Rat pulmonary microsomal cytochrome P-450 enzymes involved in the activation of procarcinogens.

Rat lung microsomal cytochrome P-450 (P-450) enzymes have been characterized with regard to their catalytic specificities towards activation of several procarcinogens to genotoxic metabolites in Salmonella typhimurium TA1535/pSK1002. We first examined the roles of rat liver microsomal P-450 enzymes in the activation of benzo[a]pyrene and its 7,8-diol enantiomers to genotoxic products, and found that P-450 1A1 is a major catalyst for the activation of these potential procarcinogens in rat livers. Using lung microsomes isolated from rats treated with various P-450 inducers we obtained evidence that at least three P-450 enzymes are involved in the activation of several procarcinogens. Immunoinhibition studies support the view that benzo[a]pyrene and its 7,8-diol derivatives, other dihydrodiol derivatives of polycyclic aromatic hydrocarbons, and 3-amino-1-methyl-5H-pyrido[4,3-b]indole are activated to genotoxins mainly by rat P-450 1A1, which is inducible in rat lungs by 5,6-benzoflavone and the polychlorinated biphenyl mixture Aroclor 1254. Activation of 2-amino-3,5-dimethylimidazo[4,5-f]quinoline and 2-amino-3-methylimidazo[4,5-f]quinoline may be catalyzed by another P-450 enzyme because the activities were not induced by treatment with 5,6-benzoflavone or Aroclor 1254. The observation that both activities were inhibited by antibodies raised against P-450 1A2 and by 7,8-benzoflavone suggests a role for an enzyme of P-450 1A family, probably P-450 1A2, in rat lung microsomes. The activation of aflatoxin B1 and sterigmatocystin appears to be catalyzed by other P-450 enzyme(s) rather than the P-450 1A family as judged by the different responses of activities to the P-450 inducers and the specific antibodies in rat lung microsomes. Interestingly, lung microsomal activation of several procarcinogens was found to be suppressed in rats treated with isosafrole and pregnenolone 16 alpha-carbonitrile. Thus, the results support the roles of different P-450 enzymes in the activation of procarcinogens in rat lung microsomes.     

Feprazone: an inducer of the P450 II B family of proteins in the rat

The ability of feprazone to induce the hepatic microsomal mixed-function oxidases was investigated in the rat, with emphasis being placed on the nature of the cytochrome P-450 family induced. Treatment with feprazone enhanced the p-hydroxylation of aniline and the dealkylations of benzphetamine and pentoxyresorufin but had no effect on the O-deethylation of ethoxyresorufin. The same treatment had no major effect on total cytochrome P-450 levels but increased the spectral interaction of metyrapone with reduced cytochrome P-450. Immunoblots employing monospecific polyclonal antibodies revealed that feprazone induces the apoprotein levels of the P450 II B, but not of the P450 I, family. It is concluded that feprazone is an inducer of the rat hepatic mixed-function oxidase system showing selectivity toward the P450 II B family.     

Toxicological significance of dog liver cytochrome P-450: examination with the enzyme expressed in Saccharomyces cerevisiae using recombinant expression plasmid.

A complementary DNA (cDNA) coding for a form of beagle dog cytochrome P-450 (Dah1), which is the orthologue to the CYP1A1 cDNA of rat, mouse and human, was inserted between the alcohol dehydrogenase (ADH) promoter and terminator regions of the yeast expression vector pAAH5. On introduction of the resulting recombinant plasmid pDC-1, Saccharomyces cerevisiae strain AH22 cells synthesized up to 1.5 x 10(5) molecules per cell of cytochrome P-450 protein (P-450(Dah1)). The carbon monoxide-bound reduced form of P-450(Dah1) showed an absorption peak at 447 nm and specific content of P-450(Dah1) was about 0.1 nmole P-450 per mg of microsomal protein. P-450(Dah1) cross-reacted with antibodies to rat P-448-H (CYP1A2) and dog P-450-D2 (CYP1A2). P-450(Dah1) activated 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) most efficiently in the umu test and exhibited a high activity of aryl hydrocarbon hydroxylase toward benzo[a]pyrene.     

