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PubMed1998

Glutathione homeostasis in human hepatic cells: overexpression of gamma-glutamylcysteine synthetase gene in cell lines resistant to buthionine sulfoximine, an inhibitor of glutathione synthesis.

The synthesis of glutathione (GSH) and its conjugation to xenobiotics are essential for detoxification in liver cells. To understand how cellular levels of GSH are balanced in response to environmental stress, we cloned two cell lines, HLE/BSO1-1 and HLE/BSO1-2, from human hepatic HLE/WT cells resistant to buthionine sulfoximine (BSO), an irreversible inhibitor of gamma-glutamylcysteine synthetase (GCS). HLE/BSO1-1 and HLE/BSO1-2 showed 35- and 40-fold higher resistance respectively, than the wild type to BSO. In the absence of BSO, cellular levels of GSH were 3.0-fold higher, whereas levels of Pi class glutathione thiol transferase, GSTP1, were 2-fold lower, in the subclones than in the wild type cells. GCS heavy subunit (GCSh) mRNA level were 2.5-fold higher in HLE/BSO1-1 and HLE/BSO1-2 as compared with HLE/WT. Sequences between -315 and -241 base pairs of the 5' region, which contain an AP1 site, were shown to be responsible for the enhanced expression of GCSh in HLE/BSO1-1 cells. The expression of a dominant-negative mutant of c-Jun was found to inhibit the AP1-dependent GCSh promoter activity in HLE/WT and HLE/BSO1-1. Both protein level of c-Jun and binding activity of AP-1 were increased in both HLE/BSO1-1 and HLE/BSO1-2 cells. The up-regulation of GCSh gene appeared to be due to enhanced GCSh promoter acting through AP-1 activation in BSO-resistant hepatic cells.

Binding Sites↗
PubMed1995

Role of glutathione in macrophage activation: effect of cellular glutathione depletion on nitrite production and leishmanicidal activity.

We have examined the effects of two agents depleting the intracellular pool of glutathione (GSH) on macrophage activation induced by IFN-gamma + LPS, as measured by nitrite production and leishmanicidal activity. Diethylmaleate (DEM), which depletes intracellular GSH by conjugation via a reaction catalyzed by the GSH-S-transferase, strongly inhibited nitrite secretion and leishmanicidal activity when added before or at the time of addition of IFN-gamma + LPS; this inhibition was progressively lost when addition of DEM was delayed up to 10 hr. A close correlation was observed between levels of intracellular soluble GSH during activation and nitrite secretion. Inhibition was partially reversed by the addition of glutathione ethyl ester (GSH-Et). Buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, also inhibited macrophage activation, although to a lesser extent than DEM despite a more pronounced soluble GSH depletion. This inhibition was completely reversed by the addition of GSH-Et. DEM and BSO did not alter cell viability or PMA-triggered O2- production by activated macrophages, suggesting that the inhibitory effects observed on nitrite secretion and leishmanicidal activity were not related to a general impairment of macrophage function. DEM and BSO treatment reduced iNOS specific activity and iNOS protein in cytosolic extracts. DEM also decreased iNOS mRNA expression while BSO had no effect. Although commonly used as a GSH-depleting agent, DEM may have additional effects because it can also act as a sulhydryl reagent; BSO, on the other hand, which depletes GSH by enzymatic inhibition, has no effect on protein-bound GSH. Our results suggest that both soluble and protein-bound GSH may be important for the induction of NO synthase in IFN-gamma + LPS-activated macrophages.

Amino Acid Oxidoreductases↗
PubMed2002

Functional relationship of alpha-glutathione S-transferase and glutathione S-transferase activity in machine-preserved non-heart-beating donor kidneys.

alpha-Glutathione S-transferase (alpha-GST) is a biochemical parameter used to estimate the amount of proximal tubule damage to a kidney. In normal clinical practice, the concentration of alpha-GST in urine is determined by a rather time-consuming immunochemical test, the enzyme-linked immunosorbent assay (ELISA). Kidneys from non-heart-beating (NHB) donors are perfused prior to transplantation. The determination of alpha-GST concentration in the perfusate to monitor damage is also done by means of an ELISA test. However, because this is a time-consuming method, it would be helpful to find a parameter proportional to GST concentrations that would be available within minutes. We therefore compared the method of determining alpha-GST concentration via ELISA with that of determining the enzymatic activity of GST, which is much faster (results available within 10 min). The comparison was made using 150 preserved kidneys that had been perfused for 6 h. The correlation was found to be very good, as indicated by the linear regression data: r=0.954, P<0.001. pi-Glutathione S-transferase (pi-GST) was also determined by means of an ELISA test, and the concentration of pi-GST was compared with the enzymatic activity of the "total" GST. The precision of the enzymatic method, given by intra- and interassay variation, was 1.5% and 10.5%, respectively.

Enzyme-Linked Immunosorbent Assay↗
PubMed1991

Purification and characterization of human muscle glutathione S-transferases: evidence that glutathione S-transferase zeta corresponds to a locus distinct from GST1, GST2, and GST3.

Human muscle glutathione S-transferase isozyme, GST zeta (pI 5.2) has been purified by three different methods using immunoaffinity chromatography, DEAE cellulose chromatography, and isoelectric focusing. GST zeta prepared by any of the three methods does not recognize antibodies raised against the alpha, mu, or pi class glutathione S-transferases of human tissues. GST zeta has a blocked N-terminus and its peptide fingerprints also indicate it to be distinct from the alpha, mu, or pi class isozymes. As compared to GSTs of alpha, mu, and pi classes, GST zeta displays higher activities toward t-stilbene oxide and Leukotriene A4 methyl ester. GST zeta also expresses GSH-peroxidase activity toward hydrogen peroxide. The Kms of GST zeta for CDNB and GSH were comparable to those reported for other human GSTs but its Vmax for CDNB, 7620 mol/mol/min, was found to be considerably higher than that reported for other human GSTs. The kinetics of inhibition of GST zeta by hematin, bile acids, and other inhibitors also indicate that it was distinct from the three classes of GST isozymes. These studies suggest that GST zeta corresponds to a locus distinct from GST1, GST2, and GST3 and probably corresponds to the GST4 locus as suggested previously by Laisney et al. (1984, Human Genet. 68, 221-227). The results of peptide fingerprints and kinetic analysis indicate that as compared to the pi and alpha class isozymes, GST zeta has more structural and functional similarities with the mu class isozymes. Besides GST zeta several other GST isozymes belonging to pi and mu class have also been characterized in muscle. The pi class GST isozymes of muscle have considerable charge heterogeneity among them despite identical N-terminal sequences.

Amino Acid Sequence↗
PubMed1995

Na-dependent glutamate transport in high K and high glutathione (HK/HG) and high K and low glutathione (HK/LG) dog red blood cells.

Na-dependent glutamate (Glu) influxes in the red blood cells of normal low K (LK), high K and high glutathione (HK/HG), and high K and low glutathione (HK/LG) dogs were compared. The ranges of the influxes in LK, HK/HG and HK/LG cells were 1.0-63, 62-174 and 1.3-26 mumol/1 cells per h, respectively. Some LK and HK/LG dogs had red blood cells with extremely low Glu influxes. In cells with extremely low Glu influxes, however, there were clear Na-dependent Glu influxes. In LK, HK/HG and HK/LG cells, the Km of Na-dependent Glu influx with respect to the medium Glu concentration were 17, 20 and 19 mM, respectively, and the half-maximal activation (K1/2) with respect to medium Na concentration was 39, 40 and 42 microM, respectively. By the addition of harmaline, a hallucinogenic alkaloid, the Vmax in LK cells was not affected and the Km was increased, while the Vmax was decreased and the Km increased in HK/HG and HK/LG cells. The Ki value with harmaline by means of Dixon plot in LK cells was 5.2 mM, against 1.8 and 1.9 mM in HK/HG and HK/LG cells, respectively. These results suggest that the difference in the Na-dependent Glu influxes between 2 HK groups was due to the varying quantity, not the quality, of the transporter.

ATP-Binding Cassette Transporters↗
PubMed1990

Glutathione disulfide enhances the reduced glutathione inhibition of lipid peroxidation in rat liver microsomes.

Experiments were undertaken to examine the effects of reduced (GSH) and oxidized (GSSG) glutathione on lipid peroxidation of rat liver microsomes. Dependence on microsomal alpha-tocopherol was shown for the GSH inhibition of lipid peroxidation. However, when GSH (5 mM) and GSSG (2.5 mM) were combined in the assay system, inhibition of lipid peroxidation was enhanced markedly over that with GSH alone in microsomes containing alpha-tocopherol. Surprisingly, the synergistic inhibitory effect of GSH and GSSG was also observed for microsomes that were deficient in alpha-tocopherol. These data suggest that there may be more than one factor responsible for the glutathione-dependent inhibition of lipid peroxidation. The first is dependent upon microsomal alpha-tocopherol and likely requires GSH for alpha-tocopherol regeneration from the alpha-tocopheroxyl radical during lipid peroxidation. The second factor appears to be independent of alpha-tocopherol and may involve the reduction of lipid hydroperoxides to their corresponding alcohols. One, or possibly both, of these factors may be activated by GSSG through thiol/disulfide exchange with a protein sulfhydryl moiety.

Animals↗
PubMedDate not supplied

Effect of arsenicals on biliary excretion of endogenous glutathione and xenobiotics with glutathione-dependent hepatobiliary transport.

Sodium arsenite (25-100 mumol/kg, i.v.) and arsenate (75-300 mumol/kg, i.v.) injected into anaesthetized rats increased the biliary excretion of endogenous non-protein thiols (NPSH) in a dose-dependent fashion up to 24- and 31-fold, respectively. Simultaneously with NPSH, glutathione (GS) excretion was increased to a similar extent suggesting that the increment in biliary thiol output originated from enhanced hepatobiliary transport of GS. After administration of labelled arsenicals, biliary excretion of 74As and NPSH followed similar time-courses. Biliary excretion of 74As was more efficient after arsenite than arsenate administration corresponding to the greater potency of arsenite compared to arsenate to increase biliary output of NPSH. Coadministered sulfobromophthalein (BSP) inhibited the biliary excretion of 74As and prevented the arsenical-induced increase in biliary NPSH. Thus, hepatobiliary transport of arsenic apparently proceeds coordinately with that of GS. However, excretion of each molecule of arsenic compound generates transport of several molecules of GS. Though mercuric, methylmercuric, cadmium and zinc ions are thought to be excreted into bile as complexes with GS, the marked arsenical-induced increase in GS excretion only doubled the biliary excretion of inorganic mercury and hardly influenced the transport of other metals into bile. This finding suggests that arsenicals markedly enhance biliary excretion of GS with a free thiol group but barely or not at all that of GS with a thiol group blocked by a firmly bound metal ion. Both arsenicals diminished the biliary excretion of BSP-glutathione conjugate after BSP administration presumably because they impaired conjugation of BSP with GSH due to decreased GS availability. It is assumed that arsenite, and arsenate after reduction to arsenite, forms an unstable complex with GS that is efficiently transported into bile resulting in increased biliary output of GS. It is demonstrated that arsenite-induced perturbation of hepatobiliary disposition of endogenous GS differentially affects biliary excretion of xenobiotics with GS-dependent hepatobiliary transport.

Animals↗
PubMed1992

Increased biliary excretion of glutathione is generated by the glutathione-dependent hepatobiliary transport of antimony and bismuth.

