title: Effect of buffer solutions on activation of Shamouti orange pyrophosphate-dependent phosphofructokinase by fructose 2,6-bisphosphate.
abstract: Shamouti phosphofructokinase (PFP) activation depends on the presence of fructose 2,6-bisphosphate (Fru-2,6-P2) in the glycolytic reaction. The effect of activation by Fru-2,6-P2 differs considerably, however, according to the buffer (pH 8.0) in which the reaction is performed: Ka = 2.77 +/- 0.3 nM in Hepes-NaOH and 7.75 +/- 1.49 nM in Tris-HCl. The presence of chloride ions (39 mM) in the Tris-HCl buffer inhibits PFP. Indeed, when using a Hepes-NaOH buffer and then adding 39 mM NaCl, Ka = 8.12 +/- 0.52 nM. The Ki for chloride ions is approximately 21.7 mM. In the gluconeogenic reaction, Shamouti PFP generally showed a high endogenous activity. Addition of Fru-2,6-P2 did not modify the velocity and the Vmax of the enzyme; however, its presence increased the affinity of the enzyme for Fru-1,6-P2 from 200 +/- 15.6 microM in absence of Fru-2,6-P2 to 89 +/- 10.3 microM in its presence (10 microM). In the presence of chloride (39 mM), the affinity for the substrate decreased with K(m) = 150 +/- 14 microM. The calculated Ki for chloride ions equals 56.9 mM. In both the glycolytic and the gluconeogenic reactions, Vmax is not affected; therefore, the inhibition mode of chloride is competitive.
authors.affiliation: Kennedy-Leigh Centre for Horticultural Research, Rehovot, Israel. van.praag@bluemail.ch
authors.forename: E
authors.initials: E
authors.lastname: Van Praag
authors.forename: A
authors.initials: A
authors.lastname: Tzur
authors.forename: U
authors.initials: U
authors.lastname: Zehavi
authors.forename: R
authors.initials: R
authors.lastname: Goren
mesh_terms: D002021:Buffers; D002957:Citrus / Q000201:enzymology*; D004789:Enzyme Activation; D005635:Fructosediphosphates / Q000494:pharmacology*; D005943:Gluconeogenesis; D006019:Glycolysis; D007700:Kinetics; D010770:Phosphotransferases / Q000378:metabolism*; D012996:Solutions; D013379:Substrate Specificity
chemical_list: D002021:Buffers; D005635:Fructosediphosphates; D012996:Solutions; C027652:fructose 2,6-diphosphate; D010770:Phosphotransferases; C022502:pyrophosphate-fructose 6-phosphate 1-phosphotransferase