31P nuclear magnetic resonance spectroscopy of lipopolysaccharides from Pseudomonas aeruginosa

Intact lipopolysaccharide antigens isolated from seven different immunotypes of Pseudomonas aeruginosa have been examined by 31P-NMR spectroscopy. These macromolecular complexes contain phosphorus covalently attached to the carbohydrate residues present in the lipid A moiety and the 'core' oligosaccharide region. The spectral signals for various ortho- and pyrophosphoric esters were observed. All phosphate groups appeared to be monoesterified. Certain shifts characteristic for phosphate diester groups, observed in lipopolysaccharide complexes from other Gram-negative bacteria, were absent. Furthermore, no evidence was found to indicate that phosphate groups are involved in the covalent linkage of individual lipopolysaccharide complexes to form dimers or trimers.     

31P nuclear magnetic resonance spectroscopy study on kidney preservation. Effect of verapamil

31P NMR spectroscopy has been used to evaluate the usefulness of verapamil, a calcium channel blocker, in preventing ischemic renal damage. Phosphorylated metabolites have been investigated before, during and after 48 hrs of hypothermic storage. The rapidity in adenosine triphosphate resynthesis and the phosphomonoesters and phosphodiesters levels after reperfusion at the end of the storage period (48 hrs), were significantly higher in verapamil-treated kidneys. Phosphomonoesters to inorganic phosphate ratio, during the storage period, is even higher. These findings suggest that verapamil may protect against ischemic renal damage and so it can be useful for renal preservation. Furthermore, it has been shown that 31P NMR spectroscopy puts into evidence the biochemical recovery and allows the assessment of the viability of organs.     

31P nuclear magnetic resonance study of enzymatically phosphorylated glycophorin A

Glycophorin A was phosphorylated using protein kinases and the new protein was investigated using³¹P NMR spectroscopy. Most of these ～30 moles of phosphate were found to be attached to Ser and Thr. Some of these phosphate residues appear to be affected by the carbohydrate residues present. The phosphorylated protein appears to be in a severe state of aggregation, with the degree of aggregationpH-dependent.     

Comparison of suspended and immobilized yeast metabolism using 31P nuclear magnetic resonance spectroscopy

Summary ³¹P nuclear magnetic resonance has been employed to monitor noninvasively Saccharomyces cerevisiae anaerobic glucose metabolism in suspended and immobilized cells. Results show that cell entrapment in Ca-alginate beads alters cell metabolism compared to that in suspended cells. Assuming similar intracellular ionic strength, differences in intracellular phosphate chemical shift indicate that the internal pH of the immobilized cells is lower than the suspended cell internal pH. This result is consistent with higher ethanol production rates exhibited by immobilized yeast.     

Direct in vivo measurement of absolute metabolite concentrations using 31P nuclear magnetic resonance spectroscopy

Whilst in vivo NMR spectroscopy provides much useful biochemical information, a limitation to such studies has been the difficulty in quantitating the results to obtain absolute metabolite concentrations. We report here a simple direct method to obtain absolute metabolite concentrations when using in vivo NMR with radiofrequency surface coils. The method has been validated for nucleoside triphosphates in two tissues; rat brain and skeletal muscle. The results obtained are in close agreement with nucleoside triphosphate concentrations obtained using other methods. Precautions for the accurate application of the method are discussed. This method can be applied to other metabolites, coils and NMR nuclei.     

