Inhibition of the GlcNAc transferase of the glycosylphosphatidylinositol anchor biosynthesis in African trypanosomes.
A wide variety of eukaryotic membrane proteins are anchored to the cell surface by a covalent linkage to glycosylphosphatidylinositol. One of the best characterised examples is the variant surface glycoprotein of the protozoan parasite, Trypanosoma brucei. The pathway for the formation of the glycosylphosphatidylinositol precursor has been previously described, with the first step being the transfer of GlcNAc, from UDP-GlcNAc to endogenous phosphatidylinositol to form N-acetyl-glucosaminylphosphatidylinositol [Doering, T. L., Masterson, W. J., Hart, G. W. & Englund, P. T. (1989) J. Biol. Chem. 264, 11,168-11,173]. Here we report that low concentrations of sulphydryl alkylating reagents irreversibly inhibit this transferase in a trypanosome-derived cell-free system. The site of inactivation by N-ethylmaleimide appears to be at, or close to, the enzyme active site, since incubation of the enzyme preparation with the donor molecule UDP-GlcNAc substantially protects the enzyme from inactivation. The protection appears to be primarily dependent on the nucleotide portion of the molecule, since UMP and UDP can mimic the protection seen with UDP-GlcNAc.
Milne
K G
KG
Department of Biochemistry, Medical Sciences Institute, University of Dundee, Scotland.
Ferguson
M A
MA
Masterson
W J
WJ
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000478:Alkylation; D000818:Animals; D005033:Ethylmaleimide / Q000494:pharmacology; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors*; D006017:Glycolipids / Q000096:biosynthesis*; D017261:Glycosylphosphatidylinositols; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007700:Kinetics; D008565:Membrane Proteins / Q000096:biosynthesis; D017351:N-Acetylglucosaminyltransferases; D010716:Phosphatidylinositols / Q000096:biosynthesis*; D014346:Trypanosoma brucei brucei / Q000201:enzymology*; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000494:pharmacology; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000378:metabolism
D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D007461:Iodoacetates; D008565:Membrane Proteins; D010716:Phosphatidylinositols; D014643:Variant Surface Glycoproteins, Trypanosoma; D014537:Uridine Diphosphate N-Acetylglucosamine; D002731:4-Chloromercuribenzenesulfonate; D005964:Glucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C037649:N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase; D005033:Ethylmaleimide; D019807:Iodoacetic Acid