We have achieved technical advances and acquired tools which place our group in a unique position to study the glandular kallikrein system. We have obtained pure human and rat urinary kallikrein and rat urinary esterase A (a urokininogenase), gathered important information about the properties and active site chemistry of kallikrein, generated high titer antisera to glandular kallikreins, developed direct radioimmunoassays, localized kallikreins at light and electron microscopic levels, demonstrated the effects of sympathomimetic drugs and endotoxin on renal and submaxillary kallikrein levels, found regulation of submaxillary kallikrein and esterase A by testosterone and thyroxine and reported a direct action of kallikrein on uterine contraction independent of detectable kinin formation. The proposed research will be devoted to the purification and characterization of free active and bound glandular kallikreins in serum by antibody-affinity chromatography and compare them to urinary or tissue kallikreins. With pure kallikreins at hand, we will employ hybridoma technology to produce monoclonal antibodies against kallikreins. Monoclonal antibodies will be purified, characterized and used for cellular localization, radioimmunoassay and kallikrein cell surface orientation studies. A significant effort will be devoted to elucidate kallikrein gene structure and regulation using recombinant DNA technology. Kallikrein mRNA will be isolated by agarose gel electrophoresis and identified by in vitro translation. Purified kallikrein mRNA will be used to construct kallikrein cDNA clones for sequencing studies and for screening kallikrein gene(s) from a genomic DNA library. We will analyze kallikrein gene sequence and organization by restriction mapping and nucleotide sequencing techniques. Kallikrein cDNA clones and monoclonal antibodies will be used to probe the effects of selected hormones and drugs on kallikrein mRNA and kallikrein protein synthesis. The proposed studies will significantly advance our knowledge of the requlation and function of the glandular kallikrein-kinin system.