α<sub>3</sub>β<sub>3</sub> complex of thermophilic ATP synthase Catalysis without the γ‐subunit

Kagawa, Yasuo; Ohta, Shigeo; Otawara-Hamamoto, Yoko

doi:10.1016/0014-5793(89)80017-1

Cited by 81 publications

(27 citation statements)

References 12 publications

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“…This could result in inactivation of atpA encoding the ␣ subunit, and therefore only the ␥ and ␤ subunits would be present; although the ␤ subunit has been shown to possess ATPase activity (13), it is reasonable to assume that the amount of this enzyme would be reduced compared to the amount of the complete F 1 unit. Similarly, the ␣␤ complex without the ␥ subunit has been shown to exhibit reduced ATPase activity (9,17). These highly likely mutations are in contrast to specific up-mutations in the promoter of the glycolytic gene encoding the enzyme with the greatest control over the flux.…”

Section: Discussionmentioning

confidence: 99%

Increasing Acidification of Nonreplicating Lactococcus lactis Δ thyA Mutants by Incorporating ATPase Activity

Pedersen

Koebmann

Jensen

et al. 2002

Appl Environ Microbiol

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Lactococcus lactis MBP71 ⌬thyA (thymidylate synthase) cannot synthesize dTTP de novo, and DNA replication is dependent on thymidine in the growth medium. In the nonreplicating state acidification by MBP71 was completely insensitive to bacteriophages (M. B. Pedersen, P. R. Jensen, T. Janzen, and D. Nilsson, Appl. Environ. Microbiol. 68:3010-3023, 2002). For nonreplicating MBP71 the biomass increased 3.3-fold over the first 3.5 h, and then the increase stopped. The rate of acidification increased 2.3-fold and then started to decrease. Shortly after inoculation the lactic acid flux was 60% of that of exponentially growing MBP71. However, when nonspecific ATPase activity was incorporated into MBP71, the lactic acid flux was restored to 100% but not above that point, indicating that control over the flux switched from ATP demand to ATP supply (i.e., to sugar transport and glycolysis). As determined by growing nonreplicating cells with high ATPase activity on various sugar sources, it appeared that glycolysis exerted the majority of the control. ATPase activity also stimulated the rate of acidification by nonreplicating MBP71 growing in milk, and pH 5.2 was reached 40% faster than it was without ATPase activity. We concluded that ATPase activity is a functional means of increasing acidification by nonreplicating L. lactis.Lactic acid bacteria are employed in many types of food fermentation. In dairy fermentations the lactic acid bacteria convert the lactose of the milk into lactic acid, which in addition to its preservation effect is also important for flavor and texture development. However, during growth the cells are susceptible to bacteriophage infections, and this is one of the greatest problems in dairies today.We have previously described construction of mutant MBP71 ⌬thyA from Lactococcus lactis subsp. lactis CHCC373 (24). The thyA gene encodes thymidylate synthase, and without this enzyme thymidine must be present in the growth medium for the synthesis of dTTP (Fig. 1). Milk and M17 broth do not support propagation of MBP71; i.e., the number of CFU does not increase unless thymidine is added (24). When dTTP is not available inside the cell, DNA replication does not occur, and an infecting phage therefore is not able to replicate its genome and phage propagation is terminated. It was demonstrated previously that acidification by MBP71 was completely unaffected by phages at a multiplicity of infection of 0.1 when thymidine was not added (24). Naturally, the cells are not able to replicate their chromosome either, and they do not replicate and do not divide.Synthesis of RNA and protein is not affected directly in a thyA mutant (Fig. 1), and therefore genes may still be expressed, which increases the metabolic capacity of the cells. Even though the cells can synthesize new enzymes, they should have a lower rate of macromolecule synthesis (8) and consume less ATP than exponentially growing cells. This leads to a lower lactic acid flux.One method of increasing ATP consumption is to introduce nonspecific ATPase acti...

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Section: Discussionmentioning

confidence: 99%

Increasing Acidification of Nonreplicating Lactococcus lactis Δ thyA Mutants by Incorporating ATPase Activity

Pedersen

Koebmann

Jensen

et al. 2002

Appl Environ Microbiol

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“…The latter investigators suggested that ATP may be generated at the cell surface of the lymphocyte upon activation by the target cell. Our results demonstrating the presence of 3-H+ATPase on the surface of neoplastic cells, and the inhibitory effect of both soluble p51.5 and anti-3 suggest the involvement of an energy-using system of tumor membrane origin in lymphocyte-mediated cytotoxicity since the/~ subunit can in fact, under appropriate conditions, hydrolyze ATP to ADP (46)(47)(48). Whereas the precise mechanism by which 3-H+ATPase functions in lymphocyte-mediated cytotoxicity remains to be determined, it is tempting to speculate that p51.5, upon combination with its lymphocyte receptor(s), may provide energy for transport of H + across the plasma membrane into the tumor cell resulting in cytolysis by osmotic shock.…”

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confidence: 82%

A novel ligand in lymphocyte-mediated cytotoxicity: expression of the beta subunit of H+ transporting ATP synthase on the surface of tumor cell lines.

