PROPERTIES OF HUMAN RAS ONCOGENE ENCODED TRANSFORMING PROTEIN

项目1：扩大以往关于ras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 很难从真核细胞中获得。 为了规避这个 问题，开发了柱色谱程序来纯化正常的 以及从细菌表达系统转化p21蛋白。 从1.5 L培养液中，可获得约2 mg明显均一的蛋白质。 纯化的蛋白质对鸟嘌呤具有生物化学活性 核苷酸结合功能、自磷酸化（如适用）和 GTTT活动。 通过研究GTP结合功能获得的知识 p21蛋白的纯化。 p21 在位置59处具有苏氨酸的蛋白质显示显著的GTP结合， 与在位置59处具有丙氨酸而没有任何可检测的 在非还原条件下的活性。 然而，在还原条件下， 具有丙氨酸59的p21蛋白也显示增强的核苷酸结合， 尽管只有它们的苏氨酸对应物的四分之一。 这些结果 表明残基59影响核苷酸与蛋白质结合。 项目2：使用合成的抗肽抗体， 在H-ras p21蛋白的161-176位残基上， p21分子中的一个重要区域已经被确定，它影响了 鸟嘌呤核苷酸与p21蛋白的结合。 纯化p21的结果 蛋白质和羧基末端抗体强烈表明，氨基和 p21分子中的羧基末端区域彼此邻近， p21蛋白的天然构象。 项目3：Hs 242的特性研究， 癌源性细胞系，已经扩展。 这种细胞株，虽然 表现正常表型，发现携带激活的H-ras基因 在位置61处突变。 进一步的研究表明，H-ras特异性RNA是 在Hs 242细胞系中表达。 然而，蛋白质的分析， 免疫沉淀法或Western印迹法未能检测到任何突变的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.