Carbohydrate Sulfates - American Chemical Society

8 Enzymatic Formation and Hydrolysis of Polysaccharide Sulfates

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K A L Y A N K. DE, K A Z U H I K O Y A M A M O T O , and R O Y L . W H I S T L E R Department of Biochemistry, Purdue University, Lafayette, I N 47907

Naturally occuring polysaccharide sulfate esters are widely distributed. These high molecular weight anionic polymers possess rheological and complexing properties that have drawn the attention of industrial interests and lead to extensive commerciall i z a t i o n of several of the polysaccharides. This, in turn, has influenced fundamental and application research to bring about a better understanding of the behavior of hydrocolloids. The next step i s a rational design and development of polysaccharide sulfates to more perfectly serve practical needs. The great bulk of natural occurring polysaccharide sulfates are found in seaweeds where they serve structural functions and possibly act as ion exchange agents and natural absorbents to hold large quantities of water for proper functioning of the sea plant. While normally water soluble, the polysaccharides are retarded from escape into the enveloping sea by the matrix character of the plant c e l l w a l l . Man has extracted these polymers and made use of their highly viscous nature and their gel forming properties. In addition, use i s made of their unique characteristic of combining with protein to produce complexes such as the useful one between carrageenan and the protein in chocolate to maintain suspension uniformity in chocolate milk. Other sulfated polysaccharides are widely distributed in animal tissues where they again serve a water holding use, provide emolliency, l u b r i c i t y and complexing characteristics. Heparin serves a special function in the control of blood coagulation. It can be expected that sulfated, and perhaps phosphorylated polysaccharides, w i l l develop greater pharmaceutical and industrial applications. Applications would be f a c i l i t a t e d by finding techniques by which sulfate-groups could be inserted into and removed from polysaccharides by expectedly low cost and surely the more specific means provided by enzymes. The present review is a summary of the existing knowledge of enzymes that transfer sulfate to ester positions in polysaccharides and of those 0-8412-0426-8/78/47-077-121$06.75/0 ©

1978 A m e r i c a n C h e m i c a l Society

In Carbohydrate Sulfates; Schweiger, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

CARBOHYDRATE SULFATES


122

enzymes t h a t c a t a l y z e s u l f a t e e s t e r h y d r o l y s i s . Although l i t t l e exact i n f o r m a t i o n i s p r e s e n t l y a v a i l a b l e , the e x c e l l e n t work so f a r done points to the l i k e l i h o o d of r a p i d f u r t h e r development. With proper a p p l i c a t i o n of e v o l v i n g i n f o r m a t i o n , p r a c t i c a l use may w e l l be made of s u l f a t e e s t e r forming and h y d r o l y z i n g enzymes. In the enzymatic formation of s u l f a t e e s t e r s , the s u l f a t e group i s t r a n s f e r r e d from a donor s u b s t r a t e to a polysaccharide acceptor. Only three donor substrates have been i d e n t i f i e d . Ascorbate 2 - 0 - s u l f a t e and adenine 3'-0^-phosphate-5'-0-phosphos u l f a t e are two n a t u r a l donors and p-nitrophenyl s u l f a t e i s a s y n t h e t i c donor. A d d i t i o n a l s y n t h e t i c donors may soon be designated. S u l f a t e t r a n s f e r from j j - n i t r o p h e n y l s u l f a t e and from ascorbate 2 - 0 - s u l f a t e r e q u i r e the presence of ATP as a c o f a c t o r and, hence, adenine 3'-0-phosphate-5'-0-phosphosulfate (PAPS) may be the u n i v e r s a l designate intermediate and s o l e donor f o r a l l s u l f a t e t r a n s f e r a s e s . When other donors are present they presumably i n t e r a c t with ATP through the c a t a l y t i c i n f l u e n c e of two a d d i t i o n a l enzymes to provide PAPS. S u l f a t a s e s capable of h y d r o l y z i n g the h a l f - e s t e r s u l f a t e l i n k a g e to carbohydrates have been observed s i n c e 1931. The group o f esterases t h a t hydrolyze s u l f a t e linkages i n a v a r i e t y of simple sugar s u l f a t e s are termed g l y c o s u l f a t a s e s (sugar s u l f a t e s u l f o h y d r o l a s e s Ε C. 3.1.6.3). S u l f a t a s e s capable of h y d r o l y z i n g s u l f a t e linkages i n polysaccharide s u l f a t e s are named a f t e r t h e i r s u b s t r a t e , as f o r example, chondrosulfatases hydrolyze c h o n d r o i t i n s u l f a t e s , and c e l l u l o s e s u l f a t a s e hydrolyzes c e l l u l o s e s u l f a t e s . The s u l f a t a s e s may be completely s u b s t r a t e s p e c i f i c even as to l o c a t i o n of the s u l f a t e group, but some are not f u l l y s u b s t r a t e s p e c i f i c since apparently a s i n g l e enzyme can hydrolyze both charonin (charonan) s u l f a t e and c e l l u l o s e s u l f a t e . A l l s u l f a t a s e s are a b s o l u t e l y s p e c i f i c f o r the s u l f a t e group. The enzymes are obtained from a v a r i e t y of sources such as m i c r o b i a l , molluscan and higher animals. None have been observed i n higher p l a n t s . Enzymatic S u l f a t i o n Enzymatic s u l f a t i o n of polysaccharide i s u s u a l l y e f f e c t e d through the t r a n s f e r of s u l f a t e from a d e n i n e - 3 ' - p h o s p h o - 5 ' phosphosulfate (3'-PAPS) i n a r e a c t i o n c a t a l y z e d by s u l f o t r a n s f e r ase [E C. 2.8.2] as shown o r i g i n a l l y by F. D'Abramo and F. Lipmann (1_). The process of s u l f a t i o n w i t h i n o r g a n i c s u l f a t e proceeds i n several s t e p s . F i r s t i s the a c t i v a t i o n of s u l f a t e to form adenine-5'-phosphosulfate. This i s immediately f o l l o w e d by phosphorylation at the 3 ' - p o s i t i o n to produce adenine 3'-phospho5 ' - p h o s p h o s u l f a t e , f o l l o w e d by the t r a n s f e r of s u l f a t e from 3'-PAPS to the a c c e p t o r , c a t a l y z e d by s u l f o t r a n s f e r a s e .


8.

DE E T A L .

Polysaccharide

A c t i v a t i o n s t e p s , SO^

Sulfates

123

+ ATP

AMP-S0=

> AMP-S0 ~ + p p . " 3

+ ATP

»

3 ' - P 0 - A M P - S 0 ~ + ADP 3

3

T r a n s f e r r i n g s t e p , ROH + 3 -P0~-AMP-S0 "" (R-NH ) ,

o

6

3

2


>

R 0 - S 0 " + 3'-P0 -AMP 3

3

(RNH-S0 ') 3

S u ! f o t r a n s f e r a s e s are of common occurance i n animal t i s s u e s t h a t c o n t a i n aminoglycans and i n p l a n t t i s s u e s , e s p e c i a l l y a l g a l c e l l s , t h a t are major producers of s u l f a t e d p o l y s a c c h a r i d e s . However, evidence f o r the mechanism of the enzymatic r e a c t i o n and c h a r a c t e r i z a t i o n of the enzyme i s incomplete due to d i f f i c u l t i e s i n o b t a i n i n g homogeneous enzyme p r e p a r a t i o n s . Enzyme preparations from c h i c k embrionic c a r t i l a g e ( l _ - 3 h hen o v i d u c t ( 4 ) , hen uterus ( 5 J , r a b b i t uterus ( 6 J , beef lung ( 7 ) , beef eyes ( 8 ) , r a t b r a i n ( 9 J , mouse l i v e r (1_0) serum (11), mast c e l l tumor Τ]_2-1_4), squid c a r t i l a g e (15), molluscus (ΙβΤΤ and marine gastropod (1_7) have been reported to c a t a l y z e s u l f a t i o n of exogeneous and endogeneous a c c e p t o r s . Occurrence of s u l f o t r a n s f e r a s e and 3'-PAPS as s u l f a t e donors (18) are i n d i c a t e d i n red algae Chondrus c r i s p u s (19), Porphyridium (20, 21_) and i n the brown a l g a e , Fucus (22-25). Experiments w i t h i n c o r p o r a t i o n of r a d i o a c t i v e s u l f a t e i n t o carrageenan of Chondrus (19) and i n t o c a p s u l a r p o l y s a c c h a r i d e of Porphyridium (20) demonstrate that the i n c o r p o r a t i o n i s a remarkably r a p i d process through 3'-PAPS as a s u l f a t e p o o l . In Porphyridium 3'-PAPS was a c t u a l l y i s o l a t e d as a water s o l u b l e intermediate ( 2]_). The r a p i d i t y w i t h which s u l f a t e i n c o r p o r a t i o n takes place seems to f a v o r the idea t h a t the s u l f a t i o n of a preformed polymer occurs. During e a r l y embryogenesis of Fucus, exogeneous r a d i o a c t i v e s u l f a t e can be incorporated i n t o an a c i d - s o l u b l e fucose polymer w i t h i n 10 hr. a f t e r f e r t i l i z a t i o n . Experimental evidence i n d i c a t e s t h a t enzymatic s u l f a t i o n occurs i n the region where fucan i s formed and where i t can immediately act as an acceptor (25J). Although several attempts have been made to o b t a i n from these a l g a e , c e l l - f r e e enzyme system which can t r a n s f e r the s u l f a t e from 3'-PAPS to f r e e sugar, sugar n u c l e o t i d e , or p o l y s a c c h a r i d e , no p o s i t i v e r e s u l t s have y e t been seen. L i t t l e i s known of the s p e c i f i c i t y of s u l f o t r a n s f e r a s e and i t i s not e s t a b l i s h e d whether there i s a s i n g l e n o n - s p e c i f i c s u l f o t r a n s f e r a s e or a number of more s p e c i f i c enzymes. The l a t t e r case becomes more l i k e l y as the enzyme systems are i n v e s t i g a t e d in greater d e t a i l . Results of the e a r l i e r work on the s p e c i f i c i t y of the crude enzyme p r e p a r a t i o n are summarized i n Table I. 9



