共查询到20条相似文献,搜索用时 15 毫秒
1.
Prostaglandin A2 (PGA2) stimulates the biosynthesis of gamma-glutamylcysteine synthetase and elevates glutathione (GSH) contents in cultured mammalian cells. To clarify the importance of gamma-glutamylcysteine synthetase induction in the defence of endothelial cells against oxidative stress, the effect of PGA2 on menadione (2-methyl-1,4-naphthoquinone)-induced cell injury was examined. Incubation of porcine aorta endothelial cells with menadione produced marked loss of cellular GSH and protein sulfhydryl groups, followed by leakage of lactic dehydrogenase (LDH) into the culture medium. The LDH leakage and modification of protein thiol was, however, completely prevented by pretreatment of the cells with PGA2. The protective effect of PGA2 was more potent than that of cysteine delivery agents such as methionine, N-acetylcysteine or 2-oxo-4-thiazolidine carboxylic acid (OTC). The results suggest that cellular GSH plays an important role in the defence against oxidative stress, and induction of gamma-glutamylcysteine synthetase is effective for protecting vascular endothelial cells. 相似文献
2.
A. Z. Reznick D. Han L. Packer 《Redox report : communications in free radical research》2013,18(3):169-174
SummaryExposure of human plasma to gas-phase cigarette smoke (CS) causes loss of human plasma antioxidants, protein modification (Frei et al, Biochem J, 1991 277:133–138; Reznick et al, Biochem J, 1992 286: 607–611) and a minimal amount of lipid oxidation. Ascorbic acid was found to prevent CS-induced lipid peroxidation and glutathione (GSH) partially protected against protein modification, as determined by loss of protein -SH groups and by increases in carbonyl content as a measure of protein oxidation. In the present study we demonstrate that dihydrolipoic acid (0.25–1.0 mM) decreases CS-induced protein carbonyls, α-tocopherol loss, and lipid hydroperoxide formation in plasma. In contrast GSH (1 mM) failed to influence CS-induced loss of α-tocopherol, and was 50% as effective as dihydrolipoate in protecting against CS-induced protein carbonyl formation. On the other hand, lipoic acid (oxidized form of dihydrolipoic acid) and oxidized glutathione (GSSG) had minimal effect in protecting against the CS-induced protein modifications. These findings demonstrate that low molecular weight thiols are capable of modifying the effect of gas-phase CS on biological fluids. Dihydrolipoate appears to be particularly useful in that it was shown to conserve ascorbic acid and α-tocopherol, i.e. supporting the antioxidant network concept in protection against protein and lipid oxidation. 相似文献
3.
Klein I Nagler RM Toffler R van Der Vliet A Reznick AZ 《Free radical biology & medicine》2003,35(11):1448-1452
Peroxidase activity in human saliva is composed of salivary peroxidase (80%), of salivary glandular origin, and myeloperoxidase (20%), of leukocyte origin. The term oral peroxidase (OPO) is used here to denote the total activity of both peroxidase species. Using the 2-nitrobenzoic acid-thiocyanate assay, OPO activity was measured in the saliva of nonsmokers after exposure to gas-phase cigarette smoke (CS) in an in vitro system using three puffs of CS in 1 h. A marked decrease of 76% of activity was observed following three puffs of CS. In order to elucidate the mechanism by which CS caused loss of OPO activity, several oxidants and antioxidants were applied to saliva in vitro in the presence and absence of CS. No protection for CS-induced loss of OPO activity occurred in the presence of glutathione, N-acetylcysteine, ascorbic acid, or Desferal. Exposure of saliva to purified aldehydes present in CS did not significantly affect OPO loss of activity. Similarly, ascorbic acid in the presence of FeCl(3) and nicotine also had no effect on OPO activity. Exposure of OPO to cyanate at levels present in CS caused a 65-70% loss of OPO activity, which was reversible after 24 h of dialysis. Moreover, hydroxocobalamin, a known cyanate chelator, could prevent CS- and potassium cyanide-induced inactivation of OPO by 70-90%. The results show that hydrogen cyanide, known to be present in microgram amounts per cigarette, is likely to be the species in CS responsible for loss of salivary OPO activity. The finding of reduced salivary OPO levels after CS exposure may represent a contributory mechanism for CS-related compromises in antimicrobial defenses in the aerodigestive tract. 相似文献
4.
Lockwood TD 《Archives of biochemistry and biophysics》2003,417(2):183-193
In early starvation tissue protein degradation increases, however in later starvation proteolysis declines so as to pace gradual atrophy during synthetic failure. Secondary decline of proteolytic pathways under progressive nutritional desperation is unexplained. After several days of starvation tissue GSH is partly depleted and GSSG/GSH is increased, followed by onset of ketonemia from fat breakdown. Ketone bodies inexplicably delay net muscle protein loss. Recent studies identify a proteome subset of more than 200 proteins with reactive sulfhydryl sites as candidates for coordinate redox control of diverse cell functions. Ketones cause protein sulfhydryl oxidation and protein S-glutathionylation. Here, redox-responsive proteolytic pathways were bio-assayed by release of [3H]leucine from rat myocardium under non-recirculating perfusion. More than 75% of myocardial protein degradation was inhibited and defined by infusion of diamide (100 microM) under constant physiologic concentrations of complete amino acids. Diamide-inhibitable proteolysis includes all lysosomal and some extra-lysosomal proteolysis. Following diamide washout, the reversal of proteolytic inhibitory action was greatly enhanced by artificial repletion of GSH by supra-physiologic extra-cellular GSH (1mM) exposure. Therefore, GSH maintains much of constitutive protein degradation in a primary tissue bioassay. Physiologic acetoacetate infusion (5mM) inhibited redox-responsive protein degradation. Uniformly [3H]leucine labeled 3T3 cells exhibited similar redox-dependent and redox-independent subcomponents of protein degradation. Independent of ketones, steady state cathepsin B reaction rate ex vivo was graded in proportion to the GSH concentration without GSSG, and inversely proportional to the GSSG/GSH redox ratio with inhibitory threshold at 0.5% oxidized. Linkage of some cysteine protease reaction rates to the interplay between GSH-GSSG/GSH status and ketonemia is suggested among transcendent mechanisms coordinating and pacing proteome turnover under prolonged starvation. The possibility of pre-emptive, redox coordination of distinct proteolytic pathways is speculatively discussed. 相似文献
5.
H F Gilbert 《Biochemistry》1989,28(18):7298-7305
Protein disulfide-isomerase, a protein localized to the lumen of the endoplasmic reticulum of eukaryotic cells, catalyzes the posttranslational formation and rearrangement of protein disulfide bonds. As isolated from bovine liver, the enzyme contains 0.8 free sulfhydryl group per mole of protein monomer and 3.1 disulfide bonds. Single-turnover experiments in which the disulfide bonds of the native enzyme are reduced by glutathione reveal three distinct reduction steps corresponding to the sequential reduction of the three disulfide bonds. The fastest disulfide to be reduced undergoes a change in the rate-determining step with increasing GSH concentration from a step which is second-order with respect to GSH concentration to a step which is first-order in GSH concentration. The disulfide which is reduced at an intermediate rate displays kinetics that are first-order in GSH concentration, and the slowest disulfide to be reduced exhibits kinetics which are second-order in GSH concentration. The enzyme catalyzes the steady-state reduction of a disulfide-containing hexapeptide (CYIQNC) by GSH. Initial velocity kinetic experiments are consistent with a sequential addition of the substrates to the enzyme. Saturation behavior is not observed at high levels of both substrates (Km for GSH much greater than 14 mM, Km for CYIQNC much greater than 1 mM). Only one of the three disulfides appears to be kinetically competent in the steady-state reduction of CYIQNC by GSH. The second-order thiol/disulfide exchange reactions catalyzed by the enzyme are 400-6000-fold faster than the corresponding uncatalyzed reactions. 相似文献
6.
Heme Inhibition of [delta]-Aminolevulinic Acid Synthesis Is Enhanced by Glutathione in Cell-Free Extracts of Chlorella
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Weinstein JD Howell RW Leverette RD Grooms SY Brignola PS Mayer SM Beale SI 《Plant physiology》1993,101(2):657-665
In plants, algae, and many bacteria, the heme and chlorophyll precursor, [delta]-aminolevulinic acid (ALA), is synthesized from glutamate in a reaction involving a glutamyl-tRNA intermediate and requiring ATP and NADPH as cofactors. In particulate-free extracts of algae and chloroplasts, ALA synthesis is inhibited by heme. Inclusion of 1.0 mM glutathione (GSH) in an enzyme and tRNA extract, derived from the green alga Chlorella vulgaris, lowered the concentration of heme required for 50% inhibition approximately 10-fold. The effect of GSH could not be duplicated with other reduced sulfhydryl compounds, including mercaptoethanol, dithiothreitol, and cysteine, or with imidazole or bovine serum albumin, which bind to heme and dissociate heme dimers. Absorption spectroscopy indicated that heme was fully reduced in incubation medium containing dithiothreitol, and addition of GSH did not alter the heme reduction state. Oxidized GSH was as effective in enhancing heme inhibition as the reduced form. Co-protoporphyrin IX inhibited ALA synthesis nearly as effectively as heme, and 1.0 mM GSH lowered the concentration required for 50% inhibition approximately 10-fold. Because GSH did not influence the reduction state of heme in the incubation medium, and because GSH could not be replaced by other reduced sulfhydryl compounds or ascorbate, the effect of GSH cannot be explained by action as a sulfhydryl protectant or heme reductant. Preincubation of enzyme extract with GSH, followed by rapid gel filtration, could not substitute for inclusion of GSH with heme during the reaction. The results suggest that GSH must specifically interact with the enzyme extract in the presence of the inhibitor to enhance the inhibition. 相似文献
7.
Kenji Aoki Motoo Arai Yasuji Minoda Koichi Yamada 《Bioscience, biotechnology, and biochemistry》2013,77(12):1913-1920
The Acid-stable α-amylase and the acid-unstable α-amylase from Aspergillus niger contained one mole of sulfhydryl group per one mole of enzyme, which probably existed correlating with calcium atom that was essential for the amylase activity.Iodine reacted at acidic pH specifically with the sulfhydryl group of both enzymes and oxidized it to considerably high degree, since about 4 eq of iodine per mole of sulfhydryl group of both enzymes were consumed. The modification of the sulfhydryl group of the acid-stable α-amylase did not affect the amylase acitvity, while, that of the acid-unstable α-amylase reduced it to 70 per cents intact enzyme. It was difficult to carry out carboxy-methylation of the sulfhydryl group of the acid-stable α-amylase under mild conditions maintaining its activity, but that of the acid-unstable α-amylase was easily achieved.These facts suggested that some differences existed in the neighborhood of the sulfhydryl group of both enzymes, and that the sulfhydryl group of them was not the active site. 相似文献
8.
Oxidative stress and other effects induced by cypermethrin (CYP, 15 mM) and their amelioration by alpha-tocopherol (400 microM) was studied in the nematode Caenorhabditis elegans. The worms exposed for 4 h to CYP showed increased levels of reactive oxygen species (46%), H2O2 (37%) and protein carbonyls (29%), accompanied by decreased lifespan and brood size. However, exposure to both CYP and alpha-tocopherol resulted in diminution of above alterations with the worms exhibiting relatively lower levels of ROS (30%), H2O2 (15%), protein carbonyls (14%), altered antioxidant enzyme activities and normal lifespan and brood size. The results suggest that CYP induces oxidative stress in C. elegans and the strategy of intervention with alpha-tocopherol could be exploited to offset this induced oxidative stress. 相似文献
9.
