Counterion-Type Characteristic Effects on Intrinsic Bending Components of Calf Thymus DNA; Hydrodynamic Investigations

Abstract

This paper stresses structural differences in A · T clusters of the ammonium salt of calf thymus (et) DNA (ctNH₄DNA) and the respective sodium salt, ctNaDNA Sequence mediated intrinsic helix bends of ctNaDNA distributed along the molecule partially randomly and partially phased with the helix screw (accompanying paper), are enhanced in ctNH₄DNA. Additionally, the number of the most strongly bent segments (of A-tract character) is raised in ctNH₄NA by a counterion mediated shift of the equilibrium between at least two local DNA conformations. Nevertheless, the apparent DNA elongation, induced by the abolition of a single apparent solenoid-related DNA tertiary structure component which generates a special intrinsic DNA bend, is the same for NH₄DNA and NaDNA.

These conclusions follow from two independent sets of experimental results:

(1.) Titration viscometric measurements with ctNH₄DNA as a function of the cation concentration in comparison to ctNaDNA (KER et al. JBSD 9, 537 (1991)) and respective DNA conformational analyses.

(2.) Quantitative viscometric analysis of DNA conformational changes on netropsin (Nt) interaction of ctNHjDNA at different temperatures and comparison with the respective data for ctNaDNA (KER et al., NAR 9, 2335 (1981).     

Multimode Interaction of Hoechst 33258 with Eukaryotic DNA; Quantitative Analysis of the DNA Conformational Changes

Abstract

The interaction of the minor groove binding ligand Hoechst 33258 (Hoe) with natural DNA was investigated by high resolution titration rotational viscometry. Analysis of the concomitant DNA conformational changes was performed with two DNA samples of sufficiently different molar mass M, at 4°C, 22°C and 40°C, for Hoe/DNA-P ratios below r = 0.02. In this narrow r range several interaction modes could be resolved. The measured conformational changes were quantified in terms of relative changes of both apparent DNA persistence length, Δa/a, and hydrodynamically operative DNA contour length, ΔL/L. Δa/a(r) primarily is a measure of ligand-induced DNA helix stiffening, but both, Δa/a(r) and ΔL/L(r), generally depend also on ligand binding induced DNA bending or DNA unbending. The essential difference obviously is that Δa/a(r) is influenced by the randomly distributed helix bends and ΔL/L(r) by phased ones. The measurements performed at different temperatures deliver informations about existence and temperature dependent abolition of intrinsic helix curvature.

Both Hoe and netropsin (Nt) prefer binding to AT rich DNA segments, which are candidates for intrinsic DNA helix bends. But our data for Hoe interaction with calf thymus DNA (ctDNA) show characteristic differences to those for Nt-ctDNA interaction. Especially for Hoe, the mode of highest affinity is saturated already at a ligand concentration of roughly 1 nM (r = 0.0015 Hoe/DNA-P). It exhibits an unusually strong temperature dependence of the conformational DNA response. A Hoe-Nt competition experiment shows that Hoe binding to the sites of the very first Hoe mode is almost unaffected by bound Nt. But Hoe binding to the sites of the following Hoe modes does not occur due to the competition with Nt. Thus this mode of strongest Hoe-DNA interaction reflects a unique mechanism, possibly of high relevance for gene regulatory systems.     

Kinetic study on the effects of intracellular K++ and Na++ on Na++, K++, Cl+− cotransport of HeLa cells by Rb++ influx determination

Summary The effects of intracellular K⁺ and Na⁺ (K⁺ c, Na⁺ c) on the Na⁺,K⁺,Cl^+– cotransport pathway of HeLa cells were studied by measuring ouabain-insensitive, furosemide-sensitive Rb⁺ influx (JRb) at various intracellular concentrations of K⁺ and Na⁺ ([K⁺]c, [Na⁺]c). When [K⁺]c was increased and [Na⁺]c was decreased, keeping the sums of their concentrations almost constant, JRb as a function of the extracellular Rb⁺ or Na⁺ concentration ([Rb⁺]e, [Na⁺]e) was stimulated. However, the apparent K _0.5 for Rb⁺ e or Na⁺ e remained unchanged and the ratio of the apparent K ^+0.5 for K⁺ c and the apparent K _i for Na⁺ c was larger than 1. When JRb was increased by hypertonicity by addition of 200 mM mannitol, the apparent maximum JRb increased without change in the apparent K _0.5 for Rb⁺ e. These results show that K⁺ c stimulates and Na⁺ c inhibits JRb, without change in the affinities of the pathway for Rb⁺ e and Na⁺ e. The affinity for K⁺ c is slightly lower than that for Na⁺ c. Hypertonicity enhances JRb without any change in the affinity for Rb⁺ e. We derived a kinetic equation for JRb with respect to K⁺ c and Na⁺ c and proposed a general and a special model of the pathway. The special model suggests that, in HeLa cells, JRb takes place when Rb⁺ e binds to the external K⁺ binding site of the pathway after the binding of K⁺ c to the internal regulatory site.We thank Mr. T. Masuya for technical assistance. This study was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (No. 03202136) from the Japanese Ministry of Education, Science and Culture.     

