Regulation of microsomal and cytosolic glutathione S-transferase activities by S-nitrosylation.
ABSTRACT There is increasing evidence that S-nitrosylation is a mechanism for the regulation of protein function via the modification of critical sulfhydryl groups. The activity of rat liver microsomal glutathione S-transferase (GST) is increased after treatment with N-ethylmaleimide (NEM), a sulfhydryl alkylating reagent, and is also increased under conditions of oxidative stress. In the present study, preincubation of purified rat liver microsomal GST with S-nitrosoglutathione (GSNO) or the nitric oxide (NO) donor, 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO), resulted in a 2-fold increase in enzyme activity. This increase in activity was reversed by dithiothreitol. The initial treatment of microsomal GST with either GSNO or DEA/NO was associated with an 85% loss of free sulfhydryl groups. After removal of the nitrosylating agents over a 6-hr period, approximately 50% of the enzyme was still nitrosylated, as determined by redox chemiluminescence. Furthermore, preincubation of either purified enzyme or hepatic microsomes with GSNO or DEA/NO prevented further enzyme activation by NEM, suggesting that NEM and the NO donors interact with a common population of sulfhydryl groups in the enzyme. In contrast, both NEM and NO donors partially inhibited the activity of cytosolic GST isoforms. The inhibitory activity of NEM and NO donors was much more evident when the GST pi isoform was used instead of a mixture of GST isoforms. These data suggest that there may be differential regulation of microsomal and cytosolic GST activities under conditions of nitrosative stress.
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ABSTRACT: Ornithine decarboxylase is the initial and rate-limiting enzyme in the polyamine biosynthetic pathway. Polyamines are found in all mammalian cells and are required for cell growth. We previously demonstrated that N-hydroxyarginine and nitric oxide inhibit tumor cell proliferation by inhibiting arginase and ornithine decarboxylase, respectively, and, therefore, polyamine synthesis. In addition, we showed that nitric oxide inhibits purified ornithine decarboxylase by S-nitrosylation. Herein we provide evidence for the chemical mechanism by which nitric oxide and S-nitrosothiols react with cysteine residues in ornithine decarboxylase to form an S-nitrosothiol(s) on the protein. The diazeniumdiolate nitric oxide donor agent 1-diethyl-2-hydroxy-2-nitroso-hydrazine acts through an oxygen-dependent mechanism leading to formation of the nitrosating agents N(2)O(3) and/or N(2)O(4). S-Nitrosoglutathione inhibits ornithine decarboxylase by an oxygen-independent mechanism likely by S-transnitrosation. In addition, we provide evidence for the S-nitrosylation of 4 cysteine residues per ornithine decarboxylase monomer including cysteine 360, which is critical for enzyme activity. Finally S-nitrosylated ornithine decarboxylase was isolated from intact cells treated with nitric oxide, suggesting that nitric oxide may regulate ornithine decarboxylase activity by S-nitrosylation in vivo.Journal of Biological Chemistry 10/2001; 276(37):34458-64. · 4.65 Impact Factor
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ABSTRACT: Microsomal glutathione transferase 1 is a homotrimeric detoxication enzyme protecting against electrophiles. The enzyme can also react with electrophiles, and when modification occurs at a unique Cys49 the reaction often results in activation. Here we describe the characterization of the chemical properties of this sulfhydryl (kinetic pK(a) was 8.8 +/- 0.3 and 9.0 +/- 0.1 with two different reagents) and we conclude that the protein environment does not lower the pK(a). Upon a direct comparison of the reactivity of Cys49 and low molecular weight thiols [L-Cys and glutathione (GSH)], the protein sulfhydryl displayed a 10-fold lower reactivity. The reactivity was correlated to reagent concentration in a linear fashion with a polar reagent, whereas the reactivity toward a hydrophobic reagent displayed saturation behavior (at low concentrations). This finding indicates that Cys49 is situated in a hydrophobic binding pocket. In a series of related quinones, activation occurs with the more reactive and less sterically hindered compounds. Thus, activation can be used to detect reactive intermediates during the metabolism of foreign compounds but certain intermediates can (and will) escape undetected. The reactivities of the three cysteines in the homotrimer were shown not to differ dramatically as the reaction of the protein with 4, 4'-dithiodipyridine could be fitted to a single exponential. On the basis of this result, a probabilistic expression could be used to relate the overall degree of modification to fractional activation. When N-ethylmaleimide activation (determined by the 1-chloro-2, 4-dinitrobenzene assay) was plotted against modification (determined with 4,4'-dithiodipyridine), a nonlinear relation was obtained, clearly showing that subunits do not function independently. The contribution to activation by single-, double-, and triple-modified trimers, were 0 +/- 0.06, 0.74 +/- 0.09, and 0.97 +/- 0.06, respectively. The double-modified enzyme appears partly activated, but this conclusion is more uncertain due to the possibility of independent modification of the purified enzyme upon storage. It is, however, clear that the single-modified enzyme is not activated whereas the triple-modified enzyme is fully activated. These observations together with the fact that MGST1 homotrimers bind only one substrate molecule (GSH) strongly support the view that subunits must interact in a functional manner.Biochemistry 01/2001; 39(49):15144-9. · 3.38 Impact Factor
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ABSTRACT: Apoptotic signaling cascades converge in the activation of caspases (interleukin-1beta converting enzyme like proteases). Treatment of the human promyelocytic leukaemia cell line U937 with actinomycin D resulted in the activation of caspase-3 also known as CPP32. Protease activity was measured in cytosolic extracts by fluorometric analysis of the time-dependent cleavage of acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin (DEVD-AMC), a caspase-3 substrate. Caspase activity was inhibited by thiol modifying agents such as N-ethylmaleimide or iodoacetamide and NO donors such as S-nitrosoglutathione (GSNO), BF4NO, and spermine-NO. NO-mediated enzyme inhibition was fully reversible upon the addition of DTT (dithiothreitol). NO. itself was not primarily responsible for downregulation of caspase-3, as we found no correlation between rates of NO* release and the magnitude of enzyme inhibition. It is likely that S-nitrosation accounts for enzyme inhibition by various NO donors. SIN-1 and peroxynitrite were inhibitory as well. In this case, however, enzyme activity was not restored upon DTT addition, suggesting oxidation as an additional thiol modification mechanism. Our studies provide evidence that caspases are targeted by NO via S-nitrosation and oxidation of critical thiol groups.Biochemical and Biophysical Research Communications 10/1997; 238(2):387-91. · 2.41 Impact Factor