Effect of ethanol on the activity of the energy-dependent Ca2+-transporting system of myometrial cells

In order of estimating some regularities of ethanol direct (effectory) effect to transmembrane calcium metabolism in the myometrium the action of this substance on the energy-dependent Ca(2+)-transporting systems of the uterine myocytes subcellular structures has been studied. The systems of Mg2+, ATP-dependent Ca2+ transport regarding their sensitivity to ethanol inhibitory effect were displayed as satisfying the following sequences: endoplasmic reticulum calcium pump > plasma membrane solubilized Ca2+, Mg2+, ATP-ase > mitochondrial Ca(2+)-accumulating system = plasma membrane calcium pump. Alongside with the latter, the oxytocin-insensitive component of Mg2+, ATP-dependent Ca2+ accumulation in the endoplasmic reticulum was defined to be less resistant to inhibitory effect of ethanol if compared with the oxytocin-sensitive one. On the base of the data received some mechanisms of ethanol effectory action on the intracellular calcium homeostasis in the myometrium cells are under the discussion.     

Accumulation of Ca ions in intracellular structures of rat myometrium during subacute, acute, and chronic administration of ethanol

The effects of subacute, acute and chronic ethanol exposure on the activity of Ca(2+)-accumulating systems of mitochondria and endoplasmic reticulum in myometrial cells of nonpregnant estrogen-treated rats were studied. It has been shown that the activity of Ca(2+)-accumulating system of mitochondria was higher than the activity of Ca(2+)-accumulating system of endoplasmic reticulum in myometrial cells from control, acute and subacute treated with ethanol rats. Under ethanol chronical assumption both Ca(2+)-accumulation in mitochondria and Ca(2+)-transporting activity of endoplasmic reticulum are inhibited. In the latter ease Mg2+, ATP-dependent Ca(2+)-pump lost its sensitivity to oxytocin.     

Contribution of the Mg2+-, ATP- AND Na+-dependent Ca2+-transport systems to the regulation of Ca2+ concentration in myometrial cells

Studies with sarcolemma from cattle myometrium containing inside-out cytoplasmic vesicles, using Ca2+-EGTA buffer, showed that the affinity of ionized Ca2+ for the Mg2+- or ATP-dependent transport is higher than that for the Na+-Ca2+ exchange system (Kd = 3,2 X 10(-6) and (4.3-5.3) X 10(-5) M), respectively. The Km values for MgATP are 2.15 mM. Oxytocin added to the homogenization medium containing rabbit and cattle myometrium cells, i.e. during the formation of closed sarcolemmal fragments, resulted in inhibition of Mg2+, ATP-dependent accumulation of 45Ca2+ by plasma membranes. However, an addition of oxytocin to the incubation medium did not affect the kinetics of active accumulation of Ca2+. It was assumed that the system of non-electrogenic Na+-Ca2+ exchange in the myometrium possessing a low affinity for Ca2+ provides for the maintenance of ionized Ca2+ concentration in the myocytes at 10(-5) M. Therefore, this system cannot induce relaxation of mechanical tension of the uterus. Further decrease of Ca2+ in the myoplasm from 10(-5) to 10(-7) M and, correspondingly, the relaxation of myometrium is provided for by the Mg2+, ATP-dependent efflux of Ca2+ from the myocytes having a high affinity for this cation. The decrease of the activity of ATP-dependent Ca2+-pump by oxytocin is the cause of Ca2+ elevation in the myoplasm and, consequently, of myometrium contraction.     

Effect of oxytocin on the calcium pump in myometrium sarcolemma

Oxytocin (10(-7) M) administered inside the myometrium sarcolemma vesicles closed outward by the cytoplasmic side is shown to inhibit Mg2+, ATP-dependent Ca2+ accumulation in these structures having no effect on the passive release of cation out of them. According to these results and to the data available in literature on the inhibitory action of the peptide hormone on Mg2+, Ca2+-ATPase of myometrium sarcolemma a conclusion is drawn that oxytocin inhibits the Ca pump activity in plasma membranes of the myometrium cells.     

