Abstract
Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and oxidative damage is one of the primary functions of NADP+-dependent isocitrate dehydrogenase (ICDH) by supplying NADPH for antioxidant systems. We investigated whether the ICDH would be a vulnerable target of peroxynitrite anion (ONOO-) as a purified enzyme, in intact cells, and in liver mitochondria from ethanol-fed rats. Synthetic peroxynitrite and 3-morpholinosydnomine N-ethylcarbamide (SIN-1), a peroxynitrite-generating compound, inactivated ICDH in a dose- and time-dependent manner. The inactivation of ICDH by peroxynitrite or SIN-1 was reversed by dithiothreitol. Loss of enzyme activity was associated with the depletion of the thiol groups in protein. Immunoblotting analysis of peroxynitrite-modified ICDH indicates that S-nitrosylation of cysteine and nitration of tyrosine residues are the predominant modifications. Using electrospray ionization mass spectrometry (ESI-MS) with tryptic digestion of protein, we found that peroxynitrite forms S-nitrosothiol adducts on Cys305 and Cys387 of ICDH. Nitration of Tyr280 was also identified, however, this modification did not significantly affect the activity of ICDH. These results indicate that S-nitrosylation of cysteine residues on ICDH is a mechanism involving the inactivation of ICDH by peroxynitrite. The structural alterations of modified enzyme were indicated by the changes in protease susceptibility and binding of the hydrophobic probe 8-anilino-1-napthalene sulfonic acid. When U937 cells were incubated with 100 microM SIN-1 bolus, a significant decrease in both cytosolic and mitochondrial ICDH activities were observed. Using immunoprecipitation and ESI-MS, we were also able to isolate and positively identify S-nitrosylated and nitrated mitochondrial ICDH from SIN-1-treated U937 cells as well as liver from ethanol-fed rats. Inactivation of ICDH resulted in the pro-oxidant state of cells reflected by an increased level of intracellular reactive oxygen species, a decrease in the ratio of [NADPH]/[NADPH + NADP+], and a decrease in the efficiency of reduced glutathione turnover. The peroxynitrite-mediated damage to ICDH may result in the perturbation of the cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.
Highlights
Peroxynitrite anion (ONOOϪ) is a potent oxidant generated from the interaction of nitric oxide (NO) and superoxide (O2.) [1,2,3]
The mouse liver cytosolic isocitrate dehydrogenase (ICDH) was expressed as a fusion protein to glutathione S-transferase (GST), and it was purified as a recombinant protein, which was almost pure as estimated by SDS-PAGE
We evaluated whether the presence of product would protect the active site of ICDH from peroxynitrite- and SIN-1-mediated inactivation
Summary
Materials—Isocitrate, -NADPϩ, NADPH, mitochondrial ICDH from pig heart, GSH, cysteine, dithiothreitol (DTT), methionine, penicillamine, 2-mercaptoethanol, ebselen, selenomethionine, selenocysteine, human serum albumin, 5Ј-dithiobis-(2-nitrobenzoate) (DTNB), xylenol orange, o-phthaldehyde, lucigenin, bovine erythrocyte SOD, Pronase, trypsin, and 8-anilino-1-naphthalene sulfonic acid (ANSA) were purchased from Sigma Chemical Co. Control and peroxynitrite (10 M, 10 min)-treated ICDH samples were subjected to gel filtration and mixed with 0.1% trifluoroacetic acid. Structural Analysis—For circular dichroism (CD) spectroscopy, samples of ICDH were desalted on Econo-Pac 10 DG column (Bio-Rad) equilibrated in 20 mM Tris buffer, pH 7.4, and fractions containing the protein were pooled. ANSA (100 M) was incubated with the various forms of ICDH in 25 mM potassium phosphate buffer, pH 7.0/50 mM KCl. The fluorescence emission spectra (excitation, 370 nm) of the different mixtures were monitored on spectrofluorometer. The concentration of total glutathione was determined by the rate of formation of 5-thio-2-nitrobenzoic acid at 412 nm (⑀ ϭ 1.36 ϫ 104 MϪ1cmϪ1) as the method described by Akerboom and Sies [37], and GSSG was measured by the DTNB-GSSG reductase recycling assay after treating GSH with 2-vinylpyridine [38]. Data are presented as means Ϯ S.D. of triplicate experiments
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