A monoclonal antibody to the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle inhibits plasmalemmal (Ca2+ + Mg2+)-dependent ATPase activity.

A monoclonal antibody (2B3) directed against the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle was prepared. This antibody reacts with a 130,000-Mr protein that co-migrates on SDS/polyacrylamide-gel electrophoresis with the calmodulin-binding (Ca2+ + Mg2+)-ATPase purified from smooth muscle by calmodulin affinity chromatography. The antibody causes partial inhibition of the (Ca2+ + Mg2+)-ATPase activity in plasma membranes from pig stomach smooth muscle, in pig erythrocytes and human erythrocytes. It appears to be directed against a specific functionally important site of the plasmalemmal Ca2+-transport ATPase and acts as a competitive inhibitor of ATP binding. Binding of the antibody does not change the Km of the ATPase for Ca2+ and its inhibitory effect is not altered by the presence of calmodulin. No inhibition of (Ca2+ + Mg2+)-ATPase activity or of the oxalate-stimulated Ca2+ uptake was observed in a pig smooth-muscle vesicle preparation enriched in endoplasmic reticulum. These results confirm the existence in smooth muscle of two different types of Ca2+-transport ATPase: a calmodulin-binding (Ca2+ + Mg2+)-ATPase located in the plasma membrane and a second one confined to the endoplasmic reticulum.     

Inhibitory antibodies to plasmalemmal Ca2+-transporting ATPases. Their use in subcellular localization of (Ca2+ + Mg2+)-dependent ATPase activity in smooth muscle.

Antibodies directed against the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase [(Ca2+ + Mg2+)-dependent ATPase] from pig erythrocytes and from smooth muscle of pig stomach (antral part) were raised in rabbits. Both the IgGs against the erythrocyte (Ca2+ + Mg2+)-ATPase and against the smooth-muscle (Ca2+ + Mg2+)-ATPase inhibited the activity of the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase from smooth muscle. Up to 85% of the total (Ca2+ + Mg2+)-ATPase activity in a preparation of KCl-extracted smooth-muscle membranes was inhibited by these antibodies. The (Ca2+ + Mg2+)-ATPase activity and the Ca2+ uptake in a plasma-membrane-enriched fraction from this smooth muscle were inhibited to the same extent, whereas in an endoplasmic-reticulum-enriched membrane fraction the (Ca2+ + Mg2+)-ATPase activity was inhibited by only 25% and no effect was observed on the oxalate-stimulated Ca2+ uptake. This supports the hypothesis that, in pig stomach smooth muscle, two separate types of Ca2+-transport ATPase exist: a calmodulin-binding ATPase located in the plasma membrane and a calmodulin-independent one present in the endoplasmic reticulum. The antibodies did not affect the stimulation of the (Ca2+ + Mg2+)-ATPase activity by calmodulin.     

The effect of cardiotoxin on (Ca2+ + Mg2+)-ATPase of the erythrocyte and sarcoplasmic reticulum

The effects of cardiotoxin on the ATPase activity and Ca2+-transport of guinea pig erythrocyte and rabbit muscle sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase (E.C.3.6.1.3) were investigated. Erythrocyte (Ca2+ + Mg2+)-ATPase was inhibited by cardiotoxin in a time- and dose-dependent fashion and inhibition appears to be irreversible. Micromolar calcium prevented this inhibitory effect. Specificity for (Ca2+ + Mg2+)-ATPase inhibition by cardiotoxin was indicated since a homologous neurotoxin had no effect. Cardiotoxin did not affect (Ca2+ + Mg2+)-ATPase activity from sarcoplasmic reticulum, but Ca2+-transport was 50% inhibited. This inhibition was not due to an increased Ca2+-efflux and could be the result of an intramolecular uncoupling of ATPase activity from Ca2+-transport. Inhibition of Ca2+-transport by cardiotoxin could not be prevented by millimolar concentrations of Ca2+. It is suggested that the biological effects of cardiotoxin could be a consequence of inhibition of plasma membrane (Ca2+ + Mg2+)-ATPases.     

Smooth muscle expresses a cardiac/slow muscle isoform of the Ca2+-transport ATPase in its endoplasmic reticulum.

Smooth muscle expresses in its endoplasmic reticulum an isoform of the Ca2+-transport ATPase that is very similar to or identical with that of the cardiac-muscle/slow-twitch skeletal-muscle form. However, this enzyme differs from that found in fast-twitch skeletal muscle. This conclusion is based on two independent sets of observations, namely immunological observations and phosphorylation experiments. Immunoblot experiments show that two different antibody preparations against the Ca2+-transport ATPase of cardiac-muscle sarcoplasmic reticulum also recognize the endoplasmic-reticulum/sarcoplasmic-reticulum enzyme of the smooth muscle and the slow-twitch skeletal muscle whereas they bind very weakly or not at all to the sarcoplasmic-reticulum Ca2+-transport ATPase of the fast-twitch skeletal muscle. Conversely antibodies directed against the fast-twitch skeletal-muscle isoform of the sarcoplasmic-reticulum Ca2+-transport ATPase do not bind to the cardiac-muscle, smooth-muscle or slow-twitch skeletal-muscle enzymes. The phosphorylated tryptic fragments A and A1 of the sarcoplasmic-reticulum Ca2+-transport ATPases have the same apparent Mr values in cardiac muscle, slow-twitch skeletal muscle and smooth muscle, whereas the corresponding fragments in fast-twitch skeletal muscle have lower apparent Mr values. This analytical procedure is a new and easy technique for discrimination between the isoforms of endoplasmic-reticulum/sarcoplasmic-reticulum Ca2+-transport ATPases.     

Utilization of purified myometrium cell plasma membrane Ca2+, Mg2+-ATPase for comparative estimation of the efficiency of energy-dependent Ca2+-transport inhibitors

With the aim of comparative estimation of efficacy of well-known inhibitors of energy-dependent Ca(2+)-transporting systems their effects were investigated on the activity of purified Ca2+, Mg(2+)-ATPase of the myometrium cell plasma membranes. From the approved inhibitors (eosin Y, o-vanadate, thapsigargin, cyclopiazonic acid, ruthenium red, sodium azide) only eosin Y and o-vanadate are potent inhibitors of myometrium sarcolemma Ca(2+)-pump: the values of Ki equal 0.8 and 4.7 microM, respectively. Thapsigargin and cyclopiazonic acid as well as ruthenium red in concentrations inhibiting, respectively, endo(sarco)plasmic reticulum Ca(2+)-pump and energy-dependent Ca(2+)-transport in mitochondria had no effect on the Ca2+, Mg(2+)-ATPase of the uterus smooth muscle cell plasma membrane. Sodium azide (10 mM) blocking completely Ca(2+)-transport in mitochondria inhibited activity of the plasma membrane Ca(2+)-transporting ATPase by 14%.     

Cyclic GMP-dependent protein kinase stimulates the plasmalemmal Ca2+ pump of smooth muscle via phosphorylation of phosphatidylinositol.

The effect of phosphorylation by cyclic GMP-dependent protein kinase (G-kinase) on the activity of the plasmalemmal Ca2+-transport ATPase was studied on isolated plasma membranes and on the ATPase purified from pig erythrocytes and from the smooth muscle of pig stomach and pig aorta. Incubation with G-kinase resulted, in both smooth-muscle preparations, but not in the erythrocyte ATPase, in a higher Ca2+ affinity and in an increase in the maximal rate of Ca2+ uptake. Cyclic AMP-dependent protein kinase (A-kinase) did not exert such an effect. The stimulation of the (Ca2+ + Mg2+)-dependent ATPase activity of the purified Ca2+ pump reconstituted in liposomes depended on the phospholipid used for reconstitution. The stimulation of the (Ca2+ + Mg2+)-ATPase activity by G-kinase was only observed in the presence of phosphatidylinositol (PI). G-kinase, but not A-kinase, stimulated the phosphorylation of PI to phosphatidylinositol phosphate (PIP) in a preparation of (Ca2+ + Mg2+)-ATPase obtained by calmodulin affinity chromatography from smooth muscle, but not in a similar preparation from erythrocytes. Adenosine inhibited both the phosphorylation of PI and the stimulation of the (Ca2+ + Mg2+)-ATPase by G-kinase. In the absence of G-kinase the (Ca2+ + Mg2+)-ATPase was stimulated by the addition of PIP, but not by PI. In contrast with previous results of Furukawa & Nakamura [(1987) J. Biochem (Tokyo) 101, 287-290], no convincing evidence for a phosphorylation of the (Ca2+ + Mg2+)-ATPase was found. Evidence is presented showing that the apparent phosphorylation occurs in a contaminant protein, possibly myosin light-chain kinase. It is proposed that G-kinase stimulates the plasmalemmal Ca2+ pump of smooth-muscle cells indirectly via the phosphorylation of an associated PI kinase.     

