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Heme occupancy of catalase in hemoglobin-free perfused rat liver and of isolated rat liver catalase 总被引:8,自引:0,他引:8
Maximal heme occupancy, the maximal proportion of total catalase heme present in the form of Compound I, is found to be 0.4 both in the enzyme isolated from rat liver and in the peroxisomal enzyme as present in the intact cells of perfused rat liver. This indicates that the ratio of second order rate constants for catalatic decomposition and for formation of Compound I, , is equal in vitro and in vivo.Catalase was isolated from rat liver, and the extinction coefficients for Compound I and for cyanide-catalase at 640 minus 660 nm were determined. The measurement of heme occupancy of catalase in hemoglobin-free perfused rat liver was made possible by wavelength scanning as well as by dual wavelength absorbance photometry. Thus, Compound I and cyanide-catalase were demonstrated in the red region and in the Soret band region.Meeting the particular needs of organ photometry, specific metabolic transitions were used to visualize specific transitions of absorbing pigments. Compound I is specifically demonstrated by its decomposition by the hydrogen donor, methanol. A measure for total catalase heme is provided by formation of cyanide-catalase. The cyanide concentrations required are well below appearance of possible interference by other cyanide-binding hemoproteins at 640–660 nm. 相似文献
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W S Thayer 《FEBS letters》1986,202(1):137-140
Cardiac metabolism of H2O2 was studied by determining the concentration dependence for H2O2-stimulated release of GSSG, an indicator for flux through the glutathione peroxidase pathway, in perfused heart preparations. Treatment of rats with aminotriazole in vivo inhibited heart catalase by 83% and shifted the dose-response curve for GSSG release toward lower H2O2 concentrations. In aminotriazole-treated rats, 50 microM H2O2 elicited a maximal rate of GSSG release (about 5 nmol GSSG/min per g heart), whereas 200 microM H2O2 was necessary for obtaining a similar rate of GSSG release in control rat hearts. The results show that catalase, although present at low levels of activity in the heart compared to other organs, functions as a major route for detoxication of H2O2 in the myocardium. 相似文献
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Peroxisomal fatty acid oxidation as detected by H2O2 production in intact perfused rat liver. 总被引:2,自引:2,他引:0
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1. H2O2 formation associated with the metabolism of added fatty acids was quantitatively determined in isolated haemoglobin-free perfused rat liver (non-recirculating system) by two different methods. 2. Organ spectrophotometry of catalase Compound I [Sies & Chance (1970) FEBS Lett. 11, 172-176] was used to detect H2O2 formation (a) by steady-state titration with added hydrogen donor, methanol or (b) by comparison of fatty-acid responses with those of the calibration compound, urate. 3. In the use of the peroxidatic reaction of catalase, [14C]methanol was added as hydrogen donor at an optimal concentration of 1 mM in the presence of 0.2 mM-L-methionine, and 14CO2 production rates were determined. 4. Results obtained by the different methods were similar. 5. The yield of H2O2 formation, expressed as the rate of H2O2 formation in relation to the rate of fatty-acid supply, was less than 1.0 in all cases, indicating that, regardless of chain length, less than one acetyl unit was formed per mol of added fatty acid by the peroxisomal system. In particular, the standard substrate used with isolated peroxisomal preparations (C16:0 fatty acid) gave low yield (close to zero). Long-chain monounsaturated fatty acids exhibit a relatively high yield of H2O2 formation. 6. The hypolipidaemic agent bezafibrate led to slightly increased yields for most of the acids tested, but the yield with oleate was decreased to one-half the original yield. 7. It is concluded that in the intact isolated perfused rat liver the assayable capacity for peroxisomal beta-oxidation is used to only a minor degree. However, the observed rates of H2O2 production with fatty acids can account for a considerable share of the endogenous H2O2 production found in the intact animal. 相似文献
