Biosynthesis of monosulfogangliotriaosylceramide and GM2 by N-acetylgalactosaminyltransferase from rat brain.
Some properties of the enzyme activity that catalyzes the transfer of N-acetylgalactosamine from UDP-N-acetylgalactosamine to exogenous lactosylceramide-II3-sulfate (SM3) and N-acetylneuraminosyllactosylceramide (GM3) were studied using the enzyme preparation solubilized from the 100,000 X g pellet of 6-day-old rat brain. The products from SM3 and GM3 were identified as gangliotriaosylceramide-II3-sulfate (SM2) and N-acetylneuraminosylgangliotriaosylceramide (GM2), respectively, by TLC-autoradiography. Optimal conditions for both activities were similar: pH (Hepes-NaOH), 7.0-7.5; detergent (heptylthioglucoside), 0.64% and Mn2+, 5-10 mM. The concentrations of the detergent optimal for both enzyme activities were also examined at various concentrations of the acceptors. The lower the amounts of acceptors, the less the amounts of detergent that were required, and vice versa, for the maximum activities. The acceptor-saturation curve for SM2 synthesis was triphasic, exhibiting a sigmoidal region at lower concentrations, a hyperbolic region and finally a descending region. For GM2 synthesis, the curve was biphasic without the descending region. The donor-saturation curves were classical hyperbolic ones for both syntheses. The Km values calculated for SM3 and GM3 were 0.37 and 0.19 mM, respectively, when the data corresponding to the hyperbolic regions were used for the double-reciprocal plots. The Km values for UDP-N-acetylgalactosamine in the SM2- and GM2-synthesis were 82 and 26 microM, respectively. SM3 and GM3 were the best acceptors for this enzyme preparation. From the results of the acceptor competition study, it was suggested that the two synthetic reactions are catalyzed by a single enzyme.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D003902:Detergents / Q000494:pharmacology; D005678:G(M2) Ganglioside / Q000096:biosynthesis*; D005700:Galactosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D005732:Gangliosides / Q000096:biosynthesis*; D006017:Glycolipids / Q000096:biosynthesis*; D006028:Glycosphingolipids / Q000096:biosynthesis*; D007700:Kinetics; D017350:N-Acetylgalactosaminyltransferases; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D013865:Thioglycosides / Q000494:pharmacology
D003902:Detergents; D005732:Gangliosides; D006017:Glycolipids; D006028:Glycosphingolipids; D013865:Thioglycosides; C011117:sulfoglycolipids; D005678:G(M2) Ganglioside; C046542:heptyl 1-thioglucoside; D005700:Galactosyltransferases; D017350:N-Acetylgalactosaminyltransferases; C003742:UDPgalactosamine-galactose acetylgalactosaminyltransferase