Higashijima T, Ferguson K M, Sternweis P C, Smigel M D, Gilman A G
J Biol Chem. 1987 Jan 15;262(2):762-6.
Mg2+ interacts with the alpha subunits of guanine nucleotide-binding regulatory proteins (G proteins) in the presence of guanosine-5'-[gamma-thio]triphosphate (GTP-gamma S) to form a highly fluorescent complex from which nucleotide dissociates very slowly. The apparent Kd for interaction of G alpha X GTP gamma S with Mg2+ is approximately 5 nM, similar to the Km for G protein GTPase activity X G beta gamma increases the rate of dissociation of GTP gamma S from G alpha X GTP gamma S or G alpha X GTP gamma S X Mg2+ at low concentrations of Mg2+. When the concentration of Mg2+ exceeds 1 mM, G beta gamma dissociates from G beta gamma X G alpha X GTP gamma S X Mg2+. Compared with the dramatic effect of Mg2+ on binding of GTP gamma S to G alpha, the metal has relatively little effect on the binding of GDP. However, G beta gamma increases the affinity of G alpha for GDP by more than 100-fold. High concentrations of Mg2+ promote the dissociation of GDP from G beta gamma X G alpha X GDP, apparently without causing subunit dissociation. The steady-state rate of GTP hydrolysis is strictly correlated with the rate of dissociation of GDP from G alpha under all conditions examined. Thus, there are at least two sites for interaction of Mg2+ with G protein-nucleotide complexes. Furthermore, binding of G beta gamma and GTP gamma S to G alpha is negatively cooperative, while the binding interaction between G beta gamma and GDP is strongly positive.
在鸟苷-5'-[γ-硫代]三磷酸（GTP-γS）存在的情况下，Mg2+与鸟嘌呤核苷酸结合调节蛋白（G蛋白）的α亚基相互作用，形成一种高度荧光的复合物，核苷酸从该复合物中解离非常缓慢。Gα与GTP-γS和Mg2+相互作用的表观解离常数（Kd）约为5 nM，类似于G蛋白GTP酶活性的米氏常数（Km）。Gβγ在低浓度Mg2+时会增加GTP-γS从Gα-GTP-γS或Gα-GTP-γS-Mg2+解离的速率。当Mg2+浓度超过1 mM时，Gβγ会从Gβγ-Gα-GTP-γS-Mg2+解离。与Mg2+对GTP-γS与Gα结合的显著影响相比，该金属对GDP结合的影响相对较小。然而，Gβγ会使Gα对GDP的亲和力增加100倍以上。高浓度的Mg2+会促进GDP从Gβγ-Gα-GDP解离，显然不会导致亚基解离。在所研究的所有条件下，GTP水解的稳态速率与GDP从Gα解离的速率严格相关。因此，Mg2+与G蛋白-核苷酸复合物至少有两个相互作用位点。此外，Gβγ和GTP-γS与Gα的结合是负协同的，而Gβγ和GDP之间的结合相互作用是强正协同的。
