Cell-Free NADPH Oxidase Activation Assays: “In Vitro Veritas”

  • Edgar Pick
Part of the Methods in Molecular Biology book series (MIMB, volume 1124)


The superoxide (O2 ∙−)-generating NADPH oxidase complex of phagocytes comprises a membrane-imbedded heterodimeric flavocytochrome, known as cytochrome b 558 (consisting of Nox2 and p22 phox ) and four cytosolic regulatory proteins, p47 phox , p67 phox , p40 phox , and the small GTPase Rac. Under physiological conditions, in the resting phagocyte, O2 ∙− generation is initiated by engagement of membrane receptors by a variety of stimuli, followed by specific signal transduction sequences leading to the translocation of the cytosolic components to the membrane and their association with the cytochrome. A consequent conformational change in Nox2 initiates the electron “flow” along a redox gradient, from NADPH to oxygen, leading to the one-electron reduction of molecular oxygen to O2 ∙−. Methodological difficulties in the dissection of this complex mechanism led to the design “cell-free” systems (also known as “broken cells” or in vitro systems). In these, membrane receptor stimulation and all or part of the signal transduction sequence are missing, the accent being placed on the actual process of “NADPH oxidase assembly,” thus on the formation of the complex between cytochrome b 558 and the cytosolic components and the resulting O2 ∙− generation. Cell-free assays consist of a mixture of the individual components of the NADPH oxidase complex, derived from resting phagocytes or in the form of purified recombinant proteins, exposed in vitro to an activating agent (distinct from and unrelated to whole cell stimulants), in the presence of NADPH and oxygen. Activation is commonly quantified by measuring the primary product of the reaction, O2 ∙−, trapped immediately after its generation by an appropriate acceptor in a kinetic assay, permitting the calculation of the linear rate of O2 ∙− production, but numerous variations exist, based on the assessment of reaction products or the consumption of substrates. Cell-free assays played a paramount role in the identification and characterization of the components of the NADPH oxidase complex, the deciphering of the mechanisms of assembly, the search for inhibitory drugs, and the diagnosis of various forms of chronic granulomatous disease (CGD).


NADPH oxidase Superoxide Cell-free assays Cytochrome b558 Nox2 Noxes Cytosolic components p47phox p67phox Rac Anionic amphiphile Arachidonic acid Superoxide dismutase Isoprenylation Peptide walking 



The research described in this report was supported by the Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research, the Ela Kodesz Institute of Host Defense against Infectious Diseases, Israel Science Foundation Grants 428/01, 19/05, and 49/09, the Roberts-Guthman Chair in Immunopharmacology, the Walter J. Levy Benevolent Trust, the Roberts Fund, the Milken—Lowell Fund, the Wallis Foundation, the Rubanenko Fund, and the Joseph and Shulamit Salomon Fund. It is important to point out that the cell-free system was discovered almost simultaneously by several investigators. R.A. Heyneman and R.E. Vercauteren, in Belgium, and Linda McPhail and John Curnutte, in the USA, each independently, contributed greatly to the birth of the “cell-free” paradigm. Edgar Pick would like to thank his fellow scientists, too many to name, who provided materials and invaluable advice, for making this work possible. There is no greater satisfaction than the realization of the fact that, on so many occasions, what started as collaboration (called “networking” these days) or competition, evolved into long-lasting friendship.


  1. 1.
    Nauseef WM (2007) How human neutrophils kill and degrade microbes: an integrated view. Immunol Rev 219:88–102PubMedGoogle Scholar
  2. 2.
    Nauseef WM (2004) Assembly of the phagocyte NADPH oxidase. Histochem Cell Biol 122:277–291PubMedGoogle Scholar
  3. 3.
    Quinn MT, Gauss KA (2004) Structure and regulation of the neutrophil respiratory burst oxidase: comparison with nonphagocyte oxidases. J Leukoc Biol 76:760–781PubMedGoogle Scholar
  4. 4.
    Groemping Y, Rittinger K (2005) Activation and assembly of the NADPH oxidase: a structural perspective. Biochem J 386:401–416PubMedGoogle Scholar
  5. 5.
    Sumimoto H (2008) Structure, regulation and evolution of Nox-family NADPH oxidases that produce reactive oxygen species. FEBS J 275:3249–3277PubMedGoogle Scholar
  6. 6.
    Han C-H, Freeman JLR, Lee T, Motalebi S, Lambeth JD (1998) Regulation of the neutrophil respiratory burst oxidase. Identification of an activation domain. J Biol Chem 273:16663–16668PubMedGoogle Scholar
  7. 7.
    Sarfstein R, Gorzalczany Y, Mizrahi A, Berdichevsky Y, Molshanski-Mor S, Weinbaum C, Hirshberg M, Dagher M-C, Pick E (2004) Dual role of Rac in the assembly of NADPH oxidase, tethering to the membrane and activation of p67phox. A study based on mutagenesis of p67phox-Rac1 chimeras. J Biol Chem 279:16007–16016PubMedGoogle Scholar
  8. 8.
    Kreck ML, Freeman JL, Lambeth JD (1996) Membrane association of Rac is required for high activity of the respiratory burst oxidase. Biochemistry 35:15683–15692PubMedGoogle Scholar
  9. 9.
    Gorzalczany Y, Sigal N, Itan M, Lotan O, Pick E (2000) Targeting of Rac1 to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly. J Biol Chem 275:40073–40081PubMedGoogle Scholar
  10. 10.
    Bedard K, Krause K-H (2007) The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev 87:255–313Google Scholar
  11. 11.
    Freeman JL, Lambeth JD (1996) NADPH oxidase activity is independent of p47phox in vitro. J Biol Chem 271:22578–22582PubMedGoogle Scholar
  12. 12.
    Koshkin V, Lotan O, Pick E (1996) The cytosolic component p47phox is not a sine qua non participant in the activation of NADPH oxidase but is required for optimal superoxide production. J Biol Chem 271:30326–30329PubMedGoogle Scholar
  13. 13.
    Cheson BD, Curnutte JT, Babior BM (1977) The oxidative killing mechanism of the neutrophil. Prog Clin Immunol 3:1–65PubMedGoogle Scholar
  14. 14.
    Pick E, Keisari Y (1981) Superoxide anion production and hydrogen peroxide production by chemically elicited macrophages—Induction by multiple nonphagocytic stimuli. Cell Immunol 59:301–318PubMedGoogle Scholar
  15. 15.
    Babior BM, Curnutte JT, McMurrich BJ (1976) The particulate superoxide-forming system from human neutrophils: properties of the system and further evidence supporting its participation in the respiratory burst. J Clin Invest 58:989–996PubMedCentralPubMedGoogle Scholar
  16. 16.
    Markert M, Andrews PC, Babior BM (1984) Measurement of O2 ∙− production by human neutrophils. The preparation and assay of NADPH oxidase-containing particles from human neutrophils. Methods Enzymol 105:358–365PubMedGoogle Scholar
  17. 17.
    Babior BM, Kipnes RS (1977) Superoxide-forming enzyme from human neutrophils: evidence for a flavin requirement. Blood 50:517–524PubMedGoogle Scholar
  18. 18.
    Cross AR, Segal AW (2004) The NADPH oxidase of phagocytes—prototype of the NOX electron transport chain systems. Biochim Biophys Acta 1657:1–22PubMedCentralPubMedGoogle Scholar
  19. 19.
    Bromberg Y, Pick E (1983) Unsaturated fatty acids as second messengers of superoxide generation by macrophages. Cell Immunol 79:243–252Google Scholar
  20. 20.
    Flores J, Witkum P, Sharp GWG (1976) Activation of adenylate cyclase by cholera toxin. J Clin Invest 57:450–458PubMedCentralPubMedGoogle Scholar
  21. 21.
    Bromberg Y, Pick E (1984) Unsaturated fatty acids stimulate NADPH- dependent superoxide generation by cell-free system in macrophages. Cell Immunol 88:213–221PubMedGoogle Scholar
  22. 22.
    Heyneman RA, Vercauteren RE (1984) Activation of a NADPH oxidase from horse poymorphonuclear leukocytes in a cell-free system. J Leukoc Biol 36:751–759PubMedGoogle Scholar
  23. 23.
    McPhail LC, Shirley PS, Clayton CC, Snyderman R (1985) Activation of the respiratory burst enzyme from human neutrophils in a cell-free system. J Clin Invest 75:1735–1739PubMedCentralPubMedGoogle Scholar
  24. 24.
    Curnutte JT (1985) Activation of human neutrophil nicotinamide adenine dinucleotide phosphate reduced (triphosphopyridine nucleotide, reduced) oxidase by arachidonic acid in a cell-free system. J Clin Invest 75:1740–1743PubMedCentralPubMedGoogle Scholar
  25. 25.
    Curnutte JT, Babior BM (1987) Chronic granulomatous disease. Adv Human Genetics 16:229–297Google Scholar
  26. 26.
    Tsunawaki S, Nathan CF (1986) Release of arachidonate and reduction of oxygen. Independent metabolic bursts of the mouse peritoneal macrophage. J Biol Chem 261:11563–11570PubMedGoogle Scholar
  27. 27.
    Bromberg Y, Pick E (1985) Activation of NADPH-dependent superoxide production in a cell-free system by sodium dodecyl sulfate. J Biol Chem 260:13539–13545PubMedGoogle Scholar
  28. 28.
    Groemping Y, Lapouge K, Smerdon SJ, Rittinger K (2003) Molecular basis of phosphorylation-induced activation of the NADPH oxidase. Cell 113:343–355PubMedGoogle Scholar
  29. 29.
    Swain SD, Helgerson SL, Davis AR, Nelson LK, Quinn MT (1997) Analysis of activation-induced conformational changes in p47phox using tryptophan fluorescence spectroscopy. J Biol Chem 272:29502–29510PubMedGoogle Scholar
  30. 30.
    Doussière J, Gaillard J, Vignais PV (1996) Electron transfer across the O2 ∙− generating flavocytochrome of neutrophils. Evidence for transition from low-spin state to a high-spin state of the heme iron component. Biochemistry 35:13400–13410PubMedGoogle Scholar
  31. 31.
    Foubert TR, Burritt JB, Taylor RM, Jesaitis AJ (2002) Structural changes are induced in human neutrophil cytochrome b by NADPH oxidase activators, LDS, SDS and arachidonate: intermolecular resonance energy transfer between trisulfopyrenyl-wheat germ agglutinin and cytochrome b 558. Biochim Biophys Acta 1567:221–231PubMedGoogle Scholar
  32. 32.
    Taylor RM, Riesselman MH, Lord CI, Gripentrog JA, Jesaitis AJ (2012) Anionic lipid-induced conformational changes in human phagocyte flavocytochrome b precede assembly and activation of the NADPH oxidase complex. Arch Biochem Biophys 521:24–31PubMedGoogle Scholar
  33. 33.
    Qualliotine-Mann D, Agwu DE, Ellenburg MD, McCall CE, McPhail LC (1993) Phosphatidic acid and diacylglycerol synergize in a cell-free system for activation of NADPH oxidase from human neutrophils. J Biol Chem 268:23843–23849PubMedGoogle Scholar
  34. 34.
    Erickson RW, Langel-Peveri P, Traynor-Kaplan AE, Heyworth PG, Curnutte JT (1999) Activation of human neutrophil NADPH oxdase by phosphatidic acid or diacylglycerol in a cell-free system. Activity of diacylglycerol is dependent on its conversion to phosphatidic acid. J Biol Chem 274:22243–22250PubMedGoogle Scholar
  35. 35.
    Tauber AI, Cox JA, Curnutte JT (1989) Activation of human neutrophil NADPH-oxidase in vitro by the catalytic fragment of protein kinase-C. Biochem Biophys Res Comm 158:884–890PubMedGoogle Scholar
  36. 36.
    Park J-W, Hoyal CR, El Benna J, Babior BM (1997) Kinase-dependent activation of the leukocyte NADPH oxidase in a cell-free system—Phosphorylation of membranes and p47phox during oxidase activation. J Biol Chem 272:11035–11043PubMedGoogle Scholar
  37. 37.
    Abo A, Boyhan A, West I, Thrasher AJ, Segal AW (1992) Reconstitution of neutrophil NADPH oxidase activity in the cell-free system by four components: p67phox, p47phox, p21rac1, and cytochrome b-245. J Biol Chem 267:16767–16770PubMedGoogle Scholar
  38. 38.
    Ebisu K, Nagasawa T, Watanabe K, Kakinuma K, Miyano K, Tamura M (2001) Fused p47phox and p67phox truncations efficiently reconstitute NADPH oxidase with higher activity than the individual components. J Biol Chem 276:24498–24505PubMedGoogle Scholar
  39. 39.
    Miyano K, Ogasawara S, Han C-H, Fukuda H, Tamura M (2001) A fusion protein between Rac and p67phox (1-210) reconstitutes NADPH oxidase with higher activity and stability than individual components. Biochemistry 40:14089–14097PubMedGoogle Scholar
  40. 40.
    Alloul N, Gorzalczany Y, Itan M, Sigal N, Pick E (2001) Activation of the superoxide-generating NADPH oxidase by chimeric proteins consisting of segments of the cytosolic component p67phox and the small GTPase Rac1. Biochemistry 40:14557–14566PubMedGoogle Scholar
  41. 41.
    Mizrahi A, Berdichevsky Y, Ugolev Y, Molshanski-Mor S, Nakash Y, Dahan I, Gorzalczany Y, Sarfstein R, Hirshberg M, Pick E (2006) Assembly of the phagocyte NADPH oxidase complex: chimeric constructs derived from the cytosolic components as tools for exploring structure–function relationships. J Leukoc Biol 79:881–895PubMedGoogle Scholar
  42. 42.
    Berdichevsky Y, Mizrahi A, Ugolev Y, Molshanski-Mor S, Pick E (2007) Tripartite chimeras comprising functional domains derived from the three cytosolic components p47phox, p67phox and Rac1 elicit activator-independent superoxide production by phagocyte membranes. Role of membrane lipid charge and of specific residues in the chimeras. J Biol Chem 282:22122–22139PubMedGoogle Scholar
  43. 43.
    Pick E, Gorzalczany Y, Engel S (1993) Role of the rac1 p21-GDP-dissociation inhibitor for rho heterodimer in the actvation of the superoxide-forming NADPH oxidase of macrophages. Eur J Biochem 217:441–455PubMedGoogle Scholar
  44. 44.
    Cross AR, Erickson RW, Curnutte JT (1999) 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. Biochem J 341:251–255PubMedGoogle Scholar
  45. 45.
    Brown GE, Stewart MQ, Liu H, Ha V-L, Yaffe MB (2003) A novel assay system implicates PtdIns(3,4)P2, PtdIns(3)P. and PKCδ in intracellular production of reactive oxygen species by the NADPH oxidase. Mol Cell 11:35–47PubMedGoogle Scholar
  46. 46.
    Bissonnette SA, Glazier CM, Stewart MQ, Brown GE, Ellson CE, Yaffe MB (2008) Phosphatidylinositol 3-phosphate-dependent and -independent functions of p40phox in activation of the neutrophil NADPH oxidase. J Biol Chem 283:2108–2119PubMedCentralPubMedGoogle Scholar
  47. 47.
    Koshkin V, Pick E (1993) Generation of superoxide by purified and relipidatred cytochrome b 559 in the absence of cytosolic activators. FEBS Lett 327:57–62PubMedGoogle Scholar
  48. 48.
    Koshkin V, Pick E (1994) Superoxide production by cytochrome b 559. Mechanism of cytosol-independent activation. FEBS Lett 338:285–289PubMedGoogle Scholar
  49. 49.
