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1.
The ADP-ribosylation of nonhistone, high mobility group (HMG) proteins in intact cultured cells was investigated. Radioactively labeled adenosine was used as a precursor to detect (ADP-ribose)n on protein. A protein fraction enriched in HMG proteins and histone H1 was separated from RNA and DNA by CsCl density gradient centrifugation, 5% perchloric acid extraction, and CM-Sephadex C-50 column chromatography. Poly- and mono(ADP-ribose) were recovered following alkaline hydrolysis, and 5'-AMP and (2'-(5"-phosphoribosyl)-5'-AMP) were produced by phosphodiesterase treatment, indicating that the protein-bound radioactive material was (ADP-ribose)n. An average chain length of 1.5 to 1.8 was determined. Analysis of proteins by sodium dodecyl sulfate and acetic acid/urea polyacrylamide gel electrophoresis demonstrated that HMG 1, 2, 14, and 17 as well as histone H1 contained (ADP-ribose)n. Treatment of cells with 3-aminobenzamide, an inhibitor of (ADP-ribose)n synthetase, decreased endogenous ADP-ribosylation in both types of chromosomal proteins but that of HMG 14 and 17 was affected more. 相似文献
2.
Endogenous ADP ribosylation of high mobility group proteins 1 and 2 and histone H1 following DNA damage in intact cells 总被引:3,自引:0,他引:3
Endogenous ADP ribosylation of nonhistone high-mobility group (HMG) proteins and histone H1 was studied in cultured mouse mammary tumor cells following treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MNNG treatment of cells caused a rapid and transient increase in ADP ribosylation of histone H1 and HMG 1 and 2, whereas (ADP-ribose)n on HMG 14 and 17 was not affected. 3-Aminobenzamide, an inhibitor of (ADP-ribose)n synthetase, prevented the increase in ADP ribosylation of histone H1 and HMG 1 and 2. This inhibitor enhanced the cell-killing effect of MNNG, but had no significant effect on the removal of methylated purines. The preferential increase in ADP ribosylation of HMG 1 and 2 and histone H1 may be necessary for cell recovery from DNA damage. 相似文献
3.
The induction capacity of dexamethasone, a synthetic glucocorticoid, for the synthesis of metallothionein was about the same as that of 3-aminobenzamide, which is an inhibitor of ADP-ribosylation of chromosomal proteins, in cultured mouse mammary tumor cells. Both inductions of metallothionein were temporally correlated with a decrease in the amount of endogenous poly (ADP-ribose) on nonhistone high-mobility-group 14 and 17 proteins. In contrast, the extent of cadmium-induced metallothionein synthesis was 2-3-times that of dexamethasone or 3-aminobenzamide. However, cadmium had essentially no effect on de-ADP-ribosylation of these proteins. 相似文献
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Glucocorticoids increase expression of specific genes by a mechanism involving binding to and "activation" of a specific receptor protein. Other steroids, such as RU 486, bind to the glucocorticoid receptor but the resultant steroid-receptor complex is unable to activate glucocorticoid sensitive genes. In the present study we have observed that steroid regulation of the glucocorticoid-regulated mouse mammary tumor virus (MMTV) genome in cultured mouse mammary tumor cells is altered by treatment of the cells with inhibitors of (ADP-ribose)n synthetase. The ability of glucocorticoid agonists to increase MMTV is about 2-fold increased by the inhibitor treatment. Interestingly, RU 486 and other steroids that are normally inactive in control cells are very good inducers of MMTV in the treated cells. This alteration in MMTV expression is associated with a 37% increase in nuclear binding of the glucocorticoid, triamcinolone acetonide, and also RU 486 in the inhibitor-treated cells. Steroids that do not bind to the glucocorticoid receptor are not inducers in control or in treated cells. The results point to a role for ADP-ribosylation of proteins as a negative regulator of MMTV expression and suggest a mechanism for activation of steroid-sensitive genomes. 相似文献
7.
