[29] Assay of (2′-5′)-oligo(A) synthetase with 2′,5′-ADP-sepharose

Publisher Summary This chapter presents an assay for 2',5'-oligoadenylate synthetase that provides reproducibly high yields of (2'-5')-oligo(A) when crude cell extracts are employed, and provides an opportunity for variation of nucleic acid activator. This method is based on the binding of (2'-5')-oligo(A) synthetase from crude extracts to 2',5'-ADP-Sepharose at 4°. After binding, the resin is washed to remove unbound molecules, and the resin bound synthetase is incubated at 30° in the presence of ATP and a nucleic acid activator. The (2'-5')-oligo(A) in the reaction supernatant is then assayed by its ability to inhibit cell free protein synthesis. This method is a convenient and quantitative alternative to the assay of (2'-5')-oligo(A) synthetase from crude extracts on poly(I) . poly(C)-Sepharose. In extracts that contain high levels of inactivating enzymes, both methods may be superior to a solution assay in that both resin assays afford a high degree of purification in the binding step.