31P-NMR determination of phosphomonoesters in relation to phospholipid biosynthesis in testis of the rat at different ages

Changes in the phosphomonoester (PM) peak, as observed in in vivo 31P-NMR spectra, are often attributed to changes in phospholipid synthesis and therefore to changes in cell proliferation. However, this technique provides information about the absolute size of the phosphomonoester pool rather than its turnover rate. To investigate whether there is a good correlation between changes in PM concentration and its turnover rate, we studied the turnover rate of the two major PM compounds, phosphocholine and phosphoethanolamine, in rat testes at different stages of testis development. [3H]Choline and [3H]ethanolamine were injected intraperitoneally into rats at the age of 3, 6 and 13 weeks, respectively. Phosphorylation of these compounds and their incorporation into phospholipids, were followed up to 6 h after injection of the phospholipid precursors. When these data were compared with the changes observed in the in vivo 31P-NMR PM peak, the concentration of the PM compounds appeared to correlate linearly, both with the conversion of choline into phosphocholine, as well with the rate of phospholipid synthesis, and therefore with the rate of cell proliferation. Hence, it is suggested that cell proliferation can be monitored by determining the changes in the PM peak that is observed in in vivo 31P-NMR spectra.     

Spin-spin relaxation of the phosphodiester resonance in the 31P NMR spectrum of human brain. The determination of the concentrations of phosphodiester components

The phosphodiester peak in 31P nuclear magnetic resonance spectra of human brain in vivo is often the most prominent feature of the spectrum. We have demonstrated that this resonance exhibits bi-exponential spin-spin relaxation, giving relaxation times of 2 and 10 ms. We interpret this in terms of the two components which make up the peak, bilayer lipids and the small cytosolic phosphates glycerophosphoethanolamine and glycerophosphocholine. Using the relaxation times and the relative peak heights of the two components we have also been able to quantitate the concentration of the bilayer lipids as 20-40 times that of ATP.     

Chemical assessment of phospholipid and phosphoenergetic metabolites in regenerating rat liver measured by in vivo and in vitro 31P-NMR.

For the assessment of 31P-NMR spectroscopic data, phospholipid precursors (phosphorylethanolamine (PE) and phosphocholine) and catabolites (glycerophosphorylethanolamine (GPE) and glycerophosphorylcholine (GPC)), as well as adenosine phosphates were chemically determined in regenerating rat liver. The data were compared with those obtained by in vivo and in vitro 31P-NMR spectroscopies. Chemical assay revealed a significant increase of PE and a decrease of GPE, GPC and ATP in hepatectomy group compared to sham operation group. The values obtained by in vitro NMR were in good agreements with those of chemical assay, but significant differences between the two groups were observed only in PE and inorganic phosphate (Pi). Noticeable increase in PME was not detected by in vivo 31P-NMR spectroscopy, although the increase of PE was about 2.5-times that of the control and its constitution ratio to the whole phosphomonoester (PME) was less than 15%. On the other hand, in vivo NMR showed a large phosphodiester (PDE) peak occupying approx. 40% of the total phosphorus signal, while the contribution of its constituents, GPE and GPC was about 5% found by both chemical assay and in vitro NMR. The PDE peak in in vivo NMR seemed to reflect the membrane phospholipid itself rather than its catabolites. A slight decrease of phosphoenergetic level in regenerating rat-liver was commonly suggested by all three analytical methods.     

Short chain phospholipids in membrane protein crystallization: a 31P-NMR study of colloidal properties of dihexanoyl phosphatidylcholine

The colloidal features of short chain phospholipids can be deduced from 31P-NMR analysis by comparison with available data on phospholipid aqueous dispersion. In this study with dihexanoyl phosphatidylcholine, detergent phase separation was obtained by temperature shift and by addition of the precipitating agent polyethylene glycol. The 31P-NMR spectra indicate that the detergent micelles fuse to enter the hexagonal HII and lamellar phases. Consequences for the crystallization of membrane proteins are discussed.     