Transepithelial Na+ transport and the intracellular fluids: A computer study

Summary Computer simulations of tight epithelia under three experimental conditions have been carried out, using the rheogenic nonlinear model of Lew, Ferreira and Moura (Proc. Roy. Soc. London. B 206:53–83, 1979) based largely on the formulation of Koefoed-Johnsen and Ussing (Acta Physiol. Scand.42:298–308, 1958). First, analysis of the transition between the short-circuited and open-circuited states has indicated that (i) apical Cl^– permeability is a critical parameter requiring experimental definition in order to analyze cell volume regulation, and (ii) contrary to certain experimental reports, intracellular Na⁺ concentration (c _Na ^c ) is expected to be a strong function of transepithelial clamping voltage. Second, analysis of the effects of lowering serosal K⁺ concentration (c _K ^s ) indicates that the basic model cannot simulate several well-documented observations; these defects can be overcome, at least qualitatively, by modifying the model to take account of the negative feedback interaction likely to exist between the apical Na⁺ permeability andc _Na ^c . Third, analysis of the effects induced by lowering mucosal Na⁺ concentration (c _Na ^m ) strongly supports the concept that osmotically induced permeability changes in the apical intercellular junctions play a physiological role in conserving the body's stores of NaCl. The analyses also demonstrate that the importance of Na⁺ entry across the basolateral membrane is strongly dependent upon transepithelial potential,c _Na ^m andc _K ^s ; under certain conditions, net Na⁺ entry could be appreciably greater across the basolateral than across the apical membrane.     

On the possibility of true phase transition in heterogeneous DNA during thermal denaturation under conditions close to equal stability of A+T and G+C pairs

The DNA helix–coil transition has been studied in the presence of high concentrations of manganese ions (about 10^?3M), which corresponds to the conditions close to equal stability of the A+T and G+C pairs, at the ionic strengths of 10^?1, 10^?2, and 1.6 × 10^?3M Na⁺. With the Mn²⁺ ion effect, the transition range is significantly reduced to not more than 0.2°C at 1.2 × 10^?3M Mn²⁺ and 1.6 × 10^?3M Na⁺. The melting curves display a sharp kink at the end of the helix–coil transition, which is interpreted as an indication of the second-order phase transition. It is shown that the melting curves obtained can be approximated by a simple analytical expression 1 – θ = exp[–a(t_c - t)], where θ is the DNA helix fraction, t_c is the phase transition temperature, and a is an empirical parameter characterizing the breadth of the melting range and responsible for the magnitude of a jump of the helicity derivative with respect to the temperature at the phase transition point.     

Sodium and potassium fluxes and compartmentation in roots of atriplex and oat

K⁺ and Na⁺ fluxes and ion content have been studied in roots of Atriplex nummularia Lindl. and Avena sativa L. cv Goodfield grown in 3 millimolar K⁺ with or without 3 or 50 millimolar NaCl. Compartmental analysis was carried out with entire root systems under steady-state conditions.

Increasing ambient Na⁺ concentrations from 0 to 50 millimolar altered K⁺, in Atriplex, as follows: slightly decreased the cytoplasmic content (Q_c), the vacuolar content (Q_v), and the plasma membrane influx and efflux. Xylem transport for K⁺ decreased by 63% in Atriplex. For oat roots, similar increases in Na⁺ altered K⁺ parameters as follows: plasma membrane influx and efflux decreased by about 80%. Q_c decreased by 65%, and xylem transport decreased by 91%. No change, however, was observed in Q_v for K⁺. Increasing ambient Na⁺ resulted in higher (3 to 5-fold) Na⁺ fluxes across the plasma membrane and in Q_c of both species. In Atriplex, Na⁺ fluxes across the tonoplast and Q_v increased as external Na⁺ was increased. In oat, however, no significant change was observed in Na⁺ flux across the tonoplast or in Q_v as external Na⁺ was increased. In oat roots, Na⁺ reduced K⁺ uptake markedly; in Atriplex, this was not as pronounced. However, even at high Na⁺ levels, the influx transport system at the plasma membrane of both species preferred K⁺ over Na⁺.

Based upon the Ussing-Teorell equation, it was concluded that active inward transport of K⁺ occurred across the plasma membrane, and passive movement of K⁺ occurred across the tonoplast in both species. Na⁺, in oat roots, was actively pumped out of the cytoplasm to the exterior, whereas, in Atriplex, Na⁺ was passively distributed between the free space, cytoplasm, and vacuole.

     

Li+ counterion self-diffusion in ordered DNA

The anisotropic self-diffusion coefficient of ⁷Li⁺ (I = 3/2) counterions has been studied in hydrated, macroscopically oriented Li-(B)DNA fibers at relatively high water contents, corresponding to approximate DNA-DNA helix axis distances of 22–35 Å, using the pulsed field gradient hmr spin-echo method. Self-diffusion coefficients parallel (D_∥) and perpendicular (D_?) to the DNA helix axis increase with increasing salt content and with increasing DNA-DNA helix axis distance. The observed anisotropy D_∥/D_? decreases from 1.6 to 1.2 with the DNA-DNA separation increasing from 22 to 35 Å in the salt-free sample. This result can be understood by the obstruction effect caused by the DNA molecules themselves. The values of the Li⁺ self-diffusion coefficients in the most water-rich system with no added salt (corresponding to an approximate distance of 35 Å between the DNA helix axes) were D_∥ ～ 1.15 × 10^?10 m² s^?1 and D_? ～ 0.98 × 10^?10 m² s^?1, compared to 9.14 × 10^?10 m² s^?1 for the diffusion of Li⁺ in an aqueous solution of LiCl (～ 2.1M). The possible occurrence of restriction effects in the DNA fibers have also been studied by determining the self-diffusion coefficient at different effective diffusion times. The self-diffusion coefficient of Li⁺ in the sample with the largest DNA-DNA helix axis distance seems to be independent of the effective diffusion time, which indicates that the lithium ions are not trapped within impermeable barriers. The possibility of diffusion through permeable barriers has also been investigated, and is discussed. © 1994 John Wiley & Sons, Inc.     

Effect of sodium ion on the high-resolution melting of lambda DNA.

A series of high-resolution melting curves were obtained by the continuous direct-derivative method [Blake, R. D. & Lefoley, S. G. (1978) Biochim. Biophys. Acta 518 , 233–246] on lambda DNA (cI₈₅₇S₇ strain) under varying conditions of [Na⁺]. Examination of the denaturation patterns at close intervals of [Na⁺] indicates that frequent changes in mechanism occur below 0.04M Na⁺, while almost none occurs above 0.1M Na⁺. Changes at low [Na⁺] generally occur in an abrupt fashion, in most cases within a 3 mM change in [Na⁺], and in at least one case within 0.6 mM, indicating the balance between alternative mechanisms is frequently quite delicate. These changes involve segments of between 900 and 1500 or more base pairs in length and are therefore not insignificant. Changes at low [Na⁺] reflect a perturbation of the energetic balance between competing mechanisms by weakly screened long-range electrostatic forces. Some perturbation probably also arises from variations in the linear charge density of the double helix induced by the proximity of premelted loop segments; however, this contribution cannot be evaluated without a detailed denaturation map. At high [Na⁺] the mechanism of melting is more conserved, permitting the dependence of subtrasitional melting temperature t_m⁽ⁱ⁾ on [Na⁺] to be examined for almost all 34 ± 2 subtransitions. The G + C composition of segments responsible for each subtransition was determined by a quantitative spectral method. Analysis according to the Manning-Record expression [Manning, G. (1972) Biopolymers 11 , 937–949; Record, M. T., Jr., Anderson, C. F. & Lohman, T. M. (1978) Q. Rev. Biophysics 11 , 103–178] relating ΔH_m and dt_m⁽ⁱ⁾/d log[Na⁺] to the fraction of Na⁺ released during melting, appears to indicate almost 40% more Na⁺ is bound to the single-stranded G and/or C residues than to A and T residues. This is consistent with a much shorter mean axial spacing and higher charge density in the former, particularly single-stranded G residues, which have an extraordinary tendency to stack.     

Na2CO3 Influence on DNA Double Helix Stability: Strong Anion Destabilizing Effect

Abstract

Addition of Na₂CO₃ to almost salt-free DNA solution (5·10^?5M EDTA, pH=5.7, Tm=26.5 °C) elevates both pH and the DNA melting temperature (Tm) if Na₂CO₃ concentration is less than 0.004M. For 0.004M Na₂CO₃, Tm=58 °C is maximal and pH=10.56. Further increase in concentration gives rise to a monotonous decrease in Tm to 37 °C for 1M N₂CO₃ (pH=10.57). Increase in pH is also not monotonous. The highest pH=10.87 is reached at 0.04M Na₂CO₃ (Tm=48.3 °C). To reveal the cause of this DNA destabilization, which happens in a narrow pH interval (10.56÷10.87) and a wide Na₂CO₃ concentration interval (0.004÷1M), a procedure has been developed for determining the separate influences on Tm of Na⁺, pH, and anions formed by Na₂CO₃ (HCO₃ ^? and CO₃ ^2-). Comparison of influence of anions formed by Na₂CO₃ on DNA stability with Cl^? (anion inert to DNA stability), ClO₄ ^? (strong DNA destabilizing “chaotropic” anion) and OH^? has been carried out. It has been shown that only Na⁺ and pH influence Tm in Na₂CO₃ solution at concentrations lower than 0.001M. However, the Tm decrease with concentration for [Na₂CO₃]≥0.004M is only partly caused by high pH≈10.7. Na₂CO₃ anions also exert a strong destabilizing influence at these concentrations. For 0.1M Na₂CO₃ (pH=10.84, [Na⁺]=0.2M, Tm=42.7 °C), the anion destabilizing effect is higher 20 °C. For NaClO₄ (ClO₄ ^? is a strong “chaotropic” anion), an equal anion effect occurs at much higher concentrations ～3M. This means that Na₂CO₃ gives rise to a much stronger anion effect than other salts. The effect is pH dependent. It decreases fivefold at neutral pH after addition of HCl to 0.1M Na₂CO₃ as well as after addition of NaOH for pH>11.2.     

The dynamics of mitochondrial Ca fluxes

We have investigated the kinetics of mitochondrial Ca²⁺ influx and efflux and their dependence on cytosolic [Ca²⁺] and [Na⁺] using low-Ca²⁺-affinity aequorin. The rate of Ca²⁺ release from mitochondria increased linearly with mitochondrial [Ca²⁺] ([Ca²⁺]_M). Na⁺-dependent Ca²⁺ release was predominant al low [Ca²⁺]_M but saturated at [Ca²⁺]_M around 400 μM, while Na⁺-independent Ca²⁺ release was very slow at [Ca²⁺]_M below 200 μM, and then increased at higher [Ca²⁺]_M, perhaps through the opening of a new pathway. Half-maximal activation of Na⁺-dependent Ca²⁺ release occurred at 5-10 mM [Na⁺], within the physiological range of cytosolic [Na⁺]. Ca²⁺ entry rates were comparable in size to Ca²⁺ exit rates at cytosolic [Ca²⁺] ([Ca²⁺]_c) below 7 μM, but the rate of uptake was dramatically accelerated at higher [Ca²⁺]_c. As a consequence, the presence of [Na⁺] considerably reduced the rate of [Ca²⁺]_M increase at [Ca²⁺]_c below 7 μM, but its effect was hardly appreciable at 10 μM [Ca²⁺]_c. Exit rates were more dependent on the temperature than uptake rates, thus making the [Ca²⁺]_M transients to be much more prolonged at lower temperature. Our kinetic data suggest that mitochondria have little high affinity Ca²⁺ buffering, and comparison of our results with data on total mitochondrial Ca²⁺ fluxes indicate that the mitochondrial Ca²⁺ bound/Ca²⁺ free ratio is around 10- to 100-fold for most of the observed [Ca²⁺]_M range and suggest that massive phosphate precipitation can only occur when [Ca²⁺]_M reaches the millimolar range.     

Inhibition of bovine heart Na+, K+-ATPase by palmitylcarnitine and palmityl-CoA.

The activity of a partially purified bovine heart Na⁺,K⁺-ATPase is inhibited by DL- and L- palmitylcarnitine (I₅₀=44–48μM). Palmitylcarnitine with a I₅₀ of 25μM also markedly inhibits K⁺-phosphatase activity. Palmityl-CoA decreases Na⁺,K⁺-ATPase activity, but to a lesser extent (I₅₀=80μM). Both palmitic acid and hexanoic acid produce 10 to 15% inhibition of activity at concentrations of 70μM and 3–5mM, respectively. These free fatty acids protect the enzyme against inhibition by 40μM palmitylcarnitine. However, at 50μM palmitylcarnitine, the protective effect by hexanoic acid is no longer apparent. Addition of 40μM palmitylcarnitine to the Na⁺,K⁺-ATPase in the presence of varying concentrations of palmityl-CoA produces an additive inhibition of enzyme activity, suggesting two different sites on the enzyme susceptible to inhibition by the two ester forms of the fatty acid.     

Sodium and potassium interactions with Na+-ATPase of inside-out membrane vesicles from high-K+ and low-K+ sheep red cells

Na⁺-ATPase of high-K⁺ and low-K⁺ sheep red cells was examined with respect to the sidedness of Na⁺ and K⁺ effects, using inside-out membrane vesicles and very low ATP concentrations (?2 μM). With varying amounts of Na⁺ in the medium, i.e., at the cytoplasmic surface, Na_cyt⁺, the activation curves show that high-K⁺ Na⁺-ATPase has a higher affinity for Na_cyt⁺ compared to low-K⁺. The apparent affinity for Na_cyt⁺ is also increased by increasing the ATP concentrations in high-K⁺ but not low-K⁺. With Na_cyt⁺ present, Na⁺-ATPase is stimulated by intravesicular Na⁺, i.e., Na⁺ at the originally external surface, Na_ext⁺, to a greater extent in low-K⁺ than high-K⁺. Intravesicular K⁺ (K_ext⁺) activates Na⁺-ATPase in high-K⁺ but not in low-K⁺ vesicles and extravesicular K⁺ (K_cyt⁺) inhibits low-K⁺ but not high-K⁺ Na⁺-ATPase. Thus, the genetic difference between high-K⁺ and low-K⁺ is expressed as differences in apparent affinities for both Na⁺ and K⁺ and these differences are evident at both cytoplasmic and external membrane surfaces.     

Characterization of glutamate transport system in hydrophobic protein (H protein) of Bacillus subtilis

Hydrophobic protein (H protein) was isolated from membrane fractions of Bacillus subtilis and constituted into artificial membrane vesicles with lipid of B. substilis. Glutamate was accumulated into the vesicle when a Na⁺ gradient across the membrane was imposed. The maximum effect of Na⁺ on the transport was achieved at a concentration of about 40 mM, while the apparent K_m for Na⁺ was approximately 8 mM. On the other hand, K_m for glutamate in the presence of 50 mM Na⁺ was about 8 μM. Increasing the concentration of Na⁺ resulted in a decrease in K_m for glutamate, maximum velocity was not affected. The transport was sensitive to monensin (Na⁺ ionophore).Glutamate was also accumulated when pH gradient (interior alkaline) across the membrane was imposed or a membrane potential was induced with K⁺-diffusion potential. The pH gradient-driven glutamate transport was sensitive to carbonylcyanide m-chlorophenylhydrazone and the apparent K_m for glutamate was approximately 25 μM.These results indicate that two kinds of glutamate transport system were present in H protein: one is Na⁺ dependent and the other is H⁺ dependent.     

