Modification of ATP regulatory function in sarcoplasmic reticulum Ca2(+)-ATPase by hydrophobic molecules

The effects of the three hydrophobic molecules triphenylphosphine, trifluoperazine and 3-nitrophenol on Ca2+ uptake and ATPase activity in sarcoplasmic reticulum vesicles was investigated. When ATP was the substrate, triphenylphosphine (3 microM) increased the amount of Ca2+ accumulated by the vesicles. At high concentrations triphenylphosphine inhibited Ca2+ uptake. This effect varied depending on the ATP concentration and the type of nucleotide used. With ITP there was only inhibition and no activation of Ca2+ uptake by triphenylphosphine. On the other hand, trifluoperazine inhibited Ca2+ accumulation regardless of whether ATP or ITP was used as substrate. When 5 mM oxalate was included in the medium in order to avoid binding of Ca2+ to the low-affinity Ca2(+)-binding sites of the enzyme, both stimulation by triphenylphosphine and inhibition by trifluoperazine were reduced. In leaky vesicles at low Ca2+ concentrations, triphenylphosphine and 3-nitrophenol were competitive inhibitors of ATPase activity at the regulatory site of the enzyme (0.1-1 mM ATP). A striking difference was observed when both the high- and low-affinity Ca2(+)-binding sites were saturated. In this condition, triphenylphosphine and 3-nitrophenol promoted a 3-4-fold increase in the apparent affinity for ATP at its regulatory site.     

Quercetin interaction with the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

In sarcoplasmic reticulum vesicles or in the (Ca2+ + Mg2+)-ATPase purified from sarcoplasmic reticulum, quercetin inhibited ATP hydrolysis, Ca2+ uptake, ATP-Pi exchange, ATP synthesis coupled to Ca2+ efflux, ATP-ADP exchange, and steady state phosphorylation of the ATPase by inorganic phosphate. Steady state phosphorylation of the ATPase by ATP was not inhibited. Quercetin also inhibited ATP and ADP binding but not the binding of Ca2+. The inhibition of ATP-dependent Ca2+ transport by quercetin was reversible, and ATP, Ca2+, and dithiothreitol did not affect the inhibitory action of quercetin.     

Modulation of the low affinity Ca2+-binding sites of skeletal muscle and blood platelets Ca2+-ATPase by nordihydroguaiaretic acid

The antioxidant nordihydroguaiaretic acid (NDGA) inhibited the different sarco/endoplasmic reticulum Ca2+-ATPase isoforms found in skeletal muscle and blood platelets. For the sarcoplasmic reticulum, but not for the blood platelets Ca2+-ATPase, the concentration of NDGA needed for half-maximal inhibition was found to vary depending on the substrate used and its concentration in the assay medium. The phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase by ATP and by Pi were both inhibited by NDGA. In leaky vesicles, measurements of the ATP Pi exchange showed that NDGA increases the affinity for Ca2+ of the E2 conformation of the enzyme, which has low affinity for Ca2+. The effects of NDGA on the Ca2+-ATPase were not reverted by the reducing agent dithiothreitol nor by the lipid-soluble antioxidant butylated hydroxytoluene.     

Voltage-dependence of Ca2+ uptake and ATP hydrolysis of reconstituted Ca2+-ATPase vesicles

Ca2+-ATPase from sarcoplasmic reticulum was reconstituted into phospholipid/cholesterol (9:1) vesicles (RO). Sucrose density gradient centrifugation of the RO vesicles separated a light layer (RL) with a high lipid/protein ratio and a heavy layer (RH). RH vesicles exhibited a high rate of Ca2+-dependent ATP hydrolysis but did not accumulate Ca2+. RL vesicles, on the other hand, showed an initial molar ratio of Ca2+ uptake to ATP hydrolysis of approximately 1.0. Internal trapping of transported Ca2+ facilitated studies over periods of several minutes. Ca2+ transport and ATP hydrolysis declined concomitantly, reaching levels near 0 with external Ca2+ concentrations less than or equal to 2 microM. Ca2+ uptake was inhibited by the Ca2+ ionophore A23187, the detergent Triton X-100, and the metabolic inhibitor quercetin. Ca2+ transport generated a transient electrical potential difference, inside positive. This finding is consistent with the hypothesis that the Ca2+ pump is electrogenic. Steady state electrical potentials across the membrane were clamped by using potassium gradients and valinomycin, and monitored with voltage-sensitive dyes. Over a range of +50 to -100 mV, there was an inverse relationship between the initial rate of Ca2+ uptake and voltage, but the rate of ATP hydrolysis was nearly constant. In contrast, lowering the external Ca2+ concentration depressed both transport and ATP hydrolysis. These findings suggest that the membrane voltage influences the coupling between Ca2+ transport and ATP hydrolysis.     

