The pro-ACTH/endorphin system offers particular advantages for studying the physiology of varous opioid systems in particular and bio-active peptides in general. The same (or similar) pro-ACTH/endorphin precursor molecule is found in neurons and endocrine cells and is subjected to a series of tissue specific post-translational processing steps that generate a number of different bio-active peptides. Pro-ACTH/endorphin-derived peptides are involved in adrenal steroidogenesis, analgesia, and various behavioral phenomena; the various peptides can exert similar or antagonistic effects depending on their precise structure. This research project seeks to define precisely what pro-ACTH/-endorphin-derived peptides are present in various regions of the rat brain, anterior pituitary and intermediate pituitary. Multiple fractionation techniques (RP-HPLC, ion exchange chromatography, isoelectric focusing) coupled with immunoassay analyses and use of biosynthetically labeled peptides will be used to look at minor proteolytic modifications, acetylation, amidation, glycosylation and phosphorylation. The enzymes responsible for the postranslational processing of pro-ACTH/endorphin will be studied; particular attention will be given to those enzymes responsible for tissue-specific differences in processing and thus bioactivities produced. A bovine secretory granule associated acetyltransferase activity that Alpha-N-acetylates, Beta-endorphin and ACTH-related peptides will be further characterized and purified. In vitro assay for peptide COOH-terminal amidating activity that could be involved in the synthesis of Alpha-MSH will be devised; amidation is essential to the bioactivity of many small peptides and intermediate pituitary provides an excellent system for studying the enzymes involved. Attempts to characterize and purify granule associated proteolytic activities that could be involved in pro-ACTH/endorphin processing will be made. Appropriate conditions for high yield serum-free culture of pro-ACTH/endorphin cells from anterior pituitary, intermediate pituitary and hypothalamus (arcuate nucleus region) will be determined. Any hormonal signals involved in maintaining the tissue-specific functioning of pro-ACTH/endorphin cells will be defined. The pro-ACTH/-endorphin-derived peptides released by thes serum-free cultures under basal, stimulated or inhibited conditions will be determined.