Similarities and differences in interaction of K+ and Na+ with condensed ordered DNA. A molecular dynamics computer simulation study

Four 20 ns molecular dynamics simulations have been performed with two counterions, K⁺ or Na⁺, at two water contents, 15 or 20 H₂O per nucleotide. A hexagonal simulation cell comprised of three identical DNA decamers [d(5′-ATGCAGTCAG) × d(5′-TGACTGCATC)] with periodic boundary condition along the DNA helix was used. The simulation setup mimics the DNA state in oriented DNA fibers or in crystals of DNA oligomers. Variation of counterion nature and water content do not alter averaged DNA structure. K⁺ and Na⁺ binding to DNA are different. K⁺ binds to the electronegative sites of DNA bases in the major and the minor grooves, while Na⁺ interacts preferentially with the phosphate groups. Increase of water causes a shift of both K⁺ and Na⁺ from the first hydration shell of O1P/O2P and of the DNA bases in the minor groove with lesser influence for the cation binding to the bases in the major groove. Mobility of both water and cations in the K–DNA systems is faster than in the Na–DNA systems: Na⁺ organizes and immobilizes water structure around itself and near DNA while for K⁺ water is less organized and more dynamic.     

Determination of the Complete Correlation Between the Sugar Ring Puckers and 5′-Exocyclic Group Rotamers in Conformationally-Flexible Nucleic Acid Components from NMR Study

Abstract

Inspection of stereochemical models suggests a possible correlation between the proportion (Y_g-/Y_t) of the g^? and t rotamers and the S pucker populations irrespective of the anti-syn conformational composition of the base. Interpretation of the NMR vicinal coupling constants in terms of conformational populations shows a decline of Y_g-/Y_t with X_s approaching zero, consistent with high unfavorability of the Ng^? conformational combination in solution, a result supported by a X-ray crystallographic data survey. Hence, the underlying assumption introduced into the present study is that the g^? rotamer and the N pucker do not coexist together in solution. Therefore, the limiting value of Y_g-/Y_t corresponding to the S pucker could be determined for each compound individually. Finally, populations and relative free energies of all conformational combinations of Ng⁺, Nt, Sg⁺, St and Sg^? (except Ng^? which is not important) have been estimated.

Results of the present study suggest several interesting regularities concerning the syn-anti effect on populations and energies of the conformational combinations in ribo- and deoxyribo-nucleosides. (a) In the anti-type nucleosides, the Ng⁺ conformation is about 2 kJ/mol more stable than Nt, but in the syn-type, the Ng⁺ and Nt have comparable energy, (b) No important changes are observed in the Ng⁺ population comparing the anti-type and syn-type of ribo- and deoxyribo-nucleosides separately, (c) The Nt is considerable stabilized and simultaneously the Sg⁺ is strongly destabilized in the syn-type nucleosides relative to the anti-type, (d) Irrespective of the syn-anti composition the St is always more stable (1–2 kJ/mol) than the Sg^? conformational combination.     

5-hydroxytryptamine regulates the (Na++K+)ATPase activity in malpighian tubules of Rhodnius prolixus: Evidence for involvement of G-protein and cAMP-dependent protein kinase

5-Hydroxytryptamine (5-HT, serotonin) acts as a diuretic hormone in Rhodnius prolixus, where it increases to 0.1 μM in the haemolymph during feeding and stimulates the fluid secretion in isolated Malpighian tubules. The ouabain-sensitive (Na⁺+K⁺)ATPase activity present in homogenates of Malpighian tubules from unfed Rhodnius prolixus is inhibited 60% by 0.01 μM 5-HT. This inhibition is reversed by ketanserin, a 5-HT₂ receptor antagonist in mammals, and also by GDPβS, a competitive inhibitor of G-protein GTPase activity. GTPγS, a nonhydrolysable analog of GTP, and cholera toxin, a G_s-protein activator, also inhibit the ouabain-sensitive (Na⁺+K⁺)ATPase activity, while pertussis toxin, a G_i-protein inhibitor, has no effect. The (Na⁺+K⁺)ATPase activity is inhibited 55% by 0.4–100 μM dibutyryl-cAMP in the presence of IBMX, a phosphodiesterase inhibitor, which also potentiates the effect of a low concentration of 5-HT. The cAMP-dependent protein kinase inhibitor peptide abolishes the 5-HT effect. These data suggest that the (Na⁺+K⁺)ATPase activity in Malpighian tubules is inhibited by 5-HT through activation of G_s-protein and a cAMP-dependent protein kinase. Inhibition of the Na⁺+K⁺ pump would contribute to the diuretic effect of 5-HT. Arch. Insect Biochem. Physiol. 36:203–214, 1997. © 1997 Wiley- Liss, Inc.     

