Iodide penetration into lipid bilayers as a probe of membrane lipid organization.

The quenching efficiency of iodide as a penetrating fluorescence quencher for a membrane-associated fluorophore was utilized to measure the molecular packing of lipid bilayers. The KI quenching efficiency of tryptophan-fluorescence from melittin incorporated in DMPC bilayer vesicles peaks at the phase transition temperature (24 degrees C) of DMPC, whereas acrylamide quenching efficiency does not depend on temperature. The ability of iodide to penetrate the hydrocarbon region of the bilayer was examined by measuring the fluorescence quenching of the pyrene-phosphatidylcholine incorporated into DMPC vesicles (pyrene was attached to the 10th carbon of the sn-2 chain). The quenching efficiency of pyrene by iodide again shows a maximum at the lipid phase transition. We conclude that iodide penetrates the membrane hydrocarbon region at phase transition through an increased number of bilayer defects. The magnitude of change in quenching efficiency of iodide during lipid phase transition provides a sensitive technique to probe the lipid organization in membranes.     

Raman spectroscopy of the thermal properties of reassembled high-density lipoprotein: apolipoprotein A-I complexes of dimyristoylphosphatidylcholine

Isolated complexes of apolipoprotein A-I (apoA-I), the major apoprotein of human plasma high-density lipoproteins, and dimyristoylphosphatidylcholine (DMPC) have been prepared and studied by differential scanning calorimetry (DSC) and Raman spectroscopy. DSC studies establish that complexes having lipid to protein ratios of 200, 100, and 50 to 1 each exhibit a broad reversible thermal transition at Tc = 27 degrees C. The enthalpy of lipid melting for each of the three complexes is about 3 kcal/mol of DMPC. Raman spectroscopy indicates that the physical state of lipid molecules in the complexes is different from that in DMPC multilamellar liposomes. Analysis of the C-H stretching region (2800-3000 cm-1) of the complexes and of the pure components in water suggests that below 24 degrees C (Tc for DMPC) there is considerably less lateral order among lipid acyl chains in the complexes than in DMPC liposomes. Above 24 degrees C, these types of interactions appear to contribute equally or slightly less to the complex structure than in pure DMPC. The temperature dependence of peaks in the C-C stretching region (1000-1180 cm-1) reveals a continuous increase in the number of lipid acyl chain C-C gauche isomers over a broad range with increasing temperature. Compared to liposomes, DMPC in the complexes has more acyl chain trans isomers at temperatures above 24 degrees C; at temperatures above ca. 30 degrees C, trans isomer content is about the same for complexes and liposomes. A large change was observed in a protein vibrational band at 1340 cm-1 for pure vs. complexed apoA-I, indicating that protein hydrocarbon side chains are immobilized by lipid binding. The Raman data indicate that the reduction in melting enthalpy for complexes DMPC (approximately 3 kcal/mol) compared to that for free DMPC (approximately 6 kcal/mol) is due to reduced van der Waals interactions in the low-temperature lipid phase.     

The action of membranotropic compounds with various structures on the parameters of the phase transition of dimyristoylphosphatidylcholine

The effect of substances of different nature on the thermodynamic characteristics of dimyristoylphosphatidylcholine (DMPC) phase transition by the differential scanning microcalorimetry has been studied. The substances disposed in hydrophobic part of membrane--alpha-tocopherol, ubiquinone Q10, ionol and vitamin K3 cause the decrease of enthalpy and cooperativity of phase transition. The substances which have the side hydrocarbon chain (tocopherol and ubiquinone Q10) compared with ones without it (ionol and vitamin K3) and reduced quinones (Q10 and vitamin K3) compared with the oxidized ones have stronger influence on the enthalpy and cooperativity of transition. The inclusion of the local anesthetic dicaine disposed mainly in the zone of polar heads of phospholipids into DMPC membranes decreases the temperature of phase transition considerably and practically does not change the cooperativity. A possibility to use the method of differential scanning microcalorimetry to estimate the localization of membrane tropic substances within lipid bilayer is under discussion.     

