The cytosolic subunit p67phox contains an NADPH-binding site that participates in catalysis by the leukocyte NADPH oxidase.
The NADPH-dependent respiratory burst oxidase of human neutrophils catalyzes the reduction of oxygen to superoxide using NADPH as the electron donor and is essential for normal host defenses. To gain insight into the function of the various oxidase subunits that are required for the full expression of catalytic activity, we studied the interactions between the 2',3'-dialdehyde derivative of NADPH (NADPH dialdehyde) and neutrophil cytosol. NADPH dialdehyde treatment of cytosol resulted in the loss of the ability of the cytosol to participate in cell-free oxidase activation; this inactivation was blocked by NADPH but not by NAD, NADP, or GTP. Partial purification of neutrophil cytosol yielded a single peak which could restore the activity lost in cytosol treated with NADPH dialdehyde. This peak contained p67phox but not p47phox or Rac2. Purified recombinant p67phox was similarly able to restore the activity lost in NADPH dialdehyde-treated cytosol and bound [32P]NADPH dialdehyde in a specific fashion. The activity of recombinant p67phox in cell-free oxidase assays was lost on treatment with NADPH dialdehyde. Together, these data suggest p67phox contains the catalytic NADPH-binding site of the leukocyte NADPH oxidase.
Smith
R M
RM
The Department of Medicine, University of California, San Diego 92093, USA.
Connor
J A
JA
Chen
L M
LM
Babior
B M
BM
D000447:Aldehydes / Q000737:chemistry; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D009249:NADP / Q000737:chemistry / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010750:Phosphoproteins / Q000378:metabolism*; D016897:Respiratory Burst
D000447:Aldehydes; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; D009249:NADP; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases