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1.
The nature of the complexes and equilibria shown by solutions of protohaemin in dimethyl sulphoxide/water mixtures and in the presence of acid and base were studied by u.v.-visible spectrophotometry. In neutral solutions containing from 40 to 100% dimethyl sulphoxide, haemin is present as a monomeric complex in which the Cl-ion is not coordinated. Only a single pH-dependent equilibrium pK12 is observed over the range 40-80% dimethylsulphoxide, corresponding to formation of the mu-oxo dimer. As the dimethyl sulphoxide content is lowered below 35%, so the single equilibrium (pK12) is replaced by two equilibria (pK1 and pK2); with solutions of 5 microM-haemin, pK1 decreases (from pK12 7.55 in 65% dimethyl sulphoxide to pK1 approx. 1.5 in 0.01% dimethyl sulphoxide), whereas pK2 hardly changes (from pK12 7.55 in 65% to pK2 approx. 7.5 in 0.01%). 相似文献
2.
N G Faleev M B Saporovskaia V M Belikov I S Sakharova L N Zakomyrdina Iu M Torchinski? 《Bioorganicheskaia khimiia》1990,16(8):1019-1023
Tryptophanase from E. coli retains its ability to form quinonoid intermediate with L-alanine in water--methanol and water--dimethylformamide (1:1 v/v) solutions. Under these conditions the enzyme catalyzes decomposition of S-o-nitrophenyl-L-cysteine (SOPC) to o-nitrophenylthiol, pyruvate and ammonium ion. The enzyme's affinity for this substrate increases on going from water to water-organic solvents whereas the reaction rate decreases. In 50% methanol tryptophanase catalyzes the formation of L-tryptophan from indole and SOPC; in the mixture of 2H2O and C2H3O2H (1:1) the enzymatic isotope exchange of alpha-proton of L-phenylalanine with complete retention of configuration was observed. 相似文献
3.
Induction of haem synthesis in Hep G2 human hepatoma cells by dimethyl sulphoxide. A transcriptionally activated event.
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Exposure of cultured human hepatoma cells (Hep G2) to medium containing 2% (v/v) dimethyl sulphoxide resulted in an approximate doubling in the activity of delta-aminolaevulinate dehydratase, an increase in the haem content and a decreased growth rate; induced enzyme activity was decrease by 50% after treatment with alpha-amanitin. The findings are strikingly similar to those seen in murine Friend-virus-transformed erythroleukaemia cells. 相似文献
4.
Fatty acid utilization during development of the rat 总被引:5,自引:4,他引:1
The effects of dimethyl sulphoxide and glycerol on ox brain microsomal Na(+)+K(+)-stimulated adenosine triphosphatase (EC 3.6.1.3), K(+)-stimulated p-nitrophenyl phosphatase and K(+)-dependent muscle pyruvate kinase (EC 2.7.1.40) were studied. Dimethyl sulphoxide at concentrations below 20% (v/v) was found to stimulate the p-nitrophenyl phosphatase and pyruvate kinase by increasing their affinity for K(+) but to inhibit the Na(+)+K(+)-stimulated adenosine triphosphatase. The latter enzyme activity was also inhibited by glycerol, which like dimethyl sulphoxide, stimulated the K(+)-activated p-nitrophenyl phosphatase at a wide range of concentrations. The solvent effects were promptly reversed by dilution. Similarity was found between glycerol and dimethyl sulphoxide, on one hand, and ATP, on the other, in their stimulatory effect and their ability to increase the ouabain- and oligomycin-sensitivity of the K(+)-stimulated p-nitrophenyl phosphatase. However, only the solvents, not the ATP, increased the binding of K(+) by the microsomes. From the above findings it is suggested that solvents may act on K(+)-dependent enzymes by altering the state of solvation of the activating cation as well as by changing the enzyme structure. 相似文献
5.
