Interaction of isolated brain synaptic vesicles with flat bilayer membranes in the rat

The conductivity of planar bilayer membrane comprising asolectin and phosphatidylserine (concentration ratio 9:1) in a buffer solution increased sharply in the presence of synaptic vesicles (SV) isolated from the rat brain and added to one side of the membrane only. The bilayer remained stable upon modification, and the conductivity increment was dependent on SV concentration in the range from 4 to 16 mu of the total protein per ml. If I mM CaCl2 was present in the buffer solution, the conductivity increased by 2 to 3 orders of magnitude upon the addition of SV at a final concentration of 3-4 mu protein per ml. The membrane was unstable and its rupture occurred often at an early stage of conductivity changes. In the absence of SV addition the membrane was stable, with its conductivity remaining unchanged for 2 h and more. With I mM CaCl2 addition to the solution already containing SV, no conductivity changes were observed, the cause perhaps, being Ca2+-induced SV aggregation.     

Effects of the fraction (1-10 kDa) of the brain of a Yakut horse on kinetic parameters of Ca2+ transport system in sarcolemma vesicles of cardiomyocytes]

The effect of the fraction (1-10 kDa) obtained from the brain of cold-adapted animal (Yakut horse) on Ca2+ transport in sarcolemma vesicles of cardiomyocytes was investigated. It was shown that during insertion of Yakut horse brain fraction into incubation medium at the concentration from 10(-9) M to 3.10(-5) M at Ca2+ transport substrate concentration from 0.1 mM to 1.0 mM, the rate of Ca2+ passive penetration into vesicles slightly increased and at Ca2+ transport substrate concentration 3 mM, which is physiologic, a decrease of rate values was established for all concentrations of the fraction (1-10 kDa) of Yakut horse. While studying the kinetics of an active Ca2+ transport for all investigated concentrations of the fraction (1-10 kDa) of Yakut horse brain from 10(-9) M to 3.10(-5) M at Ca2+ concentration in incubation medium from 10(-7) to 3.10(-6) M, calcium accumulation rates by vesicles exceeded control values. So we can suppose that application of brain fraction (1-10 kDa) of genotypically cold-adapted animal, results in a decrease of intracellular Ca2+ concentration.     

Structural changes in dipalmitoylphosphatidylcholine bilayer promoted by Ca2+ ions: a small-angle neutron scattering study

Small-angle neutron scattering (SANS) curves of unilamellar dipalmitoylphosphatidylcholine (DPPC) vesicles in 1-60mM CaCl2 were analyzed using a strip-function model of the phospholipid bilayer. The fraction of Ca2+ ions bound in the DPPC polar head group region was determined using Langmuir adsorption isotherm. In the gel phase, at 20 degrees C, the lipid bilayer thickness, dL, goes through a maximum as a function of CaCl2 concentration (dL=54.4A at approximately 2.5mM of CaCl2). Simultaneously, both the area per DPPC molecule AL, and the number of water molecules nW located in the polar head group region decrease (DeltaAL=AL(DPPC))-AL(DPPC+Ca)=2.3A2 and Deltan=n(W(DPPC))-n(W(DPPC+Ca))=0.8mol/mol at approximately 2.5mM of CaCl2). In the fluid phase, at 60 degrees C, the structural parameters d(L), A(L), and n(W) show evident changes with increasing Ca2+ up to a concentration C(Ca)(2+) < or = 10mM. DPPC bilayers affected by the calcium binding are compared to unilamellar vesicles prepared by extrusion. The structural parameters of DPPC vesicles prepared in 60mM CaCl2 (at 20 and 60 degrees C) are nearly the same as those for unilamellar vesicles without Ca2+.     

