Interaction of the purified Ca2+, Mg2+-ATPase from human erythrocytes with phospholipids and calmodulin

The Ca2+-pumping ATPase has been purified in a functional form from human erythrocytes by calmodulin affinity chromatography. The purified enzyme has a specific activity at least 300-fold higher than the membrane bound enzyme. It consists of one major protein band of 140000 Dalton, and after reconstitution in liposomes it transports Ca2+ with an efficiency of at least 1 Ca2+/ATP. In the presence of calmodulin, the affinity of the enzyme for Ca2+, and its specific activity, are greatly increased. Acidic phospholipids have an unexpected effect on the isolated enzyme: ATPase isolated or reconstituted in acidic phospholipids behaves as if calmodulin were present. Acidic phospholipids mimic the effect of calmodulin.     

A note on Ca2+ binding to calmodulin

Binding of Ca2+ to calmodulin has been simulated on the basis of a model that assumes two classes, two sites in each class, of Ca2+ binding sites. With properly chosen values of binding constants for the two classes of sites, and with the assumption that certain degree of positive cooperativity exists between the two sites in each class, the overall binding isotherm can be generated so that it appears to be a single-transition, non-cooperative binding curve of four equivalent sites. Thus this model offers a resolution for some of the discrepancies among Ca2+ binding studies of calmodulin.     

Enhancement by Mg2+ of domain specificity in Ca2+-dependent interactions of calmodulin with target sequences

Mg2+ binds to calmodulin without inducing the changes in secondary structure that are characteristic of Ca2+ binding, or the exposure of hydrophobic surfaces that are involved in typical Ca2+-dependent target interactions. The binding of Mg2+ does, however, produce significant spectroscopic changes in residues located in the Ca2+-binding loops, and the Mg-calmodulin complex is significantly different from apo-calmodulin in loop conformation. Direct measurement of Mg2+ binding constants, and the effects of Mg2+ on Ca2+ binding to calmodulin, are consistent with specific binding of Mg2+, in competition with Ca2+. Mg2+ increases the thermodynamic stability of calmodulin, and we conclude that under resting, nonstimulated conditions, cellular Mg2+ has a direct role in conferring stability on both domains of apo-calmodulin. Apo-calmodulin binds typical target sequences from skeletal muscle myosin light chain kinase and neuromodulin with Kd approximately 70-90 nM (at low ionic strength). These affinities are virtually unchanged by 5 mM Mg2+, in marked contrast to the strong enhancement of peptide affinity induced by Ca2+. Under conditions of stimulation and increased [Ca2+], Mg2+ has a role in directing the mode of initial target binding preferentially to the C-domain of calmodulin, due to the opposite relative affinities for binding of Ca2+ and Mg2+ to the two domains. Mg2+ thus amplifies the intrinsic differences of the domains, in a target specific manner. It also contributes to setting the Ca2+ threshold for enzyme activation and increases the importance of a partially Ca2+-saturated calmodulin-target complex that can act as a regulatory kinetic and equilibrium intermediate in Ca2+-dependent target interactions.     

The calmodulin binding domain of the plasma membrane Ca2+ pump interacts both with calmodulin and with another part of the pump

Synthetic peptides corresponding to the calmodulin-binding domain of the human erythrocyte Ca2+ pump were prepared representing residues 2-29 (C28W), 2-21 (C20W), 2-16 (C15W), and 16-29 (C14) of the sequence (James, P., Maeda, M., Fisher, R., Verma, A. K., Krebs, J., Penniston, J. T., and Carafoli, E. (1988) J. Biol. Chem. 263, 2905-2910). Peptides C28W, C20W, and C15W bound to calmodulin with an apparent 1:1 stoichiometry in the presence of Ca2+ and inhibited the activation of the Ca2+ pump by calmodulin, while C14 was ineffective. Substituting tyrosine (C28Y) or alanine (C28A) for the tryptophan residue lowered the affinity for calmodulin. The estimated Kd values for the calmodulin-peptide complexes were 0.1 nM for C28W, 5-15 nM for C20W, C28Y, and C28A, and 700-1700 nM for C15W. The Ca2+ pump in inside-out erythrocyte membrane vesicles was activated by proteolytic removal of the endogenous calmodulin-binding domain. Addition of C20W or C28W then inhibited calmodulin-independent Ca2+ transport, while a calmodulin-binding peptide from another enzyme had no effect. The inhibition of the pump by C20W was purely competitive with Ca2+, while C28W decreased the Vmax and increased the K1/2 for Ca2+, restoring the pump activity nearly to its low basal level. The results suggest that a calmodulin-binding peptide from any enzyme has two kinds of specificity: it shares with peptides from other enzymes the ability to bind to calmodulin, but only it has the specificity to interact with its own (proteolytically activated) enzyme.     

Tb3+ binding to Ca2+ and Mg2+ binding sites on sarcoplasmic reticulum ATPase

The interactions of Tb3+ and sarcoplasmic reticulum (SR) were investigated by inhibition of Ca2+-activated ATPase activity and enhancement of Tb3+ fluorescence. Ca2+ protected against Tb3+ inhibition of SR ATPase activity. The apparent association constant for Ca2+, determined from the protection, was about 6 x 10(6) M-1, suggesting that Tb3+ inhibits the ATPase activity by binding to the high affinity Ca2+ binding sites. Mg2+ did not protect in the 2-20 mM range. The association constant for Tb3+ binding to this Ca2+ site was estimated to be about 1 x 10(9) M-1. No cooperativity was observed for Tb3+ binding. No enhancement of Tb3+ fluorescence was detected. A second group of binding sites, with weaker affinity for Tb3+, was observed by monitoring the enhancement of Tb3+ fluorescence (lambda ex 285 nm, lambda em 545 nm). The fluorescence intensity increased 950-fold due to binding. Ca2+ did not complete for binding at these sites, but Mg2+ did. The association constant for Mg2+ binding was 94 M-1, suggesting that this may be the site that catalyzes phosphorylation of the ATPase by inorganic phosphate. For vesicles, Tb3+ binding to these Mg2+ sites was best described as binding to two classes of binding sites with negative cooperativity. If the SR ATPase was solubilized in the nonionic detergent C12E9 (dodecyl nonaoxyethylene ether alcohol), in the absence of Ca2+, only one class of Tb3+ binding sites was observed. The total number of sites appeared to remain constant. If Ca2+ was included in the solubilization step, Tb3+ binding to these Mg2+ binding sites displayed positive cooperativity (Hill coefficient, 2.1). In all cases, the apparent association constant for Tb3+, in the presence of 5 mM MgCl2, was in the range of 1-5 x 10(4) M-1.     

Novel effects of calmodulin and calmodulin antagonists on the plasma membrane (Ca2+ + Mg2+)-ATPase from rabbit kidney proximal tubules.

In this work we report an unusual pattern of activation by calmodulin on the (Ca2+ + Mg2+)-ATPase from basolateral membranes of kidney proximal tubule cells. The activity of the ATPase depleted of calmodulin is characterized by a high Ca2+ affinity (Km = 2.2-3.4 microM) and a biphasic dependence on ATP concentration. The preparation responded to the addition of calmodulin by giving rise to a new Ca2+ site of very high affinity (Km less than 0.05 microM). Calmodulin antagonists had diverse effects on ATPase activity. Compound 48/80 inhibited calmodulin-stimulated activity by 70%, whereas calmidazolium did not modify this component. In the absence of calmodulin, 48/80 still acted as an antagonist, increasing the Km for Ca2+ to 5.7 microM and reducing enzyme turnover by competing with ATP at the low affinity regulatory site. Calmidazolium did not affect Ca2+ affinity, but it did displace ATP from the regulatory site. At fixed Ca2+ (30 microM) and ATP (5 mM) concentrations, Pi protected against 48/80 and potentiated inhibition by calmidazolium. At 25 microM ATP, Pi protected against calmidazolium inhibition. We propose that the effects of ATP and Pi arise because binding of the drugs to the ATPase occurs mainly on the E2 forms.     

The effect of calmodulin on the interaction of carbodiimides with the purified human erythrocyte (Ca2+ + Mg2+)-ATPase

The activity of the solubilized and purified (Ca2+ + Mg2+)-ATPase from human erythrocyte membranes was inhibited by N,N'-dicyclohexylcarbodiimide in a concentration-dependent manner. The carbodiimide prevented formation of the phosphorylated intermediate during the catalytic cycle of the enzyme. Treatment of the enzyme with N,N'-dicyclohexyl[14C]carbodiimide resulted in the formation of a 14C-labelled polypeptide corresponding to the enzyme monomer (molecular weight 136,000). The tryptic fragmentation of this 14C-labelled enzyme resulted in the formation of three major 14C-labelled fragments with molecular weights of 58,000, 36,500 and 23,000, the latter two probably representing transmembrane and calmodulin-binding domains of the enzyme, respectively. In the absence of calmodulin, 6.7 molecules of N,N'-dicyclohexyl[14C]carbodiimide covalently bound to each molecule of Ca2+-ATPase; in the presence of calmodulin, the number of molecules of carbodiimide bound was 13.1. The binding of N,N'-dicyclohexylcarbodiimide to the (Ca2+ + Mg2+)-ATPase greatly reduced its ability to bind to a calmodulin-agarose gel.     

A one-to-one Mg2+:Mn2+ exchange in rat erythrocytes

Mg2+ efflux in rat erythrocytes was stimulated by increases in external Na+ concentration following a Michaelian-like function with an apparent dissociation constant (KNa) of 11 +/- 3 mM (mean +/- S.D. of three experiments) and a variable maximal rate ranging from 150 to 1200 mumol (liter (1) cells X h)-1. Na+-stimulated Mg2+ efflux was inhibited by quinidine and by ATP depletion. In the absence of external Na+, Mg2+ efflux was stimulated by increases in external Mn2+ concentration following a Michaelian-like function with an apparent dissociation constant (KMn) of 35 +/- 15 microM (mean +/- S.D. of four experiments) and a variable maximal rate ranging from 350 to 1400 mumol (1 cells X h)-1. Mn2+-stimulated Mg2+ efflux was inhibited by quinidine, by ATP depletion, and by increasing the external Na+ concentration. Quinidine-sensitive (or ATP-dependent) Mg2+ efflux exhibited very similar values when compared with quinidine-sensitive (or ATP-dependent) Mn2+ influx. Mn2+ efflux in rat erythrocytes (loaded with total internal Mn2+ contents of 230-450 mumol/l cells) was stimulated by increases in external Na+ concentration and inhibited by quinidine. In the absence of external Na+, Mn2+ efflux was stimulated by increases in external Mg2+ concentration following a Michaelian-like function with an apparent dissociation constant (KMg) of about 35 +/- 5 microM (mean +/- range of two experiments) and a maximal rate of about 60-100 mumol (1 cells X h)-1. In conclusion, the Na+-stimulated Mg2+ carrier of rat erythrocytes may catalyze a one-to-one and reversible Mn2+:Mg2+ exchange in the absence of external Na+.     

Determinants for calmodulin binding on voltage-dependent Ca2+ channels

Calmodulin, bound to the alpha(1) subunit of the cardiac L-type calcium channel, is required for calcium-dependent inactivation of this channel. Several laboratories have suggested that the site of interaction of calmodulin with the channel is an IQ-like motif in the carboxyl-terminal region of the alpha(1) subunit. Mutations in this IQ motif are linked to L-type Ca(2+) current (I(Ca)) facilitation and inactivation. IQ peptides from L, P/Q, N, and R channels all bind Ca(2+)calmodulin but not Ca(2+)-free calmodulin. Another peptide representing a carboxyl-terminal sequence found only in L-type channels (designated the CB domain) binds Ca(2+)calmodulin and enhances Ca(2+)-dependent I(Ca) facilitation in cardiac myocytes, suggesting the CB domain is functionally important. Calmodulin blocks the binding of an antibody specific for the CB sequence to the skeletal muscle L-type Ca(2+) channel, suggesting that this is a calmodulin binding site on the intact protein. The binding of the IQ and CB peptides to calmodulin appears to be competitive, signifying that the two sequences represent either independent or alternative binding sites for calmodulin rather than both sequences contributing to a single binding site.     

The effect of Mg2+ and guanine nucleotide exchange factor on the binding of guanine nucleotides to eukaryotic initiation factor 2

A major site of regulation of polypeptide chain initiation is the binding of Met-tRNA to 40 S ribosomal subunits which is mediated by eukaryotic initiation factor 2 (eIF-2). The formation of ternary complex, eIF-2.GTP.Met-tRNA, is potently inhibited by GDP. Measurement of the parameters for guanine nucleotide binding to eIF-2 is critical to understanding the control of protein synthesis by fluctuations in cellular energy levels. We have compared the dissociation constants (Kd) of eIF-2.GDP and eIF-2.GTP and find that GDP has a 400-fold higher affinity for GDP than GTP. The Kd for GDP is almost an order of magnitude less than has been reported previously. The difference between the Kd values for the two nucleotides is the result of a faster rate constant for GTP release, the rate constants for binding being approximately equal. This combination of rate constants and low levels of contaminating GDP in preparations of GTP can explain the apparently unstable nature of eIF-2.GTP observed by others. Mg2+ stabilizes binary complexes slowing the rates of release of nucleotide from both eIF-2.GDP and eIF-2.GTP. The competition between GTP and GDP for binding to eIF-2.guanine nucleotide exchange factor complex has been measured. A 10-fold higher GTP concentration than GDP is required to reduce [32P] GDP binding to eIF-2.guanine nucleotide exchange factor complex by 50%. The relevance of this competition to the regulation of protein synthesis by energy levels is discussed.     

Calmodulin content,Ca2+-dependent calmodulin binding proteins,and testis growth: Identification of Ca2+-dependent calmodulin binding proteins in primary spermatocytes

In contrast with the transient pre-replicative increase in calmodulin (CaM) level observed in proliferative activated cells, postnatal development of rat testis was paralleled by 3 specific rises in CaM. The first one occurred between 5 and 10 days, coincident with the appearance and proliferation start of spermatogonia and Sertoli cells. Meiosis accomplishment and spermatid differentiation were paralleled by 2 additional rises, at 24 and 32 days, respectively. The plateau phase of testis growth was coincident with the appearance of maturating spermatids and spermatozoa in the germinal epithelium, and with a decrease in CaM content. Testicular DNA:g wet tissue ratio reached the highest level in 15-day-old rats and gradually decreased up to 35 days, when a constant level was reached. A similar level of Ca²⁺-CaMBPs was observed in 5- and 20-day-old rat testis. Although all subcellular fractions showed the ability to bind CaM in a Ca²⁺-dependent manner, CaM was mainly recovered in the nuclear and soluble fractions of adult and immature rat testis. Several Ca²⁺-CaMBPs with an apparent M_r of 82, 75, 64, 19, and 14 kD were purified by affinity chromatography from pachytene primary spermatocyte nuclear matrix. Ca²⁺-CaMBPs showing an M_r of 120, 78, 72, and 66 kD were also purified from the supernatant obtained after DNA and RNA hydrolysis of meiotic nuclei. Major cytosolic Ca²⁺-CaMBPs of primary spermatocytes showed an M_r of 120, 84, 44, and 39 kD. The functions that these Ca²⁺-CaMBPs might have during the first meiotic prophase is discussed. Mol. Reprod. Dev. 48:127–136, 1997. © 1997 Wiley-Liss, Inc.     

Ca2+ and Mg2+-binding and a putative calmodulin type Ca2+-binding site in Synechococcus Photosystem II

Thylakoids and Photosystem II particles prepared from the cyanobacterium Synechococcus PCC 7942 washed with a HEPES/glycerol buffer exhibited low rates of light-induced oxygen evolution. Addition of either Ca²⁺ or Mg²⁺ to both thylakoids and Photosystem II particles increased oxygen evolution independently, maximal rates being obtained by addition of both ions. If either preparation was washed with NaCl, light induced O₂ evolution was completely inhibited, but re-activated in the same manner by Ca²⁺ and Mg²⁺ but to a lower level. In the presence of Mg²⁺, the reactivation of O₂ evolution by Ca²⁺ allowed sigmoid kinetics, implying co-operative binding. The results are interpreted as indicating that not only Ca²⁺, but also Mg²⁺, is essential for light-induced oxygen evolution in thylakoids and Photosystem II particles from Synechococcus PC 7942. The significance of the reactivation kinetics is discussed. Reactivation by Ca²⁺ was inhibited by antibodies to mammalian calmodulin, indicating that the binding site in Photosystem II may be analogous to that of this protein.Abbreviation HEPES n-2-Hydroxyethylpiperazine--2-ethane sulphonic acid     

Ca2+ and calmodulin regulate the binding of filamin A to actin filaments

Filamin A (FLNa) cross-links actin filaments (F-actin) into three-dimensional gels in cells, attaches F-actin to membrane proteins, and is a scaffold that collects numerous and diverse proteins. We report that Ca(2+)-calmodulin binds the actin-binding domain (ABD) of FLNa and dissociates FLNa from F-actin, thereby dissolving FLNa.F-actin gels. The FLNa ABD has two calponin homology domains (CH1 and CH2) separated by a linker. Recombinant CH1 but neither FLNa nor its ABD binds Ca(2+)-calmodulin in the absence of F-actin. Extending recombinant CH1 to include the negatively charged region linker domain makes it, like full-length FLNa, unable to bind Ca(2+)-calmodulin. Ca(2+)-calmodulin does, however, dissociate the FLNa ABD from F-actin provided that the CH2 domain is present. These findings identify the first evidence for direct regulation of FLNa, implicating a mechanism whereby Ca(2+)-calmodulin selectively targets the FLNa.F-actin complex.     

