Biochemical Changes of Glutathione S-Transferase Activity During the Germination of Trifolium alexandrinum
American Journal of Life Sciences
Volume 3, Issue 4, August 2015, Pages: 257-267
Received: May 16, 2015; Accepted: Jun. 1, 2015; Published: Jun. 19, 2015
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Authors
Ragaa Reda Hamed, Department of Molecular Biology, Genetic Engineering and Biotechnology Division, National Research Center, Cairo, Egypt
Ehab Mostafa Mohamed Ali, Biochemistry Division, Chemistry Department, Faculty of Science, Tanta University, Egypt
Abeer Shokeer, Department of Molecular Biology, Genetic Engineering and Biotechnology Division, National Research Center, Cairo, Egypt
Rasha Awni Guneidy, Department of Molecular Biology, Genetic Engineering and Biotechnology Division, National Research Center, Cairo, Egypt
Abdul Aziz Mohamad Gad, Department of Molecular Biology, Genetic Engineering and Biotechnology Division, National Research Center, Cairo, Egypt
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Abstract
The key objective of this study was to investigate the effect of germination on biochemical and enzymatic antioxidant activities of Trifolium alexandrinum seeds. The T. alexandrinum was chosen for GST purification and characterization due to its highest GST activity and antioxidant capacity, beside its economical importance. Seeds of T. alexandrinum were germinated for 6 days. Changes in glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx) in germinating and dormant seeds of T. alexandrinum were evaluated. Also the changes in the total phenolic and flavonoid contents as well as the antioxidant capacities were monitored for 6 days of germination using spectrophotometeric methods. Simple reproducible procedures for the purification of T. alexandrinum GST from both dormant and 6 days germinated seeds were established using DEAE-Sepharose and Sephadex G-100 columns chromatography. The results showed the presence of three isoenzymes for both dormant and germinated seeds designated as GST1, GST2 and GST3. The major GST of the dormant seed was GST2 while for the germinated one was GST3. Dormant GST2 was expressed as a heterodimer with molecular weight of 27.5 and 28.5 KDa while germinated GST3 was expressed as a homodimer with molecular weight of 27 KDa. Characterization of both dormant GST1 & GST2 and germinated GST2 and GST3 including optimum pH, kinetic parameters [kmGSH of 1.17±0.39, 1.22±0.13, 0.84±0.19, 0.96±0.24mM, respectively and kmCDNB of 0.65±0.095, 0.57±0.11, 0.59±0.14, 1.02±0.18 mM, respectively]. The substrate selectivity using different electrophilic compounds and inhibitor effects were carried out. In opposite to other isoenzymes, germinated GST3 exhibited enzymatic activity towards ethacrynic acid with specific activity of 0.073±0.005 µmol/min/mg protein while cibacron blue was the most potent inhibitor for these isoenzymes.
Keywords
Antioxidant Enzymes, Inhibition, Kinetic Parameters, Seed Germination, Trifolium alexandrinum
To cite this article
Ragaa Reda Hamed, Ehab Mostafa Mohamed Ali, Abeer Shokeer, Rasha Awni Guneidy, Abdul Aziz Mohamad Gad, Biochemical Changes of Glutathione S-Transferase Activity During the Germination of Trifolium alexandrinum, American Journal of Life Sciences. Vol. 3, No. 4, 2015, pp. 257-267. doi: 10.11648/j.ajls.20150304.12
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