124

Table I


Tissue

Acceptor s p e c i f i c i t y of some crude preparations of mucopolysaccharide s u l p h o t r a n s f e r a s e s P o t e n t i a l Acceptors Utilized Not U t i l i z e d Reference

rabbit skin hen o v i d u c t chick cartilage human leimyosarcoma human mammary carcinoma

dermatan sulphate h e p a r i t i n sulphate c h o n d r o i t i n 4- and 6-sulphates dermatan sulphate dermatan c h o n d r o i t i n 4sulphate and 6-sulphates chondroitin, c h o n d r o i t i n 4and 6 - s u l p h a t e s , dermatan s u l p h a t e , h e p a r i t i n sulphate

26 27 28 29

30

More d e t a i l e d i n v e s t i g a t i o n s on the s p e c i f i c i t y of s u l f o t r a n s f e r a s e have been conducted i n an enzyme system s o l u b i l i z e d from hen o v i d u c t ( 4 J . This c e l l - f r e e system i s capable of c a t a l y z i n g the t r a n s f e r of r a d i o a c t i v e s u l f a t e from 3'-PAPS to a number of aminoglycans. The r e l a t i v e rates of t r a n s f e r are shown i n Table II. Further experiments of the system show t h a t simple 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 N-acetyl-D-galactosamine can a l s o a c t as acceptors f o r the s u l f o t r a n s f e r a s e although the o l i g o s a c c h a r i d e s are r a t h e r l e s s e f f i c i e n t than the p o l y s a c c h a r i d e s . Both mono- and d i s u l f a t e d d e r i v a t i v e s of N - a c e t y l - D - g a l a c t o samine residues i n the o l i g o s a c c h a r i d e s are formed i n the r e a c t i o n (31, 32). Measurements of r e a c t i o n v e l o c i t y at d i f f e r e n t c o n c e n t r a t i o n s show that the V and the Michael i s constant (K^) d i f f e r among carbohydrate s u b s t r a t e s . The higher V for c h o n d r o i t i n 6 - s u l f a t e as an acceptor compared to c h o n a r o i t i n 4 - s u l f a t e i s i n agreement w i t h the data shown i n Table II.


8.

DE E T A L .

Polysaccharide

Sulfates

Table

125

II

R e l a t i v e extents of sulphate t r a n s f e r to d i f f e r e n t acceptors by the amino glycan sulphotransferases of hen oviduct and of chick embryo c a r t i l a g e


Acceptor c h o n d r o i t i n 4-sulphate c h o n d r o i t i n 6-sulphate dermatan sulphate h e p a r i t i n sulphate chondroitin (natural) c h o n d r o i t i n (chemical) hyaluronic acid heparin keratosulphate

Oviduct (4)

C a r t i l a g e (2)

1.00 1.8 0.72 0.45 1.8 0.62 0.00 0.00 0.00

1.00 0.46 0.75 1.3

-

0.13 0.08 0.26 0.25

The s u l f o t r a n s f e r a s e system i n the 105000 χ g supernatant f r a c t i o n prepared from the homogenate of c h i c k embryo c a r t i l a g e [2) can use a g r e a t e r number o f polysaccharide acceptors than the oviduct enzyme (Table I I ) . An endogeneous polysaccharide acceptor occurs i n the same enzyme preparation (33). A f t e r d e s u l f a t i o n of t h i s acceptor w i t h methanolic-hydrcgen c h l o r i d e , i t no longer accepts s u l f a t e . S i m i l a r l y , c h o n d r o i t i n prepared by d e s u l f a t i o n of c h o n d r o i t i n 4 - s u l f a t e does not a c t as an acceptor. The a b i l i t y of s u l f a t e acceptors i s l i t t l e i n f l u e n c e d by t h e i r molecular s i z e but i s s t r o n g l y dependent on the charge on the molecules. Treatment of p r o t e i n - p o l y s a c c h a r i d e complex endogeneous acceptors w i t h proteinase or w i t h a l k a l i increase t h e i r a b i l i t y to accept s u l f a t e . However, there i s a s t r i k i n g change i n the type of s u l f a t i o n t h a t r e s u l t s from removal of p r o t e i n . When the aminoglycan i s combined w i t h p r o t e i n s u l f a t e , e s t e r s are formed predominantly a t a x i a l hydroxyl groups. For example, s u l f a t e groups are introduced a t p o s i t i o n 4 of N - a c e t y l D-galactosamine u n i t s . On removal of p r o t e i n , s u l f a t i o n occurs p r i n c i p a l l y a t e q u a t o r i a l or primary hydroxyl groups. One p o s s i b l e e x p l a n a t i o n f o r t h i s i s t h a t although the p r o t e i n p o r t i o n of the endogeneous p r o t e i n - p o l y s a c c h a r i d e complex i s not necessary f o r s u l f a t e i n c o r p o r a t i o n , i t aids i n determining the s i t e s f o r s u l f a t i o n . A s u l f o t r a n s f e r a s e p u r i f i e d from mouse l i v e r homogenate (10) t r a n s f e r s s u l f a t e from 3'-PAPS only to p o s i t i o n C-6 of an N-acetyl-D-galactosamine residue i n c h o n d r o i t i n s u l f a t e and i t s Michael i s constant can be c l o s e l y c o r r e l a t e d to the degree of sulfation. In the s o l u b l e enzyme systems, only a small amount of s u l f a t e i s incorporated i n t o p o l y s a c c h a r i d e . Most of the c h o n d r o i t i n s u l f a t i n g a c t i v i t y i s found to occur as microsomal enzyme r a t h e r than as s o l u b l e enzyme (34,35J. A s u l f o t r a n s f e r a s e


CARBOHYDRATE

126

i n a microsomal preparation from chick embryo c a r t i l a g e t r a n s f e r s a greater amount of s u l f a t e from 3'-PAPS to endogeneous g l y c o saminoglycan than does the s o l u b l e enzyme {3 36). Incubation of the mcirosomal preparation at pH 6.5 with 3'-PAPS r e s u l t s i n the i n c o r p o r a t i o n of s u l f a t e i n t o endogeneous c h o n d r o i t i n 6 - s u l f a t e (60-70%) and i n t o c h o n d r o i t i n 4 - s u l f a t e (30-40%) w h i l e incubation at pH 7.8 r e s u l t s i n t h e . i n c o r p o r a t i o n of s u l f a t e i n t o c h o n d r o i t i n 6 - s u l f a t e e x c l u s i v e l y (37). Although the s u l f o t r a n s f e r a s e a c t i v i t y of chick embryo c a r t i l a g e i s predominantly associated with membrane, a s i g n i f i c a n t proportion of the enzyme a c t i v i t y i s found i n the 105000 χ g supernatant f r a c t i o n (Golgi p r e p a r a t i o n ) . This preparation contains a c t i v i t y f o r 4- and 6 - s u l f a t i o n of endogeneous p o l y s a c c h a r i d e . However, heat treatment of the preparation causes more r a p i d l o s s of a b i l i t y to s u l f a t e p o s i t i o n C-4 of the polysaccharide than l o s s of a b i l i t y to s u l f a t e p o s i t i o n C-6 (38) ( F i g . 1). This suggests t h a t a s p e c i f i c enzyme i s a c t i v e f o r s u l f a t i o n at each p o s i t i o n . Moreover, by chromatography on Sephadex G-200 the enzyme f o r 6 - s u l f a t i o n can be obtained e s s e n t i a l l y f r e e from the a c t i v i t y f o r 4 - s u l f a t i o n . The enzyme e f f e c t i n g 4 - s u l f a t i o n i s not recovered from chroma tography perhaps because of i n a c t i v a t i o n . Thus, w i t h i n the l i m i t e d work on the microsomal preparation and the Golgi p r e p a r a t i o n , the s u l f a t i o n system of chick embryo c a r t i l a g e seems to c o n s i s t of at l e a s t two enzyme species t h a t are h i g h l y s p e c i f i c with regard to the p o s i t i o n s u l f a t e d . An aminoglycan s u l f o t r a n s f e r a s e u t i l i z i n g 3'-PAPS as a s u l f a t e donor and endogeneous p r o t e i n - p o l y s a c c h a r i d e complex as an acceptor has been c h a r a c t e r i z e d (1J5). This enzyme, from squid c a r t i l a g e , shows high s p e c i f i c i t y f o r p o s i t i o n C-6 of N - a c e t y l Q-galactosamine moieties t h a t already bear a s u l f a t e group at p o s i t i o n C-4. The s u l f o t r a n s f e r a s e i s separated from endogeneous p r o t e i n polysaccharide complex and p u r i f i e d some n i n e - f o l d on DEAE-Sephadex A-50. The enzyme i s a c t i v e only w i t h exogeneous acceptors. Although various mono-, d i - , and polysaccharide are s u l f a t e a c c e p t o r s , i t i s e s s e n t i a l t h a t they have a s u l f a t e at p o s i t i o n C-4 of t h e i r j^-acetyl-D-galactosamine r e s i d u e s . The p u r i f i e d enzyme s p e c i f i c a l l y c a t a l y z e s the s u l f a t i o n of l ^ - a c e t y l D-galactosamine 4 - s u l f a t e r e s i d u e s , and produces f^-acetyl-Dgalactosamine 4 , 6 - d i s u l f a t e , whether ^-acetyl-D-galactosamine 4 - s u l f a t e i s i n a p r o t e i n - p o l y s a c c h a r i d e complex or whether i t i s p r o t e i n - f r e e . Most l i k e l y , t h e r e f o r e , the s p e c i f i c i t y of t h i s enzyme does not i n v o l v e the r e c o g n i t i o n of a p a r t i c u l a r s i z e or a p a r t i c u l a r monosaccharide sequence of an aminoglycan acceptor molecule. 9


