Assessment of unscheduled DNA synthesis in a cultured line of renal epithelial cells exposed to cysteine S-conjugates of haloalkenes and haloalkanes

The ability of S-(1,2-dichlorovinyl)-L-cysteine (DCVC), S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC), S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (PCBC), S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFEC) and S-(2-chloroethyl)-L-cysteine (CEC) to induce DNA repair was investigated in LLC-PK1, a cultured line of porcine kidney tubular epithelial cells. DNA repair due to exposure of the cells to the S-conjugates was determined as unscheduled DNA synthesis (UDS) after inhibition of replicative DNA synthesis in confluent LLC-PK1 monolayers. DCVC, TCVC and PCBC induced dose-dependent UDS in LLC-PK1 at concentrations which did not impair the viability of the cells compared to untreated controls; higher concentrations were cytotoxic, resulting in lactate dehydrogenase leakage into the medium. Cell death was also induced by CTFEC, which failed to exert genotoxicity. CEC induced the highest response among these cysteine conjugates without impairing cell viability. Inhibition of cysteine conjugate beta-lyase with aminooxyacetic acid abolished the effects of DCVC, TCVC, PCBC and CTFEC but did not influence the genotoxicity of CEC.     

Cytotoxicity of cysteine S-conjugates: structure-activity relationships

The cytotoxicity of cysteine S-conjugates was investigated in freshly isolated rat renal proximal tubule cells. The study was designed to determine the contribution of the thiols and of the acylating intermediates formed by cysteine conjugate beta-lyase to the initiation of cytotoxicity. Cell viability was determined by trypan blue exclusion and by lactate dehydrogenase leakage. The S-conjugates S-(1,2,2-trichlorovinyl)-L-cysteine, S-(1,2,3,3,3-pentachloro-prop-1-enyl)-L-cysteine and S-(1,2,3,4,4-pentachlorobuta-1,3-dienyl)-L-cysteine, at a concentration of 0.2 mM, reduced cell viability compared to controls from 85% to less than 50% after 3 h. The alpha-chlorinated enethiols formed from these S-conjugates are transformed to acylating intermediates. The S-conjugate S-(2-chlorovinyl)-L-cysteine forms an enethiol, which cannot transform to an acylating intermediate and did not reduce cell viability at 0.2 mM; at 1 mM, it resulted in a very slight reduction of cell viability after 3 h. S-(pentachlorophenyl)-L-cysteine and S-benzyl-L-cysteine, which form stable thiols after metabolism by beta-lyase, were not cytotoxic at a concentration of 1 mM. The direct acting S-(2-chloroethyl)-L-cysteine (0.2 mM) reduced cell viability after 3 h from 85% to 90% (control) to 40%. The results obtained suggest that reactions of the initial thiol-metabolites with biological macromolecules do not contribute to the induction of cytotoxicity by cysteine S-conjugates and indicate that acylating intermediates formed by cysteine conjugate R-lyase induce cytotoxic effects by non-selective acylation of cellular macromolecules.     

Bacterial cysteine conjugate beta-lyase and the metabolism of cysteine S-conjugates: structural requirements for the cleavage of S-conjugates and the formation of reactive intermediates

The cysteine conjugate beta-lyase mediated metabolism and the mutagenicity of the synthetic cysteine conjugates S-(2-chloroethyl)-L-cysteine (CEC), S-(2-chlorovinyl)-L-cysteine (CVC), S-(1,2,3,3,3-pentachloroprop-1-enyl)-L-cysteine (PCPC), S-(pentachlorophenyl)-L-cysteine (PCPhC), S-(chloro-1,2,2-trifluoroethyl)-L-cysteine (CTFEC), S-benzyl-L-cysteine (SBC) and S-methyl-L-cysteine (SMC) were investigated in Salmonella typhimurium strains TA100, TA2638, TA102 and TA98 to establish structure/activity relationships. Bacterial 100,000 X g supernatants cleaved CTFEC, PCPC, CVC, PCPhC and SBC to pyruvate; pyruvate formation was inhibited by the beta-lyase inhibitor aminooxyacetic acid (AOAA) in all cases. Of the compounds tested, CEC, PCPC and CVC were mutagenic in the Ames-test. CTFEC, PCPhC and SBC failed to increase the number of revertants above control levels. The mutagenicity of PCPC and CVC could be inhibited by AOAA. CEC exerted a potent mutagenic effect in the Ames-test which was not affected by AOAA; CEC was not transformed to pyruvate by bacterial beta-lyase. Neither pyruvate formation nor mutagenicity were observed with SMC. These results indicate that the structure of the substituent on the sulfur atom is an important determinant for the biological activity of cysteine S-conjugates. Electronegative and/or unsaturated substituents are required for beta-lyase catalysed beta-elimination reactions. The formation of chemically unstable thiols, which may be converted to thioacylating intermediates, seems to be a prerequisite for beta-lyase dependent mutagenicity of S-conjugates.     

Mutagenicity of the glutathione and cysteine S-conjugates of the haloalkenes 1,1,2-trichloro-3,3,3-trifluoro-1-propene and trichlorofluoroethene in the Ames test in comparison with the tetrachloroethene-analogues

The nephrotoxic and nephrocarcinogenic potential of the haloalkenes is associated with the conjugation of the chemicals to L-glutathione. Subsequent processing of the haloalkene glutathione S-conjugates via the cysteine conjugate beta-lyase pathway in the mammalian kidney yields nephrotoxic and mutagenic species. To investigate whether S-conjugates of the model chlorofluoroalkenes 1,1,2-trichloro-3,3,3-trifluoro-1-propene (CAS # 431-52-7) and trichlorofluoroethene (CAS # 359-29-5) show comparable effects, we have synthesised the respective cysteine and glutathione S-conjugates and subjected them to the Ames test. The cysteine and glutathione S-conjugates of tetrachloroethene (CAS # 127-18-4), S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC) and S-(1,2,2-trichlorovinyl)glutathione (TCVG) were used as positive controls and reference substances. S-(1,2-dichloro-3,3,3-trifluoro-1-propenyl)-L-cysteine (DCTFPC) and S-(2,2-dichloro-1-fluorovinyl)-L-cysteine (DCFVC) showed clear dose-dependent mutagenic effects with the Salmonella typhimurium tester strains TA100 and TA98. Using TCVC as a reference substance the following ranking in mutagenic response was established: TCVC>DCTFPC>DCFVC. S-(1,2-dichloro-3,3,3-trifluoro-1-propenyl)glutathione (DCTFPG) and S-(2,2-dichloro-1-fluorovinyl)glutathione (DCFVG) showed potent dose-dependent mutagenic effects with the S. typhimurium tester strain TA100 in the presence of a rat kidney S9-protein fraction; tests carried out in the absence of the bioactivation system resulted only in background rates of revertants. Using TCVG as a reference substance the following ranking in mutagenic response was established: TCVG=DCTFPG>DCFVG.The data obtained provide a basis for further studies on the mutagenic and presumable carcinogenic potential of the substances.     

Thioacylating agents as ultimate intermediates in the beta-lyase catalysed metabolism of S-(pentachloro-butadienyl)-L-cysteine

The transformation of the hexachloro-1,3-butadiene metabolite S-(1,2,3,4,4-pentachlorobuta-1,3-dienyl)-L-cysteine (PCBC) by bacterial cysteine conjugate beta-lyase (beta-lyase) and by N-dodecylpyridoxal bromide (PLP-Br) was investigated using GC/MS to identify products formed. PCBC was transformed by both bacterial beta-lyase and PLP-Br to the major products 2,3,4,4-tetrachlorobutenoic acid and 2,3,4,4-tetrachlorothiobutenoic acid, and to the minor metabolites trichloroacetic acid and S-(1,2,3,4,4-pentachlorobuta-1,3-dienyl)-mercaptoacetic acid. In the presence of diethylamine as model nucleophile, PLP-Br transformed PCBC to yield 2,3,4,4-tetrachlorothiobutenoic acid diethylamide; attempts to trap 1,2,3,4,4-pentachlorobutadienyl thiol, the initial metabolite formed by beta-elimination from PCBC, were unsuccessful. The results obtained suggest that the formation of a thioacylating intermediate (a thioketene or a thiono acyl chloride) may be the decisive reaction during the beta-lyase dependent activation of PCBC.     

