Phenobarbital-induced Rabbit Liver Research Articles

Direct Identification of Cytochrome P450 Isozymes by Matrix-assisted Laser Desorption/Ionization Time of Flight-Based Proteomic Approach

The main targets of our investigation were cytochrome P450 isozymes (P450), the key enzymes of the hepatic drug-metabolizing system. Current research approaches to the identification of individual P450 forms include specific P450 inhibitors or substrates, antibody-based identification, and mRNA-based expression profiling. All of these approaches suffer from one common disadvantage-they all are indirect methods. On the other hand, current developments in mass spectrometry provide a direct and reliable approach to protein identification with sensitivity in the femtomole or low picomole range. In this study we have used high-accuracy, matrix-assisted laser desorption/ionization time of flight (MALDI TOF)-based peptide mapping to perform direct identification of distinct P450 isozymes in various rat and rabbit liver microsomes. For the first time, the P450 isozyme composition of clofibrate-induced rat and phenobarbital-induced rabbit liver microsomes was determined by peptide mass fingerprinting (PMF). Application of MALDI TOF-based PMF allows differential identification of such highly homologous P450s as CYP2B1 and CYP2B2. We have found that CYP2A10 previously reported only in rabbit olfactory and respiratory nasal mucosa is present in phenobarbital (PB)-induced rabbit liver microsomes. Two other rabbit P450s, earlier identified only by screening a cDNA library, were found to be present in PB-induced rabbit liver microsomes. In summary, direct identification of P450s by proteomic technique offers advantages over other methods with regard to identification of distinct P450 isozymes and should become a standard approach for characterizing microsomes.

Production of carbon monoxide by cytochrome P450 during iron-dependent lipid peroxidation

Carbon monoxide (CO) formation was studied in the process of lipid peroxidation in phenobarbital-induced rabbit liver microsomes. The reaction was NADPH-dependent and required Fe 2+, which occurs in microsomes as being protein bound and is not a consequence of heme destruction. Zn-protoporphyrin IX, an inhibitor of the heme oxygenase activity, proved to have no effect on CO production, suggesting that heme oxygenase is not involved into the CO generation reaction. At the same time, the addition of cytochrome P450 typical inhibitors SKF 525A and metyrapone to the reaction mixture had an inhibitory effect on the CO formation rate. Antioxidants such as α-tocopherol and desferal inhibited lipid peroxidation in phenobarbital-induced rabbit liver microsomes, and in this case the CO production was not registered. Thus, on the basis of the results presented here it is possible to assert that the process of NADPH, Fe 2+-dependent carbon monoxide formation in microsomes is a result of lipid peroxidation with cytochrome P450 2B4 participation.

Effect of microsome–liposome fusion on the rotational mobility of cytochrome P450IIB4 in rabbit liver microsomes

Membrane fusion of microsomes with soybean phospholipid vesicles was performed at pH 6.5 to investigate the effect of lipid-enrichment in the membrane on the rotational mobility of cytochrome P450. Rotational diffusion of cytochrome P450 in the microsomal membrane of phenobarbital-induced rabbit liver was measured by detecting the decay of absorption anisotropy after photolysis of the heme CO complex by a vertically polarized laser flash. The fusion procedures yielded three separate fractions upon sucrose density gradient centrifugation with lipid-to-protein ratio in weight (L/P) as follows: 1.5 in the bottom fraction, 2.2 in the middle fraction, and 3.9 in the top fraction. In each fraction, co-existence of mobile and immobile cytochrome P450 was observed. The percentage of rotationally mobile P450 (with the mean rotational relaxation time of φ=505–828 μs) in each of the different bands was found to be 59% in the bottom fraction, 61% in the middle fraction, and 68% in the top fraction. This increase in mobile population of P450 due to lipid-enrichment indicates that aggregated proteins in microsomal membranes dissociate with increasing L/P which is inversely proportional to the protein concentration in the membrane. With freeze-fracture electron microscopy, it was shown that the average distance increased between intramembrane particles by lipid-enrichment. Thus, the significant immobile population (32%) of P450 in microsomal membranes can be explained by nonspecific protein aggregation which is a consequence of the low L/P of 0.8. The decrease in the mobile population in the bottom fraction compared with intact microsomes was shown to be due to the pH 6.5 incubation used for fusion.

Identification and in vitro immunosuppressive activity of a SDZ-IMM-125 metabolite isolated from phenobarbital-induced rabbit liver microsomes

1. A metabolite of D-serine-cyclosporine A has been isolated from phenobarbital induced rabbit liver microsomes using hplc. 2. This metabolite was identified by FAB, electrospray mass spectrometry as well as nmr spectroscopy and is the result of metabolism of the vinylic methyl group of the 9- carbon amino acid unique to the cyclosporins, the first amino acid of this cyclic undecapeptide. This metabolite exhibits a significantly lower immunosuppressive activity than IMM-125 and CsA.

Interaction of organophosphorus analogues of amino acids with P450.

1. This study deals with the oxidation of organophosphorus amino acid analogues by phenobarbital-induced rabbit liver microsomes. It has been shown that 1-aminoalkylphosphonous and 1-aminoalkylthiophosphonic acids are converted by P450 to 1-aminoalkylphosphonic acids. 2. Phosphonous analogues of amino acids cause type I spectral changes, and thiophosphonic analogues produce reverse type I changes in different spectra. 3. In the presence of NADPH, the 1-aminoalkylphosphonous acids form the corresponding 1-aminoalkylphosphonic acids by the reaction P-H-->P-OH, as monitored using 1H nmr spectroscopy. 4. Aminoalkylthiophosphonic acids have also proven to be the substrates for the NADPH-dependent monoxygenase system. During the course of oxidative desulphuration 1-aminoalkylphosphonic acids were formed by the reaction P = S-->P = O, as monitored by 31P-nmr spectroscopy. 5. Using resonance Raman (RR) spectroscopy, the interaction of 1-aminoisobutyl-phosphonous acid with P450 was investigated, and characteristic changes in spectral frequencies in the region between 1370 and 1700 cm-1 were demonstrated. These latter changes indicate that substrate binding of organophosphorus compounds leads to alterations in haem conformation and to redistribution of the electron density.

Heterogeneity in rabbit liver cytochrome P-450 LM2 observed by cation exchange HPLC: partial biochemical characterization of the two major LM2 subfractions.

Cytochrome P450 LM2 (CYPIIB4) from phenobarbital-induced rabbit liver microsomes, purified to only one band in SDS-PAGE, was further resolved in five peaks by cation exchange HPLC. The two major peaks were partially characterized. Both of them have the amino terminal sequence Met-Glu and the same Cys content. They exhibited the same spectral absorption maximum and similar binding constants for 1-benzylimidazole and imidazole. However, binding of benzphetamine was different. One subfraction presented a Michaelis-Menten type binding curve, but the other presents a non-typical one with an additional high affinity binding site. These subfractions of cytochrome P450 LM2 slightly differed in their catalytic activities with benzyloxy- and pentoxyresorufin substrates. On the contrary, no heterogeneity was observed for P450 LM4.

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver: II. Kinetic parameters of reductase and monooxygenase reactions

The kinetic parameters of NADPH-dependent cytochrome P450 LM 2 (2B4) reduction and substrate oxidation in the monomeric reconstituted system, consisting of purified NADPH-cytochrome P450 reductase and cytochrome P450 LM 2 monomers, and in phenobarbital-induced rabbit liver microsomes were compared. In the absence of benzphetamine, NADPH-dependent reduction of cytochrome P450 LM 2 was monophasic in the monomeric reconstituted system and biphasic in the microsomes. The presence of the substrate in the monomeric reconstituted system caused the appearance of the fast phase. In this system substrate-free cytochrome P450 LM 2 was entirely low-spin, and the addition of benzphetamine shifted the spin equilibrium to a high state very weakly. No correlation between high-spin content and the proportion of the fast phase of NADPH-dependent LM 2 reduction was found in the system. V max values for the oxidation of type I substrates (benzphetamine, dimethylaniline, aminopyrine) in the monomeric reconstituted system were higher or the same as in the microsomes, whereas K m values for the substrates and NADPH were lower in the microsomes. Maximal activity of the monomeric reconstituted system was observed at a 1:1 NADPH-cytochrome P450 reductase/cytochrome P450 LM 2 ratio. Measurements of benzphetamine oxidation as a function of NADPH-cytochrome P450 reductase/cytochrome P450 LM 2 ratio at a constant total protein concentration allowed the K d of the NADPH-cytochrome P450 reductase/cytochrome P450 LM 2 complex to be estimated as 6.4 ± 0.5 μ m. Complex formation between the NADPH-cytochrome P450 reductase and cytochrome P450 LM 2 monomers was not detected by recording the difference binding spectra of the reductase monomers with LM 2 monomers or by treatment the mixture of the monomers of the proteins with the crosslinking reagent, water-soluble carbodiimide.

Stereochemisty of oxygenation of organic sulphides with pig liver microsomal FAD-containing mono-oxygenase: comparison with cytochrome P-450PB oxidations

The enantiotopic differentiating ability of pig liver microsomal FAD-containing mono-oxygenase (EC 1.14.13.8) in the oxygenation of nine unsymmetrical sulphides has been investigated. By this enzymatic oxygenation, the sulphides are converted into the corresponding optically active sulphoxides with varying degrees of enantiomeric excess (96–12%).Three cyclic sulphides have been studied in order to establish the diastereotopic differentiating ability of the enzyme. In the oxygenation of racemic 2-methyl-2,3-dihydrobenzo[b]thiophene both enantiomeric and diastereotopic differentiation appears to take place concurrently with high selectivity. The enantiotopic, diastereotopic, and enantiomeric differentiating abilities of the FAD-containing mono-oxygenase are all higher than those of the phenobarbital-induced rabbit liver microsomal cytochrome P-450.

On the Mechanism of Action of Cytochrome P-450: Spectral intermediates in the reaction with iodosobenzene and its derivatives

Abstract Cytochrome P-450 is known to catalyze the following oxygen transfer reaction: RH + PhIO----ROH + PhI where RH represents a variety of hydroxylatable substrates and PhIO a variety of iodosobenzene derivatives that serve as oxygen donors, and neither molecular oxygen nor an external electron donor is required. To determine whether the cytochrome functions in such reactions by a peroxidase-type mechanism, the kinetics of its interaction with a variety of substituted iodosobenzenes and iodobenzene diacetates have been determined by stopped flow spectrophotometry. The reaction of phenobarbital-induced rabbit liver microsomal cytochrome P-450 form 2 with iodosobenzenes or iodobenzene diacetates leads to the reversible formation of three spectral intermediates, termed E, F, and G. Complex E is characterized by a type I difference spectrum, representing the iodosobenzene-dependent partial shift of the low spin hexacoordinate form of the ferric enzyme to the high spin pentacoordinate form, F represents a transient intermediate whose spectrum cannot be determined for kinetic reasons, and G represents a blue-shifted intermediate with an absorption maximum at about 393 nm in the absolute spectrum. The striking and principal feature of these observations is that the spectrum of Complex G does not vary with structural differences in the iodosobenzene derivatives, in contrast to the transient species observed in previous studies in this laboratory in the reaction between cytochrome P-450 form 2 and aromatic peroxy compounds. Complex G exhibits the spectral properties one might anticipate for an iron-oxo intermediate containing only one oxygen atom derived from the starting iodosobenzene.

Surface enhanced resonance Raman study of phenobarbital-induced rabbit liver cytochrome P-450 LM2

Surface enhanced resonance Raman (SERR) spectroscopy has been used to study the vibrational spectra of the heme of purified rabbit liver cytochrome P-450 LM2 which was adsorbed on colloidal silver suspensions or on a silver electrode. Based on a comparison with the resonance Raman (RR) spectra of the ‘solute’ species the high sensitivity of the SERR technique is demonstrated. Two different features were chosen in order to determine the structural and functional integrity of the adsorbed P-450. Both, substrate-induced spin state changes on the oxidized P-450 and the effect of the thiolate ligand on the oxidation state marker band ν 4 in the reduced P-450 could be observed in the SERR spectra of the adsorbed as well as in the RR spectra of the dissolved enzyme. These findings indicate that the protein structure near the substrate binding site and the coordination by thiolate are not affected by the interaction with the metal surface. Both structural elements are crucial for the function of P-450. Thus the elementary processes of the enzymatic action of P-450 can be investigated by this highly sensitive version of RR spectroscopy.

The functional role of cytochrome b5 reincorporated into hepatic microsomal fractions

Incorporation of detergent-solubilized cytochrome b 5 into phenobarbital-induced rabbit liver microsomal fractions decelerates hexobarbital-dependent reduction of ferric cytochrome P-450; this is accompanied by retardation of NADPH utilization and H 2O 2 formation in the assay media. Integration of manganese-substituted cytochrome b 5 into the microsomal preparations fails to affect these parameters. Analysis of the cytochrome P-450 reduction kinetics in the presence of increasing amounts of cytochrome b 5 reveals a gradual augmentation of the amplitude of slow-phase electron transfer at the expense of the relative contribution of the fast phase; finally, a slow, apparently monophasic reaction persists. This defect in enzymatic reduction is not due to detergent effects and also does not seem to reflect cytochrome b 5-induced perturbation of anchoring of NADPH-cytochrome c( P-450) reductase to cytochrome P-450. Experiments with the highly purified cytochrome P-450 isozyme LM 2, in which amino acid residue(s) close to the heme edge had undergone suicidal inactivation through covalent attachment of chloramphenicol metabolite(s) do not exclude the possibility that cytochrome b 5 and reductase might compete for a common electron transmission site on the terminal acceptor. Hence, the inhibitory action of cytochrome b 5 on the reduction of ferric cytochrome P-450 is tentatively attributed to partial substitution of the former pigment for reductase in direct transport of the first electron to the monooxygenase.

