Choline kinase activity in Plasmodium-infected erythrocytes: characterization and utilization as a parasite-specific marker in malarial fractionation studies

Abstract

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 K m values for choline and ATP were 79 ± 20 μ M, and 1.3 ± 0.3 mM, respectively. ATP concentrations higher than 12 mM inhibited choline kinase. Maximal activity was registered with a Mg 2+ concentration of 10 mM, whereas its replacement by Mn 2+, 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.