The cytosolic components of the respiratory burst oxidase exist as a M(r) approximately 240,000 complex that acquires a membrane-binding site during activation of the oxidase in a cell-free system.

Park, Jeen‐Woo; Ma, Muchou; Ruedi, J M; Smith, Robert M.; Babior, Bernard M.

doi:10.1016/s0021-9258(18)41929-1

Cited by 139 publications

(25 citation statements)

References 27 publications

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“…Cytosolic components of phox-p40 phox , p47 phox , and p67 phox -form a M r ‫ف‬ 240,000 complex (18) in resting neuplated and stimulated with TNF. Other neutrophils from which the compounds had been washed off were reexposed to the compounds (red hatched bars).…”

Section: Resultsmentioning

confidence: 99%

Calcium-sensing soluble adenylyl cyclase mediates TNF signal transduction in human neutrophils

Han

Stessin

Roberts

et al. 2005

The Journal of Experimental Medicine

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Through chemical screening, we identified a pyrazolone that reversibly blocked the activation of phagocyte oxidase (phox) in human neutrophils in response to tumor necrosis factor (TNF) or formylated peptide. The pyrazolone spared activation of phox by phorbol ester or bacteria, bacterial killing, TNF-induced granule exocytosis and phox assembly, and endothelial transmigration. We traced the pyrazolone's mechanism of action to inhibition of TNF-induced intracellular Ca2+ elevations, and identified a nontransmembrane (“soluble”) adenylyl cyclase (sAC) in neutrophils as a Ca2+-sensing source of cAMP. A sAC inhibitor mimicked the pyrazolone's effect on phox. Both compounds blocked TNF-induced activation of Rap1A, a phox-associated guanosine triphosphatase that is regulated by cAMP. Thus, TNF turns on phox through a Ca2+-triggered, sAC-dependent process that may involve activation of Rap1A. This pathway may offer opportunities to suppress oxidative damage during inflammation without blocking antimicrobial function.

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Section: Resultsmentioning

confidence: 99%

Calcium-sensing soluble adenylyl cyclase mediates TNF signal transduction in human neutrophils

Han

Stessin

Roberts

et al. 2005

The Journal of Experimental Medicine

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“…Proteins were eluted with a 0.1-0.3 M NaCl gradient in the same buffer, eluting at a flow rate of 0.5 mL/min. Fractions were analyzed by SDS-PAGE and immunoblotting with an antibody raised against a synthetic peptide from p67 PHOX (19). The fractions containing p67 PHOX were pooled, concentrated to <0.4 mL in a Centricon concentrator (Amicon Corp.), and applied to a Superdex 75 (Pharmacia Biotech Inc.) column equilibrated with buffer A containing 0.3 M NaCl.…”

Section: Methodsmentioning

confidence: 99%

NADPH Dehydrogenase Activity of p67^PHOX, a Cytosolic Subunit of the Leukocyte NADPH Oxidase

1999

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The leukocyte NADPH oxidase catalyzes the one-electron reduction of oxygen to O2- at the expense of NADPH. It is a multicomponent enzyme comprising a membrane-bound flavocytochrome (cytochrome b558) and at least four cytosolic components: p47PHOX, p67PHOX, p40PHOX, and Rac, a small GTPase. All the oxidase components except p40PHOX are required for enzyme activity. Many aspects of their function, however, are unclear. Using the electron acceptor ferricyanide, we found that recombinant p67PHOX from baculovirus-infected Sf9 cells could mediate the dehydrogenation of NADPH. NADPH dehydrogenation was not dependent on FAD and was insensitive to superoxide dismutase. Several control experiments showed that NADPH dehydrogenation was accomplished by p67PHOX, not by a trace contaminant in the p67PHOX preparation. The NADPH dehydrogenase activity of p67PHOX was proportional to enzyme concentration, and showed saturation kinetics with NADPH (Km 92 +/- 5 microM), but was inhibited at high concentrations of ferricyanide. NADH was also used as a substrate by p67PHOX (Km 123 +/- 38 microM). Taken together, these results show that p67PHOX is able to mediate pyridine nucleotide dehydrogenation. These findings raise the possibility that p67PHOX might participate directly in electron transfer between NADPH and the oxidase flavin.

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“…Cell free systems had a major impact on the field and thus allowed the description of the minimal components of the oxidase but they still did not tell the whole story. For instance, when p67phox is purified by gel filtration chromatography, it separates as part of a complex with a mass of 250kDa [45]. One part of this complex is p47phox but Segal's lab purified another part of the complex which turned out to be p40phox [46].…”

Section: Characterisation and Function Of The Cytoplasmic Components Of The Phagocyte Nadph Oxidasementioning

confidence: 99%

The phagocyte respiratory burst: Historical perspectives and recent advances

Thomas¹

2017

Immunology Letters

134

113

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When exposed to certain stimuli, phagocytes (including neutrophils, macrophages and eosinophils) undergo marked changes in the way they handle oxygen. Firstly, their rate of oxygen uptake increases greatly. This is accompanied by (i) the production of large amounts of superoxide and hydrogen peroxide and (ii) the metabolism of large quantities of glucose through the hexose monophosphate shunt. We now know that the oxygen used is not for respiration but for the production of powerful microbiocidal agents downstream of the initial production of superoxide. Concomitantly, glucose is oxidised through the hexose monophosphate shunt to re-generate the NADPH that has been consumed through the reduction of molecular oxygen to generate superoxide. This phagocyte respiratory burst is generated by an NADPH oxidase multi-protein complex that has a catalytic core consisting of membrane-bound gp91phox (CYBB) and p22phox (CYBA) sub-units and cytosolic components p47phox (NCF1), p67phox (NCF2) and p40phox (NCF4). Finally, another cytosolic component, the small G-protein Rac (Rac2 in neutrophils and Rac1 in macrophages) is also required for full activation. The importance of the complex in host defence is underlined by chronic granulomatous disease, a severe life-limiting immunodeficiency caused by mutations in the genes encoding the individual subunits. In this review, I will discuss the experimental evidence that underlies our knowledge of the respiratory burst, outlining how elegant biochemical analysis, coupled with study of patients deficient in the various subunits has helped elucidate the function of this essential part of innate immunity. I will also discuss some exciting recent studies that shed new light on how the abundance of the various components is controlled. Finally, I will explore the emerging role of reactive oxygen species such as superoxide and hydrogen peroxide in the pathogenesis of major human diseases including auto-inflammatory diseases.

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The cytosolic components of the respiratory burst oxidase exist as a M(r) approximately 240,000 complex that acquires a membrane-binding site during activation of the oxidase in a cell-free system.

Cited by 139 publications

References 27 publications

Calcium-sensing soluble adenylyl cyclase mediates TNF signal transduction in human neutrophils

Calcium-sensing soluble adenylyl cyclase mediates TNF signal transduction in human neutrophils

NADPH Dehydrogenase Activity of p67^PHOX, a Cytosolic Subunit of the Leukocyte NADPH Oxidase

The phagocyte respiratory burst: Historical perspectives and recent advances

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The cytosolic components of the respiratory burst oxidase exist as a M(r) approximately 240,000 complex that acquires a membrane-binding site during activation of the oxidase in a cell-free system.

Cited by 139 publications

References 27 publications

Calcium-sensing soluble adenylyl cyclase mediates TNF signal transduction in human neutrophils

Calcium-sensing soluble adenylyl cyclase mediates TNF signal transduction in human neutrophils

NADPH Dehydrogenase Activity of p67PHOX, a Cytosolic Subunit of the Leukocyte NADPH Oxidase

The phagocyte respiratory burst: Historical perspectives and recent advances

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NADPH Dehydrogenase Activity of p67^PHOX, a Cytosolic Subunit of the Leukocyte NADPH Oxidase