Effects of temperature and field strength on the NMR relaxation times of 23Na in frog striated muscle

Pulsed NMR techniques have been applied to the study of the relaxation parameters characterizing ²³Na within frog striated muscle. Experiments were performed at 3°C, 22–24°C and 39°C at a Larmor frequency of 15.7 MHz; at 22–24°C, measurements were obtained both at 15.7 MHz and at 7.85 MHz.As previously reported, only a single spine-lattice relaxation time (T₁) was observed, but both slow (T₂)_I and fast (T₂)_II components of the spin-spin relaxation time were measured. The effect of temperature (θ) upon (1/T₁) was qualitatively similar to that reported for ²³Na in free solution; (θ) did not significantly affect (1/T₂) over the range of temperatures studied. (1/T₂)_I, and to a lesser degreee, (1/T₁) exhibited a modest inverse dependence of doubtful significance on the Larmor frequency.The data are examined within the framework of a simple specific model; a conservative values in assumed for the quadrupolar coupling constant characterizing immobilized intracellular Na⁺. Within this framework, the results suggest that the fraction of bound ions whose molecular tumbling is severely restricted does not exceed some few percent of the total sodium population.     

Effects of temperature and field strength on the NMR relaxation times of Na in frog striated muscle

Pulsed NMR techniques have been applied to the study of the relaxation parameters characterizing ²³Na within frog striated muscle. Experiments were performed at 3°C, 22–24°C and 39°C at a Larmor frequency of 15.7 MHz; at 22–24°C, measurements were obtained both at 15.7 MHz and at 7.85 MHz.As previously reported, only a single spine-lattice relaxation time (T₁) was observed, but both slow (T₂)_I and fast (T₂)_II components of the spin-spin relaxation time were measured. The effect of temperature (θ) upon (1/T₁) was qualitatively similar to that reported for ²³Na in free solution; (θ) did not significantly affect (1/T₂) over the range of temperatures studied. (1/T₂)_I, and to a lesser degreee, (1/T₁) exhibited a modest inverse dependence of doubtful significance on the Larmor frequency.The data are examined within the framework of a simple specific model; a conservative values in assumed for the quadrupolar coupling constant characterizing immobilized intracellular Na⁺. Within this framework, the results suggest that the fraction of bound ions whose molecular tumbling is severely restricted does not exceed some few percent of the total sodium population.     

Matrix-dependent modulation of anisotropic effects on NMR spectra from 7Li+ and 23Na+ encapsulated in cryptands

⁷Li and ²³Na NMR spectra of the respective cations in gelatin and ι-carrageenan gels containing cryptand-[2.1.1] (for Li⁺) or cryptand-[2.2.2] (for Na⁺) displayed two transitions: the one at higher frequency corresponded to the cation surrounded by gel, the other to cation inside its appropriately sized cryptand. While binding to cryptands yielded much broader lines and shorter T ₁ relaxation times, anisotropic splitting in first order ⁷Li or ²³Na NMR spectra was not detected. Stretching the gels resulted in increasing the anisotropic electric field gradient tensor; thus, the NMR transitions of the cation in the gel were split (removal of degeneracy) to display its characteristic 3:4:3 triplet for spin = 3/2 nuclei. The transitions of the cryptand-bound cations (Li⁺-cryptand-[2.1.1] and Na⁺-cryptand-[2.2.2]) showed different extents of interaction with the electric field gradient tensor depending on the composition of the gel matrix. The NMR signal for ⁷Li⁺-cryptand-[2.1.1] in stretched gelatin gel showed a five-fold increased splitting as compared to the ⁷Li⁺ signal in the reference gel. In stretched ι-carrageenan gels, no anisotropic splitting from the cryptand-bound Li⁺ was recorded. Steady-state irradiation envelopes or z-spectra showed evidence of Li⁺ exchange between isotropic (cryptand) and anisotropic (gel) sites only at higher temperatures (55 °C). For Na⁺ bound to the cryptand-[2.2.2], anisotropic splitting (three-fold smaller compared with the ²³Na signal in the reference gel) was only recorded in stretched ι-carrageenan gels, whereas gelatin gels showed only anisotropic splitting for the ²³Na signal in the reference gel.     

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.     

Fluctuations, exchange processes, and water diffusion in aqueous protein systems: A study of bovine serum albumin by diverse NMR techniques

Experimental frequency, concentration, and temperature dependences of the deuteron relaxation times T₁ and T₂ of D₂O solutions of bovine serum albumin are reported and theoretically described in a closed form without formal parameters. Crucial processes of the theoretical concept are material exchange, translational diffusion of water molecules on the rugged surfaces of proteins, and tumbling of the macromolecules. It is also concluded that, apart from averaging of the relaxation rates in the diverse deuteron phases, material exchange contributes to transverse relaxation by exchange modulation of the Larmor frequency. The rate limiting factor of macromolecular tumbling is determined by the free water content. In a certain analogy to the classical free-volume theory, a “free-water-volume theory” is presented. There are two characteristic water mass fractions indicating the saturation of the hydration shells (C_s ≈ 0.3) and the onset of protein tumbling (C₀ ≈ 0.6). The existence of the translational degrees of freedom of water molecules in the hydration shells has been verified by direct measurement of the diffusion coefficient using an NMR field-gradient technique. The concentration and temperature dependences show phenomena indicating a percolation transition of clusters of free water. The threshold water content was found to be C_c^w ≈ 0.43.     

Reverse NaCa exchange requires internal Ca and/or ATP in squid axons

Classical NaCa exchange models are based on a symmetric carrier system where Na and Ca competing from the same site, can produce net movement of the other against its electrochemical gradient. We have explored this symmetric assumption by studying the Ca_o and Na_o-dependent Na efflux in dialyzed squid axons in which proper control of both external and internal medium was achieved. The results show: (1) In axons dialyzed without Ca_i and ATP, Ca_o-dependent Na efflux cannot be detected even in the absence of Na_o. Under these conditions, the level of Na efflux (1 pmol · cm⁻² · s⁻¹) is close to that predicted by an electrical ‘leak’. (2) In axons dialyzed with Ca_i (100 μM) and without ATP, Na efflux measured in 440 mM Na_o, is about 4–5 pmol · cm⁻² · s⁻¹ and rather insensitive to Ca_o between 0 and 10 mM. However, in the absence of Na_o, a Ca_o-dependent Na efflux is observed similar in magnitude to that found in the presence of external Na. (3) In the presence of both Ca_i and ATP, Na efflux into artificial sea-water (440 mM Na, 10 mM Ca) is 18 pmol · cm⁻² · s⁻¹. In the absence of Na_o the efflux of Na is 7.5 pmol · cm⁻² · s⁻¹. In the absence of both Na_o and Ca_o the efflux is close to ‘leak’. With full Na_o but no Ca_o, the Na efflux average 12.6 pmol · cm⁻² · s⁻¹. These results indicate a marked asymmetry in the modus operandi of the NaCa exchange system with respect to Ca_i and ATP. These two substrates are required from the cis side to promote Ca_o-dependent Na efflux (reversal NaCa exchange).     

The interaction between water and the polar head in inverted phosphatidylcholine micelles A 2H and 31P relaxation study

²H and ³¹P spin-lattice relaxation times (T₁) were studied for invented egg phosphatidylcholine micelles in CCl₄ as functions of ²H₂O concentration. When the ²H₂O/phosphatidylcholine mole ratio changed from 1.0 to 18.0, T₁ of ³¹P increased by about 2.6 fold, whereas T₁ of ²H increased by about 50 fold. A quantitative analysis of the deuterium T₁ data showed that there is only one water molecule tightly bound to the polar head, and it is in rapid exchange with the rest of the water molecules. The activation energy for the deuterium T₁ was 7.1 ± 0.8 kcal/mol (30 ± 3 kJ/mol), and was independent of the ²H₂O concentration.     

Single-Base-Pair Discrimination of Terminal Mismatches by Using Oligonucleotide Microarrays and Neural Network Analyses

The effects of single-base-pair near-terminal and terminal mismatches on the dissociation temperature (T_d) and signal intensity of short DNA duplexes were determined by using oligonucleotide microarrays and neural network (NN) analyses. Two perfect-match probes and 29 probes having a single-base-pair mismatch at positions 1 to 5 from the 5′ terminus of the probe were designed to target one of two short sequences representing 16S rRNA. Nonequilibrium dissociation rates (i.e., melting profiles) of all probe-target duplexes were determined simultaneously. Analysis of variance revealed that position of the mismatch, type of mismatch, and formamide concentration significantly affected the T_d and signal intensity. Increasing the concentration of formamide in the washing buffer decreased the T_d and signal intensity, and it decreased the variability of the signal. Although T_ds of probe-target duplexes with mismatches in the first or second position were not significantly different from one another, duplexes with mismatches in the third to fifth positions had significantly lower T_ds than those with mismatches in the first or second position. The trained NNs predicted the T_d with high accuracies (R² = 0.93). However, the NNs predicted the signal intensity only moderately accurately (R² = 0.67), presumably due to increased noise in the signal intensity at low formamide concentrations. Sensitivity analysis revealed that the concentration of formamide explained most (75%) of the variability in T_ds, followed by position of the mismatch (19%) and type of mismatch (6%). The results suggest that position of the mismatch at or near the 5′ terminus plays a greater role in determining the T_d and signal intensity of duplexes than the type of mismatch.     

Compensation for measuring error produced by finite response time in determination of rates of oxygen consumption with a Clark-type polarographic electrode

In the determination of the rates of oxygen consumption with a Clark-type oxygen electrode, and experimental error is caused by finite response time of the oxygen electrode for a rapid oxidation reaction. A theoretical equation between the observed pseudo first-order rate constant (k_obs) and the true rate constant (k)

$\text{1}\text{k}{}_{\mathrm{obs}}\text{=}\text{1}\text{k}\text{+T}$

where T is a time constant for a first-order response of the oxygen electrode, was derived and found to hold up to k = 23 min^?1 in oxidation of hydroquinone at pH 7.60–8.63.     

Intracellular water in Artemia cysts (brine shrimp): Investigations by deuterium and oxygen-17 nuclear magnetic resonance

The dormant cysts of Artemia undergo cycles of hydration-dehydration without losing viability. Therefore, Artemia cysts serve as an excellent intact cellular system for studying the dynamics of water-protein interactions as a function of hydration. Deuterium spin-lattice (T₁) and spin-spin (T₂) relaxation times of water in cysts hydrated with D₂O have been measured for hydrations between 1.5 and 0.1 g of D₂O per gram of dry solids. When the relaxation rates (I/T₁, I/T₂) of ²H and ¹⁷O are plotted as a function of the reciprocal of hydration (1/H), an abrupt change in slope is observed near 0.6 g of D₂O (or H₂ ¹⁷O)/gram of dry solids, the hydration at which conventional metabolism is activated in this system. The results have been discussed in terms of the two-site and multisite exchange models for the water-protein interaction as well as protein dynamics models. The ²H and ¹⁷O relaxation rates as a function of hydration show striking similarities to those observed for anisotropic motion of water molecules in protein crystals.

It is suggested here that although the simple two-site exchange model or n-site exchange model could be used to explain our data at high hydration levels, such models are not adequate at low hydration levels (<0.6 g H₂O/g) where several complex interactions between water and proteins play a predominant role in the relaxation of water nuclei. We further suggest that the abrupt change in the slope of I/T₁ as a function of hydration in the vicinity of 0.6 g H₂O/g is due to a change in water-protein interactions resulting from a variation in the dynamics of protein motion.

