Purification and characterization of NADP+-linked isocitrate dehydrogenase from an alkalophilic Bacillus (original) (raw)

1988, Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology

We have succeeded in purifying to homogeneity a very labile NADP +-linked isocitrate dehydrogenase (isocitrate: NADP + oxidoreductase (decarboxylating), EC 1.1.1.42) from a strain of alkalophilic Bacillus, by a simple method, with an overall yield over 76% of the original activity. The molecular weight on Sephadex G-200 was around 90000; and that by electrophoresis on SDS-polyacrylamide gels was about 44 000. The sedimentation coefficient 0 (sz0,w) and isoelectric point of the enzyme were determined to be 3.22 S and pH 4.7, respectively. The enzyme required Mn 2+ for the reaction and for stability. The optimum pH for the reaction was in the range 7.8-8.4 at 30 o C; the optimum temperature at pH 8.0 was 75 o C; the activation energy of the reaction was 6.2 kcal/mol. The K m values for threo-Ds-isocitrate, DL-isocitrate, and NADP + were 5.4 #M, 9.9 pM, and 7.3 pM, respectively. This enzyme was inhibited by NADPH, glyceraldehyde 3-phosphate, 3-phosphoglycerate, phosphoenolpyruvate, c/s-aconitate, a-ketoglutarate, and oxaloacetate. In addition, it was subject to a concerted inhibition by a combination of glyoxylate and oxaloacetate, and also to a cumulative inhibition by nucleoside triphosphates.