JBC Anatrace, Inc.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mohr, S.
Right arrow Articles by Brüne, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mohr, S.
Right arrow Articles by Brüne, B.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Volume 271, Number 8, Issue of February 23, 1996 pp. 4209-4214
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Posttranslational Modification of Glyceraldehyde-3-phosphate Dehydrogenase by S-Nitrosylation and Subsequent NADH Attachment (*)

(Received for publication, July 31, 1995; and in revised form, October 16, 1995)

Susanne Mohr (1),  Jonathan S. Stamler (2), (§),  Bernhard Brüne (3)(¶)

From the  (1)Faculty of Biology, University of Konstanz, P.O. Box 5560 M612, 78434 Konstanz, Germany, the (2)Pulmonary and Cardiovascular Divisions, Duke University Medical Center, Durham, North Carolina 27710, and the (3)Faculty of Medicine, Department IV, Experimental Division, University of Erlangen-Nürnberg, Loschgestraße 8, 91054 Erlangen, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Nitric oxide (NO)-related activity has been associated with an NAD-dependent modification of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). However, the mechanism by which NO effects covalent attachment of nucleotide and its role in regulation of enzyme activity are controversial. Recent studies have shown that S-nitrosylation of GAPDH (Cys) initiates subsequent modification by the pyridinium cofactor. Here we show that NADH rather than NAD is the preferred substrate. Transnitrosation from active site S-nitrosothiol to the reduced nicotinamide ring system appears to facilitate protein thiolate attack on the enzyme-bound cofactor. This results in attachment of the intact NADH molecule. Moreover, we find that S-nitrosylation of GAPDH is responsible for reversible enzyme inhibition, whereas attachment of NADH accounts for irreversible enzyme inactivation. S-Nitrosylation may serve to protect GAPDH from oxidant inactivation in settings of cytokine overproduction and to regulate glycolysis. NADH attachment is more likely to be a pathophysiological event associated with inhibition of gluconeogenesis.

INTRODUCTION

The versatility of NO as a biological messenger reflects its participation in rich additive and redox chemistry. Pathways of NO oxidation involve reactions with O(2), O(2), and transition metals, which support the formation of surrogates retaining NO-like bioactivity(1, 2) . This is exemplified in the case of S-nitrosothiols that are formed in vivo and serve as NO-group donors(3, 4, 5) . In particular, NO donation (heterolytic decomposition) appears to be the predominant mechanism of RSNO (^1)metabolism in many biological systems (6, 7, 8) . Examples of NO-related activities include allosteric modulation of the N-methyl-D-aspartate receptor involved in neuroprotection(9) , the antimicrobial effects of RSNO(10) , the inhibition of many sulfhydryl-containing enzymes(11, 12) , the activation of p21 and tissue plasminogen activator, and the down-regulation of transcriptional activators(13, 14, 15) .

NO-related signal transduction can be broadly classified as cGMP-dependent or mediated through redox signaling events (1) . The latter is, perhaps, best exemplified in the regulation of protein function by S-nitrosylation(16) . In the case of enzymes that contain critical thiols at their active site, covalent attachment of the NO group leads uniformly to functional attenuation. Examples of enzymes in this category include cathepsin B, aldolase, -glutamylcysteinyl synthetase, aldehyde dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (see (1) and references therein). Studies on the potential regulation of GAPDH have received particular attention in view of evidence that NO-related activity (nitric oxide synthase activity or NO donors) stimulates an NAD-dependent posttranslational modification of active site thiol in association with the loss of protein function (3, 17, 18) . The demonstration of such a modification in cells has led to the proposal that NO induces an ADP-ribosylation reaction reminiscent of that catalyzed by bacterial and mammalian enzymes (19, 20, 21) . In this reaction, the ADP-ribose moiety of NAD is transferred to acceptor amino acids with the release of nicotinamide. Studies by Pancholi and Fischetti (22) may provide the strongest evidence in favor of a true thioglycosidic linkage induced by NO. More recently, however, this mechanism has been challenged by the demonstration that both the ribose and the nicotinamide moieties of NAD are incorporated by GAPDH(23) . This activity implies linkage of the intact molecule to the active site of the enzyme. Furthermore, inhibition of enzyme activity has seemed to correlate better with the extent of S-nitrosylation than the attachment of P-nucleotide, the latter representing only a small fraction of the total protein(23) .

We recently probed the mechanism of GAPDH modification using several NO donors. Our studies revealed that NO transfer to active site thiol is requisite for subsequent modification by [P]NAD(3) . These data, however, raise a fundamental paradox, as the pathway by which S-nitrosylation facilitates covalent modification by NAD is not readily apparent. We reasoned, therefore, that NADH rather than NAD is involved in this reaction, since reduction of nicotinamide would make it susceptible to activation via nitrosative attack. Here we show that 1) S-nitrosylation promotes covalent attachment of reduced nicotinamide; 2) covalent modification by [P]NADH occurs (largely) via a thionicotinic linkage; 3) S-nitrosylation of GAPDH accounts for reversible enzyme inhibition, and 4) covalent modification by NADH is responsible for irreversible protein inactivation.

