Effect of oxidizing agents and haemin on the phosphorylation of eukaryotic elongation factor 2 in rabbit reticulocyte...

Incubation of rabbit reticulocyte lysates in the absence of added haemin resulted in the phosphorylation of a 95 kDa protein. This protein was suggested to be elongation factor 2 (eEF-2) based on the following observations, (i) phosphorylation of the 95 kDa protein was Ca2+ and CaM-dependent. (ii) eEF-2 supplemented to the lysates became phosphorylated and co-migrated with the endogenous 95 kDa phosphoprotein upon electrophoresis in SDS gels. (iii) The tryptophane specific cleavage pattern obtained from the isolated 95 kDa phosphoprotein was identical to that of phosphorylated eEF-2. Phosphorylation of the 95 kDa protein was stimulated by oxidizing agents such as oxidized glutathione and NAD+ and inhibited by addition of haemin. The haemin concentration needed for 50% inhibition (IC50) was 2.5 microM. Haemin also had an inhibitory effect on eEF-2 phosphorylation in a system containing highly purified components (IC50 = 2 microM). In this system haemin inhibited phosphorylation of eEF-2 even in the presence of a 100-fold excess of beta-mercaptoethanol. Oxidizing agents had no effect on the kinase activity in the purified system.