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.     

Na+ATPase of the mammalian erythrocyte membrane. Reversibility of phosphorylation at 0 degrees.

When human erythrocyte membranes are phosphorylated with a very low concentration of [gamma-32P]ATP (0.02 muM) at 0 degrees, and then EDTA is added, rapid disappearance of the phosphoenzyme intermediate of Na+ATPase is observed. The initial rapid phase of phosphoenzyme disappearance is, for the most part, not associated with P1 release and its rate constant, kD, is severalfold greater than the ratio of Na+ATPase activity to phosphoenzyme intermediate, v:EP, at steady state. It is concluded that this rapid disappearance of phosphoenzyme is due to resynthesis of ATP via reversal of phosphorylation. In contrast, rapid reversal is not observed when excess nonradioactive ATP is added to reduce E32P formation, provided Mg2+ is present; however, K+ added with the ATP stimulates reversal. Rapid reversal following EDTA addition is unlikely also when higher ATP concentrations (greater than or equal to 10(-6) M) are used to phosphorylate the enzyme since, at higher ATP, kD congruent to v:EP. The results are compatible with the concept that the Na+ATPase enzyme is composed of two or more catalytic subunits, in which ATP at one catalytic site can regulate the reactivity at another site.     

Role of magnesium in the (Ca2+ + Mg2+)-stimulated membrane ATPase of human red blood cells.

(Ca2+ + Mg2+)-stimulated ATPase of human red cell membranes as a function of ATP concentration was measured at fixed Ca2+ concentration and at two different but constant Mg2+ concentrations. Under the assumption that free ATP rather than Mg-ATP is the substrate, a value for Km (for ATP) of 1-2 micron is found which is in good agreement with the value obtained in the phosphorylation reaction by A.F. Rega and P.J. Garrahan (1975. J. Membrane Biol. 22:313). Mg2+ increases both the maximal rate and the affinity for ATP, whereas Ca2+ increases the maximal rate without affecting Km for ATP. As a by-product of these experiments, it was shown that after thorough removal of intracellular proteins the adenylate kinase reaction at approximately 1 mM substrate concentration is several times faster than maximal rate of (Ca2+ + Mg2+)ATPase in red cell membranes.     

A Ca2+-stimulated ATPase activity in rabbit neutrophil membranes.

An ATPase activity specifically stimulated by micromolar Ca2+ concentrations has been identified in association with rabbit neurophil membranes. These studies provide the basis of further characterization of the Ca2+-ATPase activity with regard to neutrophil function.     

Ca2+-stimulated, Mg2+-dependent ATPase activity in neutrophil plasma membrane vesicles. Coupling to Ca2+ transport

Low concentrations of free Ca2+ stimulated the hydrolysis of ATP by plasma membrane vesicles purified from guinea pig neutrophils and incubated in 100 mM HEPES/triethanolamine, pH 7.25. In the absence of exogenous magnesium, apparent values obtained were 320 nM (EC50 for free Ca2+), 17.7 nmol of Pi/mg X min (Vmax), and 26 microM (Km for total ATP). Studies using trans- 1,2-diaminocyclohexane- N,N,N',N',-tetraacetic acid as a chelator showed this activity was dependent on 13 microM magnesium, endogenous to the medium plus membranes. Without added Mg2+, Ca2+ stimulated the hydrolysis of several other nucleotides: ATP congruent to GTP congruent to CTP congruent to ITP greater than UTP, but Ca2+-stimulated ATPase was not coupled to uptake of Ca2+, even in the presence of 5 mM oxalate. When 1 mM MgCl2 was added, the vesicles demonstrated oxalate and ATP-dependent calcium uptake at approximately 8 nmol of Ca2+/mg X min (based on total membrane protein). Ca2+ uptake increased to a maximum of approximately 17-20 nmol of Ca2+/mg X min when KCl replaced HEPES/triethanolamine in the buffer. In the presence of both KCl and MgCl2, Ca2+ stimulated the hydrolysis of ATP selectively over other nucleotides. Apparent values obtained for the Ca2+-stimulated ATPase were 440 nM (EC50 for free Ca2+), 17.5 nmol Pi/mg X min (Vmax) and 100 microM (Km for total ATP). Similar values were found for Ca2+ uptake which was coupled efficiently to Ca2+-stimulated ATPase with a molar ratio of 2.1 +/- 0.1. Exogenous calmodulin had no effect on the Vmax or EC50 for free Ca2+ of the Ca2+-stimulated ATPase, either in the presence or absence of added Mg2+, with or without an ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid pretreatment of the vesicles. The data demonstrate that calcium stimulates ATP hydrolysis by neutrophil plasma membranes that is coupled optimally to transport of Ca2+ in the presence of concentrations of K+ and Mg2+ that appear to mimic intracellular levels.     

