Protocols for Glycosyltransferase Assays: Ganglioside Globoside and Lewis-X Intermediate-Lactosylceramide Biosyntheses in Eukaryotic Systems

Abstract

Protocols for assay of 24 different Glycolipid-Glycosyltransferases (GSL-GLTs) of the eukaryotic systems are described. Problems of quantitating the activities in crude membranes are also described. Different separation methods (for separation of substrate, donors, and the product of the reaction) have been described based on the paper chromatography or high voltage paper electrophoresis in 1.0% NaBO. Liquid Scintillation counting system was used for quantitation of the enzymatic product. In the assay of each GSL-GLT it is recommended to compare the selected method to be used with the exact conditions used by the authors published previously. As a test case for these assays the following kinetic parameters for Lactosylceramide Synthase, GalT-2 (UDP-Gal: Glc-Cer β1-4-galactosyltransferase), (Km of glucosylceramide = 1.65 × 10 M; Km for UDP-Gal = 0.5 × 10 M; V is determined in the presence of optimum detergent concentrations (2-15 mg/ml of Cutscum-Triton X-100, 2:1); Mn and Mg, 10-20 mM) has been reported. The importance of use of GalT-2 assay method (as a model system) in the purified Golgi-rich membranes from 13-day-old embryonic chicken brains (13-ECB) is described.