Partial purification and characterisation of hepatic cytochrome P450 from possum microsomes

Cytochrome P450 enzymes (P450s) comprise a family of haemoproteins that, in their reducing form, react with carbon monoxide (CO) and exhibit an absorbance maximum at 450 nm. The cytochrome P450 enzyme system is capable of metabolising a wide variety of structurally unrelated exogenous and endogenous compounds (Nebert and Gonzalez, 1987). It is widely distributed in many mammalian tissues, but is present in the highest concentration in hepatic endoplasmic reticulum. The functional diversity of the P450 enzyme system is attributable to the presence of a large number of isoenzymes. In recent years, considerable interest has developed in the purification and characterisation of these cytochrome P450 isoenzymes in many different species, in order to get a better understanding of the functions they perform. The purpose of this study was to isolate microsomes and partially purify and characterise cytochrome P450 isoenzymes from the liver of 'control' possums and possums treated with Phenobarbital (PB) and β-naphthoflavone (BNF). To my knowledge, this is the first report of purification of possum liver cytochrome P450s. Since the P450 purification steps were time consuming, the number of the possums used in my study were limited to five. Cytochrome P450s were partially purified from 3 untreated (control) possums to determine constitutive expression of P450s and from possums treated with either PB or BNF to induce P450s. The purification methodology was based on a modification of the procedure used by Gooneratne et al. (1997a) for purification of cytochrome P450s from the rainbow trout liver. Cytochrome P450 induction usually results in an increase in the content and activity of the P450 enzymes. Most commonly, inducers used in research fall into two principal classes, PB inducible type and the 3-methycholanthrene (3-MC) or BNF inducible type. Two inducers, PB and BNF, one from each of the above mentioned groups were used in this study. The possums received either BNF or PB via intraperitoneal (IP) injection, at a dose of 50 mg/kg body weight. Possums treated with BNF and PB were killed 2 and 4 days post injection respectively. The overall cytochrome P450 content of possum microsomes induced by BNF and PB was at least 2-fold higher than in the control possums. The P450 content in the female control possum was higher than in the male controls. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins from microsomes treated with PB and BNF possums showed relatively darker bands compared to those seen in the control possums. In the P450 purification procedure, the microsomes were initially solubilised with detergents and the P450s then separated by DEAE-Sepharose CL-6B and hydroxyapatite chromatography. With DEAE-Sepharose chromatography, 3 peaks with absorbance at 417 nm (haemoproteins) and 280 nm (proteins) were observed in all possums. Seven cytochrome P450 antibodies were used in Dot blot studies to identify the specific P450s in the three Sepharose column peak fractions, but the P450 proteins were found only in the fractions from the first peak. The fractions from the control possums gave a positive signal with P450 antibodies, 2El and 3A4 indicating that these two P450s are constitutively expressed in the possum. In the PB-induced possum four P450s (CYP 2B1, 2E1, 2C11, and 3A4) and in the BNF-treated possum three P450s (CYP 1 A1, 2B1 and 2C 11) were detected with these polyclonal antibodies. The three peaks of Sepharose chromatography were also subjected to SDS-PAGE. Proteins within the molecular weight range of P450s (45 kDa to 60 kDa) were seen only in the first peak fractions. In the control possum (#2), one major polypeptide band (46 kDa) and four other minor bands (51 kDa, 55.6 kDa, 56.5 kDa, and 58.5 kDa) were seen. In the possum (#5) treated with PB, one major polypeptide band (51 kDa) and three minor polypeptides bands (54 kDa, 57.5 kDa, and 58 kDa) were seen. In the possum (#4) induced with BNF, one major band (57 kDa) and three minor polypeptides bands (54 kDa, 56.5 kDa, and 60 kDa) were seen. Western blots of Sepharose chromatographic samples cross-reacted with the polyclonal P450 antibodies and hence different isoenzymes could not be specifically identified using this technique. In conclusion, the results presented here show that several proteins within the molecular weight range of cytochrome P450s (45 kDa to 60 kDa) are present in the possum liver, with at least two isoenzymes (CYP 2E1, 3A4) constitutively expressed and several others (CYP 1A1, 2B1, 2C11, 3A4), induced when injected with PB and/or BNF. The procedures used in this investigation will have to be modified and improved if possum P450s are to be fully characterised in future studies.