Purification and characterization of glutathione reductase from Chlamydomonas reinhardtii

Abstract

Glutathione reductase was purified to homogeneity from the unicellular alga Chlamydomonas veinhavdtii. The enzyme was a monomer with a motecular mass of 54-56 kDa as judged by gel filtration and SDS-PAGE. The activity was maximal at pH 8.2 and 49The enzyme was specific for NADPH, but not for NADH. The reverse reaction with NAD(P)' and GSH (glutathione, reduced form) was not observed in the pH range 443-8.2. K, values for NADPH and GSSG (glutathione, oxidized form) were 10.6 VM and 54-1 p~, respectively. Thiol inhibitors and metal ions such as Hg2+ and Cu'' markedly inhibited the enzyme activity. Activity was lost when the apoenzyme was prepared by dialysis, but was restored to 40% of the original activity by the addition of 50 ~M -F A D .The enzyme reaction proceeded via a branching mechanism. Upon immunoprecipitation, glutathione reductase activity of C. reirtihardtii was inhibited 50 YO and 90 % by antibodies generated against spinach and Euglena giutathione reductases, respectively. Both antibodies cross-reacted with C. reinhardtii glutathione reductase in an immunoblot analysis.

The catalase-peroxidase of Synechococcus PCC 7942: purification, nucleotide sequence analysis and expression in Escherichia coli

“…A double staining for catalase-peroxidase was employed using 3,3hdiaminobenzidine, followed by the ferricyanide negative stain to reveal bands in the polyacrylamide gel by the method of Wayne and Diaz [12]. SDS\PAGE was performed on 12.5 % (w\v) polyacrylamide slab gels as previously described [13]. Proteins in the gel were stained with silver-staining reagent.…”

“…The molecular mass of native catalase-peroxidase was determined by gel filtration on a Sephacryl S-300 column (2.4 cmi90 cm) equilibrated with 10 mM potassium phosphate buffer, pH 7.0, and calibrated with molecular mass markers (MW-GF-1000) from Sigma. In determining the molecular mass of a subunit by SDS\PAGE [13], LMW kit E from Pharmacia was used for standards.…”

Enzymic and molecular characterization of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Synechococcus PCC 7942: resistance of the enzyme to hydrogen peroxide

“…Electrophoresis was carried out at a constant current (2 mA\gel) with Bromophenol Blue as the migration marker. SDS\PAGE was performed on a 12.5 % (w\v) polyacrylamide slab gel as described previously [9]. Proteins in the gel were stained with Coomassie Brilliant Blue R-250 and destained in 7 % acetic acid.…”

“…The molecular mass of GAPDH was estimated by using a Superdex 200 HiLoad 16\60 column (2.4 cmi90 cm) equilibrated with 50 mM potassium phosphate buffer, pH 7.5, containing 2.5 mM DTT, 1 mM GSH, 10 % sucrose and 0.15 M NaCl, and calibrated with Blue Dextran (2000 kDa), bovine thyroglobulin (66.9 kDa), apoferritin from horse spleen (44.3 kDa), β-amylase from sweet potato (20 kDa), alcohol dehydrogenase from yeast (15 kDa), BSA (67 kDa) and carbonic anhydrase from bovine erythrocytes (30 kDa). For the determination of the molecular mass of a subunit by SDS\PAGE [9], phosphorylase b from rabbit muscle (94 kDa), BSA (67 kDa), ovalbumin from egg white (43 kDa), carbonic anhydrase from bovine erythrocytes (30 kDa), trypsin inhibitor from soybean (20.1 kDa) and αlactalbumin from bovine milk (14.4 kDa) were used as standards.…”

Gene cloning and expression analysis of mitochondrial glutathione reductase from Arabian camel (Camelus dromedarius) liver in Escherichia coli

“…GR isoforms from both prokaryotes and eukaryotes form stable homodimers of ~110 kDa with a large subunit interface of more than 3000 Å 2 . GR has been purified from multiple sources, e.g., calf livers (14), rodent livers (15), human red blood cells (16), sheep brains (17), sheep livers (18), turkey livers (19), and Chlamydomonas reinhardtii (20), and its characteristic properties, such as its thermostability, optimal pH, and optimal temperature, have been characterized. Affinity chromatography, ion exchange chromatography, hydrophobic and reversedphase chromatography, and size exclusion chromatography have been applied in order to purify the enzyme (21)(22)(23)(24).…”