We will delineate the pathway by which the precursor protein of three different peptide-hormones is processed to the active peptides. Hypothalamic pro-gonadotropin releasing hormone precursor protein is acted on by specific proteases to form gonadotropin releasing hormone (GnRH--10 residues), gonadotropin associated peptide (GAP-56 residues) (thought to be prolactin releasing hormone inhibiting hormone), and a peptide that comprises the first 13-24 residues of GAP. These peptides help regulate mammalian reproduction and lactation by stimulating the release of gonadotrophic hormones from the pituitary. GnRH also affects the central nervous system, gonads, and placenta. GAP inhibits the release of prolactin. we will continue our studies with an enzyme that we have discovered that is associated with hypothalamic neurosecretory granule membranes that is capable of producing GAP and GnRH directly from the precursor protein (GAP releasing enzyme) . We will identify and study the hypothalamic enzyme that catalyzes the amidation of GnRH. we will synthesize peptide-substrates and peptide inhibitors that will allow us to probe the active-sites of these processing enzymes and help determine substrate specificity. We will use reverse-phase HPLC to separate the active peptides from assay mixtures and automated Edman degradation analysi to confirm the identity of the active peptides. We will assess the effect o the processed peptides on pituitary cells in culture to determine the bioactivities of the active peptides. We will raise antibodies to selected peptide fragments of the precursor protein (that we have synthesized) and develop a radioimmunoassay for measuring the precursor protein and GAP- peptide. We will raise antibodies to GAP-releasing enzyme to facilitate its purification. using these antisera, we will confirm the cellular and subcellular distribution of GAP releasing enzyme and of the precursor protein/GAP peptide.
