The Interaction of Elongation Factor 2 with Ribosomes from Silk Gland<subtitle>Formation of an EF-2⋅Ribosome⋅GDP Complex</subtitle>

Abstract

Silk gland EF-2 was found to be bound to ribosomes only in the presence of guanosine nucleotides, such as GTP, GDP or GMPPCP. Incubation of EF-2, ribosomes, and [3H]GTP resulted in the formation of a high-molecular-weight [3H]GTP complex, which was retained on nitrocellulose filters and could be isolated by gel filtration. Most of the guanosine nucleotide present in this complex was recovered in the form of GDP. The formation of the EF-2⋅ribosome⋅GDP complex was investigated. The reaction was very rapid and was completed within 5 sec at 0°. [3H]GDP could substitute for [3H]GTP in the formation of this complex. The amount of the EF-2⋅ribosome⋅GDP complex formed was stoichiometrically the same as that of the GTP hydrolysis concomitant with complex formation. In the case of the Escherichia coli system, the complex formation was stimulated 2-fold by the addition of fusidic acid (1 mM), while in the case of the silk gland system, such stimulation of complex formation was not observed. The [3H]GDP bound to the complex was exchangeable with unlabelled GTP or GDP at 37°, but not at 0°. Fusidic acid inhibited this exchange reaction. These results suggested that the hydrolysis of GTP by EF-2 and ribosomes proceeded through the intermediate formation of an EF-2⋅ribosome⋅GDP complex.