Effect of halothane on the natural-abundance 13C NMR spectra of excised rat brain

Halothane increases the intensity of the 30.5- and 129-ppm resonances in 13C nuclear magnetic resonance spectra of excised rat brain, of phospholipid vesicles prepared from chloroform-methanol extract of rat brain, and of brain excised from rats anesthetized with halothane. The 13C spin-lattice relaxation times of the 30.5- and 129-ppm resonances are increased in excised brain, or phospholipid vesicles, upon addition of halothane, and they are also increased in brain excised from rats anesthetized with halothane. Excised brain and its membrane-rich subcellular fractions interact with [14C]halothane reversibly. The interaction is virtually abolished when the phospholipids are extracted from the brain. The [14C]halothane content of the brain membranes is correlated with the halothane-induced increase in the integral of the 129-ppm resonance. From this correlation and from the phospholipid content of the membranes, a halothane concentration of 3.34 mM and a partition of 0.057 mol of halothane/mol of phospholipid may be calculated in the brain of anesthetized rats.     

Non-invasive 31P NMR study on the development of brain membrane of gerbils and jimpy mice, a myelin-deficient mutant

The broad background resonance observed in the in vivo 31P NMR spectra of adult murine heads was investigated in terms of phosphorus atoms in bone and membrane phospholipids. The broad background resonance was found to be weak in a juvenile and increase with advance of age. Fractionation of adult gerbil heads showed that the broad signal was derived from bone and membrane, of which myelin is the major component. The two origins of the broad background resonance exhibited considerably different line shapes in spectra, which enabled us to extract the membrane component from an intact murine head spectrum (sequential subtraction method). By the use of this method, the development of membrane in gerbil brain at various age grades could be estimated. The membrane component was shown to be suppressed in a mutant mouse, jimpy, which has a deficiency in myelin formation ability. Furthermore, the value of T1 of the membrane component was estimated to be 0.9 sec, which was in good agreement with previously reported values for excised brain.     

Phosphorus nuclear magnetic resonance studies of lipid-protein interactions: human erythrocyte glycophorin and phospholipids

Human erythrocyte glycophorin containing four molecules of phospholipid tightly bound to the protein was isolated from human red cell ghosts. This protein preparation was reconstituted into a digalactosyl diglyceride bilayer. The 31P NMR spectrum of this reconstituted membrane produced an axially symmetric powder pattern arising exclusively from the phospholipids bound to glycophorin. The width of the powder pattern, about 90 ppm, is about twice as broad as that normally exhibited by a phospholipid bilayer. The chemical shift tensor is perturbed relative to phospholipids in a bilayer. The spin-lattice relaxation rate of these protein-bound phospholipids is found to be nearly an order of magnitude faster than phospholipids in a bilayer. The results are consistent with phospholipids tightly bound to the membrane protein and undergoing rotational diffusion, perhaps as a complex of phospholipid and protein.     

Synthesis and use of thiophosphatidylcholine in 31P-NMR study of membranes

Thiophosphatidylcholines, i. e. phosphatidylcholine analogues in which one of phosphate oxygens is replaced by a sulfur atom, have been synthesized. The properties of aqueous dispersions of thiophosphatidylcholine and its equimolar mixture with diphosphatidylglycerol (cardiolipin) have been studied by 31P NMR. The 31P resonance of thiophospholipids dissolved in deuterochloroform was found to be shifted 19 ppm downfield from the signals of natural phospholipids. This feature allowed a complete separation of signals of thiophospholipids and natural phospholipids in the 31P NMR spectra of model membranes by using "DANTE" pulse sequence. The possibility of employing thiophospholipids in 31P NMR studies of lipid polymorphism in model membranes was demonstrated.     

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.     

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.     

On the coordination of inhibitors to the metal ion of carboxypeptidase A. A 113Cd and 31P NMR study

113Cd and 31P NMR have been used to investigate the interactions of inhibitors with the metal ion of bovine carboxypeptidase A, using 113Cd as a replacement for the native zinc atom. In the absence of inhibitor and over the pH range 6-9, no 113Cd resonance is visible at room temperature. Upon lowering the temperature to 270 K, however, a broad resonance can be seen at 120 ppm. These results are discussed in terms of possible sources for this resonance modulation. Binding of low molecular weight inhibitors containing potential metal-coordinating moieties results in the appearance of a sharp 113Cd resonance. These inhibitors all bind to the metal ion, a fact which is reflected in the chemical shift of the cadmium resonance and, for L-phenylalanine phosphoramidate phenyl ester, by two-bond 113Cd-31P spin-spin coupling of 30 Hz in the 31P resonance of the bound inhibitor. For inhibitors that coordinate to the metal ion via oxygen, the 113Cd chemical shift is in the range 127-137 ppm, whereas for sulfur coordination there is a downfield shift of approximately 210 ppm. The complexes of 113Cd-substituted carboxypeptidase A with the D and L isomers of thiolactic acid are distinguished by a difference of 11 ppm in the chemical shift of their cadmium resonances. The enzyme complex formed with the macromolecular inhibitor from potatoes, which fills the S1 and S2 subsites, shows one or possibly two closely spaced broad 113Cd resonances. Both the chemical shift and the line width of the 113Cd resonances of the [113Cd]carboxypeptidase-inhibitor complexes give valuable structural and dynamic information about the enzyme active site.     

Aluminum binding to phosphatidylcholine lipid bilayer membranes: 27Al and 31P NMR spectroscopic studies

27Al and 31P nuclear magnetic resonance (NMR) spectroscopies were used to investigate aluminum interactions at pH 3.4 with model membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). A solution state 27Al NMR difference assay was developed to quantify aluminum binding to POPC multilamellar vesicles (MLVs). Corresponding one-dimensional (1D) fast magic angle spinning (MAS) 31P NMR spectra showed that aluminum induced the appearance of two new isotropic resonances for POPC shifted to -6.4 ppm and -9.6 ppm upfield relative to, and in slow exchange with, the control resonance at -0.6 ppm. Correlation of the (27)Al and (31)P NMR binding data revealed a 1:2 aluminum:phospholipid stoichiometry in the aluminum-bound complex at -9.6 ppm and a 1:1 aluminum:phospholipid stoichiometry in that at -6.4 ppm. Slow MAS 31P NMR spectra demonstrated shifts in the anisotropic chemical shift tensor components of the aluminum-bound POPC consistent with a close coordination of aluminum with phosphorus. A model of the aluminum-bis-phospholipid complex is proposed on the basis of these findings.     

Phosphorylation of troponin I by protein kinase C: mechanism of inhibition by calmodulin and troponin C

The total phospholipid content of excised rat muscle, liver, brain and kidney and of human muscle biopsies was estimated by natural abundance 13C-NMR after complete solubilization of the tissue membranes with excess halothane. An external dioxane capillary, calibrated against pure palmitic acid and phospholipid vesicles with known phosphate concentration, was inserted into the tissues, and the repeating methylene carbon peak area in the spectra of the halothane-treated tissues was integrated versus the dioxane reference peak area. The amount of tissue used for NMR analysis was quantitated by dry weight determination after 13C spectroscopy was completed. The phospholipid content estimated by the indirect NMR method was in good agreement with that measured by direct phosphate analysis and with literature data. For human muscle biopsies, the NMR method can also estimate the fraction of the total phospholipids which are mobile without treating the muscles with halothane. In this respect human muscles could be separated into three different groups: normal and nonspecific muscle diseases, myotonia and myopathy, Duchenne dystrophy; with increasing fraction of the mobile phospholipids in this order.     

Sarcolemma phospholipid structure investigated by immunogold electron microscopy and (31)P NMR spectroscopy with lanthanide ions.

The biological functions of plasma membranes depend greatly on the biophysical properties resulting from protein and phospholipid structure. We investigated the phospholipid structure of the normal sarcolemma membrane, which is known to be highly dysfunctional in myopathies. Combining electron microscopy and (31)P nuclear magnetic resonance (NMR) spectroscopy on isolated sarcolemma vesicles, we find that (i) the sarcolemma vesicles maintain the in-vivo cellular sidedness, (ii) the phospholipid mobility is close to that observed in model membranes (similar lateral diffusion coefficients and spin-lattice T(1) relaxation times). Using broad-band and magic angle spinning (31)P NMR spectroscopy with lanthanide ions (Pr(3+)), it is possible to quantify the distribution of phospholipids between internal and external membrane layers, showing that the trans-bilayer distribution is highly asymmetrical.     

31P NMR of phosphate and phosphonate complexes of metalloalkaline phosphatases.

31P NMR spectra of phosphate and phosphonate complexes of Escherichia coli alkaline phosphatase have been obtained by Fourier transform NMR methods. One equivalent of P1i, bound to Zn(II) alkaline phosphatase, pH 8, gives rise to a single 31P resonance 2 ppm downfield from that for Pi, and assignable to the noncovalent complex, E-P. Inorganic phosphate in excess of 1 eq per enzyme dimer gives rise to a resonance at the position expected for free Pi. At pH 5.1, a second resonance appears 8.5 ppm downfield from that for free Pi, and is assignable to the covalent complex, E-P. The large downfield shift suggests that the enzyme phosphoryl group is highly strained with an O-P-O bond angle of under 100 degrees.     

Gramicidin-induced hexagonal HII phase formation in erythrocyte membranes

Using 31P nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS), and freeze-fracture electron microscopic (FFEM) techniques, it is shown that gramicidin induces a hexagonal HII phase not only in liposomes prepared from total lipids extracted from human erythrocytes but also in isolated human erythrocyte membranes (white ghosts). A 37 degrees C, HII phase formation is detected at a gramicidin to phospholipid molar ratio exceeding 1:80. At a molar ratio of 1:5, about 30% of the phospholipid is organized in the HII phase. The gramicidin-induced HII phase exhibits a very small 31P chemical shift anisotropy [(CSA) approximately 10 +/- 1 ppm], indicating decreased head-group order, and it displays a temperature-dependent increase in tube diameter from 60.2 A at 4 degrees C to 64.2 A at 37 degrees C in ghosts and from 62.8 to 69.4 A at 37 degrees C in total lipid extracts, both in the presence of 1 mol of gramicidin/10 mol of phospholipid. This anomalous temperature-dependent behavior is probably due to the presence of cholesterol. 31P NMR data indicate that the HII phase formation by gramicidin is temperature dependent and show the gradual disappearance of the HII phase at low temperatures (less than 20 degrees C), resulting in a bilayer type of 31P NMR line shape at 4 degrees C, whereas SAXS and FFEM data suggest equal amounts of HII phases at all temperatures. This apparent discrepancy is probably the result of a decrease in the rate of lateral diffusion of the membrane phospholipids which leads to incomplete averaging of the 31P CSA in the HII phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estimation of tissue phospholipids by natural abundance 13C-NMR

The total phospholipid content of excised rat muscle, liver, brain and kidney and of human muscle biopsies was estimated by natural abundance ¹³C-NMR after complete solubilization of the tissue membranes with excess halothane. An external dioxane capillary, calibrated against pure palmitic acid and phospholipid vesicles with known phosphate concentration, was inserted into the tissues, and the repeating methylene carbon peak area in the spectra of the halothane-treated tissues was integrated versus the dioxane reference peak area. The amount of tissue used for NMR analysis was quantitated by dry weight determination after ¹³C spectroscopy was completed. The phospholipid content estimated by the indirect NMR method was in good agreement with that measured by direct phosphate analysis and with literature data. For human muscle biopsies, the NMR method can also estimate the fraction of the total phospholipids which are mobile without treating the muscles with halothane. In this respect human muscles could be separated into three different groups: (1) normal and nonspecific muscle diseases, (2) myotonia and myopathy, (3) Duchenne dystrophy; with increasing fraction of the mobile phospholipids in this order.     

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.     

