共查询到20条相似文献,搜索用时 31 毫秒
1.
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
2.
Ferrochelatase activity in human lymphocytes was found to be 50% inhibited by 10.5 microM hemin under maximal velocity conditions. The inhibition was not prevented by dithiothreitol or glutathione, suggesting that the hemin was not interacting with the sulphydryl groups of ferrochelatase. Human serum albumin, but not bovine serum albumin was able to prevent the inhibition consistent with the known formation of the tightly bound methemalbumin complex with human albumin. Kinetic studies performed under initial velocity conditions with hemin concentrations ranging from 2 to 8 microM revealed the inhibition to be non-competitive with respect to the metal substrate (zinc) and competitive with respect to the porphyrin substrate (mesoporphyrin). The kinetic analysis indicated that hemin binds to both the enzyme and enzyme-metal complex at a site normally occupied by the porphyrin substrate, and a second molecule of hemin could bind to the enzyme-metal complex but with a much lower affinity than the first molecule. We conclude that the product inhibition of ferrochelatase by hemin should be considered as a possible site of regulation of heme biosynthesis. 相似文献
3.
Heme formation in the erythron is subject to end product regulation by negative feedback, but the exact point of metabolic control in human erythroid cells is unknown. To investigate the mode of action of heme on its own formation, the effects of micromolar concentrations of hemin on de novo synthesis of protoporphyrin IX and delta-aminolevulinate (delta-ALA) by intact human reticulocytes were examined in the presence of 1 mM alpha,alpha'-bipyridyl and 200 microM 4,6-dioxoheptanoate to block their further conversion by ferrochelatase or delta-ALA dehydrase, respectively. At final concentrations (25-40 microM), hemin, which is known to reduce incorporation of [2-14C]glycine into cellular heme, significantly inhibited formation of protoporphyrin IX and total delta-aminolevulinate in situ by these cells. Since synthesis of the first committed precursor, delta-aminolevulinate, as well as protoporphyrin (which is derived from it) were diminished, the effects of hemin on delta-aminolevulinate synthase (EC 2.3.1.37) were studied. Hemin, at concentrations up to 40 microM, had no direct effect on enzymatic activity, as measured with [5-14C] alpha-ketoglutarate (in hypotonically lysed cells) or [1,4-14C]succinyl coenzyme A (in deoxycholate lysates), even after preincubation. However, when intact human reticulocytes were incubated with hemin before assay for delta-ALA synthase, there was a rapid, concentration-dependent reduction in enzymatic activity (mean 42 and 23% inhibition after 60 min for these two substrates, respectively). Hemin had no effect on steady-state levels of delta-ALA synthase mRNA, as determined by Northern blot hybridization using an erythroid-specific human cDNA probe. Thus, a mechanism for inducing feedback inhibition of the tetrapyrrole pathway exists in human erythroid cells. It controls formation of the first committed precursor of protoporphyrin IX, delta-aminolevulinate, and hence regulates heme biosynthesis by limiting the availability of the porphyrin, rather than the metal substrate for the ferrochelatase reaction. Hemin interacts with constituents of the intact reticulocyte significantly to reduce delta-aminolevulinic acid synthase activity by an indirect cellular process that does not influence the abundance of erythroid-specific synthase mRNA but may either inhibit its ribosomal translation in an unknown manner or promote degradation of the enzyme itself by specific proteolysis. 相似文献
4.
In this study we have investigated the effect of ethanol on [Ca2+]c by microfluorimetry and glutamate secretion using an enzyme-linked system, in rat hippocampal astrocytes in culture. Our results show that ethanol (1-200 mM) evoked a dose-dependent increase in glutamate secretion. 50 mM ethanol, a concentration within the range of blood alcohol levels in intoxicated humans, induced a release of Ca2+ from intracellular stores in the form of oscillations. Ca2+-mobilizing effect of ethanol was not prevented by preincubation of cells in the presence of 2 mM of the antioxidant dithiothreitol. Ethanol-evoked glutamate secretion was reduced when extracellular Ca2+ was omitted (medium containing 0.5 mM EGTA) and following preincubation of astrocytes in the presence of the intracellular Ca2+ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetraacetoxy-methyl ester (10 microM). Preincubation of astrocytes in the presence of 2 mM of the antioxidant dithiothreitol significantly reduced ethanol-evoked glutamate secretion. Finally, preincubation of astrocytes in the presence of bafilomycin (50 nM) significantly reduced ethanol-induced neurotransmitter release, indicating that exocytosis is involved in glutamate secretion. In conclusion, our results suggest that ethanol mobilizes Ca2+ from intracellular stores, and stimulates a Ca2+-dependent glutamate secretion, probably involving reactive oxygen species production, and therefore creating a situation potentially leading to neurotoxicity in the hippocampus. 相似文献
5.
Supply of cadmium chloride (0.5 mM) inhibited chlorophyll formation in greening maize leaf segments, while lower concentration of Cd (0.01 mM) slightly enhanced it. Inclusion of 2-oxoglutarate (2-OG, 0.1-10 mM) in the incubation mixture increased chlorophyll content in the absence as well as presence of Cd. Substantial inhibition of chlorophyll formation by Cd was observed at longer treatment both in the absence and presence of 2-OG. When the tissue was pre-incubated with 2-OG or Cd, the inhibition (%) of chlorophyll formation by Cd was lowered in the presence of 2-OG. Treatment with Cd inhibited ALAD activity and ALA formation and the inhibition (%) of ALA formation by Cd was strongly reduced in the presence of 2-OG. Glutamate dehydrogenase (GDH) activity was increased by the supply of Cd both in the absence as well as presence of 2-OG. In the presence of 2-OG, Cd supply significantly increased glutamate synthase (GOGAT) activity and reduced inhibition (%) of glutamine synthetase (GS) activity. The results suggested the involvement of the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway of ammonia assimilation to provide the precursor, glutamate, for ALA synthesis under Cd toxicity and 2-OG supplementation. 相似文献
6.
Mutants H-14 and H-18 of Staphylococcus aureus require hemin for growth on glycerol and other nonfermentable substrates. H-14 also responds to delta-aminolevulinate. Heme-deficient cells grown in the presence of nitrate do not have lactate-nitrate reductase activity but gain this activity when incubated with hemin in buffer and glucose. Lactate-nitrate reductase activity is also restored to the membrane fraction from such cells by incubation with hemin and dithiothreitol; addition of adenosine 5'-triphosphate has no effect upon the restoration. Cells grown with nitrate in the absence of hemin have two to five times more reduced benzyl viologen-nitrate reductase activity than do those grown with hemin. The activity increases throughout the growth period in the absence of hemin, but with hemin present enzyme formation ceases before the end of growth. There was no evidence of enzyme destruction. The distribution of nitrate reductase activity between membrane and cytoplasm was similar in cells grown with and without hemin; 70 to 90% was in the cytoplasm. It is concluded that heme-deficient staphylococci form apo-cytochrome b, which readily combines in vitro with its prosthetic group to restore normal function. The avaliability of the heme prosthetic group influences the formation of nitrate reductase. 相似文献
7.
Cell-free preparations from the unicellular green alga, Chlorella vulgaris, catalyze the conversion of glutamate to delta-aminolevulinate, which is the first committed step in heme and chlorophyll biosynthesis. Most activity remains in the supernatant fraction after centrifugation at 264,000g. Additional activity can be solubilized from the high-speed pellet by treatment with 0.5 M NaCl. After gel filtration through Sephadex G-25, the reaction catalyzed by the high-speed supernatant requires glutamate, ATP, Mg2+, and NADPH. Boiled extract is inactive. The pH optimum is between 7.8 and 7.9 and the temperature optimum is 30 degrees C. Concentrations required for half-maximal activity are 0.05 mM glutamate, 0.4 mM ATP, 6 mM MgCl2, and 0.4 mM NADPH or 0.7 mM NADH. The reaction requires no additional amino donor. Involvement of pyridoxal phosphate in the catalytic mechanism is suggested by sensitivity to pyridoxal antagonists; 50% inhibition is achieved with 5 microM gabaculine or 0.4 mM aminooxyacetate. Involvement of two or more enzymes is suggested by the nonlinear reaction rate dependence on protein concentration. Evidence for the involvement of an activated glutamate intermediate was obtained by product formation after sequential addition and removal of substrates, and by inhibition (80%) with 1 mM hydroxylamine. Protoheme inhibits the activity by 50% at 1.2 microM. Preincubation of the extract with ATP causes stimulation and/or stabilization of the activity compared to preincubation without ATP or no preincubation. In preparations obtained from C. vulgaris strain C-10, which requires light for greening, dark-grown cells yield one-third as much activity as 4-h-greened cells. 相似文献
8.
