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The Biochemical Journal May 19701. The metabolism in vitro and microsomal interactions of (+)-amphetamine, (-)-amphetamine, (+)-benzphetamine and (-)-benzphetamine were studied with hepatic microsomes...
1. The metabolism in vitro and microsomal interactions of (+)-amphetamine, (-)-amphetamine, (+)-benzphetamine and (-)-benzphetamine were studied with hepatic microsomes from phenobarbitone-pretreated male rabbits. 2. (+)-Benzphetamine was N-demethylated 30-35% faster than (-)-benzphetamine, but the apparent Michaelis constants for the two enantiomers were similar. 3. (-)-Amphetamine was deaminated about 200% faster than (+)-amphetamine. 4. The benzphetamine enantiomers gave qualitatively and quantitatively identical type I microsomal difference spectra (peak, 390nm; trough, 425nm) indicating identical apparent binding affinities for microsomes and identical spectral changes at maxima (DeltaE(max.) values). 5. The amphetamine enantiomers gave qualitatively identical type II microsomal difference spectra (peak, 433nm; trough, 395nm). However, the type II spectral data indicated that (+)-amphetamine had a markedly higher apparent binding affinity than (-)-amphetamine for microsomes. The amphetamine enantiomers gave identical DeltaE(max.) values. 6. The benzphetamine enantiomers (0.5mm) enhanced the rate of microsomal cytochrome P-450 reduction by NADPH by 400-500%, (+)-benzphetamine enhancing the rate 20-25% more than (-)-benzphetamine. 7. The amphetamine enantiomers decreased the rate of microsomal cytochrome P-450 reduction by NADPH. At a concentration of 2mm, (+)-amphetamine decreased the rate more than (-)-amphetamine. 7. All four enantiomers enhanced microsomal NADPH oxidation.
Topics: Amphetamine; Animals; Appetite Depressants; Benzphetamine; Dextroamphetamine; In Vitro Techniques; Kinetics; Male; Microsomes, Liver; NADP; Oxidoreductases; Phenethylamines; Propylamines; Rabbits; Spectrum Analysis; Stereoisomerism
PubMed: 4393781
DOI: 10.1042/bj1170833 -
The Journal of Biological Chemistry May 1975During the purification of rabbit liver microsomal cytochrome P-450 (P-450LM), evidence was obtained for the occurrence of at least four distinct forms. These were...
During the purification of rabbit liver microsomal cytochrome P-450 (P-450LM), evidence was obtained for the occurrence of at least four distinct forms. These were distinguished by polyacrylamide gel electrophoresis after treatment with sodium dodecyl sulfate in the presence or absence of mercaptoethanol and were shown to have characteristic spectra as the reduced carbon monoxide complexes. They are designated by their relative electrophoretic mobilities. P-450LM2, which was purified to apparent homogeneity, is induced by phenobarbital and has a subunit molecular weight of 50,000. P-450LM4, which was also extensively purified, is induced by beta-naphthoflavone and has a molecular weight of 54,000. P-450LM1,7, which is induced neither by phenobarbital nor beta-naphthoflavone, is a mixtureMIXTURE OF ABOUT EQUAL AMOUNTS OF TWO FORMS WITH MOLECULAR WEIGHTS OF 47,000 AND 60,000 RESPECTIVELY. Some preparations were obtained containing primarily P-450LM1 or P-450LM7. Benzphetamine, ethylmorphine, and p-nitroanisole are hydroxylated preferentially by P-450LM2, and benzpyrene by P-450LM1,7. Biphenyl is hydroxylated in both positions 2 and 4 by all of the preparations, but the latter position is strongly favored by the action of P-450LM2. Testosterone is hydroxylated primarily in position 16alpha by P-450LM2 and in position 6beta by P-450LM1,7. Although the occurrence of additional forms of the cytochrome with highly similar electrophoretic behavior is not ruled out, it appears that the presence of these forms differing in subunit molecular weight may account for the variety of catalytic activities attributed to this pigment of liver microsomes.
Topics: Animals; Anisoles; Benzopyrenes; Benzphetamine; Cytochrome P-450 Enzyme System; Cytochrome Reductases; Electrophoresis, Polyacrylamide Gel; Flavonoids; Hydroxylation; Microsomes, Liver; Molecular Weight; Morphine Derivatives; Phenobarbital; Protein Conformation; Rabbits; Testosterone
PubMed: 1123353
DOI: No ID Found -
The Biochemical Journal Apr 1985Administration of allylisopropylacetamide (AIA) to phenobarbital-pretreated rats results in the destruction of several phenobarbital-inducible cytochrome P-450...
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.
Topics: Acetamides; Allylisopropylacetamide; Animals; Chromatography, DEAE-Cellulose; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Ethylmorphine-N-Demethylase; Heme; Hemin; In Vitro Techniques; Isoenzymes; Male; Microsomes, Liver; Oxidoreductases, N-Demethylating; Proteins; Rats; Rats, Inbred Strains
PubMed: 3994685
DOI: 10.1042/bj2270277 -
Journal of Biochemistry Sep 19883,4,5,3',4'-Pentachlorobiphenyl (PenCB), one of the most potent 3-methylcholanthrene (MC)-type inducers of hepatic enzymes in animals, caused a remarkable induction of...
3,4,5,3',4'-Pentachlorobiphenyl (PenCB), one of the most potent 3-methylcholanthrene (MC)-type inducers of hepatic enzymes in animals, caused a remarkable induction of liver microsomal monooxygenases, particularly 7-ethoxyresorufin (7-ER) O-deethylase, benzo(a)pyrene (BP) 3-hydroxylase, and testosterone 16 alpha-hydroxylase in chickens, but not NADPH-cytochrome c(P-450) reductase and cytochrome b5. Two forms of cytochrome P-450 (P-450) in liver microsomes of PenCB-treated chickens were purified and characterized. The absorption maxima of the CO-reduced difference spectra of both enzymes (chicken P-448 L and chicken P-448 H) were at 448 nm. From the oxidized form of their absolute spectra, chicken P-448 L was a low-spin form and chicken P-448 H was a high-spin form. They had molecular masses of 56 and 54 kDa, respectively. In a reconstituted system, 7-ER O-deethylation, BP 3-hydroxylation, and testosterone 16 alpha-hydroxylation were catalyzed at high rates by chicken P-448 L but not by chicken P-448 H. Chicken P-448 L also catalyzed N-demethylation of aminopyrine, benzphetamine, and ethylmorphine with relatively low activity. On the other hand, chicken P-448 H functioned only in catalyzing estradiol 2-hydroxylation. These results were supported by an inhibition study of microsomal monooxygenases using an antibody against each enzyme. Immunochemical studies revealed that the enzymes differ from each other but are both inducible by PenCB-treatment. Chicken P-448 L and chicken P-448 H respectively comprise about 82 and 7% of the total P-450 content in chicken liver microsomes.
Topics: Animals; Antibodies; Chickens; Cytochrome P-450 Enzyme System; Electrophoresis, Polyacrylamide Gel; Enzyme Induction; Immunochemistry; Immunodiffusion; Isoenzymes; Male; Microsomes, Liver; NADPH-Ferrihemoprotein Reductase; Polychlorinated Biphenyls; Proteins; Steroid 16-alpha-Hydroxylase; Substrate Specificity
PubMed: 3149274
DOI: 10.1093/oxfordjournals.jbchem.a122473 -
The Journal of Biological Chemistry Oct 1984Rat liver mitoplasts containing less than 1% microsomal contamination contain cytochrome P-450 at 25% of the microsomal level and retain the capacity for monooxygenase...
