Purification and Characterization of NADP+-linked Isocitrate Dehydrogenase From an Alkalophilic Bacillus

Biochim Biophys Acta. 1988 Feb 10;952(3):282-9. doi: 10.1016/0167-4838(88)90128-8.

Abstract

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 90,000; and that by electrophoresis on SDS-polyacrylamide gels was about 44,000. The sedimentation coefficient (s020,w) and isoelectric point of the enzyme were determined to be 3.22 S and pH 4.7, respectively. The enzyme required Mn2+ for the reaction and for stability. The optimum pH for the reaction was in the range 7.8-8.4 at 30 degrees C; the optimum temperature at pH 8.0 was 75 degrees C; the activation energy of the reaction was 6.2 kcal/mol. The Km values for threo-Ds-isocitrate, DL-isocitrate, and NADP+ were 5.4 microM, 9.9 microM, and 7.3 microM, respectively. This enzyme was inhibited by NADPH, glyceraldehyde 3-phosphate, 3-phosphoglycerate, phosphoenol pyruvate, cis-aconitate, alpha-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.

MeSH terms

  • Bacillus / enzymology*
  • Cell Fractionation
  • Chromatography, Gel
  • Hot Temperature
  • Hydrogen-Ion Concentration
  • Isocitrate Dehydrogenase / antagonists & inhibitors
  • Isocitrate Dehydrogenase / isolation & purification*
  • Isocitrate Dehydrogenase / metabolism
  • Manganese / metabolism
  • Molecular Weight
  • Purine Nucleotides / pharmacology
  • Substrate Specificity
  • Triazines
  • Ultracentrifugation

Substances

  • Purine Nucleotides
  • Triazines
  • Manganese
  • DEAE affigel blue
  • Isocitrate Dehydrogenase
  • isocitrate dehydrogenase (NADP+)