We have recently demonstrated that the hepatobiliary transport of arsenic is glutathione-dependent and is associated with a profound increase in biliary excretion of glutathione (GSH), hepatic GSH depletion and diminished GSH conjugation (Gyurasics A, Varga F and Gregus Z, Biochem Pharmacol 41: 937-944 and Gyurasics A, Varga F and Gregus Z, Biochem Pharmacol 42: 465-468, 1991). The present studies in rats aimed to determine whether antimony and bismuth, other metalloids in group Va of the periodic table, also possess similar properties. Antimony potassium tartrate (25-100 mumol/kg, i.v.) and bismuth ammonium citrate (50-200 mumol/kg, i.v.) increased up to 50- and 4-fold, respectively, the biliary excretion of non-protein thiols (NPSH). This resulted mainly from increased hepatobiliary transport of GSH as suggested by a close parallelism in the biliary excretion of NPSH and GSH after antimony or bismuth administration. Within 2 hr, rats excreted into bile 55 and 3% of the dose of antimony (50 mumol/kg, i.v.) and bismuth (150 mumol/kg, i.v.), respectively. The time courses of the biliary excretion of these metalloids and NPSH or GSH were strikingly similar suggesting co-ordinate hepatobiliary transport of the metalloids and GSH. However, at the peak of their excretion, each molecule of antimony or bismuth resulted in a co-transport of approximately three molecules of GSH. Diethyl maleate, indocyanine green and sulfobromophthalein (BSP), which decreased biliary excretion of GSH, significantly diminished excretion of antimony and bismuth into bile indicating that hepatobiliary transport of these metalloids is GSH-dependent. Administration of antimony, but not bismuth, decreased hepatic GSH level by 30% and reduced the GSH conjugation and biliary excretion of BSP. These studies demonstrate that the hepatobiliary transport of trivalent antimony and bismuth is GSH-dependent similarly to the hepatobiliary transport of trivalent arsenic. Proportionally to their biliary excretion rates, these metalloids generate increased biliary excretion of GSH probably because they are transported from liver to bile as unstable GSH complexes. The significant loss of hepatic GSH into bile as induced by arsenic or antimony may compromise conjugation of xenobiotics with GSH.

Animals↗
PubMed1989

Leukotriene C4 inhibits ATP-dependent transport of glutathione S-conjugate across rat heart sarcolemma. Implication for leukotriene C4 translocation mediated by glutathione S-conjugate carrier.

Sarcolemmal vesicles prepared from rat heart exhibited ATP-dependent uptake of S-(2,4-dinitrophenyl)glutathione (DNP-SG), which obeyed Michaelis-Menten kinetics with an apparent Km of 21 microM for DNP-SG and a Vmax of 0.27 nmol.10 min-1.mg protein-1. Several model glutathione S-conjugates inhibited DNP-SG uptake, but leukotriene C4 inhibited uptake much more significantly even at lower concentrations (competitive inhibition, Ki = 1.5 microM). However, leukotrienes D4 and E4, which lack the gamma-glutamyl moiety, were less effective. The results suggest that the ATP-dependent transport system has a high affinity for leukotriene C4, and may be responsible for the translocation of this compound.

Adenosine Triphosphate↗
PubMedDate not supplied

Lymphocyte proliferation in glutathione-depleted lymphocytes: direct relationship between glutathione availability and the proliferative response.

Lymphocyte proliferation in response to mitogenic lectins is directly dependent upon glutathione (GSH) availability. Thus, proliferation can be enhanced by providing lymphocytes with excess glutathione, and strongly inhibited by limiting the quantity of intracellular GSH available during the mitogenic stimulation. Exogenous GSH, cysteine and 2-mercaptoethanol (2-ME) can all significantly enhance lymphocyte proliferation and augment intracellular GSH levels. Lymphocytes depleted of GSH by buthionine sulfoximine (BSO) fail to undergo a full blast transformation response to mitogenic lectins. In lymphocytes stimulated with mitogen in the presence of BSO, the time profile of intracellular GSH levels shows a rapid decline over the first 24-48 h and a subsequent gradual decline to levels less than 0.5 nmol/10(7) lymphocytes by 72-96 h. Exogenous GSH partially sustains intracellular GSH levels and completely restores lymphocyte proliferation even in the presence of 2000 microM BSO. Other thiols, such as cysteine and 2-ME, do not significantly alter the time profile of intracellular GSH in mitogen-stimulated lymphocytes in the presence of 2000 microM BSO, and their capacity to enhance proliferation is greatly diminished albeit not completely abolished under these conditions. Ongoing GSH synthesis is clearly essential to maintain a normal proliferative response. If intracellular GSH levels are depleted initially and lymphocytes are then stimulated with mitogen in the presence of BSO, there is a diminished capacity of cysteine and 2-ME to restore proliferation relative to exogenous GSH. There is also a diminished capacity of exogenous GSH to restore proliferation with higher concentrations of BSO. This suggests that the restoration of lymphocyte proliferation by exogenous GSH is more closely linked to effects on intracellular rather than extracellular GSH. These studies confirm the importance of intracellular GSH in lymphocyte proliferation. The essential role for intracellular GSH can be demonstrated even in the presence of other exogenous thiols, such as cysteine and 2-ME. The enhancement of lymphocyte proliferation by exogenous cysteine appears to be directly linked to effects on intracellular GSH, whereas the enhancement by 2-ME is probably more complex but clearly linked to effects on intracellular GSH.

Buthionine Sulfoximine↗
PubMed1985

Biliary secretion of glutathione and of glutathione-metal complexes.

As bile is the main route of elimination of many metals, a large number of studies have been directed toward the characterization of the hepatobiliary transport of both endogenous and exogenous metals. Although some progress has been made, we still know little of the basic mechanisms involved in the hepatocellular uptake of metals, in their intracellular translocation and metabolism, or in their transport into bile. Our recent studies have focused on the last step in the hepatobiliary transport of mercury, namely, the secretion of the metal from liver cells into bile. The rate of secretion of methyl and inorganic mercury into bile was low in suckling rats and rapidly increased to adult rates soon after weaning. These changes closely followed similar developmental changes in the biliary secretion of reduced glutathione (GSH). When GSH secretion into bile was completely inhibited, without changing hepatic levels of GSH or mercury, mercury secretion was also completely blocked. Mercury secretion paralleled individual and sex-related differences in GSH secretion. At the same time, the secretion of mercury was independent of bile flow, of the thiol and mercury concentration gradients between bile and liver cells, and of those between bile and plasma. Our results, therefore, indicate a close coupling between the secretion of mercury and that of GSH. These in vivo findings, along with in vitro studies by others in vesicles isolated from the canalicular membrane of the liver cell, indicate a carrier-mediated transport system for GSH, but the nature of the linkage of this transport system with mercury secretion is not yet fully established. Our data and those in the literature are consistent with the involvement of at least two steps in the movement of mercury from liver cells to bile--the formation of a mercury-glutathione complex in the liver cell, followed by the secretion of this complex through a process closely linked to GSH secretion. The identification of GSH as an endogenous complexing agent in the transport of metals between tissues and body fluids now permits the design of therapeutic strategies aimed at exploiting this transport vehicle to effect the removal of metals via physiological routes of excretion. The present discussion considers the role of GSH in the hepatobiliary transport of metals. In doing so, a brief review is given of current understanding of hepatic GSH metabolism and transport.

Animals↗
PubMed1988

Purification of glutathione S-transferases from rat liver and Walker 256 mammary carcinoma cells by high-performance liquid chromatography and a glutathione affinity column.

A novel method for the rapid purification of glutathione S-transferases (GST) from tissue and cell culture samples is reported. A high-performance glutathione affinity column was used and produced results comparable to those obtained with classical agarose affinity columns. Experiments with purified rat liver GST standards resulted in 87% recovery of total activity. Sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) of the affinity-purified GST was identical to the GST standard and revealed three major protein bands, corresponding to the Ya, Yb, and Yc subunits. A fourth protein band (relative molecular mass 25,000), migrating slightly faster than the Ya subunit, was present in both the standard and eluted GST samples. This polypeptide was tentatively identified as the Yk subunit. Successful purification from rat liver and Walker 256 rat carcinoma cell cytosols was also performed. Recovery of total GST enzymatic activity from Walker cell and rat liver cytosol was 49 and 58%, respectively. SDS-PAGE of these samples indicated a high degree of purity. This methodology requires less than 1 h and can be performed using small quantities of tissue. These features make this technique applicable to analysis of a broad range of biological applications including human biopsy material for GST content.

