Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.25.1 (deoxyribonuclease)
1,471 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A deoxyribonuclease, which requires nucleoside triphosphate for reaction, has been purified about 150-fold from extracts of Bacillus laterosporus. Potassium phosphate and ethylene glycol stabilize the purified enzyme. The enzyme degrades double-stranded DNA about 100 times faster than heat-denatured DNA in the presence of nucleoside triphosphate. Double-stranded DNA is not degraded to any measurable extent in the absence of ATP, but the enzyme exhibits activity toward denatured DNA in the absence of nucleoside triphosphate, and this activity seems to be an intrinsic property of this enzyme protein. The optimum pH is 8.5 and the maximum activity is obtained in the copresence of Mg2+ (8.0 X 10(-3)M) and Mn2+ (7.0 X 10(-5)M). ATP and dATP are most effective and nucleoside di- or monophosphates are ineffective. ATP is converted to ADP and inorganic phosphate during the reaction and the ratio of the amount of ATP cleaved to that of hydrolyzed phosphodiester bonds of DNA is about 3:1. An inhibitor of the enzyme was observed in bacterial extracts prepared by sonic disruption; the inhibitory substance is produced in the bacteria in the later stages of cell growth. Preliminary results show that the inhibitor emerged near the void volume of a Sephadex G-200 column, and was relatively heat-stable, RNase-resistant, and DNase-sensitive.
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PMID:A nucleoside triphosphate-dependent deoxyribonuclease from Bacillus laterosporus. Purification and characterization of the enzyme. 0 Mar 73

Meta-iodobenzylguanidine (MIBG) is a guanidine analogue of the neurotransmitter norepinephrine. Radioiodinated [131I]MIBG is clinically used as a tumor-targeted radiopharmaceutical in the diagnosis and treatment of adrenergic tumors. Moreover, non-radiolabelled MIBG exerts several cell-biological effects, tentatively ascribed to interference with cellular mono(ADP-ribosyl) transferases (Smets, L.A., Bout, B. and Wisse, J. (1988) Cancer Chemother. Pharmacol. 21, 9-13; Smets, L.A., Metwally, E.A.G., Knol, E. and Martens, M. (1988) Leukemia Res. 12, 737-743). In the present study it was investigated whether MIBG could serve as an acceptor for the ribosyl transferase activity of cholera toxin and of erythrocyte membranes. MIBG appeared a substrate for the cholera toxin-catalyzed transfer of the ADP-ribose moiety of NAD to arginine-like residues with the highest affinity for this enzyme reported as yet (Km = 6.5 microM). MIBG was also ADP-ribosylated by the mono(ADP-ribosyl)transferase(s) of turkey erythrocyte membranes. Moreover, the drug appeared a potent affector of the ADP-ribose linkage to membrane proteins by these enzymes. Interference by MIBG was stronger than by related guanyltyramine, the monoamine precursors of MIBG, meta-iodobenzylamine had no effect at all. In contrast, the drug failed to affect endogenous, O-linked poly(ADP-ribose) polymerase, induced in nuclei of S49-leukemia cells by deoxyribonuclease. Since MIBG is the first described drug that specifically interferes with the cellular N-linked mono(ADP-ribosyl) transferase reactions, it may be an important tool to elucidate the physiological role of this posttranscriptional protein modification.
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PMID:Meta-iodobenzylguanidine (MIBG), a novel high-affinity substrate for cholera toxin that interferes with cellular mono(ADP-ribosylation). 210 58

