RESUMEN
Oxazolidinones are potent inhibitors of bacterial protein biosynthesis. Previous studies have demonstrated that this new class of antimicrobial agent blocks translation by inhibiting initiation complex formation, while post-initiation translation by polysomes and poly(U)-dependent translation is not a target for these compounds. We found that oxazolidinones inhibit translation of natural mRNA templates but have no significant effect on poly(A)-dependent translation. Here we show that various oxazolidinones inhibit ribosomal peptidyltransferase activity in the simple reaction of 70 S ribosomes using initiator-tRNA or N-protected CCA-Phe as a P-site substrate and puromycin as an A-site substrate. Steady-state kinetic analysis shows that oxazolidinones display a competitive inhibition pattern with respect to both the P-site and A-site substrates. This is consistent with a rapid equilibrium, ordered mechanism of the peptidyltransferase reaction, wherein binding of the A-site substrate can occur only after complex formation between peptidyltransferase and the P-site substrate. We propose that oxazolidinones inhibit bacterial protein biosynthesis by interfering with the binding of initiator fMet-tRNA(i)(Met) to the ribosomal peptidyltransferase P-site, which is vacant only prior to the formation of the first peptide bond.
Asunto(s)
Oxazolidinonas/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Puromicina/antagonistas & inhibidores , Interacciones Farmacológicas , Escherichia coli/enzimología , Escherichia coli/metabolismo , Cinética , Biosíntesis de Péptidos/efectos de los fármacos , Peptidil Transferasas/antagonistas & inhibidores , Peptidil Transferasas/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genéticaRESUMEN
Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. In a previous study, we reproduced the regulatory features of this operon observed in vivo by using an in vitro S-30 system. We also found that, under inducing conditions, the leader peptidyl-tRNA (TnaC-peptidyl-tRNA(Pro)) is not cleaved; it accumulates in the S-30 reaction mixture. In this paper, we examine the requirements for TnaC-peptidyl-tRNA(Pro) accumulation and cleavage, in vitro. We show that this peptidyl-tRNA remains bound to the translating ribosome. Removal of free tryptophan and addition of release factor 1 or 2 leads to hydrolysis of TnaC-peptidyl-tRNA(Pro) and release of TnaC from the ribosome-mRNA complex. Release factor-mediated cleavage is prevented by the addition of tryptophan. TnaC of the ribosome-bound TnaC-peptidyl-tRNA(Pro) was transferable to puromycin. This transfer was also blocked by tryptophan. Tests with various tryptophan analogs as substitutes for tryptophan revealed the existence of strict structural requirements for tryptophan action. Our findings demonstrate that the addition of tryptophan to ribosomes bearing nascent TnaC-peptidyl-tRNA(Pro) inhibits both TnaC peptidyl-tRNA(Pro) hydrolysis and TnaC peptidyl transfer. The associated translating ribosome therefore remains attached to the leader transcript where it blocks Rho factor binding and subsequent transcription termination.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Operón , Aminoacil-ARN de Transferencia/metabolismo , ARN de Transferencia de Prolina/metabolismo , Triptofanasa/genética , Proteínas Bacterianas/metabolismo , Hidrólisis , Puromicina/antagonistas & inhibidores , Triptófano/fisiologíaRESUMEN
A systematic procedure for the kinetic study of irreversible inhibition when the enzyme is consumed in the reaction which it catalyses, has been developed and analysed. Whereas in most reactions the enzymes are regenerated after each catalytic event and serve as reusable transacting effectors, in the consumed enzymes each catalytic center participates only once and there is no enzyme turnover. A systematic kinetic analysis of irreversible inhibition of these enzyme reactions is presented. Based on the algebraic criteria proposed in this work, it should be possible to evaluate either the mechanism of inhibition (complexing or non-complexing), or the type of inhibition (competitive, non-competitive, uncompetitive, mixed non-competitive). In addition, all kinetic constants involved in each case could be calculated. An experimental application of this analysis is also presented, concerning peptide bond formation in vitro. Using the puromycin reaction, which is a model reaction for the study of peptide bond formation in vitro and which follows the same kinetic law as the enzymes under study, we have found that: (i) the antibiotic spiramycin inhibits the puromycin reaction as a competitive irreversible inhibitor in a one step mechanism with an association rate constant equal to 1.3 x 10(4) M-1 s-1 and, (ii) hydroxylamine inhibits the same reaction as an irreversible non-competitive inhibitor also in a one step mechanism with a rate constant equal to 1.6 x 10(-3) M-1 s-1.
Asunto(s)
Aciltransferasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Hidroxilamina/farmacología , Puromicina/antagonistas & inhibidores , Espiramicina/farmacología , Sitios de Unión , Escherichia coli , Modelos Químicos , Peptidil Transferasas/antagonistas & inhibidores , Inhibidores de la Síntesis de la Proteína , Aminoacil-ARN de Transferencia/metabolismoAsunto(s)
Hígado/efectos de los fármacos , Biosíntesis de Proteínas , Triptófano/farmacología , Amanitinas/antagonistas & inhibidores , Animales , Tetracloruro de Carbono/antagonistas & inhibidores , Dactinomicina/antagonistas & inhibidores , Desoxiadenosinas/antagonistas & inhibidores , Etionina/antagonistas & inhibidores , Femenino , Técnicas In Vitro , Hígado/metabolismo , Microsomas Hepáticos/metabolismo , Puromicina/antagonistas & inhibidores , ARN/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Endogámicas , Esparsomicina/antagonistas & inhibidoresAsunto(s)
Arginina Vasopresina/farmacología , Dipéptidos/farmacología , Memoria/efectos de los fármacos , Recuerdo Mental/efectos de los fármacos , Factores de Terminación de Péptidos/farmacología , Péptidos Cíclicos/farmacología , Puromicina/antagonistas & inhibidores , Animales , Arginina Vasopresina/metabolismo , Encéfalo/metabolismo , Dipéptidos/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Semivida , Masculino , Muridae , Factores de Terminación de Péptidos/metabolismo , Péptidos Cíclicos/metabolismoRESUMEN
The peptide Z-Pro-Leu-Gly-NH2 attenuated puromycin-induced amnesia in mice when administered 5 days prior to training, while arginine vasopressin, lysine vasopressin and cyclo(Leu-Gly), were effective when given 24 hr before training. The activity of all peptides to inhibit puromycin-induced amnesia decreased as the interval after training and before peptide administration increased, suggesting that the peptides influence memory processes rather than generalized arousal mechanisms.
Asunto(s)
Memoria/efectos de los fármacos , Recuerdo Mental/efectos de los fármacos , Puromicina/farmacología , Vasopresinas/farmacología , Animales , Arginina Vasopresina/farmacología , Reacción de Prevención/efectos de los fármacos , Cicloleucina/farmacología , Lipresina/farmacología , Ratones , Péptidos/farmacología , Premedicación , Puromicina/antagonistas & inhibidores , Retención en Psicología/efectos de los fármacosAsunto(s)
Antibacterianos/farmacología , Nucleósidos/farmacología , Ribosomas/enzimología , Transferasas/antagonistas & inhibidores , Aminoglicósidos/farmacología , Proteínas Bacterianas/análisis , Proteínas Bacterianas/biosíntesis , Radioisótopos de Carbono , Sistema Libre de Células , Citosina/análisis , Escherichia coli/enzimología , Isoleucina/metabolismo , Cinética , Péptidos , Fenilalanina/metabolismo , Poli U/biosíntesis , Pseudomonas/enzimología , Puromicina/antagonistas & inhibidores , Puromicina/farmacología , ARN Bacteriano/metabolismo , ARN de Transferencia/metabolismo , Uracilo/metabolismoAsunto(s)
Antibióticos Antineoplásicos/farmacología , Biosíntesis de Proteínas , Aciltransferasas/antagonistas & inhibidores , Aminoácidos/metabolismo , Animales , Antibióticos Antineoplásicos/metabolismo , Bacterias/citología , Bacterias/metabolismo , Radioisótopos de Carbono , Células Cultivadas , Clortetraciclina/metabolismo , Depresión Química , Relación Dosis-Respuesta a Droga , Iniciación de la Cadena Peptídica Traduccional/efectos de los fármacos , Factores de Elongación de Péptidos , Factores de Iniciación de Péptidos , Péptidos , Puromicina/antagonistas & inhibidores , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , ARN Viral/metabolismo , Conejos , Ribosomas/metabolismo , Factores de TiempoRESUMEN
The 12,13-epoxytrichothecenes, a group of sesquiterpenoid fungal antibiotics, inhibit protein synthesis in eukaryotic cells but do not share a common mode of action. Trichodermin stabilizes polyribosomes, prevents their disaggregation by puromycin, and also prevents the release of nascent peptides from ribosomes by puromycin. Nivalenol, T-2 toxin, and verrucarin A cause rapid and almost quantitative breakdown of polyribosomes in H-HeLa cells, a process which is inhibited by anisomycin, cycloheximide, or trichodermin. Similar effects of trichodermin, nivalenol, and verrucarin A are also observed in yeast spheroplasts. We conclude that nivalenol, T-2 toxin, and verrucarin A are potent and highly selective inhibitors of polypeptide chain initiation in eukaryotes, whereas trichodermin inhibits chain elongation and (or) termination. We have compared the structural formulae of various trichothecenes and suggest that the presence of substituents on carbon-15 of the common trichothecene ring may be important in determining the precise modes of action of this group of compounds.
