A simplified method for the separation of a kirromycin‐sensitivetufB‐encoded elongation factor Tu (EF‐TuBs) from a kirromycin‐resistanttufA product (EF‐TuAr) was obtained by exploiting the specific increase of positive charges induced by the antibiotic, resulting in a retarded elution of kirromycin‐bound EF‐TuBs on ionic chromatography. The kirromycin‐free EF‐TuBs is active in poly(Phe) synthesis and shows similar properties to EF‐TuAsBs. As expected for these two distinct species, the dissociation of the EF‐TuArBs·GTP complex in the presence of kirromycin shows a biphasic curve; in contrast, a monophasic GTP dissociation rate was found for a combination of two mutated EF‐Tu species. EF‐TuArBo. revealing the existence of intermolecular interactions. These observations prove for the first time the existence of cooperative phenomena between EF‐Tu species in vitro, as suggested earlier by in vivo experiments.
