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.     

Molecular characterization of the in situ red cell membrane calcium pump by limited proteolysis

In inside-out red cell membrane vesicles active calcium transport and the formation of the enzyme-phosphate complex (EP) of the calcium pump were simultaneously investigated and the effects of a limited proteolytic digestion examined. In order to visualize the proteolyzed EP forms we have induced the formation of a maximum level EP from [gamma-32P]ATP in the presence of Ca2+ + La3+ and applied a good-resolution acidic discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. Proteolysis of inside-out vesicle membranes by trypsin, Pronase, papain, or chymotrypsin produces a calmodulin-like activation of the calcium pump, abolishes its calmodulin sensitivity, and decreases the original 140-kDa EP complex to a limit polypeptide of 80 kDa. Trypsin digestion produces another major intermediary fragment of 90 kDa, which is still a low-activity calmodulin-sensitive form of the pump. The red cell calcium pump is activated by trypsin both in the absence and presence of Ca2+ during digestion although the rate of activation and the appearance of the 80-kDa polypeptide are enhanced by Ca2+. If proteolytic digestion is carried out by chymotrypsin, a calmodulin-insensitive maximum activation of the calcium pump coincides with the formation of a 125-130-kDa EP-forming polypeptide. Chymotrypsin and carboxypeptidase A have synergistic effects on the formation of this latter high-activity species. Based on these data we suggest a probable molecular arrangement for the functional parts of the red cell membrane calcium pump.     

Dependence of the red blood cell calcium pump on the membrane potential

(1) It is shown that the rate of calcium extrusion from intact human red cells is faster at a membrane potential of approximately +50 mV (inside) than at approximately -50 mV. (2) The positive potential applied was the chloride potential of KCl cells in a K-gluconate medium when the Ca2+ sensitive K+ channel was blocked by 0.3mM quinidine. The negative potential resulted from the high K+ permeability in Ca2+ loaded cells (the cells were loaded to a Ca2+ activity in the cell water of about 50 microM). (3) It is further demonstrated that the Ca2+ affinity of the pump ATPase is decreased both at the internal (high affinity) and external (low affinity) site by increasing the proton concentration. Acidification thus inhibits internally and stimulates externally. (4) An indirect effect of the membrane potential on the pump activity via the accompanying pH shifts on either side of the membrane could be ruled out by choosing Ca2+ concentrations which are fully activating at the internal Ca2+ binding site at pH 6.5 and not yet inhibitory at the external Ca2+ binding site at pH 8. (5) The result is compatible with the assumption that the human red cell Ca-pump is exchanging Ca2+ for protons, yet is electrogenic by virtue of a stoichiometry of 1H+:1Ca2+ for this exchange.     

Inhibition of the human erythrocyte calcium pump by dimethyl sulfoxide

The action of dimethyl sulfoxide on the human red cell Ca2+ pump was studied in inside-out vesicles. In a high-K+ medium at pH 7.6, the organic solvent inhibited both Ca2+ transport and ATP hydrolysis. Half-maximal effect was obtained with about 2% (v/v). At or below 10% dimethyl sulfoxide, the inhibition was overcome by adding inorganic phosphate or oxalate. In the absence of organic solvent, Ca2+ efflux from Ca(2+)-loaded vesicles consisted of a slow and a fast component whilst in its presence, there appears additionally a leakage component. The size of the latter depended markedly on dimethyl sulfoxide concentration, being about 3% at that level where Ca2+ uptake was half-maximally inhibited. ATP hydrolysis was more sensitive to dimethyl sulfoxide (10%) when free Ca2+ was increased within the millimolar level than when it was raised within the micromolar range. On the other hand, raising Ca2+ with organic solvent greatly stimulated ATP synthesis through ATP-Pi exchange, without reaching saturation. The results suggest that dimethyl sulfoxide blocks the red cell Ca2+ pump by increasing the affinity of the Ca2+ translocating site at the releasing step. They also show that at high concentrations, this solvent increases Ca2+ permeability.     

Uncoupling the red cell sodium pump by proteolysis

In situ proteolysis of Na,K-ATPase was studied using inside-out red cell membrane vesicles. Proteolysis of the enzyme in its "E1" conformation with either trypsin or chymotrypsin inactivated cation translocation more than ATP hydrolysis. This was evident both in the absence of intravesicular alkali cations when Na-ATPase was compared to ATP-dependent 22Na+ influx, and in the presence of K+ when Na+/K+ exchange was compared to (Na+ + K+)-activated ATPase. This differential loss in pump versus hydrolysis was observed also when the activities of only intact, non-leaky vesicles were compared and therefore reflects intramolecular uncoupling rather than nonspecific leakage. Although oligomycin and thimerosal, like trypsin and chymotrypsin, inhibit the enzyme's conformational step(s), neither effect uncoupling. It is concluded that specific cleavage(s) of Na,K-ATPase, at least as it exists in situ, alters the reaction sequence with respect to the normal ordered mechanism. Accordingly, cytoplasmic Na+ and extracellular K+ bind to the enzyme, stimulate phosphorylation (ATP + E1----E1P + ADP) and dephosphorylation (E2P----E2 + Pi), respectively, but each is then released to the same side from which it had bound; presumably release occurs prior to the conformational transitions of E1P to E2P and E2 to E1. This conclusion is supported by experiments showing that, ar micromolar ATP concentration, the hydrolytic activity (Na-ATPase) of the trypsinized but not the unmodified enzyme is stimulated by K+, consistent with earlier experiments (Hegyvary, C., and Post, R. L. (1971) J. Biol. Chem. 246, 5234-5240) showing that the K X E2 to K X E1 transition is slower than the E2 to E1 transition.     

The maximal velocity and the calcium affinity of the red cell calcium pump may be regulated independently

The kinetics of active Ca2+ transport in inside-out red cell membrane vesicles and the Ca2+-ATPase activity of the purified Ca2+ pump were studied and the effects of calmodulin, acidic phospholipids, and controlled trypsinization were compared. In the presence of calmodulin the maximal rate and the apparent affinity of the pump for Ca2+ were greatly increased in both preparations. The lowest value of Km(Ca) was between 0.5 and 0.7 microM depending on the concentration of calmodulin and on the enzyme preparation. Positive cooperativity for Ca2+ activation with a Hill coefficient of 1.6-1.7 was observed in all cases. When acidic phospholipids (phosphatidylinositol 4-phosphate was routinely used) were added to the inside-out vesicles or to the purified enzyme, maximal transport rates equal to those obtained with calmodulin were measured but the Km(Ca) decreased to 0.25 microM and the positive cooperativity disappeared (the Hill coefficient approached 1). Highly active, calmodulin-independent proteolytic fragments of molecular mass of 81 and 76 kDa were produced with controlled trypsinization. When the trypsin treatment was directed to obtain primarily the 81-kDa fragment, the preparation showed characteristics similar to those of the intact Ca2+ pump in the presence of calmodulin; that is, the same Vmax was obtained, the Km(Ca2+) was 0.5-0.6 microM, and the Hill coefficient was about 1.6. Addition of phosphatidylinositol 4-phosphate or allowing further proteolysis to produce the 76-kDa fragment, shifted the Km(Ca) to 0.25 and reduced the Hill coefficient to 1, without changes in the maximal rate. Based on these results it is suggested that the maximal velocity and the Ca2+ affinity on the erythrocyte Ca2+ pump may be regulated independently and that independent polypeptide regions of the enzyme are involved in the regulations.     

The membrane topology of the amino-terminal domain of the red cell calcium pump.

A systematic study of the membrane-associated regions in the plasma membrane Ca2+ pump of erythrocytes has been performed by hydrophobic photolabeling. Purified Ca2+ pump was labeled with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazirine ([125I]TID), a generic photoactivatable hydrophobic probe. These results were compared with the enzyme labeled with a strictly membrane-bound probe, [3H]bis-phosphatidylethanolamine (trifluoromethyl) phenyldiazirine. A significant light-dependent labeling of an M(r) 135,000-140,000 peptide, corresponding to the full Ca2+ pump, was observed with both probes. After proteolysis of the pump labeled with each probe and isolation of fragments by SDS-PAGE, a common pattern of labeled peptides was observed. Similarly, labeling of the Ca2+ pump with [125I]TID, either in isolated red blood cell membranes or after the enzyme was purified, yields a similar pattern of labeled peptides. Taken together, these results validate the use of either probe to study the lipid interface of the membrane-embedded region of this protein, and sustain the notion that the conformation of the pump is maintained throughout the procedures of solubilization, affinity purification, and reconstitution into proteoliposomes. In this work, we put special emphasis on a detailed analysis of the N-terminal domain of the Ca2+ pump. A labeled peptide of M(r) 40,000 belonging to this region was purified and further digested with V8 protease. The specific incorporation of [125I]TID to proteolytic fragments pertaining to the amino-terminal region indicates the existence of two transmembrane stretches in this domain. A theoretical analysis based on the amino acid sequence 1-322 predicts two segments with high probability of membrane insertion, in agreement with the experimental data. Each segment shows a periodicity pattern of hydrophobicity and variability compatible with alpha-helical structure. These results strongly suggest the existence of a transmembrane helical hairpin motif near the N-terminus of the Ca2+ pump.     

