A calmodulin-stimulated Ca2+ pump in plasma-membrane vesicles from Trypanosoma brucei ; selective inhibition by pentamidine

• Published in: Biochemical journal Vol. 296; no. 3; pp. 759 - 763
• Main Authors: BENAIM, G, LOPEZ-ESTRANO, C, DOCAMPO, R, MORENO, S. N. J
• Format: Journal Article
• Language: English
• Published: Colchester Portland Press 15.12.1993
• Subjects: Animals; Biochemistry. Physiology. Immunology. Molecular biology; Biological and medical sciences; Calcimycin - pharmacology; Calcium - metabolism; Calcium-Transporting ATPases - antagonists & inhibitors; Calcium-Transporting ATPases - metabolism; Calmodulin - pharmacology; Cell Membrane - enzymology; Enzyme Activation; Erythrocyte Membrane - enzymology; Fundamental and applied biological sciences. Psychology; Humans; Pentamidine - pharmacology; Protozoa; Swine; Trypanosoma brucei brucei - enzymology; Protozoa; Enzymatic activity; Enzyme; Plasma membrane; Rhizoflagellata; H; Enzyme inhibitor; Hydrolases; transporting ATP synthase; Trypanosoma brucei
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2960759

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 Ca(2+)-ATPase that does not show any demonstrable dependence on Mg2+, a high-affinity (Ca(2+)-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 microM. This was decreased to 0.23 +/- 0.05 microM, 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 to addition of the Ca2+ ionophore A23187. In addition, this Ca2+ transport was stimulated by calmodulin. Addition of NaCl to Ca(2+)-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 (Ca(2+)-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 (Ca(2+)-Mg2+)-ATPase and Ca2+ transport at concentrations that had no effect on the Ca(2+)-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 Ca(2+)-ATPase by calmodulin.