Isolation of active and inactive forms of isocitrate dehydrogenase from Escherichia coli ML 308.
In Escherichia coli ML308 isocitrate dehydrogenase is partially inactivated during growth on acetate [Bennett, P.M. and Holms, W.H. (1975) J. Gen. Microbiol. 87, 37-51]. The active form of isocitrate dehydrogenase was purified to homogeneity from cells grown on glycerol. The key step in the procedure was chromatography on procion-red-Sepharose, from which the enzyme was specifically eluted with NADP+. Two forms of isocitrate dehydrogenase were purified to homogeneity from cells grown on acetate. One form did not bind to procion-red-Sepharose and was essentially inactive; this form could be resolved from the active form by non-denaturing gel electrophoresis. The other form was specifically eluted from procion-red-Sepharose and was partially active; analysis of this form by non-denaturing gel electrophoresis suggested that it was a mixture of the active and inactive forms. The three forms comigrated on denaturing gel electrophoresis and were identical by the criterion of one-dimensional peptide mapping. Analysis of the active and inactive forms by sedimentation equilibrium centrifugation and non-denaturing gel electrophoresis showed that they differed in charge but not in size. Amino acid analysis and two-dimensional peptide mapping showed that both forms were dimers of identical subunits. The active form of the enzyme contained no detectable alkali-labile phosphate, the inactive form contained 0.8 molecule/subunit and the partially active form contained an intermediate amount. The data suggest that the active and inactive forms of isocitrate dehydrogenase differ only in the presence of one phosphate group per subunit in the latter form; this is consistent with our results from phosphorylation of isocitrate dehydrogenase in vitro (Following paper in this journal). The nature of the partially active form of isocitrate dehydrogenase and the significance of the results are discussed.
D000085:Acetates; D000596:Amino Acids / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D002627:Chemistry, Physical; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D005990:Glycerol; D007521:Isocitrate Dehydrogenase / Q000145:classification / Q000302:isolation & purification*; D008970:Molecular Weight; D010710:Phosphates
D000085:Acetates; D000596:Amino Acids; D003470:Culture Media; D010710:Phosphates; D007521:Isocitrate Dehydrogenase; D005990:Glycerol