CHARACTERIZATION OF PHOSPHATIDYLINOSITOL SYNTHASE AND EVIDENCE OF A POLYPHOSPHOINOSITIDE CYCLE IN PLASMODIUM-INFECTED ERYTHROCYTES

Plasmodium knowlesi-infected erythrocytes possess a membranous cytidin e 5'-diphospho-1,2-diacyl-sn-glycerol: myoinositol 3-phosphatidyl tran sferase (PI synthase) (EC 2.7.8.11) activity of 10 +/- 1.7 nmol min(-1 ) per 10(10) infected cells. The activity was successfully solubilized with 40 mM n-octyl-beta-D-glucopyranoside in the presence of bivalent metal ions which were absolutely required for activity. The optimal p H was 8 and the apparent K-s for Mn2+ was 0.1 mM. Mg2+ allowed two-fol d higher PI synthase activity, with an optimum above 100 mM. Calcium a lone was ineffective while at 2 mM it inhibited solubilized PI synthas e activity in the presence of 100 mM Mg2+. Enzymatic activity was full y dependent on CDP-diacylglycerol and inositol with apparent K-m of 0. 16+/-0.1 mM and 1+/-0.5 mM respectively. Affinity chromatography clear ly showed CDP-diacylglycerol-dependent interactions of PI synthase wit h CDP-diacylglycerol Sepharose. However, elution of enzymatic activity in an active form was unsuccessful while SDS-PAGE of the eluate showe d one apparent band. Incubations of Plasmodium falciparum-infected ery throcytes with P-32 or [H-3]inositol revealed de novo biosynthesis of phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidy linositol 4, 5-bisphosphate which appeared to predominate in the secon d half of the asexual cellular cycle. Ionomycin, a calcium ionophore, induced Li+-sensitive production of radioactive inositol phosphates, w ith neo-synthesized inositol 1,4,5-trisphosphate accumulation being th e highest.