Glutathione Sulfhydryl Groups Research Articles

Characterization of sulfhydryl oxidase from Aspergillus tubingensis

BackgroundDespite of the presence of sulfhydryl oxidases (SOXs) in the secretomes of industrially relevant organisms and their many potential applications, only few of these enzymes have been biochemically characterized. In addition, basic functions of most of the SOX enzymes reported so far are not fully understood. In particular, the physiological role of secreted fungal SOXs is unclear.ResultsThe recently identified SOX from Aspergillus tubingensis (AtSOX) was produced, purified and characterized in the present work. AtSOX had a pH optimum of 6.5, and showed a good pH stability retaining more than 80% of the initial activity in a pH range 4-8.5 within 20 h. More than 70% of the initial activity was retained after incubation at 50 °C for 20 h. AtSOX contains a non-covalently bound flavin cofactor. The enzyme oxidised a sulfhydryl group of glutathione to form a disulfide bond, as verified by nuclear magnetic resonance spectroscopy. AtSOX preferred glutathione as a substrate over cysteine and dithiothreitol. The activity of the enzyme was totally inhibited by 10 mM zinc sulphate. Peptide- and protein-bound sulfhydryl groups in bikunin, gliotoxin, holomycin, insulin B chain, and ribonuclease A, were not oxidised by the enzyme. Based on the analysis of 33 fungal genomes, SOX enzyme encoding genes were found close to nonribosomal peptide synthetases (NRPS) but not with polyketide synthases (PKS). In the phylogenetic tree, constructed from 25 SOX and thioredoxin reductase sequences from IPR000103 InterPro family, AtSOX was evolutionary closely related to other Aspergillus SOXs. Oxidoreductases involved in the maturation of nonribosomal peptides of fungal and bacterial origin, namely GliT, HlmI and DepH, were also evolutionary closely related to AtSOX whereas fungal thioreductases were more distant.ConclusionsAtSOX (55 kDa) is a fungal secreted flavin-dependent enzyme with good stability to both pH and temperature. A Michaelis-Menten behaviour was observed with reduced glutathione as a substrate. Based on the location of SOX enzyme encoding genes close to NRPSs, SOXs could be involved in the secondary metabolism and act as an accessory enzyme in the production of nonribosomal peptides.

Two-stage ratiometric fluorescent responsive probe for rapid glutathione detection based on BODIPY thiol-halogen nucleophilic mono- or disubstitution

Abstract 3,5-dichloro-8-phenyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BOD-2Cl) (1) as a classical fluorescent dye was synthesized for the purpose of functionalization of BODIPY dye. Herein, it was applied for the purpose of achieving two-stage ratiometric fluorescent detection of glutathione based on the two-step thiol-halogen substitution reaction with the sulfhydryl group of glutathione at the 3 (mono-substitution) and 3,5-position (disubstitution) of 1 in CTAB micelles. The first stage induced fluorescent color change from green (λem = 530 nm) to yellow (λem = 562 nm) (I562nm/I530nm, mono-substitution), excess addition of glutathione caused the second stage fluorescent color change from yellow to red (λem = 597 nm) (I597nm/I562nm, disubstitution). It exhibits excellent properties with specific colorimetric and ratiometric fluorescent change to glutathione over cysteine and homocysteine. Further application to cellular ratiometric fluorescence imaging indicated that the probe was highly responsive to the glutathione in cells.

Thiol modification by bioactivated polyphenols and its potential role in skin inflammation.

In the present study, we evaluated the modifying behavior of simple phenolic compounds on the sulfhydryl groups of glutathione and proteins. The catechol-type polyphenols, including protocatechuic acid, but neither the monophenols nor O-methylated catechol, can modify the sulfhydryl groups in a phenol oxidase-dependent manner. The possible involvement of polyphenol bioactivation in the enhancement of skin inflammation was also suggested.

Ultrasound-induced formation of S-nitrosoglutathione and S-nitrosocysteine in aerobic aqueous solutions of glutathione and cysteine.

S-Nitrosocompounds are formed when aqueous solutions of cysteine or glutathione are exposed to ultrasound (880 kHz) in air. The yield of the S-nitrosocompounds was as high as 10% for glutathione and 4% for cysteine of the initial thiol concentrations (from 0.1 to 10 mM) in the aqueous solutions. In addition to the formation of S-nitrosocompounds, thiol oxidation to disulfide forms was observed. After the oxidation of over 70% of the sulfhydryl groups, formation of peroxide compounds as well as cysteic acid derivatives was recorded. The formation of the peroxide compounds and peroxide radicals in the ultrasound field reduced the yield of S-nitrosocompounds. S-Nitrosocompounds were not formed when exposing low-molecular-weight thiols to ultrasound in atmospheres of N(2) or CO. In neutral solutions, ultrasound-exposed cysteine or glutathione released NO due to spontaneous degradation of the S-nitrosocompounds. N(2)O(3), produced due to the spontaneous degradation of the S-nitrosocompounds in air, nitrosylated sulfhydryl groups of glutathione manifested in the appearance of new absorption bands at 330 and 540 nm. The nitrogen compounds formed in an ultrasound field modified the sulfhydryl groups of apohemoglobin and serum albumin. The main target for ultrasound-generated oxygen free radicals were cystine residues oxidized to cysteic acid residues.

Glutathione conjugation and conversion to mercapturic acids can occur as an intrahepatic process.

By catalyzing the reaction of electrophilic compounds with the sulfhydryl group of glutathione, the glutathione S-transferases play physiologically important roles in the detoxication of potential alkylating agents. The glutathione S-conjugates thus formed are transported out of cells for further metabolism by gamma-glutamyltransferase and dipeptidases, ectoproteins that catalyze the sequential removal of the glutamyl and glycyl moieties, respectively. These ectoproteins are not found in all cells, but are localized predominantly to the apical surface of epithelial tissues. The resulting cysteine S-conjugates can be reabsorbed by specific cell types, and acetylated on the amino group of the cysteinyl residue by intracellular N-acetyl-transferases, to form the corresponding mercapturic acids (N-acetylcysteine S-conjugates). Mercapturic acids are then released into the circulation and delivered to the kidney for excretion in urine, or they may undergo further metabolism. Mercapturic acid biosynthesis is generally considered to be an interorgan process, with the liver serving as the major site of glutathione conjugation, and the kidney as the primary site for conversion of glutathione conjugates to cysteine conjugates. Cysteine conjugates formed in the kidney appear to be transported back to the liver for acetylation. This interorgan model of mercapturic acid synthesis is based largely on the interorgan distribution of the enzymes involved in their formation, and in particular of the enzyme gamma-glutamyltransferase. Rats have relatively low hepatic and high renal activities of gamma-glutamyltransferase, the only protein known to initiate the breakdown of glutathione S-conjugates. The low gamma-glutamyltransferase activity in rat liver limits the hepatic degradation of glutathione S-conjugates, particularly after large doses of xenobiotic. In contrast, hepatic gamma-glutamyltransferase is significantly higher in species such as rabbit, guinea pig, and dog, and as a consequence, nearly all of the glutathione and glutathione S-conjugates released by liver cells of these species is degraded within the liver. Recent studies demonstrate that glutathione S-conjugates synthesized within hepatocytes are secreted preferentially across the canalicular membrane into bile, and are broken down within biliary spaces to form cysteine S-conjugates. The latter are then reabsorbed by the liver, N-acetylated to form mercapturic acids, and reexcreted into bile, completing an intrahepatic pathway for mercapturic acid biosynthesis. The contribution of this intrahepatic pathway to overall mercapturate formation is dependent on dose of the electrophile, route of exposure, and the physicochemical properties of the glutathione S-conjugate formed, as well as the tissue distribution and activity of gamma-glutamyltransferase.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of pH on the modification of thiols by carbamoylating agents and effects on glutathione levels in normal and neoplastic cells.

In previous studies, we have suggested that the selective inhibitory effect of sodium cyanate (NaOCN) on hepatoma metabolism may be due to the lower pH observed in tumors relative to normal tissues. Lower pH might enhance the action of NaOCN by increasing the formation of isocyanic acid and carbamoylation of sulfhydryl groups. In the present work, studies were conducted on the effect of pH on the carbamoylation of sulfhydryl groups. The data indicated that carbamoylation of the sulfhydryl group of glutathione by NaOCN was enhanced by decreasing the pH from 7.4 to 6.6. A less pH-dependent response was observed with organic isocyanates. However, all reactions were reversible after the pH was increased by the addition of base. Kinetic studies showed that the rate of the reaction is very rapid, a maximal effect occurring within the first 10 min. Dose-dependent modifications of cellular glutathione by NaOCN and organic isocyanates were observed in human HT29 colon tumor cells, rat HTC hepatoma cells, and rat hepatocytes. The rate of carbamoylation of the glutathione sulfhydryl group in cells was similar to that of pure glutathione (GSH). The effect of buthionine sulfoxamine on GSH levels in cells was at least as great as that of sodium cyanate, but only the latter showed inhibitory effects on macromolecular synthesis; these were very rapid, pH-dependent, and reversible in tumor cells. Our results suggest that cellular sulfhydryl group(s) other than that of GSH might be involved in the effect of NaOCN on macromolecular synthesis.

Expression and sequence analysis of rat liver glutathione S-transferase genes.

The rat liver glutathione S-transferases are a family of enzymes which catalyze the conjugation of the reduced sulfhydryl group of glutathione with various electrophiles. In addition, the transferases bind with high affinity various exogenous hydrophobic compounds as well as potentially toxic endogenous compounds such as bilirubin and heme (1–3). The enzymes are comprised of binary combinations of at least six major subunits, Yα, Ya, Ybl, Yb2, Yc and Yn, which can be separated by onedimensional SDS-polyacrylamide gel electrophoresis (4–6).

Leukotrienes.

The leukotrienes are a family of biologically active molecules, formed by leukocytes, mastocytoma cells, macrophages, and other tissues and cells in response to immunological and nonimmunological stimuli. They exhibit a number of biological effects such as contraction of bronchial smooth muscles, stimulation of vascular permeability, and attraction and activation of leukocytes. Compared to histamine, which causes constriction of airways and edema formation, the leukotrienes are three to four orders of magnitude more potent and the effects have longer duration. The leukotrienes were discovered in 1938 as a smooth muscle-contracting factor in lung perfusates. It was referred to as "slow reacting substance" (SRS) or "slow reacting substance of anaphylaxis" (SRS-A) until 1979 when its structure was reported. The term "leukotriene" was introduced at that time as a trivial name for the new type of compound. Leukotrienes C4 and D4 are glutathione and cysteinylglycine conjugates, respectively, of arachidonic acid. After hydrolytic release from phospholipids of the cell membrane, arachidonic acid is oxygenated by a lipoxygenase to 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid. This product is further converted to leukotrienes by elimination of the 10-pro-R hydrogen and OH from the hydroperoxy group to give 5,6-oxido-7,9,11, 14-eicosatetraenoic acid (leukotriene A4). Nucleophilic opening of the epoxide at C-6 by the sulfhydryl group of glutathione gives leukotriene C4, which is metabolized to leukotrienes D4 and E4 by sequential elimination of glutamic acid and glycine. The latter reactions are catalyzed by gamma-glutamyl transpeptidase and a particulate dipeptidase from kidney. Alternatively, water may add at C-12 of leukotriene A4, leading also to opening of the epoxide at C-6 with formation of 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid (leukotriene B4). Leukotriene B4 is metabolized by omega-hydroxylation to 20-hydroxy and 20-carboxy leukotriene B4. Leukotrienes are also formed from eicosatrienoic acid (n-9) and eicosapentaenoic acid (n-3) after oxygenation at C-5 and from eicosatrienoic acid (n-6) and arachidonic acid after oxygenation at C-8 (eicosatrienoic acid) and C-12 or C-15 (arachidonic acid). Although they are formed from the same and additional fatty acids as prostaglandins and thromboxanes [reviewed in this series in (1)], the structures and the reactions involved in biosynthesis and catabolism of leukotrienes are completely separate from those required for prostaglandin formation and metabolism. The leukotrienes seem to provide a new system of biological regulators that are important in many diseases involving inflammatory or immediate hypersensitivity reactions.

Synthesis and characterization of cis- and trans-2,3-epoxybutane-1,4-diol 1,4-bisphosphate, potential affinity labels for enzymes that bind sugar bisphosphates

cis- and trans-2,3-Epoxybutane-1,4-diol 1,4-bisphosphate, which can be considered reactive analogs of several sugar bisphosphates, have been synthesized in a continuing effort to develop new and diverse affinity labeling reagents for enzymes which bind phosphorylated substrates. cis-2,3-Epoxybutane-1,4-diol was obtained by epoxidation of commercially available cis-2-butene-1,4-diol with m-chloroperbenzoic acid; the trans epoxide was obtained by reduction of 2-butyne-1,4-diol with LiAlH 4 followed by epoxidation with m-chloroperbenzoic acid. The diols were phosphorylated with diphenyl chlorophosphate, and the phenyl blocking groups were then removed by Pt-catalyzed hydrogenation. By the criterion of their reaction with the sulfhydryl group of glutathione, the phosphorylated epoxides are 6000 times less electrophilic than the previously described and structurally similar reagent 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate.

Local depilatory action of some unsaturated compounds.

IN A PRELIMINARY report1we described the local depilatory effect of the intermediary polymers (dimers) of chloroprene. When painted on the skin of laboratory animals, in all species tested these compounds caused reversible baldness at the site of application. In vitro the dimers inactivated the free sulfhydryl groups of glutathione, human epidermis, and liver homogenates in concentrations which corresponded well with the in vivo depilatory concentrations.2 We attributed the —SH-inhibitory action of the dimers to the unsaturated bonds in the molecule which tie down the —SH groups according to the equation3 On the assumption that the depilatory effect was related to the —SH-inhibitory action of these compounds, we tested a number of unsaturated compounds for their depilatory effectiveness. The substances tested fulfilled the following requirements: 1. All compounds had the —CH=CH— grouping. 2. None of the compounds contained Cl and thus did not give rise to

Hair loss from squalene.

SummarySqualene, an isoprene polymer occurring in normal human sebum, caused complete reversible local depilation in rabbits and guinea pigs after a single topical application. No hair loss was observed in mice. In vitro, squalene inactivated the free sulfhydryl groups of glutathione, human epidermis and mouse liver and inhibited succinic dehydrogenase activity of mouse liver. The depilatory and sulfhydryl inactivating effects of squalene are believed to be due to alkylation of the sulfhydryl group by the unsaturated double bonds in the molecule. The possibility is raised that squalene or a related unsaturated compound in sebum may influence human hair growth.