Protein kinase A- (PKA-) catalyzed phosphorylation of phospholamban (PLN), the protein regulator of the cardiac Ca pump, mediates abbreviation of systole in response to β-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 K(m(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 V(max(Ca)) at 25 and 37 °C, and an 11-31% decrease in K(m(Ca)) with comparable changes in Ca2+-ATPase activity. An acceleration of E2P decomposition upon PLN phosphorylation may contribute to the increased V(max(Ca)) of Ca uptake at 25 °C but not at 37 °C, based on measurement of the kinetics of E2P decomposition and steady-state E2P formation from P(i) at different temperatures. Our data document almost identical increases in V(max(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.
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