EXPERIMENTAL PROCEDURES

Materials

[P]NAD (800 Ci/mmol) was purchased from DuPont NEN. SIN-1 was provided by Cassella AG (Frankfurt, Germany). BF(4)NO was purchased from Aldrich, and BF(4)NO(2) was obtained from Fluka. Rabbit muscle GAPDH (80 units/mg) and trypsin (high sequencing grade) were obtained from Boehringer Mannheim. Pertussis toxin A protomer and isocitrate dehydrogenase (NAD-specific, 31 units/mg) were obtained from Calbiochem. Other chemicals including nicotinamide 1,N^6-ethenoadenine dinucleotide were of the highest grade of purity made available by Sigma.

Preparation of [P]NADH and [nicotinamide ^14C]NADH

10 µl (50 µCi) of [P]NAD (800 Ci/mmol) was incubated with 2 mM MnCl(2), 10 mM isocitrate (pH 7.5), 1 µM cold NAD, 22 units of isocitrate dehydrogenase (NAD specific, 31 units/mg), and 100 mM Hepes (pH 7.5) in a total volume of 100 µl at 37 °C for 45 min. Samples were loaded on a nucleosil C(18) reverse phase column equilibrated with buffer A (200 mM potassium phosphate buffer, pH 6.0) at a flow rate of 1 ml/min. Nucleotides were separated by elution with buffer B (200 mM potassium phosphate buffer, pH 6.0, 5% methanol) using gradient elution: 0-10 min, 70% B; 11-26 min, 100% B; 27-35 min, 70% B at flow rate of 0.7 ml/min. On-line detection of radioactivity (Ramona) was used to localize peak fractions. Elution of NAD occurred at 15 min and NADH at 20 min. Eluted fractions were collected, and radioactivity was determined using a liquid scintillation counter (PW 4760 from RayTest). 120,000-150,000 cpm/assay were added in GAPDH labeling experiments.

For preparation of [nicotinamide-^14C]NADH, 20 µl (1 nCi) [nicotinamide-^14C]NAD (41 mCi/mmol) was incubated with 2 mM MnCl(2), 10 mM isocitrate (pH 7.5), 22 units of isocitrate dehydrogenase (NAD-specific, 31 units/mg), and 100 mM Hepes (pH 7.5) in a total volume of 100 µl at 37 °C for 45 min. Separation of ^14C-labeled NAD/NADH was performed as described above.

Covalent Modification of GAPDH by [P]NADH/[P]NAD

Radioactive labeling of GAPDH was performed as previously described(3) . Briefly, GAPDH (10 µg/assay) was incubated in 100 mM Hepes buffer (pH 7.5) containing 2.5 mM DTT, 50-200 µM of a NO donor, [P]NADH (120,000 cpm/assay), 10 µM NADH, or [P]NAD (120,000 cpm/assay), and 10 µM NAD. At indicated time points (we usually assay at 37 °C for 20 min) proteins were precipitated with 200 µl of 20% ice-cold trichloroacetic acid, left on ice for 30 min, and then centrifuged for 15 min (13,000 times g) at 4 °C. The resulting pellets were then washed twice with 1 ml of ice-cold water-saturated ether and separated in a 10% sodium dodecyl sulfate-polyacrylamide gel. Radioactivity was quantified using the PhosphorImager system (Molecular Dynamics).

Modification of GAPDH Using Nitrosonium- and Nitronium-tetrafluoroborate

Stock solutions of BF(4)NO and BF(4)NO(2) were prepared under acidic conditions (pH 2; 0.2 M HCl)(3) . Incubations were carried out with GAPDH (10 µg/assay), [P]NADH (120,000 cpm/assay), 10 µM NADH, and 50-100 µM BF(4)NO or BF(4)NO(2), as described above for the covalent modification of GAPDH. DTT (2.5 mM) was included in some assays.

Modification of GAPDH by [nicotinamide-^14C]NADH/[nicotinamide-^14C]NAD

Modification of GAPDH was performed in the presence of 800 µM SIN-1, 2.5 mM DTT, 10 µM [nicotinamide-^14C]NADH (40,000 cpm/assay), or 10 µM [nicotinamide-^14C]NAD (40,000 cpm/assay) as outlined above. For detection of radioactivity, gels were exposed (PhosphorImager exposing cassettes) for up to 30 days.

HgCl(2) Cleavage of Pertussis Toxin/[P]NAD-treated Proteins and of SIN-1/[P]NADH-treated GAPDH

Pertussis toxin-induced ADP-ribosylation was achieved by incubating human platelet microsomes (80 µg/assay) with 1 mM ATP, 0.1 mM GTP, 10 mM thymidine, 10 µM cold NAD, [P]NAD (0.5 µCi/assay), and pertussis toxin A protomer (0.5 µg/assay, activated by treatment with 10 mM DTT for 45 min at 37 °C). Preparation of platelet microsomes was performed according to published procedures(24) . Proteins were precipitated with 200 µl of 20% ice-cold trichloroacetic acid and left on ice for 30 min. Following centrifugation (10,000 times g, 15 min) protein pellets were washed twice with ice-cold water-saturated ether. Samples of either pertussis toxin/[P]NAD platelet membranes or SIN-1/[P]NADH-treated GAPDH were resuspended in 100 µl of 100 mM Hepes buffer (pH 7.5) containing 0.5-5 mM HgCl(2). Cleavage experiments were carried out for 90 min at 37 °C and followed by protein precipitation (500 µl of 20% trichloroacetic acid). Samples were then processed for detection of protein-bound radioactivity as described above. Controls were treated in the same way, but HgCl(2) was replaced by NaCl.

