Glutamine synthetase is a molecular target of nitric oxide in root nodules of Medicago truncatula and is regulated by tyrosine nitration.

Nitric oxide (NO) is emerging as an important regulatory player in the Rhizobium-legume symbiosis, but its biological role in nodule functioning is still far from being understood. To unravel the signal transduction cascade and ultimately NO function, it is necessary to identify its molecular targets. This study provides evidence that glutamine synthetase (GS), a key enzyme for root nodule metabolism, is a molecular target of NO in root nodules of Medicago truncatula, being regulated by tyrosine (Tyr) nitration in relation to active nitrogen fixation. In vitro studies, using purified recombinant enzymes produced in Escherichia coli, demonstrated that the M. truncatula nodule GS isoenzyme (MtGS1a) is subjected to NO-mediated inactivation through Tyr nitration and identified Tyr-167 as the regulatory nitration site crucial for enzyme inactivation. Using a sandwich enzyme-linked immunosorbent assay, it is shown that GS is nitrated in planta and that its nitration status changes in relation to active nitrogen fixation. In ineffective nodules and in nodules fed with nitrate, two conditions in which nitrogen fixation is impaired and GS activity is reduced, a significant increase in nodule GS nitration levels was observed. Furthermore, treatment of root nodules with the NO donor sodium nitroprusside resulted in increased in vivo GS nitration accompanied by a reduction in GS activity. Our results support a role of NO in the regulation of nitrogen metabolism in root nodules and places GS as an important player in the process. We propose that the NO-mediated GS posttranslational inactivation is related to metabolite channeling to boost the nodule antioxidant defenses in response to NO.

Tyrosine-dependent oxidative DNA damage induced by carcinogenic tetranitromethane.

Tetranitromethane (TNM) is used as an oxidizer in rocket propellants and explosives and as an additive to increase the cetane number of diesel fuel. TNM was reported to induce pulmonary adenocarcinomas and squamous cell carcinomas in mice and rats. However, the mechanisms underlying carcinogenesis induced by TNM has not yet been clarified. We previously revealed that nitroTyr and nitroTyr-containing peptides caused Cu(II)-dependent DNA damage in the presence of P450 reductase, which is considered to yield nitroreduction. Since TNM is a reagent for nitration of Tyr in proteins and peptides, we have hypothesized that TNM-treated Tyr and Tyr-containing peptides induce DNA damage by the modification of Tyr. We examined DNA damage induced by TNM-treated amino acids or peptides using (32)P-5'-end-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. TNM-treated Tyr and Lys-Tyr-Lys induced DNA damage including the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in the presence of Cu(II) and NADH. DNA damage was inhibited by catalase and bathocuproine, indicating the involvement of H(2)O(2) and Cu(I). The cytosine residue of the ACG sequence complementary to codon 273, well-known hotspots of the p53 gene, was cleaved with piperidine and Fpg treatments. On the other hand, nitroTyr and Lys-nitroTyr-Lys did not induce DNA damage in the presence of Cu(II) and NADH. Time-of-flight mass spectrometry confirmed that reactions between Lys-Tyr-Lys and TNM yielded not only Lys-nitroTyr-Lys but also Lys-nitrosoTyr-Lys. Therefore, it is speculated that the nitrosotyrosine residue can induce oxidative DNA damage in the presence of Cu(II) and NADH. It is concluded that Tyr-dependent DNA damage may play an important role in the carcinogenicity of TNM. TNM is a new type of carcinogen that induces DNA damage not by itself but via Tyr modification.

Nitration enhances the allergenic potential of proteins.

BACKGROUND: Recent investigations have shown that proteins, including Bet v 1a, are nitrated by exposure to polluted urban air. We have investigated immunogenic and allergenic properties of in vitro nitrated allergens in in vivo models. METHODS: Untreated and nitrated samples of ovalbumin or Bet v 1a were compared for their ability to stimulate proliferation and cytokine secretion in splenocytes from DO11.10 or from sensitized BALB/c mice, and for their ability to induce specific immunoglobulin (Ig)G1, IgG2a and IgE in sensitized mice. Additionally, sera from birch pollen-allergic individuals were analysed for IgE and IgG specific for nitrated Bet v 1a. RESULTS: Upon splenocyte stimulation with nitrated as compared with unmodified allergens, proliferation as well as interleukin 5 and interferon-gamma production were enhanced. Sera of mice sensitized with nitrated allergens showed elevated levels of specific IgE, IgG1 and IgG2a, compared with sera from mice sensitized with unmodified allergens. Moreover, cross-reactivity of antibodies against unrelated, nitrated allergens was observed in mice. We also found higher amounts of functional, specific IgE against nitrated than against untreated Bet v 1a in sera from birch pollen-allergic patients. CONCLUSIONS: Our findings suggest that nitration enhances allergic responses, which may contribute to an increased prevalence of allergic diseases in polluted urban environments.

Functionality of nitrated acetylcholine receptor: the two-step formation of nitrotyrosines reveals their differential role in effectors binding.

The presence of nitrotyrosines is associated with several neurodegenerative pathologies. We evaluated the functionality of the nicotinic acetylcholine receptor possessing nitrotyrosines. The spectrum of the nitrated receptor displays an absorption band characteristic of ortho-nitrophenol. The presence of carbamylcholine in the agonist site prevented the effect of nitration by tetranitromethane in some conditions. The nitration occurred with two discrete steps and pointed out the differential involvement of tyrosines in the binding of acetylcholine and neurotoxin. We concluded that at least two residues involved in agonist binding can be nitrated, which bring similar contributions to the binding energy of the neurotransmitter.

Effects of nitration on the structure and aggregation of alpha-synuclein.

Substantial evidence suggests that the aggregation of the presynaptic protein alpha-synuclein is a key step in the etiology of Parkinson's disease (PD). Although the molecular mechanisms underlying alpha-synuclein aggregation remain unknown, oxidative stress has been implicated in the pathogenesis of PD. Here, we report the effects of tyrosine nitration on the propensity of human recombinant alpha-synuclein to fibrillate in vitro. The properties of nitrated alpha-synuclein were investigated using a variety of biophysical and biochemical techniques, which revealed that nitration led to formation of a partially folded conformation with increased secondary structure relative to the intrinsically disordered structure of the monomer, and to oligomerization at neutral pH. The degree of self-association was concentration-dependent, but at 1 mg/mL, nitrated alpha-synuclein was predominantly an octamer. At low pH, small-angle X-ray scattering data indicated that the nitrated protein was monomeric. alpha-Synuclein fibrillation at neutral pH was completely inhibited by nitrotyrosination and is attributed to the formation of stable soluble oligomers. The presence of heparin or metals did not overcome the inhibition; however, the inhibitory effect was eliminated at low pH. The addition of nitrated alpha-synuclein inhibited fibrillation of non-modified alpha-synuclein at neutral pH. Potential implications of these findings to the etiology of Parkinson's disease are discussed.

Chemical modification of glycerinated stalks shows tyrosine residues essential for spasmoneme contraction of Vorticella sp.

Chemical modification of glycerinated stalks of Vorticella with TNM is used to investigate the role of tyrosine residues in the Ca(2+)-induced contraction of the spasmoneme. Tetranitromethane (TNM) is often employed as a specific reagent for the nitration of tyrosine residues in a protein at neutral and slightly alkaline pHs although TNM can also oxidize cysteine residues in the acidic and neutral pH range. Prior incubation with Ca(2+) of stalks to be treated with TNM can protect the spasmoneme from irreversible denaturation. On the other hand, TNM treatment in the absence of free Ca(2+) causes an irreversible denaturation of the spasmoneme. It was revealed by us that an isolated Ca(2+)-binding protein called spasmin could not bind with Ca(2+) after TNM treatment, even if the treatment was performed in the presence of Ca(2+). In an additional experiment, we confirmed that the chemical modification of cysteine residues in the spasmoneme with N-7-dimethyl-amino-4methyl- coumarinyl- maleimide (DACM) has no effect on the contractibility. These results suggest that tyrosine residues in spasmin are essential for spasmoneme contraction and are protected from TNM in the presence of Ca(2+) when spasmin binds with its receptor protein in the spasmoneme.

Involvement of tyrosines at fucose-binding sites of Aleuria aurantia lectin: non-equal response to site-directed mutagenesis among five sites.