SULFATES



8.

DE E T AL.

Polysaccharide

Sulfates

127

Molecular and Cellular Biochemistry

Figure 1. Heat labilities of two different sulfotransferase activities in the 105,000χ g supernatant of cartilage ho mogenate (38)




128

Another enzyme has been p u r i f i e d from a homogenate of the albumin s e c r e t i n g region of hen oviduct (39-41). The enzyme c a t a l y z e s the t r a n s f e r of s u l f a t e from 3'-PAPS to p o s i t i o n C-6 of the N-acetyl-D-galactosamine 4 - s u l f a t e moiety of u r i d i n e diphosphate-N-acetyl-D-galactosamine 4 - s u l f a t e . Although UDP-N-acetyl-D-galactosamine 4 - s u l f a t e i s the most a c t i v e carbohydrate substrate f o r the enzyme, N-acetyl-D-galactosamine 4 - s u l f a t e and i t s 1-phosphate act as a s u l f a t e acceptor at a comparable r a t e and A ' -glucurc!nido-lN-acetyl-D-galactosamine 4 - s u l f a t e [2-acetamido-2-deoxy-3-0-($-D-gluco-4-enepyranosyluronic a c i d ) - 4 - 0 - s u l f o - D - g a l a c t o s e ] and c h o n d r o i t i n accept s u l f a t e s l o w l y . A more completely p u r i f i e d enzyme has a pH optimum f o r s u l f a t e t r a n s f e r to UDP-N-acetyl-D-galactosamine 4 - s u l f a t e at pH 4.8. The k i n e t i c data f o r s u l f a t e t r a n s f e r to several substrate are summarized i n Table I I I . 4

5

Table III S p e c i f i c i t y of a sulphotransferase from hen o v i d u c t which can u t i l i z e simple sugars or sugar d e r i v a t i v e s as substrates (39) —

Acceptor

m ( m M )

UDP-N-acetylgalactosamine 4 - s u l f a t e ^-acetylgalactosamine 4-sulfate ^ - a c e t y l g a l a c t o s a m i n e 1-phosphate 4-sulfate Δ -glucuronido-N-acetylgagactosamine 4-sulfate 4

5

y (relative)

0.05 1.4

1.00 0.72

0.13

0.39

2.0

0.05

9

The s u l f o t r a n s f e r a s e s considered so f a r have a l l formed s u l f a t e e s t e r s . However, i n the formation of heparin which contains 0 - s u l f a t e and j f - s u l f a t e , a separate s u l f o t r a n s f e r a s e i s i n v o l v e d . This i s demonstrated by the separate i d e n t i f i c a t i o n of JN- and 0 - s u l f o t r a n s f e r a s e a c t i v i t y i n an enzyme preparation from mouse mastocytoma (14·). Both s u l f o t r a n s f erases can be s o l u b i l i z e d from mastocytoma microsomal f r a c t i o n . The pH optimum f o r the enzymes i s about 7.5. The Michael i s constant f o r 3'-PAPS i s estimated to be 2 χ 10" M f o r the N - s u l f o t r a n s f e r ase and 1 χ 1 0 " f o r the 0 - s u l f o t r a n s f e r a s e . The 0 - s u l f o t r a n s ferase i s more s e n s i t i v e to h e a t - i n a c t i v a t i o n ; 60% of the a c t i v i t y being l o s t a f t e r 1 min. at 50°. Under the same c o n d i t i o n s only 15% of N - s u l f o t r a n s f e r a s e i s l o s t . The ^ - s u l f o t r a n s f e r a s e i s s e l e c t i v e l y i n h i b i t e d by sodium c h l o r i d e , whereas, the 0 - s u l f o t r a n s f e r a s e i s e s s e n t i a l l y u n a f f e c t e d . Work on the acceptor s p e c i f i c i t y of the 0 - s u l f o t r a n s f e r a s e from mouse mastocytoma shows t h a t N - s u l f a t e groups i n acceptor polysaccharides are e s s e n t i a l f o r 0 - s u l f a t i o n . The 0 - s u l f a t e group i s 5

4


8.

DE E T A L .

Polysaccharide

Sulfates

129


p r e f e r e n t i a l l y introduced i n t o the N^-sulfated (and thus p r e v i o u s l y l^-deacetylated) r a t h e r than i n t o N-acetylated regions at heparan s u l f a t e (42,43). Almost a l l work on s u l f o t r a n s f e r a s e has been done on t i s s u e s i n which s u l f a t i o n of aminoglycan occurs. L i t t l e work has been done on the mechanism of s u l f a t i o n of n i t r o g e n - f r e e glycans. However, a s u l f o t r a n s f e r a s e from the mucous gland e x t r a c t s of the marine gastropod, charonia lampus, c a t a l y z e s the t r a n s f e r of s u l f a t e from 3'-PAPS to the glucan p o l y s u l f a t e , charonan s u l f u r i c a c i d (17). Charonan s u l f u r i c a c i d c o n s i s t s of two f r a c t i o n s , one a s u l f u r - p o o r f r a c t i o n w i t h a g l y c o g e n - l i k e s t r u c t u r e and the other a s u l f u r - r i c h f r a c t i o n w i t h a c e l l u l o s e l i k e s t r u c t u r e (44,45). When S i s used i n the donor, more than 95% of the a c t i v i t y i s incorporated to the s u l f u r - r i c h fraction. 3 2

In a d d i t i o n to 3'-PAPS, other s u l f a t e donors e x i s t . One i s j j - n i t r o p h e n y l s u l f a t e , an e f f e c t i v e s u l f a t e donor i n the s u l f o t r a n s f e r a s e preparation of beef cornea e p i t h e l i c a l e x t r a c t . Here, the s u l f a t e group i s t r a n s f e r r e d from j j - n i t r o p h e n y l s u l f a t e to polysaccharide i n the presence of adenosine 3 ' , 5 ' - d i p h o s p h a t e (56). The s u l f a t i o n of polysaccharide seems to occur by p o l y saccharide s u l f o t r a n s f e r a s e coupling w i t h phenol s u l f o t r a n s f e r a s e or phenol s u l f a t a s e which i s a l s o present i n the corneal e x t r a c t . Another donor i s ascorbate 2 - s u l f a t e (47). When r a d i o a c t i v e s u l f a t e i s used, the s u l f a t e i s found incorporated i n t o c h o n d r o i t i n s u l f a t e i n c h i c k embryo c a r t i l a g e epiphyses. Enzymatic D e s u l f a t i o n Glycosulfatasewas observed f i r s t i n the d i g e s t i v e organs of the t r o p i c a l marine mollusc Charonia lampas (48-52). Subsequently, the enzymewas observed (53-63) i n e x t r a c t s of the d i g e s t i v e organs of marine molluscs from B r i t i s h waters, i n the l a r g e p e r i w i n k l e L i t t o r i n a l i t t o r e a and the common limpet P a t e l l a v u l g a t a . I t i s a l s o present i n the mould Trichoderma v i r i d e ( 6 4 ) , and the b a c t e r i a Pseudomonas carrageenovora (65). A s i m i l a r g l y c o s u l f a t a s e i s observed i n the isthmus region of the hen o v i d u c t (95). From the l i v e r of the marine gastropod Charonia lampas ( T r i t o n a l i a s a u l i a e ) there can be obtained a c e l l u l o s e p o l y s u l f a t a s e (66-68) and a chondrosulfatase (79). A porphyran s u l f a t a s e can be prepared from the algae Porphyra u m b i l i c a l i s (69-72). Chondrosulfatase i s observed a l s o i n p u t r i f a c t i v e b a c t e r i a (73-75), Proteus v u l g a r i s (76,77) and Flavobacterium heparinum (78). In a d d i t i o n to occurance i n the l i v e r of (λ lampas, the enzyme i s found i n the l i v e r of squid Ommastrephes s l o a n i



130

p a c i f i c u s (81-83), i n r a t l i v e r lysosomes (84), i n bovine aorta (85) and the v i s c e r a of P a t e l l a vulgata (80).


