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 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 human retinoblastoma cells: correlation with metabolic indices

The 31P nuclear magnetic resonance spectrum of cultured human Y-79 retinoblastoma cells was obtained at 121 MHz on intact cells trapped in agarose threads. The spectrum was dominated by monoester peaks, which varied in relative concentration from preparation to preparation. Resonances from phosphocreatine, phosphodiesters and diphosphodiesters also exhibited variability relative to ATP. The main monoester was identified as phosphorylcholine by 31P-NMR of perchloric acid extracts. It was determined that the changes in monoester concentration correlated with feeding pattern. Phosphorus spectra of cells 1, 2 and 3 days post feeding showed a 40% decrease in the relative concentration of phosphorylcholine concentration over the 3 day period. Phosphocreatine, phosphodiesters and diphosphodiesters increased relative to ATP during the same period. Growth curve experiments and oxygen consumption measurements indicated that the decrease in phosphorylcholine correlated with a decrease in cellular growth and oxygen consumption. We conclude that monoester concentration may be a useful indicator of nutritional status in these cells and possibly in intact tumors.     

Phosphorus-31 nuclear magnetic resonance spectroscopy reveals two conformational forms of chloroacetol phosphate-bound triosephosphate isomerase

Chloroacetol phosphate covalently reacts with Glu-165 in the catalytic center of triosephosphate isomerase. Reaction of the enzyme with the substrate analogue results in two 31P resonances at 6.8 and 5.5 ppm. Dissociation with guanidinium chloride results in a single resonance at 4.5 ppm. Reassociation and redimerization of the triosephosphate isomerase-chloroacetol phosphate complex restores only the resonance at 5.5 ppm. The two 31P resonances appear to represent different conformations of the enzyme which are trapped upon reaction with the affinity label.     

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.     

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 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.     

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 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.     

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 double- and triple-helical nucleic acids. Phosphorus-31 chemical shifts as a probe of phosphorus-oxygen ester bond torsional angles

The temperature dependence to the 31P NMR spectra of poly[d(GC)] . poly [d(GC)],d(GC)4, phenylalanine tRNA (yeast) and mixtures of poly(A) + oligo(U) is presented. The 31P NMR spectra of mixtures of complementary RNA and of the poly d(GC) self-complementary DNA provide torsional information on the phosphate ester conformation in the double, triple, and "Z" helix. The increasing downfield shift with temperature of the single-strand nucleic acids provides a measure of the change in the phosphate ester conformation in the single helix to coil conversion. A separate upfield peak (20-60% of the total phosphates) is observed at lower temperatures in the oligo(U) . poly(A) mixtures which is assigned to the double helix/triple helix. Proton NMR and UV spectra confirm the presence of the multistrand forms. The 31P chemical shift for the double helix/triple helix is 0.2-0.5 ppm upfield from the chemical shift for the single helix which in turn is 1.0 ppm upfield from the chemical shift for the random coil conformation.     

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.     

Phosphorus-31 nuclear magnetic resonance analysis of internal pH during photosynthesis in the cyanobacterium Synechococcus

Phosphorus-31 nuclear magnetic resonance (31P NMR) spectra were obtained from actively photosynthesizing and darkened suspensions of the unicellular cyanobacterium Synechococcus. These spectra show intracellular resonances belonging to inorganic phosphate (Pi), a sugar phosphate (sugar-P), nucleotide di- and triphosphates, and poly-phosphates. The pH-dependent chemical shifts of Pi and sugar-P allowed the estimation of intracellular pH. When irradiated with high-intensity tungsten-halogen light (100 x 10(4) ergs . cm-2 . s-1, measured in the visible range), concentrated cell suspensions in the NMR spectrometer incorporated NaH14CO3 at approximately two-thirds the rate shown by a dilute suspension of cells at saturating light intensity. On the basis of NaH14CO3 incorporation, the effective light intensity obtained under NMR conditions would support growth at approximately one-fourth the maximum rate in dilute suspensions of cells. Irradiated cells maintained a cytoplasmic pH of 7.1--7.3 when exposed to an external pH from 6.4 to 8.3. At an external pH of 6.7, a darkness to light shift caused a 0.4 pH unit alkalinization of the cytoplasm. Treatment of cell suspensions with the uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), in light or darkness, collapsed the internal pH to the level of the external pH. The results suggest a strong light- or energy-dependent buffering of the cytoplasm over a range of external pH. The study demonstrates that 31P NMR can be used to investigate intracellular events in an actively photosynthesizing microorganism.     

Phosphorus-31 nuclear magnetic resonance of highly oriented DNA fibers. 1. Static geometry of DNA double helices

The static geometry of the phosphodiesters in oriented fibers of DNA and a variety of polynucleotides was investigated by solid-state 31P nuclear magnetic resonance (NMR) spectroscopy. The structural parameters of the phosphodiester backbone expressed by two Euler angles beta and gamma were estimated on the basis of the NMR spectra of natural DNA, poly(dA).poly(dT), poly(rA).poly(dT), and poly-(rA).poly(rU). The Euler angles were calculated by using the known single crystal structures of a decamer, r(GCG)d(TATACGC), and a dodecamer, d(CGCGAATTCGCG). The distribution pattern of the Euler angles was quite different between these two oligonucleotides due to the different types of conformation, and it was fully consistent with the 31P NMR results, showing that the conformation of the B form DNA is very heterogeneous while that of the A or A' form is much more invariable with regard to the base composition. The structural parameters were also calculated by using various structures determined by the X-ray fiber diffraction studies, and they were evaluated on the basis of the 31P NMR data. Notably, poly(dA).poly(dT) fibers exhibited abnormal 31P NMR spectra which were very broad in line width and were not appreciably perturbed by hydration; a coiled double-helical structure is proposed as the most plausible model for this polymer.     

Phosphorus-31 nuclear magnetic resonance studies of the two phosphoserine residues of hen egg white ovalbumin

Ovalbumin contains two phosphoserine residues that give rise to two well-resolved resonances in a 31P NMR spectrum. Ovalbumin samples that have been digested with a variety of phosphatases may give rise to only one phosphoserine resonance, indicating that one of the two phosphorylated sites is relatively inaccessible for phosphatase action. By comparison of the amino acid sequence of the peptide containing the nonsusceptible phosphate to the overall primary structure, we have assigned the resonances observed (pH 8.3) at 5.0 and 4.75 ppm to phosphoserines-68 and -344, respectively. pH titration behavior and susceptibility of the phosphoserine residues to phosphatases indicate that both are located on the surface of the protein. Both residues have a pKa = 6.00-6.04. Analysis of the Hill coefficients measured for the pH titrations and the JPH coupling constants indicate that neither residue interacts with other charged groups on the surface of the protein. Frequency dependence of 31P NMR parameters shows that at higher magnetic field strengths the contribution of chemical shift anisotropy to the line width becomes very significant. We have calculated from the field-dependent terms that phosphoserine-344 is mobile with respect to the protein surface but that phosphoserine-68 is more restricted in its motion. The latter is also involved in a pH-dependent conformational change, since it is shielded from hydrolysis by phosphatases at higher pH. A comparison of the amino acid sequence of the phosphoserine-68 site shows that it has a striking homology to the active-site peptides of a wide variety of hydrolytic enzymes. Moreover, a comparison with the primary sequences of casein suggests that both proteins are phosphorylated by a protein kinase that specifically recognizes a Ser-X-Glu peptide.