Isolation and characterization of four cytochrome P450 isozymes from untreated and phenobarbital-treated beagle dogs.
Four different forms of cytochrome P450 (P450) were purified from liver microsomes of untreated or phenobarbital (PB)-treated male beagle dogs using HPLC techniques, and designated as DUT-1, DPB-1, DPB-2 and DPB-3, respectively. Specific contents of the purified DUT-1, DPB-1, DPB-2 and DPB-3 were 13.3, 9.6, 15.6 and 12.2 nmol/mg protein, respectively. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the monomeric molecular weights of DUT-1, DPB-1, DPB-2 and DPB-3 were estimated to be 57.5, 50.0, 47.0 and 50.0 kDa, respectively. The absolute spectra of the oxidized forms indicated that they exist in the low-spin state of heme in their oxidized forms. The NH2-terminal amino acid sequence of DUT-1 was unique and different from that of any other P450 so far reported. DUT-1 was active in the omega-hydroxylation of lauric acid. The amino-terminal sequences of DPB-1, DPB-2 and DPB-3 suggested that they belong to the P450 3A, 2C and 2B gene families, respectively. DPB-3 was a major form of P450 in PB-treated dog liver microsomes. Purified DPB-1 catalyzed nifedipine and (+)- and (-)-nilvadipine oxidations, as well as testosterone 6 beta-hydroxylation in the reconstituted system. These activities were enhanced 3- to 5-fold by the addition of cytochrome b5. DPB-2 and DPB-3 catalyzed aminopyrine N-demethylation, 7-ethoxycoumarin O-deethylation, biphenyl 4-hydroxylation and testosterone 16 alpha-hydroxylation. We believe that DUT-1 is a new form not purified previously.
Shiraga
T
T
Pharmaceutical and Pharmacokinetic Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan.
Iwasaki
K
K
Nozaki
K
K
Tamura
T
T
Yamazoe
Y
Y
Kato
R
R
Takanaka
A
A
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D006900:Hydroxylation; D015151:Immunoblotting; D007527:Isoenzymes / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009543:Nifedipine / Q000031:analogs & derivatives / Q000378:metabolism; D010634:Phenobarbital / Q000494:pharmacology*; D039181:Steroid 16-alpha-Hydroxylase; D013739:Testosterone / Q000378:metabolism
D007527:Isoenzymes; C035100:nilvadipine; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System; D039181:Steroid 16-alpha-Hydroxylase; D009543:Nifedipine; D010634:Phenobarbital