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1.
Poly(ADP-ribose) polymerase with a high specific activity was obtained from Ehrlich ascites tumor cells by extraction of nuclei with 175 mM potassium phosphate, followed by chromatography on DNA-agarose. Electrophoretic analysis indicated that the preparation contained two proteins, one of which was shown to catalyze the synthesis of poly(ADP-ribose). As expected from results obtained by other workers, the synthesis was inhibited by nicotinamide and thymidine, and stimulated by DNA. Addition of histones gave inhibition of the synthesis, unless DNA was present in the reaction mixture. 相似文献
2.
The nuclear enzyme poly(ADP-ribose) polymerase has been purified about 9200-fold from pig thymus nuclei with a 46% yield. An aqueous organic solvent system was used for the isolation of the polymerase from nuclei and for its purification by chromatography at sub-zero temperatures. Electrophoretic analysis under both denaturing and non-denaturing conditions revealed a single protein band suggesting that the preparation was homogeneous and that the enzyme is composed of one polypeptide chain. The molecular weight estimated from sodium dodecyl sulphate-/polyacrylamide gel electrophoresis was 63 500 and from gel filtration through columns of Sephadex G-100, 58 000. The enzyme preparation was free from poly(ADP-ribose)-degrading enzymes and from DNA. The purified polymerase showed an absolute requirement for both DNA and histones. The maximal specific activity of the homogeneous preparation measured by the standardized assay, was 20.7 mu mol NAD+ incorporated x min-1 x mg-1 of protein at 37 degree C. Amino-terminal group analysis with dansyl chloride did not reveal a terminal amino acid suggesting that the amino-terminal group may be blocked. In the presence of histones, the Km for NAD+ was 23 micrometer. 相似文献
3.
C F Cesarone L Scarabelli P Giannoni G Gallo M Orunesu 《Biochemical and biophysical research communications》1990,171(3):1037-1043
Previous studies have demonstrated that an increase in poly(ADP-ribose) polymerase activity could be closely related to DNA replication during liver regeneration and to DNA repair synthesis in different experimental systems. This relationship was further investigated by studying the time course of endogenous and total poly(ADP-ribose) polymerase activity in cultured rat hepatocytes stimulated by epidermal growth factor. This mitogen has been shown to stimulate DNA synthesis in liver cells both in vivo and in vitro. A 6-fold increase in endogenous activity was observed early after epidermal growth factor addition, just before DNA synthesis. A subsequent 4-fold increment in total enzyme activity, concomitant with DNA synthesis, was detected. Orotic acid, which has recently shown mitoinhibitory effect, abolished the epidermal-growth-factor-induced increase in endogenous and total poly(ADP-ribose) polymerase activity, as well as DNA synthesis. On the contrary, 3-aminobenzamide inhibitor of poly(ADP-ribose) polymerase completely suppressed the endogenous activity but only partially modified the increase in total catalytic level and the overall pattern of thymidine incorporation. Taken together, these data indicate that, in cultured hepatocytes, the induction of DNA synthesis is supported by an increased poly(ADP-ribose) polymerase activity. 相似文献
4.
H Yamanaka C A Penning E H Willis D B Wasson D A Carson 《The Journal of biological chemistry》1988,263(8):3879-3883
The addition of poly(ADP-ribose) chains to nuclear proteins has been reported to affect DNA repair and DNA synthesis in mammalian cells. The enzyme that mediates this reaction, poly(ADP-ribose) polymerase, requires DNA for catalytic activity and is activated by DNA with strand breaks. Because the catalytic activity of poly(ADP-ribose) polymerase does not necessarily reflect enzyme quantity, little is known about the total cellular poly(ADP-ribose) polymerase content and the rate of its synthesis and degradation. In the present experiments, specific human autoantibodies to poly(ADP-ribose) polymerase and a sensitive immunoblotting technique were used to determine the cellular content of poly(ADP-ribose) polymerase in human lymphocytes. Resting peripheral blood lymphocytes contained 0.5 X 10(6) enzyme copies per cell. After stimulation of the cells by phytohemagglutinin, the poly(ADP-ribose) polymerase content increased before DNA synthesis. During balanced growth, the T lymphoblastoid cell line CEM contained approximately 2 X 10(6) poly(ADP-ribose) polymerase molecules per cell. This value did not vary by more than 2-fold during the cell growth cycle. Similarly, mRNA encoding poly(ADP-ribose) polymerase was detectable throughout S phase. Poly(ADP-ribose) polymerase turned over at a rate equivalent to the average of total cellular proteins. Neither the cellular content nor the turnover rate of poly(ADP-ribose) polymerase changed after the introduction of DNA strand breaks by gamma irradiation. These results show that in lymphoblasts poly(ADP-ribose) polymerase is an abundant nuclear protein that turns over relatively slowly and suggest that most of the enzyme may exist in a catalytically inactive state. 相似文献
5.
