Impact of uranium (U) on the cellular glutathione pool and resultant consequences for the redox status of U

Uranium (U) as a redox-active heavy metal can cause various redox imbalances in plant cells. Measurements of the cellular glutathione/glutathione disulfide (GSH/GSSG) by HPLC after cellular U contact revealed an interference with this essential redox couple. The GSH content remained unaffected by 10 μM U whereas the GSSG level immediately increased. In contrast, higher U concentrations (50 μM) drastically raised both forms. Using the Nernst equation, it was possible to calculate the half-cell reduction potential of 2GSH/GSSG. In case of lower U contents the cellular redox environment shifted towards more oxidizing conditions whereas the opposite effect was obtained by higher U contents. This indicates that U contact causes a consumption of reduced redox equivalents. Artificial depletion of GSH by chlorodinitrobenzene and measuring the cellular reducing capacity by tetrazolium salt reduction underlined the strong requirement of reduced redox equivalents. An additional element of cellular U detoxification mechanisms is the complex formation between the heavy metal and carboxylic functionalities of GSH. Because two GSH molecules catalyze electron transfers each with one electron forming a dimer (GSSG) two UO₂ ²⁺ are reduced to each UO₂ ⁺ by unbound redox sensitive sulfhydryl moieties. UO₂ ⁺ subsequently disproportionates to UO₂ ²⁺ and U⁴⁺. This explains that in vitro experiments revealed a reduction to U(IV) of only around 33% of initial U(VI). Cellular U(IV) was transiently detected with the highest level after 2 h of U contact. Hence, it can be proposed that these reducing processes are an important element of defense reactions induced by this heavy metal.     

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.     

Vascular Oxidant Stress and Hepatic Ischemia/Reperfusion Injury

The objective of this study was to test the hypothesis that the extracellular oxidation of glutathione (GSH) may represent an important mechanism to limit hepatic ischemia/reperfusion injury in male Fischer rats in vivo. Basal plasma levels of glutatione disulfide (GSSG: 1.5 ± 0.2μM GSH-equivalents), glutathione (GSH: 6.2 ± 0.4 μM) and alanine aminotransferase activities (ALT 12 ± 2U/I) were significantly increased during the l h reperfusion period following l h of partial hepatic no-flow ischemia (GSSG: 19.7 ± 2.2μM; GSH 36.9 ± 7.4μM; ALT: 2260 ± 355 U/l). Pretreatment with 1,3-bis-(2-chloroethyl)-I-nitrosourea (40mg BCNU/kg), which inhibited glutathione reductase activity in the liver by 60%. did not affect any of these parameters. Biliary GSSG and GSH efflux rates were reduced and the GSSG-to-GSH ratio was not altered in controls and BCNU-treated rats at any time during ischemia and reperfusion. A 90% depletion of the hepatic glutathione content by phorone treatment (300 mg/kg) reduced the increase of plasma GSSG levels by 54%, totally suppressed the rise of plasma GSH concentrations and increased plasma ALT to 4290 ± 755 U/I during reperfusion. The data suggest that hepatic glutathione serves to limit ischemialreperfusion injury as a source of extracellular glutathione, not as a cofactor for the intracellular enzymatic detoxification of reactive oxygen species.     

Glutaredoxin 2 catalyzes the reversible oxidation and glutathionylation of mitochondrial membrane thiol proteins: implications for mitochondrial redox regulation and antioxidant DEFENSE

