High pressure antagonism of alcohol effects on the main phase-transition temperature of phospholipid membranes: biphasic response.

The combined effects of high pressure (up to 300 bar) and a homologous series of 1-alkanols (ethanol C2 to 1-tridecanol C13) were studied on the main phase-transition temperature of dipalmitoylphosphatidylcholine (DPPC) vesicle membranes. It is known that short-chain alkanols depress and long-chain alkanols elevate the main transition temperature. The crossover from depression to elevation occurs at the carbon-chain length about C10-C12 in DPPC vesicle membranes coinciding with the cutoff chain-length where anesthetic potency suddenly disappears. Alkanols shorter than C8 linearly decreased the transition temperature and high pressure antagonized the temperature depression. Alkanols longer than C10 showed biphasic dose-response curves. High pressure enhanced the biphasic response. In addition, alkanols longer than the cutoff length depressed the transition temperature under high pressure at the low concentration range. These non-anesthetic alkanols may manifest anesthetic potency under high pressure. At higher concentrations, the temperature elevatory effect was accentuated by pressure. This biphasic effect of long-chain alkanols is not related to the 'interdigitation' associated with short-chain alkanols. The increment of the transition temperature by pressure was 0.0242 K bar-1 in the absence of alkanols. The volume change of the transition was estimated to be 27.7 cm3 mol-1. This value stayed constant to the limit of the present study of 300 bar.     

Differential affinity of charged local anesthetics to solid-gel and liquid-crystalline states of dimyristoylphosphatidic acid vesicle membranes

Cationic local anesthetics decreased the transition temperature of the anionic phospholipid (dimyristoylphosphatidic acid, DMPA) vesicles. The counterion concentration changes the electrical double layer effect, and affects the magnitude of temperature depression caused by anesthetics. From the counterion effect on the transition-temperature depression, the partition coefficients of cationic local anesthetics to liquid-crystalline and solid-gel DMPA membranes were separately estimated. The differences in the partition coefficients between solid-gel and liquid-crystalline membranes correlated to the nerve blocking potencies. There are at least two states in the nerve membranes: resting state at higher temperature and excited state at lower temperature. We speculate that the resting state corresponds to the liquid-crystalline state, and the excited state to the solid-gel state. The difference in the partition coefficients to the resting and excited states is the cause of local anesthesia.     

A calorimetric and spectroscopic comparison of the effects of lathosterol and cholesterol on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membranes

We performed differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopic measurements to study the effects of lathosterol (Lath) on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine (DPPC) bilayer membranes and compared our results with those previously reported for cholesterol (Chol)/DPPC binary mixtures. Lath is the penultimate intermediate in the biosynthesis of Chol in the Kandutsch-Russell pathway and differs from Chol only in the double bond position in ring B, which is between C7 and C8 in Lath and between C5 and C6 in Chol. Our DSC studies indicate that the incorporation of Lath is more effective than Chol in reducing the temperature and enthalpy of the DPPC pretransition. At lower sterol concentrations (≤10 mol %), incorporation of both Lath and Chol decreases the temperature, enthalpy, and cooperativity of the sharp component of the main phase transition of DPPC to a similar extent, but at higher sterol concentrations, Lath is more effective at decreasing the phase transition temperature, enthalpy, and cooperativity than Chol. These results indicate that at higher concentrations, Lath is more disruptive of DPPC gel-state bilayer packing than Chol is. Moreover, incorporation of Lath decreases the temperature of the broad component of the main phase transition of DPPC, whereas Chol increases it; this difference in the direction and magnitude of the temperature shift is accentuated at higher sterol concentrations. Although at sterol concentrations of ≤20 mol % Lath and Chol are almost equally effective at reducing the enthalpy and cooperativity of the broad component of the main phase transition, at higher sterol levels Lath is less effective than Chol in these regards and does not completely abolish the cooperative hydrocarbon chain melting phase transition at 50 mol %, as does Chol. These latter results indicate that Lath both is more disruptive with respect to the low-temperature state of the sterol-enriched domains of DPPC bilayers and has a lower lateral miscibility in DPPC bilayers than Chol. Our FTIR spectroscopic studies suggest that Lath incorporation produces a less tightly packed bilayer than does Chol at both low (gel state) and high (liquid-crystalline state) temperatures, which is characterized by increased H-bonding between water and the carbonyl groups of the fatty acyl chains in the DPPC bilayer. Overall, our studies indicate that Lath and Chol incorporation can have rather different effects on the thermotropic phase behavior and organization of DPPC bilayers and thus that the position of the double bond in ring B of a sterol molecule can have an appreciable effect on the physical properties of sterol molecules.     

Interaction modes of long-chain fatty acids in dipalmitoylphosphatidylcholine bilayer membrane: contrast to mode of inhalation anesthetics

The effects of long-chain fatty acids (four saturated and two unsaturated fatty acids, one derivative) on phase transitions of dipalmitoylphosphatidylcholine (DPPC) bilayer membranes were examined in the low concentration region, and the results were compared with those for an inhalation anesthetic. The effects of all fatty acids on the pre- and main-transition temperatures of the DPPC bilayer membrane appeared in the concentration range of μM order while that of the anesthetic appeared in the mM order. The appearance modes of these ligand actions were significantly different from one another. The three differential partition coefficients of the ligands between two phases of the DPPC bilayer membrane were evaluated by applying the thermodynamic equation to the variation of the phase-transition temperatures. The DPPC bilayer membranes showed the different receptivity for the ligands; the saturated fatty acids had an affinity for gel phase whereas unsaturated fatty acids and an anesthetic had an affinity for liquid-crystalline phase to the contrary. In particular, the receptivity for the ligands in the gel phase markedly changed depending on kinds of ligands. The interaction modes between the DPPC and fatty acid molecules in the gel phase were considered from the hexagonal lattice model. The disappearance compositions of the pretransition by the fatty acids coincided with the compositions at which the membrane is all covered by the units in each of which two fatty acids molecules are regularly distributed in the hexagonal lattice in a different way, and the distribution depended on the chain length and existence of a double bond for the fatty acids. The interpretation did not hold for the case of the anesthetic at all, which proved that a number of anesthetic molecules act the surface region of the bilayer membrane nonspecifically. The present study clearly implies that DPPC bilayer membranes have high ability to recognize kinds of ligand molecules and can discriminate among them with specific interaction by the membrane states.     

A statistical mechanical analysis of the effect of long-chain alcohols and high pressure upon the phase transition temperature of lipid bilayer membranes.

Long-chain n-alcohols decrease the main phase-transition temperature of lipid vesicle membranes at low concentrations but increase it at high concentrations. The nonlinear phenomenon is unrelated to the interdigitation and is analyzed by assuming that alcohols form solid solutions with solid as well as liquid phases. The biphasic response originates from the balance of the free energy difference of alcohols in the liquid and solid membranes (delta gA) and the alcohol-lipid interaction free energy difference (delta u) between the two phases. When delta gA less than 0 and delta u greater than 0, or delta gA less than delta u less than 0, the transition temperature decreases monotonously according to the increase in the alcohol concentration. When delta gA greater than 0 and delta u less than 0, or delta gA greater than delta u greater than 0, it increases monotonously. Biphasic response occurs with a minimum temperature when delta u greater than delta gA greater than 0, and with a maximum temperature when delta u less than delta gA less than 0. When the alcohol carbon-chain length becomes closer to the lipid carbon-chain length, delta u is equalized by delta gA, and the temperature minimum of the main transition is shifted to extremely low alcohol concentrations. Hence, long-chain alcohols predominantly elevate the main transition temperature and lose their anesthetic potency. High pressure decreased both delta gA and delta u. Presumably, high pressure improves the packing efficiency of liquid membranes and decreases the difference between the solid and liquid membrane properties.     

Structural isomers of tetradecenol discriminate between the lipid fluidity and phase transition theories of anesthesia

The long-chain unsaturated alcohols, cis- and trans-9,10-tetradecenol were found to be equipotent both as general anesthetics and local anesthetics. Their effects on the phase transition temperature of dipalmitoylphosphatidylcholine/water dispersions were measured spectroscopically by partitioning of the spin label 2,2,6,6-tetramethylpiperidine-1-oxyl. Also spectroscopic order parameters were obtained from spin labeled egg lecithin/cholesterol vesicles, in the absence and presence of the two alcohols. Whereas the egg lecithin/cholesterol membranes were fluidised by both alcohols to a similar extent, cis-tetradecenol lowered the transition temperature of dipalmitoylphosphatidylcholine, while the trans-isomer elevated the transition temperature. These results are thus inconsistent with the phase transition model of anesthetic action. The possible application of these alcohols to diagnose the origin of discontinuities in Arrhenius plots is discussed.     

