N5-(1-Imino-3-butenyl)-l-ornithine
1998; Elsevier BV; Volume: 273; Issue: 15 Linguagem: Inglês
10.1074/jbc.273.15.8882
ISSN1083-351X
AutoresBoga Ramesh Babu, Owen W. Griffith,
Tópico(s)Hemoglobin structure and function
ResumoNitric oxide synthase (NOS) catalyzes the NADPH- and O2-dependent conversion ofl-arginine to nitric oxide (NO) and citrulline; three isoforms, the neuronal (nNOS), endothelial, and inducible, have been identified. Because overproduction of NO is known to contribute to several pathophysiological conditions, NOS inhibitors are of interest as potential therapeutic agents. Inhibitors that are potent, mechanism-based, and relatively selective for the NOS isoform causing pathology are of particular interest. In the present studies we report that vinyl-l-NIO (N5-(1-imino-3-butenyl)-l-ornithine;l-VNIO) binds to and inhibits nNOS in competition with l-arginine (Ki = 100 nm); binding is accompanied by a type I optical difference spectrum consistent with binding near the heme cofactor without interaction as a sixth axial heme ligand. Such binding is fully reversible. However, in the presence of NADPH and O2,l-VNIO irreversibly inactivates nNOS (kinact = 0.078 min−1;KI = 90 nm); inactivation is Ca2+/calmodulin-dependent. The cytochromec reduction activity of the enzyme is not affected by such treatment, but the l-arginine-independent NADPH oxidase activity of nNOS is lost in parallel with the overall activity. Spectral analyses establish that the nNOS heme cofactor is lost or modified by l-VNIO-mediated mechanism-based inactivation of the enzyme. The inducible isoform of NOS is not inactivated byl-VNIO, and the endothelial isoform requires 20-fold higher concentrations to attain ∼75% of the rate of inactivation seen with nNOS. Among the NOS inactivating l-arginine derivatives,l-VNIO is the most potent and nNOS-selective reported to date. Nitric oxide synthase (NOS) catalyzes the NADPH- and O2-dependent conversion ofl-arginine to nitric oxide (NO) and citrulline; three isoforms, the neuronal (nNOS), endothelial, and inducible, have been identified. Because overproduction of NO is known to contribute to several pathophysiological conditions, NOS inhibitors are of interest as potential therapeutic agents. Inhibitors that are potent, mechanism-based, and relatively selective for the NOS isoform causing pathology are of particular interest. In the present studies we report that vinyl-l-NIO (N5-(1-imino-3-butenyl)-l-ornithine;l-VNIO) binds to and inhibits nNOS in competition with l-arginine (Ki = 100 nm); binding is accompanied by a type I optical difference spectrum consistent with binding near the heme cofactor without interaction as a sixth axial heme ligand. Such binding is fully reversible. However, in the presence of NADPH and O2,l-VNIO irreversibly inactivates nNOS (kinact = 0.078 min−1;KI = 90 nm); inactivation is Ca2+/calmodulin-dependent. The cytochromec reduction activity of the enzyme is not affected by such treatment, but the l-arginine-independent NADPH oxidase activity of nNOS is lost in parallel with the overall activity. Spectral analyses establish that the nNOS heme cofactor is lost or modified by l-VNIO-mediated mechanism-based inactivation of the enzyme. The inducible isoform of NOS is not inactivated byl-VNIO, and the endothelial isoform requires 20-fold higher concentrations to attain ∼75% of the rate of inactivation seen with nNOS. Among the NOS inactivating l-arginine derivatives,l-VNIO is the most potent and nNOS-selective reported to date. Nitric oxide synthase (NOS) 1The abbreviations used are: NOS, nitric oxide synthase; nNOS, neuronal (type I) NOS; eNOS, endothelial (type III) NOS; iNOS, inducible (type II) NOS; NO, nitric oxide;l-VNIO, vinyl-l-NIO (i.e. N5-(1-imino-3-butenyl)-l-ornithine);d-VNIO, vinyl-d-NIO (i.e. N5-(1-imino-3-butenyl)-d-ornithine),l-NMA,Nω-methyl-l-arginine;l-NIO, l-NIO,N5-(1-iminoethyl)-l-ornithine; BH4, (6R)-5,6,7,8-tetrahydrobiopterin; methyl-l-NIO,N5-(1-iminopropyl)-l-ornithine; ethyl-l-NIO,N5-(1-iminobutyl)-l-ornithine; FAB, fast atom bombardment; MS, mass spectrometry. catalyzes the oxidation of l-arginine to nitric oxide (NO) and citrulline; NADPH and O2 are cosubstrates (1Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar, 2Stuehr D.J. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 339-359Crossref PubMed Scopus (458) Google Scholar, 3Sessa W.C. J. Vasc. Res. 1994; 31: 131-143Crossref PubMed Scopus (406) Google Scholar). Three major isoforms of NOS have been identified to date. The neuronal (nNOS) and endothelial (eNOS) isoforms are constitutively expressed and are regulated by Ca2+/calmodulin, whereas the activity of the inducible isoform (iNOS) is controlled transcriptionally and is not affected by changes in intracellular Ca2+. Although amino acid sequence homology among the isoforms is limited (∼50%) (3Sessa W.C. J. Vasc. Res. 1994; 31: 131-143Crossref PubMed Scopus (406) Google Scholar), all are comprised of a C-terminal reductase domain that binds NADPH and the cofactors FAD and FMN and a N-terminal oxygenase domain that bindsl-arginine and the heme and tetrahydrobiopterin (BH4) cofactors (1Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar, 2Stuehr D.J. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 339-359Crossref PubMed Scopus (458) Google Scholar, 3Sessa W.C. J. Vasc. Res. 1994; 31: 131-143Crossref PubMed Scopus (406) Google Scholar). The reductase domain is related in function and amino acid sequence to cytochrome P450 reductase (4Bredt D.S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 682-685Crossref PubMed Scopus (3211) Google Scholar), whereas the oxygenase domain is related in function (but not sequence) to the cytochromes P450. Binding of Ca2+/calmodulin to a region between the domains permits electron flow from the reductase domain to the oxygenase domain and also stimulates electron flow within the reductase domain (5Abu-Soud H.M. Yoho L.L. Stuehr D.J. J. Biol. Chem. 1994; 269: 32047-32050Abstract Full Text PDF PubMed Google Scholar). The resulting reduction of the heme cofactor allows O2 to be activated, permitting the cytochrome P450-like oxidation of l-arginine to Nω-hydroxy-l-arginine and the subsequent further oxidation of that tightly bound intermediate to citrulline and NO (1Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar, 2Stuehr D.J. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 339-359Crossref PubMed Scopus (458) Google Scholar, 6Stuehr D.J. Kwon N.S. Nathan C.F. Griffith O.W. Feldman P.L. Wiseman J. J. Biol. Chem. 1991; 266: 6259-6263Abstract Full Text PDF PubMed Google Scholar). Nitric oxide synthase-derived NO is important in many physiological processes including blood pressure homeostasis (7Aisaka K. Gross S.S. Griffith O.W. Levi R. Biochem. Biophys. Res. Commun. 1989; 160: 881-886Crossref PubMed Scopus (417) Google Scholar, 8Rees D.D. Palmer R.M.J. Moncada S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3375-3378Crossref PubMed Scopus (1695) Google Scholar, 9Umans J.G. Levi R. Annu. Rev. Physiol. 1995; 57: 771-790Crossref PubMed Scopus (255) Google Scholar), neurotransmission (10Rand M.J. Li C.G. Annu. Rev. Physiol. 1995; 57: 659-682Crossref PubMed Scopus (292) Google Scholar, 11Garthwaite J. Boulton C.L. Annu. Rev. Physiol. 1995; 57: 683-706Crossref PubMed Scopus (1547) Google Scholar), and immune function (12Nathan C.F. Hibbs Jr., J.B. Curr. Opin. Immunol. 1991; 3: 65-70Crossref PubMed Scopus (1358) Google Scholar), but overproduction of NO can have pathological consequences (13Gross S.S. Wolin M.S. Annu. Rev. Physiol. 1995; 57: 737-769Crossref PubMed Scopus (825) Google Scholar). For example, excess NO resulting from overexpression of iNOS in response to endotoxin or inflammatory cytokines is a major contributor to the vascular disregulation seen in septic shock (14Kilbourn R.G. Griffith O.W. J. Natl. Cancer Inst. 1992; 84: 827-831Crossref PubMed Scopus (265) Google Scholar, 15Kilbourn R.G. Jubran A. Gross S.S. Griffith O.W. levi R. Adams J. Lodato R.F. Biochem. Biophys. Res. Commun. 1990; 172: 1132-1138Crossref PubMed Scopus (571) Google Scholar) and in patients receiving interleukin-2-based immunotherapy (16Kilbourn R.G. Owen-Schaub L.B. Cromeens D.M. Gross S.S. Flaherty M.J. Sante S.M. Alak A.M. Griffith O.W. J. Appl. Physiol. 1994; 76: 1130-1137Crossref PubMed Scopus (43) Google Scholar, 17Kilbourn R.G. Fonseca G.A. Griffith O.W. Ewer M. Price K. Striegel A. Jones E. Logothetis C.J. Crit. Care Med. 1995; 23: 1018-1024Crossref PubMed Scopus (59) Google Scholar). Inappropriate activation of nNOS is implicated in chronic visceral pain (18Meller S.T. Gebhart G.F. Pain. 1993; 52: 127-136Abstract Full Text PDF PubMed Scopus (857) Google Scholar, 19Rice A.S.C. Neurosci. Lett. 1995; 187: 111-114Crossref PubMed Scopus (50) Google Scholar), in migraine headache (20Lassen L.H. Ashina M. Christiansen I. Ulrich V. Olesen J. Lancet. 1997; 349: 401-402Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar), and in several neurodegenerative diseases (e.g. Parkinson's disease (21Przedborski S. Jackson-Lewis V. Yokoyama R. Shibata T. Dawson V.L. Dawson T.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4565-4571Crossref PubMed Scopus (597) Google Scholar, 22Hantraye P. Brouillet E. Ferrante R. Palfi S. Dolan R. Matthews R.T. Beal M.F. Nat. Med. 1996; 2: 1017-1021Crossref PubMed Scopus (396) Google Scholar)), and is thought to contribute to post-ischemic reperfusion injury in stroke (23Nishikawa T. Hirsch J.R. Koehler R.C. Bredt D.S. Snyder S.H. Traystman R.J. Stroke. 1993; 24: 1717-1724Crossref PubMed Scopus (111) Google Scholar). Appreciation of the pathological roles of NOS-derived NO has stimulated interest in the design and synthesis of NOS inhibitors for possible therapeutic use in disorders associated with overproduction of NO (24Griffith O.W. Gross S.S. Feelisch M. Stamler J.S. Methods in Nitric Oxide Research. John Wiley & Sons, New York1996: 187-208Google Scholar,25Marletta M.A. J. Med. Chem. 1994; 37: 1899-1907Crossref PubMed Scopus (180) Google Scholar). To be pharmacologically useful, compounds should be well transported into the target tissue, strongly inhibitory, NOS isoform-selective, and chemically stable under biological conditions. In attempting to meet these goals, l-arginine analogs are particularly attractive inhibitor candidates because they are effectively transported by the ubiquitous system y+ amino acid transporter (26Baydoun A.R. Mann G.E. Biochem. Biophys. Res. Commun. 1994; 200: 726-731Crossref PubMed Scopus (74) Google Scholar, 27McDonald K.K. Rouhani R. Handlogten M.E. Block E.R. Griffith O.W. Allison R.D. Kilberg M.S. Biochim. Biophys. Acta. 1997; 1324: 133-141Crossref PubMed Scopus (27) Google Scholar) and thus show good activity in vivo.Nω-Methyl-l-arginine (l-NMA), the prototypic NOS inhibitor (28Hibbs Jr., J.B. Vavrin Z. Taintor R.R. J. Immunol. 1987; 138: 550-565PubMed Google Scholar), has been shown, for example, to reverse the hypotension of septic shock (14Kilbourn R.G. Griffith O.W. J. Natl. Cancer Inst. 1992; 84: 827-831Crossref PubMed Scopus (265) Google Scholar, 15Kilbourn R.G. Jubran A. Gross S.S. Griffith O.W. levi R. Adams J. Lodato R.F. Biochem. Biophys. Res. Commun. 1990; 172: 1132-1138Crossref PubMed Scopus (571) Google Scholar, 29Vallance P. New Horizons. 1993; 1: 77-86PubMed Google Scholar) and cytokine-induced shock (17Kilbourn R.G. Fonseca G.A. Griffith O.W. Ewer M. Price K. Striegel A. Jones E. Logothetis C.J. Crit. Care Med. 1995; 23: 1018-1024Crossref PubMed Scopus (59) Google Scholar, 30Kilbourn R.G. Gross S.S. Adams J. Jubran A. Griffith O.W. Levi R. Lodato R.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3629-3632Crossref PubMed Scopus (611) Google Scholar, 31Kilbourn R.G. Gross S.S. Lodato R.F. Adams J. Levi R. Miller L.L. Lachman L.B. Griffith O.W. J. Natl. Cancer Inst. 1992; 84: 1008-1016Crossref PubMed Scopus (69) Google Scholar) in animal studies and in early clinical trials. Unfortunately, NMA and several otherl-arginine analogs includingNω-amino-l-arginine (32Fukuto J.M. Wood K.S. Byrns R.E. Ignarro L.J. Biochem. Biophys. Res. Commun. 1990; 168: 458-465Crossref PubMed Scopus (82) Google Scholar, 33Gross S.S. Stuehr D.J. Aisaka K. Jaffe E.A. Levi R. Griffith O.W. Biochem. Biophys. Res. Commun. 1990; 170: 96-103Crossref PubMed Scopus (271) Google Scholar, 34Wolff D.J. Lubeskie A. Arch. Biochem. Biophys. 1996; 325: 227-234Crossref PubMed Scopus (30) Google Scholar) and Nω-(1-iminoethyl)-l-ornithine (l-NIO) (35McCall T.B. Feelisch M. Palmer R.M. Moncada S. Br. J. Pharmacol. 1991; 102: 234-238Crossref PubMed Scopus (247) Google Scholar, 36Moore W.M. Webber R.K. Jerome G.M. Tjoeng F.S. Misk T.P. Currie M.G. J. Med. Chem. 1994; 37: 3886-3888Crossref PubMed Scopus (420) Google Scholar) show little isoform selectivity; their pharmacological use may thus cause undesirable inhibition of physiological processes controlled by nontargeted NOS isoforms. We (37Narayanan K. Griffith O.W. J. Med. Chem. 1994; 37: 885-887Crossref PubMed Scopus (116) Google Scholar,38Narayanan K. Spack L. McMillan K. Kilbourn R.G. Hayward M.A. Masters B.S.S. Griffith O.W. J. Biol. Chem. 1995; 270: 11103-11110Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) and others (39Furfine E.S. Harmon M.F. Paith J.E. Knowles R.G. Salter M. Kiff R.J. Duffy C. Hazelwood R. Oplinger J.A. Garvey E.P. J. Biol. Chem. 1994; 269: 26677-26683Abstract Full Text PDF PubMed Google Scholar) have shown that theS-alkyl-l-thiocitrullines show modest selectivity (up to 50-fold) for nNOS over eNOS and iNOS and have proposed that these compounds may be of use in treating disorders involving overstimulation of nNOS (e.g. stroke). Improved potency and isoform selectivity would, however, be highly desirable. In the present studies, we have examined several novell-arginine antagonists and find that vinyl-l-NIO (N5-(1-imino-3-butenyl)-l-ornithine,l-VNIO) (Fig. 1) is a potent, mechanism-based inhibitor that attacks the heme cofactor of NOS. It shows a marked selectivity for nNOS. Abstracts describing this work have been published (40Babu B.R. Griffith O.W. FASEB J. 1997; 11: A884Google Scholar, 41Griffith O.W. Babu B.R. Jpn. J. Pharmacol. 1997; 75: 3PCrossref Google Scholar). Most biochemicals and reagents for organic syntheses were obtained from Sigma and Aldrich, respectively.Nω-Methyl-l-arginine and BH4 were purchased from Chemical Dynamics (Plainfield, NJ) and Alexis (La Jolla, CA), respectively.l-[14C]Arginine was from NEN Life Science Products.Nω-Alkyl-l-arginines (42Corbin J.L. Reporter M. Anal. Biochem. 1974; 57: 310-312Crossref PubMed Scopus (65) Google Scholar) and l-NIO (43Scannell J.P. Ax H.A. Pruess D.L. Williams T. Demny T.C. Stempel A. J. Antibiot. 1972; 25: 179-184Crossref PubMed Scopus (29) Google Scholar) were prepared by the general methods indicated. Rat nNOS was isolated from stably transfected kidney 293 cells (44Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2240) Google Scholar) as described previously (45McMillan K. Bredt D.S. Hirsch D.J. Snyder S.H. Clark J.E. Masters B.S.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11141-11145Crossref PubMed Scopus (373) Google Scholar). Bovine eNOS (46Liu J. Garcia-Cardena G. Sessa W.C. Biochemistry. 1996; 35: 13277-13281Crossref PubMed Scopus (204) Google Scholar) and mouse iNOS expressed in Escherichia coli 2L. J. Roman and B. S. S. Masters, unpublished results. were generous gifts from Dr. Kirkwood Pritchard (Department of Pathology, Medical College of Wisconsin, Milwaukee, WI) and Dr. Bettie S. S. Masters (Department of Biochemistry, University of Texas Health Sciences Center, San Antonio, TX), respectively. 1H and13C NMR spectra were obtained using a Bruker AC 300 MHz spectrometer. FAB mass spectral analyses were generously carried out by Dr. Frank Laib at the Department of Chemistry, University of Wisconsin, Milwaukee. d- or l-Ornithine HCl (1.68 g, 10 mmol) and cupric acetate (0.5 g, 10 mmol) were dissolved in water (20 ml) and stirred for 10 min at room temperature. The solution was then filtered to remove minor impurities and cooled to 0 °C, and the pH was adjusted to 9.5 by addition of cold 10% NaOH. Alkyl or alkenyl imidate (15 mmol), prepared separately from the corresponding nitrile and HCl(g) (43Scannell J.P. Ax H.A. Pruess D.L. Williams T. Demny T.C. Stempel A. J. Antibiot. 1972; 25: 179-184Crossref PubMed Scopus (29) Google Scholar) was then added, and the mixture was allowed to stir at pH 9.0–9.5 for 1 h at 0 °C and for 2 h at room temperature. The pH was then adjusted to 7.4 with cold dilute HCl, and the mixture was stirred at room temperature overnight. Hydrogen sulfide gas (caution: toxic) was bubbled through the solution, and the resulting copper sulfide precipitate was removed by filtration through charcoal. The filtrate was passed through Chelex to remove any residual Cu2+, and the clear solution was evaporated to dryness by rotary evaporation under reduced pressure. The residue was washed with ethyl acetate, and the product was crystallized from ethanol to give pureN5-(1-iminoalkyl)ornithine orN5-(1-iminoalkenyl)ornithine. m.p. 162 °C (dec); 1H NMR (D2O): δ 1.65–2.1 (m, 4H), 3.3 (d, 2H), 3.4(t, 2H), 3.8 (t, 1H), 5.4 (m, 2H), and 5.95 (m, 1H);13C NMR (D2O): δ 25.43, 30.35, 39.37, 44.28, 56.93, 124.16, 131.55, 168.72, and 176.98; FABMS: m/e 200 (M + H). m.p. 170 °C (dec), 1H NMR (D2O): δ 1.6–2.1 (m, 4H), 3.3 (d, 2H), 3.4(t, 2H), 3.8 (t, 1H), 5.4 (m, 2H), and 5.89 (m, 1H);13C NMR (D2O): δ 25.43, 30.35, 39.37, 44.27, 56.93, 124.13, 131.55, 168.72, and 176.98; FABMS: m/e 200 (M + H). m.p. 150 °C. (dec), 1H NMR (D2O): δ 1.29 (t, 3H), 1.6–1.9 (m, 4H), 2.54 (q, 2H), 3.38 (t, 2H), and 3.84 (t, 1H); 13C NMR (D2O): δ 13.37, 25.43, 28.10, 30.35, 44.09, 56.94, 171.97, and 177.03; FABMS: m/e 188 (M + H). m.p. 145 °C (dec), 1H NMR (D2O): δ 1.01 (t, 3H), 1.65–2.1 (m, 6H), 2.5 (t, 2H), 3.4(t, 2H), and 3.8 (t, 1H); 13C NMR (D2O): δ 15.06, 22.55, 25.50, 30.44, 37.16, 44.12, 56.97, 170.78, and 177.02; FABMS: m/e 202 (M + H). Activity of NOS was determined by monitoring the conversion ofl-[14C]arginine tol-[14C]citrulline. Reaction mixtures contained in a final volume of 50 μl, 50 mmNa+ Hepes buffer, pH 7.4, 100 μm EDTA, 0.2 mm CaCl2, 10 μg/ml calmodulin, 100 μm dithiothreitol, 50 μm BH4, 1.0 μm FAD, 1.0 μm FMN, 100 μg/ml bovine serum albumin, 500 μm NADPH,l-[14C]arginine as indicated,l-VNIO as indicated, and nNOS or eNOS. Reaction mixtures for iNOS were similar, but CaCl2 and calmodulin were omitted. Reaction was initiated by the addition of enzyme, and the solutions were maintained at 25 °C for 4 min. Reaction mixtures were quenched by the addition of 200 μl of stop buffer containing 100 mm Na+ Hepes buffer, pH 5.5, and 5 mm EGTA. Those samples were heated in a boiling water bath for 1 min, chilled and centrifuged. A portion (225 μl) of the supernatant was applied to small Dowex 50 columns (Na+form, 1 ml resin), and the productl-[14C]citrulline was eluted with 2 ml of water and quantitated by liquid scintillation counting. Studies were carried out on a Shimadzu model 2501 dual beam UV-visible spectrophotometer using either nNOS as isolated (∼20% low spin heme) or nNOS pretreated with imidazole (100% low spin heme). In the former case, 0.5 ml of nNOS as isolated (432 μg) in 50 mm Tris-HCl buffer, pH 7.5, 10% glycerol, and 0.1 mm EDTA was placed in the sample and reference cuvettes at 15 °C, and the base-line spectrum was adjusted to zero. Sequential samples of buffer and l-VNIO in buffer were added to the reference and sample cuvettes, respectively, and optical difference spectra at increasing concentrations ofl-VNIO were obtained. Similar studies using imidazole in place of l-VNIO were carried out to determine theKs for that ligand ( KsImid = 86.2 μm, data not shown). The effect of l-VNIO on imidazole liganded nNOS was then determined by initially adding 1.0 mmimidazole to the cuvettes, setting the base line to zero, and then adding sequential samples of buffer and l-VNIO in buffer to the reference and sample cuvettes, respectively, as described above. For these studies KsL-VNIO was calculated from the relationship Ks (app)L-VNIO = KsL-VNIO (1 + [Imid]/ KsImid), where KsL-VNIO is the true binding constant for l-VNIO, Ks (app)L-VNIO is the apparent binding constant for l-VNIO determined in the presence of imidazole, [Imid] is the concentration of imidazole (1.0 mm) and KsImid is the binding constant for imidazole as determined in the preliminary study mentioned (i.e. 86.2 μm). Time- and concentration-dependent inactivation kinetics for nNOS and eNOS treated with various inhibitors were determined at 25 °C in reaction mixtures (final volume = 0.15 ml) containing 50 mm Na+ Hepes buffer, pH 7.4, 0.1 mmEDTA, 50 μm BH4, 2.0 mmglutathione, 1.0 μm FAD, 1.0 μm FMN, 1 mg/ml bovine serum albumin, 100 units of superoxide dismutase, 0.2 mm CaCl2, 10 μg/ml calmodulin, 1.0 mm NADPH, l-arginine or inhibitor as indicated, and ∼40 μg NOS. Residual activity was determined after various time intervals by adding a 25-μl aliquot of the reaction mixture to a cuvette containing, in a final volume of 0.5 ml, 50 mmHepes buffer, pH 7.4, 0.1 mm EDTA, 50 μmBH4, 10 μg/ml calmodulin, 0.2 mmCaCl2, 0.1 mm GSH, 1.0 μm FAD, 1.0 μm FMN, 1 mg/ml bovine serum albumin, 0.5 mm NADPH, and 0.25 mm of l-arginine and 5 μm bovine oxyhemoglobin (prepared by reduction with sodium dithionite followed by gel filtration). Reaction mixtures for iNOS were similar but lacked CaCl2 and calmodulin. Nitric oxide-mediated oxidation of oxyhemoglobin was monitored at 401 nm (ε = 0.038 μm−1) (47Feelisch M. Noack E.A. Eur. J. Pharmacol. 1987; 139: 19-30Crossref PubMed Scopus (860) Google Scholar); the reference cuvette contained a similar mixture without enzyme. The rate of NO formation was determined and used to calculate the residual activity. The ability of carbon monoxide (CO) to bind to the reduced heme cofactor of NOS and elicit a characteristic absorption maxima at 443 nm was used to determine the loss of heme cofactor after inactivation of nNOS by l-VNIO. The incubation conditions were similar to those used to determine irreversible inactivation except the final volume was 1.8 ml. After specific time intervals (0, 10, and 20 min), an aliquot (200 μl) of the inactivation reaction mixture was added to both sample and reference cuvettes containing 50 mm Na+ Hepes buffer, pH 7.4, and 10% glycerol to give final volume of 0.5 ml. The buffer in the sample cuvette was then saturated with CO, and the difference spectrum between 400 and 500 nm was determined (48Omura T. Sato R. J. Biol. Chem. 1964; 239: 2370-2378Abstract Full Text PDF PubMed Google Scholar). Reaction mixtures of 500 μl contained 50 mm Na+ Hepes buffer, pH 7.4, 100 μm EDTA, 50 μm NADPH, 50 μmbovine heart cytochrome c, and nNOS. NADPH-dependent reduction of cytochrome c was monitored at 550 nm (ε = 0.021 μm−1). Where indicated 10 μml-VNIO, 0.2 mm CaCl2, 10 μg/ml calmodulin, and/or 800 units/ml superoxide dismutase were added to the reaction mixtures. The rate of NADPH oxidation by NOS was determined spectrophotometrically by monitoring the decrease in absorbance at 340 nm with time (ε = 6.22 mm−1). The reaction mixtures contained in a final volume of 0.5 ml 50 mm Na+ Hepes buffer, pH 7.4, 0.1 mm EDTA, 50 μm BH4, 2.0 mm GSH, 1.0 μm FAD, 1.0 μmFMN, 1 mg/ml bovine serum albumin, 0.2 mmCaCl2, 10 μg/ml calmodulin, 0.25 mm NADPH, where indicated 10 μml-VNIO, and nNOS. NADPH oxidation was initiated by addition of enzyme. All NOS isoforms are inhibited by a variety ofl-arginine analogs that compete with l-arginine for the amino acid binding site; we have reported previously thatNω-alkyl-l-arginines withn-alkyl substituents up to 4 carbons and the isostericN5-(1-iminoalkyl)-l-ornithines are good to excellent inhibitors (24Griffith O.W. Gross S.S. Feelisch M. Stamler J.S. Methods in Nitric Oxide Research. John Wiley & Sons, New York1996: 187-208Google Scholar). Consistent with these findings, in initial rate studies l-VNIO was found to be a potent inhibitor of nNOS, and its binding was competitive withl-arginine (Fig. 2). A replot of the data shows that KiL-VNIOis ∼100 nm (Fig. 2, inset); this value is substantially lower than the Km forl-arginine (1.4 μm). Similar studies showedl-VNIO also inhibits eNOS and iNOS competitively with respect to l-arginine, but the Ki values are much higher (i.e. 12.0 μm for eNOS and 60 μm for iNOS) (Table I). Note that the KiL-VNIO/ KmL-Argratios for nNOS, eNOS, and iNOS are 0.07, 3.33 and 4.80, respectively, indicating that l-VNIO competes for thel-arginine binding site of nNOS particularly well (TableI). d-Arginine is neither a substrate nor an inhibitor of NOS (1Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar), and none of the NOS isoforms was inhibited byd-VNIO when tested at 100 μm in the presence of 20 μml-arginine (data not shown).Table IInhibition of NOS isoforms by l-VNIO and related N5-(1-iminoalkyl)-l-ornithinesInhibitor or substrateKinetic constants and ratiosnNOSeNOSiNOSKiKi/KmKiKi/KmKiKi/Kmμmμmμml-VNIO0.100.0712.03.33604.80l-NIO1.71.213.91.083.90.31Methyl-l-NIO3.02.1410.02.789.50.76Ethyl-l-NIO5.33.7918.05.0012.00.96l-Arginine (Km)1.43.612.5 Open table in a new tab The isoform selectivity exhibited by l-VNIO is mirrored to a degree by structurally related l-arginine analogs (Fig. 1). As shown in Table I, methyl-l-NIO and ethyl-l-NIO (the saturated analog of l-VNIO) are also more potent inhibitors of nNOS than of eNOS or iNOS. However, expressed on a Ki/Km basis, neither of these inhibitors shows biologically significant selectivity for nNOS over eNOS; they, in fact, show a very modest selectivity (3–5-fold) for iNOS over the constitutive isoforms. The prototypicN5-(1-iminoalkyl)-l-ornithine inhibitor, l-NIO, also shows biologically insignificant isoform selectivity (Table I). A variety of studies indicate that the reactive guanidinium nitrogen of substrate l-arginine is bound near the iron of the NOS heme cofactor;Nω-hydroxy-l-arginine is then formed when that guanidinium nitrogen is oxidized by O2, which has been bound and activated as a sixth, axial heme iron ligand (1Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar, 2Stuehr D.J. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 339-359Crossref PubMed Scopus (458) Google Scholar). Although l-arginine does not bind near enough to heme iron to act as a sixth axial ligand (49McMillan K. Masters B.S.S. Biochemistry. 1993; 32: 9875-9880Crossref PubMed Scopus (169) Google Scholar), other inhibitors such as l-thiocitrulline do covalently interact with heme iron (24Griffith O.W. Gross S.S. Feelisch M. Stamler J.S. Methods in Nitric Oxide Research. John Wiley & Sons, New York1996: 187-208Google Scholar, 50Frey C. Narayanan K. McMillan K. Spack L. Gross S.S. Masters B.S.S. Griffith O.W. J. Biol. Chem. 1994; 269: 26083-26091Abstract Full Text PDF PubMed Google Scholar, 51Sennequier N. Stuehr D.J. Biochemistry. 1996; 35: 5883-5892Crossref PubMed Scopus (61) Google Scholar). Such interactions can be revealed by optical difference spectroscopy in which perturbations of the heme spectrum caused by substrates or inhibitors are determined (49McMillan K. Masters B.S.S. Biochemistry. 1993; 32: 9875-9880Crossref PubMed Scopus (169) Google Scholar). As shown in Fig. 3 A, increasing concentrations of l-VNIO, when added to solutions of native nNOS, cause a type I difference spectrum. This result, which is similar to that seen with l-arginine, indicates thatl-VNIO does not interact covalently with heme iron but does bind sufficiently close to its sixth axial position to displace an endogenous heme ligand (the identity of the ligand displaced is presently unknown). The displacement of the endogenous ligand, which is present in ∼20% of nNOS as isolated (49McMillan K. Masters B.S.S. Biochemistry. 1993; 32: 9875-9880Crossref PubMed Scopus (169) Google Scholar), is responsible for the spectral change shown in Fig. 3 A. Fig. 3 B is a replot of the data in Fig. 3 A showing that thel-VNIO dissociation constant, KsL-VNIO, is 1.1 μm; the previously reported value for KsL-Arg is 2.5 μm(49McMillan K. Masters B.S.S. Biochemistry. 1993; 32: 9875-9880Crossref PubMed Scopus (169) Google Scholar). We also determined KsL-VNIOusing imidazole-saturated nNOS. These studies confirmed thatl-VNIO gives a type I optical difference spectrum and provide a potentially more accurate estimate of KsL-VNIO since the signal is larger (i.e. heme iron is initially 100% low spin) and the amounts of l-VNIO added are larger, making it unnecessary to correct for l-VNIO bound to nNOS. With imidazole saturated nNOS, KsL-VNIO, calculated as described under "Methods," was 1.4 μm(data not shown). Note that KsL-VNIO is a simple binding constant measured in the absence of NADPH whereas KiL-VNIO (Table I) is determined under conditions of substrate turnover. The two values need not agree (52Schenkman J.B. Remmer H. Estabrook R.W. Mol. Pharmacol. 1967; 3: 113-123PubMed Google Scholar). The rate measurements used to construct Fig. 2 were obtained immediately after initiation of the enzymatic reaction. If the reaction mixtures were monitored for several minutes, the progress curves were clearly concave downward indicating occurrence of a progressive irreversible inhibition of nNOS. Such inactivation was examined directly by incubating nNOS with various concentrations of l-VNIO in the presence of NADPH and monitoring the reaction mixtures for residual nNOS activity at intervals (Fig. 4 A). As shown, l-VNIO, but not d-VNIO, caused a first-order inactivation of nNOS. There was no evidence of nNOS reactivation in these studies; product formation was constant over the time of the assay. In separate studies, passage of reaction mixtures containing l-VNIO-inactivated nNOS through small gel-filtration columns di
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