A comparison of the phosphorylated and unphosphorylated forms of isocitrate dehydrogenase from Escherichia coli ML308

AbstractNADP+ can protect active isocitrate dehydrogenase against attack by several proteases. Inactive phosphorylated isocitrate dehydrogenase is much less susceptible to proteolysis than the active enzyme, and it is not protected by NADP+. The results suggest that binding of NADP+ to, or phosphorylation of, active isocitrate dehydrogenase induces similar conformational states. Fluorescence titration experiments show that NADPH can bind to active but not to inactive isocitrate dehydrogenase. It is suggested that the phosphorylation of isocitrate dehydrogenase may occur close to its coenzyme binding site

A general method for the localization of enzymes that produce phosphate, pyrophosphate, or CO2 after polyacrylamide gel electrophoresis

Previous workers have stained gels for enzymes that produce inorganic phosphate by using the insolubility of calcium phosphate. This method can also be applied to enzymes that produce pyrophosphate or CO2. The white bands of the precipitated calcium salt are clearly visible when viewed against a dark background and can be photographed or scanned. The method can be used at pH 6 and above; the level of Ca2+ required is reduced at higher pH values. The sensitivity of the method is tested by injecting the various anions into presoaked gels; as little as 10 nmol of phosphate or pyrophosphate and 100 nmol of CO2 produce clearly visible precipitates

Control of Escherichia coli isocitrate dehydrogenase: an example of protein phosphorylation in a prokaryote

In micro-organisms growing on acetate, isocitrate can be metabolized either by the tricarboxylic acid cycle or by the glyoxylate bypass. In Escherichia coli this branch-point is controlled by reversible phosphorylation and inactivation of isocitrate dehydrogenase. Both phosphorylation and dephosphorylation are catalysed by a single bifunctional kinase/phosphatase. The properties of this enzyme suggest that phosphorylation of isocitrate dehydrogenase is controlled by the concentrations of several central metabolites, including isocitrate, the adenine nucleotides and phoenolpyruvate

How to tell the time: the regulation of phosphoenolpyruvate carboxylase in Crassulacean acid metabolism (CAM) plants

Crassulacean acid metabolism (CAM) plants exhibit persistent circadian rhythms of CO2 metabolism. These rhythms are driven by changes in the flux through phosphoenolpyruvate carboxylase, which is regulated by reversible phosphorylation in response to a circadian oscillator. This article reviews progress in our understanding of the circadian expression of phosphoenolpyruvate carboxylase kinase

The purification of isocitrate dehydrogenase from Escherichia coli using immobilized dyes

No abstract available

Reversible inactivation of isocitrate dehydrogenase in Escherichia coli

No abstract available

Purification and some kinetic properties of rat liver ATP citrate lyase

A new purification procedure for rat liver ATP citrate lyase is described. The method reproducibly gives homogenous undegraded enzyme. Steady-state kinetic analysis of ATP citrate lyase was complicated by the presence of ADP, a product of the reaction, in solutions of ATP. The kinetic patterns observed were dependent on whether ADP was removed by the assay system. When assays were performed with a method in which ADP was removed, the results showed that the enzyme obeys a double-displacement mechanism with a phosphoenzyme intermediate. This resolves a controversy between the results of previous kinetic studies and those of isotope-exchange and enzyme-labelling experiments

[54] Fructose-bisphosphatase from ox liver

This chapter describes the purification and properties of the enzyme fructose-bisphosphatase from ox liver. The enzyme is assayed by coupling the production of fructose 6-phosphate to the reduction of NADP+ in the presence of excess glucosephosphate isomerase and glucose-6-phosphate dehydrogenase. The purification reaction involves extraction, pH treatment, ammonium sulfate fractionation, gradient elution from carboxymethyl (CM)-cellulose, substrate elution from CM-cellulose, and gel filtration on Sephadex G-200. The enzyme shows only one band on sodium dodecyl sulfate (SDS)-polyacrylamide gels and its native molecular weight—as judged by gel filtration—is not altered by the purification, indicating that the purified enzyme is undegraded. The enzyme contains one residue of tryptophan per subunit as judged spectrophotometrically and by amino acid analysis after hydrolysis with methanesulfonic acid. Ox liver fructose-bisphosphatase is stimulated at neutral pH by ethylenediaminetetraacetic acid (EDTA) and other chelating agents; however, EDTA does not bind directly to the enzyme

Diurnal changes in the properties of phosphoenolpyruvate carboxylase in Bryophyllum leaves: a possible co valent modification

In plants that show Crassulacean acid metabolism, phosphoenolpyruvate carboxylase catalyses the key step of CO2 fixation at night. We show here that the properties of this enzyme from Bryophyllum fedtschenkoi undergo marked changes between night and day; the night form is much less sensitive to feedback inhibition by malate than is the day form. Incubation of leaves with 32Pi followed by extraction and immunoprecipitation of phosphoenolpyruvate carboxylase showed that only the night form contained 32P. This suggests that the activity of the enzyme is controlled by a covalent modification mechanism

Persistent circadian rhythms in the phosphorylation state of phosphoenolpyruvate carboxylase from Bryophyllum fedtschenkoi leaves and in its sensitivity to inhibition by malate

Phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPCase) from Bryophyllum fedtschenkoi leaves has previously been shown to exist in two forms in vivo. During the night the enzyme is phosphorylated and relatively insensitive to feedback inhibition by malate whereas during the day the enzyme is dephosphorylated and more sensitive to inhibition by malate. These properties of PEPCase have now been investigated in leaves maintained under constant conditions of temperature and lighting. When leaves were maintained in continuous darkness and CO2-free air at 15°C, PEPCase exhibited a persistent circadian rhythm of interconversion between the two forms. There was a good correlation between periods during which the leaves were fixing respiratory CO2 and periods during which PEPCase was in the form normally observed at night. When leaves were maintained in continuous light and normal air at 15°C, starting at the end of a night or the end of a day, a circadian rhythm of net uptake of CO2 was observed. Only when these constant conditions were applied at the end of a day was a circadian rhythm of interconversions between the two forms of PEPCase observed and the rhythms of enzyme interconversion and CO2 uptake did not correlate in phase or period