An NMR self-diffusion study of the interactions between spermidine and oligonucleotides

Self-diffusion coefficients have been determined by pulsed field gradient nmr methods for spermidine in solutions of the oligonucleotides d(GC)₄ and d(GGAATTCC). The self-diffusion behavior of spermidine in solution of d(GC)₄ is very similar to that observed previously for methylspermidine (completely N-methylated spermidine). Moreover, the self-diffusion behaviors of spermidine in solutions of d(GC)₄ and d(GGAATTCC) are also quite similar, indicating that there is no significant influenceon on self-diffusion of oligonucleotide base composition. Furthermore, self-diffusion coefficients of the oligonucleotide d(GC)₈ show only a small dependence on oligonucleotide concentration, and no measurable dependence on sodium ion or magnesium ion concentration. © 1996 John Wiley & Sons, Inc.     

Competitive interactions of Co(NH3)6(3+) and Na+ with helical B-DNA probed by 59Co and 23Na NMR

59Co NMR is demonstrated to provide a useful probe of the interactions of Co(NH3)6(3+) with helical B-DNA. The association of Co(NH3)6(3+) with B-DNA produces relatively modest effects on the relaxation rate and chemical shift of 59Co, which indicate that the octahedral coordination shell remains intact and that no significant number of long-lived "outer-sphere" complexes are formed at specific sites on the DNA surface. Under conditions where essentially all of the cobalt complex is associated with DNA, the chemical shift of 59Co appears to depend on its binding density. This effect could be due to magnetic heterogeneity in the environments of Co(NH3)6(3+) adjacent to DNA. The local exchange reaction between Co(NH3)6(3+) and Na+ in the vicinity of DNA has been investigated by measuring 59Co chemical shifts and 23Na line widths concurrently. The number of sodium ions displaced by the association of one Co(NH3)6(3+) with DNA cannot be uniquely determined, but the data indicate that this number remains constant over at least the initial stage of a titration of NaDNA with NaCl. 59Co chemical shifts have been analyzed to construct binding isotherms for the association of cobalt hexaammine with DNA over a range of salt (NaCl) concentrations. The magnitudes of the resulting binding constants and their salt dependence are similar to those previously reported for the association of structurally diverse trivalent ligands, such as spermidine and trilysine, with helical nucleic acids. Therefore, these association equilibria appear to be governed primarily by electrostatic interactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Incomplete ion dissociation underlies the weakened attraction between DNA helices at high spermidine concentrations

We have investigated the salt sensitivity of the hexagonal-to-cholesteric phase transition of spermidine-condensed DNA. This transition precedes the resolubilization of precipitated DNA that occurs at high spermidine concentration. The sensitivity of the critical spermidine concentration at the transition point to the anion species and the NaCl concentration indicates that ion pairing of this trivalent ion underlies this unusual transition. Osmotic pressure measurements of spermidine salt solutions are consistent with this interpretation. Spermidine salts are not fully dissociated at higher concentrations. The competition for DNA binding among the fully charged trivalent ion and the lesser charged complex species at higher concentrations significantly weakens attraction between DNA helices in the condensed state. This is contrary to the suggestion that the binding of spermidine at higher concentrations causes DNA overcharging and consequent electrostatic repulsion.     

In vitro interactions between polyamines and pectic substances

Putrescine, spermidine and spermine induce a decrease in the pH value of 1 mM polygalacturonic acid or pectin solutions; spermidine and spermine also cause the precipitation of the polymers. The association constants between polyamines and polygalacturonic acid were in the order of 10(5) for putrescine and spermidine, and 10(6) for spermine. The number of galacturonic units per binding sites are proportional to the number of positive charges on the polyamine molecule. Low affinity binding sites appear at high polyamine concentrations. Calcium ions seem to compete weakly with spermine by lowering the association constant 4- to 6-fold. Two natural pectins tested, showed that methylation of the carboxylic groups influences only the number of galacturonic units per site but not the association constant.     

