a r and n o n - l i n e a r r e g r e s s i o n a n a l y s i s o f k i n e t i c data. I t may be d i r e c t l y a p p l i e d t o a n u m b e r o f p r o b l e m s common i n e n z y m e k i n e t i c s , and is e a s i l y modified t o handle f u r t h e r ones. The p e r f o r m a n c e o f t h e p r o g r a m i s i l l u s t r a t e d by s o m e t y p i c a l applications. I n a d d i t i o n , fundamental s t a t i s t i c a l methods are discussed t h a t allow a critical examination of the r e s u l t s obtained. Mechanisms o f A c t i o n . -- A b o o k h a s b e e n p u b l i s h e d o n e n z y m e i n h i b i t o r s w h i c h d e r i v e s f r o m t h e p a p e r s p r e s e n t e d a t t h e 1980 s p r i n g symposium of t h e S w i s s Chemical Society.8 The program c o n s i s t e d o f 21 f u l l p a p e r s a n d 3 a b s t r a c t s f r o m i n d u s t r i a l a n d u n i v e r s i t y l a b o r a t o r i e s r e p o r t i n g on theory, s t r u c t u r e s , syntheses and v i t r o a s well as 2 v i v o e f f e c t s o f mechanism-based enzyme i n h i b i t o r s . These i n c l u d e s u i c i d e s u b s t r a t e s , t r a n s i t i o n - s t a t e a n a l o g u e s , p a r a c a t a l y t i c s e l f i n a c t i v a t o r s , a n d a number o f compounds b e l o n g i n g t o t h e class o f s u b s t r a t e and coenzyme a n a l o g s . The f u l l y indexed publication o f f e r s a wealth of valuable information t o i n v e s t i g a t o r s s e e k i n g novel s t r u c t u r e s i n t h e d e s i g n o f enzyme inhibitors.
-- V o l u m e 5 o f t h e s e r i e s e n t i t l e d ‘ E c o n o m i c M i c r o b i o l o g y ’ c o v e r s m i c r o b i a l e n z y m e s a n d b i o c o n v e r ~ i o n s . ~T h i s volume d i f f e r s from t h e p r e v i o u s volumes i n t h a t i t is concerned o n l y w i t h t h e d e v e l o p m e n t f o r i n d u s t r y o f i n d i v i d u a l e n z y m e s or s h o r t s e q u e n c e s o f enzymes. P a r t i c u l a r l y r e l e v a n t s e c t i o n s are amylases, amyloglucosidases and related glucanases, Q-glucose
Applications.
6: Enzymes
349
o x i d a s e , Q - g l u c o s e d e h y d r o g e n a s e , Q - g l u c o s e i s o m e r a s e , B-Eg a l a c t o s i d a s e and i n v e r t a s e , p e c t i c enzymes, c e l l u l a s e s , immobilized enzymes. A book on c e l l u l a s e and o t h e r n a t u r a l polymer s y s t e m s c o n t a i n s , i n a d d i t i o n t o c h a p t e r s on t h e b i o s y n t h e s i s and s t r u c t u r e o f c e l l u l o s e , c h a p t e r s which d e a l w i t h B-q-glucanases i n h i g h e r p l a n t s and c e l l u l o s e d e g r a d a t i o n . lo The p r o c e e d i n g s of t h e 2 n d Symposium o n Enzyme T h e r a p y i n G e n e t i c D i s e a s e s c o v e r : human t r i a l s : c e l l and o r g a n t r a n s p l a n t a t i o n / o t h e r t h e r a p e u t i c a p p r o a c h e s ; human t r i a l s : d i r e c t enzyme r e p l a c e m e n t ; enzyme m a n i p u l a t i o n : mechanisms and t h e r a p e u t i c t r i a l s ; a n i m a l model s t u d i e s : enzyme e n t r a p m e n t , n e u r a l d e l i v e r y and t r a n s p l a n t a t i o n , enzyme r e c o g n i t i o n and m o d i f i c a t i o n ; enzyme a v a i l a b i l i t y : p u r i f i c a t i o n and c h a r a c t e r i z a t i o n . "
-- A book on t h e u s e s o f i m m o b i l i z e d enzymes f o r food p r o c e s s i n g c o v e r s t h e f o l l o w i n g a r e a s : i m m o b i l i z e d enzyme e n g i n e e r i n g ; r e q u i r e m e n t s u n i q u e t o t h e f o o d and b e v e r a g e i n d u s t r y ; manufacture of h i g h - f r u c t o s e corn s y r u p u s i n g i m m o b i l i z e d g l u c o s e i s o m e r a s e ; p o t e n t i a l and use of i m m o b i l i z e d c a r b o h y d r a s e s ; a p p l i c a t i o n o f l a c t o s e and i m m o b i l i z e d l a c t a s e ; i m m o b i l i z e d p r o t e a s e s - p o t e n t i a l a p p l i c a t i o n , a p p l i c a t i o n and p o t e n t i a l o f aminoacylase, a s p a r t a s e , o t h e r enzymes i n f o o d p r o c e s s i n g : fumarase, glucose o x i d a s e - c a t a l a s e , s u l p h y d r y l o x i d a s e , and c o n t r o l l e d - r e l e a s e enzymes. l 2 I n t h e p r o c e e d i n g s of t h e 1 8 t h Prague IUPAC Micro Symposium on Macromolecules, p u b l i s h e d i n a s e r i e s of j o u r n a l i s s u e s , t h e r e i s a s e c t i o n which d e s c r i b e s t h e i m m o b i l i z a t i o n of enzymes t o d i f f e r e n t p o l y s a c c h a r i d e s by g r a f t cop0 l y mer i za t i o n . l 3 The use of r e v e r s i b l e enzyme i m m o b i l i z a t i o n h a s been r e p o r t e d t o f a c i l i t a t e t h e r e c o v e r y o f p e p t i d e a n t i b i 0 t i ~ s . l ~P r a c t i c a l a s p e c t s o f t h e f a c i l e i m m o b i l i z a t i o n of e n z y m e s on h y d r o u s m e t a l o x i d e s , a w e l l e s t a b l i s h e d means of enzyme-movement r e s t r i c t i o n , a r e described. Various enzymes (e.g. g l u c o a m y l a s e , p e r o x i d a s e , d e x t r a n a s e ) h a v e been i m m o b i l i z e d b y c h e l a t i o n o f s e v e r a l h y d r o u s m e t a l o x i d e s , t h o s e of t i t a n i u m ( 1 V ) and zirconium(1V) p r o v i n g t o be t h e most s a t i s f a c t o r y f o r p r a c t i c a l purposes. M o d i f i c a t i o n of t h e g e l i n t o a g r a n u l a r f o r m c o u l d be a c h i e v e d s u c c e s s f u l l y w i t h good enzyme-immobilization c h a r a c t e r i s t i c s by u s i n g ion-exchange r e s i n a s an i n t e r n a l m a t r i x . The i m m o b i l i z a t i o n p r o c e s s was h i g h l y e f f i c i e n t f o r t h e r e l a t i v e p r o p o r t i o n s of hydrous o x i d e t o enzyme used, w i t h
Enzyme I m m o b i l i z a t i o n .
350
Carbohydrate Chemistry
u s u a l l y > 90% of t h e a v a i l a b l e p r o t e i n b e i n g i n s o l u b i l i z e d . R e t e n t i o n o f e n z y m e a c t i v i t y was g e n e r a l l y v e r y g o o d a n d t h e e n z y m e was s t a b l e t o r e u s e a n d t o c o n v e n t i o n a l b u f f e r c o n d i t i o n s . A c t i v i t i e s o f t h e i m m o b i l i z e d e n z y m e s were p a r t i a l l y s t a b l e t o l y o p h i l i z a t i o n or d r y i n g of the hydrous o x i d e g e l s . M o d i f i c a t i o n o f t h e h y d r o u s m e t a l o x i d e s u r f a c e by d r y i n g o r t r e a t m e n t w i t h p h o s p h a t e o r c a r b o n a t e l e d t o a decrease i n c o m p l e x i n g a b i l i t y . The e f f e c t o f c a r b o n a t e c a n b e c i r c u m v e n t e d b y l o w e r i n g t h e pH o f t h e s o l u t i o n t o a r o u n d 5 a n d r e m o v i n g a n y c a r b o n d i o x i d e f o r m e d , by a e r a t i o n . Such t r e a t m e n t a l l o w e d compounds t o c h e l a t e t o hydrous zirconium oxide(1V) i n t h e presence of carbonate, and t h e r e f o r e t h e h y d r o u s o x i d e c o u l d be a p p l i e d s u c c e s s f u l l y t o t h e c o n c e n t r a t i o n o f p e p t i d e a n t i b i o t i c s f r o m t h e f e r m e n t a t i o n medium i n w h i c h t h e y are b e i n g p r o d u c e d , i n c l u d i n g p r o d u c t i o n a t low c o n c e n t r a t i o n s . A s i m p l e a n d n o v e l m e t h o d f o r t h e i m m o b i l i z a t i o n o f e n z y m e s by their c o i m m o b i l i z a t i o n i n hen egg-white u s i n g g l u t a r a l d e h y d e h a s been described.15 Films of highly polymerized collagen prepared under i n d u s t r i a l c o n d i t i o n s by t h e C e n t r e T e c h n i q u e d u C u i r , L y o n , F r a n c e , h a v e b e e n r o u t i n e l y used after a c y l azide a c t i v a t i o n f o r the c o v a l e n t i m m o b i l i z a t i o n o f n u m e r o u s e n z y m e s f r o m d i f f e r e n t classes.16 T h e s t a b i l i t y of t h e r e s u l t i n g membranes t o o p e r a t i o n a l and s t o r a g e conditions and their e x c e l l e n t mechahical s t r e n g t h and r e s i s t a n c e t o bacterial degradation allowed their use for several purposes. Fundamental a s p e c t s of t h e i r heterogeneous enzymology, including The d i f f u s i o n a l e f f e c t s a n d s u b u n i t i n t e r a t i o n s , were e x a m i n e d . e n z y m e - e n g i n e e r i n g a s p e c t was d e v e l o p e d w i t h p o l y m e m b r a n e b i o r e a c t o r s and enzyme e l e c t r o d e s . Besides t h e protein environment p r o v i d e d by t h e c o l l a g e n m a t r i x w h i c h p r e v e n t s e n z y m e i n a c t i v a t i o n , t h e f o r m o f these m e m b r a n e s i s a d v a n t a g e o u s f o r i n d u s t r i a l p u r p o s e s and f o r fundamental research.
2
B-Q-2-Acetamido-2-deoxygalactosidases, 2-deoxyglucosidases, and
B-g-2-Acetamido-
B-Q-2-Acetamido-P-
deoxyhexosidases
The bulk of rat b r a i n n e u t r a l B-Q-2-acetamido-2d e o x y h e x o s i d a s e s h a v e b e e n shown t o be p r e s e n t i n t h e c y t o s o l f r a ~ t i 0 n . l ~ T h e y were n o t b o u n d by c o n c a n a v a l i n A - S e p h a r o s e w h i l e t h e a c i d B-Q-2-acetamido-2-deoxyhexosidases were a l l b o u n d . The
351
6: Enzymes
h a d a pH o p t i m u m o f 5.2
n e u t r a l B-~-2-acetamido-2-deoxyglucosidase andKm
of
0.57
while
mM,
the
neutral
B-Q-2-acetamido-2-
d e o x y g a l a c t o s i d a s e h a d t h e h i g h e s t r e a c t i o n r a t e a t pH 6.0 o f 0.12
No d i v a l e n t
mM.
lost
B-Q-2-acetamido-2-deoxyglucosidase a c t i v i t y
i n
min
30
deoxygalactosidase activity
after
a t
at
The
5OoC.
more
The
5OoC.
was h e a t - s t a b l e h
3
with a
i o n s a c t i v a t e d e i t h e r o f t h e enzymes. than
90% o f
5, The the
€4-D-2-acetamido-2-
and l o s t o n l y 10-20% o f neutral
i t s
B-g-2-acetamido-2-
d e o x y g l u c o s i d a s e was i n h i b i t e d b y f r e e 2 - a c e t a m i d o - 2 - d e o x y - B - Q glucose
but
by 2-acetamido-2-deoxy-B-~-galactose.
not
The r e v e r s e
was f o u n d f o r t h e n e u t r a l ~ - ~ - 2 - a c e t a m i d o - 2 - d e o x y g a l a c t o s i d a s e . Two enzymes were s e p a r a t e d a l m o s t chromatography.
completely
by h y d r o x y a p a t i t e
Heat s t a b i l i t y o f t h e separated a c t i v i t y peaks
s u g g e s t e d t h a t t h e n e u t r a l B-g-2-acetamido-2-deoxygalactosidase, w h i c h was n o t b o u n d t o h y d r o x y a p a t i t e , may,
may b e s p e c i f i c t o t h e Q -
The n e u t r a l B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e
galacto substrate.
o n t h e o t h e r h a n d , h a v e some a c t i v i t y t o w a r d t h e Q - g a l a c t o
substrate.
B o t h o f t h e n e u t r a l enzyme a c t i v i t i e s w e r e shown t o be
h i g h e s t d u r i n g t h e f i r s t p o s t n a t a l week i n r a t b r a i n , t h e a c i d i c enzyme,
i n contrast t o
w h i c h showed peak a c t i v i t i e s d u r i n g t h e second
a n d t h i r d weeks. Activity
levels
i n v e s t i g a t e d
i n
Dugesia lugubris.18
and
cytochemical
i n t a c t
localization
regenerating
have
been
p l a n a r i a n
I n 24 h o u r r e g e n e r a t i n g p l a n a r i a n s t h e 6-B-2-
acetamido-2-deoxyglucosidase lysosomes o f
and was
dedifferentiating
found t o
cells.
be
localized i n the
T h i s B-E-2-acetamido-2-
d e o x y g l u c o s i d a s e l o c a l i z a t i o n c o r r e l a t e d w i t h t h e i n c r e a s e i n B-9-2-
acetamido-2-deoxyglucosidase
and
B-D-2-acetamido-2-
deoxygalactosidase a c t i v i t i e s during the regeneration,
f i r s t
24 h o u r s o f
confirming the involvement o f the d e d i f f e r e n t i a t i o n i n
t h e o r i g i n o f blastema.
I n non-regenerating
acetamido-2-deoxyglucosidase
activity
demonstrated i n t h e lysosomes o f
has
a n i m a l s t h e B-Q-2-
been
cytochemically
g a s t r o d e r m a l c e l l s and i n t h e
mucous g r a n u l e s o f b a s o p h i l i c s e c r e t o r y c e l l s .
This l a t t e r finding
is i n t e r p r e t e d a s p r o o f o f a s i m i l a r i t y b e t w e e n p l a n a r i a n a n d v e r t e b r a t e mucous s e c r e t i o n . High lysozyme
and c h i t i n a s e a c t i v i t i e s have been f o u n d i n
L e y d i g ’ s o r g a n o f E t m o p t e r u s s p i n a x , Z m n i o s u s m i c r o c e p h a l u s , and Tonpedo n o b i l i a n a ,
i n Leydig’s
and e p i g o n a l o r g a n s and s p l e e n o f
R a j r a d i a t a , and i n t h e e p i g o n a l o r g a n o f R h i n o p t e r a bonasus.” S t r o n g c h i t i n a s e a c t i v i t y w i t h l i t t l e o r n o l y s o z y m e a c t i v i t y was
Carbohydrate Chemistry
352 n o t e d i n Leydig's i n
the
and e p i g o n a l o r g a n s o f S c y l i o r h i n u s c a n i c u l a ,
epigonal
organ
GiX.slymostom c i r r a t u m
of
----__-____---------__ Heterodontus francisci.
Very
high
and and
B-Q-2-acetamido-2-
d e o x y g l u c o s i d a s e a c t i v i t y was f o u n d i n l y m p h o m y e l o i d o r g a n s o f a l l s p e c i e s i n v e s t i g a t e d and i n t h e pancreas o f Z m n i o s u s m i c r o c e p h a l u s . Thirteen other and
glycoside hydrolases
d i g e s t i v e
t i s s u e s
were
o f
assayed i n
lymphomyeloid
cLf;at21~,
G i n g L y ~ ~ o s t o m a
H e t e-r o d o~ n t u s -f r a ~ n c i s c-i , a n-d E t m o p t e r u s s p i n a x . A c t i v i t i e s o f a-Qmannosidase, B - g a l a c t o s i d a s e , B-~-2-acetamido-2-deoxygalactosidase, B-!-glucuronidase,
and a- a n d B - n - g l u c o s i d a s e
u s u a l l y were h i g h e r i n
lymphomyeloid t i s s u e s than i n d i g e s t i v e tissues. Nine
glycoside
hydrolases
Trypanosoma b r u c e i b r u c e i S42 h a v e a-Q - -Glucosidase to
those o f
i n
bloodstream
been p a r t i a l l y
forms
o f
characterized.20
had s i m i l a r p h y s i c o c h e m i c a l and e n z y m i c p r o p e r t i e s
a-Q-galactosidase,
- -2-acetamido-2B-Q
f3-g-glucosidase,
d e o x y g l u c o s i d a s e , and B-;-2-acetamido-2-deoxygalactosidase.
I t was
s u g g e s t e d t h a t t h e g l y c o s i d a s e s s t u d i e d may p l a y a r o l e i n t h e turnover o f trypanosomal glycoproteins,
i n particular the variant-
s p e c i f i c surface antigen. a-;-Galactosidase
deficiency
h a s been d e m o n s t r a t e d i n l y m p h o i d
c e l l l i n e s e s t a b l i s h e d by E p s t e i n - B a r r v i r u s t r a n s f o r m a t i o n o f Blymphocytes from a Fabry p a t i e n t , lymphocytes.21
as i n b l o o d w h o l e l e u k o c y t e s a n d
The r e s i d u a l a c t i v i t y
was h e a t s t a b l e .
a-p-2-
A c e t a m i d o - 2 - d e o x y g a l a c t o s i d a s e was p r e s e n t a t n o r m a l l e v e l i n leukocytes, lymphocytes, o r lymphoid c e l l s from n o r m a l and Fabry p a t i e n t s a n d was h e a t s t a b l e i n a l l c a s e s .
a-e-2-Acetamido-2-
d e o x y g a l a c t o s i d a s e e l e c t r o f o c u s i n g p r o f i l e s p r e s e n t e d one m a j o r p e a k (PI 4.5)
and
one
minor
peak
Blood
(PI 5.1).
lymphocytes
and
l y m p h o i d c e l l l i n e s f r o m F a b r y d i s e a s e showed s i m i l a r d e f e c t s o f a l l the forms o f a-g-galactosidase
g r o u p A and t h e r e s i d u a l a c t i v i t y
proceeded f r o m t h e a-!-galactosidase
acetamido-2-deoxygalactosidase.
f o r m 11, w h i c h i s an a-e-2-
I n the
absence
of
animal
models,
t h o s e e s t a b l i s h e d l i n e s seemed t o be an a c c u r a t e c e l l u l a r s y s t e m f o r
-i n vitro
e x p e r i m e n t a l s t u d i e s o f Fabry disease.
The p h y s i o l o g i c a l a c t i v a t o r p r o t e i n f o r t h e d e g r a d a t i o n o f g a n g l i o s i d e GM2 by B-Q-2-acetamido-2-deoxygalactosidase employed t o assess t h e c a p a b i l i t y
of
extracts t o catabolize t h i s ganglioside.22 more
reliable
diagnosis
of
the
T h i s method p e r m i t s a
different
g a n g l i o s i d e s t h a n t h e m e t h o d s h i t h e r t o used. a r t i f i c i a l substrates or,
has been
c u l t u r e d human f i b r o b l a s t variants
of
GM2
These e i t h e r r e l y on
when n a t u r a l s u b s t r a t e s a r e used,
on
6: Enzymes
353
d e t e r g e n t s . The p r e s e n t method avoids a number of p o s s i b l e s o u r c e s o f e r r o r i n t r o d u c e d by t h e u n p h y s i o l o g i c a l d e t e r g e n t s , such a s a l t e r a t i o n o f t h e isoenzymes’ s u b s t r a t e s p e c i f i c i t y o r i n a c t i v a t i o n o f t h e enzymes. The r a n g e o f a p p l i c a t i o n of t h e new method i s discussed. The p a t t e r n s o f B - g - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e excretion a f t e r r e n a l t r a n s p l a n t a t i o n have been i n ~ e s t i g a t e d . ~8-Q-2~ A c e t a m i d o - 2 - d e o x y g l u c o s i d a s e a c t i v i t y was a s s a y e d i n 750 e a r l y morning u r i n e samples from 25 r e n a l - t r a n s p l a n t p a t i e n t s d u r i n g t h e p o s t - o p e r a t i v e p e r i o d . Eighty-four per c e n t of a l l a c u t e r e j e c t i o n e p i s o d e s were p r e c e d e d o r a c c o m p a n i e d b y a g r e a t e r t h a n t w o - f o l d Similar r i s e i n B-~-2-acetamido-2-deoxyglucosidase a c t i v i t y . i n c r e a s e s were caused by d i a l y s i s , gentamicin therapy, and u r e t e r i c dehiscence. O n l y 9% of a l l s i g n i f i c a n t i n c r e a s e s i n B-g-acetamido2 - d e o x y g l u c o s i d a s e e x c r e t i o n c o u l d n o t be a c c o u n t e d f o r b y any o f t h e s e four p r o c e s s e s . Analysis of t h e day-to-day p a t t e r n o f B-g-2a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e a c t i v i t y a s opposed t o i n d i v i d u a l B-Q2-acetamido-2-deoxyglucosidase values provided a c l u e t o t h e o c c u r r e n c e of r e j e c t i o n d u r i n g i m m e d i a t e p o s t - t r a n s p l a n t a t i o n oligur ia. Urinary e x c r e t i o n of B-~-2-acetamido-2-deoxyglucosidase and La l a n i n e a m i n o p e p t i d a s e h a s been measured i n p a t i e n t s r e c e i v i n g a m i k a c i n o r c L ~ - p l a t i n u m . ~U~r i n a r y e x c r e t i o n of a l a n i n e aminopeptidase and B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e was determined f o r 25-70 days i n f i v e p a t i e n t s r e c e i v i n g *-platinum and f o r 8-53 days i n s i x p a t i e n t s r e c e i v i n g amikacin. T h i s s t u d y was performed t o i n v e s t i g a t e i f t h e e x c r e t i o n o f u r i n a r y enzymes r e p r e s e n t s a s e n s i t i v e parameter f o r t h e e a r l y d e t e c t i o n o f t o x i c kidney damage. I n b o t h p a t i e n t g r o u p s an i n c r e a s e i n t h e e x c r e t i o n of t h e t w o enzyme a c t i v i t i e s c o u l d be d e m o n s t r a t e d . I n patients receiving a m i k a c i n , t h e e x c r e t i o n of a l a n i n e aminopeptidase was always h i g h e r t h a n t h a t o f B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e , whereas i n t h r e e p a t i e n t s r e c e i v i n g c i s - p l a t i n u m i t was t h e o p p o s i t e . I n t w o cisp l a t i n u m p a t i e n t s t h e e x c r e t i o n o f b o t h enzymes was o f t h e same s i z e . The c h a n g e s d u r i n g a m i k a c i n t h e r a p y seem t o be r e v e r s i b l e , p a t i e n t s t h e s e c h a n g e s seemed t o be whereas i n f o u r =-platinum partly irreversible. Serum c r e a t i n i n e c o n c e n t r a t i o n was l e s s s e n s i t i v e than t h e u r i n a r y enzyme e x c r e t i o n f o r d e t e c t i o n of kidney damage. P r e d i c t i v e v a l u e s of u r i n a r y B-Q - -2-acetamido-2deoxyglucosidase, i - a l a n i n e aminopeptidase,and B-2-microglobulin
3 54
Carbohydrate Chemistry
have been used i n e v a l u a t i n g t h e n e p h r o t o x i c i t y o f g e n t a m i c i n . 2 5 I-slanine
C o n c e n t r a t i o n s o f B-e-2-acetamido-2-deoxyglucosidase, a m i n o p e p t i d a s e , and 1 3 - 2 - m i c r o g l o b u l i n
were
determined
daily
i n the
u r i n e o f 28 p a t i e n t s t r e a t e d w i t h g e n t a m i c i n ( 2 - 3 mg k g - l day-') a mean o f activity
15 days.
A l l
had n o r m a l r e n a l f u n c t i o n .
i n B-Q-2-acetamido-2-deoxyglucosidase
2 o r 3 days o f t r e a t m e n t .
serum
creatinine
levels.
This
The r e s u l t s w e r e c o m p a r e d w i t h
concentrations
study
and I - a l a n i n e
e i t h e r immediately or
a m i n o p e p t i d a s e was o b s e r v e d f o r a l l p a t i e n t s , after
for
Increased
indicates
and a
urinary
B-2-microglobulin
relationship
between
the
n e p h r o t o x i c i t y o f g e n t a m i c i n and i n i t i a l u r i n a r y e n z y m i c a c t i v i t y o f
B-Q-2-acetamido-2-deoxyglucosidase p r i o r t o any t r e a t m e n t . The degree o f B-Q-2-acetamido-2-deoxyglucosidase response d u r i n g the first
t e n d a y s o f t r e a t m e n t a p p e a r e d as a s e c o n d p r o g n o s t i c f a c t o r .
Renal f a i l u r e
was o b s e r v e d f o r o n e o u t
o f the 12 p a t i e n t s w i t h
n o r m a 1 B - Q - 2 - a c e t a m i d o - 2 - d e o x y g 1u c o s id a s e deoxyglucosidasei
< 200 p m o l day").
(B-D,
- 2 - a c e t am i do - 2 -
Seven o f t h e m showed a m a r k e d
enzyme a c t i v i t y r e s p o n s e ( > 1 5 0 0 p m o l day-')
w i t h an i n c r e a s e i n B-
2-microglobulin
the
activity.
Eleven out
of
16 p a t i e n t s
with
elevated
6-Q-2-acetamido-2-de~xyglucosidase~ (6-Q-acetamido-2d e v e l o p e d r e n a l f a i l u r e and d e o x y g l u c o s i d a s e i > 2 0 0 p m o l day") showed an e l e v a t e d m a x i m a l r e s p o n s e .
aminopeptidase
appears
to
be o f
The c o n c e n t r a t i o n o f C - a l a n i n e l i t t l e prognostic value.
v a r i a t i o n i n i n d i v i d u a l maximal urinary
enzyme
The
responses observed
among t h e 28 p a t i e n t s d u r i n g t h e f i r s t t e n days o f t r e a t m e n t p o i n t s t o the existence o f individual s e n s i t i v i t i e s t o gentamicin,
the
e x a c t mechanism o f w h i c h r e m a i n s u n c l e a r . T h e c o n d i t i o n s f o r m a x i m a l a c t i v i t y (pH, substrate concentration, enzyme
activity
concentration)
i n
versus the
range o f
buffer,
saturating
l i n e a r r e l a t i o n s h i p s between
incubation
time
f l u o r i m e t r i c assay
and versus of
several
enzyme
glycoside
h y d r o l a s e s o f l y s o s o m a l o r i g i n i n human p l a s m a and serum h a v e been e ~ t a b l i s h e d . ~T~h e
following
enzymes
were
g a l a c t o s i d a s e , B-Q-2-acetamido-2-deoxyglucosidase, B-Q-glucuronidase,
a-Q-mannosidase,
studied:
a-Q-
8-~-glucosidase,
a-C-fucosidase.
A l l examined
e n z y m e s t u r n e d o u t t o b e m o r e o r l e s s u n s t a b l e u p o n s t o r a g e a t 37'C, 4OC,
a n d -2OOC i n b o t h s e r u m a n d p l a s m a .
Generally t h e degree o f
i n s t a b i l i t y was g r e a t e r i n s e r u m t h a n i n p l a s m a .
The l e v e l s o f some
enzymes, i n c l u d i n g B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e , h i g h e r i n serum t h a n i n plasma. enzymes i n p l a t e l e t - f r e e
were markedly
C o n v e r s e l y t h e l e v e l s o f t h e same
serum e q u a l l e d those i n plasma.
This
6: Enzymes
355
s t r e s s e s t h e n e c e s s i t y t o use f r e s h l y p r e p a r e d p l a s m a f o r l y s o s o m a l g l y c o h y d r o l a s e assay. for
the
assay
the
Under t h e p r o c e d u r a l c o n d i t i o n s recommended methods
for
the
determination of
g l y c o h y d r o l a s e s i n p l a s m a a p p e a r e d t o be s i m p l e ,
lysosomal
s e n s i t i v e , and
reproducible. A
spectrophotometric
assay
for
u r i n a r y B-Q-2-acetamido-2-
d e o x y g l u c o s i d a s e a c t i v i t y h a s been d e v e l o p e d . 2 7
I t i n v o l v e s (a) g e l
f i l t r a t i o n t o s e p a r a t e t h e enzyme f r o m i n h i b i t o r s i n u r i n e , enzy mi c
g l u c o p y r a n o s i d e a t pH 4.4,and 4-nitrophenate.
(c) spectrophotometry o f the l i b e r a t e d
M e a s u r e m e n t s o f a c t i v i t y o f t h e enzyme i n 58 u r i n e
specimens c o r r e l a t e d c l o s e l y e s t a b l i s h e d procedure. was 3.7%.
(b)
4 - n i t r o p h e n y l 2-acetamido-2-deoxy-B-Q-
hydrolysis of
(2
= 0.998)
The w i t h i n - r u n
The b e t w e e n - r u n
with results
by
coefficient of variation
a v e r a g e d 6.8%.
an
( 2 3
Reference values f o r
t h e a c t i v i t y w e r e e s t a b l i s h e d by a s s a y s o r u r i n e s p e c i m e n s f r o m 135 h e a l t h y persons, for
aged t w o weeks t o 5 2 y e a r s .
d e t e c t i o n o f n e p h r o t o x i c i t y was
experimental induction o f
E f f i c a c y o f t h e assay
demonstrated i n r a t s a f t e r
reversible
r e n a l i n s u f f i c i e n c y by
intraperitoneal i n j e c t i o n o f nickel chloride.
Clinical application
o f t h e a s s a y i n a p p r o x i m a t e l y 1000 p a t i e n t s c o r r o b o r a t e d i t s u t i l i t y
f o r d e t e c t i o n and m o n i t o r i n g o f r e n a l d i s o r d e r s . The d i g e s t i o n o f
I-asparagine-linked
B-Q-2-acetamido-2-deoxyglucosidase obtained
from
substrate
fucosidosis
s p e c i f i c i t y
patients
o f
o l i g o s a c c h a r i d e s by e n d o -
i n t h e human s k i n f i b r o b l a s t s has
been
reported.28
The
human m d g - B - & - Z - a c e t a m i d o - 2 -
d e o x y g l u c o s i d a s e was s t u d i e d b y u s i n g t h e h o m o g e n a t e o f c u l t u r e d s k i n f i b r o b l a s t s o f f u c o s i d o s i s p a t i e n t s as an enzyme s o u r c e . results indicate that
biantennary-complex-type
s u g a r c h a i n s as w e l l as h i g h - P - m a n n o s e - t y p e by t h e enzyme a c t i o n .
The
I-asparagine-linked
sugar chains a r e cleaved
None o f t h e s u g a r c h a i n s w i t h a f u c o s y l
r e s i d u e on t h e p r o x i m a l 2 - a c e t a m i d o - 2 - d e o x y - ~ - g l u c o s e d i a c e t y l c h i t o b i o s e m o i e t i e s was c l e a v e d .
o f t h e i r N,"-
These r e s u l t s p r o v e d
e n z y m i c a l l y t h e mechanism o f p r o d u c t i o n o f o l i g o s a c c h a r i d e s d e t e c t e d i n t h e u r i n e o f various %-glycosidase
Degradation o f
deficiencies.
mucin oligosaccharides
of
human c o l o n s y s t e m s
has been f o u n d t o be a s s o c i a t e d w i t h e x t r a c e l l u l a r , cell-bound, and
neuraminidase.29
Among
but not with
B-Q-2-acetamido-2-deoxyglucosidase,
B-P-galactosidase,
the
seven
subjects
studied,
the
e s t i m a t e d m o s t p r o b a b l e n u m b e r s (MPN) o f f a e c a l b a c t e r i a p r o d u c i n g e x t r a c e l l u l a r B-Q-galactosidase,
B-~-2-acetamido-2-deoxyglucosidase,
and n e u r a m i n i d a s e r a n g e d f r o m
106-1010g-1
dry
faecal
w t , were
Carbohydrate Chemktry
356 comparable
to
significantly
the
MPN
of
mucin-degrading
bacteria,
and
s m a l l e r t h a n t h e MPN o f t o t a l f a e c a l b a c t e r i a .
were These
f i n d i n g s were i n t e r p r e t e d as e v i d e n c e f o r t h e e x i s t e n c e o f b a c t e r i a l sub-populations
i n
the
normal
faecal
flora
that
produce
e x t r a c e l l u l a r g l y c o s i d a s e s , and t h a t t h e s e s u b - p o p u l a t i o n s have a m a j o r r o l e i n d e g r a d i n g t h e complex o l i g o s a c c h a r i d e s o f m u c i n i n t h e g u t lumen. Adjuvant-induced
a r t h r i t i s i n r a t s has been s t u d i e d by t h e
changes i n serum and u r i n a r y p r o t e i n - b o u n d
carbohydrate metabolites,
changes i n serum and t i s s u e l y s o s o m a l g l y c o h y d r o l a s e s and l y s o s o m a l fragility.30
The
investigated, & v
free
activities of
B-Q-glucuronidase,
-
lysosomal glycohydrolases
B-Q-acetamido-2-deoxygluco-
s ida s e , B -Q - a c e t a m ido 2 - d e o x y g 1u c o s id a s e , B - Q - g a l a c t 0 s idase , a-9mannosidase,and
c a t h e p s i n D , a r e i n c r e a s e d i n l i v e r and s p l e e n i n
t h e a c u t e phase.
The f r e e a c t i v i t i e s o f B - Q - g l u c u r o n i d a s e ,
acetamido-2-deoxyglucosidase, change,
whereas
increased. of
and c a t h e p s i n
those o f B-a-galactosidase
D of
kidney
B-a-
showed
and a-p-mannosidase
no are
I n t h e c h r o n i c phase o f t h e d i s e a s e t h e f r e e a c t i v i t i e s
a l l glycohydrolases
are s i g n i f i c a n t l y
Serum g l y c o h y d r o l a s e s a r e s i g n i f i c a n t l y c h r o n i c phases.
increased i n a l l tissues.
i n c r e a s e d i n b o t h a c u t e and
S t u d i e s i n l y s o s o m a l p r e p a r a t i o n s showed i n c r e a s e d
f r a g i l i t y of l y s o s o m e s d e r i v e d f r o m l i v e r and k i d n e y o f a r t h r i t i c r a t s i n b o t h phases o f t h e d i s e a s e . The s u b u n i t a n d p o l y p e p t i d e s t r u c t u r e o f B - Q - 2 - a c e t a m i d o - 2 d e o x y g l u c o s i d a s e s f r o m human p l a c e n t a has been r e p o r t e d . 3 1
Previous
r e p o r t s have i n d i c a t e d t h a t t h e B-~-2-acetamido-2-deoxyglucosidases a r e t e t r a m e r i c enzymes composed o f e i t h e r t w o d i m e r s o f @ - c h a i n s (B-
Q - 2 - a c e t a m i d o - 2 - d e o x ~ y g l u c o s i d a s e B) o r a B - c h a i n d i m e r and an a c h a i n d i m e r ( B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e A). 6-a-2Acetamido-2-deoxyglucosidase S c o n t a i n s o n l y a-chains. The a p p a r e n t m o l e c u l a r w e i g h t o f b o t h a- and B - c h a i n s i s 25,000 d a l t o n . 8-e-2Acetamido-2-deoxyglucosidase A and 6 were i s o l a t e d and p u r i f i e d more A prepared t h a n 6 0 0 0 - f o l d f r o m B-Q-2-acetamido-2-deoxyglucosidase and a more a n o d i c a l l y S,
m i g r a t i n g B-~-2-acetamido-2-deoxyglucosidase
which contained o n l y a-chains.
After either extensive reduction
and a l k y l a t i o n o r p e r f o r m i c a c i d o x i d a t i o n ,
B-Q-2-acetamido-2-
d e o x y g l u c o s i d a s e B gave o n l y a s i n g l e p r o t e i n band i n p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i n sodium dodecyl sulphate. deoxyglucosidase
A
consistently
gave
two
B-a-2-Acetamido-2-
bands,
c o n t a i n e d chains o f two d i f f e r e n t m o l e c u l a r weights.
indicating
it
Other r e p o r t s
have n o t e d t w o bands i n B-Q-2-acetamido-2-deoxyglucosidase
A but
357
6: Enzymes have d i s m i s s e d t h e h i g h - m o l e c u l a r - w e i g h t either
band as a d i m e r c a u s e d by
h y d r o p h o b i c i n t e r a c t i o n o r an u n b r o k e n d i s u l p h i d e bond.
r u l e out
hydrophobic interaction,
the
molecular
weight
d e t e r m i n e d by g e l f i l t r a t i o n i n 6 M g u a n i d i n i u m c h l o r i d e . complete disulphide-bond
breakage,
To was
To e n s u r e
a h a r s h e r r e d u c t i o n and
a l k y l a t i o n p r o c e d u r e t h a n p r e v i o u s l y e m p l o y e d b y o t h e r s was u s e d . The r e s u l t s w e r e t h e n c o n f i r m e d b y t h e u s e o f t w o p e r f o r m i c a c i d o x i d a t i o n techniques, t h e s t r o n g e r method r e s u l t i n g i n e x t e n s i v e Samples o f B-Q-2-acetamido-2-deoxygluco-
peptide-bond oxidation. sidase A
under
either
r e d u c t i o n and
a l k y l a t i o n or
the
weaker
p e r f o r m i c a c i d o x i d a t i o n p r o c e d u r e a g a i n c h r o m a t o g r a p h e d as t w o approximately
e q u a l peaks
(50,000
B-D-2-Acetamido-2-
and 25,000).
d e o x y g l u c o s i d a s e B and t h e B - Q - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e prepared from 25,000
$-~-2-acetamido-2-deoxyglucosidase
d a l t o n peak.
S chromato-
The a u t h o r s c o n c l u d e t h a t e i t h e r
i n B-~-2-acetamido-2-deoxyglucosidase w i t h one a - c h a i n
w e i g h t o f 50,000
B
r e s u l t e d i n one
B-~-2-Acetamido-2-deoxyglucosidase
g r a p h e d a s a s i n g l e 50,000 p e a k . the a-chain
A
per a-subunit
d a l t o n c h a i n s a r e u n i t e d by a n o n - d i s u l p h i d e
has a m o l e c u l a r
o r t h a t t w o 25,000 c r o s s l i n k (-
an
i s o p e p t i d e bond). The d i s a c c h a r id e 2 - a c e t a m i d o -2-deox y - B - Q - g l u c o p y r a n o s y l - ( 1 + 3 ) -
a-{ l - 3 H ) - g a l a c t i t o l ,
prepared from keratan sulphate,
h a s been f o u n d
t o be r a p i d l y h y d r o l y s e d by t h e A a n d B i s o e n z y m e s o f n o r m a l human l i v e r B-~-2-acetamido-2-deoxyglucosidase and by t h e B i s o e n z y m e p r e p a r e d f r o m t h e l i v e r o f a Tay-Sachs
disease patient.32
The
d i s a c c h a r i d e s u b s t r a t e was a l s o h y d r o l y s e d b y e x t r a c t s o f n o r m a l , cultured-skin fibroblasts, Sachs d i s e a s e ,
and f i b r o b l a s t s o f p a t i e n t s w i t h Tay-
w h e r e a s i t was n o t h y d r o l y s e d by f i b r o b l a s t e x t r a c t s
of p a t i e n t s w i t h Sandhoff disease.
Thus,
defective degradation o f
k e r a t a n sulphate, secondary t o a d e f e c t o f t h e subunits present i n the
A
and B i s o e n z y m e s o f B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e ,
contribute
to
the
appearance
of
skeletal
lesions
i n
may
patients
a f f e c t e d by Sandhoff disease. The p h o s p h o l i p i d a c y l - c h a i n
d e p e n d e n c e o f t h e membrane-bound
l y s o s o m a l B-Q-2-acetamido-2-deoxyglucosidase c o n t r o l membranes f r o m r a t
h a s been e x a m i n e d on
b r a i n p r i m a r y c e l l c u l t u r e s and on
membranes m o d i f i e d by c u l t u r i n g t h e c e l l s i n m e d i a s u p p l e m e n t e d w i t h polyunsaturated fatty
a c i d s .33
acetamido-2-deoxyglucosidase acyl-chain
The r e l a t i o n s h i p b e t w e e n $ - Q - 2 -
a c t i v i t y and t h e membrane p h o s p h o l i p i d
c o m p o s i t i o n has been e v a l u a t e d .
An i n c r e a s e i n t h e
u n s a t u r a t i o n l e v e l of p h o s p h a t i d y l e t h a n o l a m i n e s and p h o s p h a t i d y l -
Carbohydrate Chemistry cholines,
t h e m o s t a b u n d a n t p h o s p h o l i p i d s i n t h i s membrane f r a c t i o n ,
i s r e l a t e d t o the enzymic reaction.
The A r r h e n i u s p l o t o f t h e
enzyme a c t i v i t y i n m o d i f i e d membranes showed break-temperatures, s t a r t i n g f r o m a p p r o x i m a t e l y 1 5 'C. below
and above t h e
The a p p a r e n t a c t i v a t i o n e n e r g y
break-temperature
was
not correlated with
phospholipid acyl-chain unsaturation.
Q -Man n o s e , m e t h y 1- a - g
- man n o p y r a n o s i d e ,
mannose have
been f o u n d t o
inhibit
glucosidase B
p u r i f i e d from
both
and 2 - a m i n o -2-deox y -#-
B-Q-2-acetamido-2-deoxy-
porcine
placenta
and
bovine
e p i d i d y m i ~ . ~The ~ i n h i b i t i o n i s c o m p e t i t i v e and t h e i n h i b i t o r y c o n s t a n t s a r e a b o u t 20 m M f o r
g-mannose and m e t h y l - a - 1 - m a n n o p y r a n o -
s i d e and 2 m M f o r 2 - a m i n o - 2 - d e o x y - ~ - m a n n o s e . o f t h i s i n h i b i t o r y e f f e c t i s discussed. The c h a n g e s
of
sulphate-containing
The p h y s i o l o g i c a l r o l e glycosaminoglycans
and
g l y c o s a m i n o g l y c a n a s e s d u r i n g b o v i n e f o e t a l development have been
t was shown t h a t a n a l y ~ e d . ~I ~
chondroitin 6-sulphate
increases i n
c o n c e n t r a t i o n up t o t h e 5 0 t h day o f f o e t a l d e v e l o p m e n t a n d t h e n decreases p r o g r e s s i v e l y u n t i l i t s complete disappearance i n most adult tissues.
Likewise,
h y a l u r o n i d a s e a l s o r e a c h e s a p e a k on t h e
5 0 t h day and d e c r e a s e s i n a c t i v i t y u n t i l i t s d i s a p p e a r a n c e i n a d u l t tissues.
On t h e o t h e r hand,
as w e l l as B - P - g l u c u r o n i d a s e
heparan s u l p h a t e and d e r m a t a n s u l p h a t e and B-~-2-acetamido-2-deoxyglucosidase
r e m a i n w i t h o u t s i g n i f i c a n t changes d u r i n g t h e w h o l e p e r i o d . f o e t a l chondroitin 6-sulphate molecular
weights
properties of described.
depending on t h e t i s s u e o f
foetal
The
i s tissue specific with different
m u s c l e - and
origin.
brain-hyaluronidase
Some
are
The p o s s i b l e r o l e o f c h o n d r o i t i n 6 - s u l p h a t e
also and
h y a l u r o n i d a s e i n t h e p r o c e s s e s o f d i f f e r e n t i a t i o n and d i v i s i o n i s discussed i n view o f these f i n d i n g s . The a - k - f u c o s i d a s e , mannosidase,
a-l-arabinofuranosidase muscle o f
B-~-2-acetamido-2-deoxyglucosidase,
B-p-glucuronidase,
B-;-xylosidase,
activities
of
d e v e l o p i n g and a d u l t
B-Q-glucosidase,
a-p-
and
n o r m a l and a t r o p h i c s k e l e t a l
r a t s have been c o l l e c t i v e l y
i n v e s t i g a t e d . 36
B-;-2-Acetamido-2-deoxyglucosidase mouse t e s t e s .
h a s been c h a r a c t e r i z e d f r o m
Only one o f t h e t w o i s o z y m e s o f B-Q-2-acetamido-2-
d e o x y g l u c o s i d a s e i n t e s t e s was o b t a i n e d i n t h e f i n a l p r e p a r a t i o n , having a specific
activity
of
4.44
p u r i f i e d e n z y m e s h o w e d a I&, o f 0.24 mg-l
protein.
u n i t s mg-l mM and
The o p t i m u m t e m p e r a t u r e
t e m p e r a t u r e (L112)
protein.37
The
lmax o f 0.165 pM m i n - '
i s 6OoC a n d t r a n s i t i o n
f o r h e a t d e n a t u r a t i o n i s 63OC.
The o p t i m u m pH o f
359
6: Enzymes the
enzyme
i s
filtration, potent
4.5.
The
molecular
inhibitors
weight,
dalton.
c o r r e s p o n d s t o 178,000
by
Ag',
G-200
a n d PCMB a r e
o f B-~-2-acetamido-2-deoxyglucosidase,
e t h y l m a l e i m i d e and 2 - a c e t a m i d o - 2 - d e o x y - Q - g l u c o s e enzyme a c t i v i t y .
Sephadex
Hg2',
though
3-
also i n h i b i t the
P r o t e c t i v e a c t i o n by L - c y s t e i n e f o r t h e i n h i b i t i o n
by HgC12 s u g g e s t s t h e r o l e o f a t h i o l g r o u p a t t h e c a t a l y t i c s i t e o f t h e enzyme.
The p u r i f i e d e n z y m e d o e s n o t c r o s s - r e a c t i n i m m u n o -
d i f f u s i o n p l a t e s w i t h anti-mouse t e s t i c u l a r h y a l u r o n i d a s e serum, s u g g e s t i n g t h a t B-Q-2-acetamido-2-deoxyglucosidase
does n o t have
common a n t i g e n i c d e t e r m i n a n t s i n h y a l u r o n i d a s e o f h y a l u r o n i d a s e - B - g complex o f acrosome o r t e s t e s .
2- a c et am i d o - 2 - d eo x y g l u c o s i d a s e
The a c t i v i t i e s o f v a r i o u s g l y c o s i d a s e s i n h o m o g e n a t e s o f t h e small
i n t e s t i n a l mucosa o f
wallabies
eurgenii)
(M.
two
aged
adults from
6
a n d 1 8 s u c k l i n g tamrnar to
i n v e ~ t i g a t e d . ~B ~- g - G a l a c t o s i d a s e , deoxyglucosidase,
a-k-fucosidase,and
50
weeks
have
been
B-Q-2-acetamido-2-
neuraminadase a c t i v i t i e s were
h i g h d u r i n g t h e f i r s t 3 4 weeks p o s t p a r t u m and t h e n d e c l i n e d t o v e r y low levels.
a-p-Glucosidase,
aa-trehalase
a c t i v i t i e s were v e r y l o w or a b s e n t d u r i n g t h e f i r s t 34
weeks,
l i m i t dextrinase,
and t h e n i n c r e a s e d .
a-p-glucosidase,
The B - Q - g a l a c t o s i d a s e
activity
and was
unusual i n being greater i n t h e d i s t a l than t h e middle or p r o x i m a l thirds o f the intestine,
a n d i n i t s l o w pH o p t i m u m ( p H 4.6),
i n h i b i t i o n by 4-chloromercuribenzene t h e l a c k o f 6-0-glucosidase
i t s
s u l p h o n a t e b u t n o t by T r i s ,
activity.
and
These p r o p e r t i e s w e r e t h o s e
o f a lysosomal a c i d B-Q-galactosidase r a t h e r than o f a brush border n e u t r a l B-g-galactosidase. characteristics
of
a
The a - q - g l u c o s i d a s e
lysosomal
acid
a c t i v i t y had t h e
a-a-glucosidase
early
l a c t a t i o n and o f a b r u s h b o r d e r n e u t r a l a - Q - g a l a c t o s i d a s e animals.
The s i g n i f i c a n c e o f
these findings
was
in
i n adult
discussed
i n
r e l a t i o n t o changes i n d i e t a r y c a r b o h y d r a t e s d u r i n g w e a n i n g and t o t h e mode o f d i g e s t i o n o f m i l k c a r b o h y d r a t e s by t h e p o u c h young. The
bulk
of
r a t
brain
neutral
B-g-2-acetamido-2-
deoxyhexosidases
have been shown t o
be p r e s e n t
f r a ~ t i 0 n . l ~ They
w e r e n o t bound by c o n c a n a v a l i n A-Sepharose
t h e a c i d B-P-2-acetamido-2-deoxyhexosidases
i n the
cytosol while
were a l l bound.
The
n e u t r a l B-~-2-acetamido-2-deoxyglucosidase h a d a pH o p t i m u m o f 5.2 and
Em
of
0.57
mM,
while
the
neutral
B-Q-2-acetamido-2-
d e o x y g a l a t o s i d a s e h a d t h e h i g h e s t r e a c t i o n r a t e a t pH 6.0 of
0.12
mM.
B-Q-2-acetamido-2-deoxyglucosidase a c t i v i t y
with a
No d i v a l e n t i o n s a c t i v a t e d e i t h e r o f t h e enzymes.
i n
30
min
a t
5OoC.
lost The
more
than
90%
of
Em The the
B-D-2-acetamido-2-
Carbohydrate Chemistry
360 deoxygalactosidase activity
after
3
was h e a t - s t a b l e h
at
5OoC.
and l o s t o n l y
The
neutral
10-20% o f
i t s
B-Q-2-acetamido-2-
d e o x y g l u c o s i d a s e was i n h i b i t e d b y f r e e 2 - a c e t a m i d o 2 - d e o x y - B - Q-glucose but not
by
The r e v e r s e
2-acetamido-2-deoxy-B-~-galactose.
was f o u n d f o r t h e n e u t r a l B-~-2-acetamido-2-deoxygalactosidase. enzymes were s e p a r a t e d a l m o s t chromatography.
Heat s t a b i l i t y
completely
by
Two
hydroxyapatite
o f t h e separated a c t i v i t y peaks
suggested t h a t t h e n e u t r a l B-g-2-acetamido-2-deoxygalactosidase, w h i c h was n o t b o u n d t o h y d r o x y a p a t i t e , may,
on t h e o t h e r hand,
substrate.
may b e s p e c i f i c t o t h e Q -
The n e u t r a l B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e
galacto substrate. Both of
h a v e some a c t i v i t y t o w a r d t h e Q - g a l a c t o
t h e n e u t r a l enzyme a c t i v i t i e s w e r e shown t o be
h i g h e s t d u r i n g t h e f i r s t p o s t n a t a l week i n r a t b r a i n , i n c o n t r a s t t o t h e a c i d enzyme,
w h i c h showed peak a c t i v i t i e s d u r i n g t h e s e c o n d and
t h i r d weeks. The
separation
and
properties
of
B-e-2-acetamido-2-
d e o x y g l u c o s i d a s e A , B, a n d I f r o m h o r s e b r a i n h a v e b e e n r e p ~ r t e d . ~ ’ Three forms
of
B-~-2-acetamido-2-deoxyglucosidase
been s e p a r a t e d f o r chromatography
(A,
B, and
I) h a v e
t h e f i r s t t i m e f r o m h o r s e b r a i n by i o n - e x c h a n g e on
DEAE-cellulose.
Form
I has
properties
i n t e r m e d i a t e i n t h e e l u t i o n p o s i t i o n f r o m D E A E - c e l l u l o s e PI and thermal s t a b i l i t y , f o r m s A a n d B.
under t h e assay c o n d i t i o n s ,
between those o f
A f t e r i s o e l e c t r i c f o c u s i n g , e s p e c i a l l y f o r m s A and I
a r e t r a n s f o r m e d i n t o more t h e r m o s t a b l e f o r m s . deoxy-a-mannose,
Q-Mannose, 2 - a m i n o - 2 methy1-a-Q-
2-acetamido-2-deoxy-Q-g1ucose,and
mannopyranoside have been f o u n d t o i n h i b i t t h e t h r e e above-mentioned forms.
Q-Glucose
inhibitors
for
and 2-amino-2-deoxy-!-galactose
B-!-2-acetamido-2-deoxyglucosidase
w e r e weak
B a n d I.
52-
G l u c u r o n i c a c i d was an i n h i b i t o r f o r f o r m s A and I. The m e t a b o l i c r o l e o f t h e s e i n h i b i t i o n s on s p e c i f i c i t i e s o f B-e-2-acetamido-2deoxyglucosidase towards n a t u r a l s u b s t r a t e s i s discussed. The i n d u c t i o n o f B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e
activity in
t h e s u b m a x i l l a r y g l a n d s o f s t r e p t o z o t o c i n d i a b e t i c m i c e has been reported.40
S t r e p t o z o t o c i n d i a b e t e s s t r o n g l y r e d u c e d t h e B-Q-2-
a c e t a mido-2-deoxyg lucosidase mice.
a c t i v i t y i n the submaxillary glands o f
A r e c i p r o c a l change was o b s e r v e d b e t w e e n t h e enzyme a c t i v i t y
and b l o o d s u g a r i n d i a b e t e s .
I n s u l i n t r e a t m e n t o f t h e d i a b e t i c mice
returned the reduced a c t i v i t y t o t h e normal l e v e l . f o c u s i n g a n a l y s i s showed t h a t t h e s u b m a x i l l a r y - g l a n d
Isoelectrice n z y m e was
composed o f t w o i s o z y m e s h a v i n g i s o e l e c t r i c p o i n t s (PI) o f 4.8 8.8.
O n l y t h e PI 8.8 i s o z y m e was a f f e c t e d by t h e d i a b e t e s .
and
361
6: Enzymes Two
major
isoenzymes,
and
A
of
B,
B-Q-2-acetamido-2-
d e o x y g l u c o s i d a s e have been p u r i f i e d f r o m a d u l t - r a t muscle.41
gastrocnemius
S t u d i e s w e r e a l s o done o n a d u l t - r a t
a t r o p h i c muscle a f t e r
u n i l a t e r a l neurectomy o f t h e s c i a t i c nerve.
I n b o t h n o r m a l and
a t r o p h i c t i s s u e s t h r e e a d d i t i o n a l m i n o r f o r m s o f t h e enzyme w e r e identified. Sucrose
density
lysosome-containing
-----calcitrans
L.42
galactosidase,
B-e - g l u c u r
gradients
have
been
used t o
fractions from 0 h white
The a c i d i c g l y c o s i d a s e s a-;-mannosidase,
o n i dase, a n d
characterize
prepupae o f
Stomoxys
(a-Q-glucosidase,
B-g-glucosidase,
a-Q-
B-P-galactosidase,
B-Q-2-acet amido-2-deoxyglucosidase)
e q u i l i b r a t e a t t h e same d e n s i t y a s d o e s a c i d p h o s p h a t a s e .
The
m i t o c h o n d r i a 1 m a r k e r enzyme c y t o c h r o m e o x i d a s e a l s o e q u i l i b r a t e s a t t h e same d e n s i t y .
Gomori s t a i n i n g showed t h e p r e s e n c e o f
acid
phosphatase i n t h e lysosomes. B-~-2-Acetamido-2-deoxyglucosidase from
the digestive gland of
Mercenaria
h a s been p a r t i a l l y p u r i f i e d
t h e t h r e e c o a s t a l New E n g l a n d b i v a l v e s
m e r c e n a r i a , S p i s u l a s o l i d i s s i m a , a n d blya a r e n a r i a a n d
t h e i r p r o p e r t i e s h a v e been compared.
H e a t - i n a c t i v a t i o n s t u d i e s on
t h e B-Q-2-acetamido-2-deoxyglucosidase
p r e i n c u b a t e d a t 45OC r e v e a l e d
t h a t t h e p r e p a r a t i o n f r o m S. w h i l e t h a t f r o m M.
s o l i d i s s i m a was s t a b l e u p t o 6 0 min,
a r e n a r i a and M.
m e r c e n a r i a l o s t 47 a n d 9 1 % o f
t h e i r o r i g i n a l a c t i v i t i e s under these c o n d i t i o n s ,
respectively.
I n h i b i t i o n s t u d i e s i n d i c a t e d t h a t ~ - g l u c u r o n o - 6 , 3 - l a c t o n e i s more i n h i b i t o r y t o w a r d s t h e M. a r e n a r i a enzyme,
w h i l e HgC12 a p p e a r s t o be
l e s s i n h i b i t o r y t o w a r d s t h e S. s o l i d i s s i m a enzyme. for
B-~-2-acetamido-2-deoxyglucosidase Other
deoxyglucosidase
M.
The
lma value
mercenaria
was
g r e a t e r t h a n t h a t f r o m S. s o l i d i s s i r n a a n d
approximately 2.5-fold
M. a r enaria.
from
characteristics
such
as
pH
of
the
Km,
optimum,
B-Q-2-acetamido-2mol.
wt.,
energy
o f
a c t i v a t i o n , and e f f e c t o f i o n i c s t r e n g t h o n enzyme a c t i v i t y w e r e f o u n d t o be s i m i l a r f o r a l l t h r e e s p e c i e s . B-;-2-Acetamido-2-deoxyglucosidase
and B-g-2-acetamido-2-deoxy-
g a l a c t o s i d a s e a c t i v i t y l e v e l s and c y t o c h e m i c a l l o c a l i z a t i o n have been
investigated i n
Dugesia Lugubris.18
i n t a c t
and
regenerating
planarian
I n 2 4 h r e g e n e r a t i n g p l a n a r i a n s t h e B-Q-2-
acetamido-2-deoxyglucosidase
was
lysosomes of
cells.
dedifferentiating
found
to
be
localized i n the
The B-Q-2-acetamido-2-deoxy-
glucosidase l o c a l i z a t i o n i n d e d i f f e r e n t i a t i n g c e l l s , correlated w i t h a n d B-Q-2i n c r e a s e i n B-Q-2-acetamido-2-deoxyglucosidase a c e t a m i d o - 2 - d e o x y g a l a c t o s i d a s e a c t i v i t i e s d u r i n g t h e f i r s t 24 h o f
the
362
Carbohydrate Chemistry
r e g e n e r a t i o n , c o n f i r m s t h e involvement o f t h e d e d i f f e r e n t i a t i o n i n t h e o r i g i n of b l a s t e m a . I n n o n - r e g e n e r a t i n g a n i m a l s t h e B-Q-2acetamido-2-deoxyglucosidase a c t i v i t y h a s been c y t o c h e m i c a l l y d e m o n s t r a t e d i n t h e l y s o s o m e s o f g a s t r o d e r m a l c e l l s and i n t h e mucous g r a n u l e s of b a s o p h i l i c s e c r e t o r y c e l l s . T h i s l a t t e r f i n d i n g i s i n t e r p r e t e d a s p r o o f of a s i m i l a r i t y b e t w e e n p l a n a r i a n and v e r t e b r a t e mucous s e c r e t i o n . Nine g l y c o s i d a s e s i n b l o o d s t r e a m f o r m s of Trypanosoma ------------b r u c e i b r u c e i S42 have been p a r t i a l l y c h a r a c t e r i z e d . a-gG l u c o s i d a s e had s i m i l a r p h y s i o c h e m i c a l and e n z y m i c p r o p e r t i e s t o t h o s e o f a - Q - g a l a c t o s i d a s e , B - Q - g l u c o s i d a s e , B-Q-Z-acetamido-2d e o x y g l u c o s i d a s e and B - ~ - 2 - a c e t a r n i d o - 2 - d e o x y g a l a c t 0 s i d a s e . ~ ~ a-QMannosidase was c l e a r l y d i s t i n c t from t h e o t h e r g l y c o s i d a s e s w i t h r e s p e c t t o pH optimum, t h e r m o s t a b i l i t y , s p e c i f i c a c t i v i t y d u r i n g t h e c o u r s e of p a r a s i t a e m i a , and s u b c e l l u l a r l o c a t i o n . I t was s u g g e s t e d t h a t t h e g l y c o s i d a s e s s t u d i e d may p l a y a r o l e i n t h e t u r n o v e r of trypanosomal glycoproteins, i n p a r t i c u l a r t h e v a r i a n t - s p e c i f i c s u r f ace a n t i g e n . A c o m p a r a t i v e s t u d y of B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e from M e r c e n a r i a m e r c e n a r i a , Mya a r e n a r i a , a n d S p i s u l a s o l i d i s s i m a h a s been made.43 A marked i n c r e a s e i n B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e a c t i v i t y h a s b e e n o b s e r v e d i n t h e g e r m i n a t i n g c o t y l e d o n of c o t t o n s e e d s . 4 4 The enzyme was i s o l a t e d <;om c o t t o n s e e d l i n g s and p u r i f i e d t o s t u d y i t s p h y s i o l o g i c a l f u n c t i o n i n t h e g e r m i n a t i o n of c o t t o n seeds. The p u r i f i c a t i o n p r o c e d u r e i n v o l v e s ammonium s u l p h a t e f r a c t i o n a t i o n , i o n - e x c h a n g e c h r o m a t o g r a p h y , g e l f i l t r a t i o n s , and c o n c a n a v a l i n A-Sepharose 48 chromatography, and t h e p u r i f i e d B-P-2a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e was shown t o be homogeneous b y d i s c electrophoresis. The m o l e c u l a r w e i g h t was e s t i m a t e d t o b e a b o u t 1 2 5 , 0 0 0 d a l t o n b y g e l f i l t r a t i o n . The enzyme h y d r o l y s e d b o t h 4 nitrophenyl 2-acetamido-Z-deoxy-f3-~-glucopyranoside and 4nitrophenyl 2-acetamido-2-deoxy-B-P-galactopyranoside. When 4nitrophenyl 2-acetamido-2-deoxy-8-g-glucopyranoside was u s e d a s s u b s t r a t e , Em and w e r e 0.625 nM and 228 m o l e s m i n - l m g - l , r e s p e c t i v e l y , and optimum a c t i v i t y was a t pH 5.6. The enzyme l i b e r a t e d B-linked 2-acetamido-2-deoxy-q-glucopyranosyl residues from c h i t i n , ovalbumin, and p r o n a s e - d i g e s t e d wheat-germ l e c t i n . P u r i f i e d B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e and p u r i f i e d P r o t e i n a s e I , b o t h i s o l a t e d f r o m t h e c e l l u l a r s l i m e mould D i c t y o s t e l i u m d i s c o i d e u m , h a v e b e e n shown t o b e i m m u n o l o g i c a l l y
xmax
6: Enzymes
363
c r o s s - r e a c t i v e w i t h a n t i s e r u m a g a i n s t t h e p r ~ t e i n a s e . ~A ~ p e p t i d e was i s o l a t e d from a p a p a i n d i g e s t of P r o t e i n a s e I t h a t c o m p l e t e l y i n h i b i t e d t h e i m m u n o p r e c i p i t a t i o n of B - e - 2 - a c e t a m i d o - 2 deoxyglucosidase b u t o n l y partially inhibited the T h i s peptide i n h i b i t o r immunoprecipitation of Proteinase I. contained a h i g h concentration of 2-acetamido-2-deoxy-~-glucose-lphosphoryl-L-serine. I t was proposed t h a t t h e s e phosphoryl m o i e t i e s might r e p r e s e n t a common a n t i g e n i c d e t e r m i n a n t i n t h e t w o enzymes. The s e c r e t i o n o f l y s o s o m a l e n z y m e s h a s b e e n s t u d i e d i n ---D i c t y ------------------o s t e l i u m d i s ~ o i d e u m . ~U ~ s i n g c o n d i t i o n s which employ a x o n i c a l l y grown amoebae i n s u s p e n s i o n i n a s i m p l e s t a r v a t i o n b u f f e r , t h e l y s o s o m a l enzymes were s e c r e t e d a t r a t e s t h a t were a t l e a s t a s r a p i d as d u r i n g normal development. Unlike normal growth o r development, t h e r e was no a p p r e c i a b l e lysosomal-enzyme s y n t h e s i s under s t a n d a r d s e c r e t i o n c o n d i t i o n s , s o t h e enzyme a c t i v i t i e s a c t e d a s m a r k e r s f o r t h e v e s i c l e s t h a t c o n t a i n them. One group of enzymes, i n c l u d i n g B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e , B-am a n n o s i d a s e , B - q - g l u c o s i d a s e , B - Q - g a l a c t o s i d a s e - I , and 6-Qg l u c o s i d a s e - I were very e f f i c i e n t l y s e c r e t e d , w i t h 10 t o 80% of t h e t o t a l c e l l u l a r a c t i v i t y becoming e x t r a c e l l u l a r w i t h i n a few hours. A l l of t h e s e enzymes have s i m i l a r o r i d e n t i c a l s e c r e t i o n k i n e t i c s and may come from t h e same l y s o s o m a l v e s i c l e s . The k i n e t i c s of s e c r e t i o n from t h e s e v e s i c l e s a r e c o m p l e x , w i t h an i n i t i a l l a g p e r i o d f o l l o w e d b y a s h o r t p e r i o d of r a p i d s e c r e t i o n and a f i n a l p l a t e a u w i t h l i t t l e o r no s e c r e t i o n . The s e c r e t i o n o f a c i d p h o s p h a t a s e i s d i s t i n c t from t h e s e c r e t i o n of t h e e f f i c i e n t l y s e c r e t e d glycosidases. I t is secreted w i t h linear kinetics for at l e a s t 6 h w i t h o n l y 13% becoming e x t r a c e l l u l a r . T h i s d i f f e r e n c e i n s e c r e t i o n k i n e t i c s , along w i t h d i f f e r e n t i a l e f f e c t s o f i n h i b i t o r s , i n d i c a t e s t h a t t h e s e c r e t e d a c i d p h o s p h a t a s e a c t i v i t y may be l o c a l i z e d w i t h i n v e s i c l e s o t h e r t h a n t h e above g l y c o s i d a s e s . The a u t h o r s have demonstrated t h a t t h e e g e s t i o n of i n d i g e s t i b l e m a t e r i a l from r e s i d u a l o r t e r m i n a l phagosomes i s k i n e t i c a l l y d i s t i n c t from t h e s e c r e t i o n of any of t h e l y s o s o m a l e n z y m e s . A l s o B - Q-g a l a c t o s i d a s e - I 1 and perhaps o t h e r lysosomal enzymes a r e s e c r e t e d e i t h e r i n e f f i c i e n t l y o r n o t a t a l l under our s t a n d a r d s e c r e t i o n conditions. T h u s , r e s i d u a l b o d i e s , v e s i c l e s c o n t a i n i n g B-gg a l a c t o s i d a s e - 1 1 , and perhaps o t h e r v e s i c l e s may c o n s t i t u t e a t h i r d c l a s s of lysosomal v e s i c l e s i n t h i s organism which a r e e s s e n t i a l l y non-secretory under t h e s e d e f i n e d c o n d i t i o n s . Lysosomal enzymes have been f o u n d t o p o s s e s s a common a n t i g e n i c
364
Carbohydrate Chemistry
determinant
i n D i c t y o s t e l i u m discoideum.47
A n t i s e r a have been
p r e p a r e d a g a i n s t t w o o f t h e l y s o s o m a l enzymes.
The t w o p u r i f i e d
en zy me p r e p a r a t i o n s u s e d f o r i m m u n i z a t i o n , B-2-2-ace t am i d o - 2 - d e o x y g l u c o s i d a s e and B - Q - g l u c o s i d a s e - I , each o t h e r and no s i g n i f i c a n t enzymes. equally
However,
show no c r o s s - c o n t a m i n a t i o n w i t h
c o n t a m i n a t i o n by o t h e r l y s o s o m a l
antisera raised against
w e l l t o seven d i f f e r e n t
either
enzyme b i n d
l y s o s o m a l e n z y m e s a n d show
f o r t h e enzyme a g a i n s t w h i c h t h e y w e r e r a i s e d .
preference
no
A total
o f 1 0 d i f f e r e n t a n t i s e r a h a v e b e e n e x a m i n e d a n d a l l show s i m i l a r results.
P r e a d s o r p t i o n o f a n t i s e r a w i t h e i t h e r p u r i f i e d enzyme E v i d e n c e is
r e m o v e s a l l a n t i b o d y a c t i v i t y a g a i n s t t h e o t h e r enzyme.
p r e s e n t e d w h i c h i n d i c a t e s t h a t t h e same s p e c i e s o f a n t i b o d i e s a r e r e s p o n s i b l e f o r t h e p r e c i p i t a t i o n o f s e v e n l y s o s o m a l enzymes.
These
data are discussed i n terms o f the proposal t h a t the antigen t h a t i s s h a r e d by t h e l y s o s o m a l enzymes i s a p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n o f t h e enzyme p r o t e i n s . The
relationship
between
carbohydrate
t h e r m o s t a b i l i t y o f B-g-glucosidase
---___-----_ M u c o r m e i h e i YH-10
has
been
t h e r m o s t a b l e B - -Q - g l u c o s i d a s e t h e r m o s t a b l e G-b) filtrate. 23.4%
moiety
and
from t h e t h e r m o p h i l i c fungus
i n ~ e s t i g a t e d . ~T ~ wo
( a m o r e t h e r m o s t a b l e G-a
forms
of
and l e s s
were p u r i f i e d t o homogeneity f r o m t h e c u l t u r e
G-a a n d t h e G-b w e r e g l y c o e n z y m e s , a n d t h e y c o n t a i n e d
and
13.0%
carbohydrate
residues,
c a r b o h y d r a t e r e s i d u e s w e r e !-mannose glucopyranose.
respectively.
The
and 2 - a c e t a m i d o - Z - d e o x y - B - 2 -
The c a r b o h y d r a t e r e s i d u e s i n G-a h a d a h i g h c o n t e n t
2-acetamid0-2-deoxy-13-g-glucopyranose i n the culture f i l t r a t e , and a h i g h a c t i v i t y o f B-E-2-acetamido-2-deoxyglucosidase was of
observed.
The c a r b o h y d r a t e c o n t e n t o f n a t i v e G-a d e c r e a s e d t o 13.0%
when s u f f i c i e n t l y d i g e s t e d w i t h a c o m m e r c i a l p r e p a r a t i o n o f B-Q-2-
acetamido-2-deoxyglucosidase
i n s t e a d of
d e o x y g l u c o s i d a s e p r o d u c e d by t h i s f u n g u s .
B-Q-Z-acetamido-2-
The d i g e s t e d enzyme was
i d e n t i c a l w i t h n a t i v e G-b i n c a r b o h y d r a t e c o n t e n t , amino a c i d composition, cellobiose, dependency
Em v a l u e and
isoelectric point,
f o r B-Q-glucosides,
stability.
molecular weight,
hydrolysis curve for
and i n b o t h t h e r m o - and pH-
Therefore,
c a r b o h y d r a t e s f r o m m o r e t h e r m o s t a b l e G-a
the
liberation
o f
by B - a - 2 - a c e t a m i d o - 2 -
deoxyglucosidase i n c u l t u r e f i l t r a t e l e d t o t h e f o r m a t i o n o f l e s s t h e r m o s t a b l e G-b. Six
glycoside
Bacteroides f r a g i l i s galactosidase,
hydrolases
-
i n
the
a-g-glucosidase,
B-5-galactosidase,
culture
medium
B-a-glucosidase,
o f
a-Q-
B-Q-Z-acetamido-2-
365
6: Enzymes deoxyglucosidase,
-
and a - C - f u c o s i d a s e
were s y s t e m a t i c a l l y p u r i f i e d
by ammonium s u l p h a t e p r e c i p i t a t i o n ,
gel-filtration
and
focusing.49
density-gradient
isoelectric
chromatography, The
focusing resolved the glycosidases i n t o distinct,
isoelectric
w e l l separated
f r a c t i o n s a n d r e v e a l e d t h r e e d i f f e r e n t l y c h a r g e d f o r m s o f B-E-2-
a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e and o f a - L - f u c o s i d a s e . F u r t h e r m o r e , aQ - g l u c o s i d a s e and B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e w e r e shown t o possess d u a l a f f i n i t i e s f o r t h e r e s p e c t i v e Q - g a l a c t o s i d e s u b s t r a t e s , and B - 8 - g a l a c t o s i d a s e was
purified
to
a l s o h y d r o l y s e d B-Q-fucoside.
homogeneity,
as
i s o e l e c t r i c - f o c u s i n g zymogram t e c h n i q u e . e x c e p t i o n of
B-g-glucosidase
a-Q-Glucosidase
i n d i c a t e d by
w e i g h t s v a r i e d b e t w e e n 58,000 a n d 125,000. The
thin-layer
and t h e a c i d a - l - f u c o s i d a s e ,
s e p a r a t e d f r o m o t h e r g l y c o s i d i c a c t i v i t i e s t o 99%. f r o m 4.8
a
The g l y c o s i d a s e s , w i t h were a l l
The m o l e c u l a r
The pH o p t i m a r a n g e d
t o 6.9. effect
o f
tunicamycin
on
secreted
glycosidases
of
A s p e r g i l l u s n i g e r h a s b e e n d e ~ c r i b e d . ~ ' The m a i n g l y c o s i d a s e s s e c r e t e d i n t o t h e c u l t u r e medium d u r i n g g r o w t h w e r e B - e - g l u c o s i d a s e , a - Q - g a l a c t o s i d a s e , and B-P-acetamido-2-deoxyglucosidase. presence o f tunicamycin, the activities of
I n the
an i n h i b i t o r o f p r o t e i n N - g l y c o s y l a t i o n ,
t h e s e enzymes i n t h e
culture
medium were
c o n s i d e r a b l y d e c r e a s e d , whereas f u n g a l g r o w t h and t o t a l m y c e l i a l p r o t e i n c o n t e n t were n o t d i m i n i s h e d .
Intracellular glucosidase
a c t i v i t y was a l s o r e d u c e d i n t h e p r e s e n c e o f t u n i c a m y c i n .
The
a n t i b i o t i c a l s o c a u s e d a d e c r e a s e i n t h e i n c o r p o r a t i o n o f g-mannose i n t o t o t a l protein o f the culture f i l t r a t e .
Polyacrylamide gel
electrophoresis o f these proteins indicated t h a t the m a j o r i t y o f the Q-mannosylated p r o t e i n s were a f f e c t e d .
The i n c o r p o r a t i o n o f
L_-
l e u c i n e i n t o t a l c u l t u r e f i l t r a t e p r o t e i n was n o t i n h i b i t e d b y tunicamycin.
The a m o u n t o f B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e i n
t h e c u l t u r e m e d i u m was e s t i m a t e d b y i m m u n o t i t r a t i o n o f activity
w i t h s p e c i f i c antiserum.
enzyme
The r e s u l t s s u g g e s t t h a t t h e
d e c r e a s e d enzyme a c t i v i t y o b s e r v e d d u r i n g g r o w t h i n t h e p r e s e n c e o f t u n i c a m y c i n i s due t o a r e d u c e d amount o f enzyme. I n v i t r o c u l t u r e s o f Entamoeba h i s t o l y t i c a s t r a i n s HK-9 and NIH-200 h a v e been f o u n d t o c o n t a i n c o n s i d e r a b l e a m o u n t s o f t h e a c i d h y d r o l a s e B - Q - 2 - a c e t a m i d 0 - 2 - d e o x y g l u c o s i d a s e . ~ ~ The r e s u l t s i n d i c a t e t h a t t h i s enzyme i s p r o d u c e d b y t h e amoebae and s e c r e t e d i n t o t h e g r o w t h medium.
The enzyme was p u r i f i e d 1000-2000 f o l d a n d
p r e l i m i n a r i l y characterized.
acetamido-2-deoxyglucosidases
I n cultures
f r o m NIH-200
two
B-g-2-
w i t h d i s t i n c t PIS, pH o p t i m a , a n d
Carbohydrate Chemistry
366
From c u l t u r e s HK-9 o n l y one enzyme was
k i n e t i c s were d e m o n s t r a t e d .
i s o l a t e d and i t seems t h a t i t was i d e n t i c a l w i t h one o f t h e NIH-200 enzymes.
The m o l e c u l a r w e i g h t o f a l l t h r e e e n z y m e s was 1 2 5 , 0 0 0
10,000.
As
inflammatory
i t i s
known t h a t
conditions
it
acetamido-2-deoxyglucosidase
i s
h y d r o l a s e s can be suggested
that
involved
released
2 i n
f3-Q-2-
p a r t i c i p a t e s i n the pathogenesis o f
amoebiases. Sites o f
autolysis
e l e c t r o n microscopy.
i n B a c i l l u s s u b t i l i s have been a n a l y s e d by
C e l l l y s i s i n 6 . s u b t i l i s 168/s
an a u t o l y t i c - d e f i c i e n t
mutant,
B.
s u b t i l i s FJ6,
t p r t h y and i n was s t u d i e d b y
f o l l o w i n g t h e r e l e a s e o f w a l l m a t e r i a l l i b e r a t e d by t h e t w o known autolysins,
an tj-acetylmuramoyl-L-alanine
acetamido-2-deoxyglucanase.52
a m i d a s e and an endo-B-P-2-
L y s i n g organisms were examined i n
t h i n s e c t i o n s by e l e c t r o n m i c r o s c o p y , a n d measurements were made b o t h o f t h e l o c a t i o n a n d s i z e o f h o l e s p r o d u c e d b y a u t o l y s i n a c t i o n and of peripheral w a l l thickness.
the i n i t i a l s i t e o f
I n s t r a i n 168/s
and o f one o r b o t h
l y s i s involved solubilization o f the cross-wall poles d i s t a l t o the division site.
Subsequently, t h e c y l i n d r i c a l
w a l l was d e g r a d e d t h r o u g h p e r f o r a t i o n s a t an i n c r e a s i n g n u m b e r o f sites. pole,
S p e c i f i c s i t e s o f a u t o l y s i s , a t t h e c r o s s - w a l l a n d a t one were a l s o o b s e r v e d i n o r g a n i s m s p r e f i x e d w i t h f o r m a l d e h y d e i n
a b u f f e r o f low i o n i c strength, o b s e r v e d i n s t r a i n FJ6.
although these e f f e c t s were n o t
I n s t r a i n 168/s t h e b u l k o f t h e a u t o l y s i n
a p p e a r e d t o be l o c a t e d a t t h e c r o s s - w a l l a n d a t o n e d i s t a l p o l e . The p e r i p h e r a l w a l l was r e d u c e d i n t h i c k n e s s i n a u t o l y s i n g c e l l s , a l t h o u g h i t was u n c e r t a i n w h e t h e r t h e r e d u c t i o n was due t o a u t o l y t i c attack a t the outer surface,
t o contraction o f the w a l l i n the
suspending b u f f e r o r t o shrinkage d u r i n g p r e p a r a t i o n f o r e l e c t r o n microscopy.
L y s i s o f s t r a i n FJ6 i n v o l v e d t h e i n i t i a l b r e a k d o w n of
t h e c y t o p l a s m i c membrane and l e a k a g e o f c e l l u l a r c o n t e n t s t h r o u g h a l i m i t e d number o f s i t e s a l o n g t h e w a l l .
B. s ub t i l i s MB21,
I n contrast,
lysis in
t h e p a r e n t a l s t r a i n o f F J 6 , was s i m i l a r t o t h a t
d e s c r i b e d f o r s t r a i n 168/s. The t r y p a n o c i d a l d r u g s u r a m i n h a s b e e n f o u n d t o b e a p o t e n t i n h i b i t o r o f B-~-2-acetamido-2-deoxyhexosidase
A, w i t h a
K i of
about
4.5
uM, and t o a l e s s e r e x t e n t o f B - ~ - 2 - a c e t a m i d o - 2 - d e o x y h e x o s i d a s e
(Ki
31.5
uM).
B-~-2-Acetamido-2-deoxyhexosidase
i n t h e p r e s e n c e o f 1.0
B remained a c t i v e
m M s u r a m i n w h e r e a s t h e a c t i v i t y o f B-p-2-
a c e t a m i d o - 2 - d e o x y h e x o s i d a s e A was c o m p l e t e l y i n h i b i t e d a t 0.1 mM.53 the
A p r o t e i n a c t i v a t o r (GM2-activator)
specific for stimulating
hydrolysis
by
of
GM2
ganglioside
B-Q-2-acetamido-2-
367
6: Enzymes
d e o x y h e x o s i d a s e A h a s been p u r i f i e d o v e r 1 0 5 - f o l d w i t h a h i g h y i e l d from
human
adjustment
liver.54 of
the
The
pH o f
p u r i f i c a t i o n procedure liver
extract
ammonium s u l p h a t e p r e c i p i t a t i o n ,
to
S e p h a d e x G-200
and
octyl-Sepharose
48.
physical
filtration,
and
properties
of
the
GM2-
( a ) m o d e r a t e l y h e a t s t a b l e up t o 5OoC, ( b ) m o l e c u l a r
w e i g h t a b o u t 23,500, analysis
The
the
f o l l o w e d by
Matrex Gel Blue A,
c o l u m n c h r o m a t o g r a p h i e s o n DEAE-Sephadex A-50, a c t i v a t o r are:
includes
pH 4.3,
and ( c )
i s o e l e c t r i c p o i n t a b o u t 4.8.
i d e n t i f i e s t h i s a c t i v a t o r as a p r o t e i n .
Chemical
GM2-activator
i s
v e r y s p e c i f i c f o r s t i m u l a t i n g t h e h y d r o l y s i s o f GM2 g a n g l i o s i d e ,
but
only s l i g h t l y e f f e c t i v e i n s t i m u l a t i n g t h e h y d r o l y s i s o f asialo-GM2
or
globotetraosylceramide
deoxyhexosidase taurodeoxycholate,
catalysed
Unlike
A.
b i l e
B-Q - -2-acetamido-2-
by
s a l t s
such
as
sodium
t h i s a c t i v a t o r does n o t s t i m u l a t e t h e h y d r o l y s i s
o f t h e above t h r e e g l y c o s p h i n g o l i p i d s c a t a l y s e d by B - g - 2 - a c e t a m i d o 2-deoxyhexosidase
I t does
8.
not
stimulate
the
h y d r o l y s i s of
s y n t h e t i c s u b s t r a t e s s u c h as 4-met hy l u m b e l l i f e r y 1 and 4 - n i t r o p h e n y 1
2-acetamido-2-deoxy-B-~-galactopyranosides.
The o l i g o s a c c h a r i d e
d e r i v e d f r o m G M 2 g a n g l i o s i d e i s n o t h y d r o l y s e d by B - P - 2 - a c e t a m i d o - 2 deoxyhexosidase A or B i n t h e presence o f e i t h e r GM2-activator or sodium
taurodeoxycholate.
The
rate
of
the
hydrolysis
of
g a n g l i o s i d e i s a f f e c t e d by b o t h t h e amount o f G M 2 - a c t i v a t o r and
2-acetamido-2-deoxyhexosidase
A.
GM2
B-g-
The m o l a r r a t i o o f enzyme t o G M 2 -
a c t i v a t o r f o r o b t a i n i n g t h e m a x i m a l h y d r o l y s i s o f GM2 g a n g l i o s i d e i s c l o s e t o 1:1, activator
while the molar r a t i o o f
i s about
300:l.
GM2
g a n g l i o s i d e t o GM2-
T h e s e r e s u l t s may s u g g e s t t h a t
a c t i v a t o r i n t e r a c t s w i t h B-~-2-acetamido-2-deoxyhexosidase
GM2-
A rather
than the l i p i d substrate. Pulse-chase
experiments
had shown t h a t
B-a-2-acetamido-2-
d e o x y h e x o s i d a s e was s y n t h e s i z e d i n c u l t u r e d h u m a n f i b r o b l a s t s i n p r e c u r s o r f o r m and t h a t d u r i n g m a t u r a t i o n o f t h e enzyme t h e a - c h a i n s w e r e c o n v e r t e d f r o m tdr = 67,000 t o 5 4 , 0 0 0 a n d t h e B - c h a i n s f r o m 63,000
to
29,000
plus
intermediate form o f (1980)
smaller
Mr
J. B i o l . Chem.,
fragments,
= 52,000
222,
deoxyhexosidase precursor
(Hasilik,
4937).55
which
i s
probably A.
The
Mr
through
and N e u f e l d ,
=
an
E.F.
B-Q-2-acetamido-2-
present
i n NH,,+-induced
s e c r e t i o n s h a s n o t been u s e d as s u b s t r a t e t o s t u d y t h e s e c o n v e r s i o n s i n a cell-free
system.
biosynthetically, proteinases.
A c o n c e n t r a t e o f such s e c r e t i o n s ,
labelled
was i n c u b a t e d w i t h c e l l f r a c t i o n s o r p u r i f i e d
After
d e o x y h e x o s i d a s e was
the
reaction,
the
immunoprecipitated
B-Q-2-acetamido-2and i t s
constituent
3 68
Carbohydrate Chemistry
p o l y p e p t i d e s were d e n a t u r e d ,
reduced, and a n a l y s e d by p o l y a c r y l a m i d e
g fraction of
A 3,000-12,000
g e l e l e c t r o p h o r e s i s and f l u o r o g r a p h y .
f i b r o b l a s t s catalysed the conversion o f the a-chain o f the precursor 8-~-2-acetamido-2-deoxyhexosidase from
t h e 8-chain
f r o m bir = 63,000
t o 53,000.
Mr
= 67,000
t o 56,000
and o f
These p r o d u c t s w e r e s i m i l a r
t o t h e m a t u r e a - c h a i n and t h e i n t e r m e d i a t e f o r m o f t h e 8 - c h a i n .
The
r e a c t i o n o c c u r r e d a t a c i d pH,
and
was
inhibited
chymostatin,
by
was s t i m u l a t e d by d i t h i o t h r e i t o l ,
iodoacetamide,
ana a n t i p a i n .
tj-ethylmaleimide,
P h e n y l m e t h y l s u l p h o n y l f l u o r i d e gave
p a r t i a l i n h i b i t i o n w h e r e a s H4 e d t a , no
effect.
These
properties
p e p s t a t i n A,
indicate
c a t a l y s e d by a l y s o s o m a l t h i o l p r o t e i n a s e . p r o b a b l y d i s t i n c t f r o m c a t h e p s i n B, had no e f f e c t
leupeptin,
and a p r o t i n i n had
that
the
However,
reaction
was
t h e enzyme was
as p u r i f i e d c a t h e p s i n B i t s e l f
on t h e 8 - ~ - 2 - a c e t a m i d o - 2 - d e o x y h e x o s i d a s e
precursor.
A
s i m i l a r r e a c t i o n was a l s o c a t a l y s e d b y t r y p s i n , c h y m o t r y p s i n , a n d some r e l a t e d p r o t e i n a s e s . addition polypeptides o f which resembled t h e Formation o f
the
Very h i g h l e v e l s o f t r y p s i n r e l e a s e d i n
Mr
= 30,000
products o f
56,000
and
and l o w e r m o l e c u l a r w e i g h t ,
intracellular
53,000
8-chain
products
by
maturation.
the
lysosomal
f r a c t i o n , t r y p s i n and c h y m o t r y p s i n , proceeded e q u a l l y w e l l i f t h e r a d i o a c t i v e 8 - ~ - 2 - a c e t a m i d o - 2 - d e o x y h e x o s i d a s e p r e c u r s o r was u s e d as s u b s t r a t e i n t h e f o r m o f an i m m u n o p r e c i p i t a t e . p r e c u r s o r m o l e c u l e appeared t o be degraded. of i t s
the B-~-2-acetamido-2-deoxyhexosidase catalytic
activity
potential for
towards
a
The r e m a i n d e r o f t h e
The l i m i t e d p r o t e o l y s i s precursor d i d not a f f e c t
synthetic
substrate
u p t a k e m e d i a t e d by t h e P-mannose
or
i t s
6-phosphate
r e c o g n i t i o n system. The d e l i v e r y o f
B-P-2-acetamido-2-deoxyhexosidase
cerebrum a f t e r o s m o t i c m o d i f i c a t i o n o f t h e b l o o d - b r a i n
A t o the b a r r i e r has
b e e n i n ~ e s t i g a t e d . The ~ ~ s t u d i e s were undertaken t o e v a l u a t e t h e p o s s i b i l i t y t h a t B-Q-2-acetamido-2-deoxyhexosidase
A,
t h e enzyme
d e f i c i e n t i n Tay-Sachs d i s e a s e , c o u l d be e f f e c t i v e l y d e l i v e r e d t o the brain.
P r e v i o u s s t u d i e s h a v e shown t h a t h y p e r t o n i c m a n n i t o l c a n
be u s e d t o o s m o t i c a l l y p r o d u c e r e v e r s i b l e d i s r u p t i o n o f t h e b l o o d brain barrier
i n a n i m a l s ( r a t and dog) and man w i t h o u t s i g n i f i c a n t
neurotoxicity,
and t h a t s u c h b a r r i e r m o d i f i c a t i o n s i g n i f i c a n t l y
increases t h e d e l i v e r y of selected areas o f brain.
c y t o r e d u c t i v e chemotherapy agents t o By u s i n g t h e r a t m o d e l o f b l o o d - b r a i n
b a r r i e r m o d i f i c a t i o n and r a d i o l a b e l l e d enzyme, acetamido-2-deoxyhexosidase
A
delivery
d e m o n s t r a t e d i n more t h a n 85 a n i m a l s .
t o
i n c r e a s e d 8-Q-2brain
has
been
The t i m e o f i n j e c t i o n o f 8 - a -
369
6: Enzymes 2-acetamido-2-deoxyhexosidase
A a f t e r blood-brain b a r r i e r disruption
i s c r i t i c a l f o r maximum d e l i v e r y . R a p i d ( o v e r 30 s e c ) i n t r a - a r t e r i a l A immediately
a d m i n i s t r a t i o n o f B-Q-2-acetamido-2-deoxyhexosidase a f t e r blood-brain
barrier
d i s r u p t i o n r e s u l t e d i n a marked i n c r e a s e
i n enzyme d e l i v e r y t o t h e b r a i n when t h e enzyme was a d m i n i s t e r e d 15-
20
after
min
barrier was
deoxyhexosidase A barrier
modification,
c o n t r o l animals.
disruption;
50% l e s s
delivered.
When g i v e n 6 0 - 1 2 0
t h e amount
B-Q-2-acetamido-2-
d e l i v e r e d was
min a f t e r
t h e same a s i n
T h i s c r i t i c a l t i m e course i s very d i f f e r e n t from
t h a t seen i n t r i a l s o f l o w - m o l e c u l a r - w e i g h t ( m e t h o t r e x a t e and a d r i a m y c i n ) .
A can be d e l i v e r e d t o t h e
t h a t B-P-2-acetarnido-2-deoxyhexosidase b r a i n by b l o o d - b r a i n
chemotherapeutic agents
These p r e l i m i n a r y s t u d i e s s u g g e s t
b a r r i e r m o d i f i c a t i o n and may b e i n d i c a t i v e o f
t h e p o t e n t i a l f o r enzyme r e p l a c e m e n t i n p a t i e n t s who h a v e Tay-Sachs disease. The
properties of
~ - ~ - 2 - a c e t a m i d o - 2 - d e o x y h e x o s i d a s e have b e e n
i n v e s t i g a t e d i n i s o l a t e d n o r m a l and I - c e l l lysosymes.57 combination
of
differential
e l e c t r o p h o r e s i s (Harms, Proc.
Natl.
Acad.
Sci.
centrifugation
Kern,
E.,
H.,
and S c h n e i d e r ,
11, 61391,
U.S.A.,
and
1,
authors’
and
I-cell
disease
type
(1980)
J.A.
a single population of
h i g h l y p u r i f i e d l y s o s o m e s was o b t a i n e d f r o m n o r m a l , type
Using a
free-flow
2 cultured
I-cell
disease
fibroblasts.
The
f i n d i n g s i n d i c a t e t h a t most o f t h e r e s i d u a l a c i d h y d r o l a s e
a c t i v i t i e s remaining w i t h i n the I - c e l l fibroblasts are localized i n t h e lysosomes,
analogous t o n o r m a l c e l l s .
Characterization o f the
carbohydrate-dependent p r o p e r t i e s o f t h e l y s o s o m a l B-g-2-acetamido2 - d e o x y h e x o s i d a s e r e v e a l e d t h a t t h e I - c e l l and n o r m a l enzymes do n o t c o n t a i n a s i g n i f i c a n t p r o p o r t i o n o f neuraminidase-susceptible a c i d residues,
interact poorly
sialic
with the B-8-galactose-specific
l e c t i n R i c i n u s communis, and a r e h i g h l y s e n s i t i v e t o e n d o - 2 - a c e t a m i d o 2-deoxyhexosidase
H treatment,
indicating that
the oligosaccharide
u n i t s of b o t h t h e I - c e l l and n o r m a l l y s o s o m a l B-g-2-acetamido-2d e o x y h e x o s i d a s e a r e p r e d o m i n a n t l y o f t h e h i g h Q-mannose t y p e . I-cell however,
The
and n o r m a l l y s o s o m a l B-g-2-acetamido-2-deoxyhexosidaseq differed i n t h e i r endocytotic properties.
I n contrast
to
t h e h i g h r a t e o f e n d o c y t o s i s o f t h e n o r m a l l y s o s o m a l enzyme (7.8% mg-l
h-*l,
the
1-cell
type-1
endocytosed i n t o Sandhoff
l y s o s o m a l enzyme f a i l e d t o
p h o s p h o h e x y l r e c o g n i t i o n m a r k e r on t h e I - c e l l of
the
normal
extracellular
revealed the presence of
be
c e l l s , i n d i c a t i n g an a b s e n t o r a l t e r e d enzyme.
Examination
B-g-2-acetamido-2-deoxyhexosidase
predominantly
h i g h a-mannose-type
370
Carbohydrate Chemistry
oligosaccharide units, enzyme,
similar
to
the
corresponding
although p r o p e r t i e s t y p i c a l o f complex-type I n contrast,
c h a i n s were a l s o e v i d e n t .
lysosomal
oligosaccharide
t h e s e c r e t e d I - c e l l enzyme
revealed t h e presence o f o l i g o s a c c h a r i d e u n i t s predominantly o f t h e complex
type, i n d i c a t i n g t h a t
deoxyhexosidase
the
I-cell
had h i g h 0-mannose-type
m o d i f i e d t o complex type probably
B-g-2-acetamido-2-
oligosaccharide chains
i n t h e G o l g i o r GERL p r i o r t o
secretion from the c e l l . Human
fibroblasts
with
a
genetic
deficiency
of
a
single
l y s o s o m a l enzyme a n d f i b r o b l a s t s f r o m a p a t i e n t w i t h I - c e l l d i s e a s e w i t h a m u l t i p l e d e f i c i e n c y o f l y s o s o m a l h y d r o l a s e s h a v e b e e n u s e d as recipient
cells
a c e t a m id o - 2
i n s t u d i e s on r e c o g n i t i o n a n d u p t a k e o f B-Q-2-
- d e o x y h e x o s id a s e ,
galactosidase.
N o r m a l human
B - Q - g 1u c u r o n id a s e, fibroblasts
h e p a t o c y t e s , and hepatoma c e l l s f r o m be
similar
extracellular
among
these
a c t i v i t i e s of
and
B-a_-
fibroblasts,
t h e r a t were used as donor
The r e l e a s e o f B - Q - 2 - a c e t a m i d o - 2 - d e o x y h e x o s i d a s e
cells.58 to
and
different
c e l l
B-q-glucuronidase
types,
was f o u n d but
the
and B - P - g a l a c t o s i d a s e
w e r e much h i g h e r i n r a t c e l l c u l t u r e s t h a n i n c u l t u r e s o f n o r m a l human f i b r o b l a s t s .
The e n z y m e s r e l e a s e d b y r a t f i b r o b l a s t s w e r e
i n g e s t e d by d e f i c i e n t human f i b r o b l a s t s :
enzyme f r o m n o r m a l human
f i b r o b l a s t s was s h o w n t o b e t a k e n u p b y r a t f i b r o s i s b y m e a n s o f electrophoresis.
This
indicates
that
reciprocal
transfer
l y s o s o m a l h y d r o l a s e s o c c u r s b e t w e e n human and r a t f i b r o b l a s t s .
of Rat
h e p a t o c y t e s r e l e a s e d h y d r o l a s e s t h a t w e r e p o o r l y t a k e n u p by human r e c i p i e n t f i b r o b l a s t s , and u p t a k e o f human f i b r o b l a s t enzyme was n o t detected i n t h e hepatocytes.
R a t hepatoma c e l l s ,
on t h e o t h e r hand,
r e l e a s e l y s o s o m a l enzymes t h a t w e r e t a k e n up by human d e f i c i e n t c e l l s with a higher deficiency than those from fibroblasts. uptake
was
phosphate. those f o r
subject
to
c o m p e t i t i v e i n h i b i t i o n by
q-mannose
The
6-
The k i n e t i c s o f t h i s i n h i b i t i o n w e r e c o m p a r a b l e w i t h high-uptake
f o r m s o f l y s o s o m a l enzymes.
Electrophoretic
s t u d i e s showed t h a t r a t hepatoma c e l l s n o t o n l y were c a p a b l e of i n g e s t i n g B-g-2-acetamido-2-deoxyhexosidase
from
n o r m a l human
f i b r o b l a s t s b u t a l s o d e f e c t i v e l y p r o c e s s e d e n z y m e r e l e a s e b y Icells.
T h e s e f i n d i n g s make r a t h e p a t o m a c e l l s a u s e f u l m o d e l f o r
t h e s t u d y o f r e c o g n i t i o n and u p t a k e o f l y s o s o m a l enzymes. The d i s t r i b u t i o n o f t h e d i f f e r e n t t y p e s o f o l i g o s a c c h a r i d e s i n c a t h e p s i n D a n d i n B-Q-2-acetamido-2-deoxyhexosidase
synthesized i n
c u l t u r e d human f i b r o b l a s t s h a s b e e n s t u d i e d b y u s i n g endo-B-g-2acetamido-2-deoxyglucanase
H as
a probe for
h i g h Q-mannose
371
6: Enzymes o l i g o s a c ~ h a r i d e s . ~The ~ enzymes w e r e s p e c i f i c a l l y p r o t e i n or t h e c a r b o h y d r a t e m o i e t y . cleavable
oligosaccharides
labelled i n the
I n b o t h enzymes,
prevailed i n
the
r e s i s t a n t and
secreted
enzymes.
Precursor molecules of cathepsin 0 contained two oligosaccharide s i d e chains.
M u l t i p l e forms o f t h e precursor are synthesized with
b o t h , one,or
none o f t w o o l i g o s a c c h a r i d e s s e n s i t i v e t o t h e a c t i o n o f
t h e endo-B-Q-2-acetamido-2-deoxyglucanase
H.
to
(mucolipidoses
phosphorylate
lysosomal
enzymes
I n f i b r o b l a s t s unable
11) t h e
e x c e s s i v e l y s e c r e t e d l y s o s o m a l enzymes c o n t a i n e d p r e d o m i n a n t l y t o endo-B-g-2-acetamido-2-deoxyglucanase
oligosaccharides resistant
H. A s t u d y h a s b e e n made o f
lysosomal-enzyme
and l e u k o c y t e s i n c h r o n i c h e p a t i c disease.60 a c t i v i t e s (arylsulphatase A,
a c t i v i t i e s i n serum
F i v e lysosomal-enzyme
a-Q-mannosidase,
B-p-2-
a-l-fucosidase,
a c e t a m id o - 2 - d e o x y h e x o s id a s e, and B-Q-ga l a c t 0 s i d as e ) w e r e d e t e r m i n e d i n s e r u m a n d l e u c o c y t e s o f 30 c o n t r o l s a n d 1 1 4 p a t i e n t s s u f f e r i n g from
various
liver
diseases,
including
30
with
h e m o c h r o m a t o s i s and 34 w i t h a l c o h o l i c c i r r h o s i s .
idiopathic
The r e s u l t s show:
(1) a d e c r e a s e o f t h e s e r u m a r y l s u l p h a t a s e A a c t i v i t y i n d i o p a t h i c h a e m o c h r o m a t o s i s , ( 2 ) an i n c r e a s e o f t h i s a c t i v i t y i n c h o l e s t a t i c jaundice,
and (3) m a i n l y a s h a r p r i s e o f t h e l e u c o c y t e l y s o s o m a l -
enzyme a c t i v i t i e s i n t h e l i v e r d i s e a s e s s t u d i e d ( c h i e f l y i d i o p a t h i c haemochromatosis
and a l c o h o l i c
cirrhosis).
The
mechanism
and t h e
meaning o f these d i s t u r b a n c e s a r e discussed. The e f f e c t o f t u n i c a m y c i n and c y c l o h e x i m i d e on t h e s e c r e t i o n o f acid
hydrolases
investigated.61 amounts
of
from
I-cell
I-Cell
cultured
cultured
fibroblasts
fibroblasts
B-Q-acetamido-2-deoxyhexosidase
has
been
secrete excessive
and a - I = - f u c o s i d a s e
t h e c u l t u r e m e d i a as c o m p a r e d w i t h n o r m a l f i b r o b l a s t s .
into
Addition of
t u n i c a m y c i n o r c y c l o h e x i m i d e a t doses t h a t i n h i b i t t h e i n c o r p o r a t i o n o f
{ 3H)g-mannose
(60-8046) a n d { 1 4 C } & - l e u c i n e
(40-50%)
into
t r i c h l o r o a c e t i c a c i d p r e c i p i t a t e m a t e r i a l decreased t h e s e c r e t i o n o f t h e s e I - c e l l h y d r o l a s e s t o n o r m a l v a l u e s w i t h i n 24 h o u r s b u t h a d n o effect
on t h e s e c r e t i o n o f
acid hydrolases from normal fibroblasts.
These r e s u l t s i n d i c a t e d t h a t I - c e l l c u l t u r e d f i b r o b l a s t s s e c r e t e d a t l e a s t two types of a c i d hydrolases: cycloheximide-sensitive the secreted hydrolases, tunicamycin
one o f t h e m was t u n i c a m y c i n - and
and c o n s t i t u t e d t h e g r e a t e r p r o p o r t i o n o f and a s m a l l e r p r o p o r t i o n was i n s e n s i t i v e t o
and c y c l o h e x i m i d e ,
similar to
the
acid hydrolases
s e c r e t e d by n o r m a l c u l t u r e d f i b r o b l a s t s . E n z y m i c c l e a v a g e o f 2-acetamido-2-deoxy-B-P-glucosyl
residues
Carbohydrate Chemistry
372 of
k e r a t a n s u l p h a t e h a s been s t u d i e d i n v i t r o by u s i n g as s u b s t r a t e
2-am i n o -2-deoxy -{ 3H 1 g l u c o s e -1abe l l e d d e s u l p h a t e d k e r a t an s u l p h a t e c o n t a i n i n g 2-acetamido-2-deoxy-$-Q-glucose
r e s i d u e s a t t h e non-
Both lysosomal B-~-2-acetamido-2-deoxyhexosidases A
r e d u c i n g end.62
and B were p r o p o s e d t o p a r t i c i p a t e i n t h e d e g r a d a t i o n o f k e r a t a n s u l p h a t e on t h e b a s i s o f t h e f o l l o w i n g o b s e r v a t i o n s . f i b r o b l a s t s from p a t i e n t s w i t h Sandhoff p a t i e n t s w i t h Tay-Sachs amounts of
disease,
were u n a b l e t o r e l e a s e s i g n i f i c a n t
of
peaks of
keratan sulphate-degrading
B-~-2-acetamido-2-deoxyhexosidase towards
glucopyranoside. reacted
with
A
A
On i s o e l e c t r i c
from
human l i v e r
the
a c t i v i t y coincided with the
2 - a c e t a m i d o - 2 - d e o x y - B - Q --
4-nitrophenyl
m o n o s p e c i f i c a n t i b o d y a g a i n s t t h e human enzyme
both
sulphate-degrading
Em o f
Homogenates o f
b u t n o t those from
2-acetamido-2-deoxy-$-~-{3H)glucose.
focusing
a c t i v i t y
disease,
enzyme
forms
activity.
and
precipitated
the
keratan
B o t h i s o e n z y m e s h a d t h e same a p p a r e n t
4 m M , b u t t h e B f o r m was a p p r o x i m a t e l y t w i c e as a c t i v e as t h e
form
when
isoenzymes.
profiles o f
compared w i t h t h e p H - a c t i v i t y
both
T h e r m a l i n a c t i v a t i o n o f i s o e n z y m e B was l e s s p r o n o u n c e d
towards the polymeric substrate than towards
the 4-nitrophenyl
derivative.
in
the
deoxyhexosidase
and
immobilization
on
Changes
kinetic
concanavalin
acetamido-2-deoxyhexosidase
A.63
have
B-P-2-acetamido-2been
reported
upon
T h e i m m o b i l i z e d B-p-2-
w h i c h h a d a s h a r p o p t i m u m a t pH 4.5.
T h e o p t i m u m o f f r e e a r y l s u l p h a t a s e A, immobilization.
A
s h o w e d a b r o a d o p t i m u m a t pH 3.5-4.5
c o m p a r e d w i t h t h e f r e e enzyme, after
of
properties
arylsulphatase
pH 5.5,
c h a n g e d t o pH 5.25
v,a l u e s f o r t h e f r e e a n d The a p p a r e n t I&,
concanavalin A-immobilized
B - ~ - 2 - a c e t a m i d o - 2 - d e o x y h e x o s i d a s e and
arylsulphatase
A
immobilization.
The i m m o b i l i z a t i o n s i g n i f i c a n t l y i n c r e a s e d t h e
increased
s i g n i f i c a n t l y
t h e r m o s t a b i l i t y o f t h e s e enzymes. m e t a l i o n s was
less for
The e x t e n t o f
in vitro
sialylation
of
purified
on
i n h i b i t i o n by t h e
t h e f r e e enzymes. human
acetamido-2-deoxyhexosidase has been described.64 structure-function
f o l d )
t h e i m m o b i l i z e d 13-g-2-acetamido-2-
d e o x y h e x o s i d a s e and a r y l s u l p h a t a s e A t h a n f o r The
(3-4
relationships
of
liver
B-P-2-
I n order t o study
l y s o s o m a l enzymes,
human l i v e r
B-~-2-acetamido-2-deoxyhexosidase was p u r i f i e d by an e x t r a c t i o n / a f f i n i t y - c h r o m a t o g r a p h y / i o n - e x c h a n g e p r o c e d u r e . The i s o e n z y m e s A and B , n a t i v e as w e l l as n e u r a m i n i d a s e - t r e a t e d ,
were
incubated w i t h a p a r t i a l l y p u r i f i e d preparation o f bovine colostrum s i a l y l t r a n s f e r a s e (CMP-~-acetyl-neuraminate:~-galactosylglycoprotein
373
6: Enzymes N _ - a c e t y l n e u r a m i n y 1t r a n s f e r a s e , acetamido-2-deoxyhexosidases s i a l y l t r a n s f e r a s e used.
EC 2.4.99.1).
N a t i v e B-Q-2-
w e r e f o u n d t o be p o o r a c c e p t o r s f o r t h e
However,
incorporation of s i a l i c acid i n t o
n e u r a m i n id a s e - t r e a t e d 8-g - 2 - a c e t am i d o - 2 - d e o x y h e x o s i dases A a n d B amounted t o a 58 and 72% s a t u r a t i o n o f t h e t h e o r e t i c a l a c c e p t o r sites,
respectively.
The
s i a l y l t r a n s f e r a s e suggests t h a t
acceptor E-Gale-(l
*
s p e c i f i c i t y 4)-n-GlceNAc
of
the
u n i t s may
be p r e s e n t i n a t l e a s t p a r t o f t h e B - ~ - 2 - a c e t a m i d o - 2 - d e o x y h e x o s i d a s e A and B m o l e c u l e s .
H o w e v e r , o l i g o m a n n o s i d i c - t y p e c h a i n s may a l s o
o c c u r o n t h e l y s o s o m a l enzyme, t h e enzyme.
as shown by t h e s u g a r c o m p o s i t i o n o f
The p r e s e n c e and/or
amount o f s i a l i c a c i d r e s i d u e s does
n o t appear t o a f f e c t t h e k i n e t i c p r o p e r t i e s o f B-Q-2-acetamido-2deoxyhexosidases A and B t o w a r d s 4 - m e t h y l u m b e l l i f e r y l g l y c o s i d e substrate. An u n u s u a l i s o e n z y m e o f ~ - Q - 2 - a c e t a m i d o - 2 - d e o x y h e x o s i d a s e s h a s been c h a r a c t e r i z e d f r o m a human c o l o n i c c a r c i n o m a c e l l l i n e . 6 5 separation
and
characterization
acetamido-2-deoxyhexosidases
of
the
isoenzymes
the
A-isoenzyme
activity
i s o e n z y m e t h a t was t h e r m o l a b i l e ,
and
contained
The an
The
B-Q-2-
f r o m each c e l l l i n e were r e p o r t e d .
p a r e n t a l c e l l l i n e c o n t a i n e d A and B isoenzymes. lacked
of
The
sub-line
atypical
susceptible t o alkylation,
B
and o f
l o w e r molecular weight. A
structural
d i f f e r e n c e between the $-chains
i n B-E-2-
B-Q-2-
acetamido-2-deoxyhexosidases B and A has been r e p o r t e d . 6 6 Acetamido-2-deoxyhexosidase A
by
treatment
Sepharose.
with
BA was p r e p a r e d f r o m p u r i f i e d p l a c e n t a l
m e r t h i o l a t e and r e c h r o m a t o g r a p h y
o n DEAE
The h e a t s t a b i l i t y , i s o e l e c t r i c - f o c u s i n g p a t t e r n , a n d
p e p t i d e map o f t h i s i s o e n z y m e were c o m p a r e d w i t h t h o s e o f n a t u r a l l y o c c u r i n g p u r i f i e d p l a c e n t a l B-~-2-acetamido-2-deoxyhexosidase
8.
BA
p r o v e d t o be l e s s h e a t s t a b l e a t 6 O o C , h a v e a s l i g h t l y l o w e r p1,and h a v e one more p e p t i d e s p o t
t h a n d i d B.
The l o w e r PI o f B-Q-2-
a c e t a m i d o - 2 - d e o x y h e x o s i d a s e BA i s n o t c a u s e d by s i a l i c a c i d r e s i d u e s s i n c e i t was u n a f f e c t e d by n e u r a m i n i d a s e t r e a t m e n t .
The e x t r a
p e p t i d e s p o t i n t h e B - ~ - 2 - a c e t a m i d o - 2 - d e o x y h e x o s i d a s e BA map c a n n o t b e e x p l a i n e d by c a r b o h y d r a t e d i f f e r e n c e s s i n c e n o c o r r e s p o n d i n g unpaired peptide spot h e x o s i d a s e B map.
was s e e n i n t h e $ - ~ - 2 - a c e t a m i d o - 2 - d e o x y -
Though B - ~ - 2 - a c e t a m i d o - 2 - d e o x y h e x o s i d a s e s B
a r e p r o d u c t s o f t h e same g e n e ,
there i s evidence t h a t
derived from a l a r g e r precursor polypeptide chain.
and BA
both are
The r e s u l t s
i n d i c a t e d t h a t enzymes B a n d BA a r e p r o c e s s e d d i f f e r e n t l y ,
resulting
i n a s m a l l p e p t i d e p r e s e n t i n enzyme BA t h a t i s n o t f o u n d i n B-q-2-
3 74
Carbohydrate Chemistry
acetamido-2-deoxyhexosidase
B.
C u l t u r e d r a t p e r i t o n e a l macrophages h a v e been d e m o n s t r a t e d t o c o n t a i n a receptor-mediated system f o r enzyme
p r o c e s s was s a t u r a b l e (4.5 X
the uptake o f the lysosomal
1 2 5 1 } - B - ~ - 2 - a c e t a m i d o - 2 - d e o x y h e x o s i d a s e A.67
10'8M.
The
uptake
ng
was
acetamido-2-deoxyhexosidase
i n h i b i t e d 68% by 66% by
A,
m e p a c r i n e , and c h l o r o q u i n e ( b o t h 0.1
inhibited specific
Mepacrine
inhibited
t o 50%.
uptake
by
phagocytosis
24% b y
a
mM)
were f o u n d t o
I 1251}-B-~-2-acetamido-2-
w h i l e c y t o c h a l a s i n B ( 5 ug m l - l )
d e o x y h e x o s i d a s e A,
Methylamine,
and
M e t h y l a m i n e , ammonium i o n
i n h i b i t strongly the s p e c i f i c uptake of (10-5M)
u n l a b e l l e d 8-Q-2-
B-mannan,
h e x o s a m i n o f e t u i n (Q-mannose t e r m i n a t e d ) . ( b o t h 20 m M ) ,
The u p t a k e
c e l l s ) a n d h a d a K u p t a k e o f 5.9
35
of
and
and c o l c h i c i n e
28%, r e s p e c t i v e l y .
latex
beads
t o
76%.
c h l o r o q u i n e , and c y t o c h a l a s i n B i n h i b i t e d a p p r o x i m a t e l y
C o l c h i c i n e and ammonium i o n h a d a n e g l i g i b l e e f f e c t .
r e s u l t s suggested t h a t
These
a d s o r p t i v e p i n o c y t o s i s and p h a g o c y t o s i s
f u n c t i o n independently o f each other. P u r i f i c a t i o n and k i n e t i c s t u d i e s
of
B-e-2-acetamido-2-
deoxyhexosidase A f r o m p o r c i n e k i d n e y have been r e p ~ r t e d . P ~ o~r c i n e kidney B-~-2-acetamido-2-deoxyhexosidase electrophoretic
homogeneity
sulphate precipitation,
by
procedures
A
was
purified t o
involving
ammonium
g e l f i l t r a t i o n , and c o l u m n c h r o m a t o g r a p h y on
diethylaminoethylcellulose,
octyl
Sepharose
CL-4B,
and
6-
am i n o h e x a n o y 1 d e r i v a t i v e s o f 2 - a c e t a m i d o - 2 - d e o x y - B - ~ - g l u c o p y r a n o s e c o u p l e d t o S e p h a r o s e 48.
A n a l y s i s by sodium d o d e c y l s u l p h a t e g e l
e l e c t r o p h o r e s i s s u g g e s t e d t h a t t h e enzyme c o n s i s t s o f t h r e e s u b u n i t s 31,000, a n d 9 , 0 0 0 .
w i t h m o l e c u l a r w e i g h t s o f 61,000,
The m o l e c u l a r
w e i g h t o f t h e i n t a c t e n z y m e a s d e t e r m i n e d b y g e l f i l t r a t i o n was 129,000
and
a t pH 8 . 0
mixed substrates,
as
302,000 well
a t pH 5.0.
as
K i n e t i c analysis with
inhibition studies w i t h
various
s u b s t r a t e a n a l o g u e s , i n d i c a t e d t h a t t h e enzyme h y d r o l y s e s b o t h 2-
acetamido-2-deoxy-B-Q-glucoside active site.
and - g - g a l a c t o s i d e
a t t h e same
Among h e x o s e s t e s t e d f o r i n h i b i t o r y e f f e c t , o n l y Q-
mannose was an e f f e c t i v e c o m p e t i t i v e i n h i b i t o r .
A comparison o f
p-
mannos i d e s w i t h 2 - a c e t a m i d o - 2 - d e o x y -9- g l u c o s i d e s w it h r e s p e c t t o b i n d i n g a b i l i t y t o t h e enzyme i n t e r m s o f
Ki o r K,
values i m p l i e d
t h e p r e s e n c e o f a s i t e f o r p-mannose o t h e r t h a n t h e a c t i v e s i t e . t h e o t h e r hand,
B-mannose c o m p e t e s w i t h 2 - a c e t a m i d o - 2 - d e o x y - Q - g l u c o s e , 2-deoxy-Q-galactose, The
bulk
On
k i n e t i c s t u d i e s w i t h mixed i n h i b i t o r s i n d i c a t e d t h a t
o f
2-acetamido-
and a c e t a m i d e . r a t
brain
neutral
B-Q-2-acetamido-2-
6: Enzymes
375
deoxyhexosidases have
i n the
cytosol
f r a ~ t i 0 n . l ~They w e r e n o t bound by c o n c a n a v a l i n A-Sepharose
been shown t o
be p r e s e n t
whereas
t h e a c i d B-Q-2-acetamido-2-deoxyhexosidases
Em
o f
0.57
while
mM,
the
neutral
B-Q-2-acetamido-2with a
d e o x y g a l a c t o s i d a s e h a d t h e h i g h e s t r e a c t i o n r a t e a t pH 6.0 o f 0.12
mM (see f i r s t c i t a t i o n o f r e f . 1 7
The
h a d a pH o p t i m u m o f 5.2
n e u t r a l B-~-2-acetamido-2-deoxyglucosidase and
were a l l bound.
5,
for further details).
A c i d h y d r o l a s e s f r o m t h e s l i m e mould D i c t y o s t e l i u m discoideum have been r e p o r t e d t o c o n t a i n phosphomannosyl r e c o g n i t i o n markers.6g Acid hydrolases
from
mammalian sources
contain phosphorylated
o l i g o s a c c h a r i d e s w h i c h f u n c t i o n as r e c o g n i t i o n m a r k e r s f o r t h e i r r e c e p t o r - m e d i a t e d e n d o c y t o s i s by human f i b r o b l a s t s . that
The d i s c o v e r y
d i s c o i d e u m c o n t a i n Q-mannose 6 -
g l y c o p e p t i d e s d e r i v e d f r o m D.
phosphate l e d t o the suggestion t h a t
acid hydrolases from t h i s
To t e s t t h i s h y p o t h e s i s ,
source might a l s o bear t h e marker. b i n d i n g and e n d o c y t o s i s o f
p u r i f i e d B-k-glucosidase,
acetamido-2-deoxyhexosidase,and
B-p-mannosidase
were
investigated.
efficiencies
o f 8.6
These
enzymes
t o 60% m g - l h - l ,
by human f i b r o b l a s t s
underwent
endocytosis, hydrolases
sugar
phosphates,
and t h e
The s p e c i f i c i t y o f
saturation kinetics D.
discoideufi
of
acid
t o t h o s e r e p o r t e d f o r enzyme p r e p a r a t i o n s
d e r i v e d f r o m mammalian sources.
95 t o 100% o f t h e S-p-
I n addition,
o r a-D-mannosidase
glucosidase
the
binding properties of
were s i m i l a r
with
endocytosis
and 1 m M P-mannose 6 - p h o s p h a t e
m a r k e d l y i n h i b i t e d t h e i r u p t a k e ( 8 0 t o 100%). i n h i b i t i o n by
the
B-Q-2-
m o l e c u l e s f r o m D.
p r e p a r a t i o n s were competent i n i n v i t r o c l e a r a n c e .
discoideum
Furthermore,
the
t h r e e p u r i f i e d a c i d h y d r o l a s e s c o n t a i n 5 t o 7 m o l o f !-mannose
6-
p h o s p h a t e p e r m o l o f enzyme.
This indicates that,
mammalian enzyme p r e p a r a t i o n s ,
u n l i k e many
most i f n o t a l l o f t h e s e enzyme
d i s c o i d e u m c o n t a i n t h e phosphomannosyl r e c o g n i t i o n
m o l e c u l e s f r o m D. marker. Several activities
mollusc
towards
glycosidases natural
have
been
substrates.
for
their
a-I=-Fucosidases
studied
from
Chamelea g a l l i n a , T a p e s r h o m b o i d e u m , a n d M y t i l u s e d u l i s h y d r o l y s e oligosaccharides (di-, and 1
+
4 bonds,
tri-,and
penta-saccharides)
I-fucose-containing
with 1
+
2,
1
+
3,
glycopeptides from bovine
t h y r o g l o b u l i n , and p o r c i n e s u b m a n d i b u l a r m u c i n ( d e v o i d o f s i a l i c acid).
a-I=-Fucosidase
f r o m L i t t o r i n a l i t t o r e a hydrolyses fucose-
containing glycopeptides Glucuronidase chondroitin
from
L.
4-sulphate,
from
littorea and
bovine thyroglobulin.70 hydrolyses
heparin
with
a
B-a-
hyaluronic acid, very
low
activity.
Carbohydrate Chemistry
376
However, i t i s much more a c t i v e on o l i g o s a c c h a r i d e s (from t h e abovementioned macromolecules) c o n t a i n i n g non-reducing t e r m i n a l glucuronyl residues. B - Q - 2 - A c e t a m i d o - 2 - d e o x y h e x o s i d a s e from H e l i c e l l a e r i c e t o r u m a c t s mainly w i t h an endo-hydrolase a c t i v i t y on 2-acetamido-2-deoxy-B-~-hexopyranosyl-(l + 4 ) l i n k a g e s of ovalbumin, It ovomucoid, c h i t i n , h y a l u r o n i c acid, and c h o n d r o i t i n 4-sulphate. a l s o has a secondary =-hydrolase a c t i v i t y on these s u b s t r a t e s . The s u b s t r a t e s p e c i f i c i t y of d i p l o c o c c a l B - g - 2 - a c e t a m i d o - 2 deoxyhexosidase has been s t u d i e d i n d e t a i l by u s i n g o l i g o s a c c h a r i d e s o f known structure. The enzyme cannot c l e a v e g-GlcNAc-(1 + 4)-a-Man and E-GlcNAc-(l + 6)-Q-Man l i n k a g e s , although i t r e a d i l y h y d r o l y s e s P-GlcNAc-(l * 2)-P-Man, Q-GlcNAc-(l + 3)-Q-Gal, and P-GlcNAc-(l + 6 ) P-Gal linkages.71 The g-GlcNAc-( 1 + 2)-Q-Man l i n k a g e i n B-GlcNAc-Cl 4)-ig-GlcNAc-(l + 2)I-l-Man group i s cleaved b y t h e enzyme b u t t h e l i n k a g e i n !-GlcNAc-(l + 6 ) - i P - G l c N A c - ( l + 2))-Q-Man g r o u p i s n o t , p r o b a b l y b e c a u s e o f t h e s t e r i c e f f e c t of g-GlcNAc-(1 + 6)-Q-Man r e s i d u e on g-GlcNAc-(l + 2)-B-Man l i n k a g e . S i m i l a r s t e r i c e f f e c t i s a l s o observed i n t h e c a s e s o f v a r i o u s o t h e r o l i g o s a c c h a r i d e s . I t is s u g g e s t e d t h a t t h e s u b s t r a t e s p e c i f i c i t y of d i p l o c o c c a l B-Q-2a c e t a m i d o - 2 - d e o x y h e x o s i d a s e c a n be u s e d e f f e c t i v e l y f o r t h e s t r u c t u r a l s t u d i e s o f I - a s p a r a g i n e - l i n k e d sugar c h a i n s .
-+
3
a-l-Arabinofuranosidases
and a-&-Arabinopyranosidases
A c t i v e - s i t e - d i r e c t e d i r r e v e r s i b l e i n h i b i t i o n of g l y c o s i d a s e s by t h e corresponding glycosylmethyl-(4-nitrophenyl) t r i a z i n e s has been i n ~ e s t i g a t e d . ~B -~Q - G a l a c t o p y r a n o s y l m e t h y l - ( 4 - n i t r o p h e n y l ) t r i a z i n e i s an a c t i v e - s i t e - d i r e c t e d i r r e v e r s i b l e i n h i b i t o r (ASDIN) of t h e ebgo and t h e lacZ B-B-galactosidases of E s c h e r i c h i a c o l i , of t h e B - P - g a l a c t o s i d a s e o f human l i v e r l y s o s o m e s , a n d , l e s s e f f e c t i v e l y , of t h e a - g - g a l a c t o s i d a s e of green c o f f e e beans. I t has B-Qn o e f f e c t o n t h e J..c r e p r e s s o r o f El-cglA. Glucopyranosylmethyl-(4-nitrophenyl) t r i a z i n e i s an ASDIN of b o t h Bg - g a l a c t o s i d a s e a c t i v i t i e s of s w e e t - a l m o n d B-Q-glucosidase 8. I t has n o e f f e c t on y e a s t a - P - g l u c o s i d a s e , t h e l a c 2 8 - Q - g a l a c t o s i d a s e B-Qo f E. c o l i , o r g l u c o a m y l a s e from A s p e r g i l l u s n i g e r . X y l o p y r a n o s y l m e t h y l - ( 4 - n i t r o p h e n y l ) t r i a z i n e i s an ASDIN of t h e B-Qx y l o s i d a s e from P e n c i l l i u m wortmanni, b u t has no e f f e c t on t h e B-ax y l o s i d a s e of B a c i l l u s p u m i l u s , except by v i r t u e of i t s consumption of stabilizing dithiothreitol. a-&-Arabinofuranosylmethyl-(4n i t r o p h e n y l ) t r i a z i n e i s an ASDIN f o r t h e e x t r a c e l l u l a r a - & -
377
6: Enzymes
a r a b i n o f u r a n o s i d a s e o f M o n i l i n i a f r u c t i g e n a o f m o r e a l k a l i n e pH o p t i m u m a t pH 7. acidic
pHs
The o t h e r i s o e n z y m e i s i n e r t a t t h i s pH,
destroy
the
reagent.
It
i s
and more
concluded
that
g l y c o s y l m e t h y l ( a r y 1 ) t r i a z i n e s a r e ASDINs f o r g l y c o s i d a s e s f o r w h i c h b o t h s u b s t r a t e and p r o d u c t s t e r e o c h e m i s t r i e s a t t h e a n o m e r i c c e n t r e a r e t h e same a s t h a t o f t h e r e a g e n t , t h a t t h e y c a n a c t , w i t h l o w e r effectiveness,
on g l y c o s i d a s e s f o r w h i c h b o t h s u b s t r a t e and p r o d u c t
s t e r e o c h e m i s t r i e s a r e o p p o s i t e t o t h o s e o f t h e r e a g e n t s , and t h a t t h e y h a v e no e f f e c t on g l y c o s i d a s e s w i t h o p p o s i t e s t e r e o c h e m i s t r i e s o f s u b s t r a t e and p r o d u c t ,
o r p r o t e i n s o f no c a t a l y t i c f u n c t i o n .
The a - i - f u c o s i d a s e , mannosidase,
B-n-2-acetamido-2-deoxyglucosidase,
B-;-glucosidase,
B-P-xylosidase,
B-P-glucosidase,and
a-
a-
& - a r a b i n o f u r a n o s i d a s e a c t i v i t i e s o f n o r m a l and a t r o p h i c s k e l e t a l muscle o f
d e v e l o p i n g and
adult
r a t s have been c o l l e c t i v e l y
in~estigated.~~ Substrate specificity o f a-i-arabinofuranosidase
from plant
S c o p o l i a j a p o n i c a has been examined u s i n g t h r e e k i n d s o f L - a r a b i n o disaccharides prepared from n a t u r a l sources or ~ y n t h e t i c a l l y . ’ ~ T h i s enzyme h y d r o l y s e d a-L-arabinofuranosylarabinoses
which had
o r a (1 + 5 ) l i n k a g e , b u t h y d r o l y s e d a-6a r a b i n o p y r a n o s y l a r a b i n o s e h a v i n g a (1 + 5 ) l i n k a g e t o a l e s s e r a - i - A r a b i n o f u r a n o s i d a s e , w h i c h was s h o w n t o b e a n degree. (1 + 3)
either
a
enzyme,
degraded beet araban i n c o m p l e t e l y .
*-
galactose-containing o f araban,
fragments,
A r a b i n o s e o l i g o m e r s and
isolated following acid hydrolysis
w e r e b o t h i n c o m p l e t e l y d e g r a d e d by t h e enzyme.
The
r e a s o n s f o r t h e i n c o m p l e t e d e g r a d a t i o n w e r e e x p l a i n e d by t h e n o v e l finding
of
(1
2)
+
linkages
and
I-arabinopyranosides
and
the
i n c l u s i o n o f t r a c e amounts o f Q-galactose i n t o t h e carbohydrate c h a i n o f araban.
T h i s enzyme was p r a c t i c a l l y n o n - r e a c t i n g w i t h t h e
hydroxyprolyl-L-arabinose
linkage o f
glycopeptides from plant c e l l
walls. An a - L - a r a b i n o f u r a n o s i d a s e
sp.
No.
p r o d u c e d by
17-1 has been described.75
wild-type
Streptomyces
One h u n d r e d and f i f t y s t r a i n s o f
a c t i n o m y c e t e s were i s o l a t e d f r o m s o i l s on p l a t e c u l t u r e s c o n t a i n i n g b e e t a r a b i n a n as t h e s o l e c a r b o n s o u r c e .
About o n e - t h i r d o f t h e
c u l t u r e f l u i d s were f o u n d t o h a v e a - k - a r a b i n o f u r a n o s i d a s e A w i l d - t y p e s t r a i n , S t r e p t o m y c e s s p . No.
best
producer of
activity.
1 7 - 1 , was s e l e c t e d a s t h e
a-i-arabinofuranosidase.
The h i g h e s t e n z y m i c
a c t i v i t y was o b t a i n e d i n t h e c u l t u r e f l u i d when t h e i n i t i a l pH was a d j u s t e d t o 9.0. from
the
culture
An a - i - a r a b i n o f u r a n o s i d a s e f i l t r a t e of
No.
17-1
by
was h i g h l y p u r i f i e d combining
column
Carbohydrate Chemistry
378 chromatography
on D E A E - c e l l u l o s e ,
and i s o e l e c t r i c f o c u s i n g .
g e l f i l t r a t i o n on Sephadex G-100,
The m o l e c u l a r w e i g h t o f t h e p u r i f i e d
enzyme was e s t i m a t e d t o be a b o u t 92,000, was pH 4.4.
and i t s i s o e l e c t r i c
The e n z y m i c a c t i v i t y was m a x i m u m a t pH 6.0
c o m p l e t e l y i n h i b i t e d by Hg2+.
Em v a l u e
The a p p a r e n t
f o r 4 - n i t r o p h e n y l a-!=-arabinofuranoside
point
a n d was
o f t h e enzyme
was d e t e r m i n e d t o be 3.6mM.
4 B-p-Fructof'uranosidases A
c o n t i n u o u s p h o t o m e t r i c method has been d e s c r i b e d f o r t h e
d e t e r m i n a t i o n of
B-q-fructofuranosidase
The
of
a c t i v i t y
spectrophotometrically conversion
of
from the s m a l l intestine.76
B-e-fructofuranosidase by
the
B-Q-glucose
production (a
of
reaction
i s
determined
NADH
during
product
the
B-Q-
o f
f r u c t o f u r a n o s i d a s e ) by Q - g l u c o s e d e h y d r o g e n a s e t o Q - g l u c o n o - 1 , 4 lactone. A
s t u d y o f t h e d i s t r i b u t i o n o f some a d s o r b e d a n d i n t r i n s i c
enzymes b e t w e e n t h e m u c o s a l c e l l s o f t h e r a t s m a l l i n t e s t i n e and t h e apical glycocalyx
s e p a r a t e d f r o m them has i n c l u d e d w o r k
on 8-e-
f r u c t o f u r a n o s i d a s e .77 Solubilization phosphatase from been s t u d i e d papain,
of
6-Q-fructofuranosidase
guinea- p i g i n t e s t i n a l
using
various
procedures
sodium d o d e c y l s u l p h a t e , A l l
the
procedures
60% o f of
tried
activity
t h e a l k a l i n e phosphatase.
these
proteins
are
i n the
localization
of
as t r e a t m e n t s
were
quite
with
efficient
Under o p t i m u m c o n d i t i o n s ,
i n 65-
was s o l u b i l i z e d c o m p a r e d t o 30-
Differences i n the e x t r a c t a b i l i t y
presumably
t o p o l o g i c a l arrangements i n t h e
such
alkaline
membranes h a s
s o d i u m d e o x y c h o l a t e , a n d T r i t o n X-
s o l u b i l i z i n g t h e membrane p r o t e i n s . 90% B - E - f r u c t o f u r a n o s i d a s e
and
brush- border
due
t o
their
membrane m a t r i x .
differential
Such d i f f e r e n c e s
B-P-fructofuranosidase
and a l k a l i n e
p h o s p h a t a s e i n b r u s h b o r d e r s may e x p l a i n t h e o b s e r v e d d i s p a r i t i e s i n the
absorption r a t e o f
s u c r o s e and ! - g l u c o s e Crane,
R.K.
e-glucose
l-phosphate
(1961) Bio ch im.
r e l e a s e d by
the hydrolysis o f
i n the i n t e s t i n e { M i l l e r ,
Biophys.
Acta,
52,
0. and
281-2931.
The i m m o b i l i z a t i o n o f B - Q - f r u c t o f u r a n o s i d a s e o n k r i l l c h i t i n has been i n v e s t i g a t e d .
T h e o p t i m u m pH v a l u e f o r a d s o r p t i o n was
f o u n d t o b e pH 5 . 0 . ~ ~ The
separation
and p u r i f i c a t i o n o f
B-!-fructofuranosidase
an i n u l i n a s e f r o m g e r m i n a t i n g g a r l i c ( A l l i u m s a t i v u m L.)
and
b u l b s have
379
6: Enzymes b e e n d e s c r i b e d .80 o f 76,000,
B o t h t h e enzymes h a d a p p a r e n t m o l e c u l a r w e i g h t s
a s d e t e r m i n e d b y g e l c h r o m a t o g r a p h y o n S e p h a d e x G-200,
and were i n a c t i v a t e d by t r e a t m e n t w i t h 4-chloromercuribenzoate, a c i d ) , and h e a t t r e a t m e n t a t 55OC f o r 5
5,5’-dithiobis(2-nitrobenzoic m i n a t pH 7.
The B - e - f r u c t o f u r a n o s i d a s e
r a f f i n o s e b u t had no a c t i o n on i n u l i n . s u c r o s e was 26mM.
The i n u l i n a s e h y d r o l y s e d ,
s u c r o s e and r a f f i n o s e .
Em v a l u e s
The
l O m M and 25mM,respectively.
growth
(up t o
h y d r o l y s e d s u c r o s e and
The a p p a r e n t
6 days)
Em v a l u e
for
apart from inulin,
f o r i n u l i n and s u c r o s e were
While i n the e a r l y stages o f p l a n t
t h e i n u l i n a s e and B - g - f r u c t o f u r a n o s i d a s e
a c t i v i t i e s increased i n a p a r a l l e l fashion i n the bulbs.
A t later
s t a g e s t h e i n c r e a s e i n 8 - ~ - f r u c t o f u r a n o s i d a s e a c t i v i t y was more t h a n that o f inulase activity.
f 3 - ~ - F r u c t o f u r a n o s i d a s e si n sugar-cane l e a f sheaths have been They c o n s i s t o f t h r e e
f o u n d t o b e f i r m l y bound t o t h e c e l l
d i f f e r e n t enzymes d i s t i n g u i s h e d on t h e b a s i s o f o p t i m u m pH, t h e response t o i n h i b i t o r s .
This B-Q-fructofuranosidase
d i f f e r s f r o m t h e s i n g l e enzyme d e s c r i b e d f o r
Km, a n d
complex
b o t h i m m a t u r e and
mature s t a l k tissues. C y t o p l a s m i c a n d w a 1 1- b o u n d B - E - f r u c t o f u r a n o s i d a s e s h a v e b e e n p r e p a r e d f r o m t h e s e e d l i n g s o f s u g a r b e e t , and a d s o r p t i o n - r e l e a s e characteristics
of
the
enzymes
partially purified b e l o w 0.25 pH,
were
compared by
the
use
of
a
A d s o r p t i o n o f t h e bound enzyme o c c u r r e d
M i n N a C l c o n c e n t r a t i o n and was u n a f f e c t e d b y changes i n
w h i l e t h e c y t o p l a s m i c enzyme was a d s o r b e d o n l y o n a c i d i c m e d i a
o f pH 3-4.
R e l e a s e o f r e - a d s o r b e d enzymes w i t h N a C l depended on i t s
concentration,
and t h e c y t o p l a s m i c enzyme was l i b e r a t e d more r e a d i l y
t h a n t h e bound t y p e , NaC1,
respectively.
marked differences
g i v i n g t h e i r maximum v a l u e s a t 0.2 Thus,
the
two
M and 0.8
M
f 3 - ~ - f r u c t o f u r a n o s i d a s e s showed
i n a d s o r p t i o n t o and r e l e a s e f r o m t h e w a l l .
A d s o r p t i o n o f t h e c y t o p l a s m i c enzyme t o t h e w a l l was r e v e r s i b l e w i t h c h a n g e s i n pH o f
t h e medium.
bound f3-Q-fructofuranosidase
The r e s u l t s s u g g e s t e d t h a t t h e w a l l was e s s e n t i a l l y d i f f e r e n t f r o m t h e
c y t o p l a s m i c enzyme and c o n s e q u e n t l y n o t an a r t i f a c t . Optimum c o n d i t i o n s f o r B - Q - f r u c t o f u r a n o s i d a s e d e t e c t i o n i n p o l y a c r y l a m i d e and a g a r o s e g e l s h a v e b e e n d e f i n e d f r o m c o m p a r i s o n o f zymograms o b t a i n e d w i t h t w o s t a i n i n g m e t h o d s i n c l u d i n g an o r i g i n a l one.83
Under a l l c o n d i t i o n s t e s t e d i n t h i s s t u d y d e t e c t i o n h a s b e e n
i m p r o v e d w i t h t h e new s t a i n i n g p r o c e d u r e .
The new s t a i n i n g medium
d e v e l o p e d h e r e u s e s t w o i n t e r m e d i a r y enzymes, peroxidase,
3,3’-diaminobenzidine
Q - g l u c o s e o x i d a s e and
as f i n a l a c c e p t o r ,
and s u c r o s e as
Carbohydrate Chemistry
380 substrate for
B-Q-fructofuranosidase.
zymogram i s o b t a i n e d e i t h e r
by
A B-Q-fructofuranosidase
gel immersion i n t h i s staining
solution
o r , when h i g h l y c r o s s l i n k e d p o l y a c r y l a m i d e s e p a r a t i n g g e l s
a r e used,
by o v e r l a y i n g t h e s l a b w i t h i n a t h i n b u f f e r e d a g a r o s e g e l
c o n t a i n i n g s t a i n i n g chemicals (sandwich technique).
Examples a r e
p r e s e n t e d t o i l l u s t r a t e t h e g e n e r a l p r i n c i p l e s i n v o l v e d and i n d i c a t e t h e c o n d i t i o n s n e c e s s a r y f o r o p t i m a l d e v e l o p m e n t o f t h i s p r e c i s e and s p e c i f i c technique. A s o l u b l e f o r m of
B-8-fructofuranosidase
r a d i s h (Raphanus s a t i v u s ) seedlings.84
has been p u r i f i e d f r o m U s i n g Sephadex
G-25
c h r o m a t o g r a p h y , ammonium s u l p h a t e p r e c i p i t a t i o n , c o n c a n a v a l i n A Sepharose, U l t r o g e l A c A ~ ~ A, c A ~ ~ a, n d i m m u n o s o r p t i o n c h r o m a t o g r a p h i e s , t h i s e n z y m e was p u r i f i e d 7 0 0 t i m e s . Crossed immunoelectrophoresis
with a p o l y s p e c i f i c antiserum
showed an
e x t r e m e l y l o w l e v e l o f c o n t a m i n a n t s e v e n when a h i g h amount o f f i n a l p r o d u c t was t e s t e d . weight
of
48,500
hydrate moiety,
The p u r i f i e d enzyme h a s an a p p a r e n t m o l e c u l a r
and a t o t a l sugar c o n t e n t of
necessary f o r B-Q-fructofuranosidase
The c a r b o -
7.7%.
w i t h probably a poly-n-mannosidic
structure,
i n enzyme s t a b i l i t y o r i n p r o t e c t i o n a g a i n s t p r o t e o l y s i s .
B-e-fructofuranosidase
i s not
a c t i v i t y and h a s a l i m i t e d r o l e
solubility
i s
shown
to
However,
depend
on
the
carbohydrate moiety. Radish B-q-fructofuranosidase
has been shown t o become
i n a c t i v a t e d by d i l u t i n g t h e enzyme s o l u t i o n , and t h e a c t i v i t y c a n be r e s t o r e d by a d d i t i o n o f bovine serum albumin or o t h e r ~ r 0 t e i n s . O ~ The u s e o f d e t e r g e n t ,
high-molar
s a l t s o l u t i o n s , or s i l i c o n e - c o a t e d
t u b e s s h o w e d t h a t d e c r e a s e o f s p e c i f i c a c t i v i t y u p o n d i l u t i o n was n o t l i n k e d t o a d s o r p t i o n o f t h e enzyme on t o g l a s s w a l l s .
Albumin
n e i t h e r p r o t e c t e d t h e enzyme f r o m d e n a t u r a t i o n by h e a t n o r changed i t s s t a b i l i t y d u r i n g c o n s e r v a t i o n a t room t e m p e r a t u r e .
The a c t i o n
o f a d d e d p r o t e i n s was n o t d u e t o r e m o v a l o f a n i n h i b i t o r f r o m t h e enzyme s o l u t i o n s . e f f e c t as a l b u m i n ,
Some p o l y a n i o n s
o r p o l y c a t i o n s h a d t h e same
b u t d i a l y s i s o r c h r o m a t o g r a p h y showed t h a t t h e y
d i d n o t a c t by r e a s s o c i a t i o n o f i n a c t i v e p r o d u c t s f o r m e d d u r i n g d i l u t i o n o f t h e a c t i v e enzyme.
A molecular-weight
h e t e r o g e n e i t y was
o b s e r v e d i n t h e enzyme p o p u l a t i o n when c h r o m a t o g r a p h y was p e r t o r m e d w i t h o u t albumin. dilution,
It was s u g g e s t e d t h a t i n a c t i v e f o r m s ,
f o r m e d upon
d i f f e r e d s l i g h t l y i n t h e i r molecular conformation from the
a c t i v e forms obtained a t h i g h p r o t e i n concentration. Soluble B-g-fructofuranosidase
f r o m r a d i s h c o t y l e d o n s h a s been
s u b j e c t e d t o e l e c t r o f o c u s i n g and e l e c t r o p h o r e s i s . 8 6
The r e s u l t s
381
6: Enzymes s u g g e s t e d c o n s i d e r a b l e h e t e r o g e n e i t y o f t h e enzyme. A
from
serological
s t u d y has been made on a B - Q - f r u c t o f u r a n o s i d a s e
S p a c e l i a ~ o r g h i . ~A n~t i b o d i e s
f r u c t o f u r a n o s i d a s e f r o m S. B-e-fructofuranosidase
raised against
s o r g h i were c r o s s - r e a c t e d
from Claviceps purpurea.
6-Q-
a purified
with a purified
An i m m u n o d i f f u s i o n
t e s t i n d i c a t e d a h i g h degree o f s e r o l o g i c a l i d e n t i t y between the S.
s o r q h L a n d C.
p u r p u r e a enzymes.
confirmed t h i s result.
complement
A
fixation test
The s i m i l a r i t y i n t i t r e s i n d i c a t e d a v e r y
c l o s e s t r u c t u r a l s i m i l a r i t y b e t w e e n t h e enzymes. The c a r b o h y d r a t e s t r u c t u r e o f y e a s t B - Q - f r u c t o f u r a n o s i d a s e been i n v e s t i g a t e d . 8 8
B-Q-Fructofuranosidase,
c e l l s o f Saccharomyces c e r e v i s i a e X-2180
has
e x t r a c t e d from broken
mnn2 a-mannan m u t a n t ,
was
s e p a r a t e d i n t o a f r a c t i o n i n s o l u b l e i n 75% ammonium s u l p h a t e (P7S BE-fructofuranosidase,
36% c a r b o h y d r a t e ) and a s o l u b l e f r a c t i o n ( S 7 5
B-Q-fructofuranosidase, antibodies
specific for
53% c a r b o h y d r a t e ) . t h e (1
mannoprotein outer chain,
+
u n i t o f the
w h e r e a s t h e P75 B - P - f r u c t o f u r a n o s i d a s e
f a i l e d t o react w i t h t h i s antiserum, serum a g a i n s t t e r m i n a l (1
although i t d i d react w i t h
3)-linked-a-Q-mannose
-*
c h a r a c t e r i s t i c mannoprotein core. mannanase removed t h e
The l a t t e r r e a c t e d w i t h
6)-linked-a-Q-mannose
outer
units that are
A b a c t e r i a l endo-(1
chains
from
+
6)-a-Q-
t h e S75 i n v e r t a s e
and
c o n v e r t e d i t t o a f o r m t h a t was s i m i l a r i n e l e c t r o p h o r e t i c a n d i m m u n o c h e m i c a l p r o p e r t i e s t o t h e P75 B - Q - a c e t a m i d o - 2 deoxyglucosidase,
whereas t h e &-g-mannanase
-
t h e 1a t t e r B -p - a c e t a m ido 2 - d e o x y g 1u c o s id a se.
had l i t t l e e f f e c t on The r e s u 1t s s u g g e s t
t h a t t h e P75 i n v e r t a s e i s a f o r m o f t h e enzyme t o w h i c h o n l y t h e c o r e o l i g o s a c c h a r i d e u n i t s had been added,
acetamido-2-deoxyglucosidase the
polysaccharide outer
anomeric
n.m.r.
'H
a n d t h e S75
6-Q-
r e p r e s e n t s an enzyme f r a c t i o n t o w h i c h chains
signal for
were
also
attached.
u n s u b s t i t u t e d (1
+
A
strong
6l-linked-a-Q-
mannose i n t h e S75 B - Q - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e
a n d a much
r e d u c e d s i g n a l i n t h e P75 B-~-acetamido-2-deoxyglucosidase and endoQ - m a n n a n a s e - d i g e s t e d S75 B - ~ - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e these
conclusions.
endo-B-~-Acetamido-2-deoxyglucosidase
support
digestion
o f t h e S75 and P75 B - ~ - 2 - d e o x y g l u c o s i d a s e , as w e l l as o f a p u r i f i e d wild-type apparently
yeast
B-Q-acetamido-2-deoxyglucosidase,
identical
proteins that
series
of
3
to
4
produced
an
carbohydrate-containing
were s e p a r a b l e by p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s
i n sodium d o d e c y l s u l p h a t e b u t t h a t m i g r a t e d as a s i n g l e band on
i s o e l e c t r i c focusing.
The bands r a n g e d f r o m a b o u t 63,000
t o 69,000
a n d d i f f e r e d by t h e s i z e o f one o r m o r e c a r b o h y d r a t e c o r e u n i t s e a c h
382
Carbohydrate Chemistry
o f 1 5 Q-mannose and 1 B - B - a c e t a m i d o - 2 - d e o x y g l u c o p y r a n o s e
units.
The
r e s u l t s s u g g e s t e d t h a t t h e e x t e r n a l B-Q-acetamido-2-deoxyglucosidase m o l e c u l e s c o n t a i n some c o r e u n i t s w i t h o u t a t t a c h e d o u t e r c h a i n s and t h a t t h e c e l l s c o n t a i n e d a p r e c u r s o r f o r m o f t h e enzyme t o w h i c h o n l y t h e c o r e u n i t s have been added. ‘H
n.m.r.
I n support o f t h i s conclusion,
s p e c t r a and c h r o m a t o g r a p h i c p a t t e r n s showed t h a t t h e c o r e
fragments
from
the
P75,
S75,
and
wild-type
B-Q-acetamido-2-
d e o x yglucosidase were e s s e n t i a l l y i d e n t i c a l . 2-Deoxy-Q-glucopyranose isolation
of
glucosidase
mutants
plate
derepressed yields
been used
By
the
use
selective
B-Q-
enhanced
2-deoxy-Q-glucose,
a
and
hyperproductive
with
respect
B-Q-
to
and a - a m y l a s e have been o b t a i n e d i n v e r y h i g h
from
the
yeast
Saccharomyces
kononenkoae,respectively.
a p p l i e d t o T.
of
i n the
with
c a t a b o l i c r e p r e s s o r o f c e r t a i n y e a s t enzymes,
fructofuranosidase,
Lipornyces
has
Trichoderma r e e s i
production.89
non-metabolizable mutants
of
cerevisiae
and
When 2 - d e o x y - Q - g l u c o s e
was
r e e s a i QM 9414 i n a s i m i l a r
way, i t p e r m i t t e d t h e
s e l e c t i v e s o l u t i o n i n very h i g h p l a t e y i e l d s o f mutants derepressed and h y p e r p r o d u c t i v e w i t h r e s p e c t t o B - B - g l y c o s i d a s e . S t r e p t o c o c c u s s a l i v a r i u s s t r a i n 5 1 has been f o u n d t o p r o d u c e an extracellular
B-e-fructofuranosidase
containing -tetraose, inulin,
The enzyme h y d r o l y s e s l e v a n , -pentaose,
inulotriose,
S. s a l i v arius linkage. altered
when g r o w n i n a l e v a n -
strain
and -hexaose, and 51
inuloheptaose.
levan i s
and i n u l o - b i o s e ,
b u t has n e g l i g i b l e e f f e c t s on through
The a
branching
single
(2
i n
1l-B-R
+
E v i d e n c e has been p r e s e n t e d f o r t h e s e l e c t i v e b r e a k d o w n o f tomato
B-Q-fructofuranosidase
molecules
by
a
neutral
protease from B a c i l l u s s ~ b t i l i s . ~ ~ The r e c o v e r y
m e t h o d a n d some i n d u s t r i a l p r o p e r t i e s o f n o n -
s p e c i f i c B-Q-fructofuranosidase been d e s c r i b e d . 9 2
f r o m K l u y v e r o m y s e s f r a g i l i s have
The y e a s t was g r o w n i n c o n t i n u o u s c u l t u r e o n a
c o m p l e x medium c o n t a i n i n g s u c r o s e as t h e c a r b o n s o u r c e a n d l i m i t i n g nutrient. biomass,
The
inulinase
y i e l d was
7,000 p g h e x o s e m i n e ’
d e t e r m i n e d w i t h 4% s u c r o s e a t 5 O o C a n d pH 5.0.
mg”
Maximum
amount o f enzyme was f o r m e d a t t h e l o w e s t d i l u t i o n r a t e (0.09 h - l ) tested.
Any i n c r e a s e i n d i l u t i o n r a t e c a u s e d a s e v e r e r e d u c t i o n i n
a c t i v i t y p e r u n i t b i o m a s s due t o c a r b o n c a t a b o l i t e r e p r e s s i o n . F r u c t o f u r a n o s i d a s e y i e l d s w e r e c o n s t a n t i n t h e pH r a n g e 3.5 and i n t h e d i s s o l v e d o x y g e n t e n s i o n r a n g e 2.5
t o 40% o f
B-Qt o 6.0
saturation.
C o n t i n u o u s c u l t i v a t i o n on s u c r o s e p r o d u c e d t w i c e as much i n u l a s e as t h e p r e v i o u s l y used method o f b a t c h c u l t i v a t i o n on t h e expensive
383
6: Enzymes substrate inulin.
A high-quality
a u t o l y s i s o f the yeast c e l l s , acetone p r e c i p i t a t i o n .
i n d u s t r i a l g r a d e was p r e p a r e d by
u l t r a f i l t r a t i o n o f t h e supernatant,and
Under
t h e assay c o n d i t i o n s
used the
p r e p a r a t i o n showed an a c t i v i t y r a t i o t o w a r d s u c r o s e and i n u l i n o f 10.5
a s c o m p a r e d t o 1,600
f o r baker’s yeast
invertase.
Further
c o m p a r i s o n w i t h i n v e r t a s e showed t h a t i t i s a t l e a s t as r e s i s t a n t t o substrate
inhibition,
transferase
as
activity.
t h e r m o s t a b l e , and
These r e s u l t s
fructofuranosidase
may
suggest
represent
has
that
an
slightly K.
less
B-p-
fragilis
a l t e r n a t i v e
t o
Saccharomyces c e r e v i s i a e i n v e r t a s e p r e s e n t l y used i n i n d u s t r y .
5
Q-Fructose-1,6-Bisphosphatase
P-Fructose
2,6-bisphosphate,
a known p o w e r f u l s t i m u l a n t o f
phosphofructokinase
(Van S c h a f t i n g e n ,
(1980)
122,
E L o c h e m J.,
897),
micromolar concentrations,
liver
(i) i t i s much s t r o n g e r
concentration,
at
Other,
L.,
H.G.
inhibit,
at
and muscle Q-fructose-1,6-
b i s p h o ~ p h a t a s e . ~The ~ main c h a r a c t e r i s t i c s that
Hue,
E.,
has been found t o
low
of
t h i s i n h i b i t i o n are
than a t
high substrate
(ii) i t changes t h e s u b s t r a t e s a t u r a t i o n c u r v e f r o m
a l m o s t h y p e r b o l i c t o s i g m o i d a l , and (iiii) t i s synergistic w i t h the i n h i b i t i o n b y AMP.
T h i s i n h i b i t i o n may p l a y a n i m p o r t a n t r o l e i n
t h e s t i m u l a t i o n o f g l u c o n e o g e n e s i s by g l u c a g o n , i s
known
t o
decrease
the
b e c a u s e t h i s hormone
concentration of
P-fructose-2,6-
bisphosphatase i n t h e l i v e r . The c o u p l i n g b e t w e e n Q - f r u c t o s e - 1 , 6 - b i s p h o s p h a t a s e inositol
synthetase
has
and
b i o c h e m i c a l and g e n e t i c s t u d i e s w i t h ! y o - i n o s i t o l - d e f i c i e n t a
model
i s
proposed
by
~IE-
b e e n i n ~ e s t i g a t e d . ~B ~a s e d m o s t l y the
authors
which
suggests
on
systems, a
coupled
o p e r a t i o n o f t h e g l u c o n e o g e n i c enzyme P - f r u c t o s e - 1 , 6 - b i s p h o s p h a t a s e and m y o - i n o s i t o l s y n t h e t a s e , as t h e p r o b a b l e r e g u l a t o r y e v e n t f o r c e l l s u r v i v a l under c o n d i t i o n s o f m y o - i n o s i t o l d e f i c i e n c y . A B a c i l l u s s u b t i l i s m u t a t i o n , p r o d u c i n g a d e f i c i e n c y o f Qfructose-1,6-bisphosphatase purified.95
has
been
isolated
and
The m u t a n t d i d n o t p r o d u c e c r o s s - r e a c t i n g
genetically material.
It
g r e w on any c a r b o n s o u r c e t h a t a l l o w e d g r o w t h o f t h e s t a n d a r d s t r a i n e x c e p t m y o - i n o s i t o l and p - g l u c o n a t e . on Q - f r u c t o s e ,
glycerol,
o r I,-malate
Because t h e m u t a n t c o u l d grow as t h e s o l e c a r b o n s o u r c e ,
B. s u b t i l i s c a n p r o d u c e Q - f r u c t o s e 6 - p h o s p h a t e and t h e d e r i v e d c e l l w a l l precursors from
t h e s e c a r b o n s o u r c e s i n t h e a b s e n c e o f Q-
Carbohydrate Chemistry
3 84
fructose-1,6-bisphosphatase.
I n o t h e r words,
d u r i n g gluconeogenesis
8. s u b t i l i s m u s t be a b l e t o b y p a s s t h i s r e a c t i o n . Rat
Q-fructose-1,6-bisphosphatase
l i v e r
gluconeogenic e-fructose-bisphosphatases
and t h r e e o t h e r
have
been
tested
as
s u b s t r a t e s f o r t h e c a t a l y t i c s u b u n i t o f c y c l i c AMP-dependent p r o t e i n kinase.96
I n
preparations
contrast of
mouse
to
the
liver,
rat
liver
rabbit
enzyme,
liver,
and
homogeneous pig
could not
be p h o s p h o r y l a t e d
Comparative
dodecyl
sulphate-polyacrylamide
sodium
electrophoresis of
by
kidney
fructose-bisphosphatase
the
g-
kinase. gel
g-fructose-bisphosphatases
t h e above f o u r
revealed t h a t the subunit molecular weight o f the isolated r a t l i v e r e n z y m e (E. 4 0 , 0 0 0 - 4 2 , 0 0 0 ) rabbit liver, 37,000).
was g r e a t e r t h a n t h a t o f
mouse l i v e r ,
(E. 36,000-
and p i g k i d n e y D - f r u c t o s e - b i s p h o s p h a t a s e s
Treatment
o f
3FP-labelled
r a t
liver
Q-fructose-
bisphosphatase w i t h t r y p s i n r e s u l t e d i n the conversion o f the r a t l i v e r enzyme t o an a c t i v e s p e c i e s w i t h a s u b u n i t m o l e c u l a r w e i g h t i d e n t i c a l t o t h a t o f t h e t h r e e o t h e r enzymes, the 32P-labelled
site.
fructose-bisphosphatase
w i t h complete loss o f
I d e n t i c a l t r y p s i n t r e a t m e n t o f p i g k i d n e y Qc a u s e d no change i n t h e m o l e c u l a r w e i g h t o f
t h e enzyme.
6
6-a-
and a-C-Fucosidases
S p e c i f i c i t y p a t t e r n s o f d i f f e r e n t t y p e s o f human f u c o s i d a s e have been i n v e s t i g a t e d t h r o u g h h y d r o l y s i s o f f u c o s i d e s , and
arabinosides
comparative
study
by
different
of
the
types
splitting
of
of
galactosides,
f u c o ~ i d a s e . ~ ’ The
a-k-fucoside
and
B-Q-
a r a b i n o s i d e w i t h a s i m i l a r s t r u c t u r e o f Cl-C4 o f t h e p y r a n o s e r i n g and t h e p r e p a r a t i o n o f t h e enzyme by a f f i n i t y c h r o m a t o g r a p h y showed that
both substrates
fucosidase. fucoside,
were
h y d r o l y s e d by
the
same e n z y m e ,
a-i-
The a n a l o g o u s i n v e s t i g a t i o n o f t h e h y d r o l y s i s o f B - i B-Q-galactoside,
s t r u c t u r e a t Cl-C4 of split
and
a-i-arabinoside
with
a
similar
the pyranose r i n g demonstrated t h a t these
glycosides
were
a c t i v i t y of
w h i c h was d e c r e a s e d d r a m a t i c a l l y i n G M 1 - g a n g l i o s i d o s i s .
by
the
same
I t i s suggested t h a t the s p e c i f i c
enzyme, action o f
B-Q-fucosidase, different
the
types of
f u c o s i d a s e i s d u e t o r e c o g n i t i o n by t h e s e e n z y m e s o f C l - C 4 o f t h e pyranose r i n g i n t h e corresponding substrates. d i f f e r e n t i a l diagnosis o f
The p r o b l e m s o f
some g l y c o s i d o s e s ( f u c o s i d o s i s ,
GMl-
g a n g l i o s i d o s i s , and Faby d i s e a s e ) a r e d i s c u s s e d on t h e b a s i s o f t h e
6: Enzymes data obtained. I t has been d e m o n s t r a t e d t h a t 6-a-fucosidase, digestive
juice
of
Achatina balteata,
was
i s o l a t e d from the
markedly
p r e i n c u b a t i o n w i t h t h r e e d i f f e r e n t probes,
i n a c t i v a t e d by
specific for
I-tyrosine
An e f f e c t i v e
r e s i d u e s u n d e r t h e e x p e r i m e n t a l c o n d i t i o n s used.98
p r o t e c t i o n a g a i n s t i n a c t i v a t i o n o f t h e e n z y m e was o b t a i n e d i n t h e presence o f a s u b s t r a t e analogue.
These d a t a s t r o n g l y s u g g e s t e d
t h a t B - Q - f u c o s i d a s e possesses e s s e n t i a l I - t y r o s i n e r e s i d u e s . B-Q-Fucosidases
isolated
from
the
digestive
juice
fucosides, B-Q-glucosides,and
B-q-galactosides,
of
B-9-
A c h a t i n a b a l t e a t a h a v e been shown t o c a t a l y s e t h e h y d r o l y s i s o f
and t h e c a t a l y t i c
e f f i c i e n c y i s m a x i m u m t o w a r d s B - p - f u c o s i d e ~ . ~ The ~ results of mixed-substrate
i n c u b a t i o n s t u d i e s and i n h i b i t i o n by g l y c o p y r a n o s e s
indicate
there
that
substrates
are
i s
at
least
hydrolysed.
I n
one the
site
reciprocal plots exhibit a significant s u b s t r a t e analogue i s present, These
results
are
consistent
at
absence
which of
a l l
tested
inhibitor,
the
downward curvature.
If a
t h e p l o t s c a n be s t r a i g h t l i n e s . with
the
presence
molecule o f a t l e a s t two d i s t i n c t s i t e s for
on t h e
enzyme
the s u b s t r a t e molecules,
one b e i n g an a c t i v e s i t e and t h e o t h e r b e i n g e i t h e r a s e c o n d a c t i v e s i t e with d i f f e r e n t k i n e t i c parameters or a modifier s i t e .
Also,
d a t a a r e shown t o f i t q u i t e w e l l w i t h t h e mechanism p r o p o s e d f o r a mnemonic enzyme. Crude
liver
vertebrate
a-I=-fucosidase
species
ranging
has been i n v e s t i g a t e d i n 10
from
fish
to
human
using
m e t h y l u m b e l l i f e r y l a - i - f u c o p y r a n o s i d e as substrate.100 fucosidases
w e r e c h a r a c t e r i z e d by
isoelectric-focusing profiles, p r e i n c u b a t i o n a t 4 5 O C and 6OoC, human l i v e r a - L - f u c o s i d a s e l i v e r a-I=-fucosidases
determinations
Em v a l u e s ,
4-
The a - & -
o f pH o p t i m a and
thermostabilities after
and i m m u n o p r e c i p i t a t i o n by a n t i -
antibody.
R e s u l t s suggest d i f f e r e n c e s i n
w i t h r e s p e c t t o pH o p t i m a ,
and i s o e l e c t r i c - f o c u s i n g p r o f i l e s .
However,
thermostabilities,
the
5,
values were
l a r g e l y s i m i l a r , a n d 9 t o 10 s p e c i e s w e r e p r e c i p i t a t e d by t h e human
IgG p r e p a r a t i o n , s u g g e s t i n g a n t i g e n i c s i m i l a r i t y . I n a f f i n i t y chromatography o f a-l-fucosidase liver
on
6-amino
hexanoyl
derivative
f u c o p y r a n o s e c o u p l e d t o Sepharose,
of
f r o m t h e human
2-amino-2-deoxy-B-L-
t h e e l u t i o n p r o f i l e o f t h e enzyme
h a s been c h a r a c t e r i z e d by t w o c o m p o n e n t s , a r b i t r a r i l y d e n o t e d as aC-fucosidases
A and B.lol
One o f t h e c o m p o n e n t s ( a - L - f u c o s i d a s e
A),
i n t h e a b s e n c e o f s o d i u m a z i d e i n t h e b u f f e r m i x t u r e s u s e d , was r e t a i n e d on t h e a f f i n i t y s o r b e n t , w h i l e t h e o t h e r ( a - L - f u c o s i d a s e B)
386
Carbohydrate Chemistry
was adsorbed under t h e same c o n d i t i o n s . T h i s l a t t e r component was e l u t e d by a s o l u t i o n c o n t a i n i n g t h e enzyme i n h i b i t o r L-fucose. On t h e b a s i s of rechromatography d a t a , i t i s s u g g e s t e d t h a t t h e r e i s an e q u i l i b r i u m i n s o l u t i o n between a - i - f u c o s i d a s e s A and B, d i f f e r i n g i n t h e i r a f f i n i t i e s for the a f f i n i t y sorbent. P u r i f i c a t i o n a n d p r o p e r t i e s o f t w o f o r m s o f human a - L f u c o s i d a s e have been d e s c r i b e d . l o 2 High- (100,000) and low- ( 5 0 , 0 0 0 ) m o l e c u l a r - w e i g h t forms of a - i - f u c o s i d a s e ( a - l - f u c o s i d a s e s I and 11) have been p u r i f i e d t o a p p a r e n t homogeneity from human s p l e e n , l i v e r , b r a i n , and k i d n e y on t h e b a s i s of d i f f e r e n t i a l a f f i n i t y f o r 6-amino hexanoyl d e r i v a t i v e of 2 - a m i n o - 2 - d e o x y - ~ - f u c o s e coupled t o a g a r o s e b e a d c o l u m n s . a - g - F u c o s i d a s e I ( t h e bound f o r m ) c o n s i s t e d of t w o 5 0 , 0 0 0 m o l e c u l a r - w e i g h t monomers. However, both forms could a g g r e g a t e t o t e t r a m e r and hexamer forms. The bound (100,000) form was shown t o be a s i a l o g l y c o p r o t e i n , w h e r e a s t h e unbound ( 5 0 , 0 0 0 ) form was a n e u t r a l p-mannose-rich g l y c o p r o t e i n . Other d i f f e r e n c e s w i t h r e s p e c t t o amino a c i d c o m p o s i t i o n , pH optimum, e l e c t r o p h o r e t i c m o b i l i t y , I&, t h e r m a l s t a b i l i t y , and n a t u r a l s u b s t r a t e s p e c i f i c a t i o n were observed. The c o n d i t i o n s f o r m a x i m a l a c t i v i t y (pH, b u f f e r , s a t u r a t i n g s u b s t r a t e c o n c e n t r a t i o n , r a n g e of l i n e a r r e l a t i o n s h i p s b e t w e e n enzyme a c t i v i t y v e r s u s i n c u b a t i o n t i m e and v e r s u s enzyme c o n c e n t r a t i o n ) i n t h e f l u o r i m e t r i c a s s a y of s e v e r a l g l y c o h y d r o l a s e s o f l y s o s o m a l o r i g i n i n human p l a s m a a n d s e r u m h a v e b e e n e ~ t a b l i s h e d . ~The ~ f o l l o w i n g enzymes were s t u d i e d : a-Qgalactosidase, B-p-galactosidase, B-Q-glucuronidase, a-Qm a n n o s i d a s e , a - I = - f u c o s i d a s e . See i n i t i a l c i t a t i o n of r e f . 2 6 f o r further details. A l t e r a t i o n s i n t h e f u n c t i o n s and s t r u c t u r e o f a - & - f u c o s i d a s e h a v e been d e s c r i b e d i n c y s t i c f i b r o s i s . l o 3 I n t h i s c o m m u n i c a t i o n d a t a a r e p r e s e n t e d which a r e r e l e v a n t t o o t h e r r e c e n t l y p u b l i s h e d r e p o r t s on a - i - f u c o s i d a s e a c t i v i t y i n c y s t i c - f i b r o s i s plasma ( H o s l i , P. and Vogt, E. (1979) Lancet i i , 543) and serum ( A l h a d e f f , J.A. and W a t k i n s , P. ( 1 9 7 9 ) C l i n . C h i m . A c t a , I.., 1 3 1 ) . H o s l i and Vogt ( 1 9 7 9 ) r e p o r t e d an i n c r e a s e d t h e r m o l a b i l i t y o f s e v e r a l a c i d h y d r o l a s e s i n c y s t i c - f i b r o s i s p l a s m a when c o m p a r e d t o n o n - c y s t i c f i b r o s i s plasma, and they p r e d i c t e d t h a t a - l - f u c o s i d a s e from c y s t i c I n the f i b r o s i s plasma would a l s o have an i n c r e a s e d t h e r m o l a b i l i t y . o t h e r r e p o r t , A l h a d e f f and W a t k i n s w e r e u n a b l e t o d e m o n s t r a t e a d e c r e a s e d a c t i v i t y of a - & - f u c o s i d a s e i n c y s t i c - f i b r o s i s serum. In a d d i t i o n , u s i n g c o n d i t i o n s w h i c h d i f f e r e d f r o m t h o s e o f H o s l i and
387
6: Enzymes Vogt,
t h e y w e r e u n a b l e t o d e m o n s t r a t e an i n c r e a s e d t h e r m o l a b i l i t y o f
t h e enzyme.
The d a t a r e p o r t e d h e r e d e m o n s t r a t e t h a t a - l - f u c o s i d a s e
i n cystic-fibrosis
p l a s m a does n o t h a v e an i n c r e a s e d l a b i l i t y u n d e r
t h e m i l d t h e r m a l c o n d i t i o n s o f H o s l i and Vogt.
Furthermore,
it i s
e m p h a s i z e d t h a t a g e - m a t c h e d c o n t r o l s m u s t be u s e d t o d e m o n s t r a t e t h e decreased a c t i v i t y o f a-&-fucosidase
i n c y s t i c - f i b r o s i s serum o r
plasma.
I t has been f o u n d t h a t
the activity
o f a-&-fucosidase
was
r e d u c e d 2-3 f o l d i n c u l t u r e d l y m p h o b l a s t s d e r i v e d f r o m p a t i e n t s w i t h
I t was s u g g e s t e d t h a t t h i s
c y s t i c f i b r o s i s r e l a t i v e t o normals.lo4
change may be i n v o l v e d i n t h e a l t e r a t i o n s i n t h e I - f u c o s e c o n t e n t o f some
of
the
abnormal glycoproteins
which
contribute t o
the
r e s p i r a t o r y and g l a n d u l a r o b s t r u c t i o n s i n t h e d i s e a s e . The
l y s o s o m a l enzyme a - k - f u c o s i d a s e
h a s been e x a m i n e d by t h i n
l a y e r g e l and c o l u m n i s o e l e c t r i c f o c u s i n g i n s k i n f i b r o b l a s t s and A l l three
l i v e r f r o m p a t i e n t s w i t h c y s t i c f i b r o s i s and c o n t r o l s . 1 0 5
common p h e n o t y p e s o f t h e e n z y m e w e r e o b s e r v e d i n b o t h c o n t r o l a n d cystic-fibrosis
fibroblasts.
When i n d i v i d u a l s o f t h e same a - i -
f u c o s i d a s e p h e n o t y p e were compared, no m a j o r d i f f e r e n c e s b e t w e e n t h e isoenzyme p r o f i l e s o f c y s t i c - f i b r o s i s Human a - i - f u c o s i d a s e
i n t h e n o r m a l and f u c o s i d o s i s s t a t e s h a s
b e e n p u r i f i e d and s t u d i e d . a-k-fucosidase.lo6
p a t i e n t s and c o n t r o l s were
or l i v e r tissue.
detected i n either fibroblasts
An a n t i s e r u m h a s been r a i s e d t o p u r i f i e d
Levels of
with serum o f two
cross-reaction
u n r e l a t e d f u c o s i d o s i s p a t i e n t s and n o r m a l i n d i v i d u a l s w i t h l o w a c t i v i t y a r e c o n s i s t e n t w i t h t h e presence o f very low amounts o f n o r m a l enzyme.
S i m i l a r l y no c r o s s - r e a c t i n g m a t e r i a l c o u l d be f o u n d
I t was deduced
i n cultured f i b r o b l a s t s from fucosidosis patients. t h a t i n t h e s e cases t h e r e i s no p r o d u c t i o n o f q u a n t i t i e s comparable t o normal l e v e l s . i n t e r r e l a t i o n s o f a-l-fucosidases Thermostability preincubation
studies
temperatures
liver
enzyme
focusing,
which
were
crude a d u l t
neuraminidase-treated curves
were
fucosidase.
found
have
(II-VII)
enzyme.lo7 the
performed
at
different
o n human a - & - f u c o s i d a s e s ,
t h e i s o e l e c t r i c forms o f p u r i f i e d
and f o e t a l
(VIII)
been
separated
by
liver
preparative supernatant
isoelectric enzyme,
and
Very d i f f e r e n t t h e r m o s t a b i l i t y
various
The m o s t n e u t r a l f o r m
t h e most a c i d i c form forms
for
Some o b s e r v a t i o n s o n t h e
I and I 1 a r e r e p o r t e d .
(37-65OC)
p u r i f i e d s e r u m and l i v e r enzyme,
m u t a n t enzyme i n
isoelectric
forms
of
a-&-
(I) was l e a s t t h e r m o s t a b l e a n d
most t h e r m o s t a b l e ,
with the intervening
having intermediate thermostabilities.
For
the
388
Carbohydrate Chemistry
d i f f e r e n t i s o e l e c t r i c forms o f l i v e r a-&-fucosidase
t h e r e appears t o
be a s i g n i f i c a n t t r e n d o f i n c r e a s i n g t h e r m o s t a b i l i t y w i t h i n c r e a s i n g a c i d i t y (and, p r e s u m a b l y , i n c r e a s i n g amount o f s i a l i c a c i d ) . purified sialic
acid-rich
serum
enzyme
thermostable than the p u r i f i e d l i v e r
was
The
considerably
enzyme.
more
The f o e t a l l i v e r
enzyme ( w h i c h i s l e s s a c i d i c and may c o n t a i n l e s s s i a l i c a c i d t h a n t h e a d u l t l i v e r enzyme) was l e s s t h e r m o s t a b l e t h a n a d u l t l i v e r a-&-
I n c o n t r a s t , n e u r a m i n i d a s e t r e a t m e n t o f human l i v e r a-
fucosidase.
I-fucosidase
did not
change i t s t h e r m o s t a b i l i t y ,
e v e n when
c o n s i d e r a b l e d e s i a l y l a t i o n h a d o c c u r r e d as m o n i t o r e d by i s o e l e c t r i c focusing.
Several
possible
of
interpretations
the
data
were
presented. The e f f e c t
o f t u n i c a m y c i n and c y c l o h e x i m i d e on t h e s e c r e t i o n o f
acid hydrolases investigated.61
from
1-cell
I-Cell
cultured
cultured
fibroblasts
fibroblasts
amounts o f B-~-2-acetamido-2-deoxyhexosidase
has
been
secrete excessive
and a - k - f u c o s i d a s e
t h e c u l t u r e m e d i a as c o m p a r e d w i t h n o r m a l f i b r o b l a s t s .
into
For further
d e t a i l s see i n i t i a l c i t a t i o n o f r e f . 6 1 . S t u d i e s on human a - & - f u c o s i d a s e and l o w - a c t i v i t y
sera.
h a v e been p e r f o r m e d on n o r m a l -
A f t e r DEAE-Sephacel c h r o m a t o g r a p h y ,
two
m a j o r f o r m s o f enzyme h a v e been c h a r a c t e r i z e d i n b o t h t y p e s o f s e r a . These
enzymic
thermostability
forms
were
different
with
respect
and e l e c t r o f o c u s i n g b e h a v i o u r . l o 8
f o r m (I) was t h e r m o l a b i l e . was t h e r m o s t a b l e .
The
t o
their
less acidic
I n c o n t r a s t , t h e m o r e a c i d i c o n e (11)
B u t t h e f o r m s I and I 1 h a d r e s p e c t i v e l y s i m i l a r
p r o p e r t i e s when i s o l a t e d f r o m e i t h e r
normal- or
low-activity
sera.
These r e s u l t s s u g g e s t t h e p r e s e n c e i n b o t h t y p e s o f s e r a and t w o major forms o f a-l-fucosidase
but i n variable proportions.
The l o w -
a c t i v i t y sera contained a major proportion o f the thermolabile less a c i d i c f o r m when c o m p a r e d w i t h n o r m a l - a c t i v i t y s e r a . The s t e r i c f a c t o r s i n v o l v e d i n t h e a c t i o n o f g l y c o s i d a s e s a n d P-galactose
o x i d a s e h a v e been i n v e s t i g a t e d . a-(1
B - Q - g a l a c t o s i d a s e , and ; - g a l a c t o s e
+
2)-L-Fucosidase,
o x i d a s e a r e s t e r i c a l l y h i n d e r e d by
c e r t a i n t y p e s o f b r a n c h i n g i n t h e o l i g o s a c c h a r i d e c h a i n s . l o g B-QGalactosidase
w i l l
not
cleave
the g-galactosyl
p e n u l t i m a t e sugar
carries
a sialic
Galactose oxidase
w i l l not
oxidize
t r i s a c c h a r i d e (1) b u t w i l l i n (2).
2-acetamido-2-deoxy-~-galactose,
link
when
i s (1).
Q-
the
residue
i n
!-galactose
Moreover, n e i t h e r a-galactose nor g l y c o s i d i c a l l y b o u n d as i n (31,
s u s c e p t i b l e t o o x i d a t i o n w i t h D - g a l a c t o s e o x i d a s e u n t i l t h e (1 linkage
between
them
i s
the
a c i d r e s i d u e as
cleaved
by
is
* 3)
a-Q-2-acetamido-2-
389
6: Enzymes deoxygalactosidase.
a3(1+2)-L-Fucosidase
as i n (3) and (4).
action
i s
i n h i b i t e d by
or a - E - g a l a c t o s y l
(1+3) 2-acetamido-2-deoxy-a-!-galactosyl
a
residue,
Removal o f t h e t e r m i n a l s u g a r s makes t h e a - l -
fucosyl residue susceptible t o a-l-fucosidase
action.
a-NeupNG1 2
I 6
B-Q-Gal-(1+3)-g-GalNAcol
(11 a-b - F u c g 1
I 2
B-g-Gal-(
1+3) - G a l N A c o l (2)
a-Q-GalNAc-(1+3)-8-~-Gal-(l+3)-~-GalNAcol 2
I 1 a-l-Fuc (3)
The a c t i v i t i e s o f v a r i o u s g l y c o s i d a s e s i n h o m o g e n a t e s o f t h e small
intestinal
wallabies
(M.
mucosa
eugenii)
of
two
aged
adult
from
6
i n v e ~ t i g a t e d . ~8 ~ -Q-Galactosidase, deoxyglucosidase,
a-C-fucosidase,
and to
18 50
suckling weeks
tammar
have
been
B-E-2-acetamido-2-
and n e u r a m i n i d a s e a c t i v i t i e s w e r e
h i g h d u r i n g t h e f i r s t 34 weeks p o s t p a r t u m and t h e n d e c l i n e d t o v e r y low levels.
F o r f u r t h e r d e t a i l s see i n i t i a l c i t a t i o n o f r e f . 3 8 .
The a - i - f u c o s i d a s e ,
B - Q - 2 - a c e t am i d o - 2 - d e o x y g l u c o s i d a s e ,
a-Q-
390
Carbohydrate Chemistry
mannosidase, B-g-glucosidase, B-;-xylosidase, B-g-glucosidase, and a& - a r a b i n o f u r a n o s i d a s e a c t i v i t i e s o f n o r m a l and a t r o p h i c s k e l e t a l muscle of d e v e l o p i n g and a d u l t r a t s have been c o l l e c t i v e l y i n v e s t i g a t e d . 36 S e v e r a l m o l l u s c g l y c o s i d a s e s h a v e been s t u d i e d f o r t h e i r a c t i v i t i e s towards n a t u r a l s u b s t r a t e s . a - i - F u c o s i d a s e s from Chamelea g a l l i n a , T a p e s r h o m n o i d e u s , a n d M y t i l u s e d u l i z h y d r o l y s e o l i g o s a c c h a r i d e s ( d i - , t r i - , a n d p e n t a - s a c c h a r i d e s ) w i t h 1 + 2 , 1 + 3, and 1 + 4 bonds, I - f u c o s e - c o n t a i n i n g g l y c o p e p t i d e s from bovine t h y r o g l o b u l i n and t h e p o r c i n e submandibular mucin ( d e v o i d of s i a l i c acid). a-I=-Fucosidase from L i t t o r i n a l i t t o r e a h y d r o l y s e s L-fucosec o n t a i n i n g g l y c o p e p t i d e s from bovine t h y r ~ g l o b u l i n . ~ ' For f u r t h e r d e t a i l s s e e i n i t i a l c i t a t i o n of r e f . 7 0 . A novel s y n t h e t i c chromogenic s u b s t r a t e , a-b-Fuce-(l + 2)-B-!G a l e - ( l + O C 6 H 4 N 0 2 - e ) , h a s been u s e d f o r t h e r a p i d a s s a y of a - i f u c o s i d a s e s which h y d r o l y s e t h e g l y c o s i d i c l i n k a g e , a - L - F u c e - ( l + 2)-Q-Gal.'l0 The p r o c e d u r e i s based on t h e s e q u e n t i a l a c t i o n of aI - f u c o s i d a s e and an e x o g e n o u s l y a d d e d = - $ - Q - g a l a c t o s i d a s e to r e l e a s e t h e e a s i l y m e a s u r a b l e 4 - n i t r o p h e n o l m o i e t y . T h i s method d e t e c t e d a - C - f u c o s i d a s e s from A s p e r q i l l u s n i g e r , C l o s t r i d i g m p e r f r i n q e n s , and a l m o n d , w h i c h a r e known t o h y d r o l y s e t h e (1 + 2 ) linkage specifically. The a d v a n t a g e s o f t h i s p r o c e d u r e o v e r p r o c e d u r e s p r e v i o u s l y used f o r t h e d e t e c t i o n of s u b s t r a t e - s p e c i f i c a-l-fucosidases are discussed. S i x g l y c o s i d e h y d r o l a s e s i n t h e c u l t u r e medium of B a c t e r o i d e s f r a g i l i s - a - g - g l u c o s i d a s e , B - Q - g l u c o s i d a s e , a-gg a l a c t o s i d a s e , B-!-galactosidase, B-~-2-acetamido-2-deoxyglucosidase, and a - C - f u c o s i d a s e - w e r e s y s t e m a t i c a l l y p u r i f i e d b y ammonium s u l p h a t e p r e c i p i t a t i o n , g e l - f i l t r a t i o n chromatography, and d e n s i t y gradient i s o e l e c t r i c focusing.49 The i s o e l e c t r i c focusing resolved the glycosidases i n t o d i s t i n c t , well s e p a r a t e d f r a c t i o n s and r e v e a l e d t h r e e d i f f e r e n t l y c h a r g e d f o r m s of B - P - 2 - a c e t a m i d o - 2 For f u r t h e r d e t a i l s see d e o x y g l u c o s i d a s e and a - & - f u c o s i d a s e . i n i t i a l c i t a t i o n of ref.49. I -
7
a- and B-a-Galactosidases
and P - G a l a c t o l i p i d - o r i e n t e d a- and
B-P-Galactosidases
a - q - G a l a c t o s i d a s e d e f i c i e n c y has been d e m o n s t r a t e d i n lymphoid c e l l l i n e s e s t a b l i s h e d by E p s t e i n - B a r r v i r u s t r a n s f o r m a t i o n o f B -
391
6: Enzymes lymphocytes f r o m a Fabry p a t i e n t , lymphocytes.21
as i n b l o o d w h o l e l e u k o c y t e s and
I n n o r m a l - b l o o d l y m p h o c y t e s and l y m p h o i d c e l l l i n e s ,
a-e-galactosidase
was
separated
by
m o l e c u l a r f o r m s : f o r m I (PI 5.0),
electrofocusing into
f o r m I 1 (PI 4.5),and
three
f o r m 111 (PI
F o r f u r t h e r d e t a i l s see i n i t i a l c i t a t i o n o f r e f . 2 1 .
4.3).
The c o n d i t i o n s f o r m a x i m a l a c t i v i t y (pH b u f f e r , substrate concentration, enzyme
activity
and r a n g e o f
versus
saturating
l i n e a r r e l a t i o n s h i p s between
incubation
time
and
versus
enzyme
c o n c e n t r a t i o n ) i n t h e f l u o r i m e t r i c assay o f s e v e r a l g l y c o h y d r o l a s e s o f
lysosomal
origin
i n
e ~ t a b l i s h e d . ~T ~ he
human
plasma
following
and
enzymes
serum
were
have
studied:
been a-B-
galactosidase, B-Q-galactosidase, B-Q-glucosidase, B-Qg l u c u r o n i d a s e , a-Q-mannosidase, and a-C-fucosidase. A l l examined e n z y m e s t u r n e d o u t t o b e m o r e o r l e s s u n s t a b l e u p o n s t o r a g e a t 37'C, 4'C,and
-2O'C
i n b o t h serum and plasma.
For f u r t h e r d e t a i l s see
o r i g i n a l c i t a t i o n o f r e f .26. A s i m p l e a n d s e n s i t i v e f l u o r i m e t r i c m e t h o d h a s been d e s c r i b e d f o r t h e d i f f e r e n t i a l d e t e r m i n a t i o n o f t h e a c t i v i t y o f l y s o s o m a l a+galactosidase
A
and a - g - g a l a c t o s i d a s e
B.ll1
The p r o c e d u r e e m p l o y s
4-methylumbelliferyl a-Q-galactopyranoside
acetamido-2-deoxy-Q-galactose B,
but n o t a-P-galactosidase
as A,
as s u b s t r a t e and 2-
an i n h i b i t o r o f
a-g-galactosidase
t o differentiate the two activities.
T h i s m e t h o d was shown t o be a p p l i c a b l e i n t h e d i f f e r e n t i a t i o n o f t h e t w o enzyme a c t i v i t i e s i n human t i s s u e s and i n t h e d i a g n o s i s o f t h e heterozygous
and
hemizygous
genotypes
for
Fabry's
disease
i n
cultured skin fibroblasts. Two i s o e n z y m i c f o r m s (I a n d 11) o f i s o l a t e d and p u r i f i e d f o r pancreas.l12
The
purification
s u l p h at e f r a c t i o n a t i o n
filtration.
The
lysosomes
,
a - Q - g a l a c t o s i d a s e have been of
io n - e x c h a n g e
apparent
Mytilus edulis
involved
extraction,
c h r o m a t o g r a p h y,
molecular weights
f i l t r a t i o n w e r e e s t i m a t e d t o b e 70,000
thermal stabilities. while
Hg2+,
Cu2+,
determined
a n d 50,000,
B o t h f o r m s showed d i f f e r e n t pH o p t i m u m , !,,and nitrophenyl a-Q-galactoside
hepatoammonium a n d ge 1 by g e l
respectively.
lmax v a l u e s w i t h 4-
as s u b s t r a t e and d i f f e r e d
in their
The t w o f o r m s w e r e a c t i v a t e d by N a + a n d K + ,
and
Zn2+ a l l
inhibited
G a l a c t o s i d a s e I and I1 e x h i b i t e d a c t i v i t y
both
forms.
a-Q-
on r a f f i n o s e and
melibiose. Sucrose
density
lysosome-containing
gradients
have
been used t o
f r a c t i o n s from zero-hour
characterize
w h i t e prepupae o f
S t o m o x y s ~ a l c i t r a n s . ~The ~ a c i d g l y c o s i d a s e s ( a - Q - g l u c o s i dase,
a-Q-
Carbohydrate Chemistry
3 92 galactosidase,
a-g-mannosidase,
B-D-glucuronidase,
B-Q-glucosidase,
B-Q-galactosidase,
B-~-2-acetamido-2-deoxyglucosidase)
and
e q u i l i b r a t e a t t h e same d e n s i t y as d i d a c i d p h o s p h a t a s e . Nine
glycosidases
in
bloodstream
forms
have been p a r t i a l l y c h a r a c t e r i z e d . 2 0 physicochemical
and
enzymic
of
Trypanosoma b r u c e i
a-a-Glucosidase
properties
to
had s i m i l a r
those
of
a-Q-
B-~-2-acetamido-2-deoxyglucosidase, and B-~-2-acetamido-2-deoxygalactosidase. F o r f u r t h e r d e t a i l s see
galactosidase, B-Q-glucosidase, i n i t i a l c i t a t i o n o f ref.20.
The a - Q - g a l a c t o s i d a s e o f P o t e r i o o c h r o m o n a s m a l h a m e n s i s h a s been partially characteri~ed."~ I n cell-free was s t a b l e a t pH 8.
- -galactosidase a-D
was 7. steps The
e x t r a c t s o f P.
malhamensis
The pH o p t i m u m o f t h e enzyme
The e n z y m e was p u r i f i e d 1 0 - f o l d t h r o u g h c h r o m a t o g r a p h i c i n v o l v i n g DEAE-cellulose,
apparent
molecular
gradient centrifugation. enzyme
to
of
was
360,000
by
sucrose
density-
A l l a c t i v i t y was l o s t o n s u b j e c t i n g t h e
polyacrylamide
specificity
h y d r o x y a p a t i t e , and Sephadex G-200.
weight gel
electrophoresis.
The
substrate
t h e e n z y m e was e x a m i n e d a n d some k i n e t i c v a l u e s
were d e t e r m i n e d .
The enzyme d i s p l a y e d an u n u s u a l c u r v e w i t h r e s p e c t
t o isofluoridoside. Mung- bean
seeds
possess
a
tetrarneric
Q-galactose-binding
p r o t e i n t h a t d i s p l a y s two types of a c t i v i t i e s :
(a) a haemagglutinin
a c t i v i t y and ( b ) an a - Q - g a l a c t o s i d a s e a c t i v i t y . ' 1 4
T h i s p r o t e i n can
be r e v e r s i b l y c o n v e r t e d by pH changes f r o m a t e t r a m e r i c f o r m ,
which
possesses b o t h e n z y m i c and p h y t o h a e m a g g l u t i n i n a c t i v i t i e s ,
to a
monomeric
This
form,
which
possesses
enzymic
activity
s u g g e s t e d t h a t t h e e n z y m i c p h y t o h a e m a g g l u t i n i n i s an form
of
monomeric a-a-galactosidase.
The
g a l a c t o s i d a s e h a s a pH o p t i m u m o f a b o u t pH 7.0, f o r m d i s p l a y s a pH o p t i m u m o f 5 . 6 .
only.
aggregated
tetrameric
a-9-
w h i l e t h e monomeric
Circular-dichroism difference
s p e c t r a and i n h i b i t i o n s t u d i e s s u g g e s t t h a t a g g r e g a t i o n i n d u c e s c o n f o r m a t i o n changes i n t h e s u b u n i t s enzymatic properties. equilibria, activity,
sufficient
to alter their
The p o s s i b i l i t y o f i n v i v o changes i n s u b u n i t
when c o m b i n e d w i t h t h e a c c o m p a n y i n g a l t e r a t i o n s
provides
a new
concept
worthy
of
in
consideration with
respect t o the physiological r o l e o f phytohaemagglutinins. The
p u r i f i c a t i o n and p r o p e r t i e s o f
immature s t a l k s described.ll5
of
sugar
cane
The a - p - g a l a c t o s i d a s e
sulphate fractionation,
a-Q-galactosidase
(Saccharum o f f i n a r u m ) have
from been
was h i g h l y p u r i f i e d by ammonium
g e l f i l t r a t i o n o n S e p h a d e x G-100,
exchange c h r o m a t o g r a p h y on D E A E - c e l l u l o s e and C M - c e l l u l o s e ,
ion-
and h e a t
393
6: Enzymes
In
t r e a t m e n t ( 6 O o C , 1 5 m i n ) i n t h e p r e s e n c e o f 0.2 M g - g a l a c t o s e . polyacrylamide homogeneous,
gel
electrophoresis,
i t was a p p r o x i m a t e l y 47,000.
o p t i m u m a t pH 4.5
a n d a t 6OoC.
raffinose
(Ern, 1 3 . 0
melibiose,
(Em mM,
g u a r gum,
25.9mM,
Vmax 2.7
(Em 0.83 Vmax 15.4
was
Vmax
mM,
and l o c u s t - b e a n was
25.0
vmol mg-l
gum.
was
vmol mg-l
min'l),
and
i n addition to
The h y d r o l y s i s o f
markedly
4-
i n h i b i t e d by HgC12,
4-chloromercuribenzoate L-ascorbic acid,
and P - g a l a c t o s e .
In
The a c t i v i t y
p m o l m g - l min-'),
n i t r o p h e n y l a-P-galactopyranoside AgN03,
enzyme
The p u r i f i e d enzyme h y d r o l y s e d 4 -
n i t r o p h e n y l a-Q-galactopyranoside stachyose
purified
h a v i n g a m o l e c u l a r w e i g h t o f a p p r o x i m a t e l y 46,000.
gel filtration,
min-l),
the
melibiose,
stachyose,
A l s o t h e p u r i f i e d enzyme showed a l e c t i n a c t i v i t y
with trypsinized erythrocytes.
a-Q-Galactosidase
has
been
( C a s t a n a e s a t i v a ) seeds.'l6
isolated
from
sweet -chestnut
The m a j o r s u g a r s o f f r e s h s e e d s w e r e
shown t o be r a f f i n o s e , stachyose, and s u c r o s e .
D r y i n g seeds a t 25OC
f o r 1 4 weeks i n c r e a s e d t h e r a t i o r a f f i n o s e : s t a c h y o s e 3.5,
reduced sucrose
c o n t e n t b y ~ g 5046,and .
extractable a-Q-galactosidase.
The e n z y m e a c t i v i t y was r e s o l v e d
i n t o t w o peaks, a h i g h - m o l e c u l a r - w e i g h t molecular-weight
f r o m 1.1 t o
decreased t o t a l
f o r m I1 (53,000).
f o r m I (215,000)
and a low-
The l a t t e r f o r m was p r e d o m i n a n t
i n t h e e x t r a c t o f f r e s h seeds whereas t h e f o r m e r was t h e m a i n f o r m
i n t h e 14-week d r i e d seeds.
An i n c r e a s e i n t h e a m o u n t o f e n z y m e I
was a l s o o b s e r v e d when a b u f f e r e d e x t r a c t (pH 5 . 5 ) was s t o r e d a t 4OC. respectively.
o f f r e s h seeds
Enzymes I a n d I 1 h a d pH o p t i m a o f 4.5
Both
enzymes
hydrolysed
4-nitrophenyl
a n d 6, a-Q-
g a l a c t o p y r a n o s i d e a t a much g r e a t e r r a t e t h a n t h e n a t u r a l s u b s t r a t e s raffinose,
stachyose,
locust-bean
gum, a n d c a r o b gum.
However,
enzyme I showed p r e f e r e n c e s f o r s t a c h y o s e as compared t o r a f f i n o s e . The o p p o s i t e o r d e r was o b s e r v e d f o r enzyme 11. Two f o r m s o f a - e - g a l a c t o s i d a s e
which d i f f e r e d i n molecular
w e i g h t h a v e been r e s o l v e d f r o m Cucumis s a t i v u s enzymes
were
fractionation,
partially
using
Sephadex g e l f i l t r a t i o n ,
Sephadex c h r o m a t o g r a p h y . gel filtration,
purified
w e r e 50,000
a n d 25,000.
from
mature
leaves
p a r t i a l l y p u r i f i e d 25,000
and
was
The
sulphate
and d i e t h y l a m i n o e t h y l -
The m o l e c u l a r w e i g h t s o f t h e t w o f o r m s ,
by
The 50,000 f o r m c o m p r i s e d
a p p r o x i m a t e l y 84% o f t h e t o t a l a - Q - g a l a c t o s i d a s e extracts
leaves.'l7
ammonium
activity
purified
molecular-weight
i n crude
132-fold.
The
form r a p i d l y l o s t
a c t i v i t y u n l e s s s t a b i l i z e d w i t h 0.2% a l b u m i n and a c c o u n t e d f o r
16%
of
The
the t o t a l a-g-galactosidase
activity
i n t h e crude e x t r a c t .
394
Carbohydrate Chemistry
s m a l l e r - m o l e c u l a r - w e i g h t f o r m was n o t f o u n d i n o l d e r l e a v e s . The t w o f o r m s w e r e s i m i l a r i n s e v e r a l ways i n c l u d i n g t h e i r pH o p t i m a , w h i c h w e r e 5 . 2 and 5.5 f o r t h e 5 0 , 0 0 0 and 25,000 d a l t o n f o r m s , r e s p e c t i v e l y , and a c t i v a t i o n e n e r g i e s , w h i c h w e r e 1 5 . 4 and 18.9 k i l o c a l o r i e s p e r mole f o r t h e l a r g e r and s m a l l e r f o r m s , r e s p e c t i v e l y . Both enzymes were i n h i b i t e d by Q - g a l a c t o s e a s w e l l a s b y e x c e s s c o n c e n t r a t i o n s of 4 - n i t r o p h e n y l a - g - g a l a c t o s i d e s u b s t r a t e - Em v a l u e s w i t h t h i s s u b s t r a t e and w i t h r a f f i n o s e and m e l i b i o s e were d i f f e r e n t f o r each s u b s t r a t e , b u t s i m i l a r f o r both forms of t h e enzyme. W i t h s t a c h y o s e , Em v a l u e s were 1 0 and 30 m M f o r t h e 50,000 and 25,000 m o l e c u l a r - w e i g h t f o r m s , r e s p e c t i v e l y . R e d - l i g h t - and g i b b e r e l l i c a c i d - e n h a n c e d a - P - g a l a c t o s i d a s e a c t i v i t y h a s been i n v e s t i g a t e d i n g e r m i n a t i n g l e t t u c e s e e d s . D r y l e t t u c e (Lattuca s a t i v a ) seeds contain a-e-galactosidase a t a l e v e l which i s m a i n t a i n e d i n t h e i m b i b e d d o r m a n t s t a t e i n d a r k n e s s . l l 8 Both r e d l i g h t and g i b b e r e l l i c a c i d p r o m o t e an i n c r e a s e i n enzyme a c t i v i t y s e v e r a l h o u r s p r i o r t o t h e c o m p l e t i o n of g e r m i n a t i o n . Germination and enzyme a c t i v i t y a r e not e s s e n t i a l l y l i n k e d , however, a-af o r t h e l a t t e r can i n c r e a s e w h i l e t h e f o r m e r i s i n h i b i t e d . G a l a c t o s i d a s e a c t i v i t y i n c r e a s e s w i t h i n t h e c o t y l e d o n s and t h e endosperm f o l l o w i n g r e d - l i g h t s t i m u l a t i o n , b u t t h e a x i s i s e s s e n t i a l t o p e r c e i v e t h e s t i m u l u s and t o promote and m a i n t a i n t h e i n c r e a s e i n enzyme a c t i v i t y . A d i f f u s i b l e f a c t o r ( o r f a c t o r s ) i s p r o d u c e d b y a n d / o r r e l e a s e d f r o m i r r a d i a t e d a x e s , and i t m i g r a t e s t o t h e c o t y l e d o n s (and p o s s i b l y endosperm) t o promote and i n c r e a s e i n a-ggalactosidase activity. Gibberellic acid, particularly i n the p r e s e n c e of b e n z y l a d e n i n e , can r e p l a c e t h e r e q u i r e m e n t f o r i r r a d i a t e d axes. The immediate phytochrome i n d u c t i o n of a - 0 - g a l a c t o s i d a s e has The l i g h t - i n d u c e d b r e a k i n g been d e m o n s t r a t e d i n l e t t u c e s e e d s . l l 9 of dormancy o f Grand R a p i d s l e t t u c e s e e d s i m b i b e d i n d a r k n e s s a t Although i t 25'C i s a c l a s s i c a l p h y t o c h r o m e - c o n t r o l l e d phenomenon. h a s been shown t h a t i r r a d i a t i o n of s e e d s 1 - 2 h a f t e r t h e s t a r t o f i m b i b i t i o n produces t h e a c t i v e form of phytochrome ( P f r ) w h i c h a c t s r a p i d l y a f t e r i t s f o r m a t i o n , t h e c o m p l e t i o n of g e r m i n a t i o n ( o b s e r v e d Clearly a s r a d i c l e p r o t u s i o n ) i s only e v i d e n t s e v e r a l h o u r s l a t e r . t h e n r a d i c l e emergence i s t e m p o r a r i l y removed from t h e r a p i d (and p r e s u m a b l y p r i m a r y ) a c t i o n of p h y t o c h r o m e . Very l i t t l e i s known about t h e p r o c e s s e s s e t i n motion by P f r t h a t l e a d t o t h e completion of g e r m i n a t i o n . Here t h e a u t h o r r e p o r t s t h a t r e d - l i g h t - i n d u c e d s t i m u l a t i o n of a - Q - g a l a c t o s i d a s e a c t i v i t y o c c u r s s u b s t a n t i a l l y
395
6: Enzymes before germination i s completed,
and shows t h a t
the
a c t i o n of
p h y t o c h r o m e i n p r o m o t i n g an i n c r e a s e i n t h e a c t i v i t y o f t h i s enzyme
is very rapid. Six
glycoside
hydrolases
-
Bacteroides f r a g i l i s galactosidase, sidase,
i n
the
a-Q - -glucosidase,
B-e-galactosidase,
and a - i - f u c o s i d a s e
-
culture
of
a-9-
B-~-2-acetamido-2-deoxygluco-
h a v e been s y s t e m a t i c a l l y
ammonium s u l p h a t e p r e c i p i t a t i o n , g e l - f i l t r a t i o n density-gradient
medium
B-g-glucosidase,
i s o e l e c t r i c focusing.49
resolved the glycosides i n t o distinct,
p u r i f i e d by
chromatography,
and
The i s o e l e c t r i c f o c u s i n g
w e l l separated fractions.
F o r f u r t h e r d e t a i l s see i n i t i a l c i t a t i o n o f r e f . 4 9 .
The
effect
of
tunicamycin
A s p e r g i l l u s n i g e r has
on
secreted
glycosidases
b e e n d e ~ c r i b e d . ~ ' The
o f
main glycosidases
s e c r e t e d i n t o t h e c u l t u r e medium d u r i n g g r o w t h were B - 0 - g l u c o s i d a s e , a - Q - g a l a c t o s i d a s e , and B-~-2-acetamido-2-deoxyglucosidase. presence o f tunicamycin the a c t i v i t i e s o f
I n the
t h e s e enzymes i n t h e F o r f u r t h e r d e t a i l s see
c u l t u r e medium w e r e c o n s i d e r a b l y d e c r e a s e d . i n i t i a l c i t a t i o n o f ref.50. The
specificity
galactosidase
of
from
induction of
Saccharomyces
the
investigated.'*'
Besides 0-galactose
enzyme ( m e l i b i o s e ,
r a f f i n o s e , and s t a c h y o s e ) ,
arabinose, Q-tagatose,
synthesis
carlsberclensis
of
a-a-
has
been
and t h e s u b s t r a t e s o f t h e Q-galaturonic acid,
methyl a-e-galactopyranoside,
L-
lactose,and
i s o p r o p y l B-Q-thiogalactopyranoside were a b l e t o a c t as i n d u c e r s . O f these,
methyl a-Q-galactopyranoside,
t h i o g a l a c t o p y r a n o s i d e , and
i s o p r o p y l B-Q-
lactose,
C-arabinose
have
been
shown
to
be
g r a t u i t o u s inducers w i t h which k i n e t i c s t u d i e s o f i n d u c t i o n have been c a r r i e d o u t .
L a c t o s e was t h e m o s t e f f i c i e n t
inducer, g i v i n g a
m a x i m a l d i f f e r e n t i a l r a t e o f s y n t h e s i s o f t h e enzyme o f 1 1 0 mU c e l l s a t a c o n c e n t r a t i o n o f 180 m M , c e l l s a t 40 m M ) ,
c e l l s a t 6 0 mM),and c e l l s a t 150 mM). half-maximal
f o l l o w e d by C - a r a b i n o s e
i s o p r o p y l B-Q-thiogalactopyranoside methyl a-0-galactopyranoside
The c o n c e n t r a t i o n s o f
i n d u c t i o n were
similar
for
to
methyl a-Q-galactopyranoside.
compounds t o a c t
a s i n d u c e r s was
( 2 5 mU
inducer required t o obtain those
a r a b i n o s e , and i s o p r o p y l B - a - t h i o g a l a c t o p y r a n o s i d e higher
( 6 0 mU
( 4 3 mU
The
compared t o
for
lactose,
and about
property of their
I-
5-fold
ability
the to
i n t e r a c t w i t h . t h e enzyme, and t h e r e s u l t s w e r e d i s c u s s e d i n t e r m s o f t h e m o l e c u l a r s t r u c t u r e s w h i c h a r e r e c o g n i z e d by t h e enzyme and by t h e i n d u c t i o n machinery. A method has been d e s c r i b e d f o r
following continuously the
396
Carbohydrate Chemistry
action
of
B-Q-galactosidase
galactopyranoside
on
a t pH 4 . 5 ,
i n
B-Q-
4-methylumbelliferyl
which
4-methylumbelliferone
p r o d u c t i o n was m e a s u r e d a t f l u o r e s c e n c e e x c i t a t i o n a n d e m i s s i o n wavelengths studies
of
have
324 and 444 shown
that
r e s ~ e c t i v e 1 y . l ~ I~n i t i a l - r a t e
presence
activates
The enzyme was v e r y u n s t a b l e a t 37OC a n d l o w i o n i c
strength,
stability
increased
s t a b i l i t y o f t h e enzyme a t 37'C t h e r a n g e 5.9-8.0. amounts o f activity,
inhibitory
$-Q-
concentration.
mM
i s
salt
up
100
but
of
galactosidase but
to
nm,
the
with
ionic
above
that
strength.
The
d e c r e a s e d m a r k e d l y w i t h r i s i n g pH i n
The g e l - f i l t r a t i o n p a t t e r n s h o w e d d e c r e a s i n g
dimer
with
i n c r e a s i n g pH.
The c o r r e l a t i o n b e t w e e n
s t a b i l i t y , and m o l e c u l a r f o r m o f B - Q - g a l a c t o s i d a s e
discussed.
was
I t was s u g g e s t e d t h a t t h e d i m e r i c f o r m o f t h e enzyme i s
t h e m o s t s t a b l e and a c t i v e f o r m .
The i m p l i c a t i o n s o f t h i s f i n d i n g
f o r t h e assay o f B-g-galactosidase
under p h y s i o l o g i c a l c o n d i t i o n s
f o r p r e n a t a l diagnosis were discussed.
Evidence f o r the possible
o c c u r r e n c e o f a 36,000 m o l e c u l a r - w e i g h t f o r m o f B - Q - g a l a c t o s i d a s e was p r e s e n t e d .
A computer program f o r t h e c a l c u l a t i o n o f i n i t i a l
r a t e s h a s been d e p o s i t e d as S u p p l e m e n t a r y P u b l i c a t i o n SUP 50114 a t the B r i t i s h Library Lending Division,
B o s t o n Spa.
I t h a s been r e p o r t e d t h a t t h e a g e i n g o f human l i v e r c e l l l i n e s i s a c c o m p a n i e d by a d e c r e a s e i n t h e l y s o s o m a l B - Q - g a l a c t o s i d a s e activity.122
I n six c e l l lines,
b o t h c a t a l y t i c assay
u s i n g 4-
me t h y l u m b e l l i f e r y 1 B - Q - g a l a c t o p y r a n o s i d e a n d i m m u n o c h e m i c a l t i t r a t i o n by a m o n o s p e c i f i c performed.
anti-B-Q-galactosidase
anti-serum
were
The r e d u c e d c a t a l y t i c a c t i v i t y was n o t a s s o c i a t e d w i t h
t h e presence o f c r o s s - r e a c t i n g
m a t e r i a l and p r o b a b l y d i d n o t r e s u l t
from a modification o f the biosynthesis,
nor from p o s t - t r a n s l a t i o n a l
m o d i f i c a t i o n o f t h e enzyme. A r a p i d and s e n s i t i v e m e t h o d h a s been d e v i s e d f o r d e t e r m i n i n g
B-e-galactosidase
a c t i v i t y s p e c i f i c f o r g a 1 a ~ t o c e r e b r o s i d e . l ~ A~
fluorescent derivative for
galactocerebroside,
l-~-B-~-galactosyl-2-
-N - l - d i m e t h y l a m i n o n a p h t h a l e n e - 5 - s u l p h o n y l - s p h i n g o s i n e , substrate,
and
the
product,
sulphonyl-sphingosine,
was
was u s e d a s
2-N-l-dimethylaminonaphthalene-5taken
into
organic
solvent
phase.
Q u a n t i t a t i v e a n a l y s i s o f 2-N-dimethy l a m i n o n a p h t h a l e n e - 5 - s u l p h o n y l sphingosine
was
c a r r i e d out
fluorimetrically
by
use
of
high-
p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y on s i l i c a g e l c o l u m n . T h e c o n d i t i o n s f o r m a x i m a l a c t i v i t y (pH, substrate concentration, enzyme
activity
versus
range o f
buffer,
saturating
l i n e a r r e l a t i o n s h i p s between
incubation
time
and
versus
enzyme
6: Enzymes concentration)
i n
the
fluorimetric
assay
of
several
glycoside
h y d r o l a s e s o f l y s o s m a l o r i g i n i n human p l a s m a a n d s e r u m h a v e b e e n e ~ t a b l i s h e d . ' ~ The galactosidase, deoxyglucosidase, mannosidase,
following
enzymes
were
B-5-galactosidase,
B-~-glucosidase, B-~-glucuronidase,
a-b-fucosidase. For
a-9-
A l l e x a m i n e d enzymes t u r n e d o u t t o be
m o r e o r l e s s u n s t a b l e u p o n s t o r a g e a t 37'C, serum and plasma.
a-a-
studied:
B-D-2-acetamido-2-
further
4OC,and
-2O'C
i n both
d e t a i l s see i n i t i a l c i t a t i o n o f
r e f .26. I t has
been r e p o r t e d t h a t
galactosidase
and
the
neuraminidase
human
c o r r e c t e d t o n e a r l y n o r m a l values.124 addition
of
concentrated
culture
o f B-Q-
combined d e f i c i e n c y
i n
fibroblasts
can
medium
obtained
s t i m u l a t i o n o f d i f f e r e n t t y p e s o f human f i b r o b l a s t s ,
after
NH4C1
including those
w i t h an i s o l a t e d f 3 - Q - g a l a c t o s i d a s e o r n e u r a m i n i d a s e d e f i c i e n c y . c o r r e c t i v e f a c t o r i s a macromolecular g l y c o p r o t e i n , a t 60'C.
The
which i s l a b i l e
I t s u p t a k e by human f i b r o b l a s t s i s c o m p e t i t i v e l y i n h i b i t e d
by Q-mannose-6-phosphate /neur-
be
T h i s c a n be a c c o m p l i s h e d by
and i t s c o r r e c t i v e a c t i o n w i t h i n B-gal'
f i b r o b l a s t s c o n t i n u e s d u r i n g a chase o f 72 hours.
I t h a s been shown t h a t B - Q - g a l a c t o s i d a s e a c t i v i t y was p a r t i a l l y r e s t o r e d by p r o t e a s e i n h i b i t o r s l e u p e p t i n ,
c h y m o s t a t i n , and E-64
i n
c u l t u r e d f i b r o b l a s t s from three p a t i e n t s w i t h B-Q-galactosidase neuraminidase enzyme.
deficiency.lZ5
Pepstatin
did
not
activate
this
N e u r a m i n i d a s e was n o t a f f e c t e d by any o f t h e s e compounds i n
t h e c u l t u r e medium.
I t was c o n c l u d e d t h a t t h e a c t i v a t i n g e f f e c t was
p r o d u c e d by a s p e c i f i c i n h i b i t i o n o f t h i o l p r o t e a s e s . Human
fibroblasts
with
a
genetic
deficiency
of
a
single
l y s o s o m a l enzyme and f i b r o b l a s t s f r o m a p a t i e n t w i t h I - c e l l d i s e a s e w i t h a m u l t i p l e d e f i c i e n c y o f l y s o s o m a l h y d r o l a s e s h a v e been u s e d as recipient
c e l l s i n s t u d i e s on r e c o g n i t i o n and u p t a k e o f
a c e t am id o
- 2 - d e o x y h e x o s id a s e ,
g a l a c t o ~ i d a s e . ~N~o r m a l
B
human
-c - g 1u c u r o n i d a s e ,
fibroblasts
hepatocytes
and hepatoma c e l l s f r o m
cells.
extracellular
The
activities
and
B-Q-2-
and
B-9-
fibroblast
t h e r a t were u s e d as d o n o r of
B - Q - g l u c u r o n i d a s e and B-Q-
g a l a c t o s i d a s e w e r e much h i g h e r i n t h e r a t c e l l c u l t u r e s t h a n i n cultures of
n o r m a l human f i b r o b l a s t s .
