Intrinsic uncoupling in the ATP synthase of Escherichia coli. Studies on WT and ε-truncated mutants

The H+/ATP ratio in the catalysis of ATP synthase has generally been considered a fixed parameter. However, Melandri and coworkers have recently shown that, in the ATP synthase of the photosynthetic bacterium Rb.capsulatus, this ratio can significantly decrease during ATP hydrolysis when the concentration of either ADP or Pi is maintained at a low level (Turina et al., 2004). The present work has dealt with the ATP synthase of E.coli, looking for evidence of this phenomenon of intrinsic uncoupling in this organism as well. First of all, we have shown that the DCCD-sensitive ATP hydrolysis activity of E.coli internal membranes was strongly inhibited by ADP and Pi, with a half-maximal effect in the submicromolar range for ADP and at 140 µM for Pi. In contrast to this monotonic inhibition, however, the proton pumping activity of the enzyme, as estimated under the same conditions by the fluorescence quenching of the ΔpH-sensitive probe ACMA, showed a clearly biphasic progression, both for Pi, increasing from 0 up to approximately 200 µM, and for ADP, increasing from 0 up to a few µM. We have interpreted these results as indicating that the occupancy of ADP and Pi binding sites shifts the enzyme from a partially uncoupled state to a fully coupled state, and we expect that the ADP- and Pi-modulated intrinsic uncoupling is likely to be a general feature of prokaryotic ATP synthases. Moreover, the biphasicity of the proton pumping data suggested that two Pi binding sites are involved. In order to verify whether the same behaviour could be observed in the isolated enzyme, we have purified the ATP synthase of E.coli and reconstituted it into liposomes. Similarly as observed in the internal membrane preparation, in the isolated and reconstituted enzyme it was possible to observe inhibition of the hydrolytic activity by ADP and Pi (with half-maximal effects at few µM for ADP and at 400 µM for Pi) with a concomitant stimulation of proton pumping. Both the inhibition of ATP hydrolysis and the stimulation of proton pumping as a function of Pi were lost upon ADP removal by an ADP trap. These data have made it possible to conclude that the results obtained in E.coli internal membranes are not due to the artefactual interference of enzymatic activities other than the ones of the ATP synthase. In addition, data obtained with liposomes have allowed a calibration of the ACMA signal by ΔpH transitions of known extent, leading to a quantitative evaluation of the proton pumping data. Finally, we have focused our efforts on searching for a possible structural candidate involved in the phenomenon of intrinsic uncoupling. The ε-subunit of the ATP-synthase is known as an endogenous inhibitor of the hydrolysis activity of the complex and appears to undergo drastic conformational changes between a non-inhibitory form (down-state) and an inhibitory form (up-state)(Rodgers & Wilce, 2000; Gibbons et al., 2000). In addition, the results of Cipriano & Dunn (2006) indicated that the C-terminal domain of this subunit played an important role in the coupling mechanism of the pump, and those of Capaldi et al. (2001), Suzuki et al. (2003) were consistent with the down-state showing a higher hydrolysis-to-synthesis ratio than the up-state. Therefore, we decided to search for modulation of pumping efficiency in a C-terminally truncated ε mutant. A low copy number expression vector has been built, carrying an extra copy of uncC, with the aim of generating an ε-overexpressing E.coli strain in which normal levels of assembly of the mutated ATP-synthase complex would be promoted. We have then compared the ATP hydrolysis and the proton pumping activity in membranes prepared from these ε-overexpressing E.coli strains, which carried either the WT ε subunit or the ε88-stop truncated form. Both strains yielded well energized membranes. Noticeably, they showed a marked difference in the inhibition of hydrolysis by Pi, this effect being largely lost in the truncated mutant. However, pre-incubation of the mutated enzyme with ADP at low nanomolar concentrations (apparent Kd = 0.7nM) restored the hydrolysis inhibition, together with the modulation of intrinsic uncoupling by Pi, indicating that, contrary to wild-type, during membrane preparation the truncated mutant had lost the ADP bound at this high-affinity site, evidently due to a lower affinity (and/or higher release) for ADP of the mutant relative to wild type. Therefore, one of the effects of the C-terminal domain of ε appears to be to modulate the affinity of at least one of the binding sites for ADP. The lack of this domain does not appear so much to influence the modulability of coupling efficiency, but instead the extent of this modulation. At higher preincubated ADP concentrations (apparent Kd = 117nM), the only observed effects were inhibition of both hydrolysis and synthesis, providing a direct proof that two ADP-binding sites on the enzyme are involved in the inhibition of hydrolysis, of which only the one at higher affinity also modulates the coupling efficiency.

Fra il nome e la storia. Trasformazioni del discorso politico e concetto di classe al principio della monarchia di Luglio (1831-1832)

La ricerca indaga tensioni e trasformazioni che investono le principali correnti di pensiero politico in Francia nei primi anni della monarchia di Luglio, e vi osserva l’emergere del concetto di classe. Assumendo la dimensione dell’avvenimento come punto di intersezione fra storia e teoria, l’elaborato si concentra sul periodo novembre 1831-giugno 1832 per analizzare il modo in cui, nell’ordine del discorso politico repubblicano, liberale e socialista, le vicende di questi mesi vengono interpretate cercando di dar nome alle figure sociali che esse fanno irrompere nel dibattito pubblico. Il titolo Fra il nome e la storia fa dunque riferimento allo sforzo di indagare il campo di tensione che si apre fra concreto divenire storico e grandi operazioni di nominazione che segnano l’affiorare di strutture concettuali della lunga durata. L’emergere della nozione di classe operaia e delle categorie che intorno a essa si organizzano viene interpretata come una «formazione discorsiva» che pone in questione significato e confini del politico. La frattura del 1848 è assunta come orizzonte e margine esterno della ricerca nella misura in cui si ipotizza che essa segni una prima affermazione del regime di verità di tale formazione discorsiva: lo statuto politico del lavoro. L’elaborato consta di quattro capitoli. I primi tre indagano la riflessione sul politico e la funzione che in essa svolge il concetto di classe a partire dall’interpretazione di alcuni avvenimenti del tornante 1831-32 proposta nel discorso repubblicano del quotidiano «Le National» e della Société des Amis du Peuple, in quello del liberalismo dottrinario di François Guizot e in quello socialista nascente, prima del movimento sansimoniano, e poi muovendo fino al 1848 francese con l’analisi propostane da Karl Marx. Il quarto capitolo indaga infine la dimensione del «sociale», la sua elaborazione e articolazione attraverso il lavoro di studio e oggettivazione delle figure del mondo del lavoro.