Isocitrate dehydrogenase from Rhodopseudomonas spheroides: Purification and characterization

A TPN +-specific isocitrate dehydrogenase has been isolated in an apparent state of homogeneity from the photosynthetic bacterium Rhodopseudomonas spheroides. The purified enzyme has a specific activity of between 40 and 50 enzyme units per milligram protein. Equilibrium centrifugation data indicate that the enzyme has a molecular weight of 105,000. Sedimentation velocity experiments yield an s 020 w value of 5.75. Electrophoresis of the enzyme on acrylamide gels in the presence of sodium dodecyl sulfate yields a major protein component with a molecular weight of approximately 50,000 and a minor component of molecular weight of approximately 100,000. The purified enzyme is stable in phosphate buffers but rapidly loses activity in Tris or imidazole buffers. The loss in activity in Tris buffers is accompanied by a change in sedimentation behavior on sucrose gradients. The Michaelis constants for TPN + and d-isocitrate are 7.0 × 10 −6 and 8.7 × 10 −6 m, respectively, at pH 7.0 in 0.05 m potassium phosphate buffer. Hill plots of the initial velocity data show no evidence for cooperativity. The enzyme, at low magnesium ion concentrations is activated by TPNH but not by TPN +, α-ketoglutarate, isocitrate or DPNH. It has been suggested that the enzyme may exist in two forms which are interconvertible in the presence of magnesium ions. Like other isocitrate dehydrogenases the enzyme is inhibited by sulfhydryl reagents. Titration with p-hydroxymercuribenzoate indicates that 80% of activity is lost after titration of only two or 10 sulfhydryl residues.