    Leusen JHW, Meischl C, Eppink MHM, Hilarius PM, de Boer M, Weening RS, Ahlin A, Sanders L, Goldblatt D, Skopczynska H, Bernatowska E, Palmblad J, Verhoeven AJ, van Berkel WJH, Roos D (2000) Four novel mutations in the gene encoding gp91phox of human NADPH oxidase: consequences for oxidase activity. Blood 95:666–673PubMedGoogle Scholar
  50. 50.
    Hata K, Ito T, Takeshige K, Sumimoto H (1998) Anionic amphiphile-independent activation of the phagocyte NADPH oxidase in a cell-free system by p47phox and p67phox, both in C terminally truncated forms. Implications for regulatory Src himology 3 domain-mediated interactions. J Biol Chem 273:4232–4236PubMedGoogle Scholar
  51. 51.
    Peng G, Huang J, Boyd M, Kleinberg ME (2003) Properties of phagocyte NADPH oxidase p47phox mutants with unmasked SH3 (Src homology 3) domains: full reconstitution of oxidase activity in a semi-recombinant cell-free system lacking arachidonic acid. Biochem J 373:221–229PubMedGoogle Scholar
  52. 52.
    Sigal N, Gorzalczany Y, Pick E (2003) Two pathways of activation of the superoxide-generating NADPH oxidase of phagocytes in vitro—Distinctive effects of inhibitors. Inflammation 27:147–159PubMedGoogle Scholar
  53. 53.
    Gorzalczany Y, Alloul N, Sigal N, Weinbaum C, Pick E (2002) A prenylated p67phox-Rac1 chimera elicits NADPH-dependent superoxide production by phagocyte membranes in the absence of an activator and of p47phox. Conversion of a pagan NADPH oxidase to monotheism. J Biol Chem 277:18605–18610PubMedGoogle Scholar
  54. 54.
    Mizrahi A, Berdichevsky Y, Casey PJ, Pick E (2010) A prenylated p47phox-p67phox-Rac1 chimera is a quintessential NADPH oxidase activator. Membrane association and functional capacity. J Biol Chem 285:25485–25499PubMedGoogle Scholar
  55. 55.
    Mizrahi A, Molshanski-Mor S, Weinbaum C, Zheng Y, Hirshberg M, Pick E (2005) Activation of the phagocyte NADPH oxidase by Rac guanine nucleotide exchange factors in conjunction with ATP and nucleoside diphosphate kinase. J Biol Chem 280:3802–3811PubMedGoogle Scholar
  56. 56.
    Pick E (2010) Editorial: When charge is in charge—“Millikan” for leukocyte biologists. J Leukoc Biol 87:537–540PubMedGoogle Scholar
  57. 57.
    Ugolev Y, Molshanski-Mor S, Weinbaum C, Pick E (2006) Liposomes comprising anionic but not neutral phospholipids cause dissociation of [Rac(1 or 2)-RhoGDI] complexes and support amphiphile-independent NADPH oxidase activation by such complexes. J Biol Chem 281:19204–19219PubMedGoogle Scholar
  58. 58.
    Ugolev Y, Berdichevsky Y, Weinbaum C, Pick E (2008) Dissociation of Rac1(GDP)-RhoGDI complexes by the cooperative action of anionic liposomes containing phosphatidylinositol 3,4,5-triphosphate, Rac guanine nucleotide exchange factor, and GTP. J Biol Chem 283(22257–22271):2008Google Scholar
  59. 59.
    DeCoursey TE, Ligeti E (2005) Regulation and termination of NADPH oxidase activity. Cell Mol Life Sci 62:2173–2193PubMedGoogle Scholar
  60. 60.
    van Bruggen R, Anthony E, Fernandez-Borja M, Roos D (2004) Continuous translocation of Rac2 and the NADPH oxidase component p67phox during phagocytosis. J Biol Chem 279:9097–9102PubMedGoogle Scholar
  61. 61.
    Yamaguchi T, Kaneda M, Kakinuma K (1983) Essential requirement of magnesium ion for optimal activity of the NADPH oxidase of guinea pig polymorphonuclear leukocytes. Biochem Biophys Res Comm 115:261–267PubMedGoogle Scholar
  62. 62.
    Tamura M, Takeshita M, Curnutte JT, Uhlinger DJ, Lambeth JD (1992) Stabilization of human neutrophil NADPH oxidase activated in a cell-free system by cytosolic proteins and by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. J Biol Chem 267:7529–7538PubMedGoogle Scholar
  63. 63.
    Knoller S, Shpungin S, Pick E (1991) The membrane-associated component of the amphiphile-activated cytosol-dependent superoxide forming NADPH oxidase of macrophages is identical to cytochrome b 559. J Biol Chem 266:2795–2804PubMedGoogle Scholar
  64. 64.
    Borregaard N, Heiple JM, Simons ER, Clark RA (1983) Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation. J Cell Biol 97:52–61PubMedGoogle Scholar
  65. 65.
    Lundquist H, Follin P, Khalfan L, Dahlgren C (1996) Phorbol myristate acetate-induced NADPH oxidase activity in human neutrophils: only half the story has been told. J Leukoc Biol 59:270–279Google Scholar
  66. 66.
    Clark RA, Leidal KG, Pearson DW, Nauseef WM (1987) NADPH oxidase of human neutrophils. Subcellular localization and characterization of an arachidonate-activable superoxide-generating system. J Biol Chem 262:4065–4074PubMedGoogle Scholar
  67. 67.