Gel electrophoretic identity of the (Na+ + Mg-2+)- and (Na+ + Ca-2+)-stimulated phosphorylations of rat brain ATPase.

The classical E2-P intermediate of (Na+ + K+)-ATPase dephosphorylates readily in the presence of K+ and is not affected by the addition of ADP. To determine the significance in the reaction cycle of (Na+ + K+)-ATPase of kinetically atypical phosphorylations of rat brain (Na+ + K+)-ATPase we compared these phosphorylated components with the classical E2-P intermediate of this enzyme by gel electrophoresis. When rat brain (Na+ + K+)-ATPase was phosphorylated in the presence of high concentrations of Na+ a proportion of the phosphorylated material formed was sensitive to ADP but resistant to K+. Similarly, if phosphorylation was carried out in the presence of Na+ and Ca-2+ up to 300 pmol/mg protein of a K+ -resistant, ADP-sensitive material were formed. If phosphorylation was from [gamma-32-P]CTP up to 800 pmol-32-P/mg protein of an ADP-resistant, K+ -sensitive phosphorylated material were formed. On gel electrophoresis these phosphorylated materials co-migrated with authentic Na+ -stimulated, K+ -sensitive, E2-P-phosphorylated intermediate of (Na+ + K+)-ATPase, supporting suggestions that they represent phosphorylated intermediates in the reaction sequence of this 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.     

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+.     

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.     

Kinetic properties of the purified Ca2+-translocating ATPase from human erythrocyte plasma membrane

The basic kinetic properties of the solubilized and purified Ca2+-translocating ATPase from human erythrocyte membranes were studied. A complex interaction between the major ligands (i.e., Ca2+, Mg2+, H+, calmodulin and ATP) and the enzyme was found. The apparent affinity of the enzyme for Ca2+ was inversely proportional to the concentration of free Mg2+ and H+, both in the presence or absence of calmodulin. In addition, the apparent affinity of the enzyme for Ca2+ was significantly increased by the presence of calmodulin at high concentrations of MgCl2 (5 mM), while it was hardly affected at low concentrations of MgCl2 (2 mM or less). In addition, the ATPase activity was inhibited by free Mg2+ in the millimolar concentration range. Evidence for a high degree of positive cooperativity for Ca2+ activation of the enzyme (Hill coefficient near to 4) was found in the presence of calmodulin in the slightly alkaline pH range. The degree of cooperativity induced by Ca2+ in the presence of calmodulin was decreased strongly as the pH decreased to acid values (Hill coefficient below 2). In the absence of calmodulin, the Hill coefficient was 2 or slightly below over the whole pH range tested. Two binding affinities of the enzyme for ATP were found. The apparent affinity of the enzyme for calmodulin was around 6 nM and independent of the Mg2+ concentration. The degree of stimulation of the ATPase activity by calmodulin was dependent on the concentrations of both Ca2+ and Mg2+ in the assay system.     

