Molecular properties of the red cell calcium pump: I. Effects of calmodulin,proteolytic digestion and drugs on the kinetics of active calcium uptake in inside-out red cell membrane vesicles

In calmodulin-stripped inside-out human red cell membrane vesicles /IOV/ ATP + Mg²⁺-dependent active calcium uptake is stimulated by the addition of calmodulin. Calmodulin increases the maximum calcium transport rate /V_max/, decreases K_Ca, and does not affect K_ATP of calcium uptake. The action of both membrane bound and external calmodulin is competitively inhibited by phenothiazines. Drugs reacting with SH groups of proteins reversibly inhibit calcium pumping by decreasing V_max and not affecting K_Ca and K_ATP. The relative magnitude of calmodulin stimulation of calcium transport is unaltered by SH reagents.Mild proteolytic digestion of IOVs stimulates active calcium uptake and mimics the effects of calmodulin on the kinetic parameters — that is converts the system to a “high calcium-affinity” state. Proteolysis eliminates calcium-dependent calmodulin binding to IOV membranes and any further stimulation of calcium uptake by calmodulin. Based on these results the presence of a calmodulin-binding regulatory subunit of the red cell calcium pump at the internal membrane surface is postulated.     

Molecular properties of the red cell calcium pump: II. Effects of calmodulin,proteolytic digestion and drugs on the calcium-induced membrane phosphorylation by ATP in inside-out red cell membrane vesicles

In inside-out human red cell membrane vesicles /IOV/, in the absence of Mg²⁺, the only calcium-induced labelling by γ³²P-ATP occurs in a 140–150 000 molecular weight protein fraction, representing the hydroxylamine-sensitive phosphorylated intermediate /EP/ of the calcium pump. In the presence of Mg²⁺ calcium-induced phosphorylation is accelerated but several other membrane proteins are also phosphorylated through protein kinase action forming hydroxylamine-insensitive bonds. Addition of calmodulin accelerates EP formation both in the absence and presence of Mg²⁺.Treatment of the membrane with SH-group reagents significantly reduces EP formation. Mild trypsin digestion of IOVs, stimulating active calcium transport, eliminates calmodulin action and decreases the steady-state level of EP. In trypsin-digested IOVs the molecular weight of the ³²P-labelled EP is shifted to lower values /110–120 000/ We suggest that trypsin digestion cleaves off a 20–40 000 molecular weight calmodulin-binding regulatory subunit of the calcium pump molecule.     

Transcellular calcium transport by midgut of the blowfly, Calliphoravicina

Transcellular calcium transport by the internally perfused $\text{Calliphora}$ midgut has been measured by simultaneously monitoring ⁴⁵Ca removal from the perfusing saline (entry to the cells) and its appearance in the bathing saline (exit from the cells). Reduction of the Na⁺ gradient across the basolateral membranes of midgut epithelial cells by removal of bathing Na⁺ or by addition of monensin or ouabain inhibits calcium transport across the basolateral membranes. Calcium entry at the apical membranes is inhibited in parallel. The calmodulin inhibitors, trifluoperazine or calmidazolium, do not directly affect calcium transport nor do they dissociate the parallel changes in calcium entry and exit when calcium exit is inhibited. Experiments with A23187 are consistent with a role for intracellular calcium in regulating calcium entry at the apical membranes. It is suggested that calcium transport out of midgut epithelial cells is largely by Na⁺-Ca²⁺ countertransport, and that entry may be regulated by cytoplasmic calcium so that the calcium influx never exceeds the capacity of the transport mechanisms to pump it out of the cells.     

