Properties of selected S-nitrosothiols compared to nitrosylated WR-1065

WR-1065 ([N-mercaptoethyl]-1-3-diaminopropane), the active form of the aminothiol drug Ethyol/Amifostine, protects against toxicity caused by radiation, chemotherapy and endotoxin. Because WR-1065 and other thiols readily bind nitric oxide (NO), injurious conditions or therapies that induce the production or mobilization of NO could alter the effects of WR-1065. S-Nitrosothiols were prepared from various thiols by a standard method to compare properties and stability. Heteromolecular quantum correlation 2D nuclear magnetic resonance was used to characterize nitrosylated glutathione (GSH) and WR-1065; both S- and N-nitrosothiols were observed, depending on the experimental conditions. Three categories of S-nitrosothiol stability were observed: (1) highly stable, with t(1/2) > 8 h, N-acetyl-L-cysteine nitrosothiol (t(1/2) 15 h) > GSH nitrosothiol (t(1/2) 8 h); (2) intermediate stability, t(1/2) approximately 2 h, cysteamine nitrosothiol and WR-1065 nitrosothiol; and (3) low stability, t(1/2) < 1 h, cysteine nitrosothiol and Captopril nitrosothiol. Similar relative rates were observed for Hg(+2)-induced denitrosylation: WR-1065 reacted faster than GSH nitrosothiol, while GSH nitrosothiol reacted faster than N-acetyl-L-cysteine nitrosothiol. Mostly mediated by mixed-NPSH disulfide formation, the activity of the redox-sensitive cysteine protease, cathepsin H, was inhibited by the S-nitrosothiols, with WR-1065 nitrosothiol > cysteine nitrosothiol > N-acetyl-L-cysteine nitrosothiol and GSH nitrosothiol. These observations indicate that, relative to other nitrosylated non-protein thiols, the S-nitrosothiol of WR-1065 is an unstable non-protein S-nitrosothiols with a high reactive potential in the modification of protein thiols.     

Interaction of glutathione and other low-molecular-weight thiols with DNA: evidence for counterion condensation and coion depletion near DNA

The interaction of low-molecular-weight thiols with sonicated DNA was examined using spin filtration to concentrate the DNA. Cationic thiols (WR 1065 and cysteamine) behaved as counterions and were found to have increased concentrations in the DNA retentate relative to the filtrate. Anionic thiols (GSH, 2-mercaptoethanesulfonate, mercaptosuccinate) behaved as coions and were decreased in concentration in the DNA fraction. Concentrations of the uncharged thiol 2-mercaptoethanol were little influenced by DNA. The results demonstrate the importance of counterion condensation and coion depletion in determining the concentrations of charged species near DNA. They provide a rationale for enhanced effectiveness of WR 1065 and cysteamine as radioprotectors compared to neutral and anionic thiols and suggest that anionic thiols such as GSH should be poor radioprotectors of DNA.     

Radioprotection of DNA by thiols: relationship between the net charge on a thiol and its ability to protect DNA

Release of free bases from calf thymus DNA upon irradiation in aerated 0.1 mol dm-3NaClO4 at pH 7 has been measured by HPLC and shown to be markedly influenced by the presence of thiols during irradiation. The ability of thiols to protect DNA was shown to depend upon the net charge (Z) at pH 7 in the order WR 1065 (Z = +2) greater than cysteamine (Z = +1) greater than 2-mercaptoethanol (Z = 0) approximately equal to dithiothreitol (Z = 0) greater than GSH (Z = -1) approximately equal to 2-mercaptoethanesulfonic acid (Z = -1) approximately equal to 2-mercaptosuccinate (Z = -2). A similar dependence of protection upon net charge was found for disulfides: cystamine (Z = +2) greater than 2-mercaptoethyl disulfide (Z = 0) greater than GSSG (Z = -2). Protection by WR 1065, but not by 2-mercaptoethanol or GSH, was found to decrease significantly with increasing ionic strength. Protection by WR 1065 and GSH was not markedly dependent upon pH between pH 6 and 8. The results are explained in terms of electrostatic interaction of the thiols with DNA, leading to high concentrations of cations near DNA, which allow them to scavenge hydroxyl radicals and repair DNA radicals effectively and to low concentrations of anionic thiols near DNA, which limit their effectiveness as protectors. Poly(dG,dC) and calf thymus DNA exhibited comparable release of G and C upon changing from 0.1 to 0.7 mol dm-3 MgSO4. Since this change causes poly(dG,dC), but not calf thymus DNA, to undergo a change from the B-form to the Z-form of DNA, both forms must have a comparable susceptibility to radiation-induced base release.     

Chemical reactivity of some isothiazolone biocides

Chemical reactions between the isothiazolone biocides, N-methylisothiazol-3-one (MIT), benzisothiazol-3-one (BIT) and 5-chloro-N-methylisothiazol-3-one (CMIT) with cysteine have been investigated by u.v. and NMR spectroscopy. At physiological pH all three agents interacted oxidatively with thiols to form disulphides. Further interaction with thiols caused the release of cystine and formation of a reduced, ring-opened form of the biocide (mercaptoacrylamide). In an analogous fashion to the initial reaction the mercaptoacrylamide reacted with another molecule of biocide to give biocide dimers. NMR spectral studies indicated that for CMIT the mercaptoacrylamide form is capable of tautomerization to a highly reactive thio-acyl chloride. Formation of mercaptoacrylamide was in all cases highly pH-dependent. Alcohol dehydrogenase was insensitive to all three agents but was highly sensitive to CMIT when co-administered with dithiothreitol. Capacity to form a thioacyl chloride from the mercaptoacrylamide is suggested to account for much of this enhanced activity. Stopped-flow spectroscopic studies showed rates of reaction with glutathione (GSH) to directly parallel antimicrobial activity. Additionally, CMIT was able to react directly with both ionization states of GSH (pH 7-10) whilst BIT and MIT appeared only to interact when the glutamyl-nitrogen of GSH was charged (pH 8.5).     

Glutathione dependent metabolism and detoxification of 4-hydroxy-2-nonenal.

The involvement of glutathione (GSH) dependent processes in the detoxification of 4-hydroxy-2-nonenal (4HNE) was investigated using Chinese hamster fibroblasts and clonogenic cell survival. GSH reacted, in a dose-dependent fashion, with 4HNE in phosphate buffer at pH 6.5, leading to the disappearance of 4HNE. The addition of glutathione transferase activity (GST) facilitated a more rapid disappearance of 4HNE but the reaction was still dependent on the concentration of GSH. When cell cultures were exposed to the reaction mixtures, 4HNE cytotoxicity was also reduced in a manner which was dependent on the concentration of GSH. When 2.16- or 1.08-mM GSH were incubated in phosphate buffer with 1.08-mM 4HNE in the presence or absence of GST, then mixed with media and placed on cells for 1 h, the cytotoxicity associated with exogenous exposure to free 4HNE was abolished. GSH depletion (greater than 90%) using buthionine sulfoximine (BSO) was accomplished in control (HA1) and H2O2-resistant variants derived from HA1. GSH depletion resulted in enhanced cytotoxicity of 4HNE in all cell lines. This BSO-induced sensitization to 4HNE cytotoxicity was accompanied by a significant reduction in the ability of cells to metabolize 4HNE. The magnitude of the sensitization to 4HNE toxicity caused by GSH depletion was similar to the magnitude of the reduction in the ability of cells to metabolize 4HNE. These results support the hypothesis that GSH and GST provide a biologically significant pathway for protection against aldehydic by-products of lipid peroxidation.     

Relationship between phosphorylated histone H2AX formation and cell survival in human microvascular endothelial cells (HMEC) as a function of ionizing radiation exposure in the presence or absence of thiol-containing drugs

Human microvascular endothelial cells (HMEC) were exposed to ionizing radiation at doses ranging from 0 to 16 Gy in either the presence or absence of the active thiol forms of amifostine (WR1065), phosphonol (WR255591), N-acetyl-l-cysteine (NAC), captopril or mesna. Each of these clinically relevant thiols, administered to HMEC at a dose of 4 mM for 30 min prior to irradiation, is known to exhibit antioxidant properties. The purpose of this investigation was to determine the relationship(s), if any, between the frequency of radiation-induced histone H2AX phosphorylation at serine 139 (gamma-H2AX) in cells and subsequent survival, as assessed by colony-forming ability, in exposed cell populations as a function of the presence or absence of each of the five thiol compounds during irradiation. gamma-H2AX formation in irradiated cells, as a function of relative DNA content, was quantified by bivariant flow cytometry analysis with FITC-conjugated gamma-H2AX antibody and nuclear DAPI staining. gamma-H2AX formation in cells was measured as the relative fold increase as a function of the treatment conditions. The frequency of gamma-H2AX-positive cells increased with increasing dose of radiation followed by a dose- and time-dependent decay. The most robust response for gamma-H2AX formation occurred 1 h after irradiation with their relative frequencies decreasing as a function of time 4 and 24 h later. To assess the effects of the various thiols on gamma-H2AX formation, all measurements were made 1 h after irradiation. WR1065 was not only effective in protecting HMEC against gamma-H2AX formation across the entire dose range of radiation exposures used, but it was also significantly more cytoprotective than either its prodrug (WR2721) or disulfide (WR33278) analogue. WR1065 had no significant effect on gamma-H2AX formation when administered immediately or up to 30 min after radiation exposure. An inhibitory effect against gamma-H2AX formation induced by 8 Gy of radiation was expressed by each of the thiols tested. NAC, captopril and mesna were equally effective in reducing the frequency of gamma-H2AX formation, with both WR1065 and WR255591 exhibiting a slightly more robust protective effect. Each of the five thiols was effective in reducing the frequency of gamma-H2AX-positive cells across all phases of the cell cycle. In contrast to the relative ability of each of these thiols to inhibit gamma-H2AX formation after irradiation, NAC, captopril and mesna afforded no protection to HMEC as determined using a colony-forming survival assay. Only WR1065 and WR255591 were effective in reducing the frequencies of radiation-induced gamma-H2AX-positive cells as well as protecting against cell death. These results suggest that the use of gamma-H2AX as a biomarker for screening the efficacy of novel antioxidant radioprotective compounds is highly problematic since their formation and disappearance may be linked to processes beyond simply the formation and repair of radiation-induced DSBs.     

Variable regulation of glutamate cysteine ligase subunit proteins affects glutathione biosynthesis in response to oxidative stress

Glutamate cysteine ligase (GCL), composed of a catalytic (GCLC) and modulatory (GCLM) subunit, catalyzes the first step of glutathione (GSH) biosynthesis. Using 4-hydroxy-2-nonenal (4HNE), 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and tertiary-butylhydroquinone (tBHQ) as models of oxidative stress which are known to work through different mechanisms, we measured changes in cellular GSH, GCL mRNA, and GCL protein. 4HNE and tBHQ treatments increased cellular GSH levels, while DMNQ exposure depleted GSH. Furthermore, changes in the two GCL mRNAs largely paralleled changes in the GCL proteins; however, the magnitudes differed, suggesting some form of translational control. The molar ratio of GCLC:GCLM ranged from 3:1 to 17:1 in control human bronchial epithelial (HBE1) cells and all treatments further increased this ratio. Data from several mouse tissues show molar ratios of GCLC:GCLM that range from 1:1 to 10:1 in support of these findings. These data demonstrate that alterations in cellular GSH are clearly correlated with GCLC to a greater extent than GCLM. Surprisingly, both control HBE1 cells and some mouse tissues have more GCLC than GCLM and GCLM increases to a much lesser extent than GCLC, suggesting that the regulatory role of GCLM is minimal under physiologically relevant conditions of oxidative stress.     

Reactivity of thiols towards derivatives of 2- and 6-methyl-1,4-naphthoquinone bioreductive alkylating agents

The stoichiometry and kinetics of the anaerobic reactions between some thiols and derivatives of 2- and 6-methyl-1,4-naphthoquinones in water were measured using stopped flow spectrophotometry. The stoichiometry of the reaction with representative compounds was 1:2 thiol:quinone, a finding consistent with the formation of a hydroquinone as well as a thioether in the reaction. The first-order dependence of rate on thiol concentration, and the pH-dependent rate constants indicated that the thiolate anion was involved in the rate-limiting step, with rate constants at pH 7.6 generally increasing in the order glutathione (GSH) less than cysteamine less than dithiothreitol (DTT) less than cysteine. Despite the lower reactivity of GSH, the half-lives of the uncatalyzed conjugation reaction of these quinones at typical biological concentrations of GSH (e.g. 2 mM) ranged from about 2.0 to 20 s at pH 7.6 and 25 degrees C. The implications of these reactions in the use of naphthoquinones as potential bioreductive alkylating agents and as hypoxic cell radiosensitizers are discussed.     

Factors influencing the oxidation of cysteamine and other thiols: implications for hyperthermic sensitization and radiation protection

Some of the factors influencing the oxygen uptake and peroxide formation for cysteamine (MEA) and other thiols in serum-supplemented modified McCoy's 5A, a well-known medium used to cultivate a variety of cells in vitro, have been studied. The oxidation of MEA and cysteine in modified McCoy's 5A has been compared with that in Ham's F-12, MEM, and phosphate-buffered saline. All of the growth media were supplemented with 10% calf serum and 5% fetal calf serum. The rate of oxygen uptake for all of the studied thiols was greatest in McCoy's 5A. The data indicate that this medium may contain more copper than the other preparations. MEA and cysteine were found to be more effective at 0.4 mM at producing peroxide than dithiothreitol (DTT). N-acetylcysteine was the least reactive. The ability to produce peroxide is dependent upon the temperature, the concentration of thiol, the presence of copper ions, and pH of the medium. MEA and other thiol oxidation is inhibited by the copper chelator diethyldithiocarbamate. Catalase also reduces the oxygen uptake for all thiols. This inhibition involves the recycling of peroxide to oxygen. Superoxide dismutase (SOD) was found to stimulate the oxygen uptake in the case of MEA and cysteine, but had little or no effect with DTT and glutathione. The combined presence of SOD and catalase resulted in less inhibition of oxygen uptake than that obtained by catalase alone. Alkaline pH was found to enhance the oxidation of cysteine and MEA. An important observation was the inhibition of MEA oxidation at 0 degrees C and the stimulation at 42 degrees C. The results indicate that many problems may arise when thiols are added to various media. A major consideration is concerned with the production of peroxide, superoxide, and reduced trace metal intermediates. The presence of these intermediates may result in the production of hydroxyl radical intermediates as well as the eventual oxygen depletion from the medium. Oxygen depletion may alter the results of radiation sterilization and carcinogen activation. Radical production will cause cell damage that is temperature dependent. Therefore, careful consideration must be given to changes in oxygen tension when thiols are added to cells growing in complicated growth medium to protect against either chemical or radiation damage.     

para-sulfobenzoyloxybromobimane: a new membrane-impermeable reagent useful for the analysis of thiols and their export from cells.

para-Sulfonylbenzoyloxybromobimane (sBBr) was shown to be similar to the fluorescent labeling agent monobromobimane (mBBr) in reacting rapidly and selectively with thiols to produce stable derivatives which are readily separated by HPLC. Chromatography of the sBBr derivative provides a useful means of confirming the identification of an unknown thiol based upon the chromatography of its mBBr derivative and can be useful for quantitative determination of polycationic thiols for which chromatography of the mBBr derivative is unsatisfactory. Unlike mBBr, which readily penetrates cells, sBBr was found not to be taken up by cells. These characteristics allow sBBr to be used, in conjunction with mBBr, to quantify the export of thiols from cells, as illustrated for GSH and the radioprotective drug WR1065, from V79 cells. Simultaneous determination of GSH and glutathione disulfides in cell culture medium could be achieved by labeling of thiols with sBBr followed by reduction of disulfides with dithiothreitol, labeling of the resulting thiols with mBBr, and HPLC analysis for both glutathione derivatives.     

