Purification and characterization of NADP+-linked isocitrate dehydrogenase from an alkalophilic Bacillus.
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.
Shikata
S
S
Tochigi Research Laboratories, Kao Corporation, Japan.
Ozaki
K
K
Kawai
S
S
Ito
S
S
Okamoto
K
K
D001407:Bacillus / Q000201:enzymology*; D002458:Cell Fractionation; D002850:Chromatography, Gel; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007521:Isocitrate Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008970:Molecular Weight; D011685:Purine Nucleotides / Q000494:pharmacology; D013379:Substrate Specificity; D014227:Triazines; D014461:Ultracentrifugation
D011685:Purine Nucleotides; D014227:Triazines; D008345:Manganese; C038830:DEAE affigel blue; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+)