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

（1）ATP-绑定站点of the sarcoplasmic reticulum Ca^<2+>-ATPase were titrated with TNF-[^3 H]AMP or-[^3 H]AMP， and the bound TNP-nucleotides were chased with ATP。结果显示，存在1摩尔的低亲和力ATP-绑定站点，而井为1摩尔的高亲和力ATP-绑定站点（催化站点）每摩尔的可磷酸催化站点。（2）离子化、嗜酸性、对碱性纤维素钙^<2+>的影响-ATP酶被调查。该结果显示了这种离子离子块的羟基化（ADP-insiteve phosphoenzyme intermediate），并作为一种结果强烈抑制了Ca^<2+>-ATPase。（3）碱性视网膜Ca^<2+>的Cys-674-ATPase被标记为I-EDANS，而没有催化活性的损失，并且在七种物质的荧光强度上增加了七种物质的沉积物，这些变化被停止流动方法所跟踪。稳定状态的荧光强度和其他形态也是确定的。结果显示了符合性的变化，是什么导致了无约束标签的约束，是由底层绑定到Ca^<2+>激活的酶位点。这一变化过程是在催化循环中形成磷酸酶，并被基质的肾上腺素动力大大加速。（4）FITC与碱性视网膜Ca^<2+>的绑定-ATP酶已确定。结果显示，每个摩尔的特定FITC绑定站点有两个摩尔，每个摩尔的磷酸化催化站点，所有FITC绑定站点都是酶的Lys-515。These results suggest that the functional unit of the sarcoplasmic reticulum Ca^<2 +> pump is a dimer of the Ca^<2+>-ATPase。（5）电离层的影响， A23187，关于适应性变化在Ca^<2+>-诱导激活的碱性纤维素Ca^<2+>-ATP酶已被调查。它发现Ca^<2+>-依赖的一致性变化是双向的，而这一一致性变化的第二个缓慢阶段完全被抑制。

(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.