Effects of divalent metal ions on the calcium pump and membrane phosphorylation in human red cells

In inside-out red cell membrane vesicles ATP-dependent calcium transport is activated by the divalent metal ions Mg²⁺, Mn²⁺, Co²⁺, Ni²⁺ and Fe²⁺. This activation is based on the formation of Me²⁺-ATP complexes which can serve as energy-donor substrates for the calcium pump, and probably, satisfy the requirement for free Me²⁺ in this transport process. Higher Me²⁺ concentrations inhibit calcium transport with various efficiencies. Mn²⁺ directly competes with Ca²⁺ at the transport site, while other divalent metal ions investigated have no such effect. The formation of the hydroxylamine-sensitive phosphorylated intermediate (EP) of the red cell membrane calcium pump from [γ-³²P]ATP is induced by Ca²⁺ while rapid dephosphorylation requires the presence of Mg²⁺. At higher concentrations Mn²⁺ and Ni²⁺ inhibit predominantly the formation of EP, while Co²⁺ and Fe²⁺ block dephosphorylation. The possible sites and nature of the divalent metal interactions with the red cell calcium pump are discussed. Hydroxylamine-insensitive membrane phosphorylation in inside-out vesicles from [γ-³²P]ATP is significantly stimulated by Mn²⁺ and Co²⁺, as compared to that produced by Mg²⁺, Fe²⁺ and Ni²⁺. Part of this labelling is found in phospholipids, especially in phosphatidylinositol. The results presented for the metal dependency of protein and lipid phosphorylation in red cell membranes may help in the characterization of ATP consumptions directly related to the calcium pump and those involved in various regulatory processes.     

Electron paramagnetic resonance study on calmodulin: conformational change and interaction with divalent cations

Biologically active spin labelled derivatives of calmodulin were prepared and used to study CA²⁺- and Mg²⁺-induced conformational changes of the protein. The rotational correlation time of the spin labelled residues increased upon addition of divalent cations. Two calcium ions per spin labelled calmodulin were found to induce a 75% conformational change, whereas four calcium ions were necessary for a maximum conformational change. The increase in rotational correlation time induced by Mg²⁺ is less pronounced. Two different covalently attached spin labels (iodoacetamide and maleimide) were compared and marked differences were found in their chemical stability. The binding of manganese ions to calmodulin could be observed directly from the electron paramagnetic resonance spectra of these paramagnetic ions. Two specific classes of binding sites, each binding two manganese ions with $\text{k}{}_{\mathrm{<mtext>D</mtext>}}\text{= 0.6 × 10}{}^{\mathrm{?6}}\text{m}\text{and}\text{k}{}_{\mathrm{D}}\text{= 3 × 10}{}^{\mathrm{?5}}\text{m}$, respectively, were determined. Further ion binding occurs at non-specific sites.     

The presence and binding characteristics of calmodulin in microsomal preparations enriched in sarcoplasmic reticulum from rabbit skeletal muscle

ATP-dependent oxalate facilitated calcium transport in sarcoplasmic reticulum (SR) preparations obtained from rabbit vastus lateralis muscle (fast skeletal muscle; F_sr) and soleus (slow skeletal muscle; S_sr) was determined. Addition of exogenous calmodulin did not stimulate calcium transport in either F_sr or S_sr preparations. F_sr and S_sr previously washed in 1 mM EGTA demonstrated a reduced capacity to transport Ca²⁺; the exogenous addition of calmodulin (0.24 μM) under these conditions, did not restore uptake activity but significantly decreased the steady-state level of Ca²⁺ uptake. Extracts of skeletal SR prepared by treatment with 0.2 mM EDTA and boiling produced significantly more stimulation of red cell Ca²⁺ATPase activity than extracts prepared by boiling alone. This stimulation of red cell Ca²⁺-ATPase was inhibited to a significant extent by $\text{48}\text{80}$, a known anti-calmodulin agent. Radioimmunoassay revealed that extracts prepared by boiling or EDTA-treatment followed by boiling contained considerable amounts of calmodulin. Washing with 1 mM EGTA, though, did not release any calmodulin from SR. These studies reveal that calmodulin is present in both F_sr and S_sr and can only be removed by harsh treatments. The role of calmodulin in skeletal muscle Ca²⁺-transport remains to be determined.     