A theoretical model of the temperature- and pressure-induced phase transition of phospholipid bilayers

A statistical thermodynamic model of phospholipid bilayers is developed. In the model, a new concept of a closely packed system is applied, i.e., a system of hard cylinders of equal radii, the radius being a function of the average number of gauche rotations in a hydrocarbon chain. Using this concept of a closely packed system, reasonable values are obtained for the change in specific volume at the order-disorder transition of lecithin bilayers. In addition to interactions between the lipid matrix and water molecules, between the head groups themselves and between hydrocarbon chains, as well as the intramolecular energy associated with chain conformation, the Hamiltonian of the membrane also includes the energy of the pressure field. Thus, the phase transition of phospholipid membranes induced not only by temperature hut also by hydrostatic pressure is described by this model simultaneously. In accordance with the experimental results, a linear relationship is obtained between the phase transition temperature and phase transition pressure. The other calculated phase transition properties of lecithin homologues. e.g., changes in enthalpy, surface area. thickness and gauche number per chain are in agreement with the available experimental data. The ratio of kink to interstitial conduction of bilayers is also estimated.     

The effects of bilirubin on the thermal properties of phosphatidylcholine bilayers.

The thermotropic properties of multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC), as a function of the concentration of bilirubin in the range of 0.1 to 1 mol%, were measured. The exact effects of bilirubin depended on the chain length of the polymethylene chains. But the general effects of bilirubin were the same in all systems. At the lowest concentrations tested (0.1 mol bilirubin/100 mol phospholipid (0.1 mol%)), bilirubin broadened and shifted to higher temperatures the main phase transitions of all bilayers. For DPPC and DSPC, but not DMPC, this concentration of bilirubin was associated with a new transition at 25 degrees C (DPPC) or 34 degrees C (DSPC). Bilirubin at 0.2 mol% was required for the detection of a similar transition (at 13.7 degrees C) in DMPC. Higher concentrations of bilirubin (> 0.2 mol%) suppressed completely the main phase transitions in all bilayers but increased the enthalpy of the new transition. Maximal values of delta H for these transitions were reached at 0.5, 0.25, and 0.2 mol% bilirubin in DMPC, DPPC, and DSPC, respectively. Values of delta H and delta S for these transitions were far larger than for the corresponding gel-to-liquid crystal transitions in pure lipid bilayers but were equal to those expected for a transition between crystalline and liquid crystalline phases.     

Differential scanning calorimetric study of the effect of the antimicrobial peptide gramicidin S on the thermotropic phase behavior of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol lipid bilayer membranes

We have studied the effects of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of large multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylethanolamine (DMPE) and dimyristoyl phosphatidylglycerol (DMPG) by high-sensitivity differential scanning calorimetry. We find that the effect of GS on the lamellar gel to liquid-crystalline phase transition of these phospholipids varies markedly with the structure and charge of their polar headgroups. Specifically, the presence of even large quantities of GS has essentially no effect on the main phase transition of zwitterionic DMPE vesicles, even after repeating cycling through the phase transition, unless these vesicles are exposed to high temperatures, after which a small reduction in the temperature, enthalpy and cooperativity of the gel to liquid-crystalline phase transitions is observed. Similarly, even large amounts of GS produce similar modest decreases in the temperature, enthalpy and cooperativity of the main phase transition of DMPC vesicles, although the pretransition is abolished at low peptide concentrations. However, exposure to high temperatures is not required for these effects of GS on DMPC bilayers to be manifested. In contrast, GS has a much greater effect on the thermotropic phase behavior of anionic DMPG vesicles, substantially reducing the temperature, enthalpy and cooperativity of the main phase transition at higher peptide concentrations, and abolishing the pretransition at lower peptide concentrations as compared to DMPC. Moreover, the relatively larger effects of GS on the thermotropic phase behavior of DMPG vesicles are also manifest without cycling through the phase transition or exposure to high temperatures. Furthermore, the addition of GS to DMPG vesicles protects the phospholipid molecules from the chemical hydrolysis induced by their repeated exposure to high temperatures. These results indicate that GS interacts more strongly with anionic than with zwitterionic phospholipid bilayers, probably because of the more favorable net attractive electrostatic interactions between the positively charged peptide and the negatively charged polar headgroup in such systems. Moreover, at comparable reduced temperatures, GS appears to interact more strongly with zwitterionic DMPC than with zwitterionic DMPE bilayers, probably because of the more fluid character of the former system. In addition, the general effects of GS on the thermotropic phase behavior of zwitterionic and anionic phospholipids suggest that it is located at the polar/apolar interface of liquid-crystalline bilayers, where it interacts primarily with the polar headgroup and glycerol-backbone regions of the phospholipid molecules and only secondarily with the lipid hydrocarbon chains. Finally, the considerable lipid specificity of GS interactions with phospholipid bilayers may prove useful in the design of peptide analogs with stronger interactions with microbial as opposed to eucaryotic membrane lipids.     

Temperature and compositional dependence of the structure of hydrated dimyristoyl lecithin.

Differential scanning calorimetry and x-ray diffraction techniques have been used to investigate the structure and phase behavior of hydrated dimyristoyl lecithin (DML) in the hydration range 7.5 to 60 weight % water and the temperature range -10 to +60 degrees C. Four different calorimetric transitions have been observed: T1, a low enthalpy transition (deltaH approximately equal to 1 kcal/mol of DML) at 0 degrees C between lamellar phases (L leads to Lbeta); T2, the low enthalpy "pretransition" at water contents greater than 20 weight % corresponding to the transition Lbeta leads to Pbeta; T3, the hydrocarbon chain order-disorder transition (deltaH = 6 to 7 kcal/mol of DML) representing the transition of the more ordered low temperature phases (Lbeta, Pbeta, or crystal C, depending on the water content) to the lamellar Lalpha phase; T4, a transition occurring at 25--27 degrees C at low water contents representing the transition from the lamellar Lbeta phase to a hydrated crystalline phase C. The structures of the Lbeta, Pbeta, C, and Lalpha phases have been examined as a function of temperature and water content. The Lbeta structure has a lamellar bilayer organization with the hydrocarbon chains fully extended and tilted with respect to the normal to the bilayer plane, but packed in a distorted quasihexagonal lattice. The Pbeta structure consists of lipid bilayer lamellae distorted by a periodic "ripple" in the plane of the lamellae; the hydrocarbon chains are tilted but appear to be packed in a regular hexagonal lattice. The diffraction pattern from the crystalline phase C indexes according to an orthorhombic cell with a = 53.8 A, b = 9.33 A, c = 8.82 A. In the lamellae bilayer Lalpha strucure, the hydrocarbon chains adopt a liquid-like conformation. Analysis of the hydration characteristics and bilayer parameters (lipid thickness, surface area/molecule) of synthetic lecithins permits an evaluation of the generalized hydration and structural behavior of this class of lipids.     

A fluorescent sterol probe study of cholesterol/phospholipid membranes

The behavior of dehydroergosterol in -α-dimyristoylphosphatidylcholine (DMPC) unsonicated multilamellar liposomes was characterized by absorption spectroscopy and fluorescence measurements. Dehydroergosterol exhibited a lowered absorption coefficient in multilamellar liposomes whiel the steady-state fluorescence anisotropy of dehydroergosterol in these membranes decreased significantly with increasing dehydroergosterol concentration, suggesting membrane sterol-sterol interactions. The comparative steady-state anisotropy of 0.9 mole percent dehydroergosterol in multilamellar liposomes was lower than in small unilamellar vesicles suggesting different sterol environments for dehydroergosterol. Dehydroergosterol fluorescence lifetime was relatively independent of membrane sterol content and yielded similar values in sonicated and unsonicated model membranes. In multilamellar liposomes containing 5 mole percent cholesterol, the gel-to-liqui crystalline phase transition of DMPC detected by 0.9 mole percent dehydroergosterol was significantly broadened when compared to the phase transition detected by dehydroergosterol in the absence of membrane cholesterol (Smutzer, G. et al. (1986) Biochim. Biophys. Acta 862, 361–371). In multilamellar liposomes containing 10 mole percent cholesterol, the major fluorescence lifetime of dehydroergosterol did not detect the gel-to-liquid crystalline phase transition of DMPC. Time-correlated fluorescence anisotropy decays of dehydroergosterol in DMPC multilamellar liposomes in the absence and presence of 5 mole percent cholesterol exhibited a single rotational correlation time near one nanosecond that was relatively independent of temperature and low concentrations of membrane cholesterol. The limiting anisotropy of 0.9 mole percent dehydroergosterol decreased above the gel-to-liquid crystalline phase transition in membranes without cholesterol and was not significantly affected by the phase transition in membranes containing 5 mole percent cholesterol. These results suggested hindered rotational diffusion of dehydroergosterol in multilamellar liposomes. Lifetime and time-correlated fluorescence measurements of 0.9 mole percent dehydroergosterol in multilamellar liposomes further suggested this fluorophore was detecting physical properties of the bulk membrane phospholipids in membranes devoid of cholesterol and was detecting sterol-rich regions in membranes of low sterol concentration.     

Role of peptide structure in lipid-peptide interactions: high-sensitivity differential scanning calorimetry and electron spin resonance studies of the structural properties of dimyristoylphosphatidylcholine membranes interacting with pentagastrin-related pentapeptides

The effects of amino acid substitutions in the pentapeptide pentagastrin on the nature of its interactions with dimyristoylphosphatidylcholine (DMPC) are assessed by differential scanning calorimetry and electron spin resonance. In two peptide analogues, the Asp at position 4 in pentagastrin (N-t-Boc-beta-Ala-Trp-Met-Asp-Phe-NH2) is replaced by Gly or Phe. These uncharged, more hydrophobic peptides have little effect on the transition temperature of DMPC, but they broaden the transition and lower the transition enthalpy as do integral membrane proteins. These peptides also mimic the behavior of integral membrane proteins in decreasing the order of a 5-doxylstearic acid spin probe below the transition temperature and in exhibiting a second immobilized lipid component using a 16-doxylstearic acid spin probe in DMPC. Three charged peptides were studied: pentagastrin, an analogue with positions 4 and 5 reversed (i.e., ending in Phe-Asp-NH2), and one with Asp replaced by Arg at position 4. All three of these charged peptides altered the phase transition behavior of DMPC to give two components, one above and one below the transition temperature of the pure lipid. With increasing peptide concentration, the higher melting transition became more prominent. The arginine-containing peptide produced the largest shifts in melting temperature followed by pentagastrin and then the "reversed" peptide. The arginine-containing peptide also increased the enthalpy of the transition. These peptides also increased the ordering of DMPC below the phase transition as measured with both 5- and 16-doxylstearic acid. The ordering effect was most pronounced with the arginine-containing peptide using the 5-doxylstearic acid probe. The results demonstrate that even the zwitterionic DMPC can interact more strongly with positively charged peptides than with negatively charged ones. In addition, peptide sequence as well as composition is important in determining the nature of peptide-lipid interactions. The markedly different effects of these pentagastrin peptides on the phase transition and motional properties of DMPC occur despite the similar depth of burial of these peptides with DMPC.     

Apolipophorin III interaction with model membranes composed of phosphatidylcholine and sphingomyelin using differential scanning calorimetry

