Kinetic characterization of the Ca2+-pumping ATPase of cardia sarcolemma in four states of activation.

The Ca2+ dependence of the Ca2+-pumping ATPase of bovine cardiac sarcolemma was studied for four states of activation: (a) unactivated, (b) cAMP-dependent protein kinase (cAMP protein kinase C-subunit)-activated, (c) calmodulin (CAM)-activated, and (d) CAM plus cAMP protein kinase C-subunit-activated. Analysis of the Ca2+ dependence of active transport gave the following Vmax (nanomoles Ca2+/(mg x min], Km (nM) for Ca2+, and Hill coefficient values for the four states at pH 7.4, 37 degrees C: (a) 1.7 +/- 0.3, 1800 +/- 100, 1.6 +/- 0.1; (b) 3.1 +/- 0.5, 1100 +/- 100, 1.7 +/- 0.1; (c) 15.0 +/- 2.5, 64 +/- 1.4, 3.7 +/- 0.2; and (d) 36.0 +/- 6.5, 63 +/- 1.7, 3.7 +/- 0.1. CAM has the most dramatic effect, increasing the apparent Ca2+ affinity by a factor of 28, increasing the Hill coefficient 2.0 units to a value approaching 4 and increasing the Vmax by a factor of 9 or 12. The effective Ca2+ concentration (EC50) for the Ca2+-induced activation of the enzyme in the presence of 5 microM calmodulin is close to the Km for Ca2+ for the CAM-activated state (64 nM). Activation by cAMP protein kinase C-subunit had only minor effects on the Km and Hill coefficient, but increased the Vmax of both the unactivated and the CAM-activated forms of the pump by factor of 1.8 and 2.4, respectively. Analysis suggests that CAM activation is the result of direct binding of Ca2-CAM or high complexes, conferring higher Ca2+ affinity to the enzyme. Analysis suggests that regulatory phosphorylation (cAMP protein kinase C-subunit) increases the rates of processes subsequent to or distinct from Ca2+ binding. The CAM-activated form of the pump was further characterized. Unexpectedly, this form of the enzyme is stimulated a factor of 1.9 by ADP, with half-maximal stimulation between 0.4 and 0.7 mM. Analysis of the progress curves for uptake show that the CAM-activated enzyme is highly resistant to inhibition by transported Ca2+, with an IC50 of 32 mM. The implications of these findings for the pump mechanism and for its role in the regulation of cardiac contractility are discussed.