Thermodynamic Study of Interactions of Distamycin A with Chromatin in Rat Liver Nuclei in the Presence of Polyamines.

We studied the thermodynamics of melting of isolated rat liver nuclei with different degrees of chromatin condensation determined by the concentration of polyamines (PA) and the solution ionic strength, as well as the effect of the antibiotic distamycin A (DM) on melting. Differential scanning calorimetry (DSC) profiles of nuclear preparations contained three peaks that reflected melting of three main chromatin domains. The number of peaks did not depend on the degree of condensation; however, nuclei with more condensed chromatin had a higher total enthalpy. DM stabilized peaks II and III corresponding to the melting of relaxed and topologically strained DNA, respectively, but destabilized peak I corresponding to the melting of nucleosome core histones. At the saturating concentration (DM/DNA molar ratio = 0.1), DM increased Tm of peaks II and III by ~5°C and decreased Tm of peak I by ~2.5°C. Based on the dependence of ΔH on DM concentration, we established that at low DM/DNA ratio (≤0.03), when DM interacted predominantly with AT-rich DNA regions, the enthalpy of peak II decreased in parallel with the increase in the enthalpy of peak III, which indicated that DM induces structural transitions in the nuclear chromatin associated with the increase in torsional stress in DNA. An increase in free energy under saturation conditions was equal to the change in the free energy of DM interaction with DNA. However, the increase in the enthalpy of melting of the nuclei in the presence of DM was much greater than the enthalpy of titration of nuclei with DM. This indicates a significant increase in the strength of interaction between the two DNA strands apparently due, among other things, to changes in the torsional stress of DNA in the nuclei. Titration of the nuclei with increasing PA concentrations resulted in the decrease in the number of DM-binding sites and the non-monotonous dependence of the enthalpy and entropy contribution to the binding free energy on the PA content. We suggested that the observed differences in the thermodynamic parameters were due to the different width of the minor groove in the nuclear chromatin DNA, which depends on PA concentration.

Extraction of histone H1 and decondensation of nuclear chromatin with various Mg-dependent organization levels under treatment with polyglutamic acid and distamycin.

Chromatin in rat liver nuclei under conditions of low ionic strength (20-25 mM) and [Mg2+] from 2 to 5 mM has a condensed structure (100-200 nm globules) and gives the same CD signal (320-340 nm) at interaction with the antibiotic distamycin A (DM). Reducing [Mg2+] to 1 mM leads to chromatin decondensation to 30 nm structures and increases the CD signal. Poly-L-glutamic acid (PG) at weight ratio PG/DNA = 6 and in the presence of 5 mM Mg2+ extracts only about 1/8 of nuclear histone H1, preserving a condensed chromatin structure. Removal of about 1/4 of H1 at 3 mM Mg2+ leads to chromatin decondensation to 30 nm fibrils. Extraction of about half of histone H1 at [Mg2+] ≤ 2 mM results in chromatin refolding to nucleosome fibrils. PG-decondensation leads to a significant increase in the CD signal. The main H1 extraction occurs in 1-2 min, but at all Mg2+ concentrations the more slowly PG extracted fraction is found comprising 5-7% of nuclear H1. About 25% of leaving nuclear H1 can be extracted by PG in the presence of saturating DM concentration (molar DM/DNA = 0.1). H1 release depends significantly on the PG concentration. However, even at high weight ratio PG/DNA = 30 and DM/DNA = 0.1, about 5-10% of histone H1 remained in the nuclei. Decondensation of chromatin in the nucleus is not always proportional to the yield of extracted histone H1 and is weakened in the presence of positively charged DM or high concentrations of PG. Our results show that the interaction of DM with chromatin depends primarily on chromatin packaging, while PG extraction depends on [Mg2+] supporting this packaging.

Modulation of action of wheat seedling endonucleases WEN1 and WEN2 by histones.

Wheat core histones and various subfractions of histone H1 modulate differently the action of endonucleases WEN1 and WEN2 from wheat seedlings. The character of this modulation depends on the nature of the histone and the methylation status of the substrate DNA. The modulation of enzyme action occurs at different stages of processive DNA hydrolysis and is accompanied by changes in the site specificity of the enzyme action. It seems that endonuclease WEN1 prefers to bind with protein-free DNA stretches in histone H1-DNA complex. The endonuclease WEN1 does not compete with histone H1/6 for DNA binding sites, but it does compete with histone H1/1, probably for binding with methylated sites of DNA. Unlike histone H1, the core histone H2b binds with endonuclease WEN1 and significantly increases its action. This is associated with changes in the site specificity of the enzyme action that is manifested by a significant increase in the amount of low molecular weight oligonucleotides and mononucleotides produced as a result of hydrolysis of DNA fragments with 120-140-bp length. The WEN2 endonuclease binds with histone-DNA complexes only through histones. The action of WEN2 is increased or decreased depending on the nature of the histone. Histone H1/1 stimulated the exonuclease activity of WEN2. It is supposed that endonucleases WEN1 and WEN2, in addition to the catalytic domain, should have a regulatory domain that is involved in binding of histones. As histone H1 is mainly located in the linker chromatin areas, it is suggested that WEN2 should attack DNA just in the chromatin linker zones. As differentiated from WEN2, DNA hydrolysis with endonuclease WEN1 is increased in the presence of core histones and, in particular, of H2b. Endonuclease WEN1 initially attacks different DNA sites in chromatin than WEN2. Endonuclease WEN2 activity can be increased or diminished depending on presence of histone H1 subfractions. It seems that just different fractions of the histone H1 are responsible for regulation of the stepwise DNA degradation by endonuclease WEN2 during apoptosis. Modulation of the action of the endonucleases by histones can play a significant role in the epigenetic regulation of various genetic processes and functional activity of genes.

Interaction of short peptides with FITC-labeled wheat histones and their complexes with deoxyribooligonucleotides.

Judging from fluorescence modulation (quenching), short peptides (Ala-Glu-Asp-Gly, Glu-Asp-Arg, Ala-Glu-Asp-Leu, Lys-Glu-Asp-Gly, Ala-Glu-Asp-Arg, and Lys-Glu-Asp-Trp) bind with FITC-labeled wheat histones H1, H2B, H3, and H4. This results from the interaction of the peptides with the N-terminal histone regions that contain respective and seemingly homologous peptide-binding motifs. Because homologous amino acid sequences in wheat core histones were not found, the peptides seem to bind with some core histone regions having specific conformational structure. Peptide binding with histones and histone-deoxyribooligonucleotide complexes depends on the nature of the histone and the primary structures of the peptides and oligonucleotides; thus, it is site specific. Histones H1 bind preferentially with single-stranded oligonucleotides by homologous sites in the C-terminal region of the protein. Unlike histone H1, the core histones bind predominantly with double-stranded methylated oligonucleotides and methylated DNA. Stern-Volmer constants of interaction of histone H1 and core histones with double-stranded hemimethylated oligonucleotides are higher compared with that of binding with unmethylated ones. DNA or deoxyribooligonucleotides in a complex with histones can enhance or inhibit peptide binding. It is suggested that site-specific interactions of short biologically active peptides with histone tails can serve in chromatin as control epigenetic mechanisms of regulation of gene activity and cellular differentiation.

Influence of chromatin structure, antibiotics, and endogenous histone methylation on phosphorylation of histones H1 and H3 in the presence of protein kinase A in rat liver nuclei in vitro.

In vitro phosphorylation of histones H1 and H3 by cAMP-dependent protein kinase A and endogenous phosphokinases in the presence of [γ-³²P]ATP was studied in isolated rat liver nuclei with different variants of chromatin structural organization: condensed (diameter of fibrils 100-200 nm; N-1) and partly decondensed (diameter of fibrils ~30 nm; N-2). In the N-1 state histone, H1 is phosphorylated approximately twice as much than histone H3. Upon the decondensation of the chromatin in the N-2 state, 1.5-fold decrease of total phosphorylation of H1 is observed, while that of H3 does not change, although the endogenous phosphorylation of both histones is reduced by half. Changes in histone phosphorylation in the presence of low or high concentrations of distamycin and chromomycin differ for H1 and H3 in N-1 and N-2. It was found that distamycin (DM) stimulates the phosphorylation of tightly bound H1 fraction, which is not extractable by polyglutamic acid (PG), especially in N-1. Chromomycin (CM) increases the phosphorylation of both histones in PG extracts and in the nuclear pellets, particularly in N-2. At the same time, in N-1 one can detect phosphorylation of a tightly bound fraction of histones H1 whose N-termini are located on AT-rich sites that become inaccessible for protein kinase in the process of chromatin decondensation in N-2. At the same time, in N-2 the accessibility for protein kinase A of tightly bound H1 fractions, whose N-termini are located on GC-rich sites, increases dramatically. High concentrations of both CM and DM in N-1 and N-2 stimulated phosphorylation of the non-extractable by PG fraction of H1 whose N-termini are located on sites where AT ≈ GC. CM at high concentration stimulated 4-7 times the phosphorylation of a small fraction of H3, which is extracted by PG from both types of nuclei. We detected an effect of endogenous methylation of histones H1 and H3 in the nuclei on their subsequent phosphorylation depending on the chromatin structure, histone-chromatin binding strength, and concentration of DM.

Evaluation of the electrical potential on the membrane of the extremely alkaliphilic bacterium Thioalkalivibrio.

The electrical potential on the membrane was measured in cells of strains AL2 and ALJ15 of the extremely alkaliphilic bacterium Thioalkalivibrio versutus using the penetrating cation tetraphenylphosphonium (TPP(+)) and a TPP(+)-selective electrode. The potentials were -228 ± 5 and -224 ± 5 mV, respectively, i.e. higher than in most alkaliphilic bacteria. Membrane potential in the cells was estimated by measuring the inner cell volume by two independent methods: (1) estimation of total cell volume by light microscopy and (2) estimation of the inner aqueous volume of the cells with allowance for the distribution difference of tritium labeled water penetrating through the membranes and a nonpenetrating colored protein. The inner cell volume was 2.4 ± 0.2 and 2.2 ± 0.1 µl/mg of cell protein by the two methods, respectively. Computer computation was used as an alternative to manual calculation to count the number of cells for estimation of total cell volume.

