Methyl conjugation is an important pathway in the metabolism of many drugs, xenobiotic compounds, and neurotransmitters. The studies proposed in this application are designed to enhance our understanding of biological mechanisms responsible for individual variation in methylation. Special emphasis is placed on the role of inheritance, i.e., on pharmacogenetics. Our laboratory has already described genetic polymorphisms in humans for two important methyl conjugating enzymes, thiopurine methyltransferase (TPMT) and catechol O-methyltransferase (COMT). We have also developed inbred rodent models in which the pharmacologic and toxicologic implications of genetic variation in these enzyme activities can be studied. We now propose to extend our studies of the pharmacogenetics of methyl conjugation to include N-methylation catalyzed by nicotinamide N- methyltransferase (NNMT) -- an enzyme capable of catalyzing the formation of potentially toxic pyridinium ions. We have developed a sensitive new ion-pairing radiochemical assay for NNMT and have used the assay to demonstrate large individual variations in human liver NNMT activity as well as the existence of a subgroup of individuals with high enzyme activities. We now propose to study the nature and extent of NNMT- catalyzed N-methylation of the nontoxic vitamin nicotinamide in a large human population sample and in nuclear families to determine the possible contribution of inheritance to the regulation of this pathway of methylation. We will also use the NNMT assay to purify the enzyme from human liver. Purified human liver NNMT will be used to study the biochemical properties of the enzyme as well as for photoaffinity labeling and amino acid sequencing. In parallel with these experiments, we will study the possible role of inheritance in the regulation of large strain- related variation in hepatic NNMT activity among inbred mice. Finally, we propose to extend our previous studies of the pharmacogenetics of TPMT and COMT in humans to the molecular level by application of the techniques of photoaffinity labeling and amino acid sequencing. The results of this series of experiments will enhance our understanding of biological mechanisms responsible for individual variation in the methylation of drugs, xenobiotics, and neurotransmitters and may make it possible to predict individual differences in metabolism, efficacy and toxicity of drugs metabolized by methyl conjugation.
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