Interactions of Elongation Factor 2 (EF‐2) with Guanine Nucleotides and Ribosomes

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Abstract
Interactions of rat liver elongation factor 2 (EF-2) with guanine nucleotides and ribosomes were studied by equilibrium dialysis and sedimentation methods. GDP (Kd = 0.5 .mu.M) or GDP-Mg2+ (Kd = 1.57 .mu.M) displayed a higher affinity in the formation of a binary complex with EF-2 than GTP (Kd = 2.68 .mu.M), GTP-Mg2+ (Kd = 2.77 .mu.M), or guanosine 5''-[.beta.,.gamma.-methylene]triphosphate (GuoPP[CH2]P) (Kd = 24.0 .mu.M). NaIO4-oxidized guanine nucleotides (oGDP) (Kd = 38 .mu.M) and oxidized/reduced guanine nucleotides (orGDP) (Kd = 27 .mu.M) had lower affinities to the binding site on EF-2 than those of GDP or GTP. The binding of oGDP, oGTP or oGuoPP[CH2]P to EF-2 resulted in the formation of a stable product which could be recovered by the nitrocellulose filter technique or by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. In the presence of ribosomes and EF-2 the formation of a new binding site (or a different conformation of the binding site) with a higher affinity for GuoPP[CH2]P-Mg2+ (Kd = 0.26 .mu.M) than for GDP-Mg2+ (Kd = 9.3 .mu.M) became apparent. The presence of ribosomes thus appeared to favor the formation of a complex involving guanosine triphosphates. ADP ribosylated EF-2 (ADP-Rib-EF-2) in its turn could bind to the ribosome with high affinity even without guanosine nucleotides (Kd = 0.18 .mu.M). GuoPP[CH2]P increased to some extent the affinity of ADP-Rib-EF-2 for its ribosomal binding site (Kd = 0.05 .mu.M).