Comparative study of the effect of aliphatic alcohols on energy-dependent accumulation of Ca2+ in intracellular membranes of smooth muscle cells

The effects of ethanol and other aliphatic alcohols on energy-dependent Ca2+ transport in endoplasmic reticulum and mitochondria were studied in digitonin-treated myometrium cells. The Ca2+ uptake in mitochondria increased (on 15-20%) with increasing methanol, ethanol and propanol concentrations in medium, whereas further rise of concentration inhibited this process. Treatments of myometrial cells with short-chain alcohols caused an inhibition of calcium uptake in endoplasmic reticulum. Butanol inhibited both calcium uptake in mitochondria and endoplasmic reticulum. Ca2+ accumulation in intracellular pools is inhibited by aliphatic alcohols in the following order of potency: butanol > propanol > ethanol > methanol. It is concluded that modifying effect of aliphatic alcohols on energy dependent calcium accumulation in intracellular membrane structures is defined as on origin of Ca(2+)-transporting system and (or) properties of these membrane structures so on properties of alcohols.     

Cationic specificity of a Ca2+-accumulating system in smooth muscle cell mitochondria

In the experiments conducted with application of an isotopic technique (45Ca2+) on the myometrium cells suspension treated by digitonin solution (0.1 mg/ml) some properties of Ca ions accumulation system in the mitochondria--cationic and substrate specificity as well as effects of Mg2+ and some other bivalent metals ions on the Ca2+ accumulation velocity have been estimated. Ca ions accumulation from the incubation medium containing 3 mM sodium succinate Na, 2 mM Pi (as potassium K(+)-phosphate buffer, pH 7.4 at 37 degrees C), 0.01 mM (40CaCl2 + 45CaCl2) and 100 nM thapsigargin--selective inhibiting agent of endoplasmatic reticulum calcium pump were demonstrated as detected just only in presence of Mg, while not Ni, Co or Cu ions. The increase of Mg2+ concentration from 1 x 10(-6) to 10(-3) M induced the ATP dependent transport activation in the myometrium mitochondria. Under [Mg2+] increase till 40 mM this cation essentially decreased Ca2+ accumulation (by 65% from the maximal value). The optimum for Ca2+ transport in the myometrium cells suspension is Mg2+ 10 mM concentration. Ka activation apparent constant along Mg2+ value (in presence 3 mM ATP and 3 mM sodium succinate) is 4.27 mM. The above listed bivalent metals decreased Mg2+, ATP-dependent accumulation of calcium, values of inhibition apparent constants for ions Co2+, Ni2+ and Cu2+ were--2.9 x 10(-4) M, 5.1 x 10(-5) M and 4.2 x 10(-6) M respectively. For Mg2+, ATP-dependent Ca2+ transport in the uterus myocytes mitocondria a high substrate specificity is a characteristic phenomenon in elation to ATP: GTP, CTP and UTP practically fail to provide for Ca accumulation process.     

Effect of ethanol on accumulation of Ca ions in intracellular structures of rat myometrium under conditions of first creating ethanol-dependent models

In the experiments which have been conducted on digitonin-treated myometrium cell suspensions of nonpregnant rats the direct influence of ethanol in concentration 0-10% on the Ca2+ accumulation in mitochondria and endoplasmic reticulum have been studied. Studies have been conducted on the different models, such as, control (model I), subacute (model II), acute (model III) and chronic ethanol consumption (model IV). It has been shown for all models that the dependence of Ca2+ accumulation by mitochondria on the concentration of ethanol in incubation medium is bell-shaped. Acute and chronic ethanol consumption resulted in statistically reliable decrease in the amount of accumulated cations. Nevertheless the I50 values were the same for the models I-III and were 8-9%, although in the case of model IV this one was only 4.0 +/- 0.6%. The increase of ethanol concentration in the incubation medium caused of Ca2+ accumulation decreasing in the endoplasmic reticulum for all studied models, the values of I50 also decreased for models II-IV (2.8 +/- 0.2; 2.5 +/- 0.2 and 2.3 +/- 0.3% respectively) relative to the control (3.8 +/- 0.2%). At the level of model I oxytocin-inhibited component of the endoplasmic reticulum Ca2+ uptake was more stable to the ethanol effects than oxytocin-independent one. Although the sensitivity of the first one to the ethanol effects at the level of models II-IV rose, that parameter for the oxytocin-independent component was not changed. The mechanisms of ethanol effects on Ca2+ accumulation in the myometrium intracellular structures have been discussed.     

Properties of the smooth muscle cell endoplasmic reticulum calcium pump

In experiments with 45Ca2+ conducted on digitonin-treated (0.1 mg/ml) myometrium cells suspension, the properties of ruthenium red-insensitive, oxalate- or phosphate-stimulated and thapsigargin- or cyclopiasonic acid-suppressed Mg2+, ATP-dependent calcium pump of myometrium sarcoplasmic reticulum was studied. The Ca2+ accumulation increased linearly in time up to 10 min, the average initial rate was 80-130 pmol Ca2+/10(6) cells per min. In the presence of 10 mM oxalate the values of the activation constant KMg for Mg2+ and K(m) for ATP were 0.6 and 1.0 mM, respectively. The relative efficiency of the different cations in insuring of the ATP-dependent Ca2+ accumulation was Mg2+ > Mn2+ = Co2+ > Ni2+; the Ca2+ accumulation was not observed in the presence of 3 mM Zn2+ or Cu2+. We observed the suppression of calcium pump activity by different inhibitors such as thapsigargin, cyclopiazonic acid, p-chloromercuribenzoic acid, eosin Y ad Na3 VO4: the values of K0.5 were 2.0 nM, 0.3 microM, 0.6 microM, 0.8 microM and 45 microM respectively. The conclusion was made that suspension of myometrial cells treated with digitonin represent a suitable experimental model for studying the properties of myometrium sarcoplasmic reticulum calcium pump.     

The effect of oxytocin and sigetin on Ca2+ transport in the fraction of plasma membranes of myometrial cells

Oxytocin and sigetin were studied for their effect on the active and passive transport of Ca2+ in the fraction of myometrium sarcolemma in women. Oxytocin (5.10(-7) M) introduced into the sarcolemma vesicles and sigetin (5.10(-3) M) added into the incubation medium inhibit Mg2+, ATP-dependent accumulation of Ca2+ in these structures. The both agents in the mentioned concentration do not affect the passive release of cation from vesicles. A conclusion is drawn that inhibition of the calcium pump of myometrium cell plasma membranes underlies the physiological action of oxytocin and sigetin as stimulators of the contractile activity of the myometrium.     

Mechanism of the ATP-dependent phosphatidylserine synthesis in liver subcellular fractions

It has been shown that the ATP-dependent incorporation of [14C]serine into phosphatidylserine in rat liver mitochondrial and microsomal fractions is prevented by EGTA. On the other hand, at low (microM) Ca2+ concentrations, serine incorporation is strongly stimulated by ATP and Mg2+. This stimulatory effect is reduced by calcium ionophore A23187. It is therefore suggested that the ATP-dependent process is that of serine base-exchange reaction, stimulated by endogenous Ca2+ accumulated inside the microsomal vesicles by Ca2+,Mg2+-ATPase. The mitochondrial activity can be accounted for by contamination by the endoplasmic reticulum.     

Effect of chronic craving for ethanol on accumulation of calcium ions in intracellular structures of rat myometrium

In the experiments on the impregnant estrogenized rats the effect of chronic ethanol intake on Ca(2+)-accumulative mitochondrial systems and endoplasmatic systems of myometrium was estimated. It was defined that in chronic alcohol consumption the transport activity of mitochondria Ca(2+)-accumulative systems didn't prevail over endoplasmatic reticulum Ca(2+)-accumulative activity. Therewith mitochondria and endoplasmatic Ca(2+)-transport system was essentially disturbed. In the tested conditions Mg2+, ATP-dependent sensitivity of calcium pump to oxytocin inhibiting action was shown to disappear.     

ATP-dependent Ca2+-uptake by the plasma membrane fraction of the myometrium

The specific activities of Mg2+, Ca2+-ATPase in the plasma membrane fraction of rabbit and cattle myometrium are 8.30 +/- 0.80 and 2.36 +/- 0.48 mkmoles of Pi per mg of protein, respectively. This fraction possesses a higher (in comparison with other subcellular fractions) capacity for ATP-dependent uptake of 45Ca2+ (9.37 +/- 1.66 and 6.86 +/- 0.96 nmoles of 45Ca2+ per mg of protein in 15 min for rabbit and cattle myometrium, respectively); the ratio of ATP-dependent uptake of Ca2+ to adsorbed Ca2+ is also high. Phosphate increases Ca2+ uptake in the presence of ATP and Mg2+. The ionophore A-23187 added to the incubation mixture without ATP and Mg2+ sharply increases Ca2+ binding. An addition of the ionophore at the 15th min of the ATP-dependent Ca2+ uptake causes a complete and rapid release of the accumulated Ca2+. The release of Ca2+ can be also caused by an addition of Na-DS or EGTA to the incubation mixture. This suggests that Ca2+ is accumulated through the plasma membrane inside the closed structures. It was assumed that myometrial sarcolemma plays an essential role in regulation of intracellular Ca2+ concentration in the uterus at rest and that the active Ca2+ efflux from the cells is controlled by the Mg2+, Ca2+-ATPase system.     

Effect of caffeine on azide-sensitive Mg2+, ATP-dependent increase of tetracycline fluorescent response in modeling of Ca ions accumulation in mitochondria

The influence of caffeine on the Mg2+, ATP-dependent Ca(2+)-uptake was investigated in the experiments, conducted on mitochondria isolated from myometrium of nonpregnant estrogenized rats. NaN3-sensitive CTC fluorescence increasing (lambda f = = 520 nm) was used as a test for active Ca2+ transport. Kinetics of NaN3-sensitive Mg2+, ATP-dependent component of CTC fluorescence change fits to the pattern of the first-order reaction either in the absence or in the presence of caffeine (20 mM). Caffeine (0-20 mM) inhibited both the stationary level (settled on the 2-3d min. of incubation) and the initial rate V0, and rate constant k of CTC fluorescence change. Magnitude of the apparent inhibition constant I0.5 for caffeine is 10.41 +/- 1.81 mM, inhibition process has weak positive cooperativity--the value of apparent Hill coefficient for caffeine is equal to 1.2 +/- 0.3. Data obtained suggest that caffeine inhibits both stationary Ca2+ capacity of mitochondria and the rate of NaN3-sensitive Mg2+, ATP-dependent Ca(2+)-accumulation in case of myometrium. These data could be useful for further investigation of molecular and membrane mechanisms of caffeine action on the intracellular Ca2+ homeostasis in uterus smooth muscle and its contractive activity.     

Effect of Mg ions and spermine on ATP-dependent Ca2+ transport in myometrial intracellular structures. I. Comparative study of Ca2+ accumulation in mitochondria and sarcoplasmic reticulum

In experiments, which were carried out with the use of a radioactive label (45Ca2+) on the suspension of rat uterus myocytes treated by digitonin solution (0.1 mg/ml), influence of Mg ions and spermine on Mg2+, ATP-dependent Ca2+ transport in mitochondria and sarcoplasmic reticulum was investigated. Ca2+ accumulation in mitochondria (1324 +/- 174 pmol Ca2+/10(6) cells for 1 min - the control) was tested as such which was not sensitive to thapsigargin (100 nM) and was blocked by ruthenium red (10 microM). Oxalate-stimulated Ca2+ accumulation in sarcoplasmic reticulum (136 +/- 17 pmol Ca2+/10(6) cells for 1 min - the control) was tested as such which was not sensitive to ruthenium red and was blocked by thapsigargin. It has been shown, that initial speed and level of energy-dependent Ca2+ accumulation in mitochondria considerably exceeded the values of these parameters for sarcoplasmic reticulum Ca2+-accumulation system. Ca2+ accumulation kinetic in mitochondria was characterized by a steady-state phase (for 5-10 min. of incubation) while accumulation kinetic of this cation in sarcoplasmic reticulum corresponded to zero order reaction. Increase of Mg2+ concentration up to 5 mM led to activation of Ca2+-accumulation systems in mitochondria and sarcoplasmic reticulum (values of activation constants K(Mg) for Mg2+ were 2.8 and 0.6 mM, accordingly). Concentration dependence of spermine action on Ca2+ accumulation in mitochondria was described by a dome-shaped curve with a maximum at 1 mM spermine. In case of sarcoplasmic reticulum Ca2+ pump only the inhibition phase was tested at spermine concentration above 1 mM. However values of inhibition constants for both transporting systems were practically identical--5.2 +/- 0.6 and 5.7 +/- 0.7 mM, accordingly. Hence, Mg ions carry out the important role in regulation of energy-dependent Ca2+ transporting systems both in uterus smooth muscle mitochondria and sarcoplasmic reticulum. Spermine acts first of all on mitochondrial calcium uniporter.     