Studies on the interaction of anesthetic steroids with phosphatidylcholine using 2H and 13C solid state NMR

The effects of the anesthetic steroid alphaxalone and its inactive analog delta 16-alphaxalone on model phospholipid membranes were studied using 13C and 2H solid-state nuclear magnetic resonance spectroscopy. Aqueous multilamellar dispersions of dipalmitoylphosphatidylcholine (DPPC) with specific 13C and 2H labels as endogenous probes at the carbonyl and the C-7 methylene groups, respectively, of the sn-2 chain were used to study the conformational and dynamical properties of the bilayer as a function of temperature. There were no significant changes between the 13C and 2H spectra of the DPPC preparation containing the inactive steroid and that of DPPC with no drug. However, the physiologically active steroid produces significant spectral 2H and 13C changes. These changes include a reduction of the main phase transition temperature and a broadening of that transition. Alphaxalone also increases the relative number of gauche conformers in the liquid-crystalline phase of DPPC and increases the rate of axial diffusion in both the gel and liquid-crystalline phase. The thermotropic properties of the above preparations, as monitored by differential scanning calorimetry, were congruent with the spectroscopic data.     

Effect of n-alcohols and glycerol on the pretransition of dipalmitoylphosphatidylcholine

We have systematically investigated the effect of short-chain n-alcohols and glycerol on the pretransition of 1,2-dipalmitoylphosphatidylcholine (DPPC) by spectrophotometry. It is found that the n-alcohols and glycerol remove the pretransition above a critical concentration for each ligand. In addition, the short-chain n-alcohols below the critical concentration decrease the pretransition temperature. The longer the aliphatic chain length of the n-alcohol (up to butanol) the greater the decrease in the pretransition temperature, and the lower the concentration necessary to remove the pretransition. However, glycerol differs from the short-chain n-alcohols in that it has no significant effect on either the pretransition or the main transition, but it is also capable of removing the pretransition above a critical concentration. It has previously been shown that alcohols have a biphasic effect on the main transition temperature of phosphatidylcholines (Rowe, E.S. (1983) Biochemistry 22, 3299-3305). At high alcohol concentrations, the main transition is not thermodynamically reversible (Rowe, E.S. (1985) Biochim. Biophys. Acta 813, 321-330). Recently, Simon and McIntosh (Biochim. Biophys. Acta (1984) 773, 169-172) have identified that at high ethanol concentration DPPC exists in the interdigitated phase. The critical ligand concentration at which the pretransition disappears coincides with the induction of main transition hysteresis and the biphasic alcohol effect in the main transition. These three effects appear to correlate with the induction of the interdigitated gel state by alcohols and glycerol.     

Thermotropic phase behaviour of alpha-dipalmitoylphosphatidylcholine multibilayers is influenced to various extents by carotenoids containing different structural features--evidence from differential scanning calorimetry

Carotenoids are the effective modulators of physical properties of model and natural membranes. To demonstrate the relationship between the structure of carotenoids and their effect on the molecular dynamics of membranes, we have investigated the influence of five structurally different carotenoids: beta-carotene, lycopene, lutein, violaxanthin, zeaxanthin and additionally carotane--a fully saturated derivative of beta-carotene, on thermotropic phase behaviour of dipalmitoylphosphatidylcholine (DPPC) multilamellar vesicles by means of differential scanning calorimetry (DSC). The results obtained indicate that the carotenoids used modulated the thermotropic properties of multibilayers to various extents, broadening the pretransition and the main phase transition peaks and shifting them to lower temperatures. Pronounced decrease of pretransition enthalpy (DeltaH(p)) proves that carotenoids very strongly alter the membrane properties in its gel phase. Comparison of the influence of several carotenoids shows that a rigid, polyisoprenoid chain plays a basic role in altering the thermotropic properties of such membranes and the presence of rings without oxygen-containing groups has a minor significance for the observed interactions. Carotenoids containing epoxy and/or hydroxy groups attached to their rings modify the thermotropic phase behaviour of DPPC multilamellar vesicles stronger than carotenes--a result of their orientation in the DPPC bilayer.     

Thermotropic phase behaviour of α-dipalmitoylphosphatidylcholine multibilayers is influenced to various extents by carotenoids containing different structural features- evidence from differential scanning calorimetry

Carotenoids are the effective modulators of physical properties of model and natural membranes. To demonstrate the relationship between the structure of carotenoids and their effect on the molecular dynamics of membranes, we have investigated the influence of five structurally different carotenoids: β-carotene, lycopene, lutein, violaxanthin, zeaxanthin and additionally carotane- a fully saturated derivative of β-carotene, on thermotropic phase behaviour of dipalmitoylphosphatidylcholine (DPPC) multilamellar vesicles by means of differential scanning calorimetry (DSC). The results obtained indicate that the carotenoids used modulated the thermotropic properties of multibilayers to various extents, broadening the pretransition and the main phase transition peaks and shifting them to lower temperatures. Pronounced decrease of pretransition enthalpy (ΔH_p) proves that carotenoids very strongly alter the membrane properties in its gel phase. Comparison of the influence of several carotenoids shows that a rigid, polyisoprenoid chain plays a basic role in altering the thermotropic properties of such membranes and the presence of rings without oxygen-containing groups has a minor significance for the observed interactions. Carotenoids containing epoxy and/or hydroxy groups attached to their rings modify the thermotropic phase behaviour of DPPC multilamellar vesicles stronger than carotenes- a result of their orientation in the DPPC bilayer.     

Thermotropic Properties of Cellular Membranes in Dormant and Non-Dormant Echinochloa crus-galli(L.) Beauv. Seeds

Steady-state fluorescence polarization measurements with l,6-diphenyl-l,3,5-hexatriene(DPH) were used to monitor thermotropic transitions in microsomalfractions and plasma membrane vesicles isolated from barnyardgrass[Echinochloa crus-galli (L.) Beauv.] seeds during the transititionfrom dormancy to germination. The effect of dormancy-relievingor inactive alcohols on the thermotropic properties of the cellularmembranes was determined both in vivo and in vitro. Membranefractions isolated from dormant seeds showed some discontinuitiesin the Arrhenius plots. In non-dormant or germinating seedscellular membranes showed linear Arrhenius plots over the entirerange of temperature examined. Membrane preparations from imbibedseeds showed a similar pattern in their Arrhenius plots upontreatment with the various alcohols in vitro. The results suggestthat the release from dormancy in seeds is associated with somechanges in their cellular membranes. Key words: Germination, alcohols, thermotropic transition     

A differential scanning calorimetry study of the interaction of lasalocid antibiotic with phospholipid bilayers

Interaction of lasalocid sodium salt (Las-Na) with dipalmitoylphosphatidylcholine (DPPC) as a membrane model was investigated by highly-sensitive differential scanning calorimetry (DSC). The insertion properties of the antibiotic were studied both in multilamellar suspensions and unilamellar vesicles, for Las-Na/DPPC molar ratios (r) ranging from 0.005 to 0.1. The effect of the antibiotic on the lipid thermotropic behavior is concentration dependent and drastically changes at a critical r of 0.04 in both model membranes. Below this ratio, Las-Na molecules interact with DPPC bilayers without disrupting the global organization of the membrane. In the multilamellar systems only the transition cooperativity is affected whereas for the mixed vesicles, a decrease in the enthalpy change suggests a different mode of insertion. Above this ratio, implantation of the antibiotic give rise to lateral phase separation in multilamellar systems. These structural modifications have repercussions on the formation of mixed LAS-Na/DPPC vesicles which seems limited to an r value of 0.04.     