Animals↗
PubMed2004

Glutathione precursors replenish decreased glutathione pool in cystinotic cell lines.

Cystinosis is an inherited disorder due to mutations in the CTNS gene which encodes cystinosin, a lysosomal transmembrane protein involved in cystine export to the cytosol. Both accumulation of cystine in the lysosome and decreased cystine in the cytosol may participate in the pathogenic mechanism underlying the disease. We observed that cystinotic cell lines have moderate decrease of glutathione content during exponential growth phase. This resulted in increased solicitation of oxidative defences of the cell denoted by concurrent superoxide dismutase induction, although without major oxidative insult under our experimental conditions. Finally, decreased glutathione content in cystinotic cell lines could be counterbalanced by a series of exogenous precursors of cysteine, denoting that lysosomal cystine export is a natural source of cellular cysteine in the studied cell lines.

Adolescent↗
PubMed2006

Vacuolar sequestration of glutathione S-conjugates outcompetes a possible degradation of the glutathione moiety by phytochelatin synthase.

Monochlorobimane was used as a model xenobiotic for Arabidopsis to directly monitor the compartmentation of glutathione-bimane conjugates in situ and to quantify degradation intermediates in vitro. Vacuolar sequestration of the conjugate was very fast and outcompeted carboxypeptidation to the gamma-glutamylcysteine-bimane intermediate (gamma-EC-B) by phytochelatin synthase (PCS) in the cytosol. Following vacuolar sequestration, degradation proceeded to cysteine-bimane without intermediate. Only co-infiltration of monochlorobimane with Cd2+ and Cu2+ increased gamma-EC-B formation to 4% and 25%, respectively, within 60 min. The role of PCS under simultaneous heavy metal stress was confirmed by investigation of different pcs1 null-mutants. In the absence of elevated heavy metal concentrations glutathione-conjugates are therefore first sequestered to the vacuole and subsequently degraded with the initial breakdown step being rate-limiting.

Aminoacyltransferases↗
PubMed2006

Analysis of glutathione and glutathione disulfide in human saliva using hydrophilic interaction chromatography with mass spectrometry.

A sensitive method for the determination of glutathione (GSH) and glutathione disulfide (GSSG) in human saliva was developed and validated. GSH was captured and stabilized by the addition of N-ethylmaleimide (NEM). Solid-phase extraction (SPE) using an Oasis MAX extraction cartridge was employed for sample preparation and analysis was performed on a Shimadzu LCMS-2010 A that was operated in the single ion monitoring mode using positive ion electrospray ionization (ESI) as the interface. The monitored ion for GSH-NEM was m/z 433 and that for GSSG was m/z 613. Chromatography was carried out on an Atlantis HILIC silica column (150 mm x 2.1 mm, 5 microm) with acetonitrile and formate buffer as the mobile phase at the flow rate of 0.2 ml/min. The calibration curve was linear over the range of 0.1-100 microM for GSH-NEM. The extraction recoveries of GSH-NEM spiked at concentrations of 25 and 50 microM were 97.1 and 104.4%, respectively. Similar results were obtained for GSSG. The newly developed hydrophilic interaction chromatography with mass spectrometry (HILIC/MS) method showed superior sensitivity for the determination of GSH and GSSG in human saliva samples.

Calibration↗
PubMed2004

Decrease in glutathione content in boar sperm after cryopreservation. Effect of the addition of reduced glutathione to the freezing and thawing extenders.

Although glutathione content in boar spermatozoa has been previously reported, the effect of reduced glutathione (GSH) on semen parameters and the fertilizing ability of boar spermatozoa after cryopreservation has never been evaluated. In this study, GSH content was determined in ejaculated boar spermatozoa before and after cryopreservation. Semen samples were centrifuged and GSH content in the resulting pellet monitored spectrophotometrically. The fertilizing ability of frozen-thawed boar sperm was also tested in vitro by incubating sperm with in vitro matured oocytes obtained from gilts. GSH content in fresh semen was 3.84 +/- 0.21 nM GSH/10(8) sperm. Following semen cryopreservation, there was a 32% decrease in GSH content (P < 0.0001). There were significant differences in sperm GSH content between different boars and after various preservation protocols (P = 0.0102 ). The effect of addition of GSH to the freezing and thawing extenders was also evaluated. Addition of 5 mM GSH to the freezing extender did not have a significant effect on standard semen parameters or sperm fertilizing ability after thawing. In contrast, when GSH was added to the thawing extender, a dose-dependent tendency to increase in sperm fertilizing ability was observed, although no differences were observed in standard semen parameters. In summary, (i) there was a loss in GSH content after cryopreservation of boar semen; (ii) addition of GSH to the freezing extender did not result in any improvement in either standard semen parameters or sperm fertilizing ability; and (iii) addition of GSH to the thawing extender resulted in a significant increase in sperm fertilizing ability. Nevertheless, future studies must conclude if this is the case for all boars. Furthermore, since addition of GSH to the thawing extender did not result in an improvement in standard semen parameters, this suggests that during the thawing process, GSH prevents damage of a sperm property that is critical in the fertilization process but that is not measured in the routine semen analysis.

Animals↗
PubMed1997

Human mitochondrial import receptor, Tom20p. Use of glutathione to reveal specific interactions between Tom20-glutathione S-transferase and mitochondrial precursor proteins.

The cytosolic domain of the human mitochondrial protein import receptor, hTom20, has been expressed as a fusion protein with glutathione S-transferase (GST) in bacteria and the purified protein immobilized on Sepharose beads. To discriminate between specific binding of precursor proteins with the receptor and non-specific binding, precursors were recovered as a complex with GST-hTom20 following competitive elution from the beads with reduced glutathione. Here, we describe the specificity of this assay and demonstrate that the cytosolic domain of hTom20 interacts directly with the transcription-translation product of precursor proteins that bear a diverse array of targeting signals. Such proteins include a matrix protein (pODHFR), a polytopic integral protein of the inner membrane (uncoupling protein), a beta-barrel protein of the outer membrane (VDAC/porin) as well as bitopic integral proteins which are inserted into the outer membrane by either an NH2-terminal or COOH-terminal signal anchor sequence (yTom70(1-29)DHFR and Bcl-2, respectively).

Cloning, Molecular↗
PubMed1999

Affinity purification of Schistosoma japonicum glutathione-S-transferase and its site-directed mutants with glutathione affinity chromatography and immobilized metal affinity chromatography.

A C-terminally polyhistidine-tagged protein of Schistosoma japonicum glutathione-S-transferase, named as SjGST/His, and its Cys85-->Ser, Cys138-->Ser, and Cys178-->Ser site-directed mutants were prepared and highly expressed in Escherichia coli. Both immobilized metal affinity chromatography (IMAC) and glutathione (GSH) affinity chromatography were used to purify these four enzymes. All of them were purified with equal efficiency by Ni2+-chelated nitrilotriacetic acid agarose gel, but not by GSH Sepharose 4B gel. The protein amounts of wild-type and Cys85-->Ser enzymes purified by the latter gel were three to seven-fold greater than those of the other two enzymes purified by the same gel, while their specific activities were two-fold lower, presumably because of the occurrence of noncovalent aggregation. Both purification methods yielded highly pure enzymes, while there were minor amounts of inter- and intra-disulfide forms in the IMAC purified enzymes except for the Cys85-->Ser mutant. Addition of dithiothreitol to GSH-affinity purified enzymes shifted all of their mass spectra of matrix-assisted laser desorption/ionization-time of flight mass spectrometry toward low molecular-mass regions, while addition of GSH to IMAC purified enzymes shifted the spectra toward high molecular-mass regions. The shift values of wild-type enzyme were larger than those of the three mutants, indicating that the Cys85, Cys138, and Cys178 residues were S-thiolated by GSH during the GSH-affinity purification. This result was confirmed by isoelectric focusing. These findings suggest that IMAC is more efficient than the conventional GSH-affinity system for the purification of SjGST/His enzyme, especially for its mutants and fusion proteins.

Animals↗