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Purification of Brain Cytidylate Cyclase and Physiological Function of Its Enzymological Product, Cyclic CMP

脑胞苷酸环化酶的纯化及其酶产物环化CMP的生理功能

基本信息

批准号：
02670998
负责人：
MUTO Norio
金额：
$ 1.41万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

I have found that a kind of guanine nucleotide-binding protein (G-protein) is associated with the cytidylate cyclase system in rat brain and it functionally regulates the enzyme activity. This was confirmed by the significant increase of enzyme activity after treatment with G-protein-specific activators, AlF_4^- and GTPgammaS, and by the disappearance of GTPgammaS-induced activation in the presence of G-protein inactivator, GDPbetaS. However, the responsible G-protein in cytidylate cyclase system was apparently different from well-known G-proteins such as G_i and G_s in their susceptibility to ADP-ribosylation by bacterial toxins. The catalytic unit of this enzyme was partially purified through the activation by GTPgammaS, effective solubilization by CHAPS, gel filtration on Superose 6 and DEAE-cellulose column chromatography. This was the first report on its purification, though the purity was still 40-fold over its crude membrane fraction. This preparation had a very high molecular weight on gel filtration due to its aggregation, showing distinct chromatographic behavior from that of adenylate cyclase. In this study, androgen was found to be a possible endogenous factor regulating enzyme activity directly. Although the mechanism of activation is unknown, these evidence support the important role of cytidylate cyclase and its product, cCMP, in the brain and other tissues.

我发现一种鸟嘌呤核苷酸结合蛋白（G蛋白）与大鼠脑中的胞苷酸环化酶系统相关，并在功能上调节酶的活性。用 G 蛋白特异性激活剂 AlF_4^- 和 GTPgammaS 处理后酶活性显着增加，以及在 G 蛋白失活剂 GDPbetaS 存在下 GTPgammaS 诱导的激活消失，证实了这一点。然而，胞苷酸环化酶系统中负责的G蛋白与众所周知的G蛋白如G_i和G_s明显不同，它们对细菌毒素ADP-核糖基化的敏感性明显不同。该酶的催化单元通过GTPgammaS活化、CHAPS有效溶解、Superose 6凝胶过滤和DEAE-纤维素柱层析得到部分纯化。这是关于其纯化的第一份报告，尽管纯度仍是其粗膜部分的 40 倍。由于其聚集，该制剂在凝胶过滤时具有非常高的分子量，显示出与腺苷酸环化酶不同的色谱行为。在这项研究中，发现雄激素可能是直接调节酶活性的内源性因子。尽管激活机制尚不清楚，但这些证据支持胞苷酸环化酶及其产物 cCMP 在大脑和其他组织中的重要作用。