Influence of N, N-dimethylaniline on the association of phenobarbital-induced cytochrome P-450 and NADPH-cytochrome c( P-450) reductase in a reconstituted rabbit liver microsomal enzyme system

N, N-Dimethylaniline when added to reaction mixtures provokes deviation from Michaelis-Menten law of the interaction kinetics of NADPH-cytochrome c( P-450) reductase (NADPH: ferrihaemoprotein oxidoreductase, EC 1.6.2.4) with highly purified phenobarbital-induced rabbit liver microsomal cytochrome P-450 ( P-450LM 2). This phenomenon is not associated with the low-to-high spin transition in the iron-coordination sphere of the haemoprotein, as elicited by the arylamine. Substrate-triggered departure from linearity of the kinetics is abolished by inclusion into the assay media of p-chloromercuribenzoate hinting at a vital role in the process of thiols. Similarly, the parabolic progress curve ( n H = 1.7) is transformed to a straight line ( n H = 1.01) when the N-terminal reductase-binding domain in the P-450LM 2 molecule is selectively blocked through covalent attachment of fluorescein isothiocyanate (FITC); such a modification does not alter the affinity of the haemoprotein for the amine substrate. Steady-state fluorescence polarization measurements reveal that N, N-dimethylaniline perturbs the motional properties of the fluorophore-bearing reductase-binding region, suggesting the induction of a conformational change. Summarizing these results, the data possibly indicate N, N-dimethylaniline-induced cooperativity in the association of reductase with P-450LM 2.

Reconstitution of liver monooxygenase system in solution from cytochrome P-450 and NADPH-specific flavoprotein monomers

Microsomal monooxygenase system was reconstituted in the presence of non-ionic detergent Emulgen 913 from cytochrome P-450 and NADPH-specific flavoprotein isolated from phenobarbital-induced rabbit liver microsomes. At Emulgen 913 concentration of 0.05 g/l mixed complex between flavoprotein and cytochrome was formed with 5: 5 protein molar ratio and molecular weight of 700 kD. The 2-hour incubation of the enzymes with 0.25 g/l Emulgen 913 at 4 degrees C was accompanied by dissociation of protein oligomers to monomers. The reconstituted systems containing flavoprotein and cytochrome as mixed complexes or monomers were able to catalyze NADPH-dependent cytochrome P-450 reduction and benzphetamine N-demethylation. Taking into consideration the effective concentrations of the enzymes the apparent second order rate constants of these reactions with monomers were 100 times those with complexes.

Effect of a zwitterionic detergent on the state of aggregation and catalytic activity of cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase.

The zwitterionic detergent 3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonate (CHAPS) supports reconstituted cyclohexane hydroxylase activity of cytochrome P-450LM2 and NADPH-cytochrome reductase purified from phenobarbital-induced rabbit liver. Maximum activity (approximately 50% of that with phospholipid) was observed at 2 mM CHAPS. Inhibition took place at higher CHAPS, until at 20 mM CHAPS, no cyclohexane hydroxylase activity was observed. There was little denaturation of the two enzymes under these conditions. At 2 mM CHAPS, P-450LM2 was pentameric (Mr = 250,000) and reductase was dimeric (Mr = 139,500) by sedimentation equilibrium. P-450 was monomeric in 20 mM CHAPS. In addition, a stable complex between the two enzymes was not detected under conditions of maximum activity, even in the presence of saturating substrate. This confirms our previous conclusion that a stable complex between cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase is not a prerequisite for reconstituted xenobiotic hydroxylation (Dean, W. L., and Gray, R. D. (1982) J. Biol. Chem. 257, 14679-14685). Difference spectra of ferric P-450LM2 revealed that below 5 mM CHAPS, the high spin form of the cytochrome was slightly stabilized, while higher CHAPS levels stabilized the low spin form. Monomeric P-450LM2 formed with 20 mM CHAPS catalyzed the hydroxylation of toluene by cumene hydroperoxide. Thus, the reason that monomeric cytochrome P-450LM2 was inactive in NADPH-supported hydroxylation may either be because the bound detergent blocked productive interaction of the cytochrome with reductase or the monomer may be intrinsically incapable of interaction with reductase.

The covalent structure of hepatic microsomal epoxide hydrolase. I. Isolation and characterization of the cyanogen bromide fragments.

Microsomal epoxide hydrolase was purified to homogeneity from phenobarbital-induced rabbit liver for the purpose of determining the complete amino acid sequence. All of the expected 11 cyanogen bromide fragments of epoxide hydrolase were isolated by a combination of gel filtration and high pressure liquid chromatography. Each was characterized by its amino acid composition and NH2-terminal amino acid sequence. The complete amino acid sequences of the eight small fragments, from 5-29 residues, were determined.

Complete stoichiometry of free NADPH oxidation in liver microsomes

The stoichiometry of free NADPH oxidation in phenobarbital induced rabbit liver microsomes was measured by means of registering the rates of NADPH, H + and O 2 consumption and O 2 • and H 2O 2 production. ΔO 2 • :ΔH 2O 2 ratio is approximately I indicating that about half H 2O 2 results from O 2 • dismutation, the second half being formed directly. ΔNADPH:ΔH 2O 2 and ΔO 2:ΔH 2O 2 ratios exceed I and therefore another product of the reaction is water. The fact that the ratio (ΔNADPH-ΔH 2O 2):(ΔO 2-ΔH 2O 2) is 2 allows one to consider direct 4-electron O 2 reduction as the major way of water formation rather than endogenous substrate hydroxylation.

Mechanisms of Enzymatic S-Oxygenation of Thioanisole Derivatives and O-Demethylation of Anisole Derivatives Promoted by Both Microsomes and a Reconstituted System with Purified Cytochrome P-450

Abstract Oxygenations of thioanisole derivatives have been shown to be promoted with phenobarbital induced rabbit liver microsomes. In order to understand the mechanistic details of the oxygenations, a kinetic study on the S-oxygenation promoted by a reconstituted system with purified cytochrome P-450 has been carried out. Log(Vmax) were correlated with the one electron oxidation potentials of the sulfides (Ep). The oxygenation is considered to proceed via one electron transfer from the sulfides to the iron-bound “oxenoid” intermediate of the enzyme. A similar O-demethylation of anisoles has been investigated. In the oxidative O-demethylation of p-methoxy-d3-anisole with rabbit liver microsomes, an intramolecular kinetic isotope effect of 3.4 was observed. Meanwhile, a large isotope effect of 5.1 was observed in the competitive O-demethylation of p-dimethoxybenzene and p-di(methoxy-d3) benzene. In order to clarify the oxidation pathway of O-demethylation with the enzymatic system of cytochrome P-450, the rates of the O-demethylation of p-substituted anisoles have been measured. Unlike the enzymatic oxygenation of sulfides, there was no correlation between the rates of O-demethylation reaction and one electron oxidation potentials of anisoles. These observations suggest that the O-demethylation of anisoles proceeds via the initial hydrogen abstraction with the iron-bound “oxenoid” intermediate of the cytochrome P-450.

The partial purification of rabbit liver cytochrome P450-LM 2-mRNA

Summary We have partially purified the mRNA coding for the phenobarbital induced rabbit liver cytochrome P450-LM 2 approximately 90 fold relative to control liver levels. Using a combination of Sepharose 4B chromatography, denaturing and nondenaturing sucrose gradient centrifugation, we have obtained a mRNA that when translated in vitro using wheat germ lysate codes for 7.2% cytochrome P450-LM 2 .

Oxygenation of alkyl sulfides with ferrous perchlorate/ascorbic acid/oxygen system

In the oxidation of alkyl sulfides bearing acidic α-hydrogens eiher with phenobarbital induced rabbit liver microsomal cytochrome P-450, or with the Udenfriend's model system (ferous perchlorate/ascorbic acid/oxygen system), both S-dealkylation and S-monoxygenation took place concurrently. Meanwhile, in the oxidation of simple alkyl sulfides, steeoselective S-monooxygenation was found of occur predominantly.

Reduction of tertiary amine N-oxide by purified cytochrome P-450

A reconstituted system containing cytochrome P-450 and NADPH-cytochrome c reductase, purified from phenobarbital-induced rabbit and rat liver microsomes, respectively, catalyzed anaerobic reduction of tiaramide N-oxide by NADPH at a rate comparable to that observed with intact rabbit liver microsomes. The reduction was inhibited by oxygen and carbon monoxide. When reduced methyl viologen was used as electron donor, the reduction proceeded at a much higher rate in the absence of NADPH-cytochrome c reductase. Preparations of various species of cytochrome P-450 purified from rabbit and rat liver microsomes and camphor-grown Pseudomonas putida exhibited nearly the same N-oxide reducing activity with reduced methyl viologen as donor despite their widely different specificities for hydroxylatable substrates.