The long term aim of this proposal is to obtain a better understanding of physiological roles of individuals isozymes of human """"""""low Km"""""""" aldehyde dehydrogenase (EC 1.2.1.3); the short term aim is to obtain information about individual isozymes. The recently purified E3 isozyme (from liver) is involved in polyamine metabolism and conversion of putrescine to GABA- a metabolic pathway which may be affected by ethanol metabolism. The proposed experiments include completion of E3 isozyme cDNA cloning, expression and purification of E3 isozyme, characterization of the E3 isozyme via determination of its kinetic mechanism, its coenzyme binding properties and investigation of some inhibitors. Development of an antibody, which is specific for the E3 isozyme, to be used in tissue distribution studies by isoelectric focusing, crossed immunoelectrophoresis, and ELISA assay is also proposed. Tissues from normal and from alcoholic subjects will be used. Our Preliminary Data demonstrated that on isoelectric focusing gel many different aldehyde dehydrogenase isozymes superimpose. Purification and cloning of these new aldehyde dehydrogenases from human liver and from human brain constitutes another part of this proposal. The procedures employed will include: ion exchange chromatography on CM sephadex and DEAE Sephadex and affinity chromatography on 5'AMP Sepharose, Blue Sepharose and NAD Agarose. Characterization will include: MW, subunit MW, amino terminal sequence, extinction coefficient, Km and V determinations for substrates and coenzymes. The newly purified enzymes will be cloned and cDNA will be sequenced. The main purpose of characterization will be identification of physiological substrates and determination of primary structure of enzyme deduced from cDNA, in order to unequivocally establish the identity of new enzymes. Acetaldehyde may compete for aldehyde dehydrogenase with natural substrates and thereby inhibit the natural substrate metabolism. By employing specific inhibitors of individual isozymes, their metabolic importance can be tested in the future. The proposed studies will therefore provide a groundwork for studies of the interaction between natural substrates and acetaldehyde. They may also result in increased knowledge of Genetic Factors in Alcoholism.
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