Interação dos chamados antagonistas de calmodulina, composto 48/80 e calmidazol, nas curvas de substrato, ATP e cálcio, da (Ca²⁺+Mg²⁺)ATPase de membrana basolateral de túbulos contornados proximais

Abstract

In this work we investigated the effects of two so-callled calmodulin antagonists, compound 48/80 and calmidazolium, on the (Ca²⁺+Mg²⁺)ATPase from basolateral membranes derived from rabbit kidney proximal tubules. Analysis of the crude membrane preparation by polyacrylamide gel electrophoresis in the presence of SDS followed by incubation with anti-calmodulin antibody showed that it did not contain endogenous calmodulin. Furthermore, when the preparation was solubilized with the nonionic detergent C12E8 the (Ca²⁺+Mg²⁺)ATPase activity was stimulated by exogenous calmodulin at very low calcium concentrations (< 1 uM). This activity was inhibited 70% by compound 48/80, while it was not changed by calmidazolium. In addition, we analyzed the effects of the antagonists on the calcium and ATP concentration dependence of (Ca²⁺+Mg²⁺)ATPase activity. Compound 48/80 competitively inhibited the high-affinity ATP component, increasing Km from 0.91 to 2.41 uM. In the low-affinity component (Km = 0,87 mM), this drug promoted a linearization of the substrate curve in the range of ATP concentrations employed, giving a Vmax/Km ratio of 2.15 x 10¹ x mg-1 x min-1 . Assuming a oompetitive mechanism of inhibition, it. was possible to calculate an upper limit for Km of 16 mM. Compound 48/80 also induced the appearance of negative cooperativity (from n = 1 to n = 0.46) in the low-affinity Component. Calmidazolium reduced Vmax in both high- and low affinity components, and promoted an increase in Km only for the low-affinity site (from 0.87 to 2.54 mM). At a fixed ATP concentration (5 mM), activity increased with calcium concentration up to 10-30 uM and then decreased at higher calcium concentrations. Both drugs decreased Vmax. Compound 48/80 also decreased the calcium affinity in both low and high concentration ranges (from 2.15 to 5.72 uM and from 0.21 to 0.35 mM, respectively). The addition of Pi altered the inhibition by the two drugs in different ways. At a high ATP concentration (5 mM), Pi protected the ATPase against inhibition by compound 48/80 and potentiated the inhibition by calmidazolium. At a low ATP concentration (25 uM), Pi had no effect on the inhibition by compound 48/80, but protected against calmidazolium. In the absence of Pi, the effects of the two drugs were additives. These results suggest that compound 48/80 and calmidazolium act in different sites of the enzyme structure and, probably, by distinct mechanisms.