NADPH-cytochrome c reductase from human neutrophil membranes: purification, characterization and localization

Abstract

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.

Mutation at Histidine 338 of gp91 Depletes FAD and Affects Expression of Cytochrome b 558 of the Human NADPH Oxidase

“…We examined this by adding reagent FAD or a FAD-rich NBT-reductase fraction, previously demonstrated to be obligatory to reconstitute the NADPH oxidase activity of purified cytochrome b 558 without added reagent FAD (41). In this rationale, the NBT-reductase fraction providing flavoproteins with NADPH-(30) and flavin-binding sites would work like cytochrome P-450 reductase, bypassing electrons to heme(s) in cytochrome b 558 , rebuilding electron transfer machinery (41,(43)(44)(45). This approach would indicate whether or not at least interaction with an exogenous dehydrogenase could be achieved and give indications on the patient's heme functional status.…”

Identification of subdomains in NADPH oxidase-4 critical for the oxygen-dependent regulation of TASK-1 K+ channels

“…CO inhibits the major components of endogenous oxidantgenerating machinery, NADPH oxidase, and the cytochrome-c oxidase of the mitochondrial respiratory chain, and thereby blocks formation of ROS (25,27). In our present study, a slight decrease of TASK-1 current on the application of CO or ascorbic acid might reflect a positive modulation of TASK-1 by the basal production of ROS.…”

“…It was initially supposed that the replacement of heme-bound O 2 with CO might exert a hypoxia-like effect by deterring the ROS generation from NOX4 (25,27). When cells were pretreated with CO (1%, see MATERIALS AND METHODS), however, the CO treatment only weakly decreased I TASK-1 (8 Ϯ 3.5%, n ϭ 5).…”

Purification of an NADPH‐dependent diaphorase from membrane of DMSO‐induced differentiated human promyelocytic leukemia HL‐60 cells

“…We have also purified an NADPH-dependent cytochrome c reductase from the cytosolic fraction of HL-60 cells with a molecular mass of 68 kDa, and suggested that the enzyme might be released by limited proteolysis of the membrane-bound native form during its purification [7]. The isolation of the membrane-bound diaphorase to electrophoretic homogeneity was extremely difficult due to very low content and instability after solubilization from the membrane.…”

“…In previous studies we have described the presence of an NADPH diaphorase that contained FAD and FMN (1/1) as a prosthetic group in human neutrophils and differentiated HL-60 cells, and reported its characteristics [7][8][9][10]. We have also purified an NADPH-dependent cytochrome c reductase from the cytosolic fraction of HL-60 cells with a molecular mass of 68 kDa, and suggested that the enzyme might be released by limited proteolysis of the membrane-bound native form during its purification [7].…”