The redox poise of the mitochondrial glutathione pool is central in the response of mitochondria to oxidative damage and redox signaling, but the mechanisms are uncertain. One possibility is that the oxidation of glutathione (GSH) to glutathione disulfide (GSSG) and the consequent change in the GSH/GSSG ratio causes protein thiols to change their redox state, enabling protein function to respond reversibly to redox signals and oxidative damage. However, little is known about the interplay between the mitochondrial glutathione pool and protein thiols. Therefore we investigated how physiological GSH/GSSG ratios affected the redox state of mitochondrial membrane protein thiols. Exposure to oxidized GSH/GSSG ratios led to the reversible oxidation of reactive protein thiols by thiol-disulfide exchange, the extent of which was dependent on the GSH/GSSG ratio. There was an initial rapid phase of protein thiol oxidation, followed by gradual oxidation over 30 min. A large number of mitochondrial proteins contain reactive thiols and most of these formed intraprotein disulfides upon oxidation by GSSG; however, a small number formed persistent mixed disulfides with glutathione. Both protein disulfide formation and glutathionylation were catalyzed by the mitochondrial thiol transferase glutaredoxin 2 (Grx2), as were protein deglutathionylation and the reduction of protein disulfides by GSH. Complex I was the most prominent protein that was persistently glutathionylated by GSSG in the presence of Grx2. Maintenance of complex I with an oxidized GSH/GSSG ratio led to a dramatic loss of activity, suggesting that oxidation of the mitochondrial glutathione pool may contribute to the selective complex I inactivation seen in Parkinson's disease. Most significantly, Grx2 catalyzed reversible protein glutathionylation/deglutathionylation over a wide range of GSH/GSSG ratios, from the reduced levels accessible under redox signaling to oxidized ratios only found under severe oxidative stress. Our findings indicate that Grx2 plays a central role in the response of mitochondria to both redox signals and oxidative stress by facilitating the interplay between the mitochondrial glutathione pool and protein thiols.     

Seizure-induced changes in mitochondrial redox status

The aim of this study was to determine seizure-induced oxidative stress by measuring hippocampal glutathione (GSH) and glutathione disulfide (GSSG) levels in tissue and mitochondria. Kainate-induced status epilepticus (SE) in rats resulted in a time-dependent decrease of GSH/GSSG ratios in both hippocampal tissue and mitochondria. However, changes in GSH/GSSG ratios were more dramatic in the mitochondrial fractions compared to hippocampal tissue. This was accompanied by a mild increase in glutathione peroxidase activity and a decrease in glutathione reductase activity in hippocampal tissue and mitochondria, respectively. Since coenzyme A (CoASH) and its disulfide with GSH (CoASSG) are primarily compartmentalized within mitochondria, their measurement in tissue was undertaken to overcome problems associated with GSH/GSSG measurement following subcellular fractionation. Hippocampal tissue CoASH/CoASSG ratios were decreased following kainate-induced SE, the time course and magnitude of change paralleling mitochondrial GSH/GSSG levels. Cysteine, a rate-limiting precursor of glutathione was decreased following kainate administration in both hippocampal tissue and mitochondrial fractions. Together these changes in altered redox status provide further evidence for seizure-induced mitochondrial oxidative stress.     

Properties and physiological function of a glutathione reductase purified from spinach leaves by affinity chromatography

Glutathione reductase (EC 1.6.4.2) was purified from spinach (Spinacia oleracea L.) leaves by affinity chromatography on ADP-Sepharose. The purified enzyme has a specific activity of 246 enzyme units/mg protein and is homogeneous by the criterion of polyacrylamide gel electrophoresis on native and SDS-gels. The enzyme has a molecular weight of 145,000 and consists of two subunits of similar size. The pH optimum of spinach glutathione reductase is 8.5–9.0, which is related to the function it performs in the chloroplast stroma. It is specific for oxidised glutathione (GSSG) but shows a low activity with NADH as electron donor. The pH optimum for NADH-dependent GSSG reduction is lower than that for NADPH-dependent reduction. The enzyme has a low affinity for reduced glutathione (GSH) and for NADP⁺, but GSH-dependent NADP⁺ reduction is stimulated by addition of dithiothreitol. Spinach glutathione reductase is inhibited on incubation with reagents that react with thiol groups, or with heavymetal ions such as Zn²⁺. GSSG protects the enzyme against inhibition but NADPH does not. Pre-incubation of the enzyme with NADPH decreases its activity, so kinetic studies were performed in which the reaction was initiated by adding NADPH or enzyme. The Km for GSSG was approximately 200 M and that for NADPH was about 3 M. NADP⁺ inhibited the enzyme, assayed in the direction of GSSG reduction, competitively with respect to NADPH and non-competitively with respect to GSSG. In contrast, GSH inhibited non-competitively with respect to both NADPH and GSSG. Illuminated chloroplasts, or chloroplasts kept in the dark, contain equal activities of glutathione reductase. The kinetic properties of the enzyme (listed above) suggest that GSH/GSSG ratios in chloroplasts will be very high under both light and dark conditions. This prediction was confirmed experimentally. GSH or GSSG play no part in the light-induced activation of chloroplast fructose diphosphatase or NADP⁺-glyceraldehyde-3-phosphate dehydrogenase. We suggest that GSH helps to stabilise chloroplast enzymes and may also play a role in removing H₂O₂. Glucose-6-phosphate dehydrogenase activity may be required in chloroplasts in the dark in order to provide NADPH for glutathione reductase.Abbreviations GSH reduced form of the tripeptide glutathione - GSSG oxidised form of glutathione     

