Purification and characterization of a novel glycan-phosphatidylinositol-specific phospholipase C from Trypanosoma brucei.
A novel membrane-bound glycan-phosphatidylinositol-specific phospholipase C, which catalyzes the conversion of membrane form variant surface glycoproteins to soluble variant surface glycoproteins, with the release of sn-1,2-dimyristylglycerol, has been isolated from Trypanosoma brucei. The activity was solubilized from trypanosome membrane fractions in non-ionic detergent and purified by anion exchange chromatography on DEAE-cellulose followed by chromatography on phosphatidylinositol-Sepharose. The enzyme constitutes about 0.1% of the total cellular protein and has an apparent molecular weight of 39,800. The enzyme shows a head group specificity for molecules containing carbohydrate covalently linked to glycan-phosphatidylinositol, but can also act on the monoacyl derivative of membrane form variant surface glycoprotein. It shows no specific ion requirements but is stimulated by thiol-reducing agents and inhibited by ions that thiols chelate.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D002845:Chromatography; D004229:Dithiothreitol / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010716:Phosphatidylinositols / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology*; D010738:Type C Phospholipases / Q000302:isolation & purification / Q000378:metabolism*
D010716:Phosphatidylinositols; D011134:Polysaccharides; D010738:Type C Phospholipases; D002118:Calcium; D004229:Dithiothreitol