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1.
M A Correia K Yao S A Wrighton D J Waxman A E Rettie 《Archives of biochemistry and biophysics》1992,294(2):493-503
Suicide inactivation of hepatic cytochrome P450 (P450) enzymes 2C11, 2C6, and 3A1/A2 by 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) in intact rats results in prosthetic heme destruction, albeit by apparently distinct mechanisms. Such heme destruction is now shown to be associated with the loss of immunochemically detectable apoprotein of P450s 2C11 and 3A but with little of that of P450 2C6, in spite of their comparable DDEP-mediated functional inactivation. The loss of a approximately 50-kDa hepatic microsomal protein band along with the immunoreactive P450 3A loss strengthens the concept that such an in vivo loss indeed reflects proteolysis of the DDEP-inactivated P450. Furthermore, this propensity of DDEP-inactivated P450s 3A for proteolysis appears to correlate with the relative degree of prosthetic heme alkylation of their apoprotein rather than their functional inactivation per se. Thus, rapid degradation of apoP450s 3A was seen after DDEP treatment, which promoted extensive irreversible heme binding to apoP450s 3A, but not after exposure to allylisopropylacetamide (AIA), which inactivates these isozymes comparably, but induced minimal apoP450 3A heme alkylation. In addition, differences were observed in the relative sensitivities of proteolysis of DDEP-inactivated P450s 2C11 and 3A to hemin, which largely prevented the DDEP-induced proteolytic loss of P450 2C11 but apparently failed to prevent the loss of DDEP-inactivated P450s 3A, when coadministered with DDEP. This differential hemin sensitivity of the proteolysis of DDEP-inactivated P450 2C11, coupled with the observation that immunochemically detectable P450 2C11 loss occurs after its inactivation by both AIA and DDEP, provides compelling support for the existence of distinct proteolytic pathways for individual suicidally inactivated P450s. 相似文献
2.
C Decker K Sugiyama M Underwood M A Correia 《Biochemical and biophysical research communications》1986,136(3):1162-1169
Administration of antimineralocorticoid spironolactone (SPL) to rats results in modest destruction of hepatic cytochrome P-450 with parallel loss of heme. This process is accentuated by pretreatment with dexamethasone (DEX), an inducer of cytochrome P-450p and is associated with marked functional loss of cytochrome P-450p-dependent hydroxylases. Cytochrome P-450 destruction may be replicated in vitro when microsomes from DEX-pretreated rats are incubated with SPL and NADPH and is impaired when these rats are given triacetyloleandomycin, an inhibitor of cytochrome P-450p. In vitro SPL-mediated cytochrome P-450 destruction is accompanied by a loss of heme, which appears to be converted to reactive intermediates which covalently bind to microsomes or are converted to polar metabolites. 相似文献
3.
L M Bornheim D W Parish K M Smith D A Litman M A Correia 《Archives of biochemistry and biophysics》1986,246(1):63-74
The relative potential of various structural isomers (III, XIII) and various 2,4-side chain modified analogs of heme (iron-protoporphyrin IX) to incorporate into rat liver hemoproteins, cytochrome P-450(s), and tryptophan pyrrolase was examined. Such assessments for hepatic cytochrome P-450 relied on generation of reconstitutible apocytochrome(s) P-450 by suicidal alkylation of the existing prosthetic heme moiety by allylisopropylacetamide (AIA) in vivo. Subsequent replacement of the prosthetic heme was brought about by incubating the apocytochrome(s) P-450-enriched preparations with a particular heme isomer or analog. Structure-function relationships of the reconstituted isozymes were assessed in microsomal preparations by monitoring cytochrome P-450 content (structure) and its mixed function oxidase activity (function). In parallel, the relative ability of these heme isomers and analogs to functionally constitute hepatic tryptophan pyrrolase was also assessed by monitoring the relative increase in holoenzyme activity when preparations deliberately enriched in constitutible apoenzyme were incubated with each of these compounds. The findings reveal that 2,4-side chain modifications on the heme IX skeleton markedly influence the function of the constituted hemoproteins possibly by affecting their structural assembly through steric, electronic, and/or hydrophobic interactions with the corresponding apoproteins. Furthermore, these studies not only reveal that the structural specifications of the active prosthetic site of rat liver cytochrome P-450(s) differ from those of tryptophan pyrrolase, but also that the structural specifications of these mammalian hemoproteins for their prosthetic heme differ considerably from those reported for their bacterial counterparts. 相似文献
4.
D S Riddick J E Mackie T E Massey G S Marks 《Canadian journal of physiology and pharmacology》1990,68(3):370-373
Various rat liver cytochrome P-450 (P-450) isozymes are targets for mechanism-based inactivation by 3,5-diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4- dihydropyridine (4-ethyl DDC). Unlike rat liver, which contains multiple P-450 isozymes, rabbit lung contains only three major isozymes referred to as forms 2, 5, and 6. We have examined the ability of 4-ethyl DDC to destroy P-450 heme in hepatic and pulmonary microsomes from untreated and beta-naphthoflavone (beta NF)-treated rabbits. This compound destroyed 31% of the P-450 in either hepatic microsomal preparation, but was ineffective at lowering P-450 and heme levels in pulmonary microsomes when examined at a range of concentrations (0.45-5.0 mM). These data suggest that rabbit pulmonary P-450 forms 2, 5, and 6 are not targets for destruction by 4-ethyl DDC, despite the ability of this compound to inactivate rat liver P-450c, the orthologue of rabbit lung form 6. 相似文献
5.
D L Kleiman de Pisarev A M Ferramola de Sancovich H A Sancovich 《Biochimica et biophysica acta》1991,1074(1):172-177
In the rat liver, the microsomal content of cytochrome P-450 decreased by 50% after triiodothyronine (T3) administration. The molecular basis for the decreased cytochrome P-450 levels was investigated. The activities of the enzymes involved in heme synthesis or degradation were not altered by thyroid hormone administration. The incorporation of 3H-delta-aminolaevulinate into the liver microsomal heme was markedly reduced in T3-treated rats. The latter appeared not to reflect a lowered binding affinity of the apoprotein moiety of cytochrome P-450 for heme. The sodium dodecyl sulfate gel electrophoresis of the microsomal preparation showed a decrease in apocytochrome P-450. It is suggested that the amount of the apocytochrome may be the primary event affected in the formation of cytochrome P-450, by triiodothyronine treatment of thyroidectomized rats. 相似文献
6.
The catalytic mechanism of cytochrome P-450. Spin-trapping evidence for one-electron substrate oxidation 总被引:3,自引:0,他引:3
O Augusto H S Beilan P R Ortiz de Montellano 《The Journal of biological chemistry》1982,257(19):11288-11295
Cytochrome P-450 is destroyed during catalytic oxidation of several 4-substituted 3,5-bis(ethoxycarbonyl)-2,6-dimethyl-1,4-dihydropyridine substrates. A qualitative correlation has been found between the ability to destroy cytochrome P-450 and the stability of the 4-substituent as a radical. Destruction of the enzyme by the 4-ethyl (DDEP), 4-propyl, and 4-isobutyl analogues is due to transfer of the 4-alkyl group from the substrate to a nitrogen of the prosthetic heme, a process which gives rise to isolable N-alkylprotoporphyrin IX derivatives. Little enzyme destruction is observed when the 4-alkyl group is of low radical stability (methyl, phenyl) and good destruction, but no isolable heme adducts when the 4-substituent is of very high radical stability (isopropyl, benzyl). Spin-trapping studies have established that the 4-ethyl group in DDEP is lost as a radical as a result of oxidation by cytochrome P-450. Of three commonly used spin traps, only alpha-(4-pyridyl-1-oxide) N-tert-butylnitrone was found suitable for such studies. The other spin traps, 5,5-dimethyl-1-pyrroline-N-oxide and alpha-phenyl N-tert-butylnitrone, were found to be ineffective, the latter because it strongly inhibits cytochrome P-450. Hydrogen peroxide formed in situ can support a part of the cytochrome P-450-catalyzed ethyl radical formation and DDEP-dependent self-inactivation. The results provide persuasive evidence that oxidation of the nitrogen in DDEP by cytochrome P-450 proceeds in one-electron steps. Cytochrome P-450 may thus function, at least with certain substrates, as a one-electron oxidant. 相似文献
7.
R P Green-Thompson D S Riddick J E Mackie G S Marks J R Halpert 《Canadian journal of physiology and pharmacology》1991,69(4):526-530
Structural analogues of chloramphenicol (CAP) cause mechanism-based inactivation of rat liver cytochrome P-450 (P450) either via protein acylation or destruction of the heme prosthetic group. The goal of the present work was to determine whether CAP analogues that cause loss of the P450 heme moiety also cause porphyrin accumulation in chick embryo liver cell culture. The porphyrin profiles produced by exposure of cells to CAP analogues (160 microM) were determined by high-performance liquid chromatography with fluorescence detection. Of three CAP analogues that do not cause loss of the heme moiety of rat liver P450IIB1, two dichloroacetamides were not porphyrinogenic. The third compound, a chlorofluoroacetamide, caused porphyrin accumulation. This result may be due to the presence of P450 isozymes in chick embryo hepatocytes, distinct from rat liver P450IIB1, that are susceptible to destruction by this analogue. Of four CAP analogues that inactivate rat liver P450IIB1 with concomitant heme loss, a dichloroacetamide and two chlorofluoroacetamides caused porphyrin accumulation. The remaining compound, a monochloroacetamide, was not porphyrinogenic, perhaps because the P450 apoprotein cannot be reconstituted with fresh heme drawn from the regulatory "free heme pool" following inactivation by this analogue. Alternatively, there may be no P450 isozyme in chick embryo liver cell culture that is susceptible to inactivation by this compound. 相似文献
8.
M A Correia S H Davoll S A Wrighton P E Thomas 《Archives of biochemistry and biophysics》1992,297(2):228-238
The suicide substrate 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4- dihydropyridine (DDEP) inactivates rat liver cytochrome P450 (P450) 3A isozymes through prosthetic heme alkylation of the apoprotein in a mechanism-based fashion, which marks them for rapid proteolysis. In this article, through the use of epitope-specific monoclonal antibodies, we show that both 3A1 and 3A2 isozymes are targeted for proteolysis. Furthermore, using intact rats, isolated rat hepatocytes, and rat liver subcellular fractions supplemented with ATP and MgCl2, as well as various proteolytic inhibitors as probes, we now report that the hepatic cytosolic ubiquitin-dependent proteolytic system rather than hepatic lysosomes is involved in the rapid degradation of DDEP-induced heme alkylated P450s 3A. 相似文献
9.
P R Ortiz de Montellano H S Beilan K L Kunze 《The Journal of biological chemistry》1981,256(13):6708-6713
Administration of 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC) to rats causes the accumulation of N-methylprotoporphyrin IX, a potent inhibitor of ferrochelatase. To clarify the origin of the porphyrin N-methyl group, we have synthesized and administered to rats N-ethyl-3,5-dicarbethoxy-1,4-dihydrocollidine (N-ethyl DDC) and 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP), the DDC analogue with a 4-ethyl rather than 4-methyl group. Only N-methylprotoporphyrin IX is isolated from rats treated with the former agent, and only N-ethylprotoporphyrin IX from those treated with the latter. All four isomers of N-ethylprotoporphyrin IX are formed biologically. The structure of the isolated porphyrins has been confirmed by complete spectroscopic comparison with the four synthetic isomers of N-ethylprotoporphyrin IX. DDEP has been shown to cause NADPH- and time-dependent in vitro loss of hepatic microsomal cytochrome P-450. These results unequivocally establish that the 4-alkyl groups in DDC and dDEP are the source of the N-alkyl group in N-methyl- and N-ethylprotoporphyrin IX, respectively, and strongly suggest that the alkyl group is transferred to the prosthetic heme of cytochrome P-450 during catalytic processing of the substrate by the enzyme. The mechanism of the group transfer is discussed. 相似文献
10.
Rat liver microsomal cytochrome P-450 oxidizes the 4-methyl, 4-ethyl (DDEP), and 4-isopropyl derivatives of 3,5-bis(carbethoxy)-2,6-dimethyl-1,4-dihydropyridine to mixtures of the corresponding 4-alkyl and 4-dealkyl pyridines. A fraction of the total microsomal enzyme is destroyed in the process. The 4-dealkyl to 4-alkyl pyridine metabolite ratio, the extent of cytochrome P-450 destruction, and the rate of spin-trapped radical accumulation are correlated in a linear inverse manner with the homolytic or heterolytic bond energies of the 4-alkyl groups of the 4-alkyl-1,4-dihydropyridines. No isotope effects are observed on the pyridine metabolite ratio, the destruction of cytochrome P-450, or the formation of ethyl radicals when [4-2H]DDEP is used instead of DDEP. N-Methyl- and N-ethyl-DDEP undergo N-dealkylation rather than aromatization but N-phenyl-DDEP is oxidized to a mixture of the 4-ethyl and 4-deethyl N-phenylpyridinium metabolites. In contrast to the absence of an isotope effect in the oxidation of DDEP, the 4-deethyl to 4-ethyl N-phenylpyridinium metabolite ratio increases 6-fold when N-phenyl[4-2H]DDEP is used. The results support the hypothesis that cytochrome P-450 catalyzes the oxidation of dihydropyridines to radical cations and show that the radical cations decay to nonradical products by multiple, substituent-dependent, mechanisms. 相似文献
11.
Hepatic microsonal cytochrome P-450 levels are significantly decreased (46–68%) in ascorbic acid-deficient guinea pigs. Studies attempting to elucidate the mechanism responsible for decreased cytochrome P-450 demonstrated that ascorbic acid status did not influence the turnover or the degradation of hepatic cytochrome P-450 heme. Urinary excretion of Δ-aminolevulinic acid (ALA) and coproporphyrin was significantly decreased (30 and 69% respectively) in ascorbic acid-deficient guinea pigs. Injections (ip) of ALA into ascorbic acid-deficient guinea pigs were not effective in returning cytochrome P-450 levels to values found in ascorbic acid-supplemented guinea pigs. In addition, plasma and hepatic iron and blood heme were related directly, while hepatic copper and plasma copper or ceruloplasmin were related inversely, to the ascorbic-acid status of the guinea pig. These data, along with past investigations on heme synthesis in the ascorbic acid-deficient guinea pig, are consistent with mechanisms proposing that ascorbic acid may influence: 1) apocytochrome P-450 synthesis, 2) binding of heme and apo-cytochrome P-450 to form active cytochrome P-450, and/or 3) incorporation of Fe++ into the heme moiety of cytochrome P-450, perhaps via changes in copper metabolism. 相似文献
12.
D S Riddick I McGilvray G S Marks 《Canadian journal of physiology and pharmacology》1990,68(12):1533-1541
Various 4-alkyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6- trimethylpyridine (DDC) cause mechanism-based inactivation of cytochrome P-450 (P-450) via destruction of the heme prosthetic group. This is an important component of these compounds' porphyrinogenic mechanism. In an attempt to map the P-450 isozyme selectivities of DDC analogues, we have examined the effects of these compounds on the regioselective and stereoselective hydroxylation of androstenedione (AD) and progesterone (PG) in rat liver microsomal systems. In microsomes from phenobarbital-treated male rats, DDC analogues did not cause time-dependent inactivation of AD 7 alpha-hydroxylase, AD 16 beta-hydroxylase, and PG 21-hydroxylase, selective markers for P450IIA 1/2, IIB1, and IIC6, respectively. In contrast, DDC analogues were effective inactivators of PG 2 alpha-hydroxylase and steroid 6 beta-hydroxylases, selective markers for P450IIC11 and IIIA forms, respectively. We conclude that differences in porphyrinogenicity observed with various DDC analogues are not likely to be due to the selective destruction of different P-450 isozymes by different analogues, but rather to properties of the DDC analogues themselves. 4-Ethyl DDC was found to be capable of discriminating between P450IIIA subfamily forms. In microsomes from untreated male rats, which express P450IIIA2 but not IIIA1, 4-ethyl DDC inactivated both AD and PG 6 beta-hydroxylases. However, in microsomes from dexamethasone-treated female rats, which express P450IIIA1 but not IIIA2, no inactivation of the steroid 6 beta-hydroxylases was observed. Thus, 4-ethyl DDC appears to be a potentially valuable tool for differentiating between P450IIIA forms. 相似文献
13.
Effect of cis-platinum on kidney cytochrome P-450 and heme metabolism: evidence for the regulatory role of the pituitary hormones 总被引:2,自引:0,他引:2
A novel action of the gonadotropic hormones of the adenohypophysis on the regulation of kidney heme metabolism and cytochrome P-450 concentrations is described. The treatment of rats with cis-platinum for 7 days caused a greater than twofold increase in the microsomal cytochrome P-450 and heme concentrations in the kidney. The sodium dodecyl sulfate-gel electrophoresis of the microsomal preparation revealed increased levels of both apocytochrome P-450 and heme in the molecular weight region corresponding to cytochrome P-450. In hypophysectomized rats, similar increases in heme and the cytochrome contents in the kidney were observed. Conversely, the treatment of rats with human chorionic gonadotropin (hCG) fully reversed the effect of cis-platinum on heme and cytochrome P-450 concentrations. The cellular basis of increases in concentrations of heme and the hemoprotein was explored by measuring the incorporation of [14C]glycine-labeled hemoglobin heme into the kidney microsomal heme fractions. In comparison with the control rats, the specific 14C activity of heme in microsomal fraction was not increased. Moreover, the effect of cis-platinum on kidney cytochrome P-450 appeared to be unrelated to alterations in the activities of the rate-limiting enzymes of heme biosynthesis and degradation pathways, delta-aminolevulinate synthetase, and heme oxygenase, respectively. On the other hand, ferrochelatase activity and the concentration of total porphyrins in the kidney were profoundly altered by cis-platinum treatment; a twofold increase in ferrochelatase activity and a marked reduction (40%) in the total porphyrin concentration were observed. Also, the activities of uroporphyrinogen-I synthetase and delta-aminolevulinate dehydratase were decreased in cis-platinum-treated animals. The latter effects reflect a direct inhibitory action of cis-platinum. It appears that the cis-platinum-mediated increase in the microsomal heme concentrations involves an accelerated rate of heme production as a consequence of increased ferrochelatase activity. This, in turn, could increase the production of cytochrome P-450. It is suggested that the anterior pituitary hormones control the concentration of the cytochrome P-450 in the kidney, and this process may be interrupted by cis-platinum. 相似文献
14.
L M Bornheim M A Correia K M Smith 《Biochemical and biophysical research communications》1984,121(1):95-101
After allylisopropylacetamide-mediated "suicide" destruction of their prosthetic heme moieties, certain rat liver cytochrome P-450 isozymes can be effectively reconstituted by addition of exogenous hemin in vitro. We now report that two of these isozymes will equally accept mesohemin , a 2,4-diethyl heme-analog and result in a "meso-hemoprotein" with altered spectral but not functional characteristics. 相似文献
15.
G C Farrell R Schmid K L Kunze P R Ortiz de Montellano 《Biochemical and biophysical research communications》1979,89(2):456-463
Administration of allylisopropylacetamide (AIA) produces a dose-related destruction of the heme moiety of the phenobarbital-induced subspecies of hepatic cytochrome P-450. This results in delayed plasma disappearance of the inactivating agent as determined after injection of [14C]AIA. In phenobarbital-pretreated rats, infusion of heme reversed this AIA-mediated impairment of the plasma disappearance of [14C]AIA. In the absence of phenobarbital pretreatment, cytochrome P-450 destruction by AIA was minimal and heme infusion failed to enhance plasma disappearance of [14C]AIA. Since exogenously administered heme is incorporated into hepatic cytochrome P-450 , these observations suggest that the infused heme restored the functional capacity of the phenobarbital-induced mixed function oxidase system by substituting for the prosthetic heme moiety destroyed by AIA. Heme infusion is a potentially useful therapeutic modality for enhancing drug biotransformation after intoxication with compounds that inactivate cytochrome P-450. 相似文献
16.
K Kai S Kobayashi E Uchida H Sakai E Tanaka N Kurata H Yasuhara 《Comp. Biochem. Physiol. C, Comp. Pharmacol. Toxicol.》1988,90(1):13-19
1. In Sprague-Dawley (SD) rats treated for 7 days with malotilate (MAL:250 mg/kg, p.o.), cytochrome P-450 and b5 contents, aminopyrine N-demethylase and heme oxygenase activities were significantly increased. In Wistar rats, cytochrome b5 content and heme oxygenase and delta-aminolevulinic acid synthetase activities were found to be significantly increased. 2. Among the antipyrine metabolites excreted in urine during the 24 hr after antipyrine (100 mg/kg, i.p.) administration, norantipyrine increased significantly in Sprague-Dawley rats, while a significant increase of 4-hydroxyantipyrine was observed in Wistar rats. 3. The serum dimethadione/trimethadione ratio was only found to be significantly increased in Sprague-Dawley rats. 4. These results indicate that malotilate may have inducible effects on hepatic drug metabolizing enzymes, and that it affects the various cytochrome P-450 isozymes from different strains of rat in different ways. 相似文献
17.
Differential haemin-mediated restoration of cytochrome P-450 N-demethylases after inactivation by allylisopropylacetamide. 总被引:2,自引:2,他引:0
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Administration of allylisopropylacetamide (AIA) to phenobarbital-pretreated rats results in the destruction of several phenobarbital-inducible cytochrome P-450 isoenzymes and a correspondingly marked loss of benzphetamine N-demethylase and ethylmorphine N-demethylase activities. Accordingly, the ion-exchange h.p.l.c. or DEAE-cellulose-chromatographic profile of solubilized microsomal preparations from such rats revealed a marked decrease in the cytochrome P-450 content of several eluted fractions compared with that of microsomes from corresponding non-AIA-treated controls. Incubation of liver homogenates from such rats with haemin restores not only cytochrome P-450 content from 35 to 62% of original values, but also benzphetamine N-demethylase and ethylmorphine N-demethylase activities, from 23 to 67%, and from 12 to 36% of original values respectively. Moreover, the chromatographic profiles of microsomes prepared from such homogenates indicated increases of cytochrome P-450 content only in some fractions. Reconstitution of mixed-function oxidase activity of cytochrome P-450 by addition of NADPH: cytochrome P-450 reductase to these fractions indicated that incubation with haemin restored benzphetamine N-demethylase activity predominantly, but ethylmorphine N-demethylase activity only minimally. After injection of [14C]AIA, a significant amount of radiolabel was found covalently bound to protein in chromatographic fraction III, and this binding was unaffected by incubation with haemin. Furthermore, the extent of this binding is apparently equimolar to the amount of cytochrome P-450 refractory to haemin reconstitution in that particular fraction. Whether such refractoriness reflects structural inactivation of the apo-cytochrome remains to be determined. Nevertheless, the evidence presented very strongly argues for AIA-mediated inactivation of multiple phenobarbital-induced isoenzymes, only a few of which are structurally and functionally reparable by haemin. 相似文献
18.
Formation of cytochrome P-450 containing haem or cobalt-protoporphyrin in liver homogenates of rats treated with phenobarbital and allylisopropylacetamide.
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H L Bonkovsky J F Sinclair J F Healey P R Sinclair E L Smith 《The Biochemical journal》1984,222(2):453-462
The potent porphyrogen allylisopropylacetamide and related compounds decrease hepatic concentrations of cytochrome P-450. This decrease occurs particularly in phenobarbital-induced cytochrome P-450 and is caused by suicidal breakdown of the haem of cytochrome P-450. Quantitative rocket immunoelectrophoresis showed that the protein moiety of the major phenobarbital-inducible form of hepatic cytochrome P-450 was not diminished up to 1 h, but was markedly decreased (to 43% of that of the phenobarbital-treated control) at 20 h after allylisopropylacetamide treatment. In contrast, the concentration of total cytochrome P-450, measured spectrophotometrically, decreased to 30-40% of the control at both 1 and 20 h after allylisopropylacetamide. Cytochrome P-450-dependent demethylations of ethylmorphine and benzphetamine decreased to a similar extent. When liver homogenates from rats treated with allylisopropylacetamide 1 h before being killed were incubated with haem, functional holocytochrome P-450 could be reconstituted from the apoprotein. Incubation with haem increased spectrophotometrically measurable cytochrome P-450 to 69%, ethylmorphine demethylase to 64% and benzphetamine demethylase to 93% of the activities in rats treated with phenobarbital alone. At 20 h after allylisopropylacetamide treatment, however, little or no reconstitution of cytochrome P-450 occurred after incubation with haem. When liver homogenates were incubated with cobalt and protoporphyrin, and microsomal proteins were then subjected to polyacrylamide-gel electrophoresis, cobalt-protoporphyrin was found specifically associated with proteins of Mr 50 000-53 000. When homogenates from rats given allylisopropylacetamide for 1 h or 20 h were compared, it was found that the extent of this association was higher in livers from the rats containing more apocytochrome P-450, suggesting that cobalt-protoporphyrin had associated with the apocytochrome. The data provide insight into the association of haem with the protein moiety of cytochrome P-450 and factors affecting breakdown of this protein. 相似文献
19.
Metabolism of the potent hepatocarcinogen N-nitrosodimethylamine (NDMA) was evaluated in reconstituted monooxygenase systems containing each of 11 purified rat hepatic cytochrome P-450 isozymes. The reaction has an absolute requirement for cytochrome P-450, NADPH-cytochrome P-450 reductase, and NADPH, as well as a partial dependence on dilauroylphosphatidylcholine. Of the cytochrome P-450 isozymes evaluated, only cytochrome P-450j, purified from livers of ethanol- or isoniazid-treated rats, had high catalytic activity for the N-demethylation of NDMA. At substrate concentrations of 0.5 and 5 mM, rates of NDMA metabolism to formaldehyde catalyzed by cytochrome P-450j were at least 15-fold greater than the rates obtained with any of the other purified isozymes. At the pH optimum (approximately 6.7) for the reaction, the Km,app and Vmax were 3.5 mM and 23.9 nmol/min/nmol cytochrome P-450j, respectively. With hepatic microsomes from ethanol-treated rats, which contain induced levels of cytochrome P-450j, the Km,app and Vmax were 0.35 mM and 3.9 nmol/min/nmol cytochrome P-450, respectively. Inclusion of purified cytochrome b5 in the reconstituted system containing cytochrome P-450j caused a six-fold decrease in Km,app (0.56 mM) of NDMA demethylation with little or no change in Vmax (29.9 nmol/min/nmol cytochrome P-450j). Trypsin-solubilized cytochrome b5, bovine serum albumin, or hemoglobin had no effect on the kinetic parameters of the reconstituted system, indicating a specific effect of intact cytochrome b5 on the Km,app of the reaction. These results demonstrate high isozyme specificity in the metabolism of NDMA to an ultimate carcinogen and further suggest an important role for cytochrome b5 in this biotransformation process. 相似文献
20.
Characterization of a major form of rat hepatic microsomal cytochrome P-450 induced by isoniazid 总被引:5,自引:0,他引:5
D E Ryan L Ramanathan S Iida P E Thomas M Haniu J E Shively C S Lieber W Levin 《The Journal of biological chemistry》1985,260(10):6385-6393
Cytochrome P-450j has been purified to electrophoretic homogeneity from isoniazid-treated adult male rats; and this enzyme appears to be a major protein induced in hepatic microsomes after administration of isoniazid, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hemoprotein has a minimum molecular weight of approximately 51,500, and the ferrous-carbonyl complex of cytochrome P-450j has a Soret maximum at 451-452 nm. The oxidized heme iron appears to be predominately in the high spin state as deduced from the Soret maximum at 395 nm. Ethylisocyanide binds to ferrous cytochrome P-450j to yield spectral maxima at approximately 458 and 430 nm with a resultant 458/430 ratio of 0.7 at pH 7.4. Cytochrome P-450j has no measurable catalytic activity for the metabolism of benzo[a]pyrene (3- and 9-hydroxylation), hexobarbital, testosterone, and 5 alpha-androstane-3 alpha,17 beta-diol-3,17-disulfate. Low, but detectable, catalytic activity is obtained for the metabolism of 7-ethoxycoumarin, benzphetamine, p-nitroanisole, zoxazolamine, and 2-hydroxylation of 17 beta-estradiol. In contrast, cytochrome P-450j effectively catalyzes p-hydroxylation of aniline with a turnover of 12.7 nmol/min/nmol cytochrome P-450j. Hydroxyl radical scavengers, Fe-EDTA, superoxide dismutase, and catalase have no effect on aniline p-hydroxylation catalyzed by cytochrome P-450j. Cytochrome P-450j is distinct from nine other rat hepatic microsomal cytochromes P-450 (P-450a-P-450i) previously purified in this laboratory, as well as different isozymes described by other investigators, based on several parameters including minimum molecular weight, spectral properties, and catalytic activity. In Ouchterlony double diffusion plates, antibodies against cytochromes P-450a-P-450f show no cross-reaction with cytochrome P-450j. Structural differences among cytochromes P-450a-P-450j are apparent from the NH2-terminal sequence of cytochrome P-450j, as well as the electrophoretic profiles of proteolytic digests of the hemoproteins. 相似文献