Emilio Rivas

Structure of the cubic phases of lipid-water systems.

IN the course of the structure analysis of the mesomorphic phases occurring in lipid–water systems1–4 we have observed an optically isotropic phase, the X-ray reflexions of which can be indexed on a face-centred cubic lattice2,3. We assumed that its structure consists of spherical particles, packed in a face-centred cubic lattice. We further postulated that the spheres consist of micelles containing lipid2,3; but we pointed out that the alternative model, formed by water spheres embedded in a hydrocarbon matrix, could not be discarded solely on the basis of the X-ray data5. We intend to show in this communication that the latter structure is in fact more satisfactory, when other lines of evidence are taken into account.

Structure et polymorphisme des lipides: Étude par diffraction des rayons X du système formé de lipides de mitochondries de coeur de boeuf et d'Eau☆

Abstract A lipid preparation from beef heart mitochondria, carefully extracted and analysed, has been used for the X-ray diffraction study of the structure of the phases present in the lipid-water system. At high temperature, under conditions in which the conformation of the paraffin chains is “liquid”, two phases are observed. One, found at very low water content, is formed by a two-dimensional hexagonal array of narrow water channels, embedded in a paraffin matrix; this type of hexagonal structure is common in lipid-water systems involving phospholipids of biological origin. The other phase is lamellar, formed by equidistant lipid layers of constant thickness, separated by water layers the thickness of which varies from 8 to over 250 A, according to the water content of the system. The distribution of the electron density in the lipid lamellae has been analysed quantitatively, making use of the fact that, for this type of structure, the amplitudes of the reflections are at all concentrations proportional to the Fourier transform of an isolated lamella. As the temperature is lowered gradual organization of the paraffin chains takes place, without change in the thickness of the lipid lamellae. This phenomenon is interpreted by assuming that the centre of the paraffin leaflet is occupied by an ordered layer, involving a fraction of the paraffin chains, which are organized over part of their length: the conformation of the rest of the chains is assumed to remain “liquid”. At still lower temperatures, two other lamellar phases are observed. One is formed of identical lipid leaflets, each containing a double paraffin layer, in which the chains are stiff (but randomly oriented around thin long axes) and organized in a two-dimensional hexagonal lattice. The other phase consists of an alternate sequence of two types of lipid leaflet: one is the low-temperature form just described, the other is the high-temperature lamellar form, with part of the chains disordered. The agreement of the observed and calculated structure factors provides strong confirmation of the two models. The phase transitions observed in this system certainly involve a segregation of the different lipid molecules. This phenomenon is discussed with reference to the known chemical heterogeneity of the lipids of this system.

STRUCTURE AND POLYMORPHISM IN LIPID-WATER SYSTEMS, AND THEIR POSSIBLE BIOLOGICAL IMPLICATIONS

Uniform lipid layers, in biological membranes, are thought to constitute the passive diffusion barriers between cellular compartments. The question we wish to ask here is to what extent the lipid molecules could play a more active and specific role in the physiological events that occur in membranes. Our approach to this problem is based upon the X-ray scattering analysis of the structure of the various phases that exist in lipid-water systems, as a function of concentration and temperature. This procedure is formally equivalent to the construction of a phase diagram: the different phases are first characterized, their range of existence is delimited, and their structure is determined.

Polymorphisme des lipides polaires et des galacto-lipides de chloroplastes de maïs, en présence d'eau

Abstract The lipid-water phase diagrams of polar- and galacto-lipids extracted from maize chloroplasts are described and the structures of the various phases are analysed. The general conformation of the paraffin chains is found to be liquid-like. One phase is lamellar, formed by identical planar lipid leaflets, filled by the paraffin chains and separated by layers of water. In the case of the polar lipids the thickness of the water layer increases more than 150 A with increasing water concentration, whereas in the case of the galacto-lipids only a small amount of water can be incorporated between the lipid leaflets (the maximum thickness of the water layer is 20 A) and the water in excess remains as a separate phase. This property, previously observed with other lipids, is related to the presence of net electrical charges in polar lipids. In the “dry” region of the phase diagram other phases are observed: one is a hexagonal array of infinite stiff rods, filled by the polar elements of the system, the other is formed by rods of similar structure but of finite length, joined three by three to form two interwoven three-dimensional networks, organized in a body-centred cubic lattice. The hexagonal phase is observed for both the polar- and galacto-lipids and the cubic phase only in galacto-lipids. The structures of the lamellar and of the cubic phases are consistent with the analysis of the intensity of the X-ray reflections. The observations are compared to those previously made with other lipid systems, and some conclusions of general interest are drawn.

