Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: dependence of the rate on the composition of the redox buffer

The velocity of the oxidative renaturation of reduced ribonuclease A catalyzed by protein disulfide isomerase (PDI) is strongly dependent on the composition of a glutathione/glutathione disulfide redox buffer. As with the uncatalyzed, glutathione-mediated oxidative folding of ribonuclease, the steady-state velocity of the PDI-catalyzed reaction displays a distinct optimum with respect to both the glutathione (GSH) and glutathione disulfide (GSSG) concentrations. Optimum activity is observed at [GSH] = 1.0 mM and [GSSG] = 0.2 mM. The apparent kcat at saturating RNase concentration is 0.46 +/- 0.05 mumol of RNase renatured min-1 (mumol of PDI)-1 compared to the apparent first-order rate constant for the uncatalyzed reaction of 0.02 +/- 0.01 min-1. Changes in GSH and GSSG concentration have a similar effect on the rate of both the PDI-catalyzed and uncatalyzed reactions except under the more oxidizing conditions employed, where the catalytic effectiveness of PDI is diminished. The ratio of the velocity of the catalyzed reaction to that of the uncatalyzed reaction increases as the quantity [GSH]2/[GSSG] increases and approaches a constant, limiting value at [GSH]2/[GSSG] greater than 1 mM, suggesting that a reduced, dithiol form of PDI is required for optimum activity. As long as the glutathione redox buffer is sufficiently reducing to maintain PDI in an active form [( GSH]2/[GSSG] greater than 1 mM), the rate acceleration provided by PDI is reasonably constant, although the actual rate may vary by more than an order of magnitude. PDI exhibits half of the maximum rate acceleration at a [GSH]2/[GSSG] of 0.06 +/- 0.01 mM.     

Renaturation of Escherichia coli-derived recombinant human macrophage colony-stimulating factor.

Recombinant human macrophage colony-stimulating factor (rhM-CSF), a homodimeric, disulfide bonded protein, was expressed in Escherichia coli in the form of inclusion bodies. Reduced and denatured rhM-CSF monomers were refolded in the presence of a thiol mixture (reduced and oxidized glutathione) and a low concentration of denaturing agent (urea or guanidinium chloride). Refolding was monitored by nonreducing gel electrophoresis and recovery of bioactivity. The effects of denaturant type and concentration, protein concentration, concentration of thiol/disulfide reagents, temperature, and presence of impurities on the kinetics of rhM-CSF renaturation were investigated. Low denaturant concentrations (<0.5 M urea) and high protein concentrations (>0.4 mg/ml) in the refolding mixture resulted in increased formation of aggregates, although aggregation was never significant even when refolding was carried out at room temperature. Higher protein concentration resulted in higher rates but did not lead to increased yields, due to the formation of unwanted aggregates. Experiments conducted at room temperature resulted in slightly higher rates than those conducted at 4 degrees C. Although the initial renaturation rate for solubilized inclusion body protein without purification was higher than that of the reversed-phase purified reduced denatured rhM-CSF, the final renaturation yield was much higher for the purified material. A maximum refolding yield of 95% was obtained for the purified material at the following refolding conditions: 0.5 M urea, 50 mM Tris, 1.25 mM DTT, 2 mM GSH, 2 mM GSSG, 22 degrees C, pH 8, [protein] = 0.13 mg/ml.     

Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: pre-steady-state kinetics and the utilization of the oxidizing equivalents of the isomerase

At low concentrations of a glutathione redox buffer, the protein disulfide isomerase (PDI) catalyzed oxidative renaturation of reduced ribonuclease A exhibits a rapid but incomplete activation of ribonuclease, which precedes the steady-state reaction. This behavior can be attributed to a GSSG-dependent partitioning of the substrate, reduced ribonuclease, between two classes of thiol/disulfide redox forms, those that can be converted to active ribonuclease at low concentrations of GSH and those that cannot. With catalytic concentrations of PDI and near stoichiometric concentrations of glutathione disulfide, approximately 4 equiv (2 equiv of ribonuclease disulfide) of GSH are formed very rapidly followed by a slower formation of GSH, which corresponds to an additional 2 disulfide bond equiv. The rapid formation of RNase disulfide bonds and the subsequent rearrangement of incorrect disulfide isomers to active RNase are both catalyzed by PDI. In the absence of GSSG or other oxidants, disulfide bond equivalents of PDI can be used to form disulfide bonds in RNase in a stoichiometric reaction. In the absence of a glutathione redox buffer, the rate of reduced ribonuclease regeneration increases markedly with increasing PDI concentrations below the equivalence point; however, PDI in excess over stoichiometric concentrations inhibits RNase regeneration.     

Denaturation-renaturation of the fibrin-stabilizing factor XIII a-chain isolated from human placenta. Properties of the native and reconstituted protein

The denaturation-renaturation transition between the native and unfolded states of the dimeric blood coagulation factor XIIIa has been examined by far-UV circular dichroism, fluorescence spectroscopy, activity measurements, sedimentation equilibrium analysis, and size exclusion high performance liquid chromatography. Guanidine hydrochloride and urea-dependent denaturation in the absence and in the presence of 5mM dithioerythritol or glutathione (5mM GSH) exhibit biphasic transitions. The first stage represents a sharp transition characterized by a change in secondary structure without subunit dissociation. This step is accompanied by the irreversible loss of biological activity. The second transition reflects the dissociation and complete unfolding of the protein to a random coil. After loss of biological activity no reactivation can be accomplished under any of the following conditions: (i) denaturation and renaturation under reducing or non-reducing conditions, (ii) variation of the protein concentration and temperature, (iii) addition of specific ligands (Ca2+, substrate), (iv) presence of stabilizing and/or destabilizing agents. Attempts to renature the protein under standard conditions (0.1 M Tris/HCl pH 7.5-9.0, 5mM DTE, 5mM EDTA) lead to refolding intermediates which exhibit a strong tendency to aggregate. A soluble product of reconstitution can be obtained by refolding at low protein concentration, low temperature, and in the presence of small amounts of destabilizing agents such as arginine or urea in the renaturation buffer at pH 7.5 to 9. The spectroscopic and hydrodynamic characterization of the partially reconstituted (non-native inactive) protein shows that partially reconstituted factor XIIIa exhibits the fluorescence properties and the dimeric structure of the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chromatographic and mass spectrometric analysis of glutathione in biological samples

Biological thiol compounds are classified into high-molecular-mass protein thiols and low-molecular-mass free thiols. Endogenous low-molecular-mass thiol compounds, namely, reduced glutathione (GSH) and its corresponding disulfide, glutathione disulfide (GSSG), are very important molecules that participate in a variety of physiological and pathological processes. GSH plays an essential role in protecting cells from oxidative and nitrosative stress and GSSG can be converted into the reduced form by action of glutathione reductase. Measurement of GSH and GSSG is a useful indicator of oxidative stress and disease risk. Many publications have reported successful determination of GSH and GSSG in biological samples. In this article, we review newly developed techniques, such as liquid chromatography coupled with mass spectrometry and tandem mass spectrometry, for identifying GSH bound to proteins, or for localizing GSH in bound or free forms at specific sites in organs and in cellular locations.     

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.     

Insights into deglutathionylation reactions. Different intermediates in the glutaredoxin and protein disulfide isomerase catalyzed reactions are defined by the gamma-linkage present in glutathione

