Immediate objectives of the proposed research are fourfold: l. Purification and kinetic studies of BetaGalT-4 and SAT-3 from 11-day-old embryonic chicken brains. 2. Purification and substrate specificity studies with AlphaFucT-2 (GDP-Fuc:nLcOse4Cer(al-2)fucosyltransferase) from bovine spleen. 3. Biosynthesis in vitro and structure determination of disialoneolactotetraosylceramide(NeuAc-NeuGc-Gal-GlcNAc-Gal-Glc-Cer) and lacto-N-norhexaosylceramide(Gal-GlcNac-Gal-GlcNac-Gal-Glc-Cer) in bovine spleen Golgi-rich membrane fraction. Precursor of these products have been isolated and characterized from bovine erythrocytes in our laboratory. 4. In the final states of this investigation attempts will be made to compare the levels of glycolipid:glycosyltransferase activities (BetaGalT-3, BetaGalT-4, and SAT-3) in cultured Tay-Sachs diseased cells (Transformed and nontransformed) and in the subcellular fractions derived from Tay-Sachs diseased brains (post-mortem). In addition to the absence of lysosomal hexosaminidase A the accumulation of GM2 ganglioside in Tay-Sachs brains could be the result of a severe deficiency of the synthetic enzyme BetaGalT-3 (UDP-Gal:Gm2(Beta1-3)galactosyltransferase), which is suggested from our preliminary work. (The glycosphingolipid: glycosyltransferases are abbreviated as GSLTs). Present studies are concerned with the biosynthesis of gangliosides and blood group-related glycosphingolipids in normal tissues (embryonic chicken brains and bovine spleen) and pathological human brains (post-mortem tissues). The overall goal of our research is to develop an understanding of the specificities of GSLTs.
