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Electron Carrier System and Energy Metabolism in Anaerobic Microorganisms

厌氧微生物中的电子载体系统和能量代谢

基本信息

批准号：
01580187
负责人：
YAGI Tatsuhiko
金额：
$ 1.28万
依托单位：
Shizuoka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

Effort was focused on the elucidation of the high-molecularーweight cytochrome c (hmc) among various electron carrier proteins isolated from Desulfovibrio vulgaris Miyazaki.1. The hmc was highly purified in good yield, by eliminating the steps where the hmc was exposed to proteolytic digestion. It had been realized that the hmc was extremely susceptible to the action of proteinases present in the bacterial extract.2. Quantitative estimation of amino acids indicated that the hmc contained 12 hemes in a single peptide of Mr : 67000.3. The spectral properties of the hmc was similar to those of cytochrome C_3. The purity index (the alpha-peak height of the ferro-form divided by the absorbance at 280 nm) was 2.6. There was no peak at 695 nm which is a marker for the His-Met axial ligands for hemes. The reduced-minus-oxidized difference spectrum of the fully reduced hmc had a peak at 420 nm, whereas the difference spectra of partially reduced forms had double peaks at 424 and 434 nm. This indicates conformational transformation or the appearance of the high-spin form during the reduction of the hmc.4. One of 12 hemes of hmc had a relatively positive standard redox potential, and is reduced with ascorbate. The standard redox potentials of the other hemes were estimated by redox equilibrium with FMN and calculated by means of the Nernst equation. The average E^ﾟ' value was -187 mV with an appearent n valve of 0.48 (i. e., less than unity). The redox potentials of the individual hemes were calculated from these values.5. Partial sequences of the hmc were determined, which were homologous to those of hmc from the Hildenborough strain of D. vulgaris.

本研究的主要目的是阐明从宫崎脱硫弧菌中分离的各种电子载体蛋白中的高分子量细胞色素c（hmc）。通过消除将hmc暴露于蛋白水解消化的步骤，以良好的产率高度纯化hmc。已经认识到，hmc对细菌提取物中存在的蛋白酶的作用极其敏感。氨基酸的定量估计表明，hmc含有12个血红素在一个单一的肽的Mr：67000.3。其光谱性质与细胞色素C_3相似。纯度指数（铁形式的α峰高除以280 nm处的吸光度）为2.6。在695 nm处没有峰，这是血红素的His-Met轴向配体的标记。完全还原的hmc的还原-减去-氧化差光谱在420 nm处有一个峰，而部分还原形式的差光谱在424和434 nm处有双峰。这表明在hmc的还原过程中发生了构象转变或出现了高自旋形式。4. 12种血红素中有一种血红素的标准氧化还原电位相对正，可被抗坏血酸还原。其他血红素的标准氧化还原电位由FMN氧化还原平衡估算，并由能斯特方程计算。平均E_（max）值为-187mV，表观n值为0.48（i.例如，小于1）。从这些值计算单个血红素的氧化还原电位。测定了hmc的部分序列，其序列与D.普通的