Kinetics of cation-induced aggregation of Torpedo electric organ synaptic vesicles.

Synaptic vesicles from the Torpedo ray can be induced to aggregate in the presence of Ca2+ and K+ in the 4 mM and 50 mM range, respectively. The reactions are strikingly similar to those of chromaffin granule membranes reported previously (Morris, S.J., Chiu, V.C.K. and Haynes, D.H. (1979) Membrane Biochem. 2, 163-202). The Ca2+-induced reaction includes dimerization and higher order aggregation, and is shown to be due to electrostatic screening interactions and bindng to negatively-charged groups on the membrane surface. The K+-induced reaction includes only dimerization and is shown to be due to screening interactions alone. The kinetics of the dimerization reactions were studied using the stopped-flow rapid mixing technique. The Ca2+-induced reaction has a 'bimolecular' rate constant of 4.77 . 10(8) M-1 . s-1. These values are close to the limit of diffusion control (8.03 . 10(9) M-1 . s-1), indicating that no large energy barriers or structural barriers to aggregation exist. Arrhenius plots for the Ca2+-induced aggregation showed a break at 5 degrees C. Above this temperature, the activation energy is low (+0.65 kcal/mol), consistent with the above. Below this temperature, the activation energy is high, consistent with a membrane structure change increasing theenergetic and structural barriers. This information, and the observation of a high stability constant of the complex, were taken as evidence for the involvement of 'recognition sites' on the membrane surface. The results were analyzed in terms of an encounter complex model in which vesicles with separations of 26-126 A are considered capable of transformation into a stable complex. The rate constant of the transformation step is 1.4 . 10(3) s-1 for Ca2+ and approx. 1.6 . 10(5) s-1 for K+. The values are compared with previous results for chromaffin granule membranes and for phospholipid vesicles derived from chromaffin granule lipids and from acidic phospholipids. The half-time for Ca2+-induced transformation of the encounter complex into the stable complex is 435 microseconds. It is concluded that the recognition sites are almost as optimally deployed as the vesicle plasma membrane recognition sites involved in exocytotic release.     

Kinetics of cation-induced aggregation of Torpedo electric organ synaptic vesicles

Synaptic vesicles from the Torpedo ray can be induced to aggregate in the presence of Ca²⁺ and K⁺ in the 4 mM and 50 mM range, respectively. The reactions are strikingly similar to those of chromaffin granule membranes reported previously (Morris, S.J., Chiu, V.C.K. and Haynes, D.H. (1979) Membrane Biochem. 2, 163–202). The Ca²⁺-induced reaction includes dimerization and higher order aggregation, and is shown to be due to electrostatic screening interactions and binding to negatively-charged groups on the membrane surface. The K⁺-induced reaction includes only dimerization and is shown to be due to screening interactions alone.The kinetics of the dimerization reactions were studied using the stopped-flow rapid mixing technique. The Ca²⁺-induced reaction has a ‘bimolecular’ rate constant of 4.77 · 10⁸ M^?1 · s^?1 while the value for the K⁺-induced reaction is 7.05 · 10⁹ M^?1 · s^?1. These values are close to the limit of diffusion control (8.03 · 10⁹ M^?1 · s^?1), indicating that no large energy barriers or structural barriers to aggregation exist. Arrhenius plots for the Ca²⁺-induced aggregation showed a break at 5°C. Above this temperature, the activation energy is low ($\text{+0.65}\text{kcal/mol}$), consistent with the above. Below this temperature, the activation energy is high, consistent with a membrane structure change increasing the energetic and structural barriers. This information, and the observation of a high stability constant of the complex, were taken as evidence for the involvement of ‘recognition sites’ on the membrane surface.     

Transbilayer phosphatidylcholine distributions in small unilamellar sphingomyelin-phosphatidylcholine vesicles: effect of altered polar head group

The effect of the altered polar head group of phosphatidylcholine (PC) on its transbilayer distributions in small unilamellar vesicles containing sphingomyelin (SM) was ascertained with phospholipase A2 as the external membrane probe. These vesicles were formed by sonication and fractionated by centrifugation. The vesicle size was determined by gel-permeation chromatography and solute entrapment. Experiments were done to confirm that phospholipase A2 treatments did not induce fusion, lyse the vesicles, or cause PC to migrate across the vesicle bilayer. The complete degradation of external PC in intact vesicles was assured by carrying out the enzyme reactions in the absence as well as in the presence of 9.2 X 10(-5) M bovine serum albumin. In small vesicles comprised of SM and 30 mol % 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DPPC preferentially distributed in the inner monolayer. This preference of DPPC in these vesicles disappeared upon introducing one C2H5 group at the carbon atom adjacent to the quaternary ammonium residue in its polar head group and was reversed when the C2H5 group was replaced by C6H5 and C6H5CH2 substituents or when the P-N distance was increased. These results indicate that the effective polar head-group volume is an important factor in determining the phospholipid distributions across the small vesicle bilayer.     

Configuration and interactions of the polar head group in gangliosides

1. The interactions of gangliosides with Ca²⁺ and some polar-head-group requirements for establishment of particular interactions with phosphatidylcholine were studied in monolayers at the air/145mm-NaCl interface. 2. Ganglioside–Ca²⁺ interactions, as revealed by surface-potential measurements, depended on the position occupied by sialosyl residues in the oligosaccharide chain. The interactions with Ca²⁺ of the single sialosyl residue of monosialogangliosides occurred above 0.1mm-CaCl₂, whereas the interaction of the cation with additional sialosyl groups in di- or tri-sialogangliosides depended on the carbohydrate residue to which the sialosyl moiety was attached. The sialosyl residue bound in sialosyl–sialosyl linkage interacted very little with Ca²⁺. The sialosyl residue attached to the terminal galactose of the neutral tetrasaccharide chain interacted with Ca²⁺ above 1μm-CaCl₂. 3. Experiments with mixed monolayers containing dihexadecyl phosphate and hexadecyltrimethylammonium indicated that for the occurrence of interactions of polysialogangliosides with phosphatidylcholine characterized by reductions in molecular packing and surface potential both charged groups of the phospholipid and sialosyl residues with particular dipolar properties in the ganglioside are participating. 4. Possible configurations that can explain the behaviour in monolayers were inspected with space-filling molecular models. The position of the carboxylate group of sialosyl residues with respect to the interface and to the sialosyl molecular plane can explain the different orientation of the dipole-moment vector of this residue, which depends on the position to which it is linked in the oligosaccharide chain. Favoured interactions of polysialogangliosides with phosphatidylcholine may result from a configuration allowing a partial matching of two oppositely oriented electrical vectors contributed by the zwitterionic phosphocholine group and particular sialosyl groups.     

Monovalent cation-induced fusion of acidic phospholipid vesicles

Fusion of small unilamellar vesicles (SUV) consisting of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and phosphatidylglycerol (PG) from egg yolk, dipalmitoylphosphatidylserine (DPPS) and phosphatidylserine (PS) from bovine brain was studied as a function of monovalent cation concentration. Fusion was detected by measuring the changes in the excimer to monomer fluorescence intensity ratio (IE/M) of pyrene-labeled phospholipid analogues upon fusion of the pyrene-labeled and unlabeled vesicles. No fusion was observed from vesicles consisting of DMPC, PS from bovine brain or PG from egg yolk upon addition of NaCl (up to 1 M). However, considerable fusion was evident for vesicles consisting of DMPG or DPPS upon addition of monovalent cations (300 mM to 1 M). Fusion kinetics were fast reaching a plateau after 5 min of addition of cations. The order of efficiency of different monovalent cations to induce the fusion of DMPG vesicles as judged by the changes of the IE/M ratio was Li+ greater than Na+ greater than K+ greater than Cs+. DSC-scan of sonicated DMPG vesicles showed, in the absence of salt, a phase transition at 19.2 degrees C with enthalpy of 1.1 kcal.mol-1. After incubation in the presence of 600 mM NaCl the DSC scan showed a narrow phase transition at 24.1 degrees C with enthalpy of 6.9 kcal.mol-1 and a pronounced pretransition, both supporting that the fusion of the vesicles had occurred in the presence of NaCl. The results indicate that sonicated vesicles consisting of acidic phospholipids with fully saturated fatty acids fuse in the presence of monovalent cations, whereas those containing unsaturated fatty acids do not.     

Fusion of vesicles with the air-water interface: the influence of polar head group, salt concentration, and vesicle size

Fusion of vesicles with the air-water interface and consequent monolayer formation has been studied as a function of temperature. Unilamellar vesicles of DMPC, DPPC, and DODAX (X=Cl(-), Br(-)) were injected into a subphase containing NaCl, and the surface pressure (tension) was recorded on a Langmuir Balance (Tensiometer) using the Wilhelmy plate (Ring) method. For the zwitterionic vesicles, plots of the initial surface pressure increase rate (surface tension decrease rate) as a function of temperature show a peak at the phase transition temperature (T(m)) of the vesicles, whereas for ionic ones they show a sharp rise. At high concentrations of NaCl, ionic DODA(Cl) vesicles seem to behave like zwitterionic ones, and the rate of fusion is higher at the T(m). The influence of size was studied comparing large DODA(Cl) vesicles with small sonicated ones, and no significant changes were found regarding the rate of fusion with the air-water interface.     

Divalent cation-induced aggregation of chromaffin granule membranes.

Divalent cations induce the aggregation of chromaffin granule ghosts (CG membranes) at millimolar concentrations. Monovalent cations produce the same effect at 100-fold higher concentrations. The kinetics of the dimerization phase were followed by light-scattering changes observed in stopped-flow rapid mixing experiments. The rate constant for Ca2+-induced dimerization (kapp) is 0.86-1.0 x 10(9) M-1sec-1, based on the "molar" vesicle concentration. This value is close to the values predicted by theory for the case of diffusion-controlled reaction (7.02 x 10(9) M-1sec-1), indicating that there is no energy barrier to dimerization. Arrhenius plots between 10 degrees and 42 degrees C support this; the activation energy observed, +4.4 Kcal, is close to the value (4.6-4.8 Kcal) predicted for diffusion control according to theory. Artificial vesicles prepared from CG lipids were also found to have cation-induced aggregation, but the rates (values of kapp) were less than 1/100 as large as those with native CG membranes. Also, significant differences were found with respect to cation specificity. It is concluded that the slow rates are due to the low probability that the segments of membrane which approach will be matched in polar head group composition and disposition. Thus large numbers of approaches are necessary before matched segments come into aposition. The salient features of the chromaffin granule membrane aggregation mechanism are as follows: (a) In the absence of cations capable of shielding and binding, the membranes are held apart by electrostatic repulsion of their negatively charged surfaces. (b) The divalent and monovalent cation effects on aggregation are due to their ability to shield these charges, allowing a closer approach of the membrane surfaces. (c) The major determinants of the aggregation rates of CG membranes are proteins which protrude from the (phospholipid) surface of the membrane and serve as points of primary contact. Transmembrane contact between these proteins does not require full neutralization of the surface charge and surface potential arising from the negatively charged phospholipids. (d) After contact between proteins is established, the interaction between membranes can be strengthened through transmembrane hydrogen bonding of phosphatidyl ethanolamine polar head groups, divalent cation-mediated salt bridging, and segregation of phosphatidylcholine out of the region of contact.     

Retention of aqueous contents during divalent cation-induced fusion of phospholipid vesicles

The relative kinetics of intermixing and release of liposome aqueous contents during Ca²⁺-induced membrane fusion has been investigated. Fusion was monitored by the Tb-dipicolinic acid (DPA) fluorescence assay. Release was followed by the relief of self-quenching of carboxyfluorescein or by Tb fluorescence, with essentially identical results. Fusion of large unilamellar vesicles (LUV) made of phosphatidylserine (PS) in 100 mM NaCl (pH 7.4) at 25°C was initially non-leaky, whereas the fusion of small unilamellar vesicles (SUV) was accompanied by partial release of contents. After several rounds of fusion, the internal aqueous space of the vesicles collapsed. The rate of intermixing of lipids, measured by a resonance energy transfer assay, and the rate of coalescence of aqueous contents during fusion were similar over a range of Ca²⁺ concentrations. Most of the aqueous contents were retained after the fusion of SUV (PS) in 5 mM NaCl and 1 mM Ca²⁺. LUV made of a 1:1 mixture of Bacillus subtilis cardiolipin and dioleoylphosphatidylcholine went through about two rounds of fusion in the presence of Ca²⁺ at 10°C, with complete retention of contents. Similar results were obtained with vesicles composed of phosphatidate/PS/phosphatidylethanolamine/cholesterol (1:2:3:2) in the presence of Ca²⁺ and synexin at 25°C. These results emphasize the diversity of the relative kinetics of fusion and release in different phospholipid vesicle systems under various ionic conditions, and indicate that the initial events in the fusion of LUV are in general, non-leaky.     

Dielectric properties of the polar head group region of zwitterionic lipid bilayers.

A theoretical model describing the dielectric properties of the lipid membrane-water interface region was developed. The rotating polar head groups (e.g. phosphatidylcholine) were simulated as a collection of interacting dipoles imbedded in a nonhomogeneous dielectric. The interactions between the nearest neighborhood were explicitly taken into account, while the other interactions were evaluated by means of the continuum theories. The values of the dielectric constant, its anisotropy and the spontaneous polarization of the interface were evaluated. As an application, we calculated the energy of interaction between an ion and the membrane polar head group region. The results indicate a small spontaneous polarization of the interface (1-1.7 Debyes per lipid molecule) due to the tilting angle of the choline residue with respect to the membrane surface. This dipolar field partially compensates that of opposite orientation originating from the ester group region, giving calculated overall dipolar potentials in better agreement with the experimental data. Our model suggests also a very strong dielectric anisotropy of the interface region, the component of the dielectric constant perpendicular to the membrane plane being much smaller than the parallel component.     

Studies on cation-induced membrane vesicle aggregation of porcine intestinal brush borders

Protein alterations during the development of the mouse brain were studied by two-dimensional gel electrophoresis. A protein spot with a molecular weight (MW) of 68,000 and pI value of 5.6 was found in the brain of the 11th day of gestation. Between the 12th and the 14th day of gestation, spots with the same MW and lower pI values appeared progressively. Neuraminidase digestion converted the pI of these acidic spots to 5.6. Thus, increased sialylation appeared to occur during this period. This class of molecules became hardly detectable on the 15th day, and disappeared completely after the 16th day. Analogous spots were present in the heart, liver, and stomach of the embryos, although the increased sialylation was not observed in the liver. No adult organs so far examined showed these spots. On the other hand, two polypeptides (MW 55,000, pI 4.7, and 53,000, pI 4.6) appeared in the brain on the 13th day of gestation and persisted throughout the fetal period. After birth, they became hardly detectable. Furthermore, a spot (MW 48,000, pI 4.8) became newly detectable in the brain 4-5 weeks after the birth.     

Ganglioside GD3 lactones: polar head group mediated control of the intermolecular organization

The individual properties and intermolecular organization of ganglioside GD3 and of two of its lactone forms (GD3Lactone I and GD3Lactone II) were studied in lipid monolayers. The formation of the first lactone ring in GD3Lactone I eliminates one negative charge and leads to a decrease of the molecular area at all surface pressures. The intermolecular dispersion energy and collapse pressure are higher in GD3Lactone I compared to those in the parent GD3. The surface potential per unit of molecular surface density and the resultant molecular dipole moment are increased in GD3Lactone I with respect to those in GD3 at comparable values of molecular area. In GD3Lactone I the molecular parameters suggest an oligosaccharide chain oriented similarly to that of GD3. On the average, this is perpendicular to the surface, and the resultant polar head-group dipole moment points away from the interface. In GD3Lactone II the negative charges are eliminated, resulting in considerably larger molecular areas than for GD3 and GD3Lactone I at all pressures. The intermolecular dispersion energy of GD3Lactone II is also greatly diminished and the collapse pressure is further increased compared to those of GD3Lactone I. However, the surface potential per unit molecular surface density and the resultant molecular dipole moment of GD3Lactone II are higher than in GD3 Lactone I at similar values of molecular areas. This is probably due to a positive polar head-group dipole moment contribution induced by the additional lactone ring in GD3Lactone II. These changes result from a distorted conformation of the oligosaccharide chain owing to the presence of fused carbohydrate rings which require a greater intermolecular spacing compared to GD3 and GD3Lactone I.     

Modification of the polar head group of platelet-activating factor: influence on the biological activity

Racemic analogues of platelet-activating factor and its lyso derivatives have been prepared in which one methyl of the trimethylammonium group has been replaced by ethyl, propyl, allyl, or carboxymethylene. The influence of chemical modification on the biological activity was assessed by measuring platelet aggregation and desensitization. The results point to a crucial role of a positively charged polar head group for the expression of biological activity of platelet-activating factor. There are also some indications of a more non-specific interaction of the polar head group of platelet-activating factor with its platelet binding sites.     

Modulation of casein kinase II activity by the polar head group of an insulin-sensitive glycosyl-phosphatidylinositol

A phospho-oligosaccharide, whose production is stimulated by insulin, modulated the activity of partially purified casein kinase II. Whereas at 2 microM the phospho-oligosaccharide stimulated casein kinase II 1.3-fold, higher concentrations of this molecule were inhibitory. 50% inhibition of the enzyme was obtained at 15 microM phospho-oligosaccharide. This biphasic effect of the phospho-oligosaccharide on casein kinase II activity was observed using as substrate both casein or the specific peptide for casein kinase II, Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu. The effect of the phospho-oligosaccharide on casein kinase II was still observed after gel filtration. Deamination of the phospho-oligosaccharide with nitrous acid abolished both the activation and the inhibition of casein kinase II. The glycophospholipid precursor of the phospho-oligosaccharide did not affect casein kinase II activity. Moreover, modulation of casein kinase II activity was not observed with other compounds structurally related to the phospho-oligosaccharide, when used in the micro-molar range. In conclusion, the present results indicate that the phospho-oligosaccharide that mimics and might mediate some of the actions of insulin modulates casein kinase II activity in vitro.     

Inhibition of cyclic AMP-dependent protein kinase by the polar head group of an insulin-sensitive glycophospholipid

A glycophospholipid has been purified from rat liver membranes and shown to copurify with an insulin-sensitive glycophospholipid isolated from H35 hepatoma cells. The polar head group of this glycophospholipid is a phospho-oligosaccharide generated by treatment with phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. It has been proposed that this phospho-oligosaccharide, which is also generated in response to insulin, may play a role in insulin action. Incubation of the catalytic subunit of cyclic AMP-dependent protein kinase with this phospho-oligosaccharide inhibited the activity of the kinase to phosphorylate histone IIA, a purified preparation of phospholipid methyltransferase and kemptide, a phosphate-accepting peptide. Inhibition of kinase activity was dose-dependent and 50% inhibition of histone phosphorylation was demonstrated with a concentration of phospho-oligosaccharide of around 2 microM. This effect was demonstrated in the presence of ATP at concentrations up to 1 mM, indicating that the phospho-oligosaccharide acts at physiological concentrations of ATP and that it does not compete with this nucleotide for the same binding site in the kinase. Inhibition by the phospho-oligosaccharide of kinase activity could be reversed by dilution or dialysis and was not reproduced by up to 50 microM myo-inositol, glucosamine, galactose, myo-inositol 1-phosphate, glucosamine 1-phosphate, galactose 1-phosphate or phosphorylcholine. The inhibitory activity was resistant to mild acid treatment but was labile to treatment with alkali, exposure to nitrous acid or incubation with sodium periodate. The phospho-oligosaccharide had no effect on the phosphorylation of lysine-rich histone by rat brain protein kinase C and on the binding of cyclic AMP to a cyclic AMP-dependent protein kinase. In conclusion, the data in this study suggested that a phospho-oligosaccharide generated from an insulin-sensitive glycophospholipid may play a role in insulin action by modulating cyclic AMP-dependent protein kinase activity.     

Role of cysteine residues in tryptophanase for monovalent cation-induced activation

We cloned and sequenced the tryptophanase structural gene of Escherichia coli B/1t7-A strain. The results indicate that tryptophanase proteins of E. coli B/1t7-A and K-12 are identical. When cysteine residues in tryptophanase were chemically modified with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), the stabilizing effect of the active cations such as K+ and NH4+ was abolished. In consideration of our previous results that Cys-298 was selectively modified by SH reagents [Honda T. et al. (1986) J. Chromatogr. 371, 353-360], Cys-298 seems to have a close relation to the expression of the effect of monovalent cations. Fluorescence decay measurement of the holoenzyme revealed that the fluorescence lifetime derived from the coenzyme, pyridoxal 5'-phosphate (PLP), was dependent on coexisting monovalent cations, whereas that of the tryptophyl residue was not, in either the apo- or the holoenzyme preparation. The results of the synchrotron small-angle X-ray scattering measurements showed that radii of gyration which reflect the size and shape of the enzyme were constant at around 38 A irrespective of the presence or absence of the K+ ion. These results suggest that the monovalent cations interact specifically with the PLP-binding site, and that the conformational change of enzyme protein caused by the monovalent-cation binding is limited to a small range. The above results are compatible with the possibility that Cys-298 is involved in the formation of "monovalent cation binding site" in the holoenzyme.     

The manipulation of polar head group composition of phospholipids in the wheat Miranovskaja 808 affects frost tolerance

Caryopses of the frost-resistant cultivar of the wheat Triticum aestivum L., Miranovskaja 808, were germinated and grown in the presence of various concentrations of choline chloride. Changes in the composition of leaf total phospholipids and leaf total fatty acids at two extreme temperatures (25°C and 2°C) as well as changes in frost resistance were followed. A choline chloride concentration-dependent accumulation of phosphatidyl choline was observed in the leaves. Seedlings grown at 2°C accumulated more phosphatidyl choline at each choline chloride concentration than those grown at 25°C. There was an inverse relationship between the contents of phosphatidyl choline and phosphatidic acid in the leaves. Neither the temperature nor choline chloride seemed to affect fatty-acid composition. Modification of polar-head group composition of phospholipids affected frost tolerance: Seedlings grown in the presence of 15 mM choline chloride at 25°C exhibited a freezing resistance equal to that of hardened controls. The data indicate that the polar-head group composition of membrane phospholipids in plants can be easily manipulated and point to the importance of phosphatidyl choline in cold adaptation processes.     

Conformational analysis of the polar head group in phosphatidylcholine bilayers: a structural change induced by cations

The conformation of the polar head group of phosphatidylcholine in a bilayer in the liquid-crystalline state was deduced by analyzing the deuterium quadrupole splittings of the choline group and the phosphorus chemical shift anisotropy of the phosphate group in combination with the restriction of the choline conformation determined in laser Raman studies. The latter efficiently reduced the number of candidates for the actual conformation. A family of conformations was obtained for both the dynamic-structure and rigid-structure models, respectively. The polar head group is oriented roughly parallel to the membrane surface in both models. Furthermore, they are close to conformation A of the crystal structure of 1,2-dimyristoyl-sn-glycero-3-phosphocholine. The dynamic-structure model was concluded to be more reasonable in view of the fact that the polar head-group structures in most crystals comprise two conformations, which are nearly mirror images of each other. Conformational analysis was also carried out for the polar head group in the presence of multivalent cations. A possible conformational change of the polar head group induced by cations is discussed in the light of the present results.