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1.
Administration of 3-methylcholanthrene (3MC) to rats greatly enhanced the aryl hydrocarbon hydroxylase (AHH) activity of liver nuclei. However, the binding in vitro [3H]benzo[alpha]pyrene (BP) to DNA within the nuclei which occurred at the same time as hydroxylation of BP was much less enhanced. Thin layer chromatography of the metabolites of BP produced by these nuclei revealed the same metabolites in similar relative amounts as were produced by rat liver microsomes prepared from rats which had received 3MC. The binding to DNA was further analysed by hydrolysis of the DNA and fractionation on a Sephadex column. This analysis revealed that the binding to DAN in nuclei was very similar in nature to that which occurred when calf-thymus DNA was added to microsomes metabolising BP.  相似文献   

2.
To investigate whether cytochrome P-450 catalyzes the covalent binding of substrates to DNA by one-electron oxidation, the ability of both uninduced and 3-methylcholanthrene (MC) induced rat liver microsomes and nuclei to catalyze covalent binding of benzo[a]pyrene (BP) to DNA and formation of the labile adduct 7-(benzo[a]pyren-6-yl)guanine (BP-N7Gua) was investigated. This adduct arises from the reaction of the BP radical cation at C-6 with the nucleophilic N-7 of the guanine moiety. In the various systems studied, 1-9 times more BP-N7Gua adduct was isolated than the total amount of stable BP adducts in the DNA. The specific cytochrome P-450 inhibitor 2-[(4,6-dichloro-o-biphenyl)oxy]ethylamine hydrobromide (DPEA) reduced or eliminated BP metabolism, binding of BP to DNA, and formation of BP-N7Gua by cytochrome P-450 in both microsomes and nuclei. The effects of the antioxidants cysteine, glutathione, and p-methoxythiophenol were also investigated. Although cysteine had no effect on the microsome-catalyzed processes, glutathione and p-methoxythiophenol inhibited BP metabolism, binding of BP to DNA, and formation of BP-N7Gua by cytochrome P-450 in both microsomes and nuclei. The decreased levels of binding of BP to DNA in the presence of glutathione or p-methoxythiophenol are matched by decreased amounts of BP-N7Gua adduct and of stable BP-DNA adducts detected by the 32P-postlabeling technique. This study represents the first demonstration of cytochrome P-450 mediating covalent binding of substrates to DNA via one-electron oxidation and suggests that this enzyme can catalyze peroxidase-type electron-transfer reactions.  相似文献   

3.
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.  相似文献   

4.
The role of DNAs in the nuclear binding of dexamethasone-receptor complexes (DRC) was studied. The cytosolic receptors from rat liver have a sedimentation coefficient of about 7S, the Stock's radius--of about 50 A and possess a high affinity to dexamethasone (Kas = 2,6 X 10(8) M-1). Their capacity is 3 X 10(-13) and 5.5--7.0 X 10(-12) mole of dexamethasone per mg cytosolic protein and mg DNA, respectively. DRC has the ability to bind to the nuclei of rat liver. DRC binding to nuclei is increased approximately 3-fold by temperature activation of cytosol. The nuclear acceptor sites are saturated at the level of 16.2 pmoles of bound DRC per mg nuclear DNA. Free DNA has the ability to compete with nuclei for binding with DRC. Temperature-activated DRC can bind both with homo- and heterologous DNAs. Secondary DRC-DNA complexes were isolated by means of gel filtration on Sepharose 4B. Thermal denaturation of DNA decreases its ability to bind DRC approximately 2-fold. DNAs of a similar nucleotide composition, i.e. DNA from rat liver (GC = 43 mole%) and DNA from Photobacterium belozerskii (GC = 44 mole%), have a close DRC-binding ability. At the same time, these DNAs bind about 1.5-fold less DRC, as compared to DNA from Pseudomonas aeruginosa (GC = 67 mole%) and about 1.5-fold more, than does DNA from T2 phage (GC = 35 mole%). Thus the positive correlation between the GC composition of DNA and its DRC-binding ability was established. Unique sequences (Cot greater than 600) bind several times less DRC than the reiterated sequences (also denaturated) (Cot = O--600) of rate liver DNA. Thus, DNA can be considered as a nuclear acceptor of DRC. It is assumed, that DRC is able to recognise in DNA certain short GC-rich sequences, distributed in the rate genome in a non-random fashion.  相似文献   

