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

肌浆网钙泵催化位点结构及转运机制研究

基本信息

批准号：
01580181
负责人：
KANAZAWA Tohru
金额：
$ 1.6万
依托单位：
Asahikawa Medical college
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01580181/
关键词：
Sarcoplasmic Reticulum Calcium Pump Catalytic Site Structure Analysis Transport Mechamism Calcium ATPase ATPア-ゼ Ca^<2+>-ATPase 低親和性ATP結合部位蛍光プロ-ブ I-EDANS FSBεA コンホメ-ション変化

项目摘要

(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.

(1)肌浆网Ca^2+-ATP酶的ATP结合位点用TNP-[^3H]AMP或ATP [^3H]AMP滴定，结合的TNP核苷酸用ATP追踪。结果表明，每摩尔磷酸化催化位点中存在1摩尔低亲和力ATP结合位点和1摩尔高亲和力ATP结合位点（催化位点）。(2)研究了离子载体拉沙洛西对肌浆网Ca^<2+>-ATP酶的作用。结果表明，该离子载体能阻断ADP-肌醇磷酸酶中间体的水解，从而强烈抑制Ca^2+-ATPase。(3)用I-EDANS标记肌浆网Ca^<2+>-ATP酶的Cys-674而不丧失催化活性，并通过停流法跟踪加入7种底物后荧光强度的变化。测定了荧光强度和各向异性。结果表明，底物与Ca^2+激活酶的催化位点结合引起构象变化，使结合的标记物受到更少的限制。这种变化在催化循环中促进磷酸酶的形成，并被底物的腺嘌呤部分大大加速。(4)测定FITC与肌浆网Ca^<2+>-ATP酶的结合。结果表明，每摩尔酶的磷酸化催化位点有2摩尔特异性的FITC结合位点，且所有的FITC结合位点均为酶的Lys-515。这些结果表明，肌浆网Ca^<2+>泵的功能单位是Ca^<2+>-ATP酶的二聚体。(5)研究了离子载体A23187对Ca^<2 +>诱导的肌浆网Ca ^<2 +>-ATP酶激活过程中构象变化的影响。结果发现，Ca^<2+>依赖的构象变化是双相的，并且这种构象变化的第二个慢相被A23187完全抑制。