Purification and characterization of the membrane-form variant surface glycoprotein hydrolase of Trypanosoma brucei.

Abstract

The conversion of the membrane-form variant surface glycoprotein (mfVSG) of the unicellular parasitic flagellate Trypanosoma brucei to soluble variant surface glycoprotein and sn-1,2-dimyristoyl glycerol is catalyzed by an endogeneous, membrane bound phospholipase C-like hydrolase. Using a monoclonal antibody against the enzyme the hydrolase was purified 3,000-fold with a yield of 32%. The enzyme has a molecular weight of 39,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The rate with which mfVSG hydrolase cleaves phosphatidylinositol is 170 times lower than the cleavage rate for mfVSG, whereas phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine cannot serve as substrates. Reconstitution experiments into phospholipid vesicles show that the enzyme can hydrolyze mfVSG when present in the same phospholipid bilayer but not when present in separate bilayers.