Asunto(s)
Micotoxinas/farmacología , Iniciación de la Cadena Peptídica Traduccional/efectos de los fármacos , Terminación de la Cadena Péptídica Traduccional/efectos de los fármacos , Acremonium/metabolismo , Antibacterianos/farmacología , Benzoxepinas/biosíntesis , Benzoxepinas/farmacología , Compuestos de Bencilo/farmacología , Radioisótopos de Carbono , Cicloheximida/farmacología , Dactinomicina/farmacología , Éteres Cíclicos/biosíntesis , Éteres Cíclicos/farmacología , Femenino , Fusarium/metabolismo , Células HeLa/metabolismo , Humanos , Leucina/metabolismo , Hongos Mitospóricos/metabolismo , Micotoxinas/biosíntesis , Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , Polirribosomas/efectos de los fármacos , Polirribosomas/metabolismo , Biosíntesis de Proteínas , Puromicina/antagonistas & inhibidores , Puromicina/farmacología , Pirrolidinas/farmacología , ARN/biosíntesis , Sesquiterpenos/biosíntesis , Sesquiterpenos/farmacología , Relación Estructura-Actividad , Tritio , Uridina/metabolismoRESUMEN
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 spermidine, 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.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Polirribosomas/metabolismo , Puromicina/farmacología , Ribosomas/efectos de los fármacos , Aminoácidos/metabolismo , Radioisótopos de Carbono , Sistema Libre de Células , Escherichia coli/metabolismo , Ácido Fusídico/farmacología , Cinética , Biosíntesis de Péptidos , Radioisótopos de Fósforo , Puromicina/antagonistas & inhibidores , Ribosomas/metabolismo , Espermidina/farmacología , Tetraciclina/farmacologíaAsunto(s)
Iniciación de la Cadena Peptídica Traduccional , Biosíntesis de Proteínas , Aminoácidos/metabolismo , Sitios de Unión , Escherichia coli/metabolismo , Nucleótidos de Guanina/farmacología , Guanosina Trifosfato/metabolismo , Hidrólisis , Cinética , Metionina/metabolismo , Organofosfonatos/farmacología , Factores de Elongación de Péptidos/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Puromicina/antagonistas & inhibidores , Puromicina/farmacología , ARN Bacteriano/metabolismo , ARN Mensajero/metabolismo , ARN de Transferencia/antagonistas & inhibidores , ARN de Transferencia/metabolismo , Coloración y Etiquetado , Radioisótopos de Azufre , TritioRESUMEN
The incorporation of [(3)H]puromycin into nascent polypeptide chains of polyribosomes has proved to be a sensitive method of evaluating effects of inhibitors on peptide bond synthesis. Several analogues of puromycin were found to react with polyribosomes from both bacteria and rat liver. The K(m) for puromycin is 4 muM with bacterial polyribosomes; under the same conditions, the K(i) for psi-hydroxy-puromycin (6-dimethylamino-9-[3-(l-beta-phenyllactylamino)-3-deoxy-beta- d-ribofuranosyl] purine) is 240 muM and for a carbocyclic analogue of puromycin (6-dimethylamino-9- {R- [2R-hydroxy-3R- (p-methoxyphenyl-l-alanylamino)]-cyclopentyl}purine) is 1 muM. Both were found to be competitive inhibitors of puromycin. The K(m) for C-A-C-C-A(Phe) is 250 muM. In addition, the dissociation constant for C-A-C-C-A(Phe) binding to washed ribosomes was found to be 1 and 0.03 muM in the absence and presence, respectively, of 20% (vol/vol) ethanol. The results with these analogues lead to the following conclusions. Substitution of a hydroxyl group for the alpha-amino group of puromycin results in an active analogue with about one-sixtieth the affinity of puromycin in the reaction. Omission of the 