Effect of fluorophore linkage position of n-(9-anthroyloxy) fatty acids on probe distribution between coexisting gel and fluid phospholipid phases

The quenching of probe fluorescence by spin-labeled phospholipid has been used to determine the distribution of a series of n-(9-anthroyloxy) fatty acids between coexisting gel and fluid liquid-crystal phases in multilamellar phospholipid vesicles. The phase distribution ratio in every case is found to favor the fluid lipid phase, but is much greater between fluid and Ca2+-induced gel than between fluid and thermal gel. For a given gel type, n-(9-anthroyloxy)stearic acids with n = 3, 6, 9 or 12 as well as 11-(9-anthroyloxy)undecanoic acid all exhibit similar behavior, favoring the fluid phase by about a factor of 4 over thermally-induced lipid gel phase and by 18 over Ca2+-induced gel phase. 16-(9-Anthroyloxy)palmitic acid, with the bulky probe at the terminus of the 16-carbon chain, favors the fluid phase less strongly, by a factor of 1.5 or 11 over thermally-induced or Ca2+-induced gel phase, respectively, indicating better packing of this probe in phospholipid gel phases.     

Polypeptide clearing in model membranes: an analysis of the partition of gramicidin a' between cadmium ion induced gel and liquid-crystalline phases in vesicles of phosphatidic acid and phosphatidylcholine

Using a simple model for a biological membrane we examine cation-induced gel phase formation and the depletion of polypeptide from the gel phase. The model system consists of vesicles of phosphatidic acid and phosphatidylcholine which contain gramicidin A'. By use of electron spin resonance to monitor lipid phase behavior, Cd2+ is found to induce gel and liquid-crystal phase coexistence over a wide range of lipid composition. Quenching of gramicidin A' tryptophanyl fluorescence by spin-labeled phosphatidic acid or spin-labeled phosphatidylcholine is analyzed to obtain the partition coefficient, Kp, for gramicidin A' between gel and liquid-crystal phases. The value of Kp = 3 favoring the liquid-crystal phase indicates a partial clearing of the membrane-bound polypeptide from Cd2+-induced gel phase regions of the membrane.     

Partitioning of a fluorescent phospholipid between fluid bilayers: dependence on host lipid acyl chains

The partition coefficient Kp was measured for a headgroup-labeled phospholipid (12:0,12:0)-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-PE (12-NBD-PE), equilibrated between LUV of a series of phosphatidylcholines (PC). Fluorescence resonance energy transfer between the 12-NBD-PE and a headgroup-rhodamine-labeled PE was used to find the equilibrium concentration of the 12-NBD-PE in the different LUV. Reliable equilibrium concentrations were obtained by monitoring the approach to equilibrium starting from a concentration below and from a concentration above the ultimate values. Using (16:0,18:1delta9)-PC as the reference lipid, Kp ranged from a high value of 1.65 favoring (16:0,18:1delta9)-PC over (16:1delta9,16:1delta9)-PC, to a low value of 0.90, favoring (22:1delta13,22:1delta13)-PC over (16:0,18:1delta9)-PC. The Kp values enabled calculation of the acyl chain contribution to the excess free energy of mixing for (12:0,12:0) acyl chains at infinite dilution in the L alpha phase of PC having acyl chains of (16:0,18:1delta9), (16:1delta9,16:1delta9), (18:1delta9,18:1delta9), (18:1delta6,18:1delta6), (20:1delta11,20:1delta11), and (22:1delta13,22:1delta13). (14:1delta9,14:1delta9)-PC was found to transfer so rapidly between LUV as to preclude reliable Kp measurement.     

Partitioning of amphiphiles between coexisting ordered and disordered phases in two-phase lipid bilayer membranes

