Quantitative kinetic model of ATP hydrolysis-synthesis by membrane H+-ATPase

A mathematical model of fermentative reactions of synthesis-hydrolysis of ATP by H+-ATPase, coupled to the charge transport across the membrane has been constructed. Quantitative calculations of the rate of hydrolysis--synthesis of ATP were performed. The nature of the membrane electrochemical gradient delta mu H+ is discussed.     

The vacuolar H+-ATPase of lemon fruits is regulated by variable H+/ATP coupling and slip

Lemon fruit tonoplasts, unlike those of seedling epicotyls, contain nitrate-insensitive H+-ATPase activity (Müller, M. L., Irkens-Kiesecker, U., Rubinstein, B., and Taiz, L. (1996) J. Biol. Chem. 271, 1916-1924). However, the degree of nitrate-insensitivity fluctuates during the course of the year with a seasonal frequency. Nitrate uncouples H+ pumping from ATP hydrolysis both in epicotyls and in nitrate-sensitive fruit V-ATPases. Neither bafilomycin nor oxidation cause uncoupling. The initial rate H+/ATP coupling ratios of epicotyl and the nitrate-sensitive fruit proton pumping activities are the same. However, the H+/ATP coupling ratio of the nitrate-insensitive fruit H+ pumping activity is lower than that of nitrate-sensitive and epicotyl V-ATPases. Several properties of the nitrate-insensitive H+-ATPase of the fruit indicate that it is a modified V-ATPase rather than a P-ATPase: 1) insensitivity to low concentrations of vanadate; 2) it is initially strongly uncoupled by nitrate, but regains coupling as catalysis proceeds; 3) both the nitrate-sensitive and nitrate-insensitive fruit H+-pumps have identical Km values for MgATP, and show similar pH-dependent slip and proton leakage rates. We conclude that the ability of the juice sac V-ATPase to build up steep pH gradients involves three factors: variable coupling, i.e. the ability to regain coupling under conditions that initially induce uncoupling; a low pH-dependent slip rate; the low proton permeability of the membrane.     

Purification of H+-ATPase from the Plasma Membrane of Maize Roots and Preparation of Its Antibody

The plasma membrane ATPase was purified to near homogeneityfrom corn roots. Procedures included partition in an aqueouspolymer two-phase system, solubilization of the enzyme fromplasma membranes with lysolecithin, and vertical centrifugationwith a glycerol gradient. The purified enzyme had a high specificactivity [4.7 µmol.min^–1. (mgprotein)^–1] absolutelyrequiring potassium ions for catalytic function. A specificpolyclonal antibody was produced against the 90-kDa polypeptideof the enzyme. (Received March 7, 1987; Accepted July 1, 1987)     

Na+-Na+ exchange mediated by (Na+ + K+)-ATPase reconstituted into liposomes. Evaluation of pump stoichiometry and response to ATP and ADP

(Na+ + K+)-ATPase from shark rectal glands reconstituted into lipid vesicles and oriented inside out catalyses an ouabain-sensitive Na+-Na+ exchange in the absence of intravesicular K+ when ATP is added extravesicularly. Intravesicular ouabain inhibited the exchange completely. This was also the case with digitoxigenin added to the vesicles. Intravesicular oligomycin inhibited the Na+-Na+ exchange partly in a fashion which was ATP dependent. The exchange is accompanied by a net hydrolysis of ATP with an apparent Km of 2.5 microM. ADP was found to give no stimulation of the Na+-Na+ exchange, contrarily, ADP inhibited the ATP-dependent exchange of Na+ both at optimal and supraoptimal ATP concentrations. When initial influx and efflux of 22Na was measured and the hydrolysis of ATP concomitantly determined a coupling ratio of 2.8:1.3:1 was found, i.e. 2.8 moles of Na+ were taken up (cellular efflux) and 1.3 moles of Na+ extruded (cellular influx) for each mole of ATP hydrolyzed. The electrogenic Na+-Na+ exchange generated a transmembrane potential which was measured with the fluorescent probe ANS (8-anilino-1-naphthalenesulfonic acid) to be 60 mV positive inside the liposomes (extracellular).     

The acceleration of Na+,K+-ATPase activity by ATP and ATP analogues

Since Na+,K+-ATPase (EC 3.6.1.3) of pig kidney modified with a fluorescent sulfhydryl reagent, N-[p-(2-benzimidazolyl) phenyl]maleimide, at Cys-964 of the alpha-chain showed ATP-dependent, reversible, and dynamic fluorescence changes (Nagai, M., Taniguchi, K., Kangawa, K., Matsuo, S., Nakamura, S., and Iida, S. (1986) J. Biol. Chem. 261, 13197-13202), we studied the conformational change during Na+,K+-ATPase reaction using the modified enzyme. The addition of K+ to the enzyme increased the fluorescence intensity to 2% in the presence of 160 mM Na+ and 3 mM Mg2+ (K0.5 = 16.4 mM). Addition of low concentrations of ATP immediately increased the intensity to 3.2% (K0.5 less than 0.1 microM) to accumulate fully K+-bound enzyme in the presence of 43 mM K+ with Na+ and Mg2+, but further addition of higher concentrations of ATP diminished the increase (K0.5 = 120 microM). After exhaustion of ATP, the fluorescence intensity decreased to -0.4% (K0.5 = 0.3 microM) and -2% (K0.5 = 20 microM), respectively, in the presence of low and high concentrations of ADP produced from ATP. High concentrations of ATP accelerated Na+,K+-ATPase activity with a simultaneous increase in the amount of ADP-sensitive phosphoenzyme irrespective of the modification. Adenylyl imidodiphosphate and ADP accelerated Na+,K+-ATPase activity in the presence of 2.7 microM ATP by decreasing the extent of the fluorescence without affecting the amount of phosphoenzyme, irrespective of the modification. These data suggest that Na+,K+-ATPase activity was accelerated due to the acceleration of the breakdown of K+-bound enzyme by high concentrations of ATP and ATP analogues.     

The H+/ATP stoichiometry of the (H+ +K+)-ATPase of dog gastric microsomes

Gastric microsomal vesicles isolated from dog fundic mucosa were shown to be relatively ion tight and have a low level of proton permeability. The H+ translocase, basal ATPase and K+-activated ATPase activities of these vesicles were measured and the H+/ATP stoichiometry calculated using either the total K+-ATPase or the K+-stimulatable component (total K+-ATPase--basal ATPase). The former estimations consistently gave stoichiometric of approximately one, whereas the use of only the K+-stimulatable component gave widely differing values. Measurement of the dephosphorylation of the enzyme under basal conditions revealed both a labile and a stable phosphoenzyme component. The rate of decay of the labile component completely accounted for the basal ATPase activity observed. We conclude that the basal ATPase associated with our preparations is a spontaneous dephosphorylation of the phosphoenzyme occurring in the absence of K+ and that the H+/ATP stoichiometry of the gastric ATPase is one.     

The H+/ATP stoichiometry of the (H+ + K+)-ATPase of dog gastric microsomes

Gastric microsomal vesicles isolated from dog fundic mucosa were shown to be relatively ion tight and have a low level of proton permeability. The H⁺ translocase, basal ATPase and K⁺-activated ATPase activities of these vesicles were measured and the H⁺/ATP stoichiometry calculated using either the total K⁺-ATPase or the K⁺-stimulatable component (total K⁺-ATPase—basal ATPase). The former estimations consistently gave stoichiometric of approximately one, whereas the use of only the K⁺-stimulatable component gave widely differing values. Measurement of the dephosphorylation of the enzyme under basal conditions revealed both a labile and a stable phosphoenzyme component. The rate of decay of the labile component completely accounted for the basal ATPase activity observed. We conclude that the basal ATPase associated with our preparations is a spontaneous dephosphorylation of the phosphoenzyme occurring in the absence of K⁺ and that the H⁺/ATP stoichiometry of the gastric ATPase is one.     

H+ transport by reconstituted gastric (H+ + K+)-ATPase

Gastric (H+ + K+)-ATPase was reconstituted into artificial phosphatidylcholine/cholesterol liposomes by means of a freeze-thaw-sonication technique. Upon addition of MgATP, active H+ transport was observed, with a maximal rate of 2.1 mumol X mg-1 X min-1, requiring the presence of 100 mM K+ at the intravesicular site. However, in the absence of ATP an H+-K+ exchange with a maximal rate of 0.12 mumol X mg-1 X min-1 was measured, which could be inhibited by the well-known ATPase inhibitors vanadate and omeprazole, giving the first evidence of a passive K+-H+ exchange function of gastric (H+ + K+)-ATPase. An Na+-H+ exchange activity was also measured, which was fully inhibited by 1 mM amiloride. Simultaneous reconstitution of Na+/H+ antiport and (H+ + K+)-ATPase could explain why reconstituted ATPase appeared less cation-specific than the native enzyme (Rabon, E.C., Gunther, R.B., Soumarmon, A., Bassilian, B., Lewin, M.J.M. and Sachs, G. (1985) J. Biol. Chem. 260, 10200-10212).     

Sterol Modulation of the Plasma Membrane H+-ATPase Activity from Corn Roots Reconstituted into Soybean Lipids

A partially purified H+-ATPase from the plasma membrane (PM) of corn (Zea mays L.) roots was inserted into vesicles prepared with soybean (Glycine max L.) phospholipids and various concentrations of individual sterols using either a freeze-thaw sonication or an octylglucoside dilution procedure. Both methods yielded a functional enzyme that retained its native characteristics. We have investigated the effects of typical plant sterols (i.e. sitosterol, stigmasterol, and 24-methylcholesterol) on both ATP hydrolysis and H+ pumping by the reconstituted corn root PM ATPase. We have also checked the influence of cholesterol and of two unusual sterols, 24-methylpollinastanol and 14[alpha],24-dimethylcholest-8-en-3[beta]-ol. Here we present evidence for a sterol modulation of the plant PM H+-ATPase activity. In particular, cholesterol and stigmasterol were found to stimulate the pump, especially when present at 5 mol%, whereas all of the other sterols tested behaved as inhibitors at any concentration in proteoliposomes. In all situations H+ pumping was shown to be more sensitive to a sterol environment than was ATP hydrolysis. Our results suggest the occurrence of binding sites for sterols on the plant PM H+-ATPase.     

ADP binding and ATP synthesis by reconstituted H-ATPase from chloroplasts

The H⁺-ATPase from chloroplasts, CF₀F₁, was isolated, purified and reconstituted into asolectin liposomes. The enzyme was brought either into the oxidized state or into the reduced state, and the rate of ATP synthesis was measured after energisation of the proteoliposomes with an acid—base transition ΔpH (pH_in = 5.0, pH_out = 8.5) and a K⁺/valinomycin diffusion potential, Δφ (K⁺_in = 0.6 mM, K⁺_out = 60 mM). A rate of 250 s⁻¹ was observed with the reduced enzyme (85 s⁻¹ in the absence of Δφ). A rate of 50 s⁻¹ was observed with the oxidized enzyme under the same conditions (15 s⁻¹ in the absence of Δφ). The reconstituted enzyme contained 2 ATP_bound per CF₀F₁ and 1 ADP_bound per CF₀F₁. Upon energisation the enzyme was activated and 0.9 ADP per CF₀F₁, was released. Binding of ADP to the active reduced enzyme was observed under different conditions. In the absence of phosphate the rate constant for ADP binding was 10⁵ M⁻¹·s⁻¹ under energized and de-energized conditions. In the presence of phosphate the rate of ADP binding drastically increased under energized conditions, and strongly decreased under de-energized conditions.     

Proton transport-coupled unisite catalysis by the H+-ATPase from chloroplasts

Proton transport-coupled unisite catalysis was measured with the H⁺-ATPase from chloroplasts. The reaction was measured in the ATP hydrolysis direction under deenergized conditions and in the ATP synthesis direction under energized conditions. The equilibrium constant of the enzyme does not change upon energization, whereas the dissociation constants of substrates and products change by orders of magnitude. This indicates that the Gibbs free enthalpy derived from proton translocation is used to change binding affinities of substrates and products, and this results in synthesis of free ATP.     

Kinetic studies on Na+/K+-ATPase and inhibition of Na+/K+-ATPase by ATP

Na+/K+-ATPase (EC 3.6.1.3) is an important membrane-bound enzyme. In this paper, kinetic studies on Na+/K+-ATPase were carried out under mimetic physiological conditions. By using microcalorimeter, a thermokinetic method was employed for the first time. Compared with other methods, it provided accurate measurements of not only thermodynamic data (deltarHm) but also the kinetic data (Km and Vmax). At 310.15K and pH 7.4, the molar reaction enthalpy (deltarHm) was measured as -40.514 +/- 0.9kJmol(-1). The Michaelis constant (Km) was determined to be 0.479 +/- 0.020 mM and consistent with literature data. The reliability of the thermokinetic method was further confirmed by colorimetric studies. Furthermore, a simple and reliable kinetic procedure was presented for ascertaining the true substrate for Na+/K+-ATPase and determining the effect of free ATP. Results showed that the MgATP complex was the real substrate with a Km value of about 0.5mM and free ATP was a competitive inhibitor with a Ki value of 0.253 mM.     

Pi in equilibrium ATP exchange in the absence of proton gradient by the H+-ATPase from yeast plasma membranes

Purified soluble H+-ATPase from Schizosaccharomyces pombe catalyzes a Pi in equilibrium ATP exchange in the absence of a H+ gradient. When the pH of the assay medium is raised from 5.5 to 8.0 there is a decrease of the ATPase activity and an increase of the rate of Pi in equilibrium ATP exchange. At pH 7.0 the addition of the organic solvent dimethyl sulfoxide (20%, v/v) promotes a decrease of ATPase activity and an increase of the Pi in equilibrium ATP exchange reaction. The effect of the organic solvent on the Pi in equilibrium ATP exchange is related to a decrease of the apparent Km for Pi.     

Membrane-reversible H+-ATPase from Micrococcus lysodeikticus.

Treatment of phosphorylating fragments of bacterial membrane from Micrococcus lysodeikticus with trypsin leads to increase ATPase activity. As a result of this treatment, the membrane fragments acquire the ability to transform the ATP energy into transmembrane difference in potential. Dithiothreitol has a similar effect to that of trypsin on the membrane fragments from M. lysodeikticus. Dicyclohexylcarbodimide inhibits ATPase of the membrane fragments of M. lysodeikticus, and also the ATPase-reaction-coupled generation of membrane potential. It has been suggested that the increased ATPase activity of membranes from M. lysodeikticus during treatment with trypsin and dithiothreitol is connected with the effect of these agents on the protein inhibitor of ATPase.     

Plasma Membrane H+-ATPase in Maize Roots Induced for NO3- Uptake

Plasma membrane H+-ATPase was studied in maize (Zea mays L.) roots induced for NO3- uptake. Membrane vesicles were isolated by means of Suc density gradient from roots exposed for 24 h either to 1.5 mM NO3- or 1.5 mM SO4-. The two populations of vesicles had similar composition as shown by diagnostic inhibitors of membrane-associated ATPases. However, both ATP-dependent intravesicular H+ accumulation and ATP hydrolysis were considerably enhanced (60-100%) in vesicles isolated from NO3--induced roots. Km for Mg:ATP and pH dependency were not influenced by NO3- treatment of the roots. ATP hydrolysis in plasma membrane vesicles for both control and NO3--induced roots was not affected by 10 to 150 mM NO3- or Cl-. On the other hand, kinetics of NO3-- or Cl--stimulated ATP-dependent intravesicular H+ accumulation were modified in plasma membrane vesicles isolated from NO3-- induced roots. Immunoassays carried out with polyclonal antibodies against plasma membrane H+-ATPase revealed an increased steady-state level of the enzyme in plasma membrane vesicles isolated from NO3--induced roots. Results are consistent with the idea of an involvement of plasma membrane H+-ATPase in the overall response of roots to NO3-.     

The H+-ATPase from chloroplasts: effect of different reconstitution procedures on ATP synthesis activity and on phosphate dependence of ATP synthesis

The H+-ATP synthase from chloroplasts, CF0F1, was isolated, reconstituted into liposomes and ATP synthesis activity was measured after energization of the proteoliposomes with an acid-base transition. The ATP yield was measured as a function of the reaction time after energization, the data were fitted by an exponential function and the initial rate was calculated from the fit parameters. CF0F1 was reconstituted by detergent dialysis in asolectin liposomes and phosphatidylcholine/phosphatidic acid (PtdCho/PtdAc from egg yolk) liposomes. In asolectin liposomes, high initial rates of ATP synthesis (up to 400 s(-1)) were observed with a rapid decline of the rate; in PtdCho/PtdAc liposomes the initial rate is smaller (up to 200 s(-1)), but the decline of the activity is slower. CF0F1 was reconstituted into PtdCho/PtdAc liposomes either by detergent dialysis or into reverse phase liposomes. The dependence of the rate of ATP synthesis on the phosphate concentration was measured with both types of proteoliposomes. The data can be described by Michaelis-Menten kinetics with a K(M) value of 350 microM for reverse phase liposomes and a K(M) value of 970 microM for dialysis liposomes. Both K(M) values depend neither on the magnitude of DeltapH nor on the electric potential difference, whereas V(max) decreases strongly with decreasing energization. At low phosphate concentration, there are small deviations from Michaelis-Menten kinetics. The measured rates are higher than those calculated from the fitted Michaelis-Menten parameters. This effect is interpreted as evidence that more than one phosphate binding site is involved in ATP synthesis.     

磷饥饿下番茄幼苗根系液泡膜H+-ATPase活性的适应性变化

以‘世纪星’番茄为材料。研究了磷饥饿下番茄幼苗的生长状况及其根部液泡膜H^＋-ATPase活性的适应性变化。结果表明：磷饥饿下番茄幼苗的平均高度均低于对照苗,而主根长度均显著长于对照。磷饥饿提高了番茄幼苗根部液泡膜H^＋-ATPase的水解活性,随着磷胁迫时间的延长,该酶的活性逐渐增大,在磷饥饿7d时达到最大,后又略有降低;而对照番茄幼苗根部该酶的活性变化很小。动力学分析表明：磷饥饿使番茄幼苗根部液泡膜H^＋-ATPase的Km值明显降低,但对该酶的Kmax影响不大。这说明磷饥饿提高了该酶对其底物的亲和力。此外,磷饥饿并不改变液泡膜H^＋-ATPase酶的最适pH值（仍为7．5）。