Assay of 2′,5′-oligoadenylate phosphodiesterase activity in mouse L-cell extracts

A rapid and convenient assay for adenylyl(2′ → 5′)adenosine(A2′p5′A) or adenylyl(3′ → 5′)adenosine(A3′p5′A) phosphodiesterase activities is described. The dinucleotides A3′p5′A and A2′p5′A were labeled to a high specific activity by means of a catalytic-exchange procedure. Degradation studies of each of these labeled dinucleotides showed an asymmetrical distribution of label between the two adenine bases. Enzymatic degradation of [ 3 H]A3′p5′A or [ 3 H]A2′p5′A could be quantitated by first digesting the reaction products with bacterial alkaline phosphatase and then adding a slurry of DEAE-Sephadex. Under conditions described, adenosine did not adsorb to the resin, whereas dinucleotides as well as AMP did adsorb. As a consequence, when liquid scintillation fluid was added to the DEAE-Sephadex reaction mixture slurry, the radioactivity of the dinucleotides and AMP was severely quenched. This permitted a direct estimation of the amount of adenosine liberated during the phosphodiesterase degradation and subsequent alkaline phosphatase digestion. This method was applied to the measurement of A2′p5′A degrading activities in extracts of mouse L cells. Extracts from control mouse L cells were as active in degrading A2′p5′A as extracts from interferon pretreated cells.