PHOSPHATIDYLINOSITOL HYDROLYSIS BY TRYPANOSOMA-BRUCEI GLYCOSYLPHOSPHATIDYLINOSITOL PHOSPHOLIPASE-C

Detergent-solubilized glycosylphosphatidylinositol (GPI)-anchored stru ctures can be cleaved by C-type phospholipases isolated from peanuts a nd bloodstream cells of the African trypanosome, Trypanosoma brucei. T he two enzymes differ in their reported ability to hydrolyze phosphati dylinositol (PI); while the peanut enzyme readily hydrolyzes PI in vit ro, the T. brucei enzyme was reported to be virtually inactive against PI and consequently named GPI-specific phospholipase C (GPI-PLC). In this paper, we describe experiments in which we reinvestigated the sub strate specificity of T. brucei GPI-PLC by incubating the purified enz yme with Triton X-100/PI-mixed micelles and by studying PI hydrolysis. We found that PI hydrolysis occurred in a detergent-dependent fashion over the range of concentrations tested (5 mu M to 1 mM PI). At 5 mu M PI, hydrolysis was maximal at 0.005% Triton X-100, whereas at 1 mM P I, maximal hydrolysis required 0.05% Triton X-100. Hydrolysis of both PI and GPI was strongly affected by the presence of phospholipids. End ogenous PI was hydrolyzed during osmotic and detergent lysis of trypan osomes under conditions used to obtain quantitative hydrolysis of the GPI-anchored trypanosome variant surface glycoprotein. PI hydrolysis i n the lysates was inhibited by sodium p-chloromercuriphenyIsulfonate b ut unaffected by EGTA, consistent with the proposal that hydrolysis is due to GPI-PLC. These results suggest that the function of T. brucei GPI-PLC may be to regulate PI as well as (or instead of) GPI levels.