Mechanism of passive Ca2+ permeability of vesicular sarcolemmal preparations from rat hearts

Vesicular sarcolemmal preparations isolated from rat hearts were characterized by high total ATPase (4.32 +/- 0.57 mumol/min per mg), adenylate cyclase (121 +/- 11 pmol/min per mg) and creatine kinase (1.73 +/- 0.35 mumol/min per mg) activities as well as Na-Ca exchange specific to sodium. ATPase activity was inhibited with digitoxigenin by 50-70% and was not changed by ouabain, ionophore A23187 or oligomycin. Sarcolemmal vesicles bound [3H]digitoxigenin and [3H]ouabain in isotonic medium in the presence of Pi and Mg2+. The number of binding sites for hydrophobic digitoxigenin (N = 237 pmol/mg) was several-times higher than that for hydrophilic ouabain (N = 32.7 pmol/mg). These data show that sarcolemmal preparations were not significantly contaminated by mitochondria and sarcoplasmic reticulum and consisted mostly of inside-out vesicles. Incubation of these vesicles with 45Ca2+ (0.5-10 mM) led to penetration of the latter into the vesicles with the following binding characteristics: number of binding sites (N = 20.5 +/- 4.6 nmol/mg, Kd approximately equal to 2.0 mM). Ca2+ binding to the inner surface of vesicles was proved by the following facts: (1) Ca2+ ionophore A23187 increased slightly total intravesicular Ca2+ content but markedly accelerated Ca2+ efflux along its concentration gradient; (2) gramicidin and osmotic shock showed a similar accelerating effect. Ca2+ efflux from the vesicles along its concentration gradient ([Ca2+]i/[Ca2+]e = 2.0 mM/0.1 microM) was inhibited by Mn2+, Co2+, and verapamil when they acted inside the vesicles. The rate of Ca2+ efflux was hyperbolically dependent on intravesicular Ca2+ concentration (Km approximately equal to 2.9 mM). These data reveal that Ca2+ efflux from sarcolemmal vesicles is controlled by Ca2+ binding to the sarcolemmal membrane. Ca2+ efflux from the vesicles was stimulated 1.7--times after incubation of vesicles with 0.2 mM MgATP or MgADP and 15-times after treatment with 0.2 mM adenylyl beta, gamma-imidodiphosphate. Enhancement in the rate of Ca2+ efflux correlated with the increase in the intravesicular Ca2+ content. ATP-stimulated Ca2+ efflux was suppressed by verapamil and was nonmonotonically dependent upon the transmembrane potential created by the K+ concentration gradient in the presence of valinomycin, Ca2+ efflux being slower at extreme values of membrane potential (+/- 80 mV).     

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.     

Inhibitory effects of monovalent cations on the Ca2+-induced aggregation of porcine intestinal brush border membranes

The Ca2+-induced aggregation of porcine intestinal brush border membranes could be inhibited by addition of monovalent cations to the medium or by increasing the ionic strength of the medium, as measured by the change in optical density of the membrane suspension. The relative effectiveness of monovalent cations at 100 mM in the inhibition was in the order, (Na+ approximately equal to NH4+) greater than (K+ approximately equal to Rb+ approximately equal to Li+) greater than choline+. The Ca2+ concentration dependence profile of the membrane aggregation showed that the Ca2+ threshold at which the aggregation began was distinctly shifted to a higher concentration by the addition of KCl. In addition, the results of fluorometric studies with 1-anilino-8-naphthalene sulfonate suggested that the inhibition of the membrane aggregation by extravesicular KCl is due to a decrease of the binding affinity of Ca2+ for the membranes as a result of neutralization of the surface charges. On the other hand, measurements of the incorporation of 1,6-diphenyl-1, 3,5-hexatriene (DPH) into the membrane vesicles and of the anisotropy of DPH-labeled membranes suggested that the imposition of a salt gradient across the membrane vesicles (out greater than in) causes an increase of lipid fluidity of the membranes. Based on these results, a possible contribution of membrane surface charges and/or membrane fluidity to the Ca2+-induced aggregation of the membranes is discussed.     

Studies on membrane fusion. III. The role of calcium-induced phase changes.

