The existence in man of an alcohol-inducible liver cytochrome P450 isozyme, distinct in its capacity to metabolize ethanol and other compounds of pharmacological and/or toxicological importance, is now established. However, the actual role of this microsomal enzyme, termed P450-ALC, in human diseases states promoted by chronic alcohol consumption is ill-defined at present. To that end, we will expand on our initial studies concerning the characterization of purified human liver P450-ALC. The physical properties of P450-ALC proteins isolated from different human livers will first be compared in terms of NH2-terminal amino acid sequence, amino acid composition, proteolytic digestion mapping, and spectral characteristics. The capacity of these different human P450-ALC preparations to metabolize relevant substrates, including ethanol, acetaminophen, acetone, N- nitrosodimethylamine and carbon tetrachloride, will then be examined in a reconstituted system. Our objective here is to assess whether differences exist among individuals in P450-ALC catalytic properties and, if such differences exists, whether they result from alterations in enzyme structure. The mechanisms of ethanol oxidation by human P450-ALC will be studied in detail, particularly with regard to the capacity of this hemeprotein to generate reactive metabolites other than acetaldehyde. Antibodies directed against P450-ALC will then be used in immunoquantitation and immunoinhibition studies to gain insight into this protein's role in metabolism of the aforementioned substrates by human liver microsomes. These immunochemical studies may further aid in revealing functional variations in P450- ALC among individuals. We will also investigate, on a molecular basis, the expression of P450-ALC and its inducibility by ethanol consumption in alcoholic and non-alcoholic subjects using both antibodies and cloned cDNAs. Both P450-ALC cDNA and genomic DNA clones will be employed to assess whether a) restriction fragment length polymorphisms exist in or near the P450-ALC structural gene and b) if such polymorphic DNA regions encode for a catalytically-altered protein product or give rise to genetic variation in P450-ALC expression and/or inducibility. Our overall goal is to describe, in precise biochemical terms, the relation of P450-ALC to those human pathologies caused by excessive alcohol consumption and, ultimately, to the genetic predisposition to alcoholism.
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