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1.
J Chem Phys ; 159(3)2023 Jul 21.
Article in English | MEDLINE | ID: mdl-37462283

ABSTRACT

Stretching or compressing hydrogels creates anisotropic environments that lead to motionally averaged alignment of embedded guest quadrupolar nuclear spins such as 23Na+. These distorted hydrogels can elicit a residual quadrupolar coupling that gives an oscillation in the trajectories of single quantum coherences (SQCs) as a function of the evolution time during a spin-echo experiment. We present solutions to equations of motion derived with a Liouvillian superoperator approach, which encompass the coherent quadrupolar interaction in conjunction with relaxation, to give a full analytical description of the evolution trajectories of rank-1 (T^1±1), rank-2 (T^2±1), and rank-3 (T^3±1) SQCs. We performed simultaneous numerical fitting of the experimental 23Na nuclear magnetic resonance (NMR) spectra and rank-2 (T^2±1) and rank-3 (T^3±1) SQC evolution trajectories measured in double and triple quantum filtered experiments, respectively. We estimated values of the quadrupolar coupling constant CQ, rotational correlation time τC, and 3 × 3 Saupe order matrix. We performed simultaneous fitting of the analytical expressions to the experimental data to estimate values of the quadrupolar coupling frequency ωQ/2π, residual quadrupolar coupling ωQ/2π, and corresponding spherical order parameter S0*, which showed a linear dependence on the extent of uniform hydrogel stretching and compression. The analytical expressions were completely concordant with the numerical approach. The insights gained here can be extended to more complicated (biological) systems such as 23Na+ bound to proteins or located inside and outside living cells in high-field NMR experiments and, by extension, to the anisotropic environments found in vivo with 23Na magnetic resonance imaging.

2.
J Inorg Biochem ; 115: 211-9, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22658754

ABSTRACT

The change in the NMR chemical shift of (23)Na(+) induced by the shift reagent TmDOTP was examined under various experimental conditions typical of cells, including changed Na(+), K(+), PO(4)(3-), and Ca(2+) concentrations, pH and temperature. A mathematical model was developed relating these factors to the observed chemical shift change relative to a capillary-sphere reference. This enabled cation concentrations to be deduced quantitatively from experimental chemical shifts, including those observed during biological time courses with cell suspensions containing TmDOTP DOTP, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis (methylenephosphonate) [corrected]. The model was applied to a (23)Na NMR time course in which monensin, a sodium ionophore, was introduced to human erythrocytes, changing the concentration of cations which may bind TmDOTP, and also resulting in cell volume changes. Using the model with experimentally determined conditions, the chemical shift was predicted and closely followed the experimental values over time. In addition to the model, parameter fitting was achieved by calculating the likelihood distribution of parameters, and seeking the maximum likelihood with a Bayesian type of analysis.


Subject(s)
Erythrocytes/chemistry , Erythrocytes/metabolism , Free Radical Scavengers/chemistry , Models, Biological , Models, Chemical , Phthalic Acids/chemistry , Calcium/chemistry , Calcium/metabolism , Cations/chemistry , Cations/metabolism , Free Radical Scavengers/pharmacology , Humans , Ion Transport/physiology , Magnetic Resonance Spectroscopy/methods , Phosphates/chemistry , Phosphates/metabolism , Phthalic Acids/pharmacology , Potassium/chemistry , Potassium/metabolism , Sodium Ionophores/chemistry , Sodium Ionophores/pharmacology , Sodium Isotopes/chemistry , Sodium Isotopes/pharmacology
3.
J Magn Reson ; 194(1): 81-8, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18585942

ABSTRACT

Efficient J-compensated sequences that are shorter in duration and use less RF pulses have been created from short but very efficient composite 90 degrees RF pulses. The improved J-compensation transforms in-phase into antiphase magnetization and can be incorporated in any pulse sequence that involves evolution of heteronuclear J-couplings. The compensated sequences were tested and incorporated into an HMBC sequence. J-compensated experiments referred to as HMBC-J45 + 90A and HMBC-J45 + 90B, were found to be effective over a wide range of J values.


Subject(s)
Algorithms , Models, Chemical , Nuclear Magnetic Resonance, Biomolecular/methods , Signal Processing, Computer-Assisted , Computer Simulation , Reproducibility of Results , Sensitivity and Specificity
4.
Magn Reson Med ; 58(4): 656-65, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17899589

ABSTRACT

We present a novel NMR-based study of the molecular aspects of the "attack" on human red blood cells (RBCs) by growing bacteria. Staphylococcus aureus expresses virulence factors, including alpha-hemolysin, which contribute to the clinical condition known as septic shock. alpha-Hemolysin is a pore-forming toxin and its secretion increases the permeability of a range of mammalian cell types infected with S. aureus. (31)P NMR spectra of the probe molecules dimethyl methylphosphonate (DMMP) and hypophosphite (HPA) in RBC suspensions show separate intra- and extracellular resonances. These resonances coalesced over time in RBC suspensions inoculated with S. aureus or pure alpha-hemolysin, due to increasing permeability of the RBC membrane. Increased RBC permeability resulted in leakage of intracellular proteins, plus an increase in the exchange rate of the solutes between the intra- and extracellular compartments, both effects contributing to the coalescence of the split peaks. The addition of antibiotics prevented peak coalescence and enabled the minimal inhibitory concentration (MIC) for eight strains of S. aureus to be determined for oxacillin and erythromycin. The MIC values obtained by using (31)P NMR spectroscopy were within one dilution of the MICs obtained using the standard National Committee for Clinical Laboratory Standards (NCCLS) method. The results are encouraging for the use of NMR spectroscopy in clinical microbiology.


Subject(s)
Magnetic Resonance Spectroscopy/methods , Sepsis/diagnosis , Staphylococcal Infections/diagnosis , Anti-Bacterial Agents/pharmacology , Bacterial Toxins/analysis , Erythrocytes/chemistry , Erythrocytes/microbiology , Hemolysin Proteins/analysis , Humans , Microscopy, Interference , Organophosphorus Compounds , Permeability , Phosphinic Acids
5.
Antimicrob Agents Chemother ; 51(11): 4077-84, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17785514

ABSTRACT

Invasive aspergillosis remains a potentially life-threatening infection, the incidence of which is increasing. Current methods used to determine the susceptibilities of Aspergillus strains to antifungal drugs are often unreliable. Nuclear magnetic resonance (NMR) spectroscopy can identify the metabolic complement of microorganisms while monitoring nutrient utilization from the incubation medium. We used 600-MHz (1)H NMR spectroscopy to monitor the metabolic responses of five Aspergillus species cultured in RPMI 1640-2% glucose-morpholinepropanesulfonate buffer to various concentrations of the antifungal drugs amphotericin B (AMB) and caspofungin. The metabolic endpoint (MEP) was determined from nutrient and metabolite resonances, measured as a function of the drug concentration, and was defined as a > or =50% reduction in nutrient consumption or metabolite production. MICs were evaluated by a modification of Clinical and Laboratory Standards Institute broth microdilution method M27-A, and minimal effective concentrations (MECs) were determined by microscopic examination of fungal hyphae. For AMB, the MEPs coincided with the MICs. For caspofungin, the MEPs agreed with the MECs for several Aspergillus strains, but the effect of drug pressure was more complex for others. Expansion of the MEP definition to include any significant changes in metabolite production resulted in agreement with the MEC in most cases. Paradoxical metabolic responses were observed for several Aspergillus strains at either high or low caspofungin concentrations and for one Aspergillus terreus strain with AMB. NMR spectroscopy proved to be a powerful tool for detecting the subtle effects of drug pressure on fungal metabolism and has the potential to provide an alternative method for determining the susceptibilities of Aspergillus species to antifungal drugs.


Subject(s)
Aspergillus/drug effects , Echinocandins/pharmacology , Magnetic Resonance Spectroscopy/methods , Amphotericin B/pharmacology , Antifungal Agents/pharmacology , Aspergillus/metabolism , Aspergillus flavus/drug effects , Aspergillus flavus/metabolism , Aspergillus fumigatus/drug effects , Aspergillus fumigatus/metabolism , Aspergillus nidulans/drug effects , Aspergillus nidulans/metabolism , Caspofungin , Glucose/pharmacology , Lipopeptides , Microbial Sensitivity Tests/methods
6.
Biophys J ; 93(6): L32-4, 2007 Sep 15.
Article in English | MEDLINE | ID: mdl-17631536

ABSTRACT

We have studied the interaction of CnErg1, a member of the gamma-KTX subfamily of scorpion toxins with the inactivation-deficient S631A hERG channel. In the background of this mutation, we observed a mechanistic switch from turret block, characteristic of the action of gamma-KTXs on Kv11-type channels, to pore plugging, characteristic of alpha-KTX block of Kv1-type channels. We suggest this reflects destabilization of the outer pore (turret region) of hERG allowing access of the toxin molecule to directly plug the conduction pathway.


Subject(s)
Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Ether-A-Go-Go Potassium Channels/genetics , Scorpion Venoms/toxicity , Amino Acid Substitution , Animals , Biophysical Phenomena , Biophysics , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels/chemistry , Ether-A-Go-Go Potassium Channels/metabolism , Humans , In Vitro Techniques , Ion Channel Gating/drug effects , Membrane Potentials/drug effects , Mutagenesis, Site-Directed
7.
Amino Acids ; 32(1): 63-8, 2007 Jan.
Article in English | MEDLINE | ID: mdl-16729187

ABSTRACT

Platypus venom contains an isomerase that reversibly interconverts the second amino-acid residue in some peptides between the L-form and the D-form. The enzyme acts on the natriuretic peptides OvCNPa and OvCNPb, and on the defensin-like peptides DLP-2 and DLP-4, but it does not act on DLP-1. While the isomerization of DLP-2 to DLP-4 is inhibited by the amino-peptidase inhibitor amastatin, it is not affected by the leucine amino-peptidase inhibitor bestatin. The enzyme, that is only present in minute quantities in an extract of the venom gland, is thermally stable up to 55 degrees C, and it was found by anion-exchange chromatography to be acidic. Isolation of the isomerase was carried out by combined ion-exchange chromatography and reverse-phase high performance liquid chromatography (HPLC).


Subject(s)
Amino Acid Isomerases/chemistry , Natriuretic Peptide, C-Type/chemistry , Platypus , Venoms/enzymology , Amino Acid Isomerases/antagonists & inhibitors , Amino Acid Isomerases/isolation & purification , Animals , Isomerism , Peptides/chemistry , Platypus/metabolism , Protease Inhibitors/chemistry
8.
Cell Mol Biol (Noisy-le-grand) ; 52(7): 2-5, 2006 Oct 30.
Article in English | MEDLINE | ID: mdl-17543213

ABSTRACT

A critical analysis of the discovery of the first water channel protein (later called aquaporin 1) has been performed. In 1986 Benga's group in Cluj-Napoca, Romania, published in Biochemistry, a US-based journal, the results of experiments that provided the first visual and tangible evidence that the very rapid water exchange that occurs through the membranes of the human red blood cell (RBC) is mediated by a particular protein or small group of proteins. Benga and co-workers did first see bands in a gel that corresponded to water transporters, and were the first to do so. In 1988 Peter Agre and co-workers in Baltimore, USA, while working on the rhesus blood group antigens, purified a "new" membrane protein that they called CHIP 28 (channel integral membrane protein of molecular weight 28 k). At the time they had no idea what its function was. In 1992 came the definitive experiment, that was done, according to Peter Agre in his Nobel Lecture, after much discussion with colleagues about the likely candidate function of their 'orphan' protein. In a paper published in 1992 in Science Agre and his group found that CHIP28 has the properties of a water channel protein. In 1993 the name of the protein was changed from CHIP28 to aquaporin 1. It became obvious that one of the labelled peaks observed by Benga's group (the one in the region of molecular weight ~35,000 to ~60,000) corresponds to glycosylated CHIP28 (aquaporin 1). So Benga and co-workers did first see bands in a gel that corresponded to water transporters, and were the first to do so. The "mercury labelling" experiments were confirmed and extended in Cluj-Napoca by Benga's group and the results were published in 1986 in European Journal of Cell Biology, another international journal. The work was reviewed by Benga in subsequent years in international series and even as a chapter in a book on water transport edited for a wellknown US-based publisher. Agre's group did include a reference to Benga's work in their Science paper; but this reference was only to a 1983 paper on protease resistance of "water channels" (which was relevant) and not the pertinent 1986 Biochemistry paper, or even the subsequent publications. The report of the recent exciting finding of possible involvement of aquaporins in epilepsy, published in 2005 in Proc Natl Acd Sci USA by a group including Agre failed to cite Benga and Morariu's novel and startling report in Nature in 1977.


