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

Abstract

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 FPLCgel 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 GR.. The pH optimum for GRA was 7.25 to 7.75 and for GR. was 7.25. At 250C 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 GR. was reactive with both native and denatured GR., but was cross-reactive with only native GRA.Plants have demonstrated altered antioxidant levels when exposed to air pollutants, low temperature, drought, or xenobiotics such as herbicides (1, 28). These plant-stress interactions, at one time or another, have been associated with increased cellular concentrations of superoxide anion radicals (02-s-) and hydrogen peroxide (H202). Superoxide radicals and H202 can directly attack lipid membranes and certain sulfhydryl-containing enzymes of the reductive pentose cycle (24), or they can further interact to generate hydroxyl radicals (OH) which also can react with membrane lipids and proteins (2,28). Such alterations to cell metabolism and membrane biochemistry would limit normal growth and vigor. Mechanisms for the scavenging of toxic free radicals have previously been reviewed in the plant literature (1-3, 17, 28).

Changes in Isozyme Profiles of Catalase, Peroxidase, and Glutathione Reductase during Acclimation to Chilling in Mesocotyls of Maize Seedlings

“…GR isozymes were separated on nondenaturing polyacrylamide gels (7% T, 3% C) at 80 V for 17 h and 4°C using the procedures of Laemmli (1970). Gels were stained in a solution of 0.25 M Tris, pH 7.8, containing 0.24 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 0.4 mM NADPH, 0.34 mM 2,6-dichlorophenolindophenol, and 3.6 mM GSSG in darkness for 1 h (similar to the procedure of Anderson et al, 1990). Duplicate gels were also stained in the absence of GSSG to distinguish GR from other sources of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction.…”

Seasonal Variation in the Antioxidant System of Eastern White Pine Needles

“…These In our study, the two isoforms of GR (GRA and GRB) from white pine needles were isolated and purified as previously described by Anderson et al. (2). Using chromatography and electrophoresis, we observed no new isoforms of GR when comparing summer and winter tissue (data not shown), and both isoforms showed a proportional increase in activity from summer to winter.…”

“…Using chromatography and electrophoresis, we observed no new isoforms of GR when comparing summer and winter tissue (data not shown), and both isoforms showed a proportional increase in activity from summer to winter. Hence, the GR activity in both summer and winter needles can be attributed to approximately 20% GRA and 80% GRB (2).…”

“…Two isoforms of GR, GRA and GRB, were purified from eastern white pine during early summer as described by Anderson et al (2) and were used to determine the apparent Km values between 5 and 40C. The apparent minimum Km,GSSG for GRA and GRB was 1 1.1 and 29.1 Mm, respectively, and occurred at 10°C (Fig.…”

“…The assay for, and the purification of, GR was performed as previously described by Anderson et al (2). The substratebinding affinity of GR for NADPH and GSSG was determined at 10, 20, 30, and 40°C.…”