Chemically induced lipid phase separation in model membranes containing charged lipids: a spin label study.

The lipid distribution in binary mixed membranes containing charged and uncharged lipids and the effect of Ca2+ and polylysine on the lipid organization was studied by the spin label technique. Dipalmitoyl phosphatidic acid was the charged, and spin labelled dipalmitoyl lecithin was the uncharged (zwitterionic) component. The ESR spectra were analyzed in terms of the spin exchange frequency, Wex. By measuring Wex as a function of the molar percentage of labelled lecithin a distinction between a random and a heterogeneous lipid distribution could be made. It is established that mixed lecithin-phosphatidic acid membranes exhibit lipid segregation (or a miscibility gap) in the fluid state. Comparative experiments with bilayer and monolayer membranes strongly suggest a lateral lipid segregation. At low lecithin concentration, aggregates containing between 25% and 40% lecithin are formed in the fluid phosphatidic acid membrane. This phase separation in membranes containing charged lipids is understandable on the basis of the Gouy-Chapman theory of electric double layers. In dipalmitoyl lecithin and in dimyristoyl phosphatidylethanolamine membranes the labelled lecithin is randomly distributed above the phase transition and has a coefficient of lateral diffusion of D = 2.8-10(-8) cm2/s at 59 degrees C. Addition of Ca2+ dramatically increases the extent of phase separation in lecithin-phosphatidic acid membranes. This chemically (and isothermally) induced phase separation is caused by the formation of crystalline patches of the Ca2+-bound phosphatidic acid. Lecithin is squeezed out from these patches of rigid lipid. The observed dependence of Wex on the Ca2+ concentration could be interpreted quantitatively on the basis of a two-cluster model. At low lecithin and Ca2+ concentration clusters containing about 30 mol % lecithin are formed. At high lecithin or Ca2+ concentrations a second type of precipitation containing 100% lecithin starts to form in addition. A one-to-one binding of divalent ions and phosphatidic acid at pH 9 was assumed. Such a one-to-one binding at pH 9 was established for the case of Mn2+ using ESR spectroscopy. Polylysine leads to the same strong increase in the lecithin segregation as Ca2+. The transition of the phosphatidic acid bound by the polypeptide is shifted from Tt = 47.5 degrees to Tt = 62 degrees C. This finding suggests the possibility of cooperative conformational changes in the lipid matrix and in the surface proteins in biological membranes.     

Ca2+-translocation activities of phosphatidylinositol, diacylglycerol and phosphatidic acid inferred by quin-2 in artificial membrane systems

Ca2+-translocating activities of phosphatidylinositol, diacylglycerol and phosphatidic acid were investigated in phosphatidylcholine liposomes. Using a fluorescent indicator of Ca2+ concentration, quin-2, release of encapsulated Ca2+ from egg yolk phosphatidylcholine liposomes containing 2 mol% of one of these lipids was measured at 37 degrees C. The rate of Ca2+ translocation across the liposomal membrane mediated by phosphatidic acid was about 3-fold larger than those mediated by phosphatidylinositol and diacylglycerol. The result implies that phosphatidic acid has Ca2+-ionophore activity in the agonist dependent metabolism of inositol phospholipids. The ionophoretic activity depended on the degree of unsaturation of the fatty acyl chains. The Ca2+ translocation rate was smallest in dipalmitoylphosphatidic acid, and it increased in the order of dioleoyl-, dilinoleoyl- and dilinolenoyl-phosphatidic acid. Ca2+ mobilization of a stimulated cell is discussed in the light of Ca2+-ionophore activity of phosphatidic acid converted from inositol phospholipids.     

Lipid vesicles which can bind to protein kinase C and activate the enzyme in the presence of EGTA.

Maximal protein kinase C activity with vesicles of phosphatidic acid and 1,2-dioleoyl-sn-glycerol is observed in the absence of added Ca2+. Addition of phosphatidylcholine to these vesicles restores some calcium dependence of enzyme activity. 1,2-Dioleoyl-sn-glycerol eliminates the Ca(2+)-dependence of protein kinase C activity found with phosphatidic acid alone. Phorbol esters do not mimic the action of 1,2-dioleoyl-sn-glycerol in this respect. This suggests that the 1,2-dioleoyl-sn-glycerol effect is a result of changes it causes in the physical properties of the membrane rather than to specific binding to the enzyme. The effect of 1,2-dioleoyl-sn-glycerol on the phosphatidic-acid-stimulated protein kinase C activity is dependent on the molar fraction of 1,2-dioleoyl-sn-glycerol used and results in a gradual shift from Ca2+ stimulation at low 1,2-dioleoyl-sn-glycerol concentrations to calcium inhibition at higher concentrations of 1,2-dioleoyl-sn-glycerol. Phosphatidylserine-stimulated activity is also shown to be largely independent of the calcium concentration at higher molar fractions of 1,2-dioleoyl-sn-glycerol. Thus, with certain lipid compositions, protein kinase C activity becomes independent of the calcium concentration or requires only very low, stoichiometric binding of Ca2+ to high affinity sites on the enzyme. Protein kinase C can bind to phosphatidic acid vesicles more readily than it can bind to phosphatidylserine vesicles in the absence of calcium. Addition of 1,2-dioleoyl-sn-glycerol to phosphatidylserine vesicles promotes the partitioning of protein kinase C into the membrane in the absence of added Ca2+. There is no isozyme specificity in this binding. These results suggest that a less-tightly packed headgroup region of the bilayer causes increased insertion of protein kinase C into the membrane. This is a necessary but not sufficient condition for activation of the enzyme in the presence of EGTA.     

Fusion of phospholipid vesicles induced by muscle glyceraldehyde-3-phosphate dehydrogenase in the absence of calcium

Ca2+-induced fusion of phospholipid vesicles (phosphatidylcholine/phosphatidic acid, 9:1 mol/mol) prepared by ethanolic injection was followed by five different procedures: resonance energy transfer, light scattering, electron microscopy, intermixing of aqueous content, and gel filtration through Sepharose 4-B. The five methods gave concordant results, showing that vesicles containing only 10% phosphatidic acid can be induced to fuse by millimolar concentrations of Ca2+. When the fusing capability of several soluble proteins was assayed, it was found that concanavalin A, bovine serum albumin, ribonuclease, and protease were inactive. On the other hand, lysozyme, L-lactic dehydrogenase, and muscle and yeast glyceraldehyde-3-phosphate dehydrogenase were capable of inducing vesicle fusion. Glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle, the most extensively studied protein, proved to be very effective: 0.1 microM was enough to induce complete intermixing of bilayer phospholipid vesicles. Under conditions used in this work, fusion was accompanied by leakage of internal contents. The fusing capability of glyceraldehyde-3-phosphate dehydrogenase was not affected by 5 mM ethylenediaminetetraacetic acid. The Ca2+ concentration in the medium, as determined by atomic absorption spectroscopy, was 5 ppm. Heat-denatured enzyme was incapable of inducing fusion. We conclude that glyceraldehyde-3-phosphate dehydrogenase is a soluble protein inherently endowed with the capability of fusing phospholipid vesicles.     

Phosphatidylcholine and cholesterol inhibit phosphatidate-mediated calcium traversal of liposomal bilayers

Rates of phosphatidic acid- (PA-) mediated Ca2+-traversal are maximal in 'passive bilayers' void of lipid CO and OH groups: dietherphosphatidylcholine (diether-PC) or OH-blocked cholesterol liposomes. Phosphatidylcholine (PC) as bilayer matrix causes 99% inhibition, while 45 mol% cholesterol in passive bilayers inhibits by about 70%. Possibly, the absence of CO and OH groups causes a dehydration of the 'hydrogen belts', i.e., the membrane strata occupied by hydrogen bond acceptors (CO of phospholipids) and donors (OH of cholesterol, sphingosine) and thereby facilitates the formation of dehydrated Ca(PA)2, the ionophoric vehicle; or (our preferred explanation) PC engages in a (non-ionophoric) Ca(PA X PC) complex and thus reduces the concentration of the ionophore, while cholesterol competes with Ca2+ for the CO groups of phosphatidic acid by hydrogen-bonding. The Ca2+-traversal rates realized in bilayers with modified hydrogen belts lend support to the speculation that a Ca(PA)2 ferry may be of physiological importance, e.g., in membranes (such as myelin) containing much ether phospholipid (plasmalogen); and that Ca2+-membrane association and traversal may be controlled by the composition of the hydrogen belts.     

Polymyxin binding to charged lipid membranes. An example of cooperative lipid-protein interaction.

The binding of polymyxin-B to lipid bilayer vesicles of synthetic phosphatidic acid was studied using fluorescence, ESR spectroscopy and electron microscopy. 1,6-Diphenylhexatriene (which exhibits polarized fluorescence) and pyrene decanoic acid (which forms excimers) were used as fluorescence probes to study the lipid phase transition. The polymyxin binds strongly to negatively charged lipid layers. As a result of lipid/polymyxin chain-chain interactions, the transition temperature of the lipid. This can be explained in terms of a slight expansion of the crystalline lipid lattice (Lindeman's rule). Upon addition of polymyxin to phosphatidic acid vesicles two rather sharp phase transitions (width deltaT = 5 degrees C) are observed. The upper transition (at Tu) is that of the pure lipid and the lower transition (at T1) concerns the lipid bound to the peptide. The sharpness of these transitions strongly indicates that the bilayer is characterized by a heterogeneous lateral distribution of free and bound lipid regions, one in the crystalline and the other in the fluid state. Such a domain structure was directly observed by electron microscopy (freeze etching technique). In (1 : 1) mixtures of dipalmitoyl phosphatidic acid and egg lecithin, polymyxin induces the formation of domains of charged lipid within the fluid regions of egg lecithin. With both fluorescence methods the fraction of lipid bound to polymyxin-B as a function of the peptide concentration was determined. S-shaped binding curves were obtained. The same type of binding curve is obtained for the interaction of Ca2+ with phosphatidic acid lamellae, while the binding of polylysine to such membranes is characterized by a linear or Langmuir type binding curve. The S-shaped binding curve can be explained in terms of a cooperative lipid-ligand (Ca2+, polymyxin) interaction. A model is proposed which explains the association of polymyxin within the membrane plane in terms of elastic forces caused by the elastic distortion of the (liquid crystalline) lipid layer by this highly asymmetric peptide.     

Phosphatidylethanol counteracts calcium-induced membrane fusion but promotes proton-induced fusion

The susceptibility of phosphatidylethanol-containing lipid vesicles towards Ca2+- and proton-induced fusion has been investigated, using a system of interacting vesicles. The results show that phosphatidylethanol-rich vesicles are quite resistant to Ca2+-induced fusion while being highly sensitive to proton-induced fusion. Inclusion of phosphatidylethanol was also found to promote and inhibit, respectively, the proton-induced and Ca2+-induced fusion of bilayer vesicles containing also phosphatidylethanolamine and either phosphatidylserine or phosphatidic acid. Thus, phosphatidylethanol affected Ca2+- and proton-induced fusion in opposite directions, in contrast to the naturally occurring anionic phospholipids phosphatidic acid, phosphatidylserine and phosphatidylinositol, which affect the sensitivity to Ca2+- and H+-induced fusion in the same direction. However, the fusion competence of phosphatidylethanol vesicles in response to both Ca2+ and H+ was inversely related to the apparent thickness of the polar headgroup layer, determined by using lectin-glycolipid interaction as a steric probe, as previously found for vesicles containing naturally occurring anionic phospholipids.     

Calcium diphosphatidate membrane traversal is inhibited by common phospholipids and cholesterol but not by plasmalogen

Phosphatidate-mediated Ca2+ membrane traversal is inhibited by phospholipids (PL) such a phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin and lysoPC, but not by PC-plasmalogen. Kinetics of Ca2+ traversal through a 'passive' bilayer consisting of OH-blocked cholesterol show competition between PC and phosphatidic acid (PA); it appears likely that a Ca(PA.PC) complex is formed which is not a transmembrane ionophore but will reduce the amount of phosphatidic acid available for the formation of the ionophore, Ca(PA)2. PS and PI may inhibit Ca2+-traversal in the same manner by forming Ca(PA.PL) complexes. We suggest that PC-plasmalogen, with one of the Ca2+-chelating ester CO groups missing, cannot engage in calcium cages, i.e., Ca(PA.PL) complexes, and thus does not interfere with Ca(PA)2 formation. Double-reciprocal plotting of Ca2+ traversal rates in cholesterol-containing liposomes vs. calcium concentration suggests that cholesterol inhibits Ca2+ traversal by competing with Ca2+ for PA. The inhibition does not seem to be caused by a restructuring or dehydration of the membrane 'hydrogen belts' affected by cholesterol; most probably, it is due to hydrogen bonding of the cholesterol-OH group to a CO group of PA; this reduces the amount of PA available for the calcium ferry. The inhibition by sphingomyelin and lysoPC may also be explained by their OH group interacting with PA via hydrogen bonding. The pH dependence of Ca2+ traversal suggests that H[Ca(PA)2]- can serve as Ca2+ cross-membrane ferry but that at physiological pH, [Ca(PA)2]2- is the predominant ionophore. In conclusion, the results indicate that Ca2+ traversal is strongly dependent on the structure of the hydrogen belts, i.e., the membrane strata occupied by hydrogen bond acceptors (CO of phospholipids) and donors (OH of cholesterol, sphingosine), and that lipid hydrogen belt structures may regulate storage and passage of Ca2+.     

Phosphatidic acid and arachidonic acid each interact synergistically with glucagon to stimulate Ca2+ influx in the perfused rat liver.

The administration of phosphatidic acid to rat livers perfused with media containing either 1.3 mM- or 10 microM-Ca2+ was followed by a stimulation of Ca2+ efflux, O2 uptake and glucose output. The responses elicited by 100 microM-phosphatidic acid were similar to those induced by the alpha-adrenergic agonist phenylephrine. Contrary to suggestions that phosphatidic acid acts like a Ca2+-ionophore, no net influx of Ca2+ was detected until the phosphatidic acid was removed. Sequential infusions of phenylephrine and phosphatidic acid indicate that the two agents release Ca2+ from the same intracellular source. The co-administration of glucagon (or cyclic AMP) and phosphatidic acid, and also of glucagon and arachidonic acid, led to a synergistic stimulation of Ca2+ uptake of the liver, a feature similar to that observed after the co-administration of glucagon and other Ca2+-mobilizing hormones [Altin & Bygrave (1986) Biochem. J. 238, 653-661]. A notable difference, however, is that the synergistic stimulation of Ca2+ uptake induced by the co-administration of glucagon and arachidonic acid was inhibited by indomethacin, whereas that induced by glucagon and phosphatidic acid, or glucagon and other Ca2+-mobilizing agents, was not. The results suggest that the synergistic action of glucagon and arachidonic acid in stimulating Ca2+ influx is mediated by prostanoids, but that of glucagon and phosphatidic acid is evoked by a mechanism similar to that of Ca2+-mobilizing agents.     

Fusion of phosphatidic acid-phosphatidylcholine mixed lipid vesicles

Ca²⁺-induced transformation of phosphatidylcholine-phosphatidic acid vesicles to larger bilayer structures has been examined using nuclear magnetic resonance, electron microscopy, gel permeation and radioisotope tracer techniques. For concentrated vesicle preparations where phosphatidic acid content remains less than 50% of total lipid, transformation to larger well defined unilamellar structures can be induced. The size of the product formed is dependent on phosphatidic acid content and on Ca²⁺ content when Ca²⁺ levels are between 0.3 and 1.0 mol ratios with respect to phosphatidic acid. During transformation bilayer composition remains unchanged and internal contents are retained in the final structure. These properties are indicative of concerted two vesicle and multiple vesicle fusions. The controllable and concerted fusions make the phosphatidic acid system suitable for further mechanistic studies.     

Effect of cholesterol on Ca2+-induced aggregation of liposomes and calcium diphosphatidate membrane traversal

Sonicated cholesterol-phosphatidylcholine (PC) liposomes containing 4 mol % phosphatidic acid (PA) aggregate in 10 mM Ca2+, slowly at low molar fractions of cholesterol (up to 30%) and 15 times faster at higher concentrations; the inflection point is at ca. 35 mol % bilayer cholesterol. O-[[(Methoxyethoxy)ethoxy]ethyl]cholesterol (OH-blocked cholesterol) does not give this rate enhancement. If PC is replaced by diether PC (CO groups abolished), cholesterol does not accelerate aggregation at concentrations in the bilayer below 50 mol %. No change in Ca2+-induced aggregation rates was observed if the ester CO groups of the bridge-forming PA only were replaced by CH2 (diether PA) in liposomes containing PC and cholesterol. PA-mediated Ca2+ membrane traversal seems to be accelerated by the addition of cholesterol to the PC-PA membrane, but analysis shows that the effect is due to the bilayer condensation effect of cholesterol resulting in an increase in the surface concentration of PA and that membrane cholesterol in fact slightly reduces the rate of Ca(PA)2 traversal; OH-blocked cholesterol, however, increases this rate 3-fold. It appears that lipid OH and CO groups interact, directly or with the mediation of water, in establishing the structure of the membrane "hydrogen belts", i.e., the strata containing those hydrogen-bond donors and acceptors. Cholesterol hydroxyl above 33 mol % (saturation of a 2:1 PC/cholesterol complex?) causes a restructuring of the hydrogen belts that facilitates membrane-water-membrane dehydration, the prerequisite for liposome aggregation by trans-Ca(PA)2 formation. On the other hand, the formation of the dehydrated cis-Ca(PA)2 complex that precedes Ca2+ membrane traversal is not accelerated by presence of the cholesterol hydroxyl group.     

Changes in morphology and in polyphosphoinositide turnover of human erythrocytes after cholesterol depletion

Human erythrocytes were cholesterol-depleted (5-25%) by incubation with phosphatidylcholine vesicles in media containing Ca2+ at different concentrations (0, 28 nM, 5 microM or 1 mM). After removal of the vesicles, the cells were reincubated with [32P]phosphate in the same media. Control (incubated in buffer alone) and cholesterol-maintained erythrocytes (incubated with cholesterol/phosphatidylcholine vesicles) were treated similarly. Cholesterol depletion induced the conversion of the cells into stomatocytes III and spherostomatocytes and decreased the turnover rate of phosphatidylinositol phosphate and of phosphatidylinositol bisphosphate. None of these effects were observed in cholesterol-maintained cells. In cholesterol-depleted cells, they occurred without changes in the ATP specific activity or in the polyphosphoinositide concentrations. Moreover, these modifications of shape and of lipid metabolism were proportional to the extent of the cholesterol depletion and were independent of the external Ca2+ concentration. In contrast, other effects of cholesterol depletion, a decrease in the turnover rate of phosphatidic acid, a decrease in diacylglycerol and in phosphatidic acid concentrations were dependent on the external Ca2+ concentration. Thus it appears that the shape change was not correlated with a change in the concentrations of these phospholipids or of diacylglycerol and therefore cannot be explained by a bilayer couple mechanism involving these phospholipids. However, the spherostomatocytic transformation was correlated with the decrease in the turnover rate of the polyphosphoinositides, but not with the turnover rate of phosphatidic acid, suggesting a role for the turnover of the polyphosphoinositides in the maintenance of the erythrocyte shape.     

The influence of charge on phosphatidic acid bilayer membranes.

A complete titration of phosphatidic acid bilayer membranes was possible for the first time by the introduction of a new anaologue, 1,2-dihexadecyl-sn-glycerol-3-phosphoric acid, which has the advantage of a high chemical stability at extreme pH values. The synthesis of the phosphatidic acid is described and the phase transition behaviour in aqueous dispersions is compared with that of three ester phosphatidic acids; 1,2-dimyristoyl-sn-glycerol-3-phosphoric acid, 1,3-dimyristoylglycerol-2-phosphoric acid and 1,2-dipalmitoyl-sn-glycerol-3-phosphoric acid. The phase transition temperatures (Tt) of aqueous phosphatidic acid dispersions at different degrees of dissociation were measured using fluorescence spectroscopy and 90 degrees light scattering. The Tt values are comparable to the melting points of the solid phosphatidic acids in the fully protonated states, but large differences exist for the charged states. The Tt vs. pH diagrams of the four phosphatidic acids are quite similar and of a characteristic shape. Increasing ionisation results in a maximum value for the transition temperatures at pH 3.5 (pK1). The regions between the first and the second pK of the phosphatidic acids are characterised by only small variations in the transition temperatures (extended plateau) in spite of the large changes occurring in the surface charge of the membranes. The slope of the plateau is very shallow with increasing ionisation. A further decrease in the H+ concentration results in an abrupt change of the transition temperature. The slope of the Tt vs. pH diagram beyond pK2 becomes very steep. This is the result of reduced hydrocarbon interaction energy, which was demonstrated by differential scanning calorimetry (Blume, A. and Eibl, H., unpublished data).     

Distribution of phospholipids around gramicidin and D-beta-hydroxybutyrate dehydrogenase as measured by resonance energy transfer

A resonance energy transfer method was developed to study the distribution of phospholipids around integral membrane proteins. The method involved measuring the extent of energy transfer from tryptophan residues of the proteins to different phospholipids labeled with a dansyl moiety in the fatty acid chain. No specific interactions were observed between gramicidin and dansyl-labeled phosphatidylcholine, phosphatidylethanolamine, or phosphatidic acid. The results were consistent with a random distribution of each phospholipid in the bilayer in the presence of gramicidin. However, a redistribution of both gramicidin and dansyl-labeled phospholipids was easily observed when a phase separation was induced by adding Ca2+ to vesicles made up of phosphatidylcholine and phosphatidic acid. Polarization measurements showed that in the presence of Ca2+ a rigid phosphatidic acid rich region and a more fluid phosphatidylcholine-rich region were formed. Energy-transfer measurements from gramicidin to either dansylphosphatidylcholine or dansylphosphatidic acid showed gramicidin preferentially partitioned into the phosphatidylcholine-rich regions. Energy-transfer measurements were also carried out with D-beta-hydroxybutyrate dehydrogenase reconstituted in a vesicle composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid. Although the enzyme has a specific requirement for phosphatidylcholine for activity, the extent of energy transfer decreased in the order dansylphosphatidic acid, dansylphosphatidylcholine, dansylphosphatidylethanolamine. Thus, the enzyme reorganized the phospholipids in the vesicle into a nonrandom distribution.     

Influence of alkali metal cations on the transport of calcium by phosphatidate in multi-phase systems

The effects of alkali metal cations on the rates at which Ca2+ and phosphatidic acid were cotransported from aqueous to hydrocarbon medium were examined. The alkali metal cations remained in the aqueous phase yet specifically influenced the transport of Ca2+ into the hydrocarbon solvent. For the physiological cations, Na+ and K+, there were critical concentration ranges in which small changes in concentration effected sharp changes in transport rates. The maximal rate observed with Na+ was an order of magnitude greater than that with K+; however, unlike Na+, K+ promoted low levels of transport below the critical concentration range. Li+ effected only low levels of transport even at high concentrations, whereas Rb+ and Cs+ induced transport at rates proportional to their concentrations. These results are discussed in terms of a classical ionophore model for the complex composed of a neutral phosphatidic acid dimer bridged by Ca2+.     

Evidence for tight coupling of phospholipase activation and Ca2+ influx during acrosome reaction of golden hamster spermatozoa

1. Phospholipases have been proposed to play a key role in sperm acrosome reaction. To examine the activation mechanism of phospholipases and subsequently sperm fertilizing capacity. Ca2+ fluxes and phospholipid turnover (breakdown and synthesis) were investigated in golden hamster spermatozoa during acrosome reaction. 2. Upon exposure of the spermatozoa to 1.7 mM Ca2+, a net uptake by the cells occurred in two distinguishable phases. 3. Depletion of extracellular Ca2+ by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) at a time that an initial Ca2+ uptake was observed to reach almost steady-state, prevented the secondary Ca2+ uptake and acrosome reaction. 4. The time course of an initial Ca2+ uptake seemed to precede that of the acrosome reaction. 5. Incubation of the spermatozoa with Ca2+ in the presence of [3H]glycerol induced a rapid increase in labeling of phosphatidic acid, a key intermediate of phosphinositide turnover initiated by the action of phospholipase C, which appeared to parallel the time course of a first phase of Ca2+. 6. Phospholipase A2 activation, detected by lysophospholipid formation, slightly delayed the initial events of first Ca2+ uptake and phosphatidic acid production. 7. It is concluded that first Ca2+ entry into the cells, associated with phosphatidic acid production, activates a phospholipase A2, leading to the production of substances, like lysophospholipids and fatty acids, which may contribute to acrosome reaction.     

Relationship between intact cell ATP levels and glucocorticoid receptor-binding capacity in the AtT-20 cell

A study was made on the correlation between the degree of membrane fusion and surface tension increase of phosphatidic acid membranes caused by divalent cations. Membrane fusion was followed by the Tb3+/dipicolinic acid assay, monitoring the fluorescent intensity for mixing of the internal aqueous contents of small unilamellar lipid vesicles. The surface tension and surface potential of monolayers made of the same lipids as used in the fusion experiments were measured as a function of divalent cation concentration. It was found that the 'threshold' concentration to induce massive vesicle membrane fusion was the same for Ca2+ and Mg2+, and that the surface tension increase in the monolayer, induced by changing divalent cation concentration from zero to a concentration which corresponds to its threshold value, inducing vesicle membrane fusion, was approximately the same: 6.3 dyn/cm for both Ca2+ and Mg2+. Both the divalent cation's threshold concentrations as well as the surface tension change corresponding to the threshold concentration for the phosphatidic acid membrane were smaller than those for the phosphatidylserine membrane. The different fusion capability of these divalent cations for phosphatidic acid and phosphatidylserine membranes is discussed in terms of the different ion binding capabilities of these ions to the membranes.     

Phosphoinositide metabolism and the morphology of human erythrocytes

ATP-depleted human erythrocytes lose their smooth discoid shape and adopt a spiny, crenated form. This shape change coincides with the conversion of phosphatidylinositol-4,5-bisphosphate to phosphatidylinositol and phosphatidic acid to diacylglycerol. Both crenation and lipid dephosphorylation are accelerated by iodoacetamide, and both are reversed by nutrient supplementation. The observed changes in lipid populations should shrink the membrane inner monolayer by 0.6%, consistent with estimates of bilayer imbalance in crenated cells. These observations suggest that metabolic crenation arises from a loss of inner monolayer area secondary to the degradation of phosphatidylinositol-4,5-bisphosphate and phosphatidic acid. A related process, crenation after Ca2+ loading, appears to arise from a loss inositides by a different pathway.     

The bivalent-cation dependence of phosphatidylinositol synthesis in a cell-free system from lymphocytes.

The bivalent-cation requirements of two enzymes involved in phosphatidylinositol synthesis were defined for pig lymphocyte membranes using a citric acid buffer. CTP:phosphatidic acid cytidylyltransferase (EC 2.7.7.41) is activated by free Mn2+ concentrations above 20nM and by free Mg2+ concentrations above 10 microM. When activated by Mg2+, the enzyme is weakly inhibited by Ca2+ (Ki greater than 250 microM), but Ca2+ has no effect when Mn2+ is used to stimulate CDP-diacylglycerol synthesis. The synthesis of phosphatidylinositol from phosphatidic acid is also stimulated by Mn2+ and Mg2+ concentrations similar to those above and is inhibited by free Ca2+ concentrations above 500nM, probably by its action on CDP-diacylglycerol:inositol 3-phosphatidyltransferase (EC 2.7.8.11). Taken together, these studies suggest that under physiological conditions phosphatidylinositol synthesis is activated by Mg2+ and it is possible that it is further regulated by the free concentrations of Ca2+ and/or Mn2+.     

Stabilization effect of protons and divalent cations on membrane structures of lipids

Ordered-fluid phase transitions of methylphosphatidic and phosphatidic acid bilayers are discussed theoretically to explain comprehensively various observed data in the presence of Ca2+ or protons. It is shown that the observed data can be explained reasonably by taking account of the interaction among neighboring head-groups with the aid of Ca2+ or protons. As a result, a quantitative explanation is given for the dependence of the phase-transition temperature on pH as well as Ca2+ concentration with the coexistence of monovalent cations. The dependence of the cooperativity of the transition on pH and monovalent cation concentration is also well explained. It is also pointed out that in the ordered-fluid phase transitions, a hysteresis metastable state and phase separation can be expected to appear under some conditions.