Glutathione reductase (EC 1.6.4.2) activity is present in spinach (Spinacia olracea L.) chloroplasts. The pH dependence and substrate concentration for half-maximal rate are reported and a possible role in chloroplasts is proposed.Glutathione reductase (NADPH) glutathione oxidoreductase (EC 1.6.4.2) is a ubiquitous enzyme found in animals, bacteria, fungi, and higher plants (2, 4-7, 10, 13, 14). It is usually a highly specific enzyme that utilizes NADPH to reduce oxidized glutathione (GSSG)3 to 2 molecules of reduced glutathione (GSH).Glutathione reductase can maintain a high ratio of GSH/ GSSG and by this means plays an important-role in the regulation of cell metabolism (12). GSH has been implicated in a large number of reactions including the nonenzymic reduction of thial groups, enzymic reduction of disulfide bridges of protein with consequent deactivation (10) or activation of the protein, and as a substitute for GSH peroxide (12).In higher plants, GSSG reductase has been studied in pea seedlings (6), wheat germ (4), and mitochondria isolated from avocado, mung beans, and peas (14). The mitochondrial preparations were able to couple GSH oxidation with the reduction of dehydroascorbic acid (14). This paper reports the observation of GSSG reductase in spinach chloroplasts. MATERIALS AND METHODSChloroplasts and stroma enzymes were isolated from spinach (Spinacia oleracea L.) leaves by the procedures of Bassham et al. (3). Solution Z was modified by replacement of the GSH buffer with tris (tris[hydroxymethyl]aminomethane). The GSH contained at least 0.5% of GSSG, the substrate of the GSSG reductase reaction. Several buffers were tried and all were found to be satisfactory. The GSSG reductase activity when stored at 40 C was stable for more than 4 hr.A standard reaction mixture of 1 ml at 24 C contained major I This work was supported by the United States Energy Research and Development Administration.2 To whom reprint requests should be sent. RESULTS AND DISCUSSIONThe presence of a GSSG reductase activity in the stroma fralztion was first detected during attempts to determine the stoichiometry between CO2 release and NADP reduction by the glucose-6-P dehydrogenase enzyme found in spinach chloroplast preparations. In controls involving NADPH addition, pyridine nucleotide was rapidly oxidized. Replacement of the GSH buffer with tris eliminated the oxidation of NADPH. This was the result of the presence in commercial GSH of between 0.5 to 1.0% GSSG.The GSSG reductase activity was specific for NADPH and was inhibited by Zn+2 (Table I). These characteristics are similar to those of GSSG reductases from fungi (13), wheat germ (4), peas (6), bacteria (2), and animal tissues (7). The enzyme has a broad pH maximum (Fig. 1) between pH 6.5 and 8.In the crude preparation used, the estimated concentration of the substrate GSSG for half-maximal rate of 0.07 mm (Fig. 2) fell within the range of values reported in the literature (2,7,12). The presence of 50 mm GSH did not measurably affect the rate of GSSG reduction, ...

Photosynthesis in green plants requires not only photoelectron transport, oxygen evolution, and photophosphorylation, but also the synthetic reactions whereby carbon dioxide is assimilated and reduced to a number of organic compounds via the photosynthetic carbon reduction cycle1 and secondary biosynthetic pathways.2 Complete photosynthesis with isolated chloroplasts has for many years been a goal of biochemists. Photosynthetic reactions could then be isolated from reactions of the cytoplasm, and the cell wall could be eliminated as a barrier to the assimilation of various added metabolites and chemicals. This achievement would greatly facilitate the study of the mechanisms of enzymic transformations and metabolic control in the synthetic reactions of photosynthesis.The photochemical transfer of electrons from water to artificial electron acceptors by subcellular particles from green plant cells was demonstrated many years ago by Hill and Scarisbrick.3 Since that time, isolated chloroplasts and chloroplast particles have been found to be capable of photoelectron transport to NADP,4-6 and of photophosphorylation.?While very high rates, exceeding those required for in vivo photosynthesis, have been demonstrated for photoelectron transport to both natural and artificial electron acceptors and for photophosphorylation, rates of carbon reduction during photosynthesis by isolated chloroplasts have been disappointingly low. Fixation of C14-labeled carbon dioxide into intermediate compounds of the photosynthetic carbon reduction cycle by isolated chloroplasts was reported by Allen et al. in 1955.8 This ability to fix CO2 was diminished with broken chloroplasts but could be restored by various cofactors,9 and it was claimed that CO2 fixation by chloroplasts could be accomplished in the dark if the cofactors ATP and NADPH were added.'0 A report from the same laboratory (in 1960) listed fixation rates of 4.5-6 Mumoles C02/mg Chl/hr."1 Others12 found rates of about 4. It has long been assumed, and is verified by experiment in the present report, that spinach leaves should be capable of rates of CO2 fixation exceeding 200 Mmoles C02/mg Chl/hr. Fixation of CO2 has been stimulated by the addition of some intermediates of the carbon reduction cycle to broken chloroplasts,9 or whole chloroplasts.'3 Walker14 found a rate of 1.1 .tmoles C02/mg Chl/hr to be increased to 24.3 (and later,'5 36.9) upon the addition of ribose-5-phosphate to isolated chloroplasts prepared according to his method. With the addition of 71..5 ,moles of ribose-5-phosphate per mg chlorophyll to the reaction mixture, Walker's accelerated CO2 fixation rate resulted in a total fixation of 6.0 Amoles CO2 per mg chlorophyll during the 20-min period of incubation. Thus, the stimulated CO2 fixation rate could result from the operation of only part of the carbon reduction cycle utilizing ATP from the photochemical reactions to convert ribose-5-phosphate to ribulose-1,5-diphosphate, the carboxylation reaction substrate. It would appear that Walker's preparation had a ...

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