Rabbit Liver Cytochrome P-450 LM2 Research Articles

Immunochemical and sub-structural characterization of sheep lung cytochrome P45OLgM2

Sheep hmg cytochrome P45OLgM2 belonging to gene subfamily 2B, was obtained in highly purified form and antibodies against sheep lang cytochrome P45OLgM2 were produced in rabbits by uslog the previously developed methods in our laboratory. Immunolrtgical sued enzymatic studies showed that antibodies against lung cytochrome P45014M2 inhibited beazlibetamine N-demethyiadon, ethyhnorphine N-demethylation and amine 4-hydroxylation reactions In sheep lung microsomes about 99, 80 and 62%., respectively. Benzphetamine N-demethylation reaction in sheep lung microsomes was only catalyzed by cytochrome P450LgM2 isozyme while the other isozymes of P450 as well as P45OLgM2 are also involved in the metabolism of ethylmorplone and aniline. Similar to lung microsomes, benzphetamine N-demethylsae activity of the reconstituted systems containing purified sheep lung cytochrome P45K4M2 or phenobarbital (PB) treated rabbit liver cytochrome P450LM2(2B4) was also inhibited by P450LgM2 and bodies about 95 and 82%, respectively. A 50% inhibitory effect of sheep P450LgM2 andbodies was also observed in N-demethylase activity of the reconstituted system containing parified sheep lung cytochrone P450LgM2. SDS-PAGE peptide maps obtalood following the partial proteelyais of parified sheep lung cytochrome P454LgM2 and PB-rabbit Ever P450LMVB4) isoaymes, using chymotrypsin and papsin, were similar in general. However they showed some differences both qualitatively and quantitatively, suggesting that some differences exist ammo the amino acid sequences of sheep lung cytochrome P45OLgM2 and rabbit liver cytodrome P4502B4.

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.

Phosphorylation of cytochrome P450: Regulation by cytochrome b5

Rabbit liver cytochrome P450 LM2 and several forms of rat liver cytochrome P450 are phosphorylated by cAMP-dependent protein kinase (PKA) and by protein kinase C. Under aqueous assay conditions at neutral pH LM2 is phosphorylated only to a maximum extent of about 20 mol% by PKA. We show that detergents or alkaline pH greatly enhance the extent of phosphorylation of the cytochrome P450 substrates of cAMP-dependent protein kinase. In the presence of 0.05% Emulgen, PBRLM5, which appears to be the best cytochrome P450 substrate for cAMP-dependent protein kinase, incorporates phosphate up to about 84 mol% of enzyme. We reported previously (I. Jansson et al. (1987) Arch. Biochem. Biophys. 259, 441–448) that cytochrome b 5 inhibits the phosphorylation of LM2 by cAMP-dependent protein kinase. In this paper, using PBRLM5, we demonstrate, by analysis of initial rates, that the inhibition of phosphorylation by cytochrome b 5 is competitive, with a K i = 0.48 μM. We also show that a number of forms of cytochrome P450 can be phosphorylated by protein kinase C, and that the phosphorylation of these forms by protein kinase C is also inhibited by cytochrome b 5. These data suggest that the phosphorylation site(s) of cytochromes P450 may be located within or overlap the cytochrome b 5 binding domain of the enzymes.

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.

Rabbit liver cytochrome P-450 LM2: roles of substrates, inhibitors, and cytochrome b5 in modulating the partition between productive and abortive mechanisms.

Cytochrome b5 (b5) enhanced the rate of 7-ethoxycoumarin deethylation by rabbit liver cytochrome P-450 LM2. The effect was saturable and can be analyzed as the sum of two effects: a decrease in the KM for the substrate and an increase in the Vmax. When two substrates were present simultaneously, they competed in a complex way depending on the presence of b5. Various substrates at low concentrations inhibited 7-ethoxycoumarin deethylation in a competitive-like way. Only a part of the P-450 activity was found to be affected by this mode of inhibition. Higher inhibitor concentrations caused a new kind of inhibition characterized by much higher half-effect values. The pattern seemed dependent on the ability of the inhibitors to be metabolized and was dramatically changed by the addition of b5. The relative rates of P-450-dependent NADPH oxidation and hydrogen peroxide and water formation were determined as well as their dependence on substrate and b5. A steady-state kinetic model that includes two branch points for water, hydrogen peroxide, and product formation is proposed. The model allows a full prediction of the b5 effects and seems consistent with most of the steady-state and rapid kinetic data available in the literature.

