Inside-out Red Cell Membrane Vesicles Research Articles

Characterization of the pH dependence of hemoglobin binding to band 3: Evidence for a pH-dependent conformational change within the hemoglobin-band 3 complex

The pH dependence of hemoglobin binding to inside-out red cell membrane vesicles was studied using 90° light scattering (Salhany, J.M. et al., Biochemistry 19 (1980) 1447–1454). Hyperbolic binding curves were observed for high-affinity hemoglobin binding to the cytoplasmic domain of band 3 (CDB3) within the intact transporter. Analysis of these saturation curves yielded the apparent K d and the maximum light scattering signal change (ΔLS max ). The apparent K d for hemoglobin binding did not change substantially between pH 5.5 and 7.0, while at pH 8, there is no binding. In contrast, ΔLS max decreased by about 20-fold between pH 5.5 and 7.0, with an apparent p K of 6.5. These results suggest that hemoglobin binds to CDB3 with high affinity at both neutral and acid pH, a suggestion that was confirmed using a centrifugation method. Thus, the p K for the light scattering signal change is significantly lower than the p K for the actual binding process. We show that the change in ΔLS max is not related to a change in band 3 binding capacity, which remains constant as a function of pH. We also show that hemoglobin binding to non-band 3 sites contributes less than 10% to ΔLS max under our specific experimental conditions. On the basis of these and previous fluorescence quenching results in the literature, we propose a new model for hemoglobin binding to band 3, where raising the pH from 6 and 7 causes the CDB3–hemoglobin complex to undergo a conformational change leading to the movement of the bound hemoglobin away from the surface of the bilayer. The possible implication of this new mechanistic interpretation is discussed briefly.

Effects of acylphosphatase on the activity of erythrocyte membrane Ca2+ pump

Acylphosphatase, purified from human erythrocytes, actively hydrolyzes the acylphosphorylated intermediate of human red blood cell membrane Ca(2+)-ATPase. This effect occurred with acylphosphatase amounts (up to 10 units/mg membrane protein) that fall within the physiological range. Furthermore, a very low Km value, 3.41 +/- 1.16 (S.E.) nM, suggests a high affinity in acylphosphatase for the phosphoenzyme intermediate, which is consistent with the small number of Ca(2+)-ATPase units in human erythrocyte membrane. Acylphosphatase addition to red cell membranes resulted in a significant increase in the rate of ATP hydrolysis. Maximal stimulation (about 2-fold over basal) was obtained at 2 units/mg membrane protein, with a concomitant decrease in apparent Km values for both Ca2+ and ATP. Conversely, similar amounts of acylphosphatase significantly decreased (by about 30%) the rate of Ca2+ transport into inside-out red cell membrane vesicles, albeit that reduced apparent Km values for Ca2+ and ATP were also observed in this case. A stoichiometry of 2.04 Ca2+/ATP hydrolyzed was calculated in the absence of acylphosphatase; in the presence of acylphosphatase optimal concentration, this ratio was reduced to 0.9. Acylphosphatase activity, rather than just protein, was essential for all the above effects. Taken together these findings suggest that, because of its hydrolytic activity on the phosphoenzyme intermediate, acylphosphatase reduces the efficiency of the erythrocyte membrane Ca2+ pump. A possible mechanism for this effect is that the phosphoenzyme is hydrolyzed before its transport work can be accomplished.

The effect of [formula omitted], [formula omitted] and [formula omitted], [formula omitted] cellular aging on the active calcium transport in human inside-out red cell membrane vesicles

Modelling of the in , vivo and in , vitro aging processes in the human red cell has stressed the following features of the active calcium uptake by inside-out vesicles: 1) it is higher in the outdated, in , vitro aged, than in the fresh red cell (p<0.0005), and in the densest, in , vivo aged fraction than in the lightest, young fraction (p=0.08); 2) it increases following stimulation by excess calmodulin to values that are not significantly different; 3) it decreases to the same value in the absence of endogenous calmodulin and inhibitor, with and without exogenous calmodulin; 4) it is the target of a non-competitive inhibition, that is stronger in the fresh than in the outdated red cell. We conclude that the aging process does not involve neither membrane Ca-ATPase nor calmodulin, but rather the interaction of the calcium pump with the inhibitor of Ca-ATPase.

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 E 2 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 (E 1) to an E 2 conformation.

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.

ATP regulation of the human red cell sugar transporter.

Purified human red blood cell sugar transport protein intrinsic tryptophan fluorescence is quenched by D-glucose and 4,6-ethylidene glucose (sugars that bind to the transport), phloretin and cytochalasin B (transport inhibitors), and ATP. Cytochalasin B-induced quenching is a simple saturable phenomenon with Kd app of 0.15 microM and maximum capacity of 0.85 cytochalasin B binding sites per transporter. Sugar-induced quenching consists of two saturable components characterized by low and high Kd app binding parameters. These binding sites appear to correspond to influx and efflux transport sites, respectively, and coexist within the transporter molecule. ATP-induced quenching is also a simple saturable process with Kd app of 50 microM. Indirect estimates suggest that the ratio of ATP-binding sites per transporter is 0.87:1. ATP reduces the low Kd app and increases the high Kd app for sugar-induced fluorescence quenching. This effect is half-maximal at 45 microM ATP. ATP produces a 4-fold reduction in Km and 2.4-fold reduction in Vmax for cytochalasin B-inhibitable D-glucose efflux from inside-out red cell membrane vesicles (IOVs). This effect on transport is half-maximal at 45 microM ATP. AMP, ADP, alpha, beta-methyleneadenosine 5'-triphosphate, and beta, gamma-methyleneadenosine 5'-triphosphate at 1 mM are without effect on efflux of D-glucose from IOVs. ATP modulation of Km for D-glucose efflux from IOVs is immediate in onset and recovery. ATP inhibition of Vmax for D-glucose exit is complete within 5-15 min and is only partly reversed following 30-min incubation in ATP-free medium. These findings suggest that the human red cell sugar transport protein contains a nucleotide-binding site(s) through which ATP modifies the catalytic properties of the transporter.

Direct evidence for ATP modulation of sugar transport in human erythrocyte ghosts.

Sugar transport in human erythrocyte ghosts is modulated by low molecular weight factors present in red cell cytosol that induce an asymmetry in Michaelis and velocity constants for sugar entry and exit (Carruthers, A., and Melchior, D. L. (1983) Biochim. Biophys. Acta 728, 254-266). This study examines the possibility that ATP is the transport-modulating factor. The intracellular factor must satisfy at least three criteria. It must reduce Km and Vmax for sugar efflux from inside-out red cell membrane vesicles. It should increase Km for efflux from red cell ghosts. It should have a molecular weight of less than 10 kDa. These criteria are satisfied by ATP. AMP, ADP, GTP, UTP, and ITP are without effect on sugar transport. The following results support the view that the cytosolic factor is ATP. Red cell lysate (obtained by hypotonic lysis of red cells) is unable to modify transport following dialysis against ATP-free medium. The ability of lysate to modify transport is retained following acid extraction. ATP depletion of acid-extracted lysate by treatment with apyrase results in the loss of transport-modulating potency. Myokinase partly restores both the ATP content and the ability of ATP-depleted (apyrase-treated) lysate to modify transport. Addition of ATP to ATP-depleted lysate mimics the ability to myokinase to restore the transport-modulating potency of lysate. ATP is without effect on the number and molecular size of D-glucose-sensitive cytochalasin B-binding proteins in the red cell membrane. These findings demonstrate that the transport-modulating potency of red cell cytosol is quantitatively accounted for by intracellular ATP which acts to modify the catalytic activity of plasmalemmal transporters.

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.

Ca 2+ transport and permeability in inside-out red cell membrane vesicles after freezing

To evaluate the effects of freezing and thawing on Ca 2+ transport and permeability, inside-out red cell membrane vesicles (IORCMV) are examined. Exposure to the cryoprotectant Me 2SO as well as different cooling regimes on unprotected and cryoprotected vesicles do not affect the membrane Ca 2+ transport. However, freezing and thawing increase the membrane permeability to sucrose.

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.

Mode of interaction of phosphofructokinase with the erythrocyte membrane.

