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. 2000 Jul 1;349(Pt 1):369–375. doi: 10.1042/0264-6021:3490369

Deactivation of neutrophil NADPH oxidase by actin-depolymerizing agents in a cell-free system.

M Tamura 1, M Kanno 1, Y Endo 1
PMCID: PMC1221158  PMID: 10861249

Abstract

The cell-free activation of human neutrophil NADPH oxidase (O(2)(-)-generating enzyme) is enhanced by actin [Morimatsu, Kawagoshi, Yoshida and Tamura (1997) Biochem. Biophys. Res. Commun. 230, 206--210]. In an attempt to elucidate the mechanism, we examined the effect of actin-depolymerizing agents on the duration of NADPH oxidase in a cell-free system. The addition of DNase I, an F-actin-depolymerizing protein, caused an accelerated deactivation of the oxidase. The deactivation was also facilitated by latrunculin A, a sponge toxin that depolymerizes F-actin. Exogenously added actin prevented the deactivation by DNase I or latrunculin A, whereas EDTA accelerated a dilution-induced deactivation of the oxidase and Mg(2+) ions retarded it. The stability in dilution was found to correlate well with free Mg(2+) concentration. Estimation of F-actin in the system showed that F-actin increased during the oxidase activation and that DNase I or EDTA decreased F-actin content in parallel with the activity. Treatment of the cell-free mixture with a chemical cross-linker prevented the deactivation and F-actin decrease by EDTA. Taken together, these results suggest that actin filaments which grow during the activation of NADPH oxidase prolong the lifetime of the oxidase.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aoyagi K., Takeshige K., Sumimoto H., Nunoi H., Minakami S. Role of Mg2+ in activation of NADPH oxidase of human neutrophils: evidence that Mg2+ acts through G-protein. Biochem Biophys Res Commun. 1992 Jul 15;186(1):391–397. doi: 10.1016/s0006-291x(05)80820-4. [DOI] [PubMed] [Google Scholar]
  2. Babior B. M., Kuver R., Curnutte J. T. Kinetics of activation of the respiratory burst oxidase in a fully soluble system from human neutrophils. J Biol Chem. 1988 Feb 5;263(4):1713–1718. [PubMed] [Google Scholar]
  3. Babior B. M. Oxygen-dependent microbial killing by phagocytes (first of two parts). N Engl J Med. 1978 Mar 23;298(12):659–668. doi: 10.1056/NEJM197803232981205. [DOI] [PubMed] [Google Scholar]
  4. Cano M. L., Cassimeris L., Joyce M., Zigmond S. H. Characterization of tetramethylrhodaminyl-phalloidin binding to cellular F-actin. Cell Motil Cytoskeleton. 1992;21(2):147–158. doi: 10.1002/cm.970210208. [DOI] [PubMed] [Google Scholar]
  5. Cassimeris L., McNeill H., Zigmond S. H. Chemoattractant-stimulated polymorphonuclear leukocytes contain two populations of actin filaments that differ in their spatial distributions and relative stabilities. J Cell Biol. 1990 Apr;110(4):1067–1075. doi: 10.1083/jcb.110.4.1067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chuang T. H., Bohl B. P., Bokoch G. M. Biologically active lipids are regulators of Rac.GDI complexation. J Biol Chem. 1993 Dec 15;268(35):26206–26211. [PubMed] [Google Scholar]
  7. Cross A. R., Erickson R. W., Ellis B. A., Curnutte J. T. 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):229–233. [PMC free article] [PubMed] [Google Scholar]
  8. Freeman J. L., Lambeth J. D. NADPH oxidase activity is independent of p47phox in vitro. J Biol Chem. 1996 Sep 13;271(37):22578–22582. doi: 10.1074/jbc.271.37.22578. [DOI] [PubMed] [Google Scholar]
  9. Gabig T. G., English D., Akard L. P., Schell M. J. 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. 1987 Feb 5;262(4):1685–1690. [PubMed] [Google Scholar]
  10. Grogan A., Reeves E., Keep N., Wientjes F., Totty N. F., Burlingame A. L., Hsuan J. J., Segal A. W. Cytosolic phox proteins interact with and regulate the assembly of coronin in neutrophils. J Cell Sci. 1997 Dec;110(Pt 24):3071–3081. doi: 10.1242/jcs.110.24.3071. [DOI] [PubMed] [Google Scholar]
  11. Hall A. Rho GTPases and the actin cytoskeleton. Science. 1998 Jan 23;279(5350):509–514. doi: 10.1126/science.279.5350.509. [DOI] [PubMed] [Google Scholar]
  12. Kabsch W., Mannherz H. G., Suck D., Pai E. F., Holmes K. C. Atomic structure of the actin:DNase I complex. Nature. 1990 Sep 6;347(6288):37–44. doi: 10.1038/347037a0. [DOI] [PubMed] [Google Scholar]
  13. Khaitlina S. Y., Moraczewska J., Strzelecka-Gołaszewska H. The actin/actin interactions involving the N-terminus of the DNase-I-binding loop are crucial for stabilization of the actin filament. Eur J Biochem. 1993 Dec 15;218(3):911–920. doi: 10.1111/j.1432-1033.1993.tb18447.x. [DOI] [PubMed] [Google Scholar]
  14. Knaus U. G., Heyworth P. G., Kinsella B. T., Curnutte J. T., Bokoch G. M. Purification and characterization of Rac 2. A cytosolic GTP-binding protein that regulates human neutrophil NADPH oxidase. J Biol Chem. 1992 Nov 25;267(33):23575–23582. [PubMed] [Google Scholar]
  15. Kwak J. Y., Lopez I., Uhlinger D. J., Ryu S. H., Lambeth J. D. RhoA and a cytosolic 50-kDa factor reconstitute GTP gamma S-dependent phospholipase D activity in human neutrophil subcellular fractions. J Biol Chem. 1995 Nov 10;270(45):27093–27098. doi: 10.1074/jbc.270.45.27093. [DOI] [PubMed] [Google Scholar]
  16. Lamaze C., Fujimoto L. M., Yin H. L., Schmid S. L. The actin cytoskeleton is required for receptor-mediated endocytosis in mammalian cells. J Biol Chem. 1997 Aug 15;272(33):20332–20335. doi: 10.1074/jbc.272.33.20332. [DOI] [PubMed] [Google Scholar]
  17. Morimatsu T., Kawagoshi A., Yoshida K., Tamura M. Actin enhances the activation of human neutrophil NADPH oxidase in a cell-free system. Biochem Biophys Res Commun. 1997 Jan 3;230(1):206–210. doi: 10.1006/bbrc.1996.5881. [DOI] [PubMed] [Google Scholar]
  18. Nakayama S., Tomita T. Depletion of intracellular free Mg2+ in Mg2(+)- and Ca2(+)-free solution in the taenia isolated from guinea-pig caecum. J Physiol. 1990 Feb;421:363–378. doi: 10.1113/jphysiol.1990.sp017949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nauseef W. M., Volpp B. D., McCormick S., Leidal K. G., Clark R. A. Assembly of the neutrophil respiratory burst oxidase. Protein kinase C promotes cytoskeletal and membrane association of cytosolic oxidase components. J Biol Chem. 1991 Mar 25;266(9):5911–5917. [PubMed] [Google Scholar]
  20. Nunoi H., Yamazaki T., Tsuchiya H., Kato S., Malech H. L., Matsuda I., Kanegasaki S. A heterozygous mutation of beta-actin associated with neutrophil dysfunction and recurrent infection. Proc Natl Acad Sci U S A. 1999 Jul 20;96(15):8693–8698. doi: 10.1073/pnas.96.15.8693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ogata K., Nishimoto N., Uhlinger D. J., Igarashi K., Takeshita M., Tamura M. Spermine suppresses the activation of human neutrophil NADPH oxidase in cell-free and semi-recombinant systems. Biochem J. 1996 Jan 15;313(Pt 2):549–554. doi: 10.1042/bj3130549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pick E., Gorzalczany Y., Engel S. Role of the rac1 p21-GDP-dissociation inhibitor for rho heterodimer in the activation of the superoxide-forming NADPH oxidase of macrophages. Eur J Biochem. 1993 Oct 1;217(1):441–455. doi: 10.1111/j.1432-1033.1993.tb18264.x. [DOI] [PubMed] [Google Scholar]
  23. Quinn M. T., Parkos C. A., Jesaitis A. J. The lateral organization of components of the membrane skeleton and superoxide generation in the plasma membrane of stimulated human neutrophils. Biochim Biophys Acta. 1989 Dec 11;987(1):83–94. doi: 10.1016/0005-2736(89)90458-6. [DOI] [PubMed] [Google Scholar]
  24. Redmond T., Tardif M., Zigmond S. H. Induction of actin polymerization in permeabilized neutrophils. Role of ATP. J Biol Chem. 1994 Aug 26;269(34):21657–21663. [PubMed] [Google Scholar]
  25. Rossi F. The O2- -forming NADPH oxidase of the phagocytes: nature, mechanisms of activation and function. Biochim Biophys Acta. 1986 Nov 4;853(1):65–89. doi: 10.1016/0304-4173(86)90005-4. [DOI] [PubMed] [Google Scholar]
  26. Steinmetz M. O., Stoffler D., Hoenger A., Bremer A., Aebi U. Actin: from cell biology to atomic detail. J Struct Biol. 1997 Aug;119(3):295–320. doi: 10.1006/jsbi.1997.3873. [DOI] [PubMed] [Google Scholar]
  27. Sunyer T., Codina J., Birnbaumer L. GTP hydrolysis by pure Ni, the inhibitory regulatory component of adenylyl cyclases. J Biol Chem. 1984 Dec 25;259(24):15447–15451. [PubMed] [Google Scholar]
  28. Tamura M., Takeshita M., Curnutte J. T., Uhlinger D. J., Lambeth J. D. 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. 1992 Apr 15;267(11):7529–7538. [PubMed] [Google Scholar]
  29. Tamura M., Tamura T., Burnham D. N., Uhlinger D. J., Lambeth J. D. Stabilization of the superoxide-generating respiratory burst oxidase of human neutrophil plasma membrane by crosslinking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. Arch Biochem Biophys. 1989 Nov 15;275(1):23–32. doi: 10.1016/0003-9861(89)90345-7. [DOI] [PubMed] [Google Scholar]
  30. Tapon N., Hall A. Rho, Rac and Cdc42 GTPases regulate the organization of the actin cytoskeleton. Curr Opin Cell Biol. 1997 Feb;9(1):86–92. doi: 10.1016/s0955-0674(97)80156-1. [DOI] [PubMed] [Google Scholar]
  31. Thrasher A. J., Keep N. H., Wientjes F., Segal A. W. Chronic granulomatous disease. Biochim Biophys Acta. 1994 Oct 21;1227(1-2):1–24. doi: 10.1016/0925-4439(94)90100-7. [DOI] [PubMed] [Google Scholar]
  32. Wientjes F. B., Segal A. W., Hartwig J. H. Immunoelectron microscopy shows a clustered distribution of NADPH oxidase components in the human neutrophil plasma membrane. J Leukoc Biol. 1997 Mar;61(3):303–312. doi: 10.1002/jlb.61.3.303. [DOI] [PubMed] [Google Scholar]
  33. Woodman R. C., Ruedi J. M., Jesaitis A. J., Okamura N., Quinn M. T., Smith R. M., Curnutte J. T., Babior B. M. Respiratory burst oxidase and three of four oxidase-related polypeptides are associated with the cytoskeleton of human neutrophils. J Clin Invest. 1991 Apr;87(4):1345–1351. doi: 10.1172/JCI115138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yassin R., Shefcyk J., White J. R., Tao W., Volpi M., Molski T. F., Naccache P. H., Feinstein M. B., Sha'afi R. I. Effects of chemotactic factors and other agents on the amounts of actin and a 65,000-mol-wt protein associated with the cytoskeleton of rabbit and human neutrophils. J Cell Biol. 1985 Jul;101(1):182–188. doi: 10.1083/jcb.101.1.182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zigmond S. H., Joyce M., Borleis J., Bokoch G. M., Devreotes P. N. Regulation of actin polymerization in cell-free systems by GTPgammaS and Cdc42. J Cell Biol. 1997 Jul 28;138(2):363–374. doi: 10.1083/jcb.138.2.363. [DOI] [PMC free article] [PubMed] [Google Scholar]

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