Lünsdorf H, Niedrig M, Fiebig K
Gesellschaft für Biotechnologische Forschung mbH, Abteilung Mikrobiologie, Berlin, Federal Republic of Germany.
J Bacteriol. 1991 Feb;173(3):978-84. doi: 10.1128/jb.173.3.978-984.1991.
The cytological localization of the 8-hydroxy-5-deazaflavin (coenzyme F420)-reducing hydrogenase of Methanosarcina barkeri Fusaro was determined by immunoelectron microscopy, using a specific polyclonal rabbit antiserum raised against the homogeneous deazaflavin-dependent enzyme. In Western blot (immunoblot) experiments this antiserum reacted specifically with the native coenzyme F420-reducing hydrogenase, but did not cross-react with the coenzyme F420-nonreducing hydrogenase activity also detectable in crude extracts prepared from methanol-grown Methanosarcina cells. Immunogold labelling of ultrathin sections of anaerobically fixed methanol-grown cells from the exponential growth phase revealed that the coenzyme F420-reducing hydrogenase was predominantly located in the vicinity of the cytoplasmic membrane. From this result we concluded that the deazaflavin-dependent hydrogenase is associated with the cytoplasmic membrane in intact cells of M. barkeri during growth on methanol as the sole methanogenic substrate, and a possible role of this enzyme in the generation of the electrochemical proton gradient is discussed.
利用针对纯化的脱氮黄素依赖性酶制备的特异性兔多克隆抗血清，通过免疫电子显微镜确定了巴氏甲烷八叠球菌Fusaro中8-羟基-5-脱氮黄素（辅酶F420）还原氢化酶的细胞学定位。在蛋白质免疫印迹实验中，该抗血清与天然辅酶F420还原氢化酶发生特异性反应，但与从以甲醇培养的甲烷八叠球菌细胞制备的粗提物中也可检测到的辅酶F420非还原氢化酶活性不发生交叉反应。对指数生长期厌氧固定的以甲醇培养的细胞超薄切片进行免疫金标记显示，辅酶F420还原氢化酶主要位于细胞质膜附近。根据这一结果，我们得出结论，在以甲醇作为唯一产甲烷底物生长的巴氏甲烷八叠球菌完整细胞中，脱氮黄素依赖性氢化酶与细胞质膜相关，并讨论了该酶在产生电化学质子梯度中的可能作用。