H2O2 reacts with cytochrome c peroxidase in a variety of ways. The initial reaction produces cytochrome c peroxidase Compound I. If more than a 10-fold excess of H2O2 is added to the enzyme, a portion of the H2O2 will react with Compound I to produce molecular oxygen. The remainder oxidizes the heme group and various amino acid residues in the protein. If less than a 10-fold excess of H2O2 is added to the enzyme, essentially all the H2O2 is utilized by oxidation of amino acid residues in the protein. The oxidation of the amino acid residues by H2O2 substantially modifies the reactivity of cytochrome c peroxidase. The modification of reactivity could be the direct result of amino acid oxidation or an indirect result caused by a perturbation of the protein structure at the active site. The products oxidized at pH 8 lose their ability to react with H2O2. The products oxidized at pH4 react with H2O2 but their reactivity toward Fe(CN)4-6 is substantially reduced. 相似文献
6.
Mucin biosynthesis. Properties of a bovine tracheal mucin beta-6-N-acetylglucosaminyltransferase.
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We have characterized a bovine tracheal mucin beta-6-N-acetylglucosaminyltransferase that catalyses the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the C-6 of the N-acetylgalactosamine residue of galactosyl-beta 1----3-N-acetylgalactosamine. Optimal enzyme activity was obtained between pH 7.5-8.5, at 5mM-MnCl2, and at 0.06-0.08% (v/v) Triton X-100 (or Nonidet P-40), or 0.5-5.0% (v/v) Tween 20. Ba2+, Mg2+ and Ca2+ could partially replace Mn2+, but Co2+, Fe2+, Cd2+ and Zn2+ could not. Sodium dodecyl sulphate, cetylpyridinium chloride, sodium deoxycholate, octyl beta-D-glucoside, digitonin and alkyl alcohols were less effective in enhancing enzyme activity, and dimethyl sulphoxide was ineffective. The apparent Michaelis constants were 1.25 mM for UDP-N-acetylglucosamine, 0.94-3.34 mM for freezing-point-depressing glycoprotein and 0.19 mM for periodate-treated blood-group-A porcine submaxillary mucin. Asialo ovine submaxillary mucin could not serve as the glycosyl acceptor. The structure of the 14C-labelled oligosaccharide obtained by alkaline-borohydride treatment of the product was identified as Gal beta 1----3(Glc-NAc beta 1----6)N-acetylgalactosaminitol by beta-hexosaminidase treatment, gas chromatography-mass spectrometry and 1H-n.m.r. (270 MHz) analysis. The enzyme is important in the regulation of mucin oligosaccharide biosynthesis. 相似文献
7.
Production of the superoxide adduct of myeloperoxidase (compound III) by stimulated human neutrophils and its reactivity with hydrogen peroxide and chloride. 总被引:7,自引:3,他引:4
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Examination of the spectra of phagocytosing neutrophils and of myeloperoxidase present in the medium of neutrophils stimulated with phorbol myristate acetate has shown that superoxide generated by the cells converts both intravacuolar and exogenous myeloperoxidase into the superoxo-ferric or oxyferrous form (compound III or MPO2). A similar product was observed with myeloperoxidase in the presence of hypoxanthine, xanthine oxidase and Cl-. Both transformations were inhibited by superoxide dismutase. Thus it appears that myeloperoxidase in the neutrophil must function predominantly as this superoxide derivative. MPO2 autoxidized slowly (t 1/2 = 12 min at 25 degrees C) to the ferric enzyme. It did not react directly with H2O2 or Cl-, but did react with compound II (MP2+ X H2O2). MPO2 catalysed hypochlorite formation from H2O2 and Cl- at approximately the same rate as the ferric enzyme, and both reactions showed the same H2O2-dependence. This suggests that MPO2 can enter the main peroxidation pathway, possibly via its reaction with compound II. Both ferric myeloperoxidase and MPO2 showed catalase activity, in the presence or absence of Cl-, which predominated over chlorination at H2O2 concentrations above 200 microM. Thus, although the reaction of neutrophil myeloperoxidase with superoxide does not appear to impair its chlorinating ability, the H2O2 concentration in its environment will determine whether the enzyme acts primarily as a catalase or peroxidase. 相似文献
8.
