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Structure and function of the catalytic site of sarcoplasmic reticulum Ca^<2+>-ATPase.

肌浆网Ca^2-ATP酶催化位点的结构和功能。

基本信息

批准号：
09680609
负责人：
KANAZAWA Tohru
金额：
$ 2.11万
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：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

The functional role of Arg^<198> in sarcoplasmic reticulum Ca^<2+>-ATPase was investigated by site-directed mutagenesis, in which Arg^<198> was substituted with lysine, glutamine, glutamic acid, alanine, and isoleucine. Mutated cDNAs were transfected into COS-1 cells, and kinetic analysis was performed with microsomal membranes isolated from the transfected cells. The turnover rate of the mutant R198K was almost the same as that of the wild type, whereas the turnover rates of the mutants R198Q, R198A, and R198I were substantially. lower than that of the wild type. The turnover rate was further reduced in the mutant R198E.The phosphoenzyme formed from ATP in the presence of K^+ at steady state was almost completely ADP-sensitive in the mutant R198K as well as in the wild type. In contrast, the ADP-insensitive phosphoenzyme accumulated to a considerable extent in the mutant R198Q, to a larger extent in the mutants R198A and R198I, and to the largest extent in the mutant R198E.The time course of dephosphorylation of the ADP-insensitive phosphoenzyme was determined by first phosphorylating the Ca^<2+>-ATPase with ^<32>Pi in the absence of K^+ and then diluting the sample with a solution containing nonradioactive Pi and KCl (or LiCl in place of KCl). The rate of dephosphorylation in the presence or absence of K^+ was reduced substantially in the mutant R198Q, more strongly in the mutants R198A and R198I, and most strongly in the mutant R198E, but to a much lesser extent in the R198K.These results indicate that the positive charge and high hydrophilicity of Arg^<198> are critical for rapid hydrolysis of the ADP-insensitive phosphoenzyme.

通过<198>定点突变研究了Arg^在肌浆网Ca^<2+>-ATP酶中的功能作用，其中Arg^<198>被赖氨酸、谷氨酰胺、谷氨酸、丙氨酸和异亮氨酸取代。将突变的cDNA转染到COS-1细胞中，并用从转染细胞中分离的微粒体膜进行动力学分析。突变体R198 K的周转率与野生型几乎相同，而突变体R198 Q、R198 A和R198 I的周转率基本相同。低于野生型。突变体R198 E的转换率进一步降低。在K^+存在的情况下，稳定状态下由ATP形成的磷酸酶在突变体R198 K和野生型中几乎完全对ADP敏感。相反，ADP不敏感的磷酸酶在突变体R198 Q中积累到相当大的程度，在突变体R198 A和R198 I中积累到更大的程度，在突变体R198 E中积累到最大的程度。ADP不敏感的磷酸酶的去磷酸化的时间过程是通过首先在没有K^+存在的情况下用^ Pi磷酸化Ca^2+-ATP酶<32>，然后用含有非放射性Pi和KCl（或LiCl代替KCl）的溶液稀释样品来确定的。在K^+存在或不存在的情况下，突变体R198 Q的去磷酸化速率显著降低，突变体R198 A和R198 I的去磷酸化速率降低更明显，突变体R198 E的去磷酸化速率降低最明显，而R198 K的去磷酸化速率降低更<198>小。