Physiological role of aldehyde dehydrogenase in the mammalian brain is known to be in catabolism of biogenic amines. Due to its multiplicity, however, it is not quite clear if the enzymes metabolizing biogenic amines and acetaldehyde are the same. This is further complicated by the fact that similar enzymes also occur in liver. Interaction of the metabolism of alcohol with that of biogenic amines was demonstrated previously and shown by means of inhibitors (e.g., disulfiram) to occur at the level of aldehyde dehydrogenase. In this proposal new evidence is presented which suggests that metabolism of putrescine and histamine may also interact with that of ethanol at the level of the same enzyme. Up to the present time aldehyde dehydrogenase (EC 1.2.1.3) from human liver which metabolizes acetaldehyde was known to consist of two isozymes, E and E2. The recently identified third isozyme, E3, metabolizes both acetaldehyde and gamma- aminobutyraldehyde at low concentrations. Gamma-aminobutyraldehyde arises from putrescine via diamine oxidase and is converted to gamma-aminobutyric acid (a well-known inhibitory neurotransmitter) via aldehyde dehydrogenase. Detailed characterization of the E3 isozyme with regard to its catalytic characteristics, especially with regard to its substrate specificity, inhibitor interaction, organ localization and structural characterization via cloning and DNA sequence, confirmed by amino acid sequence analysis comprises a major part of this proposal. Since human aldehyde dehydrogenase consists of three known isozymes (E1, E2, E3), it is proposed to continue characterization of all three isozymes via chemical modification. The proposal includes identification of the catalytic residue, of the third residue involved in catalysis and of the coenzyme-binding site. Studies with the enzyme inhibitors are also proposed, which, in addition to kinetic studies, envisage development of isozyme specific mechanism based suicide inactivators. Such inactivators can then be effectively employed for further studies to establish the roles of individual isozymes in alcohol:hormone:neurotransmitter interaction. Suicide inactivators will also be suitable as therapeutic agents, much superior to disulfiram because of their absolute specificity. Such new enzymes that are detectable with both the hormonal metabolites and short chain aliphatic aldehydes will be purified to homogeneity and characterized. Development of monoclonal antibodies and radioimmunoassay capable to determine precisely the individual E1, E2, and E3 isozymes in the crude tissue homogenates is also envisaged.
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