Characterization of a 2 ',5 '-oligoadenylate (2-5A)-dependent 37-kDa RNaseL - Azido photoaffinity labeling and 2-5A-dependent activation

Upregulation of key components of the 2 ' ,5 ' -oligoadenylate (2-5A) synth etase/RNase L pathway have been identified in extracts of peripheral blood mononuclear cells hom individuals with chronic syndrome, including the pres ence 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-kD a forms of RNase L has been completed utilizing photolabeling/immunoprecipi tation and affinity assays, respectively. Saturation of photolabeling of th e 80- and the 37-kDa RNase L with the 2-5A azido photoprobe, [P-32]pApAp(8- azidoA), was achieved. Half-maximal photoinsertion of [P-32]pApAp(8-azidoA) occurred at 3.7 x 10(-8) M for the 80-kDa RNase L and at 6.3 x 10(-8) M fo r the 37-kDa RNase L. Competition experiments using 100-fold excess unlabel ed 2-5A photoaffinity probe, pApAp(8-azidoA), and authentic 2-5A (p(3)A(3)) resulted in complete protection against photolabeling, demonstrating that [P-32]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 t hree times faster than the 80 M)a RNase L. The data obtained from these 2-5 A binding and 2-5A-dependent activation studies demonstrate the utility of [P-32]pApAp(8-azidoA) for the detection of the 37-kDa RNase L in peripheral blood mononuclear cell extracts.