Functional analysis of 2-5A-dependent RNase and 2-5a using 2',5'-oligoadenylate-cellulose.
2-5A is an intracellular effector that has been implicated in interferon action, hormonal regulation, and cell growth control. 2-5A action is mediated through its activation of 2-5A-dependent RNase (RNase L, RNase F). Affinity resins [2-5A-cellulose and core (2-5A)-cellulose] were chemically synthesized for purification and immobilization of 2-5A-dependent RNase from mouse L cells and rabbit reticulocyte lysates. The breakdown of poly(U)-[3'-32P]Cp to acid-soluble fragments was demonstrated using the 2-5A-dependent RNase:2-5A -cellulose complex; this activity was enhanced by adding (free) 2-5A. In contrast, RNase activity was measured from the 2-5A-dependent RNase:core (2-5A)-cellulose complex only after the addition of free 2-5A. The rabbit reticulocyte 2-5A-dependent RNase is activated only by tetramer or higher oligomers of 2-5A; therefore there was breakdown of poly(U)-[3'-32P]Cp using core (2-5A)-cellulose-bound reticulocyte 2-5A-dependent RNase after addition of tetramer 2-5A but there was no poly(U) degradation in the presence of trimer 2-5A. The absence of significant general nuclease in the assays was demonstrated by the resistance to breakdown of poly(C)-[3'-32P]Cp (not susceptible to 2-5A-dependent RNase). Moreover, core (2-5A)-cellulose was used to develop a sensitive (subnanomolar) assay for the detection of authentic 2-5A. 2-5A, or the material to be tested, was added to mouse L-cell 2-5A-dependent RNase:core (2-5A)-cellulose complex in the presence of poly(U)-[3'-32P]Cp. The concentration of 2-5A in the sample could be measured from the amount of poly(U) degradation. Several closely related analogs of 2-5A were tested and found to be completely inactive. The technology described herein may be applied to the study of the regulation of 2-5A-dependent RNase, the detection of 2-5A from cells and tissues, and other aspects of the 2-5A system.
D000227:Adenine Nucleotides / Q000032:analysis*; D000818:Animals; D002482:Cellulose / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D055598:Chemical Phenomena; D002621:Chemistry; D004722:Endoribonucleases / Q000032:analysis* / Q000302:isolation & purification; D007739:L Cells; D051379:Mice; D009841:Oligonucleotides / Q000032:analysis*; D009843:Oligoribonucleotides / Q000032:analysis*; D010761:Phosphorus Radioisotopes; D011869:Radioligand Assay; D012156:Reticulocytes / Q000201:enzymology
C045415:2',5'-oligoadenylate-cellulose; D000227:Adenine Nucleotides; D009841:Oligonucleotides; D009843:Oligoribonucleotides; D010761:Phosphorus Radioisotopes; C023505:2',5'-oligoadenylate; D002482:Cellulose; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease