Purification of 2′,5′-Oligoadenylate Synthetase from Rabbit Reticulocytes

Abstract

The 2′,5′-oligoadenylate synthetase (2-5A synthetase) from rabbit reticulocytes has been purified to apparent homogeneity. The purification procedure consists of (NH₄)₂SO₄ fractionation (30-50% cut), specific binding of the 2-5A synthetase to and elution from the affinity matrix of polyinosinic-polycytidylic-cellulose, another (NH₄)₂SO₄ precipitation step, and finally chromatography on DEAE-cellulose. Upon electrophoresis in sodium dodecyl sulfate polyacrylamide gel (10%), the purified enzyme migrates as a single polypeptide with an apparent molecular weight of 110,000 daltons. A sedimentation coefficient of 5.8S is obtained by glycerol density gradient centrifugation. The synthesis of 2′,5′-oligoadenylate by the purified enzyme is dependent on the presence of double-stranded (ds) RNA, in the absence of which the enzyme is highly unstable. Biochemical characteristics of the purified enzyme have been defined.