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.     

Initial purification and characterization of hepatic microsomal cytochrome P-450 from BNF-treated perch (Perca fluviatilis)

1. A procedure was developed for isolating and purifying cytochrome P-450 from hepatic microsomes of BNF-treated perch, using modified versions of the methods of Williams and Buhler (1982. Biochim. biophys. Acta 717, 398-404) and Goks?yr (1985. Biochim. biophys. Acta 850, 409-417). 2. Following chromatography on phenyl-Sepharose CL 4B and DEAE-Sepharose CL-6B, the major peaks, fractions b and c, were resolved into five fractions, possibly representing different isoenzymes, by a FPLC with a strong anion exchange column (Mono Q). 3. These fractions have been characterized on the basis of their spectral, electrophoretic and immunological properties. 4. The purified form of cytochrome P-450 in fraction V from perch liver showed a number of similarities to cytochrome P-450c, the major BNF-inducible cytochrome P-450 in cod liver. 5. Therefore we suggest that this purified form of cytochrome P-450 is a BNF-induced form in perch and that it is closely related to the gene subfamily cytochrome P-450 IA1.     

Pulmonary microsomal cytochrome P-450 from 3-methylcholanthrene-treated hamsters: purification, characterization, and metabolism of benzo(a)pyrene

A major form of pulmonary cytochrome P-450 (pulmonary P-450MC) was purified approximately 165-fold from lung microsomes of 3-methylcholanthrene (MC)-treated hamsters. The purified preparation contained 14.2 nmol of cytochrome P-450 (P-450) per mg protein and was essentially free from NADPH-cytochrome P-450 (cytochrome c)-reductase (NADPH-reductase) and epoxide hydrolase. Pulmonary P-450MC exhibits an absorption maximum at 446.5 nm in the difference spectrum of reduced hemoprotein-CO complex, and a low-spin state of ferric iron in the heme. By sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, the molecular weight of pulmonary P-450MC was estimated to be 56,000. In a reconstituted system, pulmonary P-450MC efficiently catalyzed benzo(a)pyrene (BP) hydroxylation, but showed low activities for 7-ethoxycoumarin O-deethylation and benzphetamine N-demethylation. In Ouchterlony double diffusion analysis, hamster pulmonary P-450MC reacted to the antibody prepared against rat hepatic P-450MC to form a faint precipitation line with a spur, indicating that the two P-450MCs have a common antigenic site but are not immunologically identical. When incubated with [14C]BP in a reconstituted system containing NADPH-reductase and epoxide hydrolase, hamster pulmonary P-450MC formed much higher amounts of BP diols, especially 7,8-diol, than were formed by rat pulmonary P-450MC.     

Immobilization of liver microsomal cytochrome P-450

Rabbit liver microsomal cytochrome P-450 was immobilized by entrapment in calcium alginate gel. Aminopyrine demethylation experiments showed that the immobilized enzyme system is highly active and exhibits an unimpaired functional stability as compared with crude microsomes. The alginate entrapped microsomes were employed in a fixed bed recirculation reactor, where aminopyrine was continuously demethylated. Such model enzyme reactor can be a useful tool for studying extracorporeal drug detoxification or preparative substrate conversion with microsomal enzyme systems.     

Partial purification and characterization of cytochrome P-450 from human placenta

Two isozymes of cytochrome P-450 were partially purified to specific contents of 7.0 and 0.5 nmol/mg of protein, respectively, from placenta of non-smoking women by chromatography on octyl Sepharose, hydroxylapatite, DEAE-cellulose and CM-cellulose. NADPH-cytochrome P-450 reductase was purified from phenobarbital-induced mouse liver and from human placenta and was combined with cytochrome P-450 and dilauroylphosphatidylcholine to reconstitute the cytochrome P-450 monooxygenase system. Substrates investigated were benzo[a]pyrene, 7-ethoxycoumarin and delta 4-androstene-3,17-dione.     

Partial purification and characterization of phenobarbital-induced house fly cytochrome P-450

Two forms of phenobarbital-induced cytochrome P-450 were partially purified from the Rutgers diazinon-resistant strain of house fly using cholate solubilization, polyethylene glycol 6000 precipitation, and chromatography on DEAE cellulose. The preparation of highest purity had an absorbance maximum of 452 nm, a specific content of 10.0 nmol/mg protein, and an apparent molecular weight of 60,000 when examined by sodium dodecyl sulfate polyacrylamide electrophoresis. The yield of the highly purified cytochrome P-450 was 2–3%. This form contained proportionately less cytochrome P-420 than the original cholate solubilized microsomes, and is thus apparently more stable. A second form of cytochrome P-450 having a specific content of 0.50–0.89 nmol/mg protein was eluted from DEAE cellulose with a 0-0.25 M salt gradient. This is consistent with a previously reported elution pattern for Emulgen 913-solubilized house fly microsomes. Several methods of solubilizing house fly microsomes were examined. High salt, 2M KCI, in the absence of detergents effectively solubilized cytochrome P-450 (50–70% recovery) with little or no conversion to cytochrome P-(420).     

Adrenal microsomal hydroxylating system: purification and substrate binding properties of cytochrome P-450C-21

The substrate-cytochrome P-450C-21 binding reaction has been investigated in detail by using the purified cytochrome. The apparent substrate dissociation constant (KDapp) depended on the enzyme concentration, indicating that the binding reaction does not follow simple two-component mass action equilibrium. However, the binding data fit reasonably well to a model in which the P-450C-21 exists in a monomer-dimer equilibrium and the substrate does not bind to the dimer. The intrinsic dissociation constant (K1) and the dissociation constant for the dimerization reaction (K2) were calculated from the titration data by a pattern search procedure. K1 and K2 were found to be essentially independent of the enzyme concentration, indicating the appropriateness of the assumed model. In the present study, all factors that increased the dissociation of the dimer, as indicated by an increase in K2, decreased KDapp so that it approached the intrinsic constant K1. These results suggest that there is mutual interaction of the substrate binding and self-association reactions of cytochrome P-450C-21 in the purified preparation.     

Evidence for induction of hepatic microsomal cytochrome P-450 by cimetidine: binding and kinetic studies

The interaction of cimetidine with liver microsomes has been examined by spectral and equilibrium partition studies. First, difference spectroscopy has been used to evaluate the proportion of cytochrome P-450 in rat liver microsomes that exhibits an affinity for cimetidine in the pharmacologically relevant, low micromolar range of drug concentration. The value of 0.45 so obtained has confirmed that a substantial proportion of rat liver cytochrome P-450 has a high binding affinity for this drug. Second, a study of the binding of cimetidine to human liver microsomes by difference spectroscopy and partition equilibrium has detected a similar interaction, thus providing direct support for the postulate that the clinically observed impairment of oxidative drug metabolism may be due in part to inhibition of cytochrome P-450 monooxygenase by cimetidine. Hepatic microsomes from cimetidine-pretreated rats have been shown to exhibit elevated cytochrome P-450 specific content but a decreased proportion of sites with high affinity for the drug; this finding has been shown not to be the consequence of cimetidine-mediated, time-dependent, irreversible monooxygenase inhibition. Although cimetidine pretreatment caused enhanced specific activity of 7-ethoxyresorufin O-dealkylation, the specific activities for O-dealkylation of 7-ethoxycoumarin and 4-nitroanisole were decreased, as were those for the N-dealkylation of morphine, ethylmorphine, aminopyrine, and dimethylnitrosamine. Since cimetidine pretreatment was shown to cause no change in the Michaelis constants for oxidation of morphine or 7-ethoxyresorufin, it is argued that these results provide strong presumptive evidence for changes in the relative abundance of isoenzymes catalyzing these various oxidations. Thus, a dual role of cimetidine, acting both as inhibitor and inducer of the cytochrome P-450 system, is proposed to account for the impaired oxidative metabolism of some drugs that occurs during coadministration with this H2-receptor antagonist.     