Two kerato s u l f a t a s e s are present i n £. lampus (86). A heparin s u l f a t a s e has been i s o l a t e d from Flavobacterium heparinum (87). Cerebroside s u l f a t a s e can be i s o l a t e d from the d i g e s t i v e gland of abalone, H a l i o t i s (88) and the lysosomes of pig kidney and from other organs as l i v e r and spleen (89-91). Glycosulfatases The g l y c o s u l f a t a s e s ( s u g a r - s u l f a t e s u l f o h y d r o l a s e s , E.C.3.1.6.3) capable of h y d r o l y z i n g e s t e r s u l f a t e linkages i n a v a r i e t y of mono-, d i - , and t r i - s u l f a t e s u b s t i t u t e d monosaccharides and d i s a c c h a r i d e s have been p a r t i a l l y p u r i f i e d and i s o l a t e d by a number of d i f f e r e n t groups from a v a r i e t y of d i f f e r e n t sources. Most of the procedures are r a t h e r long and i n v o l v e d . Several are given as examples. Example 1. Washed and macerated v i s c e r e a l organs of L i t t o r i n a ( 5 7 j are placed i n volumes of acetone at 0 ° , f i l t e r e d , macerated i n f r e s h acetone and thoroughly washed i n acetone. The residue i s suspended i n c o l d water, homogenized at pH 9 and adjusted to pH 7 f o r 15 minutes, c e n t r i f u g e d and the c l e a r supernatant d i a l y z e d and the d i a l y z a t e adjusted to pH 2.3 f o r 2 min and readjusted to 4.6, c e n t r i f u g e d , d i a l y z e d , adjusted to pH 6.7, r i b o n u c l e i c a c i d and salmon roe protamine s u l f a t e added to complete p r e c i p i t a t i o n . A f t e r c e n t r i f u g a t i o n the supernatant i s d i a l y z e d , c e n t r i f u g e d and the supernatant adjusted to pH 8, c e n t r i f u g e d , d i a l y z e d and at pH 5.7 t r e a t e d w i t h ammonium s u l f a t e to 55% of s a t u r a t i o n . A f t e r c e n t r i f u g a t i o n the supernatant at pH 4.9 i s 85% saturated w i t h ammonium s u l f a t e . The p r e c i p i t a t e i s removed by c e n t r i f u g a t i o n and d i a l y z e d . The f i n a l product has a high c o n c e n t r a t i o n of g l y c o s u l f a t a s e as w e l l as a high content of a r y l s u l f a t a s e . The p u r i f i c a t i o n procedure r e s u l t s i n the e l i m i n a t i o n of the c h o n d r o i t i n a s e and most of the 3-Ji-acetylglucosaminidase but other s u l f a t a s e enzymes are s t i l l present i n a p p r e c i a b l e amounts. N u c l e i c a c i d c o n c e n t r a t i o n has been c o n s i d e r a b l y decreased and the preparation r e t a i n s a c t i v i t y when stored i n the frozen s t a t e . It is noticed that glycosulfatase a c t i v i t y in L i t t o r i n a v a r i e s c o n s i d e r a b l y w i t h season. R e l a t i v e l y small amounts of the enzyme can be detected i n the w i n t e r months, whereas, organisms c o l l e c t e d i n J u l y provide the most a c t i v e p r e p a r a t i o n . Example 2. Trichoderma v i r i d e (64) i s grown on a medium c o n t a i n i n g the 6 - 0 - s u l f a t e of D-galactose or D-glucose. The


8. DE E T A L .

Polysaccharide

Sulfates

131


washed mycelium i s frozen and ground w i t h alumina a t -15° and e x t r a c t e d with c o l d T r i s s o l u t i o n a t pH 7.9, c e n t r i f u g e d and supernatant s t o r e d . Example 3. Pseudomonas carrageenovora (65) c e l l s are harvested, and c e n t r i f u g e d . The supernatant i s t r e a t e d w i t h streptomycin s u l f a t e to remove n u c l e i c a c i d s , c e n t r i f u g e d and the supernatant i s t r e a t e d w i t h ammonium s u l f a t e . The p r e c i p i t a t e i s d i s s o l v e d i n water and d i a l y z e d . Example 4. The l i v e r of Charonia lampas (94) i s homogenized w i t h sodium c h l o r i d e , c e n t r i f u g e d , and the supernatant d i a l y z e d at pH 3.6 and c e n t r i f u g e d . The supernatant i s then s u c c e s s i v e l y passed through columns of phosphocellulose, Sephadex G-150 and Concanavalin A-Sepharose when p u r i f i e d g l y c o s u l f a t a s e I i s i s o l a t e d . G l y c o s u l f a t a s e I I , which i s s t i l l unseparated from a r y l s u l f a t a s e a t t h i s stage, i s p u r i f i e d by i s o e l e c t r i c focussing. Example 5. Hen o v i d u c t isthmus (95) i s t r e a t e d w i t h T r i s HC1 a t pH 7.2, homogenized, and c e n t r i f u g e d . The supernatant i s t r e a t e d w i t h ammonium s u l f a t e to 40% s a t u r a t i o n , c e n t r i f u g e d , and the supernatant i s t r e a t e d again w i t h ammonium s u l f a t e t o 65% s a t u r a t i o n , and c e n t r i f u g e d . The p r e c i p i t a t e i s d i s s o l v e d i n T r i s - H C l at pH 7.2 and d i a l y z e d . This i s then a p p l i e d to a column of DEAE-cellulose. The pooled f r a c t i o n i s c o n c e n t r a t e d , d i a l y z e d at pH 4.5 and then a p p l i e d to another column of C M - c e l l u l o s e a t pH 4.5. The pooled f r a c t i o n s are concentrated and d i a l y z e d a t same pH. The enzyme r e t a i n e d 95% and 90% of i t s a c t i v i t y f o r 2 and 9 months, r e s p e c t i v e l y , when stored a t - 2 0 ° . Assay of G l y c o s u l f a t a s e s S u l f a t a s e s c a t a l y z e the r e a c t i o n : R-0-S0 " + H 0 — > 3

2

R-O-H + S 0

2 4

' + H

+

Enzyme assay i s based upon measurement of unchanged d e s u l f a t e d r e s i d u e , i n o r g a n i c s u l f a t e or change o f pH.

substrate,

The e a r l i e r i n v e s t i g a t i o n of g l y c o s u l f a t a s e s has employed assay methods f o r i n d i r e c t determination of unhydrolyzed s u b s t r a t e , by g r a v i m e t r i c estimations of i n o r g a n i c s u l f a t e l i b e r a t e d on a c i d h y d r o l y s i s and the measurement o f l i b e r a t e d i n o r g a n i c s u l f a t e by p o t e n t i o m e t r i c t i t r a t i o n (50) or n e p h e l o m e t r i c a l l y (50). These methods are not capable of a high degree of accuracy unless a p p l i e d t o l a r g e q u a n t i t i e s . Methods f o r the assay o f s u l f a t a s e s based on the t u r b i d i m e t r i c determinat i o n of barium s u l f a t e have been described (96,97). C o l o r i m e t r i c methods of s u l f a t a s e assay a l s o have been used ( 9 8 ) , and a s m a l l - s c a l e v e r s i o n of each o f these methods has been devised (99).



132

The method of Dodgson and Spencer (54), i n which p r e c i p i t a t e d benzidine s u l f a t e i s estimated c o l o r i m e t r i c a l l y , although i t i s time-consuming and demands very c a r e f u l m a n i p u l a t i o n , has proved useful f o r a number of s u l f u r i c e s t e r s . A g l u c o s e - o x i d a s e p e r o x i d a s e - O - d i a n i s i d i n e method (100) i s a p p l i c a b l e a l s o .


