Vol.
185,
No.
June
30,
1992
NITRIC
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
960-966
OXIDE SYNTHASE FROM CEREBELLUM CATALYZES THE FORMATION OF EQUIMOLAR QUANTITIES OF NITRIC OXIDE AND CITRULLINE FROM LARGININE
Peggy A. Bush, Norma E. Gonzalez, Jeanette M. Griscavage and Louis J. Ignarro Department of Pharmacology, UCLA School of Medicine, Center for the Health Sciences, Los Angeles, California 90024 Received
May 13,
1992
SUMMARY: This study examined whether constitutive nitric oxide (NO) synthase from rat cerebellum catalyzes the formation of equimolar amounts of NO plus citrulline from Larginine under various conditions. Citrulline was determined by monitoring the formation of 3H-citrulline from 3H-L-arginine. NO was determined by monitoring the formation of total NO, (NO + nitrite [NO,] + nitrate [NO,]) by chemiluminescence after reduction of NO, to NO by acidic vanadium (III). Equal quantities of NO plus citrulline were generated from L-arginine and the formation of both products was linear for about 20 min at 37°C provided L-arginine was present in excess to maintain a zero order reaction rate. Deletion of NADPH, addition of the calmodulin antagonist calmidazolium, or addition of NO synthase inhibitors (NG-methyl-L-arginine, NG-amino-L-arginine) abolished or markedly inhibited the formation of both NO and citrulline. The Km for L-arginine (14 PM; 18 PM) and the Vmax of the reaction (0.74 nmol/min/mg protein; 0.67 nmol/min/mg protein) were the same whether NO or citrulline formation, respectively, was monitored. These observations indicate clearly that NO and citrulline are formed in equimolar quantities from L-arginine by the constitutive isoform of NO synthase from rat cerebellum. 0 1992?.cademc Pre**, Inc.
NO synthase catalyzes the conversion of L-arginine to NO plus L-citrulline
(l-3).
Several isoforms of NO synthase have been identified (4). Three principal isoforms are a cytosolic inducible enzyme present in activated rodent macrophages, a membrane-bound constitutive
enzyme in vascular endothelial cells, and a cytosolic constitutive enzyme in
cerebellum.
A variety of procedures have been used to monitor NO synthase activity. The
most common procedure is monitoring the formation of 3H-citrulline from 3H-L-arginine (58). A second procedure is monitoring NO formation indirectly by coupling the NO synthase reaction to a guanylate cyclase reaction either in cultured fibroblasts (6,7) or with purified guanylate cyclase (8), where cyclic GMP formation is taken as a measure of NO formation. Another indirect measure of NO formation is coupling the NO synthase reaction to a second 0006-291X/92 Copyright All rights
$4.00
0 1992 by Academic Press, of reproduction in anq’ form
Inc. reserved.
960
Vol.
185,
No.
chemical
3,
reaction
methemoglobin,
BIOCHEMICAL
1992
where
NO
AND
is allowed
BIOPHYSICAL
to react with
which is monitored spectrophotometrically
Determination
of 3H-citrulline
measure of the stoichiometry
RESEARCH
formation
oxyhemoglobin
to form
(9).
from 3H-L-arginine
of the enzymatic reaction.
COMMUNICATIONS
provides a direct
Determinations
of NO by the
coupled reactions described above, although very sensitive and reliable, do not provide a measure of the stoichiometry of NO formation from L-arginine. studies provide unequivocal
Therefore, although such
evidence that NO synthase catalyzes the conversion of L-
arginine to NO plus citrulline,
they do not indicate whether NO synthase catalyzes the
formation of equimolar quantities of NO and citrulline from L-arginine. this important
In order to answer
question, it is necessary to determine the molar conversion of L-arginine to
NO plus citrulline, avoiding the use of two or more coupled reactions. In the present study, NO was determined
by measuring the formation of total NO, from L-arginine by a novel
chemiluminescence procedure and citrulline was determined by measuring the formation of 3H-citrulline
from 3H-L-arginine.
