ATP-induced sucrose efflux from red-beet tonoplast vesicles

 Sucrose efflux from the vacuole of mobilizing red-beet (Beta vulgaris L.) hypocotyl cells was investigated using purified tonoplast vesicles. Tonoplast vesicle purity was assured by the immunoreactivity to antibodies raised against the vacuolar ATPase and by the strong inhibition exhibited by the H⁺-ATPase to bafilomycin-A and NO₃ ⁻. Inhibition of the H⁺-ATPase by vanadate and azide was negligible. Sucrose was loaded into tonoplast vesicles by using the pH-jump method of energization. Addition of ATP to sucrose-loaded vesicles in the presence of bafilomycin-A resulted in efflux of a significant amount of sucrose. During ATP-induced sucrose efflux, bafilomycin-insensitive ATPase activity increased significantly with no increase in H⁺-translocating activity. The additional bafilomycin-A insensitive ATPase activity observed in sucrose-loaded vesicles was completely inhibited by vanadate as was the efflux of sucrose. Similar to vanadate, thapsigargin was also inhibitory to sucrose efflux and to the bafilomycin-A insensitive ATPase activity. The data indicate that vacuolar sucrose can be actively mobilized by a specific ATP-dependent efflux mechanism. Received: 12 October 1999 / Accepted: 18 November 1999     

Ca2+-dependent ATPase associated with plasma membrane from a calcareous alga, Serraticardia maxima (Corallinaceae, Rhodophyta)

The plasma membrane was isolated from a calcareous red alga, Serraticardia maxima (Yendo) Silva (Corallinaceae), by aqueous two-phase partitioning. Its purity was examined with marker enzymes, Mg²⁺-dependent ATPase, inosine diphosphatase, cytochrome c oxidase and NADH-cytochrome c reductase, as well as the sensitivity of Mg²⁺-dependent ATPase to vanadate, azide and nitrate. The results showed that the isolated plasma membrane was purified enough to study its functions. Electron microscopic observations on thin tissue sections revealed that most vesicles of the isolated plasma membrane were stained by the plasma membrane specific stain, phosphotungstic acid-chromic acid. Mg²⁺- or Ca²⁺-dependent ATPases were associated with the plasma membrane. Ca²⁺-dependent ATPase was activated at physiological cytoplasmic concentrations of Ca²⁺ (0.1–10 μmol/L). However, calmodulin (0.5 μmol/L) did not affect its activity. The pH optimum was 8.0, in contrast to 7.0 for Mg²⁺-dependent ATPase. The isolated plasma membrane vesicles were mostly right side-out. To test for H⁺-translocation, right side-out vesicles were inverted; 27% of vesicles were inside-out after treatment with Triton X-100. The inside-out plasma membrane vesicles showed reduction of quinacrine fluorescence in the presence of 1 mmol/L ATP and 100 μmol/L Ca²⁺. The reduced fluorescence was recovered with the addition of 10 mmol/L NH₄Cl, or 5 μmol/L nigericin plus 50 mmol/L KCl. UTP and CTP substituted for ATP, but ADP did not. Ca²⁺-dependent ATPase might pump H⁺ out in the physiological state. The acidification by this pump might be coupled with alkalinization at the calcifying sites, which induces calcification.     

Novel Microbial Inhibitors of Angiotensin-converting Enzyme,Aspergillomarasmines A and B

Brush border membrane vesicles (BBMV) from the midgut epithelial cells of silkworm larvae were prepared. ATP hydrolyzing activity (ATPase activity) was associated with the BBMV. ATPase activity without Mg^{2 +} was not observed at pH 7 but substantial ATP hydrolyzing activity was observed at pH 7 with Mg^{2 +}. The enzyme required Mn^{2 +}, Mg^{2 +}, or Ca²⁺ ions. The enzyme also hydrolyzed ITP and GTP but not p-NPP, ADP, or AMP. KNO₃ and NEM strongly inhibited the ATPase activity. Behaviours of the ATPase against inhibitors suggested that it resembled vacuolar type ATPase.     

ATP Synthase of Chloroplast Thylakoid Membranes: A More in Depth Characterization of Its ATPase Activity

In contrast to everted mitochondrial inner membrane vesicles and eubacterial plasma membrane vesicles, the ATPase activity of chloroplast ATP synthase in thylakoid membranes is extremely low. Several treatments of thylakoids that unmask ATPase activity are known. Illumination of thylakoids that contain reduced ATP synthase (reduced thylakoids) promotes the hydrolysis of ATP in the dark. Incubation of thylakoids with trypsin can also elicit higher rates of ATPase activity. In this paper the properties of the ATPase activity of the ATP synthase in thylakoids treated with trypsin are compared with those of the ATPase activity in reduced thylakoids. The trypsin-treated membranes have significant ATPase activity in the presence of Ca²⁺, whereas the Ca²⁺-ATPase activity of reduced thylakoids is very low. The Mg²⁺-ATPase activity of the trypsinized thylakoids was only partially inhibited by the uncouplers, at concentrations that fully inhibit the ATPase activity of reduced membranes. Incubation of reduced thylakoids with ADP in Tris buffer prior to assay abolishes Mg²⁺-ATPase activity. The Mg²⁺-ATPase activity of trypsin-treated thylakoids was unaffected by incubation with ADP. Trypsin-treated membranes can make ATP at rates that are 75–80% of those of untreated thylakoids. The Mg²⁺-ATPase activity of trypsin-treated thylakoids is coupled to inward proton translocation and 10 mM sulfite stimulates both proton uptake and ATP hydrolysis. It is concluded that cleavage of the γ subunit of the ATP synthase by trypsin prevents inhibition of ATPase activity by the ε subunit, but only partially overcomes inhibition by Mg²⁺ and ADP during assay.     

Characterisation of a Ca2+-dependent ATP hydrolysis associated with the vacuolar membrane of wheat roots

Microsomal fractions from wheat tissues exhibit a higher level of ATP hydrolytic activity in the presence of Ca²⁺ than Mg²⁺. Here we characterise the Ca²⁺-dependent activity from roots of Triticum aestivum lev. Troy) and investigate its possible function. Ca²⁺-dependent ATP hydrolysis in the microsomal fraction occurs over a wide pH range with two slight optima at pH 5.5 and 7.5. At these pHs the activity co-migrates with the major peak of nitrate-inhibited Mg²⁺. Cl-ATPase on continuous sucrose gradients indicating that it is associated with the vacuolar membrane. Ca²⁺-dependent ATP hydrolysis can be distinguished from an inhibitory effect of Ca²⁺ on the plasma membrane K⁺, Mg²⁺-ATPase following microsomal membrane separation using aqueous polymer two phase partitioning. The Ca²⁺-dependent activity is stimulated by free Ca²⁺ with a K_m of 8.1 μM in the absence of Mg²⁺ ([CaATP] = 0.8 mM). Vacuoiar membrane vacuolar preparations contain a higher Ca²⁺-dependent than Mg²⁺-dependent ATP hydrolysis, although the two activities are not directly additive. The nucleotide specificity of the divalent ion-dependent activities in vacuolar membrane-enriched fractions was low. hydrolysis of CTP and UTP being greater than ATP hydrolysis with both Ca²⁺ and Mg²⁺ The Ca²⁺-dependent activity did discriminate against dinucleotides, and mononucleotides. and failed to hydrolyse phosphatase substrates. Despite low nucleotide specificity the Mg²⁺-dependent activity functioned as a bafilomycin sensitive H⁺-pump in vacuolar membrane vesicles. Ca²⁺-dependent ATP hydrolysis was not inhibited by the V-, P-, or F-type ATPase inhibitors bafilomycin. vanadate and azide, respectively. nor by the phosphatase inhibitor molybdate, but was inhibited 20% at pH 7.5 by K⁺. Possible functions of Ca²⁺-dependent hydrolysis as a H⁺-pump or a Ca²⁺-pump was investigated using vacuolar membrane vesicles. No H⁺ or Ca²⁺ translocating activity was observed under conditions when the Ca²⁺-dependent ATP hydrolysis was active.     

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.     

Properties of proton and sugar transport at the tonoplast of tomato (Lycopersicon esculentum) fruit

Tonoplast vesicles were isolated from tomato (Lycopersicon esculentum Mill.) fruit pericarp and purified on a discontinuous sucrose gradient. ATPase activity was inhibited by nitrate and bafilomycin A₁ but was insensitive to vanadate and azide. PPase hydrolytic activity was inhibited by NaF but was insensitive to nitrate, bafilomycin A₁ vanadate and azide. Kimetic studies of PPase activity gave an apparent K_m, for PP₃ of 18 μM. Identical distributions of bafilomycin- and NO₃-sensitive ATPase activities within continuous sucrose density gradients, confirmed that bafilomycin-sensitive ATPase activity is a suitable marker for the tonoplast. By comparing the distribution of bafilomycin-sensitive ATPase activity with that of PPase activity, it was possible to locate the PPase enzyme exclusively at the tonoplast. The apparent density of the tonoplast did not change during fruit development. Measurements of tonoplast PPase and ATPase activities during fruit development over a 35-day period revealed an 80% reduction in PPase specific activity and a small decrease in ATPase specific activity. ATP- and PP₁-dependent ΔpH generation was measured by the quenching of quinacrine fluorescence in tonoplast vesicles prepared on a discontinuous Dextran gradient. No H⁺ efflux was detected on the addition of sucrose to energized vesicles. Therefore a H⁺/sucrose antiport may not be the mechanism of sucrose uptake at the tomato fruit tonoplast. Similar results were obtained with glucose, fructose and sorbitol. The lack of ATP (or PP₁) stimulation of [¹⁴C]-sucrose uptake also suggested that an antiport was not involved. Initial uptake rates of radiolabelled glucose and fructose were almost double that for sucrose. The inhibition of hexose uptake by p-chloromercuribenzene sulphonate (PCMBS) implicated the involvement of a carrier. Therefore storage of hexose in the tomato fruit vacuole and maintenance of a downhill sucrose concentration gradient into sink cells is likely to be regulated by the activity of sucrose metabolizing enzymes, rather than by energy-requiring uptake mechanisms at the tonoplast.     

Calcium efflux from platelet vesicles of the dense tubular system. Analysis of the possible contribution of the Ca2+ pump

The ATP dependent Ca²⁺ uptake of platelet vesicles was inhibited by the two hydrophobic drugs trifluoperazine (TFP) and propranolol (PROP). Inhibition was significantly lowered when Pi was used instead of oxalate as a precipitant agent. When the ATPase ligands substrate (Mg²⁺ and Pi) were absent of the efflux medium, a slow release of Ca²⁺ which did not couple with ATP synthesis (passive Ca²⁺ efflux) was observed. Both, TFP and PROP enhanced the passive Ca²⁺ efflux. This enhanced efflux was partially inhibited only when Mg²⁺ and Pi were added together to the efflux reaction media, but it was not affected by spermidine, ruthenium red or thapsigargin (TG). The Ca²⁺ ionophores A23187 and ionomycin, also enhanced passive Ca²⁺ efflux. However, in this case, Ca²⁺ efflux was inhibited just by inclusion of Mg²⁺ to the medium. Ca²⁺ efflux promoted by Triton X-100 was not affected by either Mg²⁺ or Pi, included together or separately into the efflux medium. The ATP Pi measured in the presence of Triton X-100 and millimolar Ca²⁺ concentrations was inhibited by both TFP and PROP, but not by Ca²⁺ ionophores up to 4 M. The data suggest that the observed enhancement of passive Ca²⁺ efflux promoted by TFP and PROP could be attributed to a direct effect of these drugs over the platelet Ca²⁺ pump isoforms (Sarco Endoplasmic Reticulum Calcium ATPase, SERCA_2b and SERCA₃) themselves, as it was reported for the sarcoplasmic reticulum Ca²⁺ ATPase (SERCA₁).     

Characterization of a proton-translocating ATPase in microsomal vesicles from corn roots

Sealed microsomal vesicles were prepared from corn (Zea mays, Crow Single Cross Hybrid WF9-Mo17) roots by centrifugation of a 10,000 to 80,000g microsomal fraction onto a 10% dextran T-70 cushion. The Mg²⁺-ATPase activity of the sealed vesicles was stimulated by Cl⁻ and NH₄⁺ and by ionophores and protonophores such as 2 micromolar gramicidin or 10 micromolar carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP). The ionophore-stimulated ATPase activity had a broad pH optimum with a maximum at pH 6.5. The ATPase was inhibited by NO₃⁻, was insensitive to K⁺, and was not inhibited by 100 micromolar vanadate or by 1 millimolar azide.

Quenching of quinacrine fluorescence was used to measure ATP-dependent acidification of the intravesicular volume. Quenching required Mg²⁺, was stimulated by Cl⁻, inhibited by NO₃⁻, was insensitive to monovalent cations, was unaffected by 200 micromolar vanadate, and was abolished by 2 micromolar gramicidin or 10 micromolar FCCP. Activity was highly specific for ATP. The ionophore-stimulated ATPase and ATP-dependent fluorescence quench both required a divalent cation (Mg²⁺ ≥ Mn²⁺ > Co²⁺) and were inhibited by high concentrations of Ca²⁺. The similarity of the ionophore-stimulated ATPase and quinacrine quench and the responses of the two to ions suggest that both represent the activity of the same ATP-dependent proton pump. The characteristics of the proton-translocating ATPase differed from those of the mitochondrial F₁F₀-ATPase and from those of the K⁺-stimulated ATPase of corn root plasma membranes, and resembled those of the tonoplast ATPase.

     

Mechanism of zinc resistance in Pseudomonas putida strain S4

The mechanism of Zn resistance in multiple metal-resistant Pseudomonas putida strain S4 is based on inducible efflux. An ATPase in the strain S4 mediated active extrusion of Zn²⁺, which occurred during the exponential phase of growth. The ATPase activity was inhibited by micromolar concentrations (50 M) of vanadate, suggesting the involvement of a P-type ATPase. The effluxed Zn²⁺ were not ejected out of the cell but stored in the outer membrane and periplasm, which provided the required binding sites. The strain S4, thus, employs a dual strategy of efflux and binding to bring about a proper management of essential ions like Zn.     

Changes of membrane-associated Mg2+-activated ATPase of cucumber roots during calcium starvation

The properties of membrane-associated ATPase of cucumber (Cucumis sativus cv. Seiriki No. 2) roots cultured in a complete medium (complete enzyme) and in a medium lacking Ca²⁺ (Ca²⁺-deficient enzyme) were investigated. The basal activity of membrane-associated ATPase increased during Ca²⁺ starvation, while Mg²⁺-activation of the enzyme decreased and even resulted in inhibition by high Mg²⁺ concentration at the late stage of the Ca²⁺ starvation. The complete enzyme had low basal activity and showed a Mg²⁺-activated hyperbolic reaction curve in relation to ATP concentration. Ca²⁺-deficient enzyme with high basal activity showed a biphasic reaction curve and Mg²⁺-activation was seen only at high ATP concentrations. Activation of membrane-associated ATPase by various cations was decreased or lost during Ca²⁺ starvation. The basal ATPase activity of Ca²⁺-deficient enzyme increased for various substrates including pyrophosphate, p-nitrophenyl phosphate, glucose-6 phosphate, β-glycerophosphate, AMP, ADP and ATP. Mg²⁺-activation was found only for ADP and ATP in both the complete and Ca²⁺-deficient enzymes, but the activation for ATP was greatly reduced by Ca²⁺ starvation. The heat inactivation curves for basal and Mg²⁺-activated ATPase did not differ much between the complete and Ca²⁺-deficient enzyme. The delipidation of membrane-associated enzyme by acetone affected the protein content and the basal activity slightly, but inhibited the Mg²⁺-activated ATPase activity clearly with somewhat different behaviour between the complete and Ca²⁺-deficient enzyme.     

Characterisation of a salt-stimulated ATPase activity associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.)

Ion stimulation and some other properties of an ATPase activity associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.) have been determined. The ATPase had a specific requirement for Mg²⁺ and in the presence of Mg²⁺ it was stimulated by salts of monovalent cations. The degree of stimulation by monovalent salts was influenced mainly by the anion and the order of effectiveness of the anions tested was Cl^->HCO ₃ ^- >Br^->malate>acetate>SO ₄ ^2- . For any given series of anions the magnitude of the stimulation obtained was influenced by the accompanying cation (NH ₄ ⁺ Na⁺>K⁺). This cation effect was abolished by 0.01% (v/v) Triton X-100 and it is suggested that it is the result of different permeabilities of membrane vesicles to the cations. There was no evidence of synergistic stimulation of the ATPase by mixtures of Na⁺ and K⁺. KCl- and NaCl-stimulation was maximal with salt concentrations in the range 60–150 mM. The true substrate of the enzyme was shown to be MgATP. It was shown that KCl stimulation was the result of an increase in V_max rather than a change in the affinity of the enzyme for MgATP. The ATPase was inhibited by N,N-dicyclohexylcarbodiimide, diethylstilbestrol, mersalyl and KNO₃ but other inhibitors tested (azide, oligomycin, orthovanadate, K₃[Cr(oxalate)₆] and ethyl-3-[3-dimethylaminopropyl]carbodiimide) were without effect or caused only partial inhibition at the highest concentration tested. The ATPase activity was equally distributed between pellet and supernatant fractions obtained after the subfractionation of vacuoles but the properties of the ATPase in each fraction were the same. It is suggested that beet vacuoles possess only one ATPase. The properties of the ATPase are compared with those of ATPases associated with other plant membranes and organelles and its possible role in transport at the tonoplast is discussed.Abbreviations ATP_F free ATP - ATP_T total ATP - BSA bovine serum albumen - DCCD N,N-dicyclohexylcarbodiimide - DES diethylstilbestrol - DNP 2,4-dinitrophenol - EDAC ethyl-3-(3-dimethylaminopropyl)carbodiimide - K_m apparent Michaelis constant - MgATP complex of Mg²⁺ and ATP - Mg _F ²⁺ free Mg²⁺ - Mg _T ² total Mg²⁺ - MES 2-(N-Morpholino)ethanesulphonic acid - Na₂EDTA disodium ethylenediaminetetraacetic acid - NEM N-ethylmaleimide - P_i inorganic phosphate - TCA trichloroacetic acid - Tris tris(hydroxymethyl)methylamine - V_max maximum velocity     

Coupling of proteolysis to ATP hydrolysis uponEscherichia coli lon protease functioning: I. kinetic aspects of ATP hydrolysis

Some aspects of theEscherichia coli Lon protease ATPase function were studied around the optimum pH value. It was revealed that in the absence of the protein substrate the maximum ATPase activity of the enzyme is observed at an equimolar ratio of ATP and Mg²⁺ ions in the area of their millimolar concentrations. Free components of the substrate complex (ATP-Mg)²⁻ inhibit the enzyme ATPase activity. It is hypothesized that the effector activity of free Mg²⁺ ions is caused by the formation of the “ADP-Mg-form” of ATPase centers. It was shown that the activation of ATP hydrolysis in the presence of the protein substrate is accompanied by an increase in the affinity of the (ATP-Mg)²⁻ complex to the enzyme, by an elimination of the inhibiting action of free Mg²⁺ ions without altering the efficiency of catalysis of ATP hydrolysis (based on thek _cat value), and by a change in the type of inhibition of ATP hydrolysis by the (ADP-Mg)⁻ complex (without changing theK _i value). Interaction of the Lon protease protein substrate with the enzyme area located outside the peptide hydrolase center was demonstrated by a direct experiment.     

Ultracytochemical evidence for a proton-pump adenosine triphosphatase in chick osteoclasts

Summary The distribution of Mg^{+ +}-ATPase in osteoclasts along the endosteal surface of the chick tibia was investigated by neutral and alkaline pH cytochemical methods at the electron-microscopic level. Reaction product was observed in mitochondria, cytoplasmic vesicles, and ruffled-border membrane. Levamisole, ouabain, and vanadate did not affect the enzymatic activity. Para-chloromercuribenzoic acid (PCMB) prevented staining of mitochondria, ruffled border, and most cytoplasmic vesicles. Tri-n-butyltin decreased the amount of reaction product in cytoplasmic vesicles and ruffled-border membrane, but did not inhibit reaction product formation within mitochondria. Duramycin, which is a potent inhibitor for proton-pump ATPase, blocked reaction-product formation along the ruffled-border membrane, in mitochondria, and in cytoplasmic vesicles at alkaline pH, but not at neutral pH. It is concluded that the alkaline pH method for Mg^{+ +}-ATPase appears to demonstrate sites of proton-pump ATPase activity.     

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 anionized Ca²⁺ concentration of approx. 1 μM. 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.     

