Activation of the Ca2+-ATPase of human erythrocyte membrane by an endogenous Ca2+-dependent neutral protease

Limited proteolysis of the plasma membrane calcium transport ATPase (Ca2+-ATPase) from human erythrocytes by trypsin produces a calmodulin-like activation of its ATP hydrolytic activity and abolishes its calmodulin sensitivity. We now demonstrate a similar kind of activation of the human erythrocyte membrane Ca2+-ATPase by calpain (calcium-dependent neutral protease) isolated from the human red cell cytosol. Upon incubation of red blood cell membranes with purified calpain in the presence of Ca2+ the membrane-bound Ca2+-ATPase activity was increased and its sensitivity to calmodulin was lost. In contrast to the action of other proteases tested, proteolysis by calpain favors activation over inactivation of the Ca2+-ATPase activity, except at calpain concentrations more than 2 orders of magnitude higher. Exogenous calmodulin protects the Ca2+-ATPase against calpain-mediated activation at concentrations which also activate the Ca2+-ATPase activity. Calcium-dependent proteolytic modification of the Ca2+-ATPase could provide a mechanism for the irreversible activation of the membrane-bound enzyme.     

Separation of Ca2(+)-ATPase from Mg2(+)-ATPase in plasma membrane-rich fraction of bovine parotid gland

Ca2(+)-ATPase, which does not require Mg2+ for its activation, was separated from Mg2(+)-ATPase by papain treatment of a membrane-rich fraction of bovine parotid gland. The enzyme was partially purified 48-fold by subsequent chromatography on DEAE-cellulose, gel filtration on HPLC, and ion-exchange HPLC. The enzyme showed a molecular weight of 100,000, as estimated by gel filtration on HPLC. The Ca2(+)-ATPase was activated by Ca2+ but not by Mg2+, and this enzyme did not require Mg2+ for its activation by Ca2+. In fact, Mg2+ was inhibitory. p-Nitrophenyl phosphate was not hydrolyzed in the presence of Ca2+ or Mg2+, and this enzyme had no activities of other phosphatases tested. These results suggest that the Ca2(+)-ATPase is a separate enzyme from Mg2(+)-ATPase, Ca2(+)-stimulated Mg2(+)-dependent ATPase, and alkaline phosphatase, all of which are well known to be present in other tissues.     

Ca2+-mediated activation of human erythrocyte membrane Ca2+-ATPase

Ca2+-ATPase of human erythrocyte membranes, after being washed to remove Ca2+ after incubation with the ion, was found to be activated. Stimulation of the ATPase was related neither to fluidity change nor to cytoskeletal degradation of the membranes mediated by Ca2+. Activation of the transport enzyme was also unaffected by detergent treatment of the membrane, but was suppressed when leupeptin was included during incubation of the membranes with Ca2+. Stimulation of the ATPase by a membrane-associated Ca2+-dependent proteinase was thus suggested. Much less 138 kDa Ca2+-ATPase protein could be harvested from a Triton extract of membranes incubated with Ca2+ than without Ca2+. Activity of the activated enzyme could not be further elevated by exogenous calpain, even after treatment of the membranes with glycodeoxycholate. There was also an overlap in the effect of calmodulin and the Ca2+-mediated stimulation of membrane Ca2+-ATPase. While Km(ATP) of the stimulated ATPase remained unchanged, a significant drop in the free-Ca2+ concentration for half-maximal activation of the enzyme was observed.     

K(+)- and Mg2(+)-dependent hydrolysis of acetyl phosphate catalyzed by the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

The (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum catalyzes the hydrolysis of acetyl phosphate in the presence of Mg2+ and EGTA and is stimulated by Ca2+. The Mg2(+)-dependent hydrolysis of acetyl phosphate measured in the presence of 6 mM acetyl phosphate, 5 mM MgCl2, and 2 mM EGTA is increased 2-fold by 20% dimethyl sulfoxide. This activity is further stimulated 1.6-fold by the addition of 30 mM KCl. In this condition addition of Ca2+ causes no further increase in the rate of hydrolysis and Ca2+ uptake is reduced to a low level. In leaky vesicles, hydrolysis continues to be back-inhibited by Ca2+ in the millimolar range. Unlike ATP, acetyl phosphate does not inhibit phosphorylation by Pi unless dimethyl sulfoxide is present. The presence of dimethyl sulfoxide also makes it possible to detect Pi inhibition of the Mg2(+)-dependent acetyl phosphate hydrolysis. These results suggest that dimethyl sulfoxide stabilizes a Pi-reactive form of the enzyme in a conformation that exhibits comparable affinities for acetyl phosphate and Pi. In this conformation the enzyme is transformed from a Ca2(+)- and Mg2(+)-dependent ATPase into a (K+ + Mg2+)-ATPase.     

Activation of erythrocyte membrane Ca2+-ATPase by calpain

Ca2+-ATPase of erythrocyte membranes, prepared from erythrocytes substantially removed of contaminating leukocytes, was found to be activated by calpain isolated from the same source. Saponin or glycodeoxycholate treatment of membranes was essential for elicitation of the calpain response. Unlike the membrane bound ATPase, solubilized ATPase was inactivated by calpain. Digestion of membranes with the protease did not affect the Km (ATP) of Ca2+-ATPase though stimulation of the membrane ATPase by calmodulin could be partially substituted by calpain treatment. As compared with control, Ca2+-ATPase of calpain-digested membranes attained maximal activity at a lower free Ca2+ concentration.     

Characteristics of a presynaptic plasma membrane Ca2(+)-ATPase activity from electric organ

Ca2(+)-ATPase activity was measured in electric organ synaptosomal homogenates and their derived presynaptic plasma membranes using a low ionic strength medium, low in Ca2+ and Mg2+, and devoid of K+. The enzyme activity showed a high apparent affinity for Ca2+ (KCa:0.5 microM) and was: (1) 5-fold stimulated by 120 nM calmodulin, (2) highly sensitive to LaCl3 inhibition, and (3) not affected by 20 mM NaN3 or 0.1 mM ouabain. The addition of Mg2+ promoted the disappearance of Ca2(+)-ATPase activity. Incubation of synaptosomal homogenates in the above-mentioned assay medium with [gamma -32P]ATP resulted in the appearance of a 140 kDa band as revealed by SDS-gel electrophoresis. Labeling of this band with 32P was inhibited by 1 mM EGTA or 10 mM NH2OH, indicating that the isotope incorporation required the presence of Ca2+ and the formation of an acyl-phosphate derivative. The results indicate that the Ca2(+)-ATPase activity from synaptosomal homogenates had characteristics corresponding to those of the enzyme that catalyzes an outward transport of Ca2+ in nerve terminals. Preincubation of synaptosomes in Ca2+ plus K+, a depolarizing procedure, induced a large and rapid decrease in the Ca2(+)-ATPase activity, possibly mediated via Ca2+ entry through voltage-gated Ca2+ channels. Furthermore, the muscarinic cholinergic agonist oxotremorine (at 15 microM concentration) did not significantly affect either the enzyme activity or the intensity of the Ca2(+)-dependent 32P incorporation into the 140 kDa band, suggesting that the enzyme is not coupled to muscarinic binding sites.     

Biphasic kinetics of sarcoplasmic reticulum Ca2+-ATPase and the detergent-solubilized monomer

The mechanism of ATP hydrolysis was studied at 0 degrees C and pH 7.5 using purified leaky vesicles of sarcoplasmic reticulum Ca2+-ATPase and enzyme solubilized in monomeric form with high concentrations of octaethylene glycol monododecyl ether (C12E8). The enzyme reaction of membranous Ca2+-ATPase was characterized by an initial burst in the hydrolysis of ATP and modulated by millimolar concentrations of ATP. For detergent-solubilized Ca2+-ATPase no burst and moderate low affinity modulation was observed, but the reaction was activated both at low (phosphorylating) and intermediate (K0.5 = 0.06 mM) ATP concentrations. A study of the partial reactions indicated that the effects of detergent and ATP were attributable to activation of the E1P----E2P transition which was rate-limiting. E32P dephosphorylation of membranous Ca2+-ATPase and the detergent-solubilized monomer comprised both a slow and a rapid component. The inhibitory effect of high Ca2+ was correlated with the development of a dominant contribution of slow phase dephosphorylation and with ATP-induced extra binding of Ca2+ binding which presumably takes place at the phosphorylation site (ECaP). Ca2+ was bound with lesser affinity to detergent-solubilized Ca2+-ATPase but with qualitatively the same characteristics as to membranous ECaP. Either Ca2+ or Mg2+ was required for dephosphorylation, also after detergent solubilization. It is concluded that ATP hydrolysis occurs by the same steps for membranous and monomeric Ca2+-ATPase and involves formation of either EMgP or ECaP as reaction intermediates, leading to biphasic kinetics, which, therefore, cannot be taken as evidence of an oligomeric function of the enzyme.     

Effect of acylphosphatase on human erythrocyte membrane Ca2(+)-ATPase

We studied the effect of human acylphosphatase on the activity of human erythrocyte membrane Ca2(+)-ATPase. Both the acylphosphatase that is contained in hemolysate and the purified enzyme isolated from red blood cells were able to stimulate Ca2(+)-ATPase activity in erythrocyte membranes. Given the same acylphosphatase activity, however, the hemolysate showed higher stimulatory effect than the purified enzyme. Acylphosphatase stimulation was additive to that induced by calmodulin, thus indicating that acylphosphatase acts in a calmodulin-independent manner. Trifluoperazine, a calmodulin antagonist, did not inhibit acylphosphatase-induced stimulation of Ca2(+)-ATPase activity. Acylphosphatase significantly decreased the rate of Ca2+ influx into inside-out erythrocyte membrane vescicles, thus acting as Ca2+ pump inhibitor. Taken together these findings indicate that acylphosphatase is a soluble, non-calmodulin activator of erythrocyte membrane Ca2(+)-ATPase and might be involved in the control of calcium transport across the plasma membrane.     

Abnormal Ca2(+)-ATPase activity in erythrocytes of non-insulin-dependent diabetic rats

Non-insulin-dependent diabetic (NIDD) rats have an increased Ca2(+)-ATPase activity in their kidney basolateral membranes. We find that a similar increased activity occurs in erythrocytes of the NIDD animals. This alteration in membrane ATPase activity appears to be specific for the Ca2(+)-ATPase as (Na(+) + K+) and Mg2(+)-ATPase and Na, K and Mg concentrations in the erythrocyte were not affected by the diabetic condition in these animals. Thus, abnormalities in membrane Ca2(+)-ATPase activity in the NIDD rats are not restricted to one tissue and appear to be a generalized pathology in the NIDD animals.     

Regulation of the erythrocyte Ca2(+)-ATPase by mutant calmodulins with positively charged amino acid substitutions.

Four mutant calmodulins with site-specific charge alterations have been used to activate the human erythrocyte Ca2(+)-ATPase. These charge alterations were accomplished either by insertion of new Lys residues or by substitution of Lys residues for Glu in two of the seven calmodulin alpha-helices. Two enzyme preparations, purified monomeric Ca2(+)-ATPase and erythrocyte ghost membranes, were used with comparable results. At 100 nM Ca2+, the Ca2(+)-ATPase activity was lowered significantly by charge reversal from negative to positive in both the central alpha-helix and the carboxy-terminal domain. While all mutant calmodulins with charge reversal ultimately stimulated the Ca2(+)-ATPase activity to the same extent, the concentration of mutant calmodulin required for half-maximal activation was from 36-fold (central alpha-helix) to 126-fold higher (alpha-helix in the carboxy-terminal domain) than that of the control calmodulin. There was also a significant difference in the stimulation of Ca2(+)-ATPase activity by the different mutant calmodulins as a function of Ca2+ concentration, being most pronounced at submicromolar Ca2+ concentrations where enzyme activation by calmodulin appears to be a physiologically relevant mechanism. In contrast to the mutant calmodulins with charge reversal, mutant calmodulins in which two positive charges were added in the central alpha-helix activated the Ca2(+)-ATPase in a way undistinguishable from the control calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distances between functional sites of the Ca2+ + Mg2(+)-ATPase from sarcoplasmic reticulum using Co2+ as a spectroscopic ruler

