Glycosyl transferases in chondroitin sulphate biosynthesis. Effect of acceptor structure on activity.
The D-glucuronosyl (GlcA)- and N-acetyl-D-galactosaminyl (GalNAc)-transferases involved in chondroitin sulphate biosynthesis were studied in a microsomal preparation from chick-embryo chondrocytes. Transfer of GlcA and GalNAc from their UDP derivatives to 3H-labelled oligosaccharides prepared from chondroitin sulphate and hyaluronic acid was assayed by h.p.l.c. of the reaction mixture. Conditions required for maximal activities of the two enzymes were remarkably similar. Activities were stimulated 3.5-6-fold by neutral detergents. Both enzymes were completely inhibited by EDTA and maximally stimulated by MnCl2 or CoCl2. MgCl2 neither stimulated nor inhibited. The GlcA transferase showed a sharp pH optimum between pH5 and 6, whereas the GalNAc transferase gave a broad optimum from pH 5 to 8. At pH 7 under optimal conditions, the GalNAc transferase gave a velocity that was twice that of the GlcA transferase. Oligosaccharides prepared from chondroitin 4-sulphate and hyaluronic acid were almost inactive as acceptors for both enzymes, whereas oligosaccharides from chondroitin 6-sulphate and chondroitin gave similar rates that were 70-80-fold higher than those observed with the endogenous acceptors. Oligosaccharide acceptors with degrees of polymerization of 6 or higher gave similar Km and Vmax. values, but the smaller oligosaccharides were less effective acceptors. These results are discussed in terms of the implications for regulation of the overall rates of the chain-elongation fractions in chondroitin sulphate synthesis in vivo.
D000818:Animals; D002642:Chick Embryo; D002807:Chondroitin / Q000031:analogs & derivatives*; D002809:Chondroitin Sulfates / Q000096:biosynthesis*; D002851:Chromatography, High Pressure Liquid; D002854:Chromatography, Paper; D004590:Electrophoresis, Paper; D005700:Galactosyltransferases / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000378:metabolism*; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D017350:N-Acetylgalactosaminyltransferases; D009844:Oligosaccharides / Q000378:metabolism; D013379:Substrate Specificity
D009844:Oligosaccharides; D002807:Chondroitin; D002809:Chondroitin Sulfates; D005700:Galactosyltransferases; D017350:N-Acetylgalactosaminyltransferases; C003742:UDPgalactosamine-galactose acetylgalactosaminyltransferase; D014453:Glucuronosyltransferase