Melting of Oligodeoxynucleotides with Various Structures

Abstract

Effects of DNA fragments end structures on their melting profiles were studied experimentally and theoretically. We examined melting of hairpins and dumbbells obtained from 62- bp-long linear DNA duplex which is a perfect palindromic sequence. To fit theoretical melting profile to experimental ones additional theoretical parameters were incorporated into the standard statistical mechanical helix-coil transition theory. From comparison theoretical and experimental melting profiles theoretical parameters connected with end- structure effects were evaluated. Analysis revealed the stabilization effect of the hairpin loops and helix ends with respect to DNA duplex melting. Both type of ends make melting these oligodeoxynucleotides more cooperative than predicted by the standard helix-coil transition theory. At low ionic strength ([Na⁺] < 0.04 M) this effect becomes so pronounced that melting of the DNA duplexes 30–40 bp-long conforms to the two state model.

From the analysis experimental data obtained for dumbbell structures loop-weighting factor for single-stranded loop consisting of 132 nucleotides was determined. This parameter decreases 10 times with the ionic strength decreasing by an order of magnitude from 0.2 to 0.02 M Na⁺.     

Na/H Antiport in Isolated Tonoplast Vesicles from Storage Tissue of Beta vulgaris

The pH-dependent fluorescence quenching of acridine orange was used to study the Na⁺- and K⁺-dependent H⁺ fluxes in tonoplast vesicles isolated from storage tissue of red beet and sugar beet (Beta vulgaris L.). The Na⁺-dependent H⁺ flux across the tonoplast membrane could be resolved into two components: (a) a membrane potential-mediated flux through conductive pathways; and (b) an electroneutral flux which showed Michaelis-Menten kinetics relationship to Na⁺ concentration and was competitively inhibited by amiloride (K_i = 0.1 millimolar). The potential-dependent component of H⁺ flux showed an approximately linear dependence on Na⁺ concentration. In contrast, the K⁺-dependent H⁺ flux apparently consisted of a single component which showed an approximately linear dependence on K⁺ concentration, and was insensitive to amiloride. Based on the Na⁺- and K⁺-dependent H⁺ fluxes, the passive permeability of the vesicle preparation to Na⁺ was about half of that to K⁺.

The apparent K_m for Na⁺ of the electroneutral Na⁺/H⁺ exchange varied by more than 3-fold (7.5-26.5 millimolar) when the internal and external pH values were changed in parallel. The results suggest a simple kinetic model for the operation of the Na⁺/H⁺ antiport which can account for the estimated in vivo accumulation ratio for Na⁺ into the vacuole.

     

The Rapid-onset Dystonia Parkinsonism Mutation D923N of the Na+,K+-ATPase α3 Isoform Disrupts Na+ Interaction at the Third Na+ Site

Rapid-onset dystonia parkinsonism (RDP), a rare neurological disorder, is caused by mutation of the neuron-specific α3-isoform of Na⁺,K⁺-ATPase. Here, we present the functional consequences of RDP mutation D923N. Relative to the wild type, the mutant exhibits a remarkable ∼200-fold reduction of Na⁺ affinity for activation of phosphorylation from ATP, reflecting a defective interaction of the E₁ form with intracellular Na⁺. This is the largest effect on Na⁺ affinity reported so far for any Na⁺,K⁺-ATPase mutant. D923N also affects the interaction with extracellular Na⁺ normally driving the E₁P to E₂P conformational transition backward. However, no impairment of K⁺ binding was observed for D923N, leading to the conclusion that Asp⁹²³ is specifically associated with the third Na⁺ site that is selective toward Na⁺. The crystal structure of the Na⁺,K⁺-ATPase in E₂ form shows that Asp⁹²³ is located in the cytoplasmic half of transmembrane helix M8 inside a putative transport channel, which is lined by residues from the transmembrane helices M5, M7, M8, and M10 and capped by the C terminus, recently found involved in recognition of the third Na⁺ ion. Structural modeling of the E₁ form of Na⁺,K⁺-ATPase based on the Ca²⁺-ATPase crystal structure is consistent with the hypothesis that Asp⁹²³ contributes to a site binding the third Na⁺ ion. These results in conjunction with our previous findings with other RDP mutants suggest that a selective defect in the handling of Na⁺ may be a general feature of the RDP disorder.