Cobalt ion inhibits the Ca2+ + Mg2(+)-ATPase activity of sealed sarcoplasmic reticulum vesicles, of solubilized membranes and of the purified enzyme. To use Co2+ appropriately as a spectroscopic ruler to map functional sites of the Ca2+ + Mg2(+)-ATPase, we have carried out studies to obtain the kinetic parameters needed to define the experimental conditions to conduct the fluorimetric studies. 1. The apparent K0.5 values of inhibition of this ATPase are 1.4 mM, 4.8 mM and 9.5 mM total Co2+ at pH 8.0, 7.0 and 6.0, respectively. The inhibition by Co2+ is likely to be due to free Co2+ binding to the enzyme. Millimolar Ca2+ can fully reverse this inhibition, and also reverses the quenching of the fluorescence of fluorescein-labeled sarcoplasmic reticulum membranes due to Co2+ binding to the Ca2+ + Mg2(+)-ATPase. Therefore, we conclude that Co2+ interacts with Ca2+ binding sites. 2. Co2+.ATP can be used as a substrate by this enzyme with Vmax of 2.4 +/- 0.2 mumol ATP hydrolyzed min-1 (mg protein)-1 at 20-22 degrees C and pH 8.0, and with a K0.5 of 0.4-0.5 mM. 3. Co2+ partially quenches, about 10 +/- 2%, the fluorescence of fluorescein-labeled sarcoplasmic reticulum Ca2+ + Mg2(+)-ATPase upon binding to this enzyme at pH 8.0. From the fluorescence data we have estimated an average distance between Co2+ and fluorescein in the ATPase of 1.1-1.8 nm or 1.3-2.1 nm for one or two equidistant Co2+ binding sites, respectively. 4. Co2+.ATP quenches about 20-25% of the fluorescence of fluorescein-labeled Ca2+ + Mg2(+)-ATPase, from which we obtain a distance of 1.1-1.9 nm between Co2+ and fluorescein located at neighbouring catalytic sites.     

A Ca2(+)-activated, Mg2(+)-dependent ATPase with high affinities for both Ca2+ and Mg2+ in vascular smooth muscle microsomes: comparison with plasma membrane Ca2(+)-pump ATPase

A Ca2(+)-ATPase with a high affinity for free Ca2+ (apparent Km of 0.13 microM) was found and characterized in membrane fractions from porcine aortic and coronary artery smooth muscles in comparison with the plasma membrane Ca2(+)-pump ATPase purified from porcine aorta by calmodulin affinity chromatography. The activity of the high-affinity Ca2(+)-ATPase became enriched in a plasma membrane-enriched fraction, suggesting its localization in the plasma membrane. The enzyme was fully active in the absence of exogenously added Mg2+, but required a minute amount of Mg2+ for its activity as evidenced by the findings that it was fully active in the presence of 0.1 microM free Mg2+ but lost the activity in a reaction mixture containing trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid as a divalent cation chelator which has, unlike EGTA, high affinities for both Ca2+ and Mg2+. It was able to utilize a variety of nucleoside di- and triphosphates as substrates, such as ADP, GDP, ATP, GTP, CTP, and UTP, showing a broad substrate specificity. The activity of the enzyme was not modified by calmodulin (5, 10 micrograms/ml). Trifluoperazine, a calmodulin antagonist, had a partial inhibitory effect on the activity at 30 to 240 microM, but this inhibition could not be reproduced by a more specific calmodulin antagonist, W-7, indicating that this inhibition by trifluoperazine was not specific. Furthermore, the high-affinity Ca2(+)-ATPase activity was not modified either by low concentrations (0.5-9 microM) of vanadate or by 1-100 microM p-chloromercuribenzoic acid. Cyclic GMP, nitroglycerin, and nicorandil did not have any effect on the enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2(+)-induced conformational changes and location of Ca2+ transport sites in sarcoplasmic reticulum Ca2(+)-ATPase as detected by the use of proteolytic enzyme (V8)

