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 activation studies demonstrate the utility of [(32)P]pApAp(8-azidoA) for the detection of the 37-kDa RNase L in peripheral blood mononuclear cell extracts.
Highlights
Upregulation of key components of the 2,5-oligoadenylate (2–5A) synthetase/RNase L pathway have been identified in extracts of peripheral blood mononuclear cells from individuals with chronic syndrome, including the presence of a low molecular weight form of RNase L
We had previously demonstrated that the 2–5OAS/RNase L pathway is constitutively active in peripheral blood mononuclear cells (PBMC) extracts from individuals with chronic fatigue syndrome (CFS); 2–5OAS is predominantly in its activated form and levels of bioactive 2–5A and RNase L enzyme activity are elevated in CFS PBMC extracts compared with healthy control PBMC extracts [13, 14]
We utilized the methods of covalent photoaffinity labeling with the 2–5A azido photoprobe, [32P]pApAp(8-azidoA), and immunoprecipitation with a polyclonal antibody to human RNase L to determine the binding requirements for the interaction of 2–5A with 80- and 37-kDa RNase L in healthy control and CFS PBMC extracts, respectively
Summary
Upregulation of key components of the 2,5-oligoadenylate (2–5A) synthetase/RNase L pathway have been identified in extracts of peripheral blood mononuclear cells from individuals with chronic syndrome, including the presence of a low molecular weight form of RNase L. [32P]pApAp(8-azidoA), binds to the 80- and 37-kDa RNase L in healthy control and CFS PBMC extracts, respectively.
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