Selective inactivation of rabbit reticulocyte initiation factor eIF-3 by helenalin.
Helenalin, a sesquiterpene lactone which reacts primarily with exposed sulfhydryl groups, was shown to be an effective inhibitor of protein synthesis in rabbit reticulocyte lysates. Optimal inhibition required a 30 min preincubation in the absence of any added thiol compound. Beta-Mercaptoethanol was more effective than reduced glutathione in protecting enzyme sulfhydryl groups from inactivation by helenalin. Using partially fractionated systems, it was possible to show that helenalin had no effect on the elongation reactions or on the formation of the ternary initiation complex. However, the conversion of the ternary complex to the 48 S initiation complex was strongly inhibited. In this assay, only the initiation factor(s) were sensitive to helenalin. Using an assay system which requires all the initiation factors for optimal activity it was possible to show that the 0-40% ammonium sulfate cut of initiation factors (containing eIF-3 and eIF-4B) was sensitive to helenalin, while the 40-50% ammonium sulfate cut (containing eIF-2 and eIF-5) was not. Both ammonium sulfate cuts were equally sensitive to inhibition by the sulfhydryl reagent N-ethylmaleimide. Three purified rabbit reticulocyte initiation factors were then tested in the same assay system. Only eIF-3 showed appreciable sensitivity to helenalin, while eIF-2, eIF-3 and eIF-4B were all sensitive to inactivation by N-ethylmaleimide. These data suggest that helenalin may possess a relatively high degree of specificity as a sulfhydryl reagent.
D000818:Animals; D005033:Ethylmaleimide / Q000494:pharmacology; D015852:Eukaryotic Initiation Factor-2; D039621:Eukaryotic Initiation Factor-3; D039642:Eukaryotic Initiation Factors; D005978:Glutathione / Q000032:analysis; D010448:Peptide Initiation Factors / Q000037:antagonists & inhibitors*; D011506:Proteins / Q000037:antagonists & inhibitors; D011817:Rabbits; D012156:Reticulocytes / Q000032:analysis; D012717:Sesquiterpenes / Q000494:pharmacology*; D045789:Sesquiterpenes, Guaiane
D015852:Eukaryotic Initiation Factor-2; D039621:Eukaryotic Initiation Factor-3; D039642:Eukaryotic Initiation Factors; D010448:Peptide Initiation Factors; D011506:Proteins; D012717:Sesquiterpenes; D045789:Sesquiterpenes, Guaiane; C031799:eIF-4B; C001329:helenalin; D005978:Glutathione; D005033:Ethylmaleimide