    Shpungin S, Dotan I, Abo A, Pick E (1989) Activation of the superoxide forming NADPH oxidase in a cell-free system by sodium dodecyl sulfate. Absolute lipid dependence of the solubilized enzyme. J Biol Chem 264:9195–9203PubMedGoogle Scholar
  68. 68.
    Babior BM, Kipnes RS, Curnutte JT (1973) Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent. J Clin Invest 52:741–744PubMedCentralPubMedGoogle Scholar
  69. 69.
    Csányi G, Cifuentes-Pagano E, Al Gouleh I, Ranayhossaini DJ, Egana L, Lopes LR, Jackson HM, Kelley EE, Pagano PJ (2011) Nox2 B-loop peptide, Nox2ds, specifically inhibits the NADPH oxidase Nox2. Free Rad Biol Med 51:1116–1125PubMedCentralPubMedGoogle Scholar
  70. 70.
    Nisimoto Y, Jackson HM, Ogawa H, Kawahara T, Lambeth JD (2010) Constitutive NADPH-dependent electron transferase activity of the Nox4 dehydrogenase domain. Biochemistry 49:2433–2442PubMedCentralPubMedGoogle Scholar
  71. 71.
    Cross AR, Yarchover JL, Curnutte JT (1994) The superoxide-generating system of human neutrophils possesses a novel diaphorase activity. Evidence for distinct regulation of electron flow within NADPH oxidase by p47phox and p67phox. J Biol Chem 269:21448–21454PubMedGoogle Scholar
  72. 72.
    Han C-H, Nisimoto Y, Lee S-H, Kim ET, Lambeth JD (2001) Characterization of the flavoprotein domain of gp91phox which has NADPH diaphorase activity. J Biochem 129:513–520PubMedGoogle Scholar
  73. 73.
    Nisimoto Y, Ogawa H, Miyano K, Tamura M (2004) Activation of the flavoprotein domain of gp91phox upon interaction with N-terminal p67phox (1-210) and the Rac complex. Biochemistry 43:9567–9575PubMedGoogle Scholar
  74. 74.
    Marques B, Liguori L, Paclet M-H, Villegas-Mendez A, Rothe R, Morel F, Lenormand J-L (2007) Liposome-mediated cellular delivery of active gp91phox. PLoS ONE 2(9):e856. doi: 10.1371/journal.pone.0000856 PubMedCentralPubMedGoogle Scholar
  75. 75.
    Nguyen MVC, Zhang L, Lhomme S, Mouz N, Lenormand J-L, Lardy B, Morel F (2012) Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase acticvity. Biochem Biophys Res Comm 419:453–458PubMedGoogle Scholar
  76. 76.
    Takac I, Schroder K, Zhang L, Lardy B, Anilkumar N, Lambeth JD, Shah AJ, Morel F, Brandes RF (2011) The E-loop is involved in hydrogen peroxide formation by the NADPH oxidase Nox4. J Biol Chem 286:13304–13313PubMedGoogle Scholar
  77. 77.
    Zhou M, Diwu Z, Panchuk-Voloshina N, Haugland RP (1997) A stable nonfluorescent deriavative of resorufin for the fluorometric determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal Biochem 253:162–168PubMedGoogle Scholar
  78. 78.
    Li Y, Zhu H, Kuppusamy P, Roubaud V, Zweier JL, Trush MA (1998) Validation of lucigenin (bis-N-methylacridinium) as a chemilumigenic probe for detecting superoxide anion radical production by enzymatic and cellular systems. J Biol Chem 273:2015–2023PubMedGoogle Scholar
  79. 79.
    Yu L, Quinn MT, Cross AR, Dinauer MC (1998) Gp91phox is the heme binding subunit of the superoxide-generating NADPH oxidase. Proc Natl Acad Sci U S A 95:7993–7998PubMedCentralPubMedGoogle Scholar
  80. 80.
    Sha’ag D (1989) Sodium dodecyl sulphate dependent NADPH oxidation: an alternative method for assaying NADPH-oxidase in a cell-free system. J Biochem Biophys Meth 19:121–128PubMedGoogle Scholar
  81. 81.
    Nishida S, Yoshida LS, Shimoyama T, Nunoi H, Kobayashi T, Tsunawaki S (2005) Fungal metabolite gliotoxin targets flavocytochrome b 558 in the activation of the human neutrophil NADPH oxidase. Infect Immun 73:235–244PubMedCentralPubMedGoogle Scholar
  82. 82.
    Pick E, Bromberg Y, Shpungin S, Gadba R (1987) Activation of the superoxide forming NADPH oxidase in a cell-free system by sodium dodecyl sulfate. Characterization of the membrane-associated component. J Biol Chem 262:16476–16483PubMedGoogle Scholar
  83. 83.
    Tanaka T, Makino R, Iizuka T, Ishimura Y, Kanegasaki S (1988) Activation by saturated and monounsaturated fatty acids of the O2–generating system in a cell-free preparation from neutrophils. J Biol Chem 263:13670–13676PubMedGoogle Scholar
  84. 84.
    Lambeth JD, Krause K-H, Clark RA (2008) NOX enzymes as novel targets for drug development. Semin Immunopathol 30:339–363PubMedGoogle Scholar
  85. 85.
