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1.
Chromatin was prepared from mouse liver and incubated in an invitro binding assay containing 3H-benzo(a)pyrene and a NADPH-generating system. Binding to chromatin DNA was stimulated by the presence of microsomes from 3-methylcholanthrene pretreated mice. This incubation system represents an improvement over previous studies in which purified DNA is employed as the target macromolecule in that aralkylation is being investigated under conditions which better approximate those present in the cell, i.e., the genetic material is “coated” with nuclear protein.  相似文献   

2.
Natural and synthetic estrogens can be activated by rat liver microsomes to bind covalently to polyguanylic acid, single-stranded DNA, nucleotides and proteins. Incubation of polyG, estrone and liver microsomes (0.5 nmole cytochrome P-448 or P-450) from 3-methylcholanthrene-induced, phenobarbital-induced or control rats showed that the former microsomes gave better net binding of estrogens to polyG than the other two. Estradiol incubated with 3MC-induced microsomes did bind to DNA but marginally to polyG. Mestranol and estrone sulfate, both constituents of oral contraceptive formulations, bound to polyG whereas progesterone and cholesterol did not bind. We present also preliminary data on the characterization of estrogen-nucleic acid interactions using nucleases, proteinase K and high-pressure liquid chromatography.  相似文献   

3.
Nonhistone protein BAfree was purified from the 0.075 M NaCl/0.025 M EDTApH 8 extract of whole rat liver nuclei while protein BAbound was isolated from the 0.05 M Na2HPO4/8 M urea/1% β-mercaptoethanol/pH 7.6 extract of dehistonized rat liver chromatin. Chromatin associated protein BAbound was able to bind 60% of the [3H] DNA in a nitrocellulose filter binding assay while nucleoplasmic protein BAfree showed essentially no DNA binding activity. Circular dichroism analysis of the two forms of protein BA revealed substantial differences in their conformations. Protein BAfree was found to have an α-helix content of 41% while protein BAbound displayed a spectrum more typical of unordered or β-turn structures.  相似文献   

4.
Squalene epoxidation by rat liver microsomes requires a supernatant protein factor and an acidic phospholipid in addition to NADPH and molecular oxygen. This study has shown that both the protein factor and the phospholipid lipid are necessary for externally added squalene to bind to the catalytic site on microsomal membranes. The epoxidation of squalene thus bound or biosynthesized insitu from mevalonic acid proceeds effectively if the protein factor is present. Thus, the supernatant protein factor seems to play a dual function in both the binding and epoxidation of squalene in the invitro assay system. The phospholipid is not required for the epoxidation of bound squalene.  相似文献   

5.
6.
3-Hydroxy-3-methylglutaryl coenzyme A reductase activity is diminished in several in vitro liver systems preincubated in the presence of cAMP. Reductase activity in isolated, washed liver microsomes is inactivated by ATP, Mg++, and a protein fraction separated from the liver cytosol. This effect is augmented by 3′–5′ cyclic AMP. Reductase activity in previously inactivated microsomes can be partially restored by incubation with a second protein fraction of the cytosol.  相似文献   

7.
8.
Male, weanling rats divided into three groups were maintained for 15 days on a semipurified diet containing either 5% casein fed ad libitum (group 1), 20% casein pair-fed to group 1 (group 2), or 20% casein fed ad libitum (group 3). Animals on day 16 were injected i.p. with 3H-AFB1 (1.90 mg/kg) and were sacrificed six hours later. In both the control and protein deficient animals, binding of AFB1 to DNA was greater than that for chromatin protein. In the protein deficient animals, there was a consistent decrease (70%) in binding to chromatin, DNA and chromatin protein. The decrease in binding to nuclear macromolecules in protein deficient animals is correlated with carcinogenicity and mixed function oxidase (MFO) enzyme activity, and the relationships between carcinogenicity, MFO activity, and binding are discussed.  相似文献   

9.
Injection of [3H]aflatoxin B1 into rats yielded covalently bound derivatives in hepatic DNA, rRNA, and protein. Mild acid hydrolysis of the DNA and rRNA adducts formed a derivative indistinguishable from 2,3-dihydro-2,3-dihydroxy-aflatoxin B1. The data indicate that approximately 60% of the nucleic acid adducts were derived from reactions in vivo with aflatoxin B1-2,3-oxide. Acid hydrolysis of rRNA-[3Haflatoxin B1 adduct formed by human liver microsomes in vitro also liberated the dihydrodiol in significant amount. The 2,3-oxide of aflatoxin B1 is a probable ultimate carcinogenic metabolite.  相似文献   

