Characterization of a CMP-sialic acid:lactosylceramide sialyltransferase activity in cultured hamster cells.
Previous studies have shown a strong correlation between reduced levels of GM3 ganglioside and an increase in the oncogenic transformation of cultured cells. CMP-sialic acid:lactosylceramide sialyltransferase, which catalyzes GM3 synthesis, was characterized in cultured hamster fibroblasts (NIL-8) with respect to substrate binding, pH optimum, detergent requirements, metal ion requirements, activity during cell cycle phases and activity during cell growth phases. The apparent Km values for CMP-sialic acid and lactosylceramide were 0.16 and 0.11 mM, respectively. The enzyme required Mn2+ (15 mM) for maximal, but Mg2+ and Ca2+ were able to substitute to a lesser extent. Triton CF-54 (0.3%, w/v) compared to other nonionic detergents gave the greatest enzyme activation, while ionic detergents inhibited the enzyme. A broad pH optimum (4.5-8.0) was obtained, with maximum activity at pH 6.5 in cacodylate-HCl buffer. No buffer effects on enzyme activity were seen. Sialyltransferase activity was found to be highest in the M and G1 phases of the cell cycle and in the contact-inhibited phase of cell growth.
D000818:Animals; D002413:Cations, Divalent; D002460:Cell Line; D006224:Cricetinae; D003902:Detergents; D005679:G(M3) Ganglioside / Q000096:biosynthesis*; D005732:Gangliosides / Q000096:biosynthesis*; D007700:Kinetics; D012799:Sialyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014166:Transferases / Q000378:metabolism*
D002413:Cations, Divalent; D003902:Detergents; D005679:G(M3) Ganglioside; D005732:Gangliosides; D014166:Transferases; D012799:Sialyltransferases; C021343:haematoside synthetase