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.