Conversion and inactivation of selected neuropeptides will be studied with the aim of delineating some of the enzymatic mechanisms involved in their processing in the CNS. For studies on conversion two series of reactions will be emphasized: 1) components of the 'renin-angiotensin' system in brain and 2) metabolism of ACTH/Lipotropins. Angiotensin-1 converting enzyme (E.C. 3.4.15.1) will be purified further by immunoaffinity procedures using antibody to rabbit pulmonary enzyme, and an enzyme with 'iso-renin' like properties will be purified on Sepharose-pepstatin columns and its specificity and properties compared to renin of other tissues. For studies on lipotropins, the native substrates will be prepared from pituitaries by established procedures and the precursor forms identified using specific antisera for ACTH 1-24 and LHP 61-91, along with biochemical criteria (chromatographic and electrophoretic profiles, fingerprinting techniques). Breakdown of these substrates will be followed using sensitive microprocedures involving dansylated and iodinated forms, and by bioassay. Emphasis will be placed on enzymes with 'Tryptic-like' specificities involved in the release of active fragments such as beta-MSH (LPH 41-58) and beta-endorphin (LPH 61-91) using native substrates or suitable synthetic congeners. In terms of enzymes involved in inactivation, structure-activity relationships will be examined utilizing synthetic analogs of LH-RH (luteinizing hormone releasing hormone), somatostatin, endorphins and enkephalins. Rates of biodegradation will be compared with their known potencies in vivo. Enzymes inactivating hypothalamic releasing hormones by internal cleavage (endo peptidases) using affinity chromatography and quantitative isoelectric focusing techniques. The specificity of such enzymes will be compared to others previously purified in this laboratory and available for such studies.
NIH ExPorter
NIH ExPorter