Structural Studies of the Carbohydrate Moieties of Rat Kidney

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THEJOURNALU P BIOLOGICAL CHEYISTRY Vol 258, No. 2, Issue n f .January 25, pp. 109U-lIO7, 1983 Pnnled tn

U S A.

Structural Studiesof the Carbohydrate Moieties of Rat Kidney y-Glutamyltranspeptidase AN EXTREMELY HETEROGENEOUS PATTERN ENRICHED WITH NONREDUCING TERMINAL ACETYLGLUCOSAMINE RESIDUES*

N-

(Received for publication, May 10, 1982)

Katsuko Yamashita, Akira Hitoi, Yoshiko MatsudaS, Akihiko TsujiS, Nobuhiko KatunumaS, and Akira Kobata From the Departmentof Biochemistry, Kobe University School of Medicine, Chuo-ku, Kobe, J a p a n a n dthe +Department of Enzyme Chemistry, Institute for Enzyme Research, School of Medicine, Tokushima University, Tokushima, J a p a n

The carbohydrate moieties of y-glutamyltranspeptidase purified from rat kidney were released as oligosaccharides by hydrazinolysis. Fractionation of the oligosaccharide mixturebypaper electrophoresis and Bio-Gel P-4 columnchromatographyandstructural study of each component bysequential exoglycosidase digestion in combination with methylation analysis and periodate oxidation have revealed that it is composed of 23 neutral oligosaccharides, monosialyl derivatives of 67 oligosaccharides, disialyl derivatives of 62 oligosaccharides, and trisialyl derivatives of 5 oligosaccharides. Theneutral oligosaccharides are either high mannose type or biantennary complex type, and the acidic oligosaccharides are bi-, tri-, and tetranntennary complex type sugar chains. Most of the complex type sugar chains contain an N-acetylglucosamine residue at the C-4 position of the p-mannosyl residue of their trimannosylcore.Another characteristic feature of these complex type sugar chains is that they are enriched with nonreducing terminal P-N-acetylglucosamine residues.

