Phosphorylation of AMP by inorganic phosphorylating agents

AMP was phosphorylated by inorganic phosphorylating agents: cyclo-triphosphate and diphosphonate, in aqueous solution (70-80 degrees C, pH 6-12). The molecular structures of phosphorylated products were established by use of phosphorus-31 NMR and high-performance liquid chromatography (HPLC). The OH groups on AMP were phosphorylated by both phosphorylating agents to form 2'- or 3'-phosphate but an OH group on dAMP was not phosphorylated. Phosphorylation of OH group proceeds in two steps: formation of hydrogen bond between OH group and phosphorylating agent; subsequent nucleophilic attack of OH group on a phosphorus atom. Phosphate group on AMP was phosphorylated by diphosphonate but not by cyclo-triphosphate. The difference in the reactivities is explained in terms of charge repulsion between AMP and agents.     

In vivo 31P nuclear magnetic resonance investigation of tellurite toxicity in Escherichia coli

Here we compare the physiological state of Escherichia coli exposed to tellurite or selenite by using the noninvasive technique of phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy. We studied glucose-fed Escherichia coli HB101 cells containing either a normal pUC8 plasmid with no tellurite resistance determinants present or the pTWT100 plasmid which contains the resistance determinants tehAB. No differences could be observed in intracellular ATP levels, the presence or absence of a transmembrane pH gradient, or the levels of phosphorylated glycolytic intermediates when resistant cells were studied by 31P NMR in the presence or absence of tellurite. In the sensitive strain, we observed that the transmembrane pH gradient was dissipated and intracellular ATP levels were rapidly depleted upon exposure to tellurite. Only the level of phosphorylated glycolytic intermediates remained the same as observed with resistant cells. Upon exposure to selenite, no differences could be observed by 31P NMR between resistant and sensitive strains, suggesting that the routes for selenite and tellurite reduction within the cells differ significantly, since only tellurite is able to collapse the transmembrane pH gradient and lower ATP levels in sensitive cells. The presence of the resistance determinant tehAB, by an as yet unidentified detoxification event, protects the cells from uncoupling by tellurite.     

Characterization of the phosphoserine of pepsinogen using 31P nuclear magnetic resonance: corroboration of X-ray crystallographic results

The endogenous phosphoserine residue in porcine pepsinogen has been titrated with use of phosphorus-31 nuclear magnetic resonance (31P NMR). It has an observed pKa2 of 6.7 and a narrow line width (congruent to 10 Hz). The phosphate can be readily removed by an acid phosphatase from potato; however, it is resistant to hydrolysis by several alkaline phosphatases. The X-ray crystal structure of porcine pepsinogen at 1.8-A resolution [James, M. N. G., & Sielecki, A. (1986) Nature (London) 319, 33-38] shows a rather weak and diffuse region of electron density in the vicinity of the phosphorylated serine residue. This suggests considerable dynamic mobility or conformational disorder of the phosphate. In order to define more fully this behavior, the NMR data have been used to corroborate these crystallographic results. All these physical data are consistent with a highly mobile phosphoserine residue on the surface of the zymogen and freely exposed to solvent. In addition, certain properties of this phosphoserine moiety on pepsinogen are similar to those of one of the phosphorylated residues of ovalbumin. The possible significance of this is discussed.     

Lanthanide-induced phosphorus-31 NMR downfield chemical shifts of lysophosphatidylcholines are sensitive to lysophospholipid critical micelle concentration.

Lysophosphatidylcholine (lysoPC) monomers or micelles in water give rise to a narrow, isotropic phosphorus-31 NMR signal (40.6 ppm; v1/2 1.7 Hz; 32.2 MHz). Upon addition of praseodymium ions, the phosphorus signals are shifted downfield. However, the downfield shifts for the longer-chain lysophosphatidylcholines, which exist in the aggregated state, are far greater than those for the shorter-chain homologues, which exist as monomers. At a Pr3+/lysoPC molar ratio of 0.5, the signals of C12lysoPC through C18lysoPC were shifted by 12.1 ppm, whereas the signals of C6lysoPC and C8lysoPC were shifted by only 2.26 ppm. This very pronounced difference in lanthanide-induced downfield shifts between micelles and monomers can be utilized to determine with accuracy lysoPC critical micelle concentrations (CMC) from downfield shift-vs.-concentration plots. The CMC values we determined were 57 mM for C8lysoPC, 5.7 mM for C10lysoPC, and 0.6 mM for C12lysoPC. The shift reagent phosphorus-31 nuclear magnetic resonance technique particularly lends itself to the measurement of CMC values in the millimolar and high micromolar range. The method can equally be used for measuring critical micelle concentrations of short-chain phosphatidylcholines.     

