Purification of acid lipase from rabbit liver

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In [l] we demonstrated that acid lipase activity with 4-methylumbelliferyl oleate as substrate represented both acid cholesterol esterase and triacylglycerol lipase activity and that the enzyme might be associated with the lysosomal membrane or hydrophobic macromolecules in rabbit liver. The enzymes seems to be involved in catabolism of lipoproteins and to play an important role in the intracellular metabolism of cholesterol esters and triacylglycerols [2,3]. Purification of the enzyme has been attempted by several groups, but highly purified enzyme has not been obtained because of its instability and hydrophobicity [ 1,4-61. The enzyme was purified 120-fold from rat liver [4] and 2500.fold from human liver [5]. Moreover, lOOO-3000-fold purification of the enzyme from human placenta was reported in [6]. obtained from the Protein Research Foundation (Osaka). The homogenate was centrifuged at 1000 X g for 10 min and the postnuclear supernatant was centrifuged at 3300 X g for 10 min. The resulting supernatant was centrifuged at 14 000 X g for 20 min and the precipitate (lysosome-rich fraction) was suspended in the same medium. The precipitate was washed by recentrifugation at 14 000 X g for 20 min, then homogenized in the same medium containing 0.5% (v/v) digitonin in a volume equivalent to 1/3rd that of the postnuclear supematant. The mixture was stirred at 4°C for 40 min and then centrifuged at 50 000 X g for 30 min and the supematant was collected.