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 cytosol of normal rat liver. The enzymatic activity was tested by incubation 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 liquid scintillation. For the purification of enzymes, active proteins were precipitated by (NH4)2SO4 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 1000 DEAE in gradients of 0-500 mM KCl into three enzymatic activities differing in their retention and these proteins were finally purified by affinity HPLC on columns of cholesterol immobilized on HEMA BIO 1000 E-H. Final purified enzymes showed the same single band in polyacrylamide gel electrophoresis corresponding to 16.5 kDa. Combination of individual enzymes did not increase the overall yield of cholesteryl esters 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 rapid separation of active proteins, HPLC may be a method of choice for enzyme purifications.
Hradec
J
J
Department of Experimental Virology, Institute of Hematology and Blood Transfusion, Prague, Czech Republic.
Franĕk
F
F
Dufek
P
P
D000818:Animals; D002413:Cations, Divalent / Q000737:chemistry; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004951:Esterification; D005260:Female; D008099:Liver / Q000201:enzymology* / Q000648:ultrastructure; D008274:Magnesium / Q000737:chemistry; D008623:Mercaptoethanol / Q000737:chemistry; D009994:Osmolar Concentration; D051381:Rats; D017208:Rats, Wistar; D002787:Sterol Esterase / Q000302:isolation & purification* / Q000378:metabolism; D013997:Time Factors
D002413:Cations, Divalent; D008623:Mercaptoethanol; D002784:Cholesterol; D002787:Sterol Esterase; D008274:Magnesium