Purification, characterization, and immunological properties for two isoforms of glutathione reductase from eastern white pine needles.

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 (GR(A) and GR(B)). Purified GR(A) had a specific activity of 1.81 microkatals per milligram of protein and GR(B) had a specific activity of 6.08 microkatals per milligram of protein. GR(A) accounted for 17% of the total units of glutathione reductase recovered after anion-exchange separation and GR(B) accounted for 83%. The native molecular mass for GR(A) was 103 to 104 kilodaltons and for GR(B) 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 GR(A) and 57 kilodaltons for GR(B). The pH optimum for GR(A) was 7.25 to 7.75 and for GR(B) was 7.25. At 25 degrees C the K(m) for GSSG was 15.3 and 39.8 micromolar for GR(A) and GR(B), respectively. For NADPH, the K(m) was 3.7 and 8.8 micromolar for GR(A) and GR(B), respectively. Antibody produced from purified GR(B) was reactive with both native and denatured GR(B), but was cross-reactive with only native GR(A).