Activation of the ATP.Mg-dependent type 1 protein phosphatase by the Fe2+/ascorbate system

The ATP.Mg-dependent type 1 protein phosphatase is inactive as isolated but can be activated in several different ways. In this report, we show that the phosphatase can also be activated by the Fe²⁺/ascorbate system. Activation of the phosphatase requires both Fe²⁺ ion and ascorbate and the level of activation is dependent on the concentrations of Fe²⁺ ion and ascorbate. In the presence of 20 mM ascorbate, the Fe²⁺ ion concentrations required for half-maximal and maximal activation are about 0.3 and 3mM, respectively. Several common divalent metal ions, including Co²⁺, Ni²⁺, Cu²⁺, Mg²⁺, and Ca²⁺ ions, cannot cooperate with ascorbate to activate the phosphatase, and SH-containing reducing agents such as 2-mercaptoethanol and dithiothreitol cannot cooperate with Fe²⁺ ion to activate the phosphatase, indicating that activation of the phosphatase by the Fe²⁺/ascorbate system is a specific process. Moreover, H₂O₂, a strong oxidizer, could significantly diminish the phosphatase activation by the Fe²⁺/ascorbate system, suggesting that reduction mechanism other than SH-SS interchange is a prerequisite for the Fe²⁺/ascorbate-mediated phosphatase activation. Taken together, the present study provides initial evidence for a new mode of type 1 protein phosphatase activation mechanism.     

MOUSE NEUROBLASTOMA CELL ADENYLATE CYCLASE: REGULATION BY 2-CHLOROADENOSINE, PROSTAGLANDIN E1 AND THE CATIONS Mg2+, Ca2+ AND Mn

—Some basic kinetic properties of adenylate cyclase in cell free preparations of mouse neuroblastoma were investigated. Production of cAMP from ATP by the enzyme requires the presence of either Mg²⁺ or Mn²⁺ in addition to ATP. In the presence of Mg²⁺, the K_m for ATP is 120 ± 15 μM and the interaction of ATP and adenylate cyclase appears to be non-cooperative (Hill coefficient of 1). Magnesium ion concentrations in excess of the ATP concentration cause stimulation although similar excess concentrations of Mn²⁺ cause inhibition. Prostaglandin E₁ and 2-chloroadenosine activate the enzyme. The K_m of the cyclase for 2-chloroadenosine is 6 μm . Activation by 2-chloroadenosine leads to an increase in V_max but does not effect the K_m for ATP. At a fixed ATP concentration, the extent of activation caused by prostaglandin E₁ and 2-chloroadenosine is inversely related to the Mg²⁺ concentration. Calcium ion causes inhibition of adenylate cyclase from 0.1 to 4mM with a K_i of 5 ± 10^?4m . Ca²⁺ interaction with the enzyme in the absence or presence of either 2-chloroadenosine or prostaglandin E₁ appears cooperative (i.e. Hill coefficients of ?2). Ca²⁺ inhibition is non-competitive with respect to either ATP or 2-chloroadenosine but is progressively diminished by increasing Mn²⁺ concentrations. Divalent cation effects and activation by 2-chloroadenosine and prostaglandin E₁ of the neuroblastoma adenylate cyclase are compared with ion effects and hormone activation of the enzyme obtained from non-neuronal tissue.     

Oxidative damage to sarcoplasmic reticulum Ca2+-pump induced by Fe2+/H2O2/ascorbate is not mediated by lipid peroxidation or thiol oxidation and leads to protein fragmentation

The major protein in the sarcoplasmic reticulum (SR) membrane is the Ca²⁺ transporting ATPase which carries out active Ca²⁺ pumping at the expense of ATP hydrolysis. The aim of this work was to elucidate the mechanisms by which oxidative stress induced by Fenton's reaction (Fe²⁺ + H₂O₂ HO· + OH^–+ Fe³⁺) alters the function of SR. ATP hydrolysis by both SR vesicles (SRV) and purified ATPase was inhibited in a dose-dependent manner in the presence of 0–1.5 MM H₂O₂ plus 50 M Fe²⁺ and 6 mM ascorbate. Ca²⁺ uptake carried out by the Ca²⁺-ATPase in SRV was also inhibited in parallel. The inhibition of hydrolysis and Ca²⁺ uptake was not prevented by butylhydroxytoluene (BHT) at concentrations which significantly blocked formation of thiobarbituric acid-reactive substances (TBARS), suggesting that inhibition of the ATPase was not due to lipid peroxidation of the SR membrane. In addition, dithiothreitol (DTT) did not prevent inhibition of either ATPase activity or Ca²⁺ uptake, suggesting that inhibition was not related to oxidation of ATPase thiols. The passive efflux of ⁴⁵Ca²⁺ from pre-loaded SR vesicles was greatly increased by oxidative stress and this effect could be only partially prevented (ca 20%) by addition of BHT or DTT. Trifluoperazine (which specifically binds to the Ca²⁺-ATPase, causing conformational changes in the enzyme) fully protected the ATPase activity against oxidative damage. These results suggest that the alterations in function observed upon oxidation of SRV are mainly due to direct effects on the Ca²⁺-ATPase. Electrophoretic analysis of oxidized Ca²⁺-ATPase revealed a decrease in intensity of the silver-stained 110 kDa Ca²⁺-ATPase band and the appearance of low molecular weight peptides (MW < 100 kDa) and high molecular weight protein aggregates. Presence of DTT during oxidation prevented the appearance of protein aggregates and caused a simultaneous increase in the amount of low molecular weight peptides. We propose that impairment of function of the Ca²⁺-pump may be related to aminoacid oxidation and fragmentation of the protein.Abbreviations AcP acetylphosphate - BHT butylhydroxytoluene - DTT dithiothreitol - Hepes 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - SDS sodium dodecyl sulfate - SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate - SR sarcoplasmic reticulum - SRV sarcoplasmic reticulum vesicles - TBA thiobarbituric acid - TBARS thiobarbituric acid-reactive substances - TFP trifluoperazine     

Structural Organization and Energy Transduction Mechanism of Na+,K+-ATPase Studied with Transition Metal-Catalyzed Oxidative Cleavage

This chapter describes contributions of transition metal-catalyzed oxidative cleavage of Na⁺,K⁺-ATPase to our understanding of structure–function relations. In the presence of ascorbate/H₂O₂, specific cleavages are catalyzed by the bound metal and because more than one peptide bond close to the metal can be cleaved, this technique reveals proximity of the different cleavage positions within the native structure. Specific cleavages are catalyzed by Fe²⁺ bound at the cytoplasmic surface or by complexes of ATP–Fe²⁺, which directs the Fe²⁺ to the normal ATP–Mg²⁺ site. Fe²⁺- and ATP–Fe²⁺-catalyzed cleavages reveal large conformation-dependent changes in interactions between cytoplasmic domains, involving conserved cytoplasmic sequences, and a change of ligation of Mg²⁺ ions between E₁P and E₂P, which may be crucial in facilitating hydrolysis of E₂P. The pattern of domain interactions in E₁ and E₂ conformations, and role of Mg²⁺ ions, may be common to all P-type pumps. Specific cleavages can also be catalyzed by Cu²⁺ ions, bound at the extracellular surfaces, or a hydrophobic Cu²⁺-diphenyl phenanthroline (DPP) complex, which directs the Cu²⁺ to the membrane–water interface. Cu²⁺- or Cu²⁺-DPP-catalyzed cleavages are providing information on / subunit interactions and spatial organization of transmembrane segments. Transition metal-catalyzed cleavage could be widely used to investigate other P-type pumps and membrane proteins and, especially, ATP binding proteins.     

Potassium-p-nitrophenyl phosphate interactions with (Na+ + K+)-ATPase their relevance to phosphatase activity

The K⁺-dependent p-nitrophenylphosphatase activity catalyzed by purified (Na⁺ + K⁺)-ATPase from pig kidney shows substrate inhibition (K_i about 9.5 mM at 2.1 mM Mg²⁺). Potassium antagonizes and sodium favours this inhibition. In addition, K⁺ reduces the apparent affinity for substrate activation, whereas p-nitrophenyl phosphate reduces the apparent affinity for K⁺ activation. In the absence of Mg²⁺, p-nitrophenyl phosphate, as well as ATP, accelerates the release of Rb⁺ from the Rb⁺ occluded unphosphorylated enzyme. With no Mg²⁺ and with 0.5 mM KCl, trypsin inactivation of (Na⁺ + K⁺)-ATPase as a function of time follows a single exponential but is transformed into a double exponential when 1 mM ATP or 5 mM p-nitrophenyl phosphate are also present. In the presence of 3 mM MgCl₂, 5 mM p-nitrophenyl phosphate and without KCl the trypsin inactivation pattern is that described for the E₁ enzyme form; the addition of 10 mM KCl changes the pattern which, after about 6 min delay, follows a single exponential. These results suggest that (i) the shifting of the enzyme toward the E₁ state is the basis for substrate inhibition of the p-nitrophenulphosphatase acitivy of (Na⁺ + K⁺)-ATPase, and (ii) the substrate site during phosphatase activity is distinct from the low-affinity ATP site.     

