RADIOIMMUNOASSAY FOR HUMAN LIPOPROTEIN LIPASE

人脂蛋白脂酶(LPL)和肝脂酶(HL)将 对这些酶进行了序列测定和结构域鉴定。 将采用几种策略。人早幼粒白血病和红细胞性白血病的基因 将通过筛选分离LPL mRNA，并降低严密性， 大鼠和鸡的人基因组文库和 利用预期的序列同源性。蛋白水解性和 溴化氰碎片将通过反相分离 层析和测序。产生单克隆性杂交瘤 针对脂解酶的抗体(MAb)将由 改进的方法将加强单抗的选择 暴露的天然HL和LPL表位。单克隆 抗体将被分配给特定的蛋白水解物和氰基 溴化物片段和体外产生的多肽 表达载体。将对具有特征性的单抗进行评估 它们抑制脂肪酶功能特性的能力：结合 酶对底物和硫酸乙酰肝素；催化活性； 载脂蛋白CII与LPL的结合；由单体LPL形成二聚体。 HL的功能将通过以下测试进行研究 假设：HL是乳胶粒和极低密度脂蛋白的主要调节者 通过取消屏蔽载脂蛋白E上的特定结构域来移除 这是乳杆菌受体识别所必需的。 将从HL缺乏的大鼠中分离出乳糖体残留物 测定其与肝膜的结合特性 用HL逐步消化。HL对载脂蛋白E结构域的揭开 将通过与单抗、配基的反应进行监测 分离的肝膜蛋白的印迹和体内去除 修饰的脂蛋白的速率。大鼠HL的调节作用 由激素(胰岛素、胰高血糖素、甲状腺素)引起的肝细胞 在合成、降解、分泌、HL水平上进行研究 信使核糖核酸浓度和转录。激素反应 HL基因5‘侧翼区的序列将是 已确认身份。

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.