The results of this study suggest that the well-documented loss of GSH and ascorbate in organisms under oxidative stress may be mainly due to their reactions with protein radicals and/or peroxides. Protein hydroperoxides were generated in HL-60 cells exposed to radiation-generated hydroxyl radicals. We found for the first time evidence of chain peroxidation of the proteins in cells, with each hydroxyl radical leading to the formation of about 10 hydroperoxides. Protein peroxidation showed a lag, probably due to the endogenous antioxidant enzymes, with simultaneous loss of the intracellular GSH. Enhancement of the GSH levels by N-acetylcysteine decreased the formation of hydroperoxides, while treatment with l-buthionine sulfoximine had the opposite effect. The effect of variation of GSH levels on the formation of the peroxidized proteins is explained primarily by reduction of the protein hydroperoxides by GSH. Loading of the cells with ascorbate resulted in reduction of the amounts of protein hydroperoxides generated by the radiation, which was proportional to the intracellular ascorbate concentration. In contrast to the GSH, inhibition of protein hydroperoxide formation in the presence of the high (mM) intracellular ascorbate levels achieved was mainly due to the direct scavenging of hydroxyl radicals by the vitamin. 相似文献
10.
Abstract Growth hormone (GH) transgenic fish have dramatically enhanced growth rates, increased oxygen demands and reactive oxygen species production. GH-transgenic coho salmon provide an opportunity to address effects of increased metabolism on physiological aging. The objective of this study was to compare oxidative stress in wild-type (WT) and GH-transgenic (T) coho salmon (Oncorhynchus kisutch) of different ages (1 and 2 years). Antioxidant enzyme activity, protein carbonyls (PC) and glutathione (GSH, GSSG) were measured. PC correlated to growth rates in individual fish. T fish exhibited lower antioxidant enzyme activities and GSH levels compared to the WT, while levels of PC and GSSG were higher. Age affects were observed in both WT and T fish; enzyme activities and GSH decreased while PC and GSSG increased. Our results support the metabolic rate theory of aging. This study aims to be a platform for continued studies of the theories of aging using fish as model organisms. 相似文献
11.
The α‐amylase in the midgut and salivary glands of Eurygaster integriceps was isolated and characterized. The specific activity of α‐amylase in the midgut was 1.77 U/mg protein and in the salivary glands was 1.65 U/mg protein. Sodium dodecylsulfate electrophoresis showed that both midgut and salivary glands contain isozymes. Only a trace amount of α‐amylase activity was detected in the first nymphal stage (0.19 U/mg protein), whereas α‐amylase activity was highest in the third nymphal stage (1.21 U/mg protein). The results show that α‐amylase activity in the immature stages increase constantly to the third instar stage. There was no significant difference in enzyme activity between the third, fourth and fifth nymphal stages and adults. The optimum pH and temperature for the enzyme activity was determined to be 6.5 and 35°C, respectively. The enzyme activity was inhibited by addition of ethylenediaminetetraacetic acid, urea, sodium dodecylsulfate and Mg2+, but NaCl and KCl enhanced enzyme activity. 相似文献
12.
L W?odek 《Acta biochimica Polonica》1988,35(4):307-317
The sulfhydryl groups of L-cysteine and reduced glutathione (GSH) react nonenzymatically with formaldehyde (F), acrolein (Al), acetaldehyde (AA), malondialdehyde (DAM), pyruvate (P), oxoglutarate (oxo-G) and glucose (G) to form thiazolidine derivatives. These reactions show different velocities and the adducts formed show different stabilities. The equilibrium constants K, as well as the rate constants kr for the reverse reaction, show considerable variation. The carbonyls reveal higher reactivity with sulfhydryl group of L-Cys than with those of GSH, and the stability of the adducts is higher than that of GSH. Al, F and AA react more rapidly with both thiol compounds than the other carbonyls, but the adducts are less stable. The sulfhydryl groups level of bovine serum albumin as well as those of high- and low-molecular thiols of human plasma is reduced in the presence of Al, F or DAM. 相似文献
13.
Furuhata A Nakamura M Osawa T Uchida K 《The Journal of biological chemistry》2002,277(31):27919-27926
Acrolein, a representative carcinogenic aldehyde that could be ubiquitously generated in biological systems under oxidative stress, shows facile reactivity with the epsilon-amino group of lysine to form N(epsilon)-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) as the major product (Uchida, K., Kanematsu, M., Morimitsu, Y., Osawa, T., Noguchi, N., and Niki, E. (1998) J. Biol. Chem. 273, 16058-16066). In the present study, we determined the electrophilic potential of FDP-lysine and established a novel mechanism of protein thiolation in which the FDP-lysine generated in the acrolein-modified protein reacts with sulfhydryl groups to form thioether adducts. When a sulfhydryl enzyme, glyceraldehyde-3-phosphate dehydrogenase, was incubated with acrolein-modified bovine serum albumin in sodium phosphate buffer (pH 7.2) at 37 degrees C, a significant loss of sulfhydryl groups, which was accompanied by the loss of enzyme activity and the formation of high molecular mass protein species (>200 kDa), was observed. The FDP-lysine adduct generated in the acrolein-modified protein was suggested to represent a thiol-reactive electrophile based on the following observations. (i) N(alpha)-acetyl-FDP-lysine, prepared from the reaction of N(alpha)-acetyl lysine with acrolein, was covalently bound to glyceraldehyde-3-phosphate dehydrogenase. (ii) The FDP-lysine derivative reacted with glutathione to form a GSH conjugate. (iii) The acrolein-modified bovine serum albumin significantly reacted with GSH to form a glutathiolated protein. Furthermore, the observation that the glutathiolated acrolein-modified protein showed decreased immunoreactivity with an anti-FDP-lysine monoclonal antibody suggested that the FDP-lysine residues in the acrolein-modified protein served as the binding site of GSH. These data suggest that thiolation of the protein-bound acrolein may be involved in redox alteration under oxidative stress, whereby oxidative stress generates the increased production of acrolein and its protein adducts that further potentiate oxidative stress via the depletion of GSH in the cells. 相似文献
14.
Funk CJ 《Archives of insect biochemistry and physiology》2001,46(4):165-174
Alkaline phosphatase activity was histochemically localized in adult whiteflies (Bemisia tabaci B biotype, syn. B. argentifolii) with a chromogenic substrate (5-bromo-4-chloro-3-indolylphosphate) and a fluorogenic substrate (ELF-97). The greatest amount of staining was in the basal regions of adult salivary glands with additional activity traced into the connecting salivary ducts. Other tissues that had alkaline phosphatase activity were the accessory salivary glands, the midgut, the portion of the ovariole surrounding the terminal oocyte, and the colleterial gland. Whitefly nymphs had activity in salivary ducts, whereas activity was not detected in two aphid species (Rhodobium porosum and Aphis gossypii). Whitefly diet (15% sucrose) was collected from whitefly feeding chambers and found to have alkaline phosphatase activity, indicating the enzyme was secreted in saliva. Further studies with salivary alkaline phosphatase collected from diet indicated that the enzyme had a pH optimum of 10.4 and was inhibited by 1 mM cysteine and to a lesser extent 1 mM histidine. Dithiothreitol, inorganic phosphate, and ethylenediaminetetraacetic acid (EDTA) also inhibited activity, whereas levamisole only partially inhibited salivary alkaline phosphatase. The enzyme was heat tolerant and retained approximately 50% activity after a 1-h treatment at 65 degrees C. The amount of alkaline phosphatase activity secreted by whiteflies increased under conditions that stimulate increased feeding. These observations indicate alkaline phosphatase may play a role during whitefly feeding. 相似文献
15.
