共查询到20条相似文献,搜索用时 31 毫秒
1.
Paul J. Tortoriello John F. Riebow ShakuntalaAdvani Wayne R. Bidlack 《Free radical biology & medicine》1991,10(6):387-396
Carbon tetrachloride metabolism was examined in hepatic microsomes isolated from control and phenobarbital-treated Sprague-Dawley rats to determine the mechanism of pyridine nucleotide synergism. An NADPH generator increased metabolism two fold as determined by lipid peroxidation. Addition of NADP to the reaction system did not alter the maximum velocity, but did decrease the Km for NADPH from 61 μM to 7.6 μM in control and from 21 μM to 6.3 mM PB microsomes. Addition of NAD+ produced an increase in metabolism similar to NADH. Substrates and competitive inhibitors of nucleotide pyrophosphatase also enhanced CCl4 metabolism. A high correlation (r=0.947) was indicated between the percent inhibition of nucleotide pyrophosphatase and the percent synergism of NADPH-catalyzed CCl4 metabolism. Thus, pyridine nucleotiode synergism in CCl4 metabolism appears to result from the increased availability of NADPH produced by a decreased degradation of the NADPH by the nucleotide pyrophosphatase. 相似文献
2.
In vitro inhibition of rat liver mitochondrial and microsomal aldehyde dehydrogenase (ALDH) under conditions of active CCl4 metabolism was investigated. Incubation of microsomes or mitochondria in the presence of NADPH alone caused significant, time-dependent inhibition of mitochondrial and microsomal ALDH. EDTA partially protected ALDH from inhibition. Incubation of microsomes or microsomes plus mitochondria in the presence of NADPH and CCl4 resulted in marked inhibition of microsomal and mitochondrial ALDH activity. The inhibition was both dose- and time-dependent and was relatively less in the presence of EDTA. It is proposed that the inhibition of membrane-bound ALDH may be one of the early events responsible for the genesis of CCl4-hepatotoxicity. 相似文献
3.
The metabolism of chromate by rat liver microsomes has been studied. Incubation of chromate with microsomes in the presence of the enzyme cofactor NADPH, resulted in reduction of chromate. In the absence of NADPH no reduction occurred. Only a small amount of chromate reduction was seen with NADPH in the absence of microsomes. Time course studies, microsome and NADPH concentration dependence studies resulted in conditions giving complete reduction of chromate. The possible relationship of metabolism of chromate to its carcinogenicity and mutagenicity is discussed. 相似文献
4.
The ethylmorphine-N-demethylation by liver microsomes from control and phenobarbital-treated rats of different ages was investigated by means of adding NADPH in combination with NADH to the incubation medium. The rate of ethylmorphine-N-demethylation in the presence of NADPH without NADH is greater in adult than in young rats and greater in induced that in control rats. The higher the activity of ethylmorphine metabolism with NADPH alone the more it is abolsutely enhanced by NADH. The relative increase in ethylmorphine metabolism caused by NADH is equal in all groups of animals. It is concluded that there are no differences in the introduction of the second electron from NADH to the oxygenated cytochrome P-450 but there are differences in the concentration of cytochrome-substrate complex and, consequently, in the oxygenated cytochrome-substrate complex. The enhancing effect of NADH is higher at lower NADPH concentrations. In the presence of NADH, the NADPH concentrations necessary to obtain a msximum metabolic rate are lower than without NADH. 相似文献
5.
J L Poyer R A Floyd P B McCay E G Janzen E R Davis 《Biochimica et biophysica acta》1978,539(3):402-409
Utilizing the spin-trapping agent phenyl-t-butyl nitrone, a free radical has been detected which is produced from carbon tetrachloride or bromotrichloromethane during the enzymic oxidation of NADPH by rat liver microsomes. The presence of NADPH is obligatory for generation of the radical. The formation of the trichloromethyl radical-phenyl-t-butyl nitrone adduct is an enzymic process, as evidenced by the inhibition of its formation in systems containing heated microsomes and in systems containing p-hydroxymercuribenzoate. A computer-simulated ESR spectrum for the trichloromethyl adduct of phenyl-t-butyl nitrone can reproduce the essential features of the spectrum of the spin-trapped radical produced enzymically from CCl4. A mechanism is proposed for the formation of the trichloromethyl radical from CCl4 or BrCCl3. 相似文献
6.
