A deuterium NMR study of labelled n-alkanol anesthetics in a model membrane

The 2H-NMR spectra of 50 wt.% aqueous multilamellar dispersions of dipalmitoylphosphatidylcholine (DPPC) containing either selectively deuterated 1-decanol (25 mol%) or [2H17]-1-octanol (25 mol%) have been measured as a function of temperature. Both alkanols are potent anesthetics. A detailed carbon-deuterium bond order parameter profile of 1-decanol in liquid crystalline phospholipid dispersions at 50 degrees C was determined from the quadrupolar splittings of 1-decanols deuterated at eight different positions. A maximum order parameter SCD = 0.20 was obtained for [5,5-2H2]-1-decanol, with labels at both ends of the 1-decanol exhibiting reduced order parameters. Explanations for the reduced order towards the hydroxyl group of 1-decanol are discussed in terms of either increased amplitudes of motion or geometric effects due to hydrogen bonding. By comparing the order parameter profile of sn-2 chain deuterated phosphatidylcholine dispersions containing 25 mol% 1-decanol (J.L. Thewalt, S.R. Wassall, H. Gorrissen and R.J. Cushley, Biochim. Biophys. Acta, 817 (1985) 355) with the profile of deuterated 1-decanol in DPPC, we estimate that decanol is approximately parallel to the C-3 to C-13 region of the phosphatidylcholine's sn-2 chain. Variation of the spectral moments M1 with temperature indicates that both 1-decanol and 1-octanol are sensitive to the packing of the lipid in which they are dissolved. Below the phase transition temperature, the 2H-NMR spectra of either 1-decanol (selectively deuterated) or 1-octanol (perdeuterated) are broad powder patterns, characteristic of axially symmetric rotation about the alcohol's long axis. This is in contrast to the 2H-NMR spectra obtained from deuterated phosphatidylcholine under similar conditions, which implies that the phospholipid acyl chain conformations are more restricted than those of the alcohol at these temperatures. From the M1 behavior of the various alkanol chain segments with temperature, the gel to liquid crystalline phase transition is seen to initiate in the middle of the DPPC/1-alkanol bilayer.     

Deuterium NMR investigation of ether- and ester-linked phosphatidylcholine bilayers

Deuterium nuclear magnetic resonance (2H NMR) spectra of specifically head-group- and chain-deuterated ester- and ether-linked phosphatidylcholine bilayers were studied as a function of temperature over the range -33 to 50 degrees C. Head-group-deuterated dihexadecylphosphatidylcholine ([alpha-2H2]DHPC) bilayers yield line shapes and spin-lattice relaxation times similar to those observed for its ester-linked counterpart, dipalmitoylphosphatidylcholine ([alpha-2H2]DPPC), in the high-temperature ripple and L alpha bilayer phases. These results indicate the ether linkage has no effect on the dynamics or the orientational order at the alpha-C2H2 segment of the phosphocholine head group. At all temperatures, the 2H NMR spectra of chain-deuterated 1,2[1',1'-2H2]DHPC bilayers exhibit a reduced spectral width compared to 1,2[2',2'-2H2]DPPC bilayers. The most significant feature of the deuterated alkyl chain spectrum of DHPC at 45 degrees C is the observation of four separate quadrupolar splittings from the alpha-methylene segments of the alkyl chains, in comparison to the three quadrupolar splittings reported previously from the alpha-methylene segments of the acyl chains of DPPC. Spin-lattice relaxation experiments performed on DHPC suggest an assignment of the two smaller and the two larger quadrupolar splittings to separate alkyl chains, respectively. Low-temperature (T less than or equal to -20 degrees C) gel-phase spectra of deuterated head-group [alpha-2H2]DHPC remain an order of magnitude narrower than those observed for [alpha-2H2]DPPC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular motion and conformation of cholesteryl esters in reconstituted high density lipoprotein by deuterium magnetic resonance

Reconstituted high density lipoprotein has been prepared by sonication and preparative ultracentrifugation of mixtures containing the apoprotein of high density lipoprotein, egg phosphatidylcholine, cholesteryl oleate, and acyl chain deuterated cholesteryl palmitate in aqueous buffer. The resulting structures have a size and chemical composition very similar to native high density lipoprotein. Deuterium NMR spectra and longitudinal relaxation times were obtained at approximately 25 degrees C. The variation of the 2H NMR line width with chain position is consistent with an average conformation such that the ester acyl chain is extended. In addition, 2H NMR line widths and longitudinal relaxation times indicate that the ester acyl chains possess significant mobility.     