The role of mitochondrial glutathione and cellular protein sulfhydryls in formaldehyde toxicity in glutathione-depleted rat hepatocytes 总被引:4,自引:0,他引:4
Depletion of cellular GSH by diethyl maleate (DEM) potentiates CH2O toxicity in isolated rat hepatocytes and it was postulated that this increase in toxicity is due to the further decrease in GSH caused by CH2O in DEM-pretreated hepatocytes (1). The present investigation was conducted to investigate further the effects of CH2O, DEM, and acrolein (a compound which is structurally related to CH2O and DEM) on subcellular GSH pools and on protein sulfhydryl groups (PSH). CH2O caused a decrease in cytosolic GSH but had no effect on mitochondrial GSH either in previously untreated hepatocytes or in DEM-pretreated hepatocytes in which GSH was approximately 25% of control. DEM decreased both cytosolic and mitochondrial GSH but it did not produce toxicity. Neither CH2O (up to 7.5 mM) nor DEM (20 mM) decreased PSH. However, in cells pretreated with 1 mM DEM, CH2O (7.5 mM) decreased PSH and this effect preceded cell death. Acrolein decreased both cytosolic and mitochondrial GSH and it also decreased PSH significantly prior to causing cell death. CH2O and acrolein stimulated phosphorylase alpha activity, indicative of an increase in cytosolic free Ca2+, by a PSH-independent and PSH-dependent mechanism, respectively. These results suggest that the further depletion of cellular GSH by CH2O in DEM-pretreated cells is not due to the depletion of mitochondrial GSH. CH2O toxicity in DEM-pretreated cells is, however, correlated with depletion of PSH. The critical sulfhydryl protein(s) responsible for cell death remain to be more clearly defined. 相似文献
16.
《Free radical research》2013,47(10):1183-1189
AbstractGrowth hormone (GH) transgenic fish have dramatically enhanced growth rates, increased oxygen demands and reactive oxygen species production. GH-transgenic coho salmon provide an opportunity to address effects of increased metabolism on physiological aging. The objective of this study was to compare oxidative stress in wild-type (WT) and GH-transgenic (T) coho salmon (Oncorhynchus kisutch) of different ages (1 and 2 years). Antioxidant enzyme activity, protein carbonyls (PC) and glutathione (GSH, GSSG) were measured. PC correlated to growth rates in individual fish. T fish exhibited lower antioxidant enzyme activities and GSH levels compared to the WT, while levels of PC and GSSG were higher. Age affects were observed in both WT and T fish; enzyme activities and GSH decreased while PC and GSSG increased. Our results support the metabolic rate theory of aging. This study aims to be a platform for continued studies of the theories of aging using fish as model organisms. 相似文献
17.
The authors aim to establish a method that can quantitatively evaluate vital reactions to stress. We have been examining the correlation between stress and salivary amylase activity in order to verify its validity as a stress index. In order to quantify human stress, which changes over time, the relationship between stress and salivary amylase activity must be verified by fast and repeated analysis of salivary amylase activity. Standard biosensors are designed such that the enzyme immobilized on an electrode (enzyme electrode) and the substrate-dependent activity is measured. The reverse approach of measuring the alpha-amylase-dependent activity was adopted. We fabricated an amylase activity analytical system. Maltopentaose was selected as a substrate for alpha-amylase and a flow-injection-type device was used to supply maltopentaose continuously. alpha-Glucosidase, having relatively low enzyme activity, was immobilized on a pre-activated membrane so that it could be enclosed in a pre-column, Glucose oxidase, having higher enzyme activity, was immobilized on a working electrode so that it could function as an amperometric biosensor. A saliva-collecting device was fabricated to make saliva pretreatment unnecessary. As a result, an amylase activity analytical system was fabricated that enabled us to measure salivary amylase activity from 0 to 30 kU/l, with an R(2) value of 0.97. The time-course changes in the salivary amylase activities for 1 week were 5.1%, and the initial sensitivity remained nearly constant. Through this study, we were able to verify the possible development of the amylase activity analytical system. 相似文献
18.
