Chloroplast Glutathione Reductase 1

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 METHODS Chloroplasts 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. 3 Abbreviations: GSSG: oxidized glutathione; GSH: reduced glutathione; RuDP: ribulose 1,5-diphosphate. reagents at the following concentrations. TrisHCl, 0.05 M (pH 7.5); NADPH, 0.15 mM; GSSG, 0.5 mM; and MgCl. ,, 3 mM. In all cases, enough stroma enzyme preparation (50-100 ,l containing 0.15-0.3 mg protein) was added to achieve a rate of NADPH oxidation between 0.05 and 0.08 AOD units (at 340 nm)/min. Protein was determined by the Lowry method (9) using BSA as a standard. Chlorophyll content of the grana fraction was measured using the procedure of Vernon (11). Ribulose-1,5diphosphate carboxylase was obtained from Calbiochem as a lyophylized powder. RESULTS AND DISCUSSION The 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, in agreement with the observed irreversibility of the over-all enzymic process. Since GSSG reductase activity was previously reported in higher plant tissue extracts (4, 6) and plant mitochondria (14), attempts were made to localize the activity. For this, the isolated intact chloroplasts were washed three times with buffer before lysis. The specific activity of the GSSG reductase activity did not decrease with reference to the protein content, nor with regard to the Chl content of the grana fraction after lysis; in fact, there was a small increase (20%) due perhaps to removal of small amounts of contaminating chloroplast membrane material. This provides strong evidence for the localization of the GSSG reductase within the chloroplast. The chloroplast stroma enzyme preparation was also able to transfer electrons from NADPH in a ferredoxin-dependent reaction (Table II) (1). After approximately 8 min, the amount of 1011 www.plantphysiol. org on May 3, 2019 Published by Downloaded from Copyright © 1977 American Society of Plant Biologists. All rights reserved.