the cell wall glycoprotein

7/28/2019 The Cell Wall Glycoprotein

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Z b l . Bakt. Hyg., I. Abt. Orig. C 3, 161-170 (1982)

Lehrstuhl fur Biochemie I der Universitat Regensburg, Federal Republic ofGermany

The C e l l Wal l Glycoprotein o fHalobacteri a: Structural,Functional and Biosynthetic Aspects*

F E L I X W I E L A N D , J O H A N N L E C H N E R , and M A N F R E D S U M P E R

Received September 11, 1981

SummaryThe cell wall glycoprotein of Halobacteria contains sulphate covalenty bound to dif

ferent types of saccharides. Ahigh molecular weight saccharide (HMW-saccharide), whichis N-glycosidically linked to Asn was found to have the following composition: 1 gal:1 galN: 1 g l c N : 2galUA: 2 S C V ~ . This HMW-saccharide is not a heterosaccharide, butexhibits rather a repeating unit structure similar to that of the animal glycosaminoglycans.Incorporation into the cell wall glycoprotein of this HMW-saccharide is specifically inhibited by the antibiotic bacitracin {Wieland et al., 1980). This inhibition results in aremarkable change of the halobacterial shape: the normally rod shaped cells grown in thepresence of bacitracin convert to regular spheres (Mescher and Stromi nger, 1975).

This HMW-saccharide is synthesized bound to a l ip id anchor: only after completionthe fully sulphated saccharide is transferred to the core protein.Keywords: Halobacterial glycoprotein - Sulphated glycoconjugates - Glycopro

tein biosynthesis - L i p i d li nked precursor saccharide

IntroductionHalobacteria were the first bacteria found to possess a true glycoprotein, which

is the main constituent of the Halobacterial cell wall {Mescher and Strominger,1974). This glycoprotein contains saccharides bound by the most common typesof linkage known for eucaryotes (c. f. Fig. 1): oligosaccharides free of amino sugarswhich are O-glycosidically bound to threonine and one large amino sugar contain-

* Paper given at the First International Workshop on Archaebacteria, Munchen, June 27to July 1, 1981.Abbreviati ons: HMW-saccharides: high molecular weight saccharides,

SDS gel electrophoresis: dodecylsulphate polyacrylamide gel electrophoresis,galN: 2-deoxy 2-aminogalactose,g l c N : 2-deoxy 2-aminoglucose.

11 Zbl- Bakt. Hyg ., I. Abt. O r i g . C3


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T H R T H R ASN 200 KDGAL GAL GLCN AC10GLC GLC XNAC

22-24 MANUA 12-14GAL"8-9GLC

Fig. 1. Structure of the Halobacterium sali narium cell wall glycoprotein as proposedbyMescher and Strominger (1976).

in g s ac ch a ri de N - g l y c o s i d i c a l l y b o u n d to a s p a r a g i n e w h i c h has b e e n d e p i c t e d asheterosacchar i d e (Mescher a n d Strominger, 1976).In the course of i n v e s t i g a t i o n s on m e m b r a n e b i o s y n t h e s i s in Halobacterium

halobium we o b s e r v e d that t h i s o r g a n i s m is able to i n c o r p o r a t e s u l p h a t e i n t om a c r o m o l e c u l e s w i t h o u t r e d u c i n g the sul p hate res i d ue to cycs te i n e or m e t h i o n i n e[Sumper and Herrmann, 1978). The m a i n p r o d u c t b e a r i n g s u l p h a t e t u r n e d outto be the Halobacterial c e l l w a l l g l y c o p r o t e i n [Wieland et a l . , 1980). In the f o l l o w in g the i s o l a t i o n and c h a r a c t e r i z a t i o n of s u l p h a t e d g l y c o p e p t i d e s f r o m the Halobacterial ce l l w a l l g l y c o p r o t e i n w i l l be d e s c r i b e d and the f u n c t i o n of a s u l p h a t e ds a c c h a r i d e c o n t a i n i n g a m i n o s u g a r s w i l l be d i s c u s s e d . F u r t h e r m o r e we w i l l s h o wthat th is saccharide is s y n t h e s i z e d as a l i p i d - l i n k e d p r e c u r s o r and is t r a n s f e r r e d" en b l o c k " to the core p r o t e in o n l y after s y n t he s is ( i n c l u d i n g s u l p h a t io n ) is f i n i s h e d .

