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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Sheng Wu Wu Li Hsueh Bao. Author manuscript; available in PMC 2012 November 6.
Published in final edited form as:
Sheng Wu Wu Li Hsueh Bao. 2012 April 20; 28(4): 341–350.
PMCID: PMC3490496

Reversible inactivation of dihydrolipoamide dehydrogenase by Angeli’s salt


Dihydrolipoamide dehydrogenase (DLDH) is a key component of 3 mitochondrial α-keto acid dehydrogenase complexes including pyruvate dehydrogenase complex, α-ketoglutarate dehydrogenase complex, and branched chain amino acid dehydrogenase complex. It is a pyridine-dependent disulfide oxidoreductase that is very sensitive to oxidative modifications by reactive nitrogen species (RNS) and reactive oxygen species (ROS). The objective of this study was to investigate the mechanisms of DLDH modification by RNS derived from Angeli’s salt. Studies were conducted using isolated rat brain mitochondria that were incubated with varying concentrations of Angeli’s salt followed by spectrophotometric enzyme assays, blue native gel analysis, and 2-dimensional gel-based proteomic approaches. Results show that DLDH could be inactivated by Angeli’s salt in a concentration dependent manner and the inactivation was a targeting rather than a random process as peroxynitrite did not show any detectable inhibitory effect on the enzyme’s activity under the same experimental conditions. Since Angeli’s salt can readily decompose at physiological pH to yield nitroxyl anion (HNO) and nitric oxide, further studies were conducted to determine the actual RNS that was responsible for DLDH inactivation. Results indicate that it was HNO that exerted the effect of Angeli’s salt on DLDH. Finally, two-dimensional Western blot analysis indicates that DLDH inactivation by Angeli’s salt was accompanied by formation of protein s-nitrosothiols, suggesting that s-nitrosylation is likely the cause of loss in enzyme’s activity. Taken together, the present study provides insights into mechanisms of DLDH inactivation induced by HNO derived from Angeli’s salt.

Keywords: Angeli’s salt, brain, dihydrolipoamide dehydrogenase, mitochondria, nitroxyl anion (HNO), S-nitrosylation


Dihydrolipoamide dehydrogenase (DLDH) is an essential component of pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and branched chain keto acid dehydrogenase complexes [1, 2]. It belongs to the family of flavin-dependent, pyridine dinucleotide disulfide oxidoreductase including glutathione reductase, mercuric reductase, trypanothione reductase, and thioredoxin reductase. It is also the L protein in the glycine cleavage system [3, 4]. DLDH is a stable homodimer, with each monomer owning a tightly but non-covalently bound FAD molecule, a transiently bound NAD+ or NADH molecule, and a redox-sensitive center containing two cysteine residues that are directly involved in thiol-disulfide exchanges during catalysis [1, 57]. Physiologically, DLDH oxidizes the reoxidation of dihydrolipoamide that is covalently linked to acyltransferase at the expense of NAD+ [1, 2, 8]. Therefore, the enzymatic reaction also produces NADH that can enter into the electron transport chain for oxidative phosphorylation and ATP synthesis.

DLDH is redox-sensitive and is a multifunctional oxidoreductase. In vitro, DLDH can serve as a diaphorase [9] that can transfer electrons from NADH to electron acceptors such as ubiquinone [10, 11], cytochrome c [12], and artificial electron-accepting dyes including nitro-blue tetrazolium (NBT) [13, 14] and 2,6-dichlorophenolindophenol (DCPIP) [15]. Pathophysiologically, DLDH dysfunction can cause maple syrup urine disease due to accumulation of branched chain amino acids in the body [16], and is involved in arsenite poisoning due to binding of arsenite to the vicinal thiols of dihydrolipoamide, which then prevents reoxidation of dihydrolipoamide by DLDH [17].

Interestingly, as a redox-sensitive protein, DLDH can either aggravate or mitigate oxidative damage depending on experimental conditions. On one hand, DLDH can be a source of reactive oxygen species [1822] and can be oxidatively modified leading to potential impairment of mitochondrial function [2328]; on the other hand, DLDH can scavenge nitric oxide [12] and serve as an antioxidant by protecting other proteins against oxidative modifications [29]. Moreover, when its homodimeric status is perturbed, DLDH can turn itself into a protease [30] and thereby enhances oxidative damage [31].

Our research interest in DLDH is focused on the role of its oxidative modifications in aging and age-related neurodegenerative diseases. Our first stage of studying DLDH oxidative modifications mainly centered on in vitro oxidative modifications of this protein by RNS or ROS. In the present article, we report our findings of in vitro DLDH inactivation by Angeli’s salt. We chose Angeli’s salt as the donor of RNS mainly based on our previous findings that this chemical, among the RNS donors tested, is the most reactive toward DLDH [32]. Additionally, the use of Angeli’s salt in the present study was also prompted by the fact that this pharmacological reagent has therapeutic potential [3337].

Experimental rocedures

Animal and chemicals

Adult male Sprague-Dawley rats were obtained from Harlan (Indianapolis, IN). Use of animals was in adherence with the NIH Guidelines for the Care and Use of Laboratory Animals and the protocol was approved by the University of North Texas Health Science Center Animal Care and Use Committee. Dihydrolipoamide was prepared using sodium borohydride reduction of lipoamide as previously described [15, 38]. Rabbit anti-DLDH polyclonal antibodies (IgG) were from US Biological (Swampscott, MA, USA) and goat anti-rabbit IgG conjugated with horseradish peroxidase was from Zymed (South San Francisco, CA, USA). Angeli’s salt and peroxynitrite were purchased from Cayman (Ann Arbor, MI) and all other chemicals were purchased from Sigma (St. Louis, MO) unless otherwise stated.

Preparation of brain mitochondria

The isolation of mitochondria from whole brain was carried out as previously outlined [39] with modifications [32]. Brains were removed rapidly and homogenized in 15 ml of ice-cold, mitochondrial isolation buffer containing 0.32 M sucrose, 1 mM EDTA and 10 mM Tris-HCl, pH 7.1. The homogenate was centrifuged at 1,330 g for 10 min and the supernatant was saved. The pellet was resuspended in 0.5 (7.5 ml) volume of the original isolation buffer and centrifuged again under the same conditions. The two supernatants were combined and centrifuged further at 21,200 g for 10 min. The resulting pellet was resuspended in 12% Percoll solution that was prepared in mitochondrial isolation buffer and centrifuged at 6,900 g for 10 min. The resulting supernatant was then carefully removed by vacuum. The obtained soft pellet was resuspended in 10 ml of the mitochondrial isolation buffer and centrifuged again at 6,900 g for 10 min. All of the mitochondrial pellets obtained after centrifugation were immediately used. All the protein concentrations were determined by bicinchoninic acid assay [40] using a kit obtained from Pierce (Rockford, IL).

In vitro DLDH inactivation by Angeli’s salt

DLDH oxidative inactivation in isolated brain mitochondria was performed by supplementing mitochondria with various concentrations of Angeli’s salt as previously described [32]. Essentially, mitochondria (0.25 mg/ml) were incubated at 25°C for 30 min inincubation buffer (110 mM mannitol, 10mM KH2PO4, 60 mM Tris, 60 mM KCl, and 0.5 mM EGTA, pH 7.4) in the presence or absence of Angeli’s salt. At the end of the incubation, mitochondria were pelleted by centrifugation at 8,000 g for 10 min, and mitochondrial extracts were then prepared as described below. For incubation of the mitochondrial extracts with reducing reagents such as DTT, cysteine, and GSH, 10 mM (final concentration) of each of the reducing reagent was added and the sample was further incubated for 30 min before gel analysis or spectrophotometric enzyme assay.

Preparation of mitochondrial extracts for spectrophotometric enzyme assay and BN-PAGE analysis

Whole mitochondrial extracts as the source of DLDH analyses were prepared as previously described [15, 38]. Briefly, following incubation with Angeli’s salt, mitochondria were pelleted at 8,000 g for 10 min and the pellet was resuspended at a protein concentration of approximately 0.5 mg/ml in a hypotonic buffer containing 20 mM potassium phosphate (pH 7.4), 1% Triton X-100, 2 mM EDTA, 2 mM EGTA and protease inhibitors. The suspension was sonicated in a Branson Sonifier 150, four times for 30 sat 1-min intervals. The sonicated sample was kept on ice for 30 min and clarified by centrifugation at20,000 g for 30 min. The clear DLDH-containing supernatant was then used for mitochondrial DLDH assays as described below.

