The interaction of Ca2+/Mg2+ ATPase activator protein and Ca2+ with human erythrocyte membranes.

This paper presents the first unambiguous demonstration that a unique protein isolated from the hemolysate of human erythrocytes is responsible for increasing both the apparent Ca²⁺ ion affinity and maximum rate of ATP hydrolysis of the membrane-bound $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ ATPase. Unlike previous reports where an unpurified extract from red blood cells was used to activate the ATPase, our results clearly demonstrate that a single protein species, whether initially associated with or added back to the membrane is responsible for the observed changes in ATPase activity.     

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 130000 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.     

Phosphorylation of the isolated high-affinity (Ca2+ + Mg2+) ATPase of the human erythrocyte membrane

Solubilized and purified high-affinity (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) of the human erythrocyte membrane (Wolf, H.U., Dieckvoss, G. and Lichtner, R. (1977) Acta Biol. Ger. 36, 847) has been phosphorylated and dephosphorylated under various conditions with respect to Ca2+ and Mg2+ concentrations. In the range, 0.001--100 mM, the rate of phosphorylation was dependent on Ca2+ concentration, showing a maximum at 10 mM. The phosphorylation rate was nearly independent of the Mg2+ concentration within the range 0.01-1 mM. This enzyme has at least three Ca2+ binding sites with different affinities and regulatory functions: (1) binding to the high-affinity site yields phosphorylation of the enzyme; (2) binding to a low-affinity site (Ca2+ concentrations higher than 40 microM) inhibits dephosphorylation or the conformational change which is necessary for dephosphorylation; (3) by binding to an additional low-affinity site, Ca2+ at concentrations higher than 1 mM abolishes negative cooperative behaviour (shown below 1 mM Ca2+) and causes weak positive cooperativity between at least two catalytic subunits in the phosphorylation reaction. The phosphoprotein obtained at Ca2+ concentrations above 1 mM dephosphorylates spontaneously after removal of the divalent metal ions. Addition of Mg2+ accelerates the dephosphorylation rate. Affinities of the inhibitory Ca2+ binding sites are reduced by the binding of substrate or K+.     

Large-scale isolation of human erythrocyte Ca2+-transport ATPase

A rapid procedure for preparing large quantities of purified erythrocyte Ca2+-transport ATPase is presented. The method involves: (1) fast preparation of calmodulin-deficient, essentially haemoglobin-free, erythrocyte membranes by molecular filtration using Pellicon filters; (2) solubilization of membrane proteins by deoxycholate; and (3) a batch procedure using calmodulin-Sepharose 4B gel for purification of Ca2+-transport ATPase.     

Phospholipid and detergent effects on (Ca2+ + Mg2+)ATPase purified from human erythrocytes

(Ca2+ + Mg2+)ATPase (EC 3.6.1.3) was solubilized from human erythrocyte membranes by detergent extraction with Triton N-101 (0.5 mg/mg membrane protein) and purified by calmodulin affinity chromatography. ATPase activity was assayed in mixtures of Triton N-101 and phospholipid, without reconstitution into bilayer vesicles. At low levels of phospholipid (5 micrograms/ml), the ATPase activity was highly sensitive to the detergent concentration, with maximal activity occurring at or near the critical micelle concentration of the detergent. With increased amounts of phospholipid (50 micrograms/ml), detergent concentrations greater than the critical micelle concentration were required for maximal activity. Detergent alone did not support ATPase activity. Sonicated phospholipid in the form of vesicles was equally ineffective. Activity seemed to be dependent on the presence of detergent/phospholipid mixed micelles. The acidic phospholipids, phosphatidylserine and phosphatidylinositol, as well as the commercial phospholipid preparation, Asolectin, gave activities five to eight times greater than the same amount of phosphatidylcholine. Mixtures of phosphatidylserine and phosphatidylcholine produced intermediate ATPase activities, with the maximal value dependent on the phosphatidylserine concentration. Addition of phosphatidylcholine to fixed concentrations of phosphatidylserine caused a rise in activity that was independent of the ratio of the two phospholipids or the total phospholipid concentration. Phosphatidylcholine may therefore be irreplaceable for some aspect of ATPase function. The number of phospholipid molecules present in mixed micelles at maximal ATPase activity was calculated to be near 50. This value implied that the hydrophobic surface of the ATPase molecule must be completely coated by a single layer of phospholipid molecules for maximum activity to occur.     

The effect of berbamine derivatives on activated Ca2+-stimulated Mg2+-dependent ATPase in erythrocyte membranes.