Subject(s)
Aquaporins/history , Aquaporins/metabolism , Chemistry/history , Erythrocyte Membrane/metabolism , Water/metabolism , Aquaporins/blood , Biological Transport , History, 20th Century , Humans , Romania
9.
Cell Biol Int ; 29(6): 441-8, 2005 Jun.
Article in English | MEDLINE | ID: mdl-16054561

ABSTRACT

The water residence time and diffusional water permeability in colonic epithelial T84 cancer cells was measured using (1)H NMR spectroscopy; the values estimated were 35.2+/-2.8 ms and (7.4+/-0.6)x10(-3)cms(-1), respectively. Water permeability was inhibited to approximately 10% of its original value by the mercurial diuretic, p-chloromercuribenzenesulfonate (PCMBS; 1mM), and fully restored by dithiothreitol (DTT; 1mM). The permeability was also inhibited reversibly to approximately 55%, by extracellular glibenclamide (1mM), an inhibitor of some ATP-binding cassette (ABC) transporters, including the cystic fibrosis transmembrane conductance regulator (CFTR). Addition of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IMBX; 0.1-1mM) and the adenylate cyclase activator, forskolin (0.1-1mM) did not alter water permeability. It is concluded that in T84 cells water diffuses through the membrane lipid bilayer and via channels that are inhibited by PCMBS, including the channels that are known to be inhibited by glibenclamide.


Subject(s)
Cell Membrane Permeability , Colonic Neoplasms/pathology , Epithelial Cells/metabolism , Epithelial Cells/pathology , Water/metabolism , 4-Chloromercuribenzenesulfonate/pharmacology , Biological Transport/drug effects , Cation Transport Proteins/genetics , Cell Line, Tumor , Diffusion/drug effects , Dithiothreitol/pharmacology , Epithelial Cells/drug effects , Glyburide/pharmacology , Humans , Iron-Binding Proteins/genetics , Magnetic Resonance Spectroscopy , Time Factors
10.
Magn Reson Med ; 52(3): 663-8, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15334588

ABSTRACT

31P magic angle spinning NMR (MAS-NMR) spectra were obtained from suspensions of human red blood cells (RBCs) that contained the cell-volume-sensitive probe molecule, dimethyl methylphosphonate (DMMP). A mathematical representation of the spectral-peak shape, including the separation and width-at-half-height in the 31P NMR spectra, as a function of rotor speed, enabled us to explore the extent to which a change in cell volume would be reflected in the spectra if it occurred. We concluded that a fractional volume change in excess of 3% would have been detected by our experiments. Thus, the experiments indicated that the mean cell volume did not change by this amount even at the highest spinning rate of 7 kHz. The mean cell volume and intracellular 31P line-width were independent of the packing density of the cells and of the initial cell volume. The relationship of these conclusions to other non-NMR studies of pressure effects on cells is noted.


Subject(s)
Erythrocyte Volume , Magnetic Resonance Spectroscopy/methods , Computer Simulation , Humans , Organophosphorus Compounds , Phosphorus Radioisotopes , Pressure , Temperature
11.
Eur Biophys J ; 33(7): 589-95, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15138736

ABSTRACT

Small angle neutron scattering (SANS) was performed on suspensions of actively metabolising human erythrocytes in the constant shear field induced by a Couette cell. The SANS pattern recorded on a two-dimensional detector was a function of the shear rate; at zero shear, the SANS pattern had radial symmetry around the direction of the beam. The radial average of the SANS pattern consisted of a broad intensity maximum superimposed on a decay. The intensity maximum at q = 0.1 A(-1) was attributed to isotropically oriented self-associated complexes of the tetrameric oxygen transport protein hemoglobin inside the erythrocytes. A flow curve of the cell suspension was used to identify at what shear rate a suspension of uniaxially oriented ellipsoidal cells is produced. The radial symmetry of the SANS patterns persisted until the shear rate was sufficient to produce a suspension of uniaxially oriented ellipsoidal cells. Again, an intensity maximum was present in directions parallel and orthogonal to the shear axis, but this intensity maximum was superimposed upon quite different intensity decays in each direction from that of the primary neutron beam. The angular range of the SANS instrument was limited, however the results from shear-induced structural changes is consistent with a model that involves hemoglobin complexes that are aligned with respect to the plasma membranes of the elongated cells.


Subject(s)
Erythrocytes/physiology , Erythrocytes/ultrastructure , Hemoglobins/metabolism , Hemoglobins/ultrastructure , Mechanotransduction, Cellular/physiology , Cells, Cultured , Hemoglobins/analysis , Humans , Multiprotein Complexes/analysis , Multiprotein Complexes/metabolism , Multiprotein Complexes/ultrastructure , Protein Conformation , Shear Strength , Stress, Mechanical
12.
Cell Biol Int ; 27(11): 921-8, 2003.
Article in English | MEDLINE | ID: mdl-14585286

ABSTRACT

As part of a programme of comparative measurements of diffusional water permeability (Pd) the red blood cells (RBC) from Little Penguin (Eudyptula minor) were studied. The cell dimensions were measured with light and electron microscopy, and by a newly described non-invasive technique, NMR q-space analysis. In view of its relative novelty for cell biologists, an overview of this technique is presented. The RBC revealed an ellipsoidal shape that is characteristic of avian RBC, with axis lengths ("diameters") estimated to be: a=16.0 microm; b=9.6 microm; c=5.0 microm. The values of P(d)were: 2.0 x 10(-3)cm s(-1)at 5 degrees C, 3.3 x 10(-3)cm s(-1)at 10 degrees C, 4.6 x 10(-3)cm s(-1)at 15 degrees C and approximately 5.4 x 10(-3)cm s(-1)at 20, 25, 30, 37 and 42 degrees C. There was a lack of inhibition of water permeability by p-chloromercuribenzensulfonate (PCMBS), the well-known inhibitor of RBC aquaporin. It was notable that in the temperature range 5-20 degrees C the NMR parameters, and hence the permeability, varied linearly as is found for other species, but at temperatures higher than 20 degrees C there was no temperature-dependence of Pd. Consequently, there was an obvious break at approximately 20 degrees C in the Arrhenius plot, of the mean residence life time of water inside the cells, 1/Te, versus temperature. For temperatures less than 20 degrees C the activation energy E(a,d) was 45.6 +/- 6.6 kJ/mol. For temperatures higher than 25 degrees C E(a,d) was zero. The lack of inhibition of water permeability by PCMBS and the very high value of E(a,d) for diffusive water exchange suggests that the water permeation occurs primarily via the membrane bilayer per se, i.e., there is no aquaporin in Little Penguin RBC. The discontinuity at approximately 20 degrees C in the Arrhenius plot is an interesting finding, not seen before in other species, and we suggest that it reflects a phase transition in the membrane lipids.


Subject(s)
Birds/physiology , Cell Membrane Permeability/physiology , Cell Membrane/physiology , Erythrocytes/physiology , Water-Electrolyte Balance/physiology , 4-Chloromercuribenzenesulfonate/pharmacology , Animals , Body Water/metabolism , Cell Membrane/chemistry , Cell Membrane/drug effects , Cell Membrane Permeability/drug effects , Cell Size/physiology , Diffusion , Erythrocytes/chemistry , Erythrocytes/ultrastructure , Magnetic Resonance Spectroscopy , Membrane Lipids/chemistry , Microscopy, Electron, Scanning , Temperature , Water-Electrolyte Balance/drug effects
13.
Mol Biol Rep ; 29(1-2): 217-25, 2002.
Article in English | MEDLINE | ID: mdl-12241061

ABSTRACT

We show how to incorporate the membrane potential and its effects on the kinetics of ion transport processes into kinetic models.


Subject(s)
Ion Transport/physiology , Membrane Potentials/physiology , Models, Biological , Chlorides/metabolism , Diffusion , Erythrocytes/metabolism , Kinetics , Mathematics , Potassium/metabolism , Thermodynamics
14.
J Comp Physiol B ; 172(6): 513-8, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12192513

ABSTRACT

The effects of exposure of red blood cells (RBC) of three species of marsupial to a mercury-containing sulfhydryl-modifying reagent, p-chloromercuribenzene sulfonate (PCMBS), on the water diffusional permeability ( P (d)) of their membranes were monitored by using an Mn(2+)-doping (1)H nuclear magnetic resonance (NMR) technique at 400 MHz. For koala ( Phascolarctos cinereus), RBC the maximal inhibition was reached at 37 degrees C in 60 min with 1 mmol.l(-1) PCMBS or in 15-30 min with 2 mmol. l(-1) PCMBS. In contrast, in the case of red kangaroo ( Macropus rufus) or swamp wallaby ( Wallabia bicolor) RBC, maximal inhibition required an incubation of 90 min at 37 degrees C with 2 mmol.l(-1) PCMBS. For the RBC of all three species the value of maximal inhibition was very high, being 50-70% when measured at 25 degrees C, 60-80% at 30 degrees C and 60-70% at 37 degrees C. The lowest values of P (d) appeared to be around 2 x 10(-3)-3 x 10(-3) cm.s(-1) in the temperature range of 25-37 degrees C. The mean value of the activation energy of water diffusion ( E (a,d)) was approximately 20-25 kJ.mol(-1) for control and approximately 40 kJ.mol(-1) for PCMBS-inhibited RBCs. These results show that marsupial RBC have a basal permeability to water similar to that previously reported for human RBC, but a higher value of the PCMBS-inhibitable water permeability. This indicates that the higher water permeability of marsupial RBC compared with human RBC is associated with a higher fraction of protein-mediated water permeability.


Subject(s)
4-Chloromercuribenzenesulfonate/pharmacology , Erythrocyte Membrane/metabolism , Marsupialia/metabolism , Sulfhydryl Reagents/pharmacology , Water/metabolism , Animals , Biological Transport/drug effects , Cell Membrane Permeability/drug effects , Diffusion , Macropodidae
15.
J Magn Reson ; 156(2): 249-57, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12165260

ABSTRACT

The utility of the J-HSMQC experiment to detect long-range CH correlations was investigated. Two new long-range J-compensated pulse sequences, LR-J-HSMQC(80,27) and LR-J-HSMQC(27,80), were developed using the (3beta(x))beta(y) composite 90 degrees pulse sequence. These two experiments were shown to be effective for long-range coupling constants, (n)J(CH), that were greater than 3 Hz. Although the overall sensitivities of the long-range J-HSMQC experiments were slightly lower than that of the conventional decoupled HMBC experiment, their 2D maps showed additional cross peaks that could be useful in structure elucidation. LR-J-HSMQC(27,80) was very efficient in yielding two- and four-bond relay correlations. The utility of the new sequences is demonstrated with strychnine as the sample.


Subject(s)
Acetic Acid/chemistry , Acrylates/chemistry , Magnetic Resonance Spectroscopy/methods , Strychnine/chemistry
16.
Biochem J ; 360(Pt 3): 539-48, 2001 Dec 15.
Article in English | MEDLINE | ID: mdl-11736642

ABSTRACT

A high-resolution solution structure of bucandin, a neurotoxin from Malayan krait (Bungarus candidus), was determined by (1)H-NMR spectroscopy and molecular dynamics. The average backbone root-mean-square deviation for the 20 calculated structures and the mean structure is 0.47 A (1 A=0.1 nm) for all residues and 0.24 A for the well-defined region that spans residues 23-58. Secondary-structural elements include two antiparallel beta-sheets characterized by two and four strands. According to recent X-ray analysis, bucandin adopts a typical three-finger loop motif and yet it has some peculiar characteristics that set it apart from other common alpha-neurotoxins. The presence of a fourth strand in the second antiparallel beta-sheet had not been observed before in three-finger toxins, and this feature was well represented in the NMR structure. Although the overall fold of the NMR structure is similar to that of the X-ray crystal structure, there are significant differences between the two structures that have implications for the pharmacological action of the toxin. These include the extent of the beta-sheets, the conformation of the region spanning residues 42-49 and the orientation of some side chains. In comparison with the X-ray structure, the NMR structure shows that the hydrophobic side chains of Trp(27) and Trp(36) are stacked together and are orientated towards the tip of the middle loop. The NMR study also showed that the two-stranded beta-sheet incorporated in the first loop, as defined by residues 1-22, and the C-terminus from Asn(59), is probably flexible relative to the rest of the molecule. On the basis of the dispositions of the hydrophobic and hydrophilic side chains, the structure of bucandin is clearly different from those of cytotoxins.


