31P nmr spin-lattice relaxation studies of deoxyoligonucleotides

Temperature-dependent conformational transitions of deoxyoligonucleotides have been monitored by measuring ³¹P chemical shifts, spin-lattice relaxation times (T₁), and ³¹P-{H} nuclear Overhauser enhancements (NOEs). The measured NOE ranged from 30 to 80%, compared to the theoretical maximum of 124% for a dipolar relaxation mediated by rapid isotropic rotation. The observed 3′-5′ phosphate diester ³¹P T₁ showed a similar temperature dependence over the range 2–75°C for both double- and single-stranded oligonucleotides, and for dinucleotides. The results show that dipole–dipole interactions dominate the internucleotide phosphate relaxation rate in oligonucleotides. The same is true of terminal phosphate groups at low temperature; but at higher temperature another process, possibly due to contamination by paramagnetic ions, becomes dominant. The rotational correlation time τ_R calculated from the dipole–dipole relaxation rate of the internucleotide phosphate in d(pA)₂ at 16°C is τ_R = 5.0 × 10^?10 sec, implying a Stokes radius for isotropic rotation of 7.6 Å. The T₁ and NOE values for the double-helical octanucleotide d(pA)₃pGpC(pT)₃ are consistent with dominance of dipole–dipole relaxation and isotropic rotation of a sphere of radius 14 Å, a reasonable dimension for the double helix. Activation energies for the rotation of dinucleotides range from 4 to 6 kcal/mol, close to the value of 4 kcal/mol expected for isotropic rotation. In order to test the possible effect of internal motion of correlation time τ_G on the results, we considered a model in which the nucleotide chain rotates about the P-O bonds. Comparison of the calculation with our experimental results shows that internal motion with τ_G ? 10^?9 sec, as found from other studies to be present for large nucleic acids, would not influence out T₁ and NOE values enough to be distinguished from isotropic rotation. However, we can conclude that τ_G cannot be as fast as 10^?10 sec, even for dinucleotides.     

31P-NMR studies of metabolite compartmentation in Fasciola hepatica

Fasciola hepatica, the common liver fluke, is an anaerobic parasitic worm. Possible compartmentation of metabolites between different cell types, metabolic compartments, and free and macromolecule-bound species was investigated using 31P-NMR. A spectrum of the intact worm shows unusual metabolic features, among which are large amounts of glycerolphosphorylcholine, phospholipids mobile on the NMR time-scale, and free cytosolic ADP. Spectra from cells as different as those in oral sucker tissue and eggs showed similar features. Acidosis after serotonin administration was associated with parallel changes in chemical shifts of intracellular Pi and glucose 6-phosphate, suggesting that they are in the same metabolic compartment. Although 13.4 +/- 1.1 mumol/g wet wt. (n = 3) Mg2+ is present in fluke tissue, a considerable fraction is sequestered out of the cytosol. The intracellular free [Mg2+] was independently estimated from the chemical shifts of ATP and ADP as 1.6 +/- 0.5 mM and 2.9 +/- 0.7 mM, respectively. Quantitation of observable phosphate-containing metabolites in whole tissue and in perchlorate extracts demonstrated that 60% of the total ADP and 50% of the total Pi are 'NMR-invisible' in the intact fluke and therefore probably bound to macromolecules in the cells. The apparent ATP/ADP X Pi free concentration ratio is much lower in this anaerobic tissue than in mammalian oxidative tissues.     

31P-NMR studies of DNA in nucleosome core particles

³¹P Nmr parameters (δ, T₁, W_1/2, and NOE) were measured for the DNA in nucleosome core particles at three frequencies and compared with similar data for the histone-free DNA. An essentially linear relationship was found between the frequency of observation and line-width for the single broad envelope of ³¹P resonances of the DNA in the nucleosome cores. We attributed this largely to chemical shift dispersion, with smaller contributions from chemical shift anisotropy and dipolar broadening. These results suggest the presence of different environments for phosphorus atoms in the core particles. However, within the accuracy of the method, no asymmetry in the resonance could be detected, which would tend to rule out any significant degree of DNA “kinking.” To investigate the interactions of the DNA and histones within the core particles we also studied transitions induced by urea and by temperature. Urea caused two stepwise increases in linewidth, which we attributed to conformational changes. A biphasic transition was also observed in the temperature profile, consistent with previous optical studies [Weischet et. al. (1978) Nucleic Acids Res. 5 , 139]. Various models with different types of local mobility were examined by the relaxation theory. A model of isotropic motion having a broad distribution of correlation times gave a fairly good fit to the ³¹P-nmr data.     

Adaptive cellular response to hyperthermia: 31P-NMR studies

Dynamic intracellular ATP and Pi levels were measured non-invasively for Chinese hamster V79 cells by 31P-NMR under conditions of thermotolerance and heat-shock protein induction. High densities of cells were embedded in agarose strands, placed within a standard NMR sample tube, and perfused with medium maintained either at 37 or 43 degrees C at pH 7.35. Cell survival and heat-shock protein synthesis were assessed either from parallel monolayer cultures or cells dislodged from the agarose strands post-treatment. Thermotolerance (heat resistance) and heat-shock protein synthesis was induced by a 1 h exposure to 43 degrees C followed by incubation for 5 h at 37 degrees C. After the 5 h incubation at 37 degrees C, marked thermal resistance was observed in regard to survival with concomitant synthesis of two major heat-shock proteins at 70 and 103 kDa. Studies were also conducted where tolerance and heat-shock protein synthesis were partially inhibited by depletion of cellular glutathione (GSH) prior to and during heat treatment. Dynamic measurement of intracellular ATP of cells heated with or without GSH depletion revealed no change in steady-state levels immediately after heating or during the 5 h post-heating incubation at 37 degrees C where thermotolerance and heat-shock proteins develop. These data are consistent with other reported data for mammalian cells and indicate that the steady-state ATP levels in mammalian cells remain unchanged during and after the acquisition of the thermotolerant state.     

31P-NMR saturation transfer studies of aerobic Escherichia coli cells

31P-NMR measurements of saturation transfer have been used to measure the flux between Pi and ATP in Escherichia coli cells respiring on an endogenous carbon source. Measurements were made in the wild type and in cells genetically modified to give a 5-fold higher concentration of the F1F0-ATP synthase. The flux in the two cell types was not significantly different. This, together with studies using inhibitors specific for the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase and the ATP synthase, suggests that the observed flux arises predominantly from glycolytic rather than ATP synthase activity. Although this conclusion is in disagreement with previous experiments on E. coli, it is in agreement with recent experiments on yeast.     

Energy state of chondrocytes assessed by 31P-NMR studies of preosseous cartilage

The energy state of resting and hypertrophic chondrocytes from growth plate was studied by 31P-NMR spectroscopy of superfused cartilage slices. The presence of phosphocreatine was demonstrated in both cell types, using a repetition time of 3 s. By comparing the decline in the nucleoside triphosphate level after adding blockers of the glycolysis or of the mitochondrial respiration, it was deduced that resting and hypertrophic chondrocytes use both metabolic pathways for energy production, but the glycolysis dominates. Hypertrophic cells rely more on the mitochondrial respiration than the resting cells.     

31P-NMR studies of Mycoplasma gallisepticum cells using a continuous perfusion technique

31P-NMR studies of Mycoplasma gallisepticum cells have been carried out using a continuous perfusion technique; these are the first such studies with this organism. Using this technique, glucose metabolism was monitored in the intact organisms, and cell extracts were prepared to identify the intermediates. Under glycolytic conditions, high levels of fructose-1,6-diphosphate were observed, indicating that this sugar may play a key role in the regulation of metabolism. The level of phosphoenolpyruvate was low under normal glycolytic conditions, and did not increase during starvation. From the position of the internal inorganic phosphate peak, the intracellular pH was estimated. The cells were found to maintain an intracellular pH of approximately 7.1 over an investigated external pH range of 6.6-8.6.     

31P-NMR studies of glucose and glutamine metabolism in cultured mammalian cells

31P-NMR measurements of the concentrations of phosphorus-containing metabolites in mammalian cells immobilised and perifused with glucose and glutamine as sole carbon source have shown that the intracellular Pi concentration is significantly higher in cells perifused with glutamine than with glucose. The data are consistent with the proposal that the rate of glutamine utilisation may be controlled by the activity of phosphate-activated glutaminase.     

1H- and 31P-NMR studies on smooth muscle of bullfrog stomach

³¹P-NMR spectra of bullfrog stomach smooth muscle showed peaks for creatine phosphate (4.8 μmol·g⁻¹ wet wt.), ATP (3.6), inorganic phosphate (P_i, 2.4), phosphomonoesters (3.0) and phosphodiesters (3.3). The intracellular pH was 7.3, and calculated from the chemical shift of P_i. ¹H-NMR spectra of smooth muscle yielded peaks of 2.9 for lactate, 6.6 for total creatine (creatine phosphate + creatine) and methyl protons of choline tentatively assigned to glycerolphosphorylcholine or to membrane phospholipids. Creatine phosphate and ATP decreased under anaerobic conditions, and intracellular acidification was observed with the concomitant increase in lactate. ³¹P saturation transfer studies showed that saturation of the γ-ATP resonance reduced the intensity of creatine phosphate to 60% of its control value, and the measured T₁ value of creatine phosphate was 2.4 s with saturation. The calculated forward flux of the creatine kinase reaction (decomposition direction of creatine phosphate) was 0.77 μmol·g⁻¹ wet wt.·s⁻¹. The creatine kinase flux was approx. 100-times larger than the ATP turnover rate, calculated from the oxygen consumption rate with the assumption, P/O = 3. In conclusion, the creatine kinase reaction is at equilibrium in resting smooth muscle of bullfrog stomach.     

31P-NMR studies of cerebral metabolic changes during graded hypoxia in newborn lambs

We measured cerebral phosphocreatine (PCr), inorganic phosphate (Pi), ATP, and intracellular pH (pHi) with in vivo phosphorus nuclear magnetic resonance (NMR) during 10- to 15-min periods of reversible hypoxic hypoxia in 20 newborn lambs (1-11 days). There was a significant correlation between arterial O2 partial pressure (PaO2) and the PCr/Pi ratio or pHi; however, between PaO2 130-33 mmHg, metabolite changes were not significant. PCr/Pi and pHi decreased significantly when PaO2 was lowered below 33 and 28 mmHg, respectively. After recovery, metabolite ratios and pHi returned to base-line values within 5 min. During the early phases of hypoxia and recovery, there were large fluctuations in metabolites and pHi, indicating that mitochondrial reactions were not in a steady state. After several minutes of hypoxia or recovery, PCr/Pi and pHi stabilized, suggesting steady state kinetics for mitochondrial respiration. NMR is extremely sensitive to changes in mitochondrial oxygenation, and stable PCr/Pi and pHi indicate that O2 tension in cerebral mitochondria of the newborn lamb is constant between PaO2 of 30 and 140 mmHg.     

Metabolic studies of mammalian cells by 31P-NMR using a continuous perfusion technique

Levels of ATP and Pi in metabolically active Chinese hamster lung fibroblasts were monitored noninvasively by 31P-NMR over many hours and under a variety of conditions. The cells were embedded in a matrix of agarose gel in the form of fine threads which were continuously perfused in a standard NMR tube. The small diameter of the thread allows rapid diffusion of metabolites and drugs into the cells. The changes in ATP and Pi levels were followed as a function of time in response to perfusion with a glucose-containing medium, with isotonic saline and with a medium containing 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation. This gel-thread perfusion method should enable routine NMR studies of cellular metabolism, and may have other potential biological applications.     

1H-, 13C- and 31P-NMR studies of dioctanoylphosphatidylcholine and dioctanoylthiophosphatidylcholine

Coupling constants and chemical shifts were measured for dioctanoylphosphatidylcholine and its thio analogue in a CDCl3/CD3OD solvent mixture. Replacing the bridging oxygen atom of the CH-CH2-O-P portion of the phosphatidylcholine molecule with a sulfur atom affects chemical shifts and coupling constants in the glycerol backbone portion of the molecule as well as in the choline head group region. Preferred conformations about selected bonds in the phospholipids were determined from the vicinal 1H-1H, 31P-1H and 31P-13C coupling constants. A reduction of the 31P T2* (effective spin-spin relaxation time) for the thio analogue, as well as changes in the relative chemical shifts of 13C nuclei in the acyl chains, suggest a somewhat greater degree of aggregation for the thio analogue. The quadrupolar coupling constant 1J(14N-13C) for the choline methyls of either analogue, however, indicates that aggregation of these phospholipids in the CDCl3/CD3OD solvent mixture is not significant. Differences in conformation between dioctanoylphosphatidylcholine and its thio analogue may be responsible for their differences in chemical and physical properties.     

DNA-phospholipid interaction. 31P-NMR study

The phage T7 DNA complexes with various phospholipids (PL) were studied by 31P NMR at PL/nucleotide molar ratio of 2 : 1. Using a phosphatidylcholine thion analogue, the contributions of PL and DNA into the 31P NMR spectrum of the complex were estimated. It was found that PL-DNA interaction results in partial immobilizing ability of PL depends on their structure, increasing in the following row: phosphatidylcholine-phosphatidylethanolamine less than sphingomyelin less than or equal to ternary mixture of these PL. The data obtained are indicative of nonequivalent binding of DNA with various PL species.     

