Sauer F D, Mahadevan S, Erfle J D
Biochem J. 1984 Jul 1;221(1):61-9. doi: 10.1042/bj2210061.
Methanobacterium thermoautotrophicum when grown on ordinary culture medium has a tough cell wall which is lysozyme-resistant and difficult to disrupt by physical means. The cell wall, however, can be weakened by the addition of D-sorbitol to the growth medium and the organisms form protoplasts after lysozyme addition. This technique allowed the isolation of two types of intracellular small vesicles: (a) isolated by disruption of the total cell population (lysozyme-sensitive and lysozyme-resistant cells) by ultrafrequency sound and (b) isolated by osmotic lysis of protoplasts. For the first time, a small vesicle fraction isolated as in (a) was capable of synthesizing methane from CO2 and H2 without cytoplasm. There was, however, an absolute requirement for a small, heat-stable, oxygen-sensitive cofactor which was isolated from the cytoplasm. Methane synthesis with this vesicle fraction was inhibited by the detergent deoxycholate, and by the protonophores 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. Mg2+-ATPase appeared to be located on the outer or cytoplasmic surface of the small vesicle fraction isolated as in (b). The results were consistent with a previously made suggestion [Sauer, Erfle & Mahadevan (1981) J. Biol. Chem. 256, 9843-9848] that the interior of the small intracellular vesicles becomes acid during methane synthesis.
嗜热自养甲烷杆菌在普通培养基上生长时，具有一层坚韧的细胞壁，该细胞壁对溶菌酶具有抗性，且难以通过物理方法破坏。然而，通过向生长培养基中添加D -山梨醇可使细胞壁变弱，添加溶菌酶后这些微生物会形成原生质体。这项技术使得两种类型的细胞内小泡得以分离：(a) 通过超声破坏整个细胞群体（对溶菌酶敏感和对溶菌酶抗性的细胞）来分离；(b) 通过原生质体的渗透裂解来分离。首次发现，如(a)中那样分离得到的小泡部分能够在没有细胞质的情况下利用二氧化碳和氢气合成甲烷。然而，绝对需要一种从细胞质中分离出来的、小的、热稳定的、对氧敏感的辅因子。用这种小泡部分进行甲烷合成会受到去污剂脱氧胆酸盐、质子载体2,4 -二硝基苯酚和羰基氰化物间氯苯腙的抑制。Mg2 + -ATP酶似乎位于如(b)中那样分离得到的小泡部分的外表面或细胞质表面。这些结果与之前提出的观点[Sauer, Erfle & Mahadevan (1981) J. Biol. Chem. 256, 9843 - 9848]一致，即在甲烷合成过程中细胞内小泡的内部会变酸。
