Preparative isolation of peroxisomes from liver and kidney using metrizamide density gradient centrifugation in a vertical rotor |
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| Authors: | A K Hajra D Wu |
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| Affiliation: | 1. Neuroscience Laboratory, Mental Health Research Institute, University of Michigan, 1103 E. Huron, Ann Arbor, Michigan 48109 USA;2. Department of Biological Chemistry, University of Michigan, 1103 E. Huron, Ann Arbor, Michigan 48109 USA;1. State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center, Chengdu, Sichuan, 610041, PR China;2. Department of Neurology, Chongzhou People’s Hospital, Chengdu, 611230, PR China;1. Laboratory of Clinical Pharmaceutics & Therapeutics, Division of Pharmasciences, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12-jo, Nishi-6-chome, Kita-ku, Sapporo, 060-0812, Japan;2. Department of Pharmacy, Hokkaido University Hospital, Kita-14-jo, Nishi-5-chome, Kita-ku, Sapporo, 060-8648, Japan;1. Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan;2. Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan;3. Department of Obstetrics and Genecology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan;4. Laboratory of Pharmaceutical and Medical Chemistry, Gifu Pharmaceutical University, 1-25-4 Daigaku-Nishi, Gifu 501-1113, Japan;5. Department of Analytical and Bionorganic Chemistry, Kyoto Pharmaceutical University, 5 Misasagi-Nakauchi-cho, Yamashina-ku, Kyoto 607-8414, Japan |
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| Abstract: | A method for the preparative isolation of peroxisomes from the livers of rat, guinea pig, and mouse, and also from rat kidney is described. The light mitochondrial fraction, i.e., particles sedimenting between 33,000 and 250,000g-min, or the postnuclear supernatant of liver or kidney, is subjected to a 20-50% Metrizamide density gradient ultracentrifugation in a vertical rotor. After centrifugation, the peroxisomes (marker enzyme catalase and dihydroxyacetone phosphate acyltransferase) sedimented as a band near the bottom of the tube (rho = 1.22 g/ml). From the distribution of different marker enzymes and also from the morphometric examinations, it was demonstrated that the isolated peroxisomes are not contaminated with lysosomes, mitochondria, or microsomes. |
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