Stoichiometry of p22-phox and gp91-phox in phagocyte cytochrome b558.
The phagocyte NADPH oxidase complex is an unusual electron transfer system. Its principal component, cytochrome b558, is a heme-containing integral membrane protein consisting of two subunits, gp91-phox and p22-phox. We used a novel method to measure precisely the gp91-phox:p22-phox stoichiometry. Cytochrome b558 was isolated in high purity from human neutrophil membrane preparations using a novel affinity purification method. We performed direct peptide sequencing of purified cytochrome b558 and detected two amino acid sequences which matched predicted sequences for gp91-phox and p22-phox. We quantitated amounts of both amino acids released from p22-phox and gp91-phox in each sequencing cycle. Averaging over 25 cycles, the mean p22-phox:gp91-phox ratio of released amino acids was 0.93 +/- 0.01. To correct for recovery differences between individual amino acids, we measured individual p22-phox:gp91-phox ratios for the eight different amino acids common to both p22-phox and gp91-phox in the first 25 positions. The mean of individual p22-phox:gp91-phox ratios for the eight common amino acids was 0.96 +/- 0.05. The p22-phox:gp91-phox ratios for each of the eight common amino acids varied from 0.81 to 1.20. Taken together, measured ratios for total and individual amino acids are consistent with a predicted ratio of 1.0 for 1:1 p22-phox:gp91-phox stoichiometry in cytochrome b558.
Huang
J
J
Research Service, Baltimore Veterans Affairs Medical Center, Maryland 21201, USA.
Hitt
N D
ND
Kleinberg
M E
ME
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002846:Chromatography, Affinity; D003573:Cytochrome b Group / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009252:NADPH Dehydrogenase / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D009504:Neutrophils / Q000737:chemistry*; D010750:Phosphoproteins / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification
D000596:Amino Acids; D003573:Cytochrome b Group; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D010750:Phosphoproteins; C036888:cytochrome b558; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C086986:CYBA protein, human; D009252:NADPH Dehydrogenase