Purification and characterization of NAD-isocitrate dehydrogenase from chlamydomonas reinhardtii.

NAD-isocitrate dehydrogenase (NAD-IDH) from the eukaryotic microalga Chlamydomonas reinhardtii was purified to electrophoretic homogeneity by successive chromatography steps on Phenyl-Sepharose, Blue-Sepharose, diethylaminoethyl-Sephacel, and Sephacryl S-300 (all Pharmacia Biotech). The 320-kD enzyme was found to be an octamer composed of 45-kD subunits. The presence of isocitrate plus Mn2+ protected the enzyme against thermal inactivation or inhibition by specific reagents for arginine or lysine. NADH was a competitive inhibitor (Ki, 0.14 mM) and NADPH was a noncompetitive inhibitor (Ki, 0.42 mM) with respect to NAD+. Citrate and adenine nucleotides at concentrations less than 1 mM had no effect on the activity, but 10 mM citrate, ATP, or ADP had an inhibitory effect. In addition, NAD-IDH was inhibited by inorganic monovalent anions, but L-amino acids and intermediates of glycolysis and the tricarboxylic acid cycle had no significant effect. These data support the idea that NAD-IDH from photosynthetic organisms may be a key regulatory enzyme within the tricarboxylic acid cycle.