Characterization of a 2',5'-oligoadenylate (2-5A)-dependent 37-kDa RNase L: azido photoaffinity labeling and 2-5A-dependent activation.
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.
Shetzline
S E
SE
Department of Biochemistry and the Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Suhadolnik
R J
RJ
D000227:Adenine Nucleotides / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry*; D001386:Azides / Q000737:chemistry*; D001665:Binding Sites; D001667:Binding, Competitive; D002457:Cell Extracts / Q000032:analysis; D004722:Endoribonucleases / Q000378:metabolism*; D004789:Enzyme Activation; D015673:Fatigue Syndrome, Chronic / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000201:enzymology; D009843:Oligoribonucleotides / Q000378:metabolism*; D019920:Photoaffinity Labels / Q000737:chemistry*; D011072:Poly U / Q000378:metabolism
D000227:Adenine Nucleotides; D001386:Azides; D002457:Cell Extracts; D009843:Oligoribonucleotides; D019920:Photoaffinity Labels; C059244:5'-O-triphosphoryl-8-azidoadenylyl-(2'-5')-8-azidoadenylyl-(2'-5')-8-azidoadenosine; D011072:Poly U; C023505:2',5'-oligoadenylate; D000255:Adenosine Triphosphate; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease