Kinetic effects of Ca2+ and Mg2+ on ATP hydrolysis by the purified ATP diphosphohydrolase

ATP diphosphohydrolase (EC 3.6.1.5) catalyzes the hydrolysis of diphospho- and triphosphonucleosides and is sensitive to divalent cations. In this paper, we investigated the dependence of ATP hydrolysis on the concentration of free Mg2+ and Ca2+ and the cation ATP complexes. The enzyme was isolated from porcine zymogen granule membranes, solubilized in Triton X-100, and purified on a 5'-AMP-Sepharose 4B affinity column resulting in a 1500-fold purification. Free unprotonated ATP4- was hydrolyzed in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. When hydrolysis rate was measured at different concentrations of the cation-ATP complex at constant free cation concentrations, normal hyperbolic curves were obtained. In CaCl2, both Kapp and Vapp increased as free Ca2+ increased from 25 to 1000 microM. In MgCl2, Kapp increased and Vapp decreased as free Mg2+ increased from 25 to 500 microM. From the rapid equilibrium rate equation, Ks and Vmax values of the substrates were calculated. We found that free ATP4-, Ca-ATP2-, and Mg-ATP2- are substrates and free cations do not bind the enzyme.     

Kinetic mechanism of Fo x F1 mitochondrial ATPase: Mg2+ requirement for Mg x ATP hydrolysis

The initial rates of ATP hydrolysis catalyzed by Fo x F1 (bovine heart submitochondrial particles) preincubated in the presence of Pi for complete activation of the oligomycin-sensitive ATPase were measured as a function of ATP, Mg2+, and Mg x ATP concentrations. The results suggest the mechanism in which Mg x ATP complex is the true substrate of the ATPase and the second Mg2+ bound at a specific pH-dependent site is needed for the catalysis. Simple hyperbolic Michaelis--Menten dependences of the reaction rate on the substrate (Mg x ATP) and activating Mg2+ were found. In contrast to the generally accepted view, no inhibition of ATPase by free Mg2+ was found. Inhibition of the reaction by free ATP is due to a decrease of free Mg2+ needed for the catalysis. In the presence of both Ca2+ and Mg2+ the kinetics of ATP hydrolysis suggest that the Ca x ATP complex is neither hydrolyzed nor competes with Mg x ATP, and free Ca2+ does not affect the hydrolysis of Mg x ATP complex. A crucial role of free Mg2+ in the time-dependent inhibition of ATPase by azide is shown. The dependence of apparent Km for Mg x ATP on saturation of the Mg2+-specific site suggests the formal ping-pong mechanism in which bound Mg2+ participates in the overall reaction after dissociation of one product (most likely Pi) thus promoting either release of ADP (catalytic turnover) or slow isomerization of the enzyme--product complex (formation of the dead-end ADP(Mg2+)-inhibited enzyme). The rate of Mg x ATP hydrolysis only slightly depends on pH at saturating Mg2+. In the presence of limited amounts of free Mg2+ the pH dependence of the initial rate corresponds to the titration of a single group with pKa = 7.5. The simple competition between H+ and activating Mg2+ was observed. The specific role of Mg2+ as a coupling cation for energy transduction in Fo x F1-ATPase is discussed.     

Comparative study of calixarene effect on Mg2+ -dependent ATP-hydrolase enzymatic systems from smooth muscle cells of the uterus

Investigation the influence of calyx[4]arenes C-90, C-91, C-97 and C-99 (codes are indicated) on the enzymatic activity of four functionally different Mg2+ -dependent ATPases from smooth muscle of the uterus: actomyosin ATPase, transporting Ca2+, Mg2+ -ATPase, ouabain-sensible Na+, K+ -ATPase and basal Mg2+ -ATPase. It was shown that calixarenes C-90 and C-91 in concentration 100 microM act multidirectionally on the functionally different Mg2+ -dependent ATP-hydrolase enzymatic systems. These compounds activate effectively the actomyosin ATPase (Ka = 52 +/- 11 microM [Ukrainian character: see text] 8 +/- 2 microM, accordingly), at the same time calixarene C-90 inhibited effectively activity of transporting Ca2+, Mg2+ -ATPase of plasmatic membranes (I(0,5) = 34.6 +/- 6.4 microM), but influence on membrane-bound Na+, K+ -ATPase and basal Mg2+ -ATPase. Calixarene C-91 reduce effectively basal Mg2+ -ATPase activity, insignificantly activating Na+, K+ -ATPase but has no influence on transporting Ca2+, Mg2+ -ATPase activity of plasmatic membranes. Calixarenes C-97 and C-99 (100 microM), which have similar structure, have monodirectional influence on activity of three functionally different Mg2+-dependent ATPases of the myometrium: actomyosin ATPase and two ATPases, that related to the ATP-hydrolases of P-type--Ca2+, Mg2+ -ATPase and Na+, K+ -ATPase of plasmatic membranes. Basal Mg2+ -ATPase is resistant to the action of these two connections. Results of comparative experiments that were obtained by catalytic titration of calixarenes C-97 and C-99 by actomyosin ATPase (I(0,5) = 88 +/- 9 and 86 +/- 8 microM accordingly) and Na+, K+ -ATPase from plasmatic membranes (I(0,5) = 33 +/- 4 and 98 +/- 8 nM accordingly) indicate to the considerably more sensitiveness of Na+, K+ -ATP-ase to these calixarenes than ATPase of contractile proteins. Thus, it is shown that calixarenes have influence on activity of a number of important enzymes, involved in functioning of the smooth muscle of the uterus and related to energy-supplies of the process of the muscle contracting and support of intracellular ionic homeostasis. The obtained results can be useful in further researches, directed at the use of calixarenes as pharmaceutical substance, able to normalize the contractile function of the uterus at some pregnancy pathologies in women's.     

Effect of eosin Y on Ca2+ -independent, Mg2+ -dependent ATPase activity of smooth muscle cell plasma membranes

The effect of eosin Y (2',4',5',7'-tetrabromofluorescin) on basic kinetic parameters of the reaction of Mg2+ -dependent hydrolysis of ATP catalysed "basal" Mg2+ -ATPase myometrial cells plasma membrane has been studied. The eosin Y (10-100 microM) inhibited initial maximal velocity of the "basal" Mg2+ -ATPase of plasma membrane assayed for Mg2+ and ATP. At the same time the given inhibitor reduces the affinity of Mg2+ -ATPase for ATP. However, the difficult effect of the inhibitor action is observed for Mg ions: eosin Y in concentration of 10-50 microM increases the enzyme affinity for the ion-activator, while in concentration of 100 microM the affinity of Mg2+ -ATPase for Mg2+ is reduced. An analysis of eosin Y effect on catalytic efficiency of "basal" Mg2+ -ATPase of plasma membrane has shown, that at saturating concentrations of ATP (1 mM) the enzyme activity is less sensitive to the action of inhibitor. On this basis the conclusion is made that ATP in high concentrations can compete with eosin Y for active centre of Mg2+ -ATPase of smooth muscle cells plasma membrane.     

Identification and characterization of "basal" Ca2+-independent Mg2+-dependent ATP-hydrolase enzymatic activity in smooth muscle cell plasma membrane fractions

It is shown, that for correct definition of "basal" Ca(2+)-independent Mg(2+)-dependent ATPase ac-activity (10-13 mmol Pi/hour on 1 mg of protein) in a fraction of uterus smooth muscle cell plasma membranes is necessary to use in medium without calcium of an incubation not only EGTA and digitonin--of the factor of infringement in activity by this subcellular structure, but inhibitors of others Mg(2+)-dependent ATP-hydrolyse enzymatic systems localized as in plasma membrane (Na+, K(+)-ATPase) and in others subcellular frames, first of all, in mitochondria (Mg(2+)-ATPase) and endoplasmic reticulum (transport Ca2+, Mg(2+)-ATPase). In the case of a sacolemal fraction of a smooth muscle the contribution of others Mg(2+)-dependent ATP-hydrolyse systems in a common enzymatic hydrolysis ATP, which unconnected to functioning "basal" Ca(2+)-independent Mg(2+)-dependent ATPase, is very appreciable and achieves 35%. The researches, carried out in the frameworks of definition of initial velocity of enzymatic reaction, have enabled to define its some properties--cationic and anionic specificity, and also sensitivity to action of some inhibitors. It has appeared, that the "basal" Ca(2+)-independent Mg(2+)-dependent ATP-hydrolyse reaction is nonspecific rather both in relation to cations of divalent metals Me2+, and cations of monovalent metals and anions, which were utilized for support of ionic strength. The cations La--antagonist of cations Ca--practically did not influence enzymatic activity. The non-specific inhibitors transport of ATPases--p-chloromercuribenzoate, o-vanadate and eosine Y with a various degree of efficiency inhibited "basal" Ca(2+)-independent Mg(2+)-dependent ATP-hydrolyse reaction. On the basis of the analysis of the own and literary data the conclusion is made that "basal" Ca(2+)-independent Mg(2+)-dependent ATPase of a smooth muscle cell plasma membrane is considerably less sensitive to action of nonspecific inhibitors of the Ca(2+)-transporting systems, than these systems.     

Active Na+-dependent transport of Ca 2+ into the fraction of smooth muscle sarcolemma vesicles

Studies on the vesicular fraction of myometrium sarcolemma showed that in the absence of initial Ca2+ gradient the vesicles activity accumulate Ca2+ by utilizing the energy of the antiport-directed Na+ gradient. Monensin (50 microM) suppresses practically completely the Ca2+ transport. The amount of Ca2+ entering the vesicles against the concentration gradient diminishes with a decrease in the oppositely directed Na+ gradient. Cd2+ (5 mM) causes a complete inhibition of active Ca2+ transport, whereas Mn2+ and Mg2+ inhibit this process by 85% and 35%, respectively; amiloride (500 microM) is fairly ineffective. In the absence of initial Ca2+ and Na+ gradients valinomycin (0.05-1 microM) does not affect the changes in Ca2+ concentration in the intravesicular volume both with and without K+ gradient. Under conditions of initial equilibrium for Ca2+ and Na+ the magnitude and sign of the membrane potential for the K(+)-valinomycin system have no effect on Ca2+ transport regardless of value of absolute Na+ concentration inside and outside the vesicles. Depolarization of membrane vesicles does not interfere with the Na(+)-driven active Ca2+ transport into the sarcolemma which is dependent on the energy of the Na+ gradient. Using calibration curves, it was shown that the physiologically significant (6-fold) Na+ gradient increases Ca2+ concentration in the intravesicular volume from 100 to 160-170 microM. Ac active potential-independent Ca2+ transport through the smooth muscle sarcolemma requires about one third (0.3 kcal/mol) of the Na+ gradient; energy the remainder is dissipated. It is concluded that in smooth muscles the Na+ gradient can provide the active transsarcolemmal transport of Ca2+.     

Some peculiarities of the reaction kinetics of ATP hydrolysis by Ca2+-dependent ATPase of chloroplasts]

The dependence of the reaction rate of ATP hydrolysis by CF1-ATPase of chloroplasts on the substrate (CaATP) concentration is of complex nature. It is assumed that such dependence may be due to either conformational changes of the enzyme during the increase in CaATP content in the reaction medium, or consecutive binding of the substrate to its active sites, differing in their affinity to the substrate of the reaction.     

A monoclonal antibody to the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle inhibits plasmalemmal (Ca2+ + Mg2+)-dependent ATPase activity.

A monoclonal antibody (2B3) directed against the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle was prepared. This antibody reacts with a 130,000-Mr protein that co-migrates on SDS/polyacrylamide-gel electrophoresis with the calmodulin-binding (Ca2+ + Mg2+)-ATPase purified from smooth muscle by calmodulin affinity chromatography. The antibody causes partial inhibition of the (Ca2+ + Mg2+)-ATPase activity in plasma membranes from pig stomach smooth muscle, in pig erythrocytes and human erythrocytes. It appears to be directed against a specific functionally important site of the plasmalemmal Ca2+-transport ATPase and acts as a competitive inhibitor of ATP binding. Binding of the antibody does not change the Km of the ATPase for Ca2+ and its inhibitory effect is not altered by the presence of calmodulin. No inhibition of (Ca2+ + Mg2+)-ATPase activity or of the oxalate-stimulated Ca2+ uptake was observed in a pig smooth-muscle vesicle preparation enriched in endoplasmic reticulum. These results confirm the existence in smooth muscle of two different types of Ca2+-transport ATPase: a calmodulin-binding (Ca2+ + Mg2+)-ATPase located in the plasma membrane and a second one confined to the endoplasmic reticulum.     

Mg2+- or Ca2+-activated ATPase activities of plasma membranes isolated from vascular smooth muscle

Studies of ATP hydrolysis by various subcellular fractions isolated from rat mesenteric arteries and veins indicate that an apparent ATPase activity, which can be activated by Mg2+ or Ca2+, is primarily associated with the plasma membranes. Although both Mg2+-activated and Ca2+-activated ATPase activities under the optimal condition are substantially lower in venous than in arterial plasma membrane fraction, their dependence on the concentration of Mg2+ and Ca2+ are quite similar in arterial as well as venous plasma membrane fractions. No synergistic effect on ATP hydrolysis was observed in the presence of both Mg2+ and Ca2+. In addition, Mg2+-activated and Ca2+-activated ATPase activities show similar pH dependence, inhibition by deoxycholate, stability toward heat inactivation and substrate specificity. Furthermore, Mg2+-activated and Ca2+-activated ATPase activities were similarly reduced in vascular smooth muscles of spontaneously hypertensive rats. These results suggest that the activation of ATP hydrolysis by Mg2+ or Ca2+ may represent a single enzyme moiety in the plasma membrane of vascular smooth muscle. The possible involvement of such ATPase in the Ca2+ transport function of vascular smooth muscle is discussed.     

Kinetic mechanism of the Mg2+-dependent nucleotidyl transfer catalyzed by T4 DNA and RNA ligases.

The Mg(2+)-dependent adenylylation of the T4 DNA and RNA ligases was studied in the absence of a DNA substrate using transient optical absorbance and fluorescence spectroscopy. The concentrations of Mg(2+), ATP, and pyrophosphate were systematically varied, and the results led to the conclusion that the nucleotidyl transfer proceeds according to a two-metal ion mechanism. According to this mechanism, only the di-magnesium-coordinated form Mg(2)ATP(0) reacts with the enzyme forming the covalent complex E.AMP. The reverse reaction (ATP synthesis) occurs between the mono-magnesium-coordinated pyrophosphate form MgP(2)O(7)(2-) and the enzyme.MgAMP complex. The nucleotide binding rate decreases in the sequence ATP(4-) > MgATP(2-) > Mg(2)ATP(0), indicating that the formation of the non-covalent enzyme.nucleotide complex is driven by electrostatic interactions. T4 DNA ligase shows notably higher rates of ATP binding and of subsequent adenylylation compared with RNA ligase, in part because it decreases the K(d) of Mg(2+) for the enzyme-bound Mg(2)ATP(0) more than 10-fold. To elucidate the role of Mg(2+) in the nucleotidyl transfer catalyzed by T4 DNA and RNA ligases, we propose a transition state configuration, in which the catalytic Mg(2+) ion coordinates to both reacting nucleophiles: the lysyl moiety of the enzyme that forms the phosphoramidate bond and the alpha-beta-bridging oxygen of ATP.     

Ca2+-stimulated, Mg2+-dependent ATPase in bovine thyroid plasma membranes

An isolated plasma membrane fraction from bovine thyroid glands contained a Ca2+-stimulated, Mg2+-dependent adenosine triphosphatase ((Ca2+ + Mg2+)-ATPase) activity which was purified in parallel to (Na+ + K+)-ATPase and adenylate cyclase. The (Ca2+ + Mg2+)-ATPase activity was maximally stimulated by approx. 200 microM added calcium in the presence of approx. 200 microM EGTA (69.7 +/- 5.2 nmol/mg protein per min). In EGTA-washed membranes, the enzyme was stimulated by calmodulin and inhibited by trifluoperazine.     

Kinetic modeling of enzyme-catalysed reactions with participation of activators applied to ATP hydrolysis by Mg2+- ATPase

Theoretical investigation of the model of reaction of ATP hydrolysis by "basal" Mg2+-ATPase has been carried out. It has been assumed that during the reaction each of three reacting substances (Mg2+, ATP, enzyme) can combine into complexes in couples with other participants of this process. Then the third component can associate with formed complexes producing the ternary complex of enzyme-activator-substrate. Such approach allowed to take into account all possible interactions in the chosen system, to investigate overall process in detail avoiding any simplifications and to find such peculiar properties of the process which will allow to understand the reaction mechanism and to explain observed experimental data. All possible pathways of the ATP hydrolysis process have been examined separately and as a whole. It is shown that if the reaction proceeds via two or three possible pathways then maximums are observed on plots of initial reaction rate on concentration ATP or magnesium. In addition maximums are also observed when enzyme concentration is increased. It is qualitatively new result that does not follow from the existing theories. The obtained results permit to explain some experimental data of ATP hydrolysis, active ion transport and some other reactions in a new fashion. The studied model and obtained results may be applied to another enzyme-catalysed reactions which proceed in the activator presence.     

