Effects of polyamines on mitochondrial Ca(2+) transport

Mammalian mitochondria are able to enhance Ca(2+) accumulation in the presence of polyamines by activating the saturable systems of Ca(2+) inward transport and buffering extramitochondrial Ca(2+) concentrations to levels similar to those in the cytosol of resting cells. This effect renders them responsive to regulate free Ca(2+) concentrations in the physioloical range. The mechanism involved is due to a rise in the affinity of the Ca(2+) transport system, induced by polyamines, most probably exhibiting allosteric behaviour. The regulatory site of this mechanism is the so-called S(1) binding site of polyamines, which operates in physiological conditions and is located in the energy well between the two peaks present in the energy profile of mitochondrial spermine transport. Spermine is bidirectionally transported across teh inner membrane by cycling, in which influx and efflux are driven by electrical and pH gradients, respectively. Most probably, polyamine affects the Ca(2+) transport system when it acts from the outside-that is, in the direction of its uniporter channel, in order to reach the S(1) site. Important physiological functions are related to activation of Ca(2+) transport systems by polyamines and their interactions with the S(1) site. These functions include a rise in the metabolic rate for energy supply and modulation of mitochondrial permeability transition induction, with consequent effects on the triggering of the apoptotic pathway.     

Phosphorylation of recombinant human spermidine/spermine N(1)-acetyltransferase by CK1 and modulation of its binding to mitochondria: a comparison with CK2.

Cytosolic spermidine/spermine acetyltransferase (SSAT) catalyzes the acetylation of the N(1)-propylamino groups of spermine and spermidine. The enzyme has a very short half-life and is rapidly induced by various stimuli. Once acetylated, these polyamines are subjected to the action of polyamine oxidase, which, besides initiating polyamine catabolism, may produce reactive oxygen species that in turn trigger modifications in subcellular compartments such as mitochondria. The present work evaluates the ability of the cAMP-independent Ser/Thr-protein kinase CK1 to phosphorylate SSAT. Results demonstrate that SSAT is phosphorylated by CK1, in sites distinct from those phosphorylated by CK2. Moreover, both phosphorylation processes are involved in the uptake of SSAT into rat liver mitochondria. Although CK2 is less effective than CK1 in phosphorylating SSAT, CK2 phosphorylation is much more powerful in preventing binding of SSAT to mitochondrial structures. These results suggest the involvement of CK1- and CK2-mediated SSAT phosphorylation in regulating the contents of polyamines and SSAT itself within subcellular compartments and implicate SSAT and polyamines as indirect modulators of progression through the cell cycle.     

Binding of spermidine and putrescine to energized liver mitochondria.

The binding of spermidine and putrescine to mitochondrial membranes was studied by applying a thermodynamic model of ligand-receptor interactions developed both for equilibrium and far-from-equilibrium binding processes (V. Di Noto, L. Dalla Via, A. Toninello, and M. Vidali Macromol. Theory Simul. 5, 165-181, 1996). Results demonstrate the presence of two monocoordinated binding sites (S1 and S2) for spermidine and one monocoordinated binding site (S2) for putrescine, all exhibiting high capacity and low affinity. It is proposed that differences in the polyamines' flexibility and hydrophilicity perhaps contributes to the observed variations in their interactions with the two sites. A comparison of the binding parameters of these polyamines with those of spermine reveals differences in the specific function of the S1 and S2 sites, identified in studies of spermine binding (L. Dalla Via, V. Di Noto, D. Siliprandi, and A. Toninello Biochim. Biophys. Acta 1284, 247-252, 1996).     

Effects of polyamines on mitochondrial Ca transport

Mammalian mitochondria are able to enhance Ca²⁺ accumulation in the presence of polyamines by activating the saturable systems of Ca²⁺ inward transport and buffering extramitochondrial Ca²⁺ concentrations to levels similar to those in the cytosol of resting cells. This effect renders them responsive to regulate free Ca²⁺ concentrations in the physioloical range. The mechanism involved is due to a rise in the affinity of the Ca²⁺ transport system, induced by polyamines, most probably exhibiting allosteric behaviour. The regulatory site of this mechanism is the so-called S₁ binding site of polyamines, which operates in physiological conditions and is located in the energy well between the two peaks present in the energy profile of mitochondrial spermine transport. Spermine is bidirectionally transported across teh inner membrane by cycling, in which influx and efflux are driven by electrical and pH gradients, respectively. Most probably, polyamine affects the Ca²⁺ transport system when it acts from the outside-that is, in the direction of its uniporter channel, in order to reach the S₁ site. Important physiological functions are related to activation of Ca²⁺ transport systems by polyamines and their interactions with the S₁ site. These functions include a rise in the metabolic rate for energy supply and modulation of mitochondrial permeability transition induction, with consequent effects on the triggering of the apoptotic pathway.     

Electrophoretic polyamine transport in rat liver mitochondria

Summary Naturally occurring polyamines (spermidine, putrescine, cadaverine), as the well studied spermine, are transported into rat liver mitochondrial matrix provided that mitochondria are energized and the electrical membrane potential has a value of about 180 mV. This condition is achieved by the presence of inorganic phosphate, or acetate, or nigericin in the incubation medium. Valinomycin plus K⁺ almost completely blocks polyamine transport.The obtained results clearly show that all naturally occurring polyamines are transported by an electrophoretic mechanism in responce to a high negative inner electrical potential.The distribution ratio of polyamines across the mitochondrial membrane is far from the thermodynamic equilibrium by many orders of magnitude. This result might suggest the existence of a different pathway for polyamine efflux.     

The covalent attachment of polyamines to proteins in plant mitochondria.

Plant mitochondria from both potato and mung bean incorporated radioactivity into acid insoluble material when incubated with labelled polyamines (spermine, spermidine and putrescine). Extensive washing of mitochondrial precipitates with trichloroacetic acid and the excess of cold polyamine failed to remove bound radioactivity. Addition of nonradioactive polyamine stopped further incorporation of radioactivity but did not release radioactivity already bound. The radioactivity is incorporated into the membrane fraction. The labelling process has all the features of an enzymatic reaction: it is long lasting with distinctive kinetics peculiar to each polyamine, it is temperature dependent and is affected by N-ethylmaleimide. The latter inhibits the incorporation of putrescine but stimulates the incorporation of spermine and spermidine. Treatment of prelabelled mitochondria with pepsin releases bound radioactivity thus indicating protein to be the ligand for the attachment of polyamines. HPLC of mitochondrial hydrolysates revealed that the radioactivity bound to mitochondria is polyamines; traces of acetyl polyamines were also found in some samples. On autoradiograms of SDS/PAGE gels several radioactive bands of proteins were detected. Protein sequencing of labelled spots from a 2D gel gave a sequence which was 60% identical to catalase. We suggest that the attachment of polyamines to mitochondrial proteins occurs cotranslationally possibly via transglutaminases.     

Effect of 1,3,6-triaminohexane and 1,4,7-triaminoheptane on growth and polyamine metabolism in SV-3T3 cells treated with 2-difluoromethylornithine

The possibility that one or both of the synthetic triamines, 1,3,6-triaminohexane and 1,4,7-triaminoheptane, could substitute for the naturally occurring polyamines in the growth of SV-3T3 cells was investigated. It was found that these triamines did lead to a restoration of growth in cells in which spermidine content had been depleted by exposure to the ornithine decarboxylase inhibitor 2-difluoromethylornithine. This resumption of a normal growth rate occurred prior to the reduction in the content of cellular decarboxylated S-adenosylmethionine, suggesting that this nucleoside (which increases in concentration several hundred-fold in cells treated with 2-difluoromethylornithine) does not cause the reduction of cell growth. However, unlike the increase in cell growth brought about by spermidine, which continued indefinitely, the increase produced by 1,3,6-triaminohexane or 1,4,7-triaminoheptane was transient. Cell growth in the presence of 2-difluoromethylornithine and these triamines stopped after about three or four population doublings. This corresponded to the time at which the intracellular spermine content of the cells was reduced to values less than 20% of normal. It is suggested that the increased growth rate of spermidine-depleted cells in response to these triamines is due to their uptake into the cell and ability to displace spermine from intracellular sites, thus making spermine available to fulfill the polyamine function(s) essential for growth. These results indicate that the naturally occurring polyamines spermidine or spermine are essential for continued cell growth and cannot be replaced by analogues containing only primary amino groups.     

Interaction of polyamines with chromatin and DNA: formation of compact structures

We have used flow linear dichroism (LD) and light scattering at 90 degrees to study the condensation of both DNA and calf thymus chromatin induced by spermine, triamines NH3+(CH2)iNH+(CH2)jNH3+, designated as much less than i, j much greater than: much less than 3, 4 much greater than (spermidine), much less than 3, 3 much greater than, much less than 2, 3 much greater than, much less than 2, 2 much greater than; the diamines putrescine and cadaverine and MgCl2. It is found that the different polyamines affected DNA and chromatin in a similar way. The degree of compaction of the chromatin fibers induced by spermine, triamines except much less than 2, 2 much greater than and Mg2+ has been found to be identical. The triamine much less than 2, 2 much greater than and the diamines studied do not condense either chromatin of DNA. Such a big difference in the action of the triamines indicates that not only the charge, but also the structure of the polycations are important for their interactions with DNA and chromatin. The stoichiometry of polyamine binding to chromatin at which condensation occurred is found to be 2 polyamine molecules per DNA helical turn. Polyamines are supposed to bind to the exposed sites of core DNA every 10 b.p. The extent of DNA phosphate neutralization by the histones is estimated to be about 55%. It has been shown that a mixture of mono- and multivalent cations affected DNA and chromatin condensation competitively and not synergistically, as claimed in a recent report by Sen and Crothers.     

Polyamines are sufficient to drive the transport of the precursor of ornithine carbamoyltransferase into rat liver mitochondria: possible effect on mitochondrial membranes

The polyamines spermidine, spermine and putrescine, by themselves, at physiological concentrations, induce the transport of the precursor of ornithine carbamoyltransferase into isolated rat liver mitochondria. The presence of polyamines in the transport medium results in the approach of both mitochondrial membranes, suggesting a possible role of these molecules in the transport of the precursor of ornithine carbamoyltransferase into mitochondria, by the formation and/or stabilization of mitochondrial structures involved in the transport system.     

Raman study of the interaction between polyamines and a GC oligonucleotide.

The interaction between the oligonucleotide d[G(CG)(7)]. d[C(GC)(7)] and the three biogenic polyamines putrescine, spermidine, and spermine under physiological conditions has been studied by Raman spectroscopy. The results indicate the formation of highly ordered aggregated structures in solution, largely stabilized by electrostatic attractions, which have been described as cholesteric phases. Aggregation seems to be preceded by a partial B --> Z conformational transition for spermidine and spermine, which would allow for a deeper oligonucleotide-polyamine interaction. Interaction with the nucleic bases has also been evidenced for aggregates. At low polyamine concentrations the preferential binding sites are similar to those proposed for their interactions with ct-DNA. With increasing the polyamine concentration, the oligonucleotide-polyamine interactions involve both minor and major grooves, which is consistent with the formation of cholesteric phases.     

Spermidine Uptake by Mitochondria of Helianthus tuberosus

In the present work evidence is provided that spermidine, a polyamine largely present in plant tissues, may be transported, at physiological concentrations, into the matrix space of mitochondria isolated from tubers of Helianthus tuberosus L. cv OB1 (Jerusalem artichoke). It is concluded that the movement of spermidine strictly depends on membrane potential, since it is drastically blocked by valinomycin and only slightly sensitive to nigericin. Mg²⁺ and K⁺ inhibit the transport of spermidine in line with the general concept that these cations compete for the same binding sites on the mitochondrial membrane. In contrast to previous data on mammalian mitochondria, spermidine uptake by plant mitochondria does not depend on the presence of inorganic phosphate. This latter result, along with evidence that Ca²⁺ does not affect accumulation of spermidine, indicates that the control of the polyamine uptake mechanism in plant mitochondria is distinct from that of mammalian systems.     

Uptake of spermine by rat liver mitochondria and its influence on the transport of phosphate

Spermine, a polyamine present in the mammalian cells at rather high concentration, has, among other actions, a remarkable stabilizing effect on mitochondria, functions which have generally been attributed to the capability of this and other polyamines to bind to membrane anionic sites. In the present paper evidence is provided that at physiological concentrations spermine may also be transported into rat liver mitochondrial matrix space, provided that mitochondria are energized and inorganic phosphate is simultaneously transported. The close dependence of spermine transport is also demonstrated by the concurrent efflux of spermine and inorganic phosphate when mitochondria preloaded with the two ionic species are deenergized either with uncouplers or respiratory chain inhibitors. Furthermore, Mersalyl, the known inhibitor of phosphate transport, prevents both spermine uptake and release. Mg2+ inhibits the transport of spermine conceivably by competing for the some binding sites on the mitochondrial membrane. The physiological significance of these results is discussed.     