     

Base-pair exchange kinetics of the imino and amino protons of the 3′-phenazinium tethered DNA-RNA duplex,r(5′GAUUGAA3′):d(5′TCAATC3′-Pzn), and their comparison with those of B-DNA duplex

The dynamics of the opening-closing of the constituent base-pairs as well as of the exchange kinetics of the base-paired imino and amino protons with water in a DNA-RNA hybrid, [^5′r(G¹A²U³U⁴G⁵A⁶A⁷)^3′]:^5′p[d(T⁸C⁹A¹⁰A¹¹T¹²C¹³)]^3′-Pzn] duplex (I), are reported here in details for the first time. The exchange kinetics of amino and imino protons in the DNA-RNA hybrid (duplex I) have been compared with identical studies on the following B-DNA duplexes: d(C¹G²T³A⁴C⁵G⁶)₂ (II), d[p(^5′T¹G²T³T⁴T⁵G⁶ G⁷C⁸)^3′]:d[p(^5′C⁹C¹⁰A¹¹A¹²A¹³C¹⁴A¹⁵)^3′] (III), d(C⁵G⁶C⁷G⁸A⁹A¹⁰T¹¹T¹²C¹³G¹⁴C¹⁵G¹⁶)₂ (IV) and d(C¹G²C³G⁴C⁵G⁶C⁷G⁸A⁹A¹⁰T¹¹T¹²C¹³G¹⁴C¹⁵G¹⁶C¹⁷G¹⁸C¹⁹G²⁰)₂ (V). This comparative study shows that the life-times τ_o of various base-pairs in the DNA-RNA hybrid (I) varies in the range of ∼ 1 ms, and they are quite comparable to those of the shorter B-DNA duplexes (II) and (III), but very different from the τ_o of the larger duplexes (IV) and (V): the τ_o for the base pair of T¹¹ and T¹² residues in the 20-mer (duplex V) are 2.9 ± 2.3 ms and 23.2 ± 8.9 ms, respectively, while the corresponding τ_o in the 12-mer (duplex IV) are 2.8 ± 2.2 ms and 17.4 ± 5.4 ms. It has also been shown that the total energy of activation (E_a) assessed from the exchange rates of both imino and amino protons, representing energetic contributions from both base-pair and helix opening-closing as well as from the exchange process of the imino protons from the open state with the bound water, is close to the E_a of the short B-DNA duplex (E_a ≈ 28–47 kcal/mol).     

Actinopolyspora righensis sp. nov., a novel halophilic actinomycete isolated from Saharan soil in Algeria

A novel halophilic actinomycete strain, H23^T, was isolated from a Saharan soil sample collected in Djamâa (Oued Righ region), El-Oued province, South Algeria. Strain H23^T was identified as a member of the genus Actinopolyspora by a polyphasic approach. Phylogenetic analysis showed that strain H23^T had 16S rRNA gene sequence similarities ranging from 97.8 % (Actinopolyspora xinjiangensis TRM 40136^T) to 94.8 % (Actinopolyspora mortivallis DSM 44261^T). The strain grew optimally at pH 6.0–7.0, 28–32 °C and in the presence of 15–25 % (w/v) NaCl. The substrate mycelium was well developed and fragmented with age. The aerial mycelium produced long, straight or flexuous spore chains with non-motile, smooth-surfaced and rod-shaped spores. Strain H23^T had MK-10 (H₄) and MK-9 (H₄) as the predominant menaquinones. The whole micro-organism hydrolysates mainly consisted of meso-diaminopimelic acid, galactose and arabinose. The diagnostic phospholipid detected was phosphatidylcholine. The major cellular fatty acids were anteiso-C_17:0 (37.4 %), iso-C_17:0 (14.8 %), iso-C_15:0 (14.2 %), and iso-C_16:0 (13.9 %). The genotypic and phenotypic data show that the strain represents a novel species of the genus Actinopolyspora, for which the name Actinopolyspora righensis sp. nov. is proposed, with the type strain H23^T (=DSM 45501^T = CCUG 63368^T = MTCC 11562^T).     

