31P-NMR spectra of the Ha-ras p21.nucleotide complexes

Phosphorus nuclear magnetic resonance spectra of the Ha-ras oncogene product p21 and its nucleotide complexes have been obtained. It is shown that the 31P nuclear magnetic resonance spectra of a number of nucleotide-enzyme complexes show some common features. In particular, the chemical shift values of the beta-phosphorus resonance of enzyme-bound NTP and NDP (N = A, G) of hydrolases exhibit a downfield shift virtually identical for myosin, elongation factor Tu, and the Ha-ras oncogene product p21. This suggests that the stereochemistry around the beta-phosphorus might be similar in these compounds.     

Phosphocholine and phosphoethanolamine during chick embryo myogenesis: a (31)P-NMR study

Elevated contents of phosphoethanolamine (Etn-P) and/or phosphocholine (Cho-P), a common feature of most tumours with respect to normal counterparts, may also occur in non-cancerous proliferating tissues. The significance of these alterations in relation to cell proliferation, differentiation and maturation is scarcely understood. In this work, the Cho-P and Etn-P pools were measured by (31)P-NMR in extracts of chick embryo pectoral muscle at different days of development. The average concentration of these metabolites exhibited the highest values (respectively, 1.5 and 3.0 micromol/mg DNA) on days 9-11 and decreased at later stages of myogenesis. While, however, Cho-P maintained substantial levels (above 1.0 micromol/mg DNA) also during myotube formation (days 11-18) and stepwise decreased (to about 0.5 micromol/mg DNA) upon fibres' maturation, Etn-P gradually decreased between day 11 and hatching time (down to about 0.2 micromol/mg DNA). These results demonstrate that significant changes may occur in the steady-state pools of these metabolites during normal in vivo cellular development and differentiation, and are consistent with: (a) high rates of phospholipid biosynthesis reported in the literature for proliferating myoblasts; (b) sustained phosphatidylcholine synthesis maintained also during myoblast fusion; and (c) decreased requirement of phospholipid synthesis in the last phase of in ovo myofibre maturation.     

Infrared and 31P-NMR studies of the interaction of Mg2+ with phosphatidylserines: effect of hydrocarbon chain unsaturation

Infrared and 31P-NMR spectra of aqueous dispersions of 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) and ox brain phosphatidylserine in the presence of excess Mg2+ have been recorded. A consistent picture emerges from the application of infrared and 31P-NMR spectroscopy to Mg2+-PS interactions. Mg2+ forms crystalline complexes with saturated phosphatidylserines, such as DMPS, and probably with POPS. In these crystalline PS-Mg2+ complexes the phosphate group loses its water of hydration but the serine carboxylate remains hydrated. Furthermore, there is formation of an additional hydrogen bond to one of the ester carbonyl groups of DMPS, and interchain interactions appear to be enhanced as reflected by a tighter packing of the fatty acyl chains. One main conclusion of this work is that Mg2+ binding to PS bilayers shows a gradation, the binding is in the order DMPS greater than POPS greater than ox brain PS greater than DOPS. The molecular area increases in the order DMPS less than ox brain PS less than POPS less than DOPS and is apparently an important parameter determining the affinity of PS for Mg2+. The general trend is that with increasing molecular area, and hence spacing of the ligands, the binding of Mg2+ decreases. While PS with two saturated fatty acyl chains forms tightly packed, crystalline Mg2+ complexes with an immobilized headgroup, the unsaturated PS molecules such as ox brain PS and DOPS interact only weakly with Mg2+. Their interaction seems to be restricted to electrostatic shielding, since no major changes in molecular conformation, chain packing and headgroup hydration are found. The interaction of POPS with Mg2+ is intermediate between that of saturated PS and that of DOPS. POPS exhibits a higher affinity for Mg2+ than ox brain PS, although their molecular areas (and the surface charge density) are approximately the same. This apparent anomaly is proposed to be due to a discreteness of charge effect. It is proposed that a lipid surface with regularly spaced polar groups has a higher affinity for binding Mg2+.     

Interaction between beta-Purothionin and dimyristoylphosphatidylglycerol: a (31)P-NMR and infrared spectroscopic study

The interaction of beta-purothionin, a small basic and antimicrobial protein from the endosperm of wheat seeds, with multilamellar vesicles of dimyristoylphosphatidylglycerol (DMPG) was investigated by (31)P solid-state NMR and infrared spectroscopy. NMR was used to study the organization and dynamics of DMPG in the absence and presence of beta-purothionin. The results indicate that beta-purothionin does not induce the formation of nonlamellar phases in DMPG. Two-dimensional exchange spectroscopy shows that beta-purothionin decreases the lateral diffusion of DMPG in the fluid phase. Infrared spectroscopy was used to investigate the perturbations, induced by beta-purothionin, of the polar and nonpolar regions of the phospholipid bilayers. At low concentration of beta-purothionin, the temperature of the gel-to-fluid phase transition of DMPG increases from 24 degrees C to ~33 degrees C, in agreement with the formation of electrostatic interactions between the cationic protein and the anionic phospholipid. At higher protein concentration, the lipid transition is slightly shifted toward lower temperature and a second transition is observed below 20 degrees C, suggesting an insertion of the protein in the hydrophobic core of the lipid bilayer. The results also suggest that the presence of beta-purothionin significantly modifies the lipid packing at the surface of the bilayer to increase the accessibility of water molecules in the interfacial region. Finally, orientation measurements indicate that the alpha-helices and the beta-sheet of beta-purothionin have tilt angles of ~60 degrees and 30 degrees, respectively, relative to the normal of the ATR crystal.     

The 31P-NMR spectrum of the dodecamer d(GACGATATCGTC).

The resonances in the 31P-NMR spectrum of the dodecamer d(GACGATATCGTC) have been assigned by regiospecific labelling with oxygen-17. All 11 resonances are clearly resolved at 26 degrees C. Most noticeably, individual resonances of the dinucleoside phosphates d(CpG), d(TpC), d(GpA) and d(ApT) which occur more than once can clearly be distinguished. This indicates that the position of the phosphate group in the oligomer influences its 31P-NMR shift. This observation is in agreement with what has been found for the 31P-NMR spectra of d(CGCGAATTCGCG) [Ott, J. and Eckstein, F. (1985) Biochemistry 24] and d(GGAATTCC) [Connolly, B.A. and Eckstein, F. (1984) Biochemistry 23, 5523-5527]. In general, the chemical shift appears the more at higher field the more central the dinucleoside phosphate is located in the oligomer. Exceptions are the resonances of dinucleoside phosphates of the type 5'-PyPu-3' which appear at lower field than expected from this rule. A reasonable correlation between 31P-NMR chemical shifts and the sum function of the base plane roll angles derived from Calladine's rule [Calladine, C.R. (1982) J. Mol. Biol. 161, 343-352] exists.     

Microviscosity of human erythrocytes studied with hypophosphite and 31P-NMR

A 31P-NMR method, which complements earlier 13C-NMR procedures for probing the intra-erythrocyte microenvironment, is described. Hypophosphite is an almost unique probe of the erythrocyte microenvironment, since it is rapidly transported into the cell via the band 3 protein, and intra- and extracellular populations give rise to distinct resonances in the 31P-NMR spectrum. Relaxation mechanisms of the 31P nucleus in the hypophosphite ion were shown to be spin-rotation and dipole-dipole. Analysis of longitudinal relaxation rates in human erythrocytes, haemolysates and concentrated glycerol solutions allowed the determination of microviscosity using the Debye equation. Bulk viscosities of lysates and glycerol solutions were measured using Ostwald capillary viscometry. Translational diffusion coefficients were then calculated from the viscosity estimates using the Stokes-Einstein equation. The results with a range of solvent systems showed that 'viscosity' is a relative phenomenon and that bulk (i.e., macro-) viscosity is therefore not necessarily related to the NMR-determined viscosity. The intracellular NMR-determined viscosities from red cells, ranging in volume from 65.5 to 100.1 fl, varied from 2.10 to 2.67 mPa s. This is consistent with the translational diffusion coefficients of the hypophosphite ion altering by only 20%, whereas the values determined from bulk viscosity measurements conducted on lysates of these cells are consistent with a 230% change.     

Critical oxygen tension in rat brain: a combined (31)P-NMR and EPR oximetry study

The relationship between cerebral interstitial oxygen tension (Pt(O(2))) and cellular energetics was investigated in mechanically ventilated, anesthetized rats during progressive acute hypoxia to determine whether there is a "critical" brain Pt(O(2)) for maintaining steady-state aerobic metabolism. Cerebral Pt(O(2)), measured by electron paramagnetic resonance oximetry, decreased proportionately to inspired oxygen fraction. (31)P-nuclear magnetic resonance measurements revealed no changes in P(i), phosphocreatine (PCr)/P(i) ratio, or intracellular pH when arterial blood oxygen tension (Pa(O(2))) was reduced from 145.1 +/- 11.7 to 56.5 +/- 4.4 mmHg (means +/- SE). Intracellular acidosis, a sharp rise in P(i), and a decline in the PCr/P(i) ratio developed when Pa(O(2)) was reduced further to 40.7 +/- 2.3 mmHg. The corresponding Pt(O(2)) values were 15.1 +/- 1.8, 8.8 +/- 0.4, and 6.8 +/- 0.3 mmHg. We conclude that over a range of decreasing oxygen tensions, cerebral oxidative metabolism is not sensitive to oxygen concentration. Oxygen becomes a regulatory substrate, however, when Pt(O(2)) is decreased to a critical level.     

Lipid analysis of human HDL and LDL by MALDI-TOF mass spectrometry and (31)P-NMR

The analysis of HDL and LDL is important for the further understanding of atherosclerosis because changes of the protein and lipid moieties occur under pathological conditions. Because destruction of lipids leads to the formation of well-defined products such as lysophospholipids or chlorohydrins, methods that allow their fast and reliable determination would be useful. In this study, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied for the analysis of the lipid composition of human lipoproteins. These data were compared with high resolution (31)P-NMR spectroscopy. Differences between LDL and HDL in sphingomyelin and phosphatidylcholine content could be monitored by NMR and mass spectrometry, and differences with respect to the extraction efficiency were found by MALDI-TOF MS. Additionally, treatment of LDL with hypochlorite and phospholipase A(2) resulted in marked changes (formation of chlorohydrines and lysolipids). Lysophosphatidylcholines were detectable by both methods, whereas MALDI-TOF MS failed to detect chlorohydrines of phospholipids.We conclude that MALDI-TOF MS provides rapidly a reliable lipid profile of lipoproteins. However, a previous lipid separation must be performed to detect lipid oxidation products. NMR can be directly applied, but suffers from lower sensitivity, and provides only limited information on fatty acid composition.     

Short chain phospholipids in membrane protein crystallization: a 31P-NMR study of colloidal properties of dihexanoyl phosphatidylcholine

The colloidal features of short chain phospholipids can be deduced from 31P-NMR analysis by comparison with available data on phospholipid aqueous dispersion. In this study with dihexanoyl phosphatidylcholine, detergent phase separation was obtained by temperature shift and by addition of the precipitating agent polyethylene glycol. The 31P-NMR spectra indicate that the detergent micelles fuse to enter the hexagonal HII and lamellar phases. Consequences for the crystallization of membrane proteins are discussed.     

Spectroscopic characterization of nanoErythrosomes in the absence and presence of conjugated polyethyleneglycols: an FTIR and (31)P-NMR study

We have recently developed from red blood cells a new delivery system called nanoErythrosomes. These nanovesicles offer a high degree of versatility for the encapsulation of biological or nonbiological compounds and for the binding of targeting agents. In particular, polyethyleneglycols can be conjugated by a covalent link to the basic amino acid residues constitutive of the different proteins. The binding of polyethyleneglycols to the nanoErythrosome membrane could be interesting for the therapeutic use of this delivery system since it could overcome heterologous immunogenicity and reduce rapid clearance from circulation. In the present study, we have investigated the effect of temperature on the nanoErythrosome behavior in the absence and presence of conjugated polyethyleneglycols. More specifically, Fourier transform infrared (FTIR) spectroscopy has been used to evaluate the lipid order and dynamics, the hydration and the degree of protein aggregation of the nanoErythrosomes after covalent binding of polyethyleneglycols having molecular weights of 2000 and 5000 g mol(-1). The results indicate that the nanoErythrosome lipid chain order is not significantly affected by heating the nanoErythrosomes at temperatures up to 50 degrees C. They also indicate that the nanoErythrosome proteins aggregate irreversibly at temperatures above 37 degrees C, this effect being abolished in the presence of polyethyleneglycols. The presence of polyethyleneglycols decreases the accessibility of water to the lipid head groups. On the other hand, 31P-nuclear magnetic resonance (NMR) and electron microscopy results reveal that the presence of polyethyleneglycols prevents the aggregation of the nanoErythrosome structures.     

Neutral and cationic mononuclear copper(II) complexes with enrofloxacin: structure and biological activity

The mononuclear copper complexes with the quinolone antibacterial drug enrofloxacin (=Herx) in the presence or not of a nitrogen donor heterocyclic ligand 1,10-phenanthroline (=phen) and 2,2'-bipyridine (=bipy) have been prepared and characterized. Interaction of copper(II) with deprotonated enrofloxacin leads to the formation of the neutral complex Cu(erx)2(H2O), 1, while the presence of phen or bipy leads to the formation of a neutral or a cationic mononuclear complex, respectively. The crystal structures of (chloro)(1,10-phenanthroline)(enrofloxacinato)copper(II), 2, and (aqua)(2,2'-bipyridine)(enrofloxacinato)copper(II) chloride, 3, have been determined with X-ray crystallography. The complexes have been studied with X-band electron paramagnetic resonance in aqueous solutions at liquid helium temperature. The study of the interaction of the complexes with calf-thymus DNA has been performed with diverse spectroscopic techniques and has showed that all complexes are bound to DNA by the intercalative mode. The antimicrobial efficiency of the complexes has been tested on three different microorganisms and the available evidence supports that the best inhibition is provided by Cu(erx)2(H2O) (minimum inhibitory concentration=0.125 microg mL(-1)) against Escherichia coli and Pseudomonas aeruginosa.     

Bisintercalation of ditercalinium into a d(CpGpApTpCpG)2 minihelix: a 1H- and 31P-NMR study

The structure of the complex formed in aqueous solution between ditercalinium, a bisintercalating drug, and the self-complementary hexanucleotide d(CpGpApTpCpG)2 is investigated by 400-MHz 1H-nmr and 162-MHz 31P-nmr. Whatever the drug to helix ratio, ditercalinium occurred in the bound form, whereas free and complexed hexanucleotide are in slow exchange. This allows unambiguous resonance assignment through two-dimensional chemical exchange experiments. The strong upfield shifts measured on most aromatic protons on both drug and bases as well as on DNA imino protons are consistent with bisintercalation of the dimer. Nuclear Overhauser effects observed between drug and nucleotide protons give a defined geometry for complexation, and suggest a DNA conformational change upon drug binding.     

Neutral and cationic Fe(II) β-diketiminate complexes

The three-coordinate, 12-valence electron complexes [(^MeBDK)FeR] (^MeBDK = [HC(C(Me)NAr)₂]⁻, Ar = 2,6-iPr₂C₆H₃, R = CH₂Ph, CH₂SiMe₃) are reported as well as their reactivity towards Lewis bases. With perfluoroaryl borane and -borate type activators, the monoalkyls react to give alkyl-free paramagnetic cationic iron species counterbalanced by perfluorinated arylborate anions. The paramagnetic nature of the cations permits the observation of weak and dynamic interactions with these anions via ¹⁹F NMR spectroscopy.     

Comparison of the bis-intercalating complexes formed between either ditercalinium or a flexible analogue and d(CpGpCpG)2 or d(TpTpCpGpCpGpApA)2 minihelices: 1H- and 31P-NMR analyses.

The 400-MHz 1H- and 162-MHz 31P-nmr have been used to study complexes constituted by (a) the d(TpTpCpGpCpGpApA)2 or the d(CpGpCpG)2 self-complementary oligonucleotides and (b) two bifunctional 7H-pyrido [4,3-c] carbazole dimer drugs, the antitumoral ditercalinium (NSC 366241), a dimer with a rigid bis-piperidine linking chain and its pharmacologically inactive analogue, a dimer with a flexible spermine-like linking chain. Nearly all proton and phosphorus signals have been assigned by two-dimensional (2D) nmr (correlated spectroscopy, homonuclear Hartmann-Hahn, nuclear Overhauser enhancement spectroscopy, 2D 31P (1H) heteronuclear correlated spectroscopy and 31P-31P chemical exchange experiments). Both drugs bis-intercalate into the two CpG sites. The complexes show small differences in the position of the 7H-pyrido [4,3-c] carbazole ring into the intercalation site and possibly in the ribose-phosphate backbone deformation. However, the inactive analogue exhibits a longer residence lifetime in octanucleotide than the ditercalinium does. All these results are discussed in terms of differences in dimer activities.     

