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Stabilization of Folding of Sarcoplasmic Reticulum Calcium Pump by the N-terminal Domain

N 末端结构域对肌浆网钙泵折叠的稳定

基本信息

批准号：
12680602
负责人：
DAIHO Takashi
金额：
$ 2.3万
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：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

1. The functional role of Cys876 and Cys888 in the luminal loop between 7^<th> and 8^<th> transmembrane helices of sarcoplasmic reticulum calcium pump was investigated. Isolation and sequencing of disufide-containing peptides from the pepsin-digest of the calcium pump showed that a disulfide bond is formed between these cysteines. All of C876A, C888A, and C876A/C888A mutants lacked Ca^<2+> transport activity, but their ATP hydrolysis was not inhibited. These results suggest that this disulfide bond play a role in stabilizing the enzyme structure important for coupling between ATP hydrolysis and Ca^<2+> transport.2. The cytoplasmic domains of calcium pump are small cytosolic (A), nucleotide binding (N), and phosphorylation (P) domains, which are conserved among P-type cation transporting ATPases. Movement of these domains during the ATPase reaction cycle was investigated by analyzing digestion-susceptibility of the loop connecting A with transmembrane domain (A-M loop) to proteinase K or V8 and digestion-susceptibility of A to trypsin. The results suggest that P, N, and A-M loop (but not A) gather in substrate bound enzyme (CaE/ATP) and Ca^<2+>-bound phosphoenzyme, and that all 3 domains gather to form the most compact structure by the phosphoenzyme isomerization to Ca^<2+>-unbound form (Ca^<2+>-translocating step), in which A rotate by about 90 ℃.3. It is known that Ca^<2+>-unbound calcium pump is rapidly denatured if solubilized with detergent, so that structural study of this state has not been easy. I previously reported that Mg^<2+> and fluoride bind to the enzyme very tightly to form an analog of Ca^<2+>-unbound phosphoenzyme. It was found that purified and C12E8-solubilized Mg^<2+>/F-bound calcium pump is completely active for at least 20 days without Ca^<2+>, and that this analog may be useful for crystallization.

1.研究了Cys876和Cys888在肌浆网钙泵第7次和第8次跨膜螺旋之间的管腔环路中的功能作用。对钙泵胃蛋白酶消化物中含有二硫键的肽进行分离和测序表明，这些半胱氨酸之间形成了二硫键。所有C876A、C888A和C876A/C888A突变体均缺乏Ca 2+ 转运活性，但它们的ATP水解未被抑制。这些结果表明，该二硫键在稳定酶结构方面发挥着作用，该酶结构对于ATP水解和Ca 2+ 运输之间的耦合很重要。2．钙泵的胞质结构域是小胞质 (A)、核苷酸结合 (N) 和磷酸化 (P) 结构域，这些结构域在 P 型阳离子转运 ATP 酶中是保守的。通过分析连接 A 与跨膜结构域（A-M 环）的环对蛋白酶 K 或 V8 的消化敏感性以及 A 对胰蛋白酶的消化敏感性，研究了 ATP 酶反应循环期间这些结构域的运动。结果表明，P、N和A-M环（而不是A）聚集在底物结合酶（CaE/ATP）和Ca^2+结合磷酸酶中，并且通过磷酸酶异构化为Ca^2+-非结合形式（Ca^2+-转位步骤），所有3个结构域聚集形成最紧凑的结构，其中A旋转约90℃。 3．已知Ca^2+-未结合的钙泵如果用洗涤剂溶解则迅速变性，因此该状态的结构研究并不容易。我之前报道过Mg 2+ 和氟化物与酶非常紧密地结合，形成Ca 2+ -未结合的磷酸酶的类似物。发现纯化的和C12E8溶解的Mg 2+ /F结合的钙泵在没有Ca 2+ 的情况下至少20天完全有活性，并且该类似物可用于结晶。