Incorporation of ribonucleic acid in vitro into dense ribonucleoprotein-like materials by isolated rat liver nuclei.

A cell-free system of isolated rat liver nuclei is described which permits an active incorporation of newly synthesized RNA into 'dense' ribonucleoprotein-like materials. The reaction is stimulated with increasing amounts of cytosol protein isolated from rat liver. This indicates that cytosol protein plays an important role in the formation of such material.     

A special estrogen-binding protein in the rat liver: the effect of nuclear accumulation of estradiol-receptor complexes in vitro

The accumulation of [3H]estradiol-receptor complexes by liver nuclei after preliminary incubation of the hormone with rat liver cytosol was studied. It was demonstrated that addition to female rat liver cytosol of a purified preparation of the unusual estrogen-binding protein (UEBP) from male rat liver causes a dose-dependent inhibition of subsequent accumulation of specifically bound [3H]estradiol in the nuclei. Addition to male rat liver cytosol of 1.5 microM 2 alpha-hydroxytestosterone, testosterone, 1-dehydrotestosterone, 5 alpha-androstane-3 alpha, 17 beta-diol and 5 alpha-dihydrotestosterone, i. e. compounds possessing marked affinity for UEBP, resulted in a 2-5-fold increase of the subsequent nuclear accumulation of estrogen-receptor complexes. The use of UEBP-deficient female rat liver cytosol revealed that the afore-mentioned steroids are ineffective with respect to estrogen reception. It is concluded that UEBP of male rat liver is capable of modulating estrogen reception.     

Reaction between dexamethasone-receptor complexes and isolated nuclei from Zajdela hepatoma and rat liver cells]

Temperature-activation of the hormone-receptor complex (HRC) was shown to be necessary to ensure its translocation from cytoplasm to nucleus both in the rat liver and hepatoma. Hepatoma nuclei bind 20 times less HRC derived from homologous hepatoma cytosol (0.15 pmol/mg DNA), but twice as much (5.6 pmol/mg DNA) of HRC from heterologous liver cytosol, as compared with the binding of HRC from normal liver cytosol by liver nuclei (3 pmol/mg DNA), Ka of HRC with the acceptor sites in hepatoma and liver nuclei were found to be practically of the same order of magnitude. The above findings suggest an inhibition of cytosol-nucleus translocation of HRC from the cytosol of hepatoma cells as a possible cause of the nonresponsiveness of the latter to the hormone.     

Peculiarities of nuclear genome transcription in the early stages of liver regeneration. Complex nature of the activating effect of the cytoplasmic regenerating factor on transcription in isolated rat liver nuclei

The cytosol fractions from resting rat liver and from the liver 1 hour after partial hepatectomy which were not adsorbed on DEAE-cellulose were subjected to gel filtration with a view of elucidating the mechanism of its activating effect on the RNA-synthesizing ability of isolated nuclei of resting liver. The activating effect of this fraction is a complex one and includes both specific and non-specific factors. A subfraction typical for regenerating liver was detected, which activates RNA elongation in a synthesizing system with isolated nuclei of resting liver.     

Enhancement of neutral phosphatase activity in the cytosol and nuclei of regenerating rat liver: role of endogenous regucalcin.

The role of endogenous regucalcin (RC) in the regulation of neutral phosphatase activity in regenerating rat liver was investigated. The liver weight reduced by a partial hepatectomy (about 70%) was completely restored at 72 h after surgery. Phosphotyrosine, phosphoserine, and phosphothreonine were used as the substrate for the assay of phosphatase activity. Phosphatase activity toward phosphotyrosine in the hepatic cytosol and nuclei was significantly increased at 24-72 h after hepatectomy. Such an increase was not seen in the case of phosphoserine and phosphothreonine. However, the presence of anti-RC monoclonal antibody (200 ng/ml) in the enzyme reaction mixture caused a remarkable elevation of phosphatase activity toward three phosphoaminoacids in the hepatic cytosol at 24 and 48 h after hepatectomy. In the liver nuclei after sham operation or hepatectomy, phosphatase activity toward three phosphoaminoacids was significantly raised by the addition of anti-RC antibody (150 ng/ml). The nuclear phosphatase activity toward phosphothreonine in regenerating liver was significantly enhanced in the presence of anti-RC antibody (100 and 150 ng/ml). The effect of anti-RC antibody to increase phosphatase activity toward three phosphoaminoacids in the cytosol and nuclei of regenerating liver was completely blocked by the addition of exogenous RC (1.0 microM). The present study demonstrates that protein phosphatase activity in the cytoplasm and nuclei is enhanced in regenerating rat liver. This enhancement may be suppressed by endogenous RC.     

Interaction of ATP with a macromolecular translocation inhibitor of the nuclear binding of “activated” receptor-glucocorticoid complex

Incubation of 1–5 mM ATP with nuclei and partially purified “activated” receptor-[³H]triamcinolone acetonide complex from rat liver cytosol had no significant effect on association of the activated complex with the nuclei. However, when the nuclear uptake was reduced by the macromolecular translocation inhibitor in the rat liver cytosol, addition of 5 mM ATP restored the uptake to the level without inhibitor. ADP and AMP as well as other nucleotides tested could not overcome the inhibitory effect of macromolecular inhibitor.     

5alpha-Dihydrotestosterone binding protein in rat ventral prostate; purification, nuclear incorporation, and subnuclear localization.

Treatment of cytosol from the rat ventral prostate with cold acetone (-20 degrees C) evoked a 8 approximately 10-fold increase in the binding capacity with 5alpha-dihydrotestosterone (DHT). Starting from the extract of acetone-dried prostate cytosol, some 400 approximately 600-fold purification of the DHT-binding protein complex was acieved by (NH4)2504 fractionation, DEAE-cellulose chromatography and gel- filtration with Sephadex G-200. The purified 3H-DHT-binding protein complex was incorporated into the nuclei from the ventral prostate in a temperature dependent manner. The similar incorporation was also observed in nuclei from the liver and the kidney...     

Estrogen- and progestin-receptor complexes and their interactions with rat liver cell nuclei in ontogenesis

Steroid-receptor complexes (SRC) of estrogen and progestin were isolated from rat liver and purified 1500-2000-fold. The SRC within the composition of cytosol and purified 2000-fold were characterized by gel filtration of Sephadex G-100 and by DEAE-cellulose chromatography. The purified SRC from rat liver were bound to isolated liver cell nuclei of rats of various age (1.5, 6, 12 and 24 month-old). The maximal binding of progestin and estrogen SRC from rat liver was observed in homologous nuclei of 1.5-month-old animals. The binding of SRC by the nuclei decreased progressively with age, reaching its minimum in 24-month-old rats. The observed differences in the SRC binding by cell nuclei of experimental animals may be the cause of functional changes at various stages of ontogenesis.     

Age-related characteristics of RNA transport through the nuclear membrane

The effect of cytosol and ATP-regenerating system on RNA, transport was studied in isolated liver nuclei of adult and old rats. The stimulating effect of cytosol was found not to depend on the age of animals. The release of RNA from old rat liver nuclei activated by the ATP-regenerating system was more expressed compared to adult rats. It is assumed that the age changes of energy-delivering system of the RNA transport through nuclear membrane may be conditioned by the deficit of endogenous energetic substrates in the hepatic cells of old animals.     

Partial purification of the glucocorticoid receptor from rat liver: a rapid, two-step procedure using DNA-cellulose.

Glucocorticoid receptor from rat liver was purified 1800-fold by a rapid two-step procedure using DNA-cellulose. The procedure is based on increasing the affinity of the glucocorticoid-receptor complex for DNA by heating the complex. During a first chromatography step, unheated glucocorticoid-receptor complex is separated from cytosol proteins that bind to DNA-cellulose with high affinity. During a second chromatographic step, heat-treated glucocorticoid-receptor complex is separated from proteins with low affinity for DNA. The partially purified complex is functionally competent in that it is taken up by isolated rat liver nuclei.     

Studies on the translocation inhibitor of 3H-dexamethasone-receptor complex.

Recent reports on the binding of glucocorticoid-receptor complexes to rat liver nuclei suggested the presence of components which inhibited the binding. The inhibitory component(s) of the receptor translocation was observed not only in the cytosol of the liver but also in cytosols of the kidney, the spleen and the thymus. The cytoplasmic levels of the inhibitor in these tissues were not modified by the administration of Dexamthasone (DEX). The liver inhibitor was macromolecular and clearly separated from the DEX-receptor complex on DEAE-cellulose chromatography. The mechanism of the inhibition seemed to be an interaction between the inhibitor and the steroid-receptor complex. In addition, the inhibition seemed to be less specific for the bindings of different steroid-receptor complexes to nuclei. The bindings of hepatic 3H-DEX-receptor complex by nuclei derived from livers of adrenalectomized and DEX-treated rats, in the presence or absence of the translocation inhibitor, were similar.     

Relationship between the transport from isolated nuclei of two abundant cytoplasmic messengers and the source of a messenger RNA transport factor

Messenger RNA transport from isolated nuclei requires a 35×10³ dalton cytoplasmic protein(s) which is present in both the cytosol and polyribosome fractions. Recombinant DNA probes containing cDNA inserts were used to quantitate the transport of rat liver-specific albumin and male rat liver-specific ₂U-globulin messenger RNA (mRNA) from male rat liver nuclei in response to the mRNA transport factors from homologous and heterologous tissues. No mRNA transport occurs in the absence of the transport factor(s). Both messengers are transported proportionately in response to the factor(s) from male or female rat liver cytosol, or from the polyribosomes (messenger ribonucleoprotein) of male or female rat liver, or brain. The transport factor(s) do not, therefore, appear to differentiate between the coding sequences of two unrelated hepatic messenger RNA's.     

Ribonuclease H from rat liver. I. Partial purification and characterization of nuclear ribonuclease H1.

A ribonuclease H, an enzyme that specifically degrades the RNA moiety of RNA-DNA hybrid, has been partially purified from rat liver nuclei and characterized. Neither native or denatured DNA, nor single or double-stranded synthetic polyribonucleotides were degraded by the enzyme. The enzyme possesses a molecular weight of about 36,000 and requires alkaline pH, magnesium ions, and ammonium sulphate for maximum activity. The enzyme acts on the hybrid as an endonuclease, resulting in oligonucleotides with 3'-hydroxyl termini. The properties of this enzyme were distinct from those of the rat liver cytosol enzyme reported by Roewekamp and Sekeris in many respects, such as molecular weight, optimal pH and requirements for divalent cations. Preliminary experiments suggest that the nuclear enzyme is localized in the nucleoplasm and nucleoli. These results indicate that multiple forms of ribonuclease H exist in different regions of rat liver cells.     

Maximizing the purification of the activated glucocorticoid receptor by DNA-cellulose chromatography.

With heat treatment (20 degrees C for 30 min), the glucocorticoid-receptor complex becomes 'activated' and undergoes an increase in affinity for DNA. A two-stage procedure was used to separate sequentially the rat liver glucocorticoid-receptor complex from proteins with high and low affinity for DNA. DNA-cellulose column chromatography of unheated cytosol resulted in the retention of DNA-binding proteins, but not the unactivated receptor complex. Heat treatment of the column eluate resulted in increased affinity of the receptor complex to DNA, and chromatography on DNA-cellulose then yielded receptor complex free from proteins with low affinity for DNA. Removal of DNA-binding proteins during the first chromatographic step was critically dependent on ionic conditions and the ratio of cytosol chromatographed to DNA-cellulose. A purification of 11000-fold (85% yield) was achieved by this procedure. The partially purified receptor complex was taken up by rat liver nuclei.     

Ribonucleic acid synthesis in isolated rat liver nuclei under conditions of ribonucleic acid processing and transport.

A cell-free system is described which permits a significant and prolonged synthesis of RNA in isolated rat liver nuclei, under conditions previously demonstrated to support normal nuclear processing and transport of both rRNA and mRNA. The system contains cytosol but not (NH4)2SO4 or other non-physiological components. Evidence is presented for cytosol factors which stimulate ribosomal, and to a lesser degree, non-ribosomal RNA synthesis.     

Differential effect of ATP on RNA and DNA release from nuclei of normal and neoplastic liver.

Nuclei isolated from differentiated normal liver, moderately differentiated Hepatoma 5123 and poorly differentiated Novikoff hepatoma have been compared with respect to ATP-dependence of messenger-like RNA release and resistance to lysis (DNA release) in a cell-free system containing homologous cytosol. The release of RNA from the nuclei of liver, Hepatoma 5123D and the Novikoff hepatoma was totally ATP-dependent, partially ATP-dependent and ATP-independent, repectively. The sensitivity of the nuclear RNA transport in the presence of ATP to beryllium nitrate, an inhibitor of a nuclear pore phosphatase, paralleled their ATP-dependence. Although RNA release from the nuclei of both liver and Novikoff hepatoma has an absolute requirement for cytosol proteins, the structural integrity of liver, but not Novikoff hepatoma nuclei in the presence of ATP, is dependent on macromolecules in the cytosol.     

In vivo and in vitro phosphoarylation of nuclear proteins in rat liver.

The incorporation of 32P into nuclear nonhistone proteins was compared in rat liver in vivo, in liver slices incubated in vitro, and in isolated nuclei incubated with gamma-[32P]ATP. The highest specific activities of nuclear phosphorproteins were obtained by incubating isolated nuclei. However, the Radioactivity profiles of polyacrylamide gel electrophoretograms of these proteins differed from those obtained in vivo or in liver slice experiments. A group of low molecular weight nonhistone proteins exhibited a very high incporation of labelled phosphate. These proteins could be obtained from the interface when the phosphoproteins were isolated by the buffered phenol extraction procedure. Phosphorylated proteins were also obtained from three cytoplasmic fractions (mitochondria, microsomes, and cytosol). The specific activities of these proteins were much lower than of the nuclear phosphoproteins.     

Characterization of ribonucleoprotein particles released from isolated nuclei of regenerating rat liver in two different in vitro systems.

The ribonucleoprotein particles released from isolated nuclei of regenerating rat liver in two in vitro systems were studied and the following results were obtained. 1. When the isolated nuclei of regenerating rat liver labeled in vivo with [14C] orotic acid were incubated in medium containing ATP and an energy-regenerating system (medium I) release of labeled 40-S particles was observed. Analysis of these 40-S particles showed that they contained heterogeneous RNA but no 18 S or 28 S ribosomal RNAs and their buoyant density in CsCl was 1.42-1.45 g/cm3, suggesting that they were nuclear informosome-like particles released during incubation. 2. When the same nuclei were incubated in the same medium fortified with dialyzed cytosol, spermidine and yeast RNA (medium II), release of labeled 60-S and 40-S particles was observed. Using CsCl buoyant density gradient centrifugation, two components were found in the labeled ribonucleoprotein particles released from nuclei in this medium. The labeled 60-S particles were found to contain 28-S RNA as the main component and their buoyant density in CsCl was 1.61 g/cm3, suggesting that they were labeled large ribosomal subunits. The labeled 40-S particles contained both 18 S RNA and heterogeneous RNA and they formed two discrete bands in CsCl, at 1.40 and 1.56 g/cm3, suggesting that they contained small ribosomal subunits and nuclear informosome-like particles. 3. These results clearly indicate that addition of dialyzed cytosol, spermidine and low molecular yeast RNA to medium I causes the release of ribosomal subunits or their precursors from isolated nuclei in the in vitro system.