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Analysis of the Ca^<2+> release mechanism from the E2PCa intermediate of sarcoplasmic reticulum Ca^<2+> pump

肌浆网Ca^<2>泵E2PCa中间体Ca^<2>释放机制分析

基本信息

批准号：
18570119
负责人：
YAMASAKI Kazuo
金额：
$ 2.49万
依托单位：
Asahikawa Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18570119/
关键词：
Sarcoplasmic reticulum Calcium ion Ca^<2+> pump Enzyme Kinetics SERCA1a SERCA1a

项目摘要

Development of method for measurement of Ca^<2+> binding to mutants of Ca^<2+> -ATPase, and improvement of its accuracyMeasurement of bound Ca^<2+> was enabled to be done directly by devising the washing solution of the membrane filtoration method in this research, though it was very difficult in the analysis that used the mutant of sarcoplasmic reticulum Ca^<2+>-ATPase to measure Ca^<2+> binding from the low degree of the amount of appearance so far.Analysis of Ca^<2+> releasing process that uses mutantsIn mutant that changes Tyr^<122> of sarcoplasmic reticulum Ca^<2+>-ATPase into Ala (Y122A), I have already found that the E2PCa intermediate accumulates during it in the steady state. In crystal structures of E2P analogs, Tyr^<122> located at the center of the hydrophobic interaction network (Tyr^<122> Hydrophobic cluster: Y122-HC) which situated between cytoplasm domains. The Ala substitution mutants of the other residues include in Y122-HC were made and analyzed its kinetics of Ca^<2 … More +> releasing step. It was revealed from this analysis that the rates of Ca^<2+> release toward lumen side and of association had decreased remarkably compared with a wild type in each mutant. Moreover, the accumulation of the E2PCa in the steady state was observed in a part of mutants as well as Y122A. When Ca^<2+> release from the phosphorylation medium was measured directly by using the above-mentioned method about L119A in this mutant, it was shown that Ca^<2+> release from E2PCa^<2+> was obviously slower than the conversion of the phosphoenzyme (E1PCa_2⇒E2PCa_2). The role of Y122-HC in Ca^<2+> transport of Ca^<2+>-ATPase became clear from the above-mentioned result.Analysis that uses wild type Ca^<2+>-ATPaseIn a series of analysis, it was newly clarified to wild type Ca^<2+>-ATPase that the character to Ca^<2+> of E2P resembled the Y122-HC mutant very much in the absence of K^+. The result that K^+ ion plays an important role to Ca^<2+> release process of Ca^<2+>-ATPase is a finding of an important for recognize the Ca^<2+> transport mechanism of Ca^<2+>-ATPase. Less

Ca^<2 +> -ATP酶突变体结合Ca^<2+>测定方法的建立及准确性的提高本研究通过设计膜过滤法的洗涤液，使结合Ca^<2+>的测定直接进行，虽然用肌浆网Ca^<2+>-ATPase突变体测定Ca^<2+>很困难，在将<122>肌浆网Ca^<2+>-ATP酶的Tyr^变为Ala的突变体（Y122 A）中，我已经发现E2 PCa中间体在其稳态期间积累。在E2 P类似物的晶体结构中，Tyr^<122>位于疏水相互作用网络的中心（Tyr^<122>疏水簇：Y122-HC），该疏水相互作用网络位于细胞质结构域之间。对Y122-HC中的其它氨基酸残基进行了Ala取代突变，并对其Ca ~（2+）动力学进行了分析 ...更多信息 +>释放步骤。从该分析可以看出，与野生型相比，各突变体中向管腔侧释放的Ca^<2+>速率和缔合速率显著降低。此外，在部分突变体中观察到E2 PCa在稳态下的积累，以及Y122 A。用上述方法直接测定该突变体L119 A磷酸化培养基中Ca^2+的释放，发现E2 PCa ^2+释放Ca^2+的速度明显慢于磷酸化酶（E1 PCa_2 → E2 PCa_2）的转化速度。利用野生型Ca^<2 +>-ATP酶的分析在一系列分析中，新阐明了野生型Ca^<2 +>-ATP酶在缺乏K^+的情况下，E2 P对Ca^<2 +>的特性非常类似于Y122-HC突变体。K^+离子对Ca ^<2+>-ATPase的Ca ^<2 +>释放过程起重要作用，这一发现对认识Ca ^<2+>-ATPase的Ca^<2 +>转运机制具有重要意义。少