The long term objective of this research program is the understanding of the molecular mechanisms involved in the postsynaptic regulation of neuronal function, using the catecholamine mediated regulation of serotonin N- acetyltransferase (NAT) activity in the rat pineal gland as a model. This enzyme regulates the concentration of the putative pineal hormone, melatonin, in the circulation. The significance of this study derives from 1) the wide spread role of postsynaptic regulatory processes in both normal and disordered development and maintenance of nervous system functions, as well as 2) the demonstrated role of melatonin as a regulatory hormone in many vertebrates and the likelihood that it has similarly important, but incompletely documented, functions in man. In order to study the molecular mechanisms involved in the regulation of NAT itself and, indirectly, melatonin synthesis, the following immediate aims are proposed: 1) produce antibodies against NAT, 2) purify NAT to homogeneity, 3) determine whether the synthesis of NAT protein is increased when NAT activity is stimulated at night and 4) clone NAT cDNA if NAT protein synthesis is found to be regulated by light-dark cycles. Pineal NAT in the rat exhibits a circadian rhythm with 50-100 fold increase in activity at night in the dark. The increase in NAT activity is mediated by an adrenergic-camp mechanism involving both transcriptional and translational events. The PI has recently succeeded in purifying NAT from rat pineal gland and plans to continue his studies to understand the molecular mechanisms involved in NAT regulation. A three tiered approach for the production of antibodies of different quality/specificity will be proposed in order to permit the orderly investigation of the molecular biology of NAT regulation. The hypothesis that the nocturnal increase in NAT activity involves increased synthesis of NAT protein will be tested by determining the amount of NAT protein during day and night; this will be done by immunotitration of NAT activity. In addition, the rate of incorporation of radiolabelled amino acids into NAT protein will be determined during day and night with the aid of NAT antibodies. If the synthesis of NAT protein is found to be increased at night, then efforts will be focused on cloning NAT cDNA in order to subsequently study in detail the mechanisms involved in the regulation of expression of NAT gene. If it is not found to be the case, then subsequent efforts will be directed towards the analysis to possible post-translational mechanisms of NAT regulation with the aid of antibodies.