Siess W, Wilhelm B, Aepfelbacher M, Gennity J M
Institut für Prophylaxe and Epidemiologie der Kreislaufkrankheiten bei der Universität München, Federal Republic of Germany.
Eur J Biochem. 1991 Nov 15;202(1):145-50. doi: 10.1111/j.1432-1033.1991.tb16355.x.
Inhibitory guanine-nucleotide-binding proteins (Gi proteins) are substrates for pertussis toxin and the decreased pertussis-toxin-dependent ADP ribosylation of Gi proteins upon prior specific hormonal stimulation of cells is thought to reflect the receptor-mediated activation of Gi proteins, leading to their subsequent dissociation into alpha i and beta/gamma subunits. In the present study, the effect of various platelet stimuli on the subsequent pertussis-toxin-dependent ADP ribosylation of the alpha subunit of Gi (Gi alpha) in saponized platelets and platelet membranes were studied. Stimulation of intact platelets with the Ca(2+)-ionophore A23187 or thrombin, but not phorbol 12,13-dibutyrate, decreased the subsequent pertussis-toxin-dependent ADP ribosylation of Gi alpha in saponin-permeabilized platelets in a time-dependent and dose-dependent manner. Thrombin was more effective than A23187. Parallel measurements of Ca2+ mobilization and pertussis-toxin-dependent ADP ribosylation of Gi alpha in platelets showed that Ca2+ mobilization could only partly account for the decrease in pertussis-toxin-dependent ADP ribosylation in platelets stimulated by thrombin. When the ADP-ribosylation reaction was carried out in platelet membranes, a decrease in ADP ribosylation was still observed after stimulation of platelets with thrombin, but not with A23187. In addition to Gi alpha, two other proteins were found to be ADP ribosylated by pertussis toxin; their ADP ribosylation was also decreased after A23187 and thrombin stimulation of platelets. The results indicate that Ca2+ mobilization can decrease the pertussis-toxin-dependent ADP ribosylation of Gi alpha in saponized platelets; the decrease of pertussis-toxin-dependent ADP ribosylation of Gi alpha after thrombin stimulation of platelets can only, in part, be explained by Ca2+ mobilization and involves additional mechanisms; the decrease in pertussis-toxin-dependent ADP ribosylation after A23187 and thrombin stimulation is not confined to G1 alpha and involves other proteins. We conclude that the decrease in pertussis-toxin-dependent ADP ribosylation of Gi in thrombin-stimulated platelets might not be solely caused by a specific structural change, such as dissociation of Gi. It is likely that A23187 and thrombin stimulation of platelets generates substances which interfere with the ADP-ribosylating activity of pertussis toxin.
抑制性鸟嘌呤核苷酸结合蛋白（Gi蛋白）是百日咳毒素的底物，细胞经特定激素预先刺激后，Gi蛋白上依赖百日咳毒素的ADP核糖基化作用减弱，这被认为反映了受体介导的Gi蛋白激活，导致其随后解离为αi和β/γ亚基。在本研究中，我们研究了各种血小板刺激对皂化血小板和血小板膜中Gi（Giα）α亚基随后依赖百日咳毒素的ADP核糖基化的影响。用Ca(2+)离子载体A23187或凝血酶刺激完整血小板，但不是佛波醇12,13 - 二丁酸酯，会以时间和剂量依赖的方式降低皂素通透化血小板中随后依赖百日咳毒素的Giα的ADP核糖基化。凝血酶比A23187更有效。对血小板中Ca2+动员和Giα依赖百日咳毒素的ADP核糖基化的平行测量表明，Ca2+动员只能部分解释凝血酶刺激血小板后依赖百日咳毒素的ADP核糖基化的降低。当在血小板膜中进行ADP核糖基化反应时，用凝血酶刺激血小板后仍观察到ADP核糖基化降低，但用A23187刺激则没有。除了Giα，还发现另外两种蛋白质被百日咳毒素ADP核糖基化；在A23187和凝血酶刺激血小板后，它们的ADP核糖基化也降低了。结果表明，Ca2+动员可降低皂化血小板中依赖百日咳毒素的Giα的ADP核糖基化；凝血酶刺激血小板后依赖百日咳毒素的Giα的ADP核糖基化降低，只能部分由Ca2+动员解释，还涉及其他机制；A23187和凝血酶刺激后依赖百日咳毒素的ADP核糖基化降低不仅限于G1α，还涉及其他蛋白质。我们得出结论，凝血酶刺激血小板后依赖百日咳毒素的Gi的ADP核糖基化降低可能并非仅由特定的结构变化（如Gi的解离）引起。A23187和凝血酶刺激血小板可能产生了干扰百日咳毒素ADP核糖基化活性的物质。