Expression, purification, and in vitro refolding of a humanized single-chain Fv antibody against human CTLA4 (CD152)

A human-derived single-chain Fv (scFv) antibody fragment specific against human CTLA4 (CD152) was produced at high level in Escherichia coli. The scFv gene was cloned from a phagemid to the expression vector pQE30 with a N-terminal 6His tag fused in-frame, and expressed as a 29 kDa protein in E. coli as inclusion bodies. The inclusion body of scFv was isolated from E. coli lysate, solubilized in 8M urea with 10mM dithiothreitol, and purified by ion-exchange chromatography. Method for in vitro refolding of the scFv was established. The effects of refolding buffer composition, protein concentration and temperature on the refolding yield were investigated. The protein was renatured finally by dialyzing against 3mM GSH, 1mM GSSG, 150 mM NaCl, 1M urea, and 50 mM Tris-Cl (pH 8.0) for 48 h at 4 degrees C, and then dialyzed against phosphate-buffered saline (pH 7.4) to remove remaining denaturant. This refolding protocol generated up to a 70% yield of soluble protein. Soluble scFv was characterized for its specific antigen-binding activity by indirect cellular ELISA. The refolded scFv was functionally active and was able to bind specifically to CTLA4 (CD152). The epitopes recognized by refolded anti-CTLA4 scFv do not coincide with those epitopes recognized by CD80/CD86.     

人源溶菌酶在大肠杆菌中的表达与复性研究

人源溶菌酶(Human lysozyme,HLZ)是一种糖苷水解酶,具有抗菌消炎的作用,其作为抗生素的替代品,已经被广泛应用于食品业、畜牧业和医疗等领域。如何获得高产量、高活性、高纯度的人源溶菌酶一直是亟待解决的技术问题。优化人源溶菌酶编码基因密码子,提高其在大肠杆菌中的适应度和表达量;将优化的基因克隆至大肠杆菌表达质粒pET21a,并将其在大肠杆菌表达菌株BL21(DE3)中诱导表达;利用8 mol/L尿素溶液对包涵体进行溶解变性后,探究一步透析、梯度透析和梯度稀释3种复性方式以及复性液中谷胱甘肽氧化还原对(GSSG/GSH)、精氨酸、甘油等复性物的浓度对重组人源溶菌酶复性的效果,获得最佳的复性方案。研究结果表明:37℃诱导温度下,利用0.5 mmol/L IPTG成功诱导了分子量约为14.7 kD的重组人源溶菌酶的表达,包涵体表达量约为380 mg/L(湿重)。包涵体经一步透析、梯度透析和梯度稀释3种复性方式复性后,测得比活力值分别为147 U/mg、335 U/mg、176 U/mg,表明最佳复性方法为梯度透析复性法。进一步探索了复性液中GSSG/GSH比值、精氨酸浓度、甘油浓度对人源溶菌酶复性效果的影响,表明当复性液中同时添加浓度比为1∶2的GSSG/GSH、4 mmol/L精氨酸和6%甘油时,复性后人源溶菌酶的最佳比活力值为1170 U/mg,显著高于3种复性物均不加时溶菌酶335 U/mg的比活力值,但低于溶菌酶标准品1732 U/mg的比活力值。成功地将人源溶菌酶基因在大肠杆菌中表达,并通过包涵体复性体系成功获得高活性重组人源溶菌酶。     

Effects of menadione and hydrogen peroxide on glutathione status in growing Escherichia coli