Coding nucleotide sequence of 3-methylcholanthrene-inducible cytochrome P-450d cDNA from rat liver.

We determined the coding nucleotide sequence of the mRNA for a 3-methylcholanthrene-inducible cytochrome P-450, P-450d, of rat liver by sequence analysis of cloned cDNAs. The predicted amino acid sequence of the cytochrome is composed of 513 amino acids, and its NH2-terminal sequence of 30 amino acids completely coincides with that reported from analysis of the purified cytochrome P-450d. The amino acid composition of the deduced sequence also agrees well with that determined from the purified protein. Computer-aided analysis was carried out to compare the complete primary structures of five species of cytochrome P-450, two molecular species of phenobarbital-inducible rat liver cytochrome P-450 (P-450b and P-450e), phenobarbital-inducible rabbit liver cytochrome P-450LM2, 3-methylcholanthrene-inducible rat liver cytochrome P-450d, and camphor-hydroxylating P-450 of Pseudomonas putida (P-450CAM). It is concluded therefrom that the time of divergence between cytochrome P-450b (P-450e) and P-450d is much earlier than that of branching between phenobarbital-inducible cytochromes P-450 of rat and rabbit. One highly conserved cysteine-containing region that is close to the COOH terminus is found in all of these cytochrome P-450 sequences, indicating that the heme-binding site is the cysteine residue in this region.

Cytochrome b 5 as electron donor to rabbit liver cytochrome P-450 LM2 in reconstituted phospholipid vesicles

When incorporated into phospholipid vesicles containing NADPH-cytochrome P-450 reductase and P-450 LM 2, cytochrome b 5 enhanced the rate of NADPH-supported hydroxylation of 7-ethoxycoumarin or p -nitroanisole about 5-fold. Cytochrome b 5 did not affect the rate of NADPH-oxidation, nor the rate of NADPH-supported formation of the ferrous CO-complex of cytochrome P-450. However, the cytochrome b 5-mediated increase in product formation was found to be correlated with concomitant decreases in the production of H 2O 2 or O 2 − in the system, thus strongly indicating cytochrome b 5 being a more efficient donor of the second electron to cytochrome P-450 than is NADPH-cytochrome P-450 reductase.

Monoclonal antibodies to rabbit liver cytochrome P450 LM2

The mixed-function oxidases containing cytochrome P450 are the primary systems that metabolize xenobiotics including drugs and carcinogens and endogenous substrates such as steroids [l-3]. The mixed-function oxidases may be key determinants of the rates of drug metabolism [2] and carcinogen susceptibility [3-S]. A major class of carcinogens to which humans are exposed is the polycyclic aromatic hydrocarbons. A prototype and most common hydrocarbon of this class is benzo [a] pyrene (BP) [6]. Benzo [alpyrene is oxidized by the mixed-function oxidase, aryl hydrocarbon hydroxylase (AHH) and metabolically related enzymes to >40 oxygenated metabolites which include simple epoxides, phenols, quinones, dihydrodiols, diol epoxides and water-soluble conjugates of glutathione, glucuronide and sulfate [7]. The pathway leading to the benzo [a] pyrene diol epoxides is believed to be a primary pathway of carcinogen activation [7]. Different forms of cytochrome P450 have been isolated and characterized (reviewed [8]). Cytochromes P450 LM2 and LM4 have been purified to homogeneity [9-l l] and P450 LMI, LM3 and LM7 have been partially purified [9] from rabbit liver. The highly purified LM2 and LM4 exhibit different biochemical, immunological and kinetic properties [ 121. Furthermore each isozyme of P450 exhibits stereoselectivity in substrate choice and product formation with respect to both benzo[a]pyrene metabolism and the conversion of (-)t-7,8-diol benzo[a]pyrene into the highly mutagenic benzo [a] pyrene 7,&diol9,1 Oepoxides [13,14]. Immunoglobulin genes derived from mouse-spleen cells primed with specific antigens are expressed and