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The modification of reactive protein sulfhydryls by S-nitrosoglutathione and other NO donors has been studied by gel isoelectric focusing. S-nitrosylated, unmodified, and S-glutathiolated protein forms are differentiated by this method. With specific antibodies for the protein of interest, both S-nitrosylation and S-glutathiolation of the protein were analyzed in mixtures obtained as soluble tissue or cell extracts. The effect of S-nitrosoglutathione (GSNO) on purified phosphorylase b, on carbonic anhydrase III in an extract from rat liver, and on H-ras expressed in Escherichia coli was examined. When fresh GSNO reacted with pure phosphorylase b, only S-nitrosylated forms of the protein were observed. Likewise the NO donors, amyl nitrite, spermine NONOate, and diethylamine NONOate, all generated S-nitrosylated phosphorylase b. When crude mixtures of proteins from rat liver (containing carbonic anhydrase III) or from E. coli (containing an overexpressed form of H-ras) were exposed to fresh GSNO, both the S-nitrosylated and the S-glutathiolated forms of the proteins were observed. It is suggested that reactive intermediates from the breakdown of GSNO are responsible for the observed S-glutathiolation. These experiments show that both S-nitrosylated and S-glutathiolated forms of proteins may be generated by the addition of GSNO to mixtures containing proteins with reactive sulfhydryls. These protein modifications may exhibit metabolic consequences independent of the release of nitric oxide.

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The role of cysteinylglycine S-conjugate dipeptidases in the intrahepatic mercapturic acid pathway was investigated in rat liver. Subcellular compartmentation studies and liver perfusions were performed using monochlorobimane and bimane S-conjugates as model compounds. The major part (over 95%) of total hepatic cysteinylglycine S-conjugate dipeptidase activity was located in the cytosol. Lower specific activity appeared in the canalicular plasma membrane fraction. Similar hepatic localization of dipeptidase activity was seen in the guinea pig. In intact rat liver perfused with monochlorobimane, the major products were the glutathione S-conjugate (mBSG) and the cysteinylglycine S-conjugate (mBCG) in bile. Minor amounts of the cysteine S-conjugate (mBCys) and the mercapturic acid (mBNAc) were formed, indicating a limitation in further metabolism of the dipeptide S-conjugate in the biliary space. However, when the dipeptide S-conjugate was offered to the sinusoidal space in liver perfusions, substantial uptake and conversion to mBNAc was observed, and only trace amounts of the infused dipeptide appeared in bile. The data suggest that cytosolic cysteinylglycine S-conjugate dipeptidase as identified here is involved in hepatic mercapturic acid formation from sinusoidal cysteinylglycine S-conjugates. This is especially of significance for species such as guinea pig and human, in which dipeptide S-conjugates are generated in the sinusoidal domain of the liver due to the presence of high gamma-glutamyltranspeptidase activity.

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Membrane-bound GST transferase (GSTm) occurs in hepatic microsomal and plasma membranes as well as in the outer mitochondrial membrane, and it is known to be activated by N-ethylmaleimide. We recently analysed the activation by GSSG in some detail. The approximately 5-fold stimulation is reversed upon reduction of GSSG by GSSG reductase. In steady-state experiments, the Kox value was determined to be 0.05, i.e. 20 times more GSSG than GSH produces half-maximal activation. Kox is independent of the total glutathione concentration, indicating that S-thiolation by mixed disulfide formation, rather than interchain or intrachain disulfide bridge formation, is responsible for activation. In Western blots, a 17.7 kDa band, in addition to the 17.3 kDa band, was detected upon treatment with GSSG or with GSH plus t-butyl hydroperoxide. We suggest that under oxidative stress, GSTm is activated through direct S-thiolation of the enzyme. Dethiolation occurs via thiol disulfide exchange governed by the cellular glutathione redox state.