Spectrophotometric measurement of DLDH dehydrogenase activity

DLDH dehydrogenase activity was measured by DLDH catalyzed, NAD+-dependent oxidation of dihydrolipoamide [15, 38]. The final volume of reaction was 1 ml, and the mixture contained 100 mM potassium phosphate, pH 8.0, 1.5 mM EDTA, 0.6 mg/ml BSA, 3.0 mM dihydrolipoamide and 3.0 mM NAD+. A solution containing all the assay components except mitochondrial extracts was used as the blank. The reaction was initiated by the addition of appropriate amount of mitochondrial extracts (10–20 μg/ml assay solution) and the change in absorbance at 340 nm was followed at room temperature. An extinction coefficient of 6.22 mM−1 cm−1 for NADH was used for the calculation of enzyme activity [15, 38]. One unit of dehydrogenase activity was defined as 1μmol of NAD+ reduced per min.

In-gel DLDH diaphorase activity staining

In-gel staining of DLDH diaphorase activity by NBT/NADH was performed using blue native polyacrylamide gel electrophoresis (BN-PAGE) as previously described [32]. The gel buffer contained 500 mM aminocaproic acid and 50 mM Bis-Tris, pH 7.0. The cathode buffer contained 50 mM tricine, 15 mM Bis-Tris, pH 7.0, and 0.02% Serva blue G-250, whereas the anode buffer contained 50 mM Bis-Tris, pH 7.0. The sample buffer was 50 mM aminocaproic acid (final concentration) containing 0.3% Serva blue G-250 (w/v, final concentration). Following sample loading (typically, 20 μg protein/lane), the gel was run at 150 V until the front line had entered into one-third of the gel where the cathode buffer was replaced by the one that did not have Serva blue G-250 (50 mM Tricine, 15 mM Bis-Tris, pH 7.0). Gel running was then resumed at 200 V until complete. The gel was incubated in 50 mM potassium phosphate buffer (pH 7.0) containing 0.2 mg/ml NBT and 0.1 mg/ml NADH. When an appropriate color of DLDH band appeared, the staining solution was discarded and the gel was scanned immediately for gel documentation.

Labeling of protein s-nitrosothiols by ascorbate reduction/biotin switch assay

The procedure used to label protein s-nitrosothiols was performed as previously described [41] with modifications. Mitochondrial pellet, immediately following isolation or in vitro treatment with Angeli’s salt, was solubilized in a thiol-group blocking buffer containing 100 mM sodium acetate (pH 7.0), 20 mM NaCl, 1% SDS and 100 mM N-ethylmaleimide (NEM). The protein mixture was incubated on a rotator at room temperature for 2 hrs followed by clarification of the mixture by centrifugation at 13,000 g for 10 min. Excess NEM in the supernatant was removed by gel filtration using PD-10 columns. This was followed by addition of 0.1 mM biotin-maleimide and 5 mM sodium ascorbate (both final concentrations) to the eluate. The sample was further incubated in dark on a rotator at room temperature for 30 min. Proteins were then precipitated by 10% TCA (final concentration) on ice for 10 min followed by centrifugation on a bench top centrifuge at 1,000 g for 5 min. The pellet was washed three times with ethyl acetate: ethanol (1:1, v/v). Protein pellet after the third wash was used for 2D Western blot probing of the biotinylated (s-nitrosylated) proteins.

Two-dimensional Western blot detection of sulfenated proteins

Two-dimensional Western blot was performed as previously described [42] with modifications. Following biotinylation of the s-nitrosylated cysteine residues, mitochondrial pellets were resuspended in two-dimensional rehydration buffer [8 M urea, 4% CHAPS, 0.2% ampholytes (pH 3–10), and 100 mM DTT]. First-dimensional protein separation was performed with Bio-Rad Protean IEF Cell. Samples (40 μg/IPG strip) were applied to immobilized pH gradient strips (7-cm, nonlinear pH 3–10, Bio-Rad) for 1 h at room temperature. The strips were then covered with mineral oil overnight, and isoelectric focusing was performed using the preset rapid voltage ramping method. For the second dimension, the immobilized pH gradient strips were equilibrated in room temperature for 25 min in equilibration buffer (6 M urea, 2% SDS, 0.05 mM Tris-HCl, 20% glycerol) to which 2% DTT was added before use. An additional 25 min equilibration period was then used with the same equilibration buffer to which 2.5% iodoacetamide, instead of 2% DTT, was added. The strips were then embedded in 0.7% agarose on the top of 7.5% Laemmli polyacrylamide slab gels (no stacking gel) and run by Tricine SDS/PAGE running buffer [43]. One of the resulting two-dimensional gels was stained with Coomassie colloid blue as previously described [44], and the other gel underwent electrophoretic transfer to PVDF membrane followed by immunoblotting with HRP-streptavidin. Signals on the PVDF membrane were visualized with an enhanced chemiluminescence kit. Biotin-conjugated protein marker was used for the purpose of both molecular weight ladders and positive controls. For 1D Western blot analysis, gel electrophoresis under reducing conditions was conducted. All images were scanned by an EPSON PERFECTION 1670 scanner.

