Zymogen secretion is of critical relevance in the digestive process of the gastrointestinal tract and is considered to contribute to pathological processes such as reflux esophagitis, peptic ulcer disease and pancreatitis.
The aim of the present studies is to delineate and characterize aspects of the molecular mechanisms of zymogen secretion utilizing an isolated zymogen granule model. The secretory granule is the key organelle of exocytosis, therefore, studies on characterization of zymogen granules are crucial for the understanding of exocrine secretion in general. Specifically, the aim of the present proposal is to delineate the physiological role of a novel zymogen granule membrane- associated protein kinase activity in gastric chief cells, pancreatic and parotid acinar cells. First, to identifying an important signature of this novel kinase, the endogenous substrate(s) for the kinase will be identified in a gastric chief cell model, and the specific peptide sequence of the substrate will be determined by phospho-peptide sequence analysis of phosphorylated substrates. This sequence will then be utilized to construct pseudo-substrate peptide inhibitors against the kinase. Second, purification of the kinase from peptic granule membranes will be undertaken by a combination of affinity and conventional chromatography strategies in order to obtain the microsequence. Third, monoclonal antibody against the partial peptide sequence of the kinase will be produced to determine the specific cellular localization and redistribution of the kinase utilizing immunocytochemisty at the light and confocal microscopic as well as the electron microscopic level. Fourth, the effect of the kinase on enzyme secretion will be evaluated in the permeabilized gastric gland and pancreatic acinar cell system by introduction of peptide inhibitors and specific antibodies, and finally, the determination of the complete sequence of the kinase will be undertaken utilizing the molecular cloning techniques of Polymerase Chain Reaction and screening of a gastric chief cell cDNA library. The recombinant protein will be produced for raising monoclonal antibody for further functional studies. These studies will allow the characterization of a novel putative regulator of zymogen exocytosis. The elucidation of the mechanism of zymogen secretion will facilitate the identification of its relevance to human disease processes. The ability to regulate zymogen secretion may become an important therapeutic strategy.