A tetrameric allyl complex of sodium, and computational modeling of the Na-allyl chemical shift

Transmetallation of Li[A′] (A′ = [1,3-(SiMe₃)₂C₃H₃]⁻) with sodium tert-butoxide produces the corresponding sodium salt, which crystallizes from THF/toluene in the form of a cyclic tetramer, {Na[A′](thf)}₄. The Na atoms are in a square planar arrangement, bridged with π-bound allyl ligands; the Na-C distances range from 2.591(3)-2.896(3) Å, with an average of 2.70 Å. The geometries of several model organosodium complexes containing cyclopentadienyl and allyl ligands were optimized with density functional theory methods. The optimized structures were used with the gauge-including atomic orbital (GIAO) method to calculate their ²³Na NMR magnetic shielding values. Unlike the case with NaCp, the chemical shift of unsubstituted Na(C₃H₅) is very sensitive to the presence of coordinated THF (causing a 20 ppm upfield shift); silyl substitution has an even larger effect (30 ppm upfield shift). The observed ²³Na shift of δ −3.3 ppm for Na[A′] in THF-d₈, however, cannot be reliably distinguished from that calculated for the [Na(thf)₄]⁺ cation alone.     

NMR paramagnetic relaxation enhancements due to manganese in the S0 and S2 states of Photosystem II-enriched membrane fragments and in the detergent-solubilized Photosystem II complex

The NMR paramagnetic relaxation enhancement (NMR-PRE) produced in the solvent proton resonance by manganese in the S₀ and S₂ states of the oxygen evolving center (OEC) has been recorded for three Photosystem II (PS II)-enriched preparations: (1) PS II-enriched thylakoid membrane fragments (TMF-2 particles); (2) salt-washed (2M NaCl) TMF-2 particles; and (3) the octylglucopyranoside (OGP)-solubilized PS II complex. The second and third preparations, but not the first, are depleted of the peripheral 17 and 23 kD polypeptides associated with the OEC. It has been proposed that depletion of these polypeptides increases the exposure of OEC manganese to the aqueous phase. The NMR-PRE response measures the quantity (T_1m+_m)^-1, where T_1m is the spin relaxation time and _m is the mean residence time with respect to chemical exchange reactions of solvent protons in the manganese coordination sphere, and, thus, the NMR-PRE provides a direct measure of the solvent proton chemical exchange rate constant _m ^-1. This study tested whether the 17 and 23 kD polypeptides shield the OEC from the solvent phase and whether their depletion enhances the S₂ and S₀ NMR-PRE signals by removing a kinetic barrier to the solvent proton chemical exchange reaction. The amplitude of the S₂ NMR-PRE signal, measured in its chemical exchange-limited regime (_m>T_1m), is slightly decreased, rather than increased, in preparations (2) and (3) relative to (1), indicating that removal of the 17 and 23 kD polypeptides slightly slows, rather than accelerates, the rate-limiting steps of the solvent proton chemical exchange reactions. In addition, the lifetime of the S₂ state was shortened several-fold in the solubilized PS II complex and in salt-washed TMF-2 membranes relative to untreated TMF-2 control samples. The S₀ NMR-PRE signal, which is present in TMF-2 suspensions, was not detected in suspensions of the solubilized PS II complex, even though these samples contained high concentrations of active manganese centers (approximately double those of the TMF-2 control) and exhibited an S₂ NMR-PRE signal of comparable amplitude to that of the TMF-2 preparation. These results suggest that the 17 and 23 kD extrinsic polypeptides do not shield the NMR-visible water binding site in the OEC from the aqueous phase, although their removal substantially alters the proton relaxation efficiency by shortening T_1m.Abbreviations ADRY acceleration of the deactivation reactions of the water splitting enzyme Y - BBY Photosystem II-enriched membrane fragments prepared by the method of Berthold et al. (1981) - CCCP carbonyl cyanide m-chlorophenyl hydrazone - Chl chlorophyll - DCBQ 2,5-dichlorobenzoquinone - MES morpholinoethanesulfonate - NMR nuclear magnetic resonance - OEC oxygen evolving complex - OGP octylglucopyranoside - PRE paramagnetic relaxation enhancement - PS II Photosystem II - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TMF-2 Photosystem II-enriched thylakoid membrane fragments prepared by the method of Radmer et al. (1986) - T₁, T₂ longitudinal and transverse nuclear spin relaxation times     