P r o p e r t i e s of g l y c o s u l f a t a s e s Charonia lampas g l y c o s u l f a t a s e : The e a r l i e s t study of t h i s g l y c o s u l f a t a s e has been made during the years 1931 through 1948 and t h i s work has been p a r t l y reviewed (101). U n f o r t u n a t e l y , the g l y c o s u l f a t a s e used i s not pure. I t shows g r e a t e s t a c t i v i t y a g a i n s t D-glucose 6 - 0 - s u l f a t e and shows a c t i v i t y a l s o a g a i n s t a v a r i e t y of s u l f a t e e s t e r s of monosaccharides and d i s a c c h a r i d e s as w e l l as against adenosine 5 ' - s u l f a t e . Recently p u r i f i e d g l y c o s u l f a t a s e I and II from (λ lampas have been reported as a c t i v e against D-glucose 6 - 0 - s u l f a t e , showing the same K of 25.0 mM, and the same optimum pH of 5.5. m

L i t t o r i n a l i t t o r e a g l y c o s u l f a t a s e : This g l y c o s u l f a t a s e has been e x t e n s i v e l y i n v e s t i g a t e d . The s p e c i f i c i t y of the enzyme i s r e l a t i v e l y low as i t hydrolyzes D-glycose 6 - 0 - s u l f a t e , D-glucose 3 - 0 - s u l f a t e , and D-galactose 6 - 0 - s u l f a t e (Table IV). In common w i t h most other s u l f a t a s e enzymes, g l y c o s u l f a t a s e i s s t r o n g l y i n h i b i t e d by phosphate and pyrophosphate, and to a l e s s extent by f l u o r i d e i o n s . Some increase i n enzyme a c t i v i t y i s observed i n the presence of magnesium and manganous c h l o r i d e s . Table IV Optimum Substrate^ cone χ 10 (M)

Κ χ 10 m

Relative activity

Substrate

Optimum PH

Potassium D-glucose 3-0-sulfate

5.7-5.9

7.0

3.0

1.0

Potassium D-glucose 6-0-sulfate

5.5-5.6

4.0

1.7

10.0

Potassium D-galactose β-0-sulfate

5.2

>8.0

7.2

2.0

2

None of a wide range of polysaccharides of p l a n t or animal o r i g i n i s attacked by the enzyme, nor are the o l i g o s a c c h a r i d e s produced by the a c t i o n of hyaluronidase on c h o n d r o i t i n 4 - s u l f a t e or c h o n d r o i t i n 6 - s u l f a t e (102).


8.

DE E T A L .

Polysaccharide

133

Sulfates

P a t e l l a vulgata g l y c o s u l f a t a s e : This enzyme has a wide s p e c i f i c i t y f o r 6 - 0 - s u l f a t e e s t e r s of D-glucose, D-galactose (Table V ) , and D-mannose, and a l s o f o r 2 - 0 - , 3-0- and 4 - 0 - s u l f a t e e s t e r s of L-fucose. Table V Optimum pH

Optimum substrate cone (M)

Activity (units)

6-0-sulfate

6.0

0.04

3295

D-galactose δ-0-sulfate

6.0

0.04

4035


Substrate

Κ (mM)

D-glucose 1.86 28.3

The enzyme a c t i v i t y i s s t r o n g l y i n h i b i t e d by phosphate and borate i o n s , w h i l e the e f f e c t s of cyanide, f e r r i c and c u p r i c vary w i t h the s u b s t r a t e . Trichonderma v i r i d e g l y c o s u l f a t a s e : The enzyme i s a simple g l y c o s u l f a t a s e a c t i v e towards the 6 - 0 - s u l f a t e e s t e r s of D-glucose and D-galactose but not towards D-glucose 3 - 0 - s u l f a t e . A c t i v i t y decreases sharply a t e i t h e r s i d e of the optimum temp, of 2 8 ° . The pH f o r maximum a c t i v i t y i s 7.8-7.9. The g l y c o s u l f a t a s e seems to be d i f f e r e n t from analogous molluscan enzymes t h a t a l s o can hydrolyze monosaccharide s u l f a t e e s t e r s . Pseudomonas carrageenovora g l y c o s u l f a t a s e : The enzyme shows a remarkable degree of s p e c i f i c i t y f o r h y d r o l y z i n g neocarrabiose s u l f a t e , 3-0-(3,6-anhydro-a-D-galactopyranose 4 - 0 - s u l f a t e , a degradation product of K-carrageenan, w i t h h y d r o l y s i s leading to the corresponding d i s a c c h a r i d e . The enzyme a l s o acts on D-galactose 6 - 0 - s u l f a t e , but not on D-galactose 4 - Q - s u l f a t e ; a s u r p r i s i n g r e s u l t s i n c e D-galactose 4 - 0 - s u l f a t e i s a u n i t i n the neocarrabiose s u l f a t e .

Neocarrabiose 4 - 0 - s u l f a t e



134


Enzyme a c t i v i t y i s completely i n h i b i t e d i n 0.1 M a c e t a t e b u f f e r , pH 4.0, and by 25 mM mercuric i o n . P a r t i a l i n h i b i t i o n occurs w i t h 0.1 M a c e t a t e b u f f e r , pH 5.6, and 25 mM phosphate b u f f e r , pH 7.5. There i s no i n h i b i t i o n w i t h 25 mM s u l f a t e and 3 mM ethylene diamine t e t r a a c e t i c a c i d (EDTA). Hen o v i d u c t s u l f a t a s e : This enzyme removes only the s u l f a t e at p o s i t i o n C4 and shows remarkable s p e c i f i c i t y towards UDP-Na c e t y l -Q-gal actosami ne-4-0-sul f a t e , UDP-N-acetyl-D-galactosamine4 , 6 - d i - 0 - s u l f a t e , and N - a c e t y l - D - g a l a c t o s a m i n e - 4 , 6 - d i - 0 - s u l f a t e , but not towards l N - a c e t y l - Q - g a l a c t o s a m i n e - 4 - 0 - s u l f a t e . (Table V I ) . Table VI Maximum v e l o c i t y V max (units/mg p r o t e i n )

Substrate

1.

UDP-Gal NAc-4-sulfate

Κ m (M)

4,200

4 χ ΙΟ"

4

UDP-Gal NAc-4,6-disulfate

10,500

6 χ ΙΟ"

6

3.

j\[-Acetyl gal a c t o samine 4 - s u l f a t e

0

4.

^-Acetylgalacto samine 4 , 6 - d i s u l f a t e

560

2.

0 3 χ 10

The enzyme i s not a c t i v e toward £-nitrophenyl s u l f a t e , D-glucose 6 - 0 - s u l f a t e , D-galactose 3 - 0 - s u l f a t e , D-galactose 6 - 0 - s u l f a t e , or a number of s u l f a t e d polysaccharides of p l a n t and animal o r i g i n . The enzyme i s d i s t i n c t from any of the known a r y l s u l f a t a s e and g l y c o s u l f a t a s e . Polysaccharide

sulfatases

A number of n a t u r a l polysaccharide s u l f a t e s of both p l a n t and animal o r i g i n s are known, f o r example: c h o n d r o i t i n 4 - s u l f a t e , c h o n d r o i t i n 6 - s u l f a t e , dermatan s u l f a t e , shark c h o n d r o i t i n s u l f a t e , keratan s u l f a t e , heparan s u l f a t e , h e p a r i n , porphyran, τ , κ , ι and μ-carrageenan, charonan s u l f a t e and f u c o i d a n . A number of enzymes and enzyme-systems have been i s o l a t e d that d e s u l f a t e some of these s u l f a t e d p o l y s a c c h a r i d e s . C e l l u l o s e p o l y s u l f a t a s e : An enzyme h y d r o l y z i n g s u l f u r i c e s t e r bonds i n c e l l u l o s e p o l y s u l f a t e and charonan s u l f a t e occurs i n l i v e r e x t r a c t of the marine gastropod, Charonia lampas. Since


8.

DE E T A L .

Polysaccharide

Sulfates

135


c h o n d r o i t i n s u l f a t e and amylose p o l y s u l f a t e are s c a r c e l y hydrolyzed by the enzyme p r e p a r a t i o n , t h i s new s u l f a t a s e has been c a l l e d " c e l l u l o s e p o l y s u l f a t a s e " . The l i v e r of Charonia lampas (68) i s macerated a t pH 6.0, c e n t r i f u g e d , and to the supernatant i s added a c r i f l a v i n e to 1% c o n c e n t r a t i o n . P r e c i p i t a t e d m a t e r i a l i s separated by c e n t r i f u g a t i o n , ethanol i s added to the supernatant to 70% concentrat i o n , c e n t r i f u g e d , and the p r e c i p i t a t e d i a l y z e d i n water and c e n t r i f u g e d . The supernatant, at pH 5.2, i s p u r i f i e d i n a column of CMC, changing the eluent pH g r a d u a l l y from 5.2 t o 6.0. The appropriate f r a c t i o n s are pooled, and d i a l y z e d . The crude c e l l u l o s e p o l y s u l f a t a s e from C. lampas hydrolyzes a random s u l f a t e d c e l l u l o s e removing most of the s u l f a t e groups w h i l e presumably other enzymes present hydrolyze the c e l l u l o s e to D-glucose and D-glucose s u l f a t e s from which s u l f a t e groups are then hydrolyzed. The p u r i f i e d c e l l u l o s e p o l y s u l f a t a s e hydrolyzes s u l f a t e groups from the polysaccharide without degrading the c h a i n . This enzyme shows remarkable s p e c i f i c i t y towards c e l l u l o s e p o l y s u l f a t e and charonan s u l f a t e by converting these from high s u l f u r content to low s u l f u r content p o l y s a c c h a r i d e s . I t slowly a t t a c k s dextran s u l f a t e , but not amylose s u l f a t e (Table V I I ) . Regarding the e f f e c t of the p o s i t i o n of s u l f a t e bond, Takahashi suggests t h a t c e l l u l o s e p o l y s u l f a t a s e p r e f e r e n t i a l l y a t t a c k s e s t e r bonds a t the C2 and C3 p o s i t i o n s , w h i l e g l u c o s u l f a t a s e a t t a c k s p r e f e r e n t i a l l y C6 p o s i t i o n e s t e r bonds. Porphyran s u l f a t a s e : An enzyme present i n Porphyra e x t r a c t s i s capable of h y d r o l y z i n g porphyran, a complex s u l f a t e d polysaccharide c o n t a i n i n g residues of D - g a l a c t o s e , L - g a l a c t o s e , 6-0-methyl-D-galactose, and 3,6-anhydro-L-galactose. Porphyra seaweed (70) i s washed w i t h water, minced i n t o aqueous sodium carbonate a t pH 8.3, pH adjusted to 6.0-6.5 and c e n t r i f u g e d . To the supernatant i s added a calcium phosphate g e l , s t i r r e d and c e n t r i f u g e d . The gel i s washed a t pH 7.5, c e n t r i f u g e d , and the supernatant t r e a t e d w i t h s o l i d ammonium s u l f a t e to 0.8% s a t u r a t i o n . The p r e c i p i t a t e i s i s o l a t e d by c e n t r i f u g a t i o n , d i s s o l v e d i n water, and d i a l y z e d . The enzyme d e s u l f a t e s porphyran but does not a t t a c k other sulfated polysaccharides. Complete i n h i b i t i o n o f the enzyme occurs on a d d i t i o n of metal binding reagents, thus i n d i c a t i n g t h a t there i s a b i - or t e r v a l e n t c a t i o n e s s e n t i a l to the enzyme. No i n h i b i t i o n i s observed when a mixture of Zn2 or C o ^ ions (1.5 mM) and ethylenediaminetetra a c e t i c a c i d (EDTA) (0.5 mM) i s present. A 50% i n h i b i t i o n occurs i f M n i o n i s s u b s t i t u t e d f o r one of these c a t i o n s , and complete i n h i b i t i o n occurs i f +

2 +


+

136



Mg ion i s used. Borate i s a powerful a c t i v a t o r producing 60% a c t i v a t i o n at pH 7.6. Since porphyran i s i t s e l f a p o l y e l e c t r o l y t e , i t i s conceivable t h a t c e r t a i n of the a c t i v a t o r s i n f l u e n c e s the r e a c t i o n by i n t e r a c t i o n with the substrate r a t h e r than the enzyme. I t i s known t h a t s a l t s present i n s o l u t i o n w i t h polysaccharide polyanions can a l t e r the c o n f i g u r a t i o n of the polymer, and i t i s p o s s i b l e t h a t some of the c a t i o n a c t i v a t o r s operate by i n c r e a s i n g the time spent by the s u b s t r a t e i n a c o n f i g u r a t i o n f a v o r a b l e f o r the r e a c t i o n . Chondrosulfatase Chondrosulfatase of Proteus v u l g a r i s : A s t r a i n of Proteus v u l g a r i s (N.C.T.C. 4636) appears to be a p a r t i c u l a r l y potent source of chondrosulfatase (E.C.3.1.6.4). The enzyme i s a s s o c i a t e d w i t h a c h o n d r o i t i n a s e system which can degrade the chain of c h o n d r o i t i n s u l f a t e producing s u l f a t e d o l i g o s a c c h a r i d e s which are subsequently d e s u l f a t e d by chondrosulfatase. Proteus v u l g a r i s (76,77) i s c u l t u r e d , and harvested by c e n t r i f u g i n g . The c e l l s are macerated i n acetone, f i l t e r e d , homogenized at pH 7.0, incubated at 3 7 ° , and c e n t r i f u g e d . The supernatant i s d i a l y z e d , c e n t r i f u g e d , and to the supernatant added sodium s a l t of yeast n u c l e i c a c i d at pH 7.4. The pH i s adjusted to 4.0, the p r e c i p i t a t e removed by c e n t r i f u g i n g and d i s s o l v e d i n aqueous sodium hydroxide at pH 7.4. The s o l u t i o n i s adjusted to pH 6.7 and an aqueous s o l u t i o n of protamine s u l f a t e added, d i a l y z e d and c e n t r i f u g e d . The supernatant i s adjusted to pH 8.0, c e n t r i f u g e d , and the supernatant t r e a t e d w i t h calcium phosphate gel at pH 6.55. The gel-enzyme complex i s separated by c e n t r i f u g a t i o n , washed and t r e a t e d repeatedly w i t h sodium acetate s o l u t i o n at pH 8.0 u n t i l no f u r t h e r p r o t e i n i s e l u t e d from the g e l . The e l u a t e c o n t a i n i n g s u l f a t a s e are combined and d i a l y z e d . Chondrosulfatase, i n the absence of c h o n d r o i t i n a s e , i s v i r t u a l l y i n a c t i v e towards the polymerized form of c h o n d r o i t i n s u l f a t e . However, c h o n d r o i t i n s u l f a t e degraded w i t h t e s t i c u l a r hyaluronidase i s r e a d i l y hydrolyzed. The crude Proteus c o n c e n t r a t e , which i s a c t i v e towards c h o n d r o i t i n s u l f a t e , i s i n a c t i v e towards the s u l f a t e d polysaccharides agar, f u c o i d a n , carrageenan, chondrus o c e l l a t u s mucilage and s u l f a t e d laminaran. Recently, two chondrosulfatases are i s o l a t e d from the e x t r a c t s of Proteus v u l g a r i s (N.C.T.C. 4636). One of these enzymes, chondro 4 - s u l f a t a s e , i s a c t i v e against 2-acetamido-2deoxy-3-0-(3-Q-gluco-4-enepyranosyl-uronic acid)-4-0-sulfo-D-



8. DE E T A L .

Polysaccharide

Sulfates

137

g a l a c t o s e , the product from the degradation of c h o n d r o i t i n s u l f a t e A or Β by c h o n d r o i t i n a s e , and i t s saturated analogue, acetylchondrosiη 4 - s u l f a t e , but does not a t t a c k the corresponding 6 - 0 - s u l f a t e isomer, the product from the degradation of c h o n d r o i t i n s u l f a t e C by c h o n d r o i t i n a s e , and i t s saturated analogue, a c e t y l c h o n d r o s i n 6 - s u l f a t e . In c o n t r a s t , chondro 6-sulfatase desulfates disaccharide 6-sulfates andacetyl-D-galactosamine 4 , 6 - d i s u l f a t e a t p o s i t i o n 6, but does not a t t a c k tfie d i s a c c h a r i d e 4 - s u l f a t e isomers.

2-Acetamido-2-deoxy-3-0-(3-Q-gluco-4enepyranosyluronic a c i d ) - 4 - 0 - s u l f o - D galactose

2-Acetami do-2-deoxy-3-0-(3-D-gluco-4enepyranosyluronic a c i d ) - 6 - 0 - s u l f o - Q galactose The enzymes do not a t t a c k polymer c h o n d r o i t i n s u l f a t e s , hexa-, p e n t a - , t e t r a - , or t r i s a c c h a r i d e s derived from c h o n d r o i t i n s u l f a t e s A and C by d i g e s t i o n w i t h crude t e s t i c u l a r hyaluronidase, or a c e t y l g a l a c t o s a m i n e 4- and 6 - s u l f a t e s . Chondrosulfatase of P a t e l l a v u l g a t a : The v i s c e r a of P a t e l l a vulgata (60) i s reported to c o n t a i n a g l y c o s u l f a t a s e and a l s o an enzyme t h a t i s a c t i v e a g a i n s t c h o n d r o i t i n 4 - s u l f a t e . The chondrosulfatase has been p u r i f i e d by s e p a r a t i n g from the g l y c o s u l f a t a s e by f r a c t i o n a t i o n w i t h ammonium s u l f a t e .


