Gonzalez F J, Matsunaga T, Nagata K, Meyer U A, Nebert D W, Pastewka J, Kozak C A, Gillette J, Gelboin H V, Hardwick J P
DNA. 1987 Apr;6(2):149-61. doi: 10.1089/dna.1987.6.149.
Debrisoquine 4-hydroxylase (P450db1) was purified from rat liver microsomes. Polyclonal antibody was produced and, in conjunction with immunoblots, was used to identify and purify a second immunorelated P450 (P450db2) that does not have debrisoquine hydroxylating activity. The cDNA clones to db1 and db2 were isolated from a lambda gt11 expression library, sequenced, and found to share 78% nucleotide and 73% deduced amino acid similarities. These similarities are evenly dispersed along the sequence except for a region of 190 nucleotides with 99% similarity near the carboxyl terminus of the protein-coding region; this similarity is probably the remnant of a gene conversion event. Both proteins share between 38% and 43% amino acid similarity with P450a, P450b, P450e, P450f, P450PB1, and P450j; these data indicate that P450db1 and P450db2 are members of a separate subfamily within the P450II gene family. Southern blot analysis and preliminary genomic cloning suggest that at least four genes exist in the subfamily, although the present evidence suggests that only db1 and db2 are expressed in rat liver. With the use of 19 mouse X hamster somatic cell hybrids, the db1 and db2 genes were localized to mouse chromosome 15 (P450-2D locus). A polymorphism has been described for debrisoquine metabolism in the DA rat strain, adult females having markedly decreased debrisoquine 4-hydroxylase activity. Our immunoblot analysis and mRNA analysis suggest that debrisoquine 4-hydroxylase deficiency in the female DA rat is not due to a decrease in db1 protein or mRNA. The db1 and db2 proteins are differentially regulated: during development db2 is present at birth while db1 is absent, and db1 increases by 1 week of age; in addition, db1 is slightly induced by phenobarbital, 3-methyl-cholanthrene, and dexamethasone whereas db2 is marginally increased by these latter two agents. These results demonstrate that debrisoquine 4-hydroxylase is a member of a new constitutively expressed P450II sub-family containing two or more genes in the rat and establish that the debrisoquine polymorphism in the DA rat is probably due to a structurally altered db1 protein.
从大鼠肝脏微粒体中纯化出异喹胍4-羟化酶（P450db1）。制备了多克隆抗体，并结合免疫印迹法，用于鉴定和纯化另一种无异喹胍羟化活性的免疫相关P450（P450db2）。从λgt11表达文库中分离出db1和db2的cDNA克隆，进行测序，发现它们在核苷酸水平上有78%的相似性，推导的氨基酸水平上有73%的相似性。除了在蛋白质编码区羧基末端附近有一个190个核苷酸、相似性为99%的区域外，这些相似性在序列中均匀分布；这种相似性可能是基因转换事件的残余。这两种蛋白质与P450a、P450b、P450e、P450f、P450PB1和P450j的氨基酸相似性在38%至43%之间；这些数据表明P450db1和P450db2是P450II基因家族中一个独立亚家族的成员。Southern印迹分析和初步的基因组克隆表明该亚家族中至少存在四个基因，尽管目前的证据表明在大鼠肝脏中只有db1和db2表达。利用19个小鼠×仓鼠体细胞杂种，将db1和db2基因定位到小鼠15号染色体（P450-2D位点）。已描述了DA大鼠品系中异喹胍代谢的多态性，成年雌性大鼠的异喹胍4-羟化酶活性明显降低。我们的免疫印迹分析和mRNA分析表明，雌性DA大鼠中异喹胍4-羟化酶缺乏并非由于db1蛋白或mRNA减少。db1和db2蛋白受到不同的调节：在发育过程中，db2在出生时就存在，而db1不存在，db1在1周龄时增加；此外，苯巴比妥、3-甲基胆蒽和地塞米松可轻微诱导db1，而后两种药物可使db2略有增加。这些结果表明异喹胍4-羟化酶是大鼠中一个新的组成性表达的P450II亚家族的成员，该亚家族包含两个或更多基因，并确定DA大鼠中的异喹胍多态性可能是由于db1蛋白结构改变所致。