Role of hypusinated eukaryotic translation initiation factor 5A in polyamine depletion-induced cytostasis

We have earlier shown that alpha-methylated spermidine and spermine analogues rescue cells from polyamine depletion-induced growth inhibition and maintain pancreatic integrity under severe polyamine deprivation. However, because alpha-methylspermidine can serve as a precursor of hypusine, an integral part of functional eukaryotic translation initiation factor 5A required for cell proliferation, and because alpha, omega-bismethylspermine can be converted to methylspermidine, it is not entirely clear whether the restoration of cell growth is actually attributable to hypusine formed from these polyamine analogues. Here, we have used optically active isomers of methylated spermidine and spermine and show that polyamine depletion-induced acute cytostasis in cultured cells could be reversed by all the isomers of the methylpolyamines irrespective of whether they served or not as precursors of hypusine. In transgenic rats with activated polyamine catabolism, all the isomers similarly restored liver regeneration and reduced plasma alpha-amylase activity associated with induced pancreatitis. Under the above experimental conditions, the (S, S)- but not the (R, R)-isomer of bismethylspermine was converted to methylspermidine apparently through the action of spermine oxidase strongly preferring the (S, S)-isomer. Of the analogues, however, only (S)-methylspermidine sustained cell growth during prolonged (more than 1 week) inhibition of polyamine biosynthesis. It was also the only isomer efficiently converted to hypusine, indicating that deoxyhypusine synthase likewise possesses hidden stereospecificity. Taken together, the results show that growth inhibition in response to polyamine depletion involves two phases, an acute and a late hypusine-dependent phase.     

Spermidine-Deoxyribonucleic acid interaction in vitro and in Escherichia coli.

The binding of spermidine to deoxyribonucleic acid (DNA) was studied by equilibrium dialysis in a wide range of salt concentrations. The association constants ranged from 6 x 10(5) M-1 in 1 mM sodium cacodylate, pH 7.5, to 3 x 10(2) M-1 in 0.3 M NaCl. MgCl2 reduced spermidine-DNA interaction even more than NaCl so that in moderate-ionic-strength solutions (0.3 M NaCl, 0.002 M MgCl2) there was little detectable binding. Low-ionic-strength media were used to isolate DNA from Escherichia coli by a method shown to minimize loss of spermidine from the DNA. Considerable spermidine was associated with E. coli DNA, but control experiments indicated that complex formation had taken place during or after lysis of the cells. Exogenous DNA or ribonucleic acid added to spheroplasts at the time of their lysis caused most of the cellular spermidine to be scavenged by the extra nucleic acid. The data suggest that spermidine is relatively free in the cell and thereby capable of strong (high-affinity) associations with nucleic acids only after the ionic strength of the cell environment is lowered.     

Application of the Poisson Boltzmann polyelectrolyte model for analysis of thermal denaturation of DNA in the presence of Na+ and polyamine cations

The Poisson Boltzmann (PB) cell model of polyelectrolyte solution has been used for calculation of the electrostatic free energy difference, Delta G(el), between double- and single-stranded DNA. The calculations have been performed for conditions relevant to describe the DNA helix-coil transition in NaCl solution in the presence of the natural polyamines putrescine(2+), spermidine(3+), spermine(4+) and their synthetic homologs with different spacing between the charged amino groups, for which experimental values of the DNA 'melting' transition temperature (T(m)) are available. Using the PB theory and the polyamine ion radius as an adjusting parameter provides quantitative agreement between experimental and theoretical T(m)--salt concentration dependencies only by using physically unreasonable radii for the polyamine. Thus, modeling the linear and flexible polyamines as charged spheres within the PB cell model is an implausible oversimplification. We propose another explanation for the experimental observations, still within the frame of the 'primitive' PB polyelectrolyte theory. This explanation is based on an analysis of the Delta G(el) dependence on the stoichiometry of polyamine-polyanion binding to double- and single-stranded DNA.     

Characterization of the inducible polyamine transporter in bovine lymphocytes

The polyamine uptake system in bovine lymphocytes was activated by concanavalin A. The system was common to putrescine, spermidine and spermine. The Kt values for uptake activities of putrescine, spermidine and spermine were 3.7 microM, 0.38 microM and 0.23 microM in that order. The uptake activity was inhibited by carbonyl cyanide m-chlorophenylhydrazone, gramicidin D or valinomycin in the presence of 20 mM K+ suggesting that polyamine uptake depends on the membrane potential. The uptake activity appeared 10 h after addition of concanavalin A, and the maximum was reached at 28 h indicating that induction of the polyamine transporter precedes the initiation of DNA synthesis. Addition of polyamine antimetabolites, such as alpha-difluoromethylornithine and ethylglyoxal bis(guanylhydrazone), to the medium enhanced at least eightfold the induction of the polyamine transporter. The induction was repressed by addition of 50 microM spermidine or spermine, but not putrescine. We propose here that the induction of the membrane-potential-dependent polyamine transporter is regulated by the intracellular level of spermidine and spermine.     

Application of polyelectrolyte theories for analysis of DNA melting in the presence of Na+ and Mg2+ ions.

Numerical calculations, using Poisson-Boltzmann (PB) and counterion condensation (CC) polyelectrolyte theories, of the electrostatic free energy difference, DeltaGel, between single-stranded (coil) and double-helical DNA have been performed for solutions of NaDNA + NaCl with and without added MgCl2. Calculations have been made for conditions relevant to systems where experimental values of helix coil transition temperature (Tm) and other thermodynamic quantities have been measured. Comparison with experimental data has been possible by invoking values of Tm for solutions containing NaCl salt only. Resulting theoretical values of enthalpy, entropy, and heat capacity (for NaCl salt-containing solutions) and of Tm as a function of NaCl concentration in NaCl + MgCl2 solutions have thus been obtained. Qualitative and, to a large extent, quantitative reproduction of the experimental Tm, DeltaHm, DeltaSm, and DeltaCp values have been found from the results of polyelectrolyte theories. However, the quantitative resemblance of experimental data is considerably better for PB theory as compared to the CC model. Furthermore, some rather implausible qualitative conclusions are obtained within the CC results for DNA melting in NaCl + MgCl2 solutions. Our results argue in favor of the Poisson-Boltzmann theory, as compared to the counterion condensation theory.     

[3H]Dizocilpine Association Kinetics Distinguish Stimulatory and Inhibitory Polyamine Sites of N-Methyl-d-Aspartate Receptors

Abstract: Spermine and other polyamines both stimulate and inhibit N -methyl- d -aspartate receptor function, probably by interacting with two separate sites. To characterize these two actions, the effect of spermine on the binding kinetics of the channel blocker [³H]dizocilpine was studied in the presence of glutamate and glycine. Low concentrations (10 µ M ) of spermine increased the association and dissociation rates without modifying equilibrium binding, indicating that spermine increases the accessibility of [³H]dizocilpine to the channel by interacting with a high-affinity, stimulatory site. At higher concentrations (1 m M ), spermine markedly decreased equilibrium [³H]-dizocilpine binding by decreasing both affinity and B _max, indicating that spermine allosterically inhibits binding by interacting with a second, low-affinity site. The presumed polyamine antagonists arcaine, diethylenetriamine, and 1,10-diaminodecane completely inhibited equilibrium [³H]dizocilpine binding, probably by interacting with the inhibitory polyamine site or other sites, but not with the stimulatory polyamine site. Low concentrations (10 µ M ) of ifenprodil completely reversed the increase in association rate produced by spermine, whereas higher concentrations (IC₅₀ = 123 µ M ) inhibited equilibrium binding, indicating that ifenprodil is both a potent antagonist of the stimulatory site and a low-affinity ligand of the inhibitory site. The polyamine agonists spermine, spermidine, and neomycin interacted with the inhibitory site, but produced only partial inhibition of equilibrium [³H]dizocilpine binding.     

Quantitation of cation binding to wheat germ ribosomes: influences on subunit association equilibria and ribosome activity.

The binding of Mg2+, spermine, and spermidine to wheat germ ribosomes was quantitated following equilibrium dialysis. The Mg2+ binding data demonstrate that Mg2+ and K+ compete for binding to the ribosomes. Mg2+ binding saturates at approximately 0.56 positive charges per phosphate (+/P). The Mg2+, spermine and spermidine binding data indicate that either polyamine replaces Mg2+ upon binding to the ribosomes. Mg2+ and polyamine binding combined saturates at approximately 0.29 +/P under the conditions reported. When a critical number of Mg2+ ions are replaced by either polyamine, the activity of the ribosomes falls dramatically. Ribosomal subunit association increases with the degree of phosphate charge neutralization due to the binding of Mg2+. Total charge neutralization during subunit association by Mg2+ and polyamine binding combined, is much less than that achieved by Mg2+ alone.     

Intercalation of Zn(II) and Cu(II) complexes of the cyclic polyamine Neotrien into DNA: equilibria and kinetics