Turnover and functions of glutathione studied with isolated hepatic and renal cells

Suspensions of freshly isolated rat hepatocytes and renal tubular cells contain high levels of reduced glutathione (GSH), which exhibits half-lives of 3-5 and 0.7-1 h, respectively. In both cells types the availability of intracellular cysteine is rate limiting for GSH biosynthesis. In hepatocytes, methionine is actively converted to cysteine via the cystathionine pathway, and hepatic glutathione biosynthesis is stimulated by the presence of methionine in the medium. In contrast, extracellular cystine can support renal glutathione synthesis; several disulfides, including cystine, are rapidly taken up by renal cells (but not by hepatocytes) and are reduced to the corresponding thiols via a GSH-linked reaction sequence catalyzed by thiol transferase and glutathione reductase (NAD(P)H). During incubation, hepatocytes release both GSH and glutathione disulfide (GSSG) into the medium; the rate of GSSG efflux is markedly enhanced during hydroperoxide metabolism by glutathione peroxidase. This may lead to GSH depletion and cell injury; the latter seems to be initiated by a perturbation of cellular calcium homeostasis occurring in the glutathione-depleted state. In contrast to hepatocytes, renal cells metabolize extracellular glutathione and glutathione S-conjugates formed during drug biotransformation to the component amino acids and N-acetyl-cysteine S-conjugates, respectively. In addition, renal cells contain a thiol oxidase acting on extracellular GSH and several other thiols. In conclusion, our findings with isolated cells mimic the physiological situation characterized by hepatic synthesis and renal degradation of plasma glutathione and glutathione S-conjugates, and elucidate some of the underlying biochemical mechanisms.     

Mercurochrom can be used for the histochemical demonstration and microphotometric quantitation of both protein thiols and protein (mixed) disulfides

 Mercurochrom [2,7-dibromo-4-(hydroxymercuri)-fluorescein disodium salt] used for staining of protein thiols in addition binds to other groups of proteins. Experimental evidence is provided that mercurochrom bound to non-thiol groups forms a 1:1 adduct with protein (mixed) disulfides. The disulfide contents of three different types of cells determined biochemically correlated with the corresponding mean integrated optical densities determined microphotometrically after mercurochrom staining of groups other than thiols. Intracellular disulfide exchange has been studied, leading to a transformation of protein mixed disulfides to protein disulfides and an equimolar loss of protein thiols. Protein mixed disulfides were generated from protein thiols using both methyl methanethiosulfonate (MMTS) and 2,2′-dihydroxy-6,6′-dinaphthyldisulfide (DDD). Loss of thiols as well as the equimolar increase of protein mixed disulfides were followed using both mercurochrom staining for thiols and for disulfides. Generation of protein mixed disulfides due to the DDD reaction was also followed by azocoupling with Fast blue B. On the basis of the observed stoichiometry between the loss of protein thiols and the quantity, increase or conversion of protein disulfides determined microphotometrically using both mercurochrom staining and DDD Fast blue B staining, we conclude that: (1) 1 mol of mercurochrom is bound per mol of protein (mixed) disulfide; and (2) the molar absorptivity of mercurochrom bound to disulfides is ɛ₅₂₀=34940. This study demonstrates that mercurochrom can be used for the quantitative determination of the oxidative status of protein thiols in cells. Accepted: 17 December 1996     

A new method for disulfide analysis of peptides.

A simple, sensitive, reliable method for determining disulfide groups in peptides is presented. The disulfides are cleaved in a brief treatment with strong alkali. Following neutralization with phosphoric acid, thiol resulting from the alkaline cleavage is estimated colorimetrically with 5,5′-dithio-bis(2-nitrobenzoic acid). In the presence of EDTA, the color yield is stable and is linear with the concentration of oxidized glutathione. The stoichiometry with other peptide disulfides appears to be somewhat variable but not so as to interfere with detection of peptide disulfides in chromatographic fractions. The present method compares favorably with two other proposed disulfide analytical methods. The cleavage assay is chromogenic with disulfides, thiols, and with certain blocked thiols but is not chromogenic with methionine and lanthionine.     

Bacterial beta-lyase mediated cleavage and mutagenicity of cysteine conjugates derived from the nephrocarcinogenic alkenes trichloroethylene, tetrachloroethylene and hexachlorobutadiene

The metabolism of beta-lyase and the mutagenicity of the synthetic cysteine conjugates S-1,2-dichlorovinylcysteine (DCVC), S-1,2,2-trichlorovinylcysteine (TCVC), S-1,2,3,4,4-pentachlorobuta-1,3-dienylcysteine (PCBC) and S-3-chloropropenylcysteine (CPC) were investigated in Salmonella typhimurium strains TA100, TA2638 and TA98. The bacteria contained significantly higher concentrations of beta-lyase than mammalian subcellular fractions. Bacterial 100,000 X g supernatants cleaved benzthiazolylcysteine to equimolar amounts of mercaptobenzthiazole and pyruvate. DCVC, TCVC and PCBC produced a linear time-dependent increase in pyruvate formation when incubated with bacterial 100,000 X g supernatants; pyruvate formation was inhibited by the beta-lyase inhibitor aminooxyacetic acid (AOAA). CPC was not cleaved by bacterial enzymes to pyruvate. DCVC, TCVC and PCBC were mutagenic in three strains of S. typhimurium (TA100, TA2638 and TA98) in the Ames-test without addition of mammalian subcellular fractions; their mutagenicity was decreased by the addition of AOAA to the preincubation mixture. CPC was not mutagenic in any of the strains of bacteria tested. These results indicate that beta-lyase plays a key role in the metabolism and mutagenicity of haloalkenylcysteines when tested in S. typhimurium systems. The demonstrated formation in mammals of the mutagens DCVC, TCVC and PCBC during biotransformation of trichloroethylene (Tri), tetrachloroethylene (Tetra) and hexachlorobutadiene (HCBD) may provide a molecular explanation for the nephrocarcinogenicity of these compounds.     

Analysis of biological thiols: determination of thiol components of disulfides and thioesters

This report describes a method for using selective cleavage of thioesters to allow differentiation between thioesters and disulfides. The method identifies thiol components (including glutathione, coenzyme A, and cysteine) of low-molecular-weight thioesters and disulfides in cell extracts, as well as thiols bound to protein via thioester or disulfide links. Thioesters were cleaved with 200 mM hydroxylamine under a nitrogen atmosphere in the presence of monobromobimane (mBBr), which forms a fluorescent derivative with the released thiol. For analysis of disulfides, thioesters were cleaved with hydroxylamine in the presence of N-ethylmaleimide to block released thiols: disulfides were then reduced with 10 mM dithiothreitol and subsequently labeled with mBBr. The bimane derivatives were identified and quantified using previously described HPLC methods (G. L. Newton, R. Dorian, and R. C. Fahey, 1981, Anal. Biochem. 114, 383-387). Traditional methods using dithiothreitol and sodium borohydride to cleave disulfides can also cleave thioesters and thus should not be used for specific analysis of disulfides.     

Measurement of biological disulfides by postcolumn sulfitolysis following separation by HPLC

We developed a method to measure disulfides which is applicable to biological fluids. It consisted of two parts. First, certain thiols and disulfides were separated by HPLC. Second, the eluted materials were submitted to postcolumn reaction with 2-nitro-5-thiosulfobenzoate in the presence of sulfite. The resultant yellow product, 2-nitro-5-thiobenzoate, was measured by its absorbance at 412 nm. We determined the elution characteristics of the thiols and disulfides derived from cysteine, glutathione, alpha-mercaptopropionylglycine (Thiola), and cysteamine. Penicillamine and its disulfide did not react. Cystine in the urine of 22 cystinuric patients, measured by this method, was compared with results obtained by automatic amino acid analysis.     

The Erv family of sulfhydryl oxidases

The Erv flavoenzymes contain a compact module that catalyzes the pairing of cysteine thiols into disulfide bonds. High-resolution structures of plant, animal, and fungal Erv enzymes that function in different contexts and intracellular compartments have been determined. Structural features can be correlated with biochemical properties, revealing how core sulfhydryl oxidase activity has been tailored to various functional niches. The introduction of disulfides into cysteine-containing substrates by Erv sulfhydryl oxidases is compared with the mechanisms used by NADPH-driven disulfide reductases and thioredoxin-like oxidoreductases to reduce and transfer disulfides, respectively.     

Levels of sulfhydryls and disulfides in proteins from Neurospora crassa conidia and mycelia

Proteins extracted with 6 M guanidine at 90 degrees C from conidia (asexual spores) of Neurospora crassa contained ca. 25% more total protein thiol and a fivefold-higher content of disulfide bonds than proteins extracted from mycelia, as determined by labeling with iodo[14C]acetic acid. The total thiol content was 88 mumol/g of protein in conidia and 70 mumol/g of protein in mycelia. The level of protein disulfide was 18.5 mumol/g of protein in conidia and 3.5 mumol/g of protein in mycelia, by the iodo[14C]acetic acid labeling method. Confirmatory results were obtained with 5'5-dithio-bis-2-nitrobenzoic acid titration of protein thiol groups in 1% sodium dodecyl sulfate as well as by amino acid analysis of cysteic acid derivatives. Buffer-extracted proteins from conidia, but not mycelia, were found to contain enriched levels of protein thiols and disulfides per gram of protein as compared with guanidine hydrochloride extracts. It was demonstrated that the high disulfide content of crude conidial extracts was not due to measurable levels of mixed disulfides formed between protein sulfhydryl groups and cysteine. During germination of the conidia, the high disulfide levels of the conidial proteins remained constant. These data suggest that, unlike the disulfides of glutathione, the bulk of conidial protein disulfides were not reduced, excreted, or extensively degraded during germination.     

Distribution of oxidized and reduced forms of glutathione and cysteine in rat plasma

The distribution of the glutathionyl moiety between reduced and oxidized forms in rat plasma was markedly different than that for the cysteinyl moiety. Most of the glutathionyl moiety was present as mixed disulfides with cysteine and protein whereas most of the cysteinyl moiety was present as cystine. Seventy percent of total glutathione equivalents was bound to proteins in disulfide linkage. The distribution of glutathione equivalents in the acid-soluble fraction was 28.0% as glutathione, 9.5% as glutathione disulfide, and 62.6% as the mixed disulfide with the cysteinyl moiety. In contrast, 23% of total cysteine equivalents was protein-bound. The distribution of cysteine equivalents in the acid-soluble fraction was 5.9% as cysteine, 83.1% as cystine, and 10.8% as the mixed disulfide with the glutathionyl moiety. A first-order decline in glutathione occurred upon in vitro incubation of plasma and was due to increased formation of mixed disulfides of glutathione with cysteine and protein. This indicates that plasma thiols and disulfides are not at equilibrium, but are in a steady-state maintained in part by transport of these compounds between tissues during the inter-organ phase of their metabolism. The large amounts of protein-bound glutathione and cysteine provide substantial buffering which must be considered in analysis of transient changes in glutathione and cysteine. In addition, this buffering may protect against transient thiol-disulfide redox changes which could affect the structure and activity of plasma and plasma membrane proteins.     

Determination of thiols and disulfides using high-performance liquid chromatography with electrochemical detection

Low-molecular-mass thiols, such as glutathione (GSH), and their associated disulfides are ubiquitous in nature, and based upon the many known functions of these compounds, their identification and accurate measurement is essential. Our objectives were to develop a simple method for the simultaneous measurement of thiols and disulfides in biological samples using HPLC with dual electrochemical detection (HPLC-DED). Particular emphasis was placed on the applicability to a wide variety of important GSH-related thiols and disulfides, including γ-Glu-Cys, Cys-Gly, their disulfides, and the mixed disulfide of glutathione and cysteine (CSSG), validation on different types of biological samples, maintenance of chromatographic resolution and reproducibility with routine and extended use, and enhancement of assay sensitivity. To this end, optimal HPLC conditions including mobile phase, column, and electrode polishing procedures were established and the method was applied to, and validated on a variety of biological samples. This improved methodology should prove to be a useful tool in studies on the metabolism of GSH and other thiols and disulfides and their role in cellular homeostasis and disease processes.     

Analysis of saliva for glutathione and metabolically related thiols by liquid chromatography with ultraviolet detection

Summary. A method for simultaneous determination of glutathione and its precursors cysteine, cysteinylglycine and homocysteine in saliva is presented. The procedure involves reductive conversion of disulfides to thiols, derivatization to their 2-S-quinolinium derivatives with 2-chloro-1-methylquinolinium tetrafluoroborate and separation and quantitation by reversed-phase ion-pairing high performance liquid chromatography with ultraviolet detection at 355 nm. The calibration performed with saliva samples spiked with thiol disulfides, within the practical concentration ranges, showed linear response of the detector. The method applied to the saliva samples donated by volunteers showed mean concentration (SD, n = 8) of cysteine, cysteinylglycine, glutathione and homocysteine: 26.5 (31.6), 6.05 (5.12), 16.97 (7.68), 3.64 (1.34) nmol/ml respectively.     

Mutagenic activity of 2-chloro-4-nitroaniline and 5-chlorosalicylic acid in Salmonella typhimurium: two possible metabolites of niclosamide

Niclosamide is an anti-helminthic drug susceptible to being metabolized into a bacterial mutagen by the action of enzymes present in the S9 activation mixture. Additional results from genotoxic studies in rodents and humans suggest that the drug is absorbed from the gastrointestinal tract, and mutagenic metabolites are excreted both in the free form and as conjugated glucuronides. As in the case of other secondary amides, phase I metabolism of niclosamide may result in a hydrolytic cleavage of the amide bond, giving rise to 5-chlorosalicylic acid and 2-chloro-4-nitroaniline as the main metabolites. In this study, the mutagenicity of these compounds was tested using the Salmonella typhimurium assay. Bacterial mutagenicity tests with these 2 compounds reveal a non-mutagenic response with 5-chlorosalicylic acid and a mutagenic one with 2-chloro-4-nitroaniline. However, the mutagenic potency observed with this compound is lower than that of niclosamide. The role of nitroreduction in the activation of niclosamide and 2-chloro-4-nitroaniline was also investigated with the help of S. typhimurium strains TA98NR, YG1020, YG1021 and YG1024. The results show a pattern of response which is qualitatively similar for both compounds and this indicates that its mutagenicity depends on both nitroreduction and transacetylation.     

Sulfenic acid formation in human serum albumin by hydrogen peroxide and peroxynitrite

Human serum albumin (HSA), the most abundant protein in plasma, has been proposed to have an antioxidant role. The main feature responsible for this property is its only thiol, Cys34, which comprises approximately 80% of the total free thiols in plasma and reacts preferentially with reactive oxygen and nitrogen species. Herein, we show that the thiol in HSA reacted with hydrogen peroxide with a second-order rate constant of 2.26 M(-1) s(-1) at pH 7.4 and 37 degrees C and a 1:1 stoichiometry. The formation of intermolecular disulfide dimers was not observed, suggesting that the thiol was being oxidized beyond the disulfide. With the reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl), we were able to detect the formation of sulfenic acid (HSA-SOH) from the UV-vis spectra of its adduct. The formation of sulfenic acid in Cys34 was confirmed by mass spectrometry using 5,5-dimethyl-1,3-cyclohexanedione (dimedone). Sulfenic acid was also formed from exposure of HSA to peroxynitrite, the product of the reaction between nitric oxide and superoxide radicals, in the absence or in the presence of carbon dioxide. The latter suggests that sulfenic acid can also be formed through free radical pathways since following reaction with carbon dioxide, peroxynitrite yields carbonate radical anion and nitrogen dioxide. Sulfenic acid in HSA was remarkably stable, with approximately 15% decaying after 2 h at 37 degrees C under aerobic conditions. The formation of glutathione disulfide and mixed HSA-glutathione disulfide was determined upon reaction of hydrogen peroxide-treated HSA with glutathione. Thus, HSA-SOH is proposed to serve as an intermediate in the formation of low molecular weight disulfides, which are the predominant plasma form of low molecular weight thiols, and in the formation of mixed HSA disulfides, which are present in approximately 25% of circulating HSA.     

Modulation of cellular disulfide-bond formation and the ER redox environment by feedback regulation of Ero1

Introduction of disulfide bonds into proteins entering the secretory pathway is catalyzed by Ero1p, which generates disulfide bonds de novo, and Pdi1p, which transfers disulfides to substrate proteins. A sufficiently oxidizing environment must be maintained in the endoplasmic reticulum (ER) to allow for disulfide formation, but a pool of reduced thiols is needed for isomerization of incorrectly paired disulfides. We have found that hyperoxidation of the ER is prevented by attenuation of Ero1p activity through noncatalytic cysteine pairs. Deregulated Ero1p mutants lacking certain cysteines show increased enzyme activity, a decreased lag phase in kinetic assays, and growth defects in vivo. We hypothesize that noncatalytic cysteine pairs in Ero1p sense the level of potential substrates in the ER and correspondingly modulate Ero1p activity as part of a homeostatic regulatory system governing the thiol-disulfide balance in the ER.     

Molecular basis of superreactivity of cysteine residues 31 and 32 of seminal ribonuclease

The molecular basis of the high reactivity toward reducing agents of intersubunit disulfides at positions 31 and 32 of dimeric bovine seminal ribonuclease was investigated by studying in the monomeric enzyme the fast reaction kinetics with disulfides of the adjacent cysteine-31 and -32, exposed by selective reduction of the intersubunit disulfides. Negatively charged and neutral disulfide reagents were used for measuring the thiol reaction rates at neutral pH. The kinetics studied as a function of pH permitted us to define pK values for the thiols of interest and indicated the possibility of determining pK values of SH groups in proteins indirectly by measuring the kinetics of reactivity of the SH groups with a disulfide reagent. The results were compared with those obtained under identical conditions with synthetic thiol peptides and model compounds. The data indicate that the superreactivity of intersubunit disulfides of seminal ribonuclease is matched by the high reactivity at neutral pH of adjacent cysteine residues 31 and 32, as compared to all small thiol compounds tested. The synthetic hexapeptide segment of seminal ribonuclease Ac-Met-Cys-Cys-Arg-Lys-Met-OH, which includes the two cysteine residues of interest, was even more reactive. These data, and the other results reported in this paper, led to the conclusion that the superreactivity at neutral pH of cysteine residues at positions 31 and 32 of bovine seminal ribonuclease is primarily dependent on the nearby presence of positively charged groups, particularly the epsilon-NH2 of lysine-34, and is influenced by the adjacency of the two thiols and by the protein tertiary structure.