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)     

Conformational state and functional activity of NADPH-dependent cytochrome P-450 reductase isolated and incorporated into phospholipid bilayers

It was shown that the alpha-helix content in both isolated and incorporated into phospholipid bilayer NADPH-dependent cytochrome P-450 reductase is 20%. NADPH or dithionite reduction of the flavoprotein is not followed by conformational changes. The incorporation of the NADPH-dependent cytochrome P-450 reductase molecule into the phospholipid bilayer does not affect its catalytic properties. It was found that the protein does not interact with the phospholipid bilayer of phosphatidyl choline liposomes but incorporates readily into the liposomal membrane from a microsomal phospholipid mixture with a binding constant of 17.4 microM.     

Interdigitation of phosphatidylcholine and phosphatidylethanolamine mixed with complexes of acidic lipids and polymyxin B or polymyxin B nonapeptide

A fatty acid spin label, 16-doxyl-stearic acid, was used to determine the percent interdigitated lipid in mixtures of a neutral phospholipid and an acidic phospholipid. Interdigitation of the acidic lipid was induced with polymyxin B (PMB) at a mole ratio of PMB to acidic lipid of 1:5. This compound does not bind significantly to neutral lipids or induce interdigitation of the neutral lipids by themselves. The neutral lipids used were dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), or dipalmitoylphosphatidylethanolamine (DPPE), and the acidic lipids were dipalmitoylphosphatidylglycerol (DPPG) or dipalmitoylphosphatidic acid (DPPA). The percent interdigitated lipid was determined from the percent of the spin label which is motionally restricted, assuming that the spin label is homogeneously distributed in the lipid. Assuming further that 100% of the acidic lipid is interdigitated at this saturating concentration of PMB, the percentage of the neutral lipid which can become interdigitated along with it was calculated. The results indicate that about 20 mole % DPPC can be incorporated into and become interdigitated in the interdigitated bilayer of PMB/DPPG at 4 degrees C. As the temperature approaches the phase transition temperature, the lipid becomes progressively less interdigitated; this occurs to a greater degree for the mixtures than for the single acidic lipid. Thus the presence of DPPC promotes transformation of the acidic lipid to a non-interdigitated bilayer at higher temperatures. At the temperature of the lipid phase transition little or none of the lipid in the mixture is interdigitated. Thus the lipid phase transition detected by calorimetry is not that of the interdigitated bilayer. The shorter chain length DMPC can be incorporated to a greater extent than DPPC, 30-50 mol%, in the interdigitated bilayer of PMB-DPPG. This may be a result of reduced exposure of the terminal methyl groups of the shorter myristoyl chains at the polar/apolar interface of the interdigitated bilayer. Less than 29% of the total lipid was interdigitated in a DPPC/DPPA/PMB 1:1:0.2 mixture indicating that none of the DPPC in this mixture becomes interdigitated. This is attributed to the lateral interlipid hydrogen bonding interactions of DPPA which inhibits formation of an interdigitated bilayer. DPPE was found to be incorporated into the interdigitated bilayer of PMB-DPPG to a similar extent as DPPC if the amount of PMB added is sufficient to bind to only the DPPG in the mixture. Differential scanning calorimetry showed that the remaining non-interdigitated DPPE-enriched mixture phase separates into its own domain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transport of 8-anilino-1-naphthalenesulfonate as a probe of the effect of cholesterol on the phospholipid bilayer structures.

The transport of 8-anilino-1-naphthalenesulfonate in dimyristoyl-L-alpha-lecithin bilayers has been found to be extremely sensitive to the crystalline state of the phospholipid dispersions. Thus this reaction may be used for probing the membrane structures. In binary mixtures of cholesterol and phospholipid the fluorescence enhancement of the dye completely disappears when the mole fraction of cholesterol reaches 33%. At temperatures below and above the phase transition of the lipid bilayers, the rate of the probe transport increases significantly in the binary mixtures. It reaches a maximum at 17 mol % of cholestero. The rate at this cholesterol content approaches the maximum value obtained for the probe transport in pure phospholipis, e.i., the rate at the midpoint of the phase transition. These observations indicate that the effect of cholesterol in the phospholipid dispersion is to maintain the bilayer structure close to the melting temperature of the lipid phase transition. In other words, cholesterol may be an effective buffer for membrane crystalline state when its concentration is near 17 mol %.     

Substrate binding site of microsomal cytochrome P-450 directly faces membrane lipids

Cytochrome P-450, purified from liver microsomes of phenobarbital-treated rabbits, was incorporated into dimyristoylphosphatidylcholine liposomes. The binding of benzphetamine to the liposome-bound cytochrome P-450 was examined by measuring the benzphetamine-induced spectral change at various temperatures. The van't Hoff plot of the apparent spectral dissociation constant showed a distinct break at the temperature of phase transition of the synthetic lipid. On the other hand, no such break was observed for benzphetamine binding to microsomal bound cytochrome P-450. These results suggest that the substrate binding site of cytochrome P-450 is embedded in the apolar interior of phospholipid bilayer membranes.     

The effect of salinity on the phase behaviour of total lipid extracts and binary mixtures of the major phospholipids isolated from a moderately halophilic eubacterium

The effects of molar NaCl concentrations on the phase behaviour of the total lipid extracts and binary mixtures of the major phospholipids, namely phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), isolated from the moderately halophilic eubacterium, Vibrio costicola, grown in 1 M and 3 M NaCl containing media have been studied using X-ray diffraction and freeze-fracture electron microscopy. The effect of both the PE/PG ratio and alterations in fatty acid composition were examined by using binary mixtures which mimicked the PE/PG ratio found in the native bacterial membranes. We show that the samples exhibited complex phase behaviour, including the formation of non-bilayer phases, which depend upon the salinity of both the bacterial culture medium and the suspending solution. The total lipid from bacteria cultured in 1 M NaCl-containing medium and dispersed in 1 M NaCl exhibited a mixture of L alpha and hexagonal-II phases at the optimum growth temperature of the organism (i.e., 30 degrees C), whereas the same lipid dispersed in 3 M NaCl showed only a hexagonal-II phase down to a temperature of +3 degrees C. The total lipid extracted from 3 M NaCl cultures showed only lamellar phases over the temperature range studied (+50 degrees C to -50 degrees C), but the phase transition temperatures of the various lamellar phases were generally higher when the lipid was dispersed in 3 M compared with 1 M NaCl. The phase behaviour of the binary mixtures was similar but not identical to that of the corresponding total lipid extracts and it is suggested that the minor lipid components (diphosphatidylglycerol, lysophosphatidylethanolamine and lysophosphatidylglycerol) play a part in determining the phase behaviour of the native membranes. These results show that the PE/PG ratio and fatty acid composition of the individual phospholipids, which are normally regulated by Vibrio costicola in vivo in response to culture medium salinity, are both important in maintaining a stable bilayer structure within the membrane.     

The effect of microenvironment and formation of intra-molecular links on the tertiary structure of cytochrome P-450 LM2

The effect of intramolecular cross-links formation in isolated cytochrome P-450 LM2 on its reactivation after incorporation into the liposome lipid bilayer was studied. Treatment with bifunctional reagents results in the inactivation of the solubilized haemoprotein. The degree of the enzyme immobilization determines the degree of inhibition of p-nitroanisol demethylation and aniline hydroxylation. Whereas the complete inhibition of oxidation of type II substrate turnover needs two intramolecular cross-links, that of type I substrates necessitates at least seven cross-links. The incorporation of modified and native enzymes into the membrane lipid bilayer at temperatures above the phase transition point results in the enzyme activation. However, in case of the preimmobilized enzyme the activation does not reach the maximal values. Both stabilized and liposome-incorporated cytochrome P-450 can fully be reactivated via the cross-link disulfide bond reduction. No such effect is observed at temperatures below the phase transition point.     

The structure and thermotropic phase behaviour of dipalmitoylphosphatidylcholine codispersed with a branched-chain phosphatidylcholine

The structure and thermotropic phase behaviour of a fully hydrated binary mixture of dipalmitoylphosphatidylcholine and a branched-chain phosphatidylcholine, 1, 2-di(4-dodecyl-palmitoyl)-sn-glycero-3-phosphocholine, were examined using differential scanning calorimetry, synchrotron X-ray diffraction and freeze-fracture electron microscopy. The branched-chain lipid forms a nonlamellar phase when dispersed alone in aqueous medium. Mixed aqueous dispersions of the two phospholipids containing less than 33 mol% of the branched-chain lipid form lamellar phases over the whole temperature range were studied (4 degrees C to 60 degrees C). When present in proportions greater than 33 mol% it induces a hexagonal phase in mixed aqueous dispersions with dipalmitoylphosphatidylcholine at temperatures above the fluid phase transition. At temperatures below 35 degrees C a hexagonal phase coexists with a gel bilayer phase. The lamellar<-->nonlamellar transition can be explained satisfactorily on the basis of the shape of the molecule expressed in terms of headgroup and chain cross-sectional areas. At temperatures below 35 degrees C macroscopic phase separation of two gel phases takes place. Freeze-fracture electron microscopy revealed that one gel phase consists of bilayers with a highly regular, periodic superstructure (macro-ripples) whereas the other phase forms flat, planar bilayers. The macro-ripple phase appears to represent a relaxation structure required to adapt to the packing constraints imposed by the incorporation of the branched-chain lipid into the dipalmitoylphosphatidylcholine host bilayer. The data suggest that structural changes that take place on cooling the mixed dispersion below the lamellar<-->nonlamellar phase transition temperature cannot be adequately described using the molecular form concept. Instead it is necessary to take into account the detailed molecular form of the guest lipid as well as its physical properties.     

Miscibility of phosphatidylcholine binary mixtures in unilamellar vesicles: Phase equilibria

Summary Miscibility among phospholipids with different lipid chain-lengths or with different head groups has attracted a number of research efforts because of its significance in biological membrane structure and function. The general consensus about the miscibility of phosphatidylcholines with varying lipid chainlengths appears to be that binary mixtures of phospholipids with a difference of two carbon atoms in the lipid chain mix well at the main phase transition. Miscibility between phosphatidylcholines with differences of four carbon atoms appears to be inconclusive. Previous reports on the phase transition of binary phospholipid mixtures are concerned mainly with multilamellar vesicles and are mostly limited to the main transition. In the present study, unilamellar vesicles were used and miscibility in binary systems between dimyristoyl-, dipalmitoyl- and distearoyl-phosphatidylcholines at pretransition, as well as main transition temperatures was evaluated by constructing phase diagrams. Two methods were used to monitor the phase transitions: differential scanning microcalorimetry and optical absorbance methods. The optical method has the advantage that unilamellar vesicles of dilute phospholipid concentrations can be used. The liquidus and solidus phase boundaries were determined by the onset temperature of heating and cooling scans, respectively, because the completion temperature of a phase transition has no meaning in binary solutions. Dimyristoyl- and distearoyl-phosphatidylcholines. where the difference in the, lipid chain-length is four carbon atoms, mixed well even at pretransition temperature.     

Galactocerebroside-phospholipid interactions in bilayer membranes.

Differential scanning calorimetry (DSC) and x-ray diffraction have been used to study the interaction of hydrated N-palmitoylgalactosylsphingosine (NPGS) and dipalmitoylphosphatidylcholine (DPPC). For mixtures containing less than 23 mol% NPGS, complete miscibility of NPGS into hydrated DPPC bilayers is observed in both the bilayer gel and liquid-crystal phases. X-ray diffraction data demonstrate insignificant differences in the DPPC-bilayer gel-phase parameters on incorporation of up to 23 mol% NPGS. At greater than 23 mol% NPGS, additional high-temperature transitions occur, indicating phase separation of cerebroside. For these cerebroside concentrations, at 20 degrees C, x-ray diffraction shows two lamellar phases, hydrated DPPC-NPGS gel bilayers (d = 64 A) containing 23 mol% NPGS, and NPGS "crystal" bilayers (d = 55 A). On heating to temperatures greater than 45 degrees C, the mixed DPPC-NPGS bilayer phase undergoes chain melting, and on further increasing the temperature progressively more NPGS is incorporated into the liquid-crystal DPPC-NPGS bilayer phase. At temperatures greater than 82 degrees C (the transition temperature of hydrated NPGS), complete lipid miscibility is observed at all DPPC/NPGS molar ratios.     

Optical biosensor investigation of interactions of biomembrane and water-soluble cytochromes P450 and their redox partners with covalently immobilized phosphatidylethanolamine layers

A phospholipid-containing biochip was created by covalently immobilizing phospholipids on the optical biosensor's aminosilane cuvette and employed to monitor the interactions of the membrane and water-soluble proteins in cytochrome P450-containing monooxygenase systems with planary layers of dilauroylphosphatidylethanolamine (DLPE) and distearoylphosphatidylethanolamine (DSPE), differing in acyl chain length. It was shown that the full-length membrane proteins-cytochrome P4502B4 (d-2B4), cytochrome b5 (d-b5) and NADPH-cytochrome P450 reductase (d-Fp)-readily incorporated into the phospholipids. The incorporation was largely due to hydrophobic interactions of membranous protein fragments with the phospholipid layer. However, electrostatic forces were also but not always involved in the incorporation process. They promoted d-Fp incorporation but had no effect on d-b5 incorporation. In low ionic strength buffer, no incorporation of these two proteins into the DSPE lipid layer was observable. Incorporation of d-b5 into the DLPE layer was abruptly increased at temperatures exceeding phospholipid phase transition point. Incorporation of d-2B4 was dependent on its aggregation state and decreased with increasing protein aggregability. Water-soluble proteins either would not interact with the phospholipid layer (adrenodoxin) or would bind to the layer at the cost of only electrostatic (albumin) or both electrostatic and hydrophobic (P450cam) interactions.     