Data analysis

All experiments were performed independently at least three times using different mitochondrial preparations. Where applicable, results are expressed as means ± SEM. Statistical analysis was performed with Welch’s t test using Instat software (Graphad Software, San Diego, CA). A probability value less than 0.05 (p < 0.05) was considered significant.


Angeli’s salt inactivated DLDH in a concentration-dependent manner

We have previously shown that DLDH is very sensitive to inactivation by Angeli’s salt and Angeli’s salt is more powerful in inhibiting DLDH activity than other RNS donors that were tested [32]. In the present study, we further investigated the mechanisms of DLDH inactivation by Angeli’s salt. Our first experiment was to determine whether DLDH inactivation by Angeli’s salt was concentration dependent. Results in Fig. 1 demonstrate that when analyzed by blue native PAGE, loss of DLDH diaphorase activity was indeed Angeli’s salt concentration dependent. Loss of the enzyme’s dehydrogenase activity, measured spectrophotometrically, also exhibited a similar pattern (Fig. 2). Therefore, DLDH activity in whole mitochondria can be impaired by Angeli’s salt in a concentration-dependent fashion.

Fig. 1
Effect of Angeli’s salt on DLDH diaphorase activity. Mitochondria were incubated with varying concentrations of Angeli’s salt at room temperature for 30 min followed by mitochondrial extract preparation in an hypotonic buffer containing ...
Fig. 2
Effect of Angeli’s salt on DLDH dehydrogenase activity measured spectrophotometrically. Mitochondria incubation with Angeli’s salt and preparation of mitochondrial extracts were performed as described in Fig. 1. The spectrophotometric ...

DLDH in whole mitochondria was specifically targeted by Angeli’s salt

To determine whether DLDH inactivation by Angeli’s salt is a random or targeting process, we compared the effect of Angeli’s salt with that of peroxynitrite. Results in Fig. 3 show that peroxynitrite, when incubated with whole mitochondria, could not impose its inhibitory effect on DLDH activity. In contrast, Angle’s salt, under the same experimental conditions, gave a similar result as shown in Fig. 2. These results indicate that DLDH was specifically targeted by Angeli’s salt in whole mitochondria.

Fig. 3
Peroxynitrite did not inactivate DLDH when incubated with whole mitochondria. This was in contrast to that of Angle’s salt. All values are mean ± SEM of three independent experiments.

DLDH inactivation by Angeli’s salt was caused by nitroxyl anion (HNO)

It is well-known that at physiological pH, Angeli’s salt can readily decompose to yield nitroxyl anion (HNO) and nitrite [45, 46]. To determine which of the two decomposition products was responsible for the loss of DLDH activity, we first incubated mitochondria with nitrite. Result in Fig. 4A shows that nitrite did not cause any detectable loss in DLDH activity, suggesting that HNO is responsible for DLDH inactivation. We then incubated mitochondria with Angeli’s salt in the presence of ferricyanide that can eliminate HNO by converting it to nitric oxide [47]. Result in Fig. 4B shows that the ability of Angeli’s salt to inactivate DLDH was completely abolished by ferricyanide. These results indicate that it was HNO that caused the loss of DLDH activity under our experimental conditions.

Fig. 4
No changes in dehydrogenase activity could be observed after mitochondria were incubated with (A) nitrite or (B) Angeli’s salt + ferricyanide. Mitochondria incubation with the indicated chemicals, mitochondrial extract preparation, and dehydrogenase ...

