Purification and characterization of an acetone-inducible cytochrome P-450 from hamster liver microsomes.
A form of cytochrome P-450 has been purified to electrophoretic homogeneity from the hepatic microsomes of Syrian golden hamsters treated with acetone. This P-450 form, designated ha P-450j, had an M(r) of approximately 55,000, bound dimethyl sulphoxide and exhibited a CO-reduced absorbance maximum at 451 nm. The absolute spectra of its oxidized form indicated that ha P-450j was predominantly in the low-spin state. In a reconstituted system, ha P-450j showed relatively low catalytic activities towards 7-ethoxycoumarin, 7-ethoxyresorufin, aminopyrine, ethylmorphine and benzphetamine, whereas it catalysed the oxidation of aniline, acetone and thiobenzamide with a high catalytic-centre activity. In addition, ha P-450j catalysed at a high rate the high-affinity component of dimethylnitrosamine N-demethylase; in contrast, only the low-affinity component of diethylnitrosamine N-de-ethylase was efficiently catalysed. The addition of cytochrome b5 to the reconstitution system decreased the Km value for dimethylnitrosamine N-demethylase by a factor of 5 and increased the Vmax. value, and slightly enhanced the other activities. Thiobenzamide and diethyldithiocarbamate were found to be the most effective inhibitors of the ha-P-450j-dependent aniline hydroxylation. Polyclonal antibodies against rat P-450j recognized ha P-450j in immunoblots of control and treated hamster liver microsomes. Treatment of hamsters with acetone increased the apparent abundance of ha P-450j in microsomes, whereas phenobarbital and beta-naphthoflavone did not induce it. Analysis of N-terminal amino acid sequences demonstrated that ha P-450j has a high degree of sequence identity with rat P-450j. All the evidence presented in this study indicates that ha P-450j could represent the hamster orthologue of the previously described CYP2E1(s) of other species.
Puccini
P
P
Laboratory of Genetics and Biochemical Toxicology, CNR, Pisa, Italy.
Menicagli
S
S
Longo
V
V
Santucci
A
A
Gervasi
P G
PG
D000096:Acetone / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D006224:Cricetinae; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D004790:Enzyme Induction / Q000187:drug effects; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D008297:Male; D008647:Mesocricetus; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D008969:Molecular Sequence Data; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis
D000814:Aniline Compounds; D000096:Acetone; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating; C023650:aniline