Choline kinase activity in Plasmodium-infected erythrocytes: characterization and utilization as a parasite-specific...

Choline kinase (EC 2.7.1.32) was investigated in plasmodium falciparum-infected erythrocytes. Disrupted infected erythrocytes had a choline kinase activity of 1.9 +/- 0.2 nmol phosphorylcholine/10(7) infected cells per h, whereas the activity in normal uninfected erythrocytes was less than 6 pmol/10(7) cells per h. A broad alkaline optimal pH (7.9-9.2) was observed. The Km values for choline and ATP were 79 +/- 20 microM, and 1.3 +/- 0.3 mM, respectively. ATP concentrations higher than 12 mM inhibited choline kinase. Maximal activity was registered with a Mg2+ concentration of 10 mM, whereas its replacement by Mn2+, or other divalent cations, involved a decrease in choline kinase activity of at least 75%. Inhibition by products of the reaction, such as phosphorylcholine and ADP was investigated. In plasmodium knowlesi-infected erythrocytes, choline kinase had similar properties, but with a much higher specific activity of 16.4 +/- 2.1 nmol/10(7) infected cells per h. Subcellular fractionation of P. knowlesi-infected erythrocyte suspensions revealed that choline kinase was located exclusively in the cytosol of the parasite. We show that this enzyme is a useful index of parasite cytosolic content leakage, when infected erythrocytes are fractionated by saponin lysis or nitrogen decompression.