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Formation of nitrogen oxides and citrulline upon oxidation of Nω-hydroxy-L-arginine by hemeproteins
Vo1.184, No. 3,1992 May 15,1992
BIOCHEMICALAND BIOPHYSICALRESEARCHCOMMUNICATIONS Pages 1158-1164
FORMATION OF NITROGEN OXIDES AND CITRULLINE UPON OXIDATION OF Nm-HYDROXY-L-ARGININE BY HEMEPROTEINS
J.L. BOUCHER, A. GENET, S. VADON, M. DELAFORGE and D. MANSUY Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques URA 400 CNRS, Universit~ Ren~ Descartes, 45 rue des Saints-Pares, 75270 PARIS Cedex 06, FRANCE Received March 25, 1992
ABSTRACT : HRP catalyzes the oxidation of Ne-hydroxy-L-arginine (NOHA) by H202 with formation of citrulline and NO2- with initial rates of about 0.7 and 0.2 nmol per nmol HRP per min. In the same manner, cytochromes P450 from rat liver microsomes catalyze the oxidation of NOHA to citrulline and NO2- by cumylhydroperoxide. Inhibitors of these hemeproteins (N3- and CN- for HRP and miconazole for P450) strongly inhibit both citrulline and NO2- formation. Rates of NOHA oxidation by these hemeproteins markedly decrease with time presumably because of their denaturation by nitrogen oxides and of the formation of hemeprotein-iron-NO complexes. These results suggest that NO (and other nitrogen oxides) could be formed from oxidation of NOHA by other enzymes than NO-synthases. ® 1992Academic P . . . . . I n c .
Nitric Oxide, NO, is a recently discovered messenger molecule with very important functions in the physiology of mammalian cardiovascular, immune and nervous systems (1-3). A major action of NO is to activate guanylate cyclase and to raise cGMP levels in target cells, but NO has also been involved, for instance, as an effector of the antiproliferative function exerted by cytotoxic macrophages against tumor cells (1-3). Biosynthesis of NO involves an oxidation of L-arginine by NADPH and 02 catalyzed by different kinds of NO-synthases (NOS) (4). Nm-hydroxy-L arginine (NOHA) was recently shown as an intermediate in this oxidation, NOsynthases being able to catalyze both the Nm-hydroxylation of L-arginine and the oxidative denitration of NOHA by NADPH and 02 with formation of L-citrulline and NO (5). Thus various mammalian cells containing NO-synthases oxidize L-arginine to Lcitrulline and NO and its final stable oxidation products NO2- and NO3- (eq.1) (2). As at Abbreviations : NOHA : Ne-hydroxy-L-arginine ; NOS ; nitric oxide synthase ; HRP ; horseradish peroxidase ; P450 : cytochrome P450 ; RLM : rat liver microsomes ; Hb :
hemoglobin ; C u m O O H : cumylhydroperoxide ; D a b s y l c h l o r i d e : 4(N, N')dimethylaminoazobenzenesulfonyl chloride ; D M F : N,N'-dimethylformamide. 0006-291X/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights of.reproduction in any form reserved.
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least some of these cells were found to produce NOHA (6), it was interesting to know whether other enzymes than NO-synthases were able to catalyze the second step of eq.1, the oxidative cleavage of a C-N bond of NOHA with formation of citrulline and nitrogen oxides (NO, NO2-, NO3-...). NH
II
R--C NH2 L-Arginine
NADPH'O2 NOS
N--OH
II
O
II
NADPH'O2
R--C--NH 2
NOS
NOHA
~
R--C--NH CitruUine
2 +
NO O
I
(eq.1)
NO2" , NO 3-
This paper shows for the first time, that some hemeproteins, like a peroxidase (HRP), microsomal cytochrome P450, hemoglobin and catalase are able to catalyze such an oxidative cleavage of a C-N bond of NOHA by H202 or alkylhydroperoxides.
MATERIALS
AND
METHODS
Chemicals and e n z y m e s : Horseradish peroxidase (Type VIA), hemoglobin (from
bovine blood), catalase (C40, from bovine liver), dabsylchloride, sulfanilamide, N-(1naphthyl)ethylenediamine hydrochloride, ninhydrine spray reagent and authentic Lamino-acids (L-arginine, L-citrulline and L-leucine) were purchased from Sigma. Miconazole, dexamethasone, hydrogen peroxide, cumylhydroperoxide, sodium azide, potassium cyanide and citric acid were obtained from Janssen. All solvents and other reagents were of the highest purity commercially available. NCO-hydroxy-L-arginine was synthesized according to a previously described method (7) using Na-benzyloxycarbonyI-L-ornithine (Sigma) as starting material. Synthesized NOHA (hydrochloride) displayed physical and spectroscopic characteristics identical to those reported previously (7,8). Preparation of rat liver m i c r o s o m e s : Male Sprague-Dawley rats (200-250g) were provided laboratory chow and water ad libitum. After 10 days of adaptation, animals were treated with dexamethasone (50 mg/kg, in corn oil, i.p. for 4 days). Microsomes were prepared as reported (9) and stored at -80°C until use. Protein concentrations were determined by the method of Lowry (10) with bovine serum albumin as standard. Cytochrome P450 contents were determined as described by Omura and Sato (11 ). I n c u b a t i o n p r o c e d u r e s : Standard incubations (total volume : 0.5 ml) were performed at 37°C in phosphate buffer (0.1M, pH 7.4, 0.1 mM EDTA sodium salt) containing NOHA (100 I.tM) and rat liver microsomes (0.5 nmol P450, about 0.3 mg protein) or commercially available hemeproteins (0.5 nmol heine). After preincubation 1 min at 37°C the reaction was started by addition of 500 nmol H202 or CumOOH. Incubations were stopped by adding 0.5 ml cold acetonitrile and cooling at 0°C before centrifugation at 3000 rpm for 20 min at 4°C. NO?- determination : Nitrite ion formation was measured spectrophotometrically as previously reported (12). Aliquots (0.5 ml) of the above supernatants were mixed with 0.5 ml of 1% sulfanilamide in 0.4N HCI and with 0.5 ml of 0.1% N-(1naphthyl)ethylenediamine in 0.4N HCI, and their absorption at 543 nm was measured. Quantitations of NO2- formation were done from calibration curves. Citrulline d e t e r m i n a t i o n : First method : Separation and measurement of citrulline were routinely performed after derivatisation by dabsyl chloride and reverse phase HPLC analysis as oreviously described (13). Aliquots (100 I.tl) of the supernatants were mixed with 501.tl 1159
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0.05M NaHCO3 buffer pH 9 in 2 ml HPLC glass vials, and 2.5 nmol of L-leucine were added as internal standard. 60 #1 of a 10 -2 M solution of dabsyl chloride in dry the homogeneous solution were added 400 p.I of a 50:50 mixture of ethanol and phosphate buffer (0.05M, pH 7.4) and 100 p.I aliquots were analyzed using a Kontron HPLC system. Separations were performed at room temperature on a 250 x 4.6 mm column of ODS silica (5 ~m, Nucteosil C18, Soci~t~ Fran{~aise Chromato-ColonneShandon, Eragny, France). The mobile phase was a gradient between buffer A (sodium citrate 0.01M, pH 6.5 containing 0.4% DMF) and buffer B (acetonitrile : buffer A : DMF = 700:300:40) as follows : 0 min, 10% B ; 1 min, linear gradient to 50% B in 20 min ; 21 min, linear gradient to 90% B in 10 min ; 32 min, linear gradient to 0%B in 1 min ; 41 min, linear gradient to 10% B followed by reequilibration. Flow rate was 1 ml/min and the spectrophotometric absorbance was monitored at 436 mm. The retention times or the derivatives of citrulline, NOHA and leucine were respectively 19, 23.5 and 22 rain. Quantitations were done using leucine dabsylamide as a standard and calibration curves. Second meth0d : In some experiments, citrulline separation and quantitation were performed using a Biotronik LC 6000 Autoanalyser equipped with a Durrum DC-6A resin column (280 x 6 mm). Aliquots (200 #l) of the supernatants were mixed with 20 nmoles of L-norleucine as internal standard and evaporated to dryness. The samples were then redissolved in 250 #1 sodium citrate (0.18N, pH 2.0) and 200 ILl aliquots were injected. The following 0.18N sodium citrate buffers were used as eluents :0 to 10 min, pH 3.18 at 54°C, 10 to 23 min, pH 3.85 at 54°C, 23 to 60 min, pH 4.35 at 59°C and 60 to 108 min, sodium citrate 1.6N, pH 4.75 at 59°C. The flow rate was 31 ml/h. After derivatisation with nihydrine, the optical density of the reaction mixture was monitored simultaneously at 440 and 570 mm. The retention times for the derivatives of citrulline and NOHA were respectively 46 and 128 min. The results obtained for measurement of citrulline by the two methods always were in good agreement. UV-visible spectroscopy_: Experiments were performed, at ambiant temperature, with a Kontron 940 spectrophotometer, on incubations performed in 1 cm cuvette containing100 #M NOHA, 1 mM H202 or CumOOH and 1 I~M P450 or 5 #M HRP in 1ml 0.1M phosphate buffer pH 7.4.
RESULTS Incubation of 100#M NOHA, synthesized by a previously reported method (7), with 1taM Horseradish Peroxidase (HRP) and lmM H202 led to the formation of NO2(Fig. 1). Concomitant formation of citrulline could also be demonstrated after treatment of the incubates by reagents used for derivatisation of amino-acids and analysis either by HPLC or with an autoanalyser (retention times identical to those of authentic citrulline derivatives using two different kinds of derivatisation, see Materials and Methods). Formation of NO2- and citrulline was only linear for about 10 min. Initial rates of citrulline and NO2- formation were respectively 0.7 and 0.2 mol of product per mol of HRP per min, and the citrulline : NO2 molar ratio varied between 2.8 and 4 during the reaction. The rates of citrulline and NO2- formation considerably decreased after 10 min (Fig. 1). As shown in table 1, no significant formation of NO2- and citrulline was observed if one of the components of the reaction (NOHA, H202, HRP) was omitted. The initial rates of citrulline and NO2- formation were found to linearly increase with the concentration of HRP, at least between 0.5 and 41~M (data not 1160
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12 ,~ 10 -~
8
-4 4
E
2 0
'
0
'
l0
'
20
30
time (rain) Fiq.1 : Time course of formation of citrulline and NO2- by HRP-catalyzed oxidation of NOHA by H202. nmol citrulline ([ZI) and nmol NO2- (O) formed per nmol HRP (conditions : 100 gM NOHA, lp.M HRP and 1 mM H202 in phosphate buffer pH 7.4 at 37°C ; mean values + SD from four experiments).
