J. Biol. Chem., Vol. 256, Issue 4, 1636-1642, 02, 1981
Purification and properties of an endodeoxyribonuclease from nuclei of bovine small intestinal mucosa
J Nakayama, T Fujiyoshi, M Nakamura and M Anai
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.
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