Polyamine Binding Research Articles

The binding of polyamines and magnesium to DNA

1. The binding of spermine and Mg 2+ to DNA has been investigated using the dye arsenazo III to measure unbound cations. 2. The apparent dissociation constant, K d, of DNA for spermine has been found to be 7.4 ± 3.9 × 10 −8 M and that for Mg 2+, 6.5 ± 3.3 × l0 −7 M. 3. Binding of spermine in the presence of 1 mM Mg 2+ has been shown to have a K d of about 4 × 10 −6 M. 4. Magnesium ion (2 mM) halves the concentration of spermine needed to cause DNA aggregation. 5. Spermidine binds to DNA with a similar affinity to spermine but 3,3′-iminobispropylamine and 1,5,9,13-tetra-azatridecane bind with a lower affinity. The naturally occurring polyamines thus have a higher affinity for DNA than the related polyamines which do not occur naturally. 6. Binding of spermine or spermidine to DNA alters the spectrophotometric absorbance of DNA at 260 nm.

Quantification of polyamine losses during manipulation and assay procedures

AbstractLosses in polyamine recovery during manipulation and assay procedures have been detected. They are partly due to the binding of polyamines at low concentration (around 5 μM for putrescine and 100 μM for spermine) to the containers and depend on the chemical nature of the latter, on the pH of the solution and on the length of time for which they remain in contact with the container. The recovery of labelled polyamines after dansylation was about 45% of the initial radioactivity. Remarkably high losses occurred when bound polyamines, obtained by in vitro incubation with cell‐free extracts, were estimated after acidic hydrolysis. Standard free polyamines, subjected to acidic hydrolysis conditions, yielded a 50–60% recovery, both measured as radioactivity and fluorescence, compared to standard polyamines not subjected to hydrolysis. If carbohydrates are present, the recovery after hydrolysis can be even lower, depending on the sequence in which the reagents of the dansylation procedure are added. Ion exchange chromatography to remove carbohydrates improved this recovery.

Sequestered end products and enzyme regulation: the case of ornithine decarboxylase.

The polyamines (putrescine, spermidine, and spermine) are synthesized by almost all organisms and are universally required for normal growth. Ornithine decarboxylase (ODC), an initial enzyme of polyamine synthesis, is one of the most highly regulated enzymes of eucaryotic organisms. Unusual mechanisms have evolved to control ODC, including rapid, polyamine-mediated turnover of the enzyme and control of the synthetic rate of the protein without change of its mRNA level. The high amplitude of regulation and the rapid variation in the level of the protein led biochemists to infer that polyamines had special cellular roles and that cells maintained polyamine concentrations within narrow limits. This view was sustained in part because of our continuing uncertainty about the actual biochemical roles of polyamines. In this article, we challenge the view that ODC regulation is related to precise adjustment of polyamine levels. In no organism does ODC display allosteric feedback inhibition, and in three types of organism, bacteria, fungi, and mammals, the size of polyamine pools may vary radically without having a profound effect on growth. We suggest that the apparent stability of polyamine pools in unstressed cells is due to their being largely bound to cellular polyanions. We further speculate that allosteric feedback inhibition, if it existed, would be inappropriately responsive to changes in the small, freely diffusible polyamine pool. Instead, mechanisms that control the amount of the ODC protein have appeared in most organisms, and even these are triggered inappropriately by variation of the binding of polyamines to ionic binding sites. In fact, feedback inhibition of ODC might be maladaptive during hypoosmotic stress or at the onset of growth, when organisms appear to require rapid increases in the size of their cellular polyamine pools.

Characteristics of the gene for a spermidine and putrescine transport system that maps at 15 min on the Escherichia coli chromosome.

The nucleotide sequence of the gene for the spermidine and putrescine transport system that maps at 15 min on the Escherichia coli chromosome was determined. It contained four open reading frames encoding A, B, C, and D proteins. By making several subclones, we showed that expression of all the four proteins was necessary for maximal spermidine and putrescine transport activity. A single transport system was involved in the transport of both spermidine and putrescine. The A protein (Mr 43K) was found to be associated with membranes, as shown by Western blot analysis of the cell fractions. In addition, it had consensus amino acid sequences for the nucleotide binding site. B (Mr 31K) and C (Mr 29K) proteins consisted of six putative transmembrane spanning segments linked by hydrophilic segments of variable length as shown by cell localization of the proteins synthesized in maxicells and by hydropathy profiles. D protein (Mr 39K) was inferred to be a polyamine binding protein existing in a periplasmic fraction from the results of Western blot analysis of the cell fractions and from measurements of polyamine binding to the protein. These results indicate that the spermidine and putrescine transport system can be defined as a bacterial periplasmic transport system.