Subject(s)
Elapid Venoms/chemistry , Neurotoxins/chemistry , Amino Acid Sequence , Animals , Bungarus , Chromatography, Gel , Elapid Venoms/isolation & purification , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Neurotoxins/isolation & purification , Protein Conformation , Protein Structure, Secondary , Sequence Alignment , Sequence Homology, Amino Acid
17.
J Biol Chem ; 276(30): 28042-50, 2001 Jul 27.
Article in English | MEDLINE | ID: mdl-11371569

ABSTRACT

Tropoelastin is the soluble precursor of elastin that bestows tissue elasticity in vertebrates. Tropoelastin is soluble at 20 degrees C in phosphate-buffered saline, pH 7.4, but at 37 degrees C equilibrium is established between soluble protein and insoluble coacervate. Sedimentation equilibrium studies performed before (20 degrees C) and after (37 degrees C) coacervation showed that the soluble component was strictly monomeric. Sedimentation velocity experiments revealed that at both temperatures soluble tropoelastin exists as two independently sedimenting monomeric species present in approximately equal concentrations. Species 1 had a frictional ratio at both temperatures of approximately 2.2, suggesting a very highly expanded or asymmetric protein. Species 2 displayed a frictional ratio at 20 degrees C of 1.4 that increased to 1.7 at 37 degrees C, indicating a compact and symmetrical conformation that expanded or became asymmetric at the higher temperature. The slow interconversion of the two monomeric species contrasts with the rapid and reversible process of coacervation suggesting both efficiently incorporate into the coacervate. A hydrated protein of equivalent molecular weight modeled as a sphere and a flexible chain was predicted to have a frictional ratio of 1.2 and 1.6, respectively. Tropoelastin appeared as a single species when studied by pulsed field-gradient spin-echo NMR, but computer modeling showed that the method was insensitive to the presence of two species of equal concentration having similar diffusion coefficients. Scintillation proximity assays using radiolabeled tropoelastin and sedimentation analysis showed that the coacervation at 37 degrees C was a highly cooperative monomer-n-mer self-association. A critical concentration of 3.4 g/liter was obtained when the coacervate was modeled as a helical polymer formed from the monomers via oligomeric intermediates.


Subject(s)
Tropoelastin/chemistry , Water/metabolism , Humans , Hydrogen-Ion Concentration , Magnetic Resonance Spectroscopy , Proteins/chemistry , Proteins/metabolism , Software , Temperature , Thermodynamics , Tropoelastin/metabolism
18.
Eur Biophys J ; 30(1): 69-74, 2001.
Article in English | MEDLINE | ID: mdl-11372535

ABSTRACT

When lipophilic compounds like diethyl phthalate (DEP) were added to water, two sets of resonances appeared in the 1H NMR spectrum, whereas when added in concentrations above approximately 3.5 mM to erythrocytes in a high haematocrit suspension, only one set of resonances was observed at the low-frequency position. The appearance of one set of resonances at lower frequency was found to be common to a series of lipophilic compounds in erythrocytes. The appearance of the NMR spectra is ascribed to the existence of an emulsion, meaning two different phases of a compound: a "droplet" (resonances to lower frequency) and aqueous dissolved phase (resonances to higher frequency). The absence of the resonances from the dissolved phase in erythrocyte solution is ascribed to exchange broadening. The absolute chemical shift of the compound in its "droplet" phase was also measured using a cylindrical/spherical microcell. This arrangement mimicked the geometry of the dissolved versus the phase-separated species and thus obviated the effect of a difference in magnetic susceptibility between the "droplet" solute and its aqueous solution. Factors influencing the formation of emulsion phases such as erythrocytes, haemoglobin and smaller proteins were investigated; they are found to be effective in the order given.