10.
(3H) 3-Methylcholanthrene binds in vivo to a macromolecule in addition to the previously reported binding to ligandin in liver cytosol. The properties of this second molecule are identical to those of the glucocorticosteroid receptor (Binder II) through 400 fold purification over the cytosol proteins (elution position from DEAE-Sephadex A-50 columns, molecular weight by gel filtration and pI value by isoelectrofocusing). The carcinogen, probably a metabolite, binds very strongly or covalently to the macromolecule in vivo, but non-covalently in vitro in the absence of microsomes. Large amounts of unlabeled carcinogen administered in vivo do not compete significantly with subsequent (3H) dexamethasone binding to the hormone receptor fraction in vitro. Methylcholanthrene and dexamethasone do not compete for binding sites in vitro on isolated unlabeled Binder II leading to the conclusion that the glucocorticosteroid receptor and the methylcholanthrene binding protein are distinct entities.  相似文献   

11.
Isolated rat liver microsomes were subjected to enzymatic or non-enzymatic lipid peroxidation in vitro. NADPH-dependent cytochrome c reductase activity was released from the microsomes into the media during peroxidation. This activity could be recovered from the media by DEAE-cellulose chromatography. The recovered enzyme retained high activity for the reduction of cytochrome c and a lower level of activity for the reduction of cytochrome P-450. The active fractions were capable of enzymatically supporting the peroxidation of isolated mitochondria in the presence of organically complexed Fe+3 and NADPH, and in this respect the specific activity was found to be about ten times higher than in microsomes.  相似文献   

12.
In order to define the site of bioactivation of CCl4, CHCl3 and CBrCl3 in the NADPH cytochrome c reductase-cytochrome P-450 coupled systems of liver microsomes, the 14C-labeled hepatotoxins were incubated invitro with isolated rat liver microsomes and a NADPH-generating system. The covalent binding of radiolabel to microsomal protein was used as a measure of the conversion of the hepatotoxins to reactive intermediates. Omission of NADPH, incubation under CO:O2 (8:2) and addition of a cytochrome c reductase specific antisera mardedly reduced the covalent binding of all three compounds. When cytochrome P-450 was reduced to less than 25% of normal by pretreatment of rats with allylisopropylacetamide (AIA), but cytochrome c reductase activity was unchanged, the covalent binding of CCl4, CHCl3, and CBrCl3 was decreased by 63, 83, 70%, respectively. Incubation under an atmosphere of N2 enhanced the binding of CCl4, inhibited the binding of CHCl3 and did not influence the binding of CBrCl3. It is concluded that cytochrome P-450 is the site of bioactivation of these three compounds rather than NADPH cytochrome c reductase and that CCl4 bioactivation proceeds by cytochrome P-450 dependent reductive pathways, while CHCl3 activation proceeds by cytochrome P-450 dependent oxidative pathways.  相似文献   

13.
The template activity of isolated rat liver nuclei for DNA synthesis assayed with E.coli DNA polymerase was found to be dependent upon the presence of Ca2+ or Mg2+ in the incubation medium. DNA was prepared from isolated nuclei subjected to conditions which activated the template and centrifuged in an alkaline sucrose gradient. The distribution profile showed that smaller fragments were formed, suggesting enhancement of endonucleolytic activity. When isolated nuclei were incubated with NAD to induce poly(adenosine diphosphate ribose) formation and were subjected to the activation conditions, the template for DNA synthesis remained unchanged. The distribution profile in an alkaline sucrose gradient of DNA prepared from these nuclei and control nuclei was identical. The present findings suggest that the template-activating system for DNA synthesis was blocked when isolated nuclei were treated with NAD invitro.  相似文献   

14.
Sub-nuclear fractionation. I. Procedure and characterization of fractions   总被引:7,自引:0,他引:7  
A procedure for fractionation of nuclei from rat liver, Xenopus liver and Xenopus erythrocytes is described. It is based on mild sonication of isolated nuclei for 7–12 sec in a nearly isotonic medium, separation of nuclear sap and centrifugation on a discontinuous sucrose density gradient containing Na and K citrate. Nuclei are thus separated in a single operation into 8 fractions representing nucleoplasm, euchromatin, nucleoli, heterochromatin and nuclear membranes. The sub-nuclear fractions were characterized by chemical composition (DNA, protein, RNA and phospholipid), electron microscopy, thermal denaturation properties of chromatin, relative binding of 3H-actinomycin D, polyacrylamide gel electrophoresis of nuclear proteins and titration of membranes against Triton X-100. Approx. 10% of total DNA was recovered as heterochromatin associated with membranes but the bulk of nuclear membranes co-sedimented with the major euchromatin zones. Subnuclear fractions prepared in this way retain virtually all the RNA polymerase activity bound to chromatin [41].  相似文献   