Materials and MethodsMost of the materials and methods used in the experiments presented here were de

scribed by Wieland et al., 1980 andWieland et al., 1981. As yet unpublished experimentaldetails will be given in the legends under the appropriate figures.

1. Composition of glycopeptides found in the cell wall glycoproteinI solated 3 5 S 0 4 2 ~ - l a b e l l e d Halobacterial ce l l w a l l g l y c o p r o t e i n was e x h a u s t i v e l yd i ges ted w i t h P r o n a s e and a g l y c o p e p t i d e f r a c t i o n was o b t a i n e d after c h r o m a t o g r a p h y onD o w e x A G W H + ionexchange res i n inH 2 0 in a y i e l d of a b o u t 90 to95 /o of the r a d i o a c t i v i t y . T h i s m i x t u r e of g l y c o p e p t i d e s was f u r t h e r p u r i f i e d byc h r o m a t o g r a p h y on Bi o Gel P 10. E a c h f r a c t i o n was assayed for 3 5 S 0 4 2 " - r a d i o -

a c t i v i ty , for n e u t r a l s u g a r s and for u r o n i c a c i d s . Fig. 2 s h o w s the c o r r e s p o n d i n ge l u t i o n p r o f i l e : w i t h the v o i d v o l u m e of the c o l u m n a r a d i o a c t i v e p e a k a p p e a r sw h i c h c o n t a i n s b o t h n e u t r a l s u g a r s u n d u r o n i c a c i d s . A m i n o s u g a r a n a l y s i s s h o w e da h e x o s a m i n e andg l c N in a 1: 1 s t o i c h i o m e t r y . The h e x o s a m i n e c o u l d be d e m o n strated to be g a l N by c o m b i n e d gas l i q u i d c h r o m a t o g r a p h y - mass s p e c t r o m e t r y .For th i s an a l ys i s we are i n d e p t e d to Dr . Schafer, M a r t i n s r i e d . T h i s h i g h m o l e c u l a r

Results


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FRACTIONF ig . 2. Separation of glycopeptides derived from pronase digestion of 35S042~-labelledHalobacterial cell wall glycoprotein by chromatography on Bio Gel P 10.C o l u m n : 1.6X80 cm; buffer pyridine/acetate, 0.1 M . pH 5.5. O - O radioactivity, neutral hexoses (anthroneassay), uronic acids (m-hydroxydiphenylmethod).

w e i g h t s a c c h a r i d e c o n t a i n i n g a m i n o s u g a r s w i l l be referred to in the f o l l o w i n g asH M W - s a c c h a r i d e .

A n a d d i t i o n a l r a d i o a c t i v e p e a k is o b s e r v e d e l u t i n g in a v o l u m e c o r r e s p o n d i n gto a low m o l e c u l a r w e i g h t . T h i s f r a c t i o n a g a i n c o n t a i n s n e u t r a l s u g a r s and u r o n i ca c i ds , but no a m i n o s u g a r s are f o u n d . E l u t i n g b e t w e e n the two ra d i o a c t i ve pea ksa re g l y c o pept i des w h i c h c o n t a i n s o l e l y n e u t r a l s u g a r s . The c o m p o s i t i o n s of thethree f r a c t i o n s are g i v e n inT a b l e 1.

2 . The HMW-saccharide has a repeating unit structureT h e 3 5 S 0 4 2 " - l a b e l l e d H M W - s a c c h a r i d e f r ac ti o n on h i g h v o l t a g e t h i n l ayer

e l ec t ro pho res i s sho w s one s i n g l e s p o t (c. f. Fig. 8A a ) . O n the o t h e r h a n d , on SDSg e l e l ec t ro pho res i s {Laemmli, 1970) or p o l y a c r y l a m i d e gel e l ec t ro pho res i s in thepresence of urea (Maxam and Gilbert, 1977) th i s f ra c t i o n ex hi b i t s a s t r i k i n greg u l a r pa t te rn of a b o u t 15 b a n d s (Fig. 3 A and Fig. 4A ) . T h i s p a t t e r n , w h i c h iss i m i l a r to that of o l i g o n u c l e o t i d e s of di f fe ren t c ha i n l eng ths on p o l y a c r y l a m i d eg el e l ec t ro pho res i s , led us to the idea that the di f fe ren t ba nds represen t d i f f e ren t


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T a b i d . Composition of three different fractions obtained after pronase digestion of3>SO,^-labelled cell wall glycoprotein and chromatography on Bio Gel P 10

G lc Gal G l c U A G a l U A G l c N G a l N SO, 2

HMW-saccharides(void volume)

Neutral-saccharides(middle molecularweight range)

L o w molecular weightsaccharides

50-

40-

30-


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9 abcd

Fig. 4. Comparison of the fragmentation patterns of isolated homogeneous HMW-saccharides.Radioactive material was eluted from a Maxam andGi lbert gel as marked with arrows (A).The isolated HMW-saccharides were incubated with 0.25 N HC1 for 15 min at 100 C Cand subsequently subjected to high voltage thin layer electrophoresis at pH 2.0. Fluorogramafter the run (B): total 3 5 S O | 2 ~ HMW-saccharides mixture (a), isolated HMW-saccharides(b to d).

HMW-saccharides as well as the isolated homogeneous samples all gave rise toan identical fragmentation pattern. This is taken as strong evidence for a repetitivesequence structure of the HMW-sacchar ides. Further proof for a repeating unitstructure comes from separation and analysis of fragmentation products after

15A

L-0,2

FRACTIONFig. 5. Separation of HMW-saccharide fragments according to their molecular weightsS ^ - s o ^ ^ 2 - Tha;amp,e air/ i,ldacidMrolys,s M-e^d -pt 4>un eatcd SO, HMW-saccharide was added as an internal molecular weight standardThe mixture was chromatographcd on Bio Gel P 10. O O radioactivity - - muronic acids, 4> 4, n e u t r a l hexoses. Y ' *


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g r a d e d a c id h y d ro l y s is o f th e H M W - s a c c h a r i d e f r a c t i o n : a 3 5 S 0 4 2 - - l a b e l l e d s a m p l ew as i n c ub a t e d f or 15 m in a t 100 C w i t h 0 .2 5 N H C 1 a n d s u b s e q u e n t l y c h r o m a t o -g r a p h e d o n a B i o G e l P 1 0 c o l u m n ( u n t r ea t ed 3 5 S 04 2 ~ H M W - s a c c h a r i d e s w e r ei n c l u d e d as an i n t e r n a l m ole cu lar w e igh t s t an d ar d ) : a s can b e seen i n F i g . 5 m i l da c i d h yd r o lys i s g iv e s ri se t o six n e w p e ak s o f d i f f e re n t m o le cu lar w e ig h t s . P e ak s It o V a l l con t a in s u lp h at e , g a lac t ur on ic ac id an d am in o sug ar s at a constant r a t i o ,w h e r e a s p e a k V I c o n t a in s o n l y galactose a n d free sulphate . Thus the repet i t ives acch ar id e un i t p os s ib ly con s i s ts o f t h e am in o sug ar s, g a lac t u r on ic ac id s an d sul phate res idues w i t h a p e r i p h e r a l l y b o u n d galactose r e s id ue . Wor k i s i n p r ogr e s sto e lucidate th is s t ructure in mo re deta i l .

B y com p ar a t iv e p o ly acr y lam id e g e l e l e c t rop h or e s i s t h e average c h a i n l e n g t h o ft h e H M W - s a c c h a r i d e s ( b a n d o f h i g h e s t 3 5 S 0 4 2 - i n t e n s i ty ) i s f o un d t o b e e q uiv a le n tt o 4 5 r i b o n u c l e o t i d e u n i t s ( F i g . 3 B ) .

3. The HMW-saccharide has a form-keeping functionHalobacteria g r o w n i n t h e presence o f t h e a n t i b i o t i c b a c i t r a c i n a r e n o l o n g e rr od -s h ap e d b ut quan t i t a t iv e ly con v e r t t o r e gu lar spheres (Mescher an d Strominger,

