Effect of eosin Y on Ca2+-dependent activity of Ca2+-Mg2+-ATPase in smooth muscle cell membrane

With the aim to elucidate mechanism of eosin Y inhibitory effect on the Ca(2+)-transporting ATPase activity of myometrial cell plasma membrane effect of this inhibitor on the maximal initial rate of ATP hydrolysis reaction, catalyzed by Ca2+, Mg(2+)-ATPase, and on the enzyme affinity for Ca2+ was studied. It was established that eosin Y decreased the rate of Ca2+, Mg(2+)-ATPase catalitic turnover determined by Ca2+ and had no effect on enzyme affinity for this cation.     

Effect of eosin Y on Ca2+, Mg2+-ATPase of the smooth muscle sarcolemma

Eosin Y was studied with the aim to elucidate the mechanism of its inhibitory effect on the activity of Ca(2+)-transporting ATPase of myometrium cell plasma membrane. The inhibitor was studied for its effect on the maximal rate of the ATP-hydrolase reaction catalyzed by Ca2+, Mg(2+)-ATPase, on the enzyme affinity for the substrate and a possibility of enzyme activity protection under the inhibitor effect by the main reagents of ATP-hydrolase reaction. It was established that eosin Y decreased the turnover rate of this enzyme and his affinity for ATP. Preincubation of ATPase with ATP (or ATP plus MgCl2) had no effect on the extent of enzyme inhibition by eosin Y. This result proves that eosin Y and ATP do not compete for the site of binding on the enzyme.     

Effect of eosin Y on Ca2+ -independent, Mg2+ -dependent ATPase activity of smooth muscle cell plasma membranes

The effect of eosin Y (2',4',5',7'-tetrabromofluorescin) on basic kinetic parameters of the reaction of Mg2+ -dependent hydrolysis of ATP catalysed "basal" Mg2+ -ATPase myometrial cells plasma membrane has been studied. The eosin Y (10-100 microM) inhibited initial maximal velocity of the "basal" Mg2+ -ATPase of plasma membrane assayed for Mg2+ and ATP. At the same time the given inhibitor reduces the affinity of Mg2+ -ATPase for ATP. However, the difficult effect of the inhibitor action is observed for Mg ions: eosin Y in concentration of 10-50 microM increases the enzyme affinity for the ion-activator, while in concentration of 100 microM the affinity of Mg2+ -ATPase for Mg2+ is reduced. An analysis of eosin Y effect on catalytic efficiency of "basal" Mg2+ -ATPase of plasma membrane has shown, that at saturating concentrations of ATP (1 mM) the enzyme activity is less sensitive to the action of inhibitor. On this basis the conclusion is made that ATP in high concentrations can compete with eosin Y for active centre of Mg2+ -ATPase of smooth muscle cells plasma membrane.     

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%.     

Effect of organic solvents on catalytic and functional activity of transport Ca2+,Mg2+-ATPase from smooth muscle cell membrane

It was shown that organic solvents (dioxane, acetone, ethanol, dimethylsulfoxide) at concentrations of < 10% suppress the activity of transport Ca2+, Mg(2+)-ATPase solubilized from plasmatic membranes of smooth muscle cells and Mg(2+)-ATP-dependent accumulation of Ca2+ ions in inverted membrane vesicles. It was found that one of the reasons for the inhibition of enzymatic and transport activity of Ca2+, Mg(2+)-ATPase by the action of these solvents is an increase in the attractive force between oppositely charged active center of the enzyme and the product (products) of the ATP-hydrolase reaction, which is induced by a decrease in the dielectric permeability of incubation medium.     

Modulation of Na+-K+-ATPase by calcium ions

The modulatory effects of calcium ions on highly active Na+, K(+)-ATPase from calf brain and pig kidney tissues have been studied. The inhibitory action of Ca2+free on this enzyme depends on the level of ATP (but not AcP). The reduction of pH from 7.4 to 6.0 noticeably increases, but the elevation of pH to 8.0, in its turn, decreases the inhibition of ATP-hydrolyzing activity by calcium. With the increase of K+ concentration (in contrast to Na+) the sensibilization of Na+, K(+)-ATPase to Ca ions is observed. In the presence of potassium ions Mg2+free effectively modifies the inhibitory action of Ca2+free on this enzyme. Ca2+free (0.16-0.4 mM) decreases the sensitivity of Na+, K(+)-ATPase to action of the specific inhibitor ouabain in the presence of ATP. In the presence of AcP (phosphatase reaction) such a change of enzyme sensitivity to ouabain isn't observed. The influence of membranous effects of Ca2+ on the interaction of Na+, K(+)-ATPase with the essential ligands and cardiosteroids is discussed.     

Kinetic studies on the membrane-bound and the purified coupling factor-ATPase from Rhodopseudomonas sphaeroides

The activity of membrane-bound and purified ATPase (EC 3.6.1.3) was potentiated by several divalent cations. Highest rates of ATP hydrolysis were obtained when the activity was measured with the (cation-ATP)2- complex. Free ATP and free divalent cations in excess were found to be competitive inhibitors to the complex. The apparent Km (complex) values were lower than the Ki values for free ATP indicating that the (cation-ATP)2- complex is bound more tightly to the enzyme than the free ATP. Based on these results, a binding of the complex to the active site at two points is suggested, namely through the ATP and through the cation. Removal of the coupling factor from the membrane apparently caused conformational changes which resulted in a pronounced alteration of the kinetic parameters of ATPase activity. Whereas highest values in chromatophore-bound ATPase activity were observed in the presence of Mg2+, the purified enzyme became even more active in the presence of Ca2+. The Ki values for free ATP decreased upon solubilization of the enzyme. Free Mg2+ in excess was more inhibitory on the purified ATPase than Ca2+, while free Ca2+ in excess was more inhibitory on the membrane-bound enzyme if compared to Mg2+. Ki values for product inhibition by ADP and Pi were determined. Kinetic analyses of photophosphorylation activity revealed that the (cation-ADP)- complex is the functional substrate. The apparent Km values for the complex and for Pi were estimated. Excess of free cations and ADP inhibited competitively the phosphorylation. Ki(ADP), Ki(Ca2+), and Ki(Mg2+) were calculated by Dixon analyses.     

The effect of ionizing radiation on the Ca2+, Mg2(+)-ATPase activity of plasma membranes]

A study was made of a change in Ca2+, Mg(2+)-ATPase activity induced by the effect of ionizing radiation (5-10(4) Gy) on a thymocyte plasma membrane suspension. The Michaelis' constant and maximum rate of enzymic reactions were determined. With a dose of 10(3) Gy the structural changes in Ca2+, Mg(2+)-ATPase were shown to reduce the affinity of the substrate to an active enzyme center and to decrease the rate of the enzyme/substrate complex degradation.     

Inhibition of red cell Ca2+-ATPase by vanadate

1. The Mg2+- plus Ca2+-dependent ATPase (Ca2+-ATPase) in human red cell membranes is susceptible to inhibition by low concentrations of vanadate. 2. Several natural activators of Ca2+-ATPase (Mg2+, K+, Na+ and calmodulin) modify inhibition by increasing the apparent affinity of the enzyme for vanadate. 3. Among the ligands tests, K+, in combination with Mg2+, had the most pronounced effect on inhibition by vanadate. 4. Under conditions optimal for inhibition of Ca2+-ATPase, the K 1/2 for vanadate was 1.5 microM and inhibition was nearly complete at saturating vanadate concentrations. 5. There are similarities between the kinetics of inhibition of red cell Ca2+-ATPase and (Na+ + K+)-ATPase prepared from a variety of sources; however, (Na+ + K+)-ATPase is approx. 3 times more sensitive to inhibition by vanadate.     

ATP inactivates hydrolysis of the K+-sensitive phosphoenzyme of kidney Na+,K+-transport ATPase and activates that of muscle sarcoplasmic reticulum Ca2+-transport ATPase

In order to characterize low affinity ATP-binding sites of renal (Na+,K+) ATPase and sarcoplasmic reticulum (Ca2+)ATPase, the effects of ATP on the splitting of the K+-sensitive phosphoenzymes were compared. ATP inactivated the dephosphorylation in the case of (Na+,K+)ATPase at relatively high concentrations, while activating it in the case of (Ca2+)ATPase. When various nucleotides were tested in place of ATP, inactivators of (Na+,K+)ATPase were found to be activators in (Ca2+)ATPase, with a few exceptions. In the absence of Mg2+, the half-maximum concentration of ATP for the inhibition or for the activation was about 0.35 mM or 0.25 mM, respectively. These values are comparable to the previously reported Km or the dissociation constant of the low affinity ATP site estimated from the steady-state kinetics of the stimulation of ATP hydrolysis or from binding measurements. By increasing the concentration of Mg2+, but not Na+, the effect of ATP on the phosphoenzyme of (Na+,K+)ATPase was reduced. On the other hand, Mg2+ did not modify the effect of ATP on the phosphoenzyme of (Ca2+)ATPase. During (Na+,K+)ATPase turnover, the low affinity ATP site appeared to be exposed in the phosphorylated form of the enzyme, but the magnesium-complexed ATP interacted poorly with the reactive K+-sensitive phosphoenzyme, which has a tightly bound magnesium, probably because of interaction between the divalent cations. In the presence of physiological levels of Mg2+ and K+, ATP appeared to bind to the (Na+,K+)ATPase only after the dephosphorylation, while it binds to the (Ca2+)-ATPase before the dephosphorylation to activate the turnover.     

Reversible inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid of the plasma membrane (Ca(2+)+Mg2+)ATPase from kidney proximal tubules

Calcium accumulation by purified vesicles derived from basolateral membranes of kidney proximal tubules was reversibly inhibited by micromolar concentrations of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of anion transport. The inhibitory effect of this compound on Ca2+ uptake cannot be attributed solely to the inhibition of anion transport: (Ca(2+)+Mg2+)ATPase and ATP-dependent Ca2+ transport, respectively. The rate constant of EGTA-induced Ca2+ efflux from preloaded vesicles was not affected by DIDS, indicating that this compound does not increase the permeability of the membrane vesicles to Ca2+. In the presence of DIDS, the effects of the physiological ligands Ca2+, Mg2+, and ATP on (Ca(2+)+Mg2+)ATPase activity were modified. The Ca2+ concentration that inhibited (Ca(2+)+Mg2+)ATPase activity in the low-affinity range decreased from 91 to 40 microM, but DIDS had no effect on the Km for Ca2+ in the high-affinity, stimulatory range. Free Mg2+ activated (Ca(2+)+Mg2+)ATPase activity at a low Ca2+ concentration, and DIDS impaired this stimulation in a noncompetitive fashion. The inhibition by DIDS was eliminated when the free ATP concentration of the medium was raised from 0.3 to 8 mM, possibly due to an increase in the turnover of the enzyme caused by free ATP accelerating the E2----E1 transition, and leading to a decrease in the proportion of E2 forms under steady-state conditions. Alkaline pH totally abolished the inhibition of the (Ca(2+)+Mg2+)ATPase activity by DIDS, with a half-maximal effect at pH 8.3.(ABSTRACT TRUNCATED AT 250 WORDS)     

The inhibitor of liver plasma membrane (Ca2+-Mg2+)-ATPase. Purification and identification as a mediator of glucagon action

Rat liver plasma membranes contain (Ca2+-Mg2+)-ATPase sensitive to inhibition by both glucagon and Mg2+. We have previously shown that Mg2+ inhibition is mediated by a 30,000-dalton inhibitor, originally identified as a membrane-bound protein. In fact, this inhibitor is also present in the 100,000 X g supernatant of the total liver homogenate. Its purification was achieved from this fraction by a combination of ammonium sulfate washing, gel filtration, and cationic exchange chromatography. N-Ethylmaleimide (NEM) treatment caused the inactivation of the purified inhibitor, which suggested that this protein possesses at least one NEM-sensitive sulfhydryl group essential for its activity. Treatment of the liver plasma membranes with NEM resulted in a 2- and 5-fold decrease in the affinity of the (Ca2+-Mg2+)-ATPase for glucagon and Mg2+, respectively, while the basal enzyme activity remained unchanged. This effect of NEM was concentration-, pH-, and time-dependent, optimal conditions being obtained by a 60-min treatment of plasma membranes with 50 mM NEM, at pH 7 and at 4 degrees C. The presence of 0.5 mM Mg2+ during NEM treatment of the plasma membranes prevented NEM inactivation. Reconstitution experiments showed that addition of the purified inhibitor to NEM-treated plasma membranes restored the inhibitions of the (Ca2+-Mg2+)-ATPase by both magnesium and glucagon. It is proposed that the (Ca2+-Mg2+)-ATPase inhibitor not only confers its sensitivity of the liver (Ca2+-Mg2+)-ATPase to Mg2+, but also mediates the inhibition of this system by glucagon.     

Characterisation of a high affinity Ca2+-stimulated, Mg2+-dependent ATPase in the rat parotid plasma membrane

Two Ca2+-stimulated ATPase activities have been identified in the plasma membrane of rat parotid: (a) a (Ca2+ + Mg2+)-ATPase with high affinity for free Ca2+ (apparent Km = 208 nM, Vmax = 188 nmol/min per mg) and requiring micromolar concentration of Mg2+ and (b) a (Ca2+ or Mg2+)-ATPase with relatively low affinity for free Ca2+ (K0.5 = 23 microM) or free Mg2+ (K0.5 = 26 microM). The low-affinity (Ca2+ or Mg2+)-ATPase can be maximally stimulated by Ca2+ alone or Mg2+ alone. The high-affinity (Ca2+ + Mg2+)-ATPase exhibits sigmoidal kinetics with respect to ATP concentration with K0.5 = 0.4 mM and a Hill coefficient of 1.91. It displays low substrate specificity with respect to nucleotide triphosphates. Although trifluoperazine inhibits the activity of the high affinity (Ca2+ + Mg2+)-ATPase only slightly, it inhibits the activity of the low-affinity (Ca2+ or Mg2+)-ATPase quite potently with 22 microM trifluoperazine inhibiting the enzymic activity by 50%. Vanadate, inositol 1,4,5-trisphosphate, phosphatidylinositol 4,5-bisphosphate, Na+,K+ and ouabain had no effect on the activities of both ATPases. Calmodulin added to the plasma membranes does not stimulate the activities of both ATPases. The properties of the high-affinity (Ca2+ + Mg2+)-ATPase are distinctly different from those of the previously reported Ca2+-pump activity of the rat parotid plasma membrane.     

Characterization of the Ca2+- and Mg2+-Dependent ATPases in Electrophorus Electroplax Microsomes

Electrophorus electroplax microsomes were examined for Ca2+- and Mg2+-dependent ATPase activity. In addition to the previously reported low-affinity ATPase, a high-affinity (Ca2+,Mg2+)-ATPase was found. At low ATP and Mg2+ concentrations (200 microM or less), the high-affinity (Ca2+,Mg2+)-ATPase exhibits an activity of 18 nmol Pi mg-1 min-1 with 0.58 microM Ca2+. At higher ATP concentrations (3 mM), the low-affinity Ca2+-ATPase predominates, with an activity of 28 nmol Pi mg-1 min-1 with 1 mM Ca2+. In addition, Mg2+ can also activate the low-affinity ATPase (18 nmol Pi mg-1 min-1). The high-affinity ATPase hydrolyzes ATP at a greater rate than it does GTP, ITP, or UTP and is insensitive to ouabain, oligomycin, or dicyclohexylcarbodiimide inhibition. The high-affinity enzyme is inhibited by vanadate, trifluoperazine, and N-ethylmaleimide. Added calmodulin does not significantly stimulate enzyme activity; rinsing the microsomes with EGTA does not confer calmodulin sensitivity. Thus the high-affinity ATPase from electroplax microsomes is similar to the (Ca2+,Mg2+)-ATPase reported to be associated with Ca2+ transport, based on its affinity for calcium and its response to inhibitors. The low-affinity enzyme hydrolyzes all tested nucleoside triphosphates, as well as diphosphates, but not AMP. Vanadate and N-ethylmaleimide do not inhibit the low-affinity enzymes. The low-affinity enzyme reflects a nonspecific nucleoside triphosphatase, probably an ectoenzyme.     

Characterization of the plasma membrane Mg2+-ATPase from the yeast, Saccharomyces cerevisiae.

The plasma membrane of Saccharomyces cerevisiae has a Mg2+-dependent ATPase which is distinct from the mitochondrial Mg2+-ATPase and at the pH optimum of 5.5 has a Km for ATP of 1.7 mM and a Vmax of 0.42 mumol of ATP hydrolyzed/mg/min. At least three protein components of the crude membrane (Mr = 210,000, 160,000 and 115,000) are labeled with [gamma"32P]ATP at pH 5.5. These phosphoproteins form rapidly in the presence of Mg2+, rapidly turn over the bound phosphate when unlabeled ATP is added, and dephosphorylate after incubation in the presence of hydroxylamine. Vanadate, an inhibitor of the Mg2+-ATPase activity, blocks the phosphorylation of the 210,000- and 115,000-dalton proteins. At pH 7.0, only the 210,000- and 160,000-dalton proteins are phosphorylated. While these three phosphorylated intermediates have not been unambiguously identified as components of the Mg2+-ATPase, the finding of such phosphorylated components in association with that activity implies that this enzyme differs in mechanism from the mitochondrial proton pump and that it is similar in mechanism to the metal ion pumps ((Na+-K+)-ATPase and Ca2+-ATPase) of the mammalian plasma membrane.     

