The interaction of 1-anilino-8-naphthalenesulfonate with thyroid lipids and membranes: a nuclear magnetic resonance study

The proton magnetic resonance (PMR) spectra of thyroid cell membranes and their total lipid extracts, in the presence of 1-anilino-8-naphthalenesulfonate (ANS), have been studied. The addition of ANS causes an shifting of the head group PMR signal, a splitting of the signal into two components and an increase in total spectral intensity. The data suggest that ANS interacts with phospholipid in the membrane as it does in total lipid vesicles. Evidence is also presented for the removal of lipids from the membrane, by ANS, and the subsequent formation of micelles. The membrane results are compared with our earlier work on the interaction of ANS with egg phosphatidylcholine (P.C.) vesicles and the results are used in explaining the inhibition of iodide transport in isolated thyroid slices.     

The binding of 1-anilino-8-naphthalenesulfonate, heparin, salicylate and caprylate by human antithrombin III

The binding of 1-anilino-8-naphthalenesulfonate to human antithrombin III was studied by fluorescence enhancement of the fluorophor and fluorescence quenching of the protein emission. Two molecules of 1-anilino-8-naphthalenesulfonate were found to bind per antithrombin molecule with an average dissociation constant of 4.4-10(-5) M. The binding of heparin to antithrombin was studied by ultraviolet difference spectroscopy. The stoichiometry of the heparin binding indicated 1.8 binding sites with an average dissociation constant of 4.3 - 10(-6) M. Further the fluorometric competition experiments with 1-anilino-8-naphthalenesulfonate, heparin, salicylate and caprylate indicated two different classes of anion binding sites on the antithrombin molecule.     

Inhibition of assembly of bacterial cell division protein FtsZ by the hydrophobic dye 5,5'-bis-(8-anilino-1-naphthalenesulfonate)

To gain further insight into the structural relatedness of tubulin and FtsZ, the tubulin-like prokaryotic cell division protein, we tested the effect of tubulin assembly inhibitors on FtsZ assembly. Common tubulin inhibitors, such as colchicine, colcemid, benomyl, and vinblastine, had no effect on Ca2+-promoted GTP-dependent assembly of FtsZ into polymers. However, the hydrophobic probe 5, 5'-bis-(8-anilino-1-naphthalenesulfonate) (bis-ANS) inhibited FtsZ assembly. The potential mechanisms for inhibition are discussed. Titrations of FtsZ with bis-ANS indicated that FtsZ has one high affinity binding site and multiple low affinity binding sites. ANS (8-anilino-1-naphthalenesulfonate), a hydrophobic probe similar to bis-ANS, had no inhibitory effect on FtsZ assembly. Because tubulin assembly has also been shown to be inhibited by bis-ANS but not by ANS, it supports the idea that FtsZ and tubulin share similar conformational properties. Ca2+, which promotes GTP-dependent FtsZ assembly, stimulated binding of bis-ANS or ANS to FtsZ, suggesting that Ca2+ binding induces changes in the hydrophobic conformation of the protein. Interestingly, depletion of bound Ca2+ with EGTA further enhanced bis-ANS fluorescence. These findings suggest that both binding and dissociation of Ca2+ are capable of inducing FtsZ conformational changes, and these changes could promote the GTP-dependent assembly of FtsZ.     

T1 rho in nuclear quadrupole resonance: a theoretical study

A new NQR method of measuring the spectral density of slow motions in solids is proposed. It is shown that also in NQR a 90 degrees phase shift of a resonant rf magnetic field following a 90 degrees pulse locks the nuclear magnetization in a 'rotating frame' similarly as in NMR. The spin-lattice relaxation time T1 rho of the locked magnetization is calculated in general for an arbitrary spin. It is assumed that the fluctuations of the EFG tensor dominate the spin-lattice relaxation. The calculations show that T1 rho depends on the spectral density J(omega) of the electric quadrupole fluctuations at the NQR frequencies, and also at a low frequency omega. Here omega approximately gamma B1 kHz depends on the orientation of the rf magnetic field in the principal-axis system of the EFG tensor. The term containing J(omega) in the expression for T1 rho-1 depends on the orientation of the rf magnetic field in the principal-axis system of the EFG tensor, only through the orientation dependence of omega. This term vanishes when the electric quadrupole fluctuations do not modulate the frequency of the NQR transition excited by the rf magnetic field. Two particular examples: I = 1 and I = 3/2 are worked out in details.     

Rhomboencephalitis caused by Listeria monocytogenes: a case studies with magnetic nuclear resonance

Central nervous system infections due to Listeria monocytogenes result in a variety of clinical syndromes ranging from meningitis to rhomboencephalitis. We report the case of a previously healthy patient with rhomboencephalitis in whom the CT scan was normal, while magnetic resonance imaging (MRI) confirmed the diagnosis. We review the literature and emphasize the value of MRI for timely diagnosis.     

13C nuclear magnetic resonance study of 17 alpha-substituted estradiols

We report the 13C NMR data for 20 compounds bearing a substituent (alkyl, alkenyl, alkynyl, alkylamide, spiro-gamma-lactone, phenyl, benzyl, naphthyl, etc.) at the 17 alpha-position of estradiol. The carbon assignments were done using 1D and 2D NMR experiments (distortionless enhancement by polarization transfer, homonuclear correlated spectroscopy, heteronuclear shift correlation, and heteronuclear shift correlation via long-range couplings). Only the chemical shifts of carbons 12-18, which surround the substitution site, were affected by the addition of a substituent. The magnitude of the effects (shielding or deshielding) was influenced by the 17 alpha-substituent. The individual effects at these carbons were sufficiently distinctive to identify specific centers and should be valuable for signal assignment of a variety of 17 alpha-derivatives of estradiol. In addition to carbon-skeleton assignment, we also report the carbon-substituent assignments.     

Phosphorus nuclear magnetic resonance: a non-invasive technique for the study of muscle bioenergetics during exercise

Phosphorus nuclear magnetic resonance (31P NMR) spectroscopy is a non-destructive analytical laboratory technique that, due to recent technical advances, has become applicable to the study of high-energy phosphate metabolism in both animal and human extremity muscles (in vivo). 31P NMR can assay cellular phosphocreatine, ATP, inorganic phosphate, the phosphorylated glycolytic intermediates, and intra-cellular pH in either resting or exercising muscle, in a non-invasive manner. NMR uses non-perturbing levels of radio-frequency energy as its biophysical probe and can therefore safely study intact muscle in a repeated fashion while exerting no artifactual influence on ongoing metabolic processes. Compared with standard tissue biopsy and biochemical assay techniques, NMR possesses the advantages of being non-invasive, allowing serial in situ studies of the same tissue sample, and providing measurements of only active (unbound) metabolites. NMR studies of exercising muscle have yielded information regarding fatigue mechanisms at the cellular level and are helping resolve long-standing questions regarding the metabolic control of glycolysis, oxidative phosphorylation, and post-exercise phosphocreatine re-synthesis. NMR is also being utilized to measure enzymatic reaction rates in vivo. In the near future, other forms of NMR spectroscopy may also permit the non-invasive measurement of tissue glycogen and lactate content.     

Structure of a mycobacterial polysaccharide-fatty acyl-CoA complex: nuclear magnetic resonance studies

MMP, a linear alpha 1 leads to 4 linked polymer of 3-O-methylmannose, regulates the fatty acid synthetase from Mycobacterium smegmatis by forming stoichiometric complexes with the long-chain acyl-CoA synthetase products. In agreement with previous proposals [Bloch, K. (1977) in Advances in Enzymology and Related Areas of Molecular Biology, ed. Meister, A. (Wiley, New York), Vol. 45, pp. 1-84], nuclear magnetic resonance studies show that the polysaccharide, a random coil in its free form, undergoes a major conformational transition upon enclosing long-chain acyl-CoA. The polysaccharide, probably in helical conformation in the complexed form, interacts with both the paraffinic chain and the CoA moieties of the included fatty acyl thioester.     

