Purification of restriction endonuclease from Acetobacter aceti IFO 3281 (AatII) and its properties.
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.
Department of Agricultural Chemistry, Shizuoka University, Japan.
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Sato
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Suzuki
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Yamada
D000091:Acetobacter / Q000201:enzymology*; D001483:Base Sequence; D002412:Cations / Q000494:pharmacology; D002845:Chromatography; D002850:Chromatography, Gel; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D013307:Streptomycin
D002412:Cations; D004279:DNA, Viral; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; C069035:GACGTC-specific type II deoxyribonucleases; D013307:Streptomycin