Regulation of phosphofructokinase by a new mechanism. An activation factor binding to phosphorylated enzyme (original) (raw)
Related papers
An activation factor of liver phosphofructokinase
Proceedings of the National Academy of Sciences, 1980
Pure phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) from liver is strongly inhibited by ATP, whereas crude phosphofructokinase is only slightly inhibited by ATP. A factor that is removed from the enzyme during purification and can prevent the inhibition of phosphofructokinase by ATP has been isolated. The factor can be resolved into three components that differ in molecular weights, as shown by gel filtration on Sephadex G-25. These factors overcome the ATP inhibition but have no effect on the catalytic activity under the optimum assay conditions. Furthermore, AMP acts syngeristically with the activation factor in reversing ATP inhibition. It is proposed that the activation of phosphofructokinase by the activation factor and AMP is sufficient to account for the glycolytic flux in the liver.
Significance of phosphorylation of phosphofructokinase
Journal of Biological Chemistry
In order to understand the effect of phosphorylation on phosphofructokinase, the allosteric kinetic behavior, ligand binding at various pHs, and pH-dependent cold inactivation of phosphofructokinase phosphorylated to different extents were studied.
Journal of Biological Chemistry
Low phosphate and high phosphate forms of phosphofructokinase (Furuya, E., and Uyeda, K. (1980) J. Biol. Chem. 255,[11656][11657][11658][11659] from rat liver were purified to homogeneity and various properties were compared. The specific activities of these enzymes and their electrophoretic mobilities on polyacrylamide in sodium dodecyl sulfate are the same. A limited tryptic digestion yields products with no change in the enzyme activity but with a reduction in the molecular weight of about 2000. Both low and high phosphate enzymes can be phosphorylated by the catalytic subunit of CAMP-dependent protein kinase, and approximately twice as much [32P]phosphate is incorporated into the low phosphate than the high phosphate enzyme.
A radioassay for phosphofructokinase-1 activity in cell extracts and purified enzyme
Journal of Biochemical and Biophysical Methods, 2002
Phosphofructokinase-1 plays a key role in the regulation of carbohydrate metabolism. Its activity can be used as an indicator of the glycolytic flux in a tissue sample. The method most commonly employed to determine phosphofructokinase-1 activity is based on oxidation of NADH by the use of aldolase, triosephosphate isomerase, and a-glycerophosphate dehydrogenase. This method suffers from several disadvantages, including interactions of the auxiliary enzymes with phosphofructokinase-1. Other methods that have been used also require auxiliary enzymes or are less sensitive than a coupled assay. Here, we propose a direct method to determine phosphofructokinase-1 activity, without the use of auxiliary enzymes. This method employs fructose-6-phos-32 Žw 32 x . phate and ATP labeled with P in the gamma position g-P ATP , and leads to the formation 32 Žw 32 x . of ADP and fructose-1,6-bisphosphate labeled with P 1-P fructose-1,6-bisphosphate . Actiw 32 x vated charcoal is used to adsorb unreacted g-P ATP, and the radioactive product in the w 32 x supernatant, 1-P fructose-1,6-bisphosphate, is analyzed on a liquid scintillation counter. The proposed method is precise and relatively inexpensive, and can be applied to determine phosphofructokinase-1 activity in cellular extracts as well as in the purified enzyme. q M. Sola-Penna . 0165-022Xr02r$ -see front matter q 2002 Elsevier Science B.V. All rights reserved.
Chicken liver phosphofructokinase. III. Kinetics and allosteric properties
Journal of Biological Chemistry
Some kinetic and allosteric properties of crystalline chicken liver phosphofructokinase were investigated. Lineweaver-Burk plots with fructose 6.phosphate and ATP as substrates yield a series of apparently parallel lines. The liver enzyme also catalyzes the phosphorylation of fructose-l-P, and the double reciprocal plot with fructose-l-P as the substrate yields intersecting lines. Phosphorylation of both fructose-6-P and fructose-l-P are lost at 4" at equal rate. Of the many sugar phosphates examined, only glucose-6-P, 6-P.
Kinetic characterization of phosphofructokinase isolated from rat kidney cortex
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1991
Phosphofructokinase from rat kidney cortex has been purified by affinity chromatography to a final specific activity of 15 units per mg of protein, measured at 25°C and pH 8. 2. This lower spec. act., compared with that of the enzyme from other sources, shows the enzyme in proximal tubules to be less active, which would account for the main gluconeogenic role of these nephron sections. 3. The binding of fructose-6-phosphate to the enzyme is cooperative. ATP increases the Hill coefficient and produces a marked allosteric inhibition on the activity. 4. Fructose-2,6-b/s-phosphate is a potent activator of the enzyme from this source. It reduces the Hill coefficient of the enzyme and the inhibition constant of ATP. A marked difference between this and the liver enzyme is that the activation is not cooperative .
Biochimica et Biophysica Acta (BBA) - Enzymology, 1972
Yeast phosphofructokinase (ATP :D-fructose-6-phosphate I-phosphotransferase, EC 2.7.1.11) desensitized to ATP inhibition by the method of Affing et al. ((1971) Arch. Biochem. Biophys. 143 , 587) exhibits normal Michaelis kinetics as judged by a double reciprocal plot or by a Hill plot with an apparent Km of 0.095 mM for fructose 6-phosphate which is about three times less than the minimum value observed for the untreated enzyme. The desensitized phosphofructokinase is neither inhibited tc any great extent by citrate, nor activated by AMP. However, the desensitized phosphofructokinase is still activated by NHa+. The desensitized phosphofructokinase can be reconverted to the ATP-sensitive phosphofructokinase by preincubating the enzyme at 28 °C with 3 mM ATP, IO mM citrate, or both at pH 6.8 or 7.5-These results may be interpreted in terms of the model for allosteric enzymes proposed by Monod et al. ((1965) J. Mol. Biol. 12, 88) where ATP-sensitive phosphofructokinase which predominates in the absence of ligands is converted to the desensitized phosphofructokinase form in a synergistic manner by preincubation of the enzyme with the positive effectors and salts of fluoride and magnesium.
Journal of Biological Chemistry
The effect of natural "activation factor" and synthetic fructose-2,6-Pz on the allosteric kinetic properties of liver and muscle phosphofructokinases was investigated. Both synthetic and natural fructose-2,6-Pz show identical effects on the allosteric kinetic properties of both enzymes. Auctose-2,6-Pz counteracts inhibition by ATP and citrate and decreases the K,,, for fructose-6-P. This fructose ester also acts synergistically with AMP in releasing ATP inhibition. The K,,, values of liver and muscle phosphofructokinase for fructose-2,6-Pz in the presence of 1.26 IMI ATP are 12 milliunits/ml (or 24 l ũ r ) and 5 milliunits/ml (or 10 m), respectively. At near physiological concentrations of ATP (3 1 1 1 ) and fructose-6-P (0.2 m), however, the K,,, values for fructose-2,6-Pz are increased to 12 PM and 0.8 p t for liver and muscle enzymes, respectively. Thus, fructose-2,6-Pz is the most potent activator of the enzyme compared to other known activators such as fructose-1,6-Pz. The rates of the reaction catalyzed by the enzymes under the above conditions are nonlinear: the rates decelerate in the absence or in the presence of lower concentrations of fructose-2,6-Pz, but the rates become linear in the presence of higher concentrations of fructose-2,6-Pz. Fructose-2,6-Pz also protects phosphofructokinase against inactivation by heat. Fructose-2,6-Pz, therefore, may be the most important allosteric effector in regulation of phosphofructokinase in liver as well as in other tissues.