Characteristics of Ca2+-stimulated ATPase in rat heart sarcolemma in the presence of dithiothreitol and alamethicin

We have studied the activities of Ca²⁺-stimulated ATPase in rat heart sarcolemma upon modulating the redox state of membrane thiol groups with dithiothreitol (DTT). The suitability of alamethicin to unmask the latent activity of this enzyme was also investigated. The Ca²⁺-stimulated ATPase in sarcolemma exhibited two activation sites — one with low affinity (Km = 0.70 ± 0.2 mM; Vmax = 10.0 ± 2.2 mol Pi/mg/h) and the other with high affinity (Km = 0.16 ± 0.7 mM; Vmax = 4.6 ± 0.8 mol Pi/mg/h) for Mg²⁺ATP. Alamethicin at a ratio of 1:1 with the sarcolemmal protein caused a 3-fold activation of Ca²⁺-stimulated ATPase without affecting its sensitivity to Ca²⁺ or Mg²⁺ATP. Treatment of sarcolemma with deoxycholate or sodium dodecyl sulfate resulted in a total loss of the enzyme activity; high concentrations of alamethicin also showed a detergent-like action on the sarcolemmal vesicles. DTT at 5–10 mM concentrations caused a 4–5 fold activation of Ca²⁺-stimulated ATPase in sarcolemma and this effect was observed to be dependent on the concentration of Mg²⁺ATP. DTT increased the affinity of the enzyme to Mg²⁺ATP at the high affinity site and enhanced the Vmax at the low affinity site in addition to increasing the sensitivity of Ca²⁺-stimulated ATPase to Ca²⁺. DTT protected the Ca²⁺-stimulated ATPase against deterioration by detergents and restored the enzyme activity after treatment with N-ethylmaleimide. The mechanism of action of DTT on Ca²⁺-stimulated ATPase may involve the reduction of essential thiols at the active site of the enzyme or its interaction with specific DTT-dependent inhibitor protein. No changes in the sensitivity of sarcolemmal Ca²⁺-stimulated ATPase to orthovanadate was evident in the absence or presence of DTT and alamethicin. The results suggest the use of both DTT and alamethicin for the determination of Ca²⁺-stimulated ATPase activity in sarcolemmal preparations.     

Mg2+ and Mn2+ modulation of Ca2+ transport and ATPase activity in sarcoplasmic reticulum vesicles

Kinetic experimentation was used to characterize the Mg²⁺ and Mn²⁺ modulation of Ca²⁺ transport and ATPase activity in sarcoplasmic reticulum vesicles. In addition to its participation in the ATP·Mg complex as substrate for the ATPase, Mg²⁺ is an activator of phosphoenzyme progression to hydrolylic cleavage. It is shown that this activation is due to Mg²⁺ occupancy of an allosteric site easily accessible on the outer surface of the vesicles, rather than to participation in an antiport mechanism. The Mg²⁺ site is distinct from the Ca²⁺ binding sites which are involved in activation of enzyme phosphorylation by ATP, and Ca²⁺ translocation. The role of Mg²⁺ is quite specific, inasmuch as phosphoenzyme decay is much slower if the Mg²⁺ allosteric site is occupied by Ca²⁺. Conversely, competive occupancy of the Ca²⁺ sites by Mg²⁺ does not permit enzyme phosphorylation by ATP. Intermediate characteristics between Mg²⁺ and Ca²⁺ are displayed by Mn²⁺ which is well able to stimulate phosphoenzyme cleavage by occupancy of the Mg²⁺ allosteric site, and is also able (although at much slower rates) to activate enzyme phosphorylation, and undergo active transport by occupancy of the Ca²⁺ sites.     

H-ATPase Activity from Storage Tissue of Beta vulgaris: III. Modulation of ATPase Activity by Reaction Substrates and Products

Two distinct membrane fractions containing H⁺-ATPase activity were prepared from red beet. One fraction contained a H⁺-ATPase activity that was inhibited by NO₃⁻ while the other contained a H⁺-ATPase inhibited by vanadate. We have previously proposed that these H⁺-ATPases are associated with tonoplast (NO₃⁻-sensitive) and plasma membrane (vanadate-sensitive), respectively. Both ATPase were examined to determine to what extent their activity was influenced by variations in the concentration of ATPase substrates and products. The substrate for both ATPase was MgATP²⁻, and Mg²⁺ concentrations in excess of ATP had only a slight inhibitory effect on either ATPase. Both ATPases were inhibited by free ATP (i.e. ATP concentrations in excess of Mg²⁺) and ADP but not by AMP. The plasma membrane ATPase was more sensitive than the tonoplast ATPase to free ATP and the tonoplast ATPase was more sensitive than the plasma membrane ATPase to ADP.