Variation in the Number of Genes Coding for Salivary Amylase in the Bank Vole, CLETHRIONOMYS GLAREOLA
下载免费PDF全文
![点击此处可从《Genetics》网站下载免费的PDF全文](/ch/ext_images/free.gif)
J?rn T?nnes Nielsen 《Genetics》1977,85(1):155-169
A Danish population of bank voles is polymorphic for three electrophoretically different salivary amylases; A, H and S, of which A is the most common. Both single-, double- and triple banded phenotypes were observed, and in several crosses two electrophoretic forms cosegregated. In addition to the qualitative variation, some individuals show consistent quantitative variation in the relative activities of their amylase bands. This variation has been qualified by spectrophotometrical measurements of the relative amounts of amylase protein in the various bands. --Seventy wild chromosomes were analyzed by determining the amounts of amylase they produced when heterozygous with a laboratory stock chromosome known to carry two closely linked amylase genes, both coding for a fourth electrophoretic variant, B. The amount of A-protein divided by half the amount of B-protein was used as an estimate of the number of A-genes on the tested chromosomes. The wild chromosomes fell into three clearly distinguishable classes: 9 clustered around a gene number estimate of one, 45 chromosomes yielded estimates around two genes, and the gene number estimate of the remaining 16 was close to three. The integer values of the gene number estimates and the cosegregation of electrophoretically different salivary amylases are consistent with the model that the population is polymorphic for chromosomes with either one, two, or three closely linked amylase genes. It is suggested that such gene number variation may be more common than generally recognized, and some other reported cases of quantitative enzyme variation, for instance that of human red cell acid phosphatase, are interpreted in terms of variation in the number of genes involved. 相似文献
19.
A procedure for the isolation of a sulfhydryl oxidase from an Aspergillus niger cell suspension involved three major steps and yielded enzyme preparations exhibiting a single but diffuse protein-containing zone when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a subunit molecular weight estimated to be 53,000. Sedimentation equilibrium experiments indicated a native molecular weight of 106,000. Analyses for sugar residues showed that the enzyme is a glycoprotein, containing 20.3% neutral hexose and 1.9% aminohexose by weight. This enzyme catalyzed the conversion of reduced glutathione (GSH) to its disulfide form, with concomitant consumption of O2 and release of H2O2. The ratio of GSH consumed to H2O2 produced was determined to be 2:1. At 25 degrees C, the optimum pH for the oxidation of GSH was 5.5. Under these conditions, the enzyme had a Michaelis constant of 0.3 mM for GSH. Other low molecular weight thiol compounds (cysteine, dithiothreitol, and 2-mercaptoethanol) were also oxidized, but the Michaelis constants for these substrates were substantially higher than that for GSH under identical conditions of temperature and pH. The rate of reactivation of reductively denatured ribonuclease A was enhanced by the presence of sulfhydryl oxidase, indicating that the latter is capable of oxidizing protein-associated thiol groups. The UV-visible spectrum of sulfhydryl oxidase solution had absorbance maxima at 274, 364.5, and 442.5 nm and was otherwise characteristic of the spectra of known flavoproteins.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
20.
Alterations in intracellular thiol homeostasis during the metabolism of menadione by isolated rat hepatocytes 总被引:10,自引:0,他引:10
Donato Di Monte David Ross Giorgio Bellomo Lena Eklöw Sten Orrenius 《Archives of biochemistry and biophysics》1984,235(2):334-342
The effects of menadione (2-methyl-1,4-naphthoquinone) metabolism on intracellular soluble and protein-bound thiols were investigated in freshly isolated rat hepatocytes. Menadione was found to cause a dose-dependent decrease in intracellular glutathione (GSH) level by three different mechanisms: (a) Oxidation of GSH to glutathione disulfide (GSSG) accounted for 75% of the total GSH loss; (b) About 15% of the cellular GSH reacted directly with menadione to produce a GSH-menadione conjugate which, once formed, was excreted by the cells into the medium; (c) A small amount of GSH (approximately 10%) was recovered by reductive treatment of cell protein with NaBH4, indicating that GSH-protein mixed disulfides were also formed as a result of menadione metabolism. Incubation of hepatocytes with high concentrations of menadione (greater than 200 microM) also induced a marked decrease in protein sulfhydryl groups; this was due to arylation as well as oxidation. Binding of menadione represented, however, a relatively small fraction of the total loss of cellular sulfhydryl groups, since it was possible to recover about 80% of the protein thiols by reductive treatments which did not affect protein binding. This suggests that the loss of protein sulfhydryl groups, like that of GSH, was mainly a result of oxidative processes occurring within the cell during the metabolism of menadione. 相似文献