Epidermal and dermal phospholipids of the human eyelid: a 31P nuclear magnetic resonance spectroscopy study

The phospholipids of the skin are difficult to quantify because they represent only a small fraction of the skin tissue. In this study, 31P nuclear magnetic resonance, which permits precise profiling of these phospholipids, was used to compare the phospholipids of upper eyelid epidermal and dermal lipid extracts (n = 13 profiles). Phospholipid profiles included alkylacylphosphatidylcholine (AAPC), dihydrosphingomyelin (DHSM), diphosphatidylglycerol (cardiolipin), ethanolamine plasmalogen (EPLAS), lysophosphatidylcholine, phosphatidic acid, phosphatidylcholine (PC), phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, sphingomyelin, and uncharacterized phospholipids (U1 and U2, particularly enriched in the epidermis). The computed phospholipid metabolic index (n = 86 indexes) findings can be summarized as follows: a lower content of the en-ol and ether phospholipids in the epidermis relative to the dermis, internal compensation among the component phospholipids so as to maintain the choline functional group ratio, and a greater concentration of hydroxyl-containing functional groups in the epidermis. A membrane index (fmem) value of -0.37 for the epidermis deviated considerably from the value of -0.06 characteristic of living membranes and the dermis. The production of the reduced phosphatides, EPLAS and AAPC, indicates the use of alternative pathways between the two tissues. Relative to the dermis, increased PC in the epidermis coupled with decreased DHSM, EPLAS, and AAPC are factors enabling the epidermis of eyelid tissue to be an effective water barrier.     

pKa measurements from nuclear magnetic resonance for the B1 and B2 immunoglobulin G-binding domains of protein G: comparison with calculated values for nuclear magnetic resonance and X-ray structures

Two-dimensional homo- and heteronuclear nuclear magnetic resonance (NMR) spectroscopy was used to determine pKa values for all of the acidic residues in the B1 and B2 immunoglobulin G- (IgG-) binding domains of protein G. Due to the stability of protein G over a wide pH range, estimates of ionization constants were also obtained for some basic residues. These experimentally determined ionization constants were compared with values calculated from both X-ray and NMR-derived structures of B1 and B2 using the UHBD algorithm [Antosiewicz, J., et al. (1994) J. Mol. Biol. 238, 415-436]. This algorithm has been found to be predictive for pKa measurements in proteins and, in combination with experimental measurements, allowed some evaluation of the NMR and X-ray structures. Three regions where significant differences exist between the X-ray and NMR structures are (1) the position of the E56 side chain relative to the backbone amides of K10 and D40, (2) residues 33-37 in the helix, and (3) the Y45 side-chain conformation. For all three cases, the experimental pH titration curves are notably more consistent with the X-ray structures than the NMR structures. In contrast, a number of solvent-accessible side chains have experimental pKas more in agreement with mean pKas calculated from families of NMR structures. The conformations of these side chains may be susceptible to crystal packing effects. From titration experiments under basic conditions, it is noteworthy that the chemical shift of the Y45 C epsilonH resonance is invariant up to pDcorr 12. The Y45 side-chain hydroxyl group appears to maintain a nativelike hydrogen bond with D47 at pDcorr 12, even though the protein is approximately 90% unfolded. These results suggest that this short-range (i, i + 2) interaction, located in the beta3-beta4 hairpin, is present in the high-pH denatured state and may therefore form early in the folding of protein G.     

Dislocation dynamics in vanadium: A nuclear magnetic resonance and transmission electron microscopic study

Pulsed nuclear magnetic resonance proved to be a complementary new technique for the study of moving dislocations in b.c.c. metals. From the motion induced part of the spin-lattice relaxation rate the mean jump distance of mobile dislocations has been measured in Vanadium as a function of temperature. The NMR experiments are combined with transmission electron microscopic investigations to reveal the static structure of defects in the samples. The NMR experiments show that the mean jump distance is nearly constant below 230 K whereas it decreases substantially above 230 to 300 K indicating a transition that marks two different mechanisms. NMR observations in combination with TEM support the physical picture that above the transition temperature dislocation segments are stopped between localized obstacles whereas below T_c the lattice friction controls the plastic behaviour.     

A study of precipitation using nuclear magnetic resonance: Cu-13 at.% Be

We examine the nuclear magnetic resonance (nmr) intensity of one of the components in one of the phases in a two phase binary alloy undergoing precipitation. It is shown that for certain nuclei one can readily follow the course of the reaction, and ideally can determine quantitatively the composition and amount of the final matrix if the precipitate composition is known. We develop expressions relating the resonance intensity to the degree of completion of the reaction for

• 1.(a) discontinuous and
• 2.(b) continuous precipitation.

