Conventional column chromatographic procedures enabled a 1000 fold purification of a novel porcine pituitary proteinase, called IRCM-Serine Protease #1. The cleavage specificity of this enzyme towards small fluorogenic substrates and the native POMC - related polypeptide hormones ACTH 1-39, Beta-LPH 1-89 and N-terminal 1-76 and its subcellular distribution suggest that the enzyme may participate in pro-hormone processing in vivo. It is proposed to test this hypothesis, by obtaining enough enzyme for primary sequence determination in order to obtain specific antisera for immunohistochemical co-localization studies with POMC and other pro-hormones. Due to the low levels of enzyme (12 Mug/50 g wet weight) and the need of another 500-1000 fold purification, novel affinity column procedures using peptidyl-arginal columns are being developed. The latter, will be of general use in the purification of """"""""trypsinlike"""""""" Serine and Cysteinyl proteases. Furthermore, the direct microsequencing of electroblotted proteins from SDS gels should provide the necessary sensitivity at the 1-10 pmole level. Although initial experiments are being performed with proteinases purified from porcine pituitaries, we have the unique opportunity to have access during the term of this grant to nearly 15,000 frozen human pituitaries. These will be used to extend our knowledge to """"""""human maturases"""""""" and will constitute the major aim of this application. In addition the sequence information should allow the molecular cloning of this enzyme in order to obtain the cDNA sequence and the genomic organization of this protease in human. The antibodies and cDNA probes will be used to follow the variation in the levels of this enzyme during various physiological stimuli, allowing a further definition of the factors involved in its physiological regulation. The purification can be adapted for the isolation and characterization of the homologous enzyme from frozen human pituitaries and other tissues. Nucleotide probe screening can be done in human cDNA libraries. Additionally, we are currently characterizing other proteases isolated from human pituitaries in order to assess their possible involvement in pro-hormone maturation. The characterization of the enzyme system(s) involved in the conversion of precursor proteins into their end-products is a key step for the understanding and future research endeavors in the expanding field of neuropeptides, particularly since it will apply to human glands.
