Purification of the lysosomal acid lipase from human liver and its role in lysosomal lipid hydrolysis.
The lysosomal acid lipase has been purified 2,500-fold to near homogeneity from human liver. The enzyme was converted to a soluble form by extraction of frozen tissue with Triton X-100. The enzyme, which required Triton X-100 in buffers at all purification steps for optimal yields, was stabilized by the inclusion of 33% ethylene glycol during purification. Lectin chromatography on concanavalin A-Sepharose followed by chromatography on carboxymethyl-cellulose and Sephadex G-150 provided the highly purified enzyme in 17% yield. Sodium dodecyl sulfate-acrylamide gel electrophoresis indicated that the minimum molecular weight was about 29,000 +/- 1,000. Minor protein contaminants at Mr = 58,500, 14,700 and 13,900 were present in the final preparation. A single protein band, with enzyme activity, was observed in nondenaturing acrylamide gels containing Triton X-100. Gel filtration on Sephadex G-150 in the presence of Triton X-100 gave an apparent molecular weight of about 125,000 +/- 13,000. Trioleoylglycerol, cholesterol oleate, and 1,2- and 1,3-dioleoylglycerols were substrates for the purified enzyme giving apparent Vmax values of 5,400, 1,400, 19,400, and 22,100 nmol min-1 mg of protein-1, respectively, and Km values of 0.8, 0.8, 0.9, and 1.2 mM, respectively. The recoveries of both trioleoylglycerol and cholesterol oleate hydrolytic activities were nearly identical at each purification step, suggesting that the acid lipase as single enzyme is responsible for lysosomal hydrolysis of the neutral lipids. Monooleoylglycerols were not substrates for the enzyme.
D004355:Drug Stability; D006801:Humans; D007700:Kinetics; D008049:Lipase / Q000302:isolation & purification / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D008970:Molecular Weight; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D013379:Substrate Specificity
D011092:Polyethylene Glycols; D017830:Octoxynol; D008049:Lipase