Purification and Characteristics of Hybridase (Ribonuclease H) from Rat‐Liver Cytosol

Abstract

Ribonuclease H or hybridase, the enzyme cleaving the RNA moiety of DNA · RNA hybrids has been extensively purified from rat‐liver cytosol by ammonium sulphate precipitation, DEAE‐cellulose chromatography, Sephadex G‐200 and G‐150 superfine gel filtration, isoelectric focusing, phosphocellulose and hydroxyapatite chromatography. Neither native or denatured DNA, nor single or double‐stranded natural or synthetic ribopolynucleotides are degraded by the enzyme. The pH optimum of the reaction is 7.9. Enzyme activity is totally dependent on the divalent cations Mg 2+ and Mn 2+ , Mg 2+ being optimal for the reaction. The enzyme acts on the hybrid as an endonuclease resulting in end products with a chain length less than 15, having a 5′‐phosphate and a free 3′‐hydroxyl end group. Synthetic homopolymer hybrids are digested only if they are composed of purine bases. The enzyme has a molecular weight of 120000–125000 and seems to be composed of two subunits with molecular weights of 85000 and 43000.