3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine photolabels a substrate-binding site of rat hepatic cytochrome P-450 form PB-4

Hepatic microsomes isolated from untreated male rats or from rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (3-MC) were labeled with the hydrophobic, photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). [125I]TID incorporation into 3-MC- and PB-induced liver microsomal protein was enhanced 5- and 8-fold, respectively, relative to the incorporation of [125I]TID into uninduced liver microsomes. The major hepatic microsomal cytochrome P-450 forms inducible by PB and 3-MC, respectively designated P-450s PB-4 and BNF-B, were shown to be the principal polypeptides labeled by [125I]TID in the correspondingly induced microsomes. Trypsin cleavage of [125I]TID-labeled microsomal P-450 PB-4 yielded several radiolabeled fragments, with a single labeled peptide of Mr approximately 4000 resistant to extensive proteolytic digestion. The following experiments suggested that TID binds to the substrate-binding site of P-450 PB-4. [125I]TID incorporation into microsomal P-450 PB-4 was inhibited in a dose-dependent manner by the P-450 PB-4 substrate benzphetamine. In the absence of photoactivation, TID inhibited competitively about 80% of the cytochrome P-450-dependent 7-ethoxycoumarin O-deethylation catalyzed by PB-induced microsomes with a Ki of 10 microM; TID was a markedly less effective inhibitor of the corresponding activity catalyzed by microsomes isolated from uninduced or beta-naphthoflavone-induced livers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Organ selective induction of cytochrome P-448 isozymes in the rat by 2-methoxy-4-aminoazobenzene and 3-methylcholanthrene

Male Sprague Dawley rats were injected intraperitoneally with 2-methoxy-4-amino-azobenzene (2-MeO-AAB) or 3-methylcholanthrene (MC), and then the expression of microsomal cytochrome P-450 isozymes in liver and extrahepatic tissues was investigated by means of immunological methods and a bacterial mutation test. The results of protein A-enzyme-linked immunosorbent assaying and immunoblotting using anti-rat cytochrome P-448 monoclonal antibodies showed that MC induced at least two microsomal cytochrome P-448 isozymes, a high spin form (cytochrome P-448H) and a low spin form (cytochrome P-448L), in liver, but that it induced only cytochrome P-448L in extrahepatic tissues such as lung, kidney, small intestine, and colon. The results also indicated that, in contrast to MC, 2-MeO-AAB selectively induced microsomal cytochrome P-448H in liver but did not induce any cytochrome P-448 isozymes in extrahepatic tissues. The activities of 9,000 X g supernatants from the individual organs, as to the mutagenic conversion of 3 aromatic amines (3-amino-1-methyl-5H-pyrido(4,3-b)indole, 2-amino-6-methyldipyrido(1,2-a: 3',2'-d)-imidazole and 3-methoxy-4-aminoazobenzene), toward Salmonella typhimurium TA 98 bacteria were dependent upon the quantity and/or quality of the microsomal cytochrome P-448 isozymes in the organs.     

Conversion of IQ, a dietary pyrolysis carcinogen to a direct-acting mutagen by normal intestinal bacteria of humans

The dietary carcinogen, 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ) is mutagenic in the Salmonella/microsomal mutagenicity assay when activated by microsomal enzymes. IQ is found in many cooked foods, notably fried beef and pork. In laboratory rodents IQ is carcinogenic. We showed that mixed and pure cultures of human intestinal anaerobes, notably Eubacterium spp., metabolized IQ to 2-amino-3,6-dihydro-3-methyl-7H-imidazo[4,5-f]quinoline-7-one (HOIQ). Unlike IQ, both the synthetic and bacterially produced HOIQ were direct-acting mutagens, i.e. active without microsomal activation. This new direct-acting mutagen, from the bacterial metabolism of a dietary pyrolysis carcinogen, raises new concerns about the possible role of this class of genotoxins in the etiology of human cancer.     

Inducers of cytochrome P-450d: influence on microsomal catalytic activities and differential regulation by enzyme stabilization

The interaction of isosafrole, 3,4,5,3',4',5'-hexabromobiphenyl (HBB) and hexachlorobiphenyl (HCB) with cytochrome P-450d was evaluated by characterization of estradiol 2-hydroxylase activity. Displacement of the isosafrole metabolite from microsomal cytochrome P-450d derived from isosafrole-treated rats resulted in a 160% increase in estradiol 2-hydroxylase. The increase was fully reversed by incubation with 1 microM HBB. Although isosafrole is capable of forming a complex with many different cytochrome P-450 isozymes, it appears to bind largely to cytochrome P-450d in vivo as was demonstrated by measuring the enzymatic activity of microsomal cytochromes P-450b, P-450c, and P-450d from isosafrole-treated rats. When estradiol 2-hydroxylase was measured in rats treated with increasing doses of HCB, there was a gradual decrease in microsomal enzyme activity despite a 20-fold increase in cytochrome P-450d. The ability of cytochrome P-450d ligands to stabilize the enzyme was investigated in two ways. First, cytochromes P-450c and P-450d were quantitated immunochemically in microsomes from rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), at a dose which maximally induced total cytochrome P-450, followed by a single dose of a second inducer. The specific content of cytochrome P-450d was significantly increased when isosafrole or HCB was the second inducer but not when 3-methylcholanthrene was the second inducer. Second, the relative turnover of cytochrome P-450d was measured by the dual label technique. Following TCDD treatment, microsomal protein was labeled in vivo with [3H]leucine, the second inducer was given and protein was again labeled 3 days later with [14C]leucine. A higher ratio of 3H/14C in the cytochrome P-450d from isosafrole + TCDD- and HCB + TCDD-treated rats relative to TCDD (control)-treated rats suggested that isosafrole and HCB were able to retard the degradation of cytochrome P-450d, presumably by virtue of being tightly bound to the enzyme.     

Comparison of the effects of inhibitors of cytochrome P-450-mediated reactions on human platelet aggregation and arachidonic acid metabolism

Metyrapone and SKF-525A, together with amphenone B, a structural analogue of metyrapone, which are all inhibitors of cytochrome P-450-mediated reactions, were shown to inhibit the arachidonic acid-induced aggregation of human platelets. Amphenone B, like metyrapone, exhibited a type II (ligand) binding spectrum with rat liver microsomal cytochrome P-450, in contrast to SKF 525A which is a type I (substrate) binding agent. Independently of their type of binding spectra and of their maximum spectral change, however, the affinity of the three compounds for rat liver cytochrome P-450 showed a close proportional correlation with their platelet aggregation inhibitory potency. All three compounds inhibited the formation of [1-14C]thromboxane B2 from [1-14C]arachidonic acid by human platelets aggregated with collagen. The effect of metyrapone on the remaining labelled products suggested that it is a selective thromboxane synthesis inhibitor, while amphenone B exhibited activity reminiscent of cyclo-oxygenase inhibitors. SKF 525A produced complex effects possibly attributable to cyclo-oxygenase inhibition and enhanced lipid peroxidation, since it also enhanced platelet malonaldehyde formation, which the other two compounds inhibited. These data provide further support for a role of cytochrome P-450 in thromboxane synthesis and platelet aggregation.     

Hepatic microsomes from freshwater fish--I. In vitro cytochrome P-450 chemical interactions

1. Of 87 chemicals tested for their ability to interact with oxidized hepatic cytochrome P-450 from mature male brook trout (Salvelinus fontinalis), 21 formed detectable type I or type II binding spectra. 2. When 8 of these 21 chemicals were tested with cytochrome P-450 of nine other species of freshwater fish, wide species variation in hepatic microsomal cytochrome P-450 was evident, since the spectral size of chemical interactions as related to the carbon monoxide spectrum and the ratio of type II to type I binding were not alike. 3. These spectral data suggest that hepatic microsomal cytochrome P-450 of freshwater fish exists in different forms.     

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.     

Purification and properties of cytochrome P-450 generally acting as a catalyst on benzo[a]pyrene hydroxylation from liver microsomes of untreated rats

A form of cytochrome P-450 generally catalyzing benzo[a]pyrene (B[a]P) hydroxylation was purified from liver microsomes of untreated rats on the basis of the catalytic activity. The purification procedures consisted of cholate solubilization and chromatography in 3 steps, on DEAE-Toyopearl (at room temperature), hydroxylapatite, and CM-Toyopearl columns. Cytochrome P-450 purified in this way (named P-450/B[a]P) was homogeneous on SDS-polyacrylamide gel electrophoresis, and the molecular weight was estimated to be 51,000. The absorption spectra of the oxidized form of P-450/B[a]P showed a Soret peak at 417 nm, characteristic of low-spin hemoprotein, and the Soret peak of the reduced cytochrome P-450-CO complex was at 451 nm. Immunochemical analysis of P-450/B[a]P indicated that P-450/B[a]P is immunologically distinct from P-450b (a major phenobarbital-inducible form of P-450) and P-450c (a major 3-methylcholanthrene-inducible form of P-450, which highly catalyzes the hydroxylation of B[a]P). B[a]P hydroxylase activity in liver microsomes of untreated rats was inhibited to about 20% by the P-450/B[a]P antibody. These results demonstrate that P-450/B[a]P is a different form of P-450 from P-450b and P-450c, and generally catalyzes B[a]P hydroxylation in liver microsomes of untreated rats.     

Metabolism of benzo[c]phenanthrene by rat liver microsomes and by a purified monooxygenase system reconstituted with different isozymes of cytochrome P-450

Metabolism of the environmental pollutant and weak carcinogen benzo[c]-phenanthrene (B[c]Ph) by rat liver microsomes and by a purified and reconstituted cytochrome P-450 system is examined. B[c]Ph proved to be one of the best polycyclic aromatic hydrocarbon substrates for rat liver microsomes. It is metabolized by microsomes from control rats and by rats treated with phenobarbital or 3-methylcholanthrene at 3.9, 4.2 and 7.8 nmol/nmol cytochrome P-450/min, respectively. Principal metabolites are dihydrodiols along with small amounts (less than 10%) of phenols. The K-region 5,6-dihydrodiol is the major metabolite and accounts for 77-89% of the total metabolites. The 3,4-dihydrodiol with a bay-region 1,2-double bond is formed in much smaller amounts and accounts for only 6-17% of the total metabolites, the highest percentage being formed by microsomes from control rats. Highly purified monooxygenase systems reconstituted with cytochrome P-450a, P-450b and P-450c and epoxide hydrolase form predominantly the 5,6-dihydrodiol (95-97% of total metabolites) and only a small percentage of the 3,4-dihydrodiol (3-5% of total metabolites). The 3,4-dihydrodiol is formed with higher enantiomeric purity by microsomes from 3-methylcholanthrene-treated rats (88%) than by microsomes from control rats (78%) or phenobarbital-treated rats (60%). In each case the (3R,4R)-enantiomer predominates. B[c]Ph 5,6-dihydrodiol formed by all three microsomal preparations is nearly racemic.     

Macrolide antibiotics inhibit the degradation of the glucocorticoid-responsive cytochrome P-450p in rat hepatocytes in vivo and in primary monolayer culture

Treatment of rats with macrolide antibiotics such as triacetyloleandomycin (TAO) dramatically increases the hepatic concentration of a cytochrome P-450 indistinguishable from P-450p, the major liver cytochrome induced by glucocorticoids such as dexamethasone (Wrighton, S. A., Maurel, P., Schuetz, E. G., Watkins, P. B., Young, B., and Guzelian, P. S. (1985) Biochemistry 24, 2171-2178). To investigate the mechanism of induction of P-450p, we treated rats for 4 days with these agents and found that dexamethasone and TAO induced the synthesis of P-450p at least 70- and 35-fold over control values, respectively, as estimated from measurements of P-450p mRNA translatable in a cell-free system. However, the accumulation of P-450p holocytochrome (measured as TAO-metabolite spectral complex) or P-450p protein (measured by quantitative immunoblotting) increased at least 150-fold by TAO but only 32-fold by dexamethasone. The possibility that TAO decreases the degradation of P-450p was supported by the observation that administration of TAO to dexamethasone-treated rats labeled with NaH[14C]O3 and [3H]-delta-aminolevulinic acid retarded the decay of radioactive immunoprecipitable P-450p protein (t1/2 = 60 versus 14 h) and heme (t1/2 = 73 versus 10 h). To confirm these results, P-450p protein synthesis was measured as radioactivity incorporated into immunoprecipitable P-450p in primary monolayer cultures of adult rat hepatocytes incubated with [3H]leucine. Dexamethasone treatment of the cultures stimulated P-450p synthesis by at least 30-fold whereas macrolides were without effect. However, macrolide antibiotics but not dexamethasone inhibited the disappearance of radiolabeled P-450p from cultured hepatocytes similar to the results obtained in vivo. We conclude that macrolide antibiotics induce P-450p, the most rapidly turning over cytochrome yet reported, by stimulating its synthesis indirectly and by blocking its degradation, significantly.     

Effect of a single oral dose of methanol, ethanol and propan-2-ol on the hepatic microsomal metabolism of foreign compounds in the rat.

Methanol and ethanol administered to rats as a single oral dose increased aniline hydroxylation by the hepatic microsomal fraction by a maximum of 169 and 66% respectively, whereas aminopyrine demethylation was inhibited by 51 and 61%. The concentration of microsomal cytochrome P-450, and the activities of NADPH-cytochrome c reductase and NADPH-cytochrome P-450 reductase were unchanged. Propan-2-ol, administered as a single oral dose, increased microsomal aniline hydroxylation by 165% and increased aminopyrine demethylation by 83%. The concentration of cytochrome P-450 was unchanged whereas NADPH-cytochrome c reductase and NADPH-cytochrome P-450 reductase were both increased by 38%. Methanol, ethanol and propan-2-ol administration resulted in a decreased type I spectral change but had no effect on the reverse type I spectral change. Methanol administration decreased the type II spectral change whereas ethanol and propan-2-ol had no effect. Cycloheximide blocked the increases in aniline hydroxylation and aminopyrine demethylation but could not completely prevent the decreases in aminopyrine demethylation. The increases in aniline hydroxylation were due to an increase in V, but Km was unchanged. The ability of acetone to enhance and compound SKF 525A to inhibit microsomal aniline hydroxylation was decreased by the administration of all three alcohols. The decrease in the metabolism of aminopyrine may result from a decrease in the binding to the type I site with a consequent failure of aminopyrine to stimulate the reduction of cytochrome P-450. Methanol administration may lead to an increase in aniline hydroxylation because of a failure of aniline to inhibit cytochrome P-450 reduction.     

