Home LiteratureArticle Details

PMID: 10393079 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

The mechanism of activation of NADPH oxidase in the cell-free system: the activation process is primarily catalytic and not through the formation of a stoichiometric complex.

The Biochemical journal ·Vol. 341 ( Pt 2) ·1999-07-15 ·Pages 251-5

Cross AR, Erickson RW, Curnutte JT

Abstract

It is commonly assumed that activation of the superoxide-generating NADPH oxidase requires the formation of a stable complex between flavocytochrome b-245 (the gp91phox/p22phox heterodimer) and the cytosolic cofactors p47phox, p67phox and Rac2. This association is thought to convert flavocytochrome b-245, which contains the NADPH-binding site, flavin and haem centres, from an inactive into an active state. Here we provide evidence that, in the cell-free system, this activation process does not necessarily require the formation of a stable stoichiometric complex between the phox proteins. To explain this data we propose the hypothesis that p67phox (and possibly Rac2), are capable of activating flavocytochrome b-245 in a catalytic fashion, where a single molecule of p67phox (or Rac2) is capable of activating multiple flavocytochrome b-245 molecules.

MeSH Terms

Animals Binding Sites Cell-Free System Enzyme Activation Humans NADP/metabolism NADPH Oxidases/chemistry,metabolism Neutrophils/metabolism Phosphoproteins/chemistry,metabolism Protein Binding

Chemicals

Phosphoproteins neutrophil cytosol factor 67K NADP NADPH Oxidases

Authors & Affiliations

3 authors, click to expand affiliations / ORCID

Cross A R

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA. scross@scripps.edu

Erickson R W

Curnutte J T

References (32)

