Characterization of neutrophil NADPH oxidase factors p47-phox and p67-phox from recombinant baculoviruses.
Superoxide production by phagocytic blood cells involves assembly of an active NADPH oxidase complex from components found both in membrane and cytosolic locations in resting cells. We recently cloned cDNAs encoding two cytosolic components (p47-phox and p67-phox) of the oxidase that are deficient in distinct forms of autosomal recessive chronic granulomatous disease. The precise roles of p47-phox and p67-phox were explored further using purified factors produced in large quantities using recombinant baculoviruses to infect cultured Sf9 insect cells. Neither p47-phox nor p67-phox are thought to represent the flavoprotein components of the oxidase, since neither of the purified recombinant factors contained or bound FAD. Recombinant p47-phox and p67-phox are capable of restoring the deficient cytosol from chronic granulomatous disease patient neutrophils to nearly normal levels in a cell-free reconstitution system. Both p47-phox and p67-phox, used together in the absence of neutrophil cytosol, are incapable of supporting cell free production of superoxide, confirming the involvement of other soluble factor(s) in the assembly of an active oxidase in vitro.
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892.
T L
TL
Leto
M C
MC
Garrett
H
H
Fujii
H
H
Nunoi
D016367:Baculoviridae / Q000201:enzymology* / Q000235:genetics; D015153:Blotting, Western; D002850:Chromatography, Gel; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D007700:Kinetics; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D019255:NADPH Oxidases; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D004247:DNA; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases