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Study for the regulation mechanism of nitric oxide synthase activity by an autoinhibitory domain and calmodulin

自抑制结构域和钙调蛋白调节一氧化氮合酶活性的机制研究

基本信息

批准号：
12680624
负责人：
SAGAMI Ikuko
金额：
$ 2.43万
依托单位：
TOHOKU UNIVERSITY
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

In neuronal nitric-oxide synthase (nNOS), calmodulin (CaM) binding is thought to trigger electron transfer from the reductase domain to the heme domain, which is essential for O2 activation and NO formation. To elucidate the electron-transfer mechanism, we characterized a series of heterodimers consisting of one full-length nNOS subunit and one oxygenase-domain subunit. The results support an inter-subunit electron-transfer mechanism for the wild-type nNOS, in that electrons for catalysis transfer in a Ca^<2+>/CaM dependent way from the reductase domain of one subunit to the heme of the other subunit, as proposed for iNOS. This suggests that the two different isoforms form similar dimeric complexes. In a series of heterodimers containing a Ca^<2+>/CaM-insensitive mutant (delta40), electrons transferred from the reductase domain to both hemes in a Ca^<2+>/CaM independent way. Thus, in the delta40 mutant electron transfer from the reductase domains to the heme domains can occur via both inter-subunit and intra-subunit mechanisms. However, NO formation activity was exclusively linked to inter-subunit electron transfer and was observed only in the presence of Ca^<2+>/CaM. This suggests that the mechanism of activation of nNOS by CaM is not solely dependent on the activation of electron transfer to the nNOS hemes, but may involve additional structural factors linked to the catalytic action of the heme domain.Next we constructed an expression system of nNOSμ in E. coli cell. nNOSμ is an isoform of nNOS which specifically expressed in skeletal muscle cells and contains extra-33 amino acids in the autoinhibitory domain of nNOS wild-type. We succeeded to get enough amount of purified nNOSμ protein to characterize. Now we are undergoing to analyze the regulation of NO formation activity of nNOSμ by CaM and caveoline.

在神经元型一氧化氮合酶（nNOS）中，钙调素（CaM）结合被认为触发从还原酶结构域到血红素结构域的电子转移，这对于O2活化和NO形成是必需的。为了阐明电子转移机制，我们表征了一系列由一个全长nNOS亚基和一个加氧酶结构域亚基组成的异二聚体。这些结果支持野生型nNOS的亚基间电子转移机制，因为催化电子以Ca^2+/CaM依赖的方式从一个亚基的还原酶结构域转移到另一个亚基的血红素，正如对iNOS提出的那样。这表明两种不同的同种型形成相似的二聚体复合物。在一系列含有Ca^2+/CaM不敏感突变体（delta 40）的异二聚体中，电子以Ca^2+/CaM独立的方式从还原酶结构域转移到两个血红素。因此，在δ 40突变体中，从还原酶结构域到血红素结构域的电子转移可以通过亚基间和亚基内机制发生。然而，NO形成活性仅与亚基间电子转移相关，并且仅在Ca^<2+>/CaM存在的情况下观察到。这表明CaM激活nNOS的机制不仅依赖于电子传递到nNOS血红素的激活，还可能涉及与血红素结构域催化作用相关的其他结构因素。coli细胞。nNOSμ是nNOS的一种亚型，在骨骼肌细胞中特异性表达，在nNOS野生型的自抑制结构域中含有额外的33个氨基酸。我们成功地获得了足够量的纯化nNOSμ蛋白进行表征。本研究旨在探讨CaM和caveoline对nNOSμ的NO生成活性的调节作用。