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:3.1.21.3 (deoxyribonuclease)
1,528 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Whole sheets of plasma membrane, each with their attached flagellum, were purified from Trypanosoma brucei. The method devised for their isolation included a new technique of cell breakage that used a combination of osmotic stress followed by mechanical sheer and avoided the problem of extreme vesiculation as well as the trapping of organelles in cell 'ghosts'. The purified membranes all contained the pellicular microtubular array. The antigenic surface coat was completely released from the plasma membrane during the isolation procedure. The membranes had a very high cholesterol/phospholipid ratio (1.54). A large proportion (42%) of the cellular DNA was recovered in the plasma-membrane fraction unless a step involving deoxyribonuclease treatment, which decreased the DNA content to less than 13%, was included before secrose-density gradient centrifugation. This step also aided the separation of plasma membranes from other cellular components. The ouabain-sensitive Na+ + K+-stimulated adenosine triphosphatase and adenylate cyclase co-purified with the plasma membranes. Although 5'-nucleotidase was thought to be a plasma-membrane component, it was easily detached from the membrane. The purified membranes were essentially free of L-alanine-alpha-oxoglutarate aminotransferase, L-asparte-alpha-oxoglutarate aminotransferase, malate dehydrogenase, oligomycin-sensitive adenosine triphosphatase, glucose 6-phosphatase, Mg2+-stimulated p-nitrophenyl phosphatase and catalase.
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PMID:The isolation and partial characterization of the plasma membrane from Trypanosoma brucei. 48 94

The sensitivity of Streptococcus faecalis (ATTC 8043) to S. zymogenes X-14 bacteriocin depends greatly on its physiological age. Sensitivity decreases from the mid-log phase on and is completely lost in the stationary phase. The sensitivity of erythrocytes to the hemolytic capacity of the bacteriocin showed considerable species variation. The order of increasing sensitivity was goose < sheep < dog < horse < human < rabbit. However, when red cell stromata were used as inhibitors of hemolysis in a standard system employing rabbit erythrocytes the order of increasing effectiveness was sheep < rabbit < human < horse < goose. When rabbit cells were used in varying concentrations with a constant hemolysin concentration, there was a lag of about 30 min, which for a given hemolysin preparation was constant for all red cell concentrations. Furthermore, the rate of hemolysis increased with increasing red cell concentration. If red cells are held constant and lysin varied, the time to reach half-maximal lysis varies directly with lysin but is not strictly proportional. Bacterial membranes were one to three orders of magnitude more effective than red cell stromata as inhibitors. The order of increasing effectiveness seems to be Escherichia coli < Bacillus megaterium < S. faecalis < Micrococcus lysodeikticus. In addition to membranes, a d-alanine containing glycerol teichoic acid, trypsin in high concentration, and deoxyribonuclease also inhibited hemolysis. Ribonuclease, d-alanine, l-alanine, dl-alanyl-dl-alanine, N-acetyl-d-alanine, N-acetyl-l-alanine did not inhibit hemolysis.
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PMID:Bacteriocin (hemolysin) of Streptococcus zymogenes. 497 10

Cell walls were isolated by mechanical disruption of mid-log phase cells of Bacillus stearothermophilus NCA 1503-4R grown in Trypticase-yeast extract-fructose medium at 55 C. The cell walls were purified by treatment with sodium dodecyl sulfate (SDS) and incubation with deoxyribonuclease and trypsin. The cell wall peptidoglycan contained glucosamine, muramic acid, alpha, epsilon-diaminopimelic acid, and glutamic acid. Low amounts of glycine, galactosamine, serine, aspartic acid, lysine, and valine were also present. The relative mole ratios of glutamic acid-alpha, epsilon-diaminopimelic acid-glycine-alanine were 1.00:1.26:0.08:1.55. The cell walls were free from ribonucleic acid and deoxyribonucleic acid and contained less than 0.2% chloroform-methanol extractable lipid and 0.09 mumole of phosphorus per mg of cell wall. Teichoic acid was not detected in the cell walls of this organism. Cell walls isolated without treatment with SDS contained 7.5% chloroform-methanol extractable lipid, 0.24 mumole of phosphorus per mg of cell wall, and relatively high concentrations of all amino acids. These results suggest that the extracted lipid is not a cell wall component per se, but a contaminant from the lipoprotein cell membrane.
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PMID:Chemical composition of the cell walls of Bacillus stearothermophilus. 603 16