Chemical reactivity of some isothiazolone biocides

C ollier , P.J., R amsey , A., W aigh , R.D., D ouglas , K.T., A ustin , P. & G ilbert , P. 1990. Chemical reactivity of some isothiazolone biocides. Journal of Applied Bacteriology 69 , 578–584.
Chemical reactions between the isothiazolone biocides, N-methylisothiazol-3-one (MIT), benzisothiazol-3-one (BIT) and 5-chloro-N-methylisothiazol-3-one (CMIT) with cysteine have been investigated by u.v. and NMR spectroscopy. At physiological pH all three agents interacted oxidatively with thiols to form disulphides. Further interaction with thiols caused the release of cystine and formation of a reduced, ring-opened form of the biocide (mercaptoacrylamide). In an analogous fashion to the initial reaction the mercaptoacrylamide reacted with another molecule of biocide to give biocide dimers. NMR spectral studies indicated that for CMIT the mercaptoacrylamide form is capable of tautomerization to a highly reactive thio-acyl chloride. Formation of mercaptoacrylamide was in all cases highly pH-dependent. Alcohol dehydrogenase was insensitive to all three agents but was highly sensitive to CMIT when co-administered with dithiothreitol. Capacity to form a thioacyl chloride from the mercaptoacrylamide is suggested to account for much of this enhanced activity. Stopped-flow spectroscopic studies showed rates of reaction with glutathione (GSH) to directly parallel antimicrobial activity. Additionally, CMIT was able to react directly with both ionization states of GSH (pH 7–10) whilst BIT and MIT appeared only to interact when the glutamyl-nitrogen of GSH was charged (pH 8.5).     

4-Hydroxy-2,3-trans-nonenal stimulates microsomal lipid peroxidation by reducing the glutathione-dependent protection

Glutathione (GSH) protects liver microsomes against lipid peroxidation. This is probably due to the reduction of vitamin E radicals by GSH, a reaction catalyzed by a membrane-bound protein. Pretreatment of liver microsomes with 0.1 or 1mM 4-hydroxy-2,3-trans-nonenal (HNE), a major product of lipid peroxidation, reduces the GSH-dependent protection. GSH and vitamin E concentrations are not affected by this pretreatment. Pretreatment with 0.1 mM N-ethyl maleimide (NEM), a synthetic sulfhydryl reagent, resulted in a reduction similar to that with HNE of the GSH-dependent protection against lipid peroxidation. The reduction of the GSH-dependent protection by HNE and NEM is probably the result of inactivation of the membrane-bound protein by covalent binding to an essential SH group on the protein. If the GSH-dependent protection would proceed via the microsomal GSH transferase, pretreatment with NEM, which activates the microsomal GSH transferase, should enhance the GSH-dependent protection. Actually a decrease in the GSH-dependent protection is found. Apparently the GSH-dependent protection does not proceed via the microsomal GSH transferase. Also the microsomal phospholipase A2 is not involved, since addition of 0.1 mM mepacrine, an inhibitor of phospholipase A2, did not preclude the GSH-dependent protection. Once the process of lipid peroxidation, either in vivo or in vitro, has started, the protection of liver microsomes by GSH is less effective. This might be the result of formed HNE. In this way an endproduct of lipid peroxidation stimulates the process that generates this product.     

Resveratrol and 4-hydroxynonenal act in concert to increase glutamate cysteine ligase expression and glutathione in human bronchial epithelial cells

Resveratrol has been shown to protect against oxidative stress through modulating antioxidant capacity. In this study, we investigated resveratrol-mediated induction of glutathione (GSH) and glutamate cysteine ligase (GCL), and the combined effect of resveratrol and 4-hydroxynonenal (HNE) on GSH synthesis in cultured HBE1 human bronchial epithelial cells. Resveratrol increased GSH and the mRNA contents of both the catalytic (GCLC) and modulatory subunit (GCLM) of GCL. Combined HNE and resveratrol treatment increased GSH content and GCL mRNAs to a greater extent than either compound did alone. Compared to individual agent, combining exposure to HNE and resveratrol also showed more protection against cell death caused by oxidative stress. These effects of combined exposure were additive rather than synergistic. In addition, Nrf2 silencing significantly decreased the combined effect of HNE and resveratrol on GCL induction. Our data suggest that resveratrol increases GSH and GCL gene expression and that there is an additive effect on GSH synthesis between resveratrol and HNE. The results also reveal that Nrf2-EpRE signaling was involved in the combined effects.     

Fate of 4-hydroxynonenal in vivo: disposition and metabolic pathways

Due to the cytotoxicity of 4-hydroxynonenal (HNE), and to the fact that this major product of lipid peroxidation is a rather long-living compound compared with reactive oxygen species, the capability of organisms to inactivate and eliminate HNE has received increasing attention during the last decade. Several recent in vivo studies have addressed the issue of the diffusion, kinetics, biotransformation and excretion of HNE. Part of these studies are primarily concerned with the toxicological significance of HNE biotransformation and more precisely with the metabolic pathways by which HNE is inactivated and eliminated. The other aim of in vivo metabolic study is the characterisation of end-metabolites, especially in urine, in order to develop specific and non-invasive biomarkers of lipid peroxidation. When HNE is administered intravenously or intraperitoneally, it is mainly excreted into urine and bile as conjugated metabolites, in a proportion that is dependent on the administration route. However, biliary metabolites undergo an enterohepatic cycle that limits the final excretion of faecal metabolites. Only a very low amount of metabolites is found to be bound to macromolecules. The main urinary metabolites are represented by two groups of compounds. One comes from the mercapturic acid formation from (i) 1,4 dihydroxynonene-glutathione (DHN-GSH) which originates from the conjugation of HNE with GSH by glutathione-S-transferases and the subsequent reduction of the aldehyde by a member of aldo-keto reductase superfamily; (ii) the lactone of 4-hydroxynonanoic-GSH (HNA-lactone-GSH) which originates from the conjugation of HNE followed by the oxidation of the aldehyde by aldehyde dehydrogenase; (iii) HNA-GSH which originates from the hydrolysis of the corresponding lactone. The other one is a group of metabolites issuing from the omega-hydroxylation of HNA or HNA-lactone by cytochromes P450 4A, followed eventually, in the case of omega-oxidized-HNA-lactone, by conjugation with GSH and subsequent mercapturic acid formation. Biliary metabolites are GSH or mercapturic acid conjugates of DHN, HNE and HNA. Stereochemical aspects of HNE metabolism are also discussed.     

New liquid chromatographic assay with electrochemical detection for the measurement of amifostine and WR1065

A high-performance liquid chromatographic method (HPLC) was developed for the analysis of the radio- and chemo-protectant, amifostine and its active metabolite-WR1065 in deproteinized human whole blood and plasma. The two compounds were quantified by measuring WR1065 after two different sample pretreatment procedures. During these procedures, amifostine was quantitatively converted into WR1065, by incubating the sample at 37 degrees C for 4 h at pH<1.0. The resulting amounts of WR1065 were determined by HPLC with coulometric detection (analytical cell: E(1)=200 mV and E(2)=600 mV; guard cell: E(G)=650 mV). The WR1065 standard curve ranged from 0.37 to 50.37 microM. The lower limit of quantitation of WR1065 was 0.25 microM. The within- and between-day precisions were < or = 4.3% and < or = 6.0% for amifostine, < or = 4.4% and < or = 3.8% for WR1065, respectively. The within- and between-day accuracy ranged from 95.4 to 97.7% and 95.4 to 97.8% for amifostine, and from 97.1 to 101.7% and 97.2 to 99.7% for WR1065, respectively. This method minimizes WR1065 loss during sample preparation, and allows for rapid analysis of both compounds on one system. Furthermore, the application of a coulometric electrode is more efficient and requires less maintenance than previously published methods for the two compounds.     

Cell surface redox potential as a mechanism of defense against photosensitizers in fungi.