trans-4-Phenyl-3-buten-2-one (PBO), a flavoring additive, was transformed to the carbonyl-reduced product, trans-4-phenyl-3-buten-2-ol (PBOL) by rat liver microsomes, but not by liver cytosol, in the presence of NADH or NADPH. PBOL formed was identified by comparison with an authentic sample. The reductase activity was not inhibited by quercitrin, an inhibitor of cytosolic carbonyl reductase. The carbonyl reduction product of PBO by liver microsomes was identified as the R-enantiomer of PBOL by HPLC analysis. Rat blood also exhibited the carbonyl reductase activity in the presence of NADH or NADPH, but to a lesser extent. 相似文献
7.
Chromate metabolism in liver microsomes 总被引:3,自引:0,他引:3
Karen W. Jennette 《Biological trace element research》1979,1(1):55-62
The carcinogenicity and mutagenicity of various chromium compounds have been found to be markedly dependent on the oxidation
state of the metal. The carcinogen chromate was reduced to chromium(III) by rat liver microsomes in vitro. Metabolism of chromate
by microsomal enzymes occurred only in the presence of either NADPH or NADH as cofactor. The chromium(III) generated upon
metabolism formed a complex with the NADP+ cofactor. Significant binding of chromium to DNA occurred only when chromate was incubated in the presence of microsomes
and NADPH. Specific inhibitors of the mixed function oxidase enzymes, 2′-AMP, metyrapone, and carbon monoxide, inhibited the
rate of reduction of chromate by microsomes and NADPH. The possible relationship of metabolism of chromate and its interaction
with nucleic acids to its carcinogenicity and mutagenicity is discussed. 相似文献
8.
Vanadate-dependent NAD(P)H oxidation, catalyzed by rat liver microsomes and microsomal NADPH-cytochrome P450 reductase (P450 reductase) and NADH-cytochrome b5 reductase (b5 reductase), was investigated. These enzymes and intact microsomes catalyzed NAD(P)H oxidation in the presence of either ortho- or polyvanadate. Antibody to P450 reductase inhibited orthovanadate-dependent NADPH oxidation catalyzed by either purified P450 reductase or rat liver microsomes and had no effect on the rates of NADH oxidation catalyzed by b5 reductase. NADPH-cytochrome P450 reductase catalyzed orthovanadate-dependent NADPH oxidation five times faster than NADH-cytochrome b5 reductase catalyzed NADH oxidation. Orthovanadate-dependent oxidation of either NADPH or NADH, catalyzed by purified reductases or rat liver microsomes, occurred in an anaerobic system, which indicated that superoxide is not an obligate intermediate in this process. Superoxide dismutase (SOD) inhibited orthovanadate, but not polyvanadate-mediated, enzyme-dependent NAD(P)H oxidation. SOD also inhibited when pyridine nucleotide oxidation was conducted anaerobically, suggesting that SOD inhibits vanadate-dependent NAD(P)H oxidation by a mechanism independent of scavenging of O2-. 相似文献
9.
《Archives of biochemistry and biophysics》1987,255(2):267-273
An enzymatic Na3VO4-dependent system for the oxidation of reduced pyridine nucleotides in purified rat liver microsomes was characterized. The system has a pH optimum of 6.5, and appears to be specific for vanadate, since activity in the presence of a related transition metal, molybdate, was not detected. Vanadate-dependent oxidation occurred with a concomitant consumption of O2 and, contrary to previous reports, preferred NADPH over NADH. At pH 6.5, the NADPH/NADH oxidase activity ratio was greater than 2:1. Sodium vanadate-dependent oxidation of NADH was inhibited by rotenone, antimycin A, NaN3, and NaCN. Conversely, Na3VO4-dependent NADPH oxidation was slightly affected by rotenone, but was insensitive to antimycin A, NaN3, NaCN, or quinacrine. Vanadate-dependent oxidation of either pyridine nucleotide was inhibited by the addition of either Superoxide dismutase or catalase, indicating that both superoxide and hydrogen peroxide may be intermediates in the process. Linear sucrose gradient purification of the microsomes showed that the vanadate-dependent system for NADPH oxidation resides primarily in the endoplasmic reticulum. These studies indicate the existence of separate and distinct enzymatic systems for vanadate-stimulated oxidation of NADPH and NADH in mammalian microsomal membranes, and argue against an exclusive role of endogenous Superoxide in the process. 相似文献
10.
Metabolism of nitroxide spin labels in subcellular fraction of rat liver. I. Reduction by microsomes 总被引:1,自引:0,他引:1
As part of an ongoing study of the role of subcellular fractions on the metabolism of nitroxides, we studied the metabolism of a set of seven nitroxides in microsomes obtained from rat liver. The nitroxides were chosen to provide information on the effects of the type of charge, lipophilicity and the ring on which the nitroxide group is located. Important variables that were studied included adding NADH, adding NADPH, induction of enzymes by intake of phenobarbital and the effects of oxygen. Reduction to nonparamagnetic derivatives and oxidation back to paramagnetic derivatives were measured by electron-spin resonance spectroscopy. In general, the relative rates of reduction of nitroxides were similar to those observed with intact cells, but the effects of the various variables that were studied often differed from those observed in intact cells. The rates of reduction were very slow in the absence of added NADH or NADPH. The relative effect of these two nucleotides changed when animals were fed phenobarbital, and paralleled the levels of NADPH cytochrome c reductase, cytochrome P-450, cytochrome b5 and NADH cytochrome c reductase; results with purified NADPH-cytochrome c reductase were consistent with these results. In microsomes from uninduced animals the rate of reduction was about 10-fold higher in the absence of oxygen. The products of reduction of nitroxides by microsomes were the corresponding hydroxylamines. We conclude that there are significant NADH- and NADPH-dependent paths for reduction of nitroxides by hepatic microsomes, probably involving cytochrome c reductases and not directly involving cytochrome P-450. From this, and from parallel studies now in progress in our laboratory, it seems likely that metabolism by microsomes is an important site of reduction of nitroxides. However, mitochondrial metabolism seems to play an even more important role in intact cells. 相似文献
11.
Inhibition of CCl4 metabolism by oxygen varies between isoenzymes of cytochrome P-450 总被引:1,自引:0,他引:1
R F Burk K E Hill J M Lane 《Biochemical and biophysical research communications》1988,152(3):1463-1467
Oxygen inhibition of CCl4 metabolism by different isoenzymes of cytochrome P-450 was assessed by studying liver microsomes isolated from control rats and rats treated with phenobarbital or isoniazid. Rates of CCl4 metabolism were similar for all microsomes under a nitrogen atmosphere. An air atmosphere inhibited metabolism by microsomes from control rats to 12% of the value under nitrogen and metabolism by microsomes from rats treated with phenobarbital to 5%. It inhibited metabolism by microsomes from rats treated with isoniazid only to 32%. Rats treated with phenobarbital, which increases hepatic cytochrome P-450 content, or isoniazid, which does not increase hepatic cytochrome P-450 content, both metabolized more CCl4 than control rats as indicated by exhalation of greater quantities of CCl4 metabolites and by an increase in CCl4 toxicity. These results indicate that some isoenzymes of cytochrome P-450 are more effective than others in metabolizing CCl4 when oxygen is present. 相似文献
12.
The mutagenic activity of dimethylnitrosamine activated by rat-liver microsomes in the presence of NADH was compared with that obtained with NADPH. 3 histidine auxotrophic strains of Salmonella underwent reversions after activation with NADH as the sole coenzyme. All 3 tester strains showed a dose-response relationship with dimethylnitrosamine (10-125 mumoles per plate) after NADH-supported activation. With NADH as the sole coenzyme, the most sensitive strain, hisG46, showed a 105-fold increase in mutagenesis frequency as compared with the 230-fold increase obtained with NADPH. Activation of dimethylnitrosamine in the presence of NADH and NADPH, in combination, produced mutagenesis at frequencies above those seen with NADH alone, but less than or equal to those seen with NADPH as the only coenzyme during the activation step. Experiments in vitro showed that microsomal incorporation of carbon from [14C]dimethylnitrosamine was highest in the presence of NADPH, lowest with NADH and reached intermediate levels when both coenzymes were present. The source of the microsomes in all experiments was liver from rats pre-treated with Aroclor 1254. 相似文献
13.
