Chang K C, Chuang N N
Division of Biochemistry and Molecular Sciences, Institute of Zoology, Academia Sinica, Nankang, Taipei, Taiwan 11529.
J Exp Zool. 2001 Nov 1;290(6):642-51. doi: 10.1002/jez.1115.
BALB/3T3 cells were transformed by transfection with DNA encoding the mutated ras(Q(61)K) from shrimp Penaeus japonicus (Huang et al., 2000). The GTPase-activating protein (GAP) in the cytosol fraction was significantly expressed and degraded, compared to untransformed cells on the western blot. To understand this in more detail, the interaction of the bacterially expressed shrimp Ras (S-Ras) with GAP was investigated using GAP purified from mouse brains. SDS-polyacrylamide gel electrophoresis revealed the monomers of the purified GAP to have a relative mass of 65,000. Since the purified GAP was bound to the Ras conjugated affinity sepharose column with high affinity and its GTP hydolysis activity upon binding with tubulin was suppressed, the purified enzyme was concluded to be neurofibromin-like. The purified GAP enhanced the intrinsic GTPase activity of the S-Ras, to convert it into the inactive GDP-bound form, in agreement with findings for GTP-bound K(B)-Ras in vitro. To compare the effects between isoprenoids and GAP on the GTP-hydrolysis of Ras, we applied the GTP-locked shrimp mutant S-Ras(Q(61)K) and GTP-locked rat mutant K(B)-ras(Q(61)K). Radioassay studies showed that geranylgeranyl pyrophosphate at microg level catalyzed the GTP hydrolysis of S-Ras(Q(61)K) and K(B)-ras(Q(61)K) competently, but not farnesyl pyrophosphate or the purified GAP. The present study provides the view that the geranylgeranyl pyrophosphate at carboxyl terminal CAAX assists GTP hydrolysis to Ras proteins probably in a manner similar to the substrate assisted catalysis in GTPase mechanism.
用编码来自日本对虾的突变型ras（Q(61)K）的DNA转染BALB/3T3细胞（Huang等人，2000年）。与未转化细胞相比，在蛋白质印迹上，胞质溶胶部分中的GTP酶激活蛋白（GAP）显著表达并降解。为了更详细地了解这一点，使用从小鼠脑中纯化的GAP研究了细菌表达的对虾Ras（S-Ras）与GAP的相互作用。SDS-聚丙烯酰胺凝胶电泳显示纯化的GAP单体的相对分子质量为65,000。由于纯化的GAP以高亲和力结合到与Ras偶联的亲和琼脂糖柱上，并且其与微管蛋白结合时的GTP水解活性受到抑制，因此得出纯化的酶为类神经纤维瘤蛋白的结论。纯化的GAP增强了S-Ras的内在GTP酶活性，将其转化为无活性的结合GDP的形式，这与体外GTP结合的K(B)-Ras的研究结果一致。为了比较类异戊二烯和GAP对Ras的GTP水解的影响，我们应用了GTP锁定的对虾突变体S-Ras（Q(61)K）和GTP锁定的大鼠突变体K(B)-ras（Q(61)K）。放射性测定研究表明，微克水平的香叶基香叶基焦磷酸能够有效地催化S-Ras（Q(61)K）和K(B)-ras（Q(61)K）的GTP水解，但法尼基焦磷酸或纯化的GAP则不能。本研究提出这样的观点，即羧基末端CAAX处的香叶基香叶基焦磷酸可能以类似于GTP酶机制中底物辅助催化的方式协助Ras蛋白的GTP水解。
