Anion effects on in vitro sarcoplasmic reticulum function. Co-transport of anions with calcium

In isolated sarcoplasmic reticulum vesicles, calcium-chelating but non-calcium-precipitating dicarboxylates, such as maleate and succinate, stimulated ATP-dependent Ca2+ accumulation and its ensuring spontaneous Ca2+ accumulation and its ensuring spontaneous Ca2+ release, and Ca2+-dependent ATPase activity (Chu, A., Tate, C. A., Bick, R. J., Van Winkle, W. B., and Entman, M. L. (1983) J. Biol. Chem. 258, 1656-1664). We further examined the effect of dicarboxylates on enzyme turnover. The anionic buffer maleate enhanced the rate of rapid acyl phosphoenzyme hydrolysis compared to that in the zwitterionic buffer piperazine-N,N'-bis(2-ethanesulfonic acid) but had no effect on the phosphoenzyme formation. The presence of a calcium-precipitating anion, oxalate, or a Ca2+ ionophore, A23187, eliminated the differences observed in the phosphoenzyme decay between the two buffers, but accelerated the rate of decay. Furthermore, the catalytic activity of the purified Ca2+-dependent ATPase was not affected by maleate, whether oxalate was present or not. [14C]Succinate was transported into the sarcoplasmic reticulum in a manner which was dependent on Ca2+ transport, and occurred over a similar time course as Ca2+ accumulation/release. The net succinate uptake was equivalent to the amount of succinate-stimulated Ca2+ accumulation. Rapid efflux of both [14C]succinate and 45Ca2+ was induced by A23187, whereas the efflux induced by ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid was slower and less compared to A23187. Succinate accumulation exhibited saturation kinetics with positive cooperativity (Km congruent to 20 mM; Hill coefficient = 1.70). When maleate and succinate were both present, they were equipotent, and had an additive stimulatory effect on peak 45Ca2+ accumulation at low concentrations. Maleate was a competitive inhibitor of succinate accumulation (Ki approximately equal to 17 mM; Hill coefficient = 1.75). KCl in the presence or absence of valinomycin did not influence succinate accumulation or release. The data suggest that succinate accumulation is Ca2+-dependent, but occurs at a saturable, divalent, anion-specific site. While this carrier or channel requires Ca2+ transport, it may be controlled by additional factors as well.     

Inhibition of dicarboxylic anion transport by fluorescein isothiocyanate in skeletal sarcoplasmic reticulum.

It has been demonstrated previously that dicarboxylic anions are cotransported during ATP-dependent Ca2+ transport by skeletal muscle sarcoplasmic reticulum (SR) membranes, and that anion cotransport stimulates Ca2+ transport. In the current study, we present evidence that dicarboxylic anion cotransport and Ca2+ transport are kinetically distinct in SR, but both functions are mediated by the CaATPase protein. Preincubation of SR with 40 microM fluorescein isothiocyanate (FITC) (pH 7.0) inhibited essentially all of the Ca2+ ATPase activity, as well as active oxalate-supported and oxalate-independent 45Ca2+ accumulation. The addition of 1 mM beta, gamma-methyleneadenosine 5'-triphosphate (AMP-PCP) to the preincubation media fully protected the dicarboxylic anion-independent Ca2+ ATPase activity and the oxalate-independent active 45Ca2+ accumulation from the inhibitory effects of FITC; however, the ATP-associated [14C]oxalate accumulation, the oxalate-dependent 45Ca2+ accumulation, and the oxalate- and maleate-dependent stimulation of Ca2+ ATPase activity were not protected by AMP-PCP. Thus, the dicarboxylic anion accumulation and the stimulation of Ca2+ uptake by dicarboxylic anions could be functionally separated from the ATP-dependent, anion-independent Ca2+ translocation. FITC bound exclusively to the 100-kDa (CaATPase) and 92-kDa (phosphorylase) proteins in the SR membranes and to purified CaATPase in sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 1 mM AMP-PCP inhibited 50-55% of the FITC fluorescence on the 100-kDa protein, but did not significantly alter fluorescence on the 92-kDa protein. Two-dimensional gel analysis demonstrated a single 100-kDa protein in longitudinal SR membranes. FITC appears to inhibit ATP-dependent Ca2+ transport, and dicarboxylic anion translocation through interaction at separate domains of the CaATPase protein.     

A model for the uptake and release of Ca2+ by sarcoplasmic reticulum.

On addition of ATP to vesicles derived from the sarcoplasmic reticulum (SR) of skeletal muscle, Ca2+ is accumulated from the external medium. Following uptake, spontaneous release of Ca2+ occurs in the presence or in the absence of ATP. These processes of Ca2+ uptake and release were simulated by using the models derived for ATPase activity [Gould, East, Froud, McWhirter, Stefanova & Lee (1986) Biochem. J. 237, 217-227; Stefanova, Napier, East & Lee (1987) Biochem. J. 245, 723-730] and for Ca2+ release from passively loaded vesicles [McWhirter, Gould, East & Lee (1987) Biochem. J. 245, 713-722]. The simulations are consistent with measurements of the effects of pH, K+, Ca2+ and Mg2+ on uptake and release of Ca2+. The increase in maximal Ca2+ accumulation observed in the presence of maleate is explained in terms of complexing of Ca2+ and maleate within the SR. The calculated concentration of ADP generated by hydrolysis of ATP has a large effect on the simulations. The effects of an ATP-regenerating system on the measured Ca2+ uptake is explained in terms of both removal of ADP and precipitation of Ca3(PO4)2 within the vesicles. It is concluded that both the process of Ca2+ uptake and the process of Ca2+ release seen with SR vesicles can be interpreted quantitatively in terms solely of the properties of the Ca2+ + Mg2+-activated ATPase.     

Effects of anions, pH and magnesium on calcium accumulation and release by sarcoplasmic reticulum vesicles

In the absence of oxalate, Ca2+ accumulation by isolated sarcoplasmic reticulum vesicles may show a transient behavior in which the vesicles accumulate during the first 2 min of incubation as much as twice the amount of Ca2+ which is retained after 5-7 min, when Ca2+ accumulation approaches a steady state. Before Ca2+ release begins, the Ca2+ accumulation can reach 200-250 nmol/mg protein. The spontaneous release of the "extra" Ca2+ initially accumulated appears to be triggered by the attainment of a sufficiently high concentration of free Ca2+ inside the vesicles. The amplitude of the transient phase of Ca2+ accumulation reaches a high value near pH 6.0 and is increased by free Mg2+. At optimal concentrations of H+ and Mg2+, the amount of Ca2+ accumulated during the transient is augmented by various anions, in the order maleate > or = propionate > or = succinate > chloride > sulfate > acetylglycine. The divalent anions have their maximum effects at 20-40 mM and the monovalent anions, at 40-200 mM. At 200 mM, all of the carboxylic anions tested significantly reduce the amount of Ca2+ retained in the steady state.     

Effect of guanosine triphosphate on the release and uptake of Ca2+ in saponin-permeabilized macrophages and the skeletal-muscle sarcoplasmic reticulum.

The effects of GTP, with or without polyethylene glycol (PEG), on the release and uptake of Ca2+ were examined by using saponin-treated macrophages and sarcoplasmic reticulum isolated from skeletal muscles. The application of GTP in concentrations in the range 0.1-10 microM induced a gradual, small but sustained release of Ca2+ from the saponin-treated macrophages. The addition of PEG to GTP markedly enhanced the GTP-mediated Ca2+ release. GTP at the same concentration ranges used for Ca2+ release decreased the amount of Ca2+ uptake, at a steady state, but stimulated the rate of Ca2+ accumulation in the presence of oxalate, the Ca2+-precipitating anion. The addition of PEG abolished the GTP-evoked stimulation of Ca2+ accumulation in the presence of oxalate. The stimulating effect on the rate of Ca2+ accumulation by GTP and its elimination by PEG were not due to changes in the permeability of oxalate by either GTP or PEG, or both. The Ca2+-releasing effect of GTP without PEG was enhanced by eliminating the uptake activity by decreasing the content of ATP. These results indicate that GTP has an inherent activity to release Ca2+ from non-mitochondrial intracellular stores of saponin-treated macrophages, and PEG enhances the GTP-mediated Ca2+ release, partly owing to its eliminating effect on GTP-stimulated Ca2+ uptake activity. These effects of GTP observed with saponin-permeabilized macrophages were not apparent in the isolated skeletal-muscle sarcoplasmic reticulum.     

Anion dependence of Ca2+ transport and (Ca2+ + K+)-stimulated Mg2+-dependent transport ATPase in rat pancreatic endoplasmic reticulum

Anion dependence of (Ca2+ + K+)-stimulated Mg2+-dependent transport ATPase and its phosphorylated intermediate have been characterized in both "intact" and "broken" vesicles from endoplasmic reticulum of rat pancreatic acinar cells using adenosine 5'-[gamma-32P] triphosphate ([gamma-32P]ATP). In intact vesicles (Ca2+ + K+)-Mg2+-ATPase activity was higher in the presence of Cl- or Br- as compared to NO3-, SCN-, cyclamate-, SO4(2-) or SO3(2-). Incorporation of 32P from [gamma-32P]ATP into the 100-kDa intermediate of this Ca2+ATPase was also higher in the presence of Cl-, Br-, NO3- or SCN- as compared to cyclamate-, SO4(2-) or SO3(2-). When the membrane permeability barrier to anions was abolished by breaking vesicle membrane with the detergent Triton X-100 (0.015%) (Ca2+ + K+)-Mg2+ATPase activity in the presence of weakly permeant anions, such as SO4(2-) and cyclamate-, increased to the level obtained with Cl-. However, 32P incorporation into 100-kDa protein was still higher in the presence of Cl- as compared to cyclamate-, indicating a direct effect of Cl- on the Ca2+ATPase molecule. The anion transport blocker 4,4-diisothiocyanostilbene-2,2-disulfonate (DIDS) inhibited (Ca2+ + K+)-Mg2+ATPase activity to about 10% of the Cl- stimulation level, irrespective of the sort of anions present in both intact and broken vesicles. This indicates a direct effect of DIDS on (Ca2+ + K+)-Mg2+ATPase. K+ ionophore valinomycin influenced (Ca2+ + K+)-Mg2+ATPase activity according to the actual K+ gradient: Ko+ greater than Ki+ caused inhibition, Ko+ less than Ki+ caused stimulation. From these results we conclude that Ca2+ transport into endoplasmic reticulum is coupled to ion movements which must occur to maintain electroneutrality.     

Effect of the calcium-channel blockers on calcium accumulation in sarcoplasmic reticulum of skeletal muscle

Vesicular fragments of sarcoplasmic reticulum isolated from rabbit skeletal muscle were actively loaded with Ca2+ in the presence of ATP and an ATP-regenerating system using Arsenazo III as metallochromic indicator to monitor Ca2+ movements across the membrane. Once the Ca2+ release is triggered by the presence of tetraphenylboron in the reaction medium, the addition of verapamil or diltiazem gives rise to a net Ca2+ entry inside the vesicles. Preincubation in the presence of verapamil does not abolish the tetraphenylboron-induced Ca2+ release, the verapamil-induced Ca2+ accumulation being still observed. There appears to be a high-affinity site for verapamil titrated in the micromolar concentration range, whereas diltiazem demonstrates more complex behavior when its concentration is raised. This study suggests the existence of a Ca2+ pathway (putative channels) which is blocked by the drugs tested allowing Ca2+ accumulation inside the vesicles owing to the Ca2+-dependent ATPase activity.     

Covalent and non-covalent inhibitors of the phosphate transporter of sarcoplasmic reticulum.

The sarcoplasmic reticulum (SR) of skeletal muscle contains a Pi transporter which transports Pi into the lumen of the SR, increasing the level of accumulation of Ca2+ by SR by forming insoluble salts with Ca2+. Phosphonocarboxylic acids inhibit the transport of Pi by the transporter, phosphonoformic acid itself being transported into the SR increasing the level of accumulation of Ca2+. Phenylphosphonic acid also inhibits Pi transport, distinguishing the Pi transporter of SR from the Na+/Pi transporter of brush-border membranes. Oxalate transport is also inhibited by the phosphono-carboxylic acids, consistent with the suggestion that oxalate and phosphate are carried on the same transporter. The effects of maleate are, however, not inhibited, suggesting a separate carrier for the dicarboxylic acids. Acetic anhydride and phenylglyoxal inhibit the transporter, Pi providing protection against the effects of acetic anhydride, suggesting the presence of a lysine residue at the Pi binding site. ATP provides protection against the effects of acetic anhydride and phenylglyoxal, suggesting the presence of an ATP binding site on the transporter.     

The interaction of lanthanides with isolated sarcoplasmic reticulum vesicles from rabbit skeletal muscle.

The interaction of lanthanides with isolated sarcoplasmic reticulum (SR) vesicles from rabbit skeletal muscle and the effects of lanthanides on 45Ca2+ uptake by the vesicles were studied. 153Gd3+ was taken up by the vesicles in the absence of ATP and oxalate in a time-dependent manner, reaching a maximum total accumulation of 380 nmol 153Gd3+/mg protein after 20 min with 200 microM 153Gd3+. This 153Gd3+ accumulation was not washed out by 1 mM EGTA. The addition of ATP induced the release of 87% of the bound 153Gd3+, leaving behind irreversibly-accumulated 153Gd3+. Pre-incubation of the vesicles with lanthanides in the absence of ATP and oxalate inhibited 45Ca2+ uptake without affecting Ca2+-ATPase activity. The percent inhibition of 45Ca2+ uptake increased with length of pre-incubation of the vesicles with lanthanides, reaching 33% after 20 min of pre-incubation. Increasing the 45Ca2+ concentration or adding ATP or oxalate to the preincubation medium abolished these inhibitory effects on 45Ca2+ uptake.     

Inactivation of a Ca2+-induced Ca2+ release channel from skeletal muscle sarcoplasmic reticulum during active Ca2+ transport

ATP-dependent Ca2+ uptake by subfractions of skeletal muscle sarcoplasmic reticulum (SR) was studied with the Ca2+ indicator dye, antipyrylazo III. Ca2+ uptake by heavy SR showed two phases, a slow uptake phase and a fast uptake phase. By contrast, Ca2+ uptake by light SR exhibited a monophasic time course. In both fractions a steady state of Ca2+ uptake was observed when the concentration of free Ca2+ outside the vesicles was reduced to less than 0.1 microM. In the steady state, the addition of 5 microM Ca2+ to the external medium triggered rapid Ca2+ release from heavy SR but not from light SR, indicating that the heavy fraction contains a Ca2+-induced Ca2+ release channel. During Ca2+ uptake, heavy SR showed a constant Ca2+-dependent ATPase activity (1 mumol/mg protein X min) which was about 150 times higher than the rate of Ca2+ uptake in the slow uptake phase. Ruthenium red, an inhibitor of Ca2+-induced Ca2+ release, enhanced the rate of Ca2+ uptake during the slow phase without affecting Ca2+-dependent ATPase activity. Adenine nucleotides, activators of Ca2+ release, reduced the Ca2+ uptake rate. These results suggest that the rate of Ca2+ accumulation by heavy SR is not proportional to ATPase activity during the slow uptake phase due to the activation of the channel for Ca2+-induced Ca2+ release. In addition, they suggest that the release channel is inactivated during the fast Ca2+ uptake phase.     

Enhanced Ca2+ uptake and ATPase activity of sarcoplasmic reticulum in the presence of diethyl ether

The presence of diethyl ether enhances the rates of both Ca2+ uptake and ATPase activity in sarcoplasmic reticulum vesicles (SR) isolated from rabbit skeletal muscle. Stopped-flow measurements of Ca2+ transport in SR show that, in the absence of oxalate and other calcium-complexing anions, the initial velocity of the ATP-dependent Ca2+ uptake increases from 60 to 107 nmol of Ca2+/s/mg of protein when 5% (v/v) diethyl ether is present. Similar concentrations of diethyl ether increase steady state levels of Ca2+ accumulation by over 80%. Parallel to the enhancement of the rate of Ca2+ transport, diethyl ether induces an increased rate of Ca2+-dependent ATPase activity. Among four other ether compounds tested, three enhanced the rate of Ca2+ uptake, but none as effectively as diethyl ether, and a fourth reduced the rate of Ca2+ transport by the SR. These results contrast with previous observations concerning the effect of diethyl ether on ATP-dependent Ca2+ transport by SR and are now consistent with a direct pharmacological action of ether as a muscle relaxant at the level of SR Ca2+ transport.     

Properties of epsilon-ATP hydrolysis by CF1-ATPase from pea chloroplasts

The ATPase activity of CF1 isolated from pea chloroplasts with epsilon-ATP, the fluorescent analog of ATP and ATP used as substrates, in the presence of Mg2+, Ca2+ and sodium sulfite (stimulator of the ATPase activity) was studied. The rate of epsilon-ATP hydrolysis in the presence of Mg2+ is nearly two times as low as that of ATP; an addition of sodium sulfite to the reaction mixture increases the reaction rate without changing the above ratio. The rate of Ca2+-dependent hydrolysis of epsilon-ATP is rather low as compared to that in the presence of Mg2+. epsilon-ADP is a competitive inhibitor of Mg2+-dependent ATPase reaction and inhibits this process in the presence of Ca2+, the inhibition being of a mixed type. Modification of CF1 by covalent binding of epsilon-ADP results in a 70-80% decrease of the Mg2+-dependent ATPase activity, the Ca2+-dependent ATPase activity is changed only insignificantly thereby. The differences in the activation of ATP and epsilon-ATP hydrolyses by Ca2+ and Mg2+ can be accounted for by the existence of two sites in the active center of CF1, which are specific for Mg2+ and Ca2+, respectively. It is concluded that the binding of epsilon-ADP occurs in the Mg2+-dependent ATPase site of the active center.     

Chemical modification of sarcoplasmic reticulum. Stimulation of Ca2+ release.

Pretreatment of sarcoplasmic membranes with acetic or maleic anhydrides, which interact principally with amino groups, resulted in an inhibition of Ca2+ accumulation and ATPase activity. The presence of ATP, ADP or adenosine 5'-[beta, gamma-imido]triphosphate in the modification medium selectively protected against the inactivation of ATPase activity by the anhydride but did not protect against the inhibition of Ca2+ accumulation. Acetic anhydride modification in the presence of ATP appeared to increase specifically the permeability of the sarcoplasmic reticulum membrane to Ca2+ but not to sucrose, Tris, Na+ or Pi. The chemical modification stimulated a rapid release of Ca2+ from sarcoplasmic reticulum vesicles passively or actively loaded with calcium, from liposomes reconstituted with the partially purified ATPase fraction but not from those reconstituted with the purified ATPase. The inactivation of Ca2+ accumulation by acetic anhydride (in the presence of ATP) was rapid and strongly pH-dependent with an estimated pK value above 8.3 for the reactive group(s). The negatively charged reagents pyridoxal 5-phosphate and trinitrobenzene-sulphonate, which also interact with amino groups, did not stimulate Ca2+ release. Since these reagents do not penetrate the sarcoplasmic reticulum membranes, it is proposed that Ca2+ release is promoted by modification of internally located, positively charged amino group(s).     

Effect of compound 48/80 and ruthenium red on the Ca2+-ATPase of sarcoplasmic reticulum

The effects of the condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde (compound 48/80) and ruthenium red on the partial reactions of the catalytic cycle of the sarcoplasmic reticulum Ca2+-ATPase of skeletal muscle were studied. The ATPase activity and both Ca2+ and Sr2+ uptake were inhibited by compound 48/80 when oxalate was used as a precipitating agent. The degree of inhibition decreased when oxalate was replaced by orthophosphate as the precipitating anion. Both the fast Ca2+ efflux and the synthesis of ATP observed during reversal of the Ca2+ pump were inhibited by compound 48/80. Inhibition of the reversal of the Ca2+ pump was caused by a competition between compound 48/80 and orthophosphate for the phosphorylation site of the enzyme. The fast Ca2+ release promoted by arsenate was impaired by compound 48/80. Ruthenium red competes with Ca2+ for the high affinity binding site of the Ca2+-ATPase, but did not interfere with the binding of Ca2+ to the low affinity binding site of the enzyme. In presence of Ca2+ concentrations higher than 5 microM, ruthenium red in concentrations up to 200 microM had no effect on both ATPase activity and Ca2+ uptake. However, the fast Ca2+ efflux promoted by arsenate and the fast Ca2+ efflux coupled with the synthesis of ATP observed during the reversal of the Ca2+ pump were inhibited by ruthenium red, half-maximal inhibition being attained in presence of 10-20 microM ruthenium red. In contrast to the effect of compound 48/80, ruthenium red did not inhibit the phosphorylation of the enzyme by orthophosphate. The ATP in equilibrium with Pi exchange catalyzed by the Ca2+-ATPase in the absence of transmembrane Ca2+ gradient was also inhibited by ruthenium red.     

