Purification and Characterization of a Nitric-oxide Synthase from Rat Liver Mitochondria*

Anahit Tatoyan and Cecilia GiuliviDagger

From the Department of Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles, California 90033

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

The biosynthesis of nitric oxide (NO·) in different cell types occurs concomitantly with the conversion of L-arginine to L-citrulline by the enzyme nitric-oxide synthase (NOS). NO· has been identified as a major participant in a number of basic physiological functions such as neurotransmission, vasodilation, and immune response. At the subcellular level, mitochondria have been identified as targets for NO·; however, to date, no unambiguous evidence has been presented to identify these organelles as sources of NO·. In this study, a NOS was isolated to homogeneity from Percoll-purified rat liver mitochondria. Kinetic parameters, molecular weight, requirement of cofactors, and cross-reactivity to monoclonal antibodies against macrophage NOS suggest similarities to the inducible form. However, the constitutive expression of the mitochondrial enzyme and its main membrane localization indicate the presence of either a distinctive isoform or a macrophage isoform containing posttranslational modifications that lead to different subcellular compartments. The detection of NADPH-oxidizing activities and a production of superoxide anion catalyzed by mtNOS and recombinant cytochrome P450 reductase were consistent with the sequence homology reported for these two proteins. Given the role of NO· as cellular transmitter, messenger, or regulator, the presence of a functionally active mitochondrial NOS may have important implications for the intermediary metabolism.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

The paramagnetic molecule nitric oxide (NO·1) is a colorless, monomeric gas that has been used for over 50 years as a spectroscopic probe to examine the metal ligand environment of a variety of metalloproteins (1). The biosynthesis of NO· from L-arginine (L-Arg) occurs via a specific biochemical pathway that includes the enzyme nitric-oxide synthase (EC 1.14.23; NOS) in a wide variety of cell types (2-4). NO· has been identified as a major participant in a number of basic physiological functions such as neurotransmission, vasodilation, and immune response (2-4).

There are at least two types of NOS: a calmodulin-dependent isozyme present constitutively in tissues such as brain (5, 6), endothelial cells (7), platelets (8), and adrenal glands (9); and a calmodulin-independent isoenzyme, induced in liver by treatment of rats with Escherichia coli lipopolysaccharides (10-12) and in macrophages following their activation with lipopolysaccharides or lymphokines (13, 14).

At the subcellular level, a protein antigenically related to NOS has been found in mitochondria from different tissues detected by immunocytochemistry (15-17). However, to our knowledge, no reports have indicated an actual production of NO· by intact mitochondria, despite the presence in mitochondria of substrate/cofactors required for the NOS-catalyzed synthesis of NO·: availability of L-Arg (18, 19), L-Arg transporters (20-22), calmodulin (23, 24), calmodulin-binding proteins (25-27), Ca2+, Ca2+ transporters (28), O2, and NADPH.

Recently, we (29) and others2 have reported that rat liver mitochondria produce NO·. This production has been unequivocally attributed to mitochondria, supported by the low contamination of the mitochondrial preparations with other subcellular fractions and the detection of NO· by two spectroscopic techniques: the controlled oxidation of oxymyoglobin and spin-trapping/electron paramagnetic resonance, both of which were sensitive to NMMA inhibition. However, the role of a NOS was suggested by the modulation of the NO· production by substrates and inhibitors of NOS, as well as by the kinetic constants, which were similar to those of other well characterized NOSs (29).

In this study, a NOS located in the membranes of rat liver mitochondria (mtNOS) was purified to homogeneity, and the enzymatic characteristics of this isoform were studied, yielding unambiguous evidence for the NOS-mediated production of NO· by intact mitochondria. This generation of NO· may have implications for oxygen consumption, ATP production, and oxygen free radical production by mitochondria.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Chemicals and Biochemicals

EDTA, EGTA, sodium succinate, sodium malate, sodium glutamate, mannitol, sucrose, HEPES, bovine serum albumin (fatty-acid free), horse heart myoglobin, horse heart cytochrome c, inhibitors of proteolysis, NADPH, FAD, FMN, L-Arg, and CHAPS were purchased from Sigma. Catalase and superoxide dismutase were obtained from Boehringer Mannheim. Recombinant cytochrome P450 reductase was obtained from Oxygene (Dallas, TX). The spin trap N-methyl-D-glucamine-dithiocarbamate-FeII was purchased from the Oklahoma Medical Research Foundation (Oklahoma City, OK). Oxymyoglobin was obtained as described before for oxyhemoglobin (30). Dimethoxynaphthoquinone was kindly provided by Dr. E. Cadenas. The antibodies against inducible, neuronal, and endothelial NOS were purchased from Transduction Laboratories (Lexington, KY). The antibodies against iNOS were obtained using a 21-kDa protein fragment corresponding to amino acids 961-1144 of mouse macNOS as immunogen. All other reagents were of analytical grade.