Isolated nuclei incubated with [14C]protein hydrolysate are shown to incorporate labelled amino acids into the acid-insoluble fraction. Purified chromatin and the complex of DNA with firmly bound proteins possess similar ability. The optimum pH of the reaction is 6.5-7.0, 2 mM MgCl2 stimulates incorporation, the temperature optimum is 37-40 degrees C. Chloramphenicol depresses incorporation by 70%, puromycin by 40%, cycloheximide does not affect the chromatin activity. Incorporation does not depend on the presence of ATP or GTP, and is substantially inhibited by deoxyribonuclease but not by ribonuclease treatment of chromatin or of the nuclei. Specific activity of firmly bound chromatin non-histone proteins is higher than that of labile bound ones; histones are not labelled. After pronase treatment of proteins radioactivity changes to an acid-soluble state. The molecular weight of isolated labelled polypeptides is about 6000 as shown by gel filtration and the analysis of NH2-terminal amino acids. Labelled polypeptides firmly bound to DNA consist of 7-10 amino acids. Specific activity of proteins firmly bound to DNA increases linearly with the time of incubation of chromatin with [14C]protein hydrolysate, the activity curve of labile bound non-histone proteins has a distinct sygmoid character. The polypeptide-synthesizing activity of rat liver chromatin increases between 9 h and 21 h after partial hepatectomy. Irradiation with 800 rads 30 min before the operation prevents activation of amino acid incorporation. From nine amino acids studied alanine, methionine, lysine, tyrosine and arginine are not incorporated in the system described. Glutamic acid is polymerized most effectively. Glutamine, asparagine and glycine are incorporated 7-8 times less. The data are given indicating that the incorporation is not random when an amino acid mixture is present. Preincubation of chromatin with NAD+ but not with its analogues increases the polypeptide-synthesizing activity of chromatin. The activation is prevented by thymidine and nicotinamide. Storage (18 h at 2-4 degrees C) brings about a complete loss of the polypeptide-synthesizing activity of chromatin. The ability of 'old' chromatin to incorporate amino acids can be restored by preincubating it with NAD+. Storage of chromatin in the presence of 5 mM adenosine 3',5'-monophosphate (cAMP) does not result in decrease of the polypeptide-synthesizing activity. It is assumed that poly-(ADP-ribose) is the energy source for amino acid activation in the system described.
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PMID:Polypeptide-synthesizing activity of eukaryotic chromatin. Properties, dependence on poly(ADP-ribose) and connection with the cell cycle. 737 37

A RecA/Rad51 homologue from Pyrococcus kodakaraensis KOD1 (Pk-REC) is the smallest protein among various RecA/Rad51 homologues. Nevertheless, Pk-Rec is a super multifunctional protein and shows a deoxyribonuclease activity. This deoxyribonuclease activity was inhibited by 3 mM or more ATP, suggesting that the catalytic centers of the ATPase and deoxyribonuclease activities are overlapped. To examine whether these two enzymatic activities share the same active site, a number of site-directed mutations were introduced into Pk-REC and the ATPase and deoxyribonuclease activities of the mutant proteins were determined. The mutant enzyme in which double mutations Lys-33 to Ala and Thr-34 to Ala were introduced, fully lost both of these activities, indicating that Lys-33 and/or Thr-34 are important for both ATPase and deoxyribonuclease activities. The mutation of Asp-112 to Ala slightly and almost equally reduced both ATPase and deoxyribonuclease activities. In addition, the mutation of Glu-54 to Gln did not seriously affect the ATPase, deoxyribonuclease, and UV tolerant activities. These results strongly suggest that the active sites of the ATPase and deoxyribonuclease activities of Pk-REC are common. It is noted that unlike Glu-96 in Escherichia coli RecA, which has been proposed to be a catalytic residue for the ATPase activity, the corresponding residual Glu-54 in Pk-REC is not involved in the catalytic function of the protein.
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PMID:A unique DNase activity shares the active site with ATPase activity of the RecA/Rad51 homologue (Pk-REC) from a hyperthermophilic archaeon. 1006 83

