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:3.1.21.1 (DNase)
7,655 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have made a computer-assisted search for homology among polymerases or putative polymerases of various viruses and a transposable element, the Drosophila copia-like element 17.6. The search revealed that the putative polymerase (second open reading frame) of the copia-like element 17.6 bears close resemblance in overall structural organization to the pol gene product of Moloney murine leukaemia virus (M-MuLV): they show significant homology to each other at both the N- and C-terminal portions, suggesting that the 17.6 putative polymerase carries two enzymatic activities, related to reverse transcriptase and DNA endonuclease. The putative polymerase of cauliflower mosaic virus (CaMV) shows striking homology with the putative polymerase of 17.6 over almost its entire length, but it lacks the DNA endonuclease-related sequence. Furthermore, it was shown that the N-terminal ends of the M-MuLV pol product and the CaMV and 17.6 putative polymerases exhibit strong sequence homology with the gag-specific protease (p15) of Rous sarcoma virus (RSV) as well as the amino acid sequence predicted from the gag/pol spacer sequence of human adult T-cell leukaemia virus (HTLV). These p15-related sequences contain a highly conserved stretch of amino acids which show a close similarity with sequences around the active site amino acids Asp-Thr-Gly of the acid protease family, suggesting that they have an activity similar to acid protease. On the basis of the alignment of reverse transcriptase-related sequences, a dendrogram representing phylogenetic relationships among all the viruses compared together with 17.6 was constructed and its evolutionary implication is discussed.
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PMID:Close structural resemblance between putative polymerase of a Drosophila transposable genetic element 17.6 and pol gene product of Moloney murine leukaemia virus. 240 86

The protease subtilisin has been reported to cleave skeletal muscle G-actin between Met 47 and Gly 48 generating a core fragment of 33 kDa and a small N-terminal peptide, which remains attached to the core fragment [Schwyter, D. Phillips, M., & Reisler, E. (1989) Biochemistry 28, 5889-5895]. However, amino acid sequencing and mass spectroscopy of subtilisin cleaved-actin revealed two cleavage sites, one between Met 47 and Gly 48 and a second between Gly 42 and Val 43, generating an actin core of 37 kDa and a nicked 4.4 kDa N-terminal peptide. Here we describe a procedure for purifying the actin core fragment and the attached N-terminal peptide from the linking pentapeptide comprising amino acid residues 43-47 under native conditions by anion exchange chromatography. After removal of the pentapeptide, the salt-induced polymerization of actin was abolished. However, the purified fragments could be polymerized by addition of salt plus myosin subfragment 1 or salt plus phalloidin as shown by sedimentation and fluorescence increase using N-(1-pyrenyl)iodoacetamide labeled actin. These results confirm earlier reports proposing that cleavage in the DNase I binding loop is affecting the ion induced polymerization of actin [Higashi-Fujime, S., et al. (1992) J. Biochem. (Tokyo) 112, 568-572; and Khaitlina, S., et al. (1993) Eur. J. Biochem. 218, 911-920]. Monomeric and filamentous subactin exhibited reduced abilities to inhibit deoxyribonuclease I (DNase I) and to stimulate the myosin subfragment 1 ATPase activity. Direct binding of subactin to DNase I was verified by gel filtration and to myosin subfragment 1 by affinity chromatography, chemical cross-linking, and electron microscopy.
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PMID:Purification and characterization of subtilisin cleaved actin lacking the segment of residues 43-47 in the DNase I binding loop. 757 93

Four aspartic acid residues (Asp126, Asp156, Asp233 and Asp295) of Bacillus cereus sphingomyelinase (SMase) in the conservative regions were changed to glycine by in vitro mutagenesis, and the mutant SMases [D126G (Asp126-->Gly etc.), D156G, D233G and D295G] were produced in Bacillus brevis 47, a protein-producing strain. The sphingomyelin (SM)-hydrolysing activity of D295G was completely abolished and those of D126G and D156G were reduced by more than 80%, whereas that of D233G was not so profoundly affected. Two mutant enzymes (D126G and D156G) were purified and characterized further. The hydrolytic activities of D126G and D156G toward four phosphocholine-containing substrates with different hydrophobicities, SM, 2-hexadecanoylamino-4-nitrophenylphosphocholine(HNP), lysophosphatidylcholine (lysoPC) and p-nitro-phenylphosphocholine (p-NPPC), were compared with those of the wild-type. The activity of D126G toward water-soluble p-NPPC was comparable with that of the wild-type. On the other hand, D156G catalysed the hydrolysis of hydrophilic substrates such as HNP and p-NPPC more efficiently (> 4-fold) than the wild-type. These results suggested that Asp126 and Asp156, located in the highly conserved region, may well be involved in a substrate recognition process rather than catalytic action. Haemolytic activities of the mutant enzymes were found to be parallel with their SM-hydrolysing activities. Two regions, including the C-terminal region containing Asp295, were found to show considerable sequence identity with the corresponding regions of bovine pancreatic DNase I. Structural predictions indicated structural similarity between SMase and DNase I. An evolutionary relationship based on the catalytic function was suggested between the structures of these two phosphodiesterases.
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PMID:Mutation in aspartic acid residues modifies catalytic and haemolytic activities of Bacillus cereus sphingomyelinase. 763 90

De novo design and synthesis by a solid phase technique of linear and cyclic 26-residues peptides are reported. The peptides use beta-strand-turn-beta -strand motif for sequence recognition on DNA. Amino acid sequences in the two peptides are identical, but the structure of the cyclic peptide is constrained by S-S bridge between two cysteine residues. A 28-residue peptide containing at the N-terminus a copper-chelating peptide Gly-Gly-His is also synthesized which can be used as a potential DNA-cleaving reagent. Binding of these peptides to various natural and synthetic DNAs and DNA fragment with a known base pair sequence has been studied by CD spectroscopy, fluorescence methods and DNAse I footprinting technique. By means of CD spectroscopy it is shown that 26-residue linear and cyclic peptides are partially in disordered and beta-conformations in aqueous solution in absence and in presence of 20% trifluoroethanol (TFE), but assume partially an alpha-helix conformation in the presence of 50% TFE. It is shown that linear and cyclic peptides bind to DNA. The binding approaches saturation level when one peptide molecule is bound approximately per three or four DNA base pairs. We found that antibiotic distamycin A, binding in the minor DNA groove, competes effectively with the 26-residue linear and cyclic peptides for binding to poly(dA).poly (dT). According to the CD spectroscopy data the linear and cyclic peptides undergo conformation changes upon binding to DNA, whereas the DNA structure is not markedly altered. Difference CD spectra obtained by subtracting the spectrum of the free DNA from the spectrum of the peptide-DNA mixture differ from the spectrum of the free peptide. The shapes of difference CD spectra are consistent with a conformation transition from a disordered conformation into a beta-like conformation upon binding of peptide to DNA. DNAase I footprinting diagrams show that there is a specific protection by linear and cyclic peptides of the nucleotide sequences on two ends of operators OR1, OR2 and OR3 and pseudooperators within the cro gene of 434 phage.
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PMID:[Design of de novo specific DNA-binding peptides, using the motif beta-chain-turn-beta-chain for recognizing a nucleotide sequence in DNA]. 788 38

Cytochalasin E (CE) was used in this study to evaluate relationships between platelet shape change, pseudopod extension, internal transformation, actin filament assembly, fibrinogen receptor expression, aggregation, clot tension and secretion. Various ratios of G- to F-actin, measured by the DNase inhibition assay, were achieved by using several concentrations of CE in stirred and unstirred thrombin-stimulated platelets. An increased rate of actin polymerization was found in "aggregating" conditions as compared to "activating" conditions, the latter achieved by either absence of stirring or stirring in the presence of the tetrapeptide Arg-Gly-Asp-Ser (RGDS). Larger concentrations of CE were required in aggregated than in activated platelets to obtain G-actin levels analogous to those in resting platelets. Concentrations of polymerizing actin above 20% and 55% for activated and aggregated platelets, respectively, trigger platelet shape change and the extension of pseudopods. Prevention of de novo actin polymerization by CE, added either before or after thrombin stimulation, inhibits fibrinogen binding to platelets. This suggests an involvement of the new actin polymers in fibrinogen receptor exposure. As expected, platelet aggregation and clot tension, which are dependent on fibrinogen receptor exposure, were inhibited by CE. Granular secretion was not dependent on polymerizing actin.
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PMID:Role of actin in platelet function. 792 78

