B-type cytochromes in plasma membranes isolated from rat liver, in comparison with those of endomembranes

Fractions of plasma membranes, Golgi apparatus, endoplasmic reticulum (ER), and nuclear envelope were isolated from rat liver and were characterized by electron microsocpe and biochemical methods. The purity of the fractions was controlled by morphometry and by marker enzyme activities. Amounts of cytochromes b5, P-450, and P-420 were measured, as well as the NADPH- and NADPH-cytochrome c reductase activities. The pigments of the microsomal electron transport system were found in all membrane fractions in relatively high amounts, thus excluding an origin by microsomal contamination. Purified preparations of plasma membrane and Golgi apparatus contained approximately 30% of the cytochrome b5 and cytochrome P-450 + P-420 found in ER membranes. Plasma membranes were also characterized by a high ratio of P-420/450. Degradation of cytochromes P-450 and P-420 was relatively rapid in all fractions, except in the ER. Cytochrome b5 extracted from plasma membranes was spectrophotometrically and enzymatically indistinguishable from ER cytochrome b5. However, immunnlogical characterization with rabbit antibodies against the trypsin-resistant core of microsomal cytochrome b5 showed the presence of at least two types of cytochrome b5 in ER membranes, in contrast to the plasma membranes in which only one of these components was detected. This immunological differentiation also demonstrates that the plasma membrane-bound cytochrome b5 is endogenous to this membrane and does not reflect contamination by ER elements. We conclude that cytochromes b5, P-450, and P-420 are not confined only to ER and nuclear membranes but also occur in signficant amounts in Golgi apparatus and plasma membranes. The findings are discussed in relation to observations of similar redox components in Golgi apparatus, secretory vesicles, and plasma membranes of other cells.     

Evidence for a sex pheromone metabolizing cytochrome P-450 mono-oxygenase in the housefly

Direct evidence is presented for the role of a cytochrome P-450 monooxygenase (called mixed-function oxidase, or polysubstrate mono-oxygenase, PSMO) in the metabolism of the sex pheromone (Z)-9-tricosene to its corresponding epoxide and ketone in the housefly. A secondary alcohol, most likely an intermediate in the conversion of the alkene to the ketone, was also tentatively identified. The results of in vivo and in vitro experiments showed that the PSMO inhibitors, piperonyl butoxide (PB) and carbon monoxide, markedly inhibited the formation of epoxide and ketone from (9,10-³H) (Z)-9-tricosene. An examination of the relative rates of (Z)-9-tricosene metabolism showed that males exhibited a higher rate of metabolism than females with the antennae of males showing the highest activity of any tissue/organ examined. The major product from all tissues/organs was the epoxide. Data from experiments with subcellular fractions showed that the microsomal fraction had the majority of enzyme activity, which was strongly inhibited by PB and CO and required NADPH and O₂ for activity. A carbon monoxide difference spectrum with reduced cytochrome showed maximal absorbance at 450 nm and allowed quantification of the cytochrome P-450 in the microsomal fraction of 0.410-nmol cytochrome P-450 mg^?1 protein. Interaction of (Z)-9-tricosene with the cytochrome P-450 resulted in a type I spectrum, indicating that the pheromone binds to a hydrophobic site adjacent to the heme moiety of the oxidized cytochrome P-450.     

Dimethylnitrosamine demethylation by reconstituted liver microsomal cytochrome P-450 enzyme system.

Oxidative demethylation of dimethylnitosamine was studied with both reconstituted and unresolved liver microsomal cytochrome P-450 enzyme systems from rats and hamsters. Proteinase treatment of liver microsomal preparations yielded cytochrome P-450 particulate fractions. Both cytochrome P-450 and NADPH- cytochrome c reductase fractions were required for optimum demethylation activity. Particulate cytochrome P-450 fractions were more effecient than either Triton X-100- or cholatesolubilized preparations of these particles in demethylation activity with rat and hamster liver preparations appear to be due to differences in specificity in their cytochrome P-450 fractions.     

The cytochromes in microsomal fractions of germinating mung beans.

Detailed studies of microsomal cytochromes from mung-bean radicles showed the presence of cytochrome P-420, particularly in dark-grown seedlings, accompanied by smaller quantities of cytochrome P-450. Similar proportions of cytochrome P-420 to cytochrome P-450 were found spectrophotometrically in vivo with whole radicles and hypocotyls. Assayed in vitro, maximum concentrations of both cytochromes were attained after 4 days of growth, before undergoing rapid degradation. Illumination of seedlings stabilized cytochrome P-450 and decreased the amount of cytochrome P-420. Three b cytochromes were present in the microsomal fraction, namely cytochromes b-562.5 (Em + 105 +/- 23 mV), b-560.5 (Em + 49 +/- 13 mV) and b5 (Em - 45 +/- 14 mV), all at pH 7.0. Of the b cytochromes, cytochrome b5 alone undergoes a rapid degradation after day 4, Changes in cytochrome b concentrations were confined to the microsomal fraction: mitochondrial b cytochrome concentrations were unaltered with age. Protohaem degradation (of exogenous methaemalbumin) was detected in microsomal fractions of mung beans. The rates of degradation were highest in extracts of young tissue and declined after day 4. The degradation mechanism and products did not resemble those of mammalian haem oxygenase.     

Effect of liposomal antitumor preparation 5-(5',6'-benzocoumarin-3')-methylaminouracil hydrobromide on cytochrome P-450 in the microsomal liver fraction of tumor bearing rats

Cytochrome P-450 thermal inactivation rate, and content of protein sulfhydryl groups and cytochrome P-450 in the microsomal liver fraction of rats at different stages of Huerin's carcinoma growth were investigated. Liposomal form of BCU administration on the background of preliminary (for 2 hours) administration of phosphatidylcholine liposomes suspension was performed. The low level of cytochrome P-450, protein SH-groups in microsomal liver fraction and increase of the rate of transition of microsomal cytochrome P-450 in P-420 was shown in the dynamics of Huerin's carcinoma growth in an organism. Low microsomal cytochrome P-450 distraction was shown in the rat liver under conditions of antitumor liposomal preparation BCU injection on the 21st day after the transplantation of Huerin's carcinoma. At the same time nonliposomal BCU caused the opposite effect. The preliminary administration of phosphatidylcholine liposomes favours the approach of the investigated parameters to the control values on the terminal stages of tumour growth.     

Transformation of an arachidonic acid hydroperoxide into epoxyhydroxy and trihydroxy fatty acids by liver microsomal cytochrome P-450

In the absence of NADPH, the addition of an arachidonic acid hydroperoxide, 15-hydroperoxyeicosa-5,8,11,13-tetraenoic acid, to liver microsomes, prepared from phenobarbital-treated rats, resulted in the formation of two major metabolites and several minor products, some of which have been purified by reverse-phase high-performance liquid chromatography. We propose the structures of the two major products to be 13-hydroxy-14,15-epoxyeicosa-5,8,11-trienoic acid and 11,14,15-trihydroxyeicosa-5,8,12-trienoic acid based on spectral characteristics and mass spectral analysis of derivatives of the compounds. A potential heterolytic cleavage product, 15-hydroxyeicosa-5,8,11,13-tetraenoic acid, was not a product of the reaction. Ferric cytochrome P-450 catalyzed the formation of these products as shown by the inability of boiled microsomes to support the reaction, the inhibition of epoxyhydroxy and trihydroxy fatty acid formation by imidazole derivatives which bind tightly to the ferric heme iron of cytochrome P-450, and the inability of carbon monoxide (which binds to ferrous P-450) and free iron chelators (EDTA and diethylenetriaminepentaacetic acid) to inhibit product formation. These results show that liver microsomal cytochrome P-450, in addition to its role in the NADPH-dependent metabolism of arachidonic acid, can utilize a hydroperoxide to produce an interesting series of potentially important arachidonic acid metabolites.     

Induction of liver microsomal monoxygenases in experimental cholestasis]

The dependence of expressiveness of microsomal mono-oxygenase induction by phenobarbital upon the amount of binding sites at cytochrome P-450 active center(s) has been studied. The experimental cholestasis is accompanied by accumulation of hydroxylated derivatives of cholesterol, which possess the detergent characteristics and destruct the substrate binding sites in P-450 molecule. The possibility has been demonstrated of phenobarbital induction under conditions when the inducer-monooxygenase primary binding and metabolic steps are not involved. It is assumed that the activation of de novo microsomal protein synthesis is effected by the molecule of phenobarbital itself and not by the products of its primary hydroxylation in the microsomes.     

Activity of NADPH-dependent monooxygenase hydroxylating system of rat liver under the effect of tetramethylthiuramdisulfide

Changes in the activity of a NADPH-dependent monooxygenase system of the rat liver are studied under the effect of tetramethylthiuramdisulphide. Under these conditions aniline hydroxylation is shown to be inhibited to a higher extent than amidopyrine demethylation. Besides a decrease in the level of cytochrome P-450, the central component of the microsomal system of hydroxylation, there appears cytochrome P-420--an inactivated form of cytochrome P-450.     

The involvement of cytochrome P-450 in hepatic microsomal steroid hydroxylation reactions supported by sodium periodate, sodium chlorite, and organic hydroperoxides.

The mechanism of steroid hydroxylation in rat liver microsomes has been investigated by employing NaIO4, NaClO2, and various organic hydroperoxides as hydroxylating agents and comparing the reaction rates and steroid products formed with those of the NADPH-dependent reaction. Androstenedione, testosterone, progesterone, and 17beta-estradiol were found to act as good substrates. NaIO4 was by far the most effective hydroxylating agent followed by cumene hydroperoxide, NADPH, NaClO2, pregnenolone 17alpha-hydroperoxide, tert-butyl hydroperoxide, and linoleic acid hydroperoxide. Androstenedione was chosen as the model substrate for inducer and inhibitor studies. The steroid was converted to its respective 6beta-, 7alpha, 15-, and 16alpha-hydroxy derivatives when incubated with microsomal fractions fortified with hydroxylating agent. Evidence for cytochrome P-450 involvement in androstenedione hydroxylation included a marked inhibition by substrates and modifiers of cytochrome P-450 and by reagents which convert cytochrome P-450 to cytochrome P-420. The ratios of the steroid products varied according to the type of hydroxylating agent used and were also modified by in vivo phenobarbital pretreatment. It was suggested that multiple forms of cytochrome P-450 exhibiting different affinities for hydroxylating agent are responsible for these different ratios. Horse-radish peroxidase, catalase, and metmyoglobin could not catalyze androstenedione hydroxylation. Addition of NaIO4, NaClO2, cumene hydroperoxide and other organic hydroperoxides to microsomal suspensions resulted in the appearance of a transient spectral change in the difference spectrum characterized by a peak at about 440 nm and a trough at 420 nm. The efficiency of these oxidizing agents in promoting steroid hydroxylation in microsomes appeared to be related to their effectiveness in eliciting the spectral complex. Electron donors, substrates, and modifiers of cytochrome P-450 greatly diminished the magnitude of the spectral change. It is proposed that NaIO4, NaClO2, and organic hydroperoxides promote steroid hydroxylation by forming a transient ferryl ion (compound I) of cytochrome P-450 which may be the common intermediate hydroxylating species involved in hydroxylations catalyzed by cytochrome P-450.     

Nuclear ADP-ribosyl transferase activity correlates with induction of P-450 monooxygenases by phenobarbital in rat liver microsomes

The effect of phenobarbital treatment on the nuclear ADP-ribosyl transferase activity has been studied in parallel with microsomal cytochrome P-450 concentration and related mono-oxygenase activities, in rat liver. A marked activation of the ADP-ribosyl transferase was observed 24 h after phenobarbital administration. The chronological study performed between 0-6 days after phenobarbital treatment showed a sharp increase in this nuclear enzyme activity, to approximately equal to 270% of the control value produced in 48 h. The administration of 5'-methylnicotinamide in vivo, an inhibitor of ADP-ribosyl transferase activity in vitro, produced a decrease both of the induction of liver microsomal cytochrome P-450 mono-oxygenases and nuclear ADP-ribosyl transferase activity. The role of nuclear ADP-ribosyl transferase in the adaptative response of the liver cell to phenobarbital is discussed.     

Rat liver cytochrome P-450b, P-420b, and P-420c are degraded to biliverdin by heme oxygenase

In this report we provide data, for the first time, demonstrating the conversion of the heme moiety of certain cytochrome P-450 and P-420 preparations, to biliverdin, catalyzed by heme oxygenase. We have used purified preparations of cytochromes P-450c, P-450b, P-450/P-420c, or P-450/P-420b as substrates in a heme oxygenase assay system reconstituted with heme oxygenase isoforms, HO-2 or HO-1, NADPH-cytochrome c (P-450) reductase, biliverdin reductase, NADPH, and Emulgen 911. With cytochrome P-450b or P-450/P-420b preparations, a near quantitative conversion of degraded heme to bile pigments was observed. In the case of cytochrome P-450/P-420c approximately 70% of the degraded heme was accounted for as bilirubin but only cytochrome P-420c was appreciably degraded. The role of heme oxygenase in this reaction was supported by the following observations: (i) bilirubin formation was not observed when heme oxygenase was omitted from the assay system; (ii) the rate of degradation of the heme moiety was at least threefold greater with heme oxygenase and NADPH-cytochrome c (P-450) reductase than that observed with reductase alone; and (iii) the presence of Zn- or Sn-protoporphyrins (2 microM), known competitive inhibitors of heme oxygenase, resulted in 70-90% inhibition of bilirubin formation.     

Unique properties of NADPH- and NADH-dependent metabolism of p-nitroanisole catalyzed by cytochrome P-450 isozyme 2 in pulmonary and hepatic microsomal preparations from rabbits

Approximately 90% of the NADPH- and NADH-dependent O-demethylation of p-nitroanisole (PNA) in the hepatic microsomal fraction from phenobarbital (PB)-treated rabbits and in the pulmonary microsomal fraction from untreated rabbits is catalyzed by the same isozyme of cytochrome P-450. This isozyme of cytochrome P-450 catalyzes less than 60% of this reaction in the hepatic microsomal fraction from untreated rabbits. Antibodies to NADPH-cytochrome P-450 reductase inhibit NADPH-dependent metabolism of p-nitroanisole by about 90% but have no effect on NADH-dependent metabolism. Hepatic NADPH-dependent metabolism of pNA and reduction of cytochrome c are inhibited to the same extent with varying amounts of antibodies to NADPH cytochrome P-450 reductase. The same relationship between inhibition of monooxygenase and reductase activities is observed for the hepatic and pulmonary metabolism of benzphetamine and 7-ethoxycoumarin. In contrast, the relationship between inhibition of the pulmonary NADPH-dependent metabolism of pNA and reductase activity is biphasic; at 75% inhibition of reductase activity, metabolism of pNA is inhibited by less than 25%. For NADH-dependent metabolism of pNA, our results indicate that both electrons are transferred to cytochrome P-450 from cytochrome b5.     

In vitro effects of trans-4-hydroxy-2-alkenals on mouse liver cytochrome P-450

Under in vitro conditions, trans-4-hydroxy-2-hexenal (t-4HH), trans-4-hydroxy-2-nonenal (t-4-HN) and trans-2-hexenal (t-2H) significantly reduced the levels of mouse liver microsomal cytochrome P-450. Incubation of trans-4-hydroxy-alkenals, under anaerobic conditions in the absence of an NADPH-generating system indicated that these compounds were converting cytochrome P-450 to cytochrome P-420. Prior activation by the mixed function oxidase system was not required for trans-4-hydroxy-alkenals to alter cytochrome P-450 concentrations. trans-4-Hydroxy-alkenals and non-hydroxylated alpha,beta-unsaturated aldehydes may be exerting their effects on cytochrome P-450 by binding to sulfhydryl groups in a similar manner as reported for sulfhydryl reagents such as p-chloromercuriphenylsulfonic acid and p-chloromercuribenzoate.     

Interaction of the complex copper compound Cu-2 with liver monooxygenases

It has been shown that the antitumour drug Cu-2 (copper complex compound) inhibited the activity of liver monooxygenases in male CBA mice. The in vivo experiments have revealed a considerably increased duration of sleep in mice treated with hexenal after the administration of different Cu-2 doses. In vitro, after the incubation of intact mouse liver microsomal fractions with different concentrations of Cu-2 the level of cytochrome Y-450 was decreased and a non-active form of hemoprotein--cytochrome P-420--appeared. At the same time, after the incubation of Cu-2 with liver microsomal fractions stabilized by 20% glycerol type I spectral changes (Ks 330 microM) were registered. This shows the possible metabolism of Cu-2 by cytochrome P-450. The role of the revealed interaction of Cu-2 with liver microsomes is being discussed for the chemotherapy of cancer.     

Inhibition of the mono-oxygenase system by butylated hydroxyanisole and butylated hydroxytoluene

The commonly used food-additive antioxidants, butylated hydroxyanisole and butylated hydroxytoluene, are inhibitors of the hepatic microsomal mono-oxygenase system, as assayed by benzpyrene hydroxylase activity and demethylase activities. Generally, butylated hydroxyanisole is a more potent inhibitor than butylated hydroxytoluene. Both inhibitors bind to cytochrome P-450 and induce “type I” binding spectra. Cytochrome P-450 is tentatively assigned as the site of inhibition.     

Microsomal cytochrome P-450 from neonatal pig testis. Purification and properties of A C21 steroid side-chain cleavage system (17 alpha-hydroxylase-C17,20 lyase)

A cytochrome P-450 from neonatal pig testicular microsomes was purified to homogeneity as judged by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels and by double diffusion on agar against antiserum raised in rabbits against the protein. The enzyme shows both 17 alpha-hydroxylase (Vmax = 4.6 nmol of product/min/nmol of P-450, Km = 1.5 microM) and C17,20 lyase (Vmax = 2.6 nmol of product/min/nmol of P-450, Km = 2.4 microM) activities. Both activities require NADPH and a flavoprotein P-450 reductase; microsomal P-450 reductase from pig and rat livers was used in these studies. The enzyme possesses a single subunit of molecular weight 59,000 +/- 1,000 as determined by electrophoresis on polyacrylamide with sodium dodecyl sulfate and by chromatography on sodium dodecyl sulfate-Sephadex. The enzyme is a glycoprotein and contains 8 nmol of heme/mg of protein and 40 nmol of phospholipid/mg of protein. All heme detected by pyridine hemochromogen is accounted for as P-450 by difference spectroscopy of the reduced P-450.carbon monoxide complex. This complex shows an absorbance maximum at 448 nm with no evidence of P-420. These studies raise the possibility that one microsomal protein (cytochrome P-450) may possess two enzymatic activities (hydroxylase and lyase).     

High activity of cytochrome P-450-linked aminopyrine N-demethylase in mouse brain microsomes, and associated sex-related difference.

The presence of cytochrome P-450 and associated mono-oxygenase activities was examined in brain microsomes from male and female mice. Although the cytochrome P-450 level in male mouse brain was very low as compared with mouse liver, the aminopyrine N-demethylase and morphine N-demethylase specific activities in male mouse brain were much higher than those observed in mouse liver. Ethoxycoumarin O-de-ethylase and aniline hydroxylase activities were, however, not detected in mouse brain. Sex-related differences were observed in both the cytochrome P-450 levels and aminopyrine N-demethylase activity in mouse brain, the levels of both being higher in male mouse brain as compared with female mouse brain. Aminopyrine N-demethylase activity in mouse brain microsomes was dependent on the presence of oxygen and NADPH and could be inhibited by piperonyl butoxide, N-octyl imidazole and carbon monoxide. Antiserum raised to the phenobarbital-inducible form of rat liver cytochrome P-450 [P-450(b+e)] inhibited mouse brain aminopyrine N-demethylase activity by around 80+ mouse brain microsomal protein exhibited cross-reactivity against this antiserum when examined by Ouchterlony double diffusion and immunoblotting. The present results indicate the presence of a phenobarbital-inducible form of cytochrome P-450 (or a form of cytochrome P-450 that is similar immunologically) in mouse brain microsomes, which is associated with a sex-related difference.     

