RESUMO
The F(1)F(O) and F(1)-ATPase complexes of Paracoccus denitrificans were isolated for the first time by ion exchange, gel filtration, and density gradient centrifugation into functional native preparations. The liposome-reconstituted holoenzyme preserves its tight coupling between F(1) and F(O) sectors, as evidenced by its high sensitivity to the F(O) inhibitors venturicidin and diciclohexylcarbodiimide. Comparison and N-terminal sequencing of the band profile in SDS-PAGE of the F(1) and F(1)F(O) preparations showed a novel 11-kDa protein in addition to the 5 canonical alpha, beta, gamma, delta, and epsilon subunits present in all known F(1)-ATPase complexes. BN-PAGE followed by 2D-SDS-PAGE confirmed the presence of this 11-kDa protein bound to the native F(1)F(O)-ATP synthase of P. denitrificans, as it was observed after being isolated. The recombinant 11 kDa and epsilon subunits of P. denitrificans were cloned, overexpressed, isolated, and reconstituted in particulate F(1)F(O) and soluble F(1)-ATPase complexes. The 11-kDa protein, but not the epsilon subunit, inhibited the F(1)F(O) and F(1)-ATPase activities of P. denitrificans. The 11-kDa protein was also found in Rhodobacter sphaeroides associated to its native F(1)F(O)-ATPase. Taken together, the data unveil a novel inhibitory mechanism exerted by this 11-kDa protein on the F(1)F(O)-ATPase nanomotor of P. denitrificans and closely related alpha-proteobacteria.
Assuntos
Inibidores Enzimáticos/isolamento & purificação , Subunidades Proteicas/metabolismo , ATPases Translocadoras de Prótons/química , Inibidores Enzimáticos/metabolismo , Peso Molecular , Paracoccus denitrificans/enzimologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/isolamento & purificação , Rhodobacter sphaeroides/enzimologiaRESUMO
Amphipterygium adstringens (Schltdl.) Standl. (Anacardiaceae) is widely used in traditional Mexican medicine for the treatment of gastritis and ulcers. In this work, we studied the anti-Helicobacter pylori activity of its bark, this Gram-negative bacterium is considered the major etiological agent of chronic active gastritis and peptic ulcer disease, and it is linked to gastric carcinoma. From a bio-guided assay of the fractions obtained form a continuous Soxhlet extraction of the bark, we identified that petroleum ether fraction had significant antimicrobial activity against Helicobacter pylori. From this fraction, we isolated an anacardic acids mixture and three known triterpenes: masticadienonic acid; 3alpha-hydroxymasticadienonic acid; 3-epi-oleanolic; as well as the sterol beta-sitosterol. Only the anacardic acids mixture exhibits a potent dose-dependent antibacterial activity (MIC=10 microg/ml in broth cultures). It is enriched in saturated alkyl phenolic acids (C15:0, C16:0, C17:0 C19:0) which represents a novel source of these compounds with potent anti-Helicobacter pylori activity. The promising use of anacardic acids and Amphipterygium adstringens bark in the development of an integral treatment of Helicobacter pylori diseases is discussed.
Assuntos
Anacardiaceae/química , Ácidos Anacárdicos/farmacologia , Antibacterianos/farmacologia , Helicobacter pylori/efeitos dos fármacos , Extratos Vegetais/farmacologia , Ácidos Anacárdicos/administração & dosagem , Ácidos Anacárdicos/isolamento & purificação , Antibacterianos/administração & dosagem , Antibacterianos/isolamento & purificação , Relação Dose-Resposta a Droga , Humanos , Hidroxibenzoatos/administração & dosagem , Hidroxibenzoatos/isolamento & purificação , Hidroxibenzoatos/farmacologia , Medicina Tradicional , México , Testes de Sensibilidade Microbiana , Ácido Oleanólico/isolamento & purificação , Ácido Oleanólico/farmacologia , Casca de Planta , Extratos Vegetais/administração & dosagem , Extratos Vegetais/isolamento & purificação , Sitosteroides/isolamento & purificação , Sitosteroides/farmacologia , Triterpenos/isolamento & purificação , Triterpenos/farmacologiaRESUMO
The physiological role of the membrane-bound pyrophosphatase of Rhodospirillum rubrum was investigated by the characterization of a mutant strain. Comparisons of growth levels between the wild type and the mutant under different low-potential conditions and during transitions between different metabolisms indicate that this enzyme provides R. rubrum with an alternative energy source that is important for growth in low-energy states.