Nuclear magnetic resonance of tissue 23Na correlation time

Shporer and Civan (Biochim. Biophys. Acta (1974) 354, 291–304) reported the effect of magnetic-field strength on the NMR relaxation times of ²³Na in frog skeletal muscle. From these data, they estimated the correlation time τ_c for bound ²³Na whose tumbling is severely restricted, and they suggested that the fraction of bound ²³Na does not exceed some few percent of the total ²³Na population. However, a step in their theoretical approach seems oversimplified. With an improved approach, we obtained an effective τ_c of 4–9 ns for bound ²³Na. This value is some 10 times shorter than the corresponding value estimated by them from the same data. On the other hand, their conclusion concerning the amount of bound ²³Na seems to remain valid. The origin of the observed difference between the two transverse relaxation times of tissue ²³Na is also discussed.     

Direct measurement of increased myocardial cellular23Na NMR signals in perfused guinea-pig heart induced by dihydroouabain and grayanotoxin-I

The effects of the cardiac glycoside dihydroouabain (DHO), and the ericaceous toxin grayanotoxin-I (GTX-I) on myocardial cellular sodium (Na_i) concentrations were investigated using sodium-23 nuclear magnetic resonance (²³Na NMR) spectroscopy at 30°C in isolated perfused guinea-pig hearts. The Na_i NMR signals from perfused Langendorff heart preparations were obtained by the modified inversion recovery (IR) method based on the previous observation that the spin-lattice relaxation time (T₁) of the Na_i (25 or 34 msec at 8.46 Tesla (T)) is much faster than that of extracellular sodium (64 msec at 9.4 T). Na_i was estimated from the calibration curve of the frequency area of the²³Na NMR FT spectra plotted against the standard Na concentration. The Na_i concentration of the heart increased concomitantly with the positive inotropic effects (PIE) of DHO, GTX-I and monensin (MON). The cumulative sequential addition of DHO (5×10^–6 M), GTX-I (7×10^–8 M) and MON (5×10^–6 M), each of which alone induced no appreciable PIE, produced a 22% elevation in Na_i concentration relative to that of the control (100%) accompanying a PIE of 44%. The mechanism of this Na_i elevation induced by combinational addition of DHO, GTX-I and MON may be mediated as follows: GTX-I increases the net Na-influxvia Na⁺ channels; DHO inhibits the pumping out of Na⁺ from the cell; and MON transports external Na⁺ into the cell, acting as a sodium ionophore. Consequently, these drugs act synergistically to increase the Na_i, thereby increasing the intracellular Ca²⁺ concentrationvia Na⁺–Ca²⁺ exchange.     

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.     

Compartment analysis of plant cells by means of turgor pressure relaxation: II. Experimental results onChara corallina

Summary The three-compartment model (paper I) described turgor pressure relaxations as a sum of two exponential functions. The predicted shape of the curves could be confirmed inChara corallina by improving the recording and processing of data. An evaluation on the basis of the three-compartment model provided values for the hydraulic conductivity of the plasmalemma ofLp _p=2×10^–5 to 4×10^–5 cm sec^–1 bar^–1 and ofLp _i=3×10^–5 to 1×10^–4 cm sec^–1 bar^–1 for the tonoplast (assuming the area to be 90% of the plasmalemma area). The mean proportion of the total volume occupied by the cytoplasm was calculated to be 9%. This value is consistent with the concept of a highly vacuolated cell. Other explanations for the biphasic relaxation curves are discussed.     

Kinetics of electron transfer between soluble cytochrome c-554 and purified reaction center complex from the green sulfur bacterium Chlorobium tepidum

Soluble cytochrome c-554 (M _r 10 kDa) is purified from the green sulfur bacterium Chlorobium tepidum. Its midpoint redox potential is determined to be +148 mV from redox titration at pH 7.0. The kinetics of cytochrome c-554 oxidation by a purified reaction center complex from the same organism were studied by flash absorption spectroscopy at room temperature, and the results indicate that the reaction partner of cytochrome c-554 is cytochrome c-551 bound to the reaction center rather than the primary donor P840. The second-order rate constant for the electron donation from cytochrome c-554 to cytochrome c-551 was estimated to be 1.7×10⁷ M^–1 s^–1. The reaction rate was not significantly influenced by the ionic strength of the reaction medium.This revised version was published online in October 2005 with corrections to the Cover Date.     

