Identification of endogenous calcium channel activator and develpment of clinical analysis method by spedific antibodies.

内源性钙通道激活剂的鉴定及特异性抗体临床分析方法的开发。

基本信息

批准号：
03557108
负责人：
MAKI Masatoshi
金额：
$ 8.38万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03557108/
关键词：
Calcium Calcium-channel Calpastatin Calpain Calmodulin スプライシング PCR パッチクランプ

项目摘要

We have analyzed the endogenous calcium channel activating protein (CCAP) which suppresses the rapid run-down of the L-type Ca channel in the inside-out patch mode in guineapig ventricular myocytes. CCAP has been found in cytoplasmic fraction of the myocytes and has ability to restore the Ca channel activity after run-down. Biochemical properties of the partially purified CCAP showed those of the endogenous inhibitor protein (calpastatin) of Ca-dependent protease (calpain). A polyclonal antibody against calpastatin inhibited the CCAP activity. Moreover, calpastatin purified from pig heart and produced in E.coli by gene engineering exhibited CCAP activity, suggesting calpastatin is CCAP.The activity of calpastatin as CCAP, however, was low and could restore only 20% of the channel activity.(i) Since the CCAP requires ATP or other nucleotides, co-factors such as protein kinase A might me necessary for full activity.(ii) Analysis of the human calpastatin gene and its mRNA has revealed occurrence of multiple isoforms generated by alternative splicing. Presence of diverse calpastatin isoforms were confirmed by Western blot using calpastatin antibodies. Thus, it may be possible that specific isoform of calpastatin acts as CCAP of higher activity.(iii) Structure-function analysis of calpastatin has suggested that conserved subdomains A and C is dispensable for calpain inhibition but important for interaction with calmodulin-like domain of calpain, while subdomain B is essential for the inhibition.Since the low molecular weight calpain inhibitor leupeptin could not suppress the channel activity and the CCAP could resore the activity, proteolysis by calpain may not be involved in the system. Further works are necessary to understand the mechanism of suppression of run-down and restoration of the Ca-channel by CCAPs.

我们已经分析了内源性钙通道激活蛋白（CCAP），该蛋白（CCAP）抑制了Guineapig心室心肌细胞的内而外贴片模式下L型CA通道的快速分解。在肌细胞的细胞质级分中发现了CCAP，并且具有拆卸后CA通道活性的能力。部分纯化的CCAP的生化特性显示了CA依赖性蛋白酶（CALPAIN）的内源性抑制剂蛋白（calpastatin）的生化特性。抗calpastatin的多克隆抗体抑制了CCAP活性。此外，基因工程中从猪心脏纯化并在大肠杆菌中纯化的钙蛋白酸钙蛋白酸脂蛋白表现为CCAP活性，这表明钙蛋白酸钙蛋白酸钙蛋白酸盐是CCAP。然而，Calpastatin作为CCAP的活性很低，但是仅恢复了20％的通道活性。人Calpastatin基因及其mRNA揭示了通过替代剪接产生的多种同工型的出现。使用Calpastatin抗体通过蛋白质印迹证实了多种calpastatin同工型的存在。因此，钙钙蛋白钙蛋白钙蛋白酸的特异性同工型可能充当较高活性的CCAP。（iii）结构 - 钙蛋白酸的结构 - 函数函数分析分析表明，保守的子域A和C可以抑制钙蛋白酶抑制作用，但对于与钙蛋白酶的钙质样子相互作用很重要，而对于calpain becain的体重而言，cal是calsion的体重。抑制通道活性，CCAP可以重新获得活性，系统的蛋白水解可能不参与系统。需要进一步的工作来了解CCAP抑制破败和恢复CA通道的机理。