Hydrolysis of triolein in phospholipid vesicles and microemulsions by a purified rat liver acid lipase.
An acid lipase was purified from rat liver lysosomes. Lipase purification involved affinity chromatography, gel filtration, and stabilization of the purified preparation using ethylene glycol and Triton X-100. A molecular weight of 67,000-69,000 was determined independently using density gradient centrifugation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel filtration. To study enzyme action, model substrates were prepared by incorporating radiolabeled triolein into either unilamellar vesicles or microemulsions. Substrates were prepared by cosonicating aqueous dispersions of lecithin and triolein. Formation of vesicles or emulsions depended on the relative amount of each lipid and on sonication conditions. Vesicles were prepared at molar ratios between 70:1 and 26:1 (lecithin:triolein) and the microemulsion preparation at a molar ratio of 1:1. The substrate particles were of similar size (220-250 A) as determined by Bio-Gel A-15m chromatography. Hydrolysis of triolein contained in vesicles or emulsions was similar with respect to pH, temperature, and reaction products. Kinetic studies on vesicles with increasing triolein content showed progressively greater Vmax values (0-0.6 mumol/min/mg), and Vmax for the emulsion was 3.1 mumol/min/mg. Addition of human very low or low density lipoprotein produced a dose-dependent inhibition with both substrates. The results show that synthetically prepared microemulsions are stable and effective substrates for the acid lipase and indicate that surface-oriented triolein is hydrolyzed in both preparations.
D000818:Animals; D003902:Detergents / Q000494:pharmacology; D004655:Emulsions; D007700:Kinetics; D008049:Lipase / Q000378:metabolism*; D008081:Liposomes; D008099:Liver / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D008970:Molecular Weight; D017830:Octoxynol; D010713:Phosphatidylcholines / Q000378:metabolism; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats; D014304:Triolein / Q000378:metabolism*
D003902:Detergents; D004655:Emulsions; D008081:Liposomes; D010713:Phosphatidylcholines; D014304:Triolein; D011092:Polyethylene Glycols; D017830:Octoxynol; D008049:Lipase