Yamamoto T, Kaibuchi K, Mizuno T, Hiroyoshi M, Shirataki H, Takai Y
Department of Biochemistry, Kobe University School of Medicine, Japan.
J Biol Chem. 1990 Sep 25;265(27):16626-34.
Novel regulatory proteins for smg p21A and -B, ras p21-like GTP-binding proteins (G proteins) having the same putative effector domain as ras p21s, were purified to near homogeneity from bovine brain cytosol and characterized. These regulatory proteins, designated as GDP dissociation stimulator (GDS) 1 and -2, stimulated the dissociation of both [3H]GDP and [35S] guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) from smg p21s to the same extent. smg p21 GDS1 and -2 also stimulated the binding of [35S]GTP gamma S to the GDP-bound form of smg p21s but not that to the guanine nucleotide-free form. These actions of smg p21 GDS1 and -2 were specific for smg p21s and inactive for other ras p21/ras p21-like G proteins including c-Ha-ras p21, rhoB p20, and smg p25A. Neither smg p21 GDS1 nor -2 stimulated the GTPase activity of smg p21s and by itself showed [35S]GTP gamma S-binding or GTPase activity. smg p21 GDS1 and -2 showed very similar physical and kinetic properties and were indistinguishable by peptide map analysis. The Mr values of smg p21 GDS1 and -2 were estimated to be about 53,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and from the S values, indicating that smg p21 GDS1 and -2 are composed of a single polypeptide without a subunit structure. smg p21 GDS1 and -2 were distinguishable from GTPase activating proteins (GAPs) for the ras and rho proteins, and smg p21B, and GDP dissociation inhibitors for smg p25A and the rho proteins previously identified in bovine brain cytosol. These results indicate that bovine brain contains regulatory proteins for smg p21s that stimulate the dissociation of GDP from and thereby the subsequent binding of GTP to smg p21s in addition to smg p21 GAP. It is likely that the conversion from the GDP-bound inactive form of smg p21s to the GTP-bound active form is regulated by smg p21 GDS and that its reverse reaction is regulated by smg p21 GAP.
从牛脑细胞质中纯化出了与 smg p21A 和 -B 相关的新型调节蛋白，它们是具有与 ras p21 相同假定效应结构域的 ras p21 样 GTP 结合蛋白（G 蛋白），并对其进行了表征。这些调节蛋白被命名为 GDP 解离刺激因子（GDS）1 和 -2，它们能同等程度地刺激 [3H]GDP 和 [35S] 鸟苷 5'-(3-O-硫代)三磷酸（GTPγS）从 smg p21 上解离。smg p21 GDS1 和 -2 还能刺激 [35S]GTPγS 与 GDP 结合形式的 smg p21 结合，但不能刺激其与无鸟嘌呤核苷酸形式的 smg p21 结合。smg p21 GDS1 和 -2 的这些作用对 smg p21 具有特异性，对其他 ras p21/ras p21 样 G 蛋白（包括 c-Ha-ras p21、rhoB p20 和 smg p25A）无活性。smg p21 GDS1 和 -2 均不刺激 smg p21 的 GTP 酶活性，且自身不表现出 [35S]GTPγS 结合或 GTP 酶活性。smg p21 GDS1 和 -2 表现出非常相似的物理和动力学性质，通过肽图分析无法区分。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳并根据 S 值估计，smg p21 GDS1 和 -2 的 Mr 值约为 53,000，这表明 smg p21 GDS1 和 -2 由单一多肽组成，无亚基结构。smg p21 GDS1 和 -2 与 ras 和 rho 蛋白的 GTP 酶激活蛋白（GAP）、smg p21B 以及先前在牛脑细胞质中鉴定出的 smg p25A 和 rho 蛋白的 GDP 解离抑制剂不同。这些结果表明，除了 smg p21 GAP 外，牛脑中还含有能刺激 GDP 从 smg p21 上解离从而促进随后 GTP 与 smg p21 结合的调节蛋白。很可能从 GDP 结合的无活性形式的 smg p21 向 GTP 结合的活性形式的转化受 smg p21 GDS 调节，而其逆向反应受 smg p21 GAP 调节。
