Gibbs J B, Schaber M D, Marshall M S, Scolnick E M, Sigal I S
J Biol Chem. 1987 Aug 5;262(22):10426-9.
We have analyzed the guanine nucleotides bound to mammalian ras and yeast RAS proteins overexpressed in [32P]orthophosphate-labeled cultures of exponentially growing Saccharomyces cerevisiae cells. Whereas S. cerevisiae RAS1 and RAS2 proteins were immunoprecipitated bound entirely to GDP, mammalian Harvey ras was isolated with GTP and GDP bound in near-equimolar proportions. In a strain overexpressing a RAS2 variant where the RAS unique C-terminal domain was deleted, both GTP and GDP were detected in a ratio of 3:97. Increased amounts of GTP (16-75% of total guanine nucleotide) were observed bound to all ras proteins containing mutations that inhibit GTP hydrolytic activity. Increasing proportions of GTP bound to the various ras proteins correlated with increasing biological potency to bypass cdc25 lethality in yeast.
我们分析了在指数生长的酿酒酵母细胞的[32P]正磷酸盐标记培养物中过表达的哺乳动物ras和酵母RAS蛋白所结合的鸟嘌呤核苷酸。酿酒酵母RAS1和RAS2蛋白经免疫沉淀后完全结合GDP，而哺乳动物哈维ras分离时结合的GTP和GDP比例接近等摩尔。在过表达RAS2变体（其RAS独特的C末端结构域被删除）的菌株中，检测到GTP和GDP的比例为3:97。在所有含有抑制GTP水解活性突变的ras蛋白中，观察到结合的GTP量增加（占总鸟嘌呤核苷酸的16 - 75%）。与各种ras蛋白结合的GTP比例增加与绕过酵母中cdc25致死性的生物活性增加相关。