In vivo (31)P-NMR diffusion spectroscopy of ATP and phosphocreatine in rat skeletal muscle

The aim of this study was to measure the diffusion of ATP and phosphocreatine (PCr) in intact rat skeletal muscle, using (31)P-NMR. The acquisition of the diffusion-sensitized spectra was optimized in terms of the signal-to-noise ratio for ATP by using a frequency-selective stimulated echo sequence in combination with adiabatic radio-frequency pulses and surface coil signal excitation and reception. Diffusion restriction was studied by measuring the apparent diffusion coefficients of ATP and PCr as a function of the diffusion time. Orientation effects were eliminated by determining the trace of the diffusion tensor. The data were fitted to a cylindrical restriction model to estimate the unbounded diffusion coefficient and the radial dimensions of the restricting compartment. The unbounded diffusion coefficients of ATP and PCr were approximately 90% of their in vitro values at 37 degrees C. The diameters of the cylindrical restriction compartment were approximately 16 and approximately 22 microm for ATP and PCr, respectively. The diameters of rat skeletal muscle fibers are known to range from 60 to 80 microm. The modelling therefore suggests that the in vivo restriction of ATP and PCr diffusion is not imposed by the sarcolemma but by other, intracellular structures with an overall cylindrical orientation.     

1H and 31P-NMR Assignments of the Non-exchangeable Protons of the Consensus Acceptor Exon: Intron Junction d(CpTpApCpApGpGpT)

Abstract

The consensus acceptor exon: intron junction d(CpTpApCpApGpGpT) has been synthesized by a modified phosphotriester method. The non-self complementary octamer exists in the single strand form in aqueous buffer at 20°C as evidenced by temperature variable ¹H-NMR and NOE measurements. The non-exchangeable proton assignments were secured using a combination of techniques including two-dimensional COSY, NOESY and the double quantum technique ¹H-¹H-INADEQUATE as well as inversion recovery T1 experiments. The new technique of ³¹P-¹H shift correlation is particularly valuable in removing certain ambiguities in the sugar proton assignments. Characteristic chemical shifts for the base protons which are determined by their immediate molecular environments are also useful in assignments. The consensus acceptor exon: intron junction adopts a random coil conformation in solution under the experimental conditions employed.     

1H-NMR and (31)P-NMR analysis of energy metabolism of quiescent and motile turbot (Psetta maxima) spermatozoa

31P-NMR and (1)H-NMR were used to monitor changes of several compounds with high-energy bonds and metabolites prior to and after the initiation of motility of turbot spermatozoa (Psetta maxima). The obtained (31)P-NMR spectra revealed the presence of phosphomonoesters, phosphodiester, intracellular inorganic phosphate (Pi), phosphocreatine (PCr), and free nucleotide triphosphate. Following the activation of motility, the di- and tri-phosphate nucleotides, PCr, phosphomonoesters levels dropped while Pi levels increased. A significant increase of lactate was also seen at the end of the swimming phase. The compositions of seminal fluid and urine were also determined. Lipoproteins, formic acid, amino acid, and citric acid were detected in seminal fluid. Dimethyl amine, trimethylamine, and trimethylamine oxyde were found in urine. These data suggest that at least a part of the energy required during the swimming phase results from anaerobic fermentation and oxidative phosphorylation. J. Exp. Zool. 286:513-522, 2000.     

31P-NMR study of the orotate phosphoribosyltransferase equilibrium with thiopyrophosphate as substrate

31P-nuclear magnetic resonance spectroscopy was used to directly determine the equilibrium of the reaction catalysed by yeast orotate phosphoribosyltransferase, using orotidine monophosphate and inorganic pyrophosphate as substrates. A Keq value of 0.71 was determined, in good agreement with that of 0.49 calculated by Victor, Greenberg and Sloan (J. Biol. Chem. 254 (1979) 2647-2655), from kinetic data. Substitution of thiopyrophosphate as the substrate shifted the position of the equilibrium 55-fold, to yield a Keq value of 39. Only the beta S analogue of 5-phosphoribosyl 1-diphosphate appeared to be synthesized in this reaction.     

1H and 31P-NMR assignments of the non-exchangeable protons of the consensus acceptor exon:intron junction d(CpTpApCpApGpGpT)

The consensus acceptor exon:intron junction d(CpTpApCpApGpGpT) has been synthesized by a modified phosphotriester method. The non-self complementary octamer exists in the single strand form in aqueous buffer at 20 degrees C as evidenced by temperature variable 1H-NMR and NOE measurements. The non-exchangeable proton assignments were secured using a combination of techniques including two-dimensional COSY, NOESY and the double quantum technique 1H-1H-INADEQUATE as well as inversion recovery T1 experiments. The new technique of 31P-1H shift correlation is particularly valuable in removing certain ambiguities in the sugar proton assignments. Characteristic chemical shifts for the base protons which are determined by their immediate molecular environments are also useful in assignments. The consensus acceptor exon:intron junction adopts a random coil conformation in solution under the experimental conditions employed.