Bell J D, Biltonen R L, Brunton L L
Department of Pharmacology, University of Virginia School of Medicine, Charlottesville 22908.
Mol Pharmacol. 1990 Apr;37(4):535-45.
The time course of cAMP production by S49 cell membranes in the presence of forskolin and a nonhydrolyzable GTP analog can yield information about the regulation of adenylate cyclase by both the inhibitory and stimulatory GTP-binding proteins (Gi and Gs). The time courses are complex and interpretation in terms of the activities of G1 and Gs requires a quantitative hypothesis. We present a general quantitative hypothesis that defines adenylate cyclase as existing in a distribution of two states, active and inactive. Gi and Gs, in their active states, alter the equilibrium of this distribution. Two distinct models are derived based on this hypothesis to accommodate two different proposed mechanisms for the action of Gi to inhibit adenylate cyclase: 1) a direct interaction between Gi and the catalytic subunit of adenylate cyclase and 2) a direct interaction between Gi and Gs. Perturbations of the regulation of adenylate cyclase by pertussis toxin and phorbol ester are simulated and interpreted using the models. The effect of pertussis toxin is quantitatively reconciled by decreases in the guanine nucleotide-independent adenylate cyclase activity and in the apparent rate of activation of Gi from 2.0/min to 0.01/min. The effect of phorbol ester is best accommodated by the model as a change in the distribution of active and inactive adenylate cyclase from 36% initially active to 47% active after phorbol ester treatment, without postulating any effect of phorbol ester on Gi or Gs. Both of these interpretations are independent of the model used. The effect of forskolin is also examined within the context of the two models. The results of this examination suggest an experimental approach for testing the models. These examples illustrate the usefulness of quantitative analysis of time course data using a model for the regulation of adenylate cyclase. We propose that, with this combined experimental and theoretical approach, one can address the relevance of hypotheses generated from experimental studies with isolated components to the molecular mechanisms of adenylate cyclase regulation in cellular membranes.
在存在福斯高林和一种不可水解的GTP类似物的情况下，S49细胞膜产生cAMP的时间进程能够提供有关抑制性和刺激性GTP结合蛋白（Gi和Gs）对腺苷酸环化酶调节的信息。时间进程很复杂，根据G1和Gs的活性进行解释需要一个定量假设。我们提出了一个通用的定量假设，将腺苷酸环化酶定义为以活性和非活性两种状态分布存在。Gi和Gs在其活性状态下会改变这种分布的平衡。基于该假设推导出两种不同的模型，以适应关于Gi抑制腺苷酸环化酶作用的两种不同提出机制：1）Gi与腺苷酸环化酶催化亚基之间的直接相互作用；2）Gi与Gs之间的直接相互作用。使用这些模型模拟并解释了百日咳毒素和佛波酯对腺苷酸环化酶调节的干扰。通过使鸟嘌呤核苷酸非依赖性腺苷酸环化酶活性降低以及Gi的表观激活速率从2.0/分钟降至0.01/分钟，定量地解释了百日咳毒素的作用。佛波酯的作用在模型中最好解释为，佛波酯处理后活性和非活性腺苷酸环化酶的分布从最初的36%活性变为47%活性，而无需假定佛波酯对Gi或Gs有任何影响。这两种解释均独立于所使用的模型。在这两种模型的背景下也研究了福斯高林的作用。该研究结果提出了一种测试模型的实验方法。这些例子说明了使用腺苷酸环化酶调节模型对时间进程数据进行定量分析的有用性。我们提出，通过这种实验与理论相结合的方法，可以探讨从对分离成分的实验研究中产生的假设与细胞膜中腺苷酸环化酶调节的分子机制的相关性。