The substitution of calcium for magnesium in H+,K+-ATPase catalytic cycle. Evidence for two actions of divalent cations

In order to determine the role of divalent cations in the reaction mechanism of the H+,K+-ATPase, we have substituted calcium for magnesium, which is required by the H+,K+-ATPase for phosphorylation from ATP and from PO4. Calcium was chosen over other divalent cations assayed (barium and manganese) because in the absence of magnesium, calcium activated ATP hydrolysis, generated sufficiently high levels of phosphoenzyme (573 +/- 51 pmol.mg-1) from [gamma-32P]ATP to study dephosphorylation, and inhibited K+-stimulated ATP hydrolysis. The Ca2+-ATPase activity of the H+,K+-ATPase was 40% of the basal Mg2+-ATPase activity. However, the Ca2+,K+-ATPase activity (minus the Ca2+ basal activity) was only 0.7% of the Mg2+,K+-ATPase, indicating that calcium could partially substitute for Mg2+ in activating ATP hydrolysis but not in K+ stimulation of ATP hydrolysis. Approximately 0.1 mM calcium inhibited 50% of the Mg2+-ATPase or Mg2+,K+-ATPase activities. Inhibition of Mg2+,K+-ATPase activity was not competitive with respect to K+. Inhibition by calcium of Mg2+,K+ activity p-nitrophenyl phosphatase activity was competitive with respect to Mg2+ with an apparent Ki of 0.27 mM. Proton transport measured by acridine orange uptake was not detected in the presence of Ca2+ and K+. In the presence of Mg2+ and K+, Ca2+ inhibited proton transport with an apparent affinity similar to the inhibition of the Mg2+, K+-ATPase activity. The site of calcium inhibition was on the exterior of the vesicle. These results suggest that calcium activates basal turnover and inhibits K+ stimulation of the H+,K+-ATPase by binding at a cytosolic divalent cation site. The pseudo-first order rate constant for phosphoenzyme formation from 5 microM [gamma-32P]ATP was at least 22 times slower in the presence of calcium (0.015 s-1) than magnesium (greater than 0.310 s-1). The Ca.EP (phosphoenzyme formed in the presence of Ca2+) formed dephosphorylated four to five times more slowly that the Mg.EP (phosphoenzyme formed in the presence of Mg2+) in the presence of 8 mm trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) or 250 microM ATP. Approximately 10% of the Ca.EP formed was sensitive to a 100 mM KCl chase compared with greater than 85% of the Mg.EP. By comparing the transient kinetics of the phosphoenzyme formed in the presence of magnesium (Mg.EP) and calcium (Ca.EP), we found two actions of divalent cations on dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Influence of pH and sodium on the inhibition of guinea-pig heart (Na+ + K+)-ATPase by calcium.

The inhibition of guinea-pig heart (Na+ + K+)-ATPase (ATP phosphohydrolase EC 3.6.1.3) by calcium has been studied at pH 7.4, 6.8 and 6.4. 1. A decrease in pH reduced the threshold inhibitory concentration of calcium and the calcium concentration producing an inhibition of 50% of the enzyme activity. 2. Calcium reduced the apparent affinity of the enzyme of Na+, this effect occurred only at pH 7.4. 3. Calcium increased the apparent affinity of the enzyme for K+, this effect was enhanced at acidic pH. 4. Activation of the enzyme by Na+ for a constant Na+ : K+ ratio has been studied at pH 7.4 and at pH 6.8 in the absence and in the presence of 3.10(-4) M Ca 2+; the results of this experiment indicate that Ca2+ effect at pH 7.4 was not influenced by Na+ -- K+ competition and was probably due to a Na+ -- Ca2+ interaction. 5. At pH 7.4, the calcium inhibitory threshold concentration and the concentration producing 50% inhibition were reduced when Na+ was low; at pH 6.8, the calcium inhibition was not markedly modified by the change of Na+ concentration. 6. The Ca2+ -activated ATPase of myosin B which is related to the contractile behaviour of muscle and the Ca2+ -ATPase of the sarcoplasmic reticulum which is related to the ability of this structure to accumulate calcium were activated in a range of calcium concentration producing an inhibition of (Na2+ + K+) -ATPase. The present results indicate that the increase by acidity of the (Na2+ + K+) -ATPase sensitivity to calcium might be due to a suppression of a Na+ -Ca2+ interaction. On the basis of these observations, it is proposed that calcium might inhibit the Na+ -pump during the repolarization phase of the action potential and that, by this effect, it might control cell excitability.     

Identification of Ca(2+)-activated Mg(2+)-dependent ATP-hydrolase activity of membrane microsomal fraction of loach embryo (Misgurnus fossilis L.)

The functional confirmation of availability of Ca2+ transport initially-active systems in the embryo cells of loach Misgurnus fossilis L. has been obtained. Using thapsigargin, the specific inhibitor of endoplasmic reticulum of Ca2+, Mg(2+)-ATPase, this enzyme activity was divided into thapsigargin-sensitive (actually endoplasmic reticulum Ca2+, Mg(2+)-ATPase) and thapsigargin-insensitive (plasma membrane Ca2+, Mg(2+)-ATPase) constituents. The Ca(2+)-independent Mg(2+)-dependent ATPase activity makes above 39.7% of the common Ca2+, Mg(2+)-ATPase activity of embryo loach. The periodic changes of Ca2+, Mg(2+)-ATPase activity (except for the changes of plasma membrane Ca2+, Mg(2+)-ATPase activity) were found out, which coincide with periodic [Ca2+]i oscillations during the synchronous divisions of loach blastomers embryos.     

ATP utilizing reactions of human erythrocyte membranes and the influence of modulator proteins

The ATP production of human erythrocytes in the steady state (approximately 2 mmoles . 1 cells-1 . h-1, 37 degrees C, pHi 7.2) is maintained by glycolysis and the ATP consumption is essentially limited to the cell membrane. About 25% of the ATP consumption is used for ion transport ATPases. The bulk of the ATP consuming processes in intact erythrocytes remains poorly understood. "Isotonic" erythrocyte membranes prepared under approximate intracellular conditions after freeze-thaw hemolysis have high (Ca2+, Mg2+)-ATPase activities (80% of the total membrane ATPase activity). There is a great discrepancy between the high capacity of the (Ca2+, Mg2+)-ATPase in isotonic membranes and the actual activity in the intact cell. The (Ca2+, Mg2+)-ATPase of isotonic membranes has a "high" Ca2+-affinity (Ka less than 0.5 microM) and a "low" Mg-ATP affinity (Km approximately 760 microM). This state of (Ca2+, Mg2+)-ATPase is caused by the association of calmodulin and 30000 Dalton polypeptides (ATP affinity modulator protein). Hypotonic washings of isotonic membranes result in a loss of the 30 kD polypeptides. EGTA (0.5 mM) extracts derived from isotonic membranes contain the 30 kD modulator protein and restore the properties of the (Ca2+, Mg2+)-ATPase of hypotonic membrane preparations to the isotonic characteristics. The Mg-ATP affinity modulator protein is assumed to form a complex with calmodulin and (Ca2+, Mg2+)-ATPase.     

High affinity Ca2+-stimulated Mg2+-dependent ATPase in rat brain synaptosomes, synaptic membranes, and microsomes

High affinity Ca2+-stimulated Mg2+-dependent ATPase activity of nerve ending particles (synaptosomes) from rat brain tissue appears to be associated primarily with isolated synaptic plasma membranes. The synaptic membrane (Ca2+ + Mg2+)-ATPase activity was found to exhibit strict dependence on Mg2+ for the presence of the activity, a high affinity for Ca2+ (K0.5 = 0.23 microM), and relatively high affinities for both Mg2+ and ATP (K0.5 = 6.0 microM for Mg2+ and KM = 18.9 microM for ATP). These kinetic constants were determined in incubation media that were buffered with the divalent cation chelator trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid. The enzyme activity was not inhibited by ouabain or oligomycin but was sensitive to low concentrations of vanadate. The microsomal membrane subfraction was the other brain subcellular fraction with a high affinity (Ca2+ + Mg2+)-ATPase activity which approximated that of the synaptic plasma membranes. The two membrane-related high affinity (Ca2+ + Mg2+)-ATPase activities could be distinguished on the basis of their differential sensitivity to vanadate at concentrations below 10 microM. Only the synaptic plasma membrane (Ca2+ + Mg2+)-ATPase was inhibited by 0.25-10 microM vanadate. The studies described here indicate the possible involvement of both the microsomal and the neuronal plasma membrane (Ca2+ + Mg2+)-ATPase in high affinity Ca2+ transport across membranes of brain neurons. In addition, they suggest a means by which the relative contributions of each transport system might be evaluated based on their differential sensitivity to inhibition by vanadate.