Ca2+-stimulated Mg2+-dependent ATPase (Ca2+ + Mg2+-ATPase) stimulated by calmodulin, by partial proteolysis or by oleic acid in erythrocyte membranes was inhibited by various derivatives of the naturally occurring alkaloid berbamine. The ability of these derivatives to inhibit trypsin-activated Ca2+ + Mg2+-ATPase correlated well with their ability to inhibit the calmodulin-stimulated enzyme. Inhibition of the trypsin-activated Ca2+ + Mg2+-ATPase by O-4-(ethoxybutyl)berbamine (EBB) was competitive with respect to ATP. The Ki for inhibition was about 8 microM. These results suggest that the binding site of EBB on the activated Ca2+ + Mg2+-ATPase may bear structural similarity to that on calmodulin, and may be closely related to the ATP-binding site on the enzyme.     

A protein activator of Mg2+-dependent, Ca2+-stimulated ATPase in human erythrocyte membranes distinct from calmodulin

The intracellular localization of aryl acylamidase (aryl-acylamide amidohydrolase, EC 3.5.1.13) in chicken kidney was investigated. By separation on density gradients of the silica sol Ludox AM, the enzyme was localized in the mitochondrial fraction. This mitochondrial fraction was shown to be substantially free of lysosomal contamination. Subfractionation of the purified mitochondria indicates that the enzyme is located on the outer membrane, can be solubilized, and may be a suitable marker enzyme for kidney mitochondria.     

Effects of calmodulin on the (Ca2+ + Mg2+)ATPase partially purified from erythrocyte membranes.

The stimulation of the (Ca²⁺ + Mg²⁺)ATPase of erythrocyte ghosts by calmodulin was observed not only in intact ghosts, but also in the solubilized (Triton X-100) and partially purified, reconstituted (phosphatidylserine liposomes) forms. Since the solubilized form of the enzyme migrated on Sepharose 6B at a position corresponding to a molecular weight of about 150,000, these results show that calmodulin stimulates by direct interaction with the ATPase complex. Additionally, the effects of calmodulin on erythrocyte ghosts prepared by the Dodge-EDTA method (hypotonic ghosts) and by the method of Ronner et al. (involving lysis followed by an isotonic wash repeated several times) were compared (P. Ronner, P. Gazzotti, and E. Carafoli, 1977, Arch. Biochem. Biophys. 179, 578–583). The (Ca²⁺ + Mg²⁺)ATPase of the hypotonic ghosts was low and was stimulated by added calmodulin while that of the isotonic ghosts was high and changed only slightly upon calmodulin addition; this difference in response to calmodulin persisted in the solubilized and reconstituted forms. Hypotonic ghosts bound ¹²⁵I-labeled calmodulin, while isotonic ghosts did not. This comparison of two types of ghosts showed that isotonic ghosts possess an intact calmodulin-(Ca²⁺ + Mg²⁺)ATPase complex, and that the calmodulin remained with the ATPase during solubilization and reconstitution. The isotonic preparation is a particularly useful method of preparing ghosts with an intact calmodulin-ATPase complex, since it requires no special equipment and produces an enzyme activity which is stable to freezing.     

A lipid requirement for the (Ca2+ + Mg2+)-activated ATPase of erythrocyte membranes.

The lipid requirement of the (Ca²⁺ + Mg²⁺)-stimulated ATPase of human erythrocytes has been studied. The enzyme activity was lost after removal of the phospholipids using phospholipase A₂ from Naja naja and serum albumin. Optimal restoration of the (Ca²⁺ + Mg²⁺)-ATPase activity in the partially lipid-depleted membranes was obtained with oleate. The reactivation was not due to the removal of a permeability barrier for ATP, since lysolecithin or cholate did not show latent activity. Reactivation was also obtained with several negatively charged phospholipids. Among the ones normally found in the erythrocyte membranes, only phosphatidyl serine reactivated significantly.     

Modulation of human erythrocyte Ca2+/Mg2+ ATPase activity by phospholipase A2 and proteases. A comparison with calmodulin

The human erythrocyte membrane $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ ATPase responded to the presence of an acidic phospholipase A₂ and to low levels of trypsin (and chymotrypsin) in much the same way as it did to calmodulin isolated from human erythrocytes. The increased concentration of ATP hydrolyzed in 1 hour was similar to that observed when calmodulin had been added to a suspension of membranes during the assay. The observations reported here strongly suggest that activation of the $\text{Ca}{}^{\mathrm{2+}}\text{M}{}^{\mathrm{2+}}$ ATPase can proceed by introducing apparently distinct perturbations either to the protein or to phospholipid domains of the erythrocyte membrane.     

