THE INFLUENCE OF PROTEINS ON THE REACTIVATION OF YEAST INVERTASE

1. Acid-inactivated yeast invertase could not be regenerated in the presence of the proteolytic enzymes trypsin, pepsin, and chymotrypsin. 2. Certain foreign proteins of non-enzymatic nature partially inhibited the reactivation of acid-inactivated invertase. 3. Certain proteins as gelatin, lacto-globulin, and carbohydrate-free horse crystalbumin did not prevent the reactivation of invertase at all. 4. Highly purified reactivated invertase was shown to exhibit an effect typical of original native invertase; that is, acceleration of its activity in presence of foreign protein at pH 3.0. 5. Native invertase was not digested by trypsin and chymotrypsin. 6. The addition of trypsin and chymotrypsin to reactivating invertase did not affect the invertase which had already reverted to the active form, but prevented further reactivation of inactive invertase.     

Configuration of thiols dictates their ability to promote iron-induced reactive oxygen species generation

Abstract

Iron catalyzes the production of reactive oxygen species (ROS) through the Fenton reaction. The modification of this phenomenon in the presence of various thiol compounds that are nominally reducing agents has been studied. Using the synaptosomal/mitochondrial (P2) fraction of rat cerebral cortex as a biological source of reactive oxygen species (ROS) production, we studied the influence of four compounds, glutathione (GSH), cysteine, N-acetyl-cysteine (NAC), and homocysteine on iron-induced ROS production. None of the thiol compounds alone, at the concentrations used, affected the basal rate of ROS production in the P2 fraction. GSH, homocysteine and NAC did not alter Fe-induced ROS generation, while cysteine greatly potentiated ROS formation. Measurement of the rate of ROS production in the presence of varying concentrations of cysteine together with 20 µM ferrous iron revealed a dose-response relationship. The mechanism whereby free cysteine, but not the cysteine-containing peptide GSH, homocysteine or NAC with a blocked amino group, exacerbates the prooxidant properties of ferrous iron probably involves formation of a complex between iron, a sulfhydryl and a free carboxyl residue located at a critical distance from the –SH group. Cysteine-iron interactions may, in part, account for the excessive toxicity of free cysteine in contrast to GSH and NAC.     

A novel diazonium-sulfhydryl reaction in the inactivation of yeast alcohol dehydrogenase by diazotized 3-aminopyridine adenine dinucleotide.

Diazotized 3-aminopyridine adenine dinucleotide has been found to modify four sulfhydryl groups per molecule of enzyme during the complete inactivation of yeast alcohol dehydrogenase. The reaction of sulfhydryl groups was indicated by titration studies with 5,5-dithiobis(2-nitrobenzoic acid) as well as isolation and quantitation of the cysteinyl derivative released by acid hydrolysis of the modified enzyme. The cysteinyl derivative was identified as S-(3-pyridyl)cysteine. Authentic S-(3-pyridyl)cystein was synthesized and structurally characterized for these studies. Diazonium-sulfhydryl reactions were demonstrated for a number of diazonium derivatives with cysteine, homocysteine, glutathione, and mercaptoethanol at 0-4 degrees and neutral pH. Second order rate constants were determined in reactions of these sulfhydryl compounds with diazotized 1-methyl-3-aminopyridinium chloride, diazotized 3-aminopyridine adenine dinucleotide, and diazotized 3-aminopyridine adenine dinucleotide phosphate.     

Effect of Cysteine and Its Derivatives on 1-Methyladenine Production by Starfish Follicle Cells

Spawning of the gametes in the starfish, Asterina pectinifera , induced by a gonad-stimulating substance (GSS) was inhibited by cysteine. This inhibitory effect of cysteine was due to the reduction of GSS activity and the inhibition of 1-methyadenine (1-MeAde) production in follicle cells. Homocysteine and cysteamine also reduced GSS activity, but cystamine had no effect on the activity. This suggests that the loss of GSS activity is related to a SH group. Furthermore, the effect of cysteine on 1-MeAde production was investigated using isolated follicle cells. The half maximum decrease of the GSS-dependent 1-MeAde production in follicle cells was obtained with the concentration of 2.0 mM L-cysteine. A similar effect was observed with D-cysteine. Homo cysteine and cysteamine also reduced the GSS-dependent 1-MeAde production. But methionine, serine, glutamic acid and aspartic acid did not affect the 1-MeAde production of follicle cells. In addition, cystamine also inhibited the GSS-dependent 1-MeAde production. These suggest that both a SH group and a SS bond of these compounds are effective on the inhibition of the GSS-dependent 1-MeAde biosynthesis. Cysteine and homocysteine also inhibited concanavalin A (Con A).induced 1-MeAde production of follicle cells, but they had no effect on binding of Con A to the surface of follicle cells.     

SOME FACTORS WHICH INFLUENCE THE OXIDATION OF SULFHYDRYL GROUPS

1. Cyanide inhibits the oxidation of the SH groups of cysteine and denatured egg albumin by the uric acid reagent. 2. At pH 4.8 cysteine is oxidized by the uric acid reagent and by ferricyanide in the presence but not in the absence of added copper sulfate. 3. In neutral solution, the uric acid reagent oxidizes the SH groups of denatured egg albumin in the presence of urea but not in the presence of alkyl sulfate or in the absence of denaturing agents. 4. Ferricyanide oxidizes the SH groups of neutral denatured egg albumin even in the presence of alkyl sulfate or, if precautions are taken to avoid aggregation, in the absence of denaturing agents. 5. In acid solution, ferricyanide does not oxidize the SH groups of denatured egg albumin completely. The oxidation is more complete, however, in the presence of urea than in the presence of alkyl sulfate, and more complete in the presence of guanidine hydrochloride than in the presence of urea. 6. The uric acid reagent which does not oxidize the SH groups of neutral denatured but unhydrolyzed egg albumin in the absence of denaturing agents does, under the same conditions, oxidize the SH groups of egg albumin partially hydrolyzed by pepsin. 7. At pH 4.8 in alkyl sulfate solution ferricyanide oxidizes the SH groups of digested egg albumin more completely than the SH groups of denatured but undigested egg albumin.     

Changes in the intracellular homocysteine and glutathione content associated with aging

Since moderate hyperhomocysteinemia is an independent risk factor for vascular disease by mean of its oxidant effect and glutathione plays a main role as intracellular redox-regulating agent, we have studied for the first time the total intracellular content of homocysteine in aging. Plasma homocysteine concentration, total intracellular and plasma glutathione, and other related thiol compounds such as cysteine and the glutathione catabolite cysteinglycine were also studied. Forty three healthy elderly subjects and twenty seven healthy young ones were studied. The total intracellular peripheral blood mononuclear cell content was higher for homocysteine, cysteine and cysteinglycine, whereas that of the total glutathione was greatly decreased in elderly people with respect to young ones. Elderly subjects showed significantly higher levels than young ones of total plasma homocysteine and cysteinglycine, but not cysteine, whereas total plasma glutathione levels were increased. In addition, elderly subjects showed significantly decreased plasma vitamin E levels and increased concentrations of serum lipid peroxides measured as TBARS (reaction product of malondialdehyde with thiobarbituric acid). The intracellular glutathione content presented significantly negative correlation with serum TBARS, and intracellular and plasma homocysteine levels. These findings show an increase of homocysteine synthesis associated with aging, which in turn can produce an augmented oxidant effect on endothelium, and an impaired intracellular antioxidant capacity leading to an enhanced lipid peroxidation and decreased total intracellular glutathione content.     

Oxidation of active center cysteine of bovine 1-Cys peroxiredoxin to the cysteine sulfenic acid form by peroxide and peroxynitrite.

Peroxiredoxins are antioxidant enzymes whose peroxidase activity depends on a redox-sensitive cysteine residue at the active center. In this study we investigated properties of the active center cysteine of bovine 1-Cys peroxiredoxin using a recombinant protein (BRPrx). The only cysteine residue of the BRPrx molecule was oxidized rapidly by an equimolar peroxide or peroxynitrite to the cysteine sulfenic acid. Approximate rates of oxidation of BRPrx by different peroxides were estimated using selenium glutathione peroxidase as a competitor. Oxidation of the active center cysteine of BRPrx by H2O2 proceeded only several times slowly than that of the selenocysteine of glutathione peroxidase. The rate of oxidation varied depending on peroxides tested, with H2O2 being about 7 and 80 times faster than tert-butyl hydroperoxide and cumene hydroperoxide, respectively. Peroxynitrite oxidized BRPrx slower than H2O2 but faster than tert-butyl hydroperoxide. Further oxidation of the cysteine sulfenic acid of BRPrx to higher oxidation states proceeded slowly. Oxidized BRPrx was reduced by dithiothreitol, dihydrolipoic acid, and hydrogen sulfide, and demonstrated peroxidase activity (about 30 nmol/mg/min) with these reductants as electron donors. beta-Mercaptoethanol formed a mixed disulfide and did not support peroxidase activity. Oxidized BRPrx did not react with glutathione, cysteine, homocysteine, N-acetyl-cysteine, and mercaptosuccinic acid.     

Configuration of thiols dictates their ability to promote iron-induced reactive oxygen species generation

Iron catalyzes the production of reactive oxygen species (ROS) through the Fenton reaction. The modification of this phenomenon in the presence of various thiol compounds that are nominally reducing agents has been studied. Using the synaptosomal/mitochondrial (P2) fraction of rat cerebral cortex as a biological source of reactive oxygen species (ROS) production, we studied the influence of four compounds, glutathione (GSH), cysteine, N-acetyl-cysteine (NAC), and homocysteine on iron-induced ROS production. None of the thiol compounds alone, at the concentrations used, affected the basal rate of ROS production in the P2 fraction. GSH, homocysteine and NAC did not alter Fe-induced ROS generation, while cysteine greatly potentiated ROS formation. Measurement of the rate of ROS production in the presence of varying concentrations of cysteine together with 20 microM ferrous iron revealed a dose-response relationship. The mechanism whereby free cysteine, but not the cysteine-containing peptide GSH, homocysteine or NAC with a blocked amino group, exacerbates the pro-oxidant properties of ferrous iron probably involves formation of a complex between iron, a sulfhydryl and a free carboxyl residue located at a critical distance from the -SH group. Cysteine-iron interactions may, in part, account for the excessive toxicity of free cysteine in contrast to GSH and NAC.     

The determination of glutathione, cyst(e)ine, and other thiols and disulfides in biological samples using high-performance liquid chromatography with dual electrochemical detection

A determination of glutathione, cysteine, and their disulfides using HPLC and dual electrochemical detection (HPLC-DEC) was described previously but was not validated in biological tissues for these and other important thiols and disulfides (SH/SS). Thus, our objectives were to develop this method to quantify simultaneously reduced and oxidized glutathione, cysteine, cystine, and other SH/SS in various tissues, including human blood and plasma, rat liver and hippocampus, mosquito, and spinach leaf. Optimal conditions were determined for sample processing and analysis using metaphosphoric acid and HPLC-DEC. Authentic standards of 10 common SH/SS compounds were resolved and eluted within 15 min, and all standard curves were linear from 5 to 1600 pmol. Validation was based on the following: First, tissue sample sizes were proportional to peak areas over an eightfold range. Second, recovery of SH/SS added to samples before processing was 96-101%. Finally, the results were equivalent and correlated highly with values for total SH by 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) assay (r2 = 0.996) and for total glutathione by DTNB-GSSG reductase assay (r2 = 0.998). The life span of the Au/Hg electrode was limited to 200-500 samples based on the lineal range of standard curves. On the basis of these results, we believe that this method will fill a long-time need for the simultaneous determination of SH/SS in biological tissues.     

Saccharomyces cerevisiae secretes and correctly processes human interferon hybrid proteins containing yeast invertase signal peptides.

Synthetic oligonucleotides coding for the yeast invertase secretion signal peptide were fused to the gene for the mature form of human interferon (huIFN-alpha 2). Two plasmids (E3 and F2) were constructed. E3 contained the invertase signal codons in a reading frame with the mature huIFN-alpha 2 gene. F2 had a deletion of the codon for alanine at amino acid residue-5 in the invertase signal and an addition of a methionine codon located between the coding sequences for the invertase signal and mature huIFN-alpha 2. Both hybrid genes were located adjacent to the promoter from the 3-phosphoglycerate kinase gene on the multicopy yeast expression plasmid, YEp1PT. Yeast transformants containing these plasmids produced somewhat more IFN than did the same expression plasmid containing the IFN gene with its human secretion signal sequence. HuIFN-alpha 2, purified from the medium of yeast cells containing E3, was found to be processed at the correct site. The huIFN-alpha 2 made by plasmid F2 was found to be completely processed at the junction between the invertase signal (a variant) and the methionine of methionine-huIFN-alpha 2. These results strongly suggested that the invertase signal (or its variant) attached to huIFN was efficiently recognized by the presumed signal recognition particle and was cleaved by the signal peptidase in the yeast cells. These results also suggested that amino acid changes on the right side of the cleavage site did not necessarily prevent cleavage or secretion.     

Effect of glycosylation on the mechanism of renaturation of invertase from yeast

N-Glycosylation occurs cotranslationally as soon as the growing polypeptide chain enters the endoplasmic reticulum, before the final native-like folded state is reached. We examined the role of the carbohydrate chains in the mechanism of protein folding. The in vitro folding and association of yeast invertase are used as an experimental system. External invertase contains approximately 50% carbohydrate, whereas cytoplasmic invertase is not glycosylated. The functional native state of both proteins is a homodimer. At pH greater than or equal to 6.5 and at protein concentrations below 3 micrograms/ml, the kinetics of reactivation and the final yields are similar for the two invertases. For both proteins, reactivation is a sequential reaction with a lag phase at the beginning. The nonglycosylated protein tends to aggregate during reactivation at low pH and at protein concentrations above 3 micrograms/ml. After separation of inactive material, the renatured protein is indistinguishable from the original native state by a number of physicochemical and functional criteria. The results suggest that the carbohydrate moiety does not affect the mechanism of folding and association of invertase. However, glycosylation improves the solubility of unfolded or partially folded invertase molecules and hence leads to a suppression of irreversible aggregation. Such a protective effect may also be important for the in vivo maturation of nascent glycosylated protein chains.     

Free Sulfhydryl Groups in Ripening Fruits

Ripening was found to be accompanied by an increase in sulfhydryl (SH) group content in several fruits. In ripening, climacteric,tomato fruits, this increase was due mostly to an increase inthe glutathione level. In orange, a non-climacteric fruit, therelatively high SH levels did not change during developmentand maturation. Non-ripening tomato mutants, e.g., rin and nor,were characterized by low and constant SH levels during fruitgrowth and senescence. Ripening-inducing storage conditions,such as high temperature (30?C) and ethylene treatment, concomitantlyhastened the increase in SH level in fast-ripening tomato varieties.Storage conditions that slowed down the ripening process, suchas low temperatures (2, 12?C) and low oxygen levels (5%), sloweddown the increase in SH content. Storage of red tomatoes at30?C caused an increase in the SH content in comparison withlower temperature treatments (2, 12, 20?C). SH compounds (reducedglutathione, cysteine), dithiothreitol and an SH-binding compound(n-ethylmaleimide), did not affect the ripening process of greentomatoes. The results suggest that the increase in SH groupsaccompanies the ripening processes rather than regulating them. (Received April 26, 1982; Accepted September 6, 1982)     

Intensification of tumor affinity of 99mTc—dl-homocysteine by cooperative use of SH-containing compounds

The enhancement of tumor-specific distribution was investigated on ^99mTc—dl-homocysteine (^99mTc-Hcy), a possible tumor-imaging agent. In vitro HPLC analysis showed that ^99mTc-Hcy did not bind to nonmercaptalbumin but to mercaptalbumin in blood. When glutathione, homocysteine or cysteine, which converts albumin from the mercapto- to nonmercapto-form, was intravenously injected into Tc-Hcy predosed mice bearing Ehrlich solid tumor, the SH-containing compounds did not affect the radioactivity distribution in tumor, but decreased that in blood to less than half that of untreated animals. In vitro gel filtration analysis showed that glutathione released ^99mTc-Hcy from albumin. Free ^99mTc-Hcy produced in blood by the treatment was rapidly excreted into urine. These results suggest that the combination of ^99mTc-Hcy and SH-containing compounds is useful for tumor-imaging.     

THE ESTIMATION OF SULPHATE-REDUCING BACTERIA (D. DESULPHURICANS)

SUMMARY: Sodium sulphide or cysteine stimulated the growth of sulphate-reducing bacteria; small populations often did not grow without such supplements. Ascorbic acid, glutathione or thiolacetic acid had similar properties but thiolacetic acid was sometimes inhibitory, Dilution counts in liquid media or colony counts in agar media did not bear any regular relation to the total count unless one of these supplements was present. With suitable precautions colony counts reaching 50 to 60% of the total count were obtained in media incorporating cysteine and a ferrous salt (as an indicator of sulphide formation).
Samples of natural origin containing sulphate-reducing bacteria gave greater viable counts in cysteine-iron media than in unsupplemented media. Blackend culture tubes with natural populations were sometimes due to cysteine-decomposing organisms; further examination of positive tubes was therefore necessary.     

Stimulation of h(2)s emission from pumpkin leaves by inhibition of glutathione synthesis

The effect of inhibitors of glutathione (GSH) synthesis, namely gamma-methyl glutamic acid, d-glutamic acid, cystamine, methionine-S-sulfoximine (MSX), buthionine-S-sulfoximine, and GSH itself, on the emission of H(2)S was investigated. All these compounds stimulated H(2)S emission from pumpkin (Cucurbita pepo L. cv Small Sugar Pumpkin) leaf discs in response to sulfate. MSX and GSH were the most effective compounds, stimulating H(2)S emission from leaf discs of mature pumpkin leaves by about 80% in response to sulfate. Both inhibitors did not appreciably enhance H(2)S emission in response to l-cysteine and inhibited H(2)S emission in response to sulfite.Treatment with MSX or GSH enhanced the uptake of sulfate by pumpkin leaf discs, but did not affect the incorporation of sulfate into reduced sulfur compounds. Inhibition of GSH synthesis by MSX or GSH caused an increase in the pool size of cysteine, and, simultaneously, reduced the incorporation of labeled sulfate into cysteine. The incorporation of labeled sulfate into the sulfite and sulfide pools of the cells are stimulated under these conditions.These observations are consistent with the idea that inhibition of GSH synthesis leads to an elevated cysteine pool that inhibits further cysteine synthesis. The H(2)S emitted under these conditions appears to arise from diversion of a precursor of the sulfur moiety of l-cysteine. Therefore, stimulation of H(2)S emission in response to sulfate upon inhibition of GSH synthesis may reflect a role of H(2)S emission in keeping the cysteine concentration below a critical level.     

