Isolation of active and inactive forms of isocitrate dehydrogenase from Escherichia coli ML308

Abstract

1 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]. 2 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⁺. 3 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. 4 The three forms comigrated on denaturing gel electrophoresis and were identical by the criterion of one-dimensional peptide mapping. 5 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. 6 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. 7 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). 8 The nature of the partially active form of isocitrate dehydrogenase and the significance of the results are discussed.