Comparative studies of cardiac and skeletal sarcoplasmic reticulum ATPases. Effect of a phospholamban antibody on enzyme activation by Ca2+.
Vesicular fragments of skeletal (LSR) and cardiac (CSR) sarcoplasmic reticulum were compared with the aim of defining the effect of a monoclonal phospholamban (Pl) antibody (Ab). The Pl Ab has no effect on LSR, while enhancing the Ca2+ transport rates of CSR at Ca2+ concentrations below saturation. We found no direct effect of the Pl Ab on Ca2+ binding by the ATPase in the absence of ATP. Equilibrium measurements of Ca2+ binding yield positively cooperative isotherms which are best fit with a two-interacting sites equation. LSR and CSR display nearly identical affinities for Ca2+, and no effect of the Pl Ab is observed. Taking advantage of a stable CrATP-enzyme complex, we demonstrated that the stoichiometric ratio of occluded Ca2+ to catalytic sites is 2 in either LSR or CSR and that the addition of Pl Ab does not affect the Ca2+ concentration dependence of Ca2+ occluded after equilibration of the system. Interestingly, the cooperative interaction between the two Ca2+ sites is lost in the occluded state, with only one of the two sites acquiring lumenal exposure. The concentration dependence of Ca2+ inhibition of CSR ATPase phosphorylation with Pi is also unaffected by the Pl Ab. Contrary to the lack of Pl Ab effect on reactions measured at equilibrium, enhancement of phosphorylated intermediate formation by the Pl Ab is obtained in kinetic experiments in which nonsaturating Ca2+ and ATP are added to CSR preincubated with EGTA. Therefore, Ab binding to Pl reduces specifically the activation energy for a slow transition triggered by Ca2+ binding, with consequent enhancement of overall kinetics under conditions enhancing the rate-limiting contribution of this transition.
Cantilina
T
T
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201-1503.
Sagara
Y
Y
Inesi
G
G
Jones
L R
LR
D000251:Adenosine Triphosphatases / Q000276:immunology / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000276:immunology; D004789:Enzyme Activation; D007700:Kinetics; D009132:Muscles / Q000201:enzymology* / Q000648:ultrastructure; D009206:Myocardium / Q000201:enzymology* / Q000648:ultrastructure; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D000099211:Phospholamban
D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D000911:Antibodies, Monoclonal; D002118:Calcium; D002135:Calcium-Binding Proteins; D000099211:Phospholamban