Determination of the transmembrane proton gradient in the anaerobic bacterium Desulfovibrio desulfuricans by 31P nuclear magnetic resonance

The transmembrane proton gradient of the sulfate-reducing bacterium Desulfovibrio desulfuricans strain CSN has been determined by in vivo³¹P nuclear magnetic resonance (NMR) spectroscopy in the absence of dioxygen. At pH 7.0 in the medium (pH_ex) the intracellular pH (pH_in) was 7.5. By lowering pH_ex to 5.9 pH_in decreased to 7.1. At pH_ex greater than 7.7 the transmembrane proton gradient (pH) was zero. The uncouplers 3,3,4,5-tetrachlorosalicylanilide (TCS) and carbonylcyanide-m-chlorophenylhydrazone (CCCP), or the permeant anion thiocyanate caused complete dissipation of pH.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - TCS 3,3,4,5-tetrachlorosalicylanilide - MOPS 3-(N-morpholino)-propanesulfonic acid - P _i inorganic phosphate - pH _in (pH_ex) intracellular (extracellular) pH - pH transmembrane proton gradient (pH_in-pH_ex) - electrochemical membrane potential - chemical shift in parts per million - NMR nuclear magnetic resonance     

Phosphorus-31 nuclear magnetic resonance spectroscopy of toad retina.

Phosphorus-31 nuclear magnetic resonance (31P-NMR) spectra were obtained from living toad retinae and toad retinal extracts at 4 degrees C. Several phosphorus metabolites--nucleoside di- and triphosphates (NTP), phosphocreatine, phosphodiesters, inorganic phosphate, and phosphomonoesters--were identified from the spectra of whole retinae. The intracellular pH was determined to be 7.27 +/- 0.06 at 4 degrees C and the intracellular MgNTP/NTP ratio was at least 0.77. These results are consistent with those reported by other techniques, and they show that 31P-NMR spectroscopy can be used for noninvasively and quantitatively studying the metabolism of living toad retinae, and for monitoring its changes over time.     

Intracellular pH in Dictyostelium discoideum: a 31P nuclear magnetic resonance study

We have used phosphorus-31 nuclear magnetic resonance to determine intracellular pH in the cellular slime mold Dictyostelium discoideum. We devised an air-lift circulator to maintain the dense cell suspensions in a well-oxygenated and well-stirred state while causing minimal perturbation to the sample flowing through the detector coils. Cells continued to develop normally in this set-up. Spectra acquired under these conditions typically show two peaks in the inorganic phosphate region corresponding to pH values of 7.16 +/- 0.03 and 6.48 +/- 0.02. These peaks are believed to represent the mitochondrial and cytosolic compartments respectively, based on a comparison of these values with published data and the collapse of the two compartments upon addition of the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone. Dictyostelium cells show a remarkable degree of intracellular pH homeostasis. Both mitochondrial and cytosolic pH remained unchanged as extracellular pH was varied from 4.3 to 8.1. There was also no apparent change in the pH of either compartment after up to 13.5 hours' development in suspension.     

Application of31P nuclear magnetic resonance spectroscopy to investigate plasmid effects onEscherichia coli metabolism

Summary Glucose metabolism inE. coli strain HB101, as a plasmid-free cell and as a host to two plasmids of different copy numbers, has been characterized using³¹P NMR. While the low-copy-number strain was found to behave very similarly to the plasmid-free strain, dramatically different behavior was exhibited by the high-copy-number strain. This strain maintained a nearly constant intracellular pH after addition of glucose to a starved suspension while intracellular pH of the other strains dropped considerably. The inorganic phosphate level in the high-copy-number strain was substantially higher than in the other strains, and the NTP level was much lower. Glycolytic rates of all three strains, however, were nearly identical. The trend in glycolytic rate strongly suggests that transport of glucose into the cell is the rate-limiting step under these conditions.     

Estimation of intracellular sugar phosphate concentrations in Saccharomyces cerevisiae using 31P nuclear magnetic resonance spectroscopy

