Kao Mou-Chieh, Matsuno-Yagi Akemi, Yagi Takao
Division of Biochemistry, Department of Molecular and Experimental Medicine, MEM-256, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Biochemistry. 2004 Mar 30;43(12):3750-5. doi: 10.1021/bi049927s.
The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of 14 different subunits (designated Nqo1-14), seven of which are located in the membrane domain and the other seven in the peripheral domain. It has been previously reported that membrane domain subunit Nqo7 (ND3) directly interacts with peripheral subunit Nqo6 (PSST) by using a cross-linker, m-maleimidobenzoyl-N-hydrosuccinimide ester, and heterologous expression [Di Bernardo, S., and Yagi, T. (2001) FEBS Lett. 508, 385-388]. To further explore the near-neighbor relationship of the subunits, a zero-length cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), and the Paracoccus membranes were used, and the cross-linked products were examined with antibodies specific to subunits Nqo1-11. The Nqo6 subunit was cross-linked to subunit Nqo9 (TYKY). In addition, a ternary product of Nqo3 (75k), Nqo6, and Nqo7 and binary products of Nqo3 and Nqo6 and of Nqo6 and Nqo7 were observed, but a binary product of Nqo3 and Nqo7 was not detected. The Nqo4 (49k) subunit was found to be associated with the Nqo7 subunit. Furthermore, Paracoccus subunits Nqo3, Nqo6, and Nqo7 were heterologously coexpressed in Escherichia coli, and EDC cross-linking experiments were carried out using the E. coli membranes expressing these three subunits. The results were the same as those obtained with Paracoccus membranes. On the basis of the data, subunit arrangements of NDH-1 were discussed.
反硝化副球菌的质子转运型NADH-醌氧化还原酶（NDH-1）由14种不同的亚基（命名为Nqo1 - 14）组成，其中7个位于膜结构域，另外7个位于外周结构域。此前有报道称，膜结构域亚基Nqo7（ND3）通过使用交联剂间马来酰亚胺苯甲酰-N-羟基琥珀酰亚胺酯和异源表达与外周亚基Nqo6（PSST）直接相互作用[迪·贝尔纳多，S.，和矢木，T.（2001年）《欧洲生物化学学会联合会快报》508，385 - 388]。为了进一步探究亚基之间的近邻关系，使用了零长度交联剂1-乙基-3-[3-(二甲基氨基)丙基]碳二亚胺（EDC）和反硝化副球菌膜，并使用针对亚基Nqo1 - 11的特异性抗体检测交联产物。Nqo6亚基与Nqo9（TYKY）亚基发生了交联。此外，观察到了Nqo3（75k）、Nqo6和Nqo7的三元产物以及Nqo3和Nqo6、Nqo6和Nqo7的二元产物，但未检测到Nqo3和Nqo7的二元产物。发现Nqo4（49k）亚基与Nqo7亚基相关联。此外，反硝化副球菌亚基Nqo3、Nqo6和Nqo7在大肠杆菌中进行了异源共表达，并使用表达这三个亚基的大肠杆菌膜进行了EDC交联实验。结果与用反硝化副球菌膜得到的结果相同。基于这些数据，对NDH-1的亚基排列进行了讨论。
