Aldehyde Dehydrogenase 1B1: Molecular Cloning and Characterization of a Novel Mitochondrial Acetaldehyde-Metabolizing Enzyme
Aldehyde Dehydrogenase 1B1: Molecular Cloning and Characterization of a Novel Mitochondrial Acetaldehyde-Metabolizing Enzyme
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DOI：
10.1124/dmd.110.034678
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发表时间：
2010-10-01
影响因子：
3.9
通讯作者：
Vasiliou, Vasilis
中科院分区：
文献类型：
--
作者：
Stagos, Dimitrios;Chen, Ying;Vasiliou, Vasilis
乙醇引起的损伤主要归因于它的有毒代谢物乙醛。乙醛的清除是通过其氧化实现的，主要由线粒体II类醛脱氢酶（ALDH2）催化。ALDH1B1是另一种线粒体醛脱氢酶（ALDH），与ALDH2具有75%的肽序列同源性。最近的白人人群研究表明ALDH1B1在乙醇代谢中的作用。然而，到目前为止，还没有关于ALDH1B1生化特性的正式文献。在本研究中，我们克隆并在Sf9昆虫细胞中表达了重组人ALDH1B1。所得酶经亲和层析纯化，达到均匀性。纯化的人ALDH1B1的动力学性质被评估使用广泛的醛底物。人ALDH1B1对NAD（+）作为辅助因子具有特异性，对短链和中链脂肪醛、芳香醛、脂质过氧化产物、4-羟基壬烯醛和丙二醛具有催化活性。最重要的是，人ALDH1B1对乙醛表现出55 μ M的表观K-m，使其成为该底物代谢的第二个低K-m的ALDH。ALDH1B1的脱氢酶活性对双硫仑抑制敏感，这一特征与ALDH2相同。采用定量聚合酶链反应、Western blotting和免疫组织化学方法检测ALDH1B1在C57BL/6J小鼠和人体内的组织分布。肝脏中表达量最高，其次是肠道，这意味着ALDH1B1在这些组织中具有潜在的生理作用。本研究首次报道了人ALDH1B1蛋白的表达、纯化和生化特性。
Ethanol-induced damage is largely attributed to its toxic metabolite, acetaldehyde. Clearance of acetaldehyde is achieved by its oxidation, primarily catalyzed by the mitochondrial class II aldehyde dehydrogenase (ALDH2). ALDH1B1 is another mitochondrial aldehyde dehydrogenase (ALDH) that shares 75% peptide sequence homology with ALDH2. Recent population studies in whites suggest a role for ALDH1B1 in ethanol metabolism. However, to date, no formal documentation of the biochemical properties of ALDH1B1 has been forthcoming. In this current study, we cloned and expressed human recombinant ALDH1B1 in Sf9 insect cells. The resultant enzyme was purified by affinity chromatography to homogeneity. The kinetic properties of purified human ALDH1B1 were assessed using a wide range of aldehyde substrates. Human ALDH1B1 had an exclusive preference for NAD(+) as the cofactor and was catalytically active toward short- and medium-chain aliphatic aldehydes, aromatic aldehydes, and the products of lipid peroxidation, 4-hydroxynonenal and malondialdehyde. Most importantly, human ALDH1B1 exhibited an apparent K-m of 55 mu M for acetaldehyde, making it the second low K-m ALDH for metabolism of this substrate. The dehydrogenase activity of ALDH1B1 was sensitive to disulfiram inhibition, a feature also shared with ALDH2. The tissue distribution of ALDH1B1 in C57BL/6J mice and humans was examined by quantitative polymerase chain reaction, Western blotting, and immunohistochemical analysis. The highest expression occurred in the liver, followed by the intestinal tract, implying a potential physiological role for ALDH1B1 in these tissues. The current study is the first report on the expression, purification, and biochemical characterization of human ALDH1B1 protein.