Utilizing these two procedures, the stoichiometry
of the
enzymatic conversion of L-arginine to NO plus citrulline could be determined. MATERIALS
AND METHODS
L-Arginine, L-citrulline, NADPH, calmodulin, calmidazolium, Chemicals and solutions: dithiothreitol, phenylmethylsulfonyl fluoride, pepstatin A, leupeptin, EDTA, and EGTA were purchased from Sigma Chemical Co. Dowex AG50W-X8 (H+ form) 100-200 mesh, Dowex AG l-X8, acetate form, 100-200 mesh, and Tris base (electrophoresis grade) were purchased from Bio-Rad Laboratories. Vanadium (III) chloride was obtained from Aldrich Chemical Co. Sodium nitrite and sodium nitrate were obtained from Fisher Chemical Co. Aquasol- was purchased from Du Pont Company/NEN Research Products. NG-Methyl-Larginine (10) and NG-amino-L-arginine (11) were synthesized as described previously. NO determination: NO was determined as total NO, generated in the enzymatic reaction, which includes NO + NO,‘ + NO,. Almost all of the NO, was present as the oxidized species, NO,- t NO,‘. NO in oxygen-containing solutions is chemically unstable and undergoes rapid oxidation to NO,. The presence of biological tissue catalyzes this oxidation and promotes further oxidation of NO and NO, to NO, (13). Measurement of all three species is necessary in order to determine NO accurately. NO, + NO, were measured by chemiluminescence after sample reduction in boiling acidic vanadium (III) by a modification of a method described previously (12). Acidic vanadium (III) at 98°C quantitatively reduces both NO; and NO, to NO, which is quantified by a chemiluminescence detector (Dasibi Chemiluminescence NO, Analyzer, model 2108; Glendale, CA) after reaction with ozone. A schematic diagram of the apparatus is illustrated in Fig. 1. Samples (100 ~1) were injected with a gas-tight syringe into 100 ml of 0.1 M vanadium (III) chloride in 2 N HCl at 98°C under an atmosphere of nitrogen. Signals from the detector were analyzed with a Hewlett Packard HP 3396 Series II Integrator and recorded as areas under the curve. Extensive experimentation was conducted to validate and standardize this procedure for the quantification of varying proportions of NO; and NO; (Fig. 2). Standard curves for NO;, NO;, and combinations of both anions were linear over the range of 100 pmol to 4 nmol of NO,- (NOz- and/or NO,). All sample concentrations of NO,- fell within this range. 961
Vol.
185, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Schematic diagram of the apparatus used to measure NO,. Code: A, oxygen-free nitrogen cylinder; B, nitric oxide (825 ppb) in oxygen-free nitrogen cylinder; C, heating mantle; D, 3-neck round bottom flask; E, condenser; F, refrigerated circulator; G, liquid trap; H, acidic vapor trap (sodium hydroxide pellets); I, NO, chemiluminescence analyzer including detector; J, integrator and recorder.
JggJ.
Citrulline determination: Citrulline was determined by monitoring the formation of 3Hcitrulline from 3H-L-arginine by a modification of a procedure described previously (5).
Samples (2 ml) prepared as described below were applied to columns (1 cm diameter) containing 1 ml of Dowex AG50W-X8, Nat form (prepared from the H+ form), that had been pre-equilibrated with 20 mM sodium acetate, pH 5.5, containing 1 mM L-citrulline, 2
0 10
15
20
25
10
Total
[NO,-]
15
20
25
PM
Bar graph showing the quantification of either nitrite or nitrate in mixtures of the two at 98°C. Data are expressed as nitric oxide (AUC; area under the curve in relative units). Left panel: four different concentrations of NO,‘ (NaNO,: 0,5 PM, 10 PM, 15 PM) were added to a single concentration of NO, (10 PM NaNO,). Right Panel: four different concentrations of NO, (NaNO,: 0, 5 PM, 10 PM, 15 MM) were added to a single concentration of NO, (10 PM NaNO,). Total [NO;] signifies the sum of the final concentrations of NO,’ plus NO,. The PM concentration values on the x-axis represent absolute quantities of 1, 1.5,2.0, and 2.5 nmol present in the 100 ~1 of solution injected into the reduction flask. Data represent the mean 1 S.E.M. of duplicate determinations from 4 separate experiments for each test condition.
F&J.
962
Vol.
185, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
mM EDTA, and 0.2 mM EGTA (Stop Buffer). The eluate (2 ml) was collected into a liquid scintillation vial. Columns were eluted with 2 ml of water and collected into another vial. Aquasol- (10 ml) was added to each vial and samples were counted in a Beckman LS 3801 liquid scintillation spectrometer. Citrulline was recovered in the first 4 ml of Dowex column eluate to the extent of 96%, and data were corrected to account for such recovery. Protein concentrations in cerebellum supernatant fractions Protein determination: were determined by the Bradford, Coomassie brilliant blue method as described by Bio-Rad. Bovine serum albumin was used as the standard. Rat cerebellum was used as the source of NO synthase. Rats NO svnthase assav: (Sprague Dawley, males, 150-175 g) were sacrificed by decapitation and the cerebella were excised, rinsed and stored frozen at -75°C. Homogenates (25% w/v) of cerebellum were prepared in 50 mM Tris HCl, pH 7.4, containing 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 PM pepstatin A, and 2 PM leupeptin at 0-4°C with the aid of a tissue grinder fitted with a ground glass pestle. Homogenates were centrifuged at 20,000 x g for 60 min at 4°C and the supematant was used as the source of NO synthase. Enzymatic reactions were conducted at 37°C in 50 mM Tris HCl, pH 7.4, containing 100 PM L-arginine, 100 PM NADPH, 2 mM CaCl,, 1 pg calmodulin, 0.20 - 0.40 mg supernatant protein, and other test agents as indicated, in a finai incubation volume of 100 ~1. Samples that were analyzed for citrulline also contained approximately 200,000 dpm of L-[2,3,4,5-3H]arginine HCl (77 Ci/mmol; Amersham) that was previously purified by anionic exchange chromatography on columns of Dowex AG l-X8, OH- form (prepared from the acetate form), 100-200 mesh in order to remove traces of contaminating 3Hcitrulline (6). Enzymatic reactions for the determination of citrulline were terminated by addition of 2 ml of ice-cold Stop Buffer, and samples were chromatographed as described above. Enzymatic reactions for the determination of NO did not contain 3H-L-arginine and were terminated by addition of 200 1.11of ice-cold 50 mM Tris HCl, pH 7.4, containing 10 mM EDTA. Aliquots of 100 ~1 were assayed for NO, by chemiluminescence after chemical reduction to NO as described above. RESULTS AND DISCUSSION The constitutive conversion of L-arginine
isoform of NO synthase from rat cerebellum to equimolar
catalyzed the
quantities of NO plus citrulline,
and product
formation was linear for about 20 min at 37°C under the defined assay conditions (Fig. 3). Since the chemical half-life of NO is only several seconds under assay conditions in the required presence of oxygen, it is not possible to monitor the formation of NO itself. NO undergoes spontaneous oxidation to NO, in the presence of oxygen and catalyzed oxidation to NO;
in the presence of superoxide anion, contaminating
oxyhemoproteins, quantitatively
and other tissue-derived
oxidants (13).
