Some properties of the purified (Ca2+ + Mg2+-ATPase from human red cell membranes

The (Ca²⁺ + Mg²⁺-ATPase from red cell membranes, purified by means of a calmodulin-containing affinity column according to the method of Gietzen et al. (Gietzen, K., Tej?ka, M. and Wolf, H.U. (1980) Biochem. J. 189, 81–88) with either phosphatidylcholine or phosphatidylserine as phospholipid is characterized. The phosphatidylcholine preparation can be activated by calmodulin, while the phosphatidylserine preparation is fully activated without calmodulin. The enzyme shows a biphasic ATP dependence with two K_m values of 3.5 and 120 μM. The enzyme is phosphorylated by ATP in the presence of Ca²⁺ only.     

Enhanced activation of human erythrocyte Ca2+-ATPase by calmodulin after storage or brief exposure to disulfite

Ca²⁺-ATPase of human erythrocyte membranes which are prepared from freshly drawn human blood can be activated by the calmodulin present in the hemolysate to 1.5-times the basal level. However, when the membranes are prepared from blood stored for 5–14 days the activation by calmodulin reaches 2.5-times the basal level. An enhanced reactivity to calmodulin of similar magnitude was produced by brief exposure of fresh erythrocytes to 25 mM Na₂S₂O₅ prior to isolation of the membranes. Reincubation of the activated cells in a disulfite-free medium restored the membrane-bound Ca²⁺-ATPase to a state of normal reactivity to calmodulin. It is hypothesized that these results are related to the level of cytoplasmic Ca²⁺ which is partly controlled by complex formation with 2,3-diphosphoglycerate, the concentration of which is diminished when its specific phosphatase is activated by Na₂S₂O₅.     

Differentiation between Ca2+ transport and ATP-induced Ca2+ binding by sarcoplasmic reticulum

The Ca²⁺ actively accumulated by sarcoplasmic reticulum isolated from skeletal muscle is composed of two fractions; one represented by intravesicular free Ca²⁺ and another represented by Ca²⁺ selectively bound to the membranes. Both of these Ca²⁺ fractions depend on ATP, although it is not clear whether ATP hydrolysis is essential for accumulation of the second Ca²⁺ fraction. The existence of the membrane-bound Ca²⁺ induced by ATP is clearly shown in experiments in which the Ca²⁺ retention by sarcoplasmic reticulum is measured in the presence and in the absence of X-537A, a Ca²⁺ ionophore, which makes the membrane permeable to Ca²⁺. Thus, in the presence of X-537A all Ca²⁺ accumulated due to ATP is bound to the membranes. This membrane-bound Ca²⁺ represents about 30 nmol/mg protein in the range of external $\text{p}\text{Ca}$ values of 7 to 3.5. The magnitude of this Ca²⁺ fraction is slightly higher whether or not the experiments are performed in the presence of oxalate, which greatly increased the intravesicular Ca²⁺ accumulation. Furthermore, taking advantage of the impermeability of sarcoplasmic reticulum to EGTA, it is possible to show the existence of the membrane-bound Ca²⁺ as a distinct fraction from that which exists intravesicularly.     

Studies on the mechanism of regulation of the red-cell Ca2+ pump by calmodulin and ATP

(1) The effects of calmodulin binding on the rates of Ca²⁺-dependent phosphorylation and dephosphorylation of the red-cell Ca²⁺ pump, have been tested in membranes stripped of endogenous calmodulin or recombined with purified calmodulin. (2) In Mg²⁺-containing media, phosphorylation and dephosphorylation rates are accelerated by a large factor (at 0°C), but the steady-state level of phosphoenzyme is unaffected by calmodulin binding (at 0°C and 37°C). In Mg²⁺-free media, slower rates of phosphoenzyme formation and hydrolysis are observed, but both rates and the steady-state phosphoenzyme level are raised following calmodulin binding. (3) At 37°C and 0°C, the rate of (Ca²⁺ + Mg²⁺)-ATPase activity is stimulated maximally by 6–7-fold, following calmodulin binding. At 37°C the apparent Ca²⁺ affinity for sustaining ATP hydrolysis is raised at least 20-fold, K_m(Ca) ? 10 μM (—calmodulin) and K_m(Ca) < 0.5 μM (+ calmodulin), but at 0°C the apparent Ca²⁺ affinity is very high in calmodulin-stripped membranes and little or no effect of calmodulin is observed (K_m(Ca) ? 3–4 · 10^-8 M). (Ca²⁺ + Mg²⁺)-ATPase activity in calmodulin activated membranes and at saturating ATP levels, is sharply inhibited by addition of calcium in the range 50–2000 μM. (4) A systematic study of the effects of the nucleotide species MgATP, CaATP and free ATP on (Ca²⁺ + Mg²⁺)-ATPase activity in calmodulin-activated membranes reveals: (a) In the 1–10 μmolar concentration range MgATP, CaATP and free ATP appear to sustain (Ca²⁺ + Mg²⁺)-ATPase activity equally effectively. (b) In the range 100–2000 μM, MgATP accelerates ATP hydrolysis (K_m(MgATP) ? 360 μM), and CaATP is an inhibitor (K_i(CaATP) ? 165 μM), probably competing with MgATP fo the regulatory site. (5) The results suggest that calmodulin binding alters the conformational state of the Ca²⁺- pump active site, producing a high (Ca²⁺ + Mg²⁺)-ATPase activity, high Ca²⁺ affinity and regulation of activity by MgATP.     

Regulation of the Ca2+-pump by calmodulin in intact cells

ATP-enriched human red cells display high rates of Ca²⁺-dependent ATP hydrolysis (16 mmol·litre cells^?1·h^?1) with a high Ca²⁺ affinity ($\text{K}{}_{0.5}\text{～0.2 μ}\text{M}$). The finding suggests a mechanism for regulation of cell Ca²⁺ levels, involving highly-cooperative stimulation of active Ca²⁺ extrusion following binding of calmodulin to the $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$.     

On the substrate specificity of the red cell calcium pump

ATP-dependent active calcium transport in inside-out human red cell membrane vesicles is stimulated by magnesium essentially parallel with an increase in MgATP concentration. At a constant, low (1 μM) calcium concentration, increasing ATP and magnesium increase the maximum calcium transport rate irrespective of the constant or decreasing concentrations of CaATP present. K_Ca for calcium pumping is practically unchanged at variable ATP and magnesium concentrations. Free magnesium above 1–2 mM inhibits active calcium transport, probably through a direct interaction with the transport enzyme. Based on the experimental findings reported we suggest that the true, physiological substrate of the red cell calcium pump is MgATP.     

Calmodulin and the target size of the (Ca2+ + Mg2+)-ATPase of human red-cell ghosts

An average target size of 251 kDa has been obtained for the (Ca²⁺ + Mg²⁺)-ATPase of calmodulin-depleted erythrocyte ghosts by radiation inactivation with 16 MeV electrons. This is close to twice the size of the purified calcium-pump polypeptide. When calmodulin was included during the ATPase assay, a component of about 1 MDa appeared in addition to the activated dimer.     

Studies on the Ca2+ transport mechanism of human erythrocyte inside-out plasma membrane vesicles V. Chlortetracycline fluorescence

The measurement of chlortetracycline fluorescence was employed as a probe for measuring the process to calcium transport by human erythrocyte inside-out vesicles. Chlortetracycline is a divalent metal chelator which increases its fluorrescence when bound to calcium in the presence of a membrane. Addition of calcium and ATP to inside out vesicles in the presence of chlortetracycline increased the chlortetracycline fluorescence as a function of time following an initial delay. Only after a threshold level of calcium had been accumulated did the fluorescence increase. The presence of both ATP and calcium were required. The addition of calmodulin increased the rate and absolute magnitude of the chlortetracycline fluorescence change. Similarly, calmodulin stimulated the rate and extent of ⁴⁵Ca transport by inside-out vesicles. Moreover, the presence of saponin abolished both chlortetracycline fluorescence change and ⁴⁵Ca uptake; a non-hydrolyzable ATP analog would not substitute for ATP in either ⁴⁵Ca transport or chlortetracycline fluorescence experiments. Comparison between the slopes of the linear portions of chlortetracycline fluorescence change and calcium transport time courses at varied free calcium concentrations showed a consistent ratio between the slopes. This suggests that calcium transport change can be calibrated by employing chlortetracycline fluorescence. Based on this data, it is concluded that chlortetracycline fluorescence is a rapid and accurate method for monitoring calcium transport by human erythrocyte inside-out vesicles.     