138



Chondrosulfatase from t h i s source i s reported to be capable of d e s u l f a t i n g c h o n d r o i t i n s u l f a t e without p r i o r d e p o l y m e r i z a t i o n . S u l f a t e i s a l s o l i b e r a t e d by the enzyme from a range of c h o n d r o i t i n s u l f a t e s from d i f f e r e n t sources, i n c l u d i n g a commercial preparation b e l i e v e d to be predominately c h o n d r o i t i n 6-sulfate. Chondrosulfatase of Squid l i v e r : The l i v e r e x t r a c t s of the squid (Ommastrephes s l o a n i p a c i f i c u s ) (83) contains chondros u l f a t a s e along w i t h three other enzymes, hyaluronidase (E.C. 3.2.1.35), 3-N-acetyl-hexosaminidase (E.C. 3.2.1.30), and ^-glucuronidase (E.C. 3.2.1.31). Crude enzyme i s prepared from squid l i v e r by e x t r a c t i o n w i t h acetone and then p u r i f i e d by f r a c t i o n a t i o n w i t h ammonium s u l f a t e and by using a column of Sephadex G-100_. The p u r i f i c a t i o n achieved i s about 1 5 - f o l d from the crude enzyme. The p a r t i a l l y p u r i f i e d chondrosulfatase i s stored at - 2 0 ° , without a p p r e c i a b l e l o s s of a c t i v i t y f o r a month. The squid chondrosulfatase a t t a c k s c h o n d r o i t i n 4 - s u l f a t e at g r e a t e r r a t e than i t does c h o n d r o i t i n 4 , 6 - d i s u l f a t e . Chondroitin 6 - s u l f a t e i s a l s o d e s u l f a t e d to a s l i g h t e x t e n t , but keratan s u l f a t e , dermatan s u l f a t e and heparin are not d e s u l f a t e d w i t h t h i s enzyme. A s u l f a t e d t e t r a s a c c h a r i d e and a mixture of s u l f a t e d o l i g o s a c c h a r i d e s , both prepared from c h o n d r o i t i n 4 - s u l f a t e by t e s t i c u l a r hyaluronidase d i g e s t i o n , are r e s p e c t i v e l y d e s u l f a t e d to the extent of about 32% and 56% of the s u l f a t e released from c h o n d r o i t i n 4 - s u l f a t e polymer. D e s u l f a t i o n from the s u l f a t e d di s a c c h a r i d e , 2-acetami do-2-deoxy-3-0-($-Q-gluco-4-enopyranosyluronic a c i d ) - 4 - 0 - s u l f o - Q - g a l a c t o s e , i s n e g l i g i b l e . These s t u d i e s show t h a t the squid chondrosulfatase i s capable of d e s u l f a t i n g mainly 4 - s u l f a t e i n c h o n d r o i t i n s u l f a t e s without p r i o r depolymerization. Chondrosulfatase a c t i v i t y i s extremely i n h i b i t e d by c u p r i c s u l f a t e , sodium phosphate, sodium f l u o r i d e , borax and heparin. Chondrosulfatase i s a c t i v a t e d by sodium c h l o r i d e , c u p r i c c h l o r i d e , EDTA, and c y s t e i n e h y d r o c h l o r i d e . The apparent 1^ of t h i s chondrosulfatase i s estimated as 0.49 mM f o r r e a c t i o n s at pH 5.0 and 37° f o r 3 h w i t h c h o n d r o i t i n 4 - s u l f a t e as the s u b s t r a t e . Chondrosulfatase of bovine a o r t a : I t has been known (103, 104) t h a t r a t s d e s u l f a t e c h o n d r o i t i n s u l f a t e i n v i v o and i t has been claimed (105) t h a t chondrosulfatase can be detected h i s t o c h e m i c a l l y i n human sweat glands, but attempts have been unsuccessful (56) to prepare i t from mammalian t i s s u e , e x t r a c t s w i t h chondrosulfatase a c t i v i t y . On the other hand, crude preparations from p i g kidney are known to l i b e r a t e s u l f a t e from chondroitin sulfate.


8.

DE E T A L .

Polysaccharide

Sulfates

139


Bovine a r t e r i a l t i s s u e (aorta) contains a chondrosulfatase (85). The enzyme i s p u r i f i e d by ammonium s u l f a t e f r a c t i o n a t i o n , p r e c i p i t a t i o n a t pH 4.6, and by gel f i l t r a t i o n . An 85 f o l d p u r i f i c a t i o n i s achieved. The enzyme i s a c t i v e against c h o n d r o i t i n 4 - s u l f a t e , but i t does not a t t a c k c h o n d r o i t i n 6 - s u l f a t e , dermatan s u l f a t e , and keratan s u l f a t e . In acetate b u f f e r , the pH optimum i s 4.4, and Km i s 0.735 mM. K e r a t o s u l f a t a s e : Two forms o f k e r a t o s u l f a t a s e (86) (I and I I ) are i s o l a t e d from the marine gastropod, charonia lampas. Both forms r e l e a s e a l l the s u l f a t e from kerato s u l f a t e s and n e i t h e r appears i d e n t i c a l w i t h g l y c o s u l f a t a s e or c h o n d r o s u l f a t a s e , both of which are a l s o present i n charonia lampas. The crude l i v e r e x t r a c t i s p u r i f i e d by f r a c t i o n a t i o n w i t h ammonium s u l f a t e and then by successive column chromatography of CM-Sephadex C-50 and DEAE-Sephadex A-50. K e r a t o s u l f a t a s e preparations I and II are stored a t -20° i n the presence of 0.1 M sodium c h l o r i d e without a p p r e c i a b l e l o s s of a c t i v i t y f o r several weeks. Keratosulfatases desulfate keratopolysulfate, keratosulfate and h o r a t i n s u l f a t e , a s u l f a t e d polysaccharide composed of L-fucose, g-mannose, D-glucose, D - g a l a c t o s e , D-glucosamine and D-galactosamine, i s o l a t e d from the l i v e r of £. lampas. K e r a t o s u l f a t a s e I does not l i b e r a t e s u l f a t e from h o r a t i n s u l f a t e (Table V I I I ) . Table V I I I Enzyme preparation

Substrate

Incubation time

SO^ libérât

(h) 1. 2. 3.

I II I II I II

Keratopolysulfate Same , Kerator S]sulfate Same Horatin s u l f a t e Same 7

D

96 96 96 76 48 48

(%) 80 100 95 100 < 3 100

I t a l s o appears that k e r a t o s u l f a t a s e f i r s t l i b e r a t e s s u l f a t e from k e r a t o s u l f a t e s and the d e s u l f a t e d polymer i s then degraded to D-galactose and N-acetyl-D-glucosamine by the a c t i o n of 3-D-galactosidase and 3-u-acetyl-D-glucosaminidase, enzymes a l s o present i n the p a r t i a l l y p u r i f i e d k e r a t o s u l f a t a s e .


140


Potassium dihydrogen phosphate, sodium f l u o r i d e and £-chloromercuribenzoate i n h i b i t both fkeratosulfatases I and as they do most other known £. lampus s u l f a t a s e s .


The optimum pH values of k e r a t o s u l f a t a s e s I and II 4.5 and 6.0, r e s p e c t i v e l y .

II,

are

Heparin degrading multienzyme system: No s i n g l e enzyme has been i s o l a t e d which can d e s u l f a t e heparin or h e p a r i t i n s u l f a t e s . However, f i v e enzymes from Flavobacterium heparinum (87), capable of degrading heparin to i t s b a s i c c o n s t i t u e n t 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 . The p u r i f i e d heparinase degrades heparin to t r i s u l f a t e d d i s a c c h a r i d e . This i s d e s u l f a t e d f u r t h e r by a d i s a c c h a r i d e s u l f a t a s e y i e l d i n g the d i s u l f a t e d d i s a c c h a r i d e . This product i s a s u b s t r a t e f o r glycuronidase g i v i n g g l u c o s a m i n e - 2 , 6 - d i s u l f a t e and a , 3 - k e t o a c i d . D-glucos a m i n e - 2 , 6 - d i s u l f a t e i s d e s u l f a t e d by a s u l f a t a s e and a sulfamidase y i e l d i n g f r e e glucosamine and i n o r g a n i c s u l f a t e . The crude e x t r a c t from heparinum i s p u r i f i e d by s u c c e s s i v e l y using a column chromatography of Sephadex G-200 and then by agarose gel e l e c t r o p h o r e s i s . The hepa'rinase has no a c t i v i t y upon c h o n d r o i t i n s u l f a t e s A, Β and C or on h e p a r i t i n s u l f a t e s A and B, but degrades h e p a r i t i n s u l f a t e s C and D, y i e l d i n g s u l f a t e d d i s a c c h a r i d e s w i t h the same chromatographic m o b i l i t y as the ones obtained from heparin. The s p e c i f i c i t y of the enzyme, d i s a c c h a r i d e s u l f a t a s e , i s l i m i t e d . The enzyme acts only upon the t r i s u l f a t e d d i s a c c h a r i d e , forming d i s u l f a t e d d i s a c c h a r i d e and i n o r g a n i c s u l f a t e . Heparin, s u l f a t e d hexa- and t e t r a - s a c c h a r i d e s , d i s u l f a t e d d i s a c c h a r i d e , and g l u c o s a m i n e - 2 , 6 - d i s u l f a t e are not substrates f o r t h i s enzyme. Glycuronidase acts only upon the d i s u l f a t e d d i s a c c h a r i d e , y i e l d i n g glucosamine-2,6-disulfate. This enzyme does not a c t upon the t r i s u l f a t e d d i s a c c h a r i d e . Monosaccharide s u l f a t a s e and sulfamidase a c t only upon the s u f l a t e d hexosamine monosaccharides. No a c t i v i t y upon heparin or the d i s a c c h a r i d e s has been d e t e c t e d . The pathway of enzymatic degradation of heparin may proceed as shown.


