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Analysis of the Expression and Activity of Nitric Oxide Synthase from Marine Photosynthetic Microorganisms

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1424)

Abstract

Nitric oxide (NO) functions as a signaling molecule in many biological processes in species belonging to all kingdoms of life. In animal cells, NO is synthesized primarily by NO synthase (NOS), an enzyme that catalyze the NADPH-dependent oxidation of l-arginine to NO and l-citrulline. Three NOS isoforms have been identified, the constitutive neuronal NOS (nNOS) and endothelial NOS (eNOS) and one inducible (iNOS). Plant NO synthesis is complex and is a matter of ongoing investigation and debate. Despite evidence of an Arg-dependent pathway for NO synthesis in plants, no plant NOS homologs to animal forms have been identified to date. In plants, there is also evidence for a nitrate-dependent mechanism of NO synthesis, catalyzed by cytosolic nitrate reductase. The existence of a NOS enzyme in the plant kingdom, from the tiny single-celled green alga Ostreococcus tauri was reported in 2010. O. tauri shares a common ancestor with higher plants and is considered to be part of an early diverging class within the green plant lineage.

In this chapter we describe detailed protocols to study the expression and characterization of the enzymatic activity of NOS from O. tauri. The most used methods for the characterization of a canonical NOS are the analysis of spectral properties of the oxyferrous complex in the heme domain, the oxyhemoglobin (oxyHb) and citrulline assays and the NADPH oxidation for in vitro analysis of its activity or the use of fluorescent probes and Griess assay for in vivo NO determination. We further discuss the advantages and drawbacks of each method. Finally, we remark factors associated to the measurement of NOS activity in photosynthetic organisms that can generate misunderstandings in the interpretation of results.

Key words

Nitric oxide Nitric oxide synthase Oxyhemoglobin assay Citrulline detection Griess assay DAF-FM diacetate 

References

  1. 1.
    Muller U (1997) The nitric oxide system in insects. Prog Neurobiol 51(3):363–381CrossRefPubMedGoogle Scholar
  2. 2.
    Klessig DF, Durner J, Noad R, Navarre DA, Wendehenne D, Kumar D, Zhou JM, Shah J, Zhang S, Kachroo P, Trifa Y, Pontier D, Lam E, Silva H (2000) Nitric oxide and salicylic acid signaling in plant defense. Proc Natl Acad Sci U S A 97(16):8849–8855CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Golderer G, Werner ER, Leitner S, Grobner P, Werner-Felmayer G (2001) Nitric oxide synthase is induced in sporulation of Physarum polycephalum. Genes Dev 15(10):1299–1309. doi: 10.1101/gad.890501 Google Scholar
  4. 4.
    Agapie T, Suseno S, Woodward JJ, Stoll S, Britt RD, Marletta MA (2009) NO formation by a catalytically self-sufficient bacterial nitric oxide synthase from Sorangium cellulosum. Proc Natl Acad Sci U S A 106(38):16221–16226. doi: 10.1073/pnas.0908443106 Google Scholar
  5. 5.
    Foresi N, Correa-Aragunde N, Parisi G, Calo G, Salerno G, Lamattina L (2010) Characterization of a nitric oxide synthase from the plant kingdom: NO generation from the green alga Ostreococcus tauri is light irradiance and growth phase dependent. Plant Cell 22(11):3816–3830. doi: 10.1105/tpc.109.073510 Google Scholar
  6. 6.
    Gusarov I, Starodubtseva M, Wang ZQ, McQuade L, Lippard SJ, Stuehr DJ, Nudler E (2008) Bacterial nitric-oxide synthases operate without a dedicated redox partner. J Biol Chem 283(19):13140–13147. doi: 10.1074/jbc.M710178200 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Andreakis N, D’Aniello S, Albalat R, Patti FP, Garcia-Fernàndez J, Procaccini G, Sordino P, Palumbo A (2011) Evolution of the nitric oxide synthase family in metazoans. Mol Biol Evol 28(1):163–179. doi: 10.1093/molbev/msq179 CrossRefPubMedGoogle Scholar
  8. 8.
    Alderton WK, Cooper CE, Knowles RG (2001) Nitric oxide synthases: structure, function and inhibition. Biochem J 357(Pt 3):593–615CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Salerno JC, Harris DE, Irizarry K, Patel B, Morales AJ, Smith SM, Martasek P, Roman LJ, Masters BS, Jones CL, Weissman BA, Lane P, Liu Q, Gross SS (1997) An autoinhibitory control element defines calcium-regulated isoforms of nitric oxide synthase. J Biol Chem 272(47):29769–29777CrossRefPubMedGoogle Scholar
  10. 10.
    Wever RM, van Dam T, van Rijn HJ, de Groot F, Rabelink TJ (1997) Tetrahydrobiopterin regulates superoxide and nitric oxide generation by recombinant endothelial nitric oxide synthase. Biochem Biophys Res Commun 237(2):340–344. doi: 10.1006/bbrc.1997.7069 CrossRefPubMedGoogle Scholar
  11. 11.
    Derelle E, Ferraz C, Rombauts S, Rouze P, Worden AZ, Robbens S, Partensky F, Degroeve S, Echeynie S, Cooke R, Saeys Y, Wuyts J, Jabbari K, Bowler C, Panaud O, Piegu B, Ball SG, Ral JP, Bouget FY, Piganeau G, De Baets B, Picard A, Delseny M, Demaille J, Van de Peer Y, Moreau H (2006) Genome analysis of the smallest free-living eukaryote Ostreococcus tauri unveils many unique features. Proc Natl Acad Sci U S A 103(31):11647–11652. doi: 10.1073/pnas.0604795103 Google Scholar
  12. 12.
    Studier FW (1991) Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J Mol Biol 219(1):37–44CrossRefPubMedGoogle Scholar
  13. 13.
    Sorensen HP, Mortensen KK (2005) Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microb Cell Factories 4(1):1. doi: 10.1186/1475-2859-4-1 Google Scholar
  14. 14.
    Martasek P, Liu Q, Liu J, Roman LJ, Gross SS, Sessa WC, Masters BS (1996) Characterization of bovine endothelial nitric oxide synthase expressed in E. coli. Biochem Biophys Res Commun 219(2):359–365. doi: 10.1006/bbrc.1996.0238 Google Scholar
  15. 15.
    Fossetta JD, Niu XD, Lunn CA, Zavodny PJ, Narula SK, Lundell D (1996) Expression of human inducible nitric oxide synthase in Escherichia coli. FEBS Lett 379(2):135–138Google Scholar
  16. 16.
    Wu C, Zhang J, Abu-Soud H, Ghosh DK, Stuehr DJ (1996) High-level expression of mouse inducible nitric oxide synthase in Escherichia coli requires coexpression with calmodulin. Biochem Biophys Res Commun 222(2):439–444. doi: 10.1006/bbrc.1996.0763 CrossRefPubMedGoogle Scholar
  17. 17.
    Forstermann U, Closs EI, Pollock JS, Nakane M, Schwarz P, Gath I, Kleinert H (1994) Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions. Hypertension 23(6 Pt 2):1121–1131CrossRefPubMedGoogle Scholar
  18. 18.
    Adak S, Aulak KS, Stuehr DJ (2002) Direct evidence for nitric oxide production by a nitric-oxide synthase-like protein from Bacillus subtilis. J Biol Chem 277(18):16167–16171. doi: 10.1074/jbc.M201136200 Google Scholar
  19. 19.
    Wang ZQ, Lawson RJ, Buddha MR, Wei CC, Crane BR, Munro AW, Stuehr DJ (2007) Bacterial flavodoxins support nitric oxide production by Bacillus subtilis nitric-oxide synthase. J Biol Chem 282(4):2196–2202. doi: 10.1074/jbc.M608206200 Google Scholar
  20. 20.
    Ghafourifar P, Asbury ML, Joshi SS, Kincaid ED (2005) Determination of mitochondrial nitric oxide synthase activity. Methods Enzymol 396:424–444. doi: 10.1016/S0076-6879(05)96036-9 CrossRefPubMedGoogle Scholar
  21. 21.
    Rodriguez-Crespo I, Ortiz de Montellano PR (1996) Human endothelial nitric oxide synthase: expression in Escherichia coli, coexpression with calmodulin, and characterization. Arch Biochem Biophys 336(1):151–156Google Scholar
  22. 22.
    Griffith OW, Stuehr DJ (1995) Nitric oxide synthases: properties and catalytic mechanism. Annu Rev Physiol 57:707–736. doi: 10.1146/annurev.ph.57.030195.003423 CrossRefPubMedGoogle Scholar
  23. 23.
    Knowles RG, Salter M (1998) Measurement of NOS activity by conversion of radiolabeled arginine to citrulline using ion-exchange separation. Methods Mol Biol 100:67–73PubMedGoogle Scholar
  24. 24.
    Ward TR, Mundy WR (1999) Measurement of the nitric oxide synthase activity using the citrulline assay. Methods Mol Med 22:157–162. doi: 10.1385/0-89603-612-X:157 PubMedGoogle Scholar
  25. 25.
    Combet S, Balligand JL, Lameire N, Goffin E, Devuyst O (2000) A specific method for measurement of nitric oxide synthase enzymatic activity in peritoneal biopsies. Kidney Int 57(1):332–338. doi: 10.1046/j.1523-1755.2000.00839.x CrossRefPubMedGoogle Scholar
  26. 26.
    Tischner R, Galli M, Heimer YM, Bielefeld S, Okamoto M, Mack A, Crawford NM (2007) Interference with the citrulline-based nitric oxide synthase assay by argininosuccinate lyase activity in Arabidopsis extracts. FEBS J 274(16):4238–4245. doi: 10.1111/j.1742-4658.2007.05950.x CrossRefPubMedGoogle Scholar
  27. 27.
    Xu J, Xu X, Verstraete W (2000) Adaptation of E. coli cell method for micro-scale nitrate measurement with the Griess reaction in culture media. J Microbiol Methods 41(1):23–33CrossRefPubMedGoogle Scholar
  28. 28.
    Verdon CP, Burton BA, Prior RL (1995) Sample pretreatment with nitrate reductase and glucose-6-phosphate dehydrogenase quantitatively reduces nitrate while avoiding interference by NADP+ when the Griess reaction is used to assay for nitrite. Anal Biochem 224(2):502–508. doi: 10.1006/abio.1995.1079 CrossRefPubMedGoogle Scholar
  29. 29.
    Guevara I, Iwanejko J, Dembinska-Kiec A, Pankiewicz J, Wanat A, Anna P, Golabek I, Bartus S, Malczewska-Malec M, Szczudlik A (1998) Determination of nitrite/nitrate in human biological material by the simple Griess reaction. Clin Chim Acta 274(2):177–188CrossRefPubMedGoogle Scholar
  30. 30.
    Arita NO, Cohen MF, Tokuda G et al (2007) Fluorometric detection of nitric oxide with diaminofluoresceins (DAFs): applications and limitations for plant NO research. In: Polaco JC, Lamattina L (eds) Nitric oxide in plant growth, development and stress physiology. Springer, Berlin, pp 269–280CrossRefGoogle Scholar
  31. 31.
    Fiddler RN (1977) Collaborative study of modified AOAC method of analysis for nitrite in meat and meat products. J Assoc Off Anal Chem 60(3):594–599PubMedGoogle Scholar
  32. 32.
    Foissner I, Wendehenne D, Langebartels C, Durner J (2000) In vivo imaging of an elicitor-induced nitric oxide burst in tobacco. Plant J 23(6):817–824Google Scholar
  33. 33.
    Kojima H, Nakatsubo N, Kikuchi K, Kawahara S, Kirino Y, Nagoshi H, Hirata Y, Nagano T (1998) Detection and imaging of nitric oxide with novel fluorescent indicators: diaminofluoresceins. Anal Chem 70(13):2446–2453CrossRefPubMedGoogle Scholar
  34. 34.
    Kojima H, Urano Y, Kikuchi K, Higuchi T, Hirata Y, Nagano T (1999) Fluorescent indicators for imaging nitric oxide production. Angew Chem 38(21):3209–3212CrossRefGoogle Scholar
  35. 35.
    Lim MH, Lippard SJ (2006) Fluorescent nitric oxide detection by copper complexes bearing anthracenyl and dansyl fluorophore ligands. Inorg Chem 45(22):8980–8989. doi: 10.1021/ic0609913 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Instituto de Investigaciones Biológicas, Facultad de Ciencias Exactas y NaturalesUniversidad Nacional de Mar del PlataMar del PlataArgentina
  2. 2.Laboratoire Stress Oxydant et DétoxicationCNRSGif-sur-YvetteFrance
  3. 3.iBiTec-SCEAGif-sur-YvetteFrance

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