Estimation of polyamine binding to macromolecules and ATP in bovine lymphocytes and rat liver.

To estimate the polyamine distribution in bovine lymphocytes and rat liver, the binding constants (K) for DNA, RNA, phospholipid, and ATP were determined under the conditions of 10 mM Tris-HCl, pH 7.5, 2 mM Mg2+, and 150 mM K+. The binding constants of spermine for calf thymus DNA, Escherichia coli 16 S rRNA, phospholipid in rat liver microsomes and ATP were 1.15 x 10(2), 6.69 x 10(2), 2.22 x 10(2), and 5.95 x 10(2) M-1, respectively. From these binding constants and experimentally determined cellular concentrations of macromolecules, ATP, and polyamines, spermine distribution in the cells was estimated. In bovine lymphocytes, the mols of spermine bound to DNA, RNA, phospholipid, and ATP were 0.79, 3.7, 0.23, and 4.3 per 100 mol of phosphate of macromolecules or ATP, respectively. In rat liver, they were 0.19, 1.0, 0.05, and 0.97/100 mol of phosphate of macromolecules or ATP, respectively. The binding constants of spermidine for macromolecules and ATP were smaller than those of spermine, but a similar tendency was observed with spermidine distribution among macromolecules and ATP in the above two cells. The amount of polyamine bound to DNA and phospholipid was significantly lower than that to RNA. When either the Mg2+ or K+ concentration increased, the amount of free spermine and that bound to RNA and ATP increased, but the amount of spermine bound to DNA and phospholipid decreased. The results indicate that most polyamines exist as a polyamine-RNA complex in cells. Under the conditions that globin synthesis is stimulated by spermine in a rabbit reticulocyte cell-free system, the amount of spermine bound to RNA was very close to the value estimated in the cells.

Polyamine binding activity of casein kinase II

Protein phosphorylation by the ubiquitous casein kinase II (CKII) is known to be sensitive to naturally occurring polyamines. Using isolated recombinant alpha and beta subunits of the kinase, as well as the alpha 2 beta 2 oligomeric enzyme, it is shown that (i) CKII binds [3H]-spermine with Kds in the micromolar range. (ii) The beta subunit appears mostly responsible for this binding activity. (iii) The isolated alpha (catalytic) subunit is not activated by polyamines. (iv) The polyamine-dependent activation of the oligomeric CKII requires the beta subunit, which appears as a regulatory component in the native kinase. These observations suggest that there may be a functional interaction between polyamines and CKII in living cells, especially in the response to cell growth factors and trophic hormones.

Polyamine effects upon N-methyl- D-aspartate receptor functioning: differential alteration by glutamate and glycine site antagonists

Polyamines such as spermidine potentiate activation of the N-methyl- D-aspartate (NMDA)-type excitatory amino acid receptor. The goal of the present study was to investigate interactions between the putative polyamine binding site and previously described sites for glutamate and glycine. Binding of the high-potency PCP receptor ligand [ 3H]MK-801 to well-washed rat brain membranes was used as an in vitro probe of NMDA receptor activation. Spermidine concentration-response studies were performed in the absence and presence of both glutamate and glycine, with and without D-(−)-2-amino-5-phosphonovaleric acid ( D(−)AP-5) or 7-chlorokynurenic acid (7Cl-KYN). Incubation in the presence of spermidine alone induced a 20.4-fold increase in [ 3H]MK-801 binding with an EC 50 value of 13.3 μM. The mean concentration of spermidine which induced maximal stimulation of binding was 130 μM ( n = 10,S.E.M.= 24.66,range= 25–250 μM). Glutamate (10 μM) decreased the EC 50 value for spermidine-induced stimulation of [ 3H]MK-801 binding to 3.4 μM. Glycine (10 μM) did not significantly alter either maximum spermidine-induced [ 3H]MK-801 binding or the EC 50 value for spermidine-induced stimulation of [ 3H]MK-801 binding. Incubation in the presence of the specific glutamate antagonist D(−)AP-5 attenuated [ 3H]MK-801 binding in a glutamate-reversible fashion. The competitive glycine antagonist 7Cl-KYN decreased maximum spermidine-induced [ 3H]MK-801 binding in a glycine-reversible fashion. In addition, 7Cl-KYN increased the EC 50 value for spermidine-induced stimulation of [ 3H]MK-801 binding while D(−)AP-5 was without effect. These findings suggest that glutamate and glycine regulate the polyamine binding site differentially. PCP-like agents induce a psychotomimetic state closely resembling schizophrenia by inhibiting NMDA receptor-mediated neurotransmission. The ability of polyamines to modulate NMDA receptor functioning suggests a potential site for pharmacological intervention.

