Characterization of a glycosphingolipid beta-N-acetylgalactosaminyl-transferase activity in cultured hamster (nil) cells.
The activity of a glycosphingolipid N-acetylgalactosaminyltransferase (GalNAc transferase) in cultured hamster fibroblasts (NIL-8) was characterized with respect to substrate binding, acceptor specificity, pH optimum and detergent requirements. Of the glycosphingolipid acceptors tested, transferase activity was observed only with globotriaosylceramide. The apparent Km values for uridinediphosphate-N-acetylgalactosamine and globotriasylceramide were 0.14 and 0.42 mM, respectively. The enzyme required Mn2+ for maximum activity (4 mM), and Mg2+ was not able to replace Mn2+. Of the detergents tested, sodium taurodeoxycholate gave the greatest activation of the enzyme at 1 mg/ml. A broad pH optimum (4.5-8.0) was obtained, with maximum activity at pH 6.0 in 2-(N-morpholino)ethanesulfonic acid. Globotetraosylceramide and II3-alpha-N-acetylneuraminyl-lactosylceramide inhibited transferase activity with globotriaosylceramide as substrate, but lactosylceramide had no effect on the activity with this acceptor. The major product of the assay was shown to be a tetraglycosylceramide with a terminal beta-N-acetylgalactosamine moiety by co-migration with authentic globotetraosylceramide on TLC plates and by cleavage of the labeled N-acetylgalactosamine from the product by jack bean beta-hexosaminidase.
D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D005347:Fibroblasts / Q000201:enzymology; D005679:G(M3) Ganglioside / Q000494:pharmacology; D005700:Galactosyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005915:Globosides / Q000494:pharmacology; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D017350:N-Acetylgalactosaminyltransferases; D014536:Uridine Diphosphate N-Acetylgalactosamine / Q000378:metabolism
D005679:G(M3) Ganglioside; D005915:Globosides; C024032:globotetraosylceramide; D008345:Manganese; D014536:Uridine Diphosphate N-Acetylgalactosamine; D005700:Galactosyltransferases; D017350:N-Acetylgalactosaminyltransferases; C003742:UDPgalactosamine-galactose acetylgalactosaminyltransferase