Tryptic Digestion of NAD-modified GAPDH

GAPDH (200 µg) was modified as described above using 20 mM DTT, 5 mM SIN-1, and 200 µM NAD/[P]NAD (500,000 cpm/assay). Reaction mixtures (0.5 ml) were incubated for 60 min at 37 °C. Protein was precipitated with 500 µl 50% ice-cold trichloroacetic acid, and incubates were left at 4 °C for 45 min. After centrifugation, pellets were washed twice with ice-cold water-saturated ether. Samples were then resuspended in 1 ml of 50 mM NH(4)HCO(3) buffer (pH 7.4) and incubated with 20 µg of trypsin at 37 °C for 4 h. Peptide fragments were separated by reversed phase high pressure liquid chromatography analysis (Nucleosil C(18)) using gradient elution: 0-10 min, 100% A; 10-60 min, 0-100% B at a flow rate of 1 ml/min. Buffer A contained H(2)O and 0.04% trifluoroacetic acid, while B consisted of 40% H(2)O, 60% acetonitrile, and 0.03% trifluoroacetic acid. Fragments were detected by serial UV absorbance and on-line radioactivity measurements. Radioactive fractions were concentrated, and radioactivity was allowed to decline. The fractions were then subjected to commercial peptide sequencing analysis. Based on theoretical predictions, tryptic digestion of rabbit muscle GAPDH should give three fragments containing cysteine residues. The following peptide contains the active site thiol Cys: IVSNASCTTNCLAPLAK.

GAPDH Activity

GAPDH (1 µg/assay), 1 mM DTT, 10 µM NAD, or 10 µM NADH, and up to 200 µM of a NO donor were incubated in 50 mM triethylammonium buffer (pH 7.5) in a total volume of 50 µl at 37 °C. Incubation times are indicated below. Samples were diluted in 950 µl of 50 mM triethylammonium buffer (pH 7.5) containing 50 mM arsenate, 2.4 mM glutathione, and 100 µg/ml glyceraldehyde 3-phosphate, at 37 °C. The enzymatic reduction of NAD to NADH was initiated by the addition of 250 µM NAD. GAPDH activity was monitored by recording the fluorescence emission above 430 nm after excitation at 313 and 366 nm, respectively. Samples without NO donors served as controls.

ADP-ribosylation and GAPDH Modification Using Nicotinamide 1,N^6-Ethenoadenine Dinucleotide

Nicotinamide 1,N^6-ethenoadenine dinucleotide (-NAD), a fluorescent cofactor analog, can substitute for NAD in dehydrogenase reactions, including those catalyzed by GAPDH(25) . Cleavage of the beta-nicotinamide linkage results in an increase in fluorescence(25) . When -NAD was used in pertussis toxin-induced ADP-ribosylation reactions, we observed an increase in fluorescence intensity (compared with controls, in the absence of toxin). -NAD (10 µM) was used during GAPDH (10 µg) modification by 200 µM SIN-1 and 2.5 mM DTT. After 40 min we determined GAPDH activity and changes in fluorescence relative to controls lacking the NO donor.

Stoichiometry of GAPDH Modification Achieved with NADH

GAPDH (1 µg/assay) was incubated in 100 mM Hepes buffer (pH 7.5) containing 2.5 mM DTT, 200 µM SIN-1, [P]NADH (200,000 cpm/assay), and 10 µM NADH at 37 °C for 20 min. Samples (whole assay) were transferred to microconcentrators (Amicon, Microcon, for volumes below 500 µl) and centrifuged at 10,000 times g for 10 min. Proteins were washed three times by the addition of 200 µl of 100 mM Hepes buffer (pH 7.5) and centrifuged, according to published protocol(23) . Samples were then counted in the liquid scintillation counter. A standard curve derived from [P]NADH/cold NADH ratios served as the basis for stoichiometry calculations.

RESULTS

Covalent Modification of GAPDH: NADH versus NAD

In the presence of [P]NAD, SNP stimulated thiol-dependent covalent radiolabeling of GAPDH, confirming previous reports. However, the incorporation of radioactivity was more pronounced when using the reduced pyridinium nucleotide, i.e. [P]NADH in place of [P]NAD (Fig. 1). Although some nonspecific NO-independent labeling occurred with [P]NADH, the site of protein attachment was found to be distinct from that supported by S-nitrosylation and could be readily controlled for (see below).

Figure 1: Modification of GAPDH by [P]NAD and [P]NADH. Modification of GAPDH was performed with 10 µM reduced or oxidized P-labeled nucleotide cofactor (each 200,000 cpm/assay), SNP, and DTT. Detection of radioactivity was made by PhosphorImager analysis. Experimental details are described under ``Experimental Procedures.'' Results are representative of three similar assays.