In this study, we have mapped and characterized a B cell epitope of sulfated glycoprotein ZP2 (ZP2) as a step toward the development of a multi-epitope zona pellucida (ZP) vaccine. Recombinant polypeptides expressed by random deoxyribonuclease-digested fragments of ZP2 cDNA were screened for binding to IE-3, a monoclonal antibody to murine ZP2. Positive clones contained cDNA inserts encoding polypeptide corresponding to ZP2(103-134). When normal or ovariectomized female mice were immunized with three overlapping peptides that span this region of ZP2 (101-120, 111-130, 121-140), only ZP2(121-140) elicited IgG antibodies that reacted with mouse ovarian ZP, indicative of the presence of native B epitope and helper T cell epitope in ZP2(121-140). To more finely map the ZP2 B cell epitope, a random peptide display library was screened with the IE-3 antibody, and a consensus tetramer sequence VxYK that matched the ZP2(123-126) sequence VRYK was located. Competitive immunofluorescence analysis with single alanine-substituted VxYK peptides ranked the relative contribution of the three critical B cell epitope residues as Y > V > K. A chimeric peptide was constructed that contained the YRYK motif of ZP2 and a bovine RNase T cell epitope. Although (C57BL/6xA/J) F1 (B6AF1) female mice immunized with the chimeric peptide developed ZP antibody response, this peptide elicited antibody only in mice of the histocompatibility complex (MHC) H-2(k or b) haplotype. In contrast, ZP2(121-140) peptide elicited antibody in inbred mice with three additional mouse MHC haplotypes. Moreover, although ZP2(121-140) contained a T cell epitope, no oophoritis was observed after immunization of B6AF1 mice with ZP2(121-140) in complete Freund's adjuvant (CFA). In a preliminary trial, female B6AF1 mice immunized with ZP2(121-140) in CFA had reduced litter sizes as compared with mice injected with CFA alone.
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PMID:A contraceptive peptide vaccine targeting sulfated glycoprotein ZP2 of the mouse zona pellucida. 1008 64

Two temperature-sensitive mutations in the hsdS gene, which encodes the DNA specificity subunit of the type IA restriction-modification system EcoKI, designated Sts1 (Ser(340)Phe) and Sts2 (Ala(204)Thr) had a different impact on restriction-modification functions in vitro and in vivo. The enzyme activities of the Sts1 mutant were temperature-sensitive in vitro and were reduced even at 30 degrees C (permissive temperature). Gel retardation assays revealed that the Sts1 mutant had significantly decreased DNA binding, which was temperature-sensitive. In contrast the Sts2 mutant did not show differences from the wild-type enzyme even at 42 degrees C. Unlike the HsdSts1 subunit, the HsdSts2 subunit was not able to compete with the wild-type subunit in assembly of the restriction enzyme in vivo, suggesting that the Sts2 mutation affects subunit assembly. Thus, it appears that these two mutations map two important regions in HsdS subunit responsible for DNA-protein and protein-protein interactions, respectively.
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PMID:Two temperature-sensitive mutations in the DNA binding subunit of EcoKI with differing properties. 1061 39

