Isolation and immunological characterization of the four non-identical subunits of the soluble NAD-linked hydrogenase from Alcaligenes eutrophus H16 |
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| Authors: | K Schneider B Piechulla |
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| Affiliation: | 1. Institut für Mikrobiologie, Universität Göttingen, Griesbachstrasse 8, 3400 Göttingen, Germany;2. Max-Planck-Institut für experimentelle Medizin, Hermann-Rein-Strasse 3, 3400 Göttingen, FRG, Germany;1. Department of Immunology, Blavatnik Institute, Harvard Medical School, and Evergrande Center for Immunologic Diseases, Harvard Medical School and Brigham and Women’s Hospital, Boston, MA 02115, USA;2. Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA;3. Institute for Immunology and Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA;4. Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02115, USA;5. Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA;6. Division of Hematology/Oncology, Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA;7. Department of Pathology, Brigham and Women’s Hospital, Boston, MA 02115, USA;1. Department of Microbiology, Center for Research on Coronavirus and Other Emerging Pathogens, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA;2. Grupo de Inmunología Celular e Inmunogenética, Instituto de Investigaciones Médicas, Facultad de Medicina, Universidad de Antioquia, Medellín 050010, Colombia;3. Division of Hematology-Oncology, Department of Medicine, Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA;4. Duke Human Vaccine Institute, Duke University School of Medicine, Durham, NC 27710, USA;5. Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA;6. Division of Protective Immunity, Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA;7. Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA;8. Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA;9. Acuitas Therapeutics, Vancouver, BC V6T 1Z3, Canada;10. The Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA;11. Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA;1. Department of Microbiology, University of Pennsylvania Perelman School Medicine, Philadelphia, PA 19104, USA;2. Institute for Immunology, University of Pennsylvania Perelman School Medicine, Philadelphia, PA 19104, USA;3. Tumor Microenvironment Center, Department of Immunology, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15232, USA;1. Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA;2. Division of Protective Immunity, Children’s Hospital of the University of Pennsylvania, Philadelphia, PA, USA;3. Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA;4. School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA;5. The Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA;6. Grupo de Inmunología Celular e Inmunogenética, Instituto de Investigaciones Médicas, Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia;7. Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA;8. Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA;9. Duke Human Vaccine Institute, Duke University School of Medicine, Durham, NC, USA;10. Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL, USA;11. BioNTech RNA Pharmaceuticals, Mainz, Germany;12. BIOQUAL Inc., Rockville, MD, USA;13. Acuitas Therapeutics, Vancouver, BC, Canada;14. Department of Pathology, Duke University Medical Center, Durham, NC, USA;1. Department of Immunology, University of Washington School of Medicine, Seattle, WA 98109, USA;2. Center for Lung Biology, University of Washington School of Medicine, Seattle, WA 98109, USA;3. Department of Microbiology, Center for Immunology, University of Minnesota Medical School, Minneapolis, MN 55455, USA;4. Center for Immunology and Inflammatory Diseases, and Division of Pulmonary and Critical Care Medicine, Massachusetts General Hospital, and Harvard Medical School, Charlestown, MA 02129, USA;1. Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St., Melbourne 3000, Victoria, Australia;2. Department of Immunology and Pathology, Monash University, 89 Commercial Rd., Melbourne 3004, Victoria, Australia |
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| Abstract: | The soluble NAD-linked hydrogenase of Alcaligenes eutrophus H16 is a tetramer consisting of 4 non-identical subunits with molecular weights of 63,000, 56,000, 30,000 and 26,000. Conditions have been elaborated to separate and isolate each of these subunits as a single polypeptide by a preparative scale of polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS). Against each of the 4 subunits, polyclonal antibodies were produced. From the crude sera isolated from rabbits, the antibodies (IgG fractions) were purified by Protein A-Sepharose chromatography. By the double immunodiffusion method, comparison of the 4 types of subunits revealed that they are in fact different polypeptides. Subunit 1 (Mr = 63,000) and subunit 2 (Mr = 56,000) only reacted with their own specific antibodies and showed no cross-reaction whatsoever with the antibodies raised against the other subunits. The only immunological relationship among the different subunits was observed with subunit 3 (Mr = 30,000) and subunit 4 (Mr = 26,000); the type of cross-reaction indicated that they are partially identical. A. eutrophus H16 contains, in addition to the soluble hydrogenase, a membrane-bound hydrogenase which is a dimer composed of 2 subunits with Mr of 61,000 and 30,000. Whereas the 2 native enzymes did not show any immunological cross-reaction with the respective antibodies, it was demonstrated by double immunofluorescence labeling on nitrocellulose filters that the larger subunit of the membrane-bound hydrogenase cross-reacted significantly with the antibodies raised against subunit 2 of the soluble hydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS) |
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