A Deoxyribonuclease Which Requires Nucleoside Triphosphate from Micrococcus lysodeikticus
I. PURIFICATION AND CHARACTERIZATION OF THE DEOXYRIBONUCLEASE ACTIVITY
Motoaki Anai 1, Takakata Hirahashi 1, and Yasuyuki Takagi 1
From the
1 From the Department of Biochemistry, Kyushu University School of Medicine, Fukuoka, Japan
A novel deoxyribonuclease, which requires the presence of nucleotide for the reaction, has been purified approximately 2300-fold from cell-free extracts of Micrococcus lysodeikticus. This enzyme degrades native, double-stranded DNA at about 40-fold greater rate than thermally denatured DNA. Evidence for complete dependence on ATP as well as Mg++ ion has been found. The optimum pH is around 9.4. The enzyme behaves as an endonuclease, yielding primarily oligonucleotides consisted of di-, tri-, tetra-, and pentanucleotides along with some larger fragments terminated by 5'-phosphoryl group. The average chain length of a limit digest is approximately 5.5.
The rate of decrease in viscosity of the DNA substrate relative to the appearance of an acid-soluble form is not so rapid with this enzyme as that observed with pancreatic DNase. The kinetic analysis by means of sedimentation in sucrose density gradient indicates clearly that initial DNA added as substrate disappears progressively during the reaction with the enzyme, being replaced by much more slowly sedimenting material, and the products intermediate in size between these two components are not detectable. These results suggest that the mechanism of DNA digestion by the enzyme appears to be the one-by-one type.
Submitted on October 6, 1969
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