This proposal is submitted for the purpose of continuing the functional and structural analyses of components of the human renal/urinary kinin-forming system, clarifying their relationships to similar enzymes from other human tissues and cells and examining their possible roles in renal function or pathophysiology. Kinetic and structural characterization of the interaction of urinary, pancreatic and salivary kallikreins with fully functionally active, intact high and low molecular weight kininogens; purification and characterization of latent (trypsin-activatable) kallikrein from human urine; and immunochemical localization of kallikreins in human tissue will all be continued in studies that have developed from the findings of the previous project period. The purification of latent urinary kallikrein and elucidation of its relationships to the active urinary enzyme will permit the purification, characterization, and immunohistochemical localization of a latent kallikrein activator, as well as the development and application of a radioimmunoassay to determine the role of the activator in renal function. The purification and analytical procedures developed for latent kallikrein will be applied to the purification and comparative structural and functional analysis of human renal prokallikrein. The elicitation and characterization of specific anti-kallikrein antibodies and the use of one of them to develop a kallikrein radioimmunoassay lay the groundwork for the preparation and analysis of anti-urinary kallikrein monoclonal antibodies to be used for further structural analyses of human tissue kallikreins and their related latent or precursor forms; in continued immunohistochemical localization studies; and in the development of immunoaffinity purification techniques. These monoclonal anti-kallikreins together with polyclonal antibodies specific for renal kallikrein activator(s) or kininases will be used in the development of specific radioimmunoassays to further examine the role of these components of the renal kinin-forming system in regulating salt and water clearance in normal individuals and in patients with altered renal function.
