Interaction of guanosine nucleotides with elongation factor 2. I. Equilibrium dialysis studies.
Binding of the guanosine nucleotides, GDP and GTP, to elongation factor 2 (EF-2) from rat liver was studied by equilibrium dialysis. It was found that the enzyme has one binding site for GDP with a dissociation constant of 4 times 10--7 M. The examination of GTP binding was difficult due to the simultaneous presence of GDP and GTP even in purified GTP preparations. This problem was further magnified by traces of GTPase in the enzyme preparation. However, by analyzing the incubation mixtures by thin layer chromatography the fraction of the total nucleotide binding to EF-2 which was due to GDP could be determined and corrected for. A GTP binding curve, corrected for GDP binding, and GTP hydrolysis extrapolated to one binding site with a dissociation constant of approximately 2 times 10--6 M. The nonhydrolyzable GTP analogue, theta, gamma-methylene-guanosine-5-triphosphate, also bound to EF-2 in a 1:1 ratio. During the studies of GTP binding to EF-2 it was observed that the enzyme preparation contained a GTP-GDP transphosphorylase activity. It was initially thought that this was a novel property of EF-2, but when the activity was followed during purification of EF-2 it was whown that it was an impurity in the EF-2 preparation. ATP as well as GTP can serve as a phosphate donor in the transphosphorylation reaction; this might suggest that regeneration of GTP from GDP can take place via this pathway.
D000818:Animals; D001665:Binding Sites; D003956:Dialysis; D004589:Electrophoresis, Disc; D006150:Guanine Nucleotides / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007525:Isoelectric Focusing; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008433:Mathematics; D010445:Peptide Elongation Factors / Q000302:isolation & purification; D010770:Phosphotransferases / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D012270:Ribosomes / Q000378:metabolism
D006150:Guanine Nucleotides; D010445:Peptide Elongation Factors; D006160:Guanosine Triphosphate; D010770:Phosphotransferases