Thin-layer chromatographic method for the determination of glycosyltransferase activity.
To survey glycosyltransferase activities and specificities we have developed a TLC method to separate various nucleotide sugars from both high- and low-molecular-weight sugar acceptors. Here, we report details of the procedure and its application for galactosyltransferase and fucosyltransferase detected in mouse spermatogenic cells. The assay method involves sample separation using polyethyleneimine cellulose plastic-backed thin-layer plates, developed in sodium phosphate buffer for 30 min. Nucleotide sugars, including UDP-Gal, GDP-Fuc, CMP-NeuNAc, and GDP-Man, remain at the origin, while both high- and low-molecular-weight sugar acceptors migrate within 2 cm of the solvent front. Assays for galactosyltransferase and fucosyltransferase are linear with time and yield results comparable to other methods such as gel permeation chromatography and micropartitioning filtration. The TLC protocol should be useful for determinations of many different glycosyltransferases.
Ram
P A
PA
Department of Anatomy and Cellular Biology, Harvard Medical School, Boston, Massachusetts 02115.
Fung
M
M
Millette
C F
CF
Armant
D R
DR
D000818:Animals; D001345:Autoradiography; D002849:Chromatography, Gas; D002855:Chromatography, Thin Layer; D005647:Fucosyltransferases / Q000032:analysis*; D005700:Galactosyltransferases / Q000032:analysis*; D006020:Glycopeptides / Q000378:metabolism; D006602:Hexosyltransferases / Q000032:analysis*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains
D006020:Glycopeptides; D005647:Fucosyltransferases; D005700:Galactosyltransferases; D006602:Hexosyltransferases