Characterization of phosphatidylinositol synthase and evidence of a polyphosphoinositide cycle in Plasmodium-infected erythrocytes

Abstract

Plasmodium knowlesi-infected erythrocytes possess a membranous cytidine 5′-diphospho-1,2-diacyl- sn-glycerol: myo-inositol 3-phosphatidyl transferase (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-β- d-glucopyranoside in the presence of bivalent metal ions which were absolutely required for activity. The optimal pH was 8 and the apparent K s for Mn 2+ was 0.1 mM. Mg 2+ allowed two-fold higher PI synthase activity, with an optimum above 100 mM. Calcium alone was ineffective while at 2 mM it inhibited solubilized PI synthase activity in the presence of 100 mM Mg 2+. Enzymatic activity was fully dependent on CDP-diacylglycerol and inositol with apparent K s of 0.16 ± 0.1 mM and 1 ± 0.5 mM respectively. Affinity chromatography clearly showed CDP-diacylglycerol-dependent interactions of PI synthase with CDP-diacylglycerol Sepharose. However, elution of enzymatic activity in an active form was unsuccessful while SDS-PAGE of the eluate showed one apparent band. Incubations of Plasmodium falciparum-infected erythrocytes with 32P or [ 3H]inositol revealed de novo biosynthesis of phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate which appeared to predominate in the second half of the asexual cellular cycle. Ionomycin, a calcium ionophore, induced Li +-sensitive production of radioactive inositol phosphates, with neo-synthesized inositol 1,4,5-trisphosphate accumulation being the highest.