Deoxyribonuclease II purified from Euglena gracilis SM-ZK, a chloroplast-lacking mutant: comparison with porcine spleen deoxyribonuclease II

Abstract

An acid deoxyribonuclease was extracted from Euglena gracilis SM-ZK, a chloroplast-lacking strain, by homogenizing the cells in 50 mM sodium acetate (pH 4.6). The enzyme was then purified by heat treatment and a series of chromatographic separations. The molecular mass of the Euglena acid DNase was estimated to be 45 kDa by sensitive activity staining in an SDS-polyacrylamide gel using SYBR Green. Treatment of the Euglena enzyme with a reducing agent prior to electrophoresis destroyed its DNase activity in the gel, indicating that disulfide bridging is essential for its enzyme activity. Nucleolytic properties of this enzyme are essentially the same as to those of porcine DNase II. The Euglena enzyme acts on both double-stranded (ds) and single-stranded DNA, but acts preferentially on dsDNA with an optimum pH at approximately 5.3. EDTA did not inhibit its enzyme activity. Euglena DNase makes double-strand breaks in circular DNA substrate and generates a terminus with 3′-phosphate and 5′-OH. These results indicate that the Euglena acid DNase is in fact a member of the DNase II family.