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1.
1. We have investigated the origin of proteolytic activity which causes degradation of histones in chromatin isolated from Xenopus liver and the rat liver at neutral pH. Polyacrylamide disc gel electrophoresis was used for detection of proteolytic products of histones. 2. No proteolytic degradation of histones occurs in chromatin isolated from Xenopus erythrocytes and rat liver according to our procedure even after prolonged incubation at pH 8.0 and pH 5.0. However with chromatin isolated from Xenopus liver a high level of histone degradation is observed under similar conditions. 3. Mixing isolated nuclei from Xenopus erythrocytes with a crude cytoplasmic fraction from Xenopus liver causes histone proteolysis in isolated chromatin at pH 8.0. In similar experiments with corresponding fractions from rat liver histone proteolysis can be introduced only after repeated freezing and thawing of the cytoplasmic fraction. 4. A purified lysosomal preparation from rat liver causes a similar type of histone degradation upon incubation with chromatin from Xenopus erythrocytes and rat liver. 5. The neutral proteolytic activity that can be introduced in isolated chromatin by a crude cytoplasmic fraction and by a purified lysosomal erythrocytes and rat liver. 5. The neutral proteolytic activity that can be introduced in isolated chromatin by a crude cytoplasmic fraction and by a purified lysosomal fraction from rat liver is inhibited by sodium bisulphite. 6. We conclude that the neutral proteolytic activity which causes degradation of histones in isolated chromatin is due to a contamination with neutral protease(s) originating from cytoplasmic organelles. 相似文献
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Effects of DNA and urea on the specificity for H1 histone of the neutral protease B partially-purified from rat liver chromatin 总被引:1,自引:0,他引:1
A neutral protease, named protease B in the previous report (Tsurugi, K. & Ogata, K. (1982) J. Biochem. 92, 1369-1381), was partially purified from rat liver chromatin by gel filtration through Sepharose 6B followed by DE-Sephadex column chromatography. The proteolytic activity on total histones of the partially-purified protease B was increased about two fold by addition of DNA and again increased by further addition of 2 M urea. Analysis of the hydrolysed products showed that out of five species of histones, only H1 was degraded in the presence of an amount of DNA equivalent to the amount of histones, whereas core histones were also degraded in the absence or presence of one-tenth amount of DNA. Urea accelerated the selective degradation of H1 histone because H1 histone was preferentially degraded in the presence of even a low amount of DNA. In contrast, core histones became resistant to the protease B in the presence of DNA and/or urea. Heat-denatured DNA stimulated the degradation of H1 histone even in the absence of urea to almost the same extent that native DNA did in the presence of urea. Thus, protease B efficiently degrades H1 histone when its association with DNA is destabilized by either addition of urea or pretreatment of DNA with heat. 相似文献
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Displacement of histones from calf thymus chromatin has been studied in an attempt to postulate the mechanisms involved in the total removal of somatic-type histones during transformation of spermatid chromatin. When chromatin is saturated with protamine (protamine/DNA, 0.5), histone I becomes displaceable at 0.15-0.3 M NaCl, suggesting that direct replacement by highly basic sperm histone could be a mechanism for its removal. While histone I is the only histone which is extensively degraded upon incubation of chromatin and, therefore, proteolysis might provide an additional mechanism for the removal of this histone, acetylation of chromatin by acetic anhydride greatly increases suscpetibility of histones IIb1, IIb2, and III to the chromosomally associated protease. These histones are extensively degraded and displaced from the DNA upon incubation of the acetylated chromatin. Although histone IV is not appreciably degraded, the proteolytic removal of acetylated histone III from chromatin weakens the interaction of acetylated histone IV to the DNA, and this histone becomes dissociable at 0.3 M NaCl. A comparison of the extent of chemical acetylation of individual histones observed in this investigation with that of enzymatic acetylation which can be achieved in vivo suggests that acetylation and proteolysis could be a mechanism for the removal of histone IIb2 and III. The displacement of histones IIb1 and IV could be explained on the basis of decreased binding to DNA as a result of their acetylation together with the proteolytic removal of their respective partner histones, IIb2 and III. 相似文献
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A protease activity associated with the micrococcal nuclease-solubilized chromatin from mouse seminiferous tubules has been characterized. Proteolysis of histone H1 and core histones is stimulated in the presence of 3 M urea. The pH optimum of this protease is between pH 8 and 9, and the activity is not inhibited by trypsin or chymotrypsin-active site inhibitors. Leupeptin is an effective inhibitor of the protease at low concentrations. Soluble chromatin from neonatal and prepubertal mice lacks this proteolytic activity until three to four weeks after birth. That the protease activity is localized in the dinucleosomes and higher oligomers but is lacking in mononucleosome populations suggests its association with the linker DNA. Rat testis-soluble chromatin apparently lacks such a protease activity. The developmental expression of this protease and its in situ localization are consistent with a role in histone displacement during mouse spermiogenesis. 相似文献
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Maria Imschenetzky Fredy Díaz Martin Montecino Felipe Sierra Marcia Puchi 《Journal of cellular biochemistry》1997,67(3):304-315
We have identified a 60-kDa cysteine protease that is associated with chromatin in sea urchin zygotes. This enzyme was found to be present as a proenzyme in unfertilized eggs and was activated shortly after fertilization. At a pH of 7.8–8.0, found after fertilization, the enzyme degraded the five sperm-specific histones (SpH), while the native cleavage-stage (CS) histone variants remained unaffected. Based on its requirements for reducing agents, its inhibition by sulfhydryl blocking compounds and its sensitivity to the cysteine-type protease inhibitors (2S,3S)-translator-epoxysuccinyl-L-leucyl-amido-3-methylbutane-ethyl-ester (E-64 d), cystatin and leupeptin, this protease can be defined as a cysteine protease. Consistently, this protease was not affected by the serine-type protease inhibitors phenylmethylsulfonyl fluoride (PMSF) and pepstatin. The substrate selectivity and pH modulation of the protease activity strongly suggest its role in the removal of sperm-specific histones, which determines sperm chromatin remodeling after fertilization. This suggestion was further substantiated by the inhibition of sperm histones degradation in vivo by E-64 d. Based on these three lines of evidence, we postulate that this cysteine protease is responsible for the degradation of sperm-specific histones which occurs during male pronucleus formation. J. Cell. Biochem. 67:304–315, 1997. © 1997 Wiley-Liss, Inc. 相似文献
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1. Suspensions of avian erythroid nuclei, of high purity, were prepared. Acetylation of histones was observed when nuclei were incubated in the presence of [1-14C]acetyl CoA, but not in the presence of sodium [3H]acetate. 2.The acetylation reaction was very heat labile and reproduced the in vivo reaction with high fidelity. The reaction was strongly inhibited by divalent cations and cysteine. 3. Studies, in which intact cells were pre-incubated with cycloheximide prior to the isolation of nuclei, suggested that histone acetylation in isolated erythroid nuclei was largely independent of histone synthesis. 4. The pH profile suggested the presence of at least two histone acetyltransferases, with pH optima at 8.0 and 8.6. Acetylation of histone H4 appeared to be favoured at pH 8.0. 5. Studies on histone acetylation in isolated nuclei should be very useful in correlating observations on histone acetylation in vivo, with experiments using purified histone acetyltransferases. 相似文献
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Dynamics of histone acetylation in Saccharomyces cerevisiae 总被引:3,自引:0,他引:3
Waterborg JH 《Biochemistry》2001,40(8):2599-2605
Rates of turnover for the posttranslational acetylation of core histones were measured in logarithmically growing yeast cells by radioactive acetate labeling to near steady-state conditions. On average, acetylation half-lives were approximately 15 min for histone H4, 10 min for histone H3, 4 min for histone H2B, and 5 min for histone H2A. These rates were much faster than the several hours that have previously been reported for the rate of general histone acetylation and deacetylation in yeast. The current estimates are in line with changes in histone acetylation detected directly at specific chromatin locations and the speed of changes in gene expression that can be observed. These results emphasize that histone acetylation within chromatin is subject to constant flux. Detailed analysis revealed that the turnover rates for acetylation of histone H3 are the same from mono- through penta-acetylated forms. A large fraction of acetylated histone H3, including possibly all tetra- and penta-acetylated forms, appears subject to acetylation turnover. In contrast, the rate of acetylation turnover for mono- and di-acetylated forms of histones H4 and H2B, and the fraction subject to acetylation turnover, was lower than for multi-acetylated forms of these histones. This difference may reflect the difference in location of these histones within the nucleosome, a difference in the spectrum of histone-specific acetylating and deacetylating enzymes, and a difference in the role of acetylation in different histones. 相似文献
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Alteration of chromatin structure by chromatin modifying and remodelling activities is a key stage in the regulation of many nuclear processes. These activities are frequently interlinked, and many chromatin remodelling enzymes contain motifs that recognise modified histones. Here we adopt a peptide ligation strategy to generate specifically modified chromatin templates and used these to study the interaction of the Chd1, Isw2 and RSC remodelling complexes with differentially acetylated nucleosomes. Specific patterns of histone acetylation are found to alter the rate of chromatin remodelling in different ways. For example, histone H3 lysine 14 acetylation acts to increase recruitment of the RSC complex to nucleosomes. However, histone H4 tetra-acetylation alters the spectrum of remodelled products generated by increasing octamer transfer in trans. In contrast, histone H4 tetra-acetylation was also found to reduce the activity of the Chd1 and Isw2 remodelling enzymes by reducing catalytic turnover without affecting recruitment. These observations illustrate a range of different means by which modifications to histones can influence the action of remodelling enzymes. 相似文献