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Formation of compound I by the reaction of catalase with peroxoacetic acid 总被引:4,自引:3,他引:1
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1. The formation of Compound I by the reactions of bacterial and ox liver catalases with peroxoacetic acid was examined. In both cases the process occurs almost entirely by reaction of catalase with un-ionized peroxoacetic acid molecules. The result suggests an important role for the bound peroxidic proton in the enzyme-substrate interaction. 2. The peroxidatic properties of the Compounds I formed when peroxoacetic acid was used were examined by studying the oxidations of ethanol and formate; the results closely resemble those previously reported when H(2)O(2) and alkyl hydroperoxides were used. 3. Compound I formed with bacterial catalase and peroxoacetic acid is remarkably stable in the absence of added donor and the preparation has considerable potential for detailed studies of the nature of this intermediate. 相似文献
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S S Lee J H Ye D P Jones D B McCormick 《Biochemical and biophysical research communications》1983,117(3):788-793
A substantial decrease in liver peroxisomal catalase was found during riboflavin deficiency in rats. This decrease is greater than that found among other hemoproteins and seems to follow decrease in flavin-dependent peroxisomal oxidases. This is not due to a general depression of peroxisomal enzymes, since Cu-dependent urate oxidase activity was not changed. Furthermore, the level of catalase activity as well as flavin-dependent oxidases was restored by riboflavin repletion. These results suggest that hydrogen peroxide, the substrate for catalase produced by several flavoprotein oxidases, induces catalase in mammals as has been indicated for certain bacteria. 相似文献
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Antonio Díaz-Cruz Raquel Guinzberg Ruy Guerra Magdalena Vilchis Daniel Carrasco Francisco J. García-vázquez 《Free radical research》2013,47(6):663-672
It is known that adrenaline promotes hydroxyl radical generation in isolated rat hepatocytes. The aim of this work was to investigate a potential role of NADPH oxidase (Nox) isoforms for an oxidative stress signal in response to adrenaline in hepatocytes. Enriched plasma membranes from isolated rat liver cells were prepared for this purpose. These membranes showed catalytic activity of Nox isoforms, probably Nox 2 based on its complete inhibition with specific antibodies. NADPH was oxidized to convert O2 into superoxide radical, later transformed into H2O2. This enzymatic activity requires previous activation with either 3 mM Mn2+ or guanosine 5′-0-(3-thiotriphosphate) (GTPγS) plus adrenaline. Experimental conditions for activation and catalytic steps were set up: ATP was not required; S0.5 for NADPH was 44 μM; S0.5 for FAD was 8 μM; NADH up to 1 mM was not substrate, and diphenyleneiodonium was inhibitory. Activation with GTPγS plus adrenaline was dose- and Ca2+-dependent and proceeded through α1-adrenergic receptors (AR), whereas β-AR stimulation resulted in inhibition of Nox activity. These results lead us to propose H2O2 as additional transduction signal for adrenaline response in hepatic cells. 相似文献
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Díaz-Cruz A Guinzberg R Guerra R Vilchis M Carrasco D García-Vázquez FJ Piña E 《Free radical research》2007,41(6):663-672
It is known that adrenaline promotes hydroxyl radical generation in isolated rat hepatocytes. The aim of this work was to investigate a potential role of NADPH oxidase (Nox) isoforms for an oxidative stress signal in response to adrenaline in hepatocytes. Enriched plasma membranes from isolated rat liver cells were prepared for this purpose. These membranes showed catalytic activity of Nox isoforms, probably Nox 2 based on its complete inhibition with specific antibodies. NADPH was oxidized to convert O(2) into superoxide radical, later transformed into H(2)O(2). This enzymatic activity requires previous activation with either 3 mM Mn(2+) or guanosine 5'-0-(3-thiotriphosphate) (GTPgammaS) plus adrenaline. Experimental conditions for activation and catalytic steps were set up: ATP was not required; S(0.5) for NADPH was 44 microM; S(0.5) for FAD was 8 microM; NADH up to 1 mM was not substrate, and diphenyleneiodonium was inhibitory. Activation with GTPgammaS plus adrenaline was dose- and Ca(2+)-dependent and proceeded through alpha(1)-adrenergic receptors (AR), whereas beta-AR stimulation resulted in inhibition of Nox activity. These results lead us to propose H(2)O(2) as additional transduction signal for adrenaline response in hepatic cells. 相似文献