shown), and classical inhibitors of HRP like CN- and N3- greatly reduced both citrulline and NO2- formation (table 1). Oxidation of NOHA with formation of citrulline and NO2- also occurred if alkylhydroperoxides like cumylhydroperoxide, CumOOH, were used instead of H202 in the presence of catalytic amounts of HPR or other hemeproteins. For instance, rat liver microsomes, which contain cytochromes P450, were found to catalyze the oxidation of NOHA by CumOOH with formation of citrulline and NO2 (initial rates of 0.8 and 0.3 nmol product formed per nmol P450 per min) (table 1). Formation of citrulline did not occur if one component of the reaction (NOHA, CumOOH or microsomes) was omitted, or if heat-denaturated microsomes were used (table 1). Similar results were obtained for the formation of NO2 except that low amounts of NO2- were formed in the absence of NOHA or with heat-denaturated microsomes, presumably because of non specific oxidation of nitrogenous components of microsomes by CumOOH. Implication of cytochromes P450 as the active catalysts in rat liver microsomes-dependent oxidation of NOHA was shown by the clear decrease of citrulline and NO2- formation in the presence of increasing amounts of a classical inhibitor of microsomal cytochromes P450, miconazole (14) (table 1). Table 2 shows that other hemeproteins like hemoglobin (Hb) and catalase also catalyzed the oxidation of NOHA to citrulline and NO2- by hydroperoxides. It also shows that the activity of each hemeprotein was clearly dependent on the nature of the oxidant used. In the case of HRP, a higher activity was obtained with its natural cofactor, H202 , than with CumOOH. On the contrary, oxidation with catalase only occurred with CumOOH, the lack of catalase activity with H202 being due to the particular ability of this hemeprotein to dismutate H202 rather than to use it as an oxidant for substrates. In the case of liver cytochromes P450, CumOOH gave better activities than H202, which is easily understandable because of the hydrophobicity of the active site of these cytochromes. 1161
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Table 1 . Effects of various factors on the oxidation of NOHA either by HRP and H202, or by rat liver microsomes (RLM) and CumOOH Conditions (a)
Citrulline formation activity (b)
HRP + H202 + NOHA
(%)
NO2" formation activity (b)
(%)
9.6
(100)
3.8
(100)
HRP + H202
<0.1
(<1)
<0.1
(<3)
HRP + NOHA
<0.1
(<1)
<0.1
(<3)
H202 + NOHA
0.6
(6)
< 0.1
(< 3)
HRP + H202 + NOHA + CN- (1 mM)
2.4
(25)
1
(26)
HRP + H202 + NOHA + N3- (1 mM)
1.1
(13)
0.7
(18)
RLM + CumOOH + NOHA
8.1
(100)
3.2
(100)
RLM + CumOOH
< 0.1
(< 1)
0.3
(9)
RLM + NOHA
<0.1
(<1)
<0.1
(<3)
CumOOH + NOHA
<0.1
(<1)
<0.1
(<3)
den. RLM (c) + CumOOH + NOHA
0.2
(2)
0.4
(13)
RLM + CumOOH + NOHA + miconazole (10 I~M)
6.0
(74)
2.1
(65)
RLM + CumOOH + NOHA 1.5 (19) 0.6 (19) + miconazole (100 I~M) a) incubations of 100 FM NOHA in phosphate buffer pH 7.4 with either 1 I~M HRP and 1 mM H202, or dexamethasone-pretreated rat liver microsomes (1 pM P450) and 1 mM CumOOH. b) nmol product per nmol HRP after 20 min, or nmol product per nmol cytochrome P450 after 10 min (mean values from 2 to 6 independent determinations, SD = + 15%). c) den. RLM for microsomes heated at 100°C for 5 min before the experiment.
