Role of Diacylglycerol Lipase in Phospholipid Metabolism in Liver

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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.