The regulation of hepatic glutathione.
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Animals↗
Biomedical subjects
N Kaplowitz
Publications and source records attributed to N Kaplowitz.
Sinusoidal efflux of glutathione in the perfused rat liver. Evidence for a carrier-mediated process.
Turnover of hepatic glutathione in vivo in the rat is almost entirely accounted for by cellular efflux, of which 80-90% is sinusoidal. Thus, sinusoidal efflux play a major quantitative role in homeostasis of hepatic glutathione. Som preliminary observations from our laboratory (1983. J. Pharmacol. Exp. Ther. 224:141-147.) and circumstantial evidence in the literature seemed to imply that the raising of the hepatic glutathione concentration above normal was not accompanied by a rise in the rate of sinusoidal efflux. Based on these observations, we hypothesized that the sinusoidal efflux was probably a saturable process and that at normal levels of hepatic glutathione the efflux behaved as a zero-order process (near-saturation). We tested our hypothesis by the use of isolated rat livers perfused in situ, single pass, with hemoglobin-free, oxygenated buffer medium at pH 7.4 and 37 degrees C. Preliminary experiments established a range of perfusion rates (3-4 ml/min per g) for adequacy of oxygenation, lack of cell injury, and minimization of variability contributed by perfusion rates. Hepatic glutathione was lowered to below normal by a 48-h fast, diethylmaleate (0.1-1.0 ml/kg i.p.), and buthionine sulfoximine (8 mmol/kg i.p.), and raised to above normal by 3-methylcholanthrene (20 mg/kg x 3 d i.p.) and cobalt chloride (0.05-0.27 g/kg-1 subcutaneously). Steady state sinusoidal efflux from each liver was measured over a 1-h perfusion, during which the coefficient of variation of glutathione in perfusates stayed within 10%. Hepatic glutathione efflux as a function of hepatic concentration was characterized by saturable kinetics with sigmoidal (non-hyperbolic) features. The data were fitted best with the Hill model and the following parameter values were estimated: Vmax = 20 nmol/min per g, Km = 3.2 mumol/g, and n = 3 binding/transport sites. The efflux could be inhibited reversibly by sulfobromophthalein-glutathione conjugate but was not affected by the addition of glutathione to the perfusion medium. The results support our hypothesis that sinusoidal efflux of glutathione is near saturation (approximately equal to 80% of Vmax) at normal (fed and fasted) liver glutathione concentrations. The phenomenon of saturability coupled with the ability to inhibit the efflux leads us to propose that sinusoidal efflux from hepatocytes appears to be a carrier-mediated process. Some recent studies by others, using sinusoidal membrane-enriched vesicles, also support these conclusions.
Animals↗
Identification and partial purification of a unique phenolic steroid sulphotransferase in rat liver cytosol.
Phenolic steroid sulphotransferase activity for both oestradiol and oestrone was identified in male rat liver cytosol in the 30 000-40 000 Mr fractions on gel filtration when activity was assayed at pH 5.5 (pH optimum 5.5-6.0). Activity for oestradiol but not oestrone was found in the 60 000-70 000-Mr range when assayed at pH 8.0 (pH optimum biphasic, 5.5-6.0 and 7.0-8.0). Km for oestradiol (1.3 microM) was lower than published values for hydroxysteroid sulphotransferases (15-35 microM) and previously reported oestradiol sulphotransferases (71-85 microM). At above 2 microM-oestradiol phenolic sulphotransferase activity exhibited substrate inhibition. The phenolic steroid sulphotransferase activity was found to be distinct in chromatofocusing from organic-anion-binding and bile acid-binding proteins previously identified in this Mr range. Further purification on hydroxyapatite yielded a 44-fold enriched fraction that contained two monomeric bands, Mr 32 500 and 29 500.
Animals↗
Identification and purification of a 36 kDa bile acid binder in human hepatic cytosol.
We recently purified two closely related 33 kDa proteins from rat hepatic cytosol, designated bile acid binder I and II, which selectively bind bile acids with comparable affinity as glutathione S-transferase B. This work has now been extended to human liver in which we have identified a similar cytosolic binding activity in the 30-40 kDa fraction from gel filtration. Subsequent chromatofocusing and hydroxyapatite chromatography resulted in the isolation of a homogeneous monomeric protein of 36 kDa. The binding affinity of this protein for lithocholate using the displacement of 1-anilino-8-naphthalenesulfonate (ANS) was 0.1 microM, whereas human hepatic glutathione S-transferases purified from glutathione affinity chromatography demonstrated no competitive displacement of ANS.
Amino Acids↗
Comparison of the binding affinities of five forms of rat glutathione S-transferases for bilirubin, sulfobromophthalein and hematin.
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Animals↗
Comparison of binding of thyroid hormone analogues to hepatic organic anion binding proteins.
Dv protein and ligandin are two hepatic cytosolic proteins which bind organic anions, including endogenous thyroid hormones. Binding studies were performed using the ANS displacement technique to compare the binding of a variety of thyroid hormone analogues to purified organic anion binder and ligandin. Inhibition of ANS binding by these compounds was competitive. Both proteins bound L- and D-thyroxine with comparable affinity (Kd 30-45 microM), whereas ligandin bound 3',3',5-triiodo-L-thyronine, 3',3',5-triiodo-L-thyronine and most analogues with greater affinity. Nevertheless, the order of ligand affinities for both binders was highly correlated, suggesting that the nature of the binding site on both proteins is similar. The binding affinities of these organic anion binders are 2-3 orders of magnitude lower than an hepatic cytosolic thyroid binder reported by others, suggesting that ligandin and organic anion binder may not be important in intracellular thyroid hormone transfer.
Animals↗
Inhibition of glutathione efflux from isolated rat hepatocytes by methionine.
A substantial inhibition (50-70%) of GSH efflux by methionine was demonstrated in hepatocytes isolated from fed rats. Concurrent measurements of intracellular GSH revealed maintenance of a higher concentration in methionine-supplemented cells over the 1-h incubation. Analysis of total GSH suggested that maintenance of higher intracellular GSH by methionine could be quantitatively accounted for by inhibition of GSH efflux rather than by net GSH synthesis. This conclusion was supported by studies with propargylglycine, a potent inhibitor of cysteine synthesis from methionine. Identical results were obtained in incubations containing either propargylglycine and methionine or methionine alone, thereby suggesting that net synthesis of GSH from methionine was minimal under the assay conditions. Similar decreases (40-60%) in the rate of extracellular accumulation of GSH were observed with ethionine and buthionine, two higher homologs of methionine, but not with a wide range of other naturally occurring and synthetic amino acids. The inhibition of GSH efflux by methionine was not dependent on the presence of sodium in the medium and did not correlate with metabolic consumption of ATP.
Alkynes↗
Quantitation and characterization of a newly described organic anion binder by radioimmunoassay.
We have recently identified a new group of proteins in rat liver cytosol which possess binding properties comparable to those of the glutathione-S-transferases, yet lack transferase activity. The bulk of organic anion binding in this fraction was associated with the tetrameric protein, designated the organic anion binder, molecular weight 34 000 (Sugiyama, Y., Yamada, T. and Kaplowitz, N. (1982) Biochim. Biophys. Acta 709, 342-352). In order to determine its tissue distribution and molecular forms, a sensitive and specific radioimmunoassay was developed. Gel filtration of rat liver and kidney cytosol demonstrated only one peak of organic anion binder-like immunoreactivity corresponding to a molecular weight of 35 000. Organic anion binder-like immunoreactivity was present in 14 separate tissues. The greatest concentration of the organic anion binder was found in the liver and kidney cytosol, where it constituted 0.5% of the cytoplasmic proteins. Similar hepatic contents were noted for female and male rats. The content of the organic anion binder in the liver and kidney were low in the fetus, but approached adult levels by 2-4 weeks of age. Treatments known to augment hepatic glutathione-S-transferase content had minimal effect on organic anion binder content. In summary, the organic anion binder is a newly recognized cytosolic binding protein, distinct from the glutathione-S-transferases, present in greatest concentration in the liver and kidney. The combination of the protein's binding properties along with its location and ontogeny suggests its possible role in intracellular organic anion transport.
Animals↗
Role of glutathione in gastric mucosal cytoprotection.
Exogenous thiol compounds have been reported to protect the stomach from ethanol-induced necrotic lesions. The gastric mucosa contains high levels of an endogenous thiol, glutathion (GSH). Because of the known role of glutathione in protecting against hepatic injury, its role in gastric mucosal cytoprotection was of interest. By use of an animal model for acute gastric injury from ethanol, a close parallel relation between depletion of endogenous mucosal GSH and induction of mucosal protection was demonstrated. Surprisingly, mucosal protection varied inversely with the level of mucosal GSH obtained after treatment with specific GSH-depleting agents (diethyl maleate and cyclohexene-1-one). Depletion of gastric mucosal GSH was associated with an increase in the mucosal content of prostaglandins 6-keto F1 alpha and F2 alpha but not E2. The protective effect induced by GSH-depleting agents was partially reversed by indomethacin in some but not all studies. Although GSH depletors increased gastric juice volume, protection with these agents persisted after the volume and mucosal GSH had returned to control levels and also was not reversed by increasing the dose of ethanol threefold to overcome a possible dilutional effect. We conclude that, contrary to apparent predictions, depletion of endogenous gastric GSH protects the stomach from acute ethanol-induced injury. Although the mechanism of this protection is unknown, a mediation by endogenous release of prostaglandins seems to play a minor role since diethyl maleate was protective even in indomethacin-treated animals.
