Phosphatidylinositol hydrolysis by Trypanosoma brucei glycosylphosphatidylinositol phospholipase C.
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 microM to 1 mM PI). At 5 microM 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.
Bütikofer
P
P
Institute of Biochemistry and Molecular Biology, University of Bern, CH-3012 Bern, Switzerland.
Boschung
M
M
Brodbeck
U
U
Menon
A K
AK
D000110:Acetylcholinesterase / Q000378:metabolism; D000818:Animals; D002417:Cattle; D054800:Glycosylphosphatidylinositol Diacylglycerol-Lyase; D007700:Kinetics; D008823:Micelles; D017830:Octoxynol / Q000494:pharmacology; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000378:metabolism; D010743:Phospholipids / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology*; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000378:metabolism
D008823:Micelles; D010716:Phosphatidylinositols; D010743:Phospholipids; D014643:Variant Surface Glycoproteins, Trypanosoma; D012967:Sodium Dodecyl Sulfate; D017830:Octoxynol; D000110:Acetylcholinesterase; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054800:Glycosylphosphatidylinositol Diacylglycerol-Lyase