oxyhemoglobin
and other
Both NO,- and NO;
are
reduced back to NO by refluxing in acidic vanadium (III), as discussed in
Materials and Methods.
Thus, the determination
of both NO, and NO, accumulation in
a sample represents a direct measure of any NO that had been formed in that sample. In order to ascertain whether NO formation and citrulline formation are influenced in a quantitatively
identical manner by changes in assay conditions, experiments were
conducted in which complete enzyme reaction mixtures were compared with reaction 963
Vol.
185,
No.
3,
BIOCHEMICAL
1992
16 5, .= .5 t; q
2 ‘ri ii
AND
0
Citrulline
0
NO
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
12.
0
5
10
15
20
Time
25
30
(min)
Time course of NO and citrulline formation from L-arginine by NO synthase from cerebellum. Enzymatic reactions were conducted at 37°C in 50 mM Tris HCI, pH 7.4, containing 100 /.LM L-arginine, 100 PM NADPH, 2 mM CaCI,, 1 pg calmodulin, and supernatant containing 0.22 - 0.40 mg protein in a final volume of 100 ~1. Reaction mixtures that -were assayed for 3H-citrulline contained approximately 200,000 dpm of L-[2,3,4,53H]arginine. Data points represent mean values of 4 - 8 determinations from 2 - 4 separate experiments.
JQgJ*
mixtures deficient in NADPH, containing calmodulin antagonists, or containing NO synthase inhibitors.
The constitutive isoform of NO synthase from cerebellum requires the presence
of NADPH for reducing equivalents and calmodulin for formation of the calcium-calmodulin complex, which activates the enzyme (7,14). NG-substituted analogs of L-arginine act as competitive NADPH
inhibitors of NO synthase activity (11,15).
nearly abolished the formation
calmodulin formation
of both NO and citrulline.
antagonist, nearly abolished the formation
Methyl-L-arginine,
an NO synthase inhibitor
of both NO and citrulline.
inhibitor than No-methyl-L-arginine
Fig. 4 illustrates that deletion of Calmidazolium,
of both NO and citrulline.
(15), produced a 83% - 87% inhibition
NG-Amino-L-arginine,
a NGof
a more potent NO synthase
(1 l), produced a 94% - 96% inhibition
of formation of
both NO and citrulline. Another experimental approach taken to ascertain whether the stoichiometry of the NO synthase reaction is identical for both NO and citrulline was to determine the Km for L-arginine and the Vmax of the enzymatic reaction on the basis of measurements of each of the two reaction products. Kinetic experiments were conducted under initial velocity conditions, where the substrate concentration was not limiting. Double reciprocal plots of velocity versus substrate concentration citrulline
were measured.
were constructed for reactions where NO and
The curves were nearly superimposable
similar values for Km and Vmax.
and yielded closely
The Km for L-arginine was 14 PM when NO was
measured and 18 PM when citrulline was measured. The Vmax of the enzymatic reaction 964
Vol.
185,
No.
3,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
0.6
0.5
0
Control
- NADPH
+ CM2 -
+ L-NMA
0.1 mM Calmodulin
0.3 mht
+
L-NAA 0.3 mt.t
Dependence of NO and citrulline formation from L-arginine by NO synthase from cerebellum on NADPH and calmodulin, and inhibition by NG-methyl-L-arginine and NGamino-L-arginine. Enzymatic reactions were conducted at 37°C for 1.5min in 50 mM Tris HCl, pH 7.4, containing 100 PM L-arginine, 100 I.IM NADPH (except -NADPH), 2 mM CaCl,, 1 pg calmodulin (except -calmodulin), and supernatant containing 0.40 mg protein in a final volume of 100 ~1. Certain reaction mixtures contained calmidazolium (CMZ), NGmethyl-L-arginine (L-NMA), or NG-amino-L-arginine (L-NAA) as indicated. Reaction mixtures that were assayed for 3H-citrulline contained approximately 200,000 dpm of L[2,3,4,S3H]arginine. Data represent the mean + S.E.M. of 4 - 8 determinations from 2 - 4 separate experiments.