in regardto theirisoelectric points by DEAE-cellulose column chromatography showed identical molecular weights, amino acidcompositions, and carboxyl termini. Furthermore, the isoelectric points of the isozymes were reversibly correlated with their sialic acid contents. Therefore, protein skeletons of y-glutamyltranspeptidase isozymes might beidentical and the difference of the isozymic forms is assumed to be mainly due to the difference in the degree of sialylation of the enzymes (3). However, it is still an open question whether the degree of sialylation only causes such a remarkable heterogeneity of rat kidney y-glutamyltranspeptidase or the whole structures of sugar chains bound to enzyme the are heterogeneous among isozymic forms. Although y-glutamyltranspeptidases purified from various tissues are immunologically identical, the degree of their heterogeneities varied by tissues (4).The most extreme case is human bile y-glutamyltranspeptidase, which is composed of a single component with an isoelectric point of pH 6.0 ( 5 ) .The physicochemical characteristics of y-glutamyltranspeptidase may also change according to the physiological state of cells, especially in malignant transformation (6-10). It has already been well documented that the structuresof sugar chains of y-Glutamyltranspeptidase is a membrane-bound glycopro- glycoprotein change with cell transformation (11, 12). Theretein widely distributed in various organs suchas kidney, liver, fore, y-glutamyltranspeptidases could be useful materials to small intestine, pancreas, andbrain. It is solubilized from the study the tissue-specific sugar chain formation of glycoproof sugarchains of membrane, with either detergent or proteinase treatment. y- teinsandthetransformationalchange light chains of yGlutamyltranspeptidase solubilized with detergent is a hy- glycoproteins. Since both the heavy and glutamyltranspeptidase are glycoproteins and since an NH2drophobic protein containing the domain that anchors the enzyme to the membrane. On the other hand, y-glutamyl- terminal portionof heavy chainis associated with the plasma membrane and the light chain carries a catalytic site, the transpeptidase solubilized with proteinase is a hydrophilic protein that retained the complete catalytic activity butlost study of the sugar structure of y-glutamyltranspeptidase is the membranebinding segment. Thedifferences of the molec- another interesting project for the future from the viewpoint ular weights of y-glutamyltranspeptidase solubilized with de- of the biosynthesis and anchoring mechanisms of y-glutamyltranspeptidase (1).As the first step of this line of study, the tergent and y-glutamyltranspeptidase solubilized withpromoieties of y-glutamylteinase is approximately 5,000 (l), which is about 7% of the structures of thecarbohydrate molecular weight of y-glutamyltranspeptidase solubilized with transpeptidase solubilized with proteinase from rat kidney (which will be called y-glutamyltranspeptidase in the remaindetergent calculated fromtheamino acidcomposition. yGlutamyltranspeptidase solubilized with proteinase contained ing part of this paper) will be reported in this paper. 92%of total hexose of y-glutamyltranspeptidase solubilized MATERIALS AND METHODS AND RESULTS’, with detergent. Rat kidney y-glutamyltranspeptidase is composed of many ’ Portions of thispaper (including “MaterialsandMethods,” isozymic forms (2). Several isozymic forms which were purified “Results,” Tables 1-111, and Figs. 1-10) are presented in miniprint at the endof this paper. Miniprintis easily read with the aid of standard magnifying glass. Full size photocopies are available from the Journal Research and for Cancer Research, the Ministry of Education, Sci- of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. includea ence, and Culture of Japan and by a research grant from the Yaman- Request Document No. 82M-1231, cite the authors, and check or money order for $13.60 per set of photocopies.Full size ouchi Foundation for research on metabolic disorders. The costs of publication of this article were defrayed in part by the payment of photocopies are also included in the microfilm edition of the Journal page charges. This article must therefore be hereby marked “aduer- that is available from Waverly Press. Theabbreviations used are: NeuAc, N-acetylneuraminic acid; tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate GlcNAc, N-acetylglucosamine;XylNAc,N-acetylxylosamine; SDS, this fact.

* This work was supported in part by grants-in-aid for Scientific

1098

1099

Carbohydrate of Rat y-Glutamyltranspeptidase Neutral

Oligosaccharides

tFucal

iGlcNAcR1 4

J-

6 (Ga101+4)0 2 1 2

Acidic

*GlcNAcOl+2Manall 4 6ManB1+4GlcNAcB1+4G1cNACoT *GlcNAc(31+2Manalf3

Oligosaccharides 2

[NeuAca2+3 (6)GalB1+4]

1 % ~

IGalOl+4) Z % O

t

t Fuca 1

I

J.

*Gl~NAcBl+ZManal\~ 6 4 ManB1+4GlcNAcB1+4GlcNAcOT *GlcNAcB1+2Manalf3 Fucal

[NeuAcct2+3 (6)GalB1+4]1 1 1 3

G ~ C N A BC1

GlcNAcBl

+

J-

*GlcNAcBl+2Manal\ 6 4 *GlcNAcBlh 6Man~1+4GlcNAcB1+4GlcNACoT Mana~f3 *G~CNACBV~

(*Gal~1+4GlcNAcf31+3Gal~l+4) 1212

[NeuAca2+3 ( 6 )

1 12.3 (*GalBi+4)z'(ro

i

*GlcNAcB1\6

(*Gal~1+4GlcNAcB1+3Gal51+4)12,

[NeuAca2+3(6)]1~3 (*GalBl+4)2'(ro

GlcNAcB1

Manal * G I ~ N A ~ B I ~ ~ *GlCNACBl >ManB1 +4RI \Manal * G ~ ~ N A ~ B I M ~

+

FIG. 11. Summary of the structures of all oligosaccharides liberated from rat kidney y-glutamyltranspeptidase by hydrazinolysis. RI = G l c N A c ~ ~ 4 ( F u c a l ~ 6 ) G l c N A c 0 ~ . synthesizing y-glutamyltranspeptidase. Another characteristic feature of the sugar chains of yIt was found that the carbohydratemoiety of rat kidney yglutamyltranspeptidase is that most of them have the bisect glutamyltranspeptidase is extremely heterogeneous as docuN-acetylglucosamine residue.," Harpaz and Schachter (14)inmented in Figs. lA and 2, D to I . This evidence will explain dicated that addition of the bisect N-acetylglucosamine to the the basis for the heterogeneityof this enzyme. The structural hybrid type sugar chain such as studies of each fraction, as described in detail in Miniprint, indicated that it is a mixture of neutral and acidic oligosacManolh6 charides assummarized in Fig. 11. ,panu1\6 Manal ,3ManBl+4GlcNAc~l~4GlcNAc+Asn --One of the characteristic featuresof the carbohydratemoiGlcNAcgl+ZManal ety of y-glutamyltranspeptidase is that it is enriched with nonreducing terminal /3-N-acetylglucosamine residues. This inhibitstheremoval of itsa-mannosyl residuesby Golgi result indicates that the addition of /?-galactosyl residues is a membrane a-mannosidase probably because of the steric efrate-limiting step in the formation of the sugar chains of rat fect of the newly introduced N-acetylglucosamineresidue. kidney y-glutamyltranspeptidase, probably because the level Such a steric effect of the bisect N-acetylglucosamine residue of galactosyltransferase is relatively low in the kidney cells may also inhibit the addition of /?-galactosyl residue in the DISCUSSION

sodium dodecyl sulfate. Subscript OT used is in this paper to indicate The P-N-acetylglucosamine residue linked at the C-4 position of NaB['H]4-reduced oligosaccharides. All sugars mentioned in this pa- the P-mannosyl residue of the trimannosyl core will be called bisect per were of D-configuration except for fucose which has an L-config- N-acetylglucosamine according to the suggestion of Carver and Grey uration. (37) in order to discriminate from other N-acetylglucosamine residues.

Carbohydrate of Rat y-Glutamyltranspeptidase

1100

outer chain moieties of sugar chains. Actually many sugar chains with the bisect N-acetylglucosamine residue and incomplete outer chains have been found in hen egg albumin (15, 16), ovotransferrin (17), and ovomucoid (18).Therefore, this possibility is worth studyingenzymologically in the future. The bisect N-acetylglucosamine residue is also found in the asparagine-linked sugar chains of human erythrocyte membrane glycoproteins (19-21) and immunoglobulins(22). It must be recalled that kidney, oviduct, and blood cells are all derived from mesenchyme. Since y-glutamyltranspeptidase is widely distributed in various organs, a comparative study of their sugar chains may indicate moreclearly whether the bisect N-acetylglucosamine residue is specifically distributed in the tissue of mesenchymal origin or not. The presence of the bisect N-acetylglucosamineresidue created severalcomplicated results inexoglycosidase digestion of the oligosaccharide. Baenziger and Kornfeld (23) reported that the bisect N-acetylglucosamine residue in the following sugar chain cannot be removed by jack bean P-N-acetylhexosaminidase digestion: GlcNAcBl i

GlcNAC61+2Manoh, 4 ~anB1+4GlcNAcgli4GlcNAc+Asn GlcNAcgl+2Manol

However, the bisect N-acetylglucosamine residues in various complex-typeasparagine-linked sugar chains are not completely resistant to jack bean P-N-acetylhexosaminidase digestion but are hydrolyzed at a different rate. Therefore, special care as to the concentration of the enzyme and substrate is required in studying the structuresof such sugar chains. For example, when 5 nmol of the following oligosaccharide were incubated with 3units of jack beanP-N-acetylhexosaminidase under the condition described under “Materials and Methods,”