Table 2 . Oxidation of NOHA to citrulline and NO2" by H202 or CumOOH catalyzed by hemeproteins (a) H202 Hemeprotein
Citrulline (b)
CumOOH NO2- (b)
Citrulline (b)
NO2" (b)
HRP
9.6
3.8
2.4
1.1
Hb
4.0
1.7
2.8
1.9
< 0.1
< 0.1
2.3
1.9
Catalase
RLM (c) 2.3 1.5 11 3.9 a) incubations of 100 IIM NOHA in phosphate buffer pH 7.4 with 1 p.M hemeprotein (0.25 t~M in the case of Hb) and 1 mM H202 or CumOOH. b) nmol product formed per nmol hemeprotein (0.25 nmol Hb) after 15 min (mean values from 2 to 6 independent determinations, SD = + 15 %). c) RLM = liver microsomes from dexamethasonepretreated rats (1 IIM P450). 1162
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DISCUSSION The above results show that several hemeproteins, like HRP and microsomal cytochromes P450, catalyze the oxidative cleavage of a C-N bond of N c°hydroxyarginine by H202 or alkylhydroperoxides, with formation of citrulline and NO2. Citrulline and NO2- are formed simultaneously and appear to derive from a common reaction as their formation is similarly affected by various factors like the use of inhibitors (table 1). This common reaction should be the oxidative cleavage of a C-N bond of NOHA as previously found for NO-synthase-dependent oxidation of NOHA (5). The NO2- : citrulline molar ratio, which was found clearly inferior to 1, is expected for such oxidative denitrations since other nitrogen oxides like NO3- are formed. In that respect, it is noteworthy that in most in vitro NO-synthase-dependent oxidations of Larginine, the major stable products observed, which derive from a further oxidation of NO in the medium, are NO2- and NO3- (2). The most efficient systems found for the oxidative denitration of NOHA were HRP and H202 (initial rates of 0.7nmol per nmol HRP per min) and rat liver cytochromes P450 and CumOOH (0.8nmol citrulline per nmol P450 per rain). However the activity of both systems rapidly decreases with time. This could be due to denaturing effects of NO or other nitrogen oxides on the hemeprotein catalysts. In that regard, the formation of a HRP complex characterized by peaks at 422, 543 and 575nm in visible spectroscopy could be observed upon reaction of HRP with H202 and NOHA. This spectrum is almost identical to that reported for HRP-Fe(III)-NO (15). Similarly, addition of NOHA and CumOOH to rat liver microsomes led to a transient difference visible spectrum exhibiting a peak at 437nm (data not shown), as expected for a P450-Fe(III)-NO complex (16). The decrease of activity for NOHA oxidation observed for HRP and rat liver microsomes could thus be due, at least in part, to the formation of inactive hemeprotein-iron-NO complexes. This ability of some hemeproteins to catalyze the oxidation of NOHA with formation of nitrogen oxides and citrulline suggests a possible way of NO formation in living organisms different from that shown in eq.1 which only involves NO-synthase. This new possible way of NO formation (eq.2) would involve two steps : (i) the formation of NOHA by oxidation of L-arginine catalyzed by NO-synthase in cells or cell compartments containing this enzyme, and (ii) the oxidation of NOHA to NO by some hemeproteins in cells (or cell compartments) not containing NO-synthases. This would be particularly important for the formation of NO in sites very close to those of its biological activity but not containing NO-synthases. NOHA would thus act as a relatively stable and transportable precursor of NO. The physiological significance of this pathway of NO formation remains to be established.
L-arginine
NADPH,O2 ~-NOS
ROOH NOHA
# Citrulline + nitrogen oxides Hemeprotein 1163
(eq. 2)
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: We thank Dr. D. Migliore (URA 1188, Paris) and C. Chopard (URA 400, 1Saris) for their help in the use of aminoacid auto-analyser and in EPR experiments. Acknowledaments
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Moncada, S., Palmer, R.M.J. and Higgs, E.A. (1989) Biochem. Pharm. 38, 1709-1715. Marietta, M.A. (1988) Chem. Res. Toxicol. 1,249-257. Moncada, S., Palmer, R.M.J. and Higgs, E.A. (1991) Pharmacol. Rev. 43, 109141. FSrstermann, U., Schmidt, H.H.H.W., Pollock, J.S., Sheng, H., Mitchell, J.A., Warmer, T.D., Nakane, M. and Murad, F. (1991) Biochem. Pharmacol. 42, 1849-1857. Stuehr, D.J., Kwon, N.S., Nathan, C.F., Griffith, O.W., Feldman, P.L. and Wiseman, J. (1991) J. Biol. Chem. 266, 6259-6263. Chenais, B., Yapo, A., Lepoivre, M. and Tenu, J.P. (1991) J. Chrom. 539, 433441. Wallace, G.C. and Fukuto, J.M. (1991) J. Med. Chem. 34, 1746-1748. Feldman, P.L. (1991) Tetrahedron Lett. 32, 875-878. Kremers, P., Beaune, P., Cresteil, T., De Graeve, J., Columelli, S., Leroux, J.P. and Gielen, J. (1981) Eur. J. Biochem. 118, 599-606. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Ramdall, R.J. (1951) J. Biol. Chem. 193, 265-575. Omura, T. and Sato, R. (1964) J. Biol. Chem. 239, 2370-2385. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S. and Tannenbaum, S.R. (1982)Anal. Biochem. 126, 131-138. Chang, J.Y., Knecht, R. and Braun, D.G. (1981) Biochem. J. 199, 547-555. Ortiz de Montellano, P.R. and Reich, N.O. (1986) Cytochrome P-450, (Ortiz de Montellano, P.R. Ed.) 273-314. Plenum Press, New York. Yonetani, T., Yamamoto, H., Erman, J.E., Leigh, J.S. and Reed, G.H. (1972) J. Biol. Chem. 247, 2447-2455. Ebel, R.E., O'Keeffe, D.H. and Peterson, J.A. (1975) FEBS Lett. 55, 198-201.
