Static and magic angle spinning (31)P NMR spectroscopy of two natural plasma membranes

Static and magic angle spinning (31)P NMR spectroscopy was used for the first time in natural plasma membranes from erythrocytes and skeletal muscle to study phospholipid arrangement and composition. Typical static powder-like spectra were obtained showing that phospholipids were in a bilayer arrangement. Magic angle spinning narrowed spectra into two components. The first one corresponded to phosphatidylcholine and the second one to the other phospholipids with intensities in agreement with the known phospholipid composition. These findings show that NMR data previously acquired using model membranes can be transposed to studies on phospholipids in their natural environment.     

31P NMR spectroscopic studies on purified, native and cloned, expressed forms of NADPH-cytochrome P450 reductase.

31P NMR spectroscopy has been utilized in conjunction with site-directed mutagenesis and phospholipid analysis to determine structural aspects of the prosthetic flavins, FAD and FMN, of NADPH-cytochrome P450 reductase. Comparisons are made among detergent-solubilized and protease (steapsin)-solubilized preparations of porcine liver reductases, showing unequivocally that the 31P NMR signals at approximately 0.0 ppm in the detergent-solubilized, hydrophobic form are attributable to phospholipids. By extraction and TLC analysis, the phospholipid contents of detergent-solubilized rat liver reductase, both tissue-purified and Escherichia coli-expressed, have been determined to reflect the membranes from which the enzyme was extracted. In addition, the cloned, wild-type NADPH-cytochrome P450 reductase exhibits an additional pair of signals downfield of the normal FAD pyrophosphate resonances reported by Otvos et al. [(1986) Biochemistry 25, 7220-7228], but these signals are not observed with tissue-purified or mutant enzyme preparations. The Tyr140----Asp140 mutant, which exhibits only 20% of wild-type activity, displays no gross changes in 31P NMR spectra. However, the Tyr178----Asp178 mutant, which has no catalytic activity and does not bind FMN, exhibits no FMN 31P NMR signal and a normal, but low intensity, pair of signals for FAD. The latter experiments, taking advantage of mutations in residues putatively on either side of the FMN isoalloxazine ring, suggest subtle to severe changes in the binding of the flavin prosthetic groups and, perhaps, cooperative interactions of flavin binding to NADPH-cytochrome P450 reductase.     

31P NMR spectra of rod outer segment and sarcoplasmic reticulum membranes show no evidence of immobilized components due to lipid-protein interactions

31P NMR studies of rod outer segment (ROS) and sarcoplasmic reticulum (SR) membranes have been performed under conditions where broad and narrow spectral components can be clearly resolved. Control studies of an anhydrous, solid powder of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), as well as aqueous binary mixtures of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), demonstrate clearly that broad spectral components can be detected. For the codispersions of DSPC and DOPC in the mixed-phase region at 22 degrees C, the 31P NMR spectra consist of a superposition of a broad component and a narrow, axially symmetric component, due to coexisting solid and liquid-crystalline domains, which are in slow exchange on the 31P NMR time scale. The 31P NMR spectra of the native ROS and SR membranes, however, consist of only a narrow component, to within experimental error, indicating that most or all of the phospholipids are in the liquid-crystalline (L alpha) phase at 22 degrees C. The above conclusions are in agreement with many, but not all, previous studies [see, e.g., Yeagle, P.L. (1982) Biophys. J. 37, 227-239]. It is estimated that at most 10% of the phospholipids in the ROS and SR membranes could give rise to broad 31P NMR spectral components, similar to those seen for anhydrous or solid-phase lipids, corresponding to approximately 7 phospholipids/rhodopsin molecule and approximately 11 phospholipids/Ca2+-ATPase molecule, respectively.     

31P nuclear magnetic resonance studies of the phospholipid-protein interface in cell membranes.

Both native and recombined membrane systems from the human erythrocyte membrane and the rabbit sarcoplasmic reticulum have been studied with 31P Nuclear Magnetic Resonance (NMR). We compare intensities of the anisotropic 31P resonance exhibited by these membranes with the intensity expected from the known phospholipid content of the membranous sample. In a recombinant with human erythrocyte glycophorin, a component of the phospholipid is "missing" from the 31P NMR resonance, apparently due to a severe broadening of the resonance of that component. Approximately 29 phospholipid molecules were found immobilized per glycophorin molecule in the membrane, regardless of the phospholipid:protein ratio. Cholesterol may inhibit the immobilization of phospholipids by glycophorin. Recombinants with band three from the human erythrocyte membrane contain an immobilized phospholipid component, analogous to the results with glycophorin. 31P NMR data from the native sarcoplasmic reticulum membrane also revealed an immobilized phospholipid component whose magnitude is independent of temperature between 30 degrees C and 45 degrees C. Extensive papain proteolysis of the membrane completely digests the Ca++ Mg++ ATPase and removes the immobilization of phospholipids noted in the intact membrane. Limited trypsin cleavage, however, does not completely remove the immobilized component; salt reduces the immobilized component.     

Mechanism of polymorphic transformations in phosphatidylcholine membranes

Based on the 31P NMR, PMR, and EPR data on the thermally induced behavior of water dispersions of natural and synthetic phospholipids in the presence of membranotropic agents: the neuropeptide adrenocorticotropic hormone and beta-(4-oxy,3,5-ditretbutyl-phenylpropionic acid), a new mechanism of the interaction of membranotropic substances with the molecules of hydrate shells of membrane phospholipids was proposed, which underlies polymorphic transitions in phosphatidylcholine membranes.     

Detergent-like properties of magainin antibiotic peptides: a 31P solid-state NMR spectroscopy study

(31)P solid-state NMR spectroscopy has been used to investigate the macroscopic phase behavior of phospholipid bilayers in the presence of increasing amounts of magainin antibiotic peptides. Addition of >1 mol% magainin 2 to gel-phase DMPC or liquid crystalline POPC membranes respectively, results in (31)P NMR spectra that are characterized by the coexistence of isotropic signals and line shapes typical for phospholipid bilayers. The isotropic signal intensity is a function of temperature and peptide concentration. At peptide concentrations >4 mol% of the resulting phospholipid (31)P NMR spectra are characteristic of magnetically oriented POPC bilayers suggesting the formation of small disk-like micelles or perforated sheets. In contrast, addition of magainin to acidic phospholipids results in homogenous bilayer-type (31)P NMR spectra with reduced chemical shift anisotropies. The results presented are in good agreement with the interfacial insertion of magainin helices with an alignment parallel to the surface of the phospholipid bilayers. The resulting curvature strain results in detergent-like properties of the amphipathic helical peptides.     

Detergent-like properties of magainin antibiotic peptides: A P solid-state NMR spectroscopy study

³¹P solid-state NMR spectroscopy has been used to investigate the macroscopic phase behavior of phospholipid bilayers in the presence of increasing amounts of magainin antibiotic peptides. Addition of >1 mol% magainin 2 to gel-phase DMPC or liquid crystalline POPC membranes respectively, results in ³¹P NMR spectra that are characterized by the coexistence of isotropic signals and line shapes typical for phospholipid bilayers. The isotropic signal intensity is a function of temperature and peptide concentration. At peptide concentrations >4 mol% of the resulting phospholipid ³¹P NMR spectra are characteristic of magnetically oriented POPC bilayers suggesting the formation of small disk-like micelles or perforated sheets. In contrast, addition of magainin to acidic phospholipids results in homogenous bilayer-type ³¹P NMR spectra with reduced chemical shift anisotropies. The results presented are in good agreement with the interfacial insertion of magainin helices with an alignment parallel to the surface of the phospholipid bilayers. The resulting curvature strain results in detergent-like properties of the amphipathic helical peptides.     

31P NMR and freeze fracture studies of sarcoplasmic reticulum membranes from normal and malignant hyperthermic pigs: effect of halothane and dantrolene.

The effects of halothane and dantrolene on sarcoplasmic reticulum membranes isolated from normal and malignant hyperthermia pig muscle have been investigated using 31P NMR and freeze fracture electron microscopy. The dynamical and structural changes are estimated from the second moment, as calculated from 31P NMR spectra. For both membranes, addition of halothane induces a similar decrease in the spectral second moment. At high concentration of halothane, freeze fracture replicas show small unilamellar vesicles or mixed micelles, uniformly sprayed in the case of malignant hyperthermia membranes but mainly aggregated for the normal ones. The effect of halothane on both membranes is partially inhibited by adding dantrolene. These results suggest that (i) the malignant hyperthermia syndrome is not directly related to the polar heads of phospholipids and (ii) dantrolene counteracts unspecifically the disturbing effect of halothane at the lipid level.     

Phospholipid organization in H,K-ATPase-containing membranes from pig gastric mucosa

The transverse distribution of the phospholipids in vesicular H+-translocating membranes prepared from pig gastric mucosa was investigated with the aid of phospholipase C, sphingomyelinase, and trinitrobenzenesulfonic acid. The major part (80-90%) of the phosphatidylcholine and the phosphatidylethanolamine, 60% of the phosphatidylserine, and 45% of the sphingomyelin was located on the external, cytoplasmic side of the vesicle membranes. After treatment with phospholipase C the vesicles still behaved as osmometers and appeared as closed vesicles on the electron micrographs. 31P NMR indicated that the phospholipids in untreated vesicles as well as the unhydrolyzed phospholipids in phospholipase C-treated vesicles were arranged in lamellar structures. The 31P NMR spectrum of untreated vesicles to which Pr3+ ions had been added supported the conclusion that the major part of the membrane phospholipids was located on the external surface of the vesicles. A small fraction of the lipids, 3.6 mol %, was found to consist of glycosphingolipids which occurred at a concentration of 52 nmol/mg of protein.     

Principles of multiparametric optimization for phospholipidomics by 31P NMR spectroscopy

Phospholipids have long been known to be the principal constituents of the bilayer matrix of cell membranes. While the main function of cell membranes is to provide physical separation between intracellular and extracellular compartments, further biological and biochemical functions for phospholipids have been identified more recently, notably in cell signaling, cell recognition and cell–cell interaction, but also in cell growth, electrical insulation of neurons and many other processes. Therefore, accurate and efficient determination of tissue phospholipid composition is essential for our understanding of biological tissue function. ³¹P NMR spectroscopy is a quantitative and fast method for analyzing phospholipid extracts from biological samples without prior separation. However, the number of phospholipid classes and subclasses that can be quantified separately and reliably in ³¹P NMR spectra of tissue extracts is critically dependent on a variety of experimental conditions. Until recently, little attention has been paid to the optimization of phospholipid ³¹P NMR spectra. This review surveys the basic physicochemical properties that determine the quality of phospholipid spectra, and describes an optimization strategy based on this assessment. Notably, the following experimental parameters need to be controlled for systematic optimization: (1) extract concentration, (2) concentration of chelating agent, (3) pH value of the aqueous component of the solvent system, and (4) temperature of the NMR measurement. We conclude that a multiparametric optimization approach is crucial to obtaining highly predictable and reproducible ³¹P NMR spectra of phospholipids.     

Model membrane morphology and crosslinking of oxidized lipids with proteins

Model membranes composed of thion-phosphatidylcholine, cardiolipin, and cytochrome c have been studied by 31P NMR, polyacrylamide gel electrophoresis, gel filtration, fluorescence, and freeze-fracturing. Covalent binding of oxidized phospholipids to cytochrome c was shown to result in the formation of high-molecular-weight oligomeric complexes via Schiff base formation between a protein molecule and aldehydes produced upon peroxidation of phospholipids. The initial steps of the protein oligomerization lead to the appearance of intramembranous particles (IMPs) of various size and distribution on freeze-fractured faces of these model membranes. In the final phase of the crosslinking between cytochrome c and oxidized products of cardiolipin there is a breakdown of membrane vesicles and formation of globular lipoprotein complexes which are seen as globular particles. It is believed that the covalent linking between the products of phospholipid peroxidation and membrane proteins causes the oligomerization of membrane proteins and structural alteration in the hydrophobic region of other models also and, perhaps, in biological membranes.     

Accumulation of N-acyl-ethanolamine phospholipids in rat brains during post-decapitative ischemia: a 31p NMR study

Phosphorus-31 nuclear magnetic resonance (31P NMR) spectroscopy has been used to study accumulation of N-acyl-ethanolamine phospholipids in rat brains during post-decapitative ischemia. Lipids were extracted from rat brain homogenates and the extracts were thoroughly washed with aq. potassium ethylenediaminetetraacetic acid (EDTA). The lower organic phases were isolated and evaporated to dryness under a stream of nitrogen and the lipids were redissolved in CDCl3-CH3OH-H2O 100.0:29.9:5.2 (v/v/v) for NMR analysis. Increasing the period of post-decapitative ischemia resulted in an accumulation of two signals in the NMR spectra at 0.18 and 0.22 ppm (relative to the chemical shift of 1,2-diacyl-sn-glycero-3-phosphocholine (PCDIACYL) at -0.84 ppm). These signals were identified as originating from 1,2-diacyl-sn-glycero-3-phospho-(N-acyl)-ethanolamine (NAPEDIACYL) and 1-(1'-alkenyl)-2-acyl-sn -glycero-3-phospho-(N-acyl)-ethanolamine (NAPEPLAS), respectively, by spiking with authentic materials. Additionally, the identification was verified by thin-layer chromatography, which also showed the accumulation of N-acyl-ethanolamine phospholipids. The use of K-EDTA instead of the commonly used Cs-EDTA in the preparation of the NMR samples allowed the separation of the chemical shifts of N-acyl-ethanolamine phospholipids from those of the ethanolamine phospholipids. Moreover, the chemical shift of cardiolipin was moved from 0.15 ppm observed with Cs-EDTA to about 0.31 ppm with K-EDTA.The present study demonstrates that it is possible to detect and quantify post-decapitative accumulation of NAPE subclasses (NAPEDIACYL and NAPEPLAS) in rat brains by the use of 31P NMR spectroscopy.     

2H and31P nuclear magnetic resonance studies of membranes containing bovine rhodopsin

Summary Purified, delipidated rhodopsin is recombined with phospholipid using octyl-glucoside (OG) and preformed vesicles. Normal egg phosphatidylcholine, phosphatidylcholine in which the N-methyl groups are fully deuterated, and dioleoyl phosphatidylcholine labeled with deuterium at carbons 9 and 10 were used.³¹P nuclear magnetic resonance (NMR) and²H NMR measurements were obtained of the pure phospholipids and of the recombined membranes containing rhodopsin.³¹P NMR of the recombined membrane (containing the deuterated phospholipid) showed two overlapping resonances. One resembled a normal phospholipid bilayer, and the other was much broader, representing a motionally restricted phospholipid headgroup environment. The population of phospholipids in the motionally restricted environment can be modulated by conditions in the media.²H NMR spectra of the same recombined membranes showed only one component. These experimental results agree with a theoretical analysis that predicts an insensitivity of²H NMR to lipids bound to membrane proteins. A model containing at least three different phospholipid environments in the presence of the membrane protein rhodopsin is described.Deceased.     

31P NMR of phospholipid glycerol phosphodiester residues

Saponification of extracted tissue phospholipids yields a set of isolated glycerol 3-phosphoryl phospholipid polar headgroups from which semi-quantitative 31P NMR spectra can be obtained. The resonance signals from these molecules, which frequently have been reported as uncharacterized phosphate signals observed in perchloric acid extracts of tissue, can be used as an aid in the characterization of isolated phospholipids and of tissue phospholipid 31P NMR profiles. 31P NMR chemical-shift values of the resonances at pH 7 in water and relative to 85% phosphoric acid are: glycerol 3-phosphocholine (-0.13 delta), glycerol 3-phosphoethanolamine (0.42 delta), glycerol 3-phospho(monomethyl)ethanolamine (0.29 delta), glycerol 3-phospho(dimethyl)ethanolamine (0.16 delta), glycerol 3-phosphoserine (0.14 delta), glycerol 3-phosphoinositol (-0.07 delta), glycerol 3-phosphoglycerol (0.92 delta), bis(glycerol 3-phospho)glycerol (0.79 delta), serine ethanolamine phosphodiester (-0.46 delta), glycerol 3-phosphate (0.60 delta; 4.29 delta at pH 10) glycerol 2-phosphate (0.15 delta; 3.92 delta at pH 10). In addition, analysis of extracted cancer tissue phospholipid samples yielded a new and uncharacterized polar headgroup fragment with a chemical-shift value of 0.29 delta that is independent of sample pH.     

Interaction of the antitumor Au(I) complex [Au(Ph2P(CH2)2PPh2)2]Cl with human blood plasma, red cells, and lipoproteins: 31P and 1H NMR studies

The bis-chelated tetrahedral gold(I) complex [Au(dppe)2]Cl, where dppe is Ph2P(CH2)2PPh2, is active in several animal tumor models. When added to human blood plasma in vitro it appears to bind to lipoproteins, giving a slightly broadened 31P NMR signal, and 1H NMR resonances which are too broad to detect. Some lipoprotein is denatured. 31P NMR studies suggest that some [Au(dppe)2]+ is transferred from plasma to red cells with a half-life of ca. 2 hr. The complex binds within red cell membranes and the 1H resonances of intracellular glutathione are unaffected. The 31P NMR resonance from [Au(dppe)2]+ in red cell membranes is observable only when the complex is mobilized by addition of sodium dodecyl sulphate, which also mobilizes membrane phospholipids.     

Penetration of a cardiotoxin into cardiolipin model membranes and its implications on lipid organization

The interaction of cardiotoxin II of Naja mossambica mossambica with cardiolipin model membranes was investigated by binding, fluorescence, resonance energy transfer, fluorescence quenching, 31P NMR, freeze-fracture, and small-angle X-ray experiments. An initially electrostatic binding appeared to be accompanied by a deep penetration, most likely into the acyl chain region of the phospholipids, indicating a hydrophobic contribution to the strong interaction (KD congruent to 5 X 10(-8) M). This binding results in a fusion of unilamellar vesicles as indicated by a fluorescence-based fusion assay, freeze-fracture, and X-ray diffraction. In these fused structures freeze-fracture electron microscopy reveals the appearance of particles, which is accompanied by the induction of an isotropic component in 31P NMR. The well-defined particles are interpreted as inverted micelles, and the localization of the cardiotoxin molecule in these structures is discussed.     

Reincorporation of adenosine 5'-diphosphate/adenosine 5'-triphosphate carrier into phospholipid membranes. Phospholipid-protein interaction as studied by phosphorus-31 nuclear magnetic resonance and electron microscopy

Combined phosphorus-31 nuclear magnetic resonance (31P NMR) and electron microscopic studies were performed on the ADP/ATP carrier protein from beef heart mitochondria. The protein was incorporated into phospholipids by addition of Triton-protein micelles to a lipid suspension or to the dry lipid. All of the phospholipid (egg phosphatidylcholine or mixtures of egg phosphatidylcholine and egg phosphatidylethanolamine) that contributed to the observed 31P NMR signal under these conditions appeared to be in a bilayer configuration. Freeze-fracturing and negative-staining electron microscopy showed unilamellar vesicles and multilayers. An isotropic signal could be attributed to vesicle rotation, judging from its sensitivity to increasing viscosity. The presence of small vesicles was also noticeable in the 31P NMR spectra of planar oriented membranes. In the presence of phosphatidylethanolamine, aggregation of protein particles was observed. Gel chromatography of the protein-Triton-phospholipid mixture revealed that, before Triton removal, large amounts of protein are associated with multibilayers. Separation of loaded and unloaded membranes by centrifugation in D2O showed that, upon stepwise addition, protein incorporates preferentially into unloaded liposomes. From these findings a mechanism of protein reincorporation was deduced.     

