Brandt D R, Ross E M
J Biol Chem. 1985 Jan 10;260(1):266-72.
The GTPase activity of the stimulatory guanine nucleotide-binding regulatory protein (Gs) of hormone-sensitive adenylate cyclase was investigated using purified rabbit hepatic Gs and either [alpha-32P]- or [gamma-32P] GTP as substrate. The binding of [35S]guanosine 5'-O-(thiotriphosphate) (GTP gamma S) was used to quantitate the total concentration of Gs. 1) GTPase activity was a saturable function of the concentration of GTP, with Km = 0.3 microM. MgCl2 monotonically increased the activity. The maximum observed turnover number was about 1.5 min-1. 2) During steady-state hydrolysis, 20-40% of total Gs could be trapped as a Gs-GDP complex and 1-2% could be trapped as Gs-GTP. The hydrolysis of Gs-GTP to Gs-GDP occurred with t 1/2 less than or equal to 5 s at 30 degrees C and t 1/2 approximately 1 min at 0 degrees C. Hydrolysis of Gs-GTP was inhibited by 1.0 mM EDTA in the absence of added Mg2+. 3) The rate of formation of Gs-GDP and the initial GTPase rate varied in parallel as functions of the concentrations of either GTP or MgCl2 (above 0.1 mM Mg2+). The ratio of the rate of accumulation of Gs-GDP to the GTPase rate was constant at 0.3-0.4. 4) The rate of dissociation of assayable Gs-GDP was biphasic. The initial phase accounted for 60-80% of total assayable Gs-GDP and was characterized by a t 1/2 of about 1 min. 5) Lubrol 12A9 potently inhibited the GTPase reaction and the dissociation of Gs-GDP in parallel, and inhibition of product release may account for the inhibition of steady-state hydrolysis. 6) The beta and gamma subunits of Gs markedly inhibited the dissociation of GDP from Gs in contrast to their ability to stimulate the dissociation of GTP gamma S. 7) GDP, GTP gamma S, and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) competitively inhibited the accumulation of Gs-GDP. GTP gamma S and Gpp(NH)p inhibited the GTPase reaction noncompetitively, GDP displayed mixed inhibition, and Pi did not inhibit. These data are interpretable in terms of the coexistence of two specific mechanistic pathways for the overall GTPase reaction.
利用纯化的兔肝刺激性鸟嘌呤核苷酸结合调节蛋白（Gs），以[α-32P]-或[γ-32P] GTP作为底物，研究了激素敏感腺苷酸环化酶的刺激性鸟嘌呤核苷酸结合调节蛋白（Gs）的GTP酶活性。使用[35S]鸟苷5'-O-（硫代三磷酸）（GTPγS）的结合来定量Gs的总浓度。1）GTP酶活性是GTP浓度的饱和函数，Km = 0.3μM。MgCl2单调增加活性。观察到的最大周转数约为1.5 min-1。2）在稳态水解过程中，20-40%的总Gs可以捕获为Gs-GDP复合物，1-2%可以捕获为Gs-GTP。在30℃时，Gs-GTP水解为Gs-GDP的t 1/2小于或等于5秒，在0℃时t 1/2约为1分钟。在没有添加Mg2+的情况下，1.0 mM EDTA抑制Gs-GTP的水解。3）Gs-GDP的形成速率和初始GTP酶速率作为GTP或MgCl2浓度（高于0.1 mM Mg2+）的函数平行变化。Gs-GDP积累速率与GTP酶速率之比在0.3-0.4恒定。4）可检测的Gs-GDP的解离速率是双相的。初始阶段占可检测的总Gs-GDP的60-80%，其特征是t 1/2约为1分钟。5）Lubrol 12A9有效抑制GTP酶反应和Gs-GDP的解离，并且产物释放的抑制可能解释稳态水解的抑制。6）与它们刺激GTPγS解离的能力相反，Gs的β和γ亚基显著抑制GDP从Gs的解离。7）GDP、GTPγS和鸟苷-5'-基亚氨基二磷酸（Gpp（NH）p）竞争性抑制Gs-GDP的积累。GTPγS和Gpp（NH）p非竞争性抑制GTP酶反应，GDP表现出混合抑制，而Pi不抑制。这些数据可以根据总体GTP酶反应的两种特定机制途径的共存来解释。
