Simultaneous isolation of human kidney cathepsins B, H, L and C and their characterisation |
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| Authors: | Tatjana Popovi? Vida Puizdar Anka Ritonja Jo?e Brzin |
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| Affiliation: | 1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China;2. Shanghai Engineering Research Center of Maricultured Animal Vaccines, Shanghai 200237, China;3. Shanghai Collaborative Innovation Center for Biomanufacturing, Shanghai 200237, China;1. Faculty of Fisheries, Kagoshima University, Kagoshima, Japan;2. The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima, Japan;3. Faculty of Science, Kagoshima University, Kagoshima, Japan;1. Department of Pathogen Biology, School of Medicine, Jinan University, Guangzhou, China;2. Department of Epidemiology, School of Medicine, Jinan University, Guangzhou, China;3. Key Laboratory of environmental exposure and health in Guangzhou, Jinan University, Guangzhou, China;4. Guangzhou Key Laboratory of Environmental Exposure and Health in Guangzhou, Guangdong Key Laboratory of Environmental Pollution and Health, Jinan University, Guangzhou, Guangdong, China;5. The key laboratory for virology of Guangzhou, College of life science and technology, Jinan University, China;1. Department of Applied Biochemistry, School of Engineering, Tokai University, 4-1-1 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan;2. Department of Biology, Faculty of Education, Tokyo Gakugei University, 4-1-1 Nukuikita-machi, Koganei, Tokyo 184-8501, Japan;1. Department of Cell Biology, Medical School of Ribeirão Preto, University of São Paulo (FMRP/USP), Ribeirão Preto SP 14049-900, Brazil;2. Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520, USA;3. Howard Hughes Medical Institute, Yale University, New Haven, CT 06520, USA |
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| Abstract: | A procedure for the simultaneous isolation of four cysteine proteinases, cathepsins B, H, L and C, from human kidney is described. The method includes concentration of the acidified homogenate by ammonium sulphate precipitation. The resuspended and dialysed precipitate was chromatographed on DEAE-cellulose DE-32, to allow separation of cathepsins H and C from cathepsins B and L. The main isoform of cathepsin H was separated from cathepsin C by cation-exchange chromatography on CM-Sephadex C-50. These two enzymes were further purified by covalent chromatography on thiopropyl Sepharose and gel permeation on Sephacryl S-200. The last step allowed separation of cathepsin C and the minor isoform of cathepsin H. Purification of the other two enzymes, cathepsins B and L, was carried out on thiol Sepharose, followed by chromatography on CM-Sepharose C-50. In this step, pure cathepsin L was obtained, while two isoforms of cathepsin B had to be finally purified on Sephacryl S-200 columns. The purity of each enzyme was analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis, isoelectric focusing on polyacrylamide gels and N-terminal sequencing. The activities of the purified cathepsins B, H and L were determined in terms of kcat/KM for three substrates, Z-Phe-Arg-MCA, Z-Arg-Arg-MCA and Arg-MCA. The method produced 25 mg of cathepsin B, 6.5 mg of cathepsin H, 1.5 mg of cathepsin L and 3.8 mg of cathepsin C from 3.5 kg of human kidney. |
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| Keywords: | Cathepsins Enzymes |
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