2-5A synthetase: assay, distribution and variation with growth or hormone status

Abstract

PROTEIN SYNTHESIS in cell-free systems from interferontreated cells and rabbit reticulocyte lysates is exceptionally sensitive to inhibition by double-stranded RNA (dsRNA)^1,2. Two distinct mechanisms seem to be involved: (1) inactivation of initiation factor eIF2 by dsRNA-mediated phosphorylation and (2) degradation of mRNA by one or more nucleases which are activated by sub-nanomolar levels of an unusual oligonucleotide, pppA2′p5′A2′p5′A (refs 3–10). The enzyme responsible for synthesising pppA2′p5′A2′p5′A requires dsRNA for activity, binds to poly (I)·poly(C)-Sepharose, and can be used conveniently in this insoluble form to synthesise pppA2′p5′A2′p5′A and related oligomers^4,11, collectively referred to as 2-5A. The discovery of relatively high levels of 2-5A synthetase in rabbit reticulocyte lysates¹² raised the possibility that the 2-5A system might be found in other cells which have not been treated with interferon and might, therefore, have a wider significance. We have developed a more convenient assay for the synthetase based on its activity when bound to poly(I)·poly(C)-paper rather than poly(I)·poly(C)-Sepharose and show here that this enzyme is distributed widely in mammalian cells. Moreover, different cells or tissues have widely differing levels of this enzyme and in the case of chick oviducts there is a substantial increase in synthetase level after withdrawal from stimulation by oestrogen.