Caclium uptake and associated adenosine triphosphatase activity in fragmented sarcoplasmic reticulum. Requirement for potassium ions.

The effects of monovalent cations on calcium uptake by fragmented sarcoplasmic reticulum have been clarified. Homogenization of muscle tissue in salt-containing solutions leads to contamination of this subcellular fraction with actomyosin and mitochondrial membranes. When, in addition, inorganic cations are contributed by the microsomal suspension and in association with nucleotide triphosphate substrates there is an apparent inhibition of the calcium transport system by potassium and other cations. However, when purified preparations were obtained after homogenization in sucrose medium followed by centrifugation on a sucrose density gradient in a zonal rotor, calcium uptake and the associated adenosine triphosphatase activity were considerably activated by potassium and other univalent cations. When plotted against the log of the free calcium concentration there was only a slight increase in calcium uptake and ATPase activity in the absence of potassium ions but sigmoid-shaped curves were obtained in 100 mM K+ with half-maximal stimulation occurring at 2 muM Ca2+ for both calcium uptake and ATPase activity. The augmentation in calcium uptake was not due to an ionic strength effect as Tris cation at pH 6.6 was shown to be inactive in this respect. Other monovalent cations were effective in the order K+ greater than Na+ greater than NH4+=Rb+=Cs+ greater than Li+ with half-maximal stimulation in 11 mM K+, 16 mM Na+, 25 mM NH4+, Rb+, and Cs+ and in 50 mM Li+. There was nos synergistic action between K+ AND Na+ ions and both calcium uptak and associated ATPase were insensitive to ouabain. Thallous ions stimulate many K+-requiring enzymes and at one-tenth the concentration were nearly as effective as K+ ions in promoting calcium uptake. The ratio of Ca2+ ions transported to P1 released remained unchanged at 2 after addition of K+ ions indicating an effect on the rate of calcium uptake rather than an increased efficiency of uptake. In support of this it was found that during the stimulation of calcium uptake by Na+ ions there was a reduction in the steady state concentration of phosphorylated intermediate formed from [gamma-32P]ATP. It is considered that there is a physiological requirement for potassium ions in the relaxation process.     

Interaction of sodium and potassium ions with Na+,K+-ATPase. II. General properties of ouabain-sensitive K+ binding

General properties of ouabain-sensitive K+ binding to purified Na+,K+-ATPase [EC 3.6.1.3] were studied by a centrifugation method with 42K+. 1) The affinity for K+ was constant at pH values higher than 6.4, and decreased at pH values lower than 6.4. 2) Mg2+ competitively inhibited the K+ binding. The dissociation constant (Kd) for Mg2+ of the enzyme was estimated to be about 1 mM, and the ratio of Kd for Mg2+ to Kd for K+ was 120 : 1. The order of inhibitory efficiency of divalent cations toward the K+ binding was Ba2+ congruent to Ca2+ greater than Zn2+ congruent to Mn2+ greater than Sr2+ greater than Co2+ greater than Ni2+ greater than Mg2+. 3) The order of displacement efficiency of monovalent cations toward the K+ binding in the presence or absence of Mg2+ was Tl+ greater than Rb+ greater than or equal to (K+) greater than NH4+ greater than or equal to Cs+ greater than Na+ greater than Li+. The inhibition patterns of Na+ and Li+ were different from those of other monovalent cations, which competitively inhibited the K+ binding. 4) The K+ binding was not influenced by different anions, such as Cl-, SO4(2-), NO3-, acetate, and glycylglycine, which were used for preparing imidazole buffers. 5) Gramicidin D and valinomycin did not affect the K+ binding, though the former (10 micrograms/ml) inhibited the Na+,K+-ATPase activity by about half. Among various inhibitors of the ATPase, 0.1 mM p-chloromercuribenzoate and 0.1 mM tri-n-butyltin chloride completely inhibited the K+ binding. Oligomycin (10 micrograms/ml) and 10 mM N-ethylmaleimide had no effect on the K+ binding. In the presence of Na+, however, oligomycin decreased the K+ binding by increasing the inhibitory effect of Na+, whether Mg2+ was present or not. 6) ATP, adenylylimido diphosphate and ADP each at 0.2 mM decreased the K+ binding to about one-fourth of the original level at 10 microM K+ without MgCl2 and at 60 microM K+ with 5 mM MgCl2. On the other hand, AMP, Pi, and p-nitrophenylphosphate each at 0.2 mM had little effect on the K+ binding.     

