Sendai virus membrane fusion: time course and effect of temperature, pH, calcium, and receptor concentration

The conditions that optimize Sendai virus membrane fusion with liposomes have been studied. No fusion occurs in the absence of ganglioside receptors. Maximum fusion occurs when the molar ratio of ganglioside GD1a to phospholipid is 0.02 or greater. The amount of fusion at 37 degrees C increases with time up to at least 6.5 h. The rate of fusion increases from the lowest temperature tested, 10 degrees C, to 40 degrees C. Above 43 degrees C the amount of fusion decreases because of thermal inactivation of the viral proteins. There is a broad pH maximum between pH 7.5 and pH 9.0. At both ends of the pH range the amount of fusion increases and exceeds that found in the physiologic pH range. Neither ethylenediaminetetraacetic acid nor Ca2+ changes the amount of membrane fusion. The optimal conditions for membrane fusion of Sendai virus membranes with liposomes are the same as the optimal conditions for fusion with host cells and with red blood cells. Since the liposomes contain no proteins, the optimal conditions for Sendai virus membrane fusion must be determined by the viral proteins and be mostly independent of the nature or presence of the host proteins.     

Calcium binding properties of purified zymogen granule membrane of pig pancreas. Evidence for calcium binding proteins

Ca2+ binding properties of purified zymogen granule membranes of pig pancreas have been measured: Binding increased linearly with Ca2+ concentration in the medium up to the micromolar range; in the millimolar range a sharp rise in binding capacity was observed. Binding increased with pH both at low and high concentrations of Ca2+. It was insensitive to Na+ and K+ ions at concentrations up to 100 mM. Mg2+ was inhibitory in the millimolar range whereas La2+ and Tb3+ were inhibitory in the micromolar range. The Ca2+ binding components of zymogen granule membranes were identified by two methods: (1) by measuring 45Ca2+ binding after counter-ion electrophoresis and (2) by Stain's-all (forms a complex with Ca2+ binding proteins absorbing maximally at 600 nm), after SDS-polyacrylamide gel electrophoresis. The first method, counter-ion electrophoresis, indicated that most of the 45Ca2+ was associated with an acidic band which could be subsequently subfractionated by SDS-polyacrylamide gel electrophoresis in five bands: 66, 57, 30, 27 and 22.5 kDa. The second method, Stain's-all, revealed six positive polypeptides after SDS-polyacrylamide gel electrophoresis of native zymogen granule membranes' two were unreactive after neuraminidase treatment (130 and 92 kDa, respectively), whereas four other bands were still reactive (66, 57, 43, 30 kDa, respectively.) Ca2+ binding was also measured on intact zymogen granules: the binding capacity was higher than for zymogen granule membranes. Among the Ca2+ binding proteins of the zymogen granule membrane only one is apparently located on the granule external surface: the 30 kDa polypeptide. If Ca2+ directly facilitates fusion of zymogen granules with plasma membrane by a Ca2+-protein interaction, then this protein is a presumptive candidate to play such a key role.     

Effects of calcium, pH, and blockiness on kinetic rheological behavior and microstructure of HM pectin gels

The kinetic behavior during gel formation and the microstructure of 0.75% high methoxyl (HM) pectin gels in 60% sucrose have been investigated by oscillatory measurements and transmission electron microscopy for three comparable citrus pectin samples differing in their degree of blockiness (DB). Ca2+ addition at pH 3.0 resulted in faster gel formation and a lower storage modulus after 3 h for gels of the blockwise pectin A. For gels of the randomly esterified pectin B, the Ca2+ addition resulted in faster gel formation and a higher storage modulus at pH 3.0. At pH 3.5, both pectins A and B were reinforced by the addition of Ca2+. In the absence of Ca2+, the shortest gelation time was obtained for the sample with the highest DB. Microstructural characterization of the gel network, 4 and 20 h after gel preparation, showed no visible changes on a nanometer scale. The microstructure of pectins A and B without Ca2+ was similar, whereas the presence of Ca2+ in pectin A resulted in an inhomogeneous structure.     

