A calmodulin-stimulated Ca2+ pump in plasma-membrane vesicles from Trypanosoma brucei; selective inhibition by pentamidine
1993
Benaim, G. | Lopez-Estrano, C. | Docampo, R. | Moreno, S.N.J.
Despite previous reports [McLaughlin (1985) Mol. Biochem. Parasitol. 15, 189-201; Ghosh, Ray, Sarkar and Bhaduri (1990) J. Biol. Chem. 265,11345-11351; Mazumder, Mukherjee, Ghosh, Ray and Bhaduri (1992) J. Biol. Chem. 267, 18440-18446] that the plasma membrane of different trypanosomatids only contains Ca2+-ATPase that does not show any demonstrable dependence on Mg2+, a high-affinity (Ca2+-Mg2+)-ATPase was demonstrated in the plasma membrane of Trypanosoma brucei. The enzyme became saturated with micromolar amounts of Ca2+, reaching a Vmax of 3.45 +/- 0.66 nmol of ATP/min per mg of protein. The Km.app. for Ca2+ was 0.52 +/- 0.03 micromolar. This was decreased to 0.23 +/- 0.05 micromolar, and the Vmax was increased to 6.36 +/- 0.22 nmol of ATP/min per mg of protein (about 85%), when calmodulin was present. T. brucei plasma-membrane vesicles accumulated Ca2+ on addition of ATP only when Mg2+ was present, and released it on addition of the Ca2+ ionophore A23187. In addition, this Ca transport was stimulated by calmodulin. Addition of NaCl to Ca2+-loaded T. brucei plasma-membrane vesicles did not result in Ca2+ release, thus suggesting the absence of a Na+/Ca2+ exchanger in these parasites. Therefore the (Ca2+-Mg2+)-ATPase would be the only mechanism so far described that is responsible for the long-term fine tuning of the intracellular Ca2+ concentration of these parasites. The trypanocidal drug pentamidine inhibited the T. brucei plasma-membrane (Ca2+-Mg2+)-ATPase and Ca2+ transport at concentrations that had no effect on the Ca2+-ATPase activity of human or pig erythrocytes. In this latter case, pentamidine behaved as a weak calmodulin antagonist, since it inhibited the stimulation of the erythrocyte Ca2+-ATPase by calmodulin.
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