BIOLOGICAL METHYLATION OF CYTOCHROME C IN FUNGI & PLANTS

真菌中细胞色素 C 的生物甲基化

• 批准号: 3224629
• 负责人: WOON K PAIK
• 金额: $ 18.52万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-12-01 至 1993-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3224629
• 关键词: Euglena; NADPH cytochrome c2 reductase; Neurospora; S adenosylmethionine; affinity chromatography; affinity labeling; chemical binding; cytochrome c; cytochrome oxidase; electrofocusing; enzyme mechanism; enzyme structure; enzyme substrate; genetic transcription; genetic translation; hemoprotein structure; histones; isomer; isozymes; methylation; methyltransferase; mitochondria; mutant; peroxidases; plasmids; protein biosynthesis; protein sequence; yeasts

The general objective of this program is to understand protein methylation from three perspectives: (i) The role of methylation in the dynamics of protein biosynthesis and intracellular processing and trafficking, (ii) the physico-chemical and functional modulation of protein by enzyme-catalyzed methylation, and (iii) a study of the protein methyltransferases themselves, looking specifically at substrate-binding and/or active sites. In order to achieve the above, the following specific research objectives must be met. (1) Elucidation of possible roles for methylation in the dynamics of cytochrome c biosynthesis (elongation, termination, proteolytic resistance of the nascent protein), utilizing in vitro translation systems containing exogenously added mRNA which is transcribed in vitro from plasmid containing yeast iso-l-cytochrome c gene and purified protein-lysine N-methyltransferase. (2) Utilizing in vitro systems containing isolated mitochondria, examination of possible roles for methylation in the posttranslational uptake of cytochrome c apoprotein into the mitochondria and the subsequent attachment of the heme prosthetic group. (3) Utilizing physical methods, characterization of the precise physico-chemical effects exerted by methylation on cytochrome c, and characterization of the effects of methylation on the molecular interaction of cytochrome c with cytochrome c oxidase, reductase, and peroxidase. (4) Identification and subsequent purification of two enzymes implicated to exist from observations during the past grant period. (a) An enzyme which dealkylates methylated cytochrome c in vivo. (b) An enzyme which methylates the Res-86 lysine of cytochrome c. (5) Identification and characterization of the protein structure contributing to the active sites of highly purifiedprotein methyltransferase: Cytochrome c-specific protein-lysine N- methyltransferase from Neurospora crassa and protein-carboxyl O- methyltransferase from calf brain by photoaffinity labeling with analogues of S-adenosyl-L-methionine, and subsequent analysis of labeled peptides generated by enzymatic degradation of the modified methyltransferases.

该计划的总体目标是了解蛋白质 从三个角度分析甲基化：（一）甲基化的作用 蛋白质生物合成和细胞内动力学 加工和贩运，(ii) 物理化学和 通过酶催化甲基化对蛋白质进行功能调节， (iii) 对蛋白质甲基转移酶本身的研究， 特别关注底物结合和/或活性位点。 在 为了实现上述目的，进行如下具体研究 目标必须得到实现。 (1) 阐明甲基化在动力学中可能的作用 细胞色素 C 生物合成（延伸、终止、蛋白水解 新生蛋白的抗性），利用体外翻译 含有外源添加的 mRNA 的系统，该 mRNA 在 从含有酵母 iso-l-细胞色素 c 基因的质粒体外分离， 纯化的蛋白质-赖氨酸N-甲基转移酶。 (2) 利用含有分离线粒体的体外系统， 检查甲基化的可能作用 细胞色素 c 脱辅基蛋白的翻译后摄取 线粒体和随后的血红素假体附着 团体。 (3)利用物理方法，精确表征 甲基化对细胞色素c产生的物理化学效应， 以及甲基化对分子的影响的表征 细胞色素c与细胞色素c氧化酶、还原酶的相互作用， 和过氧化物酶。 (4) 两种酶的鉴定及后续纯化 根据过去资助期间的观察表明存在。 (a) 使甲基化细胞色素 c 脱烷基化的酶 体内。 (b) 甲基化细胞色素 Res-86 赖氨酸的酶 c. (5) 蛋白质结构的鉴定和表征 有助于高纯度蛋白质的活性位点 甲基转移酶：细胞色素c特异性蛋白-赖氨酸N- 来自粗糙脉孢菌的甲基转移酶和蛋白质-羧基O- 通过光亲和标记从小牛脑中提取甲基转移酶 S-腺苷-L-甲硫氨酸的类似物，以及随后的分析 修饰后的酶降解产生的标记肽 甲基转移酶。