Specific non-histone protein fractions associated with ‘active’ chromatin in immunized rats

Summary Non-histone proteins of normal, non-immunized rats and rats immunized with mouse spleen cells were labelled with three different amino acids: [³H]tryptophan, [³H]methionine and [³H]leucine. Chromatin was fractionated at increasing salt concentrations into three fractions: 0.35 M NaCl-soluble, 2 M NaCl-soluble and residual. Non-histone protein fractions F (M_r 12 000) and H (M_r 3 000) highly labelled with [³H]tryptophan, lower with [³H]methionine but not with [³H]leucine, were present mainly in the residual fraction. After DNAse II treatment non-histone protein fractions F and H disappeared in chromatin fractions and were present in Mg^2– soluble fractions which suggests that, similar to the fractions I (M_r below 3 000) and B (M_r 120 000) described previously (5), these fractions may be associated with active transcribed genes.     

Chromatin proteins associated with micrococcal nuclease-sensitive and nuclease-resistant chromatin fractions of Kirkman-Robbins hepatoma and hamster liver

Micrococcal nuclease-sensitive (SP) and nuclease-resistant (PP) chromatin fractions from Kirkman-Robbins hepatoma and hamster liver were obtained. The molecular distribution of three non-histone proteins (NHCP1, NHCP2 and NHCP3), histones, and chromatin-bound protease activity between SP and PP fractions of both tissues was compared. Differences, mainly of quantitative nature, among non-histone proteins of neoplastic and normal tissue were observed. Moreover, it was found that polypeptides with mol. wt 81 000 (NHCP1), 39 000 (NHCP2) and 21 000, 35 000, 37 000 (NHCP1), 70 000, 112 000, 141 000, 157 000 (NHCP2), 30 000–33 000 (NHCP3) were associated only with the nuclease-sensitive part of chromatin of hepatoma and normal tissue, respectively. A major difference in histone compostion of hamster hepatoma and liver concerns histones H2A and H1. Furthermore, an enrichment of high mobility group proteins as well as other soluble non-histone proteins in an acid extract of the SP fraction was observed. Apparently chromatin-bound protease activity can be found in both fractions of chromatin.     

Binding of the estradiol-receptor complex to reconstituted nucleoacidic protein from calf uterus

Non-histone protein-DNA complexes with acceptor activity for estradiol-receptor complexes were reconstituted from fractionated calf uterine chromatin. Acceptor activity had tissue specificity with target tissue binding exceeding non-target tissue binding. The binding of estradiol-receptor complexes to acceptor sites was dependent on intact non-histone protein-DNA complexes, reconstituted select non-histone proteins, and protein equivalent: DNA reconstitution ratios. [³H]Estradiol-receptor complexes were bound to reconstituted non-histone protein-DNA complexes (i.e., nucleoacidic protein) with a high affinity and with a limited number of binding sites. Fractionation of uterine chromatin non-histone proteins identified two major sets of non-histone proteins which had acceptor activity when reconstituted with DNA. Thus, it seems possible to reconstitute nucleoacidic protein fractions with specific acceptor activity for the calf uterine estrogen receptor.     

Effects of tryptophan on serotonin in nerve endings.

—Preparations of crude synaptosome fractions (P₂) from the telencephalon and from the diencephalon plus optic lobes of the pigeon and from the telencephalon of the rat were used to study the effects of l -tryptophan on (a) the levels of serotonin (5-HT), norepinephrinc (NE) and dopamine in nerve endings and (b) the release of radioactive 5-HT, NE and dopamine from nerve endings. The level of 5-HT was significantly higher (P < 0–05) in the P₂ fraction isolated from the telencephalon of pigeons given intramuscular injections of 300mg/kg of l -tryptophan in comparison to control values (1.11 ± 0.09 vs 0.74 ± 0.13 nmol/g original tissue wt). A smaller but not statistically significant increase in 5-HT was noted in the P₂ fractions isolated from the diencephalon plus optic lobes of pigeons given injections of l -tryptophan. In vitro studies using preparations of synaptosomes (from both pigeon and rat) labelled with [³H]5–HT demonstrated that 1.0 mm -l -tryptophan caused a 30% increase (P < 0.05) in the release of [³H]5-HT over control values. This effect by l -tryptophan was blocked when a decarboxylase inhibitor was added to the medium. Tryptophan had no effect on the levels of NE or dopamine in these nerve endings nor did it have any effect on the release of these two amines from these preparations of synaptosomes. The results are discussed in terms of the role of serotonin in producing depression in pigeons working on a certain learned behavioural task.     