Enzymatic Mechanisms Involved in Phenanthrene Degradation by the White Rot Fungus Pleurotus ostreatus

The enzymatic mechanisms involved in the degradation of phenanthrene by the white rot fungus Pleurotus ostreatus were examined. Phase I metabolism (cytochrome P-450 monooxygenase and epoxide hydrolase) and phase II conjugation (glutathione S-transferase, aryl sulfotransferase, UDP-glucuronosyltransferase, and UDP-glucosyltransferase) enzyme activities were determined for mycelial extracts of P. ostreatus. Cytochrome P-450 was detected in both cytosolic and microsomal fractions at 0.16 and 0.38 nmol min(sup-1) mg of protein(sup1), respectively. Both fractions oxidized [9,10-(sup14)C]phenanthrene to phenanthrene trans-9,10-dihydrodiol. The cytochrome P-450 inhibitors 1-aminobenzotriazole (0.1 mM), SKF-525A (proadifen, 0.1 mM), and carbon monoxide inhibited the cytosolic and microsomal P-450s differently. Cytosolic and microsomal epoxide hydrolase activities, with phenanthrene 9,10-oxide as the substrate, were similar, with specific activities of 0.50 and 0.41 nmol min(sup-1) mg of protein(sup-1), respectively. The epoxide hydrolase inhibitor cyclohexene oxide (5 mM) significantly inhibited the formation of phenanthrene trans-9,10-dihydrodiol in both fractions. The phase II enzyme 1-chloro-2,4-dinitrobenzene glutathione S-transferase was detected in the cytosolic fraction (4.16 nmol min(sup-1) mg of protein(sup-1)), whereas aryl adenosine-3(prm1)-phosphate-5(prm1)-phosphosulfate sulfotransferase (aryl PAPS sulfotransferase) UDP-glucuronosyltransferase, and UDP-glucosyltransferase had microsomal activities of 2.14, 4.25, and 4.21 nmol min(sup-1) mg of protein(sup-1), respectively, with low activity in the cytosolic fraction. However, when P. ostreatus culture broth incubated with phenanthrene was screened for phase II metabolites, no sulfate, glutathione, glucoside, or glucuronide conjugates of phenanthrene metabolites were detected. These experiments indicate the involvement of cytochrome P-450 monooxygenase and epoxide hydrolase in the initial phase I oxidation of phenanthrene to form phenanthrene trans-9,10-dihydrodiol. Laccase and manganese-independent peroxidase were not involved in the initial oxidation of phenanthrene. Although P. ostreatus had phase II xenobiotic metabolizing enzymes, conjugation reactions were not important for the elimination of hydroxylated phenanthrene.     

The rabbit pulmonary monooxygenase system. Immunochemical and biochemical characterization of enzyme components.

The enzymatic components of the rabbit pulmonary monooxygenase system, cytochromes P-450I and P-450II and NADPH-cytochrome P-450 reductase, are immunochemically distinct proteins. In pulmonary microsomes, the N-demethylation of benzphetamine, amino-pyrine, and ethylmorphine, and the O-deethylation of 7-ethoxycoumarin are dependent only on cytochrome P-450I, and the hydroxylation of coumarin is apparently catalyzed by both cytochromes. Cytochrome P-450II is immunochemically distinct from the major forms of hepatic cytochrome P-450 induced by phenobarbital or 3-methylcholanthrene, whereas cytochrome P-450I is indistinguishable from the former on the basis of physical and catalytic as well as immunochemical characteristics. Pulmonary and hepatic NADPH-cytochrome P-450 reductases also have identical physical, catalytic, and immunochemical properties. The lack of response of the lung monooxygenase system to phenobarbital, therefore, is apparently not due to an inability of the lung to synthesize the enzymes induced by phenobarbital in the liver. The relatively high proportion of cytochrome P-450I in the lung appears to be responsible for the higher rates (per nmol of P-450) of N-demethylation that have been observed in rabbit pulmonary as compared to hepatic microsomal fractions.     

A convenient assay for mephenytoin 4-hydroxylase activity of human liver microsomal cytochrome P-450

A simple and rapid method for the determination of (S)-mephenytoin 4-hydroxylase activity by human liver microsomal cytochrome P-450 has been developed. [Methyl-14C] mephenytoin was synthesized by alkylation of S-nirvanol with 14CH3I and used as a substrate. After incubation of [methyl-14C]mephenytoin with human liver microsomes or a reconstituted monooxygenase system containing partially purified human liver cytochrome P-450, the 4-hydroxylated metabolite of mephenytoin was separated by thin-layer chromatography and quantified. The formation of the metabolite depended on the incubation time, substrate concentration, and cytochrome P-450 concentration and was found to be optimal at pH 7.4. The Km and Vmax rates obtained with a human liver microsomal preparation were 0.1 mM and 0.23 nmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450, respectively. The hydroxylation activity showed absolute requirements for cytochrome P-450, NADPH-cytochrome P-450 reductase, and NADPH in a reconstituted monooxygenase system. Activities varied from 5.6 to 156 pmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450 in 11 human liver microsomal preparations. The basic system utilized for the analysis of mephenytoin 4-hydroxylation can also be applied to the estimation of other enzyme activities in which phenol formation occurs.     

pH effects on the N-demethylation and formation of the cytochrome P-450 iron II nitrosoalkane complex for erythromycin derivatives.

The effects of pH on access to the cytochrome P-450 active site, N-demethylation and formation of the cytochrome P-450 Fe(II)-RNO metabolite complex for a series of erythromycin derivatives were examined. Studies were performed with dexamethasone-treated rat liver microsomes containing large amounts of cytochrome P-450 3A isozymes. In addition to factors such as hydrophobicity or hindrance around the dimethyl-amino function, the ionisation state of the N(CH3)2 group played an important role in the recognition and metabolism of the substrate by cytochrome P-450. Esterification of the desosamine in the beta position of the N(CH3)2 group leads to lower pKa values for the R--N+ H(CH3)2 <--> [R--N (CH3)2] + H+ equilibrium. At physiological pH, the amine group is mainly in the unprotonated form. Consequently, easier access to the protein active site and significant formation of cytochrome P-450 Fe(II)-RNO metabolite complex are observed for these derivatives. These results led us to interpret the formation of cytochrome P-450 Fe(II)-RNO metabolite complex as a series of multiple steps equilibria depending on the ionisation state of the N(CH3)2 group, the partition coefficient of the substrate between the microsomal layer and the aqueous media and a series of metabolic reactions leading partially to the final inhibitory nitrosoalkane-cytochrome P-450 Fe(II) complex.     

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.     

Role of phospholipids in reconstituted cytochrome P450 3A form and mechanism of their activation of catalytic activity.

Cytochrome P-450 coded for by the 3A gene family requires specific conditions in a reconstituted system, if its catalytic activity is to be efficient. We investigated the mechanism of activation of the catalytic activity of cytochrome P450 3A by phospholipids. Rat P450 PB-1 (3A2), human P450NF (3A4), and rabbit P450 3c (3A6) were used. They had low activity in a reconstituted system (system I) with dilauroylphosphatidylcholine (DLPC) but had high activity with a mixture of phospholipids (DLPC, dioleoylphosphatidylcholine, and phosphatidylserine) and sodium cholate (system II). P450 3A forms are cationic (having a high content of lysine residues) and needed the anionic phospholipid phosphatidylserine to have sufficient activity. Double-reciprocal plots of the metabolic rate of cytochrome P-450 versus the concentration of NADPH-cytochrome P-450 reductase showed that cytochrome P-450 and the reductase interacted more in system II than in system I. P450 PB-1 did not absorb at 450 nm in the presence of reductase, CO, DLPC, and NADPH, although other cytochrome P-450s absorbed at around 450 nm in such a mixture. However, P450 PB-1 was reduced in the presence of the phospholipid mixture and sodium cholate instead of DLPC. These results suggested that the stimulation of catalytic activity by phospholipids involved increased interaction between cytochrome P-450 and the reductase. Studies of proteolytic digestion and chemical cross-linking in systems I and II showed that a P450 3A form needed disaggregation of cytochrome P-450 and/or the reductase, not the formation of an aggregated complex necessary for the catalytic activity of other cytochrome P-450s.