The polymorphic phase behaviour of phosphatidylethanolamines of natural and synthetic origin. A 31P NMR study.

1. The polymorphic phase behaviour of aqueous dispersions of phosphatidylethanolamines isolated from human erythrocytes, hen egg yolk and Escherichia coli have been investigated employing 31P NMR techniques. All species exhibit well defined, reversible bilayer to hexagonal (H11) phase transitions as the temperature is increased. The temperatures at which these transition take place (10, 25--30 and 55--60 degrees C for erythrocyte, egg yolk and E. coli phosphatidylethanolamine, respectively) are sensitive to the fatty acid composition, occurring at a temperature up to 10 degrees C above the high temperature end of the hydrocarbon phase transition as detected by differential scanning calorimetry. In some cases the bilayer to hexagonal (H11) transitions may also be detected employing calorimetric techniques. 2. The addition of equimolar concentrations of cholesterol to these naturally occurring phosphatidylethanolamines does not dramatically affect the bilayer-hexagonal (H11) transition temperature, producing changes of up to 10 degrees C. 3. 18 : 1t/18 : 1t phosphatidylethanolamine undergoes the bilayer to hexagonal (H11) phase transition as the temperature is increased through the interval 50--55 degrees C. Alternatively, hydrated 12 : 0/12 : 0 phosphatidylethanolamine remains in the bilayer phase at temperatures up to 90 degrees C (50 degrees C above the hydrocarbon phase transition temperature). 4. The presence of 100 mM NaCl or 10 mM CaCl2 in aqueous dispersions of egg yolk phosphatidylethanolamine does not alter the temperature-dependent polymorphic phase behaviour significantly. However, at 40 degrees C, increasing the p2H above 8.0 results in progressive inhibition of the hexagonal (H11) phase and the appearance of a phase possibly of cubic structure at p2H 9.0. At p2H 10.0 the bilayer phase is preferred. 5. It is suggested that in biomembranes containing phosphatidylethanolamine as a majority species (such as that of E. coli) the fatty acid composition may primarily reflect the need to maintain bilayer structure. Alternatively, it is pointed out that in mammalian membranes such as that of the erythrocyte, phosphatidylethanolamine tends to destabilize bilayer structure. The resulting possibility that transitory non-bilayer lipid configurations may occur may be directly related to many important properties of biological membranes.     

The polymorphic phase behaviour and miscibility properties of synthetic phosphatidylethanolamines

(1) The polymorphic phase behaviour of aqueous dispersions of various synthetic phosphatidylethanolamines, both singly and in mixtures, has been investigated by ³¹P-NMR. (2) $\text{14:0}\text{14:0}$ PE remains in the lamellar phase up to 90°C. $\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}$ PE exhibits a lamellar to hexagonal (H_II) transition between 60°C and 63°C. For $\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}$ PE, the lamellar to hexagonal (H_II) transition occurs between 7 and 12°C, whereas for $\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}$ PE, the hexagonal (H_II) phase is the preferred structure above ?15°C. (3) Mixtures of $\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}$ PE and $\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}$ PE exhibit near-ideal miscibility behaviour. For mixtures of $\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}$ PE and $\text{14:0}\text{14:0}$ PE there is evidence of fluid-solid immiscibility at temperatures below the gel-liquid crystalline transition temperature of the $\text{14:0}\text{14:0}$ PE component. Mixtures of $\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}$ PE and $\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}$ PE exhibit complex phase behaviour involving limited fluid-solid immiscibility at low temperatures and formation of a phase allowing isotropic motional averaging at higher temperatures. (4) ³¹P-NMR provides a graphic method for investigating the miscibility properties of mixed PE systems.     

31P NMR of DNA in eukaryotic chromosomal complexes

DNA complexed with histones in soluble chromatin and with protamines in the heads of sperm has been studied by ³¹P NMR spectroscopy. Because of the large size of these nucleoprotein structures, methods of high resolution solid state NMR were employed. Proton decoupled ³¹P NMR spectra of these complexes in solution yield anisotropic chemical shift powder patterns, which indicate that the DNA is substantially immobilized by interactions with the proteins. Rapid rotation of these samples at the magic angle gives single line spectra with an isotropic chemical shift indistinguishable from DNA in the absence of proteins or that in mononucleosome core particles; this argues that packaging of the DNA by the proteins does not introduce major distortions in a predominant fraction of the phosphodiester linkages present.     