Methanogenic archaeon Methanobrevibacter arboriphilus (strains AZ and DH1), which is a strict anaerobic microorganism not able to synthesize heme, possessed a very high catalase activity in the presence of 20-50?μM hemin in a growth medium. We investigated the effect of various oxidative stresses (hydrogen peroxide and oxygenation) on M. arboriphilus cells grown on the standard nutrient medium supplemented with 0.1?% yeast extract, and on the same medium supplemented with hemin. It was demonstrated that 30?μM hemin had a very significant positive effect on the resistance of M. arboriphilus strains to H(2)O(2) and O(2) stresses because of 30- to 40-fold increase of heme catalase activity. Thus, hydrogen peroxide (0.6-1.2?mM) or oxygen (3-5?%) had a strong negative impact on low-catalase cultures grown in the hemin-free standard medium, whereas the presence of 30?μM hemin in the medium results in a high yield of biomass even under conditions of four times stronger H(2)O(2) and two times stronger O(2) stresses. The intracellular catalase activity reached a high level in 30-60?min after hemin was added to the nutrient medium, but the activity already increased about 5-7-fold in 6?min after hemin addition. Our experimental data suggest that exogenous hemin provides an effective antioxidative defense in representatives of the genus Methanobrevibacter, specially playing an important role in the puromycin-insensitive formation of the active heme-containing catalase from presynthesized apoenzyme and heme. 相似文献
9.
The biosynthesis of phosphatidylcholine in rat liver microsomal preparations catalysed by CDP-choline-1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) was inhibited by a combination of ATP and CoA or ATP and pantetheine. ATP alone at high concentrations (20 mM) inhibits phosphatidylcholine formation to the extent of 70%. In the presence of 0.1 mM-CoA, ATP (2 mM) inhibits to the extent of 80% and in the presence of 1 mM-pantetheine to the extent of 90%. ADP and other nucleotide triphosphates in combination with either CoA or pantetheine are only 10-30% as effective in inhibiting phosphatidylcholine synthesis. AMP(CH2)PP [adenosine 5'-(alphabeta-methylene)triphosphate] together with CoA inhibits to the extent of 59% and with pantetheine by 48%. AMP-P(CH2)P [adenosine 5'-(betagamma-methylene)triphosphate] together with either CoA or pantetheine had no significant effect on phosphatidylcholine formation. Other closely related derivatives of pantothenic acid were without effect either alone or in the presence of ATP, as were thiol compounds such as cysteine, homocysteine, cysteamine, dithiothreitol and glutathione. Several mechanisms by which this inhibition might take place were ruled out and it is concluded that ATP together with either CoA or pantetheine interacts reversibly with phosphatidylcholine synthetase to cause temporarily the inhibition of phosphatidylcholine formation. 相似文献
10.
The mechanism of inhibition by hemin of the mutagenic activities of food pyrolysate aminoazaarenes, particularly that of Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole), was investigated. Hemin efficiently inhibited the metabolic activation by S9 of Trp-P-2, as demonstrated by high-performance liquid chromatographic analysis of the reaction mixtures in which Trp-P-2 had been treated with S9 in the presence or absence of hemin. N-Hydroxy-Trp-P-2, an activated form of Trp-P-2 having direct mutagenicity on Salmonella typhimurium TA98, undergoes spontaneous oxidative degradation in its aqueous solution, and the presence of hemin in the solution accelerated the degradation significantly. The presence of excess hemin with N-hydroxy-Trp-P-2 completely abolished the mutagenic activity of this mutagen towards Salmonella. A UV-visible spectroscopic study has suggested the formation of a complex between hemin and N-hydroxy-Trp-P-2/Trp-P-2. In support of this view, the fluorescence spectrum of a Trp-P-2 solution was quenched efficiently by the addition of hemin. These observations indicate that this complex formation plays a role in the observed multiple actions of hemin. Similar inhibitory actions of hemin on several other direct-acting aminoazaarene mutagens are also described, as well as the inhibition activities of protoporphyrin, chlorophyllin, biliverdin and bilirubin. 相似文献
11.
The initial reaction of tetrapyrrole formation in archaea is catalyzed by a NADPH-dependent glutamyl-tRNA reductase (GluTR). The hemA gene encoding GluTR was cloned from the extremely thermophilic archaeon Methanopyrus kandleri and overexpressed in Escherichia coli. Purified recombinant GluTR is a tetrameric enzyme with a native M(r) = 190,000 +/- 10,000. Using a newly established enzyme assay, a specific activity of 0.75 nmol h(-1) mg(-1) at 56 degrees C with E. coli glutamyl-tRNA as substrate was measured. A temperature optimum of 90 degrees C and a pH optimum of 8.1 were determined. Neither heme cofactor, nor flavin, nor metal ions were required for GluTR catalysis. Heavy metal compounds, Zn(2+), and heme inhibited the enzyme. GluTR inhibition by the newly synthesized inhibitor glutamycin, whose structure is similar to the 3' end of the glutamyl-tRNA substrate, revealed the importance of an intact chemical bond between glutamate and tRNA(Glu) for substrate recognition. The absolute requirement for NADPH in the reaction of GluTR was demonstrated using four NADPH analogues. Chemical modification and site-directed mutagenesis studies indicated that a single cysteinyl residue and a single histidinyl residue were important for catalysis. It was concluded that during GluTR catalysis the highly reactive sulfhydryl group of Cys-48 acts as a nucleophile attacking the alpha-carbonyl group of tRNA-bound glutamate with the formation of an enzyme-localized thioester intermediate and the concomitant release of tRNA(Glu). In the presence of NADPH, direct hydride transfer to enzyme-bound glutamate, possibly facilitated by His-84, leads to glutamate-1-semialdehyde formation. In the absence of NADPH, a newly discovered esterase activity of GluTR hydrolyzes the highly reactive thioester of tRNA(Glu) to release glutamate. 相似文献
12.
Rhodanese (EC 2.8.1.1.) from bovine liver contains four reduced cysteine groups. The –SH group of cysteine 247, located in a rhodanese active centre, transfers sulfane sulfur in a form of hydrosulfide (–S–SH) from appropriate donors to nucleophilic acceptors. We aimed to discover whether S-nitrosylation of critical cysteine groups in rhodanese can inhibit activity of the enzyme by covalent modification of –SH groups. The inhibition of rhodanese activity was studied with the use of a number of nitric oxide (NO) donors. We have successfully confirmed using several methods that the inhibition of rhodanese activity is a result of the formation of stable S-nitrosorhodanese. Low molecular weight NO donors, such as S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (GSNO), inactivate rhodanese and are much more effective in this regard (100% inhibition at 2.5 mM) than such known inhibitors of this enzyme, as N-ethylmaleimide (NEM) (25 mM < 50%) or sulfates(IV) (90% inhibition at 5 mM). On the other hand, sodium nitroprusside (SNP) and nitrites inhibit rhodanese activity only in the presence of thiols, which suggests that S-nitrosothiols (RSNO) also have to participate in this reaction in this case. A demonstration that rhodanese activity can be inhibited as a result of S-nitrosylation suggests the possible mechanism by which nitric oxide may regulate sulfane sulfur transport to different acceptors. 相似文献
13.
To verify a possible involvement of glutathione transferase pi in intracellular transport of hemin the interaction between the protein and the ligand was studied using three different spectroscopic techniques: intrinsic fluorescence quenching, kinetic measurements in the visible range and circular dichroism. From fluorescence experiments two binding sites for the hemin were found with Kd values of about 20 nM (high-affinity site) and 400 nM (low-affinity site). In the presence of glutathione or S-methylglutathione the high-affinity site further increased its affinity, while the second site reduced its affinity for hemin. The effect of hemin on the catalytic activity of the glutathione transferase pi was studied using two different glutathione concentrations. With 1 mM glutathione a non-linear Dixon plot was obtained, while decreased hemin inhibition and a linear pattern was observed with 2.5 mM glutathione. The Ki calculated was 4 microM and the inhibition appeared to be non-competitive with respect to 1-chloro-2,4-dinitrobenzene. CD spectra of the bilirubin-glutathione-transferase complex (350-600 nm region) at different hemin concentrations showed a common binding site for bilirubin and hemin. In conclusion, the presence of a high-affinity site for the hemin and the fact that glutathione at physiological concentrations increased the affinity of this site, suggest the involvement of glutathione transferase pi in the hemin transport. 相似文献
14.
An obligately anaerobic bacterium, Eubacterium lentum, was shown to contain cytochromes a, b, and c and a carbon monoxide-binding pigment. Extracts of cells grown with hemin gave a typical absorption spectrum for cytochrome c with maxima at 424, 525, and 553 nm. Extracts from cells grown in the absence of hemin also had an absorption peak corresponding to cytochrome b (562 nm) in their reduced versus oxidized spectrum. Extraction of hemes and formation of pyridine hemochromes allowed quantitation of protoheme IX and heme c. Large amounts of cytochrome c masked the presence of cytochrome b in cells grown in medium containing hemin. When cells were grown in the presence of 50 mM nitrate, cytochrome A (606 nm) was detected. In anaerobic extracts of cells grown either with or without nitrate, cytochromes b and c were reduced by formate and oxidized by NO3. Cytochrome a appeared to be partially oxidized by NO3 and completely oxidized by air. 相似文献
15.
The incubation of erythrocyte suspensions or isolated membranes containing a residual amount of hemoglobin (0.04% of original cellular hemoglobin) with tert-butyl hydroperoxide (tBHP, 0.5 mM) caused significant inhibition of basal and calmodulin-stimulated Ca2+ + Mg2(+)-ATPase activities and the formation of thiobarbituric acid reactive products measured as malondialdehyde. In contrast, the treatment of white ghosts (membranes not containing hemoglobin) with tBHP (0.5 mM) did not lead to appreciable enzyme inhibition within the first 20 min and did not result in malondialdehyde (MDA) formation. However, the addition of either 10 microM hemin or 100 microM ferrous chloride + 1 mM ADP to white ghosts produced hydroperoxide effects similar to those in pink ghosts (membranes with 0.04% hemoglobin). The concentrations of hemin and ferrous chloride which caused half-maximal inhibition of Ca2+ + Mg2(+)-ATPase activity at 10 min were 0.5 and 30 microM, respectively. The effects of several antioxidants (mannitol, thiourea, hydroxyurea, butylated hydroxytoluene, and ascorbate) were investigated for their protective effects against oxidative changes resulting from tBHP treatment. Over a 30-min incubation period only ascorbate significantly reduced the enzyme inhibition, MDA formation, and protein polymerization. Thiourea and hydroxyurea decreased MDA formation and protein polymerization but failed to protect against the enzyme inhibition. Butylated hydroxytoluene was similar to thiourea and hydroxyurea but with better protection at 10 min. Mannitol, under these conditions, was an ineffective antioxidant for all parameters tested. 相似文献
16.