A Ca2+-stimulated ATPase activity in rabbit neutrophil membranes

An ATPase activity specifically stimulated by micromolar Ca²⁺ concentrations has been identified in association with rabbit neurophil membranes. These studies provide the basis of further characterization of the Ca²⁺-ATPase activity with regard to neutrophil function.     

A direct colorimetric assay for Ca2+ -stimulated ATPase activity

A simple and rapid colorimetric assay for measuring the high affinity Ca2+-ATPase activity in subcellular fractions is presented. With this method a one-step addition of a malachite green/molybdate/polyvinyl alcohol reagent to the assay mixture at the end of the incubation period is all that is required for the spectrophotometric quantification of the phosphomolybdate-malachite green complex. The presence of polyvinyl alcohol allows the quantification of released phosphate without having to separate it from protein. We have validated this assay by characterizing the high affinity Ca2+-ATPase activity in isolated rat liver microsomes. Comparable Ca2+-ATPase activities in rat liver microsomes and adipocyte plasma membranes were found when measured with this colorimetric assay and an isotopic assay. This method is applicable to the measurement of other types of ATPase activities.     

ATPase activity and Ca2+ transport by reconstituted tryptic fragments of the Ca2+ pump of the erythrocyte plasma membrane

The purified Ca2+ ATPase of the erythrocyte plasma membrane has been submitted to controlled trypsin proteolysis under conditions that favor either its (putative) E1 or E2 configurations. The former configuration has been forced by treating the enzyme with Ca2+-saturated calmodulin, the latter with vanadate and Mg2+. The E1 conformation leads to the accumulation of a polypeptide of Mr 85 KDa which still binds calmodulin, the E2 conformation to the accumulation of one of Mr 81 KDa which does not. Both fragments arise from the hydrolysis of a transient 90 KDa product which has Ca2+-calmodulin dependent ATPase activity, and which retains the ability to pump Ca2+ in reconstituted liposomes. Highly enriched preparations of the 85 and 81 KDa fragments have been obtained and reconstituted into liposomes. The former has limited ATPase and Ca2+ transport ability and is not stimulated by calmodulin. The latter has much higher ATPase and Ca2+ transport activity. It is proposed that the Ca2+ pumping ATPase of erythrocytes plasma membrane contains a 9 KDa domain which is essential for the interaction of the enzyme with calmodulin and for the full expression of the hydrolytic and transport activity. This putative 9 KDa sequence contains a 4 KDa "inhibitory" domain which limits the activity of the ATPase. In the presence of this 4 KDa sequence, i.e., when the enzyme is degraded to the 85 KDa product, calmodulin can still be bound, but no longer stimulates ATPase and Ca2+ transport.     

A spectrin-dependent ATPase of the human erythrocyte membrane

Removal of spectrin from erythrocyte membranes results in the simultaneous loss of a calcium-stimulated, magnesium-dependent ATPase with an apparent KD for Ca2+ of 1 microM. This ATPase activity with high Ca2+ affinity is specifically reconstituted by addition of purified spectrin to spectrin-depleted membranes, and the reconstituted activity is directly proportional to the amount of spectrin that is reassociated with the membranes. Spectrin binding and activation of the high Ca2+ affinity Mg2+-ATPase are proportionally inhibited by thermal denaturation, trypsin digestion, or treatment of the membranes with thiol-reactive reagents. Binding of calmodulin to the Ca2+ pump ATPase requires that calmodulin contains bound ca2+. By contrast, spectrin binding to the erythrocyte membrane is Ca2+-independent. Direct assay of calmodulin is purified spectrin and absence of chlorpromazine inhibition of reconstitution demonstrate that activation of the high Ca2+ affinity ATPase resulting from spectrin binding is not a result of contamination of spectrin by calmodulin. Additional evidence that the spectrin-activated ATPase is an entity separate and distinct from the Ca2+ pump is provided by other characteristics of the activation phenomenon. It is suggested that spectrin constitutes part of an ATPase which may function as a component of the "cytoskeleton" controlling erythrocyte shape and membrane flexibility.