Importantly, NO donors of several different molecular classes were found to induce [P]NADH-dependent GAPDH labeling. However, the time course of enzyme modification varied among compounds (Fig. 2). For example, SNP-induced labeling was detected at 2.5 min and reached saturation by 5 min, whereas SIN-1 modification occurred at a much slower rate. Radiolabeling was noted after 10 min and required 40 min to achieve levels comparable with those with SNP. Nonspecific labeling was observed with NADH but required reducing conditions, i.e. DTT, and much longer reaction times (i.e. labeling was not detectable during the first 20-30 min).

Figure 2: Time-dependent modification of GAPDH by NADH in the presence of SNP and SIN-1. Modification of GAPDH was carried out as described under ``Experimental Procedures'' with 10 µM [P]NADH (120,000 cpm/assay). Detection was performed using a PhosphorImager. Data are representative of three similar experiments.


The pH optimum for nucleotide incorporation was 7.5 for NADH and above 8.5 for NAD, in agreement with involvement of enzyme (active site) thiolate (Fig. 3). In aggregate, these data are compatible with reports that S-nitrosylation of GAPDH is rate-limiting, since SNP is a better nitrosating agent than SIN-1. We speculate that the higher pH optimum for NAD may reflect its more efficient reduction by reduced thiol at alkine pH (26) . Nonspecific labeling by reduced nucleotide is also more prevalent under alkine conditions.

Figure 3: pH-dependent modification of GAPDH by NAD and NADH. Modification of GAPDH in the presence of 10 µM [P]NAD (120,000 cpm/assay) and 10 µM [P]NADH (120,000 cpm/assay) was determined under the following assay conditions: 100 mM Mes, pH 6.5; 100 mM Hepes, pH 7.5; 100 mM Tris, pH 8.5. SNP and DTT were incubated in each reaction mixture for 20 min. For experimental details see Fig. 1. One of three representative experiments is shown.


The amount of radioactivity incorporated into GAPDH (based on equivalent amounts of cold and radioactive labeled nucleotide) was much higher with NADH than NAD. Under optimal labeling conditions (10 µM NADH, 2.5 mM DTT, 20 min, n = 16), SIN-1 (200 µM) stimulation led to 1.14 ± 0.37 mol of NADH/mol of GAPDH. Similar rates of incorporation were achieved using 200 µM SNP (1.05 ± 0.15 mol of NADH/mol of GAPDH, mean ± S.D., n = 8; 2.5 mM DTT for 10 min). These results demonstrate the modification of approximately one GAPDH subunit/molecule holoenzyme (GAPDH consists of four identical 39-kDa polypeptide chains), and are to be contrasted with relatively trivial modification by NAD ((23) , Fig. 3and Fig. 4).

Figure 4: Modification of GAPDH by [nicotinamide-^14C]NAD and [nicotinamide-^14C]NADH. GAPDH labeling was performed for 20 min with SIN-1, DTT, 10 µM [nicotinamide-^14C]NAD (40,000 cpm/assay) or 10 µM [nicotinamide-^14C]NADH (40,000 cpm/assay). Further details are outlined under ``Experimental Procedures.'' Similar results were obtained in two separate assays.


Posttranslational GAPDH Modification: Mechanistic Considerations

In order to probe the mechanism of nucleotide attachment, GAPDH modification was monitored in the presence of ^14C-labeled nucleotide derivatives, specifically [nicotinamide-^14C]NAD and [nicotinamide-^14C]NADH. As shown in Fig. 4, the extent of protein labeling with [nicotinamide-^14C]NAD was modest in comparison with that induced by [nicotinamide-^14C]NADH. These data emphasize involvement of NADH and suggest that its linkage to protein occurs via the nicotinamide ring.

Experiments performed with -NAD further support the notion that binding involves the intact pyridine cofactor. Upon cleavage of the beta-nicotinamide bond in -NAD by pertussis toxin, the fluorescence of the molecule increased, much as described previously for NADase(25) . In comparison, modification of GAPDH using -NAD did not result in a change in fluorescence (Fig. 5), even though the substitution for NAD led to a comparable degree of GAPDH inhibition. For example, modification of GAPDH (10 µg) by 200 µM SIN-1 (2.5 mM DTT) in the presence of either 10 µM NAD or 10 µM -NAD resulted in a 55 ± 0.5% versus 54 ± 3.5% decrease in enzyme activity relative to controls after 40 min, respectively.

Figure 5: Pertussis toxin and GAPDH catalyzed reactions using -NAD. A, pertussis toxin-catalyzed ADP-ribosylation was carried out as described under ``Experimental Procedures'' using 10 µM -NAD at 37 °C for 40 min. B, GAPDH modification was performed as described above, using 10 µM -NAD. Changes in fluorescence were recorded as difference spectra. Experimental details are described under ``Experimental Procedures.''


