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1.
Philip Wang Patricia S. Mason F.Peter Guengerich 《Archives of biochemistry and biophysics》1980,199(1):206-219
Human liver cytochrome P-450 was isolated from autopsy samples using cholate extraction and chromatography on n-octylamino-Sepharose 4B, hydroxylapatite, and DEAE-cellulose gels. Purified preparations contained as much as 14 nmol cytochrome P-450 mg?1 protein, were free of other hemoproteins, and were active in the mixed-function oxidation of d-benzphetamine and 7-ethoxycoumarin when coupled with either rat or human liver NADPH-cytochrome P-450 reductase. Some of the preparations were apparently homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; apparent subunit Mrs estimated for several preparations were 53,000 or 55,500. The amino acid composition of one preparation was determined and found to resemble those of rat liver cytochromes P-450, although some variations were noted. Rabbit antibodies raised to phenobarbital-treated rat liver cytochrome P-450 were more effective in inhibiting d-benzphetamine N-demethylase activity in human liver microsomes than were antibodies raised to 3-methylcholanthrene-treated rat liver cytochrome P-450. These antibodies also inhibited benzo(a)pyrene hydroxylation in human liver microsomes, although the inhibition patterns did not follow a general pattern as in the case of benzphetamine demethylase activity. Microsomes prepared from three different human liver samples were more effective in eliciting complement fixation with antibodies raised to phenobarbitalthan to 3-methylcholanthrene-treated rat liver cytochrome P-450. Complement fixation in such systems appears to result from similarity of certain rat and human liver cytochrome P-450 antigenic determinants, as fixation could be inhibited by removal of cytochrome P-450-directed antibodies from the total immunoglobulin population and purified human cytochrome P-450 was more effective (on a protein basis) than liver microsomes in producing fixation. Human liver microsomes prepared from five different individuals all produced ≥ 90% complement fixation, but variations were observed in the fixation curves plotted either versus microsomal protein or versus spectrally detectable microsomal cytochrome P-450.These results indicate that human liver microsomal cytochromes P-450 can be isolated using modifications of techniques developed for laboratory animals and that human and rat liver cytochromes P-450 share certain features of structural, functional, and immunological similarity. The available data suggest the existence of multiple forms of human liver microsomal cytochrome P-450, but possible artifacts associated with the use of autopsy samples suggest caution in advancing such a conclusion. 相似文献
2.
The interactions of rat liver epoxide hydrolase (EC 3.3.2.3) with itself and with cytochromes P-450 and NADPH-cytochrome P-450 reductase were investigated in microsomal preparations and in reconstituted systems in which all of the enzymes are functionally active. Hydrodynamic measurements indicated that purified epoxide hydrolase behaves as a single aggregate of approximately 16 monomeric units and that further aggregation of the protein only occurs in the presence of high concentrations of phospholipid. Neither guanidine-HCl nor the nonionic detergent Lubrol PX was able to completely dissociate the aggregate into monomers. The interactions of epoxide hydrolase with NADPH-cytochrome P-450 reductase and the major forms of cytochrome P-450 isolated from phenobarbital- and 5,6-benzoflavone-treated rats were studied by Soret difference spectroscopy, by perturbation of the fluorescence of NADPH-cytochrome P-450 reductase and fluorescein-labeled epoxide hydrolase, and by CD spectroscopy. The spectra provided evidence that binding of the proteins to each other occurs and some of the results suggest that affinity constants are on the order of 107, m?1. The spectral perturbations were not observed with other intrinsic membrane proteins. When microsomes were treated with the crosslinking reagent dimethylsuberimidate and solubilized with detergents, epoxide hydrolase could be precipitated with antibodies raised to cytochromes P-450 or NADPH-cytochrome P-450 reductase. Transient times were determined for the conversion of 1-octene to octene-1,2-dihydrodiol in a reconstituted enzyme system and for the conversion of naphthalene to naphthalene-1,2-dihydrodiol in rat liver microscomes and compared to the transient times predicted from the enzymatic rates of hydrolysis of the intermediate epoxides. In all cases the observed transient times were shorter than expected, in support of the view that coupling of epoxide hydrolase with cytochromes P-450 occurs. These results support the view that epoxide hydrolase couples with cytochrome P-450-containing mixed-function oxidase systems and may have relevance to the metabolism of potentially harmful xenobiotics by these enzymes. 相似文献
3.
G M Bartoli T Galeotti G Palombini G Parisi A Azzi 《Archives of biochemistry and biophysics》1977,184(1):276-281
Liver microsomes of adult rats produce, by an NADPH-dependent pathway, O2? radicals, as detected by the epinephrine cooxidation to adrenochrome (24.8 nmol/min/mg of protein). This production has also been measured during liver development (from 1 to 20 days after birth) and correlated to the enzyme content (NADPH-cytochrome c reductase, cytochrome b5, and cytochrome P-450), with the aim of establishing the level at which Superoxide radicals are formed in the electron transport system. At 1 day the adrenochrome formation and the activity of NADPH-cytochrome c reductase are about 50 and 40% of those of the adult, respectively, whereas those of cytochromes b5 and P-450 are approximately 10%. After 20 days of development cytochrome b5 and the dehydrogenase reach the adult level, while cytochrome P-450 is about 80%. At this age O2? radicals have a 30% increment and reach only 60% of those of the adult; H2O2 production is also 60% and the N-demethylation of aminopyrine is only 30%. Thus, at birth the formation of O2? radicals is almost entirely dependent on the activity of the flavoprotein. The close correlation between the slight increase in the demethylase activity and adrenochrome formation from 1 to 20 days suggests that a portion of O2? radicals produced by the NADPH-dependent electron transfer is directly involved in the mixed function oxidation. Since about 50% of the radicals are formed at the flavoprotein level, these results indicate that in the adult liver the remaining amount may be generated at the level of cytochrome P-450. 相似文献
4.
Hann-Ping Wang Tokuji Kimura 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,542(1):115-127
Adrenocortical mitochondrial cytochrome P?450 specific to the cholesterol side-chain cleavage (desmolase) reaction differs from that for the 11β-hydroxylation reaction of deoxycorticosterone. The former cytochrome appears to be more loosely bound to the inner membrane than the latter. Upon ageing at 0°C or by aerobic treatment with ferrous ions, the desmolase P-450 was more stable than the 11β-hydroxylase P-450. By utilizing artificial hydroxylating agents such as cumene hydroperoxide, H2O2, and sodium periodate, the hydroxylation reaction of deoxycorticosterone to corticosterone in the absence of NADPH was observed to a comparable extent with the reaction in the presence of adrenodoxin reductase, adrenodoxin and NADPH. However, the hydroxylation reaction of cholesterol to pregnenolone was not supported by these artificial agents.Immunochemical cross-reactivity of bovine adrenal desmolase P-450 with rabbit liver microsomal P-450LM4 was also investigated. We found a weak but significant cross-reactivity between the adrenal mitochondrial P-450 and liver microsomal P-450LM4, indicating to some extent a homology between adrenal and liver cytochromes P-450. 相似文献
5.
The destruction of cytochrome P-450 by allylisopropylacetamide (2-isopropyl-4-pentenamide) in microsomes from phenobarbital-pretreated rats has been shown to require oxygen, to be inhibited by NADP through inhibition of cytochrome P-450 reductase, and to be slightly stimulated by NADH. Glutathione (1 mm) does not inhibit destruction, but methyl 4,5-epoxy-2-isopropylpentanoate (5 mm), an analog of the epoxide of allylisopropylacetamide, does. The inactivation of cytochrome P-450 is both time dependent and saturable, although no more than approximately 40% of the microsomal enzyme appears to be normally destructible. However, mechanical perturbation of the microsomal suspension by rehomogenization initiates renewed destruction. Kinetic analysis shows that the destructive process is pseudo-first-order, with an apparent inactivation rate constant of 1.4 × 10?3 s?1 and an apparent Km of 1.14 mm. Approximately 230 molecules of substrate are turned over for each destructive event. These results, in conjunction with previously reported data, clearly and unambiguously establish that inactivation of cytochrome P-450 by allylisopropylacetamide is a suicidal process. 相似文献
6.
Patricia Linko Heather N. Yeowell Thomas A. Gasiewicz Joyce A. Goldstein 《Journal of biochemical and molecular toxicology》1986,1(2):95-107
Hexachlorobenzene (HCB) differs markedly from other chlorinated benzenes (CBs) as an inducer of cytochrome P-450 (P-450) isozymes as determined by radioimmunoassay and immunoblotting. At > 99% pure, HCB induced both the phenobarbital-inducible forms, cytochromes P-450b + e (70X), and the 3-methylcholanthrene-inducible forms, cytochromes P-450c (58X) and P-450d (8X), in rat liver microsomes. The concentration of P-450d was considerably greater than that of P-450c in HCB-induced rat liver. In contrast to HCB, all lower chlorinated benzenes tested were PB-type inducers. Hexachlorobenzene increased the amounts of translatable messenger RNAs (mRNAs) for P-450b, P-450c, and P-450d in rat liver polysomes, suggesting that it increases the synthesis of these proteins. Evidence that HCB interacted with the putative Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was equivocal. Western blots of liver microsomes from Ahresponsive C57BL/6J (B6) and nonresponsive DBA/2J (D2) mice demonstrated that HCB produced a large increase in P3-450 and a very small increase in P1-450 in the responsive strain. The increase in P1-450 was not observed after HCB administration to nonresponsive mice, but a small increase in P3-450 was noted. These findings suggested that HCB may act through the Ah receptor. However, HCB was at best a very weak competitor for specific binding of [3H]-TCDD to the putative receptor in rat or mouse hepatic cytosol in vitro, producing decreases in binding of [3H]-TCDD only at very high concentrations (10?6 to 10?5 M). 相似文献
7.
Helmut R. Gutmann Peter Bell 《Biochimica et Biophysica Acta (BBA)/General Subjects》1977,498(1):229-243
The hypothesis that N-hydroxylation of arylamides is essential for carcinogenicity was examined in vivo and in vitro with N-2-fluorenylacetamide, a potent carcinogen, and with N-3-fluorenylacetamide, an isomer with marginal carcinogenicity. About 10–20% of 2-[9-14C]fluorenylacetamide administered intraperitoneally to the rat was excreted in the bile as the N-hydroxy-2-[9-14C]-derivative, whereas <0.1% of 3-[G-3H]fluorenylacetamide was found as the N-hydroxy metabolite in bile and urine. N-Hydroxylation of the 2- isomer by hepatic microsomes of untreated or 3-methylcholanthrene-treated rats was 40 to 50-fold greater than that of the 3- isomer. The role of cytochromes P-450 and P1-450 in N-hydroxylation of arylamides by rat liver microsomes was shown by inhibition of the reaction with carbon monoxide and cobaltous chloride. Interaction of the arylamides with cytochrome P1-450 was also demonstrated by binding spectra obtained on addition on 2- and 3-fluorenylacetamide to hepatic chromosomes of methylcholanthrene-treated rats. There appeared to be no correlation between the magnitude of the spectra and the extent of N-hydroxylation. N-Hydroxylation of the 2- isomer by hepatic microsomes of the guinea pig, a species resistant to the carcinogenecity of this compound, was markedly less than N-hydroxylation by rat liver microsomes, even though, as judged by the appearance of the binding spectra, both 2- and 3- isomers were bound by cytochrome P1-450 of guinea pig-liver microsomes. The results are in agreement with the view that the microsomal N-hydroxylation of arylamides parallels their carcinogenicity. 相似文献
8.
A procedure to directly measure the cytochrome P-450-dependent 7-ethoxyresorufin O-deethylase activity with a visible spectrophotometer is described and compared to the standard fluorometric method. The two assays yielded identical results with both β-naphthoflavone-treated mammalian (rat) and fish (scup, Stenotomus chrysops) liver microsomes. The assay takes advantage of a clean distinction in visible absorption spectra obtained for highly purified 7-ethoxyresorufin (substrate) and resorufin (enzymatic product). The purification and characterization of resorufin, the enzymatic product, are detailed, and its extinction coefficient (ε572 = 73 mm?1 cm?1) provides for an accurate quantitation of enzyme activity. The large visible extinction coefficient of the product chromophore provides a high sensitivity for low-activity samples. The application of this enzyme assay in a visible spectrophotometer, along with the considerable evidence that a single aromatic hydrocarbon-inducible cytochrome P-450 isozyme is responsible for the catalysis, enhances the utility of this substrate in microsomal monooxygenase assays. The utility of the visible assay is further demonstrated by the simple determinations of the coupling ratio for 7-ethoxyresorufin oxidation in scup liver microsomes and the KI for 7,8-benzoflavone and phenylimidazole inhibition of the enzymatic reaction. 相似文献
9.
The development of the stearyl-CoA desaturase system was studied in newly hatched chicks. The desaturation activity was very low in hepatic microsomes from chick embryos, less than 0.05 nmol of oleate formed min?1 (mg of protein)?1. After hatching and feeding, the desaturation activity gradually increased to 4–5 nmol of oleate formed min?1 (mg of protein)?1 in 6-day-old chicks. This increase could be prevented by administration of cycloheximide or actinomycin D. Measurement of the microsomal electron transfer components throughout the induction period showed no significant changes in the NADH- or NADPH-specific reductases or in the concentrations of cytochromes b5 and P-450. However, the activity of the terminal component of the desaturase system (the desaturase enzyme) increased in parallel with the desaturation activity. Supplementing the liver microsomes from chick embryos with isolated desaturase enzyme resulted in the formation of an active desaturation system. It is proposed that the induction of the stearyl-CoA desaturase system during development of newly hatched chicks is dependent on the synthesis of the terminal desaturase enzyme. 相似文献
10.
Mark L. Mandel Susan J. Swartz Jacob G. Ghazarian 《Biochimica et Biophysica Acta (BBA)/General Subjects》1990,1034(3):239-246
We describe the isolation of cytochrome P-4501α from chick-kidney mitochondria. Although, gel permeation HPLC yielded 41% of the total amount of P-450 present in cholate-solubilized hemeproteins, it produced a highly purified mixture from which the P-4501α could be purified to homogeneity in a final detergent-free state by a single-step application of hydrophobic interaction HPLC using hydroxypropyl silica. The purified P-4501α traveled as a single band in SDS gel electrophoresis with an apparent Mr = 57 000. The absolute spectrum of the P-4501α(Fe3+) form gave a λmax at 403 nm. This characteristic lends support to the anomalous high-spin heme electron paramagnetic resonance spectrum and the heme structure of P-4501α which we have previously reported (Ghazarian et al. (1980) J. Biol. Chem. 255, 8275–8281; Pedersen et al. (1976) J. Biol. Chem. 251, 3933–3941). In reconstitution experiments with ferredoxin-dependent NADPH-cytochrome c (P-450) reductase complexes, P-4501α catalyzed the hydroxylation of 25-hydroxy-9,10-secocholesta-5,7,10(19)-trien-3β-ol at the C-1 position exclusively with a turnover number of 0.03 min?1. This number is identical to that obtained from measurements of the catalytic activity in intact mitochondria, indicating that only one major species of cytochrome P-450 occurs in chick-kidney mitochondria. The complete responsiveness of cytochrome P-450 concentrations in intact mitochondria to the vitamin D status of chicks provided additional evidence that the major cytochrome P-450 species present in renal mitochondria is uniquely associated with vitamin D metabolism. 相似文献
11.
Addition of nifurtimox (a nitrofuran derivative used for the treatment of Chagas' disease) to rat liver microsomes produced an increase of (a) electron flow from NADPH to molecular oxygen, (b) generation of both superoxide anion radical (O2?) and hydrogen peroxide, and (c) lipid peroxidation. The nifurtimox-stimulated NADPH oxidation was greatly inhibited by NADP+ and p-chloromercuribenzoate, and to a lesser extent by SKF-525-A and metyrapone. These inhibitions reveal the function of both the NADPH-cytochrome P-450 (c) reductase and cytochrome P-450 in nifurtimox reduction. Superoxide dismutase, catalase (in the presence of superoxide dismutase), and hydroxyl radical scavengers (mannitol, 5,5-dimethyl-1-pyrroline-1-oxide) inhibited the nifurtimox-stimulated NADPH oxidation, in accordance with the additional operation of a reaction chain including the hydroxyl radical. Further evidence supporting the role of superoxide anion and hydroxyl radicals in the nifurtimox-induced NADPH oxidation resulted from the effect of specific inhibitors on NADPH oxidation by O2? (generated by the xanthine oxidase reaction) and by OH. (generated by an iron chelate or the Fenton reaction). Production of O2? by rat kidney, testes and brain microsomes was significantly stimulated by nifurtimox in the presence of NADPH. It is postulated that enhanced formation of free radicals is the basis for nifurtimox toxicity in mammals, in good agreement with the postulated mechanism of the trypanocide effect of nifurtimox on Trypanosoma cruzi. 相似文献
12.
Tetsuya Kamataki Kaori Maeda Yasushi Yamazoe Tsuneji Nagai Ryuichi Kato 《Archives of biochemistry and biophysics》1983,225(2):758-770
One of each constitutive form of cytochrome P-450 from liver microsomes of adult male and female rats was purified essentially following the same method to an apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights estimated by the electrophoresis were 52,000 and 50,000 for forms of cytochrome P-450, P-450-male, and P-450-female, purified from male and female rats, respectively. In addition, the purified preparations of P-450-male and P-450-female showed properties different from each other with respect to spectral characteristics and catalytic activities. In Ouchterlony double diffusion plates, partially purified rabbit immunoglobulin G (IgG) raised against P-450-male and P-450-female showed very weak or no cross-reactivity with P-450-female and P-450-male, respectively. From these results, P-450-male was confirmed to be a form distinct from P-450-female. The anti-P-450-male and anti-P-450-female antibodies, which had been further purified by immunoadsorption, did not form any apparent precipitation bands with liver microsomes from untreated female and male rats, respectively. Supporting this, radial immunodiffusion analysis for P-450-male and P-450-female with an agarose gel impregnated with the rabbit antibodies showed that P-450-male and P-450-female appear in liver microsomes rather specifically depending on the sex hormones. Based on these results, sex differences in drug metabolism in the rat were confirmed as explicable, at least in part, by the presence of distinct forms of cytochrome P-450 in microsomes of male and female rats. 相似文献
13.
Incubation in the presence of NADPH and molecular oxygen of 14C-labeled polychlorinated biphenyls (PCBs) and two tetrachlorobiphenyl (TCB) isomers with a reconstituted system containing NADPH-cytochrome P-450 reductase and cytochrome P-450, both purified from liver microsomes of phenobarbital(PB)-pretreated rabbits, led to covalent binding of radioactive metabolites of PCBs and TCBs to the protein components of the system. A rabbit liver cytosol fraction added to the system provided more binding sites for the activated metabolites and thus increased the extent of binding markedly. The binding reaction depended absolutely on the reductase, cytochrome P-450 and NADPH, and required dilauroyl phosphatidylcholine and sodium cholate for maximal activity. A further stimulation of the binding was attained by including cytochrome b5 in the reconstituted system. Four forms of cytochrome P-450, purified from liver microsomes of PB- and 3-methylcholanthrene(MC)-treated rabbits and rats, were used to reconstitute the PCB- and TCB-metabolizing systems, and it was found that PB-inducible forms of the cytochrome from both animals were more active than those inducible by MC in catalyzing the PCB- and TCB-binding reaction. Sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis indicated that, in the system containing the reductase, cytochrome P-450 and cytochrome b5, PCB metabolites bound to the reductase and cytochrome P-450, but not to cytochrome b5. In the presence of the liver cytosol fraction, the binding took place to many cytosolic proteins in addition to the reductase and cytochrome P-450. 相似文献
14.
Adenylate cyclase activation by GTP analogs 总被引:1,自引:0,他引:1
Benznidazole (a nitroimidazole derivative used for the treatment of Chagas' disease) is reduced by rat liver microsomes to the nitro anion radical, as indicated by ESR spectroscopy. Addition of benznidazole to rat liver microsomes produced an increase of electron flow from NADPH to molecular oxygen, and generation of both superoxide anion and hydrogen peroxide. The benznidazole-stimulated O2 consumption and O2? formation was greatly inhibited by NADP+ and p-chloromercuribenzoate but not by SKF-525-A and metyrapone. The former inhibitions indicated the involvement of NADPH-cytochrome P-450 (c) reductase, while the lack of inhibition by SKF-525-A and metyrapone ruled out any major role for cytochrome P-450 in benznidazole reduction. In contrast to nifurtimox, a nitrofuran derivative (R. Docampo and A. O. M. Stoppani, 1979, Arch. Biochem. Biophys.197, 317–321), benznidazole was not reduced to the nitro anion radical, nor did it stimulate oxygen consumption, O2? production, and H2O2 generation by Trypanosoma cruzi cells or microsomal fractions. A different mechanism of benznidazole toxicity in T. cruzi and the mammalian host is postulated. 相似文献
15.
The nitrosoureas, CCNU (1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea) and BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) are representatives of a class of N-nitroso compounds which undergo denitrosation in the presence of NAD(P)H and deoxygenated hepatic microsomes from rats to yield nitric oxide (NO) and the denitrosated parent compound. Formation of NO during microsomal denitrosation of CCNU and BCNU was determined by three methods. With one procedure, NO was measured and concentration shown to increase over time in the head gas above microsomal incubations with BCNU. Two additional methods utilized NO binding to either ferrous cytochrome P-450 or hemoglobin to form distinct Soret maxima at 444 and 415 nm, respectively. Incubation of either BCNU or CCNU in the presence of NAD(P)H and deoxygenated microsomes resulted in the formation of identical cytochrome P-450 ferrous · NO optical difference spectra. Determination of the P-450 ferrous · NO extinction coefficient by the change in absorbance at 444 minus 500 nm allowed measurement of rates of denitrosation by monitoring the increase in absorbance at 444 nm. The rates of BCNU and CCNU denitrosation were determined to be 4.8 and 2.0 nmol NO/min/mg protein, respectively, for phenobarbital (PB) induced microsomes. For the purpose of comparison, the rate of [14C]CCNU (1-(2-[14C]chloroethyl)-3-(cyclohexyl)-1-nitrosourea turnover was examined by the isolation of [14C]CCU (1-(2-[14C] chloroethyl)-3-(cyclohexyl)-1-urea) from incubations that contained NADPH and deoxygenated PB-induced microsomes. These analyses showed stoichiometric amounts of NO and [14C]CCU being formed at a rate of 2.0 nmol/min/mg protein. Denitrosation catalysis by microsomes was enhanced by phenobarbital pretreatment and partially decreased by cytochrome P-450 inhibitors, SKF-525A, α-naphthoflavone (ANF), metyrapone, and CO, suggesting a cytochrome P-450-dependent denitrosation. However, in the presence of NADPH and purified NADPH cytochrome P-450 reductase reconstituted in dilauroylphosphatidylcholine, [14C]CCNU was shown to undergo denitrosation to [14C]CCU. Thus, NADPH cytochrome P-450 reductase could support denitrosation in the absence of cytochrome P-450. 相似文献
16.
Michael J. Parnham Peter C. Bragt Aalt Bast Frederick J. Zijlstra 《Biochimica et Biophysica Acta (BBA)/General Subjects》1981,677(2):165-173
Metyrapone and SKF-525A, together with amphenone B, a structural analogue of metyrapone, which are all inhibitors of cytochrome P-450-mediated reactiors, were shown to inhibit the arachidonic acid-induced aggregation of human platelets. Amphenone B, like metyrapone, exhibited a type II (ligand) binding spectrum with rat liver microsomal cytochrome P-450, in contrast to SKF 525A which is a type I (substrate) binding agent. Independently of their type of binding spectra and of their maximum spectral change, however, the affinity of the three compounds for rat liver cytochrome P-450 showed a close proportional correlation with their platelet aggregation inhibitory potency. All three compounds inhibited the formation of [1?14C]thromboxane B2 from [1?14C]arachidonic acid by human platelets aggregated with collagen. The effect of metyrapone on the remaining labelled products suggested that it is a selective thromboxane synthesis inhibitor, while amphenone B exhibited activity reminiscent of cyclo-oxygenase inhibitors. SKF 525A produced complex effects possibly attributable to cyclo-oxygenase inhibition and enhanced lipid peroxidation, since it also enhanced platelet malonaldehyde formation, which the other two compounds inhibited. These data provide further support for a role of cytochrome P-450 in thromboxane synthesis and platelet aggregation. 相似文献
17.
Evidence presented in this report suggests that the hydroxyl radical (OH.), which is generated from liver microsomes is an initiator of NADPH-dependent lipid peroxidation. The conclusions are based on the following observations: 1) hydroxyl radical production in liver microsomes as measured by esr spin-trapping correlates with the extent of NADPH induced microsomal lipid peroxidation as measured by malondialdehyde formation; 2) peroxidative degradation of arachidonic acid in a model OH · generating system, namely, the Fenton reaction takes place readily and is inhibited by thiourea, a potent OH · scavenger, indicating that the hydroxyl radical is capable of initiating lipid peroxidation; 3) trapping of the hydroxyl radical by the spin trap, 5,5-dimethyl-1-pyrroline-1-oxide prevents lipid peroxidation in liver microsomes during NADPH oxidation, and in the model system in the presence of linolenic acid. The possibility that cytochrome P-450 reductase is involved in NADPH-dependent lipid peroxidation is discussed. The optimal pH for the production of the hydroxyl radical in liver microsomes is 7.2. The generation of the hydroxyl radical is correlated with the amount of microsomal protein, possibly NADPH cytochrome P-450 reductase. A critical concentration of EDTA (5 × 10?5m) is required for maximal production of the hydroxyl radical in microsomal lipid peroxidation during NADPH oxidation. High concentrations of Fe2+-EDTA complex equimolar in iron and chelator do not inhibit the production of the hydroxyl radical. The production of the hydroxyl radical in liver microsomes is also promoted by high salt concentrations. Evidence is also presented that OH radical production in microsomes during induced lipid peroxidation occurs primarily via the classic Fenton reaction. 相似文献
18.