A A Kandutsch 《Journal of lipid research》1966,7(5):603-611
Aerobic incubation of 7-dehydrocholesterol with mouse liver microsomes in the presence of a detergent, an iron salt, and NADH or NADPH resulted in the conversion of the sterol to more polar products. In the presence of Fe(3+) or low levels of Fe(2+) the reaction was dependent upon reduced pyridine nucleotide and a microsomal enzyme system. At high levels of Fe(2+) or in the presence of Fe(2+) or Fe(3+) and ascorbic acid, nonenzymatic oxidation of 7-dehydrocholesterol occurred in the absence of NADH or NADPH. Chromatograms of products resulting from the enzyme-dependent and enzyme-independent reactions were similar. The enzymatic reaction was inhibited by certain chelating agents, by antioxidants, and by menadione, phenazine methosulfate, and ferricyanide. Low concentrations of EDTA stimulated the reaction and high concentrations inhibited it. In the complete system sterol oxidation was correlated with the peroxidation of microsomal lipids, but peroxidation of microsomal lipids proceeded more rapidly when either the sterol, the detergent, or both were omitted. Ergosterol was resistant to oxidation under conditions that caused extensive loss of 7-dehydrocholesterol. Microsomes from tissues other than liver were relatively inactive. 相似文献
14.
Microsomal metabolism of hexavalent chromium. Inhibitory effect of oxygen and involvement of cytochrome P-450 总被引:1,自引:0,他引:1
The reduction of hexavalent chromium (Cr(VI] by rat liver microsomes was studied. With 15-120 microM Na2CrO4 microsomes (0.5 mg protein/ml) effectively reduced Cr(VI) in the presence of NADPH provided anaerobic conditions. Phenobarbital (PB) and Aroclor 1254 (PCB) pretreatment increased microsomal Cr(VI) reduction while CoCl2 reduced the rate. The rates with 30 microM Na2CrO4 were: 6.4 +/- 0.1, 7.8 +/- 0.7, 13.4 +/- 0.5, 2.95 +/- 0.09 nmol Cr.mg prot.-1 min-1 for control, PB, PCB and cobalt pretreated microsomes respectively. Kinetic studies gave a Michaeli-Menten like first-order kinetics with increases both in Km and Vmax values after pretreatment with PB or PCB. CO partly inhibited the microsomal Cr(VI) reduction. The CO-sensitive reduction rate was directly correlated to the cyt. P-450 content of the different microsomal preparations. Substituting NADH for NADPH gave approximately 27% lower activity with 30 microM Na2CrO4. This activity was neither inducible by cyt. P-450 inducers nor influenced by CO. Oxygen 1.0% and 0.10% gave approximately 100% and 30% inhibition of Cr(VI) reduction (30 microM Na2CrO4) respectively, and an uncompetitive like inhibitory pattern was found. No redox cycling of Cr(VI) was seen. 51Cr binding to the microsomes was approximately 10% after complete reduction of 30 microM Na2CrO4. Externally added FMN, Fe3+-ADP and nitrobenzen stimulated microsomal Cr(VI) reduction. A 60% higher reduction rate of Cr(VI) by isolated hepatocytes was found during anaerobic in comparison with aerobic conditions. 相似文献
15.
Incubation of rat homogeneous detergent-solubilized cytochrome b5 with rat liver microsomes resulted in specific binding of the hemoprotein which was rapidly reduced by NADH. The NADH cytochrome c reductase activity in these preparations increased in proportion to the amount of cytochrome bound. However, the extra-bound detergent-solubilized cytochrome b5 did inhibit NADPH-dependent N-demethylations, the NADH synergism and NADPH cytochrome P-450 reductase activity. Manganese protoporphyrin-apocytochrome complex when bound to microsomes in amounts equivalent to detergent-solubilised cytochrome b5 showed no effect on N-demethylation activity. Furthermore, the binding of cytochrome b5 preparations reconstituted from heme and apocytochrome b5 had no effect on either the NADPH-dependent N-demethylation of aminopyrene or ethylmorphine or the NADH synergism observed with rat liver microsomes. In addition, homogeneous cytochrome b5 eluted from three additional Sephadex G-100 columns showed no inhibitory effects when bound to liver microsomes. Spectral analyses of the acid-acetone extract of the hemoprotein showed an absorption peak at 278 nm suggesting that the homogeneous b5 contains contaminating amounts of tightly bound detergent which is responsible for the observed inhibition of mixed function oxidase activity and which is removed during extraction of the heme from the apocytochrome and during further gel filtration applications. 相似文献
16.
