THE EFFECT OF F-ACTIN ON THE BINDING AND HYDROLYSIS OF GUANINE-NUCLEOTIDE BY DICTYOSTELIUM ELONGATION-FACTOR 1A

Indirect evidence implicates actin as a cofactor in eukaryotic protein synthesis. The present study directly examines the effects of F-actin on the biochemical properties of eukaryotic elongation factor 1A (eEF 1A, formerly EF1 alpha), a major actin-binding protein, The basal mech anism of eEF1A alone is determined under physiological conditions with the critical finding that glycerol and guanine nucleotide are require d to prevent protein aggregation and loss of enzymatic activity. The d issociation constants (K-d) for GDP and GTP are 2.5 mu M and 0.6 mu M, respectively, and the k(cat) of GTP hydrolysis is 1.0 x 10(-3) s(-1). When eEF1A binds to F-actin, there is a 7-fold decrease in the affini ty for guanine nucleotide and an increase of 35% in the rate of GTP hy drolysis, Based upon our results and the relevant cellular concentrati ons, the predominant form of cellular eEF1A is calculated to be GTP eE F1A F-actin. We conclude that F-actin does not significantly modulate the basal enzymatic properties of eEF1A; however, actin may still infl uence protein synthesis by sequestering GTP eEF1A away from interactio ns with its known translational ligands, e,g, aminoacyl-tRNA and ribos omes.