The relationship between GSH, GSSG and non-GSH thiol in GSH-deficient erythrocytes from Finnish landrace and Tasmanian merino sheep.

1. Two automated colorimetric methods have been developed for assaying the GSH and total thiol in protein-free extracts of erythrocytes. They employ as chromogens 5,5'-dithiobis-(2-nitrobenzoate) (DTNB) and alloxan. 2. The concentrations of GSH, GSSG and total non-protein thiol have been estimated in high and low GSH erythrocytes from Finnish Landrace and Tasmanian Merino sheep. 3. In both breeds of sheep low GSH cells were found to have low concentrations of total non-protein thiol and GSSG as well as of GSH. 4. Nevertheless high and low GSH cells have similar values for the oxidation-reduction potential of the GSH : GSSG couple.     

人胎盘谷胱甘肽S-转移酶中巯基的研究

 用巯基试剂5.5'-二硫双(2-硝基苯甲酸)(DTNB)测得人胎盘谷胱甘肽S-转移酶(GST-π)的总巯基数为每亚基2个,均为表面巯基,,其中一个与DTNB反应快,被修饰后可导致酶活力全部丧失。另一巯基与DTNB反应较慢,可能与酶活力无关。用在12℃测定剩余巯基和Stallcup-Koshland作图法求得DTNB修饰快反应和慢反应巯基的速度常数分别为44056和162min~(-1)(mol/L)~(-1)。底物谷胱甘肽的衍生物S-正辛烷谷胱甘肽(S-o-GSH)能保护GST-π能保护的快反应巯基免受DTNB的修饰,使反应速度常数随着S-o-GSH浓度的增高而降低。S-o-GSH也能保护酶被N-乙基马来酰亚胺(NEMI)修饰失活,但不能保护慢反应巯基被DTNB修饰。另一底物2,4-二硝基氯苯(CDNB)对NEMI的修饰失活没有保护作用。上述结果提示快反应巯基参与GST-π和谷胱甘肽的结合,是组成活性中心的重要基因。     

Glutathione in Intact Vacuoles: Comparison of Glutathione Pools in Isolated Vacuoles,Plastids, and Mitochondria from Roots of Red Beet

Proportions between oxidized and reduced glutathione forms were determined in vacuoles isolated from red beet (Beta vulgaris L.) taproots. The pool of vacuolar glutathione was compared with glutathione pools in isolated plastids and mitochondria. The ratio of glutathione forms was assessed by approved methods, such as fluorescence microscopy with the fluorescent probe monochlorobimane (MCB), high-performance liquid chromatography (HPLC), and spectrophotometry with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB). The fluorescence microscopy revealed comparatively low concentrations of reduced glutathione (GSH) in vacuoles. The GSH content was 104 μM on average, which was lower than the GSH levels in mitochondria (448 μM) and plastids (379 μM). The content of reduced (GSH) and oxidized (GSSG) glutathione forms was quantified by means of HPLC and spectrophotometric assays with DTNB. The glutathione concentrations determined by HPLC in the vacuoles were 182 nmol GSH and 25 nmol GSSG per milligram protein. The respective concentrations of GSH and GSSG in the plastids were 112 and 6 nmol/mg protein and they were 228 and 10 nmol/mg protein in the mitochondria. The levels of GSH determined with DTNB were 1.5 times lower, whereas the amounts of GSSG were, by contrast, 1.5–2 times higher than in the HPLC assays. Although the glutathione redox ratios depended to some extent on the method used, the GSH/GSSG ratios were always lower for vacuoles than for plastids and mitochondria. In vacuoles, the pool of oxidized glutathione was higher than in other organelles.     

Cellular disulfide-reducing capacity: an integrated measure of cell redox capacity

To assess the disulfide reduction capacity of intact cells, EA.hy926 endothelial cells were incubated with alpha-lipoic acid in the presence of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Alpha-lipoic acid was reduced within cells to dihydrolipoic acid, which could be quantified upon efflux from the cells as reduction of DTNB. Uptake of both alpha-lipoic acid and alpha-lipoamide occurred at least in part via a medium chain fatty acid transporter, based on inhibition by octanoate. Alpha-lipoic acid was reduced within cells by pyridine nucleotide-disulfide oxidoreductases, since it is not reduced by GSH and since its reduction was inhibited by carmustine. Nonetheless, reduction was also dependent on the cellular redox environment, since it was inhibited by the redox cycling of menadione, by decreasing intracellular GSH, and by reduction of dehydroascorbate. Together, these results show that alpha-lipoic acid-dependent DTNB reduction provides a simple method to assess the disulfide-reducing capacity of intact cells, especially as determined by pyridine nucleotide-disulfide oxidoreductases.     

Selenium as a sulfhydryl redox catalyst and survey of potential selenium-dependent enzymes

The ability of selenium (Se) to act as a redox catalyst is an important factor in understanding the biological function of selenoproteins in addition to that of GSH peroxidase. Selenocystine at micromolar levels exhibited pseudothiotransferase activity by enhancing the reduction of 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) by thiols. In contrast, selenite inhibited the reduction of DTNB by thiols. Selenite was more catalytic than selenocystine in the reduction of cytochrome c by GSH, whereas GSH peroxidase was a weak catalyst. Tissues from Se-deficient and Se-supplemented rats were assayed for activities of GSH-thiotransferase, NADPH cytochrome c reductase, formaldehyde dehydrogenase, and a hypothesized GSH cytochrome c reductase. GSH-thiotransferase activity was significantly increased in the liver of Se-deficient rats. No appreciable activity of this enzyme was found in the kidney of rats from either dietary group. No enzymatic activity for cytochrome c reduction by GSH was detected in cytosols, mitochondria, or microsomes from liver and kidney of Se-deficient or Se-supplemented rats. Formaldehyde dehydrogenase was significantly higher in liver cytosols from Se-supplemented rats than from Se-deficient rats. The higher activity was not attributed to Se-containing proteins, but to an unknown small molecular-weight factor. This study did not support the hypothesis that physiological levels of Se may be involved in sulfhydryl-disulfide exchange reactions in vivo, or that selenium may enhance cytochrome c reduction by GSH in vivo.     

Exogenous GSH protection during hypoxia-reoxygenation of the isolated rat heart: impact of hypoxia duration

The objective of this study was to determine the interaction between duration of myocardial hypoxia and presence of exogenous glutathione (GSH) on functional recovery upon subsequent reoxygenation. Isolated perfused rat hearts were subjected to 20, 30, 40, or 50 min hypoxia (HYP), which resulted in a progressive decline in the amount of contractile recovery (% of normoxic rate-pressure product (RPP) and developed pressure) during 30 min reoxygenation. Supplementation with 5 mM GSH throughout normoxia, hypoxia, and reoxygenation significantly improved contractile recovery during reoxygenation after 20 and 30 min hypoxia (p < 0.05), but had no effect after longer durations of hypoxia when contractile recovery was typically below 40% of RPP and significant areas of no-reflow were observed. ECG analysis revealed that GSH shifted the bell-shaped curve for reperfusion ventricular fibrillation to the right resulting in attenuated fibrillation after 20 and 30 min hypoxia then increased incidences after 40 min when Control hearts were slow to resume electrical activity. ECG conduction velocity was well preserved in all hearts after 20 and 30 min hypoxia, but GSH administration significantly attenuated the decline that occurred with longer durations. GSH supplementation did not attenuate the 35% decline in intracellular thiols during 30 min of hypoxia. When 5 mM GSH was added only during 40 min of hypoxia, RPP recovery after reoxygenation was improved compared to unsupplemented Controls (73% vs. 55% of pre-hypoxia value, p < 0.05). Administration of GSH only during reoxygenation following 40 min of hypoxia did not alter RPP recovery compared to Control hearts. We conclude that cardioprotection by exogenous GSH is dependent on the duration of hypoxia and the functional parameter being evaluated. It is not due to an enhancement of intracellular GSH suggesting that exogenous GSH acts extracellularly to protect sarcolemmal proteins against thiol oxidation during the phase of hypoxia when oxidative stress is a major contributor to cardiac dysfunction. Furthermore, if enough damage accrues during oxygen deprivation, supplementing with GSH during reoxygenation will not impact recovery.     

A method for measuring disulfide reduction by cultured mammalian cells: relative contributions of glutathione-dependent and glutathione-independent mechanisms

A method is described for measuring bioreduction of hydroxyethyl disulfide (HEDS) or alpha-lipoate by human A549 lung, MCF7 mammary, and DU145 prostate carcinomas as well as rodent tumor cells in vitro. Reduction of HEDS or alpha-lipoate was measured by removing aliquots of the glucose-containing media and measuring the reduced thiol with DTNB (Ellman's reagent). Addition of DTNB to cells followed by disulfide addition directly measures the formation of newly reduced thiol. A549 cells exhibit the highest capacity to reduce alpha-lipoate, while Q7 rat hepatoma cells show the highest rate of HEDS reduction. Millimolar quantities of reduced thiol are produced for both substrates. Oxidized dithiothreitol and cystamine were reduced to a lesser degree. DTNB, glutathione disulfide, and cystine were only marginally reduced by the cell cultures. Glucose-6-phosphate deficient CHO cells (E89) do not reduce alpha-lipoate and reduce HEDS at a much slower rate compared to wild-type CHO-K1 cells. Depletion of glutathione prevents the reduction of HEDS. The depletion of glutathione inhibited reduction of alpha-lipoate by 25% and HEDS by 50% in A549 cells, while GSH depletion did not inhibit alpha-lipoate reduction in Q7 cells but completely blocked HEDS reduction. These data suggest that the relative participation of the thioltransferase (glutaredoxin) and thioredoxin systems in overall cellular disulfide reduction is cell line specific. The effects of various inhibitors of the thiol-disulfide oxidoreductase enzymes (1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), arsenite, and phenylarsine oxide) support this conclusion.     

Exogenous GSH protection during hypoxia-reoxygenation of the isolated rat heart: Impact of hypoxia duration

The objective of this study was to determine the interaction between duration of myocardial hypoxia and presence of exogenous glutathione (GSH) on functional recovery upon subsequent reoxygenation. Isolated perfused rat hearts were subjected to 20, 30, 40, or 50 min hypoxia (HYP), which resulted in a progressive decline in the amount of contractile recovery (% of normoxic rate-pressure product (RPP) and developed pressure) during 30 min reoxygenation. Supplementation with 5 mM GSH throughout normoxia, hypoxia, and reoxygenation significantly improved contractile recovery during reoxygenation after 20 and 30 min hypoxia (p < 0.05), but had no effect after longer durations of hypoxia when contractile recovery was typically below 40% of RPP and significant areas of no-reflow were observed. ECG analysis revealed that GSH shifted the bell-shaped curve for reperfusion ventricular fibrillation to the right resulting in attenuated fibrillation after 20 and 30 min hypoxia then increased incidences after 40 min when Control hearts were slow to resume electrical activity. ECG conduction velocity was well preserved in all hearts after 20 and 30 min hypoxia, but GSH administration significantly attenuated the decline that occurred with longer durations. GSH supplementation did not attenuate the 35% decline in intracellular thiols during 30 min of hypoxia. When 5 mM GSH was added only during 40 min of hypoxia, RPP recovery after reoxygenation was improved compared to unsupplemented Controls (73% vs. 55% of pre-hypoxia value, p < 0.05). Administration of GSH only during reoxygenation following 40 min of hypoxia did not alter RPP recovery compared to Control hearts. We conclude that cardioprotection by exogenous GSH is dependent on the duration of hypoxia and the functional parameter being evaluated. It is not due to an enhancement of intracellular GSH suggesting that exogenous GSH acts extracellularly to protect sarcolemmal proteins against thiol oxidation during the phase of hypoxia when oxidative stress is a major contributor to cardiac dysfunction. Furthermore, if enough damage accrues during oxygen deprivation, supplementing with GSH during reoxygenation will not impact recovery.     

