It has been known for some time that interaction of the metabolism of ethanol with that of biogenic amines (such as dopamine and serotonin) occurs at the level of aldehyde dehydrogenase. In this proposal additional evidence is discussed which suggests that metabolism of plutrescine and histamine may also interact with that of ethanol at the level of the same enzyme. Putrescine arises from ornithine by decarboxylation and is converted via putrescine pathway to spermine and spermidine, important in development and differentiation or via diamine oxidase and aldehyde dehydrogenase pathway to gamma-aminobutyric acid - a well-known inhibitory neurotransmitter. The recently identified third isozyme, E3, of aldehyde dehydrogenase (EC 1.2.1.3) metabolizes both acetaldehyde and gamma- aminobutyraldehyde at low concentrations. 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 (EC 1,2,1,3) 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 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. Interaction of metabolism of acetaldehyde with that of hormonal aldehyde metabolites will also be studied b y gel electrophoresis techniques. Such 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 determining precisely the individual E1, E2 and E3 isozymes in the crude tissue homogenates is also envisaged.
Showing the most recent 10 out of 16 publications
