Localization of membrane-associated (NiFe) and (NiFeSe) hydrogenases of Desulfovibrio vulgaris using immunoelectron microscopic procedures.
Localization of membrane-associated (NiFe) and (NiFeSe) hydrogenases of Desulfovibrio vulgaris using immunoelectron microscopic procedures.
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使用免疫电子显微镜程序对普通脱硫弧菌的膜相关 (NiFe) 和 (NiFeSe) 氢化酶进行定位。
DOI：
10.1111/j.1432-1033.1990.tb19134.x
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
Menon,NK
中科院分区：
文献类型：
--
作者：
Rohde,M;Fürstenau,U;Mayer,F;Przybyla,AE;PeckJr,HD;LeGall,J;Choi,ES;Menon,NK
利用包埋前和包埋后的免疫电镜方法测定了寻常型脱硫弧菌的膜相关（NiFe）和（NiFeSe）氢化酶的细胞内位置。制备了针对纯化的（NiFeSe）和（NiFeSe）氢化酶的多克隆抗血清。d .一日培养；在这些研究中，所有实验都使用生长在乳酸/硫酸盐培养基上的寻常草。在包埋后标记研究中，细胞用0.2%戊二醛和0.3%甲醛固定，用甲醇脱水，并包埋在低温树脂Lowicryl K4M中。我们使用抗体金或蛋白A金作为电子密度标记的包埋后研究揭示了两种氢化酶仅在细胞周围的位置；然后通过预包埋标记证明了精确的膜定位。球质体与抗（NiFeSe）和（NiFeSe）氢化酶的多克隆抗血清孵育，然后用铁蛋白连接的二抗孵育，然后包埋和切片。观察到的标记模式明确显示，（NiFeSe）氢化酶的抗原反应位点位于细胞质膜附近面向质周间隙，而（NiFeSe）氢化酶则与细胞质膜的细胞质侧相关。
The intracellular location of membrane‐associated (NiFe) and (NiFeSe) hydrogenases ofDesulfovibrio vulgariswas determined using pre‐embedding and post‐embedding immunoelectron microscopic procedures. Polyclonal antisera directed against the purified (NiFe) and (NiFeSe) hydrogenases were raised in rabbits. One‐day‐old cultures ofD. vulgaris, grown on a lactate/sulfate medium, were used for all experiments in these studies. For post‐embedding labeling studies cells were fixed with 0.2% glutaraldehyde and 0.3% formaldehyde, dehydrated with methanol, and embedded in the low‐temperature resin Lowicryl K4M.Our post‐embedding studies using antibody‐gold or protein‐A‐gold as electron‐dense markers revealed the location of the two hydrogenases exclusively at the cell periphery; the precise membrane location was then demonstrated by pre‐embedding labeling. Spheroplasts were incubated with the polyclonal antisera against (NiFe) and (NiFeSe) hydrogenase followed by ferritin‐linked secondary antibodies prior to embedding and sectioning. The observed labeling pattern unequivocally revealed that the antigenic reactive sites of the (NiFe) hydrogenase are located in the near vicinity of the cytoplasmic membrane facing into the periplasmic space, whereas the (NiFeSe) hydrogenase is associated with the cytoplasmic side of the cytoplasmic membrane.