Pulsed nuclear magnetic resonance studies on 23 Na, 7 Li and 35 Cl binding to human oxy- and carbon monoxyhaemoglobin

Pulsed nuclear magnetic resonance studies of the longitudinal (T₁) and transverse (T₂) quadrupolar relaxation times of ⁷Li, ²³Na, ³⁵Cl ions in the absence and presence of human oxy- and carbon monoxyhaemoglobin have been used to investigate the interaction of the ions and the macromolecule.The relaxation data show that Cl^? interacts strongly with the haemoglobin but provide no evidence for binding of Na⁺ up to concentrations of 0.5 m. In the case of Li⁺, evidence for interaction is obtained at concentrations of about 0.1 m.The dependence of relaxation rate on frequency was followed over a limited frequency range in an attempt to separate the effects of correlation times and exchange rates of the bonded ions on the relaxation. In the case of Cl^?, an upper limit for the mean lifetime divided by the number of sites can be set at about 1 × 10^?6 second, and a lower limit at about 1 × 10^?8 second.     

Cadmium-113 and magnesium-25 NMR study of the divalent metal binding sites of isocitrate dehydrogenases from pig heart

The metal activator sites of NAD⁺-dependent and NADP⁺-dependent isocitrate dehydrogenases from pig heart have been probed using ¹¹³Cd- and ²⁵Mg-NMR. In the presence of isocitrate and ADP, a broad resonance for cadmium bound to NAD-dependent isocitrate dehydrogenase was observed ( −8 ppm) arising from exchange with isocitrate (−20 ppm) and/or ADP (27 ppm) complexes. The Cd shift with ADP suggests interaction of the metal with the nucleotide ring nitrogen. Increasing shifts with excess ADP are indicative of macrochelate formation. ²⁵Mg-NMR demonstrates that, unlike manganese, magnesium has a similar dissociation constant (1.8 mM) from NADP-dependent isocitrate dehydrogenase as from the enzyme-isocitrate complex (1.1 mM). The extrapolated line width of bound magnesium increases from 674 Hz in the binary complex to 10 200 Hz in the ternary complex. The quadrupole coupling constant, calculated from relaxation rates, is larger in the ternary complex. indicative of greater distortion in the magnesium coordination sphere. The line widths of magnesium complexed to NAD-dependent isocitrate dehydrogenase are broader, as expected for the larger octamer. ¹¹³Cd- and ²⁵Mg-NMR both show that the metal sites have anisotropic octahedral symmetry. ²⁵Mg relaxation rates yield correlation times corresponding to motions of a domain with motion independent of the enzyme multimers.     

Counterion binding and hydration of hyaluronate and chondroitin in solution: An 17O, 23Na,and 25Mg nuclear-magnetic-relaxation study

Nuclear magnetic relaxation rates of H₂¹⁷O, ²³Na⁺, and ²⁵Mg²⁺ have been measured in aqueous hyaluronate solutions. The dependence on solution pH of the relaxation rates has been investigated, as well as the competition behavior of Na⁺ with Ca²⁺ and Mg²⁺. H₂¹⁷O and ²³Na⁺ relaxation rates in chondroitin and hyaluronate solutions have been compared in the interval, 2 ? pH ? 12.5. The ion binding of hyaluronate can be fully accounted for by Coulomb interactions, with no need to involve chemical specificity. The hydration is only slighly pH dependent, and is comparable in magnitude to hydration of synthetic polyelectrolytes and monosaccharides. Ion-binding and hydration properties of hyaluronate and chondroitin are quite similar, except at elevated pH. At alkaline pH, an increase in charge density with pH is seen in hyaluronate and, to a much lesser degree, in chondroitin, possibly due to the titration of hydroxy groups. H₂¹⁷O data indicate an alkali-induced transition in both glycosaminoglycans.     

Lithium-7 NMR as a probe of monovalent cation sites at the active site of (Na+ + K+)-ATPase from kidney.

Lithium-7 nuclear magnetic resonance studies are used to characterize the binding of monovalent cations and substrate analogs to the (Na⁺ + K⁺)-ATPase. Li⁺ substitutes for K⁺ in the activation of the ATPase, while the longitudinal relaxation rate, $\text{1}\text{T}{}_1$, of ⁷Li⁺ is increased upon binding of either Mn²⁺ or CrATP to the enzyme. The effects of Mn²⁺ are consistent with the existence of a Li⁺ binding site 7.2A from the single catalytically active Mn²⁺ site on the ATPase. Temperature effects on the observed relaxation rates indicate that exchange of Li⁺ at the observed site is rapid, while the effects of added Na⁺ and K⁺ suggest that the observed site is a K⁺-type site not previously observed by other methods. These experiments also demonstrate that Li⁺ should be superior to other nuclei as NMR probes of the (Na⁺ + K⁺)-ATPase.     

Na+K+-ATPase Activity and K+ Channels Differently Contribute to Vascular Relaxation in Male and Female Rats

Gender associated differences in vascular reactivity regulation might contribute to the low incidence of cardiovascular disease in women. Cardiovascular protection is suggested to depend on female sex hormones’ effects on endothelial function and vascular tone regulation. We tested the hypothesis that potassium (K⁺) channels and Na⁺K⁺-ATPase may be involved in the gender-based vascular reactivity differences. Aortic rings from female and male rats were used to examine the involvement of K⁺ channels and Na⁺K⁺-ATPase in vascular reactivity. Acetylcholine (ACh)-induced relaxation was analyzed in the presence of L-NAME (100 µM) and the following K⁺ channels blockers: tetraethylammonium (TEA, 2 mM), 4-aminopyridine (4-AP, 5 mM), iberiotoxin (IbTX, 30 nM), apamin (0.5 µM) and charybdotoxin (ChTX, 0.1 µM). The ACh-induced relaxation sensitivity was greater in the female group. After incubation with 4-AP the ACh-dependent relaxation was reduced in both groups. However, the dAUC was greater in males, suggesting that the voltage-dependent K⁺ channel (K_v) participates more in males. Inhibition of the three types of Ca²⁺-activated K⁺ channels induced a greater reduction in R_max in females than in males. The functional activity of the Na⁺K⁺-ATPase was evaluated by KCl-induced relaxation after L-NAME and OUAincubation. OUA reduced K⁺-induced relaxation in female and male groups, however, it was greater in males, suggesting a greater Na⁺K⁺-ATPase functional activity. L-NAME reduced K⁺-induced relaxation only in the female group, suggesting that nitric oxide (NO) participates more in their functional Na⁺K⁺-ATPase activity. These results suggest that the K⁺ channels involved in the gender-based vascular relaxation differences are the large conductance Ca²⁺-activated K⁺ channels (BK_Ca) in females and K_v in males and in the K⁺-induced relaxation and the Na⁺K⁺-ATPase vascular functional activity is greater in males.     

Quenching of the fluorescence of proteins by silver nitrate

The mechanisms by which Ag⁺ may quench protein tryptophanyl fluorescence have been studied. A 1:1 Ag⁺-tryptophan complex was detected spectrophotometrically and shown to have a k_a = 6.5 × 10³ M^?1. The complex was nonfluorescent. Ag⁺ and NO₃^? each caused collisional quenching which proceeded at nearly diffusion-controlled rates in a series of indole-containing compounds. Analysis of the rates by means of Stern-Volmer plots and lifetime measurements showed also that charge and the presence of salt influence the quenching rate constants.The fluorescence of nonsulfhydryl proteins was quenched by AgNO₃ only in concentrations needed for Stern-Volmer quenching of simple indole model compounds. However, the plots for protein quenching were generally nonlinear, a reflection of the heterogeneity of tryptophanyl residues. AgNO₃ quenching increased the polarization of protein fluorescence and decreased the lifetime. Rotational relaxation times were determined from Perrin plots of reciprocal polarization vs fluorescence intensity in the presence of various amounts of AgNO₃.The fluorescence of the sulfhydryl proteins ovalbumin, yeast, and equine liver alcohol dehydrogenases was strongly quenched by AgNO₃ in parallel with the formation of Ag⁺-mercaptide bonds. The quenching of fluorescence of sulfhydryl proteins was exhibited even in 8 m urea, thus ruling out conformational change as a major basis for the quenching. It was found that Ag⁺ mercaptide bond formation was accompanied by development of an ultraviolet absorption band. The reaction of Ag⁺ with cysteine, for example, could be followed spectrophotometrically. The uv absorption of different silver mercaptides varied with the compound and pH.Since the uv absorption of Ag⁺-mercaptides extended up to 340 nm, and was also found in Ag⁺-treated sulfhydryl proteins, energy transfer from excited tryptophans seemed a reasonable basis for the observed fluorescence quenching. This possibility was confirmed by calculation of Förster critical transfer distances for a variety of donor-acceptor (Ag⁺-mercaptide) pairs.The lifetime of sulfhydryl protein fluorescence was decreased by AgNO₃, but the emission spectrum was relatively little affected, in contrast to previously reported quenching by Hg²⁺. Additional mechanisms of fluorescence alteration by Ag⁺ in proteins (e.g., “heavy atom” effect, conformational changes, enhancement of sulfhydryl quenching) are also considered.The spectral effects of Ag⁺ interaction with proteins have the following practical applications:determination of —SH groups; probe of accessibility of binding sites and tryptophan-sulfhydryl distances; determination of rotational relaxation times by Perrin plots of reciprocal polarization vs lifetime; kinetic studies of Ag⁺ interaction with proteins.     

Conformations and dynamics of surfactants in micelles and liquid crystals by nuclear magnetic relaxation

The order parameters as well as the rates of overall and internal motions of aggregated surfactants can be obtained from deuteron and carbon-13 nuclear relaxation experiments. The main contribution to the relaxation is generally the quadrupolar coupling (²H) or the short range dipolar interaction with protons (¹³C). In some cases it is convenient to derive the same information from the¹³C relaxation induced by long range dipolar interactions with a paramagnetic probe exchanging rapidly among the polar heads of surfactant molecules. This paper outlines the methods of interpretation of relaxation data by means of a rotational jump model of internal motions, taking into account most of the accessible conformers. The conformational and dynamical parameters are obtained from the magnetic field dependence of the longitudinal relaxation rates (micelles) or from the simultaneous fit of these rates and of the dipolar or quadrupolar splittings (liquid crystals). Some examples of application of these methods are given from recent works on single and double detailed surfactants.     

NMR measurements of intra- and extravesicular sodium in renal microvilli

Previous attempts to separate the nuclear magnetic resonances of intra- and extravesicular Na⁺ in brush-border membrane vesicles (BBMV) were unsuccessful and led to the proposal of rapid exchange of Na⁺ via sodium channels in BBMV. However, passive conductance of Na⁺ in this membrane has been found to be relatively small. This inconsistency prompted us to use a different shift reagent to reassess the issue. In guinea pig renal BBMV (15–30 mg protein/ml) equilibrated with Na⁺ (130 mequiv. 1), using the impermeant Na⁺ shift reagent dysprosium tripolyphosphate (3 mM), the resonances of intra- (3.3%) and extravesicular (96.7%) Na⁺ were resolved by 6 ppm. Increases in Na⁺ conductance induced by gramicidin D did not alter the characteristics of intra- and extravesicular Na⁺ resonances. By contrast, addition of glucose caused a transient increase in the area of the intravesicular Na⁺ resonance. The clear separation between the intra- and the extravesicular Na⁺ resonances allowed us to measure the relaxation times of Na⁺, which depend on its interactions with its immediate environment. The longitudinal relaxation time of intravesicular Na⁺ (13 ± 1 ms) was much shorter than that of the extravesicular Na⁺ (44.0 ± 0.4 ms). Thus, in intact renal BBMV, as well as in membranes treated with the cationophore gramicidin D, the exchange of Na⁺ between the intra- and the extravesicular compartments is slow on the NMR time scale, consistent with the low Na⁺ channel density of this membrane. In contrast, the increase in intravesicular Na⁺ induced by glucose, is consistent with a significant contribution of the glucose cotransport pathway to Na⁺ flux across these membranes. The short longitudinal relaxation time of Na⁺ in the intravesicular space indicates interaction of Na⁺ with BBMV binding sites or ordering of these ions in the intravesicular compartment.     

