Study on the Structure of the Catalytic Site and the Transport Mechanism of the Sarcoplasmic Reticulum Calcium Pump

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(1) ATP-binding sites of the sarcoplasmic reticulum Ca^<2+>-ATPase were titrated with TNP-[^3H]AMP or ー[^3H]AMP, and the bound TNP-nucleotides were chased with ATP. The results showed that there exists 1 mol of low-affinity ATP-binding sites as well as 1 mol of high-affinity ATP-binding sites (catalytic sites) per mole of phosphorylatable catalytic sites.(2) The effect of an ionophore, lasalocid, on the sarcoplasmic reticulum Ca^<2+>-ATPase was investigated. The results showed that this ionophore blocks hydrolysis of the ADP-insinsiteve phosphoenzyme intermediate and as a result strongly inhibits the Ca^<2+>-ATPase.(3) Cys-674 of the sarcoplasmic reticulum Ca^<2+>-ATPase was labeled with I-EDANS without a loss of the catalytic activity, and changes in the fluorescence intensity upon addition of seven kinds of substrate were followed by the stopped-flow method. The steady-state fluorescence intensity and anisotropy were also determined. The results showed that the conformational change, which makes the bound label less constrained, is induced by substrate binding to the catalytic site of the Ca^<2+>-activated enzyme. This change procedes phosphoenzyme formation in the catalytic cycle and greatly accelerated by the adenine moiety of the substrate.(4) Binding of FITC to the sarcoplasmic reticulum Ca^<2+>-ATPase was determined. The results showed that there exist two moles of specific FITC binding sites per mole of phosphorylatable catalytic sites and that all the FITC-binding sites are Lys-515 of the enzyme. These results suggest that the functional unit of the sarcoplasmic reticulum Ca^<2+> pump is a dimer of the Ca^<2+>-ATPase.(5) The effects of an ionophore, A23187, on conformational changes involved in the Ca^<2+>-induced activation of the sarcoplasmic reticulum Ca^<2+>-ATPase were investigated. It was found that the Ca^<2+>-dependent conformational change is biphasic and that the second slow phase of this conformational change is completely inhibited by A23187.