Comparison of the Kinetic Effects of Phospholamban Phosphorylation and Anti-phospholamban Monoclonal Antibody on the Calcium Pump in Purified Cardiac Sarcoplasmic Reticulum Membranes

Abstract

Protein kinase A- (PKA-) catalyzed phosphorylation of phospholamban (PLN), the protein regulator of the cardiac Ca pump, mediates abbreviation of systole in response to beta-adrenergic agonists. Investigators previously, however, have been unsuccessful in demonstrating an effect of PLN phosphorylation or anti-PLN monoclonal antibody (mAb), which is considered to mimic phosphorylation's well-known effect on Km(Ca), on microsomal Ca uptake at the (high) Ca2+ concentrations found intracellularly at peak systole. We therefore compared the effects of the catalytic subunit of PKA and anti-PLN mAb on the kinetics of Ca uptake in sucrose gradient-purified cardiac microsomes. Both treatments produced a 33-44% increase in Vmax(Ca) at 25 and 37 degrees C, and an 11-31% decrease in Km(Ca) with comparable changes in Ca2+-ATPase activity. An acceleration of E2P decomposition upon PLN phosphorylation may contribute to the increased Vmax(Ca) of Ca uptake at 25 degrees C but not at 37 degrees C, based on measurement of the kinetics of E2P decomposition and steady-state E2P formation from Pi at different temperatures. Our data document almost identical increases in Vmax(Ca) of microsomal Ca uptake with PLN phosphorylation or addition of anti-PLN mAb and hence provide insight into the kinetic mechanism of PLN's regulation of the cardiac sarcoplasmic reticulum Ca pump protein.