The action of paraquat and diquat on the respiration of liver cell fractions 总被引:5,自引:3,他引:2
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J. C. Gage 《The Biochemical journal》1968,109(5):757-761
1. Paraquat and diquat produce only a slight increase in the oxygen uptake of rat liver mitochondria, and it is likely that they do not penetrate the mitochondrial membrane. 2. In mitochondrial fragments inhibited by antimycin A or by Amytal, both substances stimulate oxygen uptake with NADH or beta-hydroxybutyrate as substrate but not with succinate. The NADH dehydrogenase of the respiratory chain appears to be involved, at a site only partially inhibited by Amytal. 3. An NADPH oxidase activity is stimulated in rat liver microsomes by diquat, and to a smaller extent by paraquat; diquat also causes an NADH oxidase activity to develop. The effect is not inhibited by carbon monoxide or p-chloromercuribenzoate, and it is probable that a flavoprotein is involved by a mechanism not requiring thiol groups. 4. One molecule of oxygen can oxidize two molecules of NADPH in the stimulated microsomal system, the hydrogen peroxide produced being broken down by a catalase activity in the microsomes. 5. Diquat can stimulate NADH oxidase and NADPH oxidase activity in the postmicrosomal soluble fraction; the enzyme involved may be DT-diaphorase. 6. The mechanism of these reactions and their significance in relation to the toxicity of the dipyridilium compounds are discussed. 相似文献
17.
Effects of the administration of trivalent chromium (Cr(III)) to mice and the activation of carbon tetrachloride (CCl4) to form trichloromethyl radicals (.CCl3) in the liver were studied. The lipid peroxidation in liver microsomes induced in vitro by CCl4 in the presence of NADPH was decreased by the preadministration of Cr(III) to mice. The activity of NADPH-cytochrome C reductase, which presumably catalyzes the formation of .CCl3 from CCl4 in liver microsomes, was depressed by Cr(III) administration and kept at a level lower than that of the control group for at least 2 hr after CCl4 dosing. Furthermore, the frequency of appearances of ESR signals of .CCl3 in the liver homogenate of mice 1 min after CCl4 administration was markedly decreased by Cr(III) preadministration, similarly to DL-alpha-tocopherol. These results suggest that Cr(III) preadministered to mice decreases the formation of .CCl3 from CCl4, an activating process of CCl4, in the liver, presumably by scavenging the radical. 相似文献
18.
Electron-transport cytochrome P-450 system is involved in the microsomal metabolism of the carcinogen chromate 总被引:3,自引:0,他引:3
The kinetics of chromate reduction by liver microsomes isolated from rats pretreated with phenobarbital or 3-methylcholanthrene with NADPH or NADH cofactor have been followed. Induction of cytochrome P-450 and NADPH-cytochrome P-450 reductase activity in microsomes by phenobarbital pretreatment caused a decrease in the apparent chromate-enzyme dissociation constant, Km, and an increase in the apparent second-order rate constant, kcat/Km, but did not affect the kcat of NADPH-mediated microsomal metabolism of chromate. Induction of cytochrome P-448 in microsomes by 3-methylcholanthrene pretreatment did not affect the kinetics of NADPH-mediated reduction of chromate by microsomes. The kinetics of NADH-mediated microsomal chromate reduction were unaffected by the drug treatments. The effects of specific enzyme inhibitors on the kinetics of microsomal chromate reduction have been determined. 2'-AMP and 3-pyridinealdehyde-NAD, inhibitors of NADPH-cytochrome P-450 reductase and NADH-cytochrome b5 reductase, inhibited the rate of microsomal reduction of chromate with NADPH and NADH. Metyrapone and carbon monoxide, specific inhibitors of cytochrome P-450, inhibited the rate of NADPH-mediated microsomal reduction of chromate, whereas high concentrations of dimethyl-sulfoxide (0.5 M) enhanced the rate. These results suggest that the electron-transport cytochrome P-450 system is involved in the reduction of chromate by microsomal systems. The NADPH and NADH cofactors supply reducing equivalents ultimately to cytochrome P-450 which functions as a reductase in chromate metabolism. The lower oxidation state(s) produced upon chromate reduction may represent the ultimate carcinogenic form(s) of chromium. These studies provide evidence for the role of cytochrome P-450 in the activation of inorganic carcinogens. 相似文献
19.
S Kitamura Y Wada K Tatsumi 《Biochemical and biophysical research communications》1984,125(3):1117-1122
NAD (P) H-dependent reduction of nicotinamide N-oxide was investigated with rabbit liver preparations. Microsomes, microsomal NADPH-cytochrome c reductase or cytosolic aldehyde oxidase alone exhibited no nicotinamide N-oxide reductase activity in the presence of NADPH or NADH. However, when the microsomal preparations were combined with the cytosolic enzyme, a significant N-oxide reductase activity was observed in the presence of the reduced pyridine nucleotide. The activity was enhanced by FAD or methyl viologen. Cytosol alone supplemented with NADPH or NADH exhibited only a slight, but when combined with microsomes, a significant N-oxide reductase activity. Based on these facts, we propose a new electron transfer system consisting of NADPH-cytochrome c reductase and aldehyde oxidase, which exhibits nicotinamide N-oxide reductase activity in the presence of the reduced pyridine nucleotide. 相似文献
20.
1. Ethanol metabolism in slices or homogenates of transplantable hepatocellular carcinoma HC-252 (HC-252) was 50 to 60% of the rate found in host liver slices or homogenates when they were expressed per gram of tissue wet weight and 70 to 80% of the liver when the rates were expressed per milligram of tissue protein. At 10 mM ethanol, the activities of alcohol dehydrogenase in tumor and liver supernatants were comparable. 2. Tumor microsomes did not oxidize ethanol in the presence of a NADPH-generating system, indicating the absence of the microsomal ethanol-oxidizing system and catalase-mediated peroxidation of ethanol. The HC-252 microsomes were contaminated with catalase, and acetaldehyde production occurred in the presence of a H2O2-generating system (xanthine oxidase). The virtual absence of ethanol oxidation and drug metabolism (aminopyrine demethylase and aniline hydroxylase) in HC-252 microsomes may be due to the low activities of NADPH-cytochrome c reductase, NADPH oxidase, and NADPH-dependent oxygen uptake. 3. Microsomal oxidation of ethanol was present in Morris hepatoma 5123C, a well-differentiated tumor of intermediate growth rate, while activity was negligible in microsomes from Morris hepatoma 7288CTC, a less differentiated tumor. Microsomal NADPH oxidase was present in the well differentiated tumor 5123C but was lacking in the less differentiated tumor 7288CTC. Several microsomal, mitochondrial, and cytosolic properties of HC-252 are similar to those of Morris hepatoma 7288CTC but differ from those of the more differentiated 5123C tumor and normal liver. 4. The content of mitochondrial protein in HC-252 was only 25% that of liver, and oxygen consumption per gram of tumor was only 28% that of the liver. When corrected for the mitochondrial protein content, oxygen uptake in tumor HC-252 and liver homogenates was comparable. Isolated tumor and liver mitochondria displayed comparable State 4 and 3 rates of oxygen consumption with succinate and glutamate as substrates. The activities of the reconstituted malate-aspartate and alpha-glycerophosphate shuttles were only slightly lower in isolated HC-252 mitochondria compared to liver mitochondria, when shuttles were reconstituted with purified enzymes. 5. Antimycin inhibited alcohol metabolism,and pyruvate stimulated alcohol metabolism, much less in tumor slices than in liver slices, suggesting the presence of an augmented mitochondria-independent, cytosolic mechanism for oxidizing reducing equivalents in the tumor. These factors suggest that oxidation of NADH is the limiting factor in ethanol metabolism. Whereas, in the liver mitochondrial reoxidation is predominant, in HC-252, cytosolic reoxidation of NADH also plays a major role. 相似文献