The interaction of phenol with lipid bilayers

The depression of the phase-transition temperature of dimyristoyl- and dipalmitoylphosphatidylcholine vesicles induced by phenol has been investigated by fluorescence polarization. This effect is strongly pH and concentration dependent. Only the uncharged phenol molecule influences the fluidity of the bilayer so that the interaction of phenol with the bilayer can be situated in the hydrophobic acyl chain region. Direct measurements of the partitioning of phenol in the phospholipid vesicles confirm these results and show a limited and concentration-dependent solubility. Phase-transition temperature depressions, obtained from thermodynamic analysis of partition coefficient measurement, are in good agreement with the experimental values.     

Correlation between temperature range of growth and structural transitions in membranes and lipids of Escherichia coli K12

Purified cytoplasmic and outer membranes isolated from cells of wild-type Escherichia coli grown at different temperatures were labelled with 1,6-diphenyl-1,3,5-hexatriene and anlyzed using fluorescence polarization techniques. Lipids extracted from the membranes were similarly analyzed using fluorescence polarization. The thermotropic structural transition in outer membranes changed as a function of growth temperature. The structural transition in cytoplasmic membranes and lipids extracted from either cytoplasmic or outer membranes did not change with growth temperature. These data suggest that adaptive changes which occur in the outer membrane determine the temperature range of growth of E. coli. These changes apparently require alterations in outer membrane components other than phospholipids.     

Effects of gaseous anaesthetics and inert gases on the phase transition in smectic mesophases of dipalmitoyl phosphatidylcholine

The phase transition in smectic mesophases of dipalmitoyl phosphatidylcholine was studied under high pressures of helium (340 atm), nitrogen (340 atm), nitrous oxide (43 atm), cyclopropane (4.4 atm) and $\text{n-}\text{propane}$ (8.2 atm), using a turbidimetric technique. Helium and nitrogen increased the transition temperature by 0.021 and 0.006°C/atm, respectively, compared with 0.024°C/atm for hydrostatic pressure. Nitrous oxide reduced the transition by 0.58°C/atm. The hydrocarbon gases spread the transition width and lowered the transition temperature with increasing effect at higher doses. Comparisons with other membrane probes are made and the concentration of gases in the bilayer which lower the transition temperature by 1°C are estimated, in mol%: He, 10.2; N₂, 13.2; N₂O, 9.04; $\text{n-}\text{C}{}_3\text{H}{}_8$, 6.3 and cyclopropane, 12.8.     

Phosphatidylcholesterol bilayers. A model for phospholipid-cholesterol interaction

Aqueous dispersions of monovalent and divalent cation salts of O-(1,2-dipalmitoyl-sn-glycero-3-phosphoryl) cholesterol form multilamellar vesicles as shown by freeze-fracture electron microscopy, by electron micrographs of the negatively stained liposomes, and by swelling curves of liposomes in hypoosmotic medium. Differential scanning calorimetry reveals that aqueous dispersions of divalent metal salts of O-(1,2-dipalmitoyl-sn-glycero-3-phosphoryl)-cholesterol undergo a characteristic thermotropic phase transition with a relatively large cooperative unit (n > 250 for the calcium salt). In contrast, monovalent cation salts of O-(1,2-dipalmitoyl-sn-glycerol-3-phosphoryl)cholesterol do not show a thermotropic phase transition under comparable conditions. The molecular area of O-(1,2-dipalmitoyl-sn-glycero-3-phosphoryl)cholesterol in a monolayer is the same in the presence and absence of Ca²⁺, and is virtually equal to the area of an equimolar mixture of dipalmitoyl phosphatidic acid and cholesterol. To account for the novel state induced by Ca²⁺ on aqueous dispersions of O-(1,2-dipalmitoyl-sn-glycero-3-phosphoryl)cholesterol (i.e., bilayer organization and highly cooperative phase transition), a linear array model is proposed in which Ca²⁺ bridges adjacent arrays of O-(1,2-dipalmitoyl-sn-glycero-3-phosphoryl)cholesterol molecules, thus freezing the acyl chains in their normal state. One of the main corollaries of the model is that the cooperative unit for a thermotropic phase transition is essentially one-dimensional, rather than a two-dimensional matrix. O-(1,2-Dipalmitoyl-sn-glycero-3-phosphoryl)cholesterol is proposed as an orientationally and conformationally restricted analog of glycerophospholipid plus cholesterol in bilayers.     

Pressure-induced changes in the molecular organization of a lipid-peptide complex. Polymyxin binding to phosphatidic acid membranes

The effect of 100 atm pressure on the organization of the lipid-peptide complex formed between polymyxin and dipalmitoyl phosphatidic acid has been investigated. Phase transition curves were obtained by electron paramagnetic resonance by measuring the partition coefficient of the spin label, 2, 2, 5, $\text{5-}\text{tetramethylpiperidine}\text{-N-}\text{oxyl}$. The three-step phase transition curve previously obtained with fluorescence polarization measurements was confirmed, demonstrating three distinct phosphatidic acid domains in the bilayer. Pressure increases binding of polymyxin to phosphatidic acid bilayers and alters the proportions of the two domains that differ in the mode of binding between phosphatidic acid and polymyxin. The binding curves of polymyxin to phosphatidic acid bilayers were determined and it was shown that application of pressure reduces the cooperativity of the binding curve.     

Dynamic stiffness and crossbridge action in muscle

Small sinusoidal vibrations at 300 Hz were applied to frog sartorius muscle to measure the dynamic stiffness (Young's modulus) throughout the course of tetanus. For a peak-to-peak amplitude of 0.4% the dynamic Young's modulus increased from 1.5×10⁵ Nm^–2 in the resting state to 2×10⁷ Nm^–2 in tetanus. After correction for the external connective tissue, the dynamic Young's modulus of the muscle was almost directly proportional to the tension throughout the development of tetanus. The ratio of dynamic Young's modulus to tensile stress thus remained constant (with a value at 300 Hz of approximately 100), consistently with Huxley and Simmons' identification of the crossbridges as the source of both tension and stiffness.For a single crossbridge the ratio of stiffness to tension was 8.2×10⁷ m^–1 at 300 Hz; it is deduced from literature data that the limiting value at high frequencies is about 1.6×10⁸ m^–1. This ratio is interpreted on Harrington's (1971) model to show that crossbridge action can be explained by a helix-coil transition of about 80 out of the 260 residues in each S-2 myosin strand. It is also shown that a helix-coil model can account for the observed rapid relaxation of muscle without invoking any complex behaviour of the crossbridge head.     

Differential scanning calorimetry of milk fat globule membranes

Differential scanning calorimetry was employed as an aid in examining the structure of the bovine milk fat globule membrane. At least six major endotherms are observed between 10 and 90°C, corresponding to order-disorder transitions of discrete structural domains of the membrane. These endothermic transitions occur at 16, 28, 43, 58, 68, and 75°C. The transitions occurring between 10 and 50°C were reversible, suggesting the involvement of lipid. However, the high temperature transitions were irreversible. The calorimetric C transition, centered at 43°C, was shown to involve neutral lipid, since the endotherm was reversible, insensitive to proteolysis, and similar to the endotherm of the isolated neutral lipid fraction of the milk fat globule membrane. The glycolipid and phospholipid fractions of the milk fat globule membrane yielded endotherms outside of the temperature range of the C transition. Another endotherm, the D transition (58°C), was found to involve the denaturation of the major membrane coat protein, butyrophilin (band 12). Evidence for this assignment included the following observations: (i) the nearly selective proteolysis of butyrophilin resulted in the complete removal of the D transition, (ii) the butyrophilin-enriched, Triton X-100-insoluble pellet of milk fat globule membrane yielded a relatively normal D transition, and (iii) the irreversible, disulfide-stabilized aggregation of butyrophilin occurred in the membrane solely at the temperature of the D transition. Furthermore, no other prominent milk fat globule membrane polypeptide formed these non-native disulfide crossbridges during the D transition. The sources of the other major endotherms of the milk fat globule membrane have not yet been assigned.     

Protein-lipid interactions and differential scanning calorimetric studies of bacteriorhodopsin reconstituted lipid-water systems

Bacteriorhodopsin has been reconstituted at various molar concentrations into liposomes of dimyristoyl- and also of dipalmitoylphosphatidylcholine. Differential scanning calorimetry indicates that as the protein concentration within the lipid bilayer increases, the cooperativity of the lipid phase transition is reduced, i.e. the transition is broadened, while the midpoint transition temperature remains virtually unchanged. Freeze-fracture electron microscopy of our preparation shows, in agreement with previous data from other laboratories, that extensive protein aggregation occurs when the liposome is cooled below the T_c transition temperature of the lipid. Laser flash photolysis measurements of protein rotation of the bacteriorhodopsin show, especially in the case of protein-rich recombinants, that protein aggregates exist even above T_c. The perturbation caused by the presence of bacteriorhodopsin in the lipid bilayer is similar to that produced by other intrinsic proteins. The difficulty of correlating the observed calorimetric enthalpy data with a simple concept of a ‘boundary lipid layer’ based upon consideration of a single isolated protein is discussed in view of the occurrence of protein aggregates both above and below T_c. It is concluded that the reduction of enthalpy is related to the number of lipids which solvate the protein aggregates within the protein-lipid patches and are thereby removed from the cooperative melting and enthalpy of the remaining regions of pure lipid.