In Vivo 31P Nuclear Magnetic Resonance Investigation of Tellurite Toxicity in Escherichia coli

Here we compare the physiological state of Escherichia coli exposed to tellurite or selenite by using the noninvasive technique of phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy. We studied glucose-fed Escherichia coli HB101 cells containing either a normal pUC8 plasmid with no tellurite resistance determinants present or the pTWT100 plasmid which contains the resistance determinants tehAB. No differences could be observed in intracellular ATP levels, the presence or absence of a transmembrane pH gradient, or the levels of phosphorylated glycolytic intermediates when resistant cells were studied by ³¹P NMR in the presence or absence of tellurite. In the sensitive strain, we observed that the transmembrane pH gradient was dissipated and intracellular ATP levels were rapidly depleted upon exposure to tellurite. Only the level of phosphorylated glycolytic intermediates remained the same as observed with resistant cells. Upon exposure to selenite, no differences could be observed by ³¹P NMR between resistant and sensitive strains, suggesting that the routes for selenite and tellurite reduction within the cells differ significantly, since only tellurite is able to collapse the transmembrane pH gradient and lower ATP levels in sensitive cells. The presence of the resistance determinant tehAB, by an as yet unidentified detoxification event, protects the cells from uncoupling by tellurite.     

Comparisons of diisopropylfluorophosphate derivatives of chymotrypsin and chymotrypsinogen by phosphorus-31 nuclear magnetic resonance

The nature of the differences in the active sites of α-chymotrypsin and chymotrypsinogen has been investigated by phosphorus-31 NMR studies of their diisopropylfluorophosphate derivatives. The phosphorus-31 resonance of the modified zymogen occurs 2 ppm upfield from that for the enzyme. An even greater separation is seen between diisopropylphosphoryl-neo-chymotrypsinogen and -α-chymotrypsin. A plausible interpretation of the chemical shift differences is based on the known structures for α-chymotrypsin, chymotrypsinogen and diisopropylphosphoryl-trypsin.     

31P NMR chemical shifts of phosphate covalently bound to proteins

31P nuclear magnetic resonance (NMR) spectroscopy for characterizing the nature of covalently bound phosphate in proteins is relatively unexploited by the biochemist. 31P NMR chemical shifts of phosphate covalently bound to naturally occurring phosphoproteins, phosphorylated enzyme intermediates and chemically phosphorylated proteins have been compiled in this review. The chemical shifts (31P NMR) of selected reference compounds are reported to assist in the assignment of 31P resonances of phosphate covalently attached to proteins. 31P NMR chemical shifts of phosphate and phospho compounds non-covalently bound to selected proteins as well as the pH dependence of 31P NMR resonance have also been compiled.     

Reversal of enzyme regiospecificity with alternative substrates for aspartokinase I from Escherichia coli.

The substrate specificity of aspartokinase I has been examined by using both steady-state kinetic analyses and phosphorus-31 NMR spectroscopic studies. Analogues in which the alpha-amino group is either derivatized or replaced are not substrates or inhibitors for the enzyme, indicating the importance of the alpha-amino group as a binding determinant. The alpha-carboxyl group is not required for substrate recognition, and the alpha-amide or alpha-esters are competent alternative substrates. In addition, beta-derivatized structural analogues, such as the beta-hydroxamate, the beta-amide, or beta-esters, were found to be viable substrates. This was unexpected since the beta-carboxyl group is the usual site of phosphorylation. The nature of the acyl phosphate products obtained from these beta-derivatized alternative substrates has been characterized by coupled enzyme assays, oxygen-18-labeling studies, and phosphorus-31 NMR spectroscopy. These beta-derivatized analogues are capable of productive binding to aspartokinase through a reversal of regiospecificity to make the alpha-carboxyl group available as a phosphoryl acceptor. Many, but not all, of these alpha-acyl phosphates have also been shown to be viable substrates for the next two enzyme-catalyzed steps in this metabolic pathway. This raises the possibility of producing enzyme-generated alternative substrates that can serve as antimetabolites for the downstream reactions in this biosynthetic pathway.     

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.     

Interaction of the Saccharomyces cerevisiae alpha-factor with phospholipid vesicles as revealed by proton and phosphorus NMR

Proton and phosphorus-31 nuclear magnetic resonance (1H and 31P NMR) studies of the interaction between a tridecapeptide pheromone, the alpha-factor of Saccharomyces cerevisiae, and sonicated lipid vesicles are reported. 31P NMR studies demonstrate that there is interaction of the peptide with the phosphorus headgroups, and quasielastic light scattering (QLS) studies indicate that lipid vesicles increase in size upon addition of peptide. Previous solution (aqueous and DMSO) studies from this laboratory indicate that alpha-factor is highly flexible with only one long-lived identifiable structural feature, a type II beta-turn spanning the central portion of the peptide. Two-dimensional (2D) 1H nuclear Overhauser effect spectroscopy (NOESY) studies demonstrate a marked ordering of the peptide upon interaction with lipid, suggesting a compact N-terminus, in addition to a stabilized beta-turn. In contrast to our results in both solution and lipid environment, Wakamatsu et al. [Wakamatsu, K., Okada, A., Suzuki, M., Higashijima, T., Masui, Y., Sakakibara, S., & Miyazawa, T. (1986) Eur. J. Biochem. 154, 607-615] proposed a lipid environment conformation, on the basis of one-dimensional transferred NOE studies in D2O, which does not include the beta-turn.     

Phosphorylation of yeast protein: Reduction of ribonucleic acid and isolation of yeast protein concentrate

Yeast nucleoproteins were chemically phosphorylated with phosphorus oxychloride (POCL(3)). Studies using (31)P nuclear magnetic resonance (NMR) spectroscopy, stability to pH and lysine estimation all indicated that the epsilon-amino group of lysine was the principal functional group phosphorylated. Phosphorylation of ca. 30% of the lysine residues resulted in removal of more than 85% of contaminant ribonucleic acid from protein precipitated at pH 4.2. Phosphorylation did not alter the amino acid composition of yeast proteins and was reversible under acidic conditions. Based on the data, a method for the preparation of phosphorylated yeast protein with low levels of nucleic acid is proposed.     

Nuclear magnetic resonance spectroscopic analysis of myo-inositol phosphates including inositol 1,3,4,5-tetrakisphosphate

1H and 31P NMR spectra of a variety of phosphorylated myo-inositols have been analyzed using a Bruker WH-360 spectrometer. Proton and phosphorus chemical shifts and coupling constants are reported for myo-inositol 1-phosphate, myo-inositol 2-phosphate, myo-inositol 5-phosphate, myo-inositol 1,2-cyclic phosphate, myo-inositol 1,4-bisphosphate, myo-inositol 1,4,5-trisphosphate, and myo-inositol 1,3,4,5-tetrakisphosphate. These data provide the basis for the chemical identification and characterization of biologically relevant inositol phosphates.     

Molecular details of melittin-induced lysis of phospholipid membranes as revealed by deuterium and phosphorus NMR