6-Ketoprostaglandin F1 alpha↗
Binding of glutathione by rat liver cytosol.
Glutathione (GSH) binding to rat liver cytosol at two different protein concentrations and a range of GSH concentrations was determined using rapid ultrafiltration. Two binding sites and nonspecific binding were determined by computer fit of the data. The high-affinity site had a similar affinity and capacity for GSH as that of the GSH S-transferases. Using the converged parameters and an estimation of cytosolic protein content of the intact liver, simulation of the GSH-free fraction and the contribution and degree of saturation of the high-affinity binding site were estimated over a broad range of GSH concentrations. The findings predict that 70-75% of cytosol GSH is free and that the high-affinity site is saturated with GSH in the physiologic range.
Animals↗
Evidence for a common high affinity binding site on glutathione S-transferase B for lithocholic acid and bilirubin.
Binding of lithocholic acid, bilirubin, and gossypol to glutathione S-transferase B (ligandin or transferase YaYc) was compared using four methods. Tryptophan quenching revealed a single high affinity site for bilirubin and gossypol but could not be used for lithocholic acid. Both displacement of the fluorescent probe, 1-anilino-8-naphthalenesulfonate, and spectral changes induced by bilirubin binding demonstrated a common high affinity site for which all three ligands compete. Similar results were obtained by equilibrium dialysis. The dissociation constants for the binding of both bilirubin and lithocholic acid were comparable with the various methods (range 0.2-0.7 microM). Thus, lithocholic acid and bilirubin share a high affinity binding site on gluthathione S-transferase B that appears to be separate from the binding site for substrates.
Anilino Naphthalenesulfonates↗
Newly identified bile acid binders in rat liver cytosol. Purification and comparison with glutathione S-transferases.
Gel filtration of male rat liver cytosol preincubated with radiolabeled lithocholic, chenodeoxycholic, and glycochenodeoxycholic acids, and taurocholic acid revealed two major peaks of radioactivity, one co-eluting with the glutathione S-transferases and the other with a separate fraction, respectively. Chromatofocusing of the pooled fractions containing the new bile acid binding activity resulted in a separation of bile acid binding from the previously described organic anion binding activity in this fraction. Two binding peaks for lithocholic acid (pI 5.6, Binder I, and pI 5.5, Binder II) were identified on chromatofocusing and were further purified to apparent homogeneity by hydroxyapatite chromatography. The two Binders were monomers having identical molecular weight (33,000) and similar amino acid compositions. Bile acid binding to purified Binders I and II and glutathione S-transferases A, B, and C was studied by inhibition of the fluorescence of bound 1-anilino-8-naphthalenesulfonate (ANS). Confirmatory experiments using equilibrium dialysis produced comparable results. Glutathione S-transferase B had greater affinity for bile acids than transferases A or C. Binder II, which had greater affinity than Binder I for most bile acids, had greater affinity for chenodeoxycholic acid than transferase B but comparable or lower affinities for the other bile acids. All bile acids studied diminished ANS fluorescence with Binder II. Taurocholic and cholic acids increased ANS fluorescence with Binder I without affecting KANS, whereas lithocholic and chenodeoxycholic acids diminished ANS fluorescence with Binder I. In summary, we have identified and isolated two proteins (Binders I and II) which, along with glutathione S-transferase B, are the major hepatic cytosol bile acid binding proteins; these proteins have overlapping but distinct specificities for various bile acids.
Amino Acids↗
Factors influencing the efflux of hepatic glutathione into bile in rats.
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Animals↗
Newly identified organic anion-binding proteins in rat liver cytosol.
Previously, all organic anion binding activity in the Y protein (ligandin-containing) fraction of rat liver cytosol has been attributed to the glutathione S-transferases. Gel filtration on Sephadex G-75 superfine has resolved the Y protein fraction into organic anion-binding fractions of two distinct molecular weights: (a) Mr 45 000 containing the glutathione S-transferases, and (b) Mr 35 000 referred to as Y'. Two proteins (Dv and D1) were purified from the Y' fraction. Dv is a basic protein consisting of four subunits of Mr 8000-9000 and selectively binds 1-anilino-8-naphthalene sulfonate and sulfobromophthalein. D1 is a monomer (Mr 40 000) which exhibits high affinity binding for Rose Bengal. Dv protein bound a broad spectrum of organic anions. Antibody raised in rabbits to rat Dv showed no cross-reactivity with purified glutathione S-transferases A, B, or C. Thus, we have identified organic anion-binding proteins which are unrelated to the glutathione S-transferases, but which were previously associated with the crude Y-fraction. The relative abundance of these proteins and their binding characteristics suggest their role in hepatic organic anion transport.