We further reasoned that nitrosation of reduced nicotinamide was required for ring activation. This would then facilitate protein thiolate attack on the nucleotide by increasing its electrophilicity. To test the validity of this mechanism, we first examined the effects of nitrosonium-tetrafluoroborate (BF(4)NO), a strong NO donor (Fig. 6). In the presence of DTT, BF(4)NO potently induced GAPDH labeling by [P]NADH. Moreover, the NO(2)-donor nitronium-tetrafluoroborate (BF(4)NO(2)), which on theoretical grounds should be equally capable of nicotinamide activation(3) , resulted in comparable degrees of GAPDH modification (Fig. 6). With both agents, maximal labeling was achieved at concentrations of 50 µM under reducing conditions.

Figure 6: NADH-dependent covalent modification of GAPDH induced by BF(4)NO and BF(4)NO(2). Modification of GAPDH was performed with 10 µM [P]NADH (150,000 cpm/assay), DTT, BF(4)NO, and BF(4)NO(2). Details are given under ``Experimental Procedures.'' This figure is representative of three similar experiments.


Cleavage experiments with HgCl(2) were then performed in order to confirm that the NADH linkage involved protein thiol groups (i.e. Cys of GAPDH). Specifically, the enzyme preparation was treated with HgCl(2) after covalent modification had been induced with SIN-1. HgCl(2) (5 mM) was found to displace the greater part of the [P]NADH radiolabel (Fig. 7).

Figure 7: HgCl(2) cleavage of ADP-ribose following pertussis toxin treatment and of NADH following NO stimulation. Human platelet microsomes (80 µg/assay) were incubated with 10 µM [P]NAD (0.5 µCi/assay) and pertussis toxin as outlined under ``Experimental Procedures.'' Labeling of GAPDH (10 µg/assay) was performed with 200 µM SIN-1, 2.5 mM DTT, and 10 µM [P]NADH (200,000 cpm/assay) for 20 min. For HgCl(2) cleavage experiments, protein pellets were resuspended in 100 mM Hepes buffer (pH 7.5) and incubated for 90 min with HgCl(2). Following protein precipitation, the remaining radioactivity was measured as described under ``Experimental Procedures.'' Data are representative of three experiments.


Importantly, radioactivity incorporated by incubating GAPDH with 10 µM NADH and 2.5 mM DTT, i.e. nonspecific labeling, could be readily discriminated from active site modifications due to its resistance toward Hg treatment. 5 mM HgCl(2) or 10 mM DTT was unable to remove any such incorporated radioactivity. NO-independent radioactivity, however, was partially cleaved (around 40% decrease) by treatment with 5 mM NH(2)OH for 90 min. For comparison, the effects of HgCl(2) were examined on human platelet membranes that had been ADP-ribosylated by treatment with pertussis toxin. As previously shown, HgCl(2) (0.5 mM) removed all the radiolabel. Furthermore, over 90% of the radioactivity remained bound to GAPDH following treatment with hydroxylamine.

To further identify the cysteine residue involved by NO, a tryptic digestion of radiolabeled GAPDH was performed, attachment followed by sequence analysis of the single fragment that contained radioactivity. Amino acid sequencing identified the peptide IVSNAS, after which analysis resisted further cycling. The sequence matches identically with the predicted tryptic digestion fragment containing the active site Cys residue of rabbit muscle GAPDH. Thus, cleavage experiments combined with tryptic digestion strongly suggest that the modification of GAPDH occurs at Cys.

Inhibition of Enzyme Activity

In order to explore the relationship among covalent modification of GAPDH, the mechanism of nucleotide attachment, and enzyme activity, we compared the effects of GAPDH inhibition supported by NADH, NAD, beta-nicotinamide mononucleotide, nicotinamide, and nicotinamide N-propanesulfonic acid (Table 1).

In general, GAPDH inhibition appeared to correlate well with the extent of nucleotide incorporation. In particular, enzyme inhibition induced by SIN-1 was greatest with NADH. NAD was significantly less effective and equal in activity to beta-nicotinamide mononucleotide. Nicotinamide and nicotinamide N-propanesulfonic acid also exhibited inhibitory effects, although they were the least active compounds. The time- and concentration-dependent effects of NADH are detailed in Table 2. Importantly, radiolabel incorporation induced by S-nitrosylation of Cys correlated well with loss of enzyme activity, whereas NO-independent modification of GAPDH by NADH did not (data not shown). Inhibition of GAPDH clearly increased with time and with higher concentrations of the nucleotide.

Experiments were then performed to delineate the role of S-nitrosylation vis à vis covalent NADH attachment in the inhibition of GAPDH (Fig. 8). GAPDH was incubated with BFNO for 5 min followed by the addition of either DTT or NADH. GAPDH catalysis (Fig. 8, upper panel) was then monitored over the ensuing 30 min, and enzyme activity was correlated with the amount of incorporated radioactivity (Fig. 8, lower panel).

Figure 8: Action of BF(4)NO and NADH on GAPDH enzyme activity. In the upper panel GAPDH was incubated with 100 µM nitrosonium tetrafluoroborate (BF(4)NO) for 5 min, followed by the addition of 10 µM NADH or 10 mM DTT. Incubations were then continued for the times indicated. Samples treated with NADH/DTT (but not BF(4)NO) served as controls. In the lower panel GAPDH was preincubated with 100 µM BF(4)NO, followed by the addition of 10 µM NADH (150,000 cpm/assay). Incorporation of radioactivity was carried out as described above. Data are representative of four similar experiments.


