Effect of Ca2+ on thermotropic properties of saturated phosphatidylcholine liposomes

The effect of various concentrations of calcium ion (Ca²⁺) on dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) and mixed DPPC/DSPC (1:1) liposomes was studied by differential scanning calorimetry. Ca²⁺ concentrations of 10 mM and above split the main transition peak of DPPC into two distinguishable components, and, at 30 mM and above, also resulted in the disappearance of a pre-transition peak. The effect of Ca²⁺ on DSPC liposomes was even more dramatic in that it induced a more definitive split in the main transition peak and caused the loss of the pretransition peak at only 10 mM concentration. The thermograms of the DPPC/DSPC mixed liposomes were unaltered in the presence of Ca²⁺, even at a concentration of 50 mM. Whether or not Ca²⁺ is able to alter thermograms of phosphatidylcholine liposomes appears to be dependent on the degree of molecular order of the bilayer prior to interaction with Ca²⁺.     

Ca2+-transporting properties of myometrial plasma cell membrane Ca2+, Mg2+-ATPase reconstituted in liposomes

Purified myometrium cells plasma membrane Ca2+, Mg(2+)-ATPase was reconstitute in liposomes in functionally active state by the method of cholate dialysis: it showed ATP-hydrolase activity increased by 0.8 microM A23187 average 4 times and it showed Mg2+, ATP-dependent Ca(2+)-transporting activity. Reconstituted system transported Ca2+ at an initial rate of 114.4 +/- 16.3 nmol.min-1.mg-1 with the stoichiometry Ca2+: ATP = 1: (3.2-3.7). Calmodulin increased by 30% the initial rate of Ca(2+)-accumulation by the proteoliposomes with reconstituted Ca2+, Mg(2+)-ATPase; 0.1 mM orthovanadate decreased by 80% Ca(2+)-accumulation by this system. Ca2+, Mg(2+)-ATPase reconstituted in liposomes is just Ca(2+)-transporting ATPase of the plasma membrane. Obtained enzyme preparate can be utilised for study of the properties of this important energy-dependent Ca(2+)-transporting system of smooth muscle cell.     

Reconstruction of purified Ca2+,Mg2+-ATPase from the myometrium sarcolemma into liposomes and its catalytic properties

The Ca2+, Mg2(+)-ATPase of the myometrium sarcolemma purified by the method of affinity chromatography on calmodulin sepharose is reconstituted into azolectin liposomes in the functionally active form by means of cholate dialysis. The ATPase-dependent accumulation of 45Ca is shown on the obtained model system. It makes up 95% of the total accumulation and may decrease to 43% under the effect of 0.8 microM A23187. Ca2+, Mg2(+)-ATPase reconstituted into azolectin liposomes is in the high affinity to Ca2+; Km for Ca2+ is equal to 0.88 +/- 0.22 microM, calmodulin practically does not change it. The highest activity of the reconstituted enzyme is observed at pH 7.0, temperature 50 degrees C, the Mg-ATP concentration 1-2 mM. The Km for substrate is 0.45 +/- 0.02 mM.     

Calorimetric and freeze-etch study of the influence of Mg2+ on the thermotropic behaviour of phosphatidylglycerol

In the presence of Mg²⁺ ions phosphatidylglycerol shows supercooling which leads to the formation of a metastable gel phase. This contrasts with the behaviour of this negatively charged phospholipid in the presence of Ca²⁺ ions (Biochim. Biophys. Acta 339 (1974) 432). It is demonstrated that the heat content of this phospholipid is dependent on the ionic environment.     

Effect of melittin on thermotropic lipid state transitions in phosphatidylcholine liposomes.

We have examined the Raman scattering due to CH stretching vibrations, as well as to v(-C=C-) and v(=C-C=) of beta-carotene, of liposomes composed of phosphatidylcholine (egg, dimyristoyl, dipalmitoyl) +/- cholesterol, beta-carotene or melittin in the temperature range of -10 degrees C to 45 degrees C. (2) Plots vs. temperature of the intensities of the 2885 cm-1 and 2930 cm-1 CH stretching bands relative to the intensity of the thermally stable 2850 cm-1 band, i.e. the I2885/I2850 and I2930/I2850 ratios, reveal a sharp discontinuity in cholesterol-free phosphatidylcholine liposomes; this coincides with the gel leads to liquid-crystal transition temperature of the fatty acyl chains. In cholesterol/phosphatudylcholine liposomes the change in I2885/I2850 occurs over a very broad temperature range and I2930/I2850 remains stable. (3) I1527/I1158, i.e. the intensity of v(-C=C-) relative to that of v(=C-C-) in beta-carotene/phosphatidylcholine liposomes, changes discontinuously at the gel leads to liquid-crystal transition temperature. The values above the transition temperature approach those of the carotenoid in organic solution. (4) The transitions reported in I2885/I2850 for phosphatidylcholine/melittin liposomes (25-56; 1, M/M) are shifted to much higher temperatures than observed in phosphatidylcholine liposomes. In the case of dimyristoyl phosphatidylcholine/melittin the changes in I2930/I2850 also occurs at a higher temperature (28 degrees C) than without melittin (21 degrees C), but the temperature shift is less than the +13 degrees C observed for I2885/I2850. It appears that the apolar moiety of melittin organizes phospholipids adjacent to and more remote from the peptide moiety, to form complexes with an elevated lipid transition temperature. The effect of the peptide moiety is greater on the methylene segments (I2885/I2850) than on the methyl termini (I2930/I2850).     

Mechanisms of Ca2+ transport in plasma membrane vesicles prepared from cultured pituitary cells. II. (Ca2+ + Mg2+)-ATPase-dependent Ca2+ transport activity

Purified plasma membrane vesicles from GH3 rat anterior pituitary cells exhibit a Mg2+-ATP-dependent Ca2+ transport activity. Concentrative uptake of Ca2+ is abolished by exclusion of either Mg2+ or ATP or by inclusion of the Ca2+ ionophore A23187. Furthermore, addition of A23187 to vesicles which have reached a steady state of ATP-supported Ca2+ accumulation rapidly and completely discharges accumulated cation. Ca2+ uptake is unaffected by treatment of vesicles with oligomycin, the uncoupler CCCP, or valinomycin and is greatly reduced in non-plasma membrane fractions. Likewise, Ca2+ accumulation is not stimulated by oxalate, consistent with the plasma membrane origin of this transport system. (Na+, K+)-ATPase participation in the Ca2+ transport process (i.e. via coupled Na+/Ca2+ exchange) was eliminated by omitting Na+ and including ouabain in the reaction medium. Ca2+ transport activity in GH3 vesicles has a similar pH dependence as that seen in a number of other plasma membrane systems and is inhibited by orthovanadate in the micromolar range. Inhibition is enhanced if the membranes are preincubated with vanadate for a short time. A kinetic analysis of transport indicates that the apparent Km for free Ca2+ and ATP are 0.7 and 125 microM, respectively. The average Vmax is 3.6 nmol of Ca2+/min/mg of protein at 37 degrees C. Addition of exogenous calmodulin or calmodulin antagonists had no significant effect on these kinetic properties. GH3 plasma membranes also contain a Na+/Ca2+ exchange system. The apparent Km for Ca2+ is almost 10-fold higher in this system than that for ATP-driven Ca2+ uptake. When both processes are compared under similar conditions, the Vmax of the exchanger is approximately 2-3 times that of ATP-dependent Ca2+ accumulation. Similar results are obtained when purified plasma membranes from bovine anterior pituitary glands were investigated. It is suggested that both Na+/Ca2+ exchange and the (Ca2+ + Mg2+)-ATPase are important in controlling intracellular levels of Ca2+ in anterior pituitary cells.     

The influence of pH, Ca and protein on the thermotropic behaviour of the negatively charged phospholipid, phosphatidylglycerol

It is demonstrated that the transition temperature from the liquid-crystalline to gel state of a synthetic phosphatidylglycerol is influenced by pH, Ca²⁺ and A₁ basic protein from myelin.     

Ca2+ and Mg2+ Influence the Thermodynamics of Peptide-Membrane Interactions

Accurate quantitative estimates of protein-membrane interactions are critical to studies of membrane proteins. Here, we demonstrate that thermodynamic analyses based on current hydropathy scales do not account for the significant and experimentally determined effects that Ca²⁺ or Mg²⁺ have on protein-membrane interactions. We examined distinct modes of interaction (interfacial partitioning and folding and transmembrane insertion) by studying three highly divergent peptides: Bid-BH3 (derived from apoptotic regulator Bid), peripherin-2-derived prph2-CTER, and the cancer-targeting pH-Low-Insertion-Peptide (pHLIP). Fluorescence experiments demonstrate that adding 1–2 mM of divalent cations led to a substantially more favorable bilayer partitioning and insertion, with free energy differences of 5–15 kcal/mol.     

