Interferon action. Covalent linkage of (2'-5')pppApApA(32P)pCp to (2'-5')(A)n-dependent ribonucleases in cell extracts by ultraviolet irradiation.
One of the mediators of interferon action is an endonuclease system. This consists of (2'-5')(A)n synthetase, which, if activated by double-stranded RNA, converts ATP into (2'-5')(A)n and RNase L, a latent endoribonuclease, which binds (2'-5')(A)n and is thereby activated. We report here that a derivative of (2'-5')(A)n (i.e. (2'-5')pppApApA[32P]pCp) can be covalently cross-linked by UV irradiation to a protein in cytoplasmic extracts from mouse Ehrlich ascites tumor cells. This protein has an apparent molecular weight of 77,000 as determined by gel electrophoresis in sodium dodecyl sulfate. It appears to be identical with RNase L according to the following criteria: co-chromatography on DEAE-cellulose and Sephacryl S300. The gel filtration in Sephacryl S300 reveals that the apparent molecular weight of the protein is between 70,000 and 90,000, indicating that it is a monomer. The cross-linking is oligonucleotide specific. It is inhibited by 10 nM (2'-5')pppApApA or 1 microM (2'-5')ApApA, i.e, compounds known to block, even at low concentration, the binding of (2'-5')pppApApApCp to RNase L. (3'-5')ApApA inhibits only at a 0.1-1 mM concentration, and 1 mM ATP, 2'-AMP, or 5', 3'-pCp have no effect. (2'-5')pppApApApCp was also cross-linked to a protein with a molecular weight of about 78,000 (as determined by gel electrophoresis in sodium dodecyl sulfate) in cytoplasmic extracts from human (HeLa) cells and to protein(s) with molecular weight(s) of 75,000-77,000 (as determined by gel electrophoresis in sodium dodecyl sulfate) in nuclear extracts from Ehrlich ascites tumor cells.
D000227:Adenine Nucleotides / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004722:Endoribonucleases; D006367:HeLa Cells / Q000032:analysis; D006801:Humans; D007372:Interferons / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D009841:Oligonucleotides / Q000378:metabolism*; D009843:Oligoribonucleotides / Q000378:metabolism* / Q000494:pharmacology / Q000528:radiation effects; D012260:Ribonucleases / Q000378:metabolism*; D014466:Ultraviolet Rays
D000227:Adenine Nucleotides; D009841:Oligonucleotides; D009843:Oligoribonucleotides; C023505:2',5'-oligoadenylate; C016964:adenosine triphosphate adenosine monophosphate adenosine monophosphate; D007372:Interferons; D004722:Endoribonucleases; D012260:Ribonucleases; C031956:2-5A-dependent ribonuclease