The objective of the proposed research is to study the biochemical properties, subcellular distribution, and physiological regulation and significance of kiningenerating proteinases (kininogenases) I have recently detected in rat and porcine pituitaries. Rat and porcine pituitary kininogenases will be solubilized, and purified using a combination of affinity and gel-filtration chromatography. Purified kininogenases will be characterized with regard to molecular weight, specificity and kinetics of substrate hydrolysis, sensitivity to synthetic and natural inhibitors, and ability to cleave intact rat pro-opiomelanocortin into its hormonal fragments and generate opioid or melanocyte-stimulating peptides from ACTH or Beta-lipotropin. Rat and porcine pituitaries will be homogenized in sucrose buffer and various subcellular fractions prepared using a combination of differential centrifugation and discontinuous sucrose density gradients; kininogenase activity in the various subcellular fractions will be determined using synthetic and natural substrates for kininogenases. In the rat, responses of pituitary kininogenases to various endocrine and pharmacological manipulations will be examined both in vitro and in vivo in order to characterize the factors which may regulate pituitary kininogenases and their function. In addition, pituitaries of various mouse strains, amphibians, and reptiles will be compared with respect to kininogenase activity. The proposed research should elucidate the characteristics of kininogenases associated with various pituitary lobes in different species and suggest how kininogenases may regulate pituitary function. Such studies should lay the foundation for future research on the role of kininogenses and other proteases in pituitary function using techniques of pharmacology, endocrinology, and molecular biology. The pituitary promises to be a model system for studying how specific proteases regulate the post-tranalational processing of secretory proteins. Such research may ultimately illuminate the involvement of proteases in clinical syndromes characterized by an altered production of peptide hormones or othe protein products.