Biological Materials

Isolation of Rat Liver Mitochondrial Preparations-- Liver mitochondria were isolated from adult Wistar rats (180-200 g) by differential centrifugation (31). Purified mitochondria were obtained by Percoll gradient centrifugation (32) of the mitochondrial fraction, followed by two washings with high ionic strength solutions (150 mM KCl; Ref. 33). Toluene-permeabilized mitochondria were obtained as described in the accompanying paper (29). The reaction mixtures used with these preparations were supplemented with 8.5% polyethylene glycol (8 kDa).

Purification of mtNOS-- Purified mitochondria from two to four rat livers, obtained as described above, were homogenized with Buffer A (1 mM EDTA, 5 mM beta -mercaptoethanol, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml pepstatin A, 100 µg/ml phenylmethylsulfonyl fluoride, 50 mM Hepes, pH 7.5). This homogenate, or 8,000 × g pellet, was centrifuged at 150,000 × g for 1 h at 0-4 °C. The mitochondrial membranes were then washed with Buffer B (Buffer A plus 1 M KCl, 10% glycerol) and centrifuged at the same speed for 30 min. The pellet was treated with Buffer A plus 20 mM CHAPS at 4 °C. After 30 min, the suspension was centrifuged at 150,000 × g for 30 min. This supernatant, concentrated in a Centricon-30TM cartridge (Amicon, Danvers, MA), was called crude fraction (150,000 × g supernatant). This fraction was subsequently purified by affinity chromatography on a 2',5'-ADP-Sepharose column (5 ml of resin; Amersham Pharmacia Biotech) pre-equilibrated with Buffer C (10 mM CHAPS, 0.5 mM L-Arg, 0.5 mM EDTA, 0.5 mM EGTA, 2 mM dithiothreitol, 7 mM reduced glutathione, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml pepstatin, 2 µM tetrahydrobiopterin, 20 mM Hepes, pH 7.5). The column was subsequently washed with Buffer C, Buffer D (Buffer C plus 0.5 M NaCl, 3 mM malic acid, 0.2 mM NADP+), and again Buffer C. NOS activity was eluted with Buffer E (Buffer C plus 5 mM NADPH, 0.75 mM NADP+, 15 mM NaCl). Fractions that showed NOS activity were loaded onto a Sephadex G-200 column (20 ml resin) pre-equilibrated with 3 mM dithiothreitol, 1 mM L-Arg, 0.2 M NaCl, 10% glycerol, and 40 mM Hepes, pH 7.5. Fractions that exhibited NOS activity and migrated as a single band of approximately 125-130 kDa on SDS-PAGE were pooled, concentrated in Centricon-30TM cartridge to final protein concentration of 0.1-0.5 mg/ml, supplemented with 10% glycerol, 0.5-1 mM L-Arg, and 2-5 µM THB4, and stored at -80 °C.

