Purification and characterization of chloroplastic NADP-isocitrate dehydrogenase from Chlamydomonas reinhardtii

Chloroplastic NADP-isocitrate dehydrogenase isoenzyme (NADP-IDH 2 ; EC 1.1.1.42) from the eukaryotic microalga Chlamydomonas reinhardtii was purified to electrophoretic homogeneity by a procedure which included affinity chromatography on Red-Sepharose as the key step. The 70-kDa isoenzyme was found to be a dimer formed by 40-kDa subunits. Antibodies raised against a recombinant tobacco cytosolic NADP-IDH cross-reacted strongly with the cytosolic NADP-IDH 1 and weakly with the NADP-IDH 2 isoenzyme from this alga. NADPH and GTP were found to inhibit both isoenzymes, whereas intermediates of the tricarboxylic acid cycle, glycolysis or reductive pentose phosphate cycle had no significant effect. The simultaneous presence of isocitrate and Mn 2+ protected NADP-IDH 2 against thermal inactivation or inhibition by reagents specific for arginine or lysine.