Glutaredoxins are small proteins with a conserved active site (-CXX(C/S)-) and thioredoxin fold. These thiol disulfide oxidoreductases catalyze disulfide reductions, preferring GSH-mixed disulfides as substrates. We have developed a new real-time fluorescence-based method for measuring the deglutathionylation activity of glutaredoxins using a glutathionylated peptide as a substrate. Mass spectrometric analysis showed that the only intermediate in the reaction is the glutaredoxin-GSH mixed disulfide. This specificity was solely dependent on the unusual gamma-linkage present in glutathione. The deglutathionylation activity of both wild-type Escherichia coli glutaredoxin and the C14S mutant was competitively inhibited by oxidized glutathione, with K(i) values similar to the K(m) values for the glutathionylated peptide substrate, implying that glutaredoxin primarily recognizes the substrate via the glutathione moiety. In addition, wild-type glutaredoxin showed a sigmoidal dependence on GSH concentrations, the activity being significantly decreased at low GSH concentrations. Thus, under oxidative stress conditions, where the ratio of GSH/GSSG is decreased, the activity of glutaredoxin is dramatically reduced, and it will only have significant deglutathionylation activity once the oxidative stress has been removed. Different members of the protein disulfide isomerases (PDI) family showed lower activity levels when compared with glutaredoxins; however, their deglutathionylation activities were comparable with their oxidase activities. Furthermore, in contrast to the glutaredoxin-GSH mixed disulfide intermediate, the only intermediate in the PDI-catalyzed reaction was PDI peptide mixed disulfide.     

High-pressure refolding of disulfide-cross-linked lysozyme aggregates: thermodynamics and optimization

Previous exploratory work revealed that high pressure (200 MPa), in combination with oxido-shuffling agents such as glutathione, effectively refolds covalently cross-linked aggregates of lysozyme into catalytically active native molecules, at concentrations up to 2 mg/mL (1). To understand further and optimize this process, in the current study we varied the redox conditions and levels of guanidine hydrochloride (GdnHCl) in the refolding buffer. Maximum refolding yields of 80% were seen at 1 M GdnHCl; higher concentrations did not increase refolding yields further. A maximum in refolding yield was observed at redox conditions with a 1:1 ratio of oxidized to reduced glutathione (GSSG:GSH). Yields decreased dramatically at more oxidizing conditions ([GSSG] > [GSH]). Kinetics of dissolution and refolding of covalently cross-linked aggregates of lysozyme depended strongly on redox conditions. At GSSG:GSH ratios of 4:1, 1:1, and 1:16, lysozyme dissolved and refolded with time constants of 62, 20, and 8 h, respectively. Estimates of the free energy of unfolding of lysozyme in GdnHCl solutions at 200 MPa suggested that the native state of lysozyme is strongly favored (ca.18.6 kJ/mol) under the conditions used for dissolution and refolding.     

Interaction of menadione (2-methyl-1,4-naphthoquinone) with glutathione

The interaction of menadione with reduced glutathione (GSH) led to a removal of menadione and formation of menadione-GSH conjugate and glutathione disulfide (GSSG). The changes in thiol level were essentially biphasic with an initial rapid decrease in GSH and appearance of GSSG (less than 1 min) followed by secondary less pronounced changes. The interaction of menadione and GSH caused an oxygen uptake and both superoxide anion radical and hydrogen peroxide were produced during the reaction, the amount dependent on the GSH/menadione ratio. Catalase did not protect against the initial decrease in GSH level but markedly inhibited the secondary changes while superoxide dismutase had little effect. These results suggest that the initial changes in thiol level are the result in part of a redox reaction between menadione and GSH as well as conjugate formation, whilst the secondary changes reflect conjugate formation and the activity of other oxidants such as hydrogen peroxide. The potential biological significance of this reaction was investigated using hepatocytes depleted of reduced pyridine nucleotides and thus not able to perform enzyme-catalyzed reduction of menadione. In these cells menadione induced GSSG formation at a rate similar to that observed in control cells. This suggests that quinone-induced oxidative challenge caused by the chemical interactions of a quinone and glutathione may have biological relevance.     

Solid-phase synthesis of huwentoxin-I and its structure and bioactivity analysis

Huwentoxin-I, a neurotoxic peptide from the spider Selenocosmia huwena, was synthesized by sol-id-phase method with Fluorenylmethoxycarbonyl amino acid pentafluorophenyl esters (Fmoc-AA-OPfp). The carboxyl and the hydroxy groups were protected by tBu; the side chains of Lys and His were protected by Roc; the guanidine group of Arg was protected by Mtr and the mercaptan group of Cys was protected by Trt. The solid-phase carrier was ethylene diamine-polyethylene-polystyrene (DEA-PEG-PS) resin. The synthetic peptide was cleaved from the resin and deprotected by a 90% TFA solution containing 5% thioanisole, 3% ethanedithiol and 2% anisole. The product was reduced with DTT and then incubated with GSSG and GSH to form the correct disulfide bond linkages. The syn-thetic peptide was purified by HPLC and then characterized by amino acid composition and sequence analysis, peptide mapping and NMR. The biological activity of the synthetic product was tested by electrophysiological method using the isolated mouse ph     

Influence of glutathione on the reactivation of enzymes containing cysteine or cystine

Refolding of dimeric porcine cytosolic or mitochondrial malate dehydrogenases and of tetrameric pig heart and skeletal muscle lactate dehydrogenases (containing 5-7 cysteine residues), as well as reformation of the four cystine cross-bridges of bovine pancreatic ribonuclease, were studied in the presence of reduced and oxidized glutathione (GSH and GSSG). At the intracellular GSH level (5 mM) reduced ribonuclease can be reoxidized by 0.01-0.5 mM GSSG (pH 7.4) both at 20 degrees C and 37 degrees C. In this physiological range of GSSG concentrations and pH, the dehydrogenases show at least partial reactivation. With GSSG concentrations greater than 5 mM, reactivation is found to be completely inhibited for all the enzymes given. The results show that at the intracellular level of GSH and GSSG, thiol groups in reduced, unfolded ribonuclease are oxidized to form intramolecular cystine cross-bridges, while thiol groups of typical cysteine enzymes, such as lactate and malate dehydrogenase, remain in their reduced state during refolding. The rate of reactivation of lactate dehydrogenase (porcine muscle) is not affected by GSSG. In the case of ribonuclease, increasing concentrations of GSSG increase the rate of reactivation: At 20 degrees C, the halftime of the correct disulfide bond formation varies from approximately equal to 80 h in the presence of 0.01 mM GSSG to approximately equal to 10 h in the presence of 0.25 mM GSSG. A further increase in the rate of reactivation at higher GSSG concentrations is accompanied by a decrease in yield. Reactivation of ribonuclease is also observed at the low glutathione level found in blood plasma (5-25 microM GSH).     

Mechanism of enhancement of prochymosin renaturation by solubilization of inclusion bodies at alkaline pH

The renaturation efficiency of recombinant prochymosin depends on not only the renaturation condi-tions but also the solubilization (denaturation) conditions. Compared with pH 8, solubilization of prochymosin-contain-ing inclusion bodies at pH 11 (8 mol/L urea) results in onefold increase of renaturation efficiency ( ~ 40% vs. ~ 20 % ). Alkaline pH facilitates the solubilization of inclusion bodies via the breakage of intermolecular disulfide bonds. Moreover, alkaline pH renders prochymosin molecules to be in a more reduced and more unfolded state which undergoes refolding readily.     

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.     

High recovery of prochymosin from inclusion bodies using controlled air oxidation

Refolding of proteins from inclusion bodies is a field of increasing interest for obtaining large amounts of active enzymes. Consequently, the development of inexpensive and scalable processes is required. This is particularly challenging in the case of eukaryotic proteins containing cysteines, which may form disulfide bonds in the native active protein. Previous studies have shown that the formation of disulfide bonds is essential for the refolding of prochymosin. In this work we demonstrate that air oxidation can be efficiently used for the refolding of prochymosin and that 48% of the unfolded protein can be recovered as active enzyme at a final protein concentration of 0.8 mg/ml. Refolding of the protein strictly correlates with the change in pH of the refolding solution. We were able to follow the degree of oxidative renaturation of the prochymosin by simply measuring pH. Thus, the scaling up of the refolding system under controlled conditions was easily achieved. Analyses of different substances as folding aids indicate that the use of L-arginine or neutral surfactants improves the recovery of active protein up to 67% of the initial protein. The overall results indicate that prochymosin can be efficiently and inexpensively refolded with high yields by controlled air oxidation.     