A shuttle mechanism for DNA-protein interactions. The regulation of poly(ADP-ribose) polymerase 总被引:9,自引:0,他引:9
Previously it had been shown that poly(ADP-ribose) polymerase requires DNA for its activity and that this enzyme is auto-poly(ADP-ribosyl)ated. The studies reported here indicate that this self-modification inhibits the enzyme and decreases its affinity for DNA, as shown by sucrose gradient density centrifugation. The coupling of poly(ADP-ribose) polymerase with poly(ADP-ribose) glycohydrolase reactivates the polymerase by degrading poly(ADP-ribose) and restoring the polymerase-DNA complex. The assay of polymerase in the presence of glyco-hydrolase was made possible by use of a double-label assay involving release of 14C-labelled nicotinamide and the incorporation of 3H-labelled ADP-ribose from NAD+. These results provide the basis for a shuttle mechanism in which the polymerase can be moved on and off DNA by the action of these two enzymes. Mg2+ and histone H1 appear to activate the polymerase by increasing the affinity of the polymerase for DNA. 相似文献
6.
The post-translational poly ADP-ribosylation of proteins by the nuclear enzyme poly(ADP-ribose) polymerase (EC 2.4.2.30) involves a complex pattern of ADP-ribose polymers. We have determined how this enzyme produces the various polymer size patterns responsible for altered protein function. The results show that histone H1 and core histones are potent regulators of both the numbers and sizes of ADP-ribose polymers. Each histone induced the polymerase to synthesize a specific polymer size pattern. Various other basic and/or DNA binding proteins as well as other known stimulators of poly(ADP-ribose) polymerase (spermine, MgCl2, nicked DNA) were ineffective as polymer size modulators. Testing specific proteolytic fragments of histone H1, the polymer number and polymer size modulating activity could be mapped to specific polypeptide domains. The results suggest that histones specifically regulate the polymer termination reaction of poly(ADP-ribose) polymerase. 相似文献
7.
E G Miller 《Biochimica et biophysica acta》1975,395(2):191-200
Isolated nuclei from HeLa cells can incorporate labeled ADP-ribose from NAD into an acid-precipitable product, poly(ADP-ribose). This reaction is stimulated by 4-6-fold by the addition of deoxyribonuclease I to the complete reaction mixture. If the nuclei are treated first with deoxyribonuclease I, no effect is seen; the stimulation is only apparent when the two enzymes deoxyribonuclease I and poly(ADP-ribose) polymerase, are operating at the same time. After making several minor modifications in the assay mixture, it was found that another endonuclease, micrococcal nuclease, can also stimulate the poly(ADP-ribose) polymerase activity of HeLa nuclei. A comparison of the two stimulatory effects indicated that the two endonucleases activated to the poly(ADP-ribose) polymerase activity of HeLa nuclei in the same way. Overall this evidence suggests that poly(ADP-ribose) polymerase may have a functional role in the process of DNA repair. 相似文献
8.
9.
K Yoshihara T Hashida H Yoshihara Y Tanaka H Ohgushi 《Biochemical and biophysical research communications》1977,78(4):1281-1288
Bovine thymus poly(ADP-ribose) polymerase with a purity of 99% on a SDS-poly-acrylamide gel electrophoresis was able to initiate poly(ADP-ribose) synthesis without adding any exogenous acceptor protein to the reaction system. Analyses of the early reaction product synthesized without exogenous acceptor protein revealed that the product was oligo(ADP-ribose) with a mean chain length of 2.6 and was bound tightly to the enzyme protein. When the radioactive early reaction product was chased by incubating further with cold NAD+, ADP-ribose unit was found to be added to the terminal AMP-residue of the oligo(ADP-ribose) attached to the enzyme. The stability of the early reaction product in high concentration of salt, strong acid, sodium dodecyl sulfate, and urea strongly suggests a covalent nature of the binding of oligo(ADP-ribose) to the enzyme. 相似文献
10.
The 40 kDa carboxy-terminal catalytic domain (CD) of avian poly(ADP-ribose) polymerase (PARP-1) was cloned, expressed in a baculovirus expression system, and purified to homogeneity by affinity chromatography. The purified polypeptide synthesized covalent CD-poly(ADP-ribose) conjugates in the absence of DNA. Electrophoretic analysis of the ADP-ribose chain length distribution generated indicated that recombinant CD was able to catalyze the initiation, elongation, and branching reactions of poly(ADP-ribose) synthesis, although at a 500-fold lower efficiency than wild-type PARP-1. Kinetic evaluation of poly(ADP-ribose) synthesis showed that the enzymatic activities of CD increased for up to 60 minutes in a time-dependent manner. Moreover, the rates of CD auto-poly(ADP-ribosyl)ation increased with second-order kinetics as a function of the protein concentration with either betaNAD(+) or 3'-deoxyNAD(+) as a substrate. Furthermore, the formation of catalytically competent CD-[PARP-1] heterodimers was also observed in specific ultrafiltration experiments. Thus, we conclude that the 40 kDa carboxy terminus of PARP-1 forms a competent catalytic dimer in the absence of DNA, and that its automodification reaction is intermolecular. 相似文献
11.
Two polypeptides with molecular masses of 76 and 59 kDa were found to copurify with poly(ADP-ribose) polymerase from calf thymus, and to be as efficient acceptors of ADP-ribose as the polymerase itself. Analysis of their CNBr fragments by sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed that the polypeptides were derived from the 112-kDa polymerase. Isolation of poly(ADP-ribose) polymerase in the absence of protease inhibitors resulted in a loss of more than 90% of the polymerase activity and an increased proportion of the 76-kDa and 59-kDa polypeptides in the final polymerase preparation. When the polymerase and the two polypeptides were separated by gel filtration or polyacrylamide gel electrophoresis in 5% acetic acid, no polymerase activity was found associated with the two fragments. Analysis of the CNBr fragments of the three polypeptides after incubation of the enzyme preparation with [32P]NAD showed that most of the fragments were radioactive, indicating multiple ADP-ribosylation sites. Several ADP-ribosylated fragments were found to be common to all three polypeptides, or to two of them. 相似文献
12.
The physicochemical properties of the purified calf thymus poly(ADP-ribose) polymerase were investigated. The enzyme purified to homogeneity was shown to contain about 10% DNA on a weight basis and its activity to be DNA independent. After removing this fragment of DNA, called the sDNA fraction, the enzyme becomes DNA dependent. The activity of this enzyme preparation was entirely dependent on, and completely restored by, added calf thymus DNA or sDNA. However, the calf thymus DNA concentration needed was a hundred times higher than that of sDNA. The properties of the two enzyme preparations, DNA independent and DNA dependent, were essentially the same. They both reacted against the specific antibody obtained with the DNA-independent poly(ADP-ribose) polymerase. The pH optimum was around 8; the activity was stimulated by Mg2+, Mn2+ and Ca2+, and inhibited by high ionic strength, p-chloromercuribenzoate, ADP-ribose, AMP and polylysine. Nicotinamide, thymidine and NADP were shown to be competitive inhibitors. The enzymatic activity was stimulated by histone H1 when the ratio of DNA to histone H1 was 2. Histones H2A, H2B, H3 and H4 had little effect on the DNA-independent enzyme activity, but were strongly inhibitory for the DNA-dependent enzyme. This inhibitory effect could be reversed by allowing the DNA-dependent enzyme to react with the sDNA fraction before adding histone subfractions. The apparent Km for NAD of the DNA-dependent poly(ADP-ribose) polymerase was shown to vary with the DNA concentration. It was minimum when the amount of sDNA was 10% of that of the enzyme. The ratio of the apparent Km for sDNA to the enzyme concentration was constant at any enzyme concentration. The minimum estimation of the number of base pairs of sDNA required for maximal activation of one enzyme molecule was 16. For calf thymus DNA, this estimation was of 640. These results suggest that the activation of the enzyme needs the formation of some complex between the protein and a specific part of the DNA. This complex was preserved in the DNA-independent enzyme preparation. 相似文献
13.