1975). I n a d d i t i o n these aut h or s r e p or t e d that t h e ov e r a l l ca r b oh yd r a t e contentw a s s o m e w h a t d i m i n i s h e d u n d e r t he in f l u e n c e o f b a c i t r a c i n . W e f o u n d that inthe presence of b ac i t r ac in a s u lp h at e d ce l l w a l l g l y c o p r o t e i n i s s y n t h e s i z e d , w h i c hd i f f er s f r om t h e n or m al su lp h at e d ce l l w a l l g l y c o p r o t e i n b y a s m a l l increase inm o b i l i t y o n S D S - g e l s , i n d i c a t i n g a l o w e r m o l e c u l a r w e i g h t . T o i n v e st ig a t e th es t r u c t u r a l d i ff e re n c e b e t w e e n t he n o r m a l 3 5 S 0 4 2 _ - l a b e l l e d c e l l w a l l g l y c o p r o t e i nan d t h e 3 5 S 0 4 2 " - g l y c o p r o t e i n f r o m Halobacteria l ab e l l e d i n t h e presence of b ac i t r ac in , p r on as e g lycop e p t id e s w e r e an a lyze d b y h ig h p r e s sur e l i q u i d c h r o m a t o g r a p h y o n a n a n i o n exchange c o l u m n : F i g . 6 A represents t h e r ad ioac t iv i t y p r o f i l eo f 3 5 S 0 4 2 ~ - g l y c o p e p t i d e s f r o m n o r m a l c el l w a l l g l y c o p r o t e i n . Pe a k s a to i d o n o tc o n t a i n a m i n o s u g a r s a n d therefore represent t he s u l p h a t e d l o w m o l e c u l a r w e i g h ts acch arid e s , w h e r e as p e ak z is caus e d b y t h e H M W -s ac ch ar id e s f r ac t ion . S e p ar a t i o n of t h e g lycop e p t id e s d e r iv e d f r om g lycop r ot e in o f ce l l s l ab e l l e d i n t h e presenceo f b a c i t r a c in is s h o w n i n F i g . 6 B : t h e s a m p l e c o m p l e t e l y la c k s t he H M W - s a c c h a r i d ep e a k . F r o m t h e absence o f t he H M W - s a c c h a r i d e i n s p h e r i c a l c el ls w e c o n c l u d ethat i t is t h e f un c t ion o f t h is g lycocon j ug a t e t o m ain t a in t he r od f or m t o Halobacteria.

4. The HMW-saccharide is synthesized via a lipid linked precusorT h e i n h i b i t i o n b y b a c i t r a c in o f t he i n c o r p o r a t i o n o f th e r ep e a t in g u n i t s a cc h ar id e

i n t o t h e g l y c o p r o t e i n a s w e l l as the sugges t ion that l i p i d i n t e r m e d ia t e s a re i n v o lv e din t h e b ios yn t h e s i s o f t h e am in o s ugar con t a in in g s acch ar id e (Mescher a n d Strominger, 1978) lead us to search fo r a l i p i d l i n k e d p r e cur s or o f t h is s acch ar id e . Fort h i s p ur p os e Halobacteria w e r e lab e l l e d i n v i v o w i t h 3 5 S 04 2 ~ in the presence ofe t h i d i u m b r o m i d e i n a c o n c e n t r a t io n i n h i b i t o r y f o r p r o t e i n s y n t h e si s. A n a l y s i sof tota l ce l l lysates b y S DS -g e l e le c t r op h or e s is y ie ld e d a s t r ik in g r e g u lar p a t t e r no f b an d s s im i la r t o that ob t a in e d b y e xh aus t iv e d ige s t ion o f t h e i s o la t e d 3 5 S 04 2 ~l ab e l l e d ce l l w a l l g l y c o p r o t e in ( F ig . 3 C ) .

Attempts to isolate th is mater ia l revealed that t h e r ad ioac t iv i t y cop ur i f i e d w i t ht h e m e m b r a n e . A f t e r sucrose 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 h e m a t e r i a l i n q u e s t i on w a s q u a n t i t a ti v e l y r ec o v e re d i n th e m e m b r a n e f r a c t i o n ( F i g . 7 A ) i n d i c a t i n g


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a - i z

a - i ^^("^WtMtfU" S O - k W l e l f / 0 ^ C f e ; ' a j a c k *e HMW-saccharide fraction. Radioactivity profile ofS O -labelled glycopeptides from cell wall glycoprote.n after separation by high pressure^T^^ j r rod shaped e,ls a) and rom pheri cai ^

F R A C T I O N

Fig 7 Sucrose density gradient centrifugation of a crude membrane fraction nf H , / * > ,to.* after ,n vivo labelling with - S04 - (A) and of a mixture of u a b e d c ude me m

show SdT , a n f ' S f t e d i , S ( V , H M W " h a r i d e fraction as a control"> The"inserts show SDS-gels of the corresponding peak fractions.