Characterization of a High-Affinity Mg2+-Independent Ca2+-ATPase from Rat Brain Synaptosomal Membranes

A high-affinity Mg2+-independent Ca2+-ATPase (Ca2+-ATPase) has been differentiated from the Mg2+-dependent, Ca2+-stimulated ATPase (Ca2+,Mg2+-ATPase) in rat brain synaptosomal membranes. Using ATP as a substrate, the K0.5 of Ca2+ for Ca2+-ATPase was found to be 1.33 microM with a Km for ATP of 19 microM and a Vmax of 33 nmol/mg/min. Using Ca-ATP as a substrate, the Km for Ca-ATP was found to be 0.22 microM. Unlike Ca2+,Mg2+-ATPase, Ca2+-ATPase was not inhibited by N-ethylmaleimide, trifluoperazine, lanthanum, zinc, or vanadate. La3+ and Zn2+, in contrast, stimulated the enzyme activity. Unlike Ca2+, Mg2+-ATPase activity, ATP-dependent Ca2+ uptake was negligible in the absence of added Mg2+, indicating that the Ca2+ transport into synaptosomal endoplasmic reticulum may not be a function of the Ca2+-ATPase described. Ca2+-ATPase activity was not stimulated by the monovalent cations Na+ or K+. Ca2+, Mg2+-ATPase demonstrated a substrate preference for ATP and ADP, but not GTP, whereas Ca2+-ATPase hydrolyzed ATP and GTP, and to a lesser extent ADP. The results presented here suggest the high-affinity Mg2+-independent Ca2+-ATPase may be a separate form from Ca2+,Mg2+-ATPase. The capacity of Mg2+-independent Ca2+-ATPase to hydrolyze GTP suggests this protein may be involved in GTP-dependent activities within the cell.     

(Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in kidney of sea bass (Dicentrarchus labrax L.)

1. Sea bass kidney microsomal preparations contain two Mg2+ dependent ATPase activities: the ouabain-sensitive (Na+ + K+)-ATPase and an ouabain-insensitive Na+-ATPase, requiring different assay conditions. The (Na+ + K+)-ATPase under the optimal conditions of pH 7.0, 100 mM Na+, 25 mM K+, 10 mM Mg2+, 5 mM ATP exhibits an average specific activity (S.A.) of 59 mumol Pi/mg protein per hr whereas the Na+-ATPase under the conditions of pH 6.0, 40 mM Na+, 1.5 mM MgATP, 1 mM ouabain has a maximal S.A. of 13.9 mumol Pi/mg protein per hr. 2. The (Na+ + K+)-ATPase is specifically inhibited by ouabain and vanadate; the Na+-ATPase specifically by ethacrynic acid and preferentially by frusemide; both activities are similarly inhibited by Ca2+. 3. The (Na+ + K+)-ATPase is specific for ATP and Na+, whereas the Na+-ATPase hydrolyzes other substrates in the efficiency order ATP greater than GTP greater than CTP greater than UTP and can be activated also by K+, NH4+ or Li+. 4. Minor differences between the two activities lie in the affinity for Na+, Mg2+, ATP and in the thermosensitivity. 5. The comparison between the two activities and with what has been reported in the literature only partly agree with our findings. It tentatively suggests that on the one hand two separate enzymes exist which are related to Na+ transport and, on the other, a distinct modulation in vivo in different tissues.     

Role of bivalent and monovalent cations in the functioning of myosin ATPase

The effects of bivalent (Mg2+, Ca2+, Sr2+) and monovalent (K+, Na+, NH4+) cations on the ATPase activity of subfragment 1 of myosin (SI) with a decreased Mg2+ content (EDTA-SI) were studied. Mg2+ activate the EDTA-SI ATPase, but only in the absence of other activating cations. K+, NH4+, a2+ and Sr2+ have a much stronger activating effect on EDTA-SI ATPase than on Mg-SI (SI enriched with Mg2+) ATPase. Monovalent cations inhibit Mg2+-ATPase and Ca2+-ATPase of EDTA-SI, while K+ and NH4+ activate Sr2+-ATPase of EDTA-SI. Based on experimental results and literary data, a hypothesis on the participation of the cations in the functioning of myosin ATPase was postulated. This hypothesis entails the existence of two closely interconnected cation-binding sites in the vicinity of the myosin active center (one for bivalent and one for monovalent cations); the ATPase activity of myosin is at any moment dependent on the nature of cations present in these two sites. An attempt to explain the role of the cations in the accomplishment of the ATPase reaction by myosin was made.     

Characterization of gill (Na+ + K+)-ATPase in the sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain both a Na+, K+ and Mg2+-dependent ATPase, which is completely inhibited by 10(-3)M ouabain and 10(-2)M Ca2+, and also a ouabain insensitive ATP-ase activity in the presence of both Mg2+ and Na+. Under the optimal conditions of pH 6.5, 100 mM Na+, 20 mM K+, 5 mM ATP and 5 mM Mg2+, (Na+ + K+)-ATPase activity at 30 degrees C is 15.6 mumole Pi hr/mg protein. Bass gill (Na+ + K+)-ATPase is similar to other (Na+ + K+)-ATPases with respect to the sensitivity to ionic strength, Ca2+ and ouabain and to both Na+/K+ and Mg2+/ATP optimal ratios, while pH optimum is lower than poikilotherm data. The enzyme requires Na+, whereas K+ can be replaced efficiently by NH+4 and poorly by Li+. Both Km and Vm values decrease in the series NH+4 greater than K+ greater than Li+. The break of Arrhenius plot at 17.7 degrees C is close to the adaptation temperature. Activation energies are scarcely different from each other and both lower than those generally reported. The Km for Na+ poorly decreases as the assay temperature lowers. The comparison with literature data aims at distinguishing between distinctive and common features of bass gill (Na+ + K+)-ATPase.     

Mg(Ca)-ATPase of arterial muscle cells]

We could show an ATPase in mitochondrial and microsomal fractions of sheep arteria carotis communis and arteria coronaria of cattle which can be stimulated by Ca2+ of Mg2+, respectively. The enzyme has a higher affinity for Ca2+ than for Mg2+. The maximum activity of the Mg(Ca)-ATPase was found at 2-4 mM Ca2+ or Mg2+, respectively. Higher concentrations of these ions inhibit the enzyme. Mn2+, Sr2+ and Co2+ can substitute Ca2+ in splitting of ATP by the ATPase of both fractions of ateria coronaria of cattle. The ions K+ and Na+, variation of temperature and pH and a variety of pharmacological active compounds has the same effect on the ATPase stimulated by Ca2+ or Mg2+. These findings prove that Ca2+ and Mg2+ act at the same site of the ATPase of the mitochondrial and microsomal fraction of vascular smooth muscle.     

Kinetics of Na+-ATPase: influence of Na+ and K+ on substrate binding and hydrolysis

An analysis of the influence of Na+ and K+ on the kinetics of Na+-ATPase in broken membrane preparations from bovine brain is presented with particular emphasis on the effect of the cations on the binding and splitting of the substrate MgATP and on the derivation of a detailed kinetic model for that interaction. It was found that the enzyme in the absence of Na+ and K+, but in the presence of 7 mM free Mg2+, at pH 7.4 (37 degrees C) exhibits an ouabain-sensitive ATPase activity. The simplest model quantitatively compatible with all the data involves two different, interconvertible (conformational) forms of the enzyme, E1 and E'1, with the following properties: The E1 form does not bind K+ but has three independent and equivalent high-affinity sites (Kd = 5.6 mM) for Na+. It binds and hydrolyzes substrate only when two or three sodium ions are bound to it. The E'1 form binds and hydrolyzes the substrate only in the absence of monovalent cations. It is competitively inhibited by K+ (Kd = 0.23 mM), and this inhibition is further enhanced by binding of Na+ to the K+-bound form at two equivalent, independent sites (Kd = 12 mM). It is suggested that the E'1 form is the Mg2+-induced conformational state of the enzyme observed by others, which differs from the usually encountered E1 and E2 forms. The model allows the calculation of ATP-binding and ADP-releasing rate constants for the E1-form for later comparison with corresponding rate constants for the (na+ + K+)-ATPase (following paper).     

Ca2+ gradient and drugs reveal different binding sites for Pi and Mg2+ in phosphorylation of the sarcoplasmic reticulum ATPase.

The first step towards ATP synthesis by the Ca2-ATPase of sarcoplasmic reticulum is the phosphorylation of the enzyme by Pi. Phosphoenzyme formation requires both Pi and Mg2+. At 35 degrees C, the presence of a Ca2+ gradient across the vesicle membrane increases the apparent affinity of the ATPase for Pi more than 10-fold, whereas it had no effect on the apparent affinity for Mg2+. In the absence of a Ca2+ gradient, the phosphorylation reaction is inhibited by both K+ and Na+ at all Mg2+ concentrations used. However, in the presence of 1 mM Mg2+ and of a transmembrane Ca2+ gradient, the reaction is still inhibited by Na+, but the inhibition promoted by K+ is greatly decreased. When the Mg2+ concentration is raised above 2 mM, the enzyme no longer discriminates between K+ and Na+, and the phosphorylation reaction is equally inhibited by the two cations. Trifluoperazine, ruthenium red and spermidine were found to inhibit the phosphorylation reaction by different mechanisms. In the absence of a Ca2+ gradient, trifluoperazine competes with the binding to the enzyme of both Pi and Mg2+, whereas spermidine and ruthenium red were found to compete only with Mg2+. The data presented suggest that the enzyme has different binding sites for Mg2+ and for Pi.     

ATPase stimulated by Na+ or K+ in gills of the freshwater mussel Anodonta.

1. Microsomal preparations from the gills of the freshwater mussel anodonta cygnea cellensis show Mg2+ -dependent Na+ - or K+ -stimulated ATPase activity, which is not inhibited by ouabain. 2. Na+ - or Ka+ -ATPase activity is decreased by Ca2+, acetylcholine, choline, and tetramethylammonium, but slightly increased by ethyl alcohol. 3. It is tentatively suggested that Na+ - or K+ -ATPase is involved in the mechanism of active monovalent cation uptake through the gills of freshwater mussels.     

Gill (Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in the gilthead bream (Sparus auratus L.)

1. Gilthead gill 10(-3) M ouabain-inhibited (Na+ + K+)-ATPase and 10(-2) M ouabain-insensitive Na+-ATPase require the optimal conditions of pH 7.0, 160 mM Na+, 20 mM K+, 5 mM MgATP and pH 4.8-5.2, 75 mM Na+, 2.5 mM Mg2+, 1.0 mM ATP, respectively. 2. The main distinctive features between the two activities are confirmed to be optimal pH, the ouabain-sensitivity and the monovalent cation requirement, Na+ plus another cationic species (K+, Rb+, Cs+, NH4+) in the (Na+ + K+)-ATPase and only one species (Na+, K+, Li+, Rb+, Cs+, NH4+ or choline+) in the Na+-ATPase. 3. The aspecific Na+-ATPase activation by monovalent cations, as well as by nucleotide triphosphates, opposed to the (Na+ + K+)-ATPase specificity for ATP and Na+, relates gilthead gill ATPases to lower organism ATPases and differentiates them from mammalian ones. 4. The discrimination between the two activities by the sensitivity to ethacrynic acid, vanadate, furosemide and Ca2+ only partially agrees with the literature. 5. Present findings are viewed on the basis of the ATPase's presumptive physiological role(s) and mutual relationship.     

The reaction of Mg2+ with the Ca2+-ATPase from human red cell membranes and its modification by Ca2+

Media prepared with CDTA and low concentrations of Ca2+, as judged by the lack of Na+-dependent phosphorylation and ATPase activity of (Na+ +K+)-ATPase preparations are free of contaminant Mg2+. In these media, the Ca2+-ATPase from human red cell membranes is phosphorylated by ATP, and a low Ca2+-ATPase activity is present. In the absence of Mg2+ the rate of phosphorylation in the presence of 1 microM Ca2+ is very low but it approaches the rate measured in Mg2+-containing media if the concentration of Ca2+ is increased to 5 mM. The KCa for phosphorylation is 2 microM in the presence and 60 microM in the absence of Mg2+. Results are consistent with the idea that for catalysis of phosphorylation the Ca2+-ATPase needs Ca2+ at the transport site and Mg2+ at an activating site and that Ca2+ replaces Mg2+ at this site. Under conditions in which it increases the rate of phosphorylation, Ca2+ is without effect on the Ca2+-ATPase activity in the absence of Mg2+ suggesting that to stimulate ATP hydrolysis Mg2+ accelerates a reaction other than phosphorylation. Activation of the E1P----E2P reaction by Mg2+ is prevented by Ca2+ after but not before the synthesis of E1P from E1 and ATP, suggesting that Mg2+ stabilizes E1 in a state from which Mg2+ cannot be removed by Ca2+ and that Ca2+ stabilizes E1P in a state insensitive to Mg2+. The response of the Ca2+-ATPase activity to Mg2+ concentration is biphasic, activation with a KMg = 88 microM is followed by inhibition with a Ki = 9.2 mM. Ca2+ at concentration up to 1 mM acts as a dead-end inhibitor of the activation by Mg2+, and Mg2+ at concentrations up to 0.5 mM acts as a dead-end inhibitor of the effects of Ca2+ at the transport site of the Ca2+-ATPase.     

A high-affinity plasma membrane Ca2+-ATPase in Dictyostelium discoideum: its relation to cAMP-induced Ca2+ fluxes

Chemotactic stimulation of Dictyostelium discoideum induces an uptake of Ca2+ by the cells followed by a release of Ca2+. In this study we investigated the mechanism of Ca2+ release and found that it was inhibited by La3+, Cd2+ and azide. Ca2+ release occurred in the absence of external Na+, indicating that an Na+/Ca2+ exchange was not involved. Plasma membranes contained high- and low-affinity ATPase activities. Apparent K0.5 values were 8 microM for the major Mg2+-ATPase and 1.1 microM for the high-affinity Ca2+-ATPase, respectively. The Mg2+-ATPase activity was inhibited by elevated concentrations of Ca2+, whereas both Ca2+-ATPases were active in the absence of added Mg2+. The activities of the Ca2+-ATPases were not modified by calmodulin. The high-affinity Ca2+-ATPase was competitively inhibited by La3+ and Cd2+; we suggest that this high-affinity enzyme mediates the release of Ca2+ from D. discoideum cells.     

Na+/Ca2+ antiport in cultured arterial smooth muscle cells. Inhibition by magnesium and other divalent cations

Cultured smooth muscle cells from rat aorta were loaded with Na+, and Na+/Ca2+ antiport was assayed by measuring the initial rates of 45Ca2+ influx and 22Na+ efflux, which were inhibitable by 2',4'-dimethylbenzamil. The replacement of extracellular Na+ with other monovalent ions (K+, Li+, choline, or N-methyl-D-glucamine) was essential for obtaining significant antiport activity. Mg2+ competitively inhibited 45Ca2+ influx via the antiporter (Ki = 93 +/- 7 microM). External Ca2+ or Sr2+ stimulated 22Na+ efflux as would be expected for antiport activity. Mg2+ did not stimulate 22Na+ efflux, which indicates that Mg2+ is probably not transported by the antiporter under the conditions of these experiments. Mg2+ inhibited Ca2+-stimulated 22Na+ efflux as expected from the 45Ca2+ influx data. The replacement of external N-methyl-D-glucamine with K+, but not other monovalent ions (choline, Li+), decreased the potency of Mg2+ as an inhibitor of Na+/Ca2+ antiport 6.7-fold. Other divalent cations (Co2+, Mn2+, Cd2+, Ba2+) also inhibited Na+/Ca2+ antiport activity, and high external potassium decreased the potency of each by 4.3-8.6-fold. The order of effectiveness of the divalent cations as inhibitors of Na+/Ca2+ antiport (Cd2+ greater than Mn2+ greater than Co2+ greater than Ba2+ greater than Mg2+) correlated with the closeness of the crystal ionic radius to that of Ca2+.     

Activation of calcium transport in skeletal muscle sarcoplasmic reticulum by monovalent cations.

The rates of calcium transport and Ca2+-dependent ATP hydrolysis by rabbit skeletal muscle sarcoplasmic reticulum were stimulated by monovalent cations. The rate of decomposition of phosphoprotein intermediate of the Ca2+-dependent ATPase of sarcoplasmic reticulum was also increased by these ions to an extent that is sufficient to account for the stimulation of calcium transport and Ca2+-dependent ATPase activity. The order of effectiveness of monovalent cations tested at saturating concentrations in increasing rate of phosphoprotein decomposition is: K+, Na+ greater than Rb+, NH4+ greater than Cs+ greater than Li+, choline+, Tris+.     

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)     

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.     

Ca2+-dependent ATPase activity of alveolar macrophage plasma membrane.

A plasma membrane fraction was isolated from lysates of Bacillus Calmette-Guérin-induced alveolar macrophages of rabbit. On the basis of morphological and biochemical criteria this fraction appeared to be minimally contaminated by other subcellular organelles. Concentrations of Ca2+, but not of Mg2+, from 6.10(-8) to 1.10(-5) M markedly stimulated the basal ATPase (EC 3.6.1.3) activity of the plasma membrane, with an apparent Km (Ca2+) of 1.10(-6) M. The specific activity of the Ca2+-ATPase assayed at pCa = 5.5 was enriched about 8-fold in the plasma membrane fraction over the macrophage lysate. In contrast, the specific activity of the K+, EDTA-activated ATPase, associated to macrophage myosin, increased only 1.3-fold. Oligomycin and -SH group reagents exerted no influence on the Ca2+-ATPase activity, which was on the contrary inhibited by detergents such as Triton X-100 and deoxycholate. The activity of the Ca2+-ATPase was maximal at pH 7, and was decreased by 50 mM Na+ and 5 mM K+. On the contrary, the activity of Mg2+-ATPase, also present in the plasma membrane fraction, had a peak at about pH 7.8, and was stimulated by Na+ plus K+. On account of its properties, it is suggested that the Ca2+-ATPase is a component of the plasma membrane of the alveolar macrophage, and that its function may be that of participating in the maintenance of low free Ca2+ concentrations in the macrophage cytosol.     

Na+/K(+)-ATPase: modes of inhibition by Mg2+.

Adding 15 mM free Mg2+ decreased Vmax of the Na+/K(+)-ATPase reaction. Mg2+ also decreased the K0.5 for K+ activation, as a mixed inhibitor, but the increased inhibition at higher K+ concentrations diminished as the Na+ concentration was raised. Inhibition was greater with Rb+ but less with Li+ when these cations substituted for K+ at pH 7.5, while at pH 8.5 inhibition was generally less and essentially the same with all three cations: implying an association between inhibition and ion occlusion. On the other hand, Mg2+ increased the K0.5 for Na(+)-activation of the Na+/K(+)-ATPase and Na(+)-ATPase reactions, as a mixed inhibitor. Changing incubation pH or temperature, or adding dimethylsulfoxide affected inhibition by Mg2+ and K0.5 for Na+ diversely. Presteady-state kinetic studies on enzyme phosphorylation, however, showed competition between Mg2+ and Na+. In the K(+)-phosphatase reaction catalyzed by this enzyme Mg2+ was a (near) competitor toward K+. Adding Na+ with K+ inhibited phosphatase activity, but under these conditions 15 mM Mg2+ stimulated rather than inhibited; still higher Mg2+ concentrations then inhibited with K+ plus Na+. Similar stimulation and inhibition occurred when Mn2+ was substituted for Mg2+, although the concentrations required were an order of magnitude less. In all these experiments no ionic substitutions were made to maintain ionic strength, since alternative cations, such as choline, produced various specific effects themselves. Kinetic analyses, in terms of product inhibition by Mg2+, require Mg2+ release at multiple steps. The data are accommodated by a scheme for the Na+/K(+)-ATPase with three alternative points for release: before MgATP binding, before K+ release and before Na+ binding. The latter alternatives necessitate two Mg2+ ions bound simultaneously to the enzyme, presumably to divalent cation-sites associated with the phosphate and the nucleotide domains of the active site.     

Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum.

ATP and the divalent cations Mg2+ and Ca2+ regulated K+ stimulation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K+-stimulated ATPase activity of the Ca2+ pump by two mechanisms. First, ATP chelated free Mg2+ and, at low ionized Mg2+ concentrations, K+ was shown to be a potent activator of ATP hydrolysis. In the absence of K+ ionized Mg2+ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K+ the concentration of ionized Mg2+ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K+-stimulation. With a saturating concentration of ionized Mg2+, stimulation by K+ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K+ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold. Activation of K+-stimulated ATPase activity by Ca2+ was maximal at an ionized Ca2+ concentration of approx. 1 microM. At very high concentrations of either Ca2+ or Mg2+, basal Ca2+-dependent ATPase activity persisted, but the enzymic response to K+ was completely inhibited. The results provide further evidence that the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme.