Phospholipid and calmodulin activation of solubilized calcium-transport ATPase from human erythrocytes: regulation by magnesium

The effect of phospholipids on Triton X-100 solubilized (Ca2+ + Mg2+)-ATPase from human erythrocyte membranes has been examined. The enzyme activity was increased by phosphatidylinositol, phosphatidylserine, and phosphatidic acid at both low (2 micrometer) and high (65 micrometer) free Ca2+ concentrations, while phosphatidylcholine had little effect and phosphatidylethanolamine and cardiolipin inhibited the (Ca2+ + Mg2+)-ATPase activity at all Ca2+ concentrations studied. The diacylglycerol, diolein, inhibited the enzyme at high, but not low, Ca2+ concentrations. Low concentrations of phospholipase A2 (1-2 international units) also activated the solubilized enzyme, at least in part by releasing free fatty acids, as the activation was mimicked by oleic acid (1-2 mumol/mg protein) and was abolished by fatty acid depleted bovine serum albumin. The combined activation by saturating levels of phosphatidylserine and calmodulin was additive at 6.5 mM MgCl2, and probably occurred at distinct sites on a regulatory component of the enzyme. The activation by both effectors was antagonized by MgCl2 at similar concentrations. Analysis of various models suggested that phosphatidylserine had two effects on (Ca2+ + Mg2+)-ATPase activity. First, a low Ca2+ affinity form of the enzyme was converted to a high Ca2+ affinity form, which was more sensitive to Ca2+ inhibition. Second, it increased the turnover of the enzyme, probably by enhancing its dephosphorylation, which was mimicked in this study by the Ca2+-dependent p-nitrophenylphosphatase partial reaction.     

Regulation of (Ca2+, Mg2+)-ATPase in human erythrocytes dependent on calcium and calmodulin

The enzymatic basis for active Ca2+ transport in erythrocytes, and probably in many other cells, is a Mg2+-dependent Ca2+-ATPase located in the plasma membrane. The Ca2+-ATPase can be prepared in two different forms, a calmodulin-deficient (A-state) and a calmodulin-saturated (B-state). The ATPase is regulated by Ca2+-, K+, and ATP, and these effectors interact in a cooperative way, especially in the B-state. Both A-state and B-state can be solubilized by treatment with Triton X-100. The detergent activates the A-state but reduces the cooperative interactions in both states. The membrane-bound A and B state contain 5 and 25 nmoles calmodulin/g membrane protein, respectively, and the amount of Ca2+-ATPase was estimated at 10--20 nmoles/g membrane protein. It is discussed whether both A and B state represent natives states of the pump-enzymes.     

Glucose transport inhibition in human erythrocytes by calmodulin antagonists

Calmodulin antagonists (tryphtazin, lidocaine, dykain, palmitate) inhibit glucose transport from human erythrocytes. Glucose efflux inhibition is proportional to the concentration of antagonists in the medium and is of uncompetitive character. It is accompanied by a decrease in the maximum transport rate with the unchanged constant of dissociation in the complex: carrier-sugar. Calcium ionophores A23187 and divaleryldibenzo-18-crown-6 eliminated the inhibiting effect of pharmacological agents on glucose transport. The authors think that the glucose transport inhibition under the influence of calmodulin antagonists may be realized through the calmodulin-dependent chain inhibition under the influence of calmodulin antagonists in the carbohydrate transport system.     

Actin-activated ATPase from human erythrocytes

A fibrillar protein complex, possessing ouabain-insensitive Ca²⁺-ATPase activity was isolated from human erythrocyte membranes by using a low ionic strength extraction procedure. Mg²⁺-ATPase activity was revealed upon addition of rabbit skeletal muscle actin, thus demonstrating the presence of a myosin-like protein in the crude extract of the erythrocyte membrane. Upon sodium dodecylsulfate gel electrophoresis, the extract showed mainly the doublet of subunit molecular weight bands of 230 000 and 210 000, and more than 10 faster moving hands. Gel filtration of the erythrocyte membrane extract on Sepharose 4B furnished 4 fractions. Fraction I, containing the doublet and 80 000, 60 000 and 46 000 subunit molecular weight bands was 5-fold purified with respect to Ca²⁺-ATPase activity, but was devoid of actin-activated Mg²⁺-ATPase activity. Fraction II, containing only the doublet, was devoid of Ca²⁺ and actin-activated Mg²⁺-ATPase activity. The 210 000 subunit molecular weight protein could be phosphorylated in the presence of Mg²⁺ in the crude extract and Fraction I but not in Fraction II.     

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.     

Actin-activated ATPase from human erythrocytes.

A fibrillar protein complex, possessing ouabain-insensitive Ca2+-ATPase activity was isolated from human erythrocyte membranes by using a low ionic strength extraction procedure. Mg2+-ATPase activity was revealed upon addition of rabbit skeletal muscle actin, thus demonstrating the presence of a myosin-like protein in the crude extract of the erythrocyte membrane. Upon sodium dodecylsulfate gel electrophoresis, the extract showed mainly the doublet of subunit molecular weight bands of 230 000 and 210 000, and more than 10 faster moving bands. Gel filtration of the erythrocyte membrane extract on Sepharose 4B furnished 4 fractions. Fraction I, containing the doublet and 80 000, 60 000 and 46 000 subunit molecular weight bands was 5-fold purified with respect to Ca2+-ATPase activity, but was devoid of actin-activated Mg2+-ATPase activity. Fraction II, containing only the doublet, was devoid of Ca2+ and actin-activated Mg2+-ATPase activity. The 210 000 subunit molecular weight protein could be phosphorylated in the presence of Mg2+ in the crude extract and Fraction I but not in Fraction II.     

Cell-cell affinity of senescent human erythrocytes

During their 120-day life span, human red blood cells (RBC) undergo several physicochemical changes, including an increased tendency to aggregate in plasma or polymer solutions. This study was designed to examine potential associations between age-related differences in RBC mobility, aggregation, and membrane glycocalyx properties for cells suspended in buffer and in 3 g/dl solutions of 70.3 kDa dextran. A recent model for depletion-mediated RBC aggregation was employed to calculate the changes of glycocalyx properties that were consistent with experimental electrophoretic mobility (EPM) and aggregation data. Young and old cells were obtained by density separation, after which aggregation and EPM were determined versus ionic strength; old cells exhibited a two- to threefold greater aggregation in dextran. EPM of old cells was identical to young cells in polymer-free media yet was 4% greater in dextran. The greater EPM for old RBC indicates a larger polymer depletion layer, which could be explained either by a 10-15% decrease of their glycocalyx thickness or a similar percentage decrease of polymer penetration into their glycocalyx. The larger depletion layer leads to markedly elevated cell-cell affinities for old cells, with the computed affinity increases consistent with enhanced old RBC aggregation. These results provide a rational explanation for the aggregation and EPM behavior of old RBC, and raise the possibility of depletion-mediated interactions contributing to senescent cell removal from the circulation.     

Effect of phosphorylation of calmodulin on calcium binding affinity as estimated by terbium fluorescence

The effect of phosphorylation of calmodulin by casein kinase 2 on the calcium binding of the former was studied by measurement of terbium fluorescence. The binding of Tb3+ to calmodulin was followed by an increase in Tb3+ fluorescence at 545 nm. The terbium fluorescence of phosphorylated calmodulin increased at a lower concentration of Tb3+ than that of non-phosphorylated calmodulin, indicating that Tb3+ binding affinity of calmodulin was increased by phosphorylation. Our results suggest that the interaction between calcium and binding domain becomes stronger by phosphorylation.     

Regulation by calcium and calmodulin of adenylate cyclase from rabbit intestinal epithelium

The effect of calcium on adenylate cyclase from rabbit small intestine has been studied using a particulate preparation obtained from isolated epithelial cells. Both basal and vasoactive intestinal peptide-stimulated activities were inhibited by calcium concentrations in the micromolar range. In the presence of calmodulin, a biphasic response was obtained. At low calcium concentration (4 X 10(-9)-6 X 10(-8) M) the enzyme was activated up to 50%. As the Ca2+ concentration was increased, the enzyme was concomitantly inhibited. Half-maximal inhibition of calmodulin-dependent activity was obtained at 1 microM free Ca2+. The activation of the enzyme was also dependent on the concentration of Mg2+. At less than 1 microM Ca2+, the enzyme exhibited a biphasic response, being activated at below 3 mM Mg2+ and inhibited at higher concentrations. At Ca2+ concentrations that were inhibitory, the enzyme did not show the biphasic response to Mg2+. At concentrations above 3 mM, the maximal rate (Vmax) remained constant. Vmax was inversely proportional to the concentration of Ca2+ present. Calmodulin altered Vmax when acting on vasoactive intestinal peptide-stimulated enzyme. Calmodulin had no effect on the Km for hormone activation. The calmodulin-dependent activity was inhibited by incubation with trifluoperazine.     

Impairment of the calcium pump of human erythrocytes by divicine

Divicine (2,6-diamino-4,5-dihydroxypyrimidine), an aglycone implicated in the pathogenesis of favism, produces a remarkable and consistent inactivation of the Ca2+-ATPase activity of the erythrocyte calcium pump. The patterns of inactivation are similar in normal and glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes. Inactivation of Ca2+-ATPase is apparently unrelated to the cellular GSH system, to the proteolytic machinery of mature erythrocytes, and to calmodulin, and also occurs in hemoglobin-free, unsealed erythrocytes membranes at 50-100 microM concentrations of divicine. Analysis of erythrocytes that have escaped destruction during the acute hemolytic crisis of a number of favic patients revealed a dramatic elevation of erythrocyte calcium and a significant decrease of Ca2+-ATPase activity. These results support the view that divicine plays a toxic role in the pathogenesis of favism and suggest that acute electrolyte imbalances, mostly affecting calcium homeostasis, are involved in the mechanisms of erythrocyte damage and destruction in this hemolytic disease.     

High affinity binding of the mastoparans by calmodulin

Calmodulin exhibits high affinity, calcium-dependent binding of the mastoparans — a group of cytoactive tetradecapeptides. The dissociation constants for the peptide-calmodulin complexes determined in 0.20 N KCl, 1.0 mM CaCl₂, pH 7.3 are ～0.3 nM for mastoparan, ～0.9 nM for mastoparan X, and ～3.5 nM for $\text{Polistes}$ mastoparan. The dissociation constant for the mastoparan-calmodulin complex is the smallest known for any calmodulin binding protein or peptide, suggesting that some type of peptide-calmodulin interaction could be physiologically significant.     

Molecular properties of calcium-pumping ATPase from human erythrocytes

The Ca2+-pumping ATPase from human erythrocyte membranes, purified by the method previously reported [Niggli, V., Penniston, J. T., & Carafoli, E. (1979) J. Biol. Chem. 254, 9955-9958], was freed of minor impurities by extensive washing while bound to the calmodulin-Sepharose column. The pure enzyme showed a single band of Mr 138000, which contained no stainable carbohydrate. The enzyme retained calmodulin-stimulable ATPase activity; with appropriate assay conditions, an activity of 21.2 mumol/(mg x min) was obtained. Amino acid analysis showed that the ATPase had a larger proportion of polar amino acids than do other integral membrane proteins. Despite this, the ATPase showed a tendency to form dimers and higher aggregates even in the presence of sodium dodecyl sulfate and urea. The enzyme required Mg2+ but showed little activity unless a second ion was added. With regard to this second ion, the enzyme responded to alkaline earth metal ions in the order Ca2+ greater than Sr2+ much greater than Ba2+. It was highly specific for ATP and was stimulated by Na+ or K+; in all of these properties it resembled the enzyme in unfractionated membranes. Limited proteolysis using trypsin yielded, at short times, many fragments of various molecular weights; continued proteolysis resulted in two trypsin-resistant fragments of Mr 81000 and 33500. Analysis of the time course of proteolysis indicated that the ATPase existed in two or more conformations that had differing susceptibilities to proteolysis. It is suggested that these correspond to active and inactive conformers of the enzyme.     

Regulation of adenylate cyclase from Leucophaea maderae by calcium,calmodulin and forskolin

1. Adenylate cyclase was assayed in homogenates ofhindgut tissue from Leucophaea maderae (L.). The 10,000 g supernatant enzyme was stimulated by calmodulin.2. Trifluoperazine inhibited calmodulin-stimulated adenylate cyclase activity.3. Elevated calcium levels (> 100 μM) inhibited natural and calmodulin-stimulated enzymic activity.4. Forskolin (1 mM) stimulated adenylate cyclase activity by approximately 10-fold.     

Comparison of the Ca-Mg ATPase and calcium transport in rat and human erythrocytes: Evidence for an electrogenic mechanism

Inside out vesicles prepared from rat erythrocytes transport calcium by an electrogenic mechanism. Calmodulin stimulates the activity of a Ca-Mg ATPase, and also stimulates calcium transport. Permeant anions stimulate calcium transport relative to that observed when an impermeant anion (gluconate) is employed. The inside out vesicles transport phosphate anion in a calmodulin-stimulated, calcium- and ATP-dependent manner. Membrane potential sensitive fluorescent dyes demonstrate a positive interior membrane potential during calcium transport in the absence of permeant anions. The properties of the rat erythrocyte calcium transport system corresponds closely to those of the human erythrocyte; the activity of the Ca-Mg ATPase, rates and stoichiometries of calcium and phosphate transport and the degree of calmodulin stimulation are directly comparable between the two species.     

Change in cytosolic calmodulin activity of density (age) separated human erythrocytes towards membrane Ca2+/Mg2+ ATPase

The membrane $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ ATPase of density (age) separated human erythrocytes was examined for its stimulation by the cytosols of these cell groups. On the assumption that the stimulatory activity in the cytosol is only calmodulin, it was consistently observed that the young cytosol had a significantly higher activity towards the membrane $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ ATPase activity (from any age group) than did the old cell cytosol. The data clearly demonstrates decided differences in the expression of calmodulin activity in cytosols from young and old erythrocytes and would support the conclusion that calmodulin activity is altered during $\text{in vivo}$ aging of these cells. Possible mechanisms for these alterations are discussed.