Vanadate inhibition of brain (Ca+Mg)-ATPase

Vanadate was a potent inhibitor of the membrane-bound (Ca+Mg)-ATPase from rat brain, the concentration required for 50% inhibition under conditions optimal for enzymatic activity being 3 M. Vanadate inhibition increased with the MgCl₂ concentration, half-maximal inhibition occurring at 2 mM MgCl₂, near the MgCl₂ concentration required for half-maximal activation of the ATPase activity. MnCl₂ could substitute for MgCl₂, and at concentrations of 1 mM (Ca+Mn)-ATPase activity was greater than (Ca+Mg)-ATPase activity, although sensitivity to vanadate was less. Vanadate inhibition increased also with the KCl concentration, half-maximal inhibition occurring at 8 mM, again near the concentration required for half-maximal activation of ATPase activity. By contrast, NaCl stimulated (Ca+Mg)-ATPase activity without potentiating vanadate inhibition. These effects of cations on ATPase activity and vanadate inhibition resemble properties of certain transport ATPases and thus suggest mechanistic and functional similarities.     

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.     

Mn2+ prevents the Ca2+-induced inhibition of ATP synthesis in brain mitochondria

The differential scanning microcalorimetry and fluorescence methods, using probes ANS and pyrene, have been employed to study thermotropic behaviour of rat liver microsomes in the presence and absence of Mg2+. Addition of Mg2+ yields three partially reversible phase transitions at 18, 27 and 32 degrees C, respectively. A character of Mg2+-induced rearrangements in a membrane and their relation to a catalytic function of a cytochrome P-450-dependent enzymatic system is discussed.     

Mg2+ inhibition of Na2+-stimulated Ca2+ release from brain mitochondria

Magnesium has been shown to modulate the Na+-stimulated release of Ca2+ (Na/Ca exchange) from brain mitochondria. The presence of 5 mM MgCl2 extramitochondrially inhibits the Na/Ca exchange as much as 70%. Additionally, Na+-stimulated Ca2+ release is enhanced by the presence of divalent chelators, this stimulation also being inhibited by the addition of excess Mg2+. The inhibitory effect of Mg2+ and the enhancement by chelating agents were both reversible. Heart mitochondria exhibit a similar enhancement of Na/Ca exchange by chelators and inhibition by MgCl2, though not as pronounced.     

Differential dynamic behavior of actin filaments containing tightly-bound Ca2+ or Mg2+ in the presence of myosin heads actively hydrolyzing ATP.

We have investigated how Ca2+ or Mg2+ bound at the high-affinity cation binding site in F-actin modulates the dynamic response of these filaments to ATP hydrolysis by attached myosin head fragments (S1). Rotational motions of the filaments were monitored using steady-state phosphorescence emission anisotropy of the triplet probe erythrosin-5-iodoacetamide covalently attached to cysteine 374 of actin. The anisotropy of filaments containing only Ca2+ increased from 0.080 to 0.137 upon binding S1 in a rigor complex and decreased to 0.065 in the presence of ATP, indicating that S1 induced additional rotational motions in the filament during ATP hydrolysis. The comparable anisotropy values for Mg(2+)-containing filaments were 0.067, 0.137, and 0.065, indicating that S1 hydrolysis did not induce measurable rotational motions in these filaments. Phalloidin, a fungal toxin which stabilizes F-actin and increases its rigidity, increased the anisotropy of F-actin containing either Ca2+ or Mg2+ but not the anisotropy of the 1:1 S1-actin complexes of these filaments. Mg(2+)-containing filaments with phalloidin bound also displayed increased rotational motions during S1 ATP hydrolysis. A strong positive correlation between the phosphorescence anisotropy of F-actin under specific conditions and the extent of the rotational motions induced by S1 during ATP hydrolysis suggested that the long axis torsional rigidity of F-actin plays a crucial role in modulating the dynamic response of the filaments to ATP hydrolysis by S1. Cooperative responses of F-actin to dynamic perturbations induced by S1 during ATP hydrolysis may thus be physically mediated by the torsional rigidity of the filament.     

Phospholipid requirement of Ca2+-stimulated,Mg2+-dependent ATP hydrolysis in rat brain synaptic membranes

The phospholipid requirement for Ca²⁺-stimulated, Mg²⁺-dependent ATP hydrolysis (Ca²⁺/Mg²⁺-ATPase) and Mg²⁺-stimulated ATP hydrolysis (Mg²⁺-ATPase) in rat brain synaptosomal membranes was studied employing partial delipidation of the membranes with phospholipase A₂ (Hog pancreas), phospholipase C (Bacillus cereus) and phospholipase D (cabbage). Treatment with phospholipase A₂ caused an increase in the activities of both Ca²⁺/Mg²⁺-ATPase and Mg²⁺-ATPase whereas with phospholipase C treatment both the enzyme activities were inhibited. Phospholipase D treatment had no effect on Ca²⁺/Mg²⁺-ATPase but Mg²⁺-ATPase activity was inhibited. Inhibition of Mg²⁺-ATPase activity after phospholipase C treatment was relieved with the addition of phosphatidylinositol-4,5-bisphosphate (PIP₂) and to a lesser extent with phosphatidylinositol-4-phosphate (PIP) and phosphatidylcholine (PC). Phosphatidylserine (PS), phosphatidic acid (PA), PIP and PIP₂ brought about the reactivation of Ca²⁺/Mg²⁺-ATPase. Phosphatidylinositol (PI) and PA inhibited Mg²⁺-ATPase activity.K _ms for Ca²⁺ (0.47 M) and Mg²⁺ (60 M) of the enzyme were found to be unaffected after treatment with the phospholipases.     

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.     

Effects of ATP, Mg2+, and redox agents on the Ca2+ dependence of RyR channels from rat brain cortex

Despite their relevance for neuronal Ca²⁺-induced Ca²⁺ release (CICR), activation by Ca²⁺ of ryanodine receptor (RyR) channels of brain endoplasmic reticulum at the [ATP], [Mg²⁺], and redox conditions present in neurons has not been reported. Here, we studied the effects of varying cis-(cytoplasmic) free ATP concentration ([ATP]), [Mg²⁺], and RyR redox state on the Ca²⁺ dependence of endoplasmic reticulum RyR channels from rat brain cortex. At pCa 4.9 and 0.5 mM adenylylimidodiphosphate (AMP-PNP), increasing free [Mg²⁺] up to 1 mM inhibited vesicular [³H]ryanodine binding; incubation with thimerosal or dithiothreitol decreased or enhanced Mg²⁺ inhibition, respectively. Single RyR channels incorporated into lipid bilayers displayed three different Ca²⁺ dependencies, defined by low, moderate, or high maximal fractional open time (P_o), that depend on RyR redox state, as we have previously reported. In all cases, cis-ATP addition (3 mM) decreased threshold [Ca²⁺] for activation, increased maximal P_o, and shifted channel inhibition to higher [Ca²⁺]. Conversely, at pCa 4.5 and 3 mM ATP, increasing cis-[Mg²⁺] up to 1 mM inhibited low activity channels more than moderate activity channels but barely modified high activity channels. Addition of 0.5 mM free [ATP] plus 0.8 mM free [Mg²⁺] induced a right shift in Ca²⁺ dependence for all channels so that [Ca²⁺] <30 µM activated only high activity channels. These results strongly suggest that channel redox state determines RyR activation by Ca²⁺ at physiological [ATP] and [Mg²⁺]. If RyR behave similarly in living neurons, cellular redox state should affect RyR-mediated CICR. Ca²⁺-induced Ca²⁺ release; Ca²⁺ release channels; endoplasmic reticulum; thimerosal; 2,4-dithiothreitol; ryanodine receptor     

The role of Mg2+ and Ca2+ in the simultaneous binding of vanadate and ATP at the phosphorylation site of sarcoplasmic reticulum Ca2+-ATPase

The sarcoplasmic reticulum Ca²⁺-ATPase was reacted with vanadate in the presence of Mg²⁺ and EGTA, and the effect of Ca²⁺, Mg²⁺ and ATP on the kinetics of vanadate release from the enzyme vanadate complex was studied after dilution with vanadate-free media. Ca²⁺ increased, whereas ATP decreased the rate of vanadate release. In absence of free Mg²⁺ in the release media ATP was bound to the vanadate-reacted Ca²⁺-ATPase with high affinity (K_d 4–5 μM), and full saturation with ATP resulted in complete inhibition of vanadate release. In media containing free Mg²⁺, where ATP predominantly was present as MgATP, binding of the nucleotide to vanadate-reacted Ca²⁺-ATPase occurred with low apparent affinity. Mg²⁺ alone did not affect the rate of vanadate release. At saturating ATP concentrations the release rate in the presence of free Mg²⁺ was less inhibited than in its absence. These results indicate that uncomplexed ATP interacts with the same Mg²⁺ at the catalytic site, which is involved in formation of the enzyme-vanadate complex (EMgV), and thereby hinders dissociation of vanadate. Destabilization of the complex by free Mg²⁺ may be caused by the presence of an additional magnesium ion in the catalytic site together with ATP.     

Regulation of the microtubule-lysosome interaction: activation by Mg2+ and inhibition by ATP

We developed a sedimentation assay to characterize and quantify the association of purified lysosomes to reconstituted microtubules (Mithieux, G., Audebet, C. and Rousset. B. (1988) Biochim. Biophys. Acta 969, 121-130). In the present work, we have examined the potential regulatory role of ATP and Mg2+ on the microtubule-lysosome interaction. The formation of microtubule-lysosome complexes takes place in the absence of Mg2+, but is activated by the addition of Mg2+; both the rate of the interaction and the amount of complexes formed are increased. The maximal effect is observed between 1.5 and 3.5 mM free Mg2+. Measured at the plateau of the interaction, the proportion of microtubules bound to lysosomes increases as a function of free Mg2+ concentration; at optimal concentration of free Mg2+, 90% of the microtubules present in the incubation mixture are bound to lysosomes. ATP induces a concentration-dependent inhibition of the formation of microtubule-lysosome complexes. The half-maximal effect is obtained at an ATP concentration of 0.83 +/- 0.11 mM (n = 7). The effect of ATP is not related to ATP hydrolysis, since ATP exerts its inhibitory action in the presence of EDTA. The ATP effect is mimicked by GTP, p[NH]ppA and tripolyphosphate, ADP and pyrophosphate, but not by AMP or phosphate. In the presence of 1 mM ATP, a Mg2+ concentration of 3 mM (corresponding to 2 mM free Mg2+) is required to overcome the inhibition caused by ATP; above 3 mM, Mg2+ exerts its activating effect. Since the modulating effects of ATP and Mg2+ are obtained at concentrations closed to those occurring in intact cells, we conclude that the regulation of the microtubule-lysosome interaction reported in this paper could be of physiological significance.     

Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum.

ATP and the divalent cations Mg2+ and Ca2+ regulated K+ stimulation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K+-stimulated ATPase activity of the Ca2+ pump by two mechanisms. First, ATP chelated free Mg2+ and, at low ionized Mg2+ concentrations, K+ was shown to be a potent activator of ATP hydrolysis. In the absence of K+ ionized Mg2+ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K+ the concentration of ionized Mg2+ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K+-stimulation. With a saturating concentration of ionized Mg2+, stimulation by K+ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K+ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold. Activation of K+-stimulated ATPase activity by Ca2+ was maximal at an ionized Ca2+ concentration of approx. 1 microM. At very high concentrations of either Ca2+ or Mg2+, basal Ca2+-dependent ATPase activity persisted, but the enzymic response to K+ was completely inhibited. The results provide further evidence that the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme.     

Fluorescence and kinetic studies of the interactions of pyrethroids with the (Ca2(+) + Mg2+)-ATPase

The fluorescence quenching properties of a series of brominated and iodinated pyrethroids have been used to study the binding of pyrethroids to the (Ca2(+) + Mg2+)-ATPase purified from skeletal muscle sarcoplasmic reticulum. It is suggested that binding at the lipid/protein interface of the ATPase is weak but that binding can occur at other (non-annular sites) on the ATPase. Pyrethroids containing either a brominated fatty acyl or iodinated alcohol moiety quench the tryptophan fluorescence of the ATPase, suggesting that the pyrethroids bound to the ATPase adopt a folded conformation with both the acid and alcohol moieties in contact with hydrophobic regions of the ATPase. Whereas effects of the pyrethroids on the activity of the ATPase in bilayers of dioleoylphosphatidylcholine are small, large increases are observed in the activity of the ATPase reconstituted into bilayers of the short-chain phospholipid, dimyristoleoylphosphatidylcholine (DMPC). The rate of phosphorylation of DMPC-ATPase by ATP is slow, but is increased on addition of pyrethroid. The level of phosphorylation of the ATPase by Pi is reduced on reconstitution into bilayers of DMPC, and this is also increased by addition of pyrethroid.     

Insights into the regulation of the ryanodine receptor: differential effects of Mg2+ and Ca2+ on ATP binding

Calcium ions are frequently used second messengers in most living organisms. Members of the family of ryanodine sensitive calcium channels (ryanodine receptors, RyRs) are responsible for many important Ca(2+) signaling events in both excitable and nonexcitable cells. The biological activity of these membrane proteins is modulated and regulated by a great variety of different cellular and extracellular effectors, proteins, and small molecules. However, very little is still understood about how the modulators work on a molecular level. The very large size of more than 2 million Da per functional tetrameric RyR unit and its membrane association have made more detailed biochemical and structural analysis extremely challenging.     

Rat brain Mg2+-ATPase activity and Ca2+ and Mg2+ concentration in the presence of amizil and arecoline]

The effect of benactyzine (the central cholinolytic) in a dose of 40 mg/kg and arecoline (cholinomimetic) in a dose of 2.5 mg/kg on the activity of Mg2+-dependent ATP-ase and the content of Ca2+ and Mg2+ ions in the brain was studied in rats. It was shown that benactyzine and arecoline evoked a biphasic change in the activity of the enzyme and the electrolyte content. A conclusion was drawn that the enzyme inhibition was connected with the accumulation of Ca2+ ions in the brain tissue, whereas its inhibition--with the Mg2+ ion accumulation. It is supposed that throught these effects benactyzine and arecoline influenced the release and retention of the neuromediators in the tissue depot.     

