Immunochemical characterization of some monooxygenase activities in liver microsomes from untreated and phenobarbital-treated rats

Two major forms of liver microsomal cytochrome P 450, one from untreated rats (P 450 A₂NI) and the other from phenobarbital-treated rats (P 450 B₂PB), were partially purified. Reconstitution of monooxygenase activities of purified enzymes and inhibition patterns of these activities by antibodies in microsomes gave the following results: 1) aniline hydroxylase activity is mainly supported by cytochrome P 450 A₂NI. This form is the major one in microsomes from control rats, but is also found at minute amounts in microsomes from phenobarbital-treated rats. It behaves as a constitutive form. 2) 4-nitroanisole-and benzphetamine-demethylase activities are mainly supported by cytochrome P 450 B₂PB which is predominant in phenobarbital-treated rats but is also present in control microsomes at low levels. 3) 4-nitroanisole-O-demethylase activity is less specific than benzphetamine-N-demethylase activity towards cytochrome P 450 B₂PB.     

Comparative study of effects of the tyrosine-copper(II) complex on xenobiotic hydroxylation and lipid peroxidation]

It has been found that NADPH-dependent hydroxylation of dimethylaniline, aniline, p- and o-nitroanisol and lipid peroxidation is inhibited by the tyrosine-copper (II) complex (low molecular weight analog of superoxide dismutase), which is indicative of a possibility of superoxide radicals formation in these reactions. The inhibition of the above-mentioned reactions with Tyr2-Cu2+ is less pronounced or absent, if cumole hydroperoxide is used as cosubstrate instead of NADPH. Differences in the Tyr2-Cu2+ complex effects on the cumule hydroperoxide-dependent xenobiotics hydroxylation and lipid peroxidation catalyzed by various forms of cytochrome P-450, e. g. microsomal, soluble and incorporated into liposomes, have been found. The data obtained suggest that the efficiency of the inhibitory effect of the Tyr2-Cu2+ complex depends on the type of cosubstrates (NADPH, cumole hydroperoxide) and substrates used as well as on the form of cytochrome P-450.     

The conformation and thermal stability of soluble cytochrome P-450 and cytochrome P-450 incorporated into liposomal membranes

The α-helix content of the cytochrome P-450 incorporated into the liposomal membranes of either phosphatidylcholine or microsomal phospholipid insignificantly differed from that of the soluble one. The binding of both type I and type II substrates with cytochrome P-450 incorporated into phosphatidylcholine and microsomal phospholipid membranes did not change the conformation of the polypeptide chain. In contrast to this the type II substrates increased the α-helix content of soluble hemoprotein about 3–5%. Dithionite reduction of the cytochrome P-450 haem increased the degree of α spiralization up to 10% for soluble hemoprotein and up to 5% for the membrane-bound enzyme only. The investigation of the thermal stability of the soluble and liposomal forms of cytochrome P-450 showed that the enzyme incorporated into phospholipid vesicles was highly stable. The heating of the enzyme was followed by a slightly pronounced cooperative transition in contrast to the well-pronounced transition for the soluble cytochrome P-450. Hence, the incorporation of the soluble cytochrome P-450 into phospholipid bilayer does not result in significant change of α-helix content, but the increasing of rigidity and thermal stability of the membrane-bound hemoprotein molecule is observed.     

Isolation and properties of cytochrome P-450 from rabbit liver microsomes]

A purified low-spin form of cytochrome P-450 was isolated from phenobarbital-induced rabbit liver microsomes. The preparation was functionally active and free from cytochromes b5 and P-420 and phospholipids. The specific content of the cytochrome was 18 nmoles per mg of protein. At the molecular weight of the hemoprotein of 50,000, it corresponds to 90% of purification. The purified hemoprotein binds substrates of type II and some substrates of type I. The complexes formed reveal spectral properties, similar to those for the complexes of these substrates with the microsomal form of cytochrome P-450.     

Strain differences in the induction of soluble and microsomal enzymes by phenobarbital and 3-methylcholanthrene

The ability of phenobarbital and 3-methylcholanthrene (3MC) to induce liver microsomal and soluble enzymes was compared in Sprague-Dawley and Long-Evans rats. 3MC increased the V for the aniline hydroxylase and stimulated the formation of the hemoprotein P₄₄₈ to a similar extent in the 2 strains of rats. On the other hand phenobarbital increased the V for the microsomal enzyme aniline hydroxylase and aminopyrine demethylase and enhanced the activity of the soluble enzyme aldehyde dehydrogenase only in Sprague-Dawley rats. It induced a more marked increase of cytochrome P₄₅₀ in the Sprague-Dawley than in the Long-Evans strain.     

The function of the microsomal oxidative system of different organs in guinea pigs administered ethanol

The microsomal cytochrome content and enzyme activity has been determined in liver, kidney, lungs and intestinal mucous from guinea pig males which were injected 25% ethanol intraperitoneally at a dose of 2 g per 1 kg of body mass. The changes in cytochrome P-450 and b5 content, amidopyrine-N-demethylase, aniline hydroxylase, p-nitrophenol hydroxylase. NADP.H-cytochrome-c-reductase activities in investigated organs of the animals have been found depending on the ethanol intoxication period (for 3, 6 or 14 days). Changes of the same type in microsomal enzyme activities have been discovered in liver, lungs and intestine, but not in kidney that is accounted for the substrate specificity and inducibility of the cytochrome P-450 some forms in extrahepatic tissues.     

Hydroxylase activity of immobilized haemoglobin

Until now, hydroxylation of substrates for practical applications has been mostly carried out by chemical and microbial processes. The hydroxylase activity of haemoglobin could be of great help for this purpose. Hydroxylation of aniline by haemoglobin immobilized as cross-linked soluble polymers and insoluble particles was studied. Activity yields after immobilization as well as kinetic constants were estimated. Hydroxylase activities similar to those of liver microsomal cytochrome P-450 activities were obtained.     

Synthetic peptide mimics of a predicted topographical interaction surface: the cytochrome P450 2B1 recognition domain for NADPH-cytochrome P450 reductase

In order to identify the cytochrome P450-binding domain for NADPH-cytochrome P450 reductase, synthetic peptide mimics of predicted surface regions of rat cytochrome P450 2B1 were constructed and evaluated for inhibition of the P450-reductase interaction. A peptide corresponding to residues 116–134, which includes the C helix, completely inhibited reductase-mediated benzphetamine demethylation by purified P450 2B1. Replacement of Arg-125 by Glu yielded a noninhibitory peptide, suggesting that this residue significantly contributes to the reductase-P450 interaction. Additional P450 peptides were prepared which correspond to combinations of regions distant in primary sequence, but predicted to be spatially proximate. A peptide derived from segments of the C and L helices was a more potent inhibitor than peptides derived from either segment alone. This topographically designed peptide not only inhibited P450 2B1 in its purified form, but also when membrane-bound in rat liver microsomes. The peptide also inhibited microsomal aryl hydrocarbon hydroxylase, aniline hydroxylase, and erythromycin demethylase activities derived from other P450s. These results indicate that the C and L helices contribute to a reductase-binding site common to multiple P450s, and present a peptide mimic for this region that is useful for inhibition of P450-mediated microsomal activities.     

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. I. Properties of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) monomers.

Oligomers and monomers of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) isolated from the liver microsomes of phenobarbital-treated rabbits were examined for physicochemical properties and catalytic activities. As measured using laser correlation spectroscopy the particle sizes of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers were 14.8 +/- 1.7 and 19.2 +/- 1.4 nm, respectively. Twenty-four-hour incubation with Emulgen 913 at 4 degrees C at a molar ratio of 1:100 led to the monomerization of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers, the particle sizes diminishing to 6.1 +/- 1.3 and 5.2 +/- 0.4 nm, respectively. The thermal stability of NADPH-cytochrome P450 reductase monomers was the same as that of oligomers, whereas cytochrome P450 LM2 monomers were less thermostable than oligomers and cytochrome P450 in microsomes. Similar to cytochrome P450 LM2 oligomers and the microsomal hemoprotein, cytochrome P450 LM2 monomers formed complexes with type I and II substrates, but with Kd values higher than those of microsomes and cytochrome P450 LM2 oligomers. Kinetic parameters (Vmax and Km) of H2O2- and cumene hydroperoxide-dependent oxidation of benzphetamine and aniline in the presence of cytochrome P450 LM2 oligomers, monomers, and microsomes were determined. Peroxidase activities of the oligomers and monomers were the same, but were lower than those of microsomes. Thus the substitution of protein-protein interactions in cytochrome P450 LM2 oligomers with protein-detergent interactions in the monomers did not influence the catalytic properties of the hemoprotein.     

Mechanism of inhibition by carbonyl cyanide m-chlorophenylhydrazone and sodium deoxycholate of cytochrome P-450-catalysed hepatic microsomal drug metabolism.

1. Treatment of liver microsomal fraction with 0.03-0.12% sodium deoxycholate and 0.005-0.06 mM carbonyl cyanide m-chlorophenylhydrazone decreases phospholipid-dependent hydrophobicity of the microsomal membrane, assayed by the kinetics of 8-anilinonaphthalene-1-sulphonate binding and ethyl isocyanide difference spectra. 2. Sodium deoxycholate at a concentration of 0.01% lacks its detergent properties, but competitively inhibits aminopyrine binding and activates the initial rate of NADPH-cytochrome P-450 reductase. In the presence of 0.03-0.09% sodium deoxycholate the rate-limiting factor in p-hydroxylation of aniline is the content of cytochrome P-450. and that for N-demethylation of aminopyrine is the activity of NADPH-cytochrome P-450 reductase. 3. Carbonyl cyanide m-chlorophenylhydrazone has no effect on the binding and metabolism of aniline; investigation of its inhibiting effect on aminopyrine N-demethylase established that the rate-limiting reaction is the dissociation of the enzyme-substrate complex in the microsomal preparations. 4. In the mechanism of action of carbonyl cyanide m-chlorophenylhydrazone the key step may be the electrostatic interaction of its protonated form and one of the forms of activated oxygen at the catalytic centre of cytochrome P-450. 5. at least two different phospholipid-dependent hydrophobic zones are assumed to exist in the microsomal membrane, both coupled with cytochrome P-450. One of them reveals selective sensitivity to the protonation action of carbonyl cyanide m-chlorophenylhydrazone and contains the 'binding protein' for type I substrates and NADPH-cytochrome P-450 reductase; the other contains the cytochrome P-450 haem group and binding sites for type II substrates.     

Induction of drug-metabolizing enzymes and aryl hydrocarbon hydroxylase by microscope immersion oil

Microscope immersion oil when administered intraperitoneally or applied to skin in experimental animals substantially increased liver weight, microsomal protein, NADPH-cytochrome c reductase activity, cytochrome P-450 content and the metabolism of the model substrates, ethylmorphine and benzo(a)pyrene. Immersion oil caused the induction of the polycyclic hydrocarbon type of hemoprotein, cytochrome P-448. When applied to skin, the oil also caused an 11-fold increase in benzo(a)pyrene hydroxylase activity at the skin sites.     

Neonatal imprinting and hepatic cytochrome P-450 II. Partial purification of a sex-dependent and neonatally imprinted form(s) of cytochrome P-450

A new form of cytochrome P-450 was partially purified from hepatic microsomes of neonatally imprinted rats (adult male and adult male castrated at four weeks of age). This new form of cytochrome P-450 appears to have an apparent molecular weight of approximately 50,000 daltons as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It appears that this form of cytochrome P-450 is either absent or present in low concentrations in cytochrome P-450 preparations isolated from neonatally nonimprinted rats (adult female and adult male castrated at birth). Reconstitution of testosterone hydroxylase and benzphetamine N-demethylase activities of this partially purified cytochrome P-450 revealed that the presence of testosterone 16α-hydroxylase activity, an imprintable microsomal enzyme, was in parallel with the imprinting status of the animals; a significantly higher activity was detected in the neonatally imprinted than that of the nonimprinted animals. This was in contrast to the nonimprintable benzphetamine N-demethylase, testosterone 7α-and 6β-hydroxylase activities which exhibited no correlation with the imprinting status of the animals. We have prepared antisera from rabbits using the partially purified cytochrome P-450 preparations from adult male rats as antigens. These antisera inhibited microsomal testosterone 16α- and 7α-hydroxylase activities in a concentration-dependent manner, without impairing 6β-hydroxylase activity. These data suggest that the partially purified cytochrome P-450 from adult male rats consists of both imprintable (16α-) and nonimprintable (7α-) testosterone hydroxylase activities. The antisera formed immunoprecipitant lines in the Ouchterlony double diffusion plates with partially purified cytochrome P-450 from both neonatally imprinted and nonimprinted adult rats. The immunoprecipitant lines, as stained by coomassie blue, suggest the homology of the cytochrome P-450 preparations from neonatally imprinted and nonimprinted rats. Immunoabsorption of the antisera against neonatally nonimprinted, partially purified cytochrome P-450 completely removed the immunoprecipitant lines without appreciably impairing the inhibitory effects of antisera on the microsomal testosterone 16α-and 7α-hydroxylase activities. In contrast, immunoabsorption of the antisera against partially purified cytochrome P-450 from adult male rats (imprinted) abolished completely both the immunoprecipitant lines and the inhibition on microsomal testosterone hydroxylation reaction (16α and 7α). The inhibitory actin of antisera on testosterone hydroxyulation was also abolished upon boiling the antisera at 100°C for 5 minutes. The biochemical and immunochemical data in this study suggest that the neonatally imprintable form or forms of hepatic microsomal cytochrome P-450 accounts for a small fraction of the bulk of total cytochrome P-450. However, the existence of this form of cytochrome P-450 is regulated by gonadal hormones during the neonatal period and accounts for the major imprintable sex difference in drug and steroid metabolism in adulthood.     

K+-valinomycin and chloride conductance of the human red cell membrane. Influence of the membrane protonophore carbonylcyanide m-chlorophenylhydrazone

The major form of microsomal cytochrome P-450 induced by trans-stilbene oxide in the liver of male Sprague-Dawley rats was purified and characterized, and compared with the isolated cytochrome P-450 B2 forms from phenobarbital- and 3-methylcholanthrene-pretreated animals. The apparent subunit molecular weight of the trans-stilbene oxide-induced cytochrome was found to be 53 000 using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the absorbance maximum of the carbon monoxide complex of the ferrous cytochrome was 450 nm. Reconstitution of the N-demethylase activity towards three different substrates showed high and similar activities with the cytochrome P-450 B2 forms from trans-stilbene oxide or phenobarbital-treated rats, with one exception. Amino-acid analysis also showed a very high degree of similarity between these two forms. Upon proteinase treatment with three different proteinases the trans-stilbene oxide-induced cytochrome demonstrated in each case a peptide pattern identical to that obtained with the phenobarbital-induced B2 form. Furthermore, both forms are completely immunologically cross-reactive. We therefore conclude from these experiments that the liver microsomal P-450 B2 from trans-stilbene oxide and phenobarbital-treated rats are very closely related, if not identical.     

The mechanism of hydroperoxide-dependent reactions with participation of cytochrome P-450

Cytochrome P-450 destruction kinetics by cumene hydroperoxide (CHP) has been studied at 25 degrees C in phosphate buffer, pH 7.25-7.50, in various systems: intact and induced rat or rabbit microsomes, highly purified LM2- and LM2- and LM4-forms of cytochrome P-450 from rabbit liver microsomes. The destruction kinetics is characterized by three phases in all systems. The CHP-influenced cytochrome P-450 destruction is a radical chain process with linear termination of the chains. The acidic phospholipids, phosphatidylserine and phosphatidylinositol and total microsomal phospholipids containing the acidic lipid components activate cytochrome P-450 in the hydroxylation of aniline and naphthalene by CHP. Phosphatidylcholine and sphingomyelin have no effect upon the cytochrome P-450 activity in the type I and II substrates oxidation by CHP. The phase transitions of the microsomal phospholipids influence the interaction of cytochrome P-450 with its reductase, altering the activation energy of type I substrates oxidation. The type II substrate oxidation is not affected by phase transitions in the full microsomal hydroxylating system.     