Angular dependence of dipole-dipole-Curie-spin cross-correlation effects in high-spin and low-spin paramagnetic myoglobin

The ¹⁵N-HSQC spectra of low-spin cyano-met-myoglobin and high-spin fluoro-met-myoglobin were assigned and dipole-dipole-Curie-spin cross-correlated relaxation rates measured. These cross-correlation rates originating from the dipolar ¹H-¹⁵N interaction and the dipolar interaction between the ¹H and the Curie spin of the paramagnetic center contain long-range angular information about the orientation of the ¹H-¹⁵N bond with respect to the iron-¹H vector, with information measurable up to 11 Å from the metal for the low-spin complex, and between 10 to 25 Å for the high-spin complex. Comparison of the experimental data with predictions from crystal structure data showed that the anisotropy of the magnetic susceptibility tensor in low spin cyano-met-myoglobin significantly influences the cross-correlated dipole-dipole-Curie-spin relaxation rates.     

Membrane conductance changes in single nodes of ranvier,measured by laser-induced temperature-jump experiments

Temperature-jump experiments on isolated myelinated nerve fibers were done using a pulsed laser system in the Q switched mode. Voltage-clamp and temperature perturbations were used to measure the relaxing ionic conductances of both the Na⁺ and K⁺ systems. It is shown that the T jump can be used to probe the K⁺ and Na⁺ conductances during non-steady state conditions and thereby elicit relaxation times for a variety of initial states. Temperature-induced K⁺ conductance relaxation times were consistent with voltage-clamp measurements. The temperature-perturbation experiments were done as a combination of a temperature step and impulse change due to an adsorption of carbon black particles on the nerve. The experiments support the hypothesis that the relaxation times of the K⁺ system are independent of the previous history of the axon. It is concluded that the K⁺ conductance is at least a second-order system whose relaxation spectrum is composed of two exponential terms the magnitudes of which are markedly dependent on the initial conditions.     

Folic acid consumption reduces resistin level and restores blunted acetylcholine-induced aortic relaxation in obese/diabetic mice

Folic acid supplementation provides beneficial effects on endothelial functions in patients with hyperhomocysteinemia. However, its effects on vascular functions under diabetic conditions are largely unknown. Therefore, the effect(s) of folic acid (5.7 and 71 μg/kg/day for 4 weeks) on aortic relaxation was investigated using obese/diabetic (+db/+db) mice and lean littermate (+db/+m) mice. Acetylcholine-induced relaxation in +db/+db mice was less than that observed in +db/+m mice. The reduced relaxation in +db/+db mice was restored by consumption of 71 μg/kg folic acid. Acetylcholine-induced relaxation (with and without folic acid treatment) was sensitive to N^G-nitro-l-arginine methyl ester, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, geldanamycin and triciribine. In addition, acetylcholine-induced relaxation was attenuated by resistin. The plasma level of resistin in +db/+db mice was sevenfold higher than that measured in +db/+m mice, and the elevated plasma level of resistin in +db/+db mice was reduced by 25% after treatment with 71 μg/kg folic acid. Folic acid slightly increased the ratio of reduced glutathione to oxidized glutathione in +db/+db mice. Moreover, folic acid caused a reduction in PTEN (phosphatase and tensin homolog deleted on chromosome 10) expression, an increase in the phosphorylation of endothelial nitric oxide synthase (eNOS^Ser1177) and Akt^Ser473, and an enhanced interaction of heat shock protein 90 (HSP90) with eNOS in both strains, with greater magnitude observed in +db/+db mice. In conclusion, folic acid consumption improved blunted acetylcholine-induced relaxation in +db/+db mice. The mechanism may be, at least partly, attributed to enhancement of PI3K/HSP90/eNOS/Akt cascade, reduction in plasma resistin level, down-regulation of PTEN and slight modification of oxidative state.     

Ozone-induced Loss of Intracellular Potassium Ion from Chlorella sorokiniana

The unicellular algae Chlorella sorokiniana was used as a model system to investigate the interaction of ozone with plant cell membranes. Ozone induces K⁺ leakage from Chlorella sorokiniana similar to the electrolytic loss observed from many higher plants under stressful conditions. The kinetics of this leakage indicate that ozone initially interacts reversibly (within sec) with sites on membranes allowing a passive efflux of K⁺. This efflux ceases within minutes after the ozone stress is removed. This return to normal efflux is very temperature dependent. High intracellular osmolarity seems to be an important criterion of susceptibility to ozone injury in this model system, since rates of ozone-induced K⁺ leakage are less when the external osmotic potential is decreased by suspension of the cells in mannitol. Cell interaction with ozone is further complicated by a saturating-type dependence of the K⁺ efflux upon ozone concentration within the medium.     

Apamin inhibits NO-induced relaxation of the spontaneous contractile activity of the myometrium from non-pregnant women

There is now considerable evidence for the involvement of K⁺ channels in nitric oxide (NO) induced relaxation of smooth muscles including the myometrium. In order to assess whether apamin-sensitive K⁺ channels play a role in NO – induced relaxation of the human uterus, we have studied the effect of specific blockers of these channels on the relaxation of myometrium from non-pregnant women. In vitro isometric contractions were recorded in uterine tissues from non-pregnant premenopausal women who had undergone hysterectomy. Apamin (10 nM) and scyllatoxin (10 nM) did not alter spontaneous myometrial contractions. However, 15-min pretreatment of the myometrium strips with apamin completely inhibited relaxation caused by diethylamine-nitric oxide (DEA/NO). The pretreatment with scyllatoxin significantly reduced (about 2.6 times) maximum relaxation of the strips induced by DEA/NO (p < 0.05). These results strongly suggest that, beside Ca²⁺ and voltage dependent charybdotoxin-sensitive (CTX-sensitive) K⁺ channels, apamin-sensitive K⁺ channels are also present in the human non-pregnant myometrium. These channels offer an additional target in the development of new tocolytic agents.     

Specificity of Na+ binding to phosphatidylserine vesicles from A 23Na NMR relaxation rate study

²³Na NMR relaxation rate measurements show that Na⁺ binds specificially to phosphatidylserine vesicles and is displaced partially from the binding site by K⁺ and Ca²⁺ but to a considerably less extent by tetraethylammonium ion. The data indicate that tetraethylammonium ion affects the binding of Na⁺ only slightly, by affecting the surface potential through its presence in the double layer, without competing for a phosphatidylserine binding site. Values for the intrinsic binding constant for the Na⁺-phosphatidylserine complex that would be consistent with the competition experiments (and the dependence of the relaxation rate on concentration of free Na⁺) fall in the range 0.4–1.2 M^?1 with a better fit towards the higher values. We conclude that in the absence of competing cations in solution an appreciable fraction of the phosphatidylserine sites could be associated with bound Na⁺ at 0.1 M Na⁺ concentration.     

Adenosine diphosphate and calcium stimulation of respiration in mitochondria from alloxan diabetic rats

Liver mitochondria from normal and alloxan diabetic rats, isolated in 0.25 M sucrose, were assayed with an oxygen electrode for ADP/O and Ca⁺²/O ratios, respiratory ratio, and respiratory control index. Mitochondria were incubated with two substrates, succinate and β-hydroxybutyrate; two types of ionic media, Na⁺ medium (Na⁺ the major monovalent cation) and K⁺ medium (K⁺ the major monovalent cation); and two respiratory stimulants, ADP (352 μM) and Ca⁺² (187 μM). Significant differences between respiratory rates and ADP/O ratios were dependent upon the substrate and ionic medium employed. The results confirm previous studies which showed no alteration in ADP/O ratio but decreased State 3 respiratory rates under similar conditions of K⁺ medium with ADP stimulation in the diabetic. Furthermore, the State 3 respiration was prolonged compared to normal. Ca⁺² stimulation was the same in normal and diabetic mitochondria in K⁺ medium. Studies in Na⁺ media revealed more significant differences in RCI's, respiratory rates, and ADP/O ratios that were substrate dependent as well as ion dependent. The results from these various studies can be accounted for by an hypothesis linking mitochondrial K⁺ interaction with alterations in the diabetic mitochondria.     