Involvement of Amino-Acid Side Chains of Membrane Proteins in the Binding of Glutathione to Pig Cerebral Cortical Membranes

Glutathione (GSH), a general antioxidant and detoxifying compound, is the most abundant thiol-containing peptide in the central nervous system. It has been earlier shown to regulate the functions of glutamate receptors and to possess specific binding sites in both neurons and glial cells. The possible involvement of disulfide bonds, cysteinyl, arginyl, lysyl, glutamyl, and aspartyl residues in the binding of tritiated GSH to specific sites in pig cerebral cortical synaptic membranes was now studied after covalent modification of membrane proteins. Treatment of synaptic membranes with the thiol-modifying reagents 5,5-dithio-bis(2-nitrobenzoate) (DTNB) and 4,4-dithiodipyridine (DDP) dramatically enhanced the binding of [³H]GSH in a dose-dependent manner. Dithiothreitol (DTT) alone reduced the binding, but pretreatment of the membranes with DTT potentiated the enhancing effect of DTNB. On the other hand, when the modification with DTNB was followed by treatment with DTT, the enhancement by DTNB was completely reversed. N-ethylmaleimide, a thiol alkylating agent, and phenylisothiocyanate, a thiol- and amino-group modifying compound, reduced the binding, and their effects were additive. The guanidino-modifying agent phenylglyoxal reduced the binding but the carboxyl-modifying reagent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide had no significant effect. The results indicate that cysteinyl side chains and disulfide bonds are essential in the binding of GSH to membrane proteins and that arginyl and lysyl side chains may also be directly involved in this process.     

Mutagenicity of tetrachloroethene in the Ames test--metabolic activation by conjugation with glutathione

The mutagenicity of tetrachloroethene (tetra) and its S conjugate, S-(1,2,2-trichlorovinyl)glutathione (TCVG) was investigated using a modified Ames preincubation assay. TCVG was a potent mutagen in presence of rat kidney particulate fractions containing high concentrations of gamma-glutamyl transpeptidase (GGT) and dipeptidases. Purified tetra was not mutagenic without exogenous metabolic activation or under conditions favoring oxidative metabolism. Preincubation of tetra with purified rat liver glutathione (GSH) S-transferases in presence of GSH and rat kidney fractions resulted in a time-dependent formation of TCVG as determined by (HPLC) analysis and in an unequivocal mutagenic response in the Ames test. Experiments with tetra in the isolated perfused rat liver demonstrated TCVG formation and its excretion with the bile; bile collected after the addition of tetra to the isolated perfused liver was unequivocally mutagenic in bacteria in the presence of kidney particulate fractions. The mutagenicity was reduced in all cases by the GGT inhibitor serine borate or the beta-lyase inhibitor aminooxyacetic acid. These results support the suggestion that cleavage of the GSH S conjugate formed from tetra by the enzymes of the mercapturic acid pathway and by beta-lyase may be involved in the nephrocarcinogenic effects of this haloalkene in rats.     

Assay of glutathione reductase in crude tissue homogenates using 5,5'-dithiobis(2-nitrobenzoic acid)

A method for assaying glutathione reductase (GSH; EC 1.6.4.2) in crude plant extracts is described. The method is based on the increase in absorbance at 412 nm when 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) is reduced by GSH. The effects of the following parameters on the assay were tested: various buffers, pH, buffer concentration, compounds commonly present in enzyme preparations, thiols, and the presence of another NADPH-dependent enzyme. The assay is more sensitive and less subject to interference than the widely used assay where NADPH oxidation is monitored. In particular, the specificity of DTNB allows assay of glutathione reductase in the presence of other NADPH-dependent enzymes and common protein extract contaminants.     

Characterization of normal, glutathione-deficient and arginase-deficient sheep erythrocytes by 1H-NMR spectroscopy

Normal sheep erythrocytes as well as glutathione- (GSH-) deficient and arginase-deficient sheep erythrocytes have been characterized by 1H nuclear magnetic resonance spectroscopy. The GSH deficiency is a result of defective amino acid transport (lesion 1), diminished gamma-glutamylcysteine synthetase activity (lesion 2), or both (lesions (1 + 2)). 1H-NMR spectra of normal sheep erythrocytes are similar to those for human erythrocytes, and consist of resonances from a number of small intracellular molecules, including GSH. In contrast, the resonances for GSH in the GSH-deficient erythrocytes are much weaker, and strong resonances are observed for lysine, threonine and ornithine or arginine, depending on the arginase activity, in erythrocytes with lesion 1 and lesions (1 + 2). A comparison of the intensity of GSH resonances in spectra for normal and GSH-deficient erythrocytes with GSH levels determined spectrophotometrically following reaction with the nonspecific thiol reagent 5,5'-dithiobis(2-nitrobenzoate) (DTNB) indicates that either not all of the GSH determined with Ellman's reagent is free and observable by 1H-NMR or that not all of the thiol determined by Ellman's reagent is GSH. If the latter is the case, the GSH levels determined with Ellman's reagent for erythrocytes with lesions (1 + 2) are most affected, which might account for their high susceptibility to oxidative stress.     

外源谷胱甘肽对石竹幼苗镉毒害的缓解效应

为了探讨外源谷胱甘肽(GSH)对地被植物镉(Cd)毒害的缓解效应, 采用温室盆栽土培的方法, 研究了不同浓度(0、20、40、60、80、100 mg·L ^-1)的外源GSH处理对50 mg·kg ^-1 Cd胁迫下石竹(Dianthus chinensis)幼苗生长的影响。结果发现, 50 mg·kg ^-1 Cd显著抑制了石竹幼苗的生长。喷施外源GSH后, 一定浓度范围内(≤60 mg·L ^-1)的外源GSH可显著缓解石竹幼苗的Cd胁迫, 过氧化氢酶(CAT)、过氧化物酶(POD)、抗坏血酸过氧化物酶(APX)、单脱氢抗坏血酸还原酶(MDAR)、脱氢抗坏血酸还原酶(DHAR)、谷胱甘肽还原酶(GR)的活性, 抗坏血酸(AsA)和GSH含量以及生物量、株高、分蘖数都显著高于无外源GSH处理的石竹幼苗, 而丙二醛(MDA)含量、细胞膜透性、Cd含量、O₂· ^-的产生速率以及H₂O₂的积累量则显著低于无外源GSH处理的石竹幼苗, 但随着外源GSH喷施浓度的增加, 缓解效应有下降的趋势。试验表明55-65 mg·L ^-1的外源GSH缓解效果最佳。     

Recognition of opioid agonist and antagonist in the opioid receptor binding site

By treating the rat crude synaptosomal fraction with 5,5'-dithio-bis-(2-nitrobenzoic acid), DTNB, a marked decrease of stereo-specific binding of opioid agonist (dihydromorphine or D-Ala-D-Leu-enkephalin) was observed, but there was no effect in the case of the binding of opioid antagonist (naloxone or diprenorphine). The decrease of the agonist binding in the presence of 500 microM of DTNB was nearly equal to that of 100 mM of NaCl. The ability of opioids to inhibit 3H-naloxone binding in the absence of DTNB was compared to their inhibitory potency in the presence of 500 microM of DTNB to obtain DTNB response ratio. This ratio closely correlated with sodium index of each opioid. Potency of the inactivation of the agonist binding by congeners of DTNB changed with net charge of the reagents, and 2,2'-dithiobis-(5-nitropyridine), bearing a positive charge, was most effective. These results suggest that an aliphatic sulfhydryl group, being sensitive to DTNB is located to the active center of an anionic binding site for the agonist, and controls opioid agonist binding through a proton transfer mechanism.     

