Complex C [ribosome-poly(U)-AcPhe-tRNA] was formed in 400 ml of binding buffer (80 mM Tris-HCl, pH 7.4, 160 mM ammonium chloride, 11 mM magnesium acetate, 2 mM spermidine and 6 mM b-mercaptoethanol) containing 0.4 mM GTP, 500 pmol ribosomes, 0.4 mg/ml poly(U) and 700 pmol Ac-[ 14 C]Phe-tRNA. Mixtures were incubated for 20 min at 30 C and then placed on ice. Complexes were purified from free Ac-[ 14 C]Phe-tRNA Phe by centrifugation through a glycerol cushion (0.5 ml; 20% glycerol in binding buffer by centrifugation at 50 000 rpm for 2 h in MLS 50 rotor). Ribosome pellets were rinsed twice with 1 ml of binding buffer and suspended in 1.15 ml of binding buffer. For puromycin reactions, 1.15 ml of complex C extract was pre-incubated at 30 C for 5 min, and reactions were initiated by adding pH neutralized puromycin (100 mM stock) to final concentrations of 10 mM. Aliquots of 100 ml were removed, and reactions were terminated at the indicated time intervals by addition of 100 ml of 1.0 N NaOH. Reaction products were extracted with 0.4 ml of ethyl acetate, 0.2 ml of organic phase was transferred to scintillation vials, and radioactivity was determined by scintillation counting. A 50-ml aliquot of initial reaction mixture was also transferred to scintillation vials, and total radioactivity (N o ) was determined. Controls without puromycin were included in each experiment, and the values obtained were subtracted as background. The percent of the bound Ac-[ 14 C]Phe-tRNA Phe converted to Ac-[ 14 C]Phe-puromycin was corrected with the extent factor a (determined if complex C were allowed to react for 1 h; C o = aN o ), as described earlier (27, 28) . The reaction plots were fit to a first-order exponential equation, and values of K obs (the apparent rate constant of entire course of reaction at a given concentration of puromycin) were calculated by using Graphpad Prism software.