Cassel D, Selinger Z
J Cyclic Nucleotide Res. 1977 Feb;3(1):11-22.
Incubation of Gpp(NH)p-activated adenylate cyclase in the presence of isoproterenol caused the release of bound [3H]-Gpp(NH)p, and the decline of activity to the basal state. The isoproterenol-induced release of the nucleotide was proportional to the decrease in adenylate cyclase activity. Since there is a large excess of Gpp(NH)p binding sites in the membrane, the isoproterenol induced release of Gpp(NH)p, rather than binding of the nucleotide, was used to measure the amount of guanyl nucleotide binding sites coupled to the activated adenylate cyclase. This amount, 1.5-2.0 pmoles/mg membrane protein, is only approximately 1% of the total Gpp(NH)p binding sites, and is about equal to the number of beta-adrenergic receptors in the membrane. Chromatographic analysis revealed that Gpp(NH)p was released from the membrane as an intact molecule. The findings suggest that persistent activation of the adenylate cyclase is due to persistent binding of Gpp(NH)p to the regulatory site, and that this GTP analog is a better activator of the adenylate cyclase than GTP because of its resistance to hydrolysis.
在异丙肾上腺素存在的情况下，对Gpp(NH)p激活的腺苷酸环化酶进行温育，导致结合的[3H]-Gpp(NH)p释放，且活性下降至基础状态。异丙肾上腺素诱导的核苷酸释放与腺苷酸环化酶活性的降低成正比。由于膜中存在大量过量的Gpp(NH)p结合位点，因此使用异丙肾上腺素诱导的Gpp(NH)p释放而非核苷酸结合来测量与激活的腺苷酸环化酶偶联的鸟苷酸结合位点的数量。这个数量为1.5 - 2.0皮摩尔/毫克膜蛋白，仅约占总Gpp(NH)p结合位点的1%，且大约等于膜中β-肾上腺素能受体的数量。色谱分析表明，Gpp(NH)p作为完整分子从膜中释放。这些发现表明，腺苷酸环化酶的持续激活是由于Gpp(NH)p持续结合到调节位点，并且由于这种GTP类似物对水解的抗性，它是比GTP更好的腺苷酸环化酶激活剂。
