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)

Endogenous protein kinase activity was detected in the outer plasma membrane of 373 and SV40 transformed 3T3 cells. When intact cells were incubated with [gamma-32P]ATP, there was a transfer of [32P]phosphate into an acid-insoluble product. The reaction was: (a) linear as a function of time (up to 30 min), (b) proportional to the number of cells present and (c) dependent on temperature and Mg2+ concentration. The acid-insoluble product was susceptible to pronase but not RNase or DNase. More specifically, phosphomonoester bonds to serine and threonine were identified. There was less than 3% hydrolysis of the [gamma-32P]ATP during the reaction; moreover, free [32P]phosphate failed to substitute for the ATP. The reaction product was located on the cell surface, as evidenced by the fact that it could be removed by mild trypsin treatment of intact 3T3 cells. Further evidence for the surface location of the kinase was shown by its activity in phosphorlating exogenous substrate, histone, and phosvitin. The level of phosphorylation increased by 2- to 4-fold prior to the start of S phase when quiescent 3T3 cells were stimulated to reinitiate growth by the addition of serum. The SV40 3T3 cells had from 5- to 10-fold more activity per cell than the quiescent 3T3 cells. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and radioautography show at least 25 phosphorylated proteins; the surface label pattern of 3T3 cells differs from that of SV40-transformed 3T3 cells.
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PMID:Endgoenous protein kinase in outer plasma membrane of cultured 3T3 cells. Nature of the membrane-bound substrate and effect of cell density, serum addition, and oncogenic transformation. 18 98

The amino acid compositions of various fragments isolated from DNase treated with 2-nitro-5-thiocyanobenzoic acid (NTCB) show peptide bond cleavages to be at Thr14, Ser40, and Ser135. Isolation and characterization of radioactive tryptic and chymotryptic peptides of [14C]cyano-DNase reveal four points of peptide bond cleavage; in addition to Thr14, Ser40, and Ser135, cleavage occurs at the amino end of Ser72. Approximately 2.8 mol of [14C]cyano group are incorporated in the completely inactivated enzyme, in which 0.6 residue of Thr14, 0.8 of Ser40, and approximately 0.3 each of Ser72 and Ser135 are modified. The inactivation by NTCB can also be obtained by reacting the enzyme with a mixture of 5,5'-dithiobis(2-nitrobenzoic acid), KCN, and iodoacetate which generates NTCB. The mixture facilitates the uses of K[14C]N, which is readily incorporated into the enzyme as the [14C]cyano derivative. The reaction of NTCB with serine or threonine resembles that with cysteine.
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PMID:Inactivation of DNase by 2-nitro-5-thiocyanobenzoic acid. II. Serine and threonine are the sites of reaction on the DNase molecule. 48 55

An ovarian cystadenocarcinoma-associated antigen (OCAA) was found to be common to all serous and mucinous cystadenocarcinomas of the ovary. It was apparently absent in tissues of normal reproductive organs. Furthermore, OCAA was not detected in benign ovarian serous and mucinous cyst-adenomas or in any other gynecologic or nongynecologic cancers thus far tested. The antigenic determinant of OCAA was immunologically unrelated to the carcinoembryonic antigen, other known tumor antigens, or the histocompatibility antigens. We purified and partially characterized OCAA. The antigen was a high-molecular-weight glycoprotein soluble in 0.6 M perchloric acid. It consisted of about 50-60% protein (based on dry wt). Amino acid composition in OCAA was characterized by a high percentage of threonine, serine, proline, and valine. Galactose and N-acetylglucosamine were the principal carbohydrate constituents. The antigenic activity was resistant to treatment with trypsin and protease and also to treatment with DNase, RNase, and N-acetylneuraminidase. The antigenicity was considerably reduced by mild periodate oxidation.
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PMID:Tumor-associated antigen for cystadenocarcinomas of the ovary. 82 81

The proteins of the secretory granules of the rat parotid gland were characterized by sodium dodecylsulfate gel electrophoresis, by chromatography of [3-H]proline-labeled proteins on DEAE-cellulose and by amino acid analysis. Sodium dodecylsulfate gel electrophoresis of the secretory granule content showed five principal proteins and a limited number of minor components. Only two of the principal bands could be identified as known secretory enzymes of the parotid gland. One was identified as the alpha-amylase and one as deoxyribonuclease. Peroxidase and ribonuclease form minor portions of the secretory proteins. The other three major proteins constitute, together, about 60% by weight, of the secretory granule content proteins. Of these, one which represents more than 30% of the total granule protein was found to contain uniquely high amounts of leucine residues (21 mole%). Another one of these principal proteins was relatively rich in cysteine residues (7 mole%). The fifth principal protein was found to contain high amounts of proline (28 mole%) glutamic acid (17 mole%) and glycine (18 mole%) residues. Its amino acid composition was very similar to that of the proline-se granules. This protein, however, differed from the "membranous" proline-rich proteins by several criteria. Two minor glycoproteins of the secretory granule content were also found to be rich in proline residues (37 mole%). As with the other proline-rich proteins of the granule, they contained no sulphur-containing amino acids, stained faintly pink with Coomassie Blue and were underestimated by the Lowry method. They differ however, from all the other proline-rich proteins of the granule by having a significantly higher content of threonine, less glycine (9 mole%) and much less glutamic acid (3 mole%). Of the principal proteins, only the deoxyribonuclease and the half-cystine-rich proteins were positively stained by periodic acid Schiff staining. The possible functions of the leucine-rich, the half cystine-rich and the various proline-rich proteins are discussed.
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PMID:The proteins of the content of the secretory granules of the rat parotid gland. 112 45

In the course of experiments on the role of the COOH-terminal residues in pancreatic deoxyribonuclease, we undertook to ascertain whether the presence of sodium dodecyl sulfate would render the normally unavailable terminus susceptible to hydrolysis by carboxypeptidase A. When DNase A is dissolved in 0.005% sodium dodecyl sulfate the protein becomes enzymically inactive when assayed against DNA in the same sodium dodecyl sulfate concentration. The loss of activity caused by treatment with sodium dodecyl sulfate for 1 hour at 45 degrees can be fully restored if the detergent-containing solution is diluted 10-fold into 6 M guanidinium chloride and then 10-fold into a pH 7.0 buffer, 10 mM in CaCl2, prior to a 100-fold dilution for assay. The presence of Ca2+ is essential for the refolding process. If the same degree of dilution is made into sodium dodecyl sulfate-free buffer without the guanidinium chloride step, there is very little reversal of the inactivation. An almost complete loss of regenerable activity is caused by 1 hour of digestion by carboxypeptidase at 45 degrees in the presence of 0.03% sodium dodecyl sulfate. Although up to 6 amino acid residues can be removed from the COOH terminus, the loss of activity can be correlated with the removal of either 1 or 2 amino acid residues (-Leu-Thr) from the COOH-terminal sequence. Thus, DNase A is one of the several enzymes in which residues at the COOH terminus are essential to the active conformation. If the enzyme minus 2 to 6 terminal residues was mixed with a 15-residue COOH-terminal peptide (obtained by cyanogen bromide cleavage), only about 2% activity could be regenerated.
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PMID:Reversible inactivation of pancreatic deoxyribonuclease A by sodium dodecyl sulfate. Removal of COOH-terminal residues from the denatured protein by carboxypeptidase A. 116 40

Design, synthesis and DNA binding activities of two peptides containing 32 and 102 residues are reported. A nonlinear 102-residue peptide contains four modified alpha helix-turn-alpha helix motifs of 434 cro protein. These four units are linked covalently to a carboxyterminal crosslinker containing four arms each ending with an aliphatic amino group. From CD studies we have found that in aqueous buffer in the presence of 20% trifluoroethanol the peptide residues assume alpha-helical, beta-sheet and random-coiled conformations with the alpha-helical content of about 16% at room temperature. Upon complex formation between peptide and DNA, a change in the peptide conformation takes place which is consistent with an alpha - beta transition in the DNA binding alpha helix-turn-alpha helix units of the peptide. Similar conformation changes are observed upon complex formation with the synthetic operator of a linear peptide containing residues 7-37 of 434 cro repressor. Evidently, in the complex, residues present in helices alpha 2 and alpha 3 of the two helix motif form a beta-hairpin which is inserted in the minor DNA groove. The last inference is supported by our observations that the two peptides can displace the minor groove-binding antibiotic distamycin A from poly(dA).poly(dT) and synthetic operator DNA. As revealed from DNase digestion studies, the nonlinear peptide binds more strongly to a pseudooperator Op1, located in the cro gene, than to the operator OR3. A difference in the specificity shown by the non-linear peptide and wild-type cro could be attributed to a flexibility of the linker chains between the DNA-binding domains in the peptide molecule as well as to a replacement of Thr-Ala in the peptide alpha 2-helices. Removal of two residues from the N-terminus of helix alpha 2 in each of the four DNA-binding domains of the peptide leads to a loss of binding specificity.
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PMID:Design and synthesis of sequence-specific DNA-binding peptides. 187 71

