Interactions between subunits a and b in the rotary ATP synthase as determined by cross-linking

The interaction of the membrane traversing stator subunits a and b of the rotary ATP synthase was probed by substitution of a single Cys into each subunit with subsequent Cu2+ catalyzed cross-linking. Extensive interaction between the transmembrane (TM) region of one b subunit and TM2 of subunit a was indicated by cross-linking with 6 Cys pairs introduced into these regions. Additional disulfide cross-linking was observed between the N-terminus of subunit b and the periplasmic loop connecting TM4 and TM5 of subunit a. Finally, benzophenone-4-maleimide derivatized Cys in the 2–3 periplasmic loop of subunit a were shown to cross-link with the periplasmic N-terminal region of subunit b. These experiments help to define the juxtaposition of subunits b and a in the ATP synthase

O perfil semiológico do paciente portador de hemorragia digestiva alta

OBJETIVO: O seguinte estudo objetivou descrever a semiologia do paciente portador de hemorragia digestiva alta, considerando como determinante na avaliação de potencias focos hemorrágicos. METODOLOGIA: Foram realizadas buscas nas plataformas do SciELO, LILACS, PubMed, Scopus e Google Scholar,utilizando os descritores gastrointestinal bleeding, peptic ulcerous disease e varicose hemorrhage, sendo identificados 35 estudos, dos quais foram incluídos 13 artigos completos. Desses estudos, 5 avaliaram as principais etiologias, 2 o surgimento de novos testes diagnósticos, 2 analisaram os aspectos epidemiológicos e 1 a sintomatologia apresentada pelo acometimento da hemorragia digestiva alta. Observou-se inicialmente a abundâncias de informações conceituais sobre o sangramento, como um transtorno clínico comum, acompanhada de inúmeras manifestações, considerando que o foco hemorrágico pode ocorrer em qualquer porção do trato gastrointestinal. Neste estudo, todas as publicações eleitas apresentaram o quadro semiológico composto por algia abdominal, indícios de choque hipovolêmico e taquicardia, alguns exibiram quedas abruptas da pressão arterial, odinofagia, êmese, náuseas e estado ictérico. Os pacientes implicados, cronicamente, já manifestaram ocorrências prévias, devido ao caráter recidivante torna-se essencial investigar a existência de varizes, fístula aorto-entérica, angiodisplasia e doença ulcerosa. CONCLUSÃO: Elucida-se que a hemorragia digestiva alta representa a principal causa de sangramento do trato gastrointestinal, majoritamente manifesta-se como hematêmese ou melena e cursam com o quadro sintomatológico que auxilia na avaliação da gravidade deste e o embasamento de potenciais focos de sangramento e que contribuam para disseminação de informações e intervenções futuras

Analysis of an N-terminal deletion in subunit a of the Escherichia coli ATP synthase.

Subunit a is a membrane-bound stator subunit of the ATP synthase and is essential for proton translocation. The N-terminus of subunit a in E. coli is localized to the periplasm, and contains a sequence motif that is conserved among some bacteria. Previous work has identified mutations in this region that impair enzyme activity. Here, an internal deletion was constructed in subunit a in which residues 6-20 were replaced by a single lysine residue, and this mutant was unable to grow on succinate minimal medium. Membrane vesicles prepared from this mutant lacked ATP synthesis and ATP-driven proton translocation, even though immunoblots showed a significant level of subunit a. Similar results were obtained after purification and reconstitution of the mutant ATP synthase into liposomes. The location of subunit a with respect to its neighboring subunits b and c was probed by introducing cysteine substitutions that were known to promote cross-linking: a_L207C + c_I55C, a_L121C + b_N4C, and a_T107C + b_V18C. The last pair was unable to form cross-links in the background of the deletion mutant. The results indicate that loss of the N-terminal region of subunit a does not generally disrupt its structure, but does alter interactions with subunit b

Analysis of an N-terminal deletion in subunit a of the Escherichia coli ATP synthase.

Subunit a is a membrane-bound stator subunit of the ATP synthase and is essential for proton translocation. The N-terminus of subunit a in E. coli is localized to the periplasm, and contains a sequence motif that is conserved among some bacteria. Previous work has identified mutations in this region that impair enzyme activity. Here, an internal deletion was constructed in subunit a in which residues 6-20 were replaced by a single lysine residue, and this mutant was unable to grow on succinate minimal medium. Membrane vesicles prepared from this mutant lacked ATP synthesis and ATP-driven proton translocation, even though immunoblots showed a significant level of subunit a. Similar results were obtained after purification and reconstitution of the mutant ATP synthase into liposomes. The location of subunit a with respect to its neighboring subunits b and c was probed by introducing cysteine substitutions that were known to promote cross-linking: a_L207C + c_I55C, a_L121C + b_N4C, and a_T107C + b_V18C. The last pair was unable to form cross-links in the background of the deletion mutant. The results indicate that loss of the N-terminal region of subunit a does not generally disrupt its structure, but does alter interactions with subunit b

Interactions between subunits a and b in the rotary ATP synthase as determined by cross-linking.

The interaction of the membrane traversing stator subunits a and b of the rotary ATP synthase was probed by substitution of a single Cys into each subunit with subsequent Cu(2+) catalyzed cross-linking. Extensive interaction between the transmembrane (TM) region of one b subunit and TM2 of subunit a was indicated by cross-linking with 6 Cys pairs introduced into these regions. Additional disulfide cross-linking was observed between the N-terminus of subunit b and the periplasmic loop connecting TM4 and TM5 of subunit a. Finally, benzophenone-4-maleimide derivatized Cys in the 2-3 periplasmic loop of subunit a were shown to cross-link with the periplasmic N-terminal region of subunit b. These experiments help to define the juxtaposition of subunits b and a in the ATP synthase

Mutagenesis of the L, M, and N Subunits of Complex I from Escherichia coli Indicates a Common Role in Function

Background: The membrane arm of Complex I (NADH:ubiquinone oxidoreductase) contains three large, and closely related subunits, which are called L, M, and N in E. coli. These subunits are homologous to components of multi-subunit Na + /H + antiporters, and so are implicated in proton translocation. Methodology/Principal Findings: Nineteen site-specific mutations were constructed at two corresponding positions in each of the three subunits. Two positions were selected in each subunit: L_K169, M_K173, N_K158 and L_Q236, M_H241, N_H224. Membrane vesicles were prepared from all of the resulting mutant strains, and were assayed for deamino-NADH oxidase activity, proton translocation, ferricyanide reductase activity, and sensitivity to capsaicin. Corresponding mutations in the three subunits were found to have very similar effects on all activities measured. In addition, the effect of adding exogenous decylubiquinone on these activities was tested. 50 mM decylubiquinone stimulated both deamino-NADH oxidase activity and proton translocation by wild type membrane vesicles, but was inhibitory towards the same activities by membrane vesicles bearing the lysine substitution at the L236/M241/N224 positions. Conclusions/Significance: The results show a close correlation with reduced activity among the corresponding mutations

Comparison of the ability to utilize decylubiquinone for proton translocation with mutations at the second site: L_Q236, M_H241, and N_H224.

<p>The reactions were initiated by addition of deamino-NADH (dNADH) to 250 µM final concentration. Membrane preparations (150 µM/ml protein) were pre-incubated with 10 mM cyanide (KCN) or 100 µM decylubiquinone (DQ) as indicated in the figure. KCN prevents recycling of the quinones by inhibiting the quinol oxidases. The addition of FCCP (1 µM) collapses the proton gradients, while pre-incubation with capsaicin (Cap) to 300 µM final concentration prevents proton translocation (not shown for all traces). The traces shown are representative of 2–3 experiments. (<i>A</i>) wild type. (<i>B</i>) L_Q236K. (<i>C</i>) M_H241K. (<i>D</i>) N_H224K.</p

The effect of exogenous decylubiquinone on deamino-NADH oxidase activity.

<p>^<i>a</i>Activity was measured in membrane preparations as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017420#s4" target="_blank">Materials and Methods</a>”.</p><p>^<i>b</i>Activities are expressed relative to the rates measured in the absence of decylubiquinone. The means and standard errors from 3–4 measurements are shown. Typical values of the rates can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017420#pone-0017420-t001" target="_blank">Table 1</a>.</p

Two schematic views of the indirect proton translocation by Complex I.

<p>The peripheral arm is shown in thin wireframe with the flavin (FMN) and Fe-S centers in colored, space filling. The membrane subunits are shown in color: H (red), N (violet), M (cyan), L (yellow), and subunits A, J, and K are shown in blue. The protein structure is from the pdb file 3m9s for Complex I from <i>Thermus thermophilus</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017420#pone.0017420-Efremov1" target="_blank">[17]</a>. The C-terminal region of subunit L can be seen to contain a lateral helix that interacts with subunits M and N, and a final transmembrane helix that interacts with N at the junction with A, J or K subunits. (<b>A</b>) The L, M and N subunits are each suggested to translocate one proton per NADH. (<b>B</b>) An alternative view is that proton translocation occurs through the interaction of two subunits, resulting in a ratio of only 2 protons per NADH. The movement of protons would be facilitated by the conserved glutamic acids and lysines shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017420#pone-0017420-g001" target="_blank">Figure 1</a>. The proton pathways would not necessarily occur along the interfaces.</p