Influence of temperature on the preferential extraction of RNA polymerase I from hepatic nuclei of the rat

RNA polymerase I has been extracted from rat liver nuclei by three consecutive washings at 0 degrees C with a medium of relatively low ionic strength (0.15 M KCl) containing Mg++ rather than by incubating the organelles at 37 degrees C in the same medium, as originally proposed by Chesterton and Butterworth. The modified technique, which has the advantage of preventing a temperature-mediated conversion of form IB to IA, gives similar yields of RNA polymerase I and retains the capacity of preferentially extracting the enzyme with respect to the other forms of nuclear RNA polymerase.     

Animal DNA-dependent RNA polymerases. Purification and molecular structure of hen-oviduct and liver class-B RNA polymerases.

Hen ovidcut and liver class B RNA polymerases have been extensively purified and their molecular structure has been analysed. While only one enzyme B form (BIIb) was found in liver, three forms (BI, BIIa and BIIb) were resolved from oviduct. The molecular structures of the various class B RNA polymerase forms purified from hen oviduct and liver are identical to the corresponding forms previously purified from calf thymus and rat liver. At the present level of resolution the only difference lies in a slight difference in the charge of one subunit (SB2a) of enzyme form BIIa, when comparing the mammal and bird enzymes. It is unlikely that the absence of enzyme forms BI and BIIa in purified hen liver RNA polymerase B could be related to limited and specific proteolysis during the purification, since co-purification of oviduct and liver RNA polymerase B activities from a mixture of oviduct and liver nuclei does not affect the presence of either oviduct enzyme form BI or BIIa in the final purified mixture.     

DNA-dependent RNA polymerases from normal mouse liver

Three forms of DNA-dependent RNA polymerase from adult mouse liver are separable by DEAE-Sephadex A-25 chromatography. Two of the forms (IA and IB) are insensitive to inhibition by α-amanitin, while the third form is completely inhibited by 0.3 μg/ml of α-amanitin. The three enzyme forms are compared to the enzymes found in adult rat liver, and to the enzymes found in several other mouse tissues.     

Glucocorticoids modify the rate of ribosomal RNA synthesis in rat thymus cells by regulating the polymerase elongation rate

The mechanism by which glucocorticoids inhibit RNA polymerase A activity, and hence rRNA synthesis, in rat thymus cells has been investigated. Studies of the intranuclear distribution of RNA polymerase A between chromatin bound ("engaged") and unbound ("free") forms revealed that the steroid-mediated inhibition of the activity of the "engaged" form of the enzyme was not accompanied by significant changes in "free" pool activity. In the presence of rifamycin AF/0-13, an inhibitor of re-initiation of RNA polymerase A, the rate of [3H]UMP incorporation into RNA was slower in nuclei from steroid-treated cells than in those from control cells, although in both conditions similar plateau levels of UMP incorporation were attained. Direct measurements of the numbers of transcribing RNA polymerase A molecules and of elongation rates showed that the inhibition of pre-rRNA synthesis was the result of a decrease in enzyme elongation rate; no significant change was observed in the number of transcribing enzymes. The steroid-induced inhibition of pre-rRNA synthesis was selectively abolished by mild proteolysis of nuclei, suggesting the involvement of a labile, regulatory glucocorticoid-induced protein. It is concluded that glucocorticoid treatment of rat thymus cells decreases 45S rRNA synthesis primarily by decreasing the polyribonucleotide elongation rate of RNA polymerase A, possibly by modification of the enzyme.     

γ线全身照射对大鼠肝癌细胞核RNA合成的影响及其机制的研究

作者观察了8Gy γ线全身照射正常和DEN诱发的肝癌大鼠其肝(癌)细胞核RNA合成的辐射生物效应,发现:<1>正常大鼠在受照射后4h出现一过性的RNA合成增高期,而后表现为抑制,出现双相效应;而肝癌大鼠在受照后4和18h均表现为強烈抑制;<2>在抑制了内源性染色质模板后,转录外源模板的游离型RNA聚合酶出现与染色质结合型酶相似的辐射效应,提示射线可直接影响RNA聚合酶;<3>在照射后4和18h,RNA聚合酶Ⅱ的活性变化率显著高于酶Ⅰ,提示酶Ⅱ及其复合体成分对射线更敏感;<4>肝癌大鼠在受照射后其核RNA合成的抑制与转录活性RNA聚合酶分子数目的减少以及酶的催化效率(延长速度)减低有关,而正常大鼠则是通过不同的机制实现。     

Control of rRNA transcription in liver of mice exposed to nutritional changes: initiation frequency may be a regulatory mechanism

Shortly after feeding protein-depleted mice on a meal containing proteins, the RNA polymerase I activity in isolated liver nuclei shows a two fold to threefold activation over the basal value in nuclei of either normal or protein-depleted mice. This activation can be accounted for by the increase in the number of growing rRNA chains. Moreover, the template-bound RNA polymerase I fraction in nuclei from re-fed mice is about three times that from protein-depleted animals. An excess of template- unbound enzyme was found in liver nuclei from animals under either nutritional condition. Shortly after inhibition of protein synthesis by pactamycin administration to re-fed mice, the number of transcribing RNA polymerase I molecules in liver nuclei decreases to the basal level found in nuclei from protein-depleted mice, while in the latter, protein synthesis inhibition has no effect. These results support the suggestion that short-lived proteins may enhance the initiation frequency by RNA polymerase I after re-feeding.     

An improved method for the quantitative isolation of rat liver nuclear RNA polymerases.

Rat liver nuclear RNA polymerases exist in two functional states, one of which is active towards the endogenous chromatin template (engaged enzyme), while the other is inactive (free enzyme) (Yu, F.L. (1974) Nature 251, 344-346). This paper reports the direct separation of these two populations of RNA polymerases from isolated rat liver nuclei by a simple extraction procedure. It is estimated that as much as 50% of the total nuclear RNA polymerase activity in normal rat liver may exist in the form of the free enzyme. Evidence is also presented to indicate that the free enzyme activity is easily lost when the nuclear isolation procedure involves the use of an isotonic buffer medium, or when the isolated nuclei are subjected to sonication as is required for the solubilization of the nuclear RNA polymerases by the conventional method. Based on these new findings, it is proposed that nuclei be isolated directly in hypertonic sucrose and that the free enzyme be extracted before the nuclei are subjected to sonication to solubilize the engaged enzyme. This method circumvents the loss of the free RNA polymerase population and, as a result, the total yield of the nuclear RNA polymerases is greatly increased. The possible functional role of the free RNA polymerase in gene expression is discussed.     

A comparative study of mouse liver and mouse blastocyst DNA-dependent RNA polymerases.

DNA-dependent RNA polymerase has been studied in adult mouse liver and mouse blastocysts. The enzyme from mouse liver was resolved into three enzyme forms by DEAE-Sephadex chromatography. Two of the forms, IA and IB, are insensitive to α-amanitin, have low $\text{Mn}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ activity ratios, and are optimally active at low ionic strength. Form II is inhibited by α-amanitin, has a higher $\text{Mn}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ activity ratio, and is most active at high ionic strength. An optimal reaction temperature of 37 ° C was found for all enzyme forms. All of the isolated enzyme forms are inhibited by the exotoxin from Bacillus thuringiensis and the inhibition can be partially reversed by increased ATP levels. Forms IA and IB are most active with native template while form II prefers denatured DNA.The blastocyst RNA polymerase activity exhibits similar requirements for divalent metal ions and ionic strength to the purified liver enzymes. The maximum inhibition of blastocyst RNA polymerase obtained with α-amanitin and exotoxin differs from that observed for purified liver enzymes but is similar to the inhibition of liver homogenate. However, the concentrations of inhibitor required for maximum inhibition by α-amanitin and exotoxin is different for the blastocyst and liver homogenate enzymes.     

Characterization of a novel alpha-D-mannosidase from rat brain microsomes

A new alpha-D-mannosidase has been identified in rat brain microsomes. The enzyme was purified 70-100-fold over the microsomal fraction by solubilization with Triton X-100, followed by ion exchange, concanavalin A-Sepharose, and hydroxylapatite chromatography. The purified enzyme is very active towards mannose-containing oligosaccharides and has a pH optimum of 6.0. Unlike rat liver endoplasmic reticulum alpha-D-mannosidase and both Golgi mannosidases IA and IB, which have substantial activity only towards alpha 1,2-linked mannosyl residues, the brain enzyme readily cleaves alpha 1,2-, alpha 1,3-, and alpha 1,6-linked mannosyl residues present in high mannose oligosaccharides. The brain enzyme is also different from liver Golgi mannosidase II in that it hydrolyzes (Man)5GlcNAc and (Man)4GlcNAc without their prior N-acetylglucosaminylation. Moreover, the facts that the ability of the enzyme to cleave GlcNAc(Man)5GlcNAc, the biological substrate for Golgi mannosidase II, is not inhibited by swainsonine, and that p-nitrophenyl alpha-D-mannoside is a poor substrate provide further evidence for major differences between the brain enzyme and mannosidase II. Inactivation studies and the co-purification of activities towards various substrates suggest that a single enzyme is responsible for all the activities found. In view of these results, it seems possible that, in rat brain, a single mannosidase cleaves asparagine-linked high mannose oligosaccharide to form the core Man3GlcNAc2 moiety, which would then be modified by various glycosyl transferases to form complex type glycoproteins.     

Modification of rat liver RNA polymerase I after in vivo stimulation by hydrocortisone or methylisobutylxanthine

Following in vivo administration of hydrocortisone or methylisobutylxanthine to rats, higher levels (1.5- to 2.3-fold) of RNA polymerase I activity are present in liver nuclei and nucleoli of the treated animals as compared to control animals. The elevated specific activity is retained after purification of the enzyme under conditions where the enzyme is dependent on exogenous template for activity. The elevated polymerase activity in nuclei, nucleoli, and soluble enzyme can be destroyed by mild trypsin treatment which results in a rapid decay of the specific activity to the control level. Under these conditions, the control polymerase I activity is stable. The results indicate that in vivo stimulation by hydrocortisone or methylisobutylxanthine results in a conversion of the enzyme to a form that is catalytically more active but has an increased sensitivity to proteolysis.     

Transcription of isolated nuclei and nucleoli by exogenous RNA polymerase A and B.

Both forms A and B RNA polymerases solubilised from rat liver nuclei transcribed templates within these organelles when added exogenously to freshly prepared nuclei. The enzymes initiated more efficiently in the presence of KCL than ammonium sulphate and required manganese rather than magnesium as the divalent cation. Form A enzyme initiated most successfully at 375 mM KC6, activity was proportional to the amount of template added and continued linearly for at least 30 min. Form B enzyme initiated with two ionic strength optima, 125 mM and 500 mM KCl. Activity in the latter case was critically dependent on the enzyme: nuclei ratio. In both instances incorporation of nucleotide precurors was linear for less than 20 min. Form A enzyme synthesised products with a size distribution mainly larger than 18 S; form B enzyme synthesised products of mainly less than 5 S at 125 mM KCl and about 10 S at 500 mM KCl. Subfractionation of nuclei indicated that exogenous RNA polymerase A activity and form B at 125 mM KCl were occurring in nucleoli; form B activity at 500 mM KCl was nucleoplasmic. Measurements of U : G ratios in the RNA products suggested that exogenous form A was synthesising species with similar base ratios to the ribosomal RNA precurosrs. Both enzymes formed rifamycin AF/0-13 resistant complexes with nucleolar templates. Size analyses of products showed that whereas form B enzyme synthesised very small RNA species, RNA polymerase A produced a range of species of similar sizes to the ribosomal RNA precurosors.     

Specificity of cyclic GMP activation of a multi-substrate cyclic nucleotide phosphodiesterase from rat liver

Phosphoprotein phosphatase IA, which represents the major glycogen synthase phosphatase activity in rat liver cytosol, has been purified to apparent homogeneity by chromatography on DEAE-cellulose, histone - Sepharose-4B and Sephadex G-100. The molecular weight of the purified enzyme was 40 000 by gel filtration and 48 000 by sodium dodecyl sulfate gel electrophoresis, Phosphatase IA is therefore a monomeric protein. When treated with 80% ethanol at room temperature, phosphatase IA underwent an inactivation which was totally prevented by 2 mM MgCl2. Catalytically, phosphatase IA has a preference for glycogen synthase D compared with phosphatases IB and II and obligatorily requires Mg2+ or Mn2+ for activity. Maximum activity was attained at 5 mM MgCl2. Since Mg2+ does not activate other phosphoprotein phosphatases in rat liver cytosol, we propose the term 'Mg2+-dependent glycogen synthase phosphatase' for phosphatase IA.     

Characterization of multiple forms of histone phosphatase in rat liver.

By using chromatography on DEAE-cellulose, aminohexyl-Sepharose 4B and Sephadex G-200, rat liver extract was shown to contain at least three fractions, IA, IB and II, of histone phosphatase. Fractions IA and II are probably the same enzymes as the previously described glycogen synthase phosphatase and phosphorylase phosphatase, respectively, but IB exhibits noticeable activities only with phosphohistone as substrate. Approximate molecular weights of 69 000, 300 000 and 160 000 were determined by gel filtration on Sephadex G-200 for IA, IB and II, respectively.