Nisimoto Y, Otsuka-Murakami H, Iwata S
Department of Biochemistry, Aichi Medical University, Japan.
Biochem J. 1994 Feb 1;297 ( Pt 3)(Pt 3):585-93. doi: 10.1042/bj2970585.
Neutrophil-membrane-associated NADPH-cytochrome c reductase and cytochrome b558 were separately eluted and highly purified by a combination of ion-exchange Sepharose, N-amino-octylagarose, 2',5'-ADP-Sepharose and heparin-Sepharose column chromatographies. The purified cytochrome c reductase with an apparent molecular mass of 68 kDa contained FMN and FAD (FMN/FAD approx. 1). Cytochrome b558 prepared in the presence of phospholipids and FAD showed marked O2-.-producing activity (Vmax., 8.53 mumol of O2-./min per mg of cytochrome; Km for NADPH 58.8 microM) in a cell-free assay system consisting of cytosol, arachidonate and GTP[S]. However, when it was obtained without FAD added to the purification process, it had negligible FAD and little or no O2-.-forming activity in the reconstituted system. The NADPH oxidase activity was not markedly stimulated on incubation of the purified reductase with either flavinated or flavin-depleted cytochrome b558 in the cell-free system, suggesting that the reductase is not likely to be involved in neutrophil O2-. generation. The purified reductase cross-reacted with polyclonal antibodies against both hepatic NADPH-cytochrome P-450 reductase and a synthetic peptide, ILVGPGTGIAPFRSF, which indicates residues 529-543 located in the glycine-rich NADPH-binding domain of the P-450 reductase, but cytochrome b558 did not produce any immunoreactive bands to these antibodies. These antibodies also produced a positive reaction with a 76 kDa protein from dimethyl sulphoxide-induced HL-60-cell microsomes. After solubilization of the microsomal membranes, the 76 kDa protein was readily converted into a partially proteolysed form (68 kDa) even in the presence of antiproteases. In addition, the microsomal fraction shows a CO difference spectrum with a peak at about 454 nm and a trough at 476 nm in the presence of dithionite, indicating the presence of a cytochrome P-450-like haemoprotein.
中性粒细胞膜相关的NADPH-细胞色素c还原酶和细胞色素b558通过离子交换琼脂糖凝胶、N-氨基辛基琼脂糖、2',5'-ADP-琼脂糖和肝素-琼脂糖柱色谱法的组合分别洗脱并高度纯化。纯化后的细胞色素c还原酶表观分子量为68 kDa，含有FMN和FAD（FMN/FAD约为1）。在由胞质溶胶、花生四烯酸盐和GTP[S]组成的无细胞测定系统中，在磷脂和FAD存在下制备的细胞色素b558表现出显著的超氧阴离子生成活性（Vmax.，每毫克细胞色素每分钟产生8.53 μmol超氧阴离子；NADPH的Km为58.8 μM）。然而，当在纯化过程中不添加FAD而获得该细胞色素b558时，在重构系统中其FAD含量可忽略不计，且几乎没有或没有超氧阴离子形成活性。在无细胞系统中，将纯化的还原酶与含黄素或不含黄素的细胞色素b558一起孵育时，NADPH氧化酶活性并未受到明显刺激，这表明该还原酶不太可能参与中性粒细胞超氧阴离子的生成。纯化后的还原酶与针对肝NADPH-细胞色素P-450还原酶的多克隆抗体以及一种合成肽ILVGPGTGIAPFRSF发生交叉反应，该合成肽代表位于P-450还原酶富含甘氨酸的NADPH结合结构域中的529 - 543位残基，但细胞色素b558对这些抗体未产生任何免疫反应条带。这些抗体也与二甲基亚砜诱导的HL-60细胞微粒体中的一种76 kDa蛋白质发生阳性反应。微粒体膜溶解后，即使在存在抗蛋白酶的情况下，76 kDa蛋白质也很容易转化为部分蛋白水解形式（68 kDa）。此外，微粒体部分在连二亚硫酸盐存在下显示出CO差光谱，在约454 nm处有一个峰，在476 nm处有一个谷，表明存在一种细胞色素P-450样血红素蛋白。
