Immunophotochemical analysis of mineralocortin by polyclonal antibodies against the native receptor from rat kidney |
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| Affiliation: | 1. Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal;2. Department of Biochemistry and Human Biology, Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal;3. Department of Neurodegeneration and Restorative Research, University Medical Center Göttingen, Waldweg 33, 37073 Göttingen, Germany;4. Chronic Disease Research Center (CEDOC), NOVA Medical School, Campo dos Mártires da Pátria, 130, 1169-056 Lisbon, Portugal;5. Max Planck Institute for Experimental Medicine, Göttingen, Germany;6. Department of Life Sciences, Faculty of Science and Technology, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal;2. Instituto de Investigaciones Oceanológicas, Universidad Autónoma de Baja California. Km 103 autopista Tijuana-Ensenada, A.P. 453. Ensenada, Baja California, México;3. Facultad de Química Farmacéutica Biológica, Universidad Veracruzana. Circuito Gonzalo Aguirre Beltrán s/n. Zona Universitaria, C.P. 91000, Xalapa, Veracruz, México;6. Laboratorio de Genómica Humana, Facultad de Medicina, Universidad Veracruzana. Médicos y Odontólogos s/n. Col. Unidad del Bosque, C.P. 91010, Xalapa, Veracruz, México;8. Laboratorio de Neurobiología Conductual, Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad de México, México;9. Laboratorio de Neurotoxicología, Departamento de Toxicología, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, A.P. 14-740, 07300, Ciudad de México, México. |
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| Abstract: | We have obtained a polyclonal antiserum by immunizing fawn Burgundy rabbits with the mineralocorticoid receptor (MCR) purified biochemically from rat kidneys. High titers of anti-MCR activity were obtained in radioimmunoassays within 3 weeks and increased with a booster shot. In Western blot analysis, the antibody revealed a major band of 94–98 kDa in renal cytosol from rat and beef kidneys. We also developed a fluorographic procedure where the MCR linked covalently to tritiated R-5020, following ultraviolet irradiation, gave imprints superimposable on the Western blot profile. The fluorographic pattern was specific since it was largely abolished in the presence of cold RU 26752 that is specific to MCR, or mineralocortin. The immune IgG precipitated rat renal MCR-[3H]RU 26752 complexes in a dose-dependent manner and also recognized MCR bound to the natural hormone aldosterone. During gel permeation chromatography on Sephacryl, the elution profile of [3H]RU 26752 shifted to high-molecular-weight regions in the presence of immune IgG. The receptor protein could be immunolocalized primarily to the principal cells of the collecting duct in rat kidney but the intercalated cells and glomeruli were not labeled, contrary to beef kidney where a uniform pattern of immunostaining was evident. These should permit large-scale purification of the MCR for detailed physicochemical studies and for screening of the MCR-positive tissues during various pathophysiological syndromes. |
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