15.
Three day progesterone treatment of ovariectomized rabbits increased invitro uterine estrogen-receptor binding to uterine chromatin. The increased binding was traced to changes in chromatin but not the cytosol. Both the number of chromatin acceptor sites and the binding affinity were higher in treated animals. Furthermore, chromatin acidic protein to DNA ratios from treated rabbits were higher by approximately the same factor as for binding sites. A mechanism of synergistic interaction is suggested.  相似文献   

16.
Microsomes isolated from whole rat brain were found to contain cytochreme P-450 (0.025 to 0.051 nmoles/mg) and NADPH cytochrome c reductase activity (26.0 to 55.0 nmoles/mg/min). The oxidation of estradiol to a reactive metabolite that became covalently bound to rat brain microsomal protein was inhibited 63% by an atmosphere of CO:O2 (9:1), indicating the involvement of a cytochrome P-450 oxygenase. In contrast, this atmosphere had no effect on the binding of either the catechol estrogen, 2-hydroxyestradiol, or several catecholamines to rat brain microsomes. An antibody prepared against NADPH cytochrome c reductase was found to decrease significantly both the formation of 2-hydroxyestradiol from estradiol by rat brain microsomes and the covalent binding of the catechol estrogen and catecholamines to rat brain microsomal protein.  相似文献   

17.
Benzo[a]pyrene became bound to the hepatic DNA in juvenile English sole (Parophrys vetulus) force fed tritiated benzo[a]pyrene. No statistically signïficant change was observed in the level of the binding from 16 h to 2 wk after the single exposure. Specific activities of binding were similar for both DNA and protein. Moreover, a binding index was calculated to represent the number of benzo[a]pyrene molecules bound per 106 nucleotides after administration of a theoretical dose of 1 mmole of hydrocarbon per kg body weight. The value for English sole liver DNA was of the same order of magnitude as the values reported for mouse skin and mammary gland in which benzo[a]pyrene is carcinogenic.  相似文献   

18.
Beef brain microsomes bound approximately 180–220 pmoles of [3H]ouabain per mg of protein in the presence of either MgCl2 and inorganic phosphate or ATP, MgCl2 and NaCl. The ouabain-binding capacity and the ouabain-membrane complex were more stable than the (Na+,K+)-ATPase activity to treatment with agents known to affect the membrane integrity, such as, NaClO4, sodium dodecyl sulfate, p-chloromercuribenzoate, urea. ultrasonication, heating, pH and phospholinase C.The presence of binding sites that were normally inaccessible to ouabain in brain microsomes was demonstrated. These sites appeared after disruption of microsomes with 2 M NaClO4 as evidenced by increased binding of [3H]ouabain. These sites may be buried during the subcellular fractionation procedure and could be accessible in the intact cell.  相似文献   

19.
Using the procedure of Bekhor and Mirell (Biochem, 18, 609, 1979), we isolated a nonhistone protein fraction tightly bound to DNA which putatively has a role in globin gene regulation in chicken reticulocytes. This fraction was tested by gel electrophoresis and microcomplement fixation and appears by these criteria very similar to the chicken nuclear antigen previously identified in reticulocyte chromatin and structurally altered erythrocyte chromatin. This antigen is tissue and species specific (Pumo etal, Biochem. 19, 2362, 1980).  相似文献   

20.
Chrysene and the 3 metabolically possible vicinal trans dihydrodiols of chrysene were tested for mutagenicity towards S. typhimurium strain TA100 in the presence of hepatic microsomes or a highly purified hepatic microsomal monooxygenase system. The products formed during the metabolic activation of chrysene 1,2-dihydrodiol were more than 20 times as mutagenic to the bacteria than the metabolites formed from chrysene, chrysene 3,4-dihydrodiol or chrysene 5,6-dihydrodiol. When the double bond in the 3,4-position of chrysene 1,2-dihydrodiol was saturated, the resulting tetrahydrodiol could not be metabolically activated. These results, which strongly suggest that chrysene 1,2-dihydrodiol is activated by metabolism to either or both of the diastereomeric chrysene 1,2-diol-3,4-epoxides, provide additional support for the bay region theory of polycyclic hydrocarbon carcinogenicity.  相似文献   

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