Following the addition of BF(4)NO, we observed an initial drop in enzyme activity by 40% compared with untreated controls. Over the next 5 min, enzyme activity partially recovered spontaneously. Thereafter, either 10 mM DTT or 10 µM NADH were added. With the addition of DTT, GAPDH activity was restored to control values (90 ± 2.5%). In contrast, NADH caused a time-dependent irreversible inhibition of the enzyme. By 35 min, only 30% of the initial enzyme activity remained (Fig. 8, upper panel). Using [P]NADH, we confirmed that the extent of radiolabeling incorporated by GAPDH paralleled the degree of inhibition following the addition of nitrosating agent (Fig. 8, lower panel). We conclude 1) that S-nitrosylation is responsible for early, reversible enzyme inhibition and 2) that S-nitrosylation promotes subsequent irreversible attachment of NADH to active site thiol.

DISCUSSION

Nitric oxide has been associated with a mono-ADP-ribosylation-like reaction in which the pyridinium nucleotide undergoes covalent attachment to the active site thiol of GAPDH. However, the mechanism of this reaction is poorly understood, and its contribution to changes in protein function is controversial. We recently demonstrated that GAPDH modification is stimulated by S-nitrosylation, i.e. NO transfer chemistry rather than reaction of nitric oxide(3) . In particular, S-nitrosylation of the enzyme active site thiol (Cys) was found to initiate subsequent covalent modification by NAD. In order to rationalize this finding, we reasoned that NAD must first be reduced under in vitro assay conditions(1, 3) . Transnitrosation from active site RSNO to the nicotinamide ring could then facilitate protein thiolate attack on the nucleotide(1) . Here we show that NO-related activity does indeed depend on the presence of reduced nicotinamide and that NADH rather than NAD is the preferred reaction substrate. Specifically, labeling with NADH is much more efficient than with NAD, occurs more rapidly, and correlates better with changes in enzyme activity.

Mechanisms of Covalent Modification of GAPDH: S-Nitrosylation versus Nucleotide Attachment

Our results suggest that both S-nitrosylation and covalent attachment of nucleotide are relevant mechanisms of GAPDH modification. The close proximity of the cofactor binding site of GAPDH to active site thiol, evidenced by reports of their participation in a charge transfer complex(27, 28) , would be predicted to facilitate nitrosative attack at the C-5 position of the nicotinamide ring(29) . Resultant ring activation (increased electrophilicity) would then lead to protein thiolate attack at the C-6 position (Fig. 9, upper pathway).

Figure 9: Suggested reaction sequence of NO- induced NADH attachment to GAPDH. Modification of GAPDH follows the upper pathway leading to a thionicotinic linkage. For details see ``Discussion.''


This proposed mechanism is supported by three findings: 1) that several reduced nicotinamide derivatives can substitute for NADH; 2) that treatment with HgCl(2) liberates the reduced nucleotide from active site thiol; and 3) that binding and inhibition by -NAD occurs without cleavage of the beta-glycosidic bond. The involvement of reduced nicotinamide might also explain reports of a NO-associated ADP-ribosylation reaction involving a true thioglycosidic linkage. In this scenario, activation of nucleotide (via transnitrosation), engenders thiolate attack at ribose C-1` by making nicotinamide a better leaving group. This reaction (Fig. 9, lower pathway) may be more viable in other proteins where structural constraints place the RSNO in closer proximity to the sugar moiety than the nicotinamide ring.

GAPDH: Enzyme Inhibition and Covalent Modification

Our studies appear to resolve the controversy over the possible contribution of S-nitrosylation versus covalent attachment of nucleotide to enzyme inhibition. Both are likely to contribute, albeit under very different conditions. Reversible enzyme inhibition is mediated by S-nitrosylation. This posttranslational modification may be involved in the regulation of glycolysis seen in cellular systems activated with inflammatory cytokines(30) . S-Nitrosylation might also provide a means to protect such thiol-containing enzymes from oxidative inactivation. In contrast, irreversible enzyme inhibition (seen in some cellular systems(31) ) is likely to be explained by covalent attachment of NADH. This modification is more likely to be a pathophysiological event associated with inhibition of gluconeogenesis.

FOOTNOTES

*
This work was supported by grants from the Deutsche Forschungsgemeinschaft, the European Community, and the National Institutes of Health (HLO2582 and HLS2529). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
A Pew Scholar in the biomedical sciences.

To whom correspondence should be addressed. Tel.: 49-9131-85-6311; Fax: 49-9131-85-9202.

(^1)
The abbreviations used are: RSNO, S-nitrosothiol(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DTT, dithiothreitol; Mes, 2-(N-morpholino)ethanesulfonic acid; -NAD, nicotinamide 1,N^6-ethenoadenine dinucleotide; SIN-1,3-morpholinosydnonimine; SNP, sodium nitroprusside.

ACKNOWLEDGEMENTS

We thank Prof. G. Whitesides for helpful discussions.

