CO DEHYDROGENASE AND ACETOCLASTIC METHANOGENESIS IN METHANOSARCINA BARKERI

本文研究了大豆中一氧化碳脱氢酶(CODH)的活性。 巴氏甲烷链霉醋酸酯培养细胞。在高海拔条件下 离子强度该酶以聚集状态存在，与 其他蛋白质的离散集合(总摩尔。WT。约3,000,000)。 当离子强度达到一定值时，聚集体发生解离 透析后下降。《卫生与公众卫生法》的分类形式保留了全部 活性，显示出约161,000的相对分子质量。一个 开发了高效的提纯工艺，产率高 纯正的慢性前列腺炎。该方法包括以下步骤 在NIH厌氧室严格的厌氧条件下进行 实验室：1.用凝胶过滤和凝胶过滤分离CODH聚集体 随后解离；2.苯基琼脂糖层析；和3. 羟基磷灰石层析。Codh似乎没有那么疏水性。 聚集体中的任何其他蛋白质成分。它紧紧地绑在一起 羟基磷灰石，因此可能有相对大量的暴露 羧酸残留物。纯CODH由两个亚基组成 分子量分别为93,800和20,400。在测试的各种化合物中， 氧和氰化物是强有力的钝化剂。乙二醛失活 仅在酶的周转期间发生，这表明反应性 基团在催化过程中的酶中间体上形成或暴露。

A study has been conducted on carbon monoxide dehydrogenase (CODH) from acetate-grown cells of Methanosarcina barkeri. Under conditions of high ionic strength the enzyme exists in an aggregated state along with a discrete set of other proteins (total mol. wt. approx. 3,000,000). Dissociation of the aggregate occurred when the ionic strength was decreased by dialysis. The disaggregated form of CODH retained full activity and exhibited a molecular weight of approximately 161,000. An efficient purification procedure was developed which produced high yields of pure CODH. The method consisted of the following steps which were carried out under strictly anaerobic conditions in the NIH Anaerobic Laboratory: 1. Isolation of the CODH aggregate by gel-filtration and subsequent dissociation; 2. Chromatography on Phenyl Sepharose; and 3. Hydroxylapatite chromatography. CODH appeared to be less hydrophobic than any of the other protein components of the aggregate. It bound tightly to hydroxylapatite and, thus, may have a relatively large number of exposed carboxylic acid residues. Pure CODH is composed of two subunits of molecular weights 93,800 and 20,400. Among various compounds tested, oxygen and cyanide are potent inactivators. Glyoxaldehyde inactivation occurred only during enzymatic turnover, which suggests that a reactive group is formed, or exposed, on an enzyme intermediate in catalysis.