HYDROLYSIS OF VITAMIN A ESTERS IN LIVER

描述（由申请人提供）：肝脏中视黄酰基酯（RE）的水解在人体维生素a的代谢中起着关键作用。饲料中的维生素a以RE的形式进入肝脏，以乳糜微粒残体的形式被水解和再酯化储存。在动员之前，RE被水解，游离视黄醇与视黄醇结合蛋白结合，分泌到血浆中。从大鼠肝脏中分离到中性和酸性视黄酰基酯水解酶（REHs），经序列分析分别为羧酸酯酶ES-2和ES-10。该项目的目的是确定ES-2、ES-10以及相关的ES-3和ES-4在维生素A代谢中的作用。羧酸酯酶已与其他底物一起研究，但尚不清楚它们是否在维生素A代谢中起重要作用。与其他酯相比，它们催化稀土水解的效率也未知。这些知识缺口将与以下具体目标进行探讨：AIM 1: REHs酶学。酶动力学将用于研究羧酸酯酶的底物特异性，并验证这些酶催化RE和其他脂质酯水解的内在能力不同的假设。目的2：羧酸酯酶的组织分布。实时荧光定量PCR（用于mRNA）、免疫检测（用于蛋白质）和酶分析将用于确定各种组织中酶的水平和分布，并验证羧酸酯酶存在于外周组织和肝脏中的假设。目的3：肝脏细胞类型分布。显微解剖和细胞分离结合实时PCR、免疫检测和酶分析将用于确定羧酸酯酶在星状细胞和肝细胞之间的分布，并测试羧酸酯酶ES-2只定位于肝细胞和羧酸酯酶ES-10定位于肝细胞和星状细胞的假设。目标4:羧酸酯酶的生理角色再保险在培养细胞的新陈代谢,互补dna转染和RNA干扰将用于调节羧酸酯酶酶水平测试的假设不同的羧酸酯酶代谢的作用在肝脏吸收和/或代谢乳糜微粒再保险公司和测试假设羧酸酯酶代谢的作用在细胞内的营业额再保险和胆甾醇酯细胞存储它们。

DESCRIPTION (provided by applicant): Hydrolysis of retinyl esters (RE) in the liver plays a key role in the body's metabolism of vitamin A. Dietary vitamin A enters the liver as RE in chylomicron remnants, which undergo hydrolysis and reesterification for storage. Prior to mobilization, RE are hydrolyzed and free retinol binds to retinol-binding protein for secretion into the plasma. Neutral and acid retinyl ester hydrolases (REHs) have been purified from rat liver and shown by sequence analysis to be carboxylesterases, ES-2 and ES-10. The goal of this project is to define the role of ES-2, ES-10, and the related ES-3 and ES-4, in the metabolism of vitamin A. Carboxylesterases have been studied with other substrates, but it is not known if they are important in vitamin A metabolism. Their efficiencies in catalyzing the hydrolysis of RE compared with other esters are also unknown. These gaps in knowledge will be explored with the following specific aims: AIM 1: Enzymoloqy of REHs. Enzyme kinetics will be used to study the substrate specificity of the carboxylesterases and to test the hypothesis that these enzymes differ in their intrinsic ability to catalyze the hydrolysis of RE and other lipid esters. AIM 2: Tissue distribution of carboxylesterases. Real-time PCR (for mRNA), immunodetection (for protein) and enzyme assay will be used to define the levels and distributions of enzymes in various tissues and to test the hypothesis that the carboxylesterases are present in peripheral tissues as well as in liver. AIM 3: Cell type distribution in liver. Laser Capture Microdissection and cell separations coupled with real-time PCR, immunodetection, and enzyme assays will be used to define the distribution of carboxylesterases between stellate cells & hepatocytes and to test the hypotheses that carboxylesterase ES-2 is localized exclusively in hepatocytes and that carboxylesterase ES-10 is localized in hepatocytes and stellate cells. AIM 4: Physiologic roles of the carboxylesterases in the metabolism of RE in cultured cells, cDNA transfection and RNA interference will be used to modulate carboxylesterase enzyme levels to test the hypothesis that the various carboxylesterases play a metabolic role in the hepatic uptake and/or metabolism of chylomicron RE, and to test the hypothesis that the carboxylesterases play a metabolic role in the turnover of intracellular RE and cholesteryl esters in cells that store them.