Characterization of a 2',5'-oligoadenylate (2-5A)-dependent 37-kDa RNase L: azido photoaffinity labeling and 2-5A-dependent activation

The Journal of Biological Chemistry
Susan E Shetzline, Robert J Suhadolnik

Abstract

Upregulation of key components of the 2',5'-oligoadenylate (2-5A) synthetase/RNase L pathway has been identified in extracts of peripheral blood mononuclear cells from individuals with chronic fatigue [corrected] 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, [(32)P]pApAp(8-azidoA), was achieved. Half-maximal photoinsertion of [(32)P]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 for the 37-kDa RNase L. Competition experiments using 100-fold excess unlabeled 2-5A photoaffinity probe, pApAp(8-azidoA), and authentic 2-5A (p(3)A(3)) resulted in complete protection against photolabeling, demonstrating that [(32)P]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 ac...Continue Reading

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Citations

Jun 1, 2005·Experimental and Molecular Pathology·Marc FrémontPatrick Englebienne
May 1, 2002·Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America·Kenny De MeirleirPatrick Englebienne
May 30, 2002·Journal of Interferon & Cytokine Research : the Official Journal of the International Society for Interferon and Cytokine Research·Susan E ShetzlineRobert J Suhadolnik
Dec 29, 2011·Frontiers in Bioscience (Scholar Edition)·Heather J Ezelle, Bret A Hassel
Jan 14, 2016·International Journal of Molecular Sciences·Heather J EzelleBret A Hassel
Jul 16, 2002·The Journal of Biological Chemistry·Edith DemettreBernard Lebleu

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