Department of Plant Physiology, University of Umeå, S -901 87 Umeå, Sweden.
Plant Physiol. 1990 Mar;92(3):809-15. doi: 10.1104/pp.92.3.809.
Symbiotic and free-living Frankia were investigated for correlation between hydrogenase activities (in vivo/in vitro assays) and for occurrence and localization of hydrogenase protein by Western blots and immuno-gold localization, respectively. Freshly prepared nodule homogenates from the symbiosis between Alnus incana and a local source of Frankia did not show any detectable in vivo or in vitro hydrogenase uptake activity, as also has been shown earlier. However, a free-living Frankia strain originally isolated from these nodules clearly showed both in vivo and in vitro hydrogenase activity, with the latter being approximately four times higher. Frankia strain Cpl1 showed hydrogen uptake activity both in symbiosis with Alnus incana and in a free-living state. Western blots on the different combinations of host plants and Frankia strains used in the present study revealed that all the Frankia sources contained a hydrogenase protein, even the local source where no in vivo or in vitro activity could be measured. The 72 kilodalton protein found in the symbiotic Frankia as well as in the free-living Frankia strains were immunologically related to the large subunit of a dimeric hydrogenase purified from Alcaligenes latus. Recognitions to polypeptides with molecular masses of about 41 and 19.5 kilodaltons were also observed in Frankia strain UGL011101 and in the local source of Frankia, respectively. Immunogold localization of the protein demonstrated that in both the symbiotic state and the free-living nitrogen-fixing Frankia, the protein is located in vesicles and in hyphae. The inability to measure any uptake hydrogenase activity is therefore not due to the absence of hydrogenase enzyme. However, the possibility of an inactive hydrogenase enzyme cannot be ruled out.
共生和自由生活的弗兰克氏菌被研究了其氢化酶活性（体内/体外测定）之间的相关性，以及氢化酶蛋白的出现和定位分别通过 Western blot 和免疫金定位。来自 Alnus incana 和本地弗兰克氏菌来源的共生体中新鲜制备的根瘤匀浆没有显示任何可检测的体内或体外氢化酶摄取活性，这与早期的研究结果一致。然而，最初从这些根瘤中分离出来的自由生活的弗兰克氏菌菌株清楚地显示了体内和体外氢化酶活性，后者大约高出四倍。Frankia 菌株 Cpl1 在与 Alnus incana 的共生和自由生活状态下都表现出氢摄取活性。本研究中使用的不同宿主植物和弗兰克氏菌菌株的组合的 Western blot 显示，所有弗兰克氏菌来源都含有氢化酶蛋白，即使是在体内或体外都无法测量到活性的本地来源也是如此。在共生弗兰克氏菌以及自由生活的弗兰克氏菌菌株中发现的 72 千道尔顿蛋白与从 Alcaligenes latus 中纯化的二聚氢化酶的大亚基在免疫学上相关。在 Frankia 菌株 UGL011101 和本地弗兰克氏菌来源中，还观察到约 41 和 19.5 千道尔顿的多肽的识别。该蛋白的免疫金定位表明，在共生状态和自由生活的固氮弗兰克氏菌中，该蛋白都位于囊泡和菌丝中。因此，无法测量任何摄取氢化酶活性并不是由于缺乏氢化酶酶。然而，不能排除无活性氢化酶酶的可能性。
