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A rapid method is described for the resolution of proteins employing isoelectric focusing in a vertical polyacrylamide minigel system. Isoelectric focusing can be performed in only 3 h, utilizing low voltage, under either native conditions or denaturing conditions in the presence of 8 M urea. The procedure permits the application of larger sample volumes(More)
Phosphorylated NADP+-isocitrate dehydrogenase (EC has been purified to electrophoretic homogeneity from in vivo 32P-labeled Escherichia coli. The cells used as the source of phosphorylated enzyme were harvested 1 h after the addition of 5 mCi of [32P]orthophosphoric acid and 25 mM sodium acetate to cultures grown to early stationary phase on a low(More)
Addition of acetate to a stationary phase culture of Escherichia coli in glycerol mineral salts medium containing phosphorus-32-labeled orthophosphate results in rapid loss of isocitrate dehydrogenase activity and concomitant incorporation of phosphorus-32 into the enzyme. This is the first example of protein phosphorylation in a bacterium in which the(More)
Affinity chromatography on Affi-Gel Blue has been used to purify the NADP-specific isocitrate dehydrogenase (EC from Escherichia coli. The protocol permits rapid purification of the enzyme in milligram quantities with a yield of 50% and is carried out almost entirely at room temperature. The preparation was judged to be homogeneous by(More)
The specific activities of the nicotinamide adenine dinucleotide phosphate-dependent isocitrate dehydrogenase in crude cell-free extracts of 15 different microorganisms, grown aerobically in simple mineral salts media containing glucose as the sole carbon source, ranged from a maximum of 0.820 in Pseudomonas aeruginosa to a minimum of 0.145 in Thiobacillus(More)
This report describes the in vivo phosphorylation of isocitrate lyase and examines the possible consequences to the control of the Kreb's cycle and glyoxylate bypass. NADP-specific isocitrate dehydrogenase from E. coli was the first bacterial protein whose enzymic activity was shown to be modulated by reversible phosphorylation. This enzyme has been thought(More)
Escherichia coli isocitrate lyase (EC can be phosphorylated in vitro by an ATP-dependent reaction. The enzyme becomes phosphorylated by an endogenous kinase when partially purified sonic extracts are incubated with [gamma-32P]ATP. Treatment of isocitrate lyase with diethyl pyrocarbonate, a histidine-modifying reagent, blocked incorporation of(More)