    Jaquet V, Scapozza L, Clark RA, Krause K-H, Lambeth JD (2009) Small-molecule NOX inhibitors: ROS-generating NADPH oxidases as therapeutic targets. Antioxid Redox Signal 11:2535–2552PubMedGoogle Scholar
  86. 86.
    Bechtel W, Richardson R (1993) Discovering complexity: decomposition and localization as strategies in scientific research. Princeton University Press, PrincetonGoogle Scholar
  87. 87.
    Babior BM (1984) Oxidants from phagocytes: agents of defense and destruction. Blood 64:959–966PubMedGoogle Scholar
  88. 88.
    Babior BM, Woodman RC (1990) Chronic granulomatous disease. Semin Hematol 27:247–259PubMedGoogle Scholar
  89. 89.
    Dagher M-C, Pick E (2007) Opening the black box: lessons from cell-free systems on the phagocyte NADPH oxidase. Biochimie 89:1123–1132PubMedGoogle Scholar
  90. 90.
    de Mendez I, Garrett MC, Adams AC, Leto TL (1994) Role of p67phox SH3 domains in assembly of the NADPH oxidase system. J Biol Chem 269:16326–16332PubMedGoogle Scholar
  91. 91.
    Maehara Y, Miyano K, Sumimoto H (2009) Role of the first SH3 domain of p67phox in activation of superoxide-producing NADPH oxidases. Biochem Biophys Res Comm 379:589–593PubMedGoogle Scholar
  92. 92.
    Zahavi A (2013) Elucidation of the domain(s) in the cytosolic NADPH oxidase component p67phox involved in binding to flavocytochrome b 558. Ph.D. Thesis, Tel Aviv UniversityGoogle Scholar
  93. 93.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedGoogle Scholar
  94. 94.
    Light D, Walsh C, O’Callaghan AM, Goetzl EJ, Tauber AI (1981) Characteristics of the cofactor requirements for the superoxide-generating NADPH oxidase of human polymorphonuclear leukocytes. Biochemistry 20:1468–1476PubMedGoogle Scholar
  95. 95.
    Burgess RR (1991) Use of polyethyleneimine in purification of DNA-binding proteins. Meth Enzymol 208:3–10PubMedGoogle Scholar
  96. 96.
    Lapouge K, Groemping Y, Rittinger K (2002) Architecture of the p40-p47-p67phox complex in the resting state of the NADPH oxidase: a central role for p67phox. J Biol Chem 277:10121–10128PubMedGoogle Scholar
  97. 97.
    Knaus UG, Heyworth PG, Kinsella BT, Curnutte JT, Bokoch GM (1992) Purification and characterization of Rac2. A cytosolic GTP-binding protein that regulates human neutrophil NADPH oxidase. J Biol Chem 267:23575–23582PubMedGoogle Scholar
  98. 98.
    Abo A, Pick E, Hall A, Totty N, Teahan CG, Segal AW (1991) Activation of the NADPH oxidase involves the small GTP-binding protein p21 rac 1. Nature 353:668–670PubMedGoogle Scholar
  99. 99.
    Zhao X, Carnevale KA, Cathcart MK (2003) Human monocytes use Rac1, not Rac2, in the NADPH oxidase complex. J Biol Chem 278:40788–40792PubMedGoogle Scholar
  100. 100.
    Kreck ML, Uhlinger DJ, Tyagi SR, Inge KL, Lambeth JD (1994) Participation of the small molecular weight GTP-binding protein Rac1 in cell-free activation and assembly of the respiratory burst oxidase. Inhibition by a carboxyl-terminal Rac peptide. J Biol Chem 269:4161–4168PubMedGoogle Scholar
  101. 101.
    Bromberg Y, Shani E, Joseph G, Gorzalczany Y, Sperling O, Pick E (1994) The GDP-bound form of the small G protein rac1 p21 is a potent activator of the superoxide forming NADPH oxidase of macrophages. J Biol Chem 269:7055–7058PubMedGoogle Scholar
  102. 102.
    Sigal N, Gorzalczany Y, Sarfstein R, Weinbaum C, Zheng Y, Pick E (2003) The guanine nucleotide exchange factor Trio activates the phagocyte NADPH oxidase in the absence of GDP to GTP exchange on Rac. “The emperor’s new clothes”. J Biol Chem 278:4854–4861PubMedGoogle Scholar
  103. 103.
    Xu X, Wang Y, Barry DC, Chanock SJ, Bokoch GM (1997) Guanine nucleotide binding properties of Rac2 mutant proteins and analysis of the responsiveness to guanine nucleotide dissociation stimulator. Biochemistry 36:626–632PubMedGoogle Scholar
  104. 104.
    Bordier C (1981) Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem 256:1604–1607PubMedGoogle Scholar
  105. 105.
    Babior BM, Kuver R, Curnutte JT (1988) Kinetics of activation of the respiratory burst oxidase in a fully soluble system from human neutrophils. J Biol Chem 23:1713–1718Google Scholar
  106. 106.
    Pilloud Dagher M-C, Doussiere J, Vignais PV (1989) Parameters of activation of the membrane-bound O2 ∙−-generating oxidase from neutrophils in a cell-free system. Biochem Biophys Res Comm 159:783–790Google Scholar
  107. 107.
