Phenobarbital-induced Rabbit Liver Microsomes 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.

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.

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.

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.

Hydrogen peroxide formation and stoichiometry of hydroxylation reactions catalyzed by highly purified liver microsomal cytochrome P-450

The stoichiometry of hydroxylation reactions catalyzed by cytochrome P-450 was studied in a reconstituted enzyme system containing the highly purified cytochrome from phenobarbital-induced rabbit liver microsomes. Hydrogen peroxide was shown to be formed in the reconstituted system in the presence of NADPH and oxygen; the amount of peroxide produced varied with the substrated added. NADPH oxidation, oxygen consumption, and total product formation (sum of hydroxylated compound and hydrogen peroxide) were shown to be equimolar when cyclohexane, benzphetamine, or dimethylaniline served as the substrate. The stoichiometry observed represents the sum of two activities associated with cytochrome P-450. These are (1) hydroxylase activity: NADPH + H + + O 2 + RH → NADP + + H 2O + ROH; and (2) oxidase activity: NADPH + H + + O 2 → NADP + + H 2O 2. Benzylamphetamine (desmethylbenzphetamine) acts as a pseudosubstrate in that it stimulates peroxide formation to the same extent as the parent compound (benzphetamine), but does not undergo hydroxylation. Accordingly, when benzylamphetamine alone is added in control experiments to correct for the NADPH and O 2 consumption not associated with benzphetamine hydroxylation, the expected 1:1:1 stoichiometry for NADPH oxidation, O 2 consumption, and formaldehyde formation in the hydroxylation reaction is observed.

Cytochrome P-450 purified to apparent homogeneity from phenobarbital-induced rabbit liver microsomes: Catalytic activity and other properties

Cytochrome P-450 was purified to a content of over 17 nmoles per mg of protein from liver microsomes of phenobarbital-treated rabbits by fractionation with polyethylene glycol 6000, DEAE-cellulose column chromatography, and hydroxylapatite column chromatography in the presence of Renex 690, a nonionic detergent. The purified preparation exhibited a single polypeptide band (molecular weight, 49,000 daltons) when submitted to SDS-polyacrylamide gel electrophoresis. Cytochromes P-420 and b 5 and NADPH-cytochrome c reductase were absent. The reconstituted system containing purified cytochrome P-450, reductase, and phosphatidylcholine catalyzed the hydroxylation of benzphetamine, cyclohexane, aniline, and laurate.

An Affinity Column Method for Partial Purification of Cytochrome P-450 from Phenobarbital-induced Rabbit Liver Microsomes

A simple method has been developed for partial purification of cytochrome P-450 from phenobarbital-induced rabbit liver microsomes. The cytochrome was solubilized with 0.1 M phosphate buffer (pH 7.25) containing 0.4% sodium cholate, 20% glycerol, 1 mM dithiothreitol, and 1 mM EDTA. The solubilized hemoprotein was adsorbed on a column of an ω-amino-n-octyl derivative of Sepharose 4B and then eluted with 1 mM Emalgen 913 (a polyoxyethylene nonylphenyl ether) in the same buffer. This method permitted us to purify cytochrome P-450 to a specific content of 8–10 nmoles per mg of protein (40–50% pure assuming a molecular weight of 50,000 for the cytochrome) with an overall yield of about 35%. The purified preparation was free from both cytochromes b5 and P-420. Its spectral properties were consistent with those reported for the microsomal bound form of cytochrome P-450 and superior in several respects to those of the partially purified preparations of the cytohcrome so far described.