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J. Biol. Chem., Vol. 276, Issue 37, 34458-34464, September 14, 2001

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Nitric Oxide Inhibits Ornithine Decarboxylase via S-Nitrosylation of Cysteine 360 in the Active Site of the Enzyme^*

Philip M. Bauer, Georgette M. Buga, Jon M. Fukuto, Anthony E. Pegg^§, and Louis J. Ignarro^¶

From the  Department of Molecular and Medical Pharmacology, UCLA School of Medicine, Los Angeles, California 90095-1735 and the ^§ Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033

Received for publication, June 6, 2001, and in revised form, July 16, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ornithine decarboxylase is the initial and rate-limiting enzyme in the polyamine biosynthetic pathway. Polyamines are found in all mammalian cells and are required for cell growth. We previously demonstrated that N-hydroxyarginine and nitric oxide inhibit tumor cell proliferation by inhibiting arginase and ornithine decarboxylase, respectively, and, therefore, polyamine synthesis. In addition, we showed that nitric oxide inhibits purified ornithine decarboxylase by S-nitrosylation. Herein we provide evidence for the chemical mechanism by which nitric oxide and S-nitrosothiols react with cysteine residues in ornithine decarboxylase to form an S-nitrosothiol(s) on the protein. The diazeniumdiolate nitric oxide donor agent 1-diethyl-2-hydroxy-2-nitroso-hydrazine acts through an oxygen-dependent mechanism leading to formation of the nitrosating agents N₂O₃ and/or N₂O₄. S-Nitrosoglutathione inhibits ornithine decarboxylase by an oxygen-independent mechanism likely by S-transnitrosation. In addition, we provide evidence for the S-nitrosylation of 4 cysteine residues per ornithine decarboxylase monomer including cysteine 360, which is critical for enzyme activity. Finally S-nitrosylated ornithine decarboxylase was isolated from intact cells treated with nitric oxide, suggesting that nitric oxide may regulate ornithine decarboxylase activity by S-nitrosylation in vivo.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ornithine decarboxylase (ODC),¹ the rate-limiting enzyme in putrescine synthesis, catalyzes the conversion of ornithine to putrescine and is essential for polyamine synthesis in mammalian cells. Despite the fact that polyamines are present in all mammalian cells, extensive research efforts have failed to fully elucidate their physiological functions. That polyamines are required, however, to maintain cell growth and function is clearly established (1-3). Many early studies indicate that polyamine synthesis is enhanced during growth and that growth-promoting stimuli lead to increases in polyamine biosynthesis. Direct evidence for this has been provided by experiments in which polyamine synthesis was prevented in mammalian cells in culture by mutations to key enzymes (such as ODC) or by the application of enzyme inhibitors (2, 4-6). This led to cessation of growth unless exogenous polyamines were provided. More recent studies have shown that polyamines stimulate DNA synthesis and increase the transcription of growth-related genes (7-10).

Increases in nitric-oxide synthase activity and addition of exogenous NO have been widely recognized to result in inhibition of cell proliferation (11-15). In many of these studies, cyclic GMP did not account for the cytostatic effect of NO, and the evidence for cyclic GMP-dependent inhibition of cell proliferation is at best inconsistent and indirect. We have consistently observed cyclic GMP-independent cytostatic effects of NO in a variety of mammalian cell types including Caco-2 human tumor cells, murine macrophages, and rat aortic endothelial and smooth muscle cells (15, 16).² One such mechanism appears to be the inhibition of ODC by NO. In our studies, the cytostatic effect of NO on tumor cells or rat aortic smooth muscle cells was reversed by the addition of exogenous polyamines but not by ornithine, suggesting that NO inhibited ODC.