Poly(ADP-ribosylation) of nuclear proteins in rat thymocytes 总被引:1,自引:0,他引:1
Specific antibodies to poly(ADP-ribose) were obtained and characterized. Using these antibodies, the tissue specificity of poly(ADP-ribose) modified nuclear proteins from rat thymocytes and hepatocytes was studied. The differences in the levels of poly(ADP-ribosylation) of nuclear proteins from both tissues were found to be quantitative rather than qualitative. Analysis of intranuclear distribution of poly(ADP-ribose) acceptor proteins revealed that the bulk of them is localized in the nuclear sap and matrix. A comparison of spectral properties of poly(ADP-ribosylated) proteins, using specific antibodies and label incorporation from [14C]NAD showed the existence of two protein groups. Some of those were modified in a great degree but exchange poly(ADP-ribose) at a slow rate, whereas others (e.g., histones and HMG proteins) modified in a small degree exchanged poly(ADP-ribose) at a much higher rate. The results obtained by different methods are discussed. 相似文献
8.
NAD-dependent ADP-ribosylation of arginine and proteins by Escherichia coli heat-labile enterotoxin.
J Moss S Garrison N J Oppenheimer S H Richardson 《The Journal of biological chemistry》1979,254(14):6270-6272
Escherichia coli heat-labile enterotoxin (labile toxin, LT) catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide and the ADP-ribosylation of arginine (Moss, J., and Richardson, S.H. (1978) J. Clin. Invest. 62, 281-285). Analysis of the product of the ADP-ribosylation of arginine by nuclear magnetic resonance spectroscopy indicated that the reaction was stereospecific and resulted in the formation of alpha-ADP-ribosyl-L-arginine. This reaction product rapidly anomerized to yield a mixture of the alpha and beta forms. In the presence of [adenine-U-14C]NAD, E. coli enterotoxin catalyzed the transfer of the radiolabel to proteins; the ADP-ribosylation of proteins was inhibited by arginine methyl ester, an alternative substrate. Digestion of the 14C-protein with snake venom phosphodiesterase released predominantly 5'-AMP. No product was obtained with a mobility similar to that of 2'-(5'-phosphoribosyl)-5'-AMP. This result is consistent with the covalent attachment by the enterotoxin of ADP-ribose rather than poly(ADP-ribose) to protein. Thus, LT is catalytically equivalent to choleragen, an enterotoxin of Vibrio cholerae, and activates adenylate cyclase through a similar stereospecific ADP-ribosylation reaction. 相似文献
9.
G G Poirier C Niedergang M Champagne A Mazen P Mandel 《European journal of biochemistry》1982,127(3):437-442
Poly(ADP-ribosylation) of histones H1, H5 and non-histone chromosomal high-mobility-group proteins HMG 1, 2, 14 and 17 from chicken erythrocytes by purified calf thymus poly(ADP-ribose) polymerase was studied using acid/urea/Triton gel electrophoresis and autoradiography. With histone H1, besides ADP-ribosylated H1 supporting short chains of polymer, the appearance of H1 'dimer' was observed and this reaction was dependent on NAD concentration and incubation time. In addition, highly modified and/or aggregated species of histone H1 were observed. Histone H5 was slightly ADP-ribosylated at low NAD concentrations. At higher NAD concentrations or after longer incubations the formation of H5 'dimer' and of more modified forms of H5 could be observed. HMG 1 and HMG 2 were found to be ADP-ribosylated, the reaction being dependent on NAD concentration and time. Here again some discrete intermediates appeared. HMG 14 and HMG 17 were only slightly ADP-ribosylated under our experimental conditions. These results indicate that the purified DNA-independent poly(ADP-ribose) polymerase can catalyse the formation of H1 'dimer' as in nuclei and nucleosomes and that H5 and HMG proteins can also be ADP-ribosylated and produce well-defined higher complexes. These modifications of nuclear proteins may provide a means of localized conformational changes of the chromatin structure in vivo. 相似文献
10.