Frequency decoding of fast calcium oscillations by calpain

We report the development of a novel procedure for generating fast, high-frequency Ca2+ oscillations in vitro and the frequency-dependent activation of m-calpain, the Ca2+-activated intracellular cysteine protease. The procedure is based upon liberating Ca2+ from a cage, DM-Nitrophen, by repetitive UV laser pulses and its concomitant binding by a 'slow' chelator, 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetate (DOTA). It is shown that a full control over the pattern of oscillations can be readily achieved because the half-life of individual spikes is determined by DOTA concentration and pH, whereas peak amplitude can be adjusted by light intensity. Frequency is only limited by the physical parameters of the light source. The sensitivity of calpain activation to the frequency of Ca2+ oscillations was monitored by the cleavage of microtubule-associated protein 2, a very sensitive physiological substrate of the enzyme. One hundred transients at a peak Ca2+ concentration of 10 microM were presented at various pH values and frequencies ranging from 1 to 50 Hz. At pH 6.0 and 7.0 significant activation occurred at high frequencies (20 and 50 Hz), but here Ca2+ accumulated due to the overlap of transients; at low frequencies (1 and 3 Hz) where Ca2+ accumulation was negligible, there was no calpain activation. At pH 8.0, where individual transients do not overlap even at 50 Hz, frequency-dependence of activation is seen when calpain is sensitized to Ca2+ by autolysis and by the addition of a phospholipid, phosphatidylinositol-4,5-bisphosphate. Our results show that calpain is sensitive to the frequency of fast Ca2+ oscillations in vitro, which is of potential physiological significance.     

Presynaptic calcium and control of vesicle fusion

Vesicle fusion and transmitter release at synapses is driven by a highly localized Ca2+ signal that rapidly builds up around open Ca2+-channels at and near presynaptic active zones. It has been difficult to estimate the amplitude and the kinetics of this 'microdomain' signal by direct Ca2+-imaging approaches. Recently, Ca2+ uncaging at large CNS synapses, among them the calyx of Held, has shown that the intrinsic cooperativity of Ca2+ in inducing vesicle fusion is high, with 4-5 Ca2+ ions needed to trigger vesicle fusion. Given the Ca2+-sensitivity of vesicle fusion as determined by Ca2+-uncaging, it was found that a surprisingly small (10-25 microM) and brief (<1 ms) local Ca2+ signal is sufficient to achieve the amount, and the kinetics of the physiological transmitter release. The high cooperativity of Ca2+ in inducing vesicle fusion and the non-saturation of the Ca2+-sensor for vesicle fusion renders small changes of the local Ca2+-signal highly effective in changing the release probability; an insight that is important for our understanding of short-term modulation of synaptic strength.     

New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures

A new family of high-affinity buffers and optical indicators for Ca2+ is rationally designed and synthesized. The parent compound is 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), a relative of the well-known chelator EGTA [ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] in which methylene links between oxygen and nitrogen are replaced by benzene rings. BAPTA and its derivatives share the high (greater than 10(5)) selectivity for Ca2+ over Mg2+ of EGTA but are very much less affected by pH changes and are faster at taking up and releasing Ca2+. The affinity of the parent compound for Ca2+ (dissociation constant 1.1 x 10(-7) M in 0.1 M KCl) may be strengthened or weakened by electron-releasing or -withdrawing substituents on the aromatic rings. The Ca2+ and Mg2+ affinities may further be altered by replacing the ether oxygens by heterocyclic nitrogen atoms. The compounds described are fluorescent Ca2+ indicators absorbing in the ultraviolet region; the very large spectral shifts observed on binding Ca2+ fit the prediction that complexation should hinder the conjugation of the nitrogen lone-pair electrons with the aromatic rings. Derivatives with quinoline nuclei are notable for their high sensitivity of fluorescent quantum yield to the binding of Ca2+ but not of Mg2+. Preliminary biological tests have so far revealed little or no binding to membranes or toxic effects following intracellular microinjection.     

Conformational studies on parvalbumins by circular dichroism.

