Weiner H, Wang T T, Farrés J
Biochemistry Department, Purdue University, W. Lafayette, IN 47907.
Alcohol Alcohol Suppl. 1991;1:91-5.
The tools of molecular biology were applied to study different facets of liver aldehyde dehydrogenase. One point investigated was the sequence of the various liver forms of the enzyme and the structure of the genes coding for the enzyme. It was found that an intron existed between the gene sequence coding for amino acids 21 and 22. The presence of the intron could explain why no sequence identity exists between the first 21 residues of the cytosol and mitochondrial isozymes while there is 70% identity between the remaining amino acids. Having the cDNA coding for the mitochondrial enzyme allowed us to produce the precursor form of the enzyme in rabbit reticulocytes and perform in vitro import into isolated mitochondria where it was found that alcohols caused an inhibition of import. The active enzyme was expressed in E. coli and site directed mutagenesis was performed to probe for the active site residues. The role of cysteine and position 302 and glutamate at position 487 was studied.
分子生物学工具被用于研究肝脏醛脱氢酶的不同方面。研究的一个要点是该酶各种肝脏形式的序列以及编码该酶的基因结构。结果发现，在编码氨基酸21和22的基因序列之间存在一个内含子。内含子的存在可以解释为什么胞质溶胶和线粒体同工酶的前21个残基之间不存在序列同一性，而其余氨基酸之间存在70%的同一性。拥有编码线粒体酶的cDNA使我们能够在兔网织红细胞中产生该酶的前体形式，并在体外导入分离的线粒体，在那里发现醇类会抑制导入。活性酶在大肠杆菌中表达，并进行定点诱变以探测活性位点残基。研究了302位的半胱氨酸和487位的谷氨酸的作用。