F&&.
was 0.74 nmol/min/mg when citrulline
protein when NO was measured and 0.67 nmol/min/mg
was measured.
protein
These observations, like those shown in Figs. 3 and 4,
indicate that equimolar quantities of NO plus citrulline were generated from L-arginine by NO synthase. The Km values for L-arginine in the present study were slightly higher than the Km value of 2.2 PM published using purified presumably because the unpurified endogenous L-arginine,
NO synthase from cerebellum
supernatant fractions contained low concentrations
synthase from rat cerebellum catalyzes the formation from L-arginine
isoform of NO
of equimolar quantities of NO plus
under the defined assay conditions.
These observations
consistent with the hypothesis that NO synthase catalyzes the monohydroxylation the two equivalent NADPH-dependent
guanidinium
nitrogen atoms of L-arginine to N-hydroxy-L-arginine
mechanisms (16-18). The NG-hydroxy-L-arginine
intermediate
likely NG-nitroso-L-arginine
are
of one of by
must be
further oxidized prior to the ultimate formation of NO. Thus, a second intermediate,
L-citrulline
of
which was not removed prior to enzyme assays.
The data presented in this study reveal clearly that the constitutive
citrulline
(7),
most
(16), may be formed, which undergoes further oxidation to form
in addition to NO. This reaction sequence catalyzed by NO synthase would
result in the conversion of L-arginine to equimolar quantities of NO and L-citrulline, major finding of the present study. 965
the
Vol.
185,
No.
3,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
ACKNOWLEDGMENTS
This work was supported in part by U.S.P.H.S. grants HL35014 and HI.40922, and a grant from the Laubisch Fund for Cardiovascular Research. The authors are grateful to Russell E. Byrns for his expert technical assistance in conducting the NO chemiluminescence experiments and preparing the illustrations. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Tayeh, M.A. and Marletta, M.A. (1989) J. Biol. Chem. 264: 19654-19658. Kwon, N.S., Nathan, C.F. and Stuehr, D.J. (1989) J. Biol. Chem. 264: 20496-20501. Forstermann, U., Gorsky, L.D., Pollock, J.S., Ishii, K., Schmidt, H.H.H.W., Heller, M. and Murad, F. (1990) Mol. Pharmacol. 38: 7-13. Forstermann, U., Schmidt, H.H.H.W., Pollock, J.S., Sheng, H., Mitchell, J.A., Warner, T.D., Nakane, M. and Murad, F. (1991) Biochem. Pharmacol. 42: 1849-1857. Bredt, D.S. and Snyder, S.H. (1989) Proc. Natl. Acad. Sci. USA 86: 9030-9033. Pollock, J.S., Forstermann, U., Mitchell, J.A., Warner, T.D., Schmidt, H.H.H.W., Nakane, M. and Murad, F. (1991) Proc. Natl. Acad. Sci. USA 88: 10480-10484. Schmidt, H.H.H.W., Pollock, J.S., Nakane, M., Gorsky, L.D., Forstermann, U. and Murad, F. (1991) Proc. Natl. Acad. Sci. USA 88: 365-369. Mayer, B., John, M. and Bohme, E. (1991) J. Cardiovasc. Pharmacol. 17: S46-S51. Hevel, J.M., White, K.A. and Marletta, M.A. (1991) J. Biol. Chem. 266: 22789-22791. Gold, M.E., Wood, K.S., Byrns, R.E., Fukuto, J. and Ignarro, L.J. (1990) Proc. Natl. Acad. Sci. USA 87: 4430-4434. Fukuto, J.M., Wood, K.S., Byrns, R.E. and Ignarro, L.J. (1990) Biochem. Biophys. Res. Commun. 168: 458-465. Braman, R.S. and Hendrix, S.A. (1989) Anal. Chem. 61: 2715-2718. Ignarro, L.J. (1990) Annu. Rev. Pharmacol. Toxicol. 30: 535-560. Bredt, D.S. and Snyder, S.H. (1990) Proc. Natl. Acad. Sci. USA 87: 682-685. Palmer, R.M.J., Rees, D.D., Ashton, D.S. and Moncada, S. (1988) Biochem. Biouhys. Res. Commun. 153: 1251-1256. Marletta, M.A., Yoon, P.S., Iyengar, R., Leaf, C.D. and Wishnok, J.S. (1988) Biochemistry 27: 8706-8711. Marletta, M.A. (1989) T&S 14: 488-492. Stuehr, D.J., Cho, H.J., Kwon, N.S. and Nathan, C.F. (1991) Proc. Natl. Acad. Sci. USA 88: 7773-7777.
