Pancreatic zymogen activation in a reconstituted system

重建系统中胰腺酶原的激活

• 批准号: 6855024
• 负责人: Edwin C Thrower
• 金额: $ 13.04万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6855024
• 关键词: adenosinetriphosphatase; calcium ion; calmodulin; cell component structure /function; cell components; cell free system; disease /disorder model; enzyme activity; enzyme induction /repression; enzyme mechanism; intermolecular interaction; laboratory rat; membrane channels; molecular pathology; nucleoside triphosphate; pancreas enzyme; pancreatitis; protein localization; proteolysis; western blottings; zymogens

DESCRIPTION (provided by applicant): Premature activation of pancreatic digestive zymogens within the acinar cell is a pivotal early step in initiation of acute pancreatitis, but the mechanisms underlying this phenomenon are largely unknown. Cytosolic events, particularly an increase in intracellular Ca2+, regulate the activation of zymogens within intracellular compartments, although the specific identity of these compartments remains unclear. To examine the identity of the compartment(s) and the molecular mechanism of this activation, we have performed the following preliminary studies. First, using a caerulein-hyperstimulation model of acute pancreatitis, we find that activated zymogens are found in both zymogen granule and microsomal fractions. Next, to directly examine pathways that regulate the activation, we have developed a reconstituted system using isolated pancreatic cytosol and a 100,000xg particulate fraction. Using this system we find that zymogen activation requires the presence of both cytosol and the particulate fraction. This activation is prominently enhanced by ATP (5 mM) but not by the inactive ATP analogue, AMP-PNP. The ATP-dependent activation is further enhanced by the addition of Ca2+ to the incubation medium and reduced by calcium chelation. When the particulate fraction was separated into zymogen granule and microsomal fractions, ATP and Ca2+ -dependent zymogen activation was observed in both. These studies are the first to report reconstitution of zymogen activation using acinar cell fractions. They demonstrate that activation occurs in relevant compartments, requires cytosolic factors and ATP and is enhanced by Ca2+. We plan to use this system to identify the compartment(s) responsible for zymogen processing and to examine the ATP and calcium-dependent mechanism of activation.

描述（由申请人提供）：腺泡细胞内胰腺消化酶的过早激活是启动急性胰腺炎的重要早期一步，但是这种现象的基本机制在很大程度上是未知的。胞质事件，尤其是细胞内Ca2+的增加，调节了细胞内室内酶原的激活，尽管这些隔室的特定身份尚不清楚。为了检查隔室的身份和这种激活的分子机制，我们进行了以下初步研究。首先，使用急性胰腺炎的囊蛋白 - 高型刺激模型，我们发现在Zymogen颗粒和微粒体馏分中都发现了活化的Zymogens。接下来，为了直接检查调节激活的途径，我们使用孤立的胰腺细胞质和100,000xG颗粒分数开发了一个重构系统。使用该系统，我们发现酶原激活需要胞质和颗粒分数的存在。 ATP（5 mM）显着增强了这种激活，但没有通过非活动的ATP类似物AMP-PNP来增强这种激活。通过在孵育培养基中添加Ca2+并通过钙螯合降低，可以进一步增强ATP依赖性激活。当颗粒分数分为酶原颗粒和微粒体级分时，在两者中都观察到ATP和Ca2+依赖性酶原激活。这些研究是第一个使用腺泡细胞级分报告酶原激活的重构的研究。他们证明激活发生在相关隔室中，需要胞质因子和ATP，并且通过Ca2+增强。我们计划使用该系统来识别负责酶原处理的车厢，并检查ATP和钙依赖性激活机制。