Inhibition of plasma membrane Ca2+-ATPase by heparin is modulated by potassium

Heparin is related to several protein receptors that control Ca2+ homeostasis. Here, we studied the effects of heparin on the plasma membrane Ca2+-ATPase from erythrocytes. Both ATP hydrolysis and Ca2+ uptake were inhibited by heparin without modification of the steady-state level of phosphoenzyme formed by ATP. Calmodulin did neither modify the inhibition nor the binding of heparin. Inhibition by heparin was counteracted by K+ but not by Li+. This effect was extended to other sulfated polysaccharides with high number of sulfate residues. Hydrolysis of p-nitrophenylphosphate was equally inhibited by heparin. No evidence for enzyme uncoupling was observed: Ca2+ uptake and ATP hydrolysis remained tightly associated at any level of heparin, and heparin did not increase the passive Ca2+ efflux of inside-out vesicles. Vanadate blocked this efflux, indicating that the main point of Ca2+ escape from these vesicles was linked to the Ca2+ pump. It is discussed that sulfated polysaccharides may physiologically increase the steady-state level of Ca2+ in the cytosol by inhibiting the Ca2+ pumps in a K+ (and tissue) regulated way. It is suggested that heparin regulates the plasma membrane Ca2+-ATPase by binding to the E2 conformer.     

Fast efflux of Ca2+ mediated by the sarcoplasmic reticulum Ca2(+)-ATPase.

The Ca2(+)-ATPase found in the light fraction of sarcoplasmic reticulum vesicles can be phosphorylated by Pi, forming an acylphosphate residue at the catalytic site of the enzyme. This reaction was inhibited by the phenothiazines trifluoperazine, chlorpromazine, imipramine, and fluphenazine and by the beta-adrenergic blocking agents propranolol and alprenolol. The inhibition was reversed by raising either the Pi or the Mg2+ concentration in the medium and was not affected by the presence of K+. Phosphorylation of the Ca2(+)-ATPase by Pi was also inhibited by ruthenium red and spermidine. These compounds compete with Mg2+, but, unlike the phenothiazines, they did not compete with Pi at the catalytic site, and the inhibition was abolished when K+ was included in the assay medium. The efflux of Ca2+ from loaded vesicles was greatly increased by the phenothiazines and by propranolol and alprenolol. In the presence of 200 microM trifluoperazine, the rate of Ca2+ efflux was higher than 3 mumol of Ca2+/mg of protein/10 s. The activation of efflux by these drugs was antagonized by Pi, Mg2+, K+, Ca2+, ADP, dimethyl sulfoxide, ruthenium red, and spermidine. The increase of Ca2+ efflux caused by trifluoperazine was not correlated with binding of the drug to the membrane lipids. It is concluded that the Ca2+ pump can be uncoupled by different drugs, thereby greatly increasing the efflux of Ca2+ through the ATPase. Displacement of these drugs by the natural ligands of the ATPase blocks the efflux through the uncoupled pathway and limits it to a much smaller rate. Thus, the Ca2(+)-ATPase can operate either as a pump (coupled) or as a Ca2+ channel (uncoupled).     

Characteristics of the Ca2+ pump and Ca2+-ATPase in the plasma membrane of rat myometrium.

A plasma membrane-enriched fraction from rat myometrium shows ATP-Mg2+-dependent active calcium uptake which is independent of the presence of oxalate and is abolished by the Ca2+ ionophore A23187. Ca2+ loaded into vesicles via the ATP-dependent Ca2+ uptake was released by extravesicular Na+. This showed that the Na+/Ca2+ exchange and the Ca2+ uptake were both occurring in plasma membrane vesicles. In a medium containing KCl, vanadate readily inhibited the Ca2+ uptake (K1/2 5 microM); when sucrose replaced KCl, 400 microM-vanadate was required for half inhibition. Only a slight stimulation of the calcium pump by calmodulin was observed in untreated membrane vesicles. Extraction of endogenous calmodulin from the membranes by EGTA decreased the activity and Ca2+ affinity of the calcium pump; both activity and affinity were fully restored by adding back calmodulin or by limited proteolysis. A monoclonal antibody (JA3) directed against the human erythrocyte Ca2+ pump reacted with the 140 kDa Ca2+-pump protein of the myometrial plasma membrane. The Ca2+-ATPase activity of these membranes is not specific for ATP, and is not inhibited by mercurial agents, whereas Ca2+ uptake has the opposite properties. Ca2+-ATPase activity is also over 100 times that of calcium transport; it appears that the ATPase responsible for transport is largely masked by the presence of another Ca2+-ATPase of unknown function. Measurements of total Ca2+-ATPase activity are, therefore, probably not directly relevant to the question of intracellular Ca2+ control.     

Regulation of Ca2+-pumping ATPase of heart sarcolemma by a phosphorylation-dephosphorylation Process

The Ca2+-ATPase of dog heart sarcolemma (1, 2) is affected by phosphorylation. As normally prepared, sarcolemmal vesicles are phosphorylated to a high degree, resulting in a relatively low additional incorporation of hydroxylamine resistant [32P]phosphate from [gamma-32P]ATP. The 32P incorporation is increased up to 20-fold by pretreating the vesicles with phosphorylase phosphatase and is inhibited by an inhibitor of cAMP-dependent protein kinases. The phosphatase treatment inhibits markedly the Ca2+-ATPase and the ATP-dependent Ca2+ uptake. The inhibition is more evident at relatively higher levels of free Ca2+ and is reversed by preincubation with ATP. The Ca2+-pumping activity is stimulated markedly by phosphorylase b kinase and inhibited by the (cAMP-dependent) protein kinase inhibitor. Both the protein kinase inhibitor and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prevent the rephosphorylation of sarcolemmal vesicles, but the effects are not additive. The Ca2+ dependence curve of the Ca2+ uptake in phospho- and dephosphorylated vesicles suggests that the phosphorylation might affect the efficiency of the enzyme (turnover rate) rather than its affinity for Ca2+.     

Effects of arsenate on the Ca2+ ATPase of sarcoplasmic reticulum

The effect of arsenate on the partial reactions of the catalytic cycle of the Ca2+ ATPase of skeletal muscle of sarcoplasmic reticulum was studied. With the use of native vesicles it was found that arsenate accelerates the rate of ITP hydrolysis and inhibits both Ca2+ or Sr2+ uptake. These effects were not observed when ATP was used as substrate or, with the use of ITP, when leaky vesicles were assayed. Activation of ITP hydrolysis is related to an increase of the enzyme's apparent affinity for ITP. Arsenate increases the steady-state level of the phosphoenzyme formed from ITP. This depends on the concentration of both Pi and Ca2+, in the medium. Ca2+ and Sr2+ efflux were accelerated by arsenate. The fast Ca2+ efflux promoted by arsenate is impaired by external Ca2+. Arsenate competes with Pi for the phosphorylating site of the enzyme.     

Mg2,Ca2+-ATPase activity of sarcolemmas of intestinal smooth muscle cells in the rabbit

Preparations of rabbit small intestine smooth muscle cell sarcolemma are capable of hydrolyzing ATP in the presence of millimolar concentrations of Mg2+ and Ca2+ and possess the activity of Mg2+,Ca2+-ATPase having a high affinity for Ca2+ (Km = 5.8 X 10(-6) M). The optimal conditions for the Mg2+,Ca2+-ATPase reaction were established. It was demonstrated that sarcolemmal preparations hydrolyze ATP, GTP, ITP and UTP almost at the same rates. The enzyme contains SH-groups that are unequally exposed to the water phase and are inhibited by 50% by p-chloromercurybenzoate and by 90% by dithionitrobenzoate. The Mg2+,Ca2+-ATPase activity is highly sensitive to oxytocin: at the concentration of 10(-7) MU/ml, the hormone completely inhibits the enzyme without affecting its Mg2+-, Ca2+- and Na+,K+-ATPase activities.     

ATP in equilibrium with 32Pi exchange catalyzed by plasma membrane Ca(2+)-ATPase from kidney proximal tubules.

The Ca(2+)-stimulated adenosine 5'-triphosphate-orthophosphate (ATP in equilibrium with 32Pi) exchange reaction was studied using a vesicular preparation derived from plasma membrane of kidney proximal tubules. With native inside-out vesicles, ATP in equilibrium with 32Pi was stimulated by micromolar Ca2+ concentrations. Treatment of the vesicles with the Ca2+ ionophore A23187 that abolished Ca2+ accumulation, strongly inhibited ATP in equilibrium with 32Pi. When Ca(2+)-ATPase was solubilized with the nonionic detergent octaethylene glycol mono n-dodecyl ether, maximal activation of ATP in equilibrium with 32Pi required millimolar Ca2+ concentrations. These Ca2+ concentrations inhibited ATP hydrolysis. ATP in equilibrium with 32Pi exhibited a Michaelian dependence on Pi and Mg2+, was stimulated by ATP, and depended on the ATP/ADP ratio. ATP in equilibrium with 32Pi was modified by the osmolytes urea, trimethylamine-N-oxide, and sucrose, which are representative of the methylamines and polyols that normally accumulate in renal tissue. These compounds did not modify the apparent affinity for Pi; they affected the response to ADP in the same fashion as the overall rate of ATP in equilibrium 32Pi, and their effects depended on medium pH. These data show that the Ca(2+)-ATPase from plasma membrane kidney proximal tubules can operate simultaneously in forward and backward directions. They also show that ATP in equilibrium with 32Pi is modulated by the ligands Ca2+, ATP, ADP, Pi, Mg2+, and H+, and by organic solutes found in renal tissue.     

Uncoupling of Ca2+ transport in sarcoplasmic reticulum as a result of labeling lipid amino groups and inhibition of Ca2+-ATPase activity by modification of lysine residues of the Ca2+-ATPase polypeptide

Limited labeling of amino groups with fluorescamine in fragmented sarcoplasmic reticulum vesicles inhibits Ca2+-ATPase activity and Ca2+ transport. Under the labeling conditions used, 80% of the label reacts with phosphatidylethanolamine and 20% with the Ca2+-ATPase polypeptide. This degree of labeling does not result in vesicular disruption or in loss of vesicular proteins and does not increase the membrane permeability to Ca2+. Fluorescamine labeling of a purified Ca2+-ATPase devoid of aminophospholipids also inhibits Ca2+-ATPase activity, suggesting that labeling of lysine residues of the enzyme polypeptide is responsible for the inhibition of Ca2+-ATPase activity in sarcoplasmic reticulum. Fluorescamine labeling interferes with phosphoenzyme formation and decomposition in both the native vesicles and the purified enzyme; addition of ATP during labeling, and with less effectiveness ADP or AMP, protects both partial reaction steps. Addition of a nonhydrolyzable ATP analog protects phosphoenzyme formation but not decomposition. The inhibition of Ca2+ transport but not of Ca2+-ATPase occurs in sarcoplasmic reticulum vesicles labeled in the presence of ATP, indicating that the transport reaction is uncoupled from the Ca2+-ATPase reaction. The inhibition of Ca2+ transport but not of Ca2+-ATPase activity is also found in sarcoplasmic reticulum vesicles in which only phosphatidylethanolamine has reacted with fluorescamine. Furthermore, the extent of labeling of phosphatidylethanolamine is correlated with the inhibition of Ca2+ transport rates. The inhibition of Ca2+ transport is a reflection of the inhibition of Ca2+ translocation and is not due to an increase in Ca2+ efflux. We propose that labeling of phosphatidylethanolamine perturbs the lipid environment around the enzyme, producing a specific defect in the Ca2+ translocation reaction.     

Interaction of myotoxin a with the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum

Myotoxin a is a muscle-damaging toxin isolated from the venom of Crotalus viridis viridis. Its interaction with the Ca2+-ATPase of sarcoplasmic reticulum (SR) vesicles purified from rabbit skeletal muscle was investigated. Myotoxin a inhibited Ca2+ loading and stimulated Ca2+-dependent ATPase without affecting unidirectional Ca2+ efflux. Its action was dose, time, and temperature dependent. Myotoxin a partially blocked the binding of specific anti-(rabbit SR Ca2+-ATPase) antibodies. It is concluded that myotoxin a attaches to the SR Ca2+-ATPase and uncouples Ca2+ uptake from Ca2+-dependent ATP hydrolysis. Myotoxin a also prevented the formation of decavanadate-induced two-dimensional crystalline arrays of the SR Ca2+-ATPase.     

Effect of Ca2+ on the dimeric structure of scallop sarcoplasmic reticulum

Scallop sarcoplasmic reticulum (SR), visualized in situ by freeze-fracture and deep-etching, is characterized by long tubes displaying crystalline arrays of Ca2+-ATPase dimer ribbons, resembling those observed in isolated SR vesicles. The orderly arrangement of the Ca2+-ATPase molecules is well preserved in muscle bundles permeabilized with saponin. Treatment with saponin, however, is not needed to isolate SR vesicles displaying a crystalline surface structure. Omission of ATP from the isolation procedure of SR vesicles does not alter the dimeric organization of the Ca2+-ATPase, although the overall appearance of the tubes seems to be affected: the edges of the vesicles are scalloped and the individual Ca2+-ATPase molecules are not clearly defined. The effect of Ca2+ on isolated scallop SR vesicles was investigated by correlating the enzymatic activity and calcium-binding properties of the Ca2+-ATPase with the surface structure of the vesicles, as revealed by electron microscopy. The dimeric organization of the membrane is preserved at Ca2+ concentrations where the Ca2+ binds to the high affinity sites (half-maximum saturation at pCa approximately 7.0 with a Hill coefficient of 2.1) and the Ca2+-ATPase is activated (half-maximum activation at pCa approximately 6.8 with a Hill coefficient of 1.84). Higher Ca2+ concentrations disrupt the crystalline surface array of the SR tubes, both in the presence and absence of ATP. We discuss here whether the Ca2+-ATPase dimer identified as a structural unit of the SR membrane represents the Ca2+ pump in the membrane.     

Structural and functional properties of a Ca2+-ATPase from human platelets

An antibody prepared against highly purified rabbit muscle Ca2+-ATPase from sarcoplasmic reticulum has been observed to cross-react with proteins in human platelet membrane vesicles. The antibody specifically precipitated Ca2+-ATPase activity from solubilized human platelet membranes and recognized two platelet polypeptides denatured in sodium dodecyl sulfate with Mr = 107,000 and 101,000. Ca2+-ATPase activity from Brij 78-solubilized platelet membranes was purified up to 10-fold. The purified preparation consisted mainly of two polypeptides with Mr approximately 100,000, and 40,000. The lower molecular weight protein appeared unrelated to Ca2+-ATPase activity. The Ca2+-ATPase in human platelet membrane vesicles exhibited "negative cooperativity" with respect to the kinetics of ATP hydrolysis. The apparent Km for Ca2+ activation of ATPase activity was 0.1 microM. Ca2+-dependent phosphorylation of platelet vesicles by [gamma-32P]ATP at 0 degrees C yielded a maximum of 0.2-0.4 nmol of PO4/mg of protein that was labile at pH 7.0 and 20 degrees C. This result suggests that only about 2-4% of the total protein in platelet membrane vesicles is the Ca2+-ATPase, which agrees with an estimate based on the specific activity of the Ca2+-ATPase in platelet membranes (20-50 nmol of ATP hydrolyzed/min/mg of protein at 30 degrees C). Calmodulin resulted in only a 1.6-fold stimulation of Ca2+-ATPase activity even after extensive washing of membranes with a calcium chelator or chlorpromazine. It is concluded that human platelets contain a Ca2+-ATPase immunochemically related to the Ca2+ pump from rabbit sarcoplasmic reticulum and that the enzymatic characteristics and molecular weight of the platelet ATPase are quite similar to those of the muscle ATPase.     