These are used to interpret the intensity vs ageing time at 280°C for a Cu-13 at.% Be alloy. The ratio of the intensity of the ⁶³Cu in the matrix in the fully aged alloy to that in the as quenched alloy is also examined and discussed in light of our experimental results. It is argued that interfacial effects at phase boundaries cause significant deviations from expected intensities.     

Kinetics of binding of methyl alpha- and beta-D-galactopyranoside to peanut agglutinin: a carbon-13 nuclear magnetic resonance study

The binding kinetics of methyl alpha- and methyl beta-D-galactopyranoside to the anti-T lectin from peanuts were studied by 13C NMR, employing methyl galactopyranosides specifically enriched in 13C at C-1. Association and dissociation rate constants, as well as their activation parameters, are reported. The association rate constants, 4.6 X 10(4) M-1 s-1 for the alpha-galactopyranoside and 3.6 X 10(4) M-1 s-1 for the beta-galactopyranoside, are several orders of magnitude below those expected for a diffusion-controlled process. For both anomers, the association rate constant was temperature independent, implying that the association process occurs without a significant activation enthalpy. However, a considerable association activation entropy was found for both ligands. The dissociation rate constants were in the range of 9-46 s-1 within a temperature range of 5-35 degrees C for the alpha-galactopyranoside, and in the range of 9-39 s-1 within a temperature range of 5-25 degrees C for the beta-galactopyranoside. A considerable dissociation activation enthalpy of ca. 10 kcal mol-1 was found for both anomers. A two-step binding model, consistent with the present NMR data and with previous UV and CD spectroscopic data, is presented.     

A calbindin D9k mutant containing a novel structural extension: 1H nuclear magnetic resonance studies

Calbindin D9k is a small, well-studied calcium-binding protein consisting of two helix-loop-helix motifs called EF-hands. The P43MG2 mutant is one of a series of mutants designed to sequentially lengthen the largely unstructured tether region between the two EF-hands (F36-S44). A lower calcium affinity for P43MG was expected on the basis of simple entropic arguments. However, this is not the case and P43MG (-97 kJ.mol-1) has a stronger calcium affinity than P43M (-93 kJ.mol-1), P43G (-95 kJ.mol-1) and even wild-type protein (-96 kJ.mol-1). An NMR study was initiated to probe the structural basis for these calcium-binding results. The 1H NMR assignments and 3JHNH alpha values of the calcium-free and calcium-bound form of P43MG calbindin D9k mutant are compared with those of P43G. These comparisons reveal that little structure is formed in the tether regions of P43MG(apo), P43G(apo) and P43G(Ca) but a helical turn (S38-K41) appears to stabilize this part of the protein structure for P43MG(Ca). Several characteristic NOEs obtained from 2D and 3D NMR experiments support this novel helix. A similar, short helix exists in the crystal structure of calcium-bound wild-type calbindin D9k-but this is the first observation in solution for wild-type calbindin D9k or any of its mutants.     

Solution hardening in aluminium-magnesium alloys: A nuclear magnetic resonance and transmission electron microscopic study

Pulsed nuclear magnetic resonance techniques as well as transmission electron microscopy have been applied to study dislocation motion in aluminium magnesium alloys (0.2–1.6 at.% Mg). The spin lattice relaxation rate in the rotating frame of ²⁷A1 has been been measured at 77 K as a function of strain at constant plastic strain rate ϵ. For finite strain rates, the movement of dislocations induces an additional relaxation rate arising from time fluctuations in the nuclear quadrupole interactions. From the motion-induced part of the relaxation rate the mean free path of mobile dislocations can be calculated. The NMR experiments are combined with transmission electron microscopic investigations to reveal the static structure of defects in the samples. The NMR measurements clearly indicate that fluctuations in the quadrupolar field caused by moving dislocations in AlMg are different compared to those in ultra pure Al. From the NMR data it could be concluded that moving dislocations advance over a number of solute atoms (order of 7) as described by Mott-Nabarro's model. On the other hand, Mott-Nabarro's model does not predict the effective solute spacing as a function of the concentration of solute atoms in accordance with NMR experiments.