Tomatidine promotes the inhibition of 24-alkylated sterol biosynthesis and mitochondrial dysfunction in Leishmania amazonensis promastigotes.

Leishmaniasis is a set of clinically distinct infectious diseases caused by Leishmania, a genus of flagellated protozoan parasites, that affects ~12 million people worldwide, with ~2 million new infections annually. Plants are known to produce substances to defend themselves against pathogens and predators. In the genus Lycopersicon, which includes the tomato, L. esculentum, the main antimicrobial compound is the steroidal glycoalkaloid α-tomatine. The loss of the saccharide side-chain of tomatine yields the aglycone tomatidine. In the present study, we investigated the effects of tomatidine on the growth, mitochondrial membrane potential, sterol metabolism, and ultrastructure of Leishmania amazonensis promastigotes. Tomatidine (0·1 to 5 µM) inhibited parasite growth in a dose-dependent manner (IC(50)=124±59 nM). Transmission electron microscopy revealed lesions in the mitochondrial ultrastructure and the presence of large vacuoles and lipid storage bodies in the cytoplasm. These structural changes in the mitochondria were accompanied by an effective loss of mitochondrial membrane potential and a decrease in ATP levels. An analysis of the neutral lipid content revealed a large depletion of endogenous 24-alkylated sterols such as 24-methylene-cholesta-5, 7-dien-3ß-ol (5-dehydroepisterol), with a concomitant accumulation of cholesta-8, 24-dien-3ß-ol (zymosterol), which implied a perturbation in the cellular lipid content. These results are consistent with an inhibition of 24-sterol methyltransferase, an important enzyme responsible for the methylation of sterols at the 24 position, which is an essential step in the production of ergosterol and other 24-methyl sterols.

CrATP interferes in the promastigote-macrophage interaction in Leishmania amazonensis infection.

Recent have shown the relationship between Ecto-Nucleoside-Triphosphate-Diphosphohydrolases (Ecto-NTPDases or ecto-nucleotidases) and virulence and infectivity in trypanosomatids. In this work, the inhibition of the ecto-ATPase activities and promastigote growth of Leishmania amazonensis by CrATP was characterized. Furthermore, this compound was used to investigate the role of ecto-nucleotidase in the interaction of L. amazonensis with resident peritoneal macrophages obtained from BALB/c mice. CrATP partially inhibits the ecto-ATPase activity, presenting Ki values of 575·7±199·1 and 383·5±79·0 µm, in the presence or absence of 5 mm MgCl2, respectively. The apparent Kms for ATP (2·9±0·5 mm to Mg2+-dependent ecto-ATPase and 0·4±0·2 mm to Mg2+-independent ecto-ATPase activities) are not significantly altered by CrATP, suggesting a reversible non-competitive inhibition of both enzymes. When CrATP was added to the cultivation medium at 500 µm, it drastically inhibited the cellular growth. The interaction of promastigote forms of L. amazonensis with BALB/c peritoneal macrophages is strongly affected by CrATP. When the parasites were treated with 500 µm CrATP before interacting with macrophages, the adhesion and endocytic indices were strongly reduced to 53·0±14·8% and 39·8±1·1%, respectively. These results indicate that ecto-nucleotidase plays an important role in the infection process caused by Leishmania amazonensis.

CrATP as a new inhibitor of ecto-ATPases of trypanosomatids.

Trypanosomatid protozoa include heteroxenic species some of them pathogenic for men, animals and plants. Parasite membrane contains ecto-enzymes whose active sites face the external medium rather than the cytoplasm. Herpetomonas sp. displayed a Mg2+-dependent ecto-ATPase activity, a Mg-independent ecto-ADPase and an ecto-phosphatase activity. Both, the ecto-ADPase and phosphatase activities were insensitive to CrATP (chromium(III) adenosine 5'-triphosphate complex). Ecto-ATPase activity was reversibly inhibited. At 2 mm ATP the apparent Ki was 4 x 7+/-1 x 0 microm but a fraction of about 40-50% was insensitive to CrATP. Remarkably, at low substrate concentration (0 x 2 mm) more than 90% of the ecto-ATPase was inhibited with Ki=0 x 33+/-0 x 10 microm. These parameter dependences are interpreted as the presence of 2 ecto-ATPases activities, one of them with high ATP apparent affinity and sensitivity to CrATP. DIDS (4,4 diisothiocyanatostilbene 2,2' disulfonic acid), suramin and ADP were also effective as inhibitors. Only ADP presented no additive inhibition with CrATP. The pattern of partial inhibition by CrATP was also observed for the ecto-ATPase activities of Leishmania amazonensis, Trypanosoma cruzi and Trypanosoma rangeli. CrATP emerges as a new inhibitor of ecto-ATPases and as a tool for a better understanding of properties and role of ecto-ATPases in the biology of parasites.

Characterization of Ca2+ uptake in a subcellular membrane fraction of Herpetomonas sp. promastigotes.

ATP-dependent Ca2+ uptake was studied in a subcellular fraction from Herpetomonas sp. prepared by mechanical disruption and using 45Ca2+ as a tracer. The uptake was stimulated by Ca2+ with a K0.5 of 0.1 microm and a Hill number (nH)=2.8+/-0.4. The Ca2+-dependent ATP hydrolysis was optimal at pH 7.0 and had a Ca2+ dependence identical to uptake. The uptake was highly stimulated by oxalate whereas calmodulin had no activating effect. ATP stimulated Ca2+ uptake with a biphasic pattern that resembled the curves described for the purified preparations of rabbit sarcoplasmic reticulum. The ATP stimulation is described as the sum of two Michaelis-Menten curves with Km1=0.25+/-0.19 microm and Km2=29.6+/-6.8 microm. GTP or UTP could also promote Ca2+ uptake, but with less efficiency than ATP. Vanadate inhibited the uptake with low apparent affinity. Thapsigargin and cyclopiazonic acid were almost ineffective. The Ca2+ uptake was insensitive to H+ ionophores and to bafilomycin suggesting no participation of acidocalcisomes. The results are comparable to those obtained using cells permeabilized with digitonin and using arsenaze III as Ca2+ indicator. The Ca2+ uptake activity described here seems to belong to the endoplasmic reticulum of Herpetomonas sp. and is suitable for further studies on the mechanisms of calcium homeostasis in parasites.

The crustacean gill (Na+,K+)-ATPase: allosteric modulation of high- and low-affinity ATP-binding sites by sodium and potassium.

The blue crab, Callinectes danae, tolerates exposure to a wide salinity range employing mechanisms of compensatory ion uptake when in dilute media. Although the gill (Na+,K+)-ATPase is vital to hyperosmoregulatory ability, the interactions occurring at the sites of ATP binding on the molecule itself are unknown. Here, we investigate the modulation by Na+ and K+ of homotropic interactions between the ATP-binding sites, and of phosphoenzyme formation of the (Na+,K+)-ATPase from the posterior gills of this euryhaline crab. The contribution of the high- and low-affinity ATP-binding sites to maximum velocity was similar for both Na+ and K+. However, in contrast to Na+, a threshold K+ concentration triggers the appearance of the high-affinity binding sites, displacing the saturation curve to lower ATP concentrations.Further, a low-affinity site for phosphorylation is present on the enzyme. These findings reveal notable differences in the catalytic mechanism of the crustacean (Na+,K+)-ATPase compared to the vertebrate enzyme.

Regulation by the exogenous polyamine spermidine of Na,K-ATPase activity from the gills of the euryhaline swimming crab Callinectes danae (Brachyura, Portunidae).

Euryhaline crustaceans rarely hyporegulates and employ the driving force of the Na,K-ATPase, located at the basal surface of the gill epithelium, to maintain their hemolymph osmolality within a range compatible with cell function during hyper-regulation. Since polyamine levels increase during the adaptation of crustaceans to hyperosmotic media, we investigate the effect of exogenous polyamines on Na,K-ATPase activity in the posterior gills of Callinectes danae, a euryhaline swimming crab. Polyamine inhibition was dependent on cation concentration, charge and size in the following order: spermine>spermidine>putrescine. Spermidine affected K(0.5) values for Na(+) with minor alterations in K(0.5) values for K(+) and NH(4)(+), causing a decrease in maximal velocities under saturating Na(+), K(+) and NH(4)(+) concentrations. Phosphorylation measurements in the presence of 20 microM ATP revealed that the Na,K-ATPase possesses a high affinity site for this substrate. In the presence of 10 mM Na(+), both spermidine and spermine inhibited formation of the phosphoenzyme; however, in the presence of 100 mM Na(+), the addition of these polyamines allowed accumulation of the phosphoenzyme. The polyamines inhibited pumping activity, both by competing with Na(+) at the Na(+)-binding site, and by inhibiting enzyme dephosphorylation. These findings suggest that polyamine-induced inhibition of Na,K-ATPase activity may be physiologically relevant during migration to fully marine environments.

ATPase and phosphatase activities are differentially inhibited by photo-oxidation of the sarcoplasmic reticulum Ca(2+)-ATPase.

We have already described that photo-oxidation of the sarcoplasmic reticulum Ca(2+)-ATPase with the halogenated dye erythrosin B produces inhibition of the ATPase activity (J.A. Mignaco et al., Biochemistry 35 (1996) 3886-3891). We now show that the Ca(2+)-dependent and Ca(2+)-independent p-nitrophenylphosphatase activities are also inhibited by this treatment. Modification of rapidly (< 10 min) oxidized residue(s) is responsible for the major loss of ATPase activity, whereas photo-inhibition of the phosphatase activities occurs more slowly (t1/2 20-30 min). Here we have focused on photo-inhibition of the Ca(2+)-independent pNPPase activity, and the counteracting effects of ATP and FITC. Following photo-oxidation, the Ca(2+)-independent pNPPase activity decreases monotonically. ATP partially protects against the inactivation of the pNPPase, whereas labeling the enzyme with FITC does not. However, the protective effect of ATP is completely abolished by the attached FITC. These data are interpreted in terms of two different sites that are susceptible to photo-oxidation and are involved in different events related to substrate hydrolysis.

Inhibition and labeling of the Ca2(+)-ATPase from sarcoplasmic reticulum by periodate oxidized ATP.

The analog of ATP obtained by oxidation of the ribose ring of ATP with periodate (oxATP) was used as a reagent for the inhibition and labeling of the Ca2(+)-ATPase purified from sarcoplasmic reticulum membranes. The substrate concentration dependence for hydrolysis showed a biphasic pattern for both ATP and oxATP as substrates. Preincubation of Ca2(+)-ATPase in the presence of 0.05 mM CaCl2, 5 mM MgCl2, 100 mM KCl and oxATP led to an irreversible inhibition. This inhibition occurred faster at alkaline pH. The presence of ADP, adenyl-5'-imidodiphosphate (AMP-PNP) or EGTA in the preincubation medium decreased the rate of inhibition. OxATP covalently labels the enzyme: the labeling was decreased by ADP. This ADP-protected labeling increased with time until it reached approx. 1 mol [3H]oxATP per mol ATPase. The rate of labeling of the ADP-protected group correlated with the rate of loss of ADP-protected activity. Trypsin digestion of oxATP-labeled ATPase followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that fragment A1 contained a high degree of label that is displaced by ADP. We propose that the A1 fragment is situated close to the ribose ring when the adenosine moiety of ATP is bound to the catalytic site of the Ca2(+)-ATPase.

Vanadate inhibition of the Ca2+-ATPase from human red cell membranes.

(1) VO3(-) combines with high affinity to the Ca2+-ATPase and fully inhibits Ca2+-ATPase and Ca2+-phosphatase activities. Inhibition is associated with a parallel decrease in the steady-state of the Ca2+-dependent phosphoenzyme. (2) VO3(-) blocks hydrolysis of ATP at the catalytic site. The sites for VO3(-) also exhibit negative interactions in affinity with the regulatory sites for ATP of the Ca2+-ATPase. (3) The sites for VO39-) show positive interaactions in affinity with sites for Mg2+ and K+. This accounts for the dependence on Mg2+ and K+ of the inhibition by VO3(-). Although, with less effectiveness, Na2+ and K+ substitutes for K+ whereas Li+ does not. The apparent affinites for Mg24 and K+ for inhibiton by VO3(-) seem to be less than those for activation of the Ca2+-ATPase. (4) Inhibition by VO3(-) is independent of Ca2+ at concentrations up to 50 microM. Higher concentrations of Ca2+ lead to a progressive release of the inhibitiory effect of VO3(-).

Calcium dependence of Pi phosphorylation of sarcoplasmic reticulum Ca2+-ATPase at low water content: water dependence of the E2-->E1 conversion.

Enzymes entrapped in reverse micelles can be studied in low-water environments that have the potential of restricting conformational mobility in specific steps of the reaction cycle. Sarcoplasmic reticulum Ca2+-ATPase was incorporated into a reverse-micelle system (TPT) composed of toluene, phospholipids, Triton X-100 and varying amounts of water (0.5-7%, v/v). Phosphorylation of the Ca2+-ATPase by ATP required the presence of both water and Ca2+ in the micelles. No phosphoenzyme (EP) was detected in the presence of EGTA. Phosphorylation by Pi (inorganic phosphate) in the absence of Ca2+ was observed at water content below that necessary for phosphorylation by ATP. In contrast to what is observed in a totally aqueous medium, EP formed by Pi was partially resistant to dephosphorylation by Ca2+. However, the addition of non-radioactive Pi to the EP already formed caused a rapid decrease in radiolabelled enzymes, as expected for the isotopic dilution, indicating the existence of an equilibrium (E+Pi<-->EP). Phosphorylation by Pi also occurred in TPT containing millimolar Ca2+ concentrations in a range of water concentrations (2-5% v/v). The substrates p-nitrophenyl phosphate, acetyl phosphate, ATP and GTP increased the EP level under these conditions. These results suggest that: (1) the rate of conversion of the ATPase conformer E2 into E1 is greatly reduced at low water content, so that E2-->E1 becomes the rate-limiting step of the catalytic cycle; and (2) in media of low water content, Pi can phosphorylate both E1Ca and E2. Thus, the effect of enzyme hydration is complex and involves changes in the phosphorylation reaction at the catalytic site, in the equilibrium between E2 and E1 conformers, and in their specificity for substrates.

Pseudosubstrate hydrolysis by the erythrocyte plasma membrane Ca(2+)-ATPase: kinetic evidence for a modified E1 conformation in dimethylsulfoxide.

The purified Ca(2+)-ATPase of pig red cells displays a phosphatase activity towards p-nitrophenylphosphate which is inhibited by Ca2+ in the absence of solvents, and activated by calmodulin. This activity has been attributed to the E2 conformation of the enzyme. Here we show that the pNPPase activity in the absence of Ca2+ is stimulated 10-25-fold by the presence of the organic solvent dimethylsulfoxide (Me2SO). This is an activation that surpasses by severalfold that induced by calmodulin in the absence of the solvent. At 30% Me2SO, activation by calmodulin disappears. In the absence of calmodulin and at pH 7.2, the Ca2+ concentration needed for half-maximal inhibition of the pNPPase activity (K1) increases from 130 microM in the absence of Me2SO to 860 microM at 30% Me2SO. This effect of Me2SO is enhanced at pH 8.0: the K for Ca2+ increases from 2.7 microM in the absence of the solvent to 2.0 mM in its presence. However, the K0.5 for Ca2+ activation of the ATPase activity decreases from 8.3 to 2.6 microM following addition of the same Me2SO concentration. This indicates that, even in the presence of Me2SO, microM Ca2+ concentrations shift the equilibrium towards E1 but the decrease in activity that would be expected if pNPP hydrolysis were catalysed exclusively by the E2 conformation is not observed. The affinity for pNPP as a substrate increases from 2.6 mM in the absence of Me2SO to 1.6 mM in the presence of 20% Me2SO. These results suggest that Me2SO induces multiple effects in the Ca(2+)-ATPase that (i) increase the reactivity of E2 towards substrate: (ii) surpass the activation by calmodulin and, (iii) allow the enzyme to hydrolyze pNPP even when Ca2+ is bound to the high-affinity sites of the enzyme. The change in reactivity is attributed to an increase on substrate catalysis rather than on pNPP binding.

Stoichiometric and electrostatic characterization of calcium binding to native and lipid-substituted adenosinetriphosphatase of sarcoplasmic reticulum.

The stoichiometry of calcium binding to specific sites (i.e., those producing enzyme activation) was found to be 8-10 nmol/mg protein in native sarcoplasmic reticulum vesicles, and 13.9-15.4 nmol/mg of ATPase purified by non-ionic detergent solubilization and anion exchange chromatography. Parallel measurements of phosphoenzyme yielded levels of 4.0-4.9 and 6.0-7.7 nmol/mg of protein in the two preparations, respectively, demonstrating that each 115 kDa ATPase chain includes one catalytic site and two calcium binding sites. The apparent association constant, K = (6 +/- 2) X 10(5) M-1, and the binding cooperativity, nH = 1.9, were unchanged when measurements were carried out with native sarcoplasmic reticulum vesicles and when the membrane surface charge was altered by lipid substitution with phosphatidylcholine or phosphatidylserine, at neutral pH in the presence of 10 mM MgCl2 and 80 mM KCl. On the other hand, the apparent association constant was increased in the absence of Mg2+ or, to a lesser extent, in the absence of monovalent cations. It was also observed that the cooperative character of the calcium binding isotherms was reduced in low ionic-strength media. Analysis of the electrostatic effects indicates that the calcium-binding domain is shielded from the membrane phospholipid surface charge by virtue of its location within the ATPase protein. The effects of various electrolytes are attributed to monovalent-cation binding in the calcium-binding domain. The apparent loss of cooperativity of the calcium binding isotherms at low ionic strength is attributed to a progressive displacement of the titration curve which is minimal at low degrees of saturation and becomes larger at higher degrees of saturation. This behavior is described quantitatively by the progressive effect of calcium binding on an electrostatic potential generated by localized protein charge densities within, or near, the calcium-binding domain.

Phosphorylation of Na+, K(+)-ATPase by ATP in the presence of K+ and dimethylsulfoxide but in the absence of Na+.

Purified Na+, K(+)-ATPase was phosphorylated by [gamma-32P]ATP in a medium containing dimethylsulfoxide and 5 mM Mg2+ in the absence of Na+ and K+. Addition of K+ increased the phosphorylation levels from 0.4 nmol phosphoenzyme/mg of protein in the absence of K+ to 1.0 nmol phosphoenzyme/mg of protein in the presence of 0.5 mM K+. Higher velocities of enzyme phosphorylation were observed in the presence of 0.5 mM K+. Increasing K+ concentrations up to 100 mM lead to a progressive decrease in the phosphoenzyme (EP) levels. Control experiments, that were performed to determine the contribution to EP formation from the Pi inevitably present in the assays, showed that this contribution was of minor importance except at high (20-100 mM) KCl concentrations. The pattern of EP formation and its KCl dependence is thus characteristic for the phosphorylation of the enzyme by ATP. In the absence of Na+ and with 0.5 mM K+, optimal levels (1.0 nmol EP/mg of protein) were observed at 20-40% dimethylsulfoxide and pH 6.0 to 7.5. Addition of Na+ up to 5 mM has no effect on the phosphoenzyme level under these conditions. At 100 mM Na+ or higher the full capacity of enzyme phosphorylation (2.2 nmol EP/mg of protein) was reached. Phosphoenzyme formed from ATP in the absence of Na+ is an acylphosphate-type compound as shown by its hydroxylamine sensitivity. The phosphate radioactivity was incorporated into the alpha-subunit of the Na+, K(+)-ATPase as demonstrated by acid polyacrylamide gel electrophoresis followed by autoradiography.

The role of Mg2+ and K+ in the phosphorylation of Na+,K(+)-ATPase by ATP in the presence of dimethylsulfoxide but in the absence of Na+.

We have previously demonstrated that Na+,K(+)-ATPase can be phosphorylated by 100 microM ATP and 5 mM Mg2+ and in the absence of Na+, provided that 40% dimethylsulfoxide (Me2SO) is present. Phosphorylation was stimulated by K+ up to a steady-state level of about 50% of Etot (Barrabin et al. (1990) Biochim. Biophys. Acta 1023, 266-273). Here we describe the time-course of phosphointermediate (EP) formation and of dephosphorylation of EP at concentrations of Mg2+ from 0.1 to 5000 microM and of K+ from 0.01 to 100 mM. The results were simulated by a simplified version of the commonly accepted Albers-Post model, i.e. a 3-step reaction scheme with a phosphorylation, a dephosphorylation and an isomerization/deocclusion step. Furthermore it was necessary to include an a priori, Mg(2+)- and K(+)-independent, equilibration between two enzyme forms, only one of which (constituting 14% of Etot) reacted directly with ATP. The role of Mg(2+) was two-fold: At low Mg2+, phosphorylation was stimulated by Mg2+ due to formation of the substrate MgATP, whereas at higher concentrations it acted as an inhibitor at all three steps. The affinity for the inhibitory Mg(2+)-binding was increased several-fold, relative to that in aqueous media, by dimethylsulfoxide. K+ stimulated dephosphorylation at all Mg(2+)-concentrations, but at high, inhibitory [Mg2+], K+ also stimulated the phosphorylation reaction, increasing both the rate coefficient and the steady-state level of EP. Generally, the presence of Me2SO seems to inhibit the dephosphorylation step, the isomerization/deocclusion step, and to a lesser extent (if at all) the phosphorylation reaction, and we discuss whether this reflects that Me2SO stabilizes occluded conformations of the enzyme even in the absence of monovalent cations. The results confirm and elucidate the stimulating effect of K+ on EP formation from ATP in the absence of Na+, but they leave open the question of the molecular mechanism by which Me2SO, inhibitory Mg2+ and stimulating K+ interact with the Na+,K(+)-ATPase.

