Premium
Purification and characterization of glutathione reductase from corn mesophyll chloroplasts
Author(s) -
Mahan James R.,
Burke John J.
Publication year - 1987
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1987.tb04355.x
Subject(s) - glutathione , biochemistry , glutathione reductase , chemistry , enzyme , chloroplast , enzyme assay , phenylglyoxal , amino acid , glutathione peroxidase , arginine , gene
Differences in the apparent molecular weights of the subunits of glutathione reductase (EC 1.6.4.2) from pea chloroplasts and corn mesophyll chloroplasts have been recently reported. In order to more fully describe the differences between the enzymes from these two sources, glutathione reductase from the mesophyll chloroplasts of corn seedlings ( Zea mays L. cv. G‐4507) has been purified 200‐fold by affinity chromatography using adenosine 2′,5′‐disphosphate agarose. The purified enzyme had a specific activity of 26 μmol NADPH oxidized (mg protein) ‐1 min ‐1 . The native enzyme had a relative molecular weight of 190 ± 30 kDa and exhibited polypeptides of 65, 63, 34, and 32 kDa when separated on sodium dodecylsulfate‐polyacrylamide gels. Comparisons of the results from electroblotting, native molecular weight and subunit molecular weight analyses suggest that the enzyme exists as a heterotetramer. Optimal enzyme activity was obtained at pH 8 in N‐2‐hydroxyethyl‐piperazine‐N′‐2‐ethanesulfonic acid (HEPES‐NaOH) buffer. The sulfhydryl reagent, n ‐ethylmaleimide, inhibited enzymatic activity when incubated in the presence of NADPH while no inhibition was detected with oxidized glutathione in the incubation mixture. Reduced glutathione (5 m M ) inactivated the enzyme by 50%. This inactivation followed first order kinetics with a rate constant of 0.0028 s ‐1 . The enzyme was also inactivated by NADPH. The inactivation reached ca 90% within 30 min and followed first order kinetics with a rate constant of 0.0015 s ‐1 .