Determination of apo and holo retinoic acid-binding protein levels in retinoid-responsive transformed cells by high-performance size-exclusion chromatography |
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| Authors: | H E Shubeita M D Patel A M McCormick |
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| Institution: | 1. University of Belgrade, Innovation Center of Faculty of Technology and Metallurgy, Karnegijeva 4, 11120 Belgrade, Serbia;2. University of Belgrade, Faculty of Technology and Metallurgy, Karnegijeva 4, 11120 Belgrade, Serbia;1. Biofuels and Bioprocessing Research Center, Institute of Technical Education and Research, Siksha ‘O’ Anusandhan [Deemed to be University], Bhubaneswar 751030, India;2. Department of Chemistry, Institute of Technical Education and Research, Siksha ‘O’ Anusandhan [Deemed to be University], Bhubaneswar, Odisha, India;3. Department of Chemistry, College of Basic Science and Humanities, Odisha University of Agriculture and Technology, Bhubaneswar, India;1. Department of Environmental Engineering, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung, Taiwan;2. Research Center of Sustainable Energy and Nanotechnology, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung, Taiwan;3. Innovation and Development Center of Sustainable Agriculture, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung, Taiwan;4. Department of Environmental and Safety Engineering, Ajou University, Suwon 16499, Republic of Korea;1. Mycotoxicology Research Unit, Department of Biosciences and Biotechnology, Babcock University, Ilishan Remo, Nigeria;2. Pathology/Mycotoxin Laboratory, International Institute of Tropical Agriculture, P.M.B. 5320, Ibadan, Nigeria;3. Laboratory of Pharmacology and Toxicology, University of Yaounde I, Yaounde, Cameroon;4. Department of Food Technology, Faculty of Science, University of Johannesburg, South Africa;5. Faculty of Agriculture, Nasarawa State University Keffi, Lafia Campus, Nasarawa State, Nigeria;6. Center for Analytical Chemistry, Department of Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences, Vienna (BOKU), Konrad Lorenzstr. 20, A-3430 Tulln, Austria;1. Chair for Food Process Engineering and Dairy Technology, Technische Universität München, Weihenstephaner Berg 1, 85354 Freising, Germany |
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| Abstract: | A method to measure the endogenous levels of apo and holo cellular retinoic acid-binding proteins was developed using calf testis cytosol as the source of retinoic acid-binding protein. 3H]Retinoic acid-retinoic acid-binding protein complexes were assayed by high-performance size-exclusion chromatography. Preincubation of cytosol with 10 mM p-hydroxymercuribenzoate at 4 degrees C resulted in complete inhibition of retinoic acid binding to apo retinoic acid-binding protein. In addition, total dissociation of preformed holo retinoic acid-binding protein complexes was noted within 20 min after mercurial addition. Thus, p-hydroxymercuribenzoate converted the total pool of cellular retinoic acid-binding protein (apo plus holo) to mercurial-protein complexes unable to bind retinoic acid in vitro. Mercurial inhibition of retinoic acid-retinoic acid-binding protein complex formation was totally reversed upon the addition of 50 mM dithiothreitol. Total cytosolic retinoic acid-binding protein was determined from specific retinoic acid binding after treatment with p-hydroxymercuribenzoate and dithiothreitol. Apo cellular retinoic acid-binding protein concentration was measured by determining specific radioligand binding prior to p-hydroxymercuribenzoate treatment, and correcting for exchange of endogenously bound retinoid with exogenous tritiated retinoic acid. Holo cellular retinoic acid-binding protein concentration was derived from the difference between total and apo retinoic acid-binding protein concentrations. Using this method, we have demonstrated that retinoid-responsive EJ and T24 human bladder carcinoma cell lines and AT3A and AT3B rat pancreatic acinar carcinoma cell lines lack detectable levels of either apo or holo cellular retinoic acid-binding protein. These results established that retinoid inhibition of transformed bladder and acinar cell proliferation in culture was mediated by a cellular retinoic acid-binding protein-independent mechanism. |
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