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1.
A new class of enzyme acting on damaged ribosomes: ribosomal RNA apurinic site specific lyase found in wheat germ 总被引:4,自引:0,他引:4
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A new enzyme, which we named ribosomal RNA apurinic site specific lyase (RALyase), is described. The protein was found in wheat embryos and has a molecular weight of 50 625 Da. The enzyme specifically cleaves the phosphodiester bond at the 3' side of the apurinic site introduced by ribosome-inactivating proteins into the sarcin/ricin domain of 28S rRNA. The 3' and 5' ends of wheat 28S rRNA at the cleavage site are 5'-GUACG-alpha-hydroxy-alpha, beta-unsaturated aldehyde and pGAGGA-3', demonstrating that the enzyme catalyzes a beta-elimination reaction. The substrate specificity of the enzyme is extremely high: it acts only at the apurinic site in the sarcin/ricin domain of intact ribosomes, not on deproteinized rRNA or DNA containing apurinic sites. The amino acid sequences of five endopeptidase LysC-liberated peptides from the purified enzyme were determined and used to obtain a cDNA sequence. The open reading frame encodes a protein of 456 amino acids, and a homology search revealed a related rice protein. Similar enzyme activities were also found in other plants that express ribosome-inactivating proteins. We believe that RALyase is part of a complex self-defense mechanism. 相似文献
2.
An endonuclease activity that acts on alkali-labile lesions in x-irradiated PM2 DNA and recognizes apurinic lesions in heat/acid treated DNA has been partially purified from Drosophila melanogaster embryos and its specific activity monitored throughout early development. The enzyme activity also showed a low level of activity on UV-irradiated DNA. The saturation kinetics observed with both x-irradiated and apurinic PM2 DNA substrates were similar. The endonuclease activity exhibited a broad pH optimum between pH 6 and 8.5 and was almost completely inhibited by 100 mM NaCl, 0.1 mM EDTA, 2 mM CaCl12 and 10 mM NEM. The reaction was not completely dependent on the presence of Mg++cation, but optimum activity was obtained at a concentration of 0.1 mM; concentrations greater than 1 mM Mgs++ were inhibitory. The specific activity of the apurinic endonuclease, partially purified from several stages of embryonic and early larval development, remained the same. Unfertilized eggs exhibited a reduced level of this presumptive repair activity.Abbreviations AP endonucleases
Apurinic/apyrimidinic endonucleases 相似文献
3.
An endonuclease which hydrolyzes depurinated DNA has been isolated from Phaseolus multiflorus enbryos; it has a molecular weight around 40,000. The enzyme is specific for apurinic sites; it has no action on normal DNA strands or on alkylated sites, and is without exonulcease activity. The rate of phosphoester bond hydrolysis near apurinic sites is far greater in native than in denatured DNA. The endonuclease is not inactivated by 10 mM EDTA, but is activity is however stimulated by Mg2+ or Mn2+. Its optimum pH is 7.5 to 8.0, and its optimum temperature 40degrees although, at this temperature, it is rapidly denatured; even low NaCl concentrations inhibit the enzyme activity. The endonuclease for apurinic sites of P. multiflorus is a non-histone protein of chromatin; the properties (like thermosensitivity of susceptibility to ionic strength) of the enzyme in situ, working on chromatin DNA, might be different from those described for the isolated endonuclease in homogenous aqueous solution. 相似文献
4.
Two endonucleases specific for DNA-containing apurinic or apyrimidinic sites (AP-endonucleases A and B) have been isolated from Micrococcus luteus and highly purified. These enzymes have no exonuclease activity. Both AP-endonucleases hydrolyze DNA-containing apurinic or apyrimidinic sites at the 5' end of the lesion, thus generating 3'-hydroxyl and 5'-phosphoryl end groups. DNA-containing pyrimidine dimers, introduced at low doses of UV, are not hydrolyzed, whereas DNA-containing lesions, introduced at high doses of UV or by gamma irradiation are nicked by either AP-endonuclease. During hydrolysis of apurinic DNA, neither of the AP-endonucleases acts as a processive enzyme. 相似文献
5.
Three cationic proteins from the granules of human neutrophil granulocytes were obtained in a high degree of purity be means of affinity chromatography on 4-phenylbutylamine-Sepharose. Together with lysozyme, the three cationic proteins exhibit the highest electrophoretic mobility toward the cathode in acrylamide gels at moderately acid pH, among the granule constituents that are solubilized in 0.1 M phosphate buffer, pH 7.0, containing 1 M NaCl. The three cationic proteins represent a group of "neutral proteases" distinct from elastase and collagenase. They hydrolyze casein, azocasein and the chymotrypsin substrate N-acetyl-L-tyrosine ethyl ester. Optimal activity is found at pH 7.4-7;5. The enzymes are inhibited by the specific chymotrypsin inhibitor N-tosyl-L-phenylalanylchloromethane and by the naturally occurring inhibitors alpha-antichymotrypsin, alpha-1-antitrypsin, as well as by the trypsin inhibitors from soy beans and limabeans. 相似文献
6.