ODC is a homodimer and forms two active sites at the interface between the two monomers. It contains 2 cysteine residues (Cys-70 and Cys-360) in each active site (19) and a total of 12 cysteine residues/monomer. Cys-360 is required for enzymatic activity because mutating Cys-360 to alanine reduces enzyme activity by 98% (20, 21). Nitrogen oxide species have been shown to readily react with both protein and low molecular weight thiols to form S-nitrosothiols (22, 23). An increasing number of proteins such as albumin, p21^ras, caspase 3, glyceraldehyde 3-phosphate dehydrogenase, hemoglobin, and NF-B as well as the low molecular weight thiols, cysteine and glutathione, have been found to be S-nitrosylated in vivo (22, 24-28). In addition, many proteins, including ODC, have been shown to be S-nitrosylated in vitro (29).

The objective of this study was 3-fold: 1) to provide further evidence for the S-nitrosylation of ODC and the chemical mechanism by which it is S-nitrosylated, 2) to determine whether Cys-360, the critical cysteine residue in the active site of ODC, is S-nitrosylated, and 3) to determine whether ODC is S-nitrosylated in intact cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chemicals and Solutions-- Unless otherwise noted all chemicals and reagents were purchased from Sigma. L-[1-¹⁴C]Ornithine was purchased from PerkinElmer Life Sciences. Ecolite scintillation mixture was purchased from ICN, Costa Mesa, CA. S-Nitrosoglutathione (GSNO) (30) and S-nitrosocysteine (CysNO) (31) were synthesized as described previously. 1-Diethyl-2-hydroxy-2-nitroso-hydrazine (DEA/NO) was a kind gift from David A. Wink, National Institutes of Health, Bethesda, MD. Saturated NO solutions were prepared by bubbling pure NO gas through water under anaerobic conditions.

Preparation of Plasmids-- pHIS-ODC was prepared as described previously (29). The resulting plasmid codes for an ODC protein with the following amino terminus: MRGSHHHHHHGS. For the pHIS-ODC C360A mutant, cysteine 360 was mutated to alanine in pGEM-ODC using the Chameleon mutagenesis kit (Stratagene, La Jolla, CA). The pGEM construct was then digested with SphI and SalI, and the fragment containing the mutation was isolated and inserted into pHIS-ODC. pCMVZeo-ODC was prepared as described previously (32). The plasmid codes for ODC truncated at amino acid residue 425. The truncated protein maintains activity comparable with wild type ODC but is no longer degraded by the proteasome.

Expression and Purification of HIS-ODC-- pHIS-ODC or pHIS-ODC C360A was expressed and purified from XL1-Blue Escherichia coli (Stratagene) as described previously (33).

Ornithine Decarboxylase Assay-- ODC activity was determined by monitoring the formation of [¹⁴C]CO₂ from L-[1-¹⁴C]ornithine (29, 34). Dithiothreitol (DTT) was separated from the enzyme by gel filtration through a Sephadex G-25 column in all experiments. For anaerobic experiments all solutions were deoxygenated either by bubbling argon through the solution or by purging the headspace with argon for at least 20 min. All anaerobic experiments were performed in a glove bag (I²R, Cheltenham, PA) under positive pressure argon.

Photolysis-Chemiluminescence-- Samples were injected directly into a photolysis chamber that consists of borosilicate glass capillary tubing coiled around a 200-watt high-pressure mercury vapor lamp (Hanovia, Newark, NJ). A carrier stream of nitrogen carries the effluent through the photolysis chamber where the photolabile S-NO bond is broken resulting in the formation of NO and thiyl radical (S^·). NO, in the gas phase, is then carried through a cold trap (75 °C) and swept into the chemiluminescence detector. Chemiluminescence was performed as described previously (15). Authentic GSNO was used to make a standard curve, and the concentration of the GSNO stock solution was confirmed by spectrophotometry. NO release from GSNO was not detectable when the light source was off.

Determination of Protein Concentration-- Protein concentrations were determined by the Bradford Coomassie Brilliant Blue method (Bio-Rad) using bovine serum albumin as the standard.

Determination of Protein SNO Content-- Purified ODC or C360A ODC was gel filtered on a Sephadex G-25 column preequilibrated with 50 mM Tris-HCl, 0.1 mM diethylenetriaminepentaacetic acid, pH 7.5 (assay buffer) to remove DTT. The buffer was then exchanged twice using Microcon centrifugal filter devices (Amicon) with a 30,000-kDa cut-off to further remove DTT and concentrate the protein. ODC, C360A ODC, or bovine serum albumin was incubated for 15 min at room temperature with 1 mM sodium cyanoborohydride, a mild reducing agent, to assure that thiols were in the reduced state. The samples were then incubated 1:1 (v/v) with a saturated NO solution (~2 mM NO as determined by spectrophotometry) in the dark at room temperature for 30 min. Excess NO was removed from the solution by purging the headspace with argon for at least 20 min and then analyzed by photolysis-chemiluminescence.

Cell Culture-- NIH 3T3 cells were grown in Dulbecco's modified Eagle's medium-Hepes supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cell cultures were grown at 37 °C in a humidified atmosphere of 5% CO₂, 95% air and were subcultured by trypsinization.

Plasmid Transfection-- 5 × 10⁵ cells were seeded in 10-cm plates and transfected the next day with 10 µg of pCMVZeo-ODC or pCMVZeo using SuperFect transfection reagent (Qiagen, Valencia, CA) according to the methods of the manufacturer. The cells were then allowed to grow for another 48 h before use.

Western Blot Analysis-- Cell lysates from untransfected, pCMVZeo-transfected, or pCMVZeo-ODC-transfected NIH 3T3 cells were prepared using ice-cold lysis buffer containing 50 mM Tris HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 µg/ml aprotinin. 20 µg of total protein from each sample were subjected to SDS-polyacrylamide (10%) gel electrophoresis, and proteins were transferred to a nitrocellulose membrane. The membrane was then probed with rabbit anti-ODC antiserum (0.5 µg/ml) followed by goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:2000 dilution) (Cell Signaling Technologies, Beverly, MA). Blots were then visualized using LumiGlo chemiluminescent detection reagent (Cell Signaling Technology) as described by the manufacturer.

Immunoprecipitation of ODC-- Cell lysates from 1-2 × 10⁸ pCMVZeo-transfected or pCMVZeo-ODC-transfected NIH 3T3 cells were prepared as described above. The lysates were aliquoted into five tubes so that immunoprecipitations were carried out on lysates from 2-4 × 10⁷ cells. Immunoprecipitations were performed using control rabbit IgG or rabbit anti-ODC antiserum and protein G-Sepharose (Amersham Pharmacia Biotech) according to the instructions of the manufacturer. Before dissociation of the immunocomplexes, the original five aliquots of each sample were again pooled. The antigen-antibody complexes were then dissociated from the beads with 500 µl of 100 mM glycine buffer (pH 2.5). The protein G-Sepharose beads were removed by centrifugation, and the supernatant was kept on ice prior to analysis.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Effects of NO Donor Agents on ODC Activity-- We previously reported that NO donor agents inhibit purified ODC via S-nitrosylation (29). We show here that the NO donor agents GSNO and DEA/NO inhibited purified ODC in a concentration-dependent manner under aerobic conditions (Fig. 1). Furthermore, the inhibition of ODC by both NO donor agents was reversible by the addition of 2.5 mM DTT and to a lesser extent by 5 mM GSH, providing evidence that NO acts to inhibit the enzyme by S-nitrosylation (Fig. 1).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   The inhibitory effects of NO donor agents on purified ODC and its reversibility by thiols. DTT was separated from the enzyme by gel filtration through a Sephadex G-25 column in all experiments. ODC was preincubated with various concentrations of GSNO or DEA/NO at 37 °C for 15 min. ODC activity was measured after a 30-min incubation at 37 °C in the presence or absence of 2.5 mM DTT or 5 mM GSH as described under "Experimental Procedures." Data represent the mean ± S.E. of duplicate determinations from three separate experiments.

GSNO was more potent than DEA/NO as an inhibitor of ODC despite the fact that GSNO does not readily release NO. DEA/NO, on the other hand, has a half-life of NO release of ~2 min. This led us to investigate the mechanism of S-nitrosylation by the two NO donor agents. We set forth the hypothesis that GSNO acts by participating in an S-transnitrosation reaction with cysteine residue(s) on ODC, whereas DEA/NO likely acts through the reaction of NO with O₂ to form N₂O₃ and N₂O₄, both of which are nitrosating agents (Fig. 2).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Reaction mechanisms involving GSNO and NO.