Rat liver mitochondria isolated in sucrose-N-tris(hydroxymethyl)methyl-2-aminoethane-sulphonic acid (TES) incorporated [(3)H]UTP into RNA for 1h. Incorporation was inhibited 50% by 1mug of actinomycin D/ml, 1mug of acriflavine/ml and 0.5mug of ethidium bromide/ml but was insensitive to rifampicin, rifamycin SV, streptovarcin and deoxyribonuclease. After the first 10min of incubation, the synthesis was insensitive to ribonuclease. RNA synthesis by mitochondria isolated in sucrose-EDTA was insensitive to actinomycin D and sensitive to ribonuclease during the first 10min of the incubation but thereafter the sensitivities were the same as for mitochondria isolated in sucrose-TES. In a hypo-osmotic medium the relative extent of incorporation of the four ribonucleoside triphosphates into RNA was CTP>UTP=ATP>>GTP. In an iso-osmotic medium the incorporation of CTP and GTP decreased. All four nucleotides were incorporated into RNA in a DNA-dependent process, as indicated by the inhibition by actinomycin D. In addition, CTP and ATP were incorporated into the CCA end of mitochondrial tRNA. ATP was also incorporated into an unidentified acid-insoluble compound, which hydrolysed in alkali to a product that was not ATP, ADP or 5'- or 2(3')-AMP. Atractyloside inhibited the incorporation of ATP into RNA with 50% inhibition at 2-3nmol/mg of protein. The [(3)H]UTP-labelled RNA had peaks of 16S and 13S characteristic of mitochondrial rRNA. In addition a peak at 20-21S was observed as well as heterogeneous RNA sedimenting throughout the gradient. The synthesis of all these species was inhibited by actinomycin D, indicating that rat liver mitochondrial DNA codes for mitochondrial rRNA as well as other as yet unidentified species.
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PMID:Synthesis of ribonucleic acid by isolated rat liver mitochondria. 440 94

An endodeoxyribonuclease from HeLa cells acting on apurinic/apyrimidinic (AP) sites has been purified to apparent homogeneity as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The presence of Triton X-100 was necessary throughout the purification for stabilization and stimulation of activity. The endonuclease has an apparent native molecular weight of 32,000 determined by molecular sieving and an apparent subunit molecular weight of 41,000 as judged by its electrophoretic mobility in SDS-polyacrylamide gels. The activity has an absolute requirement for Mg2+ or Mn2+ and a broad pH optimum between 6.7 and 9.0 with maximal activity near pH 7.5. The enzyme has no detectable exonuclease activity, nor any endonuclease activity on untreated duplex or single-stranded DNA. It is inhibited by adenine, hypoxanthine, adenosine, AMP, ADP-ribose, and NAD+, but it is unaffected by caffeine, the pyrimidine bases, ADP, ATP, or NADH. The use of a variety of damaged DNA substrates provided no indication that the enzyme acts on other than AP sites. The enzyme appears to cleave AP DNA so as to leave deoxyribose-5-phosphate at the 5' terminus and a 3'-OH at the 3' terminus; it also removes deoxyribose-5-phosphate from AP DNA which has deoxyribose at the 3' terminus. Specific antibody has been produced in rabbits which interacts only with a 41,000-dalton protein present in the purified enzyme (presumably the enzyme itself), as well as with partially purified AP endonuclease fractions from human placenta and fibroblasts.
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PMID:Purification and characterization of an apurinic/apyrimidinic endonuclease from HeLa cells. 625 65

The effect of thyrotropin (TSH) on the ADP-ribosylation of endogenous thyroid cell acceptor proteins was examined. Cells were "permeabilized" at 4 degrees C in hypotonic medium and then exposed to [(32)P]- or [(3)H-adenine]NAD(+). The net incorporation of labeled ADP-ribose was measured by trichloroacetic acid precipitation. TSH (100 mU/ml) enhanced ADP-ribosylation with a maximum effect after 30-60 min in the majority of experiments. TSH stimulation was observed even when the incubation contained 1,000-fold more exogenous NAD(+) than the amount of NAD(+) contributed by the permeabilized cells, indicating an effect on enzymatic activity rather than an alteration in NAD(+) pool size or specific activity. No incorporation of radioactivity from labeled NAD(+) was observed in cells not rendered permeable to NAD(+) by hypotonic shock. TSH did not increase the rate of disappearance of trichloroacetic-precipitable radioactivity and did not contain intrinsic NAD(+) glycohydrolase activity. Alkali and snake venom phosphodiesterase, but not ribonuclease or deoxyribonuclease digestion of trichloroacetic acid precipitable thyroid cell radioactivity, revealed primarily 5'-AMP, consistent with an effect of TSH on mono-ADP ribosylation. Nicotinamide and thymidine (50 mM) inhibited both basal and TSH-stimulated ADP-ribosylation of thyroid cell protein. Dibutyryl cyclic (c)AMP (0.1 mM) inhibited endogenous ADP-ribosylation by approximately 35% but had no effect at lower concentrations. 0.5 mM isobutylmethylxanthine inhibited this reaction by approximately 60%. We suggest that TSH enhances thyroid cell ADP-ribosylation by a mechanism independent of cAMP as a second messenger, and that ADP-ribosylation plays a role in the expression of TSH.
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PMID:Hormonal stimulation of eucaryotic cell ADP-ribosylation. 626 5

ATP-dependent deoxyribonuclease from Micrococcus luteus was purified to near homogeneity by a procedure involving gentle cell lysis, ammonium sulfate fractionation, TEAE-cellulose chromatography, Sephadex G-150 gel filtration and DNA-cellulose chromatography. Treatment of the enzyme with 2,3-butanedione, which binds specifically to arginyl residues, caused rapid loss of enzyme activities and the effect was enhanced by borate ion. The reaction obeyed first order kinetics with respect to the butanedione concentration, indicating that at least one functional arginyl residue is involved in the inactivation reaction. The enzyme was protected from inactivation by the presence of a low concentration of ATP, but not of ADP, AMP or adenosine. These results indicate that ATP-dependent deoxyribonuclease of Micrococcus luteus has functional arginyl residue(s) at an ATP-binding site.
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PMID:Inactivation of ATP-dependent deoxyribonuclease of Micrococcus luteus by 2,3-butanedione. 629 67

Epithelial cells from the lens equator differentiate into elongated fiber cells. In the final steps of differentiation, the chromatin appears quite condensed and chromatin breakdown into nucleosomes occurs. DNA breaks due to an endodeoxyribonuclease activity corresponding to at least two polypeptides of 30 and 40 kDa have been identified. To identify the nature and the developmental appearance of initial breaks, nick translation reaction was followed both biochemically and in situ in fiber and epithelial cells from chick embryonic lenses. There is no accumulation of single-strand breaks (SSB) with 3'OH ends in lens fiber cells during embryonic development. Such damage can be increased in these cells by treatment with DNAase I indicating the absence of an inhibitor of the nick translation reaction in fiber cells. However, there are indications of the presence of DNA breaks with blocked termini when the phosphatase activity of nuclease P1 is used. The presence of breaks is also indicated by the large amounts of (ADP-ribose)n found in lens fibers particularly at 11 days of embryonic development (E11) as ADP-ribosyl transferase binds to and is activated by DNA strand breaks. Incubation of lens cells in vitro, which causes nucleosomal fragmentation only in fiber cells, produces SSB with 3'OH ends in both epithelia and fibers. Incubation for short periods, observed in experiments in situ, induces SSB first in the central fiber nuclei, which are late in differentiation. This may indicate that these SSB play a physiological role. Long incubations produce larger numbers of SSB in epithelia than fibers. The SSB in the fibers may have been converted into double-strand breaks (D SB), seen as nucleosomal fragments, and therefore no longer act as substrates for nick translation. The nuclease activity responsible for SSB production is independent of divalent cations and could be implicated in lens terminal differentiation.
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PMID:DNA strand breakage during physiological apoptosis of the embryonic chick lens: free 3' OH end single strand breaks do not accumulate even in the presence of a cation-independent deoxyribonuclease. 810 72

CRM45 is a mutant form of diphtheria toxin (DTx) that lacks a 17-kDa carboxyl-terminal segment of the receptor-binding B subunit (DTB). The missing segment is a discrete structural domain of DTB that normally rests against the NAD binding pocket of the enzymically-active A subunit (DTA). Proteolytic cleavage and disulfide bridge reduction in the DTA-DTB linker region of DTx are required for optimal ADP-ribosylation of elongation factor 2 (EF-2). Here, we show that cleaved and uncleaved preparations of X-ray crystal grade CRM45 both exhibit an ADP-ribosyltransferase activity similar to that of cleaved DTx. Crystal-grade preparations of CRM45 also display a potent deoxyribonuclease activity. However, as observed with DTx, cleavage and reduction of CRM45 are not required for expression of this nuclease activity. After SDS-PAGE in a gel that contains DNA embedded in the matrix, renaturable Ca++/Mg(++)-dependent nuclease-active bands co-migrate with intact CRM45 (45 kDa) as well as with the DTA subunit (24 kDa) of CRM45. Because the 45-kDa nuclease-active band is unique to the CRM45 form of DTx, it offers direct proof that this activity is intrinsic to the DTA domain of DTx and its homologues.
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PMID:Characterization of the deoxyribonuclease and ADP-ribosyltransferase activities of CRM45, a truncated homologue of diphtheria toxin. 978 63

A spectrophotometric method for quantification of linear DNA is described. The assay measures ADP produced following digestion of linear DNA by an ATP-dependent deoxyribonuclease. Cleavage of the phosphodiester bond of the DNA substrate is proportional to ADP formed in the reaction which follows typical Michaelis-Menten kinetics (K(m) of 0.6 microM, and a V(max) of 30 nmol/min/mg). The enzyme requires Mg(2+)-ATP and Mg(2+)-DNA as substrates, although the results suggest a requirement for yet another metal ion which may be enzyme bound. Both single-stranded and double-stranded linear DNA are substrates, as demonstrated by comparable initial velocity measurements. However, covalently closed circular (CCC) and nicked open circular DNA are not substrates for the enzyme. The rate of hydrolysis of ATP is not inhibited by 1 microg RNA or covalently closed circular DNA. The product (ADP) formed in the reaction is coupled to NADH oxidation using pyruvate kinase and lactate dehydrogenase. NAD formed in the reaction is monitored spectrophotometrically as a loss in absorbance at 340 nm. This assay directly measures the amount of linear DNA present in preparations of supercoiled (CCC) plasmid DNA, and has direct utility for monitoring the quality of plasmid preparations for gene therapy.
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PMID:A spectrophotometric method to quantify linear DNA. 1260 67

Catalytic DNA cleavage reactions by an ATP-dependent deoxyribonuclease (DNase) from Micrococcus luteus were monitored directly with a DNA-immobilized 27-MHz quartz-crystal microbalance (QCM). The 27-MHz QCM is a very sensitive mass-measuring device in aqueous solution, as the frequency decreases linearly with increasing mass on the electrode at a nanogram level. Three steps in ATP-dependent DNA hydrolysis reactions, including (1) binding of DNase to the end of double-stranded DNA (dsDNA) on the QCM electrode (mass increase), (2) degradation of one strand of dsDNA in the 3' --> 5' direction depending on ATP (mass decrease), and (3) release of the enzyme from the nonhydrolyzed 5'-free-ssDNA (mass decrease), could be monitored stepwise from the time dependencies of QCM frequency changes. Kinetic parameters for each step were obtained as follows. The binding constant (K(a)) of DNase to the dsDNA was determined as (28 +/- 2) x 10(6) M(-)(1) (k(on) = (8.0 +/- 0.3) x 10(3) M (-)(1) s(-)(1) and k(off) = (0.29 +/-0.01) x 10(-)(3) s(-)(1)), and it decreased to (0.79 +/- 0.16) x 10(6) M(-)(1) (k'(on) = (2.3 +/- 0.2) x 10(3) M (-)(1) s(-)(1) and k'(off) = (2.9 +/- 0.1) x 10(-)(3) s(-)(1)) for the completely nonhydrolyzed 5'-free ssDNA. This is the reason the DNase bound to the dsDNA substrate can easily release from the nonhydrolyzed 5'-free-ssDNA after the complete hydrolysis of the 3' --> 5' direction of the complementary ssDNA. K(a) values depended on the DNA structures on the QCM, and the order of these values was as follows: the dsDNA having a 4-base-mismatched base-pair end (3) > the dsDNA having a 5' 15-base overhanging end (2) > the dsDNA having a blunt end (1) > the ssDNA having a 3'-free end (4) >> the ssDNA having a 5'-free end (5). Thus, DNase hardly recognized the free 5' end of ssDNA. Michaelis-Menten parameters (K(m) for ATP and k(cat)) of the hydrolysis process also could be obtained, and the order of k(cat)/K(m) was as follows: the dsDNA having a blunt end (1) approximately the dsDNA having a 4-base-mismatched base-pair end (3) > the ssDNA having a free 3' end (4) >> the ssDNA having a free 5' end (5). Thus, DNase could not recognize and not hydrolyze the free 5' end of ssDNA. The DNA hydrolysis reaction could be driven by dATP and GTP (purine base) as well as ATP, whereas the cleavage efficiency was very low driven with UTP, CTP (pyrimidine base), ADP, and AMP.
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PMID:Kinetic studies of DNA cleavage reactions catalyzed by an ATP-dependent deoxyribonuclease on a 27-MHz quartz-crystal microbalance. 1570 38


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