Menadione (MD) and H2O2 caused distinct effects on glutathione status in growing Escherichia coli. Treatment of E. coli AB1157 with 1-25 mM H2O2 did not result in an appreciable decrease in intracellular total glutathione (reduced glutathione [GSH] + oxidized glutathione [GSSG]). Only when cells were treated with 25 mM H2O2 an increase in GSSG and a decrease in the GSH:GSSG ratio were observed. In cells deficient in catalase HPI, such effect was observed even at 10 mM H2O2. The exposure of E. coli AB1157 to MD caused a dose-dependent decrease in intracellular total glutathione, an increase in GSSG, and a decrease in the ratio of GSH:GSSG. In E. coli deficient in cytosolic superoxide dismutase activity, a decrease in total glutathione after incubation with 0.2 mM MD was not accompanied by an increase in GSSGin, and the ratio of GSHin:GSSGin was three times higher than in the wild-type cells. The changes in the redox status of extracellular glutathione under the action of both oxidants were similar. Although the catalase activity increased several times after exposure to both oxidants, there were little or no changes in the activity of enzymes related to glutathione metabolism. A possible role of changes in redox status of glutathione under oxidative stress is discussed.     

Characterization of Glutathione Uptake in Broad Bean Leaf Protoplasts

Transport of reduced glutathione (GSH) and oxidized glutathione (GSSG) was studied with broad bean (Vicia faba L.) leaf tissues and protoplasts. Protoplasts and leaf discs took up GSSG at a rate about twice the uptake rate of GSH. Detailed studies with protoplasts indicated that GSH and GSSG uptake exhibited the same sensitivity to the external pH and to various chemical reagents. GSH uptake was inhibited by GSSG and glutathione conjugates. GSSG uptake was inhibited by GSH and GS conjugates, and the uptake of metolachlor-GS was inhibited by GSSG. Various amino acids (L-glutamic acid, L-glutamine, L-cysteine, L-glycine, L-methionine) and peptides (glycine-glycine, glycine-glycine-glycine) affected neither the transport of GSH nor GSSG. Uptake kinetics indicate that GSH is taken up by a single saturable transporter, with an apparent Km of 0.4 mM, whereas GSSG uptake exhibits two saturable phases, with an apparent Km of 7 [mu]M and 3.7 mM. It is concluded that the plasma membrane of leaf cells contains a specific transport system for glutathione, which takes up GSSG and GS conjugates preferentially over GSH. Proton flux measurements and electrophysiological measurements indicate that GSH and GSSG are taken up with proton symport. However, a detailed analysis of these measurements suggests that the ion movements induced by GSSG differ from those induced by GSH.     

Depression of biliary glutathione excretion by chronic ethanol feeding in the rat

The effects of chronic alcohol feeding on biliary glutathione excretion were studied in rats pair fed diets containing either ethanol (36% of total energy) or isocaloric carbohydrate for 4-6 weeks. An exteriorized biliary-duodenal fistula was established and total glutathione (GSH) and oxidized glutathione (GSSG) were measured. A significant decrease was observed in rats fed alcohol chronically compared to their pair fed controls in the biliary excretion of GSH (55.7 +/- 37.0 vs 243.1 +/- 29.0 micrograms/ml bile, p less than 0.025) as well as biliary GSSG (12.5 +/- 5.0 vs 49.9 +/- 8.0 micrograms/ml bile, p less than 0.05) and in bile flow (23.1 +/- 1.6 vs 29.2 +/- 1.3 micrograms/min, p less than 0.05). An acute dose of ethanol tended to exaggerate the decrease on biliary GSH and GSSG in the two groups of animals. The depression in biliary GSH could not be attributed to decreased GSH synthesis since S35-L-methionine incorporation into hepatic and biliary GSH was unchanged or even increased after chronic ethanol feeding.     

Expression of proteins containing disulfide bonds in an insect cell-free system and confirmation of their arrangements by MALDI-TOF MS

Escherichia coli alkaline phosphatase (AP) and human lysozyme (h-LYZ), which contain two and four disulfide bonds, respectively, were expressed in a cell-free protein synthesis system constructed from Spodoptera frugiperda 21 (Sf21) cells. AP was expressed in a soluble and active form using the insect cell-free system under non-reducing conditions, and h-LYZ was expressed in a soluble and active form under non-reducing conditions after addition of reduced glutathione (GSH), oxidized glutathione (GSSG), and protein disulfide isomerase (PDI). The in vitro synthesized proteins were purified by means of a Strep-tag attached to their C termini. Approximately 41 microg AP and 30 microg h-LYZ were obtained from 1 mL each of the reaction mixture. The efficiency of protein synthesis approached that measured under reducing conditions. Analysis of the disulfide bond arrangements by MALDI-TOF MS showed that disulfide linkages identical to those observed in the wild-type proteins were formed.     

Expression, purification, and refolding of a novel immunotoxin containing humanized single-chain fragment variable antibody against CTLA4 and the N-terminal fragment of human perforin

Immunotoxins might be potential in treatment of cancer for their ability to kill selected cell populations. We constructed a novel immunotoxin hS83P34 by fusing N-terminal 34 amino acid fragment of human perforin to the C-terminus of humanized single-chain fragment variable antibody against CTLA4. The fusion protein was inductively expressed as inclusion bodies at a high level about 30% of total bacterial proteins. After washing with buffer containing 2 M urea, the purity of inclusion body was about 71%. The washed inclusion bodies were solubilized in 8 M urea and further purified to homogeneity (approximately 92% purity) by cation-exchange chromatography and Ni-agarose affinity chromatography under denaturing condition. The inclusion body refolding conditions were optimized following Pro-Matrix Protein Refolding Guide. After refolded in Tris buffer (pH 8.0) containing 1M urea, 0.8 M l-arginine, and 2 mM GSH:0.2 mM GSSG or 2 mM GSH:0.4 mM GSSG for 18h at 4 degrees C, over 90% proteins were recovered from inclusion bodies. In vitro dose-dependent cytotoxicity assay demonstrates that hS83P34 is only toxic to CTLA4-positive cells. IC(50) of hS83P34 for leukemic cells Raji and 6T-CEM are about 0.85 and 1.3 microM individually. Whereas, CTLA4-negative endothelial cell ECV-304 is resistant to hS83P34.     

In vivo and in vitro oxidative regulation of rat aryl sulfotransferase IV (AST IV)

Sulfotransferase catalyzed sulfation is important in the regulation of different hormones and the metabolism of hydroxyl containing xenobiotics. In the present investigation, we examined the effects of hyperoxia on aryl sulfotransferase IV in rat lungs in vivo. The enzyme activity of aryl sulfotransferase IV increased 3- to 8-fold in >95% O2 treated rat lungs. However, hyperoxic exposure did not change the mRNA and protein levels of aryl sulfotransferase IV in lungs as revealed by Western blot and RT-PCR. This suggests that oxidative regulation occurs at the level of protein modification. The increase of nonprotein soluble thiol and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios in treated lung cytosols correlated well with the aryl sulfotransferase IV activity increase. In vitro, rat liver cytosol 2-naphthol sulfation activity was activated by GSH and inactivated by GSSG. Our results suggest that Cys residue chemical modification is responsible for the in vivo and in vitro oxidative regulation. The molecular modeling structure of aryl sulfotransferase IV supports this conclusion. Our gel filtration chromatography results demonstrated that neither GSH nor GSSG treatment changed the existing aryl sulfotransferase IV dimer status in cytosol, suggesting that oxidative regulation of aryl sulfotransferase IV is not caused by dimer-monomer status change.     

Effects of Exogenous Proline and Glycinebetaine on the Salt Tolerance of Rice Cultivars

Glutathione reductase was purified from iron-grown Thiobacillus ferrooxidas AP19-3 to an electrophoretically homogeneous state. The enzyme had an apparent molecular weight of 100,000 and was composed of two identical subunits of molecular weight (M_rs, 52,000) as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. A purified enzyme reduced one mole of the oxidized form of glutathione (GSSG) with one mole of NADPH to produce two moles of the reduced form of glutathione (GSH) and one mole of NADP⁺. The glutathione reductase was most active at pH 6.5 and 40°C, and had an isoelectric point at 5.1. The Michaelis constants of glutathione reductase for GSSG, NADPH, and NADH were 300, 26, and 125 μM, respectively.     

