NAD+-LINKED ISOCITRATE DEHYDROGENASE - ISOLATION, PURIFICATION, AND CHARACTERIZATION OF THE PROTEIN FROM PEA MITOCHONDRIA

The NAD+-dependent isocitrate dehydrogenase from etiolated pea (Pisum sativum L.) mitochondria was purified more than 200-fold by dye-ligand binding on Matrix Gel Blue A and gel filtration on Superose 6. The enzyme was stabilized during purification by the inclusion of 20% glycerol. In crude matrix extracts, the enzyme activity eluted from Superose 6 with apparent molecular masses of 1400 +/- 200, 690 +/- 90, and 300 +/- 50 kD. During subsequent purification steps the larger molecular mass species disappeared and an additional peak at 94 +/- 16 kD was evident. The monomer for the enzyme was tentatively identified at 47 kD by sodium dodecyl-polyacrylamide gel electrophoresis. The NADP+-specific isocitrate dehydrogenase activity from mitochondria eluted from Superose 6 at 80 +/- 10 kD. About half of the NAD+ and NADP+-specific enzymes remained bound to the mitochondrial membranes and was not removed by washing. The NAD+-dependent isocitrate dehydrogenase showed sigmoidal kinetics in response to isocitrate (S0.5 = 0.3 mM). When the enzyme was aged at 4-degrees-C or frozen, the isocitrate response showed less allosterism, but this was partially reversed by the addition of citrate to the reaction medium. The NAD+ isocitrate dehydrogenase showed standard Michaelis-Menten kinetics toward NAD+ (K(m) = 0.2 mM). NADH was a competitive inhibitor (K(i) = 0.2 mM) and, unexpectedly, NADPH was a noncompetitive inhibitor (K(i) = 0.3 mM). The regulation by NADPH may provide a mechanism for coordination of pyridine nucleotide pools in the mitochondria.