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

Design, synthesis and DNA binding activity of a nonlinear 102 residue peptide are reported. The peptide contains four sequence-specific DNA binding domains of 434 Cro protein. These four domains were linked covalently to a symmetrical carboxyterminal crosslinker that contains four arms each ending with an aliphatic aminogroup. From CD studies we have found that in aqueous buffer in the presence of 20% trifluoroethanol the peptide residues assume alpha helical, beta-sheet and random coiled conformations with an alpha helical content of about 16% at room temperature. The alpha helicity is increased up to 40% in the presence of 40% trifluoroethanol. Upon complex formation between the peptide and DNA a change in the peptide conformation takes place which is consistent with an alpha-beta transition in the DNA binding, helix-turn-helix motif of 434 Cro repressor. Evidently residues present in helices alpha(2) and alpha(3) form a beta hairpin which is inserted in the minor DNA groove. The latter inference is supported by our observations that the peptide can displace minor groove binding antibiotic distamycin A from a complex with poly(dA).poly(dT). As revealed from DNase protection studies the peptide exhibits preferences for binding to operator and pseudooperator sites recognized by 434 Cro repressor. It binds strongly to operator sites OR1, OR2 and OR3 and exhibits a greater affinity for pseudooperator site Op1. From analysis of nucleotide sequences in the strong affinity binding sites for the peptide on DNA a conclusion is drawn that it binds to pseudosymmetrical nucleotide sequences 5'-ACAA(W)nCTGT-3', where W is an arbitrary nucleotide. n is equal to six or seven. In the strongest affinity binding site for the peptide on DNA (Op1) motif 5'-ACAA-3' is replaced by sequence 5'-ACCA-3'. A difference in binding specificity shown by the peptide and 434 Cro protein could be attributed to a flexibility of the connecting chains between DNA-binding domains in the peptide molecule as well as to a replacement of Thr - Ala in the alpha 2 helix. Removal of two residues from the N-terminal end of helix alpha 2 in each of the four DNA binding domains of 434 Cro present in the peptide leads to a loss of binding specificity, although the modified peptide binds to DNA unspecifically.
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PMID:[Synthesis of nonlinear DNA-binding peptide with binding specificity determinants close to those of 434 Cro-repressor]. 263 35

The major nonreduced mucus glycoproteins (mucins) from sputa of cystic fibrosis (CF) and asthmatic patients have been purified to electrophoretic homogeneity and subjected to physical and chemical characterization. The sputum specimens were solubilized in buffer containing 0.22 M KSCN and fractionated on Bio-Gel A-5m, followed by digestion with DNase, rechromatography on the same column, and chromatography on hydroxylapatite. Sodium dodecyl sulfate gel electrophoresis of purified mucins gave a single band. Carbohydrate analyses of the purified mucins showed no significant differences in the sugar components from the two mucins. However, the CF mucin contained substantially higher (11%) sulfate content than that observed for the asthmatic mucin (5.9%). Amino acid analyses indicated that the CF mucin had higher levels of serine plus threonine (35%) as compared to the asthmatic mucin (29%). In contrast, CF mucin contained a lower content of aspartate, glutamate, and glycine than that observed for the asthmatic mucin. Molecular weights of 3.8 X 10(6) and 3.5 X 10(6) were obtained for CF and asthmatic mucins, respectively, from light-scattering studies of mucins in the presence of 6 M guanidine hydrochloride. Reduction of the disulfide bonds of the two mucins did not alter their molecular weights. Liquid chromatographic studies on Sepharose CL2B showed that CF mucin forms aggregates sufficiently large to be excluded from the gel. As compared to the CF mucin, the asthmatic mucin formed fewer of these large aggregates under identical experimental conditions. Reduction and alkylation of the mucins resulted in their inability to form aggregates. The higher state of aggregation of CF mucin may influence the viscoelastic properties of the CF lung's mucus secretions.
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PMID:Comparison of physicochemical properties of purified mucus glycoproteins isolated from respiratory secretions of cystic fibrosis and asthmatic patients. 300 52

Based on the published bovine DNase sequence (Liao, T.-H., Salnikow, J., Moore, S., and Stein, W. H. (1973) J. Biol. Chem. 248, 1489-1495), the ovine DNase sequence is derived from the amino acid compositions of isolated short peptides covering all regions of the intact polypeptide. The sequence is substantiated by results of automated Edman degradation of the intact polypeptide and of the two middle CNBr fragments, and by elucidation of the complete sequence of the COOH-terminal CNBr peptide. The 12 changes from bovine to ovine DNase are at residues 22 (Ala to Ser), 29 (Val to Leu), 35 (Val to Ala), 54 (Tyr to Asp), 62 (Thr to Ser), 83 (Leu to Val), 121 (His to Pro), 127 (Glu to Ala), 132 (Ala to Pro), 159 (His to Asp), 163 (Val to Ile), and 231 (Ala to Val). A minor genetic variant form of ovine DNase has Val at residue 163. The data from automated Edman degradation of the largest CNBr peptide of bovine DNase show that the published bovine DNase sequence is in error and that an Ile-Val-Arg tripeptide must be inserted between Arg-27 and Arg-28. The corrected sequence is substantiated by two peptides covering this region each with three amino acids more than the published sequence. Comparison of the bovine, ovine, and porcine DNase sequences reveals the following: with the revised bovine sequence, all three DNase sequences can be aligned without a gap; all three DNases have a carbohydrate side chain at Asn-18, but only porcine DNase has carbohydrate at Asn-106; there are 12 changes between bovine and ovine DNases, 56 between bovine and porcine, and 50 between ovine and porcine; there are six highly variable regions and four invariable ones; bovine and ovine DNases have the same length while porcine DNase is longer by 2 amino acid residues at the COOH terminus; the residues around the nucleotide-binding site, the four pairs of salt bridges, and the essential His-134 groups are not changed.
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PMID:Comparison of the three primary structures of deoxyribonuclease isolated from bovine, ovine, and porcine pancreas. Derivation of the amino acid sequence of ovine DNase and revision of the previously published amino acid sequence of bovine DNase. 378 5


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