Aromatic amines are believed to induce tumors in humans and experimental animals because of their conversion to N-hydroxylated derivatives which are further metabolized toreactive metabolites that are capableof altering nucleic acid. The formation of N-acetoxyarylamines has been implicated as the latter metabolic activation step that is crucial to the carcinogenic and mutagenic activities of many of these agents. In the rat, mouse and rabbit, and perhaps other species such as the human and hamster, the terminal activation reaction, O-acetylation, involves the transfer of an acetyl group to the hydroxylamine from either acetyl coenzyme A or an arylhydroxamic acid. The same enzyme is also capable of N-aetylation to yield arylacetamides or arylhydroxamic acids from arylamines or arylhydroxylamines, respectively. The N-, O- and N,O-acetylation activities of the enzymes from different species vary in their rates and relative abilities to carry out these reactions. These differences are significant determinants of the susceptibilities of the target organs of the aromatic amines. The objective of this project is to explore the molecular features responsible for the differences in activation by O-acetylation and, consequently, how these factors mayinfluence the carcinogenic and mutagenic responses to these agents.
The specific aims of the project are to: (1. Determine the primary sequence of the rat enzyme bymolecular cloning. Oligonucleotide probes and monoclonal antibodies will be used to screen cDNA libraries. (2. Identify the active site(s) of the enzyme by labelling with reagents specific for sulfhydryl groups and N-hydroxy-chloroacetylaminoarene substrates to identify amino acids that are crucial to the catalytic activity. the monoclonal antibodies will be used to help in establishing the areas of the molecule that are involved in catalysis. (3. Localize the acetyltransferase and mRNA in rat tissues by use of monoclonal antibodies and cDNA probes to identify those cells that may be at particular risk from these compounds. (4. Express acetyltransferase in bacterial and mammalian cells to verify the identity of the sequence and to provide a means by which cells can be provided with a metabolic activation system. (5. Compare the structures of acetyltransferases from other species that differ in their abilities to carry out N-,O- and N,O- acetyltransfer.
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