8.

DE E T A L .

Polysaccharide

Sulfates

141

Heparin

I

heparinase

T r i s u l f a t e d Disaccharide


disaccharide sulfatase D i s u l f a t e d Disaccharide + SO^ glycuronidase G l u c o s a m i n e - 2 , 6 - d i s u l f a t e + a,3-Keto A c i d monosaccharide sulfatase

sulfamidase Glucosamine-6-sulfate + S0

Glucosamine-N-sulfate

2-

+ so4

4

2-

sulfamidase

monosaccharide sulfatase Glucosamine + SO.

Cerebroside s u l f a t a s e : Presence of a cerebroside s u l f a t a s e has been f i r s t reported i n the d i g e s t i v e gland of the abalone, H a l i o t i s (88). This enzyme, i n a d d i t i o n to h y d r o l y z i n g c e r e b r o s i d e , can a l s o d e s u l f a t e c h o n d r o i t i n s u l f a t e but not p h e n y l s u l f a t e nor glucose 6 - s u l f a t e . Cerebroside s u l f a t a s e has been p u r i f i e d from the lysosomes of p i g kidney (90) and i t s presence has been shown i n other organs, p a r t i c u l a r l y l i v e r and spleen. The p u r i f i c a t i o n i s achieved by high voltage e l e c t r o p h o r e s i s of the crude e x t r a c t . This enzyme d e s u l f a t e s only cerebroside 3 - s u l f a t e s . Cerebroside 6 - s u l f a t e s are not d e s u l f a t e d . D-Galactose 3 - s u l f a t e i s hydrolyzed a t about 20% the r a t e of c e r e b r o s i d e 3 - s u l f a t e but D-galactose 6 - s u l f a t e i s not hydrolyzed. The enzyme a l s o d e s u l f a t e s cerebron and kerasin s u l f a t e s . The optimum pH i s 4.5, and i s 0.1 mM w i t h cerebroside 3 - s u l f a t e s . Enzyme a c t i v i t y i s i n h i b i t e d by s u l f i t e , s u l f a t e , phosphate, and f l u o r i d e w h i l e a c t i v a t e d by hydroxylami ne.



142

The enzyme has s t r i k i n g resemblance to a r y l s u l f a t a s e A and the enzymes could be i d e n t i c a l (107). Both cerebroside s u l f a t a s e and a r y l s u l f a t a s e A have been i s o l a t e d from human l i v e r (108) and perhaps t h e i r absence could cause metachromatic leucodystrophy i n humans.


Hunter's

Syndrome

Hunter's Syndrome i s a genetic d i s o r d e r a s s o c i a t e d w i t h f a i l u r e to degrade dermatan s u l f a t e and heparan s u l f a t e . Lysosomal storage of these polymers leads to numerous c l i n i c a l problems. F i b r o b l a s t s c u l t u r e d from the s k i n of Hunter's p a t i e n t s do not adequately degrade s u l f a t e d mucopolysaccharides because of a d e f i c i e n c y of a s p e c i f i c p r o t e i n t h a t i s present i n c e l l s e c r e t i o n s , c e l l s , and urine of i n d i v i d u a l s who do not have Hunter's Syndrome (113). Recent s t u d i e s show (109) t h a t a d e f i c i e n c y of a s u l f a t a s e , L - i d u r o n o - s u l f a t e s u l f a t a s e , which s p e c i f i c a l l y cleaves the e s t e r s u l f a t e of i d u r o n i c a c i d , to be r e s p o n s i b l e f o r the d e f e c t i n Hunter's Syndrome. This s u l f a t a s e , when added exogenously to Hunter c e l l s , a c c e l e r a t e s the degradat i o n of s u l f a t e d mucopolysaccharides. Sanfilippo

Syndrome

S a n f i l i p p o Syndrome i s a f a m i l i a l d i s o r d e r of mucopolysaccharide metabolism, t r a n s m i t t e d i n autosomal r e c e s s i v e f a s h i o n . Excessive u r i n a r y e x c r e t i o n of heparan s u l f a t e i s the major d i a g n o s t i c c r i t e r i o n . F i b r o b l a s t s from p a t i e n t s w i t h S a n f i l i p p o Syndrome f a l l i n t o two subgroups, each m a n i f e s t i n g a d e f i c i e n c y of a s p e c i f i c f a c t o r r e q u i r e d f o r normal metabolism of s u l f a t e d mucopolysaccharide. The f a c t o r d e f i c i e n t i n the A subgroup has been i s o l a t e d from normal human u r i n e . The f a c t o r a c c e l e r a t e s degradation of stored mucopolysaccharide i n S a n f i l i p p o A f i b r o b l a s t s . I t has been suggested t h a t S a n f i l i p p o A f a c t o r i s a heparan s u l f a t e s u l f a t a s e (110). When the f a c t o r i s administered exogenously to S a n f i l i p p o f i b r o b l a s t s , a c o r r e c t i o n of the abnormal metabolism occurs. Metachromatic Leucodystrophy

(MLD)

Metachromatic leucodystrophies are a group of human genetic d i s o r d e r s which are c h a r a c t e r i z e d by the d e p o s i t i o n of cerebroside s u l f a t e r i c h granules i n the c e n t r a l and p e r i p h e r a l nervous systems with r e s u l t a n t progressive n e u r o l o g i c a l degeneration. I t has been suggested t h a t the turnover of cerebroside s u l f a t e s to cerebrosides might be blocked as a r e s u l t of d e f i c i e n c y of cerebroside s u l f a t a s e (111, 112). Cerebroside s u l f a t a s e a c t i v i t y i s demonstrated i n normal mammalian t i s s u e s such as kidney and b r a i n , where i n MLD p a t i e n t s the cerebroside s u l f a t e accumulation i s e s p e c i a l l y high.


8.

DE E T A L .

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Sulfates

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Biogenesis Recent work has shown t h a t prophyran contains residues of both 3,6-anhydro-L-galactose and L - g a l a c t o s e 6 - s u l f a t e (69, 71). When e x t r a c t s of porphyran c o n t a i n i n g seaweed are incubated w i t h the i s o l a t e d p o l y s a c c h a r i d e , f r e e s u l f a t e i s l i b e r a t e d and s y n t h e s i s of 3,6-anhydro-L-galactosyl residues occurs. Equimolar q u a n t i t i e s of the two products are present i n the m i x t u r e . These r e s u l t s s t r o n g l y i n d i c a t e t h a t L-galactose 6 - s u l f a t e i s an immediate b i o l o g i c a l precursor of 3,6-anhydro-L-galactose, the residue present i n commercial agar. This enzymic h y d r o l y s i s can provide a way of making commercial agar from 6 - s u l f a t e d p o l y saccharides. S t r u c t u r a l work of polysaccharides Enzymic h y d r o l y s i s of polysaccharides can provide i n t e r e s t i n g s t r u c t u r a l i n f o r m a t i o n . Thus, the s u b s t r a t e s p e c i f i c i t y of f i v e enzymes leads to the f o r m u l a t i o n of a pathway f o r the s e q u e n t i a l degradation of heparin (87). L i k e w i s e , the demonstration of the presence of chondroitinase-ABC, chondroitinase-AC, chondro4 - s u l f a t a s e , c h o n d r o - 6 - s u l f a t a s e , and unsaturated d i s a c c h a r i d e glucuronidase provides an enzymatic mechanism f o r the degradation of c h o n d r o i t i n s u l f a t e s (78). Another obvious f e a t u r e of these enzymes i s the use to which they may be put as reagents i n studying the s t r u c t u r e of c h o n d r o i t i n s u l f a t e s as w e l l as the composition of a given mixture of isomeric c h o n d r o i t i n s u l f a t e s . Conclusion Work on enzymatic s u l f a t i o n and d e s u l f a t i o n of polysaccharides i s i n i t s formative stages but has a l r e a d y reached a p o i n t where c e r t a i n b e n e f i c i a l a p p l i c a t i o n s are apparent. C l i n i c a l c o n t r o l of several syndromes may be f a c i l i t a t e d by a p p r o p r i a t e manipulation of enzymes e f f e c t i n g s u l f a t e groups. On a broader b a s i s there i s i n d i c a t i o n of the p o t e n t i a l use of enzymes f o r s u l f a t i o n of at l e a s t c e r t a i n polysaccharides and even a more immediate use of enzymes f o r d e s u l f a t i o n and perhaps d e s u l f a t i o n w i t h r e s u l t a n t i n t r o d u c t i o n of anhydro r i n g s . Consequently, f u r t h e r i n d u s t r i a l examination of these enzyme systems can be expected.

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