The peptide nature of pituitary hormones and hypothalamic releasing factors necessitates the involvement of proteolytic enzymes in their formation and degradation. Our work during the past years has led to the identification, isolation and characterization of several pituitary and brain endopeptidases likely to be involved in the metabolism of peptide hormones and neuropeptides. These enzymes include: a membrane-bound metalloendopeptidase that degrades enkephalins (""""""""enkephalinase"""""""") and also other peptide hormones and neuropeptides, ans soluble metalloendopeptidase that degrades LBRH and neuropeptides and rapidly coverts the opioid peptides dynorphin, Alpha and Beta-neoendorphin and BAM-12P into enkephalins; prolyl endopeptidase, an enzyme apparently involved in the metabolism of proline-containing peptides (including LHRH and TRH); and a multicatalytic protease complex potentially involved in the metabolism of several peptide hormones. Our studies on these enzymes have led to the synthesis of several potent active-site-directed inhibitors that can be used as probes in studies on the role of these enzymes in the metabolism of peptide hormones. The objective of this proposal is to extend our work on the biochemistry and substrate recognition site of these enzymes, to continue and extend our synthetic work on active-site-directed inhibitors with the lowest possible Ki, and to use the inhibitors presently available, as well as those to be synthesized, as probes in in vivo studies and in studies with cell preparations on the role of these enzymes in the metabolism of peptide hormones and biologically active peptides in rats. Inhibitors of the membrane-bound metalloendopeptidase will be used to study the role of this enzyme in degradation of opioid peptides in the posterior pituitary, a tissue rich in these peptides. Inhibitors of the soluble metalloendopeptidase will be used in studies on the role of this enzyme in the conversion of dynorphins, neoendorphins and rimorphin (dynorphin B) into Leu-enkephalin. Indirect evidence suggests that prolylendopeptidase and the two metalloendopeptidases may control the in vivo degradation of LHRH. Inhibitiors of these enzymes will therefore be studied for their effect on: (1) endogenous LHRH and LH levels, (2) their in vivo effect on degradation of exogenous LHRH given by intravenous injection, (3) on the LHRH-stimulted secretion of LH in vivo and (4) on the basal and LHRH-stimulated secretion of LH by cultured pituitary cells in vitro. Studies will be continued on the biochemistry of the multicatalytic protease complex.