Apolipophorin III (apoLp-III) from Locusta migratoria was employed as a model apolipoprotein to gain insight into binding interactions with lipid vesicles. Differential scanning calorimetry (DSC) was used to measure the binding interaction of apoLp-III with liposomes composed of mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and sphingomyelin (SM). Association of apoLp-III with multilamellar liposomes occurred over a temperature range around the liquid crystalline phase transition (L_α). Qualitative and quantitative data were obtained from changes in the lipid phase transition upon addition of apoLp-III. Eleven ratios of DMPC and SM were tested from pure DMPC to pure SM. Broadness of the phase transition (T_1/2), melting temperature of the phase transition (T_m) and enthalpy were used to determine the relative binding affinity to the liposomes. Multilamellar vesicles composed of 40% DMPC and 60% SM showed the greatest interaction with apoLp-III, indicated by large T_1/2 values. Pure DMPC showed the weakest interaction and liposomes with lower percentage of DMPC retained domains of pure DMPC, even upon apoLp-III binding indicating demixing of liposome lipids. Addition of apoLp-III to rehydrated liposomes was compared to codissolved trials, in which lipids were rehydrated in the presence of protein, forcing the protein to interact with the lipid system. Similar trends between the codissolved and non-codissolved trials were observed, indicating a similar binding affinity except for pure DMPC. These results suggested that surface defects due to non-ideal packing that occur at the phase transition temperature of the lipid mixtures are responsible for apolipoprotein-lipid interaction in DMPC/SM liposomes.     

Antioxidant activity of probucol and its effects on phase transitions in phosphatidylcholine liposomes

The effect of probucol on the phase behavior of dimyristoylphosphatidylcholine (DMPC) was examined by fluorescence polarization and differential scanning calorimetry (DSC). Probucol broadens and shifts the temperature of the main phase transition of DMPC liposomes as measured by fluorescence polarization with diphenylhexatriene and trimethyl-ammonium-diphenylhexatrine at concentrations as low as 5 mole%. As measured by DSC, probucol reduces the transition temperature of the gel----liquid-crystalline phase transition of DMPC by approx. 2 C degrees at all concentrations above about 5 mole% probucol and eliminates the pretransition at less than 1 mole%. In addition, the phase transition of DMPC is broadened and the enthalpy of the transition reduced by approx. 50%. Even at high concentrations of probucol, the gel----liquid-crystalline phase transition of DMPC is not eliminated. Similar effects are observed with dipalmitoylphosphatidylcholine liposomes. Based on these DSC measurements, measurements of the melting of probucol in dry mixtures with DMPC and observations of probucol mixtures with DMPC under polarizing optics, the maximum solubility of probucol in DMPC is approx. 10 mole%. This concentration exceeds that required (approx. 0.5 mole%) to prevent peroxidation of 10 mole% arachidonic acid in DMPC liposomes for 30 min in the presence of 0.05 mM Fe(NH4)(SO4)2 at 4 degrees C. Thus, probucol has a limited solubility in saturated phosphatidylcholine bilayers, but is an effective antioxidant at concentrations lower than its maximum solubility.     

Magnetic anisotropy of lecithin membranes. A new anisotropy susceptometer

Cylindrical giant vesicles prepared from egg lecithin and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) are oriented in an external magnetic field and observed by phase contrast microscopy. The anisotropic part of the diamagnetic susceptibility of the lecithin membrane is determined from the distribution of angles between the magnetic field and the long cylinder axis due to thermal fluctuations. The anisotropy of DMPC is found to be larger by a factor of 2 than that of egg lecithin. This is attributed to the presence of unsaturated acyl chains in egg lecithin.     

A fluorescence anisotropy study on the phase behavior of dimyristoylphosphatidylcholine/cholesterol mixtures

The phase behavior of L-alpha-dimyristoylphosphatidylcholine/cholesterol mixtures was studied in multilamellar vesicles by fluorescence polarization of the sterol molecule dehydroergosterol and of the polyene molecule alpha-parinaric acid. In the absence of cholesterol, dehydroergosterol exhibited an increase in polarization as DMPC vesicles were heated through the phase transition. This rise in polarization anisotropy was observed over a 0.6-1.0 degrees C increase in temperature with the midpoint of the phase transition occurring at 23.6 degrees C. Addition of 5 mol% cholesterol completely obliterated this change in polarization anisotropy through the phase transition of DMPC. alpha-Parinaric acid underwent a characteristic decrease in polarization anisotropy through the phase transition of DMPC. The change in anisotropy through the phase transition was over 4-fold greater than the values observed with dehydroergosterol. Vesicles containing 5 mol% cholesterol in the presence of alpha-parinaric acid underwent a decrease in polarization anisotropy that was over 75% of the original decrease in amplitude observed in the absence of any membrane cholesterol. The difference in sensitivity of the two fluorescent probes to the phase transition of DMPC as a function of membrane cholesterol content may be explained by a preferential partitioning of dehydroergosterol (and cholesterol) into a sterol-rich phase at low sterol concentrations. This partitioning allows dehydroergosterol to detect sterol-rich regions in the membrane bilayer.     

Insights into the dynamics of DMSO in phosphatidylcholine bilayers.

The solvation effects of dimethyl sulfoxide (DMSO) on the phase stability of dimyristoylphosphatidylcholine (DMPC) have been fully characterized using differential scanning calorimetry (DSC) and fluorescence spectroscopy with 1,6-diphenyl-1,3,5-hexatriene (DPH). The temperatures of the sub-, pre-, and main transitions of DMPC were found to increase linearly with increasing mole fraction of DMSO up to mole fraction X=0.13 DMSO/H(2)O. Beyond X=0.13, the pre-transition peak started to merge with the peak representing the main transition. Simultaneously, the subtransition peak began to disappear as its transition temperature also decreased. At X=0.18, with both the subtransition and pre-transition absent, the main transition between the planar gel and the liquid-crystalline phase was observed at 30.3 degrees C. Transition enthalpy values indicated that the subgel, planar gel and rippled gel phases are most stable at X=0.11, 0.16 and 0.20 DMSO/H(2)O, respectively. This demonstrates that DMSO exerts distinct effects on each respective phase and corresponding transition. Temperature-dependent fluorescence emission scans show an increase in hydration as the system proceeds from the subgel phase all the way to the liquid-crystalline phase and correlated well with the effects of DMSO on the transition temperatures of DMPC observed in our calorimetry data. Initial observations for the sub- and main transition are further confirmed by fluorescence anisotropy using DPH as a probe. The results illustrate the differences in the microviscosity of each phase and how DMSO affects the phase transitions. Ultimately, our results suggest the most likely mechanism governing the biological actions of DMSO may involve the regulation of the solvation effects of water on the phospholipid bilayer.     

Pressure effects on the physical properties of lipid bilayers detected by trans-parinaric acid fluorescence decay.

The effects of hydrostatic pressure on the physical properties of large unilamellar vesicles of single lipids dipalmitoyl phosphatidylcholine (DPPC) and dimyristoyl phosphatidylcholine (DMPC) and lipid mixtures of DMPC/DPPC have been studied from time-resolved fluorescence of trans-parinaric acid. Additional experiments were carried out using diphenylhexatriene to compare the results extracted from both probes. Fluorescence decays were analyzed by the maximum entropy method. Pressure does not influence the fluorescence lifetime distribution of trans-parinaric acid in isotropic solvents. However, in pressurized lipid bilayers an abrupt change was observed in the lifetime distribution which was associated with the isothermal pressure-induced phase transition. The pressure to temperature equivalence values, dT/dP, determined from the midpoint of the phase transitions, were 24 and 14.5 degrees C kbar-1 for DMPC and POPC, respectively. Relatively moderate pressures of about 500 bar shifted the DMPC/DPPC phase diagram 11.5 degrees C to higher temperatures. The effects of pressure on the structural properties of these lipid vesicles were investigated from the anisotropy decays of both probes. Order parameters for all systems increased with pressure. In the gel phase of POPC the order parameter was smaller than that obtained in the same phase of saturated phospholipids, suggesting that an efficient packing of the POPC hydrocarbon chains is hindered.     

