Spectrin-phospholipid interaction. A monolayer study.

(1) The interaction of synthetic and natural phospholipids with spectrin, purified from human erythrocyte membranes, was studied using the monolayer technique at constant surface pressure. Spectrin penetration into the lipid monolayer was recorded as the rate of surface area increase on a two-compartment trough. (2) High spectrin penetration rates were observed with negatively charged phospholipids while zwitterionic or neutral lipids showed only poor spectrin affinity. This penetration rate was strongly affected by the subphase pH. At pH 5.5, maximal pentration rates wre obsreved for phosphatidylglycerol and phosphatidylserine but not for phosphatidylcholine. (3) In comparing the penetration rates for phospholipids with a natural fatty acid composition and the dimyristoyl species of phosphatidic acid, phosphatidylglycerol, phosphatidylserine and phosphatidylcholine, the lipid fatty acid composition proved to be an important parameter. The differences are collelated with the area per lipid molecule. (4) Other parameters affecting the area per lipid molecule such as surface pressure, pH and salt concentration also strongly influenced spectrin penetration rates for negatively charged phospholipids. Spectrin penetration into phosphatidylcholine monolayers is only slightly affected by variation of these conditions. (5) The effect of Ca2+ on spectrin-lipid interactions was studied for several phosphatidylglycerol and phosphatidylserine species. Both lipids condensed upon the addition of Ca2+, but only in the case of the phosphatidyleserine was this accompanied by extrusion of the spectrin from the interface, which is in agreement with earlier calorimetric experiments with bilayer systems of analogous composition (Mombers, C., Verkleij, A.J., de Gier, J. and van Deenen, L.L.M. (1979) Biochim. Biophys. Acta 551, 271-281). For this phenomenon a model is presented.

The interaction of spectrin-actin and synthetic phospholipids. II. The interaction with phosphatidylserine.

Sonicated vesicles of phosphatidylserine and phosphatidylserine/phosphatidylcholine mixtures were recombined with spectrin-actin from human erythrocyte ghosts. Morphological properties and physicochemical characteristics of the recombinates were studied with freeze etch electron microscopy, 31P NMR and differential scanning calorimetry. Sonicated dimyristoyl phosphatidylserine vesicles show a decrease in enthalpy change of the lipid phase transition upon addition of spectrin-actin. These vesicles collapse and fuse, into multilamellar structures in the presence of spectrin-actin, as demonstrated by freeze fracturing and NMR. Spectrin-actin cannot prevent the salt formation between phosphatidylserine and Ca2+, all phosphatidylserine is withdrawn from the lipid phase transition. In contrast a protection against the action of Mg2+ could be observed. Mixed bilayers of dimyristoyl phosphatidylserine/dimyristoyl phosphatidylcholine show phase separations at molar ratios above 1/1 (van Dijck, P.W.M., de Kruijff, B., Verkleij, A.J., van Deenen, L.L.M. and de Gier, J. (1978) Biochim. Biophys. Acta 512, 84--96). These phase spearations can be prevented by spectrin-actin. Ca2+-induced lateral phase separations in cocrystallizing phosphatidylserine/phosphatidylcholine mixtures, can be reduced by spectrin-actin. Formation of the Ca2+-phosphatidylserine salt, occurring in addition to lateral phase separation when mixtures contain more than 30 mol % phosphatidylserine, cannot be prevented by spectrin-actin.

The interaction of spectrin - actin and synthetic phospholipids.

Using differential scanning calorimetry and freeze fracture electron microscopy interactions were studied between lipids and a spectrin - actin complex isolated from human erythrocyte membranes. With dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol and mixtures of these two compounds, which for experimental reasons were chosen as the lipid counterpart, such an interaction could clearly be deduced from changes in the temperature and the enthalpy of the phase transition. Furthermore it was demonstrated that the interaction with this membrane protein protects the bilayer against the action of Ca2+ and Mg2+ and prevents fusion of lipid vesicles which easily occurs in some of the systems when divalent ions were added to the pure lipid vesicles.

Fusion of phospholipid vesicles in association with the appearance of lipidic particles as visualized by freeze fracturing.

The addition of Ca2+ to small unilamellar vesicles of an equimolar mixture of egg phosphatidylcholine and cardiolipin induces fusion of these vesicles in association with the appearance of lipidic particles on the fusion sites.

The occurrence of lipidic particles in lipid bilayers as seen by 31P NMR and freeze-fracture electron-microscopy.

A new type of lipid organization is observed in mixtures of phosphatidyl-choline with cardiolipin (in the presence of Ca2+), monoglycosyldiglyceride and phosphatidylethanolamine (in the presence of cholesterol). This phase is characterised by an isotropic 31P NMR signal and is visualised by freeze-fracturing as particles and pits on the fracture faces of the lipid bilayer. As the most favourable model for this phase we propose the inverted micelle sandwiched in between the two monolayers of the lipid bilayer.

Lipidic particles.

A new type of lipid organization is observed in mixtures of phosphatidylcholine with cardiolipin in the presence of Ca++, monoglucosyldiglyceride and phosphatidylethanolamine (in the presence of cholesterol). This phase is characterized by an isotropic 31P NMR signal and is visualized by freeze fracturing as particles and pits on the fracture faces of the lipid bilayer. As the most favourable model for this phase we propose the inverted micelle sandwiched in between the two monolayers of the lipid bilayer. It will be shown that such particles and pits appear on the fusion site during fusion of unilamellar vesicles of an equimolar mixture of phosphatidylcholine and cardiolipin in the presence of Ca++.