The renaturation of reduced chymotrypsinogen A in guanidine HCl. Refolding versus aggregation

The refolding and reoxidation of fully reduced and denatured chymotrypsinogen A have been studied in the presence of low concentrations of guanidine HCl or urea. Renaturation yields of 60 to 70% were observed when the reoxidation was facilitated by mixtures of reduced and oxidized glutathione. Refolding occurred within a narrow range of denaturant concentration (1.0 to 1.3 M guanidine HCl and 2 M urea) in which the native protein was shown to be stable, and the reduced protein was shown to regain the correct disulfide pairing. Renatured chymotrypsinogen is indistinguishable from the native zymogen in chromatographic behavior, potential chymotryptic activity, sedimentation coefficient, and spectral properties. The kinetics of renaturation were determined. Some of the protein species obtained at various times of renaturation were characterized as incorrectly oxidized molecules which could be renatured by thiol-catalyzed interchange of disulfide bonds.     

Redox control of platelet aggregation

Sulfhydryl and disulfide metabolism in platelet function has recently reemerged as a focus of platelet research. In this study we tested the effect of redox buffer on platelet aggregation and the effect of reduced glutathione (GSH) and platelet activation on sulfhydryl exposure in the platelet fibrinogen receptor, alpha IIb beta 3. In the presence of subthreshold concentrations of agonist, physiologic concentrations of GSH (10 microM) stimulated platelet aggregation and secretion. These effects were found with more than one platelet agonist and with different low molecular weight thiols, including homocysteine. The effect of low molecular weight thiols was reproduced with the peptide LSARLAF which directly activates platelets through alpha IIb beta 3, suggesting that the mechanism is at the level of this integrin. After determining optimal sulfhydryl labeling conditions for alpha IIb beta 3 (5 mM EDTA, 37 degrees C, 60 min), we found that GSH (10 microM) generated sulfhydryls in the beta 3 subunit. To determine if the requirement was for reducing equivalents or for a redox potential (ratio of GSH to GSSG), aggregation was further studied with the addition of low concentrations of GSSG to the GSH. With a ratio of GSH/GSSG of 5/1, similar to that of blood, the addition of GSSG potentiated the stimulatory effect as compared to GSH alone. This indicates that, for potentiation of aggregation, GSH is not simply reducing disulfide bonds; there is rather a requirement for a certain redox potential. Additional studies performed in the absence of added glutathione showed an increase in sulfhydryl labeling in the beta 3 subunit during platelet activation. Finally, we show that vicinal dithiols of platelet surface proteins are involved in the sulfhydryl-dependent pathways of platelet activation. In summary, these data imply that the redox potential of blood regulates activation of the alpha IIb beta 3 integrin and together with other reports in the literature suggest that disulfide bond cleavage with sulfhydryl generation in beta 3 is involved in activation of this receptor.     

Glutathione disulfide reduction in tumor mitochondria after t-butyl hydroperoxide treatment.

Treatment of isolated mitochondria from rat hepatoma tumor cells (AS-30D) with the oxidant, t-butyl hydroperoxide (tBuOOH, 1 or 5 mumol/ml) resulted in the oxidation of glutathione (GSH to GSSG) and the formation of protein-glutathione mixed disulfides (ProSSG). The GSSG was retained inside of the hepatoma mitochondria. In the presence of ADP+succinate (5 or 10 mM), or ketoglutarate (10 mM) or malate (5 mM), the GSSG was reduced to GSH, but the amount of ProSSG stayed constant. With saline or ADP+glutamate (10 mM)/malate (0.1 mm) no reduction of GSSG to GSH occurred. The presence of antimycin (5 micrograms/ml) with ADP+succinate inhibited reduction. At a concentration of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, 0.5 mM) which inhibited a major portion of the glutathione reductase activity, the reduction of GSSG to replenish GSH was also inhibited. NADPH may play a critical role as well, for the addition of 2.4 mM NADPH to permeabilized hepatoma mitochondria fostered the reduction of GSSG after tBuOOH treatment. Therefore, hepatoma mitochondria possess a glutathione reductase-dependent system to reduce GSSG to GSH. The reaction only occurs with actively respiring mitochondria.     

Thiol/disulfide redox equilibrium and kinetic behavior of chicken liver fatty acid synthase

Chicken liver fatty acid synthase is rapidly inactivated and cross-linked at pH 7.2 and 8.0 by incubation with low concentrations of common biological disulfides including glutathione disulfide, coenzyme A disulfide, and glutathione-coenzyme A-mixed disulfide. Glutathione disulfide inactivation of the enzyme is accompanied by the oxidation of a total of 4-5 enzyme thiols per monomer. Only one glutathione equivalent is incorporated per monomer as a protein-mixed disulfide, and its rate of incorporation is significantly slower than the rate of inactivation. The formation of protein-SS-protein disulfides results in significant cross-linking of enzyme subunits. The inactive enzyme is rapidly and completely reactivated, and the cross-linking is completely reversed by incubation of the enzyme with thiols (10-20 mM) including dithiothreitol, mercaptoethanol, and glutathione. In a glutathione redox buffer (GSH + GSSG), disulfide bond formation comes to equilibrium. The enzyme activity at equilibrium is dependent both on the ratio of glutathione to glutathione disulfide and on the total glutathione concentration. The equilibrium constant for the redox equilibration of fatty acid synthase in a glutathione redox buffer is 15 mM (Ered + GSSG in equilibrium Eox + 2GSH). The formation of at least one protein-protein disulfide per monomer dominates the redox properties of the enzyme while the formation of one protein-mixed disulfide with glutathione (Kmixed = 0.45) has little effect on activity. The oxidation equilibrium constant suggests that there would be no significant cycling between the reduced and the oxidized enzyme in response to likely physiological variations in the hepatic glutathione status. The possibility that changes in the concentration of cellular glutathione may act as a mechanism for metabolic control of other enzymes is discussed.     

Effects of glutathione-related compounds on increased caspase-3 and caspase-6-like activities in ricin-treated U937 cells

Both caspase-3 and -6-like activities increased in the cytosolic extract from ricin-treated U937 cells that were inhibited by glutathione disulfide (GSSG) in a dose-dependent manner, but reduced glutathione (GSH) had no effect. Interestingly, caspase-6 like activity was more sensitive to GSSG than caspase-3 like activity. The IC50 of GSSG against caspase-3 and caspase-6 like activities were estimated to be 2.8 mM and 0.8 mM, respectively. Cystine but not cysteine also showed similar inhibitory effect on caspase-3-like activity. The inhibitory effect of GSSG on these caspase-like activities was prevented by the addition of DTT to the assay mixture. These results suggest that an intact disulfide portion of GSSG is required for the effective inhibition of caspase activity.     

Nonaggregating refolding of ribonuclease A using reverse micellar dialysis

A hydrophilic ultrafiltration membrane, regenerated cellulose, facilitates the size-selectable permeability of hydrophilic solutes in reverse micellar solution. By using an ultrafiltration membrane with a molecular weight cutoff of 3,500, we demonstrate a nonaggregating protein refolding technique based on the dialysis of reverse micellar solution. This realizes concurrent removal of denaturants, urea and 2-mercaptoethanol, and the supply of redox reagents, reduced and oxidized glutathione (GSH, GSSG), to promote renaturation of proteins. Two mg/ml ribonuclease A (RNase A) was refolded completely without any dilution and aggregation for 60 h. The refolding behavior of RNase A is strongly influenced by the ratio of GSH and GSSG. Moreover, we recovered 90% of the refolded RNase A from AOT reverse micellar solution with acetone precipitation and beta-cyclodextrin washing. These findings should facilitate the production of a continuous protein refolding membrane reactor.