Search

Publisher Summary The rate of calcium uptake by the cardiac sarcoplasmic reticulum Ca2+, Mg2+-ATPase is regulated by both cAMP-dependent phosphorylation and Ca2+-calmodulin-dependent phosphorylation. The target of this covalent concerted regulation is phospholamban, a proteolipid of Mr 11,000. The short stretch of phospholamban exposed at the cytosolic side of the membrane contains both sites of phosphorylation, perhaps in a double-headed configuration. The membrane-bound kinase acts as a sensor of the cytosolic free Ca2+ concentration. Phospholamban seems to be strongly associated with its calmodulin-dependent kinase and with the calcium pump. The canine cardiac sarcoplasmic reticulum (SR) is prepared from dog hearts arrested in diastole after pentobarbital injection. Both the Ca2+-calmodulin - and cAMP-dependent phosphorylations are followed. Each type of phosphorylation can be studied without interference from the other: the cAMP-dependent phosphorylation is performed in the presence of ethylenebis(oxyethylenenitrilo) tetraacetic acid (EGTA) and catalytic subunit (C subunit) and in the absence of calmodulin, whereas the Ca2+-CaM-dependent phosphorylation is done in the presence of Ca2+, PKI, and exogenous calmodulin and in the absence of cAMP or added C subunit.