Activation of 2-5A-dependent RNase by analogs of 2-5A (5'-O-triphosphoryladenylyl(2'----5')adenylyl(2'----5')adenosine ) using 2',5'-tetraadenylate (core)-cellulose.
A variety of 2-5A (px(A2'p)nA; x = 2 or 3, n greater than or equal to 2) analogs were assayed for their abilities to activate murine 2-5A-dependent RNase (subsequently "the nuclease") using a recently developed method. This technique consists of immobilizing and partially purifying the nuclease using core-cellulose [A2'p)3A-cellulose) and then monitoring the breakdown of poly(U)-3'-[32P]Cp into acid-soluble fragments. Several 5'-adenosinecapped analogs of 2-5A (containing a tetra-, tri-, or diphosphate) were analyzed, and it was found that reducing the number of phosphoryl groups between the 5' to 5'-diadenosine linkages resulted in a progressive loss of activity. Because A5' pppp(A2'p)3A was a potent activator of the nuclease yet stable during the assay these results suggested that a free 5'-phosphoryl group may not be required for the activation of the nuclease. A number of 8-bromoadenosine-substituted analogs of 2-5A were also studied. Curiously, the brominations decreased the activities of the 5'-di- and triphosphorylated molecules while substantially increasing the activities of the 5'-monophosphorylated species. The results indicated that a tri- or diphosphate moiety on the 5'-end of 2-5A or the presence of ATP is not absolutely required for the nuclease to be active. Furthermore, the ATP analog, beta, gamma-methylene ATP, did not inhibit the activity of the nuclease. Finally, a 3',5'-phosphodiester linkage isomer of 2-5A and a 3'-deoxy (cordycepin) analog of 2-5A were tested, and both were found to be completely without activity.
Krause
D
D
Lesiak
K
K
Imai
J
J
Sawai
H
H
Torrence
P F
PF
Silverman
R H
RH
D000227:Adenine Nucleotides / Q000494:pharmacology*; D000818:Animals; D002482:Cellulose / Q000031:analogs & derivatives; D002846:Chromatography, Affinity; D004722:Endoribonucleases / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D007739:L Cells / Q000201:enzymology; D051379:Mice; D009843:Oligoribonucleotides / Q000494:pharmacology*; D013329:Structure-Activity Relationship
C045415:2',5'-oligoadenylate-cellulose; D000227:Adenine Nucleotides; D009843:Oligoribonucleotides; C023505:2',5'-oligoadenylate; D002482:Cellulose; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease