Distribution of aldehyde dehydrogenase (EC 1.2.1.3) in different subcellular locales suggests specific function for the isozymes. Purification of new isozymes from human liver and further characterization of the E1 and E2 isozymes, which are at present available in a homogeneous form, are the objectives of this proposal. The work with the homogeneous isozymes E1 and E2 will involve identification of their active and/or regulatory site(s) by chemical modification with group specific reagents, as well as with the affinity reagents, which will be developed on the basis of known substrate specificity of aldehyde dehydrogenase. Standard techniques of protein purification, including ion exchange and affinity chromatography, will be used. Characterization of the new isozymes will include: amino acid analysis, molecular weight of the isozymes and their subunits, peptide mapping, interaction with antibodies, substrate and coenzyme specificity and kinetic properties with acetaldehyde as substrate. Concurrently, aldehyde dehydrogenase content and isozyme distribution in crude extracts of different human tissues (post-mortem) from alcoholics and non-alcoholics will be investigated and isozymes from post-mortem liver of a diagnosed alcoholic will be purified for comparison with normal isozymes. The results will provide information about structural relationships, catalytic properties, human species-specific properties, and possibly, ways in which the isozymes from the normal individual differ from those of the alcoholic. Understanding of the role of aldehyde dehydrogenase in metabolic regulation also will be advanced. The chemical modification of the active site will, in addition to increasing knowledge, provide a means for designing specific inhibitors or activators of the individual isozymes, thus providing grounds for development of therapeutic agents useful for treatment of the alcoholic. In alcoholism aldehyde dehydrogenase functions in converting toxic acetaldehyde to a harmless product. Its role is essential for the health of the alcoholic, and knowledge of its properties is a basic requirements for rational interpretation of the research carried out under the Institutes Priorities of: """"""""Genetic Factors in Alcoholism"""""""", """"""""Fetal Alcohol Syndrome"""""""", and """"""""Alcoholism in Women"""""""".
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