Two ribonucleases H from cultured plant cells

Two forms of enzyme with ribonuclease H (RNase H) [EC 3.1.4.34] activities, have been par tially purified from cultured plant cells, strain GD-2, derived from carrot root. One is an Mn2+ -dependent RNase H, and the second is an Mg2+ -dependent RNase H. These enzymes degrade RNA specifically in RNA-DNA hybrid structures. They were eluted at around 0.2 M and 0.4 M potassium chloride in phosphocellulose chromatography, and were further purified using blue Sepharose. Mg2+ RNase H exhibits maximal activity at pH 9.0, and requires 10 to 15 mM Mg2+ for maximal activity, whereas the Mn2+ enzyme is most active at pH 8.0, is maximally active at an Mn2+ concentration of 0.4 m and has some activity with Mg2+ Both enzymes require a sulfhydryl reagent for maximal activity. The enzymes liberate a mixture of oligonucleotides with 5′-phosphate and 3′-hydroxyl terinini. The appar ent molecular weight of the Mg2+-dependent RNase H was estimated to 18–20 × 104 and that of the Mn2+ RNase H was estimated to be 14 × 10 by gel filtration.