The adenosine triphosphate inhibition of the pyruvate kinase reaction and its dependence on the total magnesium ion concentration

Abstract

1. The effects of ATP, PP(i) and EDTA on the skeletal-muscle pyruvate kinase reaction at various concentrations of magnesium (where ;magnesium' refers to total Mg(2+), both free and in the form of complexes) were investigated. The reaction rate was determined as the amount of pyruvate formed in a recorded time of incubation. 2. At 44mm-magnesium the K(m) values for ADP and phosphoenolpyruvate were unaltered by the presence of ATP up to 6.8mm in systems buffered with either tris-hydrochloric acid or glycylglycine-sodium hydroxide, but the K(m) values were different in these systems. The K(m) for one substrate was independent of the concentration of the second substrate. 3. At 10mm-magnesium in the tris-hydrochloric acid system ATP inhibited the reaction competitively with respect to ADP and phosphoenolpyruvate. In the glycylglycine-sodium hydroxide system the inhibition appeared to be non-competitive. At 10mm-magnesium the K(m) values were lower than at 44mm-magnesium and dependent on the system used. 4. In the tris-hydrochloric acid system the reaction rate rose with increasing magnesium concentration up to a maximum at a concentration 10-20 times that of ADP. Further increase inhibited the reaction and at 44mm-magnesium the rate was 25-50% of its maximum. This inhibition paralleled that produced by increasing trimethylammonium chloride concentrations and was not due to a specific effect of the Mg(2+) ion. 5. In the presence of 6.8mm-ATP no reaction occurred below 4-6mm-magnesium, and further increase apparently abolished the inhibition as the reaction rate increased and became equal to those obtained in the absence of ATP at 10-25mm-magnesium. Further increase in magnesium concentration gave reaction rates that were slightly higher in the presence of ATP than in its absence. The maximal rate in the presence of ATP was distinctly lower than in its absence. When 6.8mm-PP(i) or 6.8mm-EDTA was present the variations in reaction rate with rising magnesium concentration were similar to that obtained in the presence of ATP below 6-8mm-magnesium but further increase in the magnesium concentration resulted in an increase in the rate up to a maximum comparable with that of the control. The effect of pure chelation was thus a displacement of the reaction maximum to higher magnesium concentrations without changing the maximal rate. When correction had been made for this effect, ATP gave inhibition at 44mm-magnesium that was competitive with respect to ADP (K(i) 2.1x10(-2)m). This degree of inhibition is far less than was reported earlier and its importance for the mechanism of the pyruvate kinase reaction is discussed.

Secretory mechanisms. Behaviour of adenine nucleotides during the platelet release reaction induced by adenosine diphosphate and adrenaline

“…ADP (disodium salt from equine muscle; Sigma Chemical Co., St. Louis, Mo., U.S.A.), was stored in small portions (400,ug/ml in 0.15M-NaCl) at -60°C and diluted with 0.15M-NaCl to the desired concentrations imnediately before use. The 1972 exact concentration of ADP stock solutions was determined as described by Holmsen & Storm (1969). L-Adrenaline bitartrate from Winthrop Laboratories (New York, N.Y., U.S.A.) was stored at a concentration of Smg/ml in 0.15M-NaCl at -60°C and thawed immediately before use.…”

Kinetic Analysis of Proton Transport by the Vanadate-Sensitive ATPase from Maize Root Microsomes

“…3). The more sensitive coupled enzyme assay could not be used in this instance because the EDTA would remove divalent cations required for pyruvate kinase ( 12), one of the constituents of the assay medium.…”

Modulation of mitochondrial metabolism as a biochemical trait in blood feeding organisms: the redox vampire hypothesis redux

“…The energy demand is a key mechanism to maintain cellular energy homeostasis (Liesa and Shirihai, ; Wilson, , ). The regulatory control posed by the energy state (or the ATP/ADP balance) targets multiple enzymes in metabolic pathways such as glycolysis, tricarboxylic acid (TCA) cycle, OXPHOS, among others (Holmsen and Storm, ; Hansford, ; Bulos et al, ; Dobson et al, ; Brown, ; Bender and Kadenbach, ; Wilson, ). Thus, in conditions where energy demand increases, lower ATP and high ADP levels activate pathways involved in ATP production.…”