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Glutathione transferase (GST)-catalysed S-nitrosoglutathione (GSNO) formation from alkyl nitrites was determined with the homodimers 1-1, 2-2, 3-3, and 4-4 isolated from rat liver. The 4-4 isoform showed a high specificity for the alkyl nitrites. Total GST activities were studied in homogenates from different organs. The liver showed highest GST activity both with amyl nitrite and with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, the activity ratio of amyl nitrite over CDNB being 3.8. In lung and heart, these ratios were 6.2 and 5.7, respectively, indicating a selectivity of these organs for alkyl nitrite metabolism and GSNO formation.

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Microsomal glutathione transferase (GSTm) is activated up to fivefold by incubation with glutathione disulfide (GSSG). The process is reversed by the addition of an NADPH-regenerating system consisting of glutathione reductase and glucose 6-phosphate/glucose-6-phosphate dehydrogenase. By treating the microsomes at different GSH/GSSG ratios a Kox value of 0.047 is found, i.e., 21 times more GSSG than GSH is necessary to produce half-maximal activation. The Kox is independent of the total glutathione concentration, indicating that S-thiolation by GSH rather than interchain or intrachain disulfide bridge formation is responsible for activation. Further evidence for S-thiolation of GSTm comes from SDS-PAGE under nonreducing conditions and Western blotting. Treating microsomes with GSSG or with GSH and t-butyl hydroperoxide or cumene hydroperoxide results in the appearance of a second GSTm band at approximately 17.7 kDa in addition to the native band at 17.3 kDa, the size difference approximately corresponding to the molecular mass of glutathione. The 17.7-kDa band is not seen in the presence of mercaptoethanol. Microsomal preparations from rat livers perfused with t-butyl hydroperoxide or cumene hydroperoxide also contain both GSTm forms. We suggest that under oxidative stress the microsomal GST in the cell can be activated through direct hydroperoxide-mediated S-thiolation of the enzyme with GSH, its reversal occurring via a thiol exchange-mediated dethiolation imposed by the intracellular glutathione redox state.

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The formation of S-nitrosoglutathione (GSNO) from amyl nitrite and n-butyl nitrite was studied in rat liver microsomes, employing N-ethylmaleimide (MalNEt) as an activator and indomethacin as an inhibitor of microsomal glutathione S-transferase (GST). Rates were compared with GST activity measured with 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. MalNEt stimulated GST activity and the formation of GSNO from amyl nitrite and n-butyl nitrite about 10-fold. Increasing concentrations of indomethacin inhibited both reactions in parallel. N-Acetyl-L-cysteine but not L-cysteine could substitute for GSH. It is concluded that rat liver microsomal GST catalyses the formation of GSNO from amyl nitrite and n-butyl nitrite. The activity of the MalNEt-stimulated microsomal GST is calculated to be about 17 units/mg of enzyme with the alkyl nitrites and about 16 units/mg of enzyme with CDNB as a substrate, assuming that 3% of microsomal protein is GST. These rates are comparable with those obtained for cytosolic GSTs. Thus microsomal GST may play a significant role in the metabolism of alkyl nitrites in biological membranes.

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The oxidation of 2-(methylseleno)benzanilide and 2-selenylbenzanilide, metabolites of the antioxidant drug ebselen, was examined in reactions catalyzed by rat, pig, and guinea pig liver microsomes and in perfused rat liver. Microsomes from all three species catalyzed NADPH- and oxygen-dependent oxidation of the selenide and the selenol to thiol-reactive metabolites. The oxidation product of the selenide was similar in properties to the chemically synthesized selenoxide [2-(methylseleninyl)benzanilide]. The selenoxide oxidized GSH and thiocholine at rate constants of 1.2 x 10(2) and 7.2 x 10(2) M-1 s-1, respectively at pH 7.4, 37 degrees C. n-Octylamine stimulated the oxidation of the ring-opened metabolites of ebselen catalyzed by pig and guinea pig liver microsomes but it had essentially no effect on these activities in rat liver microsomes. The selenoxidase activity of microsomes from all three species was partially (30-50%) sensitive to N-benzylimidazole. The effects of n-octylamine and the imidazole suggest that the oxidation of the selenide was catalyzed primarily by enzymes with the properties of flavin-containing and P450-dependent monooxygenases, but the nature of enzymes responsible for a small fraction of the N-benzylimidazole-sensitive activity was not fully resolved. The 2-(methylseleno)benzanilide oxidase activity of pig liver microsomes sensitive to N-benzylimidazole was only partially sensitive to antisera to pig liver NADPH-cytochrome P450 reductase. While neither 2-(methylseleno)benzanilide nor ebselen affected bile flow, the biliary efflux of GSSG was stimulated about fourfold in rat liver perfused with either of these selenium compounds. The increased GSSG efflux produced by 5 microM ebselen or its methyl metabolite was abolished by N-benzylimidazole.

