PROPERTIES OF HUMAN RAS ONCOGENE ENCODED TRANSFORMING PROTEIN

项目1：扩大以前对资源需求评估系统结构和功能的研究 癌基因编码的p21蛋白需要大量的纯化蛋白 从真核细胞中获得极其困难。为了绕过这一点 问题是，开发了柱层析程序来纯化正常 以及转化细菌表达系统中的p21蛋白。从1.5开始 L培养，可获得约2 mg的明显均一的蛋白质。 纯化的蛋白对鸟嘌呤具有生化活性。 核苷酸结合功能、自动磷酸化(如适用)和 GTPase活性。对GTP结合功能研究的认识 已经扩展到纯化的p21蛋白。第21页 在59位含有苏氨酸的蛋白质显示出显著的GTP结合 与丙氨酸在59位没有任何可检测到的变异体相比 在非还原条件下的活动。然而，在还原条件下， P21蛋白与丙氨酸59还表现出增强的核苷酸结合， 虽然只有苏氨酸的四分之一。这些结果 提示残基59影响核苷酸与蛋白质的结合。 项目2：使用合成的抗肽抗体 H-ras p21蛋白结构和功能上的161-176残基 已经确定了p21分子中的重要区域，该区域影响 鸟嘌呤核苷酸与p21蛋白的结合。纯化的p21的结果 蛋白质和羧基末端抗体强烈提示氨基和 P21分子中的羧基末端区域在 P21蛋白的天然构象。 项目3：肺组织Hs242的特性研究 肿瘤来源的细胞系，已经被延伸。这个细胞系，尽管 表型正常，发现含有激活的H-ras基因 在61位发生突变。进一步研究表明，H-ras特异性RNA是 在Hs242细胞系中表达。然而，蛋白质的分析要么是通过 免疫沉淀或Western blotting均未检测到任何突变的p21。

Project 1: Extension of previous studies on structure and function of ras oncogene-encoded p21 proteins required quantities of purified protein extremely difficult to obtain from eukaryotic cells. To circumvent this problem, column chromatographic procedures were developed to purify normal and transforming p21 proteins from bacterial expression systems. From 1.5 L culture, about 2 mg of apparently homogeneous protein could be obtained. The purified proteins were biochemically active for guanine nucleotide-binding function, autophosphorylation (where applicable) and GTPase activities. Knowledge gained by the studies on GTP-binding function of eukaryotic protein has been extended to purified p21 proteins. p21 proteins with threonine at position 59 show significant GTP binding as compared to the variants with alanine at position 59 without any detectable activity under nonreducing condtions. However, under reducing conditions, p21 proteins with alanine 59 also show enhanced nucleotide binding, although only one-fourth of their threonine counterpart. These results suggest that residue 59 affects the binding of nucleotide to protein. Project 2: Using synthetic antipeptide antibodies corresponding to residues 161-176 in H-ras p21 protein, a structurally and functionally important region in the p21 molecule has been identified which affects the binding of guanine nucleotide to p21 protein. Results with purified p21 proteins and carboxy terminal antibody strongly suggest that the amino and carboxy terminal regions in the p21 molecule are proximal to each other in the native conformation of p21 protein. Project 3: Studies on the characterization of Hs242, a lung carcinoma-derived cell line, have been extended. This cell line, although exhibiting normal phenotype, was found to harbor activated H-ras gene mutated at position 61. Further studies show that H-ras-specific RNA is expressed in the Hs242 cell line. However, analysis of proteins either by immunoprecipitation or by Western blots failed to detect any mutated p21.