A calorimetric study of binary mixtures of dihydrosphingomyelin and sterols,sphingomyelin, or phosphatidylcholine

The thermotropic properties of binary mixtures of D-erythro-n-palmitoyl-dihydrosphingomyelin (16:0-DHSM), D-erythro-n-palmitoyl-sphingomyelin (16:0-SM), cholesterol, lathosterol, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were studied by differential scanning calorimetry. Addition of sterol to 16:0-DHSM and 16:0-SM bilayers resulted in a progressive decrease in both the T(m) and the enthalpy of the main transition. The sterol-induced broad components in 16:0-DHSM endotherms had markedly lower enthalpies than those induced in 16:0-SM. Pretransitions recorded in 16:0-DHSM and 16:0-SM membranes responded differently to low concentrations of cholesterol. The presence of 5 mol % cholesterol increased the pretransition temperature in 16:0-SM bilayers, whereas it decreased the temperature in 16:0-DHSM membranes. Lathosterol behaved in general as cholesterol with regard to its effects on the thermotropic behavior of both sphingolipids, but it appeared to form more stable sterol-rich domains, as seen from the higher T(m) of the broad component, in comparison to cholesterol. Thermograms recorded on binary mixtures of 16:0-SM:16:0-DHSM and DPPC:16:0-DHSM showed that 16:0-SM mixed nearly ideally with 16:0-DHSM, whereas DPPC mixing was less ideal in a 16:0-DHSM membrane. In conclusion, we observed that 16:0-DHSM interactions with sterols differed from that seen with 16:0-SM, and that 16:0-DHSM mixed better with 16:0-SM than DPPC, which indicates that DHSM could function as a membrane organizer within laterally condensed domains.     

Calorimetric study on a dipalmitoylphosphatidylcholine-propylgallate mixture

The effect of propylgallate (PrG, an antioxidant) on the thermotropic behavior of dipalmitoylphosphatidylcholine (DPPC) was studied by means of differential scanning calorimetry. A DPPC/PrG mixture displayed distinctive thermotropic behavior that was significantly different from that of a DPPC/cholesterol or DPPC/vitamin E mixture. Although the enthalpy of the phase transition (delta H) for DPPC decreased at a low concentration of the PrG and the transition peak became broadened, delta H increased again and the peak became sharper on the addition of more PrG. The same was observed for DPPC/methylgallate and DPPC/ethylgallate mixtures, but not for a DPPC/butylgallate mixture. On the other hand, the transition temperature (Tm) of the DPPC/gallate derivative mixtures decreased with an increase in the chain length of the acyl moiety of the gallate derivatives. The pre-transition and subtransition of the DPPC/PrG mixture were eliminated on the addition of a PrG, and Tm of the DPPC/PrG mixture approached about 26 degrees C. These results suggested that the chain length of the acyl moiety must be C1 to C3 for the unique effect of the gallate derivatives described above, and that DPPC forms a complex with PrG as a pure component.     

Effect of n-alcohols and glycerol on the pretransition of dipalmitoylphosphatidylcholine

We have systematically investigated the effect of short-chain n-alcohols and glycerol on the pretransition of 1,2-dipalmitoylphosphatidylcholine (DPPC) by spectrophotometry. It is found that the n-alcohols and glycerol remove the pretransition above a critical concentration for each ligand. In addition, the short-chain n-alcohols below the critical concentration decrease the pretransition temperature. The longer the aliphatic chain length of the n-alcohol (up to butanol) (a) the greater the decrease in the pretransition temperature, and (b) the lower the concentration necessary to remove the pretransition. However, glycerol differs from the short-chain n-alcohols in that it has no significant effect on either the pretransition or the main transition, but it is also capable of removing the pretransition above a critical concentration. It has previously been shown that alcohols have a biphasic effect on the main transition temperature of phosphatidylcholines (Rowe, E.S. (1983) Biochemistry 22, 3299–3305). At high alcohol concentrations, the main transition is not thermodynamically reversible (Rowe, E.S. (1985) Biochim. Biophys. Acta 813, 321–330). Recently, Simon and McIntosh (Biochim. Biophys. Acta (1984) 773, 169–172) have identified that at high ethanol concentration DPPC exists in the interdigitated phase. The critical ligand concentration at which the pretransition disappears coincides with the induction of main transition hysteresis and the biphasic alcohol effect in the main transition. These three effects appear to correlate with the induction of the interdigitated gel state by alcohols and glycerol.     