Effects of calcium and strontium on divalent ion content of refractive granules in Tetrahymena pyriformis

Quantitative electron probe analysis was employed to analyse refractile granules of T. pyriformis individually and in situ. The mean ratios of $\text{Ca}\text{P}$, $\text{Mg}\text{P}$, $\text{K}\text{P}$ and $\text{(}\text{Ca + Mg + K}\text{)}\text{P}$ in granules were similar in cells grown in three different nutrient media. Supplementing the calcium content of proteose-peptone medium with 0.3 and 3 mM calcium depressed the mean $\text{Ca}\text{P}$ ratio and increased the $\text{Mg}\text{P}$ ratio of the granules. The mean $\text{K}\text{P}$ ratio and $\text{(Ca + Mg)}\text{P}$ ratio were not significantly altered. When medium M was supplemented with 0.3 mM calcium, there was no significant change in the $\text{Ca}\text{P}$, $\text{Mg}\text{P}$, $\text{K}\text{P}$ nor $\text{(Ca + Mg)}\text{P}$ ratios. When supplemented with 3 mM Ca, the $\text{Ca}\text{P}$ ratio increased slightly, and the $\text{Mg}\text{P}$ ratio decreased slightly. There was no significant change in the $\text{K}\text{P}$ and $\text{(Ca + Mg)}\text{P}$ ratio. When each medium was supplemented with strontium, all granules incorporated this element, probably at the expense of calcium. The $\text{(Ca + Mg + Sr)}\text{P}$ ratios in granules in each strontium-containing medium were comparable to the $\text{(Ca + Mg)}\text{P}$ ratio in the granules in strontium-free media, indicating that the mix of divalent ions in granules may vary, but the proportion of divalent ions to phosphorus tends to be uniform.     

Characterization of ionomycin as a calcium ionophore.

The ionophorous properties of a new antibiotic, ionomycin, have been studied. It was found that the antibiotic is capable of extracting calcium ion from the bulk of an aqueous phase into an organic phase. The antibiotic also acts as a mobile ion carrier to transport the cation across a solvent barrier. The divalent cation selectivity order for ionomycin as determined by ion competition experiments was found to be: Ca greater than Mg greater than Sr = Ba, where the binding of strontium and barium by the antibiotic is insignificant. The antibiotic also binds La3+ to some extent, but its complexation with monovalent alkali metal ions is negligible. Measurement of the binding of ionomycin with Ca2+ indicates that ionomycin complexes and transports calcium ion in a one to one stoichiometry.     

Studies on the Ca2+ transport mechanism of human erythrocyte inside-out plasma membrane vesicles V. Chlortetracycline fluorescence

The measurement of chlortetracycline fluorescence was employed as a probe for measuring the process to calcium transport by human erythrocyte inside-out vesicles. Chlortetracycline is a divalent metal chelator which increases its fluorrescence when bound to calcium in the presence of a membrane. Addition of calcium and ATP to inside out vesicles in the presence of chlortetracycline increased the chlortetracycline fluorescence as a function of time following an initial delay. Only after a threshold level of calcium had been accumulated did the fluorescence increase. The presence of both ATP and calcium were required. The addition of calmodulin increased the rate and absolute magnitude of the chlortetracycline fluorescence change. Similarly, calmodulin stimulated the rate and extent of ⁴⁵Ca transport by inside-out vesicles. Moreover, the presence of saponin abolished both chlortetracycline fluorescence change and ⁴⁵Ca uptake; a non-hydrolyzable ATP analog would not substitute for ATP in either ⁴⁵Ca transport or chlortetracycline fluorescence experiments. Comparison between the slopes of the linear portions of chlortetracycline fluorescence change and calcium transport time courses at varied free calcium concentrations showed a consistent ratio between the slopes. This suggests that calcium transport change can be calibrated by employing chlortetracycline fluorescence. Based on this data, it is concluded that chlortetracycline fluorescence is a rapid and accurate method for monitoring calcium transport by human erythrocyte inside-out vesicles.     