Interaction of prostaglandins with adrenal microsomal cytochrome P-450 in the guinea pig.

Studies were carried out to investigate the effects of prostaglandins (PG) in vitro on adrenal microsomal steroid and drug metabolism in the guinea pig. The addition of PGE1, PGE2, PGA1, PGF1 alpha or PGF2 alpha to isolated adrenal microsomes produced typical type I difference spectra. The sizes of the spectra (delta A385-420) produced by prostaglandins were smaller than those produced by various steroids including progesterone, 17-hydroxyprogesterone and 11 beta-hydroxyprogesterone. However, the affinities of prostaglandins and steroids for adrenal microsomal cytochrome P-450, as estimated by the spectral dissociation constants, were similar. Prior addition of prostaglandins to isolated adrenal microsomes did not affect steroid binding to cytochrome P-450 or the rate of steroid 21-hydroxylation. In contrast, prostaglandins inhibited adrenal metabolism of ethylmorphine and diminished the magnitude of the ethylmorphine-induced spectral change in adrenal microsomes. The results indicate that prostaglandins inhibit adrenal drug metabolism by interfering with substrate binding to cytochrome P-450. Since 21-hydroxylation was unaffected by PG, different cytochrome P-450 moieties are probably involved in adrenal drug and steroid metabolism.     

Heterogeneity of liver microsomal cytochrome P-450: the spectral characterization of reactants with reduced cytochrome P-450.

The aerobic metabolism of benzphetamine by liver microsomes, during a cytochrome P-450-catalyzed mixed-function oxidation reaction, results in the formation of an easily detected spectral complex with an absorption band maximum at 456 nm. Electron paramagnetic resonance studies, as well as studies with the chemical reductant, sodium dithionite, or the oxidant, potassium ferricyanide, indicate that the spectral complex results from the formation of a product adduct with reduced cytochrome P-450. The spectral properties of this product complex of cytochrome P-450 have been compared to those observed with carbon monoxide, metyrapone, and ethylisocyanide. The reaction of these reagents to specific pools of microsomal cytochrome P-450 permits the identification of at least two major and two minor types of cytochrome P-450 in liver microsomes prepared from phenobarbital-treated rats.     

Hepatic mixed function oxidase system and effect of phenobarbital, 3-methylcholanthrene and 1, 1, 1-trichloro-2, 2-bis(p-chlorophenyl)ethane in developing chickens.

Contents of hepatic microsomal protein, aminopyrine N-demethylase, acetanilide hydroxylase, aniline hydroxylase, hydrogen peroxide formation, cytochrome-c-reductase, cytochrome b5 and cytochrome P-450 were examined in control, phenobarbital (PB), 3-methylcholanthrene (3-MC) and 1, 1, 1-trichloro-2, 2-bis(p-chlorophenyl)ethane (DDT) treated group of 1-28 days old chickens. Increase in aminopyrine N-demethylase, acetanilide hydroxylase, aniline hydroxylase, cytochrome-c-reductase, cytochrome b5 and cytochrome P-450 was noticed at all stages of development during administration of PB and 3-MC. But these enzyme activities were not always paralleled by increase in age. Aminopyrine N-demethylase was increased in early stages only during DDT administration, which indicates that the form of cytochrome P-450, responsible for aminopyrine N-demethylation is present in early stages only. However, acetanilide hydroxylase was decreased in all stages of development, in postnatal development the basal activities of the enzymes for various substrates do not exhibit identical pattern, the degree of inducibility by inducers varied in relation to age of animal. Hydrogen peroxide formation increased in all stages of developing chickens due to the administration of PB and DDT. It however decreased due to 3-MC administration which may be due to induction of high spin cytochrome P-450.