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it s l i p i d char ac t e r . The m a t e r i a l is best s o l u b i l i z e d w i t h a q u e o u s p r o p a n o l afterd e l i p i d a t i o n of t he p u r i f i e d m e m b r a n e s w i t h c h l o r o f o r m / m e t h a n o l . It is c o m p l e t e l yi n s o l u b l e in aq ueo us buf f e r s . T o i n v e s t i g a t e the s u g g e s t e d s t r u c t u r a l r e l a t i o n s h i po f the l i p i d - b o u n d r a d i o a c ti v e m a t e ri a l and the 8 5 S 0 4 - " - H M W - s a c c h a r i d e s , thep a t t e r n o b t a i n e d after sub j ec t io n to d i ff e re n t f r a g m e n t a t io n m e t h o d s w e r e c o m p a r e d . F i g . 8 s h o w s a h i g h v o l t a g e t h i n l a y e r e l e c t r o p h o r e s i s after m i l d a c i d h y d t o -lysis of the m e m b t a n e - b o u n d r a d i o a c t i v e m a t e r i a l (c) a n d of the H M W - s a c c h ar id e s ( d) . A s m e n t i o n e d a b o v e u n t re a te d H M W - s a c c h a t i d e s m i g r a te as a n s i n g l espo t (a), w h e r e a s u n t r e a t e d l ip id l i n k e d m a t e r i a l d u e to its l i p i d character doesn o t m i g r a t e at a l l in the aq uo us buf f e r sy s t em used (b). Iden t i t y of the sacchar idep a r t of b o t h m a t e r ia l s i s c o n f i r m e d b y th e p a tt e rn o b t a i n e d after h y d r a z i n o l y s i s a n dn i tr o u s a c id d e a m i n a t i o n ( F i g . 8 B , a to c).

Fie S T h e - S O , ' - l ip id -l in k ed material is a precursor of the HMW-saccharide.Sate1-SO7 lipid linked material and " S O , - HMW-saccharides after fragment on byS e n t metnods'were subjected to high voltage thin layer^e^phoresis J Oi k n A u u w wrrWides (al and 3 : ' S O , 2 - abelled hpid-hnked material (r>), untrearcu^ m ^ S S ^ S S iydrolysis (c)*and (d), respectively. B: after hydrazinolysis andS o u s acid dirmmation. HMW-saccharides (a), mixture of HMW-saccharides and l ,p,d-linked material (b), lipid linked material (c).

D i s c u s s i o nF i n g e r p r in t a n a ly se s a c c o r d i n g to Cleveland et a l . (1977) d e m o n s t r a t e d i d e n t i t y

of the cel l w a l l g l y c o p r o t e i n s f r o m Halobacterium halobium a n d Halobacteriumsalinarium. Ina d d i t i o n , : , r 'S04-' _ is i n c o r p o r a t e d i n t o t h e Halobacterium salinariumg l y c o p r o t e i n as w e l l , a n d the f r a g m e n t a t i o n p a t t e r n s of b o t h r a d i o a c t i v e g l y c o p r o t e i n s w e r e i d e n t i c a l (Dompert a n d Sumper, u n p u b l i s h e d ) . T h e r e f o r e o u r d a t am a y be c o m p a r e d w i t h the resul ts on the Halobacterium salinarium ce l l w a l l g l y c o p r o t e i n .

In agreement w i t h Mescher a n d Strominger (1976) we f o u n d a n e u t r a l d i s a c -c h a r i d e l i n k e d to the ce l l w a l l g l y c o p r o t e i n w h i c h c o n t a i n e d gle a n d g a l . We w e r en o t a b l e , h o w e v e r , to f i n d t ri sa c ch a r id e s c o n t a i n i n g g l e, gal a n d an u r o n i c a c i d .O n the o t h e r h a n d there w as a g l y c o p e p t i d e f r a c t i o n c o n t a i n i n g g l e , g l c U A and


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s u l p h a t e . I n v e s t i g a t i o n s c o n c e r n i n g the n a t u r e of the l i n k a g e and the s t ru c t u re ofthese saccharides are in p r o g r e s s .

