The structure and morphology of the abnormal serum lipoprotein-X

The structure and morphology of an abnormal lipoprotein particle present in the serum of patients with obstructive jaundice has been investigated by gel filtration, electron microscopy and NMR spectroscopy. Lipoprotein-X is a spherical lipoprotein particle with an average Stokes diameter of approximately 40 nm and a wide size distribution ranging from 20 to 70 nm. Different from all lipoprotein structures known so far lipoprotein-X is a hollow particle (= vesicle) with a water-filled internal cavity surrounded by a continuous, single bilayer which is impermeable to cations and K3Fe(CN)6. The packing of the bilayer is tighter and the segmental motion of both the polar group and the hydrocarbon chains are significantly reduced relative to typical phosphatidylcholine bilayers. In terms of segmental motion and anisotropy of packing the lipoprotein-X bilayer closely resembles a model bilayer system consisting of phosphatidylcholine, lysophosphatidylcholine, sphingomyelin and cholesterol mixed in the same molar ratio as in lipoprotein-X. In that model system the phospholipid distribution between the two layers of the bilayer is asymmetric with (sphingomyelin + lysophosphatidylcholine) being preferentially located on the inner layer and phosphatidylcholine preferentially on the outer layer of the bilayer. By analogy with the model system the phospholipid distribution in lipoprotein-X bilayers is proposed to be also asymmetric.     

Secretory immunoglobulins in colonic neoplasms

1. The phosphonium analogues of choline, phosphorylcholine, CDPcholine and phosphatidylcholine were synthesized chemically and characterized by 1H-NMR and 31P-NMR; in 1,2-distearoyl-DL-glycero-3-phosphorylphosphocholine, the 31P-NMR chemical shift of phosphonium relative to phosphate was--28.2 ppm. 2. A comparison was made of the rates of reaction of choline kinase, cholinephosphate cytidyltransferase, cholinephosphotransferase and phospholipase C on natural and phosphonium substrates. Enzyme reaction rates were similar for all but the cytidyltransferase, which exhibited a 3-fold preference for the normal substrate. 3. Weanling rats were maintained for 6 weeks on a diet in which choline was fully replaced by phospho[1,2-14C2]choline mixed with a trace of [Me-3H] choline. Incorporation of phosphocholine into liver lipids was detectable by 31P-NMR even in crude tissue homogenates. Choline-based phospholipids of liver, kidney, lung and brain were extracted, and phosphocholine incorporation calculated from 31P-NMR peak area ratios. The phosphatidylcholine analogues were separated by preparative thin-layer chromatography. Incorporation of phosphocholine ranged from 33% in lung phosphatidylcholine to 6% in kidney sphingomyelin. Variations in 14C/3H ratio between feed and phospholipid extracts indicated preferences for exogenous choline over phosphocholine varying from 1.3: 1 in brain to 3.2: 1 in liver. The results indicated that phosphocholine is a potentially useful 31P-NMR probe for the study of membrane lipids.     

In vitro evidence that phospholipid secretion into bile may be coordinated intracellularly by the combined actions of bile salts and the specific phosphatidylcholine transfer protein of liver

Using model systems, we explored a potential function of hepatic phosphatidylcholine transfer protein to extract biliary-type phosphatidylcholines from intracellular membranes (e.g., smooth endoplasmic reticulum) and deliver them to canalicular plasma membranes where biliary secretion occurs. We measured transfer rates of parinaroyl phosphatidylcholine, a naturally fluorescent phospholipid, from small unilamellar vesicles composed of sn-1 palmitoyl, sn-2 parinaroyl phosphatidylcholine, and egg yolk phosphatidylcholine (molar ratio 75:25) wherein the fluorophore is self-quenched to small unilamellar vesicles composed of phosphatidylethanolamine, sphingomyelin, phosphatidylserine, phosphatidylinositol, and cholesterol (molar ratios 22:22:10:8:38) representing model microsomal and canalicular plasma membranes, respectively. Following addition of phosphatidylcholine transfer protein (purified from bovine liver), fluorescence intensity increased exponentially indicating net phosphatidylcholine transfer from donor to acceptor vesicles. Submicellar concentrations of a wide hydrophobicity range of common and uncommon taurine and glycine conjugated bile salts species (anionic steroid detergent-like molecules), sodium taurofusidate (a conjugated fungal bile salt analog), and sodium dodecyl sulfate and octylglucoside, anionic and nonionic straight chain detergents, respectively, markedly stimulated phosphatidylcholine transfer protein activity. This 40-115-fold effect was most pronounced for the common bile salts and correlated positively with bile salt hydrophobicity. Thermodynamic analysis of net transfer revealed that the rate-limiting step was extraction of phosphatidylcholine molecules from donor vesicles and that bile salts facilitated their capture by enhancing both phosphatidylcholine transfer protein binding as well as perturbing phospholipid packing in vesicle bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

The impact of treatment time and smoking on local control and complications in T1 glottic cancer

Low frequency impedance measurements of pure egg lecithin (phosphatidylcholine) bilayers have revealed the presence of four layers which can be attributed to the acyl chain, carbonyl, glycerol bridge and phosphatidylcholine regions of the lecithin molecule. Measurements on bilayers formed in the presence of unoxidised-cholesterol revealed that cholesterol molecules were located in the hydrocarbon region of the bilayer with its hydroxyl groups aligned with the carbonyl region of the lecithin molecules. Measurements of oxidised-cholesterol lecithin bilayers revealed that these molecules protruded less into the hydrocarbon region and their polar hydroxyl group aligned with the glycerol bridge region of the lecithin molecule.     

Protein mediated magnetic coupling between lactate and water protons

The magnetic coupling between methyl lactate protons and water protons in samples of cross-linked bovine serum albumin (BSA) is studied. Cross-relaxation spectroscopy shows efficient magnetization transfer from immobilized BSA to both water and methyl lactate protons. Transient and steady-state NOE experiments reveal a negative intermolecular NOE between methyl lactate and water protons. Lactate is indirectly detected by selectively saturating the methyl lactate protons and measuring the decrease in water proton magnetization. Indirect detection of methyl lactate protons is an order of magnitude more sensitive than direct detection in these model systems. Lactate was indirectly imaged, via the water proton resonance, with 1.1-microliter voxels in 2 min. Immobilized BSA reduces the intermolecular correlation time between water and lactate protons into the spin-diffusion limit where the NOE is negative. Possible molecular mechanisms for this coupling and applications to in vivo spectroscopy are discussed.     

Separation and characterization of the unknown phospholipid in human lens membranes

PURPOSE: The major component of human lens membranes was thought to be sphingomyelin until 1991, when a study by phosphorus-31 (31P) nuclear magnetic resonance (NMR) spectroscopy revealed the presence of an unknown phospholipid that constituted approximately half the human lens phospholipids. The objective of this work was to isolate this phospholipid and to elucidate its identity. METHODS: The separation of sphingomyelin from the unknown was accomplished using high-performance liquid chromatography (HPLC) and an amino-bound column. Sphingomyelin standard and the membranes from human lenses were chromatographed. Chromatographic fractions were collected and spectrally characterized by proton (1H) NMR and 31P NMR spectroscopy. RESULTS: The chromatographic method did not affect the integrity of the sphingomyelin. Besides the bands corresponding to the unknown components, the chromatogram of the human lens membranes showed three large peaks, the central one with a shoulder, with elution times similar to that for sphingomyelin. The 1H NMR spectra for the fractions collected during the elution of these peaks showed differences. The study by 31P NMR indicated that the first peak contained the unknown phospholipid. The subsequent fractions showed the presence, in different relative levels, of both the unknown and sphingomyelin. By comparison and interpretation of the two-dimensional 1H NMR spectra for sphingomyelin and for the fraction containing the unknown, the unknown phospholipid is proposed to be 4,5 dihydrosphingomyelin, in which the site of unsaturation present in the sphingosine moiety is no longer present. CONCLUSIONS: The ability to separate the unknown from sphingomyelin and the power of 1H NMR spectroscopy allowed the proposition of the identity of the major component of human lens membranes as 4,5-dihydrosphingomyelin. Although the synthetic compound is known to be involved in the formation of extended hydrogen-bonding networks, its biologic and physicochemical properties need further study.     

The interaction of 1-anilino-8-naphthalenesulfonate with lipids: a nuclear magnetic resonance study

The proton magnetic resonance (PMR) and phosphorus magnetic resonance (PhMR) spectra of egg phosphatidylcholine in the presence of 1-anilino-8-nahthalenesulfonate (ANS) have been studied. At low ratios of ANS to phospholipid, the spectra indicate that ANS molecules are in the lipid interface region where they interact with the head-group protons. ANS also penetrates into the hydrocarbon region to some extent. As the ANS/phospholipid ratio approaches one, a significant splitting of the head-group signal occurs. This splitting is associated with head-group signals from inner and outer molecules of the phospholipid vesicles. As the ANS/phospholipid ratio is further increased, a gel phase often occurs. The spectra for this gel phase suggest a highly mobile head-group. Further ANS addition results in a PMR spectrum suggestive of ANS-phospholipid micelle formation. The results for a phospholipid-cholesterol complex and for the total lipid extract from a cell membrane show that the ANS effect is more complicated in these cases.     

Lipid composition of platelets in patients suffering from migraine without aura

Patients with migraine have a platelet hyperaggregability. As this alteration could be the consequence of an abnormal lipid composition of platelet membranes, we studied the phospholipid specimens and the cholesterol/phospholipid ratio in platelet of neuron patients suffering from migraine. The cholesterol/phospholipid ratio was 0.7 +/- 0.1 (normal 0.6 +/- 0.1, molar ratio). The proportion of five main platelet phospholipids components including phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phosphatidylserine, and phosphatidylinositol, were also normal. These data suggest that platelet hyperactivity in patients with migraine is not due to an altered lipid content of those cells.     

Membrane structure and dynamics as viewed by solid-state NMR spectroscopy

The purpose of the present study is the investigation of the structure and dynamics of biological membranes using solid-state nuclear magnetic resonance (NMR) spectroscopy. Two approaches are used in our laboratory. The first involves the measurement of high-resolution 13C and 1H spectra obtained by the magic angle spinning (MAS) technique while the second approach involves the measurement of 31P and 2H powder spectra in static samples. This paper will present some recent results obtained by high-resolution solid-state 1H NMR on the conformation of gramicidin A incorporated in a phosphatidylcholine bilayers. More specifically, we were able to observe changes in the gramicidin spectra as a function of the cosolubilization solvent initially used to prepare the samples. The interaction between lipid bilayers and an anticancer drug derived from chloroethylurea was also investigated using proton NMR spectroscopy. Finally, we have studied the interaction between cardiotoxin, a toxic protein extracted from snake venom, and negatively charged lipid bilayers using 31P solid-state NMR spectroscopy.     

Effects of pH and cholesterol on DMPA membranes: a solid state 2H- and 31P-NMR study

The effect of pH and cholesterol on the dimyristoylphosphatidic acid (DMPA) model membrane system has been investigated by solid state 2H- and 31P-NMR. It has been shown that each of the three protonation states of the DMPA molecule corresponds to a 31P-NMR powder pattern with characteristic delta sigma values; this implies additionally that the proton exchange on the membrane surface is slow on the NMR time scale (millisecond range). Under these conditions, the 2H-labeled lipid chains sense only one magnetic environment, indicating that the three spectra detected by 31P-NMR are related to charge-dependent local dynamics or orientations of the phosphate headgroup or both. Chain ordering in the fluid phase is also found to depend weakly on the charge at the interface. In addition, it has also been found that the first pK of the DMPA membrane is modified by changes in the lipid lateral packing (gel or fluid phases or in the presence of cholesterol) in contrast to the second pK. The incorporation of 30 mol% cholesterol affects the phosphatidic acid bilayer in a way similar to what has been reported for phosphatidylcholine/cholesterol membranes, but to an extent comparable to 10-20 mol % sterol in phosphatidylcholines. However, the orientation and molecular order parameter of cholesterol in DMPA are similar to those found in dimyristoylphosphatidylcholine.     

Current problems of neutron radiobiology

The molecular organization, interactions, phase state and membrane-membrane interactions of model membranes containing cerebroside (GalCer), sulfatide (Sulf) and myelin basic protein (MBP) were investigated. Sulf shows a larger cross-sectional area than GalCer, in keeping with the lateral electrostatic repulsions in the negatively charged polar head group. The interactions of GalCer with different phospholipids are similar while those with Sulf depend on the phosphoryl choline moiety in the phospholipid. MBP induces a decrease of the phase transition temperature in both lipids but with Sulf this occurs at lower proportions of MBP. In mixtures of Sulf with phosphatidylcholine MBP induces phase separation among Sulf-rich and PC-rich domains. Extensive apposition of bilayers containing Sulf is induced by MBP while GalCer interferes with this process. Few membrane interactions proceed to bilayer merging or whole bilayer fusion and the glycosphingolipids help preserve the membrane integrity.     

Depth profiles of pulmonary surfactant protein B in phosphatidylcholine bilayers, studied by fluorescence and electron spin resonance spectroscopy

Pulmonary surfactant-associated protein B (SP-B) has been isolated from porcine lungs and reconstituted in bilayers of dipalmitoylphosphatidylcholine (DPPC) or egg yolk phosphatidylcholine (PC) to characterize the extent of insertion of the protein into phospholipid bilayers. The parameters for the interaction of SP-B with DPPC or PC using different reconstitution protocols have been estimated from the changes induced in the fluorescence emission spectrum of the single protein tryptophan. All the different reconstituted SP-B-phospholipid preparations studied had similar Kd values for the binding of the protein to the lipids, on the order of a few micromolar. The depth of penetration of SP-B into phospholipid bilayers has been estimated by the parallax method, which compares the relative efficiencies of quenching of the protein fluorescence by a shallow or a deeper spin-labeled phospholipid probe. SP-B tryptophan was found to be located 10-13 A from the center of bilayers, which is consistent with a superficial location of SP-B in phosphatidylcholine membranes. Parallax experiments, as well as resonance energy transfer from SP-B tryptophan to an acceptor probe located in the center of the bilayer, indicate that there are significant differences in the extent of insertion of the protein, depending on the method of reconstitution. SP-B reconstituted from lipid/protein mixtures in organic solvents is inserted more deeply in PC or DPPC bilayers than the protein reconstituted by addition to preformed phospholipid vesicles. These differences in the extent of insertion lead to qualitative and quantitative differences in the effect of the protein on the mobility of the phospholipid acyl chains, as studied by spin-label electron spin resonance (ESR) spectroscopy, and could represent different functional stages in the surfactant cycle.     

Membrane lipid composition and susceptibility to bile salt damage

Erythrocyte membranes with low sphingomyelin : choline-containing phospholipid ratios haemolyse at low concentrations of the bile salt, glycocholate. Erythrocytes with higher sphingomyelin : choline-containing phospholipid ratios require progressively greater concentrations of the bile salt for lysis. Sublytic concentrations of glycocholate remove phospholipid and acetylcholinesterase from the membranes. Membranes with low sphingomyelin : choline-containing phospholipid ratios lose both particulate (microvesicles of distinct composition) and 'solubilized' material, the particulate form predominating. The proportion of particulate material falls with increase of the membrane sphingomyelin : choline-containing phospholipid ratio and those membranes of highest sphingomyelin : choline-containing phospholipid ratio lose material predominantly in 'solubilized' form. Sheep erythrocytes treated to increase their content of phosphatidylcholine (and thereby reduce their membrane sphingomyelin : choline-containing phospholipid ratio) become more susceptible to lysis by glycocholate. These observations indicate a correlation between membrane lipid composition and the perturbation of membranes with bile salt; they also point to possible features of membranes capable of surviving exposure to the high bile salt concentrations of the biliary tract.     

2D 1H-NMR conformational study of phosphatidylserine diluted in perdeuterated dodecylphosphocholine micelles. Evidence for a pH-induced conformational transition

The conformation of phosphatidylserine (DMPS) diluted in perdeuterated dodecylphosphocholine micelles (DPC) has been investigated by 1D and 2D proton NMR spectroscopy. Chemical shift pH dependence showed that the pK relative to the serine carboxyl titration (3.4 +/- 0.05) was nearly identical to that measured in bilayers. Chemical shift and NOE data revealed that the phosphatidylserine molecule undergoes a conformational transition upon titration of the serine carboxyl group. The NOE network observed between the different parts of the molecule was sufficiently abundant to allow, in combination with molecular modeling methods, an assessment of the conformational changes. The conformational changes mainly involve the glycerol backbone, which is parallel to the whole molecule, that is, to the layer normal, at low pH and becomes perpendicular to the whole molecule at neutral pH. In both cases, the conformations are remarkably close to those observed for the crystal forms of zwitterionic and negatively charged phospholipids. Two-dimensional proton NMR study of phospholipids, diluted in perdeuterated DPC micelles, appears to be a simple and relevant method to obtain complete and direct information on their conformations in a model membrane-solution interface.     