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In vivo transport in plasma and in vitro transfer of ebselen to binding sites in the hepatocyte were studied. More than 90% of intravenously administered ebselen in mouse plasma is bound by selenium-sulfur bonds to reactive thiols in serum albumin. In in vitro experiments the uptake of [14C]-ebselen from a complex prepared with bovine serum albumin (BSA) was determined in isolated perfused rat liver. Radioactive ebselen metabolites were excreted into bile. In isolated hepatocytes, radioactivity was bound to all subcellular organelles. Ebselen is transferred from the BSA complex to membrane-associated proteins after reductive cleavage of the Se-S bond effected by endogenous protein thiols. In contrast, when proteins were separated by dialysis membranes, ebselen transfer from its BSA complex occurred only in the presence of externally added reductants. Among the physiological reductants tested, ebselen release from the BSA complex was highest with glutathione (75%) and lowest with ascorbic acid (less than 10%). Quantitative release of ebselen from its BSA complex was only achieved by the combined action of reductant, notably 2-mercaptoethanol, and guanidine thiocyanate, suggesting that ebselen interacts with proteins by covalent Se-S bonds as well as by ionic charge interactions.

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Glutathione transferases purified from plasma membrane and microsomal fractions from rat liver share similar enzymic properties. The activity of both proteins with 1-chloro-2,4-dinitrobenzene can be stimulated about 10-15-fold by N-ethylmaleimide. No activation is observed using p-nitrobenzylchloride as a substrate. The enzymes are immunologically related as indicated by Western-blot analysis using antibodies against the microsomal glutathione transferase or against a synthetic peptide corresponding to the amino acid positions 55-64 of microsomal glutathione transferase. Isolated plasma membrane and microsomal glutathione transferases possess the same amino-terminal amino acid sequence and digestion with different proteases results in identical fragment patterns as displayed by SDS/PAGE. These data suggest that plasma membrane and microsomal glutathione transferase are identical proteins.

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The low-Km and high-Km components of S-dinitrophenyl-glutathione (DNPSG) uptake by inside-out vesicles of human erythrocytes show different pH profiles and inhibition properties with organic anions. Both components are competitively inhibited by various polyvalent anions, including glutathione conjugates, conjugated steroid hormones and bile salts, and bilirubin ditaurate. A variety of monovalent anions, including glucuronidated and sulphated drugs and taurocholate, inhibit the high-Km system only. Taurocholate is taken up by the erythrocyte vesicles in an ATP-dependent manner. The anionic dyes fluorescein, Indocyanine Green and bromosulphophthalein inhibit the low-Km system competitively and the high-Km system non-competitively. The study shows that interactions between different types of biologically occurring conjugates can occur at the level of the transport step out of erythrocytes. The kinetic properties suggest overlapping substrate specificities for the two systems, in which the low-Km component is physiologically more important for transport of glutathione conjugates and polyvalent organic anions, whereas the high-Km component is of significance for transport of monovalent organic anions. Low- and high-Km transport of DNPSG is also observed in plasma membrane vesicles from rat, pig and bovine erythrocytes.

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Rat liver plasma membranes exhibit membrane-bound glutathione S-transferase activity. The specific activity in isolated canalicular membranes was 83 +/- 8 mU/mg protein and 50 +/- 3 mU/mg protein in the sinusoidal membranes. Whereas microsomal and outer mitochondrial glutathione S-transferases were stimulated seven and four-fold with N-ethylmaleimide, respectively, the plasma membrane activity was activated two-fold. Western blot analysis, using an antibody against the microsomal glutathione S-transferase, shows the presence of a 17 kDa protein in canalicular and sinusoidal membrane fractions. The antibody reaction was about three-fold higher in the canalicular compared to the sinusoidal membrane fraction. These data support the conclusion that the plasma membrane glutathione S-transferase is closely related to the microsomal and outer mitochondrial membrane enzyme.

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Kinetic studies on the low- and high-Km transport systems for S-2,4-dinitrophenyl glutathione (DNP-SG) present in erythrocyte membranes were performed using inside-out plasma membrane vesicles. The high-affinity system showed a Km of 3.9 microM a Vmax of 6.3 nmol/mg protein per h, and the low-affinity system a Km of 1.6 mM and a Vmax of 131 nmol/mg protein per h. Both uptake components were inhibited by fluoride, vanadate, p-chloromercuribenzoate (pCMB) and bis(4-nitrophenyl)dithio-3,3'-dicarboxylate (DTNB). The low-Km uptake process was less sensitive to the inhibitory action of DTNB as compared to the high-Km process. N-Ethylmaleimide (1 mM) inhibited the high-Km process only. The high-affinity uptake of DNP-SG was competitively inhibited by GSSG (Ki = 88 microM). Vice versa, DNP-SG inhibited competitively the low-Km component of GSSG uptake (Ki = 3.3 microM). The high-Km DNP-SG uptake system was not inhibited by GSSG. The existence of a common high-affinity transporter for DNP-SG and GSSG in erythrocytes is suggested.

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Uptake of the thioether S-(2,4-dinitrophenyl)glutathione (DNPSG) in canalicular plasma membrane vesicles from rat liver is enhanced in the presence of ATP and exhibits an overshoot with a transient 5.5-fold accumulation of DNPSG. Stimulation by ATP is not caused by the generation of a membrane potential, based on responses of the indicator dye oxonol V. ATP-dependent uptake has an apparent Km of 71 microM for DNPSG and a Vmax of 0.34 nmol.min-1.mg of vesicle protein-1. Protein thiol groups are essential for transport activity as indicated by the sensitivity of DNPSG transport to sulfhydryl reagents. There is competitive inhibition with other thioethers, S-hexylglutathione (Ki = 66 microM), the photoaffinity label S-(4-azidophenacyl)glutathione (Ki = 56 microM), as well as with glutathione disulfide (Ki = 0.44 mM) and with the bile acid taurocholate (Ki = 0.61 mM). GSH (2 mM) or cholate (0.4 mM) does not inhibit. Both glutathione disulfide and taurocholate show ATP-dependent transport in the canalicular membrane vesicles which is inhibited by DNPSG. No ATP-dependent transport is found for GSH. Transport of DNPSG is also inhibited competitively by alpha-naphthyl-beta-D-glucuronide (Ki = 0.42 mM) but not by alpha-naphthylsulfate (2 mM), and there is substantial inhibition with the glucuronides from ebselen and p-nitrophenol. The results indicate that the canalicular transport system for DNPSG is directly driven by ATP and that the biliary transport of other classes of compounds may also proceed via this system.

