Effects of fatty acids on activity and calmodulin binding of Ca2+-ATPase of human erythrocyte membranes.

Activation and inhibition of Ca2+-ATPase of calmodulin-depleted human erythrocyte membranes by oleic acid and a variety of other fatty acids have been measured. Low concentrations of oleic acid stimulate the enzyme activity, both in the presence and in the absence of calmodulin. Concomitantly, the affinity of the membrane bound enzyme to calmodulin progressively decreases due to competitive interactions of calmodulin and oleic acid with the enzyme. Removal of oleic acid from the membrane by serum albumin extinguishes the activating effect of oleic acid and restores the ability of the enzyme to bind calmodulin with high affinity. High concentrations of oleic acid induce an almost complete and irreversible loss of enzyme activity which cannot be abolished by removal of oleic acid. Despite a complete loss of enzyme activity, binding of calmodulin to membranes is approximately normal after removal of oleic acid. Activities of (Na+ + K+)-ATPase, Mg2+-ATPase and acetylcholine esterase, as well as the total protein content, show no gross changes upon treatment of membranes with increasing amounts of oleic acid, which seems to exclude that membrane solubilisation by oleic acid causes an inactivation of the enzyme.

Relation between Ca2+-ATPase and endogenous calmodulin of human erythrocyte membranes.

Short incubation of erythrocyte membranes with oleic acid releases Ca2+-independently bound endogenous calmodulin together with a minor fraction of membrane-associated proteins without destruction of the membranes. The released endogenous calmodulin is similar if not identical to cytosolic calmodulin reversibly bound to ghosts in a Ca2+-dependent manner. The release of endogenous calmodulin proceeds without affecting the activity of Ca2+-ATPase when ghosts are incubated with oleic acid in the presence of Ca2+ plus ATP and thereafter freed from oleic acid by washings with serum albumin. Kinetic parameters of Ca2+-ATPase of ghosts with and without endogenous calmodulin are identical as are amounts of exogenous calmodulin bound to these ghosts. Thus, endogenous calmodulin does not function as an essential part of Ca2+-ATPase.

Endogenous calmodulin and Ca2+-ATPase activity of human erythrocyte membranes.

Treatment of calmodulin-depleted erythrocyte membranes with oleic acid solubilizes only a minor fraction of membrane proteins which, however, contains more than 90% of the total endogenous calmodulin. Concomitantly, Ca2+-ATPase is completely and irreversibly inactivated. By treatment with oleic acid in the presence of Ca.ATP the inactivation of Ca2+-ATPase is abolished to a great extent, the loss of endogenous calmodulin, however, is not affected. Thus, a major participation of endogenous calmodulin in the regulation of Ca2+-ATPase of erythrocyte membranes seems to be improbable.

The effects of calcium on glycolysis and ATP concentration in complete and membrane-poor hemolyzates of human erythrocytes.

Hemolyzates prepared from packed human red cells with 30 micrometer total calcium were employed as a means to examine the relationship between ATP consumption and lactate formation. Hemolyzates exposed to ultracentrifugation accumulate membrane fragments in the top layer yielding membrane-poor fractions in the buttom layers of the centrifuge tube. Lactate formation accompanied by ATP depletion amounts to 12 mumoles per ml and hour in complete hemolyzates fortified with NAD. Complexation of calcium results in about 50% inhibition of the lactate formation with a concomitant increase of ATP. Lactate formation is reduced in membrane-poor hemolyzates approximately concurrently to the extent of membrance removal which produces no discernible change in the glyceral-dehydephosphate dehydrogenase activity. 50--200 micrometer total calcium has no effect on the membrane-independent lactate formation which amounts to 1--2 mumoles per ml and hour. Triton X-100 seems to solubilise also the membrane components responsible for the high calcium-dependent ATP consumption which governs the lactate formation.

Dependence on intracellular Ca2+ on mass and turnover of phosphoinositides and phosphatidate in human erythrocytes.

Effects of a calcium-load on mass and turnover of phosphoinositides and phosphatidate were investigated in human erythrocytes by short-term labeling with [32P]Pi. The labeling of phosphatidate was accelerated at normal mass by short-term elevation of free intracellular [Ca2+] up to 1 microM and inhibited by the reduction of normal free [Ca2+]. Thus, the labeling of phosphatidate is a Ca2+-regulated process and not only the consequence of a net synthesis of diacylglycerol by other Ca2+-dependent reactions. Persisting elevation of free intracellular [Ca2+] between 1-40 microM induced an increase of the mass of phosphatidylinositol 4-phosphate with a concomitant decrease of the mass of phosphatidylinositol 4,5-bisphosphate. Under these conditions, the normal steady-state turnover of phosphoinositides was not altered by Ca2+, but mass and turnover of phosphatidate continuously rose. The increase in phosphatidate mass by far exceeded the decrease of the mass of phosphoinositides, indicating that phosphatidate was generated to a great extent by hydrolysis of other phospholipids in addition to the action of phosphoinositidase C with subsequent phosphorylation of diacylglycerol to phosphatidate. The results demonstrate that different phospholipid phosphodiesterases of human erythrocytes are activated by Ca2+-concentrations in the microM range as is known from various other cell types. In contrast to current explanations, Ca2+-dependent phospholipid phosphodiesterases of human erythrocytes did not exhibit an unusually low affinity against rising cytosolic Ca2+-concentrations.

[Determination of the concentration of free magnesium ions in hemolysates of oxygenated and deoxygenated packed human erythrocytes]. / Bestimmung der Konzentrationen freier Magnesiumionen in Hämolysaten oxygenierter und desoxygenierter gepackter menschlicher Erythrozyten.

Concentrations of ionized magnesium in hemolyzates of oxygenated and deoxygenated human erythrocytes were measured directly by means of an ion exchange method. At total concentrations of hemoglobin of 8 mmol/l H2O and total magnesium of 3.6 mmol/l H2O, mean values of [Mg2+] were found to be 0.45 in the oxygenated state and 0.57 mmol/l H2O in the deoxygenated state. The difference of 0.12 +/- 0.1 mmol/l H2O is considerably smaller than corresponding literature data obtained from equilibrium calculation.

Binding of magnesium and chloride ions to human hemoglobin A. Mg2+ concentrations in solutions simulating red cell conditions.

Ultrafiltration and direct determination of [Mg2+] by ion exchange were used to study the binding of Mg2+ and Cl- to isoionic human hemoglobin. A value of 58.8 +/- 1.7 l/mol was determined for the volume of hydrated hemoglobin from ultrafiltration of hemoglobin solutions containing 0.15-0.8 M glucose. In solutions with 5.7 mmol hemoglobin, 150 mmol KCl and 0.5-3.5 mmol MgCl2/l total water, 0.6 mol and 2 mol Cl- were bound/mol oxygenated and deoxygenated hemoglobin respectively. A value of about 11 l/mol was determined for the association constant of Mg2+ to hemoglobin monomer. Free Mg2+ concentrations were measured in hemoglobin solutions containing KCl, MgCl2, ATP and D-glycerate-2,3-bisphosphate at concentrations close to those of red cells. The experiments yielded 0.65 mmol/l free Mg2+ after oxygenation and 0.82 mmol/l after deoxygenation. The data indicate that only small changes of free Mg2+ levels in red cells are caused by physiological changes of pO2.

Relations between ion shifting, ATP depletion and lactic acid formation in human red cells during moderate calcium loading using the ionophore A 23187.

Keeping constant cellular magnesium an A 23 187 mediated moderate calcium loading of human red cells causes isoosmotic cell shrinkage, potassium efflux, slight decrease of cellular pH, ATP depletion connected with an increase of AMP, ADP and Pi and enhanced lactic acid formation. The calcium loading and accompanying effects can be abolished by EGTA or by extracellular magnesium, the latter kept more than two orders of magnitude above that of calcium which was 30 micrometer. Inhibition of the (Mg2+ + Ca2+)-dependent ATPase by ruthenium red or lanthanum decreases the calcium stimulated lactic acid formation after a lag phase. However, the ATP depletion proceeds faster and is much more pronounced under these conditions. (Mg+2 + Na+ +K+)-dependent ATPase, hexokinase, phosphofructokinase and cell shrinkage are ruled out, too, as mediators of the ATP depletion. This suggests that an unknown ATP consuming reaction, apparently not being related to the calcium pump, causes the calcium induced ATP depletion.

Dependence on intracellular pH and Mg2+ of metabolic compartmentation of phosphoinositides in human erythrocytes.

Effects of intracellular pH and Mg2+ on turnover and extent of metabolic compartmentation of phosphomonoester groups of phosphoinositides and phosphatidate were investigated in human erythrocytes by short-term and equilibrium labeling with [32P]Pi under steady-state conditions. At pH 6.7, the specific radio-activities of phosphoinositides reached apparent equilibrium values, in the range of 70% of that ATP-gamma-P after long-term labelling. At pH 7.2, these values were in the range of 40-50% of that ATP-gamma-P. This demonstrates a decreased accessibility of phosphoinositides to enzymatic phosphorylation and dephosphorylation at the transition from acidic to normal incubation conditions. These changes were more pronounced at pH 7.8. High intracellular [Mg2+] initially activated the turnover of phosphoinositides at pH 7.2. The activation changed into an inhibition and an increase of metabolic compartmentation after three hours preincubation of erythrocytes at high [Mg2+]. The long-term Mg2+ effects are reversible to a great extent by a subsequent re-reduction of [Mg2+]. In conclusion, deprotonation of phosphoinositides by low [H+] or high intracellular [Mg2+] at normal pH induces a decreased accessibility for their specific lipid kinases and phosphatases. This effect may be the result of lateral phase separation of acidic phospholipids as a consequence of divalent cation complexation under both experimental conditions, high pH and high [Mg2+] at normal pH.

