Isolation of a complex of respiratory burst oxidase components from resting neutrophil cytosol.
The respiratory burst oxidase of neutrophils is a multicomponent enzyme, dormant in resting cells, that catalyzes the reduction of oxygen to O2- at the expense of NADPH. In the resting neutrophil, some of the components of the oxidase, including proteins p47phox and p67phox, are in the cytosol, while the rest are in a fraction that usually copurifies with plasma membrane. Recent evidence has suggested that at least some of the cytosolic oxidase components exist as a complex. We have now purified such a complex from the cytoplasm of resting neutrophils using an affinity column prepared with an antibody that recognizes the C-terminal decapeptide of p47phox. Immunoblotting showed that the complex contained both p47phox and p67phox. When supplemented with recombinant p67phox, the complex displayed considerable activity in a cell-free oxidase-activating system, and even without added p67phox, the complex could more than double O2- production in an oxidase-activating system supplemented with suboptimal amounts of cytosol. Isolation of the complex was blocked by preincubating the affinity column with CFSTKRKLASAV, the peptide against which the antibody was raised. On gel filtration, the complex migrated with a molecular weight of 240-300K, similar to that observed with whole neutrophil cytosol. The p47phox/p67phox ratio in the gel-filtered complex was approximately 1 to 1. These results indicate that in resting neutrophil cytosol, p47phox and p67phox exist as a complex.
Park
J W
JW
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037.
Benna
J E
JE
Scott
K E
KE
Christensen
B L
BL
Chanock
S J
SJ
Babior
B M
BM
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D002846:Chromatography, Affinity; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000097:blood* / Q000302:isolation & purification*; D009247:NADH, NADPH Oxidoreductases / Q000097:blood* / Q000302:isolation & purification*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D009842:Oligopeptides / Q000138:chemical synthesis / Q000276:immunology; D011994:Recombinant Proteins / Q000378:metabolism; D013481:Superoxides / Q000097:blood
D000906:Antibodies; D009097:Multienzyme Complexes; D009842:Oligopeptides; D011994:Recombinant Proteins; D013481:Superoxides; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C022709:superoxide-forming enzyme