고초균에서 3 - 메칠아데닌 DNA 글아이코실라아제의 분리 및 특성

• Published in: BMB reports Vol. 22; no. 3; pp. 282 - 291
• Main Authors: 고용송, 한범희, 양철학, Yong Song Gho, Bom He Han, Chul Hak Yang
• Format: Journal Article
• Language: Korean
• Published: 생화학분자생물학회 01.01.1989
• ISSN: 1976-6696

An inducible enzyme which releases primarily 3-methyladenine from alkylated DNA was partially purified from Bacillus subtilis by using DEAE-cellulose and DNA-cellulose chromatography and designated as 3-methyladenine DNA glycosylase. It released 3-methyl-adenine and 7-methylguanine from DNA methylated with N-methyl-N-nitrosourea (MNU). 3-methyladenine (3-meA) was released 30 times faster than 7-methylguanine (7-meG). No detectable amount of O^6-methylguanine (O^6-meG) was released. The glycosylase activity was inhibited by neither free 3-meA nor 7-meG but was stimulated by spermidine. Apparent molecular weight was 30,000 on SDS polyacrylamide gel electrophoresis and 23,000 on Sephadex G-100 gel filtration. Its pI was 4.7 on column electrofocusing. Unlike the 3-methyladenine DNA glycosylases in Escherichia coli, no evidence for the presence of two different enzymes was found. The enzyme exhibited a preference for double-stranded alkylated DNA to the single stranded one. Apparent K_m for substrate DNA was 5.15×10^(-8)M. Addition of 1 mM EDTA slightly decreased the activity. Addition of 2 mM Mg^(2+) and Ca^(2+) stimulated base release, while 2 mM Co^(2+) and Mn^(2+) inhibited it. Optimum pH for the enzyme reaction was pH 6.7-7.5. The enzyme activity was quite sensitive to temperature and had a sharp optimum at 37℃. The enzyme was inactivated by N-ethylmaleimide and p-chloromercuribenzoate which was known as a sulfhydryl reagents.