Cholinephosphotransferase and ethanolaminephosphotransferase activities in Plasmodium knowlesi-infected erythrocytes. Their use as parasite-specific markers.
CDPcholine: 1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and CDPethanolamine: 1,2-diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) activities were investigated in Plasmodium knowlesi-infected erythrocytes obtained from Macaca fascicularis monkeys. Disrupted infected erythrocytes possess a cholinephosphotransferase activity (1.3 +/- 0.2 nmol phosphatidylcholine/10(7) infected cells per h) 1.5-times higher than the ethanolaminephosphotransferase activity. Optimal activities of both enzymes were observed in the presence of 12 mM MnCl2, which was about 3-times as effective as 40 mM MgCl2 as a cofactor. The two activities had similar dependences on pH and thermal inactivation. Their Arrhenius plots show an identical break at 17 degrees C and the corresponding activation energies below and above the critical temperature were similar for the two activities. Sodium deoxycholate, sodium dodecyl sulfate, Triton X-100, beta-D-octylglucoside and lysophosphatidylcholine strongly inhibited the two activities above their critical micellar concentration, but the first three detergents stimulated the activities at lower concentrations. Saponin (0.004-0.5%) either did not affect the two activities or else increased them. Cholinephosphotransferase and ethanolaminephosphotransferase activities had apparent Km values for the CDP ester of 23.4 and 18.6 microM, respectively. CDPcholine and CDPethanolamine competitively inhibited the ethanolaminephosphotransferase and cholinephosphotransferase activities, respectively. The high selectivity of these activities for individual molecular species of diradylglycerol suggests that substrate specificity is responsible for the various molecular species of Plasmodium-infected erythrocyte phospholipids. However, cholinephosphotransferase and ethanolaminephosphotransferase had different dependences on 1,2-dilauroylglycerol and 1-oleylglycerol, which were substrates for cholinephosphotransferase but not for ethanolaminephosphotransferase under our conditions. These data provide the first characterization of an enzyme involved in the intense lipid metabolism in Plasmodium-infected erythrocytes, and the presence of cholinephosphotransferase demonstrates a biosynthesis of phosphatidylcholine by the Kennedy pathway after infection. Our data suggest that cholinephosphotransferase and ethanolaminephosphotransferase activities could be catalyzed by the same enzyme. Furthermore, since host erythrocytes are devoid of these enzymatic activities, cholinephosphotransferase is a parasite-specific membrane-associated enzyme which can be used as a probe or marker.