2',5'-8-azidoadenylate trimer 5'-triphosphate (2,5-8-N3p3A3): enzymatic synthesis, biological properties and photoaffinity labelling of RNase L

Photoreactive analogs of 2-5A have been enzymatically synthesized from 8-azidoATP by 2-5A synthetase from lysed rabbit reticulocytes (LRR) in 1% average yield, but not from 100,000-fold purified 2-5A synthetase. The structure of the 2,5-8-N3p3A3 was established by enzymatic hydrolyses and HPLC. The ability of the 2,5-8-N3p3A3 to bind to and activate RNase L was compared to authentic p3A3 in radiobinding, core-cellulose and rRNA cleavage assays using RNase L in L929 cell extracts. The 2,5-8-N3p3A3 can bind to activate RNase L as well as does p3A3. Furthermore, [α-32P]2,5-8-N3p3A3 is covalently cross-linked to a single protein with a M/sub r/ of ∼80,000 in L929 cell extracts following 1 min. ultraviolet irradiation, 00C. This cross-linking is inhibited by p3A3 but not by ATP or 8-azidoATP suggesting that the protein that was photolabeled was RNase L. The use of the 2,5-azido analog provides a way to further characterize the binding and activation processes of RNase L. [α-32P]8-AzidoATP crosslinked to 2-5A synthetase; 2,5-8-N3p3A3 did not compete with 8-azidoATP for this cross-linking. The mild conditions needed for the covalent linkage to 2-5A synthetase and RNase L makes it possible to determine the subcellular distribution and role of RNase L in cell growth in the intact cell