The effect of dimethylsulfoxide on the substrate site of Na+/K(+)-ATPase studied through phosphorylation by inorganic phosphate and ouabain binding.

To obtain further information on the role of H2O at the substrate site of Na+/K(+)-ATPase, we have studied the enzymes reaction with P(i) and ouabain in 40% (v/v) Me2SO (dimethylsulfoxide). When the enzyme (E) was incubated with ouabain (O) for 5 min in a 40% (v/v) Me2SO-medium with 5 mM MgCl2 and 0.5 mM KCl (but no phosphate), ouabain was bound (as EO). Subsequent incubation with P(i) showed that E, but not EO, was rapidly phosphorylated (to EP). Long-time phosphorylation revealed that EO is also phosphorylated by P(i) albeit very slowly (t1/2 about 60 min) and that binding of ouabain to EP also is very slow. The EOP complex is stable, i.e., the t1/2 for the loss of P(i) is >> 60 min in contrast to about 1 min in water. These results in 40% Me2SO are distinctly different from what would be obtained in a watery milieu: ouabain would bind slowly and inefficiently in the absence of P(i), and ouabain would catalyse phosphorylation from P(i) rather than retard it. Equilibrium binding of [3H]ouabain to E and EP in water or 40% Me2SO confirmed these observations: Kdiss in water is 11 microM and 12 nM for EO and EOP, respectively, whereas in Me2SO they are 112 nM and 48 nM. It is suggested that the primary effect of the lowered water activity in 40% Me2SO is a rearrangement of the substrate site so that it also in the absence of P(i) attains a transition state configuration corresponding to the phosphorylated conformation. This would be sensed by the ouabain binding site and lead to high affinity ouabain binding in the absence of P(i).

Isolation and characterization of the Mg2(+)-ATPase from rabbit skeletal muscle sarcoplasmic reticulum membrane preparations.

Preparations of sarcoplasmic reticulum vesicles, obtained according to the method of Eletr and Inesi (Biochim. Biophys. Acta (1972) 282, 174), contained both Mg2(+)-ATPase and Ca2+, Mg2(+)-ATPase activity. The two enzymes were solubilized by a mixture of digitonin and lysophosphatidylcholine and separated on a DEAE-cellulose column eluted with a discontinuous gradient of NaCl. The Mg2(+)-ATPase activity was eluted with 0.43 M NaCl. The Ca2+,Mg2(+)-ATPase was obtained by increasing the NaCl concentration of the elution medium to 0.40 M. The fraction eluted with 0.043 M NaCl was insensitive to micromolar concentrations of calcium, resistant to oligomycin, ouabain, orthovanadate and thiocyanate, and was inhibited by low concentrations of Triton X-100. The enzyme showed a single apparent Km for MgATP in the range of 0.2 mM and a Vm of 2.9 mumol Pi.min-1.mg-1 protein. Activity was maximal over a broad peak between pH 6.0-8.0. Hydrolysis of ATP was unaffected by dimethylsulfoxide concentrations up to 20% (v/v) and was inhibited at higher concentrations. The enzyme was not phosphorylated by either 32Pi or [gamma-32P]ATP at significant levels when compared with the Ca2+,Mg2(+)-ATPase in an EGTA-containing medium. The kinetic pattern of the Mg2(+)-ATPase was distinctly different from that of the Ca2+,Mg2(+)-ATPase under the same conditions. The fraction eluted from the DEAE-cellulose column was subjected to electrophoresis under non-denaturing conditions. Only one band with Mg2(+)-ATPase activity was detected. The Mg2(+)-ATPase migrated much slower than the Ca2+,Mg2(+)-ATPase under non-denaturing conditions, whereas both enzymes had a molecular mass of 105 kDa on SDS gel electrophoresis.

