Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1981 Dec 11;9(23):6635–6644. doi: 10.1093/nar/9.23.6635

Purification and properties of a new DNase activity from KB cells.

G D Frenkel, K Randles, N Berns
PMCID: PMC327628  PMID: 7322924

Abstract

A deoxyribonuclease activity with specificity towards single-stranded DNA has been purified approximately four hundred-fold from KB cells, by chromatography on DEAE-cellulose, phosphocellulose and hydroxylapatite. The last step of the purification results in separation of the enzyme from a DNase activity which has been described previously (Wang, E.C., Furth, J.J. and Rose, J.A., (1978) Biochemistry 17: 544-549). The properties of the new DNase activity are significantly different from those of the enzymes which have previously been identified in these cells. The activity sediments at approximately 7.5S in a glycerol gradient. The DNase activity is optimal at pHs between 6.0 and 6.5. It cleaves DNA endonucleolytically and hydrolyzes single-stranded DNA at about 11 times the rate of double-stranded DNA and at twice the rate of Poly (dA). The activity is moderately sensitive to inhibition by N-ethylmaleimide and is inhibited 80% by 50 mM NaCl. It is stimulated twenty-fold by Mn++ at an optimal concentration of approximately 0.7 mM. It is stimulated by a lesser extent by Mg++, but not by Ca++.

Full text

PDF
6635

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Clark A. J. Recombination deficient mutants of E. coli and other bacteria. Annu Rev Genet. 1973;7:67–86. doi: 10.1146/annurev.ge.07.120173.000435. [DOI] [PubMed] [Google Scholar]
  2. EAGLE H. Propagation in a fluid medium of a human epidermoid carcinoma, strain KB. Proc Soc Exp Biol Med. 1955 Jul;89(3):362–364. doi: 10.3181/00379727-89-21811. [DOI] [PubMed] [Google Scholar]
  3. Grossman L., Braun A., Feldberg R., Mahler I. Enzymatic repair of DNA. Annu Rev Biochem. 1975;44:19–43. doi: 10.1146/annurev.bi.44.070175.000315. [DOI] [PubMed] [Google Scholar]
  4. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  5. Naas K., Frenkel G. D. A deoxyribonucleic acid binding protein from KB cells which inhibits deoxyribonuclease activity on single-stranded DNA. J Biol Chem. 1979 May 10;254(9):3407–3410. [PubMed] [Google Scholar]
  6. Nass K., Frenkel G. D. Adenovirus-induced inhibition of cellular DNase. J Virol. 1978 May;26(2):540–543. doi: 10.1128/jvi.26.2.540-543.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Padmanabhan R., Stabel S., Winterhoff U., Doerfler W. Specificity and mode of cleavage of the pH 4.0 endonuclease from adenovirus type 2 - infected KB cells. Nucleic Acids Res. 1979 Jul 11;6(9):3161–3176. doi: 10.1093/nar/6.9.3161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Reif U. M., Winterhoff U., Lundholm U., Philipson L., Doerfler W. Purification of an endonuclease from adenovirus-infected KB cells. Eur J Biochem. 1977 Mar 1;73(2):313–325. doi: 10.1111/j.1432-1033.1977.tb11321.x. [DOI] [PubMed] [Google Scholar]
  9. Reif U., Winterhoff U., Doerfler W. Characterization of the pH 4.0 endonuclease from adenovirus-type-2-infected KB cells. Eur J Biochem. 1977 Mar 1;73(2):327–333. doi: 10.1111/j.1432-1033.1977.tb11322.x. [DOI] [PubMed] [Google Scholar]
  10. Roberts R. J. Restriction endonucleases. CRC Crit Rev Biochem. 1976 Nov;4(2):123–164. doi: 10.3109/10409237609105456. [DOI] [PubMed] [Google Scholar]
  11. Tsuruo T., Arens M., Padmanabhan R., Green M. An endodeoxyribonuclease of human KB cells. Purification and properties of the enzyme. J Biol Chem. 1978 May 25;253(10):3400–3407. [PubMed] [Google Scholar]
  12. Wang E. C., Furth J. J., Rose J. A. Purification and characterization of a DNA single strand specific endonculease from human cells. Biochemistry. 1978 Feb 7;17(3):544–549. doi: 10.1021/bi00596a027. [DOI] [PubMed] [Google Scholar]
  13. Wang E. C., Rose J. A. Deoxyribonucleic acid single-strand-specific endonucleases in human cells: partial purification of a salt-resistant endonuclease with an acidic isoelectric point. Biochemistry. 1981 Feb 17;20(4):755–758. doi: 10.1021/bi00507a015. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES