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Characterization of a pancreatic DNase from pyloric caeca of atlantic cod (Gadus morhua L.)
K O Strætkvern1, A J Raae, B T Walther
1Laboratory of Marine Molecular Biology, University of Bergen, N-5020, Bergen, Norway.
Abstract:
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 Mg(2+), 0.63 mM Mn(2+) and 0.075 mM Ca(2+). However, Ca(2+) 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, Mn(2+) 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.
Insights
Cod pancreatic deoxyribonuclease (DNase) is a DNase I-type enzyme that effectively degrades DNA. It functions optimally at 42°C and is inhibited by actin and certain chemicals, offering insights into DNA processing.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Deoxyribonucleases (DNases) are crucial enzymes involved in DNA metabolism and degradation.
- Characterizing novel DNases provides insights into enzymatic mechanisms and potential applications.
Purpose of the Study:
- To characterize an alkaline deoxyribonuclease (DNase) isolated from cod pancreatic tissue.
- To determine the enzymatic properties, substrate specificity, and kinetic parameters of the cod DNase.
Main Methods:
- Enzyme purification and characterization.
- DNA hydrolysis assays using native and denatured DNA.
- Kinetic analysis (Michaelis-Menten kinetics).
- Investigation of cofactor requirements (divalent cations) and inhibitor effects.
Main Results:
- The cod enzyme is a DNase I-type endonuclease active on both native and denatured DNA.
- It exhibits Michaelis-Menten kinetics with an apparent Km of 33 µg/ml for native linear duplex DNA.
- Optimal activity is observed at 42°C, with Mn(2+) enhancing thermostability.
- The enzyme is inhibited by actin, actinomycin D, and ethidium bromide, and by histidine-modifying reagents.
Conclusions:
- Cod pancreatic DNase is a versatile endonuclease with properties similar to DNase I.
- Its nicking activity on supercoiled DNA produces substrates for DNA polymerase and ligase.
- The enzyme's characteristics suggest potential roles in DNA repair or processing, with divalent cations like Mg(2+) and Mn(2+) being important for activity.
