Twu J S, Garfinkel A S, Schotz M C
Biochim Biophys Acta. 1984 Mar 7;792(3):330-7. doi: 10.1016/0005-2760(84)90201-7.
Hepatic lipase has been purified to homogeneity from rat liver homogenates. The purified enzyme exhibits a single band on SDS-polyacrylamide gel electrophoresis. The molecular size of the native hepatic lipase is 200 000, while on SDS-polyacrylamide gel electrophoresis the apparent minimum molecular weight of the enzyme is 53 000, suggesting that the active enzyme is composed of four subunits. The relationship between triacylglycerol, monoacylglycerol and phospholipid hydrolyzing activities of the purified rat liver enzyme was studied. All three activities had a pH optimum of 8.5. The maximal reaction rates obtained with triolein, monoolein and dipalmitoylphosphatidylcholine were 55 000, 66 000 and 2600 mumol fatty acid/mg per h with apparent Michaelis constant (Km) values of 0.4, 0.25 and 1.0 mM, respectively. Hydrolysis of triolein and monoolein probably takes place at the same site on the enzyme molecule, since competitive inhibition between these two substrates was observed, and a similar loss of hydrolytic activity occurred in the presence of diisopropylfluorophosphate. Addition of apolipoproteins C-II and C-I had no effect on the hydrolytic activity of the enzyme with the three substrates tested. However, the triacylglycerol hydrolyzing activity was inhibited by the addition of apolipoprotein C-III. Monospecific antiserum to the pure hepatic lipase has been raised in a rabbit.
肝脂酶已从大鼠肝脏匀浆中纯化至同质。纯化后的酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上呈现单一条带。天然肝脂酶的分子大小为200000，而在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上该酶的表观最小分子量为53000，这表明活性酶由四个亚基组成。研究了纯化的大鼠肝脏酶的三酰甘油、单酰甘油和磷脂水解活性之间的关系。所有这三种活性的最适pH均为8.5。用三油精、单油精和二棕榈酰磷脂酰胆碱获得的最大反应速率分别为55000、66000和2600μmol脂肪酸/毫克每小时，表观米氏常数（Km）值分别为0.4、0.25和1.0毫摩尔。三油精和单油精的水解可能发生在酶分子的同一部位，因为观察到这两种底物之间存在竞争性抑制，并且在存在二异丙基氟磷酸的情况下发生了类似的水解活性丧失。添加载脂蛋白C-II和C-I对该酶对所测试的三种底物的水解活性没有影响。然而，添加载脂蛋白C-III可抑制三酰甘油水解活性。已在兔中制备了针对纯肝脂酶的单特异性抗血清。
