Plasmodium falciparum CTP:phosphocholine cytidylyltransferase expressed in Escherichia coli: purification, characterization and lipid regulation.
The Plasmodium falciparum CTP:phosphocholine cytidylyltransferase (PfCCT) 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 upon the removal of urea, and the protein was subsequently purified to homogeneity on a Q-Sepharose column. Approx. 1.4 mg of pure enzyme was obtained from a 250 ml culture of E. coli. Biochemical properties, including in vitro substrate specificity and enzymic characterization, were assessed. The lipid regulation of the recombinant plasmodial CCT activity was characterized for the first time. The Km 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 phosphatidylcholine vesicles), whereas the Km values were 0.66+/-0.07 mM for phosphocholine and 28.9+/-0.8 mM for CTP in the absence of lipid activators. The 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 slightly sensitive to PC vesicles containing neutral lipids, such as diacylglycerol and monoacylglycerol. Furthermore, the stimulated enzyme activity by oleic acid was antagonized by the cationic aminolipid sphingosine. These lipid-dependence properties place the parasite enzyme intermediately between the mammalian enzymes and the yeast enzyme.
Yeo
H J
HJ
Laboratoire de Dynamique Moléculaire des Interactions Membranaires, CNRS UMR 5539, Université Montpellier II, case 107, 34095 Montpellier Cedex 5, France.
Larvor
M P
MP
Ancelin
M L
ML
Vial
H J
HJ
D000818:Animals; D019916:Choline-Phosphate Cytidylyltransferase; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D008055:Lipids / Q000494:pharmacology*; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
D008055:Lipids; D011994:Recombinant Proteins; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase