Light-Dependent Reduction of Dehydroascorbate by Ruptured Pea Chloroplasts

Abstract

Glutathione dehydrogenase (EC 1.8.5.1) was partialHy purified from pea shoots. The pH optimum was 7.6. The Km values for GSH and dehydroascorbate were 4.4 and 0.44 millimolar, respectively. The enzyme was inhibited by iodoacetate and CuS04 but not significantly by ZnCI2 or NaN3. Part of the total enzyme activity was associated with isolated chloroplasts.Illuminated ruptured chloroplasts, in the presence of 50 micromolar NADP(H) and substrate concentrations of GSH or GSSG, catalyzed (dehydroascorbate plus glutathione)-dependent 02 evolution with the concomitant reduction of dehydroascorbate to ascorbate. Oxidation of ascorbate by ascorbate oxidase activity associated with the chloroplasts was relatively insignificant. ZnCI2 inhibited (dehydroascorbate plus glutathione)-dependent 02 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 concentrations of GSH.chloroplasts, especially as light-induced alkalization of chloroplast stroma (10) provides favorable conditions for the nonenzymic reaction.We reported previously (11) that illuminated ruptured chloroplasts catalyze the reduction of GSSG with the concomitant evolution of 02. The properties of these reactions were consistent with the operation of light-coupled GSSG reductase (reactions I and II). Taken in conjunction with the proposal of Foyer and Halliwell (5), this suggests that the reduction of DHA, and ultimately H202, could be light coupled via GSSG reductase. This mechanism predicts that in the presence of catalytic amounts of GSH or GSSG, chloroplasts will exhibit DHA-dependent 02 evolution with the concomitant reduction of DHA to ascorbate. Previously, we reported that ruptured chloroplasts did not support DHA-dependent 02 evolution in the presence of 40 tLM GSSG, 50 /LM NADPH, and 1 mm DHA (11). In this paper we report that illuminated ruptured chloroplasts in the presence of 50 UM NADPH, 0.94 mmDHA, and >0.15 nm GSH oraO.l m GSSG exhibit characteristics consistent with the operation of reactions I to IV and that reaction III involves a chloroplast GSH dehydrogenase.Chloroplasts reduce 02 in the light to H202 (2, 4). Foyer and Halliwell (5) have proposed a mechanism for the reduction of H202 in chloroplasts involving ascorbate as the reductant. Their model also postulates that the DHA3 produced in this reaction is successively reduced by GSH and NADPH, the latter involving GSSG reductase (EC 1.6.4.2) MATERIALS AND METHODS Chemicals. DHA was obtained from Koch-Light, Bucks., England; the purity as estimated by oxidation of GSH in the presence of purified GSH dehydrogenase was 94%. Cyt c (horse heart), ascorbate oxidase (lyophilizate), and GSSG were obtained from Boehringer, Mannheim, Germany. GSH was obtained from Sigma and contained 0.06 mol GSSG/mol GSH by assay ...

A novel dehydroascorbate reductase from spinach chloroplasts homologous to plant trypsin inhibitor

“…We have purified to homogeneity an active DHA reductase from spinach chloroplasts, thus substantiating that catalytic reduction of dehydroascorbate proceeds in the organelles [9,11,12]. The measured activity was similar to that of an earlier chloroplast preparation [ 121 but the enzyme has a lower specific activity than other DHA reductase fractions isolated from whole leaves [3].…”

Reactive oxygen species and antioxidant enzymes activity of Anabaena sp. PCC 7120 (Cyanobacterium) under simulated microgravity

“…According to Jablonski [8], the change of OD 240 was recorded in spectrophotometer (UV-160, Shimadizu corporation). The reduction of H 2 O 2 in reaction system was showed with units= g chl.…”

Light-Dependent Reduction of Hydrogen Peroxide by Ruptured Pea Chloroplasts

“…A number of lines of evidence support this proposed reaction sequence: (a) chloroplasts contain a light-coupled glutathione reductase (reactions 1 + 2) (15); (b) GSH dehydrogenase, catalyzing reaction 3, occurs in chloroplasts ( 16); (c) ascorbate peroxidase, catalyzing reaction 4, also occurs in chloroplasts (12); however, reactions 3 and 4 may also proceed nonenzymically (I 1, 12); and (d) illuminated chloroplasts, under specific conditions, can reduce I mol of H202 with the concomitant evolution of 0.5 mol of 02 (…”

“…Chemicals were obtained from the sources described previously (16). H202 and t-butyl hydroperoxide concentrations were determined by titration with standardized KMnO4.…”