AlF4- reversibly inhibits ''P''-type cation-transport ATPases, possibly by interacting with the phosphate-binding site of the ATPase.

The only known cellular action of AlF4- is to stimulate the G-proteins. The aim of the present work is to demonstrate that AlF4- also inhibits 'P'-type cation-transport ATPases. NaF plus AlCl3 completely and reversibly inhibits the activity of the purified (Na+ + K+)-ATPase (Na+- and K+-activated ATPase) and of the purified plasmalemmal (Ca2+ + Mg2+)-ATPase (Ca2+-stimulated and Mg2+-dependent ATPase). It partially inhibits the activity of the sarcoplasmic-reticulum (Ca2+ + Mg2+)-ATPase, whereas it does not affect the mitochondrial H+-transporting ATPase. The inhibitory substances are neither F- nor Al3+ but rather fluoroaluminate complexes. Because AlF4- still inhibits the ATPase in the presence of guanosine 5'-[beta-thio]diphosphate, and because guanosine 5'-[beta gamma-imido]triphosphate does not inhibit the ATPase, it is unlikely that the inhibition could be due to the activation of an unknown G-protein. The time course of inhibition and the concentrations of NaF and AlCl3 required for this inhibition differ for the different ATPases. AlF4- inhibits the (Na+ + K+)-ATPase and the plasmalemmal (Ca2+ + Mg2+)-ATPase noncompetitively with respect to ATP and to their respective cationic substrates, Na+ and Ca2+. AlF4- probably binds to the phosphate-binding site of the ATPase, as the Ki for inhibition of the (Na+ + K+)-ATPase and of the plasmalemmal (Ca2+ + Mg2+)-ATPase is shifted in the presence of respectively 5 and 50 mM-Pi to higher concentrations of NaF. Moreover, AlF4- inhibits the K+-activated p-nitrophenylphosphatase of the (Na+ + K+)-ATPase competitively with respect to p-nitrophenyl phosphate. This AlF4- -induced inhibition of 'P'-type cation-transport ATPases warns us against explaining all the effects of AlF4- on intact cells by an activation of G-proteins.     

Alkalinization stimulates the purified plasma-membrane Ca2+ pump by increasing its Ca2+ affinity.

The finding that negatively charged phospholipids activate the plasma-membrane (Ca2+ + Mg2+)-ATPase and that polycations counteract this stimulation suggest that negative charges in the environment of the ATPase protein could be important for its function. The aim of the present work was to investigate whether changing the charges on the ATPase protein itself by modifying the pH within the physiological range affects the activity of the purified plasma-membrane Ca2+ pump from stomach smooth muscle. Increasing the pH from 6.9 to 7.4 and using 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA) as a Ca2+ buffer, doubled the ATPase activity at 0.3 microM-Ca2+ in the presence of 100% phosphatidylcholine (PC) or after substituting 20% of the PC by negatively charged phospholipids PtdIns, PtdIns4P, phosphatidylserine and phosphatidic acid. This stimulatory effect was due to an increased affinity of the enzyme for Ca2+, while the Vmax. remained unaffected. In the case of PtdIns(4,5)P2, a stimulatory effect upon alkalinization was only observed at a PtdIns(4,5)P2 concentration of 10%. When a concentration of 20% was used, alkalinization decreased the Vmax. and no stimulatory effect on the ATPase at 0.3 microM-Ca2+ could be observed. Alkalinization not only stimulated the purified Ca2+ pump, but it also increased the activity of the enzyme in a plasma-membrane-enriched fraction from stomach smooth muscle by a factor of 2.06. The ionophore A23187-induced Ca2+ uptake in closed inside-out vesicles also increased by a factor of 2.54 if the pH was changed from 6.9 to 7.4. This finding indicates that the effect of pH is most likely to be exerted at the cytoplasmic site of the Ca2+ pump protein.     

Antibodies to the calmodulin-binding Ca2+-transport ATPase from smooth muscle

Antibodies were raised against a calmodulin-binding CaMg-ATPase (Ca2+-transport ATPase) from smooth muscle. The binding of these antibodies to a number of related Ca2+-transport ATPases was studied. Antibodies to the calmodulin-binding ATPase from porcine antrum (stomach) smooth muscle do not only bind to this CaMg-ATPase, but also to the corresponding enzyme in porcine erythrocytes. However, they do not bind to the CaMg-ATPase from sarcoplasmic reticulum of porcine skeletal muscle. The binding of these antibodies to the CaMg-ATPase of smooth muscle, does not inhibit the enzyme activity.     

