p-Nitrophenylphosphatase activity catalyzed by plasma membrane (Ca(2+) + Mg(2+)ATPase: correlation with structural changes modulated by glycerol and Ca(2+)

The plasma membrane (Ca²⁺+Mg²⁺)ATPase hydrolyzes pseudo-substrates such as p-nitrophenylphosphate. Except when calmodulin is present, Ca²⁺ ions inhibit the p-nitrophenylphosphatase activity. In this report it is shown that, in the presence of glycerol, Ca²⁺ strongly stimulates phosphatase activity in a dose-dependent manner. The glycerol- and Ca²⁺-induced increase in activity is correlated with modifications in the spectral center of mass (average emission wavenumber) of the intrinsic fluorescence of the enzyme. It is concluded that the synergistic effect of glycerol and Ca²⁺ is related to opposite long-term hydration effects on the substrate binding domain and the Ca²⁺ binding domain.     

Kinetic properties of the ATP-dependent Ca2+ pump and the Na+/Ca2+ exchange system in basolateral membranes from rat kidney cortex

Summary Basolateral plasma membranes from rat kidney cortex have been purified 40-fold by a combination of differential centrifugation, centrifugation in a discontinuous sucrose gradient followed by centrifugation in 8% percoll. The ratio of leaky membrane vesicles (L) versus right-side-out (RO) and inside-out (IO) resealed vesicles appeared to be LROIO=431. High-affinity Ca²⁺-ATPase, ATP-dependent Ca²⁺ transport and Na⁺/Ca²⁺ exchange have been studied with special emphasis on the relative transport capacities of the two Ca²⁺ transport systems. The kinetic parameters of Ca²⁺-ATPase activity in digitonin-treated membranes are:K _m =0.11 m Ca²⁺ andV _max=81±4 nmol P_i/min·mg protein at 37°C. ATP-dependent Ca²⁺ transport amounts to 4.3±0.2 and 7.4±0.3 nmol Ca²⁺/min·mg protein at 25 and 37°C, respectively, with an affinity for Ca²⁺ of 0.13 and 0.07 m at 25 and 37°C. After correction for the percentage of IO-resealed vesicles involved in ATP-dependent Ca²⁺ transport, a stoichiometry of 0.7 mol Ca²⁺ transported per mol ATP is found for the Ca²⁺-ATPase. In the presence of 75mm Na⁺ in the incubation medium ATP-dependent Ca²⁺ uptake is inhibited 22%. When Na⁺ is present at 5mm an extra Ca²⁺ accumulation is observed which amounts to 15% of the ATP-dependent Ca²⁺ transport rate. This extra Ca²⁺ accumulation induced by low Na⁺ is fully inhibited by preincubation of the vesicles with 1mm ouabain, which indicates that (Na⁺–K⁺)-ATPase generates a Na⁺ gradient favorable for Ca²⁺ accumulation via the Na⁺/Ca²⁺ exchanger. In the absence of ATP, a Na⁺ gradient-dependent Ca²⁺ uptake is measured which rate amounts to 5% of the ATP-dependent Ca²⁺ transport capacity. The Na⁺ gradient-dependent Ca²⁺ uptake is abolished by the ionophore monensin but not influenced by the presence of valinomycin. The affinity of the Na⁺/Ca²⁺ exchange system for Ca²⁺ is between 0.1 and 0.2 m Ca²⁺, in the presence as well as in the absence of ATP. This affinity is surprisingly close to the affinity measured for the ATP-dependent Ca²⁺ pump. Based on these observations it is concluded that in isolated basolateral membranes from rat kidney cortex the Ca²⁺-ATPase system exceeds the capacity of the Na⁺/Ca²⁺ exchanger four- to fivefold and it is therefore unlikely that the latter system plays a primary role in the Ca²⁺ homeostasis of rat kidney cortex cells.     

Streptozotocin-induced diabetes increases (Ca2+-Mg2+)-ATPase activity in hepatic plasma membranes of rats: Involvement of protein kinase C

The alteration in calcium transport in the liver of rats with streptozocin(STZ)-diabetic state was investigated. STZ (6 mg/100 g body weight) was subcutaneously administered in rats, and 1 or 2 weeks later they were sacrificed by bleeding. STZ administration caused a remarkable elevation of serum glucose concentration. Liver calcium content was significantly increased by STZ administration. Hepatic plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity was markedly elevated by STZ administration. This increase was completely abolished by the presence of staurosporine (10^-7-10^-5 M), an inhibitor of protein kinase C, in the enzyme reaction mixture, suggesting an involvement of protein kinase C signalling. Moreover, the STZ-induced increase in liver plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity was significantly raised by the presence of okadaic acid (10^-5 and 10^-4 M). Meanwhile, the STZ-increased (Ca²⁺-Mg²⁺)-ATPase activity was not appreciably altered by the presence of anti-regucalcin IgG in the reaction mixture, indicating that the activatory protein regucalcin does not participate in the elevation of the enzyme activity. The present study demonstrates that STZ-induced diabetes causes the increase in hepatic plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity of rats.     

Activation of Ca2+/Mg2+ ATPase in heart sarcolemma upon electrical stimulation

Electrical stimulation of the rat heart sarcolemmal membranes with a square wave current was found to increase Ca²⁺-ATPase activity. This activation of the enzyme was dependent upon the voltage of the electric current, frequency of stimulation and duration of stimulation of the sarcolemmal membranes. The increase in ca²⁺-ATPase was reversible upon terminating the electrical stimulation. The activation of sarcolemmal Ca²⁺-ATPase due to electrical stimulation was markedly depressed when the reaction was carried out at high pH (7.8 to 8.2), low pH (6.6 to 7.0), high temperatures (45 to 50°C) and low temperatures (17 to 25°C) of the incubation medium. Ca²⁺-antagonists, verapamil and D-600, unlike other types of inhibitors such as propranolol and ouabain, were found to reduce the activation of sarcolemmal Ca²⁺-ATPase by electrical stimulation. These results support the view that Ca²⁺/Mg²⁺ ATPase may be involved in the gating mechanism for opening Ca²⁺-channels in the sarcolemmal membrane upon excitation of the cardiac muscle.     

Erythrocyte membrane (Ca2++Mg2+)-ATPase in human protein-energy malnutrition

Calmodulin-free ghost membranes were prepared from erythrocytes of kwashiorkor children and from healthy children in the same age bracket. In the absence of calmodulin, the specific activity of Mg²⁺-dependent Ca²⁺-pumping ATPase (Ca²⁺+Mg²⁺-ATPase) of kwashiorkor membranes was more than 40 percent lower than the specific activity of the normal enzymes, whose maximum velocity was increased by at least four-fold by the modulator protein. In constrast, the maximum velocity of the enzymes of kwashiorkor membranes was enhanced by calmodulin by about 11/2 times the basal activity of the normal enzymes and by 2 times the basal activity of the kwashiorkor enzymes. The affinity of the pump for ATP was lower in the membranes of kwashiorkor children (K_m for ATP=30.6±2.8 M ATP) in comparison to normal membranes (K_m for ATP=21.7±2.0 M ATP). Similarly, calmodulin-affinity of the enzymes, was lower in kwashiorkor membranes than in the normal membranes irrespective of source of calmodulin. Calmodulin from haemolysates of kwashiorkor red cells activated the enzymes of normal and kwashiorkor membranes to the same degree as calmodulin partially purified from the haemolysate of healthy children. A determination of the dependence of the activity of the pump on calcium in the absence and presence of calmodulin reveals that the affinity of the kwashiorkor enzymes for Ca²⁺ is at least 70 percent lower than that of enzymes of normal membranes. Altogether, these findings suggest that the Ca²⁺-pumping ATPase of kwashiorkor membranes is less functional than the enzymes of healthy erythrocytes.     

Specificity of ligand binding to transport sites: Ca2+ binding to the Ca2+ transport ATPase and its dependence on H+ and Mg2+

Ligand binding to transport sites constitutes the initial step in the catalytic cycle of transport ATPases. Here, we consider the well characterized Ca²⁺ ATPase of sarcoplasmic reticulum (SERCA) and describe a series of Ca²⁺ binding isotherms obtained by equilibrium measurements in the presence of various H⁺ and Mg²⁺ concentrations. We subject the isotherms to statistical mechanics analysis, using a model based on a minimal number of mechanistic steps. The analysis allows satisfactory fits and yields information on occupancy of the specific Ca²⁺ sites under various conditions. It also provides a fundamental method for analysis of binding specificity to transport sites under equilibrium conditions that lead to tightly coupled catalytic activation.     

Opiates inhibit calmodulin activation of a high-affinity Ca2+-stimulated Mg2+-dependent ATPase in synaptic membranes

A high affinity Ca²⁺/Mg²⁺ ATPase has been identified and localized in synaptic membrane subfractions. This enzyme is stimulated by low concentrations of Ca²⁺ (1 M) believed to approximate the range of Ca²⁺ in the synaptosomal cytosol (0.1 to 5.0 M). The opiate agonist levorphanol, in a concentration-dependent fashion, inhibited Ca²⁺-stimulated ATP hydrolysis in lysed synaptic membranes. This inhibition was reversed by naloxone, while dextrorphan, the inactive opiate isomer, was without effect. Inhibition by levorphanol was most pronounced in a subfraction of synaptic membranes (SPM-1). The inhibition of Ca²⁺-stimulated ATP hydrolysis was characterized by a reduction inV _max for Ca²⁺. Levorphanol pretreatment reduced the Hill coefficient (H_N) of 1.5 to 0.7, suggesting cooperative interaction between the opiate receptor and the enzyme protein. Levorphanol, but not dextrorphan, also inhibited (28%) ATP-dependent Ca²⁺ uptake by synaptic membranes. Opiate ligand stereoisomers were tested for their effects on calmodulin stimulating of high affinity Ca²⁺/Mg²⁺ ATPase in synaptic membranes. Levorphanol (10 M), but not the inactive stereoisomer (+)dextrorphan, significantly inhibited (35%) the calmodulin-activated Ca²⁺-dependent ATP hydrolysis activity in a preparation of lysed synaptic membranes. Both Ca²⁺-dependent and calmodulin-dependent stimulation of the enzyme in the presence of optimal concentrations of the other co-substrate were inhibited by levorphanol (35–40%) but not dextrorphan. Inhibition of ATP hydrolysis was characterized by a reduction inV _max for both Ca²⁺ and calmodulin stimulation of the enzyme. Calmodulin stimulation of enzyme activity was most pronounced in SPM-1, the membrane fraction which also exhibits the maximal opiate inhibition (40%) of the Ca²⁺-ATPase. The results demonstrate that opiate receptor activation inhibits a high affinity Ca²⁺/Mg²⁺ ATPase in synaptic plasma membranes in a stereospecific fashion. The inhibition of the enzyme may occur by a mechanism involving both Ca²⁺ and calmodulin. Inhibition of calmodulin activation may contribute to the mechanism by which opiate ligands disrupt synaptosomal Ca²⁺ buffering mechanisms. Changes in the cytosolic distribution of synaptosomal Ca²⁺ following inhibition of Ca²⁺/Mg²⁺ ATPase may underlie some of the pharmacological effects of opiate drugs.     

Enhancement of plasma membrane (Ca2+-Mg2+)-ATPase activity in regenerating rat liver: Involvement of endogenous activating protein regucalcin

The alteration of (Ca²⁺-Mg²⁺)-ATPase activity in the plasma membranes of regenerating rat liver after a partial hepatectomy was investigated. Liver was surgically removed about two thirds of that of sham-operated rats. The reduced liver weight by partial hepatectomy was restored about 50% at 24 h after the surgery, and it was completely restored at 72 h. Regenerating liver significantly increased calcium content and plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity between 12–48 h after hepatectomy. Those increases were maximum at 24 h after the surgery. The regenerating liver-induced increase in hepatic plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity was completely abolished by the presence of anti-regucalcin IgG (1.0–4.0 g/ml). The regenerating liver-induced increase in hepatic plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity was clearly inhibited by N-ethylmaleimide (2.5 and 5.0 mM) addition into the enzyme reaction mixture. This NEM effect was also seen for the activatory effect with regucalcin (0.25 M) addition on the enzyme activity in the plasma membranes from normal rat liver. The endogenous regucalcin may play a cell physiological role in the activation of the plasma membrane (Ca²⁺-Mg²⁺)-ATPase to maintain the intracellular calcium level in regenerating rat liver.     

Purification and composition of Ca2+/Mg2+ ATPase from rat heart plasma membrane

The Ca²⁺/Mg²⁺ ATPase, which is activated by millimolar concentrations of Ca²⁺ or Mg²⁺, was solubilized from rat heart plasma membrane by employing lysophosphatidylcholine, CHAPS, Nal, EDTA and Tris-HCI at pH 7.4. The enzyme was purified by sucrose density gradient, Affi-Gel Blue column and Sepharose 6B column chromatography. The purified enzyme was seen as a single peptide band in the sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular weight of about 90,000. The apparent molecular weight of the holoenzyme as determined under non-dissociating conditions by gel filtration on Sepharose 6B column was about 180,000 indicating two subunits. The enzyme was insensitive to ouabain, verapamil, vanadate, oligomycin, N,N-dicyclohexylcarbodiimide and NaN₃, but was markedly inhibited by 20 µM gramacidin S and 50 µM trifluoperazine. Analysis of the purified Ca²⁺/Mg²⁺ ATPase revealed the presence of 17 amino acids where leucine, glutamic acid and aspartic acid were the major components and histidine, cysteine and methionine were the minor components. The purified enzyme was associated with 19.7 µmol phospholipid/mg protein which was 60 times higher than the phospholipid content in plasma membrane. The cholesterol content in the purified enzyme preparation was 0.75 µmol/mg protein and this represented an 8-fold enrichment over plasma membrane. The glycoprotein nature of the enzyme was evident from the positive periodic acid-Schiff staining of the purified Cau2+/Mg^ATPase in the sodium dodecyl sulfate polyacrylamide gel. The polysaccharide content of the enzyme was enriched 8-fold over plasma membrane; neurominidase treatment decreased the polysaccharide content. Concanavalin A prevented the ATP-dependent inactivation of the purified Ca²⁺/Mg²⁺ ATPase and was found to bind to the purified enzyme with a K_D of 576 nM and Bmax of 4.52 nmol/mg protein. The results indicate that Ca²⁺/Mg²⁺ ATPase is a glycoprotein and contains a large amount of lipids.     

Calcium uptake and Ca2+-ATPase activity in goat spermatozoa membrane vesicles do not require Mg2+

Microsomal membrane vesicles isolated from goat spermatozoa contain Ca²⁺-ATPase, and exhibit Ca²⁺ transport activities that do not require exogenous Mg²⁺ .The enzyme activity is inhibited by calcium-channel inhibitors,e.g. verapamil and diltiazem, like the well known Ca²⁺ , Mg²⁺-ATPase. The uptake of calcium is ATP (energy)-dependent and the accumulated Ca²⁺ can be completely released by the Ca²⁺ ionophore A23187, suggesting that a significant fraction of the vesicles are oriented inside out     

Renal handling of Ca2+ in diabetes

Ca²⁺ transport in kidney has gained considerable attention in the recent past. Our laboratory has been involved in understanding the regulatory mechanisms underlying Ca²⁺ transport in the kidney across the renal basolateral membrane. We have shown that ANP, a cardiac hormone, mediates its biological functions by acting on its receptors in the kidney basolateral membrane. Furthermore, it has been established that ANP receptors are coupled with Ca²⁺ ATPase, the enzyme that participates in the vectorial translocation of Ca²⁺ from the tubular lumen to the plasma. It is possible that a defect in the ANP-receptor-effector system in diabetes (under certain conditions such as hypertension) may be associated with abnormal Ca²⁺ homeostasis and the development of nephropathy. Accordingly, future studies are needed to establish this hypothesis.     

Calcium mobilizing hormones activate the plasma membrane Ca2+ pump of pancreatic acinar cells

Summary ⁴⁵Ca fluxes and free-cytosolic Ca²⁺ ([Ca²⁺] _i ) measurements were used to study the effect of Ca²⁺-mobilizing hormones on plasma membrane Ca²⁺ permeability and the plasma membrane Ca²⁺ pump of pancreatic acinar cells. We showed before (Pandol, S.J., et al., 1987.J. Biol. Chem. 262:16963–16968) that hormone stimulation of pancreatic acinar cells activated a plasma membrane Ca²⁺ entry pathway, which remains activated for as long as the intracellular stores are not loaded with Ca²⁺. In the present study, we show that activation of this pathway increases the plasma membrane Ca²⁺ permeability by approximately sevenfold. Despite that, the cells reduce [Ca²⁺]i back to near resting levels. To compensate for the increased plasma membrane Ca²⁺ permeability, a plasma membrane Ca²⁺ efflux mechanism is also activated by the hormones. This mechanism is likely to be the plasma membrane Ca²⁺ pump. Activation of the plasma membrane Ca²⁺ pump by the hormones is time dependent and 1.5–2 min of cell stimulation are required for maximal Ca²⁺ pump activation. From the effect of protein kinase inhibitors on hormone-mediated activation of the pump and the effect of the phorbol ester 12-0-tetradecanoyl phorbol, 13-acetate (TPA) on plasma membrane Ca⁺ efflux, it is suggested that stimulation of protein kinase C is required for the hormone-dependent activation of the plasma membrane Ca²⁺ pump.     

Role of Na+/Ca2+ exchange and the plasma membrane Ca2+ pump in hormone-mediated Ca2+ efflux from pancreatic acini

Summary The relative contributions of the Na⁺/Ca²⁺ exchange and the plasma membrane Ca²⁺ pump to active Ca²⁺ efflux from stimulated rat pancreatic acini were studied. Na⁺ gradients across the plasma membrane were manipulated by loading the cells with Na⁺ or suspending the cells in Na⁺-free media. The rates of Ca²⁺ efflux were estimated from measurements of [Ca²⁺] _i using the Ca²⁺-sensitive fluorescent dye Fura 2 and⁴⁵Ca efflux. During the first 3 min of cell stimulation, the pattern of Ca²⁺ efflux is described by a single exponential function under control, Na⁺-loaded, and Na⁺-depleted conditions. Manipulation of Na⁺ gradients had no effect on the hormone-induced increase in [Ca²⁺] _i . The results indicate that Ca²⁺ efflux from stimulated pancreatic acinar cells is mediated by the plasma membrane Ca²⁺ pump. The effects of several cations, which were used to substitute for Na⁺, on cellular activity were also studied. Choline⁺ and tetramethylammonium⁺ (TMA⁺) released Ca²⁺ from intracellular stores of pancreatic acinar, gastric parietal and peptic cells. These cations also stimulated enzyme and acid secretion from the cells. All effects of these cations were blocked by atropine. Measurements of cholecystokinin-octapeptide (CCK-OP)-stimulated amylase release from pancreatic acini, suspended in Na⁺, TMA⁺, choline⁺, or N-methyl-d-glucamine⁺ (NMG⁺) media containing atropine, were used to evaluate the effect of the cations on cellular function. NMG⁺, choline⁺, and TMA⁺ inhibited amylase release by 55, 40 and 14%, respectively. NMG⁺ also increased the Ca²⁺ permeability of the plasma membrane. Thus, to study Na⁺ dependency of cellular function, TMA⁺ is the preferred cation to substitute for Na⁺. The stimulatory effect of TMA⁺ can be blocked by atropine.     

Cell cycle-related fluctuations in transcellular ionic currents and plasma membrane Ca2+/Mg2+ ATPase activity during early cleavages of Lymnaea stagnalis embryos

Summary During the first four mitotic division cycles of Lymnaea stagnalis embryos, we have detected cell cycle-dependent changes in the pattern of transcellular ionic currents and membrane-bound Ca²⁺-stimulated ATPase activity. Ionic currents ranging from 0.05 to 2.50 A/cm² have been measured using the vibrating probe technique. Enzyme activity was detected using Ando's cytochemical method (Ando et al. 1981) which reveals Ca²⁺/Mg²⁺ ATPase localization at the ultrastructural level, and under high-stringency conditions with respect to calcium availability, it reveals Ca²⁺-stimulated ATPase. The ionic currents and Ca²⁺-stimulated ATPase localization have in common that important changes occur during the M-phase of the cell cycles. Minimal outward current at the vegetal pole coincides with metaphase/anaphase. Maximal inward current at the animal pole coincides with the onset of cytokinesis at that pole. Ca²⁺-stimulated ATPase is absent from one half of the embryo at metaphase/anaphase of the two- and four-cell stage, whereas it is present in all cells during the remaining part of the cell cycle. Since fluctuations of cytosolic free calcium concentrations appear to correlate with both karyokinesis and cytokinesis, we speculate that part of the cyclic pattern of Ca²⁺-stimulated ATPase localization and of the transcellular ionic currents reflects the elevation of cytosolic free calcium concentration during the M-phase. Offprint requests to: D. Zivkovic     

Effect of lead on the erythrocyte (Ca2+,Mg2+)-ATPase activity Calmodulin involvement

Summary I have investigated the effect of lead on the erythrocyte ghosts (Ca²⁺,Mg²⁺)-ATPase, with special attention to the role of calmodulin in this phenomena. Under regular incubation conditions, lead inhibits the enzyme with an IC₅₀ of 6.0 µM. The presence of exogenously added calmodulin apparently does not change this inhibitory value. DTT added during the incubation period does not affect the inhibitory action of lead. However, when the membranes are preincubated with DTT, an important IC₅₀ displacement is observed, either with or without added calmodulin. Since [¹²⁵I]calmodulin binding to the membranes is enhanced when lead is used, the possibility of a lead/calmodulin complex that optimally stimulates the enzyme using lead concentrations between 1.0 and 10.0 µM, is suggested. Based on the experimental data, I propose two well defined actions of lead; first, an inhibitory action upon the ATPase above 1.0 µM lead, most probably related to essential sullphydryl groups in the enzyme; and second, a direct action of lead upon calmodulin at lead concentrations below 1.0 µM.A preliminary report has been presented at the 5^th European Bioenergetics Conference. Aberystwyth, Wales. 20–26 July 1988. EBEC Reports. vol 5; p294 (1988).     

Disruption of energy transduction in sarcoplasmic reticulum by trypsin cleavage of (Ca2++Mg2+)-ATPase

Summary Proteolytic digestion of sarcoplasmic reticulum vesicles with trypsin has been used as a structural modification with which to examine the interaction between the ATP hydrolysis site and calcium transport sites of the (Ca²⁺+Mg²⁺)-ATPase. The kinetics of trypsin fragmentation were examined and the time course of fragment production compared with ATP hydrolytic and calcium uptake activities of the digested vesicles. The initial cleavage (TD 1) of the native ATPase to A and B peptides has no effect on the functional integrity of the enzyme, hydrolytic and transport activities remaining at the levels of the undigested control. Concomitant with the second tryptic cleavage (TD 2) of the A peptide to A₁ and A₂ fragments, calcium transport is inhibited. Kinetic analysis demonstrates that the rate constant for inhibition of calcium uptake is correlated with the rate constant of a fragment disappearance. Both Ca²⁺-dependent and total ATPase activities are unaffected by this second cleavage. Passive loading of vesicles with calcium and subsequent efflux measurements show that transport inhibition is not due to increased permeability of the membrane to calcium even at substantial extents of digestion. Steady-state levels of acidstable phosphoenzyme are unaffected by either TD 1 or TD 2, indicating that uncoupling of the hydrolytic and transport functions does not increase the turnover rate of the enzyme and that TD 2 does not change the essential characteristics of the ATP hydrolysis site. Sarcoplasmic reticulum (SR) vesicles were examined for the presence of tightly bound nucleotides and are shown to contain 2.8–3.0 nmol ATP and 2.6–2.7 nmol ADP per mg SR protein. The ADP content of SR remains essentially unchanged with TD 1 cleavage of the ATPase enzyme to A and B peptides, but declines upon TD 2 in parallel with the digestion of the A fragment and the loss of calcium uptake activity of the vesicles. The ATP content is essentially constant throughout the course of trypsin digestion. The results are discussed in terms of current models of the SR calcium pump and the molecular mechanism of energy transduction.     