The influence of structure and redox state of prenylquinones on thermotropic phase behaviour of phospholipids in model membranes

Our study was aimed to investigate the significance of the isoprenoid side chain size as well as redox state of the quinone ring for interaction of two main classes of prenylquinones: plastoquinones (PQ) and ubiquinones (UQ) with lipid bilayers. By use of differential scanning calorimetry (DSC) we have followed the thermotropic behaviour of multilamellar vesicles prepared from dipalmitoylphosphatidylcholine (DPPC) upon incorporation of increasing amount (1.3-12 mol%) of quinone (quinol) molecules. Our studies reveal that as the side chain is shorter (from 9 to 2 isoprenoid units) the height of the calorimetric profiles is reduced and the temperature of the main transition of DPPC (T(m)) decreases (T(m)=39.4 degrees C for a sample with 12 mol% of PQ-2), and then increases up to 39.8 degrees C for PQ-1. For the samples containing quinols the effect is more pronounced even at lower concentration. The greater influence of the added prenylquinones on the pretransition demonstrates a stronger distortion of the DPPC packing in the gel state. It seems that this is the isoprenoid side chain length rather than the redox state of prenylquinones that determines their effectiveness in perturbation of thermotropic properties of lipid bilayer.     

Stages of the bilayer-micelle transition in the system phosphatidylcholine-C12E8 as studied by deuterium- and phosphorous-NMR, light scattering, and calorimetry.

The perturbation of phospholipid bilayer membranes by a nonionic detergent, octaethyleneglycol mono-n-dodecylether (C12E8), was investigated by 2H- and 31P-NMR, static and dynamic light scattering, and differential scanning calorimetry. Preequilibrated mixtures of the saturated phospholipids 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), and 1,2-dilauroyl-sn-glycero-3-phosphorylcholine (DLPC) with the detergent were studied over a broad temperature range including the temperature of the main thermotropic phase transition of the pure phospholipids. Above this temperature, at a phospholipid/detergent molar ratio 2:1, the membranes were oriented in the magnetic field. Cooling of the mixtures below the thermotropic phase transition temperatures of the pure phospholipids led to micelle formation. In mixtures of DPPC and DMPC with C12E8, a narrow calorimetric signal at the onset temperature of the solubilization suggested that micelle formation was related to the disorder-order transition in the phospholipid acyl chains. The particle size changed from 150 nm to approximately 7 nm over the temperature range of the bilayer-micelle transition. The spontaneous orientation of the membranes at high temperatures enabled the direct determination of segmental order parameters from the deuterium spectra. The order parameter profiles of the phospholipid acyl chains could be attributed to slow fluctuations of the whole membrane and to detergent-induced local perturbations of the bilayer order. The packing constraints in the mixed bilayers that eventually lead to bilayer solubilization were reflected by the order parameters of the interfacial phospholipid acyl chain segments and of the phospholipid headgroup. These results are interpreted in terms of the changing average shape of the component molecules. Considering the decreasing cross sectional areas in the acyl chain region and the increasing hydration of the detergent headgroups, the bilayer-micelle transition is the result of an imbalance in the chain and headgroup repulsion. A neutral or pivotal plane can be defined on the basis of the temperature dependence of the interfacial quadrupolar splittings.     

Effect of alcohols on the phase transitions of dihexadecylphosphatidylcholine

We have systematically investigated the effect of short chain alcohols (methanol to n-propanol) on the phase transitions of 1,2-dihexadecylphosphatidylcholine (DHPC), a lipid that forms a stable interdigitated gel phase (L beta I) in aqueous solution. The temperature of the low-temperature L beta I to P beta' phase transition of DHPC was found to increase with alcohol concentration, showing that alcohol interacts preferentially with the interdigitated phase relative to the non-interdigitated gel. The main transition of DHPC exhibited a biphasic effect of alcohol concentration similar to that previously observed with DPPC (Rowe, E.S. (1983) Biochemistry 22,3299-3305). As alcohol concentration is increased the lower L beta I to P beta' and main P beta' to L alpha transitions of DHPC merge at the threshold concentration of the biphasic effect, so that above this concentration there is one phase transition from L beta I directly to L alpha. This is analogous to DPPC above its biphasic threshold. Similar to DPPC, the transition between L beta I and L alpha exhibits marked hysteresis.     