Ammazzalorso A Amoroso R Bettoni G De Filippis B Fantacuzzi M Giampietro L Maccallini C Tricca ML 《Chirality》2008,20(2):115-118
Candida rugosa lipase-catalysed hydrolysis of three different 2-substituted-aryloxyacetic esters was performed in aqueous buffer containing dimethyl sulphoxide and isopropanol from 0 to 80% v/v as additives, in order to obtain an enhancement of the enantioselectivity. For 2-(p-chlorophenoxy)acetic acid and 2-n-butyl-2-(p-chlorophenoxy)acetic acid ethyl esters, DMSO enhanced enzyme enantioselectivity more than IPA with an opposite enzymatic enantiopreference. The cosolvents moderately improved Candida rugosa lipase enantioselectivity for 2-phenyl-2-(p-chlorophenoxy)acetic acid ethyl ester. 相似文献
9.
In this work the thermal properties of diethyl sulphoxide (Et2SO), as well as its cryoprotective ability are studied and related to other well-known cryoprotectant substances, like dimethyl sulphoxide (Me2SO). We have investigated the thermal properties of Et2SO/water systems using Differential Scanning Calorimetry at a very low heating/cooling rate (2 degrees C/min). Liquid/solid or glassy/crystalline transitions have been observed only for the solutions with content of Et2SO ranging from 5 up to 40% w/w and/or greater than 85%. In the 45-75% w/w Et2SO range we have found a noticeable glass-forming tendency and a great stability of the amorphous state to the reheating. In samples with Et2SO content ranging from 80 to 85%, we observed a great stability of the glass forming by cooling, but a lesser stability to the subsequent reheating. The glass-forming tendency of these solutions is discussed in terms of existing competitive interactions between molecules of Et2SO, on the one hand, and Et2SO and water molecules, on the other hand. The results are well explainable on the basis of the model structure of water/Et2SO solutions, deduced by Raman and infrared studies [J. Mol. Struct. 665 (2003) 285-292]. The cryoprotective ability of Et2SO on Escherichia coli survival has been also investigated, and a comparison among Et2SO and other widely used cryoprotectants, like Me2SO and glycerol has been done. Survival of E. coli, determined after freezing-thawing process, was maximal at 45% w/w Et2SO (more than 85% viability). It should be noted that at the same concentration the survival is only about 35% in the presence of Me2SO and not more than 15% in the presence of glycerol. These features are well consisted with the glass-forming properties of Et2SO. 相似文献
10.
Solution structures of ferrihaem in some dipolar aprotic solvents and their binary aqueous mixtures 总被引:7,自引:4,他引:3
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1. Conductivity and u.v. and visible spectroscopic techniques were used to investigate the solution structure of the prosthetic group of the ferric haemoproteins (ferrihaem) in dimethyl sulphoxide, NN-dimethylacetamide, NN-dimethylformamide and sulpholane, and certain of their aqueous mixtures. 2. In neutral or acid dimethyl sulphoxide, chlorohaemin is monomeric and completely dissociated into Cl−ion and a ferrihaem species with dimethyl sulphoxide molecules in the fifth and sixth co-ordination positions on iron. 3. In neutral NN-dimethylacetamide and NN-dimethylformamide chlorohaemin is monomeric but is largely undissociated, giving different spectra from that of chlorohaemin in dimethyl sulphoxide. On acidification, dissociation occurs and the dimethyl sulphoxide type of spectrum results. 4. Studies in a fourth solvent, sulpholane, indicate that solvent co-ordinating power (ligand strength) rather than bulk dielectric constant is responsible for dissociation of chlorohaemin. 5. In neutral dimethyl sulphoxide–water mixtures chlorohaemin remains monomeric and completely dissociated, and spectra are independent of mixture composition, except at high water concentrations, when precipitation occurs. In alkaline dimethyl sulphoxide–water mixtures, where the complete solvent mixture range is accessible, ferrihaem is polymeric (probably dimeric) and spectra are dependent on solvent composition. A quantitative analysis indicates that the spectral changes are due to replacement by water of one molecule of co-ordinated dimethyl sulphoxide per ferrihaem aggregate, and do not involve a two-molecule replacement as has been suggested for the alkaline pyridine–water system. 相似文献
11.
