NOVEL PROTEIN KINASE IN DIGESTIVE ENZYME SECRETION

酶原分泌在消化过程中至关重要， 胃肠道，并被认为有助于 病理过程，如反流性食管炎、消化性溃疡病 和胰腺炎 本研究的目的是描述和 表征酶原分泌的分子机制 利用分离的酶原颗粒模型。 分泌颗粒是 胞吐作用的关键细胞器，因此， 酶原颗粒的形成对于了解外分泌至关重要 一般的分泌物。 具体而言，本提案的目的是 描绘一种新的酶原颗粒膜的生理作用- 相关蛋白激酶活性在胃主细胞，胰腺和 腮腺腺泡细胞 第一，识别一个重要的签名， 这种新的激酶，激酶的内源性底物， 在胃主细胞模型中鉴定，并且特异性肽 底物的序列将通过磷酸肽序列测定 磷酸化底物的分析。 这个序列将是 用于构建针对所述抗体的假底物肽抑制剂。 激酶。 第二，从消化颗粒中纯化激酶 膜将通过亲和性和 常规色谱策略，以获得 微序列 第三，针对部分肽段的单克隆抗体 将产生激酶的序列以确定特异性的 激酶的细胞定位和再分布， 免疫细胞化学在光镜和共聚焦显微镜以及 电镜水平。 第四，激酶对酶的影响 将在透化的胃腺中评价分泌， 胰腺腺泡细胞系统，通过引入肽抑制剂， 特异性抗体，最后，确定完整的 将利用分子克隆进行激酶的测序 聚合酶链反应技术与胃总管的筛选 细胞cDNA文库。 重组蛋白将被生产用于饲养 单克隆抗体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剂 胞吐作用 酶原分泌机制的阐明将有助于 有助于确定其与人类疾病的相关性 流程. 调节酶原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.