Mechanisms of degradation of 2'-5' oligoadenylates.
We have studied the mechanisms of breakdown of 2'-5' oligoadenylates. We monitored the time-courses of degradation of ppp(A2'p5')nA (dimer to tetramer) and of 5'OH-(A2'p5')nA (dimer to pentamer) in unfractionated L1210 cell extract. The 5' triphosphorylated 2'-5' oligoadenylates are converted by a phosphatase activity. However, 2'-5' oligoadenylates are degraded mainly by phosphodiesterase activity which splits the 2'-5' phosphodiester bond sequentially at the 2' end to yield 5' AMP and one-unit-shorter oligomers. The nonlinear least-squares curve-fitting program CONSAM was used to fit these kinetics and to determine the degradation rate constant of each oligomer. Trimers and tetramers, whether 5' triphosphorylated or not, are degraded at the same rate, whereas 5' triphosphorylated dimer is rapidly hydrolyzed and 5'-OH dimer is the most stable oligomer. The interaction between degradation enzymes and the substrate strongly depends on the presence of a 5' phosphate group in the vicinity of the phosphodiester bond to be hydrolyzed; indeed, when this 5' phosphate group is present, as in pp/pA2'p5'A/or A2'/p5'A2'p5'A/, affinity is high and maximal velocity is low. Such a degradation pattern can control the concentration of 2'-5' oligoadenylates active on RNAse L either by limiting their synthesis (5' triphosphorylated dimer is the primer necessary for the formation of longer oligomers) and/or by converting them into inhibitory (e.g., monophosphorylated trimer) or inactive (e.g., nonphosphorylated oligomers) molecules.
Trujillo
M A
MA
Centre d'Immunologie INSERM-CNRS de Marseille-Luminy, Marseille, France.
Barbet
J
J
Cailla
H L
HL
D000227:Adenine Nucleotides / Q000378:metabolism*; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D005095:Exoribonucleases / Q000378:metabolism; D005786:Gene Expression Regulation; D006868:Hydrolysis; D007700:Kinetics; D008223:Lymphoma / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D009843:Oligoribonucleotides / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000227:Adenine Nucleotides; D009843:Oligoribonucleotides; C023505:2',5'-oligoadenylate; D005095:Exoribonucleases; C029364:2'-phosphodiesterase