Regulatory interaction between calmodulin and ATP on the red cell Ca2+ pump

The interactions between calmodulin, ATP and Ca²⁺ on the red cell Ca²⁺ pump have been studied in membranes stripped of native calmodulin or rebound with purified red cell calmodulin. Calmodulin stimulates the maximal rate of $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ by 5–10-fold and the rate of Ca²⁺-dependent phosphorylation by at least 10-fold. In calmodulin-bound membranes ATP activates $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ along a biphasic concentration curve ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>1</mtext>}}\text{≈ 1.4 μ}\text{M}\text{, K}{}_{\mathrm{<mtext>m</mtext><mtext>2</mtext>}}\text{≈ 330 μ}\text{M}$), but in stripped membranes the curve is essentially hyperbolic ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{≈ 7 μ}\text{M}$). In calmodulin-bound membranes Ca²⁺ activates $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ at low concentrations ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{< 0.28 μ}\text{M}$) in stripped membranes the apparent Ca²⁺ affinities are at least 10-fold lower.The results suggest that calmodulin (and perhaps ATP) affect a conformational equilibrium between E₂ and E₁ forms of the Ca²⁺ pump protein.     

Compound 4880 is a selective and powerful inhibitor of calmodulin-regulated functions

Compound $\text{48}\text{80}$, a condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde, is composed of a family of cationic amphiphiles differing in the degree of polymerization. Compound $\text{48}\text{80}$ was found to be a potent inhibitor of the calmodulin-activated fraction of brain phosphodiesterase and red blood cell Ca²⁺-transport ATPase, with IC₅₀ values of 0.3 and 0.85 μg/ml, respectively. However, the basal activity of both enzymes is not at all suppressed by the drug at concentrations up to 300 μg/ml. Inhibition of Ca²⁺ transport into inside-out red blood cell vesicles by compound $\text{48}\text{80}$ follows a similar pattern in that basal, calmodulin-independent, transport is also not affected by the drug. Kinetic analysis revealed that the stimulation of Ca²⁺-transport ATPase induced by calmodulin is inhibited by compound $\text{48}\text{80}$ according to a competitive mechanism. It was demonstrated that the inhibitory constituents of compound $\text{48}\text{80}$ bind to calmodulin in a Ca²⁺-dependent fashion. Comparison of the specificity of several anti-calmodulin drugs showed that compound $\text{48}\text{80}$ is the most specific inhibitor of the calmodulin-dependent fraction of red blood cell Ca²⁺-transport ATPase that has been described hitherto. In addition, compound $\text{48}\text{80}$ was found to be a rather specific inhibitor of the calmodulin-induced activation of Ca²⁺-transport ATPase when compared with the stimulation induced by an anionic amphiphile or by limited proteolysis. Half-maximal inhibition of the activity stimulated by oleic acid or mild tryptic digestion required 8- and 32-times higher concentrations of compound $\text{48}\text{80}$, respectively, compared with the calmodulin-dependent fraction of the ATPase activity. Moreover, calmodulin-independent systems as rabbit skeletal muscle sarcoplasmic reticulum Ca²⁺-transport ATPase or calf cardiac sarcolemma (Na⁺ + K⁺)-transport ATPase are far less influenced by compound $\text{48}\text{80}$ as compared with trifluoperazine and calmidazolium. Because of its high specificity compound $\text{48}\text{80}$ is proposed to be a promising tool for studying calmodulin-dependent processes.     

