Partial purification and properties of isocitrate dehydrogenase kinase/phosphatase from Escherichia coli ML308.
Isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase were purified over 1000-fold from Escherichia coli ML308 by procedure involving fractionation with (NH4)2SO4 and chromatography on DEAE-cellulose, blue-dextran-Sepharose and Sephadex G150. The kinase and phosphatase activities copurified, in agreement with the observation [Laporte, D.C. and Koshland, D.E. (1982) Nature (Lond.) 300, 458-460] that a single protein bears both activities. Isocitrate dehydrogenase kinase catalysed the phosphorylation of homogeneous active isocitrate dehydrogenase with a stoichiometry of just under one phosphate group incorporated per subunit. This almost completely inactivated the dehydrogenase. There was a good correlation between phosphorylation and inactivation. Analysis of a partial acid hydrolysate of phosphorylated isocitrate dehydrogenase showed that the only phosphoamino acid present was phosphoserine. Isocitrate dehydrogenase phosphatase catalysed the release of 32P from 32P-phosphorylated isocitrate dehydrogenase; it required either ADP or ATP for activity. In the presence of ADP, or ATP plus an inhibitor of the kinase, the phosphatase catalysed full reactivation of isocitrate dehydrogenase and there was a good correlation between reactivation and the release of phosphate. In the presence of ATP alone the phosphatase catalysed the release of 32P from phosphorylated isocitrate dehydrogenase but the activity of the dehydrogenase remained low, indicating that the kinase and phosphatase were active simultaneously in these conditions. The active and inactive forms of isocitrate dehydrogenase can be resolved by non-denaturing gel electrophoresis; the two forms of the enzyme were interconverted by phosphorylation and dephosphorylation in vitro. The extent of the interconversion correlated well with the changes in isocitrate dehydrogenase activity.
D000596:Amino Acids / Q000032:analysis; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D002845:Chromatography / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology*; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification*; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification*; D017346:Protein Serine-Threonine Kinases
D000596:Amino Acids; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; C042301:isocitrate dehydrogenase kinase; C042300:isocitrate dehydrogenase phosphatase; D010749:Phosphoprotein Phosphatases