31P-NMR and ESR studies of the oxidation states of manganese in Staphylococcus aureus

High-resolution 31P-NMR and ESR spectroscopies are used to probe the role of manganese in oxygen metabolism, in vivo, by Staphylococcus aureus. The linewidth of the intracellular orthophosphate resonance in the 31P-NMR spectrum and the amplitude of the ESR sextet of signals due to Mn2+ hexaquo ions are found to be sensitive to the oxygenation state of the cells. These results are attributed to changes in the oxidation state of the manganese. It is concluded that manganous ions are oxidized to Mn3+ in oxygenated cells. Mn3+ is in turn reduced to Mn2+ under anaerobic conditions. The Mn2+ is also oxidized to Mn3+ by hydrogen peroxide probably as a result of the disproportionation of H2O2 to H2O and O2 by an active catalase in S. aureus. Addition of mercaptoethanol to a suspension of oxygenated cells results in the reduction of Mn3+ to Mn2+.     

Quantitative analysis of phospholipids by 31P-NMR

High-field 31P nuclear magnetic resonance spectroscopy was used to quantitate phospholipids in mixtures in organic solvents. The sample is dissolved in chloroform-methanol and analyzed at 161.7 MHz with decoupling of the protons. Signals were identified using authentic compounds, and their relative distribution was measured in mole percent. The method has good accuracy and reproducibility, and was used to analyze phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylinositol, cardiolipin, and phosphatidic acid in egg lecithin. Four commercial egg phospholipids and the phospholipids from a total lipid extract of rat liver were analyzed. The method could be utilized to analyze phospholipids from other sources.     

31P-NMR study of natural phytoplankton samples

A new fixation method was developed for the Nuclear Magnetic Resonance (NMR) study of natural phytoplankton samples collected in situ. To test NMR reliability, a Chlorella continuous culture was used in a phosphorus deficiency recovery experiment. The method was then applied to natural metalimnetic cyanobacterial plankton. The maximum Entropy Method was used to enhance the generally poor signal to noise ratio resulting from the low amount of available material and NMR sensitivity. Suggestions are made on how to improve reliability.     

31P-NMR studies of the oral pathogen Streptococcus mutans: Observation of lipoteichoic acid

We have used ³¹P-nuclear magnetic resonance spectroscopy to identify phosphorus-containing compounds in whole cells of two serotype c strains of the oral pathogen Streptococcus mutans. The major resonance, centered at 0 ppm in whole cells, was attributed to lipoteichoic acid on the basis of its chemical shift, insensitivity to pH changes, cellular localization and a comparison with spectra obtained with purified lipoteichoic acid from S. mutans. The linewidths of resonances observed for intact cells and purified lipoteichoic acid were moderately narrowed by increasing the ionic strength, and substantially broadened in the presence of the lectin concanavalin A. Experiments with purified lipoteichoic acid suggest that this compound in whole cells is complexed with divalent cations such as Mg²⁺. Intracellular pools of other phosphorus-containing metabolites were found to be low when compared to the lipoteichoic acid concentration in both starved and glycolyzing cells.     

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.     

31P-NMR studies of the freeze-tolerant larvae of the gall fly, Eurosta solidaginis

31P NMR was applied to an examination of the freeze-tolerant larvae of the gall fly, Eurosta solidaginis. Resonances from sugar phosphates, inorganic phosphate, adenylates and arginine phosphate were identified. Two peaks of Pi were identified corresponding to intracellular and extracellular Pi. Anoxia produced an expected decrease in peak intensities of ATP and arginine phosphate while the peak of intracellular Pi was enhanced and shifted to indicate intracellular acidification during anoxia. Spectra of whole larvae were monitored over a temperature range from -30 degrees to +25 degrees C. No abrupt alterations in the spectra were seen at the point of extracellular freezing which occurs at about -8 degrees C but temperature had dramatic effects upon the peak intensities of ATP and arginine phosphate. A reversible increase/decrease in peak intensities, relative to Pi, was observed as temperature was raised/lowered. At 15 degrees and -20 degrees C, the beta peak of ATP was 64% and 2% of the peak intensity of Pi while that of arginine phosphate was 78% and 11%, respectively. This temperature effect was not an artifact of instrumentation (as model solutions containing Pi, ATP and arginine phosphate did not show this effect) or a result of changes in the total amounts of these compounds in the cell with temperature. Rather it is apparent that these molecules become restricted in their rotational movement as temperature is lowered perhaps via binding to subcellular components. Changes in the amounts of freely soluble ATP and arginine phosphate with temperature could have important implications for metabolism and its control. Analysis of the effect of temperature on the chemical shift of Pi was also used to determine pH in the intracellular and extracellular compartments. Temperature change had no effect on extracellular (hemolymph) pH which remained constant at 6.1-6.3. Intracellular pH varied with temperature, however, from pH 6.8 at 15 degrees C to pH 7.3 at -12 degrees C with a change, delta pH/delta 0, of -0.0185 degrees C consistent with alphastat regulation.