Binding of [3H]triamcinolone acetonide-receptor complexes to chromatin from the B-cell leukemia line,BCL1

The binding characteristics of partially purified glucocorticoid receptor complexes from hormone sensitive, non-differentiating BCL₁ cells to sequentially deproteinized BCL₁ chromatin-cellulose was investigated. [³H]Triamcinolone acetonide (TA)-receptor complexes were purified (approx. 30-fold) from DEAF-cellulose columns by salt elution which allowed receptor activation only in the absence of molybdate. Addition of 10 mM molybdate completely blocked salt activation. The binding pattern of the activated [³H]TA-receptor complexes to chromatin-cellulose extracted with 0–8 M guanidine hydrochloride revealed three regions of increased binding activity (acceptor sites), at 2, 5 and 7 M guanidine hydrochloride. Acceptor site binding was markedly reduced for chromatin extracted with 3, 6 and 8 M guanidine hydrochloride. Non-activated receptor complexes demonstrated very low binding to deproteinized chromatin. It was also shown that chromatin binding required glucocortical receptors and that free ligand or ligand bound to other proteins did not bind significantly to chromatin. In addition, binding of [³H]TA-receptor complexes to partially deproteinized chromatin was competable by unlabeled TA-receptor complexes. Scatchard analysis demonstrated that chromatin from non-differentiating BCL₁ cells possesses multiple, high-affinity binding sites which differ in their affinity for the glucocorticoid receptor. Partially deproteinized chromatin from lipopolysaccharide-stimulated BCL₁ cells demonstrated a different pattern of receptor binding, i.e., receptor binding was significantly greater to chromatin previously extracted with 6–8 M guanidine hydrochloride. These results suggest that differentiation alters the state of chromatin and the interaction of non-histone protein/DNA acceptor sites with glucocorticoid receptors. These alterations may play a role in the acquisition of hormone resistance.     

Acetylation of histones of rat testis.

When [1-¹⁴C]acetate was injected into rats intratesticularly in the presence of cycloheximide to inhibit protein synthesis, the label was incorporated into histone fractions F2a1 and F3 and into non-histone chromosomal proteins of each of the following stages of spermatogenesis: spermatogonia-preleptotene spermatocytes, leptotene-zygotene-pachytene-diplotene primary spermatocytes, and spermatids. Acetylation of histones was particularly active in the spermatid stages. There was no significant incorporation of acetate into the lysine-rich histone fractions F1 and X₁.In early periods of in vivo incorporation of [³H]amino acids into histones the acetylated histone F2a1 fractions had higher specific activities than the main band of F2a1, but with the passage of time the label moved into the principal band to the extent that specific activities in the acetylated and principal bands were approximately equal at 6 days. However, at 24–36 days the specific activities were again higher in the acetylated bands than in the principal band of F2a1. These data support the conclusions of Candido, Louie, and Dixon, from experiments with trout testis, that acetylation of histone F2a1 may be important in the process of combination of this protein with DNA in chromatin at the spermatogonia-primary spermatocyte stage and also in the subsequent removal of this histone for replacement by protamines at the spermatid stage.[³H]Amino acids were incorporated into histone fractions X₁ and F1 at approximately equal rates, and there was no evidence that one of these fractions was a precursor of the other.Chromatin of the seminiferous epithelial cells of rat testis has a firmly bound acetylase which catalyzes the in vitro acetylation of histones F3 and F2a1 by acetyl CoA.     

