Katada T, Oinuma M, Ui M
J Biol Chem. 1986 Jun 25;261(18):8182-91.
Two proteins serving as substrates for ADP-ribosylation catalyzed by islet-activating protein (IAP), pertussis toxin, and binding guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) with high affinities were purified from the cholate extract of rat brain membranes. The purified proteins had the same heterotrimeric structure (alpha beta gamma) as the IAP substrates previously purified from rabbit liver and bovine brain and differed from each other in alpha only; the molecular weight of alpha was 41,000 (alpha 41 beta gamma) and 39,000 (alpha 39 beta gamma). Both were further resolved into alpha (alpha 41 or alpha 39) and beta gamma which were also purified to homogeneity to compare the activities of alpha-monomers with the original trimers. The maintenance of the rigid trimeric structure by combining alpha 41 or alpha 39 with beta gamma in the absence of Mg2+ was essential for the alpha-subunit to be ADP-ribosylated by IAP. The alpha-subunit was very stable but displayed the only partial GTP gamma S-binding activity under these conditions. Isolated alpha-monomers exhibited high GTPase activities when assayed in the presence of submicromolar Mg2+ but were very unstable at 30 degrees C and not ADP-ribosylated by IAP. The most favorable conditions for the GTP gamma S binding to alpha-subunits were achieved by combining alpha 41 or alpha 39 with beta gamma in the presence of millimolar Mg2+, probably due to the increase in stability and unmasking of the GTP-binding sites. There was no qualitative difference in these properties between alpha 41 beta gamma (alpha 41) and alpha 39 beta gamma (alpha 39). But alpha 39 beta gamma (or alpha 39) was usually more active than alpha 41 beta gamma (or alpha 41), at least partly due to its higher affinity for Mg2+ and lower affinity for beta gamma. Relation of these differences in activity between alpha 41 beta gamma and alpha 39 beta gamma to their physiological roles in signal transduction is discussed.
从大鼠脑膜的胆酸盐提取物中纯化出两种蛋白质，它们是胰岛激活蛋白（IAP）催化的ADP核糖基化反应的底物，IAP即百日咳毒素，这两种蛋白质还能与鸟苷5'-（3-O-硫代）三磷酸（GTPγS）高亲和力结合。纯化后的蛋白质与先前从兔肝和牛脑中纯化出的IAP底物具有相同的异源三聚体结构（αβγ），彼此仅在α亚基上存在差异；α亚基的分子量分别为41,000（α41βγ）和39,000（α39βγ）。两者进一步解离为α（α41或α39）和βγ，βγ也被纯化至同质，以比较α单体与原始三聚体的活性。在没有Mg2+的情况下，通过将α41或α39与βγ结合来维持刚性三聚体结构，对于α亚基被IAP进行ADP核糖基化至关重要。α亚基非常稳定，但在这些条件下仅表现出部分GTPγS结合活性。在亚微摩尔浓度的Mg2+存在下进行测定时，分离出的α单体表现出高GTP酶活性，但在30℃时非常不稳定，且不能被IAP进行ADP核糖基化。在毫摩尔浓度的Mg2+存在下，通过将α41或α39与βγ结合，可实现GTPγS与α亚基结合的最有利条件，这可能是由于稳定性增加以及GTP结合位点的暴露。α41βγ（α41）和α39βγ（α39）在这些特性上没有质的差异。但α39βγ（或α39）通常比α41βγ（或α41）更具活性，至少部分原因是它对Mg2+的亲和力更高，对βγ的亲和力更低。文中讨论了α41βγ和α39βγ之间这些活性差异与其在信号转导中的生理作用的关系。
