Kinetic differences in the phospholamban-regulated calcium pump when studied in crude and purified cardiac...

Phospholamban (PLN) phosphorylation contributes largely to the inotropic and lusitropic effects of beta-adrenergic agonists on the heart. The mechanical effects of PLN phosphorylation on the heart are generally attributed solely to an increase in the apparent affinity of the Ca pump in the sarcoplasmic reticulum (SR) membranes for Ca2+ with little or no effect on Vmax(Ca). In the present report, we compare the kinetic properties of the cardiac SR Ca pump in commonly studied crude microsomes with those of our recently developed preparation of light SR vesicles. We demonstrate that in crude microsomes, the increase in the apparent affinity of the pump for Ca2+ is larger, while the increase in Vmax(Ca) is smaller, than in purified vesicles. The greater phosphorylation-induced increase in apparent Ca2+ affinity in crude microsomes may be further enhanced by an ATP-sensitive inhibitory effect of ruthenium red on the activity of the pump at subsaturating, but not saturating, Ca2+ concentrations as a result of a greater inhibition in unphosphorylated microsomes. Upon increasing the ATP concentration from 1 to 5 mm, an inhibition by 10 micrometer ruthenium red is eliminated in phosphorylated microsomes and reduced in control microsomes. Addition of the phosphoprotein phosphatase inhibitor okadaic acid produces a considerable increase in the phosphorylation-induced effects in both crude and purified microsomes. We conclude that the use of purified cardiac SR vesicles is critical for the demonstration of a major increase in Vmax(Ca) in addition to an increase in the pump's apparent affinity for Ca2+ in response to phosphorylation of PLN by protein kinase A.