Inhibition by glucagon of the calcium pump in liver plasma membranes

The ATP-dependent calcium transport in plasma membrane vesicles prepared from rat liver was inhibited by 0.1 to 10 microM glucagon. Inhibition of the high affinity (Ca2+-Mg2+)-ATPase was observed concomitantly. This effect was neither mimicked by cyclic AMP nor by dibutyryl cyclic AMP. A study of the structure-activity relationships of six glucagon derivatives demonstrated the specificity of glucagon action since only one or two analogs markedly altered the (Ca2+-Mg2+)-ATPase activity. The study also demonstrated the total absence of correlation between adenylate cyclase activation and (Ca2+-Mg2+)-ATPase inhibition induced by these glucagon derivatives. The decrease in the maximal velocities induced by glucagon of both calcium transport and (Ca2+-Mg2+)-ATPase activity were related to a reduction in the rate of dephosphorylation of the Ca-dependent phosphorylated intermediate of the enzyme. This phosphorylated intermediate was characterized as a 32P-labeled 110,000-dalton protein which accumulated to 50 to 150% over the basal level in the presence of glucagon. The present results demonstrate a novel aspect of the role of glucagon as a calcium-mobilizing agent.     

Inhibition of the phosphatase activity of the red cell membrane Ca2+ pump by acidic phospholipids.

The effect of phospholipids was tested on the p-nitrophenylphosphatase activity of the Ca2+ pump. Acidic phospholipids like phosphatidylserine and phosphatidylinositol inhibited the phosphatase activity, while neutral phospholipids like phosphatidylcholine did not. This result contrasts sharply with the known activating effect of acidic phospholipids on the Ca2(+)-ATPase activity of the pump. It is known that the phosphatase activity of the Ca2+ pump can be elicited either by calmodulin and Ca2+ or by ATP and Ca2+. Unlike calmodulin, acidic phospholipids failed to stimulate the phosphatase activity. Furthermore, calmodulin-activated phosphatase was completely inhibited by acidic phospholipids. Maximal inhibition of the ATP-activated phosphatase was only 70%. Inhibition by acidic phospholipids was non-competitive regarding to calmodulin, suggesting that acidic phospholipids and calmodulin do not bind to the same domain of the pump. The presence of Ca2+ was essential for the inhibition, and the apparent affinity for Ca2+ for this effect was increased by acidic phospholipids. Results are consistent with the idea that acidic phospholipids stabilize an enzyme-Ca complex lacking phosphatase activity.     

Activation of the human red cell calcium ATPase by calcium pretreatment

Some kinetic parameters of the human red cell Ca²⁺-ATPase were studied on calmodulin-free membrane fragments following preincubation at 37°C. After 30 min treatment with EGTA(1 mm) plus dithioerythritol (1 mm), a V _max of about 0.4 μmol P_i/mg × hr and a K _s of 0.3 μm Ca²⁺ were found. When Mg²⁺ (10 mm) or Ca²⁺(10 μm) were also added during preincubation, V _maxbut not Kwas altered. Ca²⁺ was more effective than Mg²⁺, thus increasing V _max to about 1.3 μmol P_i/mg × hr. The presence of both Ca²⁺ and Mg²⁺ during pretreatment decreasedKto 0.15 μm, while having no apparent effect on V _max. Conversely, addition of ATP (2 mm) with either Ca²⁺ or Ca²⁺ plus Mg²⁺increased V_max without affecting K. Preincubation with Ca²⁺ for periods longer than 30 min further increased V_maxand reduced Kto levels as low as found with calmodulin treatment. The Ca²⁺ activation was not prevented by adding proteinase inhibitors (iodoacetamide, 10 mm; leupeptin, 200 μm; pepstatinA, 100 μm; phenylmethanesulfonyl fluoride, 100 μm). The electrophoretic pattern of membranes preincubated with or without Mg²⁺, Ca²⁺ or Ca²⁺ plus Mg²⁺ did not differ significantly from each other. Moreover, immunodetection of Ca²⁺-ATPase by means of polyclonal antibodiesrevealed no mobility change after the various treatments. The above stimulation was not altered by neomycin (200 μm), washing with EGTA (5 mm) or by both incubating and washing with delipidized serum albumin (1 mg/ml), or omitting dithioerythritol from the preincubation medium. On the other hand, the activation elicited by Ca²⁺ plus ATP in the presence of Mg²⁺ was reduced 25–30% by acridine orange (100 μm), compound 48/80 (100 μm) or leupeptin (200 μm) but not by dithio-bis-nitrobenzoic acid (1 mm). The fluorescence depolarization of 1,6-diphenyl-and l-(4-trimethylammonium phenyl)-6-phenyl 1,3,5-hexatriene incorporated into membrane fragments was not affected after preincubating under the different conditions. The results show that proteolysis, fatty acid production, an increased phospholipid metabolism or alteration of membrane fluidity are not involved in the Ca²⁺ effect. Ca²⁺ preincubation may stimulate the Ca²⁺-ATPase activity by stabilizing or promoting the E₁ conformation.     

Inhibition of red cell Ca-ATPase by vanadate

1. 1. The Mg²⁺- plus Ca²⁺-dependent ATPase (Ca²⁺-ATPase) in human red cell membranes is susceptible to inhibition by low concentrations of vanadate.

2. 2. Several natural activators of Ca²⁺-ATPase (Mg²⁺, K⁺, Na⁺ and calmodulin) modify inhibition by increasing the apparent affinity of the enzyme for vanadate.

3. 3. Among the ligands tested, K⁺, in combination with Mg²⁺, had the most pronounced effect on inhibition by vanadate.

4. 4. Under conditions optimal for inhibition of Ca²⁺-ATPase, the K for vanadate was 1.5 μM and inhibition was nearly complete at saturating vanadate concentrations.

5. 5. There are similarities between the kinetics of inhibition of red cell Ca²⁺-ATPase and (Na⁺ + K⁺)-ATPase prepared from a variety of sources; however, (Na⁺ + K⁺)-ATPase is approx. 3 times more sensitive to inhibition by vanadate.

Conformational changes of the in situ red cell membrane calcium pump affect its proteolysis

In inside-out red cell membrane vesicles trypsin digestion reduces the molecular mass of the 32P-labeled acyl-phosphate intermediate of the calcium pump from the original 140 kDa to about 80 kDa with a simultaneous activation of the calcium uptake. This process is slightly stimulated by the presence of calcium, as compared to EGTA, or EGTA + vanadate, but the proteolytic pattern is similar under all these conditions. However, trypsin degradation of the 80 kDa polypeptide, resulting in the loss of calcium transport activity and 32P-phosphoenzyme formation, is rapid in the presence of calcium, inhibited by EGTA and almost fully blocked by EGTA + vanadate. In the presence of these latter ligands, probably locking the calcium pump in an E2 conformation, the 80 kDa protein becomes insensitive even to excessive digestion by the non-specific protease, pronase. The data indicate major changes in the molecular arrangement of the calcium pump protein when transformed from a calcium-liganded (E1) to an E2 conformation.     

Inhibition by quercetin of thyroid hormone stimulation in vitro of human red blood cell Ca2+-ATPase activity

Human red blood cell membrane Ca2+-ATPase activity is stimulated in vitro by physiological concentrations of thyroid hormone. Quercetin, a flavonoid that inhibits several membrane-linked ATPases, suppressed thyroid hormone action on red cell Ca2+-ATPase activity and also interfered with binding of the hormone by red cell membranes. These effects of quercetin were dose-dependent over a range of concentrations (1-50 microM). In contrast, in the absence of thyroid hormone, quercetin at low concentrations stimulated Ca2+-ATPase activity and at 50 microM inhibited the enzyme. The effects of quercetin at low concentrations (1-10 microM), namely, stimulation of Ca2+-ATPase and inhibition of membrane-binding of thyroid hormone, mimic those of thyroid hormone and are consistent with the thyronine-like structure of quercetin. At high concentrations, quercetin is generally inhibitory of Ca2+-ATPase activity. Chalcone, fisetin, hesperetin and tangeretin are other flavonoids shown to reduce susceptibility of membrane Ca2+-ATPase to hormonal stimulation.     

Reversal of the calcium pump in human red cells

Summary Human red cells containing low ATP and high P_i concentrations were suspended in media with and without 2mm Ca²⁺, and the incorporation of (³²P)P_i into ATP was measured. There was some incorporation whatever the medium, but in every experiment there was an extra incorporation when the cells were in the Ca²⁺-containing medium. This extra incorporation was abolished by the ionophore A23187, which collapses the Ca²⁺ concentration gradient across the membranes, or by LaCl₃, which blocks the Ca²⁺ pump. Starved and phosphate-loaded cells also show an uptake of Ca²⁺ which is not apparent in fresh cells. Results are consistent with the idea that Ca²⁺-dependent incorporation of P_i into ATP is catalyzed by the Ca²⁺ pump using energy derived from the Ca²⁺ concentration gradient.     