Effects of sulfite on glutathione S-sulfonate and the glutathione status of lung cells

A mechanistic study was performed to elucidate the biochemical events connected with the cocarcinogenic effect of sulfur dioxide (SO2). Glutathione S-sulfonate (GSSO3H), a competitive inhibitor of the glutathione S-transferases, forms in lung cells exposed in culture to sulfite, the hydrated form of SO2. Changes in glutathione status (total GSH) were also observed during a 1-h exposure. Some cells were pretreated with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to inhibit glutathione reductase. In human lung cells GSSO3H formed in a concentration-dependent manner, while glutathione (GSH) increased and glutathione disulfide (GSSG) decreased as the extracellular sulfite concentration was increased from 0 to 20 mM. The ratio of GSH/GSSG increased greater than 5-fold and the GSH/GSSO3H ratio decreased to 10 with increasing sulfite concentration. GSSO3H formed in rat lung cells exposed to sulfite, with no detectable effect on GSH and GSSG. GSSO3H also formed from cellular GSH mixed disulfides. GSSO3H formed rapidly, reaching its maximum value in 15 min. The viability of both cell types was unaffected except at 20 mM sulfite. GSSO3H incubated with human lung cells did not affect cellular viability. BCNU inhibited cellular GSSO3H reductase to the same extent as GSSG reductase. These results indicate that GSSO3H is formed in cells exposed to sulfite, and could be the active metabolite of sulfite responsible for the cocarcinogenic effect of SO2 by inhibiting conjugation of electrophiles by GSH.     

Expression, purification, and characterization of an immunotoxin containing a humanized anti-CD25 single-chain fragment variable antibody fused to a modified truncated Pseudomonas exotoxin A

Vascular leak syndrome (VLS) is the major dose-limiting toxicity of immunotoxin therapy. In our previous study, a modified PE38KDEL, denoted PE38KDELKQK, was engineered to eliminate VLS. The PE38KDELKQK-based immunotoxin has been proved to retain potent anti-tumor activity but with a remarkable attenuation in VLS. In the present study, we have constructed and expressed a recombinant immunotoxin CD25-PE38KDELKQK containing humanized anti-CD25 single-chain antibody (scFv) genetically fused to PE38KDELKQK in Escherichia coli. After washing with buffer containing 2 M urea, the purity of inclusion body was about 82%. The denatured inclusion bodies were refolded on-column in Tris buffer (pH 8.0) containing 4mM of GSH and 1 mM of GSSG using a gradient of decreasing urea. We found that the presence of GSH/GSSG (4:1) in the on-column refolding buffer was important for efficient refolding. In addition, slow flow rate was another important factor could increase refolding. Under these conditions, the activity of the refolded protein could reach about 90% of that of the native protein. The refolded proteins were purified to homogeneity ( approximately 95% purity) by a combination of His-Ni(2+) metal affinity chromatography and gel filtration chromatography. The in vitro cytotoxicity assay indicated the purified immunotoxin CD25-PE38KDELKQK had specific cytotoxicity to CD25-positive leukemic cells comparable to wild-type CD25-PE38KDEL (wt). In contrast, CD25-PE38KDELKQK was shown to be much weaker in inducing VLS in mice than wt. The protein expression, purification, and refolding system established in this paper is important for further study on immunotoxin CD25-PE38KDELKQK.     

Cellular effects of N(4-ethoxyphenyl)p-benzoquinone imine, a p-phenetidine metabolite formed during peroxidase reactions

The interaction of N-(4-ethoxyphenyl)p-benzoquinone imine (NEPBQI), a metabolite formed during peroxidase catalyzed metabolism of p-phenetidine, with GSH and its effects in isolated rat hepatocytes were investigated.

When reacted with GSH NEPBQI formed both a mono- and a diglutathione conjugate as well as GSSG. Formation of glutathione conjugates and GSSG also occurred when NEPBQI was added to isolated hepatocytes. The formation of GSSG was, however, only detectable if the hepatocytes had been pretreated with the GSSG reductase inhibitor BCNU (1,3-bis-(2-chloroethyl-1-nitrosourea).

Similarly, NEPBQI caused a rapid decrease in cellular free protein thiols when added to hepatocytes, however this was expressed at higher concentrations than for effects on GSH. The protein thiol decrease was correlated with protein binding of NEPBQI.

NEPBQI was also shown to be toxic to isolated hepatocytes. At a concentration of 400 μM extensive bleb formation was followed by loss of cell membrane integrity and cell death.

To assess further the subcellular metabolism of NEPBQI microsomes and cytosol was used. NEPBQI was found to be preferentially reduced by cytochrome P-450 reductase in the microsomes whereas DT-diaphorase catalyzed its reduction in cytosol. NEPBQI did not undergo significant redox cycling since no formation of O was observed. Thus, the cytotoxic effect of NEPBQI appears to be due to protein arylation rather than redox cycling.     

Upregulation of gamma-glutamate-cysteine ligase as part of the long-term adaptation process to iron accumulation in neuronal SH-SY5Y cells

Reactive iron is an important prooxidant factor, whereas GSH is a crucial component of a long-term adaptive system that allows cells to function during extended periods of high oxidative stress. In this work, the adaptive response of the GSH system to prolonged iron loads was characterized in human dopaminergic SH-SY5Y neuroblastoma cells. After the initial death of a substantial portion of the cell population, the surviving cells increased their GSH content by up to fivefold. This increase was traced to increased expression of the catalytic and modulatory subunits of -glutamate-cysteine ligase. Under conditions of high iron load, cells maintained a low GSSG content through two mechanisms: 1) GSSG reductase-mediated recycling of GSSG to GSH and 2) multidrug resistant protein 1-mediated extrusion of GSSG. Increased GSH synthesis and low GSSG levels contributed to recover the cell reduction potential from –290 mV at the time of cell death to about –320 mV. These results highlight the fundamental role of GSH homeostasis in the antioxidant response to cellular iron accumulation and provide novel insights into the adaptive mechanisms of neurons subjected to increased iron loads, such as those observed in Parkinson's disease. oxidative stress; glutathione; multidrug resistance protein 1; oxidixed glutathione reductase; neurodegenerative diseases     

Nonenzymic reactivation of reduced bovine pancreatic ribonuclease by air oxidation and by glutathione oxidoreduction buffers.

With the glutathione system that leads to rapid regeneration of reduced lysozyme (Saxena, V. P., and Wetlaufer, D. B. (1971) Biochemistry 9, 5015), reduced pancreatic ribonuclease (RNase) regenerated activity in high yield (greater than 90%) but at a considerably lower rate (t1/2 approximately 75 min). Systematic examination of the effects upon regeneration of the concentrations and ratios of reduced and oxidized glutathione (GSH and GSSG) showed the same broad optima for RNase as were earlier found for lysozyme: [GSSG] = 5 X 10(-4) M, [GSH] = 5 X 10(-3) M. Regeneration of reduced RNase by air oxidation was shown to be inhibitable by 10(-4) M EDTA, whereas the glutathione regeneration was unaffected by EDTA. In addition the air-oxidative regeneration showed a strong temperature dependence, in contrast with the glutathione system. The mechanisms of these two kinds of regenerations are therefore different. Six potentially catalytic metal ions were tested in the air-oxidative regeneration of RNase: Cu2+, Co2+, Mn2+, Fe3+, Zn2+, and Ni2+. Of these, only Cu2+ enhanced the rate of regeneration of RNase activity, although both Cu2+ and Co2+ catalyzed thioloxidation of reduced RNase. The rates and yields of RNase regenerations were independent of protein concentration from 3 X 10(-7) M to 1.2 X 10(-5) M in the glutathione system. Preincubation of freshly dissolved reduced RNase under nonoxidizing conditions before adding glutathione did not change the rate or extent of regeneration. Studies of its pH dependence showed that the glutathione regeneration depends on the deprotonation of prototropic groups with 7.5 less than pK less than 8.0. The major ion exchange chromatographic peaks from glutathione and air-oxidative regenerations appeared to be identical with native RNase, by the criteria of specific activity, chromatographic mobility, and circular dichroic spectra. The glutathione system permits regeneration at much higher RNase concentration than the air regeneration, with rates and yields comparable to the greatest reported for air regeneration.