Polyamine-DNA interactions. Condensation of chromatin and naked DNA

We have used flow linear dichroism (LD) and light scattering at 90 degrees to study the condensation of both DNA and calf thymus chromatin by polyamines, such as spermine, spermidine and its analogs designated by formula NH3+(CH2)iNH2+(CH2)jNH3+, where i = 2,3 and j = 2,3, putrescine, cadaverine and MgCl2. It has been found that the different polyamines affect DNA and chromatin in a similar way. The level of compaction of the chromatin fibers induced by spermine, spermidine and the triamines NH3+(CH2)3NH2+(CH2)3NH3+ and NH3+(CH2)3NH2+(CH2)2NH3+ and MgCl2 is found to be identical. The triamine NH3+(CH2)3NH2+(CH2)2NH3+ and the diamines studied condense neither chromatin nor DNA. This drastic difference in the action of the triamines indicates that not only the charge, but also the structure of the polycations might play essential roles in their interactions with DNA and chromatin. It is shown that a mixture of mono- and multivalent cations affect DNA and chromatin condensation competitively, but not synergistically, as claimed in a recent report by Sen and Crothers (Biochemistry 25, 1495-1503, 1986). We have also estimated the extent of negative charge neutralization produced by some of the polyamines on their binding to chromatin fibers. The stoichiometry of polyamine binding at which condensation of chromatin is completed is found to be two polyamine molecules per DNA turn. The extent of neutralization of the DNA phosphates by the histones in these compact fibers is estimated to be about 55%. The model of polyamine interaction with chromatin is discussed.     

Interaction of polyamines with fragments and whole molecules of yeast phenylalanine-specific transfer RNA

Binding of the polyamines spermidine (∼-+3) and spermine (∼-+4) to yeast tRNA^phe has been investigated by equilibrium dialysis under the same conditions used to study Mn²⁺-tRNA^phe interactions (Schreier & Schimmel, 1974). The polyamines bind to tRNA^phe in a co-operative and a non-co-operative phase, which is analogous to the behavior found with Mn²⁺. In the co-operative phase, the empirical index of co-operativity is somewhat greater for the polyamines, however. Binding constants for both the co-operative and non-co-operative phases are similar for Mn²⁺ and spermidine, and are strongest for spermine. Estimates of the total number of ligand binding sites indicate that these numbers are inversely proportional to the charge on the ligand for all three ligands. The interaction of polyamines with four large fragments of tRNA^phe shows no evidence for co-operativity. These results, together with recent kinetic studies, collectively suggest that polyamine binding to the co-operative sites is associated with tertiary structure formation and that polyamine and divalent metal ion interactions with tRNA occur by phenomenologically similar mechanisms, in spite of their structural diversity.     

Modulation of the NMDA receptor by polyamines.

Results of recent biochemical and electrophysiological studies have suggested that a recognition site for polyamines exists as part of the NMDA receptor complex. This site appears to be distinct from previously described binding sites for glutamate, glycine, Mg++,Zn++, and open-channel blockers such as MK-801. The endogenous polyamines spermine and spermidine increase the binding of open-channel blockers and increase NMDA-elicited currents in cultured neurons. These polyamines have been termed agonists at the polyamine recognition site. Studies of the effects of natural and synthetic polyamines on the binding of [3H]MK-801 and on NMDA-elicited currents in cultured neurons have led to the identification of compounds classified as partial agonists, antagonists, and inverse agonists at the polyamine recognition site. Polyamines have also been found to affect the binding of ligands to the recognition sites for glutamate and glycine. However, these effects may be mediated at a site distinct from that at which polyamines act to modulate the binding of open-channel blockers. Endogenous polyamines may modulate excitatory synaptic transmission by acting at the polyamine recognition site of the NMDA receptor. This site could represent a novel therapeutic target for the treatment of ischemia-induced neurotoxicity, epilepsy, and neurodegenerative diseases.     

Do mammalian amine oxidases and the mitochondrial polyamine transporter have similar protein structures?

Polyamine transport across the mitochondria membrane occurs by a specific, common uniporter system and appears controlled by electrostatic interactions as for polyamine oxidative deamination by bovine serum and mitochondrial matrix amine oxidases was found. In fact in all the cases, while the catalytic constants or the maximum uptake rate values show little changes with the number of the positive charges of the substrates, Michaelis–Menten constant values demonstrate exponential dependence, confirming that electrostatic forces control the docking of the substrate into the enzyme active site or polyamine channel. By the treatment of the kinetic data in terms of Gibbs equation or Eyring theory, the contribution of each positive charge of the polyamine to the Gibbs energy values for the oxidative deamination of polyamines by two mammalian amine oxidase and for polyamine transport, are obtained. These values were comparable and in good accordance with those reported in literature. Previous studies demonstrated that two negative functional groups in the active site of bovine serum and mitochondrial matrix amine oxidases interact electrostatically with three positive charges of the polyamines in the formation of the enzyme–substrate complex. Remembering the structure–function relationship of proteins, our results suggest analogous interactions in the polyamine transporter and, as a consequence, a partial structural similitude between two proteins. It follows that the primary sequences of the amino oxidases and the mitochondrial transport may, in part, be conserved.     