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.     

Cytochrome P-450 and aryl hydroxylase activity in cells of a long-term transplantable tumor

A functionally active system of microsomal monooxygenases has been found in a long-term transplanted tumor MC-II of C57B1/6j mice. In microsomal fraction of the tumor, one could detect cytochrome P-450 and benzo(a)pyrene hydroxylase (BP hydroxylase) activity. The latter one increased more than 2 times after the animals received 3-MC and aroclor 1254. In in-vitro experiments, the microsomal monooxygenase inhibitors, SKF 525-A and metyrapone, did not affect BP hydroxylation, whereas alpha-naphthoflavone inhibited the enzyme. It is assumed that tumor MC-II contains hemoprotein that is similar to cytochrome P1-450.     

Modification by cyanide of the aniline-binding reaction with cytochrome P-450.

Hydroxylation of aniline to p-aminophenol catalyzed by the cytochrome P-450-containing monooxygenase system of liver microsomes is inhibited by cyanide, but microsomal NADPH-cytochrome c reductase is insensitive to this inhibitor. The interaction of aniline with membrane-bound cytochrome P-450, according to spectrophotometric analyses, consists of two phases with respect to aniline concentration, and cyanide interferes differently with these two reaction phases. Noncompetitive and competitive (or mixed type) inhibitions of the aniline-binding reaction by cyanide are observed in reaction systems containing low and, high concentrations of aniline, respectively, a situation similar to the inhibitory action of cyanide on aniline hydroxylase activity. Abnormal aniline-induced difference spectra appeared when cyanide was added as the spectral modifier, and the magnitude of the spectral change in the presence of both aniline and cyanide was a nonadditive change. These results suggest the dissociation of the cytochrome P-450·cyanide complex by aniline. A similar result indicating dissociation of the complex was also obtained by epr spectroscopy. We therefore suggest that addition of a high concentration of substrate causes insensitivity of the microsomal hydroxylase system to cyanide.     

Differing microsomal monoxygenase activity in the cells of ascitic and solid forms of transplantable tumors

Aryl hydroxylase activity has been demonstrated to depend on the pattern of tumor cell structural organization. The activity of microsomal monoxygenases in the ascitic forms of sarcoma MC-11, hepatoma 22a and Ehrlich's tumor was much lower than in the corresponding solid tumors. Aryl hydroxylase was activated after the animals received 3-MC, but the magnitude of the activity induced did not correlate with the basic activity in the different tumors. In in vitro experiments, 7,8-benzoflavone inhibited the enzyme in all the tumors, whereas metyrapone did not affect BP-hydroxylation. It is assumed that all the tumors investigated contain hemoprotein that is similar to cytochrome P1-450.     

The role of the parasympathetic nervous system in the regulation of microsomal monooxygenase activity in the liver of rats

The effect of vegetative nervous system activation or depression (subdiaphragmatic vagotomy, atropine, proserine and acetylcholine treatments) on the hepatic microsomal enzymes activities has been studied on Wistar male rats. It is found, that hepatic denervation and atropine treatment decreased cytochrome P450 content and aniline hydroxylase activity. Proserine and acetylcholine induced an opposite effect. It is considered that these different changes in the microsomal enzyme activities with variations in the vegetative nervous system state have proved the nervous control of these processes.     

Isolation and partial characterization of a rifampicin induced rabbit liver microsomal cytochrome P-450

Rifampicin administration to New Zealand male rabbits increased the concentration of an LM3 form of cytochrome P-450 to up to 30% of the microsomal P-450 concentration. This enzyme was purified to electrophoretic homogeneity with a yield of 8% of the original total microsomal P-450 concentration. Isolated as a low spin hemoprotein in its substrate free oxidized form, it displays in its reduced CO-complexed form an absorption maximum at 449 nm. Immunological assays, as well as activity measurements, in particular its stereospecific progesterone hydroxylation in the 6 beta-position, show a relationship between LM3,Rif and LM3c (from untreated rabbits).