Detection of (2'-5')oligoadenylate binding proteins by nondenaturing polyacrylamide gel electrophoresis and affinity blotting onto nitrocellulose.
A latent endoribonuclease, RNase L, binds to and is activated by (2'-5')oligoadenylates ((2'-5')(A)n, n = 2-15). Binding to a labeled derivative of (2'-5')(A)n, [32P](2'-5')(A)3pCp, is detected as a protein-ligand complex observed following nondenaturing polyacrylamide gel electrophoresis. One major binding complex and two minor binding complexes are readily seen in cytoplasmic extracts from Ehrlich ascites tumor cells, murine tissue extracts and rabbit liver tissue extracts. At least one of the more rapidly migrating complexes appears to be a proteolytic degradation product of the larger [32P](2'-5')(A)3pCp binding protein. Cell and tissue extracts containing [32P](2'-5')(A)3pCp binding activity can be immobilized onto nitrocellulose filters and [32P](2'-5')(A)3pCp binding activity detected using a simple, rapid, economical affinity blot assay. Detection of [32P](2'-5')(A)3pCp binding proteins following electrophoresis on nondenaturing polyacrylamide gels and the affinity blot assay significantly improve and simplify the analysis of (2'-5')(A)n binding proteins.
Floyd-Smith
G
G
Department of Zoology, Arizona State University, Tempe 85287.
D000227:Adenine Nucleotides / Q000378:metabolism*; D000818:Animals; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000302:isolation & purification / Q000378:metabolism; D004789:Enzyme Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D009843:Oligoribonucleotides / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D013997:Time Factors; D014407:Tumor Cells, Cultured
D000227:Adenine Nucleotides; D009843:Oligoribonucleotides; D011506:Proteins; C023505:2',5'-oligoadenylate; D003101:Collodion; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease