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Plant Physiology 94:1402-1409 (1990)
© 1990 American Society of Plant Biologists

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Metabolism and Enzymology

Purification, Characterization, and Immunological Properties for Two Isoforms of Glutathione Reductase from Eastern White Pine Needles 1

James V. Anderson, John L. Hess and Boris I. Chevone

Department of Plant Pathology, Physiology and Weed Science, Virginia Polytechnic Institute and State University Blacksburg, Virginia 24061, Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University Blacksburg, Virginia 24061

Glutathione reductase (EC 1.6.4.2) was purified from Eastern white pine (Pinus strobus L.) needles. The purification steps included affinity chromatography using 2', 5'-ADP-Sepharose, FPLC-anion-exchange, FPLC-hydrophobic interaction, and FPLC-gel filtration. Separation of proteins by FPLC-anion-exchange resulted in the recovery of two distinct isoforms of glutathione reductase (GRA and GRB). Purified GRA had a specific activity of 1.81 microkatals per milligram of protein and GRB had a specific activity of 6.08 microkatals per milligram of protein. GRA accounted for 17% of the total units of glutathione reductase recovered after anion-exchange separation and GRB accounted for 83%. The native molecular mass for GRA was 103 to 104 kilodaltons and for GRB was 88 to 95 kilodaltons. Both isoforms of glutathione reductase were dimers composed of identical subunit molecular masses which were 53 to 54 kilodaltons for GRA and 57 kilodaltons for GRB. The pH optimum for GRA was 7.25 to 7.75 and for GRB was 7.25. At 25°C the Km for GSSG was 15.3 and 39.8 micromolar for GRA and GRB, respectively. For NADPH, the Km was 3.7 and 8.8 micromolar for GRA and GRB, respectively. Antibody produced from purified GRB was reactive with both native and denatured GRB, but was cross-reactive with only native GRA.

1 This research was supported by Environmental Protection Agency grant No. R-814224-01-0 to B. I. C. and J. L. H. This article has not been subjected to EPA peer and administrative review and may not necessarily reflect the views of EPA and no official endorsement should be inferred.






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Copyright © 1990 by the American Society of Plant Biologists