Biochemical Assays

SDS-PAGE, Native PAGE, and Western Blot-- Different fractions obtained during the purification of mtNOS (aliquots from 8,000 × g pellet, 50,000 × g supernatant, and the eluate from the 2', 5'-ADP Sepharose 4B column) were separated by SDS-PAGE under denaturing and reducing conditions using polyacrylamide gels (precast 10% polyacrylamide mini-gels from Novex, San Diego, CA or 4-15% polyacrylamide PhastGels from Amersham Pharmacia Biotech). Samples were heated for 5 min at 95 °C in a sample buffer that contained 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 2.5% (w/v) SDS, 2.5% mercaptoethanol. After cooling the samples, 0.01% (w/v) bromphenol blue was added, and 1-1.5 µg of protein was loaded per lane. Novex gels were run with Laemmli buffer at 125 V for 90 min. PhastGels were run with PhastGel SDS buffer strips containing 0.2 M Tricine, 0.2 M Tris, 0.55% SDS, pH 8.1, and were electrophoresed using a PhastSystem apparatus (Amersham Pharmacia Biotech), at 250 V, 10 mA, at 15 °C for 70 V-h. The separated proteins were stained with Coomassie Blue. The molecular weight markers (Amersham Pharmacia Biotech) underwent the same treatment as the samples prior to electrophoresis. For Western blot analysis, the gradient gels were washed and equilibrated in transfer buffer (25 mM Tris, 192 mM glycine, pH 8.3) for 5 min and the proteins were blotted by diffusion for 2 h on nitrocellulose membranes (0.2-µm pore size; ProtranTM from Schleicher & Schuell). The membranes were blocked with 5% nonfat dry milk, 0.05% Tween 20, in Tris-buffered saline (TBS, 150 mM NaCl, 10 mM Tris-HCl, pH 7.6) for 30 min. The membranes were thoroughly washed with 0.05% Tween 20 in TBS, and incubated with mouse monoclonal antibody against macNOS (1/2, 500 in 0.05% Tween 20 in TBS) for 2 h. The membranes were extensively washed with 0.05% Tween 20 in TBS, and subsequently incubated with goat antibodies against mouse IgG conjugated with horseradish peroxidase (1/30,000; Bio-Rad) for 1 h. After washing the membranes with 0.05% Tween 20 in TBS, the immunocomplexes were developed using enhanced horseradish peroxidase/luminol chemiluminescence reaction, detected with photographic film (Hyperfilm ECL; Amersham Pharmacia Biotech) recorded after 30 s to 7 min of exposure. The positions of various molecular weight markers (in kDa) are indicated in Fig. 1. A lysate of mouse macrophage RAW 264.7 cell line, provided by Transduction Laboratories, was used as a positive control. Native PAGE was performed using 4-15% gradient polyacrylamide gels as described above, and the PhastGel native buffer strips containing 0.88 M alanine and 0.25 M Tris, pH 8.8. The proteins were loaded at 1-2 µg/lane and separated under the following conditions: 400 V, 10 mA, 2.5 watts at 15 °C for 120 V-h. The proteins were stained with Coomassie Blue.

Measurement of NO· Production-- The reaction medium used to follow NO· production contained 1 mM L-Arg, 1 mM magnesium acetate (added only in crude preparations), 1 mM CaCl2, 0.1 mM NADPH, 12 µM THB4, 10 mM CHAPS, in 0.1 M Hepes buffer, pH 7.5 (34), and 0.05-0.5 mg of protein. If required, the equivalent of 50-200 nM FMN were added to the preparations. The enzyme activity was monitored by absorption spectrophotometry by following the controlled oxidation of oxymyoglobin as described in the accompanying article (29). The oxidation of oxymyoglobin to metmyoglobin, sensitive to NMMA inhibition and in the presence of 1 µM superoxide dismutase and catalase, was followed at 581-592 nm in a double-beam spectrophotometer Hitachi U-3110 with a multiple wavelength program at 22 °C in the presence of 50 µM oxymyoglobin.

Measurement of Superoxide Anion Production-- The rates of cytochrome c reduction were measured by following the increase in the absorbance at 550-540 nm in a Hitachi U-3110 spectrophotometer with a multiple wavelength program. The reaction mixtures contained either 0.75 µg of cytochrome P450 reductase/ml or 0.56 µg of purified mtNOS/ml, 20 µM cytochrome c, 0.2 mM NADPH, in 0.2 M potassium phosphate buffer, without or with 10 µM Cu,Zn-superoxide dismutase. The rates of Obardot 2 production were calculated as the rates of cytochrome c reduction sensitive to superoxide dismutase inhibition (35).

    RESULTS AND DISCUSSION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Purification of mtNOS from Rat Liver Mitochondria

The starting material for the isolation of mtNOS was a highly purified preparation of rat liver mitochondria. The isolation procedure (differential centrifugation (31), followed by Percoll centrifugation (32), and washings with high ionic strength solutions (33)) yielded a preparation with high respiratory control ratios (4 to 6 with 10 mM succinate and 0.25 mM ADP; Ref. 29) indicating intact and functional organelles, and a low degree of nonmitochondrial contamination (1-4%; Ref. 29). Using these preparations, a modified procedure for isolating mtNOS was developed.

The purification procedure consisted of lysis of the organelles, followed by ultracentrifugation, solubilization of the enzyme with CHAPS, and separation by affinity chromatography (Table I). This procedure yielded a preparation with a specific activity of 250-350 nmol of NO·/min/mg of protein (inhibitable by NMMA) with a Km for L-Arg of 3 ± 0.4 µM. Although this procedure did not allow the assignment of the enzyme activity to a particular mitochondrial membrane, the finding of enzymatic activity in submitochondrial particles (29), the contribution of the inner membrane protein (80-90%; Ref. 36) to the total membrane protein, and the small NOS activity (10-20%) observed in soluble fractions (matrix and intermembrane compartment), suggested that mtNOS was localized mainly in the inner membrane.

