Thioacetamide-treated Rat Liver Research Articles

Evaluation of Bioinformatics Approaches for Next-Generation Sequencing Analysis of microRNAs with a Toxicogenomics Study Design.

MicroRNAs (miRNAs) are key post-transcriptional regulators that affect protein translation by targeting mRNAs. Their role in disease etiology and toxicity are well recognized. Given the rapid advancement of next-generation sequencing techniques, miRNA profiling has been increasingly conducted with RNA-seq, namely miRNA-seq. Analysis of miRNA-seq data requires several steps: (1) mapping the reads to miRBase, (2) considering mismatches during the hairpin alignment (windowing), and (3) counting the reads (quantification). The choice made in each step with respect to the parameter settings could affect miRNA quantification, differentially expressed miRNAs (DEMs) detection and novel miRNA identification. Furthermore, these parameters do not act in isolation and their joint effects impact miRNA-seq results and interpretation. In toxicogenomics, the variation associated with parameter setting should not overpower the treatment effect (such as the dose/time-dependent effect). In this study, four commonly used miRNA-seq analysis tools (i.e., miRDeep2, miRExpress, miRNAkey, sRNAbench) were comparatively evaluated with a standard toxicogenomics study design. We tested 30 different parameter settings on miRNA-seq data generated from thioacetamide-treated rat liver samples for three dose levels across four time points, followed by four normalization options. Because both miRExpress and miRNAkey yielded larger variation than that of the treatment effects across multiple parameter settings, our analyses mainly focused on the side-by-side comparison between miRDeep2 and sRNAbench. While the number of miRNAs detected by miRDeep2 was almost the subset of those detected by sRNAbench, the number of DEMs identified by both tools was comparable under the same parameter settings and normalization method. Change in the number of nucleotides out of the mature sequence in the hairpin alignment (window option) yielded the largest variation for miRNA quantification and DEMs detection. However, such a variation is relatively small compared to the treatment effect when the study focused on DEMs that are more critical to interpret the toxicological effect. While the normalization methods introduced a large variation in DEMs, toxic behavior of thioacetamide showed consistency in the trend of time-dose responses. Overall, miRDeep2 was found to be preferable over other choices when the window option allowed up to three nucleotides from both ends.

Ameliorative effects of D-glucuronolactone on oxidative stress and inflammatory/fibrogenic responses in livers of thioacetamide-treated rats

D-Glucuronolactone (C6H8O6, lactone), naturally found in plant gums, is commercially acclaimed for its hepatoprotective effects. This study was to investigate whether lactone can attenuate thioacetamide (TAA)-induced liver fibrosis in a rat model. Results showed that lactone supplementation (75 mg kg−1 bw glucuronolactone) alleviated AST values in TAA-intraperitoneally-injected rats (100 mg kg−1 bw TAA) and increased antioxidant capacity of liver via elevations of antioxidant enzymes activities [superoxide dismutase (SOD) and glutathione peroxidase (GPx)], glutathione (GSH) and trolox equivalent antioxidative capacity (TEAC) levels (p < 0.05). Down-regulated (p < 0.05) expression of inflammation including interleukin-6 (IL-6), nuclear factor-kappa B (NF-κB), activator protein 1 (AP-1), krüppel-like factor 6 (KLF-6), and fibrosis related fibrotic factors, i.e., alpha smooth muscle actin (α-SMA), and collagen alpha1 (I) (COLα1) through lactone supplementation underlay the lower collagen contents and less severe liver damage on histopathology observations. Therefore, hepatoprotection of lactone against TAA-induced liver fibrosis can be attributed to the amelioration of oxidative stress and inflammation.

Rat HMGCoA reductase activation in thioacetamide-induced liver injury is related to an increased reactive oxygen species content

In thioacetamide-induced liver injury a modification of isoprenoid content and an increase of reactive oxygen species has been described. We have examined how reactive oxygen species influence the 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate limiting enzyme of the isoprenoid biosynthetic pathway, to verify if changes of that enzyme activity are involved in the changed lipid composition of the liver. In chronic and acute thioacetamide-treated rat liver we measured the reactive oxygen species content, the activation state and K(M), the level and degradation rate of the hepatic reductase, its short term regulatory enzymes and the liver lipid profile. In thioacetamide-treated rat liver, the reactive oxygen species content is high and the reductase is fully activated with no modifications in its K(M) and its short term regulatory enzymes. The reductase level is reduced in chronic thioacetamide treated rats and its degradation rate is altered. The data show a relationship between reactive oxygen species production and altered 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. It is suggested that reducing the levels of reactive oxygen species may improve the altered lipid profile found in liver injury.

The influence of cyclosporine on lipid peroxidation and superoxide dismutase in adriamycine nephropathy in rats

The changes in the activity of nicotinamide: S-adenosylmethionine methyltransferase (nicotinamide methylase) were studied in rat liver which was subjected to different rates of cellular proliferation. The cytosolic enzyme activity increased 3–4-fold in the first 24–48 h after partial hepatectomy and decreased again to the basal levels until 4 days post-operatively, whereas it remained unchanged in the livers of sham-operated animals. A single administration of thioacetamide at a dose of 50–250 mg/kg body weight, a treatment which induces hepatocellular proliferation as well, also enhanced the enzyme activity 2–3-fold 24 h after drug administration. This activity increase was associated with a marked lowering of intracellular NAD content of as much as 50% of the control levels. d-Galactosamine, a known hepatotoxic agent causing acute hepatitis in experimental animals and preventing DNA synthesis in regenerating liver, blocked the activity increase in regenerating rat liver. The rate of 1-methylnicotinamide synthesis, as measured by incubating liver slices in the culture medium supplemented with [14C]nicotinamide as a precursor, was found to be 2–4 times higher in the slices from regenerating liver and thioacetamide-treated rat liver than those from non-proliferating control liver. These results, together with our previous finding on the enhancement by 1-methylnicotinamide of the growth of cultured rat liver cells (Hoshino, J., Kühne, U. and Kröger, H. (1982) Biochem. Biophys. Res. Commun. 105, 1446–1452), support the view that nicotinamide methylase and its product, 1-methylnicotinamide, are involved in the control of hepatocellular DNA synthesis and proliferation.

L-ornithine-induced inactivation of mammalian ornithine decarboxylase in vitro.

Partially purified ornithine decarboxylase, isolated from the liver of thioacetamide-treated rats, is stable in the absence of added low-molecular-mass thiols or other reducing agents. However, under these conditions, the enzyme is rapidly inactivated upon incubation with L-ornithine or L-2-methylornithine. The inactivation process follows first-order kinetics, and saturation kinetics are observed. Rapid recovery of activity is observed after subsequent addition of dithiothreitol. As distinct from L-ornithine, D-ornithine, putrescine, spermidine, or spermine do not produce inactivation of ornithine decarboxylase. Very similar results are obtained with pure ornithine decarboxylase isolated from androgen-stimulated mouse kidney, stabilized with a rat liver extract.

Cell-free synthesis of ornithine decarboxylase. Changes in mRNA activity in the liver of thioacetamide-treated rats.

Ornithine decarboxylase (ODC)mRNA associated with free polysomes of rat liver was translated in a reticulocyte lysate cell-free system. Newly synthesized ODC protein was identified by specific immunoprecipitation, molecular size as determined by polyacrylamide gel electrophoresis with sodium dodecyl sulfate, and competition by excess unlabeled ODC in the immunoprecipitation. A single injection of thioacetamide was found to cause several fold increases in both immunotitratable ODC protein and polysomal ODC-mRNA activity, while it provoked a much larger increase in ODC activity in rat liver. The results indicate that the induction of hepatic ODC activity by thioacetamide treatment is due not only to an increase in the activity of polysomal ODC-mRNA but also to a translational and/or posttranslational control.

Effect of 1,3-diaminopropane on ornithine decarboxylase enzyme protein in thioacetamide-treated rat liver.

A radioimmunoassay for ornithine decarboxylase was used to study the regulation of this enzyme in rat liver. The antiserum used reacts with ornithine decarboxylase from mouse, human or rat cells. Rat liver ornithine decarboxylase enzyme activity and enzyme protein (as determined by radioimmunoassay) were measured in thioacetamide-treated rats at various times after administration of 1,3-diaminopropane. Enzyme activity declined rapidly after 1,3-diaminopropane treatment as did the amount of enzyme protein, although the disappearance of enzyme activity slightly preceded the loss of immunoreactive protein. The loss of enzyme protein after cycloheximide treatment also occurred rapidly, but was significantly slower than that seen with 1,3-diaminopropane. When 1,3-diaminopropane and cycloheximide were injected simultaneously, the rate of disappearance of enzyme activity and enzyme protein was the same as that seen with cycloheximide alone. These results show that the rapid loss in enzyme activity after 1,3-diaminopropane treatment is primarily due to a loss in enzyme protein and that protein synthesis is needed in order for 1,3-diaminopropane to exert its full effect. A macromolecular inhibitor of ornithine decarboxylase that has been termed antizyme is induced in response to 1,3-diaminopropane, but our results indicate that the loss of enzyme activity is not due to the accumulation of inactive ornithine decarboxylase-antizyme complexes. It is possible that the antizyme enhances the degradation of the enzyme protein. Control experiments demonstrated that the antiserum used would have detected any inactive antizyme-ornithine decarboxylase complexes present in liver since addition of antizyme to ornithine decarboxylase in vitro did not affect the amount of ornithine decarboxylase detected in our radioimmunoassay. Anti-(ornithine decarboxylase) antibodies may be useful in the purification of antizyme since the antizyme-ornithine decarboxylase complex can be immunoprecipitated, and antizyme released from the precipitate with 0.3 M-NaCl.

Restoration of normal ornithine decarboxylase antizyme activity in rat liver after acute carcinogen treatment.

This study was undertaken to see whether or not the decrease in ornithine decarboxylase antizyme activity caused in rat liver by a hepatocarcinogen could be reversed. Thioacetamide was administered only once, in a single i.p. injection and at a non-carcinogenic, non-necrogenic dose. The activities of both hepatic ornithine decarboxylase and hepatic ornithine decarboxylase antizyme were measured at intervals of hours after the injection of thioacetamide. The hepatic ornithine decarboxylase antizyme in thioacetamide-treated rats was minimal at 40 and 80 h after carcinogen administration. The reversal process requires a very long time, namely 450 h for normal levels of hepatic ornithine decarboxylase antizyme activity to be restored in treated rats. This time is much longer than that required to restore normal ornithine decarboxylase activity in liver of thioacetamide-treated rats. The results of this study, combined with those of the preceding paper, demonstrate that hepatocarcinogens cause a relative inability of rat liver cells to make the ornithine decarboxylase antizyme and that the irreversibility of this defect in cellular control of ornithine decarboxylase activity may be a constant feature in the neoplastic transformation of the rat liver.

Methylation of nicotinamide in rat liver cytosol and its correlation with hepatocellular proliferation

The changes in the activity of nicotinamide: S-adenosylmethionine methyltransferase (nicotinamide methylase) were studied in rat liver which was subjected to different rates of cellular proliferation. The cytosolic enzyme activity increased 3–4-fold in the first 24–48 h after partial hepatectomy and decreased again to the basal levels until 4 days post-operatively, whereas it remained unchanged in the livers of sham-operated animals. A single administration of thioacetamide at a dose of 50–250 mg/kg body weight, a treatment which induces hepatocellular proliferation as well, also enhanced the enzyme activity 2–3-fold 24 h after drug administration. This activity increase was associated with a marked lowering of intracellular NAD content of as much as 50% of the control levels. d-Galactosamine, a known hepatotoxic agent causing acute hepatitis in experimental animals and preventing DNA synthesis in regenerating liver, blocked the activity increase in regenerating rat liver. The rate of 1-methylnicotinamide synthesis, as measured by incubating liver slices in the culture medium supplemented with [ 14C]nicotinamide as a precursor, was found to be 2–4 times higher in the slices from regenerating liver and thioacetamide-treated rat liver than those from non-proliferating control liver. These results, together with our previous finding on the enhancement by 1-methylnicotinamide of the growth of cultured rat liver cells (Hoshino, J., Kühne, U. and Kröger, H. (1982) Biochem. Biophys. Res. Commun. 105, 1446–1452), support the view that nicotinamide methylase and its product, 1-methylnicotinamide, are involved in the control of hepatocellular DNA synthesis and proliferation.

Nucleolar localization of protein B23 ( [formula omitted]) by immunocytochemical techniques

Highly acidic phosphoprotein B23 ( 37 5.1 ; M.W. x 10 3/ pI ) which is in preribosomal RNP particles in nucleoli of Novikoff hepatoma cells (1) was found to be one of the two major silver staining nucleolar proteins (2). An improved isolation method was developed for protein B23 which included 4 M urea/3 M LiCl extraction of nucleoli, dialysis of the extract against 4 M urea/20 mM Tris-malate/pH 5.5 and DEAE-cellulose chromatography. For studies on cellular localization of this protein, highly purified protein B23 was used to produce anti- B23 antibodies in rabbits. The specificity of the anti- B23 antibodies was demonstrated by formation of immunoprecipitin bands with the purified antigen and crude nucleolar extracts from Novikoff hepatoma cells. With the indirect peroxidase immunostaining method, a specific localization of protein B23 was demonstrated in the nucleoli of normal rat liver, thioacetamide-treated rat liver and Novikoff hepatoma cells.

Protein A24 lyase activity in nucleoli of thioacetamide-treated rat liver releases histone 2A and ubiquitin from conjugated protein A24.

Isolated liver nucleoli from rats treated for 3 days with thioacetamide contained an enzyme activity which specifically degraded conjugate protein A24. Two-dimensional polyacrylamide gel electrophoresis indicated that the amount of protein A24 in chromatin decreased during incubation at 37 degrees C for 60 min with these nucleoli. Concomitantly, a marked increase was found in the content of free ubiquitin, the nonhistone component of protein A24. Incubation of 3H-labeled protein A24 with the thioacetamide-treated liver nucleoli resulted in the linear release of 3H-labeled histone 2A and 3H-free ubiquitin in the presence of phenylmethanesulfonyl fluoride (PMSF) for 2 h. Pretreatment of the nucleoli with trypsin or by heating at 80 degrees C for 10 min inhibited their ability to cleave protein A24. Protein A24 lyase catalyzes the reaction: protein A24 leads to histone 2A plus ubiquitin.

Acetylation of spermidine in polyamine catabolism

Treatment with thioacetamide (150 mg/kg)_ was used to enhance polyamine metabolism in rat liver. The increased uptake and catabolism of [ 14C]spermine and the changes of putrescine, spermidine and spermine concentrations indicated enhanced polyamine turnover rates. The increase of hepatic putrescine concentration was accompanied by an increase of monoacetylputrescine and N 1-monoacetylspermidine concentration. In control animals, the latter compound was below detection levels. Thioacetamide treatment also enhanced putrescine excretion, which again was concomitant with an increased excretion of N 1-acetylspermidine. The close time-dependent correlation between induced putrescine formation and enhanced formation of N 1-acetylsperimidine at a time when liver spermidine and spermine concentrations are not changed, favors the notion that acetylation is an essential step in polyamine degradation and elimination. The increase of polyamine oxidase and decrease of acetylpolyamine deacetylase activities in the liver of thioacetamide-treated rats is in line with an increased polyamine turnover, but these enzymes. although essential, are not rate-limiting in the catabolic reactions.

Differential effects of dimethylsulfoxide on S-adenosylmethionine synthetase from rat liver and hepatoma

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Studies on the protein components of 110S and total ribonucleoprotein particles of rat liver nucleoli.

Proteins of rat liver nucleolar RNP, especially 110S RNP containing 45S RNA, were investigated and the following results were obtained. 1. Nucleolar extract was prepared from thioacetamide-treated rat liver nucleoli in the presence of 1 mg PVS per ml. Sucrose-density gradient centrifugation of the nucleolar extract showed RNP distributed between 60S and 110S. The 110S particles (110S RNP) contain 45S RNA as judged from the radioactivity profiles of nucleolar extract labelled in vivo with [3H]orotic acid for 10 min and 2 h. The protein components of 110S RNP and total RNP heavier than 60S (total RNP) were analyzed and compared. 2. Since the effects of PVS treatment on the extraction and the electrophoretic pattern of ribosomal proteins were removed by increasing the Mg2+ concentration during acetic acid extraction, as described in our preceding paper (1), proteins of 110S RNP as well as total RNP from PVS-pretreated nucleoli were extracted with 67% acetic acid containing 334 mM Mg2+ and analyzed by two-dimensional acrylamide gel electrophoresis. Ribosomal proteins masked by contaminating histone spots on the gel were identified by SDS-acrylamide gel electrophoresis. 3. 110S RNP contained a large portion of ribosomal proteins from large subunits; 24 protein spots were distinct, 6 protein spots were faint and 5 proteins (L3, L8, L30, L35, and L36) were missing completely. On the other hand, 110S RNP contained a small number of small subunit proteins; only 6 proteins showed distinct spots, 7 proteins showed faint spots and 12 protein spots were missing. 110S particles contained 11 non-ribosomal proteins, although it is difficult to rule out the possibility that some of them were contaminating chromatin proteins. 4. Total RNP showed electrophoretic patterns of 60S ribosomal proteins similar to those of 110S RNP, although L3 protein was present as a faint spot and 26 proteins showed distinct spots. Total RNP contained more small subunit proteins than 110S RNP; 7 proteins spots were distinct, 10 protein spots were faint and 8 protein spots were missing. The results suggest that some kinds of 40S proteins and L3 protein are attached to 110S RNP during the processing of 110S RNP.

Stabilization of ornithine decarboxylase in rat liver

The response of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity to cyloheximide has been measured in rat liver after various stimuli known to enhance the enzyme activity. While a single injection of cycloheximide almost totally (> 95%) inhibited the activity of ornithine decarboxylase in livers of growth-hormone treated rats at 45 min after the administration of the drug, this inhibition was much less striking in livers of partially hepatectomized (80%), of thioacetamide-treated (60%) and especially of carbon-tetrachloride treated (15%) animals thus suggesting a stabilization of the enzyme after the latter treatments. The measurement of the apparent half-life of ornithine decarboxylase after cycloheximide revealed a substantial decrease in the decay rate of the enzyme in livers of thioacetamide-treated rats. The apparent half-life of S- adenylsyl- l - methionine decarboxylase (EC 4.1.1.50) likewise appeared to be longer in rats previously treated with thioacetamide or carbon tetrachloride than in partially hepatctomized animals. Injection of thioacetamide also resulted in an initial stabilization of liver ornithine decarboxylase against 1,3-diaminopropane, a potent indirect inhibitor of the enzyme. However, 30 min after diaminopropane injection the enzyme activity started to fall extremely rapidly. This sudden drop in ornithine decarboxylase activity following diaminopropane could be largely prevented with cycloheximide. This finding clearly indicates that a protein-synthesis dependent formation of macromolecular inhibitors of ornithine decarboxylase may also be an early event after amine treatments. The striking stabilization of ornithine decarboxylase activity following these treatments, which are known to produce an initial liver necrosis (especially carbon tetrachloride and thioacetamide), may indicate that the system(s) responsible for the rapid inactivation of liver ornithine decarboxylase is more sensitive to tissue necrosis than the enzyme itself.

Differences in chromatin proteins of growing and non-growing tissues

The non-histone chromatin proteins of growing and non-growing tissues were compared by two-dimensional polyacrylamide gel electrophoresis. The tissues studied were normal rat liver, regenerating rat liver, thioacetamide-treated rat liver, normal rat kidney, Novikoff hepatoma and Walker 256 carcinosarcoma. Although most of the protein components were common to all of the tissues studied, the densities and sizes of spots C18, CP, C21, C25, CQ, CR, CS and CT were greater in the growing tissues than in the non-growing tissues, including the thioacetamide-treated liver. In the latter, the increased densities and sizes of spots C18, C21 and CQ are presumably related to the markedly increased nucleolar size rather than to cell division. Accordingly the increases in sizes and densities of spots C25, CP, CR, CS and CT are apparently of importance to the growth processes of normal and tumor tissues. The number of tissue specific proteins was small compared with the number of proteins in this fraction and includes BP and CBL for normal liver, BJ′ for kidney and CG′, CH′ and CP $ ́ for the tumors.

Studies on the processing of 45-S RNA in rat liver nucleolus with specific reference to 29.5-S RNA

Nucleolar RNAs prepared from rat liver labelled with [ 14C]- or [ 3H]orotic acid with cold and hot sodium dodecyl sulphate-phenol extraction, were analysed by means of polyacrylamide gel electrophoresis and the following results have been obtained. 1. 1. In nucleolar RNAs which are prepared with cold sodium dodecyl sulphatephenol extraction not only from normal, but also from thioacetamide-treated and regenerating rat livers, 29.5-S RNA is found in addition to 45-, 37-, 36-, 32-, 28-, 21.5- and 18-S RNAs, whereas in those with hot sodium dodecyl sulphate-phenol extracttion, 29.5-S RNA is not observed in all cases. Nucleolar 28-S RNA and cytoplasmic 28-S RNA of rat liver co-migrate in acrylamide gel electrophoresis and do not change their electrophoretic mobilities with hot sodium dodecyl sulphate-phenol extraction or heat treatment, indicating that the presence of 29.5-S RNA is not due to the conformational change of 28-S RNA. The 37-S minor component is very unstable in the case of normal rat liver, but stable in the case of thioacetamide-treated rat liver. 29.5-S and 37-S RNAs are thought to be newly found RNAs using the cold sodium dodecyl sulphate-phenol extraction method. 2. 2. Taking the kinetics of labelling, the results of actinomycin D chase experiment and the molecular weights of the nucleolar RNA components together, may indicate that 29.5-S RNA is the metabolic intermediate between 32-S and 28-S RNAs, and 37-S RNA is suggested to be the direct precursor of 36-S RNA. The following scheme for the processing of 45-S RNA in rat liver nucleoli is proposed: ▪

Conversion of rat liver nucleolar 29.5-S RNA to 28-S RNA in vitro

By employing nucleolar RNA, especially 28-S and 29.5-S RNAs, isolated from thioacetamide-treated rat liver nucleoli which were labelled with [ 14C]- or [ 3H]orotic acid, the conversion of 29.5-S RNA to 28-S RNA was investigated and the following results have been obtained. 1. 1. Nucleolar 29.5-S RNA disappears with incubation for 13 min, in the sodium acetate buffer (pH 7.0) above 42 °C, in sodium dodecyl sulphate-phenol above 37 °C and in 6 M urea above 25 °C. The slow cooling after heat treatment of 29.5-S RNA does not result in the reappearance of the RNA, suggesting the disappearance to be an irreversible process. The disappearance caused by heat treatment is inhibited by divalent metal ions (Mg 2+, Ca 2+, Ba 2+, Co 2+ and Mn 2+). 2. 2. With incubation in 10 mM sodium acetate buffer (pH 7.0) at 45 °C for 13 min, nucleolar 28-S RNA decreases to 60–75 %. The decrease is accompanied by the appearance of two low molecular weight RNAs (5.3-S and 6.7-S RNAs) in addition to the heterogeneous RNAs distributed from the 28-S to 12-S regions on acrylamide gel electrophoresis. 3. 3. Under the same conditions as above, the conversion of 29.5-S to 28-S RNAs occurs, which accompanies the release of a specific x component with a molecular weight of 180 000 corresponding to the difference in the molecular weight between 29.5-S and 28-S RNAs, although the heterogeneous RNAs distributed from the 28-S to 12-S regions are also observed. The x component has a similar specific activity to that of 29.5-S RNA after labelling periods of 2 and 6 h. The possible mechanism of the conversion of 29.5-S RNA to 28-S RNA is discussed.

Studies on ornithine decarboxylase from the liver of thioacetamide-treated rats Purification and some properties

Ornithine decarboxylase ( l-ornithine carboxy-lyase, EC 4.1.1.17) was purified about 5400-fold from the soluble fraction of liver from thioacetamide-treated rats. The purified enzyme showed only a single protein band on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be approx. 100 000. Its isoelectric point was found to be at pH 4.1 and its optimal pH around 7.0. The K m value for l-ornithine was 2·10 −4 M at pH 7.0. The enzyme required thiol compounds for maximal activity and was inhibited by inhibitors of pyridoxal enzymes, such as l-canaline and isonicotinic acid hydrazide. Among the various amino acids and amines tested, putrescine and d-ornithine caused a weak inhibition, while other compounds had little effect. The properties of ornithine decarboxylase from the liver of thioacetamide-treated rats were compared with those of enzyme from regenerating rat liver.

Isolation of an electrophoretically homogeneous nonhistone nucleolar protein

The first nucleolar protein to be isolated in an electrophoretically homogeneous state was extracted with 0.4 N H 2SO 4 from isolated nucleoli of Novikoff hepatoma ascites cells and separated as a slow moving distinct band by slab gel electrophoresis from the remaining nucleolar proteins. This protein, which has a molecular weight of approximately 65,000, accounts for 3–5% of the total acid-extractable nucleolar proteins. Its ratio of acidic to basic amino acids is 1.7 and serine is its amino terminal amino acid, as shown by thin layer chromatography of its dinitrophenyl derivative. The same band was also found in electrophoretic patterns of acid-extractable proteins in nucleoli of normal and thioacetamide-treated rat liver.