Enzymatic removal of phenols from aqueous solutions by Coprinus cinereus peroxidase and hydrogen peroxide. 总被引:1,自引:0,他引:1
The fungal enzyme Coprinus cinereus peroxidase (CIP) can be used for the removal of toxic phenols from water. After treating aqueous solutions of phenols with CIP and H2O2 the phenols polymerized and precipitated. The decrease in phenol concentration was investigated for 10 different phenols. At neutral pH, the investigated phenols were in general removed with high efficiency. 相似文献
12.
Detection of secondary structure in glycosaminoglycans via the H n.m.r. signal of the acetamido NH group. 总被引:1,自引:1,他引:0
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Two simple methods for dissolving salts of acid glycosaminoglycans with inorganic cations (e.g. Li+ and Na+) in dry dimethyl sulphoxide are described. Complete n.m.r. spectra of, e.g., Na+ and Li+ salts of chondroitin sulphate and keratan sulphate were obtained on these solutions. In [2H6]dimethyl sulphoxide the NH resonance of 2-acetamido-2-deoxy hexosides is in the range 7.2-8.0 delta, but is downfield (8.3-9.3 delta) when the NH is H-bonded to -CO2-. Heparan sulphate shows two NH resonances, of which one (at 8.3 delta) is probably indicative of H-bonding. Space-filling models show that a very close approach of NH to -CO2- across the alpha-glucosaminidic bond is possible, and a solution configuration for heparan sulphate is proposed. The n.m.r. results are entirely compatible with interpretations of periodate-oxidation kinetics, based on H-bonded secondary structures present in hyaluronate and chondroitin sulphates, but not in dermatan (or keratan) sulphate. 相似文献
13.
Uppu RM 《Analytical biochemistry》2006,354(2):165-168
A method for the synthesis of peroxynitrite is described. It involves nitrosation of H2O2 at pH> or = 12.5 by isoamyl or butyl nitrite in mixed solvents of isopropyl alcohol (IPA) and water at 25+/-1 degrees C. Maximum yields of peroxynitrite are obtained after 15 min of incubation at IPA concentrations of 30-70% (v/v). The solutions of peroxynitrite are processed for removal of IPA and isoamyl alcohol by solvent extraction. Unreacted H2O2 is removed by catalytic decomposition on granular MnO(2). The post processed solutions of peroxynitrite are useful in several chemical and biochemical investigations where bolus additions are required. The method as reported is amenable for large scale synthesis as it involves sequential mixing of solvents (water and IPA) to alkali followed by the addition of H2O2 and alkyl nitrite. 相似文献
14.
Characterization of two constitutive forms of rat liver microsomal heme oxygenase. Only one molecular species of the enzyme is inducible 总被引:30,自引:0,他引:30
The present report describes, for the first time, the identification of two constitutive forms of heme oxygenase, designated as HO-1 and HO-2, in rat liver microsomal fractions. HO-1 was purified to homogeneity and exhibited a specific activity of up to 4000 nmol of bilirubin/mg of protein/h. HO-2 was partially purified to a specific activity of 250 nmol of bilirubin/mg of protein/h. In the native state, the relative activity of HO-2 surpassed that of HO-1 by 2-3-fold. However, a remarkable difference existed in the regulatory mechanism(s) for the production of the two enzyme forms. Whereas the activity of HO-1 was increased up to 100-fold in response to cobalt, cadmium, hematin, phenylhydrazine, and bromobenzene, that of HO-2 was fully refractory to these agents. The two forms differed in their apparent Km, thermolability, ammonium sulfate precipitation, antigenicity, electrophoretic mobility under nondenaturing conditions, and chromatographic behavior. Specifically, for HO-1 the apparent Km value was 0.24 microM, whereas that for HO-2 was 0.67 microM. HO-2 preparation was more susceptible to heat inactivation; nearly 65% activity was retained by HO-1 preparation after exposure to 60 degrees C for 10 min, whereas under the same conditions only about 25% of HO-2 activity was retained. When subjected to ammonium sulfate precipitation the bulk of HO-1 activity precipitated between 0 and 35% saturation, whereas that of HO-2 was precipitated between 35 and 65% saturation. The two forms appeared as immunologically different entities, in so far as a crossreactivity between antibody raised against HO-1 in rabbit and HO-2 could not be detected. Similarities were observed in respect to cofactor requirements for activity, sensitivity to inhibitors, as well as their reactivity towards the substrates used in this study, i.e. hematin, hematoheme, and cytochrome c. Specifically both forms of the enzyme required NADPH-cytochrome c (P-450) reductase, NADPH or NADH, and O2 for activity, and reactions were inhibited by KCN, NaN3, and CO. Both forms cleaved the tetrapyrrole molecule exclusively at the alpha-meso bridge to form biliverdin IX alpha isomer. HO-1 and HO-2 utilized hematin and hematoheme as substrates but not intact cytochrome c. 相似文献
15.
