Graves P E, Elhag G A, Ciaccio P J, Bourque D P, Halpert J R
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson 85721.
Arch Biochem Biophys. 1990 Aug 15;281(1):106-15. doi: 10.1016/0003-9861(90)90419-y.
The nucleotide sequence of a cDNA that codes for the major phenobarbital (PB)-inducible male beagle dog hepatic cytochrome P450 has been determined. Using a rabbit P450IIB cDNA probe (R. Gasser, M. Negishi, and R. M. Philpot, 1988, Mol. Pharmacol, 32, 22-30), a cDNA clone with a 2.6-kilobase pair insert was isolated from a lambda gt11 library prepared from hepatic mRNA from a PB-treated dog. The cloned insert was sequenced and found to contain an open reading frame coding for a polypeptide of 494 amino acids (Mr 56,183). The encoded protein can be assigned to the P450IIB subfamily on the basis of homology to cytochromes P450 from other species. The deduced amino acid sequence is 79% identical to that reported for rabbit P450 BO (P450IIB4) and 75% identical to that for rat P450b (P450IIB1). The sequence identity decreases to less than 52% when the dog sequence is compared with other P450II subfamilies. The deduced NH2-terminal 30 amino acids encoded by the dog cDNA are identical to those determined by sequence analysis of purified dog cytochrome P450 PBD-2, and the amino acid composition concurs with that determined for the PBD-2 protein (D. B. Duignan, I. G. Sipes, T. B. Leonard, and J. R. Halpert, 1987, Arch. Biochem. Biophys. 255, 290-303). Northern blots revealed two mRNA species of approximately 1.9 and 2.9 kilobases in length, which hybridized to the coding region of the dog P450IIB cDNA. The level of total hybridizable mRNA was increased approximately sixfold in livers from PB-treated dogs compared with that in untreated animals. This increase correlates well with the reported nearly sixfold increase in the level of PBD-2 protein and the fivefold increase in the rate of hepatic metabolism of 2,2',4,4',5,5'-hexachlorobiphenyl following PB treatment. The two mRNA species may result from the use of different polyadenylation signals located in the 3'-noncoding region or from transcription of more than one gene for PBD-2. Southern blot analysis indicated that the dog P450IIB subfamily contains at least two closely related genes.
已确定编码主要苯巴比妥（PB）诱导的雄性比格犬肝细胞色素P450的cDNA的核苷酸序列。使用兔P450IIB cDNA探针（R. Gasser、M. Negishi和R. M. Philpot，1988年，《分子药理学》，32卷，22 - 30页），从用PB处理的犬肝脏mRNA制备的λgt11文库中分离出一个带有2.6千碱基对插入片段的cDNA克隆。对克隆的插入片段进行测序，发现其包含一个编码494个氨基酸多肽（Mr 56,183）的开放阅读框。基于与其他物种细胞色素P450的同源性，可将编码的蛋白质归为P450IIB亚家族。推导的氨基酸序列与报道的兔P450 BO（P450IIB4）的氨基酸序列有79%的同一性，与大鼠P450b（P450IIB1）的氨基酸序列有75%的同一性。当将犬的序列与其他P450II亚家族进行比较时，序列同一性降至低于52%。犬cDNA编码的推导NH2末端30个氨基酸与通过纯化的犬细胞色素P450 PBD - 2的序列分析确定的氨基酸相同，并且氨基酸组成与为PBD - 2蛋白确定的氨基酸组成一致（D. B. Duignan、I. G. Sipes、T. B. Leonard和J. R. Halpert，1987年，《生物化学与生物物理学报》，255卷，290 - 303页）。Northern印迹显示有两种长度约为1.9和2.9千碱基的mRNA，它们与犬P450IIB cDNA的编码区杂交。与未处理动物相比，PB处理犬肝脏中可杂交的总mRNA水平增加了约六倍。这种增加与报道的PB处理后PBD - 2蛋白水平增加近六倍以及2,2',4,4',5,5'-六氯联苯肝脏代谢速率增加五倍密切相关。这两种mRNA可能是由于使用了位于3'-非编码区的不同聚腺苷酸化信号，或者是由于PBD - 2的多个基因转录所致。Southern印迹分析表明犬P450IIB亚家族至少包含两个密切相关的基因。
