Significance of phosphorylation of phosphofructokinase (original) (raw)
Journal of Biological Chemistry
Liver phosphofructokinase has been separated into three fractions by DEAE-cellulose chromatography. Chromatography of 32P-labeled enzyme reveals that the first fraction contains an average of 1.2 mol of phosphate/mol of enzyme (320,000 daltons), while the second and third fractions contain 3.3 mol of phosphate/ mol of enzyme. The high phosphate forms are much more sensitive to ATP inhibition than the low phosphate form.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1988
The temperature-and pH-induced transitions in F-protein (phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11)) have been studied by means of microcalorimetry and fluorescence and CD spectroscopy. An increase in pH from approx. 6.0 to approx. 8.0 causes a change in the protein state which seems to correspond to a shift of the dimer-tetramer equilibrium in favour of the tetramers. In the absence of phosphate, stability of the protein to temperature-and urea-induced denaturation at pH 6.0 is higher than that at pH 8.0. An addition of 150 mM phosphate results in a pronounced increase in the protein's stability in such a way that the protein becomes more stable at pH 8.0 than at pH 6.0. The shift of the denaturationai heat capacity peak induced by the phosphate binding exceeds 25 o C at pH 8.0 and 9 o C at pH 6.0.
Chicken liver phosphofructokinase. II. Cold inactivation
The Journal of biological chemistry, 1973
All organic and inorganic chemicals were reagent grade. Bio-Gel A-1.5m and A-0.5m were obtained from Bio-Rad Laboratories (Richmond, Calif.). Microgranular DEAE-cellulose (DE-52) was purchased from H. Reeve Angel and Co. (New York, N.Y.); guanidine HCl was the product of HEICO, Inc. (Delaware Water Gap, Penna.). Trypsin and P-galactosidase were purchased from Worthington Biochemicals. Rabbit liver glycogen and dithiothreitol were purchased from Sigma. Other enzymes were purchased from Boehringer Mannheim, New York. Methods Assay of Phosphojructokinase Activity-Phosphofructokinase activity was determined by measuring the rate of formation of fructose-l ,6-bis-P at 25". The reaction mixture contained 50 8592 This is an Open Access article under the CC BY license.
The structure of “activation factor” for phosphofructokinase
Journal of Biological Chemistry
The "activation factor" for phosphofructokinase was shown by chemical analysis, by synthesis, and by 13C NMR spectroscopy to be /h~-fructose-2,6-P~. This compound was prepared from ~-fructose-1,2-cyclic 6-Pz by alkaline hydrolysis. ~-Fructose-1,2-cyclic 6-Pz is ineffective in activating phosphofructokinase while synthetic ~-fructose-2,6-P~ has the same specific activity toward phosphofructokinase as the "activation factor" isolated from rat liver, and it exhibits the same characteristics on paper and ion exchange chromatography. Acid treatment of both the synthetic and the natural product destroys the biological activity and yields 1 mol each of fructose-6-P and Pi; alkaline phosphatase treatment of the compound followed with acid hydrolysis yields fructose. The natural abundance 13C NMR spectra of the synthetically prepared and purified D-
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.
Changes in allosteric properties of phosphofructokinase bound to erythrocyte membranes
The Journal of biological chemistry, 1977
Human and rabbit erythrocyte membranes prepared by hypotonic hemolysis contained 5 to 15% of the phosphofructokinase in the erythrocytes. The membrane-bound phosphofructokinase can be eluted by a saline wash. Human erythrocyte and rabbit muscle phosphofructokinase bind to the saline-washed membranes. This binding is specific for the inner surface of the membrane. The amount of phosphofructokinase bound is dependent on pH; at pH 7, 6 times more enzyme is bound than at pH 7.5. Unlike free phosphofructokinase, the membrane-bound phosphofructokinase is not inhibited by ATP or 2,3-diphosphoglycerate, and its fructose-6-P saturation curve is nonsigmoidal.
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.
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.