Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.22.1 (DNase II)
 429 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An acid DNase (DNase II) from porcine spleen was purified by sequential chromatography over carboxymethyl-cellulose, blue dextran-Sepharose, hydroxylapatite, and sulfoxyethyl-cellulose. The purified enzyme shows two polypeptide bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at Mr 35,000 (alpha chain) and 10,000 (beta chain). The sum of the two molecular weights is that of the native enzyme (45,000). Thus, the DNase II molecule is an alpha,beta dimer. The two polypeptides are not joined by disulfide bonds, but can be cross-linked chemically with dimethyl suberimidate. They are dissociable in 8 M urea, after which they can be isolated by gel filtration on Sephadex G-100, eluting with 1 M acetic acid. Once dissociated, the two polypeptides cannot be reassociated to regenerate DNase II activity. The sum of the amino acid compositions of the two polypeptides is that of the native enzyme, and both contain carbohydrate. The beta chain is devoid of histidine, half-cystine, valine, and methionine. The NH2-terminal amino acid of the alpha chain is leucine, while that of the beta chain cannot be identified by either dansylation or Edman degradation. Alkylation of an essential histidine residue of DNase II occurs on incubation of the enzyme with [2-14C] ICH2COOH (Oshima, R. G., and Price, P. A. (1973) J. Biol. Chem. 248, 7522-7526). Radioactivity is found only in the alpha chain. After hydrolysis of the alpha chain with trypsin, chymotrypsin, and thermolysin, radioactive peptides were isolated by gel filtration on Sephadex G-25 and reversed-phase high performance liquid chromatography. Sequence analyses of the radioactive peptides show alkylation of 1 of 9 histidines in the entire amino acid sequence of DNase II. The sequence around this histidine, determined by manual microsequencing and by the release of amino acids with carboxypeptidases A and B, is Ala-Thr-Glu-Asp-His-Ser-Lys-Trp.
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PMID:The subunit structure and active site sequence of porcine spleen deoxyribonuclease. 403 Jul 66

DNase II is a well-known deoxyribonuclease (DNase) that catalyzes the hydrolysis of DNA into oligonucleotides under acidic conditions. We have identified a novel DNase that shows homology to DNase II, named DLAD, from a search of an expressed sequence tag data base. The full-length cDNA for rat DLAD cloned by polymerase chain reaction encodes a 356-amino acid polypeptide containing a putative N-terminal signal peptide and 5 potential N-glycosylation sites; there is a predicted catalytic domain resemblance to rat DNase II. The predicted DLAD translation product shares 32.9% identity with DNase II. Interestingly, expression of the DRAD mRNA is highly restricted to the liver. A Myc-His tagged recombinant DLAD recovered mainly from the cytoplasm of transfected HeLa S3 cells has a divalent cation-independent DNase activity. The DLAD activity prefers acidic conditions to neutral. The recombinant protein expressed in HeLa S3 cells inhibits the expression of GFP- and lac Z-expression vectors, suggesting that DLAD may play a role in elimination of exogenous DNA. Identification of the full-length cDNA for DLAD would lead to an understanding of the physiology of this DNase II-like molecule.
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PMID:Cloning of a cDNA encoding a rat DNase II-like acid DNase. 1055 78

The 134 amino acid DNase domain of colicin E9 contains a zinc-finger-like HNH motif that binds divalent transition metal ions. We have used 1D 1H and 2D 1H-15N NMR methods to characterise the binding of Co2+, Ni2+ and Zn2+ to this protein. Data for the Co2+-substituted and Ni2+-substituted proteins show that the metal ion is coordinated by three histidine residues; and the NMR characteristics of the Ni2+-substituted protein show that two of the histidines are coordinated through their N(epsilon2) atoms and one via its N(delta1). Furthermore, the NMR spectrum of the Ni2+-substituted protein is perturbed by the presence of phosphate, consistent with an X-ray structure showing that phosphate is coordinated to bound Ni2+, and by a change in pH, consistent with an ionisable group at the metal centre with a pKa of 7.9. Binding of an inhibitor protein to the DNase does not perturb the resonances of the metal site, suggesting there is no substantial conformation change of the DNase HNH motif on inhibitor binding. 1H-15N NMR data for the Zn2+-substituted DNase show that this protein, like the metal-free DNase, exists as two conformers with different 1H-15N correlation NMR spectra, and that the binding of Zn2+ does not significantly perturb the spectra, and hence structures, of these conformers beyond the HNH motif region.
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PMID:NMR studies of metal ion binding to the Zn-finger-like HNH motif of colicin E9. 1083 Aug 90

DNase II alpha (EC 3.1.22.1) is an endonuclease, which is active at low pH, that cleaves double-stranded DNA to short 3'-phosphoryl oligonucleotides. Although its biochemistry is well understood, its structure-activity relationship has been largely unexamined. Recently, we demonstrated that active DNase II alpha consists of one contiguous polypeptide, heavily glycosylated, and containing at least one intrachain disulphide linkage [MacLea, Krieser and Eastman (2002) Biochem. Biophys. Res. Commun. 292, 415-421]. The present paper describes further work to examine the elements of DNase II alpha protein required for activity. Truncated forms and site-specific mutants were expressed in DNase II alpha-null mouse cells. Results indicate that the signal-peptide leader sequence is required for correct glycosylation and that N-glycosylation is important for formation of the active enzyme. Despite this, enzymic deglycosylation of wild-type protein with peptide N-glycosidase F reveals that glycosylation is not intrinsically required for DNase activity. DNase II alpha contains six evolutionarily conserved cysteine residues, and mutations in any one of these cysteines completely ablated enzymic activity, consistent with the importance of disulphide bridging in maintaining correct protein structure. We also demonstrate that a mutant form of DNase II alpha that lacks the purported active-site His(295) can still bind DNA, indicating that this histidine residue is not simply involved in DNA binding, but may have a direct role in catalysis. These results provide a more complete model of the DNase II alpha protein structure, which is important for three-dimensional structural analysis and for production of DNase II alpha as a potential protein therapeutic for cystic fibrosis or other disorders.
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PMID:Structural requirements of human DNase II alpha for formation of the active enzyme: the role of the signal peptide, N-glycosylation, and disulphide bridging. 1255 98

Deoxyribonuclease IIalpha (DNase IIalpha) is an acidic endonuclease found in lysosomes and nuclei, and it is also secreted. Though its Caenorhabditis elegans homolog, NUC-1, is required for digesting DNA of apoptotic cell corpses and dietary DNA, it is not required for viability. However, DNase IIalpha is required in mice for correct development and viability, because undigested cell corpses lead to lesions throughout the body. Recently, we showed that, in contrast to previous reports, active DNase IIalpha consists of one contiguous polypeptide. To better analyze DNase II protein structure and determine residues important for activity, extensive database searches were conducted to find distantly related family members. We report 29 new partial or complete homologs from 21 species. Four homologs with differences at the purported active site histidine residue were detected in the parasitic nematodes Trichinella spiralis and Trichinella pseudospiralis. When these mutations were reconstructed in human DNase IIalpha, the expressed proteins were inactive. DNase II homologs were also identified in non-metazoan species. In particular, the slime-mold Dictyostelium, the protozoan Trichomonas vaginalis, and the bacterium Burkholderia pseudomallei all contain sequences with significant similarity and identity to previously cloned DNase II family members. We report an analysis of their sequences and implications for DNase II protein structure and evolution.
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PMID:A family history of deoxyribonuclease II: surprises from Trichinella spiralis and Burkholderia pseudomallei. 1259 37

DNase II is an acid endonuclease that is involved in the degradation of exogenous DNA and is important for DNA fragmentation and degradation during cell death. In an effort to understand its catalytic mechanism, we constructed plasmids encoding nine different histidine (H)-to-leucine (L) mutants for porcine DNase II and examined the enzyme properties of the expressed mutant proteins. Of the mutants, all but H132L were secreted into the medium of expressing cells. Six of the mutated DNase II proteins (H41L, H109L, H206L, H207L, H274L and H322L) showed enzyme activity, whereas the H115L, H132L and H297L mutants exhibited very little activity. The H115L and H297L mutants were found to undergo correct protein folding, but were inactive. To further examine these mutants, we expressed H115A and H297A DNase II mutants; these mutants were inactive, but their DNase activities could be rescued with imidazole, indicating that His115 and His297 are likely to function as a general acid and a general base respectively in the catalytic centre of the enzyme. In contrast with the secreted mutants, the H132L mutant protein was found in cell lysates within 16 h after transfection. This protein was inactive, improperly folded and was drastically degraded via the proteosomal pathway after 24 h. The polypeptide of another substitution for His132 with lysine resulted in the misfolded form being retained in endoplasmic reticulum.
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PMID:Identification of three crucial histidine residues (His115, His132 and His297) in porcine deoxyribonuclease II. 1673 90