Purification and properties of an endodeoxyribonuclease from nuclei of bovine small intestinal mucosa.
An endodeoxyribonuclease has been purified from nuclei of bovine small intestinal mucosa to a homogeneous state by a procedure involving affinity chromatography on heparin-agarose. The endonuclease, which was found to be bound to chromatin, has a pH optimum of 5.4. It requires Mn2+ or Co2+ for activity and its maximum activity with Mg2+ is about 80% of that with Mn2+. Its activity is strongly inhibited by sulfhydryl-blocking agents, and by ethidium bromide. The enzyme does not attack RNA and is inhibited by it. Its isoelectric point is 8.5 +/- 0.1, and its molecular weight is 49,000 +/- 3,000, determined by sucrose gradient sedimentation and gel filtration on Sephadex G-100. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the enzyme is composed of two nonidentical subunits with molecular weights of 30,000 and 23,000. The enzyme catalyzes the endonucleolytic cleavage of circular duplex ColE1 DNA via single strand scissions from the initial stage of degradation. The average size of the limit products of native phage T7 or ColE1 DNA is about 2,000 to 1,500 base pairs, estimated by neutral sucrose gradient sedimentation or agarose gel electrophoresis. The enzyme degrades denatured DNA about 20 times faster than native DNA. The products contain 5'-phosphoryl and 3'-hydroxyl termini, and all four deoxymononucleotides are present in almost equal amounts at the 5'-termini.
D000818:Animals; D002413:Cations, Divalent; D002417:Cattle; D002467:Cell Nucleus / Q000201:enzymology*; D004269:DNA, Bacterial; D003850:Deoxyribonuclease I; D003851:Deoxyribonucleases / Q000302:isolation & purification* / Q000378:metabolism; D004720:Endonucleases / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli; D007413:Intestinal Mucosa / Q000201:enzymology*; D007421:Intestine, Small / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D002413:Cations, Divalent; D004269:DNA, Bacterial; D046911:Macromolecular Substances; D013439:Sulfhydryl Reagents; D003851:Deoxyribonucleases; D004720:Endonucleases; D003850:Deoxyribonuclease I