Neomycin Is an Agonist at a Polyamine Site on the N-Methyl-D-Aspartate Receptor

Neomycin appears as a full agonist and spermidine as a partial agonist at the site where polyamines enhance 1-[1-(2-thienyl)cyclohexyl][3H]piperidine ([3H]TCP) binding on the N-methyl-D-aspartate (NMDA) receptor. Other aminoglycosides also enhance [3H]TCP binding with efficacies roughly proportional to the number of primary amine groups. The polyamine antagonists ifenprodil and arcaine inhibit enhancement of [3H]TCP binding by spermidine or neomycin. The inhibition of [3H]TCP binding by arcaine is apparently competitively reduced by neomycin and spermidine, supporting a common site. Diethylenetriamine (previously described as a polyamine antagonist) may be a partial agonist. Enhancement by neomycin or spermidine is not additive to that of Mg2+, consistent with competition of Mg2+ and spermidine or neomycin at the site where these compounds enhance [3H]TCP binding. Polyamines also enhance the binding of the competitive antagonist 2-(2-carboxypiperazin-4-yl)[3H]propyl-1-phosphonic acid ([3H]CPP). Neomycin, which does not enhance [3H]CPP binding, inhibits the enhancement by spermidine. That this site is distinct from the site where spermidine and neomycin increase [3H]TCP binding is supported by different pharmacology. Arcaine and diethylenetriamine do not inhibit spermidine enhancement of [3H]CPP binding. Mg2+ also does not compete with the spermidine enhancement of [3H]CPP binding. Ifenprodil inhibits the spermidine enhancement of [3H]CPP binding. The data suggest two or more polyamine sites, with arcaine selective for the site that enhances [3H]TCP binding. Neomycin is an agonist at one polyamine site and antagonist to the second.     

Binding sites of the polyamines putrescine, cadaverine, spermidine and spermine on A- and B-DNA located by simulated annealing

Molecular dynamics simulations with simulated annealing are performed on polyamine-DNA systems in order to determine the binding sites of putrescine, cadaverine, spermidine and spermine on A- and B-DNA. The simulations either contain no additional counterions or sufficient Na+ ions, together with the charge on the polyamine, to provide 73% neutralisation of the charges on the DNA phosphates. The stabilisation energies of the complexes indicate that all four polyamines should stabilise A-DNA in preference to B-DNA, which is in agreement with experiment in the case of spermine and spermidine, but not in the case of putrescine or cadaverine. The major groove is the preferred binding site on A-DNA of all the polyamines. Putrescine and cadaverine tend to bind to the sugar-phosphate backbone of B-DNA, whereas spermidine and spermine occupy more varied sites, including binding along the backbone and bridging both the major and minor grooves.     

POLYAMINE TRANSPORT IN THE SEAWEED ULVA RIGIDA (CHLOROPHYTA)1

The excessive growth of Ulva rigida C. Agardh, a green seaweed present in the Northern Adriatic Sea, is a problem for the inhabitants and the economy of the region. As information about hormonal control of growth in seaweeds is scarce, our aim was to investigate the presence of endogenous polyamines and their absorption by algal cells and to correlate the findings with terrestrial plants. Free polyamines (putrescine, spermidine, and spermine) were present endogenously in the algal thallus at concentrations ranging from 4 to 134 μM. Putrescine and spermidine were also present in the seawater in which the alga usually grows at concentrations between 0 and 0.9 μM. Uptake of labeled polyamines occurred, but it was inhibited by cations present in the seawater. Uptake was investigated also by incubation in distilled water. In this case, uptake displayed characteristics similar to those observed in higher plant systems. Uptake studies in seawater showed that polyamine accumulation in algal cells occurred and that it followed a concentration gradient and displayed linear kinetics. The mechanism proposed that of a passive uptake, as indicated also by the inability of metabolic inhibitors to block transport. There was evidence for polyamine binding to external cell sites, but polyamine uptake by protoplasts as well as polyamine translocation and secretion by the whole thallus was also demonstrated. Since cultured and actively growing thallus discs displayed a higher uptake ability than freshly collected ones, a role for polyamines in sustaining growth is discussed.