Schneider K, Piechulla B
Biochimie. 1986 Jan;68(1):5-13. doi: 10.1016/s0300-9084(86)81062-8.
The soluble NAD-linked hydrogenase of Alcaligenes eutrophus H16 is a tetramer consisting of 4 non-identical subunits with molecular weights of 63,000, 56,000, 30,000 and 26,000. Conditions have been elaborated to separate and isolate each of these subunits as a single polypeptide by a preparative scale of polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS). Against each of the 4 subunits, polyclonal antibodies were produced. From the crude sera isolated from rabbits, the antibodies (IgG fractions) were purified by Protein A-Sepharose chromatography. By the double immunodiffusion method, comparison of the 4 types of subunits revealed that they are in fact different polypeptides. Subunit 1 (Mr = 63,000) and subunit 2 (Mr = 56,000) only reacted with their own specific antibodies and showed no cross-reaction whatsoever with the antibodies raised against the other subunits. The only immunological relationship among the different subunits was observed with subunit 3 (Mr = 30,000) and subunit 4 (Mr = 26,000); the type of cross-reaction indicated that they are partially identical. A. eutrophus H16 contains, in addition to the soluble hydrogenase, a membrane-bound hydrogenase which is a dimer composed of 2 subunits with Mr of 61,000 and 30,000. Whereas the 2 native enzymes did not show any immunological cross-reaction with the respective antibodies, it was demonstrated by double immunofluorescence labeling on nitrocellulose filters that the larger subunit of the membrane-bound hydrogenase cross-reacted significantly with the antibodies raised against subunit 2 of the soluble hydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)
嗜糖假单胞菌H16的可溶性NAD连接氢化酶是一种四聚体，由4个不同的亚基组成，分子量分别为63,000、56,000、30,000和26,000。已经制定了相关条件，通过在十二烷基硫酸钠（SDS）存在下的制备规模聚丙烯酰胺凝胶电泳，将这些亚基中的每一个作为单一多肽分离和分离出来。针对这4个亚基中的每一个，都制备了多克隆抗体。从兔血清中分离出的粗血清中，抗体（IgG组分）通过蛋白A-琼脂糖凝胶层析进行纯化。通过双向免疫扩散法，对这4种亚基进行比较，发现它们实际上是不同的多肽。亚基1（Mr = 63,000）和亚基2（Mr = 56,000）仅与它们自己的特异性抗体反应，与针对其他亚基产生的抗体没有任何交叉反应。在不同亚基之间观察到的唯一免疫关系是亚基3（Mr = 30,000）和亚基4（Mr = 26,000）之间的；交叉反应类型表明它们部分相同。嗜糖假单胞菌H16除了可溶性氢化酶外，还含有一种膜结合氢化酶，它是由2个Mr为61,000和30,000的亚基组成的二聚体。虽然这两种天然酶与各自的抗体没有任何免疫交叉反应，但通过硝酸纤维素滤膜上的双重免疫荧光标记证明，膜结合氢化酶的较大亚基与针对可溶性氢化酶亚基2产生的抗体有明显的交叉反应。（摘要截断于250字）