We first tested the capacity of GSNO and DEA/NO to inhibit ODC under anaerobic conditions. According to our hypothesis, GSNO should inhibit ODC under anaerobic conditions, whereas DEA/NO should not because the formation of N₂O₃ or N₂O₄ from NO is oxygen-dependent. As predicted, GSNO was equipotent under aerobic and anaerobic conditions (Fig. 3). The inhibition of ODC caused by GSNO under anaerobic conditions was, like under aerobic conditions, reversible by DTT and GSH (Fig. 4). This suggests that GSNO acts by the same mechanism under both aerobic and anaerobic conditions.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   The inhibitory effects of GSNO on purified ODC under aerobic versus anaerobic conditions. Experiments were conducted under aerobic () or anaerobic () conditions. DTT was separated from the enzyme by gel filtration through a Sephadex G-25 column in all experiments. ODC was preincubated with various concentrations of GSNO at 37 °C for 15 min and then assayed as described under "Experimental Procedures." Data represent the mean ± S.E. of duplicate determinations from four separate experiments.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   The inhibition of purified ODC by GSNO under anaerobic conditions is reversible by thiols. Experiments were conducted either under positive pressure argon (anaerobic) or in room air (aerobic). DTT was separated from the enzyme by gel filtration through a Sephadex G-25 column in all experiments. ODC was preincubated with 10 µM GSNO at 37 °C for 15 min and then assayed in the presence or absence of 2.5 mM DTT or 5 mM GSH as described under "Experimental Procedures." Data represent the mean ± S.E. of duplicate determinations from four separate experiments.

DEA/NO failed to significantly inhibit ODC under anaerobic conditions at all concentrations tested (Fig. 5), demonstrating that NO released from DEA/NO must react with oxygen to inhibit ODC. This result lends credence to the hypothesis that DEA/NO acts through the reaction of NO and O₂. To provide further evidence for this we took advantage of the reaction between NO and the NO scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide (PTIO) (35). PTIO acts to scavenge NO by oxidizing it to NO₂ (Fig. 2). However, NO₂, as shown in Fig. 2, can react with another molecule of NO or dimerize to form the nitrosating agents N₂O₃ or N₂O₄, respectively. Under anaerobic conditions, 100 µM DEA/NO in the presence of 30 µM PTIO inhibited ODC by ~80%, whereas DEA/NO or PTIO alone had no effect (Fig. 6). The inhibition of ODC by the combination of DEA/NO and PTIO was reversible by the addition of DTT or GSH, again suggesting that ODC was S-nitrosylated (Fig. 7).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   The inhibitory effects of DEA/NO on purified ODC under aerobic versus anaerobic conditions. Experiments were conducted under aerobic () or anaerobic () conditions. DTT was separated from the enzyme by gel filtration through a Sephadex G-25 column in all experiments. ODC was preincubated with various concentrations of DEA/NO for 15 min and then assayed as described under "Experimental Procedures." Data represent the mean ± S.E. of duplicate determinations from four separate experiments.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   The effects of PTIO and DEA/NO on purified ODC under anaerobic conditions. Experiments were conducted under positive pressure argon. DTT was separated from the enzyme by gel filtration through a Sephadex G-25 column in all experiments. ODC was preincubated with or without 100 µM DEA/NO and/or 30 µM PTIO at 37 °C for 15 min and then assayed as described under "Experimental Procedures." Data represent the mean ± S.E. of duplicate determinations from three separate experiments.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   The inhibition of ODC by PTIO and DEA/NO under anaerobic conditions is reversible by thiols. Experiments were conducted under positive pressure argon. DTT was separated from the enzyme by gel filtration through a Sephadex G-25 column in all experiments. ODC was preincubated with or without 100 µM DEA/NO and/or 30 µM PTIO at 37 °C for 15 min and then assayed in the absence or presence of 2.5 mM DTT or 5 mM GSH as described under "Experimental Procedures." Data represent the mean ± S.E. of duplicate determinations from four separate experiments. *, significantly different (p < 0.01) from values obtained in the absence of added DTT or GSH.

