Kresse H, Löffler B M, Kunze H
Max-Planck-Institut für Experimentelle Medizin, Göttingen, Germany.
Biochem Int. 1991 Mar;23(5):885-93.
A procedure is described for the purification of hepatic lipase (HL)4 from rat liver homogenate which results in a high yield (41%) of electrophoretically homogeneous enzyme. The method is based on that of Twu et al. (Biochim. Biophys. Acta 1984: 792, 330), but it is more efficient with respect to yield (about 4-fold) and purity (1.6-fold). It includes the preparation of a high-speed supernatant, chromatography in series on octyl-, heparin- and concanavalin A-Sepharose, and finally gel filtration. On SDS-PAGE analysis, the purified enzyme exhibited an apparent molecular mass of 63.6 +/- 3.2 kDa. Heterogeneity was observed, when purified HL was subjected to isoelectric focussing. The enzyme displayed a specific catalytic activity of 23,000 U* (mumol fatty acid released per h at 37 degrees C) per mg protein, when assayed with trioleoyl glycerol suspensions in arabic gum. A highly specific antiserum against rat liver HL, capable of inhibiting 817 mU* HL per microliter antiserum, was raised in rabbits.
本文描述了一种从大鼠肝脏匀浆中纯化肝脂肪酶（HL）4的方法，该方法可获得高产率（41%）的电泳纯酶。该方法基于Twu等人（《生物化学与生物物理学报》1984年：792, 330）的方法，但在产率（约4倍）和纯度（1.6倍）方面更高效。它包括制备高速上清液、依次在辛基-、肝素-和伴刀豆球蛋白A-琼脂糖上进行色谱分离，最后进行凝胶过滤。在SDS-PAGE分析中，纯化后的酶显示出63.6±3.2 kDa的表观分子量。当对纯化的HL进行等电聚焦时，观察到了异质性。在用阿拉伯胶中的三油酰甘油悬浮液进行测定时，该酶每毫克蛋白质表现出23,000 U*（在37℃下每小时释放的微摩尔脂肪酸）的比催化活性。在兔中制备了一种针对大鼠肝脏HL的高特异性抗血清，每微升抗血清能够抑制817 mU* HL。