Oligosaccharide I

and Oligosaccharide I1

Therefore, digestion with a mixture of diplococcal P-N-acetylhexosaminidase and &galactosidase was effectively used to determine the location of the three outer chains: Mantvl- 6 (GlcNAcPl 4Mantv 1.--, 3)Manpl- 4GlcNAcoT, andGlcNAc~I-6(GlcNAcfll-2)Manal-,6(Mantvl--f3) Manpl+4GlcNAco~ were obtained from oligosaccharides I and 11, respectively. This method,however, could not be used for the structural study of the oligosaccharides reported in this paper because the GlcNAcpl+3Gal linkages in the following oligosaccharides are not cleaved by diplococcal 0-Nacetylhexosaminidase, probably because of the stericeffect of the bisect N-acetylglucosamine residue. --f

As reported in a previouspaper (16),the bisect N-acetylglucosamine residues in the following oligosaccharides are quite resistant to periodate oxidation. ?Mano?,, G1cNAc61 ,3~ana~ -0 Manal ‘6,“-t31+4GlcNAc +GalE1+4Glc:NACOlOT ,*Manu1 GlcNACBl +

GlcNACBl..

Fuca 1

G1cNAc61 ;;Mano1 1 4

i

+

GlcNAc8l

GICNACslh

p”””‘

,~Manjl+4GlcNAcfI14C1~NAcOT

,2Manal GlcNAcBl

it was completely converted to Fucal i

Manalk6 ,3ManB1+4GlCNAcBl*4GlcNAcOT Mane 1

When the amount of the undecasaccharide was decreased to 1 nmol, only 20% was converted to thehexasaccharide. When 1nmol of the undecasaccharide was incubated with0.3 unit of the enzyme, the hexasaccharide was not produced at all. By repeatingtheincubationunderthelast conditionseveral times, only two N-acetylglucosamine residues were removed from the undecasaccharide. In contrast, the decasaccharide which is free from the bisect N-acetylglucosamine residues is completely converted to thehexasaccharide by a single incubation with the enzyme under the last condition. When the outer chain N-acetylglucosamines are substitutedby ,f3-galactosyl residues,the bisect N-acetylglucosamine residue is more resistant to the enzyme action and 4 units of jack bean p-Nacetylhexosaminidase are required to reach more than 95% hydrolysis. Therefore, this amountof enzyme was usedin the standard digestion describedunder “Materials and Methods.” As reported previously (24), the GlcNAcpl-3Gal linkages in the following oligosaccharides are hydrolyzed by diplococcal p-N-acetylhexosaminidase digestion.

However, the bisect N-acetylglucosamine residues in components a to 1 were destroyed completely by periodate oxidation as reported in this paper. Probably, the presence of N,N“ diacetylchitobiitol residue instead of N-acetylglucosaminitol at the reducing termini of these components is the cause of the difference in susceptibility to periodate oxidation. Acknowledgment-We secretarial assistance.

wish to thank Ikuko Ueda for her expert

(Tokyo) 87, 1243-1248 4. Miura, T., Matsuda, Y., Tsuji, A,, and Katunuma, N. (1981) J .

Biochem. (Tokyo) 89, 217-222 5. Tsuji, A., Matsuda, Y., and Katunuma, N. (1980) Biomed. Res. 1, 410-416 6. Taniguchi, N. (1974) J. Biochem. (Tokyo)75,473-480 7. Novogrodsky, A., Tate, S., and Meister, A. (1976) Proc. Natl. Acad. Sei. U. S. A . 73, 2414-2418 8. Jaken. S.. and Mason, M. (1978) Proc. Natl. Acad. Sei. U. S. A . 75,1750-1753 9. Tsuchida, S., Hoshino, K., Sato, T., Ito, N., and Sato, K. (1979) Cancer Res. 39,4200-4205 10. Yamamoto, H., Sumikawa, K., Hada,T., Higashino, K., and Yamamura, Y. (1981) Clin. Clim. Acta 111,229-237