Diastereomeric phosphonate ester adducts of chymotrypsin: 31P-NMR measurements

Generation of diastereomeric phosphonate ester adducts of chymotrypsin was evidenced for the first time by ³¹P NMR and spectrophotometric kinetic measurements. ³¹P NMR signals were recorded for 4-nitrophenyl 2-propyl methylphosphonate (IMN) at 32.2 ppm and for its hydrolysis product at 26.3 ppm downfield from phosphoric acid. The inhibition of α-chymotrypsin at pH > 8.0 by the faster reacting enantiomer of IMN or 2-propyl methylphosphonochloridate (IMCl), or other phosphonate ester analogs of these compounds, all caused a ～6.0 ppm downfield shift of the ³¹P signal to the 39–40 ppm region. IMN, when applied below the stoichiometric amount of chymotrypsin, under the same conditions, generated two signals, at 39.0 and at 37.4 ppm. Scans accumulated in hourly intervals showed the decomposition of both diastereomers, with approximate half-lives of 12 h at pH 8.0 and 22°C, into a species with a resonance at 35.5 ppm. The most likely reaction to account for the appearance of this new peak is the enzymic dealkylation of the isopropyl group from the covalently bound phosphonate ester. We base this conclusion mostly on the similarity of the upfield shift to the hydrolysis of phosphonate esters. Contrary to experience with phosphate ester adducts of serine proteases, no signal was detected higher than 25.0 ppm downfield from phosphoric acid for several phosphonate ester adducts of chymotrypsin and in no case did the resonance for the adduct shift further downfield in the course of the experiments. © 1993 Wiley-Liss, Inc.     

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.     

Heteronuclear NMR studies of cobalamins. 31P NMR observations of cobalamins bound to a haptocorrin from chicken serum

A vitamin B12-binding protein (haptocorrin) from chicken serum has been purified to homogeneity by photodissociative affinity chromatography and characterized by gel electrophoresis and UV-visible spectrophotometry of its aquocobalamin, hydroxocobalamin, and cyanocobalamin complexes. The haptocorrin is a glycoprotein with a molecular mass of about 70 kDa and a protein moiety of about 40 kDa. 31P NMR resonances of the haptocorrin-cobalamin complexes are relatively broad singlets (with or without proton decoupling) shifted downfield by 0.7-1.0 ppm from the position of the free cobalamin resonances. From the line width data, the relaxation of the phosphorus nucleus is found to be dominated by chemical shift anisotropy with a very minor (13%) component from dipolar interaction with the two nearest neighbor protons. The rotational correlation time of the haptocorrin at 25 degrees C is estimated to be 85 ns and the activation energy for rotational correlation 3.9 +/- 0.3 kcal mol-1. The downfield shift of the 31P resonances of cobalamins upon binding to the haptocorrin cannot be due to hydrogen bonding phosphodiester moiety or displacement of the axial base by a group on the protein. Calculations also show that the downfield shift is very unlikely to be due to dipolar deshielding of the phosphorus nucleus by the ring current of an aromatic residue of the protein. It is concluded that the downfield shift of the 31P resonance must be due to sterically induced changes in phosphodiester conformation which may, or may not, involve steric compression of the axial Co-N bond.     

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.     

Inhibition of thermolysin by phosphonamidate transition-state analogues: measurement of 31P-15N bond lengths and chemical shifts in two enzyme-inhibitor complexes by solid-state nuclear magnetic resonance

31P and 15N chemical shifts and 31P-15N bond lengths have been measured with solid-state NMR techniques in two inhibitors of thermolysin, carbobenzoxy-Glyp-L-Leu-L-Ala (ZGpLA) and carbobenzoxy-L-Phep-L-Leu-L-Ala (ZFpLA), both as free lithium salts and when bound to the enzyme. Binding of both inhibitors to thermolysin results in large changes in the 31P chemical shifts. These changes are more dramatic for the tighter binding inhibitor ZFpLA, where a approximately 20 ppm downfield movement of the 31P isotropic chemical shift (sigma iso) is observed. This shift is due to changes in the shift tensor elements sigma 11 and sigma 22, while sigma 33 remains essentially constant. We observed a similar pattern for ZGpLA, but only a approximately 5 ppm change occurs in sigma iso. The changes in the 15N chemical shifts for both inhibitors are small upon binding, amounting to downfield shifts of 2 and 4 ppm for ZGpLA and ZFpLA, respectively. This indicates that there are no changes in the protonation state of the 15N in either the ZFpLA- or the ZGpLA-thermolysin complex. NMR distance measurements yield a P-N bond length rP-N = 1.68 +/- 0.03 A for the tight binding inhibitor ZFpLA both in its free lithium salt form and in its thermolysin-ZFpLA complex, a distance that is much shorter than the 1.90-A distance reported by X-ray crystallography studies [Holden et al. (1987) Biochemistry 26, 8542-8553].(ABSTRACT TRUNCATED AT 250 WORDS)     

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.