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 phospholipiddependent 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 a-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.While isolating a heat-stable factor (HS) from reticulocyte lysates that, like a factor described from bovine heart (1), inhibits translation in hemin-supplemented lysates and promotes phosphorylation of the eukaryotic initiation factor 2 (eIF-2) a-subunit, we used Ca2" for a negative control. Ca2" is known to inhibit protein synthesis, but we did not expect this metal ion to promote eIF-2 a-subunit phosphorylation. However, much to our surprise, it did so. It also clearly promoted the phosphorylation of a polypeptide that comigrates with the 90-kilodalton (kDa) band of the heme-controlled translational inhibitor (HCI) on electrophoresis in dissociating polyacrylamide gels (2). At 30°C, translational inhibition by Ca2+ was not reversed by the Ca2+ chelator EGTA after 15 min of incubation, but at 20°C, the inhibition was fully reversed by EGTA, even after incubation for 90 min, and phosphorylation of the eIF-2 a-subunit was correspondingly decreased (2). Other metal ions, including Mg2 , Mn2 , Cd2 , Co2+, and Cu2+, inhibited the hemin-dependent translation but of these only Co2+ promoted significant phosphorylation of the eIF-2 a-subunit. The effect of Co2+ has not been further investigated.We first thought that the Ca2+-promoted activation of HCI might be mediated by calmodulin. This hypothesis had some support in the fact that reticulocyte HS, unlike the one from bovine heart (1), resembles calmodulin in molecular weight and other properties: However, calmodulin antibody had no effect on the inhibition produced by Ca2", and auth...

Primary structure and inhibition of protein synthesis in eukaryotic cell‐free system of a novel thionin, γ‐hordothionin, from barley endosperm

“…To assay for eIF-2u kinase on exogenous eIF-2, lysates were incubated as for protein synthesis; aliquots of each reaction mixture were then taken and incubated at 30 "C for 5 min with eIF-2 in the presence of [Y-~~P]ATP and MgClz as described [27]. Initiation factor eIF-2 from reticulocyte lysates was purified as described [28].…”

Mechanism of activation of the heme-stabilized translational inhibitor of reticulocyte lysates by calcium ions and phospholipid.

“…After incubation for 5 min at 30°C, histone phosphorylation was assayed as described (11). EGTA, L-a-Ptd L-Ser, and diolein were the same preparations used earlier (7). The preparation of PtdSer/diolein mixtures and the methods used for determination of PtdSer and protein concentrations have also been described (7).…”

“…Rabbit reticulocyte lysates and translation and eIF-2 phosphorylation assays were as described (7). PKC activity was assayed by the procedure of Wise et al (9) with slight modifications.…”

Purification of a novel heat‐stable translational inhibitor from rabbit reticulocyte lysates

“…Translation and eIF-2, phosphorylation assays were as described [4,7]. Treatments of reticulocyte HS with proteases or nucleases was performed under optimal conditions as follows.…”

“…There are at least two CAMP-independent eIF-2, kinases in rabbit reticulocyte lysates. One, the hemecontrolled inhibitor is activated from a precursor when lysates are incubated in the absence of added hemin or, with hemin present, upon addition of either oxidized glutathione (GSSG) [3] or Ca2+-phospholipid [4]; the other, the doublestranded RNA (dsRNA)-activated inhibitor, is activated by low concentrations of dsRNA in the Once activated, HCI and DA1 inhibit translation in reticulocyte lysate in the presence of heme, but the mechanism of activation remains obscure [ 1,2].…”