Mechanisms of Ca2+ transport in plasma membrane vesicles prepared from cultured pituitary cells. II. (Ca2+ + Mg2+)-ATPase-dependent Ca2+ transport activity

Purified plasma membrane vesicles from GH3 rat anterior pituitary cells exhibit a Mg2+-ATP-dependent Ca2+ transport activity. Concentrative uptake of Ca2+ is abolished by exclusion of either Mg2+ or ATP or by inclusion of the Ca2+ ionophore A23187. Furthermore, addition of A23187 to vesicles which have reached a steady state of ATP-supported Ca2+ accumulation rapidly and completely discharges accumulated cation. Ca2+ uptake is unaffected by treatment of vesicles with oligomycin, the uncoupler CCCP, or valinomycin and is greatly reduced in non-plasma membrane fractions. Likewise, Ca2+ accumulation is not stimulated by oxalate, consistent with the plasma membrane origin of this transport system. (Na+, K+)-ATPase participation in the Ca2+ transport process (i.e. via coupled Na+/Ca2+ exchange) was eliminated by omitting Na+ and including ouabain in the reaction medium. Ca2+ transport activity in GH3 vesicles has a similar pH dependence as that seen in a number of other plasma membrane systems and is inhibited by orthovanadate in the micromolar range. Inhibition is enhanced if the membranes are preincubated with vanadate for a short time. A kinetic analysis of transport indicates that the apparent Km for free Ca2+ and ATP are 0.7 and 125 microM, respectively. The average Vmax is 3.6 nmol of Ca2+/min/mg of protein at 37 degrees C. Addition of exogenous calmodulin or calmodulin antagonists had no significant effect on these kinetic properties. GH3 plasma membranes also contain a Na+/Ca2+ exchange system. The apparent Km for Ca2+ is almost 10-fold higher in this system than that for ATP-driven Ca2+ uptake. When both processes are compared under similar conditions, the Vmax of the exchanger is approximately 2-3 times that of ATP-dependent Ca2+ accumulation. Similar results are obtained when purified plasma membranes from bovine anterior pituitary glands were investigated. It is suggested that both Na+/Ca2+ exchange and the (Ca2+ + Mg2+)-ATPase are important in controlling intracellular levels of Ca2+ in anterior pituitary cells.     

The effect of lipid intermediates on Ca2+ and Na+ permeability and (Na+ + K+)-ATPase of cardiac sarcolemma. A possible role in myocardial ischemia

The effect of fatty acid and acylcarnitine on Ca2+ and Na+ transporting enzymes and carriers was studied in sealed cardiac sarcolemma vesicles of mixed polarity. Palmitoylcarnitine markedly reduced the Na+ gradient-induced Ca2+ uptake. Half-maximal reduction was obtained at 15 microM of the carnitine derivative. In a same concentration range palmitoylcarnitine caused a rapid release of accumulated Ca2+ when added to Ca2+-filled vesicles, which suggests that palmitoylcarnitine increases the permeability of the sarcolemma vesicles to Ca2+. A rapid release of Ca2+ was also observed if Ca2+ was taken up by action of the Ca2+ pump. The (Ca2+ + Mg2+)-ATPase, which most likely drives this active Ca2+ uptake, was 90% increased by 50 microM palmitoylcarnitine and evidence was presented that the acylcarnitine effect again was linked to an alteration of Ca2+ permeability of the vesicles. At the same concentration acylcarnitine was not able to unmask the latent protein kinase, so that probably the sarcolemma ATP permeability was not affected. Palmitoylcarnitine at 25 microM did not affect the ouabain-sensitive (Na+ + K+) -ATPase in native sarcolemma vesicles, however, it inhibited markedly if the enzyme was measured in SDS-treated vesicles. The effect of increased free fatty acid concentration on some of the sarcolemma transporting properties was tested by adding oleate-albumin complexes with different molar ratios to the sarcolemma vesicles. In contrast to molar ratios 1 and 5, the ratio of 7 was able to induce a rapid Ca2+ release and to inhibit (Na+ + K+)-ATPase in either native or SDS-treated vesicles markedly. 22Na release from 22Na-preloaded sarcolemma vesicles was shown to be stimulated by either palmitoylcarnitine (50 microM) or oleate-albumin complex (with a molar ratio of 7). The possible significance of the observed effects of lipid intermediates on ion permeability and (Na+ + K+)-ATPase activity in isolated sarcolemma vesicles for the derangement of cardiac cell function in ischemia is discussed.     

Calmodulin regulation of Ca2+ transport in human erythrocytes.