Analysis of S-Nitrosylated ODC by Photolysis-Chemiluminescence-- Photolysis-chemiluminescence is an established method for determining the S-nitrosothiol content of proteins (22). We, therefore, developed our own apparatus to use photolysis-chemiluminescence to study the S-nitrosylation of ODC by NO. The present method was somewhat less sensitive than that previously described by Stamler et al. (22, 36). This was not a limitation because we were able to repeat Stamler's earlier work showing that albumin, when in its native state, is S-nitrosylated on a single cysteine residue. 250 µl of a 100 µg/ml solution of albumin was mixed 1:1 (v/v) with a saturated NO solution and incubated for 30 min in the dark at room temperature. Excess NO was removed from the solution by purging the headspace with argon for 20 min. The sample was then analyzed by photolysis-chemiluminescence. A standard curve was prepared using authentic GSNO (Fig. 8A). The concentration of GSNO stock solution was verified by spectrophotometry. In agreement with Stamler, albumin was determined to contain 0.91 ± 0.14 mol of SNO/mol of protein (Fig. 8B). The same procedure using purified ODC and treatment of ODC with NO resulted in the S-nitrosylation of 3.89 ± 0.23 thiol residues/ODC monomer (Fig. 8B), strengthening the hypothesis that NO S-nitrosylates ODC.

View larger version (12K): [in this window] [in a new window]   Fig. 8.   ODC is S-nitrosylated at Cys-360, a critical thiol in the active site of the enzyme. A, GSNO standard curve using photolysis-chemiluminescence. The standard curve was linear between 50 pmol and 10 nmol of GSNO with 10 nmol being the highest amount of GSNO tested. The equation for the line was y = 0.4564x  12.159 with a correlation coefficient of R2 = 0.988. AUC, area under the curve. B, SNO quantitation on albumin and ODC. Albumin, ODC, or C360A ODC was incubated 1:1 (v/v) with a saturated NO solution in the dark at room temperature for 30 min and then analyzed by photolysis-chemiluminescence as described under "Experimental Procedures." *, significantly different (p < 0.01) from values for wild type ODC. Data represent the mean ± S.E. of duplicate determinations from five separate experiments.

As a means of determining if Cys-360 is one of the 4 residues in ODC S-nitrosylated by NO, C360A ODC in which Cys-360 is mutated to alanine was analyzed as above. C360A ODC was determined to contain 2.81 ± 0.17 SNO molecules/ODC monomer (Fig. 8B). This is 1 less SNO molecule/monomer than wild type ODC and was significantly different as determined by the Student's t test (p < 0.01).

Isolation of S-Nitrosylated ODC from Intact Cells-- Our final objective was to determine whether S-nitrosylated ODC could be isolated from cells that had been treated with NO. ODC was overexpressed in NIH 3T3 cells by transfection with pCMVZeo-ODC. This plasmid codes for an ODC protein that is truncated at the carboxyl terminus at amino acid residue 425. Because the carboxyl terminus is required for ODC to be degraded by the proteasome, the truncated form of the protein is stable and can accumulate to very high levels within the cell. pCMVZeo-ODC-transfected cells showed a marked increase in ODC protein as determined by Western blot analysis (Fig. 9A) and an approximate 200-fold increase in ODC activity (Fig. 9B) when cell lysates from untransfected or pCMVZeo-transfected cells were compared with pCMVZeo-ODC-transfected cells.

View larger version (24K): [in this window] [in a new window]   Fig. 9.   Overexpression of ODC in NIH 3T3 cells. NIH 3T3 cells were transiently transfected with the pCMVZeo-ODC plasmid or pCMVZeo control vector. 48 h after transfection the cells were lysed and subjected to Western blot analysis (A) for ODC in untreated cells (Control), pCMVZeo-transfected cells, and pCMVZeo-ODC-transfected cells. B, assay of ODC activity in cell lysates of untransfected, pCMVZeo-transfected, or pCMVZeo-ODC-transfected cells. Data represent the mean ± S.E. of duplicate determinations from three separate experiments.

