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.
Bilgin
N
N
Biyofizik Anabilim Dali, I.U. Istanbul Tip Fakültesi, Capa, Turkey.
Sayhan
O
O
Bermek
E
E
D000818:Animals; D006150:Guanine Nucleotides / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D010084:Oxidation-Reduction; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010504:Periodic Acid; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D012270:Ribosomes / Q000378:metabolism
D006150:Guanine Nucleotides; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D010750:Phosphoproteins; D010504:Periodic Acid; D008274:Magnesium