Among the isolated fungal species of soil, one filamentous fungus, Syncephalastrum racemosum, produces a relatively large amount of DNase. This enzyme has been purified to apparent homogeneity by column chromatography on DEAE-cellulose, hydroxylapatite, phenyl-Sepharose CL-4B, and Sephadex G-100. The active enzyme requires divalent metal ions and has an optimum pH of 7.0 with Mg2+ and 7.2 with Mn2+. This enzyme is an acidic glycoprotein with a pI 5.0 and is relatively unstable at low concentrations. The M(r) of the enzyme is 56,000 during gel filtration under nondenaturing conditions but is 28,000 during polyacrylamide gel electrophoresis in sodium dodecyl sulfate. These results suggest a structure consisting of two subunits. The subunits of the holoenzyme can be cross-linked with glutaraldehyde. The yield of N-terminal phenylthiohydantoin-alanine from the holoenzyme is 140% and that of one peptide (D-Y-V-S-S-G-Y-D-R), obtained from the tryptic digest is 160%, indicating that the native enzyme is composed of two identical subunits and probably has two active domains. Fungal DNase can be inactivated by Cu(2+)-iodoacetate under conditions that inactivate bovine pancreatic DNase. The specific activity (units/mg of protein) of fungal DNase is 6.5 times that of bovine DNase. The amino acid content of fungal DNase, relative to bovine DNase, is higher in Gly and lower in Ser and Val. The fungal N-terminal 40-residue sequence shows a high degree of homology with a consensus sequence derived from DNase of three mammalian species.
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PMID:Deoxyribonuclease of Syncephalastrum racemosum--enzymatic properties and molecular structure. 848 65

Epstein-Barr virus (EBV) DNase possesses both endonuclease and exonuclease activities and accepts both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) as substrates. To map regions of EBV DNase responsible for nuclease and DNA binding activities, a series of mutant DNase polypeptides was expressed using a bacterial system for the nuclease assay and in an in vitro transcription/translation system to assay binding activity to dsDNA or ssDNA cellulose. The results indicated that the C-terminus of EBV DNase, residues 450-460, is essential for nuclease activity but dispensable for DNA binding. However, deletion of residues 441-470 resulted in the loss of both nuclease and DNA binding activities. Substitution of Phe452 and Val458 led to inactive enzymes. In the N-terminus, deletion of residues 23-28 and residues 7-61 resulted in the loss of nuclease activity but the DNA binding activities of the deleted enzymes were intermediate and low, respectively. Mutation of Leu23 to Gly showed drastically reduced nuclease activity but its DNA binding ability was not affected. Based on the amino acid sequence alignment of various herpesvirus DNases, we chose four highly conserved and two less well conserved regions as controls for mutagenesis studies. These six internal deletion (ID) mutants were prepared using a recombinant PCR method. Each of the polypeptides was expressed in a bacterial system for the nuclease assay and using an in vitro transcription/translation system for the DNA binding assay. DNA binding and nuclease activities of all six internal deletion mutants were abolished, except that mutant ID2, with deletion of residues 138-152, retained an intermediate ability to bind DNA. These data indicate that since mutations at distinct regions within EBV DNase resulted in the loss of nuclease and/or DNA binding activities, it is suggested that these distinct regions are required for maintenance of an intact and highly ordered structure(s) for both activities.
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PMID:Distinct regions of EBV DNase are required for nuclease and DNA binding activities. 950 Oct 34