The equilibria and kinetics of the interaction of the Zn(II) and Cu(II) complexes of the macrocyclic polyamine 2,5,8,11-tetraaza[12]-[12](2,9)[1,10]-phenanthrolinophane (Neotrien) with calf thymus DNA have been investigated at pH=7.0 and T=25 degrees C by spectrophotometry, spectrofluorimetry and stopped-flow method. At low dye/polymer ratios both complexes bind to DNA according to the excluded site model. At high dye/polymer ratios the binding displays cooperative features. The logarithm of the binding constant depends linearly on -log[NaCl]. The kinetic results suggest the D + S <==> D, S <==> DS mechanism where the metal complexes (D) react with the DNA sites (S) leading to fast formation of an externally bound form (D,S) which, in turn, is converted into internally bound complex (DS) by intercalation. The binding constants, evaluated as ratios of rate constants, agree with those obtained from equilibrium binding experiments, thus confirming the validity of the proposed model. Fluorescence titrations, where the metal-Neotrien complexes were added to DNA previously saturated with ethidium bromide (EB), show that both complexes displace EB from the DNA cavities. The reverse process, i.e. the addition of excess ethidium to the DNA/metal Neotrien systems, leads to fluorescence recovery for DNA/ZnNeotrien but not for DNA/CuNeotrien. This observation suggests that the binding of CuNeotrien induces deep alterations in the DNA structure. Experiments with Poly(dA-dT)*Poly(dA-dT) and Poly(dG-dC)*Poly(dG-dC) reveal that CuNeotrien mainly affects the structure of the latter polynucleotide.     

Ligand-induced DNA condensation: choosing the model

We test and compare different models for ligand-induced DNA condensation. Using 14C-labeled spermidine3+, we measure the binding to condensed DNA at micromolar to molar polyamine concentrations. DNA aggregates at a critical polyamine concentration. Spermidine3+ binding becomes highly cooperative at the onset of aggregation. At higher concentrations, spermidine3+ binding to condensed DNA reaches a plateau with the degree of binding equal to 0.7 (NH(4+)/PO3-). Condensed DNA exists in a wide range of spermidine concentrations with the roughly constant degree of ligand binding. At greater concentrations, the degree of binding increases again. Further spermidine penetration between the double helices causes DNA resolubilization. We show that a simple two-state model without ligand-ligand interactions qualitatively predicts the reentrant aggregation-resolubilization behavior and the dependence on the ligand, Na+, and DNA concentrations. However, such models are inconsistent with the cooperative ligand binding to condensed DNA. Including the contact or long-range ligand-ligand interactions improves the coincidence with the experiments, if binding to condensed DNA is slightly more cooperative than to the starting DNA. For example, in the contact interaction model it is equivalent to an additional McGhee-von Hippel cooperativity parameter of approximately 2. Possible physical mechanisms for the observed cooperativity of ligand binding are discussed.     

外源钙和亚精胺对NaCl胁迫条件下玉米幼苗体内离子平衡和多胺水平的调节

研究了钙离子和亚精胺对盐胁迫条件下玉米幼苗生长状况、电解质渗漏、离子含量和多胺水平的影响。结果表明盐胁迫下,钠离子和腐胺含量升高,质膜受到严重伤害,而钾离子、钙离子、亚精胺以及精胺水平降低。外加氯化钙和亚精胺不但能逆转氯化钠带来的离子平衡失调、多胺代谢紊乱,而且还能减轻氯化钠导致的生长抑制和质膜伤害。在盐胁迫条件下,二环己基胺引起多胺含量严重下降的情况在一定程度上能被外加氯化钙处理所缓解。这些结果提示,钙、多胺代谢以及盐渍环境下玉米生长之间可能存在一定的关系。     

Structural basis of polyamine-DNA recognition: spermidine and spermine interactions with genomic B-DNAs of different GC content probed by Raman spectroscopy

Four genomic DNAs of differing GC content (Micrococcus luteus, 72% GC; Escherichia coli, 50% GC; calf thymus, 42% GC; Clostridium perfringens, 27% GC) have been employed as targets of interaction by the cationic polyamines spermidine {[H₃N(CH₂)₃NH₂(CH₂)₄NH₃]³⁺} and spermine {[(CH₂)₄(NH₂(CH₂)₃NH₃)₂]⁴⁺}. In solutions containing 60 mM DNA phosphate (~20 mg DNA/ml) and either 1, 5 or 60 mM polyamine, only Raman bands associated with the phosphates exhibit large spectral changes, demonstrating that B-DNA phosphates are the primary targets of interaction. Phosphate perturbations, which are independent of base composition, are consistent with a model of non-specific cation binding in which delocalized polyamines diffuse along DNA while confined by the strong electrostatic potential gradient perpendicular to the helix axis. This finding provides experimental support for models in which polyamine-induced DNA condensation is driven by non-specific electrostatic binding. The Raman spectra also demonstrate that major groove sites (guanine N7 and thymine C5H₃) are less affected than phosphates by polyamine–DNA interactions. Modest dependence of polyamine binding on genome base composition suggests that sequence context plays only a secondary role in recognition. Importantly, the results demonstrate that polyamine binding has a negligible effect on the native B-form secondary structure. The capability of spermidine or spermine to bind and condense genomic B-DNA without disrupting the native structure must be taken into account when considering DNA organization within bacterial nucleoids or cell nuclei.     

