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Studies on Structure and Function of the ATP-Binding sites of the Sarcoplasmic Reticulum Calcium Pump.

肌浆网钙泵 ATP 结合位点的结构和功能研究。

基本信息

批准号：
04670134
负责人：
SUZUKI Hiroshi
金额：
$ 1.34万
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：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04670134/
关键词：
Sarcoplasmic Reticulum Calcium Pump ATP-Binding Sites Fluorescein 5-Isothiocyanate

项目摘要

In an attempt to establish the stoichiometry of the phosphorylatable catalytic site to the specific fluorescein 5-isothiocyanate (FITC) binding site (Lys-515) in the Ca^<2+>-ATPase of sarcoplasmic reticulum vescicles (SRV), labeling by FITC or phosphorylation by ATP (or Pi) was : performed with SRV under the conditions in which almost all of the specific FITC binding sites or phosphorylatable catalytic sites can be labeled or phosphorylated. The resultant SRV were solubilized in lithium dodecyl sulfate, and then the Ca^<2+>ATPase was purified by using a size exclusion high performance liquid chromatography. The contents of bound FITC and phosphoenzyme in the ATPase isolated as above were determined. Peptide maapping of the tryptic digests and sequencing showed that Lys-515 of the Ca^<2+>-ATPase was exclusively labeled with FITC.This specific labeling was completely prevented by ATP.The content of phosphoenzyme (4.57 and 4.94 nmol/mg of protein from ATP and Pi respectively) was approximately half that of the specific FITC binding site (8.16-8.19 nmol/m of protein) and also half that of the llO-KDa Ca^<2+>ATPase chain (9.06 nmol/mg of protein) calculated on the assumption that the isolated ATPase chain was pure. These findings are consistent with a possible half-of-the-sites reactivity, being in favor of a dimeric structure of the Ca^<2+>ATPase in SRV.The extent of the specific FITC binding required for complete inhibition of ATP-induced phosphorylation corresponded to 6.4 nmol of bound FITC/mg of protein. This finding suggests that FITC somewhat preferentially binds to one half of the ATPase chains in SRV and that this binding is primarily responsible for the observed inhibition of phosphorylation.

为了确定肌浆网囊泡（SRV）Ca^2+-ATP酶中磷酸化催化位点与特异性荧光素5-异硫氰酸酯（FITC）结合位点（Lys-515）的化学计量，FITC标记或ATP（或Pi）磷酸化的结果如下：在几乎所有的特异性FITC结合位点或可磷酸化的催化位点都可以被标记或磷酸化的条件下用SRV进行。用十二烷基硫酸锂溶解得到的SRV，然后用尺寸排阻高效液相色谱法纯化Ca^2+ ATP酶。测定了上述分离的ATP酶中结合的FITC和磷酸酶的含量。胰蛋白酶肽图和测序结果表明，Ca^<2+>-ATP酶的Lys-515只被FITC标记，这种特异性标记被ATP完全阻断。（ATP和Pi分别为4.57和4.94 nmol/mg蛋白）约为特异性FITC结合位点的一半（8.16-8.19 nmol/m蛋白质），也是110-KDa Ca^2+ ATP酶链的一半（9.06 nmol/mg蛋白质），假设分离的ATP酶链是纯的。这些发现与可能的半位点反应性相一致，支持SRV中Ca^2+ ATP酶的二聚体结构。完全抑制ATP诱导的磷酸化所需的特异性FITC结合程度对应于6.4 nmol结合的FITC/mg蛋白。这一发现表明，FITC在某种程度上优先结合到SRV中的ATP酶链的一半，并且这种结合是观察到的磷酸化抑制的主要原因。