The stoichiometry of the Ca2+ pump in human erythrocyte vesicles: modulation by Ca2+, Mg2+ and calmodulin

Active Ca2+ uptake and the associated (Ca2+ + Mg2+)-ATPase activity were studied under the same conditions in an inside-out vesicle preparation of human red blood cells made essentially by the procedure of Quist and Roufogalis (Journal of Supramolecular Structure 6, 375-381, 1977). Some preparations were treated with 1 mM EDTA at 30 degrees to further deplete them of endogenous levels of calmodulin. As the Ca2+ taken up by the EDTA-treated inside-out vesicles, as well as the non-EDTA treated vesicles, was maintained after addition of 4.1 mM EGTA, the vesicles were shown to be impermeable to the passive leak of Ca2+ over the time course of the experiments. In the absence of added calmodulin, both active Ca2+ uptake and (Ca2+ + Mg2+)-ATPase were sensitive to free Ca2+ over a four log unit concentration range (0.7 microM to 300 microM Ca2+) at 6.4 mM MgCl2. Below 24 microM Ca2+ the stoichiometry of calcium transported per phosphate liberated was close to 2:1, both in EDTA and non-EDTA treated vesicles. Above 50 microM Ca2+ the stoichiometry approached 1:1. When MgCl2 was reduced from 6.4 mM to 1.0 mM, the stoichiometry remained close to 2:1 over the whole range of Ca2+ concentrations examined. In contrast to the results at 6.4 mM MgCl2, the Ca2+ pump was maximally activated at about 2 microM free Ca2+ and significantly inhibited above this concentration at 1 mM MgCl2. Calmodulin (0.5-2.0 microgram/ml) had little effect on the stoichiometry in any of the conditions examined. The possible significance of a variable stoichiometry of the Ca2+ pump in the red blood cell is discussed.     

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.     

Ca2+- and Mg2+-dependent association of phosphorylase kinase with human erythrocyte membranes

The interaction of rabbit muscle phosphorylase kinase (EC 2.7.1.38) with human erythrocyte membranes was investigated. It was found that at pH 7.0 the kinase binds to the inner face of the erythrocyte membrane (inside-out vesicles) and that this binding is Ca2+- and Mg2+-dependent. The sharpest increase in the binding reaction occurs at concentrations between 70 and 550 nM free Ca2+. Erythrocyte ghost or right-side out erythrocyte vesicles showed a significantly lower capacity to interact with phosphorylase kinase. Autophosphorylated phosphorylase kinase shows a similar Ca2+-dependent binding profile, while trypsin activation of the kinase and calmodulin decrease the original binding capacity by about 50%. Heparin (200 micrograms/ml) and high ionic strength (50 mM NaCl) almost completely blocks enzyme-membrane interaction; glycogen does not affect the interaction.     

Effects of Ca2+, Mg2+ and calmodulin on the formation and decomposition of the phosphorylated intermediate of the erythrocyte Ca2+-stimulated ATPase.

Formation of the phosphorylated intermediate (ECaP) of the human erythrocyte Ca2+-stimulated ATPase (Ca2+-ATPase) was more rapid and reached steady state sooner at 400 microM-Ca2+ than at 1 microM-Ca2+. Calmodulin increased the apparent rate of ECaP formation at 1 microM-Ca2+, whereas at 400 microM-Ca2+, calmodulin decreased the steady-state level of the ECaP without affecting its apparent rate of formation. Removal of endogenous Mg2+ with trans-1,2-diaminocyclohexane-NNN'N'-tetra-acetic acid, which decreased both the velocity and Ca2+-sensitivity of the Ca2+-ATPase, did not alter the Ca2+-sensitivity or the apparent rate of formation of ECaP. ECaP formation at high Ca2+ concentrations was not affected by Mg2+ concentrations as high as 1 mM, and the ECaP could be dephosphorylated by ADP and ATP along either the forward or reverse pathways. The results suggest that high Ca2+ concentrations inhibit Ca2+-ATPase activity by preventing dephosphorylation of the E2P complex, rather than by inhibition of the transformation from E1CaP ('high-Ca2+-affinity' ECaP) to E2CaP ('lower-energy' ECaP).     

The effects of lubrol WX on brain membrane Ca2+/Mg2+ ATPase and ATP-dependent Ca2+ uptake activity following acute and chronic ethanol

Acute administration of ethanol (2.5 gm/kg, i.p.) to rats inhibits the cytosolic buffering of Ca2+ in nerve terminals. Ca2+ ATPase and ATP-dependent Ca2+ uptake are both inhibited 30 min after a single dose of ethanol. Chronic ethanol administration (6%, 14 days) did not inhibit Ca2+ ATPase but significantly stimulated ATP-dependent Ca2+ uptake. Lubrol WX treatment of acute ethanolic membranes reverses the inhibition of Ca2+ ATPase seen following ethanol. Lubrol WX treatment of chronic ethanolic membranes prevents the increase in ATP-dependent Ca2+ uptake seen in ethanolic membranes. Both acute and chronic ethanol-induced changes in Ca2+ transport within nerve terminals may involve lipid-dependent parameters of the membrane which may underlie neuronal adaptation.