Kedishvili N Y, Gough W H, Chernoff E A, Hurley T D, Stone C L, Bowman K D, Popov K M, Bosron W F, Li T K
Department of Biochemistry and Molecular Biology, Purdue University, Indianapolis, Indiana 46202-5122, USA.
J Biol Chem. 1997 Mar 14;272(11):7494-500. doi: 10.1074/jbc.272.11.7494.
This study was undertaken to identify the cytosolic 40-kDa zinc-containing alcohol dehydrogenases that oxidize all-trans-retinol and steroid alcohols in fetal tissues. Degenerate oligonucleotide primers were used to amplify by polymerase chain reaction 500-base pair fragments of alcohol dehydrogenase cDNAs from chick embryo limb buds and heart. cDNA fragments that encode an unknown putative alcohol dehydrogenase as well as the class III alcohol dehydrogenase were identified. The new cDNA hybridized with two messages of approximately 2 and 3 kilobase pairs in the adult chicken liver but not in the adult heart, muscle, testis, or brain. The corresponding complete cDNA clones with a total length of 1390 base pairs were isolated from a chicken liver lambdagt11 cDNA library. The open reading frame encoded a 375-amino acid polypeptide that exhibited 67 and 68% sequence identity with chicken class I and III alcohol dehydrogenases, respectively, and had lower identity with mammalian class II (55-58%) and IV (62%) isozymes. Expression of the new cDNA in Escherichia coli yielded an active alcohol dehydrogenase (ADH-F) with subunit molecular mass of approximately 40 kDa. The specific activity of the recombinant enzyme, calculated from active site titration of NADH binding, was 3.4 min-1 for ethanol at pH 7.4 and 25 degrees C. ADH-F was stereospecific for the 3beta,5alpha- versus 3beta,5beta-hydroxysteroids. The Km value for ethanol at pH 7.4 was 17 mM compared with 56 microM for all-trans-retinol and 31 microM for epiandrosterone. Antiserum against ADH-F recognized corresponding protein in the chicken liver homogenate. We suggest that ADH-F represents a new class of alcohol dehydrogenase, class VII, based on its primary structure and catalytic properties.
本研究旨在鉴定在胎儿组织中氧化全反式视黄醇和类固醇醇的胞质40 kDa含锌醇脱氢酶。使用简并寡核苷酸引物通过聚合酶链反应从鸡胚肢芽和心脏中扩增醇脱氢酶cDNA的500碱基对片段。鉴定出编码未知假定醇脱氢酶以及Ⅲ类醇脱氢酶的cDNA片段。新的cDNA在成年鸡肝脏中与两条约2和3千碱基对的mRNA杂交，但在成年心脏、肌肉、睾丸或大脑中不杂交。从鸡肝脏λgt11 cDNA文库中分离出全长1390碱基对的相应完整cDNA克隆。开放阅读框编码一个375个氨基酸的多肽，该多肽与鸡Ⅰ类和Ⅲ类醇脱氢酶的序列同一性分别为67%和68%，与哺乳动物Ⅱ类（55 - 58%）和Ⅳ类（62%）同工酶的同一性较低。新cDNA在大肠杆菌中的表达产生了一种活性醇脱氢酶（ADH - F），其亚基分子量约为40 kDa。根据NADH结合的活性位点滴定计算，重组酶在pH 7.4和25℃下对乙醇的比活性为3.4 min⁻¹。ADH - F对3β,5α - 与3β,5β - 羟基类固醇具有立体特异性。在pH 7.4时，乙醇的Km值为17 mM，而全反式视黄醇为56 μM，表雄酮为31 μM。针对ADH - F的抗血清识别鸡肝脏匀浆中的相应蛋白质。基于其一级结构和催化特性，我们认为ADH - F代表一种新的醇脱氢酶类别，即Ⅶ类。