A possible mechanism for the Na,K-ATPase

A model previously described for the Ca²⁺ pump of sarcoplasmic reticulum has been modified in a thought experiment so that it has the properties of a Na,K-adenosinetriphosphatase (ATPase). When the two Ca²⁺-specific sites are changed into three Na⁺-specific sites, and the channel which opens in the actively transporting conformation made univalent- instead of divalent-cation-selective, the model has the properties of the Na-ATPase which is observed on red cell membranes in the absence of both Na⁺ and K⁺ externally. As in the model for the Ca-ATPase the driving force for transport is generated by a change in solvent structure so that a preformed ionic equilibrium is displaced in favour of less-highly hydrated species; in this case highly hydrated Mg²⁺ ions displace the less highly hydrated Na⁺ ions from binding sites; and Na⁺ diffuses out through a simultaneously opened channel. With the addition of three external K⁺-selective sites per α-polypeptide chain, and the constraint that pump units with their external sites occupied by any univalent cation cannot be phosphorylated by ATP, the model turns out to have the properties of a Na,K-ATPase. It operates in the $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ exchange, $\text{Na}{}^{\mathrm{+}}\text{Na}{}^{\mathrm{+}}$ exchange, $\text{K}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ exchange, K⁺-dependent phosphatase, uncoupled Na⁺ efflux and pump reversal modes. It is concluded that if the modified water in the cleft of the phospho-enzymes has properties similar to those of water at 5°C the pump is competent to exchange three intracellular Na⁺ ions for two extracellular K⁺ ions, and one intracellular Na⁺ ion but it is incapable of exchanging three Na⁺ ions for three K⁺ ions.     

Functional domains of the in situ red cell membrane calcium pump revealed by proteolysis and monoclonal antibodies. Possible sites for regulation by calpain and acidic lipids

The functional domains of the in situ red cell membrane calcium pump were mapped by a double labeling technique. In inside-out vesicles (IOVs) the calcium pump was phosphorylated by [gamma-32P]ATP, the proteins blotted onto nitrocellulose and tagged by monoclonal antibodies raised against the purified pump protein. After proteolytic treatment of the IOVs by trypsin, chymotrypsin, or calpain-I, the fragmentation pattern of the enzyme was followed on the double-labeled immunoblots. The changes in the kinetics of the pump were examined by parallel measurements of the active calcium uptake in IOVs. By analysis of the results of tryptic digestion, it was possible to show that the antibodies recognized three different domains of the pump: 1) a Mr = 10,000-15,000 fragment (not seen directly) which includes the calmodulin-binding domain, 2) a nonphosphorylated Mr = 35,000 tryptic fragment, and 3) a phosphorylated fragment of Mr = 76,000-81,000. Chymotrypsin or calpain-I digestion of the membranes produced one major, Mr = 125,000 fragment, which had lost antibody-binding region 1. Production of this fragment coincided with the loss of calmodulin dependence and with a calmodulin-like activation of IOV calcium uptake (high Vmax, cooperativity in calcium activation). The Mr = 125,000 fragment was further activated by acidic lipids producing high Vmax and low K 1/2 (Ca2+) with no cooperativity. Based on these data a kinetic model and a functional map of the plasma membrane calcium pump is suggested.     

Spontaneous inactivation of the Ca2+-sensitive K+ channels of human red cells at high intracellular Ca2+ activity

Ionophore A23187-mediated Ca²⁺-induced oscillations in the conductance of the Ca²⁺-sensitive K⁺ channels of human red cells were monitored with ion specific electrodes. The membrane potential was continuously reflected in CCCP-mediated pH changes in the buffer-free medium, changes in extracellular K⁺ activity were followed with a K⁺-selective electrode, and changes in the intracellular concentration of ionized calcium were calculated on the basis of cellular ⁴⁵Ca content. An increased cellular ⁴⁵Ca content at the successive minima of the oscillations where the K⁺ channels are closed indicates that the activation of the channels might be a $\text{(}\text{dCa}{}^{\mathrm{2+}}\text{/}\text{d}\text{t)-}\text{sensitive}$ process and that accommodation to enhanced levels of intracellular free calcium may occur. An incipient inactivation of the K⁺ channels at intracellular ionized calcium levels of about 10 μM and a concurrent membrane potential of about ?65 mV was observed. At a membrane potential of about ?70 mV and an intracellular concentration of about 2·10^?4M no inactivation of K⁺ channels took place. Inactivation of the K⁺ channels is suggested to be a compound function of the intracellular level of free calcium and the membrane potential. The observed sharp peak values in cellular ⁴⁵Ca content support the notion that a necessary component of the oscillatory system is a Ca²⁺ pump operating with a significant delay in the activation/inactivation process in response to changes in cellular concentration of ionized calcium.     

Interaction between calcium ions and surface charge as it relates to calcium currents

Summary Calcium ions affect the gating of Ca currents. Surface charge is involved but to what extent is unknown. We have examined this, using isolated nerve cell bodies ofHelix aspersa and the combined microelectrode-suction pipette method for voltage-clamp and internal perfusion. We found that Ba and Sr currents produced by substitution of these ions for extracellular Ca ions are activated at less positive potentials than Ca currents. Mg ions do not permeate the Ca channel and changes in [Mg]₀ produce shifts in the activation-potential curves that are comparable to the effects of changes in [Ba]₀ or [Sr]₀. Inactivation of Ba currents also occurs at less positive potentials. Perfusion intracellularly with EGTA reduced inactivation of Ca currents as a function of potential, but did not shift the inactivation-potential curve. Hence, Ca current-dependent inactivation which is blocked by intracellular EGTA probably does not involve a similar change of intracellular surface potential. The voltage shifts of activation and inactivation produced by extracellular divalent cations used singly or in mixtures can be described by the Gouy-Chapman theory for the diffuse double layer with binding (Gilbert & Ehrenstein, 1969; McLaughlin, Szabo & Eisenman, 1971). From the surface potential values and the Boltzman distribution, we have computed surface concentrations that predict the following experimental observations: 1) saturation of current-concentration relationships when surface potential is changing maximally; 2) the increase in peak current when Ca ions are replaced by Sr or Ba ions; and 3) the greater inhibitory effect of Mg onI _Ba thanI _Ca. Theory indicates that surface charge cannot be screened completely even at 1m [Mg]₀ and thus that Ca channel properties must be evaluated in the light of surface charge effects. For example, after correction for surface charge effects the relative permeabilities of Ca, Ba and Sr ions are equivalent. In the presence of Co ions, however, Ca ions are more permeable than Ba ions suggesting a channel binding site may be involved.     

Active calcium transport and calcium-dependent membrane phosphorylation in human peripheral blood lymphocytes

The characteristics of the calcium pump were investigated in intact human peripheral blood lymphocytes /PBL/ and in inside-out vesicles prepared from their plasma membranes. Intact PBL were loaded with calcium by a short exposure to A23187 ionophore. After the elimination of the ionophore, calcium-loaded PBL produced an ATP-dependent, external lanthanum sensitive, uphill calcium extrusion. Calcium pump in intact PBL was insensitive to ouabain and /until cellular ATP was provided/ to oligomycin and dinitrophenol. Maximum calcium extrusion rate and the alkali cation sensitivity of the process were similar to those in human red cells. Calcium was partially sequestered by PBL, and this calcium could be released by A23187 ionophore only.Inside-out plasma membrane vesicles prepared from hypotonically lysed PBL showed and ATP + Mg²⁺-dependent uphill calcium uptake. This calcium transport was insensitive to ouabain, oligomycin, or dinitrophenol, while blocked by lanthanum and quercetin. Calmodulin significantly stimulated calcium pumping in EDTA-washed vesicles. ATP-dependent and -independent calcium uptake rates, respectively, showed different calcium concentration dependences.When PBL membrane vesicles were phosphorylated by γ ³²P-ATP, a calcium-induced, hydroxylamine-sensitive incorporation of ³²P was found in 120–150 000 molecular weight proteins. Depending on the way of membrane preparation, the molecular weight of the phosphoprotein was shifted. Similarly to that found in red cell membranes, sensitivity to calmodulin stimulation and partial proteolysis of the calcium pump molecule showed an inverse relationship.     

Pancreatic phospholipase A2 is not regulated by calmodulin

It was previously suggested [Wong, P.Y.-K and Cheung, W.Y. (1979) Biochem. Biophys. Res. Comm. $\text{90}$, 473–480] that the Ca²⁺ activation of phospholipase A₂ is mediated by the calcium binding protein calmodulin. In the present study phospholipase A₂ from pig pancreas was shown to be absolutely Ca²⁺ dependent but the enzyme was not stimulated by exogenous calmodulin and no endogenous calmodulin was found in the preparation. The enzyme was inhibited in the absence of calmodulin by several drugs (trifluoperazine, mepacrine, promethazine and propranolol) which are known to bind to calmodulin. A kinetic analysis indicated that trifluoperazine competitively inhibited phospholipase A₂, probably by interacting with phospholipid substrate.     

Interaction of metal cations with Ca2+-transport sites of the plasma membrane Ca2+ pump of secretory cells of gastric glands

Investigation of Ca2+ transport by calcium pump of the cell plasma membrane of the gastric glands isolated from guinea pigs and its inhibition by metal cations has been performed. The mainly competitive type of Ca2+ translocation inhibition by the calcium pump by metals cations (0.025-1.00 mM) was determined. Potency of inhibition increases in such an order (I50, mM): Ba2+ (0.336) < Sr2+ (0.251) < Mn2+ (0.099) < Co2+ (0.029) < Cd2+ (0.016). It was shown by one-factor dispersion analysis that potency of inhibition depends on ionic radii and hydration enthalpy of metal cations and also on stability constants of their complexes with oxygen-containing bioligands (acetic, aspartic and glutamic acid) (hx2 = 83.73-85.95). Dependence of the inhibition constants (I50) on ionic radii is most adequately described by the parabolic equation, such a dependence on hydration enthalpy and stability constants with oxygen-containing bioligands--by exponential or multiplicative equations. The conclusion has been made that selective Ca2+ translocation by the calcium pump and its inhibition by metal cations is determined by the interaction between energy of their interaction with cation-binding sites of the transport system and energy of hydration. Energetics of such interactions depends on the steric factors. The physicochemical model of the Ca2+ selective translocation by calcium pump and its inhibition by metal cations has been proposed.     

Mechanism of the sarcoplasmic reticulum calcium pump. Fluorometric study of the phosphorylated intermediates.

After removal of calcium ions bound to the high affinity sites the sarcoplasmic reticulum calcium pump can be phosphorylated by inorganic phosphate. The intrinsic fluorescence of the protein is used to follow conformational changes of the pump and an intensity change can be observed upon addition of phosphate. This effect is activated by internal calcium ($\text{K}\text{1}\text{2}\text{= 10 mM}$) and inhibited by external calcium ($\text{K}\text{1}\text{2}\text{= 0.4 μM}$) and the apparent affinity for phosphate is high (0.2 mM). We conclude that the change observed is linked to the formation of the gradient-dependent phosphorylated intermediate. It is compared with previous results concerning the enzymatic cycle of the pump.     

Complementary metal ion specificity of the metal-citrate transporters CitM and CitH of Bacillus subtilis

Citrate uptake in Bacillus subtilis is stimulated by a wide range of divalent metal ions. The metal ions were separated into two groups based on the expression pattern of the uptake system. The two groups correlated with the metal ion specificity of two homologous B. subtilis secondary citrate transporters, CitM and CitH, upon expression in Escherichia coli. CitM transported citrate in complex with Mg(2+), Ni(2+), Mn(2+), Co(2+), and Zn(2+) but not in complex with Ca(2+), Ba(2+), and Sr(2+). CitH transported citrate in complex with Ca(2+), Ba(2+), and Sr(2+) but not in complex with Mg(2+), Ni(2+), Mn(2+), Co(2+), and Zn(2+). Both transporters did not transport free citrate. Nevertheless, free citrate uptake could be demonstrated in B. subtilis, indicating the expression of at least a third citrate transporter, whose identity is not known. For both the CitM and CitH transporters it was demonstrated that the metal ion promoted citrate uptake and, vice versa, that citrate promoted uptake of the metal ion, indicating that the complex is the transported species. The results indicate that CitM and CitH are secondary transporters that transport complexes of divalent metal ions and citrate but with a complementary metal ion specificity. The potential physiological function of the two transporters is discussed.