Allosteric modulation of Leishmania donovani plasma membrane Ca²⁺-ATPase by endogenous calmodulin

Abstract

The plasma membrane of the human pathogen Leishmania donovani possesses a high-affinity transmembrane Ca²⁺-ATPase that has its catalytic site oriented toward the cytoplasmic milieu (Ghosh, J., Ray, M., Sarkar, S., and Bhaduri, A. (1990) J. Biol. Chem. 265, 11345-11351 begin_of_the_skype_highlighting 11345-11351 end_of_the_skype_highlighting). When the enzyme is studied in its more authentic, physiologically relevant, membrane-associated form, it exhibits pronounced sigmoidal kinetics with Ca²⁺ (K_0.5 ≈ 700 nM) in a trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid buffering system that effectively complexes all available Mg²⁺. Addition of exogenous Mg²⁺ (60 μM) completely abolishes sigmoidicity and establishes strictly hyperbolic kinetics, and the Km for Ca²⁺ reduces to 100 nM. Mg²⁺ can be replaced by heterologous calmodulin. The exclusive dependence of the enzyme on only Ca²⁺ for its activity and its positive allosteric modulation by Mg²⁺ distinguish this enzyme from other well-characterized plasma membrane Ca²⁺-ATPases. Employing this Ca²⁺-ATPase as the assay system, a soluble endogenous activating protein factor was purified that, by several criteria, corresponds to authentic calmodulin. The parasite calmodulin shifts the kinetics to hyperbolic kinetics, increases the Vmax 2-fold, and most important lowers the K_m (≈ 100 nM) to a physiological level. The interaction with endogenous calmodulin thus converts the enzyme from a totally inactive to a fully active state.