Fish Physiology and Biochemistry

, Volume 9, Issue 5–6, pp 439–452 | Cite as

Characterization of a pancreatic DNase from pyloric caeca of atlantic cod (Gadus morhua L.)

  • Knut O. Strætkvern
  • Arnt J. Raae
  • Bernt T. Walther


An alkaline deoxyribonuclease (DNase) from cod pancreatic tissue has been characterized. The enzyme is a DNase I type endonuclease and hydrolyzes effectively both native and denatured DNA. Monomeric actin inhibits the enzyme reaction. The enzyme obeys Michaelis-Menten kinetics and the apparent Km value for native linear duplex DNA is 33 µg/ml. The cod DNase opens supercoiled plasmid DNA, by introducing adjacent nicks in both strands, possibly separated by 5–10 nucleotides. DNA hydrolyzed by cod DNase functions as substrates both for DNA polymerase and ligase, and the nicks therefore contain 5′-phosphoryl and 3′-hydroxyl groups. Optimum concentrations of divalent cations are 5 mM Mg2+, 0.63 mM Mn2+ and 0.075 mM Ca2+. However, Ca2+ is apparently not essential for the enzymatic functions. The enzyme has a narrow temperature optimum at 42°C and is thermolabile above 50°C; however, Mn2+ shifts the optimum slightly to 45°C by causing increased temperature stability. The cod DNase reaction is inhibited by the DNA intercalating compounds actinomycin D and ethidium bromide. Histidine-modifying reagents such as tosyl phenylalanyl chloromethylketone and diethyl pyrocarbonate inhibit the enzyme activity, but the cod DNase is insensitive to disulfide-reducing agents.


Gadus morhua L. pancreatic DNase I activators inhibitors substrates DNA modification thermal properties 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References cited

