Emnova E E, Romanova A K, Zhilina T N
Mikrobiologiia. 1979 Jan-Feb;48(1):80-9.
Spheroplasts that were osmotically stable in 0.2M Tris-HCl--0.02M EDTA were prepared from the autotrophically grown cells of Pseudomonas thermophila K-2. The spheroplasts possessed 90--95% of the hydrogenase activity of the whole cells. The half-life time of hydrogenase in the spheroplasts at 80 degrees C was 8.5 min. A spectrophotometric technique was developed for determining the membrane-bound hydrogenase in the presence of sulfhydryl compounds with methylene blue as electron acceptor. The maximal specific activity of hydrogenase in extracts prepared in the anaerobic conditions in the presence of dithiothreitol and Mg2+ and Mn2+ ions was 10 +/- 3 units per 1 mg of protein, which closely corresponded with the activity of hydrogenase in the whole cells. Almost all activity of hydrogenase assayed with methylene blue was localized in the membrane fraction. The activity of soluble NAD-specific hydrogenase was not detected. Large particles located in 60-70% sucrose had the highest hydrogenase activity upon fractionation in a continuous sucrose concentration gradient. The second, lower peak of the hydrogenase activity was detected in fractions of 40--50% sucrose. As was found by electron microscopy, the size of membrane vesicles with the hydrogenase activity varied within the range of 68--156 nm. The membrane preparations possessed the activity of NADH-dehydrogenase, NADH-oxidase and succinate dehydrogenase as well.
嗜热假单胞菌K-2自养生长的细胞制备出在0.2M Tris-HCl-0.02M EDTA中具有渗透稳定性的原生质球。原生质球具有全细胞90%-95%的氢化酶活性。氢化酶在原生质球中80℃下的半衰期为8.5分钟。开发了一种分光光度技术，用于在存在巯基化合物且以亚甲基蓝作为电子受体的情况下测定膜结合氢化酶。在厌氧条件下，在二硫苏糖醇以及Mg2+和Mn2+离子存在下制备的提取物中，氢化酶的最大比活性为每1mg蛋白质10±3单位，这与全细胞中氢化酶的活性密切对应。用亚甲基蓝测定的几乎所有氢化酶活性都定位于膜部分。未检测到可溶性NAD特异性氢化酶的活性。在连续蔗糖浓度梯度分级分离时，位于60%-70%蔗糖中的大颗粒具有最高的氢化酶活性。在40%-50%蔗糖的级分中检测到氢化酶活性的第二个较低峰。通过电子显微镜发现，具有氢化酶活性的膜泡大小在68-156nm范围内变化。膜制剂还具有NADH-脱氢酶、NADH-氧化酶和琥珀酸脱氢酶的活性。
