Lorenz B, Schneider K, Kratzin H, Schlegel H G
Institut für Mikrobiologie, Universität Göttingen, F.R.G.
Biochim Biophys Acta. 1989 Mar 16;995(1):1-9. doi: 10.1016/0167-4838(89)90225-2.
Polyclonal, monospecific antibodies were produced against the two subunits (Mr 62,000, and Mr 31,000), isolated from the membrane-bound hydrogenase of Alcaligenes eutrophus H16. The antibodies (IgG fractions) were purified from crude sera by Protein A-Sepharose CL-4B chromatography. By double immunodiffusion assays and tandem-crossed immunoelectrophoresis the large and the small subunit were demonstrated not to be immunologically related. Immunological comparison of these subunits with the four non-identical subunits (Mr 63,000, 56,000, 30,000 and 26,000) of the NAD-linked, soluble hydrogenase from A. eutrophus H16 showed that the subunits of the membrane-bound hydrogenase did not cross-react with any of the antibodies raised against the four subunits of the NAD-linked enzyme and that, vice versa, none of these four subunits cross-reacted with antibodies raised against the two subunits of the membrane-bound hydrogenase. This means that A. eutrophus H16 contains altogether six non-identical immunologically unrelated hydrogenase polypeptides. The membrane-bound hydrogenases were isolated and purified from various aerobic H2-oxidizing bacteria: A. eutrophus H16, A. eutrophus type strain, A. eutrophus CH34, A. eutrophus Z1, A. hydrogenophilus, Paracoccus denitrificans and strain Cd2/01. All these proteins resembled each other and each consisted of two non-identical polypeptides. A complete separation of these subunits was achieved at high-yield by preparative FPLC gel filtration on three Superose 12 columns connected in series, using SDS and DTT-containing sodium phosphate buffer (pH 7.0). The small subunits of these enzymes turned out to be immunologically closely related to each other; they were either identical or almost identical. The large subunits were also related, but less pronounced. Only the large subunits from Z1 and type strain reacted fully identical with the H16 subunit. Of the two isolated, homogeneous subunits of the membrane-bound hydrogenase from A. eutrophus H16, the amino acid compositions and the NH2-terminal sequences have been determined. The results confirmed the diversity of the large and the small subunit. Furthermore, for comparison also the NH2-terminal sequences of the two subunits from the hydrogenase of A. eutrophus CH34 have been analysed.
制备了针对从嗜中性产碱杆菌H16膜结合氢化酶中分离出的两个亚基（分子量分别为62,000和31,000）的多克隆单特异性抗体。抗体（IgG组分）通过蛋白A - 琼脂糖凝胶CL - 4B层析从粗血清中纯化。通过双向免疫扩散试验和串联交叉免疫电泳证明，大亚基和小亚基在免疫上无相关性。将这些亚基与嗜中性产碱杆菌H16的NAD连接的可溶性氢化酶的四个不同亚基（分子量分别为63,000、56,000、30,000和26,000）进行免疫比较，结果表明膜结合氢化酶的亚基与针对NAD连接酶的四个亚基产生的任何抗体均无交叉反应，反之亦然，这四个亚基中的任何一个与针对膜结合氢化酶两个亚基产生的抗体也无交叉反应。这意味着嗜中性产碱杆菌H16总共含有六种不同的、免疫上不相关的氢化酶多肽。从多种好氧H2氧化细菌中分离并纯化了膜结合氢化酶：嗜中性产碱杆菌H16、嗜中性产碱杆菌模式菌株、嗜中性产碱杆菌CH34、嗜中性产碱杆菌Z1、嗜氢产碱杆菌、反硝化副球菌和菌株Cd2/01。所有这些蛋白质彼此相似，均由两个不同的多肽组成。通过在串联连接的三根Superose 12柱上进行制备型FPLC凝胶过滤，使用含SDS和DTT的磷酸钠缓冲液（pH 7.0），以高产率实现了这些亚基的完全分离。结果发现这些酶的小亚基在免疫上彼此密切相关；它们要么相同，要么几乎相同。大亚基之间也有关系，但不太明显。只有来自Z1和模式菌株的大亚基与H16亚基完全相同。已测定了嗜中性产碱杆菌H16膜结合氢化酶的两个分离的、均一的亚基的氨基酸组成和NH2末端序列。结果证实了大亚基和小亚基的多样性。此外，为作比较，还分析了嗜中性产碱杆菌CH34氢化酶两个亚基的NH2末端序列。
