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标题：Isolation and Characterization of Four Cytochrome P450 Isozymes from Untreated and Phenobarbital-Treated Beagle Dogs
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作者：Toshifumi SHIRAGA ; Kazuhide IWASAKI ; Kazuyoshi NOZAKI 等
期刊名称：Biological and Pharmaceutical Bulletin
印刷版ISSN：0918-6158
电子版ISSN：1347-5215
出版年度：1994
卷号：17
期号：1
页码：22-28
DOI：10.1248/bpb.17.22
出版社：The Pharmaceutical Society of Japan
摘要：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 ω-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 6β-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 16α-hydroxylation. We believe that DUT-1 is a new form not purified previously.
关键词：dog liver microsomes;cytochrome P450;phenobarbital;testosterone;nifedipine;lauric acid ω-hydroxylation