10] In Vitro assays for enzymes of ganglioside synthesis

Abstract

Publisher Summary Glycosyltransferases of ganglioside biosynthesis transfer monosaccharide from an activated sugar (sugar nucleotide) to either ceramide or the growing oligosaccharides of the glycolipids. A number of ganglioside glycosyltransferases from different sources have been cloned and expressed in a variety of cell lines, and even knockout mice with disrupted N-acetylgalactosaminyltransferase (GalNAc-T) genes have been created. Despite the great advances in molecular biology and their important contribution to the investigation of ganglioside biosynthesis, in vitro assays for these enzymes still remain necessary for the determination of transferase properties such as substrate specificities, kinetic data, ion requirements, and identification and structure analysis of the products. Ganglioside glycosyltransferases are type II membrane proteins with active sites that face the lumen of the Golgi apparatus and utilize a membrane-resident lipid acceptor and a water-soluble activated sugar donor (probably following a two-dimensional Michaelis-Menten kinetics, as exemplified by Scheel et al. ).