A single-step method to concentrate and desalt proteins which is also useful for determination of detergent binding to membrane proteins.

Abstract The conditions for obtaining highly concentrated salt-free protein solutions have been examined. It was found that a convenient and rapid method consists of combining ion-exchange and gel-filtration chromatography in one column. We observed that a volume ratio of Sephadex G-25 to ion exchanger of 2.5 is a good compromise to obtain a good recovery (97%) of a concentrated protein solution (≥ 10 mg/ml) free of salt. The procedure is also useful and accurate for measuring the detergent-to-membrane-protein-binding ratio and for preparing solutions for scattering studies.

Isolation of rhodopsin by the combined action of cardiotoxin and phospholipase A2 on rod outer segment membranes

Freeze-fracture electron microscopy was used to follow morphological changes induced by Naja mossambica mossambica venom VII4 cardiotoxin in rod outer segment membrane preparations. The extent of the morphological changes depend on the purity of the cardiotoxin. Pure cardiotoxin had no detectable effect upon the preparation, but, when contaminated with venom phospholipase A2, led to a rapid disintegration of the membrane vesicles. With trace amounts (up to about 0.5% of the cardiotoxin) of phospholipase A2, the membrane vesicles disintegrated into smooth lamellae and particles in solution. These two components were separated by centrifugation. The pellet, which showed the presence of smooth lamellae and aggregated particles, was composed of unbleached rhodopsin, initial membrane lipids, lysolipids and cardiotoxin. The supernatant, which showed only the presence of dispersed particles, was composed of unbleached rhodopsin, lysolipids and cardiotoxin. With cardiotoxin contained larger amounts of phospholipase A2 (more than 0.5% of the cardiotoxin), membrane vesicles were disintegrated into large aggregates of amorphous material, composed of bleached rhodopsin, initial membrane lipids, lysolipids and cardiotoxin. These results confirm our previous observation on the release of integral membrane proteins from membrane vesicles by the action of cardiotoxin containing traces of phospholipase A2 (Gulik-Krzywicki, T., Balerna, M., Vincent, J.P. and Lazdunski, M. (1981) Biochim. Biophys. Acta 643, 101-114) and suggest it possible use for isolation and purification of integral membrane proteins.

Thickness measurements of single walled dimyristoyl phosphatidylcholine vesicles by neutron scattering

A method of deriving by neutron scattering thicknesses of lamellae in suspensions has been applied to single-walled vesicles of dimyristoyl phosphatidylcholine. The contrast variation method, based on data obtained for a range of isotope mixtures, has been used to extract a dimension Dw related to the lipid bilayer thickness and a measure alpha of the difference of density within the lamellae. Isotope mixtures for the lipid were used to optimize the information available. Dw is compared with results from multilayer stacks of lipid layers. The thickness for the low temperature L beta, structure has been observed to be higher than for the high temperature L alpha structure. Preliminary experiments on the kinetics of the mixing of the lipid isotope species are reported, and evidence is shown that the species are not segregated for lipids either above or below the transition temperature.

Phospholipid and pigment alterations after fusion between Rhodobacter sphaeroides chromatophores and acidic liposomes

Abstract Wild-type Rhodobacter sphaeroides chromatophores were fused at acidic pH, or by freezing and thawing, with liposomes of soybean phospholipids, phosphatidylserine, phosphatidylglycerol or diphosphatidylglycerol. Equilibrium centrifugation after fusion yielded several fractions. Freeze-fracture electron microscopy showed that fusion resulted in the formation of unilamellar vesicles of diameters larger than that of chromatophores. The lateral density of the intramembrane particles decreased; the asymmetry between the two fracture faces was lost after fusion with soybean phospholipids or phosphatidylserine or phosphatidylglycerol, but gradually disappeared in parallel with diphosphatidylglycerol enrichment. After fusion with phosphatidylserine, when the fractions were frozen from below the lipid transition temperature intramembrane particles aggregated into patches surrounded by smooth lipid zones. A massive incorporation of the fusogen phospholipid was observed in the fractions together with a strong decrease of phosphatidylglycerol and a lower decrease of phosphatidylcholine and aminolipid. The 800 nm absorption band of the B800–850 antenna complex was reduced or suppressed depending on the nature of the lipids while the spectroscopic alteration of B875 chromophore was weaker. The light-induced bandshifts of carotenoid and antenna bacteriochlorophyll were also much weaker or absent; this could result from a desorganization of the B800–850 antenna, or from an impaired capacity to sustain a photoinduced membrane potential. The reaction center was not affected by the fusion, and the polypeptide composition of the various fractions did not show qualitative differences from the chromatophore pattern. Spheroplasts did not show the same capacity of fusion as chromatophores.