Gill microsomal (Na+,K+)-ATPase from the blue crab Callinectes danae: Interactions at cationic sites.

Euryhaline crustaceans tolerate exposure to a wide range of dilute media, using compensatory, ion regulatory mechanisms. However, data on molecular interactions occurring at cationic sites on the crustacean gill (Na+,K+)-ATPase, a key enzyme in this hyperosmoregulatory process, are unavailable. We report that Na+ binding at the activating site leads to cooperative, heterotropic interactions that are insensitive to K+. The binding of K+ ions to their high affinity sites displaces Na+ ions from their sites. The increase in Na+ ion concentrations increases heterotropic interactions with the K+ ions, with no changes in K0.5 for K+ ion activation at the extracellular sites. Differently from mammalian (Na+,K+)-ATPases, that from C. danae exhibits additional NH4+ ion binding sites that synergistically activate the enzyme at saturating concentrations of Na+ and K+ ions. NH4+ binding is cooperative, and heterotropic NH4+ ion interactions are insensitive to Na+ ions, but Na+ ions displace NH4+ ions from their sites. NH4+ ions also displace Na+ ions from their sites. Mg2+ ions modulate enzyme stimulation by NH4+ ions, displacing NH4+ ion from its sites. These interactions may modulate NH4+ ion excretion and Na+ ion uptake by the gill epithelium in euryhaline crustaceans that confront hyposmotic media.

Distinct mechanisms of inhibition of purified cardiac sarcolemma Ca(2+)-ATPase by two calmodulin antagonists.

The effects of calmidazolium and compound 48/80 were studied in four different states of activation of the purified Ca(2+)-ATPase from cardiac sarcolemma: "basal" or unactivated, activated by calmodulin, activated by phosphatidylserine, and activated by controlled trypsinization. When assayed in the presence of phosphatidylcholine as the sole phospholipid (basal state), the purified enzyme was resistant to inhibition by calmidazolium (0.1 to 3 microM). In the same range, calmidazolium inhibited the enzyme activated by controlled proteolysis as well as the calmodulin-activated enzyme regardless of the calmodulin concentration. The phosphatidylserine-activated enzyme was inhibited at higher calmidazolium concentrations due to non-specific trapping of the inhibitor by the excess of phospholipid. Addition of calmidazolium did not modify the K0.5 for calcium activation of ATP hydrolysis by the enzyme. The inhibition by calmidazolium was counteracted by Pi. Compound 48/80 also had no effect on the enzyme when only phosphatidylcholine was present and, like calmidazolium, it inhibited the calmodulin-activated enzyme and the phosphatidylserine-activated enzyme. The apparent Ki for inhibition by compound 48/80 was dependent on the calmodulin concentration. However, the enzyme activated by controlled trypsinization was insensitive to compound 48/80. Binding of 48/80 to the enzyme in the presence of phosphatidylserine or calmodulin reversed the increased affinity for Ca2+ caused by these activators.

Vanadate inhibition of the Ca-ATPase activity of sarcoplasmic reticulum vesicles.

The Ca-dependent ATPase of sarcoplasmic reticulum vesicles was studied with respect to the inhibition by vanadate. The vanadate concentration for half maximal inhibition (Ki) varied with the nucleotide used and with its concentrations in the medium, being 0.7 microM in presence of 5 to 50 microM ATP and increasing progressively up to 7.0 microM when the ATP concentration was raised up to 5 mM. The Ki was about 1 microM in presence of 0.1 to 2.0 mM of either GTP or ITP. The Ki also varied depending on whether the enzyme was preincubated with vanadate in absence of Ca2+ (Ki approximately equal to approximately equal to 1 microM) or in the presence of 0.1 mM Ca2+ (Ki greater than 20 microM). This effect was only observed when the activity was measured 15s after the addition of ATP. For longer incubation intervals the Ki did no longer depend on the presence of Ca2+ in the preincubation medium. Phosphorylation of the enzyme by orthophosphate was competitively inhibited by vanadate. The ATP in equilibrium Pi exchange reaction measured in vesicles which were permeable to Ca2+ was inhibited by vanadate when the Ca2+ concentration was in the range 100-200 microM. However, inhibition of both ATPase activity and ATP in equilibrium Pi. exchange were abolished when the Ca2+ concentrations was raised to the millimolar range.