Activation of the heme-stabilized translational inhibitor of reticulocyte lysates by calcium ions and phospholipid.
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 degrees C, the inhibition produced by Ca2+ is not reversed by EGTA after 15 min. However, at 20 degrees C, Ca2+ inhibition can be fully reversed as late as 90 min from the start of incubation and phosphorylation of the eIF-2 alpha-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 pro-inhibitor in equilibrium reversible inhibitor leads to 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.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D015852:Eukaryotic Initiation Factor-2; D006418:Heme / Q000494:pharmacology*; D007700:Kinetics; D008970:Molecular Weight; D010448:Peptide Initiation Factors / Q000378:metabolism; D010743:Phospholipids / Q000494:pharmacology*; D010766:Phosphorylation; D011112:Polymyxin B / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects*; D011506:Proteins / Q000378:metabolism; D012156:Reticulocytes / Q000187:drug effects / Q000378:metabolism*; D013696:Temperature
D002147:Calmodulin; D015852:Eukaryotic Initiation Factor-2; D010448:Peptide Initiation Factors; D010743:Phospholipids; D011506:Proteins; D006418:Heme; D004533:Egtazic Acid; D011112:Polymyxin B; D002118:Calcium