title: Purification of lysosomal cholesteryl ester hydrolase from rat liver by preparative isoelectric focusing.
abstract: Ion-exchange chromatography and preparative isoelectric focusing (PIEF) were compared to produce a stable rat liver lysosomal cholesteryl ester hydrolase of high specific activity. The PIEF purification method proved to be more rapid and easier to perform. PIEF purification involved the following steps: i) osmotic shock of the lysosome fraction, ii) (NH4)2 SO4 precipitation (10-70%, w/v), iii) Sepharose CL-6B gel filtration, and iv) PIEF. The enzyme was purified 60-120-fold with a yield of 2-4%. The activity of the purified enzyme was best restored by stabilizing with a 0.5% (w/v) albumin solution. The purified enzyme produced one major band on SDS-polyacrylamide gel electrophoresis having a MW of 58,500 daltons. Gel filtration showed a MW of 58,000 daltons. The optimum pH of the enzyme was 4.5, and the isoelectric point was 6.0-6.2. The specific activity of hydrolysis of cholesteryl oleate and triolein increased by similar rates during purification.
mesh_terms: D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification*; D007525:Isoelectric Focusing / Q000379:methods; D007700:Kinetics; D008049:Lipase / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D002787:Sterol Esterase / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D002265:Carboxylic Ester Hydrolases; D002787:Sterol Esterase; D008049:Lipase