Susceptibility of glutathione peroxidase to proteolysis after oxidative alteration by peroxides and hydroxyl radicals. 总被引:3,自引:0,他引:3
Glutathione peroxidase is a key enzyme in the antioxidant system of the cells. This enzyme has been shown to be irreversibly inactivated by H2O2, tert-butyl hydroperoxide (tert-BHP) and hydroxyl radicals when incubated without GSH. We observed that in our experimental conditions glutathione peroxidase was not degraded by trypsin or chymotrypsin while degraded by pronase, papa?n, pepsin, and lysosomal proteases. Hydroxyl radicals and superoxide anions but not H2O2 or tert-BHP could also fragment the enzyme on their own. A former incubation with H2O2, tert-BHP, or hydroxyl radicals also increased the proteolytic susceptibility of glutathione peroxidase. Like superoxide dismutase (SOD) and other oxidatively denatured proteins, glutathione peroxidase inactivated by peroxides or free radicals seems to be degraded preferentially by proteases. As hydroxyl radicals can fragment the enzyme by themselves, the increased proteolytic susceptibility afterwards is easily understood while the increased susceptibility induced by H2O2 and tert-BHP seems to be more specific. 相似文献
16.
The cyclododecapeptide, (Ala1-Pro2-Gly3-Val4-Gly5-Val6)2, was synthesized and its secondary structure was evaluated from extensive studies in dimethyl sulphoxide, trifluoroethanol and water using NMR methods. A selective decoupling technique in 13C-NMR has been utilized in order to assign the C=O carbon resonances. Temperature dependence of the peptide NH protons and the solvent perturbation of the peptide NH and C=O resonances show the occurrence in all solvents of a beta-turn (a 10-membered H-bond between the Val4 NH and Ala1 C=O) and a gamma-turn, an 11-membered H-bond between the Gly3 NH and the Gly5 C=O; and a possible 14-membered H-bond between the Ala1 NH and the Val4 C=O in dimethyl sulphoxide and trifluoroethanol. These secondary structural features are compared with the linear polyhexapeptide and found the the beta-turn and the gamma-turn are the common conformational features of these peptide systems. 相似文献
17.
A Boveris H Sies E E Martino R Docampo J F Turrens A O Stoppani 《The Biochemical journal》1980,188(3):643-648
The glutathione peroxidase-glutathione reductase system, an alternative pathway for metabolic utilization of H2O2 [Chance, Sies & Boveris (1979) Physiol. Rev. 59, 527-605], was investigated in Trypanosoma cruzi, an organism lacking catalase and deficient in peroxidase [Boveris & Stoppani (1977) Experientia 33, 1306-1308]. The presence of glutathione (4.9 +/- 0.7 nmol of reduced glutathione/10(8) cells) and NADPH-dependent glutathione reductase (5.3 +/- 0.4 munit/10(8) cells) was demonstrated in the cytosolic fraction of the parasite, but with H2O2 as substrate glutathione peroxidase activity could not be demonstrated in the same extracts. With t-butyl hydroperoxide or cumene hydroperoxide as substrate, a very low NADPH-dependent glutathione peroxidase activity was detected (equivalent to 0.3-0.5 munit of peroxidase/10(8) cells, or about 10% of glutathione reductase activity). Blank reactions of the glutathione peroxidase assay (non-enzymic oxidation of glutathione by hydroperoxides and enzymic oxidation of NADPH) hampered accurate measurement of peroxidase activity. The presence of superoxide dismutase and ascorbate peroxidase activity in, as well as the absence of catalase from, epimastigote extracts was confirmed. Ascorbate peroxidase activity was cyanide-sensitive and heat-labile, but no activity could be demonstrated with diaminobenzidine, pyrogallol or guaiacol as electron donor. The summarized results support the view that T. cruzi epimastigotes lack an adequate enzyme defence against H2O2 and H2O2-related free radicals. 相似文献
18.