Carbohydrate of Rat y-Glutamyltranspeptidase 11. Takasaki, S., Ikehira. H., and Kobata, A. (1980) Biochem. Biophys. Res. Commun. 93, 735-742 12. Yamashita, K., Tachibana, Y., Takeuchi,T.,andKobata, A. (1981) J. Biochem. (Tokyo) 90, 1281-1289 13. Takasaki, S., and Kobata, A. (1978) Methods Enzymol. 50,50-54 14. Harpaz, N., and Schachter, H. (1980) J. Biol. Chern. 255,48944902 15. Tai, T.,Yamashita, K., Ito, S., and Kobata, A. (1977) J . Biol. Chem. 252,6687-6694 16. Yamashita, K., Tachibana, Y., and Kobata, A. (1978) J . Biol. Chem. 253,3862-3869 17. Spik, G., Fournet, B., and Montreuil, J. (1979) C. R.Hebd. Seances Acad. Sci. Ser. D Sci. Nat. 288, 967-970 18. Bayard, B., and Montreuil, J. (1974) inMethodologie de la Structure et du Metabolisme des Glycoconjuguts, pp. 209-218, Centre National de la Recherche Scientifique, Paris 19. Yoshima, H., Furthmayr,H.,andKobata, A. (1980) J. Biol. Chem. 255,9713-9718 20. Irimura, T., Tsuji, T., Tagami, S., Yamamoto, K., and Osawa, T. (1981) Biochemistry 20,560-566 21. Tsuji, T.,Irimura,T.,and Osawa, T. (1981) Glycoconjugates (Yamakawa, T., Osawa, T., and Handa, S., eds) pp. 277-278, Japan Scientific Society Press, Tokyo 22. Kornfeld, R., and Kornfeld, S. (1976) Annu. Reu. Biochem. 45, 217-237 23. Baenziger, J., and Kornfeld, S. (1974) J. Biol. Chem. 249, 7260-

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7269 24. Ohkura, T., Yamashita, K., and Kobata, A. (1981) J . Biol. Chem. 256, 8485-8490 25. Tate, S.S.,and Meister, A. (1974) J . Biol. Chem. 249,7593-7602 26. Yamashita, K., Liang, C.-J., Funakoshi, S., and Kobata, A. (1981) J. Biol. Chem. 256, 1283-1289 27. Liang, C.-J., Yamashita, K., and Kobata, A. (1980) J. Biochem. (Tokyo) 88, 51-58 28. Li, Y.-T., and Li, S.-C. (1972) Methods Enzymol. 28, 702-713 29. Glasgow, L. R., Paulson, J. C., and Hill, R. L. (1977) J. Biol. Chem. 252,8615-8623 30. Uchida, Y., Tsukada, Y., and Sugimori, T. (1974) Biochirn. Biophys. Acta350,425-431 31. Sugahara, K., Okumura, T., and Yamashina, I. (1972) Biochim. Biophys. Acta 268,488-496 32. Yamashita, K., Ichishima, E., Arai, M., and Kobata, A. (1980) Biochem. Biophys. Res. Commun. 96, 1335-1342 33. Ichishima, E., Arai, M., Shigematsu, Y., Kumagai, H., and Sumida-Tanaka, R. (1981) Biochim. Biophys. Acta 658,45-53 34. Endo, Y., Yamashita, K., Tachibana,Y., Tojo, S.,and Kobata, A. (1979) J. Biochem. (Tokyo) 85,669-679 35. Takasaki, S., Mizuochi, T., and Kobata, A. (1982) Methods Enzymol. 83, 263-268 36. Yamashita, K., Ohkura, T., Yoshima, H., and Kobata, A. (1981) Biochem. Biophys. Res. Commun. 100,226-232 37. Carver, J. P., and Grey, A. A. (1981) Biochemistry 20, 6607-6616

1102

Carbohydrate of Rat y-Glutamyltranspeptidase S u p p l e m e n t a lM a t e i l a l s

to

STRUCTURAL STUDIES O F THE CiiRBOHYDRATE MOIETIES O B RAT KIDNEY PEPTIDASE: AN EXTREMELY HETEROGENEOUS PATTERN ENRICHED WITH NONREDUCING TERMINAL N-ACETYLGLUCOSAMINE RESIDUE.

