Synthesis of (2'-5')(A)n from ATP. Characteristics of the reaction catalyzed by (2'-5')(A)n synthetase purified from mouse Ehrlich ascites tumor cells treated with interferon.
The treatment of Ehrlich ascites tumor cells with mouse interferon increases the level of the latent enzyme (2'-5')(A)n synthetase. If activated by double-stranded RNA, this catalyzes the synthesis from ATP of a series of 2'-5'-oligoadenylates: (2'-5')(A)n where n extends from 2 to about 15. We isolated (2'-5')(A)n synthetase in a homogeneous state. In the presence of double-stranded RNA, the purified enzyme can convert the large majority (about 97%) of the ATP into (2'-5')(A)n and pyrophosphate, although it does not cleave the pyrophosphate. The stoichiometry of the reaction can be formulated as: (n + I) ATP leads to (2'-5') pppA(pA)n + n pyrophosphate. Added pyrophosphate does not inhibit the synthesis of (2'-5')(A)n. The extent of the reverse reaction, i.e. the pyrophosphorolysis of (2'-5')(A)n, was below the level of detection under our conditions. The affinity of the enzyme for ATP is low: the rate of the reaction increases by about 10% when the concentration of ATP is increased from 5 mM to 10 mM. The optimal concentration of double-stranded RNA increases with the concentration of the enzyme. As tested at 0.4, 2, and 10 micrograms/ml of enzyme concentrations, close to maximal (2'-5')(A)n synthesis can be obtained if reovirus double-stranded RNA or poly(I) . poly(C) are used at about half the concentration (in w/v) of the enzyme. The plot of the reaction rate versus enzyme concentration is sigmoidal. It remains to be seen if this reflects on a cooperative behavior of the enzyme.
D015088:2',5'-Oligoadenylate Synthetase; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism*; D011756:Diphosphates / Q000494:pharmacology; D007372:Interferons / Q000494:pharmacology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D051379:Mice; D011061:Poly A / Q000096:biosynthesis*; D011118:Polynucleotide Ligases / Q000302:isolation & purification / Q000378:metabolism*
D011756:Diphosphates; D011061:Poly A; D000255:Adenosine Triphosphate; D007372:Interferons; D015088:2',5'-Oligoadenylate Synthetase; D011118:Polynucleotide Ligases; D008274:Magnesium