Ganglioside biosynthesis in rat liver. Characterization of UDPgalactose- glucosylceramide galactosyltransferase and UDPgalactose-GM2 galactosyltransferase

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Abstract
The conditions for the quantitative determination of UDP-Gal:glucosylceramide galactosyltransferase [GL2-synthase] and of UDP-Gal:GM2 galactosyltransferase [GM1-synthase] in Golgi-enriched preparations of rat liver were optimized. Trition X-100 was the detergent routinely used as ocytl glucoside acted as a galactose acceptor forming octyl lactoside. Mn2+ ions were required for full activity, but Co2+ and Mg2+ could substitute to some extent. The nucleotide pyrophosphatase activity of the Golgi preparations which interfered with the GL2-synthase assay was inhibited by addition of 20 mM IMP; the latter is without appreciable effect on the rate of GL2 synthesis. Apparent Km values for UDP-Gal were 130 .mu.M and 140 .mu.M with GL2-synthase and GM1-synthase, respectively. That for glucosylceramide was 80 .mu.M with GL2-synthase: for GM2 it was 10 .mu.M with GM1-synthase. Competition experiments with variable concentrations of the lipid acceptors showed that the 2 synthase activities are independent catalytic entities. The specific activity of GM1-synthase exceeds that of GL2-synthase by a factor of .apprx. 25 under the optimized conditions used here.