An Improved Assay Method for Antifouling Substances Using the Blue Mussel,Mytilus edulis |
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Authors: | Kazuo Ina Reiko Takasawa Akihito Yagi Noriyuki Yamashita Hideo Ltoh Kanzo Sakata |
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Institution: | 1. Department of Agricultural Chemistry, Shizuoka University, 836 Ohya, Shizuoka 422, Japan;2. Research Laboratory of Marine Biological Science, Faculty of Agriculture, Shizuoka University, Mochimune, Shizuoka 421–01, Japan |
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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°C and pH 8.5 in a reaction mixture (50 μl) containing 1.0 μg λDNA, 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. |
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