Control cells or cells overexpressing ODC were then treated with 1 mM CysNO and incubated for 30 min. The cells were harvested, lysed, and subjected to immunoprecipitation with control rabbit IgG or rabbit anti-ODC polyclonal antibody and protein G-Sepharose as described under "Experimental Procedures." The immunocomplexes were then eluted from the protein G-Sepharose, and the eluate was analyzed by photolysis-chemiluminescence. S-Nitrosylated protein was not detected in pCMVZeo-transfected cells immunoprecipitated with the anti-ODC antibody or pCMVZeo-ODC-transfected cells immunoprecipitated with normal rabbit IgG (Fig. 10). This suggests that in both cases there was either no S-nitrosylated ODC or that it was below the detectable limit. In pCMVZeo-ODC-transfected cells immunoprecipitated with anti-ODC antibody, 36.8 ± 4.5 pmol of S-nitrosylated protein/mg of total cell protein was detected (Fig. 10). In addition, there was no detectable S-nitrosylated ODC in immunoprecipitates from pCMVZeo-ODC-transfected cells that were not treated with CysNO (data not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 10.   Isolation of S-nitrosylated ODC from intact cells. NIH 3T3 cells were transiently transfected with the pCMVZeo-ODC plasmid or pCMVZeo control vector. 48 h after transfection the cells were treated with 1 mM CysNO. The supernatant from the lysates of the NO-treated cells was subjected to immunoprecipitation with rabbit anti-ODC antiserum (-ODC Ab) or with control rabbit IgG and protein G-Sepharose. Immunoprecipitates were then analyzed by photolysis-chemiluminescence. n.d., not detected. Data represent the mean ± S.E. of duplicate determinations from four separate experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

When ODC is inactivated by the enzyme-activated, irreversible inhibitor -difluoromethylornithine, a stoichiometric amount of a covalent adduct is formed with the protein with the major site for this reaction being Cys-360 (37). This suggests that Cys-360 must be located in the active site of ODC, which was recently confirmed by x-ray crystallography (19). The importance of this residue has been established by mutation of this cysteine to serine or alanine (20).

There is ample evidence that NO inhibits ODC. The initial evidence supporting this came from a study in which sodium nitroprusside was shown to inhibit cell proliferation by inhibiting putrescine synthesis (11). Further evidence of this has been provided by studies in our laboratory in which inhibition of Caco-2 cell proliferation or vascular smooth muscle cell proliferation by NO was shown to be reversible by the addition of excess putrescine, spermidine, or spermine but not ornithine (15, 16). It was also shown that NO could inhibit ODC in crude cell lysates of Caco-2 cells or vascular smooth muscle cells. Similar studies have since been published showing inhibition of ODC in crude cell lysates by NO donor agents (17, 18).

We recently demonstrated that NO donor agents inhibit purified ODC by S-nitrosylation (17). In the current study, NO, in the form of GSNO or DEA/NO, inhibited ODC in a concentration-dependent manner. Inhibition of ODC by both NO donor agents was reversible by the addition of DTT or GSH, supporting the hypothesis that NO inhibits ODC via S-nitrosylation of a critical cysteine residue(s) on ODC. The present data further support the hypothesis that NO and related species inhibit ODC by S-nitrosylation. Photolysis-chemiluminescence is an effective tool for determining not only whether a protein is S-nitrosylated but also for quantifying the number of residues modified by S-nitrosylation (22). It combines both specificity and sensitivity because SNOs are photolabile, and the chemiluminescence detectors used for detecting NO are both specific for NO and very sensitive. Exposure to ultraviolet light may result in the formation of NO also from nitrite, nitrosamines, and dinitrosyliron complexes and was controlled for in our experiments. Using this method we show that ~4 cysteine residues/ODC monomer are S-nitrosylated when the enzyme is treated with a saturated NO solution. In addition, we provide evidence that Cys-360, which is critical for enzyme activity, is S-nitrosylated because the C360A ODC mutant is S-nitrosylated on only 3 cysteine residues.

These findings led us to investigate whether S-nitrosylated ODC could be isolated from cells treated with NO. NIH 3T3 cells transfected with pCMVZeo-ODC and treated with 1 mM CysNO were subjected to immunoprecipitation with anti-ODC antibody. This resulted in the detection of 36.8 ± 4.5 pmol of SNO/mg of total cellular protein by photolysis-chemiluminescence. S-Nitrosylated protein was not detected in cells transfected with pCMVZeo and immunoprecipitated with anti-ODC antibody. pCMVZeo-ODC-transfected cells immunoprecipitated with normal rabbit IgG also resulted in no detection of S-nitrosylated protein, demonstrating that the positive result was not because of nonspecific protein interactions or conversion of nitrite, nitrosamines, or dinitrosyliron complexes to NO by exposure of the sample to UV light. These data demonstrate that ODC is S-nitrosylated in intact cells and suggest that the same may occur in vivo.

Both S-nitrosothiol NO donor agents and the diazeniumdiolate NO donor agents inhibit purified ODC. The S-nitrosothiol GSNO, however, is ~6-fold more potent than the diazeniumdiolate DEA/NO. Not all classes of NO donor agents release NO by common mechanisms. GSNO releases NO only under specific conditions such as in the presence of transition metals or light. DEA/NO, on the other hand, spontaneously releases NO at pH 7.4 with a half-life of 2 min at 37 °C and releases 2 mol of NO/mol of DEA/NO. The mechanisms by which NO donor agents cause S-nitrosylation of ODC are dependent on the class of NO donor agent.

The inhibition of ODC by DEA/NO was O₂-dependent, suggesting that the NO released from DEA/NO is autoxidized to N₂O₃ or N₂O₄. This was substantiated by experiments in which ODC was incubated under anaerobic conditions in the presence or absence of PTIO. PTIO is an NO scavenger that acts by oxidizing NO to NO₂, which is essentially equivalent to the reaction of NO with O₂ to form NO₂ (35). The reaction of NO with O₂ is third order overall and second order with respect to NO, indicating that the rate of formation of NO₂ is dependent on the square of the NO concentration. Therefore, as the NO concentration decreases the reaction rate slows dramatically. Under physiological conditions where the NO concentration is very low (less than 100 nM), the reaction of NO with O₂ is very slow, which allows for the diffusion of NO away from its source enabling NO to react with other biological targets. In fact, the suggestion has been made that the reaction between NO and O₂ may not be relevant under physiological conditions. However, both NO and O₂ are lipophilic molecules and tend to concentrate in biological membranes, perhaps facilitating this reaction. Indeed this reaction takes place ~1500 times faster in micelles as compared with aqueous solution (39). Therefore, whereas this reaction may not take place in the cell cytosol, it may be important in biological membranes.

The inhibition of ODC by GSNO on the other hand was independent of O₂. Furthermore, the enzyme inhibition caused by GSNO was equipotent in the presence and absence of oxygen and was reversible by the addition of DTT or GSH under both conditions. These data suggest that GSNO acts by the same mechanism under aerobic or anaerobic conditions. Furthermore, these data rule out the possibility that GSNO simply acts through release of NO because NO by itself does not readily react with thiol residues. Besides being able to release NO under the appropriate conditions, S-nitrosothiols can react with thiols resulting in either the release of nitroxyl (HNO) or donation of nitrosonium (NO⁺) to another thiol via S-transnitrosation. Generation of HNO and its subsequent attack on thiol residues would likely lead to the formation of an N-hydroxysulfenamide, sulfinamide, or sulfinic acid (40). S-Transnitrosation reactions result in the transfer of an NO⁺ equivalent from one thiol to another resulting in the formation of a new S-nitrosothiol. S-Transnitrosation is considered to be the predominant mechanism for the biological actions of GSNO (41), and S-transnitrosation has been demonstrated in vivo between S-nitrosylated albumin and low molecular weight thiols (42). S-Transnitrosation reactions are relatively fast reactions and are O₂-independent, which is consistent with the higher potency of GSNO as an inhibitor of ODC and explains why GSNO inhibits ODC under anaerobic conditions.