For
further
d e t a i l s see
i n i t i a l c i t a t i o n o f r e f .58. I s o e l e c t r i c focusing
of
t h e a c i d B-Q-galactosidases
i n normal
c r u d e l i v e r s u p e r n a t a n t f l u i d s has d e m o n s t r a t e d m u l t i p l e i s o e l e c t r i c
f o r m s i n t h e pH r a n g e 4.58-5.15,
while corresponding I - c e l l
s a m p l e s h a v e shown an absence o f
i s o e l e c t r i c f o r m s i n t h e pH r a n g e
disease
Carbohydrate Chemistry
398 4.99-5.15.126 binding of
demonstrated
a 31% and 37% d e c r e a s e i n t h e
4 - m e t h y l u m b e l l i f e r y l B-Q-galactosidase
galactosidase a c t i v i t i e s , samples.
48 c h r o m a t o g r a p h y o f t h e I-
C o n c a n a v a l i n A-Sepharose
c e l l d i s e a s e m u t a n t C.A.
respectively,
Isoelectric-focusing
p r o f i l e s on t h e concanavalin A-
S e p h a r o s e 48 m e t h y l a - g - m a n n o p y r a n o s i d e
effluents containing normal
and I - c e l l d i s e a s e a c i d B - g - g a l a c t o s i d a s e but
t h e unadsorbed I - c e l l
and G M 1 B-Q-
when c o m p a r e d w i t h n o r m a l
were g e n e r a l l y s i m i l a r ,
d i s e a s e enzyme
from
concanavalin
A-
S e p h a r o s e 4 8 d e m o n s t r a t e d m o r e a c t i v i t y i n t h e pH r a n g e 4.21-4.49 than normals.
N o r m a l and I - c e l l d i s e a s e a c i d B - Q - g a l a c t o s i d a s e A
a n d B, s e p a r a t e d b y g e l - c o l u m n c h r o m a t o g r a p h y , w e r e f o u n d t o h a v e similar
-vs.
p r o p e r t i e s with respect t o apparent molecular weights,
activity
profiles,
methylumbelliferyl
Km
apparent
B-Q-galactosidase,
a s i a l o f e t u i n substrates. I-cell
and
However,
values
for
pH
the
GMl-ganglioside,
4and
lmax values f o r the
the apparent
d i s e a s e s a m p l e s w e r e c o n s i s t e n t l y r e d u c e d when c o m p a r e d t o
the r e s u l t s obtained w i t h the corresponding normal fractions. g r e a t e s t decreases i n apparent
lmax were o b t a i n e d f o r
The
a c i d B-Q-
galactosidase a c t i v i t i e s i n I - c e l l disease crude supernatant f l u i d s and f o r t h e s e p a r a t e d I - c e l l d i s e a s e 8 enzyme. the
isoelectric-focusing
concanavalin
A-Sepharose
profiles,
48,
The w h e a t - g e r m
The d i f f e r e n c e s i n altered
and t h e r e d u c e d
n a t u r a l a n d s y n t h e t i c s u b s t r a t e s may carbohydrate composition o f
the
I-cell
binding
be r e l a t e d t o
disease
t o
imax values w i t h changes
i n
a c i d B-Q-galactosidase.
l e c t i n b i n d i n g o f B-Q-galactosidase
has been
fibroblast^.'^^
s t u d i e d i n human n o r m a l and s i a l i d o s i s t y p e - I 1
To
a s s e s s w h e t h e r t h i s r e s i d u a l a c t i v i t y i s due t o d e c r e a s e d s y n t h e s i s of
n o r m a l enzyme o r
normal synthesis
a
lectin-Sepharose
mutant 48
enzyme,
columns
has
i t s
on
studied.
The t o t a l a p p l i e d a c t i v i t y p a s s e d t h r o u g h t h e c o l u m n
unbound,
wheat-germ
of
behaviour
b u t on s u b s e q u e n t w a s h i n g and e l u t i o n w i t h 8 - Q - Z - a c e t a m i d o -
2-deoxyglucose activity,
more a c t i v i t y ,
was e l u t e d .
c o r r e s p o n d i n g t o 800% o f t h e a p p l i e d
The sum o f t h e unbound and b o u n d a c t i v i t i e s
was t h e same as t h a t w h i c h m i g h t h a v e b e e n e x p e c t e d f o r cells.
been
normal
The e x p l a n a t i o n g i v e n f o r t h i s f i n d i n g was t h a t t h e r e was an
i n h i b i t o r p r e s e n t i n s i a l i d o s i s t y p e - I 1 c e l l s t h a t was s t r o n g l y bound t o t h e enzyme and o n l y d i s s o c i a b l e u n d e r t h e r e l a t i v e l y h a r s h c o n d i t i o n s o f t h e l e c t i n b i n d i n g and e l u t i o n . that
n e u r a m i n i d a s e d e f i c i e n c y may
The a u t h o r s s u g g e s t
be t h e p r i m a r y
defect
i n
sialidosis type-I1 cells. A deficient
a c t i v i t y o f n e u t r a l B-Q-galactosidase
i n l i v e r and
399
6: Enzymes
c u l t i v a t e d f i b r o b l a s t s has been observed i n p a t i e n t s w i t h l a c t o s y l ceramidosis.128 N e u t r a l 6 - Q - g a l a c t o s i d a s e was p a r t i a l l y p u r i f i e d f r o m l i v e r s of n o r m a l c o n t r o l s and p a t i e n t s w i t h N i e m a n n - P i c k d i s e a s e type A or l a c t o s y l ceramidosis, using concanavalin AS e p h a r o s e a d s o r p t i o n and f i l t r a t i o n . The p a r t i a l l y p u r i f i e d f r a c t i o n s w e r e e s s e n t i a l l y f r e e of B - Q - g a l a c t o s y l c e r a m i d e B-Eg a l a c t o s i d a s e and GM1 B-E-galactosidase a c t i v i t i e s . The normal and Niemann-Pick f r a c t i o n s were found t o h y d r o l y s e l a c t o s y l c e r a m i d e , i n t h e p r e s e n c e of sodium t a u r o d e o x y c h o l a t e , a t a pH optimum of 5.6 a s w e l l a s a r y l B - Q - g a l a c t o s i d e s and a r y l B - e - g l u c o s i d e s a t pH 6 . 2 . The c o r r e s p o n d i n g f r a c t i o n f r o m t h e l a c t o s y l c e r a m i d o s i s l i v e r c o n t a i n e d only 1-4% of t h e normal a c t i v i t y t o w a r d s a r t i f i c i a l s u b s t r a t e s and l a c t o s y l c e r a m i d e . C r o s s - r e a c t i n g m a t e r i a l i d e n t i c a l t o t h e n o r m a l was d e m o n s t r a t e d i n t h i s f r a c t i o n w i t h a n t i s e r u m r a i s e d a g a i n s t p u r i f i e d n e u t r a l 6 - P - g a l a c t o s i d a s e , b u t no a c t i v i t y was observed i n t h e p r e c i p i t i n l i n e when s t a i n e d w i t h n a p h t h o l ASL C - @ - Q - g a l a c t o s i d e o r n a p h t h o l AS-LC-B-!-glucoside. A similar d e f i c i e n c y of n e u t r a l 6 - g - g a l a c t o s i d a s e a c t i v i t y was d e m o n s t r a t e d i n c u l t i v a t e d f i b r o b l a s t s of t h e p a t i e n t w i t h l a c t o s y l c e r a m i d o s i s . F o l l o w i n g a d s o r p t i o n on c o n c a n a v a l i n A-Sepharose and anti-GMl B-qg a l a c t o s i d a s e a n t i b o d y - S e p h a r o s e c o n j u g a t e s and c h r o m a t o g r a p h y on DEAE c e l l u l o s e , f i b r o b l a s t l y s a t e s from t h e p a t i e n t e x h i b i t e d 3% o f normal a c t i v i t y t o w a r d s 4 - m e t h y l u m b e l l i f e r y l B-Q-glucoside a t pH 6.2 and 1 2 % of n o r m a l a c t i v i t y t o w a r d s l a c t o s y l c e r a m i d e a t pH 5 . 6 . T h e s e d a t a s u g g e s t e d t h a t n e u t r a l B - Q - g a l a c t o s i d a s e may h a v e an i n v i v o r o l e i n t h e c l e a v a g e of l a c t o s y l c e r a m i d e and t h a t a d e f i c i e n c y of t h i s a c t i v i t y may b e r e l a t e d t o t h e l a c t o s y l c e r a m i d e a c c u m u l a t i o n observed i n t h e p a t i e n t w i t h l a c t o s y l c e r a m i d o s i s . The amounts of B - e - g a l a c t o s i d a s e , s u c r o s e a - g - g l u c o h y d r o l a s e , a-9-glucosidase, microvillus aminopeptidase,and d i p e p t i d y l peptidase I V i n t a n g e n t i a l l y s e c t i o n e d b i o p s i e s from jejunum have been s t u d i e d b y q u an t i t a t i ve i m m u n o e l e c t r o p h o r e s i s and enzymic a s s a y s 129 A 11 e n z y m e s had t h e i r maximum a c t i v i t i e s n e a r t h e m i d - r e g i o n o f t h e v i l l i and t h e i r l o w e s t a c t i v i t i e s a t t h e b a s e s of t h e c r y p t s . The r a t i o b e t w e e n enzyme a c t i v i t y and i m m u n o r e a c t i v e p r o t e i n was c o n s t a n t a l o n g t h e v i l l u s - c r y p t a x i s . T h i s r e s u l t was c o n s i s t e n t w i t h a c o n t i n u o u s brush-border-enzyme s y n t h e s i s a s t h e e n t e r o c y t e s migrate up t h e v i l l i . A s t u d y h a s been made of lysosomal-enzyme a c t i v i t i e s i n serum and l e u k o c y t e s i n c h r o n i c h e p a t i c d i s e a s e . 6 0 F i v e lysosomal -enzyme a c t i v i t i e s ( a r y l s u l p h a t a s e A , a-g-mannosidase, a - l - f u c o s i d a s e , B-g-
.
Carbohydrate Chemistry
400
B-e-galactosidase)
2-acetamido-2-deoxyhexosidase,and
were d e t e r m i n e d
i n s e r u m a n d l e u c o c y t e s o f 30 c o n t r o l s a n d 1 1 4 p a t i e n t s s u f f e r i n g from
various
liver
diseases,
including
30
with
idiopathic
haemochromatosis and 34 w i t h a l c o h o l i c c i r r h o s i s .
For further
d e t a i l s see i n i t i a l c i t a t i o n o f r e f . 6 0 . Degradation of
mucin oligosaccharides
of
human c o l o n
has been f o u n d t o be a s s o c i a t e d w i t h e x t r a c e l l u l a r ,
systems
but not w i t h
B - Q - g a l a c t 0 s i d a s e , B - Q - 2 - a c e tamido-2-deoxyglucosidase,
c e 11- b o u n d ,
and n e u r a m i n i d a s e . 2 9
Most p r o b a b l e numbers (MPN)
estimates
f a e c a l b a c t e r i a p r o d u c i n g e x t r a c e l l u l a r B-Q-galactosidase,
of
8-Q-2-
-
a c e t am i do 2 - d e o x y g 1u c o s id a s e, a n d n e u r a m i n id a s e r a n ge d f r o m 1 0
lo1'
g"
dry
faecal
weight.
further
For
details
see
-
initial
c i t a t i o n o f r e f .29. Adjuvant-induced
a r t h r i t i s i n r a t s has been s t u d i e d by t h e
changes i n s e r u m and u r i n a r y p r o t e i n - b o u n d c a r b o h y d r a t e m e t a b o l i t e s , changes i n s e r u m and t i s s u e l y s o s o m a l g l y c o h y d r o l a s e s , and l y s o s o m a l f r a g i l i t ~ . ~ ' The investigated,
free
I&.
activities
of
d e o x y g 1ucos idase , B-Q -ga l a c t 0s idas e , 0,
lysosomal glycohydrolases
$-Q --glucuronidase,
are increased i n l i v e r
$-e-2-acetamido-2-
a-Q -mannos idas e, and
and s p l e e n
i n the
c a t heps in,
acute phase.
For
f u r t h e r d e t a i l s see i n i t i a l c i t a t i o n o f r e f . 3 0 . The a c t i v i t i e s o f v a r i o u s g l y c o s i d a s e s i n homogenates o f t h e small
intestinal
wallabies
(M.
mucosa
eugenii)
investigated.38 deoxyglucosidase,
of
two
aged
adult
from
6
and to
$-Q-Galactosidase, a-&-fucosidase,
18 s u c k l i n g 50
weeks
tammar
have
been
B-D-2-acetamido-2-
and n e u r a m i n i d a s e a c t i v i t i e s w e r e
h i g h d u r i n g t h e f i r s t 3 4 weeks p o s t p a r t u m and t h e n d e c l i n e d t o l o w
F o r f u r t h e r d e t a i l s see i n i t i a l c i t a t i o n o f r e f . 3 8 .
levels.
The s t e r i c f a c t o r s i n v o l v e d i n t h e a c t i o n o f g l y c o s i d a s e s a n d Q-galactose Fucosidase,
oxidase
have
B-;-galactosidase,
h i n d e r e d by
certain types
been of
a-(1
+
21-1-
oxidase are s t e r i c a l l y
branching i n the
oligosaccharide
For f u r t h e r d e t a i l s see i n i t i a l c i t a t i o n of r e f . 1 0 9 .
chains.
Acid B-g-galactosidase assayed
for
i t s
galactopyranoside,
o f p o r c i n e a d r e n o c o r t i c a l lysosomes,
a c t i v i t y
towards
w e i g h t o f a p p r o x i m a t e l y 270,000 (termed form
4-nitrophenyl
B-o-
h a s been shown t o p o s s e s s t w o a c t i v i t y p e a k s on
g e l - f i l t r a t i o n p r o f i l e a t pH 7.4, 65,000
investigated.lo9
and ; - g a l a c t o s e
one c o r r e s p o n d i n g t o a m o l e c u l a r
(termed form A)
Another
and t h e o t h e r a b o u t
form o f a c i d B-n-galactosidase
w i t h a m o l e c u l a r w e i g h t o f a b o u t 130,000 ( t e r m e d f o r m A2) was f o u n d w h e n t h e h i g h - s p e e d e x t r a c t o r p a r t i a l l y p u r i f i e d f r m A 1 was
401
6: Enzymes c h r o m a t o g r a p h e d on Sephadex G-150 a t pH 4.5. M NaCl
I n t h e p r e s e n c e o f 0.1
o r s a t u r a t i n g a m o u n t s o f s u b s t r a t e a t pH 4.5 t h e h i g h - s p e e d
extract
showed
the
aggregation
of
form
A2
yielding
form
A3.
D i s s o c i a t i o n o f f o r m A3 b a c k t o f o r m A 1 was o b s e r v e d on i n c u b a t i o n a t 37OC i n 0.02
M sodium phosphate b u f f e r ,
pH 7.4,
f o l l o w e d by i r r e v e r s i b l e enzyme i n a c t i v a t i o n .
a n d t h a t was
D i s s o c i a t i o n of
A3 i n t o f o r m A 1 and enzyme i n a c t i v a t i o n i n p h o s p h a t e b u f f e r , M NaC1.
w e r e p r e v e n t e d b y a d d i t i o n o f 0.1
form
pH 7.4,
The i n t e r c o n v e r t i b l e
e n z y m i c f o r m s s h o w e d t h e same p H - a c t i v i t y p r o f i l e s a n d M i c h a e l i s constants.
These r e s u l t s s u g g e s t t h a t t h e
l y s o s o m a l a c i d B-Q-
g a l a c t o s i d a s e i n t h e p o r c i n e a d r e n a l c o r t e x e x i s t s i n v i v o as t h e dimer,
and t h a t t h e d i m e r may f u r t h e r a g g r e g a t e i n t o t h e t e t r a m e r .
F i f t e e n g l y c o s i d a s e s have been assayed i n l y m p h o m y e l o i d and d i g e s t i v e t i s s u e s o f Ginqlymostoma c i r r a t u m ,
Heterodontus f r a n c i s c i ,
and
a-Q-mannosidase
Etfiopterus
galactosidase,
spinax."
Activities
of
B-~-2-acetamido-2-deoxygalactosidase,
B-aB-Q-
g l u c u r o n i d a s e , a n d a- a n d B - Q - g l u c o s i d a s e s u s u a l l y w e r e h i g h e r i n lymphomyeloid tissues than i n digestive tissues. Sucrose
density
gradients
lysosome-containing
have
fractions
been
from
used t o
of
S t o m o x y s ~ a l c i t r a n s . The ~ ~ a c i d i c glycosidases (a-Q-glucosidase,
a-
galactosidase,
a-!-mannosidase,
B-Q-glucosidase,
B-D-glucuronidase,
deoxyglucosidase) e q u i l i b r a t e
white
characterize prepupae
&-galactosidase,
h
0
and
f3-Q-
B-Q-2-acetamido-2-
a t t h e same d e n s i t y
as d o e s a c i d
phosphatase. P u r i f i c a t i o n and p r o p e r t i e s h a v e been r e p o r t e d o f t h e t w o m a j o r isoenzymic
forms
of
B-Q-galactosidase
M y t i l u s e d u l i s hepatopancreas.131 and I V )
of
8-Q-galactosidase
lysosomes o f
M.
from
lysosomes
were
separated
e d u l i s hepatopancreas t o
p h y s i c a l and k i n e t i c p r o p e r t i e s of
and p u r i f i e d f r o m
homogeneity,
isoenzymes
F o r m s 111 and I V e x h i b i t d i f f e r e n t m o l e c u l a r
pH
Em, a n d y m a x v a l u e s
g a l a c t o p y r a n o s i d e as s u b s t r a t e . GM1-ganglioside;
form
4-nitrophenyl
were
weight,
B-p-
F o r m 111 h y d r o l y s e s l a c t o s e a n d
I V hydrolyses lactose.
a r y l B-Q-galactosidase. Hg2+,
with
and t h e
111 a n d I V
ascertained. optima,
of
11, 111, F o u r i s o e n z y m i c f o r m s (I,
Both forms hydrolyse
The t w o f o r m s were a c t i v a t e d by Cl',
while
Cu2+, and Zn2+ a l l i n h i b i t b o t h f o r m s . M u l t i p l e B-Q-galactosidase a c t i v i t i e s have been i d e n t i f i e d ,
separated,
and
characterized
isoelectrofocusing,
i n
Petunia hydrida.13*
Using
m u l t i p l e forms of Petunia B-c-galactosidase
a c t i v i t y c o u l d be d e t e c t e d .
The B - Q - g a l a c t o s i d a s e p a t t e r n s h o w e d
Carbohydrate Chemistry
402 only
minor
tissue-specific
species-specific bands
which
differed
preparations.
differences.
differences.
Zea mays,
from
the
There for
zones
were,
instance,
obtained
however, showed t w o
with
pgtgcLa
P e t u n i a and c o r n l e a v e s were m i x e d and e x t r a c t e d
commonly.
The
unchanged.
P e t u n i a p r e p a r a t i o n s were i n a c t i v a t e d by 8 M urea.
species-specific
Following dialysis,
activity
patterns remained
e n z y m a t i c a c t i v i t y and t h e P e t u n i a - s p e c i f i c
p a t t e r n were r e s t o r e d .
The same h o l d s t r u e f o r a m i x t u r e o f P e t u n i a
and E. c o l i B - g - g a l a c t o s i d a s e
preparations.
On r e f o c u s i n g i s o l a t e d
u n t r e a t e d o r i n a c t i v a t e d by 8 M u r e a and r e a c t i v a t e d
P e t u n i a zones,
i t seems
by d i a l y s i s , t h e o r i g i n a l m o b i l i t e s w e r e shown.
Therefore,
h i g h l y improbable t h a t t h e B-Q-galactosidase
p a t t e r n was d u e t o
artefacts. t o
U s i n g a P e t u n i a l i n e w h i c h was 'pure',
i t s 0-P-galactosidase
pattern,
the
four
also i n respect main bands were
p r e p a r a t i v e l y s e p a r a t e d by i s o e l e c t r o f o c u s i n g and c h a r a c t e r i z e d . They showed t h e same pH o p t i m u m (4.31, t h e same t e m p e r a t u r e o p t i m u m t h e same i n a c t i v a t i o n k i n e t i c s by u r e a ,
(55OC1,
a g a i n s t C1-,
a n d c l o s e l y r e l a t e d I&,, values.
c e n t r i f u g a t i o n they
i n v a r i a b l y showed
m u l t i p l e a c t i v i t i e s could not using
various
carrier
5
values
be s e p a r a t e d by
systems
t h e same s e n s i t i v i t y I n sucrose gradient
8
of
-
10.
The
zone e l e c t r o p h o r e s i s
o r by g e l f i l t r a t i o n .
I t seems
p o s s i b l e t h a t they represent forms which d i f f e r only i n i s o e l e c t r i c points, not i n molecular weight. B-g-Galactosidase the
organs
o f
the
a c t i v i t y has been f o u n d t o o c c u r i n a l l o f
sugar-cane
plant,
and i s
also
o c c u r r e n c e among d i f f e r e n t c u l t i v a r s a n d s p e c i e s . 1 3 3 a c t i v i t y was a s s o c i a t e d w i t h t h e c e l l w a l l , a n d o n l y was an i n t r a c e l l u l a r f o r m . pH a n d Hg2+,
Em, b o t h
of
general
Most o f t h e
G.
12
-
16%
B o t h a c t i v i t i e s possess s i m i l a r optimum
a r e a c t i v a t e d b y Mn2+ a n d e t h a n o l a n d i n h i b i t e d by
and b o t h a t t a c k t h e same s u b s t r a t e s . A process f o r production o f mould B-p-galactosidase
has been
d e v e 1 0 p e d . l ~ ~T e s t s w e r e c a r r i e d o u t i n p i l o t and i n d u s t r i a l s c a l e w i t h an A s p e r g i l l u s n i g e r s t r a i n s e l e c t e d a f t e r s c r e e n i n g a number o f moulds.
A c o m p u t e r - c o u p l e d a u t o a n a l y s e r s y s t e m was u s e d f o r
m o n i t o r i n g enzyme
formation
i n
the
p i l o t
fermentor.
B-e-
G a l a c t o s i d a s e p r o d u c t i o n was i n v e s t i g a t e d u s i n g d i f f e r e n t pH a n d temperature p r o f i l e s .
A.
n i g e r l a c t o s e h a s a n a c i d pH o p t i m u m , a
h i g h t e m p e r a t u r e optimum,and good s t a b i l i t y . any m e t a l i o n s .
l a c t o s e i n a c i d whey. p r o d u c t i o n was
I t does n o t r e q u i r e
It i s suitable for immobilization for hydrolysis of Three-fold
o b t a i n e d by
enhancement
m u t a g e n i z i n g A.
o f 8-~-galactosidase niger
u s i n g NTG as
403
6: Enzymes mutagenic agent.
The B - Q - g a l a c t o s i d a s e s p r o d u c e d b y t h e m u t a n t s
h a v e t h e same pH and t e m p e r a t u r e o p t i m a and s t a b i l i t y b u t t h e g r o w t h p r o p e r t i e s o f t h e m u t a n t s were d i f f e r e n t f r o m t h o s e o f t h e o r i g i n a l strain.
S u f f i c i e n t s p e c i f i c a c t i v i t y o f t h e enzyme p r e p a r a t i o n f o r
i m m o b i l i z a t i o n was o b t a i n e d b y p u r i f y i n g t h e e n z y m e b y s e l e c t i v e a d s o r p t i o n on Na-Ca-silicate. The s e c r e t i o n o f l y s o s o m a l enzymes h a s b e e n s t u d i e d i n t h e c e l l u l a r s l i m e m o u l d D i c t y o s t e l i u m d i s ~ o i d e u m . ~U~s i n g c o n d i t i o n s w h i c h e m p l o y a x a n i c a l l y g r o w n amoebae i n s u s p e n s i o n i n a s i m p l e starvation buffer,
t h e l y s o s o m a l enzymes a r e s e c r e t e d a t r a t e s t h a t
a r e a t l e a s t as r a p i d as d u r i n g n o r m a l d e v e l o p m e n t . growth or development, synthesis
under
Unlike normal
t h e r e i s no a p p r e c i a b l e lysosomal-enzyme
standard
secretion
conditions,
so
the
enzyme
a c t i v i t i e s a c t as m a r k e r s f o r t h e v e s i c l e s t h a t c o n t a i n them.
One
B-;a n d B-Q-
i n c l u d i n g B-~-2-acetamido-2-deoxyglucosidase,
g r o u p o f enzymes, mannosidase,
B-Q-glucosidase,
glucosidase-I,
are very e f f i c i e n t l y secreted,
total cellular
a c t i v i t y becoming e x t r a c e l l u l a r w i t h i n a few hours.
A l l o f t h e s e enzymes have s i m i l a r
8-g-galactosidase-I,
w i t h up t o 50% o f t h e
or i d e n t i c a l s e c r e t i o n k i n e t i c s
and may come f r o m t h e same l y s o s o m a l v e s i c l e s .
For f u r t h e r d e t a i l s
s e e i n i t i a l c i t a t i o n o f r e f .46. An enzyme f r o m R h i z o b i u m m e l i t o t i h a s been shown t o be a B-Qg l u c o s i d a s e which has B-e-galactosidase
activity.135
An e a r l i e r
s t u d y h a d s h o w n t h e e x i s t e n c e o f t w o e n z y m e s , A a n d 8, galactosidase
activity
i n R.
meliloti.
A mutant,
w i t h 6-p-
lacking
S-p-
g a l a c t o s i d a s e A, a l l o w e d a s t u d y t o b e made o f t h e r o l e o f e n z y m e B. I t was d e m o n s t r a t e d t h a t enzyme B,
cellobiose, and
i s able
4-nitrophenyl
respectively).
whose s y n t h e s i s was i n d u c i b l e by
t o hydrolyse 4-nitrophenyl B-g-galactopyranoside
(Km
B-c-glucopyranoside 0.135
and
Enzyme B i s a c t u a l l y a B - P - g l u c o s i d a s e
6.4
mM,
with
B-g-
galactosidase a c t i v i t y . Extracellular
B-8-galactosidase
of
Fusarium m o n i l i f o r m e grown
i n whey h a s b e e n ~ h a r a c t e r i 2 e d . l ~T~h e e n z y m e ,
p r e p a r e d as
an
e t h a n o l p r e c i p i t a t e , f u n c t i o n e d o p t i m a l l y a t pH 3.8
t o 5.0 a t 6OoC
on
lactose.
both
2-nitrophenyl
B-Q-galactopyranoside
and
The
a c t i v a t i o n e n e r g y o f t h e e n z y m i c h y d r o l y s i s o f t h e g l y c o s i d e and l a c t o s e i n t h e range of
20 t o 55OC was 8,500
a n d 7,200
cal
(s. 3.57
The Em v a l u e s w e r e 4.4 x l o 4 a n d 3.02 x l o 4 J m o l - l ) , r e s p e c t i v e l y . A t optimum a n d 12.4 m M f o r t h e g l y c o s i d e and l a c t o s e , r e s p e c t i v e l y . pH,
t h e enzyme l o s t h a l f o f i t s a c t i v i t y when i t was h e a t e d a t 5OoC
f o r 6 h.
A t t h e same pH, t h e l o s s was o n l y 5 % w h e n t h e e n z y m e was
Carbohydrate Chemistry
4 04 h e a t e d a t 37OC f o r 6 h.
A t o p t i m u m c o n d i t i o n s , 50% o f t h e l a c t o s e
i n whey was h y d r o l y s e d b y 1 0 u n i t s o f t h i s e n z y m e i n 5 0 h. The B - E - g a l a c t o s i d a s e a
homogeneous
state
o f A l t e r n a r i a t e n u i s h a s been p u r i f i e d t o
from
a
filtrate
of
the
culture
fluid
by
p r e c i p i t a t i o n w i t h a c e t o n e , i o n - e x c h a n g e c h r o m a t o g r a p h y o n DEAEcellulose,
s o r p t i o n on h y d r o x y a p a t i t e ,
and a f f i n i t y c h r o m a t o g r a p h y
on N-B-~-galactopyranosylthiocarbamoyl-s-aminohexanoyl-AN-Sepharose 48.137
Homogeneity
was
confirmed
by
ultracentrifugation
and
e l e c t r o p h o r e s i s i n p o l y a c r y l a m i d e g e l w i t h a n d w i t h o u t SDS.
The
s p e c i f i c a c t i v i t y o f t h e h o m o g e n e o u s e n z y m e was 1 6 0 u n i t s p e r mg protein.
The m o l e c u l a r w e i g h t ,
142,000-176,000, 3.8-4.4
for
PI 4.6,
2-nitrophenyl
lactose,
Em =
a n d 6.57
x
0.21
x
up
t o
lactose.
30%
chloromercuribenzoate activity.
B-;-galactopyranoside
was
pH o p t i m u m
a n d 3.6-4.8
for
M f o r 2 - n i t r o p h e n y l B-Q-galactopyranoside
M for
c o n t a i n s
d e t e r m i n e d by v a r i o u s m e t h o d s ,
t e m p e r a t u r e o p t i m u m 60-65'C,
P-Galactose
The enzyme i s a g l y c o p r o t e i n and
carbohydrates.
have
no
effect
on
H4 the
e d t a
and
4-
B-Q-galactosidase
i s a competitive inhibitor.
Q - G l u c o s e h a s no
i n h i b i t i n g e f f e c t on t h e enzyme. Aspergillus niger 6-Q-galactosidase
mutant s t r a i n s
have
Aspergillus niger mutant s t r a i n , three- t o
four-fold
i n
producing elevated levels o f
been i s o l a t e d the
and
c h a r a ~ t e r i 2 e d . l ~ An ~
VTT-D-80144,
w i t h an i m p r o v e m e n t o f
production of
extracellular
g a l a c t o s i d a s e was i s o l a t e d a f t e r m u t a g e n e s i s .
B-e-
The p r o d u c t i o n o f B-
E - g a l a c t o s i d a s e by t h i s m u t a n t was u n a f f e c t e d by f e r m e n t e r s i z e ,
and
t h e enzyme was a l s o s u i t a b l e f o r i m m o b i l i z a t i o n . The u s e o f b r u s h i t e (CaHP04.2H20) been
described
and
f o r enzyme i m m o b i l i z a t i o n h a s
exemplified
with
B-Q-galactosidase.13'
I m m o b i l i z a t i o n was c a r r i e d o u t a t 5 O C i n a 0.01 M T r i s b u f f e r , 7.6,
c o n t a i n i n g 0.01 M p h o s p h a t e .
pH
The i m m o b i l i z e d enzyme s h o w e d
h i g h a c t i v i t y o v e r a l o n g p e r i o d o f t i m e ( a f t e r 90 d a y s t h e a c t i v i t y was s t i l l 44% o f t h e i n i t i a l v a l u e ) . these
matrices i s
very
low,
The l e a k a g e o f enzyme f r o m
a m o u n t i n g t o no more t h a n
4% u p o n
w a s h i n g a c o l u m n f u l s a t u r a t e d w i t h enzyme w i t h 300 c o l u m n v o l u m e s o f buffer.
A
method i s d e s c r i b e d i n which
virtually negligible.
t h e enzyme
leakage i s
One a d v a n t a g e o f b r u s h i t e a s a m a t r i x f o r
enzyme i m m o b i l i z a t i o n i s t h a t t h e i m m o b i l i z a t i o n can be done i n a l m o s t any b u f f e r , e v e n a t h i g h s a l t c o n c e n t r a t i o n , high concentrations enzymes
of
phosphate are avoided.
can be desorbed
concentration.
easily
by
provided that
The
immobilized
increasing the
S i n c e t h e enzyme i s a d s o r b e d a t
the
phosphate t o p of
the
6: Enzymes column,
405 no p r e c o n c e n t r a t i o n o f t h e enzyme s o l u t i o n i s r e q u i r e d p r i o r
t o im mob i1iza t i o n . B-8-Galactosidase
has been i m m o b i l i z e d i n a h o l l o w - f i b r e u l t r a 2-nitrophenyl
B-Q-
g a l a c t o p y r a n o s i d e was s i g n i f i c a n t l y a f f e c t e d by enzyme l o a d i n g ,
flow
f i l t r a t i o n
module.140
The
hydrolysis
r a t e , and s u b s t r a t e c o n c e n t r a t i o n . w i t h a p r o t e i n was Residence-time the
reaction
necessary
distribution was
of
Pretreatment of hollow fibres
to
m i n i m i z e enzyme
studies
significantly
indicated
adsorbed
that
by
inactivation. the
the
product
fibres,
r e s u l t e d i n t h e r e a c t o r t a k i n g 10-30 h t o a c h i e v e s t e a d y s t a t e . e q u a t i o n b a s e d on M i c h a e l i s - M e n t e n
kinetics
and a p l u g - f l o w
of
which An
model
adequately
d e s c r i b e d t h e performance o f t h e r e a c t o r w i t h r e g a r d t o
operating
variables,
even
though
some
diffusion
effects
were
observed. N o v o b i o c i n (0.05
pg m l - l ) has been f o u n d t o r e d u c e t h e g r o w t h
r a t e o f c u l t u r e s o f E s c h e r i c h i a c o l i s t r a i n DK6 by a b o u t a f a c t o r o f 2.141
The
l a g i n appearance
of
B-E-galactosidase-for ming c a p a c i t y
was e x t e n d e d f r o m 5 0 s t o 8 5 s b y t h e d r u g .
T h i s a p p e a r e d t o be t h e
r e s u l t o f a r e d u c e d r a t e o f n a s c e n t mRNA e l o n g a t i o n . The s y n t h e s i s o f B - g - g a l a c t o s i d a s e
i n E. c o l i h a s been shown t o
be r e p r e s s e d as a r e s u l t o f i n f e c t i o n w i t h s i n g l e - s t r a n d e d DNA phage 0X174.142
An amber
m u t a n t i n OX174 c i s t r o n A,
w h i c h codes f o r t w o
p r o t e i n s , does n o t i n h i b i t t h e enzyme s y n t h e s i s , i n a l l o t h e r g e n e s do c a u s e r e p r e s s i o n .
w h i l e amber m u t a n t s
A-mutant near t h e amino-
w h i c h p r o d u c e s t h e s m a l l 35,000
t e r m i n a l end o f c i s t r o n A ,
molecular-
w e i g h t c i s t r o n A p o l y p e p t i d e , a l s o i n h i b i t s t h e s y n t h e s i s o f B-Qgalactosidase.
I n h i b i t i o n i s a l s o o b s e r v e d i n an E. c o l i
rep m u t a n t
w h i c h does n o t s u p p o r t t h e r e p l i c a t i o n o f r e p l i c a t i v e - f o r m DNA. Exogenous n u c l e o t i d e bases and a d e n o s i n e 3’,5’-phosphate
do n o t h a v e
any e f f e c t on t h e d e g r e e o f r e p r e s s i o n . The i n t e r a c t i o n b e t w e e n t h e t r a n s p o r t s y s t e m s o f m e t h y l a - g glucopyranoside K12.143
It
and B - E - g a l a c t o s i d a s e s was
shown
that
the
has
been s t u d i e d
addition
of
i n E. c o l i
methyl
a-Q-
glucopyranoside t o the c e l l s leads t o suppression o f the r a t e of accumulation of
{ 1 4 C ) l a c t o s e and t h e h y d r o l y s i s o f
E-galactopyranoside.
M u t a t i o n damage t o one
t h e system o f methyl a-g-glucopyranoside
phosphoenolpyruvate-dependent
of
the
esters
of
effectiveness
p h o s p h o r y l a t i o n i s accompanied by
m e t h y l a-q-glucopyranoside of
the
components of
transport coupled w i t h
e l i m i n a t i o n o f the i n h i b i t i n g a c t i o n o f glucoside. phosphate
2 - n i t r o p h e n y l B-
the
transmembrane
Intracellular
o r ;-glucose transport
of
lower
B-g-
Carbohydrate Chemistry
406 galactosides.
The r e s u l t s o b t a i n e d s u g g e s t t h a t s u p p r e s s i o n o f t h e
B - Q - g a l a c t o s i d e permease a c t i v i t y d u r i n g t h e t r a n s p o r t o f m e t h y l
6-
q - g l u c o p y r a n o s i d e may b e a consequence o f t w o p r o c e s s e s : t r a n s l a t i o n of
methyl
B-g-glucopyranoside
intrinsically
coupled
with
p h o s p h o r y l a t i o n and a c c u m u l a t i o n o f m e t h y l B-~-glucopyranoside-6An i n t r a m e m b r a n e i n t e r a c t i o n ( d i r e c t o r
phosphate w i t h i n t h e c e l l .
i n d i r e c t ) b e t w e e n t h e enzyme IIG and lc B - Q - g a l a c t o s i d e permease i s suggested. Oxygen-18 l e a v i n g - g r o u p been d e t e r m i n e d on b o t h galactoside-catalysed
k i n e t i c i s o t o p e e f f e c t s (KIEs)
lmax (1) a n d l m a x / K m (!/El
hydrolysis o f 4-nitrophenyl
(I) a n d 2 , 4 - d i n i t r o p h e n y l
f3-Q-galactoside
s u b s t r a t e e x h i b i t s K I E s o f 1.022
-V/K,
-+
2
have
f o r t h e 8-gB-P-galactoside
(II).144 The f o r m e r
2
0.002 a n d 1.014
0.003 o n
1
and
r e s p e c t i v e l y , w h i l e c o r r e s p o n d i n g K I E s f o r t h e l a t t e r a r e 1.002
0.009
and
scission
i s
1.030
2 0,003.
largely
These
results
rate determining for
substrate saturation.
The f i r s t
indicate
that
bond
I b u t n o t f o r I1 a t
irreversible step for
both
s u b s t r a t e s must i n v o l v e cleavage o f t h e bond t o t h e n i t r o p h e n y l leaving
group.
The
mechanism
proposed
for
this
reaction
i s
c h a r a c t e r i z e d by t w o p a r a l l e l p a t h w a y s f o r s u b s t r a t e h y d r o l y s i s . The p r e d o m i n a n t r o u t e f o r
a l l b u t t h e most r e a c t i v e s u b s t r a t e s
i n c l u d e s an SN2 n u c l e o p h i l i c d i s p l a c e m e n t o f a g l y c o n by t h e enzyme t o y i e l d a c o v a l e n t g - g a l a c t o s y l enzyme, w h i c h i n t u r n i s h y d r o l y s e d
via a
n u c l e o p h i l i c a t t a c k by w a t e r .
(e.g.
11) f o r m t r a n s i e n t l y an enzyme-bound P - g a l a c t o s y l o x o c a r b o n i u m
ion
which
partitions
between
The m o s t r e a c t i v e s u b s t r a t e s
enzyme
to
give
the
covalent
E-
g a l a c t o s y l enzyme and H20 t o y i e l d ! - g a l a c t o s e . B-P-Galactosidase
has been f o u n d t o a c t
on u - l a c t o s e
slightly
more t h a n t w i c e as r a p i d l y as on B - l a c t o s e f o r b o t h t h e h y d r o l y s i s and t r a n s g a l a c t o s y l i s r e a c t i o n s . 1 4 5 the
lmax v a l u e s ; t h e Km v a l u e s
t h e same.
The s t e p o f
The e f f e c t
was
shown t o b e on
f o r t h e d i f f e r e n t a n o m e r i c forms were
the reaction
for
which
the
enzyme has
a n o m e r i c s p e c i f i c i t y was shown t o b e g l y c o s i d i c b o n d b r e a k a g e . s t e p s i n !-glucose
The
release or i n the Q-glucose acceptor r e a c t i o n
were n o t a f f e c t e d by a n o m e r i c c o m p o s i t i o n . h y d r o l y s i s nor t r a n s p o r t o f
Neither allolactose
l a c t o s e i n t o t h e c e l l s by
permease
was s e n s i t i v e t o t h e a n o m e r i c c o m p o s i t i o n o f t h e s u b s t r a t e . i m p l i c a t i o n s o f these r e s u l t s f o r
lac
The
o p e r o n i n d u c t i o n and f o r
l a c t o s e metabolism are discussed. The p o s i t i o n s o f t h e lacZX9O m u t a t i o n and h y b r i d i z a t i o n b e t w e e n c o m p l e t e and i n c o m p l e t e B - P - g a l a c t o s i d a s e
have been i n ~ e s t i g a t e d . ' ~ ~
407
6: Enzymes
The p o s i t i o n o f t h e t e r m i n a t i o n codon i n lacZX9O was d e t e r m i n e d by isolation of a
&+ revertant
I - l y s i n e w h i c h was f o u n d t o r e p l a c e
t y r o s i n e a t p o s i t i o n 1,012 o f B - g - g a l a c t o s i d a s e , protein lacked the carboxy-terminal
L-
i n d i c a t i n g t h a t X90
10 r e s i d u e s .
A heat-
and u r e a -
s e n s i t i v e h y b r i d enzyme was f o r m e d i n v i v o when supC, w h i c h s u p p l i e s I-tyrosine
t o the p o s i t i o n i n the polypeptide corresponding t o the
n o n s e n s e c o d o n , was u s e d t o s u p p r e s s l a c Z X 9 O .
T h i s r e s u l t shows
t h a t s u p p r e s s i o n t h a t adds b a c k t h e o r i g i n a l a m i n o a c i d may n o t l e a d to
the
production of
multimeric,
the
wild-type
enzyme i f
the
latter
i s
because i n c o m p l e t e c h a i n s can be i n c o r p o r a t e d i n t o t h e
oligomer. Mechanism
of
galactosidase
the
r e 1 defect
synthesis.14’
has
been i d e n t i f i e d
Relaxed
(relA)
i n
B-g-
mutants
o f
E s c h e r i c h i a c o l i are d e f e c t i v e i n B-P-galactosidase s y n t h e s i s d u r i n g amino a c i d l i m i t a t i o n .
T h i s d e f e c t was f o u n d t o c o m p r i s e b o t h a
t r a n s c r i p t i o n a l component and a t r a n s l a t i o n a l component. coupled transcription-translation
An i n v i t r o
s y s t e m has been
a s k e d t o s y n t h e s i z e t r a n s a m i n a s e B and B - Q - g a l a c t o s i d a s e presence
of
a
deoxyribonucleic
deoxyribonucleic presence o f
a c i d under
acid
template
normal &-specific
i n the
containing
lac
c o n t r o l and i n t h e
several deoxyribonucleic acid templates containing
deoxyribonucleic
acid
fused
to
the
i l v D gene.148
Time-course
e x p e r i m e n t s r e v e a l e d t h a t t r a n s c r i p t i o n o f t h e l a c Z gene f r o m t h e fusion template required a longer time than d i d t h a t i n i t i a t e d a t the
promoter.
W i t h a phage t e m p l a t e c o n t a i n i n g an i n t a c t i l v E
gene b u t l a c k i n g t h e n o r m a l * - s p e c i f i c
promoter, synthesis o f i l v E
m e s s a g e was c o m p l e t e d b e f o r e s y n t h e s i s o f l a c Z m e s s a g e . template
that
contained
the normal =-specific
A phage
promoter but from
w h i c h p a r t o f i l v E h a d b e e n d e l e t e d a l s o a l l o w e d f o r m a t i o n o f B-Pgalactosidase.
Three p l a s m i d s c o n t a i n i n g t h e i l v - l a c
a l s o u s e d as t e m p l a t e s .
i l v E gene and t h e n o r m a l * - s p e c i f i c for
fusion
were
Two p l a s m i d s t h a t c o n t a i n e d b o t h an i n t a c t
promoter required longer times
l a c Z t r a n s c r i p t i o n b u t w e r e more e f f i c i e n t t e m p l a t e s t h a n was a
plasmid i n which the contiguous non-specific the normal *-specific
i l 1 - L ~f u~s i o n ,
t h e i l v E gene,
and t h e
p r o m o t e r were i n v e r t e d w i t h r e s p e c t t o promoter.
B-P-Galactosidase
synthesis
was
s t i m u l a t e d by g u a n o s i n e 3 ’ - p y r o p h o s p h a t e - 5 ’ - p y r o p h o s p h a t e w i t h a 11 t e m p l a t e s t e s t e d except t h a t i n which t h e i l v - l a c f u s i o n has been inverted
.
A p l a s m i d was c o n s t r u c t e d t h a t a l l o w s t h e s e l e c t i o n i n v i v o o f
gene f u s i o n s b e t w e e n t h e E s c h e r i c h i a c o l i B - 0 - g a l a c t o s i d a s e
gene and
408
Carbohydrate Chemistry
t h e y e a s t ( S a c c h a r o m y c e s c e r e v i s i a e ) URA3 gene.149
A large yeast
DNA f r a g m e n t c o n t a i n i n g URA3 gene was p l a c e d u p s t r e a m o f t e r m i n a l l y d e p l e t e d v e r s i o n of c o n t a i n s sequences t h a t
t h e l a c Z gene.
an a m i n o -
The p l a s m i d v e h i c l e
a l l o w s e l e c t i o n and m a i n t e n a n c e o f
p l a s m i d i n b o t h y e a s t a n d E.
coli.
lac+ i n
Selection for
E.
the coli
y i e l d e d n u m e r o u s d e l e t i o n s t h a t f u s e d t h e l a c Z gene t o t h e URA3 gene and
flanking
yeast
sequences,
to
the
bacterial
tetracycline-
r e s i s t a n c e g e n e f r o m t h e p a r e n t p l a s m i d pBR322, a n d t o t h e y e a s t 2 pm
Some
p l a s m i d DNA.
of
these
fusion
plasmids
8-g-
produced
g a l a c t o s i d a s e a c t i v i t y under u r a c i l r e g u l a t i o n i n yeast. Overlapping
sequences
have been d e t e c t e d i n B - Q - g a l a c t o s i d a s e
a - c o r n p 1 e m e n t a t i 0 n . l ~ ~ Enzyme a c t i v i t y i s r e s t o r e d t o t w o d e f e c t i v e 6-g-galactosidase
m o l e c u l e s (M15 p r o t e i n l a c k i n g a m i n o a c i d r e s i d u e s
1 1 - 4 1 a n d M112 p r o t e i n l a c k i n g r e s i d u e s 2 3 - 3 1 ) by i n c u b a t i o n w i t h p e p t i d e r e s i d u e s 3-92 (a-acceptors)
o f B-P-galactosidase.
are dimers.
M15 and M112 p r o t e i n s
Complemented enzyme,
a tetrameric structure.
Cleavage o f
the
l i k e w i l d type,
same
has
&-
peptide with
g l u t a m i c a c i d - s p e c i f i c p r o t e a s e y i e l d e d a much s m a l l e r a - d o n o r p e p t i d e ) w h i c h was a l s o e f f e c t i v e i n c o m p l e m e n t a t i o n ,
(3-41
indicating
t h a t t h e M15 p r o t e i n c a n s u p p l y a l l o f t h e r e s i d u e s f r o m 4 2 - 9 2 f o r t h e s t r u c t u r e o f c o m p l e m e n t e d enzyme. 41
peptide
complemented
enzyme
T r e a t m e n t o f M112 p r o t e i n / 3 -
with
trypsin
under
very
mild
c o n d i t i o n s f o l l o w e d by e x a m i n a t i o n o f t h e p r o d u c t s d e m o n s t r a t e d t h a t a-donor
peptide supplies the NH2-terminal
enzyme.
Similar
trypsin treatment o f
segment
of
complemented
M15 p r o t e i n / C N B r 2
indicated
t h a t i n t h i s c o m p l e m e n t e d enzyme t h e p o l y p e p t i d e r e g i o n b e y o n d t h o s e residues m i s s i n g i n t h e a-acceptor donor
or
the
a-acceptor.
susceptible t o
Both
mild tryptic
react
with
galactosidase.
these
The
effect
two
M112 p r o t e i n ,
anti-B-Q-galactosidase of
and
M112 p r o t e i n
proteolysis than
i n d i c a t i n g a more open s t r u c t u r e . that
c a n be p r o v i d e d b y e i t h e r t h e aM15
are
more
c o m p l e m e n t e d enzyme,
Several antipeptide antibodies
proteins
do
not
l i k e M15 p r o t e i n ,
react
with
B-q-
c a n be a c t i v a t e d by
b u t t o a much h i g h e r l e v e l .
single
amino
acid substitutions i n the
g a l a c t o s i d a s e p o l y p e p t i d e c h a i n has been s t u d i e d . l 5 l
B-g-
Amino a c i d
s u b s t i t u t i o n s w e r e made a t f o u r d i f f e r e n t p o s i t i o n s , r e s i d u e s 1 7 , 23,
36,and
41,
i n the 8-e-galactosidase
polypeptide chain.
e f f e c t s on p r o p e r t i e s o f t h e p r o t e i n w e r e s t u d i e d .
The
S u b s t i t u t i o n s of
i - t y r o s i n e f o r t h e n o r m a l l y o c c u r r i n g amino a c i d s a t these p o s i t i o n s do n o t s i g n i f i c a n t l y a f f e c t enzyme a c t i v i t y , s e n s i t i v i t y i n ureas, a b i l i t y t o r e n a t u r e from urea.
Enzymes,
l i k e the w i l d type,
or
are
6: Enzymes
409
tetrameric. B-p-Galactosidases c o n t a i n i n g L - t y r o s i n e a t 23, 36, o r 41 a r e a s h e a t s t a b l e a s o r more h e a t s t a b l e t h a n w i l d t y p e , b u t &t y r o s i n e , I - g l u t a m i n e , o r L - s e r i n e s u b s t i t u t i o n s a t p o s i t i o n 17 make t h e enzyme l e s s h e a t s t a b l e . The e f f e c t s o f a m i n o a c i d s u b s t i t u t i o n s on a-complementation were s t u d i e d a f t e r i s o l a t i n g ad o n o r p e p t i d e s ( r e s i d u e s 3 - 9 2 ) f r o m t h e s u b s t i t u t e d B-0,galactosidases. &-Tyrosine s u b s t i t u t i o n a t r e s i d u e s 23, 36, and 4 1 h a d l i t t l e e f f e c t on a - d o n o r a c t i v i t y o r h e a t s t a b i l i t y o r c o m p l e m e n t e d enzymes w i t h o t h e r M15 p r o t e i n o r M112 p r o t e i n a s a acceptor. I n c o n t r a s t , I - t y r o s i n e s u b s t i t u t i o n for t h e normally o c c u r r i n g I - g l u t a m i c a c i d a t r e s i d u e 17 l e a d s t o s i g n i f i c a n t l y d e c r e a s e d a-donor a c t i v i t y and d e c r e a s e d h e a t s t a b i l i t y of c o m p l e m e n t e d enzyme. P e p t i d e s w i t h I - g l u t a m i n e and I - s e r i n e s u b s t i t u t e d a t r e s i d u e 17 h a v e n e a r l y n o r m a l a - d o n o r a c t i v i t i e s . The p o o r a - d o n o r a c t i v i t i e s o f t h e I - t y r o s i n e 1 7 p e p t i d e can be Very a t t r i b u t e d t o i t s lower a f f i n i t y f o r t h e a - a c c e p t o r p r o t e i n s . l i t t l e secondary s t r u c t u r e was s e e n i n C N B r 2 p e p t i d e s b y c i r c u l a r d i c h r o i s m measurements. P r e d i c t i o n of secondary s t r u c t u r e b y Chou and Fasman (Chou, P.Y. and F a s m a n , G.D. ( 1 9 7 8 ) A d v . Enzymol., 47, 45-148) r u l e s s u g g e s t s t h a t p o s i t i o n 1 7 i s between two B-bends. LTyrosine s u b s t i t u t i o n s a t r e s i d u e 17 could cause a c r i t i c a l localized perturbation i n structure before or a f t e r interaction w i t h a-acceptor proteins. S i x hybridomas producing monoclonal a n t i b o d i e s E s c h e r i c h i a c o l i 6 - a - g a l a c t o s i d a s e have been d e r i v e d from two s e p a r a t e s o m a t i c c e l l fusions.152 T h r e e of t h e s e a n t i b o d i e s can a c t i v a t e d e f e c t i v e e n z y m e s p r o d u c e d b y s t r a i n s of E. c o l i c a r r y i n g Z-gene p o i n t m u t a t i o n s . I n a n t i g e n e x c e s s , one monoclonal a n t i b o d y shows s i m i l a r enzyme-binding and mutant - a c t i v a t i n g c a p a c i t y . C h a r a c t e r i s t i c a l l y, t h e former r e a c t i o n has a 200-fold higher equilibrium constant. These d a t a provide d i r e c t evidence t h a t t h e enzyme-activation r e a c t i o n i s a s i n g l e - h i t e v e n t i n w h i c h one a n t i b o d y s i t e f a v o u r s t h e c o r r e c t c o n f o r m a t i o n of one a c t i v e c e n t r e of t h e enzyme. Because each a c t i v a t i n g hybridoma i s a b l e t o a c t i v a t e s e v e r a l b u t not a l l p o i n t mutant enzymes t e s t e d , i t a p p e a r s t h a t t h e c o r r e c t i o n o f t h e g e n e t i c d e f e c t i s p r o d u c e d b y b i n d i n g key s i t e s of t h e p r o t e i n t h r e e - d i m e n s i o n a l s t r u c t u r e r a t h e r than t h e s i t e s a f f e c t e d by t h e m u t a t i o n . The mechanism b y which l a r g e p r e m a t u r e t e r m i n a t i o n f r a g m e n t s of 6 - E - g a l a c t o s i d a s e a r e degraded b y E s c h e r i c h i a c o l i has been s t u d i e d ~ different u s i n g q u a n t i t a t i v e i m m u n o p r e c i p i t a t i o n t e ~ h n i q u e s . ' ~Two
410
Carbohydrate Chemistry
l a c Z nonsense
mutants
which produced apparent p r i m a r y t r a n s l a t i o n
and 109,000
p r o d u c t s of 96,000
daltons,
respectively,
produce a second B-Q-galactosidase-related 9 0 ,0 00.
w e r e shown t o
=
p o l y p e p t i d e o f blr
These 9 0 , 0 0 0 d a l t o n p o l y p e p t i d e s a p p e a r e d t o be t h e same i n
b o t h s t r a i n s s i n c e t h e y c o - m i g r a t e d when a n a l y s e d a s a m i x t u r e o f sodium
dodecyl
sulphate-polyacrylamide
gels
and
were
i n d i s t i n g u i s h a b l e when a n a l y s e d by o n e - d i m e n s i o n a l p e p t i d e mapping. Pulse-chase experiments established a s t o i c h i o m e t r i c precursorproduct
relationship
between t h e
primary mutant
gene p r o d u c t s
( c a l l e d t h e A p o l y p e p t i d e s ) and t h e common 9 0 , 0 0 0 d a l t o n p o l y p e p t i d e (called the B polypeptide).
No i n t e r m e d i a t e s w e r e d e t e c t e d b e t w e e n
t h e A and B p o l y p e p t i d e s . common
pathway
for
the
T h e a u t h o r s p r o p o s e t h a t t h e r e is a degradation
fragments f o r B-Q-galactosidase.
of
these
different
large
A c c o r d i n g t o t h i s model, t h e f i r s t
s t e p w o u l d be a s p e c i f i c e n d o p r o t e o l y t i c c l e a v a g e o f t h e p r i m a r y t r a n s l a t i o n product
w h i c h p r o d u c e s t h e 9 0 , 0 0 0 d a l t o n p o l y p e p t i d e as
a common i n t e r m e d i a t e .
The k i n e t i c a n a l y s i s d e m o n s t r a t e d a f i r s t -
o r d e r decay o f b o t h A and B p o l y p e p t i d e s b u t , first-order
surprisingly,
the
r a t e c o n s t a n t f o r t h e decay o f A a p p e a r e d d e p e n d e n t upon
t h e i n d u c t i o n regimen.
T h i s r e s u l t s u g g e s t e d t h a t d e g r a d a t i o n may
p o s s i b l y b e a u t o r e g u l a t e d e i t h e r by t h e i n t r a c e l l u l a r l e v e l o f A o r by t h e o t h e r i n t e r m e d i a t e s i n t h e d e g r a d a t i o n p a t h w a y . Active-site-directed
i r r e v e r s i b l e i n h i b i t i o n o f g l y c o s i d a s e s by
t h e c o r r e s p o n d i n g g l y c o s y 1m e t h y 1 - ( 4 - n i t r o p h e n y l ) t r i a z e n e s h a s been i n v e s t i g a t e d .72
B-p-Galactopyr anosylmethy1 - ( 4 - n i t r o p h e n y 1 ) t r i a z i n e
i s an a c t i v e - s i t e - d i r e c t e d ebg'
i r r e v e r s i b l e i n h i b i t o r (ASDIN)
and t h e l a c Z 6 - q - g a l a c t o s i d a s e s
of the
of Escherichia c o l i o f the
6-E-
g a l a c t o s i d a s e o f human l i v e r l y s o s o m e s , and,
less effectively,
the B-Q-galactosidase
I t has n o e f f e c t on d e t a i l s see i n i t i a l
the
lac
o f g r e e n c o f f e e beans.
r e p r e s s o r o f E. c o l i .
For
further
of
c i t a t i o n o f r e f .72. The p r o d u c t s o f 8 - g - g a l a c t o s i d a s e E.
a c t i o n on l a c t o s e by i n t a c t
c o l i c e l l s h a v e b e e n f o u n d t o a p p e a r as s o o n as l a c t o s e i s added
t o t h e medium and t h e amount o f p r o d u c t i s e q u a l t o t h e l a c t o s e used.154
No d e t e c t a b l e l e v e l s o f 6 - 0 - g a l a c t o s i d a s e
t h e medium,
p e r m e a s e was
present.
The a p p e a r a n c e d i d n o t
depend upon t h e
p r e s e n c e o f any o f t h e c o m m o n l y k n o w n ! - g a l a c t o s e permease systems. equal t o the
were found i n
a n d l a c t o s e was n o t s i g n i f i c a n t l y b r o k e n d o w n u n l e s s
Kt
The
Em o f
f o r l a c t o s e t r a n s p o r t by
c e l l s were b r o k e n t h e
5,
or !-glucose
p r o d u c t a p p e a r a n c e f r o m w h o l e c e l l s was permease.
became t h e n o r m a l B - g - g a l a c t o s i d a s e
When t h e
Em.
6: Enzymes
41 1
The B - e - g a l a c t o s i d a s e s of Medicago s a t i z cells have been investigated during suspension-cultured growth of 1 a ~ t o s e . l ~ ~ D u r i n g t h e g r o w t h o f s u s p e n s i o n - c u l t u r e d M. s a t i v a c e l l s , B - c g a l a c t o s i d a s e a c t i v i t i e s were i n v e s t i g a t e d , u s i n g t h e s t a n d a r d s u b s t r a t e 2-nitrophenyl-B-Q-galactopyranoside, w i t h respect t o t h e l a c t o s e uptake and r e s i d u a l s u g a r l e v e l s i n t h e c e l l s and i n t h e b r o t h . T h e pH p r o f i l e s s h o w i n g t w o o p t i m a a t pH 4 a n d pH 7 i n c r u d e e x t r a c t s o f c e l l s s t r o n g l y s u g g e s t e d t h e o c c u r r e n c e o f t w o B-Qgalactosidase activities. O n l y o n e o p t i m u m pH was d e t e c t e d i n t h e T h e B - -Q - g a l a c t o s i d a s e a c t i v i t y a t pH 7 was b r o t h a t a b o u t pH 4 . 4 . f o u n d t o b e l o c a t e d i n s i d e t h e c e l l s w h i l e t h a t o b s e r v e d a t pH 4 was bound t o t h e c e l l wall. A t t h e o n s e t of t h e a c t i v e growth phase an i n c r e a s e i n t h e B - 2 - g a l a c t o s i d a s e a c t i v i t y o c c u r r e d i n t h e medium, p r o b a b l y due t o a release o f t h e c e l l - w a l l - l i n k e d enzyme. The s e p a r a t i o n o f t h e i n t r a c e l l u l a r B - g - g a l a c t o s i d a s e s by g e l f i l t r a t i o n o n a S e p h a d e x G - 2 0 0 c o l u m n w a s a c h i e v e d a n d t h e pH d e p e n d e n c e s o f t h e e n z y m e s were c o n f i r m e d . T h e t w o e n z y m e s were f o u n d t o b e e f f i c i e n t on l a c t o s e h y d r o l y s i s . A new p r o c e d u r e f o r i n d u c i n g a n d p u r i f y i n g e n d o - B - g - g a l a c t o s i d T h e e n z y m e was a s e f r o m E s c h e r i c h i a f r e u n d i i has b e e n d e s c r i b e d . 1 5 6 found t o be induced w i t h high e f f i c i e n c y i n c u l t u r e medium c o n t a i n i n g S m i t h - d e g r a d e d h o g g a s t r i c m u c i n , w h i c h was p r e p a r e d f r o m a c o mmer c i a 1l y a v a i l a b l e s t a r t i n g mat e r i a l . e n d o - B - g - G a l a c t 0s i d a s e was t h e n p u r i f i e d by ammonium s u l p h a t e f r a c t i o n a t i o n , DEAE-Sephadex c h r o m a t o g r a p h y , and a f f i n i t y c h r o m a t o g r a p h y on S e p h a r o s e c o n j u g a t e d w i t h t h e S m i t h - d e g r a d e d m u c i n . T h e e n z y m e t h u s p u r i f i e d by o n l y t h r e e s t e p s s h o w e d n o o t h e r g l y c o s i d a s e or p r o t e a s e a c t i v i t i e s a n d had h i g h e r s p e c i f i c a c t i v i t y compared t o t h e p r e v i o u s method. T h i s new m e t h o d h a s a g r e a t a d v a n t a g e s i n c e t h e g a s t r i c m u c i n i s a b u n d a n t l y a v a i l a b l e a n d t h e e f f i c i e n c y o f e n z y m e p r o d u c t i o n was high without s i g n i f i c a n t induction of exoglycosidase. The h y d r o l y s i s of o l i g o s a c c h a r i d e s , g l y c o s p h i n g o l i p i d , and k e r a t a n Kinetic s u l p h a t e was s t u d i e d by u s i n g t h i s n e w l y p u r i f i e d e n z y m e . data i n d i c a t e t h a t h y d r o l y s a b i l i t y of t h e s e s u b s t r a t e s is l a r g e l y a f f e c t e d by s u b s t r a t e c o n c e n t r a t i o n , e n z y m e c o n c e n t r a t i o n , a n d t h e s t r u c t u r e of s u b s t r a t e s . Based on t h e s e r e s u l t s , t h e s p e c i f i c i t y o f E. f r e u n d i i e n d o - 6 - Q - g a l a c t o s i d a s e was d i s c u s s e d . B - 0 - G a l a c t o s i d a s e a c t i v i t y i n i n t a c t c e l l s o f 21 s p e c i e s o f Streptomyces has been measured using 2-nitrophenyl B-e-galactopyranoside hydrolysis, without addition of a permeabilizing agent.157 Differences i n the induction efficiency of 2-nitrophenyl
Carbohydrate Chemistry
412
€ 3-- Q - g a l a c t o p y r a n o s i d e h y d r o l y t i c a c t i v i t y by l a c t o s e o r g - g a l a c t o s e , which c o u l d have taxomic
implications,
species. f er m ent at
Car bo hy d r at e
---S. l a c t i s
io n o f
growing i n agar g e l s has
w e r e o b s e r v e d among t h e
strep t o c o cc:u s
c r e mo r is a n d
been i n v e s t i . g a t e d . l S 8
When
l a c t i c s t r e p t o c o c c i were embedded i n a g a r g e l s and i n c u b a t e d a t
3 O o C , t h e end p r o d u c t s o f c a r b o h y d r a t e f e r m e n t a t i o n depended on t h e i n i t i a l c e l l density,
which determined t h e subsequent d i s t r i b u t i o n
and s i z e o f c o l o n i e s i n t h e g e l .
With high i n i t i a l c e l l densities,
m i c r o c o l o n i e s f o r m e d c l o s e t o g e t h e r and l a c t o s e and p - g l u c o s e converted almost e n t i r e l y t o lactate. s m a l l number of c e l l s ,
r e s u l t e d i n up t o 3 0 % d i v e r s i o n o f
usually t o formate,
low-colony-density
gel cultures,
ethanol, the
and a c e t a t e .
i n i t i a l rate of
was e x p o n e n t i a l a n d o n l y l a c t a t e was f o r m e d . then
became
linear
heterolactic.
were
inoculation with a
w h i c h t h e n grew t o f o r m w i d e l y spaced and
comparatively large colonies, end-product,
However,
and f e r m e n t a t i o n
I n these
fermentation
However,
this rate
became p r o g r e s s i v e l y
more
l a c t i s M L 8 was t h e o n l y s t r a i n among t h e 1 0 t e s t e d
S.
which remained homolactic.
I n c u b a t i o n a t t e m p e r a t u r e s e i t h e r above
o r below the optimum f o r
g r o w t h and m e t a b o l i s m
d i v e r s i o n t o end-products o t h e r than l a c t a t e . t o hetero-lactic
decreased
the
The change f r o m homo-
f e r m e n t a t i o n a p p e a r s t o be c a u s e d by c a r b o h y d r a t e so t h a t f e r m e n t a t i o n i s
d e p l e t i o n i n the v i c i n i t y o f the colony, t h e n l i m i t e d by t h e d i f f u s i o n o f s u b s t r a t e .
G r o w t h o f c e l l s on g e l
s u r f a c e s e x p o s e d t o a i r r e s u l t e d i n up t o 40% d i v e r s i o n o f e n d p r o d u c t f r o m l a c t a t e , m a i n l y t o C02, a c e t o i n , 2 , 3 - b u t a n e d i o l , acetate.
Six
of
the
12
S.
cremoris
strains
h o m o l a c t i c under these a e r o b i c c o n d i t i o n s , S.
l a c t i s s t r a i n s tested, The
general
-X--a-n t h o m o n a s
i n c l u d i n g ML8,
p r o p e r t i e s
- have
cameestris
p u r i f i e d B-p-galactosidase optimum a c t i v i t y .
(Km =
been
tested
and
remained
whereas a l l 8 of
the
were h e t e r o l a c t i c .
o f 0-g-galactosidase of reported.lS9 The p a r t i a l l y
r e q u i r e d 3 2 t o 37OC a n d pH 5.5
The enzyme h a d l o w - a f f i n i t y
t o 5.8
for
lactose hydrolysis
22 m M ) and was i n h i b i t e d by t h i o l g r o u p r e a g e n t s ,
H4 e d t a ,
p-
g a l a c t o s e , and g - g a l a c t a l . S i x g l y c o s i d e h y d r o l a s e s i n t h e c u l t u r e medium o f B a c t e r o i d e s fraqilis,
B-DB-Q-glucosidase, a-g-galactosidase, B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e , a n d a-I=-
a-P-glucosidase,
galactosidase, fucosidase,
were
precipitation,
s y s t e m a t i c a l l y p u r i f i e d by a m m o n i u m s u l p h a t e
gel-filtration
i s o e l e c t r i c focusing.49
chromatography,
and d e n s i t y - g r a d i e n t
F o r f u r t h e r d e t a i l s see i n i t i a l c i t a t i o n o f
413
6: Enzymes r e f .49. An
endo-B-a-galactosidase
has
p u r i f i e d f o r m o f a new o r g a n i s m ,
been
isolated
i n
a
highly
Flavobacterium keratolyticus.160
The p u r i f i c a t i o n p r o c e d u r e i n c l u d e d ammonium s u l p h a t e p r e c i p i t a t i o n o f t h e e n z y m e f r o m t h e c u l t u r e m e d i u m , S e p h a d e x G-100 f i l t r a t i o n , c h r o m a t o g r a p h y a t t h e PI o f t h e enzyme on a c o l u m n w h i c h c o n t a i n e d e q u a l p o r t i o n s o f CM-Sephadex
C-50
and DEAE-Sephadex
b y M a t r e x g e l b l u e A a n d DEAE-Sephadex
A-50
A-50,
followed
chromatography.
The
f i n a l p r e p a r a t i o n showed one m a j o r
but
d i f f u s e p r o t e i n band on
polyacrylamide
The
molecular
gel electrophoresis.
e n z y m e i s a b o u t 30,000,
weight
u s i n g k e r a t a n s u l p h a t e as t h e s u b s t r a t e . s t a b i l i z e d b u t n o t a c t i v a t e d by Ca2+.
Escherichia A.,
-255,
r e p e a t i n g u n i t s and m i l k o l i g o s a c c h a r i d e .
t o t h e one i s o l a t e d f r o m
o f t h i s enzyme i s s i m i l a r
f r e u n d i i ( N a k a g a w a , H.,
Kitamikado,
which i s
T h i s enzyme h y d r o l y s e s endo-
l i n k a g e s i n k e r a t a n s u l p h a t e s and g l y c o c o nj u g a t e s
w i t h N-acetyl-lactosamine The s p e c i f i c i t y
this
Hg2+, .Ag+, Cu2+, a n d 4 -
c h l o r o m e r c u r i b e n z o a t e a r e p o t e n t i n h i b i t o r s f o r t h e enzyme, B-Q-galactosyl
of
a n d t h e o p t i m a l a c t i v i t y o c c u r s a t pH 6.0
L i , S.-C.,
M.,
5955-5959).
Yamada, T.,
a n d L i , Y.-T.
J., G a r d a s ,
Chien,
(1980) J. B i o l . Chem.,
The o n l y d i f f e r e n c e i s t h a t t h e enzyme i s o l a t e d
f r o m F. k e r a t o l y t i c u s h y d r o l y s e s m i l k
oligosaccharides
t h e one i s o l a t e d f r o m E. f r e u n d i i and o t h e r
---endo-B-P-galactosidase;
F.
faster
keratolyticus
produces
endo-B-g-
g a l a c t o s i d a s e w i t h o u t i n d u c t i o n by k e r a t a n s u l p h a t e . o r g a n i s m i s t h e b e s t s o u r c e so f a r f o r B-P-galactosidase
capable
of
than
organisms t h a t produce Thus t h i s
t h e p r e p a r a t i o n o f t h e endo-
cleaving
sugar
chains
with
N-
acetyl-lactosamine repeating units. Spectrophotometric
and
f l u o r i m e t r i c
assays
o f
g a l a c t o c e r e b r o s i d a s e a c t i v i t y have been used i n t h e d i a g n o s i s o f Krabbe's
disease.161
Derivatives of
galactocerebroside were
prepared c o n t a i n i n g coloured (~-2,4,6-trinitrophenylaminolauric acid)
o r f l u o r e s c e n t (11-(9-anthroxy)undecanoic
moieties.
These
cerebrosides
galactocerebrosidase activity. with
the
radioactively
r e t a i n i n g good provided
useful
and
of
brain,
liver,
used
acid) f a t t y acid
as
substrates
for
By o v e r c o m i n g p r o b l e m s a s s o c i a t e d
labelled
enzyme-substrate
substrates normally specificity,
reliable
galactocerebrosidase a c t i v i t y .
were
these
alternative
used,
yet
derivatives
substrates
for
Enzyme a c t i v i t i e s i n w h o l e e x t r a c t s
f i b r o b l a s t s , and c u l t u r e d a m n i o t i c f l u i d c e l l s w e r e
c o m p a r e d , u s i n g s u b s t r a t e s f o r t h e n o v e l c e r e b r o s i d e s a s w e l l as {3H)galactocerebroside.
Good
correlation
of
activities
was
414
Carbohydrate Chemistry
obtained. In e x t r a c t s d e r i v e d from p a t i e n t s w i t h Krabbe's d i s e a s e marked d e f i c i e n c y of g a l a c t o c e r e b r o s i d a s e a c t i v i t y was observed a s a p a r t i a l r e d u c t i o n i n enzyme a c t i v i t y . The r e s u l t s show t h a t t h e s e c o l o u r e d and f l u o r e s c e n t g a l a c t o c e r e b r o s i d e s may be u s e d w i t h c o n f i d e n c e i n t h e d i a g n o s i s and c a r r i e r d e t e c t i o n of K r a b b e ' s disease
.
8
a- and B-g-Glucosidases
The n e u t r a l i s o e n z y m e s o f human a - e - g l u c o s i d a s e h a v e been c h a r a c t e r i z e d by d i f f e r e n c e s i n s u b s t r a t e s p e c i f i c i t y , molecular weight, and e l e c t r o p h o r e t i c mobility.162 Neutral a-g-glucosidase C was p r e c i p i t a b l e i n 40-60% ammonium s u l p h a t e , had a m o l e c u l a r weight of 9 2 , 0 0 0 , an i s o e l e c t r i c p o i n t of 5.5,and r e l e a s e d Q - g l u c o s e f r o m glycogen a s w e l l a s from low-molecular-weight a r t i f i c i a l and n a t u r a l linkages. Neutral s u b s t r a t e s which c o n t a i n e d (1 .+ 4)-a-!-glucosidic a - g - g l u c o s i d a s e AB p r e c i p i t a t e d a t 0-40% ammonium s u l p h a t e , bound t o c o n c a n a v a l i n A, had a m o l e c u l a r weight of g r e a t e r t h a n 150,000, and d i d n o t u t i l i z e (1 + 4 ) - l i n k e d a - e - g l u c o s e s u b s t r a t e s l a r g e r t h a n t h e d i s a c c h a r i d e . N e u t r a l a - e - g l u c o s i d a s e AB m i g r a t e d more r a p i d l y t o t h e a n o d e t h a n a - e - g l u c o s i d a s e C when a g a r o s e , C e l l o g e l , a c r y l a m i d e , o r s t a r c h was u s e d a s s u p p o r t medium. Both i s o e n z y m e s were e q u a l l y i n h i b i t e d b y Zn2+. A s t u d y h a s b e e n p e r f o r m e d on a - a - g l u c o s i d a s e i n f o u r human colon m a l i g n a n t tumours developed i n t o n u d e mice.163 Both a c i d and n e u t r a l a - Q - g l u c o s i d a s e s w e r e c h a r a c t e r i z e d i n f o u r human adenocarcinoma tumours o b t a i n e d from c e l l l i n e s o f d i f f e r e n t glycogen c o n t e n t , b y u s i n g e i t h e r 4 - m e t h y l u m b e l l i f e r y l a - Q - g l u c o s i d e o r glycogen and m a l t o s e as s u b s t r a t e . No obvious l i n e a r r e l a t i o n s h i p b e t w e e n a - Q - g l u c o s i d a s e a c t i v i t y and g l y c o g e n l e v e l c o u l d be f o u n d . However, t h e l o w e s t and h i g h e s t a c t i v i t i e s c o i n c i d e d w i t h t h e l o w e s t and h i g h e s t g l y c o g e n c o n t e n t s , r e s p e c t i v e l y . The h y d r o l y t i c a c t i v i t y of n e u t r a l a - Q - g l u c o s i d a s e i n v e s t i g a t e d i n t h e p r e s e n c e of t u r a n o s e , an i n h i b i t o r of t h e a c i d form, was found t o be high t o w a r d s 4 - m e t h y l u m b e l l i f e r y l - a - Q - g l u c o s i d e o r m a l t o s e b u t weak t o w a r d s glycogen. K i n e t i c p a r a m e t e r s f o r both enzymes were found t o be s i m i l a r i n t h e f o u r tumours under i n v e s t i g a t i o n and c l o s e t o what was r e p o r t e d i n normal a n i m a l t i s s u e s . The f o r m a t i o n o f monoclonal a n t i b o d i e s a g a i n s t human a c i d a-gg l u c o s i d a s e h a s been r e ~ 0 r t e d . l ~Acid ~ a-Q-glucosidase p u r i f i e d
6: Enzymes
415
from human p l a c e n t a was used t o immunize a mouse ( s t r a i n Balb/cHeA) according t o a procedure described e a r l i e r ( S t a h l i , C., S t a e h l i n , T., M i g g i a n o , V., Schmidt, J., and H a r i n g , P. ( 1 9 8 0 ) J. Immunol. -Methods, 32, 297-304). A f t e r f u s i o n of s p l e e n c e l l s w i t h myeloma c e l l s , a b o u t 1 0 % o f t h e h y b r i d c l o n e s o b t a i n e d p r o d u c e d a n t i b o d i e s a g a i n s t acid a-P-glucosidase. Finally, eight stable c l o n e s p r o d u c i n g a n t i b o d i e s a g a i n s t t h e enzyme were o b t a i n e d . When p u r i f i e d a c i d 8 - e - g l u c o s i d a s e was a n a l y s e d b y p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i n t h e p r e s e n c e of s o d i u m d o d e c y l s u l p h a t e , t w o m a j o r p r o t e i n b a n d s ( m o l . w t . 7 6 , 0 0 0 and 7 0 , 0 0 0 ) , a m i n o r band of mol. w t . 96,000, and s e v e r a l minor bands w i t h a mol. w t . o f 67,000 o r lower were s e e n . S i n c e a l l t h e s e components r e a c t w i t h t h e m o n o c l o n a l a n t i b o d i e s , t h e y m u s t h a v e a t l e a s t one a n t i g e n i c d e t e r m i n a n t i n common. a l a c t 0 s i d a s e , s u c r o s e 8-Q-glucohydr o l a s e , The amount s of B-!-g a-Q-glucosidase, microvillus aminopeptidase,and d i p e p t i d y l peptidase I V i n t a n g e n t i a l l y s e c t i o n e d b i o p s i e s fom jejunum have been s t u d i e d b y q u a n t i t a t i v e i m m u n o e l e c t r o p h o r e s i s and enzymic a s s a y s . l Z 9 F o r f u r t h e r d e t a i l s s e e i n i t i a l c i t a t i o n of ref.129. Apparent normal l e u k o c y t e a - Q - g l u c o s i d a s e a c t i v i t y i n glycogen s t o r a g e d i s e a s e t y p e I1 (Pompe’s D i s e a s e ) has been r e ~ 0 r t e d . l ~An~ a-a-glucosidase determination i n the peripheral leukocytes revealed normal a c t i v i t y . However, a - Q - g l u c o s i d a s e a c t i v i t y was c o m p l e t e l y a b s e n t i n a pre-mortem s k e l e t a l muscle biopsy. Post-mortem s t u d i e s showed a - 0 - g l u c o s i d a s e a c t i v i t y t o be a b s e n t i n a l l t i s s u e s examined, i n c l u d i n g c u l t u r e d s k i n f i b r o b l a s t s . Massive glycogen d e p o s i t i o n c o r r e s p o n d e d t o t h e l o c a l i z a t i o n of t h e e n z y m i c d e f i c i e n c y , e x c e p t i n t h e b r a i n , where glycogen c o n t e n t was w i t h i n t h e normal range. The a - Q - g l u c o s i d a s e a c t i v i t y i n mixed p e r i p h e r a l l e u k o c y t e s was due t o an i s o e n z y m e of a c i d m a l t a s e i n t h e granulocyte series. A n t e - n a t a l d i a g n o s i s was a c c u r a t e i n a s u b s e q u e n t p r e g n a n c y , b u t d i s c o r d a n c e b e t w e e n enzyme a c t i v i t y i n d i f f e r e n t c e l l l i n e s i n an i n d i v i d u a l w i t h a g e n e t i c d i s e a s e i s a c o n c e i v a b l e s o u r c e o f e r r o r i n b o t h p r e - n a t a l and p o s t - n a t a l diagnoses. I t has been found t h a t reduced g l u t a t h i o n e i s not r e q u i r e d f o r measurement of a - g - g l u c o s i d a s e i n human s e m i n a l plasma. 166 P r o p e r t i e s of t h e m o l e c u l a r forms of a - g - g l u c o s i d a s e and 8-ag l u c o c e r e b r o s i d a s e from normal human and Gaucher d i s e a s e s p l e e n have been r e ~ 0 r t e d . l ~ ’ Comparative s t u d i e s of 8 - g - g l u c o s i d a s e s and 6-gg l u c o c e r e b r o s i d a s e s f r o m n o r m a l human and Gaucher d i s e a s e s p l e e n
Carbohydrate Chemistry
416
have a l l o w e d t h e i d e n t i f i c a t i o n o f t h r e e e n z y m a t i c g r o u p s on t h e b a s i s o f t h e i r s u b c e l l u l a r l o c a l i z a t i o n ( s o l u b l e o r membrane-bound), e n z y m a t i c , and g e n e t i c p r o p e r t i e s .
B-e-Glucocerebrosidase cleaves
B-E-glucocerebrosidases, and t h e a r t i f i c i a l s u b s t r a t e , methylumbelliferyl(MeUmb) B-g-glucopyranoside.
both
the
natural
substrates,
A minor p a r t (25%) i s s o l u b l e i n water
whereas 75% o f t h e t o t a l
a c t i v i t y i s membrane bound and can b e s o l u b i l i z e d by 0.25% T r i t o n X Using
100.
preparative
electrofocusing,
the
E x a c t l y t h e same f o r m s
0.2).
glucocerebrosidases
2
and 6.4
t h e membrane-bound B-a-
are found for
s o l u b i l i z e d by
B-k-
soluble
g l u c o c e r e b r o s i d a s e shows t w o m o l e c u l a r f o r m s (PI 5.2 2 0.2 Triton
X-100.
these
A l l
m o l e c u l a r f o r m s d i s p l a y t h e same e n z y m a t i c p r o p e r t i e s : o p t i m u m pH o f 5.4,
heat s t a b i l i t y
at
42OC,
Em
values
of
2 0.5
3.7
mM f o r
the
a r t i f i c i a l s u b s t r a t e i n t h e p r e s e n c e o f s o d i u m t a u r o c h o l a t e a n d 0.06
2
0.01
mM f o r t h e n a t u r a l substrate.
I n a d d i t i o n they are activated
t o t h e same e x t e n t ( 6 0 0 % ) b y s o d i u m t a u r o c h o l a t e .
Furthermore,
t h e s e t w o m o l e c u l a r f o r m s a r e coded by t h e same gene s i n c e b o t h a r e deficient
i n type I non-neuropathic
non-specific
MeUmb-B-P-glucosidase
Gaucher
disease.
catalyses
the
The s o l u b l e ,
h y d r o l y s i s of
the
a r t i f i c i a l s u b s t r a t e b u t n o t o f t h e n a t u r a l s u b s t r a t e and i s f o c u s e d i n one s i n g l e peak (PI 4.7 2 0.2) e-glucocerebrosidase
d i f f e r e n t f r o m t h e t w o f o r m s o f B-
d i s c u s s e d above.
It i s strongly
i n h i b i t e d by
s o d i u m t a u r o c h o l a t e , w e a k l y b y T r i t o n X-100, a n d i s h e a t l a b i l e a t 42OC.
No d e f i c i e n c y i s d e t e c t e d i n t h e case o f
disease r e p o r t e d here. corresponds
to
a
gene
glucocerebrosidase.
I t i s thus different
t y p e I Gaucher
suggested t h a t from
The m e m b r a n e - b o u n d ,
that
this
coding
enzyme
f o r B-Q-
n o n - s p e c i f i c MeUmb-B-g-
glucosidase also hydrolyses the a r t i f i c i a l substrate but not the natural substrate.
T h i s enzyme d i f f e r s f r o m t h e s o l u b l e one w i t h
regard t o i t s heat
s t a b i l i t y a t 42'C
M e U m b - f 3 --Q - g l u c o s i d a s e glucocerebrosidase specificity,
and i t s s t r o n g i n h i b i t i o n by
On t h e o t h e r h a n d , t h e m e m b r a n e - b o u n d n o n - s p e c i f i c
T r i t o n X-100.
and
d i f f e r s
from
i n i t s heat s t a b i l i t y
the
sodium
taurocholate
membrane-bound
B-Q-
a t 5OoC, i t s s u b s t r a t e effect.
This
enzyme
a c t i v i t y i s a b o u t n o r m a l i n Gaucher d i s e a s e and i s t h u s coded by a gene d i f f e r e n t f r o m t h e B - e - g l u c o c e r e b r o s i d a s e S i g n i f i c a n t a-g-glucosidase human a m n i o t i c
one.
a c t i v i t y has been i d e n t i f i e d i n
f l u i d d u r i n g mid-pregnancy.168
This
d i s t i n g u i s h a b l e from t h e l y s o s m a l a c i d a-E-glucosidase, o f w h i c h i s a s s o c i a t e d w i t h Pompe's d i s e a s e . i n
o p t i m u m pH,
thermal
stability,
enzyme
i s
deficiency
The t w o enzymes d i f f e r
electrophoretic
migration,
6: Enzymes
417
isoelectric point, Amniotic
m o l e c u l a r mass,
a-Q-glucosidase
n e u t r a l form.
i s
also
and i m m u n o l o g i c a l response. different
Immuno-cross-reactions
from
the
classical
suggest t h a t the amniotic
f l u i d enzyme has a d o u b l e f o e t a l o r i g i n : r e n a l and i n t e s t i n a l .
It
seems t h a t a - Q - g l u c o s i d a s e i n a m n i o t i c f l u i d i s l i n k e d t o l i p i d s . A comparative study o f a-Q-glucosidase
r a t and t r o u t
a c t i v i t i e s i n r a t and
A l l investigated tissues of
t r o u t t i s s u e s has been performed.16’
c o n t a i n e d a c i d and n e u t r a l a - e - g l u c o s i d a s e s
which
c o u l d be d i s t i n g u i s h e d f r o m one a n o t h e r and f r o m t h e d i g e s t i v e a-gg l u c o s i d a s e bound t o t h e b r u s h b o r d e r o f t h e i n t e s t i n e by t h e i r biochemical properties.
I n both species,
a-9-
the neutral
g l u c o s i d a s e was f o u n d t o be v e r y a c t i v e i n l i v e r and k i d n e y and, a lesser extent, The
i n spleen,
to
b r a i n , and p a n c r e a s .
s k e l e t a l muscle
of
cattle
suffering
from
generalized
g l y c o g e n e s i s t y p e I1 h a s b e e n s h o w n t o l a c k a c i d a - Q - g l u c o s i d a s e activity.l7O
Furthermore,
t h e r e was n o e v i d e n c e o f e n z y m i c a l l y
i n a c t i v e proteins t h a t cross-reacted a c i d a-P-glucosidase
with antibodies raised against
from t h e muscle o f n o r m a l animals.
The a c t i v i t i e s o f v a r i o u s g l y c o s i d a s e s i n h o m o g e n a t e s o f t h e small
intestinal
wallabies
(M.
mucosa
eurenii)
of
two
aged
adult
from
and
t o
6
18
50
suckling weeks
tammar
have
been
i n ~ e s t i g a t e d . ~F o~r f u r t h e r d e t a i l s see i n i t i a l c i t a t i o n o f r e f .38. Lysosomal a-P-glucosidase
has
been p u r i f i e d
from
a lysosome-
e n r i c h e d f r a c t i o n o f r a t l i v e r u s i n g an i m p r o v e d p r o c e d u r e . 1 7 1 purification factor
o f 2900-fold
Polyacrylamide
gel
non-denaturing
conditions or
was r e a c h e d ,
electrophoresis
of
the
purified
i n t h e presence o f
s u l p h a t e showed o n l y one band.
However,
A
w i t h a y i e l d o f 35%. enzyme
i n
sodium dodecyl
a m i c r o h e t e r o g e n e i t y among
e n z y m e s u b u n i t s was d e t e c t e d b y h i g h - r e s o l u t i o n t w o - d i m e n s i o n a l electrophoresis. B r u s h - b o r d e r -membrane a - q - g l u c o s i d a s e
has been p u r i f i e d f r o m
r a t k i d n e y by a p r o c e d u r e t h a t i n c l u d e d p a p a i n h y d r o l y s i s , sulphate fractionation, i n
which
Tris,
ligand.17*
an
The
gel filtration,
inhibitor
pure
of
enzyme
a-P-glucosidase, had
a
ammonium
and a f f i n i t y chromatography specific
s e r v e d as activity
the
which
represented a 1200-fold p u r i f i c a t i o n r e l a t i v e t o the a c t i v i t y i n the c o r t e x homogenate.
T h e e n z y m e was
c o n t a i n i n g 17.5% hexose. monomors o r daltons,
Em f o r
subunits,
f o u n d t o be a g l y c o p r o t e i n ,
The n a t i v e enzyme was an a g g r e g a t e o f f o u r each w i t h a
molecular
weight
of
335,000
as j u d g e d by S D S - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s .
a-e-glucosidase
was 1.6
mM,
and t h e
Ki f o r
The
T r i s was 3.4mM.
418
Carbohydrate Chemistry
Monospecific a n t i b o d y produced i n r a b b i t s a g a i n s t t h e r a t enzyme d i d not c r o s s - r e a c t w i t h dog kidney a-;-glucosidase. Unlike i n t e s t i n a l enzyme, t h e r e n a l a - q - g l u c o s i d a s e was s p e c i f i c f o r m a l t o s e and d i d not h y d r o l y s e s t a r c h o r o t h e r d i s a c c h a r i d e s . F u r t h e r work on m i c r o s o m a l ! - g l u c o s i d a s e s of r a t l i v e r h a s confirmed t h a t a t l e a s t two enzymes a r e i n v o l v e d i n t h e removal of p - g l u c o s e f r o m t h e g - g l u c o s e - c o n t a i n i n g o l i g o ~ a c c h a r i d e . One ~~~ a c t e d on t h e o l i g o s a c c h a r i d e c o n t a i n i n g t h r e e !-glucose r e s i d u e s and a n o t h e r on t h e o l i g o s a c c h a r i d e which has one o r two g - g l u c o s e s . The p - g l u c o s i d a s e w h i c h a c t s on ( Q - G ~ C ) ~ ( Q - M ~ ~ ) ~ ( ! - G ~ C N AcCo )u~l d be p u r i f i e d w i t h a c o n c a n a v a l i n A - S e p h a r o s e column f o l l o w e d b y electrofocusing. The p u r i f i e d p r e p a r a t i o n was a c t i v e on t h e residues. Heat o l i g o s a c c h a r i d e c o n t a i n i n g o n e or t w o !-glucose i n a c t i v a t i o n and i n h i b i t i o n by d i s a c c h a r i d e s was p a r a l l e l f o r both activities. I n h i b i t i o n of t h e Q - g l u c o s i d a s e a c t i v e on (Q-GlcI3(gMan)9(g-GlcNAc)2 was o b t a i n e d w i t h k o j i b i o s e which h a s a ( 1 + 2 ) l i n k a g e , w h i l e t h e Q - g l u c o s i d a s e a c t i n g on ( g - G l ~ ) ~ ( g - M a n ) ~ ( Q GlcNAcI2 was i n h i b i t e d b y n i g e r o s e ( ( 1 + 3 ) l i n k a g e ) , m a l t o s e ( ( 1 + 4) l i n k a g e ) , and P-glucose a t a h i g h e r c o n c e n t r a t i o n . None of t h e Banomers i n h i b i t e d . These r e s u l t s were c o n s i s t e n t w i t h a c o n f i g u r a t i o n of t h e t h r e e p-glucoses o f t h e d o l i c h y l d i p h o s p h a t e linked oligosaccharide. K o j i b i o s e was f o u n d t o i n h i b i t Q g l u c o s i d a s e a c t i o n not only on t h e f r e e o l i g o s a c c h a r i d e b u t a l s o on protein-bound one. F i f t e e n g l y c o s i d a s e s w e r e a s s a y e d i n l y m p h o m y e l o i d and d i g e s t i v e t i s s u e s of Ginglymostoma c i r r a t u m , H e t e r o d o n t u s f r a n c i s c i , and Etmopterus a p i n a x . A c t i v i t i e s of a-g-mannosidase, B-P-galactos i d a s e , B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g a l a c t o s i d a s e , B-g-glucuronidase, and a- and B-q-glucosidase u s u a l l y were h i g h e r i n lymphomyeloid t i s s u e s than i n d i g e s t i v e t i s s u e s . T r e h a l o s e has been found t o behave a s a mixed i n h i b i t o r o f t h e a - g - g l u c o s i d a s e a c t i v i t y o f h o n e y - b e e h a e m 0 1 y m p h . l ~ ~I n s a m p l e s w i t h i n i t i a l l y M i c h a e l i a n k i n e t i c s , t h e maximum v e l o c i t y 1, i s d e c r e a s e d and t h e M i c h a e l i s c o n s t a n t i s i n c r e a s e d b y t h e same f a c t o r , e q u a l t o 1.7 i n emerging bees and 1.3 i n f o r a g i n g bees. The d i s s o c i a t i o n c o n s t a n t s Ki and K’i, r e s p e c t i v e l y , c o r r e s p o n d i n g t o t r e h a l o s e b i n d i n g t o t h e enzyme and t o t h e ( e n z y m e - a r t i f i c i a l s u b s t r a t e ) c o m p l e x , a r e e q u a l t o 62.5 m M and 168.7 m M i n e m e r g i n g The r a t i o b e e s and 303.9 and 7 5 4 .5 m M i n f o r a g i n g b e e s . r e m a i n s c o n s t a n t . The H i l l c o e f f i c i e n t f o r t r e h a l o s e b i n d i n g t o t h e enzyme v a r i e s f r o m 0.91 i n e m e r g i n g b e e s t o 1 . 3 8 i n f o r a g i n g b e e s ,
K’i/ci
419
6: Enzymes
which i s consistent w i t h the frequently non-Michaelian tendencies o f This t h i s e n z y m e . T h e v a l u e o f 150r i s e s f r o m 9 8 . 3 m M t o 3 5 8 . 3 m M . increase,
Ki
together w i t h those o f
Kpi,
and
i s correlated with the
i n c r e a s e i n t r e h a l o s e m i a f r o m emerging t o f o r a g i n g bees i n t h e samples s t u d i e d . Sucrose
density
gradients
have
been
used
to
of
S t o m o x y s ~ a l c i t r a n s . The ~ ~ a c i d i c glycosidases (a-Q-glucosidase,
a-
!-galactosidase,
fractions
from
galactosidase,
8-Q-
8-Q-glucosidase,
B-Q-glucuronidase,
deoxyglucosidase) e q u i l i b r a t e
h
0
a-!-mannosidase,
white
characterize prepupae
lysosome-containing
and
8-g-2-acetamido-2-
a t t h e same d e n s i t y
as d o e s a c i d
phosphatase. A
comparison o f
a-e-glucosidase
and a - e - m a n n o s i d a s e
h a s been
~e made i n b l o o d s t r e a m f o r m s o f T r y p a n o s o m a b r u c e i b r ~ c e i . ' ~T h p h y s i c o c h e m i c a l and k i n e t i c p r o p e r t i e s o f t h e t w o m a j o r t r y p a n o s o m a l glycosidases,
a-e-glucosidase
b l o o d s t r e a m f o r m s o f T.
and a - p - m a n n o s i d a s e ,
were compared i n
b r u c e i b r ~ c e i . B~o t~h enzymes a r e membrane-
bound and l o c a t e d i n t r a c e l l u l a r l y .
The r e s u l t s a r e d i s c u s s e d i n
r e l a t i o n t o t h e p o s s i b l e r o l e o f a - Q - g l u c o s i d a s e and a-!-mannosidase i n the processing or catabolism o f trypanosomal glycoproteins. N i n e g l y c o s i d a s e s i n b l o o d s t r e a m f o r m s o f T. h a v e b e e n p a r t i a l l y c h a r a c t e r i z e d .20 physicochemical
and
enzymic
b r u c e i b r u c e i S42
a-0-Glucosidase
properties
to
had s i m i l a r
those
of
a-g-
8-~-2-acetarnido-2-deoxyglucosidase, a n d B-~-2-acetamido-2-deoxygalactosidase. galactosidase, 8-9-glucosidase, Purification
and s u b s t r a t e s p e c i f i c i t y o f
g l u c o s i d a s e h a v e been d e s c r i b e d . 1 7 6 from
molecular
electrophoresis. ratios
was e s t i m a t e d t o b e a b o u t
maximum
isomaltose,
analysis.
The
96,000 b y S D S - d i s c
The enzyme showed h i g h a c t i v i t i e s t o w a r d m a l t o s e ,
phenyl a-maltoside,
of
g e l f i l t r a t i o n s on Sephadex G-100.
homogeneous i n d i s c - e l e c t r o p h o r e t i c
weight
nigerose,
a-9-
s e e d s b y f r a c t i o n a t i o n w i t h ammonium s u l p h a t e ,
sweet-corn
c h r o m a t o g r a p h i e s on Sepharose,and The enzyme was
sweet-corn
An a - Q - g l u c o s i d a s e was p u r i f i e d
velocity
and m a l t o - o l i g o s a c c h a r i d e s . for
phenyl a-glucoside,
maltose,
nigerose,
The
kojibiose,
phenyl a-maltoside,
panose,
t u r a n o s e , and s o l u b l e s t a r c h w e r e e s t i m a t e d t o be 100 : 78 : 1 7 : 11 : 28 : 1 0 0 : 3 1 : 3.4
1.5
m M , 1.4 m M , 0.48
52 mg m l - l , was h i g h , the
Em
: 126, and t h e
m M , 1 4 m M , 4.2
respectively. was
also
m M , 0.28
mM,and
The maximum v e l o c i t y f o r s o l u b l e s t a r c h
b u t t h i s a-q-glucan
value
values for these substrates m M , 1.1 m M , 5.0
very
was n o t a f a v o u r a b l e s u b s t r a t e b e c a u s e high.
The
lmax values f o r malto-
420
Carbohydrate Chemistry
oligosaccharides more ;-glucose
were
(;I.
polymerization
somewhat
5,
The
dependent
on
the
degree
of
v a l u e s f o r s u b s t r a t e s h a v i n g f o u r or
.;
u n i t s increased w i t h the increase i n
Two f o r m s of a - Q - g l u c o s i d a s e h a v e been i s o l a t e d f r o m r i c e s e e d s a t t h e m i l k y s t a g e by a p r o c e d u r e t h a t ammonium s u l p h a t e ,
included fractionation with
c o l u m n c h r o m a t o g r a p h y , and p r e p a r a t i v e d i s c g e l
e l e c t r o p h o r e s i ~ . ~The ~ ~ i s o l a t e d a-g-glucosidases
I and a - p - g l u c o s i d a s e 3.4%
carbohydrate,
respectively.
i s o e l e c t r i c p o i n t were: 47,300
a n d pH 9.3
(a-E-glucosidase
11) w e r e g l y c o p r o t e i n s c o n t a i n i n g 5 . 5 % a n d 78,900
The
and pH 9.3
f o r a-Q-glucosidase
molecular
weight
f o r a-;-glucosidase
11.
and
I, and
They w e r e f o u n d t o b e
homogeneous on p o l y a c r y l a m i d e d i s c g e l e l e c t r o p h o r e s i s .
The t w o
enzymes h y d r o l y s e d m a l t o s e , m a l t o t r i o s e , p h e n y l a - m a l t o s i d e ,
amylose,
and s o l u b l e s t a r c h t o l i b e r a t e ;-glucose The
enzymes
produced
maltotriose
and
m a l t o s e as m a i n a - ; - g l u c o s y l - t r a n s f e r
b u t d i d n o t a c t on s u c r o s e . 4-a-;-nigerosyl-glucose products,
from
and t h e y h y d r o l y s e d
a m y l o s e t o l i b e r a t e a-;-glucose. Three forms o f a-9-glucosidase
have been i s o l a t e d f r o m 5-day-
o l d green gram (Phaseolus v i d i s s i m u s ) s e e d l i n g s ,
by a p r o c e d u r e
i n c l u d i n g f r a c t i o n a t i o n w i t h ammonium s u l p h a t e and p o l y e t h y l e n e g l y c o l 6000, and
column chromatography,
preparative disc
i s o l a t e d (a-Q-glucosidase g l u c o s i d a s e 11-21 electrophoresis. 65,000,
were
I, a - Q - - g l u c o s i d a s e
homogeneous
11-1, a n d a - Q -
on p o l y a c r y l a m i d e d i s c
T h e i r m o l e c u l a r w e i g h t s were 145,000,
respectively.
maltotriose,
preparative g e l electrofocusing,
g e l e l e c t r o p h ~ r e s i s . ' ~ ~The a - a - g l u c o s i d a s e s
phenyl
l i b e r a t i n g !-glucose hydrolysed
phenyl
glucoside,
and t h e y
The
three
enzymes
a-maltoside, but did not
a-maltoside
gel
105,000, and
hydrolysed
maltose,
a m y l o s e , and s o l u b l e - s t a r c h act
into
on s u c r o s e . e-glucose
The
and
enzymes
phenyl
a-k-
hydrolysed amylose l i b e r a t i n g a-Q-glucose.
M a l t o t r i o s e was t h e m a i n a - ; - g l u c o s y l - t r a n s f e r
product formed from
m a l t o s e by t h e t h r e e a-Q-glucosidases. An
a-E-galactosidase
p u r i f i e d about
130-fold
from by
tubers
ammonium
of
S.
affinis
sulphate
has
been
fractionation,
c h r o m a t o g r a p h y , and g e l f i 1 t r a t i 0 n . l ~ ~The p u r i f i e d enzyme showed a s i n g l e p r o t e i n band on d i s c g e l e l e c t r o p h o r e s i s .
The m o l e c u l a r
w e i g h t o f t h e e n z y m e was d e t e r m i n e d t o b e a p p r o x i m a t e l y 42,000 g e l f i l t r a t i o n a n d 44,000
b y SDS d i s c g e l e l e c t r o p h o r e s i s .
o p t i m u m r e a c t i o n pH was 5.2. r a p i d l y than planteose.
by The
The enzyme h y d r o l y s e d r a f f i n o s e more
The a c t i v a t i o n e n e r g y o f r a f f i n o s e and
p l a n t e o s e b y t h e e n z y m e was e s t i m a t e d t o b e 7.89
a n d 11.4
kcal
6: Enzymes
42 1
rnol-l, r e s p e c t i v e l y . The enzyme a c t i v i t y was i n h i b i t e d b y v a r i o u s ; - g a l a c t o s i d a s e s and s t r u c t u r a l analogues of g - g a l a c t o s e . Besides h y d r o l y t i c a c t i v i t y , t h e enzyme a l s o c a t a l y s e d t h e t r a n s f e r r e a c t i o n of g - g a l a c t o s y l r e s i d u e from r a f f i n o s e t o methanol. The s e c r e t i o n o f l y s o s o m a l e n z y m e s h a s been s t u d i e d i n t h e c e l l u l a r s l i m e mould D i c t y o s t e l i u m d i s c o i d e u m . One g r o u p o f e n z y m e s , i n c l u d i n g B - e - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e , a-pmannosidase, B-e-glucosidase, B-q-galactosidase-1, and a-!g l u c o s i d a s e - 1 , a r e very e f f i c i e n t l y s e c r e t e d , w i t h 10 t o 20% of t h e t o t a l c e l l u l a r a c t i v i t y becoming e x t r a c e l l u l a r w i t h i n a few h o u r s . A l l of t h e s e enzymes have s i m i l a r o r i d e n t i c a l s e c r e t i o n k i n e t i c s and may come from t h e same l y s o s o m a l v e s i c l e s . F o r f u r t h e r d e t a i l s s e e i n i t i a l c i t a t i o n of r e f .46. A c t i v e - s i t e - d i r e c t e d i r r e v e r s i b l e i n h i b i t i o n of g l y c o s i d a s e s b y t h e c o r r e s p o n d i n g g l y c o s y l m e t h y l - [ 4 - n i t r o p h e n y l ) t r i a z i n e s h a s been i n v e s t i g a t e d .72 B-i-Glucopyranosy lmet h y l - [ 4 - n i t r o p h e n y l ) t r i a z i n e i s an a c t i v e - s i t e - d i r e c t e d i r r e v e r s i b l e i n h i b i t o r (ASDIN) of both B-Pg l u c o s i d a s e and B - q - g a l a c t o s i d a s e a c t i v i t i e s of sweet-almond B-Pg l u c o s i d a s e 8 . I t has no e f f e c t on y e a s t a - 9 - g l u c o s i d a s e , t h e &Z 8 - Q - g a l a c t o s i d a s e of E . c o l i , or g l u c o a m y l a s e f r o m A s p e r g i l l u s n i g e r . F o r f u r t h e r d e t a i l s s e e i n i t i a l c i t a t i o n of r e f . 7 2 . The i n d u c t i o n of a - p - g l u c o s i d a s e s i n S t r e p t o m y c e s v e n e z u e l a e has been i n v e s t i g a t e d . ' * ' S. venezuelae c o n t a i n s i n t r a c e l l u l a r a-Qg l u c o s i d a s e s t h a t a r e induced d u r i n g growth on m a l t o s e , i s o m a l t o s e , m a l t o t r i o s e , d e x t r i n , s t a r c h , and o t h e r a - e - g l u c o s i d e s . Induction was p r e v e n t e d b y r i f a m p i c i n and i n h i b i t e d b y c h l o r a m p h e n i c o l o r s t r e p t o m y c i n , i n d i c a t i n g t h a t de novo s y n t h e s i s of m e s s e n g e r r i b o n u c l e i c a c i d and p r o t e i n was r e q u i r e d . !-Glucose and o t h e r r e a d i l y u t i l i z a b l e s u g a r s d i d n o t r e p r e s s i n d u c t i o n of a - Q g l u c o s i d a s e a c t i v i t y , whereas c e r t a i n o r g a n i c a c i d s and amino a c i d s e f f e c t i v e l y reduced enzyme s y n t h e s i s . E x t r a c t s of mycelium grown i n t h e p r e s e n c e of m a l t o s e a s an i n d u c e r h y d r o l y s e d m a l t o s e and isomaltose rapidly. S u c r o s e and o t h e r a - P - g l u c o s i d e s w e r e l e s s s u i t a b l e s u b s t r a t e s , w h e r e a s t r e h a l o s e and s t a r c h were n o t No a c t i v i t y was o b s e r v e d w i t h B - g - g l u c o s i d e s , a - g hydrolysed. g a l a c t o s i d e s , o r methyl a-;-mannopyranoside. T h e p r o d u c t i o n of 4 - n i t r o p h e n y l a - g - g l u c o p y r a n o s i d e - h y d r o l y s i n g a - ~ - g l u c o s i d a s e b y B a c i l l u s c e r e g ~A T C C 7 0 6 4 h a s b e e n O u t o f 1 7 s t r a i n s of 1 4 d i f f e r e n t B a c i l l u s investigated.l*l s p e c i e s , B . c e r e u s A T C C 7 0 6 4 p r o d u c e d t h e h i g h e s t l e v e l s of 4n i t r o p h e n y 1 a - q - g 1u c o p y r a n o s i d e - h y d r o l y s i n g a - g - g l u c o s i d a s e
.
422
Carbohydrate Chemistry
M a x i m a l e n z y m e y i e l d was a c h i e v e d a f t e r 6 h o f c u l t i v a t i o n , a t an i n i t i a l pH o f 6.5
2% p e p t o n e ,
-
o n a m e d i u m c o n t a i n i n g 2% s o l u b l e s t a r c h ,
7.0,
0.1% KH2P04.
0 . 3 % K2HP04,and
0.05% meat e x t r a c t ,
0.3% y e a s t e x t r a c t ,
When g r o w n o n 2% s t a r c h , B.
glucosidase i n t h e cytoplasm.
c e r e u s s y n t h e s i z e d a-E-
A t a starch concentration of less
t h a n 146, h o w e v e r , t h e e n z y m e a p p e a r e d i n t h e c u l t u r e b r o t h .
This
a c c u m u l a t i o n was m a x i m a l a t 0.4% s t a r c h when t h e e x t r a c e l l u l a r enzyme amounted t o 54% o f t h e t o t a l enzyme p r o d u c e d . S i x
glycoside
hydrolases
-
B acte r o-i d~ es
fragilis
actosidase,
B-Q-galactosidase,
and a - i - f u c o s i d a s e
-
were
sulphate precipitation, gradient
the
medium
chromatography,
A been
which resolved the glycosidases i n t o
fraction^.^'
a-Q-Glucosidase
was shown t o substrate.
d e t a i l s see i n i t i a l c i t a t i o n o f r e f . 4 9 .
n o v e l a-;-glucosidase reported.182
p r o d u c e d by B a c i l l u s a m y l o l y t i c u s h a s
----------B a c i l l u s a m y--lolyticus
p u l l u l a n a s e , and a - Q - g l u c o s i d a s e . t h e g r o w t h medium,
By s e l e c t i o n
a-g-glucosidase
highly specific for inactive
carbon source i n
of
a range
n i t r o p h e n y l a-Q-glucoside
of
other
and
The a - Q - g l u c o s i d a s e was
m a l t o s e and t o a l e s s e r e x t e n t
towards
r e c o r d e d a t pH 7.0
produces a-amylase,
was p r o d u c e d p r e f e r e n t i a l l y
w i t h exclusion o f the other two a c t i v i t i e s . was
ammonium
and d e n s i t y -
possess d u a l a f f i n i t y f o r t h e r e s p e c t i v e q-galactoside For f u r t h e r
o f
a-Q-gal-
B-~-2-acetamido-2-deoxyglucosidase,
gel-filtration
w e l l separated
culture
B-g-glucosidase,
s y s t e m a t i c a l l y p u r i f i e d by
i s o e l e c t r i c focusing,
distinct,
i n
a-Q-glucosidase,
maltotriose but
s u b s t r a t e s i n c l u d i n g 4-
and i s o m a l t o s e .
O p t i m a f o r a c t i v i t y were
a n d 4OoC a n d t h e e n z y m e was i n s e n s i t i v e t o H4
edta. The
i s o l a t i o n
a n d
c e r t a i n
Saccharomyces c e r e v i s i a e a - Q - g l u c o s i d a s e T h e e n z y m e was p u r i f i e d b y i o n - e x c h a n g e Sephadex A-50
were obtained,
o f
c h r o m a t o g r a p h y o n DEAE-
and i s o e l e c t r i c f o c u s i n g .
a-Q-glucosidase
p r o p e r t i e s
I 1 have been d e s c r i b e d . l a 3
As a r e s u l t ,
w i t h PIS o f 5.35
two forms o f
a n d 5.30.
The
i s o l a t e d i s o f o r m s o f t h e enzyme d i d n o t d i f f e r i n s p e c i f i c a c t i v i t y , but
their
molecular
weights,
according
polyacrylamide gel electrophoresis,
i s
t o
equal
the to
data 60,000,
o f
SDS
while
a c c o r d i n g t o t h e d a t a f r o m g e l f i l t r a t i o n t h r o u g h Sephadex G-100
it
i s 55,000.
It
Both forms exhibited electrophoretic polydispersion.
was s h o w n t h a t t h e i s o f o r m s o f a - p - g l u c o s i d a s e c o n t a i n i n g 1.5-2% 2-amino-2-deoxy-~-glucose
2-396
(isoform
B)
neutral
c h r o m a t o g r a p h y and ! - g l u c o s e
sugars.
It
are glycoproteins
and 5 - 8 % ( i s o f o r m A) was
shown
by
o x i d a s e r e a c t i o n t h a t !-glucose
and
paper i s the
423
6: Enzymes
b a s i c c o n s t i t u e n t o f t h e n e u t r a l s u g a r s i n b o t h i s o f o r m s o f a-0glucosidase. A q u a n t i t a t i v e i n s i t u a s s a y of y e a s t a - Q - g l u c o s i d a s e i n v o l v i n g
p e r m e a b i l i z a t i o n o f t h e c e l l s by described.184 different
The
assay
was
f r e e z i n g and t h a w i n g has been
applied
physiological states
and
to
was
different shown
to
strains
give
comparable t o those o b t a i n e d w i t h t o t a l c e l l homogenates. primary
advantage o f
t h e i n s i t u assay
was
the
i n
results The
possibility
of
a n a l y s i n g l a r g e n u m b e r s o f s a m p l e s f r o m t h e same c u l t u r e d u r i n g a g r o w t h c u r v e u s i n g a v e r y r e d u c e d c e l l mass. A
new
trisaccharide,
6’-a-~-glucosylsucrose,
has
been
s y n t h e s i z e d by t r a n s g l u c o s y l a t i o n r e a c t i o n o f b r e w e r ’ s y e a s t a-Qg l u c o ~ i d a s e . ~ E n~z~y m a t i c s y n t h e s i s o f o l i g o s a c c h a r i d e t h r o u g h t h e t r a n s g l u c o s y l a t i o n r e a c t i o n o f brewer’s attempted
i n
substrate.
the
g i v i n g m.p.
The t r i s a c c h a r i d e
was
only
was
sucrose
of
139
- 142’C
as
and { a } g 2 +102’ ( i n w a t e r ) .
c o n f i r m e d t o be 6F-a-Q-glucosylsucrose,
- g l u c o p y r a n o s y 1- ( 1
glucopyranoside, +119’
yeast a-glucosidase
containing
was c h r o m a t o g r a p h i c a l l y i s o l a t e d i n c r y s t a l l i n e f o r m
(monohydrate),
2-a-0
system
As t h e t r a n s g l u c o s y l a t i o n p r o d u c t , a n e w n o n - r e d u c i n g
trisaccharide
i s
reaction
+
6 1-g-B-Q-f r u c t o f u r a n o s y 1-(2
w h i c h u n d e c a - a c e t a t e gave m.p.
( i n chloroform),
148’C
+
that
1)-g-a-a-
and
and t h i s s u g a r was named i s o m e l e r i t o s e .
The c o o r d i n a t e d r e g u l a t i o n o f r i b o f l a v i n p e r m e a s e a n d a-gg l u c o s i d a s e s y n t h e s i s i n t h e y e a s t h a s been r e p o r t e d . 1 8 6 permease
of
the
yeast
t r a n s p o r t system.
Pichia guilliermondii
Riboflavin
a regulatable
I t s s y n t h e s i s i s i n d u c e d by sucrose,
methyl a-Q-glucopyranoside, glucose,
i s
trehalose,
maltose,
m e l e z i t o s e , and r a f f i n o s e , b u t n o t Q-
or cellobiose.
The s y n t h e s i s o f r i b o f l a v i n
permease i n t h e p r e s e n c e o f s u c r o s e or m a l t o s e i s s u p p r e s s e d by cycloheximide,
a c t i n o m y c i n D, a n d 8 - h y d r o x y q u i n o l i n e a n d t h u s i s
r e g u l a t e d a t t h e l e v e l of t r a n s c r i p t i o n .
Inducers o f r i b o f l a v i n
permease a r e a l s o capable o f i n d u c i n g a-Q-glucosidase s y n t h e s i s . M u t a n t s were s e l e c t e d w i t h a c o n s t i t u t i v e permease
-
formation o f r i b o f l a v i n
t h e synthesis o f a-Q-glucosidase
occurs c o n s t i t u t i v e l y .
i n such s t r a i n s a l s o
C u l t u r i n g o f t h e y e a s t s i n a medium w i t h a
h i g h c-glucose content leads t o a p a r a l l e l decrease i n r i b o f l a v i n permease
and a - g - g l u c o s i d a s e
evidence o f co-ordinated
activity.
The
data
obtained
are
r e g u l a t i o n o f r i b o f l a v i n p e r m e a s e and a-o-
g l u c o s i d a s e i n P. g u i l l i e r m o n d i i .
An i n s u f f i c i e n t o r e x c e s s c o n t e n t
o f v i t a m i n B2 i n t h e medium h a s no e f f e c t on t h e l e v e l o f r i b o f l a v i n permease i n t h i s yeast species.
424
Carbohydrate Chemistry
The s y n t h e s i s of a - Q - g l u c o s i d e i n y e a s t c e l l s d e p l e t e d of i n t r a m i t o c h o n d r i a l ATP has been i n ~ e s t i g a t e d . ' ~ I~t was found t h a t bongkrekic a c i d d i d not p r e v e n t t h e maltose-induced s y n t h e s i s of a !-glucosidase i n d e r e p r e s s e d c e l l s of t h e w i l d - t y p e and c o r r e s p o n d i n g r e s p i r a t o r y - d e f i c i e n t mutant of Saccharomyces c e r e v i s i a e . The r e s u l t s s u g g e s t e d t h a t e x p r e s s i o n of n u c l e a r genes s p e c i f y i n g a-pg l u c o s i d a s e and m a l t o s e c a t a b o l i s m i n y e a s t i s a p p a r e n t l y n o t dependent on t h e proper f u n c t i o n of m i t o c h o n d r i a 1 a d e n i n e n u c l e o t i d e t r a n s l o c a s e and does n o t even r e q u i r e t h e p r e s e n c e of normal l e v e l s of ATP i n m i t o c h o n d r i a . To o v e r c o m e t h e d i f f i c u l t i e s e n c o u n t e r e d when k i n e t i c p a r a m e t e r s of an enzyme a r e e x t r a p o l a t e d t o h i g h i n i t i a l s u b s t r a t e c o n c e n t r a t i o n s of an enzyme r e a c t o r , e f f e c t i v e and f a s t methods t o o b t a i n s i g n i f i c a n t d a t a f o r t e c h n i c a l a p p l i c a t i o n s h a v e been developed based on o n - l i n e r a t e d e t e r m i n a t i o n s . 1 8 8 Such e x t e n s i v e t r e a t m e n t has proved n e c e s s a r y f o r t h e f o l l o w i n g enzymes: a l a n i n e dehydrogenase, f o r m a t e dehydrogenase,and a - 9 - g l u c o s i d a s e . A t h e r m o s t a b l e a c t i v a t o r of B - k - g l u c o s i d a s e i n n o r m a l human The authors demonstrate t h e s a l i v a h a s been c h a r a c t e r i z e d . ' * ' p r e s e n c e of a t h e r m o s t a b l e f a c t o r i n n o r m a l human s a l i v a which a c t i v a t e s t h e B-g-glucosidase of s a l i v a . The a c t i v a t o r i s s p e c i f i c f o r B-!-glucosidase. I t i s d i a l y s a b l e and s u s c e p t i b l e t o p r o n a s e digestion. S e p h a d e x G-25 g e l f i l t r a t i o n i n d i c a t e s t h a t t h e a c t i v a t o r i s of low m o l e c u l a r w e i g h t , and p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s shows t h e low- m o l e c u l a r - weight f r a c t i o n t o m i g r a t e A m a j o r p r o p o r t i o n of t h e a s two c l o s e l y moving p r o t e i n b a n d s . The a c t i v a t o r a c t i v a t o r does not b i n d t o c o n c a n a v a l i n A-Sepharose. a p p e a r s t o a c t b y b i n d i n g t o o r m o d i f y i n g t h e s a l i v a r y B-eg l u c o s i d a s e i n a time-dependent manner. The c o n d i t i o n s f o r m a x i m a l a c t i v i t y ( p H , b u f f e r , s a t u r a t i n g s u b s t r a t e c o n c e n t r a t i o n , r a n g e of l i n e a r r e l a t i o n s h i p s between enzyme a c t i v i t y v e r s u s i n c u b a t i o n t i m e and v e r s u s enzyme c o n c e n t r a t i o n ) i n t h e f l u o r i m e t r i c a s s a y of s e v e r a l g l y c o h y d r o l a s e s o f l y s o s o m a l o r i g i n i n human p l a s m a a n d s e r u m h a v e b e e n e ~ t a b l i s h e d . ~The ~ f o l l o w i n g enzymes were s t u d i e d : a-Egalactosidase, B-P-galactosidase, B-e-glucosidase, B-cglucuronidase, a-Q-mannosidase, a-&-fucosidase. A l l examined e n z y m e s t u r n e d o u t t o be more o r l e s s u n s t a b l e upon s t o r a g e a t 37OC, 4OC,and - 2 O O C i n b o t h s e r u m and p l a s m a . F o r f u r t h e r d e t a i l s s e e i n i t i a l c i t a t i o n of r e f .26. An i m p r o v e d f l u o r i m e t r i c l e u k o c y t e B - Q - g l u c o s i d a s e a s s a y has
425
6: Enzymes been
developed
for
Gaucher's
leukocyte B-g-glucosidase
disease.19'
Three
fluorimetric
assays were compared f o r t h e i r a b i l i t y t o
d i a g n o s e Gaucher's d i s e a s e and i d e n t i f y c a r r i e r s o f t h e d i s o r d e r : t h e a c i d B-P-glucosidase
assay o f B e u t l e r and K u h l ( B e u t l e r ,
( 1 9 7 0 ) J. L a b . C l i n .
Kuhl, W.
taurocholate-dependent
Med.,
__ 76,
747-755),
E.
and
a pH 5 . 5 - s o d i u m
assay, a n d a new p r o c e d u r e w h i c h e m p l o y s
c o n d u r i t o l B epoxide,
an a c t i v e - s i t e - s p e c i f i c
inhibitor
of
A l l t h r e e assays unambiguously i d e n t i f i e d
glucocerebrosidase.
p a t i e n t s w t h Gaucher's d i s e a s e . the b i l e salt-dependent
With regard t o i d e n t i f y i n g carriers
assay o f P e t e r s e t a l .
and t h e c o n d u r i t o l B
e p o x i d e - d e p e n d e n t p r o c e d u r e gave t h e g r e a t e s t d i s c r i m i n a t i o n b e t w e e n t h e mean B - Q - g l u c o s i d a s e
v a l u e s f o r t h e c o n t r o l and h e t e r o z y g o t e
s a m p l e s when e v a l u a t e d u s i n g S t u d e n t ' s
t
test.
The m o s t r e l i a b l e
a s s a y f o r t h e i d e n t i f i c a t i o n o f t h e c a r r i e r s t a t e was t h e c o n d u r i t o l B epoxide-dependent fewest
false
p r o c e d u r e w h i c h c a n be e x p e c t e d t o p r o v i d e t h e
n e g a t i v e r e s u l t s when
c l a s s i f y i n g h e t e r o z y g o t e s (5%).
However, t h e f a c t t h a t none o f t h e s e methods c o m p l e t e l y s e p a r a t e d control
and
heterozygote
samples
indicated
that
their
use
i n
s c r e e n i n g p r o g r a m s w i l l r e s u l t i n a s i g n i f i c a n t number o f i n c o r r e c t assignments.
0 - e - G l u c o s i d a s e - s t im u l a t i n g p r o t e i n s ( c o - B - ~ - g l u c o s i d a s e ) h a v e been i s o l a t e d f r o m b o v i n e s p l e e n by a c i d i f i c a t i o n o f h o m o g e n i z e d spleen,
heat
denaturation,
and
column
chromatography.lgl
Gel
e l e c t r o p h o r e s i s o f t h e p r o d u c t r e v e a l e d a ' t r a c e o f i n e r t p r o t e i n and t w o f a s t - m o v i n g bands, a m a j o r d i f f u s e band and a m i n o r , f a s t e r m o v i n g band.
The l a t t e r t w o bands c o u l d be e l u t e d f r o m t h e g e l and
shown t o s t i m u l a t e a D - g l u c o s i d a s e p r e p a r a t i o n f r o m b o v i n e s p l e e n . They b o t h s t a i n e d w i t h S t a i n s A l l and F a s t Green, b u t p o o r l y w i t h Coomassie Blue.
The bands c o u l d a l s o be v i s u a l i z e d by u l t r a v i o l e t
scanning,
and p e r i o d a t e - S c h i f f
band.
Mr
with
The the
s t a i n was p o s i t i v e f o r t h e m a j o r
o f t h e co-B-9-glucosidase
gel-permeation
column,
was a b o u t 20,400
but
4,900
as
as m e a s u r e d
measured w i t h
a
S e p h a c r y l S-200 c o l u m n c o n t a i n i n g g u a n i d i u m c h l o r i d e a n d r o u g h l y 6 , 2 0 0 a s m e a s u r e d b y SDS g e l e l e c t r o p h o r e s i s .
i n d i c a t e d by i s o e l e c t r i c focusing. present, but
not
b u t no s i a l i c a c i d . by
phosphatase.
lyophilization,
A
PI o f
4.3-4.4
was
N e u t r a l s u g a r was f o u n d t o b e
The p r o t e i n was d e s t r o y e d b y p r o n a s e , N-ethylmaleimide,
or
S t i m u l a t i o n o f t h e b a s a l a c t i v i t y (1 n m o l h ' l
alkaline assayed
w i t h m e t h y l u m b e l l i f e r y l Q - g l u c o p y r a n o s i d e ) was i n d u c e d 50% when 0.15
ug m l - l
o f c o g l u c o s i d a s e was i n t h e i n c u b a t i o n .
protein raised the
1
The a c t i v a t i n g
v a l u e s a n d l o w e r e d t h e I&,v a l u e s when b o t h Q -
426
Carbohydrate Chemistry
g l u c o s y l c e r a m i d e a n d t h e a r t i f i c i a l s u b s t r a t e were u s e d . In contrast, phosphatidyl serine raised both the a n d t h e Em f o r c e r e b r o s i d e h y d r o l y s i s . The a c t i v a t o r p r o t e i n was f o u n d t o o c c u r i n t h e s o l u b l e p a r t o f s p l e e n as well as i n t h e m i t o c h o n d r i a 1 and lysosomal fractions. moiety, P h l o r i z i n , l a b e l l e d w i t h t r i t i u m o n l y i n t h e !-glucose was u s e d a s s u b s t r a t e f o r t h e 1 3 - g - g l u c o s i d a s e p r e s e n t i n b r u s h b o r d e r membranes f r o m hamster i n t e s t i n e i n o r d e r t o s t u d y , s i m u l t a n e o u s l y , t h e k i n e t i c s o f h y d r o l y s i s a n d t h e r a t e o f t h e Q{ 3 H ) g l u c o s e l i b e r a t e d by t h e e n z y m e . l g 2 The ;-C3H)glucose seems t o experience t h e same hydrolase-related t r a n s p o r t i n t o t h e i n t e s t i n a l v i l l i a s t h e h e x o s e s l i b e r a t e d f r o m t h e common d i s a c c h a r i d e s by their respective hydrolases. The r e l e a s e d Q-{3H)glucose a c c u m u l a t i o n r a t e i s o n l y p a r t i a l l y i n h i b i t e d by u n l a b e l l e d Qg l u c o s e a d d e d t o t h e m e d i u m e i t h e r a s t h e f r e e s u g a r o r as t h e 1-phosphate, and t h e n o n l y p r e c u r s o r s s u c r o s e , l a c t o s e , or ! - g l u c o s e when t h e s e s u g a r s a r e p r e s e n t a t v e r y h i g h l e v e l s . F u r t h e r m o r e , !g l u c o s e o x i d a s e , a d d e d t o t h e m e d i u m as a 2 - g l u c o s e s c a v e n g e r , h a s no e f f e c t on t h e u p t a k e r a t e of t h e p h l o r i z i n h y d r o l a s e - l i b e r a t e d sugar. These and o t h e r f i n d i n g s are p r e s e n t e d a s e v i d e n c e t h a t , u n d e r c o n d i t i o n s where t h e N a + - d e p e n d e n t p - g l u c o s e c a r r i e r i s m o r e t h a n 9 7 % i n h i b i t e d by p h l o r i z i n , t h e Q - g l u c o s e d e r i v e d f r o m t h e i n h i b i t o r , l i k e t h e hexoses from d i s a c c h a r i d e s , has a k i n e t i c advantage f o r t r a n s f e r i n t o the i n t e s t i n a l tissue. T h e a - & - f u c o s i d a s e , B - Q - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e , a-amannosidase, B-Q-glucuronidase, B-Q-xylosidase, B-p-glucosidase, and a - L - a r a b i n o f u r a n o s i d a s e a c t i v i t i e s o f normal and a t r o p h i c s k e l e t a l muscle of developing and a d u l t rats have been c o l l e c t i v e l y i n v e s t i g a t e d . 36 The i m m u n o h i s t o c h e m i c a l l o c a l i z a t i o n of B-2-glucosidases i n l i g n i n a n d i s o f l a v o n e m e t a b o l i s m i n c h i c k - p e a (Cicer a r i e t i n u g ) s e e d l i n g s h a s been r e p o r t e d . l g 3 C o n i f e r i n - s p e c i f i c and i s o f l a v o n e 7-B-g-glucoside-specific B-e-glucosidases have been l o c a l i z e d i n ste m a n d r o o t s e c t i o n s o f c h i c k - p e a s e e d l i n g s b y t h e i n d i r e c t i m m u n o f l u o r i m e t r i c a l method. The c o n i f e r i n - s p e c i f i c B - Q - g l u c o s i d a s e has been f o u n d i n t h e c e l l walls o f t h e t r a c h e a r y e l e m e n t s and of t h e endo-, epi-, and exo-dermis. A l l t h e s e t i s s u e s are known t o c o n t a i n either l i g n i n or polymers, l i k e s u b r i n and c u t i n , which c o n s i s t p a r t i a l l y of phenylpropanoid elements. The l o c a l i z a t i o n o f t h i s B-g-glucosidase is t h e r e f o r e i n a g r e e m e n t w i t h i t s p o s t u l a t e d r e l a t i o n s h i p t o t h e p h e n y l p r o p a n o i d m e t a b o l i s m . The i s o f l a v o n e 7-8-
6: Enzymes
427
Q - g l u c o s i d e - s p e c i f i c f3-!-glucosidase, on t h e o t h e r h a n d , i s p r e d o m i n a n t l y l o c a t e d i n t h e p a r e n c h y m a t i c c o r t e x c e l l s , and o b v i o u s l y i n t h e c y t o p l a s m . T h e s e c e l l s a r e known t o c o n t a i n t h e i s o f l a v o n e f o r m o n o n e t i n , which h a s been shown t o undergo t u r n o v e r i n c h i c k - p e a s e e d l i n g s . The a u t h o r s t h e r e f o r e a s s e r t t h a t t h i s 8-Qg l u c o s i d a s e i s i n v o l v e d i n t h e metabolism of t h e 7-8-P-glucoside of t h i s i s o f lavone. The k i n e t i c p r i n c i p l e s of t h e f o r m a t i o n of Q - g l u c o s e and c e l l o b i o s e i n t h e h y d r o l y s i s of m i c r o c r y s t a l l i n e c e l l u l o s e under t h e a c t i o n of c e l l u l a s e complexes from e i g h t d i f f e r e n t s o u r c e s have been s t u d i e d e ~ p e r i m e n t a l l y . ’ ~B~y s u c c e s s i v e a d d i t i o n of i n d i v i d u a l components of t h e c e l l u l a s e complex { endo-(1 + 4 ) - 8 - ~ - g l u c a n a s e and 8-g-glucosidase) t o the r e a c t i o n system, the s t e p s l i m i t i n g the r a t e of e n z y m a t i c h y d r o l y s i s of m i c r o c r y s t a l l i n e c e l l u l o s e were r e v e a l e d . I t was shown t h a t i n most of t h e c a s e s s t u d i e d t h e s t e p d e t e r m i n i n g t h e r a t e o f f o r m a t i o n of g l u c o s e w i t h t h e p a r t i c i p a t i o n of Only i n i n t e r m e d i a t e c e l l o b i o s e i s t h e a c t i o n of B-g-glucosidase. o n e c a s e ( c o m p l e x from A s p e r g i l l u s f o e t i d u s , e n r i c h e d w i t h B - e g l u c o s i d a s e ) i s t h e r a t e of f o r m a t i o n o f !-glucose from m i c r o c r y s t a l l i n e c e l l u l o s e l i m i t e d by t h e a c t i o n of t h e endo-(1 + 4)-B-!-glucanase of t h e c e l l u l a s e complex. I n accordance w i t h t h e k i n e t i c p r i n c i p l e s d e v e l o p e d , i t was shown t h a t when an e x c e s s of 8P - g l u c o s i d a s e i s added t o t h e r e a c t i o n s y s t e m , t h e s t e p l i m i t i n g t h e r a t e o f h y d r o l y s i s of m i c r o c r y s t a l l i n e c e l l u l o s e under t h e a c t i o n of a l l t h e c e l l u l a s e c o m p l e x e s s t u d i e d becomes t h e a t t a c k on t h e i n i t i a l i n s o l u b l e s u b s t r a t e b y endo-(1 * 4)-B-Q-glucanase. Under t h e same c o n d i t i o n s , a l i n e a r c o r r e l a t i o n was f o u n d b e t w e e n t h e s t e a d y - s t a t e r a t e of f o r m a t i o n of ;-glucose from m i c r o c r y s t a l l i n e c e l l u l o s e under t h e a c t i o n of a l l t h e c e l l u l a s e complexes s t u d i e d , on t h e o n e h a n d , and t h e a c t i v i t y o f e n d o - ( 1 + 4 ) - B - Q - g l u c a n a s e i n t h e s e complexes, on t h e o t h e r . I t was shown t h a t t h e a c t i o n o f a l l t h e c e l l u l a s e complexes s t u d i e d ( s e l e c t e d r a t h e r a r b i t r a r i l y ) i s d e s c r i b e d b y e s s e n t i a l l y t h e same k i n e t i c p r i n c i p l e s , which i s e v i d e n c e of t h e same m e c h a n i s m s of t h e h y d r o l y s i s of i n s o l u b l e c e l l u l o s e by c e l l u l a s e p r e p a r a t i o n s o f d i f f e r e n t o r i g i n . The s u b s t r a t e s p e c i f i c i t i e s of t h r e e c e l l u l a s e s and a 8-Qglucosidase p u r i f i e d from l h e f m o a s c u s a u r a n t i a c u s have been examined.19’ A l l t h r e e c e l l u l a s e s p a r t i a l l y degraded n a t i v e c e l l u l o s e . C e l l u l a s e I , b u t n o t c e l l u l a s e I 1 and c e l l u l a s e 111, r e a d i l y h y d r o l y s e d t h e mixed ( 1 + 3 ) ( 1 + 6 ) - l i n k e d p o l y s a c c h a r i d e s s u c h a s c a r b o x y m e t h y l p a c h y m a n , y e a s t g l u c a n , and l a m i n a r i n . Both
428
Carbohydrate Chemistry
c e l l u l a s e I and t h e B - Q - g l u c o s i d a s e d e g r a d e d x y l a n , and i t was proposed t h a t t h e x y l a n a s e a c t i v i t y was an i n h e r e n t f e a t u r e of t h e s e t w o e n z y m e s ; l i c h e n i n , a ( 1 + 3 ) ( 1 + 4)-B-!-glucan, was d e g r a d e d b y a l l three cellulases. C e l l u l a s e I1 cannot degrade c a r b o x y m e t h y l c e l l u l o s e , and w i t h f i l t e r paper a s s u b s t r a t e t h e endp r o d u c t was c e l l o b i o s e , which i n d i c a t e d t h a t c e l l u l a s e I1 was an --e x o - ( 1 + 4)-B-!-glucan cellobiosylhydrolase. D e g r a d a t i o n of c e l l u l o s e ( f i l t e r p a p e r ) could be c a t a l y s e d i n d e p e n d e n t l y b y each of t h e t h r e e c e l l u l a s e s . There was no s y n e r g i s t i c e f f e c t between any of t h e c e l l u l a s e s , and c e l l o b i o s e was t h e p r i n c i p a l p r o d u c t of d e g r a d a t i o n . The mode o f a c t i o n o f o n e c e l l u l a s e ( c e l l u l a s e 111) was examined by u s i n g reduced c e l l o d e x t r i n s . The c e n t r a l l i n k a g e s o f t h e c e l l o d e x t r i n s were t h e p r e f e r r e d p o i n t s of c l e a v a g e , which, w i t h t h e r a p i d d e c r e a s e i n v i s c o s i t y of c a r b o x y m e t h y l c e l l u l o s e , c o n f i r m e d t h a t c e l l u l a s e I11 was an e n d o c e l l u l a s e . The r a t e of h y d r o l y s i s i n c r e a s e d w i t h c h a i n l e n g t h of t h e reduced c e l l o d e x t r i n s , and t h e s e k i n e t i c d a t a i n d i c a t e d t h a t t h e s p e c i f i c i t y r e g i o n of c e l l u l a s e I11 was f i v e o r s i x 0 - g l u c o s e u n i t s i n l e n g t h . Optimization experiments w i t h response s u r f a c e s t a t i s t i c a l a n a l y s i s have been performed w i t h Schizophyllum commune t o o b t a i n h i g h B - e - g l u c o s i d a s e ~ i e 1 d s . l The ~ ~ factors i n t h e optimization e x p e r i m e n t w e r e t h e c o n c e n t r a t i o n s o f c e l l u l o s e , p e p t o n e , and KH2P04. T h e i r o p t i m a l v a l u e s w e r e 3.2, 3 . 0 , a n d 0 . 2 g ( 1 0 0 m l ) - ’ , respectively. Enzyme a s s a y s r e v e a l e d v e r y h i g h B-!-glucosidase ( 2 2 . 2 U m l - l ) and c e l l o b i a s e (68.9 U m l - l ) y i e l d s . The a v i c e l a s e y i e l d was low a s c o m p a r e d w i t h t h a t f r o m T r i c h o d e r m a r e e s e i . Mixtures of S. commune and T . r e e s e i c u l t u r e f i l t r a t e s caused f a s t e r and more e x t e n s i v e s a c c h a r i f i c a t i o n o f Avicel than could be achieved by e i t h e r f i l t r a t e alone. A B-!-glucosidase was i s o l a t e d and p u r i f i e d f r o m t h e o p t i m i z e d c u l t u r e f i l t r a t e of S . commune. The e l e c t r o p h o r e t i c m o b i l i t y of t h e p u r i f i e d B-0-glucosidase i n d i c a t e d a molecular weight of 97,000. The amino a c i d c o m p o s i t i o n was s i m i l a r t o t h a t of B - ~ - g l u c o s i d a s e from T. r e e s e i . The a c i d i c ( L - a s p a r t a t e a n d i - g l u t a m a t e ) r e s i d u e s o r t h e i r a m i d e s o r b o t h made up a p p r o x i m a t e l y 2 0 % o f t h e p r o t e i n . The N H 2 - t e r m i n a l a m i n o a c i d of t h e enzyme was & - h i s t i d i n e . A new u l t r a s o n i c method has been developed f o r d e t e r m i n i n g t h e c o m p o s i t i o n and p r o p e r t i e s of i n d i v i d u a l components of polyenzyme systems without t h e i r preliminary separation.197 The method i s based on a d e t e r m i n a t i o n of t h e pH dependence of t h e r a t e c o n s t a n t s of i n a c t i v a t i o n of i n d i v i d u a l components of t h e enzyme system under
6: Enzymes
429
t h e a c t i o n of c a v i t a t i o n a l u l t r a s o u n d . The method was used t o s t u d y t h e c e l l u l a s e complex from t h e fungus Geotrichum candidum. I t was shown t h a t t h i s complex c o n t a i n s a t l e a s t f o u r c e l l u l o l y t i c enzymes - -e-n d o - ( 1 + 4 ) - B - Q- - g l u c a n a s e , e=-(l + 4 ) - B - Q- - g l u c a n a s e , B-gg l u c o s i d a s e , and a r y l - B - E - g l u c o s i d a s e . These enzymes d i f f e r i n v a l u e s of pK of t h e i o n o g e n i c g r o u p s , c o n t r o l l i n g t h e pH p r o f i l e s of u l t r a s o n i c i n a c t i v a t i o n , a s w e l l a s i n v a l u e s of t h e r a t e c o n s t a n t s of i n a c t i v a t i o n of t h e f o r m of t h e a c t i v e s i t e most s t a b l e t o t h e a c t i o n of u l t r a s o u n d . The r e l a t i o n s h i p b e t w e e n c a r b o h y d r a t e m o i e t y and t h e r m o s t a b i l i t y of B-9-glucosidase from Mucor m e i h e i Y H - 1 0 has been i n ~ e s t i g a t e d . ~Two ~ forms of t h e r m o s t a b l e B-g-glucosidase ( a more t h e r m o s t a b l e G-a and l e s s t h e r m o s t a b l e G - b ) w e r e p u r i f i e d t o homogeneity from t h e c u l t u r e f i l t r a t e . G-a and t h e G-b w e r e g l y c o e n z y m e s , and t h e y c o n t a i n e d 2 3 . 4 % and 1 3 . 0 % c a r b o h y d r a t e For further information see i n i t i a l residues, respectively. c i t a t i o n of r e f .48. The components of t h e c e l l u l o l y t i c system of T-a l a r o m y----c e s e m e r s o n i i grown on c e l l u l o s i c m a t e r i a l h a v e been identified.198 The enzyme system was found t o be comprised of f o u r t o f i v e f o r m s of =-(l + 4)-B-Q - - g l u c a n a s e , a t l e a s t t w o f o r m s of --e n d o - ( 1 + 4 ) - B - Q - g l u c a n a s e , and t h r e e enzymes e x h i b i t i n g B - e g l u c o s i d a s e a c t i v i t y . One of t h e l a t t e r , t e r m e d B - Q - g l u c o s i d a s e 111, i s induced c o n c u r r e n t l y w i t h t h e c e l l u l a s e s b u t d i s a p p e a r s from t h e medium because of t h e low pH t h a t develops d u r i n g growth. The c e l l u l a s e s b y c o n t r a s t a r e more a c i d s t a b l e . L a t e r i n t h e g r o w t h cycle B-2-glucosidase I accumulates. An i n t r a c e l l u l a r B - e g l u c o s i d a s e , B - Q - g l u c o s i d a s e I V , was a l s o d e t e c t e d . The p o s s i b l e f u n c t i o n s of t h e s e enzymes a r e d i s c u s s e d . P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of t h e e x t r a c e l l u l a r and i n t r a c e l l u l a r B - Q - g l u c o s i d a s e s of t h e t e r m o p h y l l i c f u n g u s Talaromyces e m e r s o n i i have been described.199 The organism produces t h r e e e x t r a c e l l u l a r and o n e i n t r a c e l l u l a r enzymes e x h i b i t i n g B-0glucosidase activity. T w o of t h e e x t r a c e l l u l a r f o r m s , 6 - e g l u c o s i d a s e I and B - Q - g l u c o s i d a s e 1 1 1 , h a v e b e e n p u r i f i e d , a s h a s t h e i n t r a c e l l u l a r B-Q-glucosidase I V . The pH and t e m p e r a t u r e o p t i m a , s t a b i l i t y k i n e t i c p a r a m e t e r s , and s u b s t r a t e s p e c i f i c i t y of each have been d e t e r m i n e d . I t was concluded t h a t B-E-glucosidase I a n d B - g - g l u c o s i d a s e I V a r e t r u e B - 2 - g l u c o s i d a s e s w h i l e B-;g l u c o s i d a s e I11 i s an =-(l + l)-B-D-glucanase. An enzyme from Rhizobium m e l i t o t i has been shown t o be a 8-g-
430
Carbohydrate Chemistry
glucosidase activity.135 of R.
two
enzymes,
An e a r l i e r s t u d y had shown t h e e x i s t e n c e
and
A
m e l i l o t i 2011 S t r 3 .
with
B,
B-Q-galactosidase
activity
A mutant l a c k i n g 8-g-galactosidase
a s t u d y t o b e made o f t h e r o l e o f e n z y m e 6.
i n
A allowed
I t i s demonstrated t h a t
e n z y m e 8, w h o s e s y n t h e s i s was i n d u c i b l e b y c e l l o b i o s e , i s a b l e t o h y d r o l y s e 4 - n i t r o p h e n y 1 8 - Q - g l u c o p y r a n o s i d e and 4 - n i t r o p h e n y 1 6-Eg a l a c t o p y r a n o s i d e w i t h , r e s p e c t i v e l y , I$,,v a l u e s o f 0.135 Enzyme
B i s
activity
actually
.
a 8-Q-glucosidase
The t h e r m a l - s t a b i l i t y present
i n
culture
with
and 6.4
mM.
B-Q-galactosidase
c h a r a c t e r i s t i c s o f t h e c e l l u l a s e enzymes
filtrates
Sporotrichum thermophile
have
of
the
been
thermophilic
investigated
t e m p e r a t u r e s and a t d i f f e r e n t t i m e s o f e x p o s u r e . 2 0 0
at
fungus
different
Maximum e n z y m i c
a c t i v i t i e s u n d e r a s s a y c o n d i t i o n s were f o u n d a t 68OC f o r t h e f i l t e r paper
activity
(FPA)
and t h e
Cx
w h i l e t h e maxima f o r t h e C1 activity
(cellobiose)
respectively.
activity
activity
were
(carboxymethylcellulose),
( c o t t o n ) and B-;-glucosidase
found
t o
be
at
and
55OC
72'C,
C u l t u r e f i l t r a t e s were exposed t o a g i v e n c o n s t a n t
t e m p e r a t u r e f o r v a r y i n g l e n g t h s o f t i m e t o a maximum o f 48 h o u r s and then
analysed for
conditions. 65OC.
residual
enzymic
activities
under
The e x p o s u r e t e m p e r a t u r e s s t u d i e d w e r e 5OoC,
assay
6OoC, and
A f t e r 48 h o u r s ' e x p o s u r e t i m e a t 5OoC t h e r e s i d u a l a c t i v i t i e s
f o r t h e FPA, Cx,and B - ~ - g l u c o s i d a s e w e r e f o u n d t o b e 88%, 9 8 % , a n d
93% o f t h e o r i g i n a l a c t i v i t i e s ,
respectively.
L y s o s o m a l enzymes h a v e been f o u n d t o p b s s e s s a common a n t i g e n i c determinant and
i n the
antisera
enzymes.47
cellular
slime
mould D i c t y o s t e l i u m discoideum,
have been prepared a g a i n s t
two such
lysosomal
Two p u r i f i e d enzyme p r e p a r a t i o n s u s e d f o r i m m u n i z a t i o n ,
B-9-2-ace t a m i d o -2-deoxy g l u c o s i d a s e and B-;-glucos
i d a s e -1,
showed no
c r o s s - c o n t a m i n a t i o n w i t h e a c h o t h e r and n o s i g n i f i c a n t c o n t a m i n a t i o n by
other
l y s o s o m a l enzymes.
For
further
details
see
i n i t i a l
c i t a t i o n o f r e f .47. S e p a r a t i o n and some o f t h e p r o p e r t i e s o f t w o f 3 - p - g l u c o s i d a s e s
o f S p o r o t r i c h u m ( C h r y s o p o r i u m ) t h e r m o p h i l e h a v e been d e s c r i b e d . 2 0 1 I n t r a c e l l u l a r , i n d u c i b l e B - e - g l u c o s i d a s e was f r a c t i o n a t e d b y g e l c h r o m a t o g r a p h y o r i s o e l e c t r i c f o c u s i n g i n t o c o m p o n e n t s A a n d B. E n z y m e A ( m o l e c u l a r w e i g h t 440,000)
had o n l y a r y l B-Q-glucosidase
a c t i v i t y whereas enzyme 8 ( m o l e c u l a r
weight
40,000)
hydrolysed
several B-g-glucosides b u t h a d o n l y l o w a c t i v i t y a g a i n s t 2n i t r o p h e n y l B - 9 - g l u c o p y r a n o s i d e . B o t h enzymes h a d t e m p e r a t u r e o p t i m a
o f a b o u t 5OoC.
The p H o p t i m u m was 5.6
f o r e n z y m e A a n d 6.3
for
431
6: Enzymes enzyme
respectively.
B,
enzyme 6 w e r e 0.18
2-nitrophenyl
B-n-glucopyranoside
and t h e c o r r e s p o n d i n g
mM,
values f o r
m M ( 2 - n i t r o p h e n y l B - Q - g l u c o p y r a n o s i d e ) a n d 0.28
mM (cellobiose).
glucopyranoside,
Km
The
v a l u e f o r e n z y m e A was 0.5 Enzyme 8,
c o n c e n t r a t i o n a b o v e 0.4
inhibition
at
a
substrate
m M w h i c h c o u l d be r e l e a s e d b y c e l l o b i i t o l
Enzyme A was i s o e l e c t r i c a t pH 4.48,
and o t h e r a l d i t o l s .
8-Q-
when t e s t e d w i t h 2 - n i t r o p h e n y l
showed s u b s t r a t e
B was i s o e l e c t r i c a t pH 4.64.
a n d enzyme
Several i n h i b i t o r s were t e s t e d f o r
t h e i r a c t i o n on t h e a c t i v i t y o f enzymes A and B.
B o t h enzymes w e r e
f o u n d t o be c o n c o m i t a n t l y i n d u c e d i n c u l t u r e s w i t h e i t h e r c e l l o b i o s e
o r c e l l u l o s e as c a r b o n s o u r c e . Two
forms
of
B-Q-glucosidase
from
the
culture f i l t r a t e of
M a c r o p h o g i n a p h a s e o l i n a h a v e been s e p a r a t e d and p a r t i a l l y b y (NH4)2S04 p r e c i p i t a t i o n ,
ion-exchange
The f i n a l p r e p a r a t i o n was p u r i f i e d 1 0 3 - f o l d a n d 88-
filtration.202
f o l d f o r B - Q - g l u c o s i d a s e I and B-B-glucosidase Polyacrylamide g e l e l e c t r o p h o r e s i s of a s i n g l e band a t pH 8.3. respect
to
220,000
for
molecular
11, r e s p e c t i v e l y .
t h e p u r i f i e d enzymes i m p a r t e d
The t w o f o r m s d i f f e r f r o m e a c h o t h e r w i t h weight
B-g-glucosidase
electrophoretic of
purified
chromatography, and g e l
(323,600
11),
f o r B-Q-glucosidase
pH o p t i m a ,
m o b i l i t y , and s u b s t r a t e s p e c i f i c i t y .
B-P-glucosidase
may
also
differentiated
inhibition
using
nojirimycin.
N o j i r i m y c i n i s more p o t e n t f o r B - Q - g l u c o s i d a s e
B-P-glucosidase
inhibitors
The t w o f o r m s by
experiments for
as
be
11.
The
energy
I and
temperature optima,
Q-glucono-1,4-lactone of
activation for
and
I than
the
enzymes was a l s o d i f f e r e n t . The p u r i f i c a t i o n ,
characterization,
and p r o p e r t i e s o f
two
$-a-
g l u c o s i d a s e enzymes f r o m S c l e r o t i u m r o l f s i i h a v e been d e s c r i b e d . 2 0 3 F o u r B - Q - g l u c o s i d a s e enzymes were e x t e n s i v e l y p u r i f i e d f r o m t h e c u l t u r e f i l t r a t e s o f S. properties studied.
r o l f s i i a n d some o f t h e i r p h y s i c o c h e m i c a l
A l l t h e enzymes showed a s i n g l e p r o t e i n b a n d i n
S D S g e l e l e c t r o p h o r e s i s a n d i n d i s c g e l e l e c t r o p h o r e s i s a t pH 8.9 a n d 4.3.
The p u r i f i e d B - g - g l u c o s i d a s e s
were
e-Q
f r e e from
glucanase (carboxymethylcellulose v i s c o s i t y - l o w e r i n g a c t i v i t y ) .
A l l
t h e enzymes a r e g l y c o p r o t e i n s and a r e composed o f one p o l y p e p t i d e chain.
The m o l e c u l a r w e i g h t o f t h e f o u r B - q - g l u c o s i d a s e s
b e t w e e n 9 0 , 0 0 0 a n d 107,000. four
B - Q - g l u c o s i d a s e s a r e 4.2
The i s o e l e c t r i c p o i n t s f o r 5.55,
respectively.
cellobiose
as
varies
The pH a n d t e m p e r a t u r e o p t i m a o f t h e and 68OC w i t h c e l l u l o s e as s u b s t r a t e . t h e e n z y m e s a r e 4.10,
4.55,
5.10,and
The s p e c i f i c a c t i v i t i e s o f t h e e n z y m e s w i t h
s u b s t r a t e a r e 55,
78,
175, a n d 5 1 u m o l Q - g l u c o s e
432
Carbohydrate Chemistry
released per
minute
per
milligram
protein,
respectively.
The
e n z y m e s a r e i n h i b i t e d by t h e r e a c t i o n p r o d u c t ! - g l u c o s e ,
and by
g-
glucono-1,4-lactone
group
i s
and
nojirimycin.
A
carboxylate
i m p l i c a t e d i n t h e c a t a l y s i s o f B-P-glucosidase. The
purification
-Humicola
and
properties
e x h i b i t e d h i g h B-B-glucosidase b r a n medium. extract
of
i n s o l e n s have been d e s c r i b e d . 2 0 4
by
activity
The B - Q - g l u c o s i d a s e
B-P-glucosidase
This
thermophilic
when g r o w n i n s o l i d w h e a t -
was p u r i f i e d f r o m t h e c u l t u r e
consecutive column chromatographies
homogeneous
on
molecular
polyacrylamide
weight
electrophoresis,
was
from fungus
gel
estimated
disc t o
and found t o
electrophoresis.
be
250,000
by
gel
SDS
a n d t h e i s o e l e c t r i c p o i n t w a s a t pH 4.23.
enzyme h a d an o p t i m u m pH o f 5.0,
be The The
a n o p t i m u m t e m p e r a t u r e o f 5OoC, a n d
showed s i g n i f i c a n t r e s i s t a n c e t o u r e a ,
d i m e t h y l sulphoxide,and
ethyl
alcohol. The
kinetics
cellotetraose convenient
by
o f
the
hydrolyses
B-P-glucosidase
integral
of
have
technique.205
cellotriose been
and
studied
Reaction mechanisms
and
m a t h e m a t i c a l models were p o s t u l a t e d t o d e s c r i b e t h e r e a c t i o n s . end-products hydrolysis produces
o f t h e r e a c t i o n were f o u n d t o be i n h i b i t o r y
i n a competitive cellobiose
mode.
Hydrolysis of
and h y d r o l y s i s
c e l l o b i o s e and B-glucose.
of
of
using a The
toward
cellotetraose
cellotriose
produced
B o t h s u g a r s i n h i b i t e d t h e enzyme, w i t h
cellobiose being a stronger inhibitor. Oxygen-18
leaving-group
been measured f o r
k i n e t i c i s o t o p e e f f e c t s (KIEs) have
a set of glycosyl-transfer
nitrophenyl 8-Q-glycosides
as
h y d r o l y s i s and a l k a l i n e h y d r o l y s i s e x h i b i t + 0.0015
and
1.386
2 0.0032,
reactions
substrates.206
K I E s o f &16/k18
respectively.
w i t h 4-
Acid-catalysed
Lysozyme
= 1.0355
and
B-q-
(l/s)
o f ( ~ / ~ ) 1 6 / ( ~ / ~ ) 1=8 1 . 0 4 6 7 g l u c o s i d a s e A show K I E s o n l m a x / K m 2 0.0015 a n d 1.0377 2 0.0061, r e s p e c t i v e l y . The l a r g e m a g n i t u d e o f these KIEs r e q u i r e s t h a t carbon-oxygen
i n the transition states
for
these
b o n d s c i s s i o n be f a r a d v a n c e d reactions;
therefore, i n the
t r a n s i t i o n states f o r the f i r s t i r r e v e r s i b l e steps i n these r e a c t i o n sequences,
scission of
the
glycosidic
bond must
be e s s e n t i a l l y
c o m p l e t e f o r t h e r e a c t i o n s c a t a l y s e d by l y s o z y m e a n d B - Q - g l u c o s i d a s e A,
w h i c h a r e t h o u g h t t o p r o c e e d v i a SN1 a n d SN2 m e c h a n i s m s ,
ively.
Acid-catalysed
transition
hydrolysis
s t a t e i n v o l v i n g a t l e a s t 80% C-0
bond c l e a v a g e and o n l y
p a r t i a l proton t r a n s f e r t o the leaving 4-nitrophenyl 2-Deoxy-Q-glucopyranose
has
respect-
i s shown t o proceed t h r o u g h a oxygen atom.
been used i n t h e
selective
6: Enzymes
433
i s o l a t i o n of m u t a n t s of T r i c h o d e r m a r e e s e i w i t h e n h a n c e d B - Q g l u c o s i d a s e p r o d u c t i ~ n . ~W~h e n 2 - d e o x y - Q - g l u c o s e was a p p l i e d t o ---T . r e e s e i Q M 9 4 1 4 i n a s i m i l a r way i t p e r m i t t e d t h e s e l e c t i v e i s o l a t i o n i n v e r y h i g h p l a t e y i e l d s o f m u t a n t s d e r e p r e s s e d and h y p e r p r o d u c t i v e w i t h r e s p e c t t o B-e-glucosidase. E x t r a c e l l u l a r c e l l u l a s e a c t i v i t i e s of C l o s t r i d i u m t h e r m o c e l l u m L Q R I and Trichoderma r e e s e i QM9414 have been compared.207 The c r u d e e x t r a c e l l u l a r c e l l u l a s e of C. thermocellum L Q R I ( v i r g i n s t r a i n ) was very a c t i v e and s o l u b i l i z e d m i c r o c r y s t a l l i n e c e l l u l o s e a t one-half t h e r a t e o b s e r v e d f o r t h e e x t r a c e l l u l a r c e l l u l a s e o f T. r e e s e i QM9414 (mutant s t r a i n ) . C. thermocellum c e l l u l a s e a c t i v i t y d i f f e r e d c o n s i d e r a b l y f r o m t h a t o f T . r e e s e i a s f o l l o w s : h i g h e r endo-Qglucanase/z-;-glucanase a c t i v i t y r a t i o , absence of e x t r a c e l l u l a r B-Q-glucosidase or B-Q-xylosidase a c t i v i t y , long-chain o l i g o s a c c h a r i d e s i n s t e a d of s h o r t - c h a i n o l i g o s a c c h a r i d e s a s i n i t i a l ( 1 5 m i n ) h y d r o l y t i c p r o d u c t s on m i c r o c r y s t a l l i n e c e l l u l o s e , mainly c e l l o b i o s e o r x y l o b i o s e a s long-term ( 2 4 h o u r ) h y d r o l y s i s p r o d u c t s of Avicel and MN300 o r x y l a n , and high a c t i v i t y and s t a b i l i t y a t 60 to 7OoC. Under o p t i m i z e d r e a c t i o n c o n d i t i o n s , t h e k i n e t i c p r o p e r t i e s ( l m a0 x . 4 , umol m i n - ' mg-1 of p r o t e i n , e n e r g y of a c t i v a t i o n 33 k J , t e m p e r a t u r e c o e f f i c i e n t 1 . 8 ) o f C . t h e r m o c e l l u m c e 11u l o s e - s o l u b i 1i z i ng a c t i v i t y w e r e comparable t o t h o s e r e p o r t e d f o r T . r e e s e i , e x c e p t t h a t t h e dyed A v i c e l c o n c e n t r a t i o n a t h a l f The c e l l u l o s e m a x i m a l v e l o c i t y was t w o - f o l d h i g h e r ( 1 8 2 pM). s o l u b i l i z i n g a c t i v i t y of t h e two c r u d e c e l l u l a s e s d i f f e r e d c o n s i d e r a b l y i n r e s p o n s e t o v a r i o u s enzyme i n h i b i t o r s . Most n o t a b l y , Ag2+ and Hg2+ e f f e c t i v e l y i n h i b i t e d C. thermocellum b u t not T . r e e s e i c e l l u l a s e a t 20 P M , whereas Ca2+, Mg2+, and Mn2+ i n h i b i t e d T. r e e s e i b u t not C . thermocellum c e l l u l a s e a t 10 m M . Both enzymes were i n h i b i t e d b y C u 2 + ( 2 0 m M ) , Z n 2 + (1.0 m M ) , and e t h y l e n e g l y c o l b i s - ( 6 - a m i n o e t h y l e t h e r ) - N , N - t e t r a - a c e t i c a c i d (10 m M ) . T. r e e s e i b u t n o t C . t h e r m o c e l l u m c e l l u l o s e - s o l u b i l i z i n g a c t i v i t y was 2 0 % i n h i b i t e d b y E - g l u c o s e ( 7 3 m M ) and c e l l o b i o s e ( 2 9 m M ) . Both c e l l u l a s e s p r e f e r e n t i a l l y c l e a v e d t h e i n t e r n a l g l y c o s i d i c bonds of cello-oligosaccharides. The o v e r a l l r a t e s of c e l l o - o l i g o s a c c h a r i d e d e g r a d a t i o n were h i g h e r f o r T. r e e s e A t h a n f o r C. thermocellum c e l l u l a s e , e x c e p t t h a t t h e r a t e s o f c o n v e r s i o n of c e l l o h e x a o s e t o c e l l o t r i o s e were e q u i v a l e n t . The p r o d u c t i o n o f c e l l u l a s e s and h e m i c e l l u l a s e s has been s t u d i e d w i t h Trichoderma r e e s e i R u t C-30.208 T h i s organism produced, t o g e t h e r w i t h high c e l l u l a s e a c t i v i t i e s , c o n s i d e r a b l e a m o u n t s o f x y l a n a s e s and B - Q - g l u c o s i d a s e . Three
434
Carbohydrate Chemistry
cellulose
concentrations
(1.0,
2.5,
and
were
5.0%)
examined t o
d e t e r m i n e t h e maximum l e v e l s o f c e l l u l a s e a c t i v i t y o b t a i n a b l e i n submerged c u l t u r e .
Temperature
i n c r e a s e c e l l mass
to
maximum
were
used t o
days,
thereby
a n d pH p r o f i l i n g
levels within
two
enhancing fermenter p r o d u c t i v i t y a t higher substrate l e v e l s . e f f e c t s o f temperature,
pH,
Tween-80
concentration,
c o n c e n t r a t i o n on t h e r a t i o of
and s u b s t r a t e
The
c ar bon source,
m y c e l i a l g r o w t h and
e x t r a c e l l u l a r enzyme p r o d u c t i o n w e r e d e s c r i b e d . A
mutant
enzymes
strain
has
been
with
increased production of
induced
from
T r i c h o d e r m a r e e s e i Q M 9414.209
the
good
Cellulase
cellulolytic
cellulase
activities of
producer t h e mutant
i n f e r m e n t e r c u l t i v a t i o n s w e r e i n c r e a s e d t w o - t o t h r e e - f o l d and 8 - e glucosidase
activity
up
to
six-fold
when
compared
to
the
c o r r e s p o n d i n g a c t i v i t i e s p r o d u c e d by Q M 9414. Cellulolytic
enzyme complexes o b t a i n e d f r o m m u t a n t s o f
~ T r i c-h o-d er m-a r e e s e i w i t h e n h a n c e d c e l l u l a s e p r o d u c t i o n h a v e b e e n characterized.210 T.
The c e l l u l o l y t i c e n z y m e c o m p l e x e s s e c r e t e d by
r e e s e i Q M 9414 and i t s m u t a n t s M 5, M 6 , MHC 1 5 , and MHC 22 w e r e
characterized
by
determining
their
specific
f i l t e r
c a r b o x y m e t h y l c e l l u l a s e , a n d 8 - Q - g 1u c o s i d a s e a c t i v i t i e s . characterized further :$-9-glucosidase
by m e a s u r i n g t h e i r
paper,
They w e r e
carboxymethylcellulase
c o l u m n o v e r t h e pH r a n g e 3 - 1 0 .
While
the overall
filter-paper
8-e-
a c t i v i t y was r o u g h l y e q u a l i n a l l p r e p a r a t i o n s , t h e s p e c i f i c g l u c o s i d a s e a c t i v i t y was h i g h e s t i n m u t a n t s MHC 15 and MHC 22, are distinguished
morphologically from the parent s t r a i n ,
by a h i g h e r d e g r e e o f b r a n c h i n g of glucosidase
activity
were
t h e i r hyphae.
detected
preparations
f r o m Q M 9 4 1 4 a n d M 6,
mutant
while
M
5,
and
p r o f i l e s a f t e r s e p a r a t i o n on an i s o e l e c t r i c - f o c u s i n g
3 and 4 peaks,
by
which
Q M 9414,
Two p e a k s o f
isoelectric
none i n t h e enzyme f r o m respectively,
were
i n these l a s t
a l s o r e f l e c t e d i n t h e h i g h e r !-glucose
i n the
found
p r e p a r a t i o n s f r o m m o r p h o l o g i c a l m u t a n t s MHC 1 5 a n d M H C 22. higher 8-~-glucosidase a c t i v i t y
8-g-
focusing
i n The
t w o p r e p a r a t i o n s was
t o cellobiose r a t i o i n the
i n i t i a l s t a g e s o f c e l l u l o s e h y d r o l y s i s by t h e i n d i v i d u a l enzyme preparations. Production o f
----Fusarium
sp.
extracellular
cellulase
by
an
has been s t u d i e d i n shake c u l t u r e s ,
appearance o f c e l l u l a s e components (B-P-glucosidase day,
endo-(1
+
4)-B-g-glucanase
E - g l u c a n a s e on t h e t h i r d day o f observed.211
Maximum
isolate
production
of
a l l
these
a
on t h e f i r s t
on t h e s e c o n d day, and =-(l g r o w t h on i n s o l u b l e
of
and s e q u e n t i a l +
41-8-
cellulose)
was
components
was
6: Enzymes
435
a c h i e v e d on t h e f i f t h d a y . The F u s a r i u m p r o d u c e d s i g n i f i c a n t l y h i g h e r B - -0 - g l u c o s i d a s e w i t h i n a s h o r t p e r i o d of t i m e a s c o m p a r e d w i t h Trichoderma r e e s e i . The i n f l u e n c e of d i f f e r e n t n i t r o g e n and c a r b o n s o u r c e s on c e l l u l a s e h a s b e e n i n v e s t i g a t e d . Crude c e l l u l o l y t i c enzyme was used f o r h y d r o l y s i s of common a g r i c u l t u r a l w a s t e s b o t h w i t h and w i t h o u t s o d i u m h y d r o x i d e p r e t r e a t m e n t . A n a l y s i s of h y d r o l y s a t e s i n d i c a t e d g l u c o s e a s t h e major c o n s t i t u e n t (about 83% of t o t a l r e d u c i n g s u g a r ) . The r e g u l a t i o n o f t h e c e l l u l o l y t i c system i n Trichoderma r e e s e i b y s o p h o r o s e has been i n v e s t i g a t e d through measurements o f i n d u c t i o n of c e l l u l a s e and r e p r e s s i o n of B - Q - g l u c o s i d a s e . 2 1 2 Sophorose has two r e g u l a t o r y r o l e s i n t h e p r o d u c t i o n o f c e l l u l a s e e n z y m e s i n T . r ee s e i : B - Q - g l u c o s i d a s e r e p r e s s i o n and c e l l u l a s e i n d u c t i o n . S o p h o r o s e a l s o i s h y d r o l y s e d b y t h e m y c e l i a l - a s s o c i a t e d B-Qglucosidase. R e p r e s s i o n of B-Q-glucosidase r e d u c e s s o p h o r o s e h y d r o l y s i s and t h u s may i n c r e a s e c e l l u l a s e i n d u c t i o n . The e n z y m a t i c h y d r o l y s i s o f c e l l u l o s e t o !-glucose using i m m o b i l i z e d B-e-glucosidase has been i n v e s t i g a t e d , because c e l l u l a s e s y s t e m s f r e q u e n t l y c o n t a i n i n e f f i c i e n t B-Q-glucosidase t o p r e v e n t t h e accumulation o f i n h i b i t o r y amounts o f c e l l o b i o s e . 2 1 3 This r e s e a r c h has i n v e s t i g a t e d t h e use of s u p p l e m e n t a l i m m o b i l i z e d B - e g l u c o s i d a s e t o i n c r e a s e y i e l d s of ! - g l u c o s e . Immobilized B-Qg l u c o s i d a s e from A s p e r g i l l u s p h o e n i c i s was produced by s o p t i o n on The c o n t r o l l e d - p o r e a l u m i n a w i t h abowt 9 0 % a c t i v i t y r e t e n t i o n . p r o d u c t l o s t o n l y a b o u t 1 0 % o f t h e o r i g i n a l a c t i v i t y d u r i n g an ons t r e a m r e a c t i o n p e r i o d of 5 0 0 h o u r s w i t h c e l l o b i o s e a s s u b s t r a t e . Maximum a c t i v i t y o c c u r r e d n e a r pH 3.5 and t h e a p p a r e n t a c t i v a t i o n energy w i t h Trichoderma r e e s e i c e l l u l a s e t o hydrolyse c e l l u l o s i c m a t e r i a l s , s u c h a s S o l k a F l o c , c o r n s t o v e r , and e x p l o d e d wood. I n c r e a s e d y i e l d s o f Q-glucose and g r e a t e r c o n v e r s i o n s o f c e l l o b i o s e t o !-glucose w e r e o b s e r v e d when t h e r e a c t i o n s y s t e m s c o n t a i n e d s u p p l e m e n t a l i m m o b i l i z e d B-g-glucosidase. The i n h i b i t i o n o f B - g - g l u c o s i d a s e i n T r i c h o d e r m a r e e s e i C30 c e l l u l a s e b y Q - g l u c o s e , i t s i s o m e r s , and d e r i v a t i v e s h a s been s t u d i e d s i n c e u s i n g c e l l o b i o s e and 4 - n i t r o p h e n y l B-p-glucopyranoside a s s u b s t r a t e s f o r d e t e r m i n i n g enzyme a c t i v i t y . 2 1 4 The e n z y m a t i c h y d r o l y s i s o f b o t h s u b s t r a t e s was i n h i b i t e d c o m p e t i t i v e l y b y p g l u c o s e w i t h a p p r o x i m a t e Ki v a l u e s o f 0 . 5 m M and 8.7 m M f o r c e l l o b i o s e and 4 - n i t r o p h e n y l B - Q - g l u c o p y r a n o s i d e a s s u b s t r a t e , T h i s i n h i b i t i o n by E-glucose was maximal a t pH 4.8, respectively. and no i n h i b i t i o n was observed a t pH 6.5 and above. The a-anomer o f
436
Carbohydrate Chemistry
a-glucose f3-form.
i n h i b i t e d B-Q-glucosidase t o a greater extent than d i d t h e Compared w i t h g - g l u c o s e ,
and Q - g l u c o s e l - p h o s p h a t e extent,
unlike
as P --glucose
I-glucose,
n-glucose-I=-cysteine itself
F r u c t o s e (2-100
Q - g l u c o s e 6-phosphate,
i n h i b i t e d t h e enzyme t o a much l e s s e r w h i c h was a l m o s t as i n h i b i t o r y
w h e n c e l l o b i o s e was
u s e d as
Q-
substrate.
was f o u n d t o be a p o o r i n h i b i t o r o f t h e enzyme.
mM)
I t was s u g g e s t e d t h a t h i g h r a t e s o f c e l l o b i o s e h y d r o l y s i s c a t a l y s e d by
B-Q-glucosidase
may b e p r o l o n g e d b y
converting the
reaction
p r o d u c t Q - g l u c o s e t o Q - f r u c t o s e u s i n g a s u i t a b l e p r e p a r a t i o n o f Qglucose isomerase. The h y d r o l y s i s o f c e l l o b i o s e t o ! - g l u c o s e
by B - e - g l u c o s i d a s e
has been s t u d i e d w i t h emphasis upon s u s c e p t i b i l i t y t o p r o d u c t and substrate inhibition.215
T h i s work r e p o r t s a model which combines
b o t h p r o d u c t and s u b s t r a t e i n h i b i t i o n e f f e c t s f o r B - Q - g l u c o s i d a s e i s o l a t e d from a
commercial preparation o f
Trichoderma v i r i d e .
An
i n t e g r a t e d r a t e e q u a t i o n was p r e s e n t e d w h i c h p r e d i c t e d t h e t r e n d s o f t i m e courses f o r hydrolyses o f c e l l o b i o s e a t concentrations r a n g i n g f r o m 14.6-146.0
mM cellobiose.
The c o n s t a n t s u s e d i n t h e m o d e l
( d e t e r m i n e d f r o m i n i t i a l r a t e d a t a ) were compared t o t h o s e r e p o r t e d f o r B-n-glucosidase
o b t a i n e d f r o m o t h e r s o u r c e s o f T.
viride.
Most
n o t a b l e i n t h i s c o m p a r i s o n was t h e a p p a r e n t l y h i g h e r a c t i v i t y a n d r e d u c e d i n h i b i t i o n o f t h i s enzyme c o m p a r e d t o o t h e r s o u r c e s o f 8 4 glucosidase. The r o l e o f s u g a r h y d r o x y g r o u p s i n g l y c o s i d e h y d r o l y s i s h a s been i n v e s t i g a t e d . * 1 6 and
C-4
and
of
The c o n t r i b u t i o n o f t h e h y d r o x y g r o u p s a t C-2
the
hydroxymethyl
group
a t
C-5
g l u c o p y r a n o s i d e s t o t h e i r h y d r o l y s i s by B - g - g l u c o s i d a s e Asperqillus wentii
was
investigated with
glucosides w i t h appropriate s t r u c t u r a l modifications. 2 . 4 ~ 1 0 - ~ ,2 - d e o x y - 2 - a m i n o
1 . 8 ~ 1 0 - ~ , xyloside 6 . 3 ~ 1 0 - ~ , galactoside a c t i v e s i t e as measured by t h e the
Xi
Km
Relative 2-deoxy
1 ~ 1 0 ' ~ , 4-deoxy
1 ~ 1 0 ' ~ . Binding t o
value o f these s u b s t r a t e s
the
o r by
v a l u e o f a p p r o p r i a t e i n h i b i t o r s was o n l y m o d e r a t e l y d e c r e a s e d
by t h e above m o d i f i c a t i o n s .
A t e m p e r a t u r e s t u d y w i t h t h e 2-deoxy-Q-
g l u c o s i d e showed t h a t t h e decrease i n b u t t o a more n e g a t i v e AZ.
i n A*!
t h e 2-deoxy-Q - -glucoside 0.13
from
A3
4-methylumbelliferyl-B-P-
h y d r o l y s i s r a t e s e x p r e s s e d by l(cat/~cat ( Q - g l u c o s i d e ) were: 4 ~ 1 0 ' ~ ,2 - d e o x y - 2 - a m i n o
6-Q-
of
min-l)
enzyme
kcat was
was a p p r o a c h e d w i t h a b u r s t
a t pH 6 a n d 1 m M s u b s t r a t e .
was
glycosylation
partially and
n o t due t o a c h a n g e
The s t e a d y - s t a t e h y d r o l y s i s o f
rate
limiting.
(rate
constant
D e g l y c o s y l a t i o n of Rate
deglycosylation calculated from
constants
the for
pre-steady-state
437
6: Enzymes kinetics
were i n good agreement w i t h t h e c o n s t a n t s c a l c u l a t e d f r o m
experiments for
where t h e 2 - d e o x y - B - g l u c o s i d e
the hydrolysis o f the e-glucoside
was u s e d a s
and where
an i n h i b i t o r
a slow
approach t o
t h e s t e a d y s t a t e o f t h e i n h i b i t e d r e a c t i o n was o b s e r v e d . A r a d i o a c t i v e g l y c o p e p t i d e w i t h a m o l e c u l a r w e i g h t o f 13,200 A3 a f t e r l a b e l l i n g the a c t i v e
has been i s o l a t e d f r o m B - E - g l u c o s i d a s e s i t e with The
I 3H)conduritol
glycopeptide
residues.
B e p o x i d e and c l e a v a g e w i t h t r y p ~ i n . ~ ~
consisted
of
63 a m i n o
I t s a m i n o a c i d s e q u e n c e was
acids
+_
and 29
1 sugar
derived from the r e s u l t s o f
sequence a n a l y s i s o f p e p t i c and cyanogen b r o m i d e p e p t i d e s .
The
radioactive
the
sequence;
inhibitor
was
bound
I,=-aspartic
acid
12 of
t h e sugar r e s i d u e s were p r o b a b l y bound as N - g l y c o s i d e s
I-asparagine
48 and $ - a s p a r a g i n e
B-Q-Glucosidase was
carried
out
by
Aspergillus
b i o r e a c t o r s . *18 on
to
56.
production
i n v e s t i g a t e d i n s t i r r e d - t ank
---A. w e n t i i
to
Mandels
wentii
Batch
has
been
cultivation
and Reese
(1957)
of
medium
c o n t a i n i n g 3% c e l l u l o s e a n d 0.3% p e p t o n e a t 3OoC i n 1 4 a n d 3 0 l i t r e bioreactors.
Use o f a 4 8 - h o u r s - o l d
a e r a t i o n a t 4 0 0 - 8 0 0 r.p.m.,
i n o c u l u m (10% v/v),
pH a b o v e 50% s a t u r a t i o n l e v e l and b e t w e e n 5.2 produced
g
10.56
cells
0.5
v.v.m.
and m a i n t e n a n c e o f d i s s o l v e d oxygen and
1-1 a n d
61.9
t o 4.0,
I U e-l
respectively,
h-l
cellobiase
productivity. B-Q-Glucosidase
p r o d u c t i o n by A s p e r g i l l u s p h o e n i c i s h a s been
investigated i n stirred-tank the
isolated
production
f e r m e n t e r ~ . ~T h~ i ~ s a r t i c l e r e p o r t s on of
B-e-glucosidase
t o
provide
a
s u p p l e m e n t a r y a d d i t i o n o f t h e enzyme d u r i n g t h e f e r m e n t a t i o n o f c e l l u l o s e t o E - g l u c o s e by T r i c h o d e r m a r e e s e i . The
purification
Aspergillus aculeatus glucosidases
were
and p r o p e r t i e s
have
been
isolated
s p e c i f i c i t i e s are described.
of
B-Q-glucosidases
described.220 and
Three
purified.
distinct
Their
o f
B-Q-
substrate
Two o f t h e p u r i f i e d e n z y m e s a p p e a r t o
p l a y an i m p o r t a n t r o l e i n p r o d u c i n g 9 - g l u c o s e
from cellulose,
and i t
i s s u g g e s t e d t h a t t h e s e s h o u l d b e c l a s s i f i e d a s a new t y p e o f w-Bk-glucosidase. The
effect
-A s p e r g i l l u s
niger
of
tunicamycin
has
on
secreted
been described.''
The
glycosidases
s e c r e t e d i n t o t h e c u l t u r e medium d u r i n g g r o w t h o f A . glucosidase, deoxyglucosidase.
a-c-galactosidase,
and
of
main glycosidases n i g e r were
B-g-
B-k-2-acetamido-2-
I n t h e presence of t u n i c a m y c i n ,
the a c t i v i t i e s o f
t h e s e enzymes i n t h e c u l t u r e medium were c o n s i d e r a b l y decreased, whereas f u n g a l g r o w t h and t o t a l m y c e l i a l p r , o t e i n c o n t e n t were n o t
438
Carbohydrate Chemistry For f u r t h e r d e t a i l s see i n i t i a l c i t a t i o n o f r e f . 5 0 .
diminished.
removing t h e t h r e e Q-
Q-Glucosidase a c t i v i t i e s capable o f
g l u c o s e r e s i d u e s f r o m Q - G l c 3 Q - M a n 9 Q - G l c N A c 2 o l i g o s a c c h a r i d e have been d e t e c t e d i n a c e l l - f r e e p r e p a r a t i o n o f S a c c h a r o m y c e s c e r e v i s i a e X-2180.221
The D - g l u c o s i d a s e w h i c h c l e a v e s t h e D - g l u c o s e r e s i d u e a t
t h e n o n - r e d u c i n g t e r m i n u s (g-Glc3P-Mangg-GlcNAc2
9-
oligosaccharide
g l u c o s i d a s e ) was e q u a l l y d i s t r i b u t e d b e t w e e n t h e p a r t i c u l a t e and t h e supernatant f r a c t i o n s obtained a f t e r c e n t r i f u g a t i o n o f the yeast x g f o r 30 min.
h o m o g e n a t e a t 27,000
was s t i m u l a t e d by T r i t o n X-100, not affected.
The m e m b r a n e - b o u n d a c t i v i t y
w h e r e a s t h e s u p e r n a t a n t a c t i v i t y was
e-
The s o l u b l e P - G l c 3 ~ - M a n g ~ - G l c N A C 2o l i g o s a c c h a r i d e
g l u c o s i d a s e was p a r t i a l l y p u r i f i e d f r o m t h e s u p e r n a t a n t by ammonium s u l p h a t e f r a c t i o n a t i o n f o l l o w e d by DEAE-Sephadex c h r o m a t o g r a p h y .
It
was
4-
clearly
separated
from
a-P-glucosidase,
n i t r o p h e n y l a-Q-glucopyranoside, and a-E-mannosidase
which
but s t i l l contained B-Q-glucosidase
a c t i n g on 4 - n i t r o p h e n y l B - P - g l u c o p y r a n o s i d e
a-Q-mannopyranoside,
respectively.
The
requirement f o r divalent cations. e - g l u c o s e - l a b e l l e d !-Glc3P-M g-Glc2g-Mange-GlcNAc2,
and
Q-Glc3Q-Man9Q-GlcNAc2
o l i g o s a c c h a r i d e g l u c o s i d a s e h a s a pH o p t i m u m o f 6.8
GlcNAc2.
a c t s on
a n d s h o w e d no
The enzyme was v e r y a c t i v e w i t h
angP-G l c N A c 2 ,
was s l i g h t l y
a n d showed no a c t i v i t y
active with
g-Glclg-Man9p-
with
These p r o p e r t i e s s u g g e s t t h a t t h i s enzyme i s i n v o l v e d i n
the f i r s t step o f processing o f oligosaccharides a f t e r t r a n s f e r from d o l i c h y l pyrophosphate t o p r o t e i n s . T h e f o r m a t i o n a n d l o c a t i o n o f (1 + 4 ) - B - ! - g l u c a n a s e s 4)-B-Q-glucosidases
have
been
studied
i n
and (1
cultures
+
of
P e n i c i l l i u m j a n t h i n e l l u m grown on A v i c e l ,
sodium carboxymethyl
cellulose,
and maltose.222
(1
+
cellobiose,
4)-B-Q-Glucanases
f o r m a t i o n was free,
were
P-mannose,
found
to
i n d u c e d by c e l l o b i o s e .
t h e o t h e r hand, wall.
a-glucose,
be
(1
+
were f o r m e d c o n s t i t u t i v e l y
cell
free,
endo-
and t h e i r
4)-B-~-Glucosidases,
on
and were p r i m a r i l y c e l l
b u t w i t h a s m a l l amount s t r o n g l y a s s o c i a t e d w i t h t h e c e l l Low ( 1 + 4 ) - B - Q - g l u c o s i d a s e
intracellular
o r i g i n were
also
a c t i v i t i e s o f periplasmic or
found.
A
rotational
m e t h o d was d e v e l o p e d t o measure t h e t o t a l e n d o - ( 1 a c t i v i t y o f t h e c u l t u r e ( b r o t h and s o l i d s ) .
+
viscosimetric
4)-B-Q-glucanase
By t h i s m e t h o d i t was
p o s s i b l e t o d e t e r m i n e t h e e n d o - ( 1 + 4 ) - B - Q- - g l u c a n a s e
a c t i v i t y not
o n l y i n t h e supernatant o f t h e c u l t u r e b u t a l s o on t h e surface o f t h e mycelium o r absorbed on r e s i d u a l Avicel. b a t c h c u l t i v a t i o n o f P. 4)-B-!-glucanases
by
D u r i n g a 70 l i t r e
j a n t h i n e l l u m , t h e a d s o r p t i o n of endo-(1
residual
and
newly
added
10% A v i c e l
+
was
439
6: Enzymes measured.
The a d s o r p t i o n o f s o l u b l e p r o t e i n a n d e n d o - ( 1
+
4)-B-Q-
g l u c a n a s e s by A v i c e l was f o u n d t o be l a r g e l y i n d e p e n d e n t o f t h e pH v a l u e b u t dependent on t e m p e r a t u r e . The
importance of
cellulase
--_---------------Bacteroides succinoge ---nes investigated.223
and
i n the
During growth
xylanase
release
rumen environment
of
B.
succinogenes
has
from been
i n a liquid
m e d i u m w i t h c e l l u l o s e as t h e s o u r c e o f c a r b o h y d r a t e , g r e a t e r t h a n
80% o f
the
endo-(1
+ 4)-B-E-glucanase,
xylanase,
x y l o s i d a s e and 50% o f t h e a r y l - 6 - Q - g l u c o s i d a s e c e l l s i n t o the culture f l u i d .
Less t h a n 25% o f t h e B - Q - x y l o s i d a s e A p p r o x i m a t e l y 50% o f
a c t i v i t y was d e t e c t e d i n t h e c u l t u r e f l u i d . each
of
the
released
enzymes
sedimentable s u b c e l l u l a r formation,
measured
was
membrane v e s i c l e s .
t o be r e l e a s e d f r o m t h e o u t e r
and a r y l - B - E -
was r e l e a s e d f r o m
The
membrane o f
associated vesicles
with
appeared
i n t a c t c e l l s by b l e b
p r i m a r i l y i n p o c k e t s b e t w e e n t h e c e l l s and t h e c e l l u l o s e ,
a l t h o u g h a few u n a t t a c h e d c e l l s w i t h b l e b s were seen.
Many v e s i c l e s
were seen a d h e r i n g t o c e l l u l o s e , and t h e y were a l s o seen f r e e i n t h e culture fluid.
T h e s e d a t a s u g g e s t t h a t 6.
h y d r o l y t i c enzymes i n n o n - s e d i m e n t a b l e
succinogenes releases
and p a r t i c u l a t e f o r m s d u r i n g
g r o w t h by a mechanism w h i c h has u n t i l now r e c e i v e d l i t t l e a t t e n t i o n . C e l l u l o s e i n c u b a t e d i n a p o r o u s n y l o n bag i n t h e rumen was c o l o n i z e d by b a c t e r i a r e s e m b l i n g
B. s u c c i n o g e n e s , a n d s u b c e l l u l a r v e s i c l e s
were seen p e n e t r a t i n g c h a n n e l s and f r a c t u r e s
It
i n the cellulose.
was s u g g e s t e d t h a t 6. s u c c i n o g e n e s c e l l s i n t h e rumen c o n t r i b u t e t o an e x t r a c e l l u l a r p o p u l a t i o n o f s u b c e l l u l a r
v e s i c l e s t h a t possess
ce 1l u l o l y t i c and hem i c e 1l u l o l y t ic a c t i v i t i e s w h i c h p r o b a b l y enhance polymer
digestion
and p r o v i d e a s o u r c e
l a c k i n g polymer-degrading a c t i v i t y ,
of
sugars
for
microbes
thereby c o n t r i b u t i n g t o a s t a b l e
heterogeneous m i c r o b i a l p o p u l a t i o n . Polyacrylamide gel electrophoresis
o f t h e c e l l u l o l y t i c system
f r o m c u l t u r e s u p e r n a t e s o f A c e t i v i b r i o c e l l u l o l y t i c u s has shown t h e presence
of
glucanase,
four
major
enzymes:
a
B-e-glucosidase,
and t w o W - Q - g l u ~ a n a s e s . ~The ~ ~ relative
an
w-q-
proportions
o f t h e s e enzymes i n t h e c u l t u r e s u p e r n a t e were a f f e c t e d by t h e n a t u r e o f t h e c e l l u l o s i c s u b s t r a t e and by t h e l e n g t h o f t h e incubation
period.
The
molecular
enzymes were B - a - g l u c o s i d a s e Q-glucanase glucanase
38,000, C3 1 0 , 4 0 0 ,
mobilities relative Treatment of
81,000,
weights
e-a-glucanase as
estimated
t o proteins
of
the
x-a-glucanase
of
C 2 33,000, by
their
cellulolytic 38,000, and
endo-
e*-Q-
electrophoretic
known m o l e c u l a r
weight.
t h e h i g h - m o l e c u l a r - w e i g h t m - Q - g l u c a n a s e w i t h sodium
440
Carbohydrate Chemistry
dodecyl
sulphate-mercaptoethanol
led to
reversible
dissociation of
t h e enzyme i n t o p o l y p e p t i d e s u b u n i t s s i m i l a r t o t h e l o w - m o l e c u l a r weight m - E - g l u c a n a s e .
endo-g-Glucanase
a c t i v i t y c o u l d be assayed
f o r d i r e c t l y u s i n g a n o v e l method o f i n c o r p o r a t i n g carboxymethyl c e l l u l o s e i n the polyacrylamide gels. functions
of
The m o l e c u l a r w e i g h t a n d
t h e s e enzymes a r e compared w i t h t h o s e d e t e c t e d i n
culture f i l t r a t e s o f various fungi. The p r o d u c t i o n o f
carboxymethyl
cellulase
and 6 - P - g l u c o s i d a s e
by C l o s t r i d i u m a c e t o b u t y l i c u m h a s been i n v e s t i g a t e d i n an i n d u s t r i a l fermentation
medium.225
I n
liquid
medium
the
carboxymethyl
c e l l u l a s e was i n d u c e d by m o l a s s e s , a n d i t was n o t r e p r e s s e d by Q glucose.
Optimum
carboxymethyl c e l l u l a s e a c t i v i t y
occurred a t
pH
4 . 6 and 37'C. The
mechanism
Botryodiplodia of
of
the
theobromrn
action
of
B-D-glucosidase
h a s been i n v e s t i g a t e d . 2 2 6
from
I n t h e presence
a h i g h c o n c e n t r a t i o n o f 4 - n i t r o p h e n y l B-q-glucopyranoside
(donor)
t h e r a t e s o f p r o d u c t i o n o f 4 - n i t r o p h e n y l and a t r a n s g l u c o s y l a t i o n product (1-glyceryl
B-g-glucopyranoside)
increased,
whereas t h e r a t e
o f p r o d u c t i o n o f Q-glucose decreased w i t h i n c r e a s i n g c o n c e n t r a t i o n of
g l y c e r o l i n r e a c t i o n s c a t a l y s e d by t h e h i g h - m o l e c u l a r - w e i g h t
g l u c o s i d a s e o b t a i n e d f r o m c u l t u r e f i l t r a t e s o f B. t h e o b r o m a e . {donor)
was g r e a t e r t h a n
Em,
the r a t e o f production o f 4-nitrophenol
was h i g h e r i n t h e p r e s e n c e o f g l y c e r o l t h a n i n i t s absence, when { d o n o r )
was
n i t r o p h e n o l was absence. increased enzyme
Glycerol
Em b u t
less than
lower
and 1.05
i n the
Em
the
rate o f
presence o f
a c t i v i t y .
glycerol
than
4-
i n i t s
Em a n d lmax, whereas dioxan Up t o 1 m M AgN03 h a d no e f f e c t on lmax. A
s o l v e n t - i s o t o p e - e f f e c t
*H
v a l u e o f 1.40
+_
was f o u n d a t pH ( o r
0.05
2 0.01 2 0.01 w e r e f o u n d i n t h e absence and p r e s e n c e o f g l y c e r o l , a - 2 ~k i n e t i c i s o t o p e e f f e c t
respectively. f3-Q-glucosidase
Although activity,
(kH/k2H)
v a l u e s o f 1.03
m a l t o s e was a n o n - c o m p e t i t i v e the r a t i o o f
velocity
g l y c e r o l t o t h a t i n i t s absence i n c r e a s e d , w i t h i n c r e a s i n g concentration o f maltose. which involves a solvent-separated pair,
whereas
production of
increased both
decreased
{~max.(H20)/~max.(2H20))
P*H) 5.8.
B-QWhen
i n h i b i t o r of
i n t h e presence of
a f t e r an i n i t i a l d e c l i n e , A mechanism i s s u g g e s t e d
glycosyl cation-carboxylate
ion
w h i c h has g r e a t e r a f f i n i t y f o r a l c o h o l i c E - g l u c o s y l a c c e p t o r s ,
and an i n t i m a t e i o n p a i r ,
w h i c h h a s g r e a t e r a f f i n i t y f o r w a t e r as a
E - g l u c o s y l a c c e p t o r and w h i c h c o u l d c o l l a p s e r e v e r s i b l y and r a p i d l y i n t o a p r e p o n d e r a n c e o f an u n r e a c t i v e c o v a l e n t p - g l u c o s y l enzyme. T r i c h o d e r m a v i r i d e s e c r e t e s a c e l l u l a s e complex t h a t i s r i c h i n
44 1
6: Enzymes B-Q-glucosidase
and t h e r e f o r e w e l l s u i t e d f o r
the saccharification
T h e c e l l u l a s e was i n v e s t i g a t e d w i t h
o f c e l l u l o s i c materials.227
r e s p e c t t o o p t i m u m c o n d i t i o n s o f r e a c t i o n and enzyme s t a b i l i t y . Avicelase,
carboxymethylcellulase,
considerably i n t h e i r
temperatures t h e B-E-glucosidase The e f f e c t enzymes,
the
differed higher
A t
was n o t v e r y s t a b l e .
o f B-g-glucosidase
i.2.
and $ - Q - g l u c o s i d a s e
physicochemical properties. on t h r e e a s s a y s f o r
a c t i v i t i e s
h y d r o x y e t h y l c e l l u l o s e , and f i l t e r
against
paper,
cellulolytic
dyed
Avicel,
has been s t u d i e d u s i n g
c e l l u l a s e e n z y m e d e r i v e d f r o m L r i c h o d e r m a r e e s e i VTT-D-80133.228 The d y e d A v i c e l and h y d r o x y e t h y l c e l l u l o s e a s s a y s w e r e o n l y s l i g h t l y a f f e c t e d by $-E-glucosidase,
whereas t h e f i l t e r - p a p e r
l i n e a r l y d e p e n d e n t on t h e l e v e l o f B - E - g l u c o s i d a s e
a s s a y was
over a wide range
o f a c t i v i t y o f t h i s enzyme.
9
B-Q-Glucuronidases
I m m o b i l i z e d $ - P - g l u c u r o n i d a s e has been c h a r a c t e r i z e d i n aqueous and mixed-solvent attached t o
systems.229
6-Q-Glucuronidase
alkylamine-controlled
i m m o b i l i z a t i o n scheme.
The
pore
glass
activity
of
was c o v a l e n t l y a
this
glutaraldehyde
i m m o b i l i z e d B-Q-
g l u c u r o n i d a s e was s t u d i e d w i t h r e s p e c t t o s e v e r a l k i n e t i c p a r a m e t e r s i n c o m p a r i s o n w i t h t h e b e h a v i o u r of for 4-nitrophenyl oestriol-3-
t h e s o l u b l e enzyme.
Em v a l u e s o f Q-
and o e s t r i o l - 1 6 a - g l u c o s i d e s
g l u c o p y r a n u r o n i c a c i d were determined.
F o r each s u b s t r a t e t h e
Em
was e s s e n t i a l l y t h e same, 0 . 2 m M , a n d t h i s v a l u e d i d n o t c h a n g e w h e n t h e e n z y m e was i m m o b i l i z e d .
The s o l u b l e and i m m o b i l i z e d e n z y m e s
b o t h d i s p l a y e d a r e l a t i v e l y b r o a d pH maximum c e n t r e d a t pH 6.8 f o r a l l substrates.
Several
organic-aqueous
methanol, ethanol, a c e t o n i t r i l e , a n d
mixtures
including
e t h y l e n e g l y c o l were t e s t e d ,
t h e i r e f f e c t s on t h e a c t i v i t y o f i m m o b i l i z e d B - g - g l u c u r o n i d a s e similar
t o those found for
t h e s o l u b l e enzyme.
Long-term
and were
(1 y e a r )
s t o r a g e s t a b i l i t y t e s t s o f t h e i m m o b i l i z e d enzyme w e r e c a r r i e d o u t . The i m m o b i l i z e d enzyme r e t a i n e d 40% o f i t s i n i t i a l a c t i v i t y a f t e r 1 year
a n d was
very
robust
towards
most
o f the organic solvents
tested. Human
fibroblasts
with
a
genetic
deficiency
of
a
single
l y s o s o m a l enzyme and f i b r o b l a s t s f r o m a p a t i e n t w i t h I - c e l l d i s e a s e w i t h a m u l t i p l e d e f i c i e n c y o f l y s o s o m a l h y d r o l a s e s h a v e been u s e d as recipient
cells
i n
studies
on
recognition
and
uptake
of
B-Q-
442
Carbohydrate Chemistry
a c e t a m i d o - 2 - d e o x y h e x o s i d a s e , B - Q - g l u c u r o n i d a s e , a n d B-pgalactosidase.58 F o r f u r t h e r d e t a i l s s e e i n i t i a l c i t a t i o n of r e f .58. The c o n d i t i o n s f o r m a x i m a l a c t i v i t y ( p H , b u f f e r , s a t u r a t i n g s u b s t r a t e c o n c e n t r a t i o n , r a n g e of l i n e a r r e l a t i o n s h i p s b e t w e e n enzyme a c t i v i t y v e r s u s i n c u b a t i o n t i m e and v e r s u s enzyme c o n c e n t r a t i o n ) i n t h e f l u o r i m e t r i c a s s a y of s e v e r a l g l y c o h y d r o l a s e s o f l y s o s o m a l o r i g i n i n human p l a s m a a n d s e r u m h a v e b e e n a-Qestablished.26 The f o l l o w i n g e n z y m e s w e r e s t u d i e d : g a l a c t o s i d a s e , B-Q-galactosidase, B-Q-2-acetamido-2deoxyglucosidase, B-Q-glucosidase, B-Q-glucuronidase, a-amannosidase, a - & - f u c o s i d a s e . A l l examined enzymes t u r n e d out t o be more o r l e s s u n s t a b l e upon s t o r a g e a t 3 7 " C , 4'C, and - 2 O ' C i n b o t h serum and plasma. The only e x c e p t i o n s were B-Q-glucuronidase, which was s t a b l e i n p l a s m a and s e r u m . F o r f u r t h e r d e t a i l s s e e i n i t i a l c i t a t i o n of r e f .26. The i n f l u e n c e o f c y p r o t e r o n e a c e t a t e on t h e a c t i v i t y of 8-D,g l u c u r o n i d a s e i n t h e h y p o t h a l a m u s and c e r e b r a l c o r t e x o f t h e m a l e mouse h a s been i n v e s t i g a t e d , 2 3 0 D a i l y s u b c u t a n e o u s i n j e c t i o n s o f c y p r o t e r o n e a c e t a t e g r e a t l y d e c r e a s e t h e p r o t e i n c o n t e n t and B - Q g l u c u r o n i d a s e a c t i v i t y i n t h e mouse hypothalamus. These e f f e c t s a r e r e v e r s i b l e and t h e r e c o v e r y c a p a c i t y of t h e a n i m a l s e e m s t o be i n v e r s e l y r e l a t e d t o t h e d u r a t i o n of a n t i a n d r o g e n i c t r e a t m e n t . A commercial p r e p a r a t i o n of bovine l i v e r B-Q-glucuronidase has been f o u n d t o c o n t a i n t w o d i s t i n c t enzyme s p e c i e s , b o t h o f which c a t a l y s e t h e h y d r o l y s i s of 4 - m e t h y l u m b e l l i f e r y l a - I i d o p y r a n o s y l u r o n i c acid.231 T h e s p e c i e s w i t h a m o l e c u l a r weight of about 290,000 was a c t i v e t o w a r d s p h e n y l a - & - i d o p y r a n o s y l u r o n i c a c i d b u t lacked t h e l a t t e r a c t i v i t y . S t u d i e s of t h e k i n e t i c s of i n h i b i t i o n and h e a t i n a c t i v a t i o n s u g g e s t e d t h a t t h e h y d r o l y s i s of 4 m e t h y l u m b e l l i f e r y l a - l - i d o p y r a n o s y l u r o n i c a c i d i s due t o t h e 6-Qg l u c u r o n i d a s e i n t h e c a s e of t h e 290,000 d a l t o n s p e c i e s . The h i g h l y p u r i f i e d B-q-glucuronidase p r e p a r a t i o n s d e r i v e d from r a t p r e p u t i a l gland and l i v e r lysosomes a l s o e x h i b i t e d 4 - m e t h y l u m b e l l i f e r y l a-biduronidase activity. T h e s e f i n d i n g s s u p p o r t t h e view t h a t B-gg l u c u r o n i d a s e can h y d r o l y s e c e r t a i n a - I = - i d o p y r a n o s y l u r o n i c a c i d bonds and r a i s e t h e p o s s i b i l i t y t h a t B - Q - g l u c u r o n i d a s e may p l a y a r o l e i n t h e catabolism of I - i d u r o n i c a c i d - c o n t a i n i n g glycosaminoglycans A c o m p a r a t i v e s t u d y h a s been p e r f o r m e d upon h e p a t i c and i n t e s t i n a l B-p-glucuronidase from a d u l t male f i s c h e r r a t s . 2 3 2 The
.
443
6: Enzymes
s u b c e l l u l a r d i s t r i b u t i o n p a t t e r n s o f B-Q-glucuronidase i n d i c a t e d t h a t 80% o f h e p a t i c B - Q - g l u c u r o n i d a s e a c t i v i t y i s p r e s e n t i n t h e lysosomal-mitochondria1 glucuronidase supernate).
activity
pellet
i s
i n
and
the
60% of
soluble
intestinal
fraction
B-P-
(105,000
g
p u r i f i c a t i o n w i t h a y i e l d o f 45% o f B-Q-
A 55-fold
g l u c u r o n i d a s e a c t i v i t y f r o m e a c h t i s s u e was o b t a i n e d b y a m m o n i u m sulphate fractionation, Isoelectric-focusing
g e l f i l t r a t i o n , and column chromatography. r e v e a l s a u n i q u e B-Q-
gel electrophoresis
g l u c u r o n i d a s e i s e n o z y m e f o r t h e i n t e s t i n e (PI 5.4) a n d t w o f o r t h e l i v e r ( P I 5.6 a n d 6.7). Levels
of
collagenolytic
collagen p r o l y l hydroxylase adjuvant
arthritis
have
activity,
been r e p o r t e d . 2 3 3
associated with insoluble
activity
B-Q-glucuronidase,
i n paws f r o m r a t s
with
The
and
developing
collagenolytic
collagen fibres
separated
from
h o m o g e n a t e s o f i n f l a m e d paws f r o m r a t s w i t h a d j u v a n t a r t h r i t i s was q u a n t i t a t e d u s i n g H4 e d t a - s e n s i t i v e s o l u b i l i z a t i o n o f h y d r o x y p r o l i n e as a measure o f a c t i v i t y .
Approximately
60% o f t h e s o l u b i l i z e d
h y d r o x y p r o l i n e was a s s o c i a t e d w i t h d i a l y s a b l e p r o d u c t s .
The l e v e l
o f c o l l a g e n o l y t i c a c t i v i t y i n t h e paws i n c r e a s e d w i t h t i m e a f t e r t h e i n d u c t i o n o f a d j u v a n t a r t h r i t i s and p a r a l l e l e d t o a l a r g e e x t e n t t h e development o f i n f l a m m a t i o n i n b o t h t h e adjuvant i n j e c t e d ( r i g h t ) h i n d paw and i n t h e n o n - i n j e c t e d c o n t r a l a t e r a l paw. level of
free
collagenolytic
activity
i n the
By day 26,
the
i n j e c t e d paw h a d
i n c r e a s e d t o a l e v e l 30 t i m e s n o r m a l w h i l e t h a t i n t h e c o n t r a l a t e r a l paw h a d i n c r e a s e d t o a l e v e l 1 0 t i m e s n o r m a l .
Treatment o f t h e
residues from
resulted
the
i n j e c t e d paws
with
trypsin
a c t i v a t i o n o f a l a t e n t c o l l a g e n o l y t i c a c t i v i t y which, a c c o u n t e d f o r a p p r o x i m a t e l y 50% o f t h e t o t a l a c t i v i t y . level of that
The e l e v a t e d
c o l l a g e n p r o l y l h y d r o x y l a s e i n t h e i n f l a m e d paw s u g g e s t e d
the
rate of
collagen
synthesis
a c t i v i t y of B-e-glucuronidase time
i n the
o n d a y 26,
was
also
increased.
The
i n c r e a s e d i n t h e i n f l a m e d paw w i t h
a f t e r the induction o f adjuvant a r t h r i t i s .
The i n f l a m e d paw
o f t h e a d j u v a n t r a t may r e p r e s e n t a u s e f u l s y s t e m i n w h i c h t o s t u d y t h e r o l e o f c o l l a g e n o l y t i c enzymes i n t h e d e s t r u c t i o n o f c o n n e c t i v e t i s s u e by i n f l a m m a t o r y l e s i o n s . The c h a n g e s o f s u l p h a t e d g l y c o s a m i n o g l y c a n s and o f enzymes active
upon
them
during
bovine
foetal
a n a l y ~ e d . ~B ~- Q - G l u c u r o n i d a s e
and
development
have
been
B-e-2-acetamido-2-
deoxyglucosidase remains without s i g n i f i c a n t
changes d u r i n g the,
F o r f u r t h e r d e t a i l s see i n i t i a l c i t a t i o n o f r e f . 3 5 . whole period. A d j u v a n t - i n d u c e d a r t h r i t i s i n r a t s has been s t u d i e d by t h e
444
Carbohydrate Chemistry
changes i n serum and u r i n a r y p r o t e i n - b o u n d c a r b o h y d r a t e m e t a b o l i t e s , changes i n s e r u m and t i s s u e l y s o s o m a l g l y c o h y d r o l a s e s , and l y s o s o m a l fragility.30
T h e r e i s no change i n t h e t o t a l a c t i v i t y o f l y s o s o m a l
glycohydrolases,
of
lysosomal
glucuronidase, The
free
i n c l u d i n g B-Q-glucuronidase. glycohydrolases
The
investigated,
free
activities
including
B-Q-
a r e i n c r e a s e d i n l i v e r and s p l e e n i n t h e a c u t e phase.
activities
of
B-Q-glucuronidase,
B-Q-2-acetamido-2-
d e o x y g l u c o s i d a s e , and c a t h e p s i n D o f k i d n e y showed no change.
For
f u r t h e r d e t a i l s see i n i t i a l c i t a t i o n o f r e f . 3 0 .
$-Q-2-acetamido-2-deoxyglucosidase,
The a - L - f u c o s i d a s e , mannosidase,
B-!-glucuronidase,
a-&-arabinofuronosidase muscle o f
B-Q-xylosidase,
activities
of
d e v e l o p i n g and a d u l t
n o r m a l and a t r o p h i c
rats
a+-
B - Q - g l u c o s i d a s e , and
have been
skeletal
collectively
i n v e s t i g a t e d . 36 Diphenylhydantoin
induction of
intracellular
B-g-glucuronidase
and a l k a l i n e p h o s p h a t e a c t i v i t y has been r e p o r t e d i n c u l t u r e d b o v i n e dental pulp cells.234
D i p h e n y l h y d a n t o i n , a d r u g commonly used i n
the treatment
o f grande-ma1 e p i l e p t i c s e i z u r e s ,
side
which
effects
may
result
from
the
i s responsible for
induction
of
enzymes,
p a r t i c u l a r l y those found i n the microsomal f r a c t i o n o f the l i v e r . T h i s s t u d y d e s c r i b e s an e f f e c t o f t h i s d r u g upon b o v i n e d e n t a l p u l p cells,
the
induction of
B-Q-glucuronidase
and a l k a l i n e
phosphatase
activity. The p r e s e n c e o f a p r e c u r s o r f o r m o f B - ! - g l u c u r o n i d a s e , subunit molecular
weight
kidney.235
was
subunit that
This
later
molecular weight
the precursor
o f 75,000,
was
has been d e m o n s t r a t e d
processed
o f 71,500.
to
the
Tissue
associated w i t h the
mature
form,
fractionation
with
revealed
m i c r o s o m e s whereas
m a t u r e f o r m was a s s o c i a t e d w i t h t h e l y s o s o m e s . egasyn b o t h f o r m s o f B - a - g l u c u r o n i d a s e
with a i n mouse
the
I n mice l a c k i n g
were p r e s e n t , b u t t h e r a t e o f
p r o c e s s i n g was e l e v a t e d compared t o n o r m a l . C o m p a r a t i v e s t u d i e s h a v e b e e n made o f a n C - a s p a r a g i n e - l i n k e d o l i g o s a c c h a r i d e s t r u c t u r e o f r a t l i v e r m i c r o s o m a l and l y s o s o m a l B - i glucuronidases.236
The
carbohydrate
chains
of
microsomal
and
lysosomal B-Q-glucuronidases
o f r a t l i v e r w e r e s t u d i e d b y endo-$-!-
2-acetamido-2-deoxyglucanase
H d i g e s t i o n and by h y d r a z i n o l y s i s .
a
part
of
the
oligosaccharides
released
g l u c u r o n i d a s e was an a c i d i c component. hydrolysed
either
by
sialidase
from
microsomal
Only
B-g-
The a c i d i c component was n o t
or
by
E s c h e r i c h i a c o l i a l k a l i n e phosphatases,
calf but
was
intestinal
and
converted t o
a
n e u t r a l component by p h o s p h a t a s e d i g e s t i o n a f t e r m i l d a c i d t r e a t m e n t
6: Enzymes
445
i n d i c a t i n g t h e presence of a phosphodiester group. The n e u t r a l o l i g o s a c c h a r i d e p o r t i o n o f m i c r o s o m a l e n z y m e was a m i x t u r e o f f i v e high g-mannose-type s u g a r chains. F i f t e e n g l y c o s i d a s e s were a s s a y e d i n l y m p h o m y e l o i d a n d d i g e s t i v e t i s s u e s of Ginglymostoma c i r r a t u m , Heterodontus f r a n c i s c i , and E t m o p t e r u s spinax.19 A c t i v i t i e s o f a-Q-mannosidase, B-ggalactosidase, B-Q-2-acetamido-2-deoxygalactosidase, B-gg l u c u r o n i d a s e , a n d a - a n d B - E - g l u c o s i d a s e u s u a l l y were h i g h e r i n lymphomyeloid t i s s u e s t h a n i n d i g e s t i v e t i s s u e s . Sucrose density gradients have been used t o c h a r a c t e r i z e lysosome-containing fractions from 0 h white prepupae of S t o m o x y s ~ a l c i t r a n s . ~ ’ The a c i d i c g l y c o s i d a s e s a - g - g l u c o s i d a s e , aQ-galactosidase, a-Q-mannosidase, B-Q-glucosidase, B-Qg a l a c t o s i d a s e , B - P- - g l u c u r o n i d a s e , a n d B - Q - 2 - a c e t a m i d o - 2 d e o x y g l u c o s i d a s e e q u i l i b r a t e a t t h e s a m e d e n s i t y as d o e s a c i d phosphatase. S e v e r a l m o l l u s c g l y c o s i d a s e s have been s t u d i e d f o r their a c t i v i t i e s t o w a r d s n a t u r a l s ~ b s t r a t e s . ~B -~g - G l u c u r o n i d a s e f r o m -L--i-t-t o r i n a l i t t o r e a hydrolyses hyaluronic acid, c h o n d r o i t i n 4s u l p h a t e , and h e p a r i n w i t h a v e r y low a c t i v i t y . H o w e v e r , i t i s much more a c t i v e on o l i g o s a c c h a r i d e s (from the above-mentioned m a c r o m o l e c u l e s ) c o n t a i n i n g n o n - r e d u c i n g t e r m i n a l g l u c o s y l u r o n i c acid residues.
10 a-l-Iduronidases
M a t u r a t i o n of a-L-iduronidase has been s t u d i e d i n c u l t u r e d { 3 H ) L e u c i n e was a d m i n i s t e r e d t o t h e human d i p l o i d f i b r o b l a s t s . 2 3 7 c e l l s , a n d t h e enzyme was i s o l a t e d by i m m u n o p r e c i p i t a t i o n w i t h a n t i s e r u m t o human k i d n e y a - l - i d u r o n i d a s e . Radioactive polypeptides were s e p a r a t e d by p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s a n d v i s u a l i z e d by f l u o r o g r a p h y . Three r a d i o a c t i v e , i m m u n o p r e c i p i t a b l e p o l y p e p t i d e s o f a p p a r e n t m o l e c u l a r w e i g h t o f 75,000, 72,000, a n d 6,000 were f o u n d i n t r a c e l l u l a r l y a n d o n e o f 76,000 i n t h e m e d i u m . Pulse-chase e x p e r i m e n t s s h o w e d t h e 75,000 i n t r a c e l l u l a r s p e c i e s t o b e a p r e c u r s o r w h i c h was t r i m m e d t o t h e i n t e r m e d i a t e f o r m i n a f e w h o u r s and t h e l a t t e r t o t h e smaller form o v e r a p e r i o d o f 4 t o 5 days. T h e e x t r a c e l l u l a r f o r m was b a r e l y d e t e c t a b l e u n l e s s t h e f i b r o b l a s t s h a d b e e n l a b e l l e d i n t h e p r e s e n c e o f 10 m M NH4C1 o r h a d b e e n d e r i v e d f r o m p a t i e n t s w i t h I - c e l l disease ( t w o c o n d i t i o n s known t o c a u s e
Carbohydrate Chemistry
446 e n h a n c e d s e c r e t i o n of the
four
l y s o s o m a l enzymes).
radioactive
p o l y p e p t i d e s as
The i d e n t i f i c a t i o n o f different
forms
of
a-L-
i d u r o n i d a s e was e s t a b l i s h e d n o t o n l y by t h e i r t e m p o r a l r e l a t i o n s h i p but
also
by
(genetically
their
absence from
deficient
fibroblasts
i n a-i-iduronidase
of Hurler
patients
a c t i v i t y ) and by t h e
s i m i l a r i t y i n p a t t e r n s g e n e r a t e d b y p a r t i a l p r o t e o l y s i s w i t h V8
All species o f a-i-iduronidase
p r o t e a s e from Staphylococcus aureus. were p h o s p h o r y l a t e d , polypeptides.
as shown by i n c o r p o r a t i o n o f 33P i n t o t h e f o u r
The p h o s p h a t e l a b e l c o u l d b e r e m o v e d b y t r e a t m e n t
w i t h endo-B-~-2-acetamido-2-deoxyglucanase
There appeared t o be
H.
no d i f f e r e n c e i n c a t a l y t i c a c t i v i t y between t h e l a r g e and s m a l l f o r m s o f t h e enzyme. t h e i r uptake.
On t h e o t h e r h a n d , t h e r e was a d i f f e r e n c e i n
The 66,000
s p e c i e s appeared t o be o f l o w - u p t a k e form,
i n c o n t r a s t t o t h e 76,000
s e c r e t e d f o r m w h i c h was r e a d i l y t a k e n up
by t h e q-mannose 6 - p h o s p h a t e
receptor.
11 a- and B-p-Mannosidases T h e c o n d i t i o n s f o r m a x i m a l a c t i v i t y (pH, substrate concentration, enzyme
activity
range o f
versus
linear
incubation
buffer,
saturating
r e l a t i o n s h i p s between
time
and
versus
enzyme
c o n c e n t r a t i o n ) i n t h e f l u o r i m e t r i c assay o f s e v e r a l g l y c o h y d r o l a s e s of
lysosomal
origin
e ~ t a b l i s h e d . ' ~ The galactosidase, glucuronidase,
i n
human
following
plasma
B-c-galactosidase,
a-9-mannosidase,
and
enzymes
serum
were
have
studied:
B-Q-glucosidase,
a-l-fucosidase.
been
a-a-
B-Q-
For f u r t h e r d e t a i l s
s e e i n i t i a l c i t a t i o n o f r e f .26. A s t u d y h a s been made o f
l y s o s o m a l enzyme a c t i v i t i e s i n s e r u m
and l e u k o c y t e s i n c h r o n i c h e p a t i c d i s e a s e . 6 0 a c t i v i t i e s (arylsulphatase A,
a-P-mannosidase,
F i v e l y s o s o m a l enzyme a-l-fucosidase,
&a-
2 - a c e t a m i d o d e o x y h e x o s i d a s e , and B - Q - g a l a c t o s i d a s e ) w e r e d e t e r m i n e d i n s e r u m and l e u k o c y t e s o f c o n t r o l s and p a t i e n t s s u f f e r i n g f r o m v a r i o u s
For f u r t h e r d e t a i l s see i n i t i a l c i t a t i o n o f r e f . 6 0 .
l i v e r diseases.
An u n u s u a l c a s e o f m a n n o s i d o s i s w i t h s e v e r e d e f i c i e n c y o f a c i d a-Q-mannosidase
i n l e u k o c y t e s and h i g h r e s i d u a l e n z y m a t i c a c t i v i t y
i n s k i n f i b r o b l a s t s has been reported.238 t h e r m o s t a b l e and has an a l m o s t n o r m a l mild
clinical
manifestations,
oligosaccharides
was
very
high.
but
T h e m u t a n t e n z y m e was
Em v a l u e . excretion
This
case o f
compared w i t h t h o s e r e p o r t e d i n l i t e r a t u r e .
The p a t i e n t h a d of
mannose-rich
mannosidosis i s
447
6: Enzymes
I n t r a c e l l u l a r and e x t r a c e l l u l a r a-Q-mannosidase a c t i v i t y of c u l t u r e d s k i n f i b r o b l a s t s has been i n v e s t i g a t e d w i t h r e s p e c t t o a r e l a t i o n t o cystic fibrosis.239 F i b r o b l a s t i n t r a c e l l u l a r a-gm a n n o s i d a s e was i n a c t i v a t e d b y 3 0 - 5 0 % a f t e r 1 2 0 m i n a t 5 O o C . S e p a r a t i o n o f a - a - m a n n o s i d a s e by i s o e l e c t r o f o c u s i n g a n d w h e a t - g e r m l e c t i n - S e p h a r o s e i n t o n e u t r a l and acid components showed t h e f o r m e r t o be h e a t l a b i l e and t h e c a u s e of t h e o b s e r v e d l o s s . F o e t a l - c a l f s e r u m a c i d a - Q - m a n n o s i d a s e c o u l d b e a c t i v a t e d a t 6 O o C i f k e p t a t pH 9.0. F i b r o b l a s t e x t r a c e l l u l a r a - g - m a n n o s i d a s e w a s i n a c t i v a t e d by 50-70% a f t e r 120 m i n a t 5 O o C , r e f l e c t i n g t h e g r e a t e r l e v e l o f t h e n e u t r a l component. C o n t r o l and c y s t i c - f i b r o s i s a-P-mannosidase showed no d i f f e r e n c e . The s p e c i f i c a c t i v i t y o f a-2-mannosidase i n s e r u m o f I-cell d i s e a s e p a t i e n t s has b e e n f o u n d t o be c o n s i d e r a b l y i n c r e a s e d d u e t o i n c r e a s e d a m o u n t s o f t h e c o m p o n e n t w i t h o p t i m a l a c t i v i t y a t pH 4.6 ( a c i d i c form).240 T h e i n t e r m e d i a t e f o r m w i t h pH o p t i m u m o f 6.0 remains unaltered. These c o n c l u s i o n s were r e a c h e d by u s i n g o p t i m a l c o n d i t i o n s f o r d i f f e r e n t i a l a s s a y o f t h e a - a - m a n n o s i d a s e s checked by p a r t i a l s e p a r a t i o n o f t h e c o m p o n e n t s i n s e r u m by s u c r o s e centrifugation. A d j u v a n t - i n d u c e d a r t h r i t i s i n r a t s h a s b e e n s t u d i e d by t h e changes i n serum and u r i n a r y protein-bound c a r b o h y d r a t e m e t a b o l i t e s , changes i n serum and t i s s u e l y s o s o m a l glycohydrolases, and l y s o s o m a l fragility.30 T h e f r e e a c t i v i t i e s of l y s o s o m a l g l y c o h y d r o l a s e s i n v e s t i g a t e d i n c l u d i n g a-q-mannosidase are i n c r e a s e d i n l i v e r and s p l e e n i n t h e a c u t e p h a s e . F o r f u r t h e r d e t a i l s see i n i t i a l c i t a t i o n o f r e f .30. The a - & - f u c o s i d a s e , B-q-acetamido-2-deoxyglycosidase, a-Qmannosidase, B-Q-glucuronidase, B-Q-xylosidase, B-Q-glucosidase, and a - i - a r a b i n o f u r a n o s i d a s e a c t i v i t i e s of normal and a t r o p h i c s k e l e t a l muscle of developing and a d u l t rats have been c o l l e c t i v e l y i n v e s t i g a t e d 36 An a - g - m a n n o s i d a s e s p e c i f i c f o r t h e h y d r o l y s i s o f 2-g-a-Qmannosyl l i n k a g e s t o Q-mannose has been s o l u b i l i z e d and p a r t i a l l y p u r i f i e d from r a b b i t l i v e r microsomes.241 The e n z y m e i s i n h i b i t e d by H 4 e d t a a n d h a s o p t i m a l a c t i v i t y i n t h e p r e s e n c e o f c a l c i u m i o n s . The p u r i f i e d enzyme has a r e q u i r e m e n t f o r n o n - i o n i c d e t e r g e n t s or f o r specific phospholipids. A t d e t e r g e n t c o n c e n t r a t i o n s appreciably below t h e c r i t i c a l micelle c o n c e n t r a t i o n , t h e enzyme is a c t i v e i n t h e p r e s e n c e o f p h o s p h a t i d y l c h o l i n e or phosphatidylethanolamine b u t not w i t h p h o s p h a t i d y l i n o s i t o l , phosphatidylglycerol, or phosphatidic
.
Carbohydrate Chemistry
448 acid.
A t
concentrations
optimal
a c t i v i t y ,
of
the
phosphat i d y l i n o s i t o l or s p e c i f i c i t y of
phosphatidylcholine
enzyme
i s
strongly
phosphatidylglycerol.
t h e a-g-mannosidase
which
provide
i n h i b i t e d
by
The s u b s t r a t e
toward oligosaccharide substrates
s u g g e s t s t h a t t h e e n z y m e may b e i n v o l v e d i n t h e p r o c e s s i n g o f t h e o l i g o s a c c h a r i d e c h a i n s of m a m m a l i a n g l y c o p r o t e i n s . E v i d e n c e has been p r e s e n t e d f o r t h e b i o c h e m i c a l d i a g n o s i s o f t h e f i r s t c a s e o f f e l i n e m a n n o s i d o s i ~ . ~A ~m~a r k e d d e f i c i e n c y o f a c i d i c a-g-mannosidase
i n the brain,
e x c r e t i o n o f g-mannose-rich
k i d n e y , and l i v e r and e x c e s s i v e
o l i g o s a c c h a r i d e s i n t h e u r i n e were found
i n a k i t t e n s u f f e r i n g from a nervous d i s o r d e r .
m a n n o s i d a s e , r a n g i n g f r o m 2.0 observed i n t h e tissues
R e s i d u a l a c i d i c a-9-
t o 5.5% o f t h e n o r m a l a c t i v i t y , was
I t had s i m i l a r
of the affected kitten.
k i n e t i c and p h y s i c o c h e m i c a l p r o p e r t i e s t o t h e n o r m a l a c t i v i t y .
The
amount o f a-mannose i n t h e u r i n e o f t h e a f f e c t e d k i t t e n was 1 9 - f o l d g r e a t e r than i n a comparable c o n t r o l ,
and t h e m o l a r
mannose t o 2 - a c e t a m i d o - 2 - d e o x y - Q - g l u c o s e
a-
ratio of
was a p p r o x .
6:l.
High
c o n c e n t r a t i o n s o f n e u t r a l o l i g o s a c c h a r i d e s were d e t e c t e d i n t h e urine.
The
predominant
hexasaccharide.
oligosaccharide
appeared
The b i o c h e m i c a l f e a t u r e s o f b o v i n e ,
human m a n n o s i d o s i s w e r e compared,
to
be
a
feline,and
and i t was c o n c l u d e d t h a t f e l i n e
m a n n o s i d o s i s may b e a u s e f u l a n i m a l m o d e l f o r s t u d y i n g t h e human disease.
of
Kinetics
a-Q-mannosidase
action
on
various
0-9-
m a n n o p y r a n o s y l l i n k a g e s i n hen o v a l b u m i n g l y c o p e p t i d e s have been m o n i t o r e d by c a r b o n n u c l e a r m a g n e t i c r e s o n a n c e s p e c t r o s c o p y . 2 4 3 authors suggest t h a t 1 3 C for
n.m.r.
spectroscopy
The
i s a p r a c t i c a l method
f o l l o w i n g the k i n e t i c s o f enzymatic digestion o f i n d i v i d u a l
carbohydrate residues o f
glycopeptides
and f o r
structures o f the products of p a r t i a l digestions.
t h a t j a c k -be an a linkages
t o
-a - m a n n o s id as e
Q-mannose
at
corresponding 3-2-a-linkages
M.,
K o i d e , N.,
M u r a m a t s u , T.,
A.
(1975)
Biol.
J.
Chem.,
determining the The r e p o r t e d r u l e
h y d r o l y s e s 2- and 6 - 2 - a - g - m an no s y 1
least (Tai,
T.,
15
Iwashita,
220,
times
faster
Yamashita, S.,
K.,
I n o u e , Y.,
8569-8575)
than
the
Ogata-Arakawa,
i s not
and K o b a t a , of
general
validity. F i f t e e n g l y c o s i d a s e s h a v e b e e n a s s a y e d i n l y m p h o m y e l o i d and digestive
tissues
of
---E t m o e------t e r u s s e---inax.lg
Ginglymostoma c i r r a t u m ,
H e t e r o d o n t u s , and
8-QB-Q-2-acetamido-2-deoxygalactosidase, 6-Qg l u c u r o n i d a s e , a n d a- and 8 - n - g l u c o s i d a s e u s u a l l y were h i g h e r i n galactosidase,
Activities
of
a-Q-mannosidase,
449
6: Enzymes lymphomyeloid tissues than i n digestive tissues. Sucrose
density
gradients
lysosome-containing
have
fractions
been
from
used h
0
to
white
characterize prepupae
of
Stomoxys ~ a l c i t r a n s . ~ The ~ a c i d i c glycosidases a-g-glucosidase, !-galactosidase, galactosidase,
a-!-mannosidase, 8-Q-glucuronidase,
deoxyglucosidase e q u i l i b r a t e a t
the
and same
a-
B-;-
B-!-glucosidase,
B-Q-2-acetamido-2density
as
does
acid
phosphatase. N i n e
g l y c o s i d a s e s
i n
b l o o d s t r e a m
T r y p a n o s o m a b r u c e i b r u c e i S42 h a v e a-Q-Mannosidase
been p a r t i a l l y
course
of
o f
was c l e a r l y d i s t i n c t f r o m t h e o t h e r g l y c o s i d a s e s
w i t h r e s p e c t t o pH o p t i m u m t h e r m o s t a b i l i t y , the
f o r m s
characterized.20
parasiaemia,
and
specific a c t i v i t y during
subcellular
location.
It
was
s u g g e s t e d t h a t g l y c o s i d a s e s s t u d i e d may p l a y a r o l e i n t h e t u r n o v e r o f trypanosomal glycoproteins,
i n p a r t i c u l a r the variant-specific
s u r f ace a n t i g e n . The p h y s i c o c h e m i c a l a n d k i n e t i c p r o p e r t i e s o f t h e t w o m a j o r trypanosomal
glycosidases
a-E-glucosidase
and a - Q - m a n n o s i d a s e
were
compared i n b l o o d - s t r e a m f o r m s o f Trypanosoma b r u c e i b r u c e i ~ 4 2 . l ~ ~ F o r f u r t h e r d e t a i l s see i n i t i a l c i t a t i o n o f r e f . 1 7 5 .
Attempts
have
been
---D i c t y ------------------ostelium discoideum
made which
t o
i s o l a t e
express
mutants
o f
a-Q-mannosidase-2
c o n ~ t i t u t i v e l y . E~ v~i d~e n c e was s o u g h t f o r a g e n e - s p e c i f i c n e g a t i v e regulatory
element
repressor/operator and t h e i r v i r u s e s .
o f
D.
discoidggm
s i m i l a r
M u t a n t s o f D.
the
discoideum that constitutively
e x p r e s s t h e s t a g e - s p e c i f i c enzyme a-g-mannosidase-2 such evidence.
t o
s y s t e m s t h a t c o n t r o l some o p e r o n s i n p r o k a r y o t e s could provide
Reconstruction experiments demonstrated t h a t t h e
screening technique developed would detect mutants o f t h a t type. Over 2 X lo5 s u r v i v o r s o f h e a v i l y m u t a g e n i z e d c e l l p o p u l a t i o n s w e r e g r o w n and t h e i r p r o g e n y t e s t e d . obtained.
No m u t a n t s o f t h e d e s i r e d t y p e w e r e
The p o s s i b l e i m p l i c a t i o n s o f t h i s f i n d i n g a r e d i s c u s s e d .
Acid
hydrolases
from
D i c t y o s t e l i u m d i s c o i d e u m have been
r e p o r t e d t o c o n t a i n phosphomannosyl r e c o g n i t i o n markers.69 this
hypothesis,
the
b i n d i n g and e n d o c y t o s i s
B-c-2-acet amido-2-deoxyhexosidase,
glucos idase,
b y human f i b r o b l a s t s w e r e i n v e s t i g a t e d . endocytosis. An
mannosidase
To t e s t
p u r i f i e d B-Q-
and a - g - m a n n o s i d a s e
These enzymes u n d e r w e n t
F o r f u r t h e r d e t a i l s see i n i t i a l c i t a t i o n o f r e f . 6 9 .
acidic
f i l t r a t e s
of
of was
a-e-mannosidase Aspergillus
has
been
isolated The
from
culture
e x t r a c e l l u l a r a-Q-
homogeneous i n p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s .
450
Carbohydrate Chemistry
T h e m o l e c u l a r w e i g h t o f t h e e n z y m e was 5 1 , 0 0 0 a n d t h e i s o e l e c t r i c T h e p u r i f i e d e n z y m e h a s a pH o p t i m u m o f 5.0, a Em o f y e a s t g-mannan, and h a s no a c t i v i t y t o w a r d s 4-
p o i n t pH 4.5. 0.45
mM w i t h baker’s
n i t r o p h e n y l a-P-mannopyranoside.
The mode o f a c t i o n o f t h e enzyme
has been s t u d i e d w i t h baker’s
y e a s t Q-mannan and sake y e a s t Q-
mannan. chain,
The e n z y m e c l e a v e s s p e c i f i c a l l y t h e ( 1
+
2)-linked
side
p r o d u c i n g f r e e Q-mannose.
12 N e u r a m i n i d a s e s ( S i a l i d a s e s ) The
extracellular
n e u r a m i n i d a s e p r o d u c t i o n by
a e r u g i n o s a i s o l a t e d f o r m c y s t i c f i b r o s i s has The
cellular
localization
of
Pseudomonas
been i n ~ e s t i g a t e d . ’ ~ ~
glycoprotein
and
ganglioside
n e u r a m i n i d a s e s i n n o r m a l and I - c e l l d i s e a s e c u l t u r e d f i b r o b l a s t s h a s been i n v e s t i g a t e d . 2 4 7
C e l l u l a r o r g a n e l l e s h a v e been s e p a r a t e d on a
c o l l o i d a l s i l i c a gradient. enzymes i n d i c a t e d t h a t lysosomal hydrolase
the
The s u b c e l l u l a r d i s t r i b u t i o n o f t h e s e glycoprotein neuraminidase i s mainly a
whereas
the
ganglioside neuraminidase
p r i m a r i l y l o c a t e d i n t h e p l a s m a membranes.
i s
The l a t t e r i s o e n z y m e i s
t i g h t l y bound t o t h e s e membranes and t h u s c o u l d n o t be e x t r a c t e d by h o m o g e n i z a t i o n i n t h e p r e s e n c e o f T r i t o n X-000. The w h e a t - g e r m
l e c t i n b i n d i n g of B - P - g a l a c t o s i d a s e has been
s t u d i e d i n human a n d n o r m a l and s i a l i d o s i s t y p e I 1 f i b r 0 b 1 a s t s . l ~ ~ The a u t h o r s s u g g e s t t h a t n e u r a m i n i d a s e d e f i c i e n c y may be t h e p r i m a r y defect i n s i a l i d o s i s type I1 cells.
F o r f u r t h e r d e t a i l s see i n i t i a l
c i t a t i o n o f r e f .127. I t has been r e p o r t e d t h a t galactosidase
and
the
neuraminidase
combined d e f i c i e n c y o f
i n
human
fibroblasts
B-g-
can
be
c o r r e c t e d t o n e a r l y n o r m a l ~ a 1 u e s . l ~T ~ h i s c a n be a c c o m p l i s h e d by addition of
concentrated
culture
medium
obtained
s t i m u l a t i o n o f d i f f e r e n t t y p e s o f human f i b r o b l a s t s .
after
NH4Cl
For f u r t h e r
d e t a i l s see i n i t i a l c i t a t i o n o f r e f . 1 2 4 . The c h a r a c t e r i s t i c s o f t h e n e u r a m i n i d a s e o f human l e u k o c y t e s , fibroblasts,and
a m n i o t i c f l u i d c e l l c u l t u r e s have been d e t e r m i n e d
w i t h a r a d i o a c t i v e assay t h e enzyme s u b s t r a t e . 2 4 8
m e t h o d u t i l i z i n g n e u r a m i n - 1 3 H ) l a c t i t o l as F i b r o b l a s t cultures from p a t i e n t s w i t h the
i n h e r i t e d neuraminidase deficiency diseases,
including mucolipidosis
I, s i a l i d o s i s 1,and s i a l i d o s i s 11, j u v e n i l e t y p e , h a v e l e s s t h a n 10% n o r m a l n e u r a m i n i d a s e a c t i v i t y u s i n g e i t h e r t h i s s u b s t r a t e , 2-(3’met ho x y p h e n y 1) -N-ac e t y 1-a- n e u r a m i n ic ac id , o r 2’ ( 4 met h y 1u m be 11if
- -
-
45 1
6: Enzymes acid.
ery1)-N-acetyl-a-neuraminic
The t o t a l s i a l i c a c i d c o n t e n t o f
f i b r o b l a s t s and l e u k o c y t e s f r o m m u c o l i p i d o s i s I and s i a l i d o s i s I patients
is g r e a t l y e l e v a t e d .
establishing a diagnosis o f content
of
sialidosis
This
sialidase
juvenile
11,
n e u r a m i n i d a s e and B - 8 - g a l a c t o s i d a s e e l e v a t e d above n o r m a l l e v e l s . 16% o f
type,
The s i a l i c a c i d with
deficiencies,
coexistent
i s only s l i g h t l y
A patient with mucolipidosis
normal neuramin-{ 3 H ) l a c t i t o l
peripheral leukocytes.
parameter i s useful i n
deficiency.
neuraminidase a c t i v i t y
I has
i n his
H i s p a r e n t s were c l e a r l y d i s t i n g u i s h e d f r o m
t h e n o r m a l r a n g e u s i n g l e u k o c y t e enzyme l e v e l s , was i d e n t i f i e d as a p o s s i b l e c a r r i e r .
and a m a t e r n a l a u n t
The p r e s e n c e o f t h i s enzyme
i n a m n i o t i c f l u i d c e l l c u l t u r e s , b o t h f i b r o b l a s t i c and m i x e d - c e l l t y p e , makes p o s s i b l e t h e p r e n a t a l d e t e c t i o n o f t h e s e d i s e a s e s . pregnancy
from
mucolipidosis
a
family
at
risk
for
having
I was m o n i t o r e d b y a m n i o c e n t e s i s
a
child
A
with
and subsequent
n e u r a m i n i d a s e measurement o f t h e a m n i o t i c f l u i d c e l l c u l t u r e . Degradation of
mucin oligosaccharides
of
human c o l a e
has been f o u n d t o be a s s o c i a t e d w i t h e x t r a c e l l u l a r , cell-bound
B-g-galactosidase,
systems
but not with
B-~-2-acetamido-2-deoxyglucosidase,and
n e ~ r a m i n i d a s e . ~The ~ m o s t p r o b a b l e n u m b e r s (MPN) o f f a e c a l b a c t e r i a producing
extracellular
deoxyglucosidase,and
B-g-galactosidase,
B-B-2-acetamido-2For further
neuraminidase were estimated.
d e t a i l s see i n i t i a l c i t a t i o n o f r e f . 2 9 . An a d v e r s e e f f e c t surfaces
o f n e u r a m i n i d a s e on t h e a n a l y s i s of
by b o r o h y d r i d e t r i t i a t i o n h a s been r e p o r t e d . 2 4 9
e r y t h r o c y t e s u r f a c e as a m o d e l i t was t r e a t m e n t has a d e l e t e r i o u s e f f e c t
cell
Using the
found t h a t neuraminidase
on t h e m o b i l i t y a n d r e s o l u t i o n o f
l a b e l l e d g l y c o p r o t e i n s i n sodium dodecyl s u l p h a t e p o l y a c r y l a m i d e g e l electrophoresis.
The
authors
suggest
that
t h i s undesirable
c o n s e q u e n c e o f n e u r a m i n i d a s e needs t o be c a r e f u l l y the
enzyme
i s
used t o
enhance
g-galactose
c o n s i d e r e d when
oxidase/borohydride
t r i t i a t i o n o f c e l l surfaces. I t h a s been shown t h a t a t l e a s t t w o c o m p o n e n t s o f n e u r a m i n i d a s e c a n be d i s t i n g u i s h e d i n human l e u c o c y t e s o n t h e b a s i s o f pH o p t i m u m , thermolability
.
g l u c o s i d e 250 substrate,
a t 3O0C,and
the effect o f the detergent octyl-Q-
W i t h 4-me t hy l u m b e l l i f e r y 1 a - g - 3 - a c e t y l n e u r a m i n a t e as
t h e A c o m p o n e n t h a d a pH o p t i m u m o f 5.0,
was l a b i l e a t
3O0C,and was u n a f f e c t e d b y 0.2 M o c t y l 6 - Q - g l u c o p y r a n o s i d e . c o m p o n e n t h a d a pH o p t i m u m o f 4.0-4.2, most
of
i t s
The B lost
o c t y l 6-QB o t h A and B components were membrane bound b u t
activity
glucopyranoside.
was s t a b l e a t 3 0 ° C , b u t
i n
the
presence
of
0.2
M
452
Carbohydrate Chemistry
o n l y t h e A component was s o l u b i l i z e d by o c t y l B - Q - g l u c o p y r a n o s i d e i n an a c t i v e
form.
Molecular
of
weights
neuraminidases
2
r a d i a t i o n i n a c t i v a t i o n w e r e f o u n d t o b e 240,000 component, for
203,000
2
17,000
by X-ray
19,000 f o r t h e B
t h e A component, and 238,000
for
2
8,000
t h e o c t y l 8 - ~ - g l u c o p y r a n o s i d e - ~ o l u b i l i z e Ad c o m p o n e n t .
Gel
i n the presence o f o c t y l B-e-
f i l t r a t i o n o f s o l u b l e A component
g l u c o p y r a n o s i d e showed a s i n g l e peak o f a c t i v i t y e l u t e d a t
o r near
t h e v o i d volume,
w h i c h s u g g e s t e d t h a t t h e e n z y m e was s t i l l i n a n
aggregated form.
P r o f o u n d d e f i c i e n c y o f n e u r a m i n i d a s e a c t i v i t y was
f o u n d f o r b o t h A and B c o m p o n e n t s i n l e u c o c y t e s o f p a t i e n t s a f f e c t e d with
sialidoses
types
1 and
(less
2
than
15% n o r m a l )
t h e A and B c o m p o n e n t s o f
that
r e l a t e d from
l e u c o c y t e neuraminidase
t h e g e n e t i c p o i n t o f view
and
I t was s u g g e s t e d
intermediate a c t i v i t y i n o b l i g a t e heterozygotes.
are closely
and t h a t r a p i d d i a g n o s i s o f
s i a l i d o s e s c a n be d o n e by f l u o r i m e t r i c a s s a y o f n e u r a m i n i d a s e i n leucocytes. The c o n d i t i o n s o f a l i n e a r assay f o r 4 - m e t h y l u m b e l l i f e r y l a-Q-
-N - a c e t y l n e u r a m i n i c
a c i d neuraminidase
been
and an
determined
acidic
activity
liver
i n human l i v e r
have
neuraminidase has
been
c h a r a c t e r i z e d w i t h a pH o p t i m u m b e t w e e n 4.4 M i c h a e l i s c o n s t a n t o f 0.11
2
a c e t y l n e u r a m i n i c acid.251 freezing, majority
h e a t i n g , and of
human
and an a p p a r e n t
mM
various
liver
and 4.8,
f o r 4 - m e t h y l u m b e l l i f e r y l a-9-lThe a c i d i c n e u r a m i n i d a s e i s l a b i l e t o 0.02
storage
conditions.
neuraminidase
activity
The
great
found
i n
r e s u s p e n d e d p e l l e t s a f t e r h o m o g e n i z a t i o n and c e n t r i f u g a t i o n .
A
s m a l l amount o f t h i s r e s u s p e n d e d p e l l e t a c t i v i t y with the non-ionic liver
d e t e r g e n t T r i t o n X-100.
neuraminidase are
compared t o
i s
can be s o l u b i l i z e d
The p r o p e r t i e s o f human
those
of
other
mammalian
neuraminidases. The a c t i v i t i e s o f v a r i o u s g l y c o s i d a s e s i n h o m o g e n a t e s o f t h e small
intestinal
wallabies (M. gated.38
mucosa
of
two
adult
and
18
suckling
tammar
e u r e n i i ) a g e d f r o m 6 t o 50 w e e k s h a v e b e e n i n v e s t i -
8-P-Galactosidase,
L-fucosidase,and
B-B-2-acetamido-2-deoxyglucosidase,
ci-
n e u r a m i n i d a s e a c t i v i t i e s were h i g h d u r i n g t h e f i r s t
34 w e e k s p o s t p a r t u m a n d t h e n d e c l i n e d t o v e r y l o w l e v e l s .
For
f u r t h e r d e t a i l s see i n i t i a l c i t a t i o n o f ref.38. O l i g o s a c c h a r i d e s w i t h a new t y p e o f c o r e s t r u c t u r e , B-Q-GalNAc(1
+
4)-B-g-Gal-(l
+ 4)-{NGNA-(2
have been i s o l a t e d f r o m
.+
3)}-8-!-Gal-(l
+ 3)-!-GalNAcol,
t r o u t e g g ~ j l y c o p r o t e i n . ~The ~ ~ occurrence
o f t j - g l y c o l y l n e u r a m i n i c a c i d (NGNA) l i n k e d ( 2
2-acetamido-2-deoxy-Q-galactose
residue
is
not
* 3)
t o the i n t e r n a l
known
i n
any
other
453
6: Enzymes g l y c o p r o t e i n s and g l y c o l i p i d s .
T h i s NGNA r e s i d u e was f o u n d t o show
some a n o m a l o u s n a t u r e s u c h as r e s i s t a n c e t o n e u r a m i n i d a s e s a n d unusual chemical-shift feature
i s
GalNAc-(1
s t r u c t u r e B-Q-
asialo-oligosaccharide
*
4)-B-!-Gal-(l
+
A new
v a l u e s o f H-3 p r o t o n s i n NMR s p e c t r a .
a l s o found i n t h e
4)-g-GalNAc,
l i n k a g e r e g i o n d i s a c c h a r i d e B-g-Gal-(l
-+
linked
(1
3)
+
to
the
3)-!-GalNAc.
S t u d i e s have been c a r r i e d o u t on t h e e f f e c t o f b i l e s a l t s on the
rates
of
hydrolysis
of
the N-acetylneuraminyl
linkages o f
s e v e r a l s i a l i c a c i d - c o n t a i n i n g c o m p o u n d s by t h e n e u r a m i n i d a s e o f
perf ring en^.^^^
Clostridium
When G M 3 - g a n g l i o s i d e ,
two
and n e u r a m i n y l - l a c t o s e
glycolipids
(glycophorin
and o r o s o m u c o i d ) ,
substrates,
h y d r o l y s i s was o b t a i n e d e v e n i n t h e a b s e n c e o f b i l e
s a l t s , but a d d i t i o n o f these detergents, concentration,
were
u s e d as
below i t s c r i t i c a l m i c e l l a r
increased the reaction rates.
Above t h e c r i t i c a l
m i c e l l a r c o n c e n t r a t i o n of t h e detergent r a t e s decreased again. a second g a n g l i o s i d e ,
f o r b i l e s a l t s was a b s o l u t e ; w i t h o u t t h i s detergent.
h y d r o l y s i s was n o t o b s e r v e d a t a l l
With increasing concentrations o f b i l e s a l t
and i n t h e p r e s e n c e o f hydrolysis increased,
When
was u s e d a s s u b s t r a t e , t h e r e q u i r e m e n t
GM17
high concentrations
of
rates of
enzyme,
reaching maximal values a t f i x e d r a t i o s o f
b i l e s a l t t o GMl-ganglioside.
P h y s i c a l measurements showed t h a t
m i x t u r e s o f b i l e s a l t and G M 1 - g a n g l i o s i d e
formed mixed m i c e l l e s t h a t
had a higher c r i t i c a l m i c e l l a r concentration,
a lower molecular
weight, and g r e a t e r a x i a l r a t i o t h a n t h e c o r r e s p o n d i n g m i c e l l e s o f pure GM1-ganglioside. N e u r a m i n i d a s e o f C o r y n e b a c t e r i u m u l c e r a n s h a s been p u r i f i e d by affinity
chromatography
preparations.254 protein,
molecular
independent of
using
immobilized
Neuraminidase mass
appears
70,000.
t h e s u b s t r a t e used,
The
to pH
be
colominic a
acid
thermolabile
optimum
of
5.5
i s
t h e o p t i m a l t e m p e r a t u r e i s 37'C,
t h e M i c h a e l i s c o n s t a n t t o w a r d s t j - a c e t y l n e u r a m i n o s y l - l a c t o s e i s 5.2 10-4M.
Ca2+ a n d B a 2 + a c t i v a t e d t h e e n z y m e ,
chelating
agent
H4
edta
were
6) o r ( 2
inhibitory.
b u t Zn2+, The
Fe2+,
enzyme
did
x
and not
hydrolyse the (2
+
+
8) bonds o f s u b m a x i l l a r y p i g m u c i n and
colominic
respectively,
b u t i t h y d r o l y s e d s u c h s u b s t r a t e s as
fetuin,
acid,
ovomucin,
orosomucoid,and h o r s e serum g l y c o p r o t e i n s .
P a r t i a l p u r i f i c a t i o n and p r o p e r t i e s Bifidobacterium lactentis
a Gram-positive non-spore-forming isolated
from
the
of
neuraminidase from
have been d e s c r i b e d . 2 5 5
faeces
of
B.lactentis
659,
anaerobic bacterium o r i g i n a l l y breast-fed
infants,
produces
n e u r a m i n i d a s e a f t e r enzyme i n d u c t i o n w i t h 2mM 2 - a c e t a m i d o - 2 - d e o x y - e -
454
Carbohydrate Chemistry
mannose added t o t h e c u l t u r e medium.
B a c t e r i a were t r a n s f e r r e d and
g r o w n i n a medium c o n t a i n i n g c a s e i n h y d r o l y s a t e , acetate,
amino a c i d s ,
37OC u n d e r N2/C02 original because
lactentis
B.
different
taxonomic i d e n t i t y from
salts,and
atmosphere.
strain of
vitamins,
was
not
sodium
Two s u b c u l t u r e s d e r i v e d f r o m t h e 659
growth
lactose,
2% human m i l k f o r 48 h a t
were
investigated
characteristics.
doubtful.
separately
However,
N e u r a m i n i d a s e was
t h e b a c t e r i a l s e d i m e n t s by u l t r a s o n i c t r e a t m e n t
their
liberated
o f 0.15M
and was i s o l a t e d b y 60% ammonium s u l p h a t e p r e c i p i t a t i o n ,
NaCl
dialysis,
concentration,
a n d c o l u m n c h r o m a t o g r a p h y o n S e p h a r o s e CL-6B a n d
S e p h a d e x G-100.
T h e e n z y m e e x h i b i t s a m o l e c u l a r w e i g h t o f 38,000
a n d a pH o p t i m u m i n t h e r a n g e o f pH 5 - 6 i n b a r b i t a l / a c e t a t e b u f f e r s . The n e u r a m i n i d a s e has
activity
been i n v e s t i g a t e d . 2 5 6
o f Pasturella haemolytica isolates
Type
isolates
of
the
12 established
s e r o t y p e s and b o v i n e and o v i n e f i e l d i s o l a t e s w e r e i n c l u d e d i n t h e study.
N e u r a m i n i d a s e a c t i v i t i e s r a n g e d f r o m 0 t o 0.87
weight)
o f P.
haemolytica.
Activity
U p e r mg ( d r y
l e v e l s among t h e
isolates
s t u d i e d were s e r o t y p e a s s o c i a t e d . The c o m p l e t e n e u r a m i n i d a s e o f i n f l u e n z a A/PR8/34 been d e t e c t e d
i n
l o c a t e d on t h e
i t s
recombinant
virus.257
influenza recombinant
r e l a t i v e l y a poor
antigen
A/E(Heql)
A/PR8/34
(HoN1)
v i r u s was e x p l o r e d .
r a b b i t anti-A/PR8/34
(HoN1)
and o n l y
antibody
by
recombinant
precipitable
virus.
was the
( N I t i t r e 160).
the neuraminidase o f
R e s u l t s o b t a i n e d showed t h a t
serum c o n t a i n e d two d i s t i n c t t y p e s of
antineuraminidase antibody, was
A/PR8(N1)
compared w i t h
(HoNl)
This difference i n the antigenic behaviour o f
has n o t
neuraminidase
x
( N I t i t r e < 1 0 ) as
e n z y m e p r e s e n t o n t h e p a r e n t v i r u s A/PR8/34
(HoN1)
The
the
one t y p e o f a n t i n e u r a m i n i d a s e neuraminidase
Evidence suggests t h a t
located on
t h e p a r e n t enzyme(s)
the i s
n o t d e t e c t a b l e i n i t s e n t i r e t y on i t s r e c o m b i n a n t v i r u s . N e u r a m i n i d a s e h a s been p u r i f i e d f r o m t h e i n f l u e n z a v i r u s A/Hong Kong/68
(H3N2)
by
laurylsarcosinate, o n DEAE-Sephadex
treatment of
the
centrifugation at
purified 11O,OOOxg,
a n d S e p h a d e x G-200.258
virus
with
I t m i g r a t e d as a s i n g l e
component d u r i n g e l e c t r o p h o r e s i s on p o l y a c r y l a m i d e g e l , molecular weight
was e s t i m a t e d a b o u t
sodium
and chromatography
270,000.
The
and i t s
e n z y m e was
t h e r m o l a b i l e , t h e a c t i v i t y b e i n g r e d u c e d t o 6 0 % i n 1 0 m i n a t 5OoC. The p u r i f i e d n e u r a m i n i d a s e h a d a n a p p a r e n t
Em v a l u e
o f 4.1xlO-’
M
f o r 5 - t j - a c e t y 1- 2 - g - ( 3 - m e t h o x y p h e n y 1)- a - Q - n e u r am i n i c a c i d a n d was able t o release s i a l i c acid with linkages (2 8)
( w i t h very
different
efficiency) from
+
3),
fetuin,
(2
+
6),and
(2
+
gangliosides,
455
6: Enzymes colominic acid,
and b o v i n e and p o r c i n e s u b m a x i l l a r y mucins.
enzymic a c t i v i t y
was
m e a s u r e d by
The
several procedures which are
discussed.
13
B-9-X y losidases
The a - k - f u c o s i d a s e , mannosidase,
a-k-arabinofuranosidase muscle o f
B-~-2-acetamido-2-deoxyglucosidase,
B-g-glucuronidase,
$-g-xylosidase,
activities
of
d e v e l o p i n g and a d u l t
a-Q-
6-Q-glucosidase, and
n o r m a l and a t r o p h i c s k e l e t a l
r a t s have been
collectively
i n v e s t i g a t e d .36 Asperqillus niqer,
s t r a i n 110.42
producer o f high x y l a n o l y t i c x y l a n a s e and B - Q - x y l o s i d a s e
(CBS),
activities.259
h a s been s e l e c t e d as a The t i m e - c o u r s e
p r o d u c t i o n as w e l l as t h e e f f e c t
of
p-
o f pH
and t e m p e r a t u r e on t h e a c t i v i t y o f t h e s e enzymes were s t u d i e d . H.p.1.c.
a n a l y s i s of
I - a r a b i n o - Q-- x y l a n
t h e enzymatic degradation of
showed a n e a r l y c o m p l e t e c o n v e r s i o n t o p e n t o s e s u g a r s . discuss
the
use
of
crude
Q-xylanase
The a u t h o r s
preparations
for
the
s a c c h a r i f i c a t i o n o f Q-xylans. Cultures
of
Streptomyces f l a v o g r i s e u s have
p r o d u c e c o n s i d e r a b l e a m o u n t s o f Q - x y l a n a s e when c o n t a i n i n g media.260
been
found
to
g r o w n on Q - x y l a n -
C o m p a r a t i v e l y l o w e r y i e l d s o f t h i s enzyme w e r e
o b t a i n e d when h a y o r a v i c e l s e r v e d a s m a i n c a r b o n s o u r c e .
B-g-
Xylosidase
less
dependent
was
synthesized
on t h e
simultaneously
intracellularly
fermentation
substrate.
and The
appeared
strain
produced
v a r i o u s enzymes o f t h e c e l l u l a s e c o m p l e x and t h e Q-
x y l o s e - i n d u c e d !-glucose
isomerase.
Active-site-directed
i r r e v e r s i b l e i n h i b i t i o n o f g l y c o s i d a s e s by
t h e c o r r e s p o n d i n g glycosylmethyl-(4-nitrophenyl)triazines
has been
i n v e ~ t i g a t e d . ~B -~~ - X y l o p y r a n o s y l m e t h y l - ( 4 - n i t r o p h e n y l ) t r i a z i n e i s an
active-site-directed
irreversible
x y l o s i d a s e from P e n i c i l l i u m wortmanni,
inhibitor
(ASDIN)
of
B - Q--
b u t h a s no e f f e c t o f t h e B-Q-
x y l o s i d a s e o f B a c i l l u s p u m i l u s , e x c e p t by v i r t u e o f i t s c o n s u m p t i o n of stabilizing dithiothreitol.
For
further
d e t a i l s see i n i t i a l
c i t a t i o n o f r e f .72. release
from
B a c t e r o i d e s s u c c i n o q ---enes i n t h e rumen e n v i r o n m e n t has -------------------
The
importance
been
investigated.223
of
cellulase
During growth of
and 8.
g-xylanase
succinogenes i n a l i q u i d
medium w i t h c e l l u l o s e as t h e s o u r c e o f c a r b o h y d r a t e , g r e a t e r t h a n 80% o f t h e e n d o - ( 1 * 4 ) - B - Q - g l u c a n a s e , Q - x y l a n a s e , and a r y l - 8 - Q -
Carbohydrate Chemistry
456 x y l o s i d a s e and 50% o f t h e a r y l - 6 - Q - g l u c o s i d a s e
For
c e l l s i n t o the culture f l u i d .
was r e l e a s e d f r o m t h e
further
d e t a i l s see
i n i t i a l
c i t a t i o n o f r e f .223.
In
a
study
of
the
extracellular
cellulase
LQRI and T r i c h o d e r m a r e e s i
e x t r a c e l l u l a r B-e-xylosidase
a c t i v i t y was f o u n d t o be a b s e n t
14
endo-B-Q-2-acetamido-2-deoxyglucanases
h a v e been i s o l a t e d f r o m f i g l a t e x . 2 6 1
r e t a i n e d b y t h e DEAE-Sephadex and t y p e F - I 1
A-50
column,
Type F - I
p H 5.4.
mannosyl d e r i v a t i v e s o f
type F-I
was
w h e r e a s F - I 1 was n o t was f o u n d t o be
enzyme h y d r o l y s e s t h e
tri-gI-
o r h e x a - Q - m a n n o s y l compounds.
h y d r o l y s e s t h e p e n t a - and h e x a - D - m a n n o s y l
not the tri-e-mannosyl
and t y p e
F-I
di-2-acetamido-2-deoxy-Q-glucosyl
asparagine faster than the pentaType F - I 1
(type
A t pH 7.0,
The o p t i m u m pH o f t y p e F - I
a b s o r b e d by t h e column. pH 5.9
QM9414
endo-B-~-2-Acetamido-2-deoxyglucanases
Two
F-11)
of
activities
Clostridium thermocellum ........................
derivatives
but
compound.
The d i s t r i b u t i o n o f t h e d i f f e r e n t t y p e s o f o l i g o s a c c h a r i d e s i n c a t h e p s i n D and i n B - q - 2 - a c e t a m i d o - 2 - d e o x y h e x o s i d a s e
synthesized i n
c u l t u r e d human f i b r o b l a s t s h a s b e e n s t u d i e d b y u s i n g endo-B-Q-2-
acetamido-2-deoxyglucanase saccharides.262
The
H as
enzymes
a probe f o r
p r o t e i n or t h e carbohydrate moiety. cleavable
oligosaccharides
h i g h p-mannose
were s p e c i f i c a l l y were
I n b o t h enzymes, found.
The
s a c c h a r i d e s p r e v a i l e d i n t h e s e c r e t e d enzymes. of
r e s i s t a n t and
resistant
cathepsin D contained two oligosaccharide s i d e chains. sensitive to
d e o x y g l u c a n a s e H. enzymes
t h e a c t i o n of
oligo-
Precursor molecules
forms o f t h e precursor are synthesized w i t h both, saccharides
oligo-
labelled i n the
one,or
Multiple
more o l i g o -
t h e endo-B-E-2-acetamido-2-
I n f i b r o b l a s t s unable t o phosphorylate lysosomal
(mucolipidosis
11) t h e
enzymes c o n t a i n e d p r e d o m i n a n t l y
B-~-2-acetamido-2-deoxyglucanase
excessively
secreted
lysosomal
o l i g o s a c c h a r i d e s r e s i s t a n t t o endoH.
An a u t o l y t i c e n d o - B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c a n a s e ,
capable o f
c l e a v i n g t h e g l y c o s i d e l i n k a g e s o f N - u n s u b s t i t u t e d g-2-amino-2deoxy-1-glucose
i n t h e g l y c a n m o i e t y o f c e l l - w a l l p e p t i d o g l y c a n , has
been p u r i f i e d 4 7 0 - f o l d precipitate resistant
fraction
strain
of
from
obtained
a
salt
after
extract
o f
sonication of
B a c i l l u s cereus.263
The
the a
20,000
g
lysozyme-
purified
enzyme
p r e p a r a t i o n was a l s o a c t i v e t o w a r d s t h e g l y c a n c h a i n o f f u l l y
E-
457
6: Enzymes acetylated c e l l - w a l l peptidoglycan.
The e n d o - B - Q - Z - a c e t a m i d o - 2 -
d e o x y g l u c a n a s e was i n a c t i v e t o w a r d s t h e c e l l - w a l l p e p t i d o g l y c a n u n l e s s t h e p e p t i d e p o r t i o n o f t h i s p o l y m e r was r e m o v e d e i t h e r by t h e a c t i o n o f N-acetylmuramyl-l-alanine
a m i d a s e o r by t h e t r e a t m e n t w i t h
a l k a l i i n aqueous d i m e t h y l s u l p h o x i d e . t h i s enzyme t o w a r d s
chemically
S t u d i e s on t h e a c t i o n o f
modified glycans revealed t h a t
the
carboxy groups o f muramic a c i d r e s i d u e s were i n d i s p e n s a b l e t o a s u b s t r a t e f o r t h i s enzyme.
endo-B-~-2-Acetamido-2-deoxyglucanase
has been d e m o n s t r a t e d i n
human t i s s u e s and h a s b e e n p a r t i a l l y ~ h a r a c t e r i z e d . ’ ~ ~The p r e s e n c e of
enzyme a c t i v i t y t o w a r d s
and o f
o l i g o s a c c h a r i d e s of
the N-acetyl-lactosaminic
tissues.
type
the oligomannosidic
i s d e m o n s t r a t e d i n human
F u r t h e r m o r e , some p r o p e r t i e s o f t h e e n z y m e ( p H o p t i m u m ,
i n f l u e n c e of
t h i o l r e a g e n t s , and s t a b i l i t y )
i n human k i d n e y
are
described. A new
method has been d e v i s e d f o r
acetamido-2-deoxyglucanase
a s s a y i n g t h e endo-B-p-2-
a c t i v i t y by u s i n g t h e d a n s y l a s p a r a g i n y l
o l i g o s a c c h a r i d e (e-Man)5(g-GlcNAc)2-&-Asn-DNS
Q -G l c N A c - & - A s n -DNS
analysing the product,
,
as t h e s u b s t r a t e a n d by r e v e r s e - p h a s e h i g h -
pressure l i q u i d chromatography
using a silica-based
bonded o c t a d e c y l column.265
The
c o l u m n was
a c e t o n i t r i l e i n 25 m M s o d i u m b o r a t e b u f f e r , pH 7 . 5 . was m o n i t o r e d by a
U.V.
DNS
and
the
Under t h e s e c o n d i t i o n s ,
c o u l d be
c o n d i t i o n s d e s c r i b e d above,
p-
(Q-Man)5(P-GlcNAc)2-l-Asn-
was w e l l s e p a r a t e d f r o m
analysis
The e f f l u e n t
m o n i t o r a t 2 1 3 nm and a f l u o r e s c e n c e m o n i t o r
( e x c i t a t i o n 3 1 3 nm, e m i s s i o n 5 4 0 nm). GlcNAc-l-Asn-DNS
chemically
e l u t e d w i t h 8%
completed
i n
5
the lower l i m i t of
min.
Under
the
d e t e c t i o n was 0.1
nmol o f dansyl glycopeptides.
15 Agarases Agarase
has
been
concentrated
and
purified
from
culture
f i l t r a t e s o f an a g a r - d e g r a d i n g P s e u d o m o n a s - l i k e b a c t e r i a by a f f i n i t y c h r o m a t o g r a p h y on agarose.266 which,
d i v i n y l sulphone
cross-linked
a g a r a s e s I a n d 11, h a d a g a r a s e a c t i v i t y .
f u r t h e r p u r i f i e d by i s o e l e c t r i c f o c u s i n g , I I b , was i s o e l e c t r i c a t pH 5.1. I I b
was
63,000
as
A g a r a s e I 1 was
and t h e m a i n peak,
agarose
Molecular-weight determinations
i n d i c a t e d a g a r a s e I t o be a dirner agarase
macroporous
By g e l f i l t r a t i o n t h r e e f r a c t i o n s w e r e o b t a i n e d , t w o o f
with
determined
Mr by
210,000.
The
analytical
Mr
of
ultra-
458
Carbohydrate Chemistry
c e n t r i f u g a t i o n , p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i n sodium dodecyl s u l p h a t e , and m o l e c u l a r - s i e v e c h r o m a t o g r a p h y i n 6 M g u a n i d i n i u m c h l o r i d e . T h e amino a c i d c o m p o s i t i o n s of t h e two p r o t e i n s were very s i m i l a r and both were found t o b e g l y c o p r o t e i n s . The pH optimum of t h e enzymes was 6.7 and t h e o p t i m a l t e m p e r a t u r e was 38OC f o r a g a r a s e I and 43OC f o r a g a r a s e I I b . M e l t e d a g a r o s e and a g a r o s e g e l w e r e u s e d a s s u b s t r a t e s f o r t h e e n z y m e s . The r a t i o of t h e a c t i v i t i e s t o w a r d s t h e d i f f e r e n t s u b s t r a t e s was 4 . 3 f o r a g a r a s e I and 1.0 f o r Agarase I hydrolysed t h e g l y c o s i d i c l i n k a g e s i n agarase I I b . n e o a g a r o - o c t a o s e s o a s t o produce two moles of n e o a g a r o t e t r a o s e f o r one mole of neoagarohexaose and one mole of n e o a g a r o b i o s e . Agarase I I b h y d r o l y s e d only t h e c e n t r a l g l y c o s i d i c l i n k a g e t o form two moles of neoagar o t e t r aose.
16
A l g i n a s e s and A l g i n a t e Lyases
The p r o p e r t i e s of isoenzymes of a l g i n a t e l y a s e i n t h e mid-gut gland of Turbo c o r n u t u s have been i n ~ e s t i g a t e d . ~ ~ A' l g i n a t e l y a s e SP2 was s e p a r a t e d on an SP-Sephadex C-50 c o l u m n f r o m S P 1 , whose p r o p e r t i e s have a l r e a d y been r e p o r t e d , and p u r i f i e d a c c o r d i n g t o t h e method employed f o r SP1, t o o b t a i n i n f o r m a t i o n on SP2. The p r o f i l e s of t h e o p t i m a l pH, pH s t a b i l i t y , t h e r m a l i n a c t i v a t i o n , and molecular s i z e o f S P 2 w e r e e n t i r e l y t h e same a s t h o s e o f S P 1 . The i s o e l e c t r i c p o i n t of S P 1 and SP2 was 7.5 and 7 . 7 , r e s p e c t i v e l y . The a c t i o n o f SP2 on a l g i n a t e c a u s e d a r a p i d d e c r e a s e i n s o l u t i o n v i s c o s i t y . A n a l y s i s of d i g e s t i o n p r o d u c t s of a l g i n a t e w i t h S P 2 showed t h a t t h e enzyme had an a f f i n i t y toward t h e p-mannuronate-rich domains of t h e a l g i n a t e molecule and r e l e a s e d u n s a t u r a t e d o l i g o m e r s mostly composed of p-mannuronic a c i d as f i n a l p r o d u c t .
17 a-Amylases A k i n e t i c e q u a t i o n has been d e r i v e d w h i c h r e p r e s e n t s a s y n e r g y s t i c a c t i o n of endo- and --enzyme upon p o l y s a c c h a r i d e s . 2 6 8 I t s adequacy was t e s t e d by e x p e r i m e n t s u s i n g i m m o b i l i z e d a-amylase and glucoamylase f o r h y d r o l y s i s of s o l u b l e s t a r c h and amylose. An a s s a y on a m y l a s e a c t i v i t i e s u s i n g NAD-dependent m a l t o s e dehydrogenase has been d e s c r i b e d . 2 6 9 I t was d e m o n s t r a t e d t h a t t h e a c t i v i t i e s o f a - a m y l a s e and B-amylase c o u l d b e m e a s u r e d w i t h N A D -
459
6: Enzymes
d e p e n d e n t m a l t o s e d e h y d r o g e n a s e by t h e e n d - p o i n t a s s a y m e t h o d and b y t h e r a t e a s s a y method.
One u n i t o f t h e a c t i v i t y o f a - a m y l a s e or B-
a m y l a s e was d e f i n e d as t h e amount o f t h e enzyme w h i c h p r o d u c e d one p m o l o f m a l t o s e e q u i v a l e n t p r o d u c t f r o m s u b s t r a t e p e r m i n u t e a t 25OC under t h e assay c o n d i t i o n . The e f f e c t o f a l b u m i n on a - a m y l a s e polymer has been i n v e s t i g a t e d . 2 7 0
assay u s i n g b l u e s t a r c h
The b i n d i n g o f a l b u m i n t o t h e
s t a r c h p o l y m e r was r e s p o n s i b l e f o r t h e s t i m u l a t i o n o f a - a m y l a s e activity,
s i n c e t h e s t i m u l a t i o n was o b s e r v e d w i t h t h e a l b u m i n - b o u n d
b l u e s t a r c h p o l y m e r as s u b s t r a t e i r r e s p e c t i v e o f t h e p r e s e n c e o r absence o f f r e e a l b u m i n i n t h e r e a c t i o n m i x t u r e .
When t h e a l b u m i n -
b o u n d b l u e s t a r c h p o l y m e r was h y d r o l y s e d b y a - a m y l a s e ,
the blue
o l i g o s a c c h a r i d e f r a g m e n t s were r e l e a s e d w i t h t h e a l b u m i n a t t a c h e d . These o l i g o s a c c h a r i d e f r a g m e n t s
were
about
1.3
times
larger
i n
a v e r a g e m o l e c u l a r s i z e t h a n t h e f r a g m e n t s r e l e a s e d i n t h e absence o f a l b u m i n , s u g g e s t i n g t h a t t h e a p p a r e n t s t i m u l a t i o n o f a - a m y l a s e by a l b u m i n ( a b o u t 1.3
t i m e s ) i s due t o t h e l i b e r a t i o n o f t h e l a r g e r
o l i g o s a c c h a r i d e fragments. The s u i t a b i l i t y o f c o n t r o l m a t e r i a l s f o r d e t e r m i n a t i o n o f a a m y l a s e a c t i v i t y has been assessed i n c o m p a r i s o n w i t h r e f e r e n c e groups
of
a u t h e n t i c human
serum specimens c o n t a i n i n g a-amylase
e i t h e r pancreatic or s a l i v a r y o r i g i n , no p a n c r e a t i c 'pathology,
of
specimens from p a t i e n t s w i t h
and n o r m a l s p e c i m e n s t o w h i c h p o r c i n e
p a n c r e a t i c a - a m y l a s e was added.271
A f t e r d e t e r m i n a t i o n o f a-amylase
a c t i v i t y by 11 commonly u s e d t e c h n i q u e s ( f i v e d i f f e r e n t p r i n c i p l e s ) , t h e r e s u l t s were p r o c e s s e d by b o t h c l a s s i c a l ( l i n e a r r e p r e s e n t a t i o n , r e g r e s s i o n ) and m u l t i v a r i a t e ( c o r r e s p o n d e n c e a n a l y s i s , components
a n a l y s i s ) s t a t i s t i c a l techniques.
Specimens
principalcontaining
p o r c i n e p a n c r e a t i c a - a m y l a s e d i d n o t b e h a v e l i k e any o f t h e o t h e r groups.
I t was c o n c l u d e d t h a t p o r c i n e enzyme s h o u l d n o t be u s e d f o r
interlaboratory quality-control studies.
surveys or
inter-method
comparison
D e t e r m i n a t i o n o f human s a l i v a r y and p a n c r e a t i c a - a m y l a s e
showed i n t e r m e t h o d b i a s e s s i m i l a r specimens.
t o those
Human s a l i v a r y a - a m y l a s e ,
for
because of
authentic patients' both i t s behaviour
and i t s c o m m e r c i a l a v a i l a b i l i t y , i s a s a t i s f a c t o r y s o u r c e f o r aamylase a c t i v i t y of q u a l i t y - c o n t r o l specimens. matrix (polyvinylpyrrolidone, serum,
The n a t u r e o f t h e
albumin, d e l i p i d a t e d serum, bovine
o r human s e r u m ) l i t t l e i n f l u e n c e d t h e b e h a v i o u r o f t h e
s p e c i m e n s f o r any o f t h e m e t h o d s s t u d i e d .
A
p o l a r i m e t r i c method
for
a c t i v i t y h a s been d e s c r i b e d . 2 7 2
the
determination
of
a-amylase
I n the study o f the r e l a t i v e l y slow
460
Carbohydrate Chemistry
amylase-catalysed h y d r o l y s i s of o l i g o s a c c h a r i d e s , the
subsequent
k i n e t i c s of
m u t a r o t a t i o n has
the t o t a l reaction.
essentially
Relatively
i t i s assumed t h a t no
effect
on
the
l a r g e s a m p l e s and l o n g
measurement t i m e s ( s l o w r a t e o f change i n a n g l e o f r o t a t i o n ) a r e necessary
for
t h e p o l a r i m e t r i c d e t e r m i n a t i o n o f a-amylase a c t i v i t y .
For these reasons,
sample sources.
o n l y u r i n e a n d d u o d e n a l j u i c e a r e s u i t a b l e as Owing t o t h e absence o f c o u p l e d r e a c t i o n s and
aux i l l i a r y enzymes, t h e p o l a r i m e t r i c a l l y d e t e r m i n e d k i n e t i c s a r e g e n e r a l l y more r e p r e s e n t a t i v e o f t h e t r u e k i n e t i c s o f t h e amylase r e a c t i o n than addition,
those
obtained
with
fully
enzymic
s u b j e c t t o i n t e r f e r e n c e by sample components. times
methods.
I n
s i n g l e d e t e r m i n a t i o n s by t h e p o l a r i m e t r i c m e t h o d a r e l e s s
i n the
order
of
minutes,
the
W i t h measurement
polarimetric
m e t h o d shows
e x c e l l e n t l i n e a r i t y , very good p r o p o r t i o n a l i t y between a n a l y t i c a l r e s p o n s e and 1.3%).
q u a n t i t y o f enzyme,
(c=
and good p r e c i s i o n i n s e r i e s
C o i n p a r i s o n o f t h e p o l a r i m e t r i c w i t h a c h r o m o g e n i c and a f u l l
e n z y m i c m e t h o d showed an a c c e p t a b l e c o r r e l a t i o n
(z
for
each method
was a b o u t 0.98).
A number o f enzymes, i n c l u d i n g a m y l a s e s , dehydrogenases, and p r o t e a s e s , have been shown t o be r e n a t u r a b l e a f t e r p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i n t h e presence o f Enzyme
activity
was
detected
introduced i n t o the product
gel
and
o f
enzyme
by
subsequent
or unreacted substrate.
advantage
sodium dodecyl ~ u l p h a t e . * ~ ~
i n situ This
action
on
substrates
staining
of
either
the
combines
the
technique
i d e n t i f i c a t i o n w i t h t h e r e s o l u t i o n and
m o l e c u l a r - w e i g h t dependence o f o f sodium dodecyl sulphate.
g e l e l e c t r o p h o r e s i s i n t h e presence
Enzymes a p p e a r e d t o r e c o v e r a c t i v i t y as
soon as t h e d e t e r g e n t d i f f u s e d o u t o f
the gel.
Most
monomeric
enzymes c o u l d b e r e n a t u r e d even a f t e r d i s r u p t i o n o f t h e i r d i s u l p h i d e bonds,
but
several proteases,
O l i g o m e r i c enzymes renaturable.
including trypsin,
composed o f
Renatured
i d e n t i c a l subunits
enzymes
were
retained
i n
could not. were gels
poorly after
e l e c t r o p h o r e s i s l o n g e r t h a n n a t i v e enzymes w h i c h h a d b e e n s u b j e c t e d t o e l e c t r o p h o r e s i s i n t h e absence o f d e t e r g e n t .
Re-electrophoresis
o f t h e r e n a t u r e d enzymes showed t h a t p a r t o f t h e r e t a i n e d a c t i v i t y was
physically
anchored t o t h e gel,
p o s s i b l y by
the folding of
p o l y p e p t i d e s a r o u n d t h e g e l m a t r i x as t h e enzymes w e r e r e n a t u r e d . An i n h i b i t o r trypsin,
1-1,
capable o f
a c t i n g on b o t h a - a m y l a s e
and
has been p u r i f i e d t o homogeneity f r o m r a g i ( f i n g e r - m i l l e t )
grains.274
The f a c t o r was f o u n d t o b e s t a b l e t o h e a t t r e a t m e n t a t
100°C f o r 1 h i n t h e p r e s e n c e o f N a C l a n d a l s o was s t a b l e o v e r a
46 1
6: Enzymes w i d e pH r a n g e 1-10.
P e p s i n and p r o n a s e t r e a t m e n t
r e s u l t e d i n gradual loss o f
both the
Formation o f
trypsin-inhibitor
1-1 c o m p l e x ,
complex, and
trypsin-inhibitor
o f i n h i b i t o r 1-1
inhibitory
activities.
amylase-inhibitor
I-1-amylase
trimer
d e m o n s t r a t e d by c h r o m a t o g r a p h y on a B i o - G e l P-200 c o l u m n . indicated that
t h e i n h i b i t o r was d o u b l e - h e a d e d
i n h i b i t o r was r e t a i n e d on a t r y p s i n - S e p h a r o s e Elution at
acidic
pH r e s u l t e d
i n
almost
a m y l a s e - i n h i b i t o r y and t r y p s i n - i n h i b i t o r y
1-1
complex
was This
i n nature.
The
48 c o l u m n a t pH 7.0.
complete
activities.
recovery
of
a - A m y l a s e was
r e t a i n e d o n a t r y p s i n - S e p h a r o s e c o l u m n t o w h i c h i n h i b i t o r 1 - 1 was bound, b u t n o t on t r y p s i n - S e p h a r o s e a l o n e .
M o d i f i c a t i o n o f amino
groups o f t h e i n h i b i t o r w i t h 2,4,6-trinitrobenzenesulphonic
acid
r e s u l t e d i n complete loss o f a m y l a s e - i n h i b i t o r y a c t i v i t y b u t o n l y
40% loss
i n
antitryptic
activity.
Modification of
a c t i v i t y a f t e r 5 h b u t no e f f e c t o n a m y l a s e - i n h i b i t o r y T h e r e s u l t s show responsible for
that
&-arginine
l e d t o 85% loss o f a n t i t r y p t i c
r e s i d u e s by c y c l o h e x a n e - 1 , 2 - d i o n e
a single bifunctional
both amylase-inhibitory
activity.
protein factor
was
and t r y p s i n - i n h i b i t o r y
a c t i v i t i e s w i t h two d i f f e r e n t reactive sites. a-Amylase as f a c t o r s
i n h i b i t o r s f r o m wheat k e r n e l s have been i n v e s t i g a t e d
i n resistance t o post-harvest
insects.275
a-Amylase
i n h i b i t o r s were e x t r a c t e d f r o m k e r n e l s o f f i v e h a r d w i n t e r - w h e a t varieties that
were grown a t d i f f e r e n t
locations i n two crop years
and t h a t h a d been e v a l u a t e d f o r s u s c e p t i b i l i t y r i c e weevil,
S i t o p h i l u s oryzae.
or r e s i s t a n c e t o t h e
I n h i b i t o r a c t i v i t y was a s s a y e d w i t h
l a r v a l a-amylase from two species of s t o r e d g r a i n pests, t h e r i c e w e e v i l and t h e y e l l o w some wheat
mealworm,
Tenebrio molitor.
Correlation
in
v a r i e t i e s was o b s e r v e d b e t w e e n i n v i v o r e s i s t a n c e t o t h e
i n s e c t and t h e e x t e n t o f i n v i t r o i n h i b i t i o n o f t h e i n s e c t l a r v a l aa m y l a s e by t h e e x t r a c t e d i n h i b i t o r s .
These r e s u l t s i n d i c a t e d t h a t
a - a m y l a s e i n h i b i t o r s i n wheat c o u l d b e i n v o l v e d i n t h e r e s i s t a n c e o f wheat t o p o s t - h a r v e s t
infestation.
The c h e m i c a l s t r u c t u r e
of
an a m y l a s e
i n h i b i t o r S-A1 p r o d u c e d
by S t r e p t o m y c e s d i a s t a t i c u s h a s b e e n i n v e s t i g a t e d . 2 7 6 Some
properties
-S t r e e -tomyces No.
280 p r o d u c e d s e v e r a l
i n h i b i t o r A, AI-A1
of
amylase
inhibitor
s p . No. 280 h a v e b e e n r e p o r t e d . 2 7 7 6 , B’, a n d
and A I - A 2 )
C).
kinds of
amylase
A
produced
Streptomyces
by sp.
i n h i b i t o r s (amylase
Two a m y l a s e i n h i b i t o r s ( d e s i g n a t e d as
w e r e o b t a i n e d f r o m an a m y l a s e i n h i b i t o r A f r a c t i o n
AI-A1 i n h i b i t e d muscle phosphorylase 2 by paper chromatography. much more t h a n A I - A 2 and was h y d r o l y s e d b y s w e e t - p o t a t o a - a m y l a s e
Carbohydrate Chemistry
462 whereas A I - A 2
was n o t .
B o t h amylase i n h i b i t o r s had a c a r b o h y d r a t e
m o i e t y and were h y d r o l y s e d by some k i n d s o f a m y l a s e s lost
their
inhibitory
activity
against
t r e a t m e n t w i t h a c i d s o r hog p a n c r e a t i c a-amylase, increased
inhibitory
sucrase.
Both A I - A 1
basic
moiety
molecular
and A I - A 2
which
gave
weights
of
-
a p p r o x i m a t e l y 1,300 column.
activity a
toward
AI-A1
porcine
and
after
b u t t h e y showed
glucose a n d a
ninhydrin
AI-A2
They
5
small-intestinal
were composed o f
positive
or a c i d s .
phosphorylase
were
reaction.
The
estimated to
be
1,500 by g e l f i l t r a t i o n on a Sephadex G - 1 5
The n i t r o g e n c o n t e n t o f t h e a m y l a s e i n h i b i t o r s was f o u n d t o
be a b o u t 1.3% by e l e m e n t a l a n a l y s i s . The
sequence
-Streptomyces
of
the
i n h i b i t o r i s o l a t e d f r o m S. c h r o m a t o g r a p h y on CM-
467 S of
(with
the
tendae,
s t r a i n 4158,
a-amylase
&-serine
was
Hoe-467
as
N-terminal
was r e - p u r i f i e d
residue)
The
digestions o f the performic oxidized,
peptides,
(with
A
L-
i n h i b i t o r Hoepreliminary
d e t e r m i n e d by a u t o m a t i c E d m a n - d e g r a d a t i o n
i n h i b i t o r , respectively.
by
Two i n h i b i t o r s
and a - a m y l a s e
a z i r a n i z e d i n h i b i t o r and t r y p t i c
from
An a - a m y l a s e
i n h i b i t o r Hoe-457
a c i d as N - t e r m i n a l r e s i d u e )
structure
inhibitor
been e l u c i d a t e d . 2 7 8
and D E A E - c e l l u l o s e column.
c o u l d be c h a r a c t e r i z e d : aspartic
a-amylase
t e n d a e 4158 has
procedures
derived from
aziranized,and
maleylated
The a - a m y l a s e i n h i b i t o r Hoe-467 A c o n s i s t s
o f 7 4 r e s i d u e s and h a s a c a l c u l a t e d m o l e c u l a r w e i g h t o f 7958.
It
i s c o m p o s e d o f a l l common a m i n o a c i d s e x c e p t I - m e t h i o n i n e a n d
I-
phenylalanine.
D i g e s t i o n w i t h p e p s i n was c a r r i e d o u t t o d e t e r m i n e
t h e d i s u l p h i d e bonds. one L - c y s t i n e e a c h , d i s u l phi d e b r i d g e s
.
Two f r a c t i o n s c o u l d b e i s o l a t e d ,
containing
g i v i n g i n f o r m a t i o n about t h e p o s i t i o n o f t h e
The c o m p l e t e a m i n o a c i d s e q u e n c e o f one o f t h e m a j o r w h e a t protein iso-inhibitors
o f a-amylase has been determined.279
The
s e q u e n c e was d e d u c e d f r o m a n a l y s i s o f f r a g m e n t s a n d p e p t i d e s f r o m t h e p r o t e i n by c l e a v a g e w i t h c y a n o g e n b r o m i d e and b y d i g e s t i o n w i t h trypsin,
chymotrypsin,
protease.
123 residues. 65,
t h e r m o l y s i n , and t h e S t a p h y l o c o c c u s a u r e u s V8
The m o l e c u l e c o n s i s t s o f a s i n g l e p o l y p e p t i d e c h a i n o f B o t h I,=-serine a n d & - a l a n i n e w e r e f o u n d i n p o s i t i o n
and f u r t h e r
m i n o r examples o f
m i c r o h e t e r o g e n e i t y were observed
i n four other residues. The i n t e r a c t i o n o f wheat
flour
f o u r p u r i f i e d a-amylase
i n h i b i t o r s from
w i t h human s a l i v a r y a n d p a n c r e a t i c a - a m y l a s e s
investigated.280 towards s a l i v a r y
The
inhibitory
a-amylase
activity
of
was s i g n i f i c a n t l y
the
four
h a s been proteins
i n c r e a s e d by p r e -
463
6: Enzymes
i n c u b a t i o n o f t h e enzyme w i t h i n h i b i t o r b e f o r e a d d i n g s u b s t r a t e . T h i s e f f e c t was n o t o b s e r v e d w i t h t h e i n h i b i t i o n o f p a n c r e a t i c aa m y l a s e b y i n h i b i t o r s 1 a n d 2.
I n h i b i t i o n o f b o t h a m y l a s e s was
a f f e c t e d t o d i f f e r e n t degrees by i n c u b a t i n g s t a r c h w i t h i n h i b i t o r p r i o r t o t h e a d d i t i o n o f enzyme.
Maltose,
a t concentrations which
o n l y s l i g h t l y a f f e c t e d a m y l a s e a c t i v i t y , p r e v e n t e d t h e i n h i b i t i o n of b o t h enzymes by a l l f o u r i n h i b i t o r s . salivary amylase-inhibitor
G e l - f i l t r a t i o n s t u d i e s on
m i x t u r e s showed t h e f o r m a t i o n o f E I
complexes on a m o l e - t o - m o l e
ratio.
similar
A
complex between
p a n c r e a t i c a - a m y l a s e a n d i n h i b i t o r 4 was o b s e r v e d , t h o u g h c o m p l e x f o r m a t i o n b e t w e e n p a n c r e a t i c a - a m y l a s e and t h e o t h e r i n h i b i t o r s was n o t c l e a r l y demonstrated
.
A s e a r c h h a s b e e n made f o r n a t u r a l p l a n t e n z y m e i n h i b i t o r s o f
human s a l i v a r y a-amylase.281 vicolor),
T w e l v e v a r i e t i e s o f sorghum (Sorqhum
1 4 v a r i e t i e s o f p e a r l m i l l e t (Pennisetum typhpoideum),
varieties
of
setaria
(Setaria intalica),
(Eleucjng-cgfgcgfig), (Echinocloa colona), varieties
11 v a r i e t i e s
13 v a r i e t i e s of
o f
4
varieties
echinocloa
of
12
ragi
m i l l e t
proso (Panicium miliaceum),
o f k o d o ( P a s p a l u m s c o r b i c u l a t u m ) , a n d 11 v a r i e t i e s
11
of
m i l i a r e (Panicium m i l i a r e ) were screened f o r i n h i b i t o r y a c t i v i t y a g a i n s t human s a l i v a r y a m y l a s e . h a d no d e t e c t a b l e a c t i v i t y .
E c h i n o c l o a , p r o s o , kodo, and m i l i a r e
Two s t r a i n s o f
s o r g h u m and one s t r a i n
o f p e a r l m i l l e t d i d n o t show a - a m y l a s e i n h i b i t o r y a c t i v i t y . o t h e r seeds had a c t i v i t y , Setaria
h a d n o a c t i o n on human,
amylases.
A l l
t h e h i g h e s t b e i n g o b s e r v e d i n sorghum. b o v i n e , and p o r c i n e p a n c r e a t i c
Sorghum i n h i b i t o r d i d n o t
act
on b o v i n e and p o r c i n e
p a n c r e a t i c amylases.
P e a r l m i l l e t and r a g i e x t r a c t s i n h i b i t e d a l l
t h e f o u r a-amylases.
The i n h i b i t o r s w e r e n o n - d i a l y s a b l e a n d w e r e
i n a c t i v a t e d by p e p s i n t r e a t m e n t . were r e l a t i v e l y t h e r m o l a b i l e
S e t a r i a and sorghum i n h i b i t o r s
compared t o r a g i and p e a r l m i l l e t
inhibitors. An a - a m y l a s e been
p r o t e i n i n h i b i t o r from A r a c h i s hypohaea seeds has
p u r i f i e d and i t s p r o p e r t i e s have been
described.282
The
p r o t e i n , w h i c h showed a h i g h l y s p e c i f i c i n h i b i t o r y a c t i v i t y t o w a r d s h o g p a n c r e a t i c and human s a l i v a r y a - a m y l a s e s and b a c t e r i a l a - a m y l a s e s ,
but not towards p l a n t
h a s been p u r i f i e d 1 9 7 - f o l d f r o m an aqueous
e x t r a c t of p e a n u t c o t y l e d o n s u s i n g h e a t t r e a t m e n t ,
ammonium s u l p h a t e
p r e c i p i t a t i o n , and i o n - e x c h a n g e
o n OEAE-
cellulose. gel
chromatography
and CM-
The p u r i f i e d i n h i b i t o r was homogeneous by p o l y a c r y l a m i d e
electrophoresis.
Sephadex G-100
I t s
molecular
gel filtration,
weight,
as
determined
and i t s e l e c t r o p h o r e t i c
mobility
by at
464 pH
Carbohydrate Chemistry 8
relative
respectively.
t o
Bromophenol
The i n h i b i t o r y
thermal treatment
were
was
25,000
and
0.14,
relatively resistant
to
a n d m a r k e d l y i n c r e a s e d when t h e i n h i b i t o r was
preincubated
with
Further,
i n h i b i t i o n was
the
Blue
activity
the
enzyme
before
found
to
the
addition
of
b e pH d e p e n d e n t
starch. and non-
competitive i n nature. Two p r o t e i n a c e o u s a - a m y l a s e i s o l a t e d f r o m r a g i (E. sodium c h l o r i d e , CM-cellulose,
1-1 and 1-2,
inhibitors,
h a v e been
c o r a c a n a ) g r a i n s by e x t r a c t i o n w i t h 0.15
ammonium s u l p h a t e f r a c t i o n a t i o n ,
M
c h r o m a t o g r a p h y on
g e l f i l t r a t i o n on Sephadex G-50, and r e c h r o m a t o g r a p h y
o n C M - c e l l ~ l o s e . ~The ~ ~ i n h i b i t o r s 1-1 a n d 1 - 2 w e r e p u r i f i e d 5 0 -
f o l d and 1 2 - f o l d ,
respectively,
be homogeneous
by
electrophoresis. chromatography inhibitors
were
cellulose
and t h e p r e p a r a t i o n s w e r e f o u n d t o acetate
and p o l y a c r y l a m i d e g e l
The e s t i m a t e d m o l e c u l a r
w e r e a r o u n d 9,100 active
weights
f o r 1-1 a n d 8,300
a g a i n s t a-amylase
b a s e d on g e l
f o r 1-2.
f r o m hog pancreas,
The human
p a n c r e a s , and human s a l i v a b u t h a d no a c t i o n on B a c i l l u s s u b t i l i s a amylase.
B o t h t h e i n h i b i t o r s w e r e b a s i c p r o t e i n s and were s t a b l e
o v e r a w i d e pH r a n g e o f 1 - 1 0 .
Sodium c h l o r i d e i n c r e a s e d t h e heat
s t z b i l i t y and s o l u b i l i t y o f t h e i n h i b i t o r s . treatments
cause i n a c t i v a t i o n o f
i n a c t i v a t e d 1-2 chymotrypsin.
alone.
both the
B o t h 1-1
P e p s i n and p r o n a s e inhibitors.
and 1 - 2
were
Trypsin
resistant
t o
The i n h i b i t o r y p a t t e r n s o b s e r v e d w i t h 1-1 a n d 1 - 2
w e r e n o n - c o m p e t i t i v e when h o g p a n c r e a t i c a m y l a s e was used. i n h i b i t o r y a c t i v i t y was
Amylase
l o s t c o m p l e t e l y on g e r m i n a t i o n o f r a g i f o r
48 h. F o u r a - a m y l a s e i n h i b i t o r s h a v e been i s o l a t e d f r o m an a l b u m i n fraction
o f
wheat
flour
by
ion-exchange
and
c h r ~ m a t o g r a p h y . ~The ~ ~ purified inhibitors according t o carbohydrate
their
electrophoretic
content,
mobilities,
sulphydryl
gel-filtration
were
content,
characterized
molecular
weights,
susceptibility to
p r o t e o l y t i c d i g e s t i o n , and s p e c i f i c i t i e s i n i n h i b i t i n g human s a l i v a r y and p a n c r e a t i c a - a m y l a s e s .
The p r o p e r t i e s o f t h e s e i n h i b i t o r s were
c o m p a r e d t o s i m i l a r p r o t e i n s i s o l a t e d by o t h e r w o r k e r s . A s t r u c t u r a l s t u d y h a s b e e n made o f t h e s u g a r c h a i n s o f a amylases produced e c t o p i c a l l y i n tumours.285 o f
two
a-amylases
produced
from
About t h i r t y p e r c e n t a
serous
p a p i l l a r y
c y s t a d e n o c a r c i n o m a o f t h e o v a r i u m ( c a s e 1) and a b r o n c h i o l o a l v e o l a r adenocarcinoma o f t h e mol of
l u n g ( c a s e 2) was g l y c o p r o t e i n s c o n t a i n i n g 1
C-asparagine-linked
sugar
chain,
respectively.
The
s t r u c t u r e s o f t h e sugar m o i e t i e s were f o u n d by s e q u e n t i a l e n z y m a t i c
465
6: Enzymes d e g r a d a t i o n and
methylation analysis.
Structures of
L-asparagine-
l i n k e d s u g a r c h a i n s w e r e t h e same i n t h e t u m o u r o f c a s e s 1 a n d 2 a n d were i n c o m p l e t e i n c o m p a r i s o n w i t h t h o s e o f t h e p a r o t i d amylase. Amylases
have been
purified
and
a m y l a s e - p r o d u c i n g human t u m o u r s . 2 8 6 the
amylases
was
characterized
estimated t o
be
54,000
sulphate p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s , s a l i v a r y a m y l a s e (61,000 and 64,000) (60,000).
The
tumour
amylases
a n t i g e n i c i t i e s w i t h human s a l i v a r y antibody
t o human s a l i v a r y amylases
on
i s o e l e c t r i c focusing,
two of
m a j o r p e a k a t pH 6.4, s a l i v a r y amylase;
on
sodium
dodecyl
d i f f e r e n t f r o m human
had
completely
identical
and p a n c r e a t i c a m y l a s e s
amylase,
single
three
a n d human p a n c r e a t i c a m y l a s e against
while the intensity of the
t u m o u r a m y l a s e s was l e s s t h a n 2 0 % o f t h a t o f pancreatic
from
The r e l a t i v e m o l e c u l a r mass o f
radial
human s a l i v a r y and
immunodiffusion.
On
t h e t h r e e tumour amylases showed a
w h i c h c o r r e s p o n d e d t o a m a j o r p e a k o f human
t h e o t h e r s h o w e d a m a j o r p e a k a t pH 6.4,
which
c o r r e s p o n d e d t o a m i n o r peak o f human s a l i v a r y a m y l a s e .
The t h r e e
tumour
different
from
amylases those
findings
showed s i m i l a r
of
human
amino a c i d compositions,
salivary
and
p a n c r e a t i c amylases.
These
suggest t h a t tumour amylases have a t e r t i a r y s t r u c t u r e
s i m i l a r t o t h a t o f n o r m a l human a m y l a s e s
b u t d i f f e r f r o m them i n
amino a c i d composition. The p r e p a r a t i o n and c h a r a c t e r i z a t i o n o f m a t e r i a l s c o n t a i n i n g human p a n c r e a t i c and s a l i v a r y u - a m y l a s e s h a v e been d e s c r i b e d and t h e i r r e l a t i o n s h i p t o endogenous a m y l a s e i n human serum examined.287 A m y l a s e was p u r i f i e d f r o m human p a n c r e a s and s a l i v a by s o l v e n t and s a l t f r a c t i o n a t i o n and c o l u m n c h r o m a t o g r a p h y t o s p e c i f i c a c t i v i t i e s of
6 3 a n d 279 kU g - l ,
only i n activity,
respectively.
a matrix containing, sodium chloride,
Four l i q u i d pools, d i f f e r i n g
were p r e p a r e d f r o m e a c h s o u r c e o f a m y l a s e , per l i t r e ,
1 mmol o f c a l c i u m c h l o r i d e ,
hydrochloride buffer,
pH 7.4.
each i n
30 g o f h u m a n a l b u m i n , 50 m m o l o f
and 50 mmol o f T r i s
C h a r a c t e r i z a t i o n of
t h e p o o l s showed
t h a t t h e a m y l a s e a c t i v i t y i n t h e m a t e r i a l s was s t a b l e f o r a t l e a s t s i x m o n t h s a t 25'C, 0.5%
(2
Cl),
and
among-vial
v a r i a b i l i t y o f a m y l a s e a c t i v i t y was
the
were
c o n t a m i n a t i n g enzymes.
pools P l o t s of
free
from
eight
possible
s a l i v a r y versus p a n c r e a t i c amylase
a c t i v i t y measured i n m a t e r i a l s with e i g h t c o m m e r c i a l l y a v a i l a b l e m e t h o d s showed l e a s t - s q u a r e s
s l o p e s r a n g i n g f r o m 0.51
intermethod commutability of the m a t e r i a l s
t o 1.0.
(h. how c l o s e l y
The they
m i m i c e n d o g e n o u s s e r u m a m y l a s e ) was e x a m i n e d i n r e l a t i o n s h i p t o a p p r o x i m a t e l y 1 0 0 human s e r a .
466
Carbohydrate Chemistry
An e n z y m a t i c a s s a y f o r t h e d e t e r m i n a t i o n of a-amylase i n serum employed a s o l u b l e s u b s t r a t e , r n a l t o h e p t a o s e , and a coupled e n z y m a t i c i n d i c a t o r r e a c t i o n c o n s i s t i n g of B-g-glucosidase and t h e h e x o k i n a s e B-!-glucose 6-phosphate dehydrogenase system.288 H.p.1.c. was used t o e s t a b l i s h t h e a c t i o n p a t t e r n of m a l t o h e p t a o s e u n d e r t h e t e s t c o n d i t i o n s : (A) t h e a c t i o n p a t t e r n of a-amylase a l o n e , (B) t h a t o f t h e combined a c t i o n o f a-amylase and B-g-glucosidase. Conducive t o t h e e f f o r t was: t h e a v a i l a b i l i t y o f p u r e m a l t o h e p t a o s e and human p a n c r e a t i c a - a m y l a s e , t h e development of an a d e q u a t e procedure f o r s a m p l e p r e t r e a t m e n t ( p a r t i t i ' o n c h r o m a t o g r a p h y on a mixed-bed i o n exchanger) and of an h.p.1.c. s y s t e m f o r s e p a r a t i o n of s u b s t r a t e and r e a c t i o n p r o d u c t s w i t h o u t i n t e r f e r e n c e from by-products of t h e assay ( p a r t i t i o n c h r o m a t o g r a p h y on a c a t i o n - e x c h a n g e c o l u m n w i t h a c e t o n i t r i l e - w a t e r ) , and t h e u s e of a new, v e r y s e n s i t i v e r e f r a c t o m e t r i c d e t e c t o r r e v e a l i n g s u g a r a m o u n t s a s low a s 40 ng. The f o l l o w i n g s t o i c h i o m e t r i c e q u a t i o n s were d e r i v e d :
a-amy l a s e m a l t o h e p t a o s e --------- > 0.10 m a l t o p e n t a o s e + 0.79 m a l t o t e t r a o s e + 0.87 m a l t o t r i o s e + 0.29 m a l t o s e a-amy l a s e m a l t o h e p t a o s e --------- > 0.10 m a l t o p e n t a o s e + 0.77 m a l t o t e t r a o s e B-q-glucosidase + 0.06 m a l t o t r i o s e + 0.07 m a l t o s e + 2.94 g l u c o s e T h e s t a n d a r d d e v i a t i o n o f t h e r a t e c o e f f i c i e n t s i s about 5 % . A s t u d y of t h e a c t i o n of human a - a m y l a s e s on r e d u c e d m a l t o o l i g o s a c c h a r i d e s h a s been r e p o r t e d . 2 8 9 K i n e t i c s t u d i e s on t h e r e a c t i o n s of human p a n c r e a t i c and s a l i v a r y a-amylases w i t h a s e r i e s of reduced m a l t o - o l i g o s a c c h a r i d e s ( m a l t o t e t r a o i t o l t h r o u g h m a l t o h e p t a o i t o l ) were conducted. M a l t o t e t r a o i t o l was b a r e l y h y d r o l y s e d b y e i t h e r a - a m y l a s e u n d e r t h e c o n d i t i o n s u s e d , and t h e m i n i m u m s i z e o f s u b s t r a t e s u s c e p t i b l e t o h y d r o l y s i s was m a l t o p e n t a o i t o l . Among t h e s e s u b s t r a t e s , m a l t o h e x a o i t o l showed t h e h i g h e s t r e a c t i v i t y w i t h each a-amylase, e s p e c i a l l y w i t h s a l i v a r y aa m y l a s e , compared w i t h m a l t o p e n t a o i t o l . F o r t h e c o m b i n a t i o n o f each enzyme w i t h each s u b s t r a t e , t h e predominant p o i n t of c l e a v a g e of t h e s u b s t r a t e s was found t o be t h e t h i r d (1 + 4 ) - a - g - g l u c o s i d i c l i n k a g e from t h e p - g l u c i t o l r e s i d u e . Antihuman p a r o t i d a - a m y l a s e a n t i b o d i e s r a i s e d i n r a b b i t s and h o r s e s were used a s t h e primary a n t i b o d i e s i n both t h e p e r o x i d a s e a n t i p e r o x i d a s e and sandwich t e c h n i q u e s f o r t h e l o c a l i z a t i o n of human
467
6: Enzymes a-arnyla~e.~” acinar
I m m u n o r e a c t i v e enzyme was d e m o n s t r a t e d i n t h e n o r m a l
cells
of
salivary
transformation,
p r o d u c t i o n o f a-amylase o f a-amylase
glands
and
pancreas.
which has o c c a s i o n a l l y by v a r i o u s t i s s u e s ,
Malignant
resulted i n ectopic a c t u a l l y c a u s e d a loss
s y n t h e s i s by t h e t r a n s f o r m e d a c i n a r c e l l s o f s a l i v a r y
g l a n d s and d i d n o t r e s u l t i n e l a b o r a t i o n o f a - a m y l a s e by t r a n s f o r m e d ductal cells. B i o c h e m i c a l and k i n e t i c s t u d i e s
have been p e r f o r m e d on
r e c r y s t a l l i z e d u r i n a r y a - a m y l a s e o f n o r m a l s and o f p a t i e n t s w i t h cancer o f t h e bladder.*’l
Measurement of t h e r e a c t i o n r a t e a t a
r a n g e o f pH v a l u e s i n d i c a t e d t h a t pH 6.8 f o r t h e enzyme o f n o r m a l s u b j e c t i o n s , i t s
optimum
supporting
at
pH
interaction sites
The
8.
evidence
i n
kinetic
favour
behaviour
of
i n t h e enzyme o f
was t h e o p t i m u m pH v a l u e
w h i l e t h e a b n o r m a l enzyme h a s the
provides
presence
of
strong
multiple
p a t i e n t s w i t h cancer o f t h e
bladder. The m u l t i p l e - a t t a c k m o d e l o f t h e a - a m y l a s e
mechanism h a s been
a n a l y s e d and t h e t h e o r e t i c a l r a t e c o e f f i c i e n t s dependent on t h e degree o f They a r e :
of h y d r o l y s i s
and t h e a v e r a g e number o f values.
(2) h a v e (El, w h i c h
polymerization of substrate effectiveness
u n i t a r y movements (Sm),
been a p p l i e d . 2 9 2 affects
1
values,
which a f f e c t s
Km
The m o d e l e x p l a i n s t h e d e p e n d e n c e o f 1 a n d E m o n t h e d e g r e e
of p o l y m e r i z a t i o n o f s u b s t r a t e s w i t h 2 v a l u e s h i g h e r t h a n t h e number of
s u b s i t e s i n t h e enzyme-binding s i t e .
of
p o l y m e r i z a t i o n of
hog pancreas
D i s t r i b u t i o n o f t h e degree
a-amylase
products
(np) a n d
average n v a l u e has been f o u n d e x p e r i m e n t a l l y and a p p l i e d t o -P c a l c u l a t e t h e m a x i m a l number o f u n i t a r y movements (1)o f t h e enzyme d u r i n g t h e s i n g l e enzyme-substrate meeting.
1
and
Em v a l u e s
of hog
p a n c r e a s a-amylase f o r amylose and d i f f e r e n t b r a n c h e d s u b s t r a t e s h a v e been d e t e r m i n e d and d i s c u s s e d i n t e r m s o f t h e m u l t i p l e - a t t a c k theory.
T h e i r dependence on t h e o r e t i c
Yn
a n d 5,
v a l u e s has been
f o u n d i n s u p p o r t o f t h e model. The s e q u e n c e s o f
four
cyanogen bromide-cleaved
p o r c i n e p a n c r e a t i c a-amylase have been determined.293
peptides o f The s e q u e n c e
s t r a t e g y h a s i n v o l v e d t h e c l e a v a g e by CNBr and t h e s e p a r a t i o n o f t h e
9 CNBr:peptides
i n t o 7 f r a c t i o n s by g e l f i l t r a t i o n .
The c o m p l e t e
p u r i f i c a t i o n and p a r t i a l - s e q u e n c e d e t e r m i n a t i o n o f 3 p e p t i d e s (CNBr4,
CNBr-5c, a n d C N B r - 5 d )
p r e s e n t work, 1, CNBr-3b2,
have p r e v i o u s l y been r e p o r t e d .
In the
t h e p u r i f i c a t i o n o f 4 a d d i t i o n a l CNBr p e p t i d e s (CNBrCNBr-7b1, a n d CNBr-7b2)
has been c a r r i e d o u t .
and o f s m a l l e r t r y p t i c p e p t i d e s
The c o m p l e t e sequence and t h e o r d e r o f CNBr-
468
Carbohydrate Chemistry
4, C N B r - 5 c ,
CNBr-3b2,
s e q u e n c e of
CNBr-1,
ordering
has
homologous
been
CNBr-7b1,and
C N B r - 7 b 2 a s w e l l as t h e p a r t i a l
CNBr-2, and C N B r - 3 b l possible
by
h a v e been d e t e r m i n e d .
taking
mouse p a n c r e a t i c a - a m y l a s e
advantage
just
of
the
b e i n g deduced
The
highly
from
the
n u c l e o t i d i c sequence o f mouse a - a m y l a s e mRNA. A s i n g l e - s t e p p u r i f i c a t i o n o f r a t p a n c r e a t i c a n d s a l i v a r y a-
amylase
by
affinity
chromatography
amylases were p u r i f i e d 20-
has
single-step affinity-chromatographic was a n a - Q - g l u c o h y d r o l a s e
The
a-
procedure.
The a f f i n i t y l i g a n d
i n h i b i t o r coupled t o 6-aminohexyl-Seph-
P a n c r e a t i c a - a m y l a s e w a s e l u t e d as a s i n g l e p e a k a t pH
a r o s e 48. 7.4
been d e s c r i b e d . 2 9 4
t o 5 0 - f o l d w i t h a y i e l d above 75% by a
w i t h 0.1% g l y c o g e n o r a t pH 5.8
without
glycogen.
Salivary
a m y l a s e c o u l d b e e l u t e d o n l y w i t h 0.1% g l y c o g e n - c o n t a i n i n g b u f f e r a t pH 7.4.
P a n c r e a t i c a-amylase
m i g r a t e d as a s i n g l e homogeneous b a n d
on sodium dodecyl s u l p h a t e p o l y a c r y l a m i d e gels,
w h e r e a s s a l i v a r y a-
a m y l a s e m i g r a t e d as a d o u b l e t . a-Amylase
a c t i v i t y h a s been d e t e c t e d i n u n p r o c e s s e d p r e a m y l a s e
produced i n t h e c e l l - f r e e Preamylases,
t r a n s l a t i o n o f p o r c i n e p a n c r e a t i c RNA.295
s y n t h e s i z e d i n t h e RNA-dependent r a b b i t r e t i c u l o c y t e
l y s a t e t r a n s l a t i o n s y s t e m s u p p l e m e n t e d w i t h p o r c i n e p a n c r e a t i c RNA, were i d e n t i f i e d by t h e i r s p e c i f i c i m m u n o p r e c i p i t a t i o n w i t h a n t i amylase.
The p r e a m y l a s e s h a v e a p p a r e n t
c o m p a r e d t o 52,000 isoenzymes.
and 55,000
I n order
t o
Mr
= 5 5 , 0 0 0 a n d 5 8 , 0 0 0 as
for the purified, establish
whether
s e c r e t e d a-amylase the
unprocessed
p r e c u r s o r s may assume e n z y m a t i c a l l y a c t i v e c o n f o r m a t i o n s , sensitive a c t i v i t y g e l electrophoresis technique, q u a n t i t i e s o f enzyme c a n b e d e t e c t e d , a-amylase
a highly
by w h i c h p i c o g r a m
was e x p l o r e d .
When s t a n d a r d
and t r a n s l a t i o n p r o d u c t s a r e s u b j e c t e d t o e l e c t r o p h o r e s i s
o n p o l y a c r y l a m i d e g e l c o n t a i n i n g 0.01% s t a r c h a n d CaC12,
active
a m y l a s e w h i c h b i n d s t i g h t l y t o s t a r c h c a n o n l y m i g r a t e as t h e s t a r c h
is hydrolysed. appearance
of
proportional t o
When t h e g e l i s s u b s e q u e n t l y s t a i n e d w i t h 12, t h e clear the
tracks,
the
logarithm of
t h e presence o f amylase a c t i v i t y .
lengths amylase
of
which
are
concentration,
By t h i s a p p r o a c h ,
roughly signifies
i t was p o s s i b l e
t o d e t e c t amylase a c t i v i t y i n a r a n g e c o r r e s p o n d i n g t o a b o u t 100 pg of p u r e a m y l a s e (10
v l ) - l o f translation mixture.
T h i s value agrees
w e l l w i t h an e s t i m a t e f r o m r a d i o a c t i v i t y i n c o r p o r a t i o n o f t o t a l preamylase i n the t r a n s l a t i o n mixture,
a n d i t was c o n s e q u e n t l y
c o n c l u d e d t h a t u n p r o c e s s e d p r e a m y l a s e c a n assume t h e a p p r o p r i a t e conformation t o give enzymatic a c t i v i t y . The c h a r a c t e r i z a t i o n o f t h e a m i n o t e r m i n i o f mouse s a l i v a r y and
469
6: Enzymes pancreatic sequences
amylases
has
been
and i n f o r m a t i o n
reported.296
concerning
Mouse
amylase
s i g n a l sequences
from
mRNA other
p r o t e i n s have been used t o l o c a t e p o t e n t i a l c l e a v a g e p o i n t s f o r r e m o v a l o f t h e s i g n a l p e p t i d e s f r o m t h e mouse a m y l a s e s .
That i n
t u r n allowed the authors t o predict the biochemical c h a r a c t e r i s t i c s o f t h e N - t e r m i n a l t r y p t i c p e p t i d e f o r e a c h enzyme and t h e n u s e t h o s e characteristics t o
l o c a t e and p u r i f y t h e p e p t i d e .
Analysis o f the
N - t e r m i n a l p e p t i d e s s h o w e d t h a t t h e s e c r e t e d mouse a m y l a s e s h a v e p y r o g l u t a m i c ( p y r r o l i d o n e c a r b o x y l i c ) a c i d r e s i d u e s a t t h e i r Ntermini. The
formation of
a complex
of
proteinous
a n i m a l a-amylase
i n h i b i t o r ( H a i m ) w i t h h o g p a n c r e a t i c a - a m y l a s e h a s been r e p o r t e d . 2 9 7 Novel
proteinous
i n h i b i t o r s
myces g r i s e o s p o r e u s YM-25
(Haim
I, 1 1 )
o f
Stre@gZ
i n h i b i t a n i m a l a-amylase s p e c i f i c a l l y .
H a i m i s a p r o t e i n c o n t a i n i n g no s u g a r m o i e t y ,
As
t h e i n h i b i t i o n o f Haim
a g a i n s t t h e a m y l a s e was c o n s i d e r e d t o be a t t r i b u t e d t o t h e r e s u l t o f protein-protein interaction. i n h i b i t o r p r o d u c e d by S.
On t h e o t h e r h a n d , S - A I ,
d i a s t a t i c u s subsp.
an a m y l a s e
~ylostaticus, inhibits
v a r i o u s a-amylases o f a n i m a l , p l a n t , and m i c r o b i a l o r i g i n .
S-A1
c o n s i s t s o f 6 m o l o f g l u c o s e w i t h unknown m o i e t y t e n t a t i v e l y named as
S-AI-X.
S-A1
inhibitor.
may
I n t h i s paper,
be
classified
a m y l a s e , p r o t e i n o u s i n h i b i t o r (Haim), substrate
analogue,
as
a
substrate
analogue
t h e i n t e r a c t i o n s among hog p a n c r e a t i c aand X G d e r i v e d f r o m S - A I ,
are described.
t r i p l e i n t e r a c t i o n among Haim,
XG,
as a
I n f e r r i n g the r e s u l t s from the and a - a m y l a s e ,
Haim s h o u l d j o i n
a t a n e i g h b o u r h o o d o f a c a t a l y t i c s i t e o r a t a s i t u a t i o n l i k e an a l l o s t e r i c s i t e o f a-amylase. An a n t i s e r u m a g a i n s t p u r i f i e d r a t p a r o t i d a - a m y l a s e h a s b e e n u s e d t o l o c a l i z e t h e p r o t e i n i n p a r o t i d g l a n d s o f d e v e l o p i n g and The u n l a b e l l e d a n t i b o d y p e r o x i d a s e - a n t i p e r o x i d a s e
a d u l t rats.298
method and t h e p r o t e i n A-gold l i g h t and e l e c t r o n - m i c r o s c o p e a-amylase
c o l l o i d technique were used a t t h e levels,
was d e t e c t e d i n a few
day-old rats.
respectively.
Immunoreactive
s c a t t e r e d c e l l s i n t h e g l a n d s o f 2-
D u r i n g t h e f o l l o w i n g days t h e number o f c e l l s s t a i n e d
immunocytochemically f o r a-amylase i n c r e a s e d r a p i d l y .
A t 15 days o f
age
intensity
a l l acinar
cells
r e v e a l e d a-amylase,
i m m u n o s t a i n i n g v a r i e d from
c e l l to cell.
but
the
of
Electron microscopically,
a - a m y l a s e was l o c a l i z e d i n t h e s e c r e t o r y g r a n u l e s a n d , b y u s i n g a more c o n c e n t r a t e d a n t i s e r u m , G o l g i complex.
i n t h e r o u g h e n d o p l a s m i c r e t i c u l u m and
A t e a r l y stages of development t h e a c i n a r c e l l s
c o n t a i n e d f e w e r and s m a l l e r s e c r e t o r g r a n u l e s t h a n i n a d u l t a n i m a l s .
Carbohydrate Chemistry
470 The g o l d p a r t i c l e s i n d i c a t i v e o f over the secretory granules. was
distributed
inhomogeneously
t h e m a j o r i t y of
l o c a t e d over t h e electron-dense
In
p o r t i o n s o f t h e granules.
However,
s h e l l s u r r o u n d e d an a-
o r a - a m y l a s e - n e g a t i v e c o r e were n o t i n f r e q u e n t .
invertebrates
study
has
invertebrates, Annelida,
granules.
g o l d c o l l o i d p a r t i c l e s were
i n w h i c h an a m y l a s e - r i c h
comparative
A
within the secretor
secretory granules
secretory granules amylase-poor
amylase were r a n d o m l y d i s t r i b u t e d
I n t h e glands of a d u l t r a t s , amylase
been
of
carbohydrase
performed.299
species
i n
marine
of
marine
ZeomgLi, C o e l e n t e r a t a ,
belonging t o 7 types,
Arthropoda,
activities
103
Mollusca, Echinodermata, Chordata,
were t e s t e d
f o r l a m i n a r i n a s e , c e l l u l a s e , and a m y l a s e a c t i v i t i e s . A n e p h e l o m e t r i c method f o r d e t e r m i n i n g c e r i a l a-amylase has
been e ~ a l u a t e d . ~ " A n e p h e l o m e t r i c assay adapted f r o m a c l i n i c a l m e t h o d was e v a l u a t e d f o r s u i t a b i l i t y i n d e t e r m i n i n g a - a m y l a s e l e v e l s i n sprouted soft operation,
w h i t e wheat.
I n the
m a n u a l mode o f
p r o p o r t i o n a l t o t h e amount o f enzyme a s s a y e d .
of
machine
t h e v e l o c i t y o f d e c l i n e i n l i g h t s c a t t e r i n g was d i r e c t l y The a c c u r a t e
limits
t h e a s s a y p e r f o r m e d i n t h e a u t o m a t e d mode w e r e d e f i n e d as 0-720
machine u n i t s of a c t i v i t y , substrate.
w i t h a commercial B - l i m i t
d e x t r i n as
T h i s s u b s t r a t e was m o r e r e a c t i v e t h a n a m y l o p e c t i n a n d
l e s s s u b j e c t t o B-amylase i n t e r f e r e n c e .
The a u t o m a t e d a s s a y y i e l d e d
d a t a t h a t c o r r e l a t e d h i g h l y w i t h f a l l i n g number r e s u l t s . The p r o p e r t i e s o f t w o a m y l a s e a c t i v i t i e s w h i c h d i f f e r i n t h e i r substrate
specificity
and
subcellular
c h l o r o p l a s t - a s s o c i a t e d R-enzyme reported.301 filtration daltons.
location
as
well
as
a
( d e b r a n c h i n g a c t i v i t y ) have been
An e x t r a c h l o r o p l a s t i c a m y l a s e was r e s o l v e d b y g e l -
chromatography
i n t o t w o a c t i v i t i e s o f 80,000
Both extrachloroplastic
and s h e l l f i s h g l y c o g e n glycogen, B - l i m i t
and o n l y
amylopectin,
activities slowly
and 40,000
hydrolyse amylopectin
hydrolyse rabbit
and a m y l o s e .
liver
I n c o n t r a s t , t h e major
c h l o r o p l a s t i c amylase a t t a c k s a l l o f these glucans a t comparable rates.
Glucan
hydrolysis
by
both
the
extrachloroplastic
and
c h l o r o p l a s t i c amylase generates n o t only maltose b u t appreciable amounts o f o t h e r o l i g o s a c c h a r i d e s ,
whereas m a l t o t e t r a o s e h y d r o l y s i s
produces Q-glucose,
maltose,
and m a l t o t r i o s e .
d i s p l a y e d by
amylase
activities indicate
the
The a c t i o n p a t t e r n s that
both are
a l t h o u g h t h e y l a c k t h e t y p i c a l Ca2+ r e q u i r e m e n t o r s t a b i l i t y o f seed endosperm a-amylases. Dithiothreitol,
endoamylases, heat
g l u t a t h i o n e ( o x i d i z e d or reduced), dithiothreitol
plus
thioredoxin
ascorbate, have
no
dehydroascorbate, effect
on
either
and the
471
6: Enzymes chloroplastic
o r e x t r a c h l o r o p l a s t i c a m y l a s e a c t i v i t i e s . The
c h l o r o p l a s t i c R-enzyme pullulan,
and a - l i m i t
debranches dextrins,
amylopectin,
but not rabbi&
i n c r e a s e i n e x t i n c t i o n c o e f f i c i e n t and Xmax d e b r a n c h e d a m y l o p e c t i n and B - l i m i t
B l i m i t amylopectin, An
l i v e r glycogen.
i s d e t e c t e d when t h e
a m y l o p e c t i n form a complex w i t h
Based o n t h e s e p r o p e r t i e s , t h e c h l o r o p l a s t i c R-enzyme i s
12-KI.
s i m i l a r i n e n z y m i c a c t i v i t y t o t h e R-enzyme o b s e r v e d i n endosperm tissue' Endogenous g i b b e r e l l i n s
and amylase a c t i v i t y have been
i n v e s t i g a t e d i n t a l l and d w a r f s t r a i n s o f r i c e (Oryza ~ a t i v a ) . ~ ~ * Two t a l l and s e v e n d w a r f s t r a i n s o f r i c e w e r e s p r a y e d w i t h GA3. d w a r f s t r a i n s r e s p o n d e d t o exogenous
endogenous g i b b e r e l l i n c o n t e n t t h a n t h e t a l l s t r a i n s , dwarf
strains
did
not
respond
Four
G A 3 and showed a m a r k e d l y l o w
to
GA3
while two
application
and
had
c o n s i d e r a b l y h i g h e r endogenous g i b b e r e l l i n l e v e l s t h a n t h e t a l l ones.
Amylase a c t i v i t y i n g e r m i n a t i n g seeds showed a s i g n i f i c a n t
n e g a t i v e c o r r e l a t i o n w i t h t h e endogenous g i b b e r e l l i n c o n t e n t . Hard r e d winter-wheat
c u l t i v a r s have been f o u n d t o d i f f e r
m a r k e d l y i n s p r o u t i n g and i n a-amylase s y n t h e s i s i n s p r o u t e d g r a i n , w h i c h i n c r e a s e d as t i m e b e t w e e n m a t u r i t y and e x p o s u r e t o s i m u l a t e d r a i n was i n c r e a s e d . 3 0 3 falling
number
correlated.
S p r o u t i n g and a - a m y l a s e a c t i v i t y m e a s u r e d b y
and l a b e l l e d s t a r c h d e g r a d a t i o n
were
highly
However, c u l t i v a r and t e m p o r a l d e v i a t i o n s f r o m t h a t
r e l a t i o n s h i p occurred. susceptible than
Hard winter-wheat
l i n e s are generally
more
cultivars,
but
e x c e p t i o n s o c c u r r e d and d i f f e r e n c e s w e r e n o t more q u a n t i t a t i v e .
The
simulated
were hard r e d winter-wheat
sprouting
environment
clearly
identified
sprouting-
r e s i s t a n t p h e n o t y p e s , and i t w o u l d be u s e f u l f o r s e l e c t i n g d e s i r a b l e g e r m o p l a s m i n wheat - i m p r o v e m e n t p r o g r a m m e s . The p r o p e r t i e s o f p a r t i a l l y p u r i f i e d a - a m y l a s e o f p e a r l m i l l e t h a v e b e e n i n ~ e s t i g a t e d . ~ ' T~h e p u r i f i c a t i o n m e t h o d a f f e c t e d t h e physicochemical c h a r a c t e r i s t i c s of
purified
millet
a-amylases.
P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s showed t h r e e p r o t e i n bands f o r aa m y l a s e p u r i f i e d b y g l y c o g e n c o m p l e x f o r m a t i o n a n d s e v e n f o r aa m y l a s e p u r i f i e d by s t a r c h c o l u m n p r o c e d u r e s . exhibited weights,
a-amylase
a-Amylase
A l l p r o t e i n bands
i s o e n r y m e s had m o l e c u l a r
d e t e r m i n e d by s o d i u m d o d e c y l s u l p h a t e g e l e l e c t r o p h o r e s i s ,
r a n g i n g f r o m 22,000 4.8
activity.
t o 6.2.
t o 53,000 a n d i s o e l e c t r i c p o i n t s r a n g i n g f r o m
The pH o p t i m a w e r e b e t w e e n 4.4
o p t i m u m was 55'C.
and 4.8,
and t e m p e r a t u r e
Many o f t h e above c h a r a c t e r i s t i c s a r e s i m i l a r t o
t h o s e r e p o r t e d f o r amylases p u r i f i e d from immature c e r e a l g r a i n s .
472
Carbohydrate Chemistry The
d i s t r i b u t i o n and e f f e c t o f a - a m y l a s e
w h e a t s h a s been t e s t e d i n J a p a n e s e - t y p e p h y s i c a l and c h e m i c a l t e s t s . 3 0 5 s p r o u t i n g on q u a l i t y , dextrinizing
units
To c l a r i f y
amylase a c t i v i t y per
{DU)
gram)
i n
i n field-sprouted
s p o n g e c a k e and by r e l a t e d the effect of f i e l d
soft
white
fractions
of
flour
seven
a n d r e d d o g 1.0%. typical.
wheat
lo%,
composites
clear flour
were:
patent
1 5 % , b r a n 25.8%,
flour
a c t i v i t y was r e l a t i v e l y l o w f o r 4 5 % p a t e n t ,
and c l e a r f l o u r s and r e l a t i v e l y h i g h f o r b r a n ,
and r e d dog.
45%,
s h o r t s 3.2%,
Ash a n d p r o t e i n c o n t e n t s o f t h e f r a c t i o n s w e r e
a-Amylase
midpatent,
36.2%) and i n
Average y i e l d s o f t h e corresponding m i l l
m i l l fractions.
midpatent
4.13
winter-wheat
-
composites w i t h t y p i c a l l e v e l s o f f i e l d s p r o u t i n g (0 their
-
was d e t e r m i n e d ( 0
shorts,
When a - a m y l a s e a c t i v i t y i n DU p e r g r a m o f e a c h m i l l
f r a c t i o n was c a l c u l a t e d as DU p e r g r a m o f w h e a t ,
based on y i e l d ,
a c t i v i t y i n c r e a s e d l i n e a r l y i n each m i l l f r a c t i o n w i t h i n c r e a s i n g wheat
a-amylase.
When e x p r e s s e d a s p e r c e n t o f w h e a t DU,
bran
a c c o u n t e d f o r 4 2 % o f t h e a - a m y l a s e a c t i v i t y , p a t e n t f l o u r f o r 32%, s h o r t s f o r 9%, c l e a r f l o u r f o r 8%, m i d p a t e n t f l o u r f o r 7 % , a n d r e d d o g f o r 2%.
S p o n g e - c a k e v o l u m e i n c r e a s e d f r o m 1,280
c o n t r o l t o 1,315 cm3 f o r t h e f l o u r DU g-'.
cm3 f o r t h e
m i l l e d f r o m wheat c o n t a i n i n g 0.35
T h e r e a f t e r , v o l u m e d e c r e a s e d r a p i d l y t o 9 0 8 c m 3 a s DU p e r
gram o f wheat i n c r e a s e d t o
4.13.
A t
l e a s t 0.2
DU g ' l
of
wheat
( a b o u t 2.5% s p r o u t e d w h e a t ) was c o n s i d e r e d a d o u b l y s a f e l e v e l t h a t w o u l d have no a d v e r s e e f f e c t on sponge-cake q u a l i t y . volume per cent o f sprouted wheat, number, field
falling
and gas p r o d u c t i o n w e r e a l l f u n c t i o n s o f t h e a - a m y l a s e
a c t i v i t y o f wheat o r f l o u r . of
Sponge-cake
amylograph v i s c o s i t y ,
sprouting
should
I t was c o n c l u d e d t h a t d i f f e r e n t l e v e l s not
be
simulated
u n s p r o u t e d w h e a t w i t h a h i g h l y s p r o u t e d one,
by
supplementing
especially not w i t h a
highly malted barley. A m o d i f i e d p r o c e d u r e has been r e p o r t e d f o r use o f t h e P e r k i n Elmer Model 191 g r a i n amylase analyser i n d e t e r m i n i n g low l e v e l s o f a-amylase i n wheats and f l o u r s . 3 0 6
The a n a l y s e r was e m p l o y e d a t i t s
h i g h e s t p o s s i b l e s e n s i t i v i t y t o determine t h e a-amylase L i m i t d e x t r i n (0.5%) was u s e d as s u b s t r a t e ,
levels.
B-
and t h e r a t e o f d e c r e a s e
i n t u r b i d i t y e f f e c t e d b y t h e e n z y m e was f o l l o w e d o n a r e c o r d e r a t t a c h e d t o t h e g r a i n amylase analyser.
Straight-line relationships
were f o u n d b e t w e e n i n c r e a s e s i n c o n c e n t r a t i o n o f enzyme a n d r a t e s o f decrease i n nephelos p e r minute. for
a s s a y i n g a-amylase
The m e t h o d was f o u n d s a t i s f a c t o r y
l e v e l s i n wheats r a n g i n g i n Hagberg f a l l i n g
n u m b e r s f r o m 240 t o 460 s and i n a m y l o g r a p h v i s c o s i t i e s f r o m 125 t o
473
6: Enzymes 925
The
BU.
method
has
some
advantages
over
an
automated
f l u o r i m e t r i c p r o c e d u r e f o r a-amylase. An i n t e r l a b o r a t o r y i n v e s t i g a t i o n of
the Perkin-Elmer
model 191
g r a i n a m y l a s e a n a l y s e r h a s been c o n d u c t e d i n t h e U n i t e d Kingdom.307 The i n s t r u m e n t was c a l i b r a t e d a g a i n s t t h e F a r r a n d m e t h o d f o r b r e a d f l o u r s on t h e amylase-2 s c a l e o n l y . amylase-2 u n i t s t o 1 F a r r a n d unit. time
T h e c o n v e r s i o n f a c t o r was 1 7 The c a l i b r a t i o n was s t a b l e w i t h
and between t h r e e l a b o r a t o r i e s .
excellent,
The r e p r o d u c i b i l i t y
w i t h a s t a n d a r d d e v i a t i o n o f 0.035P
was
a t P amylase-2 u n i t s .
l e v e l c o u l d b e o b t a i n e d i n 1 0 m i n compared t o 2-5 h by
An a - a m y l a s e
t h e F a r r a n d method. Plant
a-amylases
have
been f o u n d t o
be s e n s i t i v e t o
high
c a l c i u m - i o n c o n c e n t r a t i o n i n ~ i t r o . ~ 'A ~t 250 m M n e a r l y 70% o f t h e a c t i v i t y o f maize, i n contrast
wheat,
and b a r l e y a - a m y l a s e s was i n h i b i t e d ,
but
h o g p a n c r e a t i c and b a c t e r i a l enzymes w e r e n o t a f f e c t e d .
I n h i b i t i o n o f m a i z e a - a m y l a s e was c o m p e t i t i v e , r e v e r s e d b y d i a l y s i s , a n d m a r k e d l y i n c r e a s e d by t h e p r e - i n c u b a t i o n o f t h e enzyme w i t h Ca2+ before
t h e a d d i t i o n of
a p p a r e n t number of
the substrate.
85 m M and 1.1, r e s p e c t i v e l y . of a co-operativity
The a p p a r e n t
Ki
and t h e
Ca2+ bound p e r enzyme m o l e c u l e w e r e f o u n d t o b e The l a t t e r v a l u e i n d i c a t e s t h e a b s e n c e
phenomenon.
The i n t e r a c t i o n b e t w e e n t h e m a i z e
a - a m y l a s e a n d Ca2+ was a l s o s t u d i e d a s a f u n c t i o n o f t e m p e r a t u r e . The
efficiency
of
Ca2+
interaction
decreases
with
increasing
t e m p e r a t u r e and as e x p e c t e d t h e o v e r a l l r e a c t i o n was e x o t h e r m i c w i t h a A t j v a l u e o f -13.8 k c a l m o l - l . The v a l u e s f o r A G a n d A 2 for the f o r m a t i o n o f C a - e n z y m e c o m p l e x a t 25OC w e r e f o u n d t o b e 1.6 k c a l mo1-l
and -51.6
negative
A2
c a l mol'l
deg",
respectively.
value suggested t h a t
The r e l a t i v e l y l a r g e
t h e enzyme m o l e c u l e u n d e r g o e s
m a r k e d c o n f o r m a t i o n a l change d u r i n g i t s i n t e r a c t i o n w i t h Ca2+. A d e t a i l e d s t u d y o f t h e i n v i t r o i n t e r a t i o n b e t w e e n Z n 2 + a n d a-
a m y l a s e s f r o m d i f f e r e n t o r i g i n s h a s shown t h a t u n d e r p h y s i o l o g i c a l c o n d i t i o n s p l a n t a-amylases
a r e s t r o n g l y i n h i b i t e d b y z i n c ions.309
T h e i n h i b i t i o n i s pH d e p e n d e n t a n d c a n b e r e v e r s e d b y e x t e n s i v e d i a l y s i s against buffer. t r e a t m e n t w i t h H4 e d t a ,
I n addition,
i t can a l s o be removed by t h e
p a r t i c u l a r l y a t l o w pH v a l u e s .
However,
e d t a i t s e l f does n o t have any e f f e c t o n e n z y m a t i c a c t i v i t y . inhibition
for
maize
c o m p e t i t i v e and t h e
Ki
a-amylase was 1 0 mM.
at
i t s
optimum
pH
The H i l l c o e f f i c i e n t
(5.5)
H4 The
was
v a l u e was 1,
s u g g e s t i n g t h a t Zn2+ i n t e r a c t s w i t h a - a m y l a s e i n a s i m p l e manner w i t h no c o - o p e r a t i v i t y .
The p u r i f i c a t i o n by a f f i n i t y
chromatography o f a-amylase
from
474
Carbohydrate Chemistry
t h e c o t y l e d o n s o f g e r m i n a t i n g V i q n a munqo s e e d s h a s b e e n r e p o r t ed.310
mungo c o t y l e d o n s , t h e a - a m y l a s e a c t i v i t y i n c r e a s e d
I n V.
i n t h e dark,
m a r k e d l y d u r i n g g e r m i n a t i o n a t 27'C o f o t h e r a m y l a s e s was v e r y l o w .
while the a c t i v i t y
The a - a m y l a s e was p u r i f i e d f r o m 4-
d a y - o l d c o t y l e d o n s by a f f i n i t y c h r o m a t o g r a p h y on e p o x y - a c t i v a t e d Sepharose 68 s u b s t i t u t e d w i t h B - c y c l o h e p t a - a m y l o s e c h r o m a t o g r a p h y on B i o - G e l P-200.
and by column
G e l f i l t r a t i o n and p o l y a c r y l a m i d e
g e l e l e c t r o p h o r e s i s showed t h a t t h e enzyme e x i s t s m o s t l y as monomer (43,000
daltons),
further
inactivation. dialysis
Ca2+ p r o t e c t e d t h e a - a m y l a s e
Incubation of
against
activity. of
b u t p a r t i a l l y aggregates t o form dimer,
multimers. 10
mM
the
enzyme
H4 e d t a
with
resulted
i n
5 a
trimer,and
against
mM
heat
or loss o f
H4 e d t a
50-90%
The i n a c t i v a t i o n was p a r t i a l l y r e v e r s e d by t h e a d d i t i o n
Ca2+.
Other p r o p e r t i e s ,
s u c h as t h e a m i n o a c i d c o m p o s i t i o n ,
Em
v a l u e , pH o p t i m u m , a n d a c t i v a t i o n e n e r g y w e r e s i m i l a r t o t h o s e o f o t h e r p l a n t a-amylases. A B a c i l l u s s u b t i l i s t r a n s f o r m a n t p r o d u c i n g t h e r m o s t a b l e a-
a m y l a s e h a s been i s o l a t e d u s i n g DNA f r o m a t h e r m o p h i l i c b a c t e r i u m , T h e r m o p h i l e V2.311 with
a
rabbit
s t r u c t u r a l gene f o r different
The e x t r a c e l l u l a r
antiserum
against
a-amylase B.
t h e t h e r m o s t a b l e a-amylase
l o c u s f r o m B. s u b t i l i s a - a m y l a s e
The t r a n s f o r m a n t was n o t t h e r m o p h i l i c ,
d i d not
subtilis
crossreact
a-amylase.
The
was i n t e g r a t e d a t a
and was
l i n k e d t o pyrA.
and i t s u p p e r t e m p e r a t u r e f o r
g r o w t h was s i m i l a r t o t h a t o f t h e h o s t b a c t e r i u m . The N - t e r m i n a l a m i n o a c i d s e q u e n c e o f a s a c c h a r i f y i n g - t y p e amylase f r o m B a c i l l u s s u b t i l i s var.
a-
a m y l o s a c c h a r i t i c u s has been
i n v e s t i g a t e d a n d a c o m p a r i s o n made w i t h t h a t o f l i q u e f y i n g - t y p e
a-
amy l a s e . 312
i n vitro
The
synthesis
of
a-amylase-like
products
i n
Bacillus s u b t i l i s RNA-directed heterologous protein-synthesizing system
has
been described.313
a-Amylase-like
synthesized i n a heterologous c e l l - f r e e prepared from Escherichia c o l i . anti-a-amylase
p r o t e i n s were
p r o t e i n - s y n t h e s i z i n g system
The p r o t e i n s w e r e p r e c i p i t a b l e w i t h
s e r u m a n d d e t e c t e d o n l y w h e n RNA e x t r a c t e d f r o m a-
a m y l a s e p r o d u c i n g 8. i n v i t r o a-amylase-like
s u b t i l i s c e l l s was u s e d a s m e s s e n g e r .
h a v i n g m o l e c u l a r w e i g h t s o f 30,000 By t h e u s e o f
The
p r o d u c t s seemed t o c o n s i s t o f t w o c o m p o n e n t s and 13,000.
2-deoxy-Q-glucose,
a non-metabolizable
catabolic
o f c e r t a i n y e a s t enzymes, m u t a n t s d e r e p r e s s e d and hyperproductive w i t h r e s p e c t t o a-amylase have been o b t a i n e d i n v e r y h i g h p l a t e y i e l d s f r o m t h e y e a s t and L i p o m y c e s k o n ~ n e n k o a e . ~ ~ repressor
475
6: Enzymes A
method
for
the
automatic
measurement
of
a-amylase
and
g l u c o a m y l a s e a c t i v i t i e s d u r i n g f e r m e n t a t i o n h a s b e e n developed.314 S o l u b l e s t a r c h d y e d w i t h R e m a z o l B r i l l i a n t O r a n g e was u s e d a s t h e s u b s t r a t e f o r a-amylase glucoamylase.
and 4 - n i t r o p h e n y l a-P-glucopyranoside
for
The same a u t o m a t i c a n a l y s i s s y s t e m c o u l d b e u s e d f o r
b o t h o f t h e s e enzymes b e c a u s e t h e r e a c t i o n p r o d u c t s w e r e m e a s u r e d a t the
same
wavelength.
Simultaneous
respective substrate presence
of
determination.
was
a-amylase
enabled
did not
pick-up
by
of
enzyme
using two
interfere
and
samplers.
with the
the The
glucoamylase
A b s o l u t e v a l u e s f o r a-amylase a c t i v i t y were o b t a i n e d
using a mathematical correction.
M o n i t o r i n g of
t h e s e enzymes was
accomplished during m i c r o b i a l fermentation. B a c i l l u s a m y l o l y t i c u s produced a-amylase,
p u l l u l a n a s e , and a-e-
glucosidase.
By s e l e c t i o n o f c a r b o n s o u r c e i n t h e g r o w t h medium, a-
p-glucosidase
was p r o d u c e d p r e f e r e n t i a l l y and w i t h e x c l u s i o n o f t h e
other two activities.18* The e f f e c t o f
glucose o n t h e e n z y m e s i n v o l v e d i n t h e
d e g r a d a t i o n o f a r e s e r v e Q - g l u c a n i n P o l y p o r u s c i r c i n a t u s h a s been studied.315
The l e v e l s o f p h o s p h o r y l a s e a c t i v i t y , a m y l a s e ,
amylo-1,6-Q-glucosidase
and
were f o u n d t o be r e g u l a t e d by Q - g l u c o s e
concentration. The c o p r o d u c t i o n o f s e v e r a l exoenzymes i n B a c i l l u s s u b t i l i s h a s been described.316 particularly
M u t a n t s d e f e c t i v e i n many o f t h e s e e n z y m e s ,
a-amylase
and a l k a l i n e and n e u t r a l p r o t e a s e ,
were
i s o l a t e d i n o r d e r t o p r o v i d e t o o l s f o r a study o f t h e mechanism o f secretion o f exoproteins. The
cloning
Bacillus subtilis
of
thermostable
phage
p l l
has
a-amylase
been
achieved
gene
using
a
using method
c o n s i s t i n g o f t w o r e p e t i t i o n s o f prophage t r a n s f ~ r m a t i o n . ~ ~ ’ The m o l e c u l a r w e i g h t o f A s p e r q i l l u s o r y z a e a - a m y l a s e ( T a k a a m y l a s e A ) h a s b e e n e s t i m a t e d a t 51,000 high-pressure
silica-gel
s c a t t e r i n g technique.318
+,
500 by t h e c o m b i n e d u s e o f
c h r o m a t o g r a p h y and a l o w - a n g l e
laser-light-
The s t u d y was c a r r i e d o u t p a r t l y t o a s s e s s
t h e performance o f t h e combined technique,
and r e s u l t s o b t a i n e d
i n d i c a t e t h a t i t i s p r o m i s i n g as a m e t h o d t o d e t e r m i n e p r o t e i n m o l e c u l a r w e i g h t b o t h a c c u r a t e l y and q u i c k l y . An a - a m y l a s e
from
S t r e p t o m y c e s p r a e c o x h a s b e e n p u r i f i e d and
i t s c h a r a c t e r i s t i c a c t i o n , t h e c o n v e r s i o n o f m a l t o t r i o s e (G3) t o m a l t o s e (G2) w i t h o u t a p p r e c i a b l e f o r m a t i o n o f P - g l u c o s e ( G l ) , i n ~ e s t i g a t e d . ~ ~ I’ s o e l e c t r i c preparation
after
f o c u s i n g showed
chromatographic
separation
that
the
comprises
was
enzyme three
476
Carbohydrate Chemistry
isoenzymes.
The p r e p a r a t i o n f r o m t h e g l y c o g e n - a d s o r p t i o n
procedure
s h o w e d t h e h i g h e s t s p e c i f i c a c t i v i t y o f any p r e p a r a t i o n o f t h i s enzyme e v e r o b t a i n e d .
Product a n a l y s i s w i t h u n i f o r m l y
l a b e l l e d G3
r e v e a l e d t h a t a t a h i g h c o n c e n t r a t i o n ( 1 8 mM) o f G3 much more G2 i s produced than G1 ( t h e p r o d u c t r a t i o G2/G1
i s o v e r 201, w h i l e a t a
l o w e r c o n c e n t r a t i o n ( 1 0 u M ) t h e r e a c t i o n m i x t u r e was c o m p o s e d o f n e a r l y e q u i m o l a r amounts o f Q - g l u c o s e and m a l t o s e .
Based on t h e
G3 i n a d d i t i o n t o t h e
product analysis o f reducing end-labelled
a b o v e f i n d i n g s , t h e f o l l o w i n g c o n v e r s i o n m e c h a n i s m was p r o p o s e d : Streptomyces a-amylase
catalyses t r a n s g l y c o s y l a t i o n t o produce
m a l t o t e t r a o s e (G4) as a t r a n s i e n t p r o d u c t degraded reaction,
into
two
2. two
molecules
of
G2 b y
which i s immediately
a subsequent
hydrolytic
m o l e c u l e s o f G3 a r e c o n v e r t e d i n t o t h r e e
molecules o f maltose w i t h o u t a p p r e c i a b l e f o r m a t i o n o f @-glucose. T h e p r o d u c t i o n o f e x t r a c e l l u l a r a m y l a s e b y P. been
s t u d i e d under
different
cultural
omnivorum has
conditions.320
Maximum
m y c e l i a l g r o w t h and a m y l a s e s y n t h e s i s w e r e f o u n d i n b a s a l s y n t h e t i c m e d i u m c o n t a i n i n g 2% g l y c o g e n a s t h e c a r b o n s o u r c e .
Significant
amounts o f a m y l a s e and g r o w t h w e r e o b t a i n e d i n m e d i a c o n t a i n i n g other
carbon
compounds
(1 + 4 ) - a - ~ - g l u c o s i d i c l i n k a g e s .
with
O p t i m u m t e m p e r a t u r e s f o r m y c e l i a l g r o w t h and a m y l a s e s y n t h e s i s w e r e 28'
and 3 O o C ,
tested,
respectively.
O f
a l l the nitrogenous
compounds
ammonium n i t r a t e s u p p o r t e d t h e maximum m y c e l i a l g r o w t h and
amylase p r o d u c t i o n .
Increasing the concentration of starch i n the
b a s a l medium i n c r e a s e d g r o w t h as w e l l as t h e enzyme s y n t h e s i s . S c h w a n n i o m y c e s c a s t e l l i i and E n d o m y c o p s i s f i b u l i g e r o h a v e b e e n f o u n d t o p r o d u c e e x t r a c e l l u l a r a m y l a s e ( s ) when g r o w n o n v a r i o u s c a r b o n s o u r c e s a n d a t d i f f e r e n t pH v a l u e s . 3 2 1 showed s i g n i f i c a n t
maltose or soluble starch. glucose, cellobiose, ethanol,
trehalose,
(a-
meletitose, raffinose,
F r e e p - g l u c o s e i n t h e c u l t u r e medium a p p a r e n t l y
i n h i b i t e d enzyme s y n t h e s i s . and a m y l a s e p r o d u c t i o n was 4.5 S.
O f the other substrates tested
sucrose,
g l y c e r o l ) d i f f e r e n c e s were f o u n d r e g a r d i n g g r o w t h and
amylase p r o d u c t i o n .
for
Both yeast species
amylase s y n t h e s i s i n t h e presence o f e i t h e r
The pH r a n g e a l l o w i n g m a x i m a l g r o w t h
-
6.0
f o r E.
f i b u l i q e r a and 5 . 5
-
7.0
castellii.
The p r o d u c t i o n o f a - a m y l a s e b y a s t r a i n o f B a c i l l u s s t e a r o -
--thermophilus
isolated
tryptone-maltose a-Amylase
from
leaf
medium a t 55'12
litter
was
investigated i n a
i n b a t c h and c h e m o s t a t c u l t u r e . 3 2 2
p r o d u c t i o n i n c h e m o s t a t c u l t u r e was i n f l u e n c e d b y t h e
g r o w t h r a t e t h r o u g h o u t t h e d i l u t i o n r a t e r a n g e used.
477
6: Enzymes B-Amylases
18
Some
properties
S t r e p t o m y c e s sp.
280
of
produced s e v e r a l k i n d s o f B, B',and
C).
amylase
inhibitor
h a v e been r e p o r t e d . 2 7 7
A
produced
by
S t r e p t o m y c e s sp.
amylase i n h i b i t o r s
280
( a m y l a s e i n h i b i t o r A,
Two a m y l a s e i n h i b i t o r s ( d e s i g n a t e d a s A I - A 1
and A I -
A2) w e r e o b t a i n e d f r o m an a m y l a s e i n h i b i t o r A f r a c t i o n b y p a p e r chromatography. than AI-A2
AI-A1
i n h i b i t e d muscle phosphorylase
A 2 was n o t .
5
i n h i b i t o r y a c t i v i t y against phosphorylase
or
hog
inhibitory
pancreatic
a-amylase,
but
They l o s t t h e i r
after treatment with
they
showed
-
Both
w e r e composed o f q - g l u c o s e and a b a s i c m o i e t y w h i c h
gave a p o s i t i v e n i n h y d r i n r e a c t i o n (1.3% n i t r o g e n ) . w e i g h t s of
increased
a c t i v i t y toward p o r c i n e s m a l l i n t e s t i n a l sucrase.
and A I - A 2
AI-A1
much more
B o t h a m y l a s e i n h i b i t o r s c o n t a i n e d c a r b o h y d r a t e and were
h y d r o l y s e d b y some k i n d s o f a m y l a s e s o r a c i d s . acids
a
and was h y d r o l y s e d by s w e e t - p o t a t o @ - a m y l a s e w h e r e a s A I -
The
molecular
and A I - A 2
w e r e e s t i m a t e d t o b e a p p r o x i m a t e l y 1,300
An a s s a y m e t h o d f o r
a m y l a s e a c t i v i t i e s u s i n g NAD-dependent
AI-A1
1,500.
m a l t o s e dehydrogenase has
It
been d e s c r i b e d . 2 6 9
was
demonstrated
t h a t t h e a c t i v i t i e s o f a-amylase and,B-amylase c o u l d be measured w i t h dependent m a l t o s e dehydrogenase by t h e e n d - p o i n t and by t h e r a t e assay method.
assay
method
One u n i t o f t h e a c t i v i t y o f a - a m y l a s e
o r B-amylase was d e f i n e d as t h e amount of t h e enzyme w h i c h p r o d u c e d one u m o l o f 25'C
maltose equivalent product from s u b s t r a t e per minute a t
u n d e r t h e assay c o n d i t i o n s . A
comparative
invertebrates invertebrates, Arthropoda,
has
study been
of
carbohydrase a c t i v i t i e s
performed.299
belonging t o 7 types,
Mollusca,
103
Spongia,
Echinodermata,
species
i n
marine
of
marine
Coelenterata,
Chordata,
were
Annelid,
tested for
l a m i n a r i n a s e , c e l l u l a s e , and a m y l a s e a c t i v i t i e s . The m o d i f i c a t i o n of
wheat B - a m y l a s e by p r o t e o l y t i c enzymes h a s
been investigated.323
Two B - a m y l a s e c o m p o n e n t s ,
separated from
flour
wheat
by
ion-exchange
I a n d 11,
were
chromatography.
C o m p o n e n t Ic o n t a i n e d t h r e e m a i n f o r m s a n d c o m p o n e n t I 1 c o n t a i n e d o n e f o r m when
a n a l y s e d by p o l y a c r y l a m i d e s l a b e l e c t r o f o c u s i n g .
P a p a i n o r a m a l t e d wheat e x t r a c t
c o n v e r t e d t h e t h r e e B-amylases
i n
c o m p o n e n t I t o f i v e f o r m s w i t h m o r e b a s i c PI v a l u e s a n d t h e o n e Ba m y l a s e i n component I 1 t o t h r e e f o r m s t h o s e a r i s i n g from
degradation of
with P vI alues i d e n t i c a l t o
component
I.
The new B - a m y l a s e s
478
Carbohydrate Chemistry
r e s u l t i n g from p r o t e o l y t i c a t t a c k were i d e n t i c a l i n P I v a l u e s t o 6a m y l a s e s i n g e r m i n a t i n g wheat, w h i c h i n d i c a t e d t h a t t h e mechanism of f o r m a t i o n of such enzymes i s 2 l i m i t e d p r o t e o l y t i c d e g r a d a t i o n . An i m m u n o c h e m i c a l s t u d y h a s been made of t w o B - a m y l a s e d e f i c i e n t i n b r e d l i n e s of r y e ( S e c a l e c e r e a l e ) , t h e k e r n e l s of which d i s p l a y e d a very low l e v e l of 6-amylase a c t i v i t y (1-3% of t h e l e v e l s g e n e r a l l y found i n rye) i n comparison w i t h a t h i r d normal l i n e . 3 2 4 An a n t i - w h e a t B-amylase i m m u n e serum which c r o s s - r e a c t e d w i t h t h e r y e enzyme a n t i - w h e a t @-amylase immune serum absorbed t h e 6-amylase a c t i v i t y i n t h e t h r e e l i n e s . Comparably s m a l l amounts of e n z y m a t i c a n t i g e n corresponded t o t h e s m a l l l e v e l s of a c t i v i t y d e t e c t e d i n t h e enzyme-deficient l i n e s . N e i t h e r t h e l e v e l of a c t i v i t y nor t h e amount of e n z y m a t i c a n t i g e n were n o t a b l y changed upon g e r m i n a t i o n . The r e s u l t s i n d i c a t e d t h a t t h e r e d u c e d a c t i v i t y i s d u e n e i t h e r t o t h e p r e s e n c e of an i n h i b i t o r n o r t o t h e p r o d u c t i o n o f i n a c t i v e enzymes. Germination can proceed n o r m a l l y w i t h o u t l a t e r p r o d u c t i o n of B-amylase. The b e h a v i o u r of s u l p h y d r y l g r o u p s of s o y b e a n 6 - a m y l a s e h a s Two s u l p h y d r y l been i n v e s t i g a t e d b y c h e m i c a l m o d i f i c a t i o n . 3 2 5 g r o u p s o u t of a t o t a l of f i v e i n t h e n a t i v e enzyme r e a c t e d w i t h 5 , 5 ’- d i t h i o b i s - ( 2 - n i t r o b e n z o i c a c i d ) , i o d o a c e t am i d e , o r monoiodoacetate a t high i o n i c s t r e n g t h , accompanied b y i n a c t i v a t i o n of t h e enzyme. A t low i o n i c s t r e n g t h , only one s u l p h y d r y l group was a c c e s s i b l e t o 5,5’-dit h i o b i s -( 2-ni t r oben z o i c a c i d 1 w i t h o u t inactivation. By u s i n g the reaction w i t h iodoacetamide, the d i s s o c i a t i o n c o n s t a n t s of t h e s e t w o s u l p h y d r y l g r o u p s and r a t e c o n s t a n t s f o r t h e r e a c t i o n were determined. The most r e a c t i v e s u l p h y d r y l g r o u p , which was i n d e p e n d e n t of t h e i n a c t i v a t i o n , r e a c t e d w i t h m o n o i o d o a c e t a t e 185 t i m e s f a s t e r t h a n t h e e s s e n t i a l s u l p h y d r y l group. Next, s e l e c t i v e m o d i f i c a t i o n of one s u l p h y d r y l group w i t h monoiodoacetate was c a r r i e d o u t . T h e modified enzyme, having a c t i v i t y e q u a l t o t h a t of t h e n a t i v e enzyme, was p u r i f i e d b y i o n -exchange column chromatography. I t was s u c c e s s i v e l y t r e a t e d w i t h 5,5’-dithiobis-(2-nitrobenzoic a c i d ) or iodoacetamide i n order t o a c h i e v e s e l e c t i v e m o d i f i c a t i o n of t h e e s s e n t i a l s u l p h y d r y l group. The e n z y m e m o d i f i e d f u r t h e r w i t h 5 , 5 ’ - d i t h i o b i s - ( 2 n i t r o b e n z o i c a c i d ) was f u l l y r e a c t i v a t e d by 2 - m e r c a p t o e t h a n o l t r e a t m e n t . I t a l s o r e c o v e r e d 65% of t h e o r i g i n a l e n z y m a t i c a c t i v i t y on t r e a t m e n t w i t h c y a n i d e b u t o n l y 7 % w i t h s u l p h i t e . M a l t o s e and c y c l o h e x a - a m y l o s e p r o t e c t e d t h e e s s e n t i a l s u l p h y d r y l g r o u p from modification, the former being effective. The u l t r a v i o l e t
479
6: Enzymes a b s o r p t i o n s p e c t r u m o f soybean
was changed by m o d i f i c a t i o n o f t h e
e s s e n t i a l s u l p h y d r y l g r o u p , a n d t h e s p e c t r u m was a l s o c h a n g e d b y the binding of
maltose.
The
change i n d u c e d by
maltose
was
It
i n f l u e n c e d by m o d i f i c a t i o n o f t h e e s s e n t i a l s u l p h y d r y l g r o u p .
was c o n c l u d e d t h a t t h e e s s e n t i a l s u l p h y d r y l g r o u p o f s o y b e a n Ba m y l a s e does n o t p a r t i c i p a t e i n t h e c a t a l y s i s b u t i s s i t u a t e d n e a r t h e b i n d i n g s i t e o f maltose. The c o n f o r m a t i o n a l p r o p e r t i e s o f s o y b e a n 8-amylase have been i n v e s t i g a t e d by t h e c i r c u l a r - d i c h r o i s m p r o b e and measurement of enzyme a c t i v i t y . 3 2 6
The enzyme e x h i b i t e d a p o s i t i v e c i r c u l a r -
d i c h r o i s m b a n d a t 1 9 2 nm, n e a r 2 1 0 nm.
a n e g a t i v e b a n d a t 2 2 2 nm,
and a s h o u l d e r
Analysis o f the spectrum i n the f a r - u l t r a v i o l e t
i n d i c a t e d t h e p r e s e n c e o f a p p r o x i m a t e l y 30% o f a - h e l i x B-pleated sheet,
zone
and 5-10% o f
t h e r e s t o f t h e p o l y p e p t i d e main chain possessing
aperiodic structure.
I n t h e n e a r - u l t r a v i o l e t r e g i o n , t h e enzyme
p r o t e i n s h o w e d a t l e a s t s i x p o s i t i v e p e a k s a t 259, 265, 273, 292; a n d 2 9 7 nm.
281,
T h e p o s i t i v e b a n d s a t 2 9 2 a n d 2 9 7 nm r e m a i n e d
u n a l t e r e d on a c e t y l a t i o n o f t h e enzyme b y N - a c e t y l i m i d a z o l e and w e r e a s s i g n e d t o I - t r y p t o p h a n y l chromophores.
These b a n d s w e r e a f f e c t e d
i n i n t e n s i t y i n t h e presence o f m a l t o s e or cyclohepta-amylose,
which
i n d i c a t e s t h a t some t r y p t o p h a n r e s i d u e s a r e s i t u a t e d a t t h e b i n d i n g sites.
The n a t i v e c o n f o r m a t i o n o f s o y b e a n B-amylase was f o u n d t o b e
s e n s i t i v e t o pH v a r i a t i o n ( b e l o w dodecyl sulphate,
sodium
pH 5 a n d a b o v e pH 101,
guanidinium chloride,
and h e a t i n g t o 50-55'C.
C o m p l e t e d i s o r g a n i z a t i o n o f t h e s e c o n d a r y s t r u c t u r e was a t t a i n e d b y 6 M guanidinium chloride.
S o d i u m d o d e c y l s u l p h a t e was e f f e c t i v e i n
d i s t u r b i n g t h e t e r t i a r y s t r u c t u r e o f t h e enzyme b u t d i d n o t significantly
t h e secondary s t r u c t u r e .
affect
E n z y m a t i c i n a c t i v a t i o n was
p a r a l l e l e d by t h e d e c r e a s e o f c i r c u l a r - d i c h r o i s m
bands i n t h e n e a r -
u l t r a v i o l e t r e g i o n as p r o d u c e d b y t h e d e n a t u r a n t s .
I t was c o n c l u d e d
t h a t t h e u n i q u e l y f o l d e d s t r u c t u r e o f t h e enzyme c o n t a i n s some l e s s r i g i d
domains
interactions,
and
a
r i g i d
core
stabilized
electrostatic interactions,
Subsite a f f i n i t i e s
of
by
hydrophobic
and h y d r o g e n bonds.
s o y b e a n B - a m y l a s e h a v e been e v a l u a t e d by
a new m e t h o d t o e l u c i d a t e t h e e f f e c t o f m u l t i p l e a t t a c k . 3 2 7 a n a l y s i s of
the reducing-end-labelled
t h a t G Z and G 3 w e r e d e g r a d e d t h r o u g h t h e m u l t i p l e - a t t a c k 0.02
M acetate buffer,
pH 5.4
r a t e constants,
(ko/Km)eo,
obtained
the
from
a t 25'C.
f o r 2-mer
decrease
of
Product
m a l t o - o l i g o s a c c h a r i d e s showed pathway,
in
The a p p a r e n t f i r s t - o r d e r substrates
substrate
c h r o m a t o g r a p h y under t h e c o n d i t i o n o f { S ) <
Em.
(2
= 3
followed
-
7) were
by
paper
Using the constants
480
Carbohydrate Chemistry
and t h e c l e a v a g e d i s t r i b u t i o n o f G ? ,
t h e s u b s i t e a f f i n i t i e s (A1,
and A s > o f B-amylase
I t was r e v e a l e d t h a t t h e f i r s t
s u b s i t e of
were e v a l u a t e d .
soybean B - a m y l a s e h a d a l a r g e r s u b s i t e a f f i n i t y t h a n any
s u b s i t e s o f o t h e r amylases so f a r evaluated. seems
to
A4,
play
an
important
role
for
the
Thus t h e f i r s t s u b s i t e characteristic
action
p a t t e r n o f t h i s enzyme, n a m e l y e x c l u s i v e m a l t o s e - f o r m i n g a c t i o n . The frequency of
m u l t i p l e a t t a c k d e c r e a s e d a s e i t h e r o r b o t h pH a n d
t e m p e r a t u r e become u n f a v o u r a b l e f o r e n z y m i c a c t i v i t y . Endogenous g i b b e r e l l i n s
and amylase
a c t i v i t y have been
i n v e s t i g a t e d i n t a l l and d w a r f s t r a i n s o f r i c e (Oryza ~ a t i v a ) . ~ ' ~ Two t a l l a n d s e v e n d w a r f s t r a i n s o f r i c e w e r e s p r a y e d w i t h 0 pg m l - l
GA3.
Four dwarf s t r a i n s responded t o
o r 40
exogenous GA3 and
showed a m a r k e d l y l o w e r endogenous g i b b e r e l l i n c o n t e n t t h a n t h e t a l l strains,
w h i l e two dwarf
s t r a i n s d i d n o t respond t o GA3 a p p l i c a t i o n
and h a d c o n s i d e r a b l y h i g h e r t a l l ones.
Amylase
endogenous g i b b e r e l l i n l e v e l s t h a n t h e
activity
i n g e r m i n a t i n g seeds
showed a
s i g n i f i c a n t n e g a t i v e c o r r e l a t i o n w i t h t h e endogenous g i b b e r e l l i n content. B-Amylase-producing
micro-organisms
have been e f f e c t i v e l y
i s o l a t e d by u s i n g an a m y l a s e i n h i b i t o r ( S - A I ) . 3 2 8
Among f o r t y - t w o
s t r a i n s i s o l a t e d , o n e was f o u n d t o b e t h e m o s t p o t e n t B - a m y l a s e producing rnicro-organism, Two k i n d s o f B - a m y l a s e ,
and was
i d e n t i f i e d as B a c i l l u s p o l y m y x a .
I a n d 11, o b t a i n e d f r o m c u l t u r e b r o t h as
e l e c t r o p h o r e t i c a l l y homogeneous f o r m s ( m o l .
wts.
44,0001,
were v e r y
s i m i l a r t o each o t h e r i n t h e i r e n z y m a t i c p r o p e r t i e s e x c e p t t h e s m a l l difference
i n
isoelectric
point
( I pH 8.35,
A m y l a s e s I a n d I1 w e r e m o s t a c t i v e a t pH 7.5 b e t w e e n pH 4 a n d 9 f o r 1 5 h a t 37'C. i n h i b i t e d by 4 - c h l o r o n e s c u r i b e n z o a t e ,
B-
11 pH 8 . 5 9 ) . a t 45'C,
and s t a b l e
B o t h enzymes were s t r o n g l y and r e a c t i v a t e d by I - c y s t e i n e .
The a m i n o a c i d c o m p o s i t i o n s o f t h e enzymes w e r e s t u d i e d . The u s e o f u l t r a f i l t r a t i o n membrane s y s t e m s i n s t i r r e d - c e l l i n thin-channel
systems
for
i m m o b i l i z i n g enzyme
i n t r i n s i c and c r y s t a l l i n e B-amylase)
and
(sweet-potato
i n t h e h y d r o l y s i s of
sweet
p o t a t o t h r o u g h a c o n t i n u o u s o p e r a t i o n mode h a s b e e n s t u d i e d . 3 2 9 B o t h t h e f i l t r a t i o n r a t e and r e d u c i n g s u g a r s , p r o d u c e d as t h e r e s u l t o f enzymatic h y d r o l y s i s , decreased w i t h the f i l t r a t i o n time.
The
i m m o b i l i z e d enzymes i n t h e t h i n - c h a n n e l s y s t e m showed a much b e t t e r p e r f o r m a n c e compared t o t h a t o f c r y s t a l l i n e sweet-potato
i n t h e s t i r r e d - c e l l system.
6-amylase
Addition
t o t h e sweet p o t a t o i n c r e a s e d
both t h e f i l t r a t i o n r a t e and r e d u c i n g - s u g a r s c o n t e n t .
Alcoholic
f e r m e n t a t i o n o f t h e f i l t r a t e r e s u l t e d i n an a l c o h o l c o n t e n t o f 4.2%.
481
6: Enzymes This
represented
fermentation of
e f f i c i e n c y of 88%. The e f f e c t o f
Q-glucose
an
i n P o l y p r u s c i r c i n a t u s has been
The l e v e l s o f p h o s p h o r y l a s e a c t i v i t y ,
amylo-1,6-~-glucosidase
with
on t h e e n z y m e s i n v o l v e d i n t h e
d e g r a d a t i o n o f a r e s e r v e 6-8-glucan studied.315
95% o f t h e s u g a r s
amylase,
and
were f o u n d t o be r e g u l a t e d by Q - g l u c o s e
concentration.
19
Amylo-1,6-~-glucosidases
R e a c t i o n o f r a b b i t m u s c l e amylo-1,6-!-glucosidase/4-a-Qg l u c a n o t r a n s f e r a s e w i t h an a c t iv e s it e - d i r e c t e d i r r e v e r s i b l e i n h i b i t o r , 1- ? - d i m e t h y l a r s i n o - 1 - t h i o - B - ; - g l u c o p y r a n o s i d e , has been
-
i n ~ e s t i g a t e d . ~ ~ The ’ i n h i b i t o r r e a c t s w i t h t h e d e b r a n c h i n g enzyme t o i n a c t i v a t e b o t h t h e ;-glucosidase The
two
activities
are
lost
at
and t h e t r a n s f e r a s e a c t i v i t i e s . different
rates.
The
rate
of
i n a c t i v a t i o n o f each a c t i v i t y i s f i r s t o r d e r i n b o t h enzyme and i n h i b i t o r concentration.
a-Glucosidase
and c o m b i n e d B - g l u c o s i d a s e -
t r a n s f e r a s e a c t i v i t i e s a r e l o s t a t t h e same r a t e , i n d i c a t i n g t h a t the Q-glucosidase glycogen
i s rate-limiting
phosphorylase
l i m i t
for
the
dextrin.
combined a c t i o n
The
relative
rate
i n a c t i v a t i o n o f t h e n - g l u c o s i d a s e and t r a n s f e r a s e a c t i v i t i e s b y
dimethylarsino-1-thio-6-Q-glucopyranoside protein
concentration.
A t
high
enzyme
on of
1-5-
i s a function of
the
concentration,
the
t r a n s f e r a s e i s i n a c t i v a t e d t w i c e as r a p i d l y a s t h e c - g l u c o s i d a s e . A t l o w enzyme c o n c e n t r a t i o n t h e t r a n s f e r a s e i s i n a c t i v a t e d o n l y o n e -
h a l f as f a s t .
The r a t e o f i n a c t i v a t i o n o f b o t h a c t i v i t i e s a t h i g h
enzyme c o n c e n t r a t i o n i s much s l o w e r ,
i n d i c a t i n g t h a t aggregation o f
t h e p r o t e i n a t t h e s e c o n c e n t r a t i o n s a f f e c t s t h e a c c e s s i b i l i t y o f 1-
S-dimethylarsino-1-thio-B-Q-glucopyranoside The s i t e s o f
with t h e debrancher
series
of
t o the active sites.
1- S - d i m e t hy l a r s i n o -1 -t h io -6-Q -g l u c o p y r anos i d e r e a c t i o n
were deduced from t h e p r o t e c t i v e e f f e c t o f a
substrates
and
substrate
analogues.
Glycogen
phosphorylase l i m i t d e x t r i n p r o t e c t s both t h e Q-glucosidase
and
t r a n s f e r a s e a c t i v i t i e s f r o m i n a c t i v a t i o n by 1-2-dimethylarsino-1-
thio-B-Q-glucopyranoside. 2,2’,2’-nitrilotriethanol),
Bis-Tris,
specifically
a t the g-glucosidase
glucosidase
but
not
Cyclohexa-amylose,
the
(2,2-bis(hydroxymethyl)-
a reversible
inhibitor
active site,
transferase
which binds t o
the
against
which
binds
p r o t e c t s t h e Qinactivation.
polymer site,
has no
Carbohydrate Chemistry
482
p r o t e c t i v e e f f e c t a g a i n s t t h e a c t i o n o f 1-S-dimethylarsino-l-thio-BThe r e s u I t s i n d i c a t e t h a t 1- g - d i methy l a r s i n o - l -
Q-glucopyr anoside.
acts
thio-B-k-glucopyranoside
as
an
active-site-directed
i r r e v e r s i b l e i n h i b i t o r a t b o t h t h e Q - g l u c o s i d a s e and t h e t r a n s f e r a s e active sites
on t h e d e b r a n c h i n g enzyme b u t
binding site.
thio-B-P-glucopyranoside kcal
not
at
the
polymer-
The t e m p e r a t u r e e f f e c t on t h e l - g - d i m e t h y l a r s i n o - l -
AS+
mol-l,
inactivation r a t e i s quite large,
54
=
e.u.
These
values
suggest
= 35
A+!
that
d i m e t h y l a r s i n o -1- t h i o - B - ~ - g l u c o p y r a n o s i d e i n a c t i v a t i o n o f
1-2the
d e b r a n c h e r o c c u r s w i t h a s i g n i f i c a n t c o n f o r m a t i o n change.
20
Cellulases
A review of
c e l l u l a s e s and t h e i r b i o s y n t h e s i s and a p p l i c a t i o n s
.
h a s been p r o d u c e d 331 Two d i f f e r e n t
c e l l u l a s e s f r o m T r i c h o d e r m a v i r i d e have been
i m m o b i l i z e d on Sepharose by d e r i v a t i z a t i o n o f t h e enzyme w i t h a bifunctional graft
vinyl
( g l y c i d y l m e t h y l a c r y l a t e ) and s u c c e s s i v e
monomer
c o p o l y m e r i z a t i o n o f v i n y l enzyme on t h e s u p p o r t . 3 3 2
yields for 18OC,
with
a
vinyl
monomer:enzyme
r e a c t i o n t i m e o f 6 hours.
ratio
and i m m o b i l i z e d enzyme o f
b e e n c o m p a r e d ( E m v a l u e s 30 m M ) , f a c t o r o f 1.8. of
of
10.2
and
X
a
F e 2 + - H 2 0 2 was u s e d a s r e d o x i n i t i a t i o n
system i n t h e c o p o l y m e r i z a t i o n r e a c t i o n . the free
The b e s t
t h e d e r i v a t i z a t i o n r e a c t i o n h a v e been o b t a i n e d a t pH 8.3,
The k i n e t i c b e h a v i o u r o f
the CM-cellulose
and t h e
h y d r o l y s i s has
lmax v a l u e s d i f f e r e d by a
The i m m o b i l i z e d enzyme h a s b e e n r e u s e d i n a s e r i e s
hydrolysis cycles,
and no s i g n i f i c a n t a c t i v i t y d e c r e a s e n o r any
i n h i b i t i o n by m e t a b o l i t e s was o b s e r v e d . The
kinetic
principles
of
the
formation
of
a-glucose
and
c e l l o b i o s e i n t h e h y d r o l y s i s o f m i c r o c r y s t a l l i n e c e l l u l o s e under t h e a c t i o n o f c e l l u l a s e c o m p l e x e s f r o m e i g h t d i f f e r e n t s o u r c e s h a v e been s t u d i e d e ~ p e r i r n e n t a l l y . ' ~ By ~ successive a d d i t i o n o f i n d i v i d u a l component
of
B-!-glucosidase)
the
cellulase
complex
(endo-(1
t o t h e r e a c t i o n system,
o f enzymic h y d r o l y s i s o f
-+
4)-B-!-glucanase
and
the steps l i m i t i n g the r a t e
microcrystalline cellulose
were r e v e a l e d .
I t was shown t h a t i n m o s t o f t h e c a s e s s t u d i e d t h e s t e p d e t e r m i n i n g the
rate of
formation
of
!-glucose
with
the
participation
i n t e r m e d i a t e c e l l o b i o s e i s t h e a c t i o n o f B-g-glucosidase. one case (complex f r o m A s p e r q i l l u s f o e t i d u s , glucosidase)
i s
the
rate
of
formation
of
Only
of in
e n r i c h e d w i t h B-D,g-glucose
from
6: Enzymes
483
m i c r o c r y s t a l l i n e c e l l u l o s e l i m i t e d b y t h e a c t i o n of t h e e n d o - ( 1 + 4)-B-!-glucanase of t h e c e l l u l a s e complex. I n accordance w i t h the k i n e t i c p r i n c i p l e s d e v e l o p e d , i t was shown t h a t when an e x c e s s of Bt - g l u c o s i d a s e i s added t o t h e r e a c t i o n system t h e s t e p l i m i t i n g t h e r a t e of h y d r o l y s i s of m i c r o c r y s t a l l i n e c e l l u l o s e under t h e a c t i o n of a l l t h e c e l l u l a s e c o m p l e x e s s t u d i e d becomes t h e a t t a c k on t h e Under i n i t i a l i n s o l u b l e s u b s t r a t e b y e n d o - ( 1 * 4)-B-!-glucanase. t h e same c o n d i t i o n s , a l i n e a r c o r r e l a t i o n was f o u n d b e t w e e n t h e s t e a d y - s t a t e r a t e of f o r m a t i o n of ;-glucose from m i c r o c r y s t a l l i n e c e l l u l o s e under t h e a c t i o n of a l l t h e c e l l u l a s e complexes s t u d i e d , on t h e o n e h a n d , and t h e a c t i v i t y o f e n d o - ( 1 + 4 ) - B - Q - g l u c a n a s e i n t h e s e complexes, on t h e o t h e r . I t was shown t h a t t h e a c t i o n of a l l t h e c e l l u l a s e complexes s t u d i e d ( s e l e c t e d r a t h e r a r b i t r a r i l y ) i s d e s c r i b e d b y e s s e n t i a l l y t h e same k i n e t i c p r i n c i p l e s , which i s e v i d e n c e o f t h e same m e c h a n i s m s of t h e h y d r o l y s i s of i n s o l u b l e c e l l u l o s e b y c e l l u l a s e p r e p a r a t i o n s of d i f f e r e n t o r i g i n . A simple procedure t h a t uses a cellulose-enriched c u l t u r e s t a r t e d from s e w a g e s l u d g e h a s been d e v e l o p e d f o r p r o d u c i n g c e l l u l o l y t i c enzymes and c o n v e r t i n g c e l l u l o s e t o a c e t i c a c i d r a t h e r t h a n t o m e t h a n e and c a r b o n d i o x i d e . 3 3 3 I n t h i s procedure, t h e c u l t u r e w h i c h c o n c e r t s c e l l u l o s e t o C H 4 and C02 was mixed w i t h a s y n t h e t i c m e d i u m and c e l l u l o s e and h e a t e d t o 8OoC f o r 15 m i n b e f o r e i n c u b a t i o n . The e n d - p r o d u c t s f o r m e d w e r e a c e t i c a c i d , p r o p i o n i c a c i d , C02, and t r a c e s of e t h a n o l and H2. S u p e r n a t a n t s from 6 - t o 10-day-old c u l t u r e s c o n t a i n e d 1 6 t o 36 m M a c e t i c a c i d . Cellulolytic e n z y m e s i n t h e s u p e r n a t a n t were s t a b l e a t 2 O C u n d e r a e r o b i c c o n d i t i o n s f o r u p t o 4 weeks and had t h e a b i l i t y t o h y d r o l y s e carboxymethylcellulose, microcrystalline cellulose, cellobiose, x y l a n , and f i l t e r paper t o r e d u c i n g s u g a r s . The e f f e c t s o f t h e s u r f a c t a n t Tween 8 0 on t h e e n z y m i c h y d r o l y s i s of n e w s p a p e r h a v e b e e n t e s t e d . 3 3 4 B y m o n i t o r i n g s u g a r p r o d u c t i o n i t was found t h a t t h e s u r f a c t a n t i n c r e a s e d t h e r a t e and e x t e n t o f c e l l u l o s e s a c c h a r i f i c a t i o n . The r a t e of enzyme u s a g e i n t h e h y d r o l y s i s r e a c t o r was i m p r o v e d b y 33%. I n a d d i t i o n , i n t h e p r e s e n c e of s u r f a c t a n t t h e r e c o v e r y of enzymes was h i g h e r . Analysis o f t h e enzyme s o l u t i o n showed t h a t w i t h Tween 80 p r e s e n t l a r g e r It f r a c t i o n s of enzyme remained i n s o l u t i o n t h r o u g h o u t h y d r o l y s i s . a p p e a r e d t h a t t h e s u r f a c t a n t h i n d e r e d t h e i m m o b i l i z a t i o n of t h e enzymes on t h e s u b s t r a t e b y r e d u c i n g t h e s t r e n g t h of a d s o r p t i o n . The e f f e c t o f m i l d sodium hydroxide t r e a t m e n t s on s u g a r -cane c e l l u l o s i c w a s t e s ( b a g a s s e , p i t h , and s t r a w ) t o i n c r e a s e t h e i r
484
Carbohydrate Chemistry
b i o l o g i c a l d e g r a d a b i l i t y has been s t u d i e d . 3 3 5 A t a l e v e l of 8% NaOH (on a d r y - m a t t e r b a s i s ) 60% d i g e s t i b i l i t y measured by t h e i n v i t r o t e c h n i q u e was achieved f o r a l l m a t e r i a l s t e s t e d , I n d i r e c t methods t o p r e d i c t t h e d i g e s t i b i l i t y of t r e a t e d m a t e r i a l s s u c h a s t h e b a c t e r i a l d e g r a d a b i l i t y , e n z y m a t i c d e g r a d a b i l i t y , hot -water s o l u b i l i t y , and c h e m i c a l oxygen demand w e r e t r i e d a s a l t e r n a t i v e methods t o t h e rumen f l u i d t e c h n i q u e . High c o r r e l a t i o n c o e f f i c i e n t s f o r a l l m a t e r i a l s were o b t a i n e d w i t h a l l a l t e r n a t i v e t e c h n i q u e s . An i m p o r t a n t r e d u c t i o n of t i m e and r e a g e n t s was a c h i e v e d b y t h e u t i l i z a t i o n of t h e s o l u b i l i t y and chemical-oxygen-demand t e s t s . I t h a s been shown t h e o r e t i c a l l y and e x p e r i m e n t a l l y t h a t p o l y e n z y m e c e l l u l a s e c o m p l e x e s can c a t a l y s e r e a c t i o n s i n k i n e t i c s y s t e m s t h a t a r e c h a r a c t e r i z e d b y t h e a b s e n c e of any o n e d e f i n i t e s t e p l i m i t i n g t h e r a t e of t h e e n z y m a t i c h y d r o l y s i s of c e l l u l o s e , even when t h e r a t e s of t h e i n d i v i d u a l s t e p s of t h e p r o c e s s d i f f e r s u b s t a n t i a l l y . 3 3 6 Such a p e c u l i a r i t y of t h e k i n e t i c b e h a v i o u r o f c e l l u l a s e complexes i s due t o t h e p r e s e n c e of s h u n t i n g pathways i n a s e r i e s of s u c c e s s i v e - p a r a l l e l r e a c t i o n s of e n z y m a t i c d e g r a d a t i o n of I n s u c h s y s t e m s t h e e q u i l i b r i u m r a t e of c e l l u l o s e t o !-glucose. f o r m a t i o n of t h e e n d - p r o d u c t i s d e t e r m i n e d , a s a r u l e , b y t h e a g g r e g a t e of t h e r a t e s of s e v e r a l s t e p s s i m u l t a n e o u s l y . A c o n s e q u e n c e of t h i s r e a c t i o n mechanism i s t h e w i d e v a r i e t y o f t h e k i n e t i c p r i n c i p l e s of t h e a c c u m u l a t i o n of Q - g l u c o s e ( t h e e n d p r o d u c t ) when t h e c o m p o s i t i o n o f i n d i v i d u a l c o m p o n e n t s of t h e An i l l u s t r a t i o n o f t h e polyenzyme c e l l u l a s e s y s t e m i s v a r i e d . k i n e t i c p r i n c i p l e s of t h e e n z y m a t i c h y d r o l y s i s of i n s o l u b l e c e l l u l o s e ( c o t t o n l i n t e r ) is c i t e d , u s i n g t e n c e l l u l a s e complexes from f u n g i o f t h e g e n e r a Trichoderma, Geotrichum, and A s p e r g i l l u s . I t was shown t h a t t h e v a l u e of t h e e q u i l i b r i u m r a t e of ! - g l u c o s e f o r m a t i o n i s d e t e r m i n e d , a s a r u l e , b y t h e a c t i o n of two or t h r e e c e l l u l o l y t i c c o m p o n e n t s o f t h e c o m p l e x . The p r e s e n c e o f a s i n g l e mechanism o f t h e h y d r o l y s i s of c e l l u l o s e f o r a l l t h e c e l l u l a s e c o m p l e x e s s t u d i e d , r e g a r d l e s s of t h e i r c o m p o s i t i o n o r o r i g i n , was d e m o n s t r a t e d t h e o r e t i c a l l y and e x p e r i m e n t a l l y . Modes o f a c t i o n o f 2x2- a n d e n d o - c e l l u l a s e s h a v e b e e n i n v e s t i g a t e d i n t h e d e g r a d a t i o n of c e l l u l o s e s I and II.337 Cotton and Valonia c e l l u l o s e ( c e l l u l o s e I ) were r e a d i l y a t t a c k e d b y endoc e l l u l a s e of a h i g h l y e n d o w i s e - h y d r o l y s i s t y p e , w i t h a s h a r p decrease i n t h e degree o f polymerization, while the simultaneous p r o d u c t i o n of r e d u c i n g s u g a r was l o w . I n c o n t r a s t , v i s c o s e r a y o n and a l k a l i c e l l u l o s e ( c e l l u l o s e 11) showed l i t t l e l o w e r i n g o f t h e
485
6: Enzymes
d e g r e e o f p o l y m e r i z a t i o n b y e n d o - c e l l u l a s e , t h o u g h t h e e f f e c t was s l i g h t l y greater than with exo-cellulase,
while the simultaneous
p r o d u c t i o n o f r e d u c i n g s u g a r was v e r y h i g h .
The s y n e r g i s t i c e f f e c t
of
9and
higher
endo-cellulases
with cellulose
on t h e h y d r o l y s i s o f c e l l u l o s e was much
I t h a n w i t h c e l l u l o s e 11.
An e x p l a n a t i o n o f
t h e s e r e s u l t s was o f f e r e d i n t e r m s o f d i f f e r e n c e s i n t h e p o l a r i t y o f c e l l u l o s e chains
i n the
c r y s t a l l i n i t y and/or
ultrastructure
of
the
cellulose fibres. The k i n e t i c s o f t h e h y d r o l y s i s o f c o t t o n ) by
eight
Trichoderma,
cellulase
Geotrichum,
conditions.338
r a t e o f !-glucose
content o f e - Q - g l u c a n a s e observed
for
enrichment
seven
cotton l i n t (short-fibred from
fungi
of
of
I t was f o u n d t h a t t h e
i n t h e c e l l u l a s e p r e p a r a t i o n s ( w h i c h was
the
complexes studied
and
with other
also
A
according t o
i s t h e f i r s t enzyme t o a c t on t h e i n s o l u b l e
i s p r o p o s e d on t h e b a s i s o f a k i n e t i c a n a l y s i s o f t h e
multienzyme
cellulase
system
and s u b s t a n t i a t e d .
under c e r t a i n e x p e r i m e n t a l c o n d i t i o n s
a linear
The
concept
that
c o r r e l a t i o n must be
observed between t h e r a t e o f h y d r o l y s i s o f n a t i v e c e l l u l o s e , one hand,
after
components).
mechanism o f t h e e n z y m a t i c h y d r o l y s i s o f c e l l u l o s e , which endo-glucanase
genera
formation i s proportional t o the
o f t h e c e l l u l a s e complex
substrate,
the
and A s p e r i g u l l u s w e r e s t u d i e d u n d e r s t e a d y -
s t a t e and n o n - s t e a d y - s t a t e steady-state
complexes
on t h e
and i t s s o l u b l e p o l y m e r i c , d e r i v a t i v e s ( i n p a r t i c u l a r ,
carboxymethylcellulose), theoretically hypothesis
on
the
and e x p e r i m e n t a l l y .
discussed
i n
the
literature
concerning t h e presence i n c e l l u l a s e C1 e n z y m e ,
other,
i s
substantiated
The d a t a o b t a i n e d r e f u t e t h e for
the
last
30 y e a r s
of a non-hydrolytic
complexes
which presumably f i r s t attacks n a t i v e c e l l u l o s e .
The
r e s u l t s o f t h e w o r k show t h a t t h e r o l e o f t h e h y p o t h e t i c a l C1 enzyme i s i n f a c t p e r f o r m e d by an & - P - g l u c a n a s e C e l l u l o s e from t h e Gram-negative
o f s t a t i s t i c a l action.
bacterium Acetobacter xylinum
has been used as a m o d e l s u b s t r a t e f o r
visualizing the action o f
c e l l u l a s e e n z y m e s f r o m t h e f u n g u s T r i c h o d e r m a r e e ~ e i . H~i g ~h - ~ r e s o l u t i o n e l e c t r o n m i c r o s c o p y r e v e a l s t h a t A.
xylinum normally
produced a r i b b o n o f c e l l u l o s e t h a t i s a composite o f bundles o f crystalline
microfibrils.
Visual
patterns
c e l l u l o s e d e g r a d a t i o n h a v e been e s t a b l i s h e d . o b s e r v e d bound t o t h e c e l l u l o s e r i b b o n . i s split
along i t s axis
i n t o bundles
of
the
W i t h i n 10 m i n , of
process
microfibrils
the ribbon which are
subsequently thinned u n t i l they are completely dissolved w i t h i n minutes.
of
Enzymes a r e i n i t i a l l y
30
I n c u b a t i o n s w i t h p u r i f i e d components o f t h e c e l l u l a s e
486
Carbohydrate Chembtry
enzyme s y s t e m produced l e s s d r a m a t i c c h a n g e s i n r i b b o n s t r u c t u r e . P u r i f i e d 1,4-B-Q-glucan c e l l o b i o h y d r o l a s e I p r o d u c e d n o v i s i b l e change i n c e l l u l o s e s t r u c t u r e . P u r i f i e d endo-1,4-B-Q-glucanase I n both produced some s p l a y i n g of r i b b o n s i n t o m i c r o f i b r i l bundles. c a s e s , whole r i b b o n s were p r e s e n t e v e n a f t e r 60 m i n u t e s of i n c u b a t i o n , v i s u a l l y c o n f i r m i n g t h e s y n e r g i s t i c mode of a c t i o n of t h e s e enzymes. The i n f l u e n c e o f major s t r u c t u r a l f e a t u r e s o f c e l l u l o s e on r a t e o f e n z y m a t i c h y d r o l y s i s , h a s been i n ~ e s t i g a t e d . ~ ~ C ' ellulosic s a m p l e s w i t h a wide r a n g e of c r y s t a l l i n i t y i n d i c e s and s p e c i f i c s u r f a c e a r e a s showed a r e l a t i o n s h i p between t h e i r r a t e s o f h y d r o l y s i s , and t h e t w o s t r u c t u r a l p a r a m e t e r s , c r y s t a l l i n i t y index and s p e c i f i c s u r f a c e a r e a , were examined. The b i n d i n g of c e l l u l a s e s t o t h e i r s u b s t r a t e a t low t e m p e r a t u r e h a s been used t o p u r i f y t h e s e enzymes.341 The c e l l u l a s e s a r e adsorbed a t O°C and then r e l e a s e d from t h e enzyme-substrate complex a t 50'C. F o r t h e a d s o r p t i o n s t e p a g r e a t e x c e s s of enzyme i s used t o reduce b i n d i n g of c l o s e l y r e l a t e d enzymes t o t h e c e l l u l o s e . Nonb o u n d p r o t e i n s a r e removed b y e x t e n s i v e w a s h i n g . The a d s o r b e d enzymes a r e r e l e a s e d b y t h e i r own c a t a l y t i c a c t i o n on t h e s u b s t r a t e . The s m a l l p o l y s a c c h a r i d e s formed from c e l l u l o s e i n t h i s r e a c t i o n a r e e a s i l y s e p a r a t e d from t h e enzyme. The p u r i t y o f enzymes i s o l a t e d b y t h e a d s o r p t i o n method was assayed b y i m m u n o e l e c t r o p h o r e s i s and t h e course of enzyme a c t i o n on s u b s t r a t e d u r i n g t h e i s o l a t i o n procedure followed b y e l e c t r o n microscopy. A comparative investigation o f various c e l l u l a s e assay p r o c e d u r e s h a s been p e r f o r m e d . 3 4 2 The c e l l u l o l y t i c a c t i v i t y o f c r u d e enzyme p r e p a r a t i o n s from d i f f e r e n t c e l l u l o l y t i c f u n g i was a s s a y e d c o m p a r a t i v e l y w i t h s e v e r a l common a n a l y t i c a l p r o c e d u r e s d e s c r i b e d i n t h e l i t e r a t u r e . The i n v e s t i g a t i o n was c a r r i e d out w i t h t h e o b j e c t i v e of e v a l u a t i n g , w i t h raw c u l t u r e f i l t r a t e s , t h e d i f f e r e n t c e l l u l a s e t e s t s i n r e l a t i o n t o t h e i r s p e c i f i c i t y f o r endoand = - c e l l u l a s e a c t i o n as w e l l as t o allow comparisons t o be made b e t w e e n r e s u l t s from d i f f e r e n t r e s e a r c h g r o u p s u s i n g d i f f e r e n t methods. A new u l t r a s o n i c method has been developed f o r d e t e r m i n i n g t h e composition and p r o p e r t i e s of i n d i v i d u a l components of polyenzyme s y s t e m s w i t h o u t t h e i r p r e l i m i n a r y s e p a r a t i o n . 1 9 7 The method i s based on a d e t e r m i n a t i o n o f t h e pH dependence of t h e r a t e c o n s t a n t s o f i n a c t i v a t i o n of i n d i v i d u a l components of t h e enzyme system under t h e a c t i o n o f c a v i t a t i o n a l u l t r a s o u n d . The method was used t o S t u d y
487
6: Enzymes t h e c e l l u l a s e complex from
t h e fungus G e o t r i c h u m candidum.
shown t h a t t h i s c o m p l e x c o n t a i n s a t l e a s t f o u r
-endo-(1 * ase,
g?-(l * 4 ) - B - ! - g l u c a n a s e ,
4)-B-;-glucanase,
B-P-glucosidase,
f t was
c e l l u l o l y t i c enzymes:
and a r y 1-6-Q-glucosidase.
B-e-glucanThese enzymes
d i f f e r i n v a l u e s o f p K o f t h e i o n o g e n i c g r o u p s , c o n t r o l l i n g t h e pH profiles of ultrasonic inactivation,
as w e l l a s i n v a l u e s o f t h e
r a t e c o n s t a n t s o f i n a c t i v a t i o n o f t h e f o r m o f t h e a c t i v e s i t e most stable t o the action o f ultrasound. There
are
many
methods
cellulolytic
enzymes
activity.343
Since,
and
of
determining the
numerous
ways
of
activity
expressing
of
this
however, t h e r e s u l t s obtained i n d i f f e r e n t
research centres are almost completely incomparable,
an a t t e m p t has
b e e n made t o i n t r o d u c e a u n i f o r m s y s t e m f o r e x p r e s s i n g a u n i t o f enzyme a c t i v i t y .
T h i s s y s t e m i s b a s e d on t h e r e l a t i o n s h i p b e t w e e n
t h e d e g r e e o f enzyme d i l u t i o n and t h e enzyme a c t i v i t y . coefficient method
a,
The s l o p e
determined experimentally, i s constant for a given
o f d e t e r m i n i n g enzyme
a c t i v i t i e s by
i r r e s p e c t i v e o f t h e s u b s t r a t e used,
reducing sugars
t i m e o f reaction, or composition
o f c e l l u l o l y t i c complex. A
radiometric
microassay f o r
cellulase activity
r e l e a s e o f 14C products from {U-14C)cellulose.344 s i m p l e and u t i l i z e s v e r y s m a l l i n c u b a t i o n volumes. e v i d e n t w i t h i n t h e f i r s t 30 m i n u t e s o f r e a c t i o n .
measures t h e The a s s a y i s Activity i s
T h i s method s h o u l d
be p a r t i c u l a r l y s u i t e d t o a c t i v i t y s u r v e y s o f c u l t u r e f i l t r a t e s f r o m f e r m e n t a t i o n s a s w e l l as t h e a n a l y s i s o f
f r a c t i o n s f r o m enzyme
p u r i f i c a t i o n studies. Extensively ball-milled
c e l l u l o s e f i b r e s have been u s e d as
natural substrate for the determination o f cellulase activity.345 T h i s p h y s i c a l treatment breaks t h e l a r g e c e l l u l o s e f i b r e s
t o small
but i n s o l u b l e p a r t i c l e s y i e l d i n g a s u b s t r a t e accessible f o r complete e n z y m a t i c breakdown.
The a c t i v i t y o f c e l l u l a s e s was e s t i m a t e d f r o m
t h e decrease i n o p t i c a l d e n s i t y o f b a l l - m i l l e d suspension o f f i b r e s and s i m u l t a n e o u s measurement o f l i b e r a t e d s u g a r s d u r i n g h y d r o l y s i s . A g o o d c o r r e l a t i o n was f o u n d b e t w e e n t h e i n i t i a l r a t e o f r e a c t i o n
and t h e amount o f s u g a r r e l e a s e d a t g i v e n t i m e s . A
convenient
reported.346
The
zymogram release of
stain dye
c e l l u l o s e a z u r e by e n d o - c e l l u l a s e s
for
from
cellulases
phospheric
and = - c e l l u l a s e s
has
been
acid-swollen i s the basis
o f a t e c h n i q u e f o r l o c a t i n g t h e s e enzymes on g e l s . An a u t o m a t i c o n - l i n e
c e l l u l a s e assay i n c o m p u t e r - c o u p l e d p i l o t
f e r m e n t a t i o n has been reported.347
The m e a s u r e m e n t was b a s e d on t h e
488
Carbohydrate Chemistry
u s e o f dyed A v i c e l c e l l u l o s e a s s u b s t r a t e . The c o m p u t e r was programmed t o c a l c u l a t e t h e c e l l u l a s e a c t i v i t y d u r i n g f e r m e n t a t i o n . The m e a s u r e d a c t i v i t y v a l u e s w e r e c o r r e c t e d a g a i n s t a s t a n d a r d s a m p l e of known a c t i v i t y b y means o f a m a t h e m a t i c a l model f o r t h e c a l i b r a t i o n c u r v e which was s t o r e d i n t h e computer. F o u r s p e c i e s o f t r o p i c a l e a r t h w o r m h a v e been f o u n d t o d i f f e r w i t h r e g a r d t o t h e i r enzyme a c t i v i t y . 3 4 8 The maximum a c t i v i t y of p r o t e a s e and of c e l l u l a s e o c c u r r e d i n t h e p o s t e r i o r r e g i o n of t h e g u t of t h e e a r t h w o r m s . On a v e r a g e O c t o c h a e t o n a s u r e n s i s s h o w s maximum a c t i v i t y and Drawida c a l e b i shows m i n i m u m a c t i v i t y f o r a l l t h e enzymes s t u d i e d . I n a c o m p a r a t i v e s t u d y of c a r b o h y d r a s e a c t i v i t i e s i n m a r i n e i n v e r t e b r a t e s 103 s p e c i e s of marine i n v e r t e b r a t e s , b e l o n g i n g t o 7 t y p e s , Spongia, C o e l e n t e r a t a , Annelida, Arthropodg, Mollusca, Echinoderm-, Chordaya, were t e s t e d f o r l a m i n a r i n a s e , c e l l u l a s e , a n d am y l a s e a c t i v i t i e s .*99 The a p p l i c a t i o n of c a r b o h y d r a s e s t o t h e e x t r a c t i o n of p r o t e i n s from commercial f u l l - f a t bran u s i n g a wet a l k a l i n e p r o c e d u r e a t pH 8.5 o b t a i n e d an e x t r a c t c o n t a i n i n g 30% of t h e t o t a l n i t r o g e n o r i g i n a l l y p r e s e n t i n t h e b r a n . 3 4 9 However, t h e y i e l d of t o t a l n i t r o g e n i n t h e e x t r a c t c o u l d be i n c r e a s e d t o 38.5% w i t h a p r e t r e a t m e n t c o n t a i n i n g c a r b o h y d r a s e s ( c e l l u l a s e , x y l a n a s e , and poly9 - g a l a c t u r o n a s e ) f o l l o w e d b y t h e normal e x t r a c t i o n p r o c e d u r e . The p u r i f i c a t i o n o f a c e l l u l a s e i s o e n z y m e w i t h a P I o f 9 . 5 f r o m k i d n e y - b e a n a b s c i s s i o n z o n e s h a s been d e s c r i b e d . 3 5 0 An i m p o r t a n t s t e p i n t h e p u r i f i c a t i o n i n v o l v e d t h e a d s o r p t i o n of t h e c e l l u l a s e i s o e n z y m e o n t o an a f f i n i t y column of C F - 1 1 c e l l u l o s e and t h e subsequent e l u t i o n w i t h c e l l o b i o s e . N a t i v e and SDS p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s e s t a b l i s h e d t h a t t h e r e was only one component i n t h e p u r i f i e d c e l l u l a s e samples. Antibodies r a i s e d against the p u r i f i e d P I 9.5 c e l l u l a s e p r e c i p i t a t e d t h i s isoenzyme from c r u d e o r p u r i f i e d s o l u t i o n s b u t d i d n o t c r o s s - r e a c t w i t h P I 4.5 c e l l u l a s e from 2 , 4 - g - t r e a t e d a b s c i s s i o n zones. The a n t i b o d y was shown t o be m o n o s p e c i f i c by i m m u n o e l e c t r o p h o r e s i s and b y t h e f a c t t h a t i t p r e c i p a t e d only a s i n g l e 14C-labelled p r o t e i n from an a d s c i s s i o n zone e x t r a c t h e a v i l y l a b e l l e d w i t h 1 4 C amino a c i d s . The h y d r o l y s e s of c e l l u l o s e s u b s t r a t e s b y c e l l u l a s e c u l t u r e f i l t r a t e s from t h r e e mutant s t r a i n s of Trichoderma r e e s e i grown on l a c t o s e and on c e l l u l o s e have been compared.351 Cellulose culture f i l t r a t e s c o n t a i n e d f i v e t o s i x t i m e s a s much c e l l u l a s e as l a c t o s e culture filtrates. Unconcentrated c e l l u l o s e c u l t u r e f i l t r a t e s
489
6: Enzymes
p r o d u c e d up t o 10% sugar s o l u t i o n s f r o m 15% c e l l u l o s e i n 24 hours. Specific
a c t i v i t y
i n
enzyme
assays
and
efficiency
i n
s a c c h a r i f i c a t i o n t e s t s were l o w f o r enzymes f r o m a l l t h e m u t a n t s . Over a w i d e r a n g e t h e p e r c e n t s a c c h a r i f i c a t i o n o f a s u b s t r a t e i n a g i v e n t i m e was d i r e c t l y p r o p o r t i o n a l t o t h e l o g a r i t h m o f o f i n i t i a l c o n c e n t r a t i o n s o f enzyme and s u b s t r a t e . this,
the r a t i o
As a r e s u l t o f
d i l u t e enzyme was more e f f i c i e n t t h a n c o n c e n t r a t e d enzyme, b u t
i f h i g h sugar c o n c e n t r a t i o n s a r e d e s i r e d
very
large quantities of
enzyme a r e r e q u i r e d .
Since the slopes o f these p l o t s varied,the
relative activity of
c e l l u l a s e on d i f f e r e n t
substrates
may
be
a f f e c t e d b y enzyme c o n c e n t r a t i o n . A
mutant
enzymes
strain with
has
been
increased production of c e l l u l o l y t i c
induced
from
T r i c h o d e r m a r e e s e i QM 9414.209
the
good
cellulase
producer
C e l l u l a s e a c t i v i t i e s o f t h e mutant
i n f e r m e n t e r c u l t i v a t i o n s w e r e i n c r e a s e d t w o - t o t h r e e - f o l d and B-Q-
gluaosidase
activity
up
t o
six-fold
when
compared
to
the
c o r r e s p o n d i n g a c t i v i t i e s p r o d u c e d by Q M 9414. C e l l u l o l y t i c enzyme complexes o b t a i n e d f r o m
mutants o f
T r i c h o d e r m a r e e s e i w i t h enhanced c e l l u l a s e p r o d u c t i o n have been characterized.210 f u n g u s T.
The c e l l u l o l y t i c enzyme c o m p l e x e s s e c r e t e d by t h e
r e e s e i Q M 9 4 1 4 a n d i t s m u t a n t s M 5 , M 6, MHC 1 5 , a n d MHC
22 w e r e
c h a r a c t e r i z e d by
determining t h e i r
c a r b o x y m e t h y l c e l l u l a s e , and B-P-glucosidase
specific
filter-paper,
activities.
They w e r e
c h a r a c t e r i z e d f u r t h e r by m e a s u r i n g t h e i r c a r b o x y m e t h y l c e l l u l a s e and €3-g-glucosidase
p r o f i l e s a f t e r s e p a r a t i o n on an
was r o u g h l y e q u a l i n a l l p r e p a r a t i o n s , activity
was
highest
isoelectrofocusing
While the o v e r a l l filter-paper
c o l u m n o v e r pH r a n g e 3-10.
i n mutants
MHC 1 5 a n d MHC 22,
d i s t i n g u i s h e d m o r p h o l o g i c a l l y from t h e p a r e n t s t r a i n , h i g h e r degree o f branching o f t h e i r glucosidase a c t i v i t y
were
activity
t h e s p e c i f i c B-Q-glucosidase
hyphae.
d e t e c t e d by
which
QM 9414,
Two p e a k s o f
are by a
6-g-
isoelectrofocusing
i n
p r e p a r a t i o n s f r o m QM 9414 and M 6 , none i n t h e enzyme f r o m t h e mutant
M
5,
w h i l e 3 and 4 peaks,
respectively,
were
found
p r e p a r a t i o n s f r o m m o r p h o l o g i c a l m u t a n t s M H C 1 5 a n d MHC 22. h i g h e r B-P-glucosidase
a c t i v i t y i n t h e s e l a s t t w o p r e p a r a t i o n s was
a l s o r e f l e c t e d i n t h e higher 2-glucose stages
of
i n The
cellulose
t o cellobiose i n the i n i t i a l
hydrolysis
by
cellulases
and
the
individual
enzyme
preparations. The
p r o d u c t i o n of
studied with Trichoderma r e e s e i Rut produced,
together
hemicellulases C-30.208
This
with high c e l l u l a s e a c t i v i t i e s ,
has
been
organism
considerable
4 90
Carbohydrate Chemistry
a m o u n t s o f x y l a n a s e s a n d B - -8 - g l u c o s i d a s e . Three c e l l u l o s e c o n c e n t r a t i o n s ( 1 . 0 , 2 . 5 , and 5 . 0 % ) w e r e e x a m i n e d t o d e t e r m i n e t h e maximum l e v e l s o f c e l l u l a s e a c t i v i t y o b t a i n a b l e i n s u b m e r g e d c u l t u r e . T e m p e r a t u r e and pH p r o f i l i n g w e r e u s e d t o i n c r e a s e c e l l mass t o maximum l e v e l s w i t h i n two d a y s and t h e r e b y e n h a n c i n g fermentor productivity a t higher s u b s t r a t e l e v e l s . The e f f e c t s of t e m p e r a t u r e , p H , Tween-80 c o n c e n t r a t i o n , c a r b o n s o u r c e , and s u b s t r a t e c o n c e n t r a t i o n on t h e r a t i o o f m y c e l i a l g r o w t h and e x t r a c e l l u l a r enzyme p r o d u c t i o n were d e s c r i b e d . The p o t e n t i a l f o r i n c r e a s e d p r o d u c t i o n o f c e l l u l a s e o f ----T r i c h o d e r m a s p . b y pH c y c l i n g and t e m p e r a t u r e p r o f i l i n g h a s been i n v e s t i g a t e d . 3 5 2 C u l t i v a t i o n of 1. Q M 9414 on 3% c e l l u l o s e medium (C/bJ r a t i o = 8 . 5 ) p r o d u c e d 4.5 1 U m l - l c e l l u l a s e i n 1 8 0 h o u r s a t a c e l l g r o w t h o f 8.0 g l i t r e " ( 0 . 2 6 6 g c e l l g" cellulose). It corresponded t o an a v e r a g e c e l l u l a s e p r o d u c t i v i t y of 25.0 I U l i t r e - l h" (3.5 IU g'l c e l l h - ' ) . I n t h e same m e d i u m 9.5 g l i t r e - ' c e l l mass (0.316 g c e l l g" c e l l u l o s e ) , 6 . 2 I U m l - l c e l l u l a s e , and 38.75 I U litre" h'l (4.0 I U g'l c e l l h - l ) c e l l u l a s e p r o d u c t i v i t y could be o b t a i n e d u s i n g pH-cycling c o n d i t i o n s d u r i n g c u l t i v a t i o n . C e l l mass, c e l l u l a s e y i e l d , and p r o d u c t i v i t y were f u r t h e r i n c r e a s e d t o 10.0 g litre-', 7.2 I U rnl-', and 44.0 I U l i t r e ' ' h - l (4.5 I U g" c e l l h-'), r e s p e c t i v e l y , by s i m u l t a n e o u s pH c y c l i n g and t e m p e r a t u r e - p r o f i l i n g strategy. An enzyme p r e p a r a t i o n from a mutant s t r a i n of c e l l u l o m o n a s CS11 7 h a s b e e n f o u n d t o a c t s y n e r g i s t i c a l l y w i t h low l e v e l s o f -----------------Trichoderma r e e s e i c e l l u l a s e i n s a c c h a r i f i c a t i o n o f alkalip r e t r e a t e d sugar-cane bagasse and i n a s s a y s of f i l t e r - p a p e r activity.353 S u p p l e m e n t a t i o n of t h e Cellulomonas p r e p a r a t i o n w i t h T. r e e s e i c e l l u l a s e provided a p a r t i c u l a r l y a c t i v e p r e p a r a t i o n . The r e g u l a t i o n of t h e c e l l u l o l y t i c system i n Trichoderma r e e s e i by sophorose has been i n v e s t i g a t e d t h r o u g h measurement of i n d u c t i o n o f c e l l u l a s e and r e p r e s s i o n o f B - Q - g l u c o s i d a s e . 2 1 2 Sophorose has t w o r e g u l a t o r y r o l e s i n t h e p r o d u c t i o n of c e l l u l a s e e n z y m e s i n T. r e e s e i : B - P - g l u c o s i d a s e r e p r e s s i o n and c e l l u l a s e i n d u c t i o n . S o p h o r o s e a l s o i s h y d r o l y s e d by t h e m y c e l i a l - a s s o c i a t e d B-Qglucosidase. Repression of B-Q-glucosidase reduces sophorose h y d r o l y s i s and t h u s may i n c r e a s e c e l l u l a s e i n d u c t i o n . The i n a c t i v a t i o n o f t h e c e l l u l a s e of T r i c h o d e r m a r e e s e i b y sheer f o r c e s i s of s u f f i c i e n t m a g n i t u d e t o merit c o n s i d e r a t i o n i n t h e d e s i g n of equipment f o r t h e e n z y m a t i c h y d r o l y s i s of c e l l u l o s e . 3 5 4 The i n a c t i v a t i o n c o n s t a n t , lid, i s a f u n c t i o n o f t h e
6: Enzymes
491
f l o w r a t e o f t h e enzyme s o l u t i o n t h r o u g h a f i n e c a p i l l a r y t u b e ; k d i n c r e a s e d s l o w l y a t l o w s h e a r s t r e s s , and much more r a p i d l y when t h e s h e a r s t r e s s was g r e a t e r than 15 dynes cm-2. In t h e l i g h t of t h e p o t e n t i a l a p p l i c a t i o n of Trichoderma r e e s e i m u t a n t s i n t h e c o m m e r c i a l s a c c h a r i f i c a t i o n of c e l l u l o s e , t h e b i o c h e m i c a l n a t u r e of t h e c e l l u l a s e s from t h e s e m u t a n t s h a s been examined i n an e f f o r t t o d e f i n e t h e enzyme complex i n r e l a t i o n t o i t s s a c c h a r i f i c a t i o n e f f i c i e n c y and t o g a i n knowledge which w i l l c o n t r i b u t e t o w a r d a b e t t e r u n d e r s t a n d i n g of h o w t o i s o l a t e more e f f i c i e n t mutants f o r enzyme production.355 The e x t r a c e l l u l a r c e l l u l a s e activities o f C l o s t r i d i u m thermocellum L Q R I and Trichoderma r e e s e i Q M 9414 have been compared.207 The c r u d e e x t r a c e l l u l a r c e l l u l a s e o f C. thermocellum LQRI ( v i r g i n s t r a i n ) was very a c t i v e and s o l u b i l i z e d m i c r o c r y s t a l l i n e c e l l u l o s e a t one-half t h e r a t e observed f o r t h e e x t r a c e l l u l a r c e l l u l a s e o f T . r e e s e i Q M 9414 ( m u t a n t s t r a i n ) . C. thermocellum c e l l u l a s e a c t i v i t y d i f f e r e d c o n s i d e r a b l y from t h a t of T. r e e s e i as f ollo ws: h i g h e r * - a g l u can a s e/=-Q - g l u can a s e a c t i v i t y r a t i o , absence of e x t r a c e l l u l a r $-2-glucosidase o r x y l o s i d a s e a c t i v i t y , long-chain o l i g o s a c c h a r i d e s i n s t e a d o f s h o r t c h a i n o l i g o s a c c h a r i d e s a s i n i t i a l (15 m i n ) h y d r o l y t i c p r o d u c t s o n m i c r o c r y s t a l l i n e c e l l u l o s e , mainly c e l l o b i o s e or x y l o b i o s e as l o n g t e r m ( 2 4 h ) h y d r o l y s i s p r o d u c t s of A v i c e l and MN300 o r x y l a n , and high a c t i v i t y and s t a b i l i t y a t 60 t o 7 O o C . Under o p t i m i z e d r e a c t i o n c o n d i t i o n s , t h e k i n e t i c p r o p e r t i e s (ymax0.4 p m o l m i n - l p e r mg of p r o t e i n , energy o f a c t i v a t i o n 33 k J , t e m p e r a t u r e c o e f f i c i e n t 1.8) of C. thermocellum c e l l u l o s e - s o l u b i l i z i n g a c t i v i t y were comparable t o t h o s e r e p o r t e d f o r T . r e e s e i , e x c e p t t h a t t h e dyed A v i c e l c o n c e n t r a t i o n a t half-maximal v e l o c i t y was two-fold high (182 u M ) . The c e l l u l o s e - s o l u b i l i z i n g a c t i v i t y o f t h e t w o c r u d e c e l l u l a s e s d i f f e r e d c o n s i d e r a b l y i n r e s p o n s e t o v a r i o u s enzyme i n h i b i t o r s . Most n o t a b l y , Ag2+ and Hg2+ e f f e c t i v e l y i n h i b i t e d C. thermocellum b u t n o t T . r e e s e i c e l l u l a s e a t <20pM, whereas Ca2+, Mg2+, and Mn2+ i n h i b i t e d T . r e e s e i b u t n o t C.thermocellum c e l l u l a s e a t >10 mM. B o t h enzymes were i n h i b i t e d b y C u 2 + (>20 m M ) , Z n 2 + (>1.0 mM),and e t h y l e n e g l y co 1- b i s ( $ -a m i n oe t h y 1 e t her -N,N- t e t r a-ace t i c a c i d ( > l o mM). T. r e e s e i b u t n o t C. thermocellum c e l l u l o s e - s o l u b i l i z i n g a c t i v i t y was 20% i n h i b i t e d by !-glucose (73 m M ) and c e l l o b i o s e (29 mM). B o t h c e l l u l a s e s p r e f e r e n t i a l l y cleaved t h e i n t e r n a l g l y c o s i d i c b o n d s of c e l l a o l i g o s a c c h a r i d e s . The o v e r a l l r a t e s of c e l l o o l i g o s a c c h a r i d e d e g r a d a t i o n were h i g h e r f o r T . r e e s e i t h a n f o r
$-a-
Carbohydrate Chemistry
492
C . t h e r m o c e l l u m c e l l u l a s e , e x c e p t t h a t t h e r a t e s of c o n v e r s i o n o f c e 1l o hex aose t o ce l l o t r i o s e were e q u i valen t A method has been developed f o r t h e s e l e c t i v e p u r i f i c a t i o n of a ( b i r 1 3 , 0 0 0 1 of t h e ce l l u l a s e low -mo l e c u l a r - w e i g h t &-g-glucanase complex from Trichoderma k ~ n i n g i i . ~The ~ ~enzyme was o b t a i n e d i n t h e form of a l y o p h i l i z e d p r e p a r a t i o n c o m p l e t e l y f r e e from cellobiase activity. I t was f o u n d t h a t c e l l o b i o s e and m e t h y l cellobioside a c t i v a t e (6-fold a t a s a t u r a t i n g concentration) the low -mo 1e.cular-weigh t endo-Q-glucanase and a l m o s t c o m p l e t e l y i n h i b i t t h e a c t i v i t y of h i g h - m o l e c u l a r - w e i g h t endo-Q-glucanases. C e l l o b i o s e had a weak i n h i b i t o r y e f f e c t on t h e u n f r a c t i o n a t e d c e l l u l a s e complex f r o m T . k o n i n g i i . A k i n e t i c a n a l y s i s o f t h i s phenomenon showed t h a t t h e a c t i v a t i n g e f f e c t of c e l l o b i o s e or i t s a n a l o g u e s on t h e m - Q - g l u c a n a s e t a k e s p l a c e b y a t r a n s g l y c o s y l a t i o n mechanism. The m u l t i p l e k i n e t i c m a n i f e s t a t i o n s of c e l l o b i o s e , which a c t s a s an a c t i v a t o r o r i n h i b i t o r of endo-Q-glucanases from v a r i o u s s o u r c e s , a r e e x p l a i n e d b y t h e v a r y i n g amounts of t h e low- and high-molecularw e i g h t ~ ~ ~ o - Q - g l u c a n a s eass w e l l a s b y t h e i r c a p a c i t y f o r t r a n s g l y c o s y l a t i o n and t h e t r a n s f e r of t h e r e d u c i n g g r o u p s of t h e reaction products to effectors acting as additional nucleophilic agents i n enzymatic c a t a l y s i s . The t h e r m a l - s t a b i l i t y c h a r a c t e r i s t i c s of t h e c e l l u l a s e enzymes p r e s e n t i n c u l t u r e f i l t r a t e s of t h e t h e r m o p h i l i c f u n g u s S p o r o t r i c h u m t h e r m o p h i l e h a v e been i n v e s t i g a t e d a t d i f f e r e n t t e m p e r a t u r e s and a t d i f f e r e n t t i m e s of exposure.200 Maximum enzymic a c t i v i t i e s under a s s a y c o n d i t i o n s were found a t 6 8 O C f o r t h e f i l t e r paper a c t i v i t y ( F P A ) and t h e Cx a c t i v i t y ( c a r b o x y m e t h y l c e l l u l o s e ) , w h i l e t h e maxima f o r t h e C1 a c t i v i t y ( c o t t o n ) and B - Q - g l u c o s i d a s e a c t i v i t y ( c e l l o b i o s e ) w e r e f o u n d t o be a t 5 5 O C a n d 7 2 ' C , r e s p e c t i v e l y . A f t e r 48 h exposure t i m e of c u l t u r e f i l t r a t e s t o 5 O o C t h e r e s i d u a l a c t i v i t i e s f o r t h e FPA, Cx,and B - Q - g l u c o s i d a s e w e r e f o u n d t o be 8 8 % , 9846, a n d 9 3 % o f t h e o r i g i n a l a c t i v i t i e s , respectively. Q - G l u c o s e , c e l l o b i o s e , A v i c e l , and S o l k a F l o c h a v e been u t i l i z e d a s s u b s t r a t e s f o r g r o w t h o f T h e r m o m o n o s p o r a s p . i n o r d e r t o s t u d y t h e i n d u c t i o n - r e p r e s s i o n c h a r a c t e r i s t i c s of i t s a s s o c i a t e d c e l l u l a s e system.357 W h i l e P - g l u c o s e p r o v e d t o be an e f f e c t i v e r e p r e s s o r of t h e c e l l u l a s e enzymes, t h e o t h e r t h r e e s u b s t r a t e s i n d u c e d r e l a t i v e l y h i g h l e v e l s o f enzyme a c t i v i t y a s measured by t h e f i l t e r - p a p e r a s s a y . On a u n i t - c e l l - m a s s b a s i s t h e h i g h e s t v a l u e s of c e l l u l a s e a c t i v i t y were o b t a i n e d when Avicel was u t i l i z e d a s t h e carbon energy s o u r c e . The n a t u r e of t h e c e l l u l o s i c
I _ -
.
493
6: Enzymes
m a t e r i a l and i t s i n i t i a l c o n c e n t r a t i o n w e r e i d e n t i f i e d as t w o v e r y i m p o r t a n t parameters o f t h e i n d u c t i o n process.
Humicola
insolens,
and c o m p o s t heaps,
a t h e r m o p h i l i c f u n g u s i s o l a t e d f r o m manure
h a s been f o u n d t o p r o d u c e s i g n i f i c a n t a m o u n t s o f
t h e r m o s t a b l e c e l l u l a s e s i n c u l t u r e s o n w h e a t - b r a n medium (5OoC, 4 days).358
The m o u l d b r a n e x t r a c t h y d r o l y s e d A v i c e l ,
c e l l u l o s e , a n d n e w s p r i n t a t 90%, 4546,and glucose.
Then
Avicelase
and
carboxymethyl-
35%, r e s p e c t i v e l y ,
carboxymethylcellulase
were
f r o m t h e c u l t u r e e x t r a c t by a d s o r p t i o n o n t o A v i c e l , t r e a t m e n t , and c o n s e c u t i v e c o l u m n c h r o m a t o g r a p h i e s state
on polyacrylamide
cellulases,
h i g h l y t h e r m o s t a b l e ( t e m p e r a t u r e o p t i m a 5OoC). a f t e r h e a t i n g a t 65OC f o r
h e a t and a c i d
t o a homogeneous
gel electrophoresis.
especially carboxymethylcellulase,
t o Q-
purified
The
purified
w e r e f o u n d t o be
A v i c e l a s e was s t a b l e
5 m i n and c a r b o x y m e t h y l c e l l u l a s e r e t a i n e d
45% o f t h e o r i g i n a l a c t i v i t y a f t e r h e a t i n g a t 95OC f o r 5 min. An
i n h i b i t o r
Dictyostelium
of
cellulase
has
been
isolated
from
d i s c o i d e u m i n t h e c u l m i n a t i o n s t a g e and p a r t i a l l y
_ _ I -
purified.359 weight
D.
T h i s i n h i b i t o r was shown t o be a p r o t e i n o f m o l e c u l a r
20,000;
i t i n h i b i t e d the two c e l l u l a s e s present i n
a t pH 6.5
discoideum b u t not c e l l u l a s e s from
o t h e r organisms.
Inhibition
was p r e v e n t e d A t pH 8.0 b u t c o u l d be r e c o v e r e d by a d j u s t m e n t o f t h e pH t o 6.5.
The i n h i b i t o r was p r e s e n t i n a l l s t a g e s o f d e v e l o p m e n t
f r o m a m o e b a e t o s p o r e s b u t d i s a p p e a r e d a s t h e s p o r e s a g e d a n d was absent from g e r m i n a t i n g c e l l s , of
this
inhibitor
indicating that specific destruction
may b e r e s p o n s i b l e f o r
the activation of the
cellulases.
I t h a s been f o u n d t h a t t h e w h i t e f o r m o f T a l a r o m y c e s e m e r s o n i i produces
more
cellulase
activity
than
the
of
or
green
v a r i a n t s when g r o w n on c e l l u l o s e - p e p t o n e media.360
red/brown
The m a i n t e n a n c e
t h e l e v e l o f e n z y m e p r o d u c t i o n was a c h i e v e d w i t h c o n c o m i t a n t
r e d u c t i o n i n medium c o s t s . The c e l l u l o s e d e g r a d a t i o n and c a r b o x y m e t h y l c e l l u l a s e p r o d u c t i o n by S p o r o c y t o p h a g a m y x o c o c c o i d e s h a v e b e e n i n v e s t i g a t e d b y g r o w i n g t h e organism i n a 31 a i r - l i f t w/v
i n s o l u b l e cellulose.361
reduced the
f e r m e n t e r u s i n g a medium c o n t a i n i n g 2% The c e l l u l o s e c o n t e n t o f t h e m e d i u m
of the fermenter but during growth the dissolved
oxygen c o n c e n t r a t i o n d i d n o t f a l l b e l o w 75% s a t u r a t i o n .
Rates o f
c e l l u l o s e d e g r a d a t i o n and e x t r a c e l l u l a r enzyme p r o d u c t i o n w e r e s i m i l a r t o those reported f o r a s t i r r e d - t a n k
---a t ~
A
new
c e l l u l a s e has
s . ~ ~ T h*e
enzyme
been p u r i f i e d
was
less
or
fermenter.
f r o m A s p e r g i l l u s acule:
only
slightly
active
on
494
Carbohydrate Chemistry
c o n v e n t i o n a l s u b s t r a t e s such as A v i c e l , c e l l o b i o s e , and s a l i c i n . amorphous
cellulose
However, such
as
carboxymethylcellulose,
t h e enzyme was p o t e n t l y a c t i v e on insoluble
cello-oligosaccharides,
p h o s p h a t e - s w o l l e n c e l l u l o s e , and a l k a l i - s w o l l e n c e l l u l o s e . e n z y m e was c l a s s i f i e d a s a c e l l u l a s e .
Thus t h e
I t was s u g g e s t e d t h a t t h i s
enzyme may p l a y an i m p o r t a n t r o l e i n s a c c h a r i f i c a t i o n o f c e l l u l o s e . A
new e n d o - c e l l u l a s e
component
of
carboxymethylcellulase
type
(En-l)
h a s been o b t a i n e d by g e l f i l t r a t i o n a n d c o l u m n c h r o m a t o g r a p h y
from
Driselase,
a
commercial
enzyme
preparation
from
The e n z y m e b e h a v e d a s a s i n g l e p r o t e i n on SDS
I r p e x lacteus.363
polyacrylamide disc
electrophoresis
(mol.
wt.
c o n t a i n e d o n l y 0.73% c a r b o h y d r a t e as g - g l u c o s e .
and
15,000)
it
The p a t t e r n o f i t s
amino a c i d c o m p o s i t i o n i s s i m i l a r t o those o f o t h e r c e l l u l a s e s i n respect o f high contents o f I-glycine,
i-serine,and
L-threonine.
T h e c e l l u l a s e was m o s t a c t i v e a t pH 4.0 a n d was v e r y s t a b l e i n t h e pH r a n g e o f 3.0
t o 6.0,
10 min.
7OoC f o r
b u t was c o m p l e t e l y i n a c t i v a t e d by h e a t i n g a t
A series of cello-oligosaccharides,
including
c e l l o b i o s e , was f o r m e d by t h i s enzyme f r o m c a r b o x y m e t h y l c e l l u l o s e as w e l l as f r o m w a t e r - i n s o l u b l e
celluloses.
carboxymethylcellulose,
increase
the
i n
I n the hydrolysis of the
fluidity
of
the
s u b s t r a t e was r e l a t i v e l y l a r g e a s c o m p a r e d w i t h t h e s i m u l t a n e o u s i n c r e a s e i n r e d u c i n g power.
From t h i s r e s u l t and t h e p a t t e r n o f
h y d r o l y s i s p r o d u c t s , E n - 1 was e l u c i d a t e d t o b e a n e n d o - c e l l u l a s e , and i t showed t h e h i g h e s t r a n d o m n e s s among t h e c e l l u l a s e c o m p o n e n t s o b t a i n e d so f a r f r o m I r p e x l a c t e u s . A
cellulolytic
A c etivibrio
enzyme p r e p a r a t i o n s cotton
batting,
cellulose,
enzyme
system
has
been
identified
c e l l u l o l y t i c u s , a newly i s o l a t e d anaerobe.364 from t h e organism
filter
and
tissue
carboxymethylcellulose,
converted paper,
a
i n
Crude
ball-milled
pulp,
microcrystalline
c e l l o b i o s e , and x y l a n t o r e d u c i n g
The p r e p a r a t i o n s showed maximum a c t i v i t y b e t w e e n pH 5 and 6
sugars.
a n d a t a t e m p e r a t u r e b e t w e e n 37 a n d 5 O o C , d e p e n d i n g o n t h e s u b s t r a t e used.
The e n z y m e a c t i v i t y was f a i r l y s t a b l e a t Z 0 C f o r 4 w e e k s .
