EFFECT OF OXIDIZING-AGENTS AND HEMIN ON THE PHOSPHORYLATION OF EUKARYOTIC ELONGATION-FACTOR-2 IN RABBIT RETICULOCYTE LYSATES

Incubation of rabbit reticulocyte lysates in the absence of added haem in resulted in the phosphorylation of a 95 kDa protein. This protein w as suggested to be elongation factor 2 (eEF-2) based on the following observations, (i) phosphorylation of the 95 kDa protein was Ca2+ and C aM-dependent. (ii) eEF-2 supplemented to the lysates became phosphoryl ated and co-migrated with the endogenous 95 kDa phosphoprotein upon el ectrophoresis in SDS gels. (iii) The tryptophane specific cleavage pat tern obtained from the isolated 95 kDa phosphoprotein was identical to that of phosphorylated eEF-2. Phosphorylation of the 95 kDa protein w as stimulated by oxidizing agents such as oxidized glutathione and NAD (+) and inhibited by addition of haemin. The haemin concentration need ed for 50% inhibition (IC50) was 2.5 mu M. Haemin also had an inhibito ry effect on eEF-2 phosphorylation in a system containing highly purif ied components (IC50 = 2 mu M). In this system haemin inhibited phosph orylation of eEF-2 even in the presence of a 100-fold excess of beta-m ercaptoethanol, Oxidizing agents had no effect on the kinase activity in the purified system.