966
185,
No.
June
30,
1992
NITRIC
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
960-966
OXIDE SYNTHASE FROM CEREBELLUM CATALYZES THE FORMATION OF EQUIMOLAR QUANTITIES OF NITRIC OXIDE AND CITRULLINE FROM LARGININE
Peggy A. Bush, Norma E. Gonzalez, Jeanette M. Griscavage and Louis J. Ignarro Department of Pharmacology, UCLA School of Medicine, Center for the Health Sciences, Los Angeles, California 90024 Received
May 13,
1992
SUMMARY: This study examined whether constitutive nitric oxide (NO) synthase from rat cerebellum catalyzes the formation of equimolar amounts of NO plus citrulline from Larginine under various conditions. Citrulline was determined by monitoring the formation of 3H-citrulline from 3H-L-arginine. NO was determined by monitoring the formation of total NO, (NO + nitrite [NO,] + nitrate [NO,]) by chemiluminescence after reduction of NO, to NO by acidic vanadium (III). Equal quantities of NO plus citrulline were generated from L-arginine and the formation of both products was linear for about 20 min at 37°C provided L-arginine was present in excess to maintain a zero order reaction rate. Deletion of NADPH, addition of the calmodulin antagonist calmidazolium, or addition of NO synthase inhibitors (NG-methyl-L-arginine, NG-amino-L-arginine) abolished or markedly inhibited the formation of both NO and citrulline. The Km for L-arginine (14 PM; 18 PM) and the Vmax of the reaction (0.74 nmol/min/mg protein; 0.67 nmol/min/mg protein) were the same whether NO or citrulline formation, respectively, was monitored. These observations indicate clearly that NO and citrulline are formed in equimolar quantities from L-arginine by the constitutive isoform of NO synthase from rat cerebellum. 0 1992?.cademc Pre**, Inc.
NO synthase catalyzes the conversion of L-arginine to NO plus L-citrulline
(l-3).
Several isoforms of NO synthase have been identified (4). Three principal isoforms are a cytosolic inducible enzyme present in activated rodent macrophages, a membrane-bound constitutive
enzyme in vascular endothelial cells, and a cytosolic constitutive enzyme in
cerebellum.
A variety of procedures have been used to monitor NO synthase activity. The
most common procedure is monitoring the formation of 3H-citrulline from 3H-L-arginine (58). A second procedure is monitoring NO formation indirectly by coupling the NO synthase reaction to a guanylate cyclase reaction either in cultured fibroblasts (6,7) or with purified guanylate cyclase (8), where cyclic GMP formation is taken as a measure of NO formation. Another indirect measure of NO formation is coupling the NO synthase reaction to a second 0006-291X/92 Copyright All rights
$4.00
0 1992 by Academic Press, of reproduction in anq’ form
Inc. reserved.
960
Vol.
185,
No.
chemical
3,
reaction
methemoglobin,
BIOCHEMICAL
1992
where
NO
AND
is allowed
BIOPHYSICAL
to react with
which is monitored spectrophotometrically
Determination
of 3H-citrulline
measure of the stoichiometry
RESEARCH
formation
oxyhemoglobin
to form
(9).
from 3H-L-arginine
of the enzymatic reaction.
COMMUNICATIONS
provides a direct
Determinations
of NO by the
coupled reactions described above, although very sensitive and reliable, do not provide a measure of the stoichiometry of NO formation from L-arginine. studies provide unequivocal
Therefore, although such
evidence that NO synthase catalyzes the conversion of L-
arginine to NO plus citrulline,
they do not indicate whether NO synthase catalyzes the
formation of equimolar quantities of NO and citrulline from L-arginine. this important
In order to answer
question, it is necessary to determine the molar conversion of L-arginine to
NO plus citrulline, avoiding the use of two or more coupled reactions. In the present study, NO was determined
by measuring the formation of total NO, from L-arginine by a novel
chemiluminescence procedure and citrulline was determined by measuring the formation of 3H-citrulline
from 3H-L-arginine.
Utilizing these two procedures, the stoichiometry
of the
enzymatic conversion of L-arginine to NO plus citrulline could be determined. MATERIALS
AND METHODS
L-Arginine, L-citrulline, NADPH, calmodulin, calmidazolium, Chemicals and solutions: dithiothreitol, phenylmethylsulfonyl fluoride, pepstatin A, leupeptin, EDTA, and EGTA were purchased from Sigma Chemical Co. Dowex AG50W-X8 (H+ form) 100-200 mesh, Dowex AG l-X8, acetate form, 100-200 mesh, and Tris base (electrophoresis grade) were purchased from Bio-Rad Laboratories. Vanadium (III) chloride was obtained from Aldrich Chemical Co. Sodium nitrite and sodium nitrate were obtained from Fisher Chemical Co. Aquasol- was purchased from Du Pont Company/NEN Research Products. NG-Methyl-Larginine (10) and NG-amino-L-arginine (11) were synthesized as described previously. NO determination: NO was determined as total NO, generated in the enzymatic reaction, which includes NO + NO,‘ + NO,. Almost all of the NO, was present as the oxidized species, NO,- t NO,‘. NO in oxygen-containing solutions is chemically unstable and undergoes rapid oxidation to NO,. The presence of biological tissue catalyzes this oxidation and promotes further oxidation of NO and NO, to NO, (13). Measurement of all three species is necessary in order to determine NO accurately. NO, + NO, were measured by chemiluminescence after sample reduction in boiling acidic vanadium (III) by a modification of a method described previously (12). Acidic vanadium (III) at 98°C quantitatively reduces both NO; and NO, to NO, which is quantified by a chemiluminescence detector (Dasibi Chemiluminescence NO, Analyzer, model 2108; Glendale, CA) after reaction with ozone. A schematic diagram of the apparatus is illustrated in Fig. 1. Samples (100 ~1) were injected with a gas-tight syringe into 100 ml of 0.1 M vanadium (III) chloride in 2 N HCl at 98°C under an atmosphere of nitrogen. Signals from the detector were analyzed with a Hewlett Packard HP 3396 Series II Integrator and recorded as areas under the curve. Extensive experimentation was conducted to validate and standardize this procedure for the quantification of varying proportions of NO; and NO; (Fig. 2). Standard curves for NO;, NO;, and combinations of both anions were linear over the range of 100 pmol to 4 nmol of NO,- (NOz- and/or NO,). All sample concentrations of NO,- fell within this range. 961
Vol.
185, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Schematic diagram of the apparatus used to measure NO,. Code: A, oxygen-free nitrogen cylinder; B, nitric oxide (825 ppb) in oxygen-free nitrogen cylinder; C, heating mantle; D, 3-neck round bottom flask; E, condenser; F, refrigerated circulator; G, liquid trap; H, acidic vapor trap (sodium hydroxide pellets); I, NO, chemiluminescence analyzer including detector; J, integrator and recorder.
JggJ.
Citrulline determination: Citrulline was determined by monitoring the formation of 3Hcitrulline from 3H-L-arginine by a modification of a procedure described previously (5).
Samples (2 ml) prepared as described below were applied to columns (1 cm diameter) containing 1 ml of Dowex AG50W-X8, Nat form (prepared from the H+ form), that had been pre-equilibrated with 20 mM sodium acetate, pH 5.5, containing 1 mM L-citrulline, 2
0 10
15
20
25
10
Total
[NO,-]
15
20
25
PM
Bar graph showing the quantification of either nitrite or nitrate in mixtures of the two at 98°C. Data are expressed as nitric oxide (AUC; area under the curve in relative units). Left panel: four different concentrations of NO,‘ (NaNO,: 0,5 PM, 10 PM, 15 PM) were added to a single concentration of NO, (10 PM NaNO,). Right Panel: four different concentrations of NO, (NaNO,: 0, 5 PM, 10 PM, 15 MM) were added to a single concentration of NO, (10 PM NaNO,). Total [NO;] signifies the sum of the final concentrations of NO,’ plus NO,. The PM concentration values on the x-axis represent absolute quantities of 1, 1.5,2.0, and 2.5 nmol present in the 100 ~1 of solution injected into the reduction flask. Data represent the mean 1 S.E.M. of duplicate determinations from 4 separate experiments for each test condition.
F&J.
962
Vol.
185, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
mM EDTA, and 0.2 mM EGTA (Stop Buffer). The eluate (2 ml) was collected into a liquid scintillation vial. Columns were eluted with 2 ml of water and collected into another vial. Aquasol- (10 ml) was added to each vial and samples were counted in a Beckman LS 3801 liquid scintillation spectrometer. Citrulline was recovered in the first 4 ml of Dowex column eluate to the extent of 96%, and data were corrected to account for such recovery. Protein concentrations in cerebellum supernatant fractions Protein determination: were determined by the Bradford, Coomassie brilliant blue method as described by Bio-Rad. Bovine serum albumin was used as the standard. Rat cerebellum was used as the source of NO synthase. Rats NO svnthase assav: (Sprague Dawley, males, 150-175 g) were sacrificed by decapitation and the cerebella were excised, rinsed and stored frozen at -75°C. Homogenates (25% w/v) of cerebellum were prepared in 50 mM Tris HCl, pH 7.4, containing 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 PM pepstatin A, and 2 PM leupeptin at 0-4°C with the aid of a tissue grinder fitted with a ground glass pestle. Homogenates were centrifuged at 20,000 x g for 60 min at 4°C and the supematant was used as the source of NO synthase. Enzymatic reactions were conducted at 37°C in 50 mM Tris HCl, pH 7.4, containing 100 PM L-arginine, 100 PM NADPH, 2 mM CaCl,, 1 pg calmodulin, 0.20 - 0.40 mg supernatant protein, and other test agents as indicated, in a finai incubation volume of 100 ~1. Samples that were analyzed for citrulline also contained approximately 200,000 dpm of L-[2,3,4,5-3H]arginine HCl (77 Ci/mmol; Amersham) that was previously purified by anionic exchange chromatography on columns of Dowex AG l-X8, OH- form (prepared from the acetate form), 100-200 mesh in order to remove traces of contaminating 3Hcitrulline (6). Enzymatic reactions for the determination of citrulline were terminated by addition of 2 ml of ice-cold Stop Buffer, and samples were chromatographed as described above. Enzymatic reactions for the determination of NO did not contain 3H-L-arginine and were terminated by addition of 200 1.11of ice-cold 50 mM Tris HCl, pH 7.4, containing 10 mM EDTA. Aliquots of 100 ~1 were assayed for NO, by chemiluminescence after chemical reduction to NO as described above. RESULTS AND DISCUSSION The constitutive conversion of L-arginine
isoform of NO synthase from rat cerebellum to equimolar
catalyzed the
quantities of NO plus citrulline,
and product
formation was linear for about 20 min at 37°C under the defined assay conditions (Fig. 3). Since the chemical half-life of NO is only several seconds under assay conditions in the required presence of oxygen, it is not possible to monitor the formation of NO itself. NO undergoes spontaneous oxidation to NO, in the presence of oxygen and catalyzed oxidation to NO;
in the presence of superoxide anion, contaminating
oxyhemoproteins, quantitatively
and other tissue-derived
oxidants (13).