REFERENCES

  1. Stamler, J. S. (1994) Cell 78, 931-936 [CrossRef][Medline] [Order article via Infotrieve]
  2. Stamler, J. S., Singel, D. J., and Loscalzo, J. (1992) Science 258, 1898-1902 [Abstract/Free Full Text]
  3. Mohr, S., Stamler, J. S., and Brüne, B. (1994) FEBS Lett. 348, 223-227 [CrossRef][Medline] [Order article via Infotrieve]
  4. Stamler, J. S., Jaraki, O., Osborne, J., Simon, D. I., Keaney, J., Vita, J., Singel, D., Valeri, C. R., and Loscalzo, J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7674-7677 [Abstract/Free Full Text]
  5. Oae, S., and Shinhama, K. (1983) Org. Prep. Proc. Int. 15, 165-198
  6. Meyer, D. J., Kramer, H., Özer, N., Coles, B., and Ketterer, B. (1994) FEBS Lett. 345, 177-180 [CrossRef][Medline] [Order article via Infotrieve]
  7. Arnelle, D. R., and Stamler, J. S. (1995) Arch. Biochem. Biophys. 318, 279-285 [CrossRef][Medline] [Order article via Infotrieve]
  8. DeGroote, M. A., Granger, D., Xu, Y., Campbell, C., Prince, R., and Fang, F. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6399-6403 [Abstract/Free Full Text]
  9. Lipton, S. A., and Stamler, J. S. (1994) Neuropharmacology 33, 1229-1233 [CrossRef][Medline] [Order article via Infotrieve]
  10. Morris, S. L., and Hansen, J. N. (1981) J. Bacteriol. 148, 465-471 [Abstract/Free Full Text]
  11. Henry, Y., Lepoivre, M., Drapier, J. C., Durocq, C., Boucher, J. L., and Guissani, A. (1993) FASEB J. 7, 1124-1134 [Abstract]
  12. Gopalakrishna, R., Chen, Z. H., and Gundimeda, U. (1993) J. Biol. Chem. 268, 27180-27185 [Abstract/Free Full Text]
  13. Tabuchi, A., Sano, K., Oh, E., Tsuchiya, T., and Tsuda, M. (1994) FEBS Lett. 351, 123-127 [CrossRef][Medline] [Order article via Infotrieve]
  14. Henderson, S. A., Lee, P. H., Aeberhard, E. E., Adams, J. W., Ignarro, L. J., Murphy, W. J., and Sherman, M. P. (1994) J. Biol. Chem. 269, 25239-25242 [Abstract/Free Full Text]
  15. Mannick, J. B., Asano, K., Izumi, K. Kieff, E., and Stamler, J. S. (1994) Cell 79, 1137-1146 [CrossRef][Medline] [Order article via Infotrieve]
  16. Stamler, J. S., Simon, D. I., Osborne, J. A., Mullins, M. E., Jaraki, O., Michel, T., Singel, D. J., and Loscalzo, J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 444-448 [Abstract/Free Full Text]
  17. Brüne, B., and Lapetina, E. G. (1989) J. Biol. Chem. 264, 8455-8458 [Abstract/Free Full Text]
  18. Brüne, B., Dimmeler, S., Molina, L., and Lapetina, E. G. (1994) Life Sci. 54, 61-70 [CrossRef][Medline] [Order article via Infotrieve]
  19. Dimmeler, S., Lottspeich, F., and Brüne, B. (1992) J. Biol. Chem. 267, 16771-16774 [Abstract/Free Full Text]
  20. Kots, A. Y., Skurat, A. V., Sergienko, E. A., Bulargina, T. V., and Severin, E. S. (1992) FEBS Lett. 300, 9-12 [CrossRef][Medline] [Order article via Infotrieve]
  21. Zhang, J., and Snyder, S. H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9382-9385 [Abstract/Free Full Text]
  22. Pancholi, V., and Fischetti, V. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8154-8158 [Abstract/Free Full Text]
  23. McDonald, L. J., and Moss, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6238-6241 [Abstract/Free Full Text]
  24. Brüne, B., Molina y Vedia, L., and Lapetina, E. G. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3304-3308 [Abstract/Free Full Text]
  25. Barrio, J. R., Secrist, J. A., III, and Leonard, N. J. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 2039-2042 [Abstract/Free Full Text]
  26. Dimmeler, S., and Brüne, B. (1992) Eur. J. Biochem. 210, 305-310 [Medline] [Order article via Infotrieve]
  27. Racker, E., and Krijmsky, I. (1952) J. Biol. Chem. 198, 731-743 [Free Full Text]
  28. Klepp, J., Oberfrank, M., Retey, J., Tritsch, D., Biellmann, J-F., and Hull, W. E. (1989) J. Am. Chem. Soc. 111, 4440-4447 [CrossRef]
  29. Oppenheimer, N. J. (1987) in Pyridine Nucleotide Coenzymes , part A (Dolphin, D., Poulson, R., and Avramovic, O., eds), pp. 323-365, John Wiley and Sons, Inc., New York
  30. Horton, R. A., Ceppi, E. D., Knowles, R. G., and Titheradge, M. A. (1994) Biochem. J. 299, 735-739
  31. Molina y Vedia, L., McDonald, B., Reep, B., Brüne, B., Di Silvio, M., Billiar, T. R., and Lapetina, E. G. (1992) J. Biol. Chem. 267, 24929-24932 [Abstract/Free Full Text]
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 CirculationHome page
L. Tao, X. Jiao, E. Gao, W. B. Lau, Y. Yuan, B. Lopez, T. Christopher, S. P. RamachandraRao, W. Williams, G. Southan, et al.
Nitrative Inactivation of Thioredoxin-1 and Its Role in Postischemic Myocardial Apoptosis
Circulation, September 26, 2006; 114(13): 1395 - 1402.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. Batthyany, F. J. Schopfer, P. R. S. Baker, R. Duran, L. M. S. Baker, Y. Huang, C. Cervenansky, B. P. Branchaud, and B. A. Freeman
Reversible Post-translational Modification of Proteins by Nitrated Fatty Acids in Vivo
J. Biol. Chem., July 21, 2006; 281(29): 20450 - 20463.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Z. Yu, T. Kuncewicz, W. P. Dubinsky, and B. C. Kone
Nitric Oxide-dependent Negative Feedback of PARP-1 trans-Activation of the Inducible Nitric-oxide Synthase Gene
J. Biol. Chem., April 7, 2006; 281(14): 9101 - 9109.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
I. Gusarov and E. Nudler
NO-mediated cytoprotection: Instant adaptation to oxidative stress in bacteria
PNAS, September 27, 2005; 102(39): 13855 - 13860.
[Abstract] [Full Text] [PDF]

