Purification and characterization of glutathione reductase from Scots pine needles

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Abstract
Glutathione reductase (EC 1.6.4.2) was purified from Scots pine (Pinus sylvestris L.) needles. The enzyme had a specific activity of 3.65 μkat (mg protein)−1, and the apparent molecular weight of the polypeptide was 59 kDa. Sulfhydryl‐modifying reagents such as N‐ethylmaleimide and iodoacetate inactivated the enzyme in the presence of NADPH. Oxidized glutathione protected the enzyme against this inactivation. NADPH inactivated the enzyme by ca 35% within 5 min. High concentrations of hydrogen peroxide (32 μM) had no effect on enzyme activity. High concentrations of sulfite (5 μM) inhibited the enzyme but low concentrations (60 μM) did not affect the activity. The Km for oxidized glutathione and NADPH was 28 and 1 μM, respectively. The pi was 4.7 and the amino‐acid composition was similar, with minor exceptions, to that of glutathione reductase obtained from rice embryos, human erythrocytes and E. coli.