Conformational transitions in the Ca2+ + Mg2+-activated ATPase and the binding of Ca2+ ions.

We have studied the fluorescence of the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum labelled with fluorescein isothiocyanate. The change in intensity of fluorescein fluorescence caused by addition of Ca2+ to the labelled ATPase can be interpreted in terms of a two-conformation model for the ATPase, one conformation (E1) having a high affinity for Ca2+, the other (E2) a low affinity. Effects of Ca2+ as a function of pH allow an estimate of the effect of pH on the E1/E2 ratio, consistent with kinetic studies. A model is presented for binding of Ca2+ to the ATPase as a function of pH that is consistent both with the data on the E1/E2 equilibrium and with literature data on Ca2+ binding.     

Effect of calmodulin on myometrial cell membrane Ca2+,Mg2+-ATPase activity

Myometrium cell plasma membrane Ca2+, Mg(2+)-ATPase purified by an affinity chromatography on calmodulin-sepharose 4B is calmodulin-dependent enzyme. Concentration of calmodulin required for half-maximal activation of enzyme was about 26 nM. By unlike to the enzymes originated from other tissues sensitivity to the calmodulin of the myometrial sarcolemma Ca(2+)-transporting ATPase was lower: calmodulin increased Vmax of ATPase about 1.25-fold, the apparent constant of the activation of enzyme by Ca2+ failed to alter independently on the phospholipid presenting at the enzyme isolation.     

Oxidation of the skeletal muscle Ca2+ release channel alters calmodulin binding

This study presents evidence for a close relationship betweenthe oxidation state of the skeletal muscleCa²⁺ release channel (RyR1) andits ability to bind calmodulin (CaM). CaM enhances the activity of RyR1in low Ca²⁺ and inhibits itsactivity in high Ca²⁺. Oxidation,which activates the channel, blocks the binding of ¹²⁵I-labeled CaM at bothmicromolar and nanomolar Ca²⁺concentrations. Conversely, bound CaM slows oxidation-induced cross-linking between subunits of the RyR1 tetramer. Alkylation ofhyperreactive sulfhydryls (<3% of the total sulfhydryls) on RyR1with N-ethylmaleimide completelyblocks oxidant-induced intersubunit cross-linking and inhibitsCa²⁺-free¹²⁵I-CaM but notCa²⁺/¹²⁵I-CaMbinding. These studies suggest that1) the sites on RyR1 for bindingapocalmodulin have features distinct from those of theCa²⁺/CaM site,2) oxidation may alter the activityof RyR1 in part by altering its interaction with CaM, and3) CaM may protect RyR1 fromoxidative modifications during periods of oxidative stress.

     

Hypertonicity activates Na+/H+ exchange through Janus kinase 2 and calmodulin

The type 1 sodium-hydrogen exchanger (NHE-1) is a ubiquitous electroneutral membrane transporter that is activated by hypertonicity in many cells. NHE-1 may be an important pathway for Na(+) entry during volume restoration, yet the molecular mechanisms underlying the osmotic regulation of NHE-1 are poorly understood. In the present study we conducted a screen for important signaling molecules that could be involved in hypertonicity-induced activation of NHE-1 in CHO-K1 cells. Hypertonicity rapidly activated NHE-1 in a concentration-dependent manner as assessed by proton microphysiometry and by measurements of intracellular pH on a FLIPR (fluorometric imaging plate reader). Inhibitors of Ca(2+)/calmodulin (CaM) and Janus kinase 2 (Jak2) attenuated this activation, whereas neither calcium chelation nor inhibitors of protein kinase C, the Ras-ERK1/2 pathway, Src kinase, and Ca(2+)/calmodulin-dependent enzymes had significant effects. Hypertonicity also resulted in the rapid tyrosine phosphorylation of Jak2 and STAT3 (the major substrate of Jak2) and CaM. Phosphorylation of Jak2 and CaM were blocked by AG490, an inhibitor of Jak2. Immunoprecipitation studies showed that hypertonicity stimulates the assembly of a signaling complex that includes CaM, Jak2, and NHE-1. Formation of the complex could be blocked by AG490. Thus, we propose that hypertonicity induces activation of NHE-1 in CHO-K1 cells in large part through the following pathway: hypertonicity --> Jak2 phosphorylation and activation --> tyrosine phosphorylation of CaM --> association of CaM with NHE-1 --> NHE-1 activation.