Department of Systems Design and Informatics, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka, Fukuoka 820-8502, Japan; Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka, Fukuoka 820-8502, Japan.
J Biosci Bioeng. 2022 Jan;133(1):76-82. doi: 10.1016/j.jbiosc.2021.10.004. Epub 2021 Nov 6.
To clarify the precise subunit composition of the respiratory supercomplex of Corynebacterium glutamicum, several wash conditions were examined. MEGA (9 + 10) wash-buffer (0.5%) was used for this purpose and two-step column chromatography was performed. Almost equal amounts of cytochrome c, b, and a were observed in the purified fraction, estimated by their different absorption spectra. The 833 kDa and 685 kDa bands were observed in the clear native polyacrylamide gel electrophoresis (CN-PAGE) of the purified fraction. Both bands were stained using N,N',N',N-tetramethyl-p-phenylenediamine (TMPD) oxidase dye, and the 833 kDa band was also stained using NADH oxidase dye. The 3D map reconstructed from the 833 kDa band indicated that the bcc complex and aa oxidase are heterodimers. Lastly, electron transfer from NADH to the bcc-aa supercomplex was observed. The 833 kDa band is the supercomplex, which includes the heterodimer cytochrome bcc complex and cytochrome aa oxidase, as well as the monomer NDH-II. Hence, we termed the 833 kDa band the extended supercomplex (ESC).
为了阐明谷氨酸棒杆菌呼吸超级复合物的确切亚基组成，研究了几种洗涤条件。为此目的使用了 MEGA（9+10）洗涤缓冲液（0.5％），并进行了两步柱层析。通过它们不同的吸收光谱估计，在纯化部分中观察到几乎等量的细胞色素 c、b 和 a。在纯化部分的清晰天然聚丙烯酰胺凝胶电泳（CN-PAGE）中观察到 833 kDa 和 685 kDa 带。使用 N，N'，N'，N-四甲基对苯二胺（TMPD）氧化酶染料对这两个带进行染色，并且 833 kDa 带也使用 NADH 氧化酶染料进行染色。从 833 kDa 带重建的 3D 图谱表明 bcc 复合物和 aa 氧化酶是异二聚体。最后，观察到 NADH 向 bcc-aa 超级复合物的电子转移。833 kDa 带是超级复合物，其包括异二聚体细胞色素 bcc 复合物和细胞色素 aa 氧化酶，以及单体 NDH-II。因此，我们将 833 kDa 带称为扩展超级复合物（ESC）。