T M Zaalishvili K M Kolkhidashvili D O Margiani R D Michilashvili 《Biokhimii?a (Moscow, Russia)》1988,53(6):931-935
The distribution of (ADP-ribose)n synthesized from [14C]NAD labeled at the adenyl ring in several protein fractions of isolated rat brain nuclei was studied. Preferential ADP-ribosylation of nonhistone nuclear proteins was shown to occur. It was demonstrated that pol (ADP-ribose)polymerase and DNA-topoisomerase II are located spatially close to each other. A correlation between ADP-ribosylation and the activity of nuclear matrix DNA-topoisomerase II was established. 相似文献
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The relationship between the cellular uptake of glucocorticoid hormones, the binding of these hormones to specific in vitro receptors, and the induction of mouse mammary tumor viruses in an established mouse mammary tumor cell line was highly correlated. These results suggest that the induction of mouse mammary tumor virus by glucocorticoid hormones is a physiological process acting through a mechanism of high affinity, saturable steroid-receptors. A temperature-sensitive or salt-dependent step following glucocorticoid-receptor interaction was required for nuclear uptake of the steroid. Induction studies with different adrenocorticoids indicate that the synthetic glucocorticoid, dexamethasone (1,4-pregnadiene-9-fluor-16alpha-methyl-11beta,17alpha,21-triol-3,20-dione), is the most potent inducer of mouse mammary tumor viruses and all steroids which caused significant induction were glucocorticoids. Other glucocorticoids appear to stimulate murine mammary tumor virus production by a mechanism similar to that of dexamethasone; for example, corticosterone competes with dexamethasone for binding to the glucocorticoid receptor and blocks the uptake of dexamethasone into cells. Progesterone also blocks the cellular uptake of dexamethasone and can bind to the glucocorticoid receptor at low concentrations (10-7 to 10-8 M) but progesterone does not consistently induce virus at hormone concentrations even as high as 10-4 M. Thus, in this system, binding to a cytoplasmic receptor is necessary but not sufficient for induction by glucocorticoids. Estrogens and androgens interfere with receptor binding and cellular uptake of dexamethasone but only at much higher concentration (10-4 M) than progesterone, and do not induce mammary tumor virus production. Although there was a positive correlation between steroid structure, binding, and biologic induction, other factors clearly affect the physiological manifestations of steroid actions. Mouse cells with comparable cytoplasmic receptor levels and comparable nuclear uptake differed absolutely in their degree of murine mammary tumor virus induction following hormone treatment. Although all mouse cells examined contain comparable levels of murine mammary tumor virus DNA, only cells producing constitutive levels of murine mammary tumor virus RNA could be induced to higher levels by a variety of glucocorticoids. 相似文献
12.
The ability of rat liver submitochondrial particles to catalyze NAD+ hydrolysis with a transfer of ADP-ribose residues to protein membranes has been demonstrated ADP-ribosylation is directly dependent on NAD+ concentration upon saturation with 1 mM NAD+ and is inhibited by physiological compounds (e.g., ATP, 10 mM; nicotinamide, 10 mM); besides, it is an artificial acceptor of ADP-ribose, arginine methyl ester. It was found that ADP-ribose is accepted by inner mitochondrial membrane protein, whose molecular masses amount to 25-30 kDa. The fact that 5'-AMP is a product of ADP-ribose degradation by snake venom phosphodiesterase suggests that the inner membrane vesiculate proteins are modified by mono(ADP-ribose). Covalent modification of membrane proteins by ADP-ribose leads to citrate transport inhibition in inner membrane vesicles the [14C]citrate uptake is significantly decreased thereby. The ability of ADP-ribosylation inhibitors to restore the citrate transport rate is suggestive of a direct regulatory effect of NAD+-dependent ADP-ribosylation on the activity of citrate-translocating system of inner mitochondrial membranes. 相似文献
13.
Poly ADP-ribosylation of histones in tumor promoter phorbol-12-myristate-13-acetate treated mouse embryo fibroblasts C3H10T1/2 总被引:2,自引:0,他引:2
The tumor promoter phorbol-12-myristate-13-acetate (PMA) increases the poly ADP-ribosylation of acid extractable (0.2N H2SO4) nuclear proteins in mouse embryo fibroblasts C3H10T1/2. Catalase suppresses the reaction by approximately 50%. Polyacrylamide gel electrophoresis reveals that the core histones H2B, A24 and H3d serve as major poly ADP-ribose acceptors. Smaller amounts of poly ADP-ribose are associated with histones H2A/H3 and H1. Poly ADP-ribosylation of histones may change the nucleosomal structure and function and play a role in PMA induced modulation of gene expression in promotion. 相似文献
14.