Phosphofructokinase is known to associate with the human erythrocyte membrane both in vitro and in vivo. Such association activates the enzyme in vitro by relieving the allosteric inhibition imposed by ATP (Karadsheh, N.S., and Uyeda, K. (1977) J. Biol. Chem. 252, 7418-7420). We now demonstrate that ADP, ATP, and NADH, all of which are known to bind to the enzyme's adenine nucleotide activation site, are particularly potent in eluting the enzyme from the membrane. In addition, both inside-out red cell membrane vesicles and a 23-kDa fragment containing the amino terminus of the membrane protein, band 3, cause a slow, partial, and reversible inactivation of phosphofructokinase. The dependence of the residual phosphofructokinase activity on phosphofructokinase concentration demonstrates that inactivation occurs through the dissociation of active tetramers to inactive dimers. Dimers of phosphofructokinase associate with the membrane more avidly than tetramers. The kinetics of phosphofructokinase inactivation are consistent with the dissociation of tetramers in solution followed by the binding of dimers to the membrane. There is no indication of an association equilibrium between tetramers and dimers of phosphofructokinase bound to the membrane. Taken together, these results suggest that the amino-terminal segment of band 3 binds to the adenine nucleotide activation site, which is thought to be located in a cleft between the dimeric subunits of phosphofructokinase. As a result, band 3 not only rapidly activates the phosphofructokinase tetramer but also slowly inactivates the enzyme by preferentially binding its dissociated subunits.

Proton-activated rubidium transport catalyzed by the sodium pump.

Although the sodium pump normally exchanges three sodium for two potassium ions, experiments with inside-out red cell membrane vesicles show that the stoichiometry is reduced when the cytoplasmic sodium concentration is decreased to less than 1 mM. The present study was designed to gain insight into the question whether other monovalent cations, particularly protons, can act as sodium congeners in effecting pump-mediated potassium transport (ATP-dependent rubidium efflux from inside-out vesicles). The results show that at low cytoplasmic sodium concentration, an increase in proton concentration effects a further reduction in sodium:rubidium stoichiometry, to a value less than the minimal expected (1Na+:3Rb+). Furthermore, when vesicles containing 86RbCl are incubated in nominally sodium-free medium. ATP-dependent net rubidium efflux (normal influx) occurs when the pH is reduced from approximately 7.0 to 6.2 or less. This efflux is inhibited by strophanthidin and vanadate. These experiments support the notion that the sodium pump can operate as an ATP-dependent proton-activated rubidium (potassium) pump without obligatory countertransport of sodium ions.

Sodium pump-catalyzed sodium-sodium exchange associated with ATP hydrolysis.

Inside-out red cell membrane vesicles have been used to study sodium interactions with the ATP-dependent sodium pump at sites accessible to both membrane surfaces. ATP-dependent 22Na+ influx (equivalent to efflux from cells) shows sigmoid dependence on extravesicular Na+ concentration. This is observed both in the absence of intravesicular cations and in the presence of intravesicular K or Rb ions. The kinetic behavior is similar to that observed earlier with intact cells, (Garay, R. P., and Garrahan, P. J. (1973) J. Physiol. (Lond.) 231, 297-325) and is consistent with a ratio of close to three Na ions transported per molecule of ATP hydrolyzed. With vesicles having relatively high intravesicular sodium concentration, (approximately 50 mM NaCl), the sodium pump effects an ATP-dependent sodium efflux coupled to sodium influx and to strophanthidin-sensitive ATP hydrolysis. The influx:efflux stoichiometry is approximately 1:1, and the influx:ATP hydrolysis ratio is close to 3. This ATP-dependent exchange has a higher affinity for vanadate than ATP plus ADP-dependent sodium exchange. It is concluded that this sodium-sodium exchange mode resembles sodium-potassium exchange whereby intravesicular sodium, i.e. sodium at the extracellular surface, at relatively high concentration, behaves like potassium.

Effects of divalent metal ions on the calcium pump and membrane phosphorylation in human red cells

In inside-out red cell membrane vesicles ATP-dependent calcium transport is activated by the divalent metal ions Mg 2+, Mn 2+, Co 2+, Ni 2+ and Fe 2+. This activation is based on the formation of Me 2+-ATP complexes which can serve as energy-donor substrates for the calcium pump, and probably, satisfy the requirement for free Me 2+ in this transport process. Higher Me 2+ concentrations inhibit calcium transport with various efficiencies. Mn 2+ directly competes with Ca 2+ at the transport site, while other divalent metal ions investigated have no such effect. The formation of the hydroxylamine-sensitive phosphorylated intermediate (EP) of the red cell membrane calcium pump from [γ- 32P]ATP is induced by Ca 2+ while rapid dephosphorylation requires the presence of Mg 2+. At higher concentrations Mn 2+ and Ni 2+ inhibit predominantly the formation of EP, while Co 2+ and Fe 2+ block dephosphorylation. The possible sites and nature of the divalent metal interactions with the red cell calcium pump are discussed. Hydroxylamine-insensitive membrane phosphorylation in inside-out vesicles from [γ- 32P]ATP is significantly stimulated by Mn 2+ and Co 2+, as compared to that produced by Mg 2+, Fe 2+ and Ni 2+. Part of this labelling is found in phospholipids, especially in phosphatidylinositol. The results presented for the metal dependency of protein and lipid phosphorylation in red cell membranes may help in the characterization of ATP consumptions directly related to the calcium pump and those involved in various regulatory processes.

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 + Mg 2+-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.

Molecular properties of the red cell calcium pump: II. Effects of calmodulin, proteolytic digestion and drugs on the calcium-induced membrane phosphorylation by ATP in inside-out red cell membrane vesicles

In inside-out human red cell membrane vesicles /IOV/, in the absence of Mg 2+, the only calcium-induced labelling by γ 32P-ATP occurs in a 140–150 000 molecular weight protein fraction, representing the hydroxylamine-sensitive phosphorylated intermediate /EP/ of the calcium pump. In the presence of Mg 2+ calcium-induced phosphorylation is accelerated but several other membrane proteins are also phosphorylated through protein kinase action forming hydroxylamine-insensitive bonds. Addition of calmodulin accelerates EP formation both in the absence and presence of Mg 2+. Treatment of the membrane with SH-group reagents significantly reduces EP formation. Mild trypsin digestion of IOVs, stimulating active calcium transport, eliminates calmodulin action and decreases the steady-state level of EP. In trypsin-digested IOVs the molecular weight of the 32P-labelled EP is shifted to lower values /110–120 000/ We suggest that trypsin digestion cleaves off a 20–40 000 molecular weight calmodulin-binding regulatory subunit of the calcium pump molecule.

Interactions of K+ with (Na,K)-ATPase orientation of K+-phosphatase sites studied with inside-out red cell membrane vesicles.

Inside-out membrane vesicles from human red cells were used to investigate the side specificity of K+ interactions with the K+-activated phosphatase, a partial reaction of the (Na, K)-ATPase. In the absence of Na+ and ATP, K+ at moderate affinity sites at the extravesicular surface (cytoplasmic K+) stimulates activity, whereas intravesicular K+ (K+ normally at the extracellular surface) is without effect. In contrast, under conditions of phosphorylation of (Na, K)-ATPase (Na+ and ATP present), K+ ions acting at high affinity sites at both surfaces are required. It is concluded that an enzyme x K complex is involved in K+-activated phosphatase activity and that it is formed either by interaction of cytoplasmic K+ with the dephosphoenzyme, or as a consequence of extracellular K+ binding and dephosphorylation of the phosphoenzyme formed in the presence of Na+ plus ATP.

Red cell sodium fluxes catalysed by the sodium pump in the absence of K+ and ADP.

In the absence of extracellular Na+ or K+, the sodium pump catalyses an ouabain-sensitive "uncoupled" Na+ efflux1-4. With red cell ghosts Glynn and Karlish5 showed that this Na+ efflux is accompanied by ATP hydrolysis and that extracellular sodium ions, at low concentrations, inhibit this efflux as well as the associated ATP hydrolysis. At higher concentrations, extracellular sodium ions restore the hydrolysis of ATP3,6 but it is not known whether there is an associated increase in Na+ efflux and, perhaps, an influx. To answer this question we have used inside-out red cell membrane vesicles which are specially suitable for controlling the composition of the medium at the two membrane surfaces while measuring 22Na+ fluxes in both directions. We report here that the sodium pump can operate in a mode in which influx and efflux of sodium are associated with ATP hydrolysis. This mode is different from the Na-Na exchange described by Garrahan and Glynn7, and Glynn and Hoffman8, which requires ADP as well as ATP9 and is probably associated with ADP-ATP exchage rather than ATP hydrolysis10,11.

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 Mg 2+-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 Mg 2+, 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.

Effects of calcium-EGTA buffers on active calcium transport in inside-out red cell membrane vesicles.

In inside-out red cell membrane vesicles, the free calcium concentration half-maximally stimulating active calcium uptake is about 2 orders of magnitude smaller in a calcium-EGTA buffer than in media containing unbuffered calcium. In calcium-EGTA buffer, the maximum rate of calcium uptake is determined by the total calcium concentration present. A possible model for explaining these findings is presented.