All rotary ATPases catalyze the interconversion of ATP and ADP-Pi through a mechanism that is coupled to the transmembrane flow of H+ or Na+. Physiologically, however, F/A-type enzymes specialize in ATP synthesis driven by downhill ion diffusion, while eukaryotic V-type ATPases function as ion pumps. To begin to rationalize the molecular basis for this functional differentiation, we solved the crystal structure of the Na+-driven membrane rotor of the Acetobacterium woodii ATP synthase, at 2.1 Å resolution. Unlike known structures, this rotor ring is a 9:1 heteromer of F- and V-type c-subunits, and therefore features a hybrid configuration of ion-binding sites along its circumference. Molecular and kinetic simulations are used to dissect the mechanisms of Na+ recognition and rotation of this c-ring, and to explain the functional implications of the V-type c-subunit. These structural and mechanistic insights indicate an evolutionary path between synthases and pumps involving adaptations in the rotor ring.
The Na(+) F(1)F(O) ATP synthase of the anaerobic, acetogenic bacterium Acetobacterium woodii has a unique F(O)V(O) hybrid rotor that contains nine copies of a F(O)-like c subunit and one copy of a V(O)-like c(1) subunit with one ion binding site in four transmembrane helices whose cellular function is obscure. Since a genetic system to address the role of different c subunits is not available for this bacterium, we aimed at a heterologous expression system. Therefore, we cloned and expressed its Na(+) F(1)F(O) ATP synthase operon in Escherichia coli. A Δatp mutant of E. coli produced a functional, membrane-bound Na(+) F(1)F(O) ATP synthase that was purified in a single step after inserting a His(6)-tag to its β subunit. The purified enzyme was competent in Na(+) transport and contained the F(O)V(O) hybrid rotor in the same stoichiometry as in A. woodii. Deletion of the atpI gene from the A. woodii operon resulted in a loss of the c ring and a mis-assembled Na(+) F(1)F(O) ATP synthase. AtpI from E. coli could not substitute AtpI from A. woodii. These data demonstrate for the first time a functional production of a F(O)V(O) hybrid rotor in E. coli and revealed that the native AtpI is required for assembly of the hybrid rotor.
50 ml of a 10% fish oil emulsion (41% ω-3 fatty acids of total fatty acids) were infused for 1 h into the arm vein of young, healthy, male volunteers. The fatty acid composition of the plasma, aggregation of the blood platelets as well as the thromboxane synthesis, were measured before the beginning of infusion, 20, 60, 120, 360 and 1,440 min after the start of the fat infusion. In the first 60 min, the fatty acid composition of the plasma changed in correspondence with the supplied fatty acid pattern. At the end of the investigation it was again within the normal range. As a result of fat application thromboxane synthesis was reduced and the aggregation of the platelets was inhibited but it was normalized by the 1,440-min value. Fish oil emulsions might be beneficial for parenterally fed patients with a high risk of thrombosis. Therefore the performance of further investigations using a varying dosage and multiple application can be recommended.
Background:The peripheral stator stalk of Escherichia coli ATP synthase contains two b subunits. Results: Using disulfide bond formation, one b subunit was cross-linked to a, ␣, and ␦ and the other to .
Conclusion:The b subunits adopt distinct positions within the stator to generate stability. Significance: The different positions imply different roles in counteracting the torque generated by the rotor.
The aim of this study is to investigate anti-arthritis activity using natural eggshell membrane (NEM). Methods: NEM was administered at 52 mg/kg, 200 mg/kg, and 400 mg/kg to SD-Rat, where arthritis was induced by monosodium iodoacetate (MIA) at 3 mg. NO production in serum was measured using Griess reagent. Cytokines including IL-1β, and IL-6 were measured by Luminex and PGE 2, MMP-2, MMP-9, TIMP-1, LTB 4, and hs-CRP were measured by ELISA. The cartilage of patella volume was examined and 3-D high-resolution reconstructions of the cartilage of patella were obtained using a Micro-CT system. Results: Production of NO, IL-1β, IL-6, PGE 2, MMP-2, MMP-9, TIMP-1, LTB 4 , and hs-CRP in serum was decreased, respectively, in comparison with control. The cartilage of patella volume increased significantly. In addition, the NEM group showed a decrease in the cartilage of patella, synovial membrane, and transformation of fibrous tissue. Conclusion: The results for NEM showed significant anti-arthritis activity. These results may be developed as a raw material for new health food to ease the symptoms mentioned above.
The ion‐translocating c ring of the Na+ F1Fo ATP synthase of the anaerobic bacterium Acetobacterium woodii is the first heteromeric c ring found in nature that contains one V‐ (c1) and two F‐type‐like c subunits (c2/c3), the latter of identical amino acid sequence. To address whether they are of equal or different importance for function, they were deleted in combination or individually. Deletion of c1 was compensated by incorporation of two c2/c3 subunits but the enzyme was unstable and largely impaired in Na+ transport. Deletion of c2 was compensated by incorporation of c3 but also led to a reduction of Na+ transport. Deletion of c3 had no effect. In contrast, deletion of both c2 and c3 led to a complete loss of ATPase activity at the cytoplasmic membrane. Mass spectrometric analysis of c2+1 Ala and c2+2 Ala variants revealed a copy number of 8 : 1 for c2/c3 which is consistent with the biochemical characteristics of the variants. These data indicate a role of c1 in assembly and a function of c2 as the predominant c ring constituent.
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