Primary structure and analysis of the location of the regulatory disulfide bond of pea chloroplast NADP-malate dehydrogenase |
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| Institution: | 1. Biotechnology Program, Faculty of Agro- and Bio-Industry, Thaksin University, Phatthalung Campus, Phatthalung 93210, Thailand;2. Department of Food Science and Agricultural Chemistry, McGill University, Macdonald Campus, Ste. Anne de Bellevue, Quebec H9X 3V9, Canada;3. Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand;4. National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Phahonyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand;5. Graduate School of Fisheries Science and Environmental Studies, Nagasaki University, 1-14 Bunkyo, Nagasaki 852-8521, Japan;6. Department of Food Science and Technology, Faculty of Agro- and Bio-Industry, Thaksin University, Phatthalung Campus, Phatthalung 93210, Thailand;1. Department of Biophysics, Faculty of Science, P. J. Šafárik University in Košice, Jesenná 5, 04154 Košice, Slovakia;2. Department of Biochemistry, Faculty of Science, P. J. Šafárik University in Košice, Moyzesova 11, 04154 Košice, Slovakia;3. Center for Interdisciplinary Biosciences, P. J. Šafárik University in Košice, Jesenná 5, 04154 Košice, Slovakia;4. Chemistry Department, Faculty of Arts and Science, Aydın Adnan Menderes University, 09010 Aydın, Turkey;5. Department of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, 040 01 Košice, Slovakia;6. Faculty of Science, University of South Bohemia in České Budějovice, Branišovská 1645/31A, 37005 České Budějovice, Czech Republic;7. Laboratory of Structural Biology and Bioinformatics, Institute of Microbiology of the Czech Academy of Sciences, Zámek 136, 37333 Nové Hrady, Czech Republic |
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| Abstract: | Purified pea chloroplast NADP-malate dehydrogenase (S)-malate: NADP+ oxidoreductase, EC 1.1.1.82) was digested with trypsin and the resulting peptides were separated by HPLC and sequenced. Together with the information from earlier work (Fickenscher, K. et al. (1987) Eur. J. Biochem. 168, 653–658) the total sequence is now known to an extent of 78%. Comparison with the sequence of the corn NADP-malate dehydrogenase deduced from its cDNA (Metzler, M.C. et al. (1989) Plant Mol. Biol. 12, 713–722) showed 84% agreement; however, the 11 N-terminal residues exhibit only 27% similarity. The N- and C-terminal extrapeptides of the pea NADP-malate dehydrogenase when aligned with non-regulatory NAD-malate dehydrogenases from bacteria or mammals consist of 30 and 17 amino acids, respectively. Since all cysteine-containing peptides were sequenced, the number of eight cysteines per subunit of the pea enzyme was established. The native, oxidized enzyme ss characterized by an extremely slow reactivity of two thiols. Titration of the thiols of the denatured, oxidized enzyme both with DTNB and with pCMB resulted in six thiols not involved in disulfide formation. Therefore, one disulfide bridge must be present per 38.9 kDa subunit. Analysis of disulfide bonds by urea gel electrophoresis confirmed this finding. Using digestion products of NADP-malate dehydrogenase with aminopeptidase K, the location of the single disulfide bridge was established to be on the N-terminal arm (Cys-12 and Cys-17) of the polypeptide chain. |
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