Protocols for glycosyltransferase assays: Ganglioside globoside and lewis-X intermediate-lactosylceramide biosyntheses in eukaryotic systems

Subhash Basu*, Arun Agarwal, Manju Basu, Rui Ma, Joseph R Moskal

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

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% Na 2 B 4 O 7 . 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 −4  M; Km for UDP-Gal = 0.5 × 10 −4  M; V max 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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc
Pages189-205
Number of pages17
DOIs
StatePublished - Jan 1 2018

Publication series

NameMethods in Molecular Biology
Volume1804
ISSN (Print)1064-3745

Keywords

  • GalT-2
  • Glycosyltransferases
  • Lactosylceramide synthase

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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