Condensation and precipitation of chromatin by multivalent cations.

The condensation and the precipitation of rat liver chromatin upon addition of spermine4+, spermidine3+, hexamminecobalt(III)3+ and Mg2+ cations have been studied using solubility, fluorescence, circular dichroism, melting curves, electric dichroism and spermidine binding measurements, made on both soluble and precipitated complexes. The soluble complexes obtained with tetra- and trivalent cations were depleted from all histones and enriched in other proteins, particularly high mobility group proteins 1 and 2, which brings about an important enhancement of tryptophan fluorescence without modification of its two lifetimes 5.1 and 1.2 ns. In the precipitates the non-histone proteins are eliminated. Under precipitation by Mg2+ ions, the distribution of proteins remains practically unchanged. The electric dichroism and the melting curves indicate that the soluble complexes between polyamines and chromatin undergo important condensation and, at high ratios of cation over phosphate, are constituted by heterogeneous assemblies of non-histone proteins and DNA. On the contrary, the insoluble complexes seem to retain the main features of original chromatin. Precipitation by Mg2+ ions reveal much less drastic changes than those produced by polyamines. Precipitation by spermidine occurs when one cation is bound per eight nucleotides, which in addition to the histone positive charges brings about a complete neutralization of chromatin phosphates.

Effect of various 6-dehydro-corticosteroids, 9, 11-dehydro-DOCA and 9 alpha-fluoro-DOCA on the fluxes of sodium and potassium.

The introduction of a double bond at carbons 6 and 7 (6-dehydro-derivatives) of deoxycorticosterone acetate (DOCA), cortisol-21-acetate, 9 alpha-fluorocortisol-21-acetate (9 alpha-F-C-ac) and aldosterone-21-acetate substantially reduces affinity for Type II receptors but not for Type I receptors. Such a modification changes the effect of these steroids on urinary excretion of Na+ and K+. 6-Dehydro-derivatives will thus bind preferentially to receptor Type I inducing the retention of sodium and compete with mineralocorticoids for such receptors. The increase in both natriuresis and kaliuresis when corticosteroids and their 6-dehydro-derivatives are administered together may be interpreted as evidence for a Type II receptor mediation of those ion fluxes. The ionic changes are not mediated by the (Na+ + K+)-ATPase system. The fluoration at 9 and the dehydrogenation at C9C11 of DOCA result in a strong increase of binding to Type I receptor and of sodium retention.

Glycine and other amino compounds prevent chromatin precipitation at physiological ionic strength.

Glycine, proline and taurine, when present in the range 0.1-0.60 M, inhibit chromatin precipitation by sodium chloride. Histone gel electrophoresis revealed that the linker histones H1 and H5 were largely depleted from the supernatant chromatin fraction at 0.2 M NaCl, while this depletion was absent in the presence of glycine. These observations are discussed in relation with the various factors which may be involved in the precipitation process.

Activation of cytosol aldosterone receptors in rat kidney.

Cytosolic aldosterone-protein complexes are isolated from rat kidney slices after incubation with [3H]aldosterone and dexamethasone. Activated and unactivated forms of the complex are characterized by gel electrophoresis and hydroxyapatite chromatography after incubation at 4 degrees C and 25 degrees C respectively. It is found that the activated form reaches a maximum after 30 min at 25 degrees C and can be separated as an homogeneous peak by electrophoresis. Intermediate forms can also be identified. In the presence of 10 mM ATP, activation immediately occurs at 4 degrees C and is almost complete. In the presence of 10 mM molybdate, the activation is strongly enhanced and the increase in activated form may be about fifteen-fold whether molybdate is added during kidney homogenization or just before incubation at 25 degrees C. On the other hand molybdate reduces to one third the binding of the aldosterone-receptor complexes to nuclei. In the presence of the steroid RU 26988 which is a pure glucocorticoid, experiments done on aldosterone-receptors complexes and their binding to nuclei are confirmed. This proves that aldosterone is specific for mineralocorticoid sites. The general pattern of the mineralocorticoid receptor activation is discussed and its resemblance to the case of other steroid hormones is emphasized.

Alteration in the nucleosome and chromatin structures upon interaction with platinum coordination complexes.

The interaction of various platinum coordination complexes with nucleosomes and chromatin has been investigated by ultraviolet absorption spectrophotometry, circular and electric linear dichroism, and thermal denaturation, at low binding ratios (r less than 0.1-0.2). The general trend of the changes in these physicochemical properties is similar to that observed for the DNA-platinum complexes, which indicates that the same binding sites are involved in the platinum interaction with DNA and with its nucleoprotein complex. The cis-bidentate ligands, cis-dichlorodiammine, diaminocyclohexane and ethylenediamine platinum(II), showed a distinct behavior, with a more important destabilization of the DNA structure in the nucleoprotein than the trans-bidentate ligand, trans-dichlorodiammine-Pt(II), and monodentate ligand, diethylenetriamine-Pt(II). The drastic decrease of the negative electric dichroism in the 260 nm absorption band of the bases, observed with the five ligands, indicates a profound alteration of the DNA arrangement in chromatin and nucleosomes, attributed to a condensation of its superhelical structure. Some differences with previous observations on DNA complexes with the same platinum compounds indicate the possible formation of protein-DNA crosslinks in chromatin and nucleosomes. These could have some importance for the biological effects.

Preparation and some properties of strongly acidic proteins from calf-thymus nucleohistone.

The isolation of acidic proteins from calf-thymus nucleohistone (starting from purified nuclei) is reported. The method involved dissociation in 1 M KCl solution. Denaturating agents were not used at all. After electrophoresis on polyacrylamide gel, fractions containing a small number of components were obtained. The fractions display high ratios of acidic to basic amino acids, the ratios ranging from 4.0 to 1.5. In all fractions, the major components were of molecular weights in the ranges 12000-15000 and 24000-28000 as determined by gel-disc electrophoresis in dodecylsulphate and by equilibrium ultracentrifugation. Minor components of high molecular weights were also present. Amino-acid analyses are also reported. The tryptophan content was determined by a fluorometric method. Circular dichroism spectra depict a very low content of alpha-helicity that did not increase at higher ionic strength. A marked RNA-polymerase activity was found in one fraction.