Metal-ATP complexes as substrates and free metal ions as activators of the red cell calcium pump

The plasma membrane calcium pump in most mammalian cells is the basic mechanism for assuring a low cytoplasmic calcium concentration. In inside-out human red cell membrane vesicles /IOVs/ the substrate and metal specificity as well as the intracellular protein /calmodulin/ regulation of the ATP-dependent active calcium transport can be investigated $\text{in}$$\text{situ}$. In this paper we demonstrate that Me²⁺. ATP^4? /in the following MeATP/ complexes, including MgATP, MnATP, CoATP, FeATP, and NiATP, can serve as substrates for the calcium pump in IOVs. Calcium pumping is activated by the above metals, while Sr, Ba, Cu, Cd ions or the trivalent cations are ineffective in this respect. Calmodulin-stimulation of the calcium transport is present independent of the metal ions used for the activation of the pump. Based on kinetic studies we suggest that divalent metal ions interact with the red cell calcium pump at four different sites: 1./ MeATP complex is the true substrate of the pump; 2./ Ca or Sr ions activate the system by binding to the transport site/s/ and other metal ions competitively inhibit this binding; 3./ the presence of free divalent metal ions /Mg, Mn, Co, Fe, or Ni, but not Ca, Sr, Ba/ is required for activating calcium translocation; 4./ interaction with a Ca — calmodulin complex specifically stimulates calcium pumping.     

Hysteretic activation of the Ca pump revealed by calcium transients in human red cells

The enzymatic basis for the Ca²⁺ pump in human red cells is an ATPase with hysteretic properties. The Ca²⁺-ATPase shifts slowly between a ground state deficient in calmodulin and an active state saturated with calmodulin, and rate constants for the reversible shifts of state were recently determined at different Ca²⁺ concentrations (Scharff, O. and Foder, B. (1982) Biochim. Biophys. Acta 691, 133–143). In order to study whether the Ca²⁺ pump in intact red cells also exhibits hysteretic properties we have analysed transient increases of intracellular calcium concentrations (Ca_i), induced by the divalent cation ionophore A23187. The time-dependent changes of Ca_i were measured by use of radioactive calcium (⁴⁵Ca²⁺) and analysed with the aid of a mathematical model, based partly on the Ca²⁺-dependent parameters obtained from Ca²⁺-ATPase experiments, partly on the A23187-induced Ca²⁺ fluxes determined in experiments with intact red cells. According to the model a delay in the activation of the Ca²⁺ pump is a prerequisite for the occurrence of A23187-induced calcium transients in the red cells, and we conclude that the Ca²⁺ pump in human red cells responds hysteretically. It is suggested that Ca²⁺ pumps in other types of cell also have hysteretic properties.     

Activation by calcium of AMP deaminase from the human red cell

We have investigated the effects of Ca2+ on AMP deaminase from human red cells. At variance with the other known modulators, Ca2+ increased the apparent affinity for AMP without modifying the characteristic positive cooperativity of the enzyme towards the substrate. Ca2+ sensitivity was not modified by dialysis, but dilution of the haemolysate produced an activation of the enzyme similar to that induced by Ca2+. Simultaneously, the Ca2+ dependence was lost. The sensitivity to other modulators, such as ATP, diphosphoglycerate or phosphate, was not modified by dilution. Partial purification of the enzyme produced the same effects as haemolysate dilution. These results may be interpreted to mean that Ca2+ acts by antagonizing an endogenous inhibitor present in red cell lysates.     

Molecular properties of the red cell calcium pump: I. Effects of calmodulin,proteolytic digestion and drugs on the kinetics of active calcium uptake in inside-out red cell membrane vesicles

In calmodulin-stripped inside-out human red cell membrane vesicles /IOV/ ATP + Mg²⁺-dependent active calcium uptake is stimulated by the addition of calmodulin. Calmodulin increases the maximum calcium transport rate /V_max/, decreases K_Ca, and does not affect K_ATP of calcium uptake. The action of both membrane bound and external calmodulin is competitively inhibited by phenothiazines. Drugs reacting with SH groups of proteins reversibly inhibit calcium pumping by decreasing V_max and not affecting K_Ca and K_ATP. The relative magnitude of calmodulin stimulation of calcium transport is unaltered by SH reagents.Mild proteolytic digestion of IOVs stimulates active calcium uptake and mimics the effects of calmodulin on the kinetic parameters — that is converts the system to a “high calcium-affinity” state. Proteolysis eliminates calcium-dependent calmodulin binding to IOV membranes and any further stimulation of calcium uptake by calmodulin. Based on these results the presence of a calmodulin-binding regulatory subunit of the red cell calcium pump at the internal membrane surface is postulated.