Regeneration of enzyme activity after western blot: activation of RNase L by 2-5A on filter--importance for its detection.
A rapid and convenient new procedure for detecting RNase L activity following Western blot by renaturation of the enzyme on the nitrocellulose sheets is described. This method allows the simultaneous analysis of enzymatic activity (e.g., cleavage of poly(uridylic acid)-3'-[32P]pCp) and RNase L binding to radioactivE probes (e.g., 2-5A-3'-[32P]pCp) in the same sample. Unlike previously published methods, this procedure eliminates interference from proteases or other RNases during the analysis of RNase L activity. The detection of RNase(s) L is also affected by the presence of endogenous 2-5A, 2-5A derivatives, or other possible "inhibitors" in cell extracts; this Western blot assay allows of RNase(s) L to be detected independently of intracellular 2-5A or analogs. Differences between the procedures used so far and this Western blot technique can indeed be demonstrated. It is shown with this Western blot assay that although RNase L has been described as a protein of 185-200 kDa under nondenaturating conditions, its 80-kDa (and 40-kDa) component is able to bind 2-5A and to cleave poly(uridylic acid) in a 2-5A-dependent way, independently of other subunit(s) or cofactor(s).
Salehzada
T
T
Laboratoire de Biochimie des Proteines, URA CNRS 1191, Université Montpellier II-Sciences et Techniques du Languedoc, France.
Silhol
M
M
Lebleu
B
B
Bisbal
C
C
D000227:Adenine Nucleotides / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D003101:Collodion; D003565:Cytidine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D004722:Endoribonucleases / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D008297:Male; D051379:Mice; D009843:Oligoribonucleotides / Q000378:metabolism / Q000494:pharmacology*; D010761:Phosphorus Radioisotopes; D011072:Poly U / Q000378:metabolism
D000227:Adenine Nucleotides; D009843:Oligoribonucleotides; D010761:Phosphorus Radioisotopes; D011072:Poly U; C032883:cytidine 3',5'-diphosphate; C023505:2',5'-oligoadenylate; D003565:Cytidine Diphosphate; C042702:triadenylyl-(2'-3')-adenylyl-cytidylic acid; D003101:Collodion; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease