PURIFICATION AND CHARACTERIZATION OF GLUTATHIONE-REDUCTASE FROM CHLAMYDOMONAS-REINHARDTII

Glutathione reductase was purified to homogeneity from the unicellular alga Chlamydomonas reinhardtii. The enzyme was a monomer with a molecular mass of 54-56 kDa as judged by gel filtration and SDS-PAGE. The activity was maximal at pH 8.2 and 49-degrees-C. The 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 4.8-8.2. K(m) values for NADPH and GSSG (glutathione, oxidized form) were 10.6 muM and 54.1 muM, respectively. Thiol inhibitors and metal ions such as Hg2+ and Cu2+ 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 muM-FAD. The enzyme reaction proceeded via a branching mechanism. Upon immunoprecipitation, glutathione reductase activity of C. reinhardtii was inhibited 50% and 90% by antibodies generated against spinach and Euglena glutathione reductases, respectively. Both antibodies cross-reacted with C. reinhardtii glutathione reductase in an immunoblot analysis.