Purification and characterization of the hepatic CYP2C and 3A isozymes from phenobarbitone pretreated rhesus monkey.
Hepatic P450s, named M-3 and M-4 were purified from phenobarbitone pretreated rhesus monkey. These demonstrated polypeptide molecular mass of 50 and 52.5 kDa and specific content of 12 and 20 nmol P450/mg protein, respectively. Both the isozymes demonstrated low spin state of heme. Antibodies raised against M-3 inhibited the activity of aminopyrine, erythromycin and ethylmorphine N-demethylase in the microsomes obtained from PB pretreated rhesus monkey by 76, 40 and 35%, respectively. M-4 did the same by 69, 85 and 79%, respectively. These observations indicated M-3 and M-4 to be the members of CYP2C and 3A subfamilies, respectively. These results were substantiated by the observations that M-3 metabolized aminopyrine whereas M-4 metabolized aminopyrine, erythromycin and ethylmorphine in the reconstituted system. Microsomal lipids and cytochrome b5 enhanced the rate of these reactions. Further confirmation to the identity of these isozymes was provided by N-terminal amino acid sequences. The first 10 N-terminal amino acid residues of M-3 were 90% similar to CYP2C20 and 2C9 and that of M-4 were 100 and 90% similar to CYP3A8 and 3A5, respectively. In conclusion, two isozymes of hepatic P450 purified from PB pretreated rhesus monkey belong to CYP2C and 3A subfamilies.
Ramana
K V
KV
Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
Kohli
K K
KK
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008253:Macaca mulatta; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects* / Q000201:enzymology; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010449:Peptide Mapping; D010634:Phenobarbital / Q000494:pharmacology*; D013057:Spectrum Analysis
C400322:cytochrome P-450 CYP2C subfamily; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D010634:Phenobarbital