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The nuclei from fertilized sea urchin eggs, obtained 80 min after fertilization, contains a neutral proteolytic activity. Optimal action on casein was observed at pH 7-8 and a Km value of 1.2 mg/ml was determined for this substrate. The proteolytic activity was stimulated 1.5 fold by the addition of 3 M urea and decreased at higher urea concentrations. NaCl and CaCl2 were inhibitory whereas MgCl2 increased the enzyme activity. Isolated histones from sea urchin sperms, and especially histones H1, H2A, H2B and H3, were degraded by the nuclear activity. A partial inhibition of histones degradation was caused by sodium bisulfite and NaCl. The proteolytic activity was found associated to the chromatin of fertilized sea urchin eggs. 相似文献
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In this report, we introduce the use of DNA-cellulose chromatography for evaluating the strength of binding of histones to DNA under a variety of conditions. We have found that histones added directly to DNA-cellulose at physiological salt concentrations bind relatively weakly, with all histones eluting together at about 0.5 M NaCl when a salt gradient is applied. However, much tighter binding of the four nucleosomal histones to DNA-cellulose is obtained if gradual histone-DNA reconstitution conditions are used. In this case, the binding of histones H2A, H2B, H3, and H4 to DNA-cellulose closely resembles their binding to native chromatin. The nativeness of the binding is indicated both by the distinctive sodium chloride elution profile of these histones from DNA-cellulose and by their relative resistance to trypsin digestion when DNA-bound. The binding to DNA-cellulose of histones H2A, H2B, H3, and H4, which have had the first 20 to 30 amino acid residues removed from their NH2 termini, is indistinguishable from the binding to DNA-cellulose of the same intact histones, as judged by their salt elution profile. Thus, even though the NH2 termini contain 40 to 50% of the positively charged amino acid residues (thought to interact with the DNA backbone), a major contribution to the DNA binding comes from the remainder of the histone molecule. Finally, we have discovered that histones can form a "nucleosome-like" complex on single-stranded DNA. The same complex does not appear to form on RNA. Histones H3 and H4 play a predominant role in organizing this histone complex on single-stranded DNA, as they do on double-stranded DNA in normal nucleosomes. We suggest that, in the cell nucleus, nucleosomal structures may form transiently on single strands of DNA, as DNA and RNA polymerases traverse DNA packaged by histones. 相似文献
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W Krajewska A Lipińska Z Kiliańska L Klyszejko-Stefanowicz 《Acta biochimica Polonica》1977,24(2):127-132
1. The activity of serine protease was studied in chromatin and in histone preparations from rat liver and Morris hepatomas, lines 5123 D and 7777. 2. Electrophoretic patterns of histones and the amounts of acid-soluble peptides released during incubation of chromatin and histones showed that there was no significant correlation between protease activity and tumour differentiation and its growth rate. 相似文献
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We had previously reported that a cysteine-protease catalyzes the sperm histones (SpH) degradation associated to male chromatin remodeling in sea urchins. We found that this protease selectively degraded the SpH leaving maternal cleavage stage (CS) histone variants unaffected, therefore we named it SpH-protease. It is yet unknown if the SpH-protease catalyzes the SpH degradation while these histones are organized as nucleosomes or if alternatively these histones should be released from DNA before their proteolysis. To investigate this issue we had performed an in vitro assay in which polynucleosomes were exposed to the active purified protease. As shown in this report, we found that sperm histones organized as nucleosomes remains unaffected after their incubation with the protease. In contrast the SpH unbound and free from DNA were readily degraded. Interestingly, we also found that free DNA inhibits SpH proteolysis in a dose-dependent manner, further strengthening the requirement of SpH release from DNA before in order to be degraded by the SpH-protease. In this context, we have also investigated the presence of a sperm-nucleosome disassembly activity (SNDA) after fertilization. We found a SNDA associated to the nuclear extracts from zygotes that were harvested during the time of male chromatin remodeling. This SNDA was undetectable in the nuclear extracts from unfertilized eggs and in zygotes harvested after the fusion of both pronuclei. We postulate that this SNDA is responsible for the SpH release from DNA which is required for their degradation by the cysteine-protease associated to male chromatin remodeling after fertilization. 相似文献