Non-histone chromatin protein fractions associated with ‘active’ chromatin in embryonic chicken liver

Non-histone chromatin proteins synthesized during chicken embryonic liver development were labeled with [3H]tryptophan and [3H]methionine and characterized by electrophoresis. During embryonic development protein/DNA ratio in chromatin was low (1.30-1.62) but synthesis of non-histone protein was high. Especially one characteristic fraction K (MW 18 000), tightly bound with DNA was preferentially associated with DNAase II sensitive, active transcribed sequences. In 7-day old and adult chicken synthesis of all non-histone proteins was low, fraction K was absent or synthesized only in small amounts in association with non-active sequences, however protein/DNA ratio in chromatin was high (2.30-2.33).     

Localization of some molecules within the grooves of DNA by modification of their complexes with dimethyl sulphate

The comparative methylation with [³H] dimethyl sulphate (DMS) of free DNA and DNA in chromatin and nuclei within the minor and major grooves of the DNA double helix and its single stranded regions was measured.The results suggest that histones lie partly inside the major groove and partly out of the grooves of DNA in chromatin leaving the minor groove open; most of the non-histone proteins of chromatin are not buried in the DNA grooves; the content of single stranded DNA in chromatin does not exceed 0.5%.     

Involvement of L-Type-Like Amino Acid Transporters in S-Nitrosocysteine-Stimulated Noradrenaline Release in the Rat Hippocampus

Abstract: Nitrogen oxides, such as nitric oxide, have been shown to regulate neuronal functions, including neurotransmitter release. We investigated the effect of S-nitroso-l -cysteine (SNC) on noradrenaline (NA) release in the rat hippocampus in vivo and in vitro. SNC stimulated [³H]NA release from prelabeled hippocampal slices in a dose-dependent manner. SNC stimulated endogenous NA release within 30 min to almost five times the basal level in vivo (microdialysis in freely moving rats). In a Na⁺-containing Tyrode's buffer, SNC-stimulated [³H]NA release was inhibited 30% by the coaddition of l -leucine. In the Na⁺-free, choline-containing buffer, SNC-stimulated [³H]NA release, which was similar to that in the Na⁺-containing buffer, was inhibited markedly by l -leucine, l -alanine, l -methionine, l -phenylalanine, and l -tyrosine. The effects of the other amino acids examined were smaller or very limited. The effect of l -leucine was stronger than that of d -leucine. A specific inhibitor of the L-type amino acid transporter, 2-aminobicyclo[2.2.1]-heptane-2-carboxylate (BCH), inhibited the effects of SNC on [³H]NA release in the Na⁺-free buffer. Uptake of l -[³H]leucine into the slices in the Na⁺-free buffer was inhibited by SNC, BCH, and l -phenylalanine, but not by l -lysine. The effect of SNC on cyclic GMP accumulation was not inhibited by l -leucine, although SNC stimulated cyclic GMP accumulation at concentrations up to 25 µM, much less than the concentration that stimulates NA release. These findings suggest that SNC is incorporated into rat hippocampus via the L-type-like amino acid transporter, at least in Na⁺-free conditions, and that SNC stimulates NA release in vivo and in vitro in a cyclic GMP-independent manner.     

Non-histone chromatin proteins of B lymphocytes stimulated by lipopolysaccharide.

The synthesis of non-histone chromatin proteins and nucleoplasmic proteins has been followed during lipopolysaccharide-induced division and differentiation of murine B lymphocytes. Synthesis was measured by pulse labelling with [3H]leucine, extraction of proteins was under conditions designed to prevent proteolysis and analysis of labelled proteins was by polyacrylamide gel electrophoresis. The average specific activity of non-histone chromatin proteins increased 3-fold, to a maximum, after culture for 24 h with lipopolysaccharide. Comparison of the relative synthesis of individual proteins (stimulation index) reveals three distinct responses: (1) those in the largest group show low stimulation indices, generally less than two; (2) a group of four proteins have indices between 4 and 5; (3) two proteins (molecular weights 21 000 and 22 000) both show an index of 5 at 24 h rising to between 7 and 8 by 48 h when the average specific activity is falling, coinciding with the period of rapid differentiation to high rate IgM secretion. Additionaly at this time, a newly labelled protein (Mr = 36 500) appears in the nucleoplasm followed by a second protein (Mr = 63 000) appearing between 48 and 72 h. The patterns of change are consistent with an overall increase in non-histone chromatin proteins synthesis, necessary for cell division, with superimposed specific changes in synthesis of non-histone chromatin proteins which could be related to regulation of cell differentiation.     

Evidence for the mannosylation of a non-histone protein in monkey liver chromatin

Summary Mannose is incorporated in monkey liver chromatin by the means of a nuclear membrane mannosyl-transferase.¹⁴C-labelled chromatin is dissociated either by sulfuric acid or 6 M urea and 0.4 M GuCl. The fractions then enriched in non-histone¹⁴C-labelled proteins are excluded from Ultro-gel AcA 202, their analysis in SDS-polyacrylamide gel electrophoresis shows that radioactivity fits with one major protein band, confirming the presence of at least a non-histone protein labelled with mannose in monkey liver chromatin, with an apparent molecular weight of 13 000.     

Studies on the mode of action of calciferol. XVIII. Evidence for a specific high affinity binding protein for 1,25 dihydroxyvitamin D3 in chick kidney and pancreas.

Cytosol fractions prepared from rachitic chick kidney and pancreas were analyzed for binding of vitamin D₃ metabolites by sucrose density gradient centrifugation. Both cytosol fractions were found to contain a 3.6S macromolecule which specifically binds 1,25-dihydroxy[³H] vitamin D₃ and in addition a 5 to 6S macromolecule which binds 25-hydroxy[³H]vitamin D₃. Sucrose gradient analysis of a KCl extract prepared from kidney or pancreas chromatin resulted in a peak (3.6S) of bound 1,25-dihydroxyvitamin D₃ which could not be distinguished from the cytoplasmic binding component. The interaction of 1,25-dihydroxy[³H]vitamin D₃ with the cytoplasmic binding component of both tissues occurred at low concentrations of hormone with high affinity.     

Nuclear proteins : IV. Deficiency of non-histone proteins in condensed chromatin of Drosophila virilis and mouse

Drosophila virilis egg nuclei were fractionated by a technique of multiple sonication and centrifugation in an isotonic buffer containing 0.15 M NaCl, Mg²⁺ and Ca²⁺. This allowed the condensed chromatin to remain tightly condensed. By Hoechst 33258 staining this procedure resulted in brightly fluorescing and poorly fluorescing fractions. The brightly fluorescing fraction was enriched in satellite DNA. Examination of the non-histone proteins by SDS slab gel electrophoresis showed that this fraction was markedly deficient in non-histone proteins and contained no unique major non-histone proteins. The poorly fluorescing fractions were enriched in non-histone proteins. Similar results were obtained with mouse liver nuclei. Comparison of the non-histone proteins of D. virilis (40% satellite DNA), D. americana (35% satellite DNA), and D. ezoana (no satellites) confirmed the absence of major, satellite specific, non-histone proteins. These results, suggesting condensed chromatin is primarily a DNA-histone complex, agree with published cytochemical studies.     

Glucosamine-labelled envelope proteins of Escherichia coli K-12 I. Electrophoretic studies and partial fractionation of the phenol-soluble proteins

Hydrophobic envelope proteins were extracted by phenol from a glucosamine- and leucine-requiring mutant of Escherichia coli K-12 (E-110). Three protein fractions labelled with D-[1-^{1 4}C]glucosamine and L-[4,5-³H]leucine were obtained by electrophoretic separation. Envelope were isolated from cells labeleed with D-[1-^{1 4}C]glucosamine—HCL and acid hydrolyzed. At least 68% of the radioactivity was recovered as glucosamine and glucose with no random distribution of label. Fingerprinting of pronase digests of glucosamine-labelled proteins showed four radioactive spots associated with peptides. Te glycoproteins were pronase- and trypsin-sensitive and had apparent molecular weights of 11 000 (fast mobility), 35 000 (intermediate mobility) and 62 000 (slow mobility) as estimated by sodium dodecyl sulfate-polyacrylamide disc electrophoresis. The two heavier fractions were labelled with meso-diamino[1,7-^{1 4}C₂]pimelic acid, while ortho[^{3 2}P]phosphate was not incorporated into any fraction. The glucosamine radioactivity of the fast fraction underwent rapid changes upon a chase with non-radioactive glucosamine. Using a Sephadex LH-20 column, the radioactive proteins were separated from the phenol and subsequently fractionated on a DEAS-cellulose column. The DEAE-cellulose fractions were distinct from each other in the number and composition of protein bands, when analyzed by sodium dodecyl sulfate-polyacrylamide disc electrophoresis. Radioactive bands with intermediate and fast electrophoretic mobilities were found in separate DEAE-cellulose fractions.     

