EVIDENCE FOR AN EFFECT OF PHOSPHOLAMBAN ON THE REGULATORY ROLE OF ATP IN CALCIUM-UPTAKE BY THE CALCIUM-PUMP OF THE CARDIAC SARCOPLASMIC-RETICULUM

The purpose of this study was to investigate the functional relationship between phospholamban and the nucleotide site of the calcium pump protein of the cardiac sarcoplasmic reticulum. We used control and trypsin-treated cardiac microsomes in which cleavage of the inhibitory cytoplasmic domain of phospholamban is associated with an activation of the calcium pump similar to that produced by protein kinase A catalyzed phospholamban phosphorylation. Phenylglyoxal was shown to inactivate the calcium pump in a pseudo-first-order reaction by binding to a single Arg at the nucleotide binding site. No differences upon trypsin treatment of microsomes were observed in the kinetics of phenylglyoxal inactivation or the ability of millimolar ATP to protect against inactivation. In subsequent kinetic studies, Ca-uptake rates measured at saturating Ca2+ and 5 muM-1 mM MgATP2- were increased 15-32% by trypsin treatment in each of three different microsome preparations. Double-reciprocal plots of the data showed marked downward curvature indicating an acceleratory effect associated with ligand binding to a lower affinity site. At 0.32 muM Ca2+, Ca-uptake rates were lower than at 11 muM Ca2+ but were stimulated to a greater extent by trypsin treatment; control microsomes showed reduced evidence of apparent negative cooperativity. At 0-2 muM MgATP2- and saturating Ca2+, there was a 50% increase in V(max(app)) when the Hill coefficient (N) was 1. At 0-10 muM MgATP2-, second-site binding was evident. At both 0-10 muM and 5 muM-1 mM MgATP2-, trypsin-treated microsomes showed greater activation of Ca uptake attributable to second-site binding than did control microsomes. Trypsin treatment produced a significant decrease in turbidity in cardiac microsome suspensions. Our data suggest that cleavage of the cytoplasmic segment of phospholamban from the membrane produces marked changes in the physical and functional properties of cardiac SR membranes that may be associated with a shift in the E* <-> E equilibrium toward E. The data, furthermore, are consistent with effects of phospholamban at two or more sites in a branched reaction pathway of the catalytic cycle, one of which is likely to interfere with the acceleratory effect of ATP at concentrations above those necessary for binding to the high-affinity catalytic site.