The s a c c h a r i f y i n g a b i l i t y o f t h e p r e p a r a t i o n was c o m p a r a b l e t o t h a t o f
commercially
available
cellulase
preparations
from
A s p e r g i l l u s n i g e r and T r i c h o d e r m a v i r i d e . The
-A c e t i v i b r i o
r e g u l a t i o n
o f
c e l l u l a s e
s y n t h e s i s
i n
cellulolyticus isolated from a cellulose-enrichment
c u l t u r e d e g r a d e d c e l l u l o s e by t h e s e c r e t i o n o f c e l l u l o l y t i c enzymes
i n t o t h e c u l t u r e medium.365 B o t h exo-p- a n d e n d o - Q - g l u c a n a s e a c t i v i t i e s w e r e d e t e c t e d and shown t o be r e g u l a t e d by i n d u c t i o n a n d
495
6: Enzymes catabolite repression.
e n d o - Q - G l u c a n a s e s y n t h e s i s was i n d u c e d by
c e l l u l o s e , c e l l o b i o s e , and s a l i c i n . Synthesis induced d u r i n g growth on c e l l o b i o s e was i n h i b i t e d , a l t h o u g h n o t c o m p l e t e l y r e p r e s s e d , by p-glucose. grown
on
z-Q-Glucanase insoluble
activity
was
polyB-q-glucoside
enhanced
when c e l l s
polymers.
were
Activity
was
p r o g r e s s i v e l y i n h i b i t e d by s u p p l e m e n t i n g m i c r o c r y s t a l l i n e s u b s t r a t e s w i t h i n c r e a s i n g amounts o f c e l l o b i o s e . The f e r m e n t a t i o n o f v a r i o u s s a c c h a r i d e s d e r i v e d f r o m c e l l u l o s i c b i o m a s s t o e t h a n o l h a s b e e n e x a m i n e d i n mono- a n d c o - c u l t u r e s o f C l o s t r i d i u m thermocellum strain
39E.
cellobiose,
s t r a i n L Q R I and C.
thermohydrosulphuricum
thermgrnydrosulphuricum f e r m e n t e d ; - g l u c o s e , and x y l o s e , b u t n o t c e l l u l o s e o r x y l a n , and y i e l d e d C.
ethanol/acetate
ratios
of
l.0.366
s u b s t r a t e c o n c e n t r a t i o n s (l%),
A t
non-limiting
cellulosic
thermocellum c e l l u l a s e h y d r o l y s i s
C.
p r o d u c t s accumulated d u r i n g monoculture f e r m e n t a t i o n o f Solka F l o c c e l l u l o s e and i n c l u d e d Q - g l u c o s e , A
stable
co-culture
C.
thermocellum
that
and C.
cellobiose,
xylose,
contained nearly
thermohydrosulphuricum
fermented a v a r i e t y o f c e l l u l o s i c substrates, o b s e r v e d was t w o f o l d h i g h e r The m e t a b o l i c b a s i s f o r
t h a n i n C.
was
thermocellum
t h e c o - c u l t u r e on S o l k a F l o c c e l l u l o s e i n c l u d e d :
hemicellulose,
.
to
established that
and t h e e t h a n o l y i e l d
t h e enhanced f e r m e n t a t i o n
-------------__ C. t h e r m o c e l l u m c e l l u l a s e
and x y l o b i o s e .
e q u a l numbers o f
hydrolyse
fermentations.
effectiveness
of
the a b i l i t y of
a-cellulose
and
t h e enhanced u t i l i z a t i o n o f mono- and d i - s a c c h a r i d e s
b y C t h e r m o h y d r o s u 1p h u r ic u m in c r e’a s e d c e 11u 1o s e c o n s u m p t io n , t h r e e f o l d i n c r e a s e i n t h e e t h a n o l p r o d u c t i o n r a t e , and t w o f o l d decrease i n the acetate production rate.
The c o - c u l t u r e a c t i v e l y
f e r m e n t e d MN300 c e l l u l o s e , A v i c e l , S o l k a F l o c , S 0 2 - t r e a t e d wood, and s t e a m - e x p l o d e d wood. The h i g h e s t e t h a n o l y i e l d o b t a i n e d was 1.8 m o l o f e t h a n o l p e r m o l o f k - g l u c o s e u n i t i n MN 3 0 0 c e l l u l o s e . The
degradation
lignocelluloses
-S.-- v i r i d o s p o r u s hardwood,
and
of
softwood,
by t w o S t r e p t o m y c e s T7A a n d S. grass
hardwood,
and
grass
s t r a i n s has been i n v e s t i g a t e d .
s e t o n i i 75V12 were g r o w n on s o f t w o o d ,
lignocelluloses,
and
the
lignocellulose
d e c o m p o s i t i o n was f o l l o w e d by m o n i t o r i n g s u b s t r a t e w e i g h t ,
lignin,
and c a r b o h y d r a t e l o s s e s o v e r t i m e . 3 6 7 R e s u l t s showed t h a t b o t h S t r e p t o m y c e s s t r a i n s s u b s t a n t i a l l y degraded b o t h t h e l i g n i n and t h e c a r b o h y d r a t e components of each l i g n o c e l l u l o s e . actinomycetes
were
more
efficient
However,
decomposers
of
these grass
l i g n o c e l l u l o s e s t h a n of hardwood o r softwood l i g n o c e l l u l o s e s . A
synthetic
medium
for
the
c e l l u l o l y t i c
anaerobe
Carbohydrate Chemistry
496
Ruminococcus
a l b u s has been d e s c r i b e d 3 6 8
study
A
Chaetomium -
of
been r e p o r t e d . 3 6 9 on
c e l l u l a s e
and
p r o t e i n
production
by
c e l l u l o l y t i c u m s t r a i n s g r o w n on c e l l u l o s i c s u b s t r a t e s has
media
F e r m e n t a t i o n w i t h C.
containing
P r o d u c t i o n of
free
either
c e l l u l y t i c u m was c a r r i e d o u t
Avicel
or
cellulose
c e l l u l o l y t i c enzymes,
cellulose
newspaper.
d e g r a d a t i o n , and
t h e f o r m a t i o n on c e l l p r o t e i n were s t u d i e d w i t h t h e o r i g i n a l and mutant s r a i n s . The
variation
during
the
of
bagasse
fermentation
investigated.370
A t
the
c r y s t a l l i n i t y and c e l l u l o s e a c t i v i t y
of
Cellulomonas
early
stage
of
c r y s t a l l i n i t y i n d e x o f bagasse i n c r e a s e d ,
bacteria
the
been the
s u g g e s t i n g t h a t t h e major
metabolized f r a c t i o n corresponded t o t h e h e m i c e l l u l o s e stage.
has
fermentation
during t h i s
L a t e r t h e c r y s t a l l i n i t y a c h i e v e d a steady s t a t e and t h e n
decreased,
which
indicated that
b a g a s s e was
being attacked.
activity of
extracellular
increase
followed
by
an
the
The
enzyme abrupt
most
i n the
structure of
complex
analysis
of
the
cellulolytic
medium showed a s h a r p
levelling
off
and
decline
i n
activity.
These r e s u l t s a l o n g w i t h t h e r e d u c t i o n o f c r y s t a l l i n i t y
index
bagasse
and
u t i l i z a t i o n (70%) l e d t h e
authors
to
the
c o n c l u s i o n t h a t t h e C1 c o m p o n e n t was p r e s e n t i n c e l l u l a s e c o m p l e x s y n t h e s i z e d by t h e b a c t e r i a . An enzyme p r e p a r a t i o n f r o m a C e l l u l o m o n a s s t r a i n has been shown p r e v i o u s l y t o be a c t i v e i n r e l e a s i n g r e d u c i n g s u g a r s f r o m a l k a l i p r e t r e a t e d sugar-cane
bagasse.371
T h i s enzyme p r e p a r a t i o n h a s been
d e m o n s t r a t e d t o be v e r y r e s i s t a n t t o e n d - p r o d u c t xylose,
!-glucose,
D u r i n g growth o f B a c t e r o i d e s succinoqenes +
4)-B-Q-glucanase,
i n a
l i q u i d medium
g r e a t e r t h a n 80% o f
the
x y l a n a s e , and a r y l - B - a - x y l o s i d a s e
and
w i t h c e l l u l o s e as t h e c a r b o h y d r a t e s o u r c e ,
--endo-(1
i n h i b i t i o n by Q -
c e l l o b i o s e , and e t h a n o l .
50% o f t h e aryl-B-Q-glucosidase
was r e l e a s e d f r o m c e l l s i n t o t h e
Less t h a n 25% o f t h e B - Q - g l u c o s i d a s e a c t i v i t y was
culture fluid.227
detected i n the culture fluid. r e l e a s e d enzymes
A p p r o x i m a t e l y 50% o f
each o f t h e
m e a s u r e d was a s s o c i a t e d w i t h s e d i m e n t a b l e
s u b c e l l u l a r membrane v e s i c l e s .
Many v e s i c l e s were seen a d h e r i n g t o
c e l l u l o s e , and t h e y were a l s o seen f r e e i n t h e c u l t u r e f l u i d .
These
d a t a s u g g e s t e d t h a t 6. s u c c i n o g e n e s r e l e a s e s h y d r o l y t i c enzymes non-sedimentable
in
and p a r t i c u l a t e f o r m s d u r i n g g r o w t h by a mechanism
w h i c h has u n t i l now r e c e i v e d l i t t l e a t t e n t i o n .
C e l l u l o s e incubated
i n a p o r o u s n y l o n b a g i n t h e r u m e n was c o l o n i z e d b y b a c t e r i a r e s e m b l i n g 6. s u c c i n o g e n e s , a n d s u b c e l l u l a r v e s i c l e s w e r e s e e n
497
6: Enzymes
p e n e t r a t i n g c h a n n e l s and f r a c t u r e s i n t h e c e l l u l o s e . I t was s u g g e s t e d t h a t 6. s u c c i n o g e n e s c e l l s i n t h e r u m e n c o n t r i b u t e t o a n extracellular population of subcellular vesicles t h a t possess c e l l u l o l y t i c and h e m i c e l l u l o l y t i c a c t i v i t i e s which probably enhance polyment d i g e s t i o n and provide a source of sugars f o r microbes lacking polymer-degrading a c t i v i t y , thereby contributing t o a s t a b l e heterogeneous microbial population. T h e p r o d u c t i o n o f c a r b o x y m e t h y l c e l l u l a s e a n d 8 - Q - g l u c o s i d a s e by Clostridium acetobutylicum has been i n v e s t i g a t e d i n a n i n d u s t r i a l medium.225 T h e c e l l u l a s e was i n d u c e d b y m o l a s s e s , a n d i t w a s n o t Optimum c a r b o x y m e t h y l c e l l u l a s e a c t i v i t y r e p r e s s e d by Q - g l u c o s e . o c c u r r e d a t DH 4.6 a n d 37’C.
21
Chitinases
An i n v i t r o s y s t e m d e s c r i b e d f o r t h e s t u d y o f c h i t i n a s e i n v o l v e s s o l u b l e enzyme p r o t e i n ( s ) and an i n s o l u b l e s u b s t r a t e ~ r e p a r a t i o n . ~ ’ ~W i t h i n s e c t m o l t i n g f l u i d c h i t i n a s e , i t s h o w s p r o p e r t i e s t h a t p a r a l l e l t h o s e observed d u r i n g i n vivo breakdown of For example, m o l t i n g f l u i d c h i t i n a s e c u t i c l e during t h e molt. a c t i v i t y not p r e v i o u s l y exposed t o c h i t i n is s t r o n g l y and s p e c i f i c a l l y adsorbed t o the substrate, i n contrast t o other e n z y m a t i c a c t i v i t i e s i n c l u d i n g B-~-2-acetamido-2-deoxyglucosidase present i n molting fluid. This leads t o pa r t i a l purification of molting fluid chitinase activity reflected i n increased specific activity of chitinase associated with the insoluble chitin substrate. M o l t i n g f l u i d c h i t i n a s e a c t i v i t y may i n v o l v e a n u m b e r o f p o l y p e p t i d e s r a n g i n g i n m o l e c u l a r w e i g h t f r o m 145,000 t o less t h a n 20,000 d a l t o n s . The s y s t e m d e s c r i b e d g i v e s r e s u l t s c o n s i s t e n t w i t h a p r o c e s s i v e mechanism f o r m o l t i n g f l u i d c h i t i n a s e , d a t a are g i v e n d e m o n s t r a t i n g t h a t molting f l u i d c h i t i n a s e c o n t i n u e s t o a c t on t h e same c h i t i n p a r t i c l e ( s ) w i t h w h i c h i t i n i t i a l l y a s s o c i a t e s r a t h e r than d i f f u s i n g f r e e l y from s u b s t r a t e p a r t i c l e , and t h e product of its a c t i o n appears t o be a monosaccharide r a t h e r than a m i x t u r e of o l i g o s a c c h a r i d e s . Processive behaviour f o r chitinase would be p r e d i c t e d f r o m t h e known s t r u c t u r e , a n d t h e i n v i v o measured rate of breakdown, of c u t i c l e c h i t i n d u r i n g t h e molt. The p r e l i m i n a r y n a t u r e o f t h i s c o n c l u s i o n , b a s e d o n w h a t i s s o f a r known a b o u t t h e s t r u c t u r e of t h e s u b s t r a t e u s e d i n t h e i n v i t r o s y s t e m , i s b r i e f l y discussed.
-.
498
Carbohydrate Chemistry High chitinase
and l y s o z y m e
a c t i v i t i e s have
been f o u n d i n
Leydig’s organ o f E t m o p t e r u s spinax, Somniosus microcephalus,and Torpedo n o b i l i a n s , i n L e y d i g ’ s and e p i g o n a l o r g a n s and s p l e e n o f R a j a r a d i a t a , and i n t h e e p i g o n a l o r g a n o f R h i n o p t e r a bonasus.” S t r o n g c h i t i n a s e a c t i v i t y w i t h l i t t l e o r n o l y s o z y m e a c t i v i t y was noted i n Leydig’s i n
the
and e p i g o n a l o r g a n s o f
epigonal
organ
Scyliorhinus canicula
cleslymontoma_cLrratum
of
and and
Heterodontus f r a n c i s c i . Chitinase
-C---o n i d i o b o l u s chitinase
i s
production
s p e c i e s has reported
substrate specificity colloidal chitin,
by
Conidiobolus lamprauges
been d e s c r i b e d . 3 7 3
in a
even
medium
free
of
chitin.
The
o f t h e e n z y m e was t e s t e d w i t h c h i t i n g e l ,
c h i t i n powder,and
ethylene glycol c h i t i n .
A chitinase-overproducing mutant o f z e r r a t i a
been i s o l a t e d . 3 7 4
and o t h e r
The p r o d u c t i o n o f
After
me t h a n e sulphonate, o r !-me
marcescens has
mutagenesis w i t h u l t r a v i o l e t thyl-”-n
it r o -!-nit
light,
ethyl
r o s o g u a n i d i ne , 1 9 , 9 40
c o l o n i e s were s c r e e n e d f o r p r o d u c t i o n o f e n l a r g e d zones o f c l e a r i n g [ i n d i c a t i v e o f c h i t i n a s e a c t i v i t y ) on c h i t i n - c o n t a i n i n g Forty-four
agar p l a t e s .
c h i t i n a s e h i g h p r o d u c e r s were t e s t e d f u r t h e r i n shake
flask cultures.
M u t a n t I M R - 1 E 1 was i s o l a t e d , w h i c h , d e p e n d i n g o n
medium c o m p o s i t i o n ,
p r o d u c e d t w o t o t h r e e t i m e s more e n d o - c h i t i n a s e
a c t i v i t y and o t h e r components o f t h e c h i t i n o l y t i c enzyme s y s t e m t h a n the
wild
type.
After
chitobiase a c t i v i t y QMB1466,
i n d u c t i o n by
chitin,
appeared a t s i m i l a r
endo-chitinase
times for
suggesting possible co-ordinate
both IMR-1E1
control of
and and
t h e s e enzymes.
The r e s u l t s w e r e c o n s i s t e n t w i t h I M R - 1 E 1 c o n t a i n i n g a r e g u l a t o r y mutation
which
increased production
c h i t i n o l y t i c enzyme s y s t e m and/or
d u p l i c a t i o n o f t h e c h i t i n a s e genes. IMR-1E1 t o d e c r e a s e d l e v e l s o f
of
the
components
of
the
w i t h IMR-1E1 c o n t a i n i n g a tandem
The h i g h r a t e o f r e v e r s i o n o f
c h i t i n a s e p r o d u c t i o n suggested t h a t
t h e o v e r p r o d u c t i o n o f c h i t i n a s e by IMR-1E1 i s due t o a tandem gene duplication.
22 Dextranases A
l i m i t
dextrinase
ungerminated
peas
h y d r o l y s e s (1
+
by
has
affinity
6)-a-P-glucosidic
c o n t a i n i n g a t l e a s t one ( 1
*
been
purified
2,700-fold
chromatography.375 linkages
The
i n alpha-limit
4 ) - l i n k e d a-g-glucose
from enzyme
dextrins
r e s i d u e on e i t h e r
6: Enzymes side
of
499 the
susceptible
linkage.
The
dextrin,
glycogen b e t a - l i m i t
dextrin,
dextrinase
l i m i t
hydrolyses the polysaccharides amylopectin,
also
amylopectin beta-limit
and p u l l u l a n ,
b u t has no
a c t i v i t y towards glycogen. A t e c h n i q u e h a s been d e s c r i b e d f o r t h e s c r e e n i n g o f d e x t r a n a s e -
p r o d u c i n g r n i c r o - ~ r g a n i s m s , ~T h~e~ c u l t u r e o f f u n g i ( 5 5 6 s t r a i n s ) and s t r e p t o m y c e s (115 s t r a i n s ) was p e r f o r m e d by t h e k o j i m e t h o d on wheat b r a n a n d s u b m e r g e d m e t h o d i n l i q u i d media. were
e x t r a c t e d and t h e
aqueous e x t r a c t s
The c u l t u r e m e d i a
screened for
endo-
d e x t r a n o l y t i c a c t i v i t y by i n c u b a t i o n w i t h a 5 % d e x t r a n s o l u t i o n . The
activity
of
t h e e n z y m e was
measured by v i s c o m e t r y .
Eight
s t r a i n s w e r e s e l e c t e d and t h e pH a c t i v i t i e s and s t a b i l i t i e s o f t h e i r d e x t r a n a s e s were d e s c r i b e d . The g e n e r a l p r o p e r t i e s a n d s p e c i f i c i t y o f a d e x t r a n ( 1
*
2)-
d e b r a n c h i n g enzyme f r o m F l a v o b a c t e r i u m h a v e b e e n e x a m i n e d i n o r d e r t o apply
t h i s enzyme t o t h e s t r u c t u r a l a n a l y s i s o f
h i g h l y branched
d e ~ t r a n s . The ~ ~ ~o p t i m u m pH r a n g e and t e m p e r a t u r e w e r e pH 5.5 and 45'C,
respectively.
f o r 1 0 min, f o r 2 4 h.
-
a n d o v e r a pH r a n g e o f 6.5
have a l s o been examined.
9.0
o n i n c u b a t i o n a t 4OC
for
t h e (1
*
The d e b r a n c h i n g e n z y m e h a s a s t r i c t
2)-a-P-glucosidic
linkage a t branch points
d e x t r a n s and r e l a t e d b r a n c h e d o l i g o s a c c h a r i d e s ,
g l u c o s e as t h e o n l y r e d u c i n g s u g a r . d e x t r a n s by t h i s enzyme and t h e 8 - 1 2 9 8 s o l u b l e , 25.2%, 0.21, 1397,
6.5
The e f f e c t s o f v a r i o u s m e t a l i o n s a n d c h e m i c a l r e a g e n t s
specificity of
-
The enzyme was s t a b l e up t o 4 O o C o n h e a t i n g
11.8%,
0.91.
and p r o d u c e s Q-
The d e g r e e o f h y d r o l y s i s o f t h e
Em v a l u e
(mg m l - l )
w e r e as f o l l o w s :
8 - 1 2 9 9 s o l u b l e , 31.5%,
0.27,
and 8-
The d e b r a n c h i n g enzyme t h u s h a s a n o v e l t y p e o f
s p e c i f i c i t y as a d e x t r a n h y d r o l a s e . enzyme a d e x t r a n a-(1
+
The a u t h o r s h a v e t e r m e d t h i s
2 ) - d e b r a n c h i n g enzyme, and i t s s y s t e m a t i c
name i s a l s o d i s c u s s e d .
23
(1
* 3)-a-g-Glucanases
I n a new c o l o r i m e t r i c m e t h o d f o r t h e d e t e c t i o n and assay o f (1 + 3)-a-~-glucanases,
t h e enzyme s u b s t r a t e c o n s i s t s o f C i b r a c r o n B l u e
F3GA c o m p l e x e d w i t h a d e x t r a n a s e - t r e a t e d
The
method
i s
especially
convenient
streptococcal g l ~ c a n . ~ ~ '
for
tests
involving
large
n u m b e r s o f s a m p l e s , a n d c a n b e a d a p t e d t o q u a n t i t a t i v e a s w e l l as qualitative applications.
The a s s a y i s s u f f i c i e n t l y s e n s i t i v e f o r
Carbohydrate Chemistry
5 00
s c r e e n i n g b a c t e r i a l s a m p l e s a s p o t e n t i a l s o u r c e s of ( 1 + 3 ) - a - p glucanase. The p u r i f i c a t i o n and p r o p e r t i e s o f e n d o - ( l + 3 ) - a - g - g l u c a n a s e An from a S t r e p t o m y c e s c h a r t r e u s i s s t r a i n have been r e p o r t e d . 3 7 9 enzyme h y d r o l y s i n g t h e w a t e r - i n s o l u b l e g l u c a n s produced from s u c r o s e b y S t r e p t o c o c c u s m u t a n s was p u r i f i e d 6 . 4 - f o l d f r o m t h e c u l t u r e c o n c e n t r a t e of S. c h a r t r e u s i s s t r a i n F 2 b y i o n - e x c h a n g e chromatography and g e l f i l t r a t i o n . E l e c t r o p h o r e s i s of t h e p u r i f i e d enzyme p r o t e i n gave a s i n g l e band on a s o d i u m d o d e c y l s u l p h a t e polyacrylamide g e l slab. I t s m o l e c u l a r weight was e s t i m a t e d t o be approximately 68,000, b u t t h e r e i s a p o s s i b i l i t y t h a t t h e n a t i v e enzyme e x i s t s i n an a g g r e g a t e d form o r i s an o l i g o m e r of t h e p e p t i d e s u b u n i t s , having a m o l e c u l a r weight l a r g e r t h a n 300,000. The pH optimum of t h e enzyme was 5.5 - 6.0, and i t s t e m p e r a t u r e optimun was The 55OC. The enzyme l o s t a c t i v i t y on h e a t i n g a t 6 5 O C f o r 10 m i n . enzyme s c t i v i t y was c o m p l e t e l y i n h i b i t e d b y t h e p r e s e n c e of 1 m M Mn2+, Hg2+, C u 2 + , Ag2+, o r m e r t h i o l a t e . The Em v a l u e f o r t h e w a t e r i n s o l u b l e g l u c a n o f S . m u t a n s O M Z 1 7 6 was an amount o f g l u c a n e q u i v a l e n t t o 1.54 m M !-glucose, 0.89 m M i n t e r m s of t h e a - ( 1 + 3)-linked-p-glucose residue. The p u r i f i e d enzyme was s p e c i f i c f o r g l u c a n s c o n t a i n i n g an ( 1 + 3 ) - a - ! - g l u c o s i d i c l i n k a g e a s t h e major bond. The enzyme h y d r o l y s e d t h e S. mutans w a t e r - i n s o l u b l e g l u c a n s e n d o l y t i c a l l y , and t h e p r o d u c t s were o l i g o s a c c h a r i d e s .
e.
24
endo-(1
* 3)-B-!-Glucanases
A c t i o n p a t t e r n s of (1 + 3 ) - B - Q - g l u c a n a s e s from z a n t h i n e o l y t i c a h a v e been i n v e s t i g a t e d on l a m i n a r a n , l i c h e n a n , and y e a s t g l u c a n . 3 8 0 Four Q - g l u c a n a s e s i n t h e c r u d e culture broth a c t s y n e r g i s t i c a l l y t o degrade the w a l l s o f viable y e a s t . Enzyme I (mol. w t . 18,000) does not l y s e v i a b l e y e a s t c e l l s , e v e n t h o u g h i t d i s p l a y s h i g h a c t i v i t y a g a i n s t l a m i n a r a n , and w i l l r e a d i l y d e p o l y m e r i z e l i c h e n a n . Enzyme I1 i s a l s o an endo-glucanase (mol. w t . 29,000) and i s a c t i v e a g a i n s t both v i a b l e y e a s t c e l l s and laminaran, b u t i t e x h i b i t s r e s t r i c t e d a c t i v i t y against lichenan. Enzymes I I I a and I I I b have m o l e c u l a r w e i g h t s o f 27,000 and 29,000, r e s p e c t i v e l y , and e x h i b i t h i g h l y t i c a c t i v i t y a g a i n s t v i a b l e y e a s t c e l l s , b u t r e l e a s e few r e d u c i n g g r o u p s f r o m l a m i n a r a n o r y e a s t glucan. They e x h i b i t h i g h e r s p e c i f i c a c t i v i t y a g a i n s t y e a s t glucan
O-e-r-s-k o v i a
6: Enzymes
501
t h a n a g a i n s t l a m i n a r a n and y i e l d an o l i g o s a c c h a r i d e of d.p. 5 a s t h e I n t h e i n i t i a l s t a g e s of h y d r o l y s i s , eventual hydrolysis product. t h e y a c t i n an e n d o m a n n e r , b u t t h e y e f f e c t c l e a v a g e of a d . p . 1 4 s u b s t r a t e i n an a p p a r e n t m g manner and h a v e h i g h s p e c i f i c i t y f o r s t r a i g h t - c h a i n B-( 1 + 3 ) - l i n k e d q-glucans. The proposed s y s t e m a t i c name f o r e n z y m e s I I I a and I I I b i s ( 1 * 3 ) - B - Q - g l u c a n p e n t a o s e h y d r o l a s e . When y e a s t glucan was hydrolysed by enzyme I I I b , 40% o f t h e glucan was r e c o v e r e d a s t h e p r o d u c t s of d.p. 5 and 9 , 20% was s o l u b i l i z e d b u t r e m a i n e d a s o l i g o s a c c h a r i d e s h a v i n g d.p. o f b e t w e e n 15 and 3 5 , 2 4 % had a d . p . > 3 5 , and 2 0 % was i n s o l u b l e m a t e r i a l t h a t r e s e m b l e d yeast-bud s c a r s m i c r o s c o p i c a l l y . C e l l - f r e e e x t r a c t s , membranous f r a c t i o n s , and c e l l - w a l l p r e p a r a t i o n s from Schizosaccharomyces p o m k were examined f o r t h e p r e s e n c e of (1 + 3 ) - a - and (1 + 6)-B-D-glucanase a c t i v i t i e s . 3 8 1 The v a r i o u s g l u c a n a s e s were assayed i n c e l l s a t d i f f e r e n t growth s t a g e s . O n l y ( 1 + 3)-B-;-glucanase a c t i v i t y was f o u n d , a n d t h i s w a s associated with the cell-wall fraction. Chromatographic f r a c t i o n a t i o n of t h e c r u d e enzyme r e v e a l e d t w o e n d o - ( 1 + 3>-8-Qg l u c a n a s e s , d e s i g n a t e d a s g l u c a n a s e I and g l u c a n a s e 11. Glucanase I c o n s i s t e d o f two s u b u n i t s o f m o l e c u l a r w e i g h t s 7 8 , 5 0 0 and 8 2 , 0 0 0 , and g l u c a n a s e I 1 was a s i n g l e p o l y p e p t i d e o f 7 5 , 0 0 0 . A l t h o u g h b o t h enzymes had s i m i l a r s u b s t r a t e s p e c i f i c i t i e s and s i m i l a r h y d r o l y t i c a c t i o n on l a m i n a r i n , g l u c a n a s e I 1 had much h i g h e r h y d r o l y t i c On t h e b a s i s of a c t i v i t y on i s o l a t e d c e l l w a l l s of S. pombe. d i f f e r e n t i a l l y t i c a c t i v i t y on c e l l w a l l s , g l u c a n a s e I1 was shown t o be p r e s e n t i n c o n j u g a t i n g c e l l s and h i g h e s t i n s p o r u l a t i n g cells. Glucanase I1 appeared t o be s p e c i f i c a l l y i n v o l v e d i n c o n j u g a t i o n and s p o r u l a t i o n s i n c e v e g e t a t i v e c e l l s and n o n - c o n j u g a t i n g and nons p o r u l a t i n g c e l l s d i d n o t c o n t a i n t h i s enzyme. The a p p e a r a n c e o f g l u c a n a s e I 1 i n c o n j u g a t i n g c e l l s may be due t o de novo enzyme s y n t h e s i s s i n c e no a c t i v a t i o n c o u l d b e d e m o n s t r a t e d b y c o m b i n i n g e x t r a c t s from v e g e t a t i v e and c o n j u g a t i n g c e l l s . I n c r e a s e d g l u c a n a s e a c t i v i t y o c c u r r e d when w a l l s from c o n j u g a t i n g c e l l s were combined w i t h w a l l s from s p o r u l a t i n g c e l l s . S t u d i e s w i t h t r y p s i n and p r o t e o l y t i c i n h i b i t o r s s u g g e s t t h a t g l u c a n a s e 11 e x i s t s a s a zymogen i n conjugating c e l l s . A t e m p e r a t u r e - s e n s i t i v e mutant of S. pombe was i s o l a t e d which l y s e d a t 37OC. Glucanase a c t i v i t y was h i g h e r i n v e g e t a t i v e c e l l s h e l d a t 3 7 O C t h a n c e l l s h e l d a t 25'C. Unlike t h e w i l d - t y p e s t r a i n , t h i s mutant c o n t a i n e d g l u c a n a s e I1 a c t i v i t y d u r i n g v e g e t a t i v e growth and may be a r e g u l a t o r y mutant.
502
Carbohydrate Chemistry 25
=-(
1
+
3)-8-g-Glucanases
I t h a s been r e p o r t e d t h a t g l y c o s y l a t i o n i s n o t n e c e s s a r y f o r the secretion of gxg-(l + 3 ) - B - D = - g l u c a n a s e by ----Saccharomyces c e r e v i s i a e p r o t o p l a s t s . 3 8 2 I t was shown t h a t t h e normal s y n t h e s i s and s e c r e t i o n of t h e major z - ( l + 3)-B-D-glucana s e s e c r e t e d b y S. c e r e v i s i a e p r o t o p l a s t s w e r e a f f e c t e d b y t u n i c a m y c i n , and a new, presumably n o n - g l y c o s y l a t e d form i s e x p o r t e d t o t h e c e l l u l o s e medium. A s i m i l a r f o r m was a l s o s e c r e t e d i n t h e p r e s e n c e of 2-deoxy-q-glucose. P a r t i a l p u r i f i c a t i o n of e n d o - and a - ( l + 3 ) - B - Q - g l u c a n a s e enzymes from Zea mays s e e d l i n g s and t h e i r involvement i n c e l l - w a l l a u t o h y d r o l y s i s have been i n v e s t i g a t e d . 3 8 3 Molecular-sieve chromatography of t h e c e l l - w a l l p r o t e i n r e s o l v e d endo-B-Q-glucanase and g ~ - ( +l 3 ) - B - Q - g l u c a n a s e a c t i v i t i e s when Avena g l u c a n and l a m i n a r a n , r e s p e c t i v e l y , were employed a s s u b s t r a t e s . The exoenzyme ( m o l . w t . 6 0 , 0 0 0 ) was s t r o n g l y i n h i b i t e d b y i n o r g a n i c m e r c u r y a t a c o n c e n t r a t i o n which s u p p r e s s e d t h e r e l e a s e of monosaccharide from a u t o l y t i c a l l y a c t i v e c e l l w a l l . The e n d o - B - Q - g l u c a n a s e ( m o l . w t . 26,000) showed a marked p r e f e r e n c e f o r s u b s t r a t e s of mixed-linkage and e x h i b i t e d f e a t u r e s i n d i c a t i n g t h a t i t i n i t i a t e s t h e a u t o l y t i c s o l u b i l i z a t i o n of w a l l glucan. * 3)-B-EThe p u r i f i c a t i o n and some p r o p e r t i e s of an = - ( l g l u c a n a s e f r o m b a s i d i o m y c e t e s p e c i e s h a v e been d e s c r i b e d . 3 8 4 An e x o - ( 1 + 3)-B-!-glucanase was p u r i f i e d f r o m t h e c o m m e r c i a l enzyme K i t a l a s e , w h i c h i s a y e a s t c e l l - w a l l l y t i c enzyme p r e p a r a t i o n , b y ammonium s u l p h a t e f r a c t i o n a t i o n , ion-exchange chromatography, and gel filtration. The optimum pH v a l u e was 5 . 8 , and t h e optimum t e m p e r a t u r e was 5 5 ' C . The enzyme was s t a b l e i n t h e pH r a n g e of 5.1 - 9.8 and a t t e m p e r a t u r e s below 53'C. The i s o e l e c t r i c p o i n t and t h e m o l e c u l a r w e i g h t w e r e e s t i m a t e d t o b e pH 9 . 3 a n d 7 3 , 0 0 0 , respectively. The enzyme was shown t o b y p a s s ( 1 -* 6 ) - l i n k a g e d b r a n c h e s t o c l e a v e ( 1 + 3 ) - l i n k a g e s when s c l e r o g l u c a n was u s e d a s s u b s t r a t e . T h e Em v a l u e s f o r l a m i n a r i n , l a m i n a r i p e n t a o s e , l a m i n a r i t e t r a o s e , and l a m i n a r i t r i o s e w e r e 0 . 1 6 , 2 . 0 1 , 2.24, a n d 1.34 m M , respectively.
503
6: Enzymes 26
endo-( 1
*
4)-B-p-Glucanases
The s p e c i f i c p r o p e r t i e s h a v e been e x a m i n e d o f t h e ( 1
4)-8-n-
+
g l u c a n a s e component o f T r i c h o d e r m a k o n i n q i i t h a t p a r t i c i p a t e s i n an e a r l y a n d e f f e c t i v e s t a g e o f random b r e a k d o w n o f n a t i v e c e l l u l o s e t o s h o r t fibres.385 components that
of
The enzyme was p u r i f i e d and f r e e d f r o m a s s o c i a t e d t h e c e l l u l a s e complex
s u c h enzymes.
B-q-glucosidase)
P u r i f i c a t i o n increased the s p e c i f i c a c t i v i t y 25-fold
over c u l t u r e f i l t r a t e s .
The enzyme h y d r o l y s e d C M - c e l l u l o s e
t h a n t h e p u r i f i e d B-g-glucosidase any
(particularly
i n t e r f e r e w i t h and c o m p l i c a t e i n t e r p r e t a t i o n o f t h e a c t i o n o f
of
i t s
substrates
specificity
of
d e r i v a t i v e s of
the
(cellobiose
glucanase
cellulose.
faster
f r o m t h e same o r g a n i s m h y d r o l y s e d was
or
cellodextrins).
directed
towards
The
soluble
The g l u c a n a s e ( t e m p e r a t u r e o p t i m u m 6 O o C )
attacked larger c e l l o d e x t r i n s (cellohexaose t o cellotetraose, i n t h a t o r d e r ) much m o r e r e a d i l y t h a n s m a l l d e x t r i n s ( c e l l o b i o s e a n d c e l l o t r i o s e ) and r e l e a s e d a m i x t u r e o f p r o d u c t s , cellopentaose.
Q - g l u c o s e up t o
S i m i l a r examination o f hydrolysates o f t h e reduced
c e l l o d e x t r i n s showed c l e a r l y t h e h i g h s p e c i f i c i t y o f t h e enzyme f o r t h e c e n t r a l bond o f i t s n a t u r a l s u b s t r a t e s ( t h e c e l l o d e x t r i n s ) , whatever t h e i r chain length,
and i n d i c a t e d t h e n a t u r e o f t h e enzyme
as an e n d o - g l u c a n a s e . A new u l t r a s o n i c m e t h o d h a s been d e v e l o p e d f o r
c o m p o s i t i o n and p r o p e r t i e s of
determining the
i n d i v i d u a l components o f
s y s t e m s w i t h o u t t h e i r p r e l i m i n a r y separation.’’’
polyenzyme
The m e t h o d i s
b a s e d on d e t e r m i n a t i o n o f t h e pH dependence o f t h e r a t e c o n s t a n t s o f i n a c t i v a t i o n o f i n d i v i d u a l c o m p o n e n t s o f t h e enzyme s y s t e m u n d e r t h e a c t i o n of c a v i t a t i o n a l ultrasound.
T h e m e t h o d was u s e d t o s t u d y
c e l l u l a s e complex from t h e fungus G e o t r i c h u m candidum. contains at glucanase,
least
exo-(l
-+
four
c e l l u l o l y t i c enzymes,
4)-B-!-glucanase,
T h i s complex
endo-(1
B-Q-glucosidase,
+
4)-B-g-
and a r y l - B - g -
g l u c o s i d a s e , w h i c h d i f f e r i n v a l u e s o f pK o f t h e i o n o g e n i c g r o u p s , c o n t r o l l i n g t h e pH p r o f i l e s o f
ultrasonic inactivation,
as w e l l as
i n values o f t h e r a t e constants o f i n a c t i v a t i o n o f the form o f the a c t i v e s i t e most s t a b l e t o t h e a c t i o n o f u l t r a s o u n d .
endo-(1
-------mocellum
-+
by
4)-B-!-Glucanase
was p u r i f i e d f r o m C l o s t r i d i u m t h e r -
centrifugation,
p r e c i p i t a t i o n , ion-exchange,
u l t r a f i l t r a t i o n ,
selective
Sephadex c h r o m a t o g r a p h y , a n d p r e p a r a t i v e
g e l e l e c t r o p h o r e ~ i s . T~h~e ~2 2 - f o l d - p u r i f i e d
enzyme behaved as a
homogeneous p r o t e i n under n o n - d e n a t u r i n g c o n d i t i o n s . The e n z y m e r e p r e s e n t e d a s i g n i f i c a n t component ( > 25%) o f t o t a l e x t r a c e l l u l a r
5 04
Carbohydrate Chemistry
e n d o - g l u c a n a s e a c t i v i t y , b u t was p u r i f i e d i n low y i e l d b y t h e p r o c e d u r e s employed. The n a t i v e m o l e c u l a r weight of t h e endo-(1 + 4 ) - B - e - g l u c a n a s e was d e t e r m i n e d b y u l t r a c e n t r i f u g a t i o n a l a n a l y s i s , amino a c i d c o m p o s i t i o n , and p o l y a c r y l a m i d e - g e l e l e c t r o p h o r e s i s and T h e enzyme c o n t a i n e d 11.2% v a r i e d b e t w e e n 8 3 , 0 0 0 and 9 4 , 0 0 0 . c a r b o h y d r a t e and was i s o e l e c t r i c a t pH 6.72. The pH and t e m p e r a t u r e o p t i m a of t h e endo-glucanase were 5.2 and 62'C, r e s p e c t i v e l y . The enzyme l a c k e d B - c y s t e i n e and was low i n s u l p h u r - c o n t a i n i n g a m i n o acids. The p u r i f i e d e n d o - ( 1 + 4 ) - B - Q - g l u c a n a s e d i s p l a y e d : h i g h a c t i v i t y towards carboxymethylcellulose, celloheptaose, cellohexaose, and c e l l o p e n t a o s e , low a c t i v i t y t o w a r d s A v i c e l m i c r o c r y s t a l l i n e c e l l u l o s e and c e l l o t e t r a o s e , no d e t e c t a b l e a c t i v i t y t o w a r d s increased a c t i v i t y towards c e l l o c e l l o t r i o s e or cellobiose, The o l i g o s a c c h a r i d e s w i t h i n c r e a s i n g d e g r e e of p o l y m e r i z a t i o n . i n t e r n a l g l y c o s i d i c bonds of c e l l o - o l i g o s a c c h a r i d e s were c l e a v e d b y t h e enzyme i n p r e f e r e n c e t o e x t e r n a l l i n k a g e s . K m and l m a f ox r c e l l o p e n t a o s e and c e l l o h e x a o s e h y d r o l y s i s were 2.30 m M and 39.3 pmol m i n - l p e r mg o f p r o t e i n and 0.56 m M and 58.7 umol m i n - ' p e r mg of protein, respectively. The k i n e t i c p r i n c i p l e s of t h e f o r m a t i o n of !-glucose and c e l l o b i o s e i n t h e h y d r o l y s i s of m i c r o c r y s t a l l i n e c e l l u l o s e under t h e a c t i o n of c e l l u l a s e complexes from e i g h t d i f f e r e n t s o u r c e s have By s u c c e s s i v e a d d i t i o n of been s t u d i e d e x p e r i m e n t a l l y . 1 9 4 i n d i v i d u a l c o m p o n e n t s of t h e c e l l u l a s e c o m p l e x ( e n d o - ( 1 + 4 ) - B - q g l u c a n a s e and B - a - g l u c o s i d a s e ) t o t h e r e a c t i o n s y s t e m , t h e s t e p s l i m i t i n g t h e r a t e of e n z y m a t i c h y d r o l y s i s of m i c r o c r y s t a l l i n e c e l l u l o s e were r e v e a l e d . I t was shown t h a t i n most o f t h e c a s e s s t u d i e d t h e s t e p d e t e r m i n i n g t h e r a t e of f o r m a t i o n o f Q - g l u c o s e w i t h t h e p a r t i c i p a t i o n of i n t e r m e d i a t e c e l l o b i o s e i s t h e a c t i o n of O n l y i n one c a s e (complex from A s p e r g i l l u s f o e t i B-g-glucosidase. &, e n r i c h e d w i t h B-g-glucosidase) i s t h e r a t e o f f o r m a t i o n of g l u c o s e from m i c r o c r y s t a l l i n e c e l l u l o s e l i m i t e d by t h e a c t i o n of t h e I n accordance endo-(1 * 4)-B-Q-glucanase of t h e c e l l u l a s e complex. w i t h t h e k i n e t i c p r i n c i p l e s d e v e l o p e d , i t was shown t h a t when an e x c e s s of B-2-glucosidase i s added t o t h e r e a c t i o n s y s t e m t h e s t e p l i m i t i n g t h e r a t e of h y d r o l y s i s of m i c r o c r y s t a l l i n e c e l l u l o s e under t h e a c t i o n of a l l t h e c e l l u l a s e complexes s t u d i e d becomes t h e a t t a c k on t h e i n i t i a l i n s o l u b l e s u b s t r a t e b y e n d o - ( 1 + 4 ) - B - Q - g l u c a n a s e . U n d e r t h e same c o n d i t i o n s , a l i n e a r c o r r e l a t i o n was f o u n d b e t w e e n t h e s t e a d y - s t a t e r a t e o f f o r m a t i o n of Lj-glucose f r o m m i c r o c r y s t a l l i n e c e l l u l o s e u n d e r t h e a c t i o n of a l l t h e c e l l u l a s e c o m p l e x e s
e-
6: Enzymes
505
s t u d i e d , on t h e one h a n d , and t h e a c t i v i t y of e n d o - ( 1 + 4 ) - 8 - Q g l u c a n a s e i n t h e s e c o m p l e x e s , on t h e o t h e r . I t was shown t h a t t h e a c t i o n of a l l t h e c e l l u l a s e c o m p l e x e s s t u d i e d ( s e l e c t e d r a t h e r a r b i t r a r i l y ) i s d e s c r i b e d by e s s e n t i a l l y t h e same k i n e t i c p r i n c i p l e s , which i s e v i d e n c e o f t h e same m e c h a n i s m s of t h e h y d r o l y s i s of i n s o l u b l e c e l l u l o s e b y c e l l u l a s e r e p a r a t i o n s of different origin. During growth of B a c t e r i o d e s s u c c i n o g e n e s i n a l i q u i d medium w i t h c e l l u l o s e a s t h e s o u r c e of c a r b o h y d r a t e , g r e a t e r t h a n 8 0 % of t h e endo-( 1 + 4)-B-Q-glucanase , x y l a n a s e , and a r y l - B - a - x y l o s i d a s e and 5 0 % o f t h e a r y l - 6 - Q - g l u c o s i d a s e was r e l e a s e d from c e l l s i n t o t h e c u l t u r e fluid.227 B y u s i n g QAE-Sephadex A50 chromatography i n t h e p r e s e n c e of 6 M u r e a , i t was p o s s i b l e t o s p l i t t h e g l u c a n a s e complex o f C l o s t r i d i u m thermocellum i n t o d i s t i n c t p r o t e i n f r a c t i o n s . 3 8 7 One of t h e s e f r a c t i o n s c o n t a i n e d an e n d o - ( 1 + 4 ) - B - e - g l u c a n a s e which was i s o l a t e d a t a h i g h d e g r e e of p u r i t y and was i d e n t i f i e d b y i t s a b i l i t y t o h y d r o l y s e t r i n i t r o p h e n y l a t e d c a r b o x y m e t h y l c e l l u l o s e . The enzyme i s o f monomeric n a t u r e , w i t h a m o l e c u l a r w e i g h t o f 5 6 , 0 0 0 . I t h a s an i s o e l e c t r i c pH of 6 . 2 and an optimum pH of 6.0. It h y d r o l y s e d c a r b o x y m e t h y l c e l l u l o s e and, a t a s l o w e r r a t e , c e l l u l o s e powder. The m a j o r e n d - p r o d u c t s o f c e l l u l o s e d e g r a d a t i o n w e r e 9 g l u c o s e , c e l l o b i o s e , and c e l l o t r i o s e . C e l l o t e t r a o s e i s formed a s an i n t e r m e d i a t e product. T h e f o r m a t i o n and l o c a t i o n of (1 + 4 ) - B - Q - g l u c a n a s e s and ( 1 + 4)-B-!-glucosidases h a v e been s t u d i e d i n c u l t u r e s of P e n i c i l l i u m j a n t h i n e l l u m grown on A v i c e l , sodium carboxymethyl c e l l u l o s e , c e l l o b i o s e , g l u c o s e , mannose, and m a l t o s e . 2 2 2 e n d o - ( 1 + 4 ) - B - k Glucanases were c e l l f r e e , and t h e i r f o r m a t i o n was induced by c e l l o biose. (1 + 4)-B-P-Glucosidases, on t h e o t h e r h a n d , w e r e f o r m e d c o n s t i t u t i v e l y and were p r i m a r i l y c e l l f r e e , b u t w i t h a s m a l l amount strongly associated w i t h the c e l l wall. A rotational viscosimetric method was developed t o measure t h e t o t a l endo-(1 + 4)-B-Q-glucanase a c t i v i t y o f t h e c u l t u r e ( b r o t h and s o l i d s ) . B y t h i s method, i t was p o s s i b l e t o d e t e r m i n e t h e endo-(1 + 4)-B-Q-glucanase a c t i v i t y not o n l y i n t h e s u p e r n a t a n t o f t h e c u l t u r e b u t a l s o on t h e s u r f a c e of t h e m y c e l i u m o r a b s o r b e d on r e s i d u a l A v i c e l . D u r i n g a 70 l i t r e b a t c h c u l t i v a t i o n of P. j a n t h i n e l l u m , t h e a d s o r p t i o n of e n d o - ( 1 + 4 ) - B - Q - g l u c a n a s e s b y r e s i d u a l and newly a d d e d 1 0 % A v i c e l was m e a s u r e d . The a d s o r p t i o n o f s o l u b l e p r o t e i n and e n d o - ( 1 + 4)-B-Qg l u c a n a s e b y A v i c e l was f o u n d t o be l a r g e l y i n d e p e n d e n t of t h e pH
Carbohydrate Chemktry
506 v a l u e b u t d e p e n d e n t on t e m p e r a t u r e . The k i n e t i c s o f
g r o w t h and e x t r a c e l l u l a r
cellulase production
by Talaromyces e m e r s o n i L grown on c e l l u l o s i c measured.198
material
were
The enzyme s y s t e m was f o u n d t o b e c o m p r i s e d o f f o u r t o
f i v e f o r m s o f ~ g - (+l 4 ) - B - Q - g l u c a n a s e a n d a t l e a s t t w o f o r m s o f + 4)-B-P-glucanase and t h r e e 6 - Q - g l u c o s i d a s e s . One o f t h e
endo-(1
l a t t e r , t e r m e d B - Q - g l u c o s i d a s e 111, i s i n d u c e d c o n c u r r e n t l y w i t h t h e c e l l u l a s e s b u t d i s a p p e a r s f r o m t h e medium b e c a u s e o f t h e l o w pH t h a t develops d u r i n g growth. stable.
The c e l l u l a s e s b y c o n t r a s t a r e m o r e a c i d
The p o s s i b l e f u n c t i o n s o f t h e s e enzymes a r e d i s c u s s e d .
Production o f
-F-u-s a r i u m
sp.
extracellular
cellulase
by
an
has been s t u d i e d i n shake c u l t u r e s ,
isolate of
appearance o f c e l l u l a s e components (8-g-glucosidase day,
endo-(1
third
day
*
4)-B-;-glucanase
of
growth
on
and
on t h e f i r s t
s-(l + 4I-B-g-glucanase
insoluble
cellulose)
a
and s e q u e n t i a l
was
on t h e
observed.211
Maximum p r o d u c t i o n o f a l l t h e s e c o m p o n e n t s was a c h i e v e d on t h e f i f t h day.
The i n f l u e n c e o f d i f f e r e n t n i t r o g e n a n d c a r b o n s o u r c e s on
c e l l u l a s e p r o d u c t i o n was
investigated.
Crude
cellulolytic
enzyme
was u s e d f o r h y d r o l y s i s o f common a g r i c u l t u r a l w a s t e s b o t h w i t h and w i t h o u t sodium hydroxide pretreatment.
Analysis o f hydrolysates
i n d i c a t e d g l u c o s e as t h e m a j o r c o n s t i t u e n t ( a b o u t
83% o f
total
r e d u c i n g s u g a r 1. P u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f t h e e x t r a c e l l u l a r and intracellular
--Talaromyces
6-g-glucosidases
t h a t B-g-glucosidase glucosidases
of
the
thermophillic
e m e r s o n i i have been d e s c r i b e d . while
fungus
The a u t h o r s c o n c l u d e
I a n d B - g - g l u c o s i d a s e I V a r e t r u e B-a-
B-g-glucosidase
111
i s
an
=-[l + 4)-B-Q-
g l u c a n a s e .199
27
endo-( 1
An e n d o - ( 1 the
*
+
6)-B-P-Glucanases 6)-B-Q-glucanase
o f F l a v o b a c t e r i u m M64 h y d r o l y s e s
octasaccharide repeating u n i t
tetra saccharide^.^^^
An
endo-(1
+
of
succinoglycan
6)-B-Q-glucanase
t o
capable
two of
hydrolysing the octasaccharide repeating u n i t o f succinoglycan t o t w o t e t r a s a c c h a r i d e s h a s been i s o l a t e d f r o m c e l l s o f F l a v o b a c t e r i u m . One t e t r a s a c c h a r i d e was c o m p o s e d o f Q - g l u c o s e , p y r u v i c a c i d (4:1:1,
molar r a t i o ) ,
g l u c o s e and P - g a l a c t o s e (3:1, t h e (1 + 6 ) - B - Q - g l u c o s i d i c
s u c c i n i c acid, and
and t h e o t h e r was composed of
molar r a t i o ) .
l i n k a g e t o t h e (1
a-
T h i s enzyme h y d r o l y s e d
*
6 ) - l i n k e d B-Q-glucose
507
6: Enzymes
r e s i d u e i n t h e o c t a s a c c h a r i d e r e p e a t i n g u n i t o f s u c c i n o g l y c a n and also
hydrolysed
the
octasaccharide
p o l y s a c c h a r i d e s p r o d u c e d by
many
repeating units
strains
of
of
similar
Agrobacterium
and
Rhizobium species. The
structure
succinoglycan
of
the
extracellular
from Alcaligenes
faecal&
acidic
polysaccharide
has been e l u c i d a t e d by
s u c c e s s i v e f r a g m e n t a t i o n o f t h e p o l y s a c c h a r i d e w i t h e x t r a c e l l u l a r Be - g l y c a n a s e ( s u c c i n o g l y c a n d e p o l y m e r a s e ) and i n t r a c e l l u l a r e n d o - ( 1 + 6)-8-P-glucanase tetrasaccharides
from
12
Flavobacterium
sp.
M64
i n t o
two
i t s o c t a s a c c h a r i d e r e p e a t i n g u n i t and t h e n
m e t h y l a t i o n a n a l y s i s and e n z y m i c h y d r o l y s i s o f t h e p r o d u c t s . 3 8 9
P-Glucanases (Miscellaneous)
28 The
use
of
dye-polysaccharide
interactions
has
i n v e s t i g a t e d f o r a $-!-glucanase
assay.390
i n p a r t i c u l a r c e r e a l B-8-glucans
and s u b s t i t u t e d c e l l u l o s e s ,
been
Certain polysaccharides, induce
b a t h o c h r o m i c s h i f t s i n t h e a d s o r p t i o n s p e c t r a o f s u c h d i r e c t dyes as Congo
Red.
interaction,
As
cello-oligosaccharides
showed
it
followed
of
that
formation
complexes m i g h t be u s e f u l i n s t u d i e s of
l i t t l e
or
no
dye-polysaccharide
B-e-glucanase
action.
This
p o s s i b i l i t y h a s been i n v e s t i g a t e d by m o n i t o r i n g t h e d i g e s t i o n o f sample of o a t B-Q-glucan w i t h p u r i f i e d B-Q-glucan-endohydrolase S o l u t i o n s o f oat B-Q-glucan
Bacillus subtilis.
a
from
(pH 6.5)
(0.5% w/v)
w e r e t r e a t e d w i t h e n z y m e a n d t h e i r v i s c o s i t y was m o n i t o r e d ,
and
aliquots
dye
were removed a t
intervals,
i n t e r a c t i o n and r e d u c i n g s u g a r . r e l a t i o n s h i p between
The
heated,
and t e s t e d f o r
results
clearly
showed a
loss i n v i s c o s i t y and l o s s i n d y e i n t e r a c t i o n ,
whereas measurements of
r e d u c i n g sugar were n o t u s e f u l u n t i l l a t e r
s t ages o f d e g r a d a t i o n . P a r t i a l p u r i f i c a t i o n o f endo-
and E-B-Q-glucanase
enzymes
f r o m Zea mays s e e d l i n g s h a s b e e n d e s c r i b e d and t h e i r i n v o l v e m e n t i n c e 11- w a 1 1 a u t o h y d r o l y s is
i n v e s t i g a t e d . 391
chromatography o f t h e c e l l - w a l l and exo-B-g-glucanase
a c t i v i t i e s when A v e n a g l u c a n and l a m i n a r a n ,
r e s p e c t i v e l y , were e m p l o y e d as s u b s t r a t e s . w e i g h t 60,000)
M o l e c u l a r - s i eve
p r o t e i n r e s o l v e d endo-B-g-glucanase The exoenzyme ( m o l e c u l a r
was s t r o n g l y i n h i b i t e d b y H g 2 + a t a c o n c e n t r a t i o n
which suppressed t h e r e l e a s e o f active c e l l wall.
monosaccharide f r o m
The e n d o - B - Q - g l u c a n a s e
(mol.wt.
autolytically
26,000),
which
Carbohydrate Chemistry
508 showed
a
marked
exhibited
for
preference
features
substrates
it
indicating that
of
mixed
initiates
the
linkage, autolytic
s o l u b i l i z a t i o n o f w a l l glucan. (1
+
3),(1
+
4 ) - B - ~ - G l u c a n a s e has been p u r i f i e d 1 4 2 - f o l d f r o m
B a c i l l u s s u b t i l i s by c h r o m a t o g r a p h y on c e l l u l o s e and SE-Sephadex
C-
50 f o l l o w e d b y g e l f i l t r a t i o n t o h o m o g e n e i t y t h r o u g h A c r y l e x P SOS
60.392
electrophoresis
w e i g h t s f o r t h e enzyme o f
and
30,000
gel
filtration
a n d 33,000,
4.5) does n o t c o n t a i n t r y p t o p h a n , enzyme (PI
gave
molecular
respectively.
The
f r e e s u l p h y d r y l groups,
or carbohydrates. I t h a s been shown t h a t c e l l o b i o s e , i n c o n t r a s t t o a - g l u c o s e ,
is
a r e g u l a t o r o f t h e a c t i v i t y o f endoglucanases o f c e l l u l a s e complexes f r o m v a r i o u s sources.393 molecular-weight
C e l l o b i o s e i s an a c t i v a t o r o f t h e l o w -
m-;-glucanase
f r o m T.
k o n i n g i i and an i n h i b i t o r
o f t h e h i g h - m o l e c u l a r - w e i g h t endo-p-glucanases Kinetic
of
analysis
c e l l o b i o s e or
the
reaction
showed
rnethylcellobiose t o the
f r o m t h e same s o u r c e . that
the
binding
low-molecular-weight
of
e - 9 -
g l u c a n a s e l e a d s t o a s i x f o l d i n c r e a s e i n t h e r a t e o f d e g r a d a t i o n of carboxymethylcellulose.
The a c t i v a t i n g a c t i o n o f c e l l o b i o s e i s due
t o i t s a b i l i t y t o play t h e r o l e o f a supplementary n u c l e o p h i l i c agent
i n
the
reaction
of
transglycosylation
of
intermediate
oligosaccharides i n the enzymatic hydrolysis o f cellulose. A new Q - g l u c a n a s e p r o d u c e d b y a m a r i n e B a c i l l u s s p e c i e s h a s been r e p o r t e d . 3 9 4
An i s o l a t e o f b a c t e r i a f r o m m a r i n e mud was f o u n d
t o p r o d u c e a new enzyme c a p a b l e o f h y d r o l y s i n g i n s o l u b l e g - g l u c a n s produced by o r a l s t r e p t o c o c c i .
The s u b s t r a t e s p e c i f i c i t y o f t h e
enzyme i n d i c a t e d t h a t t h e enzyme was a new a - Q - g l u c a n a s e . The
structure
of
the
extracellular
s u c c i n o g l y c a n f r o m A l c a l i g e n e s f a e c a l i s var. e l u c i d a t e d by
successive
ex t r a c e 1l u l a r intracellular
fragmentation
a-Q-gly canase
endo-B-(1
+
i n t o two tetrasaccharides
polysaccharide
myxoqenes 10C3 h a s been the
polysaccharide
with
( s u c c i n o g l y c a n d e p o l y m e r a s e ) and
6)-Q-glucanase
via
of
acidic
of
F l a v o b a c t e r i u m sp.
M64
i t s octasaccharide r e p e a t i n g unit.389
Polyacrylamide g e l electrophoresis o f
t h e c e l l u l o l y t i c system
f r o m t h e c u l t u r e s u p e r n a t e s o f A c e t i v i b r i o c e l l u l o l y t i c u s h a s shown t h e p r e s e n c e o f f o u r m a j o r enzymes:
a B-Q - -glucosidase,an
g l u c a n a s e , and t w o & - ~ - g l u ~ a n a s e s . ~ ~ ~
w-Q-
509
6: Enzymes
Glucoamylases
29
method
A
for
the
automatic
measurement
of
a-amylase
and
g l u c o a m y l a s e a c t i v i t i e s d u r i n g f e r m e n t a t i o n h a s b e e n d e v e l o p e d . 314 F o r f u r t h e r d e t a i l s see i n i t i a l c i t a t i o n o f
endo-
ref.314.
A k i n e t i c equation which represents a s y n e r g i s t i c action of
and exo-enzyme
tested
by
upon p o l y s a c c h a r i d e s ,
experiments
using
adequacy
immobilized
has been
a-amylase
and
g l u c o a m y l a s e .268 P h y s i c a l e n t r a p m e n t h a s b e e n u s e d a s an a p p r o a c h t o a c h i e v e t h e r m a l s t a b i l i z a t i o n o f Q - g l u c o s e o x i d a s e and g l u ~ o a m y l a s e . ~The ~~
-t
values
for
the t h e r m o i n a c t i v a t i o n of
!-glucose
oxidase
and
g l u c o a m y l a s e were i n c r e a s e d s e v e r a l - f o l d by t h e i r e n t r a p m e n t i n polyacrylamide
gels.
I n polyacrylate
behaved d i f f e r e n t l y ,
probably
gels
owing t o
the
i n d i v i d u a l enzymes
microenvironmental effects
a r i s i n g by t h e p o l y e l e c t r o l y t e n a t u r e o f t h e c a r r i e r . I n t r i n s i c k i n e t i c constants
for
pore-diffusion-limited
i m m o b i l i z e d enzyme r e a c t i o n s have been e s t i m a t e d . 3 9 6
A simple
method u s i n g t h e A r i s - B i s c h o f f
intrinsic
rate
parameters
when
slow
modulus
that
establishes
pore d i f f u s i o n
of
substrate
limits
i m m o b i l i z e d enzyme r e a c t i o n s t h a t obey M i c h a e l i s - M e n t e n k i n e t i c s has been p r e s e n t e d .
Data a t h i g h s u b s t r a t e c o n c e n t r a t i o n s , where t h e
enzyme w o u l d b e s a t u r a t e d i n t h e absence o f d i f f u s i o n l i m i t a t i o n , and a t l o w s u b s t r a t e c o n c e n t r a t i o n s ,
where e f f e c t i v e n e s s f a c t o r s a r e
i n v e r s e l y p r o p o r t i o n a l t o r e a c t i o n modulus, maximum
rate
and
Michaelis
constant,
were used t o d e t e r m i n e respectively.
Because
M i c h a e l i s - M e n t e n and L a n g m u i r - H i n s h e l w o o d k i n e t i c s a r e f o r m a l l y identical,
this
parameters of
m e t h o d may
be used t o e s t i m a t e
many h e t e r o g e n e o u s
catalysts.
demonstrated u s i n g experimental data
from
intrinsic rate
T h e t e c h n i q u e was of
maize
d e x t r i n w i t h d i f f u s i o n - l i m i t e d immobilized glucoamylase.
This
system y i e l d e d a M i c h a e l i s constant of
the hydrolysis
0.14%,
c o m p a r e d t o 0.11% f o r
s o l u b l e g l u c o a m y l a s e and 0.14% f o r i m m o b i l i z e d g l u c o a m y l a s e f r e e o f diffusional effects. The e n t r a p m e n t calcium
alginate
of
chemical derivatives o f
g e l s has
retained i n calcium alginate gels, enzyme was i n c r e a s e d , intermolecular
glucoamylase
been i n ~ e s t i g a t e d . ~ ~ G ’ lucoamylase
when t h e m o l e c u l a r w e i g h t o f t h e
9. by a t t a c h m e n t
cross-linking.
t o p o l y m e r i c s u p p o r t s o r by
Alternatively,
the
enzyme
r e t a i n e d by c o v a l e n t a t t a c h m e n t t o t h e a l g i n a t e m a t r i x . majority
of
the
i n was
c o u l d be I n the
g l u c o a m y l a s e d e r i v a t i v e s m o r e t h a n 50% o f
the
510
Carbo hy d ra re Chemistry
i n i t i a l a c t i v i t y d e t e r m i n e d w i t h m a l t o s e or p a r t i a l l y h y d r o l y s e d s t a r c h s u c h as s u b s t r a t e was r e c o v e r e d , w h i l e u p o n e n t r a p m e n t i n g e l p a r t i c l e s l e s s t h a n 40% a n d 6% o f t h e i n i t i a l a c t i v i t y t o w a r d s m a l t o s e a n d p a r t i a l l y h y d r o l y s e d s t a r c h , r e s p e c t i v e l y , was r e c o v e r e d . Thus, d i f f u s i o n a l and s t e r i c l i m i t a t i o n s on s u b s t r a t e access a p p e a r e d t o i n f l u e n c e t h e a p p a r e n t e n z y m i c a c t i v i t y , i n s p i t e of t h e high permeability of calcium a l g i n a t e g e l s . Dextran-coupled g l u c o a m y l a s e e n t r a p p e d i n c a l c i u m a l g i n a t e g e l s h a s b e e n u s e d as a packed-bed r e a c t o r f o r c o n t i n u o u s h y d r o l y s i s of o l i g o d e x t r i n s d u r i n g a p e r i o d of t w o months w i t h no change i n a c t i v i t y . The t h e r m a l s t a b i l i t y o f i m m o b i l i z e d g l u c o a m y l a s e from R h i z o p u s n i v e u s e n t r a p p e d i n p o l y a c r y l a m i d e g e l s a n d b o u n d t o SPS e p h a d e x C-50 h a s b e e n i n v e s t i g a t e d . 3 9 8 The t h e r m a l s t a b i l i t y o f i m m o b i l i z e d g l u c o a m y l a s e e n t r a p p e d i n p o l y a c r y l a m i d e g e l s was e n h a n c e d s l i g h t l y c o m p a r e d w i t h g l u c o a m y l a s e i n f r e e s o l u t i o n , and was i n d e p e n d e n t o f t h e a c r y l a m i d e m o n o m e r c o n c e n t r a t i o n a n d N,"methylene-bis(acrylamide1 c o n t e n t . To e x p l a i n t h i s p h e n o m e n o n , t h e c e l l u l a r s t r u c t u r e o f p o l y a c r y l a m i d e g e l was t a k e n i n t o c o n s i d e r a t i o n i n a d d i t i o n t o i n t e r a c t i o n s between g l u c o a m y l a s e and g e l a n d a d e c r e a s e i n d i e l e c t r i c c o n s t a n t i n t h e g e l . On t h e o t h e r h a n d , i m m o b i l i z e d g l u c o a m y l a s e b o u n d t o S P - S e p h a d e x by i o n i c i n t e r a c t i o n s h o w e d l o w e r s t a b i l i t y t h a n f r e e g l u c o a m y l a s e , a n d much greater stability than glucoamylase i n the presence of dextran s u l p h a t e , a c o n s t i t u e n t of SP-Sephadex. T h e rm al s t a b i l i t i e s f o r t h e . f r e e a n d i m m o b i l i z e d e n z y m e s were a l s o c o m p a r e d a t t h e pH n o t i n t h e b u l k s o l u t i o n b u t i n t h e SP-Sephadex. The thermal s t a b i l i t y of i m m o b i l i z e d g l u c o a m y l a s e i n t h e p r e s e n c e o f a s u b s t r a t e h a s b e e n i n v e s t i g a t e d u s i n g a mass b a l a n c e The a p p l i c a b i l i t y o f t h i s method t o a m a l t o s e - i m m o b i l i z e d g l u c o a m y l a s e s y s tem was e x a m i n e d . The p H - d e p e n d e n t u n f o l d i n g o f g l u c o a m y l a s e f r o m r a b b i t s m a l l i n t e s t i n e by m e t h a n o l h a s b e e n d e s c r i b e d . 4 0 0 Glucoamylase from the b r u s h b o r d e r s o f t h e r a b b i t s m a l l i n t e s t i n e w a s s o l u b i l i z e d by p a p a i n a n d T r i t o n X-100. T h e T r i t o n - s o l u b i l i z e d f r a c t i o n , when In t h e presence f r e e d of T r i t o n , assumed a m i c e l l e - l i k e s t r u c t u r e . o f m e t h a n o l , t h e enzyme micelle as well as t h e p a p a i n - s o l u b i l i z e d e n z y m e monomer u n f o l d e d , o b e y i n g t h e f i r s t - o r d e r r a t e law. U n f o l d i n g o f t h e m i c e l l e was d e s c r i b e d b y t w o t i m e c o n s t a n t s (k = s-l), whereas p a p a i n - s o l u b i l i z e d enzyme had s-l a n d k = s-'). T h e r a t e was s i n g l e - p h a s e k i n e t i c s o f u n f o l d i n g (k = d e p e n d e n t on pH a n d t h e v a l u e o f a c t i v a t i o n e n e r g y l i e s b e t w e e n 1-6
511
6: Enzymes k c a l mol"
a t v a r i o u s pHs.
M e t h a n o l was d e v o i d o f a n y e f f e c t o n t h e
a c t i v i t y o f p a p a i n - s o l u b i l i z e d g l u c o a m y l a s e a t v a r i o u s pHs, w h e r e a s b o t h T r i t o n - 1 0 0 - s o l u b i l i z e d enzyme a n d enzyme m i c e l l e w e r e i n h i b i t e d t o t a l l y b y 3 0 % m e t h a n o l b e t w e e n pHs 4 a n d 8 . An u n u s u a l t y p e o f
g l y c o p r o t e i n s t r u c t u r e h a s been i d e n t i f i e d
f o r a glucoamylase.401
Glucoamylase occurs i n two isoenzyme forms
( g l u c o a m y l a s e I and g l u c o a m y l a s e 11) i n e x t r a c t s f r o m c e r t a i n f u n g i . The i s o e n z y m e s f r o m A s p e r g i l l u s n i g e r a r e g l y c o e n z y m e s C o n t a i n i n g Qmannose,
Q-glucose, and
components.
Q-galactose
as
integral structural
8-
New d a t a f r o m e x p e r i m e n t s o n r e d u c t i v e a l k a l i n e
e l i m i n a t i o n and f r o m m e t h y l a t i o n a n a l y s i s show t h a t t h e c a r b o h y d r a t e chains o f glucoamylase I a r e l i n k e d Q - g l y c o s i d i c a l l y
f r o m c-mannose
- - s e r i n e or I - t h r e o n i n e r e s i d u e s o f t h e p r o t e i n m o i e t y . residues t o L t h e c a r b o h y d r a t e r e s i d u e s a r e p r e s e n t as 20 s i n g l e
I n t h i s enzyme,
p-mannose
residues,
11 d i s a c c h a r i d e c o m p o n e n t s h a v i n g t h e s t r u c t u r e
2-Q-~-mannopyranosyl-~-mannose, 8
trisaccharides,
5
and
0-
t e t r a s a c c h a r i d e s composed o f v a r i o u s c o m b i n a t i o n s o f Q-mannose, glucose,
and e - g a l a c t o s e
glycosidic linkages.
r e s i d u e s j o i n e d by ( 1 + 3 ) a n d ( 1
+
6)
Such an a r r a y o f c a r b o h y d r a t e c h a i n s i n a
g l y c o p r o t e i n i s unusual,
a n d may a c c o u n t f o r some o f t h e u n i q u e
p r o p e r t i e s e x h i b i t e d by g l u c o a m y l a s e . A m y l o l y t i c enzymes p r o d u c e d by a s t r a i n o f A s p e r g i l l u s n i g e r c u l t i v a t e d on c a s s a v a s t a r c h i n l i q u i d o r s o l i d c u l t u r e w e r e f o u n d t o b e m a i n l y g l u c o a m y l a ~ e s . ~F o ~ r~ t h e same i n i t i a l a m o u n t o f s u b s t r a t e , t h e g l u c o a m y l a s e a c t i v i t y i n c r e a s e d e v e n a f t e r 60 h o f c u l t u r e o n s o l i d medium w h e r e a s i t d e c r e a s e d i n l i q u i d c u l t u r e .
The
pH o p t i m a a n d t h e r m o s t a b i l i t i e s f o r e n z y m e s p r o d u c e d i n s o l i d a n d l i q u i d c u l t u r e s were
different.
s o l u b l e s t a r c h (100 m l ) "
The
Xm
values
e x p r e s s e d as
c u l t u r e and 0.057% f o r c r u d e enzyme f r o m l i q u i d c u l t u r e . active-site-directed
mg
w e r e 0.1% f o r c r u d e e n z y m e f r o m s o l i d Studies o f
i r r e v e r s i b l e i n h i b i t i o n o f g l y c o s i d a s e s by t h e
c o r r e s p o n d i n g glycosylmethyl-(4-nitrophenyl)triazines
show t h a t t h e y
h a v e no a c t i o n on g l u c o a m y l a s e f r o m A s p e r g i l l u s r ~ i g e r . ~ ~ A m a j o r g l u c o a m y l a s e o f A s p e r g i l l u s n i g e r h a s been p u r i f i e d 2 3 fold
(21% y i e l d )
by
ultrafiltration
c h r o m a t o g r a p h y on DEAE-Sephadex, The
purified
enzyme
polyacrylamide
gel
was
f o l l o w e d by
successive
U l t r o g e l AcA 44, a n d S P - S e p h a d e ~ . ~ ~ ~
proved
homogeneous
electrophoresis,
as
isoelectric
judged
by
focusing,
u l t r a c e n t r i f u g a t i o n , and a l s o f r o m t h e absence o f t h e g l y c o s i d a s e activities.
The enzyme was a g l y c o p r o t e i n c o n t a i n i n g n e u t r a l s u g a r
(18%) and 2-amino-2-deoxy-Q-glucose
(0.77%),
and i t s
molecular
512
Carbohydrate Chemistry
weight
was
(90,000)
estimated
by
polyacrylamide
SDS
gel
e l e c t r o p h o r e s i s and a m i n o a c i d c o m p o s i t i o n . The N - t e r m i n a l a m i n o a c i d was I - a l a n i n e . The pH o p t i m u m o f t h e p u r i f i e d enzyme was 4.5 w i t h s o l u b l e s t a r c h as a s u b s t r a t e .
The enzyme was s t a b l e b e t w e e n
p H 2.5
a n d 7.5
5OoC.
The e n z y m e a c t i v i t y was i n h i b i t e d b y H g 2 + a n d ,
extent,
and r e t a i n e d f u l l a c t i v i t y
by Pb2+ and Mn2+.
The
vmax v a l u e
c.
value w i t h
xi
thus resulting i n the
The k i n e t i c p a r a m e t e r s f o r
o t h e r s u b s t r a t e s s u c h as s o l u b l e s t a r c h , w e l l as t h e
t o a lesser
s u b s t r a t e i n glucose u n i t
2,
increased with
lmax/Km
increase i n the
up t o
value f o r m a l t o - o l i g o m e r ,markedly
decreased w i t h i n c r e a s i n g c h a i n l e n g t h of
( 5 ) and t h e
a t temperatures
g l y c o g e n , a n d i s o m a l t o s e as
v a l u e s f o r some s a c c h a r i d e s w e r e a l s o d e t e r m i n e d .
A t h e r m o s t a b l e g l u c o a m y l a s e has been p u r i f i e d from
the culture
f i l t r a t e s o f Thermomyces l a n u g i n o s u s a n d h a s b e e n e s t a b l i s h e d t o be homogeneous
by
glycoprotein
(mol.
10.12%.
a
number wt.
of
criteria.404 with
57,000)
a
The
enzyme
carbohydrate
The enzyme h y d r o l y s e d s u c c e s s i v e
9-glucose
was
content
residues
a of
from
t h e n o n - r e d u c i n g ends o f t h e s t a r c h m o l e c u l e b u t d i d n o t e x h i b i t any glucosyltransferase activity. m a l t o t r i o s e by t h e m u l t i - c h a i n optimum linkages
7 O o C ) was of
unable
isomaltose
increase i n the
The e n z y m e a p p e a r e d t o h y d r o l y s e mechanism.
to h y d r o l y s e
and
dextran,
The enzyme ( t e m p e r a t u r e
(1
and
-*
the
enzyme
lmax a n d d e c r e a s e i n Em v a l u e s
chain l e n g t h o f t h e s u b s t r a t e molecule.
exhibited
with increasing
The enzyme was i n h i b i t e d by
t h e s u b s t r a t e analogue q-glucono-l,4-lactone manner.
6)-a-Q-glucosidic
i n a non-competitive
The enzyme e x h i b i t e d r e m a r k a b l e r e s i s t a n c e t o w a r d s c h e m i c a l
and t h e r m a l d e n a t u r a t i o n . The
-R h
thermal
stability
and
kinetics
of
glucoamylase
i z w ---s n i v e u s has been i n v e s t i g a t e d i n t h e presence of
DEAE-dextran,
dextran sulphate,
t e m p e r a t u r e r a n g e f r o m 52.0
p o l y e t h y l e n e glyco1,and
t o 60.0°C.405
from
dextran,
Ficoll i n a
Protective effects of
t h e s e p o l y m e r s a g a i n s t t h e r m a l i n a c t i v a t i o n o f t h e enzyme i n c r e a s e d i n t h e order polyethylene g l y c o l < F i c o l l < dextran.
The o r d e r was
t h e same a s t h a t o f a f f i n i t i e s b e t w e e n g l u c o a m y l a s e a n d e a c h o f polymers two-phase
d e t e r m i n e d by
partition of
glucoamylase i n l i q u i d - l i q u i d
systems c o n s i s t i n g o f these polymers.
glucoamylase
with
dextran
sulphate
On t h e o t h e r hand,
e x h i b i t e d an i n t e r e s t i n g
phenomenon w h i c h was a t y p i c a l e l e c t r o s t a t i c i n t e r a c t i o n e x p l i c a b l e i n t e r m s o f an e l e c t r i c a l d o u b l e l a y e r . A F l a v o b a c t e r i u m s p e c i e s which produces c y c l o d e x t r i n - d e g r a d i n g g l u c o a m y l a s e has been i s o l a t e d . 4 0 6
The i n d u c i b l e , c e l l - b o u n d
enzyme
513
6: Enzymes was p u r i f i e d a b o u t 1 0 - f o l d t o 7 5 % p u r i t y i n 5 7 % y i e l d . o f t h e enzyme w i t h c y c l o h e x a - a m y l o s e , octa-amylose
and t y p i c a l
The a c t i o n
cyclohepta-amylose,and c y c l o -
glucoamylase s u b s t r a t e s
always
gave
D-
g l u c o s e as t h e f i n a l d e g r a d a t i o n p r o d u c t .
S m a l l amounts o f m a l t o s e ,
w h i c h c o u l d be d e t e c t e d i n t h e c o u r s e o f
cycloamylose degradation,
were h y d r o l y s e d a t a l o w e r r a t e .
A p p a r e n t l y t h e enzyme p r e f e r r e d
s h o r t e r a-Q-glucopyranosyl chains, p r o v e d t o be
and a m y l o p e c t i n and g l y c o g e n
very poor substrates.
30 Glycanases ( M i s c e l l a n e o u s ) The
occurrence
of
high
and
Mr
low
forms
p h o s p h o r y l a s e i n e x t r a c t s o f human b r a i n h a s Accorinding t o gel-exclusion
of
glycogen
been r e p ~ r t e d . ~ ”
c h r o m a t o g r a p h y and s u c r o s e d e n s i t y
g r a d i e n t s e d i m e n t a t i o n b o t h p h o s p h o r y l a s e s a and b a p p e a r i n a high
Mr
f o r m o f 400,000 a n d a l o w
d i f f e r s w i t h t h e method o f
Mr
to exist
f o r m whose a p p a r e n t s i z e
determination.
The
l o w blr
form
i s
p r o b a b l y an e q u i l i b r i u m m i x t u r e o f d i m e r a n d monomer w h i c h g i v e s different
apparent
Yr
values
depending
upon
the
position
of
equilibrium. The
assignment
of
the
human
gene
for
liver-type
6-
p h o s p h o f r u c t o k i n a s e i s o e n z y m e t o chromosome 2 1 h a s been a c h i e v e d by u s i n g s o m a t i c c e l l h y b r i d s and m o n o c l o n a l a n t i - L a n t i b o d y . 4 0 8 Substrate specificty
o f t h e glycanase a c t i v i t y associated w i t h
p a r t i c l e s o f K l e b s i e l l a b a c t e r i o p h a g e N0.6 h a s b e e n i n v e s t i c ~ a t e d . ~ ’ ~ The g l y c a n a s e c a t a l y s e s c l e a v a g e o f g - B - Q - g l u c o p y r a n o s y l - ( l
+
3)-
4 , 6 - ~ - ( 1 - ~ a r b o ~ y e t h y l i d e n e ) - ~ - ~ - m a n n o p y r a n o sl ei n k a g e s i n K l e b s i e l l a serotype-6
capsular
polysaccharide.
O f
74 h e t e r o l o g o u s
Klebsiella
p o l y s a c c h a r i d e s and t w o d e r i v a t i v e s o f t h e t y p e - 6 g l y c a n o n l y t h e t y p e - 1 a n d t y p e - 5 7 p o l y m e r s w e r e a d d t i o n a l l y d e g r a d e d by t h e phage-6 enzyme. 3eq,
The r e p e a t i n g u n i t s i n t h e t h r e e s u b s t r a t e s h a v e a l a x -
leg-eq-linked
chain g-gluco-
or Q-galacto-pyranosyl
common ( w h i c h c o n s t i t u t e s t h e r e d u c i n g e n d a f t e r
residue i n
glycanase a c t i o n )
and a c a r b o x y l group on t h e n e x t h e x o p y r a n o s y l r e s i d u e .
O f t h e 72
p o l y s a c c h a r i d e s n o t a f f e c t e d by t h e v i r a l enzyme, a t l e a s t t h e t y p e 11 a n d t y p e - 2 1 g l y c a n s a l s o c o n t a i n t h e same h o m o l o g y o f p r i m a r y s t r u c t u r e . T h i s i n d i c a t e s t h a t t h e c o n f o r m a t i o n of recognition site substrates.
also
constitutes
an
important
the glycanase
feature
of
the
514
Carbohydrate Chemistry 31 H e p a r i n Hydrolases
Purification
of
heparinase
and
heparitinase
c h r o m a t o g r a p h y on g l y c o s a m i n o l y c a n - b o u n d A H - S e p h a r o s e reported.410 heparinase
The and
respectively,
recoveries
of
heparitinase
the
were
purified
estimated
to
by
affinity
48 has been
preparations be
and
39
of 50%,
f r o m t h e c r u d e enzyme f r a c t i o n s o b t a i n e d by t h e f i r s t
column chromatography on h y d r o x y a p a t i t e . An e n & - Q - g l y c o s i d a s e
( h e p a r i n l y a s e ) a c t i n g on h e p a r i n and
h e p a r a n s u l p h a t e was p a r t i a l l y p u r i f i e d ( - 3 0 0
t i m e s ) f r o m human
p l a t e l e t s by a f f i n i t y c h r o m a t o g r a p h y on h e p a r a n s u l p h a t e - s u b s t i t u t e d S e p h a r ~ s e . ~ O n~l y~ h e p a r i n - l i k e t h e enzyme. intermediates requirement
p o l y s a c c h a r i d e s were degraded by
The s u s c e p t i b i l i t y indicated
for
that
sulphamino
of
the
but
various
biosythetic heparin
platelet
not
ester
heparitanase sulphate
had
groups.
a
No
a c t i v i t y toward other uronic acid-containing glycosaminoglycans A l t e r n a t e l i n k a g e s i n h e p a r i n or h e p a r a n
c o u l d be d e m o n s t r a t e d .
s u l p h a t e w e r e a t t a c k e d by t h e e n z y m e a s s h o w n b y a n a l y s i s o f
the
reducing
The
sugar
moiety
i n
oligosaccharide
anticoagulant a c t i v i t y o f heparin, a c t i v a t i o n assay, heparin lyase.
products.
d e t e r m i n e d i n an a n t i t h r o m b i n I 1 1
was m a r k e d l y r e d u c e d a f t e r
treatment with the
T h e e n z y m e was r e l e a s e d f r o m i t s s t o r a g e s i t e i n
platelets a f t e r induction of the platelet-release reaction. physiological function of
p l a t e l e t heparin lyase i s not
may be t o m o d i f y e x t r a c e l l u l a r h e p a r i n - l i k e
The
known b u t
polysaccharides i n the
v a s c u l a r system. H e p a r i n h y d r o l a s e p r o d u c t i o n by F l a v o b a c t e r i u m h e p a r i n u m i n c o m p l e x p r o t e i n d i g e s t medium,
w i t h h e p a r i n e m p l o y e d as t h e i n d u c e r ,
h a s b e e n i m p r o v e d 1 5 6 - f 0 l d . ~ l ~R a p i d d e a c t i v a t i o n o f h e p a r i n a s e activity,
b o t h s p e c i f i c and t o t a l ,
s t a t i o n a r y phase.
p r o d u c t i o n showed an o b l i g a t e vitamin
requirement.
h i s t i d i n e requirement. ammonium s u l p h a t e ,
was o b s e r v e d a t t h e o n s e t o f
requirement for
L-Methionine
I-histidine
partially
t h i s m e d i u m was 0.21 h - l ,
relieved
and the
no
I-
A d e f i n e d medium c o n t a i n i n g P_-glucose,
basal salts,
L-methionine,
and L - h i s t i d i n e
d e v e l o p e d f o r g r o w t h and h e p a r i n a s e p r o d u c t i o n . medium.
the
N u t r i t i o n a l s t u d i e s on g r o w t h and h e p a r i n a s e
was
The g r o w t h r a t e i n
w h i c h i s 40% h i g h e r t h a n t h a t i n c o m p l e x
The maximum v o l u m e t r i c p r o d u c t i v i t y
d e f i n e d m e d i u m was i n c r e a s e d t o 1,475
o f heparinase i n the
U 1-1 h - l ,
p r o v i d i n g a 640-
f o l d i n c r e a s e o v e r t h a t a c h i e v e d by p r e v i o u s l y p u b l i s h e d methods.
313
6: Enzymes No r a p i d d e a c t i v a t i o n was o b s e r v e d .
An e x a m i n a t i o n o f a l t e r n a t e
i n d u c e r s f o r h e p a r i n a s e showed t h a t h e p a r i n d e g r a d a t i o n p r o d u c t s , h y a l u r o n i c acid, h e p a r i n monosulphate, B-g-2-acetamido-2-deoxyg l u c o p y r a n o s e , and m a l t o s e , i n d u c e h e p a r i n a s e i n c o m p l e x medium.
An
A z u r e A a s s a y was d e v e l o p e d t o m e a s u r e t h e h e p a r i n c o n c e n t r a t i o n d u r i n g f e r m e n t a t i o n and t h e h e p a r i n a s e s p e c i f i c a c t i v i t y of c r u d e extracts of
heparinum obtained from sonication,
thus negating the
need f o r f u r t h e r p u r i f i c a t i o n t o measure a c t i v i t y .
32
Hyaluronidases
P u r i f i e d bovine t e s t i c u l a r butane-2,3-dione
h y a l u r o n i d a s e i s i n a c t i v a t e d by
i n e i t h e r b o r a t e o r H e p e s b u f f e r , pH 8.3.413
p r e s e n c e o f b o r a t e enhanced t h e i n a c t i v a t i o n p r o c e s s , pseudo-first-order
k i n e t i c s w i t h a c a l c u l a t e d second-order
c o n s t a n t o f 13.54 M - l
min-l.
The
which f o l l o w e d rate
U s i n g k i n e t i c d a t a i t was e s t i m a t e d
t h a t t h e m o d i f i c a t i o n o f 1 m o l L - a r g i n i n e p e r m o l e n z y m e was t h a t s u f f i c i e n t f o r i n a c t i v a t i o n t o occur, whereas amino a c i d a n a l y s i s i n d i c a t e d t h a t 4 m o l & - a r g i n i n e h a d been m o d i f i e d . process
was
partially
prevented
by
using
i n h i b i t o r s o r s u b s t r a t e s o f t h e enzyme, essential
L-arginine
hyaluronidase.
residue
i s
The i n a c t i v a t i o n
either
competitive
thus indicating that the
close
to
the
active
site
of
A f u l l k i n e t i c a n a l - y s i s o f t h e enzyme w i t h e i t h e r
hyaluronic acid or chondroitin 6-sulphate
as s u b s t r a t e showed t h a t
t h e a c t i v i t y o f h y a l u r o n i d a s e was u n c o m p e t i t i v e l y a c t i v a t e d b y either
H+ or
NaC1.
The
product
obtained
by
r e d u c t i o n of
the
c a r b o x y l groups o f h y a l u r o n i c a c i d t o t h e corresponding a l c o h o l groups
was
a competitive
inhibitor.
The p o s s i b i l i t y t h a t
the
m i c r o e n v i r o n m e n t o f h y a l u r o n i c a c i d was r e s p o n s i b l e f o r t h e o b s e r v e d k i n e t i c e f f e c t s o f pH and i o n i c s t r e n g t h was d i s p e l l e d . conclude t h a t these i n v o l v e s an
data are compatible
with a
The a u t h o r s
mechanism t h a t
i o n i c i n t e r a c t i o n between a c a r b o x y l group on t h e
s u b s t r a t e and an L - a r g i n i n e r e s i d u e on t h e enzyme. A
procedure has been d e s c r i b e d f o r
testicular
hyaluronidase
a d m i n s t r a t i o n of
the
enzyme.414
the reported non-specific presence of
t h e assay
i n human b l o o d f o l l o w i n g I n h i b i t a t i o n of
of
bovine
intravenous
h y a l u r o n i d a s e by
serum i n h i b i t o r i s m i n i m a l .
However,
the
human s e r u m does a l t e r t h e pH p r o f i l e o f h y a l u r o n i d a s e
and enhances
the
activity
of
the
enzyme
at
low
pH
values.
P r e l i m i n a r y d a t a i n d i c a t e t h e e f f e c t s c a u s e d b y s e r u m o n t h e pH
516
Carbohydrate Chemistry
o p t i m u m and t h e p r e s e n c e o f endogenous serum h y a l u r o n i d a s e .
The
a c t i v a t i o n e f f e c t i s n o t s p e c i f i c f o r any p a r t i c u l a r b l o o d t y p e and A i s i n d e p e n d e n t o f w h e t h e r serum o f c i t r a t e d p l a s m a i s used. similar
effect
produced
by
to
that
of
different It
concentration.
serum
on
buffer
i s
hyaluronidase
or
mixtures
recommended
h y a l u r o n i d a s e be m e a s u r e d a t pH 4.0
that
activity
increased
bovine
i s
NaCl
testicular
i n 0.1 M sodium c i t r a t e b u f f e r
M NaC1, as u n d e r t h e s e c o n d i t i o n s t h e a d d i t i o n o f
c o n t a i n i n g 0.15
human serum o r c i t r a t e d p l a s m a does n o t a l t e r t h e pH o p t i m u m o f t h e enzyme. C o m p a r a t i v e k i n e t i c s t u d i e s h a v e b e e n p e r f o r m e d o n mouse a n d b o v i n e t e s t i c u l a r h y a l ~ r o n i d a s e . ~The ~ ~ mouse enzyme has an o p t i m u m pH o f
4.3
testicular
with
a b r o a d pH s p e c t r u m
hyaluronidase.
h y a l u r o n i d a s e s show energy
of
While
maximum a c t i v i t y
activation
are
mg m l - l
that
mouse
different
Arrhenius
f r m
of
for the
values
b o v i n e enzyme. The m o s t
HgC12 a t
M concentration.
the
value
testicular
mouse t e s t i c u l a r
that
Xm
for
enzyme i s m o r e s t a b l e t h a n t h e b o v i n e e n z y m e a t 55OC. for
reveal
for
plots
mouse
potent i n h i b i t o r s
experiments
bovine
testicular
Mouse h y a l u r o n i d a s e has a
as c o m p a r e d t o 2.1 mg m l - '
Thermal-denaturation
to
and
a t 37OC,
obtained
degradation o f hyaluronic acid. o f 0.9
similar
bovine
h y a l u r o n i d a s e a r e CuS04 and
L-Cysteine
appeared t o p r o t e c t t h e
enzyme i n h i b i t i o n caused by HgC12. A
r a p i d p u r i f i c a t i o n o f b o v i n e t e s t i c u l a r h y a l u r o n i d a s e by
c h r o m a t o g r a p h y on d e r m a t a n s u l p h a t e - s u b s t i t u t e d
AH-Sepharose
46
f o l l o w e d by c h r o m a t o g r a p h y o n a c e t y l a t e d A H - S e p h a r o s e 48 h a s b e e n reported.416
The p r o c e d u r e y i e l d e d a p u r i f i e d h y a l u r o n i d a s e w i t h a
specific activity of SDS-polyacrylamide
19.1
u n i t s pmol m i n - l
g e l electrophoresis of
r e v e a l e d t h e p r e s e n c e o f t w o c l o s e bands w i t h w e i g h t s o f 61,000
mg-l
i n high yield.
the purified material approximate molecular
and 67,200.
C h a r a c t e r i z a t i o n o f B-c-2-acetamido-Z-deoxyglucosidase
from
mouse t e s t e s s h o w e d t h a t t h e e n z y m e d o e s n o t c r o s s - r e a c t i n immunodif f u s i o n p l a t e s with anti-mouse t e s t i c u l a r hyaluronidase serum,
suggesting t h a t
have common a n t i g e n i c
% - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e does n o t determinants i n hyaluronidase or
hyalurono-
g l u c o s a m i n i d a s e complex o f acrosome o r t e s t e s . 3 7 H y a l u r o n i d a s e h a s b e e n p u r i f i e d 9 4 - f o l d f r o m mouse t e s t e s b y ion-exchange
chromatography,
chromatography.417 10-20% o f
gel
filtration,
and
affinity
The enzyme was r e l a t i v e l y h e a t s t a b l e and l o s t
i t s a c t i v i t y a t 50-55OC
for
10 min.
La
f o r heat
517
6: Enzymes d e n a t u r a t i o n o f e n z y m e was 4 2 - 4 5
k c a l b e t w e e n 45 a n d 63OC.
The
M i c h a e l i s c o n s t a n t o f mouse t e s t i c u l a r h y a l u r o n i d a s e was 1.1 mg m l - I hyaluronic acid. A n t i b o d i e s t o t h e p u r i f i e d enzyme showed a s i n g l e precipitin
line
by
Ouchterlony
h y a l u r o n i d a s e i n h i b i t e d enzyme mouse
testicular
gel
diffusion.
activity
hyaluronidase
by
was
Antiserum
to
Immunologically,
25%.
species
specific.
Tissue
e x t r a c t s o f mouse v i t a l o r g a n s , e x c e p t t e s t e s a n d e p i d i d y m i s , d i d not react w i t h the antisera, occur
though n o n - s p e c i f i c
precipitation did
between i n t e s t i n a l e x t r a c t s and a n t i - h y a l u r o n i d a s e
H y a l u r o n i d a s e was
localized i n testis
immunof luorescence. localized
on
spermatids
cell
and
A
specific
boundaries
appeared
on
sections
dark-green
extending
the
sperm
by
serum.
indirect
f l u o r e s c e n c e was
from
spermatogonia
acrosome.
to
Cytoplasm o f
s p e r m a t o g o n i a and s p e r m a t o c y t e s showed l i g h t - g r e e n f l u o r e s c e n c e w h e r e a s i n t e r s t i t i a l t i s s u e was d e v o i d o f f l u o r e s c e n c e . I n a s i m p l e p l a t e assay
for
hyaluronidase a c t i v i t y hyaluronic
a c i d i s i n c o r p o r a t e d i n t o a g a r o s e g e l s a n d t h e enzyme i s a l l o w e d t o d i f f u s e f r o m punched wells.418
The u n d i g e s t e d h y a l u r o n i c a c i d i s
t h e n p r e c i p i t a t e d w i t h c e t y l p y r i d i n i u m c h l o r i d e and t h e d i a m e t e r s o f t h e c l e a r c i r c l e s a r e p r o p o r t i o n a l t o t h e l o g a r i t h m o f t h e enzyme concentration applied t o
the
well.
The
assay
e x a m i n e c o m m e r c i a l l y a v a i l a b l e h y m e n o p t e r a venoms, use
i n
allergy
diagnosis
and
h y a l u r o n i d a s e as a measure o f permits
the
analyses
reproducibility,
of
without
a
treatment, lot-to-lot
large
for
ut'ilized t o
manufactured f o r their
content
consistency.
number
t h e need f o r
was
of
of
The a s s a y
samples
with
good
any s p e c i a l i n s t r u m e n t a t i o n .
Based on t h e q u a n t i t y o f p u r i f i e d h y a l u r o n i d a s e r e p o r t e d i n h o n e y b e e venom
(T.P.
Lichenstein,
King,
1976, A r c h .
i s estimated t o
detect
A.K.
Sobotka,
Biochem.
L.
70 ng m l - l
of
Kochoumain,
172, 661-671),
Biophys.,
a n d L.M. t h e assay
p u r i f i e d honey-bee
venom
hyaluronidase. The d e g r a d a t i o n p r o c e s s o f h y a l u r o n i c a c i d b y S t r e p t o m y c e s h y a l u r o n i d a s e has been i n v e s t i g a t e d . 4 1 9
S a t u r a t e d and
unsaturated
h y a l u r o n a t e o l i g o s a c c h a r i d e s w e r e p r e p a r e d f r o m human u m b i l i c a l - c o r d h y a l u r o n i c a c i d by p a r t i a l d i g e s t i o n w i t h b o v i n e t e s t i c u l a r
-Streptomyces
hyaluronidase, respectively.
Streptomyces hyaluronidase,
the d i s t r i b u t i o n o f degradation products
f r o m t h e s e o l i g o s a c c h a r i d e s was d e t e r m i n e d . s a t u r a t e d or
unsaturated,
T e t r a - and h e x a - s a c c h a r i d e s as f i n a l p r o d u c t s .
and
After treatment with Octasaccharides, e i t h e r
were s u b s t r a t e s o f were n o t
t h e minimum size.
degraded f u r t h e r
and r e m a i n e d
This i n d i c a t e s t h a t a t l e a s t f o u r succeeding
3-
518
Carbohydrate Chemistry
a c e t y l h y a l o b i u r o n o s y l r e s i d u e s were r e q u i r e d f o r t h i s e n z y m a t i c degradation. S i n c e u n s a t u r a t e d o l i g o s a c c h a r i d e s were more s u s c e p t i b l e t o t h i s enzyme t h a n s a t u r a t e d o n e s of t h e same p o l y m e r i z a t i o n d e g r e e , and i n n e r l i n k a g e s were c l e a v e d i n p r e f e r e n c e t o t h o s e a t t h e o u t e r m o s t s i t e s , some g r o u p s a d j a c e n t t o t h i s segment seemed t o i n f l u e n c e t h e s u s c e p t i b i l i t y of t h e o l i g o s a c c h a r i d e t o t h i s enzyme. Some p r o p e r t i e s of bovine f o e t a l b r a i n h y a l u r o n i d a s e have been described.35 The p o s s i b l e r o l e of c h o n d r o i t i n s u l p h a t e C and h y a l u r o n i d a s e i n t h e p r o c e s s e s o f d i f f e r e n t i a t i o n and d i v i s i o n i s discussed.
33
Inulinases
T h e s e p a r a t i o n and p u r i f i c a t i o n of B - Q - f r u c t o f u r a n o s i d a s e and an i n u l i n a s e f r o m g e r m i n a t i n g g a r l i c ( A l l i u m s a t i v u m ) b u l b s have been d e s c r i b e d . 8 0 Both t h e e n z y m e s ( m o l . wts. 7 6 , 0 0 0 b y g e l chromatography) were i n a c t i v a t e d b y 4 - c h l o r o m e r c u r i b e n z o a t e , 5,5'dithiobis(2-nitrobenzoic a c i d ) , and h e a t t r e a t m e n t a t 5 5 O C f o r 5 m i n a t pH 7.0. The i n u l i n a s e h y d r o l y s e d i n u l i n , s u c r o s e , and r a f f i n o s e . The K m v a l u e s f o r i n u l i n a n d s u c r o s e w e r e 1 0 m M a n d 25 m M , respectively. While i n t h e e a r l y s t a g e s of p l a n t growth ( u p t o 6 days) t h e i n u l i n a s e and B - Q - f r u c t o f u r a n o s i d a s e a c t i v i t i e s i n c r e a s e d i n a p a r a l l e l fashion i n t h e b u l b s ; a t l a t e r stages the increase i n a c t i v i t y was more t h a n t h a t of i n u l i n a s e a c t i v i t y . The p r o d u c t i o n o f a l c o h o l f r o m f e r m e n t a b l e e x t r a c t s o f J e r u s a l e m a r t i c h o k e h a s been i n v e s t i g a t e d u s i n g y e a s t s w i t h i n u l i n a s e a c t i v i t y .420
34
Isoamylases
Immunoreactive t r y p s i n and p a n c r e a t i c i s o a m y l a s e a c t i v i t y i n s e r u m of p a t i e n t s w i t h c h r o n i c r e n a l f a i l u r e o r h e p a t i c c i r r h o s i s h a s been i n v e s t i g a t e d . 4 2 1 I n 121 p a t i e n t s w i t h e i t h e r l i v e r c i r r h o s i s o r c h r o n i c r e n a l f a i l u r e p a n c r e a t i c enzymes i n serum were I n r e n a l i n s u f f i c i e n c y a d e c r e a s e d r a t e of a frequent finding. enzyme e l i m i n a t i o n i s t h e most l i k e l y c a u s e of t h e a b o v e - n o r m a l v a l u e s observed f o r serum and p a n c r e a t i c i s o a m y l a s e .
519
6: Enzymes 35
Laminaranases comparative
A
invertebrates
has
study been
of
carbohydrase a c t i v i t i e s
performed.299
invertebrates belonging t o 7 types, Arhropoda,
Mollusca,
103
species
Spongia, Coelenterata,
Echinodermata,
Chordata,
were
i n
marine
of
marine
Annelida,
tested
for
l a m i n a r i n a s e , c e l l u l a s e , a n d amylase a c t i v i t i e s .
36
Lysozymes
A s i m p l e method f o r t h e u l t r a s e n s i t i v e q u a n t i t a t i o n o f l y s o z y m e
has
been
developed.422
The
l y t i c
a c t i v i t y
against
M i c r o c o c c u s l y s o d e i k t i c u s was m e a s u r e d s p e c t r o p h o t o m e t r i c a l l y a f t e r an 18 h i n c u b a t i o n p e r i o d .
The m e t h o d i s c a p a b l e o f q u a n t i t a t i n g
l y s o z y m e a t c o n c e n t r a t i o n s as l o w as 5 pg m l - l
a n d is a p p l i c a b l e t o
d e t e r m i n a t i o n s o f t h e enzyme i n c o m p l e x b i o l o g i c a l m i x t u r e s . A radioimmunoassay d e v e l o p e d f o r serum and u r i n a r y l y s o z y m e i n v o l v e s use o f an a n t i b o d y , u r i n e from
a patient
d i l u t e d 4000-fold,
raised i n rabbits,
with monocytic
leukemia.423
t o lysozyme from This antiserum,
i s incubated w i t h r a d i o l a b e l l e d lysozyme f o r
2 h
and a n t i b o d y - b o u n d l y s o z y m e is s e p a r a t e d f r o m f r e e l y s o z y m e w i t h dextran-coated charcoal.
V a l i d a t i o n o f t h e assay i n c l u d e d p r e c i s i o n
and p a r a l l e l i s m s t u d i e s w i t h s e r u m a n d u r i n e s a m p l e s f r o m p a t i e n t s w i t h m o n o c y t i c l e u k e m i a and a n a l g e s i c n e p h r o p a t h y .
Results o f the
radioimmunoassay and t h o s e o b t a i n e d w i t h a M i c r o c o c c u s l y s o d e i k t i c u s l y t i c assay c o r r e l a t e d w e l l assay
are
i t s
(r =
0.94).
The m a i n a d v a n t a g e s o f t h e
good p r e c i s i o n and r e p r o d u c i b i l i t y and i t s h i g h
sensitivity. A new l y s o z y m e a s s a y b a s e d o n f l u o r e s c e n c e p o l a r i z a t i o n o r fluorescence
-M i c r o c o c c u s
intensity u t i l i z i n g a peptidoglycan substrate from l y s o d e i k t i c u s subsequently l a b e l l e d w i t h fluorescein
i s o t h i o c y a n a t e (FITC) a t t h e amino group o f t h e p e p t i d e has been reported.424
When t h e F I T C - l a b e l l e d
lysozyme digestion, decrease of
an i n c r e a s e o f
fluorescence
s u b s t r a t e was s u b j e c t e d t o fluorescence intensity or a
p o l a r i z a t i o n value
(p
value)
was a p p a r e n t
i n f i v e m i n u t e s a t a l y s o z y m e c o n c e n t r a t i o n a s l o w a s 0.1 o r 0 . 0 1 p g ml-', respectively. The e f f e c t o f o t h e r h y d r o l y t i c enzymes, i n c l u d i n g a-P-mannosidase, p r o t e a s e s , a n d r i b o n u c l e a s e , o n t h e p v a l u e was f o u n d t o be n e g l i g i b l e . The m e a s u r e d v a l u e s r e p r e s e n t e d t h e s p e c i f i c i t y
Carbohydrate Chemistry
520 and dose o f l y s o z y m e added.
Apparent
lmax and K m v a l u e s f o r t w o
d i f f e r e n t lysozymes, chicken egg-white d e t e r m i n e d by t h i s method.
and human,
c o u l d be
A s t u d y h a s b e e n made o f t h e r a t e s o f s t r u c t u r a l f l u c t u a t i o n s
of lysozyme i n t h e range of t h e r m a l - u n f o l d i n g t r a n s i t i o n . 4 2 5 hydrogen-deuterium
exchange
reaction for
The
the &-tryptophan residues
i n l y s o z y m e h a s b e e n f o l l o w e d i n 4.5M L i B r a t p H 7 . 2 i n t h e t e m p e r a t u r e range of
the unfolding t r a n s i t i o n
a n c e c h a n g e a t 2 9 3 nm.
by m e a s u r i n g t h e t r a n s m i t t -
The e x c h a n g e r e a c t i o n p r o c e e d e d i n t h r e e
r e l a t i v e l y exposed t r y p t o p h a n r e s i d u e s on t h e m o l e c u l a r s u r f a c e . The t h i r d p h a s e c o r r e s p o n d e d t o t h e H - D three buried i-tryptophan
residues.
exchange r e a c t i o n o f t h e
The H-D
exchanges
o f three
I-
t r y p t o p h a n r e s i d u e s b u r i e d i n f o l d e d m o l e c u l e s w e r e c a u s e d by f l u c t u a t i o n between t h e folded
and u n f o l d e d s t r u c t u r e o f t h e p r o t e i n
The r a t e s o f s u c h a f l u c t u a t i o n w e r e d e t e r m i n e d f r o m t h e
molecule.
r a t e s of t h e exchange r e a c t i o n a t v a r i o u s t e m p e r a t u r e s .
These r a t e s
agreed very w e l l w i t h those determined from the temperature-jump method.
T h i s means t h a t a p r o t e i n m o l e c u l e i n s o l u t i o n f l u c t u a t e s
b e t w e e n t h e Nion region,
and D - s t a t e s
a t every temperature w i t h i n t h e t r a n s i t -
where t h e N-form
i s the t i g h t l y folded native structure
and t h e D - f o r m t h e r a n d o m l y c o i l e d c h a i n . thermal unfolding of
ester-108-lysozyme
From measurements o f
and t h e b i n d i n g c o n s t a n t o f
i t was f o u n d t h a t a l m o s t a l l
(Q-GlcNAc)3,
t o ester-108-lysozyme,
cross-linked
m o l e c u l e s a r e i n t h e f o l d e d s t a t e n e a r 5OoC and pH 7.2
i n 4.5M
LiBr,
where i n t a c t m o l e c u l e s a r e u n f o l d e d .
A s t u d y was a l s o
In the
made o f t h e H - D e x c h a n g e r e a c t i o n o f e s t e r - 1 0 8 - l y s o z y m e . temperature
region
of
43-50°C,
about
t r y p t o p h a n r e s i d u e s of ester-108-lysozyme immediately after
the
m i x i n g of
D20,
70% o f
the
exchangeable
were exchanged w i t h i n 1 s i n spite of the fact that
almost a l l molecules are i n the folded state.
T h i s was c o n s i d e r e d
the p r e m e l t i n g o f t h e surface o f a cross-linked molecule. The
interaction of
,
lysozyme
with
mixed 1,2-dipalmitoyl-Q-
-I-phosph a t idy l c h o 1i n e
p h o s p h a t id ic a c id /1 2 - d im y r is t oy 1 has been i n v e s t i g a t e d by
laser
Raman s p e c t r o s c o p y . 4 2 6
l i p o s o mes
Substantial
changes were observed i n t h e s p e c t r a o f b o t h t h e l i p i d and p r o t e i n i n t h e m i x e d l i p o s o m e s o v e r t h e r a n g e 10-62OC. b e l o w 27OC,
A t temperatures
i n t e r a c t i o n w i t h l i p i d appears t o i n c r e a s e s l i g h t l y the
amount o f h e l i c a l s t r u c t u r e i n l y s o z y m e a t t h e expense o f random conformation.
A t t e m p e r a t u r e s above 3OoC, c o n s i d e r a b l e B-sheet i s Onset o f B - f o r m a t i o n a p p e a r s t o c o i n c i d e w i t h
i r r e v e r s i b l y formed. the
formation
of
disordered
lipid
side
chains
in
the
acidic
52 1
6: Enzymes component o f t h e l i p i d .
A t a l l temperatures,
t h e 0-P-0
diester
s t r e t c h i n g mode a t 7 8 2 cm-' i s much m o r e i n t e n s e i n t h e l i p i d p r o t e i n m i x t u r e than i n l i p i d alone. I t i s observed t h a t the d i m y r i s t o y l phosphatidylcholine chain-disorder t r a n s i t i o n i s lowered by 3 O C ,
w h i l e t h a t o f t h e p h o s p h a t i d i c a c i d i s l o w e r e d by 12'C,
yet
the post-transition conformation contains a s i g n i f i c a n t l y higher p r o p o r t i o n o f t r a n s segments i n t h e presence of lysozyme.
These
(1) a p o l a r i n t e r a c t i o n between
r e s u l t s are interpreted i n terms o f
a c i d i c p h o s p h o l i p i d and l y s o z y m e a t t e m p e r a t u r e s b e l o w e i t h e r c h a i n disorder transition,
i n which lysozyme i s e s s e n t i a l l y excluded from
the hydrophobic p o r t i o n o f
the
higher
involves
temperatures
dipalmitoyl
which
phosphatidic
acid
l i p i d , and (2) i n
the
the
an i n t e r a c t i o n a t
lipid
side
disordered
chains
state
and
of i s
m a n i f e s t e d by a s u b s t a n t i a l c o n f o r m a t i o n a l change. The e f f e c t s o f d e t e r g e n t s o r f a t t y a c i d s o n p r o t e o l y s i s o f lysozymes
have
been
studied.427
Low
concentrations
of
ionic
d e t e r g e n t s w i t h 0 o r more c a r b o n s i n t h e a l k y l c h a i n w e r e p r e d i c t e d t o s h i f t t h e native-denatured t r a n s i t i o n i n the lysozyme t o the denatured state.
F a t t y a c i d s were f o u n d t o e x e r t a s i m i l a r e f f e c t ,
a n d t h e i r p a r t i c i p a t i o n was p r o p o s e d i n p r o t e o l y s i s o f a l i m e n t a r y c a n a l d i g e s t i o n and i n t r a c e l l u l a r c a t a b o l i s m . Lysinoalanine
formation
i n
lysozyme has
dependent on a l k a l i c o n c e n t r a t i o n , time.428
The u p p e r
limits of
pH,
been found
to
be
t e m p e r a t u r e , and e x p o s u r e
lysinoalanine
f o r m a t i o n i n lysozyme
and a - l a c t a l b u m i n were r e l a t e d t o t h e number o f I - l y s i n e r e s i d u e s w i t h a c y s t i n e d i s u l p h i d e bond i n t h e a d j a c e n t p o s i t i o n r a t h e r t h a n t o the i n d i v i d u a l contents o f these residues. P r e p a r a t i o n s and i m m u n o l o g i c a l c h a r a c t e r i z a t i o n s o f lysozyme d e r i v a t i v e s d i n i t r o p h e n y l a t e d a t I - l y s i n e - 3 3 96,
r e s p e c t i v e l y , have been r e p o r t e d . 4 2 9
egg-white
Two d e r i v a t i v e s o f h e n
lysozyme w i t h s i n g l e s u b s t i t u t i o n s of
(DNP) r e s i d u e w e r e p r e p a r e d . f o l d molar excess of mono-DNP-substituted by i o n - e x c h a n g e presence of derivative
2,4-dinitrobenzene
chromatographies.
cellulose.
a 7-fold This
After
s u l p h o n i c a c i d p r o v i d e d one lysozyme),
amino
material
dinitrobenzene sulphonic
was
lysozyme)
m a l e y l a t i o n o f lysozyme i n the maleic anhydride,
the
g r o u p was p u r i f i e d o n OE-52 dinitrophenylated with
a c i d and t h e
d e r i v a t i v e was p u r i f i e d o n DE-52.
w h i c h was p u r i f i e d
The o t h e r one (DNP1-96
molar excess of
w i t h one f r e e
a 2,4-dinitrophenyl
T h e r e a c t i o n o f l y s o z y m e w i t h a 10-
l y s o z y m e (DNP1-33
was p r e p a r e d a s f o l l o w s .
two
and I - l y s i n e -
DNP1-96
2,4-
mono-DNP-substituted l y s o z y m e was f i n a l l y
Carbohydrate Chemistry
522 p u r i f i e d on SE-Sephadex
C-25,
a f t e r d e m a l e y l a t i o n a t pH 3.5,
a t 37OC
f o r 5 days. DNP1-33 l y s o z y m e and DNP1-96 l y s o z y m e b o t h m i g r a t e d as On a s i n g l e band w i t h s l o w e r m o b i l i t y t h a n t h a t o f n a t i v e l y s o z y m e . reduction,
c a r b o x y m e t h y l a t i o n , a n d c h y m o t r y p s i n d i g e s t i o n , b o t h mono-
DNP-substituted lysozymes y i e l d e d a s i n g l e yellow peptide. amino a c i d compositions o r indicated
that
p a r t i a l sequence
C-lysine-33
and
of
these
I-lysine-96
were
the
d i n i t r o p h e n y l a t e d r e s i d u e s i n DNP1-33 l y s o z y m e and DNP1-96 respectively. antigenic antisera
DNP1-33
lysozyme
reactivities to
lysozyme.
a c c e s s i b l e t o anti-DNP lysozyme t o anti-DNP lysozyme.
equal
This
The
to
and
DNP1-96
that
DNP
of
residues
antibodies,
on
the
only
lysozyme,
lysozyme
native
The
peptides
showed
lysozyme
with
protein
were
b u t t h e a f f i n i t i e s o f DNP1-33
a n t i b o d i e s w e r e l o w e r t h a n t h o s e o f DNP1-96
result
i s
discussed with respect t o
the
local
e n v i r o n m e n t s o f t h e DNP r e s i d u e s i n t h e s e p r o t e i n s . Lysozymes f r o m hen,
duck 11, and goose e g g - w h i t e
lysozyme were compared a t t h e l e v e l o f using the solvent
a n d human
t h e i r L-tryptophyl
perturbation technique
and f l u o r e s c e n c e
residues emission
i n a d d i t i o n t o t w o c h e m i c a l m o d f i c a t i ~ n s . ~The ~ ~three C-tryptophyl r e s i d u e s o f goose l y s o z y m e were found
completely exposed t o the
solvent.
The s t u d y o f t h e s i t u a t i o n o f t h e a r o m a t i c r e s i d u e s o f hen,
duck 11,
and human l y s o z y m e s p r o v i d e d a d d i t i o n a l e v i d e n c e t h a t t h e
three-dimensional although
an
s t r u c t u r e s o f t h e s e enzymes a r e v e r y s i m i l a r ,
unexpected r e a c t i v i t y
of
~-tryptophan-28 of
human
lysozyme t o w a r d s N - b r o m o s u c c i n i m i d e c o u l d be evidenced.
As p a r t o f a s t u d y o f t h e whey p r o t e i n s o f v a r i o u s m a m m a l s a c o m p a r i s o n h a s been made o f t h e a - l a c t a l b u m i n s and l y s o z y m e s o f t h e kangaroo and horse.431 rufa)
there i s only
lactation,
b u t no
I n t h e m i l k o f t h e r e d kangaroo (Megaleia and i t o c c u r s
one a - l a c t a l b u m i n
l y s o z y m e has been d e t e c t e d .
throughout
T h e r e a r e t w o a-
l a c t a l b u m i n s i n t h e m i l k o f t h e g r e y kangaroo (Macropus g i g a n t e u s ) : one,
designated
lactation,
a-lactalbumin
and t h e second,
Zone
B,
i s
present
designated a-lactalbumin
present only i n l a t e lactation.
throughout Z o n e A,
One l y s o z y m e i s a l s o p r e s e n t .
i s The
m i l k o f t h e h o r s e (Equus c a b a l l u s ) c o n t a i n s one a - l a c t a l b u m i n and a t l e a s t one l y s o z y m e .
P a r t i a l amino a c i d sequences a r e p r o p o s e d f r o m
sequence
determination
compared
w i t h t h e known sequences o f
and
from
analyses other
of
tryptic
peptides
a-lactalbumins
and
lysozymes. M u l t i p l e forms of
l y s o z y m e found i n t h e r a t l i v e r have been
i s o l a t e d and c h a r a c t e r i z e d f r o m
cellular
organelles.432
Isolation
523
6: Enzymes of
the
e n z y m e s was a c h i e v e d b y Sephadex
gel
f i l t r a t i o n and
chromatography on carboxymethylcellulose. The p u r i t y o f t h e p r e p a r a t i o n s was e x a m i n e d by e l e c t r o p h o r e s i s on p o l y a c r y l a m i d e g e l . The n u c l e a r l y s o z y m e moved as a s i n g l e band, i n d i c a t i n g h o m o g e n e i t y , whereas
other
subcellular
lysozymes
t h e p r e s e n c e o f m o r e t h a n o n e band,
appeared heterogeneous
due
to
thus showing p a r t i a l p u r i t y .
Although t h e s u b c e l l u l a r lysozymes were s i m i l a r w i t h r e s p e c t t o e n z y m a t i c p r o p e r t i e s , pH, mobility,
b u f f e r m o l a r i t y optima,and
electrophoretic
d i f f e r e n c e s were o b s e r v e d i n e l u t i o n p a t t e r n s ,
t o n u c l e a r i n h i b i t o r , and h e a t s e n s i t i v i t y . distinctly
different
by
these
criteria
responses
N u c l e a r l y s o z y m e was as
compared
to
other
s u b c e l l u l a r lysozymes. E v o l u t i o n a r y i m p l i c a t i o n s h a v e b e e n made a b o u t t h e r e l a t i o n b e t w e e n h e n e g g - w h i t e l y s o z y m e and b a c t e r i o p h a g e T4 l y ~ o z y m e . ~ ~ Hen e g g - w h i t e
l y s o z y m e a n d T 4 b a c t e r i o p h a g e l y s o z y m e h a v e t h e same
c a t a l y t i c f u n c t i o n , b u t have non-homologous amino a c i d sequences. Notwithstanding the differences i n t h e i r primary structures,
the two
l y s o z y m e s have s i m i l a r i t i e s i n t h e i r o v e r a l l backbone c o n f o r m a t i o n s , in their
modes
of
b i n d i n g substrates, and p r o b a b l y
mechanisms o f a c t i o n . t h e f o l d i n g of
By d i f f e r e n t c r i t e r i a ,
in their
t h e s i m i l a r i t y between
t h e t w o enzymes can be shown t o be s t a t i s t i c a l l y
significant.
A l s o t h e t r a n s f o r m a t i o n w h i c h o p t i m i z e s t h e agreement
between t h e
backbones
the t w o
of
accurately t h e i r active-site clefts, i n the A,
8,
C,and
molecules
i s
D s u b s i t e s o f hen egg-white
w i t h i n 0.1
t o 0.2
lysozyme.
Furthermore,
shown t o
align
so t h a t s a c c h a r i d e u n i t s bound lysozyme coincide
nm w i t h a n a l o g o u s s a c c h a r i d e s
bound t o phage
a number o f t h e s p e c i f i c i n t e r a c t i o n s
b e t w e e n enzyme and s u b s t r a t e w h i c h w e r e o b s e r v e d f o r h e n e g g - w h i t e l y s o z y m e , and t h o u g h t t o be i m p o r t a n t f o r c a t a l y s i s , a r e f o u n d t o o c c u r i n a s t r u c t u r a l l y a n a l o g o u s way i n t h e phage enzyme. four
atoms from
equivalent,
i n c l u d i n g saccharides
superimpose w i t h a root-mean-square structural
and
Fifty-
t h e r e s p e c t i v e a c t i v e s i t e s w h i c h a p p e a r t o be
functional
bound i n t h e
and C
B
d i s c r e p a n c y o f 1.35
similarities
suggest
sites,
nm.
that
These
the
two
l y s o z y m e s h a v e a r i s e n b y d i v e r g e n t e v o l u t i o n f r o m a common precursor.
This
i s
completely different probability,
the
first
case
i n which
two
proteins
a m i n o a c i d s e q u e n c e h a v e b e e n shown,
of
with high
t o h a v e e v o l v e d by d i v e r g e n t r a t h e r t h a n c o n v e r g e n t
evolution. The b i n d i n g mode o f s o d i u m d o d e c y l s u l p h a t e t o l y s o z y m e a n d t h e accompanying s t r u c t u r a l change o f
l y s o z y m e by b i n d i n g h a v e been
524
Carbohydrate Chemistry
i n v e s t i g a t e d b y means o f t h e b i n d i n g i s o t h e r m ,
the precipitation
and t h e CD s p e c t r a i n p u r e w a t e r ,
and b o r a t e b u f f e r
curve,
solutions.434
NaC1,
The p r e c i p i t a t i o n p h e n o m e n a c o u l d b e e x p l a i n e d i n
t e r m s o f t h e n e u t r a l i z a t i o n o f t h e n e t c h a r g e o f l y s o z y m e due t o dodecyl s u l p h a t e - i o n b i n d i n g .
The a n a l y s i s o f t h e b i n d i n g i s o t h e r m s
by t h e u s e o f t h e B E T e q u a t i o n g a v e t h e s i t e n u m b e r o f t h e f i r s t l a y e r c o r r e s p o n d i n g t o t h e p o s i t i v e l y c h a r g e d r e s i d u e s a t t h e pH studied. helix
The c o n f o r m a t i o n a l change f r o m t h e B - s t r u c t u r e
has
been
environmental
observed
change
of
i n
the
the
second-layer
side-chain
t o t h e a-
binding.
residues
has
also
The been
observed. F l u o r e s c e n c e p o l a r i z a t i o n s t u d i e s h a v e been made o f s a c c h a r i d e binding
to
wheat-germ
- -glucopyranose deoxy-Q i n
the
polarization
saturating
levels
agglutinin
and
l y s o ~ y m e . ~2 ~ - A~c e t a m i d o - 2 -
has been shown t o c a u s e o n l y s l i g h t i n c r e a s e s of
wheat-germ
whereas
the
agglutinin
disaccharide
fluorescence
and
p r o d u c e d i n c r e a s e s i n t h e p o l a r i z a t i o n v a l u e f r o m 0.116 t o 0.151
0.154, r e s p e c t i v e l y .
at
trisaccharide and
These i n c r e a s e s have s u g g e s t e d t h a t r o t a t i o n a l
m o t i o n s o f t h e & - t r y p t o p h a n r e s i d u e a t t h e b i n d i n g s i t e s were b e i n g r e s t r i c t e d by an i n t e r a c t i o n b e t w e e n t h e s e t r y p t o p h a n s and t h e bound sugars.
A m o d e l o f t h e n a t u r e and l o c a t i o n o f t h e s e i n t e r a c t i o n s
was d i s c u s s e d .
Comparable r e s u l t s were o b t a i n e d w i t h l y s o z y m e ,
w h i c h showed a l a r g e r e f f e c t deoxy-P-glucopyranose
but
a
upon t h e b i n d i n g o f 2 - a c e t a m i d o - 2 -
maximal
increase
in
p o l a r i z a t i o n upon
b i n d i n g the corresponding disaccharide or trisaccharide. Bindings o f
calcium t o
l y s o z y m e and i t s d e r i v a t i v e s h a v e been
The s t u d i e d by UV d i f f e r e n c e s p e c t r o s c o p y a t v a r i o u s b i n d i n g c o n s t a n t was 40 M ' l a t n e u t r a l pH. The b i n d i n g c a u s e d p r o t o n r e l e a s e f r o m l y s o z y m e and d i d n o t i n h i b i t t h e b i n d i n g o f t r i -
2 - a c e t a m i d o - 2 - d e o x y - B - ~ - g l ~ ~ 0 p y r a n o s et o l y s o z y m e .
I n t h e presence
o f 0.2M Ca2+, l y s o z y m e s h o w e d 2 6 % o f t h e a c t i v i t y o f t h e f r e e e n z y m e t o w a r d h e x a - 2 - a c e t am id o -2-deox y - B - P - g l u c o p y r anose b u t t h e c l e a v a g e I t was p r e d i c t e d
p a t t e r n was s i m i l a r t o t h a t o f t h e f r e e e n z y m e .
t h a t c a l c i u m bound n e a r t h e c a t a l y t i c c a r b o x y l s c a u s e d i n h i b i t i o n o f lysozyme a c t i v i t y . digestion
that
I t was
calcium
found from
the r e s u l t s o f
binding shifted
the
protease
native-denatured
t r a n s i t i o n i n lysozyme t o w a r d t h e n a t i v e s t a t e . Oxindolealanine-62
lysozyme e q u i l i b r i u m ,
calorimetric,
and
k i n e t i c s of i t s r e a c t i o n w i t h B-~-acetamido-2-deoxyglucopyranose oligosaccharides
have
been
studied.437
r e a c t i o n w i t h N-bromosuccinimide
The
enzyme,
formed
by
and p u r i f i e d by u s i n g a f f i n i t y
525
6: Enzymes chromatography,
was
examined
i n
i t s
binding of
homologous
o l i g o s a c c h a r i d e s o f B-a-acetamido-2-deoxyglucopyranose catalysis
o f
hydrolysis
and
of
and i n i t s
transglycosylation
h e x a s a c c h a r i d e o f B-Q-acetamido-2-deoxyglucopyranose. binding constants
were
determined
by
changes
fluorescence associated with ligand binding.
of
the
Equilibrium
i n absorbance
or
Enthalpies of binding
The p a t t e r n o f v a r i a t i o n o f AGO a n d
were measured c a l o r i m e t r i c a l l y .
A H o o f b i n d i n g w i t h l i g a n d c h a i n l e n g t h a n d pH was d i f f e r e n t f o r oxindolealanine-62
lysozyme compared w i t h
native
lysozyme.
These
r e s u l t s i n d i c a t e t h a t t h e i n t e r a c t i o n s o f t h e ABC r e g i o n o f active
site
with
substrates
tryptophan-62
oxidation,
tryptophan-62
interactions.
intermediate
between
are
substantially
more t h a n e x p e c t e d f o r
oxidation o f I-tryptophan-62. hexasaccharide,
by
the
I-
l o s s o n l y o f t h e L-
The p a r t i t i o n i n g o f t h e g l y c o s y l enzyme
reaction
with
( t r a n s g l y c o s y l a t i o n ) and r e a c t i o n the
altered
a
with
Similarly,
predominantly t o
saccharide
water
i s
the p a t t e r n of
t e t r a - and
acceptor
unaffected
by
cleavage o f
di-saccharide,
is
u n a f f e c t e d by o x i d a t i o n o f I - t r y p t o p h a n - 6 2 .
The 2 0 0 0 - f o l d s l o w r a t e
of
enzyme
catalytic reaction
(relative
to
free
apparently r e f l e c t s a s t e r i c a l l y hindered f i t of t h e a c t i v e s i t e of
and
substrate)
the substrate i n t o
t h e m o d i f i e d enzyme and n o t a s p e c i a l c a t a l y t i c
i m p o r t a n c e o f I=-tryptophan-62.
The g e o m e t r y o f t h e t r a n s i t i o n s t a t e
f o r o l i g o s a c c h a r i d e h y d r o l y s i s i s i n f e r r e d t o be t h e same f o r n a t i v e and o x i d i z e d enzymes. Oxygen-18 l e a v i n g - g r o u p been measured f o r a s e t o f
kinetic isotope effects glycosyl transfer
n i t r o p h e n y l B-Q-glycosides
as
substrates.206
h y d r o l y s i s and a l k a l i n e h y d r o l y s i s e x h i b i t
-+
0.0015
and
1.0386
g l u c o s i d a s e A show
-+
0.0015
and 1.0377
0.0032,
K I E s on
2
Acid-catalysed
K I E s o f k1&18
respectively.
= 1.0355
Lysozyme and B-a-
respectively.
lmax/srn(!/K)o f
0.0061,
( K I E s ) have
r e a c t i o n s w i t h 4-
(2/5)1,/(1/5)18
= 1.0467
The l a r g e m a g n i t u d e o f
t h e s e K I E s r e q u i r e s t h a t c a r b o n - o x y g e n bond s c i s s i o n be f a r a d v a n c e d i n the t r a n s i t i o n states for these reactions.
Therefore,
i n the
t r a n s i t i o n states for the f i r s t i r r e v e r s i b l e steps i n these reaction sequences,
s c i s s i o n of
t h e g l y c o s i d i c bond must
be e s s e n t i a l l y
c o m p l e t e f o r t h e r e a c t i o n s c a t a l y s e d by l y s o z y m e and B - P - g l u c o s i d a s e A,
which
are
respectively.
thought
to
proceed
Acid-catalysed
fia
gN1
a t r a n s i t i o n s t a t e i n v o l v i n g a t l e a s t 80% C - 0 p a r t i a l proton transfer
and SN2
mechanisms,
h y d r o l y s i s i s shown t o p r o c e e d t h r o u g h bond c l e a v a g e and o n l y
t o t h e l e a v i n g 4 - n i t r o p h e n y l o x y g e n atom.
Binding o f 4-methylumbelliferyl
c h i t o t e t r a o s i d e t o hen lysozyme
526
Carbohydrate Chemistry
h a s been s t u d i e d by m e a s u r i n g changes i n f l u o r e s c e n c e a t 375 nm.438 4-methylumbelliferyl lj-acetyl-chitotetraoside
Hydrolysis of
c a t a l y s e d b y l y s o z y m e was s t u d i e d b y m e a s u r i n g t h e r e l e a s e o f 4 methylumbelliferone
from
4-methylumbelliferyl
chitotetraoside fluorimetrically,
N-acetyl-
and t h e k i n e t i c c o n s t a n t s were
d e t e r m i n e d i n t h e pH r a n g e o f 2 t o 8 a t 0.1
i o n i c s t r e n g t h and 42’C.
The b i n d i n g a n d k i n e t i c d a t a s h o w e d t h a t 4 - m e t h y l u m b e l l i f e r y l acetyl-chitotetraoside
y-
binds t o subsites A t o E (productive binding)
and s u b s i t e s A t o D w i t h t h e n o n - r e d u c i n g s u g a r r e s i d u e e x t e n d i n g beyond s u b s i t e A (non-productive k i n e t i c constants
binding).
a t p H 8.5.
p r o d u c t i v e c o m p l e x was 0.77
The f r a c t i o n o f t h e
T h e pH d e p e n d e n c e o f t h e
was a n a l y s e d a s s u m i n g t h a t t h e m o l e c u l a r s p e c i e s
w i t h i o n i z e d I - a s p a r t i c a c i d - 5 2 and p r o t o n a t e d I - g l u t a m i c a c t i v e and I - a s p a r t i c a c i d - 1 0 1
pK v a l u e s o f t h e s e g r o u p s were d e t e r m i n e d . aspartic-52, a n d 4.20,
!=-glutamic-35,
respectively,
respectively, respectively,
acid-35 i s
participates i n the binding,
and t h e
The pK v a l u e s o f
a n d L - a s p a r t i c - 1 0 1 a c i d s w e r e 3.60, for
free
lysozyme,
3.40,
f o r t h e p r o d u c t i v e c o m p l e x , a n d 3.95, f o r t h e n o n p r o d u c t i v e complex.
6.55,
I-
6.20,
a n d 3.40,
6.55,and
3.30,
The pK v a l u e s f o r f r e e
lysozyme were i n e x c e l l e n t agreement w i t h t h o s e o b t a i n e d by a n a l y s i s of
for 4-methylumbelliferyl N-acetyl-
the kinetic constants
chitotrioside. pH 5.2.
The f r e e e n e r g y o f a c t i v a t i o n was 24 k c a l m o l ”
Comparison w i t h
the
corresponding
value
obtained
at for
h y d r o l y s i s o f c h i t o h e x a o s i d e suggests t h a t t h e i n t e r a c t i o n s o f 2-
acetamido-2-deoxy-~-glucopyranose r e s i d u e s w i t h s u b s i t e s E and F i n t h e t r a n s i t i o n s t a t e are i m p o r t a n t i n lysozyme c a t a l y s i s . Binding
of
4-methylumbelliferyl
methylumbelliferyl studied
by
chitotetraoside to
fluorescence
methylumbelliferyl
chitotrioside human
measurement.439
chitotrioside
and
and
4-
l y s o z y m e has been Hydrolysis
of
4-
4-methylumbelliferyl
c h i t o t e t r a o s i d e c a t a l y s e d by human l y s o z y m e was s t u d i e d by m e a s u r i n g the
release of
4-methylumbelliferone
fluorimetrically,
and t h e
k i n e t i c c o n s t a n t s w e r e d e t e r m i n e d i n t h e pH r a n g e o f 2 t o 8 a t 0.1 i o n i c s t r e n g t h a n d 42’C.
On t h e b a s i s o f b i n d i n g and k i n e t i c d a t a ,
i t was shown t h a t 4 - m e t h y l u m b e l l i f e r y l c h i t o t r i o s i d e b i n d s m a i n l y t o s u b s i t e s A t o D w i t h t h e t e r m i n a l m e t h y l u m b e l l i f e r y l g r o u p bound t o subsite D (non-productive
b i n d i n g ) and t h a t 4 - m e t h y l u m b e l l i f e r y l
c h i t o t e t r a o s i d e b i n d s t o s u b s i t e s A t o E [ p r o d u c t i v e b i n d i n g ) and s u b s i t e s A t o D w i t h t h e n o n - r e d u c i n g s u g a r r e s i d u e e x t e n d i n g beyond subsite A kinetic
(non-productive constants
for
binding). hydrolysis
The o f
pH d e p e n d e n c e s o f t h e 4-methylumbelliferyl
527
6: Enzymes chitotrioside
and
4-methylumbelliferyl
c h i t o t e t r a o s i d e were
analysed, assuming t h a t non-productive b i n d i n g occurs c o m p e t i t i v e l y , that
an i o n i z a b l e g r o u p i n a d d i t i o n t o t h e c a t a l y t i c g r o u p s
a s p a r t i c - 5 2 and & - g l u t a m i c - 3 5 ) that
the
molecular species
(L-
p a r t i c i p a t e s i n t h e c a t a l y s i s , and
with ionized &-aspartic
acid-52
and
protonated I-glutamic acid-35 i s active.
Analyses of t h e k i n e t i c
constants
chitotrioside
for
4-methylumbelliferyl
and
4-
m e t h y l u m b e l l i f e r y l c h i t o t e t r a o s i d e b o t h gave t h e same p E v a l u e s o f the
c a t a l y t i c groups
s t r e n g t h a n d 42OC).
( p K 5 2 = 3.63
a n d p 5 3 5 = 6.68
a t 0.1
ionic
These p E v a l u e s were v e r y c l o s e t o t h e v a l u e s
d e t e r m i n e d p r e v i o u s l y by s p e c t r o s c o p i c methods. A lysozyme-catalysed r e a c t i o n o f c h i t o - o l i g o s a c c h a r i d e s has
been d e s c r i b e d . 4 4 0
The t i m e - c o u r s e s
of
substrate
c o n s u m p t i o n and
p r o d u c t f o r m a t i o n i n t h e l y s o z y m e - c a t a l y s e d r e a c t i o n were d e t e r m i n e d w i t h c h i t o t e t r a o s e and c h i t o p e n t a o s e as s u b s t r a t e t o a c c u m u l a t e d a t a s u i t a b l e f o r t h e e s t i m a t i o n o f r a t e c o n s t a n t s by n u m e r i c a l a n a l y s i s .
o r h.p.1.c.
The l y s o z y m e - c a t a l y s e d r e a c t i o n s w e r e f o l l o w e d by t.1.c.
C h i t o t e t r a o s e decomposed a p p a r e n t l y t o s m a l l o l i g o s a c c h a r i d e s w i t h i n 5 h,
and c h i t o p e n t a o s e decomposed w i t h i n 15 m i n a t pH 5.0
The t e m p e r a t u r e
dependence o f
the rate of
i n i t i a l s u b s t r a t e showed a d i f f e r e n t
and 5 O o C .
disappearance of
the
p r o f i l e from t h a t observed w i t h
g l y c o l c h i t i n as s u b s t r a t e by t h e r e d u c i n g p o w e r method.
The o r d e r
( o r d i s t r i b u t i o n ) o f t h e amount o f p r o d u c t f o r m e d f r o m c h i t o p e n t a o s e i n the r e a c t i o n time-course d e t e r m i n e d by h.p.1.c. t h a n t h a t i n t.l.c., thought
d e t e r m i n e d by t.1.c.
d i f f e r e d from that
The r e l a t i v e e r r o r i n h.p.1.c. and t h e t i m e - c o u r s e
was much l e s s
d e t e r m i n e d by h.p.1.c.
was
t o be o f s u f f i c i e n t a c c u r a c y f o r t h e e s t i m a t i o n o f r a t e
c o n s t a n t s by c o m p u t e r a n a l y s i s . High
l y s o z y m e and c h i t i n a s e a c t i v i t i e s have been f o u n d i n
Leydig's organ of Etmopterus spinax
-Tompedo
nobiliana,
i n Leydig's
, Somniosus
m i c r o c e p h a i u s , and
and e p i g o n a l o r g a n s and s p l e e n o f
R a j a r a d i a t a , and i n t h e e p i g o n a l o r g a n o f R h i n o p t e r a bonasus.19 An
enzyme
s t r e p t o c o c c u s of
actively
lysing the
c e l l walls
of
a haemolytic
g r o u p A was shown t o be a l y s o z y m e mucopeptide-l\l-
acetylmuranoylhydrolase.441
I t was i s o l a t e d f r o m t h e c u l t u r e l i q u i d
of
ammonium
A c t i n o m y c e s l e v o r i s by
further
purification
chromatography.
by
gel
sulphate
f i l t r a t i o n
The m o l e c u l a r w e i g h t (12,5001,
p r e c i p i t a t i o n and and
ion-exchange
isoelectric point
(10.61, and a m i n o a c i d c o m p o s i t i o n o f t h e enzyme w e r e d e t e r m i n e d .
A
peptidoglycan
A
obtained
from
the
c e l l
walls
o f
a
group
s t r e p t o c o c c u s was u s e d a s t h e s u b s t r a t e i n a d e t e r m i n a t i o n o f t h e
Carbohydrate Chemistry
528
s p e c i f i c i t y o f t h e enzyme. Lysozyrne a c t i v i t y h a s b e e n o b s e r v e d i n b a c t e r i o p h a g e T4 ghosts.442 T h i s enzyme is p r o b a b l y r e s p o n s i b l e f o r the l y s i s from w i t h o u t , observed a t high m u l t i p l i c i t y of i n f e c t i o n , a p r o c e s s independent of the p r e s e n c e of the e gene product which is a l s o a lysozyme. The g h o s t l y s o z y m e a n d e l y s o z y m e d i f f e r e d w i t h r e s p e c t t o t h e i r r e q u i r e m e n t s f o r maximal c a t a l y t i c a c t i v i t y and t o some extent in substrate specificity. The g h o s t l y s o z y m e w a s r e l e a s e d f r o m p h a g e p a r t i c l e by t h e a c t i o n o f T r i t o n X-100. B a c i l l u s c e r e u s peptidoglycan w i t h !-unsubstituted glucosamine r e s i d u e s has b e e n f o u n d t o b e i n s e n s i t i v e t o t r e a t m e n t w i t h b a c t e r i o p h a g e T 4 l y s o ~ y m e . ~A f~t e~r N - a c e t y l a t i o n w i t h a c e t i c a n h y d r i d e , T 4 l y s o z y m e - c l e a r e d s o l u t i o n s of t h e p e p t i d o g l y c a n and T h e d i g e s t i o n p r o d u c t s were m a i n l y r e d u c i n g s u g a r s were l i b e r a t e d . o f h i g h m o l e c u l a r w e i g h t , s i n c e t h e p e p t i d o g l y c a n is p e p t i d e c r o s s linked t o a great extent. !-Propylation d i d not convert the p a r t i a l l y !-unsubstituted p e p t i d o g l y c a n t o a s e n s i t i v e form. I t is concluded t h a t the acetarnido g r o u p s are r e q u i r e d f o r b i n d i n g and/or c a t a l y s i s by T4 l y s o z y m e .
37
~~~gO-1,6-~-Glucosidases
Detergent-solubilized pig intestinal sucrose-a-P- - g l u c o s i d a s e h a s b e e n p u r i f i e d 40 t o glucohydrolase-oligo-(1 + 6)-Q 100 t i m e s w i t h a y i e l d o f 1 0 t o 20% by a r a p i d i m m u n o a d s o r b e n t technique.444 T h e p u r i f i e d e n z y m e was s h o w n t o b e h o m o g e n e o u s by i m m u n o e l e c t r o p h o r e s i s a n d was e s s e n t i a l l y f r e e f r o m o t h e r k n o w n brush-border peptidases and disaccharides. It c o n s i s t e d o f two p o l y p e p t i d e c h a i n s w i t h a p p a r e n t m o l e c u l a r w e i g h t s o f 140,000 and 150,000, r e s p e c t i v e l y . I n c o n t r a s t , t h e enzyme isolated from p i g s i n w h i c h t h e p a n c r e a s was c o m p l e t e l y d i s c o n n e c t e d f r o m t h e duodenum 3 days before k i l l i n g migrated in polyacrylamide gel e l e c t r o p h o r e s i s i n d o d e c y l s u l p h a t e as a s i n g l e p o l y p e p t i d e c h a i n w i t h a n a p p a r e n t m o l e c u l a r w e i g h t o f 260,000. Treatment with pancreatic proteases i n v i t r o converted t h e large polypeptide chain i n t o bands w i t h m o l e c u l a r w e i g h t s e q u a l t o or somewhat l a r g e r t h a n t h o s e o f sucrose-a-e-glucohydrolase-oligo-(1 + 6 ) - P - g l u c o s i d a s e from normal pigs. I t was s u g g e s t e d t h a t t h e s i n g l e c h a i n r e p r e s e n t s a precursor, which is converted t o the f i n a l sucrose-a-kg l u c o h y d r o l a s e - o l i g o - ( 1 + 6 ) - Q - g l u c o s i d a s e i n v i v o by p a n c r e a t i c p r o t e o l y t i c e n z y m e s . T h i s i s o n e o f t h e few e x a m p l e s i n v e r t e b r a t e s
6: Enzymes of
529
a single polypeptide chain carrying two enzymatically active
sites.
The s i g n i f i c a n c e o f t h e r e s u l t f o r of
b i o s y n t h e s is
t h e mechanism o f t h e
s u c r 0 s e - a - Q - g l u c o h y d r o l a s e - 0 1ig o - ( 1
-c
6)-Q-
glucosidase i s discussed.
Pectate,
38
Pectin,
and P o l y - Q - G a l a c t u r o n a t e Lyases
P e c t a t e l y a s e h a s been p u r i f i e d t o a n e a r l y homogeneous s t a t e from the c u l t u r e f i l t r a t e o f Streptomyces nitrosporeus.445 m o l e c u l a r w e i g h t was e s t i m a t e d t o b e a b o u t 4 1 , 0 0 0 . p o i n t was pH 4.6.
The
Isoelectric
The e n z y m e was m o s t a c t i v e a t pH 10.0 a n d 5OoC,
a n d was r e l a t i v e l y s t a b l e a t a pH r a n g e o f 4 - 1 1 ( a t 2OC f o r 48 h ) and b e l o w
4OoC ( a t
pH 7.0
maximum a c t i v i t y .
for
10
min).
T h e e n z y m e was a n
Ca2+ was
required for
e n d o p e c t a t e l y a s e w h i c h was
more a c t i v e o n l o w m e t h o x y l p e c t i n t h a n on p o l y - P - g a l a c t u r o n i c
acid
and h a d m a c e r a t i n g a c t i v i t y on p o t a t o t i s s u e and G a n p i b a r k . The d e g r a d a t i o n o f p o l y - Q - g a l a c t u r o n i c a c i d b y r u m e n c i l i a t e p r o t o z o a has been i n v e s t i g a t e d . 4 4 6
The d e p o l y m e r a s e a c t i v i t y o f
c e l l - f r e e e x t r a c t s o f n i n e s p e c i e s o f r u m e n c i l i a t e p r o t o z o a and t w o mixed protozoal preparations, poly-Q-galacturonic
grown i n v i v o
a c i d was e x a m i n e d .
and i n v i t r o ,
towards
The h i g h e s t a c t i v i t y was
found w i t h E r e m o p l a s t r o n b o v i s and O s t r a c o d i n i u m o b t u s u m b i l o b u m while
there
was
none
i n
the
spined
or
spineless
forms
E n t o d i n i u m c a u d a t u m and l i t t l e i n P o l y p l a s t r o n m u l t i v e s i c u l a t u m . the b a s i s of t h e r a p i d drop i n v i s c o s i t y , p r o d u c t i o n of
u.v.-absorbing
material,
i n h i b i t i o n by EDTA,and
t h e enzymes
from
o f On the
a l l active
s p e c i e s were d e s i g n a t e d as e n d o p e c t a t e l y a s e s a l t h o u g h some p o l y - P galacturonase
may
be
present.
Neither
pectin
nor
poly-Q-
g a l a c t u r o n i c a c i d s u p p o r t e d t h e s u r v i v a l o r g r o w t h o f any o f t h e protozoal species tested. High-performance investigate
pectic
liquid
chromatography
enzymes.447
preparations Pectinex
Technical
has
been
pectic
used
to
multienzyme
U l t r a and Rohament P w e r e c h r o m a t o g r a p h e d on
an a n a l y t i c a l s c a l e u s i n g m e d i u m - p r e s s u r e l i q u i d c h r o m a t o g r a p h y on a g l y c o l methacrylate r i g i d macroreticular gel, ion-exchange derivatives. gradient
elution
(with
e m p l o y e d and f r a c t i o n s
S p h e r o n 1000 and i t s
A c o m b i n a t i o n o f i s o c r a t i c and l i n e a r -
gradients
i n
ionic
strength or
w e r e m o n i t o r e d by m e a s u r e m e n t s o f
c o n d u c t i v i t y , pH, and enzyme a c t i v i t y . (A285 a n d A_254), f o r r a p i d s e p a r a t i o n s of p e c t i c enzymes a r e e l a b o r a t e d .
pH)
was
absorbance Conditions The r e s u l t s
530
Carbohydrate Chemistry
i n d i c a t e t h e p o s s i b i l i t i e s of s e p a r a t i n g t h e t e c h n i c a l l y undesirable p e c t i n - e s t e r a s e a c t i v i t y f r o m t h e o t h e r enzyme a c t i v i t i e s , and o f a more d e t a i l e d b i o c h e m i c a l i n v e s t i g a t i o n o f t h e s e enzymes, i m p o r t a n t f o r the .food i n d u s t r y . The s i m u l t a n e o u s s y n t h e s i s o f p e c t i n l y a s e a n d c a r o t o v o r i c i n n a l i d i x i c acid, or u l t r a v i o l e t l i g h t
has been i n d u c e d by m i t o m y c i n C,
irradiation i n e i n i a c a r o t ~ v o r a . ~ When ~ ~ K i n i a carotovora Er, a bacteriocinogenic ultraviolet mitomycin
l i g h t or
or
nalidixic
C
(designated
strain,
(UV)
was
induced a f t e r
inhibitors
carotovoricin)
acid,
of
pectin
activity
i r r a d i a t i o n by
DNA s y n t h e s i s , lyase
and
such as
bacteriocin
appeared i n t h e c u l t u r e
fluid.
The o p t i m a l d o s e o f e a c h o f t h e s e a g e n t s f o r p r o d u c i n g t h e e n z y m e o r b a c t e r i o c i n was essentially
identical,
t h e same.
and t h e
time-courses
for
b o t h were
The s y n t h e s e s o f t h e enzyme and b a c t e r i o c i n
w e r e a s s u m e d t o b e r e g u l a t e d b y t h e same m e c h a n i s m ,
i n which a
r e p r e s s o r i n a c t i v a t e d by UV l i g h t ,
m i t o m y c i n C , o r n a l i d i x i c a c i d was
involved.
bacteriocinogenic
E.
The
other
three
carotovora a l s o formed p e c t i n lyase,
i n
the
presence o f
syntheses
of
mitomycin
pectic
lyase
indicating that
C,
and
strains
of
i n addition t o carotovoricin
carotovoricin
simultaneous
were
widespread
phenomena i n b a c t e r i o c i n o g e n i c s t r a i n s o f E. c a r o t o v o r a . The p e c t i c enzyme a c t i v i t i e s o f b a c t e r i a a s s o c i a t e d w i t h r o t t e d o n i o n s h a v e been i n v e s t i g a t e d . 4 4 9 with soft
as a V i b r i o sp., Acinetobacter
-----Bacillus
The
aerobic bacteria associated
r o t i n o n i o n s ( A l l i u m cepa) were Micrococcus epidermidis,
sp.,
a
megaterium.
Xanthomonas
sp.,
With the cup-plate
i s o l a t e d and i d e n t i f i e d Pseudomonas c e p a c i a ,
and
B a c i l l u s polymyxa,
and
assay method, no p e c t i n
h y d r o l a s e c o u l d b e d e t e c t e d f r o m any o f t h e s e i s o l a t e s w h e n t h e y w e r e c u l t u r e d i n p e c t i n medium, detectable. for
P.
b u t l y a s e a n d p e c t i n e s t e r a s e s were
O n i o n t i s s u e c u l t u r e s showed p e c t i n h y d r o l a s e a c t i v i t y
c e p a c i a a n d B.
polyfiyza
and l y a s e and p e c t i n e s t e r a s e
a c t i v i t i e s for a l l of the isolates,
u s u a l l y a t h i g h e r l e v e l s of
a c t i v i t y t h a n t h o s e o f t h e p e c t i n medium c u l t u r e f i l t r a t e s . culture
media,
Vibrio
sp.
pectinesterase activities.
showed
the
highest
In the viscometric test,
I n both
lyase
i s o l a t e s a c h i e v e d a t l e a s t a 50% d e c r e a s e i n v i s c o s i t y f o r enzyme,
w i t h L e p i d e r m i d i s a n d V i b r i o sp.
d e c r e a s e s a s h i g h as 83%.
lyase
recording viscosity
The a b i l i t y t o c a u s e s o f t r o t i n o n i o n
b u l b s was d e m o n s t r a t e d b y P. a c i d a t a c o n c e n t r a t i o n o f 0.8 enzyme p r o d u c t i o n ,
and
a l l o f the
c e p a g a and Xanthomonas sp. mg m l - l
Benzoic
caused t o t a l suppression of
whereas sodium benzoate a t t h i s c o n c e n t r a t i o n
53 1
6: Enzymes
r e d u c e d p e c t i n e s t e r a s e p r o d u c t i o n by 71% and l y a s e p r o d u t i o n b y 72%. The p o s s i b l e u s e o f t h e s e p r e s e r v a t i v e s i n t h e c o n t r o l o f s o f t r o t i n o n i o n s is n o t e d .
P o l y-Q-Galacturonases
39
The d e g r a d a t i o n o f p o l y - Q - g a l a c t u r o n i c p r o t o z o a has been i n v e s t i g a t e d . 4 4 6 cell-free
a c i d by rumen c i l i a t e
The d e p o l y m e r a s e a c t i v i t y o f
e x t r a c t s of n i n e s p e c i e s o f rumen c i l i a t e p r o t o z o a and t w o
mixed protozoal preparations, poly-Q-galacturonic
g r o w n i n v i v o and i n v i t r o ,
a c i d was e x a m i n e d .
towards
The h i g h e s t a c t i v i t y was
f o u n d w i t h E r e m o p l a s t r o n b o v i s and O s t r a c o d i n i u m o b t u s u m b i l o b u r n , while
there
was
none
i n
the
or
spined
spineless
forms
E n t o d i n i u m c a u d a t u m and l i t t l e i n P o l y p a s t r o n m u l t i c e s i c u l a t u m . the basis o f the r a p i d drop i n viscosity, t h e p r o d u c t i o n o f u.v.-adsorbing
i n h i b i t i o n by H4 e d t a ,
material,
may b e p r e s e n t .
On and
t h e enzymes f r o m a l l
a c t i v e s p e c i e s w e r e d e s i g n a t e d as e n d o p e c t a t e l y a s e s , poly-Q-galacturonase
of
a l t h o u g h some
Neither p e c t i n nor poly-p-
g a l a c t u r o n i c a c i d s u p p o r t e d t h e s u r v i v a l o r g r o w t h o f any o f t h e protozoal species tested. The a p p l i c a t i o n o f c a r b o h y d r a s e s i n c l u d i n g p o l y - B - g a l a c t u r o n a s e t o
the
extraction
investigated.349
of
For
proteins further
from
details
wheat see
bran
has
been
i n i t i a l citation of
r e f .349. The e f f e c t o f i m m o b i l i z a t i o n o f A s p e r g i l l u s n i q e r e x t r a c e l l u l a r poly-a-galacturonase i n v e s t i g a t e d .450
on
kinetics
and
action
pattern
The e x t r a c e l l u l a r p o l y - B - g a l a c t u r o n a s e
was c o v a l e n t l y bound t o 4 - a m i n o b u t a n o i c a c i d , a m i n o h e x a n o i c a c i d , r e s p e c t i v e l y , as s p a c e r s . enzyme i n v a r i a b l y
has
been
o f A.
glycyl-glycine,
niqer and 6 -
Immobilization o f the
l e d t o decreased a c t i v i t y ,
the extent of
the
decrease being i n v e r s e l y p r o p o r t i o n a l t o t h e chain l e n g t h o f t h e spacer.
This fact,
as w e l l a s a p p a r e n t k i n e t i c p a r a m e t e r s o f t h e
i m m o b i l i z e d enzyme, i n d i c a t e d t h a t diffusion
s t e r i c hindrance
and,
probably,
e f f e c t s a r e r e s p o n s i b l e f o r t h e decrease i n a c t i v i t y .
c o v a l e n t b i n d i n g a l s o c a u s e d an a l t e r a t i o n o f
The
the action pattern o f
t h e enzyme on p o l y m e r i c and o l i g o m e r i c s u b s t r a t e s .
I n t h e case o f
t h e p o l y m e r s t h e r a n d o m n e s s i n d e g r a d a t i o n was l o w e r e d b e c a u s e o f restriction
of
the
s u b s t r a t e molecule.
enzyme
action
to
peripheral
Among o l i g o m e r i c s u b s t r a t e s ,
c h a n g e was o b s e r v e d i n t e t r a - ( g - g a l a c t o s i d u r o n i c
areas
of
the
t h e most i m p o r t a n t acid),
i n which (2
532 +
Carbohydrate Chemistry
2) d e g r a d a t i o n
occurred
as
well
as
the
(1 +
3)
degradation
c h a r a c t e r i s t i c o f t h e s o l u b l e enzyme. The changes i n P o l Y - q - g a l a c t u r o n a s e ripening
of
normal
investigated.451
and
mutant
is
There
a
a c t i v i t y which occur d u r i n g tomato
sequential
have
been
of
two
during ripening.
These
i s o e n z y m e s h a v e been p u r i f i e d and t h e i r p r o p e r t i e s compared.
Poly-
isoenzymes,
1 and 2,
f r u i t
appearance
poly-Q-galacturonases
g-galacturonase
1 h a s a btr
and has a d e n s i t y
of
galacturonase 2 has a has a d e n s i t y of
1.343
Mr
1.300
1,000,000,
of
g cm-3
m
Poly-Q-
i s 5 0 % i n a c t i v a t e d a t 57'C,and
i ' n ~c a e s i u m c h l o r i d e .
i s o g e n i c l i n e s homozygous f o r ripen normally
i n caesium c h l o r i d e .
o f 42,000, g ~
i s 50% i n a c t i v a t e d a t 78OC
and c o n t a i n r e d u c e d a m o u n t s o f
F r u i t s from
m u t a t i o n do n o t
the ever-ripe (Nr)
poly-n-galacturonase.
O n l y p o l y a - g a l a c t u r o n a s e 1 was p r o d u c e d i n N r f r u i t .
Tomatoes f r o m
i s o g e n i c l i n e s homozygous f o r t h e r i p e n i n g i n h i b i t o r ( r i n ) m u t a t i o n do n o t r i p e n n o r m a l l y a n d p r o d u c e v e r y l i t t l e d e t e c t a b l e p o l y - a galacturonase. properties,
Although poly-0-galacturonases they
b o t h gave
rise
t o
a
1 and 2 had d i f f e r e n t
single
p o l y p e p t i d e on
e l e c t r o p h o r e s i s i n polyacryamide g e l s i n t h e presence o f sodium dodecylsuphate
(Mr
- 46,000).
p r o d u c e d by l i m i t e d with
chymotrypsin
A comparison o f
the major
fragments
p r o t e o l y s i s of poly-e-galacturonases suggests t h a t
isoenzymes were s i m i l a r .
the
polypeptides
from
1 and 2 the
two
The same c o n c l u s i o n was r e a c h e d f r o m a
comparison o f poly-P-galacturonases
1 and 2 by r a d i o i m m u n o a s s a y ,
using antibody prepared against poly-E-galacturonase
2 a n d 1251-
l a b e l l e d p o l y -g-ga l a c t u r o n a s e 2. Some p r o p e r t i e s o f
t h e p o l y g-galacturonase-elicitor
from
the
f i l t r a t e s o f R h i z o p u s s t o l o n i f e r c u l t u r e s h a v e been e x a m i n e d i n an a t t e m p t t o u n d e r s t a n d i t s mode o f a c t i o n as an e l i c i t o r o f casbene synthetase a c t i v i t y
i n castor-bean
seedlings.452
Both t h e poly-Q-
g a l a c t u r o n a s e a c t i v i t y and t h e e l i c i t o r a c t i v i t y a r e h e a t l a b i l e with
similar
heat-sensitivity
profiles.
a c t i v i t y o f t h e enzyme i s l o s t on t r e a t m e n t as h a d b e e n s h o w n p r e v i o u s l y f o r
Also,
the
the e l i c i t o r
activity.
o p t i m u m o f t h e enzyme a c t i v i t y w i t h p o l y - g - g a l a c t u r o n i c s u b s t r a t e i s 4.9.
catalytic
w i t n sodium periodate, T h e pH
a c i d as t h e
Exposures o f g e r m i n a t i n g castor-bean s e e d l i n g s t o
the e l i c i t o r for short-term
p e r i o d s of
w a s h i n g and i n c u b a t i o n i n s t e r i l e ,
1 t o 1 0 m i n u t e s f o l l o w e d by
d i s t i l l e d water are p a r t i a l l y
e f f e c t i v e i n e l i c i t a t i o n i n comparison w i t h t h e continuous exposure
o f t h e s e e d l i n g s o v e r 11 h o u r s t o t h e same a m o u n t o f t h e e l i c i t o r . The i n i t i a l r a t e o f r e a c t i o n c a t a l y s e d b y t h e enzyme is a b o u t 3
6: Enzymes
533 a c i d as a s u b s t r a t e t h a n w i t h
times faster w i t h poly-g-galacturonic partially
(50%) m e t h y l a t e d p o l y - e - g a l a c t u r o n i c
Em v a l u e
acid
o f t h e enzyme f o r p o l y - q - g a l a c t u r o n i c
(pectin).
The
a c i d i s a b o u t 4.2
m i l l i m o l a r i n t e r m s o f m o n o m e r i c u n i t s and a b o u t 0.07
millimolar i n
terms
the
of
polymer
concentration.
Examination of
types
of
p r o d u c t s f o r m e d by t h e a c t i o n o f t h e enzyme s u g g e s t s t h a t i t i s an
-endo-hydrolase.
The
amino
acid composition of
this
enzyme i s
s i m i l a r t o those o f other e x t r a c e l l u l a r fungal proteins reported. The c a r b o h y d r a t e m o i e t y o f t h e g l y c o p r o t e i n p o l y - ! - g a l a c t u r o n a s e e l i c i t o r i s c o m p o s e d o f 9 2 % q - m a n n o s e a n d 8 % 0 - g l u c o s a m i n e by g a s chromatography-mass
spectrometry
analysis.
The
linkage-group
a n a l y s i s o f t h e c a r b o h y d r a t e m o i e t y showed t h a t q - m a n n o s y l r e s i d u e s which
are
1,2-linked
comprise
about
70% o f
the
total
r e s i d u e s a n d d e m o n s t r a t e d t h e p r e s e n c e o f some 1 , 3 , 6 -
glycosyl
a n d 1,2,6-
l i n k e d b r a n c h i n g D-mannosyl r e s i d u e s .
t ur o n a s e e l i c i t or p u r i f i e d
A p p a r e n t 1y h o m o g e n e o u s p o 1y -;-galac
f r o m t h e f i l t r a t e s o f R h i z o p u s s t o l o n i f e r c u l t u r e s has been f o u n d t o stimulate
germinating castor-bean
increased
levels
of
casbene
seedlings t o produce g r e a t l y
synthetase
activity.453
The
p u r i f i c a t i o n procedure i n v o l v e d g e l - f i l t r a t i o n chromatography on Sephadex
G-25
a n d G-75
columns
followed
c h r o m a t o g r a p h y o n a Sephadex CM C-50 column. purified preparation
was
i n d i c a t e d by
the
by
cation-exchange
Homogeneity o f t h e results of
cationic
p o l y a c r y l a m i d e d i s c g e l e l e c t r o p h o r e s i s and i s o e l e c t r i c f o c u s i n g (PI = 8.0).
The i d e n t i t i e s o f t h e casbene e l i c i t o r a c t i v i t y and p o l y - Q -
galacturonase
were
i n d i c a t e d by
the
coincidence o f
the
two
a c t i v i t i e s a t a l l stages of p u r i f i c a t i o n , the coincidence o f both activities
with the single protein-staining
band d e t e c t e d on a
c a t i o n i c p o l y a c r y l a m i d e d i s c g e l and an i s o e l e c t r i c - f o c u s i n g g e l , and t h e i d e n t i c a l b e h a v i o u r o f b o t h a c t i v i t i e s on an agarose g e l affinity
column.
The p u r i f i e d p o l y - P - g a l a c t u r o n a s e
elicitor
i s
a
g l y c o p r o t e i n w i t h a p p r o x i m a t e l y 20% c a r b o h y d r a t e c o n t e n t a n d a n estimated molecular weight of
32,000
by p o l y a c r y l a m i d e d i s c g e l
electrophoresis. The
variability
isoelectric-focusing ~
of
poly-g-galacturonase
patterns
has
been
and
protein
investigated
i n
B o t 9 t i s cinerea isolates.454
Acetone p r e c i p i t a t e s f r o m c u l t u r e
filtrates
of
I _ I _
of
three
isolates
B. c i n e r e a w e r e r e s o l v e d b y
i s o e l e c t r i c f o c u s i n g on p o l y a c r y l a m i d e g e l s t o d e t e c t d i f f e r e n c e s i n the poly-Q-galacturonase four
and p r o t e i n p a t t e r n s .
Only a few
bands
-
i n t h e p r o t e i n p a t t e r n s and t w o i n t h e p o l y - E - g a l a c t u r o n a s e
534
Carbohydrate Chemistry
p a t t e r n s - were common t o a l l t h e i s o l a t e s . D i f f e r e n c e s were a l s o detected i n poly-;-galacturonase and p r o t e i n p a t t e r n s o f a l l t h e s a m e i s o l a t e a t d i f f e r e n t a g e s of c u l t u r e ( 7 , 1 4 , a n d 21 d ) . I d e n t i c a l p o l y - g - g a l a c t u r o n a s e p a t t e r n s w e r e o b t a i n e d when i s o e l e c t r i c f o c u s i n g was a p p l i e d t o an a c e t o n e p r e c i p i t a t e e i t h e r d i r e c t l y o r a f t e r f u r t h e r p u r i f i c a t i o n by ion-exchange chromatography.
40
exo-Poly-Q-Galacturonate
Lyases
An m g - p o l y - Q - g a l a c t u r o n a t e l y a s e h a s been p u r i f i e d t o a homogeneous s t a t e from t h e c u l t u r e f i l t r a t e of S t r e p t o m y c e s m a s s a s p ~ r e u s . ~The ~ ~m o l e c u l a r weight was e s t i m a t e d t o be a b o u t 5 4 , 0 0 0 and t h e i s o e l e c t r i c p o i n t was pH 5 . 5 . The enzyme was most a c t i v e a t pH 9.5 and 4 O o C , and was r e l a t i v e l y s t a b l e a t pH Ca2+ was r e q u i r e d f o r maximum a c t i v i t y . The enzyme 3.0 f o r 10 m i n . was most a c t i v e on t r i - q - g a l a c t u r o n i c a c i d and had h i g h e r a c t i v i t y on low m e t h o x y l p e c t i n s r a t h e r t h a n on p o l y - q - g a l a c t u r o n i c a c i d . The enzyme removed t e r m i n a l u n s a t u r a t e d d i - Q - g a l a c t u r o n a t e u n i t s from t h e Q - g a l a c t u r o n i d e c h a i n s .
41
Pullulanases
The p r o p e r t i e s of two a m y l a s e s which d i f f e r i n t h e i r s u b s t r a t e s p e c i f i c i t y and s u b c e l l u l a r l o c a t i o n a s w e l l a s a c h l o r o p l a s t a s s o c i a t e d R-enzyme ( d e b r a n c h i n g a c t i v i t y ) have been r e p o r t e d . 3 0 1 The c o n v e r s i o n o f a c t i v e and i n a c t i v e d e b r a n c h i n g e n z y m e s i n r i c e s e e d s h a s been r e p o r t e d . 4 5 6 D e b r a n c h i n g - e n z y m e a c t i v i t y i n r i c e s e e d s i n c r e a s e d d u r i n g t h e e a r l y s t a g e of r i p e n i n g and t h e n d e c r e a s e d , and i n c r e a s e d a g a i n d u r i n g g e r m i n a t i o n . The i n a c t i v e enzyme accumulated r a p i d l y i n r i p e n i n g from t h e t w e n t i e t h day a f t e r flowering. R a d i o a c t i v e amino a c i d s were r e a d i l y i n c o r p o r a t e d i n t o t h e a c t i v e d e b r a n c h i n g enzyme a f t e r t h e i r a b s o r p t i o n i n t o immature r i c e seeds. Subsequently, t h e r a d i o a c t i v i t y increased i n the It i n a c t i v e enzyme, accompanying a d e c r e a s e i n t h e a c t i v e enzymes. was c o n c l u d e d t h a t t h e d e b r a n c h i n g enzyme i n r i c e s e e d s i s s y n t h e s i z e d d u r i n g r i p e n i n g i n a c t i v e form and t h a t i t a c c u m u l a t e s i n i n a c t i v e form, which can be r e a c t i v a t e d d u r i n g g e r m i n a t i o n . The i d e n t i f i c a t i o n o f an i n a c t i v e d e b r a n c h i n g enzyme i n r i c e
6: Enzymes
535
seeds
and i t s a c t i v a t i o n have been r e p o r t e d . 4 5 7
flour
with
1,4-dithiothreitol
pullulanase
activity.
Maximum
i n c u b a t i o n w i t h 6mM t h i o l .
I n c u b a t i o n of r i c e
(dithiothreitol) activity
generated
occurred
after
high 24
h
A l l of t h e t h i o l s a t l O m M a c t i v a t e d t h e
d e b r a n c h i n g enzyme, s u g g e s t i n g t h a t t h e p r o c e s s i n v o l v e d c l e a v a g e o f t h e d i s u l p h i d e bonds. other reductants. effective,
T h i s v i e w was c o n f i r m e d by t h e e f f e c t
b u t s o d i u m b o r o h y d r i d e was m o d e r a t e l y e f f e c t i v e i n s p i t e
o f t h e pH (-10) w h i c h was u n f a v o u r a b l e f o r e n z y m e s t a b i l i t y . debranching proteases,
of
S o d i u m s u l p h i t e and s o d i u m d i t h i o n i t e w e r e v e r y
enzyme
was
9. papain,
also
activated
a-chymotrypsin,
to
similar
extents
The by
and t r y p s i n .
D e b r a n c h i n g enzyme h a s been p u r i f i e d f r o m m a t u r e r i c e seeds.458 The enzyme was e x t r a c t e d f r o m
a supernatant s o l u t i o n o f r i c e f l o u r
f o l l o w i n g i t s homogenization i n l O m M sodium d i t h i o n i t e s o l u t i o n . DEAE-cellulose enzyme
was u s e d a s t h e e x t r a c t i o n m e d i u m f r o m w h i c h t h e
protein
was
eluted
with
P u r i f i c a t i o n was a c h i e v e d by t h e u s e o f a C M - c e l l u l o s e column.
sodium
phosphate
buffer.
a Sephadex G-100 c o l u m n a n d
The p u r i f i e d enzyme h a d no a m y l a s e ,
maltase,
and Q-enzyme a c t i v i t i e s and was homogeneous i n g e l f i l t r a t i o n , d i s c electrophoresis,
(c. 70,000) were
and i s o e l e c t r i c
focusing.
The
molecular
weight
d e t e r m i n e d by g e l f i l t r a t i o n and t h e o p t i m u m pH o f 5 . 6
i d e n t i c a l t o those of
the milky-stage
enzyme.
Substrate
s p e c i f i c i t i e s o f t h e enzyme w e r e a l s o s i m i l a r t o t h o s e o f t h e m i l k y s t a g e enzyme.
The enzyme r a p i d l y h y d r o l y s e d p u l l u l a n b u t s c a r c e l y
hydrolysed phytoglycogen, enzymes.
s i m i l a r t o other higher-plant
debranching
K i n e t i c d a t a s i m i l a r t o t h o s e o f t h e m i l k y - s t a g e enzyme
were a l s o obtained. B a c i l l u s a m y l o l y t i c u s produces a-amylase, glucosidase.
By s e l e c t i o n o f
p u l l u l a n a s e , and a-g-
c a r b o n s o u r c e i n t h e g r o w t h medium a-
Q - g l u c o s i d a s e was p r o d u c e d p r e f e r e n t i a l l y a n d w i t h e x c l u s i o n o f t h e other two activities.la2
42
Sucrose-a-g-Glucohydrolase
The a c t i v i t i e s o f a-!-glucohydrolase
various glycosidases
e.i n c l u d i n g
i n homogenates o f t h e s m a l l - i n t e s t i n a l
t w o a d u l t and 18 s u c k l i n g tammar w a l l a b i e s ( M .
e u q e n i i ) aged f r o m 6
t o 5 0 w e e k s h a v e b e e n i n ~ e s t i g a t e d . ~F ~o r f u r t h e r i n i t i a l c i t a t i o n o f ref.38. The
amounts
of
sucrosemucosa o f
sucrose-a-Q-glucohydrolase
d e t a i l s see
i n tangentially
Carbohydrate Chemistry
536
s e c t i o n e d b i o p s i e s from j e j u n u m have been s t u d i e d by q u a n t i t a t i v e i m m u n o e l e c t r o p h o r e s i s and e n z y m i c a s s a y s . 1 2 9
For further
details
see i n i t i a l c i t a t i o n o f r e f . 1 2 9 . S u c r o s e -cr-q-g l u c o h y d r o l a s e -0 l i g o - ( 1 been p u r i f i e d from i n j e c t i o n of
rat
+
6)-a-!-glucosidase
intestinal microvillus
;-(2-3H)mannose
and & - ( 6 - 3 H ) f u c o s e ,
has
membranes
after
u s i n g a column o f
m o n o c l o n a l a n t i b o d y p r o t e i n A - S e p h a r ~ s e . ~A~f t~e r p r o n a s e d i g e s t i o n and
gel
f i l t r a t i o n
precursors,
of
the
glycopeptides
a major p a r t of
l a b e l l e d from
the radioactivity
a s p a r a g i n e - l i n k e d complex o l i g o s a c c h a r i d e s ,
was
both
recovered i n
a n d a s m a l l e r amount i n
p a r t i a l l y a l k a l i - l a b i l e high-molecular-weight
glycopeptides.
Only a
s m a l l a m o u n t o f ( 3 H ) m a n n o s e was f o u n d i n e n d o - B - Q - 2 - a c e t a m i d o - 2 d e o x y g l u c o s i d a s e H - s e n s i t i v e high-a-mannose
oligosaccharides.
D e t e r g e n t - s o l u b i l i z e d p i g i n t e s t i n a l sucrose-a-g-glucohydrolase oligo-(1
6 ) - Q - g l u c o s i d a s e h a s been p u r i f i e d 40 t o 100 t i m e s w i t h a
+
y i e l d o f 1 0 t o 20% by a r a p i d i m m u n o a d s o r b e n t t e c h n i q u e . 4 4 4
For
f u r t h e r d e t a i l s see i n i t i a l c i t a t i o n o f r e f . 4 4 4 .
43
aa- and B B - T r e h a l a s e s
The a c t i v i t i e s o f v a r i o u s g l y c o s i d a s e s i n h o m o g e n a t e s o f t h e small-intestinal wallabies
mucosa
(M. e u g e n i i )
of
two
aged
adult
from
and
6
t o
18 50
suckling weeks
tammar
have
been
i n ~ e s t i g a t e d . ~a a~- T r e h a l a s e a c t i v i t y was v e r y l o w o r a b s e n t d u r i n g t h e f i r s t 34 weeks,
and t h e n i n c r e a s e d .
I n order t o establish the s p e c i f i c i t y o f aa-trehalases t h e i r a c t i o n on m o n o m o d i f i e d a s y m m e t r i c a l d e r i v a t i v e s o f the
purification of
may-bug
(Melolontha vulgaris coleopterae)
t r e h a l a s e h a s been p u r i f i e d 1 6 , 0 0 0 - f 0 l d . ~ ~ The ~ was 307 u n i t s mg-'
of proteins,
previously reported. studied:
and
trehalose,
specific
z-
activi;ty
a p p r o x i m a t i v e l y 78 t i m e s h i g h e r t h a n
Some o f t h e p r o p e r t i e s o f t h e enzyme h a v e been
molecular weight,
isoelectric point,
a c t i o n of
various
e f f e c t o r s and i n h i b i t o r s . Effects
o f
a c r y l o n i t r i l e
T r i b o l i u m castaneum
I _ -
studied.461
and
on
trehalase
Trogoderma g r a n a r i u m
a c t i v i t y
everts
has
i n been
A c r y l o n i t r i l e i n h i b i t e d t r e h a l a s e and p h o s p h o r y l a s e s i n
l a r v a e and a d u l t s o f T r i b o l i u m c a s t a n e u m . l a r v a e p h o s p h o r y l a s e s a l o n e were i n h i b i t e d .
I n Trogoderma q r a n a r i u m
537
6: Enzymes 44
endo-( 1
The
mode o f
4)-B-a-Xylanases
+
action
of
an e n d o - ( 1
4)-B-Q-xylanase
-+
from
a
B a s i d i o m y c e t e S p o r o t r i c h u m d i m o r p h o s p o r u m h a s been r e p o r t e d and i t s utility
for
pattern
the structural investigation including substitution
o f
the
branched
me t h y 1g 1u c u r o no ) -9 been s t u d i e d . 4 6 2 region
of
the
- x y 1a n
L-afabing-(4-g-
water-soluble
f r o m r e d w o o d ( S e q u o ia s e mper v i r e n s ) h a s
The a c t i o n o f
t h e P-xylanase
polysaccharide
backbone
appears t o i n v o l v e a
having
three
P_-xylosyl
A mode o f a c t i o n t h a t r e q u i r e s u n s u b s t i t u t e d h y d r o x y l
residues.
C-3,
g r o u p s a t C-2,
a n d C-2’
o f a x y l o b i o s y l r e s i d u e was p r o p o s e d .
The b i n d i n g s i t e s e e m s t o c o r r e s p o n d t o a s h a l l o w
cavity.
The
c o m p o s i t i o n and s t r u c t u r e o f t h e f i n a l r e s i d u e o f a t t a c k shows t h a t the
enzyme
through
h a s no are
0-2
a c t i o n when t h e 2 - x y l o s y l
separated
by
only
residue.
This pattern o f action,
products,
and
the
production of
one
residues
branched
unsubstituted
P_-xylose
the nature of a
final
the dialysable
residue
i n
which
the
s u b s t i t u e n t s a r e a c c u m u l a t e d s u g g e s t t h a t t h e C - a r a b i n o s y l and
9-
g l u c o s y l u r o n i c groups a r e i r r e g u l a r l y d i s t r i b u t e d on t h e main c h a i n o f t h e 9 - x y l a n f r o m r e d w o o d a n d t h a t i n some r e g i o n s t h e y a r e i n c l o s e v i c i n i t y when n o t a c t u a l l y o n a d j a c e n t g - x y l o s y l r e s i d u e s . The
extracellular
endo-(1
+
4 ) - B - Q- - x y l a n a s e
of
the
yeast
C r y p t o c o c c u s a l b i d u s has been f o u n d t o c a t a l y s e t h e d e g r a d a t i o n o f aryl
B-q-xylosides
cleavage.463
by
reactions
Liberation
of
corresponding B-P-xylosides
other
phenol
or
than
simple
hydrolytic
4-nitrophenol
from
the
was a c c o m p a n i e d by f o r m a t i o n o f g - x y l o s e
o l i g o s a c c h a r i d e s and o n l y s m a l l a m o u n t s o f 2 - x y l o s e . of p h e n y l B-g-{U-14C)-xyloside
With the a i d
it
s y n t h e s i z e d f r o m P-{U-14C)-xylose,
was e s t a b l i s h e d t h a t t h e r e a c t i o n f o l l o w e d a c o m p l e x p a t t e r n w i t h t h e r a t e of
phenyl B-g-xyloside
d i g e s t i o n and a p p e a r a n c e o f v a r i o u s
products v a r y i n g markedly w i t h time.
The r e a c t i o n i n v o l v e d m u l t i p l e
transglycosylic reactions leading f i r s t t o phenyl-g-glycosides xylo-oligosaccharides,
of
9-
which are subsequently hydrolysed m a i n l y t o
g - x y l o b i o s e and P - x y l o t r i o s e . The a c t i o n p a t t e r n and r e a c t i o n mechanism o f t h e e n d o - ( 1 B-e-xylanase
of
the
yeast
i n v e s t i g a t e d u s i n g reducing-end
(1
+
was
4)-B-Q-xylo-oligosaccharides found
to
catalyse
Cfyptococcus
I l-3H)-labelled
albidus
xylotriose,
-+
4)-
been
and { U - 1 4 C ) - l a b e l l e d
up t o x y l ~ p e n t a o s e . ~The ~ ~ enzyme
degradation o f
oligosaccharides
pathways other than a simple h y d r o l y t i c cleavage. frequency of
have
xylotetraose,and
also
by
Bond-cleavage
x y l o p e n t a o s e was f o u n d t o
538 be
Carbohydrate Chemistry concentration
dependent.
r e a c t i o n s s u c h as x y l o s y l ,
high
A t
substrate
concentration
x y l o b i o s y l , and x y l o t r i o s y l t r a n s f e r o c c u r
and r e s u l t i n t h e f o r m a t i o n o f p r o d u c t s l a r g e r t h a n t h e s t a r t i n g substrate. reaction
g - X y l o s e and x y l o b i o s e t o a s i g n i f i c a n t e x t e n t e n t e r t h e p a t h w a y s as
glycosyl
acceptors.
None
of
the
g l y c o s y l i c r e a c t i o n s observed w i t h reducing-end-labelled
trans-
substrates
o r a c c e p t o r s was a c c o m p a n i e d by a s i g n i f i c a n t l a b e l r e d i s t r i b u t i o n from t h e reducing-end u n i t , intermediates effective
suggesting t h a t the enzyme-glycosyl
i n t h e t r a n s f e r r e a c t i o n s can be f o r m e d f r o m
the non-reducing-end u n i t s o f oligosaccharides.
Evidence for the
f o r m a t i o n o f a t e r m o l e c u l a r s h i f t e d complex o f 6-Q-xylanase
with
x y l o t r i o s e has a l s o been o b t a i n e d .
A l l features o f the degradation
o f o l i g o s a c c h a r i d e s by 6 - P - x y l a n a s e
were f o u n d t o be c o n s i s t e n t w i t h
t h e l y s o z y m e - t y p e r e a c t i o n mechanism. The s u b s t r a t e - b i n d i n g
s i t e o f endo-(1 + 4)-B-Q-xylanase
y e a s t C r y p t o c o c c u s a l b i d u s h a s been i n v e s t i g a t e d u s i n g (1 xylo-oligosaccharides Evaluation of
{ l-3H)-labelled
at
the
of the
+
reducing
4)-6-Qend.465
the a f f i n i t i e s o f t e n imaginary subsites pointed out
t h a t t h e s u b s t r a t e - b i n d i n g s i t e o f t h e enzyme i s composed o f f o u r s u b s i t e s and t h a t t h e c a t a l y t i c groups a r e l o c a l i z e d i n t h e c e n t r e . The i m a g i n a r y s u b s i t e s o n t h e l e f t - h a n d s i d e o f t h e b i n d i n g s i t e (non-reducing-end x y l o s y l residues.
s i d e ) s h o w e d l i t t l e o r no a f f i n i t y t o b i n d
b i n d i n g s i t e ('reducing-end' obtained,
9-
F o r t h e s u b s i t e s on t h e r i g h t - h a n d s i d e of the
s i d e ) n e g a t i v e v a l u e s o f a f f i n i t y were
w h i c h means t h i s r e g i o n o f t h e enzyme i s u n f a v o u r a b l e f o r
complexing
with
Q-xylosyl residues.
As
a
consequence
of
the
asymmetric d i s t r i b u t i o n o f n e g a t i v e values o f a f f i n i t y around t h e binding site,
t h e enzyme d i s p l a y s a s t r o n g p r e f e r e n c e f o r a t t a c h i n g
n e a r t h e r e d u c i n g end o f t h e s u b s t r a t e . {
l-3H)-xylo-oligosaccharides,
Regardless of t h e l e n g t h of
{ l-3H)-xylobiose
was t h e p r e v a i l i n g
r e a c t i o n p r o d u c t a t an e a r l y s t a g e o f h y d r o l y s i s ,
and frequency
d i s t r i b u t i o n o f bond c l e a v a g e decreased f r o m t h e second g l y c o s i d i c b o n d t o w a r d s t h e n o n - r e d u c i n g end. A d d i t i o n a l i n f o r m a t i o n on t h e s u b s t r a t e - b i n d i n g s i t e o f C. a l b i d u s B - p - x y l a n a s e w a s o b t a i n e d b y evaluating the
efficiency
of
p-xylose,
xylobiose,
m e t h y l 8-e-
x y l o p y r a n o s i d e , and p h e n y l 6 - Q - x y l o p y r a n o s i d e t o s e r v e as g l y c o s y l acceptors
i n
the
transglycosylic
concentrations of xylotriose.
reaction
proceeding
at
high
539
6: Enzymes 45
Xylanases (Miscellaneous)
The a c c u r a c y improved.466
of
a Q-xylanase
assay
has been t e s t e d and
The assay was u s e d t o m o n i t o r Q - x y l a n a s e p r o d u c t i o n by
a C e l l u l o m o n a s i s o l a t e and t o d e m o n s t r a t e t h a t t h i s a c t i v i t y i s d i s t i n c t f r o m t h e f 3 --p - x y l o s i d a s e
a c t i v i t y o f t h e organism.
The a p p l i c a t i o n o f c a r b o h y d r a s e s t o t h e e x t r a c t i o n o f p r o t e i n s from
wheat
b r a n has i n c l u d e d t h e e f f i c a c i o u s p r e t r e a t m e n t w i t h
xy l a n a s e .349 An e x t r a c e l l u l a r
9-xylanase
f r o m a s o i l fungus
( F u s a r i u m sp.)
g r o w n o n a medium c o n t a i n i n g g r o u n d n u t h e m i c e l l u l o s e B was p u r i f i e d 76-fold
by
ammonium
sulphate
fractionation,
c h r o m a t o g r a p h y , and g e l f i l t r a t i o n . 4 6 7
ion-exchange
The enzyme was homogeneous by
d i s c g e l e l e c t r o p h o r e s i s a t pH 8 a n d showed o p t i m a l a c t i v i t y a t pH 5.6
and
It
37'C.
hemicellulose B respectively)
by
was
were
observed degraded
that
groundnut
considerably
t h e p u r i f i e d Q-xylanase,
and
sesame
and
(-80
58%,
w h e r e a s g-gluco-!-mannan
and g - x y l a n f r o m g r o u n d n u t were c o m p a r a t i v e l y p o o r l y h y d r o l y s e d (-30-40%) The p r o d u c t i o n and c h a r a c t e r i z a t i o n o f t h e r m o s t a b l e R - x y l a n a s e f r o m T a l a r o m y c e s b y s s o c h l a f i y d o i d e s YH-50 h a v e b e e n d e s c r i b e d . 4 6 8 T h i s s t r a i n i s o l a t e d f r o m c o m p o s t heaps p r o d u c e d t h e h i g h e s t amount of
t h e r m o s t a b l e e - x y l a n a s e among 1 8 0 i s o l a t e s t e s t e d .
cultivated i n solid
wheat-bran
a d d i t i v e carbon source, was p r o d u c e d a f t e r
When i t was
medium c o n t a i n i n g
xylan
as
an
t h e m a x i m a l amount o f t h e r m o s t a b l e x y l a n a s e
3 d a y s a t 5OoC.
90% o f q - x y l a n t o g - x y l o s e .
This culture f i l t r a t e hydrolysed
The enzyme (pH o p t i m u m 5.5,
o p t i m u m 7 O o C ) was q u i t e s t a b l e a f t e r h e a t i n g a t 65'
temperature
f o r 5 min a n d
r e t a i n e d 55% o f o r i g i n a l a c t i v i t y a f t e r h e a t i n g a t 95OC f o r 5 min. T r i c h o d e r m a r e e s e i R u t C-30 high cellulase activities,
B-Q - -glucosidase. concentration, r a t i o of
The
organism
produced,
together
c o n s i d e r a b l e amounts o f i - x y l a n a s e s effect
of
temperature,
pH,
with and
Tween-80
c a r b o n s o u r c e , and s u b s t r a t e c o n c e n t r a t i o n on t h e
m y c e l i a l g r o w t h and e x t r a c e l l u l o s e enzyme p r o d u c t i o n was
d e s c r i b e d .208 A s p e r g i l l u s n i g e r s t r a i n 110.42 h a s been s e l e c t e d as a p r o d u c e r
of h i g h 0-xylanolytic a c t i v i t i e s . 4 6 9 The t i m e - c o u r s e o f x y l a n a s e a n d B - Q - x y l o s i d a s e p r o d u c t i o n a s w e l l a s t h e e f f e c t o f pH a n d t e m p e r a t u r e o n t h e a c t i v i t y o f t h e s e enzymes w e r e s t u d i e d . a n a l y s i s of
the enzymatic degradation of
H.p.1.c.
a r a b i n o x y l a n showed a
n e a r l y complete conversion t o pentose sugars.
The a u t h o r s d i s c u s s
540
Carbohydrate Chemistry
t h e use o f c r u d e p - x y l a n a s e p r e p a r a t i o n s f o r t h e s a c c h a r i f i c a t i o n o f Q - - x y l a ns. The enhanced c e l l u l o l y t i c a c t i v i t y o f a C e l l u l o m o n a s m u t a n t has been shown t o
apply
also to
t o hydrolyse g-xylan-
i t s ability
c o n t a i n i n g h e m i c e l l u l o ~ e s . ~The ~ ~ h y d r o l y t i c a c t i v i t y was d i r e c t l y proportional t o the g-xylose content i n the hemicellulose substrate. A Cellulomonas s t r a i n , sugar-cane
bagasse,
has
carbohydrase a c t i v i t i e s
with potential for saccharification o f been
found
t o
possess
a
w h i c h c o u l d be n e c e s s a r y f o r
range
of
effective
h y d r o l y s i s o f t h e h e m i c e l l u l o s e f r a c t i o n o f bagasse.471 Inducible c o n s t i t u e n t of
B-xyloside
permease
has
been
reported
as
a
the e-xylan-degrading
enzyme s y s t e m o f t h e y e a s t
C r y p t o c o c c u s a l b i d ~ s . The ~ ~ y~e a s t ,
depending on whether i t i s
g r o w n on Q - x y l a n o r !-glucose, t a k e up i n d u c e r s synthesis.
of
d i f f e r s remarkably i n the a b i l i t y t o
extracellular
I n washed,
endo-(1
2-glucose-grown
*
4)-B-g-xylanase
c e l l s the i n i t i a l l y low
a b i l i t y t o t a k e up x y l o b i o s e o r m e t h y l B - 0 - x y l o p y r a n o s i d e
increases
d u r i n g i n c u b a t i o n w i t h t h e s e compounds a f t e r a l a g phase s h o r t e r than t h e i n d u c t i o n t i m e o f t h e e x t r a c e l l u l a r B-Q-xylanase.
Using
m e t h y l B - ~ - { U - 1 4 C ~ - x y l o p y r a n o s i d e as a v e r y s l o w m e t a b o l i z a b l e i n d u c e r o f B - 0 - x y l a n a s e i t h a s been e s t a b l i s h e d t h a t t h e i n c r e a s e i n t h e r a t e o f x y l o b i o s e o r m e t h y l x y l o s i d e u p t a k e i s due t o i n d u c t i o n o f an a c t i v e t r a n s p o r t s y s t e m f o r m e t h y l B - g - x y l o s i d e
P-xylo-oligosaccharides. permease. i t s
syste'm
The p e r m e a s e a c t i v i t y o f
absence o f B - 0 - x y l a n a s e
as
The
inducers.
inactivation
cycloheximide;
and ( 1
* 41-8-
c a l l e d B-e-xylosidase
induced c e l l s decreases i n t h e
The i n d u c t i o n o f p e r m e a s e as w e l l
(degradation)
can
be
prevented
with
t h u s b o t h e v e n t s a p p e a r t o b e d e p e n d e n t o n de n o v o
p r o t e i n synthesis. f3-e-xyloside
i s
I n analogy w i t h o t h e r a c t i v e t r a n s p o r t systems,
permease
function
can
be
blocked
effectively
by
i n h i b i t o r s o f energy metabolism i n t h e c e l l s . The
importance
of
cellulase
------------------B a c t e r o i d e s s u c c i n o g ---enes investigated.223
and
p-xylanase
release
from
i n t h e rumen e n v i r o n m e n t has been
During growth o f
B.
succinogenes i n a l i q u i d
medium w i t h c e l l u l o s e as t h e s o u r c e o f c a r b o h y d r a t e , g r e a t e r t h a n 80% o f t h e x y l a n a s e was r e l e a s e d f r o m c e l l s i n t o t h e c u l t u r e f l u i d .
F o r f u r t h e r d e t a i l s see i n i t i a l c i t a t i o n o f r e f . 2 2 3 . Cultures
of
Streptomyces f l a v o g r i s E have
p r o d u c e c o n s i d e r a b l e amounts o f c o n t a i n i n g media.260
p-xylanase
been
found
to
when g r o w n on Q - x y l a n -
C o m p a r a t i v e l y l o w e r y i e l d s o f t h i s enzyme w e r e
o b t a i n e d when h a y o r A v i c e l s e r v e d as m a i n c a r b o n s o u r c e .
54 1
6: Enzymes Q - X y l a n a s e i n d u c t i o n by B - P - x y l o s i d e
was i n v e s t i g a t e d i n non-
g r o w i n g c o n d i t i o n s u s i n g non-induced m y c e l i a o f Streptomyces s p e c i e s h a r v e s t e d f r o m i - g l u c o s e medium.473 Q-xylanase w i t h o u t of
P-xylanase synthesis
x y l o s i d e added t o constants
of
the
various
inducing culture
mM,
mM,
The
induction
calculated from
i s o p r o p y l , b u t y l , and
Some a - D - -xylosides
respectively.
The r a t e
w e r e 10.53 m M , 3.83 m M , 0.55
mM,
repressed g-xylanase
Q-xylanase synthesis decreased suddenly
a f t e r the a d d i t i o n o f a-p-xyloside. methyl,
were
and t h o s e o f m e t h y l ,
The r a t e o f
synthesis.
medium.
B-g-xylopyranosides
Lineweaver-Burk p l o t s ,
was added.
d e p e n d e n t on t h e c o n c e n t r a t i o n o f 6-E-
was
ethylenecyanohydrin B-g-xylosides a n d 0.25
The m y c e l i a s t a r t e d t o p r o d u c e
l a g t i m e when B-;-xyloside
The i n h i b i t i o n c o n s t a n t s o f
e t h y l , and i s o p r o p y l a - q - x y l o p y r a n o s i d e s
a n d 33.33 m M , r e s p e c t i v e l y .
w e r e 8.80
mM,
12.50
T h e x y l a n a s e i n d u c t i o n was a l s o
r e p r e s s e d by P - g l u c o s e u n t i l c o n s u m p t i o n o f t h e a d d i t i o n a l g - g l u c o s e was c o m p l e t e . The p r o d u c t i o n o f x y l a n a s e by a S t r e p t o m y c e s s p e c i e s u s i n g n o n -
I t was f o u n d t h a t a
m e t a b o l i z a b l e i n d u c e r has been described.474 variety
of
non-metabolizable
B-g-xylosides
possessed a
marked
i n d u c i n g a b i l i t y i n comparison t o Q-xylan or i t s r e l a t e d m a t e r i a l s . I n t h e p r o d u c t i o n of difficulty basis.
P-xylanase u s i n g such a s y n t h e t i c i n d u c e r ,
i n preparing the inducer
the
i s c o n s i d e r e d on an e c o n o m i c a l
A s u i t a b l e c u l t u r e c o n d i t i o n f o r 0-xylanase p r o d u c t i o n by
methyl 6-g-xyloside
i n S t r e p t o m y c e s s p e c i e s No. 3 1 3 7 a n d a m e t h o d
f o r r e - u s i n g t h e i n d u c e r were i n v e s t i g a t e d .
46
C a r b o h y d r a t e Isomerases
Q-Xylose I s o m e r a s e s @ - G l u c o s e o f some p o l y o l s ( g - m a n n i t o l , x y l i t o l )
on
A-ctinomyces
the
-
Isomerase).
a c t i v i t y
~
--
The i n h i b i t o r y e f f e c t
Q-arabitol, Q-glucitol,
-
o f
--xylose Q
o l i v o c i n e r e u s has been studied.475
f o r g-mannitol,
0.200,
0.140,
g-arabitol,
0.030,
0.024,
g-glucitol, a n d 0.020
ribitol, M,
from
A l l the polyols
studied are purely competitive reversible inhibitors.
Xi
r i b i t o l , and
isomerase
The v a l u e s o f and x y l i t o l a r e
respectively.
The
i n h i b i t i o n i s p a r t i a l l y e l i m i n a t e d b y a n i n c r e a s e i n t h e Mg2+ a n d Co2+ c o n c e n t r a t i o n t o 5 x 1 0 - 2 and l ~ l O -M,~ r e s p e c t i v e l y .
The k i n e t i c p r o p e r t i e s o f
i m m o b i l i z e d and n o n - i m m o b i l i z e d
Q-
x y l o s e i s o m e r a s e c o n t a i n i n g A r t h r o b a c t e r s p e c i e s c e l l s have been i n ~ e s t i g a t e d . ~ ’ ~I n b o t h c a s e s t h e k i n e t i c s c o u l d b e d e s c r i b e d by a
Carbohydrate Chemistry
542 modified
Michaelis-Menten
expression.
that i t was shown t h a t t h e p e r m e a b i l i t y o f t h e c e l l membrane was i n c r e a s e d by h e a t and toluene treatments. g - X y l u l o s e , an i n t e r m e d i a t e o f k - x y l o s e c a t a b o l i s m , h a s been i m m o b i l i z a t i o n c a u s e d no d e a c t i v a t i o n .
It
appeared
Furthermore,
o b s e r v e d t o be f e r m e n t a b l e t o e t h a n o l and c a r b o n d i o x i d e i n a y i e l d o f g r e a t e r t h a n 80% by y e a s t s ( i n c l u d i n g i n d u s t r i a l b a k e r ’ s y e a s t )
condition^.^"
under f e r m e n t a t i v e
T h i s c o n v e r s i o n a p p e a r s t o be
c a r r i e d o u t by many y e a s t s k n o w n f o r Q - g l u c o s e f e r m e n t a t i o n . some y e a s t s , xylulose. g-xylose
xylitol,
i n addition t o ethanol,
I n
was p r o d u c e d f r o m
!-
Fermenting yeasts are a l s o able t o produce e t h a n o l from when ! - x y l o s e - i s o m e r i z i n g
enzyme i s p r e s e n t .
The r e s u l t s
i n d i c a t e d t h a t e t h a n o l c o u l d be p r o d u c e d f r o m P - x y l o s e i n a y i e l d o f greater
than
8 0 % by
a
converted t o g-xylulose
two-step
process.
by g - x y l o s e
First,
isomerase.
Q-xylose
g-Xylulose
i s
i s then
f e r m e n t e d t o e t h a n o l by y e a s t s . C u l t u r e s o f S t r e p t o m y c e s f l a v o g r i s e u s has been f o u n d t o p r o d u c e i n t e r a l i a 9 - x y l o s e i s o m e r a s e when i n d u c e d by g - x y l o s e . 2 6 0 The e f f e c t o f g a m m a - i r r a d i a t i o n on Q - x y l o s e i s o m e r a s e p u r i f i e d f r o m t h e c e l l s o f S t r e p t o m y c e s phoeochromogenus i n d i l u t e s o l u t i o n has been i n ~ e s t i g a t e d . ~ ’ ~ The a c t i v i t y o f t h e e n z y m e d e c r e a s e d e x p o n e n t i a l l y w i t h t h e dose u n d e r a l l c o n d i t i o n s i n v e s t i g a t e d . i n a c t i v a t i o n y i e l d s (Go v a l u e ) i n n e u t r a l s o l u t i o n w e r e 0.069 a n d 0.115
i n nitrogen.
The
in air
The r o l e o f t h e r a d i c a l s p r o d u c e d by w a t e r
r a d i o l y s i s i n t h e i n a c t i v a t i o n o f a - g l u c o s e i s o m e r a s e was e s t i m a t e d by u s i n g n i t r o u s o x i d e o r t e r t - b u t a n o l scavengers.
as s e l e c t i v e r a d i c a l
Under t h e s e c o n d i t i o n s , t h e h y d r o x y l r a d i c a l and t h e
h y d r o g e n a t o m w e r e f o u n d t o be i m p o r t a n t i n t h e enzyme i n a c t i v a t i o n , and t h e h y d r a t e d e l e c t r o n c o n t r i b u t e d v e r y l i t t l e . The
radiosensitivity
i n v e s t i g a t e d under
i n a c t i v a t i o n o f !-glucose was e x p o n e n t i a l , an
oxygenated
of
various
E-xylose
irradiation
isomerase
has
been
conditions.479
The
isomerase i r r a d i a t e d i n a cell-bound s t a t e
and an i n c r e a s e i n i n a c t i v a t i o n was r e c o g n i z e d i n condition.
The
cell-free
enzyme
was
highly
r a d i o s e n s i t i v e and h a d a s m a l l o x y g e n e f f e c t c o m p a r e d t o t h a t i n a cell-bound state.
The o x y g e n enhancement r a t i o (OER) d e c r e a s e d w i t h
a d e g r e e i n enzyme p u r i f i c a t i o n .
R e l e a s e d Q - g l u c o s e i s o m e r a s e was
p r o t e c t e d by t h e a d d i t i o n o f g l u t a t h i o n e , and t h e i n a c t i v a t i o n c u r v e i n n i t r o g e n almost agreed w i t h t h a t i n t h e c e l l .
The p r o t e c t i v e
e f f e c t o f g l u t a t h i o n e i n oxygen d e c r e a s e d a t h i g h e r doses because g l u t a t h i o n e i n o x y g e n was e a s i l y decomposed b y i r r a d i a t i o n .
6: Enzymes
543
The s u b u n i t s t r u c t u r e h a s been d e t e c t e d i n g - x y l o s e
isomerase
f r o m S t r e p t o m y c e s q r i s e o f u s c u s by u s i n g d e n a t u r a n t s . 4 8 0 The enzyme was q u i t e s t a b l e t o s o d i u m d o d e c y l s u l p h a t e u n d e r m i l d c o n d i t i o n s , a n d d i s s o c i a t i o n i n t o s m a l l e r s u b u n i t s was d e p e n d e n t A t a c i d i c pH and h i g h t e m p e r a t u r e ,
temperature.
rapid dissociation
and i n a c t i v a t i o n .
The
o n pH a n d
t h e enzyme showed
dissociation
into
c o n s t i t u e n t s u b u n i t s p a r a l l e l e d t h e loss o f e n z y m a t i c a c t i v i t y .
The
enzyme was a l s o d i s s o c i a t e d i n t o a s i n g u l a r t y p e o f s u b u n i t b y 6 M g u a n i d i n i u m c h l o r i d e w h i c h gave a s i n g l e s y m m e t r i c a l e l u t i o n p e a k o n Sepharose 68 column chromatography i n t h e p r e s e n c e o f 6 M g u a n i d i n e hydrochloride,
and t h e e l u a t e showed n o a c t i v i t y .
estimations of
the subunit,
Molecular-weight
u s i n g both sodium dodecyl sulphate
polyacry1ami.de g e l e l e c t r o p h o r e s i s and g e l f i l t r a t i o n , was
w e r e t h e same
The N - t e r m i n a l a m i n o a c i d was i d e n t i f i e d as L - s e r i n e
(43,000).
estimated t o
derivatives.
be 43 p e r
cent
from
analyses
of
The s e q u e n c e f r o m t h e N - t e r m i n a l was f o u n d t o b e
....
Ser-L-Asp-L-Gln
enzyme as 180,000
and
PTH a n d DNP
i-
Considering the molecular weight o f the n a t i v e
and t h e r e s u l t s o b t a i n e d i n t h e e x p e r i m e n t s h e r e ,
i t was c o n c l u d e d t h a t n a t i v e ! - g l u c o s e
i s o m e r a s e was c o m p o s e d o f
f o u r i d e n t i c a l or very s i m i l a r s u b u n i t s w i t h e q u a l m o l e c u l a r w e i g h t s and d i m e n s i o n s . Some p h y s i c o c h e m i c a l p r o p e r t i e s o f
p u r i f i e d g-xylose
f r o m S t r e p t o m y c e s g r i s e o f u s c u s h a v e been examined.481
(gi&,)
coefficient 11.4
and
4.0,
coefficient
and i s o e l e c t r i c p o i n t
respectively.
( 5 j O w ) ,p a r t i a l
The
(PI) were d e t e r m i n e d t o be
values
specific
for
sedimentation
(I), d i f f u s i o n
volume
c o e f f i c i e n t ( g 2 0 w ) , a n d i n t r i n s i c v i s c o s i t y ({tj))
lo7
isomerase
The e x t i n c t i o n
w e r e 8.50 S, 0.73
m l g-l, respectively. The m o l e c u l a r w e i g h t was e s t i m a t e d t o b e 1 8 0 , 0 0 0 b y t h e s e d i m e n t a t i o n e q u i l i b r i u m m e t h o d a n d 185,000 by a g e l e l e c t r o p h o r e t i c a l method.481 Cm3
g-l,
4.6
x
cm2 S-l,
a n d 3.7
T h e e n z y m e was f o u n d t o c o n t a i n f o u r
cobalt ions per molecule i n
atomic-absorption spectrophotometrical analysis. d i c h r o i s m spectrum i n t h e f a r - u l t r a v i o l e t 280 nm a n d a m a x i m u m a t 1 9 8 nm.
The c i r c u l a r -
r e g i o n showed a m i n i m u m a t
Mean r e s i d u e e l l i p t i c i t y ( ( 0 ) )
at
220 nm was e s t i m a t e d t o b e - 1 1 , 2 0 0 deg.cm2/d mol. The c o m p u t e d v a l u e s f o r t h e c o n t e n t s o f t h e s e c o n d a r y s t r u c t u r e w e r e as f o l l o w s : a-helix
40%,
8-form
36%,
a n d r a n d o m c o i l 24%.
The enzyme showed a
v i s i b l e c i r c u l a r - d i c h r o i s m s p e c t r u m h a v i n g n e g a t i v e p e a k s a t 530 nm a n d 4 3 0 nm.
This suggested t h e f o r m a t i o n of a co-ordinated m e t a l
compound, Co-amino a c i d complex.
The m o l e c u l a r d i m e n s i o n o f t h e
enzyme was e x a m i n e d by m e a s u r i n g t h e h y d r o d y n a m i c p a r a m e t e r s .
The
544
Carbohydrate Chemistry
(a/b)
frictional ratio
was 5 . 0
f o r p r o l a t e e l l i p s o i d a n d 0.2
for
0
S t o k e s ' r a d i u s was c a l c u l a t e d t o b e 47 A f o r a
oblate ellipsoid.
h y d r a t e d h y p o t h e t i c a l sphere. The p u r i f i c a t i o n a n d e n z y m a t i c p r o p e r t i e s o f E - x y l o s e i s o m e r a s e f r o m S t r e p t o m y c e s g r i s e o f u s c u s h a v e been d e s c r i b e d . 4 8 2 was p u r i f i e d 4 . 3 - f o l d ammonium
sulphate,
electrophoresis a c t i v i t y was 8.5
was
and
homogeneous
on
ultracentrifugation.
i n a c t i v e i n t h e absence o f
respectively,
!-glucose,
while
Vmax
values
The enzyme was q u i t e The enzyme c a t a l y s e d
b y
The
17.6
umol min-'
c o n t e n t t o !-glucose
s u g a r s ,
Em
x 10-1 M and 5.4 x
a t reaction equilibrium.
1.0
i n h i b i t e d
pH
and ! - r i b o s e .
were
The r a t i o o f [-I - f r u c t o s e
was a p p r o x i m a t e l y was
for
optimum
m e t a l i o n s b u t was r e m a r k a b l y a c t i v a t e d
v a l u e s f o r Q - g l u c o s e a n d c - x y l o s e w e r e 2.2 M,
gel
The
magnesium o r c o b a l t i o n s .
t h e i s o m e r i z a t i o n o f !-xylose,
respectively.
polyacrylamide
and o p t i m u m t e m p e r a t u r e 85OC.
by t h e a d d i t i o n o f
The enzyme
a n d o b t a i n e d i n c r y s t a l l i n e f o r m , by a d d i n g
mg-l,
content
The enzyme a c t i v i t y
s u g a r
a l c o h o l s ,
a n d
The Ei Q - s o r b i t o l 1.1 x 10-2
tris(hydroxymethy1)aminomethane i n a c o m p e t i t i v e manner. v a l u e s w e r e as f o l l o w s : g - m a n n i t o l 8.3
M,
3.2
x 10-1 M ,
g - x y l i t o l 1.2 x
x 1 0 - 2 M,
I - a r a b i n o s e 2.3
Chloromercuribenzoate, cyanide, effect
M,
x 10-1 M,
x 1 0 - 1 M, a n d T r i s 6.2
n-galactose x
i o d o a c e t i c a c i d , sodium azide,
sodium fluoride,and
M.
4-
potassium
2 - m e r c a p t o e t h a n o l h a d no i n h i b i t o r
on t h e a c t i v i t y o f !-glucose
a s i g n i f i c a n t l o s s of
lo3
n-mannose 3.4
isomerase,
w h i l e H4 e d t a c a u s e d
activity.
The i n v e s t i g a t i o n b y
'H
n.m.r.
spectroscopy o f the s i t e o f
p r o t o n e x c h a n g e c a t a l y s e d by p o l y ( g - m a n n u r o n i c
a c i d ) C-5 e p i m e r a s e ,
which c a t a l y s e s t h e conversion o f g-mannuronic a c i d r e s i d u e s i n t o
L-
g u l u r o n i c a c i d r e s i d u e s p r o v i d e d t h e s u b s t r a t e i s a glycuronan of
at
least
10 u n i t s ,
has been r e p o r t e d . 4 8 3
I n f o r m a t i o n i s l a c k i n g as t o
t h e enzyme's s p e c i f i c i t y f o r a p a r t i c u l a r l o c a l a r r a n g e m e n t o f u n i t s i n t h e chain.
I-Ribose
Isomerases.
--
I n d u c t i o n o f I - r i b o s e i s o m e r a s e by I - r i b o s e
i n M y c o b a c t e r i u m smegmatis has been i n v e s t i g a t e d . 4 8 4 which u n l i k e a-ribose
a growth substrate for isomerase possibly
i-Ribose,
was n o t a s u b s t r a t e o f t h e enzyme a n d a l s o n o t the organism,
i n d u c e d t h e same Q - r i b o s e
due t o t h e c h a r a c t e r i s t i c
conformation o f t h e r i b o s e molecule.
c o n f i g u r a t i o n and
545
6: Enzymes
Carbohydrate O x i d a s e s
47
--
0-Glucose O x i d a s e s .
e-Glucose o x i d a s e and an o x i d i z e d v e r s i o n o f
t h e enzyme h a v e been i m m o b i l i z e d c o v a l e n t l y t o t w o b a s i c t y p e s o f
-
sorbents
g l y c i d y l m e t h a c r y l a t e c o p o l y m e r s and bead c e l l u l o s e . 4 8 5
The p r o p e r t i e s o f t h e s a m p l e s t h u s o b t a i n e d were compared w i t h t h o s e o f i m m o b i l i z e d a-glucose
o x i d a s e bound on t o some common c a r r i e r s .
Samples w h i c h p o s s e s s e d n o t o n l y a h i g h a b s o l u t e a c t i v i t y adequate
m e c h a n i c a l and
flow
properties
were
but also
characterized
in
g r e a t e r d e t a i l w i t h r e s p e c t t o t h e i m m o b i l i z a t i o n e f f i c i e n c y and k i n e t i c p r o p e r t i e s o f bound i - g l u c o s e o x i d a s e . New
findings
i n
the
oxidation
of
glucose
by
means
i m m o b i l i z e d Q - g l u c o s e o x i d a s e have been r e p o r t e d . 4 8 6 p r o c e e d i n g p u b l i c a t i o n s t h e paper shows r e c e n t a s p e c t s , to
be
of
B a s e d on w h i c h seem
f o r an e c o n o m i c a l and t e c h n i c a l u s e o f t h e
important
i m m o b i l i z e d !-glucose
oxidase-catalase
system.
t r i a l s i n c o n t i n u o u s p r o c e s s i n g were n e g a t i v e ,
The r e s u l t s
of
since the conversion
r a t e s w e r e s u f f i c i e n t and t e c h n i c a l d i f f i c u l t i e s a r o s e i n k e e p i n g back
the
enzyme
processing
with
particles
i n
the
r e p e a t e d enzyme
reaction
vessel.
application
Batchwise
showed
the
major
i m p o r t a n c e o f o p t i m a l oxygen s u p p l y and k e e p i n g l o w t e m p e r a t u r e s o f a b o u t 2OC.
D i f f e r e n t types o f s t i r r e d - t a n k
r e a c t o r s were t e s t e d and
c l a s s i f i e d concerning t h e i r s u i t a b i l i t y .
The c h a n c e s f o r l a r g e -
scale a p p l i c a t i o n o f the immobilized g-glucose oxidase-catalase s y s t e m a r e j u d g e d t o be good. A m o d i f i e d l e c t i n m a t r i x has been u s e d t o s t u d y t h e t h r e e -
dimensional structure of
a dimeric glycoprotein,
g-glucose
w h i c h was a l l o w e d t o r e a c t w i t h I - l y s i n e - s p e c i f i c both
when
immobilized
on
a
succinoylated
oxidase,
cross-linkers,
lectin
matrix
at
a
c r i t i c a l l y l o w d e n s i t y and a l s o a t a h i g h d e n s i t y i n s o l u t i o n . 4 8 7 Analysis of the cross-linked following inferences Of
complexes t h u s o b t a i n e d l e d t o t h e
with regard t o the s t r u c t u r e o f t h i s protein.
t h e 1 5 I - l y s i n e r e s i d u e s on each g - g l u c o s e
oxidase protomer,
i s a v a i l a b l e on t h e n o n - i n t e r f a c i a l s u r f a c e s . protein
possesses
subunits,
C2
symmetry
with
none
Assuming t h a t t h i s
isologous
bonding
between
i t may be i n f e r r e d t h a t o n e a c h p r o t o m e r t h e r e a r e a t
least two I - l y s i n e clusters along or close t o the interprotomeric interface.
These i n t e r f a c i a l I - l y s i n e r e s i d u e s on each p r o t o m e r a r e
so o r i e n t e d t h a t t h e € - a m i n o g r o u p s o f L - l y s i n e r e s i d u e s 2 and protomer 1 face, lysine residues
and a r e very c l o s e t o ,
b'
and
s',
respectively,
t h e &-amino groups o f o n p r o t o m e r 2.
on
I-
General
546
Carbohydrate Chemistry
i n f e r e n c e s on t h e g e o m e t r y o f d i m e r i c p r o t e i n s d e r i v a b l e f r o m an a n a l y s i s o f t h e c r o s s - l i n k e d c o m p l e x e s o b t a i n e d ( a s w e l l as t h o s e n o t seen) by u s i n g t h i s low - d e n s i t y m a t r i x c r o s s - l i n k i n g approach were enumerated. prove
useful
glycoproteins,
I t was s u g g e s t e d t h a t m o d i f i e d l e c t i n m a t r i c e s may
i n
studying
the
three-dimensional
structure
of
p a r t i c u l a r l y non-crys t a l l i z a b l e oligomers.