Since the involvement of Tyr residues in the fucose-binding of Aleuria aurantia lectin (AAL) was proved by chemical modification using the Tyr-specific reagent tetranitromethane, site-directed mutagenesis was attempted. Since the tertiary structure of AAL was determined recently to be a six-bladed beta-propeller fold, and five fucose-binding sites per subunit were found, based on positions of Tyr residues in the tertiary structure, three classes of mutants were constructed: 1) Tyr on the 2nd beta-strand of each blade (beta-2 mutants), 2) Tyr or Trp on the 3rd beta-strand (beta-3 mutants), and 3) Tyr outside of binding sites (other-Y mutants). The mutagenized cDNA was expressed in Escherichia coli as His-tag-AAL, and the hemagglutinating activity was assayed. Among 14 mutants, three beta-2 mutants (Y26A, Y79A, and Y181A), and three beta-3 mutants (Y92A, W149A, and Y241A) showed decreased activity. These mutated residues resided at Sites 1, 2, and 4, at the same locations relatively in the binding sites. Mutagenesis of Tyr or Trp at the corresponding locations in Sites 3 and 5 did not lead to a reduction in activity. Results indicate that the properties of Sites 1, 2, and 4 are different from those of Sites 3 and 5, and that the contribution of these two sites to the hemagglutination reaction was minor.

Nitration inhibits fibrillation of human alpha-synuclein in vitro by formation of soluble oligomers.

The aggregation of alpha-synuclein in dopaminergic neurons is a critical factor in the etiology of Parkinson's disease (PD). Oxidative and nitrative stress is also implicated in PD. We examined the effect of nitration on the propensity of alpha-synuclein to fibrillate in vitro. Fibril formation of alpha-synuclein was completely inhibited by nitration, due to the formation of stable soluble oligomers (apparently octamers). More importantly the presence of sub-stoichiometric concentrations of nitrated alpha-synuclein led to inhibition of fibrillation of non-modified alpha-synuclein. These observations suggest that nitration of soluble alpha-synuclein may be a protective factor in PD, rather than a causative one.

Mechanism-based partial inactivation of glutathione S-transferases by nitroglycerin: tyrosine nitration vs sulfhydryl oxidation.

Liver glutathione-S-transferases (GSTs) are responsible for the detoxification of electrophiles, and specifically for the metabolism of orally administered organic nitrates such as nitroglycerin (NTG). Recent studies showed that reactive nitrogen species produced by tetranitromethane (TNM), peroxynitrite, or the myeloperoxidase/H2O2/nitrite system can inactivate GST. It is not known whether NTG can similarly inactivate liver GSTs, and if shown, by what mechanism(s). We incubated purified GSTs with NTG, S-nitroso-N-acetylpenicillamine (SNAP), TNM, or vehicle (5% dextrose, D5W), followed by determination of GST activity. Incubation of GST with NTG and TNM caused significant decreases in GST activity whereas no changes were observed with SNAP or D5W. The relative GST activity (vs preincubation) was 73+/-14% for NTG, 37+/-8% for TNM, 98+/-13% for SNAP, and 98+/-9% for D5W, respectively. Exogenous glutathione (GSH) prevented both NTG- and TNM-induced changes in GST activity, consistent with the observed oxidative modification of GST, such as -SH oxidation and dimerization of oxidized GST. In contrast, NTG and TNM exhibited substantial differences in their ability to nitrate tyrosine (TYR) sites in GST. These results demonstrated that NTG can reduce the activity of its own metabolizing enzyme such as GST and this inhibitory effect of NTG was unlikely to be mediated through NO, as such, since SNAP had no effect on GST activity. The partial inactivation of GST by NTG appeared to involve -SH oxidation, but not TYR nitration. These findings provided the first evidence of mechanism-based protein inactivation by NTG, and may lend insight into the hepatic metabolism of NTG and other organic nitrates after repeated oral exposure.

Activation of microsomal glutathione s-transferase by peroxynitrite.

