Title

Calcium(2+)-magnesium(2+)-ATPase of cardiac sarcolemma: Operation and regulation

Date of Award

1987

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Pharmacology

First Committee Member

Duncan H. Haynes, Committee Chair

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.)

Keywords

Biology, Animal Physiology

Link to Full Text

http://access.library.miami.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:8813683