Localization and identification of the compound causing peak ‘X’ in the 31P-NMR spectrum of Saccharomyces cerevisiae

The present study is aimed at identifying the unidentified compound which gives rise to the so-called resonance ‘X’ in the ³¹P-NMR spectra of yeast cells. In addition, it is attempted to determine the localization of X (inside or outside the cell). Enzymic removal of the cell wall causes resonance ‘X’ to disappear in the spectra of the cells. This observation indicates an extracellular localization of X. The ³¹P-NMR spectrum of the phosphomannan extracted from the yeast shows a single resonance at exactly the same position as that of resonance ‘X’. Extraction of the phosphomannan from delipidized cells causes resonance ‘X’ to disappear from the ³¹P-NMR spectrum of the cells. The intensity of resonance ‘X’ in the spectrum of the intact cells can be almost quantitatively attributed to the amount of phosphomannan present in the yeast. The present results demonstrate that the resonance ‘X’ in the ³¹P-NMR spectrum of yeast cells is caused by phosphomannan in the cell wall.     

A 31P and 1H-NMR investigation in vitro of normal and abnormal human liver

Spectral changes in human hepatic tumours and possible systemic effects of tumour on host liver were assessed by ³¹P amnd ¹H in vitro NMR spectroscopy. The ¹H and ³¹P spectra from liver tumour biopsies showed significant elevation in phosphoethanolamine, phosphocholine, taurine, citrate, alanine, lactate and glycine, and significant reduction in GPE (glycerophosphoethanolamine), GPC (glycerophosphocholine), creatine and threonine compared to histologically normal tissue. ³¹P-NMR spectra obtained from histologically normal tissue within tumour-bearing livers showed significant elevation in phosphoethanolamine and phosphocholine compared to data from liver biopsies from nontumour-bearing patients (pancreatitis). These results suggest that alterations in membrane metabolism in host liver can be detected by ³¹P-NMR.     

Multicomponent spectra from 31P-NMR studies of the phase equilibria in the system dioleogylphosphatidylcholine-dioleoylphosphatidylthanolamine-water

The phase equilibria in mixtures of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and water were studied by ³¹P-NMR and ²H-NMR. The chemical shift anisotropy is greater for DOPC than for DOPE (6–9 ppm in the lamellar phase). This difference can most probably be ascribed to different order parameters for the two lipid head groups. ³¹P-NMR spectra recorded from a lamellar phase formed by DOPC-DOPE-water below maximum hydration exhibit two resolved, superimposed powder spectra. The chemical shift anisotropy for both phospholipids has greater values at excess water contents than below maximum hydration, and the spectral resolution between DOPC and DOPE in the lamellar phase is strikingly diminished at excess water contents. From ³¹P-NMR spectra it is possible to observe relative differences in composition between different lipid phase existing in equilibrium. The proportion of DOPE is decreased in the lamellar phase, and is increased in the reversed hexagonal phase, when these phases exist in equilibrium.     

The adsorption of divalent cations to phosphatidylglycerol bilayer membranes

The ability of the Stern equation to describe the adsorption of divalent cations to phosphatidylglycerol membranes was tested by combining ³¹P-NMR and electrophoretic mobility measurements. In 0.1 M sodium chloride both the ³¹P-NMR and the zeta potential data are well described by the Stern equation. ³¹P-NMR and ¹³C-NMR results indicate that cobalt forms inner-sphere complexes only with the phosphate group of phosphatidylglycerol molecules and that a substantial fraction of the adsorbed cobalt ions form outer-sphere complexes. Evidence is presented that suggests the alkaline earth cations also bind to phospholipids mainly by forming outer sphere complexes. Electrophoretic mobility measurements were performed with several different divalent cations. In all cases the zeta potentials in 0.1 M sodium chloride were well described by the Stern equation. The intrinsic 1 : 1 association constants (M^?1) for the phosphatidylglycerol complexes decreased in the sequence: Mn²⁺, 11.5; Ca²⁺, 8.5; Ni²⁺, 7.5; Co²⁺, 6.5; Mg²⁺, 6.0; Ba²⁺, 5.5 and Sr²⁺, 5.0.     

Phospholipid Motional Characteristics in a Dry Biological System : A P-Nuclear Magnetic Resonance Study of Hydrating Typha latifolia Pollen

Analysis of the proton-decoupled ³¹P-nuclear magnetic resonance (NMR) spectrum of fully hydrated Typha latifolia pollen revealed the presence of two main peaks: A broad asymmetrical component of a `bilayer' lineshape and a much narrower symmetrical component originating from phosphorus compounds undergoing rapid isotropic motion. From (a) ³¹P-NMR experiments on the hydrated total pollen phospholipids, (b) saturation transfer ³¹P-NMR experiments, and (c) the fraction of lipid phosphate in the pollen, it can be concluded that the great majority of the endogenous phospholipids are arranged in extended bilayers in which the lipid phosphates undergo fast (τ_c < 10⁻⁶ second) long axis rotation. This bilayer arrangement of phospholipids was observed in the pollen down to hydration levels of at least 10.9% moisture content. At the lowest level of pollen hydration examined (5.2%) the ³¹P-NMR spectrum had a solid state lineshape demonstrating that all the phosphorus-containing compounds (including the phospholipids) were virtually immobile.     

A possible role of rhodopsin in maintaining bilayer structure in the photoreceptor membrane

³¹P-NMR measurements demonstrate that at 37°C, independent of the photolytic state of the photopigment rhodopsin, the lipids in the photoreceptormembrane are almost exclusively organised in a bilayer. In strong contrast, the ³¹P-NMR spectra of the extracted lipids are characteristic for the hexagonal H_II phase and an isotropic phase. The isotropic phase is characterised by freeze-fracture electron microscopy as particles and pits on smooth surfaces, possibly indicating inverted micelles. These results suggest a structural role for rhodopsin in maintaining the photoreceptor membrane lipids in a bilayer configuration.     

Effects of divalent cations and pH on phosphatidylserine model membranes: A 31P NMR study

The influence of divalent cations, and pH on the behaviour of phosphatidylserine, derived from egg phosphatidylcholine, has been examined employing ³¹P-NMR techniques. The addition of Ca²⁺ results in the observation of a “rigid lattice” ³¹P-NMR spectra and more than an order of magnitude increase in the spin-lattice relaxation time T₁. This corresponds to a strong and specific headgroup immobilization by Ca²⁺, similar to that observed for anhydrous phosphatidylserine. At pH 7.4 the hydrated sodium salt of (egg) phosphatidylserine adopts the bilayer phase, whereas when the pH is decreased through 3.5 a bilayer to hexagonal (H_II) polymorphic phase transition is observed at 50°C, which is unaffected by equimolar cholesterol. The same transition is shown to occur at 37°C for phosphatidylserine isolated from human erythrocytes.     

Continuous-flow NMR culture system for mammalian cells

A continuous-flow NMR culture system for mammalian cells has been developed on which ³¹P-NMR experiments under complete and strictly physiologic conditions have been performed. Observations on the response of the cellular metabolism to stresses such as starvation, low temperature and changes in environmental pH monitored by ³¹P-NMR are reported. The response of the intracellular pH relative to the external pH of the growth medium is studied. We find that under the experimental conditions used there exists a ΔpH varying between less than 0.2 and more than 0.6 pH units. These results are compatible with those obtained using other techniques.     

Phosphate accumulation and the occurrence of polyphosphates and cyclic 2,3-diphosphoglycerate in Methanosarcina frisia

Methanosarcina frisia accumulates phosphate up to 14% of its dry weight. The phosphate is stored as long-chain polyphosphates as shown by ³¹P-NMR investigations. Further results show that the accumulation of phosphates is substrate-dependent. In the presence of H₂ and CO₂ as the only carbon and energy source 180 mg of PO _inf4 ^sup3- /g protein were accumulated, whereas 260 mg PO _inf4 ^sup3- /g protein were accumulated in the presence of methanol. This is far more than necessary for the maintenance of essential metabolic pathways. In addition, the ³¹P-NMR studies show the occurrence of cyclic 2,3-diphosphoglycerate in Methanosarcina frisia. The role of the phosphate metabolites in cell metabolism are discussed.Abbreviations M. Methanosarcina - CCP cyclic 2,3-diphosphoglycerate     

Phase behavior of the major lipids of Tetrahymena ciliary membranes

The major lipids of Tetrahymena membranes have been purified by thin-layer and high pressure liquid chromatography and the phosphatidylethanolamine and aminoethylphosphonate lipids were examined in detail. ³¹P-NMR, X-ray diffraction and freeze-fracture electron microscopy were employed to describe the phase behavior of these lipids. The phosphatidylethanolamine was found to form a hexagonal phase above 10°C. The aminoethylphosphonate formed a lamellar phase up to 20°C, but converted to a hexagonal phase structure at 40°C. Small amounts of phosphatidylcholine stabilized the lamellar phase for the aminoethylphosphonate. ³¹P-NMR spectra of the intact ciliary membranes were consistent with a phospholipid bilayer at 30°C, suggesting that phosphatidylcholine in the membrane stabilized the lamellar form, even though most of the lipid of that membrane prefers a hexagonal phase in pure form at 30°C. ³¹P-NMR spectra also showed a distinctive difference in the chemical shift tensor of the aminoethylphosphonolipid, when compared to that of phosphatidylethanolamine, due to the difference in chemical structure of the polar headgroups of the two lipids.     

Mixtures of gramicidin and lysophosphatidylcholine from lamellar structures

It is shown by ³¹P-NMR and freeze-fracture electron microscopy that in aqueous dispersions of mixtures of gramicidin and palmitoyllysophosphatidylcholine lamellar structures are formed which contain four lysophosphatidylcholine molecules per gramicidin monomer.     

Interaction of nonionic detergents with phospholipids in hepatic microsomes at subsolubilizing concentrations as studied by 31P-NMR

The effect of low concentrations of nonionic detergents with different critical micelle concentrations such as Triton X-100, Brij 35 and octylglucoside on rabbit liver microsomes is studied by means of ³¹P-NMR, ¹H-NMR, dynamic light scattering and functional investigations. Hexane phosphonic acid diethyl ester was used as a phosphorus membrane probe molecule to monitor the interaction of detergent molecules with microsomal phospholipids by ³¹P-NMR. This method is more sensitive than ³¹P-NMR of phospholipids alone and permitted the estimation of the maximum number of detergent molecules which can be incorporated in microsomes without the formation of mixed micelles outside the membrane. These membrane saturation concentrations were determined to be 0.07 (Brij 35), 0.1 (Triton X-100) and 0.4 (octylglucoside) (molar ratio of detergent/total phospholipids). Above these detergent concentrations, mixed micelles consisting of detergent and membrane constituents are formed, coexisting with the microsomes up to the membrane solubilization concentration. The results indicate a dependence of the membrane saturation concentration on the critical micelle concentration of the detergent and a preferential removal of phosphatidylcholine over phosphatidylethanolamine from the microsomes by all detergents studied.     

13C- and 31P-NMR studies of the conformation of carcinogen-modified nucleic acid dimers

The effect of the carcinogen acetylaminofluorene (AAF) on nucleic acid structure was examined using ¹³C- and ³¹P-NMR spectroscopies. Conformational effects were compared in two AAF-modified dinucleoside monophosphates (ApG and GpA) and two AAF-modified deoxydinucleotides (dpApG and dpGpA). Changes in adenine ¹³C chemical shifts on formation of the AAF-adduct and as a function of temperature provided evidence of base stacking. Differences in fluorene ¹³C chemical shifts between the AAF-modified dimer and AAF-modified monomer provided evidence of fluorene stacking. The effect of forming the adduct on the phosphate backbone was examined using ³¹P-NMR. A correlation was demonstrated between the degree of adenine-fluorene stacking on one hand and the change in conformation of the backbone conformation on the other.     

P-NMR Spectroscopy of Roots of Intact Corn Seedlings

The application of ³¹P nuclear magnetic resonance spectroscopy to the study of metabolism in roots of intact corn seedlings is described. ³¹P-NMR spectra of developmentally distinct parts of primary roots of whole seedlings are presented. The spectra are of quality comparable to those of excised pieces of plant tissue.     

The effect of calcium on the bilayer stability of lipids from bovine rod outer segment disk membranes

The phase behavior of bovine rod outer segment disk lipids has been investigated using freeze-fracture and ³¹P nuclear magnetic resonance (NMR) techniques. ³¹P-NMR spectra of isolated disk membranes were taken as a function of temperature between 25°C and 45°C. The ³¹P-NMR spectrum characteristic of phospholipid bilayers was observed at all temperatures both in the absence of Ca²⁺ and in the presence of 10 mM and 50 mM Ca²⁺. A similar study was performed on lipids isolated from the disk membranes. In the absence of Ca²⁺ only lamellar phase behavior was observed. In the presence of less than 10 mM Ca²⁺, however, there was a change in morphology to non-lamellar structures. Removal of the Ca²⁺ caused the system to reassume the lamellar form.     

A saturation transfer phosphorus nuclear magnetic resonance study of arginine phosphokinase in the muscle of a marine mollusc

The forward and reverse fluxes of arginine phosphokinase were measured in vitro preparations of the phasic adductor muscle of the scallop Argopecten irradians concentricus using saturation transfer ³¹P-NMR techniques. The respective fluxes were 4.72 ± 1.46 and 6.98 ± 1.58 μmol/s per g wet weight. The forward-to-reverse flux ratio was near unity, indicating that the arginine phosphokinase reaction is near equilibrium under resting conditions. ³¹P-NMR spectra showed no evidence of ADP in scallop muscles. Using an experimentally determined apparent equilibrium constant for arginine phosphokinase, the free ADP level was estimated to be 51 nmol/g wet wt. It appears that at least 90% of the ADP pool is bound in this muscle.     

The synthesis and use of thionphospholipids in 31P-NMR studies of lipid polymorphism

(1) Dipalmitoyl- and dioleoylthionphosphatidylcholine, which are phosphatidylcholine analogues in which the double bonded oxygen of the phosphate group is replaced by a sulfur atom, have been synthesized in 50–60% yields by condensation of diacylglycerol with phosphorus thionchloride in the presence of choline toluene-sulfonate. Dioleoylthionphosphatidylethanolamine has been prepared by the phospholipase D-catalyzed base exchange reaction. (2) Freeze-fracturing of aqueous dispersions of the thionphospholipids reveals that the thionphosphatidylcholines are organized in extended bilayers whereas dioleoylthionphosphatidylethanolamine above 0°C forms the hexagonal H_II phase similar to dioleoylphosphatidylethanolamine. The gel → liquid crystalline phase transition of the dipalmitoylthionphosphatidylcholine occurs at 44°C which is only slightly higher than the transition temperature of dipalmitoylphosphatidylcholine which together with other data demonstrates that the thionphospholipids closely resemble the natural phospholipids in physicochemical behaviour. (3) Proton decoupled ³¹P-NMR spectra of aqueous dispersions of thionphosphatidylcholines have the characteristic asymmetrical line-shape with a low-field shoulder and a high-field peak typical of phospholipids organized in extended bilayers in which the phosphate group can undergo fast axial rotation. The ³¹P-NMR spectrum of the thionphosphatidylethanolamine in the hexagonal H_II phase has a line-shape with a reversed asymmetry and an effective chemical shift anisotropy half of that of thionphospholipids organized in bilayers which is caused by fast lateral diffusion of the lipids around the cylinders of the hexagonal H_II phase as has been observed for the corresponding phosphatidylethanolamines. (4) Since the ³¹P-NMR resonance of the thionphospholipids is completely separated from that of natural phospholipids, these lipids can be used to study by ³¹P-NMR the motional and structural properties of individual lipids in mixed systems. This is demonstrated for various lipid mixtures in which non-bilayer lipid structures have been induced by variations in composition, temperature and presence of divalent cations. It is shown that bilayer → non-bilayer transitions can be modulated by gel → liquid crystalline phase transitions and that typical bilayer forming lipids can be incorporated into non-bilayer structures such as the hexagonal H_II phase.     

31P-NMR DIFFERENTIATION BETWEEN INTRACELLULAR PHOSPHATE POOLS IN COSMARIUM (CHLOROPHYTA)1

³¹P nuclear magnetic resonance (NMR) spectroscopy of intact Cosmarium sp. cells is presented as a suitable tool for the differentiation of intracellular accumulation pools of polyphosphates. The cold trichloroacetic acid (TCA) insoluble fraction is shown to contain most of the total cellular phosphate in the phosphate rich Cosmarium cells. Moreover, evidence from a ³¹ P-NMR study and electron microscopic observations of cold TCA treated Cosmarium cells indicate that this fraction consists mostly of polyphosphates which seem to retain the native morphological structure observed in the untreated cells. The determination of orthophosphate in the hot water extract of Cosmarium cells did not measure the polyphosphate pools. Determination of total phosphorus content in the hot water extract rendered a value three times higher than the frequently used orthophosphate determination procedure. However, as revealed by the ³¹P-NMR spectra and the chemical analyses of the extract and of the treated cells, even total phosphorus in the extract measured only 30% of the total cellular phosphorus. ³¹P-NMR enabled the unequivocal chemical identification of the major phosphate compounds in the hot water extract (“Surplus P”) as orthophosphate and polyphosphates of about 10 phosphate units chainlength. More than 70% of the accumulation pool of polyphosphates was still in the cells after extraction. However, the electron microscopy study revealed that the native granular structure of polyphosphates had been destroyed by the hot water extraction procedure.