Electron transfer reactions in the NADPH oxidase system of neutrophils--involvement of an NADPH-cytochrome c reductase in the oxidase system.
Membrane-bound NADPH oxidase of pig blood neutrophils was solubilized with heptylthioglucoside in a high yield. The solubilized preparation from myristate-stimulated cells (sample S) showed high O2- generating activity, and the preparation from resting cells (sample R) had no activity, but the two samples had equal amounts of flavins and cytochrome b-558 (cyt b-558). The electron transfer reactions to exogenous cytochrome c (cyt c) or cyt b-558 in samples S and R were examined. Under anaerobic conditions, NADPH-dependent cyt c reductase activity appeared higher in sample S than in sample R, and the addition of FMN and FAD greatly enhanced the reductase activity of sample S, but not that of sample R. No marked difference between the reductase activities of samples S and R was seen with NADH. Photoreduction of the NADPH oxidase system was examined in the absence of NADPH under anaerobic conditions by monitoring the reduction rates of exogenous cyt c using a flashlight with cut-off filters between 400 and 500 nm. Cyt c reduction was much higher in sample S than in sample R on photoexcitation at about 450 nm. Photoreduction was carried out with a band-pass filter for selective irradiation at 450 nm. Marked reduction of exogenous cyt c was observed only in sample S: the small reduction of cyt c by sample R was independent of the light wavelength and was equal to the blank level. In contrast, no difference in the reduction of cyt b-558 by the two samples was found by either NADPH or photoreduction. Under aerobic conditions, no direct reduction of either cyt c or cyt b-558 was observed. These results suggest that an NADPH-cyt c reductase (a membrane-bound flavoprotein) is involved in the NADPH oxidase system of stimulated neutrophils.
Fujii
H
H
Department of Inflammatory Research, Tokyo Metropolitan Institute of Medical Sciences, Japan.
Kakinuma
K
K
D000693:Anaerobiosis; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D003573:Cytochrome b Group / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism*; D010084:Oxidation-Reduction; D013057:Spectrum Analysis; D013481:Superoxides / Q000378:metabolism; D013552:Swine
D003573:Cytochrome b Group; D003574:Cytochrome c Group; D013481:Superoxides; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; C036888:cytochrome b558; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; D019255:NADPH Oxidases