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. C...

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Bibliographic Details
Published in:Biochemistry (Easton) 1995-12, Vol.34 (51), p.16753-16757
Main Authors: Huang, Jin, Hitt, Nancy D, Kleinberg, Michael E
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amino Acids - analysis
Chromatography, Affinity
Cytochrome b Group - chemistry
Cytochrome b Group - genetics
Cytochrome b Group - isolation & purification
Humans
Membrane Glycoproteins - analysis
Membrane Glycoproteins - genetics
Membrane Glycoproteins - isolation & purification
Membrane Transport Proteins
Molecular Sequence Data
NADPH Dehydrogenase - analysis
NADPH Dehydrogenase - genetics
NADPH Dehydrogenase - isolation & purification
NADPH Oxidase 2
NADPH Oxidases
Neutrophils - chemistry
Phosphoproteins - analysis
Phosphoproteins - genetics
Phosphoproteins - isolation & purification
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Summary: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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00051a024