Effect of fluoroaluminate on the ATP-hydrolysing and Ca2+-transporting activity of the purified Ca2+, Mg2+-ATPase of myometrium cell plasma membranes

Fluoroaluminate, known modulator of G-proteins, inhibits ATP-hydrolase activity of purified solubilized Ca2+, Mg(2+)-ATPase from myometrium cell plasma membranes and Ca(2+)-transporting activity of this enzyme reconstituted into azolectin liposomes: 10 mM NaF plus 10 microM AlCl3 inhibited the primary activity by 95% and--by 81%. Inhibition of purified both solubilized and reconstituted Ca2+, Mg(2+)-ATPases by fluoroaluminate evidences for the possibility of direct interaction AlF4- with this enzyme without involvement of G-protein. The sensitivity to fluoroaluminate of sarcolemmal Ca2+, Mg(2+)-ATPase from myometrium is similar to that of Ca2+, Mg(2+)-ATPase from stomach smooth muscle.     

Ca2+-transporting properties of myometrial plasma cell membrane Ca2+, Mg2+-ATPase reconstituted in liposomes

Purified myometrium cells plasma membrane Ca2+, Mg(2+)-ATPase was reconstitute in liposomes in functionally active state by the method of cholate dialysis: it showed ATP-hydrolase activity increased by 0.8 microM A23187 average 4 times and it showed Mg2+, ATP-dependent Ca(2+)-transporting activity. Reconstituted system transported Ca2+ at an initial rate of 114.4 +/- 16.3 nmol.min-1.mg-1 with the stoichiometry Ca2+: ATP = 1: (3.2-3.7). Calmodulin increased by 30% the initial rate of Ca(2+)-accumulation by the proteoliposomes with reconstituted Ca2+, Mg(2+)-ATPase; 0.1 mM orthovanadate decreased by 80% Ca(2+)-accumulation by this system. Ca2+, Mg(2+)-ATPase reconstituted in liposomes is just Ca(2+)-transporting ATPase of the plasma membrane. Obtained enzyme preparate can be utilised for study of the properties of this important energy-dependent Ca(2+)-transporting system of smooth muscle cell.     

Mechanism of eosin Y action of activity of solubilized Ca2+, Mg2+- ATPase from smooth muscle sarcolemma

The eosin Y inhibitory effect on the activity of smooth muscle plasma membrane Ca(2+)-transporting ATPase was studied: effect of this inhibitor on the maximal initial rate of ATP-hydrolase reaction, catalyzed by Ca2+, Mg(2+)-ATPase, on the affinity of enzyme for the reaction reagents (Ca2+, Mg2+, ATP). Dependence of eosin Y inhibitory effect on some physicochemical factors of incubation medium was studied too. It was determined that eosin Y inhibited reversibly and with high specificity purified Ca2+, Mg(2+)-ATPase solubilized from myometrial cell plasma membrane (Ki--0.8 microM), decreased the turnover rate of this enzyme determined both by Mg2+, ATP and Ca2+. This inhibitor had no effect on the enzyme affinity for Ca2+, increased affinity for Mg2+ and decreased affinity for ATP. It was determined that inhibition of Ca2+, Mg(2+)-ATPase by eosin Y depended on pH and dielectric permeability of the incubation medium: increasing of pH from 6.5 to 8.0 reduced the apparent Ki, decreasing of dielectric permeability from 74.07 to 71.19 increased the apparent Ki.     

Reconstitution of the purified calmodulin-dependent (Ca2+ + Mg2+)-ATPase from smooth muscle

The purified calmodulin dependent (Ca2+ + Mg2+)-ATPase (CaMg ATPase) from porcine antral smooth muscle transports Ca2+ after reconstitution in lipid vesicles indicating that this enzyme is indeed a Ca2+-transport ATPase. For CaMg ATPase reconstituted in asolectin vesicles a good correlation was found between the time course of Ca2+ accumulation and the corresponding changes in CaMg ATPase activity. The ATPase activity was stimulated 8-fold by A23187, which further indicates a tight coupling between ATP hydrolysis and Ca2+ transport. Asolectin vesicles with incorporated enzyme accumulated Ca2+ with a ratio approaching one Ca2+ ion transported for each ATP hydrolyzed. For CaMg ATPase reconstituted in phosphatidylcholine vesicles on the other hand, Ca2+ transport and CaMg ATPase were poorly coupled as is shown by the approximately 3.5 fold stimulation by A23187. The activity of the CaMg ATPase when reconstituted in asolectin vesicles was stimulated 1.25 fold by calmodulin while in phosphatidylcholine a value of 4.25 was obtained. The CaMg ATPase activity of the enzyme reconstituted either in asolectin or phosphatidylcholine was, after its stimulation by A23187, still further stimulated by detergent by a factor of 5.     

Evidence for the presence in smooth muscle of two types of Ca2+-transport ATPase.

Membrane fractions prepared from smooth muscle of the pig stomach (antral part) contain two Ca2+-dependent phosphoprotein intermediates belonging to different Ca2+-transport ATPases. These alkali-labile phosphoproteins can be separated by electrophoresis in acid medium. The 130 kDa phosphoprotein resembles a corresponding protein in the erythrocyte membrane, whereas the 100 kDa protein resembles that of the Ca2+-transport ATPase in sarcoplasmic reticulum from skeletal muscle. These resemblances are expressed in terms of Mr, reaction to La3+ and in a similar proteolytic degradation pattern. The presence of the calmodulin-stimulated ATPase in mixed membranes from smooth muscle is confirmed by its binding of calmodulin and antibodies against erythrocyte Ca2+-transport ATPase, whereas such binding does not occur with proteins present in the presumed endoplasmic reticulum from smooth muscle.     

A Mg2+-independent high-affinity Ca2+-stimulated adenosine triphosphatase in the plasma membrane of rat stomach smooth muscle. Subcellular distribution and inhibition by Mg2+

Plasma membrane enriched fraction isolated from the fundus smooth muscle of rat stomach displayed Ca2+-stimulated ATPase activity in the absence of Mg2+. The Ca2+ dependence of such an ATPase activity can be resolved into two hyperbolic components with a high affinity (Km = 0.4 microM) and a low affinity (Km = 0.6 mM) for Ca2+. Distribution of these high-affinity and low-affinity Ca2+-ATPase activities parallels those of several plasma membrane marker enzyme activities but not those of endoplasmic reticulum and mitochondrial membrane marker enzyme activities. Mg2+ also stimulates the ATPase in the absence of Ca2+. Unlike the Mg2+-ATPase and low-affinity Ca2+-ATPase, the plasmalemmal high-affinity Ca2+-ATPase is not sensitive to the inhibitory effect of sodium azide or Triton X-100 treatment. The high-affinity Ca2+-ATPase is noncompetitively inhibited by Mg2+ with respect to Ca2+ stimulation. Such an inhibitory effect of Mg2+ is potentiated by Triton X-100 treatment of the membrane fraction. Calmodulin has little effect on the high-affinity Ca2+-ATPase activity of the plasma membrane enriched fraction with or without EDTA pretreatment. Findings of this novel, Mg2+-independent, high-affinity Ca2+-ATPase activity in the rat stomach smooth muscle plasma membrane are discussed with those of Mg2+-dependent, high-affinity Ca2+-ATPase activities previously reported in other smooth muscle plasma membrane preparations in relation to the plasma membrane Ca2+-pump.     

Partial purification of (Ca2+ + Mg2+)-dependent ATPase from pig smooth muscle and reconstitution of an ATP-dependent Ca2+-transport system.

(CaMg)ATPase [(Ca2+ + Mg2+)-dependent ATPase] was partially purified from a microsomal fraction of the smooth muscle of the pig stomach (antrum). Membranes were solubilized with deoxycholate, followed by removal of the detergent by dialysis. The purified (CaMg)ATPase has a specific activity (at 37 degrees C) of 157 +/- 12.1 (7)nmol.min-1.mg-1 of protein, and it is stimulated by calmodulin to 255 +/- 20.9 (7)nmol.min.mg-1. This purification of the (CaMg)ATPase resulted in an increase of the specific activity by approx. 18-fold and in a recovery of the total enzyme activity of 55% compared with the microsomal fraction. The partially purified (CaMg)ATPase still contains some Mg2+-and (Na+ + K+)-dependent ATPase activities, but their specific activities are increased relatively less than that of the (CaMg)ATPase. The ratios of the (CaMg)ATPase to Mg2+- and (Na+ + K+)-dependent ATPase activities increase from respectively 0.14 and 0.81 in the crude microsomal fraction to 1.39 and 9.07 in the purified preparation. During removal of the deoxycholate by dialysis, vesicles were reconstituted which were capable of ATP-dependent Ca2+ transport.     

Modulation of the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by pentobarbital

The dependence of the (Ca2+ + Mg2+)-ATPase activity of sarcoplasmic reticulum vesicles upon the concentration of pentobarbital shows a biphasic pattern. Concentrations of pentobarbital ranging from 2 to 8 mM produce a slight stimulation, approximately 20-30%, of the ATPase activity of sarcoplasmic reticulum vesicles made leaky to Ca2+, whereas pentobarbital concentrations above 10 mM strongly inhibit the activity. The purified ATPase shows a higher sensitivity to pentobarbital, namely 3-4-fold shift towards lower values of the K0.5 value of inhibition by this drug. These effects of pentobarbital are observed over a wide range of ATP concentrations. In addition, this drug shifts the Ca2+ dependence of the (Ca2+ + Mg2+)-ATPase activity towards higher values of free Ca2+ concentrations and increases several-fold the passive permeability to Ca2+ of the sarcoplasmic reticulum membranes. At the concentrations of pentobarbital that inhibit this enzyme in the sarcoplasmic reticulum membrane, pentobarbital does not significantly alter the order parameter of these membranes as monitored with diphenylhexatriene, whereas the temperature of denaturation of the (Ca2+ + Mg2+)-ATPase is decreased by 4-5 C degrees, thus, indicating that the conformation of the ATPase is altered. The effects of pentobarbital on the intensity of the fluorescence of fluorescein-labeled (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum also support the hypothesis of a conformational change in the enzyme induced by millimolar concentrations of this drug. It is concluded that the inhibition of the sarcoplasmic reticulum ATPase by pentobarbital is a consequence of its binding to hydrophobic binding sites in this enzyme.     

Effect of spermine on activity of purified Ca2+, Mg2+-ATPase from plasma membranes of myometrium cells

Effect of endogenous polyamine spermine, a relaxant of smooth muscle, on the activity of myometrium cell plasma membrane Ca2+, Mg(2+)-ATPase was studied. It was observed a tendency to activation of enzyme at the spermine concentrations 0.1-0.5 mM, the increase of the polyamine concentrations up to 10 mM inhibited. ATPase by 80% (I50 = 5.5 +/- 0.3 mM). Spermine inhibited enzyme decreasing its turnover rate and affinity for Ca2+. The ATPase affinity for Mg2+ increased in the presence of spermine. It was revealed, that the inhibitory effect of spermine is changed by the stimulatory effect under the increase of Ca2+ concentration (up to 2.6 microM), that correlates with the relaxing effect of this polyamine on the smooth muscle.     

Mechanism of inhibition of Ca2+-transport activity of sarcoplasmic reticulum Ca2+-ATPase by anisodamine

The mechanism of inhibition of Ca2+-transport activity of rabbit sarcoplasmic reticulum Ca 2+-ATPase (SERCA) by anisodamine (a drug isolated from a medicinal herb Hyoscyamuns niger L) was investigated by using ANS (1-anilino-8-naphthalenesulfonate) fluorescence probe, intrinsic fluorescence quenching and Ca 2+-transport activity assays. The number of ANS binding sites for apo Ca2+-ATPase was determined as 8, using a multiple-identical binding site model. Both anisodamine and Ca2+ at millimolar level enhanced the ANS binding fluorescence intensities. Only anisodamine increased the number of ANS molecules bound by SERCA from 8 to 14. The dissociation constants of ANS to the enzyme without any ligand, with 30 mM anisodamine and with 15 mM Ca 2 were found to be 53.0 microM, 85.0 microM and 50.1 microM, respectively. Both anisodamine and Ca2+ enhanced the ANS binding fluorescenc with apparent dissociation constants of 7.6 mM and 2.3 mM, respectively, at a constant concentration of the enzyme. Binding of anisodamine significantly decreased the binding capacity of Ca2+ with the dissociation constant of 9.5 mM, but binding of Ca2+ had no obvious effect on binding of anisodamine. Intrinsic fluorescence quenching and Ca2+-transport activity assays gave the dissociation constants of anisodamine to SERCA as 9.7 and 5.4 mM, respectively, which were consistent with those obtained from ANS-binding fluorescence changes during titration of SERCA with anisodamine and anisodamine + 15 mM Ca2+, respectively. The results suggest that anisodamine regulates Ca2+-transport activity of the enzyme, by stabilizing the trans-membrane domain in an expanded, inactive conformation, at least at its annular ring region.