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

我发现一种鸟嘌呤核苷酸结合蛋白（G蛋白）与大鼠脑内的胞苷酸环化酶系统相关，并在功能上调节该酶的活性。用G蛋白特异性激活剂AlF_4^-和GTP γ S处理后，酶活性显著增加，而在G蛋白灭活剂GDP β S存在下，GTP γ S诱导的激活消失，证实了这一点。然而，胞苷酸环化酶系统中的G蛋白与已知的G蛋白如G_i和G_s对细菌毒素的ADP核糖基化的敏感性明显不同。经GTP γ S活化、CHAPS有效增溶、Superose 6凝胶过滤和DEAE-纤维素柱层析，部分纯化了该酶的催化单元。这是关于其纯化的首次报道，尽管其纯度仍然是其粗膜级分的40倍。由于其聚集，该制剂在凝胶过滤上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素。虽然激活机制尚不清楚，但这些证据支持胞苷酸环化酶及其产物cCMP在脑和其他组织中的重要作用。

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.