69-KDA AND 100-KDA ISOFORMS OF INTERFERON-INDUCED (2'-5')OLIGOADENYLATE SYNTHETASE EXHIBIT DIFFERENTIAL CATALYTIC PARAMETERS

The (2'-5')oligoadenylate synthetase represents a family of interferon -induced proteins which when activated by double-stranded (ds)RNA poly merizes ATP into 2'-5'-linked oligomers with the general formula pppA( 2'p5'A)(n), where n>1, which for convenience are referred to as 2-5A. We studied here the influence of pH, dsRNA concentration and time on o ligomeric composition of 2-5A synthesized by purified 69-kDa and 100-k Da isoforms of (2'-5')oligo(adenylate) synthetase. In optimal conditio ns for activity, the 69-kDa form synthesized higher oligomers of 2-5A molecules whereas the 100 kDa form synthesized preferentially dimeric molecules, which are known not to be functional for the activation of RNase L. This difference does not reflect a differential affinity of t he enzymes for the preformed 2-5A dimer, which is found to be a very p oor substrate for both enzymes. This latter strongly suggests that the mechanism of elongation is more likely processive. Moreover, we show that both isoforms have efficient nucleotidyl-transferase activity and provide evidence that, in optimized conditions, GTP can be used alone as substrate by these enzymes to generate pppG2'p5'G. Our results cle arly demonstrate that the 69-kDa and 100-kDa forms of (2'-5')oligoaden ylate synthetase manifest various differential catalytic activities, a nd favor the hypothesis that these enzymes might have other functions in the cell besides those in the 2-5A system.