The effect of binding of spider-derived antimicrobial peptides, oxyopinins, on lipid membranes

Oxyopinins (Oxki1 and Oxki2) are antimicrobial peptides isolated from the crude venom of the wolf spider Oxyopes kitabensis. The effect of oxyopinins on lipid bilayers was investigated using high-sensitivity titration calorimetry and (31)P solid-state NMR spectroscopy. High-sensitivity titration calorimetry experiments showed that the binding of oxyopinins was exothermic, and the binding enthalpies (DeltaH) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) small unilamellar vesicles (SUVs) were -18.1 kcal/mol and -15.0 kcal/mol for Oxki1 and Oxki2, respectively, and peptide partition coefficient (K(p)) was found to be 3.9x10(3) M(-1). (31)P NMR spectra of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) membranes in the presence of oxyopinins indicated that they induced a positive curvature in lipid bilayers. The induced positive curvature was stronger in the presence of Oxki2 than in the presence of Oxki1. (31)P NMR spectra of phosphaditylcholine (PC) membranes in the presence of Oxki2 showed that Oxki2 produced micellization of membranes at low peptide concentrations, but unsaturated PC membranes or acidic phospholipids prevented micellization from occurring. Furthermore, (31)P NMR spectra using membrane lipids from E. coli suggested that Oxki1 was more disruptive to bacterial membranes than Oxki2. These results strongly correlate to the known biological activity of the oxyopinins.     

Effect of micellar lipids on rabbit intestinal brush-border membrane phospholipid bilayer integrity studied by31P NMR

Summary The effect of biliary salts and fatty acids on the bilayer structure of rabbit intestinal brush-border membranes was studied using the nonperturbing probe³¹P NMR. The broad. asymmetric lineshape of the³¹P NMR spectrum of isolated brush-border vesicles demostrates that their component phospholipids are organized in extended bilayers. These membranes are not significantly perturbed by incubation with physiological concentrations of biliary salts (3, 9, 18mm), demonstrating that the vesicles are highly stable, corresponding to their biological function. However, the emergence of a narrow peak superimposed on the broad lineshape indicates that a small proportion of the membrane phospholipids has reached isotropic motion, which may correspond to external or internal micellar structures. Incubation with mixed micelles of fatty acids and taurochlorate show that long-chain fatty acids enhance the membrane-perturbing effect of taurocholate while short-chain, watersoluble fatty acids do not, suggesting a difference in the absorption mechanisms.     

Phosphorus NMR analysis of human white matter in mixed non-ionic detergent micelles

In order to study the lipid composition of human white matter, we have developed a 31P NMR spectroscopy method, which allows the determination and quantitation of the main phospholipids found in biological membranes. The technique is based upon the use of a non-ionic detergent (Triton X-100) which induces, in aqueous media, the formation of mixed micelles that are magnetically isotropic. The linewidths and chemical shifts depend on both the molar ratio detergent/phospholipid and the pH of the suspension. After determination of the optimum values for these two parameters, 31P NMR spectra were recorded, in which all phospholipid resonances were resolved. After determining precise chemical shifts for each phospholipid, concentrations were measured by comparing the peak areas with that of an internal standard. Analysis of the complex phospholipid composition of human white matter using this method gave values very close to that found in the literature for such tissue. Moreover this nondestructive method proved to be very sensitive since less than 1 mg of a mixture of phospholipids was needed.