Characterization of a 2′,5′-Oligoadenylate (2–5A)-dependent 37-kDa RNase L AZIDO PHOTOAFFINITY LABELING AND 2–5A-DEPENDENT ACTIVATION

Abstract Upregulation of key components of the 2′,5′-oligoadenylate (2–5A) synthetase/RNase L pathway have been identified in extracts of peripheral blood mononuclear cells from individuals with chronic syndrome, including the presence of a low molecular weight form of RNase L. In this study, analysis of 2′,5′-Oligoadenylate (2–5A) binding and activation of the 80- and 37-kDa forms of RNase L has been completed utilizing photolabeling/immunoprecipitation and affinity assays, respectively. Saturation of photolabeling of the 80- and the 37-kDa RNase L with the 2–5A azido photoprobe, [32P]pApAp(8-azidoA), was achieved. Half-maximal photoinsertion of [32P]pApAp(8-azidoA) occurred at 3.7 × 10−8 m for the 80-kDa RNase L and at 6.3 × 10−8 m for the 37-kDa RNase L. Competition experiments using 100-fold excess unlabeled 2–5A photoaffinity probe, pApAp(8-azidoA), and authentic 2–5A (p3A3) resulted in complete protection against photolabeling, demonstrating that [32P]pApAp(8-azidoA) binds specifically to the 2–5A-binding site of the 80- and 37-kDa RNase L. The rate of RNA hydrolysis by the 37-kDa RNase L was three times faster than the 80-kDa RNase L. The data obtained from these 2–5A binding and 2–5A-dependent activation studies demonstrate the utility of [32P]pApAp(8-azidoA) for the detection of the 37-kDa RNase L in peripheral blood mononuclear cell extracts.