Identification of membrane-contacting loops of the catalytic domain of cytochrome P450 2C2 by tryptophan fluorescence scanning

The catalytic domain of cytochrome P450 is thought to contact the lipid core of the endoplasmic reticulum membrane based on antibody epitope accessibility, protease susceptibility, and hydrophobic surfaces present on P450 structures of solubilized forms of the proteins. Quenching by nitroxide spin label-modified phospholipids of the fluorescence of tryptophan residues substituted into cytochrome P450 2C2, modified to contain tryptophan only at position 120, was used to identify regions of P450 inserted into the lipid core and to estimate the depth of penetration. Consistent with the proposed models of cytochrome P450-membrane interaction, the fluorescence of tryptophans inserted at residues 36 and 69 in the two segments of P450 2C2 flanking the A-helix and at residue 380 in the beta2-2 strand was quenched by nitroxide spin labels on carbon 5 of the fatty acid tails of the phospholipids within the lipid bilayer. The fluorescence of tryptophan at 380 was also strongly quenched by a spin label on carbon 12 of the fatty acids suggesting it was deepest in the membrane. However, fluorescence of tryptophan substituted at residue 225 in the F-G loop, which was predicted to be in the lipid bilayer, was not quenched by the spin labels at carbons 5 and 12 of the fatty acids. The pattern of quenching of fluorescence for tryptophans at the other positions tested, 80, 189, 239, and 347, was similar to the parent protein indicating they were not inserted into the lipid bilayer as expected. The results are consistent with an orientation of cytochrome P450 2C2 in the membrane in which positions 36, 69, and 380 are inserted into the lipid bilayer and residues 80 and 225 are near or within the phospholipid headgroup region. In this orientation, the F-G loop, which contains residue 225, could form a dimerization interface as was observed in the P450 2C8 crystal structure (Schoch, G. A., et al. (2004) J. Biol. Chem. 279, 9497).     

The partition of cholesterol between ordered and fluid bilayers of phosphatidylcholine: a synchrotron X-ray diffraction study

The structure and composition of coexisting bilayer phases separated in binary mixtures of dipalmitoylphosphatidylcholine and cholesterol and ternary mixtures of equimolar proportions of dipalmitoyl- and dioleoylphosphatidycholines containing different proportions of cholesterol have been characterized by synchrotron X-ray diffraction methods. The liquid-ordered phase is distinguished from gel and fluid phases by a disordering of the hydrocarbon chains intermediate between the two phases as judged from the wide-angle X-ray scattering profiles. Electron density distribution calculated in coexisting bilayer phases shows that liquid-ordered phase is enriched in dipalmitoylphosphatidylcholine and cholesterol and a higher electron density in the methylene chain region of the bilayer ascribed to the location of the sterol ring of cholesterol. The ratio of the two constituents in the liquid-ordered phase is not constant because the stoichiometry is temperature-dependent as seen by respective changes in bilayer thickness over the range 20 degrees to 36 degrees C where coexisting phases are observed. Three coexisting phases were deconvolved in the ternary mixture at 20 degrees C. From an analysis of the ternary mixtures containing mole fractions of cholesterol from 0.09 to 0.15 it was found that the liquid-crystal and gel phases each contained about 10% of the cholesterol molecules and the liquid-ordered phase was comprised of 30% cholesterol molecules.     

Thermotropic behavior of liposomes from egg yolk lecithin, hydrogenized egg yolk lecithin and their mixtures

The thermotropic properties of multilamellar liposomes from egg yolk lecithin, hydrogenized egg yolk lecithin and several mixtures of these two lipids were studied with the application of excimer--forming optical probe pyrene and microcalorimetry. It was discovered that when the proportion of the egg yolk lecithin in the lipid mixture was raised the temperature of the main phase transition reduced. For all this, independent of the lipid mixture composition when the temperature was raised, apparently, polarity of pyrene microenvironment in the liposomes bilayers decreased. On the basis of the analysis of solidus and liquidus curves obtained from calorimetric studies of the lipid mixtures and bend points of Arrhenius anamorphose obtained during the pyrene excimer formation measurements some conclusions were made about the role of unmodified and hydrogenized egg yolk lecithin cluster formation in the determination of thermotropic properties of the liposomes from the above two lipids mixtures. High temperature phase transition discovered for the egg yolk lecithin while measuring the pyrene excimer formation is proposed to be closely connected with temperature-dependent changes in the organization of phospholipid heads on the interphase bilayer/H2O solution.     

2H and 13C nuclear magnetic resonance study of N-palmitoylgalactosylsphingosine (cerebroside)/cholesterol bilayers.

13C- and 2H-NMR experiments were used to examine the phase behavior and dynamic structures of N-palmitoylgalactosylsphingosine (NPGS) (cerebroside) and cholesterol (CHOL) in binary mixtures. 13C spectra of 13C=O-labeled and 2H spectra of [7,7-2H2] chain-labeled NPGS as well as 3 alpha-2H1 CHOL indicate that cerebroside and CHOL are immiscible in binary mixtures at temperatures less than 40 degrees C. In contrast, at 40 degrees C < t < or = T(C) (NPGS), up to 50 mol% CHOL can be incorporated into melted cerebroside bilayers. In addition, 13C and 2H spectra of melted NPGS/CHOL bilayers show a temperature and cholesterol concentration dependence. An analysis of spectra obtained from the melted 13C=O NPGS bilayer phase suggests that the planar NH-C=O group assumes an orientation tilted 40 degrees-55 degrees down from the bilayer interface. The similarity between the orientation of the amide group relative to the bilayer interface in melted bilayers and in the crystal structure of cerebroside suggests that the overall crystallographic conformation of cerebroside is preserved to a large degree in hydrated bilayers. Variation of temperature from 73 degrees to 86 degrees C and CHOL concentration from 0 to 51 mol% results in small changes in this general orientation of the amide group. 2H spectra of chain-labeled NPGS and labeled CHOL in NPGS/CHOL bilayer demonstrate that molecular exchange between the gel and liquid-gel (LG) phases is slow on the 2H time scale, and this facilitates the simulation of the two component 2H spectra of [7,7-2H2]NPGS/CHOL mixtures. Simulation parameters are used to quantitate the fractions of gel and LG cerebroside. The quadrupole splitting of [7,7-2H2]NPGS/CHOL mixtures and 2H simulations allows the LG phase bilayer fraction to be characterized as an equimolar mixture of cerebroside and CHOL.     

Phospholipid interactions with cytochrome P-450 in reconstituted vesicles Preference for negatively-charged phosphatidic acid

Cytochrome $\text{P-450}$ LM2 was reconstituted by the cholate-dialysis method into vesicles containing a mixture of either phosphatidylcholine or phosphatidylethanolamine with up to 50 mol% of phosphatidic acid. Phase transition curves in the presence or absence of cytochrome $\text{P-450}$ were obtained from electron paramagnetic resonance experiments by measuring the partitioning of 2,2,6,6-tetramethylpiperidine-1-oxyl. Protein-free phospholipid vesicles exhibit a phase separation into domains of gel phase enriched in phosphatidic acid in a surrounding fluid matrix containing mainly phosphatidylcholine. The phase transition of the phosphatidic acid domains disappeared following incorporation of cytochrome $\text{P-450}$ into the bilayers. In contrast, in vesicles containing mixtures of egg-phosphatidic acid and dimyristoyl phosphatidylcholine, the phase transition of the domains enriched in dimyristoyl phosphatidylcholine was less sharp than in the corresponding vesicles containing cytochrome $\text{P-450}$. The results of both of these experiments could be explained by a redistribution of the mol fraction of the two phospholipids in the gel phase due to preferential binding of the egg-phosphatidic acid to the cytochrome $\text{P-450}$. For comparison, incorporation of cytochrome $\text{P-450}$ into uncharged vesicles of dimyristoyl phosphatidylcholine and egg-phosphatidylethanolamine did not alter the     

Adenylate cyclase and a fatty acid spin probe detect changes in plasma membrane lipid phase separations induced by dietary manipulation of the cholesterol:phospholipid ratio