Effect of the membrane potential on the Mg2+,ATP-dependent transport of Ca2+ across smooth muscle sarcolemma

The effect of the membrane potential (K(+)-valinomycin system) on the Mg2+, ATP-dependent transport of Ca2+ in inside-out vesicles of myometrium sarcolemma has been studied. The membrane potential was identified by using a cyanine potential-sensitive probe, diS-C3-(5). In the presence of valinomycin (5.10(-8) M) the inside-out directed K+ gradient (delta psi = -86 mV, with a negative charge inside) stimulated the initial rate of the energy-dependent accumulation of Ca2+ transfer whereas the oppositely directed K+ gradient (delta psi = +72 mV, with a positive charge inside) had no effect on this process. The K+ gradient was formed by isotonic substitution of K+ in intra- or extravesicular space for choline +. At the same time, in the absence of K+ gradient the Mg2+, ATP-dependent accumulation of Ca2+ in membrane vesicles did not depend on the chemical nature of the cations (K+ or choline+) used for isotonicity. The decrease of delta psi from 0 to -86 mV affects the initial rate of Ca2+ accumulation but not the maximal content of the accumulated cation. Preliminary dissipation of the membrane potential (delta psi = -86 mV) in Mg2(+)-free isotonic (with respect of K+ and choline+) media containing ATP and Ca2+ resulted in the inhibition of Mg2+, ATP-dependent Ca2+ transport induced by subsequent addition of Mg2+. These results indicate that the negative (intravesicular) electrical potential activates the Ca-pump of smooth muscle sarcolemma. This activation is based on the increase in the turnover number of the Ca2+ transporting system but not on its affinity for the transfer substrate. The use of the absolute reaction rates theory made it possible to establish that the Ca-pump effectuates the transport of a single positive charge in inside-out vesicles of smooth muscle plasma membranes, i.e., the energy-dependent transport of Ca2+ occurs either as a symport (with an anion (Cl-) or an antiport with a monovalent cation (K+) or a proton. It is assumed that the potential dependence of the Ca-pump in the smooth muscle plasma membrane plays a role in the realization of effects of mediators and physiologically active substances that are manifested as stimulation of the contractile response and depolarization of the sarcolemma. In is quite probable that the delta psi-dependent Ca-pump is also responsible for the maintenance of intracellular homeostasis of monovalent cations (K+, H+, Cl-) in smooth muscle tissues.     

Rat brain endoplasmic reticulum calcium pools are anatomically and functionally segregated.

Autoradiographic imaging can localize 45Ca2+ selectively accumulated via the Ca2+, Mg2(+)-ATPase into endoplasmic reticulum stores in rat brain slices. 45Ca2+ accumulation is markedly stimulated by oxalate and displays a heterogeneous distribution which resembles the mRNA distribution for a sarcoendoplasmic reticulum Ca2+, Mg2(+)-ATPase. Inositol 1,4,5-triphosphate [I(1,4,5)P3] inhibits 45Ca2+ accumulation selectively into regions corresponding to those enriched in I(1,4,5)P3 receptor-binding sites and in Ca2+, -Mg2(+)-ATPase mRNA. Thus rat brain endoplasmic reticulum calcium stores are anatomically and functionally differentiated.     

ATP-dependent transport of Ca2+ in myocardium sarcolemma vesicles and its activation by phorbol esters

Highly purified pig myocardium sarcolemma vesicles possess the Ca2+,Mg2+-ATPase activity (4.1 mumol Pi/mg protein/hour) and induce the ATP-dependent accumulation of 45Ca2+ (6.0 nmol/mg protein/min). This reaction is not stimulated by oxalate; Ca2+ are released from the vesicles by saponin and Na+ treatment, which suggests that Ca2+ transport against the concentration gradient is induced by myocardium sarcolemma vesicles and not by sarcoplasmic reticulum fragments. The phorbol ester possessing a biological activity of a growth-promoting factor and activating membrane-bound protein kinase C stimulates the Ca2+,Mg2+-ATPase activity and the ATP-dependent accumulation of Ca2+, whereas its counterpart devoid of biological activity does not influence Ca2+ transport. Polymixin B, a specific inhibitor of protein kinase C, prevents the activating effect of phorbol esters on Ca2+ accumulation inside the vesicles. It is suggested that the ATP-dependent transport of Ca2+ in myocardium sarcolemma is controlled by Ca2+-phospholipid-dependent phosphorylation catalyzed by protein kinase C.     