The properties of poly(G) polymerase and poly(A) polymerase activities in the DNA-dependent RNA polymerase [nucleosidetriphosphate: RNA nucleotidyltransferase EC 2.7.7.6] I fraction from cauliflower (Brassica oleracea var. botrytis) were comparatively investigated. The pH optimum, the effect of ionic strength, the effect of substrate concentration on the rate of synthesis, the effect of divalent metal ion concentration, and the time course of synthesis at different temperatures were all different for the three polymerase activities. The enzyme fraction preferentially utilized denatured DNA. Synthetic poly(C) and poly(U) were more effectively utillized for the synthesis of polyguanylate and polyadenylate, respectively. Further, it was found that poly(G) and poly(A) formed in vitro by the enzyme fraction had chain length of 25-28 and 84-89 nucleotides, respectively, and that poly (adenylate-gluanylate) chain was hardly formed when ATP and GTP were added together as substrates in the same reaction medium. 相似文献
14.
Diadenosine 5', 5"'-P1,P4-tetraphosphate stimulates processing of adp-ribosylated poly(ADP-ribose) polymerase 总被引:4,自引:0,他引:4
The effect of diadenosine 5', 5"'-P1,P4-tetraphosphate (Ap4A) on the time course and acceptors of poly(ADP-ribose) synthesis was studied in undamaged and N-methyl-N'-nitro-N-nitrosoguanidine-treated human lymphocytes. Analysis of protein acceptors of poly(ADP-ribose) revealed that treatment with Ap4A stimulated ADP-ribosylation of bands at molecular weights of 96,000, 79,000, and 62,000. Pulse-chase studies showed that these bands were produced as a result of an effect of Ap4A on the processing of ADP-ribosylated proteins rather than on the synthesis of newly ADP-ribosylated proteins. By incubating permeabilized cells in the absence or presence of Ap4A and purified poly(ADP-ribose) polymerase auto-ADP-ribosylated with [32P]NAD+, we showed that the Mr = 96,000, 79,000, and 62,000 bands were derivatives of the prelabeled enzyme. Our results indicate that normal human lymphocytes process auto-ADP-ribosylated poly(ADP-ribose) polymerase to specific lower molecular weight products and that this processing is stimulated by Ap4A. 相似文献
15.
We have characterized the effect of poly(ADP-ribose) polymerase automodification on the enzyme's activities, which include poly(ADP-ribose) synthesis and NADase activity. The apparent Km of the enzyme for NAD+ during polymer synthesis is higher than the one measured for alternate NADase activity. Furthermore, we have found that there are 28 automodification sites, in contrast to the 15 sites (postulated to be on the 15 glutamic acids) reported to be present in the automodification domain. For the first time, we show that some of these acceptor sites are outside the reported automodification domain (15 kDa); we demonstrate automodification in the NAD+ binding domain (55.2 kDa) and the DNA binding domain (42.5 kDa). We have analyzed the relationship between the number of sites modified on poly(ADP-ribose) polymerase and its effect on the polymerization activity and its alternate NADase activity. Automodification greatly altered both enzyme activities, decreasing both polymer synthesis and alternate NADase activity. 相似文献
16.
Poly(ADP-ribose) polymerase auto-modification and interaction with DNA: electron microscopic visualization. 总被引:5,自引:1,他引:4
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The interaction between purified calf thymus poly(ADP-ribose) polymerase and its activating co-purified DNA (sDNA) was investigated by electron microscopy. We have shown that the enzyme-DNA complex possesses a nucleosome-like structure. The enzyme-bound DNA (sDNA) was found to be enriched in single-stranded regions and branched structures, presumed to be replication forks. The auto-ribosylated polymerase as well as the branched poly(ADP-ribose) formed were visualized by dark field electron microscopy during the auto-ADP-ribosylation reaction and the possible mechanism of this phenomenon is discussed. 相似文献
17.
K Yoshihara A Itaya Y Tanaka Y Ohashi K Ito H Teraoka K Tsukada A Matsukage T Kamiya 《Biochemical and biophysical research communications》1985,128(1):61-67
Incubation of DNA polymerase alpha, DNA polymerase beta, terminal deoxynucleotidyl transferase, or DNA ligase II in a reconstituted poly(ADP-ribosyl)ating enzyme system markedly suppressed the activity of these enzymes. Components required for poly(ADP-ribose) synthesis including poly(ADP-ribose) polymerase, NAD+, DNA, and Mg2+ were all essential for the observed suppression. Purified poly(ADP-ribose) itself, however, was slightly inhibitory to all of these enzymes. Furthermore, the suppressed activities of DNA polymerase alpha, DNA polymerase beta, and terminal deoxynucleotidyl transferase were largely restored (3 to 4-fold stimulation was observed) by a mild alkaline treatment, a procedure known to hydrolyze alkaline-labile ester linkage between poly(ADP-ribose) and an acceptor protein. All of these results strongly suggest that the four nuclear enzymes were inhibited as a result of poly(ADP-ribosyl)ation of either the enzyme molecule itself or some regulatory proteins of these enzymes. 相似文献
18.
Isolation and expression of cDNA clones encoding mammalian poly(A) polymerase. 总被引:29,自引:3,他引:26
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cDNA clones encoding mammalian poly(A) polymerase were isolated with probes generated by the polymerase chain reaction based on amino acid sequences derived from the purified enzyme. A bovine cDNA clone was obtained encoding a protein of 82 kDa. Expression in Escherichia coli resulted in the appearance of a poly(A) polymerase activity that was dependent on the addition of the purified specificity factor CPF and the presence of the polyadenylation signal AAUAAA in the RNA substrate. The activity copurified with a polypeptide of the expected size. A second class of cDNAs encoded a polypeptide of 43 kDa which was closely related to the N-terminal half of the 82 kDa protein. Northern blots showed two mRNAs of 4.2 and 2.4 kb that probably correspond to the two classes of cDNAs, as well as a third band of 1.3 kb. The sequence of the N-terminal half of bovine poly(A) polymerase is 47% identical with the amino acid sequence of the corresponding part of yeast poly(A) polymerase. Homologies to other proteins are of uncertain significance. 相似文献
19.
Y Tanaka 《Journal of biochemistry》1982,91(6):2029-2037
The effects of polyamines on reactions catalyzed by bovine thymus poly(ADP-ribose) polymerase were examined under various conditions, and the following results were obtained. (1) Spermine and spermidine, and putrescine to a lesser degree can stimulate the synthesis of poly(ADP-ribose) covalently bound to the enzyme without Mg2+ and histones. (2) A part of the above stimulation can be explained by the Mg2+-sparing effect of polyamines. (3) The other part of the stimulation is shown to be through protection of the enzyme against the formation of an abortive complex of the enzyme and denatured DNA, which contaminates some native DNA preparations used for enzyme activation. Similar protection was shown earlier in this laboratory with histones. (4) Putrescine seems to lack this enzyme-protecting activity. (5) The polyamine effect observed in the Mg2+-dependent reaction is variable depending on the DNA preparations used. (6) Chemical analysis shows that the average chain lengths of the products synthesized with Mg2+ and spermine are similar, and the products are covalently bound to the enzyme, indicating that the reaction supported by polyamines is essentially the same as that by Mg2+. (7) Under the histone H1-dependent reaction conditions where ADP-ribosylation of histone H1 is predominant, both Mg2+ and polyamines are inhibitory on the reaction and both cations decrease the number of product molecules without affecting the size of the product. These data suggest that polyamines can at least partially replace Mg2+ in terms of effect on the ADP-ribosylation reaction. The other effect of polyamines is the protection of the enzyme from abortive binding to denatured DNA, as has also been shown to occur with histones. 相似文献