Differentiation of 3T3-L1 pre-adipocytes induced by inhibitors of poly(ADP-ribose) polymerase and by related noninhibitory acids 总被引:1,自引:0,他引:1
To analyze a possible involvement of ADP-ribosylation reactions in 3T3-L1 pre-adipocyte differentiation. ADP-ribosyltransferase activities is permeabilized cells as well as endogenous amounts of protein-bound mono- and poly(ADP-ribose) residues were determined. Also, in vivo labeling with [3H]adenosine of ADP-ribose residues linked to high-mobility-group (HMG) proteins was performed. As an additional probe, the effects of ADP-ribosylation inhibitors and non-inhibitory analogs were studied. Basal and total poly(ADP-ribose) polymerase activities markedly increased prior to the appearance of the differentiation marker glycerol-3-phosphate dehydrogenase. Despite these apparent changes in activity, however, neither protein-bound poly(ADP-ribose) residue nor mono(ADP-ribosyl) groups in histones, nor the NAD content, changed significantly under these conditions. Furthermore, although HMG protein-associated [3H]ADP-ribose was reduced in differentiating [3H]adenosine-labeled cells, the data suggest altered precursor pool labeling rather than a specific decrease in ADP-ribosylated HMG proteins. Non-participation of ADP-ribosylation reactions in 3T3-L1 differentiation is further supported by experiments with inhibitors and non-inhibitory analogs. Benzamide at 0.3-3 mM per se without effect on differentiation, was able to induce specific gene expression when combined with insulin (10(-12)-10(-7) M). Similar effects were seen with benzoate as well as with nicotinamide, 3-aminobenzamide and their corresponding acids. The data indicate that benzamide and analogs have profound effects on chromatin functions that are not mediated by ADP-ribosylation reactions. 相似文献
15.
ADP-ribosylation in permeable HeLa S3 cells 总被引:2,自引:0,他引:2
ADP-ribosylation in permeabilized metaphase and interphase cells using [32P]NAD at pH 8.0 have been compared. Incorporation into trichloroacetic acid insoluble material was 4-5-times greater in metaphase cells. 17-22% was in the soluble fraction which contained material released from the cells, 16-22% in the 0.2 M HCl extract (histones) of the cell ghosts and the remaining activity in the residual fraction. Fractions were analyzed using dodecylsulphate/polyacrylamide gel electrophoresis at pH 6.0. The soluble fractions from metaphase and interphase cells exhibited three common unidentified ADP-ribosylated proteins corresponding to 78 000, 54 000 and 36 000 Da. In addition metaphase cells contained several other ADP-ribosylated proteins not present in interphase cells. The 0.2 M HCl extracts gave from metaphase cells radioactivity in the 32 000-39 000-Da region suggesting ADP-ribosylation of histone H1 with up to 10 residues of ADP-ribose and in the 17 000-20 000-Da region indicating ADP-ribosylation of core histones. The pattern of ADP-ribosylation of core histone in metaphase and interphase cells was qualitatively similar whereas the number of ADP-ribose residues per H1 molecule was higher in metaphase cells. The residual fraction contained free poly(ADP-ribose) and oligo(ADP-ribose). The results do not lend support to a special function of ADP-ribosylated histones in the mitotic event while certain ADP-ribosylated non-histone proteins may be specific for metaphase cells. 相似文献
16.
Purification of non-histone acceptor proteins for ADP-ribose from mouse testis nuclei. 总被引:2,自引:1,他引:1
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M R Faraone Mennella P Quesada B Farina E Leone R Jones 《The Biochemical journal》1984,221(1):223-233
Acceptor proteins for poly(ADP-ribose) have been purified from mouse testis nuclei. Nuclear proteins were labelled in vitro with [14C]ribose and [3H]adenine, extracted with 5% (v/v) HClO4 and 0.25 M-HCl and separated by ion-exchange chromatography. Non-histone proteins were found to be the major acceptors in both the 5% (w/v)-HClO4-soluble and 5%-HClO4-insoluble HCl-extractable fractions. Of the two groups of non-histone proteins associated with chromatin, the LMG (low-mobility-group) proteins were preferentially ADP-ribosylated. HMG (high-mobility group) proteins were labelled to lower specific radioactivity. Six LMG proteins were purified to approx. 90% homogeneity and were identified from their mobility on polyacrylamide gels at pH 2.9 and from their amino acid composition. The average length of the poly(ADP-ribose) chain was estimated to be four to six repeating ADP-ribose units. It is suggested that ADP-ribosylation of LMG proteins, a long-neglected group of chromatin-associated proteins, is important during spermatogenesis for the production of spermatozoa with intact and competent DNA. 相似文献
17.
