The proposed studies are designed to achieve a systematic and comprehensive analysis of the multigene family of salivary kallikreins in the rat and human. This analysis will focus upon gene expression and the regulation and distribution of salivary kallikreins at the cellular and subcellular levels. Tissue kallikreins are closely related serine proteases which specifically cleave peptide precursors to release biologically active peptides. Although kallikreins are found in many diverse tissues, their regulation and expression in salivary glands are important because of their potential role in processing a wide variety of substrates which may be of major importance in the maintenance of oral health. Recently developed tools have allowed us to sequence the complete rat kallikrein gene, the tonin gene, and to partially sequence several additional kallikrein-related genes. These sequences have provided valuable oligonucleotide probes to study the regulation of this family of enzymes. Further definition of the role of tissue kallikrein-related enzymes in salivary glands will be approached by: 1) complete mapping and sequencing of the cDNA for rat and human salivary kallikrein and other kallikrein gene family members, which will enable specific probe design for identifying and quantifying salivary kallikrein gene transcripts; 2) analyzing salivary kallikrein expression in vitro by deletion mapping, gel retardation assays and foot-printing techniques; 3) purifying and characterizing human and rat kallikreins to gain insight into their enzymatic properties and substrate specificities and to generate polyclonal and monoclonal antibodies for their individual identification and quantification; 4) elucidating the hormonal regulation of rat salivary kallikrein gene expression by specific RIA, Northern blotting, in situ hybridization, and immunocytochemical methods; 5) evaluating the subcellular compartmentalization of salivary kallikreins to gain insight into the sorting and secretory mechanisms of individual kallikrein gene family members.
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