Inhibition of 5-amino levulinic acid dehydratase activity by arsenic in excised etiolated maize leaf segments during greening

In vivo as well as in vitro supply of sodium arsenate inhibited the 5-Amino levulinic acid dehydratase (5-aminolevulinate-hydrolyase EC 4.2.1.24, ALAD) activity in excised etiolated maize leaf segments during greening. The percent inhibition of enzyme activity by arsenate (As) was reduced by the supply of KNO3, but it was increased by the glutamine and GSH. Various inhibitors, such as, chloramphenicol, cycloheximide and LA, decreased the % inhibition of enzyme activity by As. The % inhibition of enzyme activity was also reduced by in vivo supply of DTNB. The enzyme activity was reduced substantially by in vitro inclusion of LA, both in the absence and presence of As. In vitro inclusion of DTNB and GSH inhibited the enzyme activity extracted from leaf segments treated without arsenate (-As enzyme) and caused respectively no effect and stimulatory effect on arsenate treated enzyme (+As enzyme). Increasing concentration of ALA during assay increased the activity of -As enzyme and +As enzyme to different extent, but double reciprocal plots for both the enzymes were biphasic and yielded distinct S0.5 values for the two enzymes (-As enzyme, 40 micromol/L and +As enzyme, 145 micromol/L) at lower concentration range of ALA only. It is suggested that As inhibits ALAD activity in greening maize leaf segments by affecting its thiol groups and/or binding of ALA to the enzyme.     

Effects of 5,5''-Dithiobis-(2-Nitrobenzoic Acid) on Opiate Binding to Both the Membrane-Bound Receptor and the Partially Purified Opiate Receptor

Pretreatment of partially purified opiate receptor from rat brains with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) decreased opiate agonist binding more effectively than that of antagonist. This agent, at a concentration that inhibits only 3H-agonist binding, increases the IC50 values of agonists but not those of antagonists. We also observed similar effects of DTNB on opiate binding to the membrane-bound receptor that are in good agreement with the published data. Moreover, there was an excellent correlation between the IC50 values of the two different preparations. However, opiate binding to the partially purified receptor was about a thousandfold more sensitive to DTNB than binding to this membrane-bound receptor. Dithiothreitol, a sulfide bond reducing agent, reversed the effects of DTNB on the opiate binding.     

Insulin regulation of glutathione and contractile phenotype in diabetic rat ventricular myocytes

Cardiovascular complications of diabetes mellitus involve oxidative stress and profound changes in reduced glutathione (GSH), an essential tripeptide that controls many redox-sensitive cell functions. This study examined regulation of GSH by insulin to identify mechanisms controlling cardiac redox state and to define the functional impact of GSH depletion. GSH was measured by fluorescence microscopy in ventricular myocytes isolated from Sprague-Dawley rats made diabetic by streptozotocin, and video and confocal microscopy were used to measure mechanical properties and Ca(2+) transients, respectively. Spectrophotometric assays of tissue extracts were also done to measure the activities of enzymes that control GSH levels. Four weeks after injection of streptozotocin, mean GSH concentration ([GSH]) in isolated diabetic rat myocytes was approximately 36% less than in control, correlating with decreased activities of two major enzymes regulating GSH levels: glutathione reductase and gamma-glutamylcysteine synthetase. Treatment of diabetic rat myocytes with insulin normalized [GSH] after a delay of 3-4 h. A more rapid but transient upregulation of [GSH] occurred in myocytes treated with dichloroacetate, an activator of pyruvate dehydrogenase. Inhibitor experiments indicated that insulin normalized [GSH] via the pentose pathway and gamma-glutamylcysteine synthetase, although the basal activity of glucose-6-phosphate dehydrogenase was not different between diabetic and control hearts. Diabetic rat myocytes were characterized by significant mechanical dysfunction that correlated with diminished and prolonged Ca(2+) transients. This phenotype was reversed by in vitro treatment with insulin and also by exogenous GSH or N-acetylcysteine, a precursor of GSH. Our data suggest that insulin regulates GSH through pathways involving de novo GSH synthesis and reduction of its oxidized form. It is proposed that a key function of glucose metabolism in heart is to supply reducing equivalents required to maintain adequate GSH levels for the redox control of Ca(2+) handling proteins and contraction.     

