Rapid kinetics of alpha 2-adrenergic inhibition of adenylate cyclase. Evidence for a distal rate-limiting step.
Rapid kinetics of alpha 2-adrenergic inhibition of adenylate cyclase. Evidence for a distal rate-limiting step.
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腺苷酸环化酶的 α2-肾上腺素能抑制的快速动力学。
DOI：
10.1021/bi00448a015
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Neubig,RR
中科院分区：
文献类型：
--
作者：
Thomsen,WJ;Neubig,RR
摘要：天然鸟嘌呤核苷酸调节剂GTP存在时，腺苷酸环化酶的激活和抑制速度太快，无法用标准的生化方法进行研究。为了确定腺苷酸环化酶调节的限速步骤，我们使用一种新的快速混合淬火技术在亚秒到秒的时间尺度上测量了酶的刺激和抑制动力学。即使使用我们的快速混合淬火方法，PGE和福斯克林的激活也是瞬时的（cAMP积累在0.5到30秒之间呈线性）。相比之下，我们发现肾上腺素介导的抑制滞后期为1.2-10秒。滞后时间的长短取决于GTP和单价阳离子的浓度。在没有NaCl的情况下，抑制开始的速率常数（kinh）仅略有增加，GTP浓度在1µGTP下达到饱和值0.16 s ' 1 （11 /24.3 s）。在100 mM NaCl存在下，kinh与GTP浓度密切相关，在100µGTP下达到最大值0.57 s ' 1（1/2 1.2 s）。因此，在完整的血小板膜中，G和G的激活比先前报道的重组系统要快得多（tl/2< 5 s）。此外，腺苷酸环化酶抑制速率对GTP浓度的强烈依赖表明，抑制的限速步骤远低于GTP结合。由于KC1对kinh没有影响，所以NaCl对最大抑制率的提高是钠所特有的。相比之下，氯化钠和氯化钾都增加了肾上腺素和GTP的稳态EC50，但都没有影响最大百分比抑制。我们使用这些动态和稳态数据来定量测试a2受体和g介导的腺苷酸环化酶抑制的几种模型。在没有NaCl的情况下，两个不同的模型给出了相似的拟合实验数据。其中一个限速步骤是GDP的释放，而另一个限速步骤是gtp配体G蛋白的激活。只有后一种模型也解释了我们之前的观察，即在没有GTP的情况下，用a2激动剂对血小板膜进行预孵育会增加抑制的效力[Thomsen等人（1988）Mol. Pharmacol. 34,814 -822]。因此，在存在和不存在氯化钠的情况下，gpp配位G′的构象变化而不是GDP释放是限速的。根据该模型，氯化钠增加了三个反应步骤的速率：(1)激动剂与受体的解离；(2) GTP与G的分离；(3) gtp配体G蛋白的限速构象变化。本文报道的快速动力学研究为氯化钠和天然核苷酸调节剂GTP调控腺苷酸环化酶提供了新的信息。/激动剂结合a2-肾上腺素能受体，抑制腺苷酸环化酶（Limbird, 1981），激活其他细胞反应，如磷脂酶和离子通道（Limbird, 1988）。这些反应涉及至少三种不同的蛋白质：a2受体本身，称为G *的抑制性鸟嘌呤核苷酸结合蛋白和腺苷酸环化酶或其他效应酶的催化亚基（Gilman, 1987）。由于组分的数量及其相互作用的复杂性，激动剂介导的G蛋白活化的确切分子机制尚不清楚。利用质膜制备激素和鸟嘌呤核苷酸介导的腺苷酸环化酶刺激和抑制的瞬态动力学研究有助于我们理解这些过程的机制。对腺苷酸环化酶的刺激和抑制作用
Revised Manuscript Received June 13, 1989 abstract: Activation and inhibition of adenylate cyclase in the presence of GTP, the natural guanine nucleotide regulator, are too fast to study by standard biochemical methods. In order to identify the rate-limiting steps in adenylatecyclase regulation, we measured the kinetics of stimulation and inhibition of the enzyme on a subsecond to second time scale using a novel rapid-mix quench technique. Even using our rapid-mix quench method, activation by PGE, and forskolin was instantaneous (cAMP accumulation was linear between 0.5 and 30 s). In contrast, we found a lag period of 1.2-10 s for epinephrine-mediated inhibition. The length of the lag depended on the concentration of GTP and monovalent cations present. In the absence of NaCl, the rate constant for the onset of inhibition (kinh) increased only slightly with GTP concentration saturating at a value of 0.16 s" 1 (i1/24.3 s) at 1 µ GTP. In the presence of 100 mM NaCl, kinh was strongly dependent on GTP concentration, reaching a maximum value of 0.57 s" 1(1/2 1.2 s) at100 µ GTP. Thus, activation of both G¡ and Gs in intact platelet membranes is much faster(tl/2< 5 s) than previously reported for reconstituted systems. Also, the strong dependence of the rate of adenylate cyclase inhibition on GTP concentration implies that the rate-limiting step in inhibition is distal to GTP binding. The effect of NaCl to increase the maximal rate of inhibition is specific for sodium since KC1 has no effect on kinh. In contrast, both sodiumand potassium chloride increased the steady-state EC50 for epinephrine and GTP while neither affected the maximum percentage inhibition. We used these kinetic and steady-state data to test several models of a2-receptor and G-mediated adenylate cyclase inhibition in a quantitative manner. Two distinct modelsgave similar fits of experimental data in the absence of NaCl. In one, the rate-limiting step is GDP release while in the other, activation of the GTP-liganded G protein is limiting. Only the latter model also explains our prior observation that preincubation of platelet membranes with a2 agonist in the absence of GTP increases the potency for inhibition [Thomsen et al.(1988) Mol. Pharmacol. 34, 814-822]. Thus, both inthe presence and in the absence of sodium chloride, the conformational change of GTP-liganded G¡ rather than GDP release is rate limiting. According to this model, sodium chloride increases the rate of three reaction steps:(1) agonist dissociation from receptor;(2) GTP dissociation from G¡; and (3) the rate-limiting conformational change of GTP-liganded G protein. The rapid kinetic studies reported here provide new information regarding regulation of adenylate cyclase by sodium chloride and the natural nucleotide regulator, GTP../Agonist binding to a2-adrenergic receptors results in in-hibition of adenylate cyclase (Limbird, 1981) and activation of other cellular responses such as phospholipases and ion channels (Limbird, 1988). These responses involve at least three distinct proteins: the a2-receptor itself, an inhibitory guanine nucleotide binding protein termed G¡* and the catalytic subunit of adenylate cyclase or other effector enzyme (Gilman, 1987). Because of the number of components and the complex nature of their interactions, the exact molecular mechanism of agonist-mediated G protein activation remains unclear. Studies of the transient kinetics of hormone and guanine nucleotide mediated stimulation and inhibition of adenylate cyclase using plasma membrane preparations have contributed to our understanding of the mechanism of these processes. Both stimulation and inhibition of adenylate cyclase by non-