730 CHARACTERIZATION OF TWO GOLGI GALACTOSYLTRANSFERASE ISOZYMES WHICH CATALYZE THE BIOSYNTHESIS OF Gm GANGLIOSIDE

Abstract

Biosynthesis of gangliosides, a critical process in early development of neurological function,was studied in 3 week old rats. Analysis of the glycosylation steps in the biosynthetic pathway has been impeded due to the difficulty in solubilizing the membrane bound glycosyltransferase enzymes. We have solubilized UDP-gal Gm2 ganglioside galactosyltransferase from rat liver Golgi by detergent extraction. Two isozymes which catalyzed the synthesis of Gml ganglioside were separated by DEAE chromatography. Peak I, the more basic form, and Peak II, the more acidic form were highly unstable following chromatography but could be stored at -20°C in 50% glycerol. pH optima for Peaks I and II were 7 and 6. The Kms for UDP-gal for Peaks I and II were 7.9 × 10-4 M and 4.7 × 10-4 M; Kms for Gm2 were 2.6 × 10-5 M and 1.5 × 10-5 M. Mn was essential for both activities. Peak I activity could be demonstrated in the presence of phospholipids or nonionic detergent. Peak II was active in the presence of nonionic detergent, but little or no activity could be demonstrated with phospholipid. Human liver Hex A activator protein could replace detergent for Peaks I and II. Only Peak II activity catalyzed the addition of gal to monosaccharide amino sugars. Both Peaks I and II catalyzed the addition of galactose to glycoprotein substrates. In summary, 2 distinct membrane bound Gm2 ganglioside galactosyltransferase isozymes were resolved and characterized.