Das

Mondragon

Sadeghian

et al. 1994

The Journal of Experimental Medicine

126

109

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SummaryExtracellular adenosine triphosphate (eATP) has been suggested to play a role in lymphocyteinduced tumor destruction. We now provide evidence that a protein responsible for ATP synthesis in mitochondria may also play a physiologic role in major histocompatibility complex-independent, lymphocyte-mediated cytotoxicity. A 51.5-kD protein (p51.5) beating structural and immunologic characteristics of the B subunit of H + transporting ATP synthase (E.C. 3.6.1.34, B-H +ATPase, published molecular mass of 51.6 kD) was detected on the plasma membrane of three different human tumor cell lines studied. NH2-terminal amino acid sequence analysis of purified p51.5 from K562 tumor cells revealed 100% homology of 16 residues identified in the first 21 positions to the known sequence of human mitochondrial B-H § ATPase. Antibody directed against a 2l-iner peptide in the ATP binding region of B-H+ATPase (anti-B) reacted with only one band on Western blots of whole tumor extracts and tumor membrane extracts suggesting that the antiserum reacts with a single species of protein. Anti-B reacted with the cell membranes of tumor ceils as determined by fluorescence-activated flow cytometry and immunoprecipitated a 51.5-kD protein from surface-labeled neoplastic cells (but not human erythrocytes and lymphocytes). Purified p51.5 bound to human lymphocytes and inhibited natural killer (NK) cell-mediated cytotoxicity. Furthermore, anti-B treatment of the K562 and A549 tumor cell lines inhibited NK (by >95%) and interleukin 2-activated killer (LAK) cell (by 75%) cytotoxicity, respectively. Soluble p51.5 upon binding to lymphocytes retained its reactivity to anti-B suggesting that the ATP binding domain and the lymphocyte-receptor binding domain reside in distinct regions of the ligand. These results suggest that B-H +ATPase or a nearly identical molecule is an important ligand in the effector phase (rather than the recognition phase) of a cytolytic pathway used by naive NK and LAK cells.

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“…When separated from F 0 as a soluble complex, F 1 is composed of five different subunits in a stoichiometry of ␣ 3 ␤ 3 ␥␦⑀ and functions as an ATPase (2). The ␣ 3 ␤ 3 , ␣ 3 ␤ 3 ␥, and ␣ 3 ␤ 3 ␦ subcomplexes reconstituted from the isolated subunits of TF 1 1 are active as ATPases (3)(4)(5)(6). Both the ␣ 3 ␤ 3 and ␣ 3 ␤ 3 ␦ subcomplexes differ from TF 1 in that they are less specific for divalent cations and are insensitive to inhibition by azide (4,5).…”

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confidence: 99%

The α3β3γ Subcomplex of the F1-ATPase from the Thermophilic Bacillus PS3 with the βT165S Substitution Does Not Entrap Inhibitory MgADP in a Catalytic Site during Turnover

Jault

Dou

Grodsky

et al. 1996

Journal of Biological Chemistry

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The hydrolytic properties of the mutant ␣ 3 (␤T165S) 3 ␥ and wild-type ␣ 3 ␤ 3 ␥ subcomplexes of TF 1 have been compared. Whereas the wild-type complex hydrolyzes 50 M ATP in three kinetic phases, the mutant complex hydrolyzes 50 M ATP with a linear rate. After incubation with a slight excess of ADP in the presence of Mg 2؉ , the wild-type complex hydrolyzes 2 mM ATP with a long lag. In contrast, prior incubation of the mutant complex under these conditions does not affect the kinetics of ATP hydrolysis. The ATPase activity of the wild-type complex is stimulated 4-fold by 0.1% lauryl dimethylamine oxide, whereas this concentration of lauryl dimethylamine oxide inhibits the mutant complex by 25%. Compared with the wild-type complex, the activity of the mutant complex is much less sensitive to turnoverdependent inhibition by azide. This comparison suggests that the mutant complex does not entrap substantial inhibitory MgADP in a catalytic site during turnover, which is supported by the following observations. ATP hydrolysis catalyzed by the wild-type complex is progressively inhibited by increasing concentrations of Mg 2؉ in the assay medium, whereas the mutant complex is insensitive to increasing concentrations of Mg

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α₃β₃ complex of thermophilic ATP synthase Catalysis without the γ‐subunit

Cited by 81 publications

References 12 publications

Increasing Acidification of Nonreplicating Lactococcus lactis Δ thyA Mutants by Incorporating ATPase Activity

Increasing Acidification of Nonreplicating Lactococcus lactis Δ thyA Mutants by Incorporating ATPase Activity

A novel ligand in lymphocyte-mediated cytotoxicity: expression of the beta subunit of H+ transporting ATP synthase on the surface of tumor cell lines.

The α3β3γ Subcomplex of the F1-ATPase from the Thermophilic Bacillus PS3 with the βT165S Substitution Does Not Entrap Inhibitory MgADP in a Catalytic Site during Turnover

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α3β3 complex of thermophilic ATP synthase Catalysis without the γ‐subunit

Cited by 81 publications

References 12 publications

Increasing Acidification of Nonreplicating Lactococcus lactis Δ thyA Mutants by Incorporating ATPase Activity

Increasing Acidification of Nonreplicating Lactococcus lactis Δ thyA Mutants by Incorporating ATPase Activity

A novel ligand in lymphocyte-mediated cytotoxicity: expression of the beta subunit of H+ transporting ATP synthase on the surface of tumor cell lines.

The α3β3γ Subcomplex of the F1-ATPase from the Thermophilic Bacillus PS3 with the βT165S Substitution Does Not Entrap Inhibitory MgADP in a Catalytic Site during Turnover

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α₃β₃ complex of thermophilic ATP synthase Catalysis without the γ‐subunit