Affinity blotting assay for 2-5A-dependent RNase.
The intracellular effectors known as 2-5A (ppp-(A2'p)nA) regulate the cleavage of single-stranded RNA by activating a latent endoribonuclease (2-5A-dependent RNase or RNase L). Accordingly this enzyme may exist in either an inactive form, free of 2-5A, or an active, 2-5A-bound form. Previously, a radiobinding assay for 2-5A-dependent RNase was developed that measured the amount of labeled ppp(A2'p)2A3'-[32P]Cp, a derivative of ppp(A2'p)nA, that bound the inactive enzyme form. Because 2-5A-dependent RNase has a particularly high affinity for 2-5A the radiobinding assay may not measure the 2-5A activated form of the enzyme. Therefore an efficient procedure to facilitate the detection of total 2-5A-dependent RNase (i.e., 2-5A-free and 2-5A-bound enzyme) in mouse spleen extracts was developed. Denaturing conditions were used to ensure that all 2-5A-dependent RNase was in the 2-5A-free form. After denaturation on polyacrylamide gel electrophoresis, optimal blotting conditions onto nitrocellulose and renaturation of the 2-5A binding site of 2-5A-dependent RNase were developed. This procedure allowed a population of enzyme that otherwise is not accessible by the classical radiobinding assay to be assayed, thus leading to an increased measurement of 15-17% in cytoplasmic spleen extracts.
Bayard
B
B
CNRS U.A. 530, USTL, Montpellier, France.
Zhou
A
A
D000345:Affinity Labels; D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D003432:Cross-Linking Reagents; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008297:Male; D051379:Mice; D010777:Photochemistry; D011869:Radioligand Assay; D013154:Spleen / Q000201:enzymology*
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D003902:Detergents; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease