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.     

Phosphorus-31 Fourier transform nuclear magnetic resonance study of mononucleotides and dinucleotides. 1. Chemical shifts.

A phosphorus-31 nuclear magnetic resonance (NMR) study of adenine, uracil, and thymine mononucleotides, their cyclic analogues, and the corresponding dinucleotides is reported. From the pH dependence of phosphate chemical shifts, pKa values of 6.25-6.30 are found for all 5'-mononucleotides secondary phosphate ionization, independently from the nature of the base and the presence of a hydroxyl group at the 2' position. Conversely, substitution of a hydrogen atom for a 2'-OH lowers the pKa of 3'-monoribonucleotides from 6.25 down to 5.71-5.85. This indication of a strong influence of the 2'-hydroxyl group on the 3'-phosphate is confirmed by the existence of a 0.4 to 0.5 ppm downfield shift induced by the 2'-OH on the phosphate resonance of 3'-monoribonucleotides, and 3',5'-cyclic nucleotides and dinucleotides with respect to the deoxyribosyl analogues. Phosphate chemical shifts and titration curves are affected by the ionization and the type of the base. Typically, deviations from the theoretical Henderson-Hasselbalch plots are observed upon base titration. In addition, purine displays a more deshielding influence than pyrimidine on the phosphate groups of most of the mononucleotides (0.10 to 0.25 ppm downfield shift) with a reverse situation for dinucleotides. These effects together with the importance of stereochemical arrangement (furanose ring pucker, furanose-phosphate backbone conformation, O-P-O bond angle) on the phosphate chemical shifts are discussed.     

Phosphorus-31 nuclear magnetic resonance of insect hemolymph sera

Sera from larval and pupal stages of the tobacco hornworn, Manduca sexta, have been investigated using phosphorus-31 pulsed Fourier transform nuclear magnetic resonance. Spectra of larval and pupal sera containing 5 mm EDTA were characterized by four major peaks and one or more minor resonances. A phosphorus-31 spectrum of dialyzed larval serum showed several weak signals which indicated the presence of some higher-molecular-weight phosphorylated compounds as well. None of those signals, however, corresponded to any of the ones seen with undialyzed sera. Three of the four prominent peaks and one minor peak in the whole larval serum had the same chemical shifts as those in the pupal samples. The pupal sera, in addition, displayed an extra peak well upfield from those of the larval stage. All of the low-molecular-weight resonances detectable in the hemolymphs have been identified and included four compounds not previously reported; trehalose-6-phosphate, phosphoarginine, phosphatidylcholine, and phosphatidylethanol-amine. The phosphometabolites found at millimolar or higher concentrations in larval hemolymph were α-glycerolphosphate, phosphorylcholine, phosphorylethanolamine, inorganic phosphate, trehalose-6-phosphate, phosphatidylcholine, and phosphatidylethanolamine. All of the above compounds were found in pupal sera as well except for the addition of phosphoarginine and the deletion of phosphorylethanolamine. The levels of the phosphometabolites in common between the two stages of development, however, were quite different as were their stabilities after extraction. While the intensities of the larval phosphates remained virtually constant in the presence of EDTA at pH 7.8, those of the pupal sera changed rapidly. This was especially true for arginine phosphate which disappeared quickly.     

Phosphorus-31 nuclear magnetic resonance spectroscopy of extracellular, yeast O-phosphonohexoglycans.

P nuclear magnetic resonance spectra of a number of purified yeast O-phosphonohexoglycans were recorded. The data therefrom were correlated with established chemicals aspects of individual and collective polymer structures, permitting (a) conclusions to be drawn regarding the chemical environment of the phosphate groups of these polymers, and (b) assignment of anormeric configurations to the hexosyl phosphate residues.     

Phosphorus-31 nuclear magnetic resonance study of the thiamine diphosphate-magnesium interaction

The results obtained from a phosphorus NMR study of the interactions of Mg²⁺ with thiamine diphosphate confirm the existence of a Mg-thiamine diphosphate complex with a $\text{1}\text{1}$ stoichiometry in which the α phosphorus seems to be the most influenced by the interaction. The variations of δ and J_αβ with various concentrations of Mg²⁺ are described.     

A conformational study of nucleic acid phosphate ester bonds using phosphorus-31 nuclear magnetic resonance.

A systematic phosphorus-31 nuclear magnetic resonance study of some nucleic acid constituents (6-N-(dimethyl)adenylyl-(3',5')-uridine and some nucleotide methyl esters) is presented. The temperature dependent phosphorus-31 chemical shifts were analyzed by standard thermodynamic procedures. It is shown that gt conformations about the P-O ester bonds have a lower free energy content relative to gg conformers.     

Phosphorus-31 nuclear magnetic resonance studies of active proton translocation in chromaffin granules.

ATP hydrolysis and proton translocation in chromaffin granules were followed using 31P nuclear magnetic resonance. The intragranular pH affects the resonance frequency of the gamma-phosphate of granular ATP. By measuring frequency vs. pH in solutions which simulate the intragranular matrix, this may be calibrated to give quantitative pH measurements. The pH in the resting granule is 5.65 +/- 0.15. This drops by 0.4 to 0.5 pH unit when ATP is added externally and protons are actively pumped into the granules. Because of differences in the composition and pH of the internal and external solutions, the resonances of internal and external nucleotides and Pi can be distinguished. Consequently, ATP hydrolysis and changes in internal pH may be observed simultaneously and continuously in a single sample of chromaffin granules. From the measured buffering capacity of a reconstituted intragranular solution, pH changes were converted into an absolute number of protons translocated. The net proton flux (protons translocated/ATP hydrolyzed) was about 1.0 immediately after external ATP addition but fell toward zero as the pH gradient increased to a new steady state. These 31P NMR results agree with intragranular pH measurements determined from methylamine distribution and with H+/ATP stoichiometries calculated from pH changes observed in the external medium.     

NMR in cancer: VIII. Phosphorus-31 as a nuclear probe for malignant tumors

Spin-lattice relaxation times (T1) for 31P were determined in normal and malignant tissues by a saturation technique employing a 90 degree -tau-90 degrees pulse sequence. Results for five normal tissues from rat were (in seconds): 2.33 +/- .14 for liver; 2.19 +/- .05 for muscle; 1.13 +/- .05 for brain; 1.43 +/- .15 fro kidney; and 1.97 +/- .12 for intestine. Results for two rat malignancies, Novikoff hepatoma and Walker sarcoma, were 5.98 +/- .57 and 5.38 +/- .68, respectively, and for Crocker sarcoma of mouse, 5.19 +/- 1.42. No individual measurement of malignant tissue overlapped any of the normal measurements; probabilities of insignificance ranged from .029 for Crocker sarcoma to .000184 for Novikoff hepatoma. The data call attention to another nucleus of potential value for NMR detection of internal malignancies in humans. Also suggested, because of the strategic placement of the 31P nucleus in the nucleic acid molecule, is a possible new probe for exploring the mechanism of carcinogenesis.     

Phosphorus-31 nuclear magnetic resonance of aspartate aminotransferase from chicken heart cytosol

31P-nuclear magnetic resonance and absorption spectra of cytosolic chicken aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) have been recorded in the pH range from 5 to 8.5. The 31P chemical shift was found to be pH-dependent with a pK of 6.85; the chemical shift change was 0.35 ppm. The pK value found by spectrophotometric titration of the enzyme proved to be about 6.0. The monoanion-dianion transition of the 5'-phosphate group of a model Schiff base of pyridoxal phosphate with 2-aminobutanol in methanol is accompanied by a change in the 31P chemical shift of 5.2 ppm. It is inferred that the phosphate group of the protein-bound coenzyme is in a dianionic form throughout the investigated pH range; the pH-dependence of the 31P chemical shift may be due to a conformational change at the active site. In the presence of 100 mM succinate, 6 mM aminooxyacetate or 25 mM cycloserine, the 31P chemical shift is insensitive to pH variations.     

Phosphorus-31 nuclear magnetic resonance study of D-serine dehydratase: pryridoxal phosphate binding site.

The pyridoxal phosphate dependent enzyme D-serine dehydratase has been investigated using 31P nuclear magnetic resonance (NMR) at 72.86 MHz. In the native enzyme, the pyridoxal phosphate 31P chemical shift is pH dependent with pKa = 6.4, indicating exposure of the phosphate group to solvent. Binding of the competitive inhibitor isoserine results in the formation of the isoserine-pyridoxal phosphate complex. This transaldimination complex is fixed to the enzyme via the phosphate group of the cofactor as the dianion, independent of pH. At pH 6.6 the dissociation constant KD for isoserine determined by NMR is 0.43 mM. Reconstitution of the apoenzyme with pyridoxal phosphate monomethyl ester produces an inactive enzyme. NMR and fluorescence measurements show that this enzyme does not form the transaldimination complex, indicating that the fixation of the dianionic phosphate (probably via a salt bridge with an arginine residue) observed in the native enzyme is required for the transaldimination step of the catalytic mechanism.     

Phosphorus-31 nuclear magnetic resonance studies of wild-type and glycolytic pathway mutants of Saccharomyces cerevisiae.