Solid-state deuterium and phosphorus-31 nuclear magnetic resonance (2H and 31P NMR) studies of deuterium-enriched phosphatidylcholine [( 3',3'-2H2]DPPC, [sn-2-2H31]DPPC) and ditetradecylphosphatidylglycerol (DMPG-diether), as water dispersions, were undertaken to investigate the action of melittin on zwitterionic and negatively charged membrane phospholipids. When the lipid-to-protein ratio (Ri) is greater than or equal to 20, the 2H and 31P NMR spectral features indicate that the system is constituted by large bilayer structures of several thousand angstrom curvature radius, at T greater than Tc (Tc, temperature of "gel-to-liquid crystal" phase transition of pure lipid dispersions). At T approximately Tc, a detailed analysis of the lipid chain ordering shows that melittin induces a slight disordering of the "plateau" positions concomitantly with a substantial ordering of positions near the bilayer center. At T much greater than Tc, an apparent general chain disordering is observed. These findings suggest that melittin is in contact with the acyl chain segments and that its position within the bilayer may depend on the temperature. On a cooling down below Tc, for Ri greater than 20, two-phase spectra are observed, i.e., narrow single resonances superimposed on gel-type phosphorus and deuterium powder patterns. These narrow resonances are characteristic of small structures (vesicles, micelles, ... of a few hundred angstrom curvature radius) undergoing fast isotropic reorientation, which averages to zero both the quadrupolar and chemical shift anisotropy interactions. On an increase of the temperature above Tc, the NMR spectra indicate that the system returns reversibly to large bilayer structures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of cholesterol on the polar region of phosphatidylcholine and phosphatidylethanolamine bilayers

The structural changes in the polar head group region of unsonicated bilayer membranes of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine produced by addition of cholesterol have been determined using deuterium and phosphorus-31 NMR. Incorportion of up to 50 mol percent cholesterol produces little change in the phosphorus-31 chemical shielding anisotropies, compared with the values in pure bilayers above the phase transition temperatures, while some of the deuterium quadrupole splittings are reduced by almost a factor of two. Adjustment of the head group torsion angles by only a few degrees accounts for the observed spectral changes. Addition of cholesterol therefore has opposite effects on the hydrocarbon and polar regions of membranes: although cholesterol makes the hydrocarbon region gel-like, with an increased probability of trans conformations, the conformation of the polar head groups is very similar to that found in the liquid crystalline phase of pure phospholipid bilayers.     

肝脏肿瘤的31P 磁共振波谱研究进展

磁共振渡谱（MRS）在基础医学和临床医学中起着越来越重要的作用,是目前对人体唯一无创的研究活体组织器官代谢、生化变化及化合物定量分析的方法,能显示肿瘤和正常组织之间的不同代谢。能在分子水平上反映病理情况。磷是能量代谢的重要要素,肝脏中许多化合物都含有31P,本文就31P磁共振渡谱在肝癌的研究及,临床应用方面的近况进行综述。     

A 31P nuclear magnetic resonance study of the hydrothermal vent tube worm Riftia pachyptila.

31P nuclear magnetic resonance (NMR) was used to study the major phosphorylated compounds visible in perchloric extracts of three body regions of the vestimentiferan worm Riftia pachyptila: winged vestimentum, trunk and segmented posterior opisthosome. Two phosphagens (PGs) were present in vestimentum and opisthosome. The major resonance corresponded to those of phosphoarginine and phosphotaurocyamine, which cannot be discriminated on 31P NMR spectra. We have identified four distinct phosphodiesters (PDEs) in these tissues: glycerophosphorylethanolamine (GPE), serine ethanolamine phosphodiester (SEP), glycero-phosphorylcholine (GPC) and threonine ethanolamine phosphodiester (TEP). Three phosphonates or derivates (PAs) were observed in the three body regions. The minor one was identified as 2-aminoethyl phosphonate (2-AEP). The phosphorus profile of the trunk was appreciably different: one additional resonance in the PDE region and only one phosphagen peak were observed.     

Noninvasive NMR studies of metabolism in cultured Catharanthus roseus cells

Summary Nuclear magnetic resonance (NMR) spectroscopy provides a unique modality for the study of tissue-cultured plant cells. One of its major attractions is that it allows noninvasive studies of plant material. In addition, it can provide insight into the pH in the vacuole and cytoplasm, and into the compartmentalization of certain metabolites. In this review we show how phosphorus-31 NMR is used to study intracellular pH, phosphate uptake and storage, and energy metabolism in suspension cells of Catharanthus roseus. In addition, multinuclear NMR studies of the uptake of ammonium and the gradients of K⁺ over the membrane are discussed as well. The use of two-dimensional NMR for the study of whole cell extracts is also described. Finally, we show how nitrogen-14 and nitrogen-15 NMR are used to obtain information about the assimilation of inorganic sources in developing carrot somatic embryos. These NMR studies provide a unique insight into the metabolism of tissue-cultured plant cells.     

Reincorporation of adenosine 5'-diphosphate/adenosine 5'-triphosphate carrier into phospholipid membranes. Phospholipid-protein interaction as studied by phosphorus-31 nuclear magnetic resonance and electron microscopy

Combined phosphorus-31 nuclear magnetic resonance (31P NMR) and electron microscopic studies were performed on the ADP/ATP carrier protein from beef heart mitochondria. The protein was incorporated into phospholipids by addition of Triton-protein micelles to a lipid suspension or to the dry lipid. All of the phospholipid (egg phosphatidylcholine or mixtures of egg phosphatidylcholine and egg phosphatidylethanolamine) that contributed to the observed 31P NMR signal under these conditions appeared to be in a bilayer configuration. Freeze-fracturing and negative-staining electron microscopy showed unilamellar vesicles and multilayers. An isotropic signal could be attributed to vesicle rotation, judging from its sensitivity to increasing viscosity. The presence of small vesicles was also noticeable in the 31P NMR spectra of planar oriented membranes. In the presence of phosphatidylethanolamine, aggregation of protein particles was observed. Gel chromatography of the protein-Triton-phospholipid mixture revealed that, before Triton removal, large amounts of protein are associated with multibilayers. Separation of loaded and unloaded membranes by centrifugation in D2O showed that, upon stepwise addition, protein incorporates preferentially into unloaded liposomes. From these findings a mechanism of protein reincorporation was deduced.     

Presence of phosphorus in Nephila clavipes dragline silk.

Solid-state 31P-NMR of Nephila clavipes dragline silk indicates the presence of phosphorus in at least two chemically distinct environments. Amino acid analyses of acid-hydrolyzed silk confirm the presence of phosphotyrosine as one of the phosphorus-containing components. The unusual chemical shift (18.9 ppm downfield from 85% H3PO4), proton chemical shift, and acid lability of a second component suggest that it is part of a strained five-membered cyclic phosphate that might be found on a beta-D-ribose. The five-membered cyclic phosphate is not removed from the silk fibers by exhaustive aqueous extraction. It is absent in nascent silk fibroin from the glands, suggesting that its formation is part of the fiber processing that occurs in the ducts leading to the spinnerets. High-resolution NMR spectra of silk dissolved in propionic acid/12 N HCl (50:50 v/v) show five phosphorus sites assigned to phosphorylated tyrosine residues, phosphorylated serine residues, inorganic phosphate, and two hydrolysis products of the cyclic phosphate compound. The observed posttranslational phosphorylation may be important in the processing and modulation of the physical properties of dragline silk.     

Observations of aerobic, growing escherichia coli metabolism using an on-line nuclear magnetic resonance spectroscopy system

An experimental system has been constructed which enables on-line measurements of phosphorus-31 ((31)P) nuclear magnetic resonance (NMR) spectra for growing bacterial suspensions under anaerobic or aerobic conditions. A sample stream from a laboratory bioreactor is circulated to the NMR sample chamber in a gas exchange system which permits maintenance of aerobic conditions for high-cell-density cultures. (31)P NMR spectra with resolution comparable with those obtained traditionally using dense, concentrated, nongrowing cell suspensions can be obtained at cell densities above 25 g/L with acquisition times ranging from 14 to 3 minutes which decline as cell density increases. This system has been employed to characterize the changes in intracellular state of a stationary phase culture which is subjected to a transition from aerobic to anaerobic conditions. Both intracellular NTP level and cytoplasmic pH are substantially lower under anaerobic conditions. Also, the system has been employed to observe the response of a growing culture to external addition of acetate. Cells are able to maintain pH difference across the cytoplasmic membrane at extracellular acetate concentrations of 5 and 10 g/L. However, acetate concentrations of 20 g/L cause collapse of the transmembrane DeltapH and sharp reduction of the growth rate of the culture. The experimental configuration described should also permit NMR observations of many other types of microbial cultures and of other nuclei. (c) 1993 John Wiley & Sons, Inc.