The interaction of phosphatidylserine vesicles with Ca2+ and Mg2+ has been examined by several techniques to study the mechanism of membrane fusion. Data are presented on the effects of Ca2+ and Mg2+ on vesicle permeability, thermotropic phase transitions and morphology determined by differential scanning calorimetry, X-ray diffraction, and freeze-fracture electron microscopy. These data are discussed in relation to information concerning Ca2+ binding, charge neutralization, molecular packing, vesicle aggregation, phase transitions, phase separations and vesicle fusion. The results indicate that at Ca2+ concentrations of 1.0-2.0 mM, a highly cooperative phenomenon occurs which results in increased vesicle permeability, aggregation and fusion of the vesicles. Under these conditions the hydrocarbon chains of the lipid bilayers undergo a phase change from a fluid to a crystalline state. The aggregation of vesicles that is observed during fusion is not sufficient range of 2.0-5.0 mM induces aggregation of phosphatidylserine vesicles but no significant fusion nor a phase change. From the effect of variations in pH, temperature, Ca2+ and Mg2+ concentration on the fusion of vesicles, it is concluded that the key event leading to vesicle membrane fusion is the isothermic phase change induced by the bivalent metals. It is proposed that this phase change induces a transient destabilization of the bilayer membranes that become susceptible to fusion at domain boundaries.     

Kinetics of rapid Ca2+ release by sarcoplasmic reticulum. Effects of Ca2+, Mg2+, and adenine nucleotides

A radioisotope flux-rapid-quench-Millipore filtration method is described for determining the effects of Ca2+, adenine nucleotides, and Mg2+ on the Ca2+ release behaviour of "heavy" sarcoplasmic reticulum (SR) vesicles. Rapid 45Ca2+ efflux from passively loaded vesicles was blocked by the addition of Mg2+ and ruthenium red. At pH 7 and 10(-9) M Ca2+, vesicles released 45Ca2+ with a low rate (k = 0.1 s-1). An increase in external Ca2+ concentration to 4 microM or the addition of 5 mM ATP or the ATP analogue adenosine 5'-(beta,gamma-methylenetriphosphate) (AMP-PCP) resulted in intermediate 45Ca2+ release rates. The maximal release rate was observed in media containing 4 microM Ca2+ and 5 mM AMP-PCP and had a first-order rate constant of 30-100 s-1. Mg2+ partially inhibited Ca2+- and nucleotide-induced 45Ca2+ efflux. In the absence of AMP-PCP, 45Ca2+ release was fully inhibited at 5 mM Mg2+ or 5 mM Ca2+. The composition of the release media was systematically varied, and the flux data were expressed in the form of Hill equations. The apparent n values of activation of Ca2+ release by ATP and AMP-PCP were 1.6-1.9. The Hill coefficient of Ca2+ activation (n = 0.8-2.1) was dependent on nucleotide and Mg2+ concentrations, whereas the one of Mg2+ inhibition (n = 1.1-1.6) varied with external Ca2+ concentration. These results suggest that heavy SR vesicles contain a "Ca2+ release channel" which is capable of conducting Ca2+ at rates comparable with those found in intact muscle. Ca2+, AMP-PCP (ATP), and Mg2+ appear to act at noninteracting or interacting sites of the channel.     

Abnormal rapid Ca2+ release from sarcoplasmic reticulum of malignant hyperthermia susceptible pigs.

Using the rapid filtration technique to investigate Ca2+ movements across the sarcoplasmic reticulum (SR) membrane, we compare the initial phases of Ca2+ release and Ca2+ uptake in malignant hyperthermia susceptible (MHS) and normal (N) pig SR vesicles. Ca2+ release is measured from passively loaded SR vesicles. MHS SR vesicles present a 2-fold increase in the initial rate of calcium release induced by 0.3 microM Ca2+ (20.1 +/- 2.1 vs. 6.3 +/- 2.6 nmol mg-1 s-1). Maximal Ca2+ release is obtained with 3 microM Ca2+. At this optimal concentration, rate of Ca2+ efflux in absence of ATP is 55 and 25 nmol mg-1 s-1 for MHS and N SR, respectively. Ca(2+)-induced Ca2+ release is inhibited by Mg2+ in a dose-dependent manner for both MHS and N pig SR vesicles (K1/2 = 0.2 mM). Caffeine (5 mM) and halothane (0.01% v/v) increase the Ca2+ sensitivity of Ca(2+)-induced Ca2+ release. ATP (5 mM) strongly enhances the rate of Ca2+ efflux (to about 20-40-fold in both MHS and N pig SR vesicles). Furthermore, both types of vesicles do not differ in their high-affinity site for ryanodine (Kd = 12 nM and Bmax = 6 pmol/mg), lipid content, ATPase activity and initial rate of Ca2+ uptake (0.948 +/- 0.034 vs. 0.835 +/- 0.130 mumol mg-1 min-1 for MHS and N SR, respectively). Our results show that MH syndrome is associated to a higher rate of Ca2+ release in the earliest phase of the calcium efflux.     

Adenine nucleotide stimulation of Ca2+-induced Ca2+ release in sarcoplasmic reticulum

Rabbit skeletal muscle sarcoplasmic reticulum was fractionated into a "Ca2+-release" and "control" fraction by differential and sucrose gradient centrifugation. External Ca2+ (2-20 microM) caused the release of 40 nmol of 45Ca2+/mg of protein/s from Ca2+-release vesicles passively loaded at pH 6.8 with an internal half-saturation Ca2+ concentration of 10-20 mM. Ca2+-induced Ca2+ release had an approximate pK value of 6.6 and was half-maximally inhibited at an external Ca2+ concentration of 2 X 10(-4) M and Mg2+ concentration of 7 X 10(-5) M. 45Ca2+ efflux from control vesicles was slightly inhibited at external Ca2+ concentrations that stimulated the rapid release of Ca2+ from Ca2+-release vesicles. Adenine, adenosine, and derived nucleotides caused stimulation of Ca2+-induced Ca2+ release in media containing a "physiological" free Mg2+ concentration of 0.6 mM. At a concentration of 1 mM, the order of effectiveness was AMP-PCP greater than cAMP approximately AMP approximately ADP greater than adenine greater than adenosine. Other nucleoside triphosphates and caffeine were minimally effective in increasing 45Ca2+ efflux from passively loaded Ca2+-release vesicles. La3+, ruthenium red, and procaine inhibited Ca2+-induced Ca2+ release. Ca2+ flux studies with actively loaded vesicles also indicated that a subpopulation of sarcoplasmic reticulum vesicles contains a Ca2+ permeation system that is activated by adenine nucleotides.     

Ricinus communis agglutinin-mediated agglutination and fusion of glycolipid-containing phospholipid vesicles: effect of carbohydrate head group size, calcium ions, and spermine

The glycolipids galactosylcerebroside (GalCer), lactosylceramide (LacCer), and trihexosylceramide (Gb3) were inserted into phospholipid vesicles, consisting of phosphatidylethanolamine and phosphatidic acid. The extent to which their carbohydrate head groups protruded beyond the vesicle surface and their interference with membrane approach were examined by determining vesicle susceptibility toward type I Ricinus communis agglutinin (RCA1) induced agglutination and Ca2+- and spermine-induced aggregation and fusion either in the presence or in the absence of the lectin. The initial agglutination rates increased in the order GalCer much less than LacCer less than Gb3, while a reversed order was obtained for Ca2+- and spermine-induced aggregation and fusion, indicating an enhanced steric interference on close approach of bilayers with increasing head group size. The lectin-mediated agglutination rates for LacCer- and Gb3-containing vesicles increased by an order of magnitude when Ca2+ was also included in the medium, at a concentration that did not induce aggregation per se. Charge neutralization could not account for this observation as the polyvalent cation spermine did not display this synergistic effect with RCA1. Addition of Ca2+ to preagglutinated vesicles substantially reduced the threshold cation concentration for fusion (micromolar vs. millimolar). Quantitatively, this concentration decreased with decreasing carbohydrate head group size, indicating that the head group protrusion determined the interbilayer distance within the vesicle aggregate. The distinct behavior of Ca2+ vs. spermine on RCA1-induced agglutination on the one hand and fusion on the other indicated that Ca2+ regulates the steric orientation of the carbohydrate head group, which appears to be related to its ability to dehydrate the bilayer. As a result, lectin agglutinability becomes enhanced while fusion will be interrupted as the interbilayer distance increases, the threshold head group size being three carbohydrate residues (Gb3). Finally, GalCer-containing vesicles were not agglutinated by RCA1 at ambient temperature, irrespective of the presence of Ca2+. Above 25 degrees C, RCA1 facilitated Ca2+-induced fusion of the vesicles, which was abolished by the haptenic sugar lactose. Since Gb3- and LacCer-containing vesicles displayed a similar behavior, a temperature-induced alteration in the supporting lipid matrix is suggested, which apparently affects lectin/glycolipid interaction.     

