Purification and properties of glutathione reductase from liver of the anoxia-tolerant turtle, Trachemys scripta elegans
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Glutathione reductase (GR) is a homodimeric flavoprotein that catalyzes the reduction of oxidized glutathione (GSSG) using NADPH as a cofactor. The enzyme is a major component of cellular defense mechanisms against oxidative injury. In this study, GR was purified from the liver of the anoxia-tolerant turtle, Trachemys scripta elegans. The overall fold purifications were 13.3- and 12.1-fold with final specific activities of 5.5 and 1.44 U/mg of protein for control and anoxic turtle GR, respectively. SDS-PAGE of purified turtle liver GR showed a single protein band at approximately 55 kDa. Reverse phase HPLC of turtle GR revealed a single peak that had the same retention time as yeast GR. No new isoform of GR was detected in liver of T. s. elegans during anoxia. The K m values of turtle GR for GSSG and NADPH was 44.6 and 6.82 μM, respectively, suggesting a substantially higher affinity of turtle GR toward GSSG than most other vertebrates. Unlike other human GR, NADP+ did not inhibit turtle GR activity. The activation energy of turtle GR, calculated from the slope of the Arrhenius plot, was 32.2 ± 2.64 kJ/mol. Turtle GR had high activity under a broad pH range (having activity between pHs 4 and 10; optimal activity at pH 6.5) and the enzyme maintains activity under the pH drop that occurs under anoxic conditions. The high affinity of turtle GR suggests that turtles have high redox buffering capacity of tissues to protect against oxidative stress encountered during anoxia/reoxygenation.
Keywordsadaptation anoxia glutathione reductase turtle
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Thanks to J.M. Storey and A. Ima for editorial revisions to the manuscript. This work was supported by a postgraduate scholarship to W.G.W. and a discovery grant to K.B.S. from the NSERC Canada; K.B.S. holds the Canada Research Chair in Molecular Physiology.
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