Affinity blotting assay for 2-5A-dependent RNase.

The intracellular effectors known as 2-5A (ppp-(A2'p)nA) regulate the cleavage of single-stranded RNA by activating a latent endoribonuclease (2-5A-dependent RNase or RNase L). Accordingly this enzyme may exist in either an inactive form, free of 2-5A, or an active, 2-5A-bound form. Previously, a radiobinding assay for 2-5A-dependent RNase was developed that measured the amount of labeled ppp(A2'p)2A3'-[32P]Cp, a derivative of ppp(A2'p)nA, that bound the inactive enzyme form. Because 2-5A-dependent RNase has a particularly high affinity for 2-5A the radiobinding assay may not measure the 2-5A activated form of the enzyme. Therefore an efficient procedure to facilitate the detection of total 2-5A-dependent RNase (i.e., 2-5A-free and 2-5A-bound enzyme) in mouse spleen extracts was developed. Denaturing conditions were used to ensure that all 2-5A-dependent RNase was in the 2-5A-free form. After denaturation on polyacrylamide gel electrophoresis, optimal blotting conditions onto nitrocellulose and renaturation of the 2-5A binding site of 2-5A-dependent RNase were developed. This procedure allowed a population of enzyme that otherwise is not accessible by the classical radiobinding assay to be assayed, thus leading to an increased measurement of 15-17% in cytoplasmic spleen extracts.