PMID: 8947464Nov 15, 1996Paper

Stoichiometry of the subunits of flavocytochrome b558 of the NADPH oxidase of phagocytes

The Biochemical Journal
T M Wallach, A W Segal

Abstract

Flavocytochrome b558, the membrane-spanning component of the NADPH oxidase system of phagocytic cells, is composed of two subunits, p22phox and gp91phox (where phox stands for phagocyte oxidase). The stoichiometry of the subunits has been determined for purified flavocytochrome b556 by: (1) densitometry of Coomassie Blue-stained proteins separated by SDS/PAGE, (2) aromatic absorbance at 280 mm by the subunits after separation by gel filtration under denaturing conditions, (3) crosslinking studies with bis[sulphosuccinimidyl]suberate, where the molecular mass of the cross-linked complex was determined by Western blotting, and (4) radiolabelling of pure flavocytochrome b556 on lysine residues with 125I-labelled Bolton-Hunter reagent (N-succinimidyl-3-(4-hydroxy-5-[125I]iodophenyl)propionate), followed by SDS/PAGE and determination of the radioactivity on each subunit. The ratio of p22phox to gp91phox in the purified flavocytochrome b556 was related back to that in the neutrophil membrane by quantitative Western and dot-blotting to ensure that the stoichiometry was maintained during purification. These measurements showed that the two subunits were present in neutrophil membranes in a molar ratio of 1:1.

Citations

Nov 26, 2003·The Journal of General Physiology·Gabor L PetheöNicolas Demaurex
Jul 9, 2004·Biochimica Et Biophysica Acta·Andrew R Cross, Anthony W Segal
Oct 13, 2004·Free Radical Biology & Medicine·Ibrahim R HannaKathy K Griendling
Feb 8, 2003·Antioxidants & Redox Signaling·Ibrahim R HannaKathy K Griendling
Apr 30, 2003·Antioxidants & Redox Signaling·Uwe RueckschlossHenning Morawietz
Mar 4, 2006·Circulation Research·Peter L Hordijk
Jun 20, 2007·European Journal of Clinical Investigation·M Y KökerD Roos
Mar 6, 2010·Journal of Medical Microbiology·John P FallonKevin Kavanagh
Jun 10, 1998·Springer Seminars in Immunopathology·C Meischl, D Roos
Jul 8, 1998·Proceedings of the National Academy of Sciences of the United States of America·L YuM C Dinauer
Sep 30, 2000·Clinical and Experimental Immunology·D Goldblatt, A J Thrasher
Feb 23, 2010·Biochimica Et Biophysica Acta·Mariano A OstuniLaura Baciou

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