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Alcohol metabolism via alcohol dehydrogenase (ADH) and catalase was studied in perfused rat livers by measuring the oxidation of methanol and butanol, selective substrates for catalase and ADH, respectively. In livers from fasted rats, basal rates of methanol uptake of 15 +/- 1 mumol/g/h were decreased significantly to 8 +/- 2 mumol/g/h by addition of butanol. Concomitantly, pyridine nucleotide fluorescence detected from the liver surface was increased by butanol but not methanol. Both effects of butanol were blocked by an inhibitor of ADH, 4-methylpyrazole, consistent with the hypothesis that elevation of the NADH redox state by butanol inhibited H2O2 production via NAD+-requiring peroxisomal beta-oxidation, leading indirectly to diminished rates of catalase-dependent methanol uptake. In support of this idea, both butanol and butyraldehyde inhibited H2O2 generation. The NADH redox state was also elevated by xylitol, causing a 75% decrease in rates of methanol uptake by livers from fasted rats. This effect was not observed in livers from fed rats unless malate-aspartate shuttle activity was reduced by infusion of the transaminase inhibitor aminooxyacetate. Taken together, these data indicate that generation of reducing equivalents from ADH in the cytosol inhibits H2O2 generation leading to significantly diminished rates of peroxidation of alcohols via catalase. This phenomenon may represent an important physiological mechanism of regulation of ethanol oxidation in intact cells. 相似文献
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Nozomu Oshino Britton Chance Helmut Sies 《Archives of biochemistry and biophysics》1973,159(2):704-711
The possibility of the occurrence of the secondary catalase-peroxide complex (Compound II) in the isolated peroxisomal-mitochondrial fraction of rat liver and in the perfused rat liver has been examined under various conditions. The steady state of Compound I is maintained by either an endogeneous or a urate and glycolate-supplemented H2O2 generation in both systems, but Compound II is not detectable. Significant accumulation of Compound II, which is identified by the measurement of its difference spectrum and by its response to hydrogen donors, is observed only when Compound I is converted to Compound II by an appropriate concentration of p-cresol. The properties of Compound II observed in the perfused liver are similar to those observed with isolated catalase. 相似文献
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Treatment of rats with noradrenaline stimulated H2O2 generation in liver mitochondria using succinate, choline or glycerol 1-phosphate as substrate. The dehydrogenase activity with either succinate or choline as substrate showed no change, whereas that with glycerol 1-phosphate increased. The effect was obtained with noradrenaline, but not with dihydroxyphenylserine. Phenoxybenzamine and yohimbine, but not propranolol, prevented the response to noradrenaline treatment. Phenylephrine could stimulate H2O2 generation, whereas isoprenaline had only a marginal effect. Theophylline treatment slightly decreased the generation of H2O2 in liver mitochondria, but treatment with pargyline, Ro4-1284 and dibutyryl cyclic AMP had little effect. These studies showed that noradrenaline might possibly be acting through the alpha 2-adrenergic system. 相似文献
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Scott MD 《Free radical biology & medicine》2006,40(7):1264-1272