Hormone-stimulated Mg(2+) accumulation into rat hepatocytes: a pathway for rapid Mg(2+) and Ca(2+) redistribution

Many diseases such as cardiac arrhythmia, diabetes, and chronic alcoholism are associated with a marked decrease of plasma and parenchymal Mg(2+), and Mg(2+) administration is routinely used therapeutically. This study uses isolated rat hepatocytes to ascertain if and under which conditions increases in extracellular Mg(2+) result in an increase in intracellular Mg(2+). In the absence of stimulation, changing extracellular Mg(2+) had no effect on total cellular Mg(2+) content. By contrast, carbachol or vasopressin administration promoted an accumulation of Mg(2+) that increased cellular Mg(2+) content by 13.2 and 11.8%, respectively, and stimulated Mg(2+) uptake was unaffected by the absence of extracellular Ca(2+). Mg(2+) efflux resulting from stimulation of alpha- or beta-adrenergic receptors operated with a Mg(2+):Ca(2+) exchange ratio of 1. These data indicate that cellular Mg(2+) uptake can occur rapidly and in large amounts, through a process distinct from Mg(2+) release, but operating only upon specific hormonal stimulation.     

Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves

Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca²⁺ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.⁴⁵Ca²⁺ transport was assayed by membrane filtration technique. Results showed that Ca²⁺ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca²⁺ transport system had a high affinity for Ca²⁺(K _m (Ca²⁺)=0.4 m) and ATP(K _m (ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca²⁺ uptake in the plasma membrane vesicles was stimulated in the presence of Cl^– or NO ₃ ^– . Quenching of quinacrine fluorescence showed that these anions also induced H⁺ transport into the vesicles. The Ca²⁺ uptake stimulated by Cl^– was dependent on the activity of H⁺ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO ₄ ^3– which is known to inhibit the H⁺ pump associated with the plasma membrane, canceled almost all of the Cl^–-stimulated Ca²⁺ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca²⁺ uptake into the vesicles. These results suggest that the Cl^–-stimulated Ca²⁺ uptake is caused by the efflux of H⁺ from the vesicles by the operation of Ca²⁺/H⁺ antiport system in the plasma membrane. In Cl^–-free medium, H⁺ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca²⁺ uptake into the vesicles. These results suggest that two Ca²⁺ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca²⁺ transport system (Ca²⁺ pump) and the other is a Ca²⁺/H⁺ antiport system. Little difference in characteristics of Ca²⁺ transport was observed between the plasma membranes isolated from etiolated and green corn leaves.     

Ca2+/H+ exchange,lumenal Ca2+ release and Ca2+/ATP coupling ratios in the sarcoplasmic reticulum ATPase

The Ca²⁺ transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca²⁺ in intracellular membrane bound compartments, thereby lowering cytosolic Ca²⁺ to induce relaxation. The stored Ca²⁺ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca²⁺, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca²⁺. We review here biochemical and biophysical evidence demonstrating that release of bound Ca²⁺ into the lumen of SR requires Ca²⁺/H⁺ exchange at the low affinity Ca²⁺ sites. Rise of lumenal Ca²⁺ above its dissociation constant from low affinity sites, or reduction of the H⁺ concentration by high pH, prevent Ca²⁺/H⁺ exchange. Under these conditions Ca²⁺ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca²⁺pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.