                              
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Table I
Purification of rat liver mtNOS
The activities of the fractions were measured with oxymyoglobin (50 µM) at 581-592 nm using the experimental conditions described under "Materials and Methods" plus 5 µg of calmodulin/ml.

SDS-PAGE gels (37) performed with preparations following affinity and gel exclusion chromatographies resulted in a major band (Fig. 1A). The molecular weight of this band, interpolated in a plot of R F versus log Mr of protein markers (r = 0.98), was 125-130 kDa, similar to that of macNOS monomer (Fig. 1A; Refs. 13 and 38). Purified mtNOS was highly unstable, especially if frozen without glycerol, L-Arg, and tetrahydrobiopterin. The loss of activity could be explained in part by the occurrence of other bands of both lower Mr and intensity produced after several cycles of freeze-thawing, suggesting the presence of proteolytic fragments (data not shown). PAGE of the ADP-Sepharose concentrate performed under nondenaturing conditions resulted in a band of 230 kDa, thus indicating that the enzyme was dimeric under native conditions (Fig. 1B).


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Fig. 1.   SDS-PAGE, native PAGE, and Western blot analysis of mtNOS. Panel A, SDS-PAGE was performed using a 10% polyacrylamide precast gel (Novex, San Diego, CA) under reducing conditions. The proteins were stained with Coomassie Blue. The mitochondrial fractions were, from left to right: I, 8,000 × g pellet; II, 150,000 × g supernatant; III, NADPH eluate from the 2',5'-ADP Sepharose 4B column. Mac-Lysate, a lysate of the mouse macrophage RAW 264.7 cell line. The molecular mass of protein markers is indicated in kDa. Panel B, native PAGE was performed using 4-15% gradient polyacrylamide gel stained with Coomassie Blue using PhastSystem from Amersham Pharmacia Biotech. In the same gel, catalase (232 kDa) was run under identical conditions. Panel C, for Western blot analysis, proteins were separated with SDS-PAGE gel under the conditions described under "Materials and Methods." The proteins were transferred to nitrocellulose membranes, and later were incubated with mouse monoclonal antibodies against macNOS. The immunocomplexes were developed using enhanced horseradish peroxidase/luminol chemiluminescence reaction, detected with photographic film.

Western blotting analysis of the sample, performed with specific monoclonal antibodies to iNOS, demonstrated that mtNOS was antigenically related to the macrophage isoform (Fig. 1C). No immunoreactivity was observed with monoclonal antibodies against either brain or endothelial NOSs.

Characterization of mtNOS Enzymic Activity

Cofactor Requirement of mtNOS-- mtNOS, purified in the presence of L-Arg and THB4 and later subjected to extensive dialysis, showed an absolute requirement for L-Arg, NADPH, THB4, and FAD (Fig. 2). (The sample was dialyzed using a Mr cut-off membrane of 50 kDa against 20 mM Hepes, pH 7.5, 20% glycerol for 24 h with three changes of buffer in a volume ratio 1/4,000.) The strict dependence of the mtNOS activity on micromolar concentrations of FAD conflicted with the tight binding of this prosthetic group to NOS (as it is described for cytochrome P450 reductase, the homologous protein of NOS; Ref. 39). The reconstitution of the activity by FAD addition was explained by the presence of 1-3% FMN contamination in the commercial preparations of FAD, evidenced by high performance liquid chromatography analysis. Thus, the supplementation of the enzyme with 5 µM FAD, which carried a contribution of 50-150 nM FMN, or the addition of purified FMN (at concentrations similar to those required for the other isoforms to exhibit optimal activity) were sufficient to reconstitute the enzymatic activity.


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Fig. 2.   Requirement of cofactors for mtNOS's activity. mtNOS, isolated according to the purification procedure described under "Materials and Methods," was dialyzed (main panel) or chromatographed (inset) through a Sephadex G-25 column. The requirement of cofactors was studied by omitting one compound at a time from the following reaction mixture: 1 mM L-Arg, 0.1 mM NADPH, 0.1 mM CaCl2, 5 µg/ml calmodulin, 5 µM THB4, 5 µM FAD in 20 mM Hepes, pH 7.5. The activities were monitored by the controlled oxidation of oxymyoglobin using 2-3 µg of enzyme/ml at room temperature.

A dependence on free Ca2+ was observed when either Ca2+ addition was omitted or EGTA was added to the reaction mixture, resulting in 25% and 7% of the control activities, respectively (Fig. 2). A 70% loss of enzymatic activity was observed when calmodulin was omitted from the reaction mixture (Fig. 2). This indicates that, even under conditions of extensive dialysis, calmodulin was still tightly bound to the enzyme, thus suggesting that the enzyme may exist as a calmodulin-independent form under physiological conditions.

Consistent with this possibility, when mtNOS was purified in the presence of L-Arg and THB4 and desalted through Sephadex G-25 chromatography (5-ml column, elution buffer 20 mM Hepes, pH 7.6, 20% glycerol; Fig. 2, inset), the activity increased 30-40% associated with a variable dependence on exogenous THB4, Ca2+, and calmodulin in the assay mixture (Fig. 2, inset). It may be surmised that this partial dependence of NO· production on Ca2+/calmodulin was related to the modified desalting procedure (Fig. 2).

The instability of pterin-deficient and/or L-Arg-free enzyme over time was demonstrated by assaying mtNOS after storing the purified enzyme for 24 h at -80 °C. Supplementation of the medium with either 5 µM tetrahydrobiopterin or 20 µM L-Arg recovered 30-40% of the original activity. However, longer storage periods under these suboptimal conditions led to a rapid decrease in the enzymatic activity.

Cytochrome P450 Reductase-like Activity of mtNOS-- All the isoforms so far isolated share 50-60% amino acid homology, diverging most in the calmodulin binding domain. They also exhibit a C-terminal domain somewhat homologous to cytochrome P450 reductase, which has a reductase-like activity (39, 40). Given the similarities between NOS and cytochrome P450 reductase, it was interesting to study the NADPH-oxidizing activity and the production of Obardot 2 by mtNOS and recombinant cytochrome P450 reductase.

NADPH-oxidizing Activity of mtNOS-- mtNOS showed a cytochrome P450 reductase-like activity (superoxide dismutase-insensitive) in the presence of either cytochrome c or dimethoxynaphthoquinone as electron acceptors (Table II). These rates were 4-14 times faster than the formation of NO· (about 0.3 µmol of NO·/min/mg of protein), consistent with the values reported for brain NOS (10.2 µmol of cytochrome c2+/min/mg of protein versus 0.1-1.3 µmol of NO·/min/mg of protein; Ref. 40). Negligible rates of NADPH oxidation were observed if Ca2+ and calmodulin were omitted from the reaction mixture (data not shown) indicating an absolute requirement for Ca2+ and calmodulin to exhibit this activity, in contrast with cytochrome P450 reductase activity. Compared with the activity of recombinant cytochrome P450 reductase under identical experimental conditions, the mtNOS showed 4-7 times lower reductase activity.

                              
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Table II
NADPH-oxidizing activities of cytochrome P450 reductase and purified mtNOS
The activities were measured using 1.4 µg of enzyme/ml in 0.2 M potassium phosphate buffer, pH 7.4, in the presence of 200 µM NADPH at 340 nm. The rates of NADPH consumption were calculated in the linear range with the electron acceptor concentration.

Superoxide Anion Production by mtNOS-- At suboptimal concentrations of L-Arg, porcine brain NOS generated H2O2 (41, 42), likely originated from the dismutation of Obardot 2. Although the mechanism of Obardot 2 production by NOS is still not clear, this radical species is probably produced during the autoxidation of the flavin semiquinones and/or the reduction of O2 by the reduced heme. The production of Obardot 2, detected by the spin trapping/EPR technique, was observed with purified brain NOS (43). Similarly, purified mtNOS produced Obardot 2, as determined by monitoring the superoxide dismutase-inhibitable reduction of cytochrome c (Table III). A negligible production was observed in the absence of Ca2+, calmodulin, or NADPH. Cytochrome P450 reductase also produced Obardot 2, albeit at a rate 10 times faster (Table III). The rates of Obardot 2 production by mtNOS accounted for 13% of the rate of NADPH consumption (Table II) and 22% of the total rate of cytochrome c reduction (Table III, in the absence of superoxide dismutase).

                              
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Table III
Production of superoxide anion by cytochrome P450 reductase and purified mtNOS
The rates of cytochrome c reduction were measured by following the increase in the absorbance at 550-540 nm in a reaction mixture containing 20 µM cytochrome c, 0.2 mM NADPH, in 0.2 M potassium phosphate buffer (- SOD), plus 10 µM Cu,Zn-superoxide dismutase (+ SOD). The rates of superoxide anion production were calculated as the rates of cytochrome c reduction sensitive to SOD inhibition (35).

