Light-Dependent Reduction of Dehydroascorbate by Ruptured Pea Chloroplasts

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Abstract
Glutathione dehydrogenase (EC 1.8.5.1) was partially purified from pea shoots. The pH optimum was 7.6. The Km values for GSH [glutathione] and dehydroascorbate were 4.4 and 0.44 mM, respectively. The enzyme was inhibited by iodoacetate and CuSO4 but not significantly by ZnCl2 or NaN3. Part of the total enzyme activity was associated with isolated chloroplasts. Illuminated ruptured chloroplasts, in the presence of 50 .mu.M NADP(H) and substrate concentrations of GSH or GSSG [oxidized glutathione], catalyzed (dehydroascorbate plus glutathione)-dependent O2 evolution with the concomitant reduction of dehydroascorbate to ascorbate. Oxidation of ascorbate by ascorbate oxidase activity associated with the chloroplasts was relatively insignificant. ZnCl2 inhibited (dehydroascorbate plus glutathione)-dependent O2 evolution but not ascorbate formation. The reaction was attributed to light-dependent reduction of GSSG (involving glutathione reductase) coupled to the reduction of dehydroascorbate (involving glutathione dehydrogenase). Light-dependent reduction of GSSG appears to be the rate-limiting step in this reaction sequence at physiological concenrations of GSH.