5.
The interaction of nickel(II) with chromatin was studied in vitro and in isolated nuclei from rat liver and kidney. Nickel(II) bound to chromatin, polynucleosomes (DNA + histone octamer protein complex), and to deproteinized DNA both in intact nuclei and in vitro. The amount of nickel(II) bound depended on the concentration of nickel(II), the presence of chromosomal proteins and the binding sites on DNA which provide a stable coordination environment for nickel(II). The binding of nickel(II) to chromatin and to DNA in whole nuclei was much slower than in vitro indicating that assessibility of the DNA binding sites was influenced by the presence of the nuclear membrane, nuclear matrix and nuclear proteins and/or by the condensed nuclear structure of chromatin. Since DNA containing bound nickel(II) was isolated from chromatin, nickel(II) directly interacted with stable binding sites on the DNA molecule in chromatin. Nickel(II) was associated with the histone and non-histone nuclear proteins as well as the DNA in rat liver and kidney chromatin. Nickel(II) was found to bind to calf thymus histones in vitro. Nickel(II)-nuclear protein and -DNA interactions were investigated by gel electrophoretic analysis of in vitro incubation products. Although nickel-histone and nickel-non-histone protein interactions were completely disrupted by the electrophoretic conditions, fluorography revealed the presence of inert nickel(II)-DNA and/or nickel(II)-DNA-protein complexes.  相似文献   

6.
A large sedimentation device was developed that allows separation of 5 × 108 rat liver nuclei by velocity sedimentation at unit gravity. Using the apparatus isolated rat liver nuclei were separated into classes of diploid stromal (Von Kuppfer, sinusoidal lining) nuclei, diploid parenchymal nuclei and tetraploid parenchymal nuclei respectively. DNA content and volume of the nuclei were measured. Diploid nuclei were 100% pure; tetraploid nuclei 98%.The in vivo binding of the liver carcinogen [3H]-N-hydroxy-AAF to these classes of nuclei was determined (total binding to protein, DNA and RNA). Binding and the subsequent removal of the fluorene derivatives was registered as a function of time. At all stages diploid stromal nuclei bound 2.6–5 times less carcinogen than did diploid parenchymal nuclei. Tetraploid parenchymal nuclei bound more than twice (2.3–3.95) the amount, that was present in their diploid counterpart. This effect became more pronounced 11 days after application of N-hydroxy-N-acetyl-2-aminofluorene.DNA was enzymatically purified from pooled classes of the various nuclear types. For purified DNA also it was found that DNA derived from diploid stromal nuclei bound 2.6–2.8 times less carcinogen than did DNA derived from diploid parenchymal nuclei.  相似文献   

7.
The rate of [14C]NAD incorporation into chicken liver nuclear histones was studied under conditions of DNA damage by N-methyl-N-nitrosourea and pancreatic DNAase I. With an increase in N-methyl-N-nitrosourea concentration from 8.5 X 10(-2) to 34.0 X 10(-2) mM, the ADP ribosylation of histones increases by 20% as compared to the control. In DNAase I-treated nuclei, the binding by histones of [14C]NAD sharply increases, reaching its maximum (18.3 X 10(-8) mM) at 30% cleavage of DNA. When 50% of DNA was cleaved, the rate of [14C]NAD incorporation into the histones was 8.0 X 10(-8) mM as compared to 6.1 X 10(-8) mM/mg protein in control samples. The poly(ADPR)polymerase activity was increased in both cases. It was shown that the NAD-pyrophosphorylase activity in chicken liver nuclei treated with N-methyl-N-nitrosourea does not differ from the control one, while in DNAase I-treated nuclei the maximum of the NAD-pyrophosphorylase activity was achieved, as well as the maximum of [14]NAD incorporation into the histones within the range of DNA damage of 25-35%, being equal to 37 X 10(-8) mM NAD/min/mg protein as compared to 26.0 X 10(-8) mM/min/mg protein in the control. At different degrees of DNA damage, the average length of the poly-ADP-ribose chain did not practically alter, thus suggesting the increase in the number of polymer binding sites in the histones.  相似文献   