Influence of Ca2+ and Mg2+ on the turnover of the phosphomonoester group of phosphatidylinositol 4-phosphate in human erythrocyte membranes.

In isolated erythrocyte membranes, increasing the free Mg2+ concentration from 0.5 to 10 mM progressively activates the membrane-bound phosphatidylinositol (PtdIns) kinase and leads to the establishment of a new equilibrium with higher phosphatidylinositol 4-phosphate (PtdIns4P) and lower PtdIns concentrations. The steady-state turnover of the phosphomonoester group of PtdIns4P also increases at high Mg2+ concentrations, indicating a simultaneous activation of PtdIns4P phosphomonoesterase by Mg2+. Half-maximum inhibition of PtdIns kinase occurs at 10 microM free Ca2+ in the presence of physiological free Mg2+ concentrations. Increasing free Mg2+ concentrations overcome Ca2+ inhibition of PtdIns kinase. In the presence of Ca2+, calmodulin activates Ca2+-transporting ATPase 5-fold, but does not alter pool size and radiolabelling of PtdIns4P. In intact erythrocytes, adding EGTA or EGTA plus Mg2+ and the ionophore A23187 to the external medium does not exert significant effects on concentration and radiolabelling of polyphosphoinositides when compared with controls in the presence of 1.4 mM free Ca2+.

Calmodulin binding proteins in human erythrocyte membranes.

Human erythrocyte membranes reveal different calmodulin-binding proteins determined by a 125I-calmodulin gel overlay procedure. Beside the well-established Ca2+-transport ATPase, other proteins (205, 91, 72 and 42 kDa) bind calmodulin in a Ca2+-dependent manner. Two proteins of the human erythrocyte membrane are able to bind calmodulin only in the absence of Ca2+. One of them (76 kDa) is probably an integral, the other (240 kDa) a peripheral protein.

Turnover of phosphomonoester groups and compartmentation of polyphosphoinositides in human erythrocytes.

The turnover of phosphomonoester groups of phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] was investigated in human erythrocytes by short-term labelling with [32P]Pi. The procedure applied ensured a quantitative extraction of erythrocyte polyphosphoinositides as well as their reliable separation for the determinations of pool sizes and specific radioactivities. The pool sizes of phosphatidylinositol (PtdIns), PtdIns4P and PtdIns(4,5)P2 are 25, 11 and 44 nmol/ml of cells respectively. Under steady-state conditions, the phosphorylation fluxes from [gamma-32P]ATP into PtdIns4P and PtdIns(4,5)P2 are in the ranges 14-22 and 46-94 nmol X h-1 X ml of cells-1 respectively. Only 25-60% of total PtdIns4P and 6-10% of total PtdIns(4,5)P2 take part in the rapid tracer exchange, i.e. are compartmentalized. In isolated erythrocyte ghosts, the turnover of PtdIns4P approximately corresponds to that in intact erythrocytes, although any compartmentation can be excluded in this preparation. Under the conditions of incubation employed, the turnover of PtdIns(4,5)P2 is more than one order of magnitude smaller in isolated ghosts than that obtained for intact erythrocytes.

Purification and characterization of phosphatidylinositol 4-kinase from human erythrocyte membranes.

Two species of PtdIns 4-kinase with molecular masses of 50 kDa and 45 kDa were detected in human erythrocyte membranes using SDS/PAGE. These enzymes were purified to near homogeneity and found to display very similar enzymatic characteristics. The purification scheme consisted of solubilization from erythrocyte membranes in the presence of Triton X-100, followed by Cibacron-blue-Sephadex, phosphocellulose and Mono Q anion-exchange chromatography. The final step in the purification protocol was preparative SDS/PAGE, followed by electroelution and renaturation of the enzyme. This procedure afforded an about 4000-fold purification of the enzyme from erythrocyte membranes. Characterization of the [32P]PtdInsP products formed by the purified PtdIns kinases indicated that these enzymes specifically phosphorylated the D-4 position of the inositol ring. The Km values of both PtdIns 4-kinase species for PtdIns and ATP were found to be 0.2 mM and 0.1 mM, respectively. The enzymes are both activated by Mg2+, and inhibited by Ca2+ and by adenosine. The potential importance of these effectors for the regulation of PtdIns phosphorylation in cells is discussed.