Purification and Characterization of Acc I Endonuclease

Acc I endonuclease의 정제와 효소적 특성에 관한 연구

Acc I endonuclease has been isolated from 300g (wet weight) cells of Acinetobacter calcoaceticus. The cells were broken by using French press at 20, 000p.s.i. After ammonium sulfate fractionation, the enzyme was further purified by heparin agarose, DEAE-sephades, Affi.-gel Blue, phosphocellulose, and hydroxylapatite column chromatography. The purified Acc I endonudlease has a single polypeptide species and its subunit molecular weight was 45,000 ${\pm}$ 1,000 daltons as judged by 10% SDS-polyacrylamide gel electrophoresis. The isolated enzyme was essentially free of contaminating nucleases as judged by homochromatography by using a $^{32}P-labeled$ oligonucleotide. The enzyme showed maximum activity at pH values between 8.0 and 11.0 and in the presence of $MgCl_2$. Acc I endonuclease was maximally active in the absence of NaCl and was completely inhibited at 200 mM NaCl.

제한효소 Acc I을 정제하고 그 효소적 특성을 연구하였다. 300g(wet weight)의 Acinetobacter calcoaceticus 로부터 얻은 crude extract를 sample로 하여 ammonium sulfate fractionation을 거쳐 Heparin-agarose, DEAE-se-phades, Affi,-gel Blue, phosphoceIIulose, hydroxylapatite의 순서로 chromatography를 수행한 결고 0 .28mg의 AccI 제한효소를 얻었다. 효소의 specific activity는 mg당 $1.1{\times}10^{s}$ unit 이었다. 정제된 Acc [제한효소는 10% SDS-polyacrylamide gel electrophoresis에서 한개의 band로 나타났으며 그 분자량은 45,000~1,000이었다. 이 효소는 $MgCl_2$ 존재하에, pH 8.0에서 11.0사이에서 최대의 활성을 보였다. NaCl은 이 효소의 활성에는 필요하지 않았으나 150mN이상에서는 급격한 효소 활성의 감소가 있었다.