In rat liver microsomes, all-trans-[11,12-3H]retinoic acid was found to be metabolized to polar products in the presence of NADPH. One of the metabolites was coeluted with 4-hydroxyretinoic acid on reverse-phase high-pressure liquid chromatography (HPLC). This reaction required oxygen and was inhibited by carbon monoxide as well as aminopyrine, aniline, and ethanol, suggesting the involvement of cytochrome P-450. Isolated rat hepatocytes also metabolized all-trans[3H]retinoic acid to polar compounds, with an elution pattern on HPLC similar to that in microsomal preparations. Microsomal activity was compared in rats pair-fed with diets containing either ethanol or isocaloric carbohydrate for 4–6 weeks. Ethanol-fed rats showed enhanced microsomal retinoic acid metabolism (50%, P < 0.01) accompanied by increased microsomal cytochrome P-450 content (34%, P < 0.005). On the other hand, microsomal β-glucuronidation of retinoic acid in the presence of uridine diphosphoglucuronic acid (UDPGA) was not affected by chronic ethanol feeding. The increased hepatic microsomal cytochrome P-450-dependent metabolism of retinoic acid after chronic ethanol consumption may contribute to the accelerated catabolism of retinoic acid in vivo. 相似文献
19.
W E Fahl G Michalopoulos G L Sattler C R Jefcoate H C Pitot 《Archives of biochemistry and biophysics》1979,192(1):61-72
Cytochrome P-450, NADPH-cytochrome c reductase, biphenyl hydroxylase, and epoxide hydratase have been compared in intact rat liver and in primary hepatocyte cultures. After 10 days in culture, microsomal NADPH-cytochrome c reductase and epoxide hydratase activities declined to a third of the liver value, while cytochrome P-450 decreased to less than a tenth. Differences in the products of benzo[a]pyrene metabolism and gel electrophoresis of the microsomes indicated a change in the dominant form(s) of cytochrome P-450 in the cultured hepatocytes. Exposure of the cultured cells to phenobarbital for 5 days resulted in a threefold induction in NADPH-cytochrome c reductase and epoxide hydratase activities which was typical of liver induction of these enzymes. Exposure of the cells to 3-methylcholanthrene did not affect these activities. Cytochrome P-450 was induced over two times by phenobarbital and three to four times by 3-methylcholanthrene. The λmax of the reduced carbon monoxide complex (450.7 nm) and analysis of microsomes by gel electrophoresis showed that the phenobarbital-induced cytochrome P-450 was different from the species induced by 3-methylcholanthrene (reduced carbon monoxide λmax = 447.9 nm). However, metabolism of benzo[a]pyrene (specific activity and product distribution) was similar in microsomes of control and phenobarbital- and 3-methylcholan-threne-induced hepatocytes and the specific activity per nmole of cytochrome P-450 was higher than in liver microsomes. The activities for 2- and 4-hydroxylation of biphenyl were undetectable in all hepatocyte microsomes even though both activities were induced by 3-methylcholanthrene in the liver. Substrate-induced difference spectra and gel electrophoresis indicated an absence in phenobarbital-induced hepatocytes of most forms of cytochrome P-450 which were present in phenobarbital-induced rat liver microsomes. It is concluded that the control of cytochrome P-450 synthesis in these hepatocytes is considerably different from that found in whole liver, while other microsomal enzymes may be near to normal. Hormonal deficiencies in the culture medium and differential hormonal control of the various microsomal enzymes provide a likely explanation of these effects. 相似文献
20.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》1993,1161(2-3):124-130
Cytochrome P-450scc (P-450 XIA1) from bovine adrenocortical mitochondria was investigated using a suicide substrate: [14C]methoxychlor. [14C]Methoxychlor irreversibly abolished the activity of the side-chain cleavage enzyme for cholesterol (P-450scc) and the inactivation was prevented in the presence of cholesterol. The binding of [14C]methoxychlor and cytochrome P-450scc occurred in a molar ratio of 1:1 and the cholesterol-induced difference spectrum of cytochrome P-450scc was similar with the methoxychlor-induced difference spectrum. [14C]Methoxychlor-binding peptides were purified from tryptic-digested cytochrome P-450scc modified with [14C]methoxychlor. Determination of the sequence of the amino-acid residues of a [14C]methoxychlor-binding peptide allowed identification of the peptide comprising the amino-terminal amino-acid residues 8 to 28. 相似文献