Molecular reorganization induced by Ca2+ of plant photosystem I reconstituted into phosphatidylglycerol liposomes

The interaction of divalent cations with biomembranes is important for a number of biological processes. In this study, the regulatory effect of Ca2+ on the interaction between plant spinach photosystem I (PSI) particles and negatively charged lipid phosphatidylglycerol (PG) was investigated by circular dichroism (CD) spectroscopy. It was found that in the absence of CaCl2, PG causes an increase in alpha-helix and a decrease in disordered conformations of protein secondary structures of PSI, the beta-sheet and turns being almost unaffected. Meanwhile, the same effect also enhances the excitonic interactions relating to Chl a and Chl b from the PSI core complex and external antenna light-harvesting complex (LHCI). By contrast, in the presence of CaCl2, PG hardly interferes with the structure of the proteins' skeleton of PSI, but it can depress the excitonic interactions for Chl b of LHCI and for PSI core complex Chl a at (-) 433.5 nm of the CD signal which is accompanied by a blue shift of its peak. It is most likely that the neutralization of the phosphate groups in the PSI-PG complex and the negative surface charges of PSI, and partial dehydration in the vicinity of the ester CO region of the PG polar head group by the Ca-ions modify the interaction between PSI and PG, thereby inducing molecular reorganization of protein and pigments within both the external antenna LHCI and PSI core complex in proteoliposomes.     

Influence of dodecyltrimethylammonium halides on thermotropic phase behaviour of phosphatidylcholine/cholesterol bilayers

Effects of dodecyltrimethylammonium chloride (DTAC), dodecyltrimethylammonium bromide (DTAB) and dodecyltrimethylammonium iodide (DTAI) on thermotropic phase behaviour of phosphatidylcholine bilayers containing cholesterol as well as on 1H NMR spectra were studied. Two series of experiments were performed. In the first one the surfactants were added to the water phase while in the other directly to the lipid phase (a mixed film from cholesterol, surfactant and phosphatidylcholine was formed). The effects of particular surfactants on the main phase transition temperature, Tm, were more pronounced when added to the lipid phase (2nd method) than to the water phase (1st method); the opposite happened when cholesterol was absent (Rózycka-Roszak and Pruchnik 2000, Z. Naturforsch. 55c, 240-244). Furthermore, in the case of the first method the transitions were asymmetrical while in the second method nearly symmetrical. It is suggested that surfactant poor and surfactant rich domains are formed when surfactants are added to the water phase.     

The effect of Mg2+ on the Ca2+-binding properties of non-activated phosphorylase kinase.

The calcium binding properties of non-activated phosphorylase kinase at pH 6.8 have been studied by the gel filtration technique at calcium concentrations from 50 nM to 50 muM. Taking into account the subunit structure alpha4beta4gamma4 the enzyme binds 12 mol Ca2+ per mol with an association constant of 6.0 X 10(7) M-1, 4 mol with an association constant of 1.7 X 10(6) M-1 and 36 mol with a binding constant of 3.9 X 10(4) M-1 at low ionic strength. In buffer of high ionic strength, i.e. 180 mM NH4Cl or 60 mM (NH4)2SO4, only a single set of eight binding sites with a binding constant of 5.5 X 10(7) M-1 is left. In a buffer containing 155 mM NH4Cl and 10 mM MgCl2, the calcium affinity of these sites is reduced to a KCa of 3.0 X 10(6) M-1, indicating competition between Ca2+ and Mg2+. From these measurements, the binding constant of Mg2+ for these sites is calculated to be 1.7 X 10(3) M-1 is left. In a buffer containing 155 mM NH4Cl and 10 mM MgCl2, the calcium affinity of these sites is reduced to a KCa of 3.0 X 10(6) M-1, indicating competition between Ca2+ and Mg2+. from these measurements, the binding constant of Mg2+ for these sites is calculated to be 1.7 X 10(3) M-1. Additionally, 10 mM Mg2+ induces a set of four new Ca2+ binding sites which show positive cooperativity. Their half-saturation constant under the conditions described is 3.5 X 10(5) M-1, and they, too, exhibit competition between Ca2+ and Mg2+. Since this set of sites is induced by Mg2+ a third group of binding sites for the latter metal must be postulated.     

Properties of (Mg2+ + Ca2+)-ATPase of erythrocyte membranes prepared by different procedures: Influence of Mg2+, Ca2+, ATP,and protein activator

Erythrocyte membranes prepared by three different procedures showed (Mg²⁺ + Ca²⁺)-ATPase activities differing in specific activity and in affinity for Ca²⁺. The (Mg²⁺ + Ca²⁺)-ATPase activity of the three preparations was stimulated to different extents by a Ca²⁺-dependent protein activator isolated from hemolystes. The Ca²⁺ affinity of the two most active preparations was decreased as the ATP concentration in the assay medium was increased. Lowering the ATP concentration from 2 mM to 2–200 μM or lowering the Mg:ATP ratio to less than one shifted the (Mg²⁺ + Ca²⁺)-ATPase activity in stepwise hemolysis membranes from mixed “high” and “low” affinity to a single high Ca²⁺ affinity. Membranes from which soluble proteins were extracted by EDTA (0.1 mM) in low ionic strengh, or membranes prepared by the EDTA (1–10 mM) procedure, did not undergo the shift in the Ca²⁺ affinity with changes in ATP and MgCl₂ concentrations. The EDTA-wash membranes were only weakly activated by the protein activator. It is suggested that the differences in properties of the (Mg²⁺ + Ca²⁺)-ATPase prepared by these three procedures reflect differences determined in part by the degree of association of the membrane with a soluble protein activator and changes in the state of the enzyme to a less activatable form.     

Effect of encapsulated Ca2+ on the surface properties of curved phosphatidylcholine bilayers

In this paper, the influence of Ca2+ on the adsorption properties of 1,8-anilinonaphthalenesulfonate (ANS) and analogous probes to sonicated vesicles of phosphatidylcholine was studied by means of spectrofluorometry. The fluorescence of ANS added to the vesicle dispersion increases with the Ca2+ concentration in the inner media but remains constant when Ca2+ concentration is changed in the outside solution. However, the fluorescence decreases when large anions such as ClO4- are present in the external solution. Ca2+ inside large liposomes promotes a similar behaviour to that found in sonicated vesicles when they are osmotically contracted in hypertonic media. The results can be interpreted in terms of Ca2+ adsorption on the inner interface and a cooperative interaction between the monolayers.     

The effects of Mg2+ on the Ca2+-binding properties and Ca2+-induced tyrosine-fluorescence changes of calmodulin isolated from rabbit skeletal muscle

Calmodulin from phosphorylase kinase (the delta subunit) was obtained as a homogeneous protein in a spectroscopically pure form, and its interaction with Ca2+ and Mg2+ was studied. 1. Determination of the binding of Ca2+ to calmodulin in a buffer of low ionic strength (0.001 M) show that it contained six binding sites for this divalent cation. 2. Employment of a buffer of high ionic strength (0.18 M) allowed two Ca2+/Mg2+-binding sites (KdCa2+ = 4.0 microM), which showed Ca2+ - Mg2+ competition (KdMg2+ = 0.75 mM), to be distinguished from two Ca2+-specific binding sites (KdCa2+ = 40 microM). The remaining two Ca2+-binding sites are not observed under these conditions and are probably Mg2+-specific binding sites. Thus, the binding sites on calmodulin are remarkably similar to those of the homologous Ca2+-binding protein, troponin C [Potter and Gergely (1975) J. Biol. Chem. 250, 4628, 4633]. 3. The conformational states of calmodulin are defined by Ca2+, Mg2+ and salt concentrations, which can be differentiated by their Ca2+ affinity and their relative tyrosine fluorescence intensity. In a buffer of high ionic strength, Mg2+ induces a conformation which enhances the apparent affinity for Ca2+. Addition of Ca2+ leads to an enhancement of the tyrosine fluorescence intensity, which remains enhanced even upon removal of Ca2+ by chelation with EGTA. Only additional chelation of Mg2+ with EDTA reduces the tyrosine fluorescence intensity. 4. Comparison of the Ca2+-binding parameters of phosphorylase kinase, which were previously determined under identical experimental conditions [Kilimann and Heilmeyer (1977) Eur. J. Biochem. 73, 191-197], with those reported here on calmodulin isolated from this enzyme, allows the conclusion that Ca2+ binding to the holoenzyme occurs by binding to the delta subunit exclusively. 5. Ca2+ binding and Ca2+ activation of phosphorylase kinase are compared and discussed in relation to the Ca2+ and Mg2+-induced conformation changes of calmodulin.     