Effect of SH-Specific Reagent 5,5′-Dithobis(2-Nitrobenzoic Acid) on Functional Activity of Components of Adenylyl Cyclase Signal System

Sensitivity of adenylyl cyclase signal system to 5,5′-dithobis(2-nitrobenzoic acid) (DTNB) oxidizing SH-groups of cystein residues to disulfide bonds was studied. It was shown that treatment of plasma membranes fractions of smooth muscles of the mollusc Anodonta cygnea and of rat skeletal muscles as well as of homogenate of mouse fibroblasts culture of L strain with micromole concentrations of DTNB led to a decrease of activity of adenylyl cyclase (AC) stimulated by GIDP, sodium fluoride, and, to a lesser degree, forskolin. Dithiothreitol (DTT) partly restored the stimulating effects of GIDP, NaF, and forskolin, the effect of this dithiol being dose-dependent. AC stimulated by biogenic amines—serotonin in mollusc muscles, isoproterenol in rat muscles, and both hormones in mouse fibroblasts—is more sensitive to DTNB than the enzyme stimulated by non-hormonal agents. Thus, the stimulatory effects of hormones decreased dose-dependently in the presence of 10–100 μM DTNB and were almost completely blocked by 250 μM reagent. These effects were partly restored in the presence of 5 mM DTT. The obtained data indicate a high sensitivity of the hormone-stimulated AC to action of the reagents interacting specifically with SH-groups of the proteins components of the AC system. In the rat muscle membranes treated with 25 μM DTNB, no significant rightward shift was observed of the curve of competitive replacement of the β-adrenergic receptor antagonist [³H]-dihydroalprenolol by the β-agonist isoproterenol in the presence of GTP and the affinity of the agonist to the receptor somewhat decreased, which indicates a disturbance of functional coupling of the β-adrenergic receptor with G-protein after treatment with DTNB.     

Sulfurtransferases and the content of cysteine, glutathione and sulfane sulfur in tissues of the frog Rana temporaria

L-cysteine desulfuration was examined in tissues of Rana temporaria, in October and January. The activities of 3-mercaptopyruvate sulfurtransferase (MPST), cystathionine gamma-lyase (CST) and rhodanese were primarily concentrated in frog liver and kidney. The values of CST and rhodanese activity, as well as sulfane sulfur compounds levels fell in the range characteristic of rat. For each of the investigated tissues changes noted in the enzymatic activities and in the level of glutathione (GSH), protein-bound cysteine (PbCys) and sulfane sulfur compounds were dependent on the month in which the determination was performed, and on the character of the tissue. In such tissues as the liver or gonads, high GSH levels and high activities of MPST (in the liver) or MPST and rhodanese (in the gonads) seemed to accompany protein biosynthesis during hibernation. PbCys, the level of which was consequently diminished in all tissues in January, compensated the absence of exogenous cysteine. A significantly reduced GSH level in the brain in January seemed to be correlated with decreased requirements of the tissue for this important natural antioxidant at diminished thyroid hormones levels in the serum and minimal oxygen consumption during the hibernation. In the kidney, the possible participation of sulfane sulfur compounds in detoxification processes requires elucidation, similarly as in protection against cellular oxidative stress at extremely low levels of GSH.     

Calcium- and phosphate-dependent release and loading of glutathione by liver mitochondria

The status of glutathione (GSH) was studied in isolated rat liver mitochondria under conditions which induce a permeability transition. This transition, which is inhibited by cyclosporin A (CyA), requires the presence of Ca2+ and an inducing agent such as near physiological levels (3 mM) of inorganic phosphate (Pi). The transition is characterized by an increased inner membrane permeability to some low molecular weight solutes and by large amplitude swelling under some experimental conditions. Addition of 70 microM Ca2+ and 3 mM Pi to mitochondria resulted in mitochondrial swelling and extensive release of GSH that was recovered in the extramitochondrial medium as GSH. Both swelling and the efflux of mitochondrial GSH were prevented by CyA. Incubation of mitochondria in the presence of Ca2+, Pi, and GSH followed by addition of CyA provided a mechanism to load mitochondria with exogenous GSH that was greater than the rate of uptake by untreated mitochondria. Thus, GSH efflux from mitochondria may occur under toxicological and pathological conditions in which mitochondria are exposed to elevated Ca2+ in the presence of near physiological concentrations of Pi through a nonspecific pore. Cyclical opening and closing of the pore could also provide a mechanism for uptake of GSH by mitochondria.