An assay of membrane-bound Trypanosoma brucei phospholipase using an integral membrane protein substrate and detergent phase separation.
The technique of phase separation in a solution of the non-ionic detergent Triton X-114 was used to measure the enzymatic conversion of a membrane protein to a soluble product via removal of a hydrophobic moiety. The substrate was the major surface protein (p63), of Leishmania promastigotes and the enzyme was a phospholipase C purified from Trypanosoma brucei. This membrane-bound enzyme is responsible for the cleavage of the hydrophobic lipid membrane anchor of the variant surface glycoprotein (VSG), of T. brucei. The assay is fast, simple and uses small amounts of reagents. It has been used to determine the pH optimum, thermal resistance, and the sensitivity to inhibitors of the trypanosomal phospholipase.
D000818:Animals; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007895:Leishmania tropica; D008565:Membrane Proteins / Q000378:metabolism*; D017830:Octoxynol; D011092:Polyethylene Glycols; D013379:Substrate Specificity; D013696:Temperature; D014346:Trypanosoma brucei brucei / Q000201:enzymology*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D008565:Membrane Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D010738:Type C Phospholipases