Bimodal action of spermine on ribosomal peptidyltransferase at low concentration of magnesium ions

Drainas, Denis; Kalpaxis, Dimitrios L.

doi:10.1016/0167-4838(94)90159-7

Cited by 18 publications

(30 citation statements)

References 34 publications

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“…For example, SP (50 µM) stimulates by 3-4-fold, hydroxylation of benzphetamine and 7-ethoxycoumarin by liver microsomes [Andersson et al, 1981], and, at 1 mM, stimulates the oxidation of certain xenobiotics [Osimitz and Kulkarni, 1985]. Low concentrations (100 µM) of PA stimulate, and high concentrations (1 mM) inhibit, ribosomal peptidyltransferase [Drainas and Kalpaxis, 1994]. The K d /K s /K i values for HA and PA determined in the spectral and radioligand binding assays reported in the present study may represent a composite of interactions with multiple P450 isozymes.…”

Section: Discussionmentioning

confidence: 99%

Potent interaction of histamine and polyamines at microsomal cytochrome P450, nuclei, and chromatin from rat hepatocytes

1998

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Histamine and polyamines have been implicated in the mediation of cell proliferation. Our previous work linked the growth-modulatory effects of histamine with its binding to intracellular sites in microsomes and nuclei of various tissues. In this study, we identify cytochrome P450 enzymes as a major component of microsomal intracellular sites in hepatocytes and demonstrate that polyamines compete with high affinity for histamine binding to them. Spectral measurement of histamine binding to P450 in liver microsomes resolved high and intermediate affinity binding sites (Ks1 = 2.4 +/- 1.6 microM; Ks2 = 90 +/- 17 microM) that corresponded to microsomal binding sites (Kd1 = 1.0 +/- 0.9 microM; Kd2 = 57 +/- 13 microM) resolved by 3H-histamine binding; additional low affinity (Kd3 approximately 3 mM), and probably physiologically irrelevant, sites were resolved only by 3H-histamine radioligand studies. As determined spectrally, treatment of microsomes with NADPH/carbon monoxide decreased histamine binding to P450 by about 90% and, as determined by 3H-histamine binding, abolished the high affinity sites and reduced by 85% the number of intermediate sites. Spermine competed potently for 3H-histamine binding: in microsomes, Ki = 9.8 +/- 5.8 microM; in nuclei, Ki = 13.7 +/- 3.1 microM; in chromatin, Ki = 46 +/- 33 nM. Polyamines inhibited the P450/histamine absorbance complex with the rank order of potency: spermine > spermidine >> putrescine. In contrast, histamine did not compete for 3H-spermidine binding in nuclei or microsomes, suggesting that polyamines modulate histamine binding allosterically. We propose that certain P450 isozymes that modulate gene function by controlling the level of oxygenated lipids, represent at least one common intracellular target of growth-regulatory endogenous bioamines and, as shown previously, of exogenous growth-modulatory drugs including antiestrogens, antiandrogens, and certain antidepressants and antihistamines.

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Section: Discussionmentioning

confidence: 99%

Potent interaction of histamine and polyamines at microsomal cytochrome P450, nuclei, and chromatin from rat hepatocytes

1998

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“…Samples were then cooled to 0°C and the Mg 2+ concentration was normalized to 6 mM. Ac[ 3 H]Phe-tRNA was prepared from heterogeneous E.coli tRNA, as described elsewhere (28). It was charged to 77%, 100% being 28 pmol of [ 3 H]Phe per A 260 U (Sigma).…”

Section: Methodsmentioning

confidence: 99%

“…Also, it has been indicated by immuno-electron microscopy that polyamines are predominantly located in both free and attached ribosomes of the rough endoplasmic reticulum in mammalian cells (27). We have previously demonstrated in a cell-free system derived from E.coli that spermine not only promotes the formation and stabilization of the initiator ribosomal complex at 6 mM Mg 2+ , but also causes a concentration-dependent allosteric biphasic effect on PTase activity; at low concentrations (<100 μM) stimulates the PTase activity, whereas at higher concentrations (>200 μM) causes partial noncompetitive inhibition (28). Recently, we found that the inhibition effect is at least partially due to a perturbation by spermine of the functional conformation of the donor substrate (29).…”

Section: Introductionmentioning

confidence: 99%

Localization of spermine binding sites in 23S rRNA by photoaffinity labeling: parsing the spermine contribution to ribosomal 50S subunit functions

Xaplanteri

Πετρόπουλος

Dinos

et al. 2005

Nucleic Acids Research

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Polyamine binding to 23S rRNA was investigated, using a photoaffinity labeling approach. This was based on the covalent binding of a photoreactive analog of spermine, N1-azidobenzamidino (ABA)-spermine, to Escherichia coli ribosomes or naked 23S rRNA under mild irradiation conditions. The cross-linking sites of ABA-spermine in 23S rRNA were determined by RNase H digestion and primer-extension analysis. Domains I, II, IV and V in naked 23S rRNA were identified as discrete regions of preferred cross-linking. When 50S ribosomal subunits were targeted, the interaction of the photoprobe with the above 23S rRNA domains was elevated, except for helix H38 in domain II whose susceptibility to cross-linking was greatly reduced. In addition, cross-linking sites were identified in domains III and VI. Association of 30S with 50S subunits, poly(U), tRNAPhe and AcPhe-tRNA to form a post-translocation complex further altered the cross-linking, in particular to helices H11–H13, H21, H63, H80, H84, H90 and H97. Poly(U)-programmed 70S ribosomes, reconstituted from photolabeled 50S subunits and untreated 30S subunits, bound AcPhe-tRNA in a similar fashion to native ribosomes. However, they exhibited higher reactivity toward puromycin and enhanced tRNA-translocation efficiency. These results suggest an essential role for polyamines in the structural and functional integrity of the large ribosomal subunit.

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“…Complex C, i.e. the Ac[ 3 H]Phe-tRNA⅐poly(U)⅐70 S ribosome complex, was prepared in buffer A (100 mM Tris/HCl, pH 7.2, 6 mM magnesium acetate, 100 mM NH 4 Cl, and 6 mM 2-mercaptoethanol) containing partially purified translation factors and separated from excess Ac[ 3 H]Phe-tRNA as reported previously (21). When desired, 100 M spermine or 50 M spermine and 2 mM spermidine were also included in buffer A.…”

Section: Methodsmentioning

confidence: 99%

Polyamines Affect Diversely the Antibiotic Potency

Πετρόπουλος

Xaplanteri

Dinos

et al. 2004

Journal of Biological Chemistry

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The effects of spermine on peptidyltransferase inhibition by an aminohexosylcytosine nucleoside, blasticidin S, and by a macrolide, spiramycin, were investigated in a model system derived from Escherichia coli, in which a peptide bond is formed between puromycin and AcPhe-tRNA bound at the P-site of poly(U)-programmed ribosomes. Kinetics revealed that blasticidin S, after a transient phase of interference with the A-site, is slowly accommodated near to the P-site so that peptide bond is still formed but with a lower catalytic rate constant. At high concentrations of blasticidin S (>10 ؋ K i ), a second drug molecule binds to a weaker binding site on ribosomes, and this may account for the onset of a subsequent mixed-noncompetitive inhibition phase. Spermine enhances the blasticidin S inhibitory effect by facilitating the drug accommodation to both sites. On the other hand, spiramycin (A) was found competing with puromycin for the A-site of AcPhe-tRNA⅐poly(U)⅐70 S ribosomal complex (C) via a two-step mechanism, according to which the fast formation of the encounter complex CA is followed by a slow isomerization to a tighter complex, termed C*A. In contrast to that observed with blasticidin S, spermine reduced spiramycin potency by decreasing the formation and stability of complex C*A. Polyamine effects on drug binding were more pronounced when a mixture of spermine and spermidine was used, instead of spermine alone. Our kinetic results correlate well with cross-linking and crystallographic data and suggest that polyamines bound at the vicinity of the antibiotic binding pockets modulate diversely the interaction of these drugs with ribosomes.Blasticidin S and spiramycin are representatives of two distinct antibiotic families, the aminohexosylcytosine nucleosides and the 16-membered lactone ring macrolides, respectively, both of which have been proposed to inhibit protein synthesis by binding to the large ribosomal subunit. Blasticidin S consists of a cytosine bonded to a pyranose ring, to which an amino acid-like substituent is attached (see Fig. 1). Therefore, it is not surprising that blasticidin S and puromycin have been considered as iso-structural, both with one another and with the 3Ј-end of aminoacyl-tRNA (1, 2). Consistently, blasticidin S has been found to inhibit the binding of CACCA (Phe) to the A-site of ribosomes (3) and to compete with designated A-site inhibitors (4). In addition, the inhibition of peptidyl-or AcPhe 1 -puromycin synthesis by this antibiotic shows a competitive phase in concert with noncompetitive or mixed-noncompetitive phases (5, 6), a fact supporting the notion that blasticidin S influences, at least transiently, the affinity of the A-site. Further support derives from chemical probing studies in Escherichia coli ribosomes (7), suggesting that blasticidin S protects A2439, one of the three nucleosides in domain V of 23 S rRNA, which show altered chemical reactivity on removal of the aminoacyl group from A-site-bound tRNAs (8). Moreover, A2439 is one of the preferable cross-linking...

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Bimodal action of spermine on ribosomal peptidyltransferase at low concentration of magnesium ions

Cited by 18 publications

References 34 publications

Potent interaction of histamine and polyamines at microsomal cytochrome P450, nuclei, and chromatin from rat hepatocytes

Potent interaction of histamine and polyamines at microsomal cytochrome P450, nuclei, and chromatin from rat hepatocytes

Localization of spermine binding sites in 23S rRNA by photoaffinity labeling: parsing the spermine contribution to ribosomal 50S subunit functions

Polyamines Affect Diversely the Antibiotic Potency

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