Interaction of alkaline metal ions with Ca(2+)-binding sites of Ca(2+)-ATPase of sarcoplasmic reticulum: 23Na-NMR studies.

The analysis of the 23Na-NMR signal shape variations in the presence of vesicles of light sarcoplasmic reticulum (SR) shows the existence of sodium sites on the membranes with Kd values of about 10 mM. Other monovalent cations displace Na+ from SR fragments in a competitive manner according to the row K+ greater than Rb+ greater than Cs+ greater than Li+. Calcium ions also reduce Na+ binding, the Na+ desorption curve being of a two-stage nature, which, as suggested, indicates the existence of two types of Ca(2+)-sensitive Na+ binding sites (I and II). Sites of type I and II are modified by Ca2+ in submicromolar and millimolar concentrations, respectively. Analysis of sodium (calcium) desorption produced by calcium (sodium) allowed us to postulate the competition of these two cations for sites I and identity of these sites to high-affinity Ca(2+)-binding ones on the Ca(2+)-ATPase. Sites I weakly interact with Mg2+ (KappMg approximately 30 mM). Reciprocal effects of sodium and calcium on binding of each other to sites II cannot be described by a simple competition model, which indicates nonhomogeneity of these sites. A portion of sites I (approximately 70%) interacts with Mg2+ (KappMg = 3-4 mM). The pKa value of sites II is nearly 6.0. The number of sites II is three times greater than that of sites I. In addition, sites with intermediate affinity for Ca2+ were found with Kd values of 2-5 microM. These sites were revealed due to the reducing of the sites II affinity for Na+ upon Ca2+ binding to SR membranes. It can thus be concluded that in nonenergized SR there are binding sites for monovalent cations of at least three types: (1) sites I (which also bind Ca2+ at low concentrations), (2) magnesium-sensitive sites II and (3) magnesium-insensitive sites II.     

Ca2+ binding to chromaffin vesicle matrix proteins: effect of pH, Mg2+, and ionic strength

Recently we found that Ca2+ within chromaffin vesicles is largely bound [Bulenda, D., & Gratzl, M. (1985) Biochemistry 24, 7760-7765]. In order to explore the nature of these bonds, we analyzed the binding of Ca2+ to the vesicle matrix proteins as well as to ATP, the main nucleotide present in these vesicles. The dissociation constant at pH 7 is 50 microM (number of binding sites, n = 180 nmol/mg of protein) for Ca2+-protein bonds and 15 microM (n = 0.8 mumol/mumol) for Ca2+-ATP bonds. When the pH is decreased to more physiological values (pH 6), the number of binding sites remains the same. However, the affinity of Ca2+ for the proteins decreases much less than its affinity for ATP (dissociation constant of 90 vs. 70 microM). At pH 6 monovalent cations (30-50 mM) as well as Mg2+ (0.1-0.5 mM), which are also present within chromaffin vesicles, do not affect the number of binding sites for Ca2+ but cause a decrease in the affinity of Ca2+ for both proteins and ATP. For Ca2+ binding to ATP in the presence of 0.5 mM Mg2+ we found a dissociation constant of 340 microM and after addition of 35 mM K+ a dissociation constant of 170 microM. Ca2+ binding to the chromaffin vesicle matrix proteins in the presence of 0.5 mM Mg2+ is characterized by a Kd of 240 microM and after addition of 15 mM Na+ by a Kd of 340 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of monovalent cations to phosphatidylserine and modulation of Ca2+- and Mg2+-induced vesicle fusion