The Type IIS restriction endonuclease MnlI recognizes the non-palindromic nucleotide sequence 5'-CCTC(N)7/6 downward arrow and cleaves DNA strands as indicated by the arrow. The genes encoding MnlI restriction-modification system were cloned and sequenced. It comprises N6-methyladenine and C5-methylcytosine methyltransferases and the restriction endonuclease. Biochemical studies revealed that MnlI restriction endonuclease cleaves double- and single-stranded DNA, and that it prefers different metal ions for hydrolysis of these substrates. Mg2+ ions were shown to be required for the specific cleavage of double-stranded DNA, whereas Ni2+ and some other transition metal ions were preferred for nonspecific cleavage of single-stranded DNA. The C-terminal part of MnlI restriction endonuclease revealed an intriguing similarity with the H-N-H type nucleolytic domain of bacterial toxins, Colicin E7 and Colicin E9. Alanine replacements in the conserved sequence motif 306Rx3ExHHx14Nx8H greatly reduced specific activity of MnlI, and some mutations even completely inactivated the enzyme. However, none of these mutations had effect on MnlI binding to the specific DNA, and on its oligomerisation state as well. We interpret the presented experimental evidence as a suggestion that the motif 306Rx3ExHHx14Nx8H represents the active site of MnlI. Consequentially, MnlI seems to be the member of Type IIS with the active site of the H-N-H type.
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PMID:MnlI--The member of H-N-H subtype of Type IIS restriction endonucleases. 1602 1

The activity of human TREX2-catalyzed 3' --> 5'-deoxyribonuclease has been analyzed in steady-state and single turnover kinetic assays and in equilibrium DNA binding studies. These kinetic data provide evidence for cooperative DNA binding within TREX2 and for coordinated catalysis between the TREX2 active sites supporting a model for communication between the protomers of a TREX2 dimer. Mobile loops positioned adjacent to the active sites provide the major DNA binding contribution and facilitate subsequent binding into the active sites. Mutations of three arginine residues on these loops cause decreased TREX2 activities by up to 60-fold. Steady-state kinetic assays of these arginine to alanine TREX2 variants result in increased K(m) values for DNA substrate with no effect on k(cat) values indicating contributions exclusively to DNA binding by all three of the loop arginines. TREX2 heterodimers were prepared to determine whether exonuclease activity in one protomer is communicated to the opposing protomer. Evidence for communication across the dimer interface is provided by the 7-fold lower catalytic activity measured in the TREX2(WT/H188A) heterodimer compared with the TREX2(WT) homodimer, contrasting the 2-fold lower activity measured in the TREX2(WT/R163A,R165A,R167A) heterodimer. The measured activity in TREX2(WT/H188A) heterodimer indicates that defective catalysis in one protomer reduces activity in the opposing protomer. A DNA binding analysis of TREX2 and the heterodimers indicates a cooperative binding effect within the TREX2 protomer. Finally, single turnover kinetic assays identify DNA binding as the rate-limiting step in TREX2 catalysis.
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PMID:Cooperative DNA binding and communication across the dimer interface in the TREX2 3' --> 5'-exonuclease. 1853 78

Nuclear targeting of bacterial proteins is an emerging pathogenic mechanism whereby bacterial proteins can interact with nuclear molecules and alter the physiology of host cells. The fully sequenced bacterial genome can predict proteins that target the nuclei of host cells based on the presence of nuclear localization signal (NLS). In the present study, we predicted bacterial proteins with the NLS sequences from Klebsiella pneumoniae by bioinformatic analysis, and 13 proteins were identified as carrying putative NLS sequences. Among them, HsdM, a subunit of KpnAl that is a type I restriction-modification system found in K. pneumoniae, was selected for the experimental proof of nuclear targeting in host cells. HsdM carried the NLS sequences, (7)KKAKAKK(13), in the N-terminus. A transient expression of HsdM-EGFP in COS-1 cells exhibited exclusively a nuclear localization of the fusion proteins, whereas the fusion proteins of HsdM with substitutions in residues lysine to alanine in the NLS sequences, (7)AAAKAAA(13), were localized in the cytoplasm. HsdM was co-localized with importin o in the nuclei of host cells. Recombinant HsdM alone methylated the eukaryotic DNA in vitro assay. Although HsdM tested in this study has not been considered to be a virulence factor, the prediction of NLS motifs from the full sequenced genome of bacteria extends our knowledge of functional genomics to understand subcellular targeting of bacterial proteins.
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PMID:Prediction of bacterial proteins carrying a nuclear localization signal and nuclear targeting of HsdM from Klebsiella pneumoniae. 1985 38


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