Glucosamine-labelled envelope proteins of Escherichia coli K-12 II. Location in inner and outer membranes

Hydrophobic envelope proteins were extracted by phenol from a glucosamine- and leucine-requiring mutant of Escherichia coli K-12 (E-110). Three protein fractions labelled with D-[1-^{1 4}C]glucosamine and L-[4,5-³H]leucine were obtained by electrophoretic separation. Envelope were isolated from cells labeleed with D-[1-^{1 4}C]glucosamine—HCL and acid hydrolyzed. At least 68% of the radioactivity was recovered as glucosamine and glucose with no random distribution of label. Fingerprinting of pronase digests of glucosamine-labelled proteins showed four radioactive spots associated with peptides. Te glycoproteins were pronase- and trypsin-sensitive and had apparent molecular weights of 11 000 (fast mobility), 35 000 (intermediate mobility) and 62 000 (slow mobility) as estimated by sodium dodecyl sulfate-polyacrylamide disc electrophoresis. The two heavier fractions were labelled with meso-diamino[1,7-^{1 4}C₂]pimelic acid, while ortho[^{3 2}P]phosphate was not incorporated into any fraction. The glucosamine radioactivity of the fast fraction underwent rapid changes upon a chase with non-radioactive glucosamine. Using a Sephadex LH-20 column, the radioactive proteins were separated from the phenol and subsequently fractionated on a DEAS-cellulose column. The DEAE-cellulose fractions were distinct from each other in the number and composition of protein bands, when analyzed by sodium dodecyl sulfate-polyacrylamide disc electrophoresis. Radioactive bands with intermediate and fast electrophoretic mobilities were found in separate DEAE-cellulose fractions.     

The effect of sodium butyrate on acetylation in vitro of chromosomal proteins in three classes of liver nuclei from different ages of rats

The optimum conditions of in vitro incorporation of sodium [³H]acetate into sliced rat liver were studied. The incubations with sliced liver from three different ages of rats were performed in the presence of sodium n-butyrate. It was found that butyrate decreases the incorporation of sodium [³H]acetate into the homogenate, isolated nuclei, non-histone chromosomal proteins and histones for all age groups. The acetylations of non-histone chromosomal proteins and histones increase with age upto 2-months and decrease in 4-month-old rats both in the absence and presence of butyrate. Liver nuclei were fractionated by the simple method of zonal centrifugation into three classes, namely diploid stromal, diploid parenchymal and tetraploid parenchymal nuclei. The acetylations of non-histone chromosomal proteins and histones in three classes of nuclei of three ages of rats were studied in the presence and absence of butyrate. Butyrate can decrease the overall acetylations of non-histone chromosomal proteins and histones but increase the amount of polyacetylated histone H4 in all classes of nuclei of the three ages.     

Changes in the phosphorylation of non-histone chromatin proteins during the cell cycle of HeLa S 3 cells

The phosphorylation of non-histone chromatin proteins in synchronized HeLa S₃ cells was studied in 5 phases of the cell cycle: mitosis, G₁, early and late S, and G₂. The rate of non-histone chromatin protein phosphorylation was found to be maximal during G₁ and G₂, somewhat decreased during S phase, and almost 90% depressed during mitosis. Analysis of the phosphorylated non-histone chromatin proteins by SDS-acrylamide gel electrophoresis showed a heterogeneous pattern of phosphorylation as measured by labeling with ³²P. Significant variations in the labeling pattern were seen during different stages of the cell cycle, and particular unique species appeared to be phosphorylated selectively during certain stages of the cycle.