IRREVERSIBLE STEPS IN THE FERRITIN SYNTHESIS INDUCTION PATHWAY

The ability of cells to re-repress ferritin synthesis after removal of an inducing agent (iron or heme) was investigated. Re-repression was found to be a slow process, requiring approximately 4 (after iron remo val) to 10 h (after heme removal) for completion. Desferrioxamine mesy late (Desferal) had only a slight effect on the rate of re-repression, whereas cycloheximide was strongly inhibitory, indicating that new pr otein synthesis is re quired for re-repression. Re-repression occurred at a slow but significant rate in the presence of both Desferal and c ycloheximide. These results indicate that, in the absence of an iron c helator, the induction of ferritin synthesis is essentially irreversib le. The kinetics of the previously reported covalent modification of I RE-binding protein (IRE-BP) were then examined, to see whether this ph enomenon might account (at least in part) for the irreversibility of i nduction. It was found that the heme- or iron dependent disappearance of 98-kDa IRE-BP occurred rapidly (within 1 h), and was equally rapidl y reversed upon removal of heme after a 1-h exposure. By contrast, aft er a 4-h exposure to heme, little 98-kDa IRE-BP could be regenerated a fter heme removal. These results suggest that the slow, irreversible c ovalent modification of IRE-BP correlates closely over time with the i nduction of ferritin synthe sis. The covalent modification of IRE-BP d epends on cell growth rate, and is most readily detected in rapidly gr owing cells.