  1. Ásgeirsson, B., Fox, J.W. and Bjarnason, J.B. 1989. Purification and characterization of trypsin from the poikilothermGadus morhua. Eur. J. Biochem. 180: 85–94.CrossRefPubMedGoogle Scholar
  2. Campbell, V.W. and Jackson, D.A. 1980. The effect of divalent cations on the mode of action of DNase 1. The initial reaction products produced from covalently closed circular DNA. J. Biol. Chem. 255: 3726–3735.PubMedGoogle Scholar
  3. Davis, L.G., Dibner, M.D. and Battey, J.F. 1988. Basic Methods in Molecular Biology. Elsevier Publisher, Amsterdam.Google Scholar
  4. Douvas, A. and Price, P.A. 1975. Some effects of calcium and magnesium ions on the activity of bovine pancreatic deoxyribonuclease A. BBA 395: 201–212.PubMedGoogle Scholar
  5. Drew, H.R. 1984. Structural specificities of five commonly used DNA nucleases. J. Mol. Biol. 176: 535–557.CrossRefPubMedGoogle Scholar
  6. Frei, E., Baumgartner, S., Edström, J.E. and Noll, M. 1985. Cloning of theextra sex combs gene ofDrosophila and its identification by P-element-mediated gene transfer. EMBO J. 4: 979–987.Google Scholar
  7. Georgatsos, J.G. and Antonoglou, O. 1964. Deoxyribonuleases in tissues of marine origin. Enzymologia 27: 141–150.PubMedGoogle Scholar
  8. Junowicz, E. and Spencer, J.H. 1973a. Studies on bovine pancreatic deoxyribonuclease A. I. General properties and activation with different bivalent metals. BBA 312: 72–84.PubMedGoogle Scholar
  9. Junowicz, E. and Spencer, J.H. 1973b. Studies on bovine pancreatic deoxyribonuclease A. II. The effect of different bivalent metals on the specificity of degradation of DNA. BBA 312: 85–102.PubMedGoogle Scholar
  10. Kleppe, K., Ohtsuka, E., Kleppe, R., Molineux, I. and Khorana, H.G. 1971. Studies on polynucleotides. XCVI. Repair replication of short synthetic DNA's as catalyzed by DNA polymerases. J. Mol. Biol. 56: 341–361.PubMedGoogle Scholar
  11. Kowalski, P. and Laskowski, M., Sr. 1976. Functional characterization of nucleophosphodiesterases.In Handbook of Biochemistry and Molecular Biology, Nucleic Acids, vol. II. pp. 491–531. Edited by G.D. Fasman. CRC Press, Cleveland.Google Scholar
  12. Laskowski, M., Sr. 1971. Deoxyribonuclease I.In The Enzymes, Vol. 4. pp. 289–311. Edited by P.D. Boyer. Academic Press, New York.Google Scholar
  13. Liao, T.-H., Salnikow, J., Moore, S. and Stein, W.H. 1973. Bovine pancreatic deoxyribonuclease A. Isolation of cyanogen bromide peptides: complete covalent structure of the polypeptide chain. J. Biol. Chem. 248: 1489–1495.PubMedGoogle Scholar
  14. Linn, S. 1981. Deoxyribonucleases: Survey and perspectives.In The Enzymes, Vol. 14. pp. 121–135. Edited by P.D. Boyer. Academic Press, New York.Google Scholar
  15. Lizarraga, B., Sanchez-Romero, D., Gil, A. and Melgar, E. 1978. The role of Ca2+ on pH-induced hydrodynamic changes of bovine pancreatic deoxyribonuclease A. J. Biol. Chem. 253: 3191–3195.PubMedGoogle Scholar
  16. Lizarraga, B., Bustamante, C., Gil, A. and Melgar, E. 1979. Multiple conformations of deoxyribonuclease A. Their separation at alkaline pH and low ionic strength in the presence of Ca2+. BBA 579: 298–302.PubMedGoogle Scholar
  17. Mannherz, H.G., Goody, R.S., Konrad, M. and Nowak, E. 1980. The interaction of bovine pancreatic deoxyribonuclease I and skeletal muscle actin. Eur. J. Biochem. 104: 367–379.PubMedGoogle Scholar
  18. Melgar, E. and Goldthwait, D.A. 1968. Deoxyribonucleic acid nucleases. II. The effect of metals on the mechanism of action of deoxyribonuclease I. J. Biol. Chem. 243: 4409–4416.PubMedGoogle Scholar
  19. Moore, S. 1981. Pancreatic Dnase.In The Enzymes, Vol. 14, pp. 281–296. Edited by P.D. Boyer. Academic Press, New York.Google Scholar
  20. Nagae, S., Nakayama, J., Nakano I. and Anai M. 1982. Purification and properties of a neutral endodeoxyribonuclease from rat small intestinal mucosa. Biochemistry 21: 1339–1344.PubMedGoogle Scholar
  21. Oefner, C. and Suck, D. 1986. Crystallographic refinement and structure of DNase I at 2Å resolution. J. Mol. Biol. 192: 605–632.PubMedGoogle Scholar
  22. Paudel, H.K. and Liao, T.-H. 1986. 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. J. Biol. Chem. 261: 16012–16017.PubMedGoogle Scholar
  23. Pohl, F.M., Thomae, R. and Karst, A. 1982. Temperature dependence of the activity of DNA-modifying enzymes: Endonucleases and DNA ligase. Eur. J. Biochem. 123: 141–152.CrossRefPubMedGoogle Scholar
  24. Price, P.A. 1972. Characterization of Ca++ and Mg++ binding to bovine pancreatic deoxyribonuclease A. J. Biol. Chem. 247: 2895–2899.PubMedGoogle Scholar
  25. Price, P.A. 1975. The essential role of Ca2+ in the activity of bovine pancreatic deoxyribonuclease. J. Biol. Chem. 250: 1981–1986.PubMedGoogle Scholar
  26. Price, P.A., Liu, T.-Y., Stein, W.H. and Moore, S. 1969a. Properties for chromatographically purified bovine pancreatic deoxyribonuclease. J. Biol. Chem. 244: 917–923.PubMedGoogle Scholar
  27. Price, P.A., Moore, S. and Stein, W.H. 1969b. Alkylation of a histidine residue at the active site of bovine pancreatic deoxyribonuclease. J. Biol. Chem. 244: 924–928.PubMedGoogle Scholar
  28. Price, P.A., Stein, W.H. and Moore, S. 1969c. Effect of divalent cations on the reduction and re-formation of the disulfide bonds of deoxyribonuclease. J. Biol. Chem. 244: 929–932.PubMedGoogle Scholar
  29. Raae, A.J. 1990. Effect of low and high temperatures on chymotrypsin from Atlantic cod (Gadus morhua L.); Comparison with bovine α-chymotrypsin. Comp. Biochem. Physiol. 97B: 145–149.Google Scholar
  30. Rasskazov, V.A., Pirozhnikova, V.V. and Galkin, V.V. 1975. Some properties and specificity of deoxyribonucleases from marine invertebrates and fishes. Comp. Biochem. Physiol. 51B: 343–347.CrossRefGoogle Scholar
  31. Robson, B. and Garnier, J. 1986. The special case of metalloproteins.In Introduction to Proteins and Protein Engineering. pp. 195–206. Elsevier Publishers, Amsterdam.Google Scholar
  32. Simpson, B.K. and Haard, N.F. 1987. Cold-adapted enzymes from fish.In Food Biotechnology. pp. 495–527. Edited by D. Knorr. Marcel Dekker Inc., New York.Google Scholar
  33. Somero, G.N. 1978. Temperature adaptation of enzymes: Biological optimization through structure-function compromises. Ann. Rev. Ecol. Syst. 9: 1–29.CrossRefGoogle Scholar
  34. Strætkvern, K.O. and Raae, A.J. 1990. Deoxyribonuclease zymography adapted to the precast PhastGel electrophoresis media. Electrophoresis 11: 347–349.CrossRefPubMedGoogle Scholar
  35. Strætkvern, K.O., Raae, A.J. and Walther, B.T. 1990. Purification and physicochemical properties of deoxyribonuclease from pyloric caeca of Atlantic cod (Gadus morhua L.). Fish Physiol. Biochem. 8: 529–539.Google Scholar
  36. Suck, D., Oefner, C. and Kabsch, W. 1984. Three-dimensional structure of bovine pancreatic DNase I at 2.5Å resolution. EMBO J. 3: 2423–2430.PubMedGoogle Scholar
  37. Suck, D. and Oefner, C. 1986. Structure of DNase I at 2.0A resolution suggests a mechanism for binding to and cutting DNA. Nature, Lond. 321: 620–625.Google Scholar
  38. Suck, D., Lahm, A. and Oefner, C. 1988. Structure refined to 2Å of a nicked DNA octanucleotide complex with DNase I. Nature, Lond. 332: 464–468.Google Scholar
  39. Yarnall, M., Rowe, T.C. and Holloman, W.K. 1984. Purification and properties of nuclease γ fromUstilago maydis. J. Biol. Chem. 259: 3026–3032.PubMedGoogle Scholar
  40. Zimmer, C., Luck, G. and Triebe, H. 1974. Conformation and reactivity of DNA. IV. Base binding ability of transition metal ions to native DNA and effect on helix conformation with special reference to DNA-Zn(II) complex. Biopolymers 13: 425–453.CrossRefPubMedGoogle Scholar

Copyright information

© Kugler Publications bv 1991

Authors and Affiliations

  • Knut O. Strætkvern
    • 1
  • Arnt J. Raae
    • 1
  • Bernt T. Walther
    • 1
  1. 1.Laboratory of Marine Molecular BiologyUniversity of BergenBergenNorway

Personalised recommendations