Structural variations of two parvalbumins, Whiting III and Pike III, in various denaturing conditions, have been studied by circular dichroism. CD signals are depressed from 4 urea. For Pike III, acidic pH, sodium dodecyl sulfate or complete removal of Ca2+ show little effect in the far ultraviolet region but rather strong effects in the near ultraviolet. For Whiting III similar results are obtained at acidic pH. Carboxymethylated Whiting III (0.15 Ca2+/mol) shows, on the contrary, decreased CD signals in the far and in the near ultraviolet spectra. Addition of Ca2+ fully restores the native CD spectra in both proteins. Ca2+ binding produces structural modifications which are found to vary according to parvalbumin and which seem in any case different from those described for troponin C.     

Membrane contact, fusion, and hexagonal (HII) transitions in phosphatidylethanolamine liposomes

The behavior of phosphatidylethanolamine (PE) liposomes has been studied as a function of temperature, pH, ionic strength, lipid concentration, liposome size, and divalent cation concentration by differential scanning calorimetry (DSC), by light scattering, by assays measuring liposomal lipid mixing, contents mixing, and contents leakage, and by a new fluorometric assay for hexagonal (HII) transitions. Liposomes were either small or large unilamellar, or multilamellar. Stable (impermeable, nonaggregating) liposomes of egg PE (EPE) could be formed in isotonic saline (NaCl) only at high pH (greater than 8) or at lower pH in the presence of low ionic strength saline (less than 50 mOsm). Bilayer to hexagonal (HII) phase transitions and gel to liquid-crystalline transitions of centrifuged multilamellar liposomes were both detectable by DSC only at pH 7.4 and below. The HII transition temperature increased, and the transition enthalpy decreased, as the pH was raised above 7.4, and it disappeared above pH 8.3 where PE is sufficiently negatively charged. HII transitions could be detected at high pH following the addition of Ca2+ or Mg2+. No changes in light scattering and no lipid mixing, mixing of contents, or leakage of contents were noted for EPE liposomes under nonaggregating conditions (pH 9.2 and 100 mM Na+ or pH 7.4 and 5 mM Na+) as the temperature was raised through the HII transition region. However, when aggregation of the liposomes was induced by addition of Ca2+ or Mg2+, or by increasing [Na+], it produced sharp increases in light scattering and in leakage of contents and also changes in fluorescent probe behavior in the region of the HII transition temperature (TH). Lipid mixing and contents mixing were also observed below TH under conditions where liposomes were induced to aggregate, but without any appreciable leakage of contents. We conclude that HII transitions do not occur in liposomes under conditions where intermembrane contacts do not take place. Moreover, fusion of PE liposomes at a temperature below TH can be triggered by H+, Na+, Ca2+, or Mg2+ or by centrifugation under conditions that induce membrane contact. There was no evidence for the participation of HII transitions in these fusion events.     

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.     

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.     

Effects of pH, temperature, and calcium concentration on the stoichiometry of the calcium pump of sarcoplasmic reticulum

Coupling of Ca2+ transport to ATP hydrolysis by isolated skeletal muscle sarcoplasmic reticulum vesicles has been investigated by means of ATP pulse methods. The stoichiometric amounts of Ca2+ transported per pulse of ATP were measured by Ca2+-stat methods, using either a Ca2+ electrode or arsenazo III as end point detectors, or by means of 45CaCl2. Maximum coupling ratios (Ca2+/ATP), of 1.82 +/- 0.13 occurred at pH 6.8, 25 degrees C, and in the presence of saturating Ca2+ concentrations. Ca2+/ATP values decreased at alkaline pH, with an apparent pK alpha of 7.9. The coupling ratio was unaltered between 6 and 30 degrees C, but decreased to 0.4 at 42 degrees C. Uncoupling by alkaline pH and high temperatures was reversible. The coupling process was Ca2+-dependent, with a K0.5 value for Ca2+ of 0.12 microM and a Hill coefficient of 2.0. Ca2+ ions, which were transported into vesicles under conditions resulting in low coupling ratios, were retained as the calcium oxalate precipitate, following complete hydrolysis of substrate. Passive Ca2+ efflux and Ca2+ exchange, were independent of pH. The observed variations in Ca2+/ATP ratio cannot readily be explained on the basis of a pump-leak model. Rather, the Ca2+-ATPase appears to be capable of pumping Ca2+ ions, under physiological conditions, with variable stoichiometry that is dependent upon its thermodynamic loading.     

Calcium dependence of the binding of synexin to isolated chromaffin granules

The calcium dependence of the binding of synexin to isolated chromaffin granules has been investigated. The calcium dependence was found to be pH sensitive, binding occurring at higher Ca2+ concentrations at lower values of pH. At pH 7.2 half-maximal binding occurred at 4 microM Ca2+. This is a lower Ca2+ concentration than the 200 microM that is required to give half-maximal self-association of synexin or membrane aggregation by synexin. The data therefore suggest that in the chromaffin cell stimulated to release catecholamines and proteins by exocytosis synexin first binds to membranes and then associates with itself to draw membranes together in preparation for fusion.     

Fusion of small unilamellar vesicles with viable EDTA-treated Escherichia coli cells.

Fusion characteristics of EDTA-treated Escherichia coli cells with small unilamellar vesicles were investigated, using a membrane fusion assay based on resonance energy transfer. Ca2+-EDTA treatments of Escherichia coli O111:B4 (wild type), E. coli C600 (rough), and E. coli D21f2 (deep rough) which permeabilize the outer membrane by inducing the release of lipopolysaccharide and outer membrane proteins resulted in fusion activity of the intact and viable bacteria with small unilamellar vesicles. No fusion activity was observed when the EDTA treatment was omitted. Fusion could be elicited at low pH and by a combination of a higher pH and Ca2+. The low-pH-induced fusion was composed of a fast and a slow reaction. The latter and the Ca2+-induced fusion could be completely inhibited by trypsin treatments of the EDTA-treated cells, which also resulted in the simultaneous disappearance of two outer membrane protein bands (50 and 58 kilodaltons) and the appearance of proteins banding at 22, 52, and 54 kilodaltons. The most efficient fusion was obtained with negatively charged liposomes composed of cardiolipin. In contrast to the Ca2+-induced fusion, fusion was observed at low pH with small unilamellar vesicles containing lipids with decreased negative charge (phosphatidylserine). Fluorescent and phase-contrast microscopy revealed that essentially all bacteria were engaged in fusion. We propose that a Ca2+-EDTA treatment of E. coli cells results in the appearance of phospholipids and the exposure of a protein(s) in the outer leaflet of the outer membrane, both of which could mediate fusion with liposomes.     

Interaction of calcium-bound C-reactive protein with fibronectin is controlled by pH: in vivo implications

C-reactive protein (CRP) binds with high affinity to fibronectin (Fn), a major component of the extracellular matrix (ECM), but at physiological pH the binding is inhibited by calcium ions (Ca2+). Because CRP circulates in the blood in Ca2+ -bound form, the occurrence of CRP-Fn interactions in vivo has been doubtful. To define the basis of inhibition of CRP-Fn interaction by Ca2+ at pH 7.0, we hypothesized that Fn-binding site on CRP consisted of amino acids co-ordinating Ca2+. Site-directed mutagenesis of amino acids co-ordinating Ca2+ drastically decreased the binding of CRP to Fn, indicating that the Ca2+ -binding site indeed formed the Fn-binding site. To determine the requirements for possible interaction between Ca2+ -bound CRP and Fn, we investigated inhibition of CRP-Fn interaction by Ca2+ as a function of pH. Ca2+ did not inhibit binding of CRP to Fn at pH 6.5 and lower. The contrasting Fn binding properties of CRP at physiological and mildly acidic pH indicated that the interaction of Ca2+ -bound CRP with Fn was controlled by pH. We conclude that the inhibition of binding of CRP to Fn by Ca2+ at pH 7.0 is a mechanism to prevent CRP-Fn interactions under normal conditions. CRP, in its Ca2+ -bound state, is capable of binding Fn but only at the inflammatory sites and tumors with low pH. CRP, Fn, and the ECM all have been implicated in cancer. Taken together our data raise the possibility that CRP-Fn interactions may change the architecture of ECM to modify the development of tumors.     