Subdomain 2 of actin is a dynamic segment of the molecule. The cross-linking of Gln-41 on subdomain 2 to Cys-374 on an adjacent monomer in F-actin inhibits actomyosin motility and force generation (Kim et al., 1998; Biochemistry 37, 17,801-17,809). To shed light on this effect, additional modifications of the Gln-41 site on actin were carried out. Both intact G-actin and G-actin cleaved by subtilisin between Met-47 and Gly-48 in the DNase 1 binding loop of subdomain 2 were treated with bacterial transglutaminase. According to the results of Edman degradation, transglutaminase introduced an intramolecular zero-length cross-linking between Gln-41 and Lys-50 in both intact and subtilisin cleaved actins. This cross-linking perturbs G-actin structure as shown by the inhibition of subtilisin and tryptic cleavage in subdomain 2, an allosteric inhibition of tryptic cleavage at the C-terminus and decrease of modification rate of Cys-374. The cross-linking increases while the subtilisin cleavage dramatically decreases the thermostability of F-actin. The Mg- and S1-induced polymerizations of both intact and subtilisin cleaved actins were only slightly influenced by the cross-linking. The activation of S1 ATPase by actin and the sliding speeds of actin filaments in the in vitro motility assays were essentially unchanged by the cross-linking. Thus, although intramolecular cross-linking between Gln-41 and Lys-50 perturbs the structure of the actin monomer, it has only a small effect on actin polymerization and its interaction with myosin. These results suggest that the new cross-linking does not alter the intermonomer interface in F-actin and that changes in actomyosin motility reported for the Gln-41-Cys-374 intrastrand cross-linked actin are not due to decreased flexibility of loop 38-52 but to constrains introduced into the F-actin structure and/or to perturbations at the actin's C-terminus.
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PMID:Effect of intramolecular cross-linking between glutamine-41 and lysine-50 on actin structure and function. 1112 31

The soybean (Glycine max L. Merr. cv Williams 82) genes VspA and VspB encode vacuolar glycoprotein acid phosphatases that serve as vegetative storage proteins during seed fill and early stages of seedling growth. VspB expression is activated by jasmonates (JAs) and sugars and down-regulated by phosphate and auxin. Previous promoter studies demonstrated that VspB promoter sequences between -585 and -535 mediated responses to JA, and sequences between -535 and -401 mediated responses to sugars, phosphate, and auxin. In this study, the response domains were further delineated using transient expression of VspB promoter-beta-glucuronidase constructs in tobacco protoplasts. Sequences between -536 and -484 were identified as important for phosphate responses, whereas the region from -486 to -427 mediated sugar responses. Gel-shift and deoxyribonuclease-I footprinting assays revealed four DNA-binding sites between -611 and -451 of the soybean VspB promoter: one in the JA response domain, two in the phosphate response domain, and one binding site in the sugar response domain. The sequence CATTAATTAG present in the phosphate response domain binds soybean homeodomain leucine zipper proteins, suggesting a role for these transcription factors in phosphate-modulated gene expression.
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PMID:Homeodomain leucine zipper proteins bind to the phosphate response domain of the soybean VspB tripartite promoter. 1116 Oct 37

Effects of proteolytic modifications of the DNase-I-binding loop (residues 39-51) in subdomain 2 of actin on F-actin dynamics were investigated by measuring the rates of the polymer subunit exchange with the monomer pool at steady state and of ATP hydrolysis associated with it, and by determination of relative rate constants for monomer addition to and dissociation from the polymer ends. Cleavage of actin between Gly-42 and Val-43 by protease ECP32 resulted in enhancement of the turnover rate of polymer subunits by an order of magnitude or more, in contrast to less than a threefold increase produced by subtilisin cleavage between Met-47 and Gly-48. Probing the structure of the modified actins by limited digestion with trypsin revealed a correlation between the increased F-actin dynamics and a change in the conformation of subdomain 2, indicating a more open state of the filament subunits relative to intact F-actin. The cleavage with trypsin and steady-state ATPase were cooperatively inhibited by phalloidin, with half-maximal effects at phalloidin to actin molar ratio of 1:8 and full inhibition at a 1:1 ratio. The results support F-actin models in which only the N-terminal segment of loop 39-51 is involved in monomer-monomer contacts, and suggest a possibility of regulation of actin dynamics in the cell through allosteric effects on this segment of the actin polypeptide chain.
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PMID:Role of the DNase-I-binding loop in dynamic properties of actin filament. 1175 19


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