Crude extracts of pea shoots (Pisum sativum) catalyzed oxidized glutathione (GSSG)-dependent oxidation of NADPH which was atributed to NADPH-specific glutathione reductase. The pH optimum was 8 and the Km values for GSSG and NADPH were 23 jM and 4.9 jzM, respectively. Reduced glutathione (GSH) inhibited the reaction. Crude extracts also catalyzed NADPH-dependent reduction of GSSG; the ratio of the rate of NADPH oxized to GSH formed was 0.49. NADH and various substituted mono-and disulfides would not substitute for NADPH and GSSG respectively. Per mg of chlorophyll, enzyme activity of isolated chloroplasts was 69% of the activity of crude extracts.Iuminated sonicated pea chloroplasts, in the presence of catalytic amounts of NADPH, catalyzed GSSG-dependent 02 evolution (mean of 10 determinations, 10.4 jLmol per mg chlorophyil per hour, SD 1.4) with the concomitant production of GSH. The molar ratio of GSH produced to O2 evolved was 3.8 and the highest ratios for 02 evolved to GSSG added were 0.46 and 0.44. The Km value for GSSG was 26 ,LM. GSH inhibited the reaction. The reaction was attributed to photosyntheticafly coupled glutathione reductase.Ruptured chloroplasts, in the presence of catalytic amounts of GSSG and NADPH, did not catalyze sustained 02 evolution in the presence of substrate amounts of hydrogen peroxide, dehydroascorbate, L-cystine, sulfite, or sulfate.Glutathione reductase (glutathione oxidoreductase NADPHspecific, EC 1.6.4.2) has been described in a number of organisms (16). In animals the main function of glutathione reductase appears to be the reduction of GSSG3 which is formed during the reduction of H202 in a reaction catalyzed by glutathione peroxidase (15). Glutathione reductase has been reported in crude extracts from several plant species (10) and purified from germinating pea seeds (11). It has also been described in avocado mitochondria (18) and more recently in chloroplasts (6,13 3 Abbreviations: GSSG: oxidized glutathione; GSH: reduced glutathione; DTNB: 5,5'-dithiobis (2-nitrobenzoic acid).mutase for the removal of free radicals of 02 produced by the light reactions (6,8). Another suggestion for the chloroplast enzyme is that GSH participates in the light-dependent regulation of the activity of various chloroplast enzymes by controlling the degree of oxidation of sulfhydryl groups of these enzymes (17). Part of this proposal is based on the requirement for light for the synthesis of NADPH and therefore the reduction of GSSG. Here, we report that ruptured (but not intact) pea chloroplasts, in the presence of catalytic amounts of NADPH, catalyze the reduction of GSSG to GSH in the light with the concomitant evolution of 02. This system, which we attribute to photosynthetically coupled4 glutathione reductase activity, provides a means for testing some of the hypotheses concerning the physiological role of GSH and glutathione reductase in chloroplasts. MATERIALS AND METHODSPlant Material. Peas (Pisum sativum cv. Massey Gem) were soaked in water for 24 hr (1.6 ml/g of dry seeds) a...

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 ...

Ruptured pea (Pisum sativum cv. Massey Gem) chloroplasts exhibited ascorbate peroxidase activity as determined by H202-dependent oxidation of ascorbate and ascorbate-dependent reduction of H202. The ratio of ascorbate peroxidase to NADP-glyceraldehyde 3-phosphate dehydrogenase activity was constant during repeated washing of isolated chloroplasts. This To date, detailed studies on the mechanism(s) of 02 evolution coupled to H202 reduction have not been presented. Here, we report such studies using ruptured chloroplasts. Reaction 5 is also consistent with catalase activity. Although catalase is predominantly a peroxisomal enzyme (13), residual catalase activity is invariably associated with isolated chloroplast preparations (1). Accordingly, evidence is presented which excludes the possibility that catalase activity is involved in the operation of reaction 5 in ruptured chloroplasts, in the light. MATERIALS AND METHODSChemicals. Chemicals were obtained from the sources described previously (16). H202 and t-butyl hydroperoxide concentrations were determined by titration with standardized KMnO4. The GSH contained 6% GSSG (16).Chloroplast Preparation. Unwashed, sonicated, or osmotically shocked chloroplasts were prepared as described previously (15, 16) from pea seedlings (Pisum sativum cv. Massey Gem). In the studies reported here, the unwashed chloroplasts were repeatedly washed (4 times) with a washing medium (16)

Protein modulase and ferredoxin/thioredoxin reductase are soluble proteins that have been suggested to catalyze the light-dependent modulation of enzyme activity in the stromal compartment of the chloroplast. Protein modulase is active in vitro without additional ferredoxin and thioredoxin, whereas ferredoxin/thioredoxin reductase requires additional ferredoxin and thioredoxin. We hypothesize that protein modulase is a complex protein composed of ferredoxin/thioredoxin reductase, ferredoxin, and thioredoxin. In recenstituted chloroplast systems, antiserum directed against ferredoxin, at concentrations sufficient to inhibit the photoreduction of NADP, had no effect on light modulation. Antiserum directed against thioredoxin gave variable results: one batch of polyclonal antibodies inhibited light modulation, another was stimulatory, and another was without effect. These results suggest that the ferredoxin and thioredoxin active in light modulation are not free in solution. Furthermore, molecular sieve chromatography of stromal proteins results in the elution of four species that catalyze light modulation. Based on whether or not ferredoxin and/or thioredoxin must be added for activity, these four species have been tentatively identified as protein modulase, a complex of ferredoxin/thioredoxin reductase and ferredoxin, a complex of ferredoxin/thioredoxin reductase and thioredoxin, and ferredoxin/ thioredoxin reductase. That is, the four correspond to all the possible combinations of ferredoxin, ferredoxin/thioredoxin reductase, and thioredoxin. We suggest that buffer ionic strength affects the interactions among these proteins and in part determines the fate of the protein modulase complex in vitro.The activity of several chloroplastic enzymes is modulated during the dark/light transition. Three different systems (the 'light effect mediator,' the 'ferredoxin/thioredoxin,' and the 'ferralterin' systems) have been proposed to account for this phenomenon. One difference among these is the nature of the stromal factor(s) that putatively shuttle(s) electrons from PSI to the target enzymes (1,8,15).The activity of these stromal factors is assayed in reconstituted chloroplasts (created by mixing washed thylakoids with stromal extracts) by observing the change in activity of a target enzyme.Of the differences among the stromal factors that catalyze light 'Supported by National Science Foundation Grant DBM 84 17081. modulation, the most significant is whether or not thioredoxin and ferredoxin need be included in the assay mixture. Protein modulase and ferralterin, the stromal factors of the LEM' and ferralterin systems, are active in the absence of added Td and Fd. Ferredoxin/thioredoxin reductase activity requires exogenous Td and Fd. We hypothesize that PM, and likely ferralterin, is a complex protein composed of FTR, Fd, and Td. The soluble components of the proposed systems, then, would simply differ in the extent to which this complex has broken down. The results we report here are consistent with this hypothe...

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