Mephenytoin-type polymorphism of drug oxidation: purification and characterization of a human liver cytochrome P-450 isozyme catalyzing microsomal mephenytoin hydroxylation

A genetic polymorphism causing deficient metabolism of the anticonvulsant drug mephenytoin occurs in 5% of the Caucasian and 23% of the Japanese population. By monitoring the activities of the two major oxidative pathways of mephenytoin metabolism in the column eluates, we have purified from human livers a cytochrome P-450 isozyme, P-450 meph, which exclusively and stereoselectively catalyzes the 4-hydroxylation of (S)-mephenytoin, the major pathway affected by the polymorphism, whereas P-450 meph was virtually devoid of catalytic activity for N-demethylation of mephenytoin, the pathway remaining unaffected by the genetic deficiency. P-450 meph had an apparent Mr of 55 000 and a lambda max in the reduced CO-binding spectrum of 450 nm. Polyclonal rabbit antibodies against purified human P-450 meph almost completely inhibited the 4-hydroxylation of mephenytoin but had little effect on N-demethylation in human liver microsomes. In microsomes of liver biopsies of two subjects characterized in vivo as 'poor metabolizers' of mephenytoin, immunocrossreactive and immunoinhibitable material was observed with similar or identical properties to those of P-450 meph. There was no difference in the extent of the immunochemical reaction between microsomes of in vivo phenotyped poor metabolizers and extensive metabolizers of mephenytoin. These data suggest that P-450 meph is the target of the genetic deficiency and support the concept that a functionally altered variant form of P-450 meph causes this polymorphism.     

Isolation and characterization of a constitutive form of rabbit liver microsomal cytochrome P-450

A heretofore unrecognized form of cytochrome P-450 was purified from rabbit liver microsomes with an average yield and purity similar to that of other highly purified forms of cytochrome P-450. Several properties of this cytochrome are contrasted with those of form 2, the major phenobarbital-inducible cytochrome P-450, form 4, the major 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible cytochrome, and form 6, a cytochrome that is selectively induced in liver microsomes by 2,3,7,8-tetrachlorodibenzo-p-dioxin during the perinatal period. Thes four forms can be distinguished by virtue of their molecular weights as determined using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, by their respective peptide fingerprints, and by the monospecificity of their antisera. Since the enumerated properties are thought to reflect the primary structure of the cytochromes and since the observed differences are extensive, we suggest that these four forms are not derived from a common protein precursor.     

Purification and characterization of hepatic microsomal prostaglandin omega-hydroxylase cytochrome P-450 from pregnant rabbits

Prostaglandin omega-hydroxylase, designated as cytochrome P-450 LPG omega (P-450 LPG omega), has been purified, to a specific content of 15 nmol of cytochrome P-450/mg of protein, from liver microsomes of pregnant rabbits. The purified P-450 LPG omega was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and to have an apparent molecular weight of 52,000. The enzyme showed a maximum at 450 nm in the carbon monoxide (CO)-difference spectrum for its reduced form. This cytochrome P-450 efficiently catalyzed the omega-hydroxylation of prostaglandin E1 (PGE1), prostaglandin E2 (PGE2), prostaglandin D2 (PGD2), prostaglandin F2 alpha (PGF 2 alpha), prostaglandin A1 (PGA1), and prostaglandin A2 (PGA2), as well as the omega- and (omega-1)-hydroxylation of myristate and palmitate, in a reconstituted system containing cytochrome P-450, NADPH-cytochrome P-450 reductase, phospholipid, and cytochrome b5. Various monovalent and divalent cations further stimulated these reactions in the presence of cytochrome b5. In addition, the reactions were also markedly enhanced by various organic solvents, such as ethanol and acetone. This cytochrome P-450 showed no detectable activity toward several xenobiotics tested. P-450 LPG omega was very similar or identical to the pulmonary prostaglandin omega-hydroxylase (P-450p-2) (Yamamoto, S., Kusunose, E., Ogita, K., Kaku, M., Ichihara, K., & Kusunose, M. (1984) J. Biochem. 96, 593-603) in its molecular weight, absorption spectra, catalytic activity, peptide mapping pattern, and N-terminal amino acid sequence. However, P-450 LPG omega was more unstable than P-450p-2 on storage. In sharp contrast to P-450p-2, P-450 LPG omega was not induced by progesterone.     

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).     

A phenobarbital-inducible hepatic mitochondrial cytochrome P-450 immunochemically related to microsomal P-450b

We have purified and characterized a phenobarbital (PB)-inducible hepatic mitochondrial cytochrome P-450 (P-450), termed P-450mt4, which is distinctly different from the previously characterized mitochondrial isoforms. The level of induction of P-450mt4 by PB in the male livers is nearly 20-fold, as against a marginal induction in the female livers, suggesting that it may be a male predominant isoform. P-450mt4 shows a close resemblance to microsomal P-450b (the major PB-inducible form) with respect to electrophoretic migration (apparent molecular mass of 50 kDa) and immunological cross-reactivity, although it exhibits a distinct isoelectric pH (pI 6.9 vs 6.5 for P-450b), peptide fingerprint pattern, and amino acid composition. Further, the N-terminal sequence analysis shows over 90% positional identity (39 out of 42) between P-450mt4 and P-450b, suggesting that it is a close relative of the P-450 IIB gene family. In vitro reconstitution experiments show that P-450mt4 can metabolize a wide range of substrates such as benzphetamine, (dimethylamino)antipyrine, aflatoxin B1, and vitamin D3, exclusively in the presence of mitochondrial-specific ferredoxin and ferredoxin reductase as electron carriers. P-450mt4 is translated as a 53-kDa precursor, which is transported into mitochondria under in vitro conditions and processed into a mature 50-kDa protein. These results provide conclusive evidence for the occurrence of a male-specific P-450 belonging to the IIB gene family in rat liver mitochondria.     

Interaction of purified microsomal cytochrome P-450 with cytochrome b5

The interactions between purified microsomal cytochrome P-450 and cytochrome b₅ has been demonstrated by aqueous two-phase partition technique. Major forms of cytochrome P-450 induced by phenobarbital (P-450_LM2) and β-naphthoflavone (P-450_LM4) are almost exclusively distributed in the dextran-rich bottom phase (partition coefficient, K = 0.06), whereas NADPH-cytochrome P-450 reductase and cytochrome b₅ are mainly distributed in the polyethylene glycol-rich top phase (K = 3.5 and 2.5, respectively), when these enzymes were partitioned separately in the dextran-polyethylene glycol two-phase system. The mixing of P-450_LM with cytochrome b₅ changes the partition coefficients of both P-450_LM and cytochrome b₅ indicating that molecular interaction between P-450_LM and cytochrome b₅ occurred. Complex formation was also confirmed by optical absorbance difference spectral titration, and the stimulation of the P-450_LM-dependent 7-ethoxycoumarin and p-nitrophenetole O-deethylase activities by equal molar quantity of detergent-solubilized cytochrome b₅, but not trypsin-solubilized enzyme, in the reconstituted system. Cytochrome b₅ decreases the K_m's of both substrates for P-450_LM2-dependent O-deethylations and increases the V's of both reactions by two- to three-fold. This stimulatory effect requires the presence of phospholipid in the reconstituted enzyme system. These results suggest that cytochrome b₅ plays a role in some reconstituted drug oxidation enzyme systems and that molecular interactions among cytochrome P-450, reductase, and cytochrome b₅ are catalytically competent in the electron transport reactions.