Indirect inactivation of rabbit reticulocyte initiation factor eIF-2 by helenalin and bis(helenalinyl) malonate

Helenalin and bis(helenalinyl) malonate, sesquiterpene lactones that react primarily with exposed sulfhydryl groups, were shown to be equally effective inhibitors of endogenous protein synthesis in rabbit reticulocyte lysates. By use of partially fractionated systems, it was possible to show that helenalin preferentially inhibited the conversion of the ternary initiation complex to the 48S preinitiation complex. Previous experiments have shown that this preferential inhibition is due to selective inactivation of eIF-3 [Williams, W. L., Chaney, S. G., Willingham, W., Considine, R. T., Hall, I. H., & Lee, K.-H. (1983) Biochim. Biophys. Acta 740, 152-162]. Bis(helenalinyl) malonate was much less active as an inhibitor of 48S complex formation than helenalin and clearly did not possess sufficient activity in that assay to explain its effectiveness as a protein synthesis inhibitor in whole lysates. Kinetic studies also showed a clear difference between the mechanism of action of these two drugs. Bis(helenalinyl) malonate inactivated protein synthesis in reticulocyte lysates only after a lag of 10 min, and the inhibition of protein synthesis could be completely reversed by the addition of 5 mM cAMP. Helenalin showed more complex kinetics. While full inhibition only occurred after a lag of 10-15 min, a partial inhibition was observed from very early times. cAMP at 5 mM was only partially able to reverse inhibition by helenalin. Phosphorylation studies showed that both helenalin and bis(helenalinyl) malonate were equally effective at activating eIF-2 alpha kinase and indirectly causing phosphorylation of eIF-2.(ABSTRACT TRUNCATED AT 250 WORDS)