Light-induced membrane protein phosphorylation in the bovine rod outer segment. A magic angle spinning 31P-NMR study

Magic angle spinning 31P-NMR (MAS 31P-NMR) spectra of bovine rod outer segments, unphosphorylated and phosphorylated, were obtained. In the phosphorylated samples the spectra showed new resonances not assignable to phospholipids. These signals were present only when stimulation of receptor phosphorylation occurred. These resonances were not due to exogenous, soluble phosphorus-containing compounds. Limited proteolysis to remove the carboxyl-terminal region of the photoreceptor that contains the phosphorylation sites removed these resonances. The chemical shifts were in the usual range for serine phosphate and threonine phosphate. The pKa obtained from a pH titration of the 31P chemical shift was typical of serine phosphate. Therefore, these 31P-NMR resonances were assigned to the phosphorylation sites on membrane proteins in the rod outer segment disk membranes. Static 31P-NMR measurements revealed that at least some of these sites gave rise to relatively narrow resonances, indicative of considerable motional freedom of the carboxyl-terminal segment of the photoreceptor when phosphorylated. These data indicate that it is possible to study phosphorylation sites on membrane proteins using MAS 31P-NMR, and that using in vivo 31P 'spin labelling' one can study directly and selectively regions of receptors crucial to receptor function.     

Application of 31P-NMR saturation transfer techniques to investigate phospholipid motion and organization in model and biological membranes

The potential of ³¹P-NMR saturation transfer experiments for determining motional characteristics (in the millisecond to second time scale) of phospholipids in model and biological membranes is demonstrated. A technique to separate membrane phospholipid ³¹P-NMR signals from those of small water-soluble phosphates in intact cells in liver tissue is also illustrated.     

31P NMR studies of excised gray and white calf brain

1. 31P NMR examination of isolated calf gray and white matter reveals that white matter contains higher levels of the phosphodiester glycerolphosphoryl choline (GPC) than gray. 2. It is suggested that GPC may play a role in maintaining the level of phospholipids present by inhibition of phospholipases. 3. The spectra also reveal a skewed peak whose maximum is at -11 ppm which is inferred to arise from myelin-like structures. 4. The results show that phosphorus spectra from the brain must be carefully considered whether they arise from the same type tissue or represent a mixed sample since variation in results may represent anatomy as well as physiology.     

Structural properties of phospholipids in the rat liver inner mitochondrial membrane. A P-NMR study

1. 1. The ³¹P-NMR characteristics of intact rat liver mitochondria, mitoplasts and isolated inner mitochondrial membranes, as well as mitochondrial phosphatidylethanolamine and phosphatidylcholine, have been examined.

2. 2. Rat liver mitochondrial phosphatidylethanolamine hydrated in excess aqueous buffer undergoes a bilayer-to-hexagonal (H_II) polymorphic phase transition as the temperature is increased through 10°C, and thus prefers the H_II) arrangement at 37°C. Rat liver mitochondrial phosphatidylcholine, on the other hand, adopts the bilayer phase at 37°C.

3. 3. Total inner mitochondrial membrane lipids, dispersed in an excess of aqueous buffer, exhibit ³¹P-NMR spectra consistent with a bilayer arrangement for the majority of the endogeneous phospholipids; the remainder exhibit spectra consistent with structure allowing isotropic motional averaging. Addition of Ca²⁺ results in hexagonal (H_II) phase formation for a portion of the phospholipids, as well as formation of ‘lipidic particles’ as detected by freeze-fracture techniques.

4. 4. Preparations of inner mitochondrial membrane at 4 and 37°C exhibit ³¹P-NMR spectra consistent with a bilayer arrangement of the large majority of the endogenous phospholipids which are detected. Approx. 10% of the signal intensity has characteristics indicating isotropic motional averaging processes. Addition of Ca²⁺ results in an increase in the size of this component, which can become the dominant spectral feature.

5. 5. Intact mitochondria, at 4°C, exhibit ³¹P-NMR spectra arising from both phospholipid and small water-soluble molecules (ADP, P_i, etc.). The phospholipid spectrum is characteristic of a bilayer arrangement. At 37°C the phospholipids again give spectra consistent with a bilayer; however, the labile nature of these systems is reflected by increased isotropic motion at longer (at least 30 min) incubation times.

6. 6. It is suggested that the uncoupling action of high Ca²⁺ concentrations on intact mitochondria may be related to a Ca²⁺-induced disruption of the integrity of the inner mitochondrial phospholipid bilayer. Further, the possibility that non-bilayer lipid structures such as inverted micelles occur in the inner mitochondrial membrane cannot be excluded.

Cerebral metabolism in brain tumor of mice studied by in vivo 31P-NMR spectroscopy

We now report a mouse model system of brain tumor for 31P-NMR spectroscopic study of in vivo cerebral metabolism. In vivo 31P-NMR (109 MHz) spectra were taken on the 9th day by the Faraday shield method of the brain of mice (3-week-old) transplanted intracerebrally with mKS X A tumor cells. In tumor-bearing mice, the amount of creatine phosphate decreased markedly and that of inorganic phosphate plus sugar phosphate increased accordingly. Furthermore, the broadening and splitting of individual signals were also noted with tumor-bearing mice; this is interpreted as indicating a variety of changes in chemical shift occurring in the brain of the animals due to heterogeneous distribution of pH. Binding or detaching of divalent cations to and from phosphometabolites may also be responsible for these changes.     

The glycophorin-phospholipid interface in recombined systems. A 31P-nuclear magnetic resonance study.

Glycophorin, the MN glycoprotein from the erythrocyte membrane, was recombined with egg phosphatidylcholine and with the total lipid extract from human erythrocyte membranes in a membranous form. 31P-nuclear magnetic resonance (NMR) spectra of the recombinants resembled spectra obtained from unsonicated phospholipid dispersions and biological membranes. The glycophorin/phospholipid ratio in these recombinants was varied from approximately 50:1 (lipid/protein) to 200:1, and 31P-NMR spectral intensities were obtained. Comparison of these intensities to that expected based on a pure phospholipid standard revealed that there were two phospholipid environments in the recombinants: one immobilized by the protein, and one slightly disordered and nonimmobilized. A relatively constant number of phospholipids were immobilized per glycophorin at all lipid/protein ratios studied.     

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.     

Phospholipid composition and organization of cytochrome c oxidase preparations as determined by 31P-nuclear magnetic resonance

The molecular organization as well as the composition of the phospholipids in cytochrome c oxidase preparations (bovine heart) were investigated by 31P-nuclear magnetic resonance. In the so-called 'lipid-rich' preparation the lipids were found to form a fluid bilayer around the enzyme since the 31P-NMR spectrum was characteristic of a fast, axially symmetric motion of the phosphate groups with a chemical shift anisotropy of delta sigma = -45 ppm. In contrast, the 'lipid-depleted' cytochrome c oxidase gave rise to a broader spectrum where the motion of the phospholipids was no longer axially symmetric. Nevertheless, the total width of the spectrum was still considerably narrower than observed for immobilized phospholipids in solid crystals. Both enzyme preparations were dissolved in 1% detergent solution and used for high-resolution 31P-NMR spectroscopy. Narrow lines of about 20 Hz linewidth were obtained for both types of enzyme preparations, and well-resolved resonances could be assigned to cardiolipin, phosphatidylethanolamin and phosphatidylcholine. The major differences between lipid-rich and lipid-depleted cytochrome c oxidase were the absolute amount of phospholipid associated with the protein and the relative contribution of the individual lipid classes to the 31P-NMR spectrum. For lipid-rich cytochrome c oxidase about 130 molecules phospholipid were bound per enzyme (approx. 11 cardiolipins, 54 phosphatidylethanolamines and 64 phosphatidylcholines). For lipid-depleted cytochrome c oxidase only 6-18 lipids were bound per enzyme (1 or 2 cardiolipins, 3-8 phosphatidylethanolamines and 2-8 phosphatidylcholines). In contrast to earlier suggestions that cardiolipin is the only remaining lipid in lipid-depleted cytochrome c oxidase, the 31P-NMR studies demonstrate that all three lipids remain associated with the protein.     

In vivo and in vitro 31P-NMR spectroscopy of rat liver treated with halocarbons

Both in vivo and in vitro 31P-NMR spectroscopy were used to demonstrate metabolic changes in rat liver as a function of time after exposure to either carbon tetrachloride (CCl4) or bromotrichloromethane (BrCCl3). The inorganic phosphate resonance, measured in vivo, moves upfield, which is associated with a decrease in cytosolic pH over a 12 or 20 h period (for BrCCl3 or CCl4, respectively). Intoxication by CCl4 or BrCCl3 causes an intracellular acidosis to pH 7.05 or 6.82 (+/- 0.05), respectively. Also, it has been found that halocarbon exposure increases the amounts of phosphomonoesters (PME) detected. High resolution in vitro 31P-NMR spectroscopy studies of perchloric acid extracts of CCl4-treated rat livers indicated a significant increase in the height of the phosphocholine resonance in the PME region 4-5 h after CCl4 exposure.     

Sodium binding to and protonation of ATP: a multinuclear magnetic double resonance study at 8.46 tesla

We show that the interaction of ATP with Na+ and H+, whether binding or dissociation, gives rise to exchange broadened 31P-NMR spectra at 8.4 T, pH 6.7 and 310 K. We interpret the effect as being due to a two-step conversion between two NMR-differentiated ATP pools. A quantitative analysis yields all involved equilibrium constants and some of the dynamic parameters. Our results help to understand previous studies of magnesium binding to ATP and the appearance of high-field in vivo 31P-NMR spectra.     

