The intermolecular complex of erythromycin and ribosome

Abstract

The binding of erythromycin A to ribosomes was studied by four different methods: sucrose gradient centrifugation, membrane filtration, ultrafiltration and equilibrium dialysis. All the methods consistently showed that erythromycin A is bound specifically to 50 s ribosomal subunits in a ratio of one molecule per ribosome. The binding requires either NH 4 + or K + ions; Na + ions cannot substitute for them. Mg 2+ ions slightly inhibit the binding. The rate of binding of erythromycin A increases with an increase in temperature for 0 to 37 °C but the extent does not. The association constant of the erythromycin-ribosome complex, determined by membrane filtration and equilibrium dialysis, is 4.7 × 10 5 M −1 and 2.8 × 10 5 M −1, respectively. Although the erythromycin-ribosome complex is very stable, a covalent bond is not involved since the complex is dissociable by dialysis, and erythromycin A can be extracted from the complex by organic solvents. From studies of the inhibition of erythromycin A binding by various derivatives, it was found that the 2′-, 11-, 12-hydroxyl, 3′-dimethylamino, 9-keto and 3′'-methoxy groups are important for the formation of the complex. Ribosomes treated with p-chloromercuribenzoate, dinitrofluorobenzene or nitrogen mustard could still bind erythromycin. However, those treated with formaldehyde or perphthalic acid were unable to bind erythromycin. These results suggest that the nucleotide bases in ribosomal RNA are essential for binding while no specific residues on the protein have been implicated. Nevertheless protein is essential for the erythromycin binding since 42 s ribonucleoprotein particles isolated from the 50 s subunit of ribosomes have no binding capacity. It is proposed that those essential groups of erythromycin form hydrogen bonds with primary amino groups and the N(1) of nucleotide bases. Protein stabilizes the complex by forming hydrophobic bonds with the hydrophobic region of erythromycin and by holding ribosomal RNA in a geometry favorable to the formation of the complex.