Hydrolysis of vitamin A esters in liver

维生素A酯在肝脏中的水解

基本信息

批准号：
7289291
负责人：
EARL Howard HARRISON
金额：
$ 33.55万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-09-30 至 2011-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7289291
关键词：
1,2-diacylglycerol Accounting Acids Air All-Trans-Retinol Bile Acids and Salts Binding Biological Assay Carboxylic Ester Hydrolases Cell Line Cell Separation Cells Cholesterol Esters Chylomicrons Complementary DNA Complex Coupled Cultured Cells Diglycerides Drug or chemical Tissue Distribution Emulsions Enzymatic Biochemistry Enzyme Kinetics Enzymes Esters Facility Construction Funding Category Goals Hepatic Hepatic Stellate Cell Hepatocyte Hydrolysis In Vitro Incubated Kidney Kinetics Knowledge Lipids Lipoproteins Liposomes Liver Liver Microsomes Localized Lysophospholipids Messenger RNA Metabolic Metabolism Mono-S Mus Peripheral Pharmaceutical Preparations Phenotype Phospholipids Physiological Plasma Play Polymerase Chain Reaction Primary carcinoma of the liver cells Protein Secretion Proteins RNA Interference Rate Rattus Relative (related person)Retinoids Retinol Binding Proteins Role Sequence Analysis Serum Site Small Interfering RNA Smooth Muscle Myocytes Substrate Specificity System Testing Testis Time Tissues Transfection Triglycerides Vitamin A Water bile salts carboxylesterase cell type chylomicron remnant enzyme substrate hepatoma cell in vivo laser capture microdissection macrophage monolayer retinyl esterase stellate cell uptake

项目摘要

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.

描述（申请人提供）：视黄酯（RE）在肝脏中的水解在人体维生素 A 的代谢中起着关键作用。膳食维生素 A 以乳糜微粒残留物中的 RE 形式进入肝脏，经过水解和再酯化进行储存。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。中性和酸性视黄酯水解酶 (REH) 已从大鼠肝脏中纯化出来，并通过序列分析显示为羧酸酯酶 ES-2 和 ES-10。该项目的目标是明确 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。已经用其他底物研究了羧酸酯酶，但尚不清楚如果它们对维生素 A 代谢很重要。与其他酯相比，它们催化 RE 水解的效率也是未知的。这些知识空白将通过以下具体目标进行探讨：目标 1：REH 的酶学。酶动力学将用于研究羧酸酯酶的底物特异性，并检验这些酶催化 RE 和其他脂酯水解的内在能力不同的假设。目标 2：羧酸酯酶的组织分布。实时 PCR（针对 mRNA）、免疫检测（针对蛋白质）和酶测定将用于确定各种组织中酶的水平和分布，并检验羧酸酯酶存在于外周组织和肝脏中的假设。目标 3：肝脏中的细胞类型分布。激光捕获显微切割和细胞分离与实时 PCR、免疫检测和酶测定相结合，将用于定义星状细胞和肝细胞之间的羧酸酯酶分布，并测试羧酸酯酶 ES-2 仅位于肝细胞中以及羧酸酯酶的假设ES-10 定位于肝细胞和星状细胞。目标 4：培养细胞中羧酸酯酶在 RE 代谢中的生理作用，将使用 cDNA 转染和 RNA 干扰来调节羧酸酯酶水平，以检验各种羧酸酯酶在肝脏摄取和/或代谢中发挥代谢作用的假设乳糜微粒 RE，并检验羧酸酯酶在细胞内 RE 和胆固醇酯的周转中发挥代谢作用的假设在储存它们的细胞中。

项目成果

期刊论文数量（49）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Proteomic analysis of rat plasma by two-dimensional liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

通过二维液相色谱和基质辅助激光解吸电离飞行时间质谱法对大鼠血浆进行蛋白质组分析。

DOI：
发表时间：
2006-08-11
期刊：
影响因子：
0
作者：
Linke, Thomas;Ross, A Catharine;Harrison, Earl H
通讯作者：
Harrison, Earl H

Mechanisms of provitamin A (carotenoid) and vitamin A (retinol) transport into and out of intestinal Caco-2 cells.

维生素原 A（类胡萝卜素）和维生素 A（视黄醇）转运进出肠道 Caco-2 细胞的机制。

DOI：
发表时间：
2007-10
期刊：
影响因子：
6.5
作者：
During, Alexandrine;Harrison, Earl H
通讯作者：
Harrison, Earl H

Naturally occurring eccentric cleavage products of provitamin A β-carotene function as antagonists of retinoic acid receptors.

天然存在的维生素原A β-胡萝卜素的偏心裂解产物充当视黄酸受体的拮抗剂。

DOI：
发表时间：
2012-05-04
期刊：
影响因子：
0
作者：
Eroglu, Abdulkerim;Hruszkewycz, Damian P;dela Sena, Carlo;Narayanasamy, Sureshbabu;Riedl, Ken M;Kopec, Rachel E;Schwartz, Steven J;Curley Jr, Robert W;Harrison, Earl H
通讯作者：
Harrison, Earl H

Purification and characterization of a neutral, bile salt-independent retinyl ester hydrolase from rat liver microsomes. Relationship To rat carboxylesterase ES-2.

从大鼠肝微粒体中纯化和表征中性、不依赖胆汁盐的视黄酯水解酶。