RADIOIMMUNOASSAY FOR HUMAN LIPOPROTEIN LIPASE

人脂蛋白脂肪酶（LPL）和肝脂肪酶（HL）将在 这些酶的结构域被鉴定。 将采用几种策略。 人HL的cDNA和 LPL mRNA将通过筛选分离，严格性降低， 具有大鼠和鸡cDNA的人类基因组文库， 利用预期的序列同源性。 蛋白水解和 溴化氰碎片将被反相分离 层析并测序。 产生杂交瘤的单克隆 脂肪分解酶的抗体（mAb）将通过以下方法产生： 改进的方法，其将增强mAb的选择， 天然HL和LPL的暴露表位。 单克隆 抗体将被分配到特定的蛋白水解和氰 溴片段和在体外产生的肽， 表达载体。 将评价表征的mAb， 它们抑制脂肪酶功能特性的能力： 酶对底物和硫酸乙酰肝素的催化活性; Apo CII与LPL的结合;单体LPL形成二聚体。 将通过以下测试研究HL的功能 假设：HL是乳糜微粒和VLDL的主要调节剂 通过暴露Apo E上的特定结构域进行清除， 乳糜微粒受体识别所必需的。 将从HL缺陷大鼠中分离乳糜微粒残留物， 它们与肝膜的结合特性， 用HL逐渐消化。 HL对Apo E结构域的解蔽 将通过与单克隆抗体、配体 分离的肝膜蛋白的印迹和体内去除 脂蛋白修饰率。 最后，大鼠中的HL调节 肝细胞激素（胰岛素，胰高血糖素，甲状腺素）将 在合成、降解、分泌、HL mRNA浓度和转录。 激素应答 HL基因的5 '侧翼区中的序列将是 鉴定

Human lipoprotein lipase (LPL) and hepatic lipase (HL) will be sequenced and structural domains on these enzymes identified. Several strategies will be employed. cDNA's for human HL and LPL mRNA will be isolated by screening, with reduced stringency, human genomic libraries with rat and chicken cDNA's and exploiting the expected sequence homologies. Proteolytic and cyanogen bromide fragments will be separated by reversed phase chromatography and sequenced. Hybridoma producing monoclonal antibodies (mAb) to lipolytic enzymes will be generated by improved methods which will enhance the selection of mAb's to exposed epitopes of the native HL and LPL. Monoclonal antibodies will be assigned to specific proteolytic and cyanogen bromide fragments and to peptides generated in vitro in expression vectors. Characterized mAb's will be evaluated for their ability to inhibit functional properties of lipases: Binding of enzyme to substrate and heparan sulfate; catalytic activity; binding of Apo CII to LPL; dimer formation from monomeric LPL. The function of HL will be investigated by testing the following hypothesis: HL is a major regulator of chylomicron and VLDL removal by unmasking specific domains on Apo E which are necessary for recognition by the chylomicron receptor. Chylomicron remnants will be isolated from HL-deficient rats and their binding characteristic to liver membranes measured after gradual digestion with HL. Unmasking of Apo E domains by HL will be monitored by reactivity with monoclonal antibodies, ligand blotting of separated liver membrane proteins and in vivo removal rate of modified lipoproteins. Finally HL regulation in rat hepatocytes by hormones (insulin, glucagon, thyroxine) will be studied at the level of synthesis, degradation, secretion, HL mRNA concentration and transcription. Hormone responsive sequences in the 5'-flanking region of the HL gene will be identified.