SEARCH · Search PubMed

Results for “Benzphetamine”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 109 records · Page 6Linked to original sources

PubMed1989

Protein-protein interactions in microsomal cytochrome P-450 isozyme LM2 and their effect on substrate binding.

The effects of protein-protein interactions and substrate binding on the structure of the active site of rabbit liver microsomal cytochrome P-450 LM2 have been analyzed by resonance Raman spectroscopy of the monomeric and oligomeric protein in solution. Also H2O2-dependent catalytic activities of the two states have been compared. The two vinyl substituents of the heme exhibit different orientations, as indicated by the frequencies and intensities of their stretching vibrations. One group lies in the plane of the heme and remains unchanged in the two states of cytochrome P-450 LM2, the other is tilted out of the plane. The tilting angle in oligomers was smaller than in monomers. These vinyl stretching modes together with some porphyrin modes, were found to be sensitive indicators of the quaternary structure and of substrate binding. In both the oligomer and the monomer, substrate binding causes changes of the relative intensities of some porphyrin modes and the vinyl stretching vibrations which may reflect modifications of the electronic transitions due to hydrophobic interactions between the bound substrate and the heme. In contrast to the monomeric cytochrome P-450 LM2, benzphetamine binding to the oligomers of this isozyme additionally produces a shift of the spin-state equilibrium. This indicates that in the oligomer the substrate-binding pocket is converted by protein-protein interaction to a structure that forces substrates to interfere with the sixth ligands, inducing an increase of the five-coordinated high-spin configuration. In the monomer the substrate-binding pocket can accommodate benzphetamine without affecting the spin state. Binding of imidazole to the monomeric and oligomeric cytochrome P-450 LM2 produces essentially the same resonance Raman spectra. Apparently the replacement of the native sixth ligand by imidazole disturbs the structure of the active site in such a way that it becomes insensitive to protein-protein interactions. H2O2-dependent N-demethylation of benzphetamine and aniline p-hydroxylation by cytochrome P-450 LM2 did not depend on its state of aggregation.

Animals↗

PubMed1990

Hepatic effects of ketoconazole in the male Swiss Webster mouse: temporal changes in drug metabolic parameters.

There have been conflicting observations regarding the effects of ketoconazole on hepatic metabolism. The objectives of these studies were to determine whether ketoconazole was an enzyme inducer or inhibitor in the mouse and then to establish the time frame of these ketoconazole-induced enzyme changes. Ketoconazole was administered (150 mg/kg p.o. X 4 days) to male Swiss Webster mice. Biochemical observations over a period of 6 days following treatment indicated that ketoconazole had a temporal biphasic effect on the liver. Although liver weight and microsomal protein were elevated, all other parameters monitored were lower at 2 h following ketoconazole treatment. At 24 h after the last dose of ketoconazole, hepatic biochemical parameters (liver wt., % liver wt./body wt., microsomal protein, and cytochrome P-450) were statistically elevated, while enzyme activities (benzphetamine N-demethylation, 6 beta- and 7 alpha-hydroxylation of testosterone, formation of androstenedione and UDP-glucuronyltransferase) were inhibited. At 72 h the ketoconazole-induced changes in the hepatic biochemical parameters were comparable to those observed at 24 h, and enzymatic parameters generally appeared to be induced by ketoconazole, with the exception of benzphetamine N-demethylase and UDP-glucuronyltransferase, which exhibited lower enzyme activities. Ethoxyresorufin O-deethylase, 7 alpha-hydroxylation of testosterone and glutathione S-transferase, on the other hand, were unaltered by ketoconazole treatment. The opposing effects of ketoconazole on benzphetamine N-demethylase and ethylmorphine N-demethylase at 72 h were further examined. Enzyme kinetics studies indicated that ketoconazole did not effect the Michaelis constants (Km) of the two substrates, but the maximum velocity (Vmax) of the reactions was altered.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

N-aralkylated derivatives of 1-aminobenzotriazole as isozyme-selective, mechanism-based inhibitors of guinea pig hepatic cytochrome P-450 dependent monooxygenase activity.

The mechanism-based inactivation of hepatic cytochrome P-450 by the suicide inhibitor 1-aminobenzotriazole and two of its derivatives, N-benzyl-1-aminobenzotriazole and N-alpha-methylbenzyl-1-aminobenzotriazole, was investigated in microsomes from untreated, phenobarbital-induced, and beta-naphthoflavone-induced guinea pigs. Microsomal 7-ethoxyresorufin O-deethylase, 7-pentoxyresorufin O-dealkylase, and benzphetamine N-demethylase activities, and cytochrome P-450 content were determined following incubation with 1-aminobenzotriazole and its analogues. The loss of hepatic cytochrome P-450 content and monooxygenase activity was dependent on inhibitor concentration and required NADPH. N-Benzyl-1-aminobenzotriazole and N-alpha-methylbenzyl-1-aminobenzotriazole were more potent inhibitors of monooxygenase activity than the parent compound in microsomes from untreated and phenobarbital-induced guinea pigs. In microsomes from phenobarbital-induced guinea pigs, N-alpha-methylbenzyl-1-aminobenzotriazole (10 microM) was highly selective for the inactivation of the major cytochrome P-450 isozyme catalyzing 7-pentoxyresorufin O-dealkylation (the guinea pig ortholog of P-450IIB1) compared with those isozymes catalyzing 7-ethoxyresorufin O-deethylation or benzphetamine N-demethylation (88 +/- 3% loss of activity vs. 35 +/- 11 and 13 +/- 7%, respectively). N-Benzyl-1-aminobenzotriazole was also selective for the inactivation of 7-pentoxyresorufin O-dealkylase activity, but to a lesser degree (56 +/- 6 vs. 31 +/- 8 and 21 +/- 8%, respectively). In hepatic microsomes from untreated guinea pigs, the two N-substituted analogues were selective for the inhibition of 7-pentoxyresorufin O-dealkylation compared with benzphetamine N-demethylation, but not 7-ethoxyresorufin O-deethylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Cytochrome P-450 spin state and leakiness of the monooxygenase pathway.

1. The monooxygenase and oxidase activities of liver microsomes from phenobarbital (PB)-treated rabbits were investigated for their dependence on the high spin shift (delta alpha) of the ferric cytochrome P-450 induced by a series of benzphetamine analogues. 2. The spin shift activity of the substrate determines, via the first electron transfer kinetics, the steady-state level of the reaction intermediate oxycytochrome P-450. Correlation of the amount or oxycytochrome P-450 with delta alpha can be experimentally proved. 3. The spin-state-dependent formation of oxycytochrome P-450 regulates quantitatively the rates of NADPH oxidation and substrate N-demethylation. Both activities correlate with delta alpha. Oxycytochrome P-450 is substrate-stabilized towards decay with the formation of O2- which, upon dismutation, gives rise to H2O2. 4. The ratio of N-demethylase to NADPH oxidase activity (coupling ratio) also increases with the spin shift, delta alpha. Concomitantly, the proportion of NADPH accounted for by H2O2 and H2O formation via two- and four-electron reduction of dioxygen decreases. This indicates that the substrate-induced structural changes in the enzyme active centre which give rise to spin transition may likewise modify the coupling properties. 5. Perfluorinated compounds, which fail to undergo monooxygenation, fall in line with the benzphetamine derivatives with respect to the dependence of NADPH oxidation rate and steady-state oxycytochrome P-450 level on delta alpha. The increased oxidase activity results mostly in H2O formation. 6. The leakiness of the PB-induced monooxygenase pathway in the biotransformation of oxygen in the presence of the benzphetamines and perfluorinated compounds does not result in marked increases in H2O2 formation. Therefore, the increase of NADPH oxidase activity by these substrates does not significantly enhance H2O2-mediated oxygen tissue toxicity.

Animals↗

PubMed1996

Effects of roxithromycin, erythromycin and troleandomycin on their N-demethylation by rat and human cytochrome P450 enzymes.

1. The effects of treatment of rat with roxithromycin, erythromycin and troleandomycin as well as other chemicals including typical cytochrome P450 inducers were examined in rat and human liver microsomes. 2. Erythromycin and troleandomycin but not roxithromycin caused slight increases in CYP3A1 levels and the N-demethylation of roxithromycin, erythromycin and troleandomycin and oxidation of nifedipine in rat, but none of these chemicals induced significantly CYP2B1 levels or benzphetamine N-demethylation activities. 3. Dexamethasone and pregnenolone 16 alpha-carbonitrile induced CYP3A1 levels and N-demethylation of roxithromycin, erythromycin and troleandomycin but not of benzphetamine, in rat liver microsomes. Treatment of rat with phenobarbital caused increases in both CYP2B1 and 3A1 levels and all of the N-demethylation activities examined. Phenytoin and metyrapone produced increases in contents of 2B1 and activities of benzphetamine N-demethylation as well as of roxithromycin, erythromycin and troleandomycin, although these two inducers did not induce 3A1 protein significantly. 4. In man, a liver sample that was high in CYP3A4 and nifedipine oxidation activity was found to be the most active in N-demethylation activities towards these substrates examined. In addition, recombinant 3A4 catalysed very efficiently the N-demethylation of roxithromycin, erythromycin and troleandomycin in reconstituted monooxygenase systems. 5. These data suggest that erythromycin and troleandomycin, but not roxithromycin, were able to induce CYP3A1 in rat liver microsomes, and that N-demethylation of roxithromycin, erythromycin and troleandomycin were catalysed mainly by 3A1 (and partly by 2B1) in rat and by 3A4 in man.

Animals↗

PubMed1989

Pharmacologic effects of 4-chlorophenol in rats: comparison to clofibrate.

4-Chlorophenol (4-CP) is an identified trace contaminant in commercial clofibrate preparations and the pharmacologic effects of 4-CP have not yet been widely established. We have examined the dose-dependent effects of oral 4-CP and clofibrate administration on selected hepatic parameters and on serum glucose, cholesterol, and triglyceride concentrations in male rats. 4-CP treatment (0.00125-0.08 mmol/kg, twice a day) of rats for 2 weeks increased hepatic microsomal protein (20-30%) and cytochrome P-450 (20-190%) contents without changing liver/body weight ratios. Both 4-CP (0.0025 mmol/kg body wt, twice a day) and CPIB (0.4 mmol/kg body wt, twice a day) treatment to rats for 2 weeks caused significant elevations in microsomal cytochrome P-450 content and in the maximal activities of ethylmorphine, aminopyrine, and benzphetamine N-demethylase, but not in the activity of zoxazolamine 6-hydroxylase. With the same dose of 4-CP, time-dependent increases in hepatic microsomal protein, cytochrome P-450, and the activity of benzphetamine N-demethylase were observed for a 4-week period, and the induction of hepatic microsomal benzphetamine N-demethylase activity by 4-CP was associated with an increased enzyme synthesis. 4-CP treatment produced a marked morphologic change in liver cell ultrastructure, including a proliferation of mitochondria and endoplasmic reticulum at lower 4-CP doses. A clustering of intracellular organelles (mitochondria and endoplasmic reticulum) and a foamy cytoplasm were seen at doses greater than 0.01 mmol/kg, twice a day for 2 weeks, and at 0.0025 mmol/kg, twice a day for greater than 4 weeks. The effects of 4-CP and clofibrate on fasting blood glucose and fasting serum lipid levels were also monitored throughout an 8-week period. Both 4-CP (0.005 mmol/kg body wt, twice a day) and clofibrate (0.2 mmol/kg body wt, twice a day) produced significant elevations in fasting serum glucose levels, but this dosage of 4-CP did not alter serum lipid and lipoprotein parameters, whereas clofibrate significantly reduced serum total cholesterol and high density lipoprotein cholesterol levels. These results lead us to conclude that 4-CP does not contribute to the antilipidemic effects of clofibrate.

Animals↗

PubMed2001

Differential effects of phenobarbital on the constitutive and inducible expression of P450 2B and 3A subfamilies in sheep tissues.

The activity and expression of cytochromes P450 were determined in liver, kidneys, lungs, duodenum, jejunum, ileum, and caecum of adult Lacaune sheep. High expression of total P450, benzphetamine and erythromycin demethylase activities, and P450 2B isoforms, as two distinct proteins that were detected and called P4502 Bm and P4502 Bx, was found in the lungs (in addition to liver). By contrast, the P450 3A subfamily was only expressed in liver and duodenal mucosa of untreated sheep. Phenobarbital (PB) treatment led to significant increases in all measured hepatic parameters and in total P450 of each investigated organ with the exception of ileum and caecum. Benzphetamine demethylase activity increased in liver and kidneys, correlating with the expression of the two P450 2B proteins, which were also induced in duodenum and ileum. By contrast, benzphetamine demethylase activity and expression of the P450 2B isoforms in lungs were unchanged by PB treatment. Erythromycin demethylation activity and P450 3A subfamily expression was increased only in liver of PB-treated sheep.

Animals↗

PubMed1990

Age-dependent toxicity of acorn extract in young and old male rats.

Intraperitoneal administration of acorn extract of dosage levels of 200, 400 and 600 mg/kg body weight did not produce significant change in the hepatic microsomal cytochrome P-450 levels and the activities of NADPH-cytochrome c reductase, benzphetamine N-demethylase and aniline hydroxylase in young, adult rats (weighing 200-250 g), with the exception of the activity of benzphetamine N-demethylase at the 600 mg/kg dose which was decreased significantly. On the other hand, a dose of only 100 mg/kg body weight ip to old rats (weighing 400-450 g) caused significant decreases in the microsomal cytochrome P-450, benzphetamine N-demethylase and NADPH-cytochrome c reductase activities. However, there was no significant change in the activity of aniline hydroxylase in these rats, indicating selective inhibition of the microsomal enzymes and higher susceptibility of old rats than young ones to acorn toxicants. When the serum samples from the treated young rats were analyzed for sorbitol dehydrogenase (SDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities as markers of liver toxicity, these activities were significantly higher in the treated rats than the corresponding control values. Similar changes were noted for old rats receiving a dose of 100 mg/kg body weight of acorn extract. The results indicate that acorn extract affects old rats more than young rats as measured by its effect on liver and liver microsomal enzymes.

Aging↗

PubMedDate not supplied

Reconstitution of the rabbit pulmonary microsomal mixed-function oxidase system from solubilized components.

Lung microsomal cytochrome P-450 was solubilized and purified 2-fold. NADPH-cytochrome c reductase (EC 1.6.2.3) was solubilized and purified 4-6 fold by three methods with use of sonication and detergent digestion followed by either DEAE-cellulose chromatography or ammonium sulfate fractionation. Benzphetamine N-demethylase and 7-ethoxycoumarin deethylase activities were reconstituted when NADPH-cytochrome c reductase and cytochrome P-450 fractions were combined. Reductase fractions prepared by sodium cholate digestion of microsomes were highly active in supporting the hydroxylation activity in the reconstituted systems, whereas those prepared with sodium deoxycholate were not. About twice as much NADPH-cytochrome c reductase was required for saturation of the 7-ethoxycoumarin deethylase activity as for saturation of the benzphetamine N-demethylase activity. A heat-stable lipid fraction was necessary for maximum hydroxylation activity. NADH did not support benzphetamine N-demethylation in the reconstituted system or increase the rate of reaction when added with NADPH.

Animals↗

PubMed1990

The effect of alloxan diabetes on the activity of some mixed function oxidases in male rats.

The effect of alloxan-induced diabetes on the duration of hexobarbital sleep (HB sleep) the activity of ethylmorphine-N-demethylase (EMND), aniline hydroxylase (AH), the content of microsomal cytochrome P-450 and b5, on the activity of ethoxycumarine-0-deethylase (ECOD) and ethoxyresorufine-0-deethylase (EROD) after induction with beta naphthoflavone (beta-NF), as well as the activity of benzphetamine-N-demethylase and pentoxyresorufine-O-dealkylase (PROD) after induction with phenobarbital (PB), was studied in experiments on male Wistar rats. In rats with alloxan diabetes there was a significant prolongation of HB sleep (by 106%) and inhibition of the liver EMND (by 54%), while the AH activity increased by 131%, with a parallel rise in the content of microsomal cytochromes P-450 (by 67%) and b5 (by 113%). In rats with alloxan diabetes the enzyme-inducing effect of beta-NF with respect to the activities of EROD and ECOD is reduced, although diabetes by itself causes a rise in the ECOD activity in untreated animals. When induced with PB, the PROD and benzphetamine-N-demethylase activity in diabetic rats is lower than in the healthy animals. However, if the enzyme activity after the application of inducers is referred to the respective starting enzyme activities of the two groups of animals, it is found that the enzyme-inducing effect of PB is preserved and even slightly potentiated in the diabetic rats compared with the healthy ones: the increases in the benzphetamine-N-demethylase activity is by 60% in the diabetic rats, compared with a rise of 28% in the healthy animals, of the PROD activity 19 times for the diabetic compared with 16 times increase for the healthy rats.

Animals↗

PubMedDate not supplied

6-Fluoro-2-methylspiro(chroman-4,4'-imidazolidine)-2',5'-dione and related compounds as inducers of monooxygenase in rat liver microsomes.

The relationship between the structure of spirohydantoin derivatives and their inducing effects on hepatic monooxygenase system was studied. At the dose of 1 mumol/kg (0.3 mg/kg), 6-fluoro-2-methylspiro(chroman-4,4'-imidazolidine)-2',5'-dione (M79175) was found to exhibit an inducing effect on benzphetamine N-demethylase. On the contrary, the inducing effect of Sorbinil, in which the 2-methyl group on the chroman ring of M79175 was replaced by hydrogen atom, on benzphetamine N-demethylase was 100 times less than that of M79175. The substitution of the methyl group of M79175 with the dimethyl group did not affect the inducing effect, whereas the substitution with the hexyl group resulted in the loss of the inducing effect on drug oxidation activities tested at a dose of 10 mg/kg. Furthermore, cyclohexane spiro-2,6-chloro-1'-(3-dimethylaminopropyl)spiro(chroman-4,4'-imidazolid ine)-2' , 5'-dione (M79193) had an inducing effect on total cytochrome P-450 content, but had no inducing effect on benzphetamine N-demethylase. In addition, M79175 was found to induce cytochrome P-450 which is immunochemically related to one of the major forms of phenobarbital-inducible cytochrome P-450 (P-450(PB-1], but not cytochrome P-450 which is immunochemically related to the major form of 3-methylcholanthrene-inducible cytochrome P-450 (P-450(MC-1]. On the other hand, M79193 was found to induce neither P-450(PB-1) nor P-450(MC-1).

Animals↗

PubMed1988

The effect of NADPH concentration on the reduction of cytochrome P-450 LM2.

Cytochrome P-450 LM2 reduction was measured at a series of NADPH concentrations in the absence of substrate and in the presence of 1 mM benzphetamine. In the absence of substrate reduction could be described as a biphasic process with 55% of the reaction occurring in the first phase (at 20 microM NADPH). When benzphetamine was present, the fraction of the reaction occurring in the first phase was increased to 91%. When examined either in the absence or presence of benzphetamine, the rate constant and fraction of LM2 reduced in the fast phase were decreased as the NADPH concentration was decreased. In each case the fraction of LM2 reduced in the second phase was not substantially altered over the NADPH concentrations examined. To explain the effect of NADPH concentration on the initial rate of LM2 reduction, the effect of NADPH on the reduction of NADPH-cytochrome P-450 reductase was examined. Due to the presence of two flavins within each reductase molecule, there would be nine possible oxidation-reduction states of the reductase which may be present at a given NADPH concentration. Based on the redox potentials for the flavin half-reactions and for NADPH oxidation, the relative concentrations of each of the reductase subspecies could be determined. Rate constants were assigned for the reduction of LM2 by the various reductase subspecies, and the theoretical initial rates of LM2 reduction at various NADPH concentrations were compared with values obtained experimentally. The experimental data are consistent with a model where, under the conditions of this assay, the fully reduced reductase is the form primarily responsible for the reduction of LM2.

Animals↗

PubMed1988

Separation and characterization of hepatocytes from immature and adult rats into distinct subpopulations by centrifugal elutriation.

Hepatocytes were isolated from immature and adult rat liver by retrograde perfusion with calcium free buffer, followed by enzymic digestion, and separated into subpopulations by centrifugal elutriation. Several subpopulations with increasing cell diameters were distinguished. The smaller cells were attributed to the periportal area, the larger ones to the perivenous (centrilobular) region. Profiles of total cytochrome P-450 concentration, benzphetamine N-demethylation and ethoxyresorufin O-deethylation, NADPH-cytochrome c-reductase, glucose-6-phosphatase and glutamate-pyruvate-transaminase activities were determined in all subpopulations. With adult hepatocytes an increasing cytochrome P-450 concentration with increasing cell diameter (increasing from periportal to perivenous hepatocytes) could be observed, paralleled by increasing activities of benzphetamine N-demethylation and ethoxyresorufin O-deethylation activities. While NADPH-cytochrome c-reductase did not show a distinct zonation, glucose-6-phosphatase and glutamate-pyruvate-transaminase revealed increasing activities with increasing cell diameter. Immature hepatocytes (rats aged 11-15 days) were smaller, and more fragile. They could not be isolated with the same enzyme solution as adult hepatocytes and they did not show any zonation of cytochrome P-450 concentration, although the zonation of benzphetamine N-demethylation and ethoxyresorufin O-deethylation was almost fully developed. For NADPH-cytochrome c-reductase a zonation with higher activities in the perivenous cells could be demonstrated, in contrast to the lack of zonation in adult rats. Glucose-6-phosphatase activity showed a decline with increasing cell diameter in immature hepatocytes, whereas glutamate-pyruvate-transaminase activity did not show any zonation. In rats aged 20 days the zonation of these parameters in liver was in between that of younger and older animals. Zonation of the liver lobule develops postnatally with individual patterns for the different parameters.

Aging↗

PubMed1988

[Comparison of cytochrome P-450 forms induced by phenobarbital and 1,4-bis[3,5-dichloropyridyloxy)]benzene in the mouse and rat liver].

A form of cytochrome P-450 (P-450PB) with a molecular weight of 53.5-54.0 kD possessing a high benzphetamine-N-demethylase activity (100-120 nmol formaldehyde/min/nmol cytochrome) was isolated from liver microsomes of phenobarbital-induced C57Bl/6 mice. This cytochrome P-450 form is immunologically identical to its rat liver counterpart-P-450b (Mr = 52 kD) which is also characterized by a high rate of benzphetamine-N-demethylation. It was shown that 1.4-bis[2-(3.5-dichloropyridyloxy])benzene (TCPOBOP) induces in mouse liver the synthesis of the monoxygenase form whose substrate specificity and immunologic properties are identical to those of cytochromes P-450PB and P-450b. The immunochemically quantitated content of this form makes up to 20% of the total P-450 pool in liver microsomes of phenobarbital- or TCPOBOP-induced mice. Immunochemical analysis of microsomes with the use of antibodies to cytochromes P-450PB and P-450b revealed the presence on the electrophoregrams of phenobarbital-induced rat liver microsomes of two immunologically identical forms of cytochrome P-450, i.e., P-450b and P-450e (the latter had a low ability to benzphetamine N-demethylation). Liver microsomes of phenobarbital- or TCPOBP-induced mice gave only one precipitation band corresponding to cytochrome P-450PB.

Animals↗

PubMed1985

Chemical characterization of protein-protein interactions between cytochrome P-450 and cytochrome b5.

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.

Animals↗

PubMed1982

Relationship between state of aggregation and catalytic activity for cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase.

The detergent 1-O-n-octyl-beta-D-glucopyranoside (octylglucoside) was found to replace the phospholipid requirement in the demethylation of benzphetamine by cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase purified from phenobarbital-treated rabbit liver. At low enzyme concentration (0.1 microM) in the absence of glycerol and phosphate, the maximum rate of benzphetamine-specific NADPH oxidation was approximately 35% of that observed in the presence of dilauroylglyceryl-3-phosphoryl choline. At higher enzyme concentration (2.5 microM) and in the presence of 0.15 M phosphate, 20% glycerol, octylglucoside was as effective as phospholipid in stimulating the production of formaldehyde from benzphetamine. The detergent concentration required for maximal enzymatic activity was 2.5-4.0 g/liter, depending on the cytochrome preparation used. At higher octylglucoside concentrations (5-7 g/liter), activity decreased to zero, although neither enzyme appeared to be irreversibly denatured at these detergent concentrations. Sedimentation equilibrium experiments with P-450LM2 alone or in the presence of equimolar reductase showed that increasing octylglucoside levels promoted disaggregation of the cytochrome. Pentamers and hexamers predominated at detergent concentrations where maximal activity was observed, while higher levels of detergent where activity was absent produced cytochrome dimers and, ultimately, monomers. The reductase was monomeric at detergent levels between at least 3 and 7 g/liter. Moreover, both gel filtration and sedimentation equilibrium experiments demonstrated that a stable complex between P-450LM2 and its reductase was not formed at octylglucoside concentrations where high activity was evident. These results are consistent with a model of P-450/reductase interaction in which functional aggregates of three to six cytochrome polypeptides move laterally in the microsomal membrane and interact with the reductase by random collision.

Animals↗

PubMed1983

Kinetics and mechanism of CO binding to cytochromes P-450LM2 and P-450LM4. Effect of phospholipid, nonionic detergent, and substrate binding.

The kinetics of carbon monoxide binding to purified, soluble cytochrome P-450LM2 from liver microsomes of phenobarbital-induced rabbits and P-450LM4 from liver microsomes of 5,6-benzoflavone-induced rabbits was determined in the presence of catalytically essential phospholipid, d-benzphetamine or nonionic detergent (0.1% Tween 80, 0.1% Triton N-101, or 0.3% n-octylglucoside). The kinetics under these conditions was consistent with a two-step binding mechanism consisting of bimolecular association followed by a unimolecular rearrangement step. However, when d-benzphetamine was added to P-450LM2 in the presence of sonicated dilauroylglyceryl-3-phosphorylcholine (0.15 mM) or any of the above detergents, CO binding followed simple second order kinetics with a bimolecular rate constant of approximately 3 x 10 M-1 s-1 at 20 degrees C. P-450LM4-CO binding kinetics was not affected by concurrent addition of phospholipid and d-benzphetamine. These results imply that simultaneous binding of amphiphile and substrate induces a conformation of reduced P-450LM2 which is different from that found when either substance is bound alone.

Animals↗

PubMed1982

Drug-metabolizing enzymes in human foetal liver: partial resolution of multiple cytochromes P 450.

Cytochrome P 450 concentration, related monooxygenase activities (towards aniline, p-nitroanisole, benzphetamine, ethoxycoumarin, benzo(a)pyrene, testosterone, and dehydroepiandrosterone), epoxide hydrolase, and glutathione S-transferase activities were measured in the liver of human foetuses aged from 15 to 38 weeks and compared to adult activities. Different ontogenic patterns seem to exist between monooxygenase activities: if the overall cytochrome P 450 concentration, aniline hydroxylase, benzphetamine demethylase, epoxide hydrolase, and glutathione S-transferase activities reach about the half of adult values as early as 15-25 weeks of gestational age, the metabolism of benzo(a)pyrene, ethoxycoumarin, and testosterone in position 6 beta is very low in these foetuses, whereas the 16 alpha hydroxylation of dehydroandrosterone is higher than in adult human liver. Foetal and adult cytochromes P 450 were resolved by DEAE cellulose chromatography into three different fractions: in reconstitution experiments, the major fraction (A) was active toward aniline, whereas benzphetamine and ethoxycoumarin were mainly metabolized by the two other fractions (Ba and Bb). Results show that multiple cytochromes 450 are present in foetal liver and are able to catalyze, with a lower molecular activity, the same reactions as in adult human liver.

Aryl Hydrocarbon Hydroxylases↗