Oligopeptidase B from Serratia proteamaculans. III. Inhibition analysis. Specific interactions with metalloproteinase inhibitors.

Inhibition of the novel oligopeptidase B from Serratia proteamaculans (PSP) by basic pancreatic trypsin inhibitor, Zn2+ ions, and o- and m-phenanthroline was investigated. A pronounced effect of calcium ions on the interaction of PSP with inhibitors was demonstrated. Inversion voltamperometry and atomic absorption spectrometry revealed no zinc ions in the PSP molecule. Hydrophobic nature of the enzyme inhibition by o- and m-phenanthroline was established.

Influence of distamycin, chromomycin, and UV-irradiation on extraction of histone H1 from rat liver nuclei by polyglutamic acid.

Rat liver nucleus histone H1 was fractionated by polyglutamic acid (PG) in the presence of distamycin A (DM) or chromomycin A(3) (CM). In the absence of the antibiotics, PG extracts from the nuclei about half of the nuclear H1. DM or CM added to the nuclei in saturating concentrations weakens the binding potential of most of H1. Titration of nuclei with DM shows that the number of binding sites for DM in the nuclei is less than in isolated DNA by only 20-25%, and this difference disappears after treatment of nuclei with PG. The lower CD value of DM complexes with nuclei compared to that of DM complexes with free DNA is evidence of a change in the DM-DNA binding mode in nuclear chromatin. About 25% of total histone H1 is sensitive only to DM and ~5% is sensitive only to CM. Half of the DM-sensitive H1 fraction seems to have a different binding mode in condensed compared relaxed chromatin. A small part of H1 (~3%) remains tightly bound to the nuclear chromatin independent of the presence of the antibiotics. Subfraction H1A is more DM-sensitive and H1B is more CM-sensitive. UV irradiation of nuclei results in dose-dependent cross-linking of up to 50% of total H1, which is neither acid-extractable nor recovered during SDS electrophoresis. PG with DM extracts only about 3% of H1 from UV-stabilized chromatin. DM treatment of the nuclei before UV irradiation results in extraction of the whole DM-sensitive H1 fraction (~25%), which in this case is not stabilized in the nucleus. A hypothesis on possible roles of the found H1 fractions in chromatin structural organization is discussed.

H1 histone modulates DNA hydrolysis with WEN1 and WEN2 endonucleases from wheat coleoptiles.

We show that total H1 histone from wheat seedlings or rat liver enhances hydrolysis of lambda phage DNA with plant endonucleases WEN1 and WEN2 isolated from wheat coleoptiles. Optimal DNA/protein weight ratio in the hydrolysis reaction is 1 : 1. The action of fractions I and IV (obtained from total wheat H1 histone by electrophoresis) on DNA hydrolysis with WEN1 and WEN2 enzymes depends on the DNA methylation status. Fraction IV of wheat histone H1 stimulates hydrolysis of unmethylated lambda phage DNA with WEN1 and WEN2 enzymes. Hydrolysis of methylated lambda phage DNA (it contains 5-methylcytosine in Cm(5)CWGG sequences and N(6)-methyladenine in Gm(6)ATC sites) with WEN1 is inhibited with fractions I and IV of wheat H1 histone. Fractions II and III of wheat H1 histone do not influence DNA hydrolysis with WEN1 and WEN2. S-Adenosyl-L-methionine (SAM) stimulates activity of these plant enzymes. But in the presence of H1 histone, SAM does not add to the ability of the enzyme to hydrolyze more DNA compared with that induced with H1 histone itself. Therefore, the stimulating effects of SAM and H1 histone on DNA hydrolysis with plant endonucleases may be similar. It could be suggested that SAM and H1 histone can induce more or less analogous allosteric transformations in the structure of the investigated plant endonucleases. Thus, DNA hydrolysis with plant endonucleases is modulated with total H1 histone. H1 histone fractions affect DNA hydrolysis in a different fashion; they enhance or inhibit hydrolysis depending on the DNA methylation status. We suggest that H1 histone changes site specificity of endonucleases or it might be responsible for formation of new or masking of old sites available for these enzymes due to changes in DNA structure induced in a DNA-histone complex.

H1 histone in developing and aging coleoptiles of etiolated wheat seedlings.

It has been established that the DNA and H1 histone contents in aged coleoptile of 8-day-old etiolated wheat seedling are about 40 and 30%, respectively, lower than those in young seedlings. H1 histone in wheat seedlings is represented as six electrophoretically different subfractions. The ratios of H1 histone subfractions in wheat coleoptile and initial leaf are similar. In contrast to some animal cells, apoptosis in wheat coleoptile is not accompanied by changes in the set and ratios of H1 histone subfractions. Aging of coleoptiles is associated with a progressive diminution of the H1 histone and DNA contents. H1 histone/DNA ratio in aged coleoptile is 1.5-2-fold higher than that in the young organs. Therefore, the content of H1 histone in chromatin of coleoptile decreases with age more slowly than DNA content.