Lang M A, Juvonen R, Järvinen P, Honkakoski P, Raunio H
Department of Pharmacology and Toxicology, University of Kuopio, Finland.
Arch Biochem Biophys. 1989 May 15;271(1):139-48. doi: 10.1016/0003-9861(89)90264-6.
Genetic experiments with two inbred strains of mice, AKR/J and DBA/2N, show a single major gene inheritance of additive mode for pyrazole-inducible coumarin 7-hydroxylase. Intragroup variation in the enzyme activity further suggests the contribution of minor modifying genes to the final enzyme activity. Western blot analysis with a polyclonal antibody raised against the purified isozyme P450Coh (highly active in the 7-hydroxylation of coumarin) showed that a difference in the amounts of P450Coh protein between the D2 and AKR mice is the reason for the differences in the enzyme activity between the two mouse strains. Accordingly, changes at the regulatory level rather than at the structural gene would explain the genetic difference in the activity of coumarin 7-hydroxylase. This hypothesis is further supported by the identical Km values of the basal and induced enzyme. The inducibility of coumarin 7-hydroxylase by phenobarbital (PB) and its genetic regulation have been previously studied by A. W. Wood and colleagues ((1974) Science 185, 612-614; (1979); J. Biol. Chem. 254, 5641-5646 and 5647-5651). Our present experiments show that the regulation is the same for the pyrazole-inducible enzyme. Furthermore the experiments with anti-P450Coh antibody show that the PB- and pyrazole-inducible proteins have the same molecular weight and are immunologically indistinguishable. This suggests that PB and pyrazole may induce the same enzyme. Immunoinhibition of microsomal coumarin 7-hydroxylase is practically 100% for control animals and after pretreatment with pyrazole or PB. This suggests that in each case the same or immunologically closely related proteins are metabolizing coumarin and that the P450Coh may be the only P450 isoenzyme in mouse liver microsomes catalyzing the 7-hydroxylation of coumarin. The N-terminal amino acid sequence of P450Coh was found to be identical with those from Type I and Type II genes of the mouse P45015 alpha family for the first 21 amino acids. With rat PB-inducible P450b the homology is only 33%. Also the immunological properties of P450Coh are different from those of P450b. This may suggest that P450Coh has a closer association to the steroid 15 alpha-hydroxylase gene family than to the P450IIB subfamily of phenobarbital-inducible isoenzymes.
对两种近交系小鼠AKR/J和DBA/2N进行的遗传学实验表明，吡唑诱导型香豆素7-羟化酶呈单一位点主要基因的加性模式遗传。酶活性的组内变异进一步表明，次要修饰基因对最终酶活性有贡献。用针对纯化的同工酶P450Coh（在香豆素7-羟化反应中高度活跃）制备的多克隆抗体进行的蛋白质印迹分析表明，D2和AKR小鼠之间P450Coh蛋白量的差异是两种小鼠品系酶活性差异的原因。因此，调控水平而非结构基因的变化可以解释香豆素7-羟化酶活性的遗传差异。基础酶和诱导酶相同的米氏常数进一步支持了这一假说。A. W. Wood及其同事先前已对苯巴比妥（PB）诱导香豆素7-羟化酶的诱导作用及其遗传调控进行了研究（（1974年）《科学》185, 612 - 614；（1979年）；《生物化学杂志》254, 5641 - 5646和5647 - 5651）。我们目前的实验表明，吡唑诱导型酶的调控方式相同。此外，用抗P450Coh抗体进行的实验表明，PB诱导型和吡唑诱导型蛋白具有相同的分子量，且在免疫上无法区分。这表明PB和吡唑可能诱导相同的酶。对于对照动物以及用吡唑或PB预处理后的动物，微粒体香豆素7-羟化酶的免疫抑制率实际上为100%。这表明在每种情况下，代谢香豆素的是相同或免疫上密切相关的蛋白，并且P450Coh可能是小鼠肝微粒体中催化香豆素7-羟化反应的唯一P450同工酶。发现P450Coh的N端氨基酸序列在前21个氨基酸上与小鼠P45015α家族的I型和II型基因相同。与大鼠PB诱导型P450b的同源性仅为33%。此外，P450Coh的免疫特性也与P450b不同。这可能表明P450Coh与类固醇15α-羟化酶基因家族的关联比与苯巴比妥诱导型同工酶的P450IIB亚家族更密切。