Ca2+ - and Mg2+-dependent ATPase activities in the deoxycholate-treated rat heart sarcolemma

• 1.1. Isolated rat heart sarcolemma was treated with different concentrations of an ionic detergent, deoxycholate (DOC) and ATP hydrolysis in the presence of Ca²⁺ or Mg²⁺ was determined.
• 2.2. Both Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase activities were decreased in the DOC-treated membranes; however, the depression of Mg²⁺-dependent ATPase activity was greater than that of Ca²⁺-dependent ATPase.
• 3.3. The differential changes in Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase activities were apparent when incubations with DOC were carried out for different time intervals and at different temperatures.
• 4.4. In DOC-treated preparations, the K_m value for Ca²⁺-dependent ATPase was decreased whereas that for Mg²⁺-dependent ATPase was increased. The half maximal velocities of the Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase enzyme reactions in the treated preparations were obtained at a DOC: membrane protein ratio of 3.0 and 0.6, respectively.
• 5.5. In the DOC-treated membranes exhibiting the half maximal velocities of enzyme reactions, the K_i value for Ca²⁺-dependent ATPase was drastically reduced but remained unchanged for Mg²⁺-dependent ATPase.
• 6.6. The DOC treatment was associated with a loss of protein as well as phospholipids and resulted in changes in the ultrastructural integrity of the membrane.
• 7.7. Varying degrees of decreases in the activities of sarcolemmal adenylate cyclase. (Na⁻-K⁺)-ATPase. 5'-nucleotidase and calcium binding were seen upon DOC treatment.
• 8.8. The extent of reduction in Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase activities were also different when the membrane was treated with a non-ionic detergent, Lubrol PX.
• 9.9. These data suggest that Ca²⁺-dependent ATPase in heart sarcolemma is more resistant than Mg²⁺-dependent ATPase to detergent treatments and further indicate some differences in the properties of these enzymes.

     

t-Butylhydroperoxide-induced Ca2+ efflux from liver mitochondria in the presence of physiological concentrations of Mg2+ and ATP

Isolated rat liver mitochondria, energized either by succinate oxidation or by ATP hydrolysis, present a transient increase in the rate of Ca2+ efflux concomitant to NAD(P)H oxidation by hydroperoxides when suspended in a medium containing 3 mM ATP, 4 mM Mg2+ and acetate as permeant anion. This is paralleled by an increase in the steady-state concentration of extramitochondrial Ca2+, a small decrease in delta psi and an increase in the rate of respiration and mitochondrial swelling. With the exception of mitochondrial swelling all other events were found to be reversible. If Ca2+ cycling was prevented by ruthenium red, the changes in delta psi, the rate of respiration and the extent of mitochondrial swelling were significantly diminished. In addition, there was no significant decrease in the content of mitochondrial pyridine nucleotides. Mitochondrial coupling was preserved after a cycle of Ca2+ release and re-uptake under these experimental conditions. It is concluded that hydroperoxide-induced Ca2+ efflux from intact mitochondria is related to the redox state of pyridine nucleotides.     

Localization of (Ca2+ + Mg2+)-ATPase, Ca2+ pump and other ATPase activities in cardiac sarcolemma

N-Ethylmaleimide was employed as a surface label for sarcolemmal proteins after demonstrating that it does not penetrate to the intracellular space at concentrations below 1.10(-4) M. The sarcolemmal markers, ouabain-sensitive (Na+ +K+)-ATPase and Na+/Ca2+-exchange activities, were inhibited in N-ethylmaleimide perfused hearts. Intracellular activities such as creatine phosphokinase, glutamate-oxaloacetate transaminase and the internal phosphatase site of the Na+ pump (K+-p-nitrophosphatase) were not affected. Almost 20% of the (Ca2+ +Mg2+)-ATPase and Ca2+ pump were inhibited indicating the localization of a portion of this activity in the sarcolemma. Sarcolemma purified by a recent method (Morcos, N.C. and Drummond, G.I. (1980) Biochim. Biophys. Acta 598, 27-39) from N-ethylmaleimide-perfused hearts showed loss of approx. 85% of its (Ca2+ +Mg2+-ATPase and Ca2+ pump compared to control hearts. (Ca2+ +Mg2+)-ATPase and Ca2+ pump activities showed two classes of sensitivity to vanadate ion inhibition. The high vanadate affinity class (K1/2 for inhibition approx. 1.5 microM) may be localized in the sarcolemma and represented approx. 20% of the total inhibitable activity in agreement with estimates from N-ethylmaleimide studies. Sucrose density fractionation indicated that only a small portion of Mg2+-ATPase and Ca2+-ATPase may be associated with the sarcolemma. The major portion of these activities seems to be associated with high density particles.