T h e a m i n o su g ar c o n t a in i n g H M W - s a c c h a r i d e in our h a n d s s h o w e d q u i t e ad i f f e r e n t c o m p o s i t i o n t h a n t h a t d e s c r i b e d for the a m i n o s u g a r c o n t a i n i n g Halo-bacterium salinarium sa c c ha r i d e : we f o u n d n e i t h e r man nor glc but g a l a c t u r o n i ca c i d and su l pha t e w h i c h o c c u r in a p p r e c i a b l e a m o u n t s in the H M W - s a c c h a r i d e s(c . f. T a b l e 1). F r o m the i d e n t i t y of the f r a g m e n t a t i o n p a t t e r n s of H M W - s a c c h a r i d e s of 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 (c. f. F i g . 4) and f r o m the c o m p o s i t i o n ofi s o l a t e d f r a g m e n t s of 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 (c. f. Fig. 5) we c o n c l u d e t h a tth e so c a l l e d " h e t e r o s a c c h a r i d e " is a g l y c o c o n j u g a t e w h i c h is m a d e up by a s u l p h a t e d r e p e a t i n g u n i t : a s t ru c t u re w h i c h is s i m i l a r to that of the a n i m a l g l y c o -s a m i n o g l y c a n s . The a b o u t 15 b a n d s f o u n d on SDS gels of a 3 5 S 0 4 2 ~ - l a b e l le d H M W -sa c c ha r i d e f ra c t i o n t hu s represen t a b o u t 15 d i f f e ren t c ha i n l eng t hs of the r e p e a t i n gu n i t sa c c ha r i d e , the d i s t a nc e b e t ween two adjacent b a n d s b e i n g due to one rep e a t i n g u n i t .

F r o m the f inding that Halobacterial s p h e r e s g r o w n in the presence of b a c i t ra c i nc o m p l e t e l y l a c k the H M W - s a c c h a r i d e f r a ct io n we c o n c l u d e t h a t t h i s g l y c o s a m i n o -g l y c a n - l i k e s t ru c t u re is i n v o l v e d inm a i n t a i n i n g the rod f o r m of n o r m a l Halobac-teria.T h e b i o s y n t h e s i s of t h i s su l pha t ed repea t i ng u n i t sa c c ha r i d e seems to be d i f f e ren tf r o m t h a t k n o w n for the g l y c o s a m i n o g l y c a n s (for a r e v i e w see Roden, 1980): thesa c c ha r i d e c ha i n is not e l o n g a t e d step by step on a p r o t e i n b o u n d l i n k e r r e g i o n ,b u t the H M W - s a c c h a r i d e s are s y n t h e s i z e d on and t r a n s f e r r e d f r o m a l i p i d c a rr i e r .R e m a r k a b l y , the sulphate res idues are detectable a l rea d y in the l i p i d l i n k e d pre c u r s o r at all degrees of p o l y m e r i z a t i o n . T h i s a g a i n seems to be u n u s u a l , as in thesy n t hes i s of h e p a r i n , for ex a mpl e , su l pha t e res i d u es are i n t r o d u c e d i n t o the m o l e c u l e o n l y after g l y c o s id i c p o l y m e r i s a t i o n is f i n i s h e d (Lindbal andHook, 1978).T h e l i p i d l i n k e d to the H M W - s a c c h a r i d e p r e cu r so r a c c or d i n g to Mescber et al.(1976) and Mescber and Strominger (1978) m o s t p r o b a b l y is a p o l y p r e n y l of 11to 12 i s o p r e n e u n i t s . S o l u b i l i t y of the p r e c u r s o r in a s o l v e n t m o r e h y d r o p h i l i c( p r o p a n o l / w a t e r , 1/1) t ha n the s o l v e n t s n o r m a l l y u s e d for p o l y p r e n y l - s u g a r c o m p o u n d s (CHCI3/CH3OH/H2O = 10/10/3, for e x a m p l e ) is e a s i l y e x p l a i n e d by thepresence of n e g a t i v e charges i n t r o d u c e d by the sulphate res idues .W e c o u l d not yet u l t i ma t e l y d ec i d e whet her the o b served he t e ro g ene i t y in cha inl e n g t h of the H M W - s a c c h a r i d e is c a u sed a r t i f i c i a l l y by the very low su l pha t ec o n c e n t r a t i o n necessary in 3 5 S 0 4 2 _ - l a b e l l i n g ex pe rim e n t s or w h e t h e r it s i m p l yreflects a re l a t i ve l y b ro a d c ha i n l eng t h spec i f i c i t y of the c o r r e s p o n d i n g t r a n s f e r a s e .T he l a t t e r is p r o b a b l y t r u e , as the pa t t e rn onSDS gels of the H M W - s a c c h a r i d e sis not i n f l u e n c e d by v a r y i n g the s u l p h a t e c o n c e n t r a t i o n in3 5 S 0 4 2 " ~ - l a b e l l i n g e x p e r i m e n t s b e t w e e n a b o u t 7 1 0 ~ 6 M to 5 1 0 ~ 3 M .

Acknow ledgment. This work was supported by the Deutsche Forschungsgemeinschaft(SFB 43, Regensburg).

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