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Volume 271, Number 26, Issue of June 28, 1996 pp. 15533-15541
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Phosphatidylinositol Hydrolysis by Trypanosoma brucei Glycosylphosphatidylinositol Phospholipase C

(Received for publication, January 19, 1996, and in revised form, April 4, 1996)

Peter Bütikofer , Monika Boschung , Urs Brodbeck and Anant K. Menon ^¶

From the  Institute of Biochemistry and Molecular Biology, University of Bern, CH-3012 Bern, Switzerland and the ^¶ Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706

Detergent-solubilized glycosylphosphatidylinositol (GPI)-anchored structures can be cleaved by C-type phospholipases isolated from peanuts and bloodstream cells of the African trypanosome, Trypanosoma brucei. The two enzymes differ in their reported ability to hydrolyze phosphatidylinositol (PI); while the peanut enzyme readily hydrolyzes PI in vitro, 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 substrate specificity of T. brucei GPI-PLC by incubating the purified enzyme 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 µM to 1 mM PI). At 5 µM PI, hydrolysis was maximal at 0.005% Triton X-100, whereas at 1 mM PI, maximal hydrolysis required 0.05% Triton X-100. Hydrolysis of both PI and GPI was strongly affected by the presence of phospholipids. Endogenous PI was hydrolyzed during osmotic and detergent lysis of trypanosomes under conditions used to obtain quantitative hydrolysis of the GPI-anchored trypanosome variant surface glycoprotein. PI hydrolysis in the lysates was inhibited by sodium p-chloromercuriphenylsulfonate but 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.

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