Increased substitution of phosphate groups in lipopolysaccharides and lipid A of the polymyxin-resistant pmrA mutants of Salmonella typhimurium: a 31P-NMR study

De-O-acylated lipopolysaccharides (LPS) of three polymyxin-resistant Salmonella typhimurium pmrA mutants and their parent strains were analysed by ³¹P-NMR (nuclear magnetic resonance) in order to assess, in relation to polymyxin resistance, the types and degree of substitution of phosphates of the LPS and lipid A. in the pmrA mutant LPS phosphate diesters predominated over phosphate monoesters, whereas the latter were more abundant in the parent wild-type LPS. The increase in the proportion of phosphate diesters was traced to both the core oligosaccharide and the lipld A part. In the latter, the ester-linked phosphate at position 4’was to a large extent (79–88%) substituted with 4-amino-4-deoxy-l -arabinose, whereas in the wild-type LPS the 4′-phosphate was mainly present as monoester. In each LPS, regardless of the pmrA mutation, the glycosidically linked phosphate of lipid A was largely unsubstituted.     

A th-1 Mutant of Arabidopsis thaliana Is Defective for a Thiamin-Phosphate-Synthesizing Enzyme: Thiamin Phosphate Pyrophosphorylase

We have examined the activity of the thiamin phosphate pyrophosphorylase in Arabidopsis thaliana wild type and in a mutant (th-1) which requires exogenous thiamin for growth. Mutant and wild-type plants grown in 1 × 10⁻⁷ molar thiamin were used for the examination of the production of thiamin and thiamin monophosphate (TMP) using 4-methyl-5-hydroxyethylthiazole phosphate and 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate as substrates. While the wild-type strain formed both thiamin and TMP, the th-1 mutant did not. When TMP was added to the extracts, the th-1 mutant, as well as wild type, produced thiamin. Accordingly, it was concluded that the th-1 mutant was defective in the activity of TMP pyrophosphorylase. Some of the characteristics of the enzyme from the wild-type plant were examined. The optimum temperature for the reaction is 45°C, and the K_m values for the substrates are 2.7 × 10⁻⁶ molar for 4-methyl-5-hydroxyethylthiazole phosphate and 1.8 × 10⁻⁶ molar for 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate.     

Phosphate accumulation and the occurrence of polyphosphates and cyclic 2,3-diphosphoglycerate in Methanosarcina frisia

Methanosarcina frisia accumulates phosphate up to 14% of its dry weight. The phosphate is stored as long-chain polyphosphates as shown by ³¹P-NMR investigations. Further results show that the accumulation of phosphates is substrate-dependent. In the presence of H₂ and CO₂ as the only carbon and energy source 180 mg of PO _inf4 ^sup3- /g protein were accumulated, whereas 260 mg PO _inf4 ^sup3- /g protein were accumulated in the presence of methanol. This is far more than necessary for the maintenance of essential metabolic pathways. In addition, the ³¹P-NMR studies show the occurrence of cyclic 2,3-diphosphoglycerate in Methanosarcina frisia. The role of the phosphate metabolites in cell metabolism are discussed.Abbreviations M. Methanosarcina - CCP cyclic 2,3-diphosphoglycerate     

Pyrophosphate amplification reaction for measuring amino acid concentrations with high sensitivity using aminoacyl-tRNA synthetase from Escherichia coli

ABSTRACT

To measure amino acid concentrations with high sensitivity, the pyrophosphate amplification reaction conditions of histidyl-tRNA synthetase (HisRS) and tyrosyl-tRNA synthetase (TyrRS) were examined. The amount of pyrophosphate produced by reactions involving HisRS and TyrRS was amplified compared with the amount of the initial substrate L-amino acid after the addition of excess adenosine-5′-triphosphate and magnesium ions, with incubation at 50°C in an alkaline pH. The amount of pyrophosphate produced in the HisRS and TyrRS reactions was approximately 24- and 16-fold higher than the initial amount of L-His and L-Tyr, respectively. The pyrophosphate amplification reactions involving HisRS and TyrRS showed high substrate specificity for L-His and L-Tyr, respectively. Products of pyrophosphate amplification were identified as p¹, p⁴-di(adenosine) 5′-tetraphosphate, and adenosine-5′-monophosphate using high-performance liquid chromatography. A strong positive correlation was observed for 0 to 50 μM of L-His and L-Tyr in the pyrophosphate amplification reaction (R = 0.98 and R = 1.00, respectively).

Abbreviations: L-His: L-histidine; L-Tyr: L-tyrosine; aaRSs: aminoacyl-tRNA synthetases; ATP: adenosine-5′-triphosphate; aminoacyl-AMP-aaRS: aminoacyl-adenylate intermediate; Ap₄A, P¹, P⁴-di(adenosine) 5?-tetraphosphate; AMP: adenosine-5′-monophosphate; PAR: pyrophosphate amplification rate     

Multinuclear magnetic resonance studies of monomers and dimers containing 2'-fluoro-2'-deoxyadenosine

A series of 2′-fluorinated adenosine compounds, dAfl, dAflp, pdAfl, dAfl-A, A-dAfl, and dAfl-dAfl, have been investigated by nmr spectroscopies. The ¹H-, ¹⁹F-, and ³¹P-nmr data provide structural information from different parts of these moleucles. The pK_a of the phosphate group of these two 2′-fluoro-2′-deoxyadenosine monophosphates was found to be the same as that of hte parent adenosine monophosphate. As for the pentose conformation, the ³E population is greatly increased as a result of the fluorine substitution at the C_2′ position. However, the populations of conformers of gg (C_4′-C_5′) and g′g′ (C_5′-O_5′) and the average angle ?′(C_3′-O_3′) of the 2′-fluoro compounds remain unchanged as compared to the natural riboadenosine monomer and dimer (A-A). Thefefore, the backbone conformation of the 2′-fluoro-2′-deoxy-adenosine, its monophosphates and dimers, resembles that of RNA. The extent of base-base overlapping in these 2′-fluoro-2′-deoxy-adenosine-containing dimers is also found to be similar to or even greater than A-A. Thus, the conformations of these compounds can be considered as those in the RNA family. These fluorocompounds also serve as models for a careful study on the ¹⁹F-nmr in nucleic acid. The ¹⁹F chemical-shift values are sensitive to the environment of the fluorine atom such as ionic structure of the neighboring group(s) (phosphate of base), solvation, and ring-ruccent anisotropic effect from the base(s). Qualitatively, the change of the ¹⁹F chemical-shift values (up to 2 ppm) is much larger than that of ¹H-nmr (up to 0.5 ppm) in the dimers. Using dAfl·poly(U), poly(dAfl)·poly(dAfl), and poly(dAfl)·poly(U) helix–coil transition as model systems, the linewidth of ¹⁹F in dAfl- residues reflects effectively the mobility of the unit in the nucleic acid complex as calibrated by uv data and by ¹H-nmr. Therefore, application of ¹⁹F-nmr spectroscopy on fluorine-substituted nucleic acid can also be used to detect nucleic acid-nucleic acid interaction in complicated systems.     

The Occurrence of 4-Amino-4-Deoxy-Arabinose in LPS of Supersusceptible Strain Pseudomonas aeruginosa Z61: Noncorrelation with Polymyxin Resistance

Lipopolysaccharides (LPS) extracted from the supersusceptible strain Pseudomonas aeruginosa Z61 were compared with LPS from other strains with varying antimicrobial susceptibilities. The presence of 4-amino-4-deoxy-arabinose (4-AraN) in P. aeruginosa Z61 LPS was confirmed by gas-liquid chromatography/mass spectrometry (GLC-MS) and quantitated by high-performance liquid chromatography (HPLC). Z61 LPS (compared with wild-type strain PAO1) has reduced amounts of rhamnose and higher concentrations of hydroxy fatty acids, 4-AraN, and phosphates. ³¹P Nuclear magnetic resonance revealed that Z61 LPS phosphates are configured in monophosphates, phosphodiesters, pyrophosphomonoesters, and glycosidic pyrophosphodiester groups. The presence of 4-AraN in P. aeruginosa LPS did not correlate with antimicrobial resistance. Received: 31 August 1998 / Accepted: 5 November 1998     

Effect of aPMR1 disruption on the processing of heterologous glycoproteins secreted in the yeastSaccharomyces cerevisiae

TheSaccharomyces cerevisiae PMR1 gene encodes a Ca²⁺-ATPase localized in the Golgi. We have investigated the effects ofPMR1 disruption inS. cerevisiae on the glycosylation and secretion of three heterologous glycoproteins, human α₁-antitrypsin (α₁-AT), human antithrombin III (ATHIII), andAspergillus niger glucose oxidase (GOD). Thepmr1 null mutant strain secreted larger amounts of ATHIII and GOD proteins per a unit cell mass than the wild type strain. Despite a lower growth rate of thepmr1 mutant, two-fold higher level of human ATHIII was detected in the culture supernatant from thepmr1 mutant compared to that of the wild-type strain. Thepmr1 mutant strain secreted α₁-AT and the GOD proteins mostly as core-glycosylated forms, in contrast to the hyperglycosylated proteins secreted in the wild-type strain. Furthermore, the core-glycosylated forms secreted in thepmr1 mutant migrated slightly faster on SDS-PAGE than those secreted in themnn9 deletion mutant and the wild type strains. Analysis of the recombinant GOD with anti-α1,3-mannose antibody revealed that GOD secreted in thepmr1 mutant did not have terminal α1,3-linked mannoses unlike those secreted in themnn9 mutant and the wild type strains. The present results indicate that thepmr1 mutant, with the super-secretion phenotype, is useful as a host system to produce recombinant glycoproteins lacking high-mannose outer chains.     

Formation of polyprenol-linked mono- and oligosaccharides in Phaseolus aureus

A crude membrane preparation from Phaseolus aureus hypocotyls catalyzes the incorporation of mannose from GDP-[¹⁴C]mannose into a acid labile glycolipid and a methanol insoluble fraction. Addition of dolichyl monophosphate to the incubation mixture stimulated the formation of both the mannolipid and the methanol insoluble endproduct. Thin-layer chromatography of endogenous lipid and of the stimulated lipid fraction revealed that both compounds run identical. Ficaprenyl monophosphate also stimulates the incorporation of mannose; however, the ficaprenyl monophosphate mannose formed is not identical to the endogenous mannolipid. This suggests that the endogenous acceptor has the properties of an α-saturated polyprenyl monophosphate rather than those of the ficaprenyl phosphate type. The same membrane preparation also incorporates N-acetylglucosamine into an acid labile glyolipid as well as into a polymer fraction. Evidence is presented that the N-acetylglucosamine containing lipid consists of a mixture of dolichyl pyrophosphate N-acetylglucosamine and dolichyl pyrophosphate di-N-acetylchitobiose. It seems likely that the two compounds have a precursor-product relationship. Incubation of dolichyl pyrophosphate di-N-acetylchitobiose together with GDP-mannose gives rise to lipid-bound mannosyl-di-N-acetylchitobiose. Radioactivity from either the [¹⁴C]mannolipid or the N-acetyl[¹⁴C]glucosamine containing lipid is incorporated into a methanol insoluble product to 3.4 and 6.3%, respectively; it seems, at least in part, to be a glycoprotein.     

Crystal Structure of 2′-Deoxycytidine Hemidihydrogenphosphate Reveals C+·C Base Pairs and Tight,Hydrogen-Bonded (H2PO4 −)∞ Columns (1)

Abstract

2′-Deoxycytidine hemidihydrogenphosphate has been crystallized in the hexagonal space group P6₂ with α=25.839(3), c = 12.529(1) Å. The structure has been solved using the Patterson search method. The asymmetric unit contains two protonated, base-paired 2′-deoxycytidine dimers and two H₂PO₄ ^? anions. The C⁺·C base pairs are composed of a protonated and a neutral species each and are triple H-bonded, the central N(3)…N(3) bonds being 2.850(7) and 2.884(5) Å. The conformations of the four nucleosides fall in the same category (sugar puckers 2·-endo, glycosidic links anti) but in one of them the glycosidic torsion angle is quite low with consequences in other geometrical parameters. The H₂PO₄ ^? anions are located on twofold axes and form two types of tight columns with P…P separations about 4.18 Å The neighboring units along a column are linked via two very short O…H…O hydrogen bonds (O…O about 2.49 Å) leading to effective equalization of the P-O bonds. The base pairs of the two dC⁺·dC cations are coplanar and form layers perpendicular to the phosphate columns repeating every c/3. Within the layers, the dimers form a network through 0(5′)…O(2) hydrogen bonds but their primary intermolecular interactions have the form of H-bond anchors [N(4)-H…O-P and 0(3′)-H…O-P] to the phosphate groups.     

Factors Influencing the Formation of the Carbon Dioxide Radical Anion (CO2−) Spin Adduct of Pbn in the Rat Liver Metabolism of Halocarbons

Spin trapping techniques have been used to detect free radicals generated from the in vitro metabolism by rat liver microsomes of carbon tetrachloride (CCl₄) and bromotrichloromethane (BrCCI) under conditions of varying oxygen tension and pH. Dispersions of rat liver microsomes incubated with ¹²CCl₄, ¹³CCl₄ or Br¹²CCl₃, α-phenyl-tert-butyl nitrone (PBN) and NADPH/NADH in a phosphate buffer varying in pH from 6.6 to 8.0 under varying oxygen tensions produced various amounts of four different PBN adducts: PBN-CCl₃, PBN-L, PBN-OL and PBN-CO^?₂ where L is a carbon-centered lipid type radical and LO is an oxygen-centered lipid type radical. The relative amount of PEN-CO; increases with the absence of oxygen. With the use of ³¹P-NMR in vivo spectroscopy it was possible to detect a pH change from 7.4 to 6.8 in the livers of rats treated with CCl₄, or BrCCl₃. These results suggest that halocarbon metabolism in biological systems may depend on both oxygen tension as well as pH.     

Phospholipid Motional Characteristics in a Dry Biological System : A P-Nuclear Magnetic Resonance Study of Hydrating Typha latifolia Pollen

Analysis of the proton-decoupled ³¹P-nuclear magnetic resonance (NMR) spectrum of fully hydrated Typha latifolia pollen revealed the presence of two main peaks: A broad asymmetrical component of a `bilayer' lineshape and a much narrower symmetrical component originating from phosphorus compounds undergoing rapid isotropic motion. From (a) ³¹P-NMR experiments on the hydrated total pollen phospholipids, (b) saturation transfer ³¹P-NMR experiments, and (c) the fraction of lipid phosphate in the pollen, it can be concluded that the great majority of the endogenous phospholipids are arranged in extended bilayers in which the lipid phosphates undergo fast (τ_c < 10⁻⁶ second) long axis rotation. This bilayer arrangement of phospholipids was observed in the pollen down to hydration levels of at least 10.9% moisture content. At the lowest level of pollen hydration examined (5.2%) the ³¹P-NMR spectrum had a solid state lineshape demonstrating that all the phosphorus-containing compounds (including the phospholipids) were virtually immobile.     

Characterization of mannosyl transferases during the pupal instar of Stomoxys calcitrans (L)

Particulate fractions (10,000g) from pupae of Stomoxys calcitrans transfer [¹⁴C]-mannose from GDP-[¹⁴C]-mannose to dolichol monophosphate and proteins. Production of the mannosyl lipid was inhibited by Mn²⁺, UDP, GMP, GDP, and EDTA. The insect growth regulator diflubenzuron had no effect on mannosyl transferase activity. Dolichol monophosphate and Mg²⁺ stimulated mannosyl transferase activity. The mannosyl lipid product was identified as mannosyl-phosphoryl-dolichol (Man-P-Dol). The apparent K_m and V_max values for the formation of Man-P-Dol using GDP-[¹⁴C]-Man while holding dolichol phosphate constant were 2.4 ± 0.9 μM and 9.4 ± 2.3 pmol Man-P-Dol·min^?1·mg^?1 protein, respectively. The apparent K_m and V_max values using dólichol phosphate while holding GDP-Man constant were 2.2 ± 1.2 μM and 18.5 ± 1.7 pmol Man-P-Dol·min^?1·mg^?1 protein.     

31P NUCLEAR MAGNETIC RESONANCE STUDY OF INTRACELLULAR PHOSPHATE POOLS AND POLYPHOSPHATE METABOLISM IN HETEROSIGMA AKASHIWO (HADA) HADA (RAPHIDOPHYCEAE)1

The effects of starvation and subsequent addition of phosphate-containing medium on the phosphate metabolic intermediates were studied by ³¹P-NMR spectroscope of perchloric acid extracts and intact cells of Heterosigma akashiwo (Hada) Hada. When orthophosphate in the medium was completely depleted the medium was enriched with orthophosphate (4.5 μM). In the phosphate starved condition, the P cell quota was 76 fmol·cell⁻¹ and the major components of phosphate intermediates were phosphodiester, sugar phosphate and orthophosphate (P_i). After addition of P_i, rapid uptake of P_i was observed and the P cell quota increased to 108 fmol·cell⁻¹ in 2 h, 134 fmol·cell⁻¹ in 5 h and 222 fmol·cell⁻¹ in 1 day after addition of phosphate. The ³¹P-NMR spectrum indicated that a major portion of P was stored as polyphosphate, in which the average chain length of polyphosphate increased from 10 to 20 phosphate residues in one day after addition of P_i.     

Adrenergic regulation of Rana balcanica erythrocyte pyruvate kinase

The regulation of pyruvate kinase activity by noradrenaline was investigated in Rana balcanica red cells. Thirty minutes of noradrenaline incubation induced a significant increase in the V _o/V _max ratio of pyruvate kinase. The S _0.5 for phosphoenolpyruvate of the enzyme significantly increased in the presence of noradrenaline while the K _m for ADP decreased. In response to hormonal stimulation the Na⁺/H⁺ exchange was activated as was shown by the increase in Na⁺ and cyclic adenosine monophosphate from the 3rd min of incubation. All these effects were specific to α₁ and β antagonists. High concentrations of fructose diphosphate significantly activated the enzyme in the presence of noradrenaline but not in its absence. Furthermore, the presence of noradrenaline partially released the inhibition of the enzyme by adenosine triphosphate, inorganic phosphate and 2,3-diphosphoglycerate. The results suggest that noradrenaline stimulates glycolysis through pyruvate kinase activation. The mechanism of stimulation may is through Na⁺/H⁺ exchange activation, cyclic adenosine monophosphate concentration and Na⁺-K⁺-ATPase activation. Accepted: 3 October 1999     

Investigating the candidacy of LPS-based glycoconjugates to prevent invasive meningococcal disease: chemical strategies to prepare glycoconjugates with good carbohydrate loading

In previous studies protective antibodies that could facilitate bactericidal killing of Neisseria meningitidis (Nm) serogroup B strains were derived from immunisation with glycoconjugates prepared from O-deacylated lipopolysaccharide (LPS-OH) via direct reductive amination between the reducing end of the oligosaccharide molecule, created by treatment with alkaline phosphatase, and amino functionalities on the CRM₁₉₇ carrier protein. These glycoconjugates proved difficult to prepare because the presence of amide linked fatty-acyl groups results in glycolipids that are relatively insoluble and aggregate. Therefore, we have examined several strategies to prepare glycoconjugates in order to identify a robust, consistently reproducible strategy that produces glycoconjugates with a high loading of LPS derived oligosaccharides. Initially we used completely deacylated LPS molecules, but lacking phosphoethanolamine (PEtn) from the core OS as the strong basic conditions required to completely deacylate the LPS would modify the PEtn residue. We utilised a squarate linker and conjugated via the reducing end of the carbohydrate antigen following removal of the glycosidic phosphate to amino groups on CRM₁₉₇, however carbohydrate loading on the carrier protein was low. Glycoconjugates were then produced utilising amidases produced by Dictyostelium discoideum (Dd), which partially remove N-linked fatty acids from the lipid A region of the Nm LPS molecule, which enabled the retention of the PEtn residue. LPS-OH was treated with Dd amidase, the reducing glycosidic phosphate removed, and using a cystamine linker strategy, conjugated to the carrier protein. Carbohydrate loading was somewhat improved but still not high. Finally, we have developed a novel conjugation strategy that targets the amino functionality created by the amidase activity as the attachment point. The amino functionality on the PEtn residue of the inner core was protected via a novel blocking and unblocking strategy with t-butyl oxycarbonyl. A maleimide-thiol linker strategy, targeting lysine residues on the carrier protein did not result in high loading of the carbohydrate molecules, however when we targeted the carboxyl residues we have consistently obtained a high loading of carbohydrate antigens per CRM₁₉₇, which can be controlled by variation in the amount of activated carbohydrate utilised in the conjugation reaction.     