Stimulation of an exocytotic event, the hamster sperm acrosome reaction, by cis-unsaturated fatty acids

The influence of extracellular Mg2+ on Ca2+ ionophore (A23187 and ionomycin) induced secretion and changes in the cytosol pH of rabbit neutrophils suspended in Ca2+-free buffer has been investigated. While extracellular Ca2+ is obligatory for ionomycin induced secretion, we have defined conditions under which A23187 can induce secretion in Ca2+-free media. The different behaviour of these two Ca2+ ionophores is discussed on the basis of their different counter cation specificities.     

Expression, partial purification and functional properties of themuscle-specific calpain isoform p94.

The muscle-specific calpain isoform p94 has high propensity to autocatalytic degradation, thus no significant amounts of the intact active protein have been available so far. As a result, aspects like its regulation (via Ca2+ and other factors) and its intracellular localization are unknown or obscure. In this work, large amounts of human p94 have been produced in insect cells using a recombinant baculovirus expression system. Although most of the protease was recovered in an insoluble and catalytically inactive form, the soluble fraction contained amounts of intact active p94 adequate for its characterization. His-tagged recombinant p94, obtained by the same expression system, was partially purified as an active product. Both the unmodified and the partially purified His-tagged p94 bound calcium with high affinity, and their autolytic activity required Ca2+. The sensitivity of the catalytic activity of the recombinant protease to Ca2+ was very high. In fact, p94 in soluble cell extracts autolysed to a significant extent even in the presence of submicromolar Ca2+ levels. Thus, in analogy to what demonstrated for the ubiquitous m- and micro-calpain isoforms, intracellular Ca2+ might be one of the factors controlling the activity of this muscle-specific calpain isoform.     

Effects of pH conditions on Ca2+ transport catalyzed by ionophores A23187, 4-BrA23187, and ionomycin suggest problems with common applications of these compounds in biological systems.

Phospholipid vesicles loaded with Quin-2 and 2'',7''-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) have been used to investigate the effects of pH conditions on Ca2+ transport catalyzed by ionophores A23187, 4-BrA23187, and ionomycin. At an external pH of 7.0, a delta pH (inside basic) of 0.4-0.6 U decreases the rate of Ca2+ transport into the vesicles by severalfold under some conditions. The apparent extent of transport is also decreased. In contrast, raising the pH by 0.4-0.6 U in the absence of a delta pH increases both of these parameters, although by smaller factors. The relatively large effects of a delta pH on the transport properties of Ca2+ ionophores seem to reflect a partial equilibration of the transmembrane ionophore distribution with the H+ concentration gradient across the vesicle membrane. This unequal distribution of ionophore can cause a very slow or incomplete ionophore-dependent equilibration of delta pCa with delta pH. A second factor of less certain origin retards full equilibration of delta pCa when delta pH = 0. These findings call into question several ionophore-based methods that are used to investigate the regulatory activities of Ca2+ and other divalent cations in biological systems. Notable among these are the null-point titration method for determining the concentration of free cations within cells and the use of ionophores plus external cation buffers to calibrate intracellular cation indicators. The present findings also indicate that the transport mode of Ca2+ ionophores is more strictly electroneutral than was thought, based upon previous studies.     

Fig1p facilitates Ca2+ influx and cell fusion during mating of Saccharomyces cerevisiae

