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.

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.

Magnesium-dependent ecto-ATP diphosphohydrolase activity in Herpetomonas muscarum muscarum.

In the present work we characterized the ecto-ATP diphosphohydrolase activity of the trypanosomatid parasite Herpetomonas muscarum muscarum. This parasite hydrolyzed ATP at a rate of 15.52 nmol Pi/mg protein/min and this activity reached a maximum at pH 7.5. Classical inhibitors of acid phosphatases, such as sodium orthovanadate (NaVO3), sodium fluoride (NaF), and ammonium molybdate presented no effect on this activity. MgCl2, ZnCl2, and MnCl2 stimulated the ATP hydrolysis by H. m. muscarum. The ecto-ATPase activity was insensitive to oligomycin and sodium azide, two inhibitors of mitochondrial Mg-ATPase, bafilomycin A1, a V-ATPase inhibitor, ouabain, a Na(+)+K+-ATPase inhibitor and to levamizole, an inhibitor of alkaline phosphatase. An extracellular impermeant inhibitor 4,4'-diisothiocyanostylbene 2',2'-disulfonic acid (DIDS) and a inhibitor of some ecto-ATPases, suramin, which is also a competitive antagonist of P2-purinergic receptors, promoted a great inhibition on the ATP hydrolysis. This enzyme is able to hydrolysis ATP, ADP, UTP, and UDP, but not GTP, GDP, CTP, or CDP. ADP inhibited the enzymatic activity in a concentration dependent manner, reaching 70% inhibition.

Characterization of the intracellular Ca(2+) pools involved in the calcium homeostasis in Herpetomonas sp. promastigotes.

Trypanosomatids of the genus Herpetomonas comprises monoxenic parasites of insects that present pro- and opisthomastigotes forms in their life cycles. In this study, we investigated the Ca(2+) transport and the mitochondrial bioenergetic of digitonin-permeabilized Herpetomonas sp. promastigotes. The response of promastigotes mitochondrial membrane potential to ADP, oligomycin, Ca(2+), and antimycin A indicates that these mitochondria behave similarly to vertebrate and Trypanosoma cruzi mitochondria regarding the properties of their electrochemical proton gradient. Ca(2+) transport by permeabilized cells appears to be performed mainly by the mitochondria. Unlike T. cruzi, it was not possible to observe Ca(2+) release from Herpetomonas sp. mitochondria, probably due to the simultaneous Ca(2+) uptake by the endoplasmic reticulum. In addition, a vanadate-sensitive Ca(2+) transport system, attributed to the endoplasmic reticulum, was also detected. Nigericin (1 microM), FCCP (1 microM), or bafilomycin A(1) (5 microM) had no effect on the vanadate-sensitive Ca(2+) transport. These data suggest the absence of a Ca(2+) transport mediated by a Ca(2+)/H(+) antiport. No evidence of a third Ca(2+) compartment with the characteristics of the acidocalcisomes described by A. E. Vercesi et al. (1994, Biochem. J. 304, 227-233) was observed. Thapsigargin and IP(3) were not able to affect the vanadate-sensitive Ca(2+) transport. Ruthenium red was able to inhibit the Ca(2+) uniport of mitochondria, inducing a slow mitochondrial Ca(2+) efflux, compatible with the presence of a Ca(2+)/H(+) antiport. Moreover, this efflux was not stimulated by the addition of NaCl, which suggests the absence of a Ca(2+)/Na(+) antiport in mitochondria.

Characterization of nucleotide binding sites of the isolated H(+)-ATPase from spinach chloroplasts, CF(0)F(1).

Soluble purified CF(0)F(1) from chloroplasts was either oxidized or reduced and then incubated with [alpha-(32)P]ATP in the presence or in the absence of Mg(2+). Depending on the conditions of incubation, the enzyme showed different tight-nucleotide binding sites. In the presence of EDTA, two sites bind [alpha-(32)P]ATP from the reaction medium at different rates. Both sites promote ATP hydrolysis, since equimolar amounts of [alpha-(32)P]ATP and [alpha-(32)P]ADP are bound to the enzyme. In the presence of Mg(2+), only one site appears during the first hour of incubation, with characteristics similar to those described in the absence of Mg(2+). However, after this time a third site appears also permitting binding of ATP from the reaction medium, but in this case the bound ATP is not hydrolyzed. Covalent derivatization by 2-azido-[alpha-(32)P]ATP was used to distinguish between catalytic and noncatalytic sites. In the presence of Mg(2+), there are at least three distinct nucleotide binding sites that bind nucleotide tightly from the reaction medium: two of them are catalytic and one is noncatalytic.

Protection against thermal denaturation by trehalose on the plasma membrane H+-ATPase from yeast. Synergetic effect between trehalose and phospholipid environment.

Yeast cells have had to develop mechanisms in order to protect themselves from chemical and physical agents of the environment to which they are exposed. One of these physical agents is thermal variation. Some yeast cells are known to accumulate high concentrations of trehalose when submitted to heat shock. In this work, we have studied the effect of trehalose on the protection against thermal inactivation of purified plasma membrane H+-ATPase from Schizosaccharomyces pombe, in the solubilized and in the reconstituted state. We observed that after 1 min of incubation at 51 degrees C in the presence of 1 M trehalose, about 50% of soluble enzyme remains active. In the same conditions, but in the absence of trehalose, the activity was completely abolished. The t0.5 for the enzyme inactivation increased from 10 to 50 s after reconstitution into asolectin liposomes. Curiously, in the presence of 1 M trehalose, the t0.5 for inactivation of the reconstituted enzyme was further increased to higher than 300 s, regardless of whether trehalose was added inside or outside the liposome. Additionally, the concentration that confers 50% for the protection by trehalose (K0.5) decreased from 0.5 M, in the solubilized state, to 0.04 M in the reconstituted state, suggesting a synergetic effect between sugar and lipids. Gel electrophoresis revealed that the pattern of H+-ATPase cleavage by trypsin changed when 1 M trehalose was present in the buffer. It is suggested that both in a soluble and in a phospholipid environment, accumulation of trehalose leads to a more heat-stable conformation of the enzyme, probably an E2-like form.

A novel role of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid as an activator of the phosphatase activity catalyzed by plasma membrane Ca2+-ATPase.

The hydrolysis of p-nitrophenyl phosphate catalyzed by the erythrocyte membrane Ca2+-ATPase is stimulated by low concentrations of the compound 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a classic inhibitor of anion transport. Enhancement of the phosphatase activity varies from 2- to 6-fold, depending on the Ca2+ and calmodulin concentrations used. Maximum stimulation of the pNPPase activity in ghosts is reached at 4-5 microM DIDS. Under the same conditions, but with ATP rather than pNPP as the substrate, the Ca2+-ATPase activity is strongly inhibited. Activation of pNPP hydrolysis by DIDS is equally effective for both ghosts and purified enzyme, and therefore is independent of its effect as an anion transport inhibitor. Binding of the activator does not change the Ca2+ dependence of the pNPPase activity. Stimulation is partially additive to the activation of the pNPPase activity elicited by calmodulin and appears to involve a strong affinity binding or covalent binding to sulfhydryl groups of the enzyme, since activation is reversed by addition of dithiothreitol but not by washing. The degree of activation of pNPP hydrolysis is greater at alkaline pH values. DIDS decreases the apparent affinity of the enzyme for pNPP whether in the presence of Ca2+ alone or Ca2+ and calmodulin or in the absence of Ca2+ (with 5 microM DIDS the observed Km shifts from 4.8 +/- 1.4 to 10.1 +/- 2.6, from 3.8 +/- 0.4 to 7.0 +/- 0.8, and from 9.3 +/- 0.7 to 15.5 +/- 1.1 mM, respectively). However, the pNPPase rate is always increased (as above, from 3.6 +/- 0.6 to 11.2 +/- 1.7, from 4.4 +/- 0.5 to 11.4 +/- 0.9, and from 2.6 +/- 0.6 to 18.6 +/- 3.9 nmol mg-1 min-1, in the presence of Ca2+ alone or Ca2+ and calmodulin or in the absence of Ca2+, respectively). ATP inhibits the pNPPase activity in the absence of Ca2+, both in the presence and in the absence of DIDS. Therefore, kinetic evidence indicates that DIDS does more than shift the enzyme to the E2 conformation. We propose that the transition from E2 to E1 is decreased and a new enzyme conformer, denoted E2*, is accumulated in the presence of DIDS.

Stimulation of ouabain binding to Na,K-ATPase in 40% dimethyl sulfoxide by a factor from Na,K-ATPase preparations.

In 40% dimethyl sulfoxide (Me2SO) high-affinity ouabain (O) binding to Na,K-ATPase (E) is promoted by Mg2+ in the absence of inorganic phosphate (Pi) (Fontes et al., Biochim. Biophys. Acta 1104, 215-225, 1995). Furthermore, in Me2SO the EO complex reacts very slowly with Pi and this ouabain binding can therefore be measured by the degree of inhibition of rapid phosphoenzyme formation. Here we found that, unexpectedly, the ouabain binding decreased with the enzyme concentration in the Me2SO assay medium. We extracted the enzyme preparation with Me2SO or chloroform/methanol and demonstrated that the extracted (depleted) enzyme bound ouabain poorly. Addition of such extracts to assays with low enzyme concentration or depleted enzyme fully restored the high-affinity ouabain binding. Dialysis experiments indicated that the active principle had a molecular mass between 3.5 and 12 kDa. It was highly resistant to proteolysis. It was suggested that the active principle could either be a low-molecular-weight, proteolysis-resistant-peptide (e.g., a proteolipid) or a factor with a nonproteinaceous nature. A polyclonal antibody raised against the C-terminal 10 amino acids of the rat kidney gamma-subunit was able to recognize this low-molecular-weight peptide present in the extracts. The previously depleted enzyme displayed lower amounts of the gamma-proteolipid in comparison to the native untreated enzyme, as demonstrated by immunoreaction with the antibody.

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.

Ca(2+)-ATPase from chicken (Gallus domesticus) erythrocyte plasma membrane: effects of calmodulin and taurine on the Ca(2+)-dependent ATPase activity and Ca2+ uptake.

In the present report we describe a method for purifying plasma membranes from chicken erythrocytes using sonication under conditions that facilitate preferential lysis of plasma membrane, followed by centrifugation through a sucrose gradient. The Ca(2+)-dependent ATP hydrolysis by plasma membranes is activated by nanomolar levels of calmodulin, similarly to that from anucleated erythrocytes. Inside-out vesicles display a calmodulin-activated Ca2+ uptake. Purified Ca(2+)-ATPase is obtained from the plasma membrane by Sepharose-calmodulin affinity chromatography, and exhibits an apparent molecular mass of 150 kDa on SDS-polyacrylamide gel electrophoresis, clearly showing that the enzyme is distinct from that described in anucleated erythrocytes (140 kDa). The enzyme is insensitive to physiological concentrations of taurine, a beta-amino acid that has been proposed to be involved in Ca2+ homeostasis of nucleated erythrocytes, suggesting that the effect of taurine is not mediated by the Ca(2+)-ATPase. Taken together, these data suggest that the enzyme may be an isoform that resembles the previously described plasma membrane Ca(2+)-ATPase from anucleated erythrocytes in its regulation by calmodulin, but differs in its apparent molecular weight.

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.

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.

Effects of photo-oxidizing analogs of fluorescein on the sarcoplasmic reticulum Ca2+-ATPase. Functional consequences for substrate hydrolysis and effects on the partial reactions of the hydrolytic cycle.

Erythrosin B was used to photo-oxidize the sarcoplasmic reticulum Ca2+-ATPase. The ATPase activity is rapidly and irreversibly inhibited by photo-oxidation with erythrosin. This inhibition is protected by the presence of ATP during the photo-oxidation period. After photo-oxidation, the steady-state phosphorylation by ATP remains almost unchanged, whereas phosphorylation by inorganic phosphate is impaired. The pseudo-first order rate constants for phosphorylation by 15 microM ATP at 25 degrees C are strongly inhibited when starting from either a Ca2+-bound or a Ca2+-free enzyme form, decreasing from 145 to 23 s-1 for the Ca2+-bound form and from 50 to 18 s-1 for the Ca2+-free form. Concurrently, the rate constants for dephosphorylation are also severely inhibited, changing from a fast double exponential to a very slow single exponential decay in the reverse direction and from a moderately slow single to a very slow single exponential decay in the forward direction. Ca2+ binding data show that the phosphorylated intermediate formed by the photo-oxidized enzyme contains two occluded Ca2+, and TNP-ATP fluorescence measurements indicate that it accumulates in a E1-P.Ca2-like conformation. Protection by ADP against glutaraldehyde-induced cross-linking indicates that ADP binding to Ca2+-ATPase is not impaired by photo-oxidation nor by free erythrosin. These data support the view that an ADP-insensitive, Ca2+-bound, slowly interconverting phosphoenzyme is formed. Thus, photo-oxidation with erythrosin B leads to impairment of phosphoryl transfer reactions and related conformational changes.

Two simultaneous binding sites for nucleotide analogs are kinetically distinguishable on the sarcoplasmic reticulum Ca(2+)-ATPase.

Erythrosin B and eosin Y stimulate p-nitrophenyl phosphate hydrolysis by purified sarcoplasmic reticulum Ca(2+)-ATPase by nearly 2-3 fold in the presence of Ca(2+). This stimulation is not due to the change on the apparent affinity for substrate but is indeed due to acceleration of the turnover rate of the enzyme. Stimulation reaches a maximum at approximately 5 microM erythrosin or 20 microM eosin and is strictly dependent on the presence of Ca(2+) in reaction media, while higher concentrations of dye progressively inhibit phosphatase activity. Labeling with fluorescein isothiocyanate (FITC) largely shifts the Km for p-nitrophenyl phosphate (pNPP) and completely abolishes the stimulation of phosphatase activity induced by erythrosin in the presence of Ca(2+), apparently by FITC impairing dye binding to an activator site and allowing only manifestation of an inhibitory binding site. In the absence of Ca(2+), both erythrosin and eosin inhibit pNPP hyrolysis with Ic50 values 3-4 fold higher than the maximally stimulatory enzyme with FITC, which by its turn does not affect pNPPase activity in absence of Ca(2+). It is suggested that stimulation and inhibition of phosphatase activity are related to two simultaneous and physically different nucleotide analog binding sites.

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).

Regulation of the erythrocyte Ca(2+)-ATPase at high pH.

The activation of the Ca(2+)-ATPase from erythrocyte membranes at high pH has been investigated. Following alkalinization and in the absence of regulators, the enzyme exhibits a very high affinity for Ca2+ and a decreased maximal velocity. Either addition of calmodulin, addition of acidic phospholipids, or controlled trypsinization decreases the concentration of effector required to elicit half-maximal activation of the enzyme for calcium to similar values. The increase in affinity for Ca2+, however, is smaller than that observed at neutral pH. The maximal velocity at high pH becomes insensitive to both calmodulin and controlled proteolysis, although calmodulin binds to the protein with similar affinities at pH 7.0 and 8.0, as indicated by similarity in binding to a calmodulin-Sepharose resin and in dependence on calmodulin concentrations when the pH is increased. In contrast to the attenuated effects of calmodulin and proteolysis, at pH 8.0 the enzyme is susceptible to stimulation by phospholipids, indicating that the pathway for transduction of the signal from phospholipids is distinct from that pathway engaged by calmodulin and/or trypsinization. At pH 8.0, phosphatidylinositol induces the modulatory effect of ATP at the regulatory site but calmodulin does not. We suggest that the intraenzymic connection between the calmodulin-binding, autoinhibitory peptide and the nucleotide domain of the enzyme is impaired upon alkalinization, which would account for the differing abilities of the activators to modulate the ATP effects.