The partition coefficients (K(P)) of a series of single-chain and double-chain fluorescent amphiphiles, between solid ordered (P(beta') and L(beta)) and liquid disordered (L(alpha) of the type l(d)) lipid phases coexisting in the same lipid bilayer, was studied using steady-state fluorescence emission anisotropy. The single-chain amphiphiles were N-(7-nitrobenzoxa-2, 3-diazol-4-yl)-alkylamines, and the double-chain amphiphiles were N-(7-nitrobenzoxa-2, 3-diazol-4-yl)-phosphatidylethanolamines with chain lengths of 12-18 carbon atoms. Saturated 18-carbon alkyl/acyl chain compounds were also compared with Delta(9)-cis unsaturated chains of the same chain length. The fluorescence anisotropy of the probes was examined in lipid bilayers (multilamellar vesicles) prepared from an equimolar mixture of dilauroylphosphatidylcholine and distearoylphosphatidylcholine and studied as a function of temperature through the entire temperature range of coexistence of ordered gel phases and a disordered fluid phase in this system. The unsaturated chain amphiphiles partitioned exclusively into the fluid phase whenever this phase was present, as did the saturated chain amphiphiles with the shortest chains (C(12:0)), while K(P) ranges between 1 and 2, in favor of the L(beta) solid phase, for the amphiphiles with long saturated (C(18:0)) alkyl/acyl chains, with intermediate behavior for the intermediate chain lengths. All probes appeared to be totally excluded from P(beta') solid (gel) phases. The technique was also used to determine partitioning of some of the probes between coexisting liquid ordered (cholesterol-containing) (l(o)) and liquid disordered (l(d)) L(alpha) phases. In this case the ratio of signal amplitude to noise allowed us to obtain a qualitative, but not quantitative, measure of the phase partitioning of the probes. We conclude that the partitioning behavior of the probes examined between coexisting l(o) and l(d) phases is qualitatively similar to that observed between solid ordered and liquid disordered phases.     

Effect of ethanol-induced lipid interdigitation on the membrane solubility of Prodan, Acdan, and Laurdan.

The effect of ethanol-induced lipid interdigitation on the partition coefficient (Kp) of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and its two derivatives, 6-acetyl-2-(dimethylamino)naphthalene (Acdan) and 6-lauroyl-2-(dimethylamino)naphthalene (Laurdan), in L-alpha-dipalmitoylphosphatidylcholine (DPPC) vesicles has been examined by a precipitation method over the ethanol concentration range of 0-1.8 M. At 20 degrees C and in the absence of ethanol, the Kp values for Acdan, Prodan, and Laurdan are 2.0 x 10(3), 2.8 x 10(4), and 4.7 x 10(6), respectively. This result suggests that the Kp of Prodan and its derivatives is not simply a linear function of the polymethylene units. As DPPC undergoes the ethanol-induced phase transition from the noninterdigitated to the fully interdigitated gel state, Kp for Prodan and Acdan decreases by a factor of 5 and 2, respectively, whereas Kp for Laurdan exhibits no detectable changes with ethanol. The differences in Kp are in parallel with the differences in the fluorescence emission spectra of these probes over the ethanol concentration range examined. Previous fluorescence and infrared data indicated that membrane perturbation caused by the probes increases in the order: Laurdan > Prodan > Acdan. Thus, the degree of membrane perturbation also seems to be in parallel with Kp. Among these three probes, Prodan fluorescence reflects most correctly the ethanol-induced lipid interdigitation. In conclusion, the partitioning of small solutes in lipid membranes is significantly reduced by ethanol-induced lipid interdigitation, probably as a result of an increased membrane surface density due to the increased intramolecular lipid acyl chain ordering and a tighter overall intermolecular packing.     

The septate junction limits mobility of lipophilic markers in plasma membranes ofHydra vulgaris (attenuata)

Summary Fluorescent lipophilic probes were used to study the role of septate junctions in maintaining distinct apical and basolateral domains of plasma membranes in epithelial cells of hydra. In short-term experiments, a 16-carbon chain aminofluorescein probe (AFC₁₆) was localized to the apical plasma membranes of ectodermal and endodermal epithelial cells when presented in the culture medium or injected into the gastric lumen, but did not demarcate basolateral membranes. In longer term experiments, basolateral membranes were stained and the staining was independent of temperature conditions. A dual 18-carbon chain indocarbocyanine probe (DiIC₁₈) gradually diffused across the septate junction to label basolateral membranes at room temperature, but not at 4°C. DiIC₁₈ also filled and stained certain mounted nematocytes. The results indicate that in hydra, lipophilic probes may be limited in mobility within the membrane plane by the septate junctions in a manner similar to vertebrate tight junctions, and that apical membranes of mature nematocytes are differentially permeable.     

Nonequilibrium phenomena in the phase separation of a two-component lipid bilayer.

Lipid bilayers composed of two phospholipids with significant acyl-chain mismatch behave as nonideal mixtures. Although many of these systems are well characterized from the equilibrium point of view, studies concerning their nonequilibrium dynamics are still rare. The kinetics of lipid demixing (phase separation) was studied in model membranes (large unilamellar vesicles of 1:1 dilauroylphosphatidylcholine (C(12) acyl chain) and distearoylphosphatidylcholine (C(18) acyl chain)). For this purpose, photophysical techniques (fluorescence intensity, anisotropy, and fluorescence resonance energy transfer) were applied using suitable probes (gel phase probe trans-parinaric acid and fluid phase probe N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dilauroylphosphatidylethanolamine). The nonequilibrium situation was induced by a sudden thermal quench from a one-fluid phase equilibrium situation (higher temperature) to the gel/fluid coexistence range (lower temperature). We verified that the attainment of equilibrium is a very slow process (occurs in a time scale of hours), leading to large domains at infinite time. The nonequilibrium structure stabilization is due essentially to temporarily rigidified C(12) chains in the interface between gel/fluid domains, which decrease the interfacial tension by acting as surfactants. The relaxation process becomes faster with the increase of the temperature drop. In addition, heterogeneity is already present in the supposed homogeneous fluid mixture at the higher temperature.     

Glycosphingolipid acyl chain orientational order in unsaturated phosphatidylcholine bilayers.

The glycosphingolipid, galactosyl ceramide (GalCer), was studied by 2H nuclear magnetic resonance (NMR) in fluid phospholipid bilayer membranes, with regard to arrangement of its acyl chain. For this purpose, species with perdeuterated 18-carbon fatty acid (18:0[d35]GalCer) or with perdeuterated 24-carbon fatty acid (24:0[d47] GalCer) were dispersed in bilayers of the 18-carbon phospholipid, 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC). For 18:0[d35] GalCer, smoothed profiles of the order parameter, SCD, were found to be very similar to one another over the range of glycolipid concentration, 5-40 mol%. In addition, they were very similar to orientational order parameter profiles well known from the literature on phospholipid and glycolipid acyl chains (which deals in general with membranes of homogeneous chain length in the range 14-18 carbons). Corresponding order parameter profiles for the long-chain species, 24:0[d47] GalCer, were also similar to one another for glycolipid concentrations between 5 and 40 mol%. Their shapes, however, were distinctly different from those of the shorter chain analogues. SCD profiles for the two species were quantitatively similar to a membrane depth of C15. SCD values at C16 and C17 were approximately 20 and 30%, respectively, higher for the long-chain glycosphingolipid than for its short-chain analogue in SOPC. Nitroxide spin labels attached rigidly to C16 of the long-chain glycolipid in SOPC gave electron paramagnetic resonance (EPR) order parameters that were twice as high as for a spin label at C16 on the shorter chain glycolipid. Comparison was made between spectra of 24:0[d47] GalCer in SOPC and fully hydrated bilayers of the pure 24:0[d47] GalCer, a system that is considered to be partially interdigitated in fluid and gel phases. The resultant 2H NMR order parameter profiles displayed similar features, indicating that related organizational properties exist in these fluid systems. Effective chain length of 24:0[d47] GalCer within the SOPC membrane was calculated using the method of Schindler and Seelig (1975. Biochemistry, 14:2283-2287). The result suggested that the long-chain fatty acid should protrude roughly one third of the host matrix chain length across the bilayer midplane. However, a treatment of the same order parameters making very few assumptions about chain conformation indicated a high degree of orientational flexibility for the "extra" length of the long chain fatty acid.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular conformations of cerebrosides in bilayers determined by Raman spectroscopy.