Spontaneous fusion of phosphatidylcholine small unilamellar vesicles in the fluid phase

Using a high-sensitivity differential scanning microcalorimeter capable of performing cooling scans, we have examined the phase behavior of small unilamellar vesicles (SUV) as a function of time of storage above their order-disorder phase transition. Vesicles composed of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were examined. Cooling scans on fresh (5-7-h postsonication) samples revealed broad, relatively simple heat capacity peaks (peak temperatures: 19.9 degrees C for DMPC, 37.8 degrees C for DPPC) free of high-temperature spikes or shoulders. Subsequent heating scans displayed a sharp peak characteristic of previously described fusion products formed below the phase transition. SUV samples stored for 1 or more days above their phase transition displayed a moderately broad, high-temperature shoulder (23.8 degrees C for DMPC and 40.2 degrees C for DPPC) in the cooling profile. For DMPC, the enthalpy associated with this peak increased in a first-order fashion with time. Hydrolysis products were not detected until 12-20 days of storage. Both the rate and extent of shoulder appearance increased with temperature (k = 0.0017 h-1, fraction of total enthalpy = 0.1 at 36 degrees C; k = 0.0037 h-1, fraction = 0.2 at 42 degrees C). Freeze-fracture electron micrographs confirmed that an intermediate-sized vesicle population (diameters 400-500 A) appeared in SUV samples stored above their phase transition. Also, the trapped volume of DMPC SUV increased from 0.26 microL/mumol after 17 h of storage to 0.54 microL/mumol after storage for 16 days at 36 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation of enzyme activities with fluorescence anisotropy of dansyl-labeled cytochrome b5/NADH-cytochrome-b5 reductase systems in phosphatidylcholine vesicles

The changes in steady-state fluorescence lifetimes and anisotropy decay parameters, as well as enzyme activities, of dansyl-labeled cytochrome b5 (DNS-cytochrome b5), on interaction with NADH-cytochrome-b5 reductase in DMPC vesicles, have been measured as a function of temperature. Steady-state fluorescence of DNS-cytochrome b5 in DMPC vesicles with and without cholesterol was increased on interaction with reductase at temperatures both above and below the DMPC phase transition. In all systems three fluorescence decay components of the dansyl label in DNS-cytochrome b5 were observed. In the reductase-containing system, the long (major) decay time component of DNS-cytochrome b5 and the fraction of the total fluorescence associated with this component increased over the temperature range 15-30 degrees C. In time-resolved anisotropy measurements, the order parameters of DNS-cytochrome b5 in DMPC vesicles increased on interaction with reductase at temperatures above the DMPC phase transition, and this increase was even more pronounced in cholesterol-containing vesicles, at temperatures from 15-30 degrees C. The enzyme activity of the DNS-cytochrome-b5 reductase system in DMPC vesicles was also greatly increased in the presence of cholesterol. These results show that interaction of vesicle-bound DNS-cytochrome b5 and NADH-cytochrome-b5 reductase leads to an increased degree of order of the dansyl-labeled cytochrome with little change in its rotational flexibility, and suggests that the increased order can be correlated with increased enzyme activity.     

Protein and lipid structural transitions in cytochrome c oxidase-dimyristoylphosphatidylcholine reconstitutions

The thermotropic behavior of the mitochondrial enzyme cytochrome c oxidase (EC 1.9.3.1) reconstituted in dimyristoylphosphatidylcholine (DMPC) vesicles has been studied by using high-sensitivity differential scanning calorimetry and fluorescence spectroscopy. The incorporation of cytochrome c oxidase into the phospholipid bilayer perturbs the thermodynamic parameters associated with the lipid phase transition in a manner analogous to other integral membrane proteins: it reduces the enthalpy change, lowers the transition temperature, and reduces the cooperative behavior of the phospholipid molecules. Analysis of the dependence of the enthalpy change on the protein:lipid molar ratio indicates that cytochrome c oxidase prevents 99 +/- 5 lipid molecules from participating in the main gel-liquid-crystalline transition. These phospholipid molecules presumably remain in the same physical state below and above the transition temperature of the bulk lipid, thus providing a more or less constant microenvironment to the protein molecule. The effect of the phospholipid bilayer matrix on the thermodynamic stability of the cytochrome c oxidase complex was examined by high-sensitivity differential scanning calorimetry. Detergent (Tween 80)-solubilized cytochrome c oxidase undergoes a complex, irreversible thermal denaturation process centered at 56 degrees C and characterized by an enthalpy change of 550 +/- 50 kcal/mol of enzyme complex. Reconstitution of the cytochrome c oxidase complex into DMPC vesicles shifts the transition temperature upward to 63 degrees C, indicating that the phospholipid bilayer moiety stabilizes the native conformation of the enzyme. The lipid bilayer environment contributes approximately 10 kcal/mol to the free energy of stabilization of the enzyme complex. The thermal unfolding of cytochrome c oxidase is not a two-state process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential scanning calorimetry on mixtures of lecithin,lysolecithin and cholesterol

The effect of increasing concentrations of lysolecithin (1-palmitoyl-sn-glycerol-3-phosphorylcholine) on the gel → liquid crystal thermal transition of lecithin (1,2-dipalmitoyl-sn-glycerol-3-phosphorylcholine) in the aqueous phase was studied by differential scanning calorimetry (DSC). Lysolecithin showed an endothermic transition at 3.4°C whereas the transition of the lecithin occurred at 42°C. No phase separation could be observed calorimetrically at lysolecithin concentrations up to 60 mol%. Freeze etch electron microscopy showed that mixtures containing as much as 50 mol% lysolecithin exist in a lamellar phase. The lysolecithin was found to cause an initial slight increase in the enthalpy of transition followed by a gradual decrease. The enthalpy increased again at very high lysolecithin concentrations. The lysolecithin also caused a non-linear decrease in the temperature at which the lecithin transition took place.Cholesterol was found to decrease the enthalpy of transition of the lysolecithin, eliminating it at a concentration of 50 mol%. Cholesterol caused an increase in the temperature at which the lysolecithin transition took place.     

Phospholipid structure determines the effects of peptides on membranes. Differential scanning calorimetry studies with pentagastrin-related peptides

The effect of phospholipid structure on the interaction between small peptides and phospholipid membranes has been studied by high-sensitivity differential scanning calorimetry. The peptides used, N-Boc-beta-Ala-Trp-Met-Arg-Phe-NH2 and N-Boc-beta-Ala-Trp-Met-Lys-Phe-NH2, are basic analogs of the hormone pentagastrin. These peptides split the gel-to-liquid crystalline phase transition of synthetic phosphatidylcholines into two components. For dimyristoyl (DMPC), dipalmitoyl (DPPC) and 1-stearoyl-2-oleoyl (SOPC) phosphatidylcholines, one component remains at the temperature corresponding to that of pure lipid and the other one is shifted towards higher temperatures. With increasing peptide concentration there is a gradual increase in the enthalpy of the high-temperature component at the expense of the low-temperature one, and there is also an increase in the total enthalpy of the transition. A mixture of the peptide with distearoylphosphatidylcholine (DSPC) behaves differently, with the transition occurring at a temperature below that of the pure lipid increasing with peptide concentration. The susceptibility of various phosphatidylcholines to perturbation by the peptides increases in the order DMPC greater than SOPC greater than DPPC greater than DSPC. The effect of these peptides on the phase transitions of acidic phosphatidylglycerols is generally greater than with the corresponding phosphatidylcholines, but the dependence on the length of lipid hydrocarbon chains is similar. Perturbation of the thermotropic phase transition is strongest for dimyristoylphosphatidylglycerol, followed by the dipalmitoyl and the distearoyl analogs. The effect of the peptides on the phase transition of dimyristoylphosphatidylserine is significantly smaller compared to that observed with dimyristoylphosphatidylglycerol and it is further reduced for dimyristoylphosphatidic acid. The phase transition of this latter lipid remains virtually unchanged, even in the presence of high concentrations of the peptide. Similar resistance to the perturbation of the phase transitions by the peptides is observed for synthetic phosphatidylethanolamine. The different susceptibility of various phospholipids to perturbation by the peptides is suggested to be related to different degrees of intermolecular interaction between phospholipid molecules, and particularly to different abilities of phospholipids to form intermolecular hydrogen bonding.