Purification of cholesterol-esterifying enzymes from rat liver cytosol by high-performance liquid chromatography |
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| Institution: | 1. Department of Experimental Virology, Institute of Hematology and Blood Transfusion, Prague, Czech Republic;2. Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic;1. Division of Respirology, Department of Medicine, McMaster University, Hamilton, Ontario, Canada;2. Guangzhou Institute of Respiratory Diseases, Guangzhou Medical University, Guangzhou, China;3. Institut universitaire de cardiologie et de pneumologie de Québec, Quebec City, Quebec, Canada;4. Division of Pneumologie, University of Montreal, Montreal, Quebec, Canada;5. Meakins-Christie Laboratories, Research Institute of the McGill University Health Center, Hamilton, Ontario, Canada;1. Department of Computer Engineering and Computer Science, California State University, Long Beach, 1250 Bellflower Boulevard-MS 8302, Long Beach, CA 90840, USA;2. Department of Electrical Engineering, University of California, Los Angeles, 56-125B Engineering IV Building, Box 951594, Los Angeles, CA 90095, CA;3. Department of Electrical Engineering, California State University, Long Beach, 1250 Bellflower Boulevard, Long Beach, CA 90840, CA;1. Dep. of Physics, College of Education for Pure Science, University of Babylon, Iraq;2. Dep. of Chemistry, College of Woman Science, University of Babylon, Iraq;3. Dep. Of Physics, Faculty of Science, University of Kufa, Iraq;1. Biofuels Institute, School of Environment and Safety Engineering, Jiangsu University, Zhenjiang 212013, China;2. Institute of Medicine & Chemical Engineering, Zhenjiang College, Zhenjiang 212000, China;3. School of Materials Science and Engineering, Jiangsu University, Zhenjiang 212013, China |
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| Abstract: | 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 (Mr 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. |
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