The in vivo formation of nonbilayer lipid phase in E. coli membranes during the development of Ca2-dependent competence

31P-NMR studies on E. coli cells reveal the in vivo formation of nonbilayer lipid structures, presumably of H11 phase, during Ca2-dependent competence induction. The data suggest the involvement of these structures in the exogenous DNA transfer into the cells during genetic transformation and transfection. The suggestion is supported by in vitro experiments in which the liposomes composed of different phospholipid species bind 14C-DNA in DNase and wash resistant form in conditions promoting the hexagonal phase formation.     

Regulatory aspects of mitochondrial phospholipase A2: correlation of hydrolysis rates with substrate configuration as evidenced by 31P-NMR

The influence of variation of the phospholipid composition in model membranes composed of phosphatidylcholine and phosphatidylethanolamine on the hydrolysis of these phospholipids by rat liver mitochondrial phospholipase A2 was investigated. With the pure phospholipids, phosphatidylethanolamine was hydrolyzed over 30-times faster than phosphatidylcholine. Upon increasing the mole percentage of phosphatidylethanolamine in mixtures, a gradual, though non-linear, increase in the initial rate of hydrolysis of this phospholipid was observed. By contrast, phosphatidylcholine hydrolysis remained constant up to about 50 mol% phosphatidylethanolamine, whereafter a sudden fall-off of activity was observed. This drop in the hydrolysis rate coincided with a transition of the phospholipid structure from bilayer to an as yet unidentified organization characterized by an isotropic signal in the 31P-NMR spectra recorded in the presence of Ca2+. The occurrence of this phase was clearly dependent on Ca2+, since mixtures with identical composition in the absence of Ca2+ remained largely in bilayer configuration. That the structure adopted by phospholipids is of importance for their susceptibility to attack by this intracellular phospholipase A2 became evident also in studies with the single phospholipids in the absence or presence of Triton X-100 above the critical micellar concentration. While phosphatidylcholine hydrolysis was inhibited in mixed micelles as compared to its bilayer organization, the hydrolysis of phosphatidylethanolamine in mixed micelles was 3-fold that in the hexagonal HII phase.     

Infrared and 31P-NMR studies of the effect of Li+ and Ca2+ on phosphatidylserines

Infrared and 31P-NMR spectra of solid samples of 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) have been recorded. Comparison of the spectra of the Na+ salts of these phospholipids with those of complexes formed with Li+ and Ca2+ ions allows the characterization of conformational changes induced by complexation with Li+ and Ca2+. Ca2+ forms tight, crystalline complexes with these phosphatidylserines (PS), irrespective of the degree of unsaturation in the hydrocarbon chains. In these PS-Ca2+ complexes the torsion angles of the two P-O ester bonds exhibit the antiplanar-antiplanar conformation which is significantly different from the standard gauche-gauche conformation commonly found in phosphodiesters. In contrast, complexation with Li+ does not induce this conformational change in the phosphodiester group. It is shown that the degree of unsaturation in the hydrocarbon chains, and related to it, the cross-sectional area of the phospholipid or the surface charge density, determine the affinity of the phosphatidylserine for the metal ion. In general, the affinity of phosphatidylserines for both Li+ and Ca2+ decreases with increasing unsaturation in the hydrocarbon chains or decreasing surface charge density; it is in the order DMPS greater than POPS greater than DOPS.     

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.     

Detection of individual phospholipids in lipid mixtures by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: phosphatidylcholine prevents the detection of further species

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is an established tool for the analysis of proteins, whereas it gained by far less interest in the field of lipid analysis. This method works well with phospholipids as well as organic cell extracts and provides high sensitivity and reproducibility. The aim of the present paper is to extend our previous studies to the analysis of lysophospholipids and phospholipid mixtures. To study the suitability of MALDI-TOF mass spectrometry for the analysis of lysophospholipids, different phospholipids like phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, and phosphatidylinositol as well as their mixtures were digested with phospholipase A(2). Positive and negative ion mass spectra of all phospholipids before and after digestion were recorded. In all these cases, the molecular ions of the expected digestion products could be detected and only a very small extent of further fragmentation was observed. On the other hand, spectra of phospholipid mixtures containing phosphatidylcholine were strongly dominated by phosphatidylcholine and lysophosphatidylcholine signals, which prevented the detection of further phospholipids even if those lipids were present in comparable amounts. This is of paramount interest for the analysis of tissue and cell extracts.