Dynamic structure of the lower density lipoproteins. II. Deuterium NMR studies of the monolayer of very low and low density lipoproteins

The order of phosphatidylcholine (PC) acyl chains in the surface monolayer of very low density lipoproteins (VLDL) and low density lipoproteins (LDL) has been determined from 2H nuclear magnetic resonance order parameters, SCD, using selectively deuterated PC or palmitic acids. From the computer simulated line shapes, we find two distinct phospholipid domains within the amphiphilic monolayer of both VLDL and LDL. In the more ordered domain of LDL, SCD was approximately 0.3 for the "plateau" chain region. The SCD values of VLDL particles are similar to those of LDL for the 5,6- and 11,12-positions, hence we suggest the organization of the more ordered region of VLDL and LDL are similar. The domain of low order in LDL comprises less than 10% of the phospholipid molecules (we do not distinguish between PC and sphingomyelin), having approximately the same order (SCD less than 0.1) as egg PC - sphingomyelin unilamellar vesicles. In VLDL, the domain of low order comprises between approximately 10 and approximately 20% of the phospholipid molecules and the entire acyl chain is in an essentially isotropic environment (SCD less than 0.02). We prepared VLDL-sized microemulsions composed of egg PC, deuterated PC, and triolein to test whether the apoproteins were responsible for creating the two differently organized domains in VLDL and LDL. Surprisingly, these protein-free particles also showed two domains of different order at two temperatures. The high order region, however, is less ordered than in VLDL and LDL. We explain two surface domains of PC in terms of lipid organization and the unique interactions of lipids in the various lipoprotein particles.     

The mitochondrial precursor protein apocytochrome c strongly influences the order of the headgroup and acyl chains of phosphatidylserine dispersions. A 2H and 31P NMR study

Deuterium and phosphorus nuclear magnetic resonance techniques were used to study the interaction of the mitochondrial precursor protein apocytochrome c with headgroup-deuterated (dioleoylphosphatidyl-L-[2-2H1]serine) and acyl chain deuterated (1,2-[11,11-2H2]dioleoylphosphatidylserine) dispersions. Binding of the protein to dioleoylphosphatidylserine liposomes results in phosphorus nuclear magnetic resonance spectra typical of phospholipids undergoing fast axial rotation in extended liquid-crystalline bilayers with a reduced residual chemical shift anisotropy and an increased line width. 2H NMR spectra on headgroup-deuterated dioleoylphosphatidylserine dispersions showed a decrease in quadrupolar splitting and a broadening of the signal on interaction with apocytochrome c. Addition of increasing amounts of apocytochrome c to the acyl chain deuterated dioleoylphosphatidylserine dispersions results in the gradual appearance of a second component in the spectra with a 44% reduced quadrupolar splitting. Such large reduction of the quadrupolar splitting has never been observed for any protein studied yet. The lipid structures corresponding to these two components could be separated by sucrose gradient centrifugation, demonstrating the existence of two macroscopic phases. In mixtures of phosphatidylserine and phosphatidylcholine similar effects are observed. The induction of a new spectral component with a well-defined reduced quadrupolar splitting seems to be confined to the N-terminus since addition of a small hydrophilic amino-terminal peptide (residues 1-38) also induces a second component with a strongly reduced quadrupolar splitting. A chemically synthesized peptide corresponding to amino acid residues 2-17 of the presequence of the mitochondrial protein cytochrome oxidase subunit IV also has a large perturbing effect on the order of the acyl chains, indicating that the observed effects may be a property shared by many mitochondrial precursor proteins. In contrast, binding of the mature protein, cytochrome c, to acyl chain deuterated phosphatidylserine dispersions has no effect on the deuterium and phosphorus nuclear magnetic resonance spectra, thereby demonstrating precursor-specific perturbation of the phospholipid order. The inability of holocytochrome c to perturb the phospholipid order is due to folding of this protein, since unfolding of cytochrome c by heat or urea treatment results in similar effects on dioleoylphosphatidylserine bilayers, as observed for the unfolded precursor. Implications of these data for the import of apocytochrome c into mitochondria will be discussed.     

Deuterium nuclear magnetic resonance study of the interaction of branched chain compounds (phytanic acid, phytol) with a phospholipid model membrane

Deuterium nuclear magnetic resonance (2H-NMR) spectra have been determined for 50 wt% aqueous dispersions of 1-palmitoyl(stearoyl)-2-[2H31]palmitoyl-sn-glycero-3-phosphocho lin e (PC-d31) containing 20 mol% of the isoprenoid compounds phytol or phytanic acid over the temperature range -5-55 degrees C. Concentration effects of the isoprenoid compounds are also reported. First moments (M1) and order parameters were calculated from the spectra. 20 Mol% of either branched chain compound causes an approximate 9% increase in the mean order parameter SCD. Significant effects are seen on the PC-d31 phase behavior. 20 Mol% of either branched chain compound causes the gel to liquid crystalline onset temperature (Ts) to drop to 28 degrees C from 38 degrees C for PC-d31 alone, as seen from the temperature dependent M1 values. The melting range ([Tl--Ts]) is congruent to 1.5 degrees C for PC-d31 and congruent to 11 degrees C for PC-d31 containing 20 mol% of the branched chain compounds. This is in direct contrast to their straight chain analogues, hexadecanol and palmitic acid, which have been shown to elevate the phase transition temperature. The isoprenoid compounds cause significant disruption of the gel phase, forcing nearest neighbor phospholipid chains apart. Transverse relaxation times (T2e, the time constant for decay of the quandrupolar echo) have been determined over the temperature range -5-50 degrees C. Possible explanation for the effect of the isoprenoid compounds on the dynamic structure of phospholipids in the bilayer are proffered.     

Deuterium magnetic resonance of triacylglycerols in phospholipid bilayers

Aqueous dispersions of egg-phosphatidylcholine and egg-phosphatidylcholine/30 mol% cholesterol containing deuterated tripalmitin or triolein were studied at approx. 25°C by ²H-NMR. Incorporation of tripalmitin into egg-phosphatidylcholine bilayers was found to be less than 0.1 mol%, while the incorporation of trolein is approx. 2.5 mol% in the absence and approx. 0.7 mol% in the presence of cholesterol. The profile of order parameter versus chain position for deuterated triolein suggests that the oleoyl chains of the triacylglycerol have an average orientation such that the C2 chain segments and the segments in the vicinity of the C9–C10 double bond are tilted with respect to the bilayer normal, while all other segments are parallel to the bilayer normal. Longitudinal relaxation times were also determined and indicate that the acyl chains of triolein have a motional behaviour similar to that of phospholipid acyl chains in the bilayer.     

Simultaneous observation of order and dynamics at several defined positions in a single acyl chain using 2H NMR of single acyl chain perdeuterated phosphatidylcholines

Deuterium nuclear magnetic resonance (2H NMR) spectra from aqueous dispersions of phosphatidylcholines in which perdeuterated palmitic acid is esterified at the sn-1 position have several very useful features. The powder spectra show six well-resolved 90 degree edges which correspond to the six positions closest to the methyl end of the acyl chain. The spectral overlap inherent in the multiple powder pattern line shape of these dispersions can be removed by using a "dePaking" procedure [Bloom, M., Davis, J.H., & Mackay, A. (1981) Chem. Phys. Lett. 80, 198-202] which calculates the spectra that would result if the lipid bilayers were oriented in the magnetic field. This procedure produces six well-resolved doublets whose NMR properties can be observed without interference from the resonances of other labeled positions. The presence of a single double bond in the sn-2 chain increases the order of the saturated 16:0 sn-1 chain at every position in the bilayer compared with a saturated sn-2 chain at the same reduced temperature. Surprisingly, addition of five more double bonds to the sn-2 chain only slightly reduces the order of the 16:0 sn-1 chain at many positions in the bilayer compared with the single double bond. Calculating oriented spectra from a spin-lattice (T1) relaxation series of powder spectra allows one to obtain the T1 relaxation times of six positions on the acyl chain simultaneously. As an example of the utility of these molecules, we demonstrate that the dependence of the spin-lattice (T1) relaxation rate as a function of orientational order for two unsaturated phospholipids differs significantly from the corresponding fully saturated analogue. Interpreting this difference using current models of acyl chain dynamics suggests that the bilayers containing either of the two unsaturated phospholipids are significantly more deformable than bilayers made from the fully saturated phospholipid.     

Effect of doxorubicin on the order of the acyl chains of anionic and zwitterionic phospholipids in liquid-crystalline mixed model membranes: absence of drug-induced segregation of lipids into extended domains.

We investigated the effect of the antineoplastic drug doxorubicin on the order of the acyl chains in liquid-crystalline mixed bilayers consisting of dioleoylphosphatidylserine (DOPS) or -phosphatidic acid (DOPA), and dioleoylphosphatidylcholine (DOPC) or -phosphatidylethanolamine (DOPE). Previous 2H-NMR studies on bilayers consisting of a single species of di[11,11-2H2]oleoyl-labeled phospholipid showed that doxorubicin does not affect the acyl chain order of pure zwitterionic phospholipid but dramatically decreases the order of anionic phospholipid [de Wolf, F. A., et al. (1991) Biochim. Biophys. Acta 1096, 67-80]. In the present work, we studied mixed bilayers in which alternatively the anionic or the zwitterionic phospholipid component was 2H-labeled so as to monitor its individual acyl chain order. Doxorubicin decreased the order parameter of the mixed anionic and zwitterionic lipids by approximately the same amount and did not induce a clear segregation of the lipid components into extended, separate domains. The drug had a comparable disordering effect on mixed bilayers of unlabeled cardiolipin and 2H-labeled zwitterionic phospholipid, indicating the absence of extensive segregation also in that case. Upon addition of doxorubicin to bilayers consisting of 67 mol% DOPE and 33 mol% anionic phospholipid, a significant part of the lipid adopted the inverted hexagonal (HII) phase at 25 degrees C. This bilayer destabilization, which occurred only in mixtures of anionic phospholipid and sufficient amounts of DOPE, might be of physiological importance. Even upon formation of extended HII-phase domains, lipid segregation was not clearly detectable, since the relative distribution of 2H-labeled anionic phospholipid and [2H]DOPE between the bilayer phase and HII phase was very similar. Our findings argue against a role of extensive anionic/zwitterionic lipid segregation in the mechanism of action and toxicity of doxorubicin.     

Dynamic structure of the lower density lipoproteins. I. Incorporation of high levels of labelled lipids into very low and low density lipoproteins

Selectively labelled lipids have been incorporated into the surface monolayer of human serum low density lipoprotein (LDL) and very low density lipoprotein (VLDL). From 3 to 17 mol% of phosphatidylcholine, selectively deuterated at various positions along the sn-2-acyl chain, was transferred from unilamellar vesicles to VLDL using a partially purified phosphatidylcholine transfer protein. Selectively deuterated palmitic acids were incorporated into LDL (6-20 mol%) and into VLDL (7-10 mol%). Electron microscopy, light scattering, and 31P nuclear magnetic resonance indicated that particle size remained unchanged. Gel exclusion chromatography and chemical analysis showed no difference in hydrodynamic properties and only slight alteration to particle component ratios. Biological activity of labelled VLDL was measured from the rate of cholesterol esterification by cultured J774A.1 cells. Effect of labelling LDL was evaluated by monitoring LDL uptake and degradation by cultured human skin fibroblasts. In all cases the lipoproteins containing labels were indistinguishable from their native counterparts.     

Role of carnitine and carnitine palmitoyltransferase as integral components of the pathway for membrane phospholipid fatty acid turnover in intact human erythrocytes.

The deacylation and reacylation process of phospholipids is the major pathway of turnover and repair in erythrocyte membranes. In this paper, we have investigated the role of carnitine palmitoyltransferase in erythrocyte membrane phospholipid fatty acid turnover. The role of acyl-L-carnitine as a reservoir of activated acyl groups, the buffer function of carnitine, and the importance of the acyl-CoA/free CoA ratio in the reacylation process of erythrocyte membrane phospholipids have also been addressed. In intact erythrocytes, the incorporation of [1-14C]palmitic acid into acyl-L-carnitine, phosphatidylcholine, and phosphatidylethanolamine was linear with time for at least 3 h. The greatest proportion of the radioactivity was found in acyl-L-carnitine. Competition experiments using [1-14C]palmitic and [9,10-3H]oleic acid demonstrated that [9,10-3H]oleic acid was incorporated preferentially into the phospholipids and less into acyl-L-carnitine. When an erythrocyte suspension was incubated with [1-14C]palmitoyl-L-carnitine, radiolabeled palmitate was recovered in the phospholipid fraction, and the carnitine palmitoyltransferase inhibitor, 2-tetradecylglycidic acid, completely abolished the incorporation. ATP depletion decreased incorporation of [1-14C]palmitic and/or [9,10-3H]oleic acid into acyl-L-carnitine, but the incorporation into phosphatidylcholine and phosphatidylethanolamine was unaffected. In contrast, ATP depletion enhanced the incorporation into phosphatidylcholine and phosphatidylethanolamine of the radiolabeled fatty acid from [1-14C]palmitoyl-L-carnitine. These data are suggestive of the existence of an acyl-L-carnitine pool, in equilibrium with the acyl-CoA pool, which serves as a reservoir of activated acyl groups. The carnitine palmitoyltransferase inhibition by 2-tetradecylglycidic acid or palmitoyl-D-carnitine caused a significant reduction of radiolabeled fatty acid incorporation into membrane phospholipids, only when intact erythrocytes were incubated with [9,10-3H]oleic acid. These latter data may be explained by the differences in rates and substrates specificities between acyl-CoA synthetase and the reacylating enzymes for palmitate and oleate, which support the importance of carnitine palmitoyltransferase in modulating the optimal acyl-CoA/free CoA ratio for the physiological expression of the membrane phospholipids fatty acid turnover.     

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)     

Rapid metabolism of fatty acids covalently bound to myelin proteolipid protein

Proteolipid protein (PLP), the major protein of central nervous system myelin, contains approximately 2 mol of covalently bound fatty acids. In this study, the in vivo turnover rate of the acyl chains bound to PLP was determined in 40-day-old rats after a single intracranial injection of [3H]palmitic acid. The apparent half-life of total fatty acids bound to PLP was approximately 7 days. After correction for acyl chain interconversion, the half-life of palmitate bound to PLP was only 3 days. This turnover rate is much more rapid than that of the protein moiety calculated under the same experimental conditions (t1/2 = 1 month). Additional evidence for the dynamic metabolism of acyl groups was provided by experiments in brain tissue slices which showed that acylation of PLP occurs in adult animals as well as during active myelination. Acylation of endogenous PLP in purified myelin and its subfractions was also studied during rat brain development using either [3H]palmitoyl-CoA or [3H]palmitic acid plus ATP and CoA. Labeling of endogenous PLP with [3H]palmitoyl-CoA was observed as early as 10 days postnatal and continued at the same rate throughout development. When [3H]palmitic acid was used as precursor in the presence of both ATP and CoA, esterification of myelin PLP occurred rapidly in adult animals, indicating that both nonacylated PLP and acyl-CoA ligase are present in myelin. Finally, pulse-chase experiments in a cell-free system showed that PLP-bound fatty acids turn over with a half-life shorter than 10 min. These observations are consistent with the concept that acylation of myelin PLP is a dynamic process involved mainly in myelin maintenance and function.     

Octyl-beta-D-glucopyranoside partitioning into lipid bilayers: thermodynamics of binding and structural changes of the bilayer.

The interaction of the nonionic detergent octyl-beta-D-glucopyranoside (OG) with lipid bilayers was studied with high-sensitivity isothermal titration calorimetry (ITC) and solid-state 2H-NMR spectroscopy. The transfer of OG from the aqueous phase to lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) can be investigated by employing detergent at concentrations below the critical micellar concentration; it can be defined by a surface partition equilibrium with a partition coefficient of K = 120 +/- 10 M-1, a molar binding enthalpy of delta H degrees D = 1.3 +/- 0.15 kcal/mol, and a free energy of binding of delta G degrees D = -5.2 kcal/mol. The heat of transfer is temperature dependent, with a molar heat capacity of delta CP = -75 cal K-1 mol-1. The large heat capacity and the near-zero delta H are typical for a hydrophobic binding equilibrium. The partition constant K decreased to approximately 100 M-1 for POPC membranes mixed with either negatively charged lipids or cholesterol, but was independent of membrane curvature. In contrast, a much larger variation was observed in the partition enthalpy. delta H degrees D increased by about 50% for large vesicles and by 75% for membranes containing 50 mol% cholesterol. Structural changes in the lipid bilayer were investigated with solid-state 2H-NMR. POPC was selectively deuterated at the headgroup segments and at different positions of the fatty acyl chains, and the measurement of the quadrupolar splittings provided information on the conformation and the order of the bilayer membrane. Addition of OG had almost no influence on the lipid headgroup region, even at concentrations close to bilayer disruption. In contrast, the fluctuations of fatty acyl chain segments located in the inner part of the bilayer increased strongly with increasing OG concentration. The 2H-NMR results demonstrate that the headgroup region is the most stable structural element of the lipid membrane, remaining intact until the disordering of the chains reaches a critical limit. The perturbing effect of OG is thus different from that of another nonionic detergent, octaethyleneglycol mono-n-dodecylether (C12E8), which produces a general disordering at all levels of the lipid bilayer. The OG-POPC interaction was also investigated with POPC monolayers, using a Langmuir trough. In the absence of lipid, the measurement of the Gibbs adsorption isotherm for pure OG solutions yielded an OG surface area of AS = 51 +/- 3 A2. On the other hand, the insertion area AI of OG in a POPC monolayer was determined by a monolayer expansion technique as AI = 58 +/- 10 A2. The similar area requirements with AS approximately AI indicate an almost complete insertion of OG into the lipid monolayer. The OG partition constant for a POPC monolayer at 32 mN/m was Kp approximately 320 M-1 and thus was larger than that for a POPC bilayer.     

Physicochemical characterization of 1,2-diphytanoyl-sn-glycero-3-phosphocholine in model membrane systems.

We report here on a series of studies aimed at characterization of the structural and dynamical properties of the synthetic lipid diphytanoyl phosphatidylcholine, in multilamellar dispersions and vesicle suspensions. The lipid exhibits no detectable gel to liquid crystalline phase transition over a large temperature range (-120 degrees C to +120 degrees C). Examination of proton nuclear magnetic resonance (NMR) free induction decays obtained from multilayer dispersions of diphytanoyl phosphatidylcholine provided an estimate of the methylene proton order parameter. The estimated magnitude of 0.21 is comparable to those determined for other phospholipids. Sonication of aqueous dispersions of diphytanoyl phosphatidylcholine led to formation of bilayer vesicles as determined by the measurement of the outer/inner choline methyl proton resonances, vesicle sizes in electron micrographs, and comparison of proton NMR linewidths between multilayer and sonicated dispersions. Ultracentrifugation studies of diphytanoyl phosphatidylcholine vesicles in H2O and 2H2O media yielded a value of 1.013 +/- 0.026 ml/g for the partial specific volume of this lipid. We have measured spin lattice relaxation rates for the methyl and methylenemethyne protons of the hydrocarbon chains of diphytanoyl phosphatidylcholine in bilayer vesicles over a range of temperatures and at two NMR frequencies (100 and 220 MHz). The observed relaxation rates for the methylene protons in this system were approximately twice those previously reported for dipalmitoyl phosphatidylcholine at comparable temperatures and resonance frequencies, whereas the relaxation rates measured for the methyl protons were greater than those of the straight chain lipid by an order of magnitude. Measurement of the spin lattice relaxation rates of the hydrocarbon protons of the diphytanoyl phosphatidylcholine in a 10 mol% mixture of the branched-chain lipid in a deuterated host lipid, diperdeuteropalmitoyl phosphatidylcholine, showed a discontinuity in the temperature dependence of the proton NMR longitudinal relaxation rates of the branched-chain lipid in the region of the gel to liquid crystalline phase transition temperature of the deuterated dipalmitoyl phosphatidylcholine host lipid. This result may be taken as evidence of lateral phase separation of a liquid cyrstalline phase enriched in diphytanoyl phosphatidylcholine from a gel phase enriched in diperdeuteropalmitoyl phosphatidylcholine at temperatures below the phase transition temperature of deuterated host lipid. This conclusion is supported by the observation of an abrupt change in the hydrocarbon methylene linewidth (at 100 MHz) of 10 mol% diphytanoyl phosphatidylcholine in diperdeuteropalmitoyl phosphatidylcholine over the temperature range where lateral phase separation is taking place according to differential thermograms.     

Deuterium nuclear magnetic resonance study of the interaction of substrates and inhibitors with soybean lipoxygenase

Deuterium NMR spectra for a series of selectively deuterated substrates and inhibitors in the presence of lipoxygenase-1 (EC 1.13.11.12) are presented. Extrapolation of the 2H NMR line widths yielded transverse relaxation rates for the bound inhibitors [2H21]dodecanoic acid (protonated at the 2,2-position), [2,2-2H]dodecanoic acid, and [12,12,12-2H]dodecanoic acid which are 1/T2,bd = 5.0 X 10(3), 1.12 X 10(4), and 1.16 X 10(3) s-1, respectively. The substrates [9,10,12,13-2H]linoleic acid and [11,11-2H]linoleic acid had 1/T2,bd = 8.2 X 10(3) and 7.95 X 10(3) s-1, respectively. Kinetic measurements established Ki = 1.5 X 10(-3) M for dodecanoic acid (lauric acid) inhibition of lipoxygenase when the substrate is linoleic acid (Km = 2.6 X 10(-5) M). Lipoxygenase, with Mr 102,000, is predicted to have a rotational correlation time tau c - 1.2 X 10(-7) s, yielding a 1/T2,bd = 1.56 X 10(4) s-1 for tightly bound ligand. Hence, the correlation times of the selectively deuterated inhibitors indicate internal motions are present in the bound species.     

The membrane interaction of amphiphilic model peptides affects phosphatidylserine headgroup and acyl chain order and dynamics. Application of the "phospholipid headgroup electrometer" concept to phosphatidylserine

Deuterium nuclear magnetic resonance (2H NMR) was used to study the interaction of amphiphilic model peptides with model membranes consisting of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine deuterated either at the beta-position of the serine moiety ([2-2H]DOPS) or at the 11-position of the acyl chains ([11,11-2H2]DOPS). The peptides are derived from the sequences H-Ala-Met-Leu-Trp-Ala-OH (AX, one-letter code with X = MLWA) and H-Arg-Met-Leu-Trp-Ala-OH (RX+) and contain a positive charge of +1 (AXme+) or +2 (RXme2+) at the amino terminus or one positive charge at each end of the molecule (AXetN2+). Upon titration of dispersions of DOPS with the peptides, the divalent peptides show a similar extent of binding to the DOPS bilayers, which is larger than that of the single charged peptide. Under these conditions the values of the quadrupolar splitting (delta vq) of both [2-2H]DOPS and [11,11-2H2]DOPS are decreased, indicating that the peptides reduce the order of both the DOPS headgroup and the acyl chains. The extent of the decrease depends on the amount of peptide bound and on the position of the charged moieties in the peptide molecule. The effects exerted by the peptides on the delta vq value of [2-2H]DOPS are consistent with the PS headgroup responding as a molecular electrometer to the surface charge resulting from the presence of the peptides in the lipid-water interface. The effects on the acyl chain deuterons are in agreement with a localization of the peptides intercalated in between the lipid headgrouops.(ABSTRACT TRUNCATED AT 250 WORDS)     

The pH dependence of headgroup and acyl chain structure and dynamics of phosphatidylserine, studied by 2H-NMR

By varying the pH, the influence of the ionization degree on the structure and dynamics of aqueous dispersions of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) was studied, using 2H-NMR methods. For this purpose DOPS was synthesized with deuterium labels incorporated either stereospecifically at the beta-position of the serine headgroup ([2-2H]DOPS) or at the 11-position of both acyl chains ([11,11-2H2]DOPS), allowing the effects of pH on headgroup and acyl chains to be measured in parallel. A large scale synthesis procedure of stereospecific 1,2-dioleoyl-sn-glycero-3-phospho-[2-2H]-L- serine is described. The quadrupolar splitting (delta nu q) of [2-2H]DOPS is shown to be a sensitive sensor for the degree of protonation of the molecule. Whereas the delta nu q of [2-2H]DOPS decreases upon lowering the pH, that of [11,11-2H2]DOPS gradually increases, indicating an increase in acyl chain ordering. In the pH range below the pKa value, DOPS exhibits a temperature-dependent bilayer to hexagonal HII phase transition, apparent from the 31P-NMR spectra and the occurrence of a second component in the [11,11-2H2]DOPS 2H-NMR spectrum, with a much smaller delta nu q. The HII phase component in spectra from [2-2H]DOPS coincides with the isotropic position and has no defined delta nu q. In the bilayer organization delta nu q and spin-lattice relaxation time (T1) values for the acyl chain deuterated DOPS are similar to those obtained for other lipid systems. In contrast the PS headgroup region displays a relatively rigid structure as evidenced by a large delta nu q and very small T1 values. Upon adopting the HII phase the T1 values of the acyl chain deuterons are hardly affected. The uniqueness of the PS headgroup with respect to structure and motional properties is reinforced by the occurrence of a T1 minimum at 45 degrees C in the measurement of the temperature dependence of T1 for [2-2H]DOPS in the hexagonal HII configuration. Quantitative analysis yields a correlation time (tau c) for the motions determining T1 under these conditions, of 3.45 ns.     

Deuterium NMR study of the effect of n-alkanol anesthetics on a model membrane system

The effects of 25 mol% incorporation of two anesthetics, 1-octanol and 1-decanol, on a deuterated, saturated phospholipid in 50 wt% aqueous multilamellar dispersions have been studied by 2H-NMR spectroscopy and differential scanning calorimetry (DSC). The phospholipid used is sn-2 substituted '[2H31]-palmitoylphosphatidylcholine' (PC-d31). DSC thermograms demonstrate that PC-d31 has phase behavior qualitatively similar to that of dipalmitoylphosphatidylcholine, with a pretransition at 31 degrees C and a main gel to liquid crystalline transition at 40 degrees C. Analysis of the temperature-dependent 2H-NMR spectra in terms of the first moment, which is extremely sensitive to the phospholipid phase, shows that 1-octanol and 1-decanol depress and broaden the main transition. This is confirmed by DSC, which shows that the pretransition is eliminated by the 1-alkanols. The carbon-deuterium bond order of the phospholipid deuterated acyl chains, in the presence and absence of 1-alkanols, was determined from deuterium quadrupolar splittings. Spectra were analyzed using the depaking technique. A 1-alkanol concentration of 25 mol% had no significant effect on the profile of the carbon-deuterium bond order parameter SCD along the phospholipid acyl chain at 50 degrees C. Thus, it appears that the liquid crystalline phase is able to accommodate large amounts of linear anesthetic molecules without substantial effect on molecular ordering within the membrane bilayer. Preliminary results show that the transverse relaxation rates of the acyl chain segments are significantly decreased by the presence of 1-octanol or 1-decanol.     

Combined influence of cholesterol and synthetic amphiphillic peptides upon bilayer thickness in model membranes.

Deuterium (2H) NMR was used to study bilayer hydrophobic thickness and mechanical properties when cholesterol and/or synthetic amphiphillic polypeptides were added to deuterated POPC lipid bilayer membranes in the liquid-crystalline (fluid) phase. Smoothed acyl chain orientational order profiles were used to calculate bilayer hydrophobic thickness. Addition of 30 mol% cholesterol to POPC at 25 degrees C increased the bilayer thickness from 2.58 to 2.99 nm. The peptides were chosen to span the bilayers with more or less mismatch between the hydrophobic peptide length and membrane hydrophobic thickness. The average thickness of the pure lipid bilayers was significantly perturbed upon addition of peptide only in cases of large mismatch, being increased (decreased) when the peptide hydrophobic length was greater (less) than that of the pure bilayer, consistent with the "mattress" model of protein lipid interactions (Mouritsen, O.G., and M. Bloom. 1984. Biophys. J. 46:141-153). The experimental results were also used to examine the combined influence of the polypeptides and cholesterol on the orientational order profile and thickness expansivity of the membranes. A detailed model for the spatial distribution of POPC and cholesterol molecules in the bilayers was proposed to reconcile the general features of these measurements with micromechanical measurements of area expansivity in closely related systems. Experiments to test the model were proposed.