Ganglioside biosynthesis in rat liver. Characterization of UDP-N-acetylgalactosamine -- GM3 acetylgalactosaminyltransferase.
UDP-N-acetylgalactosamine--GM3 acetylgalactosaminyltransferase (GM2-synthase) was studied in a Golgi-rich fraction from rat liver. Activity in a cell-free system required the presence of detergents; octyl glucoside was found to be the most effective in stimulating the enzyme. Optimal activity of GM2-synthase was obtained at pH 7.2, in the presence of 0.8% octyl glucoside, 10 mM Mn2+ and 5 mM CDP-choline. The latter was used to counteract the rapid sugar nucleotide hydrolysis caused by a nucleotide pyrophosphatase activity in the Golgi fraction. The apparent Km values for UDP-N-acetylgalactosamine and added GM3 were 0.035 mM and 0.1 mM, respectively. Different results were obtained if endogenous GM3 only was used as the glycolipid acceptor. In this case, the apparent Km value for UDP-N-acetylgalactosamine was 0.18 mM and Co2+ and Fe2+ exceeded Mn2+ in activating GM2-synthase. Under optimal assay conditions and in the presence of added GM3 and 5 mM CDP-choline, the specific activity of the enriched Golgi fraction was measured to be 25-30 nmol X mg protein-1 X h-1; with endogenous GM3 as the sole glycolipid acceptor, V was calculated to be 9 nmol X mg protein-1 X h-1.
D000818:Animals; D003566:Cytidine Diphosphate Choline / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D005679:G(M3) Ganglioside / Q000378:metabolism; D005700:Galactosyltransferases / Q000378:metabolism*; D005732:Gangliosides / Q000096:biosynthesis*; D005960:Glucosides / Q000494:pharmacology; D006056:Golgi Apparatus / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D017350:N-Acetylgalactosaminyltransferases; D009711:Nucleotides / Q000037:antagonists & inhibitors; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors; D051381:Rats; D014536:Uridine Diphosphate N-Acetylgalactosamine / Q000378:metabolism; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D003566:Cytidine Diphosphate Choline; D003902:Detergents; D005679:G(M3) Ganglioside; D005700:Galactosyltransferases; D005732:Gangliosides; D005960:Glucosides; D017350:N-Acetylgalactosaminyltransferases; D009711:Nucleotides; D011755:Pyrophosphatases; D014536:Uridine Diphosphate N-Acetylgalactosamine; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C018619:octyl-beta-D-glucoside; C023220:(N-acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase; C021899:nucleotide pyrophosphatase