J. Biochem, 1974, Vol. 76, No. 6 1269-1280
© 1974 Japanese Biochemical Society
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Studies on Polypeptide Elongation Factor 2 from Pig Liver
III. Interaction with Guanine Nucleotides in the Presence and Absence of Ribosomes
Institute of Medical Science, University of Tokyo Takanawa, Minato-ku, Tokyo 108
The interaction of pig liver EF-2 with guanine nucleotides has been investigated by several methods in the absence and presence of ribosomes.
When the formation of EF-2 guanine nucleotide complex was studied by the nitrocellulose membrane technique, only EF-2·[3H]GDP was detected, with a Kd value of 1.4×10–6M. Although binary complexes between EF-2 and other guanine nucleotides could not be directly demonstrated by this method, the presence of EF-2·GTP and EF-2·Gpp(CH2)p was suggested by several experiments. Direct evidence for the formation of EF-2·[3H]GDP as well as EF-2·[3H]/[
-32P]GTP was obtained by gel filtration of EF-2 on a Sephadex G-50 column equilibrated with a buffer containing labeled GDP or GTP. From Scatchard plots of the values obtained in these experiments, the Kd values for GDP and GTP were estimated as 4.1×10–7 and 1.4×10–5M, respectively. The number of binding sites for these nucleotides was one per EF-2 molecule.
Studies on the formation of the ribosome·EF-2·guanine nucleotide complex by the nitrocellulose membrane technique revealed that [3H]GTP, [3H]GDP, and [3H]Gpp(CH2)p were bound to the membrane to almost the same extent in the presence of both ribosomes and EF-2. However, when [-32P]GTP was used as a labeled nucleotide no radioactivity was retained on the membrane, indicating that the ribosome·EF-2·GTP complex formed was rapidly converted to ribosome·EF-2·GDP complex with a release of inorganic phosphate. The formation of the ternary complex was directly demonstrated by ultracentrifugation. The complex isolated by ultra centrifugation was found to contain ribosomes, EF-2 and the labeled nucleotide in a molar ratio of 1 : 1: 1. Although the binding of EF-2 to ribosomes occurred in the absence of guanine nucleotides, this interaction was found to be non-specific. Finally, [3H]GDP in the ternary complex was readily exchanged with unlabeled GTP, GDP, or Gpp(CH2) whereas [3H]Gpp(CH2)p in the complex was not exchanged with any of the unlabeled guanine nucleotides even at 37°.
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