Periplasmic phosphorylation of lipid A is linked to the synthesis of undecaprenyl phosphate

One-third of the lipid A found in the Escherichia coli outer membrane contains an unsubstituted diphosphate unit at position 1 (lipid A 1-diphosphate). We now report an inner membrane enzyme, LpxT (YeiU), which specifically transfers a phosphate group to lipid A, forming the 1-diphosphate species. (32)P-labelled lipid A obtained from lpxT mutants do not produce lipid A 1-diphosphate. In vitro assays with Kdo(2)-[4'-(32)P]lipid A as the acceptor shows that LpxT uses undecaprenyl pyrophosphate as the substrate donor. Inhibition of lipid A 1-diphosphate formation in wild-type bacteria was demonstrated by sequestering undecaprenyl pyrophosphate with the cyclic polypeptide antibiotic bacitracin, providing evidence that undecaprenyl pyrophosphate serves as the donor substrate within whole bacteria. LpxT-catalysed phosphorylation is dependent upon transport of lipid A across the inner membrane by MsbA, a lipid A flippase, indicating a periplasmic active site. In conclusion, we demonstrate a novel pathway in the periplasmic modification of lipid A that is directly linked to the synthesis of undecaprenyl phosphate, an essential carrier lipid required for the synthesis of various bacterial polymers, such as peptidoglycan.     

Contribution of G Protein Activation to Fluoride Stimulation of Phosphoinositide Hydrolysis in Human Neuroblastoma Cells

Abstract: To examine the possibility that NaF enhances phosphoinositide-specific phospholipase C (PIC) activity in neural tissues by a mechanism independent of a guanine nucleotide binding protein (G_p), we have evaluated the contribution of G_p activation to NaF-stimulated phosphoinositide hydrolysis in human SK-N-SH neuroblastoma cells. Addition of NaF to intact cells resulted in an increase in the release of inositol phosphates (450% of control values; EC₅₀ of ～ 8 mM). Inclusion of U-73122, an aminosteroid inhibitor of guanine nucleotide-regulated PIC activity in these cells, resulted in a dose-dependent inhibition of NaF-stimulated inositol lipid hydrolysis (IC₅₀ of ～ 3.5 μM). When added to digitonin-permeabilized cells, NaF or guanosine-5′-O-thiotriphosphate (GTPγS) resulted in a three- and sevenfold enhancement, respectively, of inositol phosphate release. In the combined presence of optimal concentrations of NaF and GTPγS, inositol phosphate release was less than additive, indicative of a common site of action. Inclusion of 2–5 mM concentrations of guanosine-5′-O-(2-thiodiphosphate) (GDPβS) fully blocked phosphoinositide hydrolysis elicited by GTPγS, whereas that induced by NaF was partially inhibited (65%). However, preincubation of the cells with GDPβS resulted in a greater reduction in the ability of NaF to stimulate inositol phosphate release (87% inhibition). Both GTPγS and NaF-stimulated inositol phosphate release were inhibited by inclusion of 10 μM U-73122 (54–71%). The presence of either NaF or GTPγS also resulted in a marked lowering of the Ca²⁺ requirement for activation of PIC in permeabilized cells. These results indicate that in SK-N-SH cells, little evidence exists for direct stimulation of PIC by NaF and that the majority of inositol phosphate release that occurs in the presence of NaF can be attributed to activation of G_p.     

Distribution of polyphosphates in cell-compartments of Chlorella fusca as measured by 31P-NMR-spectroscopy

In suspensions of the green alga Chlorella fusca the influence of high pH and high ethylene-diamine-tetraacetic acid concentrations in the external medium, of French-press and perchloric acid extraction of the cells and of alkalization of the intracellular pH on the polyphosphate signal in ³¹P-nuclear magnetic resonance (³¹P NMR) spectra was investigated.The results show that part of the polyphosphates of asynchronous Chlorella cells are located outside the cytoplasmic membrane and complexed with divalent metal-ions. These polyphosphates are tightly bound to the cell wall and/or the cytoplasmic membrane and are not susceptible to hydrolyzation by strong acid at room temperature, in contrast to the intracytoplasmic polyphosphates.Upon alkalization of the internal pH of Chlorella cells, polyphosphates, previously not visible in the spectra become detectable by ³¹P-NMR-spectroscopy. ³¹P-NMR spectroscopic monitoring of polyphosphates during gradual alkalization of the extra-and intracellular space is proposed as a quick method for the estimation of the cellular polyphosphate content and distribution.Abbreviations CCCP Carbonylcyanide-m-chlorophenyl-hydrazone - NTP/NDP Nucleotide triphosphate/-diphosphate - PCA Perchloric acid - ³¹P-NMR ³¹P-nuclear magnetic resonance - PolyP polyphosphates - PP₁, PP₂, PP₃ terminal, second and third phosphate residue of polyphosphates, respectively - PP₄ core phosphate residues of polyphosphates     

The synthesis and use of thionphospholipids in 31P-NMR studies of lipid polymorphism

(1) Dipalmitoyl- and dioleoylthionphosphatidylcholine, which are phosphatidylcholine analogues in which the double bonded oxygen of the phosphate group is replaced by a sulfur atom, have been synthesized in 50–60% yields by condensation of diacylglycerol with phosphorus thionchloride in the presence of choline toluene-sulfonate. Dioleoylthionphosphatidylethanolamine has been prepared by the phospholipase D-catalyzed base exchange reaction. (2) Freeze-fracturing of aqueous dispersions of the thionphospholipids reveals that the thionphosphatidylcholines are organized in extended bilayers whereas dioleoylthionphosphatidylethanolamine above 0°C forms the hexagonal H_II phase similar to dioleoylphosphatidylethanolamine. The gel → liquid crystalline phase transition of the dipalmitoylthionphosphatidylcholine occurs at 44°C which is only slightly higher than the transition temperature of dipalmitoylphosphatidylcholine which together with other data demonstrates that the thionphospholipids closely resemble the natural phospholipids in physicochemical behaviour. (3) Proton decoupled ³¹P-NMR spectra of aqueous dispersions of thionphosphatidylcholines have the characteristic asymmetrical line-shape with a low-field shoulder and a high-field peak typical of phospholipids organized in extended bilayers in which the phosphate group can undergo fast axial rotation. The ³¹P-NMR spectrum of the thionphosphatidylethanolamine in the hexagonal H_II phase has a line-shape with a reversed asymmetry and an effective chemical shift anisotropy half of that of thionphospholipids organized in bilayers which is caused by fast lateral diffusion of the lipids around the cylinders of the hexagonal H_II phase as has been observed for the corresponding phosphatidylethanolamines. (4) Since the ³¹P-NMR resonance of the thionphospholipids is completely separated from that of natural phospholipids, these lipids can be used to study by ³¹P-NMR the motional and structural properties of individual lipids in mixed systems. This is demonstrated for various lipid mixtures in which non-bilayer lipid structures have been induced by variations in composition, temperature and presence of divalent cations. It is shown that bilayer → non-bilayer transitions can be modulated by gel → liquid crystalline phase transitions and that typical bilayer forming lipids can be incorporated into non-bilayer structures such as the hexagonal H_II phase.