Mickevicius C K, Harrison J K, Gnegy M E
Mol Pharmacol. 1986 Nov;30(5):469-75.
The effect of cholera toxin on activation of adenylate cyclase by the endogenous Ca2+-binding protein, calmodulin, GTP, dopamine, and forskolin was investigated in bovine striatum. Adenylate cyclase activity was measured in washed membrane fractions prepared from homogenates that had been preincubated with cholera toxin. Pretreatment of striatal membranes with cholera toxin increased the response of adenylate cyclase to GTP, calmodulin, and forskolin as compared to vehicle controls. After cholera toxin pretreatment, the maximal response of adenylate cyclase to GTP was increased 4.7-fold and the apparent Ka for GTP was reduced 3-fold. The apparent Vmax for calmodulin was doubled after cholera toxin pretreatment. The activation of adenylate cyclase by forskolin was increased by cholera toxin, but the effect on kinetic parameters was not determined due to solubility considerations. In contrast, dopamine-stimulated adenylate cyclase activity was abolished after cholera toxin pretreatment. Examination of a concentration-response curve for cholera toxin in altering these activities revealed that calmodulin-stimulated adenylate cyclase was maximally affected at lower concentrations of cholera toxin than was activation by GTP and forskolin. Cholera toxin also affected the interaction between calmodulin and GTP. In the absence of cholera toxin, calmodulin decreased the apparent Ka for GTP nearly 10-fold. After cholera toxin pretreatment, however, calmodulin could not further decrease the apparent Ka for GTP but increased the maximal response to GTP by 30%. Calmodulin could potentiate GTP activation by stabilizing the interaction between Ns and the catalytic subunit, an action which could be negated by prior treatment with cholera toxin. ADP-ribosylation of the striatal homogenates with [32P]NAD demonstrated predominant labeling of a band of Mr 45,000 which corresponds to the known molecular weight of the alpha-subunit of the stimulatory GTP-binding protein, Ns. These results suggest that the activational state of Ns can affect the stimulation of adenylate cyclase by calmodulin and forskolin. Calmodulin and forskolin may act at separate sites on the catalytic subunit that can allosterically interact with Ns.
在牛纹状体中研究了霍乱毒素对由内源性钙结合蛋白钙调蛋白、GTP、多巴胺和福斯高林激活腺苷酸环化酶的影响。在与霍乱毒素预孵育的匀浆制备的洗涤膜部分中测量腺苷酸环化酶活性。与载体对照相比，用霍乱毒素预处理纹状体膜增加了腺苷酸环化酶对GTP、钙调蛋白和福斯高林的反应。霍乱毒素预处理后，腺苷酸环化酶对GTP的最大反应增加了4.7倍，GTP的表观Ka降低了3倍。霍乱毒素预处理后，钙调蛋白的表观Vmax增加了一倍。霍乱毒素增加了福斯高林对腺苷酸环化酶的激活，但由于溶解性考虑，未确定对动力学参数的影响。相反，霍乱毒素预处理后，多巴胺刺激的腺苷酸环化酶活性被消除。检查霍乱毒素改变这些活性的浓度-反应曲线发现，钙调蛋白刺激的腺苷酸环化酶在比GTP和福斯高林激活更低浓度的霍乱毒素下受到最大影响。霍乱毒素还影响钙调蛋白和GTP之间的相互作用。在没有霍乱毒素的情况下，钙调蛋白使GTP的表观Ka降低了近10倍。然而，霍乱毒素预处理后，钙调蛋白不能进一步降低GTP的表观Ka，但使对GTP的最大反应增加了30%。钙调蛋白可以通过稳定Ns与催化亚基之间的相互作用来增强GTP激活，这种作用可以被预先用霍乱毒素处理所抵消。用[32P]NAD对纹状体匀浆进行ADP核糖基化显示，主要标记的一条带的Mr为45,000，这与刺激性GTP结合蛋白Ns的α亚基的已知分子量相对应。这些结果表明，Ns的激活状态可以影响钙调蛋白和福斯高林对腺苷酸环化酶的刺激。钙调蛋白和福斯高林可能作用于催化亚基上可以与Ns变构相互作用的不同位点。