The phytotoxin cercosporin, a singlet oxygen-generating photosensitizer, is toxic to plants, mice, and many fungi, yet the fungi that produce it, Cercospora spp., are resistant. We hypothesize that resistance to cercosporin may result from a reducing environment at the cell surface. Twenty tetrazolium dyes differing in redox potential were used as indicators of cell surface redox potential of seven fungal species differing in resistance to cercosporin. Resistant fungi were able to reduce significantly more dyes than were sensitive fungi. A correlation between dye reduction and cercosporin resistance was also observed when resistance levels of Cercospora species were manipulated by growth on different media. The addition of the reducing agents ascorbate, cysteine, and reduced glutathione (GSH) to growth media decreased cercosporin toxicity for sensitive fungi. None of these agents directly reduced cercosporin at the concentrations at which they protected fungi. Spectral and thin-layer chromatographic analyses of cercosporin solutions containing the different reducing agents indicated that GSH, but not cysteine or ascorbate, reacted with cercosporin. Resistant and sensitive fungi did not differ in endogenous levels of cysteine, GSH, or total thiols. On the basis of data from this and other studies, this report presents a model which proposes that cercosporin resistance results from the production of reducing power at the surfaces of resistant cells, leading to transient reduction and detoxification of the cercosporin molecule.     

Cell surface redox potential as a mechanism of defense against photosensitizers in fungi.

The phytotoxin cercosporin, a singlet oxygen-generating photosensitizer, is toxic to plants, mice, and many fungi, yet the fungi that produce it, Cercospora spp., are resistant. We hypothesize that resistance to cercosporin may result from a reducing environment at the cell surface. Twenty tetrazolium dyes differing in redox potential were used as indicators of cell surface redox potential of seven fungal species differing in resistance to cercosporin. Resistant fungi were able to reduce significantly more dyes than were sensitive fungi. A correlation between dye reduction and cercosporin resistance was also observed when resistance levels of Cercospora species were manipulated by growth on different media. The addition of the reducing agents ascorbate, cysteine, and reduced glutathione (GSH) to growth media decreased cercosporin toxicity for sensitive fungi. None of these agents directly reduced cercosporin at the concentrations at which they protected fungi. Spectral and thin-layer chromatographic analyses of cercosporin solutions containing the different reducing agents indicated that GSH, but not cysteine or ascorbate, reacted with cercosporin. Resistant and sensitive fungi did not differ in endogenous levels of cysteine, GSH, or total thiols. On the basis of data from this and other studies, this report presents a model which proposes that cercosporin resistance results from the production of reducing power at the surfaces of resistant cells, leading to transient reduction and detoxification of the cercosporin molecule.     

Sensor specific imaging of proteomic Zn2+ with zinquin and TSQ after cellular exposure to N-ethylmaleimide

The impact of the thiol binding reagent N-ethylmaleimide (NEM) on proteomic Zn(2+) availability was investigated in rat glioma cells. Zinquin (ZQ) or TSQ, two related fluorescent sensors, were used to observe reactive Zn(2+). Control cells contained proteomic Zn(2+) but no detectable low molecular weight (LMW) Zn(2+). With either sensor, basal cellular fluorescence emission centered near 470 nm, indicative of sensor-Zn-proteins. ZQ sequestered 13% of proteomic Zn(2+) as Zn(ZQ)(2); TSQ reacted only with the Zn-proteome. NEM (100 μM) abolished LMW thiols, including glutathione (GSH) and lowered proteomic sulfhydryl content about 30%. In ZQ-treated cells, NEM exposure enhanced fluorescent intensity and the formation of Zn(ZQ)(2) (λ(MAX), 492 nm). Cells incubated with TSQ and NEM also displayed increased fluorescence without a spectral shift in wavelength maximum, consistent with increased formation of TSQ-Zn-protein adducts but not Zn(TSQ)(2). In neither experiment was Zn(2+) lost from cells. NEM altered Zn(2+) accessibility to sensors in membrane-nuclear and cytosolic fractions, but Zn(ZQ)(2) was only generated in the cytosol. Similar results were obtained when cell supernatant replaced cells. In contrast, when isolated proteome was reacted with ZQ and 100 μM NEM in the absence of GSH, 70% of the proteomic thiols underwent reaction. As a consequence, most of the ZQ-Zn-protein adducts were converted to Zn(ZQ)(2). Substituting TSQ for ZQ, only increased TSQ-Zn-proteins were observed. Evidently, the results of imaging cells with Zn(2+) sensors are dependent upon the specific chemical properties of the sensors and can only be understood after detailed chemical analysis.     

Irreversible thiol oxidation in carbonic anhydrase III: protection by S-glutathiolation and detection in aging rats

Proteins with reactive sulfhydryls are central to many important metabolic reactions and also contribute to a variety of signal transduction systems. In this report, we examine the mechanisms of oxidative damage to the two reactive sulfhydryls of carbonic anhydrase III. Hydrogen peroxide (H2O2), peroxy radicals, or hypochlorous acid (HOCl) produced irreversibly oxidized forms, primarily cysteine sulfinic acid or cysteic acid, of carbonic anhydrase III if glutathione (GSH) was not present. When GSH was approximately equimolar to protein thiols, irreversible oxidation was prevented. H202 and peroxyl radicals both generated S-glutathiolated carbonic anhydrase III via partially oxidized protein sulfhydryl intermediates, while HOCl did not cause S-glutathiolation. Thus, oxidative damage from H202 or AAPH was prevented by protein S-glutathiolation, while a direct reaction between GSH and oxidant likely prevents HOCl-mediated protein damage. In cultured rat hepatocytes, carbonic anhydrase III was rapidly S-glutathiolated by menadione. When hepatocyte glutathione was depleted, menadione instead caused irreversible oxidation. We hypothesized that normal depletion of glutathione in aged animals might also lead to an increase in irreversible oxidation. Indeed, both total protein extracts and carbonic anhydrase III contained significantly more cysteine sulfinic acid in older rats compared to young animals. These experiments show that, in the absence of sufficient GSH, oxidation reactions lead to irreversible protein sulfhydryl damage in purified proteins, cellular systems, and whole animals.     

Free thiols of platelet thrombospondin. Evidence for disulfide isomerization

The free thiols of platelet thrombospondin (TSP) were derivatized with labeled N-ethylmaleimide (NEM) or iodoacetamide (IAM). When Ca2+ was chelated with EDTA, 2.9 mol of NEM or 2.6 mol of IAM reacted/mol of native TSP. No additional thiols were found after denaturation with urea. Since TSP has three apparently identical polypeptide chains, this suggests one free thiol/polypeptide chain. Ca2+ protected all of the thiols from reaction with IAM. In Ca2+ about half the thiols reacted normally with NEM and the others were unreactive, indicating that the thiols of TSP are not identical. The number of reactive thiols as a function of [Ca2+] revealed a sigmoidal curve with a transition midpoint of 207 microM. The ability of analogs of NEM to compete for derivatization of the thiols with labeled NEM was greater with larger, more hydrophobic agents. Gel electrophoretic separation of labeled TSP that had been partially digested with thrombin and trypsin indicated that some of the label was in the C-terminal tryptic fragment but that most was in the adjacent trypsin-sensitive region. After cyanogen bromide cleavage of the labeled and reduced protein, four labeled fractions were obtained from a gel filtration column. With subsequent combinations of tryptic digestion and reversed-phase high performance liquid chromatography, labeled peptides were purified from these four fractions, and the amino acid sequences were determined. Twelve labeled cysteines were identified, each with a specific radioactivity less than that of the thiol labeling reagent, indicating that only a fraction of that cysteine in a population of TSP molecules was a free thiol at the time of derivatization. While 2 labeled cysteines are in the non-repeating C-terminal portion of the molecule, the other 10 labeled cysteines are in the adjacent trypsin-sensitive type 3 repeats proposed (Lawler, J., and Hynes, R. O. (1986) J. Cell. Biol. 103, 1635-1648) as the calcium-binding region of the molecule. The disulfide bonds most sensitive to reduction by dithioerythritol were also stabilized by Ca2+, implying location in the Ca2(+)-sensitive part of the molecule. It is proposed that one equivalent of free thiol/polypeptide chain is distributed among 12 different cysteine residues through an intramolecular thioldisulfide isomerization.