The effect of several monovalent cations on the Ca2+-induced aggregation and fusion of sonicated phosphatidylserine (PS) vesicles is studied by monitoring the mixing of internal compartments of the fusing vesicles using the Tb/dipicolinic acid assay. The dissociation of the fluorescent Tb-dipicolinate complex which accompanies Ca2+-induced vesicle fusion is determined directly and is due to leakage of contents and entry of medium into vesicles. PS vesicles do not fuse when the medium contains only monovalent cations (at pH 7.4), regardless of the cation concentration or whether there is aggregation of the vesicles. A mass-action kinetic analysis of the data provides estimates for the rate of aggregation, C11, and for the rate of fusion per se, f11. Values of f11 increase dramatically with reduction in monovalent cation concentration and are primarily determined by binding ratios of Ca2+ or Mg2+ per PS. With 300 mM of monovalent cations, the fusion per se is essentially rate-limiting to the overall fusion process and values of f11 are significantly larger with the monovalent cations which bind the least, i.e., according to the sequence tetramethylammonium greater than K+ greater than Na+ greater than Li+. With monovalent cations in concentrations of 100 mM or less, the aggregation is rate-limiting to the fusion and the overall initial fusion rates are determined by an interplay between aggregation and fusion rates. Under conditions of fast aggregation, the Ca2+-induced fusion of small PS vesicles can occur within milliseconds or less.     

Involvement of Ca2(+)-induced Ca2+ release in the volume regulation of human epithelial cells exposed to a hypotonic medium

Exposure of cultured human epithelial cells (Intestine 407) to a hypotonic solution results in initial osmotic swelling and in a subsequent volume decrease near to the original level. The regulatory volume decrease was inhibited by reduction of the extracellular free Ca2+ concentration to 90 nM. Single epithelial cells responded to a hypotonic challenge with a biphasic increase in the cytosolic free Ca2+ level from about 90 to 200 nM. Both phases of the Ca2+ rise were abolished by reducing the extracellular Ca2+ to 90 nM. In the presence of caffeine (20 mM), the second-phase Ca2+ response to a hypotonic challenge occurred earlier immediately after the first-phase response. The second-phase Ca2+ response was selectively impaired by adenine (10 mM), procaine (1 mM) or ryanodine (5 to 10 microM). These blockers for Ca2(+)-induced Ca2+ release channels inhibited volume regulation after osmotic swelling. It is concluded that Ca2(+)-induced Ca2+ release from a ryanodine-sensitive store is a prerequisite for the volume regulation of human intestinal epithelial cells under hypotonic conditions.     

The electrophoretic properties and aggregation of mouse lymphoma cells, chinese-hamster fibroblasts and a somatic-cell hybrid.

1. The electrophoretic mobilities of a mouse lymphoma cell, a Chinese-hamster fibroblast and a somatic-cell hybrid (also fibroblastic), produced by fusion of the hamster cell and a mouse lymphoma cell, were measured at 25 degrees C over a range of pH, concentration of Ca2+ ions and concentration of La3+ ions. 2. All the cells have pI at pH3.5. 3. Ca2+ ions decrease the mobilities and zeta potentials of the cells to zero in the range 1-100mM. 4. La3+ ions lower the mobilities and zeta potentials in the range 10 muM-1 mM, and the cells become positively charged above 1 mM. 5. The data are consistent with specific adsorption of La3+ ions on approx. 2 X 10(14) sites/m2 of cell surface with a free energy of approx. -37kJ/mol. 6. The effects of Ca2+, La3+ and ionic strength on the extent of aggregation of the cells and of neuraminidase-treated cells were studied. 7. Ca2+ ions do not markedly increase aggregation, whereas La3+ ions gave rise to extensive aggregation in the range 10 muM-1 mM, corresponding to the region of La3+ adsorption. 8. Both fibroblastic cell lines are aggregated at high ionic strength. 9. The fibroblastic cells have larger amounts of trypsin-sensitive carbohydrate than does the lymphoma cell; the possible role of this material in cellular aggregation is discussed.     

Calcium-induced aggregation of archaeal bipolar tetraether liposomes derived from the thermoacidophilic archaeon Sulfolobus acidocaldarius

Previously, we showed that the proton permeability of small unilamellar vesicles (SUVs) composed of polar lipid fraction E (PLFE) from the thermoacidophilic archaeon Sulfolobus acidocaldarius was remarkably low and insensitive to temperature (Komatsu and Chong 1998). In this study, we used photon correlation spectroscopy to investigate the time dependence of PLFE SUV size as a function of Ca2+ concentration. In the absence of Ca2+, vesicle diameter changed little over 6 months. Addition of Ca2+, however, immediately induced formation of vesicle aggregates with an irregular shape, as revealed by confocal fluorescence microscopy. Aggregation was reversible upon addition of EDTA; however, the reversibility varied with temperature as well as incubation time with Ca2+. Freeze-fracture electron microscopy showed that, after a long period of incubation (2 weeks) with Ca2+, the PLFE vesicles had not just aggregated, but had fused or coalesced. The initial rate of vesicle aggregation varied sigmoidally with Ca2+ concentration. At pH 6.6, the threshold calcium concentration (Cr) for vesicle aggregation at 25 and 40 degrees C was 11 and 17 mM, respectively. At pH 3.0, the Cr at 25 degrees C increased to 25 mM. The temperature dependence of Cr may be attributable to changes in membrane surface potential, which was -22.0 and -13.2 mV at 25 and 40 degrees C, respectively, at pH 6.6, as determined by 2-(p-toluidinyl)naphthalene-6-sulfonic acid fluorescence. The variation in surface potential with temperature is discussed in terms of changes in lipid conformation and membrane organization.     

Influence of glycophorin incorporation on Ca2+-induced fusion of phosphatidylserine vesicles

The effect of incorporation of glycophorin, the major integral sialoglycoprotein of the erythrocyte membrane, into bovine brain phosphatidylserine (PS) vesicles on the Ca2+-induced fusion of these vesicles has been investigated. Fusion was monitored by the terbium-dipicolinic acid fluorescence assay for the mixing of aqueous contents of the vesicles and by a resonance energy transfer assay that follows the intermixing of membrane lipids. The Ca2+-induced fusion of PS vesicles is completely prevented by incorporation of glycophorin (molar ratio of PS/glycophorin = 400-500:1) for Ca2+ concentrations up to 50 mM. The ability to fuse is partially restored after treating the glycophorin-containing vesicles with neuraminidase, which removes the negatively charged sialic acid residues of glycophorin. Fusion is further facilitated by trypsin treatment, removing the entire extravesicular glycosylated head group of glycophorin. However, Ca2+-induced fusion of enzyme-treated glycophorin-PS vesicles proceeds at a slower rate and to a smaller extent than fusion of protein-free PS vesicles. The influence of the aggregation state of the glycophorin molecules on fusion has been investigated in experiments using wheat germ agglutinin (WGA). Addition of WGA to the glycophorin-PS vesicles does not induce fusion. However, upon subsequent addition of Ca2+, distinct fusion occurs concomitantly with release of vesicle contents. The inhibition of Ca2+-induced fusion of PS vesicles by incorporation of glycophorin is explained by a combination of steric hindrance and electrostatic repulsion between the vesicles by the glycosylated head group of glycophorin and a direct bilayer stabilization by the intramembranous hydrophobic part of the glycophorin molecule.     

Dissociation of calcium from the phosphorylated calcium-transporting adenosine triphosphatase of sarcoplasmic reticulum: kinetic equivalence of the calcium ions bound to the phosphorylated enzyme.

The internalization of 45Ca by the calcium-transporting ATPase into sarcoplasmic reticulum vesicles from rabbit muscle was measured during a single turnover of the enzyme by using a quench of 7 mM ADP and EGTA (25 degrees C, 5 mM MgCl2, 100 mM KCl, 40 mM MOPS.Tris, pH 7.0). Intact vesicles containing either 10-20 microM or 20 mM Ca2+ were preincubated with 45Ca for approximately 20 s and then mixed with 0.20-0.25 mM ATP and excess EGTA to give 70% phosphorylation of Etot with the rate constant k = 300 s-1. The two 45Ca ions bound to the phosphoenzyme (EP) become insensitive to the quench with ADP as they are internalized in a first-order reaction with a rate constant of k = approximately 30 s-1. The first and second Ca2+ ions that bind to the free enzyme were selectively labeled by mixing the enzyme and 45Ca with excess 40Ca, or by mixing the enzyme and 40Ca with 45Ca, for 50 ms prior to the addition of ATP and EGTA. The internalization of each ion into loaded or empty vesicles follows first-order kinetics with k = approximately 30 s-1; there is no indication of biphasic kinetics or an induction period for the internalization of either Ca2+ ion. The presence of 20 mM Ca2+ inside the vesicles has no effect on the kinetics or the extent of internalization of either or both of the individual ions. The Ca2+ ions bound to the phosphoenzyme are kinetically equivalent. A first-order reaction for the internalization of the individual Ca2+ ions is consistent with a rate-limiting conformational change of the phosphoenzyme with kc = 30 s-1, followed by rapid dissociation of the Ca2+ ions from separate independent binding sites on E approximately P.Ca2; lumenal calcium does not inhibit the dissociation of calcium from these sites. Alternatively, the Ca2+ ions may dissociate sequentially from E approximately P.Ca2 following a rate-limiting conformational change. However, the order of dissociation of the individual ions can not be distinguished. An ordered-sequential mechanism for dissociation requires that the ions dissociate much faster (k greater than or equal to 10(5) s-1) than the forward and reverse reactions for the conformational change (k-c = approximately 3000 s-1). Finally, the Ca2+ ions may exchange their positions rapidly on the phosphoenzyme (kmix greater than or equal to 10(5) s-1) before dissociating. A Hill slope of nH = 1.0-1.2, with K0.5 = 0.8-0.9 mM, for the inhibition of turnover by binding of Ca2+ to the low-affinity transport sites of the phosphoenzyme was obtained from rate measurements at six different concentrations of Mg2+.     

Purification and Na+ uptake by human placental microvillus membrane vesicles prepared by three different methods

Three methods were used to prepare microvillus membrane vesicles from each of six human placentas. Two of these incorporated an agitation stage to preferentially remove microvilli and either Ca2+ (Method 1) or Mg2+ (Method 2) aggregation of non-microvillus membrane. The third method involved homogenisation of the tissue followed by Mg2+ aggregation of non-microvillus membrane (Method 3). Enrichment of alkaline phosphatase activity (27.6 +/- 1.9, 25.3 +/- 2.7) and ouabain binding (5.9 +/- 2.6, 5.3 +/- 2.2, respectively) was similar in vesicles prepared by Methods 1 and 2, respectively. Method 3 vesicles showed a significantly (P less than 0.01) lower alkaline phosphatase enrichment (18.1 +/- 1.2), but ouabain binding enrichment (6.3 +/- 1.3) was not different and vesicle protein recovery (mg/g placenta) was 5-fold greater. Na+ uptake in the presence of an outwardly directed proton gradient was significantly inhibited in all microvillus membrane vesicles by amiloride (0.5 mM). However, the amiloride sensitive component of Na+ uptake was 3-6-fold greater in Method 3 vesicles than in Method 1 and 2 vesicles, and showed overshoot above equilibrium in the former but not the latter. Further experiments using the pH sensitive dye, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein suggested that the proton gradient dissipated faster from Method 1 than from Method 3 vesicles. Thus methodological differences can have a marked effect on transport processes in microvillus membrane vesicles prepared from the human placenta.