Peroxynitrite (ONOO-) toxicity is associated with protein oxidation and/or tyrosine nitration, usually resulting in inhibition of enzyme activity. We examined the effect of ONOO- on the activity of purified rat liver microsomal glutathione S-transferase (GST) and found that the activity of reduced glutathione (GSH)-free enzyme was increased 4- to 5-fold by 2 mM ONOO-; only 15% of this increased activity was reversed by dithiothreitol. Exposure of the microsomal GST to ONOO- resulted in concentration-dependent oxidation of protein sulfhydryl groups, dimer and trimer formation, protein fragmentation, and tyrosine nitration. With the exception of sulfhydryl oxidation, these modifications of the enzyme correlated well with the increase in enzyme activity. Nitration or acetylation of tyrosine residues of the enzyme using tetranitromethane and N-acetylimidazole, respectively, also resulted in increased enzyme activity, providing additional evidence that modification of tyrosine residues can alter catalytic activity. Addition of ONOO--treated microsomal GST to microsomal membrane preparations caused a marked reduction in iron-induced lipid peroxidation, which raises the possibility that this enzyme may act to lessen the degree of membrane damage that would otherwise occur under pathophysiological conditions of increased ONOO- formation.

Evidence that light modulates protein nitration in rat retina.

As part of ongoing efforts to better understand the role of protein oxidative modifications in retinal pathology, protein nitration in retina has been compared between rats exposed to damaging light or maintained in the dark. In the course of the research, Western methodology for detecting nitrotyrosine-containing proteins has been improved by incorporating chemical reduction of nitrotyrosine to aminotyrosine, allowing specific and nonspecific nitrotyrosine immunoreactivity to be distinguished. A liquid chromatography MS/MS detection strategy was used that selects all possible nitrotyrosine peptides for MS/MS based on knowing the protein identity. Quantitative liquid chromatography MS/MS analyses with tetranitromethane-modified albumin demonstrated the approach capable of identifying sites of tyrosine nitration with detection limits of 4-33 fmol. Using two-dimensional gel electrophoresis, Western detection, and mass spectrometric analyses, several different nitrotyrosine-immunoreactive proteins were identified in light-exposed rat retina compared with those maintained in the dark. Immunocytochemical analyses of retina revealed that rats reared in darkness exhibited more nitrotyrosine immunoreactivity in the photoreceptor outer segments. After intense light exposure, immunoreactivity decreased in the outer segments and increased in the photoreceptor inner segments and retinal pigment epithelium. These results suggest that light modulates retinal protein nitration in vivo and that nitration may participate in the biochemical sequela leading to light-induced photoreceptor cell death. Furthermore, the identification of nitrotyrosine-containing proteins from rats maintained in the dark, under non-pathological conditions, provides the first evidence of a possible role for protein nitration in normal retinal physiology.

Characterization of a 95 kDa high affinity human high density lipoprotein-binding protein.

A new human 95 kDa high density lipoprotein (HDL)-binding protein (HBP) corresponding to a high affinity HDL-binding site with K(d) = 1.67 microg/mL and a capacity of 13.4 ng/mg was identified in human fetal hepatocytes. The HDL binding with the 95 kDa HBP plateaus at 2.5-5 microg/mL under reducing and nonreducing conditions. The association of HDL(3) with the 95 kDa HBP plateaued in 15-30 min while dissociation was complete in 30 min. HDL(3), apoA-I, and apoA-II were recognized by the 95 kDa HBP while low density lipoproteins (LDL) and tetranitromethane-modified HDL were not. The 95 kDa HBP predominantly resides on the surface of cells since trypsin treatment of HepG2 cells eliminated nearly 70% of HDL binding. All studied human cells and cell lines (HepG2, Caco-2, HeLa, fibroblasts, SKOV-3, PA-I) demonstrated the presence of the 95 kDa protein. Both RT-PCR and Western blotting for HB-2/ALCAM were negative in human fetal hepatocytes while Gp96/GRP94 was clearly differentiated from the 95 kDa HBP by two-dimensional electrophoretic mobility. Moreover, deglycosylation of HepG2 membrane preparations did not affect either HDL binding to the 95 kDa HBP or its size, while in contrast it affected the molecular weights of HB-2/ALCAM and SR-BI/CLA-1. We conclude that the 95 kDa HBP is a new HDL receptor candidate widely expressed in human cells and cell lines.

Determination of 3-nitrotyrosine by high-pressure liquid chromatography with a dual-mode electrochemical detector.

3-Nitrotyrosine, a product of tyrosine nitration, is useful as a marker for the generation of reactive nitrogen oxide species with short half-lives such as peroxynitrite. A reverse-phase high-pressure liquid chromatographic method using a dual-mode electrochemical detector in series with a photodiode array detector has been developed to determine the levels of 3-nitrotyrosine in biological samples. The principle of this method involves reduction of 3-nitrotyrosine at an upstream gold amalgam electrode and oxidation of the resulting product(s) at a downstream glassy carbon electrode. 3-Nitrotyrosine is quantified by the amount of the current generated at the downstream electrode, and a femtomole detection level can be achieved. The disappearance of the corresponding peak when the electrochemical detector is used only in the single oxidative mode provides additional evidence for the identity of 3-nitrotyrosine in the sample. Tyrosine from the same sample is determined by its UV absorption at 280 nm, thus eliminating the need for an internal standard. With this method a dose-dependent increase of 3- to 10-fold in the levels of protein 3-nitrotyrosine was observed in the blood plasma, and a 2- to 4-fold increase in the lung cytosols, of rats treated with the lung carcinogen and nitrating agent tetranitromethane.

Critical role of tyrosine 277 in the ligand-binding and transactivating properties of retinoic acid receptor alpha.

Retinoic acid receptors specifically bind all-trans-retinoic acid (RA) and function as RA-inducible transcriptional regulatory factors. Binding of RA to RARalpha, beta, and gamma is sensitive to nitration with tetranitromethane, a tyrosine-specific modifying reagent. To identify tyrosine residue(s) that are important for RA binding, we carried out chemical modification experiments with purified RARalpha ligand-binding domain (RARalpha-LBD) subjected to partial acid hydrolysis and selective proteolysis. The chemically modified peptides containing each of the three Tyr residues present in the RARalpha-LBD sequence were then analyzed and identified by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC/ESI-MS). We found that RA binding to RARalpha-LBD protected Tyr(277)-containing peptides from nitration. Protection of Tyr(277) could result either from direct masking by the bound ligand or from ligand-induced changes in receptor conformation and tyrosine accessibility. The role of Tyr residues was further documented by site directed mutagenesis using three site-specific RARalpha mutants: Y208A, Y277A, and Y362A. The affinity for RA of these mutant receptors was in the range of that of the wild-type protein, except for the Y277A receptor mutant, which displays a 15-20-fold reduction in affinity and transactivation activity for RA. Whereas mutation of Tyr(277) into alanine had a variable effect on different agonists and antagonists binding, it caused a dramatic decrease of retinoid-dependent transactivation activity. This later effect was also observed with mutation of Tyr(277) into phenylalanine. It is unlikely that major conformational changes are responsible for the lower affinity of RA binding and RA-dependent transactivation since these mutants displayed wild-type dimerization and DNA-binding activities. Limited proteolysis revealed that upon ligand binding, the Y277A mutant induced a conformational change slightly different from that obtained with the wild-type protein. These data could suggest that Tyr(277) play a critical role in the ligand-induced conformational changes required for the activation of RARalpha.

Identification of an essential tyrosine residue in nitroalkane oxidase by modification with tetranitromethane.

The flavoprotein nitroalkane oxidase from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to the respective aldehydes or ketones with production of nitrite and hydrogen peroxide. The enzyme is irreversibly inactivated by incubation with tetranitromethane, a tyrosine-directed reagent, at pH 7.3. The inactivation is time-dependent and shows first-order kinetics for two half-lives of inactivation. Further inactivation can be achieved upon a second addition of tetranitromethane. A saturation kinetic pattern is observed when the rate of inactivation is determined versus the concentration of tetranitromethane, indicating that a reversible enzyme-inhibitor complex is formed before irreversible inactivation occurs. Values of 0.096 +/- 0.013 min(-1) and 12.9 +/- 3.8 mM were determined for the first-order rate constant for inactivation and the dissociation constant for the reversibly formed complex, respectively. The competitive inhibitor valerate protects the enzyme from inactivation by tetranitromethane, suggesting an active-site-directed inactivation. The UV-visible absorbance spectrum of the inactivated enzyme is perturbed with respect to that of the native enzyme, suggesting that treatment with tetranitromethane resulted in nitration of the enzyme. Comparison of tryptic maps of nitroalkane oxidase treated with tetranitromethane in the presence and absence of valerate shows a single peptide differentially labeled in the inactivated enzyme. The spectral properties of the modified peptide are consistent with nitration of a tyrosine residue. The amino acid sequence of the nitrated peptide is L-L-N-E-V-M-C-(NO(2)-Y)-P-L-F-D-G-G-N-I-G-L-R. The possible role of this tyrosine in substrate binding is discussed.