Pulsed nuclear magnetic resonance study of39K within Halobacteria

Summary The³⁹K contents of isolated pellets and supernatant solutions from suspensions ofHalobacterium halobium were studied at 21–22°C by pulsed NMR spectroscopy. The rates of transverse relaxation were measured directly from the free induction decay (FID). The rate of longitudinal relaxation was measured by studying the FID after pairs of pulses of approximately 90°. Care was exercised to minimize the effect of magnetic field inhomogeneity; its contribution to the FID was approximately 25–30 sec^–1. The transverse relaxation process was found to consist of at least two components, whose rates were 321–449 sec^–1 and 1,122–2,067 sec^–1. In one preparation where the longitudinal relaxation process was studied, the data could be well fit to a single exponential relaxing at 253±33 (mean ±95% confidence limits) sec^–1. Comparison of the relative intensities of the NMR signals with the results of atomic absorption photometric analyses indicated that the great bulk of the intracellular³⁹K was detected by the NMR techniques used. The data obtained from the current NMR ofH. halobium are consistent with: (1) fractional binding of <3% of the total intracellular K⁺, (2) a small ordering factor characterizing all of the intracellular K⁺, or (3) some combination of the two.     

Relaxation studies on complex formation of macrocyclic and open chain antibiotics with monovalent cations

The stability constants for the 1 : 1 complexes of macrocyclic antibiotics (nonactin, monactin, dinactin and trinactin) with Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, NH⁺₄ and for the Na⁺-complexes with the open chain compounds nigericin and monensin in methanol solution have been determined. The relaxation amplitude method was employed to obtain both the equilibrium constants and the enthalpies of reaction. The kinetics were studied with the help of temperature-jump, electric-field pulse and ultrasonic absorption techniques. Although complex formation of the metal ions with the antibiotics involves multidentate ligand chelation, the formation rates are in general very high, i.e. close to the limits imposed for diffusion controlled processes. The data for the macrotetrolides indicate the existence of conformational transition prior to complexation. A sequential substitution or “redressing” mechanism is proposed which is in accord with the high rates of complex formation. The selectivity patterns, as expressed by the equilibrium constants, are similar to those observed for the transport of metal ions across membranes in presence of the antibiotics. Selectivity results from an optimal balance between the strength of metal ion solvation and the stability of the individual metal complex, which in turn is governed by the conformational flexibility of the antibiotics.     

Monovalent and divalent promoted GAAA tetraloop-receptor tertiary interactions from freely diffusing single-molecule studies

Proper assembly of RNA into catalytically active three-dimensional structures requires multiple tertiary binding interactions, individual characterization of which is crucial to a detailed understanding of global RNA folding. This work focuses on single-molecule fluorescence studies of freely diffusing RNA constructs that isolate the GAAA tetraloop-receptor tertiary interaction. Freely diffusing conformational dynamics are explored as a function of Mg²⁺ and Na⁺ concentration, both of which promote facile docking, but with 500-fold different affinities. Systematic shifts in mean fluorescence resonance energy transfer efficiency values and line widths with increasing [Na⁺] are observed for the undocked species and can be interpreted with a Debye model in terms of electrostatic relaxation and increased flexibility in the RNA. Furthermore, we identify a 34 ± 2% fraction of freely diffusing RNA constructs remaining undocked even at saturating [Mg²⁺] levels, which agrees quantitatively with the 32 ± 1% fraction previously reported for immobilized constructs. This verifies that the kinetic heterogeneity observed in the docking rates is not the result of surface tethering. Finally, the K_D value and Hill coefficient for [Mg²⁺]-dependent docking decrease significantly for [Na⁺] = 25 mM vs. 125 mM, indicating Mg²⁺ and Na⁺ synergy in the RNA folding process.     

Effect of morphine analogues on the conformational state of polyriboadenylic acid in solution: An NMR study

The interaction of morphine analogues with polyadenylic acid in aqueous solution at pH 7 has been investigated. The NMR spectra measured at several temperatures were shown to give evidence of a lowering, of the transition temperature from the helix to the random-coil structure. The measured chemical shifts and relaxation times were interpreted in terms of an ionic interaction between the ⁺N-CH₃ group of the drug and the phosphate residue of the polynucleotide.