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:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The properties of the enzyme ribonuclease N were investigated. By comparing the distribution in the cell of RNase N with the bonafide intracellular beta-galactosidase, and the periplasmic alkaline phosphatase enzymes, we showed that RNase N is an intracellular enzyme. Since previous studies suggested that it is an endoribonuclease, it was compared to RNase III, the only other known intracellular endoribonuclease in Escherichia coli. Using homopolymers and co-polymers we found that, while RNase III could digest double-stranded RNA only, RNase N digested single-stranded and double-stranded RNA with similar efficiency. Furthermore, all RNAs used, natural as well as synthetic, were substrates for the enzyme. Using 5 S rRNA as a substrate it was confirmed that the enzyme is an endonuclease. The final products of the reaction of this enzyme are 5'-mononucleotides. The molecular weight of the enzyme is about 120,000 and it seems to contain two subunits which are similar in size. These properties thus differentiate this enzyme from all other known ribonucleases in E. coli.
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PMID:Characterization of an endoribonuclease, RNase N, from Escherichia coli. 9

Phosphodiesterase I from the venom of Bothrops atrox has been purified by successive chromatography on phosphocellulose P-11, hydroxyapatite, and DEAE-cellulose DE 52. The final product gave a single band on sodium dodecylsulfate-polyacrylamide gels and was free of endonuclease, 5' -nucleotidase, and unspecific alkaline phosphatase activity. It was concentrated in an Amicon ultrafiltrator without loss of activity and could be stored in 10 mM magnesium acetate and 10% glycerol at 4 degrees C for at least a year. Under optimal conditions, the enzyme reaction required 15 mM Mg2+ and a pH of 9.2. Phosphodiesterase I is relatively thermostable and, in the presence of a macromolecular substrate, was not denatured after 4 h at 55 degrees C. The pure enzyme offers new possibilities for sequence studies on highly structured nucleic acids at elevated temperatures.
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PMID:Purification and characterization of phosphodiesterase I from Bothrops atrox. 17 76

A rapid batch procedure is described for purification of T4 polynucleotide kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) to near homogeneity using Blue Dextran-Sepharose chromatography. The enzyme preparation is sufficiently free of contaminating endonuclease and alkaline phosphatase activities to be suitable for radioactively labeling nucleic acids in vitro. Kinetic measurements indicate that the chromophore of Blue Dextran, Cibacron Blue F3GA, inhibits the activity of T4 polynucleotide kinase competitively with respect to single stranded DNA substrate and non-competitively with respect to the rATP substrate.
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PMID:A rapid purification of T4 polynucleotide kinase using Blue Dextran-Sepharose chromatography. 21 25

Py pyrimidine dimers Py correndonucleases I and II from Micrococcus luteus act exclusively on thymine-thymine, cytosine-cytosine, and thymine-cytosine cyclobutyl dimers in DNA, catalyzing incision 5' to the damage and generating 3'-hydroxyl and 5'-phosphoryl termini. Both enzymes initiate excision of pyrimidine dimers in vitro by correxonucleases and DNA polymerase I. The respective incised DNAs, however, differ in their ability to act as substrate for phage T4 polynucleotide ligase or bacterial alkaline phosphatase, suggesting that each endonuclease is specific for a conformationally unique site. The possibility that their respective action generates termini which represent different degrees of single strandedness is suggested by the unequal protection by Escherichia coli binding protein from the hydrolytic action of exonuclease VII.
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PMID:Micrococcus luteus correndonucleases. II. Mechanism of action of two endonucleases specific for DNA containing pyrimidine dimers. 33 May 26

Extracts of interferon-treated HeLa cells adsorbed to poly(I) . poly(C)-agarose have been used to synthesize 2'5'oligo(A). This oligonucleotide has been characterized by enzymatic digestion with alkaline phosphatase, snake venom phosphodiesterase, T2 ribonuclease and chromatography on DEAE, and PEI-cellulose. The oligonucleotide inhibits protein synthesis in vitro and activates an endonuclease present in extracts of control and interferon-treated cells. The metabolic stability of 2'5'oligo(A) has been investigated in these cell extracts. The oligonucleotide undergoes rapid degradation, particularly in the absence of ATP and of an energy regenerating system. Furthermore, the 2'5'oligo(A)-activated endonuclease reverts to an inactive state under these conditions, but can be reactivated upon further addition of 2'5'oligo(A). A possible role for the degradation of 2'5'oligo(A) in the mechanism of interferon action is discussed.
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PMID:Metabolic stability of 2' 5'oligo (A) and activity of 2' 5'oligo (A)-dependent endonuclease in extracts of interferon-treated and control HeLa cells. 42 14

Poly(A)-containing messenger RNA isolated from rabbit reticulocytes as estimated by periodate oxidation and condensation with [3H]isoniazid has two oxidizable end groups per molecule of mol. wt. 220000. When the mRNA is subjected to stepwise degradation by beta-elimination, only one oxidizable end-group is found. This indicates that one of the 2',3' hydroxyl end-groups is linked through the normal 3'--5' phosphodiester bond, but that the other is linked in such a way that after stepwise degradation no new 2',3 hydroxyl group is revealed. This structure could be a 5'-linked 5'-phospho di- or tri-ester. On digestion with ribonuclease the isoniazid-labelled RNA produced oligonucleotide hydrazones consistent with a poly(A) sequence at the 3' end plus fragments that are not found after stepwise degradation. These fragments have a charge of --6 and --8 from pancreatic ribonuclease or --7 from ribonuclease T1 digestion. These charges are changed to --3.4 and --4.1 after pancreatic ribonuclease, ribonuclease T2 and alkaline phosphatase digestion. methyl-3H-labelled-poly(A)-containing RNA isolated from late erythroid cells contain a methyl-labelled fragment resistant to endonuclease and phosphodiesterase II digestion. After digestion with phosphodiesterase I this fragment produces methyl-3 H-labelled nucleotides with the electrophoretic mobility of pm7G and pAm. It is concluded that globin mRNA has the 5' sequences m7G(5')ppp'AmpYpGp ... and m7G(5')pppAmpApGpYp.
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PMID:The nature of the 5'-linked 5' nucleotide sequence at the 5' end of rabbit globin messenger ribonucleic acid. 94 25

DNA was extracted from rat liver of non-irradiated animals, and was irradiated in vitro, and from animals which received whole body doses of X-radiation. Sedimentation on neutral and alkaline sucrose gradients as well as measurements of 32P release after sequential treatment with endonuclease and alkaline phosphatase and determination of triphosphate incorporation after the sequential treatment with endonuclease, alkaline phosphatase and DNA polymerase indicated that DNA irradiated in vivo and in vitro were effective substrates for the mammalian repair endonuclease. The experiments suggest that in addition to strand breaks, X-radiation causes base damage and they have provided a plausible explanation for the formation of double strand breaks in DNA irradiated in vivo.
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PMID:The effect of a mammalian repair endonuclease on x-irradiated DNA. 116 20

