The exchange of ribosomal subunits during the release of growing polypeptide chains by puromycin has been investigated in a bacterial cell-free system engaged in protein synthesis. The addition of sperinidine, used as a stabilizing agent of 70S monomers, caused a strong inhibition of the subunit exchange. This result led us to conclude that upon premature release of unfinished protein chains by the antibiotic, the ribosomes fall off mRNA as 70S particles. This behavior is different from that occurring during physiological termination of translation, where the ribosomes detach in a dissociated form. Some implications of the postulated mechanism are also discussed.Puromycin is a well-known inhibitor of protein synthesis in vivo as well as in cell-free systems. Its structure, analogous to aminoacyl-tRNA, leads to the premature release of unfinished polypeptide chains as polypeptidyl-puromycin derivatives (1-4).However, the fate of ribosomes upon release of growing protein chains by puromycin is still not well understood. Schlessinger et al. (5) reported that ribosomes engaged in polyphenylalanine synthesis were dissociated into subunits after treatment with puromycin, because the free 30S-50S couples were unstable. More recently Kohler et al. (6) could not confirm these results; they found that 708 particles bearing polyphenylalanyl-tRNA remained intact when peptide chains were liberated by the antibiotic.Both experiments lead to opposite conclusions, but neither of them can be used as a good model of the puromycin reaction at the polysomal level, which constitutes a more physiological system.

Paromomycin, an aminoglycoside antibiotic having low mammalian cell toxicity, is one of the drugs currently used in the chemotherapy of cutaneous and visceral leishmaniasis. In order to understand the mode of action of this antibiotic at the molecular level, we have investigated the effects of paromomycin on protein synthesis in Leishmania and its mammalian hosts. We were able to demonstrate that in vivo protein synthesis in the promastigote stage of the parasite and its proliferation rate are markedly inhibited by paromomycin while being only slightly affected by other aminoglycoside antibiotics, such as streptomycin and neomycin B. Furthermore, both in vitro polypeptide synthesis induced by poly(U) as mRNA and accuracy of translation are significantly decreased by paromomycin in cell-free systems containing ribosomal particles of Leishmania promastigotes. Conversely, when ribosomes from mammalian cells are used instead of the protozoan particles, polyphenylalanine synthesis is only barely reduced by the antibiotic and the translation misreading remains almost unaltered. Surface plasmon resonance analysis of the interaction between paromomycin and protozoan or mammalian cell ribosomal RNAs shows a strong binding of antibiotic to the parasite ribosomal decoding site and practically no interaction with the mammalian cell counterpart. Our results indicating differential effects of paromomycin on the translation processes of the Leishmania parasite and its mammalian hosts can explain the therapeutic efficiency of this antibiotic as an antileishmaniasis agent.

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