Involvement of P2X receptors in the NAD+-induced rise in [Ca2+]i in human monocytes

In the present study, we show that the extracellular addition of nicotinamide adenine dinucleotide (NAD⁺) induces a transient rise in [Ca²⁺]_i in human monocytes caused by an influx of extracellular calcium. The NAD⁺-induced Ca²⁺ response was prevented by adenosine triphosphate (ATP), suggesting the involvement of ATP receptors. Of the two subtypes of ATP receptors (P2X and P2Y), the P2X receptors were considered the most likely candidates. By the use of subtype preferential agonists and antagonists, we identified P2X₁, P2X₄, and P2X₇ receptors being engaged in the NAD⁺-induced rise in [Ca²⁺]_i. Among the P2X receptor subtypes, the P2X₇ receptor is unique in facilitating the induction of nonselective pores that allow entry of ethidium upon stimulation with ATP. In monocytes, opening of P2X₇ receptor-dependent pores strongly depends on specific ionic conditions. Measuring pore formation in response to NAD⁺, we found that NAD⁺ unlike ATP lacks the ability to induce this pore-forming response. Whereas as little as 100 μM ATP was sufficient to activate the nonselective pore, NAD⁺ at concentrations up to 2 mM had no effect. Taken together, these data indicate that despite similarities in the action of extracellular NAD⁺ and ATP there are nucleotide-specific variations. So far, common and distinct features of the two nucleotides are only beginning to be understood.     

Mechanisms of Saccharomyces Cerevisiae PMA1 H+-ATPase inactivation by Fe2+, H2O2 and Fenton reagents

Although considerably more oxidation-resistant than other P-type ATPases, the yeast PMA1 H⁺-ATPase of Saccharomyces cerevisiae SY4 secretory vesicles was inactivated by H₂O₂, Fe²⁺, Fe- and Cu-Fenton reagents. Inactivation by Fe²⁺ required the presence of oxygen and hence involved auto-oxidation of Fe²⁺ to Fe³⁺. The highest Fe^2- (100 μM) and H₂O₂ (100 mM) concentrations used produced about the same effect. Inactivation by the Fenton reagent depended more on Fe²⁺ content than on H₂O₂ concentration, occurred only when Fe²⁺ was added to the vesicles first and was only slightly reduced by scavengers (mannitol, Tris, NaN₃, DMSO) and by chelators (EDTA, EGTA, DTPA, BPDs, bipyridine, 1, 10-phenanthroline). Inactivation by Fe- and Cu- Fenton reagent was the same; the identical inactivation pattern found for both reagents under anaerobic conditions showed that both reagents act via OH^·. The lipid peroxidation blocker BHT prevented Fenton-induced rise in lipid peroxidation in both whole cells and in isolated membrane lipids but did not protect the H⁺-ATPase in secretory vesicles against inactivation. ATP partially protected the enzyme against peroxide and the Fenton reagent in a way resembling the protection it afforded against SH-specific agents. The results indicate that Fe²⁺ and the Fenton reagent act via metal-catalyzed oxidation at specific metal-binding sites, very probably SH-containing amino acid residues. Deferrioxamine, which prevents the redox cycling of Fe²⁺, blocked H⁺-ATPase inactivation by Fe²⁺ and the Fenton reagent but not that caused by H₂O₂, which therefore seems to involve a direct non-radical attack. Fe-Fenton reagent caused fragmentation of the H⁺-ATPase molecule, which, in Western blots, did not give rise to defined fragments bands but merely to smears.     

The lactate-dependent enhancement of hydroxyl radical generation by the Fenton reaction

The effect of lactic acid (lactate) on Fenton based hydroxyl radical (^·OH) production was studied by spin trapping, ESR, and fluorescence methods using DMPO and coumarin-3-carboxylic acid (3-CCA) as the ^·OH traps respectively. The ^·OH adduct formation was inhibited by lactate up to 0.4mM (lactate/iron stoichiometry = 2) in both experiments, but markedly enhanced with increasing concentrations of lactate above this critical concentration. When the H₂O₂ dependence was examined, the DMPO-OH signal was increased linearly with H₂O₂ concentration up to 1 mM and then saturated in the absence of lactate. In the presence of lactate, however, the DMPO-OH signal was increased further with higher H₂O₂ concentration than 1 mM, and the saturation level was also increased dependent on lactate concentration. Spectroscopic studies revealed that lactate forms a stable colored complex with Fe³⁺ at lactate/Fe³⁺ stoichiometry of 2, and the complex formation was strictly related to the DMPO-OH formation. The complex formation did not promote the H₂O₂ mediated Fe³⁺ reduction. When the Fe³⁺-lactate (1:2) complex was reacted with H₂O₂, the initial rate of hydroxylated 3-CCA formation was linearly increased with H₂O₂ concentrations. All the data obtained in the present experiments suggested that the Fe³⁺-lactate (1:2) complex formed in the Fenton reaction system reacts directly with H₂O₂ to produce additional ^·OH in the Fenton reaction by other mechanisms than lactate or lactate/Fe³⁺ mediated promotion of Fe³⁺/Fe²⁺ redox cycling.     

A comparative study of the rat heart sarcolemmal Ca2+-dependent ATPase and myosin ATPase

In order to gain some information regarding Ca²⁺-dependent ATPase, the enzyme was purified from cardiac sarcolemma and its properties were compared with Ca²⁺-ATPase activity of myosin purified from rat heart. Both Ca²⁺-dependent ATPase and myosin ATPase were stimulated by Ca²⁺ but the maximal activation of Ca²⁺-dependent ATPase required 4 mM Ca²⁺ whereas that of myosin ATPase required 10 mM Ca²⁺. These ATPases were also activated by other divalent cations in the order of Ca²⁺ > Mn²⁺ > Sr²⁺ > Br²⁺ > Mg²⁺; however, there was a marked difference in the pattern of their activation by these cations. Unlike the myosin ATPase, the ATP hydrolysis by Ca²⁺-dependent ATPase was not activated by actin. The pH optima of Ca²⁺-dependent ATPase and myosin ATPase were 9.5 and 6.5 respectively. Na⁺ markedly inhibited Ca²⁺-dependent ATPase but had no effect on the myosin ATPase activity. N-ethylmaleimide inhibited Ca²⁺-dependent ATPase more than myosin ATPase whereas the inhibitory effect of vanadate was more on myosin ATPase than Ca²⁺-dependent ATPase. Both Ca²⁺-dependent ATPase and myosin ATPase were stimulated by K-EDTA and NH₄-EDTA. When myofibrils were treated with trypsin and passed through columns similar to those used for purifying Ca²⁺-ATPase from sarcolemma, an enzyme with ATPase activity was obtained. This myofibrillar ATPase was maximally activated at 3–4 mM Ca²⁺ and 3 to 4 mM ATP like sarcolemmal Ca²⁺-dependent ATPase. K⁺ stimulated both ATPase activities in the absence of Ca²⁺ and inhibited in the presence of Ca²⁺. Both enzymes were inhibited by Na⁺, Mg²⁺, La³⁺, and azide similarly. However, Ca²⁺ ATPase from myofibrils showed three peptide bands in SDS polyacrylamide gel electrophoresis whereas Ca²⁺ ATPase from sarcolemma contained only two bands. Sarcolemmal Ca²⁺-ATPase had two affinity sites for ATP (0.012 mM and 0.23 mM) while myofibrillar Ca²⁺-ATPase had only one affinity site (0.34 mM). Myofibrillar Ca²⁺-ATPase was more sensitive to maleic anhydride and iodoacetamide than sarcolemmal Ca²⁺-ATPase. These observations suggest that Ca²⁺-dependent ATPase may be a myosin like protein in the heart sarcolemma and is unlikely to be a tryptic fragment of myosin present in the myofibrils.     

The calmodulin-activated form of the Ca2+-pumping ATPase of the cardiac sarcolemmal membrane produces Ca2+ gradients with a thermodynamic efficiency of 100%

The thermodynamic efficiency of the calmodulin-activated form of the Ca²⁺-pumping ATPase of the bovine cardiac sarcolemma (SL) was evaluated in sealed vesicles under reversible conditions. The free internal Ca²⁺ concentration ([Ca²⁺]_i) established in the SL vesicle lumen by action of the ATPase was determined as a function of the [ATP]/([ADP][P_i]) ratio for the following experimental conditions: 250mM sucrose, 100mM KCI, 0.1mM Mg²⁺, 25mM HEPES, 25mM Tris, pH 7.40, at 37°C, [Ca²⁺]_o=50nM (1mM Ca/EGTA buffer), 0.75mM Mg-ATP, 0.1mM P_i, variable [ADP]. Under these conditions, with the pump working near itsK _m of 64nM, the [Ca²⁺]_i achieved was 18mM, decreasing with increasing [ADP] for [ADP] 0.84mM. A plot of the square of the [Ca²⁺]_i/[Ca²⁺]_o ratio against [ATP]/([ADP][P_i]) gave a straight line with a slope of 1.5×10⁷M. This was in agreement, within the experimental error, with the equilibrium constant for ATP hydrolysis under these conditions (1.09×10⁷M). These results demonstrate (1) tight coupling between Ca²⁺ transport and ATP hydrolysis with a stoichiometry of 2 Ca²⁺ moved per ATP split and (2) a low degree of passive leakage. Analysis at low [ADP] (<0.83mM) showed the unexpected result that ADP increases the rate of theforward reaction of the pump. The maximal effect on the initial rate is a 96±5% increase, with an EC₅₀ of approximately 0.4mM (ADP). Similar but lesser stimulation was observed with CDP. The implications of the above results for the energetics of the pump and for its physiological function in the beating heart are discussed.     

Effect of Mg2+ concentrations on phosphorylation/activation of phosphorylase b kinase by cAMP/Ca2+-independent, autophosphorylation-dependent protein kinase

In a previous report (Yu and Yang,Biochem. Biophys. Res. Commun. 207, 140–147 (1995)], phosphorylase b kinase from rabbit skeletal muscle was found to be phosphorylated and activated by a cyclic nucleotide- and Ca²⁺-independent protein kinase previously identified as an autophosphorylation-dependent multifunctional protein kinase (autokinase) from brain and liver (Yanget al, J. Biol. Chem. 262, 7034–7040, 9421–9427 (1987)]. In this report, the effect of Mg²⁺ ion concentration on the auto-kinase-catalyzed activation of phosphorylase b kinase is investigated. The levels of phosphorylation and activation of phosphorylase b kinase catalyzed by auto-kinase are found to be dependent on the concentration of Mg²⁺ ion used. Phosphorylation of phosphorylase b kinase at high Mg²⁺ ion (>9 mM) is 2–3 times higher than that observed at low Mg²⁺ ion (1 mM) and this results in a further 2- to 3-fold activation of the enzyme activity at high Mg²⁺ ion. Analysis of the phosphorylation stoichiometry of and subunits of phosphorylase b kinase at different Mg²⁺ ion concentrations further reveals that the phosphorylation level of the subunit remains almost unchanged, whereas the phosphorylation level of the subunit increases dramatically and correlates with the increased enzyme activity. In similarity with the subunit, phosphorylations of myelin basic protein and histone 2A by auto-kinase are also unaffected by Mg²⁺ ion. Taken together, the results provide initial evidence that Mg²⁺ ion may specifically render thea subunit a better substrate for auto-kinase to cause further phosphorylation/activation of phosphorylase b kinase, representing a new mode of control mechanism for the regulation of auto-kinase involved in the phosphorylation and concurrent activation of phosphorylase b kinase.     

Regulation of Na/Mg antiport in rat erythrocytes

In rat erythrocytes, the regulation of Na⁺/Mg²⁺ antiport by protein kinases (PKs), protein phosphatases (PPs), intracellular Mg²⁺, ATP and Cl⁻ was investigated. In untreated erythrocytes, Na⁺/Mg²⁺ antiport was slightly inhibited by the PK inhibitor staurosporine, slightly stimulated by the PP inhibitor calyculin A and strongly stimulated by vanadate. PMA stimulated Na⁺/Mg²⁺ antiport. This effect was completely inhibited by staurosporine and partially inhibited by the PKC inhibitors Ro-31-8425 and BIM I. Participation of other PKs such as PKA, the MAPK cascade, PTK, CK I, CK II, CAM II-K, PI 3-K, and MLCK was excluded by use of inhibitors. Na⁺/Mg²⁺ antiport in rat erythrocytes can thus be stimulated by PKCα.In non-Mg²⁺-loaded erythrocytes, ATP depletion reduced Mg²⁺ efflux and PMA stimulation in NaCl medium. A drastic activation of Na⁺/Mg²⁺ antiport was induced by Mg²⁺ loading which was not further stimulated by PMA. Staurosporine, Ro-31-8425, BIM I and calyculin A did not inhibit Na⁺/Mg²⁺ antiport of Mg²⁺-loaded cells. Obviously, at high [Mg²⁺]_i Na⁺/Mg²⁺ antiport is maximally stimulated. PKC_α or PPs are not involved in stimulation by intracellular Mg²⁺. ATP depletion of Mg²⁺-loaded erythrocytes reduced Mg²⁺ efflux and the affinity of Mg²⁺ binding sites of the Na⁺/Mg²⁺ antiporter to Mg²⁺. In non-Mg²⁺-loaded erythrocytes Na⁺/Mg²⁺ antiport essentially depends on Cl⁻. Mg²⁺-loaded erythrocytes were less sensitive to the activation of Na⁺/Mg²⁺ antiport by [Cl⁻]_i.     

Implications for in vitro studies of the autoxidation of ferrous ion and the iron-catalyzed autoxidation of dithiothreitol

The influences of buffers and iron chelators on the rate of autoxidation of Fe²⁺ were examined in the pH range 6.0–7.4. The catalysis by Fe²⁺ and Fe³⁺ of the autoxidation of dithiothreitol was also investigated. In buffers which are non- or poor chelators of iron, 0.25 mM Fe²⁺, and 0.3 mM dithiothreitol when present with iron, oxidize within minutes at pH 7.4 and 30°C. The stability of each increases as the pH is decreased and more than 90% of each remains after 1 h at pH 6.0. In the presence of buffers or oxy-ligands which preferentially and strongly chelate Fe³⁺ over Fe²⁺, Fe²⁺ autoxidizes rapidly in the pH range 6.0–7.4 while dithiothreitol is protected. Ligands which preferentially bind strongly to Fe²⁺ stabilize both Fe²⁺ and dithiothreitol at pH 7.4. Dithiothreitol readily reduces Fe³⁺ in non-chelating buffers or in the presence of strong chelators of Fe²⁺, however, the ferrous ions produced are prone to reoxidation at higher pH values. These results show that Fe²⁺ and dithiothreitol are very susceptible to autoxidation in the neutral pH range, and that the rates are strongly influenced by the presence of chelators of Fe²⁺ and Fe³⁺. The rapid autoxidations of these species need to be taken into account when designing and interpreting experiments involving Fe²⁺ or both dithiothreitol and iron.     

Role of the deinhibitor protein in the interconversion of the ATP,Mg-dependent protein phosphatase

The small molecular weight (± 9,000) heat stable deinhibitor protein, isolated from dog liver, not only protects the multisubstrate protein phosphatase from inhibition by inhibitor-1 and the modulator protein. It prevents the conversion of the active enzyme to the ATP,Mg-dependent enzyme form brought about by the modulator protein, and also affects the activation of the ATP,Mg-dependent protein phosphatase, probably by stabilizing the enzyme in its active conformation during the reversible activation by protein kinase F_A. Therefore the deinhibitor protein could be an important factor in the process of glycogen synthesis, which requires glycogen synthase and phosphorylase as dephosphorylated enzymes.     

Active transport of Ca in membrane vesicles from pea: Evidence for a H/Ca antiport

Two non mitochondrial systems involved in ATP-dependent Ca²⁺ accumulation have been described and characterized in two membrane fractions from pea internodes purified on a metrizamide-sucrose discontinuous gradient. In the lighter membrane fraction an ATP-dependent Ca²⁺ accumulation system, which shows the characteristics of an ATP-dependent H⁺/Ca²⁺ antiport, predominates. This system is inhibited by FCCP and nigericin and stimulated by 50 mM KCl. It is saturated by 0.8–1.0 mM MgSO₄-ATP, strictly requires ATP and is severely inhibited by an excess of free Mg²⁺ or Mn²⁺. A second system of ATP-dependent Ca²⁺ accumulation, recovered mainly in the heavier membrane fraction, is insensitive to FCCP, is saturated by 8–10 mM MgSO₄-ATP, can utilize also ITP or other nucleoside triphosphates although at lower rate than ATP and is only scarcely affected by an excess of free Mg²⁺ or Mn²⁺. This system is interpreted as corresponding to the (Ca²⁺ + Mg²⁺)-ATPase described by Dieter, P. and Marmé, D. ((1980) Planta 150, 1–8).     