oxyhemoglobin
and other
Both NO,- and NO;
are
reduced back to NO by refluxing in acidic vanadium (III), as discussed in
Materials and Methods.
Thus, the determination
of both NO, and NO, accumulation in
a sample represents a direct measure of any NO that had been formed in that sample. In order to ascertain whether NO formation and citrulline formation are influenced in a quantitatively
identical manner by changes in assay conditions, experiments were
conducted in which complete enzyme reaction mixtures were compared with reaction 963
Vol.
185,
No.
3,
BIOCHEMICAL
1992
16 5, .= .5 t; q
2 ‘ri ii
AND
0
Citrulline
0
NO
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
12.
0
5
10
15
20
Time
25
30
(min)
Time course of NO and citrulline formation from L-arginine by NO synthase from cerebellum. Enzymatic reactions were conducted at 37°C in 50 mM Tris HCI, pH 7.4, containing 100 /.LM L-arginine, 100 PM NADPH, 2 mM CaCI,, 1 pg calmodulin, and supernatant containing 0.22 - 0.40 mg protein in a final volume of 100 ~1. Reaction mixtures that -were assayed for 3H-citrulline contained approximately 200,000 dpm of L-[2,3,4,53H]arginine. Data points represent mean values of 4 - 8 determinations from 2 - 4 separate experiments.
JQgJ*
mixtures deficient in NADPH, containing calmodulin antagonists, or containing NO synthase inhibitors.
The constitutive isoform of NO synthase from cerebellum requires the presence
of NADPH for reducing equivalents and calmodulin for formation of the calcium-calmodulin complex, which activates the enzyme (7,14). NG-substituted analogs of L-arginine act as competitive NADPH
inhibitors of NO synthase activity (11,15).
nearly abolished the formation
calmodulin formation
of both NO and citrulline.
antagonist, nearly abolished the formation
Methyl-L-arginine,
an NO synthase inhibitor
of both NO and citrulline.
inhibitor than No-methyl-L-arginine
Fig. 4 illustrates that deletion of Calmidazolium,
of both NO and citrulline.
(15), produced a 83% - 87% inhibition
NG-Amino-L-arginine,
a NGof
a more potent NO synthase
(1 l), produced a 94% - 96% inhibition
of formation of
both NO and citrulline. Another experimental approach taken to ascertain whether the stoichiometry of the NO synthase reaction is identical for both NO and citrulline was to determine the Km for L-arginine and the Vmax of the enzymatic reaction on the basis of measurements of each of the two reaction products. Kinetic experiments were conducted under initial velocity conditions, where the substrate concentration was not limiting. Double reciprocal plots of velocity versus substrate concentration citrulline
were measured.
were constructed for reactions where NO and
The curves were nearly superimposable
similar values for Km and Vmax.
and yielded closely
The Km for L-arginine was 14 PM when NO was
measured and 18 PM when citrulline was measured. The Vmax of the enzymatic reaction 964
Vol.
185,
No.
3,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
0.6
0.5
0
Control
- NADPH
+ CM2 -
+ L-NMA
0.1 mM Calmodulin
0.3 mht
+
L-NAA 0.3 mt.t
Dependence of NO and citrulline formation from L-arginine by NO synthase from cerebellum on NADPH and calmodulin, and inhibition by NG-methyl-L-arginine and NGamino-L-arginine. Enzymatic reactions were conducted at 37°C for 1.5min in 50 mM Tris HCl, pH 7.4, containing 100 PM L-arginine, 100 I.IM NADPH (except -NADPH), 2 mM CaCl,, 1 pg calmodulin (except -calmodulin), and supernatant containing 0.40 mg protein in a final volume of 100 ~1. Certain reaction mixtures contained calmidazolium (CMZ), NGmethyl-L-arginine (L-NMA), or NG-amino-L-arginine (L-NAA) as indicated. Reaction mixtures that were assayed for 3H-citrulline contained approximately 200,000 dpm of L[2,3,4,S3H]arginine. Data represent the mean + S.E.M. of 4 - 8 determinations from 2 - 4 separate experiments.
