PLASMODIUM-FALCIPARUM CTP-PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE EXPRESSED IN ESCHERICHIA-COLI - PURIFICATION, CHARACTERIZATION AND LIPID REGULATION

The Plasmodium falciparum CTP:phosphocholine cytidylyltransferase PfCC T) has been isolated from an overexpressing strain of Escherichia coli . The plasmid pETPfCCT mediated the overexpression of the full-length polypeptide directly. The recombinant protein corresponded to 6-9% of the total cellular proteins and was found essentially in the insoluble membrane fraction. Urea at 6 M was used to solubilize the recombinant protein from the insoluble fraction. The CCT activity was restored up on the removal of urea, and the protein was subsequently purified to h omogeneity on a Q-Sepharose column. Approx. 1.4 mg of pure enzyme was obtained from a 250 ml culture of E. coli. Biochemical properties, inc luding in vitro substrate specificity and enzymic characterization, we re assessed. The lipid regulation of the recombinant plasmodial CCT ac tivity was characterized for the first time. The K-m values were 0.49/-0.03 mM (mean+/-S.E.M.) for phosphocholine and 10.9+/-0.5 mM for CTP in the presence of lipid activators (oleic acid/egg phosphatidylcholi ne vesicles), whereas the K-m values were 0.66+/-0.07 mM for phosphoch oline and 28.9+/-0.8 mM for CTP in the absence of lipid activators. Th e PfCCT activity was stimulated to the same extent in response to egg phosphatidylcholine vesicles containing anionic lipids, such as oleic acid, cardiolipin and phosphatidylglycerol, and was insensitive or sli ghtly sensitive to PC vesicles containing neutral lipids, such as diac ylglycerol and monoacylglycerol. Furthermore, the stimulated enzyme ac tivity by oleic acid was antagonized by the cationic aminolipid sphing osine. These lipid-dependence properties place the parasite enzyme int ermediately between the mammalian enzymes and the yeast enzyme.