A simple and defined method to study calcification by isolated matrix vesicles effect of ATP and vesicle phosphatase

A simplified and defined system was developed to study in vitro calcium phosphate deposition by isolated matrix vesicles from rabbit growth plate cartilage, and to examine the relationship between vesicle phosphatase and calcium deposition. Samples of suspended vesicles containing 25 μg of protein, were incubated for 2 h in a ⁴⁵Ca-labelled solution with 2.2 mM Ca²⁺, 1.6 mM PO₄^3? and 1 mM ATP at pH 7.6. Calcium deposition was related to the amount of PO₄ hydrolysed by matrix vesicle phosphatases from ATP and other phosphate esters. Ca²⁺ or Mg²⁺ was found to stimulate matrix vesicle. ATPase, but the hydrolysis of phosphoenolpyruvate, glucose 1-phosphate, β-glycerol phosphate and AMP was independent of either cation. All of the above substrates supported calcium deposition. 1 mM ATP was more effective than 5 mM in supporting calcium deposition, indicating inhibition of mineralization at higher ATP concentrations. Our results suggest that, in addition to concentrating calcium, veiscles provide phosphate from ATP for mineral formation and at the same time remove the inhibitory effect of ATP upon mineral deposition.     

Reversible sporicidal action of cuprous ions

At 10 mM, Cu⁺ was highly protective against killing of spores of Bacillus megaterium ATCC 19213 by H₂O₂, while at higher concentrations, from 15–100 mM, killing was augmented. In contrast, Cu²⁺, Fe²⁺, Fe³⁺, Co²⁺ or Co³⁺ ions acted only protectively. Cu⁺ itself was sporicidal in the absence of H₂O₂ or ascorbate, and its sporicidal action did not depend on generation of highly reactive oxygen species. It appeared that killing involved either inhibition of germination or copper toxicity to germinated cells in that Cu⁺-inactivated spores did not germinate readily but chemical decoating of the cells prior to plating on a solid medium resulted in reversal of the sporicidal effect. Received 12 July 1996/ Accepted in revised form 03 November 1996     

Acid Metallophosphatase from the Ameba Amoeba proteus

Tartrate-resistant acid phosphatase (TR-AcPh) from the ameba Amoeba proteus is represented by 3 bands (electromorphs) revealed after disk-electrophoresis in PAAG, using 2-naphthylphosphate as substrate. The presence of 50 mmol/l MgCl₂ or CaCl₂ in the incubation mixture increases activities of all electromorphs of TR-AcPh, while of ZnCl₂, of two of them. The activity of the TR-AcPh electromorphs also rose after the 30-min incubation of the gels in MgCl₂, CaCl₂ or ZnCl₂ (10 and 100 mM) before gel staining. However, 1 M ZnCl₂, unlike 1 M CaCl₂ or 1 M MgCl₂, partly inactivated two out of three TR-AcPh electromorphs. The TR-AcPh electromorphs were inhibited by 1,10-phenanthroline (1,10-Ph), EDTA, and EGTA (all at a concentration of 5 mM) faster than by H₂O₂ (10 mM). The inactivation of the TR-AcPh electromorphs by the chelating agents did not depend (EGTA) or nearly did not depend (EDTA, 1,10-Ph) on their concentration (0.05, 0.5, and 5 mM). Out of 5 tested ions (Mg²⁺, Ca²⁺, Fe²⁺, Fe³⁺, and Zn²⁺), only Zn ions reactivated the TR-AcPh electromorphs inactivated by 1,10-Ph, EDTA or EGTA. The TR-AcPh electromorphs were reactivated worse after inactivation by EGTA than by EDTA or 1,10-Ph. It is suggested that the active site of TR-AcPh contains the zinc ion essential for catalytic activity of this enzyme, i.e., TR-AcPh of A. proteus is a metallophosphatase performing the phosphomonoesterase activity in acidic medium.     