Inhibition of both ATPases by free ATP was complex. Inhibition of the plasma membrane ATPase by ADP was competitive whereas the tonoplast ATPase demonstrated a sigmoidal dependence on MgATP²⁻ in the presence of ADP. Inorganic phosphate moderately inhibited both ATPases in a noncompetitive manner.

Calcium inhibited the plasma membrane but not the tonoplast ATPase, apparently by a direct interaction with the ATPase rather than by disrupting the MgATP²⁻ complex.

The sensitivity of both ATPases to ADP suggests that under conditions of restricted energy supply H⁺-ATPase activity may be reduced by increases in ADP levels rather than by decreases in ATP levels per se. The sensitivity of both ATPases to ADP and free ATP suggests that modulation of cytoplasmic Mg²⁺ could modulate ATPase activity at both the tonoplast and plasma membrane.

     

Characterization of an ecto-ATPase activity in Fonsecaea pedrosoi

In this work, we characterized an ecto-ATPase activity in intact mycelial forms of Fonsecaea pedrosoi, the primary causative agent of chromoblastomycosis. In the presence of 1 mM EDTA, fungal cells hydrolyzed adenosine-5′-triphosphate (ATP) at a rate of 84.6 ± 11.3 nmol Pi h⁻¹ mg⁻¹ mycelial dry weight. The ecto-ATPase activity was increased at about five times (498.3 ± 27.6 nmol Pi h⁻¹ mg⁻¹) in the presence of 5 mM MgCl₂, with values of V _max and apparent K _m for Mg-ATP²⁻corresponding to 541.9 ± 48.6 nmol Pi h⁻¹ mg⁻¹ cellular dry weight and 1.9 ± 0.2 mM, respectively. The Mg²⁺-stimulated ecto-ATPase activity was insensitive to inhibitors of intracellular ATPases such as vanadate (P-ATPases), bafilomycin A₁ (V-ATPases)_, and oligomycin (F-ATPases). Inhibitors of acid phosphatases (molybdate, vanadate, and fluoride) or alkaline phosphatases (levamizole) had no effect on the ecto-ATPase activity. The surface of the Mg²⁺-stimulated ATPase in F. pedrosoi was confirmed by assays in which 4,4′-diisothiocyanostylbene-2,2′-disulfonic acid (DIDS), a membrane impermeant inhibitor, and suramin, an inhibitor of ecto-ATPase and antagonist of P₂ purinoreceptors. Based on the differential expression of ecto-ATPases in the different morphological stages of F. pedrosoi, the putative role of this enzyme in fungal biology is discussed.     

Effect of magnesium and ATP on ATPase of sugarcane vacuoles

Kinetic analysis of the Mg²⁺-dependence of tonoplast ATPase from suspension-cultured cells of sugarcane showed that the enzyme activity increased with increasing magnesium concentrations till 1–3 mM and then decreased consideably for higher concentrations. This kinetic could be explained by the assumption that MgATP^2- is the substrate of ATPase: MgATP^2- concentration increases with increasing concentration of magnesium till, at high concentrations of magnesium, Mg₂ATP is formed. No evidence for a direct role of Mg²⁺ as activator or inhibitor was found. These data corroborate previous findings that MgATP^2- is the sole substrate of the vacuolar ATPase of sugarcane (Thom and Komor 1984). High concentrations of ATP seemed to inhibit the ATPase. This result, however, could be traced back to interference of ATP with the Fiske-Subbarow method of phosphate determination. After adjustment of the test conditions, inhibition by ATP was no longer found. Reported data for ATPases of other plant materials, showing inhibition of enzyme activity with high magnesium or ATP concentrations, might be explicable in a similar way.Abbreviation Mes 2-(N-morpholino)ethane+Sulfonic acid