Redox-mediated injury is an important pathway in the destruction of beta thalassemic red blood cells (RBC). Because of the autoxidation of the unstable hemoglobin chains and subsequent release of globin free heme and iron, significant amounts of superoxide (O2-) and, more importantly, hydrogen peroxide (H2O2) are generated intracellularly. Hence, catabolism of H2O2 is crucial in preventing cellular injury. Removal of H2O2 is mediated via two primary pathways: GSH-dependent glutathione peroxidase or catalase. Importantly, both pathways are ultimately dependent on NADPH. In the absence of any exogenous oxidants, model thalassemic RBC demonstrated significantly decreased GSH levels (P < 0.001 at 20 h). Perhaps of greater pathophysiologic importance, however, was the finding that the model thalassemic RBC exhibited significantly (P < 0.001) decreased catalase activity. Following 20 h incubation at 37 degrees C only 61.5 +/- 2.9% of the initial catalase activity remained in the alpha-hemoglobin chain-loaded cells versus 104.6 +/- 4.5 and 108.2 +/- 3.2% in the control and control-resealed cells, respectively. The mechanism underlying the loss of both catalase activity and GSH appears to be the same in that both catabolic pathways require adequate NADPH levels. As shown in this study, model beta thalassemic cells are unable to maintain a normal ( approximately 1.0) NADPH/NADP(total) ratio and, after 20 h, the model beta thalassemic cells have a significantly (P < 0.001) lower ratio ( approximately 0.5) which is quite similar to a G6PD-deficient RBC. In support of these findings, direct inactivation of catalase gives rise to significantly increased oxidant damage. In contrast, GSH depletion is not closely associated with oxidant sensitivity. Indeed, the consumption of GSH noted in the thalassemic RBC may be via a prooxidant pathway as augmentation of cellular GSH levels actually enhances alpha-hemoglobin chain-mediated injury. 相似文献
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A study was made of generation of O2- in NADPH-dependent chain of oxidation of liver microsomes in irradiated rats (7 and 10 Gy). The rate of O2- generation sharply increased at early times after irradiation. The data obtained prompt an assumption that the increase in the rate of O2- generation is perhaps connected with the changes in functioning of both NADPH-cytochrome P-450-reductase and cytochrome P-450. 相似文献
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Thiol modification in H2O2- and thromboxane-induced vaso- and bronchoconstriction in rat perfused lung. 总被引:1,自引:0,他引:1
L Atzori K Olafsdóttir A M Corriga G Bannenberg A Ryrfeldt P Moldéus 《Journal of applied physiology》1991,71(4):1309-1314
Hydrogen peroxide (H2O2), arachidonic acid (AA), and U-44069, a thromboxane analogue, all induced vaso- and bronchoconstriction in the isolated perfused rat lung. The role of protein sulfhydryl modifications in these processes was investigated. The thiol oxidizing agent diamide inhibited both vaso- and bronchoconstriction induced by H2O2, AA, or U-44069. Diamide had only a marginal effect on glutathione and protein thiol levels and no effect on lung mechanics. The diamide inhibition was reversible, and H2O2-induced vaso- and bronchoconstriction was almost maximal after 10 min of perfusion with buffer. The recovery was more rapid if dithiothreitol, a thiol reducing agent, was used in the buffer. H2O2- and AA-induced vaso- and bronchoconstriction is caused by thromboxane release. Diamide did not influence H2O2- or AA-dependent thromboxane formation, indicating that neither AA release nor AA metabolism to thromboxane is sensitive to thiol oxidation. Thus our results indicate that the site of diamide-induced thiol oxidation is the thromboxane receptor or its signal transduction. 相似文献
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M L Kremer 《European journal of biochemistry》1989,185(3):651-658
The kinetics of formation of the dominant intermediate (CII) formed between hemin and H2O2 has been studied by the stopped-flow method. CII is preceded by a precursor (CI) for which a steady state is established at an early stage of the reaction. The formation of CI from hemin and H2O2 causes only a marginal change in the optical absorbance (A). The transition CI----CII is accompanied by a substantial decrease of A in the Soret region. Relevant rate constants (or combinations of them) and the molar absorption coefficients of the intermediates at 400 nm have been determined. The absorption spectrum of CII in the Soret region has been evaluated. Aspects of the catalysis of decomposition of H2O2 by hemin in relation to the Fe3+ ion and catalase are discussed. 相似文献