STUDIES ON THE IODINATION OF A RAS PROTEIN AND THE DETECTION OF RAS POLYMERS
STUDIES ON THE IODINATION OF A RAS PROTEIN AND THE DETECTION OF RAS POLYMERS
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DOI：
10.1007/bf00926042
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发表时间：
1994-08-17
影响因子：
4.3
通讯作者：
BARRITT, GJ
中科院分区：
文献类型：
--
作者：
CHATAWAY, TK;BARRITT, GJ
比较了重组v-H-ras蛋白的几种碘化方法。Iodobead方法得到最大的放射性掺入，ras蛋白的修饰最小。用[I-125] Nal和Iodobead处理ras蛋白后，放射性最初掺入22 kDa物质中，pi为5.2，然后主要掺入23 kDa物质中，pi为5.4。[I-125]ras的比活性为6 × 10（6）cpm/pmol总ras蛋白。电离没有改变ras蛋白的生物活性，判断其结合GTP γ S和诱导非洲爪蟾卵母细胞成熟的能力。它的结论是，虽然碘化改变的表观分子量和PI的ras，大概是由一个或多个类的氨基酸的氧化，这并不影响蛋白质的生物学功能。使用Iodobead方法用碘放射性标记的ras蛋白，应该适合于研究涉及ras的蛋白质-蛋白质相互作用。用化学交联剂辛二酸二琥珀酰亚胺酯处理碘化ras，发现存在几种次要的高分子量蛋白质。该结果表明，在纯化ras蛋白的稀溶液中，单体形式与少量聚合形式处于平衡。
Several methods for the iodination of recombinant v-H-ras protein were compared. The Iodobead method gave greatest incorporation of radioactivity with minimal modification of the ras protein. Upon treatment of the ras protein with [I-125] Nal and an Iodobead, radioactivity was initially incorporated into a 22 kDa species with a pi of 5.2, then predominantly into a 23 kDa species with a pi of 5.4. The specific activity of [I-125]ras was 6 x 10(6) cpm/pmol total ras protein. Iondination did not alter the biological activity of the ras protein as judged by its ability to bind GTP gamma S and induce maturation of Xenopus laevis oocytes. It is concluded that while iodination alters the apparent molecular weight and pi of ras, presumably by the oxidation of one or more classes of amino acids, this does not affect the biological function of the protein. The ras protein, radioactively-labelled with iodine using the Iodobead method, should be suitable for studies of protein-protein interactions involving ras. Treatment of iodinated ras with the chemical cross-linking agent disuccinimidyl suberate revealed the presence of several minor high molecular weight protein species. This result shows that, in a dilute solution of purified ras protein, the monomeric form is in equilibrium with small amounts of polymeric forms.