Purification and kinetic characterization of the liverwort Pallavicinia lyelli (Hook.) S. Gray. cytosolic ascorbate peroxidase |
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| Authors: | S Sajitha Rajan K Murugan |
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| Institution: | 1. Department of Genetics, Federal University of Rio Grande do Sul, Brazil;2. Biotechnology Center, Federal University of Rio Grande do Sul, Brazil;3. Department of Botany, Federal University of Rio Grande do Sul, Brazil;4. Department of Biochemistry and Molecular Biology, Federal University of Ceará, Brazil;5. Department of Biophysics, Federal University of Rio Grande do Sul, Brazil;1. Laboratório de Fisiologia Vegetal “Coaracy M. Franco”, Instituto Agronômico, CP 28, CEP 13012-970 Campinas, SP, Brazil;2. Departamento de Biologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas, CP 6109, CEP 13083-970 Campinas, SP, Brazil;3. Laboratório de Metabolismo de Plantas, Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, CEP 60455-970 Fortaleza, CE, Brazil;1. Centre for Vascular Research, School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia;2. Rural Clinical School, University of New South Wales, Sydney, NSW 2052, Australia;3. Centre for Free Radical Research, Department of Pathology, University of Otago, 8140 Christchurch, New Zealand;1. Programa de Pós-Graduação em Biologia Celular e Molecular, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil;2. Departamento de Genética, Universidade Federal do Rio Grande do Sul, 91501-970, Porto Alegre, RS, Brazil;3. Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, 60455-970, Fortaleza, CE, Brazil;4. Faculdade de Agronomia e Medicina Veterinária, Programa de Pós-Graduação em Agronomia, Universidade de Passo Fundo, Passo Fundo, RS, Brazil;1. School of Bioresources and Technology, King Mongkut''s University of Technology Thonburi, Bangkok 10150, Thailand;2. Pilot Plant Development and Training Institute, King Mongkut''s University of Technology Thonburi, Bangkok 10150, Thailand;1. Laboratory of Plant Biotechnology, Ecology and Ecosystem Valorization, Faculty of Sciences, University Chouaib Doukkali, 24000 El Jadida, Morocco;2. Group of Fruit Tree Biotechnology, Department of Plant Breeding, CEBAS-CSIC, PO Box 164, 30100 Murcia, Spain |
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| Abstract: | Ascorbate peroxidase (APX) of the liverwort Pallavicinia lyelli was extracted and purified through ammonium sulfate precipitation, Butyl-Toyopearl, DEAE-Cellulofine and Sephadex G-75 chromatography. The purification factor for APX was 285 with 7.9% yield. The enzyme was characterized for thermal stability, pH and kinetic parameters. The molecular mass of APX was approximately 28 kDa estimated by SDS-PAGE. The purity was checked by native PAGE, showing a single prominent band. The optimum pH was 6.0. The enzyme had a temperature optimum at 40 °C and was relatively stable at 60 °C, with 54% loss of activity. When the enzyme was diluted with the ascorbate-deleted medium, the half inactivation time was approximately 15 min. The absorption spectra of the purified enzyme and the inhibition by cyanide and azide showed that it is a hemoprotein. Spectral analysis and inhibitor studies were consistent with the presence of a heme moiety. When compared with ascorbate peroxidase activity derived from ruptured intact chloroplasts, the purified enzyme was found to have a higher stability, a broader pH optimum for activity and the capacity to utilize alternate electron donors. p-chloromercuribenzoate (pCMB), hydroxyurea and salicylic acid (SA) significantly inhibited APX activity. Ascorbate (AsA) and pyrogallol were found to be efficient substrates for Pallavicinia APX, considering the Vmax/Km ratio. We detected the activity of monodehydroascorbate reductase (MDHAR) involved in the regeneration of ascorbate, but failed to detect the dehydroascorbate reductase (DHAR) activity. The data obtained in this study may help to understand desiccation tolerance mechanism in the liverwort. |
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