Activation of the heme-stabilized translational inhibitor of reticulocyte lysates by calcium ions and phospholipid

Abstract

Hemin-supplemented reticulocyte lysates can be activated for translational inhibition by addition of Ca2+ or phospholipid. The fact that this inhibition is prevented or decreased in both cases either by the Ca2+ chelator EGTA or by polymyxin B, an inhibitor of the recently described Ca2+ - and phospholipid-dependent protein kinases, suggests the involvement of both Ca2+ and phospholipid in this activation. The inhibition by Ca2+ or phospholipid is accompanied by phosphorylation of the 38-kilodalton subunit of the eukaryotic initiation factor 2 (eIF-2) and the 90-kilodalton band of the heme-controlled translational inhibitor (HCI) and can be reversed by high concentrations of eIF-2 or GTP. When incubation is conducted at 30°C, the inhibition produced by Ca2+ is not reversed by EGTA after 15 min. However, at 20°C, Ca2+ inhibition can be fully reversed as late as 90 min from the start of incubation and phosphorylation of the eIF-2 α-subunit is correspondingly decreased. These results are consistent with the idea that, like heme deprivation, the activation by Ca2+ and phospholipid promotes the first step of the reaction proinhibitor ⇆ reversible inhibitor → irreversible inhibitor and suggest that, in the presence of hemin albeit by a different mechanism, this activation affects the same inhibitor that is activated in the absence of heme-namely, HCI. Whether this activation is direct or indirect-e.g., via a separate Ca2+-and phospholipid-dependent protein kinase-remains to be determined.