T Wurtz 《European journal of biochemistry》1985,152(1):173-178
To approach experimentally changes of chromatin structure introduced by glucocorticoids, the histone H1 compositions of hormone-treated and non-treated mouse mammary tumor cells of the GR line [Ringold, G., Lasfargues, E. Y., Bishop, J. M. and Varmus, H. E. (1975) Virology 65, 135-147] were compared. To define the biologically important hormone concentration range, the cells were exposed to different concentrations of triamcinolone, a synthetic glucocorticoid. The induction of mouse mammary tumor virus (MMTV) RNA was measured by cDNA excess hybridization, and the amount of hormone bound to nuclei was determined by a filter-binding assay. Between 0.3 nM and 30 nM triamcinolone the relative increase in nuclear bound hormone corresponded well with the relative induction of MMTV RNA. The half-life of triamcinolone in nuclei of growing cells was 1 h, as measured by a pulse-chase experiment. Reversed-phase high-performance liquid chromatography of histone H1 resulted in its separation into four subfractions. The treatment of cells with biologically active glucocorticoid, 3 nM or 30 nM triamcinolone or 1 microM dexamethasone, resulted in changes in the relative amounts of two subfractions and to a positional shift of two subfractions as compared to untreated cells. No changes were observed after exposure to 3 nM dexamethasone, a concentration which does not induce MMTV RNA [Ringold, G. M., Yamamoto, K. R., Tomkins, G. M., Bishop, J. M. and Varmus, H. E. (1975) Cell 6, 299-305]. 相似文献
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Acceptor proteins for (ADP-ribose)n in the HeLa S3 cell cycle 总被引:3,自引:0,他引:3
The acceptor proteins for (ADP-ribose)n were investigated by using nuclei or chromosomes isolated from specific phases of the cell cycle of HeLa S3 cells. Analysis of HMG proteins and histone H1 by acetic acid/urea polyacrylamide gel electrophoresis demonstrated that the (ADP-ribosyl)n-ation of HMG 14 and 17 and histone H1 increased by 12- and 5-fold, respectively, in the metaphase chromosomes as compared with that in the G1 phase cell nuclei. The degree of (ADP-ribosyl)n-ation of these proteins in the S phase cell nuclei was as low as that in G1 phase cell nuclei. In the G2 phase cell nuclei, the degrees of (ADP-ribosyl)n-ation of HMG 14 and 17 and histone H1 were about 5- and 2-fold greater, respectively, as compared with that in the G1 phase cell nuclei. The (ADP-ribosyl)n-ation of HMG 1 and 2 was constant through the cell cycle except for a slight decrease in the S phase. The data may imply that the (ADP-ribosyl)n-ation of HMG 14 and 17 and histone H1 is linked to chromatin structural changes in mitosis. 相似文献
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Bektaş M Akçakaya H Aroymak A Nurten R Bermek E 《The international journal of biochemistry & cell biology》2005,37(1):91-99
Different lines of evidence indicate that eukaryotic elongation factor 2 (eEF2) can be ADP-ribosylated endogenously. The physiological significance of this reaction has, however, remained unclarified. In order to address this issue we investigated the in vivo ADP-ribosylation of eEF2 and the effect of oxidative stress thereon. The investigation revealed that the endogenous ADP-ribosylation of eEF2 is complex and can take place in K562 cell lysates either under the action of endogenous transferase from [adenosine-14C]NAD or by direct binding of free [14C]ADP-ribose. These two types of ADP-ribosylation were distinguished by use of different treatments based on the chemical stability of the respective bonds formed. Under standard culture conditions, in vivo labeling of eEF2 in the presence of [14C]adenosine was reversed to about 65% in the presence of diphtheria toxin and nicotinamide. This finding implied that the modification that took place under physiological circumstances was, mainly, of an enzymic nature. On the other hand, H2O2-promoted oxidative stress gave rise to a nearly two-fold increase in the extent of in vivo labeling of eEF2. This was accompanied by a loss of eEF2 activity in polypeptide chain elongation. Oxidative stress specifically inhibited the subsequent binding of free ADP-ribose to eEF2. The results thus provide evidence that endogenous ADP-ribosylation of eEF2 can also take place by the binding of free ADP-ribose. This nonenzymic reaction appears to account primarily for in vivo ADP-ribosylation of eEF2 under oxidative stress. 相似文献