Reconstitution of superoxide-forming NADPH oxidase activity with cytochrome b558 purified from porcine neutrophils. Requirement of a membrane-bound flavin enzyme for reconstitution of activity.
Cytochrome b558 of pig blood neutrophils was purified from the membranes of resting cells to examine its ability to reconstitute superoxide (O2-)-forming NADPH oxidase activity in a cell-free assay system containing cytosol and fatty acid. The membrane-associated cytochrome b558 was solubilized with a detergent, n-heptyl beta-thioglucoside, and purified by DEAE-Sepharose, heparin-Sepharose, and Mono Q column chromatography. The final preparation of cytochrome containing 11.5 nmol of protoheme/mg of protein gave bands of the large and small subunits on immunoblotted gel. The cell-free system with the purified cytochrome alone as a membrane component showed little O2(-)-generating activity in the absence of exogenous FAD. However, the system showed high O2(-)-generating activity of 31.8 mol/s/mol of cytochrome b558 (52.5% of the original O2(-)-generating activity of the solubilized membranes) in the presence of a nitro blue tetrazolium (NBT) reductase fraction that was separated from the cytochrome b fraction by heparin-Sepharose chromatography. Heat treatment of the NBT reductase fraction resulted in loss of the O2(-)-generating activity in the reconstituted system. The O2(-)-forming activity of the reconstituted system was markedly decreased by removal of FAD from the NBT reductase fraction and was restored by readdition of FAD to the FAD-depleted reductase. The reconstituted system containing purified cytochrome b558 plus the NBT reductase showed approximately 100 times higher O2(-)-generating activity than a system containing rabbit liver NADPH-cytochrome P-450 reductase instead. These results suggest that both the FAD-dependent NBT reductase and cytochrome b558 are required as membrane redox components for O2(-)-forming NADPH oxidase activity. The present data are discussed in comparison with previously reported results on reconstituted systems containing added free FAD.
Miki
T
T
Department of Inflammation Research, Tokyo Metropolitan Institute of Medical Science, Japan.
Yoshida
L S
LS
Kakinuma
K
K
D000818:Animals; D015153:Blotting, Western; D002474:Cell-Free System; D002846:Chromatography, Affinity; D003573:Cytochrome b Group / Q000097:blood* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D009504:Neutrophils / Q000201:enzymology*; D011817:Rabbits; D013481:Superoxides / Q000378:metabolism*; D013552:Swine
D003573:Cytochrome b Group; D008565:Membrane Proteins; D013481:Superoxides; D005182:Flavin-Adenine Dinucleotide; C036888:cytochrome b558; D009247:NADH, NADPH Oxidoreductases; C021848:NADPH-tetrazolium reductase; D009251:NADPH-Ferrihemoprotein Reductase; D019255:NADPH Oxidases