Competitive inhibition of hydrogen peroxide-induced aggregation of calf platelets by prostaglandin H2/thromboxane A2 receptor ligands

Hydrogen peroxide (H₂O₂)-induced aggregation of calf platelets and its modification by agents with specific properties were characterized employing a spectrophotometric assay. An Arrhenius activation energy of 20 ± 1 kcal/mol was found in the temperature range of 25‡-36‡C. Rate inhibition occurred on either side of this temperature range, and under anaerobic conditions. Exogenous Ca²⁺ ions were not required but Ca²⁺ ions, at 1 mM-concentration, optimally increased rates and extent of aggregation at suboptimal H₂O₂ concentrations but only extent of aggregation at optimal H₂O₂ concentrations. Ba²⁺, Sr²⁺, Cd²⁺, Mn²⁺ and Ni²⁺ ions (1 mM) and Zn²⁺, Pb²⁺ and Hg²⁺ ions (10 mM) were inhibitory. The cyclo-oxygenase inhibitor, indomethacin (10-30 mM) exerted only mild inhibition by a competitive mechanism. Another cyclo-oxygenase inhibitor, aspirin, functioned to increase aggregation. Ligands acting directly at the prostaglandin H₂/thromboxane A, receptor (5Z. 9, 11, 13E, 15(S) 15-hydroxy 9(11) epoxy methano prosta 5, 13-dien-1-oic acid, pinane thromboxane A₂, arachidonic acid, eicosapentaenoic acid, and N-ethylmaleimide) functioned as competitive inhibitors. Another platelet-activating sulphydryl reagent, thimerosal, also inhibited competitively while the protein kinase C inhibitor, sphingosine, and the protein kinase C modulator, Zn²⁺ ions, inhibited by different mechanisms. The results indicate direct action of H₂O₂ at the prostaglandin H₂/thromboxane A₂ receptor, possibly its sulphydryls, to activate the protein kinase C pathway, independently of cyclo-oxygenase products. The results underscored the power of the kinetic approach for investigating mechanisms of platelet activation.     

Dissociation between Ca2+-ATPase and alkaline phosphatase activities in plasma membranes of rat duodenum

The presence of Ca²⁺-ATPase activities with high-affinity sites for Ca²⁺ in brush border as well as basolateral plasma membranes of rat duodenal epithelium has been reported previously (Ghijsen, W.E.J.M. and van Os, C.H. (1979) Nature 279, 802–803). Since both plasma membranes contain alkaline phosphatase (EC 3.1.3.1), which also can be stimulated by Ca²⁺, the substrate specificity of Ca²⁺-induced ATP-hydrolysis has been studied to determine whether or not alkaline phosphatase and Ca²⁺-ATPase are two distinct enzymes. In basolateral fragments, the rate of Ca²⁺-dependent ATP-hydrolysis was greater than that of ADP, AMP and $\text{p-}\text{nitrophenylphosphate}$ at Ca²⁺ concentrations below 25 μM. At 0.2 mM Ca²⁺ the rates of ATP, ADP, AMP and $\text{p-}\text{nitrophenylphosphate}$ hydrolysis were not significantly different. In brush border fragments the rates of ATP, ADP and AMP hydrolysis were identical at low Ca²⁺, but at 0.2 mM Ca²⁺, Ca²⁺-induced hydrolysis of ADP and AMP was greater than either ATP or $\text{p-}\text{nitrophenylphosphate}$. Alkaline phosphatase in brush border and basolateral membranes was inhibited by 75% after addition of 2.5 mM theophylline. Ca²⁺-stimulated ATP hydrolysis at 1 μM Ca²⁺ was not sensitive to theophylline in basolateral fragments while the same activity in brush border fragments was totally inhibited. At 0.2 mM Ca²⁺, Ca²⁺-induced ATP hydrolysis in both basolateral and brush border membranes was sensitive to theophylline. Oligomycin and azide had no effect on Ca²⁺-stimulated ATP hydrolysis, either at low or at high Ca²⁺ concentrations. Chlorpromazine fully inhibited Ca²⁺-stimulated ATP hydrolysis in basolateral fragments at 5 μM Ca²⁺, while it had no effect in brush border fragments. From these results we conclude that, (i) Ca²⁺-ATPase and alkaline phosphatase are two distinct enzymes, (ii) high-affinity Ca²⁺-ATPase is exclusively located in basolateral plasma membranes, (iii) alkaline phosphatase activity, present on both sides of duodenal epithelium, is stimulated slightly by low Ca²⁺ concentrations, but this Ca²⁺-induced activity is inhibited by theophylline and shows no specificity with respect to ATP, ADP or AMP.