DLDH inactivation by Angeli’s salt was largely reversible

To determine whether DLDH’s inactivation by Angeli’s salt was reversible or not, we evaluated the effects of DTT, cysteine, and glutathione (GSH, reduced form) on enzyme’s activity following mitochondrial incubation with Angeli’s salt (0.8 mM, in this experiment). At the concentration of 10 mM, each of the three reducing reagents could restore the dehydrogenase activity to greater than 80% of the control value (Fig. 5). Interestingly, DLDH’s diaphorase activity, when analyzed by BN-PAGE, could only be significantly restored by cysteine and GSH (10 mM each), but not DTT (Fig. 6). The reason for this discrepancy of DTT’s effect on dehydrogenase and diaphorase activities remains unknown. Nonetheless, these results demonstrate that DLDH inactivation by Angeli’s salt could largely be reversed by appropriate reducing reagents.

Fig. 5
Loss of dehydrogenase activity induced by Angeli’s salt could be restored by reducing reagents such as DTT, cysteine, and GSH (10 mM each). After mitochondria incubation with Angeli’s salt, mitochondrial extracts were prepared in the presence ...
Fig. 6
Loss of diaphorase activity induced by Angle’s salt could be restored by cysteine and GSH, but not by DTT. (A) BN-PAGE measurement of DLDH diaphorase activity. (B) Densitometric values derived from (A). Values, expressed as percentage of control ...

S-nitrosylation was likely involved in DLDH inactivation by Angeli’s salt

As it is well established that Angeli’s salt can induce protein s-nitrosylation [48] and s-nitrosylation is a reversible process [49, 50], we further determined whether DLDH inactivation by Angeli’s salt involved s-nitrosylation. To this end, we used a biotin-switch method [41] followed by localization of s-nitrosylated proteins by 2D Western blot probed with streptavidin linked with horse radish peroxidase. DLDH was localized by 2D Western blots probed with anti-DLDH antibodies. Results are shown in Fig. 7. Fig. 7A shows a 2D Western blot map in the absence of ascorbate that was used to reduce protein nitrosothiols back to free thiols for biotinylation and detection. As can be seen in Fig. 7B, more nitrosylated proteins could be visualized by the use of ascorbate and the arrow-indicated spot was that of DLDH. The identity was further confirmed by blotting the same membrane with anti-DLDH antibodies (Fig. 7C) as spot in Fig. 7C overlapped with that indicated in Fig. 7B. Moreover, when the same spot in Fig. 7B was excised and analyzed by 1D Western blot probed with anti-DLDH antibodies, a positive immunostaining could also be visualized (Fig. 7D), which further confirmed the spot’s identity in Fig. 7B as that of DLDH. These results indicate that DLDH inactivation by Angeli’s salt likely involved s-nitrosylation.

Fig. 7
Two dimensional Western blot detection of DLDH nitrosylation by Angeli’s salt. Samples were biotinylated in the absence (A) or presence (B) of ascorbate, a specific reducing reagent for protein s-nitrosothiols. In (B) the spot indicated by the ...


The main findings of the present study are as the following: 1) mitochondrial DLDH could be reversibly inactivated by Angeli’s salt; 2) the inactivation was a targeting rather than a random process as peroxynitrite, at similar concentrations, did not show any inhibitory effect on DLDH activity; 3) the RNS that actually inactivated DLDH was HNO derived from Angeli’s salt; and 4) the inactivation likely involved formation of s-nitrosothiols on DLDH.

It is noteworthy that when incubated with whole mitochondria, Angeli’s salt seemed to target DLDH. In contrast, peroxynitrite lacked this targeting ability in the presence of other mitochondrial proteins (Fig. 3); although peroxynitrite, when incubated with purified DLDH, could indeed inactivate the enzyme (unpublished data, this laboratory). Such observations suggest that peroxynitrite is consumed by other mitochondrial proteins before it can get to DLDH when whole mitochondria are treated by peroxynitrite. Alternatively, peroxynitrite, at the same concentration as that of Angeli’s salt, may not be as effective as Angeli’s salt in inactivating DLDH. Therefore, no inhibitory effect of peroxynitrite could be detected under our experimental conditions. Regardless, it is suffice to say that inactivation of DLDH by Angeli’s salt is a targeting process.