During the mating process of yeast cells, two Ca2+ influx pathways become activated. The resulting elevation of cytosolic free Ca2+ activates downstream signaling factors that promote long term survival of unmated cells, but the roles of Ca2+ in conjugation have not been described. The high affinity Ca2+ influx system is composed of Cch1p and Mid1p and sensitive to feedback inhibition by calcineurin, a Ca2+/calmodulin-dependent protein phosphatase. To identify components and regulators of the low affinity Ca2+ influx system (LACS), we screened a collection of pheromone-responsive genes that when deleted lead to defects in LACS activity but not high affinity Ca2+ influx system activity. Numerous factors implicated in polarized morphogenesis and cell fusion (Fus1p, Fus2p, Rvs161p, Bni1p, Spa2p, and Pea2p) were found to be necessary for LACS activity. Each of these factors was also required for activation of the cell integrity mitogen-activated protein kinase cascade during the response to alpha-factor. Interestingly a polytopic plasma membrane protein, Fig1p, was required for LACS activity but not required for activation of Mpk1p mitogen-activated protein kinase. Mpk1p was not required for LACS activity, suggesting Mpk1p and Fig1p define two independent branches in the pheromone response pathways. Fig1p-deficient mutants exhibit defects in the cell-cell fusion step of mating, but unlike other fus1 and fus2 mutants the fusion defect of fig1 mutants can be largely suppressed by high Ca2+ conditions, which bypass the requirement for LACS. These findings suggest Fig1p is an important component or regulator of LACS and provide the first evidence for a role of Ca2+ signals in the cell fusion step of mating.     

Increase of human spermatozoa intracellular Ca2+ concentration after fusion with prostasomes.

Prostasomes are membranous vesicles (150-200 nm diameter) present in human semen. They are secreted by the prostate gland and contain large amounts of cholesterol, sphingomyelin and calcium, and some of their proteins are enzymes. Prostasomes are involved in a number of biological functions. In previous work, we discovered that prostasomes may fuse to sperm at neutral or at slightly acidic pH values. This mechanism may deliver calcium to sperm, thereby influencing sperm functions. We measured sperm [Ca2+]i with the fura-2 AM method and found that it increased after mixing prostasomes and sperm at pH values allowing fusion (pH 5-7). The increase of [Ca2+]i was proportional to the extent of fusion as measured through the relief of R18 self-quenching. We also examined the increase of sperm [Ca2+]i and the extent of fusion as a function of sperm to prostasome ratio and, also in this case, there was proportionality between the extent of fusion and the increase of [Ca2+]i that reached its maximal values in about 10-20 min. However, a detectable increase of [Ca2+]i was attained after 2 min of fusion. This would represent a new mechanism to influence sperm [Ca2+]i besides ion-exchange systems and ATP-dependent pumps. The value of [Ca2+]i remained elevated, unless Na+ was also present in the external medium. Therefore, the mechanism of fusion might influence deeply the physiology of sperm by producing a transient increase of [Ca2+]i.     

Calcium-induced condensation-reorganization phenomena in multilamellar vesicles of phosphatidic acid. pH potentiometric and 31P-NMR, Raman and ESR spectroscopic studies

In biological membranes, the anionic characteristics of the polar headgroup of phosphatidic acids are responsible for structural changes induced by Ca2+ in many cellular processes. The very simple headgroup structure of dipalmitoylphosphatidic acid (DPPA) offers particular advantages as a model to study the interactions between Ca2+ and natural phosphatidic acids such as cardiolipin and phosphatidylserine. The effects of calcium ions on DPPA membranes have been studied as a function of temperature by potentiometry and by Raman, ESR and 31P-NMR spectroscopies. The protons in monosodic DPPA liposomes have been considered as a probe to detect pH variations resulting from introduction of Ca2+ inside the membrane. This method has also allowed us to determine the stoichiometry of this reaction: 2 DPPA(H) + Ca2+----Ca(DPPA)2 + 2H+. 31P-NMR spectroscopy has been used to detect reorganization-condensation phenomena in multilamellar vesicles of DPPA under the influence of calcium and temperature. Furthermore, the temperature profiles obtained from Raman spectra for Ca(DPPA)2 membranes provide conclusive evidence that Ca2+ induces major reorganization of the phosphatidic acid component into a highly ordered phase. Quantitative estimates of the degree of motional restriction of spin-labeled soaps embedded inside membranes composed of DPPA with or without Ca2+ have been made using ESR technique. These results are discussed and compared to those found previously for a natural phosphatidic acids such as phosphatidylserine.