8.
Estrogen receptor (ER) from chicken liver and calf uterus were used to study the capacity and the characteristics of the receptor binding sites (acceptor sites) in chicken target cell nuclei. Binding studies were performed at a physiological salt concentration of 0.15 M KCl. Binding of liver ER to liver nuclei was temperature-dependent, showing a 9-fold increase between 0 and 28 degrees C. The maximal number of acceptor sites measured in this cell-free system (280 sites/nucleus) was considerably lower than measured in nuclei after in vivo administration of estrogen (820 sites/nucleus). Moreover incubation of nuclei with the liver ER preparation resulted in a substantial breakdown of nuclear DNA, making this ER less suitable for DNA binding studies. The temperature-activated calf uterine receptor bound to liver nuclei at 0 degrees C, at which temperature no DNA degradation was measured. To all chicken cell nuclei tested, the receptor bound with a high affinity (Kd = 0.4-1.0 nM). Nuclear binding displayed tissue specificity: oviduct greater than heart, liver greater than spleen greater than erythrocytes and was salt dependent. Calf uterine ER binding in liver nuclei ranged from 3000-6000 acceptor sites per nucleus when assayed under conditions of a constant protein or a constant DNA concentration. Nuclei isolated from estrogen-treated cockerels bound a 2-fold lower number of calf uterine ER complexes when compared to control nuclei. Incubation of nuclei with a fixed concentration of [3H]ER from liver and increasing concentrations of uterine non-radioactive-ER also resulted in a reduced binding of the liver receptor. Both types of experiments suggest that liver and uterine ER compete for a common nuclear acceptor site. Our data demonstrate that the ER from calf uterus is very useful as a probe to examine the nature of the acceptor sites in heterologous chicken target cell nuclei. The assay system functions at 0 degrees C, a temperature at which no DNA degradation occurs.  相似文献   

9.
To examine the binding specificity of steroid hormone-cytoplasmic receptor complexes to nuclei, binding of 3H-dexamethasone (Dex)-liver, 3H-Dex-thymus and 3H-dihydrotestosterone (DHT)-prostate receptor complexes to nuclei from liver, prostate, thymus, spleen and kidney was studied. It was observed that a significant amount of steroid-receptor complexes was bound to any nuclei used in the present study and the extent of the binding of receptor complexes to nuclei from homologous tissues was not always greater than that to nuclei from heterogenous tissues. However, a significant portion of the 3H-Dex-liver and 3H-DHT-prostate receptor complexes was not absorbed by nuclei from kidney, spleem, and thymus, and the unabsorbed complexes were efficiently bound to liver and prostate nuclei. The results obtained indicate that two types of receptor complex with regard to nuclear binding were present in cytosols of liver and prostate; one binds to nuclei from kidney, spleen, thymus, liver and prostate and the other does not bind to nuclei from kidney, spleen and thymus but does bind to nuclei of liver and prostate. The latter type of receptor complex was not observed in the cytosol from the thymus.  相似文献   

10.
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.  相似文献   

11.
Iron-induced DNA damage and synthesis in isolated rat liver nuclei.   总被引:3,自引:1,他引:2       下载免费PDF全文
Incubation of iron with isolated rat liver nuclei stimulated fragmentation of single-stranded DNA, incorporation of [3H]thymidine into DNA and the binding of 59Fe to DNA. FeCl2 was about twice as active as FeCl3. Lipid peroxidation took place in nuclei incubated with FeCl2, but not with FeCl3. Generation of reactive forms of oxygen was required for iron-mediated DNA damage, but evidence for direct interaction of reactive oxygen with DNA was not found. Apparent adducts of iron bound to DNA seemed to be formed by an enzymic mechanism.  相似文献   

12.
The formation of benzo[a]pyrene (BP)-DNA adducts was studied in vitro in the presence of microsomes prepared from the isolated labyrinth zone of the rat placenta, the hematopoietic erythroblast cells of the fetal liver, the fetal liver, as well as the maternal liver. Pregnant rats received beta-naphthoflavone (beta NF; 15 mg/kg, i.p.) on day 17 gestation. One day later, placentae, fetal and maternal livers were obtained and hematopoietic erythroblast cells were separated from hepatocytes in the fetal livers. The respective microsomal fractions were incubated in the presence of calf thymus DNA, NADPH-regenerating system and [3H]BP (300 microCi) at 37 degrees C for 30 min. Following beta NF pretreatment, the levels of covalent binding (pmol/mg DNA/mg microsomal protein) for maternal liver, fetal liver, placenta and erythroblast cells were: 28.4, 2.4, 0.31 and 3.9, respectively, with the hematopoietic erythroblast cells being the most active among fetal tissue preparations. The extent of transplacental induction compared to control was greatest in the hematopoietic cells (18-fold) followed by fetal liver (16-fold) and labyrinth zone (5-fold). Further experiments characterized the BP-DNA adducts formed by induced microsomes. DNA was isolated, purified and digested sequentially with DNase I, snake venom phosphodiesterase type II and alkaline phosphatase type III. The deoxynucleoside-BP adducts were purified on a Sephadex LH-20 column and then separated on HPLC and the adducts were quantitated radiometrically. Seven distinct adducts were separated on HPLC and named A-G in order of elution. Adduct B was prominent in all preparations (22-55% total radioactivity). The adduct profile and retention time for peak B is similar to that reported for the adduct formed by microsomal activation of 9-hydroxy BP. Peak D constituted a major fraction (19%) in maternal liver profiles in comparison with the three fetal tissue preparations (8%). In subsequent experiments, peak D was shown to be derived from reaction of (+/-)7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) with DNA. Peak C was unique to erythroblast cell and labyrinth profiles, while peak G was specific for maternal liver and fetal liver profiles. These results demonstrate that fetal liver and its hematopoietic cells are significant sites of BP bioactivation which may contribute to the fetal toxicity of polyaromatic hydrocarbons.  相似文献   

