2- and 8-Azido photoaffinity probes. 1. Enzymic synthesis, characterization and biological properties of 2- and 8-azido photoprobes of 2-5A and photolabeling of 2-5A binding proteins

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Abstract
The 2- and 8-axido trimer 5''-triphosphate photoprobes of 2-5A have been enzymatically synthesized from [.gamma.-32P]2-azidoATP and [.alpha.-32P]8-azidoATP by 2-5A synthetase from rabbit reticulocyte lysates. Identification and structural determination of the 2- and 8-azido adenylate trimer 5''-triphosphates were accomplished by enzymatic hydrolyses with T2 RNase, snake venom phosphodiesterase, and bacerial alkaline phosphatase. Hydrolysis products were identified by HPLC and PEI-cellulose TLC analyses. The 8-azido photoprobe of 2-5A displaces p3A4 [32P]pCp from RNase L with affinity equivalent to p3A3 (IC50 = 2 .times. 10-9 M in radiobinding assays). The 8-azido photoprobe also activates RNase L to hydrolyze poly(U) [32P]pCp 50% at 7 .times. 10-9 in core-cellulose assays. The 2- and 8-azido photoprobes and autheritic p3A3 activate RNase L to cleave 28S and 18S rRNA to specific cleavage products at 10-9 M in rRNA cleavage assays. The nucleotide binding site(s) of RNase L and/or other 2-5A binding proteins in extracts of interferon-treated L929 cells were investigated by photoaffinity labeling. Dramatically different photolabeling patterns were observed with the 2- and 8-azido photoprobes. The [.gamma.-32P]2-azido adenylate trimer 5''-triphosphate photolabels only one polypeptide with a molecular weight of 185 000 as determined by SDS gel electrophoresis, whereas the [.alpha.-32P]8-azido adenylate trimer 5''-triphosphate covalently photolabels six polypeptides with molecular weights of 46 000, 63 000, 80 000, 89 000, 109 000, and 158 000. Evidence that the photolabeling by 2- and 8-azido 2-5A photoprobes was highly specific for the p3A3 allosteric binding site was obtained as follows. Addition of 1 .times. 10-4 M or 1 .times. 10-5 M authentic p3A3 to incubation mixtures containing either the [132P]2- or 8-azids photoprobe prevented the photolabeling. However, neither 2'',5''-or 3'',5''-trimer core molecules nor ATP prevented the photolabeling. This new class of 2-5A photoaffinity and their application for investigation of the nucleotide binding domain of R Nase L and/or other 2-5A binding proteins in the antiviral/antiproliferative state of mammalian cells are discussed.

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