Covalent phosphorylation of the Mg2+-dependent ATPase of yeast plasma membranes

Phosphorylation by [gamma-32P]ATP of proteins associated with the plasma membrane of Saccharomyces cerevisiae has been studied both in vivo and in vitro. Although at least nine proteins are labeled in vivo, there is only one major protein labeled in vitro. This species with an apparent molecular weight of 114,000 has been identified as the plasma membrane Mg2+-ATPase. Phosphorylation of this enzyme occurs exclusively on serine residues. This is the first report that the proton-translocating ATPase of fungal plasma membranes is subject to phosphorylation by a protein kinase.     

Influence of omeprasole and lansoprasole on Na+, K+ -ATPase and Mg2+ -ATPase activity of the plasmatic membrane of myometrium smooth muscle cells

The paper deals with the influence of the proton pump inhibitors - omeprasole and lansoprasole on the enzymatic activity of the ouabain-sensitive Na+, K+ -ATPase and the ouabain-resistant Mg2+ - ATPase in the suspension of the myometrium cell plasmatic membranes treated with 0.1% digitonin solution. It was found, that omeprasole and lansoprasole inhibited Na+, K+ -ATPase in the range from 10 to 100 microM. The maximal effect was observed at a concentration of 100 microM with the percentage of inhibition of 81 and 86% at an average as compared with the control for omeprasole and lansoprasole, respectively. The magnitudes of the inhibition coefficient I(0.5) for omeprasole and lansoprasole were 35.60 +/- 0.81 and 29.40 +/- 1.79 microM respectively. Meanwhile cooperative effects on the Na+, K+ - ATPase activity were not found, as the Hill coefficient n(H) for omeprasole was 1.00 +/- 0.08, while for lansoprasole it was 1.20 +/- 0.03. These substances had also insignificant influence on Mg2+ -ATPase: the enzymatic activity was decreased to 84 and 82% as compared with the control with omeprasole and lansoprasole, respectively, in concentration of 100 microM for each inhibitor. The inhibition of Na+, K+ -ATPase activity can evidence for the possible side effects of omeprasole and lansoprasole when they are used for treatment of acid-dependent diseases of the stomach. In addition, obtained experimental data can be useful for further research of the membrane mechanisms of omeprasole and lansoprasole action on cationic exchange in the smooth muscle cells.     

High-affinity Ca2+-stimulated and Mg2+-dependent ATPase from rat osteosarcoma plasma membranes

Transplantable rat osteosarcoma plasma membrane preparations contain high-affinity and low-affinity calcium-stimulated ATPases. The high-affinity enzyme displayed a K0.5 for calcium of 0.03 microM, a Vmax of 99.2 nmol/min/mg, and a requirement for magnesium ions. It was not inhibited by 20 microM trifluoperazine nor stimulated by the addition of 2 ng of calmodulin. Lack of stimulation with exogenous calmodulin may be related to the high endogenous calmodulin content of the membrane preparations. The low-affinity Ca2+- or Mg2+-ATPase displayed a K0.5 for calcium of approximately 2.40 mM (Vmax of 185 nmol/min/mg) and a K0.5 for magnesium of approximately 2.75 mM (Vmax of 250 nmol/min/mg).     

Ca2+-stimulated,Mg2+-independent ATP hydrolysis and the high affinity Ca2+-pumping ATPase. Two different activities in rat kidney basolateral membranes

The Mg²⁺-dependency of Ca²⁺-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg²⁺- and Ca²⁺-buffering ligands. ATP hydrolysis is strongly stimulated by Mg²⁺ with a K_m of 13 μ M in the absence or presence of 1 μ M free Ca²⁺. At free Mg²⁺ concentrations of 1 μ M and lower, ATP hydrolysis is Mg²⁺ -independent, but is strongly stimulated by submicromolar Ca²⁺ concentrations K_m  0.25 μM, V_max  24 μmol P_i/h per mg protein). The Ca²⁺-stimulated ATP hydrolysis strongly decreases at higher Mg²⁺ concentrations. The Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg²⁺ concentrations calmodulin and trifluoperazine affect the high affinity Ca²⁺-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca²⁺-pumping in renal basolaterals and on Ca²⁺-ATPase in erythrocyte ghosts suggest that the Ca²⁺-pumping enzyme requires Mg²⁺. In contrast, a role of the Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis in active Ca²⁺ transport across basolateral membranes is rather unlikely.