13.
Neuron-rich and glial nuclear preparations and liver nuclei were isolated from adult guinea pigs. These nuclei were incubated to carry out DNA-ligation and -synthesis reactions. Before and after incubation, the sizes of single-standed DNA and DNA-synthesis patterns in single strands were analysed by using alkaline sucrose-density-gradient centrifugation. Isolation of nuclei by cell-fractionation technique shortened chromatin DNA and decreased markedly the number-average molecular weight of DNA strands. Chromatin DNA in neuronal and glial nuclei was ligated at the nicks during incubation in a reaction mixture containing ATP, Mg(2+), dithiothreitol and four deoxyribonucleotides. The number-average molecular weights were estimated to increase 1.1-and 2.1-fold in neuronal and glial nuclei respectively. DNA strands in liver nuclei were shortened during incubation, but elongated under conditions that inhibit deoxyribonuclease. Since the endogenous deoxyribounuclease activity was conspicuously higher in liver nuclei than in neuronal and glial nuclei, the shortening and elongation were thought to depend on the balance between DNA ligase and deoxyribonuclease reactions. DNA synthesis occurred at the gaps in chromatin DNA and about 50% of the total synthesized DNA was found in the shorter strands having 6 to 297 bases in all species of nuclei. Based on these results, it was concluded that in nuclei isolated from non-dividing cells (neurons) and slowly dividing cells (glial and liver cells) DNA-ligation and -synthesis reactions proceeded in parallel at the breaks in single-stranded DNA, which was produced mainly by endogenous deoxyribonuclease during isolation and incubation processes.  相似文献   

14.
1. Male rats were injected intravenously with amounts ranging from 0.08 to 111.0mumoles of [(7)Be]beryllium sulphate/kg. body wt. The distribution in the rat and the subcellular distribution of beryllium in the liver were determined. 2. Within the entire dose range a higher specific activity of beryllium was present in a mitochondrial fraction containing the lysosomes. Purification of this fraction confirmed that beryllium is taken up by lysosomes. 3. With doses approaching the LD(50), beryllium was also found in increasing amounts to be present in the liver cell nuclei. Beryllium also showed affinity towards isolated cell nuclei in vitro. Evidence is presented that they have one class of binding sites for beryllium. Mitochondria have less affinity for beryllium. 4. No evidence could be obtained of an affinity of beryllium for DNA or RNA by fractionation of nuclei and dialysis experiments. 5. The presence of beryllium in liver cell nuclei may be relevant to the effects of beryllium on nuclear structure and function.  相似文献   

15.
Receptor binding in the rat liver nuclear matrix   总被引:1,自引:0,他引:1  
3H-Dexamethasone (Dex)-receptor complexes prepared from the rat liver cytosol efficiently bound to the nuclear matrix from the same tissue. The binding was increased with the concentration of the 3H-Dex-receptor complex added and reached a maximum plateau. However, when the partially purified 3H-Dex-receptor complex was used, saturation of the binding sites in the nuclear matrix was not observed in the range of concentration of 3H-Dex-receptor complex used. Therefore, it was considered that the apparent saturability observed in the binding of the unpurified receptor complexes is caused by the translocation inhibitor(s) in the cytosol. When the binding capacity was expressed on the basis of unit weight of DNA, the nuclear matrix exhibited 20 times more of that of the unfractionated nuclei. However, no line of evidence of enrichment of the binding sites in the DNA isolated from the nuclear matrix was observed. These observations show that the role of the nuclear matrix in the action of glucocorticoid is quite uncertain.  相似文献   

16.
The in vivo binding of [3H]benzo(a)pyrene (BP) and 3-[3H]methylcholanthrene (3MC) to liver and lung DNA was studied in A/J mice. Only in liver was there any reduction in total DNA-bound radioactivity between 4 h and 24 h after administration of the hydrocarbon. DNA was fractionated on Sephadex LH-20 after enzymatic digestion. A single deoxyribonucleoside-BP adduct was detected whereas two major 3MC-adducts were observed. With both BP and 3MC, three additional peaks of radioactivity eluted rapidly in the lung DNA experiments while a fourth was noted with liver DNA. The nucleoside-bound adducts from lung represented a much larger proportion of the total radioactivity than with liver. In vitro analysis of 3MC binding to DNA showed the nucleoside-bound adducts to be predominantly deoxyguanosine-dependent but that the early peaks were independent of base suggesting binding to another part of the DNA molecule, perhaps phosphate, i.e., phosphotriesters.  相似文献   