The dependence on dicarboxylic acids and energy of citrate accumulation in depleted rat liver mitochondria

The accumulation of some organic anions in the space inaccessible to sucrose of rat liver mitochondria was measured. In untreated mitochondria anions were apparently concentrated from 1mm applied concentration by between five- and 22-fold, depending on their charge. After depletion of endogenous reserves either with uncoupling agent or with oligomycin uptakes were decreased. The accumulation of citrate was restored by combinations of a dicarboxylic acid (malate, succinate, maleate or meso-tartrate) and energy. The energy could either be provided by oxidation of a suitable dicarboxylic acid or from ascorbate in the presence of tetramethylphenylenediamine, or from ATP. The restoration of citrate uptake is not necessarily accompanied by a gain of K(+), but a cation- and energy-linked citrate uptake can be induced with valinomycin. When citrate is added to mitochondria in the presence of malate the latter is competitively displaced. The anion accumulation could arise from an internal energy-linked positive potential.     

Inhibitory effect platinum and palladium complexes as indicator of conformational changes in sarcoplasmic reticulum membranes]

Inhibition of Ca2+-dependent ATPase of sarcoplasmic reticulum membranes (SRM) by platinum and palladium complexes is considerable enhanced during the incubation of these compunds with SRM preparations in the presence of small (10(-5) M) concentrations of ATP or ADP. AMP and nucleotides with non-adenine bases do not have inhibitory effect. To increase the sensitivity of Ca2+-dependent ATPase to platinum and palladium complexes under the action of ATP (but not ADP), the presence of free Ca2+-ions in the medium is required. In the absence of ATP Ca2+-ions do not affect the inhibiting effect of the complexes. The increase in pH of the medium up to 8.5 and the increase of temperature up to 45degree C sharply decrease the ATP ability to enchance the sensitivity of Ca2+-dependent ATPase to platinum and palladium compunds. It is assumed that the ATP ability to enhance Ca2+-dependent ATPase inhibition by platinum and palladium complexes is due to ATP-dependent structural changes in SRM, which increase the availability of certain groups of the enzyme to those compounds.     

Inhibition of the sarcoplasmic reticulum Ca2+ transport ATPase by thapsigargin at subnanomolar concentrations.

ATP-dependent calcium uptake by isolated sarcoplasmic reticulum vesicles is inhibited by concentrations of free thapsigargin as low as 10(-10) M. This effect is due to primary inhibition of the Ca(2+)-dependent ATPase which is coupled to active transport. When binding of calcium to the activating sites of the enzyme is measured under equilibrium conditions in the absence of ATP, addition of thapsigargin produces strong inhibition. On the other hand, if [tau-32P]ATP is added to ATPase preincubated with Ca2+ under favorable conditions, significant levels of 32P-phosphorylated intermediate are still formed transiently, even in the presence of thapsigargin. The phosphoenzyme, however, decays rapidly as the calcium-enzyme complex is destabilized as a consequence of ATP utilization, and formation of the thapsigargin-enzyme complex is favored. Formation of the thapsigargin-enzyme complex is also favored by Ca2+ chelation with EGTA, with consequent inhibition of the enzyme reactivity to Pi (i.e. reverse of the ATPase hydrolytic reaction). Neither the Ca(2+)- and ATP-induced Ca2+ release from junctional sarcoplasmic reticulum nor the Ca(2+)- and calmodulin-dependent ATPase of plasma membranes (erythrocyte ghosts) were found to be altered by thapsigargin at such low concentrations.     

Ca2+-stimulated, Mg2+-dependent ATPase activity in neutrophil plasma membrane vesicles. Coupling to Ca2+ transport

Low concentrations of free Ca2+ stimulated the hydrolysis of ATP by plasma membrane vesicles purified from guinea pig neutrophils and incubated in 100 mM HEPES/triethanolamine, pH 7.25. In the absence of exogenous magnesium, apparent values obtained were 320 nM (EC50 for free Ca2+), 17.7 nmol of Pi/mg X min (Vmax), and 26 microM (Km for total ATP). Studies using trans- 1,2-diaminocyclohexane- N,N,N',N',-tetraacetic acid as a chelator showed this activity was dependent on 13 microM magnesium, endogenous to the medium plus membranes. Without added Mg2+, Ca2+ stimulated the hydrolysis of several other nucleotides: ATP congruent to GTP congruent to CTP congruent to ITP greater than UTP, but Ca2+-stimulated ATPase was not coupled to uptake of Ca2+, even in the presence of 5 mM oxalate. When 1 mM MgCl2 was added, the vesicles demonstrated oxalate and ATP-dependent calcium uptake at approximately 8 nmol of Ca2+/mg X min (based on total membrane protein). Ca2+ uptake increased to a maximum of approximately 17-20 nmol of Ca2+/mg X min when KCl replaced HEPES/triethanolamine in the buffer. In the presence of both KCl and MgCl2, Ca2+ stimulated the hydrolysis of ATP selectively over other nucleotides. Apparent values obtained for the Ca2+-stimulated ATPase were 440 nM (EC50 for free Ca2+), 17.5 nmol Pi/mg X min (Vmax) and 100 microM (Km for total ATP). Similar values were found for Ca2+ uptake which was coupled efficiently to Ca2+-stimulated ATPase with a molar ratio of 2.1 +/- 0.1. Exogenous calmodulin had no effect on the Vmax or EC50 for free Ca2+ of the Ca2+-stimulated ATPase, either in the presence or absence of added Mg2+, with or without an ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid pretreatment of the vesicles. The data demonstrate that calcium stimulates ATP hydrolysis by neutrophil plasma membranes that is coupled optimally to transport of Ca2+ in the presence of concentrations of K+ and Mg2+ that appear to mimic intracellular levels.     

Stimulation of Ca2+ efflux from sarcoplasmic reticulum by preincubation with ATP and inorganic phosphate.

Preincubation of sarcoplasmic reticulum with 1 mM-ATP completely inhibits Ca2+ accumulation and stimulates ATPase activity by over 2-fold. This effect of ATP is obtained only when the preincubation is carried out in the presence of Pi, but not with arsenate, chloride or sulphate. The inhibition by ATP of Ca2+ accumulation is pH-dependent, increasing as the pH is increased above 7.5. Inhibition of Ca2+ accumulation is observed on preincubation with ATP, but not with CTP, UTP, GTP, ADP, adenosine 5'-[beta gamma-methylene]triphosphate or adenosine 5'-[beta gamma-imido]triphosphate. The presence of Ca2+, but not Mg2+, during the preincubation, prevents the effect of ATP + Pi on Ca2+ accumulation. The ATP + Pi inhibition of Ca2+ accumulation is not due to modification of the ATPase catalytic cycle, but rather to stimulation of a rapid Ca2+ efflux from actively or passively loaded vesicles. This Ca2+ efflux is inhibited by dicyclohexylcarbodi-imide. Photoaffinity labelling of sarcoplasmic-reticulum membranes with 8-azido-[alpha-32P]ATP resulted in specific labelling of two proteins, of approx. 160 and 44 kDa. These proteins were labelled in the presence of Pi, but not other anions.     

ATPase and phosphatase activities from human red cell membranes: I. The effects of N-ethylmaleimide.

In human red cell membranes the sensitivity to N-ethylmaleimide of Ca2+-dependent ATPase and phosphatase activities is at least ten times larger than the sensitivity to N-ethylmaleimide of (Na+ + K+)-ATPase and K+-activated phosphatase activities. All activities are partially protected against N-ethylmaleimide by ATP but not by inorganic phosphate or by p-nitrophenylphosphate. (ii) Protection by ATP of (Na+ + K+)-ATPase is impeded by either Na+ or K+ whereas only K+ impedes protection by ATP of K+-activated phosphatase. On the other hand, Na+ or K+ slightly protects Ca2+-dependent activities against N-ethylmaleimide, this effect being independent of ATP. (iii) The sensitivity to N-ethylmaleimide of Ca2+-dependent ATPase and phosphatase activities is markedly enhanced by low concentrations of Ca2+. This effect is half-maximal at less than 1 micron Ca2+ and does not require ATP, which suggests that sites with high affinity for Ca2+ exist in the Ca2+-ATPase in the absence of ATP. (IV) Under all conditions tested the response to N-ethylmaleimide of the ATPase and phosphatase activities stimulated by K+ or Na+ in the presence of Ca2+ parallels that of the Ca2+-dependent activities, suggesting that the Ca2+-ATPase system possesses sites at which monovalent cations bind to increase its activity.