Purification, properties and recognition sequence and cleavage site determinations of restriction endonuclease from Acetobacter pasteurianus IFO 13753 (ApaLI).
A new restriction endonuclease, designated as ApaLI, was purified from cell-free extracts of Acetobacter pasteurianus IFO 13753 by streptomycin treatment, ammonium sulfate fractionation, combined column chromatographies on heparin-Sepharose CL-6B and DEAE-Sepharose CL-6B and Fast Protein Liquid Chromatography on Mono Q HR 5/5. The purified enzyme was homogeneous on polyacrylamide gel disc electrophoresis. The molecular weight of the purified enzyme was calculated as 26,000 daltons by gel filtration using Sephadex G-200, and the isoelectric point of the purified enzyme was 4.8 by ampholine sucrose-density gradient isoelectric focusing. The purified enzyme cleaved lambda, Ad2, SV40, M13mp18 RF 1, psi X174 RF I and pBR322 DNAs at 4, 7, 0, 0, 1 and 3 sites, respectively. The purified enzyme worked best at 37 degrees C and pH 8.0 in a reaction mixture (50 microliters) containing 1.0 micrograms lambda DNA, 10mM Tris-HCl, 7 mM 2-mercaptoethanol, 7 mM MgCl2 and 25mM NaCl. However, the purified enzyme did not require NaCl necessarily for the enzyme reaction. The purified enzyme recognized the palindromic hexanucleotide DNA sequence, 5'-GTGCAC-3' and cut between G and T, producing a 5'-cohesive tetranucleotide extension.
Department of Agricultural Chemistry, Shizuoka University, Japan.
M
M
Murakami
Y
Y
Yamada
D000091:Acetobacter / Q000201:enzymology*; D000645:Ammonium Sulfate / Q000494:pharmacology; D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D002458:Cell Fractionation; D002853:Chromatography, Liquid / Q000379:methods; D004262:DNA Restriction Enzymes / Q000302:isolation & purification* / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006358:Hot Temperature; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011189:Potassium Chloride / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013307:Streptomycin / Q000494:pharmacology
D002413:Cations, Divalent; D004279:DNA, Viral; D012965:Sodium Chloride; D011189:Potassium Chloride; D004262:DNA Restriction Enzymes; D000645:Ammonium Sulfate; D013307:Streptomycin