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The Journal of Biological Chemistry Nov 1991Liver microsomes from phenobarbital-treated rats of four inbred strains expressing distinct allelic variants of cytochrome P450IIB1 were analyzed. The Wistar Munich (WM)... (Comparative Study)
Comparative Study
Liver microsomes from phenobarbital-treated rats of four inbred strains expressing distinct allelic variants of cytochrome P450IIB1 were analyzed. The Wistar Munich (WM) strain exhibited 5- to 10-fold lower androstenedione 16 beta-hydroxylase activity (a specific P450IIB1 marker) than the Lewis, Wistar Kyoto, and Wistar Furth strains. The androstenedione 16 beta-hydroxylase in the WM liver microsomes was refractory to inactivation by N-(2-p-nitrophenethyl)chlorofluoroacetamide, a selective P450IIB1 inactivator in the other three strains. Purified P450IIB1-WM was insensitive to the inactivator and exhibited 5-fold lower androstenedione 16 beta-hydroxylase, testosterone 16-hydroxylase, and 7-ethoxycoumarin deethylase activities but the same benzphetamine demethylase activity and slightly higher androstenedione 16 alpha-hydroxylase activity than a P450IIB1 purified from outbred Sprague-Dawley rats, which appears to correspond to the form in Lewis rats. The stereoselectivity of androstenedione 16-hydroxylation catalyzed by P450IIB1-WM (16 beta-OH:16 alpha-OH = 1.4) is thus distinct from that (16 beta-OH:16 alpha-OH = 12-15) of other P450IIB1 preparations described. A cDNA encoding P450IIB1-WM was cloned and sequenced, revealing a single amino acid substitution (Gly-478----Ala) compared with the published sequence (Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., and Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2793-2797). Heterologous expression of P450IIB1 and P450IIB1-WM confirmed the striking difference in androstenedione metabolite profiles, strongly implicating the involvement of Ala-478 in defining the distinctive catalytic properties of P450IIB1-WM.
Topics: Alleles; Animals; Cloning, Molecular; Cytochrome P-450 CYP2B1; Cytochrome P-450 Enzyme System; DNA; Genetic Variation; Kinetics; Liver; Male; Microsomes, Liver; Oxidoreductases; Phenobarbital; Plasmids; RNA; Rats; Rats, Inbred Lew; Rats, Inbred Strains; Rats, Inbred WF; Rats, Inbred WKY; Recombinant Proteins; Sequence Homology, Nucleic Acid; Species Specificity; Steroid 16-alpha-Hydroxylase; Substrate Specificity
PubMed: 1718996
DOI: No ID Found -
The Journal of Biological Chemistry Nov 1983The interactions between purified rat hepatic microsomal cytochrome P-450 and the type I ligands benzphetamine and cytochrome b5 have been studied in the presence of...
Thermodynamic studies of the protein-protein interactions between cytochrome P-450 and cytochrome b5. Evidence for a central role of the cytochrome P-450 spin state in the coupling of substrate and cytochrome b5 binding to the terminal hemoprotein.
The interactions between purified rat hepatic microsomal cytochrome P-450 and the type I ligands benzphetamine and cytochrome b5 have been studied in the presence of phospholipid using difference spectrophotometry. Cytochrome b5 was shown to interact with cytochrome P-450 to form a tight 1:1 complex (Kd = 275 nM), in which the proportion of high spin cytochrome P-450 was increased from 7 to 30%. The presence of saturating cytochrome b5 was shown to cause a decrease in the apparent Kd for benzphetamine binding from 111 microM to 40 microM. Likewise, the presence of benzphetamine was shown to cause a decrease in the apparent dissociation constant for cytochrome b5 binding to cytochrome P-450 (Kd = 90 nM). The above interactions were resolved into the basic equilibria inter-relating the various ligation states of the hemoprotein in an energetically closed eight-state free energy coupling model and the relative magnitudes of the microequilibria were analyzed to determine the degree of coupling of the interactions between cytochrome P-450 and both benzphetamine and cytochrome b5. Consequently, the spin state changes in cytochrome P-450 induced by benzphetamine and cytochrome b5 binding were shown to arise because these ligands interact 7 and 4 times more tightly with high spin cytochrome P-450, respectively. Furthermore, the data revealed that these ligands interact at independent sites on cytochrome P-450. Thus the effects of cytochrome b5 upon benzphetamine binding and vice versa were rationalized simply in terms of an increase in the proportion of a high spin (high affinity) conformation of cytochrome P-450 brought about by pre-equilibration with the effector ligand, with the intrinsic binding affinities of the two ligands for the low or high spin states remaining relatively unaltered. The thermodynamic parameters associated with the interactions between cytochrome P-450 and cytochrome b5, determined from the temperature dependence of these interactions, revealed that these protein interactions are entropy driven and probably occur by a hydrophobic mechanism.
