The in vitro binding of virginiamycin M to bacterial ribosomes and ribosomal subunits

Summary Virginiamycin M (VM), an antibiotic of type A synergimycin group of antibiotics, binds to bacterial ribosomes and subunits in vitro: the amount of linked drug is linearly dependent on ribosome and VM concentrations. The technique used to measure the association reaction is based on the finding that the unbound drug is adsorbed by norite A: this procedure is twice as sensitive as the sedimentation and filtration methods (for technical reasons, column chromatography and equilibrium dialysis are unsuitable for this study). Saturation curves with 70S and 50S particles overlap, thus indicating a comparable affinity of the inhibitor for ribosomes and large subunits; instead, very small amount of VM, if any, attaches to 30S particles. Kinetics of binding is influenced by the temperature; the 4° C and 25° C saturation curves overlap, however, upon pre-incubation of ribosomes in 10 mM Mg buffer at 37° C (reactivation). This suggests that binding of VM depends on the configura tion of the 50S particles, which is altered at low temperature. Differences in Mg⁺⁺ concentration in the range 1 to 20 mM do not modify the binding curve, nor does the replacement of K⁺ by either NH ₄ ⁺ or Na⁺. Previously bound labelled VM is slowly displaced by an excess of unlabeled VM, and the associa tion curve remains unchanged in the presence of VS. Binding of VM is inhibited (10 to 60%) in the presence of an excess (tenfold to hundredfold) of one of the 50S inhibitors: chloramphenicol, oleandomycin and erythromycin. From the Scatchard plot, an as sociation constant of 3.2 × 10⁵M^–1 has been calculated: this value is about 1/8 of that reported for VS, a component of type B synergimycin group of antibiotics. The v value is 0.85 for both ribosomes and large subunits, indicating a monomolecular association of VM with ribonucleoprotein particles.