Vibrational Raman spectra of the solid and gel phases of bovine brain cerebrosides and the component fractions, kerasin and phrenosin, provide conformational information for these glycosphingolipids in bilayer systems. The carbon-carbon stretching mode profiles (1,150-1,000 cm-1) indicate that at 22 degrees C the alkyl chains assume an almost all-trans arrangement. These spectral data, combined with those from the C-H stretching region (3,050-2,800 cm-1), show that phrenosin forms the most highly ordered polycrystalline solid and kerasin the most ordered gel phase. The conformation of the unsaturated, 24-carbon acyl chains is monitored independently by a skeletal stretching mode at 1,112 cm-1. The alkyl chains in the kerasin and phrenosin gels are sufficiently extended to allow interdigitation of the 24-carbon acyl chains across the midplane of the bilayer. The amide I vibrational mode occurs at a lower frequency in solid phrenosin than kerasin, a shift consistent with stronger hydrogen bounding. This band is broadened and shifted to higher frequencies, however, in the phrenosin gel phase. In both the solid and gel phases natural cerebroside exhibits a composite amide I mode. The disruptive effects on cerebroside chain packing and headgroup orientation arising from mixing with dimyristoyl phosphatidylcholine are examined. Vibrational data for cerebroside are also compared to those for ceramide, sphingosine, and distearoyl phosphatidylcholine structures. Spectral interpretations are discussed in terms of calorimetric and X-ray structural data.     

Electron spin resonance study of phospholipid membranes employing a comprehensive line-shape model

The electron spin resonance spectra of the 1-myristoyl-2-[6-(4,4-dimethyloxazolidine-N-oxyl)myristoyl]-sn-glycero- 3-phosphocholine spin-label in highly oriented, fully hydrated bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine have been studied as a function of temperature and magnetic field orientation. The oriented spectra show clear indications of slow motional components (rotational correlation times greater than 3 ns) even in the fluid phase (T greater than 23 degrees C), indicating that motional narrowing theory is not applicable to the spectral analysis. The spectra have been simulated by a comprehensive line-shape model that incorporates trans-gauche isomerization in addition to restricted anisotropic motion of the lipid long molecular axis and that is valid in all motional regimes. In the gel (L beta') phase the spin-label chains are found to be tilted at 28 degrees with respect to the normal of the orienting plane. In the intermediate (P beta') phase there is a continuous distribution of tilt angles between 0 degrees and 25 degrees. In fluid (L alpha) phase there is no net tilt of the lipid chains. The chains rotate at an intermediate rate about their long axis in the fluid phase (tau R,parallel = 1.4-6.6 ns for T = 50-25 degrees C), but the reorientation of the chain axis is much slower (tau R, perpendicular= 13-61 ns for T = 50-25 degrees C), whereas trans-gauche isomerization (at the C-6 position) is rapid (tau J less than or equal to 0.2 ns). Below the chain melting transition both chain reorientation and chain rotation are at the ESR rigid limit (tau R greater than or equal to 100 ns), and trans-gauche isomerization is in the slow-motion regime (tau J = 3.7-9.5 ns for T = 22-2 degrees C). The chain order parameter increases continuously with decreasing temperature in the fluid phase (SZZ = 0.47-0.61 for T = 50-25 degrees C), increases abruptly on going below the chain melting transition, and then increases continuously in the intermediate phase (SZZ = 0.79-0.85 for T = 22-14 degrees C) to an approximately constant value in the gel phase (SZZ congruent to 0.86 for T = 10-2 degrees C).(ABSTRACT TRUNCATED AT 400 WORDS)     

Conformational free energy of alkylsilanes by nonequilibrium-pulling Monte Carlo simulation

The stationary phase in supercritical fluid chromatography includes alkylsilanes, bearing typically 18-carbon alkane chains, bonded to silica. The silanes are in contact with supercritical carbon dioxide. Interaction of the stationary phase with analytes from the mobile phase depends on conformation of the silanes, whether they form a collapsed layer between the silica and the carbon dioxide or are extended into the carbon dioxide. Although equilibrium conformation of alkylsilanes can be determined by equilibrium Monte Carlo (MC) simulation, that is hampered by slow relaxation of the chains. An alternative is to pull alkylsilanes from collapsed to extended conformations, then calculate free energy change from the Jarzynski equality. This work compares conformational results from equilibrium MC simulation to free energies from nonequilibrium pulling simulations. Because both equilibrium and nonequilibrium simulations are faster for shorter silanes, this work also compares results from 8-carbon and 18-carbon silanes. Free energies from nonequilibrium pulling predict that alkylsilanes tend to bend over and form a layer between silica and carbon dioxide. Results from equilibrium simulations are qualitatively consistent with results from nonequilibrium pulling. Longer-chain silanes have greater tendency to extend slightly into the carbon dioxide.     

The localization of the local anesthetic tetracaine in phospholipid vesicles: A fluorescence quenching and resonance energy transfer study

Fluorescence quenching and resonance energy transfer have been used to determine the localization of the local anesthetic tetracaine in vesicles composed of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) as a function of both temperature and ionic strength. The fluorescence behaviour of tetracaine in vesicles can be attributed to its different partition coefficients in acid and basic solution, in gel phase and fluid phase vesicles, respectively. Using both steady-state and time-resolved fluorescence measurements we show that a saturable binding rather than a partitioning model holds for the interaction of tetracaine with gel phase bilayers. The relative quenching efficiencies of the series of n-AS dyes depend on the phase state of the bilayer and suggest a deeper incorporation of tetracaine in fluid phase than in gel phase membranes. Resonance energy transfer measurements support the view that tetracaine is incorporated predominantly in the region of the 9-AS chromophore in DMPC-bilayers.     

Klebsiella pneumoniae nitrogenase: formation and stability of putative beryllium fluoride-ADP transition state complexes.

Incubation of the MoFe protein (Kp1) and Fe protein (Kp2), the component proteins of Klebsiella pneumoniae nitrogenase, with BeF(3)(-) and MgADP resulted in a progressive inhibition of nitrogenase activity. We have shown that at high Kp2 to Kp1 molar ratios this inhibition is due to the formation of an inactive complex with a stoichiometry corresponding to Kp1.{Kp2.(MgADP.BeFx)2}2. At lower Kp2:Kp1 ratios, an equilibrium between this 2:1 complex, the partially active 1:1 Kp1.Kp2.(MgADP. BeFx)2 complex, and active nitrogenase components was demonstrated. The inhibition was reversible since incubation of the 1:1 complex in the absence of MgADP and beryllium resulted in complete restoration of activity over 30 h. Under pseudo-first-order conditions with regard to nitrogenase components and MgADP, the kinetics of the rate of inhibition with increasing concentrations of BeF(3)(-) showed a square dependence on [BeF(3)(-)], consistent with the binding of two Be atoms by Kp2 in the complex. Analytical fplc gel filtration profiles of Kp1.Kp2 incubation mixtures at equilibrium resolved the 2:1 complex and the 1:1 complex from free Kp1. Deconvolution of the equilibrium profiles gave concentrations of the components allowing constants for their formation of 2.1 x 10(6) and 5.6 x 10(5) M(-1) to be calculated for the 1:1 and 2:1 complexes, respectively. When the active site concentration of the different species was taken into account, values for the two constants were the same, indicating the two binding sites for Kp2 are the same for Kp1 with one or both sites unoccupied. The value for K(1) we obtain from this study is comparable with the value derived from pre-steady-state studies of nitrogenase. Analysis of the elution profile obtained on gel filtration of a 1:1 ratio incubation mixture containing 20 microM nitrogenase components showed 97% of the Kp2 present initially to be complexed. These data provide the first unequivocal demonstration that Fe protein preparations which may contain up to 50% of a species of Fe protein defective in electron transfer is nevertheless fully competent in complex formation with MoFe protein.     

IR spectroscopic study of the structure and phase behavior of long-chain diacylphosphatidylcholines in the gel state.

Fully hydrated dispersions of simple linear saturated diacylphosphatidylcholines with even-numbered acyl chains of lengths from 18 to 24 carbons can exist in a low-temperature, highly ordered, orthorhombic phase (G(o)) that differs from the L beta phase (Gd) normally found for shorter chains. The temperature behavior of these dispersions has been studied by infrared spectroscopy. Chain packing in the G(o) phase was found to be nearly identical to that of the orthorhombic phase of crystalline n-alkanes. With increasing temperature, the G(o) phase undergoes a transition to Gd at approximately 45 degrees C below Tm. This transition occurs at a higher temperature and becomes sharper with increasing chain length. Chain packing in the Gd phase was found to be disordered in a way that can be expressed in terms of a distribution of subcell setting angles. The Gd phase converts to a phase (Gh) with hexagonal-like chain packing at temperatures below Tm. The results support and extend those of a recent x-ray diffraction study of the 24-carbon diacyclphosphatidylcholine gel.     

Fluorescence study of the macrolide pentaene antibiotic filipin in aqueous solution and in a model system of membranes.

The polyene antibiotic filipin (a pentaene) has been studied using photophysical techniques. The polyene self-aggregates in water with a critical micellar concentration of 2 microM. Two approaches were used to evaluate the aggregate dimensions: (a) a lower limit of 10 nm for the aggregate radius was obtained from energy transfer experiments; (b) a formula for rationalizing the turbidity spectrum was derived, and from its application a spherical shape of radius about 50 nm was deduced. The low value for the fluorescence anisotropy of the aggregate (r = 0.02) is compatible with a very loose structure, i.e. the chromophore has very efficient depolarization dynamics that is not controlled by the aggregate size. The Stern-Volmer plot of aggregated filipin fluorescence quenching by iodide is non-linear, presenting a downward curvature. A model was used for the interpretation of these data, along with a study of the quenching in transient state; it was concluded that all the components of the decay are affected by the quencher, i.e. the aggregate has a very open structure with respect to the iodide ion. The partition constants of the polyene, Kp, between a model system of membranes (small unilamellar vesicles of dipalmitoylglycerophosphocholine) and the aqueous phase were determined from anisotropy measurements; the values obtained were Kp (gel phase) = (3.4 +/- 0.8) x 10(3) and Kp (liquid crystal phase) = (7.7 +/- 2.2) x 10(2). The observation that the polyene incorporation is efficient is at variance with the belief that the presence of sterols are essential for the interaction of polyene antibiotics with membranes [for review see Bolard, J. (1986) Biochim. Biophys. Acta 864, 257-304].     

Effects of hydrostatic pressure on bilayer phase behavior and dynamics of dilauroylphosphatidylcholine.

Deuterium nuclear magnetic resonance spectroscopy was used to study the thermotropic phase behavior of dilauroylphosphatidylcholine (DLPC) bilayers at pressures up to 221 MPa. Pressure was found to separate the liquid crystal to gel transition from the gel to ordered crystalline phase transition. The jump in chain order observed on cooling through the transition into the gel phase was found to be small and thus consistent with the trend in longer chain saturated diacyl phosphatidylcholines. On cooling, DLPC was observed to enter an unusual state above the transition into the gel phase. This unusual state displayed fluid-like conformational order but short transverse relaxation times. It was found to be much better pronounced and to span a broader temperature range at elevated pressure than at lower pressures. Transverse relaxation measurements of deuterons on the chain alpha-carbons revealed a substantial slowing of molecular motions within the temperature range of the unusual fluid phase. The observation of such a phase at high pressure appears to be consistent with recent reports of an unusual fluid phase, Lx, in DLPC at ambient pressure.     

Physicochemical properties, binary and ternary phase diagrams of ketoprofen

Compared to simulated moving bed (SMB) chromatography, fractional crystallization is a simple and economical method for enantioseparation. Therefore, the coupling of SMB chromatography and fractional crystallization is suggested for enantioseparation of racemic compounds. In this work, a nonsteroidal antiinflammatory drug, ketoprofen (Kp), was chosen to be studied. Kp was verified as a racemic compound by FTIR, PXRD, and thermodynamic calculations. To derive the condition where pure (S)-Kp could be crystallized from a solution, which was previously enantiomerically enriched, the binary melting phase diagram and the ternary solubility phase diagram in the mixed solvent of ethanol and water over a temperature range of 15-30 degrees C were obtained. From these phase diagrams the eutectic point was determined as 91.6% mole percent (S)-Kp from the binary phase diagram and 91% from the ternary phase diagram. The results may provide valuable experiment data for the possibility of coupling fractional crystallization with SMB for Kp separation.     

The interactions of 1-palmitoyl-2-oleylphosphatidylcholine and bovine brain cerebroside

Model membranes composed of 1-palmitoyl-2-oleylphosphatidylcholine (POPC) and bovine brain galactocerebroside (BOV-CER) have been studied by differential scanning calorimetry (DSC). POPC is a naturally occurring phospholipid, and BOV-CER is a major component of the myelin membrane. POPC and BOV-CER are immiscible in the gel state over the composition range 0-70 mol% BOV-CER. At most POPC/BOV-CER ratios, broad dual-peaked acyl chain transitions are observed, characteristic of the co-existence of a fluid POPC-rich liquid-crystalline phase and a solid BOV-CER-rich gel phase over a wide temperature range.     

Deuterium magnetic resonance study of phase equilibria and membrane thickness in binary phospholipid mixed bilayers.