A systematic procedure has been formulated for estimating the relative intracellular concentrations of sugar phosphates in Saccharomyces cerevisiae based upon (31)P nuclear magnetic resonance (NMR) measurements. The sugar phosphate region of the (31)P NMR spectrum is first decomposed by computer analysis, and the decomposition consistency and identification of individual sugar phosphate resonances are established based on in vitro chemical shift calibrations determined in separate experiments. Numerous evaluations of intracellular S. cerevisiae compositions for different strains and different cell environments provide the basis for in vivocorrelations of inorganic phosphate chemical shift with the chemical shifts of 3-phosphoglycerate, beta;-fructose 1,6-diphosphate, fructose 6-phosphate, and glucose 6 phosphate. Relative intracellular sugar phosphate concentrations are obtained by correcting peak areas for partial saturation during transient in vivo experiments. In vivo concentrations estimated by this method agree well with estimates for similar systems based on other techniques. This approach does not require costly la belled compounds, and has the advantage that other important metabolic state variables such-as internal and external pH and intracellular levels of phosphate, ATP, ADP, NAD(H), and polyphosphate may be determined from the same (31)P spectrum. Extension of this strategy to other cellular systems should be straightforward.     

A quantitative study of organic phosphate-amine interaction by 31P nuclear magnetic resonance

Interactions between the phosphate group of 4-deoxypyridoxine 5′-phosphate and different protonated amines were quantitatively measured by means of {³¹P}-¹H nuclear magnetic double resonance technique combined with pD titration. An interaction of the phosphate group with added amine resulted in a measurable difference in the ³¹P chemical shift of these phosphate-containing samples with and without amine [Δδ(³¹P)]. Basic amino acids and biogenic amines had significant measurable Δδ(³¹P) values. No interactions were observed for acidic or neutral α, β and γ-amino acids.     

31P nuclear magnetic resonance study of growth and dimorphic transition in Candida albicans

A 31P NMR study of the fungal pathogen Candida albicans was carried out. Yeast-form cells at different phases of growth, as well as germ tubes and hyphae were examined. In all cases, the NMR spectra showed well separated resonance peaks arising from phosphorus-containing metabolites, the most prominent being attributable to inorganic phosphate (Pi) polyphosphates, sugar phosphates and mononucleotides, NAD, ADP and ATP. Relevant signals were also detected in the phosphodiester region. The intensity of most signals, as measured relative to that of Pi, was clearly modulated both at the different phases of growth and during yeast-to-mycelium conversion, suggesting significant changes in the intracellular concentration of the corresponding metabolites. In particular, the intensity of the polyphosphate signal was high in exponentially growing, yeast-form cells, then progressively declined in the stationary phase, was very low in germ tubes and, finally, undetectable in hyphae. NMR spectral analysis of the Pi region showed that from early-stationary phase, Pi was present in two different cellular compartments, probably corresponding to the cytoplasm and the vacuole. From the chemical shift of Pi, the pH values of these two compartments could be evaluated. The cytoplasmic pH was generally slightly lower than neutrality (6.7-6.8), whereas the vacuolar pH was always markedly more acidic.     

Use of 31P nuclear magnetic resonance spectroscopy and electron microscopy to study phosphorus metabolism of microorganisms from wastewaters

We used 31P nuclear magentic resonance to study the transfer of phosphorus between Pis and polyphosphates in microorganisms involved in wastewater treatment. We showed that the transfer process is reversible and of the first order in accordance with the polyphosphate concentration. The presence of nitrates in the anoxic phase led to results similar to those obtained during the aerobic phase. (Anoxic implies absence of oxygen but presence of nitrate, whereas anaerobic implies absence of oxygen and nitrate. In bacteriology, the term anoxic is not common, and the term anaerobic implies absence of oxygen and includes the conditions under which nitrate is present.) We observed that carbon dioxide lowers the pH, which entails a hydrolysis of polyphosphates, and helium seems to stop the evolution of the cells. Further, 2,4-dinitrophenol decouples the oxidative phosphorylation and brings about a decrease in the polyphosphate pool.     

