NOVEL PROTEIN KINASE IN DIGESTIVE ENZYME SECRETION

酶原分泌在猪的消化过程中起着至关重要的作用 胃肠道，并被认为对 反流性食管炎、消化性溃疡等病理过程 还有胰腺炎。本研究的目的是勾勒出和 酶原分泌分子机制的研究进展 利用分离的酵母菌颗粒模型。该分泌颗粒为 因此，胞吐作用的关键细胞器的特性研究 酶原颗粒对于理解外分泌是至关重要的 一般的分泌物。具体地说，本提案的目的是 为了描述一种新型的酶原颗粒膜的生理作用- 胃主细胞、胰腺和胃壁的相关蛋白激酶活性 腮腺腺泡细胞。首先，要确定一个重要的签名 这种新的激酶，内源性底物(S)将是 在胃主细胞模型中鉴定，以及特定的多肽 底物的序列将由磷酸肽序列确定 磷酸化底物的分析。然后，该序列将是 用来构建针对该病毒的拟底物多肽抑制剂 激活剂。其次，从消化颗粒中分离纯化出一种蛋白激酶。 膜将由亲和力和亲和力的组合进行 传统的层析策略以获得 微序列。第三，抗部分肽的单抗 将产生的激酶序列来确定其特异性 利用蛋白激酶的细胞定位和重分布 光镜和共聚焦显微镜下的免疫细胞化学以及 电子显微镜水平。第四，激酶对酶的影响 分泌物将在渗透性胃腺和 胰腺腺泡细胞系统通过引入多肽抑制剂和 特定的抗体，最后，确定完整的 将利用分子克隆对该激酶进行测序 聚合酶链式反应技术及胃长的筛选 细胞c DNA文库。将生产重组蛋白用于饲养 单抗用于进一步的功能研究。这些研究将 允许表征一种新的假定的酶原调节因子 胞吐。对酶原分泌机制的阐明将 促进确定其与人类疾病的相关性 流程。调节酵素分泌的能力可能成为一种 重要的治疗策略。

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.