Inoue Y, Fishman P H, Rebois R V
Membrane Biochemistry Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1990 Jun 25;265(18):10645-51.
Fluoroaluminate had no effect on cAMP levels in cells but inhibited agonist-stimulated cAMP accumulation. In membranes, fluoroaluminate stimulated adenylate cyclase activity between 1 and 10 mM but not at higher concentrations, and it inhibited agonist-stimulated adenylate cyclase activity at concentrations greater than 1 mM. Fluoroaluminate is known to activate Gs and Gi, the guanine nucleotide-binding (G) proteins that stimulate and inhibit adenylate cyclase. G proteins are heterotrimeric, with unique alpha and common beta gamma subunits, and activation involves dissociation of alpha from beta gamma. Pertussis toxin catalyzes ADP-ribosylation of alpha i of heterotrimeric Gi but not free alpha i. Fluoroaluminate prevented pertussis toxin-catalyzed ADP-ribosylation of Gi in cells and membranes, suggesting that Gi is activated by fluoroaluminate in both. Cholera toxin catalyzes ADP-ribosylation of the alpha s subunit of Gs. In cells, agonist often increased cholera toxin-catalyzed ADP-ribosylation of Gs, but fluoroaluminate decreased ADP-ribosylation even in the presence of agonist, suggesting that Gs cannot be activated in the presence of fluoroaluminate. In membranes, both agonist and fluoroaluminate increased cholera toxin-catalyzed ADP-ribosylation, suggesting that Gs is activated by these agents. We conclude that fluoroaluminate activates Gi but not Gs in cells and activates both G proteins in membranes. The value of bacterial toxins in assessing the state of G protein in cells and membranes is demonstrated.
氟铝酸盐对细胞内的环磷酸腺苷（cAMP）水平没有影响，但能抑制激动剂刺激的cAMP积累。在细胞膜中，氟铝酸盐在1至10 mM浓度范围内刺激腺苷酸环化酶活性，但在更高浓度时则无此作用，且在浓度大于1 mM时抑制激动剂刺激的腺苷酸环化酶活性。已知氟铝酸盐可激活Gs和Gi，这两种鸟嘌呤核苷酸结合（G）蛋白分别刺激和抑制腺苷酸环化酶。G蛋白是异源三聚体，具有独特的α亚基和共同的βγ亚基，其激活涉及α亚基与βγ亚基的解离。百日咳毒素催化异源三聚体Gi的αi亚基进行ADP核糖基化，但不催化游离的αi亚基。氟铝酸盐可阻止百日咳毒素催化细胞和细胞膜中Gi的ADP核糖基化，这表明在细胞和细胞膜中Gi均可被氟铝酸盐激活。霍乱毒素催化Gs的αs亚基进行ADP核糖基化。在细胞中，激动剂通常会增加霍乱毒素催化的Gs的ADP核糖基化，但即使存在激动剂，氟铝酸盐也会降低ADP核糖基化，这表明在氟铝酸盐存在的情况下Gs无法被激活。在细胞膜中，激动剂和氟铝酸盐均可增加霍乱毒素催化的ADP核糖基化，这表明这些试剂可激活Gs。我们得出结论，氟铝酸盐在细胞中激活Gi但不激活Gs，在细胞膜中则可激活两种G蛋白。这证明了细菌毒素在评估细胞和细胞膜中G蛋白状态方面的价值。