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FORMATION OF NITROGEN OXIDES AND CITRULLINE UPON OXIDATION OF Nm-HYDROXY-L-ARGININE BY HEMEPROTEINS
J.L. BOUCHER, A. GENET, S. VADON, M. DELAFORGE and D. MANSUY Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques URA 400 CNRS, Universit~ Ren~ Descartes, 45 rue des Saints-Pares, 75270 PARIS Cedex 06, FRANCE Received March 25, 1992
ABSTRACT : HRP catalyzes the oxidation of Ne-hydroxy-L-arginine (NOHA) by H202 with formation of citrulline and NO2- with initial rates of about 0.7 and 0.2 nmol per nmol HRP per min. In the same manner, cytochromes P450 from rat liver microsomes catalyze the oxidation of NOHA to citrulline and NO2- by cumylhydroperoxide. Inhibitors of these hemeproteins (N3- and CN- for HRP and miconazole for P450) strongly inhibit both citrulline and NO2- formation. Rates of NOHA oxidation by these hemeproteins markedly decrease with time presumably because of their denaturation by nitrogen oxides and of the formation of hemeprotein-iron-NO complexes. These results suggest that NO (and other nitrogen oxides) could be formed from oxidation of NOHA by other enzymes than NO-synthases. ® 1992Academic P . . . . . I n c .
Nitric Oxide, NO, is a recently discovered messenger molecule with very important functions in the physiology of mammalian cardiovascular, immune and nervous systems (1-3). A major action of NO is to activate guanylate cyclase and to raise cGMP levels in target cells, but NO has also been involved, for instance, as an effector of the antiproliferative function exerted by cytotoxic macrophages against tumor cells (1-3). Biosynthesis of NO involves an oxidation of L-arginine by NADPH and 02 catalyzed by different kinds of NO-synthases (NOS) (4). Nm-hydroxy-L arginine (NOHA) was recently shown as an intermediate in this oxidation, NOsynthases being able to catalyze both the Nm-hydroxylation of L-arginine and the oxidative denitration of NOHA by NADPH and 02 with formation of L-citrulline and NO (5). Thus various mammalian cells containing NO-synthases oxidize L-arginine to Lcitrulline and NO and its final stable oxidation products NO2- and NO3- (eq.1) (2). As at Abbreviations : NOHA : Ne-hydroxy-L-arginine ; NOS ; nitric oxide synthase ; HRP ; horseradish peroxidase ; P450 : cytochrome P450 ; RLM : rat liver microsomes ; Hb :
hemoglobin ; C u m O O H : cumylhydroperoxide ; D a b s y l c h l o r i d e : 4(N, N')dimethylaminoazobenzenesulfonyl chloride ; D M F : N,N'-dimethylformamide. 0006-291X/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights of.reproduction in any form reserved.
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least some of these cells were found to produce NOHA (6), it was interesting to know whether other enzymes than NO-synthases were able to catalyze the second step of eq.1, the oxidative cleavage of a C-N bond of NOHA with formation of citrulline and nitrogen oxides (NO, NO2-, NO3-...). NH
II
R--C NH2 L-Arginine
NADPH'O2 NOS
N--OH
II
O
II
NADPH'O2
R--C--NH 2
NOS
NOHA
~
R--C--NH CitruUine
2 +
NO O
I
(eq.1)
NO2" , NO 3-
This paper shows for the first time, that some hemeproteins, like a peroxidase (HRP), microsomal cytochrome P450, hemoglobin and catalase are able to catalyze such an oxidative cleavage of a C-N bond of NOHA by H202 or alkylhydroperoxides.