    Cross AR, Erickson RW, Curnutte JT (1999) Simultaneous presence of p47phox and flavocytochrome b -245 are required for activation of NADPH oxidase by anionic amphiphiles. Evidence for an intermediate state of oxidase activation. J Biol Chem 274:15519–15525PubMedGoogle Scholar
  108. 108.
    Pick E, Mizel D (1981) Rapid microassays for the measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassasy reader. J Immunol Methods 46:211–226PubMedGoogle Scholar
  109. 109.
    Pick E (1986) Methods for studying the oxidative metabolism of macrophages. Microassays for O2 ∙− and H2O2 production and NBT reduction using an enzyme immunoassay microplate reader. Meth Enzymol 132:407–421PubMedGoogle Scholar
  110. 110.
    Ligeti E, Doussiere J, Vignais PV (1988) Activation of the O2 ∙−-generating oxidase in plasma membrane from bovine polymorphonuclear neutrophils by arachidonic acid, a cytosolic factor of protein nature, and nonhydrolyzable analogues of GTP. Biochemistry 27:193–200PubMedGoogle Scholar
  111. 111.
    Seifert R, Rosenthal W, Schultz G (1986) Guanine nucleotides stimulate NADPH oxidase in membranes of human neutrophils. FEBS Lett 105:161–165Google Scholar
  112. 112.
    Gabig TG, English D, Akard LP, Schell MJ (1987) Regulation of neutrophil NADPH oxidase activation in a cell-free system by guanine nucleotides and fluoride. Evidence for participation of a pertussis and cholera toxin-insensitive G protein. J Biol Chem 262:1685–1690PubMedGoogle Scholar
  113. 113.
    Aharoni I, Pick E (1990) Activation of the superoxide-generating NADPH oxidase of macrophages by sodium dodecyl sulfate in a soluble cell-free system. Evidence for involvement of a G protein. J Leukoc Biol 48:107–115PubMedGoogle Scholar
  114. 114.
    Lawson CD, Donald S, Anderson KE, Patton DT, Welch HCE (2011) P-Rex1 and Vav1 cooperate in the regulation of formyl-methionyl-leucyl-phenylalanine-dependent neutrophil responses. J Immunol 186:1467–1476PubMedGoogle Scholar
  115. 115.
    Toporik A, Gorzalczany Y, Hirshberg M, Pick E, Lotan O (1998) Mutational analysis of novel effector domains in Rac1 involved in the activation of nicotinamide adenine dinucleotide phosphate (reduced) oxidase. Biochemistry 37:7147–7156PubMedGoogle Scholar
  116. 116.
    Heyworth PG, Knaus UG, Xu X, Uhlinger DJ, Conroy L, Bokoch GM, Curnutte JT (1993) Requirement for posttranslational processing of Rac GTP-binding proteins for activation of human neutrophil NADPH oxidase. Mol Biol Cell 4:261–269PubMedCentralPubMedGoogle Scholar
  117. 117.
    Fuchs A, Dagher M-C, Jouan A, Vignais PV (1994) Activation of the O2 ∙–-generating NADPH oxidase in a semi-recombinant cell-free system. Assessment of the function of Rac in the activation process. Eur J Biochem 226:587–595PubMedGoogle Scholar
  118. 118.
    Li S, Yamauchi A, Marchal CC, Molitoris JK, Quiliam LA, Dinauer MC (2002) Chemoattractant-stimulated Rac activation in wild-type and Rac2-deficient murine neutrophils: preferential activation of Rac2 and Rac2 gene dosage on neutrophil functions. J Immunol 169:5043–5051PubMedGoogle Scholar
  119. 119.
    Glogauer M, Marchal CC, Zhu F, Worku A, Clausen B, Foerster I, Marks P, Downey GP, Dinauer MC, Kwiatkowski DJ (2003) Rac1 deletion in mouse neutrophils has selective effects on neutrophil functions. J Immunol 170:5652–5657PubMedGoogle Scholar
  120. 120.
    Sumimoto H, Hata K, Mizuki K, Ito T, Kage Y, Sakaki Y, Fukumaki Y, Nakamura M, Takeshige K (1996) Assembly and activation of the phagocyte NADPH oxidase. Specific interaction of the N-terminal src homology 3 domain of p47phox with p22phox is required for activation of the NADPH oxidase. Proc Natl Acad Sci U S A 271:22152–22158Google Scholar
  121. 121.
    Maehara Y, Miyano K, Yuzawa S, Akimoto R, Takeya R, Sumimoto H (2010) A conserved region between the TPR and activations domains of p67phox participates in activation of the phagocyte NADPH oxidase. J Biol Chem 285:31435–31445PubMedGoogle Scholar
  122. 122.
    Kwong CH, Adams AG, Leto TL (1995) Characterization of the effector-specifying domain of Rac involved in NADPH oxidase activation. J Biol Chem 270:19868–19872PubMedGoogle Scholar
  123. 123.
    El-Benna J, Dang PM-C, Périanin A (2010) Peptide-based inhibitors of the phagocyte NADPH oxidase. Biochem Pharmacol 80:778–785PubMedGoogle Scholar
  124. 124.
    Dahan I, Pick E (2012) Strategies for identifying synthetic peptides to act as inhibitors of NADPH oxidases, or “All that you did and did not want to know about Nox inhibitory peptides”. Cell Mol Life Sci 69:2283–2305PubMedGoogle Scholar
  125. 125.
    El-Benna J, Dang PM-C, Périanin A (2012) Towards specific NADPH oxidase inhibition by small synthetic peptides. Cell Mol Life Sci 69:2307–2314PubMedGoogle Scholar
  126. 126.
    Joseph G, Pick E (1995) “Peptide walking” is a novel method of mapping functional domains in proteins. Its application to the Rac1-dependent activation of NADPH oxidase. J Biol Chem 270:29079–29082PubMedGoogle Scholar
  127. 127.
    Morozov I, Lotan O, Joseph G, Gorzalczany Y, Pick E (1998) Mapping of functional domains in p47phox in volved in the activation of NADPH oxidase by “peptide walking”. J Biol Chem 273:153435–15444Google Scholar
  128. 128.
    Dahan I, Issaeva I, Gorzalczany Y, Sigal N, Hirschberg M, Pick E (2002) Mapping of functional domains in the p22phox subunit of flavocytochrome b 559 participating in the assembly of the NADPH oxidase complex by “peptide walking”. J Biol Chem 277:8421–8432PubMedGoogle Scholar
  129. 129.
    Dahan I, Molshanski-Mor S, Pick E (2012) Inhibition of NADPH oxidase activation by peptides mapping within the dehydrogenase region of Nox2—A “peptide walking” study. J Leuk Biol 91:501–515Google Scholar
  130. 130.
    Rotrosen D, Kleinberg ME, Nunoi H, Leto T, Gallin JI, Malech HL (1990) Evidence for a functional cytoplasmic domain of phagocyte oxidase cytochrome b 558. J Biol Chem 265:8745–8750PubMedGoogle Scholar
  131. 131.
    Uhlinger DJ, Tyagi SR, Lambeth JD (1995) On the mechanism of inhibition of the neutrophil respiratory burst oxidase by a peptide from the C-terminus of the large subunit of cytochrome b 558. Biochemistry 34:524–527PubMedGoogle Scholar
  132. 132.
    Joseph G, Gorzalczany Y, Koshkin V, Pick E (1994) Inhibition of NADPH oxidase activation by synthetic peptides mapping within the carboxy-terminal domain of small GTP-binding proteins. Lack of amino acid sequence specificity and importance of the polybasic motif. J Biol Chem 269:29024–29031PubMedGoogle Scholar
  133. 133.
    Seifert R, Schultz G (1987) Fatty acid-induced activation of NADPH oxidase in plasma membranes of human neutrophils depends on neutrophil cytosol and is potentiated by stable guanine nucleotides. Eur J Biochem 162:563–569PubMedGoogle Scholar
  134. 134.
    Souabni H, Thoma V, Bizouarn T, Chatgilialoglu C, Siafaka-Kapadai A, Baciou L, Ferreri C, Houée-Levin C, Ostuni MA (2012) trans arachidonic acid isomers inhibit NADPH-oxidase activity by direct interaction with enzyme components. Biochim Biophys Acta 1818:2314–2324PubMedGoogle Scholar
  135. 135.
    Diatchuk V, Lotan O, Koshkin V, Wikstroem P, Pick E (1997) Inhibition of NADPH oxidase activation by 4-(2-aminoethyl)-benzenesulfonyl fluoride and related compounds. J Biol Chem 272:13292–13301PubMedGoogle Scholar
  136. 136.
    Pick E, Gadba R (1988) Certain lymphoid cells contain the membrane-associated component of the phagocyte-specific NADPH oxidase. J Immunol 140:1611–1617PubMedGoogle Scholar
  137. 137.
    Horecker BL, Kornberg A (1948) The extinction coefficients of the reduced band of pyridine nucleotides. J Biol Chem 175:385–390PubMedGoogle Scholar
  138. 138.
    Azzi A, Montecucco C, Richter C (1975) The use of acetylated ferricytochrome c for the detection of superoxide radicals produced in biological membranes. Biochem Biophys Res Comm 65:597–603PubMedGoogle Scholar
  139. 139.
    Yamaguchi T, Kaneda M, Kakinuma K (1986) Effect of saturated and unsaturated fatty acids on the oxidative metabolism of human neutrophils. The role of calcium ion in the extracellular medium. Biochim Biophys Acta 861:440–446PubMedGoogle Scholar
  140. 140.
    Hashida S, Yuzawa S, Suzuki NN, Fujioka Y, Takikawa T, Sumimoto H, Inagaki F, Fujii H (2004) Binding of FAD to cytochrome b 558 is facilitated during activation of the phagocyte NADPH oxidase, leading to superoxide production. J Biol Chem 279:26378–26386PubMedGoogle Scholar
  141. 141.
    Quinn MT, Evans T, Loetterle LR, Jesaitis AJ, Bokoch GM (1993) Translocation of Rac correlates with NADPH oxidase activation. Evidence for equimolar translocation of oxidase components. J Biol Chem 268:20983–20987PubMedGoogle Scholar
  142. 142.
    Molshanski-Mor S, Mizrahi A, Ugolev Y, Dahan I, Berdichevsky Y, Pick E (2007) Cell-free assays. The reductionist approach to the study of NADPH oxidase assembly, or “All you wanted to know about cell-free assays but did not dare to ask”. Methods Mol Biol 412:385–428PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Edgar Pick
    • 1
  1. 1.The Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research and the Ela Kodesz Institute of Host Defense against Infectious DiseasesSackler School of Medicine, Tel Aviv UniversityTel AvivIsrael

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