Peroxynitrite inactivation of tyrosine hydroxylase: mediation by sulfhydryl oxidation, not tyrosine nitration.

Tyrosine hydroxylase (TH) is the initial and rate-limiting enzyme in the biosynthesis of dopamine (DA). TH activity is significantly diminished in Parkinson's disease (PD) and by the neurotoxic amphetamines, thereby accentuating the reductions in DA associated with these conditions. Reactive oxygen and nitrogen species have been implicated in the damage to DA neurons seen in PD and in reaction to amphetamine drugs of abuse, so we investigated the hypothesis that peroxynitrite (ONOO(-)) could interfere with TH catalytic function. ONOO(-) caused a concentration-dependent inactivation of TH. The inactivation was associated with tyrosine nitration (maximum of four tyrosine residues nitrated per TH monomer) and extensive sulfhydryl oxidation. Tetranitromethane, which causes sulfhydryl oxidation at pH 6 and 8 but which nitrates tyrosines only at pH 8, inactivated TH equally at either pH. Bicarbonate protected TH from ONOO(-)-induced inactivation and sulfhydryl oxidation but increased significantly tyrosine nitration. PNU-101033 blocked ONOO(-)-induced tyrosine nitration in TH but could not prevent enzyme inactivation or sulfhydryl oxidation. Together, these results indicate that the inactivation of TH by ONOO(-) is mediated by sulfhydryl oxidation. The coincident nitration of tyrosine residues appears to exert little influence over TH catalytic function.

Peroxynitrite inactivates tryptophan hydroxylase via sulfhydryl oxidation. Coincident nitration of enzyme tyrosyl residues has minimal impact on catalytic activity.

Tryptophan hydroxylase, the initial and rate-limiting enzyme in serotonin biosynthesis, is inactivated by peroxynitrite in a concentration-dependent manner. This effect is prevented by molecules that react directly with peroxynitrite such as dithiothreitol, cysteine, glutathione, methionine, tryptophan, and uric acid but not by scavengers of superoxide (superoxide dismutase), hydroxyl radical (Me(2)SO, mannitol), and hydrogen peroxide (catalase). Assuming simple competition kinetics between peroxynitrite scavengers and the enzyme, a second-order rate constant of 3.4 x 10(4) M(-1) s(-1) at 25 degrees C and pH 7.4 was estimated. The peroxynitrite-induced loss of enzyme activity was accompanied by a concentration-dependent oxidation of protein sulfhydryl groups. Peroxynitrite-modified tryptophan hydroxylase was resistant to reduction by arsenite, borohydride, and dithiothreitol, suggesting that sulfhydryls were oxidized beyond sulfenic acid. Peroxynitrite also caused the nitration of tyrosyl residues in tryptophan hydroxylase, with a maximal modification of 3.8 tyrosines/monomer. Sodium bicarbonate protected tryptophan hydroxylase from peroxynitrite-induced inactivation and lessened the extent of sulfhydryl oxidation while causing a 2-fold increase in tyrosine nitration. Tetranitromethane, which oxidizes sulfhydryls at pH 6 or 8, but which nitrates tyrosyl residues at pH 8 only, inhibited tryptophan hydroxylase equally at either pH. Acetylation of tyrosyl residues with N-acetylimidazole did not alter tryptophan hydroxylase activity. These data suggest that peroxynitrite inactivates tryptophan hydroxylase via sulfhydryl oxidation. Modification of tyrosyl residues by peroxynitrite plays a relatively minor role in the inhibition of tryptophan hydroxylase catalytic activity.

Regulation of the reduced-folate transporter by nitric oxide in cultured human retinal pigment epithelial cells.

The regulation of the reduced-folate transporter (RFT) by nitric oxide (NO) was analyzed in human retinal pigment epithelial (HRPE) cells. NO inhibited specifically and reversibly the uptake of N5-methyltetrahydrofolate by a cGMP-independent mechanism. The inhibition was associated with a decrease in substrate affinity. The NO-induced inhibition was prevented by antioxidants and NO scavengers. Agents capable of modifying thiol groups in proteins inhibited RFT, indicating that the likely mechanism of NO-induced inhibition is via modification of essential thiol groups in this protein. These studies suggest that NO produced during retinal disease may affect the function of RFT in adjacent RPE cells.