Treatment of Ca2(+)-ATPase from sarcoplasmic reticulum with V8 protease from Staphylococcus aureus produced appreciable amounts of a Ca2(+)-ATPase fragment (p85) in the presence of Ca2+ (E1 conformation of the enzyme), along with many other peptide fragments that were also formed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (E2 conformation). p85 was formed as a carboxyl-terminal cleavage product of Ca2(+)-ATPase by a split of the peptide bond between Glu-231 and Ile-232. Other conformation-dependent V8 splits were localized to the "hinge" region, involved in ATP binding, between the middle and COOH-terminal one-third of the Ca2(+)-ATPase polypeptide chain. Representative split products in this region (p48,p31) were identified as NH2-terminal and COOH-terminal cleavage products of p85. In the membrane p85 probably remains associated with its complementary NH2-terminal fragment(s) and retains the capacity to bind Ca2+ as evidenced by resistance to V8 degradation in Ca2+ and ability to become phosphorylated by ATP. However, the hydrolysis rate of the phosphorylated enzyme is reduced, indicating that peptide cleavage at Glu-231 interferes with Ca2+ transport steps after phosphorylation. Binding of Ca2+ to V8 and tryptic fragments of Ca2(+)-ATPase was studied on the basis of Ca2(+)-induced changes in electrophoretic mobility and 45Ca2+ autoradiography after transfer of peptides to Immobilon membranes. These data indicate binding by the NH2-terminal 1-198 amino acid residues (corresponding to the tryptic A2 fragment) and the COOH-terminal 715-1001 amino acid residues (corresponding to p31). By contrast the central portion of Ca2(+)-ATPase, including the NH2-terminal portion of p85, is devoid of Ca2+ binding. These results question an earlier proposition that Ca2(+)-binding is located to the "stalk" region of Ca2(+)-ATPase (Brandl, C. J., Green, N. M., Korczak, B., and MacLennan, D. H.) (1986) Cell 44, 597-607) but are in agreement with recent data obtained by oligonucleotide-directed mutagenesis of Ca2(+)-ATPase (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478). These different studies suggest that Ca2+ translocation sites may have an intramembranous location and are formed predominantly by the carboxyl-terminal part of the Ca2(+)-ATPase polypeptide chain.     

The high affinity (Ca2+-Mg2+)-ATPase in liver plasma membranes is a Ca2+ pump. Reconstitution of the purified enzyme into phospholipid vesicles

The purified (Ca2+-Mg2+)-ATPase from rat liver plasma membranes (Lotersztajn, S., Hanoune, J., and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215) was incorporated into soybean phospholipid vesicles, together with its activator. In the presence of millimolar concentrations of Mg2+, the reconstituted proteoliposomes displayed a rapid, saturable, ATP-dependent Ca2+ uptake. Half-maximal Ca2+ uptake activity was observed at 13 +/- 3 nM free Ca2+, and the apparent Km for ATP was 16 +/- 6 microM. Ca2+ accumulated into proteoliposomes (2.8 +/- 0.2 nmol of Ca2+/mg of protein/90 s) was totally released upon addition of the Ca2+ ionophore A-23187. Ca2+ uptake into vesicles reconstituted with enzyme alone was stimulated 2-2.5-fold by the (Ca2+-Mg2+)-ATPase activator, added exogenously. The (Ca2+-Mg2+)-ATPase activity of the reconstituted vesicles, measured using the same assay conditions as for ATP-dependent Ca2+ uptake activity (e.g. in the presence of millimolar concentrations of Mg2+), was maximally activated by 20 nM free Ca2+, half-maximal activation occurring at 13 nM free Ca2+. The stoichiometry of Ca2+ transport versus ATP hydrolysis approximated 0.3. These results provide a direct demonstration that the high affinity (Ca2+-Mg2+)-ATPase identified in liver plasma membranes is responsible for Ca2+ transport.     

Purification and characterization of (Ca2+-Mg2+)-ATPase in rat liver lysosomal membranes.

A (Ca(2+)-Mg2+)-ATPase associated with rat liver lysosomal membranes was purified about 300-fold over the lysosomal membranes with a 7% recovery as determined from the pattern on polyacrylamide gel electrophoresis in the presence of SDS. The purification procedure included: preparation of lysosomal membranes, solubilization of the membrane with Triton X-100, WGA-Sepharose 6B, Con A-Sepharose, hydroxylapatite chromatography, and preparative polyacrylamide gel electrophoresis. The molecular mass, estimated by gel filtration with Sephacryl S-300 HR, was approximately 340 kDa, and SDS-polyacrylamide gel electrophoresis showed the enzyme to be composed of four identical subunits with an apparent molecular mass of 85 kDa. The isoelectric point of the purified enzyme was 3.6. The enzyme had a pH optimum of 4.5, a Km value for ATP of 0.17 mM and a Vmax of 71.4 mumol/min/mg protein at 37 degrees C. This enzyme hydrolyzed nucleotide triphosphates and ADP but did not act on p-nitrophenyl phosphate and AMP. The effects of Ca2+ and Mg2+ on the ATPase were not additive, thereby indicating that both Ca2+ and Mg(2+)-ATPase activities are manifested by the same enzyme. The (Ca(2+)-Mg2+)-ATPase differed from H(+)-ATPase in lysosomal membranes, since the enzyme was not inhibited by N-ethylmaleimide but was inhibited by vanadate. The effects of some other metal ions and compounds on this enzyme were also investigated. The N-terminal 18 residues of (Ca(2+)-Mg2+)-ATPase were determined.     

Characterization of the high and low affinity components of the renal Ca2(+)-Mg2+ ATPase

The purpose of this study was to characterize the interrelationship between free calcium (Ca2+) and magnesium (Mg2+) in the Ca2+ ATPase enzyme cycle of kidney membranes. Experiments were performed with basolateral membranes from rat renal cortex and microdissected proximal and distal tubules from mice. Results were similar in the three types of preparations. We first investigated the effect of ATP concentration on Ca2(+)- and Mg2(+)-dependent ATP hydrolysis. With 0.2 microM Ca2+, the enzyme activity, as a function of ATP concentration, showed two saturable components: a high affinity component with a Km of 33 microM ATP and a low affinity component with a Km of 0.63 mM ATP. These components may represent either two distinct sites of ATP binding or two forms of the same site. For the sake of simplicity, it was assumed that the two components correspond to a high affinity and a low affinity substrate site. At the high affinity site (ATP = 50 microM), the Ca2+ dependence of ATP hydrolysis followed a single Michaelis-Menten kinetics with Km for Ca2+ of 0.08 microM. The addition of 1 mM Mg2+ resulted in a relatively constant increase in ATP hydrolysis at all Ca2+ concentrations, indicating that the effects of the two cations were additive. With high ATP concentration (ATP = 3 mM), Ca2+ also induced an ATP hydrolysis according to a saturable process, with a Km for Ca2+ of 0.2 microM. In contrast with what occurred with low concentrations of ATP, addition of millimolar Mg2+ completely curtailed the sensitivity of the enzyme to Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fast efflux of Ca2+ mediated by the sarcoplasmic reticulum Ca2(+)-ATPase.

The Ca2(+)-ATPase found in the light fraction of sarcoplasmic reticulum vesicles can be phosphorylated by Pi, forming an acylphosphate residue at the catalytic site of the enzyme. This reaction was inhibited by the phenothiazines trifluoperazine, chlorpromazine, imipramine, and fluphenazine and by the beta-adrenergic blocking agents propranolol and alprenolol. The inhibition was reversed by raising either the Pi or the Mg2+ concentration in the medium and was not affected by the presence of K+. Phosphorylation of the Ca2(+)-ATPase by Pi was also inhibited by ruthenium red and spermidine. These compounds compete with Mg2+, but, unlike the phenothiazines, they did not compete with Pi at the catalytic site, and the inhibition was abolished when K+ was included in the assay medium. The efflux of Ca2+ from loaded vesicles was greatly increased by the phenothiazines and by propranolol and alprenolol. In the presence of 200 microM trifluoperazine, the rate of Ca2+ efflux was higher than 3 mumol of Ca2+/mg of protein/10 s. The activation of efflux by these drugs was antagonized by Pi, Mg2+, K+, Ca2+, ADP, dimethyl sulfoxide, ruthenium red, and spermidine. The increase of Ca2+ efflux caused by trifluoperazine was not correlated with binding of the drug to the membrane lipids. It is concluded that the Ca2+ pump can be uncoupled by different drugs, thereby greatly increasing the efflux of Ca2+ through the ATPase. Displacement of these drugs by the natural ligands of the ATPase blocks the efflux through the uncoupled pathway and limits it to a much smaller rate. Thus, the Ca2(+)-ATPase can operate either as a pump (coupled) or as a Ca2+ channel (uncoupled).     