Amino Acids↗
Identification of hepatic Z-protein in a marine elasmobranch, Platyrhinoides triseriata.
Previous studies were unable to identify Z-protein in elasmobranch liver with bromosulphophthalein as ligand. By using 8-anilinonaphthalene-1-sulphonate and Rose Bengal as ligands, however, we demonstrated in hepatic cytosol from Platyrhinoides triseriata an organic-anion-binding protein with gel-filtration characteristics identical with those of rat Z-protein. By comparison with pooled rat Z-protein, Pl. triseriata Z-protein had slightly lower affinity for 8-anilinonaphthalene-1-sulphonate and Rose Bengal, greatly decreased binding affinity for bromosulphophthalein and no binding activity for oleic acid or squalene. The Pl. triseriata Z-protein binding site was less hydrophobic than that of rat Z-protein. This observation may explain the differences in binding characteristics between the Z-proteins of these species.
Anilino Naphthalenesulfonates↗
Propylthiouracil treatment for alcoholic hepatitis: should it and does it work?
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Animals↗
Glutathione S-transferases in elasmobranch liver. Molecular heterogeneity, catalytic and binding properties, and purification.
In order to gain insight into the phylogeny and physiological significance of organic-anion-binding proteins in the liver, the hepatic glutathione S-transferases of rat and a typical elasmobranch, the thorny-back shark (Platyrhinoides triseriata), were compared with respect to both glutathione S-transferase activites and organic-anion-binding properties. On gel filtration (Sephadex G-75, Superfine grade) of rat cytosol, the elution volumes of enzyme activities with 1-chloro-2,4-dinitrobenzene and p-nitrobenzyl chloride as substrates were identical (rat Y-fractions; M(r) 45000). In contrast, two peaks of enzyme activity for 1-chloro-2,4-dinitrobenzene with elution volumes corresponding to M(r) 52000 (PLAT Y(1)) and M(r) 45000 (PLAT Y(2)) were detected on gel filtration of P. triseriata cytosol. Only fraction PLAT Y(2) had enzyme activity with p-nitrobenzyl chloride. Enzyme kinetic studies showed that rat Y-fraction had higher affinities for both 1-chloro-2,4-dinitrobenzene and glutathione than PLAT Y(1)- and PLAT Y(2)-fractions. The two forms of P. triseriata glutathione S-transferases differed greatly in affinity for glutathione. At a glutathione concentration that we found to be physiological in P. triseriata, PLAT Y(2) accounted for approx. 70% of the total glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene. Binding studies revealed that PLAT Y(1) and PLAT Y(2) fractions had much lower affinities for sulphobromophthalein and bilirubin than rat Y-fraction. In contrast, binding affinities of PLAT Y(1) and PLAT Y(2) for Rose Bengal and 1-anilino-8-naphthalenesulphonate were comparable with that of rat Y-fraction. Inhibitory kinetics suggested that sulphobromophthalein and Rose Bengal were non-competitive inhibitors of glutathione S-transferase activities when 1-chloro-2,4-dinitrobenzene was used as substrate for both PLAT Y(1) and PLAT Y(2). The major glutathione S-transferase from the PLAT Y(2) fraction was purified 81-fold by sequential chromatography on Sephadex G-75, DEAE-Sephadex and hydroxyapatite, and consisted of two identical subunits with pI7.7. The highly enriched Y(2)-fraction retained high affinity binding of Rose Bengal and 1-anilino-8-naphthalenesulphonate.
Amino Acids↗
Rapid oxidation in vitro of endogenous and exogenous glutathione in bile of rats.
Biliary excretion of glutathione has recently been described but poorly characterized. Controversy has existed concerning the relative contribution of oxidized and reduced glutathione to total glutathione efflux from the liver into bile. We found that bile, unlike liver cytosol or buffer, had the unique ability to oxidize GSH rapidly (t 1/2 = 5 min) to the disulfide form by a nonenzymatic, O2, and pH-dependent chemical reaction inhibited by only certain chelating agents. Significant oxidation of GSH occurred during the collection of bile samples resulting in significant time-dependent alterations in the ratio of biliary GSH to GSSG. Thus the preponderance of GSSG in bile in the normal state reported by others represents a postexcretory in vitro artifact. Inhibition of oxidation by acidification of bile during its collection established the true contribution of GSH and GSSG to total biliary efflux.
Animals↗