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

建立了Ca^<2+>与Ca^<2+>- atp酶突变体结合的测量方法，并提高了其准确性。本研究通过设计膜过滤法的洗涤液，可以直接测量结合的Ca^<2+>，尽管目前使用肌浆网Ca^<2+>- atp酶突变体测量Ca^<2+>的结合程度从外观量的低程度来看，分析起来非常困难。利用mutantsIn突变体将肌浆网Ca^<2+>- atp酶的Tyr^<122>转化为Ala （Y122A）的Ca^<2+>释放过程分析，我已经发现在此过程中E2PCa中间体在稳定状态下积累。在E2P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），位于细胞质结构域之间。制备了Y122-HC中其他残基的Ala取代突变体，并分析了其Ca^<2…More +>释放步骤的动力学。分析表明，与野生型相比，每个突变体向管腔侧释放Ca^<2+>的速率显著降低。此外，在部分突变体和Y122A中观察到E2PCa在稳态下的积累。用上述方法直接测定该突变体L119A在磷酸化培养基中Ca^<2+>的释放量，发现E2PCa^<2+>中Ca^<2+>的释放量明显慢于磷酸化酶（E1PCa_2⇒E2PCa_2）的转化量。Y122-HC在Ca^<2+>-ATPase的Ca^<2+>转运中的作用从上述结果中得到明确。通过对野生型Ca^<2+>- atpasin的一系列分析，新明确了野生型Ca^<2+>-ATPase对E2P的Ca^<2+>的性状与缺乏K^+的Y122-HC突变体非常相似。K^+离子在Ca^<2+>-ATPase的Ca^<2+>释放过程中起重要作用的结果，对于认识Ca^<2+>-ATPase的Ca^<2+>转运机制具有重要意义。少

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