F&&.
was 0.74 nmol/min/mg when citrulline
protein when NO was measured and 0.67 nmol/min/mg
was measured.
protein
These observations, like those shown in Figs. 3 and 4,
indicate that equimolar quantities of NO plus citrulline were generated from L-arginine by NO synthase. The Km values for L-arginine in the present study were slightly higher than the Km value of 2.2 PM published using purified presumably because the unpurified endogenous L-arginine,
NO synthase from cerebellum
supernatant fractions contained low concentrations
synthase from rat cerebellum catalyzes the formation from L-arginine
isoform of NO
of equimolar quantities of NO plus
under the defined assay conditions.
These observations
consistent with the hypothesis that NO synthase catalyzes the monohydroxylation the two equivalent NADPH-dependent
guanidinium
nitrogen atoms of L-arginine to N-hydroxy-L-arginine
mechanisms (16-18). The NG-hydroxy-L-arginine
intermediate
likely NG-nitroso-L-arginine
are
of one of by
must be
further oxidized prior to the ultimate formation of NO. Thus, a second intermediate,
L-citrulline
of
which was not removed prior to enzyme assays.
The data presented in this study reveal clearly that the constitutive
citrulline
(7),
most
(16), may be formed, which undergoes further oxidation to form
in addition to NO. This reaction sequence catalyzed by NO synthase would
result in the conversion of L-arginine to equimolar quantities of NO and L-citrulline, major finding of the present study. 965
the
Vol.
185,
No.
3,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
ACKNOWLEDGMENTS
This work was supported in part by U.S.P.H.S. grants HL35014 and HI.40922, and a grant from the Laubisch Fund for Cardiovascular Research. The authors are grateful to Russell E. Byrns for his expert technical assistance in conducting the NO chemiluminescence experiments and preparing the illustrations. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Tayeh, M.A. and Marletta, M.A. (1989) J. Biol. Chem. 264: 19654-19658. Kwon, N.S., Nathan, C.F. and Stuehr, D.J. (1989) J. Biol. Chem. 264: 20496-20501. Forstermann, U., Gorsky, L.D., Pollock, J.S., Ishii, K., Schmidt, H.H.H.W., Heller, M. and Murad, F. (1990) Mol. Pharmacol. 38: 7-13. Forstermann, U., Schmidt, H.H.H.W., Pollock, J.S., Sheng, H., Mitchell, J.A., Warner, T.D., Nakane, M. and Murad, F. (1991) Biochem. Pharmacol. 42: 1849-1857. Bredt, D.S. and Snyder, S.H. (1989) Proc. Natl. Acad. Sci. USA 86: 9030-9033. Pollock, J.S., Forstermann, U., Mitchell, J.A., Warner, T.D., Schmidt, H.H.H.W., Nakane, M. and Murad, F. (1991) Proc. Natl. Acad. Sci. USA 88: 10480-10484. Schmidt, H.H.H.W., Pollock, J.S., Nakane, M., Gorsky, L.D., Forstermann, U. and Murad, F. (1991) Proc. Natl. Acad. Sci. USA 88: 365-369. Mayer, B., John, M. and Bohme, E. (1991) J. Cardiovasc. Pharmacol. 17: S46-S51. Hevel, J.M., White, K.A. and Marletta, M.A. (1991) J. Biol. Chem. 266: 22789-22791. Gold, M.E., Wood, K.S., Byrns, R.E., Fukuto, J. and Ignarro, L.J. (1990) Proc. Natl. Acad. Sci. USA 87: 4430-4434. Fukuto, J.M., Wood, K.S., Byrns, R.E. and Ignarro, L.J. (1990) Biochem. Biophys. Res. Commun. 168: 458-465. Braman, R.S. and Hendrix, S.A. (1989) Anal. Chem. 61: 2715-2718. Ignarro, L.J. (1990) Annu. Rev. Pharmacol. Toxicol. 30: 535-560. Bredt, D.S. and Snyder, S.H. (1990) Proc. Natl. Acad. Sci. USA 87: 682-685. Palmer, R.M.J., Rees, D.D., Ashton, D.S. and Moncada, S. (1988) Biochem. Biouhys. Res. Commun. 153: 1251-1256. Marletta, M.A., Yoon, P.S., Iyengar, R., Leaf, C.D. and Wishnok, J.S. (1988) Biochemistry 27: 8706-8711. Marletta, M.A. (1989) T&S 14: 488-492. Stuehr, D.J., Cho, H.J., Kwon, N.S. and Nathan, C.F. (1991) Proc. Natl. Acad. Sci. USA 88: 7773-7777.
966
