Binding of periodate-oxidized guanine nucleotides to eukaryotic elongation factor 2.

Periodate-oxidized guanine nucleotides (GTPox and GDPox) were shown to bind stoichiometrically to rat liver elongation factor 2 (EF-2). This binding was quantitatively inhibited in the presence of GTP. After binding, oxidized nucleotides remained on EF-2 despite extensive dialysis. They exchanged, however, with free quanine nucleotides in the course of prolonged (greater than 1 h) incubations. The prior reduction EF-2.GTPox with NaBH4 abolished, to a large extent, this slow exchange. Thus, a Schiff's base was implicated to be formed between EF-2 and oxidized guanine nucleotides. Mg2+ increased the GTPox concentration necessary for a stoichiometric binding to EF-2. EF-2-oxidized nucleotide conjugates bound in the presence of ribosomes a second molecule of GTP (or GTPox). GTPox bound to EF-2 in the presence of ribosomes appeared to exchange readily with free GTP. Moreover, GTPox proved to be active as substrate in EF-2 and ribosome-dependent GTPase reaction: Km values found for GTPox and GTP were 7.7 and 3.4 microM, respectively. The binding of GTPox to EF-2 inhibited only partially the subsequent ribosome-dependent GTP binding, and GTPase reaction or polyphenylalanine (polyPhe) synthesis. On the other hand, the binding of GuoPP[CH2]Pox to EF-2 inhibited all of these reactions strongly. The nature of the binding site involved in the direct interactions of EF-2 with guanine nucleotides is discussed in the light of these results.

On the nature of cellular ADP-ribosyltransferase from rat liver specific for elongation factor 2.

A cellular ADP-ribosyltransferase, specific for elongation factor 2 (EF-2), is found in extracts from rat liver. Co-migrating with EF-2 throughout purification, this activity is, moreover, located in the protein bands corresponding to EF-2 after native or sodium dodecyl sulfate polyacrylamide gel electrophoresis. The observed activity is thus implicated to be an inherent property of EF-2. Preincubation of EF-2 with GuoPPCH2Pox inhibits endogenous, but not diphtheria toxin catalyzed ADP-ribosylation.

Effect of transferrin absence on in vitro globin chain synthesis in reticulocytes of beta-thalassemic patients.

The effect of transferrin absence on in vitro globin chain synthesis and on the alpha/beta ratio were investigated in four patients with beta-thalassemia intermedia and one heterozygote with "silent" beta-thalassemia. With one exception the lack of transferrin in the incubation medium resulted in the reduction of the globin alpha/beta ratio which paralleled a reduction in overall protein synthesis. In one patient with beta-thalassemia intermedia, however, the absence of transferrin failed to affect this ratio.