Journal of Muscle Research & Cell Motility

, Volume 21, Issue 2, pp 171–181 | Cite as

S-NO-actin: S-nitrosylation kinetics and the effect on isolated vascular smooth muscle

  • Isabella Dalle-donne
  • Aldo Milzani/snm>
  • Daniela Giustarini
  • Paolo Di Simplicio
  • Roberto Colombo
  • Ranieri Rossi


We describe the modification of reactive actin sulfhydryls by S-nitrosoglutathione. Kinetics of S-nitrosylation and denitrosylation suggest that only one cysteine of actin is involved in the reactions. By using the bifunctional sulfhydryl cross-linking reagent N,N′-1,4-phenylenebismaleimide and the monofunctional reagent N-iodoacetyl-N′-(5-sulpho-1-naphthyl)ethylenediamine, we identified this residue as Cys374. The time course of filament formation followed by high-shear viscosity changes revealed that S-nitrosylated G-actin polymerizes less efficiently than native monomers. The observed decrease in specific viscosity at steady state is due mainly to a marked inhibition of filament end-to-end annealing and, partially, to a reduction in F-actin concentration. Finally, S-nitrosylated actin acts as nitric oxide donor showing a fast, potent vasodilating activity at unusually low concentrations, being comparable with that of low molecular weight nitrosothiols.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Arnelle DR and Stamler JS (1995) NO+, NO, and NO-donation by S-nitrosothiols: implications for regulation of physiological functions by S-nitrosylation and acceleration of disulfide formation. Archives Biochem Biophys 318: 279–285.Google Scholar
  2. Bani D, Failli P, Bello MG, Thiemermann C, Bani Sacchi T, Bigazzi M and Masini E (1998) Relaxin activates the L-arginine-nitric oxide pathway in vascular smooth muscle cells in culture. Hypertension 31: 1240–1247.PubMedGoogle Scholar
  3. Becker K, Gui M and Schirmer RH (1995) Inhibition of human glutathione reductase by S-nitrosoglutathione. Eur J Biochem 234: 472–478.PubMedGoogle Scholar
  4. Carlier MF, Pantaloni D and Korn ED (1984) Evidence of an ATP-cap at the end of actin filaments and its regulation of the F-actin steady state. J Biol Chem 259: 9983–9986.PubMedGoogle Scholar
  5. Clancy RM, Levartovsky D, Leszczynska-Piziak J, Yegudin J and Abramson SB (1994) Nitric oxide reacts with intracellular glutathione and activates the hexose monophosphate shunt in human neutrophils: evidence for S-nitrosoglutathione as a bioactive intermediary. Proc Natl Acad Sci USA 91: 3680–3684.PubMedGoogle Scholar
  6. Cooper JA, Walker SB and Pollard TD (1983) Pyrene actin: documentation of the validity of a sensitive assay for actin polymerization. J Muscle Res Cell Motil 4: 253–262.PubMedGoogle Scholar
  7. Dalle-Donne I, Milzani A and Colombo R (1995) H2O2-treated actin: assembly and polymer interactions with cross-linking proteins. Biophys J 69: 2710–2719.PubMedGoogle Scholar
  8. Dalle-Donne I, Milzani A and Colombo R (1999) The t-butyl hydroperoxide-induced oxidation of actin Cys-374 is coupled with structural changes in distant regions of the protein. Biochemistry 38(38): 12471–12480.PubMedGoogle Scholar
  9. Dela-Torre A, Schroeder RA and Kuo PC (1997) Alteration of NF-j B p50 DNA binding kinetics by S-nitrosylation. Biochem Biophys Res Commun 238: 703–706.PubMedGoogle Scholar
  10. Di Simplicio P, Lusini L, Giannerini F, Giustarini D, Bellelli A, Boumis G, Amiconi G and Rossi R (1998) S-nitrosylation of thiol groups in hemoglobins of various species. In: Moncada S, Nisticò G, Baggetta G and Higgs EA (eds). Nitric Oxide and the Cell: Proliferation, Dierentiation and Death. (pp. 47–59). Portland Press, London.Google Scholar
  11. Drewes G and Faulstich H (1990) The enhanced ATPase activity of glutathione-substituted actin provides a quantitative approach to filament stabilization. J Biol Chem 265: 3017–3021.PubMedGoogle Scholar
  12. Elzinga M and Phelan JJ (1984) F-actin is intermolecularly crosslinked by N,N′-p-phenylenedimaleimide through lysine 191 and cysteine 374. Proc Natl Acad Sci USA 81: 6599–6602.PubMedGoogle Scholar
  13. Elzinga M, Collins JH, Kuehl WM and Adelstein RS (1973) Complete amino-acid sequence of actin of rabbit skeletal muscle. Proc Natl Acad Sci USA 70: 2687–2691.PubMedGoogle Scholar
  14. Faulstich H, Merkler I, Blackholm H and Stournaras C (1984) Nucleotide in monomeric actin regulates the reactivity of the thiol groups. Biochemistry 23: 1608–1612.PubMedGoogle Scholar
  15. Frenkel SR, Clancy RM, Ricci JL, Dicesare PE, Rediske JJ and Abramson SB (1996) Effects of nitric oxide on chondrocyte migration, adhesion, and cytoskeletal assembly. Arthritis Rheum 39: 1905–1912.PubMedGoogle Scholar
  16. Frieden C, Lieberman D and Gilbert HR (1980) A fluorescent probe for conformational changes in skeletal muscle G-actin. J Biol Chem 255: 8991–8993.PubMedGoogle Scholar
  17. Furchgott RF (1996) Bioassay with isolated vascular tissue for endo thelium-derived relaxing factor, nitric oxide and nitric oxide donors. In: Feelisch M and Stamler JS (eds). Methods in Nitric Oxide Research. (pp. 567–581). John Wiley and Sons, Chilchester, UK.Google Scholar
  18. Gaston B, Drazen JM, Jansen A, Sugarbaker DA, Loscalzo J, Richards W and Stamler JS (1994) Relaxation of human bronchial smooth muscle by S-nitrosothiols in vitro. J Pharmacol Exp Ther 268: 978–984.PubMedGoogle Scholar
  19. Gordon DJ, Yang YZ and Korn ED (1976) Polymerization of Acanthamoeba actin. Kinetics, thermodynamics and copolymerization with muscle actin. J Biol Chem 251: 7474–7479.PubMedGoogle Scholar
  20. Holmes KC, Popp D, Gebhard W and Kabsch W (1990) Atomic model of the actin filament. Nature 347: 44–49.PubMedGoogle Scholar
  21. Ikkai T, Wahl P and Auchet J-C (1979) Anisotropy decay of labelled actin. Evidence of the flexibility of the peptide chain in F-actin molecules. Eur J Biochem 93: 397–408.PubMedGoogle Scholar
  22. Ji Y, Akerboom T, Sies H and Thomas J (1999) S-nitrosylation and S glutathiolation of protein sulfhydryls by S-nitroso glutathione. Arch Biochem Biophys 362: 67–78.PubMedGoogle Scholar
  23. Jia L, Bonaventura C, Bonaventura J and Stamler JS (1996) S nitrosohaemoglobin: a dynamic activity of blood involved in vascular control. Nature 380: 221–226.PubMedGoogle Scholar
  24. Kawamura M and Maruyama K (1970) Electron microscopic particle length of F-actin polymerized in vitro. J Biochem (Tokyo) 79: 159–171.Google Scholar
  25. Kelm M and Schrader J (1990) Control of coronary vascular tone by nitric oxide. Circ Res 66: 1561–1575.PubMedGoogle Scholar
  26. Kharitonov VG, Sundquist AR and Sharma VS (1995) Kinetics of nitrosation of thiols by nitric oxide in the presence of oxygen. J Biol Chem 270: 28158–28164.PubMedGoogle Scholar
  27. Knight P and Offer G (1978) p-N,N'-Phenylenebismaleimide, a specific cross-linking agent for F-actin. Biochem J 175: 1023–1032.PubMedGoogle Scholar
  28. Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227: 680–685.PubMedGoogle Scholar
  29. Lander HM, Hajjar DP, Hempstead BL, Mirza UA, Chait BT, Campbell S and Quilliam LA (1997) A molecular redox switch on p21(ras). Structural basis for the nitric oxide-p21(ras) interaction. J Biol Chem 272: 4323–4326.PubMedGoogle Scholar
  30. Liu Z, Rudd AM, Freedman JE and Loscalzo J (1998) S-Transnitro sation reactions are involved in the metabolic fate and biological actions of nitric oxide. J Pharmacol Exp Ther 284: 526–534.PubMedGoogle Scholar
  31. Maclean-Fletcher S and Pollard TD (1980) Mechanism of action of cytochalasin B on actin. Cell 20: 329–341.PubMedGoogle Scholar
  32. Mathews WR and Kerr SW (1993) Biological activity of S-nitrosothiols: the role of nitric oxide. J Pharmacol Exp Ther 267: 1529–1537.PubMedGoogle Scholar
  33. Millonig R, Salvo H and Aebi U (1988) Probing actin polymerization by intermolecular cross-linking. J Cell Biol 106: 785–796.PubMedGoogle Scholar
  34. Milzani A and Dalle-Donne I (1999) Effects of chlorpromazine on actin polymerisation: slackening of filament elongation and filament annealing. Arch Biochem Biophys 369: 59–67.PubMedGoogle Scholar
  35. Moncada S, Palmer RMJ and Higgs EA (1991) Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 43: 109–142.PubMedGoogle Scholar
  36. Myers PR, Minor RL Jr, Guerra R Jr, Bates JN and Harrison DG (1990) Vasorelaxant properties of the endothelium-derived relaxing factor more closely resemble S-nitrosocysteine than nitric oxide. Nature 345: 161–163.PubMedGoogle Scholar
  37. Nakaoka Y and Kasai M (1969) Behaviour of sonicated actin polymers: adenosine triphosfate splitting and polymerization. J Mol Biol 44: 319–332.PubMedGoogle Scholar
  38. Ookawa K, Sato M and Ohshima N (1992) Changes in the micro structure of cultured porcine aortic endothelial cells in the early stage after applying a fluid-imposed shear stress. J Biomech 25: 1321–1328.PubMedGoogle Scholar
  39. Pawloski RJ, Swaminathan RV and Stamler JS (1998) Cell-free and erythrocytic S-nitrosohemoglobin inhibits human platelet aggregation. Circulation 97: 263–267.PubMedGoogle Scholar
  40. Radomski MW, Rees DD, Dutra A and Moncada S (1992) S-nitrosoglutathione inhibits platelet activation in vitro and in vivo. Br J Pharmacol 107: 745–749.PubMedGoogle Scholar
  41. Rossi R, Lusini L, Giannerini F, Giustarini D, Lungarella G and Di Simplicio P (1997) A method to study kinetics of transnitrosation with nitrosoglutathione: reactions with hemoglobin and others thiols. Anal Biochem 254: 215–220.PubMedGoogle Scholar
  42. Saville B (1958) A scheme for the colorimetric determination of microgram amounts of thiols. Analyst 83: 670–672.Google Scholar
  43. Simons PC and Vander Jagt DL (1981) Purification of glutathione S-transferases by glutathione-affinity chromatography. Methods Enzymol 77: 235–237.PubMedGoogle Scholar
  44. Singh SP, Wishnok JS, Keshive M, Deen WM and Tannenbaum SR (1996) The chemistry of the S-nitrosoglutathione/glutathione system. Proc Natl Acad Sci USA 93: 14428–14433.CrossRefPubMedGoogle Scholar
  45. Spudich JA and Watt S (1971) The regulation of rabbit skeletal muscle contraction. Biochemical studies on the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem 246: 4866–4871.PubMedGoogle Scholar
  46. Stamler JS, Simon DI, Osborne JA, Mullins ME, Jaraki O, Michel T, Singel DJ and Loscalzo J (1992b) S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds. Proc Natl Acad Sci USA 89: 444–448.PubMedGoogle Scholar
  47. Stamler JS, Jia L, Eu JP, Mcmahon TJ, Demchenko IT, Bonaventura J, Gernert K and Piantadosi CA (1997) Blood flow regulation by S-nitrosohemoglobin in the physiological oxygen gradient. Science 276: 2034–2037.Google Scholar
  48. Stamler JS, Jaraki O, Osborne J, Simon DI, Keaney J, Vita J, Singel D, Valeri CR and Loscalzo J (1992a) Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin. Proc Natl Acad Sci USA 89: 7674–7677.PubMedGoogle Scholar
  49. Steinmetz MO, Goldie KN and Aebi U (1997) A correlative analysis of actin filament assembly, structure and dynamics. J Cell Biol 138: 559–574.PubMedGoogle Scholar
  50. Stournaras C, Drewes G, Blackholm H, Merkler I and Faulstich H (1990) Glutathionyl(cysteine-374) actin forms filaments of low mechanical stability. Biochim Biophys Acta 1037: 86–91.PubMedGoogle Scholar
  51. Tait JF and Frieden C (1982) Chemical modification of actin. Acceleration of polymerization and reduction of network formation by reaction with N-ethylmaleimide, (iodoacetamido)tetramethylrhodamine or 7-chloro-4-nitro-2,1,3-benzoxidiazole. Biochemistry 21: 6046–6053.PubMedGoogle Scholar
  52. Tanford C (1961) Physical chemistry of macromolecules. John Wiley, New York.Google Scholar
  53. Taylor DL, Reidler JA, Spudich JA and Stryer L (1981) Detection of actin assembly by fluorescence energy transfer. J Cell Biol 89: 362–367.PubMedGoogle Scholar
  54. Toyoshima YY, Kron SJ, McNally EM, Niebling KR, Toyoshima C and Spudich JA (1987) Myosin subfragment-1 is sufficient to move actin filaments in vitro. Nature 328: 536–539.PubMedGoogle Scholar
  55. Vandekerckhove J and Weber K (1978) Actin amino acid sequence: comparison of actins from calf thymus, bovine brain, and SV-40 transformed mouse 3T3 cells with rabbit skeletal muscle actin. Eur J Biochem 90: 451–462.PubMedGoogle Scholar
  56. Wang Y-L and Taylor DL (1980) Preparation and characterization of a new molecular cytochemical probe: 5-iodoacetamidofluoresceinlabeled actin. J Histochem Cytochem 28: 1198–1206.PubMedGoogle Scholar
  57. Wechezak AR, Viggers RF and Sauvage LR (1985) Fibronectin and F-actin redistribution in cultured endothelial cells exposed to shear stress. Lab Invest 53: 639–647.PubMedGoogle Scholar
  58. Wegner A and Savko P (1982) Fragmentation of actin filament. Biochemistry 21: 1909–1913.PubMedGoogle Scholar
  59. Wolzt M, Macallister RJ, Davis D, Feelish M, Moncada S, Vallance P and Hobbs AJ (1999) Biochemical characterization of S-nitrosohemoglobin. J Biol Chem 274: 28983–28990.PubMedGoogle Scholar
  60. Xu L, Eu JP, Meissner G and Stamler JS (1998) Activation of the cardiac calcium release channel (ryanodine receptor) by poly-S-nitrosylation. Science 279: 234–237.PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2000

Authors and Affiliations

  • Isabella Dalle-donne
    • 1
  • Aldo Milzani/snm>
    • 1
  • Daniela Giustarini
    • 2
  • Paolo Di Simplicio
    • 2
  • Roberto Colombo
    • 1
  • Ranieri Rossi
    • 2
  1. 1.Lab. Biochem. Biophys. Cytoskel., Department of BiologyUniversity of MilanMilanItaly
  2. 2.Pharmacology Section, Institute for Mental and Nervous DiseasesUniversity of SienaSienaItaly

Personalised recommendations