Relation between phosphorylation and adenosine triphosphate-dependent Ca2+ binding of swine and bovine erythrocyte membranes

The correlation between the ATP-dependent Ca²⁺ binding and the phosphorylation of the membranes from swine and bovine erythrocytes was studied. The Ca²⁺ binding was measured by using ⁴⁵CaCl₂, and the phosphorylation by [γ-³²P]ATP was studied with the technique of SDS polyacrylamide gel electrophoresis. 200 mM NaCl and KCl markedly repressed the Ca²⁺ binding of swine erythrocyte membranes. The radioactivity of ³²P-labelled membranes was revealed mainly in 250 000 dalton protein and a lipid fraction. NaCl and KCl also repressed the phosphorylation of the lipid which was identified as triphosphoinositide by paper chromatography. The membranes prepared from trypsin-digested erythrocytes completely retained the Ca²⁺-binding activity, and lost 30% of $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ activity. The Ca²⁺-binding and ATPase activity of isolated membranes decreased to 55% and to 0%, respectively, by tryptic digestion. Neither the Ca²⁺ binding nor the phosphorylation of polyphosphoinositides were detected in bovine erythrocyte membranes.These results suggest that the formation of triphosphoinositide rather than the $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ of membranes is linked to the ATP-dependent Ca²⁺ binding of erythrocyte membranes.     

Compound 48/80 and calmodulin modify the interaction of ATP with the (Ca2+ + Mg2+)-ATPase of red cell membranes

Compound 48/80, an anti-calmodulin agent, reduces the maximum effect of ATP and does not affect the apparent affinity for ATP of the high-affinity site of the Ca2+-ATPase from calmodulin-bound membranes of human red cells. In the same preparation, 48/80 reduces more than 50-times the apparent affinity for ATP of the low-affinity site with little change in the maximum effect of the nucleotide at this site of the Ca2+-ATPase. The effects of compound 48/80 are independent of the concentration of Ca2+ between 30 and 200 microM. The apparent affinity of the low-affinity site of the Ca2+-ATPase for ATP is almost 100-fold less in calmodulin-stripped membranes than in calmodulin-bound membranes. In calmodulin-stripped membranes, exogenous calmodulin increases the apparent affinity for ATP up to the control values. These results indicate that apart from increasing the apparent affinity of the transport site for Ca2+, calmodulin also increases the apparent affinity of the regulatory site of the Ca2+-ATPase for ATP. Since this effect is exerted within the physiological ranges of ATP concentrations, it may participate in the physiological regulation of Ca2+ pumping by calmodulin.     

The role of the sites for ATP of the Ca2+-ATPase from human red cell membranes during Ca2+-phosphatase activity

1. In the presence of ATP, the Ca²⁺ pump of human red cell membranes catalyzes the hydrolysis of $\text{p-}\text{nitrophenyl}$ phosphate. The requirement for ATP of the Ca²⁺-$\text{p-}\text{nitrophenylphosphatase}$ activity was studied in relation to the two classes of site for ATP that are apparent during Ca²⁺ -ATPase activity. 2. (a) The $\text{K}{}_{0.5}$ for ATP as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ extrapolated at 0 mM PNPP is equal to the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of the Ca²⁺ -ATPase. (b) PNPP competes with ATP and its effectiveness is the same regardless the nucleotide acts as the substrate of the Ca²⁺ -ATPase or as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$. 3. PNPP at the high-affinity site does not substitute for ATP as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$. 4. At ATP concentrations that almost saturate the high-affinity site, Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity increases as a function of PNPP along an S-shaped curve, while Ca²⁺ -ATPase activity is partially inhibited along a curve of the same shape and apparent affinity. The fraction of Ca²⁺ -ATPase activity which is inhibited by PNPP is that which results from occupation of the low-affinity site by ATP. 5. Activation of the Ca²⁺ -ATPase by ATP at the low-affinity site is associated with inhibition of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity. Both phenomena take place with the same apparent affinity and along curves of the same shape. 6. Experimental results suggest that: (a) the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity depends on ATP at the high-affinity site; (b) PNPP is hydrolyzed at the low-affinity site; (c) Ca²⁺ -ATPase activity at the high-affinity size persists during Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity.     

Characteristics and regulation of active calcium transport in inside-out red cell membrane vesicles

Sealed, inside-out human red cell membrane vesicles, prepared by a modified method of Steck (Steck T.L. (1974) in Methods in Membrane Biology (Korn, E.D., ed.), Vol 2, pp. 245–281, Plenum Press, New York), accomplish an ATP and Mg²⁺-dependent uphill calcium uptake with a reproducible maximum rate of 12–15 nmol/mg vesicle protein per min under physiological conditions. This maximum rate is increased by about 60–70% in the presence of a heatstable cytoplasmic activator protein (calmodulin) obtained from red cells. Calcium efflux from inside-out vesicles is smaller than 0.01 nmol/mg vesicle protein per min at intravesicular calcium concentrations between 0.1 and 20.0 mM.In the presence of Mg²⁺, active calcium uptake is supported by ATP, ITP, or UTP, but not by ADP, AMP, or p-nitrophenyl phosphate. The optimum pH for the process is 7.4–7.6, and the activation energy is 19–20 kcal/mol, irrespective of the presence or absence of calmodulin. Calcium uptake in inside-out vesicles is unaffected by ouabain or oligomycin, but blocked by low concentrations of lanthanum, ruthenium red, quercetin and phloretin. K⁺ and Na⁺, when compared to choline⁺ or Li⁺, significantly increase active calcium uptake. This stimulation by K⁺ and Na⁺ is independent of that by calmodulin.Concentrated red cell cytoplasm activates calcium uptake at low soluble protein:membrane protein ratios, while a ‘deactivation’ of the transport occurs at high cytoplasm: membrane protein ratios. A heat-labile cytoplasmic protein fraction antagonizing calmodulin activation, can be separated by DEAE-Sephadex chromatography. Based on these findings the regulation of active calcium transport in human red cells is discussed.     

Trifluoperazine inhibition of calmodulin-sensitive Ca2+-ATPase and calmodulin insensitive (Na+ + K+)- and Mg2+-ATPase activities of human and rat red blood cells

Trifluoperazine dihydrochloride-induced inhibition of calmodulin-activated Ca²⁺-ATPase and calmodulin-insensitive (Na⁺ + K⁺)- and Mg²⁺-ATPase activities of rat and human red cell lysates and their isolated membranes was studied. Trifluoperazine inhibited both calmodulin-sensitive and calmodulin-insensitive ATPase activities in these systems. The concentration of trifluoperazine required to produce 50% inhibition of calmodulin-sensitive Ca²⁺-ATPase was found to be slightly lower than that required to produce the same level of inhibition of other ATPase activities. Drug concentrations which inhibited calmodulin-sensitive ATPase completely, produced significant reduction in calmodulin-insensitive ATPases as well. The data presented in this report suggest that trifluoperazine is slightly selective towards calmodulin-sensitive Ca²⁺-ATPase but that it is also capable of inhibiting calmodulin-insensitive (Na⁺ + K⁺)-ATPase and Mg²⁺-ATPase activities of red cells at relatively low concentrations. Thus the action of the drug is not due entirely to its interaction with calmodulin-mediated processes, and trifluoperazine cannot be assumed to be a selective inhibitor of calmodulin interactions under all circumstances.     

An electrogenic Na+/Ca2+ antiporter in addition to the Ca2+ pump in cardiac sarcolemma

Vesicles isolated from rat heart, particularly enriched in sarcolemma markers, were examined for their sidedness by investigation of side-specific interactions of modulators with the asymmetric (Na⁺ + K⁺)-ATPase and adenylate cyclase complex. The membrane preparation with the properties expected for inside-out vesicles showed the highest rate of ATP-driven Ca²⁺ transport. The Ca²⁺ pump was stimulated 1.7- and 2.1-fold by external Na⁺ and K⁺, respectively, the half-maximal activation occurring at 35 mM monovalent cation concentration. In vesicles loaded with Ca²⁺ by pump action in a medium containing 160 mM KCl, a slow spontaneous release of Ca²⁺ started after 2 min. The rate of this release could be dramatically increased by the addition of 40 mM NaCl to the external medium. In contrast, 40 mM KCl exerted no appreciable effect on vesicles loaded with Ca²⁺ in a medium containing 160 mM NaCl. Ca²⁺ movements were also studied in the absence of ATP and Mg²⁺. Vesicles containing an outwardly directed Na⁺ gradient showed the highest Ca²⁺ uptake activity. These findings suggested the operation of a Ca²⁺/Na⁺ antiporter in addition to the active Ca²⁺ pump in these sarcolemmal vesicles. A valinomycin-induced inward K⁺-diffusion potential stimulated the Na⁺- Ca²⁺ exchange, suggesting its electrogenic nature. If in the absence of ATP and Mg²⁺ the transmembrane Na_i⁺/Na_o⁺ gradient exceeded 160/15 mM concentrations, Ca²⁺ uptake could be stimulated by the addition of 5 mM oxalate, indicating Na⁺ gradient-induced Ca²⁺ uptake to be a translocation of Ca²⁺ to the lumen of the vesicle. A sarcoplasmic reticulum contamination, removed by further sucrose gradient fractionation, contained rather low Na⁺-Ca²⁺ exchange activity. This result suggests that the activity can be entirely accounted for by the sarcolemmal content of the cardiac membrane preparation.     

Calmodulin an activator of human erythrocyte (Ca2+ + Mg2+)-ATPase phosphorylation

The effect of purified calmodulin on the calcium-dependent phosphorylation of human erythrocyte membranes was studied. Under the conditions employed, only one major peak of phosphorylation was observed when solubilized membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of this phosphorylated protein band was estimated to be 130 000 and in the presence of purified red blood cell calmodulin, the rate of phosphorylation of this band was increased. These data suggest that calmodulin activation of (Ca²⁺ + Mg²⁺)-ATPase could be a partial reflection of an increased rate of phosphorylation of the (Ca²⁺ + Mg²⁺)-ATPase of human erythrocyte membranes.     

Effects of Ca2+, calmodulin and cyclic AMP on the phosphorylation of endogenous proteins by homogenates of rat islets of Langerhans

Activation of Ca²⁺-calmodulin- and cyclic AMP-dependent protein kinases has been suggested to be involved in stimulus-secretion coupling in the pancreatic β-cell. To study the properties of such kinases and their endogenous protein substrates homogenates of rat islets of Langerhans were incubated with [γ-³²P]ATP. Phosphorylated proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and detected by autoradiography. The phosphorylation of certain proteins could be enhanced by Ca²⁺ plus calmodulin or by cyclic AMP. The major effect of Ca²⁺ and calmodulin was to stimulate the phosphorylation of a protein (P53) of molecular weight 53 100±500 (n = 15). Maximum phosphorylation of protein P53 occurred within 2 min with 2 μM free Ca²⁺ and 0.7 μM calmodulin. Incorporation of label into protein P53 was inhibited by trifluoperazine or W7 but not by cyclic AMP-dependent protein kinase inhibitor. Phosphorylation of a protein of similar molecular weight could be enhanced to a lesser extent in the absence of Ca²⁺ but in the presence of cyclic AMP and 3-isobutylmethylxanthine: this phosphorylation was blocked by cyclic AMP-dependent protein kinase inhibitor. Cyclic AMP also stimulated incorporation of label into polypeptides of molecular weights 55 000 and 70–80 000. The results are consistent with the hypothesis that protein phosphorylation mechanisms may play a role in the regulation of insulin secretion.     

Comparison of calmodulin binding to brain synaptic and coated vesicles

Several characteristics of calmodulin association with brain synaptic and coated vesicles were analyzed and compared. Radioimmunoassay revealed that both classes of vesicles contain approx. 1 μg of calmodulin per mg of vesicle protein. Discontinuous sucrose gradients revealed that coated and synaptic vesicles preparations were homogeneous and had different sedimentation properties. Binding of ¹²⁵I-labeled calmodulin to synaptic and coated vesicles was Ca²⁺ dependent and displaced by unlabeled calmodulin but not by troponin-C. Scatchard analysis revealed the presence of two binding sites. In both vesicle types there was one high-affinity, low-binding-capacity site ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1–39}\text{nM and B}{}_{\mathrm{<mtext>max</mtext>}}\text{= 4–16}\text{pmol}\text{/}\text{mg}$) and one low-affinity, high-binding-capacity site ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 102–177}\text{nM and B}{}_{\mathrm{<mtext>max</mtext>}}\text{= 151–202}\text{pmol}\text{/}\text{mg}$). (Ca²⁺ + Mg²⁺)-ATPase activity was stimulated in both synaptic and coated vesicles by calmodulin. Thus synaptic and coated vesicles may possess similar calmodulin binding sites.     

Effect of extracellular Ca2+, K+ and OH− on erythrocyte membrane potential as monitored by the fluorescent probe 3,3′-dipropylthiodicarbocyanine

Changes in fluorescence intensity of thiodicarbocyanine, DiS-C₃(5), were correlated with direct microelectrode potential measurements in red blood cells from Amphiuma means and applied qualitatively to evaluate the effects of extracellular Ca²⁺, K⁺ and pH on the membrane potential of human red cells. Increasing extracellular [Ca²⁺] from 1.8 to 15 mM causes a K⁺-dependent hyperpolarization and decrease in fluorescence intensity in Amphiuma red cells. Both the hyperpolarization and fluorescence change disappear when the temperature is raised from 17 to 37°C. No change in fluorescence intensity is observed in human red cells with comparable increase in extracellular Ca²⁺ in the temperature range 5–37°C. Increasing the extracellular pH, however, causes human red cells to respond to an increase in extracellular Ca²⁺ with a significant but temporary loss in fluorescence intensity. This effect is blocked by EGTA, quinine or by increasing extracellular [K⁺], indicating that at elevated extracellular pH, human erythrocytes respond to an increase in extracellular Ca²⁺ with an opening of K⁺ channels and associated hyperpolarization of the plasma membrane.