Rats fed with a cholesterol supplement to their diet exhibited an increase in their plasma membrane cholesterol phospholipid (C/P)-lipid molar ratio from 0.72 to 0.98, whereas those fed the hypocholesterolaemic drug clofibrate in their diet exhibited a decrease in this ratio to 0.62. The properties of these membranes were analysed with regard to ligand-stimulated adenylate cyclase activity and the mobility of a fatty acid spin probe which allowed lipid phase separations to be identified. Membranes with elevated C/P ratios exhibited two distinct lipid phase separations, one at around 36 degrees C that was attributed to the external half of the bilayer and one at around 22 degrees C which was attributed to the inner half of the bilayer. Membranes with lowered C/P ratios exhibited a single lipid phase separation occurring at around 21 degrees C which was attributed to the lipids of the inner half of the bilayer. These results were compared with those obtained by manipulation of C/P ratios in vitro using liposome-cholesterol exchange techniques. Dietary manipulation of the C/P ratio of plasma membranes in vivo led to alterations in the fold stimulation of adenylate cyclase by various stimulatory ligands.     

Incorporation of amphotericin B into large unilamellar vesicles composed of phosphatidylcholine and phosphatidylglycerol

The spontaneous incorporation of the polyene antibiotic amphotericin B from a micellar solution into phospholipid vesicles was examined as a function of the lipid composition of the vesicles and their physical state. Virtually no insertion of the antibiotic into egg phosphatidylcholine vesicles was observed even when cholesterol was also present in the bilayer. In contrast, rapid incorporation occurred into systems containing an anionic phospholipid such as phosphatidylglycerol or phosphatidylserine with the fastest rates observed for lipids containing the saturated dimyristoyl fatty acyl species. Insertion of amphotericin B into vesicles composed of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol (7:3 mole ratio) was rapid either above, below or within the gel-to-liquid-crystalline phase transition temperature (23 degrees C). The ability of amphotericin B to intercalate into lipid vesicles is discussed in relation to their relative bilayer stabilities.     

Temperature quenching of cytochrome P-450 fluorescence in proteoliposomes with different physical states of the lipid bilayer

Studies were carried out of temperature quenching of self-fluorescence of cytochrome P-450 in solution and liposomes from natural phosphatidylcholine, dimiristoylphosphatidylcholine, dipalmitoylphosphatidylcholine. The fluorescence spectrum of cytochrome P-450 is a superposition of triptophane and tyrosine components. During protein incorporation into liposomes a significant short-wave shift of the emission spectrum takes place. Temperature dependence of the intensity of cytochrome P-450 self-fluorescence in solution has bends at 30, 45 and 50 degrees C. When protein is incorporated into liposomes the location of bends depends on individual properties of lipids forming the bilayer. Effect of lipid surrounding on temperature conformational rearrangements of cytochrome P-450 molecule is discussed.     

Lamellar gel-lamellar liquid crystal phase transition of dipalmitoylphosphatidylcholine multilayers freeze-dried from aqueous trehalose solutions. A real-time X-ray diffraction study

The mechanism of the phase transition of dipalmitoylphosphatidylcholine multilayers freeze-dried from fully hydrated gel phase (L beta') in the presence of trehalose has been investigated by real-time X-ray diffraction methods. Sequential diffraction patterns were recorded with an accumulation time of 3 s during heating and 1.2 s during cooling between about 20 and 80 degrees C. A transition is observed in the range 47-53 degrees C that involves structural events typical of a lamellar gel-lamellar liquid-crystal (L beta--L alpha) transformation. This transition is completely reversible with a temperature hysteresis of 2-3 degrees C and thereby resembles the main phase transition of fully hydrated dipalmitoylphosphatidylcholine multilayers. The mechanism of the transition from L beta to L alpha as seen in the wide-angle scattering profiles show that the sharp peak at about 0.41 nm, characteristic of the gel phase, broadens and shifts progressively to about 0.44 nm towards the end of the transition. A temperature jump of 6C degrees/s through the phase transition region of a freeze-dried dipalmitoylphosphatidylcholine: trehalose mixture (molar ratio 1:1) showed that the phase transition had a relaxation time of about 2 s which is similar to that of the main transition in the fully hydrated lipid. X-ray diffraction studies of the melting of dipalmitoylphosphatidylcholine freeze-dried from the lamellar-gel phase in the absence of trehalose showed a transition at above 70 degrees C. The low-angle diffraction data of phospholipid/trehalose mixtures are consistent with an arrangement of trehalose molecules in a loosely packed 'monolayer' separating bilayers of phospholipid. Trehalose appears to reduce the direct interbilayer hydrogen bond coupling thereby modifying the thermal stability and the phase transition mechanism of the bilayers.