The $\underline{Eco}$ RI restriction enzyme which catalyzes the hydrolysis of a specific nucleotide linkage having the sequence of GAATTC in double strand DNA was isolated from $\underline{E·coli}$ RY13 and was purified partially by phosphocellulose column to give a homogeneous enzyme preparation.
The determination of the enzyme reaction was carried out by agarose gel electrophoresis and the quantitative analysis of the enzyme activity was assayed by a new modified spectrophotometric method emplouing bacteriophage λ DNA as the substrate. Under the standard assay condition, the enzyme exhibited its optimum activity at pH 7.5 and 37℃. The enzyme was found to be very specific toward $Mg^{++}$ ion as a cofactor for the activity. However most of the other metal ions such as $Mn^{++}$, $Cu^{++}$, $Co^{++}$, $Zn^{++}$, and $Fe^{++}$ showed rather inhibitory effect on the activity of $\underline{Eco}$ RI restriction enzyme. Magnesium ion dependent enzyme activity showed an optimum peak, i.e., the activity was increased by increasing concentration of metal ion, but after reach to a maximum the activity was rather reduced by the increasing level of the metal ion. The enzyme activity was also stimulatelyslightly by the presence of 100 mM of NaCl.
Inhibition experiments with several natural and unnatural deoxyribonucleotides were also carried out, and the results suggested a possible involvement of a charged group in the active site of the enzyme. Inhibition by pairs of complementary nucleotides for the enzyme activity was also observed.
$\underline{Eco RI}$ 제한 효소를 $\underline{E coli RY}$ 13 균주로 부터 추출하여 phosphocellulose column 크로마토그라피에 의해 부분 정제하고, 그의 성질을 연구하였다. 이 방법으로 얻은 효소는 exonuclease 및 다른 endonuclease 의 contamination 이 없었다. 이 효소는 double strand DNA 의 GAATTC sequence 를 인식하며 G 와 A 사이를 가수분해 한다.
효소 반응은 0.7% agarose 에서 DNA를 전기 이동하여 형광 band 로 확인 하였으며, 정량적인 척도는 DNA band를 perchlorate로 녹여 260 nm 에서 흡광도를 측정하여 구했다. 이 효소는 pH 7.5, 37℃ 에서 optimum activity 를 나타내었고 $Mg^{++}$ 이온을 cofactor 로 요구 하였으며, 다른 금속 이온에 의하여 활성도가 저하되었다. 그리고 높은 농도의 $Mg^{++}$ 조건에서도 활성도가 저하되었다. 이 효소는 NaCl 에 의해서 40% 의 활성도가 증가 하였다. 이 효소의 여러 종류의 nucleotide 에 의한 활성저하 실험 결과 효소의 활성부위에 양이온 전하를 가진 group 이 효소반응에 관여한다는 것이 밝혀졌다.
