Kinetic differences in the phospholamban-regulated calcium pump when studied in crude and purified cardiac sarcoplasmic reticulum vesicles

Phospholamban (PLN) phosphorylation contributes largely to the inotropic an d lusitropic effects of beta-adrenergic agonists on the heart. The mechanic al effects of PLN phosphorylation on the heart are generally attributed sol ely to an increase in the apparent affinity of the Ca pump in the sarcoplas mic reticulum (SR) membranes for Ca2+ with little or no effect on V-max(Ca) . In the present report, we compare the kinetic properties of the cardiac S R Ca pump in commonly studied crude microsomes with those of our recently d eveloped preparation of light SR vesicles. We demonstrate that in crude mic rosomes, the increase in the apparent affinity of the pump for Ca2+ is larg er, while the increase in V-max(Ca) is smaller, than in purified vesicles. The greater phosphorylation-induced increase in apparent Ca2+ affinity in c rude microsomes may be further enhanced by an ATP-sensitive inhibitory effe ct of ruthenium red on the activity of the pump at subsaturating, but not s aturating, Ca2+ concentrations as a result of a greater inhibition in unpho sphorylated microsomes. Upon increasing the ATP concentration from 1 to 5 m M, an inhibition by 10 mu M ruthenium red is eliminated in phosphorylated m icrosomes 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 d emonstration of a major increase in V-max(Ca) in addition to an increase in the pump's apparent affinity for Ca2+ in response to phosphorylation of PL N by protein kinase A.