Role of Diacylglycerol Lipase in Phospholipid Metabolism in Liver

本课题的目的是研究二酰甘油脂肪酶在大鼠肝脏磷脂代谢中的作用。在先前的研究中，我们已经发现在大鼠肝脏的各种亚细胞组分中都存在二酰甘油脂肪酶活性。该脂肪酶已被证明能有效地催化由磷脂酸或磷脂酰胆碱酶解生成的二酰甘油。在本研究中，我们尝试从大鼠肝细胞胞浆中提取脂肪酶。经DEAE-纤维素柱、肝素-琼脂糖柱和ConA-琼脂糖柱层析，得到纯度较高的双甘油脂肪酶制剂，在SDS-聚丙烯酰胺凝胶上可见31 kDa的主带。虽然制剂的纯度估计在90%以上，但在凝胶上可以检测到几条微弱的条带。部分纯化的二酰甘油的性质如下：a)最适pH=9.5b)被锌离子、氟化钾、丝氨酸酯酶抑制剂如二异丙基氟磷酸盐和苯甲基磺酰氟所抑制；c)被去污剂如Triton X-100、脱氧胆酸钠和CHAPS激活；d)在超滤、透析或-20ﾟC储存的过程中很容易失去活性，但可通过添加终浓度为0.1 mg/ml的牛血清白蛋白稳定下来。使用该抗体对脂肪酶进行免疫学表征正在进行中。

The purpose of this project is to investigate the role of diacylglyceol lipase in phospholipid metabolism in rat liver.In a previous study we have found the presence of diacylglycerol lipase activity in verious subcellular fractions of rat liver. The lipase has been shown to effectively catalized the hydrolysis of diacylglycerol generated enzymatically from phosphatidic acid or phosphatidylcholine. In this study we tried to pirified the lipase from rat liver cytosol. Afeter three step chromatography on DEAE-cellulose, Heparin-Sepharose and ConA-Sepharose columns, we obtained the highly rurified preparation of diacylglycerol lipase which showed 31kDa major band on SDS-polyacrylamide gel. Although the purity of the preparation was estimated to be higher than 90%, several faint bands could be detected on on the gel. The properties of the partially purified diacylglycerol were as follows ; a) optimal pH = 9.5 b) Inhibited by Zn^<2+>, potassium fluoride, serine esterase inhibitors such as diisoproylfluorophosphate and phenylmethylsulfonylfluoride c) Activated by detergents such as Triton X-100, sodium deoxycholate and CHAPS d) The activity is easily lost during the process of ultratiltration, dialysis or storage at-20^ﾟC but is stabilized by the addition of bovine serum albumin at final concentration of 0.1mg/ml.We have obtained rabbit anti-DG lipase antibody using 31kDa protein eluted from the polyacrylamide gel as antigen. Immunological characterization of the lipase using the antibody is undergoing.