~-GLUT-YLTRANS.

The purIfIcdtlon S t e p si n c l u d i n gs O l u b r l i z a t l O nw i t hp a p a l n , anmon~um s u l f a t e f r a c t l o n a t l o n , g e l f i l t r a t i o n on Sephadex G-200 and Con A-Sepharose column chromatography were performed accordlnq t o t h e method p r e v i o u s l y F u r t h e rP u r l f L C a t l O np r o c e d u r e s were m o d i f l e d as f o l l o w s . d e s c r l k d 131. The e l u t e from t h e Con A-Sepharole column was S u b l e c t e d to g e l f l l t r a t l o n on U l t r ' o g e l AcA 34 and t h e f r a C t l O n S c o n t a l n l n g t h e enzyme a c t l v l t y Were pooled and c o n c e n t r a t e d by Amlcon u l t r a f l l t r a t l o n s y s t e m . The a c t l v ef r a c t l o n was dialyzed aqa1Mt 5 0 MI m i d a r o l eb u f f e r pH 7 . 2 o v e r n l q h t ,a p p l l e d on t h e h y d r a x y l a p a t l t e column e q u l l r b r a r e d the same b u f f e ra, n d eluted wrth lo0 MI sodxum p h o s p h a t eb u f f e r , pH 7 . 2 . The p o l e d a c t l v e f r a c t m n was c o n c e n t r a t e d by AmlcOn f l l t r a t l o n , a p p l i e d on a Bia-Gel P-10 column w a s h e dw l t hd e z o n l z e d water extensively a n de l u t e dw l r hd e l o n l z e dw a t e r . The s a l t f r e e protein fraction was l y o p h l l l z e d . The y l e l d o f i - q l u t a m y l t r a n l p e p t i d a s e was 70 m q . The s p e c l f l c a c t l v l t y o f t h l s enzyme was 6 6 3 U/mq p r o t e m . When t h i s enzyme was s u b l e c t e d to S D S - p o l y a c r y l a m l d eq e l elecrrophores~s t h e p a t t e r n showed were 46 0 0 0 and o n l y two b a n d sa n dt h em o l e c u l a rw e r q h t so ft h e two $ & n i t s 23.000. *-Glutamyltranspeptrdaee a c t l v i t y was m e a s u r e ds p e c t r o p h o t o m e t r i c a l l y Tate and Melster (251. One Unitof enzyme a c t i v l t y w a s by t h e methodof d e f l n c d as t h e amount o f t h e enryme r e q u i r e df o r the f o r m a t i o no f 1 vmol Of p r o d u c tp e r m m from L - , - q l u t a m y l - p - n l t r o a n l l l d e .

