Effect of incubation medium dielectric permeability on the kinetics 2(+)-transporting ATPase of ATP hydrolysis catalyzed by the ca solubilized from smooth muscle cell plasma membranes

Transport Ca2+,Mg2+-ATPase was solubilized from myometrial plasma membrane and purified by calmodulin-Sepharose 4B affinity chromatography, and the effect of the dielectric permeability of the incubation medium on the kinetics of ATP hydrolysis by the enzyme was studied. Increasing concentrations of dimethylsulfoxide, ethanol, acetone, and dioxane (up to 10%) caused various dose-dependent inhibitions of enzymatic ATP hydrolysis. The correlation between the specific activity (A) of Ca2+,Mg2+-ATPase and the dielectric permeability of the incubation medium (D) did not depend on the nature of the organic solvent and was satisfactorily approximated by similar lines in the Laidler-Scatchard coordinate system, i.e., lnA versus 1/D. Decreasing polarity of the incubation medium (changing D from 73.05 to 68.80) reduced the initial maximal rate of the enzymatic reaction for ATP and Mg2+ and increased the apparent K-m values for these compounds. Decreasing the ionic strength of the incubation medium from 100 to 25 mM KCl decreased the maximal initial rate of ATP hydrolysis for Ca2+ and increased the apparent K-m value of this cation. The data are discussed in terms of a model for the transport Ca2+,Mg2+-ATPase active-site structure.