Catalytic properties of purified Ca2+,Mg2+-ATPase from the myometrium sarcolemma

The catalytic properties of myometrium sarcolemmal Ca2+, Mg2(+)-ATPase purified from plasma membrane solubilizate by affinity chromatography on calmodulin-Sepharose were investigated. The enzyme isolated in the presence of azolectin revealed a calmodulin-independent affinity for Ca2+ (Km = 0.17 microM). Purified Ca2+, Mg2(+)-ATPase displayed a strict substrate specificity, was inhibited by low concentrations of o-vanadate and was insensitive to oxytocin and prostaglandins E2 and F2 alpha. The enzyme activity was maximal at 45 degrees C, pH 7.5-8.0, and at Mg-ATP and Ca2+ concentrations of 1.5-2.5 mM and 5-20 microM, respectively.     

Effects of local anesthetics on the passive permeability of sarcoplasmic reticulum vesicles to Ca2+ and Mg2+

Sarcoplasmic reticulum vesicles are used here as model membrane system to question the hypothesis of enhancement of permeability of cations by anesthetics, particularly that of Ca2+ and of Mg2+. The effects of dibucaine (up to 800 microM), tetracaine (up to 2 mM), lidocaine (up to 10 mM) and procaine (up to 10 mM) on the permeability of these membranes to Ca2+ and Mg2+ have been measured. We have used an experimental approach based on the light scattering method (Kometani, T. and Kasai, M. (1978) J. Membrane Biol. 41, 295-308). It has been found that all the local anesthetics cited above markedly increase the permeability of sarcoplasmic reticulum vesicles to Mg2+ and, in the concentration range tested herein, only dibucaine and tetracaine increase the permeability to Ca2+. The kinetic analysis of the time dependence of the light-scattering data after the osmotic shock shows that, in the absence of local anesthetics, the Mg2+ influx can be described as proceeding through a unique type of channel. However, Ca2+ influx appears to involve two channel of different kinetic properties. Because the relative fraction of both types of Ca2+ channel is similar to the average ratio between light and heavy vesicles in unfractionated sarcoplasmic reticulum, we suggest that each type of channel can be preferentially located in one of these fractions. The determined rate constants for Ca2+ permeability through both types of channel are 0.77 +/- 0.08 min-1 (fast channels) and 0.025 +/- 0.005 min-1 (slow channels) and that for Mg2+ is 0.08 +/- 0.02 min-1. These results agree with data obtained by other groups using different experimental approaches. Dibucaine and tetracaine significantly alter the rate of Mg2+ and Ca2+ influx through the slow channels. In addition, these two local anesthetics also produce the effect that the Mg2+ influx cannot be described with only one exponential process, thus suggesting a differential effect on vesicles of different density. The increase of Ca2+ and Mg2+ permeability by dibucaine and by tetracaine is found at concentrations of these drugs that do not produce a noticeable inhibition of the (Ca2+ + Mg2+)-ATPase activity of sarcoplasmic reticulum vesicles.     

Some properties of the purified (Ca2+ + Mg2+-ATPase from human red cell membranes

The (Ca²⁺ + Mg²⁺-ATPase from red cell membranes, purified by means of a calmodulin-containing affinity column according to the method of Gietzen et al. (Gietzen, K., Tej?ka, M. and Wolf, H.U. (1980) Biochem. J. 189, 81–88) with either phosphatidylcholine or phosphatidylserine as phospholipid is characterized. The phosphatidylcholine preparation can be activated by calmodulin, while the phosphatidylserine preparation is fully activated without calmodulin. The enzyme shows a biphasic ATP dependence with two K_m values of 3.5 and 120 μM. The enzyme is phosphorylated by ATP in the presence of Ca²⁺ only.