Home page
 Plant Physiol.Home page
C. Lindermayr, G. Saalbach, and J. Durner
Proteomic Identification of S-Nitrosylated Proteins in Arabidopsis
Plant Physiology, March 1, 2005; 137(3): 921 - 930.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Interv.Home page
B. M. Gaston, J. Carver, A. Doctor, and L. A. Palmer
S-Nitrosylation Signaling in Cell Biology
Mol. Interv., August 1, 2003; 3(5): 253 - 263.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. ProteomicsHome page
T. Kuncewicz, E. A. Sheta, I. L. Goldknopf, and B. C. Kone
Proteomic Analysis of S-Nitrosylated Proteins in Mesangial Cells
Mol. Cell. Proteomics, March 1, 2003; 2(3): 156 - 163.
[Abstract] [Full Text] [PDF]

Home page
 Drug Metab. Dispos.Home page
H. Han, S.-G. Kim, M.-G. Lee, C.-K. Shim, and S.-J. Chung
Mechanism of the Reduced Elimination Clearance of Benzylpenicillin from Cerebrospinal Fluid in Rats with Intracisternal Administration of Lipopolysaccharide
Drug Metab. Dispos., November 1, 2002; 30(11): 1214 - 1220.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. Eaton, W. Fuller, and M. J. Shattock
S-Thiolation of HSP27 Regulates Its Multimeric Aggregate Size Independently of Phosphorylation
J. Biol. Chem., June 7, 2002; 277(24): 21189 - 21196.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. Eaton, H. L. Byers, N. Leeds, M. A. Ward, and M. J. Shattock
Detection, Quantitation, Purification, and Identification of Cardiac Proteins S-Thiolated during Ischemia and Reperfusion
J. Biol. Chem., March 15, 2002; 277(12): 9806 - 9811.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
C. COLUSSI, M. C. ALBERTINI, S. COPPOLA, S. ROVIDATI, F. GALLI, and L. GHIBELLI
H2O2-induced block of glycolysis as an active ADP-ribosylation reaction protecting cells from apoptosis
FASEB J, November 1, 2000; 14(14): 2266 - 2276.
[Abstract] [Full Text]

Home page
 FASEB J.Home page
H. E. MARSHALL, K. MERCHANT, and J. S. STAMLER
Nitrosation and oxidation in the regulation of gene expression
FASEB J, October 1, 2000; 14(13): 1889 - 1900.
[Abstract] [Full Text]

Home page
 FASEB J.Home page
B. A. KINGWELL
Nitric oxide-mediated metabolic regulation during exercise: effects of training in health and cardiovascular disease
FASEB J, September 1, 2000; 14(12): 1685 - 1696.
[Abstract] [Full Text]

Home page
 Cancer Res.Home page
G. Melino, F. Bernassola, M. V. Catani, A. Rossi, M. Corazzari, S. Sabatini, F. Vilbois, and D. R. Green
Nitric Oxide Inhibits Apoptosis via AP-1-dependent CD95L Transactivation
Cancer Res., May 1, 2000; 60(9): 2377 - 2383.
[Abstract] [Full Text]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
W. Hirschfield, M. R. Moody, W. E. O'Brien, A. R. Gregg, R. M. Bryan Jr., and M. B. Reid
Nitric oxide release and contractile properties of skeletal muscles from mice deficient in type III NOS
Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2000; 278(1): R95 - R100.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
M. A. Arstall, D. B. Sawyer, R. Fukazawa, and R. A. Kelly
Cytokine-Mediated Apoptosis in Cardiac Myocytes : The Role of Inducible Nitric Oxide Synthase Induction and Peroxynitrite Generation
Circ. Res., October 29, 1999; 85(9): 829 - 840.
[Abstract] [Full Text] [PDF]