The gel-fluid phase equilibrium in a two-component system formed from dimyristoylphosphatidylcholine (DMPC) and distearoylphosphatidylcholine (DSPC) was investigated using solid-state wide-line 2H NMR spectroscopy. Analysis of the spectral first moments and the quantitation of gel and fluid phases by means of difference spectroscopy provided the temperature-composition phase diagrams. Phase diagrams were constructed for mixtures of perdeuterated DMPC, DMPC-d54, with DSPC and for the complementary system comprised of DMPC and perdeuterated DSPC, DSPC-d70. The gel-fluid coexistence region was found to extend over a wider range of temperature and composition for the DMPC-d54-DSPC system than for the DMPC-DSPC-d70 system. Comparison of these data with the phase diagram for the DMPC-DSPC system showed that in the gel-fluid region the fraction of lipids in the fluid phase at a given temperature and system composition decreases for the three systems in the order DMPC-d54-DSPC greater than DMPC-DSPC greater than DMPC-DSPC-d70. While the fluid fraction varies by as much as 90% among the three systems, the composition of the fluid phase, i.e., the ratio of the concentrations of the two molecules in the fluid phase, varies by about 20% over the whole temperature and system composition range. The effective acyl chain lengths of the DMPC-d54 and DSPC-d70 molecules as a function of temperature and composition in the fluid phase, when the system is all fluid or is in the gel-fluid coexistence region, were calculated from the quadrupole splittings in the axially symmetric powder patterns obtained for the all-fluid phase. The magnitudes of the coefficient of thermal expansion for both the DMPC-d54 and the DSPC-d70 molecules were smaller in the fluid phase of binary mixtures than in one-component bilayers containing either DSPC-d70 or DMPC-d54 alone. In addition, at any given temperature in the fluid phase, the increase in the acyl chain length of DMPC-d54 with increasing DSPC content of the system was smaller than the concomitant increase in the length of DSPC-d70 in mixtures with DMPC. In the entire temperature and composition range when the binary mixtures are in the all-fluid or in the gel-fluid coexistence region, the largest value obtained for the DMPC-d54 molecule in the fluid phase was smaller than the smallest value obtained for the DSPC-d70 molecule in the fluid phase. The acyl chain lengths were used to calculate the effective weighted-average thickness, d, of the fluid phase bilayer.(ABSTRACT TRUNCATED AT 400 WORDS)     

Long-range interactions between the Fe protein binding sites of the MoFe protein of nitrogenase

We report the properties and reactivity of the catalytically active heterologous nitrogenase formed between the Fe protein from Clostridium pasteurianum (Cp2) and the MoFe protein from Klebsiella pneumoniae (Kp1). Under turnover conditions, in the presence of MgATP, a stable 2:1 (Cp2)2Kp1 electron transfer complex is formed, in which the [4Fe-4S]+ centre of Cp2 is protected from chelation by alpha,alpha'-bipyridyl. However, the two Fe protein-binding sites on Kp1 are not equivalent, since a 1:1 Cp2.Kp1 complex was isolated by gel filtration. The non-equivalence of the Fe protein binding sites was also indicated by the inhibition pattern of Klebsiella nitrogenase by Cp2. The EPR spectrum of the isolated 1:1 Cp2.Kp1 complex showed an S=1/2 signal characteristic of dithionite-reduced Cp2 and signals with g values of 4.27, 3.73, 2.01 and 4.32, 3.63, 2.00 characteristic of the high- and low-pH forms of the FeMoco centre of Kp1, respectively. The unoccupied binding site of Kp1 of the isolated 1:1 Cp2Kp1 complex was shown to be catalytically fully functional in combination with Kp2. In contrast to homologous nitrogenases, which require MgATP for detectable rates of electron transfer from the Fe protein, stopped-flow kinetic studies revealed that electron transfer from Cp2 to Kp1 occurred in the absence of MgATP with a rate constant of 0.065 s(-1). Subsequently, a slower transient decrease and restoration of absorption in the electronic spectrum in the 500-700 nm region was observed. These changes corresponded with those in the intensity of the S=3/2 EPR signal of the FeMoco centres of Kp1 and were consistent with the transient reduction of the FeMoco centre of Kp1 to an EPR-silent form, followed by restoration of the signal at longer reaction times. These changes were not associated with catalysis since no evolution of H2 was detectable.