MOLECULAR TOXICOLOGY OF PLACENTAL CARBOXYLESTRASE

申请人摘要：已知羧酸酯酶在 在外源物质代谢和农药解毒中的作用 拟议研究的目的是确定 存在多种形式的人类羧酸酯酶，并确定 这些酶的结构、功能和调节。 中央 该项目的假设是， 在人胎盘中表达的羧酸酯酶是不同的基因产物 它们具有不同的底物特异性，并且独立地 监管. 该项目的具体目标是（1）从人类中分离 胎盘文库全长cDNA编码两种羧酸酯酶（HP-1和 HP-2），和（2）通过生物化学、免疫化学和分子生物学表征 技术的结构，功能和这些酶的调节。 一部分 酶和免疫化学研究，重组酶和抗体 将产生针对重组蛋白的抗体。 为了检验这一假设 HP-1和HP-2具有不同的底物特异性， 将检测酶水解选定酯的能力， 酰胺，以及它们催化可卡因和 脂肪酸乙酯的合成。 免疫沉淀 将进行胎盘样本比较酶特性 天然酶和重组酶的区别 为了验证这个假设， HP-1和HP-2是独立调节的，mRNA和蛋白质水平将 用来自单个胎盘和培养的胎盘的样品测定 用外源性物质处理的切片。 这些研究的样本将涵盖 广泛的遗传（即，种族）和环境（即，吸烟） 因素 在实现项目目标方面取得了一些进展。 的 胎盘微粒体水解几种酯的酶活性， 确定了 两个部分cDNA（每个348 bp）显然编码两个 已经从人胎盘中分离出不同的羧酸酯酶。 在 除了水解许多异生物质如药物和杀虫剂外， 已知羧酸酯酶催化酯交换反应， 脂肪酸乙酯合成。 乙基可卡因，一种更有效的代谢物， 可卡因在介导致死率和心脏毒性方面优于母体 化合物是通过酯交换反应形成的。 的 由于怀孕期间酗酒而积累的乙酯， 显示出对胎儿酒精的发展有重要作用 综合征 因此，本项目中概述的实验将 有助于我们对羧酸酯酶的基本理解， 参与解毒和生物活化的酶家族 异生物质

APPLICANT'S ABSTRACT: Carboxylesterases are known to play an important role in xenobiotic metabolism and detoxication of pesticides The long-term objective of the proposed studies is to determine the molecular basis for the existence of multiple forms of human carboxylesterases, and to determine the structure, function and regulation of these enzymes. The central hypothesis of the proposed project is that the multiple forms of carboxylesterases expressed in human placenta are distinct gene products that have different substrate specificities, and are independently regulated. The specific aims of the project are (1) to isolate from a human placental library full-length cDNAs encoding two carboxylesterases (HP-1 and HP-2), and (2) to characterize by biochemical, immunochemical and molecular techniques the structure, function and regulation of these enzymes. As part of enzymatic and immunochemical studies, recombinant enzymes and antibodies against the recombinant proteins will be produced. To test the hypothesis that HP-1 and HP-2 have different substrate specificities, the recombinant enzymes will be examined for their ability to hydrolyze selected esters and amides, and their ability to catalyze the transesterification of cocaine and the synthesis of fatty acid ethyl esters. Immunoprecipitation from placental samples will be conducted to compare enzymatic characteristics between the natural and recombinant enzymes. To test the hypothesis that HP-1 and HP-2 are independently regulated, the MRNA and protein levels will be determined with samples from individual placenta and cultured placental slices treated with xenobiotics. Samples for these studies will cover a broad range of genetic (i.e., ethnic) and environmental (i.e., smoking) factors. Some progress has been made toward the goals of the project. The enzymatic activity of placental microsomes to hydrolyze several esters has been determined. Two partial cDNAs (348 bp each) that apparently encode two distinct carboxylesterases have been isolated from human placentas. In addition to hydrolyzing numerous xenobiotics such as drugs and pesticides, carboxylesterases are known to catalyze a transesterification reaction and fatty acid ethyl ester synthesis. Ethylcocaine, a more potent metabolite of cocaine in mediating lethality and cardiac toxicity than the parent compound, is formed through the transesterification reaction. The accumulation of ethyl esters due to alcohol abuse during pregnancy has been shown to contribute significantly to the development of fetal alcohol syndrome. Therefore, the experiments outlined in this project will contribute significantly to our basic understanding of carboxylesterases as a family of enzymes involved in the detoxication and bioactivation of xenobiotics.