Effects of divalent cations and pH on phosphatidylserine model membranes: A 31P NMR study

The influence of divalent cations, and pH on the behaviour of phosphatidylserine, derived from egg phosphatidylcholine, has been examined employing ³¹P-NMR techniques. The addition of Ca²⁺ results in the observation of a “rigid lattice” ³¹P-NMR spectra and more than an order of magnitude increase in the spin-lattice relaxation time T₁. This corresponds to a strong and specific headgroup immobilization by Ca²⁺, similar to that observed for anhydrous phosphatidylserine. At pH 7.4 the hydrated sodium salt of (egg) phosphatidylserine adopts the bilayer phase, whereas when the pH is decreased through 3.5 a bilayer to hexagonal (H_II) polymorphic phase transition is observed at 50°C, which is unaffected by equimolar cholesterol. The same transition is shown to occur at 37°C for phosphatidylserine isolated from human erythrocytes.     

31P nuclear magnetic resonance study of enzymatically phosphorylated glycophorin A

Glycophorin A was phosphorylated using protein kinases and the new protein was investigated using³¹P NMR spectroscopy. Most of these 30 moles of phosphate were found to be attached to Ser and Thr. Some of these phosphate residues appear to be affected by the carbohydrate residues present. The phosphorylated protein appears to be in a severe state of aggregation, with the degree of aggregationpH-dependent.     

The interaction of myosine S1 with phosphorothioates of ADP: An 18O exchange study by 31P NMR

The ¹⁸O exchange reaction which labeled P_i undergoes in the presence of complexes of myosin subfragment 1, MgCl₂, and the different phosphorothioates of ADP has been observed by ³¹P NMR. From these experiments it can be concluded that ADP and ADP (α-S) (A) on the one hand and ADP (β-S) and ADP (α-S) (B) on the other hand form similar complexes as far as the number of reversals of the nucleoside triphosphate formation step from the nucleoside diphosphate and P_i, is concerned. In addition, the same seems to hold for the rate constant k_?2, which describes the binding step of free P_i, to the subfragment 1 nucleoside diphosphate complex. These observations support former kinetic experiments which yielded the same similarities for the rate parameters describing association and dissociation of the subfragment 1 nucleotide complexes.     

Synthesis and polymorphic phase behaviour of polyunsaturated phosphatidylcholines and phosphatidylethanolamines

A series of phosphatidylcholines and phosphatidylethanolamines was synthesized containing two acyl chains of the following polyunsaturated fatty acids: linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4) and docosahexaenoic acid (22:6). In addition two phospholipids with mixed acid composition were synthesized: 16:0/18:1_c phosphatidylcholine and 16:0/18:1_c phosphatidylethanolamine. The structural properties of these lipids in aqueous dispersions in the absence and in the presence of equimolar cholesterol were studied using ³¹P-NMR, freeze fracturing and differential scanning calorimetry (DSC).The phosphatidylcholines adopt a bilayer configuration above 0°C. Incorporation of 50 mol% of cholesterol in polyunsaturated species induces a transition at elevated temperatures into structures with ³¹P-NMR characteristics typical of non-bilayer organizations. When the acyl chains contain three or more double bonds, this non-bilayer organization is most likely the hexagonal H_II phase, 16:0/15:1_c phosphatidylethanolamine shows a bilayer to hexagonal transition temperature of 75°C. The polyunsaturated phosphatidylethanolamines exhibit a bilayer to hexagonal transition temperature below 0°C which decreases with increasing unsaturation and which is lowered by approximately 10°C upon incorporation of 50 mol% of cholesterol. Finally, it was found that small amounts of polyunsaturated fatty acyl chains in a phosphatidylethanolamine disproportionally lower its bilayer to hexagonal transition temperature.     

A high-resolution NMR study (1H, 13C, 31P) of the interaction of paramagnetic ions with phospholipids in aqueous dispersions

¹H-, ¹³C-and ³¹P-NMR spectra of egg-yolk phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidic acid (PA) and cosonicated mixtures of these phospholipids were obtained from ultrasonicatcd dispersions containing Pr³⁺, Eu³⁺, Gd³⁺ and Mn²⁺ ions.The differences in chemical shift values. °_n, between the “inner” and “outer” resonance signals for the different nuclei of the polar head group of egg-yolk phosphatidyl choline provide information about the average distances of the paramagnetic ion within the polar groups of the phospholipid molecules. In the Pr(²H₂O)³⁺_n/egg-yolk phosphatidylcholine system the ions are nearest to the phosphate and -CH₂CH₂ group, respectively but relatively far from the N(CH₃)₃ group of the polar head group of the lipid.The integral analysis of the¹ H-NMR spectra obtained from dispersions containing Pr³⁺ and Mn²⁺ ions enables us to calculate the number of the polar groups in both sides of the egg-yolk phosphatidylcholine bilayer, the size of the lipid vesicle and to give some features of the arrangement of the phospholipid molecules in cosonicated egg-yolk phosphatidylcliotine/ phosphatidytserine vesicles. At p²H 8.3 in PC/PS mixtures an extreme asymmetry is observed with PS preferentially in the outer side of the membrane. This side contains approximately three times more PS than PC molecules.Some comments are made concerning the quantitative integral analysis of proton-noise decoupled ³¹ P-NMR spectra as obtained from similar phospholipid mixtures by Michaelson et al. and Berden et at.     

Phosphocreatine in Ehrlich ascites tumor cells detected by noninvasive 31P NMR spectroscopy

³¹P NMR spectra of intact Ehrlich ascites tumor cells of high phosphorylation potential reveal a well-defined resonance peak assignable to phosphocreatine, corresponding to 2.3 μmoles/ml cell H₂O in adenosine-treated cells containing 5.2 μmoles ATP/ml. The NMR spectrum of Ehrlich cells incubated with iodoacetate and glucose indicates depletion of phosphocreatine and ATP to undetectable levels and substantial accumulation of fructose-1,6-bisphosphate. From the difference between the chemical shifts of internal P_i and phosphocreatine resonances, the intracellular pH was estimated to be 7.1 ± 0.1 in protein-synthesizing cells suspended in a medium of pH 7.4 at 10°C. Ehrlich cells are unable to transfer the labeled amidine group from L-(guanidino-¹⁴C)-arginine to the large intracellular glycine pool to form labeled guanidinoacetate, the demethylated precursor of creatine. These results imply that the synthesis of phosphocreatine in ATP-rich Ehrlich cells is limited primarily by the extracellular free creatine supply, the extent of which depends upon the degree of cachectic perturbation of energy and nitrogen metabolism of the tumor-bearing host.     

13C and31P NMR spectroscopic studies on glucose metabolism inTribolium confusum

Nuclear magnetic resonance (NMR) technology was applied to study the glucose metabolism inTribolium confusum (Coleoptera).¹³C signals of D-(1-¹³C)glucose eaten by beetles were clearly detected in such metabolites of the glucose metabolism as glycogen, trehalose, triacylglycerol, alanine and proline by¹³C-NMR. After glucose feeding the³¹P-NMR spectra ofT. confusum showed the signal intensity increases in arginine-phosphate, sugar-phosphate and uridine diphosphoglucose. The results demonstrated the potential of NMR analysis for the study of glucose metabolism inT. confusum.     

The interaction between water and the polar head in inverted phosphatidylcholine micelles A 2H and 31P relaxation study

²H and ³¹P spin-lattice relaxation times (T₁) were studied for invented egg phosphatidylcholine micelles in CCl₄ as functions of ²H₂O concentration. When the ²H₂O/phosphatidylcholine mole ratio changed from 1.0 to 18.0, T₁ of ³¹P increased by about 2.6 fold, whereas T₁ of ²H increased by about 50 fold. A quantitative analysis of the deuterium T₁ data showed that there is only one water molecule tightly bound to the polar head, and it is in rapid exchange with the rest of the water molecules. The activation energy for the deuterium T₁ was 7.1 ± 0.8 kcal/mol (30 ± 3 kJ/mol), and was independent of the ²H₂O concentration.     

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 phosphatidylcholine with cardiolipin (in the presence of Ca²⁺), monoglucosyldiglyceride 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.     

31P NMR study of head group behavior in sonicated phosphatidylcholine liposomes in the gel and in the liquid state

We measured the ³¹P[¹H] Nuclear Overhauser Effect (NOE) as a function of temperature and of ¹H irradiation frequency, the linewidth $\text{Δν}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ as a function of temperature and the relaxation time T₁ above and below the thermal transition temperature, of the ³¹P-NMR signal in sonicated liposomes of 1,2-dimiristoyl-3-sn-phosphatidylcholine (DMPC), 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) and 1,2-dimiristoyl-3-sn-phosphatidylcholine (DSPC). The same measurements were repeated in the presence of high molecular weight dextrans. They strongly reduce the NOE and produce longer relaxation times T₁. According to the current models, we were able to evaluate, in the different situations, the correlation time of the internal motion τ_G and the distance r between interacting groups in the region of the polar head groups. While the first parameter changes abruptly through the phase transition and under the effect of dextrans, the latter does not appear modified in any case. These results are discussed in terms of a conformational change of the phosphocholine head groups.