Cheng C F, Bensadoun A, Bersot T, Hsu J S, Melford K H
J Biol Chem. 1985 Sep 5;260(19):10720-7.
Human lipoprotein lipase and hepatic triglyceride lipase were purified to homogeneity from post-heparin plasma. These enzymes were purified 250,000- and 100,000-fold with yields of 27 +/- 15 and 19 +/- 6%, respectively. Molecular weight determination by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and reducing agents yielded Mr of 60,500 +/- 1,800 and 65,200 +/- 400, respectively, for lipoprotein lipase and hepatic triglyceride lipase. These lipase preparations were shown to be free of detectable antithrombin by measuring its activity and by probing of Western blots of lipases with a monospecific antibody against antithrombin. In additions, probing of Western blots with concanavalin A revealed no glycoproteins corresponding to the molecular weight of antithrombin. Four stable hybridoma-producing distinct monoclonal antibodies (mAb) to hepatic triglyceride lipase were isolated. The specificity of one mAb, HL3-5, was established by its ability to immunoprecipitate hepatic triglyceride lipase catalytic activity. Interaction of HL3-5 with this lipase did not inhibit catalytic activity. The three other mAb interacted with hepatic triglyceride lipase only after denaturation of the enzyme with detergents. The relatedness of these two enzymes was examined by comparing under the same conditions the thermal inactivation, the sensitivity to sulfhydryl and reducing agents, amino acid composition, and the mobility of peptide fragments generated by cyanogen bromide cleavage. The results of these studies strongly support the view that the two enzymes are different proteins. Immunological studies confirm this conclusion. Four mAb to hepatic triglyceride lipase did not interact with lipoprotein lipase in Western blots, enzyme-linked immunosorbent assay, and immunoprecipitation experiments. These immunological studies demonstrate that several epitopes of the hepatic triglyceride lipase protein moiety are not present in the lipoprotein lipase molecule.
人脂蛋白脂肪酶和肝甘油三酯脂肪酶从肝素后血浆中纯化至均一。这些酶分别纯化了250,000倍和100,000倍，产率分别为27±15%和19±6%。在十二烷基硫酸钠和还原剂存在下通过聚丙烯酰胺凝胶电泳测定分子量，脂蛋白脂肪酶和肝甘油三酯脂肪酶的Mr分别为60,500±1,800和65,200±400。通过测量其活性并用抗抗凝血酶单特异性抗体探测脂肪酶的蛋白质印迹，表明这些脂肪酶制剂不含可检测到的抗凝血酶。此外，用伴刀豆球蛋白A探测蛋白质印迹未发现与抗凝血酶分子量相对应的糖蛋白。分离出了四种产生稳定杂交瘤的、针对肝甘油三酯脂肪酶的不同单克隆抗体（mAb）。一种mAb HL3-5的特异性通过其免疫沉淀肝甘油三酯脂肪酶催化活性的能力得以确立。HL3-5与该脂肪酶的相互作用不抑制催化活性。其他三种mAb仅在酶用去污剂变性后才与肝甘油三酯脂肪酶相互作用。通过在相同条件下比较热失活、对巯基和还原剂的敏感性、氨基酸组成以及溴化氰裂解产生的肽片段的迁移率，研究了这两种酶的相关性。这些研究结果有力地支持了这两种酶是不同蛋白质的观点。免疫学研究证实了这一结论。针对肝甘油三酯脂肪酶的四种mAb在蛋白质印迹、酶联免疫吸附测定和免疫沉淀实验中均不与脂蛋白脂肪酶相互作用。这些免疫学研究表明，肝甘油三酯脂肪酶蛋白部分的几个表位不存在于脂蛋白脂肪酶分子中。