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Chlorpromazine (10 mumol/L) causes a marked increase in portal pressure in perfused rat liver. Simultaneously, oxygen consumption, hepatic clearance of taurocholate and bile flow are diminished. These effects are prevented by the cyclooxygenase inhibitors indomethacin (15 mumol/L), acetylsalicylate (3 mmol/L) or ibuprofen (200 mumol/L). On addition of chlorpromazine the liver releases increased amounts of prostaglandin D2; this increase does not occur in the presence of indomethacin. At higher concentrations of chlorpromazine (100 mumol/L) the inhibition of taurocholate clearance and bile flow is accompanied by only a moderate increase of portal pressure, and indomethacin is without effect. At this high concentration, substantial cell damage, as indicated by the release of lactate dehydrogenase, is present. We conclude that arachidonic acid-derived metabolites, notably prostanoids, are involved in the inhibition of bile flow and of taurocholate clearance observed at low concentrations of chlorpromazine. The data suggest that changes in the microcirculation are responsible for the impairment of the liver functions. At higher concentrations of chlorpromazine the cell toxicity of the drug becomes prominent.

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Uptake of [35S]lipoate was studied in perfused rat liver and in isolated rat hepatocytes. During single-pass perfusion of [35S]lipoate about 30% of the radioactivity is retained in the liver. A substantial amount of 5,5'-dithiobis(2-nitrobenzoic acid)-reactive material appears in the effluent perfusate, while hepatic efflux of GSH is unchanged. The hepatic uptake of lipoate, the release of thiols, and also the biliary excretion of 35S-labeled compounds are suppressed by octanoate. In isolated hepatocytes the uptake of lipoate follows saturation kinetics showing a Km value of 38 microM and a Vmax of 180 pmol/mg X 10 s. The uptake is temperature-dependent; from the Arrhenius plot an activation energy of 14.8 kcal/mol at 20 microM lipoate is calculated. At high concentrations of lipoate (above 75 microM) a nonsaturable uptake component becomes predominant. Lipoate uptake is selectively inhibited by medium-chain fatty acids. Only slight inhibition is seen in the presence of long-chain fatty acids, and there is no inhibition with acetate or lactate. Substantial inhibition is also observed with acetylsalicylic acid, but not with taurocholate, bromosulfophthalein or biotin. Lipoate uptake can be inhibited by high concentrations of phloretin (200 microM) and is rather insensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (200 microM). The results indicate that hepatic uptake of lipoate at physiological concentrations is largely carrier-mediated.

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ATP-stimulated uptake of S-(2,4-dinitrophenyl)glutathione with a high activity of 0.35 nmol/min per mg protein is found in a rat liver plasma membrane vesicle preparation enriched in sinusoidal marker enzymes. Transport takes place into an osmotically active space. Vanadate and S-(azidophenacyl)glutathione inhibit transport, whereas Ca2+, EGTA and ouabain are without effect.

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A method for the synthesis of the glutathione conjugate S-(4-azidophenacyl)[35S]glutathione is described. The compound was used for photoaffinity labeling of proteins present in canalicular membrane vesicles (CMV), sinusoidal membrane vesicles (SMV), mitochondria and microsomes from rat liver. Most of the radioactivity introduced by photoaffinity labeling of CMV appeared in the 25-29 kDa range. Further labeled proteins were observed in bands at 37, 105 and about 120 kDa. 79% of the 25-29 kDa associated radioactivity was recovered in the supernatant after extensive revesiculation (washing) of the vesicles, together with the 37 kDa protein. CMV and SMV contained glutathione S-transferase (GST) activity which in CMV was decreased by 75% by washing. Photolabeling of a mixture of purified basic GST subunits from rat liver resulted in a band pattern at 25-29 kDa similar to that in the membrane preparations. Isoelectric focusing of the CMV indicated the presence of basic soluble GST subunits. S-Hexylglutathione-Sepharose affinity chromatography showed reversible binding of photolabeled proteins at 25-29 kDa. Difference photoaffinity labeling with GSSG, S-hexylglutathione, taurocholate and phenylmethylsulfonyl fluoride decreased the radioactivity bound by GST, but not that introduced into the 105 kDa protein band present in CMV. It is concluded that membrane-associated basic GST isoenzymes are present in standard membrane vesicle preparations. In the cell, the function may be transport of GST-bound compounds across the membrane and protection of the membranes against electrophiles.

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