Alkaline phosphatase was the first zinc enzyme to be discovered in which three closely spaced metal ions (two Zn ions and one Mg ion) are present at the active center. Zn ions at all three sites also produce a maximally active enzyme. These metal ions have center-to-center distances of 3.9 A (Zn1-Zn2), 4.9 A (Zn2-Mg3), and 7.1 A (Zn1-Mg3). Despite the close packing of these metal centers, only one bridging ligand, the carboxyl of Asp51, bridges Zn2 and Mg3. A crystal structure at 2.0-A resolution of the noncovalent phosphate complex, E.P, formed with the active center shows that two phosphate oxygens form a phosphate bridge between Zn1 and Zn2, while the two other phosphate oxygens form hydrogen bonds with the guanidium group of Arg166. This places Ser102, the residue known to be phosphorylated during phosphate hydrolysis, in the required apical position to initiate a nucleophilic attack on the phosphorous. Extrapolation of the E.P structure to the enzyme-substrate complex, E.ROPO4(2-), leads to the conclusion that Zn1 must coordinate the ester oxygen, thus activating the leaving group in the phosphorylation of Ser102. Likewise, Zn2 appears to coordinate the ester oxygen of the seryl phosphate and activate the leaving group during the hydrolysis of the phosphoseryl intermediate. Both of these findings suggest that there may be a significant dissociative character to each of the two displacements at phosphorous catalyzed by alkaline phosphatase. A water molecule (or hydroxide) coordinated to Zn1 following formation of the phosphoseryl intermediate appears to be the nucleophile in the second step of the mechanism. Dissociation of the product phosphate from the E.P intermediate is the slowest, 35 s-1, and therefore the rate-limiting, step of the mechanism at alkaline pH. Since the determination of the initial crystal structure of alkaline phosphatase, two other crystal structures of enzymes involved in phosphate ester hydrolysis have been completed that show a triad of closely spaced zinc ions present at their active centers. These enzymes are phospholipase C from Bacillus cereus (structure at 1.5-A resolution) (43) and P1 nuclease from Penicillium citrinum (structure at 2.8-A resolution) (74). Both enzymes hydrolyze phosphodiesters. Substrates for phospholipase C are phosphatidylinositol and phosphatidylcholine, while P1 nuclease is an endonuclease hydrolyzing single stranded ribo- and deoxyribonucleotides. P1 nuclease also has activity as a phosphomonoesterase against 3'-terminal phosphates of nucleotides. The Zn ions in both enzymes form almost identical trinuclear sites.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Structure and mechanism of alkaline phosphatase. 152 73

A paralyzed Rhodobacter sphaeroides mutant strain (PARA1) was isolated by a motility screening procedure following mutagenesis of wild-type R. sphaeroides WS8-N with the transposable element TnphoA (Tn5 IS50L::phoA). PARA1 synthesized a wild-type level of flagellin, as detected by Western immunoblotting with antiflagellar antiserum. Flagellar staining showed that flagellin was assembled into apparently normal external flagellar filaments. Electron micrographs of basal body structures from PARA1 showed that some ring structures that were present were similar to those in wild-type R. sphaeroides WS8-N. PARA1 cells were nonmotile under all growth conditions. No pseudorevertants to motility were seen when PARA1 was grown in the presence of kanamycin to select for the presence of the transposon. The presence of the single copy of TnphoA in the PARA1 chromosome was demonstrated by Southern blotting. Western blotting of cytoplasmic, periplasmic, and membrane fractions of PARA1 with anti-alkaline phosphatase antiserum showed that the transposon had been inserted in-frame into a gene encoding a membrane protein. A SalI restriction endonuclease fragment was cloned from the chromosome of PARA1; this fragment contained a portion of the transposon and R. sphaeroides DNA sequence 5' of the site of insertion. This flanking R. sphaeroides DNA sequence was used to probe an R. sphaeroides WS8 cosmid library. A cosmid designated c19 hybridized to the probe, and a SalI restriction endonuclease fragment derived from this cosmid restored wild-type motility to PARA1 when introduced into this mutant strain by conjugation. The significance of this finding in a bacterium with unidirectionally rotating flagella is discussed.
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PMID:Isolation, characterization, and complementation of a paralyzed flagellar mutant of Rhodobacter sphaeroides WS8. 185 Apr 1

A restriction enzyme-nick translation procedure has been developed for localizing sites of restriction endonuclease action on chromosomes. This method involves digestion of fixed chromosome preparations with a restriction enzyme, nick translation with DNA polymerase I in the presence of biotinylated-dUTP, detection of the incorporated biotin label with streptavidinalkaline phosphatase, and finally staining for alkaline phosphatase. Results obtained obtained on human chromosomes using a wide variety of restriction enzymes are described, and compared with results of Giemsa and Feulgen staining after restriction enzyme digestion. Results of nick translation are not in general the opposite of those obtained with Giemsa staining, as might have been expected. Although the nick translation procedure is believed to give a more accurate picture of the distribution of restriction enzyme recognition sites on chromosomes than Giemsa staining, it is clear that the results of the nick translation experiments are affected by accessibility to the enzymes of the chromosomal DNA, as well as by the extractability of the DNA.
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PMID:Patterns of digestion of human chromosomes by restriction endonucleases demonstrated by in situ nick translation. 196 55


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