Sodium-23 NMR studies of cation-DNA interactions

Sodium-23 NMR has been used to study the extent to which monovalent cations associate with double stranded DNA in aqueous solution (28°C, pH = 7.5). On the basis of the two site model for rapid exchange the ²³Na linewidth can be related to the fraction of sodium ions associated with DNA. To test the applicability to this system of the condensation model for the association of small counterions with polyelectrolytes, the concentration dependence of the sodium linewidth has been determined by making additions of NaCl to solutions of tetraethyl or tetrabutylammonium DNA. ([P], the DNA phosphate concentration was about 0.02M). The resulting titration curves extend over a wide range of the ratio [Na]/[P] (0.3–30). When [Na]/[P] ? 3 only sodium is associated, and the extent to which it compensates the charges on DNA does not vary with the addition of salt, at least until [Na]/[P] ≈ 30, the highest concentration examined. When [Na]/[P] ? 3 the tetraalkylammonium species is also associated with DNA; an equation has been derived to account for the effect on the ²³Na linewidth of the competition between sodium and another monovalent cation. Based on the assumption that the fraction of uncompensated charge remaining on DNA after the condensation of both species is constant, this equation fits all the linewidth data if the charge fraction is in the range 0.25 ± 0.10. The value required by the condensation model for DNA in the presence of monovalent counterions is ξ^?1 = 0.24. The reasonable agreement between experimental and theoretical values of the charge fraction and its invariance with respect to large variations in the concentration of added salt indicate that even in moderately concentrated solutions of DNA, the association of sodium can usefully be described in terms of the condensation model. If the theoretical value of the charge fraction is assumed, it follows from fitting the titration curves that the approximate relative affinities for DNA of Na⁺, Et₄N⁺, and Bu₄N⁺ are in the ratio 20:5:1, and the transverse relaxation rate of condensed sodium is 180 ± 10 s^?1.     

Propagation Speed in Myelinated Nerve: I. Experimental Dependence on External Na+ and on Temperature

Conduction speed (θ) in single myelinated Rana pipiens sciatic nerve fibers has been precisely measured using intracellular recording and on-line digital computer techniques. The dependence of relative speed on external Na concentration at 15°C has been found to be ln(θ₁/θ₂) = 0.524 (±0.018) ln ([Na⁺]₁/[Na⁺]₂) + 0.003. Thus θ has very close to a square root dependence on [Na⁺]₀ for these fibers. This experimental finding is not in complete agreement with a theoretical prediction based on a solution of the Hodgkin-Huxley (H.H.) equations. The effect of small temperature variations around 15°C on θ has also been measured for Rana fibers in Ringer's solution. θ has close to an exponential dependence on T and a Q₁₀ of 2.95 has been estimated.     

The E2P-like state induced by magnesium fluoride complexes in the Na,K-ATPase. Kinetics of formation and interaction with Rb+

The first X-ray crystal structures of the Na,K-ATPase were obtained in the presence of magnesium and fluoride as E2(K₂)Mg–MgF₄, an E2∙Pi-like state capable to occlude K⁺ (or Rb⁺). This work presents a functional characterization of the crystallized form of the enzyme and proposes a model to explain the interaction between magnesium, fluoride and Rb⁺ with the Na,K-ATPase. We studied the effect of magnesium and magnesium fluoride complexes on the E1–E2 conformational transition and the kinetics of Rb⁺ exchange between the medium and the E2(Rb₂)Mg–MgF₄ state. Our results show that both in the absence and in the presence of Rb⁺, simultaneous addition of magnesium and fluoride stabilizes the Na,K-ATPase in an E2 conformation, presumably the E2Mg–MgF₄ complex, that is unable to shift to E1 upon addition of Na⁺. The time course of conformational change suggests the action of fluoride and magnesium at different steps of the E2Mg–MgF₄ formation. Increasing concentrations of fluoride revert along a sigmoid curve the drop in the level of occluded Rb⁺ caused by Mg^2 +. Na⁺-induced release of Rb⁺ from E2(Rb₂)Mg–MgF₄ occurs at the same rate as from E2(Rb₂) but is insensitive to ADP. The rate of Rb⁺ occlusion into the E2Mg–MgF₄ state is 5–8 times lower than that described for the E2Mg–vanadate complex. Since the E2Mg–MgF₄ and E2Mg–vanadate complexes represent different intermediates in the E2-P → E2 dephosphorylation sequence, the variation in occlusion rate could provide a tool to discriminate between these intermediates.     

Direct inhibitory action of EGTA-Ca complex on reverse-mode Na/Ca exchange inMyxicola giant axons

Summary Giant axons from the marine annelidMyxicola infundibulum were internally dialyzed with solutions containing²²Na ions as tracers of Na efflux. In experiments performed in Li-substituted seawater, Na efflux that is dependent on external Ca ion concentration, [Ca²⁺] _o , was measured using dialysis to maintain [Na⁺] _i at 100mm, which enhances the [Ca²⁺] _o -dependent Na efflux component, (i.e., reverse-mode Na/Ca exchange). When dialysis fluid contained EGTA (1mm) to buffer the internal Ca concentration, [Ca²⁺] _i , to desired levels, Na efflux lost its normal sensitivity to external calcium. The inhibition was not simply due to the Ca-chelating action of EGTA to produce insufficient [Ca²⁺] _i to activate Na/Ca exchange. The addition of EGTA inhibited Ca _o -dependent Na efflux even when a large enough excess of [Ca²⁺] _i was present to saturate the EGTA and still produce elevated values of [Ca²⁺] _i . Control experiments showed that these high values of [Ca²⁺] _i resulted in normal Na/Ca exchange in the absence of EGTA. It is concluded that the presence of EGTA itself interferes with the manifestation of reverse-mode Na/Ca exchange inMyxicola giant axons.     

Use of Na-Nuclear Magnetic Resonance To Follow Sodium Uptake and Efflux in NaCl-Adapted and Nonadapted Millet (Panicum miliaceum) Suspensions

Cellular Na⁺ transport was followed in vivo by ²³Na nuclear magnetic resonance (NMR) using anionic dysprosium-based shift reagents to resolve internal and external ²³Na⁺ resonances. Proso millet (Panicum miliaceum) cell suspensions adapted for rapid growth on 130 mm NaCl had biphasic ²³Na efflux kinetics when shifted to low Na⁺ medium, while nonadapted cells had little measurable Na⁺ efflux after preloading with ²³NaCl. Uptake of ²³Na was also observed using ²³Na NMR. The resonance frequency of the external Na⁺-dysprosium (III) triphosphate, relative to that of the ²³Na in the cells, was sensitive to pH, permitting the pH of the external medium to be followed during the course of in vivo experiments.     

Experimental studies of thermal denaturation of the Na-DNA system with respect to Manning's model

The sodium and chloride activity coefficients in DNA solution were measured by selective electrodes. These experiments were performed for native and thermally denatured DNA. The ratio of activities in helix and coil states were compared with those given by Manning's model. These results are in good agreement with the theoretical values.We also compare the experimental values of the charge parameter XXX of DNA in the helix (XXX_h) and coil (XXX_c) configurations with the theoretical parameters appearing in Manning's model and which have been adjusted to correspond with the known conformation of the molecule. From this comparison, we deduce the change of enthalpy (ΔH)T_m at the temperature of denaturation (T_m) of DNA.The value of (ΔH)T_m thus calculated is smaller than the theorstical value and comparable with that observed experimentally by Privalov et al.