NADPH-CYTOCHROME-C REDUCTASE FROM HUMAN NEUTROPHIL MEMBRANES - PURIFICATION, CHARACTERIZATION AND LOCALIZATION

Neutrophil-membrane-associated NADPH-cytochrome c reductase and cytochrome b(558) were separately eluted and highly purified by a combination of ion-exchange Sepharose, N-amino-octyl-agarose, 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 b(558) prepared in the presence of phospholipids and FAD showed marked O-2(-.)-producing activity (V-max, 8.53 mu mol of O2(-.)/min per mg of cytochrome; K-m for NADPH 58.8 mu M) 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 O-2(-.)-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 b(558) in the cell-free system, suggesting that the reductase is not likely to be involved in neutrophil O-2(-.) 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 b(558) 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.