Kinetics and thermodynamics of calcium-induced lateral phase separations in phosphatidic acid-containing bilayers

Abstract

The effects of calcium on the mixing of synthetic diacylphosphatidylcholines (PC's) and diacylphosphatidylethanolamines (PE's) with the corresponding phosphatidic acids (PA's) have been examined by high-sensitivity differential scanning calorimetry and by measurements of the fluorescence of labeled PA or PC species in PA-PC bilayers. Calorimetrically derived phase diagrams for dimyristoyl- and dielaidoyl-substituted PA-PC and PA-PE mixtures indicate that these species are readily miscible in the absence of calcium but phase-separate very extensively in the presence of high levels of calcium (30 mM). The limiting solubilities of PA (Ca2+) in liquid-crystalline PC or PE bilayers are less than or equal to 10 and approximately 5 mol %, respectively, while approximately 20 mol % of PC or PE can be introduced into the "cochleate" phase of PA (Ca2+) before a distinct PC-rich (or PE-rich) phase appears. The kinetics of calcium-induced lateral phase separations were examined for dioleoyl- and dielaidoyl-substituted PA-PC unilamellar vesicles labeled with fluorescent (C12-NBD-acyl) PA or PC, whose fluorescence becomes partially quenched upon phase separation. Our results indicate that, for the PA-PC system, lateral phase separation is very rapid (approximately less than 1 s) after calcium addition and develops partially (possibly in only one face of the bilayer) when calcium is present only on one side of the bilayer. Moreover, phase separations can develop at a rate faster than that of vesicle diffusion when calcium is added to dilute suspensions of vesicles, suggesting that interbilayer contacts are not essential to promote phase separations.

Divalent Cation Induced Fusion and Lipid Lateral Segregation in Phosphatidylcholine-Phosphatidic Acid Vesicles

“…Representative thermograms for mixtures of DEPC and DEPA in the presence of high magnesium levels are shown in Figure 7A. The thermograms are qualitatively very similar to those observed previously for this lipid system in the presence of high levels of calcium (Graham et al, 1985), and they can be analyzed in an entirely similar way to give the phase diagram shown in Figure 7B. For the purposes of the present discussion, the most important feature of this phase diagram is the lefthand vertical boundary of the two-phase region existing between 22 and 94 "C, since this boundary defines the bilayer concentration of DEPA below which no phase separation will occur in the presence of magnesium.…”

“…we investigated the possibility that such fusion requires the lateral segregation of PA-enriched domains in the vesicle membranes. In a previous study (Graham et al, 1985). Aggregates of vesicles can be observed, and many bilamellar diaphragms can be observed in these aggregates in regions of extensive intervesicle contact, as Rand et al (1985) have reported previously for phosphatidylserine vesicles treated with calcium.…”

“…By observing the presence or absence and the amplitude of the endotherm at 22 "C in it appears reasonable to conclude that the solubility of DOPA in liquid-crystalline DOPC in the presence of magnesium is similar to that for the other PA-PC mixtures, i.e., -20 mol %. By contrast, PA-PC mixtures in the presence of calcium phase-separate when the bilayers contain as little as 10 mol 7% PA (Graham et al, 1985).…”

“…We have recently demonstrated that fluorescence measurements can be used to monitor reliably and specifically the lateral distribution of lipids in vesicles containing phosphatidic acid (PA)' and phosphatidylcholine (PC) in the presence and absence of divalent cations (Graham et al, 1985). In this study, we have correlated fluorometric observations of divalent cation induced mixing of lipids and aqueous contents (which we define operationally as "fusion") between PA-PC vesicles with direct electron microscopic observations of vesicle-vesicle ' Abbreviations: ANTS, 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt; C,,-NBD-PC, 1-palmitoyl-2-[ 12-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine; DO, dioleoyl; DPX, p-xylene bis(pyridinium bromide); EDTA, ethylenediaminetetraacetic acid disodium salt; egg PA, phosphatidic acid prepared from egg yolk phosphatidylcholine; egg PC, phosphatidylcholine from egg yolk; LUV, large unilamellar vesicle(s); NBD-PE, N-(7-nitro-2,1,3-benzoxadiazol-4-y1)phosphatidylethanolamine: PA, 1,2-diacyl-snglycero-3-phosphatidic acid: PC, 1,2-diacyI-sn-glycero-3-phosphocholine; PE, 1,2-diacyl-sn-glycero-3-phosphoethanolamine; PS, 1,2-diacyl-snglycero-3-phosphoserine; Rho-PE, N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine; Tes, N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo)]-tetraacetic acid; DE, dielaidoyl. interactions and with calorimetric and fluorometric measurements of PA-PC miscibility, under conditions that promote interactions between vesicles.…”

The Phase Property of Membrane Phospholipids Is Affected by the Functionality of Signal Peptides from the Escherichia coli Ribose-binding Protein

“…The fluorescence of NBD-labeled phospholipids is subject to self-quenching, providing a basis for detecting phospholipid association in the membrane (30,31). Using this property, we tested the possibility that signal peptides induce phase separation of acidic phospholipid.…”