Purification and properties of a methionine formylmethionyl-tRNA-binding initiation factor from pig liver

(i) A factor, EIF-2, that binds methionyl-tRNA_f^Met in the presence of GTP has been isolated from pig liver. (ii) Dodecylsulfate-gel electrophoresis and sedimentation equilibrium centrifugation indicate that the factor has a molecular weight of 122,000 and that it consists of three unequal subunits. (iii) The apparent K_D for binding of methionyl-tRNA_f^Met varies with factor concentration. GTP participates in the binding with a K_D of 0.5 μm. β,γ-Methylene-guanosine triphosphate supports 40% of the binding observed with GTP. GDP is a competitive inhibitor with a K_i of 0.2 μm. The optimal, free Mg²⁺ concentration is approximately 50 μm. GTP and Mg²⁺ stabilize the factor against thermal inactivation and inactivation by N-ethyl maleimide. (iv) The factor is required for the formation of a sucrose gradient-stable complex between methionyl-tRNA_f^Met and the 40S ribosomal subunit. The presence of template is not necessary, but poly(A,U,G) increases the binding observed 1.5-fold. (v) The factor markedly stimulates synthesis in a reconstituted protein-synthesizing system with globin messenger RNA as template.