Galactosyltransferases Catalyzing the Formation of the Galactosyl-Galactosyl Linkage in Glycosphingolipids

Galactosyltransferases that catalyze the transfer of galactose from UDP-galactose to galactosylceramide, lactosylceramide, and ganglioside GalNAcβ1 →4(NANα2 →3)-Galβ1 →4Glc →ceramide (Gm2) were measured in rat tissues as a function of development; the subcellular distribution of these enzymes was also determined. The novel galactosyltransferase catalyzing the transfer of galactose from UDP-galactose to galactosylceramide has been characterized. The enzyme attached the terminal galactose in an α linkage; it required Mn2+ for activity and showed a pH optimum of 6.0. The enzyme was detected in extracts obtained from kidney, but not from any other tissue tested. The galactosyltransferase attaching the terminal galactose in triglycosylceramide was also studied. The anomeric configuration of the terminal galactose in the triglycosylceramide was found to be α. Since the two transferases are both active with acceptor lipids containing terminal β-galactosyl units, the substrate specificities of these transferases must be defined by a structure larger than the ultimate glycosyl unit. These two transferases were differentiated from a third galactosyltransferase catalyzing the transfer of galactose from UDP-galactose to ganglioside Gm2. High activities of this enzyme were demonstrated in spleen and liver. A relationship between the concentration of detergent and the enzyme concentration required for optimal reaction rate was found. In view of this finding, the limited value of classical kinetics in the examination of glycolipid transferases is emphasized.