17.
Putative thyroid hormone (TH) receptors have been demonstrated in the isolated liver nuclei of Singi fish, Heteropneustes fossilis (Bloch), and their binding characteristics have been examined. Nuclear T3 saturation analyses were carried out in vitro at 27 degrees C in a sucrose-Tris-HCl buffer (pH 7.5) containing calcium (2 mM), magnesium (3 mM) and 2-mercaptoethanol (5 mM). After incubation the bound and free hormones were separated by centrifugation and the nuclei were treated with Triton X-100 (final concentration 0.25%) to reduce the non-specific binding. The binding was saturable and reached equilibrium by 20 minutes of incubation and was also stable for 2 hours. The binding was reversible and the rate of dissociation was more or less equal to the rate of association. The binding was linearly increased with the increased concentrations of the DNA (nuclei). Scatchard analyses of the equilibrium binding data revealed that only one class of binding sites for T3 did exist in the hepatic nuclei of Singi fish. The affinity of these sites or the mean dissociation constant (Kd = 0.20 +/- 0.07 x 10(-10) M) and the mean maximum binding capacity (MBC = 0.17 +/- 0.04 pmol/mg DNA) were in reasonable agreement with the values reported for other teleost fishes.  相似文献   

18.
To elucidate the role of poly(ADP-Rib) in the nucleus, DNA synthesis and DNA fragmentation were studied in isolated nuclei of rat liver and rat ascites hepatoma AH-130 cells. Liver and hepatoma cell nuclei formed the same amount of poly(ADP-Rib) per mg of nuclear DNA from NAD. Preincubation of liver nuclei with NAD repressed DNA polymerase activity to 30% of that of the control, but preincubation of hepatoma cell nuclei with NAD did not affect DNA polymerase activity. It was also found that incubation of liver nuclei with NAD prevented the fragmentation of nuclear DNA which occurred without NAD. Incubation of hepatoma cell nuclei with or without NAD did not result in fragmentation of DNA. The role of endonuclease in primer formation for DNA synthesis is discussed.  相似文献   

19.
M Yamaguchi  T Sakurai 《FEBS letters》1991,279(2):281-284
Incubation of isolated rat liver nuclei with ATP, NAD+, and micromolar Ca2+ concentrations of various metal ions resulted in extensive DNA hydrolysis. Half-maximal activity occurred with 1.0 microM Ca2+ added, and saturation of the process was observed with 10 microM Ca2+. The Ca2+ (10 microM)-activated DNA fragmentation was inhibited by the presence of Ca2(+)-binding protein regucalcin isolated from rat liver cytosol. The inhibitory effect of regucalcin was complete at 0.5 microM. At 25 microM Ca2+ added, such an effect of regucalcin (1.0 microM) was not seen. Regucalcin also inhibited Ca2(+)-activated DNA fragmentation in the presence of calmodulin (10 and 20 micrograms). The results show that regucalcin can inhibit the Ca2(+)-activated DNA fragmentation due to binding the metal, suggesting a role in regulation of liver nuclear functions.  相似文献   

20.
Insulin binding to the plasma membrane is known to be altered by modifying the membrane composition through dietary treatment. As insulin binding receptors are also present on nuclear membrane, this study was undertaken to investigate if specific binding of insulin to the liver nuclei is altered by diet. 8-wk-old female C57 B 6J lean and ob/ob mice were fed semipurified diets containing 20% (w/w) fat of either high or low polyunsaturated-to-saturated (P/S) fatty acid ratio for 4 wk. Liver nuclei were prepared, insulin binding was measured and nuclear phospholipids were isolated for lipid analysis. Insulin binding was highest in nuclei prepared from lean mice fed a high P/S diet. Specific binding of insulin to nuclei prepared from obese mice was also increased by the high P/S diet, but to a lesser extent compared to lean mice. Feeding a high P/S diet increased polyunsaturated fatty acid content of membrane phospholipids from both lean and ob/ob mice. Obese mice were characterized by higher levels of arachidonic acid and lower levels of linoleic acid in phosphatidylcholine. The present study establishes that insulin binding to liver nuclei is increased by feeding a high P/S diet, and that insulin binding to liver nuclei from obese mice is lower than from lean mice.  相似文献   

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