Inside-out vesicles of human erythrocytes took up Ca2+ against an electrochemical gradient. This Ca2+ uptake was dependent on ATP and was stimulated by calmodulin. Treatment of vesicles with 1 mM-EDTA exposed an apparent low-CA2+-affinity Ca2+-transport component with Kd of about 100 microM-Ca2+ or more. This was converted into a single high-Ca2+-affinity transport activity of Kd about 2.5 microM-Ca2+ in the presence of 2 micrograms of calmodulin/ml, showing that the decrease in transport activity after EDTA treatment was reversible. Vesicles not extracted with EDTA showed mainly apparent high-Ca2+-affinity kinetics even in the absence of added calmodulin. Trifluoperazine (30 microM) and calmodulin-binding protein (20 micrograms/ml) inhibited about 50% of the high-affinity Ca2+ uptake and (Ca2+ + Mg2+)-ATPase (Ca2+-activated, Mg2+-dependent ATPase) activity of these vesicles, indicating that the vesicles isolated by the procedure used retained some calmodulin from the erythrocytes. Comparison of Ca2+ transport and (Ca2+ + Mg2+)-ATPase activities in inside-out vesicles yielded a variable Ca2+/P1 stoichiometric ratio. At low free Ca2+ concentrations (below 20 micro-Ca2+), a Ca2+/P1 ration of about 2 was found, whereas at higher Ca2+ concentrations the stoichiometry was approx. 1. The stoichiometry was not significantly altered by calmodulin.     

Biphasic kinetics of sarcoplasmic reticulum Ca2+-ATPase and the detergent-solubilized monomer

The mechanism of ATP hydrolysis was studied at 0 degrees C and pH 7.5 using purified leaky vesicles of sarcoplasmic reticulum Ca2+-ATPase and enzyme solubilized in monomeric form with high concentrations of octaethylene glycol monododecyl ether (C12E8). The enzyme reaction of membranous Ca2+-ATPase was characterized by an initial burst in the hydrolysis of ATP and modulated by millimolar concentrations of ATP. For detergent-solubilized Ca2+-ATPase no burst and moderate low affinity modulation was observed, but the reaction was activated both at low (phosphorylating) and intermediate (K0.5 = 0.06 mM) ATP concentrations. A study of the partial reactions indicated that the effects of detergent and ATP were attributable to activation of the E1P----E2P transition which was rate-limiting. E32P dephosphorylation of membranous Ca2+-ATPase and the detergent-solubilized monomer comprised both a slow and a rapid component. The inhibitory effect of high Ca2+ was correlated with the development of a dominant contribution of slow phase dephosphorylation and with ATP-induced extra binding of Ca2+ binding which presumably takes place at the phosphorylation site (ECaP). Ca2+ was bound with lesser affinity to detergent-solubilized Ca2+-ATPase but with qualitatively the same characteristics as to membranous ECaP. Either Ca2+ or Mg2+ was required for dephosphorylation, also after detergent solubilization. It is concluded that ATP hydrolysis occurs by the same steps for membranous and monomeric Ca2+-ATPase and involves formation of either EMgP or ECaP as reaction intermediates, leading to biphasic kinetics, which, therefore, cannot be taken as evidence of an oligomeric function of the enzyme.     

Ca2+ transport by rat liver plasma membranes: the transporter and the previously reported Ca2+-ATPase are different enzymes

A rat liver plasma membrane fraction showed an ATP-dependent uptake of Ca2+ which was released by the ionophore A23187. This activity represents a plasma membrane component and is not due to microsomal contamination. The Ca2+ transport displayed several properties which were different from those of the high-affinity Ca2+-ATPase previously observed in these membranes (Lotersztajn et al. (1981) J. Biol. Chem. 256, 11209-11215; Birch-Machin, M.A. and Dawson, A.P. (1986) Biochim. Biophys. Acta 855, 277-285). These observations have shown that Ca2+-ATPase does not require added Mg2+ whereas we have demonstrated that, in the same membrane preparation, Ca2+ uptake required millimolar concentrations of added Mg2+. The Ca2+-ATPase has a broad specificity for the nucleotides ATP, GTP, UTP and ITP while Ca2+ uptake remains specific for ATP. Ca2+ uptake also displayed different affinities for free Ca2+ and MgATP compared to Ca2+-ATPase activity, with apparent Km values of 0.25 microM Ca2+, 0.15 mM MgATP and 1.0 microM Ca2+, 4 microM MgATP respectively. The apparent maximum rate of Ca2+ uptake was about 150-fold less than Ca2+-ATPase activity. These features suggest that the high-affinity Ca2+-ATPase is not the enzymic expression of the ATP-dependent Ca2+ transport mechanism.