Quantitative determination of SH groups in low- and high-molecular-weight compounds by an electron spin resonance method

To quantitatively determine SH groups in high- and low-molecular-weight compounds, a disulfide biradical (RS-SR), where R is imidazoline residue, has been used. The biradical is shown to participate in a thiol-disulfide exchange reaction with compounds containing SH groups. In this case the ESR spectra of the biradical RS-SR and the resulting monoradical R-SH are different. The reaction of the biradical with cysteine, glutathione, and human serum albumin has been studied using the ESR method and the rate constants kf of this reaction have been calculated. Studies of the pH dependence of kf indicate that the thiol-disulfide exchange occurs by reaction with mercaptidione. Protein human serum albumin and hemoglobin have been modified by RS-SR. It has been shown that the treatment of modified proteins with reduced glutathione leads to removal of the radical from the protein; such modifications are thus reversible. The method proposed has been used to quantitatively determine the SH groups of cysteine and glutathione in mouse and rat blood. The method is shown to coincide within experimental error with the determination of glutathione and cysteine by titration with p-chloromercuribenzoate or reaction with Ellman's reagent. This method allows detection of 10(-6)-10(-7) M SH compounds even in colored and highly absorbing samples. The kinetics of the SH group modification can also be determined, leading to deduction about accessibility of the SH group in protein.     

Assay of biological thiols by a combination of high-performance liquid chromatography and postcolumn reaction with 6,6'-dithiodinicotinic acid

A simple and rapid method for the determination of nanomole levels of biological thiols is described. The analysis is based on the combination of reverse-phase high-performance liquid chromatography with a postcolumn reaction with 6,6'-dithiodinicotinic acid. Thiols, including cysteine, cysteamine, thiolhistidine, homocysteine, glutathione, penicillamine, ergothioneine, and thiouracil were separated by eluting with 33 mM KH2PO4 at pH 2.2. Glutathione, cysteine, cysteamine, homocysteine, and penicillamine were quantitatively determined with detection limits of 0.1 nmol, while the quantitative detection of thiolhistidine, ergothioneine, and thiouracil was not successful. The method was applied to the assay of glutathione in human erythrocytes and Escherichia coli.     

Effect of different antioxidants and free radical scavengers on aflatoxin production

Different antioxidants and free radical scavengers on aflatoxin production are analysed. The different compounds at different concentration were used: buthylated hydroxyanisole (BHA), buthylated hydroxytoluene (BHT), α-tocopherol (vitamin E), ascorbic acid (vitamin C), reduced glutathione, cysteine, cysteamine. The above compounds were tested in culture ofAspergillus parasiticus supplemented with carbon tetrachloride, a potent stimulating agent of aflatoxin biosynthesis. Cysteamine and BHA highly inhibited the aflatoxin production induced by carbon tetrachloride, the inhibition decreased by lowering the concentration. On the contrary, vitamin E, vitamin C, reduced glutathione and cysteine further enhanced the carbon tetrachloride stimulating effect. The addition of the above compounds did not significantly affect the growth of the fungal mycelia.     

Reactivity toward oxygen radicals and antioxidant action of thiol compounds

Thiol compounds exert diverse functions in the defense network against oxidative stress in vivo. Above all, the role of glutathione in the enzymatic removal of hydrogen peroxide and lipid hydroperoxides has been well established. The scavenging of reactive free radicals is one of the many functions. In this study, the reactivities of several thiol compounds toward oxygen- and nitrogen-centered radicals were measured from their reaction with galvinoxyl and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and also from their sparing effects on the decay of fluorescein, pyrogallol red, and BODIPY induced by peroxyl radicals. Furthermore, the antioxidant capacity against lipid peroxidation was assessed in the oxidation of methyl linoleate induced by free radicals in micelle systems. Cysteine, homocysteine, and glutathione exhibited considerable reactivity toward galvinoxyl, DPPH, and peroxyl radicals in this order but methionine did not. Bovine serum albumin (BSA) was less reactive toward these radicals than cysteine on molar base. Cysteine, homocysteine, and glutathione suppressed the oxidation of methyl linoleate in micelle systems, but methionine did not. The reactivity toward free radicals and antioxidant capacity of these thiol compounds were less than that of ascorbic acid, but higher than that of uric acid.