Topics: Animals; Cytochrome P-450 Enzyme System; Cytochrome b Group; Cytochromes b5; Kinetics; Ligands; Male; Mathematics; Microsomes, Liver; Models, Biological; Protein Binding; Rats; Rats, Inbred Strains; Spectrophotometry; Thermodynamics
PubMed: 6643436
DOI: No ID Found -
Zhongguo Yao Li Xue Bao = Acta... Apr 1999To explore the capacity and characteristics of adrenal mitochondria to metabolize xenobiotics in vitro in human fetus.
AIM
To explore the capacity and characteristics of adrenal mitochondria to metabolize xenobiotics in vitro in human fetus.
METHODS
Subcellular fractions of fetal adrenal were prepared by differential centrifugation. Mitochondrial P-450 system was proved by spectral analyses and SDS-PAGE. The formaldehyde formation contents were measured with Nash reagent.
RESULTS
The erythromycin N-demethylation linearly increased in the protein concentration (1-4 mg)- and incubation time (10-30 min)-dependent manners. A typical concentration-effect relationship appeared with erythromycin 0.067-1 mmol.L-1 and a positive correlation (r = 0.641, P < 0.05) existed between erythromycin N-demethylation and gestation months. The N-demethylation values (nmol.s-1/g protein) of erythromycin (2.7 +/- 0.8), benzfetamine (1.1 +/- 0.5), and aminophenazone (0.9 +/- 0.4) in mitochondria were 89% (P > 0.05), 162% (P < 0.01), and 62% (P < 0.01), respectively, of those in microsomes. There was correlation between mitochondria and microsomes in the N-demethylation of erythromycin (r = 0.708, P < 0.05) and benzfetamine (r = 0.707, P < 0.05). Troleandomycin stimulated erythromycin N-demethylation in adrenal mitochondria as well as in adrenal and liver microsomes in vitro.
CONCLUSION
Fetal adrenal mitochondria, with multiple P-450 isoforms and greater capacity of demethylation, play a role in drug-metabolism during fetal development.
Topics: Adrenal Glands; Aminopyrine; Benzphetamine; Cytochrome P-450 Enzyme System; Erythromycin; Fetus; Formaldehyde; Humans; Microsomes, Liver; Mitochondria; Troleandomycin
PubMed: 10452125
DOI: No ID Found -
The Journal of Biological Chemistry Apr 1985Native cytochrome b5 interacts with either RLM5 or LM2 to form tight equimolar complexes (Kd = 250 and 540 nM, respectively) in which the content of high spin cytochrome...
Native cytochrome b5 interacts with either RLM5 or LM2 to form tight equimolar complexes (Kd = 250 and 540 nM, respectively) in which the content of high spin cytochrome P-450 was substantially increased. Cytochrome b5 caused 3- and 7-fold increases in the binding affinities of RLM5 and LM2 for benzphetamine, respectively, and benzphetamine decreased the apparent Kd for cytochrome b5 binding. Upon formation of the ternary complex between cytochromes P-450, b5, and benzphetamine the percentage of cytochrome P-450 in the high spin state was increased from 28 to 74 (RLM5) and from 9 to 85 (LM2). Cytochrome b5 caused 13- and 7-fold increases in the rate of RLM5- and LM2-dependent p-nitroanisole demethylation, respectively. Amino-modified (ethyl acetimidate or acetic anhydride) cytochrome b5 produced results similar to those obtained above with native cytochrome b5. In contrast, modification of as few as 5 mol of carboxyl groups/mol of amidinated cytochrome b5 resulted in both a substantial loss of the spectrally observed interactions with either cytochrome P-450 LM2 or cytochrome P-450 RLM5, and in a loss of the cytochrome b5-mediated stimulation of p-nitroanisole demethylation catalyzed by either monooxygenase. In further studies, native and fully acetylated cytochromes b5 reoxidized carbonmonoxy ferrous LM2 at least 20 times faster than amidinated, carboxyl-modified cytochrome b5 derivatives. In contrast, amidination, or acetylation of amino groups, or amidination of amino groups plus methylamidination of the carboxyl groups did not appreciably slow the rate of reduction of the cytochrome b5 by NADPH-cytochrome P-450 reductase. Collectively, the results provide strong evidence for an essential role of cytochrome b5 carboxyl groups in functional interactions with RLM5 and LM2.
Topics: Animals; Cytochrome P-450 Enzyme System; Cytochrome b Group; Cytochromes b5; Male; Microsomes, Liver; Models, Chemical; NADPH-Ferrihemoprotein Reductase; Rabbits; Spectrophotometry
PubMed: 3920211
DOI: No ID Found -
The Journal of Biological Chemistry Jan 1988In vivo administration of the alcohol dehydrogenase inhibitor pyrazole induces a cytochrome P-450 isozyme. The pyrazole-inducible cytochrome P-450 has been purified from...
In vivo administration of the alcohol dehydrogenase inhibitor pyrazole induces a cytochrome P-450 isozyme. The pyrazole-inducible cytochrome P-450 has been purified from rat livers to electrophoretic homogeneity and its biochemical, spectral, and immunological properties characterized. The final preparation had a specific content of 11 nmol of cytochrome P-450/mg of protein. A single band with an apparent molecular weight of 52,000 was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The absolute spectrum of the isolated pyrazole cytochrome P-450 displayed peaks at 648 and 396 nm, suggestive of a high spin cytochrome. The ethylisocyanide difference spectrum exhibited two maxima, one at 457 nm, the other at 428 nm. Pyrazole and dimethyl sulfoxide produced binding spectra with the purified P-450, with peaks at 425 or 419 nm and troughs at 390 or 386 nm, respectively. K8 values for dimethyl sulfoxide and pyrazole were 21 and 0.04 mM, respectively. The catalytic activity of the pyrazole cytochrome P-450 was elevated with aniline and dimethylnitrosamine (low Km) but not with aminopyrine, benzphetamine, ethoxycoumarin, or ethoxyresorufin as substrates. An antibody against pyrazole cytochrome P-450 recognized a 52,000 molecular weight protein upon reaction with saline microsomes. The intensity of the immunoblot was increased when microsomes isolated from pyrazole, 4-methylpyrazole-, acetone-, or chronic ethanol-treated rats were utilized, but not after phenobarbital or 3-methylcholanthrene treatment. Homology at the amino terminus of 19 amino acids was observed between pyrazole P-450 and the isoniazid-inducible P-450j. Based upon the above catalytic, spectral, and immunological properties, it appears that pyrazole induces a form of cytochrome P-450 which is identical to that induced by ethanol and isoniazid.
Topics: Amino Acids; Animals; Cytochrome P-450 CYP2E1; Cytochrome P-450 Enzyme System; Enzyme Induction; Isoenzymes; Kinetics; Male; Microsomes, Liver; Oxidoreductases, N-Demethylating; Pyrazoles; Rats; Rats, Inbred Strains; Sulfuric Acid Esters
PubMed: 3335529
DOI: No ID Found -
Journal of Biochemistry Mar 1984Four forms of cytochrome P-450, tentatively designated PB-1, PB-2, MC-1, and MC-2, were purified from liver microsomes of rats treated with phenobarbital (PB-1 and PB-2)...
Four forms of cytochrome P-450, tentatively designated PB-1, PB-2, MC-1, and MC-2, were purified from liver microsomes of rats treated with phenobarbital (PB-1 and PB-2) or 3-methylcholanthrene (MC-1 and MC-2). Each purified form showed a single protein-staining band on SDS-polyacrylamide gel electrophoresis giving a minimum molecular weight of 56,000 (MC-1), 53,000 (PB-1), 53,000 (MC-2), or 49,000 (PB-2). PB-1 and MC-1 were the major cytochrome P-450 components inducible by phenobarbital (PB) and 3-methylcholanthrene (MC), respectively. Antibodies prepared against each form of purified cytochrome P-450 did not cross-react with heterologous antigens in Ouchterlony double diffusion tests, confirming the immunological distinctness of the four forms. The CO-compounds of reduced PB-1 and PB-2 had an absorption maximum at 450 nm, whereas those of MC-1 and MC-2 had a maximum at 447 nm. Judging from the oxidized absolute spectra, MC-2 was of high spin type and the others were of low spin type. Amino acid analysis revealed considerable differences among the purified four forms of cytochrome P-450, and the amino acid sequences of their NH2-terminal portions confirmed that the four forms were different proteins. In a reconstituted system containing NADPH and NADPH-cytochrome P-450 reductase, PB-1 and PB-2 oxidized benzphetamine at high rates, but their oxidation of benzo(a)pyrene was much slower than that by MC-1, which catalyzed rapid hydroxylation of benzo(a)pyrene but had low activity with benzphetamine. The quantity of each form of cytochrome P-450 in microsomes was determined by quantitative immunoprecipitation, and selective induction of PB-1 and MC-1 by PB and MC, respectively, was confirmed. Some induction of PB-2 and MC-2 by the corresponding inducers was also noticed. PB group P-450's were not increased by MC treatment, nor were MC group P-450's by PB.
Topics: Amino Acids; Animals; Cytochrome P-450 Enzyme System; Electrophoresis, Polyacrylamide Gel; Immunodiffusion; Male; Methylcholanthrene; Microsomes, Liver; Phenobarbital; Rats; Rats, Inbred Strains; Substrate Specificity
PubMed: 6427199
DOI: 10.1093/oxfordjournals.jbchem.a134660 -
British Journal of Anaesthesia May 1995We have studied the effect of propofol on the cytochrome P450-dependent mono-oxygenase system in human liver microsomes by assaying mono-oxygenase activities toward...
We have studied the effect of propofol on the cytochrome P450-dependent mono-oxygenase system in human liver microsomes by assaying mono-oxygenase activities toward specific cytochrome P450 isoform test substrates, aniline, 7-ethoxycoumarin, benzphetamine and benzo(a) pyrene. Propofol inhibited benzo(a)pyrene hydroxylation to a greater extent than the oxidative metabolism of the other test substrates, even at 0.05 mmol litre-1. The degrees of inhibition of benzphetamine N-demethylation and 7-ethoxy-coumarin O-de-ethylation were similar, while aniline hydroxylation was least affected by propofol. Spectral analysis showed that propofol competed with carbon monoxide for binding to the haem moiety of haemoprotein in the P450 enzyme. The variable inhibition observed may be caused by the differential binding of propofol to P450 isoforms. Propofol 0.05-1.0 mmol litre-1 exhibited a concentration-dependent inhibitory effect on human cytochrome P450 2E1, 2B1 and 1A1. These inhibitory actions of propofol on human liver microsomal enzymes in vitro suggest that potential drug interactions may exist between propofol and other drugs administered clinically.
Topics: Aniline Compounds; Benzo(a)pyrene; Benzphetamine; Coumarins; Cytochrome P-450 Enzyme Inhibitors; Dose-Response Relationship, Drug; Humans; In Vitro Techniques; Microsomes, Liver; Oxygenases; Propofol
PubMed: 7772432
DOI: 10.1093/bja/74.5.558 -
Biochemical and Biophysical Research... Aug 1969
Topics: Aminopyrine; Animals; Appetite Depressants; Benzphetamine; Cytochromes; Depression, Chemical; Ethers; Fatty Acids; Formaldehyde; Hexobarbital; Lipid Metabolism; Liver; Microsomes; Mixed Function Oxygenases; Morphinans; NADP; Oxidoreductases; Phenethylamines; Phenobarbital; Propylamines; Rabbits; Spectrophotometry
PubMed: 4390187
DOI: 10.1016/0006-291x(69)90339-8 -
Proceedings of the National Academy of... Jan 1987A covalent complex between rabbit hepatic microsomal cytochromes P-450 isozyme 2 (LM2) and b5 was created and purified to greater than 95% homogeneity. The purified...
A covalent complex between rabbit hepatic microsomal cytochromes P-450 isozyme 2 (LM2) and b5 was created and purified to greater than 95% homogeneity. The purified complex was largely comprised of the two cytochromes covalently attached at the interface of the functional electron transfer-effector complex as shown by the following evidence. The spin state of the LM2 within the complex was greater than the spin state of free LM2, and the addition of free cytochrome b5 (cyt b5) did not further increase the spin state of the LM2 within the complex. The spectral binding parameters (Kd and delta Amax) for the association of benzphetamine with LM2 in the complex were identical to those observed with free LM2 in the presence of saturating concentrations of free cyt b5 and much different from those observed for LM2 in the absence of cyt b5. Reconstituted monooxygenase activity of the covalent LM2-cyt b5 complex (LM2-cyt b5) in the presence of NADPH-cytochrome P-450 reductase was much higher than the activity of free LM2 and approached the activity of free LM2 in the presence of optimal concentrations of free cyt b5. Furthermore, the Km for the flavoprotein in supporting either free LM2 or LM2-cyt b5-dependent p-nitroanisole demethylation were similar. (iv) Less than 20-25% of the cyt b5 within the complex could be reduced by free NADH-cytochrome b5 reductase (NADH-cyt b5 reductase) albeit at a slow rate. The implications of this data to the current understanding of the mechanism and stoichiometry of protein interactions in the hepatic mixed function oxidase system are discussed.
Topics: Animals; Cytochrome P-450 Enzyme System; Isoenzymes; Kinetics; Macromolecular Substances; Microsomes, Liver; Rabbits
PubMed: 3467342
DOI: 10.1073/pnas.84.1.11 -
The Journal of Biological Chemistry Mar 1992A rabbit cytochrome P450 which catalyzes the epoxidation of arachidonic acid to two of the four possible regioisomeric epoxyeicosatrienoic acid metabolites was purified... (Comparative Study)
Comparative Study
A rabbit cytochrome P450 which catalyzes the epoxidation of arachidonic acid to two of the four possible regioisomeric epoxyeicosatrienoic acid metabolites was purified from renal cortex. A small amount of the unresolved omega/omega-1 hydroxylated eicosatetraenoic acid products were also produced. The enzyme had a specific content of 8.4 nmol of P450/mg of protein and exhibited a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after silver staining. Sequencing revealed a single NH2-terminal amino acid sequence with the first 20 residues identical to rabbit cytochrome P450 2C2. We suggest this enzyme be termed P450 2CAA (for arachidonic acid) until the complete sequence and substrate selectivity are established. Purified P450 2CAA was in the low spin state as evidenced by an absorption maximum at 415 nm; the reduced-carbonyl complex exhibited a maximum at 451 nm. The specific activity for metabolism of 7 microM arachidonic acid was 1.1 nmol of product formed/min/nmol of P450. About 75% of the metabolites were two of the four possible epoxyeicosatrienoic acids identified as the 11,12- and 14,15-epoxyeicosatrienoic acids by coelution with synthetic and commercial standards on reversed and normal-phase high pressure liquid chromatographic separations. The ratio of the 11,12- to 14,15-epoxyeicosatrienoic acids was 1.5:1. The purified enzyme exhibited no significant activity toward 7-ethoxyresorufin or progesterone, but demethylated aminopyrine and benzphetamine. Other fatty acids were also substrates for the enzyme. Oleic, linoleic, and lauric acids, all at about 10 microM, were metabolized at rates of 0.32, 0.72, and 0.73 nmol/min/nmol of P450, respectively. Monoclonal antibody that cross-reacts with P450 2C2 inhibited 63% of the microsomal epoxidation activity from renal cortex microsomes from phenobarbital-treated rabbits. The production of the epoxide metabolites of arachidonic acid suggests that P450 2CAA may have a significant role in arachidonic acid-mediated intra- and intercellular signalling pathways.
Topics: Amino Acid Sequence; Animals; Arachidonic Acids; Chromatography, Affinity; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Fatty Acids, Nonesterified; Kidney Cortex; Kinetics; Microsomes; Molecular Sequence Data; Oxygenases; Rabbits; Sequence Homology, Nucleic Acid; Substrate Specificity
PubMed: 1544930
DOI: No ID Found