Calmodulin-dependent Ca 2+-pump ATPase of human smooth muscle sarcolemma

Abstract

An enzymatically active Ca 2+-stimulated ATPase has been isolated from the sarcolemmal sheets of human smooth muscle (myometrium). Ca 2+-ATPase activity was quantitated in an assay medium which simulated the characteristic free ionic concentrations of the cytosol. New computer programs for calculating the composition of solutions containing metals (Ca, Mg, Na, K) and ligands (EGTA, ATP), based on the updated stability constants, were used. In detergent-soluble form the enzyme has a high Ca 2+-affinity expressed by an apparent K m (Ca 2+) of 0.25 ± 0.04 μM. The maximum specific activity (about 20 nmol of P i/mg protein/min) was found in the micromolar domain of free-Ca 2+ concentrations, the same levels required for normal maximal contractions in smooth muscle. The variation of free-Ca 2+ concentration in the assay medium over 4 orders of magnitude (pCa 9 to pCa 5) resulted in a sigmoidal dependence of enzymatic activity, with a Hill coefficient of 1.4, which suggested the regulation of Ca 2+-ATPase by allosteric effectors. The presence and the activator role of endogenous calmodulin in smooth muscle sarcolemma was proved by calmodulin-depletion experiments and by using suitable anticalmodulinic concentrations of trifluoperazine. The addition of exogenous calmodulin restored the enzyme activity. Apparently, the concentration of calmodulin in isolated smooth muscle sarcolemma is about 0.1% of sarcolemmal proteins, as deduced from the comparison of calmodulin-depletion and calmodulin-readdition experiments. Calmodulin increased significantly the enzyme Ca 2+-affinity and V max (by a factor of about 10). At variance with the sarcoplasmic reticulum Ca 2+-ATPase, the sarcolemmal Ca 2+-ATPase is extremely sensitive to orthovanadate, half-maximal inhibition being observed at 0.8 μM vanadate. In conclusion, the Ca 2+-ATPase isolated from smooth muscle sarcolemma appears very similar to the well-known Ca 2+-pump ATPases of erythrocyte membrane, heart sarcolemma or axolemma. We suggest that this high-affinity Ca 2+-ATPase represents the calmodulin-regulated Ca 2+-extrusion pump of the smooth muscle sarcolemma.