Synthesis and binding of [125I2]philanthotoxin-343, [125I2]philanthotoxin-343-lysine, and [125I2]philanthotoxin-343-arginine to rat brain membranes.

125I2-iodinated philanthotoxin-343 (PhTX-343), [125I2]PhTX-343-arginine, and [125I2]PhTX-343-lysine were synthesized and evaluated as probes for glutamate receptors in rat brain synaptic membranes. It was found that these probes were not specific for the glutamate receptors but may be useful for investigating the polyamine binding site. Filtration assays with Whatman GF/B fiber glass filters were unsuitable because the iodinated PhTX-343 analogues exhibited high nonspecific binding to the filters, thus hindering detection of specific binding to membranes. When binding was measured by a centrifugal assay, [125I2]PhTX-343-lysine bound with low affinity (KD = 11.4 +/- 2 microM) to a large number of sites (37.2 +/- 9.1 nmol/mg of protein). The binding of [125I2]PhTX-343-lysine was sensitive only to the polyamines spermine and spermidine, which displaced [125I2]PhTX-343-lysine with Ki values of (3.77 +/- 1.4) x 10(-5) M and (7.51 +/- 0.77) x 10(-5) M, respectively. The binding was insensitive to glutamate receptor agonists and antagonists. Binding results with [125I2]PhTX-343-arginine were similar to those of [125I2]-PhTX-343-lysine. Considering the high number of toxin binding sites (10000-fold more than glutamate) in these membranes and the insensitivity of the binding to almost all drugs that bind to glutamate receptors, it is evident that most of the binding observed is not to glutamate receptors. On the other hand, PhTX analogues with photoaffinity labels may be useful in the isolation/purification of various glutamate and nicotinic acetylcholine receptors; they could also be useful in structural studies of receptors and their binding sites.

Implications and concepts of polyamine‐nucleic acid interactions

Modeling, x-ray diffraction, and solution studies have contributed to the understanding of interactions between polyamines and nucleic acids. Polyamines stabilize a variety of unusual DNA structures and conformations in vitro, including both the left-handed Z and the right-handed A DNA. In addition, polyamines condense DNA and may be important in bending specific sequences. Investigations into the mechanisms of these effects provide support for both specific and nonspecific interactions between polyamines and DNA. Although exact relationships between the binding of polyamines and conformational changes in nucleic acids are still being clarified, polyamines remain important candidates for regulators of DNA conformation in vivo.

Location of spermine and other polyamines on DNA as revealed by photoaffinity cleavage with poly(amino)benzenediazonium salts

Although polyamines interact strongly with nucleic acids, X-ray and NMR studies have not revealed much structural information about spermine-DNA complexes. Therefore, it was of interest to look at the binding of polyamines to 32P-labeled DNA restriction fragments by sequencing gel electrophoresis of the photoaffinity cleavage products induced by polyaminobenzendiazonium salts. The shift of cleavage patterns observed on opposite strands as well as competition experiments with distamycin shows polyamines to be located in the minor groove of B-DNA and to depend on the nucleic acid polymorphism, jumping to the major groove in the A-form. The sequence selectivities of various polycations (spermine, putrescine, and cobalt (III) hexaammine) are similar and slightly favor A,T-rich regions. Taken together, these results show that polycations which are not point charges are guided by the electronegative potential along the nucleic acid and suggest fast crawling of the polyamine within the minor groove, due to individual NH2+ jumping between multiple equidistant and isoenergetic bidentate hydrogen-bonding sites. Such a picture could be the clue to the unexpected NMR and to the frequently silent X-ray behavior of polyamines when bound to DNA.

Effect of Acetylpolyamines on In Vitro Protein Synthesis and on the Growth of a Polyamine-Requiring Mutant of Escherichia coli

The functions of acetylpolyamines were examined with respect to stimulation of protein synthesis and cell growth. Unlike polyamines, acetylpolyamines could not lower the optimal Mg2+ concentration of protein synthesis, and the degree of stimulation of protein synthesis by acetylpolyamines was small. The addition of N1-acetylspermine did not stimulate cell growth of a polyamine-requiring mutant of Escherichia coli MA261, although acetylspermine was accumulated in the cells. Acetylspermine did not interfere with polyamine stimulation of protein synthesis and cell growth of E. coli MA261. The binding of acetylpolyamines to RNA was very weak, and the binding of polyamines to RNA was not disturbed significantly by the presence of acetylpolyamines. When the growth of E. coli MA261 was stimulated by addition of polyamines, significant amounts of acetylpolyamines were also formed in the cells. These results suggest that acetylation of polyamines, together with polyamine excretion, may regulate the intracellular level of the parent polyamines when excess amounts of polyamines accumulate intracellularly.