High-resolution phosphorus-31 nuclear magnetic resonance (31P NMR) spectra of wild-type and mutant strains of Saccharomyces cerevisiae were observed at a frequency of 145.7 MHz. Levels of various phosphorus metabolites were investigated upon addition of glucose under both aerobic and anaerobic conditions. Three mutant strains were isolated and their biochemical defects characterized: pfk lacked phosphofructokinase activity; pgi lacked phosphoglucose isomerase activity; and cif had no glucose catabolite repression of the fructose bisphosphatase activity. Each mutant strain was found to accumulate characteristic sugar phosphates when glucose was added to the cell suspension. In the case of the phosphofructokinase deficient mutant, the appearance of a pentose shunt metabolite was observed. 31P NMR peak assignments were made by a pH titration of the acid extract of the cells. Separate signals for terminal, penultimate, and central phosphorus atoms in intracellular polyphosphates allowed the estimation of their average molecular weight. Signals for glycero(3)phosphochline, glycero(3)phosphoserine, and glycero(3) phosphoethanolamine as well as three types of nucleotide diphosphate sugars could be observed. The intracellular pH in resting and anaerobic cells was in the range 6.5--6.8 and the level of adenosine 5'-triphosphate (ATP) low. Upon introduction of oxygen, the ATP level increased considerably and the intracellular pH reached a value of pH 7.2--7.3, irrespective of the external medium pH, indicating active proton transport in these cells. A new peak representing the inorganic phosphate of one of the cellular organelles, whose pH differed from the cytoplasmic pH, could be detected under appropriate conditions.     

Transport and phosphorylation of choline in higher plant cells. Phosphorus-31 nuclear magnetic resonance studies

When sycamore cells were suspended in basal medium containing choline, the latter was taken up by the cells very rapidly. A facilitated diffusion system appertained at low concentrations of choline and exhibited Michaelis-Menten kinetics. At higher choline concentrations simple diffusion appeared to be the principal mode of uptake. Addition of choline to the perfusate of compressed sycamore cells monitored by 31P NMR spectroscopy resulted in a dramatic accumulation of P-choline in the cytoplasmic compartment containing choline kinase and not in the vacuole. The total accumulation of P-choline over a 10-h period exhibited Michaelis-Menten kinetics. During this period, in the absence of Pi in the perfusion medium there was a marked depletion of glucose-6-P, and the cytoplasmic Pi resonance disappeared almost completely. When a threshold of cytoplasmic Pi was attained, the phosphorylation of choline was sustained by the continuous release of Pi from the vacuole although at a much lower rate. However, when 100 microM inorganic phosphate was present in the perfusion medium, externally added Pi was preferentially used to sustain P-choline synthesis. It is clear, therefore, that cytosolic choline kinase associated with a carrier-mediated transport system for choline uptake appeared as effective systems for continuously trapping cytoplasmic Pi including vacuolar Pi entering the cytoplasm.     

Phosphorus-31 nuclear magnetic resonance study of polyphosphate metabolism in intact ectomycorrhizal fungi

Summary Phosphorus-31 nuclear magnetic resonance spectra at 36.4 MHz are presented for intact ectomycorrhizal fungi grown in pure culture. Resonances from polyphosphates and intracellular orthophosphate are identified inCenococcum graniforme, Hebeloma cylindrosporum, andH. crustuliniforme. Comparison of the NMR spectra with phosphorus fractionation of the fungi extracts leads to the statement that the NMR-observed polyphosphaes is a good part of the accumulated polyphosphates. In actively growing mycelia, this fraction account for up to 17% of total P.     

Hypophosphite ion as a 31P nuclear magnetic resonance probe of membrane potential in erythrocyte suspensions.

Hypophosphorus acid has a single pKa of 1.1 and at physiological pH values it is therefore present almost entirely as the univalent hypophosphite ion. When added to a red cell suspension the ion crosses the cell membrane rapidly, via the anion exchange protein, and the intra- and extracellular populations of the ion give rise to separate 31P NMR resonances. From a single 31P NMR spectrum it was possible to determine the relative amounts of hypophosphite in the intra- and extracellular compartments and thereby estimate the corresponding concentrations. The ratio of intracellular to extracellular hypophosphite concentration was independent of the total hypophosphite concentration for cells suspended in NaCl solutions and was independent of hematocrit. The hypophosphite distribution ratio increased as extracellular NaCl was replaced iso-osmotically with citrate or sucrose, through it remained very similar to the corresponding hydrogen ion distribution ratio. Incorporation of the hypophosphite distribution ratio into the Nernst equation yielded an estimate of the membrane potential. For cells suspended in NaCl solutions the estimated potential was consistently around -10 mV.     

