Rabbit Microsomes Research Articles

Spectral interaction of 11-hydroxy-delta-8-tetrahydrocannabinol with rabbit and rat liver microsomal cytochrome P-450.

11-Hydroxy-Δ8-tetrahydrocannabinol, which is an active metabolite of Δ8-tetrahydrocannabinol, produced a spectral change on interacting with liver microsomes of rabbits and rats (λmax 395 nm ; λmin 428 nm). The apparent dissociation constants (Ks) were found to be 402.1 and 76.6 μM for microsomes of untreated rabbits and rats, respectively. Pretreatment with phenobarbital enhanced the spectral change caused by 11-hydroxy-Δ8-tetrahydrocannabinol with microsomes of both species, as well as increasing the cytochrome P-450 content. Furthermore, 11-hydroxy-Δ8-tetrahydrocannabinol interfered with the spectral change caused by Δ8-tetrahydrocannabinol or hexobarbital, but not with that caused by aniline. These results are consistent with the view that 11-hydroxy-Δ8-tetrahydrocannabinol is further metabolized by the microsomal monooxygenase system involving cytochrome P-450.

UDP-glucuronyltransferase activities of rabbit liver microsomes for cannabinoids.

UDP-glucuronyltransferase activities for delta 8-tetrahydrocannabinol, 11-hydroxy-delta 8-tetrahydrocannabinol, cannabidiol and cannabinol were examined using liver microsomes of rabbits. The activities for cannabidiol and cannabinol were 2.5 and 19 times higher, respectively, than those for delta 8-tetrahydrocannabinol and 11-hydroxy-delta 8-tetrahydrocannabinol. Thus, the present study supports the view that glucuronide formation plays an important role in the metabolism of cannabinol, but does only a minor role in that of delta 8-tetrahydrocannabinol and 11-hydroxy-delta 8-tetrahydrocannabinol.

Interaction of 9-hydroxyellipticine with two forms of partially purified rabbit lung cytochrome P-450. Inhibition of lung microsomal benzo[a]pyrene hydroxylase.

9-Hydroxyellipticine (9-OHE), a potent inhibitor of rat liver monooxygenase activities, binds to the various forms of partially purified lung cytochromes P-450 from untreated and 3-methylcholanthrene (3-MC)-treated rabbits. The spectral data (λ max: 428 nm (ox.), 447 nm (red.), K s: 10 μM and 5 μM for cytochrome I and cytochrome II from 3-MC-treated rabbits respectively) resemble those obtained with cytochrome P-450 purified from liver of Aroclor 1254-pretreated rats (λ max: 428 nm (ox.), 445 nm (red.), K s: 8 μM). 9-OHE has been shown to inhibit the benzo[ a]pyrene hydroxylase activity of rat and rabbit lung microsomes. The inhibitory effect was higher towards the 3-MC-induced lung microsomes than with the control microsomes. However, the lung microsomes, as well as the liver microsomes of rabbits were less sensitive to inhibition by 9-OHE than the corresponding microsomes from rats. These results suggest that rabbit and rat cytochromes P-450 have subtle structural differences.

Isolation and incorporation of rabbit liver epoxide hydrase into phospholipid vesicles.

Methods are described for the incorporation into phospholipid vesicles of epoxide hydrase isolated from liver microsomes of phenobarbital-treated rabbits. Chromatography on a Sephadex G-50 column of epoxide hydrase and egg yolk phosphatidylcholine treated with sodium cholate yielded homogeneous vesicles with a diameter of about 25 nm and containing 80 to 85% of the protein applied. At high substrate concentrations, the vesicles catalyzed the hydration of benzo(a)pyrene-4,5-oxide and styrene-7,8-epoxide at a rate similar to that obtained with the enzyme in a soluble form. However, the kinetics of styrene glycol formation catalyzed by the vesicular or microsomal preparations were complex. Convex Lineweaver-Burk plots and concave Hill plots were obtained, whereas normal Michaelis-Menten kinetics characterized the hydration catalyzed by the enzyme in a soluble form. The results could be explained if reconstitution of the enzyme into the vesicles gives rise to low affinity high capacity sites for the substrate on the enzyme, or alternatively facilitates the interaction of the substrate with such sites already present. It is suggested that reconstituted liposomes containing both the liver microsomal hydroxylase system and epoxide hydrase may prove to be a good model system for evaluating substrate specificity and factors of importance in the formation of toxic and carcinogenic metabolites by these enzymes.

Chemical and biological studies of 1-(2,5-dihydroxy-4-methylphenyl)-2-aminopropane, an analog of 6-hydroxydopamine

Autoxidation of the bis(O-demethyl)-p-hydroquinone metabolite of the psychotomimetic amine 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM) at pH 7.4 leads exclusively to a bicyclic imino quinone. This imino quinone is a good alkylating agent, forming covalent adducts via 1,4 addition to thiols. The autoxidation appears to be dependent on trace metal catalysis and is dramatically inhibited by components of the 10000g supernatant fraction of rabbit liver homogenates. Incubation of tritium-labeled hydroquinone with bovine serum albumin under oxidizing conditions leads to significant amounts of nonextractable radioactivity which presumably is dependent on imino quinone alkylation of nucleophilic functionalities present on macromolecules. Incubation of tritium-labeled DOM with rabbit microsomes in the presence of NADPH leads to irreversible binding of the label to macromolecular components of the microsomes. Since this binding is NADPH dependent, it is likely that metabolic conversion of DOM to the hydroquinone is involved. The imino quinone oxidation product is highly lypophilic and is capable of crossing the blood-brain barrier. Intravenous administration of tritium-labeled imino quinone to rats resulted in significant nonextractable radioactivity in brain tissue. These properties of the hydroquinone metabolite parallel those reported for the structurally related sympatholytic compound 6-hydroxydopamine and have led to the hypothesis that the psychotomimetic properties of DOM may be mediated through 6-hydroxydopamine-type interactions of the hydroquinone with important macromolecules in the brain.

Interaction between NADPH-cytochrome p-450 reductase and cytochrome p-450 in the membrane of phosphatidylcholine vesicles

Cytochrome P-450 and NADPH-cytochrome P-450 reductase, both purified from liver microsomes of phenobarbital-pretreated rabbits, have been incorporated into the membrane of phosphatidylcholine vesicles by the cholate dialysis method. The reduction of cytochrome P-450 by NADPH in this system is biphasic, consisting of two first-order reactions. The rate constant of the fast phase, in which 80–90% of the total cytochrome is reduced, increases as the molar ratio of the reductase to the cytochrome is increased at a fixed ratio of the cytochrome to phosphatidylcholine, suggesting that the rate-limiting step of the fast phase is the interaction between the reductase and the cytochrome. The rate constant of the fast phase also increases when the amount of phosphatidylcholine, relative to those of the two proteins, is decreased. This latter observation suggests that the interaction between the two proteins is effected by their random collision caused by their lateral mobilities on the plane of the membrane of phosphatidylcholine vesicles. The rate constant of the slow phase as well as the fraction of cytochrome P-450 reducible in the slow phase, on the other hand, remains essentially constant even upon alteration in the ratio of the reductase to the cytochrome or in that of the two proteins to phosphatidylcholine. No satisfactory explanation is as yet available for the cause of the slow-phase reduction of cytochrome P-450. The overall activity of benzphetamine N-demethylation catalyzed by the reconstituted vesicles responds to changes in the composition of the system in a similar way to the fast-phase reduction of cytochrome P-450, though the latter is not the rate-limiting step of the overall reaction.

Biosynthesis of phosphatidic acid by liver and lung of maternal and fetal rabbits

1. 1. The synthesis of phosphatidic acid from labelled precursors, [ 3H]palmitic acid and [ 14C]glycerol 3-phosphate. was studied in microsomes of liver and lung from maternal and fetal rabbits. 2. 2. The rate of in vitro synthesis of phosphatidic acid by liver microsomes of pregnant rabbits was in good agreement with the levels of plasma glycerolipids. 3. 3. It agrees with previous data on the biosynthesis of phosphoglycerides and pulmonary surfactant during the perinatal development of the lung. 4. 4. On the other hand. metabolic maturity of liver in order to synthesize phosphatidic acid was achieved during the postnatal period.

The formation of phenylethane-1,2-diol 2-acetate in the metabolism of styrene oxide by rabbit liver microsomes in vitro.

1. Phenylethane-1,2-diol 2-acetate has been isolated from the incubation of styrene oxide or styrene diol with liver microsomes of phenobarbital-pretreated rabbits. 2. Observed ratios of phenylethane-1,2-diol to its 2-acetate in the incubation mixtures ranged between 19 : 1 and 5 : 1 with different microsomal preparations and different periods of incubation. 3. The formation of phenylethane-1,2-diol 2-acetate may be a source of errors in the radiometric determination of epoxide hydrase activity in microsomes.

Resonance Raman studies of hepatic microsomal cytochromes P-450: evidence for strong pi basicity of the fifth ligand in the reduced and carbonyl complex forms.

Resonance Raman spectra have been measured for cytochromes P-450 purified from liver microsomes of phenobarbital-treated rabbits (PB P-450 and PB P-448) and of 3-methylcholanthrene-treated rabbits (MC P-448). In the reduced state, all three cytochromes P-450 exhibit Raman spectra of ferrous high-spin type but show the so-called "oxidation state marker" (band IV) at unusually low frequencies, indicating extensive delocalization of electrons from the iron dpi orbital to the porphyrin II (eg) orbital and, consequently, the strong pi basicity of the fifth ligand of the heme iron. The reduced CO complexes of the cytochromes P-450 also exhibit band IV at markedly lower frequencies than CO complexes of hemoglobin and myoglobin. These anomalies observed for the reduced form and CO complex disappear upon conversion of the cytochromes to the catalytically inactive form called cytochrome P-420. Oxidized PB P-450 shows a Raman spectrum which is characteristic of typical ferric low-spin heme compounds, whereas those of PB P-448 and MC P-448 are of the ferric high-spin type. PB P-450 is also clearly distinguishable from the two P-448 preparations in the reduced state. The reduced form of cytochrome P-420, produced by laser illumination, exhibits two sets of Raman lines and, therefore, seems to be a mixture of both high- and low-spin species.

Multiple forms of cytochrome P-450: Resolution and purification of rabbit liver aryl hydrocarbon hydroxylase

We describe the resolution and partial purification of two minor forms of cytochrome P-450 from liver microsomes of rabbits treated with 2,3,7,8-tetrachlorodibenzo- p -dioxin. Both forms have different electrophoretic mobilities when compared to the major form of cytochrome P-450 isolated from this source. The two cytochromes show different activities with several substrates. One form is very active in the hydroxylation of benzo( a )pyrene when reconstituted with highly purified NADPH-cytochrome P-450 reductase.

Resolution of two forms of cytochrome P-450 from liver microsomes of rabbit treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Two forms of cytochrome P-450 with different substrate specificities were isolated from liver microsomes of rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). A specific antibody was produced toward the major form of the cytochrome. The antibody inhibits microsomal acetanilide hydroxylation (80%). It does not cross-react with the minor fraction of the cytochrome or inhibit the hydroxylation of 3,4-benzpyrene or coumarin, the N-demethylation of aminopyrine or the O-deethylation of 7-ethoxycoumarin catalyzed by rabbit liver microsomes. The major form has an estimated Mr = 54,000 and displays an n-octylamine difference spectrum with an absorption maximum at 426 nm and a minimum at 391 nm. When reconstituted, this cytochrome catalyzes acetanilide hydroxylation at a higher rate than microsomes or the minor fraction. The n-octylamine difference spectrum of the minor fraction displays an absorption maximum at 431 nm and a minimum at 410 nm. When reconstituted, this fraction catalyzes the hydroxylation of 3,4-benzpyrene and the O-deethylation of 7-ethoxycoumarin. The two cytochromes appear to be distinct entities and function in different catalytic pathways.

ENHANCEMENT OF NADPH-DEPENDENT LIPID PEROXIDATION ACTIVITY BY ETHYLENE-DIAMINETETRAACETIC ACID (EDTA) IN THE PRESENCE OF FERROUS ION IN RABBIT LIVER MICROSOMES

The addition of EDTA to the incubation mixture containing rabbit liver microsomes and ferrous ion resulted in 2-fold increase of lipid peroxidation activity. Such an enhancement was not observed in rat liver microsomes. The maximum lipid peroxidation activity seen in rabbit microsomes in the presence of EDTA and ferrous ion was about 80% that seen in rat liver microsomes. From these results, it is likely that low lipid peroxidation activity in rabbit liver microsomes may account for the insufficiency of an EDTA-LIKE FACTORS(S) IN RABBIT LIVER MICROSOMES.

Irreversible binding of 3-14C-antipyrine to hepatic protein in vivo and in metabolizing liver microsomes.

After i.p. injection of 3-14C-antipyrine (10 micronmole = 1.9 mg with 10 micronCi per 10 g of body weight) to mice radioactivity was irreversibly bound to liver proteins. The irreversible binding reached maximal values of 0.15 nmole/mg protein in liver microsomes after 30-60 min. During 60 min incubation with liver microsomes of mice and rabbits (phenobarbital pretreated) and a NAKPH-regenerating system 3-14C-antipyrine was irreversibly bound to microsomal protein at a rate of 1.5 nmole/mg protein (mouse) and 3 nmole/mg protein (rabbit). In identical incubates with rabbit liver microsomes the 4-hydroxylation of antipyrine was 24 nmole/mg protein in 60 min and formaldehyde production from antipyrine 3 nmole/mg protein in 60 min. In incubates with rabbit liver microsomes the binding rate was 80-90% inhibited by 1mM metyrapone, SKF 525-A and trichloropropene epoxide respectively; 4-hydroxylation was 70-80% inhibited by the same substances. In the presence of 1mM GSH, cysteine or ethylene diamine binding was 30-40% inhibited, whereas 4-hydroxylation showed no inhibition.

103) - Studies on the drug-metabolizing activites of cytochrome P-450S separated from liver microsomes of phenobarbital-treated rabbits; pH curve and Lineweaver-Burk plot

103) - Studies on the drug-metabolizing activites of cytochrome P-450S separated from liver microsomes of phenobarbital-treated rabbits; pH curve and Lineweaver-Burk plot

CAUSE OF DECREASE OF ETHYLMORPHINE N-DEMETHYLASE ACTIVITY BY LIPID PEROXIDATION IN MICROSOMES FROM THE RAT, GUINEA PIG AND RABBIT

There were marked differences among animal species between NADPH-dependent and ascorbic acid-Fe++-dependent lipid peroxidation. In NADPH-dependent lipid peroxidation, this activity occurred to the greatest extent in rats followed by guinea pigs and rabbits and such was much lower in rabbits than in guinea pigs. On the other hand, rabbit microsomes exhibited higher lipid peroxidation activity than guinea pigs in ascorbic acid plus Fe++ or Fe++-dependent lipid peroxidation although the activity was still lower than in rats. The ascorbic acid plus Fe++-stimulated lipid peroxidation produced a decrease in ethylmorphine N-demethylase activity which was closely related to ethylmorphine-enhanced NADPH-cytochrome P-450 reductase activity but was not related to the change of the apparent content of cytochrome P-450 in all animal species. These results indicate that decrease of NADPH-cytochrome P-450 reductase activity induces a decrease in ethylmorphine N-demethylase activity by lipid peroxidation.

The effect of pesticides on hydroxylation of testosterone in hepatic microsomes of rabbits.

L'hydroxylation de la testostérone par les microsomes du foie de lapins adultes est inhibée par l'hexachlorobenzène, par le 0,0-diméthyle-0-(3-méthyle-4-nitrophényle) thiophosphate et par le phosphate éthylmercurique administrés par voie orale, tous les deux jours, pendant 5 mois.

Comparisons of the formation of cytochrome P-450 complexes absorbing at 455 nm in rabbit and rat microsomes

The formation of 455 nm complexes during the metabolism of various drugs has previously been studied in the rat. The formation of 455 nm complexes during the metabolism of amphetamine congeners and other compounds has now been studied in the rabbit to determine the differences that exist between these two species. Rabbit microsomes, in contrast to those from rat, show stereochemical preference for the l-isomer over the d-isomer of amphetamine. In both species, benzyl and hydroxyl substitutions on the nitrogen increased the rate of 455 nm complex formation, while methyl substitution did not. Much lower rates of 455 nm complex formation from SKF 525-A and propoxyphene were observed in rabbit microsomes than in rat microsomes. Both rat and rabbit were incapable of forming a 455 nm complex from p-hydroxyamphetamine. These and other differences in 455 nm complex formation parallel, in part, the qualitative and quantitative differences observed in the metabolism of these compounds in the two species.

Preparation and Properties of Partially Purified Cytochrome P-450 and Reduced Nicotinamide Adenine Dinucleotide Phosphate-Cytochrome P-450 Reductase from Rabbit Liver Microsomes

The liver microsomal enzyme system which hydroxylates fatty acids, hydrocarbons, and a variety of drugs and other foreign compounds was previously solubilized and resolved into three components: cytochrome P-450, NADPH-cytochrome P-450 reductase, and phospholipid. The two enzymes were partially purified from pyrophosphate-treated, cholate-solubilized liver microsomes of phenobarbital-treated rabbits by fractionation with polyethylene glycol 6000 and DEAE-cellulose column chromatography in the presence of Renex-690, a nonionic detergent. These steps were followed by treatment with Amberlite XAD-2 and calcium phosphate gel. Cytochrome P-450 preparations purified to a content as high as 15 nmoles per mg of protein were free of cytochrome b5 and contained no significant amount of NADPH-cytochrome c reductase, NADH-cytochrome c reductase, nonheme iron, other metals as measured by neutron activation analysis, or labile sulfide. The molecular weight was judged to be about 280,000 by gel exclusion chromatography and sucrose density gradient centrifugation. Polyacrylamide gel electrophoresis of the partially purified cytochrome P-450 preparation treated with sodium dodecyl sulfate and mercaptoethanol showed the presence of two major polypeptide bands, one with a molecular weight of 47,000 to 49,000 and the other 52,000 to 53,000. The component of lower molecular weight corresponded to the major polypeptide induced by phenobarbital treatment, as shown by electrophoresis of the proteins in normal and induced microsomes. The partially purified preparation accepted 2 electrons from dithionite per molecule of cytochrome P-450 under anaerobic conditions, indicating the presence of another electron acceptor in addition to the iron atom of the hemeprotein. The partially purified NADPH-cytochrome P-450 reductase preparations, which catalyzed the reduction of 8.6 to 10.0 µmoles of cytochrome c per min per mg of protein at 30°, were free of cytochrome P-450 and contained only a trace of NADH-cytochrome c reductase. FMN and FAD were found to be present in equimolar amounts, and labile sulfide was absent. The reconstituted enzyme system was active in the hydroxylation of fatty acids (laurate), hydrocarbons (hexane, cyclohexane, and octane), drugs (benzphetamine, hexobarbital, and ethylmorphine), and aniline. The presence of both phosphatidylcholine and deoxycholate was necessary for maximal activity.

A gel-electrophoretically homogeneous preparation of cytochrome P-450 from liver microsomes of phenobarbital-pretreated rabbits

Cytochrome P-450 was purified from liver microsomes of phenobarbital-pretreated rabbits to a specific content of 16 to 17 nmoles per mg of protein with a yield of about 10 %. The purified cytochrome yielded only a single protein band on sodium dodecylsulfate-urea-polyacrylamide gel electrophoresis, and an apparent molecular weight of about 45,000 was estimated for the protein. The preparation was free of cytochrome b 5 , NADH-cytochrome b 5 reductase, and NADPH-cytochrome c reductase activities. Aniline hydroxylase and ethylmorphine N-demethylase activities could be reconstituted upon mixing the purified cytochrome with an NADPH-cytochrome c reductase preparation (purified by a detergent method) and phosphatidyl choline.

Cytochrome P-450 purified to apparent homogeneity from phenobarbital-induced rabbit liver microsomes: Catalytic activity and other properties

Cytochrome P-450 was purified to a content of over 17 nmoles per mg of protein from liver microsomes of phenobarbital-treated rabbits by fractionation with polyethylene glycol 6000, DEAE-cellulose column chromatography, and hydroxylapatite column chromatography in the presence of Renex 690, a nonionic detergent. The purified preparation exhibited a single polypeptide band (molecular weight, 49,000 daltons) when submitted to SDS-polyacrylamide gel electrophoresis. Cytochromes P-420 and b 5 and NADPH-cytochrome c reductase were absent. The reconstituted system containing purified cytochrome P-450, reductase, and phosphatidylcholine catalyzed the hydroxylation of benzphetamine, cyclohexane, aniline, and laurate.