Characterization of a glycosphingolipid β- N-acetylgalactosaminyl-transferase activity in cultured hamster (NIL) cells

Abstract

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 K m values for uridinediphosphate- N-acetylgalac-tosamine and globotriasylceramide were 0.14 and 0.42 mM, respectively. The enzyme required Mn 2+ for maximum activity (4 mM), and Mg 2+ was not able to replace Mn 2+. 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-niorpholino)ethanesulfonic acid. Globotetraosyl-ceramide and II 3-α-N-acetylneuraminyl-lactosylceramide inhibited transferase activity with globotriaosyl-ceramide 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 β- 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 β-hexosaminidase.