Indexed on: 01 Sep '84Published on: 01 Sep '84Published in: Lipids
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.