Polyamine uptake, binding and release in rat brain

The uptake, binding and release of the polyamines, spermidine and spermine, and of their diamine precursor, putrescine, were examined in synaptosomal preparations from rat hippocampus. The specific and relatively high-affinity uptake by synaptosomes was found only with putrescine (V max = 21.6 pmol/mg protein per h; K m =28.6 nM) and not with the other polyamines. In contrast, specific binding to membranes was found for spermidine (B max = 28.6 pmol/mg protein; K d = 42.9 nM) and for spermine (B max = 156.3 pmol/mg protein; K d = 83.3 nM), but not for putrescine. High potassium concentrations (35 mM) both induced the release of accumulated polyamines from synaptosomes and inhibited their binding. Specific polyamine binding evidently occurs selectively on the inner but not on the outer surface of synaptosomal membranes.

Reduction of NMDA receptors with dithiothreitol increases [3H]‐MK‐801 binding and NMDA‐induced Ca2+ fluxes

1. We have investigated the modulation of N-methyl-D-aspartate (NMDA) receptor activation by the sulphydryl redox reagents dithiothreitol (DTT) and 5,5-dithio-bis-2-nitrobenzoic acid (DTNB). 2. Increases in [3H]-MK-801 binding produced by glutamate, glycine and spermidine were enhanced by DTT (2mM) and diminished by DTNB (0.5 mM). 3. The inhibition of [3H]-MK-801 binding by CGS 19755 and 7-chlorokynurenate was not altered by 2 mM DTT. However, the potency of the competitive polyamine antagonist, arcaine, was decreased by DTT. 4. NMDA-induced Ca2+ fluxes into primary cultures of rat forebrain neurones were enhanced by DTT in a DTNB-reversible fashion. In addition to augmenting the magnitude of NMDA-induced increase in intracellular free Ca2+, 10 mM DTT also prolonged the duration of the Ca2+ signal. However, DTT had no effect on the increase in Ca2+ produced by depolarizing neurones with 50 mM KCl. 5. These studies show that the reduction of disulphide bonds on the NMDA receptor complex by DTT increases activation. The precise site of these groups remains unclear but they are unlikely to form an integral part of the glutamate, glycine or polyamine binding domains. The enhancement of the activation of the NMDA receptor by DTT is associated with increased Ca2+ fluxes. The possible pathophysiological consequences of receptor reduction are discussed.

Spermine and philanthotoxin potentiate excitatory amino acid responses of Xenopus oocytes injected with rat and chick brain RNA

The effects of spermine and a synthetic analogue (PhTX-343) of the polyamine amide toxin, delta-philanthotoxin, on the responses of Xenopus oocytes to application of amino acids were examined using voltage clamp. The oocytes were injected with either total rat brain RNA or chick cerebrum, poly(A+)RNA. The responses to N-methyl-D-aspartate and L-kainate were potentiated by low concentrations (10(-11)-10(-7) M) of PhTX-343 and by 10(-5)-10(-4) M spermine. There was variability between oocytes in terms of their responsiveness to these compounds and recovery from their effects was slow and often incomplete. Prolonged or repeated applications of PhTX-343 and spermine eventually resulted in inhibition. Higher concentrations of these compounds always inhibited the responses to acidic amino acids. Low concentrations of PhTX-343 and spermine also potentiated the responses to nicotine and gamma-aminobutyric acid. These results are discussed in terms of the postulated polyamine binding site on the N-methyl-D-aspartate receptor.

Polyamine Binding Sites in the Rat Brain Hippocampus Plasma Membranes: MK 801 Does Not Influence the Binding Process

The study was undertaken to characterize the polyamine binding sites in rat brain hippocampus plasma membranes. There were two types of binding sites for putrescine, Bmax 650 and 100 pmol/mg protein, with Kd1 = 39.2 and Kd2 = 6.7 microM, respectively, while those for spermidine (Spd) and spermine (Spm) represented only one type of population with Bmax 2.55 and 15 nmol/mg protein, respectively. The Kd values for Spd and Spm were 34 and 30.3 microM, respectively. The maximum binding of polyamines was found at pH 8.0. The binding capacity of these molecules was curtailed at 4 degrees C, indicating that the binding is an energy-dependent phenomenon. The specific binding was not appreciably influenced by the addition of MK 801, an antagonist of NMDA receptor, indicating that there are polyamine-specific binding sites that are different from those for MK 801. Glycine also did not significantly influence the binding of these biogenic amines. Interestingly, the addition of polyamino acids (polylysine, polyornithine, and polyglutamic acid) inhibited the polyamine binding to their receptor sites, supporting the notion that positive charge of polyamines could be important factor in the binding process.