Although the presence of low molecular weight S-nitrosothiols such as GSNO and CysNO has been demonstrated in extracellular environments such as plasma, most of the evidence for intracellular low molecular weight S-nitrosothiols is indirect. For example, Mayer et al. (43) demonstrated that in rat isolated hearts a Cu⁺-specific chelator prevented bradykinin-induced cyclic GMP accumulation but did not affect cyclic GMP accumulation due to exogenous NO sources. Because Cu⁺ is known to catalyze the release of NO from nitrosothiols, it was concluded that an S-nitrosothiol must be involved. However, Stamler et al. (44) recently described the identification of an enzyme termed "GSNO reductase" that metabolizes GSNO, therefore regulating intracellular levels of GSNO. They demonstrate that deleting the reductase gene abolishes the GSNO-consuming activity of cells and increases intracellular levels of GSNO. The regulation of GSNO within the cell supports the idea that GSNO is an important intracellular molecule and provides evidence for the formation of GSNO intracellularly.

Biologically speaking, however, the problem arises as to how S-transnitrosation reactions are targeted to specific thiols on proteins when intracellular GSH concentrations can be as high as 5-10 mM and the concentration of total cellular protein thiols is significantly higher than that. Specificity may come from the reactivity of the target thiol, which can vary by several orders of magnitude. For example thiolates, unprotonated thiols, are more reactive than the protonated form. In addition, S-transnitrosation reactions are reversible with the equilibrium favoring the more stable protein nitrosothiols over low molecular weight nitrosothiols (45). Another theory, proposed by Stamler et al. (38), is that basic and acidic amino acids in the vicinity of the target thiol residue catalyze S-transnitrosation reactions by acid-base catalysis, leading to enhanced reactivity of that cysteine residue. In fact, Stamler et al. (38) have proposed that (K/R/H)C(D/E) is a consensus motif for the S-nitrosylation of proteins by S-transnitrosation with the acidic amino acid following the cysteine residue being the most important. Although ODC does not have a basic amino acid immediately preceding Cys-360, it does have an aspartate residue immediately following Cys-360. The crystal structure of ODC further reveals that the active site contains several acidic and basic amino acids in the vicinity of Cys-360.

We provide evidence herein of the S-nitrosylation of ODC on 4 cysteine residues including Cys-360, a critical thiol residue in the active site of ODC. In addition, we demonstrate the isolation of S-nitrosylated ODC from cells overexpressing ODC, suggesting that ODC may be S-nitrosylated in vivo. We further reveal that low molecular weight S-nitrosothiols inhibit ODC with a greater potency than NO itself and by a different mechanism. The hypothesis is put forth that low molecular weight S-nitrosothiols may be physiological regulators of ODC activity and, therefore, cell proliferation. We recently demonstrated that NO inhibits vascular smooth muscle cell proliferation by inhibiting ODC. However, low molecular weight S-nitrosothiols and not NO may be the physiological regulator of vascular smooth muscle cell proliferation because the former are found in vivo (41) and are more potent inhibitors of ODC.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants HL 35014 (to L. J. I.), HL 40922 (to L. J. I.), and CA 18138 (to A. E. P.).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.

^¶ To whom correspondence should be addressed. Tel.: 310-825-9930; Fax: 310-206-0589; E-mail: lignarro@mednet.ucla.edu.

Published, JBC Papers in Press, July 18, 2001, DOI 10.1074/jbc.M105219200

² P. M. Bauer, G. M. Buga, and L. J. Ignarro, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: ODC, ornithine decarboxylase; GSNO, S-nitrosoglutathione; CysNO, S-nitrosocysteine; DEA/NO, 1-diethyl-2-hydroxy-2-nitroso-hydrazine; DTT, dithiothreitol; PTIO, 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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