A differential scanning calorimetry study of phosphocholines mixed with paclitaxel and its bromoacylated taxanes

High sensitivity differential scanning calorimetry (DSC) was used to investigate the thermotropic phase properties of binary mixtures of disaturated phosphocholines (PCs) and alpha-bromoacyl taxane derivatives. The alpha-bromoacyl taxanes were synthesized as hydrolyzable hydrophobic prodrugs of paclitaxel. The PCs used were 1, 2-dimyristoyl-sn-glycero-3-phosphatidyl-choline (DMPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1, 2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). The bromoacyl chain lengths of the taxane prodrugs were varied from 6 to 12 or 16 carbons. For comparison, paclitaxel and PC mixtures were also examined. DSC data from DPPC and bromoacyl taxane mixtures showed a complete abolition of the pretransition and significant broadening of the main phase transition with increasing amounts of bromoacyl taxane prodrugs. The effects were more pronounced with the long-chain compared to the short-chain prodrugs. Under equivalent DSC conditions, the short-chain DMPC showed greater changes in thermotropic phase behavior than with DPPC on taxane addition, suggesting an enhanced degree of association with the fluid-type bilayers. Under similar conditions, the long-chain DSPC bilayers showed a far less significant change in phase behavior on taxane addition than DPPC. These changes were also chain length-dependent for both the PCs and the taxane prodrugs. In contrast, PC and paclitaxel (lacking the acyl chain) mixtures under similar conditions showed insignificant changes in the endotherms, suggesting only slight insertion of the molecule into the PC bilayers. From the DSC data it is apparent that taxane prodrugs solvated in DMPC bilayers more than in DPPC and DSPC bilayers, and taxane prodrugs with longer acyl chains were able to associate with PCs better than those with shorter chain prodrugs. DSC data also suggest that paclitaxel was poorly associated with any of the PCs. In general, the amount of taxane association with bilayers decreased in order: DMPC > DPPC > DSPC. In contrast, the transition enthalpy (DeltaH) of DMPC, DPPC, and DSPC mixtures with paclitaxel showed significantly lower enthalpies than with taxane prodrugs. Taken together, the DSC data suggest that the acyl chains of paclitaxel prodrugs have some access into the bilayers via alignment with the acyl chain of the PC component.     

Is there a specific receptor for anesthetics? Contrary effects of alcohols and fatty acids on phase transition and bioluminescence of firefly luciferase.

Firefly luciferase emits a burst of light when mixed with ATP and luciferin (L) in the presence of oxygen. This study compared the effects of long-chain n-alcohols (1-decanol to 1-octadecanol) and fatty acids (decanoic to octadecanoic acids) on firefly luciferase. Fatty acids were stronger inhibitors of firefly luciferase than n-alcohols. Myristyl alcohol inhibited the light intensity by 50% (IC50) at 13.6 microM, whereas the IC50 of myristic acid was 0.68 microM. According to the Meyer-Overton rule, fatty acids are approximately 12,000-fold stronger inhibitors than corresponding alcohols. The Lineweaver-Burk plot showed that myristic acid inhibited firefly luciferase in competition with luciferin, whereas myristyl alcohol inhibited it noncompetitively. The differential scanning calorimetry (DSC) showed that an irreversible thermal transition occurred at approximately 39 degrees C with a transition DeltaHcal of 1.57 cal g-1. The ligand effects on the transition were evaluated by the temperature where the irreversible change is half completed. Alcohols decreased whereas fatty acids increased the thermal transition temperature of firefly luciferase. Koshland's transition-state theory (Science. 1963. 142:1533-1541) states that ligands that bind to the substrate-recognition sites induce the enzyme at a transition state, which is more stabilized than the native state against thermal perturbation. The long-chain fatty acids bound to the luciferin recognition site and stabilized the protein conformation at the transition state, which resisted thermal denaturation. Eyring's unfolding theory (Science. 1966. 154:1609-1613) postulates that anesthetics and alcohols bind nonspecifically to interfacial areas of proteins and reversibly unfold the conformation. The present results showed that alcohols do not compete with luciferin and inhibit firefly luciferase nonspecifically by unfolding the protein. Fatty acids are receptor binders and stabilize the protein conformation at the transition state.