Alternation of Two Forms of Restriction Endonuclease HindIII

Two forms of the restriction enzyme HindIII were alternated with each other under some physiological or biochemical conditions. Addition of a low amount of phase T7 to the culture of HindIII-producing Haemophilus influenzae Rd, resulted in appearance of some amounts of the P2 fraction of HindIII, which was eluted with a high concentration of KCI from a phosphocellulose column. Higher amounts of T7 caused a decrease of the P2 fraction; finally the alternative PI fraction of HindIII, which was eluted with a lower concentration of KCI, remained exclusively. Addition of disaccharides such as maltose and trehalose to the bacterial extract, yielded more P2, although the disaccharides inhibited to this enzyme. Urea showed an interesting distribution of these two forms of HindIII. Phosphocellulose chromatography in the presence of 2 m urea generated a broad peak of HindIII Activity. Addition of 4 m urea, on the contrary, showed only one active peak of this enzyme. The HindIII could be purified by the following DEAE-cellulose chromatography. These results indicate the presence of only one kind of HindIII molecule, which was alternated between free and bound forms, and a certain kind of factor that would equilibrate these two forms.