Böhm M, Gräbel C, Kirchmayr R, Lensche H, Erdmann E, Gierschik P
Klinik III für Innere Medizin, Universität zu Köln, Germany.
Biochem Pharmacol. 1993 Dec 14;46(12):2145-54. doi: 10.1016/0006-2952(93)90603-t.
Immunochemical detection of pertussis toxin-sensitive guanine-nucleotide binding proteins has been suggested to represent the most direct approach to quantitate the protein than pertussis toxin-catalysed [32P]ADP-ribosylation. The latter technique is potentially hampered by pre-existing covalent modification of the C-terminus. However, limited data exist as to whether and in what way modifications of the C-terminus affect immunoreactivity of Gi alpha (alpha-subunit of the inhibitory G-protein of adenylyl cyclase). Membranes from human myocardium, thrombocytes, adipose tissue and lung were treated with pertussis toxin or N-ethylmaleimide. Both, conditions prevented high affinity agonist binding to m-cholinoceptors and inhibited [32P]ADP-ribosylation by pertussis toxin consistent with the notion that the modifications took place at the C-terminus. Pertussis toxin treatment increased immunoreactivity to different antisera raised against the C-terminal decapeptide of transducin alpha (KENLKDCGLF, DS 1-4, AS). N-Ethylmaleimide reduced immunoreactivity towards all antisera studied. Pertussis toxin reduced the mobility of Gi alpha on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) depending on the presence of the toxin and sensitivity to inhibition of ADP-ribosylation by nicotinamide. In native membranes from none of the tissues studied, immunoreactive material comigrating with pertussis toxin-modified form of Gi alpha was detected. It is concluded that modification of the C-terminus by pertussis toxin or N-ethylmaleimide resulting in the same functional consequence, i.e. prevention of high affinity agonist receptor binding, is capable of producing opposite changes of immunoreactivity. Pertussis toxin treatment reduces the electrophoretic mobility on SDS-PAGE. Separation of the native and pertussis toxin-modified form of Gi alpha on SDS-PAGE demonstrates that endogenously ADP-ribosylated Gi alpha is lacking in membranes from human myocardium, thrombocytes, lung and adipose tissue.
免疫化学检测百日咳毒素敏感的鸟嘌呤核苷酸结合蛋白被认为是定量该蛋白比百日咳毒素催化的[32P]ADP-核糖基化更为直接的方法。后一种技术可能受到C末端预先存在的共价修饰的阻碍。然而，关于C末端的修饰是否以及以何种方式影响Giα（腺苷酸环化酶抑制性G蛋白的α亚基）的免疫反应性，现有数据有限。用人心肌、血小板、脂肪组织和肺的膜用百日咳毒素或N-乙基马来酰亚胺处理。两种情况均阻止高亲和力激动剂与毒蕈碱受体结合，并抑制百日咳毒素的[32P]ADP-核糖基化，这与修饰发生在C末端的观点一致。百日咳毒素处理增加了对针对转导素α的C末端十肽（KENLKDCGLF，DS 1-4，AS）产生的不同抗血清的免疫反应性。N-乙基马来酰亚胺降低了对所有研究抗血清的免疫反应性。百日咳毒素根据毒素的存在和对烟酰胺抑制ADP-核糖基化的敏感性，降低了Giα在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上的迁移率。在所研究的任何组织的天然膜中，均未检测到与百日咳毒素修饰形式的Giα共迁移的免疫反应性物质。结论是，百日咳毒素或N-乙基马来酰亚胺对C末端的修饰导致相同的功能结果，即阻止高亲和力激动剂受体结合，能够产生相反的免疫反应性变化。百日咳毒素处理降低了SDS-PAGE上的电泳迁移率。在SDS-PAGE上分离天然和百日咳毒素修饰形式的Giα表明，人心肌、血小板、肺和脂肪组织的膜中缺乏内源性ADP-核糖基化的Giα。
