Purification of Multiple Forms of Glutathione Reductase from Pea (Pisum sativum L.) Seedlings and Enzyme Levels in Ozone-Fumigated Pea Leaves

Abstract

Glutathione reductase was purified from pea seedlings using a procedure that included 2',5'-ADP Sepharose, fast protein liquid chromatography (FPLC)-anion exchange, and FPLC-hydrophobic interaction chromatography. The purified glutathione reductase was resolved into six isoforms by chromatofocusing. The isoform eluting with an isoelectric point of 4.9 accounted for 18% of the total activity. The five isoforms with isoelectric points between 4.1 and 4.8 accounted for 82% of the activity. Purified glutathione reductase from isolated, intact chloroplasts also resolved into six isoforms after chromatofocusing. The isoform eluting at pH 4.9 constituted a minor fraction of the total activity. By comparing the chro.natofocusing profile of the seedling extract with that of the chloroplast extract, we inferred that the least acidic isoform was extraplastidic and that the five isoforms eluting from pH 4.1 to 4.8 were plastidic. Both the plastidic (five isoforms were pooled) and extraplastidic glutathione reductases had a native molecular mass of 114 kD. The plastidic glutathione reductase is a homodimer with a subunit molecular mass of 55 kD. Both glutathione reductases had optimum activity at pH 7.8. The Km for the oxidized form of glutathione (GSSG) was 56.0 and 33.8 gm for plastidic and extraplastidic glutathione reductase, respectively, at 25°C. The Km for NADPH was 4.8 and 4.0 gm for plastidic and extraplastidic isoforms, respectively. Antiserum raised against the plastidic glutathione reductase recognized a 55-kD polypeptide from purified antigen on western blots. In addition to the 55-kD polypeptide, another 36-kD polypeptide appeared on western blots of leaf crude extracts and the purified extraplastidic isoform. The lower molecular mass polypeptide might represent GSSG-independent enzyme activity observed on activity-staining gels of crude extracts or a protein that has an epitope similar to that in glutathione reductase. Fumigation with 75 nL L1 ozone for 4 h on 2 consecutive days had no significant effect on glutathione reductase activity in peas (Pisum sativum L.). However, immunoblotting showed a greater level of glutathione reductase protein in extracts from ozonefumigated plants compared with that in control plants at the time when the target concentration was first reached, approximately 40 min from the start of the fumigation, and 4 h on the first day of fumigation.

Cloning, Sequencing, and Regulation of the Glutathione Reductase Gene from the Cyanobacterium Anabaena PCC 7120

“…For instance, GR isoenzymes seem to be products of a multigene family in soybean root nodule (39) and in red spruce (40). In contrast, the multiple forms of GR identified in other photosynthetic organisms such as pea (16,41) and Arabidopsis thaliana (17) indicate the existence of post-translational regulatory mechanisms, as only a single-copy gor gene has been detected. The Anabaena PCC 7120 gor gene examined here, as well as those from E. coli and P. aeroginosa, are also likely to be single-copy genes.…”

Amelioration of Ozone-Induced Oxidative Damage in Wheat Plants Grown under High Carbon Dioxide (Role of Antioxidant Enzymes)

“…Electrophoretic separation was performed at 4°C for 4 h with a constant current of 35 mA per gel. After completion of electrophoresis the gels were stained for the activities of SOD, POD, and GR following the procedures of Misra and Fridovich (1977), Castillo et al (1984), and Madamanchi et al (1992), respectively. Native gels were stained for SOD activity by incubation in a solution containing 2.5 mM nitroblue tetrazolium for 25 min, followed by incubation in 50 mM potassium phosphate buffer (pH 7.8) containing 28 p~ riboflavin and 28 mM tetramethyl ethylene diamine for 20 min in the dark.…”

Oxidative Stress and Acclimation Mechanisms in Plants

“…There are two known genes encoding GR (Madamanchi et al 1992;Creissen et al 1995a;Creissen et al 1995b). The redox state of the GSH/GSSG couple may act as a direct link between environmental cues and crucial molecular adaptive responses of plant cells (Hausladen and Alscher 1993;Broadbent et al 1995;Roxas et al 1997;May et al 1998a;May et al 1998b;Baginsky et al 1999).…”