Biochemical and molecular characterisation of wheat chloroplastic glutathione reductase

Wheat leaves contain two charge/mass-separable isoforms of glutathione redu ctase (GR, EC 1.6.4.2), one chloroplastic and the other probably cytosolic. The chloroplastic GR was purified to homogeneity, and its biochemical and molecular characterisation showed features very similar to the other plant GRs. In its native conformation the enzyme is composed by two subunits of 5 6 kDa and an associated polypeptide of 32 kDa, with an overall molecular ma ss of approximately 150 kDa. Optimum activity was observed at pH 8.00 and w ith an ionic strength between 60 to 100 mM. GR activity is highly sensitive to temperature changes, exhibiting an exponential increase up to 45 degree sC. It showed a high affinity for oxidised glutathione and an intermediate affinity for NADPH at pH 8.0. Inhibition tests with thiol and histidine mod ifiers demonstrated that -SH groups and histidine residues are essential fo r the catalytic properties of the enzyme. To study the origin of GR isoform s, the number of GR gene copies and the number and size of GR transcripts w ere determined. Southern analyses showed that wheat GR isoforms are encoded by multiple gene copies. However, a single size transcript of approximatel y 1.4 kb was observed, suggesting that different GR isoforms could be gener ated by posttranscriptional and/or post-translational modifications.