The long-term objective of this research project is to elucidate the enzymatic regulation of alcohol metabolism. It is our hypothesis that genetic variations in the isoenzymes of human liver alcohol dehydrogenase (ADH) account to a large extent for individual differences in alcohol elimination rate and may underlie, in part, the observed individual differences in the physiological, psychological and pathological consequences of ethanol consumption. Studies in the past 12 years have revealed that ADH in human liver can be differentiated into 3 classes on the basis of their closeness in molecular and catalytic properties. Class I contains a large group of isoenzymes composed of various dimeric combinations of Alpha, Beta and Gamma subunits, coded by three genes, designated ADH1, ADH2 and ADH3, respectively. Genetic polymorphism at the ADH2 and ADH3 loci have been detected, giving rise to Beta 1, Beta 2 and BetaIndianapolis subunits, and Gamma 1 and Gamma 2 subunits, respectively. Class II are the pi-ADH and class III in chi-ADH molecular forms. The isoenzyme forms within class I differ in Vmax and Km values for substrates and coenzymes, and in pH-optima for activity. The goals of tis continuing research program in the next 5 years are the isolation and characterization of the BetaInd-containing isoenzyme forms, the multiple forms of Pi-ADH, and the high and low activity Beta 1 Beta 1 forms; the kinetic characterization of the negative cooperativity of ethanol oxidation by Gamma 1 Gamma 1 and Gamma 2 Gamma 2; elucidation of the structural and kinetic basis for the observed differences among the Beta 1 Beta 1, Beta 2 Beta 2 and BetaInd Beta 1 Ind alleloenzymes; determination if the amounts of the Alpha, Beta, Gamma, Pi and Chi subunits produced by the five ADH genes differ in different individuals; and the determination of ADH genotypes in livers and white blood cells by use of recombinant DNA technology. Affinity and ion-exchange chromatography, HPLC, starch-gel electrophoresis, isoelectric focusing and quantitative immunoblotting will be employed to isolate and identify the specific isoenzymes and to quantify the amounts of the different subunit forms. Steady-state and transient stopped-flow kinetics will be employed to compare the effects of pH, anions and deuterated ethanol on the kinetic and rate constants of the alleloenzymes. Protein and peptide sequence analysis and chemical modification of catalytically essential Cys, Arg and His residues will be employed to elucidate structural differences. Finally, human ADH-cDNA probes will be developed to search for restriction-length-polymorphism in liver specimens of known phenotypes and in leukocytes.
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