DNA repair in Saccharomyces cerevisiae: purification and characterization of apurinic endonucleases 总被引:1,自引:1,他引:0
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Five chromatographically distinct apurinic endonucleases (D1, D2, D3, D4, and E) were purified from Saccharomyces cerevisiae 234, 122, 1,000, 4,550, and 5,490-fold, respectively. All appeared to be class II apurinic endonucleases and were not contaminated with exonuclease or nonspecific endonuclease activities under the reaction conditions used. All had similar pH optima, but endonucleases D4 and E showed higher salt requirements and endonuclease D4 had a lower Mg2+ requirement for optimal activity than the other endonucleases. Endonuclease D4 also nicked OsO4-treated DNA. The molecular weights of the apurinic endonucleases as determined by glycerol gradient sedimentation analysis were 37,000, 49,000, and 10,000, for endonucleases E, D4, and D2, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of samples of radioiodinated endonuclease E showed the presence of two proteins. 相似文献
7.
The endonuclease activity specific for apurinic sites in DNA was detected in barley embryos. The enzyme was partially purified. It reveals high activity on partially depurinated DNA but low or nil activity on intact and alkylated DNA. The method used for the detection of enzyme activity was based on the changes in the sedimentation velocity of substrate DNA in neutral sucrose gradients with 80 % formamide. 相似文献
8.
The nucleotide sequence of the Pseudomonas aeruginosa pyrE-crc-rph region and the purification of the crc gene product. 总被引:1,自引:0,他引:1
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C H MacGregor S K Arora P W Hager M B Dail P V Phibbs Jr 《Journal of bacteriology》1996,178(19):5627-5635
The gene (crc) responsible for catabolite repression control in Pseudomonas aeruginosa has been cloned and sequenced. Flanking the crc gene are genes encoding orotate phosphoribosyl transferase (pyrE) and RNase PH (rph). New crc mutants were constructed by disruption of the wild-type crc gene. The crc gene encodes an open reading frame of 259 amino acids with homology to the apurinic/apyrimidinic endonuclease family of DNA repair enzymes. However, crc mutants do not have a DNA repair phenotype, nor can the crc gene complement Escherichia coli DNA repair-deficient strains. The crc gene product was overexpressed in both P. aeruginosa and in E. coli, and the Crc protein was purified from both. The purified Crc proteins show neither apurinic/apyrimidinic endonuclease nor exonuclease activity. Antibody to the purified Crc protein reacted with proteins of similar size in crude extracts from Pseudomonas putida and Pseudomonas fluorescens, suggesting a common mechanism of catabolite repression in these three species. 相似文献
9.
Food proteins have been identified as a source of bioactive peptides. These peptides are inactive within the sequence of the parent protein and must be released during gastrointestinal digestion, fermentation, or food processing. Of bioactive peptides, multifunctional cationic peptides are more useful than other peptides that have specific activity in promotion of health and/or the treatment of diseases. We have identified and characterized cationic peptides from rice enzymes and proteins that possess multiple functions, including antimicrobial, endotoxin-neutralizing, arginine gingipain-inhibitory, and/or angiogenic activities. In particular, we have elucidated the contribution of cationic amino acids (arginine and lysine) in the peptides to their bioactivities. Further, we have discussed the critical parameters, particularly proteinase preparations and fractionation or purification, in the enzymatic hydrolysis process for producing bioactive peptides from food proteins. Using an ampholyte-free isoelectric focusing (autofocusing) technique as a tool for fractionation, we successfully prepared fractions containing cationic peptides with multiple functions. 相似文献
10.
Kenny MK Mendez F Sandigursky M Kureekattil RP Goldman JD Franklin WA Bases R 《The Journal of biological chemistry》2001,276(12):9532-9536
The interaction of human heat shock protein 70 (HSP70) with human apurinic/apyrimidinic endonuclease (HAP1) was demonstrated by coimmunoprecipitation. A combination of HSP70 and HAP1 also caused a shift in the electrophoretic mobility of a DNA fragment containing an apurinic/apyrimidinic site. The functional consequence of the HSP70/HAP1 interaction was a 10-100-fold enhancement of endonuclease activity at abasic sites. The physical and functional interaction between HSP70 and HAP1 did not require the addition of ATP. The association of HSP70 and a key base excision repair enzyme suggests a role for heat shock proteins in promoting base excision repair. These findings provide a possible mechanism by which HSP70 protects cells against oxidative stress. 相似文献
11.
The aromatic amine 9-amino-ellipticine is a synthetic DNA intercalating compound derived from the antitumor agent ellipticine, which cleaves at very low doses DNA containing apurinic sites by beta-elimination through formation of a Schiff base. This compound has been shown to potentiate the cytotoxic effect of alkylating drugs, such as dimethyl sulfate, in E. coli through a mechanism involving apurinic sites. We have studied the ability of 9-amino-ellipticine to inhibit an enzymatic repair system mimicking base-excision repair, in which E. coli exonuclease III only presents an endonuclease for apurinic/apyrimidinic site activity. 10 microM of 9-amino-ellipticine inhibits 70% of apurinic site repair. Other intercalating agents with similar affinities for DNA do not induce any inhibition. In another system designed for the direct assay of the exonuclease III-induced incisions 5' to AP sites 10 microM of 9-amino-ellipticine inhibits 65% of the endonuclease for apurinic/apyrimidinic site activity of E. coli exonuclease III. The 9-amino-ellipticine-induced formation of a 2',3'-unsaturated deoxyribose and cleavage at the 3' side of the apurinic site, and possible creation of an adduct, as suggested by Bertrand and coworkers (1989), on the 3' position of the deoxyribose seem to strongly inhibit the endonuclease for apurinic/apyrimidinic site activity. 9-Amino-ellipticine appears therefore to be the first small ligand which can inhibit, by an irreversible modification of the substrate, the repair of apurinic sites through the base excision-repair pathway at a pharmacological concentration. 相似文献
12.
Two nucleases active on alkylated-depurinated DNA have been extracted from rat liver chromatin with 1 M KCl. The major enzyme was purified to near homogeneity; it has a molecular weight of 12 500 (although some dimerization might occur), needs Mg2+ or Mn2+ for activity. The endonuclease activity is specific for apurinic/apyrimidinic sites in DNA; the enzyme has no associated exonuclease activity. 相似文献
13.
Methyl methane sulfonate-sensitive mutant of Escherichia coli deficient in an endonuclease specific for apurinic sites in deoxyribonucleic acid. 总被引:25,自引:12,他引:13
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A methyl methane sulfonate (MMS)-sensitive mutant of Escherichia coli AB 1157 was obtained by N-methyl-N'-nitro-N-nitrosoguanidine treatment. The mutant strain, AB 3027, is defective both in endonuclease activity for apurinic sites in deoxyribonucleic acid (DNA) and in DNA polymerase I, as shown by direct enzyme assays. Derivative strains, which retained the deficiency in endonuclease activity for apurinic sties (approximately 10% of the wild-type enzyme level) but had normal DNA polymerase I activity, were obtained by P1-mediated transduction (strain NH5016) or by selection of revertants to decreased MMS sensitivity. These endonuclease-deficient strains are more MMS-sensitive than wild-type strains. Revertants of these deficients strains to normal MMS resistance were isolated. They had increased levels of the endonuclease activity but did not attain wild-type levels. The data suggest that endonuclease for apurinic sites is active in repair of lesions introduced in DNA as a consequence of MMS treatment. Two different endonucleases that specifically attack DNA containing apurinic sites arepresented in E coli K-12. A heat-labile activity, sensitive to inhibition by ethylenediaminetetraacetate, accounts for 90% of the total endonuclease activity for apurinic sties in crude cell extracts. The residual 10% is due to a more heat-resistant activity, refractory to ethylenediaminetetraacetate inhibition. The AB3027 and NH5016 strains have normal amounts of the latter endonuclease but no or very little of the former activity. 相似文献
14.
15.
Purification and characterization of an endonuclease specific for apurinic sites in DNA from a permanently established mouse plasmacytoma cell line. 总被引:3,自引:3,他引:0
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I F Nes 《Nucleic acids research》1980,8(7):1575-1589
An endonuclease specific for apurinic sites in double stranded DNA has been purified 373-fold from the nuclei of mouse plasmacytoma cells (line MPC-11). The enzyme is free of any detectable amounts of aspecific nucleases. The enzyme does not act on methylated or OsO4-treated DNA. However, high doses of UV-light and gamma-rays render the DNA slightly susceptible to endonucleolytic attack, which is believed to be due to depurination of depyrimidination caused by the treatment. The molecular weight of the enzyme is determined to be 28,000 and its apparent Km of the purified enzyme is calculated to be 2.7 nM apurinic sites. The activity is not absolutely dependent upon the presence of Mg2+ in the assay mixture although metal chelating agents such as sodium citrate and EDTA abolish the activity completely. The nuclease was stimulated by moderate concentrations of potassium chloride optimizing at 50 mM, and higher concentrations inhibiting the activity. The pH optimun for the reaction was 9.5. 相似文献
16.
den V gene of bacteriophage T4 determines a DNA glycosylase specific for pyrimidine dimers in DNA. 总被引:22,自引:7,他引:15
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Endonuclease V of bacteriophage T4 has been described as an enzyme, coded for by the denV gene, that incises UV-irradiated DNA. It has recently been proposed that incision of irradiated DNA by this enzyme and the analogous "correndonucleases" I and II of Micrococcus luteus requires the sequential action of a pyrimidine dimer-specific DNA glycosylase and an apyrimidinic/apurinic endonuclease. In support of this two-step mechanism, we found that our preparations of T4 endonuclease V contained a DNA glycosylase activity that produced alkali-labile sites in irradiated DNA and an apyrimidinic/apurinic endonuclease activity that converted these sites to nicks. Both activities could be detected in the presence of 10 mM EDTA. In experiments designed to determine which of the activities is coded by the denV gene, we found that the glycosylase was more heat labile in extracts of Escherichia coli infected with either of two thermosensitive denV mutants than in extracts of cells infected with wild-type T4. In contrast, apyrimidinic/apurinic endonuclease activity was no more heat labile in extracts of the former than in extracts of the latter. Our results indicate that the denV gene codes for a DNA glycosylase specific for pyrimidine dimers. 相似文献
17.
Apurinic endonucleases from Saccharomyces cerevisiae also recognize urea residues in oxidized DNA.
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Saccharomyces cerevisiae apurinic endonucleases E cochromatographed with activity against a DNA substrate containing urea residues. The urea-recognizing activity of endonuclease E was competitively inhibited by apurinic DNA, and the heat labilities of both activities were the same. The apparent VmaxS of endonuclease E for both substrates were about the same, while the apparent Km for urea-containing DNA was about threefold greater than that for apurinic DNA. These results were similar to those obtained previously with Escherichia coli exonuclease III (Y. Kow and S. Wallace, Proc. Natl. Acad. Sci. USA 82:8354-8358, 1985) and suggest that the ability to recognize urea residues may be a general property of apurinic endonucleases. 相似文献
18.
An endonuclease which hydrolyzes depurinated DNA has been purified from extracts of Bacillus subtilis cells. The endonuclease is a monomeric protein and has a molecular weight of around 56,000. The enzyme is specific for apurinic sites in double-stranded DNA, has a pH optimum at 8.0, and is slightly stimulated with 50 mM NaCl but completely inhibited with 500 mM NaCl. It requires no divalent cations and is insensitive to EDTA; it has no associated exonuclease. These properties are very similar to those of Escherichia coli endonuclease IV, which is also insensitive to EDTA and has no exonuclease activity, and very different from those of the main endonuclease for apurinic sites (endonuclease IV) of the same bacterium. 相似文献
19.
Characterization of human enzymes specific for damaged DNA: resolution of endonuclease for irradiated DNA from an apparent N-glycosidase active on alkylated DNA.
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T P Brent 《Nucleic acids research》1977,4(7):2445-2454
An endonuclease partially purified from human lymphoblasts, and active against ultraviolet-irradiated DNA, was found to act additionally on DNA damaged by either x-radiation or methylmethanesulfonate. To determine if these activities were truly endonucleolytic, the reaction products were analyzed under conditions that prevented conversion of apurinic or apyrimidinic sites to single-strand breaks. With either ultraviolet- or x-irradiated DNA, strand breakage remained maximal, hence confirming the endonucleolytic character of the enzyme. By contrast, with DNA alkylated with methylmethanesulfonate, strand breakage was sharply reduced. Additional experiments indicated that the activity for alkylated DNA induces strand breaks only in concert with a purified endonuclease specific for apurinic sites, suggesting that it is an N-glycosidase that depurinates alkylated bases. This enzyme was separated from the endonuclease specific for irradiated DNA, by chromatography on DNA-agarose. 相似文献
20.
Cationic proteins of human granulocytes. VI. Effects on the complement system and mediation of chemotactic activity. 总被引:9,自引:0,他引:9
The chymotrypsin-like cationic proteins of human granulocytes are shown to possess the ability to produce conversion of the complement components C1s, C4, C3, and C5 as detected by crossed immuno-electrophoresis. This ability seems to be a direct proteolytic effect. Incubation of cationic proteins with serum or functionally pure preparations of C3 and C5 is shown to generate the formation of chemotactic activity which is abolished by prolonged incubation. Also, the chemotactic activity of porcine C5a or spontaneously activated C5 is abolished by incubation with cationic proteins. It is suggested that the chymotrypsin-like cationic proteins of human granulocytes after extrusion from the phagocytic cell play an important role for generation of inflammatory mediators. 相似文献