MATERIALS
AND
METHODS
Chemicals and e n z y m e s : Horseradish peroxidase (Type VIA), hemoglobin (from
bovine blood), catalase (C40, from bovine liver), dabsylchloride, sulfanilamide, N-(1naphthyl)ethylenediamine hydrochloride, ninhydrine spray reagent and authentic Lamino-acids (L-arginine, L-citrulline and L-leucine) were purchased from Sigma. Miconazole, dexamethasone, hydrogen peroxide, cumylhydroperoxide, sodium azide, potassium cyanide and citric acid were obtained from Janssen. All solvents and other reagents were of the highest purity commercially available. NCO-hydroxy-L-arginine was synthesized according to a previously described method (7) using Na-benzyloxycarbonyI-L-ornithine (Sigma) as starting material. Synthesized NOHA (hydrochloride) displayed physical and spectroscopic characteristics identical to those reported previously (7,8). Preparation of rat liver m i c r o s o m e s : Male Sprague-Dawley rats (200-250g) were provided laboratory chow and water ad libitum. After 10 days of adaptation, animals were treated with dexamethasone (50 mg/kg, in corn oil, i.p. for 4 days). Microsomes were prepared as reported (9) and stored at -80°C until use. Protein concentrations were determined by the method of Lowry (10) with bovine serum albumin as standard. Cytochrome P450 contents were determined as described by Omura and Sato (11 ). I n c u b a t i o n p r o c e d u r e s : Standard incubations (total volume : 0.5 ml) were performed at 37°C in phosphate buffer (0.1M, pH 7.4, 0.1 mM EDTA sodium salt) containing NOHA (100 I.tM) and rat liver microsomes (0.5 nmol P450, about 0.3 mg protein) or commercially available hemeproteins (0.5 nmol heine). After preincubation 1 min at 37°C the reaction was started by addition of 500 nmol H202 or CumOOH. Incubations were stopped by adding 0.5 ml cold acetonitrile and cooling at 0°C before centrifugation at 3000 rpm for 20 min at 4°C. NO?- determination : Nitrite ion formation was measured spectrophotometrically as previously reported (12). Aliquots (0.5 ml) of the above supernatants were mixed with 0.5 ml of 1% sulfanilamide in 0.4N HCI and with 0.5 ml of 0.1% N-(1naphthyl)ethylenediamine in 0.4N HCI, and their absorption at 543 nm was measured. Quantitations of NO2- formation were done from calibration curves. Citrulline d e t e r m i n a t i o n : First method : Separation and measurement of citrulline were routinely performed after derivatisation by dabsyl chloride and reverse phase HPLC analysis as oreviously described (13). Aliquots (100 I.tl) of the supernatants were mixed with 501.tl 1159
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0.05M NaHCO3 buffer pH 9 in 2 ml HPLC glass vials, and 2.5 nmol of L-leucine were added as internal standard. 60 #1 of a 10 -2 M solution of dabsyl chloride in dry the homogeneous solution were added 400 p.I of a 50:50 mixture of ethanol and phosphate buffer (0.05M, pH 7.4) and 100 p.I aliquots were analyzed using a Kontron HPLC system. Separations were performed at room temperature on a 250 x 4.6 mm column of ODS silica (5 ~m, Nucteosil C18, Soci~t~ Fran{~aise Chromato-ColonneShandon, Eragny, France). The mobile phase was a gradient between buffer A (sodium citrate 0.01M, pH 6.5 containing 0.4% DMF) and buffer B (acetonitrile : buffer A : DMF = 700:300:40) as follows : 0 min, 10% B ; 1 min, linear gradient to 50% B in 20 min ; 21 min, linear gradient to 90% B in 10 min ; 32 min, linear gradient to 0%B in 1 min ; 41 min, linear gradient to 10% B followed by reequilibration. Flow rate was 1 ml/min and the spectrophotometric absorbance was monitored at 436 mm. The retention times or the derivatives of citrulline, NOHA and leucine were respectively 19, 23.5 and 22 rain. Quantitations were done using leucine dabsylamide as a standard and calibration curves. Second meth0d : In some experiments, citrulline separation and quantitation were performed using a Biotronik LC 6000 Autoanalyser equipped with a Durrum DC-6A resin column (280 x 6 mm). Aliquots (200 #l) of the supernatants were mixed with 20 nmoles of L-norleucine as internal standard and evaporated to dryness. The samples were then redissolved in 250 #1 sodium citrate (0.18N, pH 2.0) and 200 ILl aliquots were injected. The following 0.18N sodium citrate buffers were used as eluents :0 to 10 min, pH 3.18 at 54°C, 10 to 23 min, pH 3.85 at 54°C, 23 to 60 min, pH 4.35 at 59°C and 60 to 108 min, sodium citrate 1.6N, pH 4.75 at 59°C. The flow rate was 31 ml/h. After derivatisation with nihydrine, the optical density of the reaction mixture was monitored simultaneously at 440 and 570 mm. The retention times for the derivatives of citrulline and NOHA were respectively 46 and 128 min. The results obtained for measurement of citrulline by the two methods always were in good agreement. UV-visible spectroscopy_: Experiments were performed, at ambiant temperature, with a Kontron 940 spectrophotometer, on incubations performed in 1 cm cuvette containing100 #M NOHA, 1 mM H202 or CumOOH and 1 I~M P450 or 5 #M HRP in 1ml 0.1M phosphate buffer pH 7.4.
RESULTS Incubation of 100#M NOHA, synthesized by a previously reported method (7), with 1taM Horseradish Peroxidase (HRP) and lmM H202 led to the formation of NO2(Fig. 1). Concomitant formation of citrulline could also be demonstrated after treatment of the incubates by reagents used for derivatisation of amino-acids and analysis either by HPLC or with an autoanalyser (retention times identical to those of authentic citrulline derivatives using two different kinds of derivatisation, see Materials and Methods). Formation of NO2- and citrulline was only linear for about 10 min. Initial rates of citrulline and NO2- formation were respectively 0.7 and 0.2 mol of product per mol of HRP per min, and the citrulline : NO2 molar ratio varied between 2.8 and 4 during the reaction. The rates of citrulline and NO2- formation considerably decreased after 10 min (Fig. 1). As shown in table 1, no significant formation of NO2- and citrulline was observed if one of the components of the reaction (NOHA, H202, HRP) was omitted. The initial rates of citrulline and NO2- formation were found to linearly increase with the concentration of HRP, at least between 0.5 and 41~M (data not 1160
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12 ,~ 10 -~
8
-4 4
E
2 0
'
0
'
l0
'
20
30
time (rain) Fiq.1 : Time course of formation of citrulline and NO2- by HRP-catalyzed oxidation of NOHA by H202. nmol citrulline ([ZI) and nmol NO2- (O) formed per nmol HRP (conditions : 100 gM NOHA, lp.M HRP and 1 mM H202 in phosphate buffer pH 7.4 at 37°C ; mean values + SD from four experiments).
