PURIFICATION OF CHOLESTEROL-ESTERIFYING ENZYMES FROM RAT-LIVER CYTOSOL BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Three enzymes esterifying cholesterol with long-chain fatty acids were purified approximately 31 000-fold to apparent homogeneity from the c ytosol of normal rat liver. The enzymatic activity was tested by incub ation of active fractions with tritiated cholesterol and separation of newly formed esters from non-reacted cholesterol by a passage through silica gel cartridges with subsequent assay for radioactivity by liqu id scintillation. For the purification of enzymes, active proteins wer e precipitated by (NH4)(2)SO4 to 35% saturation. The bulk of inactive proteins was removed by size-exclusion chromatography on TSK G3000 SW. The active fraction was subsequently separated on Separon HEMA BIO 10 00 DEAF in gradients of 0-500 mM KCl into three enzymatic activities d iffering in their retention and these proteins were finally purified b y affinity HPLC on columns of cholesterol immobilized on HEMA BIO 1000 E-H. Final purified enzymes showed the same single band in polyacryla mide gel electrophoresis corresponding to 16.5 kDa. Combination of ind ividual enzymes did not increase the overall yield of cholesteryl este rs but the reaction-rate was significantly accelerated. These proteins are apparently subunits of a larger complex (M(r) 65 000) that can be demonstrated by electrophoresis in the absence of 2-mercaptoethanol. Results presented in this paper indicate that because of good and rapi d separation of active proteins, HPLC may be a method of choice for en zyme purifications.