Alcohol dehydrogenase (ADH) is the major enzymatic pathway for ethanol metabolism, and as such has been studied for links to the wide between- individual variation in alcohol metabolic rates and to risk of alcoholism and its consequences. ADH isozymes at the ADH2 locus (beta1, beta2, and beta3) have very large differences in kinetic properties and significant influence on risk of alcoholism in Asians. Moreover, the beta2 isozyme appears to confer increased risk of alcoholic liver disease, possibly related to increased rates of acetaldehyde generation. However, despite the large differences in kinetic properties, the effects of ADH isozymes on alcohol metabolic rates are relatively small. We hypothesize that high activity isozymes (beta2 and beta3) are less active in vivo than predicted because of product inhibition by acetaldehyde. In addition, they may be less stable than the beta1 isozyme, and they may be subject to inhibition by the hydrated (gem-diol) form of acetaldehyde, acting as a competitive substrate. We will test the hypothesis directly by generating cell lines expressing the beta1, beta2, and beta3 enzymes. The turnover of these enzymes will be measured by the rate of decay of protein after inhibition of protein synthesis, and by pulse-chase labeling. The ability of these enzymes to metabolize ethanol will be tested in cell culture conditions that permit alterations of the substrate and product concentrations, as well as fatty acids, acetate, and adenine nucleotides. The rates of ethanol oxidation will be fit to steady-state rate equations to determine if the enzyme behavior is accurately predicted by the equations. This study should answer the question of how the presence of different isozymes of ADH modulates alcohol metabolic rates and intracellular acetaldehyde.
|Effective start/end date||1/1/99 → 12/31/03|
- National Institutes of Health: $149,386.00
- National Institutes of Health: $155,061.00
- National Institutes of Health
- National Institutes of Health: $152,055.00
Alcoholic Liver Diseases
Cell Culture Techniques