Antibodies against the fungal enzyme mannitol dehydrogenase |
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| Affiliation: | 1. Institute of Plant Biology, University of Zu¨rich, 8008 Zu¨rich, Switzerland;2. Institute of Pharmacology and Biochemistry, University of Zu¨rich, 8057 Zu¨rich, Switzerland;1. State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai, China;2. School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China;1. Division of Pulmonary, Allergy & Critical Care Medicine, Department of Medicine, Worchester, MA;2. Division of Health Informatics and Implementation Science, Department of Population Quantitative Health Service, Worchester, MA;3. Department of Medicine, Worchester, MA;4. Department of Family Medicine and Community Health, Worchester, MA;5. Division of Hematology and Oncology, Department of Medicine, Boston University School of Medicine, Boston, MA;6. Center for Outcomes Research & Evaluation, Maine Medical Center Research Institute, Portland, ME;7. Meyers Primary Care Institute, Worcester, MA;1. Department of Biological Sciences, Egerton University, Nakuru, Kenya;2. Doctoral School of Health Sciences, Faculty of Health Science, University of Pẻcs, Hungary |
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| Abstract: | Affinity-purified, electrophoretically homogeneous NADP+-mannitol dehydrogenase (MtDH; EC 1.1.1.138), isolated from fruit bodies ofAgaricus bisporus (Lange) Sing., was used to produce polyclonal antibodies. Antiserum against MtDH and the affinity-purified antibody inhibited enzyme activity in a dose-dependent manner, with about 15 mol of purified antibody required for 50% inhibition of 1 mol MtDH. Immunological specificity of the antisera was demonstrated by double immunodiffusion, the dotimmunobinding assay, and immunoblotting. Controls (preimmune sera and preadsorbed antisera) were negative. An enzyme-linked immunosorbent assay (ELISA) was established to quantitate immunobinding in various cell fractions of the fungus. The bound protein was found predominantly in the soluble fraction (150,000g), where it contributed 5% to the total protein of the fruit body and 1% to that of the mycelium. Though only to a minor extent, the anti-MtDH antiserum also bound to the supernatants obtained following treatment of a “mixed membrane fraction” and the cell wall fraction (54,000g and 4,000g sediments, respectively; both rigorously washed with buffer) with digitonin or sodium dodecyl sulfate/heat. In sporocarp extracts, the antibody bound specifically to a protein of Mr 30K (corresponding to the subunit molecular weight of MtDH) as shown by immunoblotting, whereas mycelial extracts contained three (30K, 26K, 24K) protein bands, which cross-reacted with the anti-MtDH antiserum. |
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