Some characteristics of Na/K-ATPase from rat intestinal basal lateral membranes

Summary Basal lateral membrane vesicles were isolated from rat intestinal epithelial cells. The sodium potassium triphosphatase (Na/K-ATPase) of these plasma membranes has been characterized by (1) the molecular weight of the phosphorylated intermediate, (2) the sensitivity of the phosphorylated intermediate to hydroxylamine, (3) its ouabain binding constants, and (4) its susceptibility to digestion by pronase. The phosphorylated intermediate was shown by SDS polyacrylamide gel electrophoresis to be a protein of 100,000 Daltons apparent mol wt. Its extensive hydrolysis in hydroxylamine demonstrated that it was an acyl phosphate. The isolated basal lateral membranes bound ouabain with a dissociation constant,K _m (1.5×10^–5 m), similar to the inhibitory constantK _I (3×10^–5 m), measured for ouabain inhibition of the Na/K-ATPase activity. The association rate constant measured for ouabain binding at 22°C was 1.3×10³ m ^–1 sec^–1 and is similar to the association rate constants reported for other tissues and species. The high dissociation rate constant, 3.6×10^–2 sec^–1, is consistent with the insensitivity of the rat to ouabain. Digestion of the intact cells by pronase yielded basal lateral membranes in which the Na/K-ATPase had been unaffected. The phosphorylated intermediate ran as a sharp band at 100,000 Daltons on electrophoresis, and the ouabain dissociation constant appeared to be unchanged. In these membranes, protein stains of polyacrylamide gels revealed digestion of the major high mol wt proteins including the major protein at 100,000 Daltons. This suggests that the Na/K-ATPase represent a minor component, less than 1%, of the basal lateral membrane protein. From these characteristics of the phosphorylated intermediate and the ouabain binding constants, we conclude that the Na/K-ATPase of the basal lateral membranes of rat intestinal epithelial cells is similar to that found in other tissues and species. Estimates of the number of pump sites and the turnover number predict rates of Na transport that are consistent with observed values.This paper is dedicated to the memory of Professor David H. Smyth, FRS, who died on September 10, 1979.     

Kinetic analysis of carrier-mediated ion transport by the charge-pulse technique

Summary The charge-pulse technique has been used previously for the study of quasistationary processes in membranes which required only a moderate time resolution. It is shown here that a time resolution of about 400 nsec may be achieved with this technique and that it may be applied to the kinetic analysis of carrier-mediated ion transport. By this method we have studied the transport of alkali ions through optically black monoolein membranes in the presence of the ion carrier valinomycin. All three relaxation processes that are predicted by theory have been resolved. From the relaxation times and the relaxation amplitudes the rate constants for the association (k _R ) and the dissociation (k _D ) of the ioncarrier complex, as well as the translocation rate constants of the complex (k _MS ) and the free carrier (k _S ) could be obtained. For 1m Rb⁺ at 25° C the values arek _R =3×10⁵ m ^–1 sec^–1,k _D =2×10⁵ sec^–1,k _MS =3×10⁵ sec^–1,k _S =4×10⁴ sec^–1. The activation energies of the single rate constants which have been estimated from experiments at two different temperatures range between 50 and 90 kJ/mol.     

An adaptive on-line control feed rate system in a laboratory scale bakers yeast process

Summary A new control policy for the on-line optimization of the nutrient supply in bakers yeast process is proposed. A feed rate corresponding to minimal substrate uptake time was shown to be optimal for cell yield and specific growth rate. Cultivation results of baker's yeast are presented.Nomenclature c glucose concentration in wort (mol.l^–1) - C total glucose used (mol) - c_e ethanol concentration in wort (mg.l^–1) - c_p glucose concentration in fresh medium (mol.l^–1) - dt/dc glucose consumption time (sec.mol^–1) - F substrate feed rate (litre.hr^–1) - q_c glucose uptake rate (mol.hr^–1) - Q_c specific glucose uptake rate (moll.g^–1.hr^–1) - qO₂ oxygen uptake rate (mol.hr^–1) - QO₂ specific oxygen uptake rate (mol.g^–1.hr^–1) - r_x productivity (g.l^–1.hr^–1) - t time (hr) - x biomass concentration (g.l^–1) - X total biomass (g) - Y_x/c cell yield (g.g^–1): (g.mol^–1) - Y_o/c consumed oxygen to glucose ratio (mol.mol^–1)     

Cyclic AMP-dependent regulation of activities of synthetase and phosphodiesterase of 2′,5′-oligoadenylate in NIH 3T3 cells

Summary Dihydrofolate synthetase (EC 6.3.2.12) from N. gonorrhoeae was isolated and enzyme characteristics were determined. The purified enzyme was found quite stable when stored at –60 °C. About 50% of the enzyme activity wag destroyed within 6 weeks when kept at 4 °C. Maximum velocity was observed at pH 9.3. The enzyme required a monovalent cation, K⁺ or NH₄ ⁺ , and divalent cation, Mg²⁺ or Mn²⁺ for its function. ATP at 5 mM concentration gave maximum activity. K_m values for dihydropteroate and L-glutamate at pH 9.3 were 3.5 × 10^–5 M and 6.5 × 10^–4 M, respectively. Patterns of product inhibition by dihydrofolate were found to be non-competitive with respect to dihydropteroate, having a K_i value of 5.1 ± 0.8 × 10^–4 M, and competitive with respect to L-glutamate, having a K_i value of 6.2 × 10^–4 M.     

Medium optimization for a methanol utilizing bacterium based on chemostat theory

Summary In the productions of biomass and vitamin B₁₂ using methanol as the sole carbon source, it is necessary to use a medium in which methanol is the growth limiting substrate. Other inorganic salts should be in slight excess so that the yield of cells and the intracellular content of vitamin B₁₂ do not vary. From basic principles of chemostat culture, a medium was optimized for Pseudomonas AM-1 a methanol utilizing bacterium, for the concentrations of various inorganic salts. This was done in a series of chemostat cultures at a dilution rate of 0.1 h^–1. Optimum amounts of NH₄ ⁺, PO₄ ^3- and Mg²⁺ were estimated from the minimum concentration of the salt at which methanol became growth limiting. The optimum concentrations of Ca²⁺, Fe²⁺, Mn²⁺, and Zn²⁺ as a group were determined in the same way. Cu²⁺, Mo⁶⁺, Co²⁺ and B³⁺ are required at concentrations of g/l and they were not studied as these very low level can be introduced as contaminants from other salts. The optimum medium composition (in g/l) was as follows: (NH₄)₂SO₄, 1.0; H₃PO₄, 75×10^–3; MgSO₄ · 7H₂O, 30×10^–3; CaCl₂ · 2H₂O, 3.3×10^–3; FeSO₄ · 7H₂O, 1.3×10^–3, MnSO₄ · 4H₂O, 0.13×10^–3; ZnSO₄ · 7H₂O, 0.13×10^–3; CuSO₄ · 5H₂O, 40×10^–6; Na₂MoO₄, 40×10^–6; CoCl₂ · 6H₂O, 40×10^–6; H₃BO₃, 30×10^–6 and methanol 4.     

Sulfate reduction in a cell-free system ofChlorella

Summary During sulfate reduction in a cell-free system ofChlorella activated sulfate of APS is transferred to a thiosulfonate reductase. The sulfate thus bound to the thiosulfonate reductase (i.e. bound sulfite) is reduced to bound sulfide in a ferredoxin dependent reaction. This bound sulfide can be used with O-acetylserine as acceptor for cysteine biosynthesis; serine and O-phosphoserine are not used. An assaysystem for thiosulfonate reductase activity using methylviologen dependent reduction of S₂O₄ ^2– to S^2– is developed and a procedure for isolating thiosulfonate reductase fromChlorella cells is presented. During isolation of thiosulfonate reductase a low weight molecular factor, needed for optimal enzyme activity was lost. The bound sulfite seems to be attached to this factor. Reduction of APS or GS-SO₃H to the level of S^2– is inhibited by cysteine. 50% inhibition of GS-SO₃H reduction was found at a molar cysteine concentration of 6.8×10^–5.Abbreviations APS adenosine-phosphosulfate - PAPS 3-phosphoadenosine-5-phosphosulfate - GSH reduced glutathion - GS-SO₃H S-sulfoglutathion - fd ferredoxin - Mv methylviologen - DTT dithiothreitol     

Evidence of α fluctuations in myoglobin's denaturation in the high temperature region: Average relaxation time from an Adam–Gibbs perspective

In this work, we derive an analytical expression for the relaxation time τ as a function of temperature T for myoglobin protein (Mb, PDB:1MBN) in the high temperature limit (T > T_g = 200 K). The method is based on a modified version of the Adam–Gibbs theory (AG theory) for the glass transition in supercooled liquids and an implementation of differential geometry techniques. This modified version of the AG theory takes into account that the entropic component in protein's denaturation has two major sources: a configurational contribution ΔS_c due to the unfolding of the highly ordered native state N and a hydration contribution ΔS^hyd arising from the exposure of non-polar residues to direct contact with solvent polar molecules. Our results show that the configurational contribution ΔS_c is temperature-independent and one order of magnitude smaller than its hydration counterpart ΔS^hyd in the temperature range considered. The profile obtained for log τ(T) from T = 200 K to T = 300 K exhibits a non-Arrhenius behavior characteristic of α relaxation mechanisms in hydrated proteins and glassy systems. This result is in agreement with recent dielectric spectroscopy data obtained for hydrated myoglobin, where at least two fast relaxation processes in the high temperature limit have been observed. The connection between the relaxation process calculated here and the experimental results is outlined.     

Substitution of D-Trp32 in NPY Destabilizes the Binding Transition State to the Y1 Receptor Site in SK-N-MC Cell Membranes

The retention rate of the spin label 3-isothiocyanto methyl-2,2,5,5-tetramethyl-1-pyrrolidinyl oxyl spin label (proxyl) attached to the porcine N-acetyl-NPY peptide and the porcine N-acetyl-D-Trp³²-NPY peptide at Lys⁴ was investigated using SK-N-MC neuroblastoma cell membranes containing the Y1 receptor. The release rate of the spin labeled peptides was monitored by electron spin resonance and the K_D was determined by a direct radiolabeled NPY displacement binding assay. The analyses show that for the porcine [Ac-Tyr¹N⁴-proxyl]-NPY, the K_D was 8 × 10^–10 M and k_off was 2.7 × 10^–4 sec^–1 yielding a value for k_on of 3.3 × 10⁵ sec^–1 M^–1. The [Ac-Tyr¹, N⁴-proxyl,-D-Trp³²]-NPY antagonist ligand had a value of K_D equal to 1.35 × 10^–7 M and k_off was 1.7 × 10^–4 sec^–1 leading to a value for k_on of 1.2 × 10³ sec^–1 M^–1. The difference in the k_on rates of two orders of magnitude is interpreted as demonstrating the N-acetyl-N⁴ proxyl-D-Trp³²-NPY ligand binding transition state to be of higher energy then for the unmodified NPY amino acid sequence.     

Heterotrophic glucose uptake and respiration in Lake Kinneret

The turnover times of glucose, averaged for 0–10 m in the upper waters of Lake Kinneret and measured by the addition of single or multiple concentrations of substrate, ranged from 23 to 188 hours and 1 to 87 hours respectively. Potential uptake rates (estimated as V_max) ranged from 0.095 to 1.94 µg glucose l^–1h^–1, while measured uptake rates varied from 0.09 to 1.1 µg glucose l^–1h^–1. Concentrations of dissolved carbohydrates and glucose averaged 0.71 mg glucose equivalents l^–1 and 39 µg glucose l^–1 respectively. No evident relationships between glucose cycling and any fractions of dissolved organic matter, phytoplankton biomass or primary productivity were found. Turnover times were generally most rapid immediately after the decline of the spring Peridinium bloom. The respiration percentage of incorporated glucose ranged from 25% to 61% with highest values during the summer months. Respiration may be influenced by the nature of the indigenous bacterial population as well as by temperature. Daily heterotrophic glucose carbon uptake was about 9% of the photosynthetic incorporation and could provide a bacterial yield of about 7 × 10⁴ ml^–1d^–1.     

Cell sodium activity and sodium pump function in frog skin

Summary Cell Na activity,a _Na ^c , was measured in the short-circuited frog skin by simulaneous cell punctures from the apical surface with open-tip and Na-selective microelectrodes. Skins were bathed on the serosal surface with NaCl Ringer and, to reduce paracellular conductance, with NaNO₃ Ringer on the apical surface. Under control conditionsa _Na ^c averaged 8±2mm (n=9,sd). Apical addition of amiloride (20 m) or Na replacement reduceda _Na ^c to 3mm in 6–15 min. Sequential decreases in apical [Na] induced parallel reductions ina _Na ^c and cell current,I _c . On restoring Na after several minutes of exposure to apical Na-free solutionI _c rose rapidly to a stable value whilea _Na ^c increased exponentially, with a time constant of 1.8±0.7 min (n=8). Analysis of the time course ofa _Na ^c indicates that the pump Na flux is linearly related toa _Na ^c in the range 2–12mm. These results indicate thata _Na ^c plays an important role in relating apical Na entry to basolateral active Na flux.     

Sulfate reduction and S-oxidation in a moorland pool sediment

In an oligotrophic moorland pool in The Netherlands, S cycling near the sediment/water boundary was investigated by measuring (1) SO₄ ^2– reduction rates in the sediment, (2) depletion of SO₄ ^2– in the overlying water column and (3) release of³⁵S from the sediment into the water column. Two locations differing in sediment type (highly organic and sandy) were compared, with respect to reduction rates and depletion of SO₄ ^2– in the overlying water.Sulfate reduction rates in sediments of an oligotrophic moorland pool were estimated by diagenetic modelling and whole core³⁵SO₄ ^2– injection. Rates of SO₄ ^2– consumption in the overlying water were estimated by changes in SO₄ ^2– concentration over time in in situ enclosures. Reduction rates ranged from 0.27–11.2 mmol m^–2 d^–1. Rates of SO₄ ^2– uptake from the enclosed water column varied from –0.5, –0.3 mmol m^–2 d^–1 (November) to 0.43–1.81 mmol m^–2 d^–1 (July, August and April). Maximum rates of oxidation to SO₄ ^2– in July 1990 estimated by combination of SO₄ ^2– reduction rates and rates of in situ SO₄ ^2– uptake in the enclosed water column were 10.3 and 10.5 mmol m^–2 d^–1 at an organic rich and at a sandy site respectively.Experiments with³⁵S^2– and³⁵SO₄ ^2– tracer suggested (1) a rapid formation of organically bound S from dissimilatory reduced SO₄ ^2– and (2) the presence of mainly non SO₄ ^2–-S derived from reduced S transported from the sediment into the overlying water. A³⁵S^2– tracer experiment showed that about 7% of³⁵S^2– injected at 1 cm depth in a sediment core was recovered in the overlying water column.Sulfate reduction rates in sediments with higher volumetric mass fraction of organic matter did not significantly differ from those in sediments with a lower mass fraction of organic matter.Corresponding author     

Discriminating Hepatocellular Carcinoma in Rats Using a High-Tc SQUID Detected Nuclear Resonance Spectrometer in a Magnetic Shielding Box

In this study, we report the spin-lattice relaxation rate of hepatocellular carcinoma (HCC) and normal liver tissue in rats using a high-T_c superconducting quantum interference device (SQUID) based nuclear magnetic resonance (NMR) spectrometer. The resonance spectrometer used for discriminating liver tumors in rats via the difference in longitudinal relaxation time in low magnetic fields was set up in a compact and portable magnetic shielding box. The frequency-domain NMR signals of HCC tissues and normal liver tissues were analyzed to study their respective longitudinal relaxation rate T₁ ⁻¹. The T₁ ⁻¹ of liver tissues for ten normal rats and ten cancerous rats were investigated respectively. The averaged T₁ ⁻¹ value of normal liver tissue was (6.41±0.66) s⁻¹, and the averaged T₁ ⁻¹ value of cancerous tissue was (3.38±0.15) s⁻¹. The ratio of T₁ ⁻¹ for normal liver tissues and cancerous liver tissues of the rats investigated is estimated to be 1.9. Since this significant statistical difference, the T₁ ⁻¹ value can be used to distinguish the HCC tissues from normal liver tissues. This method of examining liver and tumor tissues has the advantages of being convenient, easy to operate, and stable.     

Structural and developmental differences between three types of Na channels in dorsal root ganglion cells of newborn rats

Summary The changes in Na current during development were studied in the dorsal root ganglion (DRG) cells using the whole-cell patch-clamp technique. Cells obtained from rats 1–3 and 5–8 days after birth were cultured and their Na currents were compared. On top of the two types of Na currents reported in these cells (fast-FA current and slow-S current) a new fast current was found (FN). The main characteristics of the three currents are: (i) The voltages of activation are –37, –36, and –23 mV for the FN, FA and S currents, respectively. (ii) The activation and inactivation kinetics of FN and FA currents are about five times faster than those of the S current. (iii) The voltages at which inactivation reaches 50% are –139, –75 and –23 mV for the FN, FA and S currents, respectively.The kinetics and voltage-dependent parameters of the three currents and their density do not change during the first eight days after birth. However, their relative frequency in the cells changes. In the 1–3 day-old rats the precent of cells with S, FA, and mixed S+FN currents is 22, 18, and 60% of the cells, respectively. In the 5–8 day-old, the percent of cells with S, FA, and FN+S is 10, 66 and 22%. The relative increase in the frequency of cells with FA current during development can contribute to the ease of action potential generation compared with cells with FN currents, which are almost completely inactivated under physiological conditions. The predominance of FA cells also results in a significant decrease in the relative frequency of cells with the high-threshold, slow current.Antibodies directed against a part of the S₄ region of internal repeat I of the sodium channel (C ₁ ⁺ , amino acids 210–223, eel channel numbering) were found to shift the voltage dependence of FA current inactivation (but not of FN or S currents) to more negative potentials. The effect was found only when the antibodies were applied externally. The results suggest that FN, FA and S types of Na currents are generated by channels, which are different in the topography of the C ₁ ⁺ region in the membrane.     

Hydration-State Change of Horse Heart Cytochrome c Corresponding to Trifluoroacetic-Acid-Induced Unfolding

We investigate the hydration state of horse-heart cytochrome c (hh cyt c) in the unfolding process induced by trifluoroacetic acid (TFA). The conformation of hh cyt c changes from the native (N) state (2.9 < pH < 6.0) to the acid-unfolded (U_A) state (1.7 < pH < 2.0) to the acid-induced molten globule (A) state (pH ∼1.2). Hydration properties of hh cyt c during this process are measured at 20°C by high-resolution dielectric relaxation (DR) spectroscopy, UV-vis absorbance, and circular dichroism spectroscopy. Constrained water of hh cyt c is observed at every pH as an ∼5-GHz Debye component (DC) (DR time, τ_D ∼30 ps) and its DR amplitude (DRA) is increased by 77% upon N-to-U_A transition, when pH changes from 6.0 to 2.0. Even in the N state, the DRA of the constrained-water component is found to be increased by 22% with decreasing pH from 6.0 to 2.9, suggesting an increase in the accessible surface area of native hh cyt c. Moreover, hypermobile water around native hh cyt c is detected at pH 6.0 as a 19-GHz DC (τ_D ∼ 8.4 ps < τ_DW = 9.4 ps), but is not found at other pH values. The DRA signal of constrained water is found to return to the pH 2.9 (N-state) level upon U_A-to-A transition. Fast-response water (slightly slower than bulk) around A-state hh cyt c is detected at pH 1.2, and this suggests some accumulation of TFA⁻ ions around the peptide chain. Thus, this high-resolution DR spectroscopy study reveals that hh cyt c exhibits significant hydration-state change in the TFA-unfolding process.     

The Effect of Ionic Strength and Specific Anions on Substrate Binding and Hydrolytic Activities of Na,K-ATPase

The physiological ligands for Na,K-ATPase (the Na,K-pump) are ions, and electrostatic forces, that could be revealed by their ionic strength dependence, are therefore expected to be important for their reaction with the enzyme. We found that the affinities for ADP³⁻, eosin²⁻, p-nitrophenylphosphate, and V_max for Na,K-ATPase and K⁺-activated p-nitrophenylphosphatase activity, were all decreased by increasing salt concentration and by specific anions. Equilibrium binding of ADP was measured at 0–0.5 M of NaCl, Na₂SO₄, and NaNO₃ and in 0.1 M Na-acetate, NaSCN, and NaClO₄. The apparent affinity for ADP decreased up to 30 times. At equal ionic strength, I, the ranking of the salt effect was NaCl ≈ Na₂SO₄ ≈ Na-acetate < NaNO₃ < NaSCN < NaClO₄. We treated the influence of NaCl and Na₂SO₄ on K _diss for E·ADP as a “pure” ionic strength effect. It is quantitatively simulated by a model where the binding site and ADP are point charges, and where their activity coefficients are related to I by the limiting law of Debye and Hückel. The estimated net charge at the binding site of the enzyme was about +1. Eosin binding followed the same model. The NO₃ ⁻ effect was compatible with competitive binding of NO₃ ⁻ and ADP in addition to the general I-effect. K _diss for E·NO₃ was ∼32 mM. Analysis of V_max/K _m for Na,K-ATPase and K⁺-p-nitrophenylphosphatase activity shows that electrostatic forces are important for the binding of p-nitrophenylphosphate but not for the catalytic effect of ATP on the low affinity site. The net charge at the p-nitrophenylphosphate-binding site was also about +1. The results reported here indicate that the reversible interactions between ions and Na,K-ATPase can be grouped according to either simple Debye-Hückel behavior or to specific anion or cation interactions with the enzyme.