Study on the restriction endonuclease: preparation and charaterization of Eco RI endonuclease from E. coli RY13

Abstract

The $\mbox{\underline{Eco}}$ RI restriction enzyme which catalyzes the hydrolysis of a specific nucleotide linkage having the sequence of GAATTC in double strand DNA was isolated from $\mbox{\underline{E}}$. $\mbox{\underline{coli}}$ 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 $\lambda$ DNA as the substrate. Under the standard assay condition, the enzyme exhibited its optimum activity at pH 7.5 and 37$^\circ$C. 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 $\mbox{\underline{Eco}}$ 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 stimulately slightly 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.