Comparison of calmodulin and troponin C with and without its amino-terminal helix (residues 1-11) in the activation of erythrocyte Ca(2+)-ATPase.

Troponin C can replace calmodulin in the activation of the Ca(2+)-ATPase of pig erythrocytes provided that the reaction medium contains relatively high free Ca2+ concentrations (> 0.5 microM). In the presence of 10 microM free Ca2+, the troponin C-activated ATPase reaches a maximal velocity of approximately 70% of that attained with calmodulin. The half-maximal concentration for troponin C activation is about 200 times greater than for calmodulin. Troponin C displaces the half-maximal concentration for activation by Ca2+ to pCa 5.46 and the cooperativity between the Ca2+ binding sites to nH 1.1, compared with pCa 6.14 and nH 1.72 when calmodulin is used. Both EF-hand proteins also elicit activation by ATP at a nucleotide regulatory site, as well as a Ca(2+)-dependent p-nitrophenyl phosphatase activity. Troponin I prevents activation of the enzyme by troponin C. A mutant of troponin C with the amino-terminal helix deleted (NHdel) activates the Ca(2+)-ATPase to the same extent and with the same Ca2+ dependence as wild-type troponin C (rTnC); the half-maximal concentration for activation by NHdel is 2.5 times smaller than that for rTnC. We conclude that the structural features that distinguish the two EF-hand proteins affect their binding to the target enzyme more than their ability to transform the enzyme's response to Ca2+ or ATP. The differences in the amino-terminal domains of troponin C and calmodulin cannot account for the differences in ability of these proteins to activate the target system used as a model.

Are there different water requirements in different steps of a catalytic cycle? Hydration effects at the E1 and E2 conformers of sarcoplasmic reticulum Ca(2+)-ATPase studied in organic solvents with low amounts of water.

The Ca(2+)-ATPase from sarcoplasmic reticulum was transferred in an active form to a low-water system composed of toluene, phospholipids, and Triton X-100 (TPT). The Ca(2+)-ATPase activity in the TPT system with 4.0% water (by vol. was about 50% of the activity observed in all-aqueous mixtures. Phosphate formation was linear with time up to 20% of ATP hydrolysis and, as expected from an enzyme-catalysed reaction, activity was linear with protein concentration. No ATPase activity was detected in the presence of 3 mM EGTA, indicating that the enzyme retained its Ca2+ dependence in the TPT system. A hyperbolic response to ATP concentration was observed with a Km of 0.15 mM. There was no detectable ATPase activity at water concentrations below 1.5% (by vol.). With 2.0% water, activity became detectable and increased as the water content was progressively raised to 7.0% (by vol.). Higher amounts of water produced unstable emulsions. Enzyme phosphorylation by ATP and dephosphorylation took place in the TPT system. The velocities of both enzyme phosphorylation and dephosphorylation increased with increments in the water content. The enzyme could also be phosphorylated in the TPT system by inorganic phosphate. However, in comparison to ATP, phosphorylation by phosphate took place with significantly lower amounts of water. It is suggested that at low amounts of water, the enzyme is in a relatively rigid conformation and, as the water content is increased, the ATPase acquires more flexibility and, hence, the capacity to carry out catalysis at higher rates. Nevertheless, the release of conformational constraints of the catalytic site of the E2 conformer takes place at water concentrations much lower than those needed for the expression of catalytic activity by the E1 conformer.