Polyamines Modulate the Binding of [3H]MK-801 to the Solubilized N-Methyl-D-Aspartate Receptor

Spermine and spermidine enhance the binding of [³H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a, d]cyclohepten-5,10-imine ([³H]MK-801) to N-methyl-D-aspartate (NMDA) receptors in membranes prepared from rat brain. These polyamines also enhance binding of [³H]MK-801 to NMDA receptors that have been solubilized with deoxycholate. Other polyamines selectively antagonize this effect, a finding indicating that the polyamine recognition site retains pharmacological and structural specificity after solubilization. In the presence of spermidine, an increase in the affinity of the solubilized NMDA receptor for [³H]MK-801 is observed. However, the rates of both association and dissociation of [³H]MK-801 binding to solubilized NMDA receptors are accelerated when assays are carried out in the presence of spermidine. When kinetic data are transformed, pseudo-first-order association and first-order dissociation plots are nonlinear in the presence of spermidine, an observation indicating a complex binding mechanism. Effects of spermidine on solubilized NMDA receptors are similar to effects previously described in studies of membrane-bound receptors. The data indicate that polyamines interact with a specific recognition site that remains associated with other components of the NMDA receptor complex after detergent solubilization.     

23Na-NMR investigations of counterion exchange reactions of helical DNA

Changes in Δν_½, the nmr linewidth of ²³Na, have been determined during titrations of helical DNA with polyamines (divalent putrescine and trivalent spermidine) and with inorganic cations (Mg²⁺ and Co(NH₃)). In each case additions of a multivalent cation (M^z+) to a solution containing NaDNA and NaCl cause decreases in Δν_½, which is a population-weighted average of contributions from nuclei in bound and free environments. Thus, the binding of M^z+ to DNA displaces sodium ions from regions where the quadrupolar relaxation of ²³Na is relatively efficient. At a given extent of titration, the binding of a polyamine produces a smaller decrease in Δν_½ than does the binding of an inorganic ion of the same valence. The concentration dependence of Δν_½ during the course of a titration can be interpreted most simply as a two-state ion-exchange reaction by assuming that the binding of M^z does not alter R_B, the average relaxation rate of sodium nuclei that remain bound. On the basis of this assumption, the initial linear portions of titration curves can be analyzed to determine upper bounds for r°, the number of sodium ions bound per DNA phosphate in the absence of any competing counterion. Analyzing the titration curves for the four multivalent competitors leads to a range of upper-bound estimates for r°: 0.5–0.8. The differences in these estimates could indicate that polyamines displace fewer sodium ions from DNA than do their smaller inorganic counterparts. Alternatively, the range in upper-bound estimates for r° could also reflect specific differences in the effects of the various multivalent cations on R_B, if this relaxation rate does change during titration.     

Effects of aminooxy analogues of biogenic polyamines on aggregation and stability of calf thymus DNA

The effect of a series of aminooxy analogues of the biogenic polyamines spermidine and spermine on the conformation of calf thymus DNA is studied. These new molecules are isosteric and charge insufficient analogues that are suitable to study the roles of both charge distribution and structural requirements in the molecular physiology of the biogenic polyamines. They are also evidenced as useful tools to inhibit polyamine biosynthesis and cell growth. Circular dichroism (CD) spectra of solutions containing DNA and the aminooxy analogues at different concentrations (100-1000 microM) and different pH values, (5-7.5) are recorded. We use both sonicated and highly polymerized calf thymus DNA. The CD spectra of sonicated DNA showed the formation of Psi-DNA, a highly ordered aggregated structure similar to liquid crystals, in the presence of the aminooxy analogues. Aggregation induced by an aminooxy derivative of spermine is followed by DNA collapse when increasing the polyamine concentration. The features of Psi-DNA are not detected for highly polymerized DNA. Temperature melting measurements support a high degree of structural order of the aggregates. The CD experiments indicate that dications are unable to induce major changes on the macromolecular structure of DNA. In addition, aggregation is only observed when the trimethylene moiety is present between two adjacent positive charges. The observed differences among the CD spectra of DNA solutions with different aminooxy derivatives of spermidine indicate different roles for different amino groups of this biogenic polyamine when interacting with DNA. Our results support the idea that aminooxy analogues can be used as good models in studying the physiological functions of biogenic polyamines.