Chemlcalx ond hnlynies NaBl'H1* 1230 mCilmmol1 wag purchased from New EnglandNuclear,Boston, Mass. NaB[ Hjr 198%) was puzchasedfrom Merck C o . , Damstadt. Human c e r u l a p l a s m l n was k i n d l ys u p p h e d by Green C r o s sC o r p r a t l o n , Bovlne p a n c r e a t i c r i b o n u c l e a s e B and bovine epididymal cI-L-fucosIdase Osaka. were p u r c h a s e d from S l p a Chemlcal CO., S t . L i l u l s , Mo. J a c k bean 8 - q a l a c t o s l d a s e ,I - m a n n o s ~ d a s ea n d 8-N-acetylhexosamlnldase were p u r l f i e df r o ml a c k bean m e a la c c o r d l n qt ot h em e t h o d Of Lx and LI (281. D L ~ ~ E O S-N-acetylhexosamLnlC C ~ d a s e was p n r l f i e d from c u l t u r e f l u l d Of U ~ p l O m e ~pneunantoe d a c c o r d i n g to t h e S i a l i d a s ep u r i f l e df r o mA r t h r o b a c t e ru r e a f a c x e n s method Of Glasgov et al. ( 2 9 1 . (301 was p u r c h a s e df r o mN a k a r a l Chemicals, Ltd.,Kyoto.Snall8-mannosldase WhLCh 1311 was k i n d l y supplied by S e l k a q a k u Kogyo Co.,Kyoto.o-nannosldase c l e a v e s o n l y Manul-2Man l l n k a q e 132) was p w z f i e d from Asperglllvs s a ~ l o l a c c a r d l n q to themethod of I c h l s h l m a e t a1 1331. Bia-Gel P-4 under 4 0 0 mesh ( c o n t r o l n u m b e r 2083331 Wdh purchasedfrom 010-Rad L a h o r a t o r l e s .R i c h n d , Ca. C o l l a d l o n bag was p u r c h a s e df r o mS a r t O r I u sC a . .a t t t n q e n . Paper Chronrorogrophy and Paper EleCtmphDreSiS Descendlnq paper chromatography was ~ r f o r m e dv l t ht h ef o l l o v l n qs o l v e n t ;b u t a n o l - l / e t h a n o l / w a t e r1 4 : l : 1). H l q hV o l t a q ep a p e re l e c t r o p h a r e s l s w a s performed Y S L D ~p y r z d m e j a c e r a t e a p o t e n t x a lo f 7 3 vlcm b u f f e r . pH 5.4 ( p y r i d l n e j a c e t l c a c l d h a t e r , 3 : 1 : 3 8 7 l a t f a r 2 h or 0 . 0 6 M b o r a t eb u f f e r , pH 9.5 a t 40 V/cm f o r2 . 5 h. Whatman N0.1 was used f a ra n a l y t r c a le x p e r l m e n t sa n d Whatman N o . 3 M f o r preparative p u r p o s e s . Gel Permeatton Chmmaloqraphy Blo-Gel P-4 [ u n d e r 400 mesh) column chromataqraphy was performed u51nq column. ( 2 . 5 m x 2 cm r.d.1 equLpped v l t h a water Iacket. D u r l n q o p e r a t i o n ,t h e column v a s k e p t at 55-C by c ~ r c u l a t i n q warm w a t e r I" t h e J a c k e t . Sugar5 were e l u t e df r o mt h e column v l t h distilled water a t a f l o w rate o f 0 . 3 mlimln u 5 m g model 6000A s o l v e n t d e l i v e r y s y s t e m (Wafers A 5 5 0 c l a t e 5 , Inc., MLlfOrdl. A d l f f e r e n t l a lr e f r a c t o m e t e r , Shodex RI model SE-11 IbhowdDenko L t d . ,T o k y o ) , was ueed f o rm o n l t o r l n q sugars e l u t e d

from t h e column.

RESULTS P q e r Eleclmphoresm of OlLgosocchondes Ltberaled f m m I -Clulomylfrmspepltdase Ammo a c i d analysis o f 1 - q l u t a m y l t r a n s p e p t l d a S e I n d i c a t e d t h e presence o f qluco*amme b u tn o tg a l a c t o s a m i n e .T h e r e f o r e ,t h e Sugar c h a m so fr h l sq l y c o p r o t e l n s h o u l d a l l be a s p a r a g m - l i n k e d suqar chains. When a p a r t o ft h er a d l o a c t l v e I - q l u t a m y l t l = n s p e p t l d a s e by h y d r a r l n a l y O l l q o s a c c h a r i d ef r a c t i o nO b t a l n e df r o m SIP Was s c i b l e c t e dt op a p e re l e c t r o p h o r e s l s . I t was s e p a r a t e dI n t o a n e u t r a l IN1 and f i v ea c i d i cf r a c t i o n s (Al-ASI as shovn In F l q . IA. The peicenr molar I - ~ ~ L o f N, Al, A 2 , A I , A4 and A5 c a l c u l a t e d On t h e b a s i s o f t h e i r r a d ~ o a c f l v l t i e s was 2 9 . 8 ,1 5 . 7 ,1 3 . 8 ,2 2 . 0 , 9.6 and9.1. When d q e s t e dw i t hs l a l i d a s e , all f i v ea c l d i co l l q o s a c c h a r l d e fractions were c o m p l e t e l yc o n v e r t e d to neutral o l l q o s a c c h a r i d ef r a c t l o n s I F l q . 1 6t oF ,d o t t e d l i n e s ) . By m l l da c l d hydrolysis 10.01 N HC1 a t lO0'c f o r 3 minl whereby a p o r t r o no ft h eo r l q l n a l OllqOsaCCharLdes s t r l l remained, A1 and A2 were c o n v e r t e dt on e u t r a lo l l q o s a c c h a r i d e so n l y I F l q . 18and C . s o l l dl i n e a l . A3 and A 4 qave one, and A5 gave t w o a d d l t l o n a l a c l d l c peaks by t h e p a r t i a l d e s i a l y l a t i o n I F l q . 1D. E and F , s o l i d llnes, r e s p e c t l v e l y ) .T h e s e r e s u l t s x n d l c a t e dt h a tt h ea c l d l cn a t u r eo ft h ef l v e

O

Carbohydrate of Rat 7-Glutamyltranspeptidase

s a c c h a r l d es h o u l dh a v et h ef o l l o w l n gs t r u c t u r e .

1103

Carbohydrate of Rat y-Glutamyltranspeptidase

1104

a n

360 420 ELUTION VOLUME ( M U ~~

ELUTION

VOLUME (MU

1105

Carbohydrate of Rat y-Glutamyltranspeptidase FroQmenfs

VIII

VI1

VI

V

GlcNACBl

IV

ELUTION

VOLUME (MU

Ill

II

I

211) 300 3KI 360 ELUTION VOLUME (ML) Flq. 8 . Jack bean E-qalactasldase digestion of components 0 to d l . The radloactlve products were analyzed by 810-Gel P-4 column Chromatography. The black armwe are the Qas ~n Ag. 2 and the whtle wmws lndlcate the eluting pos~tion5of f M p m n t a I to V I . The black trlongles I" each column ~ n d l c a t e the elutlnq po51t1ons of components before the enzyme treament. A . the product from componenr a, 8 . that from CDmpOnenL b; C. that from component c l . D,that from component C I , E. that from component e , ; F . that from compoenr d l . C, that from component d i ; H , that from component d , .

Carbohydrate of Rat y-Glutamyltranspeptidase

1106

GlcNAcPl

GlcNAcgl

GlcNAcgl

2x)

xx)

330

360

ELUTION VOLUME (MU

GlCNACBl

4 le 'I h

GlcNACB1

GlcNAcg1

1

GlcNAcBl

1107

Carbohydrate of Rut y-Glutamyltranspeptidase Table I. Yerhylatlon analysls of fragments I to V I I 1 , m n p o n e n t

2 and thelr

p a r t i a l deqradatlon producCs.

Componenf

Fucltol 2.3,I-Trl-P-methyl 11.5-di-O_-acetyll

I

I1

0.8

0.9

1.1

0.9

2.1

1.8

0.9

1.1

0.8

0.9

traceb)rrace

The dotted

The peak

The peak I"

e

VI11

4D

-4 C

Fxg.

0.8

0

0

0.8

0.8

0.7

0

0

0

0

0

0

l 2.3.1.6-Tetra-0-methyl 11.5-d1i)-acetyll

0

0

0

0

0

0

0

0

3.3

1.8

0

0

3,