Angeli’s salt is a unique donor of RNS in that it can decompose readily at physiological pH with the production of two reactive nitrogen species, i.e., HNO and nitrite. Nevertheless, most of Angeli’s salt actions on proteins have been attributed to HNO [51]. In the present study, we also found that it was indeed HNO, but not nitrite, that actually inactivated DLDH (Fig. 3). This result is therefore in consistent with those by others that HNO is the actual species that imposes the biochemical and pharmacological effects of Angeli’s salt [36, 37]. It should be noted that nitric oxide can also be produced when Angeli’s salt is incubated with ferricyanide [52]. Nonetheless, nitric oxide generated this way apparently had no detectable inhibitory effect on DLDH activity (Fig. 3).

While we determined that DLDH inactivation by Angeli’s salt likely involved the formation of s-nitrosothiols, we were unable to determine which of DLDH’s redox-sensitive cysteine residues that were actually nitrosylated and led to decrease in DLDH activity. Our attempt to analyze modified cysteine residues by mass spectrometry techniques failed mainly because the peptide containing the two cysteine residues could not be recovered following HPLC and mass spectrometry analysis. Therefore, the redox cysteine residue(s) that was nitrosylated could only be speculated. DLDH has two cysteine residues at its active center that are engaged in catalysis, namely, cys45 and cys50 [53]. During catalysis, cys45 is the substrate-binding residue while cys50 is the FAD interacting residue. Studies by others have indicated that cys45 is more reactive than cys50 toward thiol-reactive reagents [6]. In glutathione reductase whose structure is similar to that of DLDH, the substrate binding cysteine residue could be nitrosylated and sulfenated upon treatment with nitric oxide donors [54]. Such studies suggest that the nitrosylated cysteine residue on DLDH is likely that of cys45 following treatment with Angeli’s salt.

It is well-established that protein s-nitrosylation is a reversible process and is involved not only in oxidative damage [55], but also in regulation and expansion of protein function [56]. For example, s-nitrosylation can contribute to protein misfolding and neuronal synaptic damage [55]; s-nitrosylation of mitochondrial proteins can also be involved in ischemic preconditioning that protects hearts against subsequent, severe ischemic injury [57]. While the physiological and pathophysiological significance of DLDH nitrosylation remains to be determined, such modification will certainly impair the enzyme’s function that may further perturb mitochondrial oxidative phosphorylation. It should be noted that while it has been previously reported that DLDH underwent s-nitrosylation [58, 59], none of these earlier studies examined the relationship between s-nitrosylation and loss of enzyme activity. In contrast, our present data suggest that nitrosylation was likely responsible, at least in part, for the loss of DLDH activity.

It should be pointed out that the ascorbate/biotin switch method may yield a false-positive signal in detection of protein S-nitrosylation [60]. Therefore, it might be argued that our observation of DLDH nitrosylation may have nothing to do with the observed reversible inactivation of DLDH function. This is the reason why we have only cautiously concluded that S-nitrosylation is probably the cause of DLDH reversible inactivation. A definitive conclusion can only be reached when both control and treated samples undergo the procedures of ascorbate reduction and biotin switch, whereby no positive or less intensive nitrosylation signals will be detected in the control samples. On the other hand, it is also possible that formation of protein sulfenic acids (S-sulfenation) could be partially responsible for the reversible inactivation of DLDH function observed in the present study as protein sulfenic acids can be readily derived from protein S-nitrosothiols [61]. If this is indeed the case, DLDH nitrosylation induced by Angeli’s salt would be only a transient form of DLDH modification and may only be considered partially responsible for the loss in DLDH activity. In other words, both S-nitrosylation and S-sulfenation may be involved in loss of DLDH activity induced by Angeli’s salt. Regardless, whether Angeli’s salt can induce DLDH sulfenic acid formation under our experimental conditions will need to be further investigated.

In summary, our study has indicated that brain mitochondrial DLDH could be inactivated in a dose-dependent manner by Angeli’s salt and the inactivation was caused by HNO. Our results have also revealed that DLDH inactivation by HNO was a targeting process and was accompanied by HNO-induced S-nitrosylation. Overall, the present study provides insights into the mechanisms of DLDH inactivation by Angeli’s salt, albeit that further studies are needed to determine which cysteine residues are nitrosylated.


This work was supported in part by the National Institutes of Health (AG022550).


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