Characterization of phosphatidylinositol synthase and evidence of a polyphosphoinositide cycle in Plasmodium-infected erythrocytes.
Plasmodium knowlesi-infected erythrocytes possess a membranous cytidine 5'-diphospho-1,2-diacyl-sn-glycerol: myoinositol 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-beta-D-glucopyranoside in the presence of bivalent metal ions which were absolutely required for activity. The optimal pH was 8 and the apparent Ks for Mn2+ was 0.1 mM. Mg2+ 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 Mg2+. Enzymatic activity was fully dependent on CDP-diacylglycerol and inositol with apparent Km 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.
Elabbadi
N
N
Interactions Membranaires, CNRS URA. 530, Université Montpellier II, France.
Ancelin
M L
ML
Vial
H J
HJ
D000818:Animals; D051607:CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D003567:Cytidine Diphosphate Diglycerides / Q000378:metabolism; D004912:Erythrocytes / Q000201:enzymology* / Q000469:parasitology*; D008252:Macaca fascicularis; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D010961:Plasmodium / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism; D016790:Plasmodium knowlesi / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism; D017855:Transferases (Other Substituted Phosphate Groups) / Q000187:drug effects / Q000302:isolation & purification*
D002413:Cations, Divalent; D003567:Cytidine Diphosphate Diglycerides; D018129:Phosphatidylinositol Phosphates; D017855:Transferases (Other Substituted Phosphate Groups); D051607:CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase; D002118:Calcium