Characterization of a putative Ca2(+)-transporting Ca2(+)-ATPase in the pellicles of Paramecium tetraurelia

In Paramecium, no Ca2(+)-ATPases with the properties of Ca2+ pumps have been identified. Here we report a pellicle associated Ca2(+)-ATPase activity and a corresponding phosphoprotein intermediate characteristic of a pump. The Ca2(+)-ATPase activity requires 3 mM Mg for optimal Ca2+ stimulation (KCa = 90 nM) and is specific for ATP as substrate (Km = 75 microM). Vanadate and calmidazolium inhibit Ca2(+)-stimulated activity with an EC50 of about 2 microM and 0.5 microM, respectively. Likewise, 10 microM trifluoperazine inhibits 80% of Ca2(+)-ATPase activity, but bovine calmodulin fails to stimulate. The Ca2(+)-ATPase is not inhibited by sodium azide (10 mM), oligomycin (10 micrograms/ml) or ouabain (0.2 mM). Incubation of pellicles with [gamma-32P]ATP specifically labels a 133 kDa protein in a Ca2(+)-dependent, hydroxylamine-sensitive manner, and the level of phosphorylation is increased by 100 microM La3+. Phosphorylation of an endoplasmic reticulum-enriched fraction labels a Ca2(+)-dependent protein different from the pellicle protein, being lower in molecular mass and unaffected by La3+. Ca2+ uptake by the alveolar sacs, integral components of the pellicle membrane complex, is poorly coupled to Ca2(+)-stimulated ATP hydrolysis (Ca2+ transported/ATP hydrolysed less than 0.2) and is much less sensitive to vanadate inhibition (EC50 approx. 20 microM) compared to the total Ca2(+)-ATPase activity. Therefore, the majority of the Ca2(+)-ATPase activity is likely to be plasma membrane associated.     

Ca2+-activated ATPase of rat liver plasma membrane.

Rat liver plasma membranes hydrolyze ATP in the presence of Ca2+. The rate of hydrolysis is different when Mg2+ions are present in the incubation system. Several parameters differentiate Ca2+-ATPase from Mg2+-ATPase: a) the Km of ATP hydrolysis for Ca2+ (2.25 x 10(-4) M) is lower than for Mg2+ (2.14 x 10(-3) M); b) the shape of the activation curve is hyperbolic in the presence of Ca2+ and sigmoid in the presence of Mg2+; c) Mg2+-ATPase shows two different values of activation energy while Ca2+-ATPase presents only a single value; d) Ca2+-ATPase is inhibited, while Mg2+-ATPase is unaffected by cyclic AMP. Ca2+-ATPase is localized on the plasma membrane and is not inhibited by cysteine. It does not hydrolyze substrates different from nucleotides triphosphate, such as glucose-1-phosphate or alpha-glycero-phosphate. The enzyme is probably related to a mechanism of calcium transport.     

The effect of berbamine derivatives on activated Ca2+-stimulated Mg2+-dependent ATPase in erythrocyte membranes.

Ca2+-stimulated Mg2+-dependent ATPase (Ca2+ + Mg2+-ATPase) stimulated by calmodulin, by partial proteolysis or by oleic acid in erythrocyte membranes was inhibited by various derivatives of the naturally occurring alkaloid berbamine. The ability of these derivatives to inhibit trypsin-activated Ca2+ + Mg2+-ATPase correlated well with their ability to inhibit the calmodulin-stimulated enzyme. Inhibition of the trypsin-activated Ca2+ + Mg2+-ATPase by O-4-(ethoxybutyl)berbamine (EBB) was competitive with respect to ATP. The Ki for inhibition was about 8 microM. These results suggest that the binding site of EBB on the activated Ca2+ + Mg2+-ATPase may bear structural similarity to that on calmodulin, and may be closely related to the ATP-binding site on the enzyme.