Basi N S, Okuya S, Rebois R V
Membrane Biochemistry Section, National Institute of Neurological Disease and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
Cell Signal. 1996 Mar;8(3):209-15. doi: 10.1016/0898-6568(95)02056-x.
The stimulatory G protein (Gs) mediates activation of adenylyl cyclase. Gs is a heterotrimeric protein (alpha beta gamma) that is activated when guanosine triphosphate (GTP) or a non-hydrolyzable GTP analogue displaces tightly bound guanosine diphosphate (GDP) from the guanine nucleotide-binding site of the alpha-subunit (Gs alpha). Divalent cations such as magnesium are also required for Gs activation. Subunit dissociation can accompany Gs activation and is thought to be critical for this process. We investigated the effects of MgCl2 and various purine nucleotides on Gs-subunit dissociation and activation. Subunit dissociation was assayed by measuring the amount of G protein beta-subunit that was co-precipitated by Gs alpha-specific antiserum. Gs activation was determined by its ability to reconstitute adenylyl cyclase activity in S49 cyc-membranes that lack Gs alpha. High concentrations of MgCl2 caused bound GDP to dissociate from Gs and inactivated the protein unless high concentrations of GDP or GTP were present in solution. MgCl2 caused a concentration-dependent dissociation of Gs subunits. GTP gamma S (a non-hydrolyzable GTP analogue) shifted the MgCl2 concentration-response curve for subunit dissociation to lower concentrations of MgCl2, suggesting that GTP gamma S promoted subunit dissociation. On the other hand, GDP and GTP were equally effective in shifting the curve to higher concentration of MgCl2. These results suggest that GTP, the compound that activates Gs in vivo, was no more effective at promoting Gs subunit dissociation than was GDP.
刺激性G蛋白（Gs）介导腺苷酸环化酶的激活。Gs是一种异源三聚体蛋白（αβγ），当鸟苷三磷酸（GTP）或不可水解的GTP类似物从α亚基（Gsα）的鸟嘌呤核苷酸结合位点取代紧密结合的鸟苷二磷酸（GDP）时，它被激活。二价阳离子如镁对于Gs的激活也是必需的。亚基解离可能伴随Gs的激活，并且被认为对这一过程至关重要。我们研究了MgCl2和各种嘌呤核苷酸对Gs亚基解离和激活的影响。通过测量被Gsα特异性抗血清共沉淀的G蛋白β亚基的量来检测亚基解离。通过其在缺乏Gsα的S49 cyc - 膜中重建腺苷酸环化酶活性的能力来确定Gs的激活。高浓度的MgCl2导致结合的GDP从Gs上解离并使该蛋白失活，除非溶液中存在高浓度的GDP或GTP。MgCl2导致Gs亚基的浓度依赖性解离。GTPγS（一种不可水解的GTP类似物）将亚基解离的MgCl2浓度 - 反应曲线移至较低的MgCl2浓度，表明GTPγS促进亚基解离。另一方面，GDP和GTP在将曲线移至更高的MgCl2浓度方面同样有效。这些结果表明，在体内激活Gs的化合物GTP在促进Gs亚基解离方面并不比GDP更有效。
