MilliporeSigma

Assembly of phagocyte NADPH oxidase: A concerted binding process?

Biochimica et biophysica acta (2014-08-12)
Gilda Karimi, Chantal Houée Levin, Marie Claire Dagher, Laura Baciou, Tania Bizouarn
ABSTRACT

The phagocyte NADPH-oxidase is a multicomponent enzyme that generates superoxide anions. It comprises a membrane redox component flavocytochrome b558 and four cytosolic proteins (p67(phox), p47(phox), p40(phox) and Rac) that must assemble to produce an active system. In this work we focused on the spatio-temporal control of the activation process of phagocyte NADPH oxidase. A wide range of techniques including fast kinetics with a stopped-flow apparatus and various combinations of the activating factors was used to test the order of assembly and the role of the p47(phox)-p67(phox) complex. The data presented here are consistent with the absence of a catalytic role of the p47(phox)-p67(phox) interacting state and support the idea of independent binding sites for the cytosolic proteins on the flavocytochrome b558 allowing random binding order. However, the formation of the active complex appears to involve a synergistic process of binding of the activated cytosolic subunits to cytochrome b558. All partners should be in the vicinity for optimal assembly, a delay or the absence of one of the partners in this process seems to lead to a decrease in the efficiency of the catalytic core. The activation and assembly of the NADPH oxidase components have to be achieved simultaneously for the formation of an efficient and optimal enzyme complex. This mechanism appears to be incompatible with continuous fast exchanges of the cytosolic proteins during the production of superoxide ion in the phagosome.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
BIS-TRIS, ≥98.0% (titration)
Sigma-Aldrich
BIS-TRIS, BioPerformance Certified, suitable for cell culture, suitable for insect cell culture, ≥98.0%
Sigma-Aldrich
BIS-TRIS, BioXtra, ≥98.0% (titration)
Sigma-Aldrich
BIS-TRIS, BioUltra, ≥99.0% (NT)
SAFC
BIS-TRIS
SAFC
BIS-TRIS
Supelco
Glutathione, Pharmaceutical Secondary Standard; Certified Reference Material
Glutathione, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
Arachidonic acid, >95.0% (GC)
Sigma-Aldrich
Arachidonic acid, from non-animal source, ≥98.5% (GC)
Sigma-Aldrich
L-Glutathione reduced, ≥98.0%
Sigma-Aldrich
L-Glutathione reduced, suitable for cell culture, BioReagent, ≥98.0%, powder
Sigma-Aldrich
L-Glutathione reduced, BioXtra, ≥98.0%
Sigma-Aldrich
Kit for Molecular Weights 14,000-500,000 Non-denaturing