32 references, click to expand

Generation of superoxide by purified and relipidated cytochrome b559 in the absence of cytosolic activators.
FEBS Lett. 1993 Jul 19;327(1):57-62 PMID: 8392946
Cytochrome b-245 is a flavocytochrome containing FAD and the NADPH-binding site of the microbicidal oxidase of phagocytes.
Biochem J. 1992 Jun 15;284 ( Pt 3):781-8 PMID: 1320378
A structural model for the nucleotide binding domains of the flavocytochrome b-245 beta-chain.
Protein Sci. 1993 Oct;2(10):1675-85 PMID: 8251942
Reconstitution of neutrophil NADPH oxidase activity in the cell-free system by four components: p67-phox, p47-phox, p21rac1, and cytochrome b-245.
J Biol Chem. 1992 Aug 25;267(24):16767-70 PMID: 1512217
The translocation of respiratory burst oxidase components from cytosol to plasma membrane is regulated by guanine nucleotides and diacylglycerol.
J Biol Chem. 1992 Oct 5;267(28):19901-6 PMID: 1328185
The respiratory burst oxidase of human neutrophils. Guanine nucleotides and arachidonate regulate the assembly of a multicomponent complex in a semirecombinant cell-free system.
J Biol Chem. 1993 Apr 25;268(12):8624-31 PMID: 8386165
Production of recombinant cytochrome b558 allows reconstitution of the phagocyte NADPH oxidase solely from recombinant proteins.
J Biol Chem. 1993 Jul 5;268(19):14256-60 PMID: 8314788
Superoxide production by cytochrome b559. Mechanism of cytosol-independent activation.
FEBS Lett. 1994 Feb 7;338(3):285-9 PMID: 8307196
The superoxide-generating system of human neutrophils possesses a novel diaphorase activity. Evidence for distinct regulation of electron flow within NADPH oxidase by p67-phox and p47-phox.
J Biol Chem. 1994 Aug 26;269(34):21448-54 PMID: 8063777
p67-phox enhances the binding of p47-phox to the human neutrophil respiratory burst oxidase complex.
J Biol Chem. 1994 Sep 2;269(35):22095-8 PMID: 8071333
Spatial and electrogenic properties of superoxide-producing cytochrome b-559 incorporated into liposomes.
Biochim Biophys Acta. 1995 May 10;1229(3):329-33 PMID: 7748884
Cytochrome b-245 of the neutrophil superoxide-generating system contains two nonidentical hemes. Potentiometric studies of a mutant form of gp91phox.
J Biol Chem. 1995 Jul 21;270(29):17075-7 PMID: 7615499
Aerobic and anaerobic functioning of superoxide-producing cytochrome b-559 reconstituted with phospholipids.
Biochim Biophys Acta. 1995 Dec 12;1232(3):225-9 PMID: 8534675
The NADPH oxidase and chronic granulomatous disease.
Mol Med Today. 1996 Mar;2(3):129-35 PMID: 8796870
NADPH oxidase activity is independent of p47phox in vitro.
J Biol Chem. 1996 Sep 13;271(37):22578-82 PMID: 8798426
Electron transfer across the O2- generating flavocytochrome b of neutrophils. Evidence for a transition from a low-spin state to a high-spin state of the heme iron component.
Biochemistry. 1996 Oct 15;35(41):13400-10 PMID: 8873608
The cytosolic component p47(phox) is not a sine qua non participant in the activation of NADPH oxidase but is required for optimal superoxide production.
J Biol Chem. 1996 Nov 29;271(48):30326-9 PMID: 8939991
Involvement of p40phox in activation of phagocyte NADPH oxidase through association of its carboxyl-terminal, but not its amino-terminal, with p67phox.
J Exp Med. 1996 Sep 1;184(3):893-902 PMID: 9064349
p40(phox) down-regulates NADPH oxidase activity through interactions with its SH3 domain.
J Biol Chem. 1997 Apr 4;272(14):9141-6 PMID: 9083043
Gp91(phox) is the heme binding subunit of the superoxide-generating NADPH oxidase.
Proc Natl Acad Sci U S A. 1998 Jul 7;95(14):7993-8 PMID: 9653128
Spontaneous activation of NADPH oxidase in a cell-free system: unexpected multiple effects of magnesium ion concentrations.
Biochem J. 1999 Feb 15;338 ( Pt 1):229-33 PMID: 9931320
Simultaneous presence of p47(phox) and flavocytochrome b-245 are required for the activation of NADPH oxidase by anionic amphiphiles. Evidence for an intermediate state of oxidase activation.
J Biol Chem. 1999 May 28;274(22):15519-25 PMID: 10336445
Cytochrome b-245 of neutrophils is also present in human monocytes, macrophages and eosinophils.
Biochem J. 1981 Apr 15;196(1):363-7 PMID: 7306076
The enzymic reduction and kinetics of oxidation of cytochrome b-245 of neutrophils.
Biochem J. 1982 May 15;204(2):479-85 PMID: 7115343
Activation of a NADPH oxidase from horse polymorphonuclear leukocytes in a cell-free system.
J Leukoc Biol. 1984 Dec;36(6):751-9 PMID: 6594417
Activation of human neutrophil nicotinamide adenine dinucleotide phosphate, reduced (triphosphopyridine nucleotide, reduced) oxidase by arachidonic acid in a cell-free system.
J Clin Invest. 1985 May;75(5):1740-3 PMID: 2987311
Activation of NADPH-dependent superoxide production in a cell-free system by sodium dodecyl sulfate.
J Biol Chem. 1985 Nov 5;260(25):13539-45 PMID: 2997168
Assembly and activation of the NADPH:O2 oxidoreductase in human neutrophils after stimulation with phorbol myristate acetate.
J Biol Chem. 1990 Jan 15;265(2):924-30 PMID: 2153119
Two cytosolic components of the human neutrophil respiratory burst oxidase translocate to the plasma membrane during cell activation.
J Clin Invest. 1990 Mar;85(3):714-21 PMID: 2155923
Cell-free translocation of recombinant p47-phox, a component of the neutrophil NADPH oxidase: effects of guanosine 5'-O-(3-thiotriphosphate), diacylglycerol, and an anionic amphiphile.
Biochemistry. 1992 Mar 17;31(10):2765-74 PMID: 1312346
Cytochrome b558: the flavin-binding component of the phagocyte NADPH oxidase.
Science. 1992 Jun 5;256(5062):1459-62 PMID: 1318579
Translocation of Rac correlates with NADPH oxidase activation. Evidence for equimolar translocation of oxidase components.
J Biol Chem. 1993 Oct 5;268(28):20983-7 PMID: 8407934

Article Info

Journal

The Biochemical journal

Abbr.

Biochem J

ISSN

0264-6021

scross@scripps.edu

Published

1999-07-15

Pages

251-5

Language

English

Region

England

NLM ID

2984726R

PMCID

PMC1220353

Subset

Grants

NIAID NIH HHS · AI24838 · United States

External Links

PubMed source PMC Full Text View journal details

Analysis Services

The mechanism of activation of NADPH oxidase in the cell-free system: the activation process is primarily catalytic and not through the formation of a stoichiometric complex.

Contact

WeChat Official Account