转染人血红素加氧酶-1基因对大鼠血管平滑肌细胞抗氧化损伤的作用 总被引:7,自引:0,他引:7
本实验构建含人血红素加氧酶-1(hHO-1)基因的逆转录病毒载体XM-6/hHO-1,将其导入离体培养的大鼠血管平滑肌细胞(vascular smooth muscle cells,VSMC),观察外源性hHO-1基因在VSMC内的表达及其抗活性氧损伤作用,结果表明:(1)hHO-1基因可在靶细胞中明显表达,转染VSMC的HO-1蛋白表达和HO酶活性分别比非转染细胞高1.8倍和2.0倍;(2)转染hHO-1的VSMC可对抗大剂量H2O2对细胞的损伤作用,表现为细胞存活率增加和乳酸脱氢酶(LDH)漏出减少,上述保护作用可被HO特异性抑制剂锌原卟啉IX(Zinc-proto-porphyrinIX,ZnPP-IX)所阻断,研究结果提示,外源性HO-1的过量表达可增加VSMC对抗氧化损伤的能力。 相似文献
19.
Spectral and catalytic parameters of peroxidase solubilized in the aerosol OT-water-octane system have been studied. The spectrum of peroxidase solubilized in octane with AOT reversed micelles, a degree of surfactant hydration being above 12, is actually identical to that of the enzyme aqueous solution. On the other hand, significant spectral changes have been detected when transferring the enzyme from water to the reversed micelle medium at low degrees of surfactant hydration, precisely [H2O]/[AOT] less than 12. The reversed micelle-entrapped peroxidase catalyses the oxidation of pyrogallol with hydrogen peroxide much more actively (at [H2O]/[surfactant] approximately 13) than that in aqueous solution. The entrapment of peroxidase into surfactant reversed micelles increases precisely the catalytic constant of the reaction, i.e. the virtual reactivity of the enzyme increases ten and hundred times depending on degrees of surfactant hydration and concentration. The systems of reversed micelles may be considered as models of biomembranes. Our findings hence show that enzymes in vivo can be much more catalytically active then it appears possible to reveal in conventional experiments in vitro in aqueous solutions. 相似文献
20.
Dennery PA Visner G Weng Yi Nguyen X Lu F Zander D Yang G 《Free radical biology & medicine》2003,34(1):124-133
In many models, a protective role for heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, has been demonstrated. Also, HO-1 null mice (KO) are more susceptible to inflammation and hypoxia and transplant rejection. Nonetheless, their response to hyperoxia (> 95% O(2)) has not yet been evaluated. Surprisingly, after acute hyperoxic exposure, KO had significantly decreased markers of lung oxidative injury and survived chronic hyperoxia as well as wild-type (WT) controls. Disrupted HO-1 expression was associated with decreased lung reactive iron and iron-associated proteins, decreased NADPH cytochrome cp450 reductase activity, and decreased lung peroxidase activity compared to WT. Injection of tin protoporphyrin, an inhibitor of HO, in the WT decreased acute hyperoxic lung injury, whereas transduction of human HO-1 in the KO reversed the relative protection of the KO to acute injury and worsened hyperoxic survival. This suggests that disruption of HO-1 protects against hyperoxia by diminishing the generation of toxic reactive intermediates in the lung via iron and H(2)O(2). 相似文献