Phosphorus-31 nuclear magnetic resonance of phosphoenzymes of sodium- and potassium-activated and of calcium-activated adenosinetriphosphatase

Prior studies identified phosphoenzyme intermediates in the turnover of sodium- and potassium-activated adenosinetriphosphatase [(Na,K)ATPase] from several sources and of the calcium-activated adenosinetriphosphatase [(Ca)-ATPase] of skeletal muscle sarcoplasmic reticulum. In both cases, the transphosphorylation is to a beta-aspartyl carboxyl group at the active site. We now report observation of a K+-sensitive phosphorylated intermediate of purified (Na,-K)ATPase from the salt gland of the duck using high-field 31P nuclear magnetic resonance. Addition of ATP to a suspension of this enzyme in the presence of Mg2+ and Na+ produced a resonance at about +17 ppm relative to 85% phosphoric acid. Addition of inorganic phosphate and Mg2+ to (Na,K)ATPase also produced a resonance at about +17 ppm which was enhanced in the presence of a saturating concentration of the inhibitor, ouabain; again, addition of K+ made this resonance disappear. These findings are consistent with earlier kinetic characterization of an acid-stable (Na,K)ATPase phosphoenzyme intermediate by 32P-labeled phosphate incorporation into a denatured precipitate of the enzyme. We attribute the +17-ppm resonance to formation of an acyl phosphate at an aspartyl residue of the catalytic site of (Na,K)ATPase. This is supported by our finding of a similar resonance at +17 ppm after phosphorylation of another membrane-bound cation transport enzyme, sarcoplasmic reticulum (Ca)ATPase, as well as by a similar resonance at about +17 ppm after phosphorylation of the model dipeptide L-seryl-L-aspartate.     

Biochemical changes during sucrose deprivation in higher plant cells. Phosphorus-31 nuclear magnetic resonance studies

An experimental arrangement was described that enables nuclear magnetic resonance spectra of compressed plant cells to be recorded while circulating a medium through the sample. The system provided a convenient arrangement for monitoring by 31P NMR the behavior of plant cells over a long period of time under different conditions such as sucrose starvation. Perfusion of compressed sycamore cells with sucrose-free culture medium triggered a progressive decrease in the glucose 6-P and uridine-5'-diphosphate-alpha-D-glucose resonances over 30 h. When almost all the intracellular carbohydrate pool had disappeared the nucleotide triphosphate resonances decline progressively. These changes were accompanied by a Pi accumulation in the vacuole and a phosphorylcholine (P-choline) accumulation in the cytoplasm. The very long lag phase observed for ATP and P-choline evolution was comparable with that observed for the progressive intracellular digestion of cytoplasmic constituents (Journet, E., Bligny, R. and Douce, R. (1986) J. Biol. Chem. 261, 3193-3199). Addition of sucrose in the circulating system after a long period of sucrose starvation led to a disappearance of the cytoplasmic Pi resonance and a marked increase in that of glucose 6-P. Under these conditions the vacuolar Pi pool did not fluctuate to buffer the Pi in the cytoplasm. The results suggest that Pi which has been sequestered in the vacuole during the course of sucrose starvation is not restored to the cytoplasm for rapid metabolic processes. Furthermore, the presence of P-choline in plant cells in large excess should be considered as a good marker of membrane utilization after a long period of sucrose starvation and is very likely related to stress.     

Phosphorus-31 nuclear magnetic resonance study on cytoplasmic aspartate aminotransferase from pig heart. A reinvestigation

An average target size of 251 kDa has been obtained for the (Ca2+ + Mg2+)-ATPase of calmodulin-depleted erythrocyte ghosts by radiation inactivation with 16 MeV electrons. This is close to twice the size of the purified calcium-pump polypeptide. When calmodulin was included during the ATPase assay, a component of about 1 MDa appeared in addition to the activated dimer.     

Phosphorus-31 nuclear magnetic resonance of highly oriented DNA fibers. 2. Molecular motions in hydrated DNA

31P nuclear magnetic resonances (NMR) of salmon sperm DNA, poly(rA).poly(rU), and poly(rA).poly(dT) fibers were measured as a function of relative humidity. The results indicated that the spectra were strongly perturbed by the molecular motions occurring in the hydrated fibers. The humidity dependence of the spectra at a number of orientations of the fibers relative to the magnetic field was reasonably explained by taking into account at least three motional modes, namely, conformational fluctuations, restricted rotation about a tilted axis, and rotational diffusion about the helical axis. The rotational diffusion about the helical axis was found to perturb the spectral line shapes most strongly, and its constants were 1.5 X 10(4) and 5.0 X 10(4) S-1 for DNA fibers at 92% and 98% relative humidities, respectively. A DNA-RNA hybrid, poly(rA).poly(dT), has been shown to adopt different conformations on two strands at high relative humidity [Zimmerman, S. B., & Pheiffer, B. H. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 78-82], which was unquestionably confirmed in the present study: that is, the 31P NMR spectra from the hydrated form of this polymer were clearly explained by assuming that one strand had an A-like conformation and the other a B-like conformation.