Purification and characterization of lysosomes from Chinese hamster ovary cells |
| |
| Authors: | E A Madden J B Wirt B Storrie |
| |
| Institution: | 1. Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, Toronto, Ontario, Canada;2. Program in Genetics and Genome Biology, Hospital for Sick Children, Toronto, Ontario, Canada;3. Department of Medical Genetics, University of Calgary, Calgary, Alberta, Canada;4. Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands;5. Department of Clinical Genetics, Liverpool Hospital, Sydney, New South Wales, Australia;6. School of Women’s and Children’s Health, University of New South Wales, Sydney, New South Wales, Australia;7. German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Ingolstaedter Landstraße, Neuherberg, Germany;8. Chair of Experimental Genetics, TUM School of Life Sciences, Technische Universität München, Freising, Germany;9. German Center for Diabetes Research (DZD), Ingolstaedter Landstraße, Neuherberg, Germany;10. Division of Neurology, The Hospital for Sick Children, Toronto, Ontario, Canada;11. Departments of Paediatrics and Biochemistry, University of Toronto, Toronto, Ontario, Canada;1. National Institute on Drug Dependence, Peking University, Beijing 100191, China;2. Institute of Mental Health, National Clinical Research Center for Mental Disorders, Key Laboratory of Mental Health and Peking University Sixth Hospital, Peking University, Beijing 100191, China;3. Hangzhou Seventh People''s Hospital, Hangzhou 310007, China;4. Department of Psychiatry, First Hospital of Hebei Medical University, Shijiazhuang 050031, China;5. Department of Psychiatry and Behavioral Sciences, University of Washington, P.O. Box 356560, Seattle, WA 98195-6560, USA;1. Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, A-1190 Vienna, Austria;2. acib - Austrian Centre of Industrial Biotechnology, A-8010 Graz, Austria;3. Department of Analytical Chemistry, University of Vienna, A-1090 Vienna, Austria;1. University of Applied Sciences Biberach, Institute of Applied Biotechnology, Hubertus-Liebrecht-Straße 35, 88400 Biberach, Germany;2. Boehringer Ingelheim Pharma GmbH & Co KG, Cell Culture Development CMB, Birkendorfer Straße 65, 88397 Biberach, Germany;3. Boehringer Ingelheim Pharma GmbH & Co KG, Early Stage Bioprocess Development, Birkendorfer Straße 65, 88397 Biberach, Germany;1. Georgia Cancer Center and Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta University, Augusta, GA, USA;2. MetCure Therapeutics LLC, Atlanta, GA, USA;1. KTH - Royal Institute of Technology, School of Engineering Sciences in Chemistry, Biotechnology, and Health, Dept. of Protein Science, SE-106 91 Stockholm, Sweden;2. KTH - Royal Institute of Technology, School of Engineering Sciences in Chemistry, Biotechnology, and Health, Dept. of Industrial Biotechnology, SE-106 91 Stockholm, Sweden;3. Cell Culture & Fermentation Sciences, BioPharmaceutical Development, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK;4. Analytical Sciences, BioPharmaceutical Development, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK and Advanced Biomanufacturing Centre, Department of Chemical and Biological Engineering, University of Sheffield, Mappin Street, Sheffield, UK |
| |
| Abstract: | Lysosomes were isolated from Chinese hamster ovary cells by fractionation of a postnuclear supernatant in consecutive density gradients. By marker enzyme analysis, the preparation was 63-fold enriched for lysosomes compared to the homogenate and contained at most trace amounts of marker activities for plasma membrane, Golgi, endoplasmic reticulum, peroxisomes, cytosol, and mitochondria. The lysosomes were intact as indicated by greater than 95% latency of beta-hexosaminidase activity, and the yield was about 12% relative to the homogenate. By electron microscopy, the lysosomal preparation contained very few mitochondrial profiles. By cytochemistry, greater than 80% of the organelle profiles were positive for the native lysosomal marker, acid phosphatase, and profiles were positive for long-term internalized horseradish peroxidase, an endocytic marker for lysosomes. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the lysosomal preparation displayed a unique pattern of polypeptides and was devoid of mitochondrial contamination. Lysosomes were fractionated into membrane and lumenal compartments by Na2CO3 treatment. Each compartment contained 20-30 distinct electrophoretic species ranging from 18 to 200 kDa. Each polypeptide could be assigned to either the membrane or lumenal compartment. A comparison of silver-stained polypeptides with those metabolically labeled with 35S]methionine indicated that, with the possible exception of an 18-kDa species, all of the major lysosomal polypeptides in both compartments were derived by endogenous synthesis in these exponentially growing fibroblasts. |
| |
| Keywords: | |
| 本文献已被 ScienceDirect 等数据库收录！ |
|