5'-hydroxymethyl group or substitution of the furanosyl ring oxygen by a carbon atom in the carbocyclic analogue reduces its activity compared with puromycin only slightly. Additionally, the relatively high K(m) for C-A-C-C-A(Phe) as an acceptor compared with puromycin suggests the existence of a protective mechanism on polyribosomes, which prevents aminoacyl-transfer ribonucleic acid (tRNA) free in solution from stripping nascent chains from polyribosomes so that only aminoacyl-tRNA bound to ribosomes through the appropriate coding mechanism can form a peptide bond.
Asunto(s)
Biosíntesis de Péptidos , Polirribosomas/metabolismo , Puromicina/farmacología , Animales , Unión Competitiva , Escherichia coli/metabolismo , Técnicas In Vitro , Cinética , Hígado/metabolismo , Puromicina/antagonistas & inhibidores , Puromicina/metabolismo , Ratas , Relación Estructura-Actividad , TritioAsunto(s)
Biosíntesis de Péptidos , Plantas/metabolismo , Propionatos/farmacología , Ribosomas/metabolismo , Compuestos de Anilina/farmacología , Isótopos de Carbono , Centrifugación por Gradiente de Densidad , Ácidos Ciclohexanocarboxílicos/farmacología , Fluoruros/farmacología , Leucina/metabolismo , Sustancias Macromoleculares , Metionina/metabolismo , Nitrocompuestos/farmacología , Iniciación de la Cadena Peptídica Traduccional/efectos de los fármacos , Fenilalanina/metabolismo , Células Vegetales , Plantas/efectos de los fármacos , Polinucleótidos/farmacología , Puromicina/antagonistas & inhibidores , Puromicina/farmacología , ARN de Transferencia/metabolismo , ARN Viral/farmacología , Ribosomas/efectos de los fármacos , Virus del Mosaico del Tabaco , Nucleótidos de Uracilo/farmacologíaRESUMEN
The puromycin-induced blockade of expression of maze learning in mice can be prevented by subcutaneous administration of "Purified Cortrophin Gel" up to 3 days prior to training. A similar protective effect was not found when highly purified corticotropin was tested, but was observed after administration of desglycinamide(9)-lysine vasopressin. It appears that pressorpeptides are more potent protective agents than corticotropin when administered subcutaneously, and that vasopressin contamination of the commercial preparation was probably responsible for the protective effects previously reported. These experiments suggest that vasopressin and its congeners modify memory consolidation in such a way that the "expression" of memory becomes insensitive to puromycin.
Asunto(s)
Hormona Adrenocorticotrópica/farmacología , Memoria/efectos de los fármacos , Puromicina/antagonistas & inhibidores , Vasopresinas/farmacología , Hormona Adrenocorticotrópica/administración & dosificación , Animales , Glicina/farmacología , Inyecciones Subcutáneas , Lipresina/análogos & derivados , Lisina/farmacología , Ratones , Puromicina/farmacología , Factores de TiempoRESUMEN
It has previously been shown that the expression of memory of maze-learning in mice is blocked by intracerebrally-injected puromycin one or more days after the training experience, and that bilateral adrenalectomy before training protects memory against this effect of puromycin. Purified cortrophin gel, injected subcutaneously up to 3 days before training, has now been found to give results like those obtained with adrenalectomy and, additionally, to provide a high degree of protection when injected up to 16 hr after training. Several possible explanations for these effects have been considered and found to be inadequate.