Purification and analysis of murine 2-5A-dependent RNase.
2-5A-dependent RNase (RNase L, RNase F) is an enzyme which mediates effects of 2-5A (px(A2'p)nA; x = 2 or 3, n greater than or equal to 2) in cells. 2-5A binding activity present in mouse liver extracts was measured using a 32P-labeled 2-5A derivative. Analysis of Scatchard plots was consistent with a single noninteracting 2-5A binding site with a Ka of 2.5 X 10(10) M-1. Similarly, affinity labeling of proteins with a 32P-labeled 2-5A derivative revealed a single, high-affinity 2-5A-binding protein of Mr 80,000. This 2-5A-binding protein was the only mouse liver protein specifically and consistently eluted by 2-5A from an affinity resin consisting of core(2-5A) covalently attached to cellulose. The 2-5A-eluted protein could degrade polyuridylic acid but not polycytidylic acid. Furthermore, when the 2-5A-eluted protein was electrophoresed into a polyuridylic acid-containing, nondenaturing gel, a band of degraded polyuridylic acid was demonstrated after incubation with 2-5A. There was no band of degraded polyuridylic acid when the elution was performed either in the absence of oligonucleotide or in the presence of low amounts of a closely related analog of 2-5A, p3I2'pA2'pA. Therefore, the Mr 80,000 2-5A-binding protein and the 2-5A-dependent RNase were almost certainly the same protein. Finally, the Mr 80,000 2-5A-binding protein was purified to homogeneity by electroelution from a polyacrylamide gel.
Silverman
R H
RH
Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
Jung
D D
DD
Nolan-Sorden
N L
NL
Dieffenbach
C W
CW
Kedar
V P
VP
SenGupta
D N
DN
D000818:Animals; D004722:Endoribonucleases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D051379:Mice; D008970:Molecular Weight; D010761:Phosphorus Radioisotopes
D010761:Phosphorus Radioisotopes; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease