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Biochemistry. 1995 Dec 26;34(51):16753-7.

Stoichiometry of p22-phox and gp91-phox in phagocyte cytochrome b558.

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1
Research Service, Baltimore Veterans Affairs Medical Center, Maryland 21201, USA.

Abstract

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.

PMID:
8527449
DOI:
10.1021/bi00051a024
[Indexed for MEDLINE]

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