shown), and classical inhibitors of HRP like CN- and N3- greatly reduced both citrulline and NO2- formation (table 1). Oxidation of NOHA with formation of citrulline and NO2- also occurred if alkylhydroperoxides like cumylhydroperoxide, CumOOH, were used instead of H202 in the presence of catalytic amounts of HPR or other hemeproteins. For instance, rat liver microsomes, which contain cytochromes P450, were found to catalyze the oxidation of NOHA by CumOOH with formation of citrulline and NO2 (initial rates of 0.8 and 0.3 nmol product formed per nmol P450 per min) (table 1). Formation of citrulline did not occur if one component of the reaction (NOHA, CumOOH or microsomes) was omitted, or if heat-denaturated microsomes were used (table 1). Similar results were obtained for the formation of NO2 except that low amounts of NO2- were formed in the absence of NOHA or with heat-denaturated microsomes, presumably because of non specific oxidation of nitrogenous components of microsomes by CumOOH. Implication of cytochromes P450 as the active catalysts in rat liver microsomes-dependent oxidation of NOHA was shown by the clear decrease of citrulline and NO2- formation in the presence of increasing amounts of a classical inhibitor of microsomal cytochromes P450, miconazole (14) (table 1). Table 2 shows that other hemeproteins like hemoglobin (Hb) and catalase also catalyzed the oxidation of NOHA to citrulline and NO2- by hydroperoxides. It also shows that the activity of each hemeprotein was clearly dependent on the nature of the oxidant used. In the case of HRP, a higher activity was obtained with its natural cofactor, H202 , than with CumOOH. On the contrary, oxidation with catalase only occurred with CumOOH, the lack of catalase activity with H202 being due to the particular ability of this hemeprotein to dismutate H202 rather than to use it as an oxidant for substrates. In the case of liver cytochromes P450, CumOOH gave better activities than H202, which is easily understandable because of the hydrophobicity of the active site of these cytochromes. 1161
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Table 1 . Effects of various factors on the oxidation of NOHA either by HRP and H202, or by rat liver microsomes (RLM) and CumOOH Conditions (a)
Citrulline formation activity (b)
HRP + H202 + NOHA
(%)
NO2" formation activity (b)
(%)
9.6
(100)
3.8
(100)
HRP + H202
<0.1
(<1)
<0.1
(<3)
HRP + NOHA
<0.1
(<1)
<0.1
(<3)
H202 + NOHA
0.6
(6)
< 0.1
(< 3)
HRP + H202 + NOHA + CN- (1 mM)
2.4
(25)
1
(26)
HRP + H202 + NOHA + N3- (1 mM)
1.1
(13)
0.7
(18)
RLM + CumOOH + NOHA
8.1
(100)
3.2
(100)
RLM + CumOOH
< 0.1
(< 1)
0.3
(9)
RLM + NOHA
<0.1
(<1)
<0.1
(<3)
CumOOH + NOHA
<0.1
(<1)
<0.1
(<3)
den. RLM (c) + CumOOH + NOHA
0.2
(2)
0.4
(13)
RLM + CumOOH + NOHA + miconazole (10 I~M)
6.0
(74)
2.1
(65)
RLM + CumOOH + NOHA 1.5 (19) 0.6 (19) + miconazole (100 I~M) a) incubations of 100 FM NOHA in phosphate buffer pH 7.4 with either 1 I~M HRP and 1 mM H202, or dexamethasone-pretreated rat liver microsomes (1 pM P450) and 1 mM CumOOH. b) nmol product per nmol HRP after 20 min, or nmol product per nmol cytochrome P450 after 10 min (mean values from 2 to 6 independent determinations, SD = + 15%). c) den. RLM for microsomes heated at 100°C for 5 min before the experiment.
Table 2 . Oxidation of NOHA to citrulline and NO2" by H202 or CumOOH catalyzed by hemeproteins (a) H202 Hemeprotein
Citrulline (b)
CumOOH NO2- (b)
Citrulline (b)
NO2" (b)
HRP
9.6
3.8
2.4
1.1
Hb
4.0
1.7
2.8
1.9
< 0.1
< 0.1
2.3
1.9
Catalase
RLM (c) 2.3 1.5 11 3.9 a) incubations of 100 IIM NOHA in phosphate buffer pH 7.4 with 1 p.M hemeprotein (0.25 t~M in the case of Hb) and 1 mM H202 or CumOOH. b) nmol product formed per nmol hemeprotein (0.25 nmol Hb) after 15 min (mean values from 2 to 6 independent determinations, SD = + 15 %). c) RLM = liver microsomes from dexamethasonepretreated rats (1 IIM P450). 1162
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DISCUSSION The above results show that several hemeproteins, like HRP and microsomal cytochromes P450, catalyze the oxidative cleavage of a C-N bond of N c°hydroxyarginine by H202 or alkylhydroperoxides, with formation of citrulline and NO2. Citrulline and NO2- are formed simultaneously and appear to derive from a common reaction as their formation is similarly affected by various factors like the use of inhibitors (table 1). This common reaction should be the oxidative cleavage of a C-N bond of NOHA as previously found for NO-synthase-dependent oxidation of NOHA (5). The NO2- : citrulline molar ratio, which was found clearly inferior to 1, is expected for such oxidative denitrations since other nitrogen oxides like NO3- are formed. In that respect, it is noteworthy that in most in vitro NO-synthase-dependent oxidations of Larginine, the major stable products observed, which derive from a further oxidation of NO in the medium, are NO2- and NO3- (2). The most efficient systems found for the oxidative denitration of NOHA were HRP and H202 (initial rates of 0.7nmol per nmol HRP per min) and rat liver cytochromes P450 and CumOOH (0.8nmol citrulline per nmol P450 per rain). However the activity of both systems rapidly decreases with time. This could be due to denaturing effects of NO or other nitrogen oxides on the hemeprotein catalysts. In that regard, the formation of a HRP complex characterized by peaks at 422, 543 and 575nm in visible spectroscopy could be observed upon reaction of HRP with H202 and NOHA. This spectrum is almost identical to that reported for HRP-Fe(III)-NO (15). Similarly, addition of NOHA and CumOOH to rat liver microsomes led to a transient difference visible spectrum exhibiting a peak at 437nm (data not shown), as expected for a P450-Fe(III)-NO complex (16). The decrease of activity for NOHA oxidation observed for HRP and rat liver microsomes could thus be due, at least in part, to the formation of inactive hemeprotein-iron-NO complexes. This ability of some hemeproteins to catalyze the oxidation of NOHA with formation of nitrogen oxides and citrulline suggests a possible way of NO formation in living organisms different from that shown in eq.1 which only involves NO-synthase. This new possible way of NO formation (eq.2) would involve two steps : (i) the formation of NOHA by oxidation of L-arginine catalyzed by NO-synthase in cells or cell compartments containing this enzyme, and (ii) the oxidation of NOHA to NO by some hemeproteins in cells (or cell compartments) not containing NO-synthases. This would be particularly important for the formation of NO in sites very close to those of its biological activity but not containing NO-synthases. NOHA would thus act as a relatively stable and transportable precursor of NO. The physiological significance of this pathway of NO formation remains to be established.
L-arginine
NADPH,O2 ~-NOS
ROOH NOHA
# Citrulline + nitrogen oxides Hemeprotein 1163
(eq. 2)
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: We thank Dr. D. Migliore (URA 1188, Paris) and C. Chopard (URA 400, 1Saris) for their help in the use of aminoacid auto-analyser and in EPR experiments. Acknowledaments
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Moncada, S., Palmer, R.M.J. and Higgs, E.A. (1989) Biochem. Pharm. 38, 1709-1715. Marietta, M.A. (1988) Chem. Res. Toxicol. 1,249-257. Moncada, S., Palmer, R.M.J. and Higgs, E.A. (1991) Pharmacol. Rev. 43, 109141. FSrstermann, U., Schmidt, H.H.H.W., Pollock, J.S., Sheng, H., Mitchell, J.A., Warmer, T.D., Nakane, M. and Murad, F. (1991) Biochem. Pharmacol. 42, 1849-1857. Stuehr, D.J., Kwon, N.S., Nathan, C.F., Griffith, O.W., Feldman, P.L. and Wiseman, J. (1991) J. Biol. Chem. 266, 6259-6263. Chenais, B., Yapo, A., Lepoivre, M. and Tenu, J.P. (1991) J. Chrom. 539, 433441. Wallace, G.C. and Fukuto, J.M. (1991) J. Med. Chem. 34, 1746-1748. Feldman, P.L. (1991) Tetrahedron Lett. 32, 875-878. Kremers, P., Beaune, P., Cresteil, T., De Graeve, J., Columelli, S., Leroux, J.P. and Gielen, J. (1981) Eur. J. Biochem. 118, 599-606. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Ramdall, R.J. (1951) J. Biol. Chem. 193, 265-575. Omura, T. and Sato, R. (1964) J. Biol. Chem. 239, 2370-2385. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S. and Tannenbaum, S.R. (1982)Anal. Biochem. 126, 131-138. Chang, J.Y., Knecht, R. and Braun, D.G. (1981) Biochem. J. 199, 547-555. Ortiz de Montellano, P.R. and Reich, N.O. (1986) Cytochrome P-450, (Ortiz de Montellano, P.R. Ed.) 273-314. Plenum Press, New York. Yonetani, T., Yamamoto, H., Erman, J.E., Leigh, J.S. and Reed, G.H. (1972) J. Biol. Chem. 247, 2447-2455. Ebel, R.E., O'Keeffe, D.H. and Peterson, J.A. (1975) FEBS Lett. 55, 198-201.
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