Non-oxidative alcohol metabolism to form fatty acid ethyl esters occurs in those extrahepatic organs most commonly injured by alcohol abuse, such as the brain, pancreas and heart. Over the last ten years, the laboratory of the principal investigator has described this pathway, particularly in the heart, and these metabolites have been identified as toxic to mitochondria. During the current grant application interval, the applicant has successfully cDNA cloned Synthase II, determined its cDNA sequence (J. Biol. Chem., manuscript in press) and mapped the gene to chromosome 11q14. These results confirm our previous contention that Synthase III is a new member of the glutathione S-transferases (GST). Because these latter enzymes are responsible for carcinogen inactivation, these results interrelate ethanol and carcinogen metabolism. Importantly, only four of 210 amino acid residues differ between Synthase III and the known GSTpi-1, which lacks alcohol-metabolizing capabilities. Thus the current application seeks to employ chemical modification and site-directed mutagenesis to determine which residues are responsible for conferring alcohol metabolic capability to the human myocardial Synthase III. Complementary studies will determine the structure of the human gene for Synthase III. Also completed during the current grant interval is the successful purification to homogeneity of human myocardial Synthases I and II. These results enabled the generation of specific antibodies to these proteins as well as determination of the N-terminal amino acid sequence for developing oligonucleotide probes for cDNA cloning. Accordingly, proposed studies also include determination of the cDNA and gene structure for Synthase I and II, as well as their chromosomal localization and mapping. The results of the proposed studies will thus provide us with a complete gene structure and location for all the known fatty acid ethyl ester synthases (I, II and III) and will establish the bases for genetic linkage studies in patients and their families with alcohol-induced end organ phenotypes, such as alcohol-induced heart muscle disease.
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