Characterization of Lysosomal Acid Lipase Purified from Rabbit Liver1

Abstract

Lysosomal acid lipase from rabbit liver was solubilized with digitonin and purified 25,000-fold by Bio-Gel A-1.5 m, DEAE Bio-Gel A and phenyl Sepharose column chromatographies, preparative slab gel electrophoresis and finally Affi-Gel Blue affinity column chromatography. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis both in the presence and absence of sodium dodecyl sulfate. The molecular weight of the acid lipase was estimated to be 42,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 40,000 by gel filtration on Bio-Gel A-0.5 m. The enzyme was a hydrophobic glycoprotein with an isoelectric point of 5.15-5.90. The purified enzyme hydrolyzed tri-, di-, and monoolein and cholesterol oleate, with apparent Vmax values of 5.41, 56.1, 21.7, and 3.25 mumol/min/mg protein, and Km values of 50, 70, 200, and 40 microM, respectively. It hydrolyzed 4-methylumbelliferyl esters with fatty acids of different lengths in the order, medium length chains greater than long chains much greater than short chains. It did not hydrolyze dipalmitoylphosphatidylcholine. Its activity was inhibited by micromolar concentrations of p-chloromercuriphenyl sulfonic acid and p-bromophenacyl bromide and millimolar concentrations of Cu2+ and diethylpyrocarbonate. The activities of the enzyme towards the five substrates listed above showed almost identical thermal stabilities, mobilities on polyacrylamide gel electrophoresis and inhibition by several inhibitors. These findings support the idea that one enzyme is involved in the hydrolysis of both acylglycerols and cholesterol esters in lysosomes.

Niemann‐Pick type C disease: molecular mechanisms and potential therapeutic approaches

“…1). The structure of LAL is yet to be elucidated; however, biochemical evidence suggests that LAL has significant preference for the length, number and the saturation of the fatty acid acyl chains, and only a weak preference for the nature of the esterified alcohol (Imanaka et al 1984). This is consistent with the ability of LAL to hydrolyze both cholesteryl esters and triacylglycerides.…”

DRY‐CURED MEDITERRANEAN HAMS: LONG PROCESS, SLOW CHANGES AND HIGH QUALITY: A REVIEW1

“…Muscular lysosomal acid lipase hydrolyses principally neutral lipids, such as cholesterol esters and primary ester-bonds of triacylglycerols at pH 4-5 (Imanaka et al 1984). Lipoprotein lipase is responsible for the hydrolysis of the di-and triacylglicerol components of the very low-density lipoproteins and chylomicrons at an optimum pH of 8-9.…”

Triglyceride Lipase Activities in Rat Liver

“…Glycerol ester li pase activities have been reported for each subcellular fraction of liver homogenates: a cell membrane lipase that may be released into plasma by heparin [1,2]; a mitochon drial lipase [3]; an acid lysosomal lipase [4,5]; an alkaline microsomal lipase [6,7], and a triglyceride lipase from the cytoplasmic soluble fraction [8,9], among others. The present paper reports a kinetic study of neu tral and alkaline triglyceride lipase activities from different liver homogenate fractions, namely plasma membrane, mitochondria and microsomes, providing new data to characterize these lipases.…”