Biological Significance

This study provides unambiguous evidence for the presence of a constitutive, active NOS in mitochondria. This evidence was furnished by the purification to homogeneity of a membrane-bound NOS from mitochondria. These latter preparations were thoroughly purified and characterized, ruling out any contribution of enzymatic activities from other subcellular compartments. Moreover, this mtNOS may be ascribed to mitochondria derived from parenchymal cells, because hepatocytes constitute 78% of the parenchymal volume. In contrast, the mitochondrial membranes of nonparenchymal cells contribute only 0.2% to the total surface area (44). However, further immunohistochemical and/or in situ hybridization studies will be required to verify this aspect.

Based on the characteristics shared by mtNOS and macNOS (cross-reactivity with monoclonal antibodies against macNOS, Mr, kinetic constants) it could be suggested that these proteins are similar. However, the constitutive expression (which excludes the inducible NOS found in soluble fractions of rat liver after lipopolysaccharides treatment; Ref. 12) and the main membrane localization of mtNOS suggest the presence of either a distinctive isoform or a macNOS containing co- or posttranslational modifications that lead to localization in different subcellular compartments similar to that seen with the skeletal muscle neuronal isoform of NOS known as µNOS (45-47). Preliminary experiments support this view based on the similar amino acid analyses of mitochondrial and macrophage isoforms, and the distinctive acylation pattern found with mtNOS.3

In this connection, it has been reported that macrophages have both constitutive and inducible NOS (48), the latter stimulated by interferon-gamma and lipopolysaccharides present in soluble and particulate fractions. Endothelial cells, apart from having a constitutive particulate isoform (endothelial NOS), also express an iNOS activity after activation with cytokines (49). Although the exact relationship of these constitutive activities to the inducible forms is not yet clear, it could be expected that the former activities are represented to a certain extent by the mitochondrial isoform.

The requirements of mtNOS activity for cofactors (FMN, calmodulin, Ca2+, tetrahydrobiopterin) have allowed the identification of this enzyme with the NOS monooxygenases. NOS isoforms are flavoproteins with a sequence homology to NADPH-cytochrome P450 reductase (39). In this regard, the observed NADPH-oxidizing activity of mtNOS with different electron acceptors, albeit at a slower rate than that of recombinant cytochrome P450 reductase, may have important implications for mitochondrial metabolism, in particular, in oxygen free radical production.

Finally, the production of NO· by mitochondria can be understood as a novel regulatory process designed to modulate cellular ATP production.4 This is based on the predominant role of mitochondria in the maintenance of energy metabolism, the ubiquitous distribution of mitochondria, the immunodetection of NOS in mitochondria from different tissues, and the inhibition of respiration in different biological systems by exogenous NO·. However, based on the reactions of NO· with both O2 and Obardot 2, the production of NO· may also play a role in biological damage. Careful kinetic considerations of the production of NO· and Obardot 2 by mitochondria under different concentrations of O2 and Ca2+ are necessary to evaluate the contribution of NO· in these deleterious reactions (50).

    ACKNOWLEDGEMENT

We are grateful to Dr. E. Cadenas for his generosity and thoughtful comments in support of the ongoing research.

    FOOTNOTES

* This work was supported by the University of Southern California Liver Disease Research Center (Grant P30DK48522 from the National Institutes of Health) and by National Science Foundation Grant MCB 9724060. This work was presented in part at the Meeting of the Society for Free Radicals Research, November 21-23, 1996.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence and reprint requests should be addressed: Dept. of Molecular Pharmacology and Toxicology, University of Southern California, 1985 Zonal Ave., Los Angeles, CA 90033. Tel.: 213-342-1420; Fax: 213-224-7473; E-mail: cgiulivi{at}hsc.usc.edu.

1 The abbreviations used are: NO·, nitric oxide; PAGE, polyacrylamide gel electrophoresis; NMMA, NG-monomethyl-L-arginine; NOS, nitric-oxide synthase; iNOS, macNOS, and mtNOS, inducible, macrophage, and mitochondrial nitric-oxide synthase, respectively; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; TBS, Tris-buffered saline; THB4, (6R)-5,6,7,8-tetrahydrobiopterin; Tricine, N-tris(hydroxymethyl)methylglycine.

2 C. Richter, personal communication.

3 A. Tatoyan and C. Giulivi, unpublished results.

4 C. Giulivi, submitted for publication.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References

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