Calcium-ion-transporting activity in two microsomal subfractions from rat pancreatic acini. Modulation by carbamylcholine

Two microsomal subfractions from isolated rat pancreatic acini were produced by centrifugation through a discontinuous sucrose density gradient and characterized by biochemical markers. The denser fraction ( SF2 ) was a highly purified preparation of rough endoplasmic reticulum; the less-dense fraction ( SF1 ) was heterogeneous and contained Golgi, endoplasmic reticulum and plasma membranes. 45Ca2+ accumulation in the presence of ATP and its rapid release after treatment with the bivalent-cation ionophore A23187 were demonstrated in both fractions. The pH optimum for active 45Ca2+ uptake was approx. 6.8 for the rough endoplasmic reticulum ( SF2 ) and approx. 7.5 for SF1 . Initial rate measurements were used to determine the affinity of the rough-endoplasmic-reticulum uptake system for free Ca2+. An apparent Km of 0.16 +/- 0.06 microM and Vmax. of 21.5 +/- 5.6 nmol of Ca2+/min per mg of protein were obtained. 45Ca2+ uptake by SF1 was less sensitive to Ca2+, half-maximal uptake occurring at 1-2 microM-free Ca2+. When fractions were prepared from isolated acini stimulated with 3 microM-carbamylcholine, 45Ca2+ uptake was increased in the rough endoplasmic reticulum. The increased uptake was due to a higher Vmax. with no significant change in Km. No effect was observed on 45Ca2+ uptake by SF1 . In conclusion, two distinct non-mitochondrial, ATP-dependent calcium-uptake systems have been demonstrated in rat pancreatic acini. One of these is located in the rough endoplasmic reticulum, but the precise location of the other has not been determined. We have shown that the Ca2+-transporting activity in the rough endoplasmic reticulum may have an important role in maintaining the cytosolic free Ca2+ concentration in resting acinar cells and is involved in Ca2+ movements which occur during stimulation of enzyme secretion.     

Isolation and characteristics of the plasma membrane fraction from the swine myometrium

An accelerated method is developed for isolating a fraction of plasma membranes of pig myometrium using ultracentrifugation within the sucrose density gradient (15% and 30%). The membranes possessed the high activity of 5'-nucleotidase and Na+, K+-ATPase and the low activity of rhotenon-insensitive NADH-cytochrome c reductase. The vesicularized preparations of plasma membranes are able of ATP-dependent accumulation of Ca2+ (7.5 +/- 0.3 nmol. 45Ca2+ per 1 mg of protein for 15 min). Phosphate increases the calcium accumulation in the presence of ATP and Mg2+. Ionophore A 23187 promotes a complete and rapid release of the previously active-accumulated calcium. The release of 45Ca2+ accumulated by the membrane fraction may be reached by introduction of 1 mM EGTA or DS-Na into the incubation medium, that evidences for the cation accumulation inside closed structures. Using concanavalin-A-sepharose 4B it is shown that 60% of membrane vesicles are turned inside out. The low saponine concentrations (0.0005%) which inhibit Ca2+-accumulation by plasma membranes but not by the endoplasmic reticulum inhibit this process by 60-70% in preparations of the isolated membrane fraction. The method has certain advantages over the previously applied methods used for isolating of plasma membrane fragments from smooth muscles.     

Kinetic characteristics of Ca2+, Mg2+-ATPases in cells of the submandibular salivary gland of rats

Kinetic properties of Ca2+, Mg2+-ATPases membranes from acinar cells of rat submandibular salivary glands have been investigated. It was found that kinetics of ATP hydrolysis dependent on Ca2+, Mg2+-ATPases corresponds to the first-order reaction during first 2 min. It was found that the initial velocity of the reaction (V0), maximal amount of the reaction product (Pmax) and characteristic time of the reaction (T) comprised 1.8 +/- 0.4 and 1.6 +/- 0.2 mmole Pi/min per 1 mg protein, 7.5 +/- 1.3 and 1.4 +/- 0.2 mmole Pi/mg protein and 4.1 +/- 0.7 min and 1.1 +/- 0.1 for Ca2+-ATPases from plasma and endoplasmic reticulum membranes, correspondingly. High- and low-affinity sites of ATP and Ca2+-binding in Ca2+-ATPases from plasma and endoplasmic reticulum membranes were identified. Negative cooperation in ATP binding to Ca2+-ATPase from plasma membrane and a positive cooperation for Ca2+-ATPase from endoplasmic reticulum has been found. Ca2+ binding to low-affinity sites of both Ca2+-ATPases showed no cooperation, while Ca2+ binding to high-affinity sites showed the positive cooperation. Using the Hill's coordinates we have found the values of the Mg2+ Michaelis constant (K(Mg)) which yielded 3.89 x 10(-5) and 3.80 x 10(-5) mole/l for Ca2+-ATPases from plasma and endoplasmic reticulum membranes, correspondingly. It is supposed that obtained data are important for further studies of molecular and membrane mechanisms involved in the regulation of intracellular calcium signalling and secretion by salivary acinar cells.