Rat liver mitoplasts containing less than 1% microsomal contamination contain cytochrome P-450 at 25% of the microsomal level and retain the capacity for monooxygenase activation of structurally different carcinogens such as aflatoxin B1 (AFB1), benzo(a)pyrene (BaP), and dimethylnitrosamine. Both phenobarbital (PB) and 3-methylcholanthrene (3-MC) induce the level of mitochondrial cytochrome P-450 by 2.0- to 2.5-fold above the level of control mitoplasts. The enzyme activities for AFB1 (3-fold) and BaP (16-fold) metabolism were selectively induced by PB and 3-MC, respectively. Furthermore, the metabolism of AFB1 and BaP by intact mitochondria was supported by Krebs cycle substrates but not by NADPH. Both PB and 3-MC administration cause a shift in the CO difference spectrum of mitoplasts (control, 448 nm; PB, 451 nm; and 3-MC, 446 nm) suggesting that they induce two different forms of mitochondrial cytochromes P-450. Mitoplasts solubilized with cholate and fractionated with polyethylene glycol exhibit only marginal monooxygenase activities. The activity, however, was restored to preparations from both PB-induced and 3-MC-induced mitochondrial enzymes (AFB1 activation, ethylmorphine, and benzphetamine deamination and BaP metabolism) by addition of purified rat liver cytochrome P-450 reductase, and beef adrenodoxin and adrenodoxin reductase. The latter proteins failed to reconstitute activity to purified microsomal cytochromes P-450b and P-450c that were fully active with P-450 reductase. Monospecific rabbit antibodies against cytochrome P-450b and P-450c inhibited both P-450 reductase and adrenodoxin-supported activities to similar extents. Anti-P-450b and anti-P-450c provided Ouchterlony precipitin bands against PB- and 3-MC induced mitoplasts, respectively. We conclude that liver mitoplasts contain cytochrome P-450 that is closely similar to the corresponding microsomal cytochrome P-450 but can be distinguished by a capacity to interact with adrenodoxin. These inducible cytochromes P-450 are of mitochondrial origin since their levels in purified mitoplasts are over 10 times greater than can arise from the highest possible microsomal contamination.
Topics: Aflatoxin B1; Aflatoxins; Animals; Benzo(a)pyrene; Biotransformation; Carcinogens; Cytochrome P-450 Enzyme System; Dimethylnitrosamine; Kinetics; Male; Microsomes, Liver; Mitochondria, Liver; Rats; Rats, Inbred Strains; Tritium
PubMed: 6436235
DOI: No ID Found -
The Journal of Biological Chemistry Dec 1982Evidence for the existence of a previously unknown rat hepatic microsomal reductase, short chain trans-2-enoyl-CoA reductase (SC reductase) is presented. This reductase...
Evidence for the existence of a previously unknown rat hepatic microsomal reductase, short chain trans-2-enoyl-CoA reductase (SC reductase) is presented. This reductase has a specific requirement for NADPH, is unable to utilize NADH, and catalyzes the conversion of crotonyl-CoA and trans-2-hexenoyl-CoA to butyric acid and hexenoic acid at a rate of 5 and 65 nmol per min per mg of microsomal protein, respectively. Highly purified NADPH cytochrome P-450 reductase incorporated into liposomes prepared from dilauroyl phosphatidylcholine in the presence or absence of cytochrome P-450 possesses no SC reductase activity. These liposomal preparations did, however, catalyze mixed function oxidations of benzphetamine and testosterone. Rabbit antibody to rat liver NADPH cytochrome P-450 reductase had little to no effect on the conversion of crotonyl-CoA and trans-2-hexenoyl-CoA, suggesting that the SC reductase accepts reducing equivalents directly from NADPH. When acetoacetyl-CoA was incubated with hepatic microsomes and either NADH or NADPH, no formation of butyrate was detected; however, when both cofactors were present, a rate of formation of 3 nmol of butyrate was determined per min per mg of microsomal protein. These results suggest the presence of a previously unknown short chain beta-ketoreductase which catalyzes the reduction of short chain beta-keto acids, only in the presence of NADH. Our results also indicate that the electrons from NADH to the beta-ketoreductase bypass cytochrome b5. The physiological significance is discussed in terms of lipogenesis and ketone body utilization by the liver.
Topics: Animals; Fatty Acid Desaturases; Fatty Acid Synthases; Immune Sera; Kinetics; Male; Microsomes, Liver; NADH, NADPH Oxidoreductases; NADP; NADPH-Ferrihemoprotein Reductase; Oxidation-Reduction; Rats; Rats, Inbred Strains
PubMed: 6815194
DOI: No ID Found -
The Biochemical Journal Nov 1992A form of cytochrome P-450 has been purified to electrophoretic homogeneity from the hepatic microsomes of Syrian golden hamsters treated with acetone. This P-450 form,...
A form of cytochrome P-450 has been purified to electrophoretic homogeneity from the hepatic microsomes of Syrian golden hamsters treated with acetone. This P-450 form, designated ha P-450j, had an M(r) of approximately 55,000, bound dimethyl sulphoxide and exhibited a CO-reduced absorbance maximum at 451 nm. The absolute spectra of its oxidized form indicated that ha P-450j was predominantly in the low-spin state. In a reconstituted system, ha P-450j showed relatively low catalytic activities towards 7-ethoxycoumarin, 7-ethoxyresorufin, aminopyrine, ethylmorphine and benzphetamine, whereas it catalysed the oxidation of aniline, acetone and thiobenzamide with a high catalytic-centre activity. In addition, ha P-450j catalysed at a high rate the high-affinity component of dimethylnitrosamine N-demethylase; in contrast, only the low-affinity component of diethylnitrosamine N-de-ethylase was efficiently catalysed. The addition of cytochrome b5 to the reconstitution system decreased the Km value for dimethylnitrosamine N-demethylase by a factor of 5 and increased the Vmax. value, and slightly enhanced the other activities. Thiobenzamide and diethyldithiocarbamate were found to be the most effective inhibitors of the ha-P-450j-dependent aniline hydroxylation. Polyclonal antibodies against rat P-450j recognized ha P-450j in immunoblots of control and treated hamster liver microsomes. Treatment of hamsters with acetone increased the apparent abundance of ha P-450j in microsomes, whereas phenobarbital and beta-naphthoflavone did not induce it. Analysis of N-terminal amino acid sequences demonstrated that ha P-450j has a high degree of sequence identity with rat P-450j. All the evidence presented in this study indicates that ha P-450j could represent the hamster orthologue of the previously described CYP2E1(s) of other species.
Topics: Acetone; Amino Acid Sequence; Aniline Compounds; Animals; Blotting, Western; Cricetinae; Cytochrome P-450 CYP2E1; Cytochrome P-450 Enzyme System; Enzyme Induction; Humans; Hydroxylation; Kinetics; Male; Mesocricetus; Microsomes, Liver; Molecular Sequence Data; Oxidoreductases, N-Demethylating; Sequence Homology, Amino Acid; Spectrum Analysis
PubMed: 1445245
DOI: 10.1042/bj2870863 -
British Journal of Clinical Pharmacology Dec 1983Drug-metabolizing enzyme activities were determined in liver microsomes from six kidney-transplant donors, one tricyclic antidepressant-treated and five untreated...
Drug-metabolizing enzyme activities were determined in liver microsomes from six kidney-transplant donors, one tricyclic antidepressant-treated and five untreated donors. The tricyclic antidepressant treatment modifies neither the overall cytochrome P-450 content of the liver, nor enzymatic activities of 4-nitroanisole demethylase, aniline hydroxylase, epoxide hydrolase and glutathione S-transferase. Only benzphetamine and ketotifen demethylation and conjugation of bilirubin with UDP-glucuronic acid are markedly augmented (more than two-fold). Separation of the different cytochrome P-450 fractions on a DEAE cellulose column indicates a modification of the elution pattern: the fraction increased by tricyclic antidepressants is responsible for the enhanced monooxygenase activity towards benzopyrene and benzphetamine.
Topics: Adult; Antidepressive Agents, Tricyclic; Cytochrome P-450 Enzyme System; Enzyme Induction; Female; Humans; Liver; Male; Pharmaceutical Preparations
PubMed: 6661349
DOI: 10.1111/j.1365-2125.1983.tb02236.x -
The Biochemical Journal Oct 1982The interaction of substrates of the microsomal mixed-function oxidases with cytochromes P-450 and P-448 was investigated by using liver microsomes from rats pretreated...
The interaction of substrates of the microsomal mixed-function oxidases with cytochromes P-450 and P-448 was investigated by using liver microsomes from rats pretreated with phenobarbital or 3-methylcholanthrene, and with purified forms of the cytochromes isolated from rabbit liver. The two forms of the cytochrome have different substrate specificities; cytochrome P-450 has one type 1 substrate-binding site that can accommodate a large variety of substrates, but in contrast cytochrome P-448 may possess two type 1 substrate-binding sites, one of which is different to that of cytochrome P-450 in that it shows a specificity for substrates such as safrole and 9-hydroxy-ellipticine. These findings explain why the two forms of the cytochrome have different substrate specificities and play contrasting roles in the activation and deactivation of xenobiotics.
Topics: Animals; Benzphetamine; Binding Sites; Cytochrome P-450 CYP1A2; Cytochrome P-450 Enzyme System; Cytochromes; Ellipticines; Fluorenes; In Vitro Techniques; Male; Methylcholanthrene; Microsomes, Liver; Phenobarbital; Rats; Rats, Inbred Strains; Safrole; Spectrophotometry; Substrate Specificity
PubMed: 7181861
DOI: 10.1042/bj2070051 -
Journal of Biochemistry Sep 1993A form of P450 [termed P450(h-1)] was purified from the liver microsomes of a male horse to electrophoretic homogeneity. The specific content of the final P450(h-1)...
A form of P450 [termed P450(h-1)] was purified from the liver microsomes of a male horse to electrophoretic homogeneity. The specific content of the final P450(h-1) preparation was 14.8 nmol/mg of protein and the recovery was 0.38% of the microsomal P450. The apparent molecular weight of P450(h-1) was 52,000 Da. The absorption spectra of P450(h-1) indicated that P450(h-1) was a low- and high-spin mixed type P450 in the oxidized form. The reconstituted system containing P450(h-1) could catalyze benzphetamine N-demethylation, 7-ethoxycoumarin O-deethylation, and testosterone 16 alpha-hydroxylation. In the horse hepatic microsomes, aniline p-hydroxylation and testosterone 6 beta-hydroxylation, in addition to the above reactions, were detected. The N-terminal amino acid sequence of P450(h-1) was highly homologous to that of rat P450 2C11. Western blot analysis using anti-P450(h-1) antibody revealed that this antibody most strongly recognized P450 2C13 among ten rat P450s belonging to eight different subfamilies involved in hepatic drug metabolism. This anti-P450(h-1) antibody inhibited the testosterone 16 alpha-hydroxylase activity in horse liver microsomes. These results suggest that P450(h-1) belongs to the P450 2C subfamily and contributes to the testosterone 16 alpha-hydroxylation in horse liver microsomes.
Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Aryl Hydrocarbon Hydroxylases; Catalysis; Cytochrome P-450 Enzyme System; Cytochrome P450 Family 2; Horses; Immunochemistry; Isoenzymes; Male; Microsomes, Liver; Molecular Sequence Data; Sequence Homology, Amino Acid; Spectrophotometry; Steroid 16-alpha-Hydroxylase; Steroid Hydroxylases
PubMed: 8282739
DOI: 10.1093/oxfordjournals.jbchem.a124195