Abramowitz J, Campbell A R
Biol Reprod. 1985 Mar;32(2):463-74. doi: 10.1095/biolreprod32.2.463.
Cholera toxin elicited 5- to 7-fold stimulation of adenylyl cyclase activity. Half-maximal activation was at 4.42 micrograms/ml cholera toxin. Cholera toxin-mediated activation was time dependent. At 0.1 mM ATP, both guanosine triphosphate (GTP) and nicotinamide adenine dinucleotide (NAD+) were required for cholera toxin activation of luteal adenylyl cyclase. The concentrations of GTP and NAD+ required for half-maximal activation were 1 and 200 microM, respectively. The GTP requirement could be eliminated by increasing the ATP concentration to 1.0 mM. Guanosine-5'-O-(2-thiodiphosphate) [GDP beta S] did not support cholera toxin activation of the luteal enzyme. Cholera toxin treatment increased GTP-stimulated activity, did not significantly alter guanyl-5'-yl imidodiphosphate [GMP-P(NH)P]-stimulated activity, and depressed NaF-stimulated activity. Furthermore, toxin treatment resulted in a 3.4-fold reduction in the Kact values for ovine luteinizing hormone (oLH) to activate adenylyl cyclase. A similar reduction in Kact values for oLH was obtained when concentration-effect curves performed in the presence of GMP-P(NH)P were compared to those performed in the presence of GTP. In addition, luteal membranes treated with cholera toxin and [32P]NAD+ were subjected to autoradiographic analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This treatment resulted in the [32P] adenosine diphospho (ADP)-ribosylation of a 45,000-dalton protein doublet, corresponding to the alpha subunit of the stimulatory guanine nucleotide-binding regulatory component (Ns). As with activation of adenylyl cyclase activity, cholera toxin-specific [32P] ADP-ribosylation was time dependent and increased with increasing concentrations of cholera toxin. GTP, GMP-P(NH)P, and NaF, but not GDP beta S, were capable of supporting [32P] ADP-ribosylation of the protein doublet. oLH did not alter the ability of cholera toxin to ADP-ribosylate the protein activation of luteal adenylyl cyclase activity is due to the ADP-ribosylation of the alpha subunit of Ns and the concomitant inhibition of a GTPase associated with adenylyl cyclase.
霍乱毒素可引起腺苷酸环化酶活性5至7倍的刺激。半最大激活浓度为4.42微克/毫升霍乱毒素。霍乱毒素介导的激活具有时间依赖性。在0.1毫摩尔/升ATP条件下，鸟苷三磷酸（GTP）和烟酰胺腺嘌呤二核苷酸（NAD+）都是黄体腺苷酸环化酶被霍乱毒素激活所必需的。半最大激活所需的GTP和NAD+浓度分别为1微摩尔/升和200微摩尔/升。将ATP浓度提高到1.0毫摩尔/升可消除对GTP的需求。鸟苷-5'-O-(2-硫代二磷酸) [GDPβS] 不能支持霍乱毒素对黄体酶的激活。霍乱毒素处理增加了GTP刺激的活性，对鸟苷-5'-基亚氨基二磷酸 [GMP-P(NH)P] 刺激的活性没有显著改变，并且降低了氟化钠刺激的活性。此外，毒素处理使绵羊促黄体生成素（oLH）激活腺苷酸环化酶的Kact值降低了3.4倍。当比较在GMP-P(NH)P存在下进行的浓度-效应曲线与在GTP存在下进行的曲线时，oLH的Kact值也有类似程度的降低。此外，用霍乱毒素和[32P]NAD+处理的黄体膜在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后进行放射自显影分析。这种处理导致一种45000道尔顿的蛋白质双峰发生[32P]腺苷二磷酸（ADP）-核糖基化，对应于刺激性鸟嘌呤核苷酸结合调节成分（Ns）的α亚基。与腺苷酸环化酶活性的激活一样，霍乱毒素特异性的[32P]ADP-核糖基化具有时间依赖性，并且随着霍乱毒素浓度的增加而增加。GTP、GMP-P(NH)P和氟化钠能够支持蛋白质双峰的[32P]ADP-核糖基化，但GDPβS不能。oLH不会改变霍乱毒素对蛋白质进行ADP-核糖基化的能力。黄体腺苷酸环化酶活性的激活是由于Ns的α亚基的ADP-核糖基化以及与腺苷酸环化酶相关的一种GTP酶的伴随抑制。
