Pseudosubstrate hydrolysis by the erythrocyte plasma membrane Ca 2+-ATPase: kinetic evidence for a modified E 1 conformation in dimethylsulfoxide

Abstract

The purified Ca 2+-ATPase of pig red cells displays a phosphatase activity towards p-nitrophenylphosphate which is inhibited by Ca 2+ in the absence of solvents, and activated by calmodulin. This activity has been attributed to the E 2 conformation of the enzyme. Here we show that the pNPPase activity in the absence of Ca 2+ is stimulated 10–25-fold by the presence of the organic solvent dimethylsulfoxide (Me 2SO). This is an activation that surpasses by severalfold that induced by calmodulin in the absence of the solvent. At 30% Me 2SO, activation by calmodulin disappears. In the absence of calmodulin and at pH 7.2, the Ca 2+ concentration needed for half-maximal inhibition of the pNPPase activity ( K i) increases from 130 μM in the absence of Me 2SO to 860 μM at 30% Me 2SO. This effect of Me 2SO is enhanced at pH 8.0: the K i for Ca 2+ increases from 2.7 μM in the absence of the solvent to 2.0 mM in its presence. However, the K 0.5 for Ca 2+ activation of the ATPase activity decreases from 8.3 to 2.6 μM following addition of the same Me 2SO concentration. This indicates that, even in the presence of Me 2SO, μM Ca 2+ concentrations shift the equilibrium towards E 1 but the decrease in activity that would be expected if pNPP hydrolysis were catalysed exclusively by the E 2 conformation is not observed. The affinity for pNPP as a substrate increases from 2.6 mM in the absence of Me 2SO to 1.6 mM in the presence of 20% Me 2SO. These results suggest that Me 2SO induces multiple effects in the Ca 2+-ATPase that: (i) increase the reactivity of E 2 towards substrate; (ii) surpass the activation by calmodulin and, (iii) allow the enzyme to hydrolyze pNPP even when Ca 2+ is bound to the high-affinity sites of the enzyme. The change in reactivity is attributed to an increase on substrate catalysis rather than on pNPP binding.