Effects of a series of homologous α,ω‐dimethylaminoalkanes on cell proliferation: binding and uptake of putrescine by a human glioblastoma cell line (U251) in culture

The first step of polyamine uptake is the binding of polyamines to the cell membrane. In order to characterize the specificity of the putrescine binding sites at the surface of the glioblastoma cells (U251), we have carried out competition experiments between putrescine bound to latex microspheres and vizualized by scanning electron microscopy and a series of N,N'-tetramethyl-alpha,omega-diaminoalkanes. N,N'-tetramethyl-1,4-butanediamine (N,N'-tetramethylputrescine) and higher homologs inhibit the latex putrescine binding to the cell surface and concomitantly cell proliferation. [14C] putrescine uptake was mainly inhibited by the lower homologs, which were devoid of antiproliferative effects. Our results suggest that putrescine uptake by the human glioblastoma cell line U251, and putrescine binding to the surface of these cells are independent processes. The potential relationship between antitumor effect of N,N'-tetramethyl-alpha,omega-diaminoalkanes and its binding to a specific putrescine acceptor site is discussed.

Affinity chromatographic isolation of a major polyamine binding protein from serum and placenta, identified as serum albumin

Affinity chromatographic isolation of a major polyamine binding protein from serum and placenta, identified as serum albumin

New photoactivatable structural and affinity probes of RNAs: specific features and applications for mapping of spermine binding sites in yeast tRNAAspand interaction of this tRNA with yeast aspartyl-tRNA synthetase

Aryldiazonium salts are shown to be useful as phototriggered structural probes for RNA mapping as well as for footprinting of RNA/protein interaction. In particular the yeast tRNA(Asp)/aspartyl-tRNA synthetase complex is shown to involve the variable loop face and the concave side of the L-shaped nucleic acid bound to a lipophilic area of the enzyme. When chemically linked to spermine, the photoactive group cleaves RNA at polyamine binding sites; 3-4 spermines have been located in the tRNA(Asp), stabilizing the central part of the molecule in regions where two ribose-phosphate strands are close to each other.

Polyamine binding to proteins in oat and Petunia protoplasts.

Previous work (A Apelbaum et al. [1988] Plant Physiol 88: 996-998) has demonstrated binding of labeled spermidine (Spd) to a developmentally regulated 18 kilodalton protein in tobacco tissue cultures derived from thin surface layer explants. To assess the general importance of such Spd-protein complexes, we attempted bulk isolation from protoplasts of Petunia and oat (Avena sativa). In Petunia, as in tobacco, fed radioactive Spd is bound to protein, but in oat, Spd is first converted to 1,3,-diaminopropane (DAP), probably by polyamine oxidase action. In oat, binding of DAP to protein depends on age of donor leaf and conditions of illumination and temperature, and the extraction of the DAP-protein complex depends upon buffer and pH. The yield of the DAP-protein complex was maximized by extraction of frozen-thawed protoplasts with a pH 8.8 carbonate buffer containing SDS. Its molecular size, based on Sephacryl column fractionation of ammonium sulfate precipitated material, exceeded 45 kilodaltons. Bound Spd or DAP can be released from their complexes by the action of Pronase, but not DNAse, RNAse, or strong salt solutions, indicating covalent attachment to protein.

Uptake, intracellular binding, and excretion of polyamines during growth of Neurospora crassa

In Neurospora crassa mycelia, the amounts of the main polyamines, putrescine and spermidine, are approximately 0.8 and 18 nmol/mg, dry weight. We wished to know what determines these pool sizes. In the growth medium, externally added polyamines enter cells largely by a nonsaturable, diffusional system. In a mutant unable to synthesize polyamines, internal and external spermidine appear to equilibrate across the cell membrane during growth. However, this was true only after an intracellular “sink,” with a capacity equal to the amount of spermidine found in wild-type cells, had been saturated. We speculate that internal anionic binding sites, detectable in permeabilized cells, sequester virtually all of the spermidine normally found in exponentially growing N. crassa. Further evidence for this view was that in mature, stationary cultures, excess spermidine is excreted. Putrescine is also excreted if its concentration in the cell is abnormally high. The control of pool size by intracellular binding and excretion may be an advantage in this pathway, because feedback inhibition does not prevail, enzyme regulation is by comparison slow, and excessive polyamines are toxic.