title: Purification and characterization of isocitrate dehydrogenase from a hyperthermophilic archaebacterium, Caldococcus noboribetus.
abstract: Isocitrate dehydrogenase from a hyperthermophilic archaebacterium Caldococcus noboribetus produced in Escherichia coli was purified. The purification was performed by heat treatment at 80 degrees C followed by single column chromatography. N-terminal amino acid sequencing analysis revealed that the N-terminal methionine is removed from the purified enzyme. Gel filtration analysis suggests that the enzyme has a homodimeric structure with a molecular weight of 90,000. The isoelectric point of the enzyme was estimated to be 5.6 by isoelectric focusing electrophoresis. The circular dichroism spectrum suggests that the enzyme has a secondary structure consisting of 23% alpha-helix and 34% beta-sheet. Enzymatic activity was observed under neutral pH, and the highest specific activity was obtained using cacodylic acid-KOH (pH 7.0) buffer. MgCl2 or MnCl2 was essential for the activity, and KCl concentrations higher than 0.33 M had an inhibitory effect on it. Apparent Km values were 72 and 43 microM for D,L-isocitrate and NADP, respectively. The enzyme showed extremely high stability against heat treatment, and no activity loss was observed by the treatment at 80 degrees C. The specific activity of the enzyme increased as temperature rose. Nearly no activity was observed at 40 degrees C or lower.
authors.affiliation: Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, Hachioji, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Aoshima
authors.forename: T
authors.initials: T
authors.lastname: Oshima
mesh_terms: D001105:Archaea / Q000201:enzymology*; D002384:Catalysis; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D007521:Isocitrate Dehydrogenase / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D008970:Molecular Weight
chemical_list: D007521:Isocitrate Dehydrogenase