Subject(s)
Erythrocytes/physiology , Water , Blood Proteins/chemistry , Blood Proteins/metabolism , Butylated Hydroxytoluene , Emulsions , Hemoglobins/chemistry , Hemoglobins/metabolism , Humans , Hydrogen , Nuclear Magnetic Resonance, Biomolecular , Phthalic Acids , Solubility
19.
Pediatr Res ; 48(6): 735-40, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11102539

ABSTRACT

The cosecretion of pancreatic lipase and colipase are important in normal fat digestion. As adsorption of phosphatidylcholine to the lipid substrate interferes with lipase activity, hydrolysis to lysophosphatidylcholine with subsequent desorption is also essential for fat digestion. There are some data regarding the secretion of pancreatic phospholipases in normal adults but none in children or patients with pancreatic disease. In the present study, we aimed a) to develop an accurate fast assay method to measure phospholipase A(2) and b) to determine the secretion rate of pancreatic phospholipase A(2) and whether it is cosecreted with lipase and colipase in children with exocrine pancreatic dysfunction. Nine male patients aged 0.5 to 16 y (seven with cystic fibrosis, two with malabsorption) underwent pancreatic stimulation tests. Their colipase and lipase secretion rates were measured by titrimetric methods and phospholipase A(2) and A(1) by phosphorus magnetic resonance spectroscopy ((31)P NMR). It was found that the phospholipases, colipase, and lipase were absent in the two patients with pancreatic insufficiency. In patients with normal absorption, there were marked inter-and intrasubject variations of lipase, colipase, and phospholipase secretion rates that were consistent with the degree of exocrine pancreatic dysfunction. However, in the three 20-min stimulation periods of the pancreatic function test, pancreatic phospholipase is cosecreted with lipase and colipase, and average colipase and phospholipase A(2) secretion rates follow a similar or parallel pattern. These findings are consistent with the important role of pancreatic phospholipases in intestinal phospholipid hydrolysis leading to the desorption of phospholipids from the lipid substrate and enhancing lipid hydrolysis and phospholipid absorption.


Subject(s)
Colipases/metabolism , Cystic Fibrosis/enzymology , Lipase/metabolism , Malabsorption Syndromes/enzymology , Pancreas/metabolism , Phospholipases A/metabolism , Adolescent , Child , Child, Preschool , Cystic Fibrosis/physiopathology , Dietary Fats/pharmacokinetics , Humans , Infant , Intestinal Absorption , Magnetic Resonance Spectroscopy , Malabsorption Syndromes/physiopathology , Male , Pancreas/enzymology , Phospholipases A/analysis , Phospholipids/metabolism , Secretory Rate
20.
Eur Biophys J ; 29(3): 221-7, 2000.
Article in English | MEDLINE | ID: mdl-10968214

ABSTRACT

The rapid exchange of water across erythrocyte membranes is readily measured using an NMR method that entails doping a suspension of cells with a moderately high concentration of Mn(2-) and measuring the rate of transverse relaxation of the nuclear magnetisation. Analysis of the data yields an estimate of the rate constant for membrane transport, from which the membrane permeability can be determined. It is assumed in the analysis that the efflux rate of the water is solely a function of the rate of membrane permeation and that the time it takes for intracellular water molecules to diffuse to the membrane is relatively insignificant. The limits of this assumption were explored by using random-walk simulations of diffusion in cells modelled as parallel planes, spheres, and biconcave discs. The rate of membrane transport was specified in terms of a transition probability but it was not initially clear what the relationship should be between this parameter and the diffusional membrane permeability P(d). This relationship was derived and used to show that the mean residence time for a water molecule is determined by P(d) when the diffusion coefficient is above a certain threshold value; it is determined by the distance to the membrane below that value.


Subject(s)
Monte Carlo Method , Permeability , Animals , Cell Membrane/metabolism , Computer Simulation , Diffusion , Elephants , Erythrocytes/metabolism , Humans , Magnetic Resonance Spectroscopy , Models, Theoretical , Probability , Time Factors , Water/metabolism
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