The e n h a n c e m e n t
of
oxygen a b s o r p t i o n by m a g n e t i t e - c o n t a i n i n g
beads of i m m o b i l i z e d g - g l u c o s e o x i d a s e has been i n v e s t i g a t e d . 4 8 8 R a t e s o f oxygen a b s o r p t i o n o f Q - g l u c o s e s o l u t i o n s were measured u s i n g an i m m o b i l i z e d - e n z y m e
reactor,
i n which m a g n e t i t e - c o n t a i n i n g
b e a d s o f i m m o b i l i z e d g - g l u c o s e o x i d a s e w e r e moved b y a r e v o l v i n g magnetic f i e l d t o reduce t h e mass-transfer l i q u i d i n t e r f a c e and a r o u n d t h e bead.
resistances at
t h e gas-
D a t a were a l s o o b t a i n e d f o r
solutions containing soluble
oxygen a b s o r p t i o n i n t o !-glucose
immobilized glucose oxidase (without magnetite),
or
as w e l l as f o r
p h y s i c a l a b s o r p t i o n f o r t h e r u n s w i t h t h e m a g n e t i t e - c o n t a i n i n g beads m e c h a n i c a l s t i r r i n g caused by s p i n n i n g o f
i n c r e a s e d because o f
beads a t t h e g a s - l i q u i d i n t e r f a c e . enhancement f a c t o r s
the
I n t h i s case t h e e x p e r i m e n t a l
were found t o be l a r g e r t h a n t h o s e p r e d i c t e d on
t h e b a s i s o f t h e f i l m t h e o r y f o r gas a b s o r p t i o n w i t h a p s e u d o - f i r s t order reaction. P h y s i c a l e n t r a p m e n t h a s b e e n u s e d as an a p p r o a c h t o a c h i e v e t h e r m a l s t a b i l i z a t i o n o f ;-glucose
oxidase.395
t h e t h e r m o i n a c t i v a t i o n o f e-glucose
for
I n polyacrylate gels the
f o l d by e n t r a p m e n t i n p o l y a c r y l a m i d e g e l s . enzyme behaved d i f f e r e n t l y ,
to.5 value
The
o x i d a s e was i n c r e a s e d s e v e r a l -
probably owing t o a microenvironmental
e f f e c t of t h e p o l y e l e c t r o l y t e n a t u r e of
the carrier.
I t has been r e p o r t e d f o u n d t h a t i n a d d i t i o n t o oxygen s i x r e d o x i n d i c a t o r s can s e r v e
as
Pencillium
The pH dependence o f t h e r a t e o f t h e r e a c t i o n
ita ale.^^'
c a t a l y s e d by g - g l u c o s e oxygen
and
substrates
for
Q-glucose
with
from
o x i d a s e was i n v e s t i g a t e d i n t h e p r e s e n c e o f
artificial
electron
I n contrast
acceptors.
r e a c t i o n w i t h o x y g e n , whose o p t i m u m pH i s 5.6, reactions
oxidase
phenazine
methosulphate,
to
the
t h e o p t i m u m pH o f
basic
dark
blue
2K,
p o l y v i o l o g e n , and t h e i o n - r a d i c a l s a l t o f b4,N,","-tetramethyl-4p h e n y l e n e d i a m i n e was o b s e r v e d a t pH 7.5. t h e r e d u c e d enzyme deprotonated)
can
exist
differing i n rate
i n of
The r e s u l t s i n d i c a t e d t h a t
two
forms
(protonated
o x i d a t i o n by
oxygen
and
and i n
a b i l i t y t o b i n d and r e d u c e a r t i f i c i a l e l e c t r o n a c c e p t o r s w i t h t h e f o r m a t i o n o f t h e semiquinone form of t h e c o f a c t o r . The u s e o f ! - g l u c o s e
oxidase-catalase
system
i n measuring
547
6: Enzymes aeration
capacity
of
fermenters
has
been
investigated
c o m p a r i s o n made o f t h e d y n a m i c a n d s t e a d y - s t a t e measurement.490
The
conditions
spontaneous h y d r o l y s i s o f
have
been
and
specified
where
l a c t o n e was s u f f i c i e n t l y r a p i d ,
thus I n Q-
e l i m i n a t i n g i n h i b i t o r y a c t i o n o f l a c t o n e on t h e o x i d a t i o n . glucose o x i d a s e - f r e e batches,
kla
the
a
kla
methods o f
v a l u e s were d e t e r m i n e d u s i n g
v a r i o u s m o d i f i c a t i o n s o f t h e dynamic method.
The d y n a m i c m e t h o d s i n
w h i c h gas i n t e r c h a n g e was e f f e c t e d w i t h o u t i n t e r r u p t i n g a e r a t i o n and
kla
agitation o f the batch yielded erroneously lower compared t o t h e r e s u l t s o f s t e a d y - s t a t e v a l u e was h i g h e r t h a n 0 . 0 3 s - ' .
v a l u e s as
methods i f t h e measured
kla
The v a l u e y i e l d e d b y t h e d y n a m i c
m e t h o d i n w h i c h t h e g a s i n t e r c h a n g e was e f f e c t e d a t t h e same t i m e w i t h t u r n i n g on a e r a t i o n and a g i t a t i o n of values r e s u l t i n g from the steady-state measured
kla
t h e b a t c h agreed w i t h
method,
provided that the
v a l u e s w e r e l o w e r t h a n 0.08 s - l a n d t h e s i m u l t a n e o u s
i n t e r f a c i a l t r a n s p o r t o f n i t r o g e n and oxygen had been t a k e n i n t o account i n the evaluation.
A t
kla
v a l u e s h i g h e r t h a n 0.08s"
kla
m o d i f i c a t i o n o f t h e dynamic method a l s o y i e l d e d l o w e r compared w i t h t h e outcome o f t h e s t e a d y - s t a t e experiments
performed
did
not,
however,
u n a m b i g u o u s l y on w h e t h e r t h e s e l o w e r
kla
allow
this
v a l u e s as
method. one
to
The
decide
v a l u e s w e r e due t o f a i l u r e
o f t h e adopted model t o descibe adequately t h e dynamic behaviour o f t h e system o r whether they were t r u e values d i f f e r i n g f r o m those y i e l d e d by t h e s t e a d y - s t a t e
m e t h o d on a c c o u n t o f d i f f e r e n t
physical
p r o p e r t i e s o f compared batches. The k i n e t i c s o f o x i d a t i o n o f ! - g l u c o s e o x y g e n i n t h e p r e s e n c e o f !-glucose been s t u d i e d b o t h t h e o r e t i c a l l y
s o l u t i o n by d i s s o l v e d
o x i d a s e and e x c e s s c a t a l a s e h a v e
and
experiment all^.^^^
The k i n e t i c
m o d e l u s e d was b a s e d o n t h e d e t a i l e d mechanism o f t h e r e a c t i o n .
The
d e s c r i p t i o n o f t h e m o d e l was g i v e n by t h e M i c h a e l i s - M e n t e n e q u a t i o n f o r t h e case o f t w o s u b s t r a t e s . a closed reactor commercial
at
enzymes.
The e x p e r i m e n t s w e r e c a r r i e d o u t i n
a t e m p e r a t u r e o f 25OC a n d p H 5 . 5 5 Kinetic
constants
of
the
using
reaction
were
e v a l u a t e d by f i t t i n g t h e oxygen c o n c e n t r a t i o n p r o f i l e s c a l c u l a t e d f r o m t h e model t o t h o s e f o u n d e x p e r i m e n t a l l y , by t h e method o f nonlinear
regression.
d i s t o r t i o n of
The r a t e o f
p r o b e were t a k e n i n t o account. the
reaction
were
i n
good
m u t a r o t a t i o n and
The d e t e r m i n e d k i n e t i c c o n s t a n t s o f agreement
l i t e r a t u r e f o r p u r i f i e d enzymes. data of
Q-glucose
d a t a caused by t h e dynamics o f t h e a p p l i e d oxygen with
those
given
i n
the
Adoption o f l i t e r a t u r e k i n e t i c
t h i s r e a c t i o n t o t h e d e t e r m i n a t i o n of
aeration capacity o f
548
Carbohydrate Chemistry
f e r m e n t e r s by t h e Q - g l u c o s e o x i d a s e s y s t e m p r o v e d p o s s i b l e . A t pH 7 . 4 , i n 0 . 1 M p h o s p h a t e b u f f e r , Q - g l u c o s e o x i d a s e a n d B l u e D e x t r a n i n t e r a c t s t r o n g l y w i t h each o t h e r . 4 9 2 Under these c o n d i t i o n s a s o l u b l e c o m p l e x i s f o r m e d , as s h o w n by u l t r a f i l t r a t i o n e x p e r i m e n t s u s i n g a D i a f l o XM-300 m e m b r a n e ( n o m i n a l m o l e c u l a r w e i g h t c u t - o f f 300,000). I n t h i s c o m p l e x , t h e e n z y m e i s a b o u t 40% m o r e a c t i v e t h a n when f r e e i n s o l u t i o n , a n d i s a l s o m o r e s t a b l e t o w a r d s a c i d denaturation. Comparative s t u d i e s with a high-molecular-weight d e x t r a n d e v o i d o f dye r e s i d u e s ( D e x t r a n T - 2 0 0 0 ) s u g g e s t e d t h a t t h e dye moiety of Blue Dextran is d i r e c t l y i n v o l v e d i n t h e b i n d i n g of t h e p r o t e i n , and p o s s i b l y a l s o i n t h e enhancement o f i t s e n z y m a t i c activity. Q-Glucose o x i d a s e p u r i f i e d from A s p e r g i l l u s n i g e r on s u b j e c t i o n t o i s o e l e c t r i c f o c u s i n g a n d g e l e l e c t r o p h o r e s i s was f o u n d t o b e c o m p o s e d o f a t l e a s t s i x c o m p o n e n t e n z y m e s ( P I 3 . 9 t o 4.3).493 They a l l p o s s e s s an i d e n t i c a l p r o t e i n moiety, s i n c e the amino acid compositions, C-terminal sequences, catalytic parameters, q u a n t i t a t i v e and q u a l i t a t i v e immunological p r o p e r t i e s , and t h e e l e c t r o p h o r e t i c p a t t e r n s o f t h e p e p t i d e f r a g m e n t s o b t a i n e d by CNBr c l e a v a g e were p r a c t i c a l l y t h e s a m e . On t h e o t h e r h a n d , t h e c a r b o h y d r a t e c o n t e n t s o f t h e e n z y m e s were f o u n d t o b e d i f f e r e n t , a n d t h e s e d i f f e r e n c e s were a s s o c i a t e d i n t h e m a i n w i t h a p a r t i c u l a r peptide fragment. L-Galactonolactone Oxidases. -- L - G a l a c t o n o l a c t o n e o x i d a s e h a s b e e n f o u n d t o b e i n a c t i v a t e d by v a r i o u s s u l p h y d r y l r e a g e n t s : t h e o r d e r o f i n a c t i v a t i o n r a t e w a s HgC12, 4 - c h l o r o m e r c u r i b e n z o a t e , 4,4’dipyridyldisulphide, 2,2’-dipyridy l d i s u l p h i d e , 5 ,S9-dithio-bis-(2nitrobenzoate), and fj-ethylmaleimide iodoacetamide.494 The i n a c t i v a t i o n by 4,4’-dipyridyldisulphide w a s s t u d i e d i n d e t a i l , a n d i t was f o u n d t h a t t h e maximum d e g r e e o f i n a c t i v a t i o n a t t a i n e d w i t h i n c r e a s i n g r e a g e n t c o n c e n t r a t i o n was 93% a n d t h a t t h e k i n e t i c s o f i n a c t i v a t i o n were f i r s t o r d e r w i t h r e s p e c t t o r e a g e n t c o n c e n t r a t i o n . T h e pH d e p e n d e n c e o f t h e s e c o n d - o r d e r r a t e c o n s t a n t o f t h e i n a c t i v a t i o n r e v e a l e d t h a t a s u l p h y d r y l g r o u p w i t h a pKa o f 9.8 was involved i n the i n a c t i v a t i o n process. T h e v a l u e o f pKa i s h i g h compared w i t h t h a t of low-molecular-weight t h i o l s , i n d i c a t i n g t h a t t h e i o n i z a t i o n o f t h e s u l p h y d r y l g r o u p i s a f f e c t e d by t h e e l e c t r i c f i e l d of a n e g a t i v e l y charged group l o c a t e d i n its v i c i n i t y . The v a l u e s o f K m a n d lmax for the C-galactonolactone oxidase reaction d i d n o t c h a n g e g r e a t l y a r o u n d pH 9.8. This is consistent with the
549
6: Enzymes
v i e w t h a t t h e s u l p h y d r y l g r o u p does n o t p a r t i c i p a t e i n t h e c a t a l y t i c process.
The v e l o c i t y o f i n a c t i v a t i o n i n t h e p r e s e n c e o f s u b s t r a t e
was c o n s i d e r a b l y g r e a t e r
It
t h a n t h a t o b s e r v e d i n i t s absence.
a p p e a r s t h a t t h e s u l p h y d r y l g r o u p becomes more r e a c t i v e d u r i n g t h e catalytic
cycle of
the
enzyme.
I t was
also noted that
the
a b s o r p t i o n spectrum of t h e f l a v i n p r o s t h e t i c group ( t h e o x i d i z e d was m o d i f i e d b y a d d i n g 4 - c h l o r o m e r c u r i b e n z o a t e .
form)
I-Gulonolactone
--
Oxidases.
The
activity
of
I-gulonolactone
o x i d a s e i n l i v e r s o f 49 s p e c i e s o f e u t h e r i a n mammals h a s been f o u n d to
vary
intraspecifically
v a r i a t i o n w e r e 0.2
t o 0.4
--
Cellobiose Oxidases. produced
cellobiose
cellulose.496
among
individuals:
coefficients
of
i n many s p e c i e s . 4 9 5 A s p e c i e s o f t h e i m p e r f e c t fungus M o n i l i a
oxidase
extracellularly
when
grown
on
The i n d u c i b l e enzyme was b o t h bound t o t h e m y c e l i u m
and r e l e a s e d i n t o t h e g r o w t h medium and showed a h i g h d e g r e e o f specificity for
cellobiose,
but
a l s o o x i d i z e d l a c t o s e and 4-;-8-!-
glucopyranosyl-q-mannose.
The s p e c i f i c i t y
was
compounds
restricted V.
+0.22
t o those
4-Benzoquinone
n o t reduced.
of
the electron acceptor
having a redox
and s e v e r a l o t h e r q u i n o n e s ,
weight
48,000
of
t e c h n i q u e was d e v e l o p e d f o r polyacrylamide gels focusing
&-Fructose
Oxidases. the
study
enzymes
i m m o b i l i z e d on
can
assessed
be
and
by
-of
a
PI of
An
runs
The
which
zymogram
and
isoelectric
c a t a l y s e d by
kinetic allow
behaviour
a
the surface kinetics,
separate and t h e
The m e t h o d h a s b e e n a p p l i e d i n t h e
s t u d y o f s u c r o s e i n v e r s i o n by 8 - & - f r u c t o s e
o x i d a s e i m m o b i l i z e d on
resin.
2-Galactose Oxidases. of
new
reactions
s u p p o r t s .497
experimental
f l u i d - p a r t i c l e mass t r a n s f e r .
A
e x p e r i m e n t a l p r o c e d u r e has been
liquid-phase
porous
The enzyme h a d a
cellobiose oxidase i n
electrophoresis
evaluation o f the internal diffusion,
IRA-93
5.4.
the detection o f
following
.
proposed for
were
O x y g e n was n o t c o n s u m e d n o r was h y d r o g e n p e r o x i d e
p r o d u c e d by c e l l o b i o s e o x i d a t i o n o f c e l l o b i o s e . molecular
potential of however,
glycosidases
!-Galactose
and
oxidase
--
The s t e r i c f a c t o r s i n v o l v e d i n t h e a c t i o n
g-galactose
o x i d a s e have been i n ~ e s t i g a t e d . ” ~
i s s t e r i c a l l y hindered by c e r t a i n types of
branching i n substrate oligosaccharide chains
and w i l l n o t o x i d i z e
550
Carbohydrate Chemistry
t h e 4 - g a l a c t o s e r e s i d u e i n t r i s a c c h a r i d e ( 5 ) b u t w i l l i n ( 6 ) . PG a l a c t o s e a s i n ( 7 ) i s not s u s c e p t i b l e t o o x i d a t i o n w i t h !-galactose oxidase u n t i l a f t e r t h e a p p l i c a t i o n of B-Q-2-acetamido-2deoxygalactosidase. a-NeuNGL 2
I 6
B-g-Gal-( 1+3)-!-GalNAcol (5)
B-Q-Gal-(1+3)-g-GalNAcol 2
I 1 ? - L-- F u C
(6)
B-Q-GalNAc-(1+3)-B-g-Gal-(l+3)-~-GalNAcol 2
I 1
a-i-Fuc (7)
48
Carbohydrate Transferases
A review of t h e a f f i n i t y chromatography of g l y c o s y l t r a n s f e r a s e s summarizes t h e use o f b i o s p e c i f i c chromatography t e c h n i q u e s i n t h e ~~ that are p u r i f i c a t i o n of mammalian g l y c o s y l t r a n ~ f e r a s e s . ~Ligands analogues of donor o r a c c e p t o r s u b s t r a t e s have been l i n k e d t o cyanogen b r o m i d e - a c t i v a t e d a g a r o s e f o r use a s a f f i n i t y a d s o r b e n t s . Immobilized l e c t i n s have been employed t o r e c o g n i z e t h e c a r b o h y d r a t e m o i e t i e s o f g l y c o s y l t r a n s f e r a s e and remove them f r o m complex m i x t u r e s . The a p p l i c a t i o n of these methods has p e r m i t t e d e x t e n s i v e p u r i f i c a t i o n of many membrane-bound g l y c o s y l t r a n s f e r a s e s , some t o homogeneity. T h e p o s s i b i l i t y t h a t murein g l y c o s y l t r a n s f e r a s e of E s c h e r i c h i a c o l i may f u n c t i o n a s an exoenzyme t o c l e a v e t h e j u r e i n
551
6: Enzymes s a c c u l u s i n a s y s t e m a t i c f a s h i o n has been i n v e s t i g a t e d . 4 9 9
Two
m o l e c u l a r s p e c i e s o f t h i s h y d r o l y t i c enzyme h a v e b e e n i s o l a t e d and characterized:
one i s a s s o c i a t e d w i t h t h e s o l u b l e f r a c t i o n
o t h e r w i t h t h e e n v e l o p e f r a c t i o n o f r u p t u r e d E.
and t h e
c o l i cells.
The
s o l u b l e enzyme was e m p l o y e d t o d i g e s t m u r e i n s a c c u l i t h a t h a d b e e n uniformly
l a b e l l e d w i t h { 3H)diaminopimelic
acid.
The
analysis
of
t h e r e a c t i o n p r o d u c t i n d i c a t e d t h a t t h e enzyme d i d n o t c l e a v e t h e glycan
c h a i n s random l y
.
To
d e t e r m i n e whet h e r
g l y c o s y l t r a n s f e r ase
from t h e 2-acetamido-2-deoxy-~-glucosyl
r e l e a s e d muropeptide f i r s t
or t h e 1 , 6 - a n h y d r o m u r a m y l e n d s o f t h e g l y c a n c h a i n s ,
t h e {3H)-
d i a m i n o p i me l a t e - l a b e l l e d s a c c u li w e r e f u r t h e r r a d i o l a b e l l e d a t t h e i r
2-acetamido-2-deoxy-~-glucosyl ends w i t h { 14)-Q-galactose galactosyltransferase reaction.
by a Q-
The g l y c o s y l t r a n s f e r a s e r e l e a s e d Q-
g a l a c t o s e - l a b e l l e d X + X’ m u r o p e p t i d e s ( w h e r e X = 2 - a c e t a m i d o - 2 deoxy-~-glucosyl-1,6-anhydro-~-acetylmuramyl-~-alanyl-~-~-glutamylmeso-diaminopimelic o f digestion,
= X-2-alanine)
a c i d , X’
e a r l y d u r i n g t h e course
suggesting exoenzymatic cleavage of t h e glycan chains
preferentially from the 2-acetamido-2-deoxy-~-glycosyl
ends.
The
k i n e t i c s o f t h e a c t i v i t y o f t h e membrane-bound enzyme w e r e f o u n d t o b e i d e n t i c a l t o t h o s e o f t h e s o l u b l e enzyme, molecular species of
indicating that both
g l y c o s y l t r a n s f e r a s e f u n c t i o n as exoenzymes
i n vitro. I n a c t i v a t i o n of
-----mutans
and S .
by
from o r a l Streptococcus
photochemical
oxidation
has
been
C e l l - f r e e Q - g l u c o s y l t r a n s f e r ase of Q-glucose-grown
r e p o r t e d .500 S.
P-glucosyltransferases
sanggL5
m u t a n s AHT was c o m p l e t e l y i n a c t i v a t e d i n t h e p r e s e n c e o f 0.002%
o f M e t h y l e n e B l u e a t 25OC a n d pH 7.0 a f t e r i l l u m i n a t i o n w i t h a 1 5 0 W i n c a n d e s c e n t lamp. v a l u e s (7.0.
T h e r a t e o f i n a c t i v a t i o n was d e c r e a s e d a t pH
i-Histidine
was t h e o n l y a m i n o a c i d r e s i d u e m o d i f i e d
t o a s i g n i f i c a n t e x t e n t , and t h e r a t e s o f o x i d a t i o n o f I - h i s t i d i n e and loss o f enzyme a c t i v i t y c l o s e l y a g r e e d .
Production o f both
w a t e r - i n s o l u b l e and - s o l u b l e
f r o m s u c r o s e by t h e
oxidized
k-glucan f r a c t i o n s
1-glucosyltransferase
inhibited.
preparations
was
significantly
P h o t o - o x i d a t i o n w i t h 0.002% Rose B e n g a l a t pH 7.0
also
induced complete i n a c t i v a t i o n o f t h e Q-glucosyltransferase. These r e s u l t s s t r o n g l y suggest t h a t t h e i m i d a z o l e p o r t i o n o f I - h i s t i d i n e may
function
as
part
glucosyltransferase responsible for
o f
the
active
isoenzymes of
t h e s y n t h e s i s o f (1
+
S.
s i t e s
o f
m u t a n s AHT,
both
Q-
which are
3 ) - and (1 + 6 ) - a - Q - g l u c o s i d i c
linkages.
I t has
been r e p o r t e d
that
the
glucosyltransferases
of
Carbohydrate Chemistry
552 S t r e e --------------tococcus mutans ----
have
been r e s o l v e d i n t o t w o components
e s s e n t i a l t o w a t e r - i n s o l u b l e glucan synthesis.501
Collagen-D=-galactotransferase
and
collagen-Q-
g l u c o s y l t r a n s f e r a s e a c t i v i t i e s h a v e been s t u d i e d i n c u l t u r e d human f o e t a l l u n g WI-38
and I M R - 9 0 d i p l o i d f i b r o b l a s t s . 5 0 2
functioned i n concert hydroxylysine membranes, UDP-!-Gal
units
t o
as
found
naturally
i n
collagens,
and c e r t a i n serum g l y c o p r o t e i n s . and UDP-g-Glc
These enzymes
s y n t h e s i z e Q-glucosyl-Q-galactosyl-L-
as g l y c o s e d o n o r s ,
basement
The t r a n s f e r a s e s u s e d c o l l a g e n s and c o l l a g e n -
d e r i v e d p e p t i d e s o r g l y c o p e p t i d e s as g l y c o s e
acceptors,
and w o r k e d
b e s t i n t h e p r e s e n c e o f m a n g a n e s e as a r e q u i r e d d i v a l e n t c a t i o n . Two pH o p t i m a , b e t w e e n pH 6 a n d 6 . 5
a n d b e t w e e n pH 7 . 5
noted for
and t h e s e o p t i m a ,
each t y p e o f
transferase,
i n t h e case o f Q - g l u c o s y l t r a n s f e r a s e , s i z e of
were e v i d e n t r e g a r d l e s s o f
a c c e p t o r employed i n t h e assay.
a c t i v i t y of
and 8, were particularly
About 35% o f t h e t o t a l
e a c h enzyme was f o u n d i n t h e s o l u b l e f r a c t i o n s o f
cell
homogenates, and about 50% of t h e p a r t i c u l a t e f r a c t i o n a c t i v i t i e s c o u l d be r e l e a s e d by m i l d s o n i c a t i o n o r by t r e a t m e n t w i t h T r i t o n X100.
Assessment o f t r a n s f e r a s e a c t i v i t i e s as a f u n c t i o n o f c e l l u l a r
a g e i n g i n c u l t u r e r e v e a l e d t h a t s i g n i f i c a n t d e c r e a s e s i n enzyme l e v e l s o c c u r r e d a s t h e c e l l a p p r o a c h e d s e n e s c e n c e ( l a t e p h a s e II), a n d t h e s e e f f e c t s w e r e r e v e r s e d when t h e c e l l a t t a i n e d s e n e s c e n c e ( p h a s e 111). conditions
A d d i t i o n of known
hydroxylation,
the
glycosyltransferases
a s c o r b i c a c i d t o young c u l t u r e s ,
increase
caused
glycosyltransferases suggested t h a t
t o
no
endogenous
on
effects
the
activities
t o w a r d exogenous a c c e p t o r s . activities
of
under
collagen-peptide of
collagen-hydroxylases
m i g h t n o t be c o - o r d i n a t e l y
regardless o f t h e h y d r o x y l a t i o n events,
the
These r e s u l t s and
r e g u l a t e d and t h a t ,
g l y c o s y l a t i o n of t h e peptide
m i g h t be l i m i t e d t o a s p e c i f i c f r a c t i o n o f i - h y d r o x y l y s i n e r e s i d u e s during the post-translational
m o d i f i c a t i o n of collagen.
Bovine g l y c o p r o t e i n B-E-galactosyltransferase have two metal-binding
h a v e been e s t a b l i s h e d by u s i n g k i n e t i c , structural
s p e c t r o s c o p i c , and a f f i n i t y -
M e t a l s i t e I,
c h r o m a t o g r a p h i c approaches.503 maintaining the
h a s b e e n shown t o
the f u n c t i o n a l properties o f which
sites,
integrity
of
which i s i n v o l v e d i n
the
protein,
must
be
l i g a n d e d p r i o r t o o t h e r s u b s t r a t e s b i n d i n g and p r i o r t o a second m e t a l b i n d i n g t o s i t e 11, w h i c h n- - g a l a c t o s e b i n d i n g .
i s shown t o be a s s o c i a t e d w i t h UDP-
B o t h m e t a l s i t e s can b i n d a v a r i e t y o f
metals.
However,
c a l c i u m and i t s f l u o r e s c e n t a n a l o g u e e u r o p i u m b i n d o n l y t o
s i t e I I.
F 1u o r es c e n t r e s o n a n c e - e n e r gy t r a n s f e r m e a s u r e m e n t s b e t w e e n
553
6: Enzymes
e u r o p i u m i n s i t e I1 and c o b a l t i n s i t e I i n d i c a t e a d i s t a n c e o f 18 0
3 A between t h e two s i t e s .
mercuric-N-dansylcysteine
2
C h e m i c a l - m o d i f i c a t i o n s t u d i e s w i t h 5i n d i c a t e t h a t one ( o f a t o t a l o f t h r e e
e x p o s e d s u l p h y d r y l g r o u p s ) c a n be s p e c i f i c a l l y d a n s y l a t e d and t h a t t h i s s u l p h y d r y l group i s i n or near t h e UDP-a-Salactose-binding site.
Resonance-energy
transfer
measurements
i n t r o d u c e d s u l p h y d r y l group and c o b a l t distance of
2
19
3
dl
between t h i s
i n metal s i t e I
between these p o i n t s ,
give
a
consistent with the
i n t e r p r e t a t i o n t h a t the UDP-a-galactose-binding
site,
which
i s
a s s o c i a t e d w i t h m e t a l s i t e 1 1 , i s l o c a t e d some d i s t a n c e f r o m t h e s t r u c t u r a l m e t a l s i t e ( s i t e 1). A !-galactose
protein),
B-galactosyltransferase (lactose synthetase A
w h i c h t r a n s f e r s Q - g a l a c t o s e f r o m UDP-D-galactose
t o 2-
a c e t a m i d o - 2 - d e o x y - ~ - g l u c o s e , was p u r i f i e d 2 8 6 , 0 0 0 - f o l d t o h o m o g e n e i t y w i t h 40% y i e l d f r o m human p l a s m a by r e p e a t e d a f f i n i t y c h r o r n a t o g r a p h y o n a - l a c t a l b u m i n - S e p h a r ~ s e . ~ ~S~o d i u m d o d e c y 1 s u l p h a t e p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of
t h e p u r i f i e d enzyme
showed a s i n g l e p r o t e i n band w i t h a m o l e c u l a r w e i g h t o f 49,000.
The
enzyme i s a g l y c o p r o t e i n w i t h 11% by w e i g h t c a r b o h y d r a t e w h i c h h a s o n l y 2-acetamido-2-deoxy-glucose-~-asparagine g l y c o p e p t i d e l i n k a g e s . The enzyme showed c h a r a c t e r i s t i c changes i n a c t i v i t y a t d i f f e r e n t alactalbumin concentrations,
i n d i c a t i n g t h a t t h e enzyme i s t h e A
Km v a l u e s f o r t h e s u b s t r a t e s were rnM f o r U D P - Q - g a l a c t o s e and 0.20 m M f o r Mn2+. The
p r o t e i n of lactose synthetase. f o u n d t o be 0.056
a c t i v i t y o f t h e enzyme was n e u t r a l i z e d b y a n t i - e n z y m e a n t i b o d y ,
but
the antibody d i d not neutralize the bovine m i l k Q-galactosyltransferase (A p r o t e i n ) a c t i v i t y . Bovine
a-lactalburnin
enzymatically f u l l y
active,
has
been
highly
dansylated
fluorescent
t o
derivative
on t h e N - t e r m i n a l I - g l u t a m i c a c i d residue.505
give
an
labelled
This fluorescent
d e r i v a t i v e of
a - l a c t a l b u m i n has been c o v a l e n t l y
glycoprotein
B-P-galactosy ltransferase
p i me lirn id a t e.
Resonance - e n e r gy t r a n s f e r
by
crosslinked t o
using
dirnethy 1
me as u r e rnents u s i n g c o b a I t
b o u n d t o t h e t r a n s f e r a s e as t h e a c c e p t o r o f e n e r g y t r a n s f e r f r o m t h e d a n s y l g r o u p on t h e a - l a c t a l b u m i n 32 of
A"
i n d i c a t e t h a t t h e d a n s y l group i s
A model o f the a c t i v e s i t e f r o m t h e c o b a l t on t h e t r a n s f e r a s e . t h e t r a n s f e r a s e and i t s i n t e r a c t i o n w i t h a - l a c t a l b u r n i n i s
proposed on t h e b a s i s of
t h e s e and p r e v i o u s s t u d i e s .
have a l l o w e d an e x t e n s i o n o f s y n t h e t a s e and g i v e g r e a t e r
the active-site
These s t u d i e s
mapping o f l a c t o s e
i n s i g h t i n t o t h e i n t e r a c t i o n o f a-
l a c t a l b u m i n w i t h t h e t r a n s f e r a s e and i t s r o l e i n l a c t o s e s y n t h e t a s e .
554
Carbohydrate Chemistry
G1ycoprotein:C-fucosyltransferase from sheep b r a i n , s o l u b i l i z e d and p r e p u r i f i e d by hydrophobic chromatography on e t h y l - a g a r o s e , has been f u r t h e r p u r i f i e d by chromatofocalization.506 T h i s p r o c e d u r e y i e l d e d f o u r isoenzymes. The s o l u b l e & - f u c o s y l t r a n s f e r a s e o f t h e r a t s m a l l - i n t e s t i n a l mucosa p u r i f i e d b y g e l f i l t r a t i o n on S e p h a d e x G-100 h a s b e e n r e s o l v e d i n t o two i s o e n z y m e s F1 ( P I = 4 . 4 7 ) and F 2 ( P I = 4.96) b y i s o e l e c t r i c focusing.S07 The u s e of D E A E - c e l l u l o s e i n s t e a d of S e p h a d e x G-100 l e a d s t o a n o t h e r component F 3 ( P I = 8 . 7 0 ) . K i n e t i c p a r a m e t e r s (5, and ymax)f o r t h e t h r e e isoenzymes were d e t e r m i n e d . The e n z y m a t i c mechanism s e e m s t o be a b i - b i random t y p e f o r t h e t h r e e isoenzymes, and F 3 a p p e a r s a s t h e most a c t i v e s p e c i e s . T h e i s o e l e c t r o f o c u s i n g p a t t e r n s o f 2 - a - i - , 3 - a - i - , and 4-a-Cf u c o s y l t r a n s f e r a s e s from human m i l k serum have been r e p o r ted.’08 The s p e c i f i c i t y of s i a l y l t r a n s f e r a s e has been i n v e s t i g a t e d w i t h r e f e r e n c e t o t h e s i a l y l a t i o n of o v i n e s u b m a x i l l a r y mucin i n v i t r o which was { 1 4 C ) s i a l y l a t e d i n v i t r o u s i n g a p o r c i n e l i v e r c e l l - f r e e p r e p a r a t i ~ n . ’ ~ ~The o l i g o s a c c h a r i d e c h a i n s were c l e a v e d from t h e p r o d u c t g l y c o p r o t e i n by B - e l i m i n a t i o n under r e d u c t i v e c o n d i t i o n s , f r a c t i o n a t e d by g e l f i l t r a t i o n on Bio-Gel P-2, and c h a r a c t e r i z e d by t h i n - l a y e r chromatography. The s t r u c t u r e o f t h e product c h a i n was s t u d i e d by p e r i o d a t e o x i d a t i o n and a n a l y s i s of t h e p e e l i n g p r o d u c t s formed i n t h e B - e l i m i n a t i o n s t e p . I t appeared t h a t { 1 4 C ) s i a l i c a c i d had been i n t r o d u c e d e x c l u s i v e l y t o t h e Q - g a l a c t o s e r e s i d u e s o f Q G a l - ( 1 + 3)-Q-GalNAc d i s a c c h a r i d e u n i t s o c c u r r i n g on t h e mucin a s minor c h a i n s . No i n d i c a t i o n f o r a t r a n s f e r t o 2-acetamido-2-deoxyIn g - g a l a c t o s y l r e s i d u e s on t h i s g l y c o p r o t e i n was o b t a i n e d . agreement w i t h t h i s r e s u l t s i a l y l t r a n s f e r a s e a c t i v i t i e s of p o r c i n e , r a t , human, and c a n i n e l i v e r w i t h B - G a l - ( l + 3 ) - Q - G a l N A c - p r o t e i n a c c e p t o r s w e r e i n v a r i a b l y much h i g h e r t h a n t h o s e w i t h o v i n e submaxillary asialomucin. When t h e a s i a l o m u c i n had been { 1 4 C ) s i a l y l a t e d b y an o v i n e s u b m a x i l l a r y g l a n d c e l l - f r e e p r e p a r a t i o n , a n a l y s i s of t h e p r o d u c t o l i g o s a c c h a r i d e c h a i n r e v e a l e d t h e i n t r o d u c t i o n o f { 1 4 C ) s i a l i c a c i d t o p o s i t i o n C - 6 on t h e 2acetamido-2-deoxy-!-galactose r e s i d u e s . The s p e c i f i c i t y o f t h i s t r a n s f e r was r e f l e c t e d by t h e very high s i a l y l t r a n s f e r a s e a c t i v i t i e s of g l a n d p r e p a r a t i o n s w i t h g-Gal-(l + 3)-Q-GalNAc-protein a s w e l l a s a-GalNAc-protein a c c e p t o r s . Mixed-enzyme e x p e r i m e n t s i n d i c a t e d t h a t t h e d i f f e r e n c e i n l i v e r and g l a n d o v i n e s u b m a x i l l a r y a s i a l o m u c i n s i a l y l t r a n s f e r a s e a c t i v i t i e s was n o t d u e t o t h e p r e s e n c e o f a s p e c i f i c i n h i b i t o r i n t h e l i v e r o r an a c t i v a t o r i n t h e g l a n d . It w i t h T r i t o n X-100
555
6: Enzymes
was c o n c l u d e d t h a t l i v e r s o f man, p i g , r a t , a n d dog c o n t a i n a a - G a l s i a l y l t r a n s f e r a s e which i s involved i n the
(1 + 3)-g-GalNAc-protein
sialylation o f g-glycosidically glycoproteins.
l i n k e d c a r b o h y d r a t e c h a i n s on serum
g-GalNAc-protein
sialyltransferase activity,
which
r i c h l y o c c u r s i n o v i n e s u b m a x i l l a r y g l a n d , h o w e v e r , a p p e a r s t o be lacking from l i v e r tissue. A t e c h n i q u e h a s been d e s c r i b e d by a f f i n i t y c h r o m a t o g r a p h y o f
o r o t a t e p h o s p h o r i b o s y l t r a n s f e r a s e f r o m E s c h e r i c h i a c o li K - 1Z?'
I-Iduronic Acid 2 - S u l p h a t e S u l p h a t a s e s
49
A m o r e s e n s i t i v e assay h a s been d e v e l o p e d f o r t h e I - i d u r o n i c 2 sulphate
sulphatase
which
i s
deficient
i n cases o f
the Hunter
The s u b s t r a t e i s 2 - ( a - C - i d o p y r a n o s y l u r o n i c
syndrome.511 su1phate)-( 1
.+
a c i d 2-
4) - 2 , 5 - a n h y d r o -Q-{ 3 H - l } r n a n n i t o 1 6 - s u l p h a t e
a f t e r incubation,
,
and,
i t i s s e p a r a t e d f r o m t h e p r o d u c t by i o n - e x c h a n g e
r a t h e r t h a n by c h r o m a t o g r a p h y o n a m i c r o - c o l u m n o f Dowex 1 x 2 (Cl') h i g h - v o l t a g e e l e c t r o p h o r e s i s o r ECTEOLA c e l l u l o s e c h r o m a t o g r a p h y . S i n c e t h e b l a n k c o r r e c t i o n i s t h e n much s m a l l e r ,
a shorter
i n c u b a t i o n t i m e c a n be u s e d and c o n v e r s i o n o f t h e s u b s t r a t e r e d u c e d f r o m a p p r o x i m a t e l y 50% down t o l e v e l s w h e r e c o m p l i c a t i o n s r e s u l t i n g f r o m s u b s t r a t e d e p l e t i o n and p r o d u c t i n h i b i t i o n a r e m i n i m a l .
Using
With w h o l e s e r u m t h e a p p a r e n t 5, f o r t h e s u b s t r a t e i s 0.2 n m o l 1-l. a n i n c u b a t i o n t i m e o f 20 m i n , s e r a f r o m h e t e r o t y g o t e s e x h i b i t e d a p p r o x i m a t e l y 35% o f t h e n o r m a l l e v e l s of L - i d u r o n a t e 2 - s u l p h a t e sulphatase carriers, females).
(0.11-0.61, 0.24-2.35,
mean 0.34, mean 0.94,
nmol h'l
n m o l h'l
mg-l
protein for
21
m g - l p r o t e i n f o r 37 n o r m a l
S e r u m a n a l y s e s c a n t h u s be u s e d t o s u p p l e m e n t t h o s e on
h a i r r o o t s i n t h e d e t e c t i o n o f c a r r i e r s o f t h e H u n t e r syndrome. The L - i d u r o n i c a c i d 2 - s u l p h a t e s u l p h a t a s e o f human p l a c e n t a h a s been s e p a r a t e d by ion-exchange and g e l - f i l t r a t i o n c h r o m a t o g r a p h y i n t o t w o components, a l e s s a c i d i c f o r m A and a more a c i d i c f o r m The t w o f o r m s have d i f f e r e n t m o b i l i t i e s on g e l e l e c t r o p h o r e s i s a n d d i f f e r e n t i s o e l e c t r i c p o i n t s ( A PI 5.0, B PI 4.5).
similar
They show t h e same pH o p t i m a i n s o d i u m a c e t a t e b u f f e r a n d
Em v a l u e s
f o r { 3H)disulphated disaccharide substrate.
f o r m i s more heat l a b i l e t h a n t h e B form.
w e i g h t s w e r e f o u n d by g e l f i l t r a t i o n w h i l e s i m i l a r estimated
by
sucrose
gradient
The A
Different molecular
centrifugation.
values were
Neuraminidase
t r e a t m e n t o f t h e t w o f o r m s g i v e s e v i d e n c e t h a t t h e s e enzymes c o n t a i n
Carbohydrate Chemistry
556 s i a l i c a c i d residues.
Human u r i n e h a s been shown t o c o n t a i n a n o v e l s u l p h a t a s e w h i c h i s
specific
2-deoxy-2-sulphamido-4-glucopyranoside
for
3-
~ u l p h a t e . O~ f ~t h~ e t h r e e i s o m e r i c s u l p h a m a t e d e r i v a t i v e s , 3-g-,
0-,
and 6 - g - s u l p h a t e
enzymatic
esters,
activity
only t h e 3-g-ester
required that
4The
t h e a m i n o g r o u p be s u l p h a t e d .
S u l p h a t e i s n o t r e l e a s e d ift h e a m i n o g r o u p
i s
free or acetylated.
I t h a d a pH o p t i m u m o f 6.3 a n d was
The e n z y m e was p u r i f i e d 7 0 - f o l d . i n h i b i t e d by
was h y d r o l y s e d .
i n o r g a n i c s u l p h a t e and p h o s p h a t e .
t h i s enzyme s u g g e s t e d t h a t a 3 - g - s u l p h a t e d
The s p e c i f i c i t y
of
2-amino-2-deoxy-~-glucose
m o i e t y may h a v e a r o l e i n t h e p h y s i o l o g i c a l a c t i v i t y o f
or
heparin
heparan sulphate.
50
Arylsulphatases
A s t u d y h a s b e e n made o f l y s o s o m a l - e n z y m e a c t i v i t i e s i n c l u d i n g
A
arylsulphatase disease.60
i n
serum
For further
and
in
leukocytes
chronic
hepatic
d e t a i l s see i n i t i a l c i t a t i o n o f r e f . 6 0 .
A v a r i a n t f o r m o f a r y l s u l p h a t a s e A h a s been i d e n t i f i e d i n human u r i n e derived d i r e c t l y from the r e n a l pelvis. t h e enzyme was f o u n d i n n e p h r o s t o m i c u r i n e , form,
The v a r i a n t
w h i c h i s t h e s o l e component o f a r y l s u l p h a t a s e A i n v o i d e d
urine.514
The
n e p h r o s t o m i c enzyme
enzyme w i t h r e s p e c t
differed
from
t o the k i n e t i c parameters,
the
p o i n t s of t h e voided-urine respectively. and
5,
The i s o e l e c t r i c
a n d n e p h r o s t o m i c e n z y m e s w e r e 4.7
and
The n e p h r o s t o m i c enzyme was more h e a t l a b i l e a t
62.5OC t h a n t h e v o i d e d - u r i n e (130,000)
voided-urine
the isoelectric
p o i n t , h e a t s t a b i l i t y , and i m m u n o l o g i c a l r e a c t i v i t y . 5.3,
form o f
i n a d d i t i o n t o the minor
values
of
s u l p h a t e as s u b s t r a t e were
enzyme. the
Although the molecular weights
two
enzymes
a l m o s t t h e same,
with the
nitrocatechol
1
value o f the
n e p h r o s t o m i c enzyme was 10% t h a t o f t h e v o i d e d - u r i n e enzyme. d e m o n s t r a t e d by v a r i o u s methods, p u r i f i e d voided-urine
enzyme,
I t was
using IgG antibody against the
t h a t the nephrostomic
e n z y m e was
a n t i g e n i c a l l y d i s t i n c t f r o m t h e v o i d e d - u r i n e enzyme. R a b b i t l i v e r a r y l s u l p h a t a s e A i s a g l y c o p r o t e i n c o n t a i n i n g 4.6% carbohydrate g l u c o s e (71, 140,000).515 chains.
c o m p r i s i n g p-mannose
(25),
2-acetamido-2-deoxy-Q-
and s i a l i c a c i d ( 3 r e s i d u e s p e r enzyme monomer m o l .
The p r o t e i n h a s a r e l a t i v e l y h i g h c o n t e n t o f
glycine,and
wt.
E a c h monomer c o n s i s t s o f t w o e q u i v a l e n t p o l y p e p t i d e I-leucine,
&-proline,
I-
and t h e a m i n o a c i d c o m p o s i t i o n i s s i m i l a r t o
557
6: Enzymes that
of
other
known
liver
sulphatases.
The
arylsulphatase A
catalyses the h y d r o l y s i s o f a wide v a r i e t y o f sulphate esters, although i t appears t h a t cerebroside sulphate i s a p h y s i o l o g i c a l s u b s t r a t e f o r t h e enzyme b e c a u s e t h e
Em i
s very low (0.06
The
mM).
turnover r a t e f o r hydrolysis of nitrocatechol sulphate or related synthetic
substrates
i s
much
higher
than
the
rate
with
most
n a t u r a l l y o c c u r r i n g s u l p h a t e e s t e r s s u c h as c e r e b r o s i d e s u l p h a t e , s t e r o i d sulphates,
$-tyrosine
sulphate, or B-Q-glucopyranose
6-
sulphate.
t h e turnover r a t e with ascorbate 2-sulphate
i s
However,
comparable t o t h e r a t e s measured u s i n g most s y n t h e t i c s u b s t r a t e s . These r e s u l t s a r e d i s c u s s e d i n r e l a t i o n s h i p t o s e v e r a l p r e v i o u s l y d e s c r i b e d s u l p h a t a s e enzymes w h i c h were c l a i m e d t o have u n i q u e specificities. A r y l s u l p h a t a s e has been p u r i f i e d 2 1 9 - f o l d species."6
and i m m u n o l o g i c a l a n a l y s i s . weight
f r o m Pseudomonas
The f i n a l p r e p a r a t i o n was homogeneous by e l e c t r o p h o r e t i c
about
51,000,
The enzyme i s a monomer o f m o l e c u l a r
w i t h a Stokes r a d i u s of
f r i c t i o n a l r a t i o o f 1.2,
3.0
x
cm,
and a s e d i m e n t a t i o n c o e f f i c i e n t o f
a
4.1 S.
I t h a s been r e p o r t e d t h a t a r y l s u l p h a t a s e a c t i v i t y i s s t i m u l a t e d by
arylamines
produced.517
and
evidence
number
A
of
for
substrate
arylamines
tryptamine) increased the i n v i t r o a c t i v i t y Pseudomonas sp.
(2.9.
s t r a i n C12B.
I-tyrosine
and
a c t i v a t i o n has
(including of
been
tyramine
arylsulphatase
and from
Amino a c i d a n a l o g u e s o f t h e s e a m i n e s
I-tryptophan)
failed
t o exert
an e f f e c t .
S t i m u l a t i o n o f a c t i v i t y by t y r a m i n e c o u l d n o t be a c c o u n t e d f o r i n t e r m s o f s u l p h o t r a n s f e r a s e a c t i v i t y f o r t h i s p h e n o l , a n d no s h i f t i n the
pH o p t i m u m
tryptamine. enzyme
for
Increased
concentration
concentration.
the
enzyme
occurred
i n
lmax due t o t h e s e a m i n e s but
varied
the
presence
of
was i n d e p e n d e n t o f
significantly
with
substrate
Evidence i s presented which suggests t h a t arylamines
enhance a r y l s u l p h a t a s e a c t i v i t y by f o r m i n g a s a l t l i n k a g e w i t h t h e s u b s t r a t e and r e n d e r i n g i t more s u s c e p t i b l e t o e n z y m a t i c and a c i d catalysed hydrolyses.
The r e c r y s t a l l i z e d t r y p t a m i n e s a l t o f t h e
s u b s t r a t e e x h i b i t e d a reduced a f f i n i t y
for
the
h y d r o l y s e d more r a p i d l y t h a n t h e p o t a s s i u m s a l t ,
enzyme
but
was
which i s n o r m a l l y
e m p l o y e d as t h e a s s a y s u b s t r a t e . The s t i m u l a t i o n o f a r y l s u l p h a t a s e s y n t h e s i s i n P s e u d o m o n a s a e r u g i n o s a by exogenous n u c l e o t i d e s has been r e p o r t e d . 5 1 8
558
Carbohydrate Chemistry 51
2-Acetamido-2-deoxy-Q-glucose 6 - S u l p h a t e S u l p h a t e s
A d e f i c i e n c y of 2-acetamido-2-deoxy-g-glucose 6-sulphate r e q u i r e d f o r heparan s u l p h a t e degradation has been r e p o r t e d i n p a t i e n t s w i t h S a n f i l i p p o d i s e a s e t y p e D.519 Skin f i b r o b l a s t s from t w o p a t i e n t s who h a d s y m p t o m s o f t h e S a n f i l i p p o s y n d r o m e ( m u c o p o l y s a c c h a r i d o s i s 111) a c c u m u l a t e d e x c e s s i v e a m o u n t s o f h e p a r a n s u l p h a t e a n d were u n a b l e t o r e l e a s e s u l p h a t e f r o m 2 - a c e t a m i d o - 2 deoxy-g-glucose 6-sulphate l i n k a g e s i n heparan sulphate-derived oligosaccharides. Keratan sulphate-derived oligosaccharides bearing t h e same r e s i d u e a t t h e n o n - r e d u c i n g e n d a n d 4 - n i t r o p h e n y l 2 acetamido-2-deoxy-Q-glucopyranoside 6 - s u l p h a t e were d e g r a d e d normally. K i n e t i c d i f f e r e n c e s between the s u l p h a t a s e activities of T h e s e o b s e r v a t i o n s s u g g e s t t h a t 2n o r m a l f i b r o b l a s t s were f o u n d . acetamido-2-deoxy-a-glucose 6-sulphate a c t i v i t i e s degrading heparan s u l p h a t e and k e r a t a n s u l p h a t e , r e s p e c t i v e l y , can be d i s t i n g u i s h e d . It is the a c t i v i t y directed towards heparan s u l p h a t e t h a t is d e f i c i e n t i n these p a t i e n t s . The a u t h o r s propose t h a t t h i s d e f i c i e n c y c a u s e s S a n f i l i p p o d i s e a s e t y p e D.
52
Miscellaneous E n z y m e s
Dextransucrase. -- D e x t r a n s u c r a s e f r o m L e u c o n o s t o c m e s e n t e r o i d e s has been p r o d u c e d i n a s e m i c o n t i n u o u s c u l t u r e w i t h s l o w a d d i t i o n o f a concentrated sucrose solution.520 The r e s u l t i n g h i g h a c t i v i t y o f t h e f e r m e n t a t i o n b r o t h a l l o w e d a o n e - s t e p p u r i f i c a t i o n m e t h o d , by g e l - p e r m e a t i o n c h r o m a t o g r a p h y i n 96.4% y i e l d . This procedure r e s u l t e d i n 140-fold purification, with s p e c i f i c a c t i v i t y of 122 U mg-l. The e n z y m e was i m m o b i l i z e d o n t o a n a m i n o - S p h e r o s i l s u p p o r t activated with glutaraldehyde. Preparations with dextransucrase a c t i v i t i e s a s h i g h a s 4 0 . 5 U g - l o f s u p p o r t were o b t a i n e d w h e n l o w s p e c i f i c a r e a s u p p o r t s were u s e d a n d m a l t o s e was a d d e d d u r i n g t h e enzyme c o u p l i n g . D i f f u s i o n a l l i m i t a t i o n s were f o u n d d u r i n g e n z y m e r e a c t i o n , a s s h o w n by a k i n e t i c s t u d y . As a c o n s e q u e n c e o f immobilization, the average molecular weight of dextrans appeared t o increase. I m m o b i l i z e d d e x t r a n s u c r a s e c o u l d be p r o m i s i n g f o r l o w molecular-weight dextran production. C l i n i c a l d e x t r a n was s y n t h e s i z e d when t h e p o l y s a c c h a r i d e s p r o d u c e d i n t h e p r e s e n c e o f The m a l t o s e were u s e d a s a c c e p t o r s o f a s e c o n d s y n t h e s i s r e a c t i o n . m o l e c u l a r - w e i g h t d i s t r i b u t i o n of t h e r e s u l t i n g p r o d u c t i o n was less
559
6: Enzymes
d i s p e r s e d t h a n when c l i n i c a l d e x t r a n was p r o d u c e d by a c i d h y d r o l y s i s
-
-
o f h i g h mo l e c u l a r w e i g h t dex t r an. S t u d i e s have been c a r r i e d o u t on t h e a c c e p t o r s p e c i f i c i t y o f
d e x t r a n s u c r a s e
w h i c h
h a d
been
i s o l a t e d
f r o m
Streptococcus ~ a n g u i s . R ~ a~d ~ i o a c t i v e a c c e p t o r s were e m p l o y e d i n r e a c t i o n s w i t h c o l d s u c r o s e and t h e c o u n t s i n c o r p o r a t e d were t a k e n as a m e a s u r e o f a c c e p t o r a c t i v i t y .
An o r d e r o f r e l a t i v e a c t i v i t y
was f o u n d t o be p o l y s a c c h a r i d e > o l i g o s a c c h a r i d e > g l y c o s i d e > monosaccharide.
An e v a l u a t i o n o f t h e t i m e - c o u r s e o f t h e r e a c t i o n
w i t h methyl a-q-glucopyranoside,
or maltose,
homologous s e r i e s o f o l i g o s a c c h a r i d e s
was f o r m e d f r o m
showed t h a t each.
a
This
s u g g e s t e d t h a t t h e i n d i v i d u a l members o f t h e s e r i e s were r e l a t e d as precursors different
and
products.
The
kinetics
a c c e p t o r s were s t u d i e d .
of
the
reaction
with
A l l a c c e p t o r s s t u d i e d caused an
a c t i v a t i o n o f t h e enzyme and changes i n t h e
5,
f o r sucrose.
The
k i n e t i c c o n s t a n t s o b t a i n e d were a l s o used t o compare t h e v a r i o u s acceptors.
I t has been d e m o n s t r a t e d t h a t a - g - 1 - f l u o r o g l u c o s e
i s a glucosyl
donor f o r d e x t r a n s u c r a s e i s o l a t e d f r o m S t r e p t o c o c c u s ~ a n q = . ~ ~ * The d e t a i l s o f t h e r e a c t i o n have been e s t a b l i s h e d as w e l l as p r o v i n g t h a t t h e mechanism o f 1 - f l u o r o g l u c o s e t r a n s f e r i s c o m p a r a b l e t o t h a t of p - g l u c o s y l t r a n s f e r f r o m s u c r o s e . the reaction i s reported,
A new p r o c e d u r e f o r m o n i t o r i n g
and i s based on t h e measurement o f p r o t o n
f o r m a t i o n u s i n g t h e pH i n d i c a t o r 6 r o m c r e s o l P u r p l e . F-
Production o f
was f o u n d t o be s t o i c h i o m e t r i c w i t h p r o t o n p r o d u c t i o n .
studies
with
the
substrate
indicate
undergoes spontaneous h y d r o l y s i s , presence of
When { 1 4 C ) m a l t o s e a n d a - q - 1 -
f l u o r o g l u c o s e o r a-l-Q-{ 1 4 C ) f l u o r o g l u c o s e observed.
a series
Rate
a-g-1-fluoroglucose
w h i c h is g r e a t l y i n c r e a s e d i n t h e
nucleophilic buffers.
w i t h dextransucrase,
that
of
and m a l t o s e were i n c u b a t e d
oligosaccharide
products
was
The r e s u l t s i n d i c a t e t h a t t h e c - g l u c o s y l m o i e t y o f a-e-1-
f l u o r o g l u c o s e t r a n s f e r r e d t o t h e acceptor.
The n a t u r e o f f o r m a t i o n
of t h e products i s c o n s i s t e n t w i t h a s e r i e s o f precursor-product reactions.
P r o d u c t a n a l y s i s of
the
r e d u c t i o n a n a l y s i s demonstrated t h a t t o t h e non-reducing
end o f
maltose.
saccharides the g-glucosyl
When e i t h e r
by
borohydride
u n i t was added
1-{1 4 C ) f r u c t o s e or
a - Q - l - { 1 4 C ~ f l u o r o g l u c o s e was i n c u b a t e d w i t h enzyme, a r e a c t i o n was o b s e r v e d w h i c h was a n a l o g o u s t o t h e i s o t o p i c - e x c h a n g e r e a c t i o n c a t a l y s e d by t h e enzyme i n t h e p r e s e n c e o f
g-{l4C}fructose
and
sucrose. S t u d i e s on d o n o r - s u b s t r a t e
s p e c i f i c i t y have been p e r f o r m e d w i t h
Carbohydrate Chemistry
560 dextrans~crase.~~P ' revious studies
-Proc.
S O C . Exp.
linked
B i o l . Med., 1 9 7 2 , f l u o r i n e atom a t C - 1 o f
activation to permit this dextransucrase.
s e r v e d as
competitive
A comparison o f
importance of
specific
several
for
a t the donorhas been
of
sucrose,
the
natural
p r o v i d e d i n f o r m a t i o n about t h e
changes i n t h e !-glucose
moiety
w i t h regard
S i m i l a r k i n e t i c s t u d i e s were c a r r i e d out and
the
corresponding free
These w e r e f o u n d t o be n o n - c o m p e t i t i v e i n h i b i t o r s ,
and t o b i n d p o o r l y . substrates
exception of
inhibitors t h e sis
l-fluoro-B-Q-sugars
monosaccharides. donor
substrate
a s e r i e s o f l - f l u o r o - a - Q -- s u g a r s
t o b i n d i n g t o t h e enzyme. with
a n a l o g u e t o be a d o n o r
I n k i n e t i c e x p e r i m e n t s , i t has been d e t e r m i n e d t h a t
synthesized. substrate.
glucose
Hehre,
h a v e s h o w n t h a t a n aprovided sufficient
I n order t o study the s p e c i f i c i t y
substrate-binding site, they
G e n g h o f f and E.J.
(D.S.
l40,1 2 9 8 )
The l - f l u o r o - a - ! - s u g a r s i n reactions
w e r e a l s o e x a m i n e d as
w i t h known
l-fluoro-a-!-glucose,
acceptors.
none o f
With the
t h e s e a n a l o g u e s was
active i n t h i s capacity. Several carbohydrates, designed, synthesized,and
i n c l u d i n g sucrose
analogues,
h a v e been
t e s t e d as d e x t r a n s u c r a s e i n h i b i t o r s . 5 2 4
C e r t a i n c a r b o h y d r a t e s c o n t a i n i n g an a m i n o g r o u p w e r e s hown t o be p o t e n t i n h i b i t o r s o f dextransucrase. M u l t i p l e f o r m s o f L e u c o n o s t o c m e s e n t e r o i d e s d e x t r a n s u c r a s e have b e e n s e p a r a t e d by g e l f i l t r a t i o n a n d e l e c t r o p h o r e t i c a n a l y s e s . 5 2 5 Two c o m p o n e n t s o f e n z y m e , h a v i n g d i f f e r e n t a f f i n i t i e s f o r d e x t r a n gel,
were
s e p a r a t e d by a c o l u m n o f
component
was
treated
with
Sephadex
dextranase
e l e c t r o p h o r e t i c a l l y homogeneous s t a t e . m o l e c u l a r w e i g h t o f 64,000-65,000, 17% o f c a r b o h y d r a t e .
The
G-100.
and
The
purified
major to
an
p u r i f i e d enzyme had a
PI v a l u e o f 4.1,
and c o n t a i n e d
H 4 - e d t a showed a c h a r a c t e r i s t i c i n h i b i t i o n on
t h e enzyme w h i l e s t i m u l a t i v e e f f e c t s w e r e o b s e r v e d by t h e a d d i t i o n o f exogenous d e x t r a n t o t h e i n c u b a t i o n m i x t u r e . was s t i m u l a t e d b y v a r i o u s d e x t r a n s a n d i t s
w i t h i n c r e a s i n g c o n c e n t r a t i o n of showed no a f f i n i t y
for
a Sephadex
dextran. G-100
The enzyme a c t i v i t y
Km v a l u e
was d e c r e a s e d
The p u r i f i e d enzyme
g e l and r e a d i l y a g g r e g a t e d
a f t e r p r e s e r v a t i o n a t 4OC i n a c o n c e n t r a t e d s o l u t i o n .
S y n t hases.
--
C o n c e n t r a t i o n s o f ADP-Q-glucose:a-1,4-Q-glucan-4-
g l u c o s y l t r ans f e r a s e ( s t a r c h s y n t h a s e
glucan-6-glycosyltransferase seeds
of
Pisum sativum
have
a n d a - 1 ,4-O_- - g l u c a n : a - l , 4 - Q --
( b r a n c h i n g enzyme) f r o m d e v e l o p i n g been
measured.526
Primed
starch
s y n t h a s e a c t i v i t y i n c r e a s e d f r o m 0 t o 1 4 d a y s a f t e r a n t h e s i s and
561
6: Enzymes d e c r e a s e d by 50% a t 26 days.
C i t r a t e - s t i m u l a t e d s t a r c h synthase
a c t i v i t y was h i g h e s t a t 10 d a y s a f t e r a n t h e s i s , d e c r e a s i n g t o l o w l e v e l s by 22 days. B r a n c h i n g - e n z y m e a c t i v i t y i n c r e a s e d f r o m 8 t o 18 d a y s a f t e r a n t h e s i s and d e c r e a s e d l i t t l e by 26 days.
Two f r a c t i o n s
o f s t a r c h s y n t h a s e w e r e r e c o v e r e d b y g r a d i e n t e l u t i o n f r o m DEAEcellulose of
extracts
fractions differed
from
12- and 1 8 - d a y - o l d
Em f o r
i n primer specificity,
r e l a t i v e amount o f c i t r a t e - s t i m u l a t e d a c t i v i t y .
seeds.
The
two
ADP-Q-glucose, a n d
A m a j o r and
a
minor
f r a c t i o n o f b r a n c h i n g enzyme w e r e o b s e r v e d i n e x t r a c t s f r o m b o t h 1 2 and 1 8 - d a y - o l d
seeds.
Marked d i f f e r e n c e s i n t h e r e l a t i v e a b i l i t i e s
of t h e two branching-enzyme f r a c t i o n s t o s t i m u l a t e phosphorylase and t o b r a n c h a m y l o s e as w e l l as i n pH o p t i m a w e r e f o u n d .
Although
t h e c o n t e n t o f t h e s t a r c h s y n t h a s e and branching-enzyme f r a c t i o n s v a r i e d w i t h s e e d age,
l i t t l e d i f f e r e n c e was s e e n i n t h e p r o p e r t i e s
o f chromatographically s i m i l a r fractions. s t a r c h synthase
and b r a n c h i n g - e n z y m e
T h e r e f o r e , t h e changes i n activity
d u r i n g pea
seed
development r e s u l t e d f r o m changes i n t h e c o n c e n t r a t i o n s o f a few enzyme f o r m s b u t n o t t h e a p p e a r a n c e of d i f f e r e n t enzyme f o r m s . The h o r m o n a l r e g u l a t i o n o f g l y c o g e n s y n t h a s e p h o s p h o r y l a t i o n i n p e r f u s e d r a t s k e l e t a l m u s c l e h a s b e e n i n v e ~ t i g a t e d . ~ ~U’ s i n g t h e r a t hind-limb p e r f u s i o n technique, k i n e t i c evidence t h a t glycogen synthase i s s u b s t a n t i a l l y
phosphorylated i n c o n t r o l s k e l e t a l muscle
a n d t h a t e p i n e p h r i n e c a u s e s f u r t h e r p h o s p h o r y l a t i o n was o b t a i n e d . The p h o s p h a t e c o n t e n t o f
t h e enzyme p u r i f i e d f r o m
control
and
h o r m o n e - t r e a t e d m u s c l e has been measured and a good c o r r e l a t i o n b e t w e e n t h e p h o s p h o r y l a t i o n s t a t e o f t h e enzyme and a c t i v i t y has been found. The a m i n o a c i d s e q u e n c e o f a r e g i o n i n r a b b i t s k e l e t a l m u s c l e g l y c o g e n s y n t h a s e p h o s p h o r y l a t e d by c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e h a s been r e p o r t e d . 5 2 8
Analogues of
t h e amino a c i d sequence
s u r r o u n d i n g s i t e l a d e m o n s t r a t e d t h a t s i t e s l a and l b a r e s e p a r a t e d by o n l y 1 3 a m i n o a c i d s i n t h e p r i m a r y s t r u c t u r e o f g l y c o g e n s y n t h a s e , w h i c h c o m p r i s e s 770 r e s i d u e s . The i n h i b i t o r y e f f e c t s
*
31-B-q-glucan
gated.529
o f p a p u l a c a n d i n B and a c u l e a c i n A o n (1
synthase from
G e o t r i c h u m l a c t i s h a v e been i n v e s t i -
The i n h i b i t i o n i s s p e c i f i c
and o c c u r s n o t o n l y i n v i t r o
but also i n vivo since the addition o f the antibiotics t o a culture leads t o c e l l - f r e e
e x t r a c t s with a p a r t i a l l y i n a c t i v e synthase.
Some c h a r a c t e r i s t i c s o f t h e i n h i b i t i o n a r e d e s c r i b e d . C h i t i n synthase i n t h e s t i p e of
--b i s e ---orus
has
been
isolated
and
the basidiomycete Agaricus i t s
properties
have
been
Carbohydrate Chemistry
562 i n v e s t i g a t e d .530
--
Lyases. has
been
Heparan s u l p h a t e p u r i f i e d
by
l y a s e from
repeated
Flavobacterium heparinum
hydroxyapatite
column
c h r o m a t o g r a ~ h y . ~The ~ ~ enzyme was u s e d t o d e g r a d e h e p a r a n s u l p h a t e o c c u r r i n g on t h e s u r f a c e s o f
a s c i t e s hepatoma c e l l s ,
AH66,
and was
f o u n d t o be more e f f e c t i v e t h a n t r y p s i n i n r e m o v i n g h e p a r a n s u l p h a t e from
the
cells.
Furthermore,
on a n a l y s i n g
g l y c o s a m i n o g l y c a n s and
g l y c o p e p t i d e s from t h e enzyme-treated c e l l s and c o n t r o l c e l l s , i t was c o n c l u d e d t h a t h e p a r a n s u l p h a t e was e x c l u s i v e l y p r e s e n t on t h e c e l l s u r f a c e and a c c e s s i b l e t o t h e h e p a r a n s u l p h a t e l y a s e whereas o t h e r c e l l - s u r f ace c o m p l e x c a r b o h y d r a t e s r e m a i n e d i n t a c t . The c h a n g e s i n t h e c a t a l y t i c a c t i v i t y o f N - a c e t y l n e u r a m i n a t e l y a s e h a v e been s t u d i e d as a f u n c t i o n o f t e m p e r a t u r e i n t h e p r e s e n c e
o r absence of
i t s substrates.532
The
l y a s e was f o u n d t o
was s u g g e s t e d t h a t t h i s p r o t e c t i v e e f f e c t
be
and i t
e f f e c t i v e l y p r o t e c t e d a g a i n s t h e a t i n a c t i v a t i o n by p y r u v a t e ,
c a n b e u s e d i n a new
s i m p l e s t e p i n t h e p u r i f i c a t i o n o f t h e enzyme. A m u l t i f u n c t i o n a l enzyme, a c t i v e o n b o t h p o l y s a c c h a r i d e s and
glycosides, P.
has
placenta.533
A
been
isolated
from
molecular weight
the
wood-decay
o f 185,000
fungus
was d e t e r m i n e d w i t h
l o w e r - m o l e c u 1a r - w e i g h t s u b u n i t s c h a r a c t e r ized on SDS po 1y a c r y l a m ide g e l electrophoresis.
An u n u s u a l PI o f 1.8
was e s t a b l i s h e d f o r t h i s
protein.
--
Dehydrogenases.
The g e n e t i c and b i o c h e m i c a l c h a r a c t e r i z a t i o n o f
Q-arabinose
dehydrogenase
reported.534
The enzyme was p u r i f i e d t o h o m o g e n e i t y f r o m w i l d - t y p e
N.
c r a s s a 74-A
from
Neurozeora crassa
and f r o m t w o c o l o n i a l m u t a n t s ,
o f t h e enzyme.
has
been
f o u n d t o c o n t a i n more
The e n z y m e s w e r e c h a r a c t e r i z e d b y m e a s u r e m e n t o f
s e v e r a l k i n e t i c and p h y s i c o c h e m i c a l p a r a m e t e r s and w e r e t h e same i n a l l characteristics
studied.
Immunological studies performed w i t h
enzyme p r e p a r a t i o n s f r o m t h e t h r e e s t r a i n s showed a n t i g e n i c i d e n t i t y and
indicated that
enzyme,
those
colonial
strains
c o n t a i n more n o r m a l
r a t h e r t h a n t h e u s u a l amount o f an a l t e r e d ,
i m p r o v e d enzyme.
Q u a n t i t a t i o n o f t h e enzyme i n c r u d e e x t r a c t s , p e r f o r m e d by s i n g l e r a d i a l immunmodiffusion, the
l e v e l
o f
enzyme
characterization, heterokaryosis,
showed t h a t t h e c o l o n i a l s t r a i n s h a v e t w i c e as
the
performed
and r e v e r s i o n s ,
by
wild-type analysis
of
strain. meiotic
Genetic products,
indicated that the difference i n
p-
a r a b i n o s e d e h y d r o g e n a s e a c t i v i t y d e t e c t e d among t h e t h r e e s t r a i n s i s
563
6: Enzymes p r o b a b l y d e t e r m i n e d by one gene.
D - F r u c t o s e dehydrogenase o f G l u c o n o b a c t e r i n d u s t r i u s has been i s o l a t e d and c h a r a c t e r i z e d f o r t h e e n z y m a t i c m i c r o d e t e r m i n a t i o n o f Q-Fructose
dehydrogenase
p u r i f i e d f r o m t h e membrane f r a c t i o n o f
was
solubilized
g l y c e r o l - g r o w n G.
and
industrius
by s o l u b i l i z a t i o n o f t h e e n z y m e w i t h T r i t o n X - 1 0 0 a n d s u b s e q u e n t f r a c t i o n a t i o n by i o n - e x c h a n g e c h r o m a t o g r a p h i e s . was
tightly
bound
to
a c-type
The p u r i f i e d enzyme
cytochrome
e x i s t i n g as a d e h y d r o g e n a s e - c y t o c h r o m e
and a n o t h e r
complex.
peptide
The p u r i f i e d enzyme
was deemed p u r e by a n a l y t i c a l u l t r a c e n t r i f u g a t i o n as w e l l as by g e l f i l t r a t i o n on a Sephadex G-200 column. 140,000)
on
sodium
electrophoresis
The enzyme complex ( m o l .
sulphate
showed t h e p r e s e n c e o f
molecular weights and 19,700
dodecyl
of
67,000
50,800
Only p - f r u c t o s e
dichlorophenolindophenol, or phazine methosulphate. dinucleotide,
n i c o t i n a m i d e adenine
and oxygen d i d n o t f u n c t i o n of
!-fructose
fructose)
was s t a b l e a t pH 4.5
The o p t i m u m pH
The e n z y m e ( K m lO’*rnM
t o 6.0.
2,6-
Nicotinamide
d i n u c l e o t i d e phosphate,
as e l e c t r o n a c c e p t o r s .
o x i d a t i o n was 4.0.
c),
(cytochrome
was r e a d i l y o x i d i z e d
b y t h e enzyme i n t h e p r e s e n c e o f d y e s s u c h as f e r r i c y a n i d e , adenine
gel
t h r e e components h a v i n g
(dehydrogenase),
(unknown f u n c t i o n ) .
wt.
polyacrylamide
for
p-
S t a b i l i t y of the p u r i f i e d
enzyme was much enhanced by t h e p r e s e n c e o f d e t e r g e n t i n t h e enzyme Removal o f d e t e r g e n t f r o m t h e enzyme s o l u t i o n f a c i l i t a t e d
solution.
t h e a g g r e g a t i o n o f t h e enzyme and caused i t s i n a c t i v a t i o n .
Levansucrase.
--
Levansucrase,
t h e p o l y s a c c h a r i d e l e v a n from
which
catalyses
the g-fructosyl
the
synthesis
of
r e s i d u e s o f sucrose,
was i s o l a t e d f r o m t h e c u l t u r e f l u i d o f G l u c o n o b a c t e r o x y d E a n d p u r i f i e d t o a homogeneous s t a t e by c h r o m a t o g r a p h y on h y d r o x y a p a t i t e and g e l f i l t r a t i o n . 5 3 6
According t o t h e data of electrophoresis,
t h e m o l e c u l a r weight of
l e v a n s u c r a s e i s e q u a l t o 58,000.
m o l e c u l e c o n t a i n s 25.86% a c i d i c a m i n o a c i d s , acids, per
and 12.77% a r o m a t i c amino a c i d s ,
mole.
The u l t r a v i o l e t
The enzyme
13.74% b a s i c a m i n o
and one m o l e o f m e t h i o n i n e
absorption spectrum
of
purified
l e v a n s u c r a s e h a s a maximum a t 280 nm a n d a m i n i m u m a t 254 nm.
By
measuring
by
the
l e v a n s u c r a s e of
i n i t i a l G.
rates
of
the
reactions
catalysed
oxydans i n t h e p r e s e n c e o f { 1 4 C } s u c r o s e ,
i t was
e s t a b l i s h e d t h a t t h i s enzyme c a t a l y s e s a r e a c t i o n o f h y d r o l y s i s o f sucrose,
a
reaction
of
synthesis
of
levan,
and
a reaction
of
exchange o f t h e l a b e l l e d Q - g l u c o s e h a l f o f s u c r o s e w i t h f r e e Qglucose. The i n i t i a l r a t e s o f l i b e r a t i o n o f E - g l u c o s e a n d p -
5 64
Carbohydrate Chemistry
f r u c t o s e d u r i n g t h e t r a n s f r u c t o s y l a t i o n r e a c t i o n were d e t e r m i n e d . levan t o the r e a c t i o n m i x t u r e a s a p r e c u r s o r of p o l y s a c c h a r i d e s y n t h e s i s l e a d s t o a d o u b l i n g of t h e i n i t i a l r a t e of l i b e r a t i o n of l a b e l l e d p_-fructose and an i n c r e a s e i n t h e y i e l d o f l e v a n from 41 t o 69%. I t was shown t h a t t h e a d d i t i o n of low-molecular-weight
P e c t i n e s t e r a s e s . -- T e c h n i c a l p e c t i c m u l t i e n z y m e p r e p a r a t i o n s , P e c t i n e x U l t r a and Rohament P , were chromatographed on an a n a l y t i c a l s c a l e u s i n g m e d i u m - p r e s s u r e l i q u i d c h r o m a t o g r a p h y on a g l y c o l m e t h a c r y l a t e r i g i d m a c r o r e t i c u l a r g e l , S p h e r o n 1 0 0 0 , and i t s i o n e x c h a n g e d e r i v a t i v e s . 4 4 7 A c o m b i n a t i o n o f i s o c r a t i c and l i n e a r g r a d i e n t e l u t i o n ( w i t h g r a d i e n t s i n i o n i c s t r e n g t h o r p H ) was employed and f r a c t i o n s were monitored by measurements o f absorbance Conditions (4285 and A2541, c o n d u c t i v i t y , pH, and enzyme a c t i v i t y . f o r r a p i d s e p a r a t i o n s of p e c t i c enzymes a r e e l a b o r a t e d . The r e s u l t s i n d i c a t e t h e p o s s i b i l i t i e s of s e p a r a t i n g t h e t e c h n i c a l l y u n d e s i r a b l e p e c t i n e s t e r a s e a c t i v i t y from t h e o t h e r enzyme a c t i v i t i e s , and o f a more d e t a i l e d b i o c h e m i c a l i n v e s t i g a t i o n of t h e s e enzymes, i m p o r t a n t f o r t h e food i n d u s t r y . The aerobic b a c t e r i a associated w i t h s o f t r o t i n onions ( A l l i u m c e p a ) w e r e i s o l a t e d and i d e n t i f i e d a s a V i b r i o s p e c i e s , M i c r o c o c c u s e p i d e r m i d i s , Eudomonas c e p a c i a , an A c i n e t o b a c t e r s p e c i e s , Xanthomonas s p e c i e s , B a c i l l u s polyrnyxx, and B a c i l l u s m e g a t e r i ~ m . ~W~i t~h t h e c u p - p l a t e assay method, no p e c t i n h y d r o l a s e could b e d e t e c t e d from any of t h e s e i s o l a t e s when they were c u l t u r e d i n p e c t i n medium, b u t l y a s e and p e c t i n e s t e r a s e s w e r e d e t e c t a b l e . Onion t i s s u e c u l t u r e s showed p e c t i n h y d r o l a s e a c t i v i t y f o r P . c e p a c i a and B. polymyxa and l y a s e and p e c t i n e s t e r a s e a c t i v i t i e s f o r a l l of t h e i s o l a t e s , u s u a l l y a t h i g h e r l e v e l s o f a c t i v i t y than I n both c u l t u r e t h o s e of t h e p e c t i n medium c u l t u r e f i l t r a t e s . m e d i a , V i b r i o s p e c i e s showed t h e h i g h e s t l y a s e and p e c t i n e s t e r a s e activities. I n t h e v i s c o m e t r i c t e s t , a l l of t h e i s o l a t e s achieved a t l e a s t a 50% d e c r e a s e i n v i s c o s i t y f o r l y a s e e n z y m e , w i t h M . e p i d e r m i d i s and V i b r i o s p e c i e s r e c o r d i n g v i s c o s i t y d e c r e a s e s a s h i g h a s 8 3 % . The a b i l i t y t o c a u s e s o f t r o t i n o n i o n b u l b s was d e m o n s t r a t e d b y P. c e p a c i a and Xanthomonas s p e c i e s . Benzoic a c i d a t a c o n c e n t r a t i o n o f 0.8 mg m 1 - l c a u s e d t o t a l s u p p r e s s i o n of enzyme p r o d u c t i o n whereas sodium b e n z o a t e a t t h i s c o n c e n t r a t i o n reduced p e c t i n e s t e r a s e p r o d u c t i o n b y 71% and l y a s e p r o d u c t i o n by 72%. The p o s s i b l e use of t h e s e p r e s e r v a t i v e s i n t h e c o n t r o l o f s o f t r o t i n o n i o n s i s noted.
6: Enzymes
565
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575
6: Enzymes 430 431 432 433 434 435 436 437 438 4 39 440 441 442 443 444 44 5 446 44 7 44 8 449 450 4 51 452 453 454 455 4 56 457 458 459 460 461 462 463 464 465 466 467 468 469 4 70 471 472 473 474 4 75
J. Biochen. ("b 0 1 , 1981, 89, 963. J.Perraudin, Y.?mze, J . V i G e n t e l l i , and J.Leonis, Comp. Biochem. Physiol., P t B, 1980, 65, 127. K.Bel1, H.A.McKensie, V.Muller, and D.C.Shaw, M o l . C e l l . Biochem., 1980, 29, 3. V. Sidhan and S. Gurnani, Agric. B i o l . Chgn., 1981, 45, 1817. B.W. Matthews, S.J. Remington, M.G. G r i i t t e r and W.F. Anderson, J. M o l . B i o l . , 1981, 147, 545. m u s h i m a , Y. Murata, N. Nishikido, G. Sugihara and M.Tanaka, Bull. Chem. Sec. Jpn., 1981, 54, 3122. P.J. S t e i n and K. C r a i g Heehn, Biochem. Biophys. R e s . C o m E . , 1980, 95, 547. T. Imoto, T. Om and H. Yamada, J. Biochen. ("bkyo),1981, 90, 335. A. Shrake and J.A. Rupley, Biochemistry, 1980, 19, 4044. Y. Yang and K. Hamaguchi, J. Biochen. (Tokyo), 1980, 88, 829. Y. Yaw, S. Kurarrtitsu and K. Hamguchi, J. Biochem. (Tokyo), 1981, 89, 1357. A. Masaki, T. Fdcamizo, A. Otakara, T. T o r i k a t a , K. Hayashi and T. Imoto, J. Biochgn. ("bkyo), 1981, 90, 527. E.P. Savel'ev, G.I. P e t r o v , Z.F. Shmakova and S.A. Bitko, Biochemistry (Engl. Transl.), 1980, 5, 247. B. Szewczyk and T. Skorko, Biochim. Biophys. A c t a , 1981, 662, 131. G. Kleppe, E. Vasstrard and H.B. Jensen, Eur. J. Biochm., 1981, 119, 589. H. Sjijstrom, 0. Nor&, L. C h r i s t i a n s e n , H. Wacker and G. Semenza, J. B i o l . Chen., 1980, 255, 11332. M. Sat0 and A. Kaji, A ric. B i o l . Chem., 1980, 44, 1345. J. Gen. Microbiol. , 1980, 120, 295. G.S. Coleman, D.C., - aS D. Mikeg, J. S e d l s k o v a , L. Rexov&Benko-v& J. Chromatoqr. , 1981, 207, 99. Y. Itoh, K. Izaki and H. Takahashi, Agric. B i o l . Chem., 1980, 44, 1135. S.K.C. Obi and G.M. Umezurike, 1. Ehvir. Microbiol., 1981, 42, 585-589. Rex~vd-Benk~vdand M. .M -okd ~ Res. I 1981, 98, 115. G.A. Tucker, N.G. Robertson and D. G r i e r s o n , Eur. J. Biochem., 1980, 119. S.C. Lee and C.A. W e s t , Plant Physiol., 1981, 67,640. S.C. Lee and C.A. W e s t , Plant Physiol., 1981, 67, 633. P. Magro, P. Dilenna, P. Marciano and C. P a l l a v i c i n i , J. Gen. Microbiol., 1980, 120, 105. M. Sat0 and A. Kaji, Agric. B i o l . Chem., 1980, 44, 717. J. Yamada, Agric. B i o l . Chen., 1981, 45, 747. J. Y m d a , Carbohydr. Res., 1981, 90, 153. J. Yamada, Agric. B i o l . Chen., 1981, 45, 1269. A. H e r s m v i c s , A. Quaroni, B. Bugge and K. Kirsch, Biochem. J., 1981, 197,
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E. Bar-Guilloux, D. Robic and J.-E. Courtois, Biochimie, 1980, 62, 719. S. Rajendran and M. Muthu, Experi e n t i a , 1981, 37, 886. J. C o m t a t and J.P. J o s e l e a u , Carboh d r . Res., 1981, 95, 101. P. B i e l y , M. Vrganskdand 2. KrdtkqIyEur. J. Biochem., 1980, 112, 375. P. Biely, M. V&anovd and 2. K r d w , Eur. J. Biochem., 1981, 119,565. and M. V&anskd, Eur. J. Biochen., 1981, 559. P. Biely, 2. -tJ@ P.A.D. R i c k a r d and T.A. Laughlin, Biotechnol. Lett., 1980, 2, 363. D.B. Wankhede, K.R. Vijayalakshmi and M.R.R. R a o , Carbohydr. Res., 1981, 96, 249. H. Yoshioka, S. Chavanich, N. Nilubol and S. Hayashida, Agric. B i o l . Chem., 1981, 45, 579. D. m r a d , Biotechnol. Lett., 1981, 2, 345. P.A.D. Rickard and S.P. P e i r i s , Biotechnol. Lett., 1981, 3, 39. P.A.D. Rickard, M.I. Rajoka and J.A. Ide, Biotechnol. Lett., 1981, 2, 487. Z. K r d w and P. Biely, Eur. J. Biochem., 1980, 112, 367. K. Nakanishi and T. Yasui, Agric. B i o l . men., 1980, 44, 1885. 2729. K. Nakanishi and T. Yasui, Aqric. B i o l . Chem., 1980, A.V. Ananichev, I.V. Ulezlo, A.M. Egorov, A.M. Bezborodov, E.V. Berezin,
119,
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Carbohydrate Chemistry
576 476 477 478
Biochemistry, 1980, 45, 753. M.A. Van K e u l e n , K. V e l l e n g a a n d G.E.M. JOOSten, B i o t e c h n o l . Bioenq., 1 9 8 1 , 23, 1437. C.-S. Gong, L-F. Chen, M.C. F l i c k i n g e r , L.-C. C h i a n g a n d G.T. T s ~ o , A p p l . Envir. Microbiol., 1981, 41, 430. T. Kume, H. Watanabe, S. Aoki a n d T. S a t o , Agric. B i o l . Chem., 1 9 8 1 , 45,
1311.
T. Kume, H. Watanabe, M. T a k e h i s a a n d T. S a t o , Agric. B i o l . Chem., 1981, 45, 1351. 480 T. Kasumi, K. H a y a s h i , N. Tsumura a n d T. T a k a g i , Agric. B i o l . Chem., 1981, 45, 1097. 481 T. Kasumi, K. H a y a s h i , N. TsUmUra a n d T. T a k a g i , Agric. B i o l . Chem., 1981, 45, 1087. 482 T. Kasumi, K. Hayashi and N. T m u r a , Agric. B i o l . Qlem., 1981, 45, 619. 483 B. Larsen and H. Grasdalen, Carboh dr. Res., 1981, 92, 163. 484 K. I z u m o r i , S. S u g i m o t o a n d B. k A g r i c . B i o l . Chem., 1980, %(1), 223. 48 5 0. Valentovd, M. Marek, F. g v e c , J. gtamberg a n d 2. V c d r & k a , B i o t e c h n o l . Bioenq., 1981, 23, 2093. 486 W. Hartmeier, S t a r c h , 1981, 33, 97. 487 S. P i l l a i , Biochan. J., 1981, 193, 825. 1981, 23, 1037. 488 E. Sada, S. Katoh and M. T e r a s h h , Biotechnol. Bioen 489 Ya.A. A l e k s a n d r o v s k i i , L.V. B e z h i k i n a a n d Yu.V. Rozionov, B i o c h e m i s t r y , 1981, 46, 593. 49 0 V. L i n r k , P. Benei?, F. Hovorka a n d 0. H o l e z e k , B i o t e c h n o l . Bioenq., 1981, 23, 1467. 49 1 V. L i n e k , P. Beneg, J. S i n k u l e , 0. H o l e z e k and V. Malq, B i o t e c h n o l . Bioenq., 1980, 22, 2515. 49 2 B. Solomon, N. L o t a n a n d E. K a t c h a l s k i - K a t z i r , J. Chromatogr., 1 9 8 1 , 215, 121. 49 3 S. Hayashi and S. Nakamura, Biochim. Biophys. A&, 1981, 657, 40. 494 E. N a p c h i , M. Nishikimi and K. Yagi, J. Biochen. (Tbkyo), 1981, 90, 33. 49 5 R. J e n n e s s , E.C. B i r n e y a n d K.L. Ayaz, Comp. Biochem. P h y s i o l . , 1980, 67, 479
c
.,
49 6 497 49 8 499 500 501 50 2 503 504 505 506 50 7 508 5 09 510
511 512 513 514 515
195.
R.F.H.Dekker, J. e n . Microbiol., 1980, 120, 309. L. Tom and D. Gaudioso, Can. J. Biochan., 1980, 58, 667. J.E. Sadler, T.A. Beyer and R.L. H i l l , J. Chrmtogr., 1981, 215, 181. E.H. Beachey, W. Keck, M.A. D e P e d r o a n d U. S c h w a r z , Eur. J. Biochem., 1981, 116, 355. T. Koga and M. I m u e , C a r b h y d r . Res., 1981, 93, 125. K. Fukushima, R. Motoda, K. Takada a n d T. I k e d a , FEBS L e t t . , 1981, 128, 213. HH. Carnicero, A.M. Adaamany and S. Ehglard, Arch. Biochem. Biophys., 1981, 210, 678. E.T. O'Keeffe, R.L. H i l l and J.E. Bell, Biochemistry, 1980, 19, 4954. Y. F'ujita-Yamaguchi and A. Yoshida, J. Biol. Chan., 1981, 256, 2701. E.T. O'Keeffe, T. Mordick and J.E. Bell, Biochemistry, 1980, 19, 4962. P. B r q u e t and P. Louisot, Biochimie, 1981, 63, 803. A, M a r t i n , M.C. B i o l , E. A r r a m b i d e , M. R i c h a r d a n d P. L o u i s o t , B i o c h i m i e , 1981, 63, 241. C. C-Mulet, J. Badet, J.P. Cartron, FEBS. L e t t . , 1981, 126, 123. D.H. van d e n E i j n d e n , M.L.E. Bergh, B. D i e l e m a n a n d W.E.C.M. S c h i p h o r s t , Hoppe-Seyler's 2, Physiol. Chan., 1981, 362, 113. G. Dodin, FEBS. L e t t . , 1981, 134, 20. I.M. A r c h e r , P.S. Harper a n d F.S. Wusteman, C l i n . Chim. Acta, 1981, 107. I.G. Leder, Biochan. Biophys. Res. (3m'mun., 1980, 94, 1183. P. D i Natale and L. R o n s i s v a l l e , Biochim. Biophys. A c t a , 1981, 661, 106. T. I s h i b a s h i , A. M a r u , Y. I m a i , A. M a k i t a a n d I. T s u j i , Biochim. Biophys. A c t a , 1980, 616, 218. A. Waheed and R.L. van E t t e n , Biochim. Biophys. A&, 1980, 2,92.
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6: Enzymes
577
516 J.R. George and J.W. F i t z g e r a l d , J. B a c t e r i o l . , 1981, 145, 1428. 517 J . R . George and J.W. F i t z g e r a l d , J. Bacteriol., 1981, 147,69. 518 J.W. F i t z g e r a l d , RB. Kellogg and G.J. S t e w a r t , FEMS Microbiol. Lett., 1981, 11, 93. F i g u r a , W. G i l b e r g a n d W. F u c h s , 519 H. Kresse, E. P a s c h k e , K.V. Proc. Natl. Acad. S c i . USA - B i o l . Sci., 1980, 77, 6822. 520 P. Monsan and A. Lopez, Biotechnol. Bioenq., 1981, 23, 2027. 5 21 M.M. M a t t h e w s , C.L. F u t e r m a n , V.K. P a r n a i k a n d S.M. J u n g , Arch. Biochem. Biophys., 1981, 208, 278. 522 S.M. Jung and R.M. Mzyer, Arch. Biochem. Biophys., 1981, 208, 288. 523 T.J. Greir and R.M. Mayer, Arch. Biochen. Biophys., 1981, 212, 651. Doyle, 524 S. T h a n i y a v a r a n , S. S i n g h , C.M. Maynard, K.G. T a y l o r a n d R.J. Carbohydr. R e s . , 1981, 96, 134. s. A c t a , 1980, 614, 46. 52 5 M. Kobayashi and K. Matsuda, Biochim. B i o 526 G.L. Matters and C.D. & y e , P h y t o c h a n i s g 1980, 20, 1805: 52 7 J.-L. C h i a s s o n , J.H. Aylward, H. Shikama a n d J.H. E x t o n , FEBS. L e t t . , 1 9 8 1 , 127, 97. 528 P.J. P a r k e r , A. A i t k e n , T. Bilham, N. Embi a n d P. Cchen., FEBS. L e t t . , 1981, 123, 332. 529 P. P a e z , R. VXOM, I. G a r c i a - M a , A. W a n , FEBS. L e t t , 1981, 129, 249. 530 G.D. Craig, D.A. W o o d and K. G u l l , FEMS Microbiol. Lett., 1981, 10, 43. 531 Y. Ohkubo, I. F u n a k o s h i and I. Yamashina, J. Biochem. (Tokyo), 1981, 89, 161. 532 F.N. Kolisis, T.G. S o t i r o u d i s a n d R E . E v a n g e l o p o u l o s , FEBS. L e t t . , 1980, 121. 533 K.E. W o l ter , T.L. H i g h l y a n d F.J. Evans, Biochem. Biophys. R e s . Commun., 1980, 97, 1499. 534 A. Carrasw, G. P h c h e i r a and T. Ureta, J. B a c t e r i o l . , 1981, 145, 164. 535 M. Ameyama, E. S h i n a g a w a , K. M a t s u s h i t a a n d 0. A d a c h i , J. Bacteriol., 1981, 145, 814. 536 fi.E l i s a s h v i l i , Biochemistry (Engl. Transl. 1, 1980, 45, 14.
7
Glycolipids and Gangliosides BY I. M. MORRISON 1 Introduction
Various aspects of glycolipids are reviewedin the second of a twovolume book on cell membranes and viral envelopes.’ The structure and function of plant lipids occupy Volume 4 of the comprehensive treatise on the biochemistry of plants.2 A review on the role of lipids in biological membranes has appeared .3 2 Analytical and General Methods 1.r.
photoacoustic spectroscopy has been used to study skin Using CHC13-MeOH (2: 1 ) extracts, the results were similar to those obtained by i.r. transmission spectroscopy,but only l o - * times the amount of material was required. The phenol-sulphuric acid method for the estimation of sugars in lipids has been modified by keeping the products at 100°C for 5 More reproducible results were obtained. A dry-column method for the quantitative extraction and simultaneous separation of lipid classes has been developed.6 The tissue sample, anhydrous sodium sulphate,and diatomaceous earth are ground together and packed in a column. Total lipid or lipid classes are then eluted by isocratic or sequential elution, respectively. A two-stage, one-dimensional t.1.c. method has been developed for the separation of lipid classes.’ Lecithin was found to influence the chromatography of steroid glucosiduronates .8 It was concluded that hydrogen bonds were formed between the phosphodiester group of lecithin and the hydroxy groups of the steroid conjugates. D-Galactose oxidase is able to oxidize the hydroxymethyl group of lipids containing 9-galactose or 2-acetamido-2-deoxy-Dg a l a c t o ~ e .These ~ sugar residues can then be labelled by reduction with radiolabelled borohydride. Conditions are described for the most efficient production of product. Methylation techniques for the structural analysis of
lipid^.^
7: Glycolipids and Gangliosides
579
glycolipids have been reviewed. It has been shown that particles, previously observed in freeze-fracture replicas of mixed phospholipid dispersions, occur in dispersions of mixed ~ - 3 - ~ - g a l a c t o p y r a n o s y l d i a c y l - g l y c e r o l s l. 1 These particles correspond to lipid micelles sandwiched within a membrane bilayer.
3 Gangliosides The gangliosides GM1 and GD have been specifically oxidized at la the +position of the sphingosine moiety with 2,3-dichloroo f Triton X-1 00.l 2 5,6-dicyanobenzoquinone in the presence Subsequent reduction with borohydride restored the ganglioside to its original form, and if a radiolabel was used 99% of the radioactivity was incorporated. Proof of the reaction was obtained by reoxidation when the label was removed. The interaction of gangliosides with Ca2+ ions and some polar head-group requirements for the establishment of particular interactions with phosphatidylcholine have been studied in monolayers at the air-0.145M NaCl interface. l 3 The gangliosideCa2+ interaction depended on the position occupied by the neuraminosyl residues in the oligosaccharide chain. Favoured interactions o f polyneuraminosylgangliosides with phosphatidylcholine may result from a configuration which allows a partial matching of two oppositely orientated electrical vectors contributed by the zwitterionic phosphocholine group and particular neuraminosyl groups. The gangliosides G D ~and GT were biosynthesized as secondary la products from lactosylceramide and GM1,respectively.l 4 By a series of reactions involving periodate oxidation, borohydride reduction, and hydrolysis, the linkage between the two neuraminic acid residues was identified as (2+8). 'H n.m.r. spectroscopy has demonstrated that N-acetyl-aacid is the first product released by the D-neuraminic neuraminidases of both Clostridium perfringens and Arthrobacter ureafaciens when hydrolysing neuraminic acid-containing carbohydrate chains. l 5 Spectrofluorimetric techniques have been used to study various The studies ganglioside and ganglioside-lecithin dispersions. l 6 indicate that the ultimate and/or penultimate carbohydrate moieties of the neutral sugar backbone of gangliosides and the
580
Carbohydrate Chemistry
topographical difference in the locations of the neuraminic acid linkage influence the integrity of the membranes including the hydrophobic region. The micellar properties of gangliosides in aqueous solutions have been investigated by quasi-elastic light - scattering The scattered intensity data allowed the measurements. l 7 derivation of an upper limit to the critical micelle concentration and the evaluation of the molecular weight of the micelle from GMl and GDla. Aqueous dispersions of G M 1 and Triton X-100, in different proportions, have been analysed for some physicochemical properties and their susceptibility to ;-galactose oxidase. l8 Differences in the molar ratio gave well defined transitions between different micellar species as measured by the physicochemical methods, and these transitions were also evident in the kinetics of the !-galactose oxidase. The properties of these micelles were further studied using light-scattering methods. The ;-galactose oxidase acted very poorly on homogeneous G *1 micelles,but at fixed G M , concentrations the rate was increased by increasing Triton X-100 concentrations. The formation of statistical micelle aggregates was followed by inhibition of the ?-galactose oxidase activity. H and 13C n.m.r. spectroscopy, laser-light scattering,and differential scanning microcalorimetry have been used to study the influence of GH, and G D l a on the structural and thermotropic properties of sonicated small 1,2-dipalmitoyl-a-~-phosphatidylcholine vesicles.” Since the average dimensions of all vesicles were the same, differences in the temperature dependence of heat capacity in the presence of the gangliosides were attributed to differences in interaction of the hydrophobic parts of the molecules. The higher content of C20 sphingosines in G D l a as compared to GM was considered to be one possible source of 1 difference. Trineuraminosylgangliosides are spontaneously incorporated into 1,2-dipalmitoyl-a-~-phosphatidylcholine vesicles and, since the ganglioside, on incorporation, is susceptible to neuraminidase action, it is concluded that incorporation occurs only on the outer face of the bilayer.*l Calorimetric studies have indicated that the ganglioside stabilizes the vesicular structures by inhibiting the fusion of small vesicles that occurs below the phase-transition temperature. Cholesterol enhanced the binding of thyrotropin to 1,2-dipalmitoyl-a-~,~-phosphatidylcholine liposomes containing G, 1
7: Glycolipids and Gangliosides
581
ganglioside but not those containing the gangliosides G, and 1 The role of gangliosides in the binding and action of GDla' thyrotropin has been re-evaluated by comparing thyrotropin binding with cholera-toxin binding in a cloned line of normal rat thyroid cells and neuraminidase-treated cells . 2 3 The results indicate that more complex gangliosides do not serve as a component of the thyrotropin receptors nor are they involved in the transmission of the hormone signal across the cell membrane of these cells. Gangliosides that bind cholera toxin have been detected by the direct binding of 1251-labelled toxin on t.l.c.24 The method is and should be applicable to other sensitive to 70 fmol G M ~ carbohydrate-binding materials. The preparation of a receptorspecific affinity chromatographic matrix for the large-scale purification of cholera toxin has been described.25 The ganglioside G M ~is hydrolysed to lyso GM,, which is coupled, via stabilized Schiff's bases, to porous silica beads. The amount of toxin bound was proportional to the amount of lyso G M ~used. Ganglioside affinity filters have been used to identify toxigenic strains of Clostridium botulinum types C and D but could be used on a general procedure for identifying botulinal toxin and toxigenic Antibodies to the gangliosides G D ~ and G M ~ have been prepared and their properties determined.27 The antibodies to G, were 3 highly specific while those to G M 1 reacted with many other A periodate-oxidized gangliosides and their derivatives. itself. derivative of GM, cross-reacted almost as readily as G, 1 The specificity of limulin and wheat-germ agglutinin towards neuraminic acids has been studied using lipid vesicles containing GM3. 2 8 Limulin binds specifically to E-glycoloyl derivatives of G but the hydroxy group at C-4 and the carboxy group must also 3, be free. The side chain is not involved in binding. Wheat-germ agglutinin only binds when the hydrophilic tail is removed. The acetamido group but not the carboxy group is involved. The ganglioside GM,, incorporated in a planar lipid bilayer, interacts specifically with Ricinus toxin.29 A fourteen-fold increase in conductance was observed. An interspecies comparison of the brain ganglioside patterns from fish, amphibians, birds,and mammals has been studied by twodimensional t. 1 .c .30 Over 30 components were detected in mammalian neural samples. Differences were observed between the vertebrate classes,but greater similarity exists between the lower and higher
**
582
Carbohydrate Chemistry
classes than had previously been noted. Various surfactants have been shown to be able to replace the requirements f o r the activator protein in the hydrolysis of G M ~ ganglioside by the B-~-2-acetamido-2-deoxy-hexosidase A from human liver.31 In the presence of saturating concentrations of the activator, the enzymatic hydrolysis of the substrate was further stimulated. The surfactants had no effect on the enzymatic 2-acetamido-2-deoxy-B-Dhydrolysis of 4-methylumbelliferyl glucose. A model for this reaction is discussed. A mononeuraminosylganglioside has been shown to be the antigen of a monoclonal antibody that is specific for cells from carcinoma of the human colon.32 The surface distribution of the ganglioside G, on human blood 1 cells and the effect of endogenous GM, and neuraminidase on cholera-toxin surface labelling have been demonstrated by an immunocytochemical study.33 Neutrophils were the most heavily labelled blood cells while lymphocytes, erythrocytes,and platelets were only labelled to a limited extent. The labelling was significantly altered on neuraminidase treatment. The lymphocytes of human peripheral mononuclear cells have been activated by oxidation with periodate .34 The neuraminosyl group was converted to a 7-carbon analogue, but the L-fucose and !-galactose residues were also oxidized. The response to periodate was reduced 5 50% by pretreatment with neuraminidase. The gangliosides from human peripheral blood lymphoctyes and neutrophils have been isolated and their structures determined by mass spectrometry and glycosidase hydrolysis results.35 The three were found in both types with ( 1 ) being the structures ( 1 ) - ( 3 ) major ganglioside in lymphocytes and ( 3 ) the major ganglioside in (2) may be a leukocyte-specific neutrophils. Structure glycosphingolipid. Large gangliosides with the general structure (4) were also isolated. The gangliosides in human leukocytes have been characterized and differences were observed between types of leukocyte .36 More ganglioside was present in normal leukemic cells than in granulocytes, while the complex gangliosides were more abundant in myelogenous cells than lymphocyte cells. Concanavalin A was shown to increase the incorporation of labelled 2-amino-2-deoxy-~-glucose into the gangliosides . 3 7 Thus, associated with cellular ganglioside synthesis is not only division but also occurs within a short time of lymphocyte activation.
583
7: Gfycolipids and Gangliosides
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Carbohydrate Chemistry
Anti-asialoganglioside sera has been shown to cross-react with human blood-group N antigen .38 An immunocytochemical study of the surface ganglioside GM in 1 haemic cell lines of normal human bone marrow has been carried out similarly to ref. 33.39 The extent of incorporation was related to specific cell types and to their stage of maturation. The ganglioside patterns of horse, donkey,and mule brains have been deter~nined.~' Forebrains contained the highest concentration with the brain stern of the mule having the lowest value. The N-glycoloylneuraminic acid content was (3% of the total neuraminic acid. G M l , GDla, and G D l b were the major gangliosides. The gangliosides from equine kidney and spleen have been characterized as haematoside, GM2, G M ~ ,and G D , ~4.1 G D ~was found in the kidney and G D l b in the spleen. The relative proportions of 1-acetyl and N-glycoloyl neuraminic acids were also determined. Liposomes, composed of gangliosides from bovine brain and dipalmitoyllecithin, have been studied by e.s.r. spectro~copy.~~ At temperatures above the transition temperature ( 4 7 ° C ) the gangliosides diffuse freely in the lipid matrix,but at 19'C, in the gel state, the gangliosides cluster together. Gangliosides from bovine brain have been modified by attaching biotin hydrazide to the aldehydo group after periodate oxidation.43 The modified gangliosides were incorporated into mature rat thymocytes,and the results suggested that gangliosides may be involved in transmembrane communication during lymphocyte stimulation. A sulphated ganglioside has been isolated from bovine gastric mucosa, and its structure (5) was elucidated by partial acid hydrolysis, specific glycosidase degradation, periodate oxidation, and methylation analyses.44 71% of the neuraminic acid was the N-acetyl derivative. A sulphated ganglioside from bovine gastric mucosa had the structure (6) confirmed by the usual procedure^.^^ 65% of its neuraminic acid was the 1-acetyl derivative. A non-specific lipid-transfer protein, purified from bovine liver, will stimulate the transfer of the ganglioside GM, and neutral glycosphingolipids from phosphatidylcholine vesicles to erythrocyte ghosts .46 E-Glycoloylhaematoside and a ganglioside, containing N-glycoloyl-neurarninic acid and having an oligosaccharide chain identical to that of erythrocyte neuraminosylparagloboside, have been isolated from both normal and leukemic bovine blood were identified in lymphocytes. 47 Three other gangliosides
7: Glycolipids and Gangliosides
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586
Carbohydrate Chemistry
leukemic lymphocytes. The gangliosides have been isolated from bovine optic nerve and were found to be GM (1291, G D (~9 8 1~, G D l b ( 9 7 1 , G T ( 8~0 1 ~, G D ~ 1 (311, and Gq, (121, the concentrations being ug g-l wet tissue.48 Together they accounted for 97% of the total neuraminic acid present. The three L-fucogangliosides ( 7 ) - ( 9 ) have been isolated from porcine nervous tissue.49 A l l three were found in dorsal-root ganglia and spinal cord but only ( 7 ) and ( 9 ) in the forebrain. The chemical analyses were confirmed by mass-spectral analysis. The specificity of the neuraminosyltransferase from bovine liver microsomes has been determined.50 The transfer was solely to C-3 of E-galactose residues with no transfer to 2-acetamido-2deoxy-;-galactose residues. Eight gangliosides have been identified as constituents of ovine testis, with GMg, GD3, GMl,and GD , respectively, being the la most abundant.5 1 A new solvent system has been developed for t.1.c. of the gangliosides of rat ~ e r e b e l l u m . ~The ~ separation takes 1 h and virtually no tailing occurs. The considerable heterogeneity of this material was demonstrated by identifying 28 separate gangliosides. 2-Acetamino-2-deoxy-~-glucose has been shown to be incorporated 2-3 times higher into undernourished rats than normal rats.53 This enhanced incorporation was found in all brain areas. The turnover of the gangliosides was slower in young undernourished rats, relative to the controls, but greater in older rats. Microsomal gangliosides in rat brain have a higher initial incorporation of 2-acetamido-2-deoxy-~-mannose than synaptosomal g a n g l i ~ s i d e s . ~ ~ The gangliosides G T l b and G q l b were more highly labelled than other gangliosides,but a decrease in incorporation was observed on treatment with the convulsant pentylenetetrazol. The role of neuraminosyl compounds on choline uptake by synaptosomes has been studied.55 Neuraminidase treatment of synaptosomal fractions brought about a reduction in the uptake of choline. The activation of (Na+, K+) ATPase by the ganglioside G M ~has been shown to follow biphasic kinetics.56 The break is at 50nM G,l with a stable complex below that value and a loosely bound one above it. It was suggested that the activation was a specific phenomenon not related to the amphiphilic and ionic properties of the ganglioside but due to a modification of the membrane lipid
587
7: Glycolipids and Gangliosides
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Carbohydrate Chemistry
environment around the enzyme. The highest degree of labelling in homogenized neuronal perikarya from rat brain with f3Hl-5-acetylneuraminic acid has been found in subcellular fractions that also showed the highest specific activities for several ganglioside glycosyltransferases.57 The neuraminic acid-containing glycoconjugates remained associated with the membrane after all treatments tried except sodium deoxycholate extraction. Some of the results suggested that the gangliosides change their accessibility to added enzymes in the process of transport through the axon and incorporation into the synaptic membrane. An increased threshold for electrical stimulation to induce somatosensory-evoked potentials in peripheral nerves followed the transection and rejoining of the nerve in rats.58 The amount of this increase was diminished by treatment with gangliosides s o it was concluded that gangliosides enhance the rate of nervous regeneration. (UDP-2-acetamid0-2-deoxy-~-galactose-G~~-2GM2-Synthase acetamido-2-deoxy-D-galactosyltransferase) has been studied in Golgi-rich fraction from rat liver.59 Many requirements of this enzyme were demonstrated along with its physical properties. Methods for the chemical detection of gangliotetraosylceramide, the rat T lympohocyte macrophage-associated antigen, have been reported along with its cellular distribution.60 The glycolipids of murine lymphocyte subpopulations have been investigated. A neuraminosyl derivative of asialo GM,, which is sensitive to neuraminidase treatment, is increased in concentration in splenic lymphocytes.61 Asialo G M , is a normally occurring lipid and not an acid-hydrolysed product of G M ~ . The total glycolipids from thymocytes of leukemic A K R / J murine thymus have been shown to be greater than those of non-leukemic thymocytes.62 Of the individual gangliosides, GM3, GM2, GDla,and G D l b contents were higher while GM and G, were lower. Parallel 1 1 with these findings were the observations that two neuraminosyltransferases were also at an elevated level. During cells from mice into the differentiation of leukemia MI macrophages, the ganglioside content has been shown to change markedly.63 A several-fold increase in GM content (confirmed by lb neuraminidase treatment) was noted with a concomitant decrease in the dineuraminosylganglioside fraction. The glycolipid content of a murine cell line ( J L S - V 9 ) and its ouabain-resistant mutant clone ( J L S - V g OR) have been compared.64 The ganglioside, G M ~ ,content of
7: Glycolipids and Gangliosides
589
the mutant cells was 1.14 times that of the parent cells but, whereas 98% of the neuraminic acid in the parent cells was in the N-acetyl form, only 69% of that in the mutants was in the same form The ganglioside pattern of rabbit brain has been studied by twodimensional t .1 .c. and a ~ t o r a d i o g r a p h y . ~ ~Numerous minor components, representing previously uncharacterized gangliosides, were identified, several of which were novel L-fucose-containing gangliosides. The gangliosides of rabbit thymus have been analysed by a ganglioside mapping procedure.66 The G M gangliosides and the for 76.6 and 23.3%, combined G, and G , species accounted respectively, of the lipid-bound neuraminic acid. The gangliosides (10) and ( 1 1 ) accounted for 38.4 and 31.3%,respectively,of the total fraction while G D 3 (11.8%) and G M ~( 1 7 % ) accounted for the remainder. The GD3 and 62% of GM3 were in the N-glycoloyl form. The lacto-N-neotetraose backbone of ( 1 0 ) and ( 1 1 ) has been shown to be tissue specific for thymus with 70% of all thymus gangliosides possessing it.67 In addition the N-glycoloyl derivative of neuraminic acid represents 90% of that species. The mononeuraminosyl gangliosides of rabbit skeletal muscle have been analysed with G M representing 73% of the fraction.68 A 3 novel ganglioside, 5-acetylneuraminosyl lacto-N-noroctaosylceramide (12),constituted 5% of the fraction. Addition of gangliosides has induced a statistically significant increase in the number of neurites in the outgrowth zone of guinea-pig spinal ganglia.69 However, only a slight increase in neurite length was observed. Three novel gangliosides (13)-(15) have been isolated from frog fat body and their structures confirmed by the usual methods. 7 0 During phylogeny and early ontogeny of higher vertebrates the number of ganglioside fractions was reduced especially with regard to the more polar ~pecies.~'Arranging the different ganglioside fractions subsequent to their paths of formation, it has become apparent that in elasmobranch fish and embryonic chicken the third pathway is used whereas in the adult chicken there is a switch to the first and second pathway. The results are taken as further evidence of the recapitulation principle according to Haeckel's biogenetic rate. An unusual pattern of myelin gangliosides has been observed in avian central nervous system. 7 2 The total concentration was
.
Carbohydrate Chemistry
590
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Carbohydrate Chemistry
5 92
considerably higher than in other mammalian species,with GM3 being present in concentrations similar to G M ~ . A new t.1.c. system has been used to separate complex gangliosides from embryonic chicken brain. 73 Ten fractions were detected, eight of them also being present in the brain of rays. Six of these were similar to the gangliosides GT39 GT2’ GTlc9 G Q l c , GPlc,and GPlb identified in cod brain. Four of these have been partially characterized and they contain neuraminic acid: sphingosine ratios of 4: 1 , 5: 1 , 6:1, and 7: 1. 7 4 Mild neuraminidase treatments yield G T and ~ G ~ D transiently ~ ~ but G M ~finally. During brain development the amounts of the forms with ratios of 6 and 7 decreased. The brain gangliosides of the ice fish Trematomus hansoni have an extremely high content of neuraminic acid which confers a high degree of polarity on the neuronal membrane and maintains their temperature^.^^ At least four gangliosides functionality at low were detected which were more highly substituted than the pentaneuraminosyl ganglioside G, 1 Two novel gangliosides have been isolated and characterized from the sea urchin Strongylocentrotus intermedius.76 The first had the structure ( 1 6 ) and the second was similar except that it contained a sulphate ester group at C-8 of the terminal neuraminic acid residue. The neuraminidation of gangliosides has been studied during maturation of cultured neurons.77 During the first 4 h GM and GD 3 3 are produced, after 12 h GD and GD are produced,and finally la lb and G T l b are produced. GD3 does not appear to be incorporated G M ~ into the membranes formed during the period of synaptogenesis in the same way as GDla and polyneuraminosylgangliosides. The ganglioside contents of SV40-transformed Balb/c3T3 cells (SV3TC cells) and concanavalin A-selected SV3T3 revertant cells have been determined and compared with untransformed cells. 7 8 There was a decrease in the concentration of higher gangliosides in transformed and revertant cells, but the content of GM3 in revertant cells was higher than in transformed or original cells. GM3 may have a role in growth control.
.
4 Animal GlvcoliDids A new solvent system coupled with
been
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a porous silica-gel column has for separating glycolipids containing mono- to
7: Glycolipids and Gangliosides
593
tetrakaidecasaccharide chains in 60 min. 79 The separation and microdetection ( 1 0 pmol) of oligosaccharide chains of glycolipids have been achieved on high-performance cellulose t.1.c. and autoradiofluorography .80 Differential scanning calorimetry and X-ray diffraction of anhydrous and hydrated N-palmitoyl-D-galactosylsphingosine have provided evidence for a complex polymorphic behaviour and interconversions between stable and metastable structural forms.8 The anhydrous glycolipid exhibits three lamellar crystal forms below 143°C and a liquid-crystal form between 1 4 3 and 180°C. The 360 MHz ' H and 90.5 MHz 13C n.m.r. spectra of Q-galactosylceramides have been recorded in DMSO and show a 4 C l configuration of the sugar ring.82 In contrast to an aqueous solution, the hydroxymethylene group at C-6 can freely rotate A series of glycolipids has been around the C-6 : C-5 bond. analysed by 360 MHz I H n.m.r. s p e c t r o ~ c o p y .The ~ ~ resonance of all H-1 and H-2 as well as most H-3 and H-4 and some H-5 protons could be assigned with the aid of spin-decoupling difference spectroscopy. Regularities were observed in the type, anomeric configuration, site of glycoside linkage,and sugar residues which could be used for the structure of more complex glycolipids. Mass spectrometry has been used for the sequencing of the oligosaccharide chain of a dodecaglycosylceramide.84 This is the largest saccharide conclusively identified. Mass spectrometry, after t.l.c., has been used to identify the structures of glycolipid mixtures from a variety of species.85 The anomalous thermotropic phase-transition behaviour of 1 , 2 distearoyl-p-galactolipids has been investigated .86 The penetration of melittin and myelin basic protein into glycosphingolipid monolayers depends on the lipid polar head group, the protein concentration, and the initial surface pressure.87 The interaction leads to a modification of the surface properties of both the glycosphingolipid and the protein. Glycolipid synthetases have been measured in a two-phase scintillation assay carried out in a small The method should be suitable for many assays but the specific enzyme determined in this investigation was ceramide : UDP-E-glucose q-glucosyltransferase. has been derivatized for use as the D-Galactosylcerebroside ligand in affinity chromatography by ozonolysis of the sphingosine The ant i -gdouble bond followed by oxidation. 89
'
Carbohydrate Chemistry
5 94
galactosylcerebroside antibodies purified on this materia.1 were highly specific. Proteins can be covalently bound to liposomes after periodate oxidation of the glycosphingolipids in the vesicle membrane. The method has potential for the antibody-mediated targeting of vesicles to cells. The synthetic glycolipids lactosyland melibiosylphosphatidylethanolamine can be incorporated into unilamellar l i p o ~ o m e s . ~ Both are agglutinated by Ricinus communis lectin but only the latter by Banderiaea simplicifolia isolectin I. Efficient fusion of phospholipid vesicles with monolayer cultures of eukaryotic cells has been accomplished by attaching glycolipid-containing vesicles to the cell surface by using a lectin displaying binding for both the cell surface and the glycol ipid Single bilayer vesicles of glycosphingolipids and phosphatidylcholine have been prepared and their physical Incubations with high-density properties determined.9 3 lipoprotein-3 resulted in the transfer o f the glycolipid, a process which did not occur in the absence of phosphatidylcholine. The complex has been further characterized,and only 69% of the lipid is transferred from the vesicles.94 The lipid compositions of axolemma-enriched fractions and myelin from human brains have been determined . 9 5 D-Galactolipid accounted for 25.8% of the lipid in the axolemma fractions, 83% of this being cerebrosides. The q-galactosylceramidase of human brain is activated by phosphatidylserine but the G M , ganglioside 8-P-galactosidase is not activated . 9 6 A review of sulpho-2-galactosylceramide has suggested that this glycolipid possesses multifunctional properties in biological membranes. The characteristic deposits of glycolipid in tissues from patients with Fabry’s disease have been studied by electron microscopy.9 8 Fourier transformation for the analysis of the distribution periodicity of the inclusions showed that, in synovial membrane and muscle, the inclusions had a monocrystalline character while in the other tissues they were amorphous or polycrystalline. The crystalline morphology of the trihexoside ceramide inclusions characteristic of Fabry’s disease have been Image processing further examined especially in synovial tissue
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7: Glycolipids and Gangliosides
595
inclusions and corroborated the proposed classification. (Gaucher's storage material) have been D-Glucosylcerebrosides shown to stimulate the release of lymphocyte activating factor from macrophages. l o o Other ceramides had no effect. The importance of these observations to Gaucher's disease was discussed. The complete structure of a trisaccharide ( 1 7 ) from a patient l3 C n.m.r. with mannosidosis has been established by spectroscopy.lol One of the features of a patient with mannosidosis has been excessive gingival hyperplasia with storage oligosaccharides . ' 0 2 The trisaccharide ( 1 7 ) was of D-mannose-linked the predominant material in the urine, but tetraand pentasaccharides were more abundant in gingiva. The neutral glycolipid compositions of human gastric and colonic cancer tissues have been compared with those from normal The cancerous tissues had elevated levels of tissues. Io3 lactosylceramide and 4-fucoglycolipids. The 4-fucoglycolipids contained both 3-0- and 4-g-substituted residues o f 2-acetamido2-deoxy-P-glucose. Similar studies have confirmed these results, especially those of the L-fucoglycolipids, and further showed that the levels of sulpho-E-galactolipids were also elevated. lo4 Blood group A-active glycolipids were present in cancer tissue from two patients with blood group 0 but not in the associated healthy tissue. The liver and kidney of a patient with I-cell disease have been di- and tri-hexoside found to contain elevated levels of This was associated with a B-P-galactosidase ceramide. lo5 deficiency. It was concluded that the B-8-galactosidase deficiency in I-cell disease is a more specific phenomenon rather than a secondary inhibition as found in mucopolysaccharidoses. Evidence has been presented that Niemann-Pick disease, type C, is caused by the deficiency of an activating factor stimulating sphingomyelin and P-glucocerebroside degradation in the spleen of these patients. Io6 The glycolipids o f the meconium of a human 0 Le (a-b+) secretor have been characterized by structural and immunological The dominating species were E-glucosyl- ( 1 5 % ) and methods . I o 7 (30%). Of the higher saccharides, the lacto Ij-galactosyl-ceramide (30% of total weight) was represented by series lactotetraosylceramide and H-active, Lea-active, and Leb-active Similar studies, involving more glycolipids of the lacto series. detailed separations and characterization methods, have suggested
596
Carbohydrate Chemistry
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7: Glycolipids and Gangliosides
that meconium is a rich source of glycolipids which allows the chemical fingerprinting of the blood-group type in individuals.' O 8 The, non-acidic glycosphingolipids of human cord blood erythrocytes from blood groups 0 and A have been characterized.lo9 Lactosylceramide and globoside were the major components. Increased amounts of penta- and hexa-glycosylceramides, which lacked L_-fucose substituents, were present and could be A and H biosynthetic precursors of ordinary blood - group glycosphingolipids. The glycosphingolipids of human plasma lipoproteins have been reviewed with specific interest in the way the different glycolipids exchange between the lipoproteins and erythrocytes. l o The identification of a blood-group Leb-active glycosphingolipid in plasma by m.s. and n.m.r. spectroscopy has suggested a new method of chemical blood-typing of human individuals.' A series of papers has been published on the immunochemistry o f the Lewis blood-group system. In the first, the complete structures of the oligosaccharide chains of the Lea-, Leb-, and H-type 1-active glycolipids have been established as (18)-(201, respectively. 1 1 * The second and third papers consider the m.s. analysis113 and 'H n.m.r. spectroscopy,ll 4 respectively, which have been used to establish these structures. Neutral glycosphingolipids have been isolated from the hairy cell leukemia and comprise cells of a patient with hairy lactosylceramide,and two higher glycolipids of g-glucosylceramide, the globo series which are similar to those found in human lymphocytes and chronic lymphocytic leukemia cells. The glycolipid pattern may be useful in classifying leukemias of uncertain origin. A high incidence of an autoantibody against the neutral glycolipid asialo G p l l has been observed in patients with systemic lupus erythematosus. l 6 Since no activity was shown against other glycolipids or gangliosides it was suggested that the antibody played a role in the pathogenesis of such neurological disorders. The glycosphingolipids of normal human lymphocytes have been separated by h.p.1.c. The major component was lactosylceramide with others being detected in smaller amount^."^ Purified B- and T-cell fractions had a similar complement of glycolipid but the B-cells contained significantly more of each component. Similar studies have been carried out on human peripheral blood lymphocytes and the lymphoid cells of a patient with B-cell
'
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598
''
Carbohydrate Chemistry
chronic lymphocytic leukemia. The glycol ipids were the same as those in ref. 114. A blood-group A-specific tetrasaccharide (21) has been isolated and characterized from human urine.ll9 The sugar had A activity almost as great as that of the most active oligosaccharide isolated from soluble blood-group A substance. The alkyl-q-galactolipids from calf brain have been separated by h .p. 1 .c . 1 2 0 The two oligosaccharides B-~-mannopyranosyl-(l+4)-2-acetamido2-deoxy-;-glucose and 42-g- 6-D-mannopyranosylchitobiose accumulated in the kidney of a goat with mannosidosis.12' The amounts represented three times those found in the brain,but while lesions of the brain caused profound neurological disorders functional impairment of the kidney was not evident. Using an epithelial cell line derived from monkey kidney, it has been shown that E-galactosylcerebroside is present in the cytoplasm in close association with colchicine-sensitive microtubule-like subcellular structures.' The distribution of glycero-g-glucolipids in the mucous barrier of dog stomach fundus, body, and antrum has been investigated. 1 2 3 The content of neutral glycero-8-glucolipid in the antral portion was higher than that in the other two areas,but the content of sulphated glycolipid was even higher. The localization of q-galactosylcerebroside in distal tubules, ascending limbs of Henle's loops and collecting tubules of kidney, periportal bile ducts and hepatic parenchyma of liver, and bronchioles and alveoli of lungs has been demonstrated by an immunological method in these organs from hamsters.l 2 4 The existence was further confirmed by chemical analysis. The presence of SV40-specific glycolipid antigens in the plasma membrane, the nuclear membrane,and probably other inner membranes of SV40-transformed cells from hamsters has been indicated by immunofluorescence methods. 2 5 Incubation of NIL 2 c l cells, a cloned hamster cell line, with an equilibrium mixture of radioactive UDP-P-glucose and UDP-9-galactose has suggested that this is a useful method for determining the glycolipid composition of cultured cells. 26 Using the same cell line, the sequential glycosylations of endogenous glycosphingolipids have been studied using nucleotide sugars. 27 Differences were noted in the sequential glycosylation between cells harvested from confluent cultures and sparse cultures.
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7: Glycolipids and Gangliosides
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Carbohydrate Chemistry
Furthermore, there was an indication that two distinct pools of each glycolipid were present. Precursor-product relationships do not exist among the main pool of each glycolipid. of UDP-q-galactosylceramide: The specific localization P-galactosyltransferase in heavy-myelin and membrane fractions of rat brain at an early age when myelination is just beginning has been suggested as a site for the myelination process.128 The turnover of glycolipids in the adult rat brain has been studied vivo by determining the half-life of the carbohydrate portion. 12’ These half-lives were much shorter than that of the total carbon pool, Since lactosylceramide had the shortest half-life, it was suggested that this glycolipid may serve as a branch point for the biosynthesis of cerebral gangliosides in vivo and not as a degradation product of more complex molecules. In various rat neural tumours, the contents of lactosylceramide were up to 80 g kg-l compared with normal neural tissue values of 10 g kg-l.130 The changes in lipid-bound surface carbohydrates during cell differentiation have been studied in the small intestine of rats. 1 3 ’ The g-glucosylceramide content increased 2-3 fold. There was little change in many of the complex glycolipids. In similar studies, the content of trihexosylceramide has been shown to increase up to about 13 days after p a r t ~ r i t i 0 n . l ~ ~ A novel sulphoglycosphingolipid ( 2 2 ) has been isolated from rat kidney and characterized by the usual methods. 3 3 The clearance of D-glucocerebrosidase by rat liver has shown the presence of two distinct enzyme forms with different recognition characteristics, 3 4 Neuraminosyl, q-galactosyl, and 2-acetamido-2-deoxy-~-glucosyl residues are important in the uptake of the enzyme. Modification of the enzyme to expose some of these residues results in increased delivery to specific cell and types. The disaccharides cx-~-mannopyranosyl-(l+2)-~-mannose a-E-mannopyranosyl-(l+3)-g-mannose have been synthesized from dolichyl-D-mannosylphosphate and g-mannose by rat liver membrane systems. 1 3 5 The proportion of each product was dependent on the assay conditions. and dolichyl The formation of dolichylphosphate-D-glucose diphosphate oligosaccharides in rat spleen lymphocytes has been studied. 136 It was observed that the phospho-oligosaccharide populations contain far fewer products which had been E-glucosylated than the dolichyldiphosphate oligosaccharides from
7: Glycolipids and Gangliosides
60 1
which they are derived, suggesting that the p-glucosyl units inhibit the reaction. Sulpho-D-galactosylacylalkyl glycerol is enriched in a plasma membrane fraction from adult rat testis.137 By comparing 11 separate tissues from rats, the presence of this lipid has been shown to be restricted to the testis.'38 Several novel sulpholipids were detected during this study. L-Fucose-terminated glycoconjugates are recognized by pinocytosis receptors on macrophages where the uptake of 6-P-glucuronidase is blocked. 3 9 An anti-glycosphingolipid antibody has been prepared and purified and has been used to demonstrate the localization of asialo G M ~ on the tips of the surface membrane of rat ascites hepatomas, 4 0 Analogues of ceramides which inhibit ;-glucocerebroside synthetase in mouse brain have been in~estigated.'~' The most potent compound tested was 2-decanoylamino-3-morpholino-1-phenylpropanol. An examination of the effects suggested that the active region of the enzyme contains four active sites, one of them being an anionic moiety that binds the 9-glucose residue in an activated form. The concentrations of myelin-characteristic D-galactolipid in organotype cultures of newborn mouse cerebellum have been determined by h . p.l.~.'~~ These lipids are detectable at 9 days and increase steadily in concentration until the explants are more than 3 weeks old. The g-galactosylceramide sulphotransferase activity of the cerebrum and cerebellum of normal and jimpy mice has been measured during postnatal development. 1 4 3 The activity was related to net sulphatide synthesis and was 25-505 lower in the brain parts of the mutants when myelination was taking place in the controls. The effective antibody in an antiserum having anti-natural killer cell activity and raised against mouse brain tissue is directed against the cell-surface glycolipid asialo G M .144 ~ The distribution of the same glycolipid in the small intestine of the mouse has been determined using an immunofluorescence staining method.145 Clear staining o f the brush border and basolateral membranes of epithelial cells, cell membranes of cryptic cells,and some secretory granules was observed. q-Glucosylceramide, on intravenous injection in mice, is stored predominantly in the liver and has a half-life of 3.5 days.146 High levels of this lipid were found in the bile of one patient
Carbohydrate Chemistry
602
and the liver of two patients with a biliary obstruction,which suggests a relationship between the biliary excretion of this lipid and Gaucher's disease. The glycolipid on the surface of natural killer cells from mouse spleen has been identified as asialo G, 147 1' A method has been developed for the extraction of carbohydrate-defined Ia antigens from murine spleen and serum cells.14* The antigen appeared to have a glycolipid structure with the antigenic activity residing in an oligosaccharide unit containing more than seven monosaccharide residues. Two marker glycolipids of alloantigen-activated murine T-lymphocytes have been partially characterized as a-E-galactosyl6-IJ-galactosyl-P-glucosylceramide and the same ceramide with a 2-acetamido-2-deoxy-~-~-glucosyl residue at the non-reducing terminal position. 149 A monoclonal antibody, directed to the stage-specific embryonic antigen in mice, has been shown to react with a branched glycosphingolipid similar in structure to Ii antigen. 150 Two related sublines of the mouse lymphoma L5178Y, which were selected for their different susceptibility to natural killer cell-mediated lysis, have different glycolipid patterns. 15' The ~ was not sensitive line contains a high level of asialo G M which detected in the resistant line. However, there was no correlation between asialo G M ~ content and sensitivity in sublines. Modification of the surface of distearoylphosphatidylcholine vesicles with synthetic glycolipids has dramatically affected the rate of uptake of these vesicles by murine peritoneal The high rate of uptake of 6-amino-6-deoxy-Pmacrophage. 15' mannose-modified vesicles is inhibited by cytochalin B and chloroquine but not by colchicine, indicating that the mechanism is phagocytosis. These vesicles remain intact after phagocytosis as aggregates of fused and intact vesicles surrounded by a single bilayer membrane structure. Several blood-group-type glycolipids from the small intestine of an individual rabbit have been identified by select-ion monitoring. 5 3 The dominating species was a hexaglycosylceramide group B-type sequence, while a second with a blood hexaglycosylceramide having a blood-group A-type sequence was also found. into lipid-linked The incorporation of [ 2-3H]-g-mannose is stimulated by oligosaccharides of rabbit mammary gland
-
7: Glycolipids and Gangliosides
603
hormones.154 An I-active ceramide decasaccharide (23) has been isolated and chacterized from rabbit erythrocyte membranes.155 The homologous series isomaltose to isomaltoheptaose can be coupled to stearylamine by reductive amination using cyanoborohydride ion. 1 5 6 The stearyl oligosaccharides from isomaltotriose to the heptasaccharide, when incorporated into rabbit liposomes, were agglutinated in the presence of specific antibodies and were lysed if complement was also present. As the disaccharide conjugate was not agglutinated, it may not protrude far enough from the surface to react with the antibody. The specificities and the sizes and shapes of the antibody combining sites in the above systems have been investigated by quantitative precipitin and precipitin-inhibition studies.157 The sizes of 15 antisera have been determined and all had combining sites in the range isomaltotriose to isomaltohexaose. One of the antisera to stearylisomaltopentaose has been separated into IgM and IgG fractions, and the antibody binding sites were studied.158 Both were similar to unfractionated antibodies with sites complementary to isomaltotetraose. Another antiserum had two site sizes, one for isomaltopentaose and the other for isomaltotetraose. The specific antibodies elicited in rabbits against D-glucosylceramide did not cross-react with E-galactosylceramide or ~ u 1 p h a t i d e . l ~ ~ They did react with the natural hapten present in red-blood-cell membranes of sheep. Very low levels of glycolipid have been found in chick embryonic brain yet nearly adult levels were reached by the 5th day. 160 The deposition of mono-D-galactosyldiglyceride in the cerebrum was unique in that all the secretion occurred between the 16th day of embryonic life and the 5th day after hatching, a period characterized by a high rate of myelination. The spermatozoa of the fresh-water bivalve Hyriopsis schlegelii have a complex spectrum of neutral glycolipids. Two of the minor components (24) and (251,which accounted for <3% of the total neutral glycolipid,were characterized. This is the first report of E-xylose residues being present in a ceramide. Vesicular stomatitis virus-infected BHK cells when deprived of !-glucose are unable to incorporate !-glucose into the oligosaccharide-lipid intermediates and, consequently, the G protein.162 A unique late effect of rubella virus infection of BHK21113s cells was the formation of a glycolipid not detected in
Carbohydrare Chemistry
604
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605
7: Glycolipids and Gangliosides
control cells or virus.163 The glycolipid contained L-fucose and glycerol in a 2:l molar ratio. The differentiation of M1 cells into macrophages has been induced by lymphokine. During differentiation, asialo GM' appeared on the surface of the M1 cells.
5 Plant Glycolipids A rapid procedure has been reported for the purification of glycolipids free from surface-active contaminants and suitable for the measurement of surface-pressure isotherms.' 6 5 A major plant glycolipid, mono-P-galactosyldiglyceride, gave very reproducible results with the method. The motional properties of four mono-P-galactosyldiacylglycerols have been determined using 13C n.m.r. relaxation times of the spectroscopy. 6 6 The longitudinal D-galactosyl carbon atoms were essentially constant. The use of stearoyl l a c t y l a t e ~ ' ~and ~ sucrose mono- and diesters 1 6 8 in breadmaking has been described. The structure of a tri-g-galactosyldiglyceride from alfalfa The non-reducing terminal leaves has been partially determined. and penultimate ;-galactose residues are linked to the next residue, one by a 1+4 bond and the other by a 1 4 bond. The residue is linked to the glycerol residue by innermost D-galactose a l+3 bond. The chemical compositions of cerebrosides in Adzuki bean seeds have been determined. 70 3-g-Mono- and 3-sn-di-;-galactosyldilinolenoylglycerol have both been isolated from bean leaves and the corresponding distearoyl derivatives prepared by
'
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hydrogenation. The unsaturated lipids formed stable monomolecular films at an air/water interface while the saturated glycolipids formed condensed monolayers which were relatively unstable. The saturated mono-D-galactosyl derivatives did not form dispersions suitable for fluorescence probe studies of the phase transition. An alkylether linkage-containing glycero-oligolipid has been isolated from germinating mung beans and the structure ( 2 6 ) confirmed. 1 7 2 It has been established that there are at least two sites for desaturation of 18:2 fatty acid to 18:3, one o f them being and which is associated with mono-P-galactosyldiglyceride
606
Carbohydrate Chemistry
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inhibited by the pyridazinone herbicide San 9785.173 Membrane preparations from developing soybean cotyledons, at the time of glycoprotein synthesis, have been shown to catalyse the assembly of lipid-linked oligosaccharides from UDP-2acetamido-2-deoxy-~-glucose and GDP-D-mannose with chain lengths of up to 10 sugar residues.174 The glycolipids do not appear to turn over in pulse-chase experiments and do not appear to produce completed storage glycoproteins. A lipid-bound oligosaccharide has been isolated from pea cotyledons incubated with E - m a n n 0 ~ e . l ~It ~ appeared to be identical to one obtained from rat liver which is known to contain three !-glucose, nine P-mannose, and two 2-acetamido-2-deoxy-D-glucose residues. Enzymes were present in the same tissue and soybean roots which catalysed the transfer of part of the oligosaccharide chain to endogenous protein. Young cassava (Manihot esculenta) leaves have a low lipid content, 25% of which is glycolipid.’76 This mixture consists of and steryl glycosides, mono- and di-E-galactosyldiglycerides, cerebrosides. Mono- and di-D-galactosyldiglycerides were the major glycolipids of ungerminated pollen from Crotalaria juncea.17’ In growing pollen tubes, glycolipid biosynthesis from acetate has been enhanced by the presence o f CAMP but not by boric acid. Spinach thylakoids have been fractionated by detergent treatment and more than 80% of the g-galactolipid was separated from the chlorophyll-protein complexes. 78 Since the integrity of these complexes was maintained on detergent treatment, the free P-galactolipid apparently originates from the lipid matrix of the thylakoid membrane. Further studies have shown that specific associations between endogenous !-galactolipid and chlorophyllprotein complexes do not occur after membrane solubilization. Other studies have been carried out on the same tissue. Alterations in the pH of isolation had no effect on peptide patterns but a great effect on 9-galactolipid composition. 17’ The changes could be explained by interlipid 9-galactosyl transfer during isolation at pH 7.2, since there was a reduction in the concentration o f the mono-derivative with corresponding increases in the tri- and tetra-derivatives. The rates and pattern of P-galactolipid synthesis have been studied at different pH values. I8O The highest rates of interlipid E-galactosyl transferase are seen at pH 6.0 in diacylglycerol-poor envelopes Intact and at pH 7.2 in diacylglycerol-rich envelopes.
608
Carbohydrate Chemistry
chloroplasts behave essentially as isolated envelopes. Using spinach and tobacco membranes, lipid-bound !-glucosecontaining oligosaccharides have been synthesized which are similar to those found in animals and yeasts.18’ These oligosaccharides were based on (Glc),(Man)g(GlcNAc)2 where is 2 or -
3.
Most of the polar lipids in tobacco leaves vary in concentration with the chlorophyll content during leaf development and senescence. Of the individual polar lipids the greatest changes were in the lipids associated with the chloroplast membranes, namely mono- and di-D-galactosyl, and sulphoquinovosyldiglycerides. The mono- and di-q-galactosyldiglyceride contents o f wheat and barley chloroplasts are reduced under water stress. 1 8 3 The reduction was greater in the more water-requiring cultivars with the less water-requiring cultivars showing a good recovery on irrigation. Two D-glycosylsterols, B-~-glucopyranosyl-(l+3)-sitosterol and B-cellotriosyl-(1+3)-sitosterol,have been isolated from the leafy stem of rice.184 A new type o f monoglycosylceramide has been isolated from the alkali-stable lipid fraction o f rice bran.185 D-Glucose was the major sugar residue present. The pollen of Oenothera missouriensis is rich in monoglycosylceramides and also in acidic glycosphingolipids. 1 8 6 Compatible pollination leads to a large increase in monoglycosylceramides whereas incompatible self-pollination leads to a decrease in the triglycosylceramide content, irrespective of the genotype of the flower. Chloroplast lipids have been extracted from varieties of common groundsel (Senecio vulgaris), common lambsquarter (Chenopodium album), and redroot pigweed (Amaranthus retroflexus) and analysed for differences between triazine-resistant and -sensitive The resistant varieties had higher biotypes. 18’ mono-D-galactosyldiglyceride contents and lower di-P-galactosyldiglyceride contents. It has been shown that in Volvox carteri the antibiotic UDP-2-acetamido-2-deoxy-D-glucose tunicamycin inhibits the d o l i c h y l p h o s p h a t e : 2 - a c e t a m i d o - 2 - d e o x y - D --g l u c o s e - l - p h o s p h a t e transferase which is the first enzyme in the dolichol cycle.188 The antibiotic showdomycin inhibits the same enzyme and dolichol-phospho-P-glucose synthetase.
7: Glycolipids and Gangliosides
609
6 Microbial Glycolipids Mono- and di-P-glucosyldiglycerides are the dominant lipids of the Acholeplasma laidlawii membrane.' 8 9 The di-D-glucosyl derivative forms a lamellar liquid-crystalline phase while the monoderivative forms a reversed hexagonal phase. In a mixture, a reversed cubic phase can be formed at physiological temperatures. The structure of the cubic phase is composed of aggregates where the lipids can diffuse over microscopical distances. These glycolipids can be extracted by different detergents and the results indicated that the mono-E-glucosyl-diglyceride was involed in anchoring the protein D12 in the membrane.lgO The membrane-associated lipoglycan from A.granularum has been shown to possess 1 2 of the repeating units ( 2 7 ) in a linear form attached to a diacylgly~erol.~~' The composition of Bdellovibrio bacteriovorus lipopolysaccharide has been determined in cells grown axenically and intraperiplasmically on Escherichia coli or Pseudomonas put ida. 9 2 The axenically grown cells contained ;-glucose and 2-amino-2,6-dideoxy-~-galactose as the sole sugar residues, while the intraperiplasmically grown cells contained the above components plus some derived from the substrate cells. However, since !-galactose was not present, it was suggested that only the lipid A component was transferred. The major sulphated glycolipid of the halophile Halobacterium salinarium has been characterized by m.s. and n.m.r. spectroscopy as a trihexosyl glycerol CZ0-diether with the sulphate group being on the non-reducing terminal hexose residue.lg3 It may be identical to a similar lipid characterized previously from H.cutirubrum. Three minor glycolipids have been isolated from the halophile H.cutirubrum and their structures characterized as ( 2 8 1 , the desulphated product from ( 2 8 ) and ( 2 9 ). l g 4 The carbohydrate contents of the lipopolysaccharide antigens from three strains of Klebsiella pneumoniae have been determined, but the presence of capsular antigens has not been demonstrated. 9 5 Two unusual phosphoglycolipids, which account for 64% of the total cellular lipids, have been isolated from the methanogenic archaebacterium Methanospirillum hunRatei and are derivatives of 1-dibiphytanyl diglycerol tetraether. l g 6 One of the free hydroxy groups is glycosidically linked to a disaccharide, the two
Carbohydrate Chemistry
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disaccharide residues being identified as ( 3 0 ) and ( 3 1 ) . 1 9 7 A new synthesis of cord factors and analogues has been reported involving benzylation of 6,6'-di-~-trityl-trehalose.' 9 8 The benzyl 6,6'-di-Q-methane-sulphonyl derivative is converted via the derivative to the cord factors. The C-mycosidic glycopeptidolipid typing antigens from all serovars in the Mycobacterium avium/M. intracellulare/ M.scrofulaceum complex have been examined to varying extent,and some of them consist of short acetylated oligosaccharides linked to a common fatty acyl-peptidyl-Q-(3,4-di-Q-methyl-L-rhamnose) 'core' 9 9 A basal rhamnosyl-6-deoxytalosyl disaccharide is always linked to allo-threonine. The glycopeptidolipid antigens in their structural principals, cellular location,and physiological role bear a striking miniscular resemblance to cell-wall components of other bacteria such as the pantigenic and R-antigenic lipopolysaccharides. A novel phenolic glycolipid has been isolated from M.leprae which is distinct from, although related to, that from M. kansasii. 2 o o The methylated glycolipid contained residues of 2,3di-g-methylrhamnose, 3-Q-methylrhamnose, and 3,6-diiQ-methylglucose. The endotoxic glycolipid from Salmonella minnesota, when injected into mice, has produced increased levels of aminopyrine-N-demethylase and aniline hydroxylase activities in the liver.201 A glycolipid has been isolated from the cellular slime mould Dictyostelium discoideum and characterized as A 22-stigmasteryl-Dglucoside.202 The amount present was higher in the vegetative-stage cells, late aggregation-stage cells, and l-day sorocarps than in cells of other stages. A new glycolipid from a strain of Nocardia caviae has been characterized as a 2',3"-di-~-acyl-~-~-glucopyranosyl-(1+2)-~glyceric acid with myristic, palmitic,and stearic acids being the principal acyl substituents. 203 A B -D-mannosylceramide has been identified in the hepatopancreas of the fresh-water bivalve Hyriopsis schlegelii and represents 5% of the monohexosylceramide fraction. * 0 4
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.'
(References begin o v e r l e a f )
Carbohydrate Chemistry
612 References 1
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361,
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128,
189,
-
665,
186,
2,
673,
674, 2,
673,
-
Carbohydrate Chemistry
616 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187
Higgins and C.R. P a r i s h , Mol. Immunol., 1980, l7, 1065. Gruner, R.V.W. Van E i j k , and P.F. MGhlradt, Biochemistry, 1981, 20, 4518. E. Nudelman, S.-I. Hakomori, B.B. Knowles, D. S o l t e r , R.C. Nawinski, M.R. Tam, and W.W. Young, jun., Biochem. Biophys. Res. Commun., 1980, 97, 443. W.W. Young, J.M. Durdik, D. Urdal, S.I. Hakomori, and C.S. Henney, J. Iunnunol., 1981, 1. P.S. Wu, G.W. Tin, and J.D. Baldeschweiler, Proc. Natl. Acad. S c i . USA, 1981, 78, 2033. M.E. Breimer, G.C. Hansson, K.A. Karlsson, H. L e f f l e r , W. P i m l o t t , and B.E. Samuelsson, Biomed. Mass Spectrom., 1979, 6, 231. J.P. Bradshaw and D.A. White, Biochem. SOC. Trans., 1981, 9 , , 6 6 . Kuhn, D. Roelcke, and J. P. Hanfland, H. Egge, U. Dabrowski, S. Dabrowski, Biochemistry, 1981, 20, 5310. C. Wood and E.A. Kabat, J. Exp. Med., 1981, 154, 432. C. Wood and E.A. Kabat, Arch. Biochem. Biophys., 1981, 212, 262. C. Wood and E.A. Kabat, Arch. Biochem. Biophys., 1981, 212, 277. B. Zalc, P. Dupouey, M . J . Coulon-Morelec, and N.A. Bamann, Immunol., 1979, 297. K.N. Singh and K.S. Rao, I n d i a n J. Biochem. Biophys., 1979, 349. M. S u g i t a , T. Yamamoto, S. Masuda, 0. I t a s a k a , and T. Hori, J. Biochem. (Tokyo), 1981, 90, 1529. S.J. Turco, Arch. Biochem. Biophys., 1980, 205, 330. G. B a r d e l e t t i and A. Voiland, Arch. V i r o l . , 1981, 68, 285. K.S. Akagawa, T. Momo, Y. Nagai, and T. Tokunaga, FEBS L e t t . , 1981, 130, 80. P. TancrLde, G. Chauvette, and R.M. Leblanc, J. Chromatogr., 1981, 3, 387. J.M. Coddington, S.R. Johns, D.R. Leslie, R.I. W i l l i n g , and D.G. Bishop, Biochim. Biophys. Acta, 1981, 653. B. Thewlis, J. S c i . Food ARric., 1981, 2, 125. H. Chung, P.A. S e i b , K.F. Finney, and C.D. Magoffin, Cereal Chem., 1981, 58, 164. S. I t o and Y. F u j i n o , Agric. B i o l . Chem., 1980, 1181. M. Ohnishi and Y. F u j i n o , Agric. Biol. Chem., 1981, 9, 1283. A. Sen, W.P. W i l l i a m s , and P.J. Quinn, Biochim. Biophys. Acta, 1981, 663, 380. Y. Kondo, Biochim. Biophys. Acta, 1981, 471. N.W. Lem and J.P. Williams, P l a n t Physiol., 1981, 68, 944. Verma, and G.A. MacLachlan, D.S. B a i l e y , V. Deluca, M. Durr, D.P.S. P l a n t Physiol., 1980, 66, 1113. R.J. S t a n e l o n i , M.E. Tolmasky, C. P e t r i e l l a , and L.F. Leloir, Plant Physiol., 1981, 68, 1175. H.T. Khor and H.L. Tan, J. S c i . Food Agric., 1981, 2,399. I.S. Bhandal and C.P. Malik, Phytochemistry, 1981, 20, 429. 105. E. Heinz and D. Siefermann-Harms, FEBS L e t t . , 1981, J.F.G.M. Wintermans, A. Van Besouw, and G. Bsgemann, Biochim. Biophys. Acta, 1981, 99. A. Van Besouw, J.F.G.M. Wintermans, and G. agemann, Biochim. Biophys. 1981, 108. L. Lehle, FEBS L e t t . , 1981, 123, 63. A. Koiwai, T. Matsuzaki, F. Suzuki, and N. Kawashima, P l a n t C e l l Physiol., 1981, 22, 1059. S. C h e t a l , D.S. Wagle, and H.S. Nainawatee, P l a n t S c i . L e t t . , 1981, 20, 225. M. Ohnishi and Y. F u j i n o , Phytochemistry, 1981, 20, 1357. M. Ohnishi, D. Park, and Y. F u j i n o , Agric. Biol. Chem., 1981, 45, 755. B. B r i s , C. D e l b a r t , D. Coustaut, and R. Linder, Phytochemistry, 1981, 20, 1255. P. P i l l a i and J.B. S t . John, P l a n t Physiol., 1981, 68, 585. T.J. K.R.
-
126,
m.
16,
16,
-
663,
*,
-
665,
129,
-
s,
-
663, 663,
7: Glycolipids and Gangliosides 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204
617
Th. Mhler, E. Bause, and L. Jaenicke, FEBS Lett., 1981, 128, 208. A. Wieslander, L. Rilfors, L.B.-A. Johansson, and G. Lindblom, Biochemistry, 1981, 20, 730. K.-E. Johansson, C. Jagersten, A. Christiansson, and 1. Wieslander, Biochemistry, 1981, 20, 6073. P.F. Smith, Biochim. Biophys. Acta, 1981, 665, 92. D.R. Nelson and S.C. Rittenberg, J. Bacteriol., 1981, 3,860. K.E. Falk, K.-A. Karlsson, and B.E. Samuelsson, Chem. Phys. Lipids, 1980, 27, 9. B.W. Smallbone and M. Kates, Biochim. Biophys. Acta, 1981, 5, 551. P. Bienvenu, B. Jouanneteau, D. Robert, R. Guilluy, G. Normier, L. Dussourd d’Hinterland, and R. Fontanges, C.R. Hebd. Seances Acad. Sci., Ser. D, 1981, 292, 1113. S.C. Kushwaha, M. Kates, G.D. Sprott, and I.C.P. Smith, Science, 1981, 211, 1163. S.C. Kushwaha, M. Kates, G.D. Sprott, and I.C.P. Smith, Biochim. Biophys. Acta, 1981, 664, 156. A. Liav and M.B. Goren, Chem. Phys. Lipids, 1980, 27, 345. P.J. Brennan, H. Mayer, G.O. Aspinall, and J.E. Nam Shim, Eur. J. Biochemistry, 1981, 9, 7. S.W. Hunter and P.J. Brennan, J. Bacteriol., 1981, 3,728. K. Egawa and N. Kasai, Microbiol. Immunol., 1979, 23, 87. A. Hase, Arch. Biochem. Biophys., 1981, 210, 280. M.-T. Pommier and G. Michel, Eur. J. Biochem., 1981, 118, 329. T. Hori, M. Sugita, and H. Shimizu, Biochim. Biophys. Acta, 1981, 665, 170.
Chemical Synthesis and Modification of Oligosaccharides, Polysaccharides, Glycoproteins, Enzymes, and Glycolipids BY C. M. STURGEON 1 Synthesis o f Polysaccharides, Oligosaccharides, G l y c o p r o t e i n s , G l y c o p e p t i d e s , and G l y c o l i p i d s
--
Polysaccharides.
A r e v i e w o f t h e s y n t h e s i s and p o l y m e r i z a t i o n
o f a n h y d r o s u g a r s h a s appeared.'
1,2-Anhydro-3,4,6-tri-~-benzyl-a-g-glucopyranose p o l y m e r i z e d w i t h a number o f L e w i s a c i d s . 2 a t -6O'C
has been
Phosphorus p e n t a f l u o r i d e
caused p o l y m e r i z a t i o n t o a p r o d u c t r i c h i n B-linkages,
while other Lewis acids a t polysaccharides
o f
stereoselectivity. yielded a n a t u r a l (1
higher
lower
gave p e r b e n z y l a t e d
weight
w i t h
less
D e b e n z y l a t i o n o f t h e most r e g u l a r d e r i v a t i v e
polysaccharide with +
temperatures
molecular
properties similar t o
those
of
2)-B-g-glucopyranans.
The i n f l u e n c e o f 4-bromo 1,6-anhydr o -2,3,4-tr
s u b s t i t u t i o n on t h e p o l y m e r i z a t i o n o f
i- 2 - b e n zy 1- 8 - g - g l u c o p y r a n o s e
h a s been i n v e s t i -
g a t e d and c o m p a r e d w i t h e f f e c t s i n t h e p o l y m e r i z a t i o n and c o - p o l y merization of other 2,3,4-tri-g-substituted Polymerization of
1,6-anhydro
sugar^.^
1 , 2 - a n h y d r o -3,4,6-tri-g-benzyl-B-g-mannopyranose
proceeds i n t h e presence o f Lewis acids, c a t a l y s t s , and s t r o n g bases.4 oligomeric saccharides or
cationic
co-ordination
Debenzylation o f the products yields
low polymers
w h i c h have been p h y s i c a l l y
characterized. The r e g u l a r h e t e r o p o l y s a c c h a r i d e g l u c o r h a m n a n { + 6 ) - ! - G l c ~ - ( l
+}n
+
3-0acetyl-4-~-(2,3,4-tri-~-acetyl-6-~-trityl-~-~-glucopyranosyl)-l,2-~4)-l=-Rhae-(
1
has been o b t a i n e d by p o l y c o n d e n s a t i o n
(l-exo-cyanoethylidene)-B-L-rhamnopyranoside
of
using a triphenyl-
methylium perchlorate ~ a t a l y s t . ~ Stereospecific
polycondensation
of
t r i t y l
trisaccharide 1,2-L-cyanoethy l i d e n e d e r i v a t i v e s
ethers
of
f o l l o w e d by
d e p r o t e c t i o n h a s a f f o r d e d r e g u l a r h e t e r o p o l y s a c c h a r i d e s b u i l t up o f repeating trisaccharide units,
e.
+
6)-B-g-Man~-(l
+
4)-a-i-Rha~-
8: Chemical Synthesis and Modification (1
+
3)-B-Q-Galp-(l
+
and
+
619
6)-a-Q-Manp-(l
+
4)-a-&-Rhap-(l
+
3)-B-
Q - G a l e - ( l +.6
--
Oligosaccharides.
The u s e o f p a r t i a l l y b e n z y l a t e d o x a z o l i n e
d e r i v a t i v e s o f 2 - a c e t a m i d o - 2 - d e o x y - q , g l u c o p y r a n o ~ i d e as s t a n d a r d ized
intermediates
for
oligosaccharide
synthesis
has
been
C o n d i t i o n s were f o u n d f o r t h e s u c c e s s f u l c o u p l i n g o f
reported.’
O - a c e t y l - d i -g-benzy
1 derivatives
(1,2)
of
2-methy 1-(1,2-dideoxy-B-E-
l i n e t o t h r e e b e n z y 1 t r i - O _ - b e n z y 1-1-
g l u c o p y r anol-{ 2,1-g1-2-oxazo
thio-B-Q-galactopyranosides (3-5) and t o a p a r t i a l l y p r o t e c t e d g l y c o s i d e ( 6 ) o f 2-acetamido-2-deoxy-~-glucose. The p r o d u c t s w e r e f u l l y s u b s t i t u t e d d i s a c c h a r i d e s o f 2-acetamido-2-deoxy-$-Q-glucose l i n k e d (1
+
3),
(1
+
6),
(1
+
41, a n d ( 1
41, r e s p e c t i v e l y ,
+
c a p a b l e o f c h a i n e x t e n s i o n f r o m p o s i t i o n 3, 3’,
4,or
4’
and
following
r e m o v a l o f t h e s i n g l e t e m p o r a r y g - a c e t y l p r o t e c t i n g g r o u p f r o m each.
OR
(1) R = A C
( 2 ) R = Bn
2-Methyl-~2-acetamido-4-~-acetyl-6-~-benzyl-3-~-(2-butenyl)-
1,2-dideoxy-B-~-glucopyrano}-{2,l-~}-2-oxazoline,
a glycosylating
reagent i n which t h e t h r e e h y d r o x y l groups a r e b l o c k e d w i t h p r o t e c t i n g groups o f d i f f e r i n g r e a c t i v i t y , has been s y n t h e s i z e d i n a t e n s t e p sequence f r o m 2-acetamido-2-deoxy-~-glucose.8 a versatile intermediate f o r the synthesis o f r i d e s of
bacterial cell-wall,
human-milk,and
R’O
The compound i s
complex
oligosaccha-
blood-group substances.
CH20R3
Bnb ( 3 ) R’ = R 3 = En, R 2 = H (4) R’
= H, R 2 = R 3 = Bn
(5) R’
= R 2 = Bn, R 3 = H
620
Carbohydrate Chemistry A 11y 1 6 -2- be n z y 1 - 3 , 4
-0-is o p r op y 1i d e n e -a -Q -ga 1a c t o p y r a no s i de
has
b e e n u s e d t o p r e p a r e a s e r i e s o f 2-g-benzoyl-3,6-di-g-benzyl-a-gg a l a c t o p y r a n o s y l h a l i d e s c a r r y i n g e i t h e r a second benzoyl group o r a s e l e c t i v e l y r e m o v a b l e t e m p o r a r y p r o t e c t i n g g r o u p a t p o s i t i o n 4.' These compounds a r e d e s i g n e d t o s e r v e a s p r e c u r s o r s o f B - l i n k e d i n t e r i o r a-galactopyranose
units having chain extension a t position
4 a n d b r a n c h i n g ifd e s i r e d a t p o s i t i o n 2.
CH,OBn
H : n - OAcNH * , ,
( 6 ) A l l = a l l y 1 = CHz=CH-CHz3 - 0 - A c e t y 1-2,4,6- t r i -0be n z y l -a-Q -mannopy r a n o s y l b r o m i d e h a s been s y n t h e s i z e d f o r use a s a key i n t e r m e d i a t e i n t h e p r e p a r a t i o n o f b r a n c h e d-ch a i n
Q-man no s y 1
o l igo s a c c h a r i de s.
N i t r o x i d e s p i n - l a b e 11e d a - Q - g l y c o p y r a n o s id e s
have
been
s y n t h e s i z e d i n g o o d y i e l d a n d i n a h i g h l y s t e r e o s e l e c t i v e m a n n e r by r e a c t i o n o f p e r - g - g l y c o p y r a n 0 sy 1 b r om i d e s w i t h 2,2 ,6,6- t e t r a m e t h y l f o l l o w e d b y h y d r o g e n a t i o n a n d ox id a t i o n.'
4-pi p e r idinol,
2 - d e o x y - D-- g l u c o s e a t
C-2,
and
and t h e
-Q-galactose
derivatives, having a
s p i n - l a b e l l e d
compound
2-Am i n o spin label
1-{4-(B-&-
galactopyranosyloxy)phenyl}-3-(2,2,6,6-tetramethylpiperidin-l-oxyl4-yl)-Z-thiourea Efficient d e s c r ib e d catalyst.12
were a l s o s y n t h e s i z e d . formation of
1,2-trans-glycosidic
u s in g t r i m e t h y 1s i 1y l
l i n k a g e s has
been
t r i f l u o r om e t h a n e s u l p h o n a t e a s
The c a t a l y s t was s u c c e s s f u l l y
employed i n reactions
in vo 1v i n g o r t h o e s t e r , o x a z o 1ine, o r 1 , 2 - t r a n s - d i a c e t a t e s t a r t i n g mate r i a l s . C r y s t a 11i n e 4 - m e t h y l u m b e l l if e r y 1 3 , 4 , 6 - t r i
-2-acetyl-2-deox
o f t h i s compound f o l l o w e d
y-2-
A c e t y 1a t i o n
ox i m i n o - a - q - a r a b i n o - h e x o p y r a n 0 si de h a s b e e n p r e p a r e d .
by r e d u c t i o n o f t h e r e s u l t i n g c r u d e
acetyloxime w i t h borane i n oxolane,
and a c e t y l a t i o n ,
0-
yields the
3,4,6- t r i - 2 - a c e t y l d e r i v a t i ve o f 4 - m e t h y l u m b e l l i f e r y l 2 - a c e t a m i d o - 2 -
d e o x y - a - Q- - g l u c o p y r a n o s i d e ,
the
substrate
i n a new
sensitive
f 1u o r o g e n i c a s s a y o f 2 - a c e t a m i d o - 2 - d e o x y - a - Q - g l u c o s i d a s e .
The s y n t h e s e s o f m e t h y l 2-2-B-Q-galactopyranosyl-B-Q-galactopy r a n 0 s i d e and m e t h y l 2 - 0 - ( 2-g-B-Q-gal a c t o p y ranosy l)-B-q-galactopy r a no s ide h a v e
b e e n r e p o r t e d u s in g 2 - 2 - be n z o y 1-3,4,6-
t r i-2- be n z y 1-
62 1
8: Chemical Synthesis and Modification 1
-0-( 4 - t o 1y 1s u l p h o n y 1) - Q - ga 1a c t o p y r a n o s i de
as s t a r t i n g m a t e r i a l
An e f f i c i e n t s y n t h e s i s o f B - ! - m a n n o p y r a n o s i d e s By a c e t a l i z a t i o n u n d e r
k i n e t i c c o n t r o l a-mannose
c r y s t a l l i n e 2,3:4,6-di
- 0 - c ~c l o h e x y l i d e n e - a - Q - m a n n o p y r a n o s e ,
reacts
i n the
presence o f
c h 1o r id e t o y ie 1d 2 , 3 : 4 , 6 - d i chloride
for
triethylamine
-0-c y c
use i n Koenigs-Knorr
i s
converted i n t o which
methanesulphonyl
l o h e x y 1id e n e - a - B - m a n n o p y r a n 0 s y 1 of
1-01 a n d o f 4 - t r i f l u o r o a c e t a m i d o p h e n o l
of
with
3 - 0-- ( 3 , 6 - D i d e o x y - a - Q -
syntheses.
x y l o - h e x o p y r a n 0 s y l ) - a - g - m a n n o p y r a n o s i de s reaction
.'
h a s b e e n d e s c r i bed.15
8 - m e t h o x y c a r b o ny l o c t a n -
have been s y n t h e s i z e d by lo-hexopy ranosy 1
2 , 4 - d i -g-benzyl-3,6-dideoxy-a-;-xy
b r o m ide w i t h s u i t a b l y p r o t e c t e d manno s i d e s.16 The s y n t h e s e s h a v e b e e n r e p o r t e d o f i s o m e r s o f t h e t e r m i n a l disaccharides
of
mithramycin.17
the
saccharide
cytostatics
component o f t h e b a c t e r i a l l i p o p o l y s a c c h a r i d e , o f B - Q -( 1+6)
-l i n k e d
deoxy-E-glucose A
synthesis o f
-a - g 1u c o s e
gram
o f
hydrogen
amounts of
pu r if ic a ti on The
a c y 1a t e d 2 - a m i n 0 - 2
-
t am i d o - 2 - d e o x y - 4 - g - B - Q -
2-ace
( al c et y 1-1a c t o s a m ine 1
g a l a c t o p y r a n o s y l - Q - a r a b i no s e . 10-20
and
has been r e p o r t e d u s i n g t h e o x a z o l i n e method.18
convenient
a d d i t i o n
A
the l i p i d A
a facile preparation
E- f a t t y
d i s a c c h a r ide s o f
g a l a c t o p y r a no s y 1 by
olivomycin
the synthesis o f
As a n a p p r o a c h t o
cyanide
t o
T h e m e t h o d a 1 1ow s p r e p a r a t i o n o f
the disaccharide without
.
h as bee n r e p o r t e d
l-N-benzyl-3-g-B-Q-
4 -S-B
p o t e n t i a l e n z ym e i n d u c e r
any c h r o m a t o g r a p h i c
-! -ga 1a c t o p y r a n 0 s y 1 - 4 -
Q - g l u c o p y r a n o s e ( t h i o l a c t o s e ) has been s y n t h e s i z e d
-
2
thio-
two routes.20
-
T h e T a n t ige n ic de t e r m in a n t actopyranosyl-a-p-galactopyranoside
2 - a c e t a m ido 2 - d e o x y 3 -0-6 -p - ga 1-
methoxycarbonyl-octyl
and
s i d e chain.22
has been s y n t h e s i z e d w i t h an 8 -
s i d e chainz1
with
a
water-soluble
Both side chains are s u i t a b l e f o r f u r t h e r
ion. T h e c h e m ic a 1 s y n t h e s is o f 2
uronic)-Q-xylose,
a
-9-( 4 - 9 -
constituent of
m e t h y 1-a
amide
derivatizat-
-D - g l u c o p y r a n o s y 1-
gymnosperm x y l a n s ,
has
been
r e p o r t e d . 23 The
synthesis o f
d i s a c c h a r i de s
t a l o p y r a n o s y l g r o u p s has system
previously described for
chloride, 3,4
disaccharides
r a n o s y 1 groups. 24
was t r e a t e d w i t h s i l v e r
3,4,6-Tri-g-
imidazolate,
mercury(I1)
and i o d i n e i n a c e t o n i t r i l e t o y i e l d a -5:l
mixture of
,6 - t r i - 9 - a c e t y l - 2 - d e o x
chlorides,
using a novel glycosylating
the synthesis of
c o n t a i n i n g 2 - d e o x y - 2 - i o d o -a-IJ-mannopy acetyl-Q-galactal
c o n t a i n i n g 2 - d e o x y - 2 - i o d o -a-D_-
been r e p o r t e d
y-2-iodo -a-Q-talo-
a n d -Q - g a 1a c t o p y r a n o s y 1
w h i c h was t h e n t r e a t e d w i t h s u i t a b l y p r o t e c t e d a l c o h o l s
622
Carbohydrate Chemistry
t o provide the required disaccharide. The r e a c t i o n o f octa-g-acetylcellobiononi t r i l e ( 7 ) w i t h s o d i u m methoxide, and a c e t y l a t i o n o f t h e r e s u l t i n g m i x t u r e , has a f f o r d e d t h e anom e r i c p e r a c e t a t e s o f
0-B-Q- g l
u c o p y r a n o s y 1-( 1+3)-!-arabino-
p y r a n o s e and - f ~ r a n o s e . ~ ~
CH~OAC I
- t r i de ox y - 4 -0-( 3,4
M e t h y 1 2,3,6
-0-a c e t y 1 - 2 , 6
-di
1yx 0- hex opy r a n 0 sy 1)-3 - ( t r i f 1uo r o a c e t am i d o ) - B a
disaccharide
having
daunosamine
and
sequence f o u n d n a t u r a l l y i n r h o d o m y c i n s , T r if 1uo r o a c e t a m ido p h e n y 1 3 side,
- d i deo x y - a - l -
-I- 1 y x o - hexopy r a n 0 s i de ,
2-deoxy-l-fucose
i n the
h a s been s y n t h e s i z e d . 2 6
4-
-0-( a -a - g l uco p y r a no sy 1) - a -! - m a n no p y r a qo -
o f w h i c h t h e d i s a c c h a r i d e m o i e t y has been s u g g e s t e d t o be t h e
immunodominant g r o u p C,),
p a r t o f 2-antigen
14 i n Salmonella bacteria
(sero-
has been ~ y n t h e s i z e d . ~ ’
4 - N i t r o p h e n y l 6-0-( 2-acetam ido-2-deox y-B-Q-gl u c o p y r a n o s y 1 ) - a - D mannopy r a n 0 s i d e has
been s y n t h e s i z e d
from
4- n i t r o pheny 1 2 , 3 - 0 - i s o -
p r o p y li d e n e - a - u - m a n n o py r a n 0 s i d e a n d 2 - m e t h y l -( 3,4,6- t r i - ) - a c e t y l -
-
1,2 - d i de o x y - a -Q g l uc o py r a no ) - { 2 , l 6-g-substituted
-c- 1 -2-ox a z o l i n e
disaccharide derivative.28
a cry s t a l li n e
S y n t h e s i s o f m e t h y l 6-0-
( 2 - a c e t am i d o - 2 - d e o x y - 6 -Q - g l u c o p y r a n 0 s y 1)-a-g-m annopy r a n 0 s i de was a l s o a c c o m p l i s h e d by a s i m i l a r r e a c t i o n sequence. during mercuric cyanide-catalysed
Acetal migration
g l y c o s y l a t i o n h a s been shown t o be
responsible f o r the synthesis o f 4-nitrophenyl
6-g-B-Q-galacto-
-
be n z o y 1 4,6 py r a no s y 1-B -9 - ga 1a c t o p y r a no s ide v i a 4 - n i t r o p h e ny 1 3 -0i s o p r o p y 1i d e n e -B-Q - -ga l a c t o p y r a no s i de i n s t e a d o f t h e ex p e c t e d
,
product,
the
(1+3)-linked
isomer.29
de s c r ib e d f o r 4 - n i t r o p h e n y 1 2 - 2 - a side,
-0-
A r a p i d s y n t h e s i s h a s been
-I- f ucopy r a no sy 1-B -E -ga 1a c t opy r a no -
a s u b s t r a t e f o r human ( 1 + 2 ) - a - l - f u c o s y l t r a n s f e r a s e . 30 P h e n y 1 2 - a c e t a m ido - 2
glucopy ran0 side
has
been
- d e o x y -4,6 -0-( 4reacted
with
m e t h ox y benz y l i d e ne 1-B-9 2,3,4-
tri-()-be n z y l - a - i -
f u c o p y r a n o s y l bromide under h a l i d e i o n - c a t a l y s e d c o n d i t i o n s t o y i e l d p h e n y 1 2 - a c e t am ido - 2 -deox y-4,6
-0-( 4-me t hpx y be n z y l i d e ne 1-3 -0-( 2,3,4-
t r i - g - b e n z y l - a - l - f u c o p y r a n 0 sy 1)- B - Q - g l u c o p y r a n 0 s i d e ( 8 ) .31
Mild
623
8: Chemical Synthesis and Modification
t r e a t m e n t o f ( 8 ) w i t h a c i d , f o l l o w e d by h y d r o g e n o l y s i s , y i e l d e d t h e d i s a c c h a r i d e p h e n y l 2-acetamido-2-deoxy-3-g-a-k-fucopyranosyl-B-Qglucopyranoside. Using a similar r e a c t i o n sequence t h e s y n t h e s i s of 6 - ( t r i f l u o r o a c e t a m i d o ) h e x y l 2-acetamido-2-deoxy-3-~-a-~-fucopyranos y l - B - P - g l u c o p y r a n o s i d e was a l s o r e p o r t e d .
BnO 1 , 2 , 3 - T r i - g - a c e t y 1- 4 - g - b e n
z y 1- B - Q - x y l o p y r a n o s e h a s b e e n u s e d
f o r t h e s t e p w i s e s y n t h e s i s o f (1+4)-B-Q-xylo-oligosaccharides.32
T h e s y n t h e s e s o f a- a n d 8 - c e l l o t r i o s e h e n d e c a - a c e t a t e s a n d o f s e v e r a l
6 , 6 ’ , 6 ’ - t r i - s u b s t i t u t e d d e r i v a t i v e s o f me t h y 1 B - c e l l o t r i o s i d e h a v e b e e n d e s c r i b e d .33 ~ - a - ~ - G l u c o p y r a n o s y l - ( l + 2 ) - ~ - a - ~ - g l u c o p y r a n o s y l (1+2)-Q-glucopyranose ( k o j i t r i o s e ) has b e e n s y n t h e s i z e d by c o n d e n s a t i o n o f 1 , 3 , 4 , 6 - t e t r a-2-acety l - a - P - g l u c o p y r a n o s e w i t h h e p t a O-acetyl-a-ko j i b i o s y l bromide under halide ion-catalysed c o n d i t i o n s , f o l l o w e d by f r a c t i o n a l r e c r y s t a l l i z a t i o n a n d d e p r ~ t e c t i o n . ~ ~ The s y n t h e s i s o f a n a l d o t r i u r o n i c acid d e r i v a t i v e r e l a t e d t o (4-0-me t h y 1 g l u c u r o n o ) - a - x y l a n s has b e e n r e p o r t e d . 35 Ace t y l a t i o n o f met h y 1 4-O_- ( 2 - g - b e n z y 1- B - Q - x y l o p y r a n o s y 1 ) - B - Q - x y l o p y r a n o s i d e f o l l o w e d by c a t a l y t i c d e b e n z y l a t i o n o f t h e p r o d u c t y i e l d e d m e t h y l
2,3-di-~-acetyl-4-~-~3,4-di-~-acetyl-~-~-xylopyranosyl)-~-~-xylo-
pyranoside. R e a c t i o n o f t h i s n u c l e o p h i l e w i t h m e t h y l 2,3-di-gb e n z y l - l - c h l o r o -1 - d e o x y -4-0-me t h y l-a,B-P-glucopyranuronate i n t h e p r e s e n c e o f s i l v e r p e r c h l o r a t e a n d 2 m - c o l l i d i n e a f f o r d e d t h e 4-0m e t h y l - a - e - g l u c u r o n i c a c i d - c o n t a i n i n g t r i s a c c h a r i d e d e r i v a t i v e (9) as t h e major product, from which t h e desired methyl ester methyl g l y c o s i d e ( 1 0 ) was r e a d i l y o b t a i n e d .
@P
R’O
OR’
(10) R’
= R2 = H
Carbohydrate Chemistry
624 A key
intermediate for
a _ - g a 1a c t o p y r a n a n s ,
solid-phase
s y n t h e s i s o f B-(1+6)-linked
- t r i- g - a c e t y 1 - 6 - C - ( c h l o r o a c e t y 1 ) - a - D
2,3,4
g a l a c t o p y r a n o s y l b r o m i d e (111,
-
has been p r e p a r e d and condensed w i t h
1 , 2 , 3 , 4 - t e t r a - g - a c e t y 1- Q - g a l a c t o p y r a n o s e t o y i e l d 1,2,3,4- t e t r a - g acetyl-6-0-2,3,4-tri-~-acetyl-6-O-(chloroacetyl)-~-~-galactosyl-Dgalactopyranose.36 group
from
the
After
adduct
selective removal o f
by
thiourea
c o n d e n s e d a g a i n w i t h (11) t o y i e l d , D-ga 1a c t o p y r a n 0 sy 1 - 6
treatment, after
the chloroacetyl the
deprotection,
product
was
6-0-(6-0-B-
-p- -ga 1a c t o p y r a n 0 sy 1) - p-- g a l a c t o p y r a n 0 s e
.
AcO AcO
(11)
2,3,4,6
- Te t r a -0a c e t y 1- a - Q - m a n n o p y r a n o s y 1 b r o m i d e h a s b e e n
r e a c t e d w i t h m e t h y 1 3 - g - b e n z y 1-4,6 -g-be n z y l ide n e - a - Q - m a n n o p y r a n 0 s i de i n t h e presence o f m e r c u r i c c y a n i d e t o y i e l d m e t h y l 3-g-benzyl-4,6-
O - b e n z y l ide n e - 2 - 2 - ( 2 , 3 , 4 , 6 mannopyranoside.37 a r id e
w i th
2,3,4,6
t e t r a - g - a c e t y 1- a - a - m a n n o p y r a n o sy 1) -a-B-
Condensation o f the
debenzylidenated disacch-
- t e t r a -0-a c e t y l - a - Q - m a n n o p y
afforded the corresponding trisaccharide, which t h e r e p e a t i n g u n i t o f an a l k a l i - s o l u b l e
ran0sy 1 bromide
is
r e p o r t e d t o be
polysaccharide
(S-Iawe)
i s o l a t e d from t h e m y c e l i a o f Epidermophyton floccosum.
p y r a n 0 sy 1-( 1+3) -0-a-L - r h amno py r a n 0 s y 1- ( 1 + 2 ) - a n d -O-a-L-Rhamno (1 + 3 ) - O - - a- L- r ham nopy ra n o se , two constituents o f b a c t e r i a l c e l l - w a l l
polysaccharides,
h a v e b e e n s y n t h e s i z e d.
U t i1iz in g 1 , 2 :3 , 4 - d i
-0-
iso p r o p y 1ide n e a-Q -ga 1a c t o p y r a no s e a s s t a r t i n g m a t e r ia 1, co n ve n i e n t s y n t h e s e s have
been r e p o r t e d f o r g-a-~-rhamnopyranosyl-(1+3) a n d
a - L - r h a m n o p y r a n o s y 1- ( 1+2) - 0 - a - k - r h a m n o w h i c h a r e components o f
flavonol
p y r a n 0 sy 1- ( 1+6) - B - g a l a c t o s e
trio side^.^'
0-
,
625
8: Chemical Synthesis and Modification The
synthesis of
2 ~ - ~ - a - ~ - f u c o p y r a n o s y l - l a c t o s he a s been r e p o r t e d ,
u s i n g 2,3: 5,6: 3’, 4’-t r i - g - i s o p r o p y l i d e n e - l a c t o s e
dimet h y l acet a 1
A s y n t h e s i s o f 2’-g-methyl-
( 1 2 ) as s t a r t i n g m a t e r i a l (Scheme l L 4 0 l a c t o s e was a l s o d e s c r i b e d . The
mercury
salt-catalysed
reaction
of
r h a m n o s i d e s has been s t u d i e d and t h e e f f e c t h a l i d e r e a c t i v i t y determined.41 part
of
the
repeating unit
Escherichia c o l i glucose,
075
been
(13)
(13)
of
the
synthesized
developed
was
for
groups of s e v e r a l of
substituents
on
Various trisaccharides i n c l u d i n g
g - g a l a c t o s e , and L-rhamnose.
c o l i tetrasaccharide has
were
a-Q-
substituted
g a l a c t o s y l h a l i d e s w i t h the r e a c t i v e 4-hydroxy
lipopolysaccharide from
from The
2-acetamido-Z-deoxy-Q-
full
synthesis
of
r e p o r t e d ~ e p a r a t e l y . ~A ~ new
the
stereoselective
o l i g o s a c c h a r i d e s c o n t a i n i n g a B - l i n k e d ;-mannose n o t a v a i l a b l e by d i r e c t s y n t h e s i s . 4 3
the
&.
method
synthesis
of
u n i t and p r e v i o u s l y
A silver silicate catalyst
p r e c i p i t a t e d on aluminium oxide i s used t o promote t h e g l y c o s i d e synthesis.
The s y n t h e s e s o f s e v e r a l Q - m a n n o s e - c o n t a i n i n g o l i g o -
s a c c h a r i d e s a r e r e p o r t e d u s i n g t h i s method. B-g-Mane-(1+4)-a-tj-Gale-(1+4)-l-Rha
1
I 3
6-Q -G lCeNAc (13) The s y n t h e s e s o f t h e t e r m i n a l d i s a c c h a r i d e a n d t r i s a c c h a r i d e a n t i g e n i c d e t e r m i n a n t s o f Groups A and C S t r e p t o c o c c i and o f t h e tetrasaccharide polysaccharide
sequence have
been
thought
to
constitute
reported.44
The
the
synthesis
variant-A has
been
r e p o r t e d o f t h e branched pentasaccharide r e p e a t i n g u n i t o f t h e 0specific side chain belonging t o the lipopolysaccharide obtained from
Shigella
halide block,
dysenteriae o f
serotype
2.45,46
o b t a i n e d from t h r e e 2-azido-sugars,
a disaccharide b l o c k t o g i v e t h e pentasaccharide. s t e p o f t h e s y n t h e s i s gave a - g l y c o s i d e s
A
trisaccharide
was c o m b i n e d w i t h Each c o m b i n a t i o n
stereoselectively.
The t r i s a c c h a r i d e s e q u e n c e 2 - a c e t a m i d o - 2 - d e o x y - a - ~ - g l u c o s y l (1+4)-B-Q-galactosyl-(l+4)-2-acetamido-2-deoxy-~-glucopyranose of blood-group-active
s u b s t a n c e s has been o b t a i n e d u s i n g a d i r e c t l y
stereoselective a-glycoside
s y n t h e t i c approach.47
A postulated
t r i s a c c h a r i d e o f human e r y t h r o c y t e membrane s i a l o g l y c o p r o t e i n s ,
0-0-
Carbohydrate Chemistry
626
4
x
v)
0
C
m
f-l ZI
a 0 0
3
ce I
N
0,
-01 I
d
r
I 4
0
0 I
I " g N$ 0
=
0" Q,
I
z
I
s = OO
c Q,
E
N
0
\ / O O N 0,
I
627
8: Chemical Synthesis and Modification
Q- - g a 1a c t o p y r a n o sy 1- ( 1+4) - 0 - 2 - a c e t am ido - 2 -de o x y -6 -Q- - g l uc o p y r a n o sy 1-
enzyl 6-0-allylh a s b e e n ~ y n t h e s i z e d . ~B ~
(1+3)-Q-mannopyranose,
-- b e n z y l - a - D - m - annopy 2,4-di -0
r a n o s i de w a s p r e p a r e d a n d t h e n c o n d e n s e d
w i t h an oxazoline derived from Q-lactosamine t o y i e l d a protected form o f t h e r e q u i r e d t r i s a c c h a r i d e .
- t e t r a -0a c e t y 1-a -Q - g a 1a c t o py r a no s y 1 b r o m i de
R ea c t io n o f 2,3,4,6 with
b e n z y l 2-acetam i d o -3,6-di-2-benzyl-2-deox
i n 1,2-dichloroethane
has p r o v i d e d a f t e r
be n z y l 2 - a c e t a m i d o -3,6 - d i s y 1)-a - Q - g l u c o p y r a n 0 s i de
.
y-a-Q-g1 ucopy r a n o s i d e
deacetylation
crystalline
-2- b e n z y l - 2 - d e o x y - 4 - ~ - ( B - ~ - g a l a c t o p y r a n o Ace t o n a t i o n f o 11ow e d by be n z y 1a t io n a n d
m i l d a c i d h y d r o l y s i s y i e l d s c r y s t a l l i n e b e n z y l 2-acetamido-4-0-(2,6-
-
-
-
d i-0 - b e n z y 1- B -R- ga 1a c t o p y r a n o s y 1) - 3 ,6 d i-0b e n z y 1 2 - d e o x y - a -B - g 1u c o R e a c t i o n o f t h i s d i o l w i t h l-g-(&-methyl)acetimidyl-
pyranoside.
-t e t r a - 2 - b e n z
2,3,4,6
y l-B-Q-gal actopy r a n 0se
r e g i o s p e c i f ic a 11y
a d e r i v a t i v e from which the blood-group-active
trisaccharide
y i e l de d
2-a-Q-
ga 1a c t o p y r a n o s y 1-( 1+3) - i - B -Q - g a l a c t o p y r a n 0 s y 1- ( 1+4 ) - 2 - a c e t am i d o - 2 d e o x y - e- - g l u c o p y r a n o s e
s a c c h a r ide.
0-B -Q - g a
was o b t a i n e d .
g l u c o p y r a n o s y 1)- ( 1+6) - Q - m a n n o s e , saccharide,
The
the tri-
synthesis o f
1a c t o p y r a n o s y 1-( 1+4) -g-( 2 - a c e t a m i d o - 2 - d e o x y-B-Q-. a b l o o d - g r o up
I i - a c t i ve o 1i g o -
has been r e p ~ r t e d . ~ ' B e n z y l 2,3,4-tri-g-benzyl-a-Q-
m a n n o p y r a n o s i d e was condensed w i t h a d i s a c c h a r i d e o x a z o l i n e ,
2-
ra-0-ace t y l -B
-B-
m e t h y l - { 3,6-di - 2 - a c e t y l - l , 2 - d i
deoxy-4-2- ( 2,3,4,6-tet
g a l a c t o p y r a n 0 s y 1)- a - Q - g l u c o p y r a n 0 1 - { 2 , l r e q u i r e d product. T h e t e t r a s a c c h a r ide de o x y -B
-a- - g l
azoli n e, t o yi e ld t h e
2-B -[1 -ga 1a c t o py r a n o s y 1- ( 1+4) - 2 - a c e t am ido - 2 -
u c o p y r a n o sy 1- ( 1+3 ) -B
amido-2-deoxy-Q-
-dl -2-ox
--; ga 1a c t 0 p y r a n o sy 1- ( 1+4 ) - 2 - a c e t -
g lucopy ranose
has
been
s y n t h e s i z e d.51
This
s t r u c t u r e has been found i n g l y c o l i p i d s i s o l a t e d f r o m e r y t h r o c y t e m e m b r a n e s a n d i s r e c o g n i z e d by s e v e r a l a n t i - I the
synthetic
route
to
the
Lewis
b
antisera.
As p a r t o f
blood-group
antigenic
t h e r a p i d s y n t h e s i s o f b e n z y l 2 - a c e t am id o - 2 - d e o x y - 3 - 0 -
determinant,
B-Q - g a l a c t o p y r a n 0 sy 1-B -Q - g l u c o p y r a n 0 s i de h a s b e e n r e p o r t e d . c o m p o u n d was d e r i v a t i z e d t o y i e l d (141, w it h 2 , 3 , 4 -
af f orde d
t r i -0be n z y l -a
the
-I -fucopy
r a n 0 sy 1 b r o m i d e a n d d e p r o t e c t i o n
r e q u i r e d t e t r a s a c c h a r i de
Q - g a l a c t o p y r a n 0 sy 1- ( 1+3)
-2- {a-L-
This
which f o l l o w i n g condensation
,
O-a-L-
f u c o p y r a n 0 sy 1-( 1+2) -6-
f u c o p y r a n 0 s y 1- ( 1 + 4 ) 1 - 2 - a c e t am i d o -2-
deoxy-Q-gl ucose. Treatment
of
b e n z y l 2-acetamido-3,6-di-g-benzyl-2-deoxy-4-{-B-
Q - g a 1a c t o p y r a n o s y 1-0:-Q - g l u c o p y r a n o s id e w i t h be n z a 1 de h y d e i n t h e presence o f
zinc
chloride
followed
by
regioselective
benzoylation
h a s p r o v i de d c r y s t a 11i n e be n z y 1 2-a c e t am ido - 4 - 2 - ( 3 - 2 - b e n z o y 1-4,6
-0-
628
Carbohydrate Chemistry
be n z y l i d e ne -$ -Q-ga 1a c t o p y r a n 0 sy 1) - 3 , 6 - d i -0-be n z y l - 2 - d e o x y - a - Q - g l uco p y r a n ~ s i d e . ~C~o n d e n s a t i o n o f be n z y l -1 ( 2,3,4
this
disaccharide
-
w i t h 2,3,4-tri-Q-
-0-(t-me t h y 1a c e t im idy 1)-B-L-fuco py r a n 0 se y i e l d e d be n z y l 0nz y l -a -C- f uco py r a n 0 sy 1 ) - ( 1+2) -0( 3-g-be n z y 1 - 4 , 6 -0-
- t r i -:-be
b e n z y l i d e ne - B -9-ga 1a c t o p y r a n 0 sy 1) - ( 1+4) -2-ace t am i d o - 3 , 6 - d i - 2 - b e n z y l 2-deoxy-a-Q-glucopyranoside. Debenzoylation, f o l l o w e d by co n de n s a t i o n w it h 2,3 ,4 ,6 - t e t r a -0be nz y 1- 1-0(y- m e t h y 1ace t im id y 1) -B 0-galactopyranose,
p r o v i d e d a t e t r a s a c c h a r i de de r i va t i v e t h a t w a s
(151, t h e a n t i g e n i c d e t e r m i n a n t o f
c a t a l y t i c a l l y hydrogenolysed t o human b l o o d g r o u p B - t y p e 2.
I
OH
NHAC (14)
I
HO
The a n t i g e n i c d e t e r m i n a n t s o f t y p e 2 o f b l o o d - g r o u p s u b s t a n c e s A and B a s w e l l as b l o o d - g r o u p
s u b s t a n c e H have been s y n t h e s i z e d by
s e l e c t i v e g l y c o s i d a t i o n r e a c t i o n s combined w i t h a s e r i e s o f b l o c k i n g and d e b l o c k i n g steps.54
The p e n t a s a c c h a r i d e c h a i n o f
t h e Forssman
a n t i g e n h a s been s y n t h e s i z e d u s i n g a n o v e l b l o c k s y n t h e s i s method i n v o l v i n g c a t a l y s e d r e a c t i o n o f s u i t a b l y p r o t e c t e d d i s a c c h a r i d e and t r i s a c c h a r i d e d e r i v a t i v e s . 55 R e g i o - and s t e r e o - c o n t r o l l e d pentasaccharides (16)
and ( 1 7 )
synthetic routes t o the branching
have
been r e p o r t e d . 5 6
Similar
syn-
t h e t i c r o u t e s t o t h e b r a n c h e d m a n n o p e n t a o s i d e (18) a n d mannohexa-
629
8: Chemical Synthesis and Modification
a-Q-Mane- ( 1+2) -a-D -Mane- ( 1+6) -a
-0 -Mane-
( 1+OMe
3
1' 1 a -Q -Mane 2
t 1
a -Q -Mane
(16)
t3 -Q-GlcQNA c - ( 1+2 ) -a -Q -Mane- ( 1+6 ) -a -Q-Mane- ( 1+0M e 3
c
1 a -p -Mane
2
? 1 B-Q-GlceNAC (17)
a-Q-Mane-( 1 + 6 ) - a - Q - M a n ~ - ( 1 + 6 ) - a - Q - M a n ~ - ( 1+OMe 3
3
t
t 1
1
a -Q -Mane
.+-Mane
2
+
1 R
(18)
R = H
( 19)
R = a-Q-Mane
o s i d e (19) have a l s o been described.57 man no py r a no sy 1-a -Q -man no py r a no sy 1)- 3 -2-a
M e t h y l 6-0-(2,6-di-g-a-Q-
-p -man no py r a no sy 1-a-Q -man no -
2,6-di -2-a-p-mannopy r a n 0 sy 1)-a-Q-mannopy r a no s ide a n d m e t h y 1 6 -2-( py r a no s i de h a v e
been sy n t h e s i z e d.
3 -0- ( 3,6 -D i-2-a
-Q -m anno py r a no -
630
Carbohydrate Chemistry
sy l-a-Q-mannopy r a n 0 sy 1) -6-2-a-p-mannopy a n d m e t hy 1 3 -2- ( 3,6 -2-a
-O-a-Q-mannopy
-p -m
r a n o sy l-a-Q-mannopy r a n o s i d e
anno py r a no sy 1- a -Q -man no py r a no sy 1)-6-0- ( 2-
r a n 0 sy l-a-Q-manno py r a n 0 sy 1)-a-Q-manno py r a n 0 s i d e ,
which
p r o v i d e models o f t h e o u t e r c h a i n o f t h e g l y c a n o f soybean agglut i n i n , have been prepared.59 of
cell-wall
e f f ic i e n t
proteoglycans
To p r o v i d e a m o d e l o f a n i n n e r c h a i n
isolated
from
Piricularia
sy n t h e s e s h a ve bee n de s c r ibe d f o r m e t h y 1 2,6 - d i
oryzae,
-2- ( 2-0-a -
p-manno py r a n 0 sy l - a -Q -m anno py r a no sy 1)-a -Q -m anno py r a no s i de a n d m e t h y 1
-2- ( 2-2-a-a-mannopy
2,4-di
o s i de. 6 o
r a n 0 sy 1 - a - p -manno py r a no sy 1) -a-Q-mannopy r a n -
m e t h y l 3 , 4 - d i -2-be nz y l - a - Q - m anno -
g l yco sy 1 a c c e p t o r s
The
py r a n o s i d e and m e t h y l 3,6-di -2-benzyl-a-Q-mannopyranoside reacted
with
suitably
protected
were
a - m anno t r i o s i de s.
S i l v e r t r i f l uo r om e t h a n e s u l p h ona t e - p r o m o t e d c o n d e n s a t i o n o f 3,6d i -2-acetyl-4-2-(
2,3,4,6-
t e t r a - 0 - a c e t y l - $ - Q - g a l a c t o p y r a n o s y l ) -2-
d e o x y - 2 - p h t h a l i m i d o - $ - Q - g l u c o p y r a no s y 1 b r om i d e w it h b e n z y l 3 - 2 be n z y l - 4 , 6 - g - b e
nz y l i d e n e -a-Q-manno py r a n 0 s i de
-
- a -Q - m a n no py r a no s ide , a n d be n z y 1 3,6 - d i mannopyranoside saccharide
has
yielded
derivatives,
,
be nz y l 3 , 6 - d i -2-be nz y l
- t r i -0- be nz y l - a -p -
-0- ( 3,4,6
p r o t e c t e d tri-,
penta-, and h e p t a -
a l l c o n t a i n i n g 2-acetamido-2-deoxy-B-Q-
l a c t o s a m i n y l residues.61
The f r e e o l i g o s a c c h a r i d e s , p a r t s o f t h e
complex-type carbohydrate m o i e t y o f g l y c o p r o t e i n s , were o b t a i n e d a f t e r e x c h a n g i n g t h e 2 -deox y-2- p h t h a 1 i m ido g r o u p s f o r 2-ace t am ido -2
-
deoxy g r o u p s a n d d e b l o c k i n g . Regio- and s t e r e o - c o n t r o l l e d s y n t h e s i s o f t h e h e x a s a c c h a r i d e ( 2 0 ) h a s been a c h i e v e d by e m p l o y i n g a p r o p e r l y p r o t e c t e d m a n n o t r i o s i d e a s a key osy 1-( 1+4)
intermediate.62
-2- ( 2-ace t am i d o -2-deox
pyranose,
part
of
The s y n t h e s i s o f 2 - B - Q - g a l a c t o p y r a n y- B - Q - g l ucopy r a n 0 sy 1) - ( 1 6 ) -Q-manno
the most highly
carbohydrate portions o f glycoproteins,
Glycoproteins.
--
branched complex
type
-
o f
has been r e p o r t e d . 6 3
The a c y l h y d r a z i d e d e r i v a t i v e o f 8 - m e t h o x y c a r b o n -
y 1 o c t y 1 2-ace tam i d o -2 -deox y- 3 -g-(B -Q -ga 1a c t o p y r a no sy 1)-a
-p -ga 1a c t o -
p y r a n o s i d e has been r e a c t e d w i t h b o v i n e serum a l b u m i n t o y i e l d an effective per
artificial
mole o f
T-antigen
protein.21
c o n t a i n i n g 22 e q u i v a l e n t s o f h a p t e n
4 - T r i fluoroacetamidopheny1 3-2-(a-Q-gluco-
py r a n o s y l ) -a-Q-mannopy r a n 0 s i d e h a s b e e n c o n v e r t e d t o i t s i s o t h i o c y a n a t e d e r i v a t i v e a n d c o u p l e d t o b o v i n e serum a l b u m i n t o . a f f o r d a n a r t i f i c i a l S a l m o n e l l a antigen.2’ P s e u d o g l y c o p r o t e i n s h a v e b e e n s y n t h e s i z e d by t r e a t i n g p r o t e i n s with
carbohydrates
a f f i n i t y
(see
e.g.
chromatography.
Tables The
1-5),
chiefly
preparation,
for
use i n
p r o p e r t i e s , and
63 1
8: Chemical Synthesis and Modification applications
of
carbohydrate
conjugates
of
proteins
have
been
reviewed.64
$-Q-Glc~NAc-(1+4)-a-Q-Manp-(l+6)-a-Q-Manp(l+OMe 2
3
t
t
1
1 6-p-GlceNAc
a-E-Mane 2
t
1 $ -Q -G 1CeNA C
(20)
Glycopeptides. prepared,
--
N-(L-B-Asparty 1 ) - a - Q - g l u c o p y r a n o s y l a m i n e h a s b e e n
as a m o d e l o f a c o r r e s p o n d i n g d e r i v a t i v e p o s s i b l y p r e s e n t
i n glomerular
basement
carbonyl-I=-aspartate
was
membrane o f condensed
rats.65
a-Ethyl
benzyloxy-
w i t h 2,3,4,6-tetra-g-acetyl-a-Q-
glucopyranosylamine i n the presence o f diethylphosphocyanidate. T h i s was f o l l o w e d by h y d r o g e n o l y s i s ,
acetylation,
and d e - e t h o x y l a t -
i o n o f t h e r e s u l t i n g 2,3,4,6-tetra-g-acetyl-N-(a-ethyl
carbonyl-I=-B-asparty1)-a-Q-glucopyranosylamine
benzyloxyt o remove t h e
p r o t e c t i n g groups. O t h e r c o m p o n e n t s o f t h e g l o m e r u l a r basement membrane, g - ( 2 - g - a -
~ - g l u c o p y r a n o s y l ) - $ - ~ - g a l a c t o p y r a n o s i d e so f hydroxy-l.=-lysylglycine aspartylglycine,
optically
pure
6-
and 6 - h y d r o x y - ~ - l y s y l g l y c y l - ~ - g l u t a m y l - ~ -
have a l s o been s y n t h e s i z e d . 6 6
The
use o f
the
l e v u l i n y l g r o u p as a t e m p o r a r y b l o c k i n g g r o u p f o r t h e 2 - h y d r o x y function
of
!-galactose
f o r m a t i o n of
a B-linkage
glycine,
proved
to
be
very
between a-galactose
efficient
for
and a l s o f o r t h e l i n k a g e o f Q - g a l a c t o s e t o Q - g l u c o s e
a - i n t e r g l y c o s i d i c bond.
the
and 6 - h y d r o x y - i - l y s y l an
S y n t h e s i s i n s o l u t i o n and i n s o l i d phase
h a s been u s e d t o p r e p a r e N - a c e t y l m u r a m y l d e r i v a t i v e s o f a number o f ~ l i g o p e p t i d e s . ~I m ~m u n o a d j u v a n t a c t i v i t i e s w e r e d e t e r m i n e d f o r t h e s e d e r iv a t i v e s . I n o r d e r t o c l a r i f y t h e s t r u c t u r a l r e q u i r e m e n t s f o r t h e immunoadjuvant a c t i v i t y o f t h e carbohydrate moiety i n tj-acetylmuramoyl-kalanyl-Q-isoglutamine,
2-acetamido-Z-deoxy-4-
and -6-g-(Q-Z-propan-
oyl-&-alanyl-P-isoglutaminel-;-glucopyranose, 2-acetamido-Z-deoxy-3-O - (-~ - 2 - p r o p a n o y l - ~ - a l a n y l - ~ - i s o g l u t a m i n e ) - ~ - a l l o p y r a n o s e ,- g - g u l o pyranose,
-!-galactopyranose,
-;-mannopyranose,
and - I - i d o p y r a n o s e ,
632
Carbohvdrate Chemistry
and 3-g-(Q-2-propanoy
l - k - a l a n y 1- P _ - i s o g l u t a m i n e 1-E-
p y r a n o s e h a v e been s y n t h e s i z e d . 6 8
and - L - g l u c o -
Immunoadjuvant a c t i v i t y o f t h e
a c e t y l m u r a m o y l d i p e p t i d e a n a l o g u e s was e x a m i n e d i n g u i n e a p i g s .
EThe
syntheses o f n o v e l 1-acylmuramoyl d i p e p t i d e s r e l a t e d t o t h e l i p i d A constituent
of
the
bacterial
lipopolysaccharide
have
reported.69
A l l
the
synthetic
muramoyl dipeptide
analogues
exhibited potent a c t i v i t y
i n i n d u c i n g delayed-type
been
hypersensitivity
i n guinea pigs. The
syntheses o f
t h r e e 4-deoxy
analogues,
and a 4,6- d i c h l o r o -4,6- d i d e o x y - 2 - g a l a c t o
a 4-ch,loro-4-deoxy-
a n a l o g u e o f !-ace
t y l m uram-
o y l - ~ - a l a n y l - ~ - i s o g l u t a m i n eh a v e b e e n r e p o r t e d a n d t h e i r i m m u n o adjuvant a c t i v i t i e s i n v e s t i g a t e d . 70 methods,
Using solid-phase synthesis
t r a - 2 - a c e t y 1-1-tj-{E-( t e r t - b u t y l o x y c a r b o n y l ) - L -
2,3,4,6-te
a s p a r t - 4 - o y l ) - Q - g l u c o p y r a n o s y l a m i n e and 2-acetamido-3,4,6-tri-gacetyl-l-~-{~-(tert-butyloxycarbonyl)-~-aspart-4-oyl~-2-deoxy-~glucopyranosylamine
have
been
s e q ue n c e 5 t o 9 o f s o m a t o s t a t in
.
introduced
into
the
amino
acid
3 -0- ( 2 - A c e t a m ido - 3,4,6 - t r i-0-
acetyl-2-deoxy-B-~-glucopyranosyl)-tj-(tert-butyloxycarbonyl)-~s e r i n e h a s been p r e p a r e d a n d c o n d e n s e d by t h e s o l i d - p h a s e to
give the
model g l y c o t r i p e p t i d e
procedure
(21) and a g l y c o s o m a t o s t a t i n
( 2 2 1 . ~ ~ G l y - { B-g-GlcpNAc-( 1+3) -Ser )-Ala-OH (21)
B-~-Glc~NAc-(1+3)-Ser-13-somatostatin (22)
Glycolipids.
--
M o r a p r e n y l phosphate has been t r e a t e d w i t h
sulphinyldi-imidazole a-g-galactopyranosyl moraprenyl,
N,tj'-
and t h e r e s u l t i n g i m i d a z o l i d a t e r e a c t e d w i t h
or a-P-glucopyranosyl phosphate t o g i v e t h e P l p y r o p h o s p h a t e s (23) or ( 24).73 The method
P2-glycosyl
was u s e d t o p r e p a r e t h e b i o l o g i c a l l y a c t i v e o l i g o s a c c h a r i d e p o l y p r e n y l d e r i v a t i v e s (25) and (261, biosynthesis o f 2-specific
which are intermediates i n the
polysaccharides o f Salmonella serological
g r o u p E. Two
disaccharide
analogues
o f
l i p i d
A
glycoside precursors of and
mono-dephospho
the palmitoyl
l i p i d A
have
been
~ y n t h e s i z e d . ~A ~ l t h o u g h p a l m i t i c a c i d was t h e s o l e f a t t y a c i d used, t h e s y n t h e t i c scheme p r o v i d e s f o r t h e s e l e c t i v e i n t r o d u c t i o n o f
any
d e s i r e d s a t u r a t e d f a t t y a c i d a t each o f t h e f i v e a c y l a t e d p o s i t i o n s
8: Chemical Synthesis and Modification
633
A c o n v e n i e n t s y n t h e s i s o f 2-deoxy-Z-Q-
i n the f i n a l product.
- L- - ( 3 - h y d r o x y t e t r a d e c a n o y l - a m i n o ) - p - g l u c o s e
diastereoisomers o f
m o n o m e r i c l i p i d A component o f t h e b a c t e r i a l l i p o p o l y s a c c h a i d e been r e p o r t e d ,
s t a r t i n g from
and the has
d i a s t e r e o i s o m e r i c benzyl 3,4,6-tri-g-
acetyl-2-deoxy-2-(~~-3-hydroxytetradecanoylamino)-~-Q-glucopyranos i d e . 75
CHqOH
II 0 -POH I
0
II
0 - P-
I 0-
0-
(23) R'
= OH,
(24) R'
= R 3 = H,
= OH,
R2 = H
Cholesterol-containing h y d r o p h i l i c spacer
R2 = R3 = H R 2 = OH
= OH, R 2 = H, R 3 = a - i - R h a e - ( l +
(25)R' (26) R '
ORm
group,
,
R 3 = B-Q-Man~-(1+4)-a-L-Rha~-(l+
B-Q-galactosyl
g l y c o l i p i d s h a v i n g a new
8-amino-3,6-dioxaoctanoic
~ y n t h e s i t e d . ~T h ~e h y d r o p h i l i c n a t u r e o f
acid,
h a v e been
t h i s spacer
group
e l i m i n a t e s many o f t h e p r o b l e m s i n h e r e n t i n t h e use o f h y d r o p h o b i c
o r c h a r g e d s p a c e r arms. Condensati o n o f Ij,"-diacet by o x i d a t i o n o f N,"-diacety
N,"-diacetylchitobionic
y l c h it obiono-1,5-lactone,
l c h it o b i o s e ,
I j - a l k y l a m i d e ~ . ~ The ~
g l y c o l i p i d s w i t h wheat - g e r m
produced
w i t h l-aminoalkanes
yields
i n t e r a c t i o n o f these
a g g l u t i n i n and G r i f f o n i a s i m p l i c i f o l i a
I 1 l e c t i n was d e s c r i b e d .
A convenient approach t o t h e s y n t h e s i s o f t r i g l y c o s y l - 2 , 3 - d i - g phytanyl-%-glycerols
2
h a s been r e p o r t e d
(Scheme 2).'*
Modification of Polysaccharides and Oligosaccharides and Uses o f Modified Polysaccharides and Oligosaccharides
Introduction.
--
P o l y s a c c h a r i d e d e r i v a t i v e s c o n t i n u e t o be w i d e l y
used i n t h e p r e p a r a t i o n o f m a t r i c e s f o r
affinity
chromatography.
A
book has been p u b l i s h e d w h i c h p r e s e n t s a t h e o r e t i c a l t r e a t m e n t o f t h i s technique together with d e t a i l e d information concerning the p r e p a r a t i o n and use proceedings of
of
the 4th
affinity-chromatographic International
media.79
The
Symposium o n A f f i n i t y
Carbohydrate Chemistry
634
fib
CH,OBn
-
BnO@Br
OBn
+, J
0 1111
HO
R =
>Si
/i,viii
& o
AcO
-
Reagents:
-tg
CHMeCH2% H
iii, iv
/
0
diq
+CH,CH,
-i
>Si’
fOAC
AcO
~
I
A
-
“‘l“‘d
-to 0
i,p h y t y l b r o m i d e ; ii,H 2 / P d ; iii,C 1 ( P r i 2 ) S i O S i ( P r 2)C 1; iv 2 - d i br omo me t h y 1be n zoy 1 c h l o r i d e ; v , Ac20 pyr; v i , s i l v e r t r i f l a t e ; v i i , 2,3,4-tri-(g-acetyll-6(2,2,2-triethoxycarbonyl)-Q-mannosyl b r o m i d e - a c e t o n i t rile/nitromethane; v i i i , Zn/THF-AcOH; ix, 2,3,4,6tetra-g-acetyl-a-a-glucopyranosyl b r o m i d e , HgBr2, Hg(CN12; x , Na’OMe-/MeOH/ether
,
Scheme
2
635
8: Chemical Synthesis and Modification Chromatography,
held i n
Veldhoven
i n
June
1981,
have
been
~ u b l i s h e d . ~ ’ They p r o v i d e a s u r v e y o f new d e v e l o p m e n t s i n t h e f i e l d and i l l u s t r a t e t h e i n c r e a s i n g i m p o r t a n c e o f a f f i n i t y i n d u s t r i a l and b i o m e d i c a l / d i a g n o s t i c
applications.
saccharide polymers i n chromatography proceedings o f molecules.81
the
techniques i n
The u s e o f p o l y -
has been d e s c r i b e d i n t h e
1 8 t h Prague IUPAC Microsymposium
The use o f p o l y s a c c h a r i d e s a s s u p p o r t
on Macro-
polymers i n the
i m m o b i l i z a t i o n o f m a c r o m o l e c u l e s w i t h b i o c h e m i c a l and b i o m e d i c a l applications
h a s been d i s c u s s e d . 8 2
Summaries o f
recent l i t e r a t u r e
o n m o l e c u l a r i m m o b i l i z a t i o n and b i o a f f i n i t y phenomena, i n v o l v e s use o f p o l y s a c c h a r i d e m a t r i c e s ,
much o f w h i c h
are being published a t
r e g u l a r i n t e r vals.83-86 The a f f i n i t y c h r o m a t o g r a p h y o f m a c r o m o l e c u l a r s u b s t a n c e s o n a d s o r b e n t s b e a r i n g c a r b o h y d r a t e l i g a n d s h a s b e e n r e ~ i e w e d . ~ ’ The use o f p o l y s a c c h a r i d e for
supports b e a r i n g i s o n i t r i l e f u n c t i o n a l groups
c o v a l e n t f i x a t i o n o f b i o l o g i c a l l y a c t i v e m o l e c u l e s has been
described.88
Reviews have
appeared concerning the affinity-
chromatographic p u r i f i c a t i o n o f
lectins
utilizing
m a t r i x -bo un d g l y co p r o t e i ns a n d g l yco pe p t ide s and
di- saccharide^,^'
and
the
use
of
,
polysaccharides,
and m a t r i x -bo un d m ono
-
affinity-chromatographic
t e c h n i q ue s i n t h e p u r if i ca t i o n o f m amm a 1 i a n g l yco sy 1t r a n s f e r a ses. ’O The a p p l i c a t i o n o f
affinity-chromatographic
separations
studies
components of A
of
and
on
molecular
techniques
interactions
for
of
the
t h e b l o o d - c o a g u l a t i o n system has been d e ~ c r i b e d . ’ ~
u n i f i e d treatment o f systems containing i m m o b i l i z e d bio-
c h e m i c a l s a n d c h r o m a t o g r a p h i c s y s t e m s has been d e v e l o p e d f r o m b a s i c thermodynamic The
considerations of
theoretical
coefficients
from
treatment readily
p a r t i t i o n i n g i n b i p h a s i c systems.’* allows
prediction
obtainable experimental
of
partition
parameters,
and
s i m p l e procedures are suggested f o r the d e t e r m i n a t i o n o f b i n d i n g c o n s t a n t s by a f f i n i t y o r a d s o r p t i o n c h r o m a t o g r a p h y . The t e c h n i q u e o f
affinity
electrophoresis
has been r e v i e ~ e d . ’ ~
I n a theoretical treatment of the subject, evaluation o f the e f f e c t s of
protein multivalency,
m i c r o d i s t r ib u t i o n ,
immobilized
ligand heterogeneity
and
and d e t e r m i n a t i o n o f e f f e c t i v e c o n c e n t r a t i o n s o f
i m m o b i l i z e d l i g a n d has been ~ o n s i d e r e d . ’ ~ The use o f a f f i n i t y
i s o e l e c t r i c focusing i n polyacrylamide gels
has been d e s c r i b e d f o r q u a l i t a t i v e
determination o f ligand-binding
p r o t e i n s and f o r e x a m i n i n g t h e f u n c t i o n a l h o m o g e n e i t y o f p u r i f i e d protein
preparation^.'^
A f f i n i t y g e l s c o n t a i n i n g i m m o b i l i z e d sugars
o r b l u e d e x t r a n were used t o d e m o n s t r a t e t h e i n t e r a c t i o n o f t h e
636
Carbohydrate Chemistry
immobilized ligands with l e c t i n s o r lactate dehydrogenase.
-- A g a r o s e g e l s c r o s s l i n k e d t o d i f f e r e n t e x t e n t s h a v e f r a c t i o n a t i o n o f human serum.96 A method b e e n u s e d f o r t h e h.p.1.c. f o r running i s o e l e c t r i c - focusing separations under denaturing The method m i n i m i z e s c o n d i t i o n s i n a g a r o s e has been d e s c r i b e d . " t h e r i s k o f c a r b a m y l a t i o n o f p r o t e i n s when u s i n g u r e a i n c o m b i n a t i o n with agarose. An i m p r o v e d r a p i d m e t h o d o l o g y f o r t h e i s o l a t i o n o f n u c l e i c a c i d s from a g a r o s e g e l s has been described.98 Hydroxy-apatite is used t o bind the deoxyribonucleic a c i d a f t e r s o l u b i l i z a t i o n o f a g a r o s e w i t h p o t a s s i u m i o d i d e o r by h e a t i n g , a n d t h e n u c l e i c a c i d c a n t h e n be e l u t e d f r o m t h e i n o r g a n i c m a t r i x free o f a g a r o s e . L i v i n g i m m o b i l i z e d c e l l s h a v e b e e n p r e p a r e d by e n t r a p m e n t i n a g a r g e l s . 99 9 l o o Agarose has been used without modification as an affinityc h r o m a t o g r a p h i c s u p p o r t f o r t h e p u r i f i c a t i o n o f a n t i - ( T h y 1.2) monoclonal a n t i b o d y c o v a l e n t l y l i n k e d t o ricin."' Acid t r e a t m e n t o f a g a r o s e t o expose !-galactose units has provided an a f f i n i t y matrix for t h e purification of a-galactose b i n d i n g l e c t i n s f r o m s o m e E r y t h r i n a specie^.^^',^^^ A mathematical model has been derived f o r the c a l c u l a t i o n o f isothermal distribution coefficients f o r the immobilization of r e s i d u e s on a g a r o s e gels.lo4 S i m i l a r c o e f f i c i e n t s have been o b t a i n e d experimentally i n chromatographic runs. Agarose h a s been o x i d i z e d w i t h p e r i o d a t e and t h e n c o u p l e d w i t h haemoglobin i n the p r e s e n c e of sodium c y a n o b o r o h y d r i d e t o y i e l d a n a f f i n i t y m a t r i x f o r t h e p u r i f i c a t i o n o f haemoglobin-binding p r o t e i n s .lo5 Gels p r e p a r e d by i m m o b i l i z a t i o n o f a c r i f l a v i n o n a g a r o s e h a v e been s u c c e s s f u l l y a p p l i e d t o t h e s e p a r a t i o n of n u c l e o t i d e s , oligonucleotides, and nucleic acids.lo6 The use o f f l a v i n d e r i v a t i v e s immobilized on agarose f o r a f f i n i t y chromatography has been reviewed."' A l k y l a n d a r o m a t i c d e r i v a t i v e s o f a g a r o s e c o n t i n u e t o be u s e d i n p r e p a r a t i v e t e c h n i q u e s i n vol vin g hydrophobic chroma t o graphy and The r e l a t i v e c o n t r i b u t i o n o f h y d r o p h o b i c f o r enzyme i m m o b i l i z a t i o n . and i o n i c interactions during chromatography of proteins on h o m o l o g o u s s e r i e s o f h y d r o c a r b o n - c o u p l e d a g a r o s e s , o b t a i n e d by c o u p l i n g o f a l k y l a m i n e s t o a g a r o s e , h a s b e e n a s s e s s e d by c o m p a r i n g these columns w i t h two similar s e r i e s o f columns devoid o f Agarose.
637
8: Chemical Synthesis and Modification charge.lo8
Using a
columns
identical
of
variety
o f pure
p r o t e i n s i t was shown t h a t
l i g a n d d e n s i t i e s have,
qualitatively
and
s i m ila r a d s o r p t i o n a n d d i s c r i m i n a t i o n p r o p e r t i e s
quantitatively,
whether charged or not.
From t h e s e r e s u l t s i t was c o n c l u d e d t h a t
hydrophobic r a t h e r than i o n i c i n t e r a t i o n s are o f primary importance i n chromatography on a l k y l - a g a r o s e columns. The e t h e r - l i n k e d h y d r o p h o b i c s u b s t i t u e n t s o f o c t y l - a g a r o s e phenyl-agarose
have
been
cleaved with
q u a n t i f i e d by g a s c h r o m a t o g r a p h y . l o 9 reproducible, liga n d s
and
.
can
easily
and
boron tribromide and
The p r o c e d u r e i s s e n s i t i v e ,
be a p p l i e d
to
other
ether-linked
The b i n d i n g o f p h y t o c h r o m e f r o m pea s e e d l i n g s t o a l k y l - a n d w am in o a 1 k y l - a g a r o s e s h a s b e e r l i n v e s t i g a t e d . l 1 0
w - A m in o b u t y l - a g a r o s e
has been a p p l i e d t o t h e i s o l a t i o n o f a n o n - h i s t o n e m o b i l i t y - group p r o t e i n from has been used f o r
for
liver
the purification o f
m u r i n e in t e s t i n e 1 1 2
-___ c o c c u s ---aureus
mouse
Decyl-agarose
glycosyl-ceramidase
from
a n d f o r h y d r o p h o b i c chroma t o graphy o f Staphylo:
delta-toxin.'l3
purification
chromosomal high-
nuclei.'"
of
w-Aminoethyl-agarose
glycogen-debranching
enzyme
h a s been used from
rabbit
m us c l e. 1' 4
6-Aminohexyl-agarose
h a s been a p p l i e d t o
n u c l e a r androgen r e c e p t o r , l l 5 (Platichtys flesus),l16 from
l i v e r o f flounder
t h e p y r u v a t e dehydrogenase component o f t h e
p y r u v a t e dehydrogenase oxidase
the p u r i f i c a t i o n s o f
phosphorylase b from
complex
f r o m E s c h e r i c h i a c o l i KIZ,ll'
bovine serum,ll8
a rat liver
p h o s p h o d i e ~ t e r a s e , a~n~d ~a g - m a n n a n - b i n d i n g
amine
c y c l i c GMP-activated p r o t e i n associated with
t h e e a r l y c h i c k e n em bryo.12'8-Amino-octyl-agarose
h a s been used f o r t h e p a r t i a l p u r i f i Commercially a v a i l a b l e
c a t i o n o f c y t o c h r o m e P-450 f r o m rats.121 octyl-agarose
has been used f o r t h e p u r i f i c a t i o n o f t h e g l y c o s y l -
ceramidase from
murine intestine,'12
o f a B-Q-hydroxybutyrate M,lZ3
o f
Staphylococcus
h e x o s a m i n i d a s e C,124 of
of
pig urinary
dehydrogenase from aureus
kallikrein,lZ2
Z o o g l o e a r a m i g e r a I-16-
delta-toxin,l13
of
bovine
brain
of a topoisomerase from U s t i l a g o maydis,lZ5
uroporphorinogen I
s y n t h a s e f r o m human e r y t h r o c y t e s . l Z 6
and
Octyl-
a g a r o s e h a s a l s o been a p p l i e d s u c c e s s f u l l y t o t h e p u r i f i c a t i o n o f a modified fluorescent heparin preparation.lZ7
A simple procedure f o r
t h e p r e p a r a t i o n o f human l y m p h o b l a s t o i d i n t e r f e r o n i n h i g h y i e l d involves
chromatography
purification.128
on
octyl-agarose
as
one
step
S p h i n g o m y e l i n h a s been i m m o b i l i z e d by
of
the
adsorption
o n t o o c t y l - a g a r o s e and t h e n used as s u b s t r a t e i n a n enzyme assay f o r
638
Carbohydrate Chemistry
three
b a c t e r i a l p h o s p h o l i p a s e s . 129
By
immobilizing labelled l i p i d
by t h e same m e t h o d t h e p e r f o r m a n c e o f t h e same enzyme a s s a y has been s i m p l i f i e d and t h e s e n s i t i v i t y
increased.l3O
H y d r o p h o b i c - i n t e r a c t i o n c h r o m a t o g r a p h y u t i l i z i n g p h e n y l - a g a r o se h a s been a p p l i e d t o t h e c o - p u r i f i c a t i o n o f a l k a l i n e p h o s p h a t a s e and 5'-AMP-specific
nucleotidase from
D i c t y o s t e l i u m d i s c ~ i d e u m , ' ~ a~n d
to the p u r i f i c a t i o n o f v a n i l l a t e hydroxylase
( d e c a r b o x y l a t i n g ) from
S p o r o t r i c h u m p u l v e r ~ l e n t u m , o~f ~ E~u g l e n a g r a c i l i s elongation
factor
and o f
chloroplast
h u m a n ~ r o 1 a c t i n . l ~M~e m b r a n e
p r o t e i n s f r o m human b r a i n h a ve been c h r o m a t o g r a p h e d o n p h e n y l agarose
after
solubilization
hydroxylase,136
with
Escherichia c o l i
Staphylococcus
Triton
a u r e u s d e l t a - t o x in,113
bo v ine b r a in B -Q - 2
Subunits o f pike-eel
host
g l uco s i da se
,
2 4 and r a t 1i v e r
been p u r i f i e d
u s i n g phenyl-
g o n a d o t r o p h i n have been s e p a r a t e d by
A Treponema R e i t e r
hydrophobic chromatography o n p h e n y l - a g a r 0 ~ e . l ~ ' RNA a n t i g e n w h i c h
Tyrosine factor,137
rat uterine p e r o ~ i d a s e , ' ~ ~
- a c e t a m ido - 2 -deox y -
p h e n y l a l a n i n e h y d r o x y l a ~ e lh~a v~e a l s o agarose.
X-100.135
integration
p r e c i p i t a t e s w i t h a n t i b o d i e s i n human s y p h i l i t i c
s e r a has been p u r i f i e d o n p h e n y l - a g a r 0 ~ e . l ~ P ~henyl-agarose also
has
been u s e d f o r t h e p u r i f i c a t i o n o f an e s t e r h y d r o l a s e w h i c h
hydrolyses phorbol diesters from murine liver,142
a n d o f a Ca2+-
a c t i v a t e d n e u t r a l protease from r a b b i t s k e l e t a l m ~ s c 1 e . l ~ ~
3 - c a r b o x y p r o p i 0 n y 1)am in o -
10-Car b o x y d e c y l a m i n o - a g a r o s e a n d !-(
o c t y l - a g a r o s e have been p r e p a r e d a n d u s e d f o r c h r o m a t o g r a p h y o f wheatgerm
aspartate
t r a n ~ c a r b a m o y 1 a s e . l ~ ~U n d e r
appropriate
e x p e r i m e n t a l c o n d i t i o n s t h e enzyme can be s p e c i f i c a l l y d e s o r b e d f r o m either
support
experimental
using
evidence
heterogeneous susceptible
sites
to
the
substrate
suggests on
each
from
These
essentially
published
.
w - A m ino a 1ky 1- aga r o s e
a g a r o s e ~ . ' ~ ~T h e
of
The
of
which
that the
are
i n i t i a l
desorption
biospecific desorption
regarded
de r iva t ive s
matrices
and t h a t
that
adsorbents
currently
p r e p a r e d by r e a c t i o n o f c h l o r o a c e t y l
some
desorption,
suggest
nonbiospecific
phosphate.
enzyme i s a d s o r b e d a t
column only
biospecific,
results
purifications
ch r om a t o g r a p h i c
the
substrate-specific
adsorption i s not e s s e n t i a l l y i s biospecific.
carbamoyl
that
may as
explain being
g l u t a t h ione
some
affinity h a ve
be e n
d e r i v a t i v e s w i t h w-aminoalkyl-
were
applied
to
the
affinity
chroma t o g r a p h y o f he pa t i c g l u t a t h i o n e S - t r a n s f e r a s e s. Benzoquinone-activated
a g a r o s e h a s been r e a c t e d w i t h f o l i c a c i d
p r e - c o u p l e d t o b o v i n e serum a l b u m i n t o p r o v i d e a n a f f i n i t y m a t r i x
8: Chemical Synthesis and Modification
639
f o r p u r i f i c a t i o n o f folate-binding p r o t e i n from porcine choroid p 1ex u s .146 R e a c t i v e c a r b o n y l g r o u p s h a v e been i n t r o d u c e d i n t o a g a r o s e g e l s by o x i d a t i o n w i t h a q u e o u s b r o m i n e . l b 7 were
coupled
amination.
to
the
oxidized gels
P r o t e i n s and o t h e r amines i n high
yield
The e f f e c t o f t h e o x i d a t i o n - r e d u c t i o n
by
reductive
procedure on the
c h r o m a t o g r a p h i c p r o p e r t i e s o f t h e a g a r o s e g e l s was i n s i g n i f i c a n t . Benzoquinone-activated agarose has p r o v i d e d a
satisfactory
matrix f o r the immobilization o f phosphotransferase.lb8 B r o m o a c e t y 1-1,6-diam i n o h ex y l - a g a r o se adenosylhomocysteine t o
2-
has' been r e a c t e d w i t h
p r o v i d e an a f f i n i t y - c h r o m a t o g r a p h i c
f o r p u r i f i c a t i o n o f 5'-methylthioadenosine
matrix
nucleosidase from Lupinus
l u t e u s seeds.149 The
activation o f
agarose
with
1, l ' - c a r b o n y l d i - i m i d a z o l e
(CDI)
has been f u r t h e r i n v e s t i g a t e d and e x t e n d e d t o o t h e r c a r b o n y l a t i n g reagents.15'
R e s u l t s c o n f i r m e d t h a t use o f C D I a l l o w s t h e f a c i l e
p r e p a r a t i o n o f a c t i v a t e d matrices devoid o f a d d i t i o n a l charge and s u i t a b l e f o r a f f in i t y- c h r o m a t o g r a p h ic s up p o r t s. 1,2,4-triazole
1 , l '-Car bo ny l d i -
(CDT) g a v e a m o r e r e a c t i v e a c t i v a t e d m a t r i x , w h i l e
1, l ' - c a r b o n y l d i - 1 inefficiently.
,2,3-benzotriazole
reacted only
c r o s s - l i n k e d a g a r o s e by g e n e r a t i n g C D I
in
pure
CDI.
convenient
of
and
s i t u from phosgene and
i m i d a z o l e gave o n e - t h i r d o f t h e l e v e l o f a c t i v a t i o n o f with
slowly
The i n t r o d u c t i o n o f i m i d a z o l y l c a r b a m a t e g r o u p s o n t o
CDI
the
i s concluded t o
be
that obtained
t h e most e f f e c t i v e
carbonylating reagents studied,
but
the
and CDT-
a c t i v a t e d m a t r i x may be u s e f u l f o r t h e c o u p l i n g o f u n s t a b l e p r o t e i n l i g a n d s where s h o r t c o u p l i n g t i m e s a r e e s s e n t i a l . Agarose
m a t r i c e s w i t h a c o n t r o l l e d degree o f s u b s t i t u t i o n c a n
be s y n t h e s i z e d u s i n g C D I ,
and a h i g h l e v e l o f a c t i v a t i o n can r e a d i l y
be a c h i e v e d ifr e q u i r e d . 1 5 1
The C D I - a c t i v a t e d a g a r o s e was f o u n d t o
h a v e a h a l f - l i f e o f m o r e t h a n f o u r t e e n weeks when s t o r e d i n d i o x a n e . Based o n t h e
r e s u l t s obtained,
o f p r o t e i n s were e s t a b l i s h e d .
conditions suitable f o r the coupling The 3 - a l k y l c a r b a m a t e
linkage of
the
a m i n o g r o u p o f t h e l i g a n d s t o t h e s u p p o r t was shown t o p o s s e s s good s t a b i l i t y o v e r a w i d e pH r a n g e . subsequently successfully
Agarose a c t i v a t e d w i t h C D I and
c o u p l e d t o a m i n o p h e n y l b o r o n i c a c i d h a s been used f o r the s e p a r a t i o n of
g l y co s y l a t e d ha emo g l o b i ns. l5
S u l p h a t e - a g a r o s e h a s been p r e p a r e d by r e a c t i o n o f a g a r o s e w i t h c h l o r o s u l p h o n i c acid.153
The m a t r i x w a s a p p l i e d t o t h e c h r o m a t o -
graphic p u r i f i c a t i o n o f bovine F a c t o r V I I I . A g a r o s e a c t i v a t e d w i t h c y a n u r i c c h l o r i d e has b e e n r e a c t e d w i t h
640
Carbohydrate Chemistry
c o l o m i n i c a c i d t o provide an a f f i n i t y - c h r o m a t o g r a p h i c p u r i f i c a t i o n of
matrix for
neuraminidase from Corynebacterium ~ 1 c e r a n s . l ~ ~
A g a r o s e a c t i v a t e d w i t h c y a n o g e n b r o m i d e c o n t i n u e s t o be u s e d extensively
for
the
immobilization
m a c r o m o l e c u l e s and f o r
affinity
have been d e v e l o p e d f o r imidocarbonates,
biologically
active
A n a l y t i c a l methods
the determination o f
cyanate e s t e r s and
the a c t i v e species present on polysaccharide resins
a c t i v a t e d w i t h cyanogen bromide.155 coupling
of
chromatography.
A procedure f o r determining the
capacity o f a r e s i n i s described:
t h e amount o f c o u p l e d
l i g a n d c a n be d e t e r m i n e d d i r e c t l y o n t h e r e s i n a n d w i t h o u t p r i o r hydrolysis.
Using
these
procedures
the
coupling
capacity
of
a c t i v a t e d r e s i n s t o w a r d s s m a l l l i g a n d s was p r e d i c t e d by d e t e r m i n i n g t h e amounts o f cyanate e s t e r s and i m i d o c a r b o n a t e s p r e s e n t on t h e resin.
C y a n a t e e s t e r s w e r e f o u n d t o be p r e d o m i n a n t l y r e s p o n s i b l e
f o r c o u p l i n g o f l i g a n d t o a c t i v a t e d agarose. cyanogen
bromide-treated
p r o t e i n h a s been shown t o
agarose
suggests
that
without
coupled
release a t h i o l protease i n a ~ t i v a t 0 r . l ~ ~
I t was i s o l a t e d a n d s h o w n t o be t h e evidence
Alkaline treatment o f
both w i t h o r
the
cyanate
carbamate
ion.
group
i n
Experimental the
activated
p o l y s a c c h a r i d e i s t h e most l i k e l y p r e c u r s o r o f t h e c y anat e i o n . Coupling o f ligands t o
cyanogen b r o m i d e - a c t i v a t e d
h i t h e r t o been assumed t o o c c u r
via
agarose has
n u c l e o p h i l i c a t t a c k o f an amino
group o n t h e t r a n s - c y c l i c i m i d o c a r b o n a t e r i n g (see V o 1 . 1 4 , p.398). Recent r e s u l t s 1 5 5 su g g e st t h a t cyanate e s t e r s a r e , i n a n t l y responsible f o r and t h a t extent.
the
cyclic
i n fact,
predom-
c o u p l i n g o f l i g a n d t o t h e a c t i v a t e d agarose
imidocarbonate i s
formed
to
a much l e s s e r
A c c o r d i n g l y t h e t e r m c y c l i c i m i d o c a r b o n a t e p r e v i o u s l y used
i n t hese volumes t o d e s c r i b e t h e p r o d u c t o f r e a c t i o n o f agar os e and o t h e r p o l y s a c c h a r i d e s w i t h cyanogen b r o m i d e i s now term cyanogen bromide a c t i v a t e d . aration of
r e p l a c e d by t h e
Recent references t o the prep-
a c t i v e immobilized forms of
enzymes ( f o r use a s i m m o b i l -
i z e d enzymes) and i m m u n o l o g i c a l l y a c t i v e ma c r om olec ules ( f o r immunoadsorbents) and o f chromatography
various affinants
use a s
( f o r use a s a f f i n i t y -
m a t r i c e s ) by s u c h m e t h o d s a r e s u m m a r i z e d i n T a b l e s
1 ,l5’-la2 2ia3-’05
and 3,2°6-293
respectively.
I m m o b i l i z e d goat a n t i - ( r a b b i t used i n column form
i m m u n o g l o b u l i n ) a n t i b o d y has b e e n
f o r t h e s e p a r a t i o n o f bound f r o m f r e e i n a
r a d i o i m m u n o a s s a y f o r human p l a c e n t a l 1 a ~ t o g e n . l ~U~s i n g c l a s s s p e c i f i c F(ab’)2 a n t i b o d y f r a g m e n t s i m m o b i l i z e d on cyanogen br om idea c t i v a t e d a g a r o s e i m m u n o g l o b u l i n G h a s b e en i s o l a t e d f r o m human serum
i n a
single chromatographic
step
with high
y i e l d and
64 1
8: Chemical Synthesis and Modification p u r i t y .l 98 S o l u b l e immune c o m p l e x e s o f
b o v i n e serum a l b u m i n - a n t i ( b o v i n e
serum a l b u m i n ) a n t i b o d y ( t h e m o d e l s y s t e m ) have been p r e p a r e d a n d
Table 1
Use o f cyanogen b r o m i d e - a c t i v a t e d agarose f o r t h e p r e p a r a t i o n o f a c ti ve immo b i l i z e d enz ymes
Enzyme c o u p l e d t o cyanogen
E. C. No.
b r om i d e - a c t iv a t e d a ga r o s e
Agarose
Ref.
in t erm e d i a t ea
A l c o h o l d e h y d r o gena se
157 3.4.21.4
158,159
Oeoxyribonuclease I
3.1.21.1
161,162
E l a s t a se
3.4.21.11
163,164
Chymot r y p s i n C y s t a t h i o n e B -sy n t h a se
160
P-Galacto s y l hydroxy-k-
165
l y s y l g l u c o s y lt r a n s f e r a s e G 1uco am y 1ase
3.2.1.3.
166
G l y c e r a l d e h y de 3-pho s p h a t e
1.2.1.12
167,168
dehy d r o gena se La c t operox idase L y s o z yme
3.2.1.17
171
N e u r a m i n i dase
3.2.1.18
172,173
Papain
3.4.22.2
174
Pep s i n
3.4.23.1
176
T r y ps i n
3.4.21.4
1 7 7 - 1 80
Tyro s i nase
1.14.18.1
169,170
175
181 182
aSee t e x t a t r e f . 299 a n d Scheme 3
t h e n bound t o i m m o b i l i z e d p r o t e i n A.2o2
The c o m p l e x e s c o u l d be
desorbed f r o m t h e p r o t e i n A m a t r i x and d i s s o c i a t e d i n t o a n t i g e n a n d ( T e x t c o n t i n u e s on page 655)
Carbohydrate Chemistry
642
Table 2
Use o f c y a n o g e n b r o m i d e - a c t i v a t e d a g a r o s e f o r t h e p r e p a r a t i o n o f immunoadsorbents
Immunologically active
Use o f p r o d u c t
Ref.
P u r i f i c a t i o n o f al-anti-
183
corn po ne n t co up 1e d t o cy ano ge n b r om idea c t iv a t ed aga r o s e
A n t i - ( h u m a n serum a l b u m i n )
c h y m o t r y p s i n f r o m human
antibody
p l e u r a l f l u i d a n d serum A n t i - ( a1 - a n t ich ymo t r y p s i n )
P u r i f i c a t i o n o f al-anti-
183
c h ymo t r y p s i n f r om h urn an
a n t i body
p l e u r a l f l u i d a n d serum A n t i - ( h u m a n or r a t a - f e t o p r o t e i n l a n t i bo dy Anti-(human
a-fetoprotein)
mono c l ona 1 a n t i bo dy Anti-(blood-group
P u r i f i c a t i o n o f human o r
184
P u r i f i c a t i o n o f human
185
r a t a - f et o p r o t e i n
a-fetopro t e in P u r if ica t i o n o f sh ee p
1)
blood-group A n t i - ( human c h o r i o n i c gonadotrophin
186
g a s t r i c mucins w i t h
antibody
1
antibody
Ii a c t i v i t y
Development o f a n enzyme
187
immunoassay f o r human c h o r i o n i c gonadotrophin
A n t i - ( h u m a n C 5 component o f complement ) a n t i b o d y
P u r i f ica t i o n o f c o m p l erne n t
188
a t t a c k complexes f r o m i n s u l i n - a c t i v a t e d human serum
A n t i - ( r a b b i t i m m uno g l o b u l i n
Radioimmunoassay
f o r human
189
placental lactogen
GI antibody A n t i- i n t e r f e r o n a n t i bo dy
P u r i f i c a t i o n o f human i n t e r f e r o n 1 2 8
Anti-( urinary k a l l i k r e i n )
Purification o f a prekallikrein
190
from r a t p anc r eas
antibody Ant i - r e n i n antibody
P u r i f i c a t i o n o f human r e n i n
191
Ant i - ( R h i z o b i u m s t r a i n
S e p a r a t i o n o f py r u v y l a t e d
192
T A 1 polysaccharide
antibody
1
p o l y s a c c h a r i d e s i nt o p y r u v a t e - r i c h and -poor
643
8: Chemical Synthesis and Modification Table 2 continued
I m m u n o l o g i c a l l y a c t i ve
Use o f p r o d u c t
Ref.
compo ne n t co up 1e d t o cyano gen b r o m i d e a c t i v a t e d agarose
fractions Ant i - ( t h e r m o l a b i l e a n t i g e n 1
P u r if i ca t i o n o f t h e r m o l a b i l e
193
a n t i g e n f r o m baker’s y e a s t
antibody A n t i - ( t u m o u r - asso c i a t e d
I s o l a t i o n o f polyoma v i r u s -
surface antigen) antibody
induced surface antigen
H uman cho r i o n i c go na do t r o p h i n
P u r if ica t i o n o f a n t i - ( h um an
194
f r o m mouse c e l l s
carbox y l t e r m i n a l tricosapeptide
(synthetic 1
I m m uno g l ob u l i n A ,
187
ch o r i o n i c go nado t r o p h i n 1 a n t i body P u r i f i c a t i o n o f membrane
195
r e c e p t o r f o r polymer
dimerica
i m m uno g l o b u l in
I m m uno g l o b u l i n A , po 1ym e r i c b Immunoglobulin E
P u r i f i ca t i o n o f f r e e s e c r e t 0 r y
196
complex f r o m r a t b i l e Purificat i o n of cell-surface
197
r e c e p t o r f o r IgE I m m uno g l o b u l i n F ( a b ’ l2
antibody
fragments
I n t r i n s i c factor
S i n g l e - s t e p i s o l a t i o n o f IgG
198
f r o m human serum Purification o f a proteolytic
199
derived i l e a l receptor f o r i n t r i n s i c f a c t o r - co b a l a m i n Protein A
I m m o b i 1i z a t ion o f mono c l ona 1
a n t i - ( s uc r a se- isom a 1 t a se )
200
antibody f o r p u r i f i c a t i o n o f t h e enzyme D e t e c t i o n o f complexes
20 1
between Ig G and i n h i b i t o r y active part o f the inter-a-
trypsin inhibitor I s o l a t i o n o f s o l u b l e immune complexes p r i o r t o i s o e l e c -
20 2
Carbohydrate Chemistry
644 T a b l e 2 continued
I m munolo g ic a l l y a c t i v e
Use o f p r o d u c t
Ref.
component c o u p l e d t o cyano gen b r om idea c t i v a t e d agarose
t r i c focusing I m m o b i l i z a t i o n o f a n t i - ( NADP
20 3
is0 c i t r a t e d e h y d r o genase 1 antibody S t u d y of d i f f e r e n c e s
i n
204
binding a f f i n i t y o f r a t IgG subclasses t o p r o t e i n A F r a c t i o n a t i o n o f n o r m a l human I g G p r o t e i n s f r o m Japanese
aSee t e x t a n d Scheme 3 ( 3 1 ) bSee t e x t a n d Scheme 3 ( 2 8 )
205
8: Chemical Synthesis and Modification Table 3
645
Use o f c y a n o g e n b r o m i d e - a c t i v a t e d a g a r o s e f o r t h e p r e p a r a t i o n o f a f f i n i ty-chroma t o grap hy m a t e r i a l s w i t h o u t linkage extension
Ligand o r a f f i n a n t coupled
Use o f p r o d u c t
Ref.
t o cyanogen bromidea c t i v a t e d agarose
2-A c e t am ido-!-(
6- am ino-
hexanoy 1)-2-deox y-B -D_-g l u co py ra no s y 1am in e 2 -Ace t am ido -2 -de ox y -Qgalactose
Affinity
20 6
chromatography
o f B-Q-2-acetamido-2-deoxyh e x o s i d a s e s A and B A f f in i t y -ch roma t o g r a p h ic
20 7
purification of a lectin from soybean ( G l y c i n e
w)
A f f i n i ty-chroma t o g r a p h i c
20 8
i d e n t i f i c a t i o n o f bloodg r o u p A - a c t i ve g l y c o p r o t e i n s i n human e r y t h r o c y t e membrane Acriflavin
A c t i n , g l u t a r a 1de hy de cross linked
A f f i n i t y chromatography of
107
o l i g o n u c l eo t i de s
A f f i n i t y chromatography o f
20 9
l i g h t - c h a i n isoenzymes o f myosin head r e g i o n
Actin,
a-actinin
A f f i n i t y chromatography o f
210
p r o t e o l y t i c fragments o f f ib r o n e c t i n
A 1 b umi n
Affinity
chromatography o f
210
p r o t e o l y t i c fragments o f
f ib r o ne c t i n 8-( 2-Aminoethylamino )
c y c l i c AMP
-N- ( 6-Am ino h ex anoy 1) -B -Ifucosylamine
F6-( Aminohex y l ) a d e n o s i n e 2’ ,5’-diphosphate
P u r i f i c a t i o n o f a novel
211
pho spho d i e s t e r a se P u r i f i c a t i o n o f porcine
2 12
t hy r o i d f uco s ida se A f f i n i t y chromatography o f g 1u t am in e s y n t h e t a s e s from r i c e
213
Carbohydrate Chemistry
646 Table 3 Continued
Ligand o r affinant
coupled
Use o f p r o d u c t
Ref.
t o cyanogen bromidea c t i v a t e d agarose
P u r i f i c a t i o n o f 3f3-hydroxyA5-C2,-steroid
-N 6 - (
6-Am i n o hex y 1)ade no s i ne
5 '-mo nopho spha t e
-
214
ox i d o r e d p c t a s e
from r a b b i t l i v e r A f f in i t y - c h rom a t o g r a ph ic
2 15
p u r i f i ca t i o n o f l a c t a t e dehy d r o g e n a se iso enz yme C4 Pu r if i c at i o n o f p l a n t
216
1a c t a t e de h y d r o g e n a s e P u r i f i c a t i o n o f 3B-hydroxyA5-C2,-steroid
2 14
ox i d o r e d u c t a s e
from r a b b i t l i v e r 4 -Amino p h eny 1
mercuric acetate
A f f i n i t y chromatography o f
217
co n s t i t ue n t e n z ym e a c t iv i t i e s o f t h e m u l t i f u n c t i o n a l enzyme p u t r e s c i n e sy n t ha se
A n t i t h r o m b i n 111
A f f i n it y -chromatographic
study
127
P u r i f i c a t i o n o f a phytohaemag-
2 18
of modified heparin A s i a 1o f e t u i n
g l u t i n i n from t h e AzollaAnabaena s y m b i o s i s Purification o f lactose-
2 19
b l o c k i n g l e c t i n a c t i v i t y from
fetal-calf Bandaeiraea s i m p l i c i f o l i a
s k e l e t a l muscle
A f f i n i t y chromatography o f
I lectin
b l o o d - g r o u p A B H - a c t i ve
Blue dextran
A f f i n i t y chromatography o f
220
po 1y g l yco sy 1 pe'p t ide s
L-
221
t h r e o n i ne de h y d r ogena se from chicken l i v e r P u r i f i c a t i o n o f glutamine syn-
222
thetase from Escherichia c o l i P u r i f i c a t i o n o f a c y l coenzyme A
223
647
8: Chemical Synthesis and Modification T a b l e 3 Continued
Ligand o r a f f i n a n t coupled
Use o f p r o d u c t
Ref.
t o cy ano gen b r o m i d e -
a c t i v a t e d agarose
synthetase from
E.
coli
A f f i n i t y chromatography of
224
g l y ce r a l d e h y d e 3-pho spha t e de h y d r o gena se A f f i n i t y chromatography o f
&-
225
conostoc phosphoglycerate mutase
A f f i n i t y - c h r o m a t o g r a p h i c p u r i f i c a - 226 t i o n o f r a t F a c t o r I1 Calmodulin
P u r i f i c a t i o n o f smooth-muscle m y o s i n lig h t - c h a i n
C a r bo x ype p t ida se A inhibitor
Carcinoscorpius rotunda cauda ( h o r s e s h o e c r a b )
22 7
kinase
P u r i f i c a t i o n o f carboxypeptidase
228.
A from r a t s k e l e t a l m u s c l e A f f i n ity-chromatographic
fraction-
229
a t i o n o f s i a l oglycoproteins
lectin Casein
P u r i f i c a t i o n of a heparin-sensi-
230
t i v e nuclear protein kinase C i t r u l 1ine
A f f i n i t y chromatography o f
217
pu tr e scine synthase Collagen
P u r i f i c a t i o n o f bovine dental
231
sac c o l l a g e n a s e Concanavalin A
P u r i f i c a t i o n o f c a t h e p s i n D from
232
p i g myometrium P u r i f i c a t i o n o f an i r r e v e r s i b l e
233
t i s s u e i n h i b i t o r o f collagenase i n human a m n i o t i c f l u i d A f f i n i t y chromatography o f
234
Trypanosoma b r u c e i a n t i g e n s Purification o f r a t a-fetoprotein
235
P u r i f i c a t i o n o f 53K g l y c o p r o t e i n
236
from r a b b i t s k e l e t a l muscle P u r i f i c a t i o n o f a c i d phosphatase
237
648
Carbohydrate Chemistry
Table 3 C o n t i n u e d
Ligand o r a f f i n a n t coupled t o c y a n o g e n bromidea c t i v a t e d agarose
Use of p r o d u c t
Ref.
f r om r a t pro s t a t i c a d e no c a r c i noma P u r i f i c a t i on o f c h o l i n e s t e r a s e 238 from c h i c k e n e g g y o l k A f f i n i t y - ch roma t o g r a p h i c f r a c 239 t i o na t i o n of g l y cope p t i de s i s o l a t e d from a r a t l i v e r biantennary glycan P u r i f i c a t i o n of a phospholipid240 d e p e n d e n t a - m a n n o s i d a s e from rabbit liver 2 41 P u r i f i c a t i o n of a concanavalin A r e c e p t o r from b o a r s p e r m a t o z o a P u r i f i c a t i o n o f human l i v e r a c i d 242 B -Q-ga 1a c t o s i da s e Pur i f i c a t i on o f two a n t i g e n i c 243 g l y c o p r o t e i n s from r y e - g r a s s (Lolium p e r e n n e ) p o l l e n P u r i f i ca t i o n o f a1 - a n t i t r y p s i n 244 A f f i n i t y - chroma t o g r a p h i c p u r i f i 2 45 c a t i o n o f human l i v e r B-e-2a c e t a m i d o - 2 - d e o x y-h exo s i d a s e Pur i f i c a t i on o f g l yco co n j uga t e s 2 46 from Sponqia o f f i c i n a l i s Affini ty-chromatographic f rac247 t i o n a t i o n o f a m i x t u r e o f 21 3 H ) -!-mannosel a b e l l e d g l ycop e p t i d e s d e r i v e d from mouse lymphoma c e l l g l y c o p r o t e i n s I s o l a t i o n o f two e&-€444-224 8 a c e t ami do -2-de ox y- g l uca n a s e s from f i g l a t e x P u r i f i c a t i o n o f a n e u t r a l pro249
649
8: Chemical Synthesis and Modification Table 3 Continued
Ligand o r affinant
coupled
Use o f p r o d u c t
Ref.
t o cyanogen bromidea c t i v a t e d agarose
tease c l e a v i n g type I V c o l l a g e n P u r i f i c a t i o n o f B-Q-2-acetamido2-deoxy-hexosidases
250
A and B
f r o m human l i v e r Purification o f sciatin
25 1
Purification of variant antigens
252
o f Trypanosoma c o n g o l e n s e P u r i f ic a t i o n o f p l a smi no ge n
253
a c t i v a t o r s e c r e t e d by human melanoma c e l l s i n c u l t u r e A f f i n i t y chromatography of
pepsin
254
f r a g m e n t s o f human p l a c e n t a l a n d r e n a l basement membrane l a m i n i n P u r i f i c a t i o n o f r a t l i v e r a+2-acetamido-2-deoxy-
25 5
glucos y l
phosphodiesterase A f f i n i t y chromatography o f glycoproteins from adult-rat
256
brain
synaptic vesicles
A f f in i t y - c h r o m a t o g r a p h i c
124
p u r i f i c a t i o n o f bovine b r a i n
B -!-2-ace
tam i d o -2-deox y -
hexosidase C C o p u r i f i c a t i o n o f a l k a l i n e phos-
131
p h a t a s e and 5’-AMP-specific n u c l e o t i d a s e from D i c t y o s t e l i u m discoideum P u r i f i c a t i on o f g l ycosy l c e r a m i -
112
dase f r o m m u r i n e i n t e s t i n e Purification o f r a t uterine
138
p e r ox ida s e P u r i f i c a t i o n from murine l i v e r
142
Carbohydrate Chemistry
650 Table 3 C o n t i n u e d
L i g a n d o r a f f i n a n t coupled
Use o f p r o d u c t
Ref.
t o cyanogen bromidea c t i va t e d a g a r o se
o f an e s t e r h y d r o l a s e a c t i v e on p h o r b o l d i e s t e r s Purification o f a root l e c t i n
20 7
f r o m soybean ( G l y c i n e Cytochrome _b5
P u r i f i c a t i o n o f l i n o l e o y l CoA
25 7
desaturase from r a t l i v e r m i c r o some s Deox y r i bo nuc 1e i c a c i d
A f f i n i t y chromatography o f
25 8
p o r c i n e pancreas deoxyribonuclease I Discoidin I
P u r i f i c a t i o n o f d i s c o i d i n I-
25 9
b i n d i n g p r o t e o g l y c a n from axenic D i c t y o s t e l i u m discoideum Dolichos b i f l o r u s l e c t i n
P u r i f i c a t i o n o f receptors f o r
260
t h e l e c t i n from embryonal carcinoma c e l l s Factor X
Affinity-chromatographic
puri-
153
f i c a t i o n o f f a c t o r VIII Fetuin
P u r i f i c a t i o n o f a neuraminic
26 1
a c i d - b i n d i n g l e c t i n from horseshoe crab ( C a r c i n o s c o r p i u s r o t u n d a caudal --
F i b r o ne c t i n
P u r i f i c a t i o n o f g l y c o s a m i n o g l y c a n s 262
Hog g a s t r i c m u c i n
Purification o f ea-B-Q-galacto-
(Smith degraded) Gelatin
263
sidase from Escherichia f r e u n d i i A f f i n i t y c h r o m a t o g r a p h y o f p r o t e o - 210 l y t i c fragments o f f i b r o n e c t i n Pur if i c a t i o n o f a g l y c o p r o t e i n
26 4
s e c r e t e d by a o r t i c e n d o t h e l i a l cells i n culture P u r i f i c a t i o n o f r a t plasma f i b r o -
265
65 1
8: Chemical Synthesis and Modification Tabl e 3 Continued
L i g a n d o r a f f i n a n t coupled
Use o f p r o d u c t
Ref.
t o cyanogen bromidea c t i v a t e d agarose
nectin Guanosine diphosp h a te hex a n o 1am ine
C o p u r i f i c a t i o n o f Lewis blood-
266
g r o u p 2-acetam i d o - 2 - d e o x y - p g l uco sy 1-a( 1+4)- L - f u c o sy 1t r a n s f e r a s e and a 2-acetamido-2de o x y
-a - g l uco sy 1- a ( 1+3 ) --I
f uco s y l t r a n s f e r a s e f r o m h um a n milk Guano s i n e mo no pho spha t e
P u r i f i c a t i o n o f human h y p o x a n -
H e l i x pomatia l e c t i n
A f f in i t y - c h r o m a t o g r a p h i c s t u d y
t h i n e - g u a n i ne ph o s p h o r i b o sy 1
267
t ransferase 26 8
of s u b u n i t s t r u c t u r e o f e n t e r o k i na se He pa r i n
P u r i f i c a t i o n o f a heparin-sensi-
230
t i v e nuclear p r o t e i n kinase P u r i f i c a t i o n o f r a t l i v e r 2-aceta-
255
m i d o -2 -deox y-Q- - g l uco sy 1 ph o sp h o dieste rase P u r i f i c a t i o n o f Xenopus l a e v i s
26 9
t y p e 1 topoisomerase Purification o f r a t hepatic
270
triglyceride lipase A f f i n i t y chromatography o f n a t i v e
271
and c h e m i c a l l y m o d i f i e d a n t i thrombin I11 Purification o f tyrosine
136
h y d r ox y l a se Histone
P u r i f i c a t i o n o f a c a t a l y t i c sub-
272
u n i t o f a p r o t e i n kinase from human e r y t h r o c y t e membranes Homoar g i n i n e
Affinity
chromatography
of
217
652
Carbohydrate Chemistry
Table 3 Continued
Ligand o r a f f i n a n t coupled t o cy ano gen br om i d e a c t i v a t e d agarose
a - L a c t a 1bumin L e n t i l (Lens culinaris) lectin Lichena n
{Q- Leu 6, o c t a pe p t i de (renin ) inhibitor Lotus tetragonolobus lectin Low-density l i p o p r o t e i n (human 1
- sine L-Ly
g-Mannan 0 Val bum i n 0 vo m uc o i d
Pea (Pisum
Use of p r o d u c t
p u t r e s cine s y ntha s e P u r i f i c a t i o n o f human serum 8- g a l a c t o s y l t r a n s f e r a s e Af f i n i t y - c h r o m a t o g r a p h i c s t u d y of b i n d i n g of g l y c o p e p t i d e s o f d i f f e r e n t st r uc t u r e s P u r i f i c a t i o n o f (1+3), (1+4)-6Q - g l u c a n a s e from B a c i l l u s P u r i f i c a t i o n o f human r e n i n Affini ty-chromatographic s t u d y of s u b u n i t s t r u c t u r e o f e n t e r o k i na se A f f i n i t y chromatography of various modified glycans A f f i n i t y chromatography of ch o n d r o i t i n 6 - s u l p h a t e Af f i n i ty-chroma t o g r a p h i c remo v a l o f p l a s m i n o g e n a n d p l a s m i n from g u i n e a - p i g serum Large-scale i s o l a t i o n o f funct i o n a l l y a c t i v e components of t h e human complement system P u r i f i c a t i o n of heparin I s o l a t i o n o f a E-mannan-binding p r o t e i n from r a t l i v e r A f f i n i t y c h r o m a t o g r a p h y of p r o t e o l y t i c fragments of fibronectin I s o l a t i o n o f a-q-mannose : 1 , 2 ( 2-a c e tam i do -2 -de o x y -8- g l uco s y 1) t r a n s f e r a s e from t r a c h e a mucosa Af f i n i t y - c h r o m a t o g r a p h i c s t u d y
Ref.
273 274
2 75 191
26 8
276 277 278
279
2 80 281 210 282
274
653
8: Chemical Synthesis and Modification
Table 3 Continued
Ligand o r a f f i n a n t coupled t o cyanogen bromidea c t i v a t e d a ga r o s e
sativum) l e c t i n
Phosvit i n P 1 asm i n o g e n
Poly(inosine), poly(cytidy1ic acid) P o l y ( u r i dy 1i c a c i d ) R i c i n u s communis agglu t in in
Ricinus sanguinis agglutinin Thy r o i d - s t i m u l a t i n g hormone (TSH) a-Tox i n Transf e r r i n Troponin T, dephospho r y l a t e d T r y p s i n i nh i b i t o r
Use o f p r o d u c t
of b i n d i n g o f g l y c o p e p t i d e s of d i f f e r e n t s t r u c t u r e s A f f i n i t y - c h r o m a t o g r a p h i c f ract i o n a t i o n o f a m i x t u r e o f 2{ 3 H ) -p-manno s e - l a b e l l e d g l y c o p e p t i d e s d e r i v e d from m o u s e 1y m p h om a c e l 1 g 1y c o p ro t e i n s P u r i f i c a t i o n o f a heparin-sensitive nuclear protein kinase P u r i f i c a t i o n o f a2 p l a s m i n in h i b i t o r P u r i f i c a t i o n o f human ( 2 F , 5 9 ) ( A ) n syn th etase P u r i f i c a t i o n o f mammalian v i r a l reverse transcriptases Purification of a glycoprotein from s e r a o f cancer p a t i e n t s A f f in i ty- chroma t o g r a p h i c s e p a r a t i o n of c e l l - s u r f a c e glycop r o t e i n s and glycolipids A f f i n i t y chromatography of glycop r o t e i n s from a d u l t - r a t b r a i n s y n a p t i c ve s i c l e s P u r i f i c a t i o n o f human TSH r e c e p t o r s from t h y r o i d membrane P u r i f i ca t i o n o f n i c o t i n i c a c e t y 1choline receptor proteins P u r i f i ca t i o n o f a u t o a n t i bo d i e s to transferrin P u r i f i c a t i o n of dog c a r d i a c troponin T kinase Purification o f Streptomyces
Ref.
2 47
230 283 2 84 2 85 2 86
2 87
256
2 88 289 2 90 2 91 2 92
654
Curbohydrute Chemistry
Table 3 Continued
Ligand o r a f f i n a n t coupled
Use o f p r o d u c t
Ref.
t o cy ano gen b r om i d e a c t i v a t e d agarose
(r i c e bran)
griseus trypsin
Trypsin i n h i b i t o r
A f f i n i t y chromatography o f p o r c i n e
258
pancreas deoxyribonuclease I
( s o y bean ) UDP-hexanolamine
Purification o f rabbit liver
2 93
o e s t r o n e a n d 4- n i t r o p h e n o l
-Vicia cracca
UDP-g1 uc u r ony 1t r a n s f e r a s e s lectin
Affinity-chromatographic a t i o n o f blood-group
fraction-
220
ABH-active
p o l y g l y c o s y l p e p t i d e s f r o m human e r y t h r o c y t e membranes A f f i n i t y chromatography o f bloodgroup A - a c t i v e
294
g l y c o p r o t e i n s from
human e r y t h r o c y t e membranes von W i l l e b r a n d f a c t o r
I s o l a t i o n o f human p l a t e l e t r e c e p -
295
t o r s f o r von W i l l e b r a n d f a c t o r
W hea t -ge r m a g g l u t in i n
Affinity
chromatography o f g l y c o c o n - 246
conjugates from Spongia o f f i c i n a l i s P u r i f i c a t i o n o f a g l y c o p r o t e i n from
286
sera of cancer p a t i e n t s P u r i f i c a t i o n o f i n t r a c e l l u l a r a-g-
296
mannosidase f r o m c u l t u r e d s k i n
f ib r o b l a s t s I s o l a t i o n o f p l a s m a membrane
297
v e s i c l e s o f human p l a t e l e t s I s o l a t i o n o f human p l a t e l e t membrane 2 9 8 fractions
655
8: Chemical Synthesis and Modification a n t i b o d y by i s o e l e c t r i c - f o c u s i n g minoglycan-lipoprotein
analysis.
interaction
c o m p o s i t i o n and charge
density
the
I n a study o f glycos-
effect
of
uronic
acid
o f t h e g l y c a n h a s been i n v e s t i g a t e d
.
u s i n g aga r o se s u b s t i t Ute d w i t h 1ow-densi t y l i p o p r o t e i n 276 Direct
attachment
of
an
enzyme o r
an a f f i n a n t
a c t i v a t e d w i t h cyanogen b r o m i d e has f r e q u e n t l y
to
agarose
been u n r e w a r d i n g ,
s i n c e a p p r o a c h t o t h e bound l i g a n d may be s t e r i c a l l y impeded.
This
d i f f i c u l t y c a n b e o v e r c o m e by i n s e r t i n g a c o v a l e n t b r i d g e b e t w e e n matrix
and l i g a n d .
B r i d g i n g i s a l s o u s e d when f u n c t i o n a l g r o u p s o n
t h e l i g a n d do n o t r e a c t w i t h t h o s e o n t h e m a t r i x , new t y p e o f f u n c t i o n a l i t y used
to
provide
chromatography
t o be i n t r o d u c e d .
bridges
i n
the
=.,f r o m
matrices,
since i t allows a
Modifications recently
preparation
agarose c y c l i c
o f
a f f i n i t y -
imidocarbonates
a r e s u m m a r i z e d i n S c h e m e s 3 a a n d 3b a n d T a b l e 4 . 2 9 9 - 3 4 5
Where
b r i d g i n g h a s been u s e d t o i m m o b i l i z e enzymes o r t o p r e p a r e immunod e t a i l s a r e g i v e n i n T a b l e 1 o r 2,
adsorbents,
p r o d u c t s o b t a i n e d by
coupling various
respectively.
The
substances t o agarose c y c l i c
i m i d o c a r b o n a t e f o r d i v e r s e u s e s a r e g i v e n i n T a b l e 5.346-376 Amine-substituted
hormone
analogues o f
tri-iodo thyronine
have
been p r e p a r e d and c o u p l e d t o 6 - a m i n o h e x y l - a g a r o s e
i n the development
of
o f t h y r o i d hormone
support
matrices f o r a f f i n i t y
chromatography
r e c e p t o r s .344
2 - Ace t am ido - 2 - d e o x y - B - Q - g 1 u c o sy 1 hex a n o l a m i n e co u p 1 e d t o a g a r o s e has been u s e d i n s t u d i e s o f t h e e n z y m a t i c s y n t h e s i s and 1 3 C n.m.r.
c o n f o r m a t i o n o f d i s a c c h a r i d e s c o n t a i n i n g B - Q - g a l a c t o s e and 8-
- { 1-13C}
- galactose
achieved
using
r e s i d u e s .346 s o l u b l e
The s o l i d - p h a s e
UDP-D=-galactose
s y n t h e s i s was and
UDP-D=-
galacto syltransferase. The use o f i m m o b i l i z e d l e c t i n s ( c o n c a n a v a l i n A and w h e a t - g e r m a g g l u t i n i n ) t o p r e p a r e serum f r e e o f g l y c o p r o t e i n hormones,
i n radioimmunoassay,
has been d e s c r i b e d . 3 6 3
p r o t e i n A - a g a r o s e has been d e v e l o p e d f o r XIIIa-catalysed
coupling
of
various
f o r use
A new m e t h o d u t i l i z i n g
quantitation of the factor
structural
proteins. 368
Using
i m m o b i l i z e d g l u t a t h i o n e a s a n a c t i v a t o r a new a n d v e r y s e n s i t i v e assay
f o r t h i o l proteases
(g. cathepsin
D) h a s been r e p o r t e d . 3 6 9
I m m o b i l i z e d h e p a r a n s u l p h a t e has been used i n s t u d i e s o f s e l f association
between
various
o l i g o s a c c h a r i d e s t h e r e o f .370 ,371 been
used t o
assess t h e
p r o t e i n k i n a s e and h e p a r i n ,
heparan
sulphate
species
and
H e p a r i n immo b i l i z e d o n a ga r o se has
i n t e r a c t i o n among p o l y a m i n e s , as w e l l as f o r
nuclear
purification of protein
k i n a s e s i n h ib i t e d by hepa r i n. 3 7 2 ( T e x t c o n t i n u e s on p a g e 6 7 0 )
8: Chemical Synthesis and Modification
657
a
I
Z
0 ON h
N
I
V
v
o=v
a
I
0
u
Z
C
Z
C
o
N
I
u
Y
I
N
o
N
I
ub -
-
o=v
OYYO
I
z
I
u
m
N
I
V
Y
6II
z
C
n
N
I
2
I
T k
-
0 I
*,
N
I
V
Y
-
h
X a D
u II
z
C
n
N
I V
Y
Carbohydrate Chemistry
658 Table 4
Use o f
cyanogen b r o m i d e - a c t i v a t e d
agarose
for
p r e p a r a t i o n o f a f f i n i ty-ch romat o graphy m a t e r i a l s
the
via
linkage e x t e n s i o n
Ligand o r a f f i n a n t
A ga ro se
c o u p l e d t o cyanogen
inter-
brom ide-a c t i va t e d
m e d i a t ea
Use o f p r o d u c t
Ref.
A f f i n i t y - c h r omato g r a p h i c
2 45
agarose
2-Acetamido-2-deoxy-
30
B -0- ga 1a c t o se
Ade no s i n e d i p h o spha t e
p u r i f i c a t i o n o f human
1ive r 8 -Q - -2 -ace t am ido 2-deox y-h exo s i dase 29
-
A f f i n i t y - c h roma t o g r a p h i c
299
p u r i f i c a t i on o f g l utamine s y n t h e t a s e f r o m Rhodos p i r i l l u m rubrum Adenosine monophosphate
29
A f f i n i ty-ch romato g r a p h i c
300
p u r i f i c a t i o n o f Simian l i v e r a 1coh ol de hy d r o ge nase Adenosine monophosphate,
29
pe r io da t e- ox id i z e d
A f f i n i ty-chroma t o g r a p h i c
301
p u r i f i c a t i o n o f two forms o f RNAse U2 29
A f f in i t y - chroma t o g r a p h i c
302
p u r i f i c a t i o n o f RNAse T 2 from Taka-diastase Adenosine t r i p h o s p h a t e
31
A f f i n i ty-chroma t o g r a p h i c
230
p u r i f i c a t i o n o f a heparinsensitive nuclear protein k i n a se A f f i n it y - chromatographi c
303
p u r i f i c a t i o n o f flavokinase f rom P h a s e o l u s a u r e u s
(m ung bean )
4- Am ino be nz am id i n e
32
A f f i n i t y - c h r oma t o g r a p h i c
26 8
659
8: Chemical Synthesis and Modification Table 4 continued
Ligand o r a f f i n a n t
A ga r o se
c o u p l e d t o cyanogen
inter-
b r om ide - a c t iva t e d
Ref.
Use o f p r o d u c t
mediatea
aga r o s e
study o f enterokinase 2 - A m i no-2-deox y -Q-
30
A f f i n i t y - ch roma t o g r a p h i c
304
p u r i f i c a t i o n o f a 2-amino-
galactose
2-deoxy-g- g a l a c t o seb i n d i n g p r o t e i n f r o m Psophocarpus tetragonolobus 6-Ami noh ex y l 2 - a c e t
( w i n g e d be a n )
-
30
A f f i n i ty-chroma t o g r a p h i c
am ido -2 -deox y - l -
p u r i f i c a t i o n o f B-Q-2-
t h i0-8 -Q-g1 uco s i de -
acetam i d o -2-deox y-hexo-
25 0
s i d a s e s A a n d B f r o m human liver
6-Aminohexyl 1 - t h i o - B -
30
A f f i n i ty-chromatographic
rem o va 1 o f B -0 - ga 1a c t o s i da se
g-galactoside
25 0
d u r i n g p u r if ic a t i o n o f h exos i dases 4-Ami no phe ny l - 2 - a c e t a -
30
b r a i n B -Q-2-ace tam i d o -
g l uco s i de
-
ace tamido-2-deox y-B -
-
2-deoxy-hexosidase
30
-
C
A f f i n i t y - c h roma t o g r a p h i c
124
p u r i f i c a t i on o f bo v i n e b r a in B -9 - 2 - ace t am ido
p-g1 uco s i de 4-Am ino phe ny 1-1 t h i o -
124
p u r i f i c a t i o n o f bovine
mido-2-deoxy-8 -Q-
4- Am i no phe n y 1-1 t h i o - 2
A f f in i t y - c h r o m a t o g r a p h i c
-
2-deox y-h exo si dase C
30
B - a - g l uco s i de
A f f i n i ty-chroma t o g r a p h ic
305
p u r i f i c a t i o n o f a f3-Qglucosyl hydrolase transferase present i n w a l l s o f soybean c e l l s
4-Ami no s a l i c y l i c a c i d
33
A f f in i t y ch r om a t o g r a p hy o f Pseu domonas sa 1i c y 1a t e
306
Carbohydrate Chemistry
660 T a b l e 4 continued
Ligand or a f f i n a a t
Agaro se
c o u p l e d t o cyanogen
inter-
b r omi de-a c t i va t e d
me d i a t e a
Use o f p r o d u c t
Ref.
agarose
hydroxylase Apro t in i n
30
Purification of pig urinary
122
ka 11ik r e i n I - A r g i n i n a l semi c a r ba-
34
zone h y d r o f 1uo r i de
L-Ar
A f f i n i t y - chroma t o g r a p h i c
307
p u r i f i ca t i on o f t r y p s i n
g inine
30
A f f i n i t y - chroma t o g r a p h i c
308
p u r i f ica t i on o f co 1l a g e n a se from Clostridium h i s t o l y t i c u m Bay 9 5 4 2 1 a m y l a s e
29
inhibitor
A f fi n i ty-chroma t o g r a p h i c
309
i s o l a t i o n of r a t pancreatic and s a l i v a r y
4-Car boxy b e n z a l d e h y d e
29
amylases
R a p i d p u r i f i c a t i o n o f human
310
p l a c e n t a l aldose reductase 2’3’-0-{1-(2-Carboxye t hy 1) e t h y 1i d e ne 1
’
29
-
deami nase
inosine 29
’-
diphosphate
2 ’ 3 ’-g- { 1-( 2-Ca r bo x y
-
-
3 12
messenger RNA c a p b i n d i n g protein 29
A f f in i t y - c h roma t o g r a p h i c
3 13
p u r if i c a t i o n of guanine am i n o h y d r o 1ase
x a n t h o s i ne
7-( 5-Car b o x y p e n t y 11-
A f f in i t y - ch r o m a t o g r a p h i c
p u r i f ica t i o n o f e u ka r y o t i c
ethyl)ethylidene1-7-
e t h y 1) e t h y 1 ide ne 1
3 11
tography o f adenosine
2 3 ’-g- { 1- ( 2-Carbo x y methylguanosine 5
Q u a n t i t a t i ve a f f in i t y chroma-
29
A f f i n i t y - c h r oma t o g r a p h i c
guano s i ne 5 ’ - d i p h o s -
p u r i f i c a t i o n o f eukaryot i c
phate
messenger RNA cap b i n d i n g
312
protein Cholic acid
29
A f f in i t y - ch r oma t o g r a p h i c
314
purification o f steroid 12a-mono-ox ygenase 29
A f f i n i t y - chroma t o g r a p h i c
315
66 1
8: Chemical Synthesis and Modification Table 4 continued
Ligand o r a f f i n a n t
Agarose
c o u p l e d t o cyanogen
inter-
brom ide-a c t i va t e d
mediatea
Use o f p r o d u c t
Ref.
agarose
p u r i f ic a t i o n of S-transferases
g l u t a t h i o ne A and C f r o m
r a t l i v e r cytosol Cortisol
35
A f f in i t y - chroma t o g r a p h i c
316
p u r if ica t i on o f human
t r a n s co r t i n Creatine
29
A
f f in i t y - ch r oma t o g r a p h i c
3 17
p u r i f i c a t i o n o f sarcosine o x i d a s e f r o m Corynebacterium species Cy ano co ba 1am in
36
L a r g e - s c a l e a f f i n i ty-chroma t o -
318
g r a p h i c p u r if i ca t io n o f human h o l o - t ranscobalamin p-Cy s t e i ny l-lp r o l i ne
29
II
A f fi n i t y - ch roma t o g r a p h i c
319
p u r i f i c a t i o n o f human serum
-
an g i o t e n s i n 1 con ve r t i n g enzyme Dermatan s u l p h a t e
29
P u r i f ica t i o n o f bo v i n e
3 20
t e s t i c u l a r h y a 1 u r o n i da se Dextran sulphate, c y ano g e n b r om ide -
37
A f f i n i t y - chroma t o g r a p h i c
3 21
p u r i f i c a t i o n o f a c i d 8-
activated
g l u c o s i d a s e f r o m human placenta
Dihydrolipoic acid
29
A f f i n i ty-chromatographic
322
p u r if i c a t i on o f py r u v a t e dehydrogenase complex f r o m E s c h e r i c h i a c o l i K12 E t h y l ?-(3-carboxy-
29
A f f in i t y - ch r oma t o g r a p h i c
3 23
isolation o f plant tubulin
ph eny 1) c a rbam a t e
f r o m A z u k i bean e p i c o t y l s Folic acid
29
A f f i n i ty-chroma t o g r a p h i c
324
Carbohydrate Chemistry
662 Table 4 continued
Ligand o r a f f i n a n t
Agarose
c o u p l e d t o cyanogen
inter-
b r o m i de-ac t i va t e d
mediatea
Use o f p r o d u c t
Ref.
agarose
p u r i f i c a t i o n o f two components o f a multienzyme system f r o m C l o s t r i d i u m t h e rmoace t icum 5-Fo r m y 1-5,6,7,8-
te tra-
29
hy dr o p t e r o y 1g l u t a m ic
3 25
study o f f o l a t e - b i n d i n g
acid
-0 - a -L-
A f fi n i ty-chroma t o g r a p h i c proteins of r a t l i v e r mitochondria
-Fuco py r a no sy 1-
-b
p y r a n o s y l - ( 1+3)-{aL - f u c o p y r a n o s y l - ( 1+4))
P u r i f i c a t i o n o f goat a n t i -
3 26
hexaose i m m u n o g l o b u l i n G
(1+2) -B-a-galacto-
-
2-acetamido-2-deoxy-BQ-91 UCO Sy 1-( 1+3 I -B -Qg a l a c t o s y 1-( 1+4)
-Q-
g l uco se
0- - G 1 uco - Q- -man na n
29
A f f i n i t y - ch roma t o g r aph ic
327
p u r i f i c a t i o n o f B-Q-
cy ano ge n b r om i d e -
manna na s e
activated
G1 y co sph i ngo 1i p i d
29
A f f i n ity-chromatographic
328
p u r i f i ca t i o n o f a n t i -
G 1 y co Sam ino g l y c a n s
g l yco sph ingo 1i p i d a n t i bo dy
,
31
p e r i oda t e- ox id i z e d 5 '-Guanosine
pha t e,
Affinity
chromatography o f
329
basement membrane l a m i n i n 29
monophos-
pe r i o d a t e- ox i d i z e d
A f fi n i t y - c h roma t o g r a p h i c
3 30
purification o f ribon u c l e a s e T1
29
A f f i n i t y - c h r o m a t o g r a p h ic purification of ribon u c l e a s e from F u s a r i u m moniliforme
331
663
8: Chemical Synthesis and Modification Table 4 continued
Ligand o r a f f i n a n t coupled t o cyanogen b r om i d e - a c t i v a t e d agarose
A g a r o se interme d i a t e a
Heparin
2 - Imi n o b i o t i n
29
29 29
Insulin
38
Methot r e x a t e
29
lO-Methy1-5,8-dideazafolate
29
4-Me t h y l - n i c o t i n a m i d e
39
ade n i ne d i n u c l eo t i d e
1 , 2 - Naph t h oq u i no n e
29
N i coti namide a d e n i n e
d i n u c l e o t i de N i co ti nami d e a d e n i n e d i n uc 1e o t i de p h o s p h a t e
Pepsta t i n
39
Use o f p r o d u c t
Ref.
A f f i n i t y - c h r oma t o g r a p h i c
3 20
p u r i f i ca t i o n o f bo v i n e t e s t i c u l a r hy a1 u r o n i da s e Af f i n i ty-chroma to g r a p h i c p u r i f i ca t i on o f a v i d i n Af f i n i ty-chroma to g r a p h i c p u r i f i ca t i o n o f s t r e p t a v i d i n Af f i n i t y - c h r o m a t o g r a p h i c p u r i f i c a t i o n o f human placental insulin receptor A f f i n i t y - c h r oma t o g r a p h i c p u r i f i ca t i o n o f d i h y d r o f o l i c acid reductase A f f i n i ty-chromato g r a p h i c p u r i f i c a t i o n o f mouse l i v e r t h y m i dy l a t e s y n t h e t a s e A f f i n i t y - ch roma t o g r a p h i c p u r i f i c a t i o n o f NADglycohydrolase from Neurospora crassa A f f i n i t y - c h roma t o g r a p h i c i s o l a t i o n o f human l i v e r d i hy d r o p t e r i d i n e r e d u c t a s e A f f i n i ty-chroma t o g r a p h i c p u r i f i ca t i o n o f f r a gm e n t of d i p h t h e r i a t o x i n A f f i n i t y - c h roma t o g r a p h i c p u r i f i c a t i o n o f aldehyde r e d u c t a s e from r a t b r a i n a n d l i v e r a n d ox b r a i n A f f in i t y - c h r o m a t o g r a p h i c
332 333 334
3 35
336
337
338
339
3 40
232
Carbohydrate Chemistry
664 Table 4 continued
Ligand o r a f f i n a n t
Agarose
c o u p l e d t o cyanogen
inter-
b r o m i d e - a c t i va t e d
mediatea
Use o f p r o d u c t
Ref.
aqarose
p u r i f ica t i o n o f ca t h e p s i n D from p i g myometrium 29
A f f i n i ty-chroma t o g r a p h i c
3 41
purification o f a proteinase from Candida a l b i c a n s c u l t u r e supernatants 29
A f f i n i ty-chroma t o g r a p h i c
191
p u r i f i c a t i o n o f human r e n i n 29
L a r ge-s c a l e a f f in i t y - ch roma t o -
342
graphic i s o l a t i o n o f rennin f r o m mouse s u b m a x i l l a r y g l a n d P t e r o g l u t am ic a c i d
40
Affinity-chromatographic
iso-
343
l a t i o n o f a folate receptor f r o m human p l a c e n t a Put rescine
30
Affinity
chromatography o f
constituent
enzyme a c t iv i
-
217
t i e s o f the multifunctional enzyme p u t r e s c i n e s y n t h a s e Riboflavin
29
Affinity-chromatographic p u r i -
303
f i c a t i o n o f f l a v o k i n a s e from Ph a se o lu s aureus 3 ,5,3’-Tri-iodothyro-
29
nine
(mung bean)
A f f i n i t y chromatography o f
344
t h y r o i d hormone r e c e p t o r s
U r i d i n e d i p h o sp ha t e
Q - g 1 uc u r o na t e
29
A f f i n i ty-chroma t o g r a p h i c p u r if ic a t i o n o f g u i ne a- p i g l i v e r m i c r o s o m a l UDP
B-
g l uc u r o n o sy 1t r a n s f e r a s e
aSee t e x t a t c i t a t i o n o f T a b l e 4 and Scheme
3
bReac t i o n w i t h 1- a m i no-2-( 4-ami nopheny 1)e t h a n e ,
followed
by r e a c t i o n w i t h cyanogen b r o m i d e - a c t i v a t e d a g a r o s e
3 45
8: Chemical Synthesis and Modification Table 5
Use o f
665
cyanogen b r o m i d e - a c t i v a t e d
agarose
for
the
p r e pa r a t i o n o f m i s c e l l a n eo us de ri v a t ive s
Species coupled t o
Use o f p r o d u c t
cy ano gen b r o m i d e -
Ref.
a c t i v a t e d agarose
2-Ace t am ido-2-deox y -
B -a-g1 - uco sy 1
Solid-phase enzymatic s y n t h e s i s
3 46
o f d i saccharide s
h ex a n o 1am ine A de no s i ne mono ph 0 s -
p ha t e
,
pe r io d a t e -
ox i d i z e d a
D e t e c t i o n and i s o l a t i o n o f s m a l l
301
amounts o f b a s e - s p e c i f i c RNAses i n crude c e l l e x t r a c t s
4-Aminophenyl 8-aglucopy ran0 s i d e
A f f i n i ty-chromatographic
studies
347
o f the specific i n t e r a c t i o n o f c o n c a n a v a l i n A and s a c c h a r i d e s
Asialo-GMl,
ozonolyseda
P u r i f i c a t i o n o f a n t i - g l y co s p h i n -
348
go 1i p i d a n t i bo dy
2- C h l o r o -10 -( 3- aminop r opy 1) p h eno t h i a z i n e
A f f i n i t y - c h roma t o g r a p h i c s t u d y o f
349
t h e Ca2+-de p e n d e n t in t e r a c t io n o f SlOOb,
t r o p o n i n C,and
calmo-
d u l i n w i t h t h e drug
Chromaffin granule
m em b r a ne s
I s o l a t i o n by Ca2+-de pen d e n t a f f i-
350
n i t y chromatography o f p r o t e i n s that bind to chromaffin granule membrane
Concanavalin A
Demo n s t r a t i o n o f g l y co p r o t e i n
35 1
c h a r a c t e r i s t i c s o f t h e s o d i um channel s a x i t o x i n - b i n d i n g
compo-
n e n t from mammalian s a r c o l e m m a C h a r a c t e r i z a t i o n o f Xenopus 00 cy t e
35 2
s u b c e l l u l a r f r a c t i o n s and i n c u b a t i o n media Study of
t r a n s f o r m a t i on-de p e n d e n t
q u a n t i t a t i v e changes i n g l y c o peptide binding t o the l e c t i n
353
666
Carbohydrate Chemistry
T a b l e 5 continued
Species coupled t o cy ano gen b r o m i d e -
Use o f p r o d u c t
Ref.
a c t i va t e d a ga r o s e
Reversible binding of antithrom-
35 4
b i n I11 f o r use i n t h e f r a c tionation o f heparin Study o f b i n d i n g o f i n s u l i n
355
receptors t o l e c t i n s R em o v a 1 o f h a em a g g 1ut ina t in g
35 6
a c t i v i t y during protease purification Study o f t h e g l y c o s y l a t i o n o f t h e
35 7
a r g i n i n e vaso p r e s s i n / n e u r o p h y s i n I1 common p r e c u r s o r Study o f m i c r o h e t e r o g e n e i t y o f
35 8
o v a l bum i n Study o f f i b r i n o g e n chains d u r i n g
35 9
sy n t h e s i s A f f in i t y - ch rom a t o g r aph ic demons t r a t i o n o f t w o al-acid
360
glyco-
protein populations i n p u r i f i e d protein preparations A f f i n i t y - c h roma t o g r a p h i c sepa r a -
t i o n o f right-side-out
361
oriented
s u b f r a c t i o n s from p u r i f i e d c a l f t h y m o c y t e p l a s m a membranes D e m o n s t r a t i o n o f two t y p e s o f
362
c o l o n y - s t i m u l a t i ng f a c t o r i n s e r um 0 f lipo p o l y saccha r i det r e a t e d mice Removal o f
l u t r o p h i n and c h o r i o -
gonadotrophin 8-subunit
363
during
preparation of glycoprotein h o r m o n e - f r e e serum Demons t ra t i o n o f g l yco p r o t e i n
364
667
8: Chemical Synthesis and Modification Table 5 continued
Species coupled t o cy an0 ge n b r om i d e
Use o f p r o d u c t
-
Ref.
a c t i v a t e d agarose
nature of chlorophyllase Demonstration o f heterogeneity
365
o f h e t e r o g a l a c t a n from f r u i t bodies o f Fomitopsis p i n i c o l a C o n c a n a v a l i n A,
s u c c i ny 1a t e d
I n v e s t i g a t i o n of
chemical c r o s s -
366
l i n k i n g o f Q-glucose oxidase on a modified l e c t i n matrix
Deox y r i bo n u c l e i c a c i d
Model system f o r development o f a
36 7
me t h o d f o r cy t o ch emi c a l hy b r i d i z a t i on w i t h f l u o r o c h romel a b e l l e d RNA Fibrin
Development o f a new m e t h o d o f
368
e xa min in g and q u a n t i t a t i n g t h e F a c t o r X I I I a - c a t a 1y se d coup 1i n g
of Gluta thione
various s t r u c t u r a l proteins
Development o f a v e r y s e n s i t i v e
369
assay f o r t h i o l p r o t e a s e s u s i n g i m m o b i l i z e d g l u t a t h i o n e as an a c t iva t o r Heparan s u l p h a t e , pe r i o d a t e - o x i d i z e d b
Demonstration o f s e l f-asso c i a t i o n o f bovine l u n g heparan
370, 3 71
sulphates Heparin
Assessment o f i n t e r a c t i o n s among polyamines,
3 72
nuclear protein
kinase, and heparin.
Purification
o f p r o t e i n k i n a s e s i n h i b i t e d by heparin M o d i f i c a t i o n o f heparin-agarose procedure for
determination o f
plasma l i p o l y t i c a c t i v i t y o f no r m a l i p i d e m i c a n d h y p e r l i p i d emic s u b j e c t s a f t e r h e p a r i n
373
Carbohydrate Chemistry
668 Table 5 continued
Species coupled t o cy ano ge n b r o m i d e
Use o f p r o d u c t
-
Ref.
a c t i va t e d a g a r o se
injection Lentil lectin
S t udy o f t h e ca r bo h y dr a t e - b i n d i n g
247
specificity of l e n t i l lectin Study o f b i n d i n g o f i n s u l i n
355
receptors t o l e c t i n s Myosin
Development o f a new method o f
368
examining and q u a n t i t a t i n g t h e Factor XIIIa-catalysed of Pea l e c t i n
coupling
various s t r u c t u r a l proteins
S t u d y o f t h e ca r bo h y d r a t e- b i n d i n g specificity
2 47
o f pea l e c t i n
Study o f b i n d i n g o f i n s u l i n
355
receptors t o l e c t i n s Peanut a g g l u t i n i n
I s o l a t i o n o f l e c t i n receptors
374
f r o m human e r y t h r o c y t e s w i t h o u t use o f d e t e r g e n t s Phy t o h a e m a g g l u t i n i n E
Support f o r immobilization o f
3 75
e r y t h r o p o i e t i n f o r attachment o f erythroid colonies i n v i t r o Protein A
Development o f a new method o f
368
examining and q u a n t i t a t i n g t h e F a c t o r X II I a - ca t a 1ys e d co up 1i n g of various s t r u c t u r a l proteins R i c i n I and r i c i n I 1
Study o f b i n d i n g o f i n s u l i n
355
receptors t o l e c t i n s R i c i n u s communis agg lu t in
Demonst r a t i o n o f g l yco p r o t e i n
35 1
c h a r a c t e r i s t i c s o f t h e sodium channel s a x i t o x i n - b i n d i n g
compo-
n e n t f r o m mammalian sarcolemma A f f i n i t y chromatography o f angiot e n s i n- co n v e r t i n g enzyme f r o m swine k i d n e y ,
serum, and c u l t u r e
172
669
8: Chemical Synthesis and Modification Table 5 continued
Species coupled t o
Use o f p r o d u c t
Ref.
I s o l a t i o n o f l e c t i n receptors
374
cy ano gen b r o m i d e a c t i v a t e d agarose
media Soybean a g g l u t i n i n
f roll; human e r y t h r o cy t e s w i t h o u t use o f d e t e r g e n t s Thrombin
I n t e r m e d i a t e g e l i n method f o r
3 76
demonstration o f thrombinb i n d i n g p l a t e l e t p r o t e i n s by c r o s se d i m m uno e l e c t r o ph o r e s i s Wheat -ge r m a g g l u t i n i n
Demonstration o f glycoprot e i n
35 1
c h a r a c t e r i s t i c s o f t h e sodium channel s a x i t o x i n - b i n d i n g
compo-
n e n t f r o m mammalian s a r c o l e m m a Removal o f l u t r o p h i n ,
chorio-
gonadotrophin B-subunit,
363
folli-
t r o p h i n , and t h y r o t r o p h i n d u r i n g preparation o f glycoprot e i n hormone-free
serum
Support f o r i m m o b i l i z a t i o n o f e r y t h r o p o i e t in f o r a t t a c h m e n t
o f erythroid colonies i n v i t r o
aSee Scheme 3 ( 2 9 ) bSee Scheme 3 ( 3 1 )
375
Carbohydrate Chemistry
670
C o l um n s o f d i e t h y 1am i n o e t h y l - aga r o s e b e a d s o f h i g h m a x i m um capacity
have
been
examined f o r
their
suitability
s e p a r a t i o n i n chromatofocusing systems.377
for
protein
L a r g e sudden c h a n g e s o f
i o n i c s t r e n g t h s h o u l d be a v o i d e d when u s i n g t h i s medium,
a n d when a n
a n i o n exchanger i s used t h e components o f t h e p r e - e q u i l i b r a t i o n b u f f e r a n d t h e e l u t i o n b u f f e r s h o u l d be o f t h e c a t i o n i c t y p e , t h e counter-ions
s h o u l d n o t be
subject
while
to association-dissociation
e q u i l i b r i a i n t h e pH r a n g e o f o p e r a t i o n .
Experimental d e t a i l s f o r
o p t i m a l use o f t h e s y s t e m a r e r e p o r t e d .
I so 1a t i o n o f e l e c t r o 1e c t in, a B-Q -ga 1a c t o s i d e - b i n d i n g l e c t in from glectrophorus electricus using
lactose
bound t o
( e l e c t r i c eel), has been a c h i e v e d
d i v i n y 1s u l p h o n e - a c t i v a t e d
agarose.378
D i v i n y l s u l p h o n e - a c t i v a t e d a g a r o s e has been u s e d t o i m m o b i l i z e d i c o um a r o 1 f o r
the
a f f in i t y
- ch r o m at o g r a p h i c
azo-
p u r if i c a t i o n o f
c y t o s ~ l i c m , ~i t~o ~~ h o n d r i a l ,a ~ n d~ m ~ icrosomal 380 DT-diaphorase f r o m l i v e r o f c o n t r o l and 3 - m e t h y l - c h o l a n t h r e n e - t r e a t e d E p o x y - a c t i v a t e d agaroses c o n t i n u e t o a r a t i o n of
affinity
matrices.
Affinants
be
rats.
used w i d e l y
for
prep-
coupled t o e p o x y - a c t i v a t e d
agarose have i n c l u d e d 3 - a m i n o p h e n y l b o r o n i c a c i d f o r i s o l a t i o n o f p o l y p e p t i d e s c o n t a i n i n g a r g i n i n e m o d i f i e d w i t h c y c l o h e x a n e 1,2d i ~ n e ,f l u ~ p~ h e~n a z i n e
for
the
rapid purification of
calmodulin i n
good y i e l d , 3 8 2
cyclohexaamylose f o r
i n g enzyme of
g e r m i n a t i n g r i c e e n d ~ s p e r m , 2~- h~y ~ droxy-3,5-di-iodo-
benzoic a c i d for affinity
t h e p u r i f i c a t i o n o f a debranch-
affinity-chromatographic
p r o t e i n s from
c o r n membranes,384
p u r i f i c a t i o n o f auxin
maltose f o r
purification
o f m a l t o s e - b i n d i n g p r o t e i n f r o m S a c c h a r o m y c e s ~ e r e v i s i a e ,a n~d ~ ~ ovomucoid f o r
purification
of
a trypsin-like
enzyme f r o m
Strepto=
myces
e r y t h r e ~ s . U ~ s~i n~g p o l y e t h y l e n e g l y c o l c o u p l e d t o e p o x y a c t i v a t e d agarose, t h e i n f l u e n c e o f mo b ile -p has e c o m p o s i t i o n on t h e chromatographic behaviour
o f human p e r i p h e r a l b l o o d c e l l s h a s been
investigated.387
P o l y e t h y l e n e i m i n e has been c o u p l e d t o epoxy-
a c t i v a t e d agarose
and i t s p r o p e r t i e s a n d s u i t a b i l i t y
f o r use a s an
have been i n ~ e s t i g a t e d . ~ ' ~The m a t r i x
exhibits high
i o n exchanger capacity,
'
fast
e q u i l i b r a t i o n on a cid -b a se
i n a pH g r a d i e n t .
titration,
and no maxima
R e s o l v i n g p o w e r c o u l d be i m p r o v e d by c o n v e r t i n g
some o f t h e i m i n o g r o u p s i n t o g u a n i d i n e g r o u p s by m o d i f i c a t i o n w i t h
0-methylisourea. purifications of coupled t o
The
matrix
epoxy-activated
p u r i f i c a t i o n of
was
applied successfully
m y o g l o b i n and a c e t y l a t e d p a p a i n . agarose
6G-fructosyltransferase
r i b o s e 5-phosphate
include
to
the
Other a f f i n a n t s
raffinose,
fro m asparagus
for
roots,389
the
Q-
f o r p u r i f i c a t i o n o f Candida u t i l i s t r a n s k e t o l a s e
67 1
8: Chemical Synthesis and Modification free
from
g-ribulose
5 - p h o ~ p h a t e - 3 - e p i m e r a s e , ~ ~and ~ tris(hydroxy-
methy1)aminomethane f o r
the
purification of
brush
border
membrane
m a l t a s e f rom r a t kidney.391 E p o x y - a c t i v a t e d a g a r o s e h a s been t r e a t e d w i t h ammonium hydroxide succinic
to
yield
anhydride
an to
amino-agarose
which
was
treated
a f f o r d an N-hydroxysuccinimide
with
ester.392
C h i t o b i o s e was c o u p l e d t o t h i s m a t r i x by r e d u c t i v e a m i n a t i o n i n t h e presence o f
sodium cyanoborohydride,
and t h e r e s u l t i n g a f f i n i t y
m a t r i x was a p p l i e d s u c c e s s f u l l y t o t h e p u r i f i c a t i o n o f a C y t i s u s t y p e U l e x e u r o p a e u s h a e m a g g l u t i n i n 11.
The d e r i v a t i z a t i o n o f epoxy-
a c t i v a t e d agarose w i t h various carbohydrates i n order t o prepare s t a b l e and h i g h - c a p a c i t y particular
relevance
a f f i n i t y a d s o r b e n t s h a s been d e s c r i b e d w i t h to
the
use
of
the
matrices 93
chroma t o g r a ph y o f ca r b o h y d r a t e - b i n d i n g p r o t e i n s
for
.
affinity
S u p p o r t s f o r m e t a l - c h e l a t e a f f in i t y c h r o m a t o g r a p h y h a v e b e e n p r e p a r e d by c o u p l i n g i m i n o d i a c e t i c a c i d w i t h e p o x y - a c t i v a t e d a g a r o s e to
yield
a
bis-carboxymethylamino-agarose,
e q u i l i b r a t e d w i t h a s u i t a b l e m e t a l ion. chromatography
has been a p p l i e d t o t h e
polymerase 8 s t i m u l a t o r y l i v e r nuclei.394
The
which
i s
then
I n t h i s manner Cu2+ c h e l a t e p a r t i a l p u r i f i c a t i o n o f RNA
f a c t o r from nonhistone p r o t e i n s o f r a t
use o f m e t a l - c h e l a t e
affinity
chromatography
f o r the separation o f nucleotides according t o t h e i r metal a f f i n i t y has been i n v e s t i g a t e d i n d e p t h u s i n g Cu2+ a n d N i 2 + i o n s . 3 9 5 adsorption
and d e s o r p t i o n o f
chromatography
p r o t e i n s i n Zn2+-chelate
The
affinity
has been s t u d i e d u s i n g p u r i f i c a t i o n o f a l b u m i n a s a
model system.396
A d s o r p t i o n was s h o w n t o be d e p e n d e n t t o a g r e a t
e x t e n t o n t h e t y p e o f b u f f e r i o n s used:
a c e t a t e and phosphate i o n s ,
w i t h a low a f f i n i t y f o r t h e chelated metal, f a c i l i t a t e adsorption w h i l e a m i n e s p r e v e n t a d s o r p t i o n o r cause d e s o r p t i o n o f a d s o r b e d U s i n g 2 n2+-ch e l a t e ch rom a t o g r a p h y s u c c e s s f u l p u r if i c a t i ons
protein.
r e p o r t e d h a v e i n c l u d e d t h o s e o f p l a s m i n o g e n a c t i v a t o r s e c r e t e d by human melanoma
cells
i n culture,253
of
Dolichos b i f l o r u s
seed
l e ~ t i n a, n d~ o~f h~u m a n f i b r o b l a s t i n t e r f e r o n . 3 9 8
Co2+, Cu2+, a n d
Z n2+
have
for
com p l exes
with
immobilization
b i s - ca r b o x ym e t h y l a m i n o - a g a r o s e of
alkaline
phosphatase,
lactate
be e n u s e d
dehydrogenase,
and m a l a t e d e h ~ d r o g e n a s e . ~ ~ ~ Commercially
available
Affi-gel
supports,
agarose
beaded g e l
i n c l u d e d i n a p o l y a c r y l a m i d e s u p p o r t h a v i n g d i f f e r e n t s p a c e r arm lengths,
have been u s e d f o r t h e p r e p a r a t i o n o f a number o f a f f i n i t y
matrices. linkages.
The
spacers are
anchored
by
extremely
stable
ether
R e a c t i o n c o n d i t i o n s have been o p t i m i z e d f o r i m m o b i l -
Carbohydrate Chemistry
672
i z a t i o n o f concanavalin A (as model p r o t e i n ) t o agarose bearing an u n c h a r g e d 1 - h y d r o x y s u c c i n i m i d e e s t e r s p a c e r arm ( A f f i - g e l 101 a n d t o agarose
derivatized
with
an N - h y d r o x y s u c c i n i m i d e
t e r t i a r y amine i n t h e spacer t h e charge o f important
(Affi-gel
15).400
group
with
a
R e s u l t s showed t h a t
t h e p r o t e i n r e l a t i v e t o t h e charge o f t h e g e l i s an
f a c t o r a f f e c t i n g t h e amount o f p r o t e i n c o u p l e d t o a c t i v e
e s t e r gels.
Ligands i m m o b i l i z e d on A f f i - g e l
c l o n a l anti-(human
1 0 have i n c l u d e d mono-
leucocyte interferon) antibody f o r the p u r i f i c a t -
i o n o f recombinant
human l e u c o c y t e i n t e r f e r o n , 4 0 1
2-chloro-10-(3-
aminopropy1)phenothiazine f o r the p u r i f i c a t i o n o f c a l m o d u l i n from bovine brain,402
i n s u l i n f o r p u r i f i c a t i o n o f the i n s u l i n receptor
p r o t e i n f r o m p o r c i n e l i v e r membranes,403 purification
of
a
chymotrypsin-like
bovine405 granulocytes,
and
chromatographic studies of
4-phenylbutylamine
enzyme
soybean
from
agglutinin
for
or for affinityhuman404
the l e c t i n p r o p e r t i e s o f herpes simplex
v i r u s type 1 glycoprotein.406 h a s been i m m o b i l i z e d o n A f f i - g e l
Tetraiodofluorescein used f o r
separation
of
heterogeneous
1 0 2 and
p r o t e i n mixtures.407
Simple
a n d r a p i d p u r i f i c a t i o n o f t r y p t o p h a n 5-mono-oxygenase f r o m r a b b i t b r a i n has
been a c h i e v e d
u s i n g 2- am i n 0 - 4 - hy d r o x y-6,7-dime t h y 1 t e t r a -
h y d r o p t e r i d i ne i m m o b i l i z e d o n A f f i - g e l 202.408 E t h y l e n e g l y c o l y l b i s ( s u c c i n i m i d y l s u c c i n a t e ) has p r o v i d e d a b i f u n c t i o n a l reagent
f o r c r o s s l i n k i n g and r e v e r s i b l e i m m o b i l i z a t i o n
o f p r o t e i n s t h r o u g h t h e i r amine
groups.409
Trypsin
i m m o b i l i z e d on
a g a r o s e u s i n g t h i s r e a g e n t r e t a i n s f u l l s p e c i f i c a c t i v i t y and c a n be r e l e a s e d from t h e m a t r i x w i t h hydroxylamine.
N- S u c c i n i m i d y 1 3 -( 2-py r i dy 1d i t h i o ) p r o p i ona t e h a s been u s e d f o r c o n t r o l l e d c o u p l i n g o f l y s o z y m e a n d b o v i n e serum a l b u m i n t o g e l a t i n agarose.410
The
protein to
be
c o u p l e d and t h e
gelatin-agarose
c o n j u g a t e w e r e s e p a r a t e l y t r e a t e d w i t h t h e c o m p o u n d t o y i e l d 3(2-py r i dy 1d i t h i o ) - p r o p i o ny 1 de r i va t i ve s. reduced t o
thiopropionyl-protein
The so 1ub 1e de r i v a t i ve w as
and t h e n c o n j u g a t e d t o
the
deriv-
a t i z e d g e l a t i n c o n j uga t e t h r o ugh s u l ph y d r y 1- d i s u l p h i de e x c h a n g e . This
new
method p e r m i t s
insoluble matrix,
controlled coupling of
proteins to
an
and t h e bond t h r o u g h w h i c h r e a c t i o n o c c u r s i s
known p r e c i s e l y . A method f o r
redox
system
as
g r a f t i n g enzymes o n t o p o l y s a c c h a r i d e s u s i n g a a
Glycidylmethacrylate
radical was
i n i t i a t o r
used as
a
has
been
vinylating
reported.411
reagent
and t h e
r e a c t i o n p r o d u c t w i t h enzymes was c o p o l y m e r i z e d w i t h a g a r o s e . Organomercurial-agarose
has
been
applied
to
the
affinity
673
8: Chemical Synthesis and Modification chromatography
o f
constituent
enzyme
activities
of
the
A new method f o r p r e p a r a t i o n o f p o l y a c r y l h y d r a z i d e - a g a r o s e
has
m u l t i f u n c t i o n a l enzyme p u t r e s c i n e ~ y n t h a s e . ~ ~ ~ been d e s c r i b e d , by
based o n t h e p e r i o d a t e o x i d a t i o n o f a g a r o s e f o l l o w e d
reacti o n w i t h
p o l y a c r y l h y d r a z ide.41
Enzymes,
antibodies,
l e c t i n s , and s m a l l l i g a n d s were c o u p l e d t o t h e s u p p o r t : o f b i n d i n g and b i o l o g i c a l a c t i v i t y combines
the
advantages
interactions with proteins,
of
both
h i g h degrees
were obtained. agarose
good f l o w
The c a r r i e r
(minimal
nonspecific
r a t e s ) and a c r y l a m i d e ( l a r g e
number o f m o d i f i a b l e g r o u p s , absence o f c h a r g e d g r o u p s ) . Differences i n the interactions of
t h e c a t a l y t i c groups o f t h e
a c t i v e c e n t r e s o f a c t i n i d i n and p a p a i n have been s t u d i e d by a f f i n i t y chromatography
on
the
2-pyridyl
disulphide
mer cap t o h y d r o x y p r o p y l e t h e r a g a r o se g e l . Thiol-agarose
derivative
of
413
the p u r i f i c a t i o n o f
al-
a n t i c h y m o t r y p s i n f r o m human p l e u r a l f l u i d and human s e r u m , l a 3
h a s been a p p l i e d t o
the
p a r t i a l p u r i f i c a t i o n o f b i o l o g i c a l l y active low-molecular-weight human a n t i - h a e m o p h i l i c
f a c t o r f r e e o f von W i l l e b r a n d f a c t o r , 4 1 4
purification of
a Ca2+-dependent
platelets,415
isolation of
casein,416
-i n vitro
the
thiol
k-casein-like
protease
from
fractions
from
the
human human
and t h e f r a c t i o n a t i o n o f n u c l e a r p r o t e i n s A O P - r i b o s y l a t e d i n t o t h i o l - and n o n - t h i o l - c o n t a i n i n g
Thiopropyl-agarose
fractions.417
h a s been shown t o p r o v i d e a s i m p l e and r a p i d
means o f i s o l a t i n g t h i o l p e p t i d e s f r o m l a r g e p r o t e i n s , p l a s m i n as a model protein.418
using cerulo-
The m a t r i x h a s a l s o been a p p l i e d t o
t h e p u r if ica t i o n s , by t h io 1- d i s u l p h i de in t e r c han ge ch r om a t o g r a p h y , b i o l o g i c a l l y a c t i v e l o w - m o l e c u l a r - w e i gh t hum an a n t i-haem o p h i l i c
of
factor,414
and a l - a n t i c h y m o t r y p s i n
f r o m human p l e u r a l f l u i d a n d
human s e r u m . l a 3 Agarose
h a s been t r e a t e d w i t h 4 - t o l u e n e s u l p h o n y l
(tosyl chloride)
with
formation
of
chloride
4-toluenesulphonic
ester
( t o ~ y l a t i o n ) . ~The ~ ~ r e a c t i o n i s r a p i d a n d c a n be c o n t r o l l e d t o g i v e a r a n g e o f s u b s t i t u t i o n s up t o v e r y h i g h l e v e l s . agarose
shows
excellent
long-term
stability
p r o p e r t i e s s i m i l a r t o t h o s e o f u n t r e a t e d agarose,
The t o s y l a t e d
and h a s
cross-linking o f the support occurs during tosylation. displacement nucleophiles,
of
the
tosyl
groups
occurs
on
.
Efficient
addition
such a s amino- o r m e r c a p t o - g r o u p - c o n t a i n i n g
a l l o w i n g ready p r e p a r a t i o n o f i m m o b i l i z e d a f f i n i t y proteins
swelling
i n d i c a t i n g t h a t no of
compounds, l i g a n d s 'and
T r i a z i n y l dyes ( u s u a l l y C i b a c r o n Blue F3GA) r e a c t w i t h a g a r o s e
674
Carbohydrate Chemistry
via the the
c h l o r i n e atoms on t h e t r i a z i n e
purification of
numerous
h a s been examined a s a method f o r phosphatase,
and have been used f o r
Dy e - l i g a n d
chromatography
the purification o f alkaline
a n d 46 dye-MatrexR g e l s w e r e assessed f o r t h e i r a b i l i t y
t o b i n d a l k a l i n e phosphatase.420
C i b a c r o n B l u e F3GA a n d P r o c i o n R e d
each i m m o b i l i z e d o n agarose,
HE-36,
ring
proteins.
investigation o f enzymes.421
their
The
have been u s e d i n a s y s t e m a t i c
interactions
r e s u l t s were
multinucleotide-dependent
with
used t o
design
specific
elution
t e c h n i q u e s f o r p u r i f i c a t i o n o f t h e s e e n z y m e s by a f f i n i t y c h r o m a t o g raphy. C i b a c r o n B l u e F3GA i m m o b i l i z e d o n a g a r o s e h a s b e e n s u c c e s s f u l l y a p p l i e d t o t h e p u r i f i c a t i o n s o f c a l f thymus
ribonuclease H I,422
cy t o s o l i c b r o a d s p e c i f i c i t y B - Q - -glucosidase n i t r a t e reductase
from
Ankistrodesmus
a
f r o m human l i v e r , 4 2 3
braunii,424
r a t
l i v e r
a s p a r a g i n e ~ y n t h e t a s e ,h~u m~a~n p l a t e l e t - d e r i v e d g r o w t h f a c t o r , 4 2 6
a
s o l u b l e g l y c o p r o t e i n from l u n g i n pulmonary a l v e o l a r p r o t e i n ~ s i s , ~ ~ ~ a
lymphocyte
ATP-dependent
deoxyribonuclease,428
B-hydroxy-
i s o b u t y r a t e d e h y d r o g e n a s e f r o m C a n d i d a r ~ g o s a p, o~t a~t o~ ( S o l a n u m
-______ t u b e r osum)
l a c t a t e d e h y d r o ge n a s e iso e n z y m e s , 4 3 0 h u m a n f i b r o b l a s t
i n t e r f e r o n ,431
p i g h e a r t n u c l eo s i d e
mine synthetase
from
d i pho s p h at e
k i n a s e ,432
n i t r a t e r e d u c t a s e a n d NADH : n i t r a t e r e d u c t a s e f r o m r a t ~ i ~ - a n t i t r y p s i n2 ,- e~n ~o y~l - C o A phytochrome
from
rye
c o r n roots,434
reductase from Mycobacterium
~ m e g m a t i s ,a~n ~N~A D - l i n k e d a c e t o a c e t y l - C o A
ramigera,437
I-g l u t a -
: t h e c y a n o b a c t e r i u m A n a b a e n a 7 1 2 0 , ~NAD(P)H ~ ~
r e d u c t a s e f r o m Zoogloea
( S e c a l e c e r e a l e ) ,438
flavokinase
f r o m m u n g b e a n ( P h a s e o l u s a ~ r e u s ) , ~ 'c ~a r n o s i n e s y n t h e t a s e f r o m a v i a n muscle,439
.
a n d NAD g l y c o h y d r o l a s e f r o m
Bungarus f a s c i a t u s
venom 440 T h e C i b a c r o n B l u e F3GA d y e b i n d i n g p r o p e r t i e s o f f r o m f o u r m a m m a l i a n s p e c i e s (human, investigated
using the
dye
rat,
bound t o
a-feto-protein
b o v i n e , o v i n e ) have been
agarose.441
The
affinity
c h r o m a t o g r a p h y o f serum a l b u m i n s f r o m human a n d a n i m a l p l a s m a s h a s b e e n i n v e s t i g a t e d u s i n g C i b a c r o n B l u e F 3 G A - a g a r 0 s e . ~ ~ ~A f f i n i t y
gel
e l e c t r o p h o r e t i c s t u d i e s were used t o examine t h e e f f e c t o f l i g a n d p r e s a t u r a t i o n with b i l i r u b i n and f a t t y serum a l b u m i n s
with
a c i d s on t h e i n t e r a c t i o n o f
i m m o b i l i z e d dye.443
The
effect
o f nucleotide
p r e sa t u r a t i o n o n t h e c h r o m a t o g r a p h ic be h a v i o u r o f 6 - p h o s p h o g 1 u c o na t e dehydrogenase
from
Bacillus stearothermophilus on
immobilized
C i b a c r o n B l u e F3GA a n d P r o c i o n R e d H E 3 6 h a s a l s o b e e n i n v e s t i g ated.444
C i b a c r o n B l u e F3GA-agarose
has
been used t o i d e n t i f y
the
m a j o r component o f t h e oestrogen-induced p r o t e i n o f r a t u t e r u s as
8: Chemical Synthesis and Modification t h e BB i s o e n z y m e o f
creatine
675 kinase.445
I m m o b i l i z e d Cibacron Blue
F3GA a n d P r o c i o n R e d HE36 h a v e b e e n u s e d f o r s t u d i e s o n t h e i n t e r action of
t h e dyes w i t h dopamine B - m o n o ~ x y g e n a s e . ~ ~ ~
Triazine-activated dihydrazide
agarose
has
and t h e n c o u p l e d w i t h
been t r e a t e d w i t h
cysteinyl-proline
provide a matrix f o r affinity-chromatographic
succinic
derivatives
to
p u r i f i c a t i o n o f human
serum a n g i o t e n s i n-conve r t i n g enzym e.447 Cibacron
B l u e F3GA i s a l s o a v a i l a b l e
immobilized on Affi-gel,
a n d t h e s e p r e p a r a t i o n s have been u s e d t o i n v e s t i g a t e t h e i n t e r a c t i o n between t r a n s c o b a l a m i n I 1 and t h e
dye,448
t o p u r i f y the
s t i m u l a t e d l i p a s e f r o m human m i l k w h e y , 4 4 9
c a s e i n k i n a s e I,450 and t o p r o b e t h e m o l e c u l a r chrome.451
Affi-gel
structure of
from
synthetase
cells,453
o a t seedlings,454
phyto-
B l u e h a s a l s o been u s e d f o r p u r i f i c a t i o n s o f an
ap u r i n i c / a p y r i d i m i n i c e n d o n u c l e a s e from
bile salt-
t o p u r i f y c a l f thymus
Ehrlich ascites
tumour
HeLa
cells,452
CTP
phytochrome
from
, n~d ~ ~ m e t h y l t r a n s f e r a s e I from B a c i l l u s ~ u b t i l i s a
a mem b r a n e - b o u n d ATP d i p h o s p h o h y d r o l a s e f r o m C i c e r a r i e t i n u m ( c h i c k pea) roots.456
C i b a c r o n B l u e F3GA i m m o b i l i z e d o n M a t r e x R g e l h a s
been u s e d f o r t h e p u r i f i c a t i o n o f k e r a t a n s u l p h a t e - d e g r a d i n g e B - 6 g a l a c t o s i d a s e f r o m F l a v o b a c t e r i u m k e r a t o l y t i ~ u s . ~M~a t~r e x
Gel
R e d h a s b e e n u s e d f o r p u r i f i c a t i o n o f CTP s y n t h e t a s e f r o m E h r l i c h a s c i t e s tumour cells.453
Procion Blue-agarose
has been used t o
remove c o n t a m i n a n t s d u r i n g p u r i f i c a t i o n o f human serum a n g i o t e n s i n l - c o n v e r t i n g enzyme,319
-Acer
and 6 - p h o s p h o g l u c o n a t e
C
pseudopxamgs
has
been
purified
dehydrogenase f r o m
on P r o c i o n
R e d HE-3B
immobilized on a g a r ~ s e . ~ ~ ~ B l u e d e x t r a n ( d e x t r a n c o u p l e d w i t h C i b a c r o n B l u e F3GA) c o u p l e d to
cyanogen
bromide-activated
agarose
has
been
used
for
the
L-
p u r i f i c a t i o n s o f chicken l i v e r L-threonine
dehydrogenase,221
glutamine synthetase from Escherichia coli,222
coenzyme A s y n t h e t a s e
&.
from
Q - g l y c e r a l d e h y d e 3-phosphate dehydrogenase from
l a c t i c a c i d b a c t e r i a , 2 2 4 L e u c o n o s t o c p h o s p h o g l y c e r a t e r n ~ t a s e ,a~ n d~ ~ r a t
Factor
.
11,226 and
t o
probe
the
molecular
structure
of
p h y t o ch r om e 451
Amylose.
--
Amylose
has
been r e a c t e d w i t h
the
methylsulfinyl
c a r b a n i o n i n d i m e t h y l s u l p h o x i d e t o form t h e a l k o x i d e o f amylose, which
r e a c t s w i t h a l l y 1 bromide
starting
material
for
to afford tri-)-allylamylose,
synthesis
of
amylose-polyester
the block
co p o l y m e r . 45 The s t r u c t u r e o f
the hydrated amylose-iodine
c o m p l e x h a s been
676
Carbohydrate Chemistry
determined
using E-ray
analysis.460 centre
of
diffraction
and s t e r e o c h e m i c a l
packing
I o d i d e was f o u n d a s an a l m o s t l i n e a r c h a i n i n t h e the
6-fold
left-handed
amylose
helix.
Eight
water
m o l e c u l e s o f h y d r a t i o n p e r u n i t c e l l w e r e l o c a t e d i n good h y d r o g e n b o n d ing p o s i t i o n s between t h e amylose h e l i c e s . C h a r a c t e r i z a t i o n o f t h e changes i n a b s o r p t i o n a n d c.d.
spectra
o f t r i - i o d i d e i o n s i n a m y l o s e w i t h c h a n g i n g DP h a s been r e p o r t e d . 4 6 1 P o s s i b l e e x p l a n a t i o n s f o r t h e s p e c t r a l changes w e r e d i s c u s s e d .
Cellulose.
--
E i g h t y - t w o compounds have been i d e n t i f i e d a s v o l a t i l e
products a f t e r Comparisons
pyrolysis of
between
cellulose
volatile
under
various
conditions.462
p y r o l y s i s p r o d u c t s and components o f
c e l l u l o s e c i g a r e t t e smoke p r o v e d t h a t t h e y w e r e q u a l i t a t i v e l y lar,
simi-
b u t t h a t m o s t p y r o l y s i s p r o d u c t s w e r e o b t a i n e d i n much l a r g e r
q u a n t i t i e s t h a n smoke c o m p o n e n t s p e r w e i g h t o f
cellulose.
Pyrene,
l - m e t h y l p y r e n e , a n d l - h y d r o x y p y r e n e have been i s o l a t e d a n d i d e n t i f i e d as mutation-enhancing p r i n c i p l e s from c e l l u l o s e p y r ~ l y s a t e . ~ ~ ~ Cellulases
from
Trichoderma
reesei
have
b i o spe c i f i c s o r p t i o n o n amorphous c e l l u l o se.464 investigation
of
cellulose
acetates
has
been
isolated
by
The r m o - chem ic a l
been
reported.465
L e u c o n o s t o c m e s e n t e r o i d e s c e l l s have been i m m o b i l i z e d by e n t r a p m e n t
i n agar
on
an
acetylcellulose
a n a 1y t i c a1 de t e r m ina t io n o f 4-Aminobenzamidine
filter,
and
I- ph e ny 1a 1a p i n e .
used f o r
the
rapid
a m i n o d o d e c y l c e l l u l o s e has been a p p l i e d t o
t h e a f f i n i t y - c h r o m a t o g r a p h i c p u r i f i ca t i o n o f a p l a s m i n o gen a c t i v a t o r f r o m m o u s e c e l l s t r a n s f o r m e d by a n o n c o g e n i c v i r u s . 4 6 6 attachment of
papain t o 4-aminobenzoyl
c e l l u l o s e has
using terephthalic diazide
as mediator.467
i m m o b i l i z e d on 4-aminobenzyl
cellulose after
matrix.468
Aminohexyl-cellulose
Covalent
been a c h i e v e d
@ - A m y l a s e h a s been d i a z o t i z a t i o n of
the
has been u s e d a s a s u p p o r t f o r
i m m o b i l i z a t i o n o f e p o x y - a c t i va t e d t a n n i n . 469 A v i d i n i m m o b i l i z e d o n c e l l u l o s e d i s c s a c t i v a t e d w i t h s o d i u m tb u t o x i d e and l - t o s y l o x y - 3 - i s o c y a n o p r o p a n e s o l i d - p h a s e assay
has been u s e d t o d e v e l o p a
for a - b i ~ t i n . ~ ~B ' enzoquinone-activated c e l l u l o s e
h a s b e e n u s e d f o r t h e i m m o b i l i z a t i o n o f p a p a i n 471 a n d o f trypsin inhibitor.472
The l a t t e r was u s e d s u c c e s s f u l l y
potato
for affinity-
chroma t o g r a p h i c p u r i f i ca t i o n o f chymot r y p s i n . T h e 1, l ' - c a r b o n y l d i - i m i d a z o l e
method o f a c t i v a t i o n o f agarose
has been e x t e n d e d t o c e l l u l o s e d e r i v a t i v e s (see f i r s t c i t a t i o n o f ref.
151) Q u a n t i t a t i v e s t u d i e s o n t h e b i n d i n g o f a v a r i e t y o f enzymes t o
677
8: Chemical Synthesis and Modification carboxymethyl-cellulose
h a v e been c a r r i e d o u t ,
and t h e m a g n i t u d e o f
the a f f i n i t y e l u t i o n e f f e c t i n the presence of enzymes
has
been
determined.473
Some
substrates o f the
calculations
were
made
d e m o n s t r a t i n g t h e r a n g e o f s t r e n g t h s o f i n t e r a c t i o n s b e t w e e n enzyme and a d s o r b e n t , molecule.
and t h e e n e r g y i n v o l v e d p e r c h a r g e on t h e p r o t e i n
C e l l u l o s e e x c h a n g e r s w i t h p y r o g a l l o l as a n c h o r g r o u p h a v e
been a p p l i e d s u c c e s s f u l l y
t o the chromatographic separation o f
antimony(1111.4~4 Cyanogen
bromide-activated
cellulose
has
been
used
t o
i m m o b i l i z e anti-(polyinosine-polycytosine)
antibody f o r s p e c i f i c
separation
and
of
double-stranded
dihydrolipoamide use
i n
the
N u c l e a s e PI
reductase
RNA’s475
t o co-immobilize
and 3 a - h y d r o x y s t e r o i d
continuous-flow
analysis
of
dehydrogenase
h a s been i m m o b i l i z e d by r e a c t i o n w i t h c y a n o g e n b r o m i d e -
a c t i v a t e d c e l l u l o s e , by c a r b o d imi ide -me d ia t e d r e a c t i o n w i t h
exchange c e l l u l o s e , complexes.477 yielding
for
3a-hydroxy~teroids.~~~
an
and
by
reaction
with
an i o n -
titanium-cellulose
The l a s t m e t h o d was f o u n d t o be t h e m o s t e f f e c t i v e , active
immobilized
enzyme
with
good
s t a b i l i t y
properties. Activati.on o f c e l l u l o s e w i t h butan-1,4-diol
d i g l y c i d y l ether
h a s p r o v i d e d a m a t r i x f o r s i m p l e and e f f i c i e n t i m m o b i l i z a t i o n o f d e o x y r i b o n u c l e i c acid.478
Development o f
t h e m e t h o d was d e s c r i b e d ,
a n d t h e a f f i n i t y m a t r i x was t h e n a p p l i e d t o t h e p u r i f i c a t i o n o f messenger
ribonucleic
cellulose
has
dependent
also
acid.
been
Deoxyribonucleic
used
deoxyribonuclease
for
the
from
acid
coupled t o
purification of
Bacillus
an
ATP-
laterosporus.479
A c t i v a t e d g l u c o c o r t i c o i d - r e c e p t o r complex f r o m r a t l i v e r c y t o s o l has been adsorbed on d e o x y r i b o n u c l e i c a c i d - c e l l u l o s e . 4 8 0 c o u l d be e x t r a c t e d i n a dose-dependent different
The c o m p l e x
manner by i n c u b a t i o n w i t h
c o n c e n t r a t i o n s o f sodium t u n g s t a t e .
Using d i a z o t i z e d paper b l o t s , obtained from d i f f e r e n t i a t e d
heterogeneous p r o t e i n samples
n e u r o b l a s t o m a c e l l s have been e l e c t r o -
p h o r e t i c a l l y t r a n s f e r r e d t o t h e b l o t s and used f o r
-
a f f i n i t y
p u r if ic a t i o n o f a h e t e r o ge neo u s a n t i m ic r o t u b u l e p r o t e in .481 Kinetic reasons for
the
i n h o m o g e n e i t y and c h a r a c t e r
o f
deformability o f the s p a t i a l network o f chemically crosslinked c e l l u l o s e e t h e r s have been discussed.482 The b e h a v i o u r o f m e t h y l o l a t e d c a r b a m o y l e t h y l a t e d c e l l u l o s e towards d i f f e r e n t
c l a s s e s o f d y e s t u f f s has been i n v e s t i g a t e d . 4 8 3
T h e d y e i n g o f c o t t o n g r a f t c o p o l y m e r s w i t h some d i r e c t , b a s i c , a n d r e a c t i v e d y e s h a s a l s o been d e s c r i b e d . 4 8 4
678
Carbohydrate Chemistry
Well d e f i n e d low- m o l e c u l a r - w e i g h t p o l y s t y r e n e has been g r a f t e d o n t o c e l l u l o s e a c e t a t e i n homogeneous s o l u t i o n . 4 8 5 The g r a f t i n g was performed b y e s t e r i f y i n g t h e f r e e hydroxyls i n t h e c e l l u l o s e a c e t a t e w i t h a n i o n i c a l l y p r e p a r e d p o l y s t y r e n e having a c a r b o x y l i c a c i d e n d group which was a c t i v a t e d e i t h e r b y c o n v e r s i o n t o t h e c o r r e s p o n d i n g a c i d c h l o r i d e o r by r e a c t i o n w i t h t r i f l u o r o a c e t i c a n h y d r i d e . G r a f t c o p o l y m e r i z a t i o n of methyl m e t h a c r y l a t e o n t o c e l l u l o s e h a s been s t u d i e d b y v a r y i n g t h e monomer, i n i t i a t o r , t h i o u r e a and a c i d c o n c e n t r a t i o n s , and t e m p e r a t u r e . 4 8 6 The g r a f t y i e l d i n c r e a s e d The e f f e c t s of s t e a d i l y w i t h an i n c r e a s e i n monomer c o n c e n t r a t i o n . d i f f e r e n t monomers and t h e n a t u r e of t h e s u b s t r a t e s were a l s o studied. P h o t o s e n s i t i z e d g r a f t p o l y m e r i z a t i o n of a v i n y l monomer onto c o t t o n h a s been i n v e s t i g a t e d . 4 8 7 M o d i f i c a t i o n of c e l l u l o s e w i t h v i n y l monomers u s i n g t h e xanthogenate-hydrogen p e r o x i d e redox system h a s a l s o been d e s c r i b e d . 4 8 8 G r a f t i n g of m e t h y l m e t h a c r y l a t e on c e l l u l o s e c o n t a i n i n g s u l p h o n i c a c i d groups has b e e n a c h i e v e d u s i n g an Fe*+-hydrogen p e r o x i d e redox system.489 A method f o r g r a f t i n g enzymes o n t o p o l y s a c c h a r i d e s u s i n g a redox system a s a r a d i c a l i n i t i a t o r h a s been reported.411 G l y c i d y l m e t h a c r y l a t e was u s e d a s a v i n y l a t i n g r e a g e n t and t h e r e a c t i o n p r o d u c t w i t h enzymes was copolymerized w i t h c e l l u l o s e . C e l l u l o s e n i t r a t e h a s been u s e d t o t r a n s f e r t i s s u e - s p e c i f i c a n t i g e n s from p o l y a c r y l a m i d e g e l s f o l l o w i n g e l e c t r o p h o r e ~ i s . ~ ~ ~ C e l l u l o s e n i t r a t e b l o t t i n g h a s a l s o been u s e d i n a s t u d y of h e p a t i t i s B s u r f a c e a n t i g e n e l e c t r o p h o r e s e d i n a g a r o s e gels.491 P o l y ( a d e n y 1 i c a c i d ) - c o n t a i n i n g r i b o n u c l e o p r o t e i n s h a v e been s e p a r a t e d b y a f f i n i t y c h r o m a t o g r a p h y on o l i g o t h y m i d y l i c a c i d cellulose.492 The c o v a l e n t b i n d i n g of t r y p s i n t o c e l l u l o s e b e a d s a f t e r p e r i o d a t e o x i d a t i o n has been examined.493 T h e d e g r e e of c e l l u l o s e derivative solubilization is directly related t o cellulose oxidation and i n c r e a s e s w i t h t h e i n c r e a s i n g pH of t h e r e a c t i o n m i x t u r e . D e x t r a n s u c r a s e from Leuconostoc m e s e n t e r o i d e s h a s been i m m o b i l i z e d on porous p h e n a x y a c e t y l c e l l u l o s e beads.494 NAD p y r o p h o s p h a t a s e has been immobilized b y a d s o r p t i o n on p h o s p h o c e l l u l o s e . 495 The p o s i t i o n a n d d e g r e e o f e s t e r s u l p h a t i o n i n n a t u r a l spectrosc e l l u l o s e s u l p h a t e h a v e been d e t e r m i n e d b y 1 3 C n . m . r . COPY
496
T h i o s u l p h a t e d e r i v a t i v e s , w h i c h c a n be r e d u c e d w i t h mercaptoacetic acid, a r e s u i t a b l e intermediates for the preparation
679
8: Chemical Synthesis and Modification o f t h i o l d e r i v a t i v e s o f polymers.497 c e l l u l o s e have been p r e p a r e d
hydroxy-propylcellulose,
1Lg
Thiosulphate d e r i v a t i v e s o f
chlorodeoxy-
or
3-chloro-2-
w h i l e mercaptodeoxy-cellulose
prepared
a c h l o r o d e o x y d e r i v a t i v e was m o r e s u i t a b l e f o r t h e i m m o b i l i z a t i o n o f no n - t h i o l enz ym es
.
4 - To 1ue ne s u l p h ony 1 ch 1o r ide - t r e a t e d c e 11u l ose h a s bee n t r e a t e d
separately
with aniline,
to y i e l d four with
be nz y l a m ine
,
1-am inob utane, a n d p i p e r a z i ne
d i f f e r e n t a m i n o c e l l u l o s e s w h i c h were f u r t h e r t r e a t e d
carbon
disulphide
to
furnish
four
dithiocarbamate
cellu-
l o s e ~ . ~ A~ *c o m p a r a t i v e s t u d y w a s made o f t h e i r p e r f o r m a n c e a s adsorbents for
Chitin.
--
several k i n d s o f m e t a l ions.
C h i t i n h a s been d i s s o l v e d i n s o d i u m h y d r o x i d e t o p r o d u c e
a h i g h l y v i s c o u s a l k a l i n e c h i t i n s o l u t i o n w h i c h was t h e n t r e a t e d w i t h 2-chloroethanol
t o y i e l d w a t e r - s o l u b l e g l y c o l ~ h i t i n . The ~ ~ ~
d e g r e e o f g l y c o l a t i o n o f c h i t i n was shown t o be p r o p o r t i o n a l t o t h e amount
o f 2-chloroethanol
used,
under t h e c o n d i t i o n s employed.
Lysozyme h y d r o l y s e d 8-30% g l y c o l a t e d c h i t i n w i t h a l m o s t c o n s t a n t efficiency,
b u t a t a l o w d e g r e e o f g l y c o l a t i o n t h e g l y c o l c h i t i n was
much l e s s e f f i c i e n t l y h y d r o l y s , e d and t h e r e f o r e i s n o t a s u i t a b l e s u b s t r a t e f o r measurement o f t h e a c t i v i t y of lysozyme. P e r i o d a t e o x i d a t i o n o f t h e n o n - r e d u c i n g end g r o u p s o f c h i t i n The r a t e s o f h y d r o l y s i s o f
h a s been r e p o r t e d . 5 0 0
the modified
s u b s t r a t e s by c h i t i n a s e a n d b y l y s o z y m e a r e i n c r e a s e d r e l a t i v e t o those o f unmodified c h i t i n . The a d s o r p t i o n o f
uranium
on c h i t i n p h o s p h a t e h a s b e e n
i n v e s t i g a t e d t o o b t a i n i n f o r m a t i o n about aqueous
systems,
especially
sea
water
uranium recovery
and uranium
mine
from waste
wa t e r .501 Chitin,
unmodified,
h a s been u s e d f o r a f f i n i t y - c h r o m a t o g r a p h i c
p u r i f i c a t i o n o f l y soz yme .502 The s u i t a b i l i t y concentrations
of
of
k r i l l chitin,
KOH
and
HC1
f o r
prepared using d i f f e r e n t deproteinization
and
demineralization, respectively,
as a s u p p o r t f o r enzyme i m m o b i l i z a t -
i o n has been i n v e s t i g a t e d . 5 0 3
The a c t i v i t y
on such chitin,
supports
depends
on t h e
t h e a v a i l a b i l i t y o f a m i n o groups,
t h e mesh s i z e ,
o f enzymes i m m o b i l i z e d
degree o f
deproteinization o f
the content o f minerals,
t h e s t r u c t u r e o f t h e s u r f a c e , and t h e c o n f o r m a t i o n o f
t h e c h i t i n molecules.
The i m m o b i l i z a t i o n o f B - a - f r u c t o f u r a n o s i d a s e
o n k r i l l c h i t i n has been d e s c r i b e d . 5 0 4
680
Carbohydrate Chemistry
--
Chitosan.
O l i g o s a c c h a r i d e s h a v e b e e n p r e p a r e d f r o m c h i t o s a n by
p a r t i a l a c i d i c h y d r o l y s i s f o l l o w e d by f r a c t i o n a t i o n on Dowex 5 0 (H' f o r m ) .505 The u l t r a s t r u c t u r e s o f c h i t o s a n a n d some g e l - f o r m i n g
branched-
c h a i n c h i t o s a n d e r i v a t i v e s have been s t u d i e d . 5 0 6 Using sodium cyanoborohydride, 2-amino
l a c t o s e h a s been c o u p l e d t o t h e
f u n c t i o n s o f c h i t o s a n t o a f f o r d a branched l - d e o x y l a c t i t - l -
y l derivative.507
The p r o d u c t e x h i b i t e d i n t e r e s t i n g a n d a l t e r e d
i t was i n s o l u b l e i n e t h a n o l ,
solution properties:
when d i l u t e aqueous s o l u t i o n s w e r e m i x e d w i t h a c i d
Cr2+, o r Sn2+
a q u e o u s s o l u t i o n s o f Ca2+, chromate, Five
derivatives
derivative
of
chitosan
was
o r base o r w i t h
chlorides,
potassium
o r combinations thereof.
b o r i c acid,
prepared from
aqueous e t h a n o l ,
b u t i t d i d n o t g e l or p r e c i p i t a t e
a n d o t h e r common o r g a n i c s o l v e n t s ,
p a r t i a l l y N-acetylated
c h i t o s a n h a v e been
by r e a c t i o n w i t h a c e t i c a n h y d r i d e . 5 0 8
exhaustively
oxidized with
periodate,
Each
reduced w i t h
borohydride, and h y d r o l y s e d w i t h d i l u t e a c i d b e f o r e g e l - c h r o m a t o graphic fractionation.
The c o m p o s i t i o n o f t h e r e a c t i o n p r o d u c t s
i n d i c a t e d t h a t f r e e amino g ro u p s were h e t e r o g e n e o u s l y p r e s e n t i n t h e p a r t i a l l y N-acetylated chitosan.
C h i t o s a n h a s been r e a c t e d w i t h
i n t r a m o l e c u l a r d i c a r b o x y l i c anhydrides t o form novel N-(carboxyacyl)
derivatives.509
Some p r o p e r t i e s o f
s o l u t i o n s o b t a i n e d were
P e r i o d a t e o x i d a t i o n o f t h e non-reducing c h i t o s a n has been r e p o r t e d . 5 0 0 modified substrate
by
t h e g e l s and v i s c o u s
reported. The
end groups o f N - a c e t y l -
rates o f
c h i t i n a s e a n d by
hydrolysis
of
the
lysozyme were i n c r e a s e d
r e l a t i v e t o those o f the u n m o d i f i e d polysaccharide. P a r t ia 1 1y
N- s u c c i n y l a t e d
of
de r i v a t ive s
c h i t o sa n
and
g l y c o l c h i t o s a n have been p r e p a r e d and c r o s s l i n k e d u s i n g a w a t e r s o l u b l e c a r b o d i - i m i d e t o f o r m g e l s w h i c h may p r o v i d e u s e f u l m o d e l systems f o r The
biochemical reactions.510
adsorption
of
uranium
on c h i t o s a n
i n v e s t i g a t e d t o o b t a i n i n f o r m a t i o n about aqueous
systems,
especially
sea
water
phosphate
uranium
and
has
been
recovery
from
uranium
mine
waste
w a t e r .501
A
new
ionotropic
m u l t i va
i m m o b i l i z a t i o n method has gelation of
1e n t
anion ic
chitosan co un t e r
(a
been d e s c r i b e d u s i n g t h e
polycation)
-ions
with
( F e (C N 164-,
p o l y p h o s p h a t e s, p o l y ( a 1 d e h y do- c a r bo n i c a c i d )
,
different
~e ( C N ) 6 3 - ,
p o l y -( 1- h y d r ox y -1 -
s u l p h o n a t e - p r o ~ - 2 - e n e ) ) . ~ l l E s c h e r i c h i a c o l i c e l l s have been i m m o b i l i z e d successfully
by
this
method and
r e t a i n e d -57%
of
their
8: Chemical Synthesis and Modification
68 1
i n i t i a l tryptophan synthetase a c t i v i t y .
P e p s i n h a s been i m m o b i l i z e d
on ~ h i t o s a n . ~ ~ ’
Cycloamyloses.
--
M a l t o - o l i g o s a c c h a r i d e s o f DP r a n g i n g f r o m 6 t o 8
have been p r e p a r e d by p a r t i a l h y d r o l y s i s o f c y c l o h e x a - , and c y c l m c t a - a m y l o s e s acid,
B-,
(u-,
cyclohepta-,
and y - c y c l o d e x t r i n s ) w i t h s u l p h u r i c
f o l l o w e d by c h r o m a t o g r a p h i c s e p a r a t i o n o n a c o l u m n o f h y d r o -
p h i l i c v i n y l p o l y m e r gel.513 Two h e x a h y d r a t e m o d i f i c a t i o n s o f
cyclohexa-amylose have
p r e v i o u s l y been r e p o r t e d i n aqueous s o l u t i o n w i t h o u t added g u e s t molecules.
A t h i r d crystal modification,
with five water molecules
l o c a t e d o u t s i d e t h e m o l e c u l a r c a v i t y and 2.57
w i t h i n the
cavity
as
d i s o r d e r e d w a t e r , h a s now been d e s c r i b e d . 5 1 4 Cy c l ohep ta-am y l o se has been c r y s t a l l i z e d w it h 2,5-di-iodo b e n z o i c a c i d a s a g u e s t molecule.515
The X - r a y
crystal structures o f t h i s
and o f complexes o f o t h e r meta-substituted guests were reported. The f u n c t i o n a l l y
i m p o r t a n t c a r b o x y l i c a c i d group appears t o l i e i n
the primary-hydroxyl
end o f the cyclohepta-amylose molecule,
which
s u g g e s t s t h a t t h e s t r u c t u r e s e e n i n t h e c r y s t a l s d e s c r i b e d does n o t correspond t o a c a t a l y t i c a l l y a c t i v e species. Formation constants f o r w it h a 1k a n o i c / a l kanoa t e,
b i n a r y complexes o f
cyclohexa-amylose
3- a n d 4-s u b s t i t U t e d p h e n o l / p h e n o l a t e ,
and
b e n z o i c / b e n z o a t e base s u b s t r a t e s h a v e been d e t e r m i n e d by pH p o t e n t i o m e t r y and v i s i b l e spectrophotometry.516
Dipolar interactions
a p p e a r t o be t h e m a i n b i n d i n g f o r c e i n t h e s e r e a c t i o n s . Two n e w l y s y n t h e s i z e d c y c l o h e p t a - a m y l o s e - n i c o t i n a m i d e s n i c o t i n a m i d e group o n t h e secondary-hydroxy amylose,
have a
side o f cyclohepta-
o n e i n an a x i a l a n d o n e i n an e q u a t o r i a l c o n f i g u r a t i o n . 5 1 7
The c o r r e s p o n d i n g r e d u c e d f o r m s c a n r e d u c e n i n h y d r i n w i t h l a r g e r a t e e n h a n c e m e n t s (40- and 6 0 - f o l d , r e s p e c t i v e l y ) r e l a t i v e t o NADH. also
show
enzyme-like
saturation
kinetics
i n the
They
reduction o f
n i n h y d r i n , i n d i c a t i n g t h a t t h e r e a c t i o n i n v o l v e s complex f o r m a t i o n . T h e r a t e o f h y d r o l y s i s o f 4 - n i t r o p h e n y l a c e t a t e c a t a l y s e d by azobenzene-capped
cyclohepta-amylose
i s
accelerated
by
photo-
irradiation. C y c l o h e p t a - a m y l o s e p o l y m e r s have been u s e d f o r t h e i n c l u s i o n c h r o m a t o g r a p h y o f some i n d o l e a l k a l o i d s . 519 An a q u e o u s s o l u t i o n o f o r a n g e I 1 d y e a n d c y c l w c t a - a m y l o s e f o r m s a l y o t r o p i c mesophase a t room t e m p e r a t u r e . 5 2 0
N.m.r.
a n d c.d.
s t u d i e s s u g g e s t t h a t t h e mesophase i s f o r m e d f r o m t h e i n c l u s i o n c o m p o u n d w h i c h h a s a 1:l
structure,
and t h a t orange I1 e n t e r s
682
Carbohydrate Chemistry
cyclo-octa-amylose mainly from t h e l o n g a x i s s i d e o f t h e molecule, The which then a l i g n s perpendicularly t o the magnetic f i e l d . e q u i l i b r i u m c o n s t a n t s and r a t e c o n s t a n t s f o r t h e f o r m a t i o n o f 1 3 azobenzene
and one p h e n y l a z o - n a p h t h a l e n e
c o m p l e x e s h a v e b e e n d e t e r m i n e d a t 26.6OC
dye-cyclohexa-amylose
and an i o n i c s t r e n g t h o f
The e f f e c t o f c h a n g i n g t h e s u b s t i t u e n t s o n t h e d y e s was
0.15.521
e x p l a i n e d i n t e r m s o f t h e s i z e s o f t h e s u b s t i t u e n t s and t h e c h a r g e s on t h e s u b s t i t u e n t s . The r a t e s o f h y d r o l y s i s o f p r o s t a c y c l i n ( P G 1 2 ) a n d i t s m e t h y l ester
(PG12Me)
i n
aqueous
solutions
s i g n i f i c a n t l y r e t a r d e d by c y c l o h e x a - , amyloses,
and showed
competitive
characteristic
inhibition.522
The
been
shown
to
be
and cyclo-octa-
saturation
kinetics of
deceleration effects
and
cycloamyl-
PG12Me w e r e a b o u t 3 t i m e s l a r g e r t h a n
oses on t h e h y d r o l y s i s of those on t h e h y d r o l y s i s of
PG12.
The i m p o r t a n c e o f t h e s p a t i a l
r e l a t i o n s h i p b e t w e e n t h e h o s t and g u e s t the
have
cyclohepta-,
k i n e t i c a l l y determined
stability
m o l e c u l e s was r e f l e c t e d i n
constant
for
these
inclusion
complexations. The
formation
and
stability
complexes
The m o d i f i e d d r u g e x h i b i t e d i n c r e a s e d p h o t o s t a b i l i t y towards
acid,
cyclohepta-amyloses
suggesting
that
the
have
of
studied.523
stability
and
inclusion
with
and
cyclohexa-
o f
guaiatulene
been
cycloamylose
d e r i v a t i v e s may b e o f g r e a t u t i l i t y i n t h e p r e p a r a t i o n o f s t a b l e drugs. Cyclohepta-amylose c i t r u s f r u i t products,
has
been used s u c c e s s f u l l y
t o
debitter
p r e s u m a b l y by c o m p l e x a t i o n w i t h n a r i n g i n and
limonin, the b i t t e r factors i n the fruits.524 Cycloocta-amylose
s l i g h t l y enhances t h e f l u o r e s c e n c e i n t e n s i t y
o f a-naphthyloxy-acetic cycloacta-amylose
a c i d i n aqueous s o l u t i o n . 5 2 5
When b o t h
and c y c l o h e x a n o l a r e p r e s e n t t h e i n t e n s i t y i s
m a r k e d l y enhanced,
s h o w i n g t h e r o l e o f c y c l o h e x a n o l as a space
regulator which narrows the c a v i t y o f cycloocta-amylose t o allow the i n c l u s i o n o f the fluorophore. T h e f o r m a t i o n o f a c h a r g e - t r a n s f e r c o m p l e x b e t w e e n s o d i u m anaphthylacetate
and p i c r i c
m a r k e d l y by c y c l o a c t a - a m y l o s e amylose,
a c i d has
been shown t o
but not significantly
p r e s u m a b l y because o n l y c y c l o o c t a - a m y l o s e
be
promoted
by c y c l o h e p t a -
can i n c l u d e b o t h
e l e c t r o n d o n o r and a c c e p t o r i n t h e same c a v i t y . 5 2 6 The i n t e r a c t i o n s b e t w e e n c y c l o a m y l o s e p o l y u r e t h a n e r e s i n s various
organic
chromatography.527
compounds
have been e s t i m a t e d by
Cycloamylose polyurethane r e s i n s
and
gas-solid
were
obtained
683
8: Chemical Synthesis and Modification by
polymerization o f
and/or
d.m. f.
with
guest
The
cycloamyloses
cycloamylose
molecules containing
r e s i n s c a n be u s e d t o xylene
di-isocyanates
i n pyridine
strong interactions
o r hetero-atoms.
?r-electrons
The
d i s t i n g u i s h between the c o n f i g u r a t i o n s o f
i s o m e r s and p y r i d i n e
behaviour o f
with
resins exhibit
cyclohexa-
derivatives.
The
and c y c l o h e p t a - a m y l o s e
chromatographic
polyurethane resins
p r e p a r e d by t h i s m e t h o d h a s been s t u d i e d u s i n g s i x a r o m a t i c a c i d s as model
com p o u n d s . 5 2 8
Phe n y l g l y c i n e ,
t y r o sine,
t r y p t o p h a n , and
p h e n y l a l a n i n e c a n be s e p a r a t e d c o m p l e t e l y , a l t h o u g h p h e n y l a l a n i n e i s very
strongly retained.
The i n t e r a c t i o n s b e t w e e n t h e
cycloamylose
u n i t s i n t h e r e s i n s and t h e amino a c i d s were discussed. The s o r p t i o n b e h a v i o u r o f o r g a n i c c o m p o u n d s o n p o l y u r e t h a n e
o r 6-deoxy-cyclohepta-amyloses,
r e s i n s c o n t a i n i n g 6-deoxy-cyclohexawhose
primary- hydroxyl
investigated.529
groups
are
a l l
deoxygenated,
Results suggested t h a t
these
has
been
r e s i n s w o u l d be
u s e f u l as c o l u m n p a c k i n g s f o r t h e c o n c e n t r a t i o n and s e p a r a t i o n o f o r g a n i c compounds Dextrans.
--
.
A m i x e d - s o l u t e e x c l u s i o n method f o r p o r e - s i z e
bution analysis of crosslinked
g e l substances A
dextran
has
been
mixed s o l u t i o n
distri-
developed of
using
polymer
and
oligomers covering a s u f f i c i e n t l y wide molecular-weight range i s brought
into
contact
with
a
gel
sample,
and t h e
resulting
d i f f e r e n t i a l d i l u t i o n o f the solute f r a c t i o n s i s determined as a f u n c t i o n o f t h e m o l e c u l a r s i z e by means o f g e l - p e r m e a t i o n c h r o m a t o g raphy.
The m e t h o d c o m p a r e d f a v o u r a b l y w i t h t w o o t h e r m e t h o d s .
The g e l - c h r o m a t o g r a p h i c crosslinked dextran gels investigated.531 was
explained
thermodynamic
The i n
behaviour
mechanism
terms
functions
for
of
a l i p h a t i c a l c o h o l s on
( S e p h a d e x R G-10 of
of
a n d G-15)
has
i n t e r a c t i o n i n aqueous
hydrophobic
been
systems
interactions
using
d i s s o l u t i o n i n w a t e r and f o r p a r t i t i o n
i n g e l chromatography. Affinity
c h r o m a t o g r a p h y on c r o s s l i n k e d - d e x t r a n - g e l
columns has
been u s e d t o d e m o n s t r a t e t w o d i s t i n c t a(1+3) s p e c i f i c i t i e s o f r a b b i t anti-dextran
81355 a n t i b o d i e s . 5 3 2
C o n c a n a v a l i n A,
m o d i f i e d by
U.V.
i r r a d i a t i o n i n t h e p r e s e n c e o f c h l o r o a c e t a m i d e , h a s been p u r i f i e d by a f f i n i t y chromatography on c r o s s l i n k e d - d e x t r a n - g e l Lipophilic gel-filtration S e p h a s o r b R HP U l t r a f i n e )
have
dex t r a n g e l s
columns.533
(Sephadex
LH-20
been a p p l i e d t o t h e f r a c t i o n a t i o n
p o l a r o r g a n i c c o n s t i t u e n t s i n e n v i r o n m e n t a l samples.534
and of
The m e t h o d
a l l o w e d e s s e n t i a l l y q u a n t i t a t i v e r e c o v e r y o f t h e d i f f e r e n t compounds
Carbohydrate Chemistry
684 investigated
and i s s u f f i c i e n t l y r a p i d and c o n v e n i e n t f o r use i n
routine analysis.
A p p l i c a t i o n o f t h e method t o a weathered E k o f i s k
c r u d e o i l was d e s c r i b e d . Dextran T 1 0
has
been o x i d i z e d w i t h
aqueous
bromine a t
pH
7 . 0 . ~ The ~ ~ r e s u l t i n g oxodextran and i t s methoximated d e r i v a t i v e w e r e a n a l y s e d by 1 3 C n.m.r.
s p e c t r o s c o p y and t h e t o t a l number o f
k e t o groups and t h e i r p o s i t i o n s were e s t a b l i s h e d .
I n accord w i t h
the
mono-
proposed mechanism
for
bromine o x i d a t i o n o f
and d i -
saccharides, the glucopyranosyl residues o f dextran were oxidized mainly
at
a n d C-4.
C-2
Over-oxidation
proportion o f a c i d i c ring-cleavage
resulted i n a
small
products.
D e x t r a n d e r i v a t i v e s have been s u c c e s s f u l l y a c t i v a t e d w i t h 1,l’carbonyldi-imidazole, first
citation
cyano gen
of
as previously this
described
reference).151
bromide- t reat e d cro s s l i nked dext ran,
coupled protein, inactivator,
for
Alkaline
agarose
(see
treatment
with
of
or without
has been shown t o r e l e a s e a t h i o l p r o t e a s e
the cyanate ion.155
Experimental evidence suggested
t h a t t h e carbamate group i n t h e a c t i v a t e d p o l y s a c c h a r i d e i s t h e most l i k e l y p r e c u r s o r o f t h e cyanate. Sephadex
activated with
cyanogen bromide has been used t o
i m m o b i l i z e t r y p ~ i n . D~E ~ A E~- d e x t r a n has been u s e d f o r t h e i s o l a t i o n of
casein
and
matrix.537
study
of
i t s
interaction with
the
ion-exchange
DEAE-dextran has been a d s o r b e d o n t o S p h e r o s i l R p o r o u s
s i l i c a beads and t h e n c r o s s l i n k e d w i t h 1 , 4 - b u t a n e d i o l ether.538
diglycidyl
Following a c t i v a t i o n o f the matrix w i t h periodate,
lyso
G M l g a n g l i o s i d e w a s i m m o b i l i z e d o n t o t h e DEAE-dex t r a n / S p h e r o s i l
matrix
and
applied
successfully
to
the
large-scale
chromatographic p u r i f i c a t i o n o f cholera t o x i n from cultures. reacted
Periodate-oxidized with
affinity-
Vibrio cholerae
d e x t r a n ( d i a l d e h y d e d e x t r a n ) h a s been
1,2-diaminaethane
to
yield
a
polyamine
dextran
d e r i v a t i v e w h i c h was t h e n c o a t e d o n t o t h e i n n e r w a l l s o f g - a l k y l a t e d n y l o n tubes.539
Following glutaraldehyde
d e r i v a t i z e d n y l o n tubes, enzymes
from
Cytophaga
periodate-oxidized
a c t i v a t i o n o f the
u r e a s e was s u c c e s s f u l l y i m m o b i l i z e d . have
b e e n m o d i f i e d by
Lytic
reaction with
d e x t r a n t o y i e l d a s o l u b l e enzyme p r e p a r a t i o n . 5 4 0
D e x t r a n s u l p h a t e h a s been a c t i v a t e d w i t h c y a n o g e n b r o m i d e a n d then coupled w i t h lysyl-agarose t o provide a m a t r i x f o r a f f i n i t y chromatographic
purification of
a c i d B-glucosidase
from
human
p l a c e n t a . 321 A s e r i e s o f dyes c o v a l e n t l y l i n k e d t o d e x t r a n T70 have been used i n a
study
of
the
relationship
between
the
structure
of
685
8: Chemical Synthesis and Modification anthraquinone-triazine lactate
compounds a n d t h e i r a f f i n i t y t o r a b b i t m u s c l e
d e h y d r o g e n a ~ e . ~ The ~~
degree
of
substitution
p o l y s a c c h a r i d e was f o u n d t o a f f e c t t h e a f f i n i t y .
of
the
The i n f l u e n c e o f
the s t r u c t u r e o f the polysaccharide c o n s t i t u e n t on the a f f i n i t y Cibacron Blue-(a-Q-glucans)
--
Q-Glucans.
of
was a l s o d i s c u s s e d .
C i b a c r o n B l u e F3GA h a s b e e n c o u p l e d t o a - ( 1 + 3 ) - g -
g l u c a n t o p r o v i d e t h e s u b s t r a t e i n a new
assay
f o r a-193-Q-glucan-
a s e ~ . ~ L~i c* h e n a n i m m o b i l i z e d o n a g a r o s e h a s b e e n u s e d f o r t h e a f f i n i t y - chrom a t o g r a p h i c p u r if ica t io n o f 6-91 uca na ses f r o m 2 75 SP.
Glycosaeinoglycans. some o f t h e i r
and
s t u d i e d.
--
The vacuum-u. v.
complexes
with
c. d.
Bacillus
spectra o f chondroitins
poly(l-arginine)
have
been
P e r io da t e - ox id i z e d c h a i n s o f g l yco Sam i n o g l y c a n s h a v e
43
been c o u p l e d t o a d i p i c a c i d d i h y d r a z i d e - s u b s t i t u t e d
agarose f o r
use
i n affinity chr~matography.~~~ Dermatan sulphate-bound
6-aminohexyl-agarose
h a s been c o a t e d
w i t h d e r m a t a n s u l p h a t e and used f o r t h e a f f i n i t y - c h r o m a t o g r a p h i c removal o f sulphatase contaminants during p u r i f i c a t i o n o f heparinase from Flavobacterium &parinurn hexyl-agarose
extracts.544
H e p a r i n - b o u n d arnino-
c o a t e d w i t h h e p a r i n was u s e d i n t h e same s t u d y f o r t h e
a f f i n i t y - c h r o m a t o g r a p h i c p u r i f i ca t i o n o f h e p a r a t i nase. P o r c i n e a n d w h a l e h e p a r i n s have been d e a m i n a t e d a n d some o f t h e r e s u l t i n g s u l p h a t e d o l igo s a c c h a r ide s s e p a r a t e d an d ide n t if i e d .5 45 The i n t e r a c t i o n s o f s u l p h a t e d h e p a r i n a n d human l o w - d e n s i t y
lipo-
p r o t e i n have been i n v e s t i g a t e d w i t h p a r t i c u l a r r e s p e c t t o t h e e f f e c t o f t h e u r o n i c a c i d c o m p o s i t i o n and charge d e n s i t y o f t h e glycan.276 Gel-filtration
s t u d i e s of methylene blue-heparin i n t e r a c t i o n s i n
aqueous s o l u t i o n s have been r e p o r t e d . 5 4 6
Q-Mannans.
--
G u a r a n h a s b e e n c o u p l e d t o P r o c i o n B l u e HB a n d t h e
m o d i f i e d p o l y s a c c h a r i d e used t o lectins.547
develop a reagent
for detection o f
The m e t h o d i n v o l v e s u s e o f t h e p r e c i p i t i n r e a c t i o n :
f o l l o w i n g i n c u b a t i o n o f dyed guaran and l e c t i n ,
t h e amount o f dye
l e f t i n t h e supernatant i s determined. A l k a l i t r e a t m e n t o f i v o r y n u t p-mannan h a s y i e l d e d 3-deoxy-2-Chydroxymethylpentonic, de o x y pe n t it 01 - 2 acids
produced,
predominated.548
3,4-dideoxy-pentonic,
- ca r bo x y 1ic
and 1,4-anhydro-3-
a c i ds a s t h e m o s t a b u n d a n t p o l y h y d r ox y
indicating
that
1,4-glycosidic
linkages
R e s u l t s demonstrate t h a t a l a r g e p r o p o r t i o n o f t h e
686
Carbohydrate Chemistry
Q-mannan m o l e c u l e s was b r o u g h t c o m p l e t e l y i n t o s o l u t i o n and t h a t ,
in
addition
Q-
to
non-reducing
mannose
end
groups,
non-reducing
g a l a c t o s e end g r o u p s were p r e s e n t .
--
M i s c e l l a n e o u s P o l y s a c c h a r i d e s , O l i g o s a c c h a r i d e s , and G l y c o l i p i d s . k-Carrageenan
beads c o n t a i n i n g s u l p h a t e g r o u p s have been a p p l i e d t o
t h e c h r o m a t o g r a p h i c p u r i f i c a t i o n o f u r o k i n a s e a n d as a m a t r i x f o r binding tanin
via
Fe3+ ions.549
c a r r a g e e n a n beads were
used f o r
C r o s s l i n k e d and d e s u l p h a t e d k gel-filtration
chromatographic
p u r i f i c a t i o n o f u r e a s e , a s p a r a g i n a s e , and b o v i n e serum a l b u m i n . C o l o m i n i c a c i d , i m m o b i l i z e d by r e a c t i o n w i t h c y a n u r i c c h l o r i d e a c t i v a t e d a g a r o s e , has been used f o r t h e a f f i n i t y - c h r o m a t o g r a p h i c p u r i f i c a t i o n of neuraminidase from Corynebacterium ulcerans.154 After
epoxy a c t i v a t i o n o f
crosslinked
pectate,
iminodiacetate
has been c o u p l e d t o t h e g e l and t h e n e q u i l i b r a t e d w i t h Fe3’
ions.550
The a c t i v a t e d g e l was used as a s u p p o r t m a t r i x f o r i m m o b i l i z a t i o n o f cells,
a n d t h e i n f l u e n c e o f pH, i o n i c s t r e n g t h , a n d
c e l l s t r a i n on
i m m o b i l i z a t i o n was s t u d i e d . The
use
of
4-nitrophenyl-2-~-(a-I-fucopyranosyl)-B-Q-
galactopyranoside f o r the r a p i d detection o f s u b s t r a t e - s p e c i f i c a-if u c o s i d a s e s has been d e s c r i b e d . 551
The p r e p a r a t i o n o f s e v e r a l 6 , 6 ’ - d i s u b s t i t u t e d methyl B-laminaribioside methyl
6-
and
Laminaribiose,
has been r e p o r t e d . 5 5 2
6’-deoxy-B-laminaribiosides
d e r i v a t i v e s of The d e r i v a t i v e s
were
also
prepared.
c e l l o b i o s e , and g e n t i o b i o s e have been a c e t o n a t e d w i t h
2,2-dimethoxy-propane
under v a r i o u s conditions.553
Laminaribiose
y i e l d e d two i s o p r o p y l i d e n e a c e t a l s i n which t h e r e d u c i n g [l-glucose r e s i d u e had t h e f u r a n o i d form,
w h i l e two i n which the reducing
a-
glucose r e s i d u e formed t h e a c y c l i c d i m e t h y l a c e t a l were o b t a i n e d from cellobiose.
G e n t i o b i o s e gave b o t h t y p e s o f i s o p r o p y l i d e n e
compound. The s e l e c t i v e 4 - t o l u e n e s u l p h o n y l a t io n o f 1,6-anhydro-4’, benzylidene-B-maltose
has been r e p o r t e d . 5 5 4
6’-0-
The f i v e t o s y l a t e s
o b t a i n e d were s e p a r a t e d and i d e n t i f i e d . Using the nitromethane-mercuric hydroxy-2-naphth-2-anisidide
cyanide procedure,
( n a p h t h o l AS-61)
7-bromo-3-
has been condensed
w i t h 2,3,4,6-tetra-~-acety1-a-~-mannopyranosyl chloride.555
product
obtained
a f t e r
deprotection,
mannopyranosyloxy)-2-naphth-g-anisidide,
The
7-bromo-3-(a-P-
has been u s e d s u c c e s s f u l l y
f o r h i s t o c h e m i c a l d e t e c t i o n o f a-mannosidase. On t r e a t m e n t w i t h s o d i u m m e t h y l s u l p h i n y l m e t h a n i d e ,
2-0-(4-0-
687
8: Chemical Synthesis and Modification
me t h y 1- a -Q-g1 uco py r a no sy 1u r o n i c a c i d ) -D_-xy l o s e h a s been show n t o be -
r a p i d l y d e g r a d e d by % - e l i m i n a t i o n t o f o r m 2-!-(4-deoxy-B-L-threoThe k i n e t i c s o f a c i d
hex-4-enopyranosyluro n i c a c i d ) - Q - x y l o se.556 hydrolysis o f
t h e s e t w o compounds have been s t u d i e d .
polyacrylamide
gel
copolymers
have
been
c.94
e l e c t r o p h o r e s i s o f l e c t i n s a n d cy t o c h r o m e Periodate-oxidized t o
immunoglobulin
used
Glycosyl-
for
a f f i n i t y
g l y c o l i p i d s i n l i p o s o m e s have been a t t a c h e d using
G
~ y a n o b o r o h y d r i d e . ~ ~ ’ The
sodium
conjugates
oar
borohydride are
of
potential
sodium use
for
antibody-mediated t a r g e t i n g o f vesicles o f c e l l s . A g l y c o s p h i n g o l i p i d o b t a i n e d from asialo-GM1
by o z o n o l y s i s a n d
s u b s e q u e n t a l k a l i t r e a t m e n t has been i m m o b i l i z e d o n a m i n o - h e x y l agarose
by
reductive
a m i n a t i ~ n . ~ A n~t i~- ( a s i a l o - G M 1 )
antibody
was
s u c c e s s f u l l y p u r i f i e d o n t h e immunoadsorbent t hus o b t a i n e d . The a n t i g e n i c i t y a n d i m m u n o l o g i c a l p r o p e r t i e s o f a s e r i e s o f g l y co 1i p i d a n t i g e n s p r e p a r e d by coup 1i n g iso m a 1 t o se o 1igo sa ccha r i de s (isomaltose- isomaltoheptaose) t o a l i p i d carrier (stearylamine) have
been i n v e s t i g a t e d . 5 5 8
The a n t i g e n i c i t i e s o f t h e d i f f e r e n t
s t e a r y l i s o m a l t o s y l o l i g o s a c c h a r i d e s t h u s o b t a i n e d w e r e compared, e i t h e r when i n j e c t e d a l o n e o r when i n c o r p o r a t e d i n t o l i p o s o m e s . Glycosyl-polyacrylamide
gel
copolymers
have
been used f o r
a f f i n i t y e l e c t r o p h o r e s i s o f l e c t i n s and c y t o c h r o m e 2.94 Starch.
--
S t a r c h has been c o u p l e d t o a g a r o s e a n d t h e n r e a c t e d w i t h
b u t y l a m i n e t o i n t r o d u c e h y d r o p h o b i c g r o u p s o n t o t h e m a t r i x .559
The
immobilized starch
the
d e r i v a t i v e was
successfully
applied
to
a f f in i t y - c h r o m a t o g r a p h ic p u r if i c a t i o n o f c h l o r o p l a s t a - l , 4 - 9 1 u c a n phosphorylase from spinach leaves. S t a r c h dyed w i t h Remazol b r i l l i a n t o r a n g e h a s been u s e d a s substrate
for
measurement
o f a-amylase
and g l u c o a m y l a s e a c t i v i t i e s
p r o d u c e d d u r i n g f e r m e n t a t i o n . 560 The p h o t o c h e m i c a l g r a f t i n g o f v i n y l monomers o n t o s t a r c h h a s been r e p o r t e d . 561
3
M o d i f i c a t i o n o f G l y c o p r o t e i n s a n d Uses o f
Mo d i f i e d Gly cop r o t e i n s The
sensitivity
s t r u c t u r a l analyses glycoproteins
has
of of
been
a
fluorescence
l a b e l l i n g method
asparagine-linked increased
by
sugar
using
moieties
h.p.1.c.
with
for of a
688
Carbohydrate Chemistry
fluorescence
detector.562
polypeptide portions exposed were reductively
aminated
--
separation o f
a c e t y l a t e d and t h e
As l i t t l e a s 0.1
Albumins.
After
by h y d r a z i n o l y s i s , with
a
sugar m o i e t i e s from
free
amino
r e d u c i n g ends
fluorescent
of
reagent,
groups sugar
thus
chains
2-aminopyridine.
pmol o f p y r i d y l a m i n o d e r i v a t i v e s c o u l d be d e t e c t e d .
The i m m u n o c h e m i s t r y o f
c o n j u g a t e s p r e p a r e d f r o m serum
a l b u m i n s a n d a c r i d i n e n i t r o g e n m u s t a r d s h a s been i n v e s t i g a t e d . 5 6 3 Bovine
serum
albumin
microspheres
containing
adriamycin
have
b e e n p r e p a r e d by h e a t s o l i d i f i c a t i o n o f a l b u m i n i n a n e m u l s i o n o f adriamycin,
albumin,and
cottonseed
Results suggest t h a t
a l b u m i n m i c r o s p h e r e s c o n t a i n i n g a d r i a m y c i n may be a p p l i c a b l e a s d r u g c a r r i e r s i n t h e a d j u v a n t chemotherapy o f l i v e r m et as t as es . The e f f e c t o f m o d i f i c a t i o n o f a l b u m i n o n t o l b u t a m i d e - s e r u m a l b u m i n b i n d i n g has been s t u d i e d . 5 6 5
Antibodies.
--
Following periodate activation,
a n t i - ( h u m a n serum
a l b u m i n ) a n t i b o d y has been c o v a l e n t l y l i n k e d t o a m y l o g l u c o s i d a s e and t h e c o n j u g a t e q u a n t i t a t e d u s i n g an e n z y m e - c y c l i n g assay.566 C o n j u g a t i o n o f enzymes t o i m m u n o g l o b u l i n s u s i n g t h e m a l e i m i d e and p e r i o d a t e methods o f c o u p l i n g h a s been compared.567
Use o f
Q’-
( o x y d i m e t h y l e n e ) d i m a l e i m i d e r a t h e r t h a n p e r i o da t e t o e f f e c t c o u p l i n g was
f o u n d t o be s u p e r i o r
for
coupling o f
peroxidase,
p e n i c i 11i n a se, a n d B - D - g a 1a c t o s i d a s e .
ox i d a se,
peroxidase-conjugated
goat a n t i - i m m u n o g l o b u l i n
B-glucose
Horser a d i sh
G antibody
has
been
a p p l i e d t o t h e d e t e r m i n a t i o n o f immune c o m p l e x e s i n human serum.568 Anti-Thy
1.2
m o n o c l o n a l a n t i b o d y has been c o v a l e n t l y l i n k e d t o
r i c i n and shown t o groups
have
been
be a p o t e n t c e l l - t y p e - s p e c i f i c introduced
into
antibody
s u c c i n i m idy 1 - 3 ( 3-py r i dy 1d i t h i o )p r o p i ona t e . 5 6 9 pyridyldisulphide dithiothreitol
groups
to y i e l d free
on
the
protein
t h i o l groups
toxin.”’
molecules
Thiol using
were
reduced w i t h
through which
oxidation-
i n d u c e d d i s u l p h i d e c o u p l i n g w i t h r i c i n A was t h e n e f f e c t e d . c o n j u g a t e s were a p p l i e d s u c c e s s f u l l y
N-
The r e s u l t i n g 2 -
The
t o the selective k i l l i n g o f
n o r m a l and n e o p l a s t i c B - c e l l s .
A s i m p l e method f o r p r e p a r a t i o n o f a n t i b o d y t a r g e t e d l i p o s o m e s has
been d e ~ c r i b e d . ~ ” F ( a b ’ ) 2
antibody
fragments
were
covalently
c o u p l e d t o p h o s p h a t i d y l e t h a n o l a m i n e and su b sequent ly a s s o c i a t e d w i t h d i m y r i s t o y l p h o spha t i d y l c h o l i n e l i p 0 some s.
Side r e a c t i o n s d u r i n g t h e
c o u p l i n g w e r e m i n i m i z e d by p r i o r c i t r a c o n y l a t i o n o f t h e F ( a b ’ l 2 fragment s
.
8: Chemical Synthesis and Modification
689
-- M e t h o t r e x a t e a n d c h l o r a m b u c i l h a v e b o t h been covalently linked t o abrus agglutinin, abrin, r i c i n u s When i n j e c t e d i n t o s a r c o m a a g g l u t i n i n , r i c i n , or c o n c a n a v a l i n A.571 b e a r i n g mice each c o n j u g a t e s h o w e d a h i g h e r i n h i b i t o r y e f f e c t o n D N A b i o s y n t h e s i s of tumour cells t h a n d i d an e q u i v a l e n t dose of t h e free chemotherapeutic agent. S u c c i n y l a t e d or a c e t y l a t e d c o n c a n a v a l i n A , b u t n o t t h e n a t i v e l e c t i n , lyses sheep e r y t h r o c y t e s i n t h e p r e s e n c e o f g u i n e a - p i g The e f f e c t appeared t o be s p e c i f i c s i n c e complement.572 s u c c i n y l a t e d wheat-germ a g g l u t i n is i n a c t i v e and h a e m o l y s i s was i n h i b i t e d s e l e c t i v e l y by m e t h y l a - ! - g l u c o p y r a n o s i d e . Succinylated concanavalin A has been immobilized on agarose and used f o r c h e m i c a l c r o s s l i n k i n g o f a d i m e r i c p r o t e i n (!-glucose ~ x i d a s e ) . ~ ~ ~ T h e e f f e c t s o f c h e m i c a l m o d i f i c a t i o n on t h e c o n f o r m a t i o n a n d b i o l o g i c a l a c t i v i t y of peanut a g g l u t i n i n have been investigated.573 F r e e a m i n o g r o u p s o n t h e l e c t i n were m o d i f i e d w i t h s u c c i n i c a n h y d r i d e a n d w i t h 1- i s 0 t h i o c y a n a t o - 4 - b e n z e n e s u l p h o n i c a c i d . A l t h o u g h t h e e x t e n t s o f m o d i f i c a t i o n were r e s p e c t i v e l y 95% a n d 85%, the d e r i v a t i v e s showed no l o s s o f s u g a r - b i n d i n g c a p a c i t y , b u t t h e i r a g g l u t i n a t i n g a n d m i t o g e n i c a c t i v i t i e s were r e d u c e d . Derivatization o f p e a n u t a g g l u t i n i n w i t h t e t r a n i t r o m e t h a n e , 4 - a m i n o p h e n y l a-eg l u c o p y r a n o s i d e , a n d 2-~-(4-aminobenzyll-a-Q-neuraminic acid was investigated: a g g l u t i n a t i n g a n d m i t o g e n i c a c t i v i t i e s were n o t seriously diminished. The i n f l u e n c e o f t h e s e m o d i f i c a t i o n s on t h e c o n f o r m a t i o n o f t h e l e c t i n was i n v e s t i g a t e d by m e a n s o f c.d. s t u d i e s i n t h e f a r - a n d near-u.v. r e g i o n s . A n o n - a g g l u t i n a t i n g d e r i v a t i v e o f wheat-germ a g g l u t i n i n has b e e n p r e p a r e d by t r e a t m e n t o f t h e l e c t i n w i t h f o r m i c a c i d a n d cyanogen bromide.574 The d e r i v a t i v e b i n d s t o p l a t e l e t s and p r e c i p i t a t e s an antibody t o the l e c t i n .
Phytohaemagglutinins.
-- P r o d u c t i o n o f a u t o a n t i b o d i e s t o t r a n s f e r r i n h a s b e e n a c h i e v e d by i m m u n i z a t i o n o f r a b b i t s w i t h t r a n s f e r r i n m o d i f i e d a t e i t h e r i t s n e u r a m i n i c a c i d or i t s n e u r a m i n i c a c i d a n d Q - g a l a c t o s e residues with methionine sulphone h y d r a ~ i d e . A ~ u~ t ~o a n t i b o d i e s o b t a i n e d r e a c t e d w i t h t h e n a t i v e r a b b i t t r a n s f e r r i n i n radioimmunoa s s a y a n d g e l - d i f f u s i o n systems. Transferrin.
M i s c e l l a n e o u s G l y c o p r o t e i n s . -- T h e e f f e c t o f t h e c a r b o h y d r a t e m o i e t y o f k - c a s e i n on c o m p l e x f o r m a t i o n w i t h B - l a c t o g l o b u l i n has
690
Carbohydrate Chemistry
been s t u d i e d . 5 7 5 formation,
The c a r b o h y d r a t e p o r t i o n d o e s i n f l u e n c e c o m p l e x
and Ca2'
Conjugates of
has an i n h i b i t o r y e f f e c t on t h e i n t e r a c t i o n . f e t u i n and a s i a l o f e t u i n w i t h d i p h t h e r i a t o x i n
f r a g m e n t A h a v e been p r e p a r e d . 5 7 6 synthesis
on
cultured
The c o n j u g a t e s i n h i b i t e d p r o t e i n
r a t
hepatocytes
and
bound
t o
a s i a l o g l y c o p r o t e i n r e c e p t o r s on t h e h e p a t o c y t e s . Sulphydryl-agarose
has been used t o remove Hg2+ f r o m Hg2+-
f i b r o n e c t i n f o r r e g e n e r a t i o n o f i t s t h i o l groups.577 Asialo-(human
c h o r i o n i c g o n a d o t r o p h i n ) has been m o d i f i e d a t i t s w i t h Q - g a l a c t o s e o x i d a s e or
Q - g a l a c t o s e r e s i d u e s by t r e a t m e n t
p e r i o d a t e and b o r ~ h y d r i d e . ~The ~ ~ effects
of
with
such m o d i f i c a t i o n
t h e b i o l o g i c a l p r o p e r t i e s o f t h e hormone were s t u d i e d .
on
Covalent
c r o s s l i n k i n g o f human c h o r i o n i c g o n a d o t r o p h i n t o i t s r e c e p t o r i n r a t testes
has
been
achieved
using
disuccinimidyl
.
suberate
or
d i t h i o b i s ( s u c c i n i m i d y l p r o p i o n a t e ) 579 D e s i a l y l a t e d ovine s u b m a x i l l a r y mucin,
i m m o b i l i z e d on a g a r o s e ,
h a s b e e n u s e d as an a c c e p t o r f o r g - g a l a c t o s e
t r a n s f e r f r o m UDP-Q-
galactose.580
Immobilized D e r i v a t i v e s o f Glycoproteins.
--
glycoproteins
immobilized derivatives
t o yield biologically
active
c o n t i n u e s t o a t t r a c t much a t t e n t i o n .
The m o d i f i c a t i o n o f
These d e r i v a t i v e s and t h e i r
u s e s a r e s u m m a r i z e d i n T a b l e 6.581-598 The
use o f
monoclonal
antibodies
i n biochemical research,
i n c l u d i n g t h e i r use i n immunoadsorption chromatography, described.599
MonOClOnal a n t i b o d i e s t o a - f e t o p r o t e i n
i m m o b i l i z e d and a p p l i e d t o p u r i f i c a t i o n o f Anti-interferon
monoclonal antibody
has been have been
human a - f e t 0 p r 0 t e i n . l ~ ~
i m m o b i l i z e d on A f f i - g e l
10 has
been u s e d t o p u r i f y r e c o m b i n a n t human l e u c o c y t e i n t e r f e r o n . 4 0 1 Anti-(human
i m m u n o g l o b u l i n GI a n t i b o d i e s i m m o b i l i z e d on S p h e r o n
g e l have been used s u c c e s s f u l l y cells.587
for
s e p a r a t i o n o f human T and B
Pregnancy-specif i c B1-glycoprotein
successfully
by f l u o r o i m m u n o a s s a y
using
has been measured
antibody
coupled t o
m a g n e t i z a b l e p a r t i c l e s .588 A r e v i e w has a p p e a r e d d e s c r i b i n g t h e p u r i f i c a t i o n o f enzymes by chromatography
on h e p a r i n - a g a r o s e
h e p a r i n w i t h enzymes i s d e s c r i b e d ,
matrices.595 methods
for
The
interaction of
covalent attachment
o f h e p a r i n t o a g a r o s e a r e r e v i e w e d , and a d v a n t a g e s and d i s a d v a n t a g e s o f t h e p u r i f i c a t i o n method a r e d i s c u s s e d . An i m m u n o a d s o r b e n t h a s b e e n p r e p a r e d b y c o u p l i n g a l b u m i n t o g e l a t i n p r e - c o u p l e d t o agarose.410 The m e t h o d p e r m i t s c o n t r o l l e d ( T e x t c o n t i n u e s on page 7 1 4 )
Development o f a d i r e c t method f o r measurement o f a c t i v i t y o f
Study of use o f g r a f t copolymers a s s u p p o r t s f o r p r o t e i n immob i 1i z a t i o n Immunoadsorption o f r e c e p t o r specific ligands
A f f i n i t y chromatography of p r o t e o l y t i c fragments of f i b r o ne c t i n D e m o n s t r a t i o n o f new m e t h o d o f protein immobilization D e v e l o p m e n t o f a new m e t h o d f o r p r e p a r a t i on o f immunoadso r b e n t s
R e a c t i o n w i t h cyanogen bromideactivated agarose
A 1 bum i n
Reaction with agarose activated by o x i d a t i o n w i t h B r 2 -N-Succi n i m i d y l - 3 - ( 2-py r i d y 1dit h i o )propionate-mediated reaction with gelatin-agarose C a r b o di-im i d e -me d i a t e d r e a c t i o n w i t h nylon-co ( a c r y l i c a c i d ) po 1ymer s G 1u t a r a 1d e h y d e o r c a r bo d i - i m i d e m e d i a t e d c o - c r o s s 1 i n k i ng w i t h a-cobra t o x i n o r a c e t y l choline receptor Glutaraldehyde-mediated co-crossl i n k i n g w i t h B-glucose oxidase
Use o f p r o d u c t
Macromolecule o r m a t r i x coupled a n d mode o f c o u p l i n g
M o d i f i c a t i o n o f g l y c o p r o t e i n s by c o u p l i n g t o i n s o l u b l e m a t r i c e s and o t h e r macromolecules
Glycoprotein
Table 6
583
5 82
5 81
410
147
Ref.
s
E'
2
B
k
*
sr;'
9
?
1
p r o t e i n ) monoclonal antibody
a n t i bo dy Anti-(human a - f e t o -
a-fetoprotein
Anti-(human o r r a t
t r y p s i n ) a n t i bo dy
A n t i ( al - a n t i c h y m o -
-
albumin 1 antibody
A n t i - ( h u m a n serum
G l y co p r o t e i n
T a b l e 6 continued
3-( 2
3 ’-
a c t i va t e d a ga ro se
R e a c t i on w i t h cy anogen b r o m i d e
a c t i va t e d aga ro se
-
R e a c t i o n w i t h cy ano gen brom i d e -
a c r y l i c beads
c r y l a t e copolymer o r o x i r a n e -
epox y p r o p o x y ) p r o p y 1-s i 1i c a epox y-a c t i va t e d g l y c i d y l met ha-
poxy ) p r o p y l - g l a s s
R e a c t i o n w i t h 3-( 2 y y 3 y - e p o x y p r o -
a n d p e r o x i d a s e o n t o a membrane
a n d mode o f c o u p l i n g
Macromolecule o r m a t r i x coupled
protein
Pur if i c a t i on o f human a - f e t o -
a-fetopro t e i n
P u r i f i c a t i o n o f human o r r a t
a n d serum
t r y p s i n f r o m human p l e u r a l f l u i d
P u r i f i c a t i o n o f al-anti-chymo-
f l u i d and serum
t r y p s i n f r o m human p l e u r a l
Pur if i c a t i o n o f a l - a n t i - c h y m o -
supports
d i f f e r e n t epox i d e co n t a i n i n g
-
o f proteins on t h e i r coupling t o
Study o f t h e e f f e c t o f t h e n a t u r e
i m m o b i l i z e d enzymes
Use o f p r o d u c t
185
184
183
183
5 84
Ref.
4
s5
2
%
k
s
2-
2
\o N
Q\
w i t h a m i n o - d e r i v a t i z e d con-
t r o l l e d pore glass R e a c t i o n w i t h cyanogen b r o m i d e -
An t i - i n t e r f e r o n
G 1 u t a r a l deh yde -me d i a t e d r e a c t i o n
a c t i v a t e d SpheronR
P u r i f i c a t i o n o f human l y m p h o b l a s t o i d
immunoadso r p t i o n c h r o m a t o g r a p h y
by r e p e t i t i v e s e m i - a u t o m a t i c
P u r i f i c a t i o n o f p o l y c l o n a l IgM
S e p a r a t i o n o f human T a n d B c e l l s
immunoadso r p t i o n chroma t o g r a p h y
-
t r o l l e d pore glass
R e a c t i o n w i t h cy anogen b r o m i d e
r e p e t i t i ve sem i - a u t oma t i c
o f t h e F a c t o r V I I I complex P u r i f i c a t i o n o f p o l y c l o n a l I g A by
activated BiogelR A G 1 u t a r a l dehy de-me d i a t e d r e a c t i o n
w i t h am i n o - d e r i va t i z e d con-
Study o f t h e s t r u c t u r e and f u n c t i o n
R e a c t i o n w i t h cyanogen b r o m i d e -
M) antibody
A n t i - ( i m m uno g l o b u l i n
globulin G) antibody
A n t i - (human immuno -
A ) antibody
antibody A n t i - ( immuno g l o b u l i n
VIII)
human serum
Anti-(Factor
com p l exe s f r om in s u l in - a c t iv a t e d
a n t ibo dy
P u r i f i c a t i o n o f complement a t t a c k
n e n t o f complement)
C 5 compo-
gonadotrophin
Anti-(human
a s s a y f o r human c h o r i o n i c
gonadotrophin)
antibody
w i t h b l o o d - g r o u p Ii a c t i v i t y D e v e l o p m e n t o f a n enzyme immuno-
chorionic
P u r i f i c a t i o n o f sheep g a s t r i c m u c i n s
Use o f p r o d u c t
a n t i bo dy
a n d mode o f c o u p l i n g
Macromolecule o r m a t r i x coupled
Anti-(human
A n t i - ( b l o o d g r o u p I)
G 1y co p r o t e i n
Table 6 continued
128
5 86
5 87
5 86
5 85
188
187
186
Ref.
%.
a
5
g
G.
2
*s
g
-.
?
immuno-
cy ano ge n b r om i d e -
saccharide) antibody Anti-(SP1 1 a n t i b o d y ,
s t r a i n TA1 poly-
c e l l u l o s e - ir o n ox ide p a r t ic l e s
Reaction w i t h magnetizable
R e a c t i G n w i t h cyanogen b r o m i d e a c t i v a t e d agarose
Anti-renin antibody
A n t i - (R h i z o b i u m
R e a c t i o n w i t h cyanogen b r o m i d e activated cellulose
agarose
antibody
Anti-(poly (I)-poly (C 1) a n t i b o d y
c r o s s l i n k i n g o n t o p r o t e i n A-
measurement o f p r e g n a n c y - s p e c i f i c
S eq ue n t ia 1 f 1uo r o immunoa s say f o r
c h a r i d e s i n t o p y r u v a t e - r i c h and -poor f r a c t i o n s
Se p a r a t i on o f py r u v y 1a t e d po 1y sa c -
P u r i f i c a t i o n o f human r e n i n
Spe c i f i c sepa r a t i o n o f d o u b l e s t r a nde d r ibo n u c l e i c a c i ds
by i m m un o ads o r p t ion
NADP-iso c i t r a t e d e h y d r o gena se
P u r i f i c a t i o n o f Escherichia c o l i
D i m e t h y l s ube r i m i d a te-me d i a t e d
de hy d r o ge na se )
-
A n t i (NA DP i s 0 c i t r a t e
-
Purification o f a prekallikrein from r a t p a n c r e a s
cental lactogen
Radioimmunoassay f o r human p l a -
1euko cy t e i n t e r f e r o n
P u r i f i c a t i o n o f r e c o m b i n a n t human
interferon i n high y i e l d
Use o f p r o d u c t
R e a c t i o n w i t h cyanogen bromidea c t i va t e d a g a r o se
a c t i v a t e d agarose
R e a c t i o n w i t h cyanogen bromide-
R e a c t i o n w i t h A f f i - g e l R 10
a c t iva t e d a g a r o se
a n d mode o f c o u p l i n g
Macromolecule o r m a t r i x coupled
Anti-(urinary k a l l i krein) antibody
globulin G) antibody
Anti-(rabbit
monoclonal antibody
-
a n t i bo dy A n t i i nt e r f e r o n
G l y c o p r ot e i n
T a b l e 6 continued
588
192
191
475
203
190
189
40 1
Ref.
2
4
s.
9
P
9
5&
6
W P
Q\
o z o no 1y s e d
Asialo-GMl,
A s i a l o fe tu i n
A n t i - t h r o m b i n I11
gen) a n t i b o d y
ciated surface a n t i -
A n t i- ( t h r om b i n-a s so -
a n t i ge n 1 a n t i bo dy
A n t i - (t h e r mo l ab i l e
activated
G 1 y cop r o t e i n
T a b l e 6 continued
o r m a t r i x coupled
a c t i v a t e d agarose
R e a c t i o n w i t h cyanogen b r o m i d e -
a c t i v a t e d agarose
R e a c t i o n w i t h cyanogen b r o m i d e -
a n d mode o f c o u p l i n g
Macromolecule
l i p i d antibody
P u r i f i c a t i o n o f anti-glycosphingo-
s k e l e t a l muscle
l e c t i n a c t i v i t y from f e t a l - c a l f
P u r i f i c a t i o n o f lactose-blocking
symbiosis
t i n i n from t h e Azolla-Anabaena
P u r i f i c a t i o n o f a phy t o h a e m a g g l u -
i n a c t i v e forms o f whale h e p a r i n
h i g h l y a c t i v e and r e l a t i v e l y
Comparative study o f s t r u c t u r e s o f
(fluorescent ) heparin
Study o f p r o p e r t i e s o f m o d i f i e d
s u r f a c e a n t i g e n f r o m mouse c e l l s
I s o l a t i o n o f polyoma v i r u s - i n d u c e d
a n t i g e n f r o m baker’s y e a s t
P u r i f i c a t i o n o f thermolabile
B1-gl y c o p r o t e i n
Use o f p r o d u c t
348
2 19
218
589
127
194
193
Ref.
o\ \o VI
3
g.
$
0
g
fs
0
s
$
a,
ii’
93
PP
I lectin
Concanavalin A
complement
C l q component o f
Casein
lectin
(horseshoe crab)
r o t u n d a cauda
--
Carcinoscorpius
cifolia
Bandeiraea s i m p l i -
G 1 y co p r o t e i n
T a b l e 6 continued
a c t i v a t e d aga r o s e
R e a c t i o n w i t h cyanogen b r o m i d e -
p o l y s t y r e n e tubes
Adsorption on inner surfaces o f
a n d mode o f c o u p l i n g
Macromolecule o r m a t r i x coupled
fractiona-
C o - p u r i f i c a t i o n o f a l k a l i n e phospha-
131
124
-
a c e t am ido 2 -deox y - h exo s i da se C
da se P u r i f i c a t i o n o f b o v i n e b r a i n 2-
112
138
from m u r i n e i n t e s t i n e
568
230
229
2 20
Ref.
Purification of r a t uterine peroxi-
'
P u r i f i c a t i o n o f glycosylcerarnidase
i n human serum
D e t e r m i n a t i o n o f immune c o m p l e x e s
n u c l e a r p r o t e i n k i nase
Purification o f a heparin-sensitive
t i on o f s i a l o g l y c o p r o t e i n s
Affinity-chromatographic
pep t i d e s
group ABH-act i v e p o l y g l yco sy 1
A f f i n i t y chroma t o g r a p h y o f b l o o d -
Use o f p r o d u c t
&
.1'
5
f
2
0
z 9
9
G 1y c o p r o t e i n
Table 6 c o n t i n u e d
or m a t r i x coupled
a n d mode o f c o u p l i n g
Macromolecule
nucleoti-
r a t p r o s t a t i c adenocarcinoma P u r i f i c a t i o n o f cholinesterase from
from
P u r i f i c a t i o n o f a c i d phosphatase
238
237
236 from r a b b i t s k e l e t a l m u s c l e
235 P u r i f i c a t i o n o f 53K g l y c o p r o t e i n
234
233
23 2
207
142
Ref.
P u r i f i c a t i o n o f r a t al-fetoprotein
Trypanosoma b r u c e i a n t i g e n s
A f f i n i t y chromatography o f
i n human a m n i o t i c f l u i d
tissue i n h i b i t o r o f collagenase
P u r i f i c a t i o n o f an i r r e v e r s i b l e
po r c i n e my ome t r i urn
P u r i f i c a t i o n o f cathepsin D from
s o y b e a n ( G l y c i n e E)
I s o l a t i o n o f a r o o t l e c t i n from
from murine l i v e r
P u r i f i c a t i o n o f an e s t e r h y d r o l a s e
dase f r o m D i c t y o s t e l i u m d i s c o i d e u m
t a s e and 5'-AMP-specific
Use o f p r o d u c t
4 v3
o\
5'
9
Q
5
a
3 2 zr'
f?,
*
K'
3
n
g
PP
G l y co p r o t e in
Table 6 c o n t i n u e d
Macromolecule o r matrix coupled a n d mode o f c o u p l i n g
chicken egg yolk A f f i n i t y - c h r o m a t o g r a p h i c f r a c t i onat i o n o f gly copepti d es is o l at e d from a r a t l i v e r b i a n t e n n a r y g l y c a n P u r i f i c a t i o n of a phospholipidd e p e n d e n t a-Q-mannosidase from rabbit liver Purification o f a concanavalin A r e c e p t o r from b o a r s p e r m a t o z o a P u r i f i c a t i o n o f human l i v e r a c i d B - -Q - g a l a c t o s i d a se P u r i f i c a t i o n o f two a n t i g e n i c g l y c o p r o t e i n s from rye-grass (Lolium p e r e n n e ) p o l l e n P u r i f i c a t i o n o f al - a n t i t r y p s i n Af f in i t y - c h r oma t o g r a p h i c p u r i f i c a t i o n o f human l i v e r B-p-2a c e t a m i d o - 2 - d e o x y- h e x o s i d a s e P u r i f i c a t i o n o f g l y c o c o n j u ga t e s
Use o f p r o d u c t
246
5 245
%
4
0
z 1.
s
k
S'&
0 244
243
242
2 41
2 40
239
Ref.
03 m
m
Gl y copro t e in
Table 6 continued
a n d mode o f c o u p l i n g
Macromolecule o r m a t r i x coupled
from f i g
a c t i v a t o r s e c r e t e d by human
P u r i f ic a t i o n o f p l a s m i no ge n
25 3
252 Trypanosoma c o n g o l e n s e
25 1 P u r i f i c a t i o n of variant antigens o f
250
Purification o f sciatin
human l i v e r
d e o x y - h e x o s i d a s e s A and €3 f r o m
P u r i f i c a t i o n o f 0-p-2-acetamido-2-
cleaving type I V collagen
P u r i f i c a t i o n o f a n e u t r a l protease
latex
mido-2-deoxy-glucanases
I s o l a t i o n o f two e S - B - p - 2 - a c e t a -
249
3
g l yco p r o t e i n s
a\
iD iD
0.
o
9
S
a
Go
2:
2
*s
d e r i v e d f r o m mouse lymphoma c e l l 248
247
Ref.
man no se- l a b e 1l e d g l yco p e p t ide s
t i o n o f a m i x t u r e o f 2-PHI-!-
from Spongia o f f i c i n a l i s Affinity-chromatographic fractiona-
Use o f p r o d u c t
$ 3 fi’
0
90
Glycoprote in
T a b l e 6 continued
o r m a t r i x coupled
a n d mode o f c o u p l i n g
Macromolecule
brain
q u a n t i t a t i v e changes i n g l y c o peptide binding t o the l e c t i n
S t u d y o f t r a n s f o r m a t io n- de p e nde n t
i n c u b a t i o n media
s u b c e l l u l a r f r a c t i o n s and
C h a r a c t e r i z a t i o n o f Xenopus o o c y t e
n e n t f r o m mammalian s a r c o l e m m a
c h a n n e l s a x i t o x i n - b i n d i n g compo-
c h a r a c t e r i s t i c s o f t h e sodium
Dem ons t r a t i o n 0 f g l yco p r o t e i n
s y n a p t i c v e s i c les
p r o t e i n s from a d u l t - r a t
A f f i n i t y chromatography o f glyco-
d e o x y - g l u c o s y l phospho d i e s t e r a s e
P u r i f i c a t i o n o f r a t l i v e r a-p-2-
r e n a l basement membrane l a m i n i n
f r a g m e n t s o f human p l a c e n t a l a n d
A f f i n i t y chromatography o f p e p s i n
melanoma c e l l s i n c u l t u r e
Use o f p r o d u c t
353
352
35 1
25 6
255
25 4
Ref.
4
&
4
: 5
2
g
e
w s
6
0
Gly c o p r o t e i n
T a b l e 6 continued
Macromolecule o r matrix coupled a n d mode o f c o u p l i n g
Re ver s i b l e b i n d i n g o f a n t i t h r o m b i n I11 f o r u s e i n t h e f r a c t i o n a t i o n of heparin Study o f binding o f i n s u l i n receptors t o lectins Removal o f h a e m a g g l u t i n a t i n g a c t i vity during protease purification Study o f t h e g l y c o s y l a t i o n o f t h e arginine vasopressin/neurophysin I1 common p r e c u r s o r Study o f microheterogeneity o f o va l b um i n Study of f i b r i n o g e n c h a i n s during synthesis A f f i n i t y - c h r o m a t o g r a p h i c demons t r a t i o n o f two a l - a c i d g l y c o protein populations in purified protein preparations A f f i n i t y - c h r oma t o g r a p h i c s e p a r a t i o n
Use o f p r o d u c t
361
3 60
359
358
357
356
g. 355
0
~
0
5 $
& 354
B
G.
s
k
b
Ref. %
Z'
3
h
y
?
G l y copr ot e in
Table 6 c o n t i n u e d
Macromolecule or m a t r i x coupled a n d mode o f c o u p l i n g
h e t e r o g a l a c t a n from f r u i t b o d i e s of Fomitopsis p i n i c o l a S u p p o r t f o r i m m o b i l i z a t i o n o f 13-p2-acetamido-2-deoxy-hexosidase and a r y l s u l p h a t a s e A
o f right-side-out oriented s u b f r a c t i o n s from p u r i f i e d c a l f thymocy t e p l a s m a membranes D e m o n s t r a t i o n o f two t y p e s o f colony-stimulating factor i n serum o f l i p o p o l y s a c c h a r i d e treated mice Removal o f l u t r o p h i n a n d c h o r i o g o n a d o t r o p h i n 13-subunit d u r i n g preparation of glycoprotein hormone-free serum Demonstration o f g l y c o p r o t e i n nature of chlorophyllase Demonstration o f h e t e r o g e n e i t y o f
Use o f p r o d u c t
590
365
364
363
362
Ref.
?f 5 4
c1
%
6 3 w
cr
4 0 N
Dolichos b i f l o r u s lectin
Discoidin I
Glycoprotein
Table 6 c o n t i n u e d
R e a c t i o n w i t h cyanogen bromidea c t i v a t e d aga r o s e
Glutaraldehyde-mediated r e a c t i o n w i t h E i o g e l R P300
R e a c t i o n w i t h c y a n o gen b r om i deagarose
R e a c t i o n w i t h A f f i g e l R 15
Macromolecule o r m a t r i x coupled a n d mode o f c o u p l i n g
591
A f f i n i t y chromatographic study o f o l i g o s a c c h a r i d e s from band 3 g l y c o p r o t e i n from human e r y t h r o c y t e mem b r a n e s Purification of proteinase inhibitors Model p r o t e i n f o r s t u d y o f e f f e c t o f p r o t e i n c h a r g e on i m m o b i l i z a t i o n I n v e s t i g a t i o n o f c r o s s l i n k i n g o f Qglucose oxidase onto t h e modified l e c t i n matrix P u r i f i c a t i o n of d i s c o i d i n I-binding p r o t e o g l y c a n from a x e n i c D i c t y o s t e l i u m discoideum I d e n t i f i c a t i o n o f endogenous binding p r o t e i n s f o r t h e l e c t i n i n Dictyos t e l ium d i s c o i d e u m Purification of receptors for the l e c t i n from e m b r y o n a l c a r c i n o m a cells
260
593
259
366
400
592
Ref.
Use o f p r o d u c t
4
w 0
2'
L"1 P c2
E
R
2
9
PP
p u r ifica-
Heparin
Purification o f a heparin-sensitive
simplex v i r u s type 1 g l y c o p r o t e i n
Study o f l e c t i n p r o p e r t i e s o f h e r p e s
subunit structure of enterokinase
230
406
268
dase f r o m E s c h e r i c h i a f r e u n d i i A f f i n i ty-chromatographic study o f
263
desialylated
H e l i x pomatia l e c t i n
262
glycosamino glycans P u r i f i c a t i o n o f endo-B-Q-galactosi-
261
153
3 75
Hog g a s t r i c m u c i n ,
(Carcinoscorpius rotunda caudal
b i n d i n g l e c t i n from horseshoe c r a b
P u r i f i c a t i o n o f a neuraminic acid-
t i o n o f F a c t o r VIII
A f f i n i ty-chromatographic
formation i n v i t r o
5 94
Ref.
P u r i f i c a t i o n of
phytohaemagglutinin
agglutinin or
S t i m u l a t i o n o f e r y t h r o i d colony
lampranges
P u r i fi c a t i o n o f c h it i n - b i n d i n g haemagglutinin from Conidiobolus
Use o f p r o d u c t
F ib r o n e c t i n
Fetuin
Factor X
wheat-germ
Ads o r p t i o n o n a ga r o s e boun d
Eryt h r o p o i e t i n
-
Formalinized
a n d mode o f c o u p l i n g
Macromolecule o r m a t r i x coupled
Erythrocytes
G l y c o p r ot e i n
Table 6 continued
$
4
G.
g3
P $
w
2
P 0
-4
Glycoprotein
T a b l e 6 continued
a c t i v a t e d agarose
R e a c t i o n w i t h cyanogen b r o m i d e -
w it h am i n o h ex y l - a ga r o s e
Car bo d i - i m i d e -rnedi a t e d r e a c t i o n
a n d mode o f c o u p l i n g
Macromolecule o r m a t r i x coupled
p u r ifica-
P u r i f i c a t i o n o f pro-
and heparin.
Modification o f heparin-agarose pro-
t e i n k i n a s e s i n h i b i t e d by h e p a r i n
nuclear p r o t e i n kinase,
palyamines,
Asses sme n t o f i n t e r a c t io n s b e t w e e n
ronidase
t i o n o f bovine t e s t i c u l a r hyalu-
Affinity-chromatographic
thrombin 111
and c h e m i c a l l y m o d i f i e d a n t i -
A f f i n i t y chromatography o f n a t i v e
glyceride lipase
373
3 72
320
271
2 70
Purification o f r a t hepatic tri-
1 topoisomerase
269
255
Ref.
P u r i f i c a t i o n o f Xenopus l a e v i a t y p e
d i e s t e r a se
mido-2-deox y-g-gluco s y l phospho-
P u r i f i c a t i o n o f r a t l i v e r 2-aceta-
n u c l e a r p r o t e i n k i na s e
Use o f p r o d u c t
v1 0
4
=. 2
0
9
5
a
n
C'
8
s
-2
3
CI.
B3
PP
a n d DNA a n d RNA p o l y m e r a s e s
t h r o m b i n I11 o n h e p a r i n i z e d b i o -
Immunoglobulin E
R e a c t i o n w i t h cyanogen b r o m i d e -
w it h am i n o hex y 1-a g a r o se
complex f r o m r a t b i l e Purification of cell-surface
f o r polymer immunoglobulin P u r i fi ca t i o n o f f r e e s e c r e t o r y
de r iva t iz e d a g a r o se G 1u t a raldehyde-me d i a t e d r e a c t i o n
dimeric Immunoglobulin A,
polymeric
P u r i f i c a t i o n o f membrane r e c e p t o r
Reaction w i t h adipic dihydrazide-
material
I n a c t i va t i o n o f t h r o m b i n by a n t i-
w i t h p o l y v i n y l a l c o h o l beads
l y s oz yme
One-step p u r i f i c a t i o n o f eggwhite
RNAse,
P u r i f i c a t i o n o f lysosomal hydrolases,
197
196
195
5 97
5 96
595
44 9
s t i m u l a t e d l i p a s e f r o m human whey
136
P u r i f i c a t i o n o f tyrosine hydroxylase
Ref.
P u r i f i c a t i o n o f the b i l e s a l t -
a f t e r h e p a r i n i n j e c ti o n
demic and h y p e r l i p i d e m i c s u b j e c t s
l i p o l y t i c a c t i v i t y o f normal l i p i -
cedure f o r d e t e r m i n a t i o n o f plasma
Use o f p r o d u c t
G l u t a r a l d e h y d e -me d i a t e d r e a c t i o n
Reaction w i t h UltrogelR
a n d mode o f c o u p l i n g
Macromolecule o r m a t r i x coupled
Immunoglobulin A,
G 1y cop r o t e in
Table 6 c o n t i n u e d
4
2. 4
D
,$
Q
k
9
$-
4 0 Q\
0 v a l b um in
Lotus tetragonolobus l e c t i n
aris) lectin
L e n t i l (Lens c u l i n -
a-Lactalbumin
a n t i body f ragme n t s
Immunoglobulin F l a b ’ )
Glycoprotein
Table 6 continued
a c t iv a t e d aga r o se
a n d mode o f c o u p l i n g
Macromolecule o r m a t r i x coupled
study o f Study o f i n t e r a c t i o n s w i t h a n i o n i c
subunit structure o f enterokinase
Affinity-chromatographic
c y t e membranes
g l y c o p r o t e i n f r o m human e r y t h r o -
171
268
5 91 o l i g o s a c c h a r i d e s f r o m band 3
study o f
A f f i n i ty-chromatographic
tors t o lectins
355
274
247
198
Ref.
Study o f b i n d i n g o f i n s u l i n r e c e p -
different structures
binding o f glycopeptides o f
A f f i n i t y - ch r oma t o g r a p h i c s t u d y o f
specificity of l e n t i l lectin
S t u d y o f t h e c a r bo h y d r a t e - b i n d i n g
Q-galacto s y lt ransferases
P u r i f i c a t i o n o f human serum
human serum
S i n g l e - s t e p i s o l a t i o n o f IgG from
receptor f o r IgE
Use o f p r o d u c t
4
s
$
s
E’
2
s
$
R s.&
cl
PP
lectin
Pea (Pisum s a t i v u m )
0 vo m uco id
G l y cop r o t e i n
T a b l e 6 continued
a c t iva t e d a ga r o se
w it h e pox y-ac t iv a t e d a ga r o se R e a c t i o n w i t h cyanogen b r o m i d e -
a c t i v a t e d agarose o r r e a c t i o n
R e a c t i o n w i t h cyanogen b r o m i d e -
and mode o f c o u p l i n g
Macromolecule o r m a t r i x coupled
-a
Study o f b i n d i n g o f i n s u l i n recep-
355
4
5
3
cs
z
a a'
Y 3-
s
274
6 &
d i f ferent structures
study o f
247
386
282
210
Ref.
b i n d i n g of glycopeptides o f
A f f i n i ty-chromatographic
f i c i t y o f pea l e c t i n
lymphoma c e l l g l y c o p r o t e i n s . S tudy of the carbohydrate-binding speci-
t u r e o f 2 { 3H } -m a nno se -1a be1l e d g l y c o p e p t i d e s d e r i v e d f r o m mouse
-
enzyme f r o m S t r e p t o m y c e s
erythreus A f f i n i t y chromatography o f a mix-
sin-like
t r a n s f e r a s e from t r a c h e a mucosa A f f i n i t y chromatography o f a t r y p -
a c e t a m i do-2-deox y-Q-gluco syl)
I s o l a t i o n o f a-p-mannose: 1,2-( 2-
l y t i c fragments o f f i b r o n e c t i n
A f f i n i t y chromatography o f p r o t e o -
detergents
Use o f p r o d u c t
00 0
4
R i c i n u s communis agglutinin
R i c i n I a n d r i c i n I1
Phy t o h a e m a g g l u t i n i n E
P e a n ut a g g l ut i n i n
tors t o l e c t i n s I s o l a t i o n of l e c t i n r e c e p t o r s from human e r y t h r o c y t e s w i t h o u t u s e o f deter g e n t s Support f o r immobilization o f ery t h r o p o i e t i n f o r a t t a c h m e n t o f e r y t h r o i d c o l o n i e s in v i t r o S t u d y o f b i n d i n g o f i n s u l i n receptors to lectins A f f i n i t y chromatography o f angiot e n s i n-co n v e r t i n g e n z y m e f r o m porcine kidney, serum, and c u l t u r e d porcine a o r t a endothelial cells P u r i f i c a t i o n o f a glycoprotein from sera of c a n c e r p a t i e n t s A f f i n i ty-ch roma t o g r a p h i c s e p a r a t i o n of c e l l - s u r f a c e g l y c o p r o t e i n s and g ly c o lip i ds Demonstration o f g l ycoprot e i n
Use o f p r o d u c t
35 1
287
286
172
355
375
374
Ref.
\c
0
4
2
,h.
0
Q.
5 $
0
*
3 r;. &
Macromolecule o r matrix coupled a n d mode o f c o u p l i n g
Glycoprot e i n
$
r?,
Table 6 continued
Thrombin
lated
(ovine),
desialy-
S ubm ax i11a r y m u c i n
Soybean a g g l u t i n i n
a g g lu t i n in
Ricinus sanguinis
G 1y co p r o t e i n
Table 6 continued
a n d mode o f c o u p l i n g
Macromolecule o r m a t r i x coupled
580
electrophoresis
l e t p r o t e i n s by c r o s s e d immuno-
s t r a t i o n o f t h r o m b i n - b i n d i n g p l a t e-
I n t e r m e d i a t e g e l i n m e t h o d f o r demon- 3 7 6
ghosts
c a t a l y s e d by a - g a l a c t o s y l t r a n s f e r a s e from human e r y t h r o c y t e
406
simplex v i r u s t y p e 1 g l y c o p r o t e i n Acceptor f o r e-galactose t r a n s f e r
374
25 6
Ref.
Study o f l e c t i n p r o p e r t i e s o f herpes
detergents
human e r y t h r o c y t e s w i t h o u t u s e o f
I s o l a t i o n o f l e c t i n receptors from
synaptic vesicles
A f f i n i t y chromatography o f g l y c o p r o t e i n s from a d u l t - r a t b r a i n
from mammalian sarcolemma
c h a n n e l sax i t o x i n - b i n d i n g component
c h a r a c t e r i s t i c s o f t h e sodium
Use of p r o d u c t
4
&
4
$!z
!$ n
c9
2
0
c.
Vicia cracca l e c t i n
--
( soy b e a n )
Trypsin i n h i b i t o r
( r i ce b r a n )
T r.y p si n in h i b i t o r
(potato )
Trypsin i n h i b i t o r
Transfer r i n
hormone ( T S H )
Thy r o i d- s t i m u l a t i n g
Glycoprotein
Table 6 continued
a c t iva t e d a ga r o s e
R e a c t i o n w i t h cyanogen b r o m i d e -
w it h amino s i 1a n i z e d L i C h r o s p h e r R
Glutaraldehyde-mediated reaction
a c t i v a t e d agarose
R e a c t i o n w i t h cyanogen b r o m i d e -
activated cellulose
R e a c t i o n w it h benzoq u i none-
Macromolecule o r m a t r i x coupled a n d mode o f c o u p l i n g
I liquid affinity
group A-active
g l y c o p r o t e i n s from
A f f i n i t y chromatography o f blood-
e r y t h r o c y t e membranes
p o 1y g l y co s y l p e p t ide s f r om human
t i o n o f bloodgroup ABH-active
A f f i n i t y - c h r oma t o g r a p h i c f r a c t i o na-
ch r om a t o g r a p hy
high-performance
Study of r e s o l u t i o n a c h i e v a b l e i n
p a n c r e a t i c d e o x y r ib o n u c l e a s e
A f f i n i t y chromatography o f p o r c i n e
qriseus trypsin
P u r i f i c a t i o n o f Streptomyces
t r y p s in
A f f i n i t y c h r o m a t o g r a p h y o f chymo-
transferrin
P u r i f i c a t i o n o f autoantibodies t o
f r o m t h y r o i d membrane
P u r i f i c a t i o n o f human TSH r e c e p t o r s
Use o f p r o d u c t
2 94
2 20
5 98
25 8
292
472
290
288
Ref.
2
2.
0
2
s
2 a
2:
B
B
*
3s.
9
PP
factor
tinin
Wheat -ge r m a g g l u -
von W i l l e b r a n d
G 1y co p r o t e i n
T a b l e 6 continued
and mode o f c o u p l i n g
Macromolecule o r m a t r i x coupled
351
Demonstration o f g l y c o p r o t e i n char-
Removal o f l u t r o p h i n ,
m amm a 1 i a n sa r c o l emm a
choriogonado-
363
298
I s o l a t i o n o f human p l a t e l e t membrane f r a c t i o n s a c t e r i s t i c s o f t h e sodium c h a n n e l s a x i t o x i n - b i n d i n g component f r o m
297
296
286
2 46
295
Ref.
I s o l a t i o n o f plasma membrane v e s i c l e s o f human p l a t e l e t s
fibroblasts
mannosidase f r o m c u l t u r e d s k i n
P u r i f i c a t i o n o f i n t r a c e l l u l a r a-p-
sera of cancer p a t i e n t s
P u r i f i c a t i o n o f a g l y c o p r o t e i n from
c o n j u g a t e s f r o m Spongia o f f i c i n a l i s
A f f i n i t y chromatography o f g l y c o -
t o r s f o r von W i l l e b r a n d f a c t o r
I s o l a t i o n o f human p l a t e l e t r e c e p -
human e r y t h r o c y t e membranes
Use o f p r o d u c t
&
4
$.
9
s
9
k
6
h,
I--
4
G 1y co pr o t e i n
Table 6 continued
a n d mode o f c o u p l i n g
Macromolecule o r m a t r i x coupled
hormone-free
serum
t r o p h i n , and t h y r o t r o p h i n d u r i n g p r e p a r a t i o n o f g l yco p r o t e i n
Use o f p r o d u c t
Ref.
$ 2
a %
Q
E'
2
s
$
E
K-
3
$
n
?
7 14
Carbohydrate Chemistry
c o u p l i n g o f p r o t e i n s t o an i n s o l u b l e m a t r i x , and t h e bond t h r o u g h w h i c h r e a c t i o n o c c u r s i s known p r e c i s e l y . Anti-albumin antibodies bound
to
the
agarose-gelatin-albumin
t i g h t l y than t o agarose-albumin,
conjugate
are
bound l e s s
permitting recovery o f p u r i f i e d
a n t i b o d y f r o m t h e column immunoadsorbent i n i m p r o v e d y i e l d .
--
I m m o b i l i z e d Cells. use b o t h
i n affinity
The i m m o b i l i z a t i o n o f c e l l u l a r m a t e r i a l f o r chromatography
and f o r
enzymatically a c t i v e preparations continues
the production of
t o receive considerable
attention.
-_---__ Acetobacter
---aceti
c e l l s adsorbed on c y l i n d r i c a l m o n o l i t h s o f
c o r d i e r i t e have been
used
production of
a c e t i c acid.600
i n
or
alginate
d e t r i t i a t i o n of
i n
fixed-cell gel
have
been
The k i n e t i c s o f
have
been
compared.602
entrapped i n agar
rapid
applied
t o
the
p-
isomerization of
g l u c o s e c a t a l y s e d by i m m o b i l i z e d and n o n - i m m o b i l i z e d cells
for
Alcaligenes eutrophus c e l l s entrapped
k-carrageenan water.601
reactors
Azotobacter
g e l have been used i n a
Arthrobacter
chroococcum batch
cells
system
for
con t in u o u s n i t r o ge n f ix a t io n .603 A
hydrogen p e r o x i d e sensor s y s t e m has been d e v e l o p e d u s i n g
Beneckea h a r v e y i c e l l s i m m o b i l i z e d on p h o t o - c r o s s l i n k a b l e w i t h i n a l g i n a t e gel.604
r e s i n s or
Brevibacterium S m o n i a q e n e s c e l l s entrapped
i n p o l y a c r y l a m i d e g e l have f o r m e d p a r t o f a r e a c t o r c e l l s y s t e m f o r p r o d u c t i o n o f g l u t a t h i o n e a n d NADP.605
L - G l U t a m i c a c i d has been
produced i n a r e c y c l e r e a c t o r employing B r e v i b a c t e r i u m f l a v u m c e l l s i m m o b i l i z e d on c o l l a g e n . 6 0 6 devised
allowed
use
of
The any
m o d i f i e d i m m o b i l i z a t i o n procedure given
pH
and
ionic
strength.
B r e v i b a c t e r i u m f u s c u m c e l l s e n t r a p p e d i n k - c a r r a g e e n a n g e l h a v e been u s e d f o r c o n t i n u o u s p r o d u c t i o n o f 1 2 - k e t o c h e n o d e o x y c h o l i c acid.607 C a l d a r i e l l a ----a c i d o e---hila -----------
glutaraldehyde-mediated Magnetite
could
also
cells
be
immobilized
demonstrate
the
immobilized
of
a
by
afford
a
C a n d i d a t r o p i c a l i s c e l l s have
i n epoxy-activated u t i l i t y
been
i n chicken eggwhite.608
entrapped i n the r e s i n t o
biocatalyst w i t h magnetic properties. been
have
co-crosslinking
new
crosslinked pectate to support
for
whole-cell
i m m o b i l i z a t i o n .550 The e f f e c t o f i m m o b i l i z a t i o n on v a r i o u s g l a s s - f o r m i n g monomers ( h y d r o p h i l i c and h y d r o p h o b i c ) on t h e s t a b i l i t y o f p h o t o s y s t e m I1 activity of
s p i n a c h c h l o r o p l a s t s h a s been i n v e s t i g a t e d . 6 0 9 , 6 1 0
thermostability immobilization.
of
the
chloroplasts
was
greatly
increased
The by
Spinach c h l o r o p l a s t s and C l o s t r i d i u m b u t y r i c u m
8: Chemical Synthesis and Modification
715
c e l l s h a v e been e n t r a p p e d i n a g a r g e l and u s e d t o g e t h e r i n a p h o t o chemical-energy
c o n v e r s i o n system.611
Immobilized g o s t r i d i u m
b u t y r i c u m s p o r e s have been used i n t h e development o f a method f o r s t a r t i n g a fermentor aseptically.612 p r o d u c t i o n by
Biophotolyt i c
hydrogen
i m m o b i l i z e d C l o s t r i d i u m p a s t e u r i a n u m c e l l s h a s been
investigated.613
The c e l l s w e r e i m m o b i l i z e d by g l u t a r a l d e h y d e -
mediated r e a c t i o n w i t h aminopropyl
glass or
amino-SpherosilR.
C o r i o l u s v e r s i c o l o r f u n g a l c e l l s e n t r a p p e d i n a l g i n a t e g e l have been
-Daucus c a r o t a
used f o r d e c o l o r i z a t i o n o f a k r a f t m i l l effluent.614
c e l l s e n t r a p p e d i n a l g i n a t e g e l have been used f o r c o n v e r s i o n o f g i t o x i g e n i n t o 5f3 - h y d r o x y g i t o x i g e n i n . 615 Neur a m i n i d a s e - t r e a t e d human e r y t h r o c y t e s have been bound t o l e c t i n - c o a t e d agarose.374
C e l l s c o u l d be s h e a r e d o f f
t h e beads by
mechanical disruption, l e a v i n g l e c t i n receptors s t i l l attached t o t h e beads.
Ery t h r o i d c o l o n i e s have been a t t a c h e d t o a g a r o s e - l e c t i n -
e r y t h r o p o i e t i n complexes and t h e mechanism o f c o l o n y s t u d i ed.
formation
75
Escherichia alcalescens c e l l s entrapped i n polyacrylamide gel have been used t o c o r r e l a t e t h e f a t e o f {14C)-~-leucine-labelled b a c t e r i a w i t h changes i n t h e enzymic a c t i v i t y
E. -coli
cells.616
o f the entrapped
c e l l s h a v e b e e n i m m o b i l i z e d by e n t r a p m e n t i n
p o l y u r e t h a n e foam ,617
by
reaction w i t h epoxy-activated crosslinked
p e ~ t a t e , ~ by ~ ' e n t r a p m e n t i n p o l y a c r y l a m i d e gel,605
a n d b y a new
i m m o b i l i z a t i o n method i n v o l v i n g i o n o t r o p i c g e l a t i o n o f c h i t o s a n w i t h a m u l t i v a l e n t a n i o n i c c ~ u n t e r - i o n . ~K~l u~y v e r o m y c e s f r a q i l i s c e l l s e n t r a p p e d i n a l g i n a t e g e l have been used f o r b a t c h p r o d u c t i o n o f e t h a n o l from lactose,618
o r f r o m J e r u s a l e m a r t i c h o k e tubers.619
L e u c o n o s t o c m e s e n t e r o i d e s c e l l s i m m o b i l i z e d by e n t r a p m e n t i n a g a r o n f i l t e r have been u s e d f o r r a p i d d e t e r m i n a t i o n o f
an a c e t y l c e l l u l o s e
I=-ph eny l a l a n i n e u s i n g a l a c t a t e e l e c t rode. 9 9
fie t h a n o s a r c i n a b a r k e r i
c e l l s e n t r a p p e d i n a l g i n a t e g e l have been a p p l i e d t o t h e c o n v e r s i o n o f m e t h a n o l t o methane.620 N o c a r d i a e r y t h r o p o l i s , l. opaca, and M y c o b a c t e r i u m p h l e i c e l l s h a v e each been i m m o b i l i z e d by e n t r a p m e n t i n
polyacrylamide
silica.621
gel
o r by a d s o r p t i o n o n D E A E - c e l l u l o s e a n d
The i m m o b i l i z e d - c e l l
p r e p a r a t i o n s have 4 - e n - 3 - o x o s t e r a c t i v i t i e s a n d have b e e n u s e d i n
oid:(acceptor)-l-en-oxidoreductase
continuous column processes t o transform 1,4-dien-3-oxo-steroids i n t o 4-en-3-oxo-steroids. alginate xylose.622 activity
Pachysolen tannophilus c e l l s entrapped i n
g e l have been u s e d f o r
of
The
stability
of
Pseudomonas dacunhae
production of
the
L-aspartate
ethanol from
9-
B-decarboxylase
c e l l s entrapped i n
k-carrageenan
Carbohydrate Chemistry
716
g e l has been i n v e s t i g a t e d . 6 2 3 The p e r f o r m a n c e o f a h o l l o w - f i b r e m i c r o b e r e a c t o r has been compared w i t h a r e a c t o r c o n t a i n i n g Pseudomonas d e n i t r i f i c a n s c e l l s e n t r a p p e d i n a l g i n a t e g e l . 6 2 4 Rhizopus n i g r i c a n s c e l l s entrapped i n polyacrylamide, and
agar
g e l s have
been
used
for
the
alginate,
llu-hydroxylation
o f
R h o d o p s e u d o m o n a s c a p s u l a t a c e l l s i m m o b i l i z e d by
progesterone.625
glutaraldehyde-mediated
c r o s s l i n k i n g w i t h a l b u m i n have been used i n
an i n v e s t i g a t i o n o f t h e i n f l u e n c e o f i m m o b i l i z a t i o n o n l i g h t - i n d u c e d phosphorylation o f p r o d u c t i o n by
5.
ADP
by
the
chromatophore
s y s t e m .626
h a s been compared.627
C e l l s w e r e i m m o b i l i z e d by g l u t a r a l d e h y d e -
mediated co-crosslinking w i t h albumin o r gelatin, a l g i n a t e o r k-carrageenan gel, prepolymers.
ATP
c a p s u l a t a c e l l s i m m o b i l i z e d by a n u m b e r o f m e t h o d s
An e l e c t r o n - m i c r o s c o p i c
-R h o d o p s e u d o m o n a s
by e n t r a p m e n t i n
o r by c o p o l y m e r i z a t i o n w i t h u r e t h a n e study
o f
immobilized
capsulata c e l l s , entrapped i n a l g i n a t e gel, cells
has
been described.628
Rhodotorula minuta
entrapped w i t h i n
photocrosslinked o r
p o l y u r e t h a n e r e s i n g e l s have been u s e d f o r
s t e r e o s e l e c t i v e h y d r o l y s i s o f !&-men t h y 1 s u c c i na t e . 6 2 9 S a c c h a r o m y c e s c e r e v i s i a e c e l l s e n t r a p p e d i n a l g i n a t e g e l have been used f o r
the
continuous production o f
Polycation alginate
gel
resulted
phosphate.634
i n
treatment
a
matrix
with
g-
higher
i n
resistance
to
The t r e a t m e n t i n h i b i t e d r e s p i r a t i o n o f e n t r a p p e d
c e l l s b u t d i d not reduce e t h a n o l production.
-S.
from
entrapment
ethanol
following
E t h a n o l p r o d u c t i o n by
c e r e v i s i a e c e l l s i m m o b i l i z e d by a d s o r p t i o n o n d i f f e r e n t i o n -
exchange r e s i n s , 6 3 5
by e n t r a p m e n t i n k - c a r r a g e e n a n g e l s , 6 3 6
and by
5. e n t r a p m e n t i n p o l y a c r y l a m i d e g e l 637 has a l s o been r e p o r t e d . c e r e v i s i a e c e l l s e n t r a p p e d i n p o l y a c r y l a m i d e g e l have been u s e d a s p a r t o f a r e a c t o r c e l l system, s i m u l a t i n g t h e g l y c o l y t i c pathway, f o r t h e p r o d u c t i o n o f g l u t a t h i o n e a n d NADP.6059638
2.
cerevisiae
c e l l s have been used a s a model system f o r i n v e s t i g a t i o n o f c e l l i m m o b i l i z a t i o n methods i n v o l v i n g entrapment i n s i l i c a hydro r e a c t i o n w i t h epox y - a c t i v a t e d c r o s s l i n k e d p e c t a te,550 gelatin,640
entrapment i n
a n d e n t r a p m e n t i n a l g i n a t e beads f o l l o w e d by t r e a t m e n t
w i t h p o l y e t h y l e n e i m i n e and g l u t a r a l d e h y d e
or reaction with
p e r i o d a t e - o r c a r bo di-im id e - a c t i va t e d a l g i n a t e
b e a d s f o l l o w e d by
p o l y e t h y l e n e i m i n e c r o s s l i n k i n g . 641 Continuous
production
achieved i n a packed-column
o f
steroid
glycoalkaloids
recycle reactor
employing
has
been
Solanum
a v i c u l a r e c e l l s i m m o b i l i z e d by g l u t a r a l d e h y d e - m e d i a t e d r e a c t i o n w i t h p o l y c p h e n y l e n e o x i d e ) . 642
717
8: Chemical Synthesis and Modification Staphylococcus aureus c e l l s ,
h e a t k i l l e d and f o r m a l i n f i x e d o n
a p l e a t membrane f i l t r a t i o n system, t u m o r i c i d a l responses i n spontaneous
have been shown t o
produce
c a n i n e neoplasms a f t e r e x t r a -
corporeal perfusion over the immobilized cells.643
The t u m o r i c i d a l
e f f e c t was p r e s u m a b l y due t o p r o t e i n A p r e s e n t i n t h e c e l l s r e m o v i n g immune c o m p l e x e s .
_--S t e m e h y---l i u m ---l o t i
cells
i m m o b i l i z e d by
treatment
with
p o l y e l e c t r o l y t e f l o c c u l a t i n g a g e n t s have been u s e d f o r b a t c h - w i s e o r continuous degradation o f cyanide.644
-S t r e p t o m y c e s
aureofaciens c e l l s
have
b e e n i m m o b i l i z e d by
g l u t a r a l d e h y d e c r o s s l i n k i n g i n t o a g e l a t i n m a t r i x and a p p l i e d t o t h e b i o t r a n s f o r m a t i o n o f daunomycinone.645
A m i l d new m e t h o d f o r c e l l
g l u t a r a l d e h y d e - , o r pe r i o d a t e -
immobilization i n v o l v i n g glyox al-,
o x i d i z e d p o 1y v i n y 1 a 1 c o h o l - c r o s s l i n k i n g o n t o h y d r a z i d e g e l s has been d e v e l o p e d u s i n g m o d e l system.646
2.
polyacrylami d e
c l a v u l i g e r u s c e l l s as the
The i m m o b i l i z e d c e l l s w e r e u s e d f o r a n t i b i o t i c
(cephalosporin) production
and y i e l d e d s i g n i f i c a n t l y h i g h e r amounts
o f p r o d u c t t h a n d i d c e l l s i m m o b i l i z e d by d i r e c t p o l y m e r i z a t i o n o f
2.
a c r y l a m i d e monomer. g e l
have
been
used
fradiae c e l l s entrapped i n polyacrylamide f o r
S. p r o d ~ c t i o n . ~ ~’
protease
phaeochromogenes c e l l s w i t h a - g l u c o s e
i s o m e r a s e a c t i v i t y have been
a d s o r b e d o n diatomaceous e a r t h and used i n a column f o r m f o r a s t u d y of
substrate
pulse
reactions.648
response
predicted theoretically. i m m o b i l i z e d by reactor.649
i n
reversible
and
consecutive
R e s u l t s o b t a i n e d were i n good agreement w i t h t h o s e
2.
phaeochromogenes c e l l s have a l s o
aggregation o f
been
c h i t o s a n and used i n a n enzyme
The 1 6 a - h y d r o x y l a t i o n o f d e h y d r o e p i a n d r o s t e r o n e h a s
been a c h i e v e d u s i n g
2.
roseochromogenes
c e l l s immobilized i n a
p h o t o c r o s s 1i n k a b l e r e s i n . 6 5 0 A s t u d y o f t h e i m m o b i l i z a t i o n o f l e t t u c e t h y l a k o i d s by s e v e r a l
methods
has
been
reported.651
microenvironments on a c t i v i t y functional
s t a b i l i t y
phosphorylation o f
was
The yield,
described.
e f f e c t
o f
storage
s t a b i l i t y , and
d i f f e r e n t
The
fermentative
n u c l e o t i d e s h a s been a c h i e v e d u s i n g y e a s t
entrapped i n polyethylene
glycol
hy d r o x y e t h y l a c r y l a t e
cells
gels. 652
Zymomonas m o b i l i s c e l l s e n t r a p p e d i n a l g i n a t e g e l have b e e n u s e d f o r e f f i c i e n t production o f ethanol i n a b i ~ r e a c t o r . ~ ~ ~ The u s e o f
i m m o b i l i z e d c e l l s h a s r e c e n t l y been r e v i e w e d . 6 5 4
A
m a t h e m a t i c a l t r e a t m e n t has been p u b l i s h e d w h i c h a l l o w s c a l c u l a t i o n of
the theoretical
m a x i m u m r e a c t o r p r o d u c t i v i t i e s w h i c h c a n be
achieved i n gas-producing immobilized-cell
systems.655
Carbohydrate Chemistry
718
4 M o d i f i c a t i o n o f Enzymes and Uses o f M o d i f i e d Enzymes
-- T h e s t a b i l i z a t i o n o f a c i d p h o s p h a t a s e by l i n e a r chain, commercially a v a i l a b l e polyacrylamides i n u l t r a f i l t r a t i o n membrane r e a c t o r s h a s been studied.656 E n z y m e s t a b i l i t i e s were c o m p a r e d w i t h t h a t o f t h e e n z y m e a f t e r i m m o b i l i z a t i o n by g l u t a r a l d e h y d e - m e d i a t e d c r o s s l i n k i n g w i t h human s e r u m a l b u m i n .
A c i d Phosphatase.
--
h a s b e e n i n a c t i v a t e d by t r e a t m e n t o r t e t r a n i t r o met h a n e , p r o v i d i n g evidence for the existence of e s s e n t i a l tyrosine residues.657 E f f e c t i v e p r o t e c t i o n a g a i n s t i n a c t i v a t i o n o f t h e e n z y m e was a c h i e v e d by i n c l u d i n g a s u b s t r a t e a n a l o g u e ( l a c t o s e ) i n t h e i n c u b a t e s . 6-Q-Fucosidase. w ith io d in e
B-Q-Fucosidase
, 3- a c e t y l i m i d a z o l e ,
-- P r o s t a g l a n d i n F 2 a h a s b e e n l a b e l l e d w i t h $-8g a l a c t o s i d a s e u s i n g t h e mixed-anhydride method o f coupling.658 The c o n j u g a t e was u s e d i n t h e d e v e l o p m e n t o f a s e n s i t i v e a n d s p e c i f i c e n z y m e i m m u n o a s s a y f o r p r o s t a g l a n d i n F2a. 6-Q-Galactosidase.
-- A n t i ( h u m a n s e r u m a l b u m i n ) i m m u n o g l o b u l i n G h a s b e e n c o v a l e n t l y l i n k e d t o glucoamylase and q u a n t i t a t e d i n an enzymec y c l i n g assay.566
Glucoamylase.
I s o m e r a s e . -- T h e e f f e c t o f y - i r r a d i a t i o n o n p u r i f i e d Qglucose isomerase i n d i l u t e s o l u t i o n has been i n ~ e s t i g a t e d . ~ ~ ’
8-Glucose
-- F u n c t i o n a l a r g i n i n e r e s i d u e s i n p u r i f i e d b o v i n e h y a l u r o n i d a s e h a v e b e e n m o d i f i e d w i t h b u t a n e 2,3-
Hyaluronidase.
testicular d i o n e . 660
Lysozyme. -- The b i n d i n g o f Ca2+ t o l y s o z y m e a n d i t s d e r i v a t i v e s h a s b e e n s t u d i e d by U.V. d i f f e r e n c e s p e c t r o s c o p y a t v a r i o u s pHps.661 Binding caused p r o t o n release from lysozyme and d i d n o t i n h i b i t t h e b i n d i n g o f c h i t o t r i o s e t o l y s o z y m e . Ca*+ was s u g g e s t e d t o b i n d n e a r t h e c a t a l y t i c c a r b o x y l groups i n t h e enzyme, t h e r e b y c a u s i n g i n h i b i t i o n of enzyme a c t i v i t y . Ion binding shifted the natived e n a t u r e d t r a n s i t i o n i n lysozyme t o w a r d the n a t i v e s t a t e . The p r e p a r a t i o n and i m m u n o l o g i c a l c h a r a c t e r i z a t i o n o f two l y s o z y m e d e r i v a t i v e s , d i n i t r o p h e n y l a t e d a t L y s - 3 3 a n d Lys-96, r e s p e c t i v e l y , have b e e n r e p o r t e d . 6 6 2 The s p e c i f i c i t y o f s i a l y l t r a n s f e r a s e s has been s t u d i e d u s i n g
8: Chemical Synthesis and Modification
719
g l y c o s y l a t e d l y s o z y m e d e r i v a t i v e s as s u b s t r a t e s . 663 Miscellaneous
--
Glycoenzymes.
Conjugation
of
horse- r a d i s h
p e r o x i d a s e t o S t a p h y l o c o c c u s aureus p r o t e i n A has been compared u s i n g benzoquinone, reagents.664
glutaraldehyde,or
p e r i o d a t e as c r o s s l i n k i n g
Benzoquinone and g l u t a r a l d e h y d e t r e a t m e n t gave h i g h
y i e l d s o f coupled p r o t e i n A i n
conjugates o f low molecular size,
whereas p e r i o d a t e t r e a t m e n t r e s u l t e d i n a h i g h y i e l d o f p o l y m e r i c conjugates of
large molecular size.
I m m o b i l i z e d D e r i v a t i v e s o f Enzymes.
continues
to
proceedings of
be t a k e n
i n
the
--
Considerable i n t e r e s t
immobilization of
enzymes.
The
t h e 5 t h I n t e r n a t i o n a l Enzyme E n g i n e e r i n g C o n f e r e n c e
h a v e been p u b l i s h e d . 6 6 5
The a p p l i c a t i o n o f i m m o b i l i z e d enzymes i n
t h e f o o d i n d u s t r y h a s been r e v i e w e d . 6 6 6 The r e v e r s i b l e i m m o b i l i z a t i o n o f enzymes has b e e n r e v i e w e d w i t h s p e c i a l r e f e r e n c e t o t h e i r use i n a n a l y t i c a l procedures.667
The
p o s s i b i l i t y o f u s i n g a low degree o f s u b s t i t u t i o n o f t h e support p e r m i t s h i g h s e n s i t i v i t y i n i n h i b i t o r assays
and may e n c o u r a g e u s e
o f b i n d i n g assays i n c o m b i n a t i o n w i t h c o n v e n t i o n a l enzyme-based a n a l y t i c a l techniques.
The a p p l i c a t i o n o f e n z y m e e l e c t r o d e s i n
a n a l y s i s 6 6 8 and t h e u s e o f enzyme t h e r m i s t o r s f o r p r o c e s s have been r e v i e w e d .
D i f f e r e n t methods f o r t h e i m m o b i l i z a t i o n o f
enzymes o n l i p o s o m e s h a v e b e e n r e v i e w e d . 6 7 0 The
use
of
polyethylene
beads
as
supports
for
enzyme
i m m o b i l i z a t i o n h a s been d i s c u s s e d . 6 7 1
Films o f highly polymerized
collagen
conditions
prepared
routinely
a f t e r
under acyl
industrial azide
activation
for
have the
been
i m m o b i l i z a t i o n o f numerous enzymes f r o m d i f f e r e n t c l a s s e s . 6 7 2 s t a b i l i t y of conditions, to
used
covalent The
t h e r e s u l t i n g membranes t o o p e r a t i o n a l and s t o r a g e
t h e i r : e x c e l l e n t m e c h a n i c a l s t r e n g t h and t h e i r r e s i s t a n c e
b a c t e r i a l degradation render
them s u i t a b l e
for
a number o f
applications. A new a m p e r o m e t r i c a n a l y t i c a l s y s t e m b a s e d on b i o c a t a l y s t s h a s
been d e s c r i b e d . 673 A
model
has
been
proposed
i n
which
p r o t e c t s an a c t i v e enzyme f r o m d e a c t i v a t i o n , and s o l u b l e enzY mes.674 o f t h e enzyme-support
a
d e a c t i v a t e d enzyme for
both immobilized
S t e r i c h i n d r a n c e s and t h e i m m o b i l e n a t u r e
l i n k o f i m m o b i l i z e d enzymes a p p e a r t o l e s s e n
the extent o f t h i s protection. A
theoretical
treatment describing approximate expressions o f
Carbohydrate Chemistry
720
e f f e c t i v e n e s s f a c t o r s f o r i m m o b i l i z e d b i o c a t a l y s t s h a s appeared.675 The e f f e c t o f i n t r a p a r t i c l e d i f f u s i o n r e s i s t a n c e o n t h e a p p a r e n t
-
s t a b i 1it y o f i m m o b i 1iz e d e n z ym e s sub j e c t t o f ir s t o r de r de a c t i va t i on has
been q u a n t i t a t i v e l y
studied.676
A
general
e x p r e s s i o n was
d e r i v e d t o describe t h e r e l a t i o n s h i p between t h e d e c r e a s i n g observed e n z y m a t i c r e a c t i o n r a t e a n d t h e i n t r i n s i c enzyme d e a c t i v a t i o n r a t e . A m e t h o d o f e s t i m a t i n g t h e i n t r i n s i c d e a c t i v a t i o n r a t e c o n s t a n t was
also
proposed.
The
experimentally.
theoretical
simple
A
treatment
e s t a b l i s h e s i n t r i n s i c r a t e p a r a m e t e r s when s l o w substrate l i m i t s immobilized-enzyme Menten k i n e t i c s . 6 7 7 and r e p l a c e m e n t
In a
was
validated
method h a s been p r e s e n t e d which
theoretical
p o l i c i e s have
pore
diffusion of
r e a c t i o n s t h a t obey M i c h a e l i s treatment optimal operation
been p r o p o s e d f o r
reactors with
i m mo b i l i z e d enzymes s u b j e c t t o deac t i va t i o n .678 A c o n v e n i e n t a p p a r a t u s h a s been d e s c r i b e d f o r t h e d e t e r m i n a t i o n o f i m m o b i l i z e d e n z y m e a c t i v i t i e s by t h e s t i r r e d b a t c h m e t h o d . 6 7 9 Advantages
o f
the
system
include
e q u i l i b r a t i o n o f temperature,
rapid working,
efficient
c o m p a c t n e s s o f t h e r e a c t i o n chamber,
t h e p o s s i b i l i t y o f a s s a y i n g t h e s a m p l e r e p e a t e d l y o r o n l y once,
and
t h e ease o f r e m o v a l o f r e a g e n t s o l u t i o n s o r i m m o b i l i z e d p r e p a r a t i o n s from t h e apparatus. A
g r a p h i c a l m e t h o d h a s been d e v e l o p e d f o r d e t e r m i n i n g t h e
Michaelis-Menten constant f r e e o f external mass-transfer f o r i m m o b i l i z e d enzymes i n a p a c k e d b e d r e a c t o r . 6 8 0
resistance
The v a l i d i t y o f
t h e t h e o r e t i c a l t r e a t m e n t was i l l u s t r a t e d w i t h e x a m p l e s f r o m t h r e e d i f f e r e n t enzyme r e a c t i o n s . Some o b s e r v a t i o n s o n t h e s i m i l a r i t y o f b a t c h a n d p l u g f l o w s y s t e m s a s a p p l i e d t o enzyme r e a c t o r s have been p u b l i s h e d w i t h p a r t i c u l a r relevance t o e x t r a c t i o n o f true values o f the k i n e t i c p a r a m e t e r s , e v e n when t h e e x t e r n a l m a s s - t r a n s f e r e f f e c t s w e r e n o t e l i m i n a t e d . 681 A theoretical treatment
g i v i n g r a t e e x p r e s s i o n s f o r enzyme
poisoning which are consistent w i t h a Michaelis-Menten main reaction h a s been published.682
The e x p r e s s i o n s c a n be u s e d t o a n a l y s e t h e
p e r f o r m a n c e o f a f i x e d b e d r e a c t o r c o n t a i n i n g i m m o b i l i z e d enzyme. An o p e r a t i o n a l scheme f o r
u s i n g i m m o b i l i z e d enzymes i n packed
bed r e a c t o r s t h a t p e r m i t s o p e r a t i o n a t a constant throughput r a t e a n d w i t h c o n s t a n t p r o d u c t q u a l i t y h a s been d e s c r i b e d . 6 8 3
The m e t h o d
i n v o l v e s use o f c o l u m n s o p e r a t e d i n s e r i e s w i t h c o n t i n u o u s enzyme addition.
The d e s i g n o f a n o n u n i f o r m l y d i s t r i b u t e d b i o c a t a l y s t ha:
been d e s c r i bed.684
72 1
8: Chemical Synthesis and Modification
Many p r e p a r a t i o n s o f i m m o b i l i z e d enzymes a n d o f enzymes c o u p l e d t o o t h e r macromolecules t o their
o r -insoluble
give water-soluble
have been r e p o r t e d d u r i n g t h e p a s t year. u s e s a r e s u m m a r i z e d i n T a b l e 7.685-743
a r e r e f e r r e d t o i n t h i s Table,
forms
These d e r i v a t i v e s a n d I m m o b i l i z e d enzymes
whether they are e n z y m a t i c a l l y a c t i v e
o r not. The f o l l o w i n g i n f o r m a t i o n o n i m m o b i l i z e d g l y c o s i d e a n d p o l y s a c c h a r i d e h y d r o l a s e s and c a r b o h y d r a t e i s o m e r a s e s a n d ox i d a s e s i s worthy o f note.
I m m o b i l i z a t i o n o f d e x t r a n s u c r a s e a'ppears t o be
favourable f o r low-molecular-weight
dextran production.707
k i n e t i c m o d e l l i n g o f a m u l t i p l e immobilized-enzyme reported.744
The
validity
of
the
model
The
s y s t e m h a s been
was
successfully
dem o n s t r a t e d u s i n g Q - g 1uco se ox i d a s e a n d B - c - f r u c t o f u r ano s i da se coimmobilized i n poly-(2-hydroxymethacrylate
g e l ) .745
furanosidase a c t i v i t y o f immobilized yeast
The B - p - f r u c t o -
c e l l s has been u t i l i z e d
i n a n enzyme t h e r m i s t o r m e t h o d f o r c o n t r o l l i n g s u c r o s e
t r a t i o n d u r i n g f e r m e n t a t i on. 7 4 6
concen-
B-Q-Fructo f urano s i dase has been
s u c c e s s f u l l y i m m o b i l i z e d by a d s o r p t i o n o n k r i l l c h i t i n . 5 0 4 observed t h a t treatment
with glutaraldehyde
reduction o f the a c t i v i t y o f
I t was
r e s u l t e d i n 10-40% B-Q - -Fructo-
t h e enzyme p r e p a r a t i o n s .
f u r a n o s i d a s e i m m o b i l i z e d by g l u t a r a l d e h y d e - m e d i a t e d
crosslinking i n
hen e g g w h i t e r e t a i n e d about 25% o f i t s o r i g i n a l a c t i v i t y . 7 0 8 P r o p e r t i e s o f t h e i m m o b i l i z e d enzyme w e r e i n v e s t i g a t e d . The enzyme h a s a l s o been i m m o b i l i z e d o n m o l e c u l a r s i e v e s 710 surfaces
of
nylon
tubes.711
and o n t h e i n n e r
B-9-Fructofuranosidase
has
been
a d s o r b e d o n bead c e l l u l o s e c o n t a i n i n g weak, b a s i c m - d i e t h y l a m i n o - 2 h y d r o x y p r o p y l groups.709
Conditions most suitable f o r adsorption
w e r e d e s c r i b e d a n d i n v e r s i o n o f s u c r o s e u s i n g t h e p r e p a r a t i o n was studied i n a s t i r r e d reactor. sucrose inversion Under
these
under
conditions
concentration and flow
Use o f t h e i m m o b i l i z e d e n z y m e f o r
flow
c o n d i t i o n s was
the
inversion
also
was
investigated.747
affected
by
the
r a t e o f t h e s u b s t r a t e a n d by t h e r e a c t i o n
temperature. The h a l f - l i f e o f t h e e n z y m e w a s 2 1 5 d a y s . I t was f o u n d t h a t t h e i m m o b i l i z a t i o n was m o r e e f f e c t i v e u n d e r f l o w conditions
r eacto r
.
The
than
under t h e
equilibrium
immobilization o f
processing
has
immobilized
by
g l u t a r a 1de h y d e
been
B-c-galactosidase
reviewed.748
adsorption
on
c r o s s l in k i n g 7 1
r e a c t i o n w i t h nylon-co(acry1ic
conditions for
of a use
6-Q-Galactosidase DEAE-cellulose a n d by
stirred i n dairy
has
followed
been by
c a r b o di-im i d e - m e d i a t e d
a c i d ) polymers.581
Adsorption o f the
( T e x t c o n t i n u e s on page 7 5 2 )
3.2.1.52
B-e-2-Acetamido-2deoxy-hexosidase
Adenosine ( p h o s p h a t e ) 3.5.4.17
3.1.3.2
-
3.1.1.7
Acetylcholinesterase
Acetobacter a c e t i cells Acid p h o s p h a t a s e
EC No.
Development o f a new method o f enzyme i m m o b i l i z a t i o n Study o f k i n e t i c p r o p e r t i e s of t h e a c t i v e immobilized enzyme Production o f a c e t i c acid i n a fixed-cell reactor Development o f a new method o f enzyme i m m o b i l i z a t i o n
Reaction w i t h thiosulphate d e r i v a t i v e s o f c e l lu l ose Reaction w i t h concanavalin A
Comparative s t u d y of enzyme s t a b i 1i z a t i o n by r e a c t i o n w i t h s o l u b l e and i n s o l u b l e macromolecules Study o f t h e k i n e t i c properties o f t h e enzymes A c t i v e immobilized enzyme
Carbodi-imide-mediated r e a c t i o n w i t h nylon-co(acry1ic a c i d ) p o l ymer R e a c t i o n w i t h commer c i a 1 1y a va i l a b l e water- s o l u b l e p o 1y acrylamides o r glutaraldehydemediated c r o s s l i n k i n g w i t h a 1 b um in Co - ge 1a t i o n o r co - po 1ymer i z at i o n and g e l a t i o n w i t h albumin o n t o membrane r e a c t o r w a l l s R e a c t i o n w i t h ion-exchange
Adsorption on c o r d i e r i t e
Use o f p r o d u c t
Matrix o r macromolecule c o u p l e d and mode o f c o u p l i n g
M o d i f i c a t i o n o f enzymes by c o u p l i n g t o insoluble m a t r i c e s and other macromolecules
Enzyme
Table 7
686
685
656
5 81
600
5 90
497
Ref.
L'
5
2.
!?
$
k
%
3
&
-4 h , h,
-
EC No.
A l k a l i n e phosphatase
de h y d r ogenase
Aldehyde
3.1.2.1
1.2.1.3
A l c o h o l d e h y d r o g e n a s e 1.1 .l. 1
Alcaligenes eutrophus cells
deamina se
E nz yme
Table 7 continued
-
reaction
PO 1y
reaction e t hy l e n e i m i n e
Reaction w i t h E(carboxymethy1-
co p o l ymer
w i t h ny l o n -
Glutaraldehyde-mediated
po 1y a c r y 1am i d e ge 1
entrapment i n
a d s o r p t i o n o n Amber-
l i t e R IRA-94,
agarose,
w i t h cyanogen b r o m i d e - a c t i v a t e d
w i t h porous glass,
G l u t araldehyde-me d i a t e d r e a c t i o n
a c t i v a t e d agarose
R e a c t i o n w i t h cy ano gen b r o m i d e
an a l b u m i n m a t r i x
D e v e l o p m e n t o f a new m e t h o d o f
ch o l es t e r o 1
Automated d e t e r m i n a t i o n o f
i m m o b i l i z e d enzyme
Comparison o f p r o p e r t i e s o f
NADPH/NA DP r e ge ne r a t i on
u s e i n a n enzyme e l e c t r o d e
A c t i v e i m m o b i l i z e d enzyme f o r
i m m o b i l i z e d enzyme
sional effects o f
Study o f k i n e t i c s and d i f f u -
Glutaraldehyde-crosslinked i n
complex m e t h o d D e t r i t i a t i o n o f water i n b a t c h t a n k o r column r e a c t o r
Ti4+
Use o f p r o d u c t
Entrapment i n a l g i n a t e o r k- ca r r a geenan ge 1s
resins & v
a n d mode o f c o u p l i n g
M a t r i x o r macromolecule coupled
399
689
157
688
687
601
Ref.
4 h)
w
u
a x.
4
0
3
R
Q
5
*
K’
z3
PP 0
EC No.
3.5.1.14
3.2.1.1
E nz yme
Am inoa c y 1a se
a-Am y 1ase
Table 7 continued
racemic m i x t u r e
693
i m m o b i l i z e d enzyme A c t ive i m m o b i 1i z e'd e n z yme
Reaction with polymethacrylate r e a c t i o n w i t h CM-
ce 11u l o s e
esters,
with zirconia-coated glass
692
411
503
691
S t u d y of p r o p e r t i e s o f a c t i v e
enz ym e i m m o b i 1i z a t io n
D e v e l o p m e n t o f a new m e t h o d o f
enzyme immo b i l i z a t i o n
D e v e l o p m e n t o f a new m e t h o d o f
enz ym e i m m o b i 1iz a t io n
D e v e l o p m e n t o f a new m e t h o d o f
6 90
Ref.
G 1 u t a r a l dehyde -medi a t e d r e a c t i o n
v i n y l a t i o n o f t h e enzyme
e n t po 1y sa c c h a r ide s f o l 1ow i n g
Graft polymerization to d i f f e r -
Adsorption on k r i l l c h i t i n
crosslinked polyvinyl alcohol
Ni2+, o r Fe2+) c o m p l e x e s o f
R e a c t i o n w i t h m e t a l (Co2+,
p h e n y l g l y c i n e from i t s
C o n t i n u o u s i s o l a t i o n o f Q-
ca r r ie r s
r egenerab l e m e t a l - chelat e
Adsorption on DEAE-cellulose
enzyme i m m o b i l i z a t i o n o n
amino )aga r o s e p r e -eq u i 1i b r a t e d
Use o f p r o d u c t
w i t h Cu2+, Zn2+, o r C o 2 + i o n s
a n d mode o f c o u p l i n g
M a t r i x o r macromolecule coupled
&
$.
n
P
k
2
4 t 4 P
3.5.3.1
Arginase
2.6.1.1
A s p a r t a t e amino-
coccum c e l l s
Azotobacter chroo-
-
1.10.3.3
Ascorbate oxidase
t r a n s f e r a se
3.1.6.1
A r y l sulphatase
-
3.2.1.2
B-Am y 1ase
Arthrobacter c e l l s
EC No.
Enzyme
~~
Table 7 continued
D e v e l o p m e n t o f a new m e t h o d o f enzyme i m m o b i l i z a t i o n Continuous n i t r o g e n f i x a t i o n
v a t e d c o l l a g e n membranes Entrapment i n agar g e l
i z e d enzyme a c t i v i t i e s
f o r measurement o f i m m o b i l -
Development o f an a p p a r a t u s
m e t r i c assay o f v i t a m i n C
Enzyme e l e c t r o d e f o r ampero-
t h e a c t i v e i m m o b i l i z e d enzyme
Study o f k i n e t i c p r o p e r t i e s o f
isomerase a c t i v i t y o f c e l l s
o f L1210 m u r i n e l e u k a e m i a S t u d y of k i n e t i c s o f e - g l u c o s e
i m m o b i l i z e d enzyme, s t u d y o f e f f e c t o f conjugate on growth
Study o f p r o p e r t i e s o f a c t i v e
A c t i v e i m m o b i l i z e d enzyme
Use o f p r o d u c t
Reaction with a c y l azide-acti-
agarose
Unspecified reaction with
with nylon netting
Glutaraldehyde-mediated r e a c t i o n
Reaction with concanavalin A
Not g i v e n
i n t e r f a c i a 1 po 1y m e r i z a t io n
membrane m i c r o c a p s u l e s by
Encapsulation within nylon
4-amino benz y l c e l l u l o s e
Reaction with diazotized
a n d mode o f c o u p l i n g
M a t r i x o r macromolecule coupled
603
696
679
695
590
602
694
468
Ref.
h)
VI
4
3
2g.
%
$
h.
2
E
*
s
-
E C No.
Ca t a l a s e
Candida t r o p i c a l i s cells
1.11.1.6
-
Brevibac t e r i u m ammoniagenes c e l l s B r e v i b a c t e r i u m flavum cells B r e v i b a c t e r i u m fuscum cells 3.1.1.8 B u t y ry l c h o l i n e e s t erase C a l d a r i e l l a acidophila cells
Beneckea h a r v e y i cells
Enzyme
T a b l e 7 continued
Reaction w i t h thiosulpha t e de r i va t i ve s of c e 11ulo s e G1 u t a ra 1de h yde -mediated r e a c t i o n w i t h chicken eggwhite, w i t h o r without magnetite present Reaction w i t h epoxy-activated pectate pre-reacted w i t h i m i n o d i a c e t i c a c i d and e q u i l i b r a t e d w i t h Fe3+ i o n s Adsorption on p o l y e t h y l e n e -
Co-casting w i t h c o l l a g e n followed by g l u t a r a l d e h y d e - t r e a t m e n t E n t rapment i n k- c a r r a g e e nan ge 1
React i o n w i t h ph o t o - c r o s s 1 i n ka b l e r e s i n s : entrapment i n a l g i n a t e gel Entrapment i n polyacry lamide
M a t r i x o r macromolecule coupled and mode o f c o u p l i n g
608
497
607
606
605
604
Ref.
A c t i v e immobilized enzyme
697
Development o f a new method f o r 550 whole c e l l i m m o b i l i z a t i o n
Production o f glutamic a c i d i n a recycle reactor Continuous p r o d u c t i o n o f 1 2 ke t o che no deoxycholi c a c i d Development of a new method o f enzyme immo b i 1i z a t i o n L i v i n g immobilized c e l l s w i t h 6 -g -ga 1a c t o s i da s e a c t i v i t y
N A DP
P r o d u c t i o n o f g l u t a t h i o n e and
Development o f a s e n s o r system f o r h y d r o gen pe r ox i de
Use of p r o d u c t
4
s
--e
e D
g
P&
m
h)
4
1.1.3.6
3.4.21.1
Chymotrypsin
-
Cholesterol oxidase
Chloroplasts
-
E C No.
Cephalexin-synthesizing en zyme
Enzyme
Table 7 continued
Use o f p r o d u c t
Oxygen-st a b i li zed enzyme e l e c t r o d e f o r Q-glucose o x i d a s e by g l u t a r a l d e h y d e mediated r e a c t i o n w i t h P t gauze analysis i n fermentation broths Adsorption on k a o l i t e , S t u d y of k i n e t i c s and s t a b i bentonite, c e l i t e , activated l i t i e s o f the active carbon o r s i l i c a immobilized enzymes y-Ray-induced p o l y m e r i z a t i o n S t u d y of s t a b i l i t i e s o f w i t h g l a s s - f o r m i n g monomers immobilized c e l l s Study o f s t a b i l i t y o f immobily-Ray i r r a d i a t i o n w i t h v i n y l ized c e l l s monomers Photochemical-energy c o n v e r s i o n Entrapment i n a g a r g e l system Glutaraldehyde-mediated reacImmobilized enzyme s e n s o r u t i l i z i n g chemiluminescence t i o n w i t h c e l l u l o s e beads f o r measurement o f c h o l e s t e r o l i n serum Glutaraldehyde-mediated r e a c t i o n A c t i v e immobilized enzyme f o r
imine-coated g l a s s microbeads Co-immobilized w i t h P-glucose
Matrix o r macromolecule coupled and mode o f c o u p l i n g
701
700
611
610
609
699
698
Ref.
4 4
N
3
62.
$
Q.
5
o
G-
E'
3
g
n
PP
Enzyme
Table 7 continued
E C No.
complex
casein hydrolysis i n stirred tank reactors Study o f e f f e c t o f p r o t e i n nature on coupling t o d i f f e r e n t epoxide-co n t a i n i n g s u p po r t s
Use of product
ferent lowing Reaction esters
polysaccharides f ole nz yme irnmo b i 1i z a t i o n v i n y l a t i o n o f t h e enzyme w i t h polymethacrylate A c t i v e immobilized enzyme o r r e a c t i o n w i t h CM-
R e a c t i o n w i t h 3 - ( 2 ’ ,3’-epoxypropox y ) propy l g l a s s , 3 ( 2 ’ , 3 9-epoxypropoxy ) p r o p y l s i l i c a , epoxy-activated gly c i d y 1 me t h a c r y l a t e copolymer o r o x i r a n e - a c r y l i c beads R e a c t i o n w i t h cyanogen bromideStudy of s t a b i l i t y of t h e a c t i v e immobilized enzyme a c t i v a t e d agarose o r dextran o r entrapment i n p o l y a c r y l a m i d e gel or sheet R e a c t i o n w i t h cyanogen bromideStudy o f autodigestion of a c t i v a t ed agaro s e c h ymo t ry p s i n Graft polymerization t o d i f Development of a new method o f
w i t h Ni-NiO-albumin
Matrix o r macromolecule coupled and mode o f c o u p l i n g
693
411
159
158
5 84
Ref.
4
5
3
2
s
P
k
S&
&
2
00
N
4
cells
Coriolus versicolor
p a s t e u r i anum c e l l s
Clostridium
cells
Clostridium butyricum
Enzyme
Table 7 c o n t i n u e d
-
-
-
EC No.
soluble poly-
glass o r amino-SpherosilR Entrapment i n a l g i n a t e g e l
r e a c t i o n with aminopropyl-
G l u t araldehyde-media t e d
Entrapment i n agar g e l
Not g i v e n
w i t h methacry l a t e copolymers
(vinyl alcohol) or reaction
Reaction w i t h derivatized poly-
mers, a n d membranes
t o f o r m monomers,
Glutaraldehyde-mediated reaction
cellulose R e a c t i o n w i t h ChromagelR A-2
M a t r i x o r macromolecule coupled a n d mode o f c o u p l i n g
Ref.
703
612
effluent
Decolorization o f a k r a f t m i l l
production
system B i o p h o t o l y t i c hydrogen
614
613
Pho t o c h em ic a l - e n e r gy con ve r s i o n 6 11
ally
s t a r t i n g a fermentor a s e p t i c -
Development o f method f o r
f o r enzyme i m m o b i l i z a t i o n
S t u d y o f new p o t e n t i a l c a r r i e r s 7 0 5
sin
chemically m o d i f i e d chymotryp-
C o m p a r a t i v e s t u d y o f n a t i v e and 704
t h i o l proteinase i n h i b i t o r s
P a r t i a l p u r i f i c a t i o n o f u r i n a r y 702,
Use o f p r o d u c t
F
h) 4
0
g
5 .$
a
9
C'
2
5
*s
.:
3
n s
I
1.6.4.3
D i hy dr o l i p o am i d e
r e d u c t a se
2.4.1.5
-
activated cellulose
R e a c t i o n w i t h cyanogen bromide-
c e l l u l o s e beads
R e a c t i o n w i t h phenox yace t y 1-
t i o n w i t h amino-SpherosilR
Glutaraldehyde-mediated reac-
g l a s s o r amino-SpherosilR
r e a c t i o n w i t h a m i n o p r o p y 1-
G 1 u t a r aldehyde-me d i a t e d
a c t i v a t e d agarose
R e a c t i o n w i t h cyanogen b r o m i d e -
3a -hy d r o x y s t e r o ids
Continuous f l o w a n a l y s i s o f
production o f dextran A c t i v e immobi 1i z e d enzyme
A c t i v e i m m o b i l i z e d enzyme f o r
p r o d u c ti o n
B i o p h o t o l y t i c hydrogen
hair follicles
I s o l a t i o n o f a c t i n f r o m human
from a c t i n
binding protein distinct
D e m o n s t r a t i o n o f a DNAase-
systemic l u p u s erythematodes
476
494
707
6 13
162
161
615 706
Bioconversion o f g i t o x i g e n i n Model system f o r p a t i e n t s w i t h
Immobilized on e r y t h r o c y t e s
-
3.1.21.1
A c t i v e i m m o b i 1i z e d enz yme
160
Ref.
a c t i va t e d a ga r o s e Entrapment i n a l g i n a t e g e l
Use o f p r o d u c t
R e a c t i o n w i t h cy ano gen brom i d e -
a n d mode o f c o u p l i n g
M a t r i x o r macromolecule coupled
-
EC No.
Dex t r a n s u c r a s e
furicans cells
D e s u l f o v i b r i o desu 1-
Deoxyribonuclease
Daucus c a r o t a c e l l s --
C y s t a t h i o n e B-synthase
Enzyme
Table 7 continued
G-
3
%
3 k
2 s-
0
w
4
-
-
-
Escherichia
alcalescens cells Escherichia c o l i cells
3.4.21.11
E l a s t a se
E r y t h r o cy t es
EC No.
E nz yme
T a b l e 7 continued
i n p o l ya c r y l am i de
ge 1 E n t r a pm e n t i n po 1y a cr y 1am i d e gel R e a c t i o n w i t h epox y - a c t i va t e d pectate pre-reacted w i t h i m i n o d i a c e t i c a c i d and e q u i l i b r a t e d w i t h Fe3+ i o n s E n t r a p m e n t i n p o l y u r e t h a n e foam
E n t r a pme n t
e r y t h r o p o i e t i n complex
Reaction w i t h agarose-lectin-
activated agarose
R e a c t i o n w i t h cyanogen bromide-
Matrix o r macromolecule coupled
in
550
617
Living immobilized c e l l s w i t h
605
616
375
164
163
L
w
~
=. ?
2
El
5
3K' Ref.
Development o f a new m e t h o d f o r whole c e l l immobilization
NADP
Production o f g l u t a t h i o n e and
t h r o i d colony formation vitro Living immobilized c e l l s
s p e c i f i c i n h i b i t o r from b r o n c h i a l mucus A c t i v e i m m o b i l i z e d enzyme f o r p r o t e o l y s i s o f human p l a s m i n o ge n Study o f s t i m u l a t i o n of ery-
P u r i f i c a t i o n o f an e l a s t a s e -
Use o f p r o d u c t
r?o n
s i da se
-
3.2.1.26
3.4.22.3
Ficin
B-g-Fruc t o f u r a no
EC No.
Enzyme
Table 7 continued
l i n k i n g w i t h hen e g g w h i t e
G 1 u t a r a l de hy de-me d i a t e d c r o s s -
A d s o r p t i o n o n an ion-exchanger
A d s o r p t i o n o n A m b e r l i t e R IRA-94
Adsorption on k r i l l c h i t i n
c e l lulose
I m m o b i l i z e d o n carboxymethyl-
sa n
Ionotropic g e l a t i o n with chito-
and mode o f c o u p l i n g
M a t r i x o r macromolecule coupled
h e n e g g w h i t e as s u p p o r t f o r
assessment o f s u i t a b i l i t y o f
A c t i v e i m m o b i l i z e d enzyme and
M i cha e l i s -Me n t e n c o n s t a n t
method f o r d e t e r m i n i n g t h e
Validation o f a modified
ilit y o f immobi 1i z e d enzymes
sistance on t h e apparent stab-
intraparticle diffusion re-
theory concerning e f f e c t o f
Experimental v a l i d a t i o n o f
enzyme immo b i l i z a t i o n
Development o f a new method o f
M ichaelis-Menten constant
f o r determining the
V a l i d a t i o n o f m o d i f i e d method
tryptophan synthetase a c t i v i t y
L i v i n g immobilized c e l l s with
p e n i c i l l i n G acylase a c t i v i t y
Use o f p r o d u c t
708
680
6 76
504
503,
680
511
Ref.
%'
Ba
8
e-
k 3
2 &
h)
w 4
-
3.2.1.23
B-Q-Gal a c t o s i d a s e
g-gal act o s y 1 h y d r o x y k-lysyl glucosylt r a n s f era s e
E C No.
Enzyme
T a b l e 7 continued
Metal-link-mediated r e a c t i o n with molecular sieves G 1 u t a r a 1de h yde - m e d i a t e d r e a c t i o n w i t h g - a l k y l a t e d nylon tubes Carbodi-imide-mediated r e a c t i o n w i t h nylon-co ( a c r y l i c a c i d ) po 1ymer Adso r p t i on o n b r u s h i t e A d s o r p t i o n on DEAE-cellulose f o l 1owed by g l u t a r a 1de h y d e c r o s s l i n king R e a c t i o n w i t h cyanogen bromidea c t i va t e d a g a r o se
A d s o r p t i o n o n "-diethylamino2- h y d r o x y p r o p y l c e l l u l o s e
Matrix o r m a c r o m o l e c u l e c o u p l e d a n d mode o f c o u p l i n g
o f a n t i - ( c h i c k-em b r y o g a l a c t o s y 1 h y d r o x y - l - l y s y 1g l uco s y l t r a n s f e r a s e ) a n t i bo dy
Immunoadsorption p u r i f i c a t i o n
71 2
A c t i v e i m m o b i l i z e d enzyme Study of p r o p e r t i e s of t h e a c t i v e i m m o b i l i z e d enzyme
165
71 3
581
711
A c t i v e immobi 1i z e d enzyme
D e v e l o p m e n t o f a new m e t h o d o f enzyme immo b i l i t a t i o n
710
709
A c t i v e i m m o b i l i z e d enzyme
enzyme i m m o b i l i z a t i o n I n v e r s i o n of s u c r o s e i n a stirred r e a c t o r
Use o f p r o d u c t
w
4
3
$.
$
3
a
c;. a
3 2
*
3 fi' Ref. k
G 1 uc o am y 1a se
e n d o - Q - G a l a c t u r o nanase -
E nz yme
T a b l e 7 continued
a n d mode o f c o u p l i n g
M a t r i x o r macromolecule coupled
3.2.1.3
g l y c y l g l y cine,
2-dime t h y l a m ino e t h y 1 m e t h a c r y-
A d s o r p t i o n o n t o a copolymer o f
cellulose
e s t e r s o r r e a c t i o n w i t h CM -
Reaction with polymethacrylate
with alkylamine glass
G 1u t a r a l d e h y d e - m e d i a t e d r e a c t i o n
Adsorption on k r i l l c h it i n
a c t i v a t e d agarose
R e a c t i o n w i t h cyanogen b r o m i d e -
o r 6 -amino h ex ano i c a c i d
butanoic acid,
w i t h SpheronR p r e - c o u p l e d t o g l y c i n e , 6 - a l a n i n e , 4-amino-
3.2.1 .15 Car bo di-im ide-me d i a t e d r e a c t i o n
EC No.
-
i m m o b i l i z e d enzyme
Study o f p r o p e r t i e s o f a c t i v e
A c t i v e i m m o b i l i z e d enzyme
f o r pore d i f f u s i o n - l i m i t e d immo b i l i t e d - e n z y m e r e a c t i o n s
i n t r i n s i c k i n e t ic co n s t a n t s
new m e t h o d f o r e s t i m a t i n g
Experimental v a l i d a t i o n o f a
enzyme i m m o b i l i z a t i o n
D e v e l o p m e n t o f a new m e t h o d o f
o f a n t i g l ucoam y 1a s e a n t i bo dy
Immunoadsorption p u r i f i c a t i o n
a c t i v i t y o f t h e enzyme
immobilization on the
Investigation of effect o f
Use o f p r o d u c t
715
693
677
503
166
714
Ref.
B' 4
9 3
3
a 2
k
2 & $
4 w P
1.1.1.47
5.3.1.18
1.1.3.4
G 1uco s e
dehydrogenase Glucose isomerase
!-Glucose
oxidase
EC No.
Enzyme
Table 7 continued
or glutaraldehyde o r
e n t polysaccharides following
Graft polymerization t o d i f f e r -
enzyme immo b i l i z a t i o n
D e v e l o p m e n t o f a new m e t h o d o f
enzyme i m m o b i l i z a t i o n 411
696
D e v e l o p m e n t o f a new m e t h o d o f
v a t e d c o l l a g e n membranes
modified gelatin gel Reaction with a c y l azide-acti-
719
696
718
717,
716
Ref.
t h e a c t i v e i m m o b i l i z e d enzyme A c t i v e i m m o b i l i z e d enzyme 7 20
Study o f k i n e t i c p r o p e r t i e s o f
enzyme immo b i l i z a t i o n
D e v e l o p m e n t o f a new m e t h o d o f
a c t i v e i m m o b i l i z e d enzyme
Study o f s t a b i l i t y o f t h e
t h e a c t i v e i m m o b i l i z e d enzyme
Study o f t h e r m a l s t a b i l i t y o f
Use o f p r o d u c t
method Reaction w i t h radiation-
I m m o b i l i z a t i o n by m e t a l - l i n k
v a t e d c o l l a g e n membranes
Reaction w i t h a c y l azide-acti-
1,6 - d i am in o h ex ane
acid,
controlled pore glass followed by c r o s s l i n k i n g w i t h t a n n i c
Reaction with Ti4+-acti vated
l a t e and m e t h y l m e t h a c r y l a t e Entrapment i n p o l y a c r y l a m i d e g e l
M a t r i x o r macromolecule coupled a n d mode o f c o u p l i n g
VI
w 4
s
$.
8
5
a
a
3
>
8
f?.
? g.
?
E nz yme
Table 7 continued
EC No.
G 1u t a raldehyde-me d i a t e d r e a c t i o n
c h l o r i d e membrane
Adsorption on porous p o l y v i n y l
w i t h n y l o n 6 6 t o f o r m a membrane
Glutaraldehyde-mediated r e a c t i o n
Entrapment i n p o l y a c r y l a m i d e g e l
t i o n w i t h c e l l u l o s e beads
G l u t a raldehyde-me d i a t e d r e a c -
t i o n w i t h P t gauze
glutaraldehyde-me d i a t e d reac-
C o - i m m o b i l i z e d w i t h c a t a l a s e by
coated glass microbeads
Adsorption on polyethyleneimine-
v i n y l a t i o n o f t h e enzyme
a n d mode o f c o u p l i n g
M a t r i x o r macromolecule coupled
analysis
o f enzymic a c t i v i t y A c t i v e i m m o b i l i z e d enzyme
,
study o f mechanical c o n t r o l
A c t i v e i m m o b i l i z e d enzyme,
o n l y when m e c h a n i c a l l y s t retched
Membrane w i t h enzyme a c t i v i t y
t h e a c t i v e i m m o b i l i z e d enzyme
Study o f t h e r m a l s t a b i l i t y o f
i n serum
f o r measurement o f q - g l u c o s e
u t i1i z i n g chem i 1um i n e sce n c e
I m m o b i l i z e d enzyme s e n s o r
i n fermentation broths
t r o d e f o r ;-glucose
O x y g e n - s t a b i l i z e d enzyme e l e c -
A c t i v e i m m o b i l i z e d enzyme
Use o f p r o d u c t
723
722
721
716
70 0
698
6 97
Ref.
2
2
2
2.
3
s
&
s
m
c1
o\
w
4
3.2.1.31
1.2.1.12
6-E-Glucuronidase
G l y c e r a l d e h y d e 3-
g i l i s cells
Kluyveromyces
Inulinase
fra-
dehydrogenase
3a-Hydroxysteroid
-
3.2.1.7
1.1.1.50
phosphate dehydrogenase
EC No.
Enzyme
Table 7 continued
or macromolecule coupled
Enbrapment i n a l g i n a t e g e l
A d s o r p t i o n on D E A E - c e l l u l o s e
activated cellulose
R e a c t i o n w i t h cyanogen b r o m i d e -
a c t i v a t e d agarose
R e a c t i o n w i t h cyanogen b r o m i d e -
with alkylamine glass
Glutaraldehyde-mediated r e a c t i o n
membrane
asymmetric a c e t y l c e l l u l o s e
w i t h t h e p o r o u s s i d e o f an
and mode o f c o u p l i n g
Matrix
Ref.
continu-
lactose t o ethanol
Continuous conversion o f
syrup from i n u l i n
ous p r o d u c t i o n o f f r u c t o s e
i m m o b i l i z e d enzyme,
Study o f p r o p e r t i e s o f a c t i v e
3a-hydroxysteroids
Continuous f l o w a n a l y s i s o f
i m m o b i l i z e d enzyme
Study o f p r o p e r t i e s o f a c t i v e
h.p.1.c.
g a t e s p r i o r t o a n a l y s i s by
Analysis o f glucuronide conju-
enzyme
i t y o f active immobilized
618
726
47 6
168
167,
725
S t u d y o f p r o p e r t i e s a n d s t a b i l - 724
ment o f E - g l u c o s e i n human serum o r w h o l e b l o o d
enzyme e l e c t r o d e f o r m e a s u r e -
Use o f p r o d u c t
w 4
3 ~
p5
a
0
i;.
2
zs
R
3
$
??
1.1.1.27
L a c t a t e dehy d r o -
L y s o z yme
Luciferase
3.2.1.17
-
-
L e u c o n o s t o c mesen-
teroides c e l l s
-
La c t o p e r o x i dase
ge na se
EC No.
E nz yme
Table 7 continued
a c t i va t e d a ga r o se
R e a c t i o n w i t h cyanogen bromide-
d i a l y s i s membrane
l i n k i n g w i t h albumin onto a
Glutaraldehyde-me d i a t e d cross-
cellulose f i l t e r
Entrapment i n a g a r on an a c e t y l -
a c t i v a t e d agarose
or with co n ca na V a l in A - a ga r o s e
interaction with a n i o n i c d e t e r ge n t s
Study o f
creatine kinase a c t i v i t y
Microscale analysis of
serum
o f I - p h e n y l a l a n i n e i n human
lactate electrode f o r analysis
Used i n c o n j u n c t i o n w i t h a
a c t i v e immobi 1i z e d enzymes
enzyme immobi 1i z a t i on Comparison o f p r o p e r t i e s o f
for
S t u d y o f new s u p p o r t
p a c k e d i n h.p.1.c. columns R e a c t i o n w i t h cyanogen bromide-
i m m o b i l i z e d enzyme
artichoke tubers Study o f p r o p e r t i e s o f a c t i v e
e t h a n o l from J e r u s a l e m
Repeated b a t c h p r o d u c t i o n o f
Use o f p r o d u c t
A d s o r p t i o n o n PTFE p a r t i c l e s
Adsorption on alumina
a n d mode o f c o u p l i n g
M a t r i x o r macromolecule coupled
171
729
99
170
72 0
727
619
Ref.
$4
23
L
oL1
w
4
-
3.6.1.22
NAD+ pyrophosphatase
Nocardia e r y t h r o p o l i s o r opaca c e l l s
3.2.1.18
-
-
-
E C No.
Neuraminidase
Naringinase
Methanosarcina barkeri c e l l s Mycobacterium p h l e i cells
Enzyme
Table 7 continued
A d s o r p t i o n on DEAE-cellulose o r s i l i c a entrapment i n p o l y acrylamide g e l
Adsorption on p h o s p h o c e l l u l o s e
R e a c t i o n w i t h cyanogen bromidea c t i vated aga r o s e
w i t h alkylamine g l a s s
Adsorption on DEAE-cellulose o r s i l i c a , e n t r a p m e n t i n polyacrylamide g e l G 1 u t a r a 1de h yde -me d i a t ed r e a c t i o n
reaction w i t h gelatin-agarose Entrapment i n a l g i n a t e g e l
d i t h i o )pro p i ona te-me d i a t ed
-N-S u c c i nim i d y 1-3 -( 2- py r i d y 1-
Matrix o r macromolecule coupled and mode o f c o u p l i n g
3
730
6 21
620
410
w 4
0
g.
g5
$
2 g.
E
k
En
Ref. k
Desi a 1 y l a t i on o f a n g i o t e n s i n172 c o n v e r t i n g enzyme D e s i a l y l a t i o n o f human 173 platelets Enzyme r e a c t o r f o r p r o d u c t i o n 4 95 o f n i co t i nam i d e mono nucl eo t i d e L i v i n g immobilized c e l l s f o r 621 steroid transformations
Act i ve i m m o b i 1i z e d e nz ym e
Development of a new method f o r p r e p a r a t i o n o f immunoadso r be n t s Conversion o f methanol t o methane L i v i n g immobilized c e l l s f o r steroid transformations
Use of p r o d u c t
3
R
? 0
philus cells Papain
Pachysolen tanno-
n u c l ea s e
M ic r o co c ca 1 e n d o-
Nuclease P
Enzyme
Table 7 continued
3.4.22.2
-
3.1.31.1
-
EC No.
carbodi-
Ti4+-complexation
c r o s s l i n k i n g w i t h 4-amino-
T e r e p h t h a 1ic d i az i d e -me d i a t e d
a c t i v a t e d agarose
R e a c t i o n w i t h cy ano gen b r om i d e
e t h y l - agarose
Reaction with succinylamino-
Entrapment i n a l g i n a t e g e l
a c t i v a t e d agarose
R e a c t i o n with cyanogen bromide-
r e s i n s y&
Reacti o n with ion-exchange
complex
reaction with Ti4+-cellulose
anion-exchange c e l l u l o s e , o r
imide-mediated r e a c t i o n w i t h
activated cellulose,
R e a c t i o n w i t h cyanogen b r o m i d e -
a n d mode o f c o u p l i n g
M a t r i x o r macromolecule coupled
Study o f p r o p e r t i e s o f a c t i v e i m m o b i l i z e d enzyme
enzyme i n a c o l u m n r e a c t o r
Use o f a c t i v e i m m o b i l i z e d
A c t i v e i m m o b i l i z e d enzyme
- -xylose Q
Production o f ethanol from
i m m o b i l i z e d enzyme
a c i d h y d r o l y s i s by a c t i v e
Study o f k i n e t i c s o f n u c l e i c
A c t J. ve i m m o b i 1iz e d e nz ym e
A c t i v e i m m o b i l i z e d enzyme
Use o f p r o d u c t
467
175
174
622
732
7 31
47 7
Ref.
3
3
2
a-
&
-
4 P 0
Pepsin
enzyme
Penicillium duponti
3.4.23.1
-
3.5.1 .ll E n t r a p m e n t i n g e l a t i n
w i t h 6-aminohexyl-agarose
G 1u t a r a 1de h y de -me d i a t e d r ea c t ion
l i n k i n g onto collagen
G1 u t a r a 1de h yde - m e d i a t e d c r o s s -
e l e c t r o l y t e complexes
t i o n w i t h w a t e r - so 1ub 1e po 1y-
Cyan u r i c c h l o r i de-me d i a t e d r e a c -
mide g r a f t c o p o l y m e r s
t i ve s o f a 1g i na t e- p o l y a c r y 1a-
Reaction w i t h hydrazide deriva-
r e a c t i o n w i t h CM- c e l l u l o s e
methacrylate esters o r
Reaction with r e a c t i v e poly-
activated cellulose
R e a c t i o n w i t h 4 -be n z o q u i no ne
benz y l - c e l l u l o s e
-
o r macromolecule coupled
a n d mode o f c o u p l i n g
P e n i c i l l i n amidase
Matrix
EC No.
Enzyme
Table 7 continued
F(ab’)2
fragments f o r
Digestion o f IgG t o obtain
A c t i v e i m m o b i l i z e d enzyme
A c t i v e i m m o b i l i z e d enzyme
M i c h a e l i s-Me n t e n c o n s t a n t
f o r determining the
V a l i d a t i o n o f m o d i f i e d method
enzyme immo b i l i z a t i o n
S t u d y o f new s u p p o r t f o r
A c t i v e i m m o b i l i z e d enzyme
A c t i v e i m m o b i l i z e d enzyme
Use o f p r o d u c t
176
7 35
734
6 80
733
693
471
Ref.
P
4
0
2.
$
R,
Q
s
$
PP n
l a t a chroma t o pho r e s -
Rhodopseudomonas c a p s u -
cells
Rhizopus n i g r i c a n s
ficans c e l l s
Pseudomonas d e n i t r i -
cells
-
-
-
-
1.11.1.7
Perox i d a s e
Pseudomonas dacunhae
EC No.
__
E nz yme
~~
T a b l e 7 continued
hyde-crosslin king
-
l i n k i n g w i t h albumin and with/
G 1 u t a r a 1de h y de -me d i a t e d c r o s s
agar g e l
Entrapment i n p o l y a c r y lamide o r
Entrapment i n a l g i n a t e g e l
Entrapment i n k-carrageenan g e l
w i t h a l k y l a m i n e g l a s s beads
625
624
6 23
736
7 00
I n v e s t i g a t i o n o f t h e i n f l u e n c e 626 o f immobilization on l i g h t -
p r o ge s t e r o n e
lla-Hydroxylation o f
Living immobilized c e l l s
Living immobilized c e l l s
A c t i v e i m m o b i l i z e d enzyme
f o r measurement o f serum
g- g l u c o se
a f i l m f o l l o w e d by g l u t a r a l d e G 1 u t a r a l d e h y d e -me d i a t e d r e a c t i o n
u t ili z in g c h em i1um ine s ce n c e
I m m o b i l i z e d enzyme s e n s o r
o f enzyme i m m o b i l i z a t i o n
crosslinkable prepolymer i n t o
Copolymerization w i t h a photo-
i n g v i n y l a t i o n o f t h e enzyme
e r e n t p o l y s a c c h a r i d e s f 0 1 1 ow-
512
411
D e v e l o p m e n t o f a new m e t h o d
Graft polymerization t o d i f f -
Ref.
A c t i v e i m m o b i l i z e d enzyme
i n t r a v e n o u s use
Use o f p r o d u c t
Reaction with chitosan
a n d mode o f c o u p l i n g
M a t r i x o r macromolecule coupled
23
n
ii:
2
a-
0
-8
P N
4
visiae cells
Saccharomyces c e r e -
R ibo n uc 1ea se
cells
Rhodotorula minuta
Enzyme
Table 7 continued
-
-
EC No.
organic solvent
gels
Continuous' p r o d u c t i o n o f ethanol
immobilization
c a r r i e r s f o r enzyme
(vinyl alcohol) o r reaction w i t h m e t h a c r y l a t e co p o l ymers Entrapment i n a l g i n a t e g e l
S t u d y of new p o t e n t i a l
Reaction w i t h derivatized poly-
succinate i n
dl-rnenthyl
S t e r e o s e l e c t i ve h y d r o l y s i s o f
a n i m m o b i l i z e d l i v i n g system l i n k e d o r polyurethane r e s i n
Entrapment w i t h i n photocross-
Entrapment i n a l g i n a t e g e l
Electron microscopic study o f
procedures. Production of ATP
o r copolymerization with ur e t h a n e p r epo 1ym e r s
se ve r a l immo b i 1i z a t i o n
tin, entrapment i n
b i o l o g i c a l p h o t o s y s t ems by
C o m p a r a t i v e s t a b i l i z at i o n o f
system
ADP by t h e c h r o m a t o p h o r e
i n d u c e d pho spho r y l a t i o n o f
Use o f p r o d u c t
a l g i n a t e o r k-carrageenan gel,
l i n k i n g with albumin o r gela-
Glutaraldehyde-med i a t e d cross-
w i t h o u t hexokinase present
a n d mode o f c o u p l i n g
M a t r i x o r macromolecule coupled
6306 34
705
629
628
Ref.
4
w P
c;.
2
s
$
L.
8
3
cb
h
?
E n z yme
Table 7 continued
EC No.
gel
f o r use
for whole c e l l immobilization
d i a c e t i c a c i d and e q u i l i b r a t e d
D e v e l o p m e n t o f a new m e t h o d
activity pectate pre-reacted with imino-
Reaction with epoxy-activated
550
640
L i v i n g immobilized c e l l s with
Entrapment i n g e l
f? -p-fr uc t o f u r a n o si da se -
6 38 639
P r o duc t i o n o f g l u t a t h i o n e
637
6 36
635
641
Ref.
L i v i n g immobilized c e l l s
ethanol
Continuous p r o d u c t i o n o f
ethanol
Continuous p r o d u c t i o n o f
e t h a no 1
Continuous p r o d u c t i o n o f
i n c e l l immobilization
c o n t a i n i n g media,
s t a b l e i n phosphate-
preparing alginate gels
Development o f methods f o r
Use o f p r o d u c t
Entrapment i n s i l i c a h y d r o g e l
Entrapment i n polyacrylamide g e l
Entrapment i n k-carrageenan
A d s o r p t i o n o n ion-exchange r e s i n s
a n d mode o f c o u p l i n g
M a t r i x o r macromolecule coupled
s
s.
z
3 (7
2
R
k
&
4 P P
-
f a c i e n s enzyme e x t r a c t
-
-
faciens c e l l s Streptomyces aureo-
loti
-
Streptomyces aureo-
Stemphylium cells
Staphylococcus aureus cells
S o r b i t o l dehydroge na se
1.1.1.14
-
Solanum a v i c u l a r e
cells
EC No.
E nz yme
Table 7 continued
1
Development o f a method o f enzyme i m m o b i 1i z a t i on
continuous production o f s t e r o i d g l y c o a l k a l o i ds
NA DP L i v i n g immobilized c e l l s f o r
P r o d u c t i o n o f g l u t a t h i o n e and
Use o f p r o d u c t
6 96
605 642
Ref.
13 d i hy d r o da un om y c i no n e
-
1 3 - d i h y d r o daunom y c i no n e C o n v e r s i o n o f daunomycinone t o
-
Co nve r s i o n o f da unom y c i none t o
-
a gelatin matrix Rea c t i o n w i t h g l u t a r a l d e h y d e a c t iv a t e d bead c e l l u l o s e
Cy a n i de de g r ada t i o n
G 1 u t a r a l de h yde c r o s s 1 i n k i n g o n t o
Treatment w i t h f l o c c u l a t i n g agents
w i t h Fe3+ions
d i a c e t i c a c i d and e q u i l i b r a t e d
645
645
644
Reaction with epoxy-activated D e v e l o p m e n t o f a new m e t h o d 550 pectate pre-reacted w i t h iminof o r whole c e l l i m m o b i l i z a t i o n
Reaction w i t h a c y l azidea c t i v a t e d c o l l a g e n membranes
w i t h poly(pheny1ene o x i d e
Glutaraldehyde-mediated r e a c t i o n
w i t h Fe3+ i o n s Entrapment i n p o l y a c r y l a m i d e g e l
a n d mode o f c o u p l i n g
M a t r i x o r macromolecule coupled
4 v1 P
z.
%
B
9
a
5.
?J
Thylakoids (lettuce)
Thermolysin
chromogenes c e l l s
Streptomyces roseo-
chromogenes c e l l s
Streptomyces phaeo-
cells
Streptomyces f r a d i a e
gerus c e l l s
Streptomyces c l a v u l i -
Enzyme
T a b l e 7 continued
~
-
3.4.24.4
-
-
-
-
EC No.
~~
G1 u t a r a l d e h yde-me d i a t e d c r o s s -
linkable resin
Entrapment i n a photocross-
c h it o s an
Reaction with aggregating
A d s o r p t i o n o n t o diatomaceous earth
Entrapment i n polyacrylamide g e l
1inea r w a t e r- so 1u b l e po 1yacrylamide gels
Cro s s l i n k i n g o n t o p r e - f o rmed
a n d mode o f c o u p l i n g
M a t r i x o r macromolecule coupled
737 651
precursor Study o f p r o p e r t i e s and
650
649
648
647
6 46
Ref.
S y n t h e s i s o f an a s p a r t a m i n e
16a -hy d r ox y l a t i on o f deh y d r oe p i a ndro s t e r o ne
L i v i n g immobilized c e l l s for
u s e i n a n enzyme r e a c t o r
L i v i n g immobilized c e l l s for
Q - -glucose isomerase a c t i v i t y
L i v i n g immobilized c e l l s w i t h
protease production
L i v i n g immobilized c e l l s for
a n t i b i o t i c production
immobilized c e l l s f o r
m i c r o b i a l c e l l s and l i v i n g
method f o r i m m o b i l i z a t i o n o f
D e v e l o p m e n t o f a m i l d new
Use o f p r o d u c t
d
y
35.
P
2
-2
Q.
w s
rr
m
P
4
EC No.
3.4.21.4
Enzyme
Tryp s i n
T a b l e 7 continued
o r macromolecule coupled
agarose
R e a c t i o n w i t h 6-aminohexyl
a c t i v a t e d aga r o s e
R e a c t i o n w i t h cyanogen b r o m i d e -
e r i z a t i o n with urethane polymers
copolyrn-
entrapment i n a l g i n a t e
o r i-carrageenan gels,
tin,
l i n k i n g w i t h albumin o r gela-
a n d mode o f c o u p l i n g
Matrix
179 180
t r y p s i n and a p r o t e a s e o n t h e
p r o pe r t i es o f i m m o b i 1iz e d
Comparative study o f i n h i b i t i o n 181
to trypsin
i n g o f complement p r o t e i n C3
i s t i c s o f the covalent bind-
the character-
I n v e s t i g a t i o n of
178
177
Ref.
P u r i f i c a t i o n o f duck ovornucoid
oliviae
embryos o f H e m i l e u c a
i n h i b i t o r s from d e v e l o p i n g
P u r i f i c a t i o n o f protease
i n h i b i t o r s from Acacia e l a t a
P u r i f i c a t i o n o f proteinase
ph o t o s y s t em
stability of biological
Use o f p r o d u c t
4 P 4
9
:g.:
35
4
r2
EL
3E'
n
PP
Enzyme
Table 7 continued
E C No.
Reaction w i t h periodate-activa t ed ce 11u l ose be a d s Reaction w i t h thiosulphate d e r i v a t i v e s of c e l l u l o s e R e a c t i o n w i t h cyanogen bromideactived agarose o r crosslinked dextran Reaction w i t h d e r i v a t i z e d poly( v i n y l alcohol) o r reaction w i t h met hacry 1a t e co po 1ymer s Reaction w i t h hydrazide derivat i ves o f a l g i n a t e - p o l y a c r y 1-
Reaction with agarose mediated by e t h y l e n e g l y c o l y l &( s u c c i n i m i d y l s u c c i na t e )
and mode o f c o u p l i n g
Matrix o r macromolecule coupled
&
Active immobilized enzyme
f o r enzyme i m m o b i l i z a t i o n 733
3
P
t;.
k
ef!
2
S &
536
497
493
Study of new p o t e n t i a l c a r r i e r s 705
409
Ref.
s u r f a c e of E h r l i c h a s c i t e s tumour c e l l s Demonstration of p o t e n t i a l o f a new c l e a v a b l e r e a g e n t f o r c r o s s l i n k i n g and r e v e r s i b l e immobilization of proteins Study o f new method f o r enzyme immobilization Development o f a new method o f enzyme i m m o b i l i z a t i o n Investigation o f the influence of e f f e c t o r s on t h e r e f o l d i n g o f immobilized t r y p s i n
Use of p r o d u c t
03
4 P
E C No.
1.14.18.1
3.5.1.5.
E n t yme
T y r o s i na s e
Urease
T a b l e 7 continued
Ref.
Active immobilized enzyme and s t u d y o f s u b u n i t i n t e r a c t i o n s 18* of t y r o s i n a s e 738 A c t i v e immobilized enzyme and s t u d y of e f f e c t s o f s u b s t r a t e flow r a t e o n immobilizedurease assays Study of use of p o l y s a c c h a r i d e 539 derivatives as coats for nylon tube-immobilized u r e a s e
Use of p r o d u c t
Glutaraldehyde-mediated r e a c t i o n w i t h 2 - a l k y l a t e d nylon t u b e s c o a t e d w i t h polyaminated d e r i v a t i v e s of s t a r c h , dextran, d i a l d e h y d e dextran, o r d i a l d e h y d e starch Reaction w i t h d e r i v a t i z e d polyStudy of new p o t e n t i a l c a r r i e r s 705 f o r enzyme i m m o b i l i z a t i o n (vinyl alcohol) o r reaction w i t h methacry l a t e copolymers Entrapment i n p o l y a c r y l a m i d e g e l Study of performance o f 739
Glutaraldehyde-mediated r e a c t i o n w i t h 2 - a l k y l a t e d nylon t u b e s
amide g r a f t copolymers R e a c t i o n w i t h cyanogen bromidea c t i va t e d a garo se
and mode o f c o u p l i n g
Matrix o r macromolecule coupled
\D
P
~
pE.
5
2b. 9A
3
r?.
*
E'
B3
?
Table 7 continued
EC No.
1.7.3.3
3.4.21.31
Enzyme
Uricase
U r o k i na se
~~
i n human serum
i n n e r w a l l o f g l a s s t u b e s used
Reaction w i t h collagen-synthetic polymer composite m a t e r i a l
by o x i d a t i o n w i t h Br2
Reaction w i t h agarose a c t i v a t e d
analysers
Study o f i n v i t r o and i n v i v o behaviour o f t h e a c t i v e
protein immobilization
D e m o n s t r a t i o n o f new m e t h o d o f
determination o f u r i c acid
as r e a c t o r s i n continuous-flow
A c t i v e i m m o b i l i z e d enzyme a n d
with s i l i c a f i l a m e n t s on t h e
urate
A u t o m a t e d a n a l y s i s o f serum
i m m o b i l i z e d enzyme
K i n e t i c study of the a c t i v e
u t i1i z ing c h em i1um ine s ce n c e
Immobilited-enzyme sensor
magnetic f i e l d s
enzyme r e a c t o r s u t i l i z i n g
f l u i d i z e d -bed immo b i l i z e d-
Use o f p r o d u c t
G 1 u t a r a l d e h y de-me d i a t e d r e a c t i o n
with n y l o n tube surfaces
G1 u t a r a l d e hyde-me d i a t e d r e a c t i o n
w i t h c e l l u l ose beads
Glutaraldehyde-mediated r e a c t i o n
i n the presence o f magnetite
a n d mode o f c o u p l i n g
M a t r i x o r macromolecule coupled
743
147
7 42
7 41
7 40
70 0
Ref.
a
$
.5
!+
k
s
2
$
0
(n
4
Zymomonas m o b i l i s cells
-
-
1.2.3.2
Xanthine oxidase
Yeast c e l l s
EC No.
E n z yme
Table 7 continued
Entrapment i n alginate g e l
h y d r o x y e t h y 1a c r y 1a t e g e 1
Copolymerization with a photoc r o s s l i n k a b l e prepolymer i n t o a f i l m , f o l l o w e d by g l u t a r a l de h y de c r o s s l i n k i n g Entrapment i n polyethyleneglycol
a n d mode o f c o u p l i n g
Matrix o r macromolecule coupled
Ref.
efficient production o f ethanol i n a bioreactor
immobilized enzyme Immobilized-enzyme s e n s o r 700 u t i 1i z i n g c h e m i l um i n e s c e n c e f o r measurement of hypoxanthine i n tuna muscle S t u d y o f p r o p e r t i e s of a n i m 652 m o b i 1i z e d g l y c o 1y s i s s y s t em o f y e a s t i n f e r m e n t a t i v e phosphorylation o f nucleotides Living immobilized cells f o r 653
Use of p r o d u c t
g
3
&
2g.
P
2 2
9 s
Z'
3
n
5?
752
Carbohydrate Chemistry
e n z y m e o n t o b r u s h i t e ( C a H P 0 4 . 2 H 2 0 ) h a s b e e n r e p o r t e d t o be a d v a n t a g e o u s a s i m m o b i l i z a t i o n can be p e r f o r m e d i n a l m o s t any b u f f e r even a t h i g h s a l t c o n c e n t r a t i o n s , p r o v i d e d h i g h c o n c e n t r a t i o n s o f phosphate a r e avoided.712 s u p p o r t was o b s e r v e d ,
Very l i t t l e leakage o f enzyme from t h e
a n d t h e enzyme c o u l d be d e s o r b e d by i n c r e a s i n g
the c o n c e n t r a t i o n o f phosphate ions. Glucoamylase h a s been i m m o b i l i z e d o n a l k y l a m i n e
g l a s s by
g l u t a r a l d e h y d e - m e d i a t e d r e a c t i o n , 6 7 7 o r by t h e m e t a l - l i n k method.71* I n t h e l a t t e r s t u d y t r e a t m e n t o f t h e i m m o b i l i z e d e n z y m e w i t h 1,6diaminohexane products.
or
glutaraldehyde
was
found
to
yield
superior
The i n f l u e n c e o f c o u p l i n g c o n d i t i o n s o n a c t i v i t y a n d
o p e r a t i o n a l s t a b i l i t y o f glucoamylase i m m o b i l i z e d on Ti4+-activated c o n t r o l l e d p o r e g l a s s h a s been discussed.717 t o be s t a b i l i z e d v e r y e f f e c t i v e l y
reagents such as g l u t a r a l d e h y d e and/or stabilities increased gels.716
of
glucoamylase
several-fold
by
Bound enzyme was f o u n d
by c r o s s l i n k i n g w i t h b i f u n c t i o n a l
and
their
tannic acid.
e-glucose entrapment
The t h e r m o -
oxidase i n
have
been
polyacrylamide
I n p o l y a c r y l a t e g e l s t h e enzymes behaved d i f f e r e n t l y ,
probably owing t o microenvironmental e f f e c t s a r i s i n g from the polyelectrolyte nature of the carrier:
the thermo-stability
of
gluco-
a m y l a s e f r o m one s o u r c e (Thermomyces l a n u g i n o s u s ) i n c r e a s e d 7 - f o l d , w h i l e t h a t f r o m R h i z o p u s sp. d e c r e a s e d c o n s i d e r a b l y .
The t h e r m o -
s t a b i l i t y o f Q-glucose oxidase from A s p e r q i l l u s n i g e r increased s l i g h t l y o n i m m o b i l i z a t i o n i n p o l y a c r y l a t e gel.
Lower t h e r m o s t a b i l -
i t y was a l s o o b s e r v e d a f t e r i m m o b i l i z i n g g l u c o a m y l a s e by a d s o r p t i o n o n t o a copolymer o f 2-dimethylaminoethyl m e t h a c r y l a t e and m e t h y l m e t h a c r y l a t e . '15 Com m e r c i a 11y ava i1a b l e i m m o b i 1i z e d i - g l uco se isom e r a s e h a s bee n used i n experiments t o v a l i d a t e a t h e o r e t i c a l l y
d e r i v e d optimum
temperature c o n t r o l p o l i c y f o r an i m m o b i l i z e d glucose isomerase r e a c t o r system.749
The t h e o r e t i c a l t r e a t m e n t a l l o w e d f o r enzyme
de a c t iva t i o n d u r in g co n t inuo us r e a c t o r o pe r a t i on. Rates o f oxygen a b s o r p t i o n i n t o g - g l u c o s e
s o l u t i o n s have
been
m e a s u r e d u s i n g Q - g l u c o s e o x i d a s e i m m o b i l i z e d by e n t r a p m e n t w i t h m a g n e t i t e i n p o l y a c r y l a m i d e gel.750
An i m m o b i l i z e d enzyme r e a c t o r
w a s e m p l o y e d i n w h i c h t h e e n z y m e b e a d s w e r e m o v e d by a r e v o l v i n g magnetic f i e l d t o reduce the mass-transfer l i q u i d i n t e r f a c e a n d a r o u n d t h e bead.
r e s i s t a n c e s a t t h e gas-
A comparison o f d i f f e r e n t
m e t h o d s o f P - g l u c o s e o x i d a s e i m m o b i l i z a t i o n has been p u b l i s h e d . 7 5 1 Immobilized 0-glucose
oxidase
e l e c t r o d e f o r measurement of
has
been u s e d i n a n enzyme
Q - g l u c o s e i n human serum o r w h o l e
753
8: Chemical Synthesis and Modification
The p r e p a r a t i o n o f a s t r e s s - s e n s i t i v e n - g l u c o s e o x i d a s e blood.723 n y l o n membrane has been reported.721 The e n z y m e , i m m o b i l i z e d o n glutaraldehyde-treated conditions,
nylon-66,
had no
activity
under
normal
b u t o n l y when t h e membrane was m e c h a n i c a l l y s t r e t c h e d .
A l i n e a r r e l a t i o n s h i p between s t r e s s s t r e n g t h and g-glucose oxidase
a c t i v i t y was found.
Mechanical c o n t r o l of t h e a c t i v i t y o f g-glucose
o x i d a s e i m m o b i l i z e d o n p o r o u s p o l y v i n y l c h l o r i d e membranes h a s a l s o been r e p o r t e d . 7 2 2
Anomalous b i n d i n g k i n e t i c s have been r e p o r t e d f o r
a h i g h - y i e l d i m m o b i 1i z e d - e n z ym e sy s t em em p l oy i n g Q - g 1 uco se ox i d a se a d s o r b e d o n n o n - p o r o u s p o l y e t h y l e n e i m i n e - c o a t e d g l a s s m i c r o beads.697 The p r e s e n c e o f
several low-molecular-weight
a p p a r e n t l y competed w i t h g - g l u c o s e
i m p u r i t i e s which
o x i d a s e f o r b i n d i n g was f o u n d t o
be r e s p o n s i b l e f o r t h e a t y p i c a l a d s o r p t i o n c u r v e s o b t a i n e d f o r t h e system.
When l a r g e e x c e s s e s o f p r o t e i n w e r e a d d e d t o t h e beads t h e
b i n d i n g o f i m p u r i t i e s became s i g n i f i c a n t and t h e amount o f enzyme a c t i v i t y p e r u n i t o f bead was reduced,
explaining the adsorption
i s o t h e r m s observed. B-Q-Glucosidase
adsorbed o n c o n t r o l l e d p o r e a l u m i n a has been
used i n c o n j u n c t i o n w i t h s o l u b l e Trichoderma r e e s e i c e l l u l a s e t o hydrolyse c e l l u l o s i c materials.752
I n c r e a s e d y i e l d s o f P-glucose
and increased conversion o f c e l l o b i o s e t o Q-glucose were observed when s u p p l e m e n t a l i m m o b i l i z e d 6 - 8 - g l u c o s i d a s e
was u s e d i n f i x e d bed,
f l u i d i z e d bed, a n d h y d r o l y s i s r e a c t o r s . I n u l i n a s e a d s o r b e d o n DEAE-cellulose h a s been a p p l i e d t o t h e c o n t i n u o u s p r o d u c t i o n of f r u c t o s e
syrup from i n u l i n ,
and p r o p e r t i e s
o f t h e i m m o b i l i z e d enzyme h a v e been s t u d i e d . 7 2 6 I m m o b i l i z e d n e u r a m i n i d a s e h a s been a p p l i e d t o t h e d e s i a l y l a t i o n o f a n g i o t e n s i n - c o n v e r t i n g enzyme t o d e t e r m i n e t h e e f f e c t s o f t h i s t r e a t m e n t o n l e c t i n binding,172
a n d t o t h e d e s i a l y l a t i o n o f human
p l a t e l e t s f o r studies o f the accumulation o f 5-hydroxytryptamine t h e p l a t e l e t s .173 (References begin o v e r l e a f )
by
754
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1981, 2, 86. 69 2 V. Ramesh and C. Singh, Eslzyme Microb. Technol., 1981, 2, 246. 693 J. KuEera and M. Kminkov3, (311. Czech. Chm. Cunnun., 1980, 5, 298. 69 4 P. O'Grady and P.Joyce, Enzyme Microb. Technol., 1981, 2, 149. 69 5 P. pcsbdka and L. h c h o l b l a n , all. Czech. Chm. Cunnun., 1979, 44, 3395. 69 6 P.R. Coulet and D.C. Gautheron, J.Chramatoqr., 1981, 215, 65. 697 B.P. Wasserman, B.S. Jacobson, and H.O. H u l t i n , Biochim. Biophys. A S , 1981, 657,52. 69 8 S.O. Ehfors, Ehzyme Microb. Technol., 1981, 3, 29. 699 W.G. Choi, S.B. Lee, and D.D.Y. Ryu, Biotechnol. Bioenq., 1981, 23, 361. 700 T. Kobayashi, K. Saga, S. Shimizu, and T. Goto, Aqric. B i o l . Chem. ( J p n ) , 1981, 45, 1403. 70 1 P.A. Munro, P. Dunnill, and M.D. L i l l y , Biotechnol. Bioenq., 1981, 2, 677. 702 K. Taniguchi, J. Ito, a n d M . Sasaki, J. Biochen. ( J p n ) , 1981, 89, 179. 703 M. Sasaki, K. Taniguchi, and K. Minakata, J. Biochm. (Jpn)., 1981, 89, 169. 704 M.H. R a y , D. Guillochon, and D. Thomas, J. Chranatoqr., 1981, 215, 87 70 5 G. Manecke and D. P o l a k m k i , J. C h r m t o q r . , 1981, 215, 13. 706 T. K i t a o , Y. K i t a o , and K. Hattori, Ekperientia, 1981, 37, 452. 707 P. Monsan and A. Lapez, Biotechnol. Bioenq., 1981, 2, 2027. 708 S.F. D'Souza and G.B. Nadkarni, Biotechnol. Bioenq., 1981, 23, 431. 709 J. Hradil and F. Svec, Eslzyme Microb. Technol., 1981, 2, 331. 710 L. Iyengar and A.V.S. Prabhakara Rao, J. Gen. Appl. Microbiol., 1981, 27, 339. 711 F.N. Onyezili and A.C. O n i t i r i , Anal. Biochan., 1981, 113,203. r., 1981, 215, 25. 712 S. HjertrSn, Y. Kunquan, and M. Ogunlesi, J. Chrcma 713 Y.K. Park and G.M. Pastore, J. Ferment. Technol., 2 1 , 2, 1 6 5 7 714 L. Rexovd-Benkovd and M. MraEkovb-Dobrotovzl, Carbohydr. Res., 1981, 9, 115. S r i n i v a s a n , K.T. Joseph, and M. Santappa, 715 V.S. Nithianandam, K.S.V. Biotechnol. Bioenq., 1981, 23, 2273. 716 V. Basaveswara R a o , N.V.S. S a s t r i , and P.V. Subba Rao, Biochem. J., 1981, 389. -193, . 717 J.M.S. Cabral, J.P. Cardoso, and J.M. Novais, IQIzyme Microb. !I!echnol., 1981, 3, 41. 718 J.M.S. C a b r a l , J.M. Novais, and J.P. Cardoso, Biotechnol. Bioenq., 1981, 23, 2083. 719 E. B i s & , A. S c h o l e r , and D.J. Von Der S c h m i t t , J. A p p l . Biochem., 1981, 3, 176 720 S . Bachman, L. Gebicka, and 2. Gasyna, S t a r c h , 1981, 33, 63. 721 Y. I s h i m o r i , I. Karube, and S. Suzuki, Eur. J. App. Microbiol. Biotechnol., 1981, 2, 19. 72 2 Y. Ishimori, I. Karube, and S. Suzuki, Biotechnol. Bioenq., 1981, 23, 2601. 1981, 3, 326. 723 T. Tsuchida and K. Yoda, Enzyme Microb. Technol 724 L.D. Bawers and P.R. JOhnson, Biochim. Biophys. A c t a , 1981, 661, 100. Biochan., 1981, 116, 111. 725 L.D. mers ard P.R. Johnson, -1. 726 J.P. Guiraud, S. Demeulle, and P. Galzy, Biotechnol. L e t t . , 1981, 3, 683. 727 G.A. Kovalenko, N.B. S h i t o v a , and V.D. S o k o l o v s k i i , Biotechnol. Bioenq., 1981, 23, 1721. 1981, 23, 1913. 728 N.D. Danielson and RW. S i e r g i e j , Biotechnol. Bioen 729 0. Rodriguez and G.G. Guilbault, Enzyme Microb. Anol.,1981, 3, 69. N.H. Park and H.N. Chang, J. Ferment. "echnol., 1979, 57, 310. 730 731 K. Rokugawa, T. F u j i s h i m a , A. Kuninaka, and H. Yoshino, J. Ferment. Technol., 1980, 58, 423. 732 J.M. G u i s a and A. Bdllesteros, Enzyme Microb Technol., 1981, 3, 313. Kumaraswamy, K. Panduranga Rao, K.T. Joseph, and M. Santappa, 733 M.D.K. 1981, 23, 1889. BiOtechnOl. B i m 734 A.L. Margolin, V%. Izumruiov, V.K. Svedas, A.B. Zezin, V.A. Kabanov, and I.V. Berezh, Biochim. Biophys. A c t a , 1981, 660, 359. 735 B. Adu-Amankwa, A. C o n s t a n t i n i d e s , and W.R. Vieth, Biotechnol. Bioenq., 1981, 23, 2609. B i o t e d m o l . Bioenq., 1981, 23, 2161. 736 J. m-zewski,
._
.,
.,
.,
8: Chemical Synthesis and Modification
77 1
737 K. Oyama, S. N i s h i m u r a , Y. Nonaka, K. K i h a r a , a n d T. H a s h i m o t o , J. Org. am., 1981, 46, 5241. 738 F.N. Onyezili and A.C. onitiri, 1. Bioenq., 1981, 23, 739 E. Sada, S. Katoh, M. Shiozawa, 2561. 740 A.B. S d l e h and W.M. Ldingham, Int. J. Biochm., 1981, 13,1113. 741 A.B. Salleh and W.M. Mingham, Anal. Biochm., 1981, 116,40. 742 A. lob and H.A. Mottola, Clin Ct~m.,1981, 27, 195. 743 S. Watanabe, Y. Shimizu, T. Teramatsu, and T. Murachi, J. Biomed. Mater. Res., 1981, 15, 553. 744 E.Merer and K.F. O'Drismll, Biotechnol. Bioenq., 1981, 23, 2447. 745 D.G. Merm and K.F. O ' D r i s c o l l , Biotechnol. Bioenq., 1981, 23, 2465. 746 C.F. Mandenius, B. Danielsson, and B. M a t t i a s s o n , A c t a Chem. Scand., 1980, 34B, 463. 747 C H r a d i l and F. Svec, Enzyme Microb. Techno1 1981, 2, 336. 748 N.A. Greenberg and R.R. Mahoney, Process Biochan., 1981, 16, 2. 749 S.H. Park, S.B. Lee, and D.D.Y. Ryu, Biotechnol. Bioenq., 1981, 23, 1237. 750 E. Sack, S. Katoh, and M. Terashirra, Biotechnol. Bioeng., 1981, 23, 1037. 751 0. Valentovd, M. Marek, F. Svec, J. Stamberg, and Z. Vodrddka, Biotechnol. Bioenq., 1981, 23, 2093. K l e i , R.W. Coughlin, G.J. Biederman, and C.A. Brouwer, 752 D.W. Sundstrom,H.E. Biotechnol. Bioenq., 1981, 23, 473.
.,
Author Index I n t h i s i n d e x t h e number g i v e n i n p a r e n t h e s e s i s t h e c h a p t e r number, which i s f o l l o w e d by t h e r e l e v a n t r e f e r e n c e n u m b e r ( s ) w i t h i n t h a t c h a p t e r .
Aaberg, A . , ( 8 ) 534 Aach, H.G., (3) 206 Aalmo, K.M., ( 2 ) 109 Aaronson, W . , (4) 95 Aarsman, M.E.G., (5) 9 34 A a s t r u p , S . , ( 3 ) 153 Abadie, B., (6) 293 Abbas, S.A., ( 8 ) 29, 40 Abdel Bari, S., ( 8 ) 489 Abdel-Hay, F . I . , (581) A b d e l l a , P.M., ( 8 ) 409 Abe. A . , ( 6 ) 7 4 Abe, J . , (6) 388, 389 Abe. S., ( 8 ) 249 Abe. Y . , (5) 148; ( 8 ) 533 Abedin, M . Z . , ( 5 ) 263 A b e l s , J . , ( 8 ) 318 Abou-Zeid, N.Y., ( 8 ) 483 Abraham, E.C., ( 5 ) 805 A c c o l l a , R.S., ( 6 ) 152 Ackerman, C . A . , ( 7 ) 33, 39 Ackerman, J . J.H I ( 3 ) 18 A d a c h i , O . , ( 6 ) 5 35 Adachi, S . , (5) 718, 719: ( 8 ) 648 Adamany, A.M., 5 ) 285, 918; ( 6 ) 502 Adams, C . A . , ( 3 66 Adams, M.E., ( 5 ) 319 Adams. M.J., (4) 234; ( 5 ) 880 Adams-Stemler. P., ( 5 ) 144 Addeo, F., ( 8 ) 416 Addis. J . , (5) 595 A d e l s t e i n , R.S., ( 8 ) 227 Ademola, J . I . , ( 8 ) 148 Adiga, P.R., ( 8 ) 217 A d k i n s , W.N., ( 5 ) 979 Adu-Amankwa, B . , ( 8 ) 735
A f f r o n t i , L.F., ( 5 ) 685 Afonso, A.M.M., (5) 967 A f t i n g . E.G., ( 8 ) 232 A g g e l e r , J . , (5) 540; ( 8 ) 233 Agneray, J.C.L., ( 5 ) 522, 603; ( 7 ) 34 Agrawal, H . C . , ( 5 ) 463 A g u i l a r , J . , ( 4 ) 182 Agwel, J . P . L . , ( 3 ) 47 Aharonowitz, Y . , ( 8 ) 646 Ahmad. Z . I . , ( 4 ) 143 Ahmed, M . , ( 3 ) 246 A i d a , K . , ( 5 ) 206, 207, 903; (6) 373; ( 8 ) 356, 594 A i r b a r a , S., ( 6 ) 325 A i t k e n , A . , (6) 528 Akagawa, K.S., ( 7 ) 164 A k a i , Y . , (5) 102; ( 8 ) 393 Akamatsu, N., ( 5 ) 1046 Akazawa, T., (3) 60 Akedo, H . , ( 6 ) 270 Akiyama, F . , ( 5 ) 344 Akiyama, Y . , ( 3 ) 144 Alais, C., (5) 717 A l a i s , J . , ( 8 ) 19, 48, 50 Al-Ani, A.T.M., ( 8 ) 588 A l a z a r d , D., ( 6 ) 402 A l b a n e s i , D . , ( 8 ) 685 A l b e r , D . , (6) 25 A l b e r s h e i m , P., ( 3 ) 160, 161, 191. 223, 232, 233; ( 4 ) 152; (8) 305 A l b e r t , A . , ( 5 ) 824 A l b e r t o , B.P. ( 4 ) 140; ( 5 ) 855; ( 8 ) 532 A l b r e c h t , G.J., ( 8 ) 163 A l d e n , J.R., ( 4 ) 143 A l e x a n d e r , R.J. (3) 49 Aleksandrovkii, Ya.A., (6) 489 A l f a n i , F . , ( 8 ) 685
A l f o l d i , J . , ( 8 ) 35 Al-Habib. A . , ( 8 ) 181 A l h a d e f f , J.A., (6) 100, 107. 251 Al-Hakeim, M . H . H . , (8) 588 A l i , I.U.. ( 5 ) 217 A l i t o l o , K . , ( 5 ) 284 A l l a i n , J.C., ( 5 ) 272 A l l c o c k , E . R . , (6) 225 A l l e n , A . , ( 5 ) 947, 950. 951, 952, 955, 956; ( 6 ) 219 Allen, A.K., ( 5 ) 152, 175, 451 A l l e n , H.J., ( 5 ) 590 A l l e r h a n d , A , , (2) 90; ( 5 ) 479, 986; ( 6 ) 243 A l l u e , L.A.Q., (5) 1038, 1049 Almeida. A.P., ( 5 ) 349 Alonso-Fernandez, J.R., (2) 24 A l p e n f e l s , W.F., ( 2 ) 39; (5) 1023 A l p e r s , D.H., ( 8 ) 199 Altamirano, G.A., (8) 196 A l v e r e z , V.L., ( 5 ) 612 Amado, R . , (3) 33, 147, 182 Aman, P., ( 4 ) 152 Amano, E . , (3) 61 Amano, F . , ( 5 ) 636 Amano, T.. (6) 429; ( 8 ) 662 Ambesi-Impiombato, F . S . , (5) 468 Amemura, A . , ( 4 ) 151, 153, 154; ( 6 ) 388, 389 Ameyama, M., ( 6 ) 535 Amin, E . , ( 6 ) 291 Aminoff. D., ( 5 ) 937, 1022; ( 6 ) 109 Amontov, S.V., ( 8 ) 160 Amrani, D . , ( 5 ) 220
Author Index A m r i , M . A . , ( 4 ) 231 Anai, M . , (8) 479 Ananichev, A.V., ( 6 ) 475 A n a s t a s s i a d e s , T., ( 2 ) 1; (5) 1026 A n d e r s e n , A.J.. ( 5 ) 95 Andersen, T.T., (5) 564 Anderson, A . J . , (3) 167; (4) 8, 80; ( 5 ) 118 Anderson, B.M., ( 5 ) 544: (8) 440 Anderson, D. J . , ( 5 ) 671 Anderson, G . N . , ( 5 ) 1068 Anderson, J . S . , ( 4 ) 1 1 Anderson, L . , ( 8 ) 7, 9, 74 Anderson, P.M.. ( 8 ) 126 Anderson, W.F., (6) 433 A n d e r s s o n , L.C., ( 5 ) 921 A n d e r s s o n , L.O., ( 5 ) 771, 774; (8) 91 A n d e r t o n , B.H., ( 5 ) 1008 Ando, S., ( 5 ) 978; ( 7 ) 72 Andre, A . , ( 5 ) 987 A n d r e o t t i , R.E., (6) 351 Andrews, J.M., ( 8 ) 568 Andrews, S.P.. (5) 1070 Andrews-Smith, F.L., (6) 107 A n i l i o n i s , A . , ( 5 ) 75 A n j a n e y a l u , Y . V . , (3) 190 Andrade, A.F.B., ( 5 ) 200 A n s a r i , A . A . , ( 8 ) 215 A n s a r i , S . , (6) 78 A n s a r t , J . F . , ( 6 ) 171 Ansbacher, R., (5) 375; ( 8 ) 262 A n s e l , S . , ( 7 ) 125 A n s t e e , D.J., (5) 907 A n t a i , S.P., ( 6 ) 367 A n t o n i , G., (2) 60 Antonijevic, A , , (8) 689 Antony, A.C., ( 5 ) 490; ( 8 ) 343 Anumula, K.R., ( 5 ) 1065; ( 8 ) 282 Anwar. W . , ( 8 ) 483 Aoki, S . , (6) 478; ( 8 ) 659 Aoyama, Y . , ( 8 ) 221 A p a k i , Y., (6) 263 Apitz-Castro, R . , (5)
773 655 A p l i n , J.D.. ( 8 ) 64 A p r i l e t t i , J.W., ( 8 ) 344 A p o r t i , F.. ( 7 ) 58 A p p a j i Rao, N . , ( 8 ) 303 A p p u k a t t a n , P.S., ( 5 ) 110; (8) 304 Arai, F., (2) 68 Arai, M . , ( 4 ) 244, 245; ( 6 ) 245, 297, 328 A r a i , Y . , (3) 61, 62; ( 8 ) 510 A r a k i , S., (4) 28 A r a k i , Y . , ( 4 ) 28, 232 Aranyi. P . , (5) 759 Arao, Y . , ( 7 ) 79 Arashima, S., (6) 28 A r c h e r , I . M . , ( 6 ) 511 Archibald, A.R., (4) 9 A r g r a v e s , W.S., ( 5 ) 447 A r i a s , M.E., ( 6 ) 157 A r i g a , T.. ( 5 ) 978; ( 7 ) 36 Armant, D.R., ( 8 ) 131 Armstrong, G . D . , (4) 179 Armtage, I . M . , ( 5 ) 913 A r n a r p , J . , (8) 61, 63 A r n d t , R . , ( 7 ) 60 Arnold, S.L., ( 6 ) 214 A r o r a , D.J.S., ( 6 ) 257 Arrambide, E., (6) 507 A r r a n g , J.M., ( 5 ) 691 A r t h u r , R . , (3) 149 Arumugham, S., ( 6 ) 30 A r z b e r g e r . A., (6) 22 Asakawa, M . , ( 6 ) 261: (8) 248 Asamizu, T., ( 4 ) 187 Asano, T . , (4) 108 A s c h a u e r , H . , ( 6 ) 278 A s e n j o , J.A., (8) 540 A s h f o r d , D., (5) 175 Ashmarina, L. I . , ( 8 ) 167 A s h r a f , J . , ( 2 ) 100 A s h r a f , M . , (3) 246 A s h w e l l , G., ( 5 ) 192 A s h w e l l , G.G., (5) 560 A s l a n , A . , ( 8 ) 467 A s p i n a l l , G.O., (4) 38, 39; ( 7 ) 199 Atha, D . H . , (5) 757 A t k i n s , E.D.T., (3) 264, 283 A t k i n s o n , D., ( 7 ) 81 A t k i n s o n , P.H., (5) 55, 982, 983 Atrat, P., (8) 621 A t t a r d i , D.G., ( 8 ) 269 A t t a r d i . G., (8) 335
A u b e r t , J . P . , ( 5 ) 1039 A u f f r e t , C . A . , (5) 997; ( 8 ) 234 A u g u s t , J.T., ( 5 ) 638, 646, 879 Auino. D., ( 5 ) 466 A u s t e n , K.F., ( 8 ) 278 A u s t i n , P.R., ( 5 ) 1004, 1005 Avaeva, S.M., ( 6 ) 299 A v i z h e n i s , V. Yu , ( 6 ) 392 Awad. 0.. ( 6 ) 291 Axelsen, N . H . , ( 8 ) 141 Ayad, S., ( 5 ) 263 Ayaz, K.L., (6) 495 A y e r s , J.S.. ( 8 ) 150, 151 Aylward, J.H., ( 6 ) 527 Azhar, A . , ( 6 ) 329 A z h a r , S., ( 5 ) 582 Azizov, Yu.M., ( 7 ) 47 Azuma, I . , ( 8 ) 68, 69, 70 Azuma, J . I . , ( 3 ) 208
.
B a a l , O.P., (5) 688 B a a t h , E . , ( 3 ) 124 Baba, T . , (3) 61, 62 B a b u l , J . , ( 8 ) 407 Bach. G., ( 6 ) 247 Bachman, S., ( 8 ) 720 Bachawat, B.K., (5) 198, 1009; ( 7 ) 91; (8) 229, 261 B a c h u r , N . R . , ( 5 ) 172 B a c i c , A . , ( 3 ) 163 Backinowsky, L. V. , ( 8 ) 6 B a c k u s , B.T., ( 5 ) 685 Bacon, J.S.D., ( 3 ) 188 B a d d i l e y , J.. ( 4 ) 2, 3. 4, 12, 13 Bade, M.L., ( 6 ) 372 B a d e t , J . , ( 5 ) 1071; ( 6 ) 508 BBckstrBm, G., (5) 361 B a e n z i g e r , J.U., ( 2 ) 33, 36; ( 5 ) 569, 1013; (8) 426 Baenziger, N . L . , (5) 651, 878 B a e r , H., ( 6 ) 418 Baez, L., ( 5 ) 214 Bagshawe, K.D., ( 5 ) 865 Bagur, S.S., ( 8 ) 199 B a h l , O.P.. ( 5 ) 689, 1007, 1037: (8) 578 B a h r , H . , ( 5 ) 178 Bailey, A.J., ( 5 ) 283 B a i l e y , A.V., ( 3 ) 199 B a i l e y , D.S., (5) 90,
774 1041; ( 7 ) 174; ( 8 ) 190 B a i l e y , M.J., ( 6 ) 209 228 B a i l l y , M. , ( 6 ) 271 Bain, A.D., ( 3 ) 95 Baines, B.S., ( 8 ) 413 Baird. M . A . , ( 6 ) 287 B a j p a i , P . , ( 8 ) 619, 653 Baker, D.A., ( 5 ) 864; ( 8 ) 21 Baker, J.R., ( 5 ) 331 Baker, R.C.F., ( 6 ) 87 Baker, T.J., ( 3 ) 97 Balachandran, N . , ( 5 ) 33 Balaram, P . , ( 5 ) 124 Balasubramanian, A.S., ( 6 ) 189 Balazs, E.A., ( 5 ) 386 Baldeschweiler, J.D., ( 7 ) 152 ( 5 ) 533; B a l i s , J.U., ( 8 ) 427 B a l l , E.M., ( 5 ) 843; ( 8 ) 166 B a l l e s t e r o s , A., ( 8 ) 7 32 Ballou, C . E . , ( 2 ) 99; ( 4 ) 227, 230: ( 5 ) 6, 7 , 893; ( 6 ) 88 Ballou, L., ( 5 ) 7 Balny, C . , ( 5 ) 559 B a l t r o s B.M., ( 5 ) 297 B a l t s c h e f f s k y , H., (8) 299 B a l t z , M.L., ( 5 ) 219 Banas-Gruszka, Z . , ( 5 ) 954; ( 7 ) 44, 45, 123 Banchereau, J.F.J., ( 5 ) 522, 603; ( 7 ) 34 Bandre, T.R., ( 3 ) 125 Banerjee, D.K., ( 5 ) 1050 ( 5 ) 151 Banerjee, K.K., Banerjee, N . , ( 4 ) 139 Banja, J.P., ( 5 ) 552 Banks, D.M., ( 8 ) 338 Banks, J . . ( 5 ) 528 Banoub, J.H., ( 4 ) 55 Bara, J . , ( 5 ) 953 Barbarash C.R., ( 5 ) 462 Barbotin, J.N., ( 8 ) 583, 628, 651 ( 5 ) 583; Barchi, R.L., ( 8 ) 351 Barclay, A.N., ( 5 ) 606 B a r d e l e t t i , G . , ( 7 ) 163 Barenholz, Y., ( 6 ) 253; ( 7 ) 21 Bar-Guilloux, E., ( 6 )
Carbohydrate Chemistry 460 B a r i n g e r , J.R., ( 5 ) 29 Barka, T . , ( 6 ) 298 (71 129 Barkai, A . I . , Barker. R.. . ( 5 ) 1069; (8) 346 Barker, S.A., ( 4 ) 170; ( 6 ) 14 Barlow, G.H., ( 5 ) 785 Barlow, J . J . , ( 6 ) 110; ( 8 ) 10, 28, 29, 30, 31. 40, 52, 551 Barmore, C . R . , ( 3 2 38 Barnes. J.A.. . ( 8 ) 198 Barnes, M.J., ( 5 ) 276, 773 Barnoud, F., ( 3 ) 222 Bar-Nun. N . , ( 5 ) 96 Baron, D.A., ( 5 ) 550 Barondes. S.H., ( 5 ) 99, 208 B a r r a , Y . , ( 8 ) 194 Barrach, H.J.. ( 5 ) 446 Barranco-Acosta, C . , ( 8 ) 196 Barranger, J.A., ( 6 ) 53, 56; ( 7 ) 100, 134 Barreto-Bergter, E.M., ( 3 ) 183 Barreto-Bergter, R . , ( 4 ) 220 B a r r e t t , A.J., ( 5 ) 740 B a r r o w c l i f f e , T.W., ( 5 ) 770 Barrowman, J.A., ( 5 ) 1077 Barsoum, A.L., ( 5 ) 873; ( 8 ) 290 B a r t e l l , P.F., ( 4 ) 75 Barth, H.G., ( 3 ) 180, 21 1 B a r t h , T . , ( 8 ) 534 Barthova, J . , ( 8 ) 216 B a r t l e s , J.R., ( 8 ) 259 Barque, J.P., ( 5 ) 64 Basak, S., ( 5 ) 40 Basaveswara Rao, V . , ( 8 ) 716 Basha, S.M.M., ( 5 ) 8 7 Basu, D . , ( 5 ) 110; ( 8 ) 304 Basu, S., ( 3 ) 260 Batavyal, L . , ( 4 ) 105 B a t e s , G.W., ( 3 ) 231 Batley, M., ( 4 ) 19, 62 B a t r a , V.I.P., ( 3 ) 57 Battabiraman, T.N., ( 6 ) 80 B a t t i s t e l , E., ( 5 ) 142 Bauer, C., ( 5 ) 147 Bauer, P . I . , ( 5 ) 759 Bauer, S., ( 6 ) 210
Baugh, R . F . , ( 5 ) 657; ( 8 ) 295 Baugher, B.W., ( 8 ) 267 Bauman, J.G. J . , (8) 367 Baumann, H., ( 5 ) 1034 Baumann, N . , ( 7 ) 97 Baumann, N.A., ( 7 ) 159 Baur, M.C., ( 3 ) 49 Bausch, J.N.. ( 5 ) 165 Bause, E., ( 6 ) 217; ( 7 ) 188 Baust, J.G., ( 2 ) 43 Bautheron, D.C., ( 6 ) 16 Bauvois, B., ( 5 ) 663 Baxter, J.D., ( 8 ) 344 Bayard, B., ( 5 ) 541; ( 8 ) 235 Bayer, E., ( 2 ) 8 B a y l i s s . G . J . , ( 5 ) 69 ( 6 ) 287 Bayse, D.D., Bazin, H., ( 5 ) 836; ( 8 ) 204 B c c h e t t i , S., ( 5 ) 33 Beachey, E.H., ( 4 ) 18; ( 6 ) 499 Beahon, S., ( 6 ) 446 Beardmore, D.R., (6) 340 Bearspark, T., ( 8 ) 206 Beauregard, G., (6) 250 Beavan, M.A., ( 5 ) 349 Beck, E . A . , ( 5 ) 775 Becker, F.F., ( 5 ) 216 Becker, M.L., ( 8 ) 232 Becker, J.U., ( 4 ) 195 Beclanan, J.M., ( 4 ) 234; ( 5 ) 880 Beckner, S.K., ( 7 ) 2 3 Beddows, C.G., ( 8 ) 581 Bedewi, S., ( 8 ) 489 Bedford, P.A., ( 5 ) 212 B e e l e r , D.L., ( 5 ) 368 Begley, J.A., ( 8 ) 448 ( 5 ) 185 Behnke, W.D., Behrens. F.. ( 5 ) 406 B e i e r , R.C., ( 2 ) 89 Beighton, D., ( 4 ) 167 Beja da Costa, M . , ( 6 ) 89 ( 5 ) 222 B e j a n i a n , M.V., Bekesi, J.G., ( 2 ) 81 B e l e i a , A . , ( 3 ) 50; ( 6 ) 304 B e l f i e l d , A . , ( 5 ) 813 B e l i a r d , R., ( 5 ) 412 B e l i s l e , M . , ( 6 ) 250 B e l l , A.E., ( 5 ) 947 B e l l , J.E., ( 6 ) 503, 505 B e l l , K . , ( 5 ) 714, 715 B e l l , L.E., ( 8 ) 230 B e l l , N.H., ( 5 ) 700
Author Index B e l l e t , D., ( 5 ) 691 Belo, P.S. jun., (3) 228 B e l t o n , P . S . , ( 3 ) 269 BeMiller, J.N., ( 3 ) 12 Ben-Bassat, A., ( 6 ) 366 Bencheva, S., ( 3 ) 13 B e n d e r , B., ( 5 ) 496 B e n d e r , H., ( 3 ) 56; ( 6 ) 406 B e n d e r , M.L., ( 8 ) 159 Benecke, I., ( 2 ) 16 B e n e d e t t i , E . , ( 4 ) 25 Benedetto, J.P.. (5) 1064 Benes, P . , ( 6 ) 490, 491 Ben-Ghedalia, D., ( 3 ) 195, 197 B e n j a n n e t , S . , ( 5 ) 704 B e n n e t t , G.M., ( 4 ) 86 B e n n e t t , J . C . , ( 2 ) 44 B e n n e t t , L.G., ( 4 ) 124 B e n n e t t , M . , ( 5 ) 749 Bensadoun, A . , ( 8 ) 270 Ben-Yoseph, Y . , ( 6 ) 128, 431 Benziman, M . , ( 3 ) 130; ( 4 ) 175 Benzing, L . , ( 4 ) 126 Beppu. K . , ( 8 ) 12 Berenson, G.S., ( 5 ) 312 B e r e n t , S . L . , ( 6 ) 191 B e r e z i n , E.V., ( 6 ) 475 B e r e z i n , I . V . , ( 8 ) 734 B e r e z i n , V . A . , ( 5 ) 37 B e r g , J . O . , ( 6 ) 49 Berg, T . , ( 5 ) 567 B e r g e l ' s o n , L.D., ( 7 ) 47 B e r g e r , E.G.. ( 8 ) 580 B e r g e r , R.L., ( 2 ) 74 B e r g e l s o n , L.D., ( 7 ) 7 6 B e r g g a r d , I . , ( 5 ) 962 Bergh, M.L.E., ( 5 ) 940; ( 6 ) 509; ( 7 ) 50 B e r g h h s e r , J . , ( 8 ) 495 Bergman, J . E . , ( 5 ) 59 Bergmann, C.W., ( 5 ) 386 B e r g n e r , U . , ( 5 ) 88 B e r j e n n e a u , C., ( 6 ) 84 B e r l i n e r , L . J . , (5) 710 Berman, D.T., ( 4 ) 5 8 Berman, E . , ( 2 ) 90; ( 5 ) 479, 986: ( 6 ) 243 B e r n a r d , M . , (6) 108 B e r n a r d i , L.R., ( 2 ) 74 B e r n h a r d t , G., ( 4 ) 184 B e r n s t e i n , A., ( 5 ) 595 B e r r o u , E . , ( 5 ) 321, 404 B e r r y , L.R.. ( 8 ) 280 B e r t h e . M.C., (4) 237
775 B e r t h i l l i e r , G., ( 5 ) 1064 Bertram. K.C., ( 4 ) 4 B e s l e y , G.T.N.. ( 6 ) 161 Betaneli, V.I., (8) 6 B e t h e l l , G . S . , ( 8 ) 150, 151 Bethenod, M . , ( 5 ) 744 B e t r a b e t , S.M., ( 3 ) 120 Beushausen, T.W., ( 2 ) 72 Beveridge, T.J., (6) 223, 227 B e v i l a c q u a , M.P., ( 5 ) 220 Bewley, J . D . , ( 3 ) 184: ( 6 ) 118, 119 B e y e r , E.M., ( 6 ) 97 Beyer, T.A., ( 2 ) 117; ( 5 ) 1028, 1072; ( 6 ) 498; ( 8 ) 90, 266 Beyreuther, K., ( 5 ) 919, 920 Bezborodov, A.M., ( 6 ) . 137, 475 B e z h i k i n a , L.V., ( 6 ) 489 B h a n d a l , I.S., ( 7 ) 177 Bhardwaj, D.. ( 5 ) 295 Bhardwaj, N . , ( 4 ) 6 8 Bhargava. M . , ( 3 ) 132 Bhandari, N.N., ( 3 ) 132 Bhat. P.G., ( 3 ) 86; ( 6 ) 80 B h a t i a , D., ( 8 ) 606 B h a t n a g a r , R . , ( 4 ) 139 B h a t t a c h a r j e e , A.K., ( 4 ) 94: ( 5 ) 888; (8) 36 B h a t t a c h a r y a , S.B.. ( 4 ) 121, 122 Bharracharyya, L., (5) 107; ( 8 ) 102 B h a t t a c h a r y y a , S.N., ( 5 ) 534, 537 Bhavanandan, V.P., ( 5 ) 444, 528 B h o g a l , B., ( 5 ) 505 Bhown, A . S . , ( 5 ) 36, 40 B i a n o , C., ( 5 ) 220 Biederman. G . J . , ( 6 ) 213: ( 8 ) 752 B i e l y , P . , ( 3 ) 201, 202; ( 6 ) 463, 464, 465, 472 Bienvenu. F., ( 5 ) 744 Bienvenu, J . , ( 5 ) 744 Bienvenu, P . , ( 4 ) 106; ( 7 ) 195 B i e t h , J.B., (5) 733 Bigelow. C.C., (5) 709 B i g u e t , J . , ( 4 ) 222
Bilham, T., ( 6 ) 528 B i l i a d e r i s , C.G., ( 3 ) 24, 25 B i l l i a u , A., ( 5 ) 514; ( 8 ) 398 B i l o d e a u , G., ( 5 ) 345 B i n a g l i a , L . , ( 6 ) 33 B i n d e r , B.R., ( 8 ) 122 B i n d e r , R., ( 8 ) 117 Bing, D.H., ( 8 ) 568 B i n i o n , S., ( 5 ) 834 B i o l , M.C., ( 6 ) 507 Biou, D.R., ( 5 ) 1021 Birnbaum, S., ( 8 ) 641 B i r n e y , E.C., ( 6 ) 495 Biserte, G.. ( 5 ) 836; ( 8 ) 204 B i s h a y e e , S . , ( 5 ) 198, 1009; ( 8 ) 229, 261 B i s h o p , C.T., ( 4 ) 125 Bishop, D.G., ( 7 ) 166 B i s h o p , P . , ( 3 ) 233 B i s h o p , P.D., ( 3 ) 234 Bisse, E . , ( 2 ) 76; ( 8 ) 719 Bissett, F . H . , ( 6 ) 351 Bisswanger, H. ( 8 ) 117, 322 B i t k o , S.A., ( 6 ) 441 Bitman, J . , (7) 7 B i t t n e r , C., ( 5 ) 97 Bizzozeroo, J.P., (6) 293 B j b r k , I., ( 5 ) 364, 365, 761, 764 B j b r l i n g , T., ( 4 ) 197 B j b r n s s o n , S., ( 5 ) 314, 325, 413 B l a c h e , D., ( 4 ) 61 B l a c i k , L . J . , ( 5 ) 297 Black, M.M., ( 5 ) 505 Blackburn, M.N., (8) 271 B l a c k b u r n , P . , ( 5 ) 931 B l a c k w e l l , J . , ( 3 ) 91 Blackwood, G.C., ( 3 ) 250 B l a d i e r , D., ( 5 ) 905 BlBckberg, L . . ( 8 ) 449 B l a k e r , F . , ( 2 ) 58 Blajchman, M.A., ( 5 ) 668 B l a k e , D.A., ( 5 ) 134, 1067 Blakemore, W.F., ( 5 ) 975: ( 6 ) 242 B l a n c h , H.W., ( 6 ) 208 B l a n c a r d , J.S., (8) 596 B l a s z c z y k , M., ( 5 ) 857; ( 7 ) 32 Blaydem, S . L . , ( 6 ) 361 B l a z e j . A., ( 3 ) 115
Carbohydrate Chemistry
776 B l e h a , T . , ( 3 ) 85 B l o b e l , G . , (5) 671 B l o c k h a u s , M . , ( 5 ) 857 Bloemendal, H., (8) 162 Blomberg, K . , ( 8 ) 379 B l o j . B . , (7) 46 B l o m q u i s t , C.H., ( 2 ) 86 Bluhm, T . L . , (3) 4: ( 8 ) 460 Blumaurerova , M. , ( 8 645 B l u m e n f e l d , 0.0.. ( 5 ) 918 B l u m e n t h a l , H. J . , ( 4 ) 158 B l u n d e l l , J.K., ( 4 ) 41, 42 B o a t , T . F . , ( 5 ) 930 B o b b i t t , T.F., (4) 208 Bocquet, J . , ( 5 ) 412 Bodwell, J . E . , (8) 228 Mgemann, G., ( 7 ) 179, 180 B o e r m a , A . , ( 2 ) 35; (5) 929, 932, 945 Bogdanov, A.A., ( 4 ) 199 Boggs, J.M., (5) 1078 Mg-Hansen, T.C., ( 5 ) 747 Bogin, E . , ( 2 ) 83 Bogwald, J . , (4) 214 Bohaun, C . , ( 5 ) 691 B o l e s , H.P., (6) 275 Bolmer, S.D., ( 5 ) 814 Bolmer, S.L., (8) 286 B o l o g n e s i , D.P., ( 5 ) 7 9 B o l t e , L . C . , ( 6 ) 305 B o l w e l l , G.P., ( 3 ) 205 Bonaly, R . , (4) 231, 237 Bonazzi, M . , ( 8 ) 189 B o n f i l s , C., (5) 559 Bonner, B.A., ( 3 ) 177 Bonora, G.M., (4) 25 B o o k s t e i n , F.L., ( 5 ) 132 Boon, J.J., ( 4 ) 7 Boos, W . , (4) 134 B o r d a s , M.C., ( 5 ) 1021 B o r g i a , P . T . , (4) 235 B o r n s t e i n , P . , ( 5 ) 504; (8) 264 B o r o n a t , A . , ( 4 ) 182 B o r r e b a e c k , C.A.K., (5) 153, 154, 155, 156, 157 158; (8) 397 B o r r i s s , R., ( 8 ) 275 B o r z a n i , W., (8) 471 B o s c h e t t i , E., ( 8 ) 596 Bose, S.M., ( 6 ) 30 B o t s t e i n , D . , ( 4 ) 198; (6) 149
Bouchard, B.G., ( 7 ) 51 B o u c h i l l o u x , S., ( 5 ) 70 1 Bouhours, D., ( 5 ) 477; (7) 132 Bouhours, J.-F., (5) 477; (7) 132 B o u l d i n g , E.T., ( 6 ) 2 9 B o u l t o n , A., (5) 994; ( 8 ) 352 B o u n i a s , M . , ( 6 ) 174 B o u q u e l e t , S., (8) 206 B o u r r i l l o n , R., ( 5 ) 531, 559, 890, 891 B o u r i o t i s , V., ( 8 ) 420 B o u r r i l l o n , R., (5) 895 B o u t r y , J.-M., ( 7 ) 69 Bovara, R . , (8) 476 Boveda, M.D., ( 2 ) 24 Bowers L.D., ( 6 ) 229: ( 8 ) 724, 725 Bowles, D . J . , ( 5 ) 122 Bowness, J.M., ( 5 ) 494 Boyd, J.. (5) 889 Boyer, C.D., ( 3 ) 37, 65; (6) 526 B o z z o l a , J.J., ( 4 ) 163 Bradbury, A.G.W., (2) 12 Bradbury, J.H., ( 5 ) 707 Bradshaw, I . J . , (4) 170, 171 Bradshaw, J . P . , (5) 726; ( 7 ) 154 Bradshaw-Rouse , J. J , ( 4 ) 138 B r a d y , R.O., ( 5 ) 687: (6) 53, 56; ( 7 ) 23, 100, 146: ( 8 ) 579 B r a e l l , W.A., ( 5 ) 925 B r a i n , A.P.R., ( 7 ) 11 Bramwell, M.E., ( 5 ) 527 Brana, A . F . , ( 4 ) 246 B r a n e f o r s - H e l a n d e r , P., (4) 100 Brannon, P.M., ( 6 ) 294; (8) 309 B r a n y i k , A., ( 6 ) 210 Brasch, D.J.. (3) 262 B r a s s e u r , R., ( 7 ) 42 B r a t t a i n , M.G., (6) 65 B r a u d e , I . A . , ( 5 ) 517 B r a u n i t z e r , G . , ( 6 ) 278 B r a u t i g a n , V.M., ( 4 ) 10 B r e a t h n a c h , S.M., ( 5 ) 505 B r e b o r o w i c z , D., ( 5 ) 818 Breborowicz, J . , (5) 818 B r e e n , A . R . , ( 3 ) 32
.
Breen, M., ( 5 ) 297 Breimer, M.E., ( 2 ) 93; (7) 84, 85, 131, 153 Breitenbach, M., ( 8 ) 108 B r e k l e , A . , ( 5 ) 393 Brennan, P . J . (4) 38, 39; ( 7 ) 199, 200 Bretaudiere, J.P., (6) 27 1 B r e t o n , M . , ( 5 ) 321, 404 B r e t t i n g , H., ( 5 ) 992 B r e u e r , W . , (5) 4 B r e v e r , W . , ( 8 ) 598 Brew, K., (5) 713 Brew, S . A . , ( 5 ) 690 B r i a n d , J . P . , ( 5 ) 1070 Brice, R.E., ( 2 ) 62 B r i g g s , R.C., (2) 55: ( 5 ) 1012; ( 8 ) 490 B r i l e s , D . E . , ( 4 ) 118; (55) 874 B r i l l o u e t , J.M., ( 3 ) 192 B r i n e , C . J . , ( 5 ) 1004, 1005 B r i n t o n , C.C., ( 8 ) 333 B r i q u e t , M . , (8) 639 Bris, B . , ( 7 ) 186 Brissot, P . , ( 6 ) 60 B r o b s t , K.M., 12) 23 Brockhaus, M . , ( 7 ) 32 B r o c k l e h u r s t , K., ( 8 ) 413 B r o c t e u r , J . , ( 5 ) 987 Broedelmann T . J . , ( 5 ) 225 B r o e z e , B., (8) 284 Broman. T.H., (3) 66 B r o o k h o u s e r . L.W., (3) 240 B r o q u e t , P . , ( 6 ) 506 Brossmer, R., ( 5 ) 131; ( 6 ) 124; ( 7 ) 15: ( 8 ) 573 B r o s s t a d , F . , ( 8 ) 376 B r o t , N., ( 8 ) 133 Brougham, M.J., ( 8 ) 125 Broughton, E.M., (5) 79 Brouwer, C.A., ( 6 ) 213, (8) 752 Brouwer-Kelder , B., ( 5 1 870; ( 6 ) 164 Brown, C.R., ( 5 ) 580 Brown, D.Mi, ( 5 ) 409 Brown, F . E . , ( 5 ) 276 Brown, G . E . , ( 3 ) 238 Brown, I . R . , ( 5 ) 532 Brown, J . A . , ( 6 ) 235 Brown, J . E . , ( 5 ) 657, 771: (8) 295
777
Author Index Brown, K.S., ( 5 ) 446 Brown, L . E . , ( 5 ) 41, 45 Brown, N.M., ( 8 ) 635 Brown, R . M . , ( 3 ) 116, 130: ( 4 ) 175; ( 6 ) 339 Brown, T.L., ( 6 ) 295 Brown, W.R.A., ( 5 ) 606 Bruce, M., ( 5 ) 930 Bruchovsky, N., ( 8 ) 115 Bruck, C., ( 5 ) 13 B r u e l , A . , ( 5 ) 381, 422 Bruneteau, M . , ( 4 ) 61 (5) Brungardt, G.S., 2 65 Bruno, E., ( 5 ) 511 Bruns, D . , ( 5 ) 89 B r u v i e r , C.,(2) 96 Bryan, A.H., ( 5 ) 682, 684 Bryon, P.A., ( 5 ) 477 Bubenzer, H.J., ( 8 ) 403 Buchala, A.J., ( 3 ) 128 Buchholz, K., ( 8 ) 598 Buchs, L., ( 3 ) 128 Buck, C.A., ( 5 ) 549, 572 Buckland, P.. ( 5 ) 872; ( 8 ) 288 Buckwalter, J . , ( 5 ) 318 ( 8 ) 691 Budanov, M.V., ( 5 ) 475 Budarf, M.L., Buddecke, E . , ( 5 ) 323, 438 BUchi, K.G., ( 5 ) 759 BUnsch. H . , ( 8 ) 45, 46 BUsen, W . , ( 8 ) 422 Buffington, L.A., ( 3 ) 179 B u g a s h e t t i , B.K., ( 2 ) 15 Bugge, B., ( 5 ) 577, 1056; ( 6 ) 459; ( 8 ) 200 B u i j s , F., ( 5 ) 870; ( 6 ) 164 B u i j s , J . , ( 3 ) 141 (5) Bullimore, D.W., 315 Bundle, D.R., ( 8 ) 44 Bunn, H.F., ( 5 ) 467, 797, 809 Bunsch, A . , ( 8 ) 55 Buonocore, V., ( 8 ) 608 Burba, P., ( 8 ) 474 Burchard, W., ( 5 ) 815 Burd, G.I., ( 6 ) 143 B u r d e t t , I.D.J., ( 6 ) 52 B u r d i t t , L.J., ( 5 ) 975; ( 6 ) 242 Burge, J., ( 8 ) 278 Burgess, A.W.. ( 8 ) 362 B u r k a r t , T., ( 7 ) 143
Burke,. J . , ( 5 ) 1041 B u r k e r t , W.G., ( 3 ) 8 3 Burlingame. A.L., ( 2 ) 99; ( 4 ) 227; ( 5 ) 893 Burlingame, R.W., ( 5 ) 428 Burmeister, G . , ( 6 ) 193 Burns, P.F., ( 8 ) 171 Burns, R . A . , ( 6 ) 46 Burny, A., (5) 13 B u r r i l l , P.H., ( 6 ) 294; ( 8 ) 309 B u r s t e i n , E.A., ( 5 ) 712 Burton, B.K., ( 6 ) 128 Burton, Z.F., ( 8 ) 222 Burzynska, M . H . , ( 5 ) 852 Busby, T.F., ( 5 ) 757 Busch, C . , ( 6 ) 411 Bushway, A.A., ( 7 ) 51 Bushway, R.J., ( 7 ) 51 Buswell, J.A., ( 8 ) 132 Butchko, A.W., ( 5 ) 79 Butkowski, R.J. , ( 5 ) 265 B u t l e r , W.T., ( 5 ) 227 (2) B u t t e r f i e l d , D.A., 100 B u t t e r s , T.D., ( 5 ) 669 Butterworth, J . , ( 6 ) 239, ( 8 ) 296 Buznikov, G., ( 7 ) 76 Byrd, J.C., ( 5 ) 476 Cabello, A., ( 6 ) 335 Cabezas, J.A., ( 6 ) 39, 70, 258; ( 7 ) 40 C a b r a l , J.M.S., ( 8 ) 717, 718 Cacan, R . , ( 5 ) 663; ( 7 ) 136 Cadenas, R.A., ( 8 ) 25 Cadenbach, J.E., (5) 398 Caen, J . , ( 5 ) 663 Caen, J.P., ( 5 ) 656 Cahan, L.D., (5) 6 3 C a h i l l , E., ( 5 ) 1070 C a i l l a u d , J.M., ( 5 ) 691 Caimi, L., ( 6 ) 26 C a j i p e , G.J.B., ( 3 ) 273; ( 4 ) 204 C a l a b r e s e , V.P., ( 7 ) 95 Calam, D.H., ( 2 ) 30 C a l c o t t , P.H., ( 4 ) 86 Caldwell, C.R., ( 8 ) 402 Caliendo, M.F., ( 6 ) 230 Callahan, S., ( 5 ) 190; ( 8 ) 219 Calvo, P., ( 6 ) 70, 258 Cambarnous, Y., ( 5 ) 696 Cameron, D.P., ( 5 ) 804
Cammarata, P . , ( 5 ) 1036 Campagnon, A.T., ( 8 ) 171 Campagnon, C.W.. ( 8 ) 171 Campbell, D.G., ( 5 ) 593, 594 Campbell, K.P.. ( 5 ) 495; ( 8 ) 236 Campbell, L.K., ( 4 ) 16 Campbell, W.H., ( 8 ) 434 Canales, M., ( 6 ) 370 Canella, R., ( 7 ) 58 Canevascini, G., ( 6 ) 201, 342 Canoy, M.W., ( 5 ) 306 C a n t a r e l l a , M., ( 8 ) 685 Cantoni, G.L., ( 8 ) 149 Capitanio, A . , (5) 654 Capka, M . , ( 8 ) 584 Cappelletti, R., (5) 445, 499; ( 8 ) 329 Caputo, C.B., ( 5 ) 402 Caputto, R . , ( 7 ) 13, 57, 87 C a r d e l i n o , B., ( 8 ) 516 C a r d i n i , C . E . , ( 3 ) 68, 69 Cardoso, E.A., ( 5 ) 12 Cardoso, J.P. , ( 8 ) 717, 718 Carducci, C . , ( 5 ) 471 Carey, A.M., ( 4 ) 45 Carey, V., ( 2 ) 74 C a r l i n , B . , ( 5 ) 496 C a r l i n i , F., ( 5 ) 471 Carlo, D., ( 4 ) 120 C a r l o , D.J., ( 4 ) 125, 126, 127 C a r l s e n , S.A., ( 5 ) 555 Carlson. R.I., ( 5 ) 894 C a r l s s o n , J . , ( 8 ) 399 C a r l s t e d t , I., ( 5 ) 338, 343 C a r l s t r t h , C., ( 5 ) 330 Carmian, F.R., ( 5 ) 676 (5) C a r n i c e r o , H.H., 285; ( 6 ) 502 Caron, M., ( 5 ) 126 Carper, D., ( 8 ) 310 Carrasco, A., ( 6 ) 534 Carraway, C.A.C., (5) 588 Carraway, K.L., ( 5 ) 588 589 C a r r e a , G., ( 8 ) 476 C a r r e l l , R . , ( 5 ) 752 C a r r o l l , M., ( 6 ) 20, 175 Carson, D.D., ( 5 ) 1042 Carson, S.D., ( 8 ) 135 C a r t e r , V.C., ( 5 ) 30
Carbohydrate Chemistry
7 78 C a r t e r , W.G., ( 5 ) 592 C a r t r o n , J.P., ( 5 ) 1071: ( 6 ) 508 Carver, J.P., ( 5 ) 982, 983, 1019 Casadaban, M.J., ( 4 ) 198: ( 6 ) 149 C a s a l i , P.. ( 5 ) 7 3 C a s e t i , C . , ( 5 ) 701. 702 Cassiman, J . J . , ( 5 ) 738, 739 Castanon. M . , ( 6 ) 334 Castellino, F.J., (5) 564 C a s t i l l a , C . , ( 6 ) 163, 169 Casu, B., ( 5 ) 369 Catley, B.J., ( 4 ) 207 (6) 23 C a t t o , G.R.D., Cawley, D.B., ( 5 ) 577: ( 8 ) 576 Cawson. R . A . , ( 4 ) 210 Cazzulo. J . J . , ( 5 ) 1038 C e c c a r i n i , C., ( 5 ) 982, 983, 1036 C e c c o r u l l i , L.M., (5) 6 82 Ceh, M . . ( 3 ) 4 5 Celada, F., ( 6 ) 152 Cerami, A . , ( 5 ) 931 ( 8 ) 514 Chacko, K.K., Chaiken, I.M., ( 8 ) 171 Chaki, S., ( 4 ) 229 Chaloupka, J . , ( 8 ) 616 Cham, D.. ( 5 ) 254 Chambat. G., ( 3 ) 96, 222 Chan, L., ( 5 ) 1036, ( 8 ) 4 78 Chan, R . , ( 4 ) 65 Chandrasekaran, E.V., ( 5 ) 845, 846, 1065: ( 8 ) 282 Chandrasekher, G., ( 6 ) 281 Chang, C.W., ( 3 ) 63 Chang, D., ( 8 ) 426 Chang, G.D., ( 8 ) 140 Chang, H.N., ( 8 ) 494, 730 Chang, L.S., ( 5 ) 845 Chang, N.C., ( 7 ) 54 Chanzy, H . , ( 3 ) 93 Chao, H., (5) 353, 354 Chapnan, A . , ( 5 ) 1058 Chapman, M . J . , (5) 823 Chard, T., ( 8 ) 588 Charlwood, P.A., (5) 967 C h a r p e n t i e r , B. , ( 5 ) 812
Charpentier, C., ( 4 ) 237 Chary, M.A.S., ( 3 ) 248 Chasis, J . , ( 5 ) 511 C h a t i s , P.A., ( 5 ) 57 C h a t t e r j e e , S., ( 6 ) 180 C h a t t e r j i , D., ( 6 ) 400 Chaubet, N., ( 6 ) 155 Chaudhry, M.A., ( 3 ) 42, 43, 44 Chauvette, G . , ( 7 ) 165 Chavanich, S.. ( 6 ) 468 Cheah, S.C., ( 4 ) 3 Cheetham, N.W. H . , ( 2 ) 32, 42 Chen, A.C., ( 2 ) 84 Chen, C . , ( 5 ) 172 Chen, C.C., ( 2 ) 6 Chen, K.C., ( 8 ) 649, 678 Chen, L.F., ( 4 ) 200: ( 6 ) 477 Cheng, P.W., ( 5 ) 930 Cheng, S.Y., ( 5 ) 817 Cherednikova, T.V., (8) 168 Cherian, R . , ( 6 ) 189 Cherniak, R., ( 2 ) 7: ( 4 ) 92 Cheron, A . , ( 5 ) 729: ( 6 ) 171 Cheryan, M . , ( 6 ) 140 Chesebro, B . , ( 5 ) 16 Chesson, A . , ( 3 ) 196 (5) Chesterman, C.N., 837 C h e t a l , S., ( 7 ) 183 C h e v a l i e r , G., ( 6 ) 163 Chiang, L.-C., ( 4 ) 183, 200: ( 6 ) 477 Chiang, P.K., ( 8 ) 149 Chiao, Y.B., ( 8 ) 423 Chiarug, V.P., ( 5 ) 378 C h i a r u g i , V., (5) 445, 499: ( 8 ) 329 Chiasson, J.-L., (5) 292; ( 6 ) 527 Chiba, H . , ( 2 ) 53: ( 5 ) 724 Chiba, S., ( 6 ) 176, 185 Chibata, I., ( 8 ) 469, 549, 605, 623, 636, 638, 654 Chicheportiche, Y., ( 6 ) 293 Chidambareswaran, P.K., ( 3 ) 92 Chien, S.F., ( 6 ) 102 C h i l d s , R . A . , ( 5 ) 597 111, ( 4 ) C h i l d s , W.C., 10 ( 3 ) 271 C h i l v e r s , G.R.,
Chin, J.14.. ( 5 ) 195 Chin, W.W., ( 5 ) 699 Chindemi, P.A., ( 5 ) 565 Chinen, I . , ( 6 ) 115 Ching, S.F., ( 8 ) 172 Chinnadurai, G., ( 5 ) 25 Chiou, S.H., ( 5 ) 467 Chiquet, M . , ( 5 ) 228 Cho, G.H., ( 8 ) 633 Cho. Y . R . , ( 8 ) 494 Choay, J . , ( 5 ) 369 Chobert. J.M.. ( 8 ) 416 Choi, C.Y.. ( 8 ) 633. 680 ’ Choi, W.G., ( 8 ) 699 ( 8 ) 633 Choi, Y.D., Choppin, P.W., ( 5 ) 54, 74 Chopra. R.K., ( 5 ) 494 Chotai, K . , ( 5 ) 975: ( 6 ) 242 Chou, K.H., ( 7 ) 128, 130 Choudhury, N., ( 6 ) 353, 37 1 Chow, P., ( 5 ) 353: ( 8 ) 13 C h r e t i e n , M., ( 5 ) 474, 695, 703, 704, 705 C h r i s t i a n s e n , L., ( 6 ) 444 (3) C h r i s t i a n s o n , D.D.,
73
C h r i s t i a n s s o n , A., ( 7 ) 190 Christornanou, H., ( 7 ) 106 Chu. F.K., ( 5 ) 115 ( 5 ) 473 Chu, L.L.H., Chu, M.I., ( 4 ) 216 Chu, T.M., ( 5 ) 835: ( 8 ) 202, 237 Chuah, C.T., ( 3 ) 262 Chumpitazi-Hermoza, B . , ( 3 ) 96 Chun, Y . Y . , ( 8 ) 650 ( 5 ) 487. Chung, A.E., 496 Chung, H., ( 7 ) 168 (6) Churilova, I . V . , 194, 197, 356, 393 Churms, S.C., ( 3 ) 140, 259 Chute, L., ( 5 ) 781 Chylack, L . T . , ( 5 ) 467 Ciborowski, C.J., ( 5 ) 374 Cimino. C.D., ( 8 ) 226 Cina, R . , ( 6 ) 152 C l a f l i n , J.L., ( 4 ) 118; ( 5 ) 874 Clamagirand-Mulet, C.,
779
Author Index ( 5 ) 1071 C l a r k , F.Y., ( 5 ) 665 Clark, J.M., ( 4 ) 178 C l a r k , S.P., ( 5 ) 578 Clarke, A.E., ( 5 ) 92, 93, 104; ( 8 ) 243 Clarke, E.M.W., ( 5 ) 171 C l a r k e , J.T.R., ( 7 ) 110 Clarke, K.J., ( 3 ) 252 Clay, M.G., ( 5 ) 480 Clegg, R . M . , ( 5 ) 140 Cleland, R.L., ( 5 ) 388 Clementi, F., ( 6 ) 321 Clement-Jones, V.V., ( 5 ) 1015 Clemetson, K.J., ( 5 ) 654, 662 Clemmensen. I., ( 8 ) 283 Clevinger, B., ( 5 ) 881 C l i n e , K., ( 3 ) 160, 161; ( 8 ) 305 C l o n i s , Y.D., ( 8 ) 421 ( 8 ) 635 Cluett, W.R., Cochet, N . , ( 8 ) 550 Cochran. F.B., ( 7 ) 72 C o c i t o , C . , ( 4 ) 29 Cockburn, C.G., ( 5 ) 780 C o c k e r i l l , P . , ( 5 ) 252 Cocquempot, M.F., ( 8 ) 65 1 Coddington, J.M., ( 7 ) 166 Codington, J.F., ( 5 ) 551 Coe, F.L., ( 5 ) 486 Cbster, L . , 333, 337, 338, 339, 343 Coffey, J.W., ( 6 ) 233 Coffman, R.L., ( 5 ) 601 Cohen, D.L., ( 6 ) 322 Cohen, F.E., ( 5 ) 594 Cohen, G . , ( 8 ) 470 Cohen. I., ( 5 ) 235; ( 8 ) 368 Cohen, M.P., (5) 373, 374 Cohen, P . , ( 5 ) 291: ( 6 ) 528 ( 5 ) 291 Cohen, P.T.W., ( 4 ) 230; Cohen, R.E., ( 6 ) 88 Cohen, S., ( 5 ) 583 Cohen, S.A., ( 8 ) 351 (2) Cohenford, M.A., 77; ( 5 ) 936 Cohen-Solal, L., ( 5 ) 2 72 Cohn, D.V., (5) 473 Colas, B., ( 6 ) 98, 99; ( 8 ) 657 Cole, A.L., ( 6 ) 195 Cole, E.S., ( 5 ) 585
Cole, W.G., ( 5 ) 254 Coleman, G.S., ( 5 ) 446 C o l e t t a , M., ( 5 ) 526 Coligan, J.E., ( 5 ) 611, 613, 614, 615, 616, 617, 618, 628 C o l l e n , D., ( 5 ) 529, 826; ( 8 ) 164, 253 ( 5 ) 799 Colley, K.J., C o l l i n s , J.F., ( 5 ) 249 C o l l i n s , J.G., ( 5 ) 707 ( 5 ) 16 C o l l i n s , J.K., C o l l i n s , J.M., ( 5 ) 1077 Colman, A., ( 5 ) 994; ( 8 ) 352 Colvin, J.R., ( 3 ) 111 Combarnous, Y., ( 5 ) 678 Comeau, T., ( 8 ) 115 Comings, D.E., ( 5 ) 218; ( 8 ) 111 Compans, R.W., ( 2 ) 44; ( 5 ) 19, 36, 40 Comtat, J . , ( 6 ) 462 Conde, J . , ( 6 ) 335 Conkerton, E.J., ( 3 ) 137 Conner, A.H., ( 3 ) 103 Conner, B.J., ( 8 ) 111 Connolly, D.T., ( 5 ) 191 Conrad, D., ( 6 ) 259, 469 Conrad, D.H., ( 5 ) 643 Conradt, P., ( 8 ) 353 C o n s i g l i o , E . , ( 5 ) 468 Constans, A . , ( 7 ) 98, 99 Constantinides, A . , ( 8 ) 606, 735 Constantopoulos, G., ( 6 ) 53 Contaxis, C.C., (5) 709 Contesso, G . , ( 5 ) 691 Converse, A., ( 3 ) 122 Conway, N . A . , ( 5 ) 1070 Conzelmann, E., ( 6 ) 22 ( 8 ) 120, Cook, G.M.W., 173 Cook, J.A., ( 3 ) 164 Cook, R.C., ( 5 ) 610 Coolbear , T., ( 5 ) 1044 Cooney, C.L., ( 6 ) 412 Cooper, A.R., ( 5 ) 500 Cooper, C . , ( 7 ) 59 ( 5 ) 208 Cooper, D.N., Cooper, E.H., ( 5 ) 747 ( 8 ) 585 Cooper, H.A., ( 3 ) 130; Cooper, K.M., ( 4 ) 175 Coplin, D.L., ( 4 ) 138 Coppa, G., ( 6 ) 32 Coppini, R . , ( 7 ) 78 Cornale, P . , ( 8 ) 189
C o r n i l l o t , P., ( 5 ) 905 C o r t i , M., ( 7 ) 17, 19 Corvino, A . , ( 6 ) 230 Cosma, S., ( 5 ) 394 Cossu, G., ( 5 ) 526 Costamagna, L., ( 6 ) 321 C o s t e r t o n , J.W., ( 4 ) 45, 65 Cothran, W.C., ( 5 ) 227 Cotmore, S.F., ( 5 ) 449 Cotner, T., ( 5 ) 627 C o t t e , J . , ( 5 ) 744 ( 8 ) 338 C o t t o n , R.G.H., Couchie, D . , ( 8 ) 119 Coughlan, M.P., ( 6 ) 198, 199, 346, 360 (6) Coughlin, R.W., 213; ( 8 ) 752 C o u l e t , P.R., ( 6 ) 16; ( 8 ) 399, 672, 696 Coulon-Morelec, M.J., ( 7 ) 159 Coupek, J., ( 8 ) 587 Courtney, H., ( 4 ) 18 Courtney, R.J., ( 5 ) 28 C o u r t o i s , J.-E., (6) 460 Couso, R.O., ( 4 ) 173, 174 C o u s t a u t , D., ( 7 ) 186 Cowing, C., ( 5 ) 605 Cowman, M . K . , (5) 386 ( 5 ) 866 Cox, C.E., Cox, G.S., ( 5 ) 677, 686 Cox, R.P., ( 5 ) 673 Coyle, P.J., ( 8 ) 423 Cragg, B.G., ( 6 ) 53 ( 6 ) 530 Craig, G.D., Craig, R . , ( 5 ) 994; ( 8 ) 352 Craig Heehn, K., ( 6 7 ) 435 C r a i n , L.R., ( 6 ) 430 Craswell, P., ( 6 ) 423 Craves, F.B., ( 7 ) 97 Crawford, D.L., ( 6 ) 367 Crawford, N . , ( 5 ) 650 Creech, H.J., ( 8 ) 563 Creese, E., ( 6 ) 200 Cremonesi, P., ( 8 ) 41 1 , 476 Crespo, A., ( 6 ) 163, 169 C r e u t z , C.E., ( 8 ) 350 Crine, P., ( 5 ) 703 C r i p p s , R.E., ( 4 ) 172 C r i s t e a , A., ( 8 ) 432 Cross, J . , ( 5 ) 266 Crow, B., ( 8 ) 443 Crowhurst, S.A., ( 5 ) 449 Crowley, J.F., ( 5 ) 106
Carbohydrate Chemistry
780 Cruickshank, W.H., (5) 788 Crumpton, M.J., ( 5 ) 625 Cruz, M.R., (5) 655 C r u z , T.F., ( 5 ) 455 C u a t r e c a s a s , P., (6) 67 C u l l e n , S.E., ( 5 ) 605 C u l l i n g , C.F.A. (5) 480 C u l p , L A . , ( 5 ) 375; ( 8 ) 262 Cumar, F.A., ( 7 ) 13, 87 Cumming, C.G., (4) 160 Cummings, R.D., ( 5 ) 1031 Cumsky,M.G., ( 5 ) 203, 204 Cunningham-Rundles, C., (5) 833 C u r l e y , W.H., ( 6 ) 232 C u r t i s , P.J., (5) 75 C u t l e r , A.N., ( 4 ) 142 C u t l e r , J . , (2) 69 Czuppon, A.B., ( 5 ) 579 Dabrowski, J . , (7) 82, 83, 114, 155 Dabrowski, U., (7) 114, 155 d a Costa, J . , (5) 802 Da Cruz, F.S., (3) 183 D a f e l d e c k e r , W.P., ( 8 ) 300 Dahl, J.B., ( 5 ) 160; (8) 207 Dahmus, M.E., (8) 450 Dahr, W . , (5) 919, 920 Dain, J.A., ( 2 ) 77; ( 5 ) 936; (6) 43 D a i r e a u x , M., ( 5 ) 412 Dale, R.M.K., (5) 170, 172 D a l e s , S., ( 5 ) 83 D a l e s s a n d r o , G., (3) 203, 204 Dallas, J . , (5) 816 Damas, J . , ( 3 ) 275, 276, 277 Damewood, P.A., ( 3 ) 37 Damsky, C.H., (5) 572 Danca, M., (5) 576 D'Angiuro, L., (3) 117; ( 6 ) 332; ( 8 ) 411 D'Angona, J . , (2) 99; ( 4 ) 227; ( 5 ) 893 Dani, M., (5) 143 D a n i e l , P.F., ( 5 ) 973 D a n i e l s , L.B., ( 6 ) 190: ( 8 ) 423 D a n i e l s , P.F., ( 7 ) 102 D a n i e l s , S.M., (8) 438 D a n i e l s o n , N.D., (8) 728
Danielsson, A., (5) 365, 764 D a n i e l s s o n , B., ( 8 ) 632, 669, 746 D a n i l o v , L.L., ( 8 ) 73 Danishefsky, I., (5) 359 Dankers, I., ( 5 ) 119 D a n k e r t , M.A., (4) 173, 174 Dannevig, B.H., (5) 567 Dand, K., (8) 466 D a n o i s , D.M., ( 5 ) 522, 603; ( 7 ) 34 D a o u s t , V., ( 4 ) 125, 127 D a r b y s h i r e , B., ( 3 ) 87 D a r v i l l , A.G., (3) 191, 223, 232, 233; ( 4 ) 152 D a r v i l l , J . E . , ( 3 ) 191 Das, A . , (3) 139, 185; ( 5 ) 507 Das, M.K., ( 5 ) 168, 888 Das. N . N . , (3) 209 Das. P.K., ( 5 ) 107; ( 8 ) 102 Das, S.C., ( 3 ) 209 Das, S.K., (7) 48 Dash, M.C., ( 6 ) 348 Da S i l v a , R.S.F., ( 6 ) 349 d a s Neves, H. J. C . , ( 2 ) 8 D a s s , H.B., ( 8 ) 373 Datema, R . , (5) 34, 1051 D a t t a , T.K., ( 5 ) 108; ( 8 ) 103 D a u g u l i s , A.J., ( 8 ) 635 D a u r e l l e s , J . , (6) 420 Daury, G., ( 7 ) 98, 99 Dauss, H., (5) 183 D a u s s a n t , J . , ( 3 ) 55; (6) 324 Dauste-Blazy, L., ( 6 ) 167 Davankov, V.A., ( 8 ) 691 Davidson, E.A., (5) 21, 528, 814, 873; ( 8 ) 286, 290 Davidson, J., ( 2 ) 30 Davidson, J.F., ( 5 ) 783, 868 D a v i e , E.W., ( 5 ) 779; (8) 153 D a v i e , J.M., ( 5 ) 881 D a v i e s , B.L., (5) 786 D a v i e s , D.R., ( 5 ) 841 D a v i e s , H.M., (5) 94 D a v i e s , M., ( 4 ) 64 D a v i l a , M . , ( 8 ) 282
D a v i l l a , M., ( 5 ) 1065 Davis, M.A.F., (3) 214 D a v i s , P.E., ( 6 ) 322 D a v i s , P.F. ( 5 ) 248 D a v o u s t , J., ( 5 ) 1018 Dawson, A., ( 3 ) 134 Dawson, G., ( 5 ) 453; ( 6 ) 102; (7) 93, 94 Day, D.G., (5) 673 Day, W . R . , (2) 32 D a y a l u , K.I., ( 4 ) 92 d'Azzo, A . , 16) 58, 124 Dean, D.C., ( 5 ) 264 Dean, P.D.G., ( 8 ) 420, 443, 444, 458 Debanne, M.T., (5) 565 Debeau Fre-Apps, R.J., (5) 780 d e Beer, F.C., ( 5 ) 219, 505 De B i e v r e , C . , ( 4 ) 225 De Boeck, H . , ( 5 ) 133 DeBoeck, S., ( 8 ) 238 De Boer, W . R . , (4) 7, 27 Debray, H., ( 5 ) 123, 150, 548, 558: ( 8 ) 2 39 De Bruyne, C.K., ( 5 ) 133 Debruyne, I., ( 8 ) 238 D e b u i r e , B., (5) 897 D e c a e n s , C . , ( 5 ) 953 Decastel, M . , ( 5 ) 130 Dechavanne, M., ( 5 ) 654 Decker, K . , (7) 59 Decout, D., ( 5 ) 123 d e Crombrugghe, B., ( 5 ) 255 D e f e u d i s , D.F., ( 5 ) 973; (7) 102 D e f i l p o , S.S., ( 7 ) 57 D e f l a n d r e , E . , ( 3 ) 275 De F r a n c o , C.L., ( 6 ) 431 Degand, P., ( 2 ) 35; (50 932, 942, 943, 944, 945 D e g i o r g i o , V . , ( 7 ) 17, 19 De Halac, I.N., ( 3 ) 158 DeHerdt, E., (8) 492 Dekker, J . , 93) 126 Dekker, R.F.H., (6) 496 De L a p e y r i e r e O., ( 8 ) 194 De La Rosa, M A., ( 8 ) 424 D e l b a r r e , F., ( 7 ) 98, 99 D e l b a r t , C., 7 ) 186 d e l Campillo, E., (5)
78 1
Author Index 184; ( 6 ) 1 1 4 d e Leo, A . B . , ( 5 ) 72 D e l e r s , F., ( 8 ) 105 Deley, M . , ( 5 ) 514; ( 8 ) 398 D e l i a , D., ( 5 ) 621 Dell, A . , ( 2 ) 99; ( 4 ) 227: ( 5 ) 893 Delmaere, F . , ( 6 ) 135 Delmelle, M., ( 7 ) 42 Deley, M . , ( 8 ) 398 Delmer, D.P., ( 3 ) 129, 163; ( 5 ) 94 Delmotte, F., ( 3 ) 287 Delmotte, F.M., ( 5 ) 133 De Loach, J.R., ( 6 ) 42 Delphech, B., ( 5 ) 389 d e l ROSSO, M . , ( 5 ) 445, 499; ( 8 ) 329 Delsorbo, F . , ( 6 ) 230 Deluca, V . , ( 5 ) 90; ( 7 ) 174 De Lumen, B.O., ( 3 ) 228 de M.Cruz, M . R . , (8) 471 Demeulle, S . , ( 8 ) 726 DeMeyer, P . , ( 8 ) 492 De Meyts, P . , ( 5 ) 599 Demnerova, K., ( 8 ) 645 Den, H., ( 5 ) 195 Denburg, J.L., ( 5 ) 193 Denton, J . , ( 8 ) 354 d e Neve, R . , ( 5 ) 163 Dennis, C . , ( 3 ) 249 Dennis, P . , ( 6 ) 147 Denton, J . , ( 5 ) 357 d e Paoli-Roach, A.A., ( 5 ) 294 De P a o l i s , A., ( 8 ) 269 d e Pedro, A . , ( 6 ) 70 De Pedro, M.A., ( 6 ) 499 Depirro, R., ( 5 ) 471 Derappe, C . , ( 7 ) 119 De Rosa, M., ( 8 ) 608 Derzko, Z., ( 7 ) 92 Desai, N.N., ( 5 ) 152 De S e r r e s , G., ( 5 ) 474 Deslandes, Y . , ( 2 ) 113; (3) 8 5 Desnick, R.J., ( 5 ) 305; ( 8 ) 126 Desomer, P . , ( 5 ) 514; ( 8 ) 398 Desrochers, M . , ( 6 ) 196; ( 8 ) 614 De Tanhoffer De Volcsey, L., ( 4 ) 31 D e t a r , C.C., ( 8 ) 100 Detroy, R.W., ( 3 ) 73 Deudon, E., ( 5 ) 404 Deuel, T.F., ( 8 ) 426 Deufel, T., ( 5 ) 808
Deugnier, Y . , ( 6 ) 60 Deutsch, E., ( 5 ) 791 Devaux, P.F., ( 5 ) 1018 De Vlieghere, M., ( 4 ) 31 De V r i e s , A.L., ( 3 831 DeVries, G.H., ( 7 ) 95 DeVries, J.A., ( 8 ) 5 67 De Wildt, P.J.M., 3) 141 Dey, P.M., ( 6 ) 113 115 Deykin, D., ( 5 ) 78 D'Hinterland, L.D. (4) 106 D i a s , J.A., ( 5 ) 697 Diaz-Maurino, T . , ( 5 ) 824 D i B l a s i o , B., ( 4 ) 25 de Cioccio, R . A . , (6) 110; ( 8 ) 551 Dick, J . , ( 2 ) 92 Dickens, M.J., ( 7 ) 1 1 Dickerson, A.G., ( 6 ) 87 Dickerson, J.W.T., ( 7 ) 53 Dickson, L.A., ( 5 ) 255 Didelez, J . , ( 8 ) 639 D i e d r i c h , D.F., ( 6 ) 192 Dieleman, B . , ( 6 ) 509 Diener, U . , ( 6 ) 24 Dieringer, H . , ( 5 ) 270 D i e t r i c h , C.P., ( 5 ) 358, 441; ( 6 ) 35 ( 4 ) 180 D i e t z l e r , D.N., Diez, T . , ( 6 ) 70 d i F e r r a n t e , D.T., ( 5 ) 3 92 d e F e r r a n t e , N., ( 5 ) 350, 392: ( 8 ) 277 Dilenna, P., (6) 454 D i l l , K . , ( 5 ) 1017 D i l l e y , W.G., ( 5 ) 866 Dimond, R.L., ( 6 ) 46, 47 Dimov, K . , ( 8 ) 488 D i Natale, P., ( 6 ) 513 D i n i , G., ( 5 ) 445 D i n t z i s , F.R., ( 3 ) 17 Dion, A.S., ( 2 ) 8 5 Dissous, C . , (6) 171 Diven, W.F., ( 6 ) 190 Divies, C . , ( 8 ) 637 Divry, P., ( 5 ) 744 D i x , R.D., ( 5 ) 29 Dlugosz, J., ( 3 ) 283 ( 4 ) 76 Dmitriev, B.A., Do, D.D., ( 8 ) 682 DOane, W.M., ( 3 ) 75 Doctor, V.M., ( 2 ) 2 Dodgson, K.S., (6) 413, 414; ( 8 ) 660 Dodin, G . , ( 6 ) 510
Dodon, M.D.,
hi, A . , Doi, H . ,
( 5 ) 842
( 5 ) 174, 390
( 5 ) 716, 722; ( 8 ) 575 Doig, M.T., ( 8 ) 336 Dolecek, V . , ( 3 ) 45 Dolhofer, R . , ( 5 ) 798 Dolmans, M . , (5) 1072; ( 8 ) 266 Dombradi. V . , ( 5 ) 290 Domek, D.B., ( 4 ) 235 Domingo, M . , ( 8 ) 105 Dominici, R., ( 5 ) 471 Donald, A.S.R., ( 5 ) 538 Donaldson, M.L., ( 6 ) 111 Doner, L.W., ( 3 ) 230 Donnelly, P.V., ( 5 ) 434; ( 6 ) 32 Donovan, J.W., ( 5 ) 224 Dopheide, T.A., ( 5 ) 42, 43, 4 4 Dorai, D.T., (5) 198, 1009; ( 8 ) 229, 261 Doran, T.I., ( 4 ) 116, 161 Dorland, L., ( 2 ) 91; ( 5 ) 558, 720, 729, 748, 929, 937, 972, 968, 990; ( 8 ) 239 Dorling, P.R., ( 6 ) 170 Dorrington, K.J., ( 5 ) 841, 847 Dougherty, J., ( 8 ) 284 Douglas, L.J., ( 4 ) 191 Douglas, S., ( 3 ) 9; ( 6 ) 307 Douste-Blazy, L., ( 6 ) 21 Downie, J.C., ( 5 ) 45 Downs, D . , ( 8 ) 373 Doyle, R.J., ( 6 ) 524 Dragsten, P.R., ( 5 ) 647 Drake, H.L., ( 8 ) 324 Drake, P., ( 6 ) 271 Dreher, T.W., ( 3 ) 293 Drews, G., ( 4 ) 8 9 Dreyfus, H., ( 7 ) 55, 77 Dreyfus, J.C., ( 6 ) 168 Drickamer, K., (5) 194 D r i s k e l l , W.J., ( 5 ) 697 Dryburgh, HG., ( 5 ) 750 D'Souza, M.P., ( 5 ) 908 D'Souza, S.F., ( 6 ) 15; (8) 708 Duarte, E.R., ( 5 ) 991 Duarte, G.R., ( 5 ) 991 Duarte, H.S., ( 5 ) 991, 993 Duarte, J.H., ( 5 ) 991, 993 Dube, D.K.. ( 6 ) 202
782 Dube, S., ( 6 ) 202 DUC, M., (8) 432 Duchet, D., (8) 49 Dudikova, E., ( 6 ) 187 Dufrane, S.P., (7) 22, 42 Duk, M., ( 5 ) 181 Duman, J.G., ( 5 ) 831 Dumont, J.E., ( 8 ) 119 h m o n t , J.M., ( 4 ) 87 Duncan, H.J., ( 3 ) 226 Duncan, M.J., ( 4 ) 155 Duncan, P.H., ( 6 ) 287 Dunlop, D.B., (8) 635 Dunn, A . J . , ( 7 ) 3 0 Dunn, N.W., ( 6 ) 353, 371 Dunn, W.L., ( 5 ) 480 Dunnhill, P., (8) 540 D u n n i l l , P., ( 8 ) 625, 701 du P l e s s i s , D.H., ( 5 ) 67 Dupoisot, H., ( 7 ) 98, 99 Dupond, J.L., ( 6 ) 25 Dupouey, P . , ( 7 ) 159 Dupuis, D., ( 5 ) 656, 906 Duran, A . , ( 4 ) 211; ( 6 ) 529 Durand, G., ( 5 ) 603; (8) 600 ( 5 ) 522, Durand, G.M., 1021 Durand, G.M.S., ( 7 ) 34 Durand, P . , ( 6 ) 250 Durbin, M.L., ( 3 ) 123; ( 6 ) 350 Durdik, J.M., ( 7 ) 151 Durette, P.L., (8) 8 Durr, M., ( 5 ) 90; ( 7 ) 174 Dussourd d ' H i n t e r l a n d , L., ( 7 ) 195 D u t e u r t r e , B., ( 4 ) 231 Duthie, M., ( 6 ) 104 D u t t , A.S., ( 3 ) 209 Dutton, G.G.S., ( 4 ) 103, 104 Dwek, R.A., ( 5 ) 889 J h r l a n d , L., ( 5 ) 890 Dyatlovitskaya, E.V., ( 7 ) 47 Dyck, R.F., ( 5 ) 505 Eaton, D.R., ( 3 ) 95 E b e r h a r d t , N.L., (8) 344 E b e r l e , R., ( 5 ) 2 8 Ebisu, S., ( 5 ) 134 Ebner, K.E., ( 5 ) 708,
Carbohydrate Chemistry 1066 Ebringerova, A . , ( 3 ) 106 Eby, R., ( 8 ) 2 , 1 4 Echigo, T . , ( 3 ) 8 9 Edge, A.S.B., ( 5 ) 926, 1014 Edmond, B.J., ( 5 ) 8 5 Edmonds, J.S., ( 3 ) 302 Edward, N., ( 6 ) 2 3 Edwards, B . , ( 4 ) 37 Edwards, H.H., ( 5 ) 660 Edwards, J.R., ( 4 ) 165 Edwards, K.G., ( 4 ) 158 (5) Edwards, P.A.W., 62 1 Egan, W., ( 4 ) 99 Egawa, K., ( 6 ) 267; ( 7 ) 201 Egge, H., ( 7 ) 82, 83, 113, 114, 155 ( 8 ) 106 Egly, J.M., Egorin, M.J., ( 5 ) 170 Egorov, A.M., ( 6 ) 475 Egorova, V.V., ( 6 ) 77 Eguchi, T . , ( 8 ) 224, 225 Eguchi, Y . , ( 8 ) 225 Ehara, M., ( 6 ) 270 Ehlerding, U . , ( 5 ) 147 Ehrismann, R . , ( 5 ) 228 Eid, P . , ( 6 ) 258 E i f l e r , R . , ( 5 ) 205 Eikenberry, ( 5 ) 255 Einarsson, M., (8) 147 E i s e n b a r t h , G.S., ( 8 ) 599 Eisenberg, D., (8) 222 E i s e n s t e i n , S.M., ( 5 ) 443 Ejzemberg, R . , ( 8 ) 572 Ek, K . , ( 5 ) 117 Ekstrdm, B., ( 5 ) 962 Ekstrdm, D., ( 5 ) 963 E l a h i , M., ( 3 ) 246 Elander, M., ( 3 ) 288; ( 6 ) 51 E l b e i n , A.D., 95) 1053, 1055 E l i s a s h v i l i , V.E., ( 6 ) 536 El-Kashouti, M . , ( 8 ) 484 E l Khadem, H.S., ( 8 ) 26 E l l i o t t , H . , ( 5 ) 355, 356 E l l i s , S., ( 5 ) 680; ( 8 ) 363 E l o d i , S., ( 5 ) 658 Elbdi, P., ( 8 ) 405 ( 8 ) 484 El-Rafie, M.H., E l s , H.J., ( 4 ) 93
ElsHsser-Beile, U., ( 4 ) 102; ( 6 ) 409 E l - S i s i , F., ( 8 ) 484 Eltekov, Y.A., ( 2 ) 18 E l v e r s , J . , ( 8 ) 17 Elyakova, L.A., ( 6 ) 299 Embi, N . , ( 6 ) 528 Emeruwa, A . C . . 94) 144 Emmerling, W.N., ( 3 ) 41 EmBd, I . , ( 8 ) 308 Emonds-Alt, X . . ( 5 ) 304 Emura, M., ( 8 ) 353 E n a r i , T.M., ( 6 ) 341, 345; (8) 464 Endo, H., ( 8 ) 372 Enfors, S.O., ( 2 ) 71; ( 8 ) 698 Engdahl, G . , ( 7 ) 51 Engelhorn, S.C., (8) 400 Engeset, J . , ( 6 ) 2 3 England, J.D., ( 5 ) 802 Englander, S.W., ( 5 ) 82 1 E n g l a r , J.R., ( 3 ) 263 Englard, S., ( 5 ) 285: ( 6 ) 502 Engvall, E., ( 5 ) 213, 227, 230, 540; (8) 2 33 Engwall, E . , ( 5 ) 210 Enikeeva, A . K . , (8) 465 Enriquez, A . , ( 6 ) 370 Epifanio, E.C., ( 3 ) 273; ( 4 ) 204 Erard, F ., ( 5 ) 817 E r d e i , S., ( 5 ) 50 Erickson, R.P.. ( 5 ) 626 E r i k s s o n , K.E., ( 8 ) 132 Erikssoon, L.C., ( 5 ) 1068 Erneux, C., ( 8 ) 119 E s c o u r o l l e , R . , ( 7 ) 69 Eshdat, Y . , ( 5 ) 901; ( 8 ) 374 Espinosa, M . , ( 4 ) 6 E s s e r , P . , ( 6 ) 255 Esteban, M., ( 6 ) 9 E t c h e b e r r i g a r a y , J.L., ( 6 ) 133 E t c h i s o n , J.R., ( 5 ) 61 Eto, Y . , ( 7 ) 105 E t z l e r , M.E., ( 5 ) 153, 154, 155, 156. 157, 158; ( 8 ) 397 Evangelatos, G . P . , ( 7 ) 9 Evangelopoulos, A.E., ( 6 ) 415, 532 Evans, F.J., ( 6 ) 533 Evans, J.O., ( 6 ) 192 Eveleigh, D.E., ( 6 ) 355
783
A uthor Index Evenson, J . P . . ( 3 ) 32 E v e r e t t , M . M . , ( 6 ) 234 Ewars, E . , (5) 1016 E w e n s t e i n , B . M . , 611, 628 E x t o n , J . H . , ( 5 ) 292; ( 6 ) 527 E v e , D . R . , ( 5 ) 257 Ezepchuk, Yu.V., ( 6 ) 254: ( 8 ) 154 F a b e r , S., ( 3 ) 154 Fabia, F., ( 5 ) 905 F a b i o , J . Di., ( 4 ) 104 F a b r e , J . W . , ( 5 ) 450, 45 1 F a c c i , L . . ( 7 ) 56 Fahey, D . , ( 5 ) 515; ( 8 ) 431 F a h n r i c h , P . . ( 6 ) 369 F a h r e n k r u g , J . , (6) 421 F a i r , W . R . , ( 5 ) 352; ( 6 ) 166 F a l a s c a , A . , ( 5 ) 100 F a l e s , F.W., (3) 11 F a l k , K . E . , ( 7 ) 111. 193 F a l l a n i , A , , (7) 78 Faltynek, C . R . , (5) 416, 1014 F a m u l a r i , N . G . , ( 5 ) 19 Fan, L . T . , (6) 340 F a n c e y , K.S., ( 3 ) 270 Fang, P . , ( 3 ) 114 Fange, R . , ( 6 ) 19 F a n n i n , F.F., ( 6 ) 192 F a r e e d , J . , ( 5 ) 755 F a r k a s , V . , ( 6 ) 210 F a r o o q u i , A . A . , ( 6 ) 63; (8) 590, 595 F a r q u h a r , M.G., ( 5 ) 400 F a r v e r , 0.. (5) 885 F a r w e l l , D . C . , ( 2 ) 85 F a s s , D . N . , (5) 869 F a s t , D . M . , ( 6 ) 287 F a u b i o n , J . M . , (3) 200 F a u r e , A . , ( 5 ) 126 Faye, L . , ( 6 ) 83, 84, 85 F e a r n , T . , ( 6 ) 307 Feeney, R.E., (5) 816 F e g e r , J . M . , ( 5 ) 1021 F e i g e , U . , (4) 82 Feighny, R . J . , ( 5 ) 68 F e i l d , J . A . . ( 5 ) 81 F e i n g o l d , D . S . , ( 2 ) 15 Feinman, R . D . , ( 5 ) 732 F e i n s t e i n , A . , ( 5 ) 219 F e i z i , T . , ( 5 ) 597, 860, 948, 949; ( 8 ) 186 Felgner, P . L . , (7) 21
F e l l i , P . , ( 5 ) 471 F e l l i n i , S . A . , ( 5 ) 426 F e l o n , M . , ( 5 ) 126 F e l s t e d , R . L . . ( 5 ) 170. 172 F e l t s , J . M . , ( 5 ) 433 F e n e l o n , S., ( 7 ) 69 F e n t o n , J . W . , ( 5 ) 764 F e r e n c i , T . , ( 4 ) 134 F e r r a g h t , J . A . , ( 8 ) 182 F e r r a n t e , N . D . , ( 6 ) 32 F e r r a r i , B., ( 5 ) 1017 F e r r a r i , T.E., ( 5 ) 89 F e s s l e r , J . H . , ( 5 ) 282 F e s s l e r , J . G . , ( 5 ) 281 F e s s l e r , L.E., ( 5 ) 281, 282 F e v r e , M., ( 3 ) 166 Fex, G., ( 5 ) 964 F i a t , A . M . , ( 5 ) 717, 720 F i b b i , G . , ( 5 ) 445 Fichtinger-Schepman , A . M . J . , ( 3 ) 297, 298 F i d e l i o , G . D . , (7) 87 F i e l d , M . J . . 95) 502 F i e t z e k , P . P . . ( 5 ) 255 Figlewicz, D.A., (5) 4 62 F i g u r a , K.V., ( 6 ) 519 F i g u r e s , W . R . , ( 4 ) 165 F i l i m o n o v , V.V., (5) 222 F i n c h e r , G.B., ( 3 ) 164 Finkelman. M . , ( 6 ) 344 F i n n , C.W., (4) 95 F i n n , F.M., ( 8 ) 333 F i n n e , J . , ( 2 ) 108, 110; ( 5 ) 904, 1025; ( 7 ) 10; ( 8 ) 220 F i n n e y , K.F., ( 6 ) 305; ( 7 ) 168 Firantene, R.K., (6) 3 92 F i r g a i r a , F.A., ( 8 ) 338 F i s c h e r , M . H . , ( 5 ) 979 F i s c h e r , W . , ( 4 ) 15 F i s h e r , L.W., ( 5 ) 335 Fishman, M . C . , ( 5 ) 647 Fishman, P . H . , (5) 687; ( 7 ) 23; ( 8 ) 579 F i t z g e r a l d , F., ( 5 ) 600 Fitzgerald, J.W., (6) 516, 517, 518 F l a h e r t y , L., ( 5 ) 610 F l a n d r o i s , J . P . , (4) 14; ( 5 ) 177 F l e e t , G.H., ( 6 ) 381 F l e i s s n e r , E., ( 5 ) 7 2 Fleming, S.E., ( 6 ) 92 F l i c k i n g e r , M.C., ( 6 ) 4 77
F l o r e z , I.G., ( 6 ) 120 F l o s s , H . G . , ( 3 ) 225 F l o u r e t , B., ( 4 ) 2 4 Flower, R . L . P . , ( 5 ) 604 F l t l c k i g e r . R . , ( 5 ) 801 F l o w e r s , H . M . , ( 3 ) 210 F o c h e r , B., ( 3 ) 117; ( 6 ) 332; (8) 411 F o g a r t y , W . M . , ( 6 ) 182 F o g l i e t t i , M. J . , (6) 108 F o i d a r t , J . M . , (5) 242 F o l a n , M.A.', ( 6 ) 360 F o l e y , D . , ( 6 ) 147 F o n e s c a , G., ( 5 ) 655 F o n t a i n e , M . , ( 5 ) 746; (8) 360 F o n t a n a , J . D . , 95) 991 F o n t a n g e s . R . , ( 4 ) 87, 106; ( 7 ) 195 Foord, S . A . , ( 3 ) 264, 283 F o r b e s , J . T . , ( 8 ) 339 F o r d , R . , ( 5 ) 216 F o r e s t e r , H . , ( 4 ) 16 F o r i e r s . A . , ( 5 ) 163 Forman, C . , ( 4 ) 118; (5) 874 F o r r y , K . R . , ( 5 ) 853; ( 8 ) 166 F o r s b e r g . B., ( 8 ) 147 F o r s b e r g , C.W., ( 6 ) 223, 227 F o r s b e r g , L . S . , ( 6 ) 401 Forsee, W . t . . ( 5 ) 419; ( 6 ) 241; ( 8 ) 240 F o s s e t t , N.G., ( 5 ) 659, 661; ( 8 ) 574 F o t h e r g i l l . J.E., (5) 984 Fothergill. L.A., (5) 984 F o u a s s i e r , J . P. , ( 8 ) 487, 561 F o u g e r e a u , M . , ( 6 ) 293 F o u l k e , R.S., ( 5 ) 71 F o u r n e t , B., ( 2 ) 96, 98, 106; (5) 558, 748, 883, 895, 937, 1024; ( 8 ) 206, 239 F o u r n i e r , J . M . , ( 4 ) 107 Fowler, A . V . , ( 6 ) 146, 150, 151 Fox, L . , ( 8 ) 133 Fox, P . C . , ( 6 ) 345 Fraga, J.M., (2) 24 F r a i s s e , M . , ( 6 ) 163 F r a n c e s c h i , C . , ( 5 ) 100 Francesconi, K.A., (3) 3 02 Franchimont, P . , ( 5 ) 299, 313
Carbohydrate Chemistry
784 F r a n c i s , C.W., ( 5 ) 785 F r a n c k e , U., (6) 408 F r a n c o i s - G e r a r d , C., (5) 987 F r a n g o u , S.A., ( 3 ) 266 F r a n k , G.H., (6) 256 Frank, H., ( 2 ) 8 F r a n k , J . F . , (6) 159 F r a n k , R . M . , ( 4 ) 159 F r a n k e l , M.E., (5) 46 F r a n s e n , A . , ( 5 ) 325 F r a n s s o n , L.A., ( 5 ) 309, 311, 333, 337, 338, 339, 376, 377, 378: ( 8 ) 276, 370, 371 F r a n t z i s , N., ( 5 ) 670 F r a n z , H . , (5) 112, 167 F r a n z e n , L.E., ( 4 ) 152 F r a s e r , B.A., (2) 102: ( 4 ) 112 F r a s e r , I . H . , ( 5 ) 1077 F r a t e r , R . , (5) 405 F r a z i e r , W.A., ( 8 ) 259 F r e d e r i c k , J.F., (3) 30 1 Fredmam, P., ( 7 ) 49 Freeman, A . , (8) 646 F r e e s e , E.B., ( 4 ) 216: (6) 95 F r e e z e , H.H., ( 6 ) 69 F r e i r e , E., (7) 21 F r e j a v i l l e , C.M., ( 3 ) 47 F r e i r e Rama, M., ( 6 ) 18, 112 F r e n c h , F.S., ( 5 ) 491 F r e n c h , G.R., (5) 71 F r e n k e l , E . , ( 6 ) 56 Frenoy, J.P.. (5) 130 F r e u n d , T.G., ( 5 ) 120 F r e y , R . , (5) 183 F r e y s s i n e t , J.M., ( 5 ) 787, 790 F r i a u f . W.S., ( 2 ) 7 4 F r i d o v i c h , S.E., ( 5 ) 563 F r i e b o l i n , H., ( 7 ) 15 F r i e d , H., (5) 63 F r i e d , V.A., ( 6 ) 153 F r i e d l a n d e r , P., (5) 481 F r i e d m a n , H.P., ( 5 ) 120, 161 F r i e d r i c h s , W.E., ( 5 ) 85 F r i e n d , J . , ( 3 ) 220 F r i s c h , A . , (6) 55 Frommelt, H., ( 3 ) 109 F r o s t , L.S., (4) 179 F r o s t , R.G., ( 8 ) 400 F r u s h , H.L., (2) 61
F u , S.C., ( 5 ) 455 Fuchs, W . , (5) 432; ( 6 ) 519 F u d e n b e r g , H.H., (4) 110 Fuhrman, H.S., ( 8 ) 516 F u j i i , H . , (5) 911 F u j i i , M., ( 6 ) 268 F u j i i , N., (5) 340 F u j i k a w a , T., ( 3 ) 294 F u j i m o t o , F.K., (8) 152 F u j i m o t o , K., ( 5 ) 1047 F u j i m u r a , T., (8) 609, 610 F u j i m u r a , Y., ( 2 ) 66 F u j i n a m i , R.S., (5) 73 F u j i n o , M., ( 5 ) 681: (8) 187 F u j i n o , Y., ( 7 ) 169, 170, 184, 185 F u j i o , H., ( 6 ) 429: ( 8 ) 6 62 F u j i s a w a , H., ( 8 ) 408 F u j i s h i m a , T., (8) 477, 686, 731 F u j i t a , H., (4) 219 Fujita-Yamaguchi, Y.. (5) 1062: (6) 504: ( 8 ) 273 F u j i y o s h i , T., ( 8 ) 479 F u j k i , H., (6) 286 F u j i t a , Y, ( 6 ) 95 Fukamizo, T., (6) 440 Fukuda, H . , ( 3 ) 285, 286; (8) 652 Fukuda, I., ( 8 ) 331 Fukuda, K., (5) 914 Fukuda, M . , ( 5 ) 223, 642, 645, 949: ( 7 ) 35: ( 8 ) 205 Fukuda, M . N . , ( 5 ) 156: ( 7 ) 35: ( 8 ) 263 Fukuda, N., ( 6 ) 115 F u k u h a r a , H., ( 4 ) 35 F u k u i , H . , (5) 784 F u k u i , S., ( 5 ) 430: ( 8 ) 629 F u k u i , T., ( 8 ) 123, 437, 739 Fukushima, K . , ( 4 ) 164 F u l l e r , G.M., (5) 825: ( 8 ) 359 F u n a k o s h i , I., ( 5 ) 379; (6) 531 F u r b i s h , F.S., ( 6 ) 56: (7) 134 F u r c h t . L.T., ( 5 ) 242 F u r l a n , M . , (5) 775 F u r l o n g , R.A., ( 5 ) 770. 786 F u r t h m a y r , H., ( 5 ) 973, 917
F u r u h a t a , K.. ( 2 ) 112 F u r u t o , D . K . , ( 5 ) 262 F u t e r m a n , C.L., ( 6 ) 521 Fuwa, H . , ( 3 ) 36, 39: ( 8 ) 383 F y f e , J.A.M., ( 4 ) 146 Gabbay, K.H., (5) 801 G a b e l , D., ( 8 ) 109 Gabler-Kolacsek, I., (2) 11 Gacesa. P., ( 6 ) 413, 414; (8) 660 Gadd, G.M., ( 5 ) 114: ( 8 ) 218 Gade, W . , ( 5 ) 160: ( 8 ) 207 Gagnon, J . , ( 5 ) 593 Gahmberg, C.G., ( 5 ) 546, 904, 921: ( 8 ) 208, 294 Gahmberg. G.G., ( 8 ) 220 Gajewski. A . , (7) 61 Gal, A.E., ( 7 ) 146 G a l a n o s , C., (4) 52 Galas, E . , ( 6 ) 343 G a l b r a i k h , L.S., ( 3 ) 48 G a l i c k i , N.I., ( 7 ) 44, 123 G a l j a a r d , H . , ( 6 ) 58 G a l l a g h e r , J.M., ( 5 ) 87 1 G a l l a n t , J . , ( 6 ) 147 G a l l a s c h , E., (5) 919 G a l l i h e r , P.M., ( 6 ) 412 G a l l o , R.C., (8) 285 G a l l o p , P.M., ( 5 ) 801 Galloway, D . R . , (5) 639 G a l u n s k y , B., ( 8 ) 598 G a l y a a r d , H . , ( 6 ) 124 G a l z y , P., ( 6 ) 420: ( 8 ) 726 Gamarra, M. . ( 5 ) 835; (8) 202 Gambacorta, A., ( 8 ) 608 Ganguly, P., (5) 659, 661: ( 8 ) 574 Ganguly, S., ( 8 ) 334 Ganno, S., ( 2 ) 48, 52 G a n s e r a , R., (5) 121 G a r b i s a , S., ( 5 ) 260: (8) 249 Garcia, I., ( 5 ) 849 Garcia-Acha, I . , ( 4 ) 211; ( 6 ) 529 Garcia-Alonso. J . , ( 7 ) 40 G a r d n e r , W.T., ( 5 ) 368 Garegg, P . J . , ( 5 ) 139: ( 8 ) 15, 16, 24, 27 Garg, H.G., (5) 442 G a r g i u l o , A.M., ( 6 ) 131
Author Index G a r n i e r , J . , (5) 678 Garon, C . F . , ( 4 ) 95 Garon, S . J . , (5) 433 Garten, W . . ( 5 ) 47, 48 Gartner, T . K . , ( 5 ) 509 Garver, F . A . , ( 5 ) 845, 846 Gasa, S . , ( 7 ) 41 Gascon, S., ( 6 ) 120 Gasyna, Z.. ( 8 ) 720 Gathmann, W . D . , ( 5 ) 937, 1022; ( 6 ) 109 G a t t , S., ( 6 ) 161, 253 G a t t e g n o , L . , ( 5 ) 905 G a t t i , R . , (6) 250 G a t t i k e r , H . , ( 5 ) 345 G a t t l e n . C . , 96) 342 Gaudioso, D., ( 6 ) 497 Gaugler, R . W . , ( 6 ) 378 ( 8 ) 542 Gaur, N . , ( 5 ) 109. 169 Gautheron, D . C . , ( 8 ) 672, 696 Gawthorne, J . M . , ( 6 ) 170 Gaye, P . , P5) 723 G a z i t , B . , (6) 253 G a z i t t , Y . , ( 5 ) 827 Gebauer, G., (5) 182 Gebicka, L . , ( 8 ) 720 G e i l i n g e r , I . , (3) 33 Geilinger. K . . (5) 70 G e i e r , T . A . , (3) 132 G e l b , R . I . , ( 8 ) 516 Gelder, F . B . , (7) 33, 37 G e l l f , G., ( 8 ) 626, 627, 651 G e l p i , M . E . , ( 8 ) 25 Gemeiner, P . , (8) 497, 54 1 G e n i e s e r , H . G . , ( 8 ) 109 Genko, Y . . (8) 157 Genton, E . , ( 5 ) 668 Geoghegan, K . F . , ( 5 ) 81 6 George, J . N . , ( 5 ) 648, 668 George, J . R . I ( 6 ) 516, 517 Georgopoulos, C . , ( 5 ) 61 Gerard, P . , ( 8 ) 639 Gerday, C . , (5) 987 Gerhard, W . , ( 5 ) 46 Gerok, W . , (5) 147 Gerrard, J . M . , ( 5 ) 509 Gershanovich, B . N . , ( 6 ) 143 G e r s t e n , D . M . , ( 8 ) 442 G e r s t n e r , E . , (4) 81 Gery, I . , ( 7 ) 100
785 G e t t i n s , P . , (5) 889 Geunet, L., ( 6 ) 122 Ghia, S.K., (4) 153, 154 Ghayman, E., ( 5 ) 336 Gherardini, F . , ( 4 ) 176 G h i d o n i , R . , ( 7 ) 12, 17, 18, 19 Ghim, Y.S., ( 8 ) 494 Ghommidh. C., (8) 600 Ghosh, P . . ( 7 ) 91 Ghosh, T . K . , ( 3 ) 155 Ghuysen, J.M. ( 4 ) 29 Gianazza, E . , ( 5 ) 117 G i a n f r e d a . L . , ( 8 ) 656 G a i n n i s , D . E . , (5) 840 Gibbons, R . J . , ( 5 ) 119 Gibey, R . , (6) 25 Gibson, K . D . , ( 5 ) 259 G i d l e y , M . J . . (3) 213, 214, 215 Giesberts, M.A.H., (5) 2 96 G i l b e r g , W . . ( 5 ) 432; (6) 519 G i l b e r t , A . S . , ( 4 ) 111 G i l b e r t , R . D . , (8) 459 Gilboa-Barber, N . , ( 5 ) 159 G i l e a d , Z., ( 5 ) 827 G i l l a r d , B . K . , ( 6 ) 330 Gilles, R . , ( 5 ) 97 Ginsberg, M . H . , ( 5 ) 233 Ginsburg, V., ( 5 ) 857; (7) 24, 32; ( 8 ) 326 G i r o t , P . , ( 8 ) 596 Gitelman, A . K . , (5) 37 Glabe, C . G . , (5) 586 Glad. M . , (2) 27 G l a i s e , D . . ( 6 ) 60 G l a n v i l l e , R . W . , (5) 270 G l a s e r , G . H . , ( 7 ) 54 G l a s s , E . , (5) 637 G l a s s , J . , 95) 585 G l a s s , M . R . , (5) 747 G l a s s , W.F., ( 2 ) 55; (5) 1012; ( 8 ) 490 Glaudemans, C . P . J . , ( 4 ) 94; (5) 856, 888, 889; ( 8 ) 36 Gleitsmann, B . , (8) 474 Glemzha, A . A . , ( 6 ) 183 Glen, R . H . , ( 6 ) 190 Glenney. J . R . , ( 5 ) 553 G l e w , R . H . , 95) 533, 751; ( 8 ) 423, 427, 435 G l i c k , M . C . , ( 6 ) 103 G l b s s l , J . , ( 5 ) 391; ( 6 ) 62; ( 8 ) 250 Glover, D.V., (3) 36
Glowacki, J . , ( 5 ) 266 Gludhova, M.A , ( 5 ) 222 Gmeiner, J . , 4 ) 43, 72 G o d e l a i n e , D. ( 5 ) 470 Goel. M . . ( 6 ) 309 Goetinck, P . F . , ( 5 ) 322, 447 Gogstad, G.O., ( 5 ) 508, 667 Goh, Y . , ( 8 ) 68, 69, 70 Goi, G . C . , (6) 26 Golan, M . D . , ( 5 ) 794 Gold, A . H . , ( 5 ) 287 Gold, A . M . , ( 6 ) 407 Gold, D . V . , (5) 958 Gold, E . W . , ( 5 ) 429 Gold, P . , (5) 481 Goldberg, E . , ( 6 ) 417 Goldberg, M . , ( 5 ) 1003 G o l d f a r b , R . H . , ( 5 ) 497 G o l d f i n e , I . D . , ( 5 ) 187 Goldstein, D.E., (5) 8 02 G o l d s t e i n , G., ( 5 ) 627 Goldstein, I . J . , (5) 106, 132, 134, 139, 1067; ( 8 ) 11, 77 G o l d s t e i n , L . , ( 8 ) 88, 470 G o l d s t e i n , S., ( 5 ) 823 Gomez, E . I . V . , (8) 471 Gomez d e G r a c i a , A . , (7) 77 Gomez-Moreno, C . , ( 8 ) 424 Gonda, S.R., ( 5 ) 221; (8) 577 Gong, C . S . , ( 4 ) 200; (6) 205, 215, 477 G o n g g r i j p , R . , (4) 73 Good, R . A . , (5) 886 G o o d e l l , W . ( 4 ) 32 Goodman, H . , (4) 49 Goodman, L., ( 8 ) 20 Gooi, H . C . , (5) 860 Goormaghtigh, E . , ( 7 ) 22, 29 Goosen, M . F . A . , ( 8 8 ) 5 97 Gopalkrishnan, K . , ( 6 ) 218 Gordon, A . H . , ( 3 ) 188 Goren, M . B . , ( 7 ) 198 Gorin, P . A . J . , ( 2 ) 87; ( 3 ) 183; ( 4 ) 220; (5) 993 Gormally, J . , ( 3 ) 134 Gormley, R . , ( 3 ) 229 Goro, A . , ( 6 ) 297 Goryachenkova, E.V., ( 8 ) 160
Carbohydrate Chemistry
786 (5) 946 Gospodarowicz, D. , ( 5 258, 277 G o s s i , G., ( 2 ) 74 Got, R., (5) 1064 G o t o , S., ( 8 ) 565 Goto, T . , (8) 700 G o t s c h l i c h , E.C., ( 4 ) 112, 113 Govan, J.R.W., ( 4 ) 146 Governman, J.M., (5) 6 92 Gowda, D.C., ( 3 ) 190 Gowda, J.P., (3) 190 Gowlland, L., ( 5 ) 680: (8) 363 G r a b e l , L.B., ( 5 ) 586 Graham, A.B., (8) 345 Graham, H.A., ( 7 ) 112 Graham, H.D., (3) 31 G r a n e t t , S., ( 4 ) 181 G r a n t , B.R., (3) 293 G r a n t , D.A.W., ( 5 ) 543: (8) 268 G r a n t , D.R., ( 3 ) 24, 25 G r a n t , G., (5) 171 Grantham, J. J., ( 5 ) 265 G r a s d a l e n , H . , (3) 265: ( 6 ) 483 Grassl, M., ( 3 ) 52; ( 6 ) 288 G r a t z e r , W.B., ( 5 ) 900 G r a v a l l e s e , E. ,(5) 809 Craw, J . , ( 8 ) 428 Gray, G . R . , (4) 221 G r a y , M.J., ( 2 ) 41: ( 5 ) 1027 G r a y , P.P., ( 6 ) 353, 37 1 G r a y , S.L., ( 5 ) 587 G r e a v e s , M., (5) 621 Greco, G., ( 8 ) 656 Green, B.G., (5) 630 Green, C . , ( 2 ) 2 Green, R.W., (5) 65 G r e e n b e r g , E . , ( 4 ) 156 G r e e n b e r g , N.A., (8) 748 G r e e n l a n d , T . , ( 5 ) 654 G r e e n w e l l , P . , (5) 1033 G r e g r , V . , ( 4 ) 190 G r e i l i n g , H . , (5) 394, 395, 398, 437 Greir, T . J . , (6) 523 G r e i s i g e r , L.M., ( 8 ) 491 Gresham, H.D., ( 5 ) 792: ( 8 ) 279 G r e s i k , E.W., ( 6 ) 298 Gressel, J . , (3) 210 G r e s s n e r , A.M., ( 5 ) 398, 410 Goso, K . ,
G r e t h l e i n , H . , ( 3 ) 122 Grey, A., (5) 982 G r e y , A.A., ( 5 ) 983, 1019 G r i e r s o n , D., ( 3 ) 244: (6) 451 G r i f f i n , B., ( 5 ) 773 G r i f f i n , M.M., ( 8 ) 181 G r i f f i t h s , R., ( 5 ) 502 G r i g o r a s h , S.Yu., ( 3 ) 121: ( 6 ) 336, 338 Grimes, W.J., (5) 9 G r i v e t , J.P., ( 3 ) 287 Groh, H., (8) 621 Groot Wassink, J.W.D., (6) 92 Grose, C., ( 5 ) 85 G r o t e , E., (6) 222 Grove, D . S . , ( 2 ) 78: (8) 212 Grubb, A . , ( 5 ) 964 GrUtter, M.G., (6) 433 Gruezo, F.G., ( 5 ) 176 Grundy, S.M., (5) 263 G r u n e r , K.R., ( 7 ) 149 Grynpas, M.D., (5) 257 G u a z z e l l i , C., ( 5 ) 445 Gudin, C . , ( 3 ) 292 Guenounou, M., ( 5 ) 522, 603; (7) 34 G u i l b a u l t , G.G., ( 8 ) 668, 729 G u i l d , B.C., ( 5 ) 619 Guillaume. J.-B., (6) 135 G u i l l e m i n , R.C., ( 8 ) 71, 72 G u i l l o c h o n , D., ( 8 ) 704 G u i l l u y , R., ( 7 ) 195 G u i r a n d , J . P . , ( 6 ) 420: (8) 726 G u i s a n , J.M., ( 8 ) 732 G u i t t o n , J . D . , (5) 503 G u l l , K., ( 6 ) 530 Gunasekaran, M . , ( 6 ) 32 0 C u p t a , G.S., ( 6 ) 37, 417 G u p t a , K.C., ( 5 ) 105: (8) 547 Gupta. M . , ( 6 ) 359 Gupta, S . , (5) 154, 157 Guranowski, A.B., ( 8 ) 149 Gurd, J.W., ( 5 ) 455, 5 32 G u r e v i t c h , R . , ( 8 ) 470 G u r l i t z , R.H.G., (3) 112 C u r n a n i , S., ( 6 ) 432 Gurov, A.N.. (8) 537 G u s l a n d i , M . , . ( 5 ) 928
G u s t a p s o n , G.L., (6) 45 G u s t a v s s o n , H., ( 5 ) 309 G u t h r i e , J.T., (8) 581 Gysen, P., ( 5 ) 299, 313 Haagensen, D.E., ( 5 ) 866 Haar, L., ( 5 ) 31 Haass, D . , ( 5 183 Habeeb, A.F.S A., ( 8 ) 410 Habener , J. F. ( 5 ) 699, 700 Habu, S., ( 7 ) 144 Hackman, R.H., ( 5 ) 1003 Hadwiger, L.A., ( 4 ) 234: (5) 880 Hadziyev, D., ( 3 ) 245 H a e f f n e r c a v a i l l o n , N., ( 5 ) 841 H a e g e l e , E.O., ( 3 ) 52: (6) 288 HBmmerling, U . , ( 5 ) 72 Hagen, I., (5) 508, 667: ( 8 ) 376 Hagen, S., ( 5 ) 463 Hahn, L . C . , ( 6 ) 431 Hahn, M.G., ( 3 ) 232 Hahn, T.R., ( 8 ) 454 Hahn-Hagerdal, B . , ( 8 ) 624 H a i g l e r , C . H . , ( 3 ) 130: (4) 175 Hakima, J . , ( 5 ) 55, 982, 983 Hakomori, S., ( 5 ) 949 Hakomori, S.I., (5) 223, 592, 859, 861, 862: ( 7 ) 150, 151 H a l a n t - P e e r s , M.C., ( 5 ) 9 90 H a l a v e n t , C., ( 5 ) 389 H a l e y , J . E . , (7) 65 H a l l , C . A . , ( 8 ) 448 H a l l , D.O., (8) 613 H a l l , L.D., ( 2 ) 88: ( 8 ) 506, 507 H a l l , M.A., ( 3 ) 220 Hall, P.K., (5) 743 H a l l , R., ( 5 ) 872; ( 8 ) 2 88 H a l l e y , D.J.J., (6) 58 Halliday, D.J., ( 2 ) 12 Halliday, M.I.. (5) 819 H a l l i m a n , F., (5) 828 H a l l i w e l l , G., ( 6 ) 385 Halmi, N.S., (5) 706 H a l p e r i n , G., ( 8 ) 108 Hamada, A . , (5) 914, 915, 916 Hamada, N . , ( 6 ) 384 Hamada. S.. ( 4 ) 168
787
Author Index Hamada T., (4) 223, 224; ( 5 ) 148; ( 8 ) 533 Hamaguchi, A . , (8) 498 Hamaguchi, K . , ( 6 ) 438, 439 Hamdy, M.K., ( 6 ) 329 Hamelin. J . , (5) 705 Hamers, R . , ( 5 ) 998 Hamilton, J . A . , (8) 515 Hamilton, J . W . , ( 5 ) 473 Hammer, C.H., ( 5 ) 792; ( 8 ) 279 Han, M.H., ( 8 ) 633, 680 Hanada, E . , (7) 96 Hanaoka, Y . , ( 5 ) 675 Hancock, I . C . , (4) 2, 4, 5, 12, 13 Hancock, J . G . , ( 3 ) 240 Hancock, R . E . W . , ( 4 ) 45 Hancock, W.S., ( 8 ) 150, 151 Handa, T., ( 3 ) 10 ( 8 ) 461 Hanfland, P . , ( 5 ) 858; (7) 82, 83, 112 113, 114, 155 Hanke, D . W . , ( 6 ) 92 Hankins, C . N . , ( 5 184; (6) 114 Hanneken, A . , ( 5 ) 801 Hannoun, C . , (6) 258 Hanover, J . A . , ( 5 ) 1032 Hansen, H.F., (5) 735, 74 1 Hansen, W . , ( 6 ) 7 6 Hansley, M., (5) 866 Hanson, B . A . , ( 2 ) 47 Hanson, C . A . . (5) 682 Hanssen, E . , ( 5 ) 189 Hansson, G . C . , (2) 93; (7) 84, 85, 131, 153 Hansson, H., (8) 396 Hansson, H . A . , ( 8 ) 664 Hansson, L . , (2) 27 Hanyu, T. , ( 5 ) 324 Hapner, K . D . , ( 5 ) 125 Hara, A . , ( 5 ) 867; ( 8 ) 184 Hara, C . , ( 4 ) 247 Hara, M . , (8) 145 Hara, T . , ( 8 ) 372 Harada, K . , 94) 50 Harada, T., ( 4 ) 151, 153, 154: (6) 388, 3 89 Haraldson, A . , (4) 197 Harano, Y . , (8) 157, 676 Harata, K . , ( 3 ) 78, 79 Hardesty, B . , (5) 294 Hardham, L . E . , (4) 40 Hardingham, T . E . , ( 5 )
327 Hardisson, C . , ( 4 ) 201, 246; (6) 157 Hardisty, R . M . , ( 5 ) 780 Hardy, L . , (4) 16 Harford, J . , ( 5 ) 560 Haris, A . , ( 6 ) 105 Harms, E . , ( 6 ) 57 Harnish, D., ( 5 ) 33 Harper, J . R . , ( 8 ) 566 Harper, P . S . , (6) 511 Harper, S.H.T., ( 3 ) 194 Harrington, P . C . , ( 5 ) 146 Harris, C . C . , ( 3 ) 17 Harris, J . E . , ( 3 ) 249 Harris, R . B . , ( 5 776, 777; (8) 319, 414, 447 Harrison, L . C . , ( 5 ) 631; (8) 355 Harrison, R . , ( 5 ) 725; (8) 289 Harth, S., ( 7 ) 55, 77 Hartig, A . , ( 4 ) 216 Hartmeier, W . , ( 6 ) 486 Hascall, N . C . , (5) 308 Ha sc a l l , V . C . , ( 5 ) 402, 407, 408, 415, 426, 427 Hase, A . , ( 7 ) 202 Hase, S., (2) 49, 115; ( 8 ) 562 Hasegawa, A . , ( 8 ) 18, 68, 69, 70, 75, 553 Hasegawa, H., ( 8 ) 139 Hasegawa, J . , (8) 429 Hasegawa, K . , ( 6 ) 428 Haselkorn, R . . (8) 433 Hashiguchi, M.. , ( 8 ) 331 Hashimoto, C . , ( 4 ) 230 Hashimoto, H . , ( 7 ) 116 Hashimoto, K . , ( 5 ) 50, 51; (8) 648 Hashimoto, T., ( 8 ) 737 Ha si l i k , A . , (5) 525; ( 6 ) 262 Ha sl i k , A . , ( 6 ) 59 Hasson, M . A . , ( 5 ) 308 Hastings, J . R . B . , ( 5 ) 252 Hata, R . I . , ( 5 ) 399 Hatakryama, K . , (6) 40 Hatanaka, C . , ( 2 ) 67 Hatano, M . , (3) 82 Hatheway, C . L . , ( 4 ) 92 Hatton, M.W.C., (8) 280, 597 Ha t t o r i , A . , ( 6 ) 376 Hattori, H., (7) 104 Ha t t o r i , K . , ( 8 ) 517;
706 Hauck, M., ( 8 ) 405 Haug, A . , (8) 402 Haugaard, N . , (2) 69 Haupt, H., ( 5 ) 189 Haustein, D., ( 8 ) 169 Hauw, J . - J , ( 7 ) 69 Haverkamp. , J . , ( 5 ) 937 Haverstick, D.M., ( 5 ) 287 Havsmark, B . , ( 5 ) 377: (8) 371 Hawthorne, D.B., ( 3 ) 2 93 Hay, G., ( 7 ) 138 Hayakawa, S . , 92) 75 Hayakawa, T . , ( 8 ) 231 Hayasgum, K . , ( 6 ) 125 Hayashi, J., ( 3 ) 111 Hayashi, K . , ( 6 ) 440, 480, 481, 482 Hayashi, M . , (5) 231 Hayashi, S., ( 6 ) 493 Hayashi, T., ( 3 ) 174, 175, 176 Hayashi, Y . , ( 8 ) 658 Hayashida, S., (6) 48, 204, 358, 468 Haydar, M., ( 3 ) 245 Hayem, A . , ( 5 ) 536; (8) 183 Hayes, L . W . , (8) 172 Hayes, S . , ( 7 ) 26 Hayes, T., ( 5 ) 553 Hayes, T.G., (5) 512 Hayman, E.G., ( 5 ) 213, 227 Hayman, M . J . , ( 5 ) 15 Hazenberg, C.A.M. , ( 3 ) 141 Hazum, E . , ( 6 ) 67 Hearn, M . T . W . , (8) 150, 151 Heasley, F . A . , ( 4 ) 88 Heath, T.D., (5) 838: ( 7 ) 90; ( 8 ) 557 Heatley, F . , (5) 385 Hebeish, A . , ( 8 ) 483, 484, 489 Hecht, S.M., ( 8 ) 312 Hechtman, P . , (7) 31 Heckley, M.J., ( 6 ) 307 Heckman, S.M., (8) 448 Hedo, J . A . , ( 5 ) 631 Hedo, J . E . , ( 8 ) 355 Heeren, R . , (8) 203 Heffernan, M . E . , ( 6 ) 182 H e i f e t z , A . , ( 5 ) 1035 Heine, J.W., (5) 514; ( 8 ) 398
788 H e i n e g a r d , D., ( 5 ) 314, 325, 330, 413, 414, 4 93 H e i n r i k s o n , R.L., ( 8 ) 433 H e i n z , E., ( 7 ) 178 Heinz, F . , ( 2 ) 72 H e l a n d e r , I., ( 4 ) 83 Helbecque, N . , 95) 1039 Held, W.A., ( 5 ) 1034 H e l d i n , C.-H., (6) 411 H e l d t , H.W., ( 3 ) 70, 71 Helena N e v a l a i n e n , K.M., ( 6 ) 138 H e l e n i u s , A . , (5) 49 Helfman, D.M., ( 8 ) 211 H e l l g r e n , L., 7 ( 4 ) H e l l s t r o m , A., ( 6 ) 223, 227 H e l m , R . M . , ( 5 ) 589 H e l s e t h , D.L., (5) 279 Hemker, H.C., ( 5 ) 789 Hemming, F.W., ( 6 ) 50 Hempstead, B.L., ( 5 ) 644; ( 8 ) 197 H e n d r i x , D.L., ( 2 ) 43 Heney, G., ( 8 ) 332 Henge, M . H . , ( 5 ) 696 Henner, J.A., (5) 1037 Henney, C.S., ( 7 ) 151 Henning, M.C., (5) 249 H e n r i c h s e n , J . , ( 4 ) 120 H e n r i k s e n , O., (5) 612 Henry, B.E., ( 5 ) 6 8 Henry, J . C . , (6) 25 Henry, R . J . , ( 3 ) 87, 165 Herbage, D., ( 5 ) 267 H e r b a u t , J . , (4) 222 H e r b e r t , E., ( 5 ) 475 H e r b e r t z , L., (8) 403 Herman, J . , ( 5 ) 581 Hermann, J . , (8) 241 Hermanson, G.T., ( 8 ) 152 Herman-Taylor, J . , ( 5 ) 543: (8) 268 H e r n e l l , O., ( 8 ) 449 Herold, R . , (8) 487, 561 H e r p , A.,(5) 941, 954 H e r r a n z . J . , (3) 99 Herrler, G., ( 5 ) 36 Herrmann, M.s., 95) 185 H e r r o n , J.N., ( 5 ) 840 Herron, M.J., (4) 221 Hers, H.G., ( 6 ) 93 Herschkowitz, N.N., (7) 143 Herschman, H.R., ( 5 ) 566; (8) 576 H e r s c o v i c s , A., ( 5 )
Carbohydrate Chemistry 577, 1056; ( 6 ) 221, 459; ( 8 ) 200 Hesford, F.J., (8) 580 Hess, D., ( 6 ) 132 Hess, R.L.. (5) 802 H e w , C.L., 5 ) 832 Hewish, D., (5) 405 Hewitt. A.T , ( 5 ) 215 Heyne, E.G. ( 6 ) 303 Heynen, G . , ( 5 ) 299, 31 3 Heyns, W . , 5 ) 492 Heyworth, C M . , ( 6 ) 121. 127: ( 8 ) 242 H i b i , - N . , (5)-867; ( 8 ) 184 H i g g i n s , P . J . , ( 5 ) 797 H i g g i n s , T . J . , 95) 547, 851; ( 7 ) 148 H i g h l y , T.L., (6) 533 H i g n i t e , C.E., ( 5 ) 587 H i j n e n , W.A.M., (3) 76 H i l b i g , R . , ( 7 ) 71, 75 H i l g e r s , J., (5) 870; ( 6 ) 164 H i l k e n s , J.. ( 5 ) 870; (6) 164 H i l l , R.L., ( 2 ) 117; (5) 564, 1028. 1029, 1072; ( 6 ) 498, 503; (8) 90, 266 H i m m e l , M.E., ( 2 ) 29 H i n n i e , J . , (5) 502 Hino, Y.. ( 8 ) 525 Hinsch, W . , (8) 689 Hinshaw, V.S., ( 5 ) 10 Hinz, H.J., (7) 20 H i n z e , W.L., ( 3 ) 83 Hirabayashi, Y., (6) 54, 261; ( 7 ) 140; ( 8 ) 248, 328, 348 H i r a i , A., ( 3 ) 94 Hirai, H . , (3) 80, 84; ( 5 ) 867; ( 8 ) 184 H i r a m a t s u , M., (6) 40 H i r a n i , S., ( 5 ) 975; (6) 242 H i r a n o , K., ( 2 ) 75 H i r a n o , S., (5) 1006; ( 8 ) 500, 508, 509, 510 H i r a n o , T., ( 7 ) 116 Hirayama, A., ( 6 ) 429; ( 8 ) 662 Hirayama, F., ( 3 ) 79; (8) 522 H i r o m i , K., ( 6 ) 319, 327; (8) 513 H i r o n . M . , ( 5 ) 746; ( 8 ) 3 60 H i r o s e , M . , ( 5 ) 724 Hirose, Y . , (6) 394
H i r s c h , J . , ( 8 ) 32, 35 H i r s c h h o r n , R . , ( 6 ) 162 H i r s h , J . , ( 5 ) 668 H i r t z e l , F . , (5) 394 H i s a m a t s u , M., ( 4 151, 153, 154; ( 6 ) 3 9 H i s a t s u n e , K . , ( 4 56 H i t s c h o l d , T . , (5 794 H i x s o n , D.C.. ( 5 ) 553 H i z u k u r i , A., (3) 34 H i z u k u r i , S., ( 3 ) 19 H j e r t e n , S., ( 6 ) 39; ( 8 ) 96. 712 H j o r t h , J.P.,. ( 6 ) 296 H n i l i c a , L.S., ( 2 ) 55; ( 5 ) 1012; ( 8 ) 490 H o a g l a n d , P. D., ( 3 ) 230 Hobbs. D.S., ( 8 ) 401 H o b i s h , M.K., ( 5 ) 191 H o c h s t r a s s e r , K., (5 1 753, 965, 966; ( 8 ) 163, 201, 592 Hodes, J . E . , ( 6 ) 290 Hodes. M.E.. ( 6 ) 290 Hodge, J.E.. ( 3 ) 73 Hodgins, L.T., (5) 777; ( 8 ) 414 Hodgkinson, S. C. , ( 8 ) 134 H B j e b e r g , B., ( 8 ) 379, 380 HBBk, M . , ( 5 ) 367. 380; (6) 411 HUrmann, H . , ( 5 ) 226, 239 HBsel, W . , ( 6 ) 193 H o e s s l i , D., (5) 623 Hof, L., ( 5 ) 1014 H o f f s t e i n , S.T., ( 5 ) 241 H o f l a c k , B., ( 7 ) 77, 136 Hofman, T., ( 5 ) 847 Hoffman, F . , (5) 511 Hofmann, H., ( 5 ) 383 Hofmann, K . , ( 8 ) 333 H o f s t a d . T., ( 4 ) 57 Hogan, B.L.M., ( 5 ) 500 Hogenauer, G., ( 4 ) 67 Holahan, J.R., (5) 653 H o l a n , Z., ( 3 ) 282 Holbrook, J . J . . ( 5 ) 328, 401 H o l e c e k , 0.. ( 6 ) 490, 491 H o l f o r d , S., ( 5 ) 219 Hollaway, W.L., (2) 44 Holloman, W.K.. ( 8 ) 125 Holmberg, L . , (5) 768 Holmer, E., ( 5 ) 370 Holmes, B . , ( 4 ) 221 Holmes. K.V., ( 3 ) 112
789
Author Index Holmgren. J . , ( 7 ) 25; ( 8 ) 538 H o l s t , 0.. (4) 81 H o l t , E.C.. ( 3 ) ) 186 H o l t , S . C . , (4) 54 H o l t z e r , H . , ( 8 ) 161 H o l z e r , G., (2) 2 Homer, L . D . , ( 5 ) 139 Homewood, T., (3) 128 Honda, S., ( 2 ) 48, 52, 53, 95 Hong, J . , ( 6 ) 215 Hong, N., (6) 250 Hongo, S., ( 8 ) 425 Honig, W . , (6) 272 Honma, K., 95) 915 Hood, L . , (5) 881 Hood, L.F., ( 3 ) 30, 31 Hoogeveen, A . , (6) 124 Hoogh Winkel, G. J . M . , (5) 296, 940; (6) 64; ( 7 ) 50; ( 8 ) 245 Hopfer, S.M., (6) 27 Hopkins, H.P.. ( 2 ) 74 Hopkins, N., (5) 17 Hoppe, C . A . , ( 5 ) 191 Hopper, J . , (5) 433 Hopper, K.E., ( 5 ) 715 Hopwood. J.J., (5) 355, 356 H o r a k , E., ( 6 ) 2 7 H o r e j s i , V . , ( 5 ) 103; ( 8 ) 93, 94, 95 H o r i , K., (8) 553 H o r i , M., ( 5 ) 681; ( 8 ) 187 H o r i , T., ( 7 ) 161, 204 Horii, F . , (3) 94 H o r i k o s h i , K., ( 6 ) 269 H o r i k o s h i , T . , ( 3 ) 290; ( 8 ) 501 H o r i o , T., ( 8 ) 258 H o r i t s u , H . , ( 8 ) 225 H o r i s b e r g e r , M., ( 5 ) 72 1 H o r i u c h i , T . , ( 6 ) 289 Horne, C . , (5) 847 H o r o w i t z , M.I., ( 2 ) 59 Horst, A . , (4) 66 H o r t o n , D., ( 4 ) 78 H o r v a t h , I . , (5) 759 H o r w i t z , A.F., ( 5 ) 821 Hoseney, R . C . , (3) 200 Hosey, M.M., ( 6 ) 96 H o s k i n s , L . C . , (6) 29 Hosokawa, S. , ( 8 ) 365 HOson, T . , (3) 146 H o s p a t t a n k a r , A.V., (7) 88 H o t t a , K., ( 5 ) 927, 946, 981; (8) 358 Hou, Y., ( 7 ) 92
Houdret, N., (51 943, 944 Houghie, C . , ( 5 ) 657; (8) 295 Hounsell, E.F., (5) 860, 948. 949; ( 8 ) 186 Hounslow, M . E . , ( 5 ) 464 Hourdry, J . , (5) 576 Hovorka, F., ( 6 ) 490 Howard, S.C., (5) 588 Howell, J.M., ( 6 ) 170 H o w l e t t , B . J . , (5) 92, 93, 104; ( 8 ) 243 H o w l e t t , G., (5) 750 Hoyer, L.W., ( 5 ) 765 H r a d i l , J . , (8) 709, 747 H r s a k , I . , ( 4 ) 21 Hseu, T,.S.. (8) 156 H s i a o , H.-Y., ( 4 ) 183 HSU, M . , ( 5 ) 54 Hsu, T . A . , ( 6 ) 205 Hu, S . I . , (8) 324 Huang, F.L., ( 8 ) 140 Huang, L.L., (8) 441 Hubbard, S . C . , ( 5 ) 1040 Huber, D . J . , (3) 162; ( 6 ) 383, 391 Huber, J . , ( 8 ) 601 Huber, R . E . , ( 6 ) 145, 154 H u b e r t , P . , ( 8 ) 395 Huckerby, T.N., ( 3 ) 105; ( 8 ) 496 Hudry-Clergeon, G., ( 5 ) 787 Hudson, B.G., ( 5 ) 264, 265. 274 Huelsmann, C., ( 4 ) 202 Huet, C . , (7) 125 H u e t h e r , G., ( 5 ) 1016 Huggins, J.W., ( 5 ) 588 Hughes, E.N., ( 5 ) 638, 646, 879 Hughes, E.W., ( 5 ) 275 Hughes, J . V . , (5) 60 Hughes, R.C., ( 5 ) 570, 669. 1061 Hugon, J.S., ( 5 ) 576 H u l d t , G., (6) 51 H u l l , B.E., ( 5 ) 575 H u l l , M.T., ( 6 ) 290 H u l l , W.E., ( 2 ) 91; ( 5 ) 968, 972 H u l t b e r g , H., ( 5 ) 139; (8) 16 H u l t i n , H.O., ( 8 ) 697 H u l t i n , H.P., ( 5 ) 1 Humphrey, A . E . , ( 6 ) 357 Humphreys, J . D . , ,(6) 14
Humphries, M., ( 4 ) 22, 37 Hung, C.H., ( 5 ) 274 Hunger, U . , (4) 81 Hunsmann, G., ( 5 ) 22 Hunt, L . A . , (5) 56, 76, 77 Hunt, R.C., ( 5 ) 545 Hunt, S., ( 3 ) 105; (8) 496 H u n t e r , G.D., ( 7 ) 3 0 H u n t e r , S.J., (5) 425 H u n t e r , S.W., ( 7 ) 200 H u n t e r , T.. ( 5 ) 217 H u r l b u r t , K.L., ( 6 ) 145, 154 H u r s t . R . E . , ( 5 ) 303 Hussey, H . , ( 4 ) 3 Hussey, R . E . , ( 5 ) 600 H u t c h i n s o n , D.W., ( 8 ) 148 H u t n e y , J.. ( 3 ) 53; ( 6 ) 2 92 H U X , R.A., ( 3 ) 95 Hynes, J . B . , ( 8 ) 336 Hynning, P.A., ( 8 ) 118 I a c o m i n i , M . , (5) 991 Iacono, V.J., ( 4 ) 49 Iborra, J.L., ( 8 ) 182 I b u k i , F., ( 5 ) 716, 722; (8) 575 I c h i h a s h i , Y., (5) 84 I c h i k a w a , T . , ( 4 ) 229 I c h i s h i m a , E., ( 6 ) 245 I d b e r g , A . , (5) 336 I d e , J . A . , ( 6 ) 471 I e l p i , L . , ( 4 ) 173, 174 I e z u i t o v a . N.N., ( 6 ) 77 I g u c h i , T., (4) 56 I i d a , M., ( 8 ) 650 I i j i m a , K., (8) 231 I i z u k a , H., ( 8 ) 650 Ikami, T., (6) 179 Ikawa, Y., ( 3 ) 36 I k e d a , M., ( 3 ) 284 I k e d a , T., ( 4 ) 164, 238; (6) 501 I k e h a r a , Y., ( 5 ) 754 I k e n a k a , T.. (2) 49, 115; 324; ( 8 ) 562 I k u t a , T., (5) 731; ( 8 ) 244 I l i n , A.A., ( 3 ) 48 I m a h o r i , K., (6) 125; ( 8 ) 143 Imai, K., (5) 639 I m a i , M., ( 5 ) 516; ( 8 ) 128 Imai, S., ( 8 ) 498 Imai, Y . , (6) 514; ( 8 ) 257
Carbohydrate Chemistry
790 Irnoto, T . , ( 6 ) 427. 436, 440; ( 8 ) 499, 661 I n a g a k i , H . , ( 5 ) 346; ( 8 ) 546 I n e r o t , S., ( 5 ) 330 I n g b e r , D . E . , ( 5 ) 575 Ingham, K . C . , ( 5 ) 690, 757 I n o k u c h i , N . , ( 6 ) 403 Inoue, M . , ( 4 ) 188; ( 6 ) 379, 500; ( 8 ) 145 Inoue, S., ( 2 ) 103, 104; ( 5 ) 506, 995; ( 6 ) 252 I n o u e , Y . , ( 3 ) 81; ( 8 ) 505 I o b , A . , 98) 742 I r i e , M . , ( 6 ) 403 I r i m u r a , T., ( 5 ) 912, 923, 924; ( 8 ) 591 I r r g a n g , K . , ( 6 ) 369 I r s h a d , M . , ( 6 ) 282, 308, 309 I r v i n . R.T., ( 4 ) 45, 65 Irwin, G.N., (5) 9 I s a k s o n , P . , ( 5 ) 877; ( 8 ) 569 I s b e l l , H.S., ( 2 ) 61 Isernura, M . , ( 5 ) 324; ( 6 ) 231 Isenman, D.E., ( 5 ) 841 I s h a q u e , M . , ( 6 ) 260 I s h i b a s h i , H . , ( 5 ) 731; ( 8 ) 244 I s h i b a s h i , K . , ( 6 ) 376 I s h i b a s h i , T . , ( 6 ) 514; ( 8 ) 257 I s h i d a , K . , ( 3 ) 74; ( 4 ) 130 I s h i g a m i , S., ( 6 ) 91 I s h i g u r o , E . E . , ( 4 ) 33 I s h i h a r a , H . . ( 5 ) 985 I s h i i , M . , ( 5 ) 542 I s h i i , N . , ( 5 ) 867 I s h i i , S.. ( 2 ) 66; ( 3 ) 221; ( 5 ) 148; ( 8 ) 533 I s h i i , S . I . , ( 5 ) 135; ( 8 ) 307, 347 I s h i k a w a , F . , ( 5 ) 206, 207; ( 6 ) 373; ( 8 ) 356, 594 I s h i m o r i , Y . , ( 8 ) 721, 722 Ishino, F., ( 4 ) 3 0 I s h i z u k a , I . , (7) 64, 133 I s h n i i , N . , ( 8 ) 184 Isles, M . , ( 5 ) 329 I s o b e , T . , ( 5 ) 845 I s s e l b a c h e r , K.J., ( 5 ) 894
I t a s a k a , 0 . . ( 7 ) 161 I t o , E . , ( 4 ) 8, 28, 232; ( 6 ) 263 I t o , H . , (8) 3 I t o , J . , ( 5 ) 985; ( 8 ) 7 02 I t o , K . , ( 4 ) 129 I t o , M . , ( 6 ) 160; ( 8 ) 457 I t o , S., ( 7 ) 169 I t o , Y . , ( 2 ) 75; ( 5 ) 102; ( 8 ) 393 I t o h , T . , ( 3 ) 61, 62; ( 5 ) 718, 719; ( 8 ) 510 I t o h , Y . , (4) 46. 47, 48; ( 6 ) 440 I t z h a k i , S., ( 8 ) 181 I v a n o f f , B . , ( 4 ) 87 I v a n y i , J . , ( 5 ) 598 I v a t t , R.J., ( 5 ) 1040 I v e l l , R . , ( 5 ) 996; ( 8 ) 357 I v e r s o n , T., ( 5 ) 139; ( 8 ) 15. 44 I v y , J . , ( 8 ) 671 I w a k i , K . , ( 8 ) 383 Iwamori, M . , ( 7 ) 66, 67, 68, 116, 124, 147 Iwamoto, N., ( 8 ) 629 Iwamoto, R., ( 3 ) 280 Iwamuro, Y . , ( 4 ) 213; ( 5 ) 187 Iwanage, S., ( 4 ) 109 I w a n i j , V . , (5) 575 I w a s a , S., ( 5 ) 681; ( 8 ) 187 I w a s a k i , M . , ( 2 ) 104; ( 5 ) 506, 995; ( 6 ) 252 I w a s a k i , Y . , ( 4 ) 239 Iwase, H . , ( 2 ) 38; ( 5 ) 981; ( 6 ) 265; ( 8 ) 358 I y e n g a r , L . , ( 8 ) 710 I y e r , R.K., ( 8 ) 213 I y e r , Y . , ( 3 ) 28 I z a k i , K . , ( 4 ) 135, 223; ( 6 ) 448 I z u m i , T., ( 6 ) 17 I z u m o r i , K . , ( 6 ) 484 Izumrudov, V . A . , ( 8 ) 734 J a c k , M . A . , ( 5 ) 160; ( 8 ) 207 J a c k s o n . D.C., ( 5 ) 41, 45 J a c o b , S.T., ( 8 ) 230 J a c o b s , J.W., ( 5 ) 700 J a c o b s , S.. ( 6 ) 67; ( 8 ) 3 34 J a c o b s o n , B . S . , ( 8 ) 697 J a c o b s o n , K . , ( 7 ) 92 J a c q u e s , N.A., ( 4 ) 16
J a c q u i n e t , J.C., ( 8 ) 22, 49 J a e n i c k e , L., ( 5 ) 97; ( 7 ) 188 J a f f e , R . , ( 5 ) 496, 533; ( 8 ) 427 J a g e r s t e n , C., ( 7 ) 190 J a h r e i s , G . P . , ( 6 ) 235 Jakobovits, A., (5) 901; ( 8 ) 374 J a l a n i c o , H . , ( 5 ) 218 J a l a n k a , H . , ( 8 ) 618 James, A . M . , ( 4 ) 40 James, E . , ( 5 ) 654 James, H . , ( 2 ) 43 J a m i e s o n , G . A . , ( 5 ) 602 J a m i e s o n , J.D., ( 5 ) 575 J a n c z u r a , E . , ( 4 ) 29 J a n d e r , R . , ( 5 ) 261 Jann, B . , ( 4 ) 96 J a n n , K., ( 4 ) 96 J a n s o n s , V.K.. ( 5 ) 839; ( 8 ) 570 J a n s s o n , P . E . , ( 4 ) 60, 119 Jarman, T.R., ( 4 ) 146 J a r n e f e l t , J . , ( 2 ) 100, 108; ( 5 ) 904, 1025; ( 7 ) 10; ( 8 ) 220 J a r r e t t . 0 . . ( 5 ) 15 J a r v i s , M . C . , ( 3 ) 220 Jasalavich, C.A., (5) 95, 118 J a s t o r f f , B., ( 8 ) 109 Jaton, J.C.. (5) 849 Jayaram, B . , ( 8 ) 284 Jayson, M.I.V., ( 5 ) 315 J e a n l o z , D . A . , ( 8 ) 555 Jeanloz, R.W., ( 5 ) 959; ( 8 ) 555 J e a n o t t e , L . , ( 5 ) 703 J e a n s s o n , S . , ( 5 ) 32; ( 8 ) 406 J e c k . R . , ( 8 ) 495 J e f f r e y , G . A . . ( 2 ) 114 J e f f r i e s , T.W., ( 6 ) 380 J e l i n i c . V . , ( 5 ) 239 J e n n e s s , R . , ( 6 ) 495 J e n n i n g s , B.R.. ( 5 ) 329 J e n n i n g s , H.J., ( 4 ) 117 J e n n i s s e n , H.P., ( 8 ) 104 J e n s e n , G.L., ( 8 ) 270 J e n s e n , H.B., ( 6 ) 443; ( 8 ) 502 J e n s e n , J.W., 9 5 ) 1059, 1060 J e n s e n , M . , (4) 83 J e n s e n , S . A . , ( 3 ) 153 Jermyn, M . A . , ( 5 ) 125; ( 8 ) 177
79 1
Author Index J e r v i s , L . , ( 8 ) 430 J e s s u p , W . , (8) 458 J e t t , M . , ( 5 ) 602 J e z e k , J . , (8) 67 Jill-Schubert, J., (6) 257 Jimbo, A . , ( 5 ) 174, 390 J i r g e n s o n s , B . , (6) 326 J i r k u , V . , ( 8 ) 642 J i z b a , J . , (8) 645 Johansson, B.G., (5) 311: (8) 276 Johansson, B . S . , (8) 299 J o h a n s s o n , K.-E., ( 7 ) 190 Johansson, L.B.-A., (7) 189 J o h a n s s o n , R . , ( 5 ) 139; (8) 15 J o h n , M . , ( 3 ) 98 J o h n c o c k , S . I . M . , (2) 51 J o h n s , S.R., ( 7 ) 166 J o h n s o n , A.J., (5) 776, 777: ( 8 ) 414 J o h n s o n , A . M . , ( 5 ) 1001 J o h n s o n , D., ( 5 ) 462 J o h n s o n , D . C . , (3) 97 J o h n s o n , E.A.Z., (5) 590 J o h n s o n , J.A., ( 3 ) 46 J o h n s o n , L . D . , (5) 244 J o h n s o n , P . , ( 8 ) 439 J o h n s o n , P . H . , (5) 1073 J o h n s o n , P . R . , ( 6 ) 229; (8) 724, 725 Johnson, R.F., ( 8 ) 516 J o h n s o n , S.D., (4) 11 J o h n s o n , T.C., ( 5 ) 60 J o k i n e n , M . , (5) 921 J o l l e s , J . , ( 5 ) 717, 730 J o l l e s , P . , (5) 717, 720 J o n e s , A . , ( 8 ) 615 J o n e s , L . M . , (4) 58 J o n e s , M . Z . , ( 5 ) 452, 453; (7) 121 J o n e s , P . , ( 4 ) 170 J o n e s , P . P . . (5) 620 J o n e s , R . , ( 5 ) 580 Jones, R.L., (3) 177, 178 J o n e s , T.H.D., ( 6 ) 359 J o o s t e n , G.E.H., (8) 602 Joosten, G.E.M., ( 6 ) 476 J o r d a n , F . , ( 5 ) 178 J o r d a n , K.B., (6) 46 J l ) r n v a l l , H . , ( 5 ) 24,
742, 764: J o s e p h , K.T., ( 8 ) 512, 715, 733 J o s e p h s o n , S., ( 8 ) 44 J o r s t a d , C.M., (6) 141 J o s e l e a u , J . P . , ( 3 ) 96, 222; (6) 462 J o s e p h , A . P . , ( 5 ) 21 Jouanneau, J . , (5) 531, 890. 891, 895 J o u a n n e t e a u , B . , (7 1 195 J o u v e r t , S., ( 4 ) 222 J o v i n , T.M., (5) 140, 91 0 J o y c e , P . , ( 8 ) 694 J o z i a s s e , D.H., ( 5 ) 1021: ( 6 ) 64; ( 8 ) 245 Jozwiak, W . , (7) 103 J u d e l . G.K., ( 3 ) 59 Jung, S.M., (6) 521, 522 Jungalwala, F . B . , (7) 128, 130 J u n q u a , S . , ( 8 ) 246 J u r a s e k , L . , (6) 196, ( 8 ) 614 J u r e n i t s c h , J . , ( 2 ) 10, 11 Kabanov, V . A . , ( 8 ) 734 Kabat, E . A . . ( 5 ) 166, 176, 852, 854, 992; (7) 156, 157, 158; ( 8 ) 558 K a c h r a , 2.. ( 7 ) 31 Kado, A . . ( 6 ) 312 Kadokura, S., ( 5 ) 346; (8) 546 Kador, P . F . , ( 8 ) 310 Ktltlrillnen, L . . (5) 50, 51, 921 Ktirkkainen, J . , ( 5 1025 K a e t s u , I . , ( 8 ) 609, 610 K a g e d a l , L . , ( 8 ) 396 Kahane. I . , (4) 90 Kahn, D . R . , ( 5 ) 235; (8) 368 K a i f u , R., ( 8 ) 77 Kainuma, K . , (3) 8 K a i s e r , E . , ( 5 ) 486 K a i t o , A . , (3) 82 K a j i , A . , ( 6 ) 75, 445, 455 K a j i u r a , T., ( 3 ) 10 K a j i w a r a , T . , (5) 411 K a k e h i , K . , ( 2 ) 48, 52, 53, 95 Kakinuma. A . , ( 4 ) 108 K a k i u c h i , S., ( 8 ) 382
K a k u t a , M . , ( 4 ) 209 Kalachev, I.Ya., ( 6 ) 143 K a l a l , J . , ( 2 ) 18; ( 8 ) 584 Kalinichenko, L . P . , (5) 712 K a l y a n , N.K., ( 5 ) 688, 689; (8) 578 Kalyanaraman , V. S., ( 8 2 85 Kamada, R . , ( 7 ) 140: (8) 328, 348 Kambayashi, J . , ( 8 ) 297 Kamataki, T . , ( 8 ) 121 Kambayashi, J . , ( 8 ) 382 Kameda, K . , ( 8 ) 223 Kamei, S., ( 2 ) 68 Kamerling, J.P., ( 2 ) 96: ( 3 ) 297, 298, ( 5 ) 935 Kameya, T., ( 6 ) 286 Kameyama, T . , ( 5 ) 207, 903; ( 8 ) 356 Kamibuko, T., ( 6 ) 398, 399, 405; ( 8 ) 158, 648 Kamimura, T., ( 5 ) 253 Kaminkova, J . , (8) 587 Kamio, Y., ( 4 ) 46, 47, 48 Kanamori, M . , ( 5 ) 716, 722; (291, 575 Kanamaru, K . , ( 4 ) 213 Kanaya, S., ( 8 ) 302, 330 Kanda, T., ( 6 ) 337, 363 Kane, C . M . , (8) 452 Kaneda, Y., ( 8 ) 68 Kannan, K . , ( 5 ) 198; ( 8 ) 261 Kano, Y., ( 3 ) 58; ( 7 ) 36 K a n s t a d , S.O., ( 7 ) 4 Kanta, S., ( 3 ) 299, 300 K a n t e r , R . , ( 7 ) 146 Kao, K.J., (5) 769 K a p a d i a , G., ( 5 ) 683 K a p i t z a , H.G., (5) 910 K a p l a n , A . I . , ( 5 ) 407 Kaplan, G . , (634) K a p l a n , N.O., ( 5 ) 186 Kapmeyer, W . , ( 5 ) 186 Kapoor, O.S., ( 5 ) 25 Kapoor, R . , ( 5 ) 333 Kappel, W . K . , ( 4 ) 136 K a p r a l , F . A . , ( 8 ) 113 K a p t e i n , R . , ( 5 ) 710 Kapur, D . K . , ( 6 ) 37 Kar, S.K., ( 8 ) 486 Karakawa, T . , ( 3 ) 58 K a r a n t h , N.G., ( 8 ) 681
Carbohydrate Chemistry
792 K a r e l i n , V.P., ( 5 ) 2 7 K a r h i , K . K . , ( 5 ) 904; K a r i k a r i , S.K., (3) 67 Karim, K.A., ( 5 ) 1027 Karitonenkov, I.G.. (5) 37 K a r k k a i n e n , J., ( 2 ) 108; ( 7 ) 10 Karkstam, B.. ( 5 ) 136 K a r l s s o n , A . , (2) 93 K a r l s s o n , K.-A., (5) 857: ( 7 ) 49, 84, 85, 107, 108, 111, 131, 153, 193 Karn, R.C., ( 6 ) 290, 296 K a r t c h n e r , R.J., ( 3 ) 16 Karube, I . . ( 8 ) 99, 603, 611, 647, 721, 722 K a r u s h , F., ( 5 ) 844 Karvonen, E . , ( 6 ) 134, 347 Kas, J . , ( 8 ) 645 Kasai, K . I . , (5) 135; ( 8 ) 307, 347 K a s a i , M . , (7) 116, 144, 147 K a s a i , N . , ( 7 ) 201 Kasche, V., ( 8 ) 598 Kasei, M . , ( 5 ) 431 K a s h l a n , N., ( 6 ) 279 Kasper, D . L . , ( 4 ) 117 Kasumi, T . , ( 6 ) 480, 481, 482 Katano, K., ( 8 ) 56 Kataoka, S., ( 6 ) 399 Katchalski-Katzir, E., ( 6 ) 492 Kates, M., ( 7 ) 5, 194, 196, 197 K a t l i c , A.W., ( 5 ) 528 Kato, G., ( 4 ) 114 K a t o , J . , ( 8 ) 605, 636, 638 K a t o , K . , ( 3 ) 144: ( 4 ) 35, 50; (5) 756; ( 6 ) 179, 231, 236: ( 8 ) 553 K a t o , M . , ( 4 ) 188 Kato, S., (5) 417, 434, 1052 K a t o , T . , ( 5 ) 724 Kato, Y . , (3) 170, 171, 174, 176; ( 5 ) 981; (8) 358, 564 K a t o h , K., 93) 236 Katoh, S . . ( 6 ) 488; ( 8 ) 739, 750 Katoh, T., (4) 187 K a t s u k i , H., ( 4 ) 135 Kattermann, R., ( 2 ) 80:
( 5 ) 822 K a t z e n , H.M., ( 5 ) 630 Katzenellenbogen, E., ( 4 ) 59 Katzmann, J.A., ( 5 ) 869 Kaufman, S., ( 8 ) 139 Kaufmann, J . F . , ( 5 ) 624 Kauppinen, V.. ( 6 ) 314: ( 8 ) 560 Kaur, H . , ( 8 ) 280 Kauss, H . , ( 6 ) 113 Kavanagh, M . I . , (5) 783 Kavanagh, M.L., ( 5 ) 868 Kawagishi, S.. ( 6 ) 263 Kawaguchi, M.. ( 6 ) 28 Kawai, K . , ( 8 ) 224, 225 Kawakami, H . , ( 5 ) 372 Kawakami, M . , ( 2 ) 65 Kawamura, F . , ( 6 ) 317 Kawamura, M., ( 2 ) 66 Kawamura, Y., ( 8 ) 158 Kawarasaki, I . , ( 4 ) 226; ( 8 ) 37 Kawasaka, S . , ( 3 ) 138 Kawasaki, A , , ( 4 ) 5 0 Kawasaki, T . , ( 5 ) 562; ( 8 ) 281 Kawasaki, Y., ( 7 ) 16 Kawashima, N., ( 7 ) 182 Kawata, T . , ( 4 ) 185 Kayano, H., ( 8 ) 611 Kaye, A.M., ( 8 ) 445 K a y i s u , K . , ( 3 ) 30 K a y s e r , G., ( 7 ) 29 K e a t i n g . B.A., ( 3 ) 32 Keck, W . , ( 6 ) 499 Kecskes, E.. (5) 759 Keefer, L . M . , ( 5 ) 599; ( 8 ) 433 Keenan, T.W., ( 7 ) 51 Keil, B . , (5) 581; ( 8 ) 241, 308 K e i l i c h , G., ( 7 ) 15 Keim, P . , ( 8 ) 433 Keinan, E . , (8) 688 K e l l e h e r , T . J . , ( 6 ) 355 Keller, F., ( 3 ) 164 Keller, R . , ( 5 ) 394, 395 K e l l e y , D.G., ( 5 ) 225 K e l l e y , W . N . , ( 8 ) 267 K e l l o g g , R.B., ( 6 ) 518 K e l l y , C.T., ( 6 ) 182 Kelman, A . , ( 4 ) 138 Kemball-Cook, G., ( 5 ) 770 Kemp, M.C., ( 2 ) 44: ( 5 ) 19 Kemp, R.G., ( 7 ) 62 Kempner, E.S., ( 5 ) 192 Kemper, J . G . , ( 5 ) 528 Kenne, L . , ( 4 ) 100
Kennedy, B.B., ( 8 ) 426 Kennedy, J . F . , ( 1 ) 1: ( 4 ) 170, 171: (6) 14: ( 8 ) 82, 83, 84, 85, 86 198 Kertlnen, S., ( 5 1 50 K e r c k a e r t , J.P., ( 5 ) 541: ( 8 ) 235 Kern, H . , ( 6 ) 57 Kern, P . , ( 5 ) 271 Keski-Oja, J., ( 5 ) 229, 230, 237: ( 8 ) 210 Kessler, A . C . , ( 2 ) 73 Kessler, M.J., 95) 1037 Kestere, A . Y . , ( 8 ) 179 Kezdy, F . J . , ( 5 ) 250, 486 K h a l i l , N . F . , 93) 226 Khan, A . R . , ( 3 ) 46 Khan, A.W., ( 6 ) 224, 333, 364, 365 Khan, J.A., ( 6 ) 36, 41, 73 Khan, M . , ( 4 ) 158 Khan, M . I . , ( 5 ) 124, 168, 169 Khan, N., ( 3 ) 246 K h a t r i , G.S., ( 5 ) 105; ( 8 ) 547 K h i r a b a d i , B.S., ( 8 ) 4 42 Khor, H.T., ( 7 ) 176 K h o r l i n a , A.Ya., ( 6 ) 137 K i b b e l a a r , M.A., ( 8 ) 162 Kiang, W . L . , ( 5 ) 332 K i c h i , J.. ( 8 ) 231 K i e f e r , C . R . , ( 5 ) 845 K i h a r a , K . , (8) 737 Kiho, T . , ( 4 ) 247 K i k u c h i , H., ( 8 ) 394 K i k u c h i , K . , ( 5 ) 329 K i k u c h i , M . , ( 5 ) 363; ( 6 ) 410; ( 8 ) 544, 589 K i k u c h i , Y.. ( 3 ) 285 K i k u t a n i , H.K., ( 5 ) 886 K i l a r a , A , , ( 8 ) 174 K i l e y , P . , ( 4 ) 54 Kilic, N . , ( 2 ) 2 5 K i l k e r , R.D., ( 6 ) 221 K i l l i n g t o n , R.A., (5) 33 K i m , I . S . , ( 8 ) 454 K i m , Y.S., ( 5 ) 554; ( 8 ) 287 K i m a t a , K . , ( 5 ) 446 K i m b a l l , E.S., ( 5 ) 614, 61 8 K i m b a l l , P.M., ( 6 ) 65 Kimmins, W.C., ( 3 ) 131 Kimura, A., ( 8 ) 652
Author Index Kimura, J.H.,
( 5 ) 402, 415, 426, 427 Kimura, S., ( 5 ) 253, 273 Kimura, T . , ( 8 ) 629 K i n d e r s . , R.J., ( 5 ) 60 K i n d l e , C.S., ( 5 ) 605 K i n d t , A. ,(5) 112 K i n d t , T.J., ( 5 ) 611, 615, 628 King, I.A., ( 5 ) 421, 1054 King, T.P., ( 5 ) 171 Kinnon, C., ( 6 ) 57, 61 K i n o s h i t a , J.H., ( 8 ) 310 K i n o s h i t a , S., ( 8 ) 607 K i n o s h i t a , T., ( 5 ) 664, 666 K i n p a r a , H . , ( 4 ) 187 Kirby, E.P., ( 5 ) 778 Kirkham, S., ( 6 ) 127 K i r s c h , J.F., ( 6 ) 206 K i r s c h , K . , ( 5 ) 577; ( 6 ) 459; (8) 200 K i r s c h n e r , D.A., ( 5 ) 2 57 K i r s o h , J.F., ( 6 ) 144 K i s a k i , T., ( 4 ) 213 K i s h i d a , T., ( 8 ) 205 Kishimoto, Y., ( 7 ) 120, 142 Kiso, M., ( 8 ) 7, 18, 68, 69, 70, 75, 553 Kiss, P . , ( 5 ) 822 K i s s o n e r g h i s , A.M., (5) 625 K i t a d a , C . , ( 5 ) 681; ( 8 ) 187 K i t a g a k i , H., ( 5 ) 102; ( 8 ) 393 Kitagawa, T.. ( 7 ) 105 Kitagawa, Y., (8) 475 K i t a j i m a , M., ( 2 ) 6 8 K i t a j i m a , S., ( 6 ) 428 Kitamaru. R . , ( 3 ) 94 Kitamikado, M., ( 6 ) 160; ( 8 ) 457 Kitamura, T . , ( 6 ) 270 K i t a o , T . , ( 8 ) 706 K i t a o , Y., (8) 706 Kivirikko, K.I., (5) 284 K i z a k i , H., ( 8 ) 453 K j e l l e n , L . , ( 5 ) 380 Kjosbakken, J . , ( 4 ) 128 Kltlmbt, D . , ( 8 ) 384 Klee, C.B., ( 8 ) 227 Klei, H.C., ( 6 ) 213 Klei, H.E., ( 8 ) 752 K l e i n , A.S., (3) 129 K l e i n , G., ( 5 ) 827 407,
793 K l e i n , J . , (8) 511, 620 K l e i n , J . P . , ( 4 ) 159 K l e i n . K., ( 8 ) 617 K l e i n , M., ( 5 ) 841, 847 K l e i n , U . , ( 5 ) 525 K l e i n e , T.O., ( 5 ) 298 Kleinman, H., ( 5 ) 240 Kleinman, H.K., ( 5 ) 215 Klenk, H.D.. ( 5 ) 8, 47, 48
Kleppe, G . , ( 6 ) 443 Klesov. A.A., ( 3 ) 121; ( 6 ) 194, 197, 336, 338, 356, 393 K l i b a n o v , A.L., ( 8 ) 670 Klibanov, A.M., ( 8 ) 601 Klimov, E.M., ( 8 ) 5 K l i n g e r , M.M., ( 5 ) 489 K l i n g h a r d t , G.W., (7) 49 Klis, F.M., ( 3 ) 141, 142 K l o c k , J.C., ( 7 ) 35, 115, 118 Klok. J . , ( 2 ) 3 K l u e p f e l . D . , ( 6 ) 260 Kluge, M., ( 8 ) 617, 620 K l y a s h c h i t s h k i i , B.A., (61, 97, 101 Kminkova, M., ( 8 ) 693 Knack, I., ( 6 ) 7 Knappert, D . , ( 3 ) 122 Knecht, D.A., ( 6 ) 47 Knepper, P . A . , ( 5 ) 297 Kneubeuhl, F., ( 5 ) 775 Knight, E., ( 5 ) 513, 515; ( 8 ) 431 Knippers. R . , ( 8 ) 428 K n i r e l , Y.A., ( 4 ) 76 K n o l l , E., ( 6 ) 24 Knowles, B.B., ( 5 ) 563. 861, 862: ( 7 ) 150 Knowles, C.J., ( 8 ) 644 Knox, K.W., (4) 16, 19 Knudsen, K.A., ( 5 ) 572 Koba, Y.. ( 6 ) 277 Kobara, Y., ( 2 ) 67 Kobata, A., (5) 53, 542, 977; ( 6 ) 28, 71, 236, 285 Kobatake, H . , ( 5 ) 716 Kobayashi. M., ( 6 ) 377, 525 Kobayashi, N . , ( 8 ) 526 Kobayashi, R., ( 6 ) 384 Kobayashi, T., ( 6 ) 368; ( 8 ) 112, 604, 700 Kobayashi, Y., ( 6 ) 269 Koch, H.U., ( 4 ) 15 Koch, N . , ( 8 ) 169 Kocharov, S.L., ( 7 ) 76 Kocharova, N.A., ( 4 ) 7 6
Kochetkov, N . K . ,
( 8 ) 5, 6, 73
( 7 ) 6;
K o c i s , P.. ( 8 ) 23 Kocourek, J . , ( 5 ) 162 Kodama, T., ( 4 ) 147 K o e f f l e r , H.P., ( 5 ) 645 K o e h l e r , D.E., ( 3 ) 123; ( 6 ) 350
Koeners, H.J., ( 8 ) 66 Kbnig, W.A., ( 2 ) 16 Kbnigsmann-Lange, K . , (5) 992
Koepp, L.H.,, ( 4 ) 75 Koerner, T.A.W., (5) 91 3
K b t t g e n , E., ( 5 ) 147 Koga, T . , ( 6 ) 500 Koga, Y., ( 5 ) 484 Kohama, K . , ( 8 ) 161 Kohama, T . , ( 5 ) 47, 48 Kohi, Y . , ( 6 ) 429; ( 8 ) 662
Kohlwey, D.E., ( 6 ) 140 Kohn, J . , ( 8 ) 155 Kohn, L.D., ( 5 ) 468, 469
Kohn, R . , 224
( 3 ) 193, 217.
Koiwai, A., ( 7 ) 182 Koj, A., ( 8 ) 280 K o j i , T., ( 5 ) 867; ( 8 ) 184
Kojima, K . ,
( 5 ) 954; ( 7 ) 44, 45, 123, 140; ( 8 ) 328, 348 Kojima, M., ( 8 ) 517 Kokubu, T . , ( 8 ) 647 Kokubun, Y., ( 7 ) 105 K o l a r , C . , ( 8 ) 54 Kolarova. N . , ( 6 ) 210 Kolb, H . , ( 5 ) 568 Kolb, W.P., ( 8 ) 188 Kolb-Bachofen, V., ( 5 ) 5 68 Kolhouse, J . F . , ( 5 ) 490; ( 8 ) 343 K o l i s i s , F.N., ( 6 ) 415, 5 32 Komano, H . , ( 5 ) 201 Komiyama, M., ( 3 ) 80, 84 Komp, M., ( 6 ) 132 Komraiah, M., ( 3 ) 237 Konami, Y . , ( 5 ) 111; ( 8 ) 392 Kondo, A., ( 2 ) 68 Kondo, H . , ( 8 ) 513 Kondo, K.. ( 5 ) 681; ( 6 ) 429; ( 8 ) 187, 492, 662 Kondo, S., ( 4 ) 56 Kondo, Y . , ( 7 ) 172; ( 8 )
Carbohydrate Chemistry
7 94 K o z u t s u m i , , Y.,
508
K o n i g s b e r g , W.H.,
135
Konno, A., Konno, H.,
236
8)
(8) 524 (3) 218
K o n r a d s s o n , P., (8) 24 Kopp, B . , (2) 10, 1 1 Koppel, D.E., (4) 85 Koppen, P.. (2) 34 Koprowski, H., (5) 857;
(7) 32
Kopylov, A.M., (4) 199 Korman, A.J., (5) 624 K o r n e r , 0.. (7) 20 K o r n f e l d , K . , (5) 164;
(8) 247, 274
Kornfeld, R.,
(5) 23, 164; (8) 247, 274 K o r n f e l d , S., (5) 1030, 1047, 1058, 1074: (8) 255 K o r p e l a , T., (8) 679 Korsmo, R., (8) 376, 508 K o r t t , A.A (8) 177 Kosaka, H., (5) 324; (6) 231 K o s a k a i , M . , (8) 545 K o s a k i , C., (8) 297, 382 K o s c i e l a k , J . , (7) 103 Kosher, R.A., (5) 396 K o s h i b a , T., (6) 310 K o s h i j i m a , T., (3) 118, 208 Koshimizu, L.H., (8) 471 K o s i k , M., (3) 115 Kossen, N.W.F., (8) 612, 655
.,
Kostereiserfunke, W.,
(5) 410
K o t a n i , S., (4) 35, 50,
168
K o t a r s k i , S.F., (4) 132 K o t e l i a n s k y , V.E., (5)
222, 238
K o t l e r , D.P., (2) 64 Kotsubo, K . , (2) 40 Kouyama, H . , (6) 130 Kovac, P., (2) 94; (3)
189; (8) 23, 32, 35 (2) 94; (3) 189 Kovalenko, G.A., (8) 727 Koyabu, K., (3) 294 Koyama, M., (8) 323, 498 Kozak, J . J . , (5) 83 K o z i o l , M.J., (2) 63 Kovacik, V.,
(5) 62;
(8) 281
K r a e h e n b u h l , J.P..
884: (8) 195
K r a k e n a i t e , R.P.,
(5) (6)
183
Kramer, G., (5) 294 Kramer, J.D., (6) 165 K r a n z , D.M., (5) 840 Kraska, B., (6) 484 K r a s o w s k i , J.A., (3) 7 K r a t k y , C . , (4) 53 K r a t k y , Z. ,(3) 201,
202: (6) 463, 464, 476, 472
K r a u s , R u p p e r t , R.,
461
(5)
Krchnak, V., (8) 67 Krembel, J . , (6) 171 Kren, V., (4) 190 Kress, B.C., (6) 57, 61 Kresse, H., (5) 391,
432; (6) 62, 519: (8) 250 K r e t c h m e r , N., (6) 294; (8) 309 K r e t s c h m e r , P. J., (4) 95 Krett, N.L., (7) 124 K r i e g e r , R., (2) 80 K r i m , K.A,.(2) 41 K r i s h n a r a j , R . , (6) 62 Krohn, R . I . , (8) 152 K r o l i c k , K.A., (5) 877; (8) 569 K r o l l . H.P., (4) 43 Kronman, M.J., (5) 711 Krouwel, P.C., (8) 612, 655 KrUger, J., (5) 919, 220 K r u g e r , J.E., (3) 51: (6) 306, 323 Kruk, I., (1) 6, 174 Krumphanzl, V., K r u s i u s , T., (2) 108,
110; (5) 904, 1025; (7) 10; (8) 220 Kruyssen, F . J . , (4) 7, 27 Ksheminskaya, C.P., (6) 186 Kubanek, V., (8) 642 Kubelka, W., (2) 10, 1 1 Kubin, V., (4) 115 Kubota, M . , (5) 253, 273 K u c e r a , J . , (8) 472, 693 K u c e r a , M., (8) 178 Kudo, J . , (5) 731; (8) 244
Kuehn, L., (8) 403 KUhn, L.C., (5) 884;
(8) 195
K u e l e n s c h m i d t , T., (8)
427
Kuga, S., Kuge, T . , Kuhn, K.. Kuhn, S., Kula, M.R., Kulczychi,
(8) 530 (8) 33 (5) 270 (7) 155 (6) 188 A . , (5) 644,
Kulp, K.,
(3) 14, 15,
(8) 197
21
K u l y s . J.J., (8) 673 Kumagai, H . , (6) 245 Kumamoto, C.A., (5) 281 Kumanotani. K., (2) 13 Kumaraswamy, M.D.K.,
(8) 733
Kume, T.,
(8) 659
96) 478, 479;
Kumom, A., (8) 291 Kundu, R . K . , (6) 202 Kundu, S.K., (7) 27 Kung, H . , (8) 401 Kung, P.C., (5) 627 Kuniak, L., (8) 541 K u n i c k i . T.J.. (5) 656,
906
Kuninaka, A.,
686, 731
(8) 477,
K u n i t a k e , N . , (3) 58 Kunquan, Y . , (6) 39,
96, 712
Kuo, J . F . , (8) 21 KUO, J.C., (2) 50 971 K u r a c h i , K., (5) 370 Kuramitsu, H.K., (4)
163
K u r a m i t s u , S., (6) 439 Kurasawa, S., (6) 394 K u r a t a , K., (6) 91 K u r e l e c , B., (5) 202 K u r i a n , P., (8) 442 K u r i h a r a , H., (8) 531 K u r i t z a , A . , (3) 149 Kuriyama, M . , (5) 978 K u r k i j H r v i , K., (8)
170, 679
K u r k i n e n , M., (5) 500 Kuroda, T., (8) 529 K u r o k i , M., (5) 484 Kurome, H., (8) 648 K u r t h , R., (5) 21 Kusaka, T., (8) 436 Kusano, T., (4) 46, 47 Kushwaha, S.C., (7) 5,
196, 197
K u s t e r , B.F.M.,
26
(2) 21,
795
Author Index Kusunose, H . , ( 3 ) 181; ( 4 ) 243 Kuul, O., ( 7 ) 128 Kvarnstrbm, I . , ( 8 ) 24 Kveder, S., ( 4 ) 2 1 Kwak, J.C.T., ( 5 ) 351 Kwok. B.C.P., ( 7 ) 93, 94 Kwon-Chung, K. J . , ( 4 ) 94 Kyosaka, S., ( 6 ) 6 8 Laas, T., ( 8 ) 97 L a b a n e i a h , M.E.O., (3) 27 Labat-Robert, J . , (5) 953 Labow, R.S., ( 8 ) 423 Labudova, I . . ( 6 ) 210 L a c e , D.A., ( 8 ) 159 L a c e l l e , N., ( 5 ) 179 Lachance. M.-A., (4) 192 L a c h e r , K.P., ( 4 ) 11 Lacks, S.A., ( 6 ) 273 Lacombe, J.M., ( 5 ) 1017 La C o r b i e r e , M . . ( 5 ) 278 L a c r o i x , L . J . , ( 3 ) 51 L a d e s i c , B., (4) 21 L a d i s c h , M.R., ( 6 ) 215 L a f i t t e , J . J . , (5) 942 L a f o n t , S., ( 4 ) 87, 106 Lanunoff. D.. ( 5 ) 371 L a h e t . c., ( 5 ) 744 Lai, C.J., (5) 35 L a i , C.Y., ( 8 ) 401 L a i , P.C.W., (8) 441 L a i n e , A., ( 5 ) 536; 8 ) 183 L a i n e , M.D., ( 5 ) 632 L a i n e , R . A . (2) 97, 100; ( 5 ) 452, 489, 640, 641, 1063; ( 7 121 Lajmanovich, A . , ( 5 ) 787 Lake, P. ( 5 ) 595 Laker, M.F., ( 2 ) 17 L a k i n , K.H., ( 5 ) 450 Lama, L . , (8) 608 Lamb, J.E., ( 5 ) 132 Lamb, M . A . , (8) 585 L a m b e r t , C . , ( 6 ) 85 L a m b e r t s , B.L., ( 6 ) 378; ( 8 ) 542 Lamblin, G . , ( 2 ) 35; ( 5 ) 929, 932, 942, 943, 944, 945 Lamed, R.J., ( 8 ) 688 LaMont, J.T., ( 5 ) 957 Lamph, W , . ( 5 ) 7 6
Lan, S.L., ( 8 ) 156 Landa, C.A., ( 7 ) 57 Landa. L., ( 8 ) 267 L a n d i s , D.A., ( 8 ) 494 Lane, C.D... ( 5 ) 994: ( 8 ) 352 L a n g e r , B., ( 6 ) 2 4 Langer, R . , ( 6 ) 412 Langone, J.L., ( 8 ) 572 L a n g r i s , M . , (5) 412 L a o u s s a d i , S., ( 7 ) 98, 99 L a r a , A . R . , ( 3 ) 119 L a r i v i e r e , N., ( 5 ) 474 Larm, O., ( 5 ) 362; ( 8 ) 147, 535 L a r g i t t e , F.C., ( 6 ) 86 L a r r e t a Garde, V., ( 8 ) 626, 627, 628 L a r r i b a , C . , ( 4 ) 217; ( 5 ) 488: ( 6 ) 382 L a r s e n , B., ( 3 ) 265; ( 6 ) 483 L a r s e n , C.J., ( 5 ) 64 L a r s o n , C., (5) 857; ( 7 ) 107, 108, 109 L a r s s o n , B., ( 3 ) 102 L a r s s o n , J., ( 5 ) 964 L a r s s o n , K., (8) 535 L a r s s o n , P.O., ( 8 ) 641 L a s a r c y k , H.. ( 5 ) 815 L a s c u , I . , ( 8 ) 432 L a s e r n a , E.C., ( 3 ) 273; ( 4 ) 204 L a t e r r a , J., ( 5 ) 375: (8) 262 Latham, K.R., ( 8 ) 344 Laube, V.M.. ( 3 ) 136; ( 4 ) 186 L a u e r , E., ( 6 ) 255 L a u f e r . D.A., (8) 516 L a u g h l i n , T.A., ( 6 ) 466 L a u r e n t , G.J., ( 5 ) 252 L a u r e n t , M., ( 5 ) 271 L a v i e l l e , S., (8) 71, 72 L a w l e r , J.W., ( 5 ) 510 Lawrence, J . A . , (5) 1077 Lazo, P.S., ( 6 ) 120 Lea, O.A. ( 5 ) 491 L e a v e r , J., ( 4 ) 12 L e a v i t t , R.D.. ( 5 ) 172 Le Bergh, M . , ( 2 ) 34 L e b l a n c , R.M., ( 7 ) 165 L e b l o v a , S., ( 8 ) 216 L e b r e t o n , J.P., ( 5 ) 746; ( 8 ) 360 Lebrun, E . , ( 5 ) 163 L e c h n e r , J . , ( 4 ) 184 L e c k i e , M.P., ( 4 ) 180 L e c o n t e d e F l o r i s , R.,
( 6 ) 25 L e d e e n , R.W., ( 7 ) 65, 72 L e d e r , I.G., (5) 361, 362; ( 6 ) 512 Ledingham, W.M., (8) 740, 741 L e d l e y , R.S., ( 8 ) 442 Lee, C., ( 8 ) 237 L e e , C.J., ( 4 ) 120 Lee, D., (8) 585 L e e , D.M., ( 3 ) 91 Lee, C.J.L., ( 2 ) 37, 45; ( 5 ) 302 Lee, G.K., ( 6 ) 396; ( 8 ) 677 L e e , K.H., ( 3 ) 296 Lee, K.S., ( 8 ) 459 Lee, R.E., ( 2 ) 43; ( 6 ) 190 L e e , S.B., ( 8 ) 684, 699, 749 L e e , S.C., ( 3 ) 241, 242; ( 6 ) 452, 453 Lee, T.H., ( 4 ) 244, 245 Lee, W.M.F., ( 7 ) 115, 118 Lee, Y.C., (5) 191, 524: ( 7 ) 139 L e e , Y.H., ( 6 ) 340 Leeuw, J.W., ( 2 ) 3 L e f f l e r , H., ( 7 ) 84, 85, 131, 153 l e G a l l , J.Y., ( 6 ) 60, 122 L e g l e r , C., ( 6 ) 216, 216 L e h l e , L., ( 6 ) 88; ( 7 ) 181 Lehmann, P., ( 3 ) 52; (6) 288 L e h r f e l d , J . , ( 2 ) 14 L e h r m i t t e , M . , ( 5 ) 942 L e h t o , V.P., ( 5 ) 591 L e i f l e r , H . , (2) 93 L e i s o l a , M., ( 6 ) 314, 347; (8) 560 L e l o i r , L.F., ( 6 ) 173; ( 7 ) 175 l e Lous, M., ( 5 ) 272 Lem, N.W., ( 7 ) 173 l e Marchand B r u s t e l , Y., (5) 291 L e m a t r e , J . , ( 4 ) 237 Lemieux, R.U., ( 5 ) 852, 864; ( 8 ) 2 1 Lemkin, M.C., ( 5 ) 400 Lemmens, P.J.M.R., (4) 73 Lemonnier , M., ( 8 ) 246 Lempart, K . , ( 5 ) 753; ( 8 ) 163, 201
Carbohydrate Chemistry
796 L e n g y e l , P . , ( 8 ) 284 L e n h o f f , H . M . , ( 8 ) 671 L e n n a r z , W.J., ( 5 ) 1032, 1042, 1043 L e n o i r , G., (6) 21 L e n s t r a , J . A . , ( 8 ) 162 Leon, A . , (7) 56 L e o n t e i n , K . , ( 4 ) 123 l e Pape. A . , (5) 283, 503 L e p r a t , R . , ( 6 ) 246 L e r a y , G., (6) 122 L e r o y , J . C . , ( 6 ) 240 L e r o y , Y., (2) 96. 98; ( 5 ) 1024 L e s c h , R . A . , ( 6 ) 396; (8) 677 L e s k o v a , Z., ( 6 ) 187 L e s k o v a r , S., (3) 45 L e s l i e , , D . R . , ( 7 ) 166 L e s t e r , R.L., (2) 47 L e s y n g , B . , ( 3 ) 77 L e t a r t e , M . , (5) 595 Le T r e u t , A . , ( 5 ) 943. 944; (6) 60, 122 L e t u n o v a , E . V . , ( 6 ) 137 Leung, D . W . M . . (3) 184; ( 6 ) 118, 119 Leung, L.L.K., (5) 664, 665, 666 L e v i , G., ( 5 ) 113; ( 8 ) 378 L e v i n e , M.J., ( 5 ) 933 Levy, P., (5) 381, 422 Lewandowski, D . G . , ( 5 ) 2 97 L e w i n , E . B . . ( 4 ) 113 Lewin, M . , (3) 100 L e w i s , B . A . , ( 5 ) 979 L e w i s , D.E., (5) 727 L e w i s , L., ( 6 ) 61 L e w i s , L.N., (3) 123; (6) 350 L e w i s , M.R.H., ( 6 ) 36, 73 L e w i s , M . H . R . ( 6 ) 41 L e w i s , W . E . , (8) 354 L e y b i n , L., ( 7 ) 97 Leyh-Bouille, M., (4) 29 L e y i s , W.E., ( 5 ) 357 L e y t i n , V.L., (5) 238 L e z i c a , R.P., ( 5 ) 1051 L h e r m i t t e , M . , (5) 929, 932, 944 L i , J . , (5) 170 L i , J.S., ( 8 ) 571 L i , S . C . , (2) 38; (6) 54, 261, 265; ( 8 ) 248 L i , Y.T., (2) 38: (6) 54, 160, 261, 265; (8) 248, 457
L i a n g , J . N . , ( 3 ) 266 L i a o , J . , (5) 852 L i a v , A . , ( 7 ) 198 L i e s e r , K.H., ( 8 ) 474 L i f e l y , M . R . , ( 4 ) 111 L i j n e n , H.R., ( 8 ) 164 L i l l y , M . D . , ( 8 ) 625, 701 Lim, V . I . , ( 5 ) 247 L i n , J.Y., (8) 571 L i n , L.C., ( 5 ) 897 L i n , L.S., (5) 517 L i n d , C . , ( 8 ) 379, 380 L i n d , J . , (3) 288; ( 6 ) 19, 51 L i n d a h l , U., ( 5 ) 362, 367 L i n d b e r g , A . A . , ( 4 ) 57, 60 L i n d b e r g , B., ( 4 ) 60, 100, 119, 123 L i n d b l a d , M . , ( 7 ) 25; ( 8 ) 538 L i n d b l o m , G., ( 7 ) 189 L i n d e m a n s , J . , (8) 318 L i n d e r , R . , ( 7 ) 186 L i n d e r , S., (5) 649; ( 8 ) 298 L i n d e r f e r , M.A., ( 5 ) 144 L i n d m a n , B., ( 4 ) 233; (5) 309 L i n d q u i s t , U., ( 4 ) 119 L i n d s a y , J . G . , (5) 908 L i n d s a y , R . M . , ( 6 ) 426 L i n d s h e i d , M . , ( 5 ) 893 L i n d s h i , U., ( 5 ) 361 L i n d s t r d m , L.A., ( 8 ) 548 L i n d q u i s t , L., ( 6 ) 49 L i n e k , V . , ( 6 ) 490, 491 L i n g , N.C., ( 8 ) 71, 72 Lingwood, C . , (7) 138 L i n h a r d t , R.J., ( 6 ) 412 L i n i n g t o n , C . , ( 5 ) 460 L i n k o , M . , ( 6 ) 134, 314; (8) 560 L i n k o , P., ( 8 ) 618, 631 L i n k o , Y.Y., (8) 618, 63 1 L i n n , S., ( 8 ) 452 L i n s c h e i d , M . , (2) 99; ( 4 ) 227 L i o t t a , L.A., (5) 260, 497; (8) 249 L i p k i n d , M . A . , ( 2 ) 83 L i p p e r t , J.L., (6) 426 L i p t a k , A . , ( 2 ) 94 L i s , H., (5) 98, 101; ( 8 ) 89 L i s , M . , ( 5 ) 705 L i s m a n , J. J.W., (6) 64,
264; ( 8 ) 124, 245 L i s o w s k a , E . , ( 5 ) 181, 962 L i s t e n , H . R . , ( 5 ) 826 L i t t l e , C . , ( 8 ) 138 L i t w i l l e r , R.D., (7) 8 L i u , D.W., (2) 45; ( 5 ) 302 L i u , T.T.Y., ( 3 ) 35 L i u , T.Y., ( 4 ) 112 L i v e r n o c h e , D . . ( 8 ) 614 L j u n g , R . , (5) 768 L l o y d , K.O., ( 5 ) 485 L l o y d , T., (8) 136 L o , T.B., ( 8 ) 140 L o a d h o l t , C.B., ( 4 ) 110 L o b a r e v a , L.S., ( 8 ) 691 Lobarzewski, J., (8) 736 L o c k h a r t , S.M., ( 5 ) 18 L o c k h o f f , 0.. (8) 41, 42, 43 L o d i s h , H.F., ( 5 ) 11, 563, 625, 925 L o e b , M . R . , ( 4 ) 98 L o e f , B.G., ( 8 ) 318 L o e f f l e r , C . , ( 5 ) 964 L b n b l a d , P.B., (5) 742 L B n n e r d a l , B., ( 5 ) 153, 158; (8) 397 L b n n g r e n , J . , ( 4 ) 123; (8) 61, 63 L U v g r e n , T., ( 8 ) 385 L b w e n d a h l , L . , ( 8 ) 548 L b w e r , J., ( 5 ) 2 1 Loh, H . , (7) 97 L o k h a n d e . H.T., ( 3 ) 92 Lombardo, A . , ( 6 ) 26 L o m b a r t , C., ( 8 ) 105 Long, W.F., ( 5 ) 758 L o n g a s , M . O . , ( 5 ) 387 L o n g i n , R . , (6) 387 Longmore, G., ( 5 ) 939 Loomis, W.F., ( 6 ) 244 L o o n t i e n s , F.G., (5) 133, 140 L o o s , P . J . , ( 3 ) 31 L o p e z , A . , (6) 520; ( 8 ) 707 L o p e z , P., (4) 6 Lopez-Planes, R., (3) 119 L o r d , J . M . , ( 5 ) 173 L o r e n z . K., (3) 14, 15, 21, 38 L o r m e a u , J . C . , ( 5 ) 369 L o r s c h e i d e r , F.L., ( 8 ) 44 1 L o s i t o , R . , ( 5 ) 345 L o t a n , N., (6) 492 L o t a n , R . , ( 5 ) 188 L o t t , I . T . , ( 5 ) 973;
7 97
Author Index ( 7 ) 102 Loucheux-Lafebvre, M.H., ( 5 ) 717, 1039 Louis, C . J . , ( 5 ) 149 ( 7 ) 55, Louis, J.-C., 77 L o u i s o t , P., ( 6 ) 506, 507 Louden, J.A., ( 6 ) 66 Louden. J.S., ( 6 ) 31 Lowe, C.R., ( 8 ) 421 Loury, P.J., ( 5 ) 1015; (8) 134 Loyau, G., ( 5 ) 412 Loyter, A . , ( 5 ) 52 Lozano, J.A., ( 8 ) 182 (8) Lozinskaya, N.V., 537 Lucas, J . J . , ( 5 ) 1045 Lucia, A , , ( 6 ) 184 L u c i e r , G.W., ( 6 ) 232 Lucu, c., ( 5 ) 202 Ludolph, T . , ( 5 ) 391; ( 6 ) 62; (8) 250 LUderitz, 0.. ( 4 ) 5 2 Luedtke, R . , ( 5 ) 844 LUscher, E.F., ( 5 ) 654, 662 Luft, A.J., ( 8 ) 441 Lugowski, C . , ( 4 ) 59, 117 Luh, B.S., ( 3 ) 27 Luknar, O., ( 3 ) 224 Lunblad, A., ( 7 ) 119 Lund, P.K., ( 5 ) 700 Lundblad, A . , ( 5 ) 962, 963 Lundblad, G . , ( 3 ) 288: ( 6 ) 19, 51 Lundblad, J.L., ( 5 ) 224 Lundquist, K . , ( 3 ) 207 Lundwall, A., ( 4 ) 214 ( 8 ) 289 Lunt, G.G., Luscombe, M., ( 5 ) 328, 401 Luzakova, V., ( 3 ) 115 Luzzana, M., ( 2 ) 74 Lycke, E., ( 5 ) 32: ( 8 ) 406 L y l e s , D.s., ( 5 ) 20 Lynch, J.M., ( 3 ) 194 Lynn, W.S., ( 5 ) 535, 537 Lyon, M., ( 6 1 , 416: ( 8 ) 320 Lyons, G., ( 5 ) 443 Ma, A . , ( 5 ) 804 MacAlister, T.J., ( 4 ) 65 McAnulty, R.J., ( 5 ) 252 (4) McArthur, H . A . I . ,
13 McArthur, J.W., ( 5 ) 959 McBride, B.C., ( 4 ) 169 Maccioni , H. J. F., ( 7 57 McClean, C . , ( 5 ) 1015 (3) McCleary, B.V., 182; ( 8 ) 327 McConnell, K.A., ( 5 ) 20 MacConnell, W.P., ( 5 ) 1 86 McCormick, D.B., ( 8 ) 107 McCourtie, J . , ( 4 ) 191 McCrate, A.J., ( 6 ) 303 McCrorie, P., ( 6 ) 20, 175 McCullough, M.S., ( 7 ) 48 McCully, M.E., ( 3 ) 252 McDonagh, J . , ( 5 ) 236 McDonagh, R.P., ( 5 ) 236 McDonald, G.G., (3) 230 McDonald, J.A., ( 5 ) 225 MacDonald, M.E., (5) 5 95 McDonald, P.J., ( 5 ) 1001 Macek, T., (8) 642 McEver, R.P., ( 5 ) 651, 878 McFeeters, R.F., ( 3 ) 239 McGarvie, D , . ( 3 ) 256, 257, 258 McGeeney, K.F., ( 6 ) 280, 284 McGinley, K.J., ( 4 ) 242 McGinnis, G.D., ( 2 ) 6 ; ( 3 ) 114 McGregor, J.L., ( 5 ) 654 McGuire, T.A., ( 3 ) 75 McHale, A . , ( 6 ) 198, 199, 346 Macher, B.A., ( 5 ) 838; ( 7 ) 35. 90, 715, 118: ( 8 ) 557 Macholalan., ( 8 ) 695 Machovich, R . , ( 5 ) 759 Maciel, G.E., ( 3 ) 18 McKee, P.A., ( 5 ) 769, 782 McKenzie, H.A., ( 5 ) 714, 715 McKenzie, J.L., ( 5 ) 451 McKeown-Longo , P. J , ( 5 ) 322, 447 Mackie, I . J . , ( 5 ) 767 Mackle, Z.M., ( 5 ) 248 Macklewicz, A . , ( 5 ) 818 McKnight, J.L., ( 6 ) 153 McKusik, V.A., (5) 256
.
MacLachan, G.A., ( 5 ) 90; ( 7 ) 174 McLaren, J . , ( 5 ) 289 ( 3 ) 274 McLean, M.W., MacLennan, D.H., ( 5 ) 495; ( 8 ) 236 Macleod, J.L., ( 6 ) 244 McMichael, J.C., ( 8 ) 491 Macmillan, J.D., ( 6 ) 3 80 McNeally, S . S . , ( 6 ) 287 McNeil, M., ( 3 ) 191, 223, 233; ( 4 ) 152; ( 6 ) 287 McNicol, D., ( 5 ) 318 McPhee, D.A., ( 5 ) 66 McPherson, J . , ( 5 ) 504; ( 8 ) 264 MacRae, R., ( 2 ) 92 Macris, B.J., ( 6 ) 136 McVeigh, D.J., ( 5 ) 837 Maddox, I.S., ( 8 ) 625 Maddux, B.A., ( 5 ) 187 Maddy, A.H., ( 6 ) 249 Madnick, H.M., ( 5 ) 688; ( 8 ) 578 Madsen, K., ( 5 ) 424 Maeda, H., ( 6 ) 424 Maeda, K., ( 8 ) 121 Maekaji, K . , ( 3 ) 187 MSkelS, P.H., ( 4 ) 8 3 Maelicke, A . , ( 8 ) 582 Mllnts818, P., ( 6 ) 316 Maffei, C., 96) 33 Magee, A.G., 95) 543: ( 8 ) 268 Maget-Dana, R . , ( 5 ) 199; ( 7 ) 28 Magid, E., ( 6 ) 421 Maggio, B . , ( 7 ) 13, 87 Magil, A.B., ( 5 ) 480 Magnani, J.L., ( 5 ) 857; ( 7 ) 24 Magnusson, K. E., ( 7 ) 92 Magnusson, S., ( 5 ) 236, 734, 736, 737, 741, 742 Magoffin, C.D., ( 7 ) 168 Magro, P., ( 6 ) 454 Mahan, L.C., ( 5 ) 899 Mahany, T., ( 8 ) 442 Maher, P., ( 5 ) 584 Mahmood. A.. ( 6 ) 78 ( 8 ) 748 Mahoney, R . R . , Mahuran, D., ( 6 ) 3 t 66 Maidment, B.W. ( 5 ) 835; ( 8 ) 202 Maier, M . , ( 8 ) 122 Majerus, P.W., ( 5 ) 651, 878 Majunder, A.L., ( 6 94
Carbohydrate Chemistry
798 Maki, M . , (5) 724 Maki, Z., ( 8 ) 292 M a k i t a , A . , (6) 514; ( 7 ) 41 Maksimov, V . I . , ( 6 ) 356, 393 Makus, D . J . , ( 3 ) 234 Malchenko, L.A., (7) 76 M a l c h i o d i , F., ( 5 ) 521 Maler, T., (6) 104 Maley, F., ( 5 ) 115 Maleszka, R . , (8) 622 M a l i k , C.P., ( 7 ) 177 M a l i k , R.K., (6) 352 M a l i n o w s k i , C.E., ( 5 ) 941 M a l l e t t , P.L.. ( 5 ) 839; (8) 570 Malley, D.J., ( 6 ) 50 M a l l i a , A.K., (8) 152 Malloy. P . , ( 8 ) 203 Malmqvist, H . , (6) 266 Malmqvist, M . , ( 8 ) 129 Malmqvist, T . , (8) 129, 130 Malmstrbm, A., ( 5 ) 338, 343, 418 Maloof, F., ( 5 ) 699 Malovikova, A . , (3) 193, 217 Maloy, W.L., ( 5 ) 613, 61 8 M a l p i e c e , Y . , ( 8 ) 583 Malthouse, J.P.G., (8) 41 3 M a l t s e v , S . D . , ( 8 ) 73 Maly, V., (6) 491 Mammerickx, M., ( 5 ) 13 Manca, F . , (5) 142, 143 Mancal, P . , ( 8 ) 587 Mandal, G . , ( 3 ) 139, 185 Mandal, P . K . , ( 3 ) 251 Mandecki, W . , (6) 146 Mandel, P . , ( 7 ) 55, 77 Mandels, G . R . , (6) 212 Mandels, M., ( 6 ) 331, 35 1 Mandenius, C . F . , ( 8 ) 632, 669, 746 Manecke, G., ( 8 ) 705 Mangnani, J.L., (7) 32 M a n j u l a , B.N., (5) 856 Mankin, A . S . , (4) 199 Manley, G., ( 5 ) 300 Manners, D . J . , (3) 20 Mannock, D . A . , ( 7 ) 86 Mansson, J.-E., (7) 49 Mansson, M.O., ( 2 ) 27 Mansson, P., (3) 104; ( 8 ) 485 M a n t l e , D., ( 5 ) 956
M a n t l e , M., ( 5 ) 952, 955, 956 Mantsch, H.H., ( 5 ) 128. 129, 179 Manzanal. M.B., ( 4 ) 246 M a r c h e s i , S., ( 1 511 M a r c h e s i , V.T., ( 5 ) 909, 922 M a r c h e s i n i , ( 6 ) 26 M a r c h e s s a u l t , R H., ( 2 ) 113; ( 3 ) 85; 4 ) 208 Marchis-Mouren, G . , (6) 293 Marchvlo. ( 3 ) 51 - . B.A.. Marciano, P., (6)-454 M a r c i p a r , A , , ( 8 ) 550 Marcus, D.M., (7) 27 Marcus, F., ( 6 ) 96 Marcus, S., ( 5 ) 122 M a r d e r , V . J . , ( 5 ) 785 Marek, M . , (6) 485; ( 8 ) 645, 751 M a r e t , A . , ( 6 ) 21, 167 M a r g a r i t e l l a , P . , (5) 791 M a r g a r i t i s , A., ( 6 ) 200; ( 8 ) 200; ( 8 ) 619. 653 M a r g o l i n , A.L., ( 8 ) 734 M a r g o l i s , H.C.. (5) 486 M a r g o l i s , R.K., ( 5 ) 332 M a r g o l i s , R.U., (5) 332, 466 M a r g o s s i a n , S. S. , ( 5 ) 510 M a r g u e r i e , G . , ( 5 ) 787 M a r i a t , F . , (4) 225 M a r i n o , M., ( 5 ) 526 Mark, H . , (3) 90 Mark, H . F . , ( 3 ) 3 M a r k a k i s , P., ( 6 ) 136 M a r k e l o n i s , G. J . , ( 8 ) 251 M a r k i n , V.A., ( 6 ) 137 Markovic, 0.. (3) 224, 243 Markowitz, S., ( 5 ) 922 Marmer, W.N., (7) 6 Marmet, D., ( 4 ) 14; ( 5 ) 177 Marossy, K . , ( 8 ) 404, 405 M a r r i o t t , J., ( 3 ) 67 Marsden, H.S., (5) 31 Marshak, D.R., ( 8 ) 349 M a r s h a l l , K., (6) 90 M a r s h a l l , P.J., ( 6 ) 72 M a r s h a l l , R.D., (5) 967 M a r s h a l l , S.E., ( 5 ) 328, 401 M a r t i n , A . , ( 6 ) 507 M a r t i n , G.R., (5) 240,
256, 586 M a r t i n , J . , ( 6 ) 258 M a r t i n , S.E., ( 5 ) 785 M a r t i n , S.M., ( 3 ) 136; ( 4 ) 186; ( 6 ) 365 M a r t i n e k . K., (8) 536 Martinez, R.J., ( 6 ) 422 M a r t i n i u k , F . , ( 6 ) 162 M a r t i n k o , J . , ( 5 ) 615 Maru, A , , (6) 514 Maru. K., ( 3 ) 253 Maruo, B., ( 6 ) 312, 313 Maruyama, S., ( 8 ) 523 Maruyama, Y.. ( 4 ) 114; ( 7 ) 144 M a r z e t t i , A . , ( 3 ) 117; (6) 332 M a s a k i , A . , ( 6 ) 440 Mashimo, H . , (5) 627 Masouredis, S.P., (5) 8 99 M a s s a r e l l i , R., ( 7 ) 55 M a s s e r i n i , M . , ( 7 ) 12, 18, 19 Massey, J . , ( 7 ) 146 M a s t e r s , V.M., ( 8 ) 173 Masuda, H . , ( 5 ) 317; (6) 82 Masuda, K . , ( 4 ) 185 Masuda, S., ( 7 ) 161 Masuda, Y.. ( 3 ) 145, 198 Masumoto, K., ( 4 ) 187 Matheson, N.K., ( 3 ) 20, 64 Mathew, M.K., ( 5 ) 124 Mathieu-Mahul, D . , ( 5 ) 64 M a t h u r , N.K., ( 8 ) 547 M a t s a s , R . , ( 8 ) 190 Matsuda, K., ( 3 ) 170, 171, 174, 175, 176; ( 4 ) 223, 224; ( 6 ) 377, 525 M a t s u d a , S., ( 8 ) 123 Matsuda, T . , (5) 988, 989 M a t s u h a s h i , M., ( 4 ) 30 M a t s u i , H . , ( 6 ) 176 M a t s u i , M., ( 8 ) 56 M a t s u i , S., ( 8 ) 145 Matsurnoto. I . , ( 5 ) 102, 111, 174, 180, 390. 392, 393 Matsumoto, M., ( 7 ) 140; (8) 328, 348 Matsumoto, U . , ( 8 ) 387 Matsumura, G . , ( 2 ) 103; ( 5 ) 995; (6') 252, 419 Matsumura, H . , ( 4 ) 243 Matsunaga, T . , ( 8 ) 99, 603, 611
799
Author Index Matsuno, R . , ( 6 ) 398, 399, 405; 8 ) 158, 648, 652 Matsuoka, Y. ( 5 ) 484 Matsura.. F.. . ( 5 ) 974 Matsusaka, K., ( 6 ) 185 Matsushima, Y . , 49, 115: ( 8 ) 562 Matsushita, K., ( 6 ) 535 Matsushita, R., ( 8 ) 498 Matsuuchi, L . , ( 5 ) 887 Matsuura, D., (8) 26 Matsuura, F., ( 7 ) 101, 121 Matsuzaki, K., ( 4 ) 141; (5) 347 Matsuzaki, T., ( 7 ) 182 ( 5 ) 939: Matta, K.L., ( 6 ) 110; ( 8 ) 10, 28, 29, 30, 31, 40, 52, 551 M a t t a i , J . , ( 5 ) 351 M a t t e r s , G.L., ( 3 ) 65; ( 6 ) 526 M a t t i a s s o n , B., ( 8 ) 667 Matthel, S., ( 8 ) 353 Matthews, B.W., ( 6 ) 433 Matthews, T.J., ( 5 ) 79 Mathewson, P.R., ( 6 ) 305 Matthieu, J.M., ( 5 ) 461 ( 2 ) 46 Mathison, G.W., ( 6 ) 521 Matthews, M.M., (3) Matthysse, A.G., 112 Matthyssens, G., ( 5 ) 998 M a t t i a s s o n , B., ( 8 ) 624, 632, 669, 746 M a t t i n g l y , S.J., ( 4 ) 116, 161 M a t t i s o n , S.L., ( 7 ) 37 (7) 8 Mattox, V.R., Matuo, Y., ( 8 ) 258 Mauchauffe, M., ( 5 ) 64 Maury, C.P.J., ( 5 ) 969, 970 Mawbry, W.J., ( 5 ) 907 Maxwell, R.J., ( 7 ) 6 ( 5 ) 96 Mayer, A.M., Mayer, H . , ( 4 ) 38, 82; ( 7 ) 199 96) 522, Mayer, R.M., 523 Mayer, R.T., ( 2 ) 84; ( 6 ) 42 Mayes, J.S., ( 6 ) 111 Maylie-Pfenniger, M.F., ( 5 ) 575 Maynard, C.M., ( 6 ) 524 Maynard, M.T., ( 4 ) 163 Maynard, Y., ( 5 ) 569
Mazumber, T., (5) 109, 169 Mazur, E.M., ( 5 ) 511 Mazurek, M.. ( 5 ) 993 Mazurier, J . , ( 5 ) 730 Mazzei, Y . , ( 6 ) 293 Mazzola, G., ( 8 ) 411, 476 Mazzuca, M . , ( 5 ) 942 Meade-Cobun , K. S. , ( 5 ) 684 Meadow, P.E., ( 8 ) 300 ( 2 ) 12 Medcalf, D.G., Medeiros, J.E., ( 6 ) 351 Meguro, H . , ( 8 ) 365 Mehta, S.L., ( 3 ) 57 Meier, H . , ( 3 ) 128 Meier-Ewert, H., ( 5 ) 36 Meis, M.E., ( 5 ) 281 Meitzner, E.P., ( 8 ) 8 Melet, J . , ( 5 ) 296 M e l l i s , S.J., ( 2 ) 36; ( 5 ) 1013 ( 5 ) 114: Mellor, R.B., ( 8 ) 218 Melnykovych, G., ( 5 ) 587 Melrose, S.M., ( 5 ) 505 Melton, L.D., ( 3 ) 262 Memeli, L., ( 6 ) 250 ( 5 ) 463 Mena, E.E., Menard, D., ( 5 ) 576 Menashi, S., ( 5 ) 650 Mendicino, A . , ( 5 ) 846 Mendicino, J . , ( 5 ) 845, 856, 1065: ( 8 ) 282 Menegus, F., ( 8 ) 337 Mengel, K . , ( 3 ) 59 Mengin-Lecreulx, D., ( 4 ) 24 Mengod, G., ( 5 ) 638, 646, 879 Menon, K.M.J., ( 5 ) 582 Menon, R . , ( 5 ) 175 Mercer, D.G., ( 8 ) 744, 745 Mercier, C., (3) 192 Meredith, P . , ( 3 ) 23 Meredith, S.C., ( 5 ) 250 Merlin, A., ( 8 ) 487 M e r r i f i e l d , E.H., ( 3 ) 259: ( 4 ) 101 M e r r i l l , A . H . , ( 8 ) 107 ( 6 ) 290 M e r r i t t , A.D., Mersmann, G., ( 5 ) 393 Merten, B., ( 5 ) 298 Merz, D.C., ( 5 ) 74 Meskanen, A., ( 6 ) 347 Messer, M., ( 5 ) 707: ( 6 ) 38 Messeter, L., ( 7 ) 119 Messmore, H.L., ( 5 ) 755
Messner, P., ( 4 ) 97 ( 8 ) 443 Metcalf, E.C., ( 8 ) 369 Metrione, R.M., M e t t l e r , L., ( 5 ) 579 Metz-Boutique, M.H., (5) 730 Metzger, J . , ( 3 ) 6 Metzger, M., (5) 753; ( 8 ) 201 ( 5 ) 224 Meunier, A.M., Meunier, F., ( 5 ) 272 Meyer, B., ( 5 ) 362: ( 8 ) 535 Meyer, H.E., ( 8 ) 403 ( 6 ) 201 Meyer, H.-P., Meyer, K., ( 5 ) 386, 387 ( 8 ) 228 Meyer, W.L., Meyland, I., ( 3 ) 261 Mezei, C., ( 5 ) 465 Michael, A.F., ( 5 ) 409 Michalski, J.C., ( 2 ) 91 ; (5) 968, 972 Michel, G . , ( 4 ) 61: ( 7 ) 203 Michel, V . , ( 5 ) 1018 (5) Michelacci, Y.M., 392 Michel-Briand, Y., ( 6 ) 246 Michelena, V., ( 5 ) 655 Michiels, F., ( 5 ) 998 Mihalov. V., ( 3 ) 189: ( 8 ) 35 Mikami, B., ( 6 ) 325, 326 Mikami, S., ( 5 ) 784 Mikami, Y . , ( 4 ) 213 Mikes, D., ( 6 ) 447 Mikes, O., ( 3 ) 235 Mikhatlov, A.T., ( 7 ) 76 (5) 532 Miki, B.L.A., M i k i , K., ( 8 ) 123 M i k i , N.K., ( 3 ) 252 M i l a t , M.L., ( 8 ) 49, 53 (5) Milenkovic, A.G., 60 M i l l e r , A . , ( 5 ) 327 Miller, A.L., ( 6 ) 57, 61, 69, 126 M i l l e r , E.J., ( 5 ) 259, 262 M i l l e r , F., ( 5 ) 958 Miller, J . A. , (5) 809 Miller, J.M., ( 5 ) 14 M i l l e r , L.L., ( 8 ) 226 M i l l e r , R.S., ( 5 ) 869 Miller, W.A., ( 6 ) 234 Miller-Anderson, M., ( 5 ) 763 Miller-Podraza, H., ( 7 ) 103 M i l l e t , J . , ( 6 ) 387
800 M i l l i g a n . L . P . , ( 2 ) 46 Millis, J . R . , (8) 687 Millman, I . , ( 8 ) 491 M i l n e r , L . A . , (6) 45 Milone, M . , ( 6 ) 230 M i l s t e i n , 0.. (3) 210 Mima, S., ( 3 ) 280 Minakata, K . , ( 8 ) 703 Minami, N., ( 6 ) 40 Minamikawa, T., ( 6 ) 310 Mininsohn, M . M . , ( 5 ) 80 1 Minoda, Y . , ( 4 ) 147 Minoo, O., (3) 115 Minowada, J . , (5) 645 Miron, J . . (3) 195, 197 Miron, T., ( 8 ) 412 M i s a k i , A . , ( 3 ) 172; ( 4 ) 209 M i s a k i , M . , ( 8 ) 524 Mishra, P . C . , ( 6 ) 348 Mislovicova, D . , ( 8 ) 54 1 Misuma, H . , ( 2 ) 65 M i t c h e l l , J.R., ( 3 ) 250 M i t c h e l l , J.W., ( 5 ) 293 M i t c h e l l , K.F., ( 7 ) 32 M i t r a , S., ( 5 ) 676 M i t r a n i c , M.M., (5) 1078 M i t s u h a s h i , S , . ( 4 ) 188 M i t s u i , K . , ( 4 ) 30 M i t s u i s h i , Y . , ( 6 ) 377 M i t s u y a s u , N . , ( 8 ) 145 Mityushova. N . M . . (6) 77 Miura. I . , ( 8 ) 18 Miya, M . , ( 3 ) 280 Miyai, K . , ( 8 ) 648 M i y a j i , H . , (4) 209 Miyamoto, I . , ( 2 ) 57: (5) 301, 334, 341 Miyamoto, T., ( 5 ) 346: (8) 546 Miyanaga, 0.. ( 5 ) 731 Miyasoto, M . , (5) 754 Miyata, Y . , ( 3 ) 81 Miyatake, T., (5) 978: ( 7 ) 36 Miyawaki, M . , ( 8 ) 524 Miyazaki, K . , (8) 258 Miyazaki, T., ( 4 ) 240, 241 Mizobuchi, Y . , ( 8 ) 527, 528, 529 M i z r a h i , L . , ( 5 ) 159 Mizukami, T . , (6) 317, 319 Mizuno. D . , ( 8 ) 201, 636 Mizuno, K., ( 8 ) 323 Mizuno, T . , (4) 69, 70,
Carbohydrate Chemistry 71, 239: ( 8 ) 365 Mizuno, Y . , (5) 562: ( 8 ) 281 Mizuochi, T., ( 5 ) 542; (6) 236 Moczar,E., ( 5 ) 1075: (8) 663 Mod, R . R . , ( 3 ) 137 Mbllby, R . , ( 8 ) 129, 130 Mohamed, N., ( 6 ) 291 Mohun, T., (5) 994; ( 8 ) 352 M o i n i e r , D . , ( 6 ) 293 Moir. A . G . , (5) 984 Molday, R.S.. ( 5 ) 584, 578 Moledina, K.H., ( 3 ) 245 Molinaro, M . , (5) 526 Molinu, C., ( 8 ) 189 Moll, M . , (4) 231 Mollenhauer, H.H., ( 6 ) 42 M o l l e r , V . , ( 8 ) 466 Momii, F . , ( 4 ) 129 Momo, T . , ( 7 ) 164 Momoi, T . , ( 7 ) 60, 80, 122 Monaco, F . , ( 5 ) 471 Monahan, J . B . , (5) 796 Moncada, R . , ( 5 ) 755 Monge, M . , (7) 97 Monner, D.A., ( 4 ) 7 2 Monnom. D . , ( 5 ) 1072; ( 8 ) 266 Mononen, I., ( 2 ) 9 Monsan, P . , (6) 520; ( 8 ) 707 Monsigny, M., ( 3 ) 287: (5) 199, 570: 97) 28 Montague, M.D., ( 4 ) 143 Montalvo, R . , ( 6 ) 370 Montelione, G . J . , (5) 190: (8) 219 M o n t e n e c o u r t , B.S., (6) 355 Montesoro, E., ( 6 ) 152 Montezinos, D . , ( 3 ) 129 Monthony, J.F., ( 8 ) 400 Montibeller, J . A . , (8) 333 M o n t r e u i l , J . , ( 2 ) 91, 96, 98, 106; (5) 123, 150, 558, 663, 729, 730. 748, 882, 883, 937, 961. 968, 972, 987, 990, 1018, 1024; ( 6 ) 264: ( 8 ) 206, 239 Mook, G . E . , (6) 56 Mookerjea, S., ( 5 ) 1044, 1077 M o o r c r o f t , D . , ( 5 ) 385
Moore, B.W., ( 5 ) 463 Moore, G.K., ( 3 ) 278. 279, 281 Moore, J . P . , ( 8 ) 478 Moore, N.F., (5) 545 Moorhouse, C . , ( 6 ) 307 Moori, K . , ( 6 ) 319 M o r a l e s , M., ( 4 ) 217 Morandi, C., ( 8 ) 335 M o r d a r s k i , M . , ( 8 ) 690 Mordick, T . , ( 6 ) 505 More, R . P . , (5) 766 M o r e i r a , A . R . , (6) 357 Morel, P., ( 5 ) 919 Morel du Boil, P . G . , (2) 5 Moreno, C . , ( 4 ) 111 Moreno, E . , (4) 58 Moreno, R., ( 5 ) 146 Morgan, A . C . , (5) 639 Morgan, R.K., ( 5 ) 648 Morgan, W.T., (5) 811 M o r g o l i s , R . K . , ( 5 ) 466 Mori, E . , ( 6 ) 326 Mori, H . , ( 5 ) 516: ( 8 ) 128 Mori, M . , ( 8 ) 225, 554 Mori. T . , (8) 469 Mori, Y., ( 5 ) 423 Morimoto, Y., (8) 564 Morin, B . P . , ( 3 ) 48 Morinaga, T., (2) 38: ( 6 ) 265 Morino, Y., ( 8 ) 145 Morioka, T . , ( 6 ) 379 M o r i t a , T., ( 4 ) 109 M o r i t a , Y . , (6) 325, 326, 327 Moriyama, S., ( 6 ) 398, 399, 405 Moriyasu, T., ( 8 ) 509 Morozova, L . A . , (5) 712 Morris, D.R., ( 6 ) 141 M o r r i s , E . , (5) 947 Morris, E . J . , ( 4 ) 93 M o r r i s , E.R., ( 3 ) 179, 213, 214, 215, 266, 267; (4) 142 M o r r i s , J.E., (5) 306 M o r r i s , V.J., ( 3 ) 269, 270, 271 M o r r i s o n , I.M., ( 2 ) 62 M o r r i s o n , S . L . , (5) 854, 887 Morrison, T.G., (5) 57 M o r r i s s e y , J. J . , (5) 473 Morrone, S., ( 7 ) 146 Morser, J . , (5) 994; ( 8 ) 352 M o r t e n s e n , S.B., ( 5 ) 735, 741
80 1
Author Index Morton, L . F . , ( 5 ) 276 Mosbach, K . , ( 2 ) 27; ( 8 ) 419, 641 Mosca, A.. ( 2 ) 74 Moscarello, M.A., ( 5 ) 1078 Moser, H.W., ( 5 ) 428 Mosesson, M . W . , (5) 220 Mosher, D.F., ( 5 ) 242, 487 Moshudis, E., ( 6 ) 272 Mosolo, V.V., ( 8 ) 179 Moss, L.G., ( 8 ) 478 Motoda, R . , ( 4 ) 164; ( 6 ) 501 Motokawa, Y . , ( 8 ) 221 M o t t o l a , H . A . , ( 8 ) 742 Moudgil, V.K., ( 8 ) 480 Mount, J.N., (2) 17 Mourao, P.A.S., (5) 350, 434, ( 8 ) 277 MOUS, J . , ( 5 ) 492 Moyes, L . , (5) 598 Mozhaev, V.V., ( 8 ) 536 Mrackova-Dobrotova, M . , ( 6 ) 450; ( 8 ) 714 MUhlradt, P . F . , (4) 72; ( 5 ) 518, 519, 520. 596; ( 7 ) 149 M t l l l e r , E . , ( 5 ) 395 MUller, I . . ( 5 ) 202 M t l l l e r , M . , ( 5 ) 815 M u e l l e r , O.T., ( 6 ) 248 MOller, Th., ( 7 ) 188 M u e l l e r , U.W., ( 8 ) 316 M u e l l e r , W.W., ( 5 ) 830 M U l l e r t z , S., ( 8 ) 283 Mugnani, G . , ( 7 ) 7 8 Muh, J . P . , ( 5 ) 283, 503 Muir, H., ( 5 ) 319, 327 Mujwid, D.K., ( 5 ) 869 Mukherjee, A.K., ( 3 ) 251 M u k h e r j e e , B.B., ( 5 ) 1041 Mukhopadhyay, S.N., ( 6 ) 3 52 M u l e t , C.-C ( 6 ) 508 M u l l e r , W.E.G.. ( 5 ) 202 M u l l i n s , D.J., ( 5 ) 456 M u l l i n s , J . T . , ( 4 ) 205, 206 Munakata, S . , ( 3 ) 113; ( 8 ) 462 Mundy, B.P., ( 2 ) 8 9 Mundy, J . P . , (5) 810 M u n j a l , D.D., ( 5 ) 483 Munnecke, D.M., ( 8 ) 630 Munro, P.A., ( 8 ) 701 Munz, E . , ( 2 ) 73 Murachi, T . , ( 8 ) 743
Murakami, K . , ( 8 ) 314, 342 Murakami, N . , ( 8 ) 480 Murakami, S., ( 6 ) 268 Muraki, E., ( 3 ) 118 Muramatsu, H . . (5) 530; ( 8 ) 260 Muramatsu, T , . ( 5 ) 530; ( 6 ) 367; ( 8 ) 260 Murano, G . , ( 5 ) 762, 763 Murao, S . , ( 4 ) 244, 245,; ( 6 ) 276, 297, 328, 362 Murase, S., ( 8 ) 75 M u r a t , J . C . , ( 1 6 ) 163, 169 M u r a t a , K . , ( 8 ) 605, 638 M u r a t a , M.. ( 4 ) 213; ( 6 ) 185 M u r a t a , Y . , ( 6 ) 434 Murayama, J . , ( 5 ) 916 Murazumi, N . , ( 4 ) 8 Murbach, N.L., ( 6 ) 300 Murdoch, J . , ( 5 ) 866 Muresan, V . , (5) 575 Muroa, S., ( 6 ) 220 Muroi, M . , ( 8 ) 498 Muronetz, V.I., ( 8 ) 167, 168 Murphy, G.P., ( 8 ) 237 Murphy, L.A., ( 5 ) 134, 1048 Murphy-Ullrich, J . E . , ( 5 ) 487 Murray, E . J . , ( 3 ) 213, 215 Murray, G . J . , ( 5 ) 573 Murty, V.L.N., ( 7 ) 44 M u s t r a n t o , A . , ( 6 ) 134 Musquera, S., ( 8 ) 105 Muthu, M . , ( 6 ) 461 Myerowitz, R . , ( 5 ) 573, 574; ( 6 ) 237 MyllylH, G . , ( 5 ) 904; ( 8 ) 220 M y l l y l B , R., ( 5 ) 284, 875; ( 8 ) 165 Nachbar, M.S., ( 4 ) 68 Nachman, R.L., ( 5 ) 664, 665, 666 Naczk, M . , ( 6 ) 79; ( 8 ) 503, 504 Nader, H. B., ( 5 ) 358 Nader, W . , ( 4 ) 195 N a d k a r n i , G.B., ( 6 ) 15; (8) 708 N a d l e r , H.L., ( 6 ) 128, 431 N a g a i , Y . , ( 5 ) 340,
399; ( 7 ) 16, 66, 67, 68, 89, 116, 122, 124, 147, 164 Nagano, M . , ( 5 ) 731; (8) 244 Nagao, M . , ( 8 ) 257 Nagasawa, K . , ( 5 ) 347, 348, 360; ( 8 ) 127 Nagase. S., ( 2 ) 57; ( 5 ) 301, 334, 341 Nagashima, F . , ( 8 ) 145 N a g a t a , K . , ( 8 ) 386 Nagata, M . , (2) 95 N a g a t a , Y., ( 6 ) 312, 313; ( 8 ) 342 N a g e l e , A . , ( 5 ) 36 Nagradova, N . K . , (8) 167. 168 Naim, H.Y., ( 5 ) 662 N a i n a w a t e e , H.S., ( 7 ) 183 N a k a b a y a s h i , S., ( 8 ) 12, 62 Nakada, H . , ( 5 ) 561 Nakada, T . , ( 8 ) 123 Nakagawa. H . , ( 6 ) 91 Nakagawa, Y . , ( 5 ) 486 Nakahara, Y . , ( 3 ) 111 Nakajima, A . , ( 3 ) 290; ( 8 ) 501 Nakajima, K., ( 5 ) 423 Nakajima, T . , ( 4 ) 223, 224 Nakamura, M . , ( 3 ) 58; ( 4 ) 114 Nakamura, N . , ( 3 ) 156, 157 Nakamura. S., ( 6 ) 493 Nakamura, T . , ( 4 ) 109; ( 6 ) 115 Nakamura, Y., ( 8 ) 342 Nakamuua, T . , ( 6 ) 268 N a k a n i s h i , K., ( 6 ) 398, 399, 405, 473, 474; ( 8 ) 158 N a k a n i s h i , M., ( 5 ) 53 N a k a n i s h i , Y . , ( 5 ) 417, 1052 Nakano, H., ( 6 ) 277 Nakasawa, S., ( 8 ) 128 Nakata. H . , ( 8 ) 408 N a k a t a n i , H . , (8) 513 Nakawawa. S., ( 5 ) 516 Nakayama. M . , ( 6 ) 425 Nakhapetyan, L.A., ( 8 ) 584 Nam S h i n , J . E . , ( 4 ) 38, 39: ( 7 ) 199 N a n a s i , P . , ( 8 ) 38, 39 Naoi, M., ( 6 ) 123 Narang, C.K., ( 5 ) 105; ( 8 ) 547
Carbohydrate Chemistry
802 Naruse, H . , (2) 66 Nash, H . A . , (8) 137 Nashed, M.A., ( 8 ) 7, 9, 74 Nasir-ud-Din, ( 5 ) 959 Nathan, D.M., (5) 803 Nathenson, S.G., ( 5 ) 611, 613, 614, 615, 616, 617, 628 Nathur, N.K., (5) 105 N a t o , F . , ( 5 ) 559 N a t o r i , S., (5) 201 Natowicz, M., ( 2 ) 33 N a t s u a k i , O., (6) 305 Naughton, M.A., ( 5 ) 804 Navarro, J . M . , (8) 600 N a v i a , M.a., ( 5 ) 841 Nayak, B.R., (3) 254, 255 Nayak, P.L., ( 8 ) 486 Nayak, S.S., (5) 800 N a z l y , N . , ( 8 ) 644 Ndulue, A . , (4) 14; ( 5 ) 177 Nedelcheva, M . , ( 3 ) 13 Negre, A . , (6) 21, 167 N e g u l i a n u , C., ( 3 ) 110 Neihaus, W.G., (2) 86 Neil, J . C . , ( 5 ) 15 Nelles, L.P., (5) 743 N e l s o n , D . R . , ( 5 ) 204; ( 7 ) 192 N e l s o n , R . D . , ( 4 ) 221 Nelson, T . E . . (6) 330; ( 8 ) 114 Nemoto, T . , ( 5 ) 835; (8) 202 Neoh, S.H., ( 5 ) 1001 Neokesariiskii, A . A . , (5) 454 Neri, P . , (2) 60 Neszmelyi, A . , ( 8 ) 38, 39 N e u b e r g e r , A . , ( 5 ) 152, 175 Neuberger , M . S. , ( 5 ) 8 98 N e u f e l d , E . F . , ( 6 ) 55, 237 Neuhaus, F . C . , ( 4 ) 1 0 Neuhoff, V . , (5) 1016 Neukom. H . , ( 3 ) 33, 147, 182 Neumann, E . F . , ( 6 ) 121; (8) 242 Neumeyer, B . , ( 7 ) 46 Neurohr, K . J . , (5) 128, 129, 179 Neuwelt, E . A . , ( 6 ) 56 Nevalainen, K.M. H . (6) 209 N e v i l l e , A.M., ( 5 ) 218
N e v i l l e , D.M., ( 5 ) 573, 876; ( 8 ) 101 Nevins. D . J . , (3) 162. 168: ( 6 ) 383, 391 Newman, C.W., (3) 159 Newman, E . S . , ( 5 ) 866 Newman, J . , (5) 776 Newton, L . E . , ( 5 ) 132 Ney, K . A . , (5) 799 Ng, T . K . , ( 4 ) 133; ( 6 ) 207, 366, 386 Ng, W.G., ( 5 ) 289 Ngo, T . T . , (8) 671 Nguyen, B . T . , ( 4 ) 147 Nguyen, T.D., (8) 433 Nicholls, A . J . , (6) 23 Nicholson-Weller, A , , ( 8 ) 278 Nickerson, J.M., (5) 825; (8) 359 N i c o l a i s e n , F.M., ( 3 ) 261 N i c o l a u , c . , ( 7 ) 20 N i c h o l a u s , B . (8) 608 N i c o l e a n u , J . , ( 3 ) 110 N i c o l l e t , I . , (5) 746; ( 8 ) 360 N i c o l o f f , J . T . , ( 5 ) 698 N i e b e r g van V e l z e n , E.H., ( 2 ) 3 Nieduszynski, I . A . , (5) 339. 357; ( 8 ) 354 Niel, C . , ( 6 ) 135 N i e l s e n , L . S . , ( 8 ) 466 N i e l s e n , J . T . , (6) 296 N i e l s e n , M . T . , ( 6 ) 303 Nieuw Amerongen , A . V. , ( 5 ) 934 N i i m u r a , Y . , ( 7 ) 64 N i k a i d o , H., (4) 63 N i k k a r i , T . , ( 5 ) 310 Nikonovich, G.V., ( 8 ) 465 Niku-Paavola, M.L., ( 6 ) 341, 345, ( 8 ) 464 N i l s s o n , I., ( 8 ) 624 N i l s s o n , K . , (8) 419 N i l u b o l , N., ( 6 ) 468 Ninomiya, H . , (2) 65 Ninomiya, Y., ( 5 ) 399 N i s b e t , A . D . , (5) 984 N i s h i , A . , ( 4 ) 187 N i s h i , S., (5) 867; ( 8 ) 184 N i s h i h o r i , K . , ( 8 ) 18 N i s h i b o r i , K., (8) 68 N i s h i g u c h i , H . , ( 8 ) 18, 75 N i s h i j i r n a , M., ( 4 ) 241 N i s h i k a t a , M . , ( 8 ) 307 N i s h i k i d o , N . , ( 6 ) 434 N i s h i k i m i . M . , ( 6 ) 494
Nishimura, O . , ( 4 ) 112 Nishimura, S., (8) 737 N i s h i m u r a , Y., ( 2 ) 52, 53; (6) 231, 236 N i s h i n o , M., ( 5 ) 784 N i s h i t a n i , K . , ( 3 ) 145 N i s i z a w a , K . , ( 6 ) 337, 363 N i s s l e y , S.P., ( 5 ) 407 Nithianandam, V.S., (8 512, 715 Niv, A . , ( 8 ) 470 Noda, A . , ( 6 ) 398 Noelken, M . E . , ( 5 ) 264 274 Noguchi, E . , ( 6 ) 494 N o j i r i , H . , (7) 63 N o l t e , F . s . , ( 8 ) 113 Nomura, 0.. (2) 82 Nonaka, Y., ( 8 ) 737 Nonnenmacher, D . , ( 5 ) 131; ( 8 ) 573 Norberg, T . , ( 8 ) 16, 27 Nord, C . E . , ( 6 ) 49 Nordal, P.-E., (7) 4 Nordenman, B . , ( 5 ) 364 Norder, H., (8) 418 N o r d i n , J . H . , ( 4 ) 208 Noren, O., (6) 444 N o r i d o , F . , ( 7 ) 58 N o r i s u y e , T . , ( 4 ) 219 N o r l a n d , S., ( 8 ) 299 Normand, F . L . , ( 3 ) 137 Normier, G., ( 4 ) 106; (7) 195 NorrlBw, 0.. ( 8 ) 419 N o r t h c o t e , D.H., ( 3 ) 203, 204, 205 N o t i , J.D., ( 6 ) 148 N o v a i s , J . M . , ( 8 ) 717, 71 8 Novak, G . E . , ( 4 ) 212 Novakova, J . , (5) 162 Novotny, J . , ( 5 ) 594 Nowinski, R.C. (5) 861, 862; ( 7 ) 150 Nudelman, E . , (5) 861, 862; ( 7 ) 150 N u e s s l e , D.W., (4) 166 Nugent, P.G., (5) 975; (6) 242 Nummi, M., ( 6 ) 341, 345; ( 8 ) 464 Nunez, H . A . , ( 5 ) 974, 1069; (7) 101: (8) 346 Nunez, M.T., ( 5 ) 585 Nunn, W.D., ( 8 ) 223 Nurden, A . T . , ( 5 ) 656, 663, 906 Nurminen, M . , ( 4 ) 83 Nustad, K . , ( 8 ) 190
803
Author Index Nygren, H., ( 8 ) 664 Oba, S., (4) 28 O b a t a , F . , ( 5 ) 145 O b e r h o l t z e r , J.C., ( 5 )
82 1
O b e r l e y , T.D.. ( 5 ) 487 O b i , S.K.C., (6) 449 O b r e n o v i t c h , A., (5)
570
O l B r i e n , M., ( 4 ) 176 O b r i n k , B.. (5) 558 O c h i a i , H., ( 5 ) 3 O c h i a i , Y . , ( 7 ) 116 Ochoa, A.G., ( 6 ) 120 Ochoa, J.L., (5) 116 O c k l i n d , C . , ( 5 ) 557 Ockman, N . , (5) 141 O ' C o n n e l l , A.P., ( 8 )
563
O ' C o n n e l l , B.T.,
300
(6)
C.M., (6) 280. 284 Oda, K., ( 5 ) 754 Oda, Y . , ( 5 ) 135; ( 8 ) 347 Oden, U . , ( 6 ) 188 O'Donnell, D.J., (3) 18 O ' D o n n e l l , P.V., ( 5 ) 7 2 O ' D r i s c o l l , K.F., (8) 744, 745 Oegema, T.R., ( 5 ) 406, 409 Oeltmann, T.N., ( 8 ) 339 Oette, K., (6) 272 O f e k , I., ( 4 ) 18 O f f o r d , R.E., (8) 381 Ogamo, A., ( 5 ) 347 Ogata, S., (5) 754 O g a t a , S.I., ( 5 ) 485 Ogawa, K . , (3) 150 Ogawa, T., ( 3 ) 8; ( 4 ) 50; (8) 12, 56, 57, 58. 59, 60, 62 O g i h a r a , Y., (4) 226; ( 8 ) 37, 65 Ogino, H., (3) 79 O'Grady, P., ( 8 ) 694 O g u n l e s i , M., (6) 139; ( 8 ) 712 O g u r a , H., ( 2 ) 112 Ogura, N . , (6) 91 Oh, T.H., ( 8 ) 251 O h a n e s s i a n , J . , (5) 126 O h a s h i , M., ( 7 ) 7 0 Ohba, R . , (8) 468 Ohkubo. A., ( 2 ) 6 8 Ohkubo, Y . , (5) 379; ( 6 ) 531 Ohkura, T., ( 5 ) 977; (6) 71
O'Connor.
O h l s o n , S., ( 2 ) 27 O h l s s o n , J.T., (8) 319,
447
O h n i s h i , M., ( 6 ) 319,
327; (7) 170, 184, 185 Ohno, H . , ( 8 ) 297 Ohrambach, A,, (5) 683 Ohsawa, T., ( 8 ) 342 Ohst. E . , (5) 395 O h t a , S., ( 5 ) 423 O h t a n i , K . , (3) 172 Ohyama, K., ( 6 ) 276, 328 Oie, M., ( 5 ) 84 O i s h i , K . , (5) 206, 207, 903; ( 6 ) 373; (8) 356, 594 Oiwa, R . , ( 4 ) 34 Ojamo, H., ( 6 ) 134, 314; ( 8 ) 560 O j i , C , . ( 8 ) 123 Okada, J . , ( 4 ) 8 Okada, S., (5) 977 Okada, Y . , ( 5 ) 53 Okami, Y . , (6) 394 Okamoto, H . , ( 8 ) 463 Okamoto, K . , (3) 60 Okamoto, N . , ( 6 ) 428 Okamoto, Y . , ( 5 ) 1046 Okamura, K., ( 3 ) 150 Okayasu, T . , ( 8 ) 257 O'Keeffe, E.T., ( 6 ) 503, 505 O k i t a , T.W., ( 4 ) 137; (6) 301 Okubo, H . , ( 5 ) 731; ( 8 ) 244 Okubo, T., ( 4 ) 188 Okubo, Y . , ( 4 ) 229; ( 5 ) 784 Okuda, K., ( 8 ) 314 Okuhara, E . , (8) 475 Okumura, H . , ( 8 ) 69 Okumura, K . , ( 7 ) 116, 144, 147 Okuyama, T., ( 5 ) 850 O l a v s e n , A.H., (5) 385 O l a v e s e n , A.H., ( 6 ) 413, 414; (8) 660 O l d b e r g , A., ( 6 ) 411 O l d f i e l d , E . , (7) 81 O l d s t o n e , M.B.S., ( 5 ) 73 O l i v e i r a , D.E.. ( 6 ) 184 Olmsted, J . B . , (8) 481 O l o f s s o n , S., ( 5 ) 32; (8) 406 O l s e n , B.R., ( 5 ) 255 O l s e n , R.L., ( 8 ) 138 O l s o n , S.T.. ( 5 ) 760, 76 1
O l s s o n , I., ( 8 ) 97 O l s t a d , R . , ( 5 ) 634 O l i v e i r a , M.B.M., ( 6 )
315
Omary, M.B.,
609
( 5 ) 608,
Omata, T., ( 8 ) 629 Omedeo-Sale. F., ( 5 )
687; ( 8 ) 579
Omelkova, J., ( 3 ) 235;
( 6 ) 447
Omori, K . , ( 5 ) 724 Omura, S . , (4) 34 Ona, Y . , (6>, 268 O ' N e i l , H.C., (5) 547,
85 1
O ' N e i l l , M.A., ( 3 ) 148 Ong, R.L., (5) 909 O n i t i r i , A . C . , ( 8 ) 539,
711, 738 ( 4 ) 166; ( 5 ) 3 ( 5 ) 324; ( 6 ) 231, 436; ( 8 ) 661 O n y e z i l i , F.N.. (8) 539, 711, 738 G n y z i l i , N . I . , (5) 502 O o r a i k u l . B., ( 3 ) 245 Ooshima, H., ( 8 ) 157, 676 O o s t a , C.M., ( 5 ) 368 Oppenheim, J.D., (4) 68 Oppenheimer, J.H., ( 5 ) 144 O r d a l , C.W., ( 8 ) 455 Orengo, A., (8) 566 Oreste, P., ( 5 ) 369 Orford, C.R., (5) 342 O r g r y d z i a k , D.M., ( 6 ) 374 O r l a c c h i o , A.. ( 6 ) 33, 131 O r l a n d o , P., ( 7 ) 12 O r l o v s k a y a , A.G., ( 6 ) 186 Oro, J . , ( 2 ) 2 O r o s z l a n , S . , ( 5 ) 18 O r p i n , C.G., ( 4 ) 203 Orr, G.A., ( 8 ) 332 Orr, J . , ( 8 ) 433 Orr. T . (4) 75 O r y , R.L., ( 3 ) 137 Osa, T., (8) 525, 526 O s a j i m a , K., ( 4 ) 129 Osawa, T . , ( 5 ) 111, 180, 912, 923. 924; (8) 272, 392, 591 O s b o r n , M.J., ( 4 ) 85 Osborne, B.G.. (3) 9; ( 6 ) 307 O s b o r n e , D., ( 3 ) 73 Oshima, G., (5) 348 Oshima. H., ( 4 ) 188
Ono, K . , Ono, T . ,
Carbohydrate Chemistry
804 Oshima, R . , (2) 13 Oshima, T., ( 6 ) 311 Osserman, E . F . , (5) 845 Ossowski, L . , ( 8 ) 466 Ota, T . , (4) 50 O t a , Y . , ( 3 ) 187 O t a g i r i . M., (3) 79; ( 8 ) 522 O t a k a r a , A . , ( 6 ) 440 O t e r o , M.A., (6) 335 Otomine, Y . , ( 8 ) 603 O t o t a n i , N . , (5) 363, 366; ( 6 ) 410; ( 8 ) 544, 589 O t s u , K . , ( 5 ) 417 O t t e s o n , M . , (6) 397 O t t o s s o n , H . , ( 8 ) 63 Ouazana, R . , (5) 267 Ovchinnikov, M.V., ( 8 ) 6 O v e r d i j k , B., ( 6 ) 264; (8) 124 Owada, M.. ( 7 ) 105 Owen, C . S . , (5) 844 Owen, M.J., ( 5 ) 624, 625 Owens, M.R., ( 8 ) 226 Owensby, C . N . , (3) 83 Oyama, K . , ( 8 ) 737 Ozawa, H . . (6) 428 Ozawa, J . , ( 3 ) 218 Ozawa, M . , ( 5 ) 530; ( 8 ) 260 Pace, M., ( 8 ) 337 P a c i f i c i , M . , (5) 526 P a c k e r , N., ( 4 ) 6 2 Pacuszka, T , . (7) 103 Pagano, J.S.. ( 5 ) 68 P a g e l , M . , (6) 56 P a i c e . M.G., ( 6 ) 196 P a i n , D . , (5) 169 P a i n t e r , T . J . , ( 2 ) 109 P a l , J . , (4) 121 P a l , S.K., ( 6 ) 142 P a l l a v i c i n i , C., (6) 454 P a l m e r , D . S . , ( 5 ) 788 Palmer, J.K., (3) 126 P a l v a . E . T . , ( 6 ) 209 Pamblanco, M., (5) 882 Panak. J . , ( 3 ) 134 Pandey, J . P . , (4) 110 P a n d u r a n g a , Rao, K., (8) 733 P a n e k , A.D., ( 6 ) 184 Panem. S.. (5) 78 Pang, D., ( 5 ) 7 Papahadjopoulos, D., ( 5 ) 838; ( 7 ) 90; ( 8 ) 557 P a p s i d e r o , L.D. , ( 5 )
835; ( 8 ) 202 P a r a l i k a r , K.M., 120
(3)
P a r a n c h y c h , W . , ( 4 ) 179 P a r a s c a n d o l a , P., ( 8 ) 640 P a r e i l l e u x , A., ( 6 ) 155 P a r e s , X . , (8) 300 P a r i s , H., ( 6 ) 169 P a r i s h , C.R., (5) 547, 809, 851: ( 7 ) 148 P a r k , D . , ( 7 ) 185 Park, J.M., ( 8 ) 680 P a r k , N.H., (8) 730 P a r k , S.H., ( 8 ) 749, 684 P a r k , Y.K., ( 8 ) 713 P a r k e r , C.W., (5) 644; ( 8 ) 197 P a r k e r , K.M., ( 5 ) 802 P a r k e r , P . J . , (6) 528 P a r n a i k , V.K., ( 6 ) 521 P a r n i a k , M., ( 8 ) 139 Parodi, A.J., ( 4 ) 228; ( 5 ) 1038 P a r o l i s , H. ( 3 ) 256, 257, 258 P a r r a d o , C . , ( 2 ) 24 Parrish, R.F., (5) 352; ( 6 ) 166 P a r r y , J.B., ( 4 ) 131 P a r r y , S., (5) 951 P a r s h k o v , E.M., ( 6 ) 77 P a r s o n s , T.F., (5) 672, 674, 693 P a r v e z , Z . , (5) 755 P a s c h e r , I . , ( 7 ) 49 Paschke, E . , (5) 391, 432; ( 6 ) 62, 519; ( 8 ) 2 50 P a s c o l i n i , R., ( 6 ) 131 P a s e r o , L., (6) 293 P a s t o r e , G.M., ( 8 ) 713 P a s t y r , J . , (3) 106 P a t e r s o n , D.S., ( 8 ) 180 P a t i l , N.B., (3) 92, 120 P a t o n , D., ( 3 ) 7 2 P a t r i c k , J . , ( 5 ) 178 P a t t , L.M., ( 5 ) 9 Pattabiraman, T.N., (3) 86, 254, 255. 800; (6) 274, 281, 283 P a t t e r s o n , J.L., ( 5 ) 831 P a t t i n s o n , N., ( 8 ) 315 P a t t o n , C . L . , (5) 999 P a u l o , J . , ( 5 ) 970 P a u l s e n , G.M., ( 6 ) 303 P a u l s e n , H . , ( 8 ) 22, 41, 42, 43, 45. 46, 47, 54, 55
P a u l s o n , J . C . , ( 2 ) 117; ( 5 ) 63, 1028 P a u l s s o n , M . , ( 5 ) 330, 493 Pav, J.W., ( 2 ) 45; ( 5 ) 3 02 P a v i a , A . A . , ( 5 ) 1017 P a v l o v , P., ( 8 ) 488 Pavone, V., ( 4 1 25 P a w a s s a r a t , V . , (5) 579 P a y s , E., ( 5 ) 998 P a z u r , J . H . , (5) 853; ( 6 ) 401; ( 8 ) 87, 166 Peake, I . R . , (5) 770, 786 P e a r c e , G., ( 3 ) 233, 234 Pearlstein, E., (5) 214, 241 P e a r s o n , J.P., ( 5 ) 950, 951, 952 P e c h t , I., ( 5 ) 885 Pecoud, A . R . , (5) 643 Peczon, B.D., ( 5 ) 264 P e d e r s e n , N.S., ( 8 ) 141 P e d e r s o n , E . D . , ( 6 ) 378 Pedone, C . , (4) 25 P e e t e r s , B., ( 5 ) 492 Peeters-Joris, C . , (5) 304 P e i r i s , S.P., ( 6 ) 470 Pekin, B., (8) 467 P e l t o n e n , L., ( 5 ) 256 Pena, J . , ( 2 ) 24 P e n d l e t o n , R., ( 8 ) 641 P e n f o l d , P., (5) 552 P e n f o r n i s , H . , ( 5 ) 412 P e n n i s i , F . , ( 8 ) 189 Pennock, C. ( 2 ) 70 P e n n y p a c k e r , J.P., ( 5 ) 446 P e n t c h e v , P.G., ( 7 ) 146 P e p p e r , D.S., (5) 328, 401, 773 P e p y s , M.B., ( 5 ) 219, 505 P e r a u d e a u . L., ( 5 ) 64 P e r c h e r o n , F . , ( 6 ) 108 P e r c i v a l , E . , ( 3 ) 295 Perdew, G . H . , (5) 727, 728 P e r e g o , L . , ( 8 ) 471 P e r e i r a . L., (5) 29 P e r e i r a , M.E.A., (5) 2 00 P e r e z , N., ( 6 ) 258 P e r e z , P., ( 4 ) 211; ( 6 ) 529 P e r e z U r e n a , M.T.. (4) 6 P e r i n i , F., ( 5 ) 682 P e r k i n s , H . R . , ( 4 ) 41,
805
Author Index 42 P e r k i n s , S.J.. ( 5 ) 327 Permyakov, E.A., ( 5 ) 712 Personen. M . , ( 5 ) 51 Persson, H . , ( 5 ) 24 P e r r e t , B.A., ( 5 ) 775 P e r r e t , G., ( 5 ) 905 P e r r y , M.B., ( 4 ) 125, 126, 127 Personne, P . , ( 8 ) 583 P e s c i o t t a , D.M., ( 5 ) 255 Peska, J . , ( 2 ) 1 8 Pestka, S., ( 8 ) 401 P e t e r s , B . P . , ( 5 ) 682 P e t e r s , C . , (5) 323 Petersen. C.S., ( 8 ) 141 P e t e r s e n , L . C . , ( 8 ) 283 P e t e r s e n , T.E.. ( 5 ) 236, 734, 736, 737, 741, 742; ( 6 ) 296 Peterson, D . I . , ( 5 ) 26 P e t e r s o n , D.M.. ( 5 ) 652 P e t e r s o n , D.W., ( 5 ) 316 P e t e r s o n , I . M . , ( 8 ) 116 Peterson, P.S., ( 5 ) 622 P e t e r s s o n , K . , ( 4 ) 100 P e t i a r d , V., ( 6 ) 155 P e t i t o u . M . , ( 5 ) 369 Petrakova, E., ( 8 ) 23 P e t r e , J . , ( 6 ) 387 P e t r i , W.A., ( 5 ) 58 P e t r i e l l a , C . , ( 7 ) 175 Petropavlovsky, G.A., ( 8 ) 482 P e t r o v , G . I . , ( 6 ) 441 P e t t e r s s o n , B . , ( 8 ) 132 P e t t e r s s o n , I., ( 5 ) 380 P e t t e r s s o n , L.G., ( 6 ) 355 P e t t e r s s o n , R.F., ( 5 ) 82 PfannemUller, B., ( 3 ) 41 P f e f f e r , P.E., ( 3 ) 230 P f e i f f e r , M . , ( 8 ) 495 P f e i l , R . , ( 5 ) 935 Pham, K . , ( 5 ) 294 P h a r r , D.M., ( 6 ) 117 Phelps, C.F., ( 5 ) 315, 333, 339, 357; ( 6 ) 416; ( 8 ) 320, 354 P h i l i p p , B., ( 3 ) 109 ( 5 ) 660 P h i l l i p s , D.R., ( 3 ) 134 P h i l l i p s , G.O., P h i l l i p s , J.A., ( 6 ) 357 P h i l l i p s , L.R., ( 2 ) 102 P h i l l i p s , M . A . , ( 5 ) 475 Pholpramool, C . , ( 5 ) 580 P i c a r d , J . , ( 5 ) 321,
381, 404. 422 Picque, M.T., ( 5 ) 836; ( 8 ) 204 P i d a r d , D., ( 5 ) 906 Pieczenik, G., ( 2 ) 56; ( 5 ) 1010 ( 5 ) 371 Piepkorn, M.W., P i e r , G.B., ( 4 ) 145 P i e r c e , J.G., ( 5 ) 672, 674, 692, 693 P i e r c e , M., ( 5 ) 1031 P i e r c e , R.J., ( 6 ) 264 Pierce-Crete1.A. ( 2 ) 106; ( 5 ) 882, 883. 987 P i e r o t t i , M . , ( 5 ) 72 P i e t i l l l , K . , ( 5 ) 310, 397 P i h l a j a n i e m i , T . , (5) 2 84 P i l l a i , P., ( 7 ) 187 P i l l a i , S., ( 5 ) 350; ( 6 ) 487; ( 8 ) 277, 366 P i m l o t t , W . , ( 2 ) 93; ( 7 ) 49, 84, 153 P i n c h e i r a , G., ( 6 ) 534 ( 4 ) 23 Pincus, M . R . , P i n t e r , A . , ( 5 ) 72 Pisko-Dubienski, R . , ( 6 ) 154 P i s k o r z , C . , ( 6 ) 110; ( 8 ) 30, 551 P i s t o l e , T.G., ( 4 ) 91 P i t c h e r , W.H., j u n . , ( 6 ) 12 Pitman, W.D., ( 3 ) 186 P i t t s , M.J., ( 5 ) 196, 197 P i z z o , S.V., ( 5 ) 769, 799 P l a c h a , P . , ( 8 ) 216 Planche, J . , ( 8 ) 194 (8) P l a s t e r k , R.H.A., 613 P l e d g e r , D.R., ( 5 ) 813 ( 8 ) 11 Plessas. N.R., Ploegh. L.L., ( 5 ) 624 Plow, E.F., (5) 233 Plummer, T.H., ( 5 ) 1011 Pober, J.S., ( 5 ) 619 Pochon, F., ( 5 ) 733 ( 6 ) 142 Poddar, R . K . , P o d l e s k i , T.R., 95) 190; ( 8 ) 219 Poduslo, J.F., ( 2 ) 54; ( 5 ) 459 Poenaru, L . , (6) 168 Pokrywka, G., ( 5 ) 170 Polakowski, D, ( 8 ) 705 Polivka, E . , ( 8 ) 122 ( 5 ) 684 P o l l a c k , V.A., Polley, M.J.. ( 5 ) 665
.
P o l l o c k , J . J . , ( 4 ) 49 Pomeranz, Y.. ( 6 ) 305 ( 7 ) 203 Pommier, M.-T., Pompecki, R . , ( 5 ) 482 Ponzetto-Zimmerman, C., ( 6 ) 407 Poole, A.R., ( 5 ) 427 Poole, R . , ( 5 ) 415 Popa, M., ( 3 ) 110 Porath, J . , ( 8 ) 395, 399 P o r c e l l a t i , G . , ( 6 ) 33 Poretz, R . D . , ( 2 ) 56; ( 5 ) 165, 176, 1010 Porro, M . , ( 2 ) 60; ( 5 ) 11 P o r t e r , S.E., ( 4 ) 180 P o r t e t e l l e , D . , ( 5 ) 13 Portoles, A., ( 4 ) 6 Portoukalian, J . , ( 5 ) 85 9 Posadka, P . , ( 8 ) 695 P o s p i s i l , M . , ( 8 ) 587 Poss, A . , ( 7 ) 22 Poteshnikh, A. V., ( 8 537 P o t i e r , M . , ( 6 ) 250 P o t t e r , H . , ( 5 ) 889 P o t t e r , J.L., ( 6 ) 165 P o t t e r , J.M., ( 5 ) 830; ( 8 ) 316 P o t t s , J.T., ( 5 ) 700 Poulain, D . , ( 4 ) 222 ( 3 ) 213, Powell, D.A., 214, 215 Powell, M.E., ( 5 ) 949 ( 4 ) 160 Poxton, I . R . , Poznansky, M.J., ( 5 ) 2 95 Pozsgay, V., ( 8 ) 38 Prabhakara Rao, A.V.S., ( 8 ) 710 Prabhu, K.A., ( 4 ) 139 Prado, F.E., ( 6 ) 81 P r a g e r , M.D., ( 5 ) 1035 ( 5 ) 660 Prasanna, H . R . , P r a t t , G.W., ( 3 ) 12 P r e i s s , J . . ( 4 ) 136, 137, 156, 157; ( 5 ) 286; ( 6 ) 301 P r e n t i c e , N., ( 3 ) 154 P r e s t e g a r d , J.H., ( 5 ) 909 P r e s t i d g e , R.L., ( 2 ) 44 P r i c e , J . , ( 4 ) 142 P r i d d l e , J.D., ( 8 ) 381 P r i e e l s , J.P., ( 5 ) 1072; ( 8 ) 266 P r i e s t , D.G., ( 8 ) 336 P r i g o z i n a , I.G., ( 6 ) 239 P r i h a r , H.S., ( 2 ) 15
806 P r i t c h a r d , D.G., ( 5 ) 40 P r o c k o p , D. J . , ( 5 ) 256. 280 P r o f f i t t , R.T., ( 8 ) 426 P r o h a s k a , R . , ( 5 ) 913 P r o k a z o v a , N.V., ( 7 ) 7 6 P r o t m o r e , J . , ( 5 ) 848 Proud, D . , ( 8 ) 190 Pueppke, S.G.. ( 5 ) 120, 127, 161 P u g l i a , K.V., ( 5 ) 918 P u j o l , J . P . , ( 5 ) 412 P u r n e l l , M . R . , ( 8 ) 417 P u s z t a i , A , , ( 5 ) 171 Putman, F.W., ( 5 ) 897 P u z i c , o., ( 2 ) 1; ( 5 ) 1026 Puzic, R., ( 2 ) 1 ; (5) 1026 PYC, K . , ( 6 ) 343 PYC, R . , ( 6 ) 343 Q a d r i , F . , ( 8 ) 444 Q a l l u e , L.A., ( 5 ) 1057 Q u a d e r , H . , ( 3 ) 127 Quarles, R . A . , ( 5 ) 462 Q u a r o n i , A . , ( 5 ) 577; ( 6 ) 459; ( 8 ) 200 Q u a s h , G . A . , (5) 842 Q u i l l , H . , ( 7 ) 135 Q u i n n , P . J . , ( 7 ) 11, 86, 171 Q u i r k , J.M., ( 6 ) 5 6 Rabin, E . M . , ( 5 ) 18 Racker, E . , ( 5 ) 232 Radhakrishnamurthy, B . , (5) 312 R a d i n , N.S., ( 6 ) 190, 191; ( 7 ) 9, 88, 141 Radoff, S., ( 5 ) 359 Rad z i e jews ka-Lebrecht , J., ( 4 ) 8 2 R a f f i , J . J . , ( 3 ) 47 Raghavan, D., (5) 218 Rahman, M.A., (3) 295 Rahman, R . , ( 8 ) 285 Rahmann, H . , ( 7 ) 71, 75 R a i m b a u l t , M . , ( 6 ) 402 Rajasingham, K . C . , (4) 21 0 R a j e n d r a n , S., ( 6 ) 461 Rajewsky, K., (5) 898 R a j n , D.S., ( 6 ) 281 Rajoka, M . I . , (6) 471 Rakowi c z S z u l c z yn s k a , E.M., (4) 66 R a l e i g h , E., ( 4 ) 149 R a l l , J.E., (5) 468 Ramachandran, C.K., (5) 5 87 Ramachandran, K . B . , ( 6 )
Carbohvdrate Chemistry 21 8 Ramesh, V . , ( 8 ) 692 Ramey, C.M., ( 5 ) 980 Ramey, W.D.. ( 4 ) 33 Ramos M a r t i n e z , I . , ( 6 ) 18, 112 Ramstorp, M . , ( 8 ) 624 Ramwell,. P.W., ( 8 ) 442 Rana, S.S., ( 8 ) 10, 28, 31, 52 Rando, R . R . , ( 8 ) 76 Rao, C . C . V . N . , ( 3 ) 155 Rao, C . S . , ( 6 ) 349 Rao. C . V . , ( 5 ) 676 Rao, C . V . N . , ( 3 ) 260 ( 4 ) 121, 122 Rao, D.R., ( 8 ) 261 Rao, K . K . , ( 6 ) 211: 8 ) 61 3 Rao, K . S . , ( 7 ) 160 Rao, M . R . R . , (6) 467 Rao, P.E., ( 5 ) 572 Rao, P.V.S., ( 6 ) 404 Rao, V.B., ( 6 ) 395. 404 Rao, P.V.S., ( 6 ) 395 Rao, V.S.R., ( 2 ) 111 Rapp, P.. ( 6 ) 222 Ras, D.R., ( 5 ) 198 Rasche, B., ( 8 ) 163 Rask, L . , ( 5 ) 622 R a s t o g i , R.K., ( 6 ) 230 R a t c l i f f e , R.M., ( 8 ) 21 Ratge. D . , ( 6 ) 2 4 R a t h a u r , B.S., ( 5 ) 105; ( 8 ) 547 Ratman, S . , ( 5 ) 1077 Rauch, J . , ( 5 ) 481 R a u n i o , V . , ( 6 ) 341: (8) 464 R a u s c h e r , E . , ( 3 ) 52; (6) 288 R a u t e n b e r g , P.. ( 5 ) 1000: ( 8 ) 252 Rautenstrauch, H., ( 6 ) 24 R a u t e r b e r g , E.W., ( 8 ) 586 R a u t e r b e r g , J . , ( 5 ) 261 Rauvala, H . , ( 2 ) 108: ( 5 ) 1025: ( 7 ) 10 R a v a n a t , G., (3) 212 R a w l i n s , G. A., ( 5 ) 865 Rawls, W.E., ( 5 ) 33 Ray, P.M., ( 3 ) 231 Raz, A . (5) 188 Read, A . , ( 5 ) 807 R e a r i c k , J . I . , ( 2 ) 117; ( 5 ) 1028: 1047, 1058 Rebois, R . V . , ( 5 ) 687; ( 8 ) 579 R e c h l e r , M . , ( 5 ) 407 Reddi, A . H . , ( 5 ) 211:
( 8 ) 265 Reddy, A . S . , ( 3 ) 237 Reddy, S.M., ( 3 ) 237, 248 Redinbaugh, M.C., ( 8 ) 4 34 Redmond, J . , ( 4 ) 19, 62 Reeber, A . , ( 5 ) 448; ( 8 ) 256 Reed, L.A., ( 8 ) 2 0 Reej, R . , ( 6 ) 271 Rees, D . A . , ( 3 ) 179, 213, 214, 215, 266, 267; ( 4 ) 142; ( 5 ) 947 Rees, S . , ( 6 ) 53 R e e s e , E.T., ( 6 ) 354 Reeves, H . C . , ( 8 ) 203 Reeves, P . , ( 4 ) 62 R e g l e r o , A . , ( 6 ) 34, 39, 70, 258; ( 7 ) 40 R e g h a u l t , F . , ( 5 ) 271 R e g o c e c z i , E . , ( 8 ) 280 R e g o e c z i , E . , ( 5 ) 565 R e i c h e l t , B.Y., ( 6 ) 381 R e i c h e r t , L.E., ( 5 ) 697, 1014 R e i c h e r t , R.D., ( 3 ) 227 R e i d , G . P . , ( 5 ) 908 R e i d , J.D., ( 6 ) 374 R e i d , J . S . , ( 3 ) 184 R e i d , K.B.M., ( 5 ) 593 Reid, P.E., ( 5 ) 480 R e i g n e r , J . , ( 5 ) 461 R e i l l y , P . J . , ( 6 ) 396: ( 8 ) 494, 677 R e i n a u e r , H . , ( 8 ) 403 R e i n e r , A . , ( 5 ) 415, 427 Reinherz, E.L., ( 5 ) 600, 607 R e i n w a l d , E . , ( 5 ) 1000; ( 8 ) 252 R e i s f e l d , R . A . , ( 5 ) 639 Reiskind, J.B., (4) 205, 206 Reiss, N.A., ( 8 ) 445 Reiss, U., ( 6 ) 172; ( 8 ) 391 Reitman, M . L . , ( 5 ) 164, 1030, 1074; ( 8 ) 247, 274 R e i t s c h e l , E.T., ( 4 ) 57 Remington, S. J . , ( 6 ) 433 Remold-O'Donnell, E., (5) 633 Remy, M.H., ( 8 ) 704 R e n f e r , L., (5) 792; ( 8 ) 279 Rennard, S.I., ( 5 ) 215 R e n n e r t , O . M . , ( 7 ) 105 R e n n i e , P.S., ( 8 ) 115
807
Author Index Rephaeli, A . , ( 5 ) 232 Resch, K., ( 8 ) 361 Reuser, A.J.J., (6) 58 Reuter, G., ( 5 ) 935 R e v i l l a r d , J.P., ( 5 ) 81 2 Revol, J.F., ( 3 ) 85; ( 4 ) 208 Rexova-Benkova, L., ( 3 ) 235; ( 6 ) 447, 450; ( 8 ) 714 Reynolds, J.T., ( 5 ) 7 8 ( 5 ) 259 Rhodes, R.K., R i a l d i , G., ( 5 ) 142, 143 Ribadeau-Dumas, B., ( 8 ) 416 R i b e i r o , J.M.C., ( 5 ) 200 R i c a r t , G., ( 5 ) 937 Ricca, G.A., ( 5 ) 745 R i c c i , R., ( 6 ) 32 ( 5 ) 241 Rich, A.M., Rich, R . R . , ( 5 ) 610 Richards, J.C., ( 5 ) 571 Richard, M . , ( 6 ) 507 Richardson, M . , ( 6 ) 279 Richey, J . , ( 5 ) 165 Richman, P.G., ( 6 ) 418 R i c h t e r , D., ( 5 ) 996; ( 8 ) 357 R i c h t e r , H., ( 5 ) 226 (6) Rlckard, P.A.D., 466, 470, 471 Rickards, C . , ( 5 ) 872; ( 8 ) 288 R i d e r , D., ( 5 ) 741 R i e s e n f e l d , J . , ( 5 ) 367 Riess-Maurer, I . , ( 8 ) 39 R i e t s c h e l , E.Th., ( 4 ) 52, 83 Riggs, J . , ( 5 ) 216 R i g h e t t i , P.G., ( 5 ) 117 Rijken, D.C., ( 5 ) 429; ( 8 ) 253 R i l e y , D.A., ( 4 ) 78 R i l f o r s , L., ( 7 ) 189 Rinaudo, M., ( 3 ) 212, 268, 272 Ring, S.G., ( 3 ) 173 ( 3 ) 66 Rinne, R.W., Riordan, J.R., ( 6 ) 104 ( 5 ) 552 Riott, I.M., ( 4 ) 107 R i o t t o t , M.M., Riquelme, M.E., ( 8 ) 147 R i s s e , H.J., ( 5 ) 1000; ( 8 ) 252 R i s t e l i , J . , (5) 251 R i s t e l i , L., ( 5 ) 498; ( 8 ) 254 R i s t e l i , J . , (5) 270 '
R i t t e n b e r g , S.C., ( 7 ) 192 R i v a t , C., ( 5 ) 841 R i v e t t , A.J., ( 8 ) 340 R i v e t z , B., ( 2 ) 83 Roach, P.J., ( 5 ) 294 ( 5 ) 573, Robbins, A.R., 574 Robbins, J . , ( 5 ) 817 Robbins, J.B., ( 4 ) 112, 113 Robbins, P.W., ( 5 ) 17, 1051 Robert, D . , ( 4 ) 106; ( 7 ) 195 Robert, L, ( 8 ) 246 Roberts, G.A.F., ( 3 ) 278, 279, 281 Roberts, K., ( 3 ) 148 Roberts, L.M., ( 5 ) 173 ( 5 ) 743 Roberts, R.C., ( 5 ) 87 Roberts, R.M., Robertson, C . A . , ( 8 ) 137 Robertson, N.G., ( 3 ) 244; ( 6 ) 451 Robey, P.G., ( 8 ) 249 Robic, D . , ( 6 ) 460 Robinson, D., ( 6 ) 106 Robinson, G . . ( 3 ) 267 Robinson, H.B.. ( 6 ) 165 Robinson, J . , ( 5 ) 621 Robinson, M . , ( 3 1 67 Robinson, R.J., ( 3 ) 46 Robinson, W.J., ( 5 ) 282 Roboz, J . , ( 2 ) 81 Rochas, C . , ( 3 ) 268, 272 Roche, A.C., ( 5 ) 199, 570; ( 7 ) 28 Roche, E . , ( 3 ) 93 Roche, J . , ( 5 ) 471 Rochemont, J . , ( 5 ) 474, 705 Rock, G . , ( 5 ) 788 Roden, L., ( 5 ) 331, 419 Rodionov, Y u . V . , ( 6 ) 489 Rodkey, L.S., ( 5 ) 834 Rodriguez, O., ( 8 ) 729 Rodriguez, R.J., ( 5 ) 10 (4) Rodriguez, R.L., 137 Rodriguez Lopex, M . , ( 6 ) 112 Roe, C.R., ( 8 ) 306 Roelcke, D . , ( 7 ) 155 R o e p s t o r f f , P., ( 6 ) 296 Roescke, D., ( 5 ) 863 RBsel, P., ( 4 ) 15 ( 6 ) 216 Roeser, K.R.. Rasner, H., ( 7 ) 71, 73,
74 Rogers, G.T., ( 5 ) 865 Rohde, H., ( 5 ) 251 (6) 7 Rohm, K.-H., Rohrbach, R.P., ( 8 ) 521 R o i t s c h , C.A., ( 5 ) 612 Rojas-Hidalgo, E . , ( 3 ) 99 Rokugawa, K., ( 8 ) 477, 686, 731 ( 5 ) 751; R o l l , D.E., ( 8 ) 435 R o l l i n , P., ( 6 ) 84 Romanowska, A . , ( 4 ) 59 Romanowska, E., ( 4 ) 59 Rombauts, W . , ( 5 ) 492 Rome, L.H., ( 6 ) 430 Romelli, P . , ( 8 ) 189 Romer, W . , ( 8 ) 586 Ronin, C . , ( 5 ) 701, 702 Ronnett, G.V., ( 5 ) 632 R o n s i s v a l l e , L., ( 6 ) 51 3 Roodman, G.D., ( 8 ) 375 ( 5 ) 71 Rosato, R.R., Rose, K., ( 8 ) 381 ( 8 ) 230 Rose, K.M., Rose, M . , ( 4 ) 198; ( 6 ) 149 Rose, M.L., ( 5 ) 521 Roseman, S., ( 5 ) 555 Rosemeyer, H., ( 8 ) 311, 313 Rosen, O.M., ( 8 ) 334 Rosen, S.D., ( 5 ) 586 (5) Rosenberg, R.D., 368 Rosenberg, S., ( 6 ) 144, 206 Rosenfeld, E.L., ( 6 ) 238 Rosenfelder , G., ( 5 1 519 Rosenweig, N.S., ( 2 ) 64 Rosi, J . , ( 6 ) 321 Rosik, J . , ( 3 ) 193 Rosing, J . , ( 5 ) 789 ( 5 ) 17 Rosner, M.R., ROSS, A . , ( 5 ) 2 Ross, P.W., ( 4 ) 160 Rossi, C . A . , ( 5 ) 100 Rossi, J . , ( 6 ) 321 Rossignol, D.P., ( 5 ) 1043 Ross-Murphy, S. B. , ( 4 1 142 Rossmanith., ( 5 ) 966 Roth, J . , ( 5 ) 631; ( 8 ) 355 Roth, N.J.L., ( 3 ) 159 ( 5 ) 187 Roth, R . A . , Roth, S., ( 5 ) 1031
Carbohydrate Chemistry
808 Rotman, A . , (5) 649: (8) 298 Rouhandeh, H., ( 5 ) 571 Roughley, P . J . , ( 5 ) 318, 439 R o u g i e r , M., ( 3 ) 166 Roukema, P.A., (5) 934 R o u r r i l l o n , R . , ( 5 ) 130 Rousseaux, J., (5) 836; ( 8 ) 204 R o u s s e l , P., ( 2 ) 35; (5) 929, 942, 943, 944, 945 R o u s s e t , M., ( 6 ) 163 Rouxhet, P.G., (8) 639 Roue, T.C., ( 8 ) 125 Rowell, P., (5) 114; ( 8 ) 218 Rowland, S.P., ( 3 ) 107 ROY, A., (3) 209; ( 5 ) 856, 888 Roy, N., (4) 105 Royer, G.P., ( 8 ) 409 Rozmarin, G . , (3) 108 R u b e n t h a l e r , G.L., ( 6 ) 300 Rubin, C.S., ( 8 ) 334 Rubin, K . , (5) 557 R u b i n s t e i n , B., ( 3 ) 178 Ruddon, R.W., (5) 682, 684 Ruddy, S., ( 5 ) 643 R k h e l , R . , (8) 341 Rlldiger, H., ( 5 ) 121, 182 R u f f , C.J., ( 8 ) 114 Ruggieri, M.R., (2) 69 R u g g i e r i , S., ( 7 ) 7 8 R u i z - h e r r e r a , J., ( 4 ) 217 RUOCCO, M.J., ( 7 ) 8 1 R u o s l a h t i , E . , (5) 210 R u p l e y , J.A., ( 6 ) 437 R u p p r e c h t , K.M., (8) 312 R u s c e t t i , S.K., ( 5 ) 81 Rusche, J.R., (8) 125 Rush, J.S., ( 5 ) 640, 64 1 R u s s e l l , R.R.B., ( 4 ) 17 R u s s i n , T.Z., (5) 1063 Russo, A., ( 6 ) 423 Ruth, J.M., (7) 7 R u t h e r f o r d , C.L., ( 8 ) 131 R u t h e r f o r d , N.G., (8) 120 R u y s s c h a e r t , J.H., ( 7 ) 22, 29, 42 Ryan, C.A., ( 3 ) 233, 234 R y b i c k a , K., ( 5 ) 288
R y c h t e r a , M., ( 4 ) 190 Ryden, L., ( 8 ) 418 Ryggvason, K.T., (8) 249 R y l a t t , D.B., ( 5 ) 810 Rynd, L.S., (5) 306 Ryu, D., ( 8 ) 649 Ryu, D.D.Y., ( 6 ) 331; ( 8 ) 684, 699, 749 Ryu, D.Y., (6) 354 S a a t , Y.A., ( 7 ) 62 S a b e s a n , M.N., (8) 515 S a c c h i , N., ( 6 ) 5 8 Sackmann, E . , (5) 910 S a c k t o r , B., ( 6 ) 172; (8) 391 S a - C o r r e i a , I . , ( 4 ) 193 Sada, E . , (6) 488; ( 8 ) 739, 750 Sadana, A . , 8) 674 Sadana, J., 6 ) 203 S a d d l e r , J. N , (6) 224, 365 S a d l e r , J.E. ( 2 ) 117: (5) 1028; 6) 498: (8) 90 S a d o f f , J.C. ( 4 ) 74, 145 S a d o w s k i , J. ( 3 ) 55: (6) 324 S a e e d , A., ( 8 ) 238 S a e n g e r , W . , (3) 77: ( 8 ) 514 Safi, A . , (5) 725 S a g a , K., ( 8 ) 604, 700 Sagawa, H., (4) 35, 50 S a g e , H., ( 5 ) 504: ( 8 ) 264 S a h a , S.C., ( 6 ) 202 Sahm, H., (8) 620 S a h u , S.C., ( 5 ) 535 Saier, Jr., M.H., (4) 162 S a i n i , H.S., ( 3 ) 64 St. John, J.B., ( 7 ) 187 S a i n t - L e b e , L.R., ( 3 ) 47 S a i r a m , M.R., ( 5 ) 694, 695 S a i t o , H., ( 6 ) 317 S a i t o , M., (7) 63 S a i t o , N., ( 6 ) 289 Saito, T . , (4) 188; ( 5 ) 718, 719: (8) 123, 437 S a i t o , Y., ( 8 ) 224 S a j j a n , S.U., ( 3 ) 40 Sakagami, T., ( 5 ) 556 S a k a g u c h i , T., (3) 290; (8) 501 S a k a i , R., ( 5 ) 145
Sakakibara, K., (7) 122, 124 Sakamoto, R . , ( 6 ) 220, 3 62 Sakamoto, S., ( 3 ) 39; ( 7 ) 36 S a k a n e , M., ( 6 ) 425 Sakano, T . , (5) 430 S a k a s h i t a , S., ( 5 ) 501 Sakon, M., ( 8 ) 297, 382 Sakuma. H . , ( 3 ) 113: (8) 462 S a k u r a b a , H., ( 6 ) 125 S a k u r a d a , I:, (3) 94 S a k u r a d a , T., ( 8 ) 453 S a k u r a i , N., ( 3 ) 198 S a l a c i n s k i , P.R.P., ( 5 ) 1015 S a l a m i d a , G., ( 6 ) 110; (8) 551 S a l a s , J . A . , ( 4 ) 201 S a l e s s e , R . , (5) 678 S a l i n a s , F.A., ( 2 ) 41: ( 5 ) 1027 S a l i s b u r y , B.G.J., (5) 436 S a l i s b u r y , J.G., ( 5 ) 3 82 S a l l e h , A.B., ( 8 ) 740, 741 S a l t m a n , R . , ( 8 ) 72 S a l t v e i t , M.E., ( 3 ) 239 S a l u n k h e , D.K., ( 3 ) 28 S a l v a d o r , R.A., ( 6 ) 233 S a l v a t o r e , G., ( 5 ) 468 S a l v a y r e . R., ( 6 ) 21, 167 S a l v e s e n , G.S., ( 5 ) 740 S a l y e r s , A . A . , ( 3 ) 149; ( 4 ) 132, 176 Samanta, H., ( 8 ) 284 Sampaio, L.O., ( 5 ) 441; (6) 35 Sampietro, A.R., ( 6 ) 81, 133 Sampson, E. J . , ( 6 ) 287 S a m u e l l , C.T., ( 5 ) 435 Samuelson, 0.. ( 3 ) 102; (8) 548 S a m u e l s s o n , B., ( 3 ) 104: ( 7 ) 49; (8) 24 S a m u e l s s o n , B.E., ( 2 ) 93; (7) 84, 109, 111, 153, 193 S a n c h e x , A . , ( 6 ) 382 S a n c h e z , J . , ( 6 ) 157 S a n d e r , M., ( 5 ) 199; (7) 28 S a n d e r s , G.T.B., (5) 806 S a n d e r s , R., ( 4 ) 149 S a n d e r s o n , K.E., (4) 84
809
Author Index Sandford, D . C . , ( 6 ) 446 Sandhoff, K . , (6) 22 San Huang, J . , ( 8 ) 426 Sankey, E . A . , ( 5 ) 276 Sann, L . , ( 5 ) 744 Sano, T . , (5) 516; ( 8 ) 128 Santappa, M., ( 8 ) 512, 715, 733 S a n t e r , V . , ( 5 ) 318, 439 S a n t o r o , B . C . , ( 8 ) 259 S a n t o r o , P . F . , (6) 43 S a n t o r o , S . A . , ( 5 ) 772 S a n t o s Neto, C . , ( 6 ) 184 Sanu, G., ( 8 ) 486 S a n y a l , A . , (6) 202 S a n y a l , D . , ( 4 ) 196 S a r a s t e , J . , (5) 50, 51 S a r f a t i , S . R . , ( 2 ) 101 Sarkar, M., (5) 166 Sarko. A . , ( 3 ) 150 Sarma, T . A . , (3) 299, 300 Sarngadharan, M.G., ( 8 ) 2 85 S a r r a s , M . P . , ( 5 ) 575 S a r t o , V . , (3) 117; ( 6 ) 332 S a r v a s , M., ( 5 ) 49 Sasajima, K . , (8) 56, 57, 58, 59, 60 S a s a k i , I . , (8) 258 S a s a k i , M., ( 8 ) 702, 703 S a s a k i , R . , ( 5 ) 724 S a s a k i , T . , (4) 209; ( 5 ) 556, 793; ( 7 ) 126, 127 S a s a k i , Y . , ( 4 ) 8; ( 8 ) 520 S a s e k , V . , ( 3 ) 125 S a s t r i , N.V.S., ( 6 ) 395, 404; (8) 716 S a t h e , S.K., (3) 28 S a t o , A . , ( 5 ) 550 S a t o , M., (6) 445, 455 S a t o , T . , ( 4 ) 219; ( 5 ) 431; (6) 478, 479; ( 8 ) 425, 549, 659 S a t o . Y . , (5) 988, 989 Satomi, D . , ( 7 ) 142 Satp, M., (6) 75 Saukkonen, J . J . , ( 4 ) 177, 178 Saumweber, H., ( 8 ) 117 Saunders, R . M . , (6) 275 Saundry, R . H . , ( 5 ) 984 S a u n i e r , B . , (6) 221 Savage, A . V . , ( 4 ) 103 Savage, D . C . , (4) 202
Savage, M . P . , ( 5 ) 396 S a v e l ' e v , E . P . , (6) 441 S a v i o n , N . , ( 5 ) 258, 277 Savitsky, M.J., (6) 413, 414; (8) 660 S a v v i d o u , G., ( 5 ) 847 Sawada, H . , (8) 607 Sawamura, M., ( 3 ) 181; (4) 243 Sawamura, T . , ( 5 ) 561 Sawitsky, A . , (7) 146 S a y e r s , C . A . , ( 5 ) 740 S c a n l i n , T . F . , (6) 103 S c a r d i , V . , ( 8 ) 640, 685 S c h a c h t e r , H . , ( 5 ) 938, 939; (7) 137, 138 S c h l f f l e r , K.J., (2) 5 Schaer, M.G., ( 5 ) 1050 S c h a f e r , I . A . , ( 6 ) 165 S c h a f f e r , P . A . , (5) 30 S c h a r f , H . D . , ( 5 ) 395 Schattenkerk, C . , ( 8 ) 66 Schauer, R . , (5) 199, 579, 935, 1075; ( 7 ) 28; ( 8 ) 663 Schaumburg, K . , (3) 261 S c h e e r e r , J . B . , ( 6 ) 111 S c h e i d , A , , (5) 54, 74 Schellekens, A.P.M., (5) 806 Schenck, P . A . , ( 2 ) 3 Schenkel-Brunner, H . , ( 5 ) 858, 902 Scheraga, H . A . , (4) 23 S c h e r e r , P . , 8 ) 620 S c h i a c h , E . , 3) 52; ( 6 ) 288 S c h i c h i j o , S. ( 5 ) 317 Schichmanter, E., ( 2 ) 83 S c h i c k , L . A . , ( 5 ) 766 S c h i l l e r , C.M , ( 6 ) 232 S c h i l l i n g , J . ( 5 ) 881 S c h i l t z , E . , 5 ) 182 S c h i n d l e r , M., ( 4 ) 85 Schink, B . , (3) 247 Sc h i p h o r s t , W. E . C. M. , (5) 1076; (6) 509 Schlaeger, E . J . , (8) 428 S c h l a g e r , S . T . , (8) 683 S c h l e b u s c h , H . , (2) 73 S c h l e i c h e r , E . , ( 5 ) 808 Schlepper-Schgfer, J . , ( 5 ) 568 Schlesinger, P.H., (5) 523, 524; ( 7 ) 139 Schlossman, S . F . , ( 5 ) 600, 607
Schmale, H . , ( 5 ) 996; (8) 357 S c h m e l l , E . , ( 5 ) 555 Schmer, G., ( 5 ) 371 Schmerr, M . J . F . , ( 5 ) 14 Schmid, K . , ( 5 ) 428, 748, 749 Schmidt, A . , ( 5 ) 323, 438 Schmidt, D . D . , ( 5 ) 307 Schmidt, D . E . , ( 2 ) 28 Schmidt, E . L . , ( 4 ) 150; (5) 160; ( 8 ) 207 Schmidt, J . , ( 3 ) 98 Schmidt, M . A . , (4) 96 Schmincke-Ott, E . , ( 8 ) 322 Schmut, O., ( 5 ) 383 Schneerson, R . , (4) 95, 99 S c h n e i d e r , A.B., ( 5 ) 4 72 S c h n e i d e r , E . M . , ( 5 ) 91 S c h n e i d e r , H . , ( 8 ) 622 Schneider, J . , ( 5 ) 22 S c h n e i d e r , J . A . , (6) 57 S c h n e i d e r , P.M., ( 5 ) 224 S c h n e i d e r , S . , ( 8 ) 361 S c h n e l l , D . , ( 8 ) 47 S c h o c h a t , D . , ( 5 ) 958 S c h b l l e r , M., (4) 159 Schoemaker, H., ( 5 ) 984 Schoemaker, J . M . , ( 4 ) 177, 178 Schoemaker, P . J . H . , ( 4 ) 177 Schbberger, O.L., (5) 753, 966; ( 8 ) 163, 201 Scholander, E . , ( 8 ) 147, 535 S c h o l e r , A . , ( 2 ) 76; (8) 719 S c h r a d e r , M., ( 4 ) 8 9 Schraer, H . , (5) 335, 42 5 S c h r e i b e r , G., ( 5 ) 750 S c h r e i b e r , M., (5) 750 S c h r e y e r , D . , ( 6 ) 76 Schrbder, J . , (8) 185 S c h r o e d e r , L . R . , ( 3 ) 97 S c h r o e r , K . , (4) 118, ( 5 ) 874 S c h u b e r t , D . , ( 5 ) 278 Schuchman, E. H . , ( 5 ) 305 Schuerch, C . , ( 8 ) 1, 2, 3, 4, 14 S c h u l t e , B . A . , ( 5 ) 550 S c h u l t z , A . M . , ( 5 ) 18 S c h u l t z , R . , ( 6 ) 426
810 S c h u l z , A . , ( 5 ) 22 S c h u l z , B . C . , ( 5 ) 120 S c h u r z , H . , (5) 121 S c h u s t e r , J . , ( 5 ) 481 S c h u t z b a c h , J.S., ( 5 )
1059, 1060; ( 6 ) 241: (8) 240 Schwarting, G.A., (7) 61, 117 Schwartz, A.L., ( 5 ) 563 Schwartz, E . R . , (2) 28; ( 5 ) 307 S c h w a r t z , L.M., ( 8 ) 516 Schwartz, R . , (5) 481 Schwarz, R . T . , (5) 34, 1051 Schwarz, U . , ( 6 ) 499 Schwermann, J . , (5) 323 S c o b e l l , H.D., (2) 23 S c o l n i c k , E.M., (5) 81 S c o p e s , R.K., ( 8 ) 473 Scott, D.L., (5) 212 S c o t t , D.M., ( 5 ) 276, 773 S c o t t , J . E . , ( 5 ) 275, 342, 385 Scouten, W.H., (8) 79 Searle, B.A., (4) 218 S e a v e r , A . , ( 4 ) 86 Secheresse, J.P., ( 8 ) 596 S e c k b a c h , J . , ( 3 ) 301 Sedlackova, J., (3) 235; ( 6 ) 447 S e e l a , F . , ( 8 ) 31 1, 313 S e e t h a r a m , B., ( 8 ) 199 S e f t o n , M.V., (8) 597 S e g a l , H.L., ( 5 ) 688; (8) 578 Segawa, S.-I., ( 6 ) 425 Sege, K . , (5) 622 Seghatchian, M.J., (5) 767 S e i b . P.A., ( 7 ) 168 S e i d , R.C., (4) 74 S e i d a h , N . G . , ( 5 ) 474, 965, 703, 704, 705 S e i d l , M., ( 5 ) 226 S e k i g u c h i , K . , (5) 223; ( 6 ) 91 S e l a , B . A . , ( 5 ) 629 S e l a n d e r , R.K., (8) 185 S e l j e l i d , R . , ( 4 ) 214 S e l s t e d , M.E., (6) 422 S e l v e n d r a n , R.R., (3) 173, 219 Semenza, G., ( 6 ) 444 Sen, A . , (5) 107, 151: ( 7 ) 11, 86, 171; ( 8 ) 1 02 S e n e , C . , ( 5 ) 570 S e n e a r , D.F., (5) 137,
Carbohydrate Chemistry 138
S e n n , H.-J., (7) 59 Seno, N . , (5) 102, 174,
344, 390; ( 8 ) 393 Senyi, A.F., (5) 668 Seong, B . L . , ( 8 ) 680 S e p p a l a , P., (5) 82 S e q u e i r a , L., ( 4 ) 138 Serafini-Fracassini, A . , ( 5 ) 502 S e r i f , G.S., (2) 78;
( 8 ) 212
S e r r a n o , R., ( 8 ) 456 Setchell, B.P., (5) 580 S e t t i n e , J.M., ( 5 ) 303 Sewell, A . C . , (5) 976 S e y d e w i t z , H . , ( 5 ) 750 Shafer, J.A., ( 5 ) 139 Shafit-Zagardo, B., (8)
32 1
S h a i n o f f , J. R.,
(5)
221; (8) 577
S h a l t i e l , S.. ( 8 ) 108 Shannon, J . C . , (3) 35 Shannon, L.M., ( 3 ) 123:
( 5 ) 184; (6) 114, 350
S h a p e r , J.H., ( 5 ) 65 S h a p i r o , M . , (5) 626 S h a r a n , M . , ( 8 ) 583 S h a r b e r t , F . , (5) 822 Sharkey, P.F., ( 8 ) 2 Sharma, B . P . , ( 8 ) 100 Sharma, C.B., ( 6 ) 282,
308, 309
( 5 ) 854 (5) 96, 98, 101, 209, 901; ( 8 ) 89, 374 Shashkov, A . S . , ( 4 ) 76; (6) 97 Shatkin, A.J., ( 8 ) 312 S h a v l o v s k i i , G.M., (6) 186 Shaw, D.H., ( 4 ) 55 S h e c h t e r , Y., (5) 629 S h e e h a n , J.K., ( 5 ) 338, 339, 377; (8) 371 S h e f f e t , Gg., ( 3 ) 197 S h e l l e y , S., (5) 533; (8) 427 S h e n , B.W., ( 7 ) 93, 94 S h e p h e r d , M.G., ( 6 ) 195 Shepherd, V.L., (5) 524; (7) 139 Sherblom, A.P., ( 5 ) 588 Sherman, J.M., (5) 930 S h e r r y , A.D., ( 5 ) 144 Sherwood, L . M . , (5) 472 Shevchenko, N . M . , ( 6 ) 299 S h e w a l e , J.G., ( 6 ) 203 S h i b a e v , V.N., ( 8 ) 73 S h a r o n , J., Sharon, N . ,
S h i b a o k a , H . , ( 8 ) 323 S h i b a t a , K . , ( 3 ) 198;
( 5 ) 731; ( 8 ) 205, 244
S h i b a t a , N . , ( 4 ) 229 S h i b a t a , S., ( 8 ) 65 S h i b a t a , T., ( 8 ) 468 S h i b a t a , Y., ( 8 ) 301 Shibusawa, Y., ( 8 ) 387 S h i b u y a , N., ( 2 ) 107 S h i d a , H. (5) 83 S h i f r i n , S., ( 5 ) 468,
469
S h i g e m a t s u , H., ( 2 ) 40 S h i g e m a t s u , Y., ( 6 ) 245 Shikama, H., ( 5 ) 292;
(6) 527
Shimada, E . , ( 6 ) 419 S h i m a h a r a , K . , ( 3 ) 284 S h i m a k a t a , T., ( 8 ) 436 Shimizu, H . , (3) 82:
( 7 ) 204
S h i m i z u , K.,
(8) 556
( 2 ) 105;
( 5 ) 417 ( 6 ) 368; ( 8 ) 604, 700 Shimizu, Y., (8) 743 Shimomura, T . , ( 6 ) 185 S h i n , K.Y., ( 5 ) 472 Shinagawa, E., ( 6 ) 535 Shinmoto, H . , (5) 5; (8) 193 S h i n o d a , T., ( 5 ) 850, 896 S h i n o h a r a , T., (2) 4 Shinomiya, S., (6) 311, 31 7 Shiokawa, H . , ( 5 ) 145 Shiokawa, Y., ( 7 ) 11.6 S h i o m i , N . , ( 3 ) 88; ( 8 ) 389 Shiozawa, M., ( 8 ) 739 S h i p l e y , G . G . , (7) 81 S h i r a k u r a , Y., ( 8 ) 437 S h i r l e y , M . A . , (7) 137 S h i r o i s h i , M., ( 5 ) 980 S h i t o v a , N . B . , (8) 727 S h i v a r a j , B., ( 6 ) 274, 283 S h i v e l y , J.E., ( 5 ) 482 S h i y a , S.D., ( 6 ) 137 Shmakova, Z.F., ( 6 ) 441 Shockman, G.D., (4) 49 S h o f s t a l l , R.E., ( 5 ) 473 Shohmori, T., ( 2 ) 65 S h o j i , M., ( 8 ) 211 Shono, T., ( 8 ) 527, 528, 529 S h o r e , J.D., ( 5 ) 760, 761 S h o s h i n a , V.J., ( 8 ) 465
Shimizu, M . , Shimizu, S.,
81 1
Author Index S h o w a l t e r , A.M.,
255
(5)
Shoyab, M . , ( 8 ) 142 S h r a k e , A . , (6) 437 S h r e f f l e r , D.C., (5)
605
S h u l ' g i n , M.N., ( 8 ) 179 Shuman, H.A., (4) 134 S h u t a , H . , ( 8 ) 437 S i a , D.Y., (5) 810 S i b i r n y i , A.A., ( 6 ) 186 Sibley, C.C., ( 8 ) 271 S i b l e y , C.H., ( 5 ) 892 S i d b e r r y , H.F., (4) 145 S i d d i q u i , I . R . , ( 3 ) 140 S i d h a n , V . , (6) 432 S i e b e r t , C.J., ( 8 ) 400 S i e f e l , T.W., (8) 334 Siefermann-Harms, D.,
(7) 178 S i e f f , C . , ( 5 ) 621 S i e f k e n , D.A., ( 8 ) 230 S i e g a l , G.P., ( 5 ) 260 S i e g e l , G . , (5) 309 S i e g e r s , H . , ( 8 ) 492 S i e g r i s t , H.P., (7) 143 S i e r g i e j , R.W., ( 8 ) 728 S i e s s , M.H., (8) 637 Sietsma, J.H., (4) 215 S i e v e r s , A . , (5) 91 S i e v e r s , S., ( 2 ) 16 S i f e r s , R.N., (6) 111 S i g n e r s , E., ( 4 ) 149 Sikder, S.K., (5) 507 S i k o r s k i , Z.E., ( 6 ) 79; (8) 503, 504 S i l b e r t , C.K., ( 5 ) 234 S i l b e r t , J.E., (5) 416 S i l v e r , F.H., ( 5 ) 539 S i l v e r , H.K.B., (2) 41: (5) 1027 S i l v e r , R.P., ( 4 ) 95 S i l v e s t r i , L., (5) 331 S i l v i a , J.S., ( 5 ) 1066 Sim, E., (5) 795: (8) 180 Sim, R.B., ( 5 ) 795: ( 8 ) 180 Simeonov, N . , ( 8 ) 488 S i m e r a l , L.S., (5) 186 S i m i z u , B., ( 5 ) 86 Simons, K . , (5) 49 Simonson, L.G., ( 6 ) 378 Simonson, R., (3) 207 Simpson, D.L., ( 5 ) 566: (6) 401: (8) 576 Simpson, E.K.G., ( 3 ) 37 Simpson, W.A., (4) 18 S i n a y , P., ( 5 ) 369: ( 8 ) 49, 53 S i n c l a i r , R., ( 5 ) 1041 S i n g e r , M.S., (5) 586
S i n g e r , S . J . , ( 5 ) 59 S i n g h , B.D., (6) 302 S i n g h , B.N., ( 5 ) 1045 Singh, C . , (8) 692 S i n g h , K.N., ( 7 ) 160 S i n g h , O.M.P., (8) 345 S i n g h , R.B., ( 6 ) 302 Singh, R.P., (6) 302 S i n g h , S., ( 6 ) 524 Sinha, S.K., (5) 713 S i n k u l e , J . . ( 6 ) 491 S i n n , W . , (5) 403, 420 S i n n o t t , M.L., (6) 72 S i p p o l a , M., (6) 316 S i r i m a n n e , P . , ( 2 ) 32,
42
S i r o , M.R., ( 8 ) 385 Sissons, J.G.P., (5) 73 S i t i a , R., ( 5 ) 886 S i u , C.H., (8) 593 S i v a k , M.N., ( 3 ) 68, 69 Sivakami, S., (6) 400 S i w i n s k a , K., ( 6 ) 343 S j b b e r g , I . , ( 5 ) 378 S j b s t r b m , H . , ( 6 ) 444 Skarjune, R.P., (7) 81 S k e h e l , J . J . , ( 5 ) 38,
39
Skoda, J . , (8) 616 Skorko, T., (6) 442 Skorstengaard, K., (5)
236
S k o t l a n d , T., ( 8 ) 446 S k o v b j e r g , H , . (6) 129 S k r i v a n e k , J.A., ( 7 ) 65 Slakey, L.L., (8) 172 Slama, J . , ( 8 ) 76 S l a t e r , E.E., (8) 191 S l a u g h t e r , D., ( 5 ) 832 S l a y t e r , H.S., (5) 510,
539, 551
(3) 288: ( 6 ) 19, 51 S l e z a r i k , A., ( 3 ) 243 S l i f e , C.W., (8) 7, 9 S l o d k i , M.E., ( 4 ) 158 Slomiany, A . , (5) 954; ( 7 ) 44, 45, 123 Slomiany, B . L . , (5) 954; ( 7 ) 44, 45, 123 S l u t z k y , B . , (3) 119 S l u y t e r m a n , L.A.A. (8) 175, 377, 388 S m a l l , D.A.P., ( 8 ) 209 Small, D.M., (7) 81 S m a l l b o n e , B.W., (7) 194 Smardo, F . L . , ( 5 ) 473 Smart, E . L . , (6) 117 S m a r t , J . E . , ( 5 ) 15 Smidsrdd, O., (3) 265 Smirnov, V.N., ( 5 ) 22,
S l e t t e n g r e n , K.,
238
Smirnova, L.F., ( 6 ) 77 Smith, A . F . , (5) 807 S m i t h , B.D., ( 5 ) 234 Smith, B . R . , (5) 872;
( 8 ) 288
S m i t h , D., ( 5 ) 242 Smith, D.A., ( 3 ) 180 S m i t h , D . F . , ( 7 ) 24,
32: (8) 326
Smith, Smith, Smith, Smith, Smith,
D.H.. E.E., G.J.,
( 4 ) 98
( 4 ) 166 ( 4 ) 41 H.S., ( 5 ) 216 I.C.P., ( 5 ) 129, 179: ( 7 ) 196, 197 S m i t h , I . L . , ( 8 ) 340 S m i t h , K.K., ( 5 ) 269 S m i t h , P., ( 5 ) 67 S m i t h , P.F., (4) 90; ( 7 ) 191 S m i t h , P.K., ( 8 ) 152, 409 Smith, P.J., ( 6 ) 72 Smith, W.O., (8) 110, 438, 451 Smonson, L.G., ( 8 ) 542 Snow, P., (5) 600 S n y d e r , B.D., ( 2 ) 86 Sobhanaditya, J., (8) 303 Sobue, K . , ( 8 ) 382 Soderman, D.D., (5) 630 S o d e t z , J.M., ( 5 ) 796 S d e r s t o b m , G., (5) 370 Soederstroem, B., (3) 124 S a l t e r , J . , (5) 189 S o k o l o v s k i i , V.D. , ( 8 ) 727 S o l i m a n , A . , ( 8 ) 299 Solomon, B . , (6) 492 S o l t e r , D., ( 5 ) 861, 862; (7) 150 S o l t e s , E . J . , ( 3 ) 186 Solum, N.O., (5) 508, 667; ( 8 ) 376 Somkuti, G.A., ( 6 ) 159 Sommers, P.B., ( 5 ) 711 Sonenberg, N., ( 8 ) 312 Song, P.S., (8) 454 Sonnino, S., (7) 12, 17, 18, 19, 56 S o r g e r , M., (2) 73 S o s u l s k i , F.W., ( 3 ) 26 S o t i r o u d i s , T.G., (6) 415, 532 S o t t r u p J e n s e n , L., ( 5 1 236, 734, 735, 736, 737, 741, 742 S o u l i e r , S . , (5) 723 Sparks, K.J., ( 5 ) 322
812 Speake, B . K . , (6) 50 S p e c t o r , I . , ( 5 647 S p e c t o r , M., (5) 232 S p e c t o r , R . , ( 8 ) 146 S p e i r s , C . I . , (3) 250 S p e n c e r , C . A . , ( 5 ) 698 S p e t h , S . L . , (4) 58 S p i c e r , S.S., ( 5 ) 550 S p i e g e l , J . , (5) 170 S p i e g e l , S., ( 7 ) 43 S p i k , G . , (2) 106; ( 5 ) 123, 729, 730, 882, 883, 987, 1018; ( 6 ) 264; ( 8 ) 206 Spiri-Nakagawa, P. , (4 1 34 S p i r o , M . J . , ( 5 ) 470 S p i r o , R . G . , (5) 213, 470, 1048 S p i v a k , J . L . , ( 8 ) 375 S p r i n g e r , G . F . , ( 7 ) 38 S p r i n g e r , T.A., (5) 635 Springfield, J.D., (5) 1059 Springhorn, S . S . , ( 6 ) 273 S p r o t t , G . D . , ( 7 ) 196, 197 Squire, P.G., (2) 29 Sramek, S . , (5) 488 S r e e n i v a s a n , S., ( 3 ) 92 S r i n i v a s a n , K. R . , (5 1 761 S r i n i v a s a n , K . S . V . , (8) 512, 715 S r i n i v a s a n , S. R. , ( 5 ) 312 Srivastava, H.C., (4) 139 Srivastava, S.K., (6) 218 S r i v a s t a v a , P.M., ( 8 ) 5 90 Srivastava, P.N., (6) 63 Srivenugopal, K . S . , ( 8 ) 217 S t a b e r , F . G . , ( 8 ) 362 S t a e h e l i n , T . , (8) 401 S t a h l , P . D . , ( 5 ) 523, 524; (7) 139 Stamberg, J . , ( 2 ) 18; (6) 485; (8) 493, 751 Stampfer, M . R . , ( 5 ) 216 Staneloni, R . J . , (6) 173, ( 7 ) 175 S t a n e s c u , V . , (5) 320 Staun-Olsen, P . , ( 6 ) 421 S t a v n e z e r , J . , ( 5 ) 886 S t e e r , C . J . , (5) 192; ( 7 ) 134
Carbohydrate Chemistry S t e g w e e , D . , ( 3 ) 141 S t e i n , P . J . , (6) 435 S t e i n , R . , ( 6 ) 57, 61 S t e i n , T., (5) 395 S t e i n e r , S., ( 5 ) 488 S t e i n e r , S.M., (5) 489 S t e i n e r t , M . , ( 5 ) 998 S t e i n h a r d t , I . , (6) 423 S t e i n h o f f , M.C., ( 4 ) 113 Steinmann, B . , ( 5 ) 256 Steinrauf, L.K., (8) 515 S t e l l w a g e n , E . , ( 8 ) 407 S t e n b e r g , S., (8) 379 Stenman, U.H., ( 8 ) 185 S t e p a n i k , T.H., (5) 742 S t e p h e n , A.M., ( 3 ) 140, 259; (4) 101 Stephenson, F . A . , ( 8 ) 289 Steplewski, Z . , (5) 857; (7) 32 S t e r n , R . , ( 5 ) 258, 277 S t e r n b e r g , D . , (6) 212, 219 Sternberg, M . J . E . , ( 5 ) 5 94 Sternberger, N.H., ( 5 ) 4 62 S t e w , M . , ( 8 ) 559 S t e v e n , F . S . , ( 8 ) 181 Stevens, B.J.H., (3) 219 S t e v e n s , E . S . , ( 3 ) 179, 151, 266; (8) 543 S t e v e n s , J . , ( 2 ) 28; (5) 307 S t e v e n s , R.L., ( 5 ) 407, 408, 428 S t e w a r t , G . G . , ( 4 ) 189 S t e w a r t , G . J . , (6) 518 S t e w a r t , J . , ( 5 ) 637 S t e w a r t , J . C . , ( 4 ) 131 S t e w a r t , W.D.P., ( 5 ) 114; (8) 218 S t e w a r t , W.E., ( 5 ) 517 Stewart-Tull, D . E . S . , (4) 20, 64 S t i n s o n , A . , ( 6 ) 372 Stipanovic, A.J., (3) 151; (8) 543 S t i r l i n g , J . , ( 8 ) 206 Stirm, S., (4) 102; ( 6 ) 409 S t i r p e , F . , ( 5 ) 100 S t i t t , M., (3) 70, 71 S t i x , D . , ( 4 ) 53 S t o c k , J . W . , (5) 249 S t o c k e r , B . A . D . , ( 4 ) 84 S t o c k s , J . , (8) 238 Stoddard, R . W . , ( 4 ) 196
S t o n e , B . A . , ( 3 ) 164, 165 S t o n e , P . R . , ( 8 ) 417 Stone, R . G . , (3) 7 S t r a c h e r , A . , (8) 415 Strahm, A . , ( 3 ) 147 S t r a k a . R . , ( 8 ) 67 Strakhova, N.M., (2) 18 S t r a p r a n s , I., ( 5 ) 433 S t r a u b , J . P . , (2) 79 S t r a u s , D . C . , ( 4 ) 116, 161 S t r e c k e r , G., ( 2 ) 91, 98, 106; ( 5 ) 123, 525, 882, 883, 960, 968, 972, 990, 1024; ( 8 ) 206 S t r i c k l a n d , D. K . , ( 5 ) 5 64 S t r i c k l a n d , S., ( 5 ) 269 Strickler, J.E., (5) 999 S t r o b e l , G . A . , ( 2 ) 89 Strbmberg, P . , (5) 813 Strominger , J . L. , ( 4 ) 26; 619, 624, 627 S t r o p n i k , C . , ( 3 ) 45 Strosberg, A . D . , ( 5 ) 163 S t r o u d , R . M . , ( 5 ) 331 S t r o u t , H . V . , ( 8 ) 191 S t u a r t , M.C., ( 5 ) 680; ( 8 ) 363 Stuhlsatz, H.W., (5) 394, 395, 437 Stumpel, J . , ( 5 ) 910 Sturgeon, C.M. (8) 83, 84, 85, 86 SU, L.-C., ( 5 ) 161 Subba Rao, P . V . , ( 8 ) 716 S u b i k , J . , ( 6 ) 187 Sudhakaran, P . R . , ( 5 ) 403 Sudhararan, P . R . , ( 5 ) 420 S u g a , K . , ( 8 ) 649 Suga, K . I . , (8) 678 Suga, S., ( 6 ) 312 Sugahara, K . , (2) 82 Suganuma, T., ( 6 ) 319, 327 Sugawara, S., ( 3 ) 113; (6) 82; ( 8 ) 462 Sugibayashi , K., ( 8 5 64 S u g i h a r a , G . . ( 6 ) 434 Sugihara, N . , ( 8 ) 292 Sugimoto, K . , ( 2 ) 82 Sugimoto, S., (6) 484 Sugimoto, Y . , ( 3 ) 36. 39
813
Author Index S u g i n o , Y., ( 4 ) 108 S u g i t a , M . , (7) 161, 204 S u g i u r a , M., ( 2 ) 75 S u g i u r a , Y., (8) 65 S u i , C.H., ( 5 ) 4 S u l e i m a n , S . A . , (8) 146 S u l i t z e a n u , D., ( 5 ) 827 Sumi, S., (6) 427 Sumida-Tanaka, R., ( 6 ) 245 Summers, D . F . , ( 5 ) 61 Sumper, M., (4) 184: ( 5 ) 1002 S u m r e l l , C., ( 3 ) 199 S u n d a r a l i n g a m , M., ( 2 ) 114 Sundaram, P . V . , (8) 582, 689 Sundaresan, R.V.S., (3) 131 Sunderman, F.W., ( 6 ) 27 Sundstrom, D.W., (6) 213; ( 8 ) 752 Sung, E . , (5) 620 Supp, M., ( 7 ) 15 Surma, M.L., (5) 373 S u r o l i a , A . , ( 5 ) 109, 124, 168, 169; (7) 91 S u r o l i a , N., ( 5 ) 124 S u t h e r l a n d , C.A., (5) 606 S u t h e r l a n d , I.W., ( 4 ) 148, 172; ( 8 ) 192 S u t h e r l a n d , T., ( 8 ) 222 S u t i n e n , M.L., ( 8 ) 185 S u t t a j i t , M., ( 2 ) 81 S u t t o n , B.C., (3) 169 S u t t o n , R . , ( 3 ) 169 S u z u k i , A . , (3) 19, 34: ( 7 ) 145 S u z u k i , F., ( 7 ) 182: (8) 342 S u z u k i , H., ( 3 ) 156, 157 S u z u k i , K., ( 5 ) 86; ( 6 ) 17, 125: (7) 96: (8) 112, 143, 272 S u z u k i , M., (5) 978: ( 8 ) 317, 520 S u z u k i , S., (4) 229: (5) 417, 1052: ( 8 ) 99, 603, 611, 647, 721, 722 S u z u k i , T., ( 5 ) 719, 843; (8) 176, 365 S v e c , F., ( 6 ) 485; ( 8 ) 584, 709, 747, 751 S v e d a , M.M., ( 5 ) 35 S v e d a s , V.K., (8) 734 Svennerholm, L., ( 7 ) 25, 49; ( 8 ) 538
S v e n s o n , A . , ( 8 ) 118 Svenson, B . , (6) 397 S v e n s s o n , S., ( 5 ) 963; ( 7 ) 119 S v e n s s o n , S.C.T., ( 8 ) 24 S v i r i d o v , D.D., ( 5 ) 238 Swaisgood, H.E.. (8) 92 Swallow, D., ( 6 ) 105 Swank, R.T., (6) 235 Swann, D.A., ( 5 ) 442, 539 S w e e l e y , C . C . , (5) 974, (7) 101 Sweet, M.B.E., ( 5 ) 320, 440, 443 S w i t z e r , C.H., ( 5 ) 864 S w i t z e r , M.E.P.. (5) 782 Sykes, J.E.C., ( 5 ) 1015 S y n o w i e c k i , J., (6) 79: ( 8 ) 503, 504 Szamel, M., (8) 361 S z e j t l i , J . , ( 8 ) 519 Szewczuk, A . , (8) 690 Szewczyk, B., ( 6 ) 442 S z i l a s i , M., (8) 519 S z o k a , F., ( 7 ) 92 s z u , s., (4) 120 S z u m i l o , T., ( 4 ) 194 S z w a j c e r , E . , (8) 690 Tabachnk, N.F., ( 5 ) 931 Tabak, L . A . , (5) 933 T a b a t a b a i , L.B., ( 6 ) 256 Tabiowo, A , , ( 5 ) 1054 T a c h i b a n a , Y . , (6) 28, 285 Tack. B.F., ( 5 ) 792 Tada, T., ( 7 ) 147 Tadano, K., ( 7 ) 133 Tagami, S., (5) 912 T a g e r , J.M., ( 5 ) 870: (6) 164 T a g u c h i , H . , ( 8 ) 607, 649, 678 T a g u c h i , M., ( 4 ) 135 T a g u c h i , S., (6) 130 T a j i r i , T., ( 6 ) 277 Takada, H . , (4) 50, 232 Takada, K., ( 4 ) . 164; (6) 501 Takado, Y., ( 6 ) 428 T a k a g a k i , Y., (6) 429: ( 8 ) 662 T a k a g i . K., ( 7 ) 140: (8) 328, 348, 523 T a k a g i , S., ( 5 ) 980 T a k a g i , T., (5) 980; ( 6 ) 318, 480, 481 T a k a h a s h i , H.. (5) 675;
( 6 ) 448 T a k a h a s h i , H.K., ( 5 ) 358 T a k a h a s h i , K.. ( 8 ) 372, 518. 525 T a k a h a s h i , M., ( 2 ) 48, 52 T a k a h a s h i , N . , ( 3 ) 208: (5) 897, 985; ( 8 ) 205 T a k a h a s h i , T., ( 6 ) 403 T a k a h a s h i , Y., (5) 784 Takahashi-Nakamura, M., (8) 143 T a k a i , M., ( 3 ) 111 Takakowa, M., ( 5 ) 5 Takaku, F., ( 7 ) 36 Takakuwa, M., ( 8 ) 193 Takamatsu, S., ( 8 ) 623 T a k a s a k i , Y . , (3) 54 T a k a s e , K., ( 5 ) 708 T a k a s h i , N . , (5) 850, 896 T a k a t s u k i , K . , ( 5 ) 472 T a k a u c h i , T., ( 6 ) 286 T a k a y a s u , T., ( 5 ) 850. 8 96 T a k e d a , T., ( 4 ) 226; ( 8 ) 37, 65 T a k e d a , Y., ( 3 ) 19, 34 T a k e h a r a , T., ( 6 ) 379 T a k e h i s a , M., ( 6 ) 479 Takema, Y . , (5) 253, 273 Takenaka, A . , ( 5 ) 207; ( 8 ) 356 T a k e n a k a , T., ( 3 ) 89 Takeo, K . , ( 8 ) 33, 34, 552 T a k e s h i t a , K., ( 5 ) 916 Taketomi, T., (7) 104 T a k e n c h i , E., ( 5 ) 985 T a k e u c h i , T., (3) 74; ( 4 ) 130: ( 6 ) 285 T a k e u c h i , Y., (4) 63 T a k i , T., ( 7 ) 140; ( 8 ) 328, 348 T a l i e r i , M.J., ( 2 ) 25 Tam, M.R., (5) 861, 862; ( 7 ) 150 Tamai, Y., (5) 5; ( 8 ) 193 Tamaki, S., ( 4 ) 30 Tamaki, Y . , ( 8 ) 205 Tampion, J . , ( 6 ) 361 Tan, H.L., ( 7 ) 176 T a n a h a s h i , E . , ( 8 ) 69, 70 Tanaka, A . , ( 8 ) 627, 629, 652 T a n a k a , H., (4) 34; ( 8 ) 258 T a n a k a , I., ( 5 ) 148;
Carbohydrate Chemistry
814 (8) 533 T a n a k a , M . , ( 2 ) 22: ( 4 ) 209: (5) 756; (6) 68, 74, 434; ( 8 ) 527, 528, 529 T a n a k a , R . , ( 3 ) 118; (6) 181 T a n a k a , S., ( 4 ) 109, 140, 141; (5) 855: ( 8 ) 532 T a n a k a , T., ( 5 ) 430: (6) 298 T a n a k a , Y . , ( 4 ) 34 T a n c r e d e , P . , (7) 165 Tandecarz. J . S . , (3) 68, 69 Tangnu, S.K., ( 6 ) 208 T a n i , K . , (8) 605, 638 Taniguchi, H . , (3) 58 T a n i g u c h i , K., (8) 702, 703 T a n i g u c h i , N . , ( 5 ) 431 Tanner, M . J . A . , (5) 907 T a n n e r , W . , ( 5 ) 88 Tans, G . , (5) 789 T a n z e r , M.L., ( 5 ) 411 T a p p e s e r , B . , (8) 384 T a r d y , M . , ( 7 ) 25: ( 8 ) 538 T a r e n t i n o , A.L., (5) 115, 1011 T a r t a k o f f , A . , ( 5 ) 623 T a s h i r o , M . , (8) 292 T a s h i r o , Y., ( 5 ) 561 T a t a r y n , D.N., (5) 481 T a t s u t o m i , Y., ( 8 ) 652 Tauber-Finkelstein, M . , ( 8 ) 108 Tavetkova, I.V., (6) 238 T a y a , M., ( 6 ) 368 T a y l o r , A . , (5) 500 T a y l o r , I.E.P., ( 2 ) 20; ( 3 ) 135, 216; (5) 2 T a y l o r , J . B . , ( 8 ) 92 T a y l o r , J . M . , (5) 745 T a y l o r , K.G., ( 6 ) 524 T a y l o r , R., (2) 70 T a y o t , J . L . , ( 7 ) 25; (8) 538 T e g e l a e r s , F.P.W., ( 5 ) 870; ( 6 ) 164 T e g t m e y e r , H . , ( 7 ) 38 T e i c h , N.M., (5) 21 Teichberg, V.I., (8) 378 T e i c h b e r g , V.L., ( 5 ) 113 T e j i m a , S.. ( 5 ) 985; (8) 554 T e l e f o n c u , A., ( 8 ) 467 T e l l e r , D.C., ( 5 ) 137,
138 Tempany, E., ( 5 ) 828, 829 T e n g b l a d , A . , ( 5 ) 326, 384 T e n n e n t , G . , ( 5 ) 505 T e n n i , R . , (5) 392 Tenovuo, J . , ( 8 ) 170 T e p f e r , M . , (3) 135, 216 T e p h l y , T.R., ( 8 ) 293 T e r a m a t s u , T., (8) 743 T e r a o , T., ( 8 ) 272 T e r a o k a , N . , (8) 99 T e r a s h i m a , M . , ( 6 ) 488; (8) 750 T e r a w a k i , Y . , ( 4 ) 46, 47, 48 Terayama, H., ( 5 ) 372 T e r h o r s t , C., (5) 600, 607 Terman, D.S., ( 8 ) 643 T e r p s t r a , W . , ( 8 ) 364 T e r r a n o v a , V.P., ( 5 ) 4 97 T e r r y , M.E., ( 3 ) 177, 178 T e t a e r t , D., ( 5 ) 897 T e t t a m a n t i , G., (6) 26; ( 7 ) 12, 17, 18, 19, 56 T e v e t h i a , S.S., ( 5 ) 30 T h a n i y a v a r a n , S., ( 6 ) 524 T h e u r e r , B . , ( 3 ) 16 T h e w l i s , B . , (7) 167 Thiel, H.J., (5) 79 Thiele, H.G., (7) 60 Thiem, H . J . , ( 5 ) 992 Thiem, J . , (8) 17, 535 T h i e s s e n , M., ( 5 ) 183 Thirmnig, R . L . , (5) 60 T h d g e r s e n , H . , ( 5 ) 852 T h o g e r s e n , H.C., (5) 236 T h o i , L.L., ( 5 ) 648 Thomas, D . , (3) 292; ( 8 ) 583, 626, 627, 651, 704 Thomas, G.H., ( 5 ) 428 Thomas, J., (8) 213 Thomas, J . A . , ( 5 ) 293 Thomas, J . J . , (6) 423 Thomas, T.D., ( 6 ) 158 Thomasset, B . , (8) 627, 628, 651 Thompson, D.M.P., ( 5 ) 481 Thompson, J . , ( 4 ) 162 Thompson, J . S . , (2) 25; ( 4 ) 22, 37 Thompson, S., ( 6 ) 249
Thompson, T.E., ( 7 ) 21 Thonar, E . J . M . , ( 5 ) 415, 427 Thonar, E.J.M.A., ( 5 ) 440 Thorleylawson, D.A., (5) 70 T h o r n , W . , ( 2 ) 58 T h o r n t o n , W . , (5) 806 T h o r p e , R . , ( 6 ) 106 Thorpe, R.C., ( 5 ) 1008 T h o r s e n , S., ( 8 ) 283 T h r e l f a l l , D.R., (3) 220 T h u n b e r g , L., ( 5 ) 361, 362, 367 Thuy, L . P . , ( 5 ) 657, 771; (8) 295 T i b i , L . , ( 5 ) 807 T i c h i , M . , ( 5 ) 103, 162; ( 8 ) 94, 95 T i e b e r , V.L., ( 4 ) 180 Tieckelmann, H., (2) 37, 45: (5) 302 T i e r n e y , A . R . , ( 2 ) 64 T i k h o m i r o v a , A.S., ( 6 ) 137 T i m o f e e v a , N . M . , ( 6 ) 77 Timpl, R . , ( 5 ) 251, 254, 270, 498 T i n , G.W., ( 7 ) 152 T i n g , I . P . , ( 3 ) 169 T i n g s v i k , K.. ( 3 ) 207 T i p p l e s , K.H., ( 6 ) 306 T i p t o n , K.F., ( 8 ) 340 T i u n o v a , N . A . , ( 6 ) 392 T j e s s e m , K . , (8) 534 Tlaskalova-Hogenova, H . , (8) 587 Tocchini-Valentini, G.P., ( 8 ) 269 T o d a , F., ( 8 ) 517 Toda, K., (5) 3 Toda, N . , ( 5 ) 174, 390 T o d a r o , G. J . , ( 5 ) 237; ( 8 ) 142 Todd, C.W., ( 5 ) 482 Toe, R . , ( 5 ) 289 T o f f a n o , G., ( 7 ) 56 Tokunaga, E., (5) 843; (8) 176 Tokunaga, T., ( 7 ) 144, 164 T o k u y a s u , K.T., ( 5 ) 59 T o l e d o , I . , ( 5 ) 268 Tolleshaug, H . , (5) 567, 820 Tolmasky, M.E., ( 7 ) 175 T o l o s a , E . A . , ( 8 ) 160 T o l s t o g u z o v , V. B . , ( 8 1 537 Toma, S., ( 5 ) 392; ( 6 )
815
Author Index
32 Toman, R . , ( 3 ) 193 Tomasi, M . , ( 5 ) 52 Tomasie, J., ( 4 ) 21 Tomasz, A . , ( 4 ) 32 Tombaccini, D., ( 7 ) 78 Tominaga, Y . , ( 4 ) 236; ( 5 ) 853; ( 6 ) 401; ( 8 ) 166 Tomioka, H . , ( 5 ) 850 Tomita, K . , ( 8 ) 123. 437 T o m i t a , M . , (5) 914, 915, 916 Tomoda, M . , ( 3 ) 253 Tomono, T . , ( 5 ) 843; ( 8 ) 176 Tong, C . C . , ( 6 ) 195 Tong, H . K . , ( 3 ) 296 Tong, J.P.K., ( 3 ) 95 Tong, N.T., ( 8 ) 308 T o n i o l o , C., ( 4 ) 25 T o n t t i , K., ( 8 ) 185 T o r c h i a , D . A . , ( 5 ) 308 T o r c h i l i n , V.P., ( 8 ) 670 T o r i g o e , A . , ( 3 ) 253 T o r i i , H . , ( 4 ) 108 T o r i i , M . , ( 4 ) 140, 141. 168; (8) 532 T o r i k a t a , T., ( 6 ) 440 T o r o , L . , (6) 497 Torp, J., (3) 82 Torre-Blanco, A , , ( 5 ) 268 T o r r i , G., ( 5 ) 369 T o r r i , M . , ( 5 ) 855 T o s a , T., ( 8 ) 469, 549, 623, 654 T o s e l l i , M., ( 5 ) 142 T o u e t , G., (3) 206 Tourbez-Perrin, M., ( 5 ) 733 T o u s t e r , 0 . . ( 5 ) 476 T r a c y , S., (8) 98 T r a c z , J . S . , ( 6 ) 221 T r a v a s s o s , L.R., ( 4 ) 220 T r a v i s , J . , ( 5 ) 743 T r a y e r , I . P . , (8) 209 T r i f n o f f , E . , ( 5 ) 707 T r i p h a u s , G.F., (55) 438 T r i v e d i , L.S., ( 6 ) 211 T r o n c h i n , C . , (4) 222 Trowbridge, I.S., ( 5 ) 608, 609 T r u g l i a , J.A., ( 8 ) 415 T r y g g v a s o n , K . , (5) 260 T s a o , D . , ( 5 ) 554; ( 8 ) 287 T s a o , C.T., ( 4 ) 183,
200; ( 6 ) 205, 215, 4 77 Tscholakowa, J . , ( 8 ) 474 T s e n g , S.C.G., ( 5 ) 258, 277 T s i e n , H.C., ( 4 ) 150 T s i v i o n , T., (5) 209 Tsuboyama, A . , ( 7 ) 36 T s u c h i d a , T . , ( 8 ) 723 T s u c h i h a s i , H.. ( 4 ) 240 T s u c h i t a n i , Y.. ( 4 ) 168 T s u c h i y a . T., ( 1 ) 2 T s u i . F.P., ( 4 ) 99 T s u j i , I., ( 6 ) 514 T s u j i , M . , ( 5 ) 417, 1052 T s u j i , S.. ( 8 ) 143 T s u j i , T., ( 5 ) 111, 180, 912, 923, 924; ( 8 ) 392, 591 T s u j i m o t o , M . , ( 4 ) 50 T s u j i s a k a , Y . , ( 4 ) 236; ( 6 ) 384 T s u k a d a , S,. ( 8 ) 505 T s u k a d a , Y . , ( 5 ) 867; ( 8 ) 184 T s u k a g o s h i , N., ( 7 ) 3 Tsumura, N., ( 6 ) 480, 481, 482 T s u n e y a s u , S., ( 8 ) 508 T s u i t s u i , Y., (6) 75 Tubb, R.S., ( 4 ) 218 Tuck, B.F., ( 8 ) 279 T u c k e r , G.A., ( 3 ) 244: ( 6 ) 451 T u c k e r , R.F., ( 8 ) 407 Tuckova, L . , (8) 587 Tuderman, L., ( 5 ) 256 TUdbs, F . , (8) 519 T u f f , S., ( 5 ) 680: ( 8 ) 3 63 Tukey, R . H . , ( 8 ) 293 T u l i , R., ( 8 ) 213 Tung, J.S., ( 5 ) 17 Tung. T.C., ( 8 ) 571 Tung, W.H., ( 5 ) 467 Turco, S., ( 5 ) 62, 640 T u r c o , S . J . , ( 2 ) 31, 47; (5) 641, 1063; ( 7 ) 162 T u r k o v a , J., ( 8 ) 493, 584 T u r n e r , B.M., ( 8 ) 321 T u r n e r , C.L., ( 6 ) 145 T u r n e r , D.C., ( 5 ) 228 T u r n e r , K.W., ( 6 ) 158 T u r n e r , M.J., ( 5 ) 997; (8) 234 T u r n e r , R.B., ( 8 ) 178 T u r n e r , S.H., ( 2 ) 7 T u r o v s k i i , V.S., ( 5 )
454 Twose, T.M., T y l e r , R.T.,
( 8 ) 180 (3) 26
U c h i d a , T., ( 6 ) 74; ( 7 ) 16, 89, 122, 124; ( 8 ) 301, 302, 330 Uchiyama, H . , (5) 347. 348, 360; ( 8 ) 127 Ueda, R., ( 5 ) 485 Ueda, S., ( 4 ) 129; ( 6 ) 277; ( 8 ) 468 U e h a r a , H.. ( 5 ) 611, 615, 616, 617, 628 Uckama, K . , ( 3 ) 79; ( 8 ) 5 22 Uemura, K . , ( 7 ) 104 Ueng, P.P., ( 4 ) 183 Ueno, A , , ( 8 ) 518, 825, 826 Ueno, K . , ( 7 ) 54 Ueno, Y . , ( 6 ) 179; ( 8 ) 553 U g a l d e , R.A., ( 6 ) 173 Ogolev, A.M., (6) 77 U g o r s k i , M., ( 3 ) 53; ( 6 ) 292 U h l e n b r u c k , G., ( 5 ) 189, 202 Uhr, J.w., ( 5 ) 877; ( 8 ) 569 U i , N., ( 2 ) 19; ( 5 ) 1020 U i t t o , J . , ( 5 ) 280 U i t t o , V.J., (5) 280 Ujimoto, K., ( 8 ) 531 Ukai, S., ( 4 1 247 U l e z l o , I.V., ( 6 ) 475 U l l a h , A . H . J . , ( 8 ) 455 Ulmanen, I., ( 5 ) 82, 92 1 Umadevi, S., ( 3 ) 29 Umbarger, H . E . , ( 6 ) 148 Umemoto, T., ( 4 ) 35, 50 Umezurike, G.M., (6) 226, 449 Unger, F . M . , ( 4 ) 51, 53, 97 Unger, P., ( 4 ) 100 Uozaki, T , (5) 431 Urbanowski, J.C., ( 2 ) 77; (5) 936 U r d a l , D . , ( 7 ) 151 Ureta, T . , (6) 534 Uryu, T., (4) 141 U s h i j i m a , A . , ( 8 ) 123 Usmanov, K.U., ( 8 ) 465 Usui, T., (2) 82; (4) 239; ( 5 ) 430, 719; ( 8 ) 365 U t l e y , C . , ( 5 ) 490; ( 8 ) 343
.
Carbohydrate Chemistry
816 Uyenco, F . , ( 3 ) 273; (4) 204 Uzuka, M . , ( 5 ) 423
Vaara, M . , (4) 83 Vaara, T . , ( 4 ) 83 Vaerman, J . P . , (8) 196 Vaes, G., ( 5 ) 304 V a h e r i , A . , (5) 243, 284 V a i r o , M.L.R., ( 8 ) 471 V a i t u k a i t i s , J., (5) 683 V a j e n e r , J . , ( 8 ) 493 Vakirtzi-Lemonias, C . , ( 5 ) 670 Valchev, V . , ( 3 ) 13 Valcheva, E . , (3) 13 V a l e n t o v a , 0.. ( 6 ) 485; (8) 645. 751 Valla, S . , ( 4 ) 128 Vallee, B.L., (8) 300 V a l u e v a , T.A., ( 8 ) 179 Vameri, A . , (5) 237 Vanagthoven, A . , ( 5 ) 607 Van Besouw, A., ( 7 ) 179, 180 Van B o e c k e l , C.A.A., (8) 78 Van Boom, J.H., ( 8 ) 66, 78 Van BOven, C.P.A., ( 4 ) 73 Vance, I . , ( 6 ) 361 V a n c h e r i , L . , (8) 189 Vancurova, D., ( 8 ) 493 Vandamme, J . , (5) 514; ( 8 ) 398 van d e n Berg, L., ( 6 ) 333 van d e n Berghe, H., ( 5 ) 738, 739 Vandenbranden, M . , ( 7 ) 29 Van den E i j n d e n , D.H., (2) 34; (5) 940, 1021, 1076; (6) 64, 509; (7) 50; (8) 245, 5 80 V a n d e r k o o i , J.M., ( 5 ) 844 Van Der K o o i j , D., ( 3 ) 76 Van d e r K r o e f , W.M.J., (6) 264; ( 8 ) 124 Van d e r Laan, W.F.M., (8) 612 van d e r Maaten, M.J., (5) 14 V a n d e r w i n k e l , E., ( 4 ) 31
van D i e i j e n , G . , ( 5 ) 789 Van D u i j n , P., ( 8 ) 367 van E i j k , R.V.W., (5) 518, 519, 420, 596; ( 7 ) 149 Vanek, T., ( 8 ) 642 V a n e l d i k , L . J . , (8) 349 Van E l s e n , A.F., ( 6 ) 240 van E t t e n , R.L., ( 6 ) 515 van G e l d e r n , H.H., (5) 2 96 van G r o e n s t e i n , T., ( 5 ) 806 Van Gylswyk, N.O., (4)
93
Van H a e c h t , J.L., ( 8 ) 639 van H a l b e e k , H . , ( 2 ) 91: (5) 720, 729, 748, 890, 929, 937, 968, 972, 990 Van H e i j e n o o r t , J . , ( 4 ) 24 Van Hoef, B, ( 5 ) 826; (8) 164 Van H o l s t , G.J., ( 3 ) 141, 142 van Horne, K.C., ( 5 ) 490: (8) 343 Van K a p e l , J., ( 8 ) 318 Van Keulen, M.A., (6) 476: ( 8 ) 602 van Landschoot, A , , ( 5 ) 140 van Leuven, F., ( 5 ) 738, 739 Vann, W.F., ( 4 ) 95, 96 Vannuchi, S . , (5) 499; ( 8 ) 329 Vannuchi, S., ( 5 ) 445 van Rapenbusch, R . , (5) 163 Van R o o s t , E., ( 8 ) 196 Vanschaftingen, E., (6) 93 Van Soest, P.J., ( 3 ) 30 Van S t e i j n , G.E.J., ( 8 ) 124 v a n T h i e n e n , G.M., ( 5 ) 870; (6) 164 van Uden, N., ( 4 ) 193; (6) 89 V a r a , F., ( 8 ) 456 V a r a d i , K.,(5) 658 V a r d a n i s , A., ( 3 ) 289; (6) 344 V a r k i , A . , ( 8 ) 255 Varona, R., (4) 211; (6) 529
.
Varriano-Marston, E., (3) 50; ( 6 ) 304 V a r t i o , T., ( 5 ) 243, 591 V a s i l i e v a . G.G., (8) 4 82 V a s i l i u - O p r e a , C., ( 3 ) 110 V a s s a l l i , P . , (5) 623 V a s s a u l t , A . , ( 6 ) 271 V a s s t r a n d , E., ( 6 ) 443 V a s s t r a n d , E.N., ( 8 ) 502 V a s s t r a n s , E.N., ( 4 ) 36 V a t t u o n e , M.A., (6) 81, 133 Vaughan, L., ( 5 ) 752 Vaz, W.L.C., (5) 910 Vazquez P e r n a s , R . , ( 6 ) 18, 112 Veatch, W.R., ( 5 ) 619 Vecchio, G.. (8) 411 Vega, J.M., ( 8 ) 424 Veh, R.W., (7) 27, 28 Vehar, G.A., ( 5 ) 779; (8) 153 Veis. A., ( 5 ) 279 V e l d i n k , G . A . , (2) 91; ( 5 ) 937, 968, 972, 990 V e l i k y , I., ( 8 ) 634 V e l i k y , I . A . , (8) 614, 615, 622 V e l l e n g a , K . , ( 6 ) 476; (8) 602 V e l u r a j a , K . , ( 2 ) 111 Vem, R.W., (5) 199 V e n t o l a , A.S.,, ( 5 ) 957 V e n t r e l l a , G., (5) 502 Venyaminov, S.Y., ( 5 ) 222 V e r b e r t , A., ( 5 ) 663; (7) 136 Vercelotti, J . R., ( 5 959 Vered, Y . , ( 3 ) 210 V e r h a a r , L.A.T., (2) 21, 26 Verhoeven, J . J . , ( 8 ) 66 V e r h o f f , F.H., (8) 683 Verma, D.P.S., ( 5 ) 90: ( 7 ) 174 Vermorken, A.J.M., (8) 162 Veroy, R.L., ( 3 ) 273; ( 4 ) 204 V e r p o o r t e , J.A., ( 5 ) 4 65 V e r s l u i s , C . , ( 3 ) 297, 2 98 V e r t e s y , L., ( 6 ) 278 V e r t i e v , Yu. V., (6)
817
Author Index 254; ( 8 ) 154 Verwilghen, R.L., (5) 478 V e y r i e r e s , A., ( 8 ) 19, 48, 50, 51 Via, D.P., ( 5 ) 488 V i c t o r i a , E.J., (5) 899 Vidal-Valverde, C., ( 3 ) 99 Vierhaus, S., ( 5 ) 437 ( 8 ) 606, Vieth, W . R . , 735 Vignon, M., ( 4 ) 103 V i i t a l a , J . , ( 5 ) 904; ( 8 ) 220 V i i t a n e n , K., ( 8 ) 385 (5) 727, Vijay, I . K . , 728 Vijayagopal, P., ( 5 ) 312 Vijayalakshmi, M.A., ( 8 ) 550 V i jayalakshmi, K. R., ( 6 ) 467 Vilarem, M.J., ( 5 ) 531 V i l l a , T.G., ( 6 ) 382 V i l l a e r e j o , M . , ( 4 ) 181 Villanueva, J.R., ( 6 ) 382 Villar-Palasi, C., ( 8 ) 291 V i l l a r r o e l , L.H., ( 3 ) 291 Villemez, C . , ( 5 ) 877; ( 8 ) 569 V i l l i g e r , B., ( 5 ) 225 Vincendon, G . , (5) 448: ( 7 ) 52: ( 8 ) 256 Vincendon, M., ( 3 ) 268 Vincent, C., ( 5 ) 812 Vincent, J . , ( 7 ) 4 Vincent, R . , ( 6 ) 385 Vindershain, G. Ya., ( 6 ) 101 V i n e t , M.C., ( 6 ) 168 Vining, L.C., ( 6 ) 180 Virkkunen, J . , ( 6 ) 314, 347: ( 8 ) 560 V i r t a n e n , I . , ( 5 ) 591 Vischer, P . , ( 5 ) 570, 1061 V i t e t t a , E.S., ( 5 ) 877; ( 8 ) 569 V i t i , M., ( 5 ) 499; ( 8 ) 329 V i t i , S., ( 2 ) 6 0 V i t i e l l o , F . , ( 7 ) 52 Vlasakova, V . , ( 3 ) 282 V l i e g e n t h a r t , J.F.G., ( 2 ) 91; ( 3 ) 558, 720, 729, 748, 890, 929, 935, 937, 968, 972,
990; ( 8 239 V lod av sky I., ( 5 ) 216 Voara, S. ( 6 ) 408 Vodrazka, Z., ( 6 ) 485; ( 8 ) 751 V8rbs, E., ( 5 ) 658 Vogel, H.J., ( 4 ) 179 ( 5 ) 316 Vogel, K.G., Voiland, A . , ( 7 ) 163 Volgin, Yu. V . , ( 7 ) 47 (4) Volleberg, M.P.W., 73 Volon, G . , ( 3 ) 276 von Bassewitz, D.B., ( 5 ) 261 von d e r Schmitt, D.J.. ( 2 ) 76 ( 8 ) 719 von F i g u r a , K., ( 5 ) 403, 420, 432, 525: ( 6 ) 59, 262 Vonlanthen, M . , ( 5 ) 721 von N i c h o l a i , H . , ( 6 ) 255 Vorlop, K.D., ( 8 ) 511 Vose, J.R., ( 3 ) 24, 25 Voss, B., ( 5 ) 261 Voss, E.W., (5) 840 Voss, T., ( 5 ) 270 Votruba, J . , ( 3 ) 282 Vreugdenhil, A.P., ( 5 ) 934 Vrsanova, M., ( 3 ) 202; (6) 464 Vrsanska, M . , ( 3 ) 201; ( 6 ) 463, 465 Vunnam, R . R . , ( 7 ) 141 Wachter, E.. ( 5 ) 965, 966; (8) 592 Wacker, H . , ( 6 ) 4 4 4 Wada, H . , ( 2 ) 6 5 Wada, M., ( 3 ) 294; ( 8 ) 636 Wada, S., ( 3 ) 146 Waechter, C.J., (5) 1043, 1050 Waehneldt, T.V., ( 5 ) 460 Wagh. P.V., ( 5 ) 1007 Wagle, D.S., ( 7 ) 183 Wagner, B., ( 4 ) 115 Wagner, C . , ( 8 ) 325 Wagner, F., ( 6 ) 222 Wagner, H . , ( 8 ) 39 Wagner, M., ( 4 ) 115; ( 7 ) 59 Wagner, R . A . , ( 5 ) 892 ( 5 ) 58 Wagner, R . R . , ( 5 ) 436 Wagner, W.D., Wagstaffe, P.J., ( 2 ) 51 Waheed, A . , (5) 525: ( 6 ) 515
Waibel, R . , ( 3 ) 182 Wakabayashi, K . , ( 6 ) 337, 363 Wakayama, N., ( 7 ) 1 6 Waki, T., ( 8 ) 649 Wakuda, T., ( 8 ) 522 ( 8 ) 585 Wagner, R . H . , Walborg, G.F., ( 5 ) 553 Walcott, P.J., ( 3 ) 8 Waldron-Edward, D., ( 5 ) 953 Walega, M.A., ( 8 ) 136 Wallace, B., ( 8 ) 653 Wallace, D.G., ( 5 ) 224 (5) 89 Wallace, D.H., Wall, D., ( 6 ) 333 ( 5 ) 286 Walsh, D.A., ( 5 ) 479 Walters, D.E., ( 5 ) 212 Walton, K.W., Waly, A . , ( 8 ) 489 Wandrey, C., ( 3 ) 98; ( 6 ) 188 Wands, J.R., ( 5 ) 894 Wang, C.S., ( 8 ) 373 Wang, D., ( 5 ) 732 Wankat, P.C., ( 6 ) 215 ( 3 ) 29, Wankhede, D.B., 40; ( 6 ) 467 ( 5 ) 42, 43, Ward, C.W., 44, 45 Ward, J.B., ( 4 ) 1 Ward, J.C., ( 3 ) 247 Ward, M., ( 6 ) 423 (6) 192 Warden, D.A., ( 8 ) 188 Ware, C . f . , ( 8 ) 326 Warner, C.V., Warnke, P.C., ( 7 ) 59 Warren, C . , ( 5 ) 300 Warren, L., ( 5 ) 549 Warren, T.C., ( 8 ) 142 Wasserman, B.P., ( 5 ) 1; ( 8 ) 697 Wasteson, A., ( 6 ) 411 Waszczynskys, N., ( 6 ) 349 Watanabe, H . , ( 5 ) 334; ( 6 ) 478, 479; ( 8 ) 659 Watanabe, K . , ( 5 ) 988, 989; ( 7 ) 79 Watanabe, PI., ( 8 ) 394 Watanabe, S . , ( 8 ) 743 Watanabe, T., ( 8 ) 469 W a t e r f i e l d , M.D., ( 5 ) 449 Watkins, P., ( 6 ) 100 (5) Watkins, W . M . , 1033, 1073 (8) Watterson, D.M., 349 W a t t s , G.H., ( 3 ) 159 Waxman, D.J., ( 4 ) 26 Weatherhead, J.C., ( 5 )
Carbohydrate Chemistry
818 481 Weaver, J . , ( 3 ) 225 Weber, G., (8) 453 Weber, P., ( 5 ) 926, 1014 W e b s t e r , G.F., ( 4 ) 242 W e b s t e r , J . , (8) 173 W e b s t e r , R., ( 5 ) 46 W e b s t e r , R.G., (5) 10 Weckesser, J . , ( 4 ) 81, 82, 89 Wedlock, D., ( 3 ) 134 Weerkamp, A.H., (4) 169 Weetall, H.H., ( 8 ) 100 Wehler, C . , (8) 568 Wehring, B., ( 5 ) 652 Weigand, K., (5) 750 W e i g e l , H., ( 3 ) 295; (6) 90 W e i g e l , P.H., ( 5 ) 555 Weiland, E . , (2) 58 Weiland, R.H., ( 8 ) 682 Weinberg, J . B . , (4) 90 Weiner, F., ( 3 ) 110 Weinhold, A.R., (3) 240 W e i n s t e i n , H.G., (5) 2 97 W e i n s t e i n , M., (5) 781 W e i n s t e i n , W.M., (5) 864 W e i n t r a u b , A., ( 4 ) 5 7 W e i n t r o u b , H., (5) 650 Weir, D.M., ( 5 ) 637 Weiss, J . B . , (5) 263 Weiss, R.A., ( 5 ) 2 1 Weiss, R.E., (5) 211: ( 8 ) 265 W e i s s b a c h , H . , ( 8 ) 133 Weissman, I . L . , (5) 601 Weissmann, B . , ( 5 ) 353. 354: ( 8 ) 13 Weissmann, G., ( 5 ) 241 Weitzman, S., (8) 48 Wellems, T., ( 8 ) 433 W e l l n i t z , D . , (8) 384 Wells, C . , ( 5 ) 747 Wells, G.B., (2) 47 Wells, S . A . , ( 5 ) 866 Welply, J . K . I (6) 150, 151 Wember, M., ( 5 ) 1075; (8) 663 Wendelschafer-Crabb, G. ( 5 ) 242 Wenger, D.A., ( 6 ) 248 Wenzl, S., ( 5 ) 1002 Wepsic, H.T., ( 5 ) 867: ( 8 ) 184 Werb, Z., ( 5 ) 540; ( 8 ) 233 Wessels, J . G . H . , (4) 215
West, C . A . , ( 3 ) 241, 242; (6) 452, 453 Westaway, E.G., ( 5 ) 66 W e s t e r d u i n , P., (8) 78 Westfelt, L . , ( 8 ) 485 Weston, P.D., (8) 567 W e s t p h a l , O., ( 4 ) 5 2 W e t e r i n g s , P. J. J.M., ( 8 ) 162 Wetsel, R.A., ( 8 ) 188 Wexler, H . , (4) 68 W h a t l e y , M.H., ( 4 ) 138 Whish, W . J . D . , (8) 417 W h i s l e r , R . L . , ( 7 ) 37 W h i t a k e r , A . , ( 6 ) 322 W h i t e , A.R., ( 3 ) 116, 130: (4) 175; (6) 339 W h i t e , C . A . , ( 8 ) 82 White C . J . B . , (5) 456, 457, 458, 464 White, D.A., (5) 726; ( 7 ) 154 W h i t e , D.O., ( 5 ) 41 White, G.C., (5) 653 W h i t e , J.G., ( 5 ) 509 White, R.C., ( 6 ) 330: ( 8 ) 114 White, R.J., ( 5 ) 439 White, W.E., (6) 65 W h i t f i e l d , C., ( 4 ) 172 W h i t i n g , P.H., (6) 23 W h i t n e r , V.S., ( 6 ) 287 Whittaker, N.F., (5) 9 92 Whyte, J.N.C., ( 3 ) 263 W i a t r o s z a k , I . , (3) 55; ( 6 ) 324 Wichmann, R., ( 6 ) 188 Wicken, A . , (4) 19 Wicken, A . J . , ( 4 ) 16 Widdows, D., (6) 72 Wiedemann, H., ( 5 ) 270 W i e d e r s c h a i n , G., Ya., ( 6 ) 97 W i e g a n d t , H., ( 7 ) 60, 80 W i e g a n t , J., ( 8 ) 367 W i e g a n t , V.M., (7) 30 W i e l a n d , F., ( 4 ) 184 Wieland, O.H., (5) 798, 808 W i e s l a n d e r , A., ( 7 ) 189, 190 W i e s l a n d e r J . , ( 5 ) 414 Wiesmann, U.N., (7) 143 W i j d e n e s , J . , ( 8 ) 175, 377, 388 W i k v a l l , K., ( 8 ) 214 Wilchek, M., (7) 43; ( 8 ) 155, 412 Wilcox, G.E., (5) 604 W i l e y , D.C., ( 5 ) 38, 39
Wilgns, H . , ( 8 ) 139 W i l k e , C . R . , ( 6 ) 208, 334 W i l k e s , C.M., ( 5 ) 240 W i l k i n s , R.G., (5) 146 W i l k i n s o n , A . E . , ( 4 ) 37 W i l k i n s o n , S.G., ( 4 ) 77, 79 Williams, A . F . , ( 5 ) 593, 594, 606 Williams, D., ( 5 ) 938, 939; (8) 630 Williams, D.C., ( 5 ) 509 Williams, J.P., ( 7 ) 173 Williams, L . , ( 5 ) 762, 763 Williams, R.E., ( 8 ) 634 Williams, T . J . , ( 5 ) 134, 139 W i l l i a m s , W.P., ( 7 ) 11, 86, 171 Williamson, F.B., (3) 274: (5) 758 W i l l i n g , R.I., ( 7 ) 166 Wilson, I.A., ( 5 ) 38, 39 Wilson, I.B., ( 8 ) 319, 447 W i l s o n , J . , ( 5 ) 780 W i l s o n , J.M., ( 8 ) 267 W i l s o n , W.W., ( 3 ) 114 W i m s , L.A., (5) 887 W i n c h e s t e r , B.G., (5) 975; (6) 242 Wind, M.L., ( 5 ) 215 Wing, R.E., (3) 75 Wingard, L.B., ( 8 ) 687 Winnick, F.M., (5) 983 W i n q u i s t , F., ( 8 ) 669 W i n s t a n l e y , M.A., ( 8 ) 209 Winterbourne, D.J., (5) 3 82 Wintermans, J.F.G.M., ( 7 ) 179, 180 Wirtz, G.H., ( 5 ) 792; (8) 279 Wisdom, G.B., ( 5 ) 819, 87 1 Wiseman, G., ( 4 ) 2 Wisser, H., (6) 24 Witmer, R.R., ( 8 ) 373 W i t t , I., (5) 750 Wittwer, A . J . , ( 8 ) 325 W o i s e t s c h l a g e r , M., ( 4 ) 67 W o j c i e s z y n , J., ( 7 ) 92 Wojcik, J.F., ( 8 ) 521 Wold, F., ( 5 ) 160; ( 6 ) 295: (8) 207 Wold, W.S.M., ( 5 ) 23, 25
819
Author Index Wolf, G . , ( 7 ) 135 Wolf, H . , (5) 69 Wolfe, S., ( 6 ) 251 Wolken, K.W., (7) 33, 39 Wollenweber, H.W., (4) 57 Wollin, R . , ( 4 ) 60 Wolter, K.E., (6) 533 Wong, H . A . , ( 3 ) 296 Wong, K.Y., (5) 187 Wong, R . , ( 5 ) 466; ( 7 ) 146 Wong, T . C . , ( 5 ) 852 Wong, T.Y., (7) 55 Wong, W . , ( 4 ) 49 Woo, P.W.K., (5) 178 Wood, C . , ( 7 ) 156, 157, 158: (8) 558 Wood, C . N . , ( 5 ) 783, 868 Wood, D . A . , ( 6 ) 530 Wood, D . L . , (7) 7 Wood, E., ( 5 ) 948, 949; (8) 186 Wood, G . C . , ( 8 ) 345 Wood, H.G., (8) 324 Wood, M . R . G . , ( 8 ) 439 Wood, P.J., (2) 116: ( 3 ) 152: ( 6 ) 390 Wood, S.W., (8) 333 Wood, T., ( 8 ) 390 Woods, A . , (4) 138 Woods, D . R . , ( 6 ) 225 Woodward, J . , (6) 214 Woodward, R.W., ( 3 ) 225 Wootton, M., (3) 42, 43, 44 Wouters, J., ( 8 ) 238 Wouters, J . T . M . , (4) 7 Wouters, T . M . , ( 4 ) 27 Wrigglesworth, R . , ( 8 ) 567 Wright, C . D . , ( 4 ) 221 Wright, S.E., (5) 76, 77 Wriston, J . C . , ( 8 ) 64 Wu, A . M . , (5) 166, 176 Wu, H . L . , ( 8 ) 159 Wu, K . , (5) 732 Wu, P.S., ( 7 ) 152 wu, V.Y., (5) 373 Wunner, W.J., ( 5 ) 75 Wusteman, F.S., (6) 511 Wyllie, R . G . , ( 5 ) 149 Wyn-Jones, E., (3) 134 Wynn, C . H . , ( 6 ) 121, 127: (8) 242 Yabuuchi, H . , ( 5 ) 977 Yadomae, T., (4) 240 Yagi, K., ( 6 ) 123, 494
Yagi, Y . , (5) 1006: ( 8 ) 500 Y a g i s h i t a , K . , ( 6 ) 427 Yago, N . , ( 6 ) 130 Yahara, S., ( 7 ) 120 Yajima, H . , (3) 10; ( 8 ) 461 Yaku, F., ( 3 ) 118 Yalpani, M., (2) 88; ( 8 ) 506, 507 Yalpani, N . , (8) 506 Yamada, H . , ( 6 ) 436; (8) 499, 661 Yamada, J., ( 6 ) 456, 457, 458 Yamada, K., ( 3 ) 39 Yamada, K.M., (5) 229, 231; ( 8 ) 210 Yamada, M . , (7) 63 Yamada, T., (51 978 Yamada, Y., (6) 268 Yamaguchi, H . , ( 8 ) 4 Yamaguchi, R . , (8) 510 Yamakawa, T., ( 7 ) 145 Yamamoto, K . , (5) 86, 180, 912; ( 8 ) 623 Yamamoto, K.T., ( 8 ) 110 Yamamoto, R . , ( 3 ) 168, 198 Yamamoto, S., ( 8 ) 658 Yamamoto, T . , (3) 62; (7) 161 Yamanaka, M . , ( 2 ) 68 Yamane, K . , ( 6 ) 311, 317 Yamane, T., ( 8 ) 675 Yamanobe, T., ( 3 ) 54 Yamasaki, Y . , ( 3 ) 218, 236; (6) 177, 178 Yamashina, I., ( 5 ) 379, 430, 562: (6) 531: ( 8 ) 281 Yamashiro, S., ( 6 ) 68 Yamashita, J . , (8) 258 Yamashita, K., ( 5 1 977: (6) 28, 71, 285; ( 8 ) 258 Yamauchi, H . , ( 6 ) 313 Yamauchi, R . , ( 6 ) 179; ( 8 ) 553 Yamazaki, R., ( 8 ) 382 Yamazaki, T., (8) 555 Yamazoe, Y . , ( 8 ) 121 Yamin, M . A . , (3) 133 Yamskov, I . A . , ( 8 ) 691 Yanase, Y . , (5) 516; ( 8 ) 128 Yang, B . H . , ( 8 ) 582 Yang, D . C . H . , (5) 196, 197 Yang, M . D . , ( 8 ) 156 Yang, M . T . , (2) 46
Yang, K . , ( 8 ) 284 Yang, Y., ( 6 ) 438, 439 Yankofsky, S . A . , ( 8 ) 470 Yano, T., ( 8 ) 658 Yanulaitene, K.K., (6) 183 Yarmolenko, V. V. , ( 5 ) 712 Yasato, T., ( 8 ) 33 Yasuda, M . , ( 3 ) 19 Yasui, T., ( 6 ) 473, 474 Yasumatsu, K . , (8) 524 Yates, A . D . , ( 5 ) 1033, 1073 Yates, A . J . , ( 7 ) 37 Yavin, 2.. (5) 468 Yazawa, I., ( 6 ) 176 Yellowlees, D . , ( 6 ) 375 Yep, J.M., (5') 553 Y e t t e r , M . A . , ( 6 ) 275 Yeung, E.S., ( 2 ) 50; (5) 971 Yewdell, J., ( 5 ) 46 Y i , C . K . , (6) 44 Yoda, K . , ( 8 ) 723 Yoder, J . M . , (5) 766 Yohe, H . C . , ( 7 ) 54 Yokogawa, K . , (6) 379 Yokoi, M., ( 3 ) 253 Yokoi, T., (5) 431 Yokota, M . , ( 3 ) 62 Yokoyama, S., ( 5 ) 486 Yon, R.J., ( 8 ) 144 Yoneda, T., ( 7 ) 144 Yonehara, S., ( 5 ) 516; ( 8 ) 128 Yonemasu, K., (5 793 Yonemitsu, O., ( 5 ) 148; ( 8 ) 533 Yonezawa, Y . , ( 8 ) 523 Yoshe, H . C . , ( 7 ) 14 Yoshida, A . , ( 5 ) 911, 1062; (6) 504; (8) 273 Yoshida, D . , ( 8 ) 463 Yoshida, H . , ( 5 ) 430: ( 6 ) 204; ( 8 ) 331 Yoshida, I., (5) 681, ( 8 ) 187 Yoshida, K., ( 2 ) 40; (3) 156 Yoshida, M . , ( 5 ) 80 Yoshida, N., (8) 386 Yoshida, T., (8) 607 Yoshii, F . , (8) 609, 61 0 Yoshikawa, A . , ( 2 ) 13 Yoshikawa, J., (8) 555 Yoshikawa, M . , ( 5 ) 724 Yoshikawa, S., ( 3 ) 280 Yoshikura, H . , ( 5 ) 80
Carbohydrate Chemistry
820 Yoshima, H . , (5) 53, 542; ( 6 ) 7 1 Yoshino, H . , ( 8 ) 477, 686, 731 Yoshioka, H., ( 6 48, 358. 468 Yoshioka, K . , ( 6 ) 270 Y o s h i t o m i , H., (8) 565 Yosizawa, Z., ( 5 ) 363, 366; ( 6 ) 410: ( 8 ) 544, 545, 589 Yost, A., (5) 544 Y o s t , D.A., ( 8 ) 440 You, K.S., (8) 306 Youle, R.J., ( 5 ) 573, 876: (8) 101 Young, J . D . , ( 5 ) 1070 Young, N . M . , (5) 128, 129 Young, W.W., ( 5 ) 859. 861, 862: (7) 150, 151 Youngs, C.G., (3) 26 Yu, N . X . , (4) 151 Yu, R.K., ( 7 ) 14, 54, 72 Yu, W., ( 4 ) 26 Yung, S . C . , (4) 157 Yusuf, H.K.M., ( 7 ) 53 Z a b i e l s k i , J . , (5) 24 Z a b i n , I., ( 6 ) 146, 150, 151 Z a b l o t s k a y a , A.E., ( 7 ) 47 Z a c a r y , A.L., ( 4 ) 98 Z a d r a l , M., (8) 67 Zahn, R.K., ( 5 ) 202 Zalc, B., (7) 97, 159 Z a l i t e , O.M., ( 5 ) 222 Zancan, G.T., (6) 315 Z a n e t t a , J.P., ( 5 ) 448: (7) 52: (8) 256 Z a n i , B., ( 5 ) 526 Z a n j a n i , E.D., (8) 375 Zanlungo, A.B., ( 3 ) 291 Z b a s z y n i a k , B., (3) 55; ( 6 ) 324 Z e i k u s , J.G., ( 3 ) 247; (4) 133: (6) 207, 366, 688 Z e i k u s , T., (6) 386 Z e i g l e r , M., ( 6 ) 247 Z e l t n e r , J.Y., (4) 125 Zeman, Z . , (5) 478 Zemanova, I., (8) 584 Zemek, J . , ( 8 ) 497, 541 Z e t u s k y , W. J . , (7) 95 Z e z i n , A.B., ( 8 ) 734 Zhang, W.-J., (4) 230; ( 5 ) 6, 7 Zhdanov, V . M . , ( 5 ) 27
Ziderman, I., (3) 101 Z i d o v e t z k i , R., ( 5 ) 885 Z i e g l e r , D., ( 7 ) 15 Z i g l e r , J.S., ( 7 ) 100 Z i l b e r s t e i n , A . , (5) 11 Z i l v e r s m i t , D.B., ( 7 ) 46 Z i n g a r o , R.A., (6) 330 Z i s k a , P., (5) 112, 167, 205 Zissis, E., (8) 36 Zmachinski, C., ( 7 ) 95 Z o l l i n g e r , W.D., (4) 110 Zopf, D.A., ( 8 ) 326 Z o p p e t t i , C . , (5) 369 Zsadon, B., ( 8 ) 519 Zugenrnaier, P., ( 3 ) 4: ( 8 ) 460 Z u r a w s k i , V.R., ( 5 ) 894 Zusman, D.R., (5) 203, 204 Z v e z d i n a , N.D., ( 7 ) 76 Zweibaum, A . , ( 6 ) 163 Zwez, W., ( 2 ) 73
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