Unsaturated Phosphatidylethanolamine Research Articles

Two types of lipidic particles

The molecular arrangements in lipidic particles (LIP) seen on freeze fracture faces of liposomes have been a subject of much discussion. Miller and Hui proposed that these particles were intermembrane attachment sites, while Cullis and de Kruijff favored a model consisting of inverted micelles between bilayer leaflets.In our study of polymorphic phase transitions in mixtures of unsaturated phosphatidylethanolamine (PE) and phosphatidylcholine (PC), We have found evidence of two distinct types of LIP. In most PE/PC mixtures such as soybean PE and egg PC, the LIP have a dimple-like appearance and are definitely conical in cross fracture (fig. 1). Both concave and convex forms are commonly found on the same fracture face (fig. 2). In mixtures of dilinoleoyl-PE and palmitoyl-oleoyl-PC, the LIP are larger and have a round, bead-like appearance. These LIP often align in rows between entrapped water space (fig. 3), or stack in a cubic arrangement (fig. 4). In less ordered areas, conical and spherical LIP can be viewed as complementary lattice.

The nature of lipidic particles and their roles polymorphic transitions

The structural transition between bilayer (L α), inverted hexagonal (H II and inverted cubic (C II) phases in mixtures of unsaturated phosphatidylethanolamines (PE) and phosphatidylcholines (PC) were investigated. Freeze fracture electron micrographs of intermediate stages of phase transitions showed that C II was a stable intermediate form between the L α-H II transition. The electron microscopic observation was supported by X-ray diffraction and 31P-NMR results. Detailed morphology revealed that during the L α-C II transition, interlamellae attachment points (conical lipidic particles) connect adjacent bilayers to form arrays of entrapped water pockets (inverted micelles). These water-containg spherical units were packed in a cubic lattice. In the C II to H II transition, these spherical units were linearly connected to form tubes. During the L α-H II transition, a ripple pattern was observed across the otherwise smooth lamellar. The troughs of the ripples were transformed into linear connections between adjacent bilayers, thereby converting multilayer structures into parallel tubes. No lipidic particles were involved in this type of transition. We show that there are different mechanisms involved in the L α, H II, C II polymorphic transitions, and that different types of ‘lipidic particles’ representing different molecular organizations may be involved in each case. Models of these transitions are proposed.

Ca 2+ and pH induced fusion of small unilamellar vesicles consisting of phosphatidylethanolamine and negatively charged phospholipids: A freeze fracture study

Fusion processes in small (sonicated) unilamellar vesicle (SUV) systems composed of 80 mol % unsaturated phosphatidylethanolamine and 20 mol % of phosphatidylserine, phosphatidylinositol, phosphatidic acid or cardiolipin have been examined by freeze-fracture techniques. All these mixtures have the property that Ca 2+ and, in some cases low pH, can trigger bilayer to hexagonal (H II) phase transitions in large multilamellar dispersions of these lipid mixtures. It is shown that when the SUV systems are subjected to similar protocols, the vesicles first fuse to form larger systems prior to H II phase formation and this fusion is accompanied by the appearance of lipidic particle structures, often localized to the fusion interface. We conclude that factors promoting H II phase structure initially result in fusion of unilamellar systems and that this fusion proceeds via intermediary formation of non-lamellar, possibly inverted micellar, structure.

Stabilization of bilayer structure for unsaturated phosphatidylethanolamines by detergents

The structural preferences of mixed lipid systems containing egg yolk or 18:1 c 18:1 c phosphatidylethanolamine and representative detergents (Triton X-100, deoxycholate, octylglucoside and lyso-phosphatidylcholine) have been examined. It is shown that all these detergents exhibit an ability to stabilize a bilayer organization for the phosphatidylethanolamine at detergent to phosphatidylethanolamine molar ratios of 0.05 to 0.5, depending on the detergent and/or phosphatidylethanolamine species. These results are interpreted in terms of molecular shape, where the ‘inverted cone’ shape detergents combine in a complementary fashion with ‘cone shaped’ phosphatidylethanolamine to result in net bilayer structure.

Rapid transbilayer movement of phosphatidylcholine in unsaturated phosphatidylethanolamine containing model membranes

Aqueous dispersions of egg phosphatidylethanolamine/18 : 1 c, 18 : 1 c-phosphatidylcholine/cholesterol/18 : 1 c, 18 : 1 c-phosphatidic acid (50 : 16 : 30 : 4) undergo a temperature-dependent transition from extended bilayers to structures characterized by isotropic 31P-NMR signals and visualized by freeze-fracturing as lipidic particles associated with the bilayer. This transition is accompanied by a 3-fold increase in the phosphatidylcholine pool which can be exchanged by phospholipid exchange protein demonstrating a direct relation between the occurrence of non-bilayer lipid structures and an increased transbilayer movement of phosphatidylcholine.

Fusion competence of phosphatidylserine-containing liposomes quantitatively measured by a fluorescence resonance energy transfer assay

A sensitive, quantitative assay has been developed which measures the extent of liposome fusion by monitoring fluorescence resonance energy transfer between two lipid analogs originally in separate membranes. This transfer of photon energy from donor to acceptor molecules occurs only if both probes are in the same membrane. Energy transfer is measured as quenching of the donor probe's fluorescence emission. The extent of fusion was estimated by comparing the quenching due to the fusion protocol with the maximum quenching from “mock-fused” vesicles. This assay was used to investigate the effects of calcium ion concentration, calcium ion permeability, and lipid composition on fusion competence. The calcium concentration threshold and extent of fusion was a function of lipid composition. At a given molar percentage of phosphatidylserine, increasing the phosphatidylcholine content raised the threshold. The extent of fusion decreased when the molar percentage of phosphatidylserine was decreased. The inclusion of either cholesterol or phosphatidylethanolamine facilitated fusion competence, but the latter was more effective. Increasing the calcium ion permeability by adding the ionophore X-537a moderately enhanced the extent of fusion in most cases, although it never appreciably affected the threshold. X-537a did not enhance fusion in the presence of unsaturated phosphatidylethanolamine. Liposomes containing unsaturated phosphatidylethanolamine had an optimum calcium ion concentration for fusion in the mid-range of the divalent cation concentrations. We conclude that it is possible for large, unilamellar vesicles with near physiological molar percentages of phosphatidylserine and phosphatidylethanolamine to undergo divalent cation-induced fusion at calcium ion concentrations in the millimolar range. This finding provides a useful model system for investigating mechanisms of such phenomena as exocytosis and cell-cell fusion.

Polymorphism of phosphatidylglycerol-phosphatidylethanolamine model membrane systems: A 31P NMR study

Summary The structural preferences of saturated and unsaturated species of phosphatidylglycerol in isolation and in mixed systems with unsaturated (H II phase) phosphatidylethanolamine have been investigated employing 31 P NMR techniques. It is shown that equimolar (with respect to change) Ca 2+ induces a “rigid lattice” (no motion) situation in the phosphate region of the polar headgroup for a saturated species, but not for unsaturated species. Further, phosphatidylglycerol can stabilize a bilayer organization in the presence of phosphatidylethanolamine which would otherwise assume the hexagonal (H II ) phase, and structural bilayer-H II transitions can then be triggered in these systems by the addition of Ca 2+ .

Barrier characteristics of membrane model systems containing unsaturated phosphatidylethanolamines

The barrier characteristics of membrane model systems containing unsaturated phosphatidylethanolamines have been investigated. (1) At increasing temperatures 18:I c/18:1 c-phosphatidylethanolamine liposomes lose their permeability barrier for K + as a consequence of the transition from a lamellar to a hexagonal orientation as detected by 31P-NMR and freeze-fracturing electron microscopy. (2) Introduction of 18:1 c/18:1 c-phosphatidylcholine in the 18:1 c/18:1 c-phosphatidylethanolamine lipid system stabilizes the bilayer structure and the permeability barrier for K + and glucose while cholesterol destabilizes. (3) Upon heating of the investigated 18:1 c/18:1 c-phosphatidylcholine-18:1 c/18:1 c-phosphatidylethanolamine-(cholesterol) mixtures, structures are formed which give rise to isotropic 31P-NMR signals and which on the basis of freeze-fracture pictures are interpreted as sponge-like structures. Lowering the temperature results in restoration of the barrier function of the lipid structures.

The role of the de novo synthetic pathway in forming molecular species of phospholipids in resting lymphocytes from human tonsils

Phosphatidylcholine of resting human tonsil lymphocytes contained dipalmitoyl (18 mol%), 1-oleoyl,2-palmitoyl (11 mol%) and dioleoyl (5 mol%) species. 1-Oleoyl and 1-linoleoyl species were also detected in other more highly unsaturated phosphatidylcholine species. The features of phospholipid synthesis in lymphocytes were investigated by incubating cells with radio-labeled precursors. The de novo synthesis of phospholipids occurring in lymphocytes was estimated to be physiologically important, in particular for supplying dipalmitoyl-glycerophosphocholine and highly unsaturated phosphatidylethanolamine. It was also suggested that diacylglycerol(s) not originating from glycerophosphate is (are) involved in the synthesis of tetraenoic phospholipids. From radioactive palmitic and oleic acids were actively synthesized dipalmitoyl, dioleoyl and 1-oleoyl,2-palmitoyl species of diacylglycerol. The mode of diacylglycerol synthesis was reflected upon phosphatidylcholine formation. The formed polyunsaturated phosphatidylethanolamine was also found to contain 1-oleoyl or 2-palmitoyl species. Radioactive linoleic and arachidonic acids were incorporated predominantly into the C-1 position of diacylglycerol, whereas the majority of the formed phospholipids was of 2-linoleoyl or 2-arachidonoyl species. Labeled stearic acid was exclusively esterified to the C-1 position of the glycerolipids. However, the labeling pattern of molecular species by stearate was considerably deviated from that observed with labeled palmitate. These results indicated that the de novo synthetic pathway operating in lymphocytes is primarily responsible for forming 1-unsaturated type of phospholids. The synthesis of 1-saturated,2-unsaturated species appeared to be due to remodeling of the once-formed phospholids.

Isolation and characterization of lipid N-methyltransferase from dog lung

A soluble protein with lipid N-methyltransferase activity has been isolated from microsomes of dog lung. This protein catalyzes the transfer of methyl groups from S-adenosylmethionine to phosphatidyl ethanolamine with the formation of phosphatidyl choline. The partially purified protein was stable at pH greater than 8.2 in the presence of cysteine. It contained 2–5% lipid and had optimal activity at pH 8.0–9.0. Reaction velocities were markedly increased by exclusion of O 2 from the reaction system. When saturating levels of S-adenosylmethionine were used, highest reaction velocities were obtained with disaturated phosphatidyl ethanolamine and lower rates with more unsaturated substrates. Reaction rate in the liver particulate-bound system are higher than lung but are the same with saturated or unsaturated phosphatidyl ethanolamine as substrate. In sucrose density-gradient centrifugation experiments the transferase protein was shown to be associated with lamellated structures peculiar to lung pneumocytes. These results suggest that the N-methyltransferase of lung has special properties and may be important in the synthesis of disaturated surface-active phosphatidyl choline.

Cofactor requirements of the L-malate dehydrogenase of Pseudomonas ovalis Chester.

1. The l-malate dehydrogenase of Pseudomonas ovalis Chester, which is independent of nicotinamide nucleotides and which is structurally and functionally bound to the cell-wall membrane, has been prepared in a soluble form and partially purified. 2. The purified dehydrogenase exhibits a triple cofactor requirement for FAD, quinone and phospholipid, and in the presence of these cofactors can utilize 2,6-dichlorophenol-indophenol as hydrogen acceptor. 3. The formation of reduced forms of FAD was not detected, but in the presence of both FAD and phospholipid the enzyme catalysed the reduction of quinone by l-malate at rates equivalent to those obtained with 2,6-dichlorophenol-indophenol as terminal acceptor. The l-malate dehydrogenase of Ps. ovalis Chester is therefore an l-malate-quinone oxidoreductase. 4. The quinone and the phospholipids present in the fragments of the cell-wall membrane from which the soluble dehydrogenase was prepared have been extracted and purified. The quinone was identified as coenzyme Q(9). At least eight phospholipids were detected, and the major component is an unsaturated phosphatidylethanolamine. 5. The nature of the phospholipid required to activate the enzyme depends on the nature of the quinone used in the assay system. When 2-methyl-1,4-naphthaquinone is used, a wide variety of phospholipids, including all those isolated from the organism, will activate the enzyme, but when coenzyme Q(9) is used the phospholipid specificity of the enzyme is much more restricted, and the most effective activator is the unsaturated phosphatidylethanolamine isolated from the organism. 6. Evidence is presented to support the view that the restricted phospholipid specificity exhibited by the enzyme in the presence of coenzyme Q(9), as opposed to the broad specificity exhibited when 2-methyl-1,4-naphthaquinone is used, is due to the fact that coenzyme Q(9) has a large substituent on position 3.