Abstract
ATP-dependent Ca$\sp{2+}$ uptake accomplished by the Ca$\sp{2+}$-Mg$\sp{2+}$-ATPase of isolated bovine cardiac sarcolemma (SL) vesicles was quantitated with the fluorescent chelate probe chlorotetracycline. The fluorescence signal is a faithful linear reporter of internal calcium concentrations generated by the SL ATPase.A biophysical analysis of this ATPase revealed the enzyme's high affinity for Ca$\sp{2+}$ and relatively slow transport velocity. Calcium is transported with 100% intrinsic thermodynamic efficiency in a coupling ratio with ATP of two. The enzyme accomplishes Ca$\sp{2+}$ transport by an electrically silent pathway, coupling the exchange of two H$\sp{+}$ per each Ca$\sp{2+}$ moved. An evaluation of the pH dependence of the transport function of the ATPase identified a decrease in the affinity of the cation binding sites for Ca$\sp{2+}$ as (H$\sp{+}$) was increased. Conversely, uptake velocity increased over the same range of acidic pH values when evaluated at saturating external calcium concentration. The binding of H$\sp{+}$ to the inwardly-oriented translocator is predicted to increase its rate of return to the outward orientation, thus completing the cycle. Since H$\sp{+}$ is the primary (if not exclusive) counter-translocated species, such a competition is expected to greatly alter uptake kinetics. However, the ATPase responds to (H$\sp{+}$) elevation at limiting (Ca$\sp{2+}$) $\sb{\rm o}$ with a sharp decrease in transport velocity. The combined effects of acidosis on affinity and on transport velocity are expected to limit Ca$\sp{2+}$ extrusion by this pump during periods of myocardial ischemia.Regulation of the pump is accomplished through two separate mechanisms: (1) calmodulin and (2) cyclic AMP-dependent protein kinase (cAMP PK) activity. Allosteric activation by calmodulin greatly increases the apparent affinity of the pump for calcium (reducing the K$\sb{\rm m}$ from 1 $\mu$M to 0.07 $\mu$M). It is the calmodulin-activated form of the pump which is predicted to "set" resting (Ca$\sp{2+}$) $\sb{\rm cyt}$.Activation of cAMP PK dependent activity results in approximately a 2-fold increase in transport velocity both in the presence and in the absence of calmodulin without affecting affinity. This mechanism of activation provides for coupling beta-adrenergic activation of the myocyte to enhanced bulk extrusion of Ca$\sp{2+}$, thus maintaining calcium homeostasis. A significant role of this enzyme in both establishing and maintaining low resting calcium concentrations is predicted. (Abstract shortened with permission of author.)