The effect of phloretin on the hydration of egg phosphatidylcholine multilayers

The effect of phloretin on the hydration, structure and interactive properties of supported phospholipid bilayers has been studied by a combination of direct water adsorption measurements and X-ray diffraction. Adsorption isotherms show that over a wide range of relative vapor pressures (from 0 to approximately 1.0) the addition of 20 or 40 mol% phloretin significantly alters the amount of water adsorbed by egg phosphatidylcholine (EPC) multilayers. X-ray diffraction analysis shows that the incorporation of phloretin decreases the width of the EPC bilayer, thereby increasing the area per lipid molecule from approximately 64 A2 for EPC to about 78 A2 for EPC:Ph, 3:2, M:M. Phloretin also decreases the distance between apposing EPC bilayers, most likely because it causes a reduction in repulsive hydration/steric pressure between apposing bilayers. Because phloretin decreases the fluid layer between bilayers by a larger amount than it increases the area per EPC molecule, phloretin has the effect of decreasing the water volume in the multilayers.     

Outside-inside distributions and sizes of mixed phosphatidylcholine-cholesterol vesicles

(1) The effect of the incorporation of cholesterol upon the distribution of various molecular species of phosphatidylcholine across the bilayers of mixed sonicated liposomes (vesicles) has been studied with 31P-MNR. (2) The outside-inside ratio of both saturated and unsaturated phosphatidylcholine species was not much affected by the incorporation of up to 30 mol% cholesterol. Above 30 mol% cholesterol the outside-inside ratio strongly increased for phosphatidylcholines with cis unsaturated fatty acid chains. In contrast the outside-inside ratio of trans unsaturated and fully saturated phophatidylcholine species was either not affected or decreased by the incorporation of more than 30 mol% cholesterol. (3) a simple relationship between the size of the vesicle and the linewidth of the 31P-NMR resonance is described. From the measured linewidths the sizes of the various cholesterol containing vesicles have been obtained. It is found that incorporation of 0-30 mol% cholesterol does not significantly affect the size of the vesicle whereas above 30 mol% cholesterol does not significantly affect the size of the vesicle whereas above 30 mol% cholesterol the size of all phosphatidylcholine vesicles sharply increases. The increase in size is the largest for the more saturated phosphatidylcholine species. (4) From the outside-inside ratio and the size of the vesicle the composition of the outer and inner layer of the mixed vesicles could be obtained. Below 30 mol% cholesterol the composition of outer and inner layer is nearly identical. Above 30 mol% cholesterol the distribution of lipid across the bilayer of all visicles becomes assymetric with a disporportionately larger amount of cholesterol present in the inside monolayer.     

Hidden anti-phospholipid antibodies in normal human sera circulate as immune complexes whose antigen can be removed by heat, acid, hypermolar buffers or phospholipase treatments

Heat treatment of normal human serum reveals otherwise masked anti-cardiolipin antibodies (aCL). We studied the mechanism of masking and the nature of the inhibitor of these aCL IgG. Other forms of treatment, besides heating for 30 min at 56 degrees C, can also unmask hidden aCL IgG. These include acid pH, hypermolar buffers and phospholipase digestion. When unmasked, these aCL recognize other anionic and zwitterionic phospholipids, but do not react with DNA, cell antigens or IgG. Using thin layer chromatography we demonstrate that the heat-labile inhibitor(s) of these aCL are phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine. These antibodies are not beta2-glycoprotein-I dependent and actually compete with this protein for phospholipid binding. The hidden antibodies are comprised of two populations of IgG autoantibodies: one reactive with cardiolipin, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine and sphingomyelin, and the other reactive almost exclusively with phosphatidylcholine and phosphorylcholine on enzyme-linked immunosorbent assay plates or when exposed by bromelain on the erythrocyte surface. Our data suggest that hidden aCL are natural oligoreactive IgG anti-phospholipid autoantibodies that circulate masked by their antigen.     

31P NMR studies of unsonicated aqueous dispersions of neutral and acidic phospholipids. Effects of phase transitions, p2H and divalent cations on the motion in the phosphate region of the polar headgroup

1. The 129 MHz (non-proton decoupled) and 36.4 MHz (proton decoupled) 31P NMR spectra arising from unsonicated aqueous dispersions of well defined species of phospholipid have been investigated. The phospholipids employed (and the parameters varied) include phosphatidylcholine (temperature), phosphatidylethanolamine (temperature), phosphatidic acid (temperature and p2H) and phosphatidylglycerol (temperature, p2H and Ca2+ (or Mg2+)) concentration. 2. At p2H = 7 the 31 P MNR spectra arising from saturated species of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol become progressively broader as the temperature is reduced below the phase transition, demonstrating reduced motion in the phosphate region of the polar headgroup. 3. In the liquid crystalline state at p2H = 7 the molecular dipolar order parameters obtained for saturated species of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol and very similar, and are independent of the acyl chain length for species derived from lauric and myristic acid. Thus the motion in the methylene-phosphate-methylene region is similar for these different liquid crystaline phospholipid species. 4. The 31 P NMR spectra of aqueous dispersions of 14:0/14:0 phosphatidic acid display anomalous temperature and p2H dependences. The effective chemical shift anistropy (delta v CSA EFF) at 5 degrees C varies from 71 ppm at p2H = 8.5 to 38 ppm at p2H = 2.5. Further, the motion in the phosphate region is relatively insensitive to the gel or liquid crystalline nature of the hydrocarbon chains. 5. The addition of 40 mol% Ca2+ (or Mg2+) to saturated species of phosphatidylglycerol causes an increase of approx. 20 degrees C in the hydrocarbon phase transition temperature as indicated by 31 P NMR. Equimolar concentrations of Ca2+ increase the transition temperature by approx. 70 degrees C, and no 31P NMR signal could be observed for the very condensed precipitate formed below this temperature. In the liquid crystalline state the motion in the phosphate region of the polar headgroup is not significantly affected by the presence of Ca+ or Mg2+. 6. The 31P NMR spectra obtained from 18 : 1c/18 : 1c phosphatidylethanolamine are consistent with a phase transition from a lamellar to an hexagonal (HII) phase in the region 10-15 degrees C. 7. The observed narrowing of the 31 P NMR spectra of aqueous dispersions of phospholipids as the temperature is raised toward the hydrocarbon transition temperature is discussed in terms of the "pretransition" observed in calorimetric studies.     

Activation of phospholipase D by phosphatidic acid. Enhanced vesicle binding, phosphatidic acid-Ca2+ interaction, or an allosteric effect?

The activity of bacterial phospholipase D (PLD), a Ca2+-dependent enzyme, toward phosphatidylcholine bilayers was enhanced 7-fold by incorporation of 10 mol % phosphatidic acid (PA) in the vesicle bilayer. Addition of other negatively charged lipids such as phosphatidylinositol, phosphatidylmethanol, and oleic acid either inhibited or had no effect on enzyme activity. Only negatively charged lipids with a free phosphate group, phosphatidylinositol 4-phosphate and lyso-PA, had the same effect as PA on enzyme activity. Changes in vesicle curvature and fusion were not the reason for PA activation; rather, a metal ion-induced lateral segregation of PA in the vesicle bilayer correlated with PLD activation. Significant PA activation was also observed with monomer phosphatidylcholine substrate upon the addition of PA vesicles. The PA activation was caused by Ca2+.PA interacting with PLD at an allosteric site other than active site.     

Interfacial conformation of dipalmitoylglycerol and dipalmitoylphosphatidylcholine in phospholipid bilayers

Diacylglycerols are minor constituents of membrane lipids, yet are essential in the activation and membrane association of protein kinase C. Solid-state 13C NMR experiments have been used to characterize the orientation of the glycerol backbone of dipalmitoylglycerol (DPG) and dipalmitoylphosphatidylcholine (DPPC) in egg phosphatidylcholine (PC) bilayers. The 13C NMR spectra of both DPG and DPPC specifically 13C-labeled at the sn-2 chain carbonyl exhibit a single narrow resonance (approximately 2 ppm) in liquid-crystalline egg PC bilayers. In contrast, specific 13C-labeling of both the sn-1 and sn-2 chain carbonyls results in an additional broad component (24-32 ppm) with an axially symmetric line shape. These data reveal that DPG has a distinct motionally-averaged structure in PC bilayers that is similar to that of DPPC and is not significantly affected by the absence of the large polar PC headgroup. The NMR line shapes are roughly consistent with the results of previous FTIR and NMR studies that indicate the sn-1 chain extends from the C1 carbon of the glycerol backbone into the hydrophobic interior of the bilayer, while the sn-2 chain first extends parallel to the bilayer surface and incorporates a bend at the ester linkage in order to keep the sn-1 and sn-2 chains parallel. However, the data suggest that the time-averaged orientation of the glycerol backbone is tilted from the bilayer normal, in contrast to the nearly parallel orientation observed in the crystal structures of phosphatidylcholines and phosphatidylethanolamines or the perpendicular orientation observed in the crystal structures of diacylglycerols.