A new guanosine nucleotide has been synthesized and characterized: guanosine 5'-O-[S-(3-bromo-2-oxopropyl)]thiophosphate (GMPSBOP), with a reactive functional group which can be placed at a position equivalent to the pyrophosphate region of GTP. This new analog is negatively charged at neutral pH and is similar in size to GTP. GMPSBOP has been shown to react with bovine liver glutamate dehydrogenase with an incorporation of 2 mol of reagent/mol of subunit. The modification reaction desensitizes the enzyme to inhibition by GTP, activation by ADP, and inhibition by high concentrations of NADH, but does not affect the catalytic activity of the enzyme. The rate constant for reaction of GMPSBOP with the enzyme exhibits a nonlinear dependence on reagent concentration with KD = 75 microM. The addition to the reaction mixture of alpha-ketoglutarate, GTP, ADP, or NADH alone results in little decrease in the rate constant, but the combined addition of 5 mM NADH with 0.4 mM GTP or with 10 mM alpha-ketoglutarate reduces the reaction rate approximately 6-fold. GMPSBOP modifies peptides containing Met-169 and Tyr-262, of which Tyr-262 is not critical for the decreased sensitivity of the enzyme toward allosteric ligands. The presence of 0.4 mM GTP plus 5 mM NADH protects the enzyme against reaction at both Met-169 and Tyr-262, but yields enzyme with 1 mol of reagent incorporated/mol of subunit which is modified at an alternate site, Met-469. In the presence of 0.2 mM GTP + 0.1 mM NADH, protection against modification of Tyr-262, but only partial protection against labeling of Met-169, is observed. In contrast, the presence of 10 mM alpha-ketoglutarate + 5 mM NADH protect only against reaction with Met-169. The results suggest that GMPSBOP reacts at the GTP-dependent NADH regulatory site [Lark, R. H., & Colman, R. F. (1986) J. Biol. Chem. 261, 10659-10666] of bovine liver glutamate dehydrogenase, which markedly affects the sensitivity of the enzyme to GTP inhibition. The reaction of GMPSBOP with Met-169 is primarily responsible for the altered allosteric properties of the enzyme. 相似文献
17.
Cyclic AMP-dependent protein kinases I and II, partially purified from rat liver cytosol, were inhibited 50% by 40 μM hemin and 100 μM hemin, respectively. With the purified catalytic subunit of cyclic AMP-dependent protein kinase, hemin caused non-competitive inhibition with respect to the peptide substrate and mixed inhibition with respect to ATP. Hemin also inhibited purified phosphorylase b kinase, indicating that hemin concentrations above 10 μM markedly inhibit multiple protein kinases. In isolated intact hepatocytes, hemin inhibited the glucagon-dependent activation of cyclic AMP-dependent protein kinases and the activation of glycogen phosphorylase. For both effects, high heme concentrations (40–60 μM) were required for 50% inhibition. Similar high levels of exogenous hemin inhibited total hepatocyte protein synthesis. By contrast, 5 μM hemin or less was sufficient to raise intracellular heme levels, as indicated by the relative heme-saturation of tryptophan oxygenase in hepatocytes. Hemin, 5 μM, completely repressed induction of 5-aminolevulinate synthase by dexamethasone in hepatocyte primary cultures. Such repression is unlikely to be mediated by inhibition of protein kinases. 相似文献
18.
The oxidation of melatonin (MEL) using the Cu(II) + H 2O 2 + HO − (the Fenton-like reaction) system was investigated by chemiluminescence (CL), fluorescence, spectrophotometric, and EPR spin trapping techniques. The reaction exhibits CL in the 400–730 nm region. The light emission from the Fenton-like reaction was greatly enhanced in the presence of MEL and was strongly dependent on its concentration. The spectrum measured with cut-off filters revealed maxima at around 460, 500, 580–590, 640–650, and 690–700 nm. The band at 460 nm may be due to the excited cleavage product, N 1-acetyl-N 2-formyl-5-methoxykynuramine, whereas the bands at 500, 580–590, 640–650, and 700 nm were similar to those observed for singlet molecular oxygen ( 1O 2). The effect of reactive oxygen species (ROS) scavengers on the light emission was studied. The CL was strongly inhibited by the 1O 2 scavengers in a dose-dependent manner; at concentration 1 mM the potency of 1O 2 scavenging was 5,5-dimethylcyclohexandione-1,3 > methionine > histidine > hydroquinone. The potency of HO • scavenging by thiourea, tryptophan, cysteine at concentration 5 mM was 79–94%, by 1 mM glutathione and trolox 75 and 94%, respectively, and by 10 mM cimetidine 18%. Specific acceptors of O 2•− such as p-nitroblue tetrazolium chloride and 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron) at concentration 5 mM decreased the CL by 51 and 95%, respectively, whereas superoxide dismutase (SOD) does not reduce the emission at concentration 2.8 U/ml. At higher concentration SOD substantially enhanced the light emission. Addition of 1360 U/ml catalase and 100 μM desferrioxamine strongly inhibited CL (96 and 90%, respectively). The increased generation of 1O 2 from the Cu/H 2O 2 system in the presence of MEL was confirmed using the spectrophotometric method based on the bleaching of p-nitrosodimethylaniline and by trapping experiments with 2,2,6,6-tetramethylpiperidine (TEMP) and subsequent electron paramagnetic (EPR) spectroscopy. These findings suggest the increased production of reactive oxygen species (O 2•−, HO •, 1O 2) from the Fenton-like reaction in the presence of MEL. This means that the hormone is not able to act as classical chain-breaking antioxidant even at low concentration, and may show clear prooxidant activity at higher concentrations. In addition, long-lived carbonyl product of the MEL transformation in the triplet state can also be toxic by transferring its energy to organelles and causing a photochemical process. 相似文献
19.
Red blood cell lysis is a common symptom following severe or prolonged oxidative stress. Oxidative processes occur commonly in sickle cells, probably mediated through denatured hemoglobin and the accumulation of ferric hemes in the membranes. Calmodulin-stimulated (Ca2+ + Mg2+)-ATPase from sickle red cell membranes is partially inactivated (Leclerc et al. (1987) Biochim. Biophys. Acta 897, 33-40). In this study (Ca2+ + Mg2+)-ATPase activity from normal adult erythrocyte membranes was measured in the presence of hemin. We report a time- and concentration-dependent inhibition of the activity of the enzyme by hemin due to a decrease in the maximum velocity. Only a mild inhibitory effect was observed in the presence of iron-free protoporphyrin IX, indicating the catalytic influence of the iron. Experiments carried out with hemin (ferric iron) liganded with imidazole or with reduced protoheme (ferrous iron) liganded with carbon monoxide, demonstrated that the inhibition requires that hemin be capable of binding additional ligands. The inhibition was not influenced by the absence of oxygen but was prevented by addition of bovine serum albumin. Addition of butylated hydroxytoluene, a protective agent of lipid peroxidation, failed to prevent the inhibition of calmodulin-stimulated (Ca2+ + Mg2+)-ATPase. As dithiothreitol partially restores the enzyme activity, we postulated that hemin interacts with the thiol groups of the enzyme. 相似文献
20.
A biosensor based on flow injection of the recognition element has been developed. As a model a pH-transducer was used, and urease was chosen as the recognition element. The pH-transducer was immersed in an internal flow-through chamber which was in contact with the sample solution via a semi-permeable membrane. The recognition element, urease, was injected into the buffer solution passing through the biosensor. The enzyme catalysed the hydrolysis of urea and the concomitant increase in pH was recorded. The biosensor response time was about three minutes at a constant flow rate of 0·05 ml/min. The linear range of the calibration curve of the biosensor was 0–5 mM. The observed detection limit was approximately 0.1 mM. The sample throughput was 6–12 per hour. The pH-response of the biosensor, for a sample solution containing urea (3·26 mM), showed a reproducibility (r.s.d) of 28% ( n = 5) and a repeatability (r.s.d.) of 8% ( n = 5). Operation at elevated temperatures (up to 50°C) was demonstrated. The presence of glucose (28 mM), acetone (6·7 mM), citric acid (0·2 mM) or sodium acetate (0·6 mM) in the sample solution did not interfere with the sensor response. A lowering of the biosensor response which was observed in the presence of copper ions (due to urease inhibition) could be completely eliminated by adding EDTA to the urease solution. Thus, this work demonstrates a new type of biosensors, based on SIRE-technology (Sensors with Injectable Recognition Elements), which show high accuracy and stability, quick response and high sample throughput. These features suggest the suitability of the system for automation. Such sensors should readily be combined with other enzymes or enzyme systems. The enzyme (urease) cost per analysis (injection) for the biosensor was estimated to be approximately US$0·02. This could be substantially reduced by further optimisation and miniaturisation. 相似文献
|