IN VITRO AND IN VIVO STRUCTURE/FUNCTION ANALYSIS OF LPL AND HL

肝脂酶（HL）和脂蛋白脂酶（LPL）是内皮结合的 在脂质代谢中起主要作用的脂解酶， 水解循环中的甘油三酯和磷脂 血浆脂蛋白尽管它们的组织和结构相似， HL和LPL在代谢中生理作用 富含甘油三酯的颗粒和HDL明显不同， 这可能部分由不同的底物特异性介导 这两种酶。与LPL相比，HL是活性更高的磷脂酶 磷脂酶活性的增强可能在 与LPL相反，HL调节HDL代谢的能力。 为了研究不同的结构基础， 磷脂酶活性之间的LPL和HL，我们已经产生了突变 其中HL和LPL盖的脂肪酶，其调节脂质的进入 将底物交换到活性位点。进行这些研究 在体内，我们已经表达了天然和突变体脂肪酶在HL-缺陷 使用重组腺病毒对血浆磷脂增加的小鼠进行检测。 因此，表达天然LPL和HL以及嵌合LPL和HL的腺病毒可以在体外被表达。 含有HL主链和LPL盖或LPL的脂肪酶 在总共16个HL缺陷的小鼠中注射了具有HL盖的骨架。 小鼠注射表达含HL lid的脂肪酶的病毒的动物 血浆磷脂显著降低（80%）， 注射含有LPL盖的脂肪酶的小鼠中的减少仅是 百分之三十因此，无论脂肪酶主链如何，HL盖的存在都是可能的。 显著增强体内磷脂酶活性，表明 脂酶盖是相对体内磷脂酶的主要决定因素 两种酶的活性。使用重组腺病毒 在体内表达突变蛋白质提供了一种强大的新方法， 在体内进行蛋白质的结构-功能分析。

Hepatic lipase (HL) and lipoprotein lipase (LPL) are endothelial-bound lipolytic enzymes that play a major role in lipid metabolism by hydrolyzing triglycerides and phospholipids present in circulating plasma lipoproteins. Despite their similar organization and structure, the physiologic role that HL and LPL play in the metabolism of triglyceride-rich particles and HDL are clearly distinct, a difference that may in part be mediated by the different substrate specificities of the two enzymes. Compared to LPL, HL is the more active phospholipase and this enhanced phospholipase activity may in fact, play a major role in the ability of HL, as opposed to LPL, to modulate HDL metabolism. In order to investigate the structural basis for the different phospholipase activities between LPL and HL we have generated mutant lipases in which the HL and LPL lids, which modulates access of lipids substrates to the active sites, are exchanged. To perform these studies in vivo, we have expressed native and mutant lipases in HL-deficient mice with increased plasma phospholipids using recombinant adenoviruses. Thus, adenovirus expressing native LPL and HL as well as chimeric lipases containing either the HL backbone with the LPL lid or the LPL backbone with the HL lid were injected in a total of 16 HL-deficient mice. Animals injected with viruses expressing lipases containing HL lid had a dramatic decrease (80%) in plasma phospholipids whereas the reduction in mice injected with lipases containing the LPL lid was only 30%. Thus, regardless of the lipase backbone, the presence of the HL lid markedly enhances in vivo phospholipase activity, indicating that the lipase lid is a major determinant of the relative in vivo phospholipase activities of the two enzymes. The use of recombinant adenovirus to express mutant proteins in vivo provides a powerful new approach for performing structure-function analysis of proteins in vivo.