87Rb, 23Na and 31P nuclear magnetic resonance spectroscopy of the perfused rat kidney

87Rb, 23Na and 31P nuclear magnetic resonance (NMR) were used to monitor changes in renal cations and energetics during the induction of hypoxia in the isolated perfused rat kidney. The NMR-determined unidirectional Rb+ flux in normoxic kidneys was shown to be a good measure of net intracellular K+ influx in the perfused rat kidney model. The changes in 87Rb, 23Na and 31P spectra following the induction of hypoxia are consistent with hypoxic depletion of intracellular adenosine triphosphate (ATP) and a subsequent decrease in Na-K-ATPase transport activity. The exponential rate constant for 87Rb+ efflux measured during Rb+ uptake in normoxic kidneys (0.12 +/- 0.01 min-1) was not significantly different to the rate constant for 87Rb+ efflux during the induction of hypoxia (0.16 +/- 0.07 min-1). We conclude that there is no direct effect of hypoxia on renal cellular membrane integrity and that renal cell sensitivity to hypoxia is due to an inability to sustain cellular ion gradients following depletion of intracellular ATP.     

Measurement of adenosine triphosphate and 2,3-diphosphoglycerate in stored blood with 31P nuclear magnetic resonance spectroscopy

Conditions for blood storage are chosen to assure adequate levels of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG). Because of the invasive nature of the techniques, biochemical assays are not routinely used to measure levels of these compounds in stored blood. However, 31P NMR spectroscopy measures phosphorylated intermediates in intact cells and could be used without disruption of the storage pack. We compared levels of ATP and 2,3-DPG measured by 31P spectroscopy and standard enzyme-linked biochemical assays in whole blood (WB) and packed red blood cells (PRBCs) at weekly intervals during a 35-day storage period. NMR demonstrated a marked decrease in 2,3-DPG and an increase in inorganic phosphate after the first week of storage. No significant differences in ATP concentrations were seen in WB during the storage period, but a significant decrease in ATP in PRBCs was documented. There was good agreement in levels of ATP and 2,3-DPG measured by NMR and biochemical techniques. 31P NMR spectroscopy is a noninvasive technique for measuring ATP and 2,3-DPG which has a potential use in quality assurance of stored blood.     

Characterization of phosphorus in algae from a eutrophic lake by solution 31P nuclear magnetic resonance spectroscopy

Limnology - The identification and quantification of phosphorus (P) compounds derived from algal biomass are crucial for a better understanding of algal P dynamics in lake ecosystems. Quantity and...     

Phosphorothioate analogues of 5-phosphoribosyl 1-diphosphate: 31P nuclear magnetic resonance study

31P nuclear magnetic resonance (NMR) has been used to study the 1-phosphorothioate analogues of 5-phosphoribosyl 1-diphosphate (P-Rib-PP). Comparison of the proton-decoupled spectra of 5-phosphoribosyl 1-O-(2-thiodiphosphate) (P-Rib-PP beta S) and the SP diastereomer of 5-phosphoribosyl 1-O-(1-thiodiphosphate) (P-Rib-PP alpha S) with the parent molecule revealed a characteristic large downfield chemical shift change for the resonance signal associated with the thiophosphate group (delta delta approximately 40-50 ppm) and an increase in the magnitude of the phosphate-thiophosphate spin-spin coupling constant (delta J alpha beta approximately 10 Hz). Both these changes are consistent with the observed effects of sulfur substitution on the behavior of the adenosine nucleotides, particularly ADP [Jaffe, E. K., & Cohn, M. (1978) Biochemistry 17, 652-657]. High-field 31P NMR has also been used to demonstrate the diastereomeric purity of P-Rib-PP alpha S (Sp diastereomer) and the greater lability of this analogue when compared with both P-Rib-PP beta S and P-Rib-PP. Sulfur substitution was found to cause a large decrease in the apparent pKa associated with the thiophosphate moiety of P-Rib-PP beta S (delta pKa approximately 1.4 units) and also to enhance the sensitivity of the thiophosphate chemical shift to protonation and, in particular, to Mg2+ binding, compared with P-Rib-PP. The potential application of the phosphorothioate analogues as probes of the reactions catalyzed by the phosphoribosyltransferase enzymes is discussed.