Purification and some properties of isocitrate dehydrogenase from Paracoccus denitrificans.
NADP-dependent isocitrate dehydrogenase (ICDH) from the bacterium Paracoccus denitrificans was purified to homogeneity. The purification procedure involved ammonium sulphate fractionation, ion exchange chromatography, and gel permeation chromatography. The specific activity of purified ICDH was 801 nkat/mg, the yield of the enzyme 58%. The purity of the enzyme was checked by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. ICDH is a dimer composed of two probably identical subunits of relative molecular weight 90,000. The pH optimum of the enzyme reaction in the direction of substrate oxidation was found to be 5.6; the presence of Mn2+ is essential for enzyme activity. The absorption and fluorescence spectra of the homogeneous enzyme were measured as well.
Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Czech Republic. janiczek@chemi.muni.cz
Oldrich
O
Janiczek
Zdenek
Z
Glatz
Michaela
M
Wimmerová
Jitka
J
Psotová
D000645:Ammonium Sulfate / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007521:Isocitrate Dehydrogenase / Q000737:chemistry* / Q000302:isolation & purification*; D007700:Kinetics; D008345:Manganese / Q000737:chemistry; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010231:Paracoccus denitrificans / Q000201:enzymology*
D001426:Bacterial Proteins; D008345:Manganese; D007521:Isocitrate Dehydrogenase; D000645:Ammonium Sulfate