Chataway T K, Barritt G J
Department of Medical Biochemistry, School of Medicine, Flinders University, Adelaide, Australia.
Mol Cell Biochem. 1994 Aug 17;137(1):75-83. doi: 10.1007/BF00926042.
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 [125I] Nal and an Iodobead, radioactivity was initially incorporated into a 22 kDa species with a pl of 5.2, then predominantly into a 23 kDa species with a pl of 5.4. The specific activity of [125I]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.
比较了几种对重组v-H-ras蛋白进行碘化的方法。碘珠法能使放射性最大程度地掺入，同时对ras蛋白的修饰最小。用[125I]碘化钠和碘珠处理ras蛋白后，放射性最初掺入到一个分子量为22 kDa、等电点为5.2的蛋白条带中，随后主要掺入到一个分子量为23 kDa、等电点为5.4的蛋白条带中。[125I]ras的比活度为6×10(6) cpm/pmol总ras蛋白。从其结合GTPγS和诱导非洲爪蟾卵母细胞成熟的能力判断，碘化并未改变ras蛋白的生物学活性。得出的结论是，虽然碘化可能通过氧化一类或多类氨基酸改变了ras的表观分子量和等电点，但这并不影响该蛋白的生物学功能。用碘珠法放射性标记碘的ras蛋白应适用于涉及ras的蛋白质-蛋白质相互作用的研究。用化学交联剂辛二酸二琥珀酰亚胺酯处理碘化的ras后，发现存在几种较小的高分子量蛋白条带。这一结果表明，在纯化的ras蛋白稀溶液中，单体形式与少量聚合物形式处于平衡状态。
