Studies on restriction endonucleases of acetic acid bacteria and allied organisms. PartXI. Purification of restriction endonuclease from Acetobacter aceti IFO 3281 (AatII)and its properties.

Abstract

The restriction endonuclease AatII was purified from cell-free extracts of Acetobacter aceti IFO 3281 by streptomycin treatment, ammonium sulfate fractionation, combined column chromatographies on DEAE-Toyopearl 650S, heparin-Sepharose CL-6B and DEAE-Sepharose CL-6B and FPLC on Mono Q and on Superose 12 (gel filtration). The purified enzyme was homogeneous on SDS-polyacrylamide gel disk electrophoresis. The relative molecular mass of the purified enzyme was 190,000 daltons by gel filtration. The SDS-polyacrylamide gel disk electrophoresis gave the relative molecular mass of 47,500 daltons. These data indicated that the purified, native enzyme is a tetramer (190,000 daltons) composed of four 47,500-dalton subunits. The isoelectric point of the enzyme was 6.0. The purified enzyme was intensely activated by manganese ion (50-fold increase or more when compared with magnesium ion). The enzyme worked best at 37 degrees C and pH 8.5 in a reaction mixture (50 microliters) containing 1.0 micrograms lambda DNA, 10 mM Tris-HCl, 7 mM 2-mercaptoethanol, 7 mM MnCl2 and 50 mM NaCl. The enzyme recognizes the same palindromic hexanucleotide sequence 5'-GACGTC-3', cuts between T and C and produces a 3'-tetranucleotide extension in the presence of MnCl2, as it does in the presence of MgCl2.