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Previously identified mutations in subunits a and b of the F0 sector of the F1F0-ATPase from Escherichia coli are further characterized by isolating detergent-solubilized, partially purified F1F0 complexes from cells bearing these mutations. The composition of the various F1F0 complexes was judged by quantitating the amount of each subunit present in the detergent-solubilized preparations. The composition of the F0 sectors containing altered polypeptides was determined by quantitating the F0 subunits that were immunoprecipitated by antibodies directed against the F1 portion. In this way, the relative amounts of F0 subunits (a, b, c) which survived the isolation procedure bound to F1 were determined for each mutation. This analysis indicates that both missense mutations in subunit a (aser206----leu and ahis245----tyr) resulted in the isolation of F1F0 complexes with normal subunit composition. The nonsense mutation in subunit a (atyr235----end) resulted in isolation of a complex containing the b and c subunits. The bgly131----asp mutation in the b subunit results in an F0 complex which does not assemble or survive the isolation. The isolated F1F0 complex containing the mutation bgly9----asp in the b subunit was defective in two regards: first, a reduction in F1 content relative to F0 and second, the absence of the a subunit. Immunoprecipitations of this preparation demonstrated that F1 interacts with both c and mutant b subunits. A strain carrying the mutation, bgly9----asp, and the compensating suppressor mutation apro240----leu (previously shown to be partially unc+) yielded an F1F0 ++ complex that remained partially defective in F1 binding to F0 but normal in the subunit composition of the F0 sector. The assembly, structure, and function of the F1F0-ATPase is discussed. 相似文献
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Pacheco-Moisés F Minauro-Sanmiguel F Bravo C García JJ 《Journal of bioenergetics and biomembranes》2002,34(4):269-278
The F1F0 complex of Paracoccus denitrificans (PdF1F0) is the fastest ATP synthase but the slowest ATPase. Sulfite exerts maximal activation of the PdF1F0-ATPase (Pacheco-Moisés, F., García, J. J., Rodríguez-Zavala, J. S., and Moreno-Sánchez, R. (2000). Eur. J. Biochem.
267, 993–1000) but its effect on the PdF1F0-ATP synthase activity remains unknown. Therefore, we studied the effect of sulfite on ATP synthesis and 32Pi ATP exchange reactions of inside-out membrane vesicles of P. denitrificans. Sulfite inhibited both reactions under conditions of maximal pH and normal sensitivity to dicyclohexylcarbodiimide. Sulfite increased by 10- and 5-fold the K
0.5 for Mg2+-ADP and Pi during ATP synthesis, respectively, and by 4-fold the IC50 of Mg2+-ADP for inhibition of the PdF1F0-ATPase activity. Thus, sulfite exerts opposite effects on the forward and reverse functioning of the PdF1F0 complex. These effects are not due to membrane or PdF1F0 uncoupling. Kinetic and structural modifications that could account for these results are discussed. 相似文献
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An approach for the deconvolution of multiexponential fluorescence decay data in which a single exponential decay is used in place of the usual excitation profile is described. For analysis by the method of moments, the resulting decay lifetimes are identical to those in the multiexponential decay, while the pre-exponential factors are a simple function of the true values and the parameters of the single exponential decay. This approach, which we call the F/F deconvolution method, is capable of eliminating the errors in decay analyses which arise from the wavelength dependence of the instrument response function. 相似文献
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Danièle C. Gautheron Catherine Godinot 《Journal of bioenergetics and biomembranes》1988,20(4):451-468
Monoclonal and polyclonal antibodies directed against peptides of F1-ATPase or F1F0-ATPase synthase provide new and efficient tools to study structure-function relationships and mechanisms of such complex membrane enzymes. This review summarizes the main results obtained using this approach. Antibodies have permitted the determination of the nature of subunits involved in the complex, their stoichiometry, their organization, neighboring interactions, and vectorial distribution within or on either face of the membrane. Moreover, in a few cases, amino acid sequences exposed on a face of the membrane or buried inside the complex have been identified. Antibodies are very useful for detecting the role of each subunit, especially for those subunits which appear to have no direct involvement in the catalytic mechanism. Concerning the mechanisms, the availability of monoclonal antibodies which inhibit (or activate) ATP hydrolysis or ATP synthesis, which modify nucleotide binding or regulation of activities, which detect specific conformations, etc. brings many new ways of understanding the precise functions. The specific recognition by monoclonal antibodies on the subunit of epitopes in the proximity of, or in the catalytic site, gives information on this site. The use of anti- monoclonal antibodies has shown asymmetry of in the complex as already shown for . In addition, the involvement of with respect to nucleotide site cooperativity has been detected. Finally, the formation of F1F0-antibody complexes of various masses, seems to exclude the functional rotation of F1 around F0 during catalysis.Abbreviations IF1
natural protein inhibitor of the ATPase-ATP synthase
- OSCP
oligomycin sensitivity-conferring protein
- DCCD
dicyclohexylcarbodiimide
- SDS-PAGE
sodium dodecylsulfate polyacrylamide gel electrophoreses
- F1
F1-ATPase, coupling factor F1 of ATPase
- F1F0
F1F0-ATP synthase, ATPase-ATP synthase complex 相似文献
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Cofactors in the biosynthesis of prostaglandins F and F 总被引:1,自引:0,他引:1
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Sergio J. Couoh-Cardel Salvador Uribe-Carvajal Stephan Wilkens José J. García-Trejo 《The Journal of biological chemistry》2010,285(47):36447-36455
The structure of the dimeric ATP synthase from yeast mitochondria was analyzed by transmission electron microscopy and single particle image analysis. In addition to the previously reported side views of the dimer, top view and intermediate projections served to resolve the arrangement of the rotary c10 ring and the other stator subunits at the F0-F0 dimeric interface. A three-dimensional reconstruction of the complex was calculated from a data set of 9960 molecular images at a resolution of 27 Å. The structural model of the dimeric ATP synthase shows the two monomers arranged at an angle of ∼45°, consistent with our earlier analysis of the ATP synthase from bovine heart mitochondria (Minauro-Sanmiguel, F., Wilkens, S., and Garcia, J. J. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 12356–12358). In the ATP synthase dimer, the two peripheral stalks are located near the F1-F1 interface but are turned away from each other so that they are not in contact. Based on the three-dimensional reconstruction, a model of how dimeric ATP synthase assembles to form the higher order oligomeric structures that are required for mitochondrial cristae biogenesis is discussed. 相似文献
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Usukura E Suzuki T Furuike S Soga N Saita E Hisabori T Kinosita K Yoshida M 《The Journal of biological chemistry》2012,287(3):1884-1891
ATP synthase (F(0)F(1)) is made of two motors, a proton-driven motor (F(0)) and an ATP-driven motor (F(1)), connected by a common rotary shaft, and catalyzes proton flow-driven ATP synthesis and ATP-driven proton pumping. In F(1), the central γ subunit rotates inside the α(3)β(3) ring. Here we report structural features of F(1) responsible for torque generation and the catalytic ability of the low-torque F(0)F(1). (i) Deletion of one or two turns in the α-helix in the C-terminal domain of catalytic β subunit at the rotor/stator contact region generates mutant F(1)s, termed F(1)(1/2)s, that rotate with about half of the normal torque. This helix would support the helix-loop-helix structure acting as a solid "pushrod" to push the rotor γ subunit, but the short helix in F(1)(1/2)s would fail to accomplish this task. (ii) Three different half-torque F(0)F(1)(1/2)s were purified and reconstituted into proteoliposomes. They carry out ATP-driven proton pumping and build up the same small transmembrane ΔpH, indicating that the final ΔpH is directly related to the amount of torque. (iii) The half-torque F(0)F(1)(1/2)s can catalyze ATP synthesis, although slowly. The rate of synthesis varies widely among the three F(0)F(1)(1/2)s, which suggests that the rate reflects subtle conformational variations of individual mutants. 相似文献
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In order to identify the subunits constituting the rat liver F0F1-ATP synthase, the complex prepared by selective extraction from the mitochondrial membranes with a detergent followed by purification on a sucrose gradient has been compared to that obtained by immunoprecipitation with an anti-F1 serum. The subunits present in both preparations that are assumed to be authentic components of the complex have been identified. The results show that the total rat liver F0F1-ATP synthase contains at least 13 different proteins, seven of which can be attributed to F0. The following F0 subunits have been identified: the subunit b (migrating as a 24 kDa band in SDS-PAGE), the oligomycin-sensitivity-conferring protein (20 kDa), and F6 (9 kDa) that have N-terminal sequences homologous to the beef-heart ones; the mtDNA encoded subunits 6 (20 kDa) and 8 (less than 7 kDa) that can be synthesized in isolated mitochondria; an additional 20 kDa protein that could be equivalent to the beef heart subunit d. 相似文献
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M A Sibileva T N Osipova A O Zalenski? M A Chebishian A I Golikova 《Molekuliarnaia biologiia》1976,10(2):514-520
Characteristic viscosity, sedimentation constant and optical anisotropy were studied of the complexes formed between DNA and histone fractions F3 and F3+F2a2. The parameters mentioned continuously change with the increase of protein content within the complex. Analysis of experimental data shows that binding of a histone bads to a decrease of size and thermodynamic rigidity of the DNA molecule. On the basis of results obtained a model of F3 histone binding with DNA is suggested, amino acid sequence of this protein being taken into account. Comparison of behaviour of nucleohistones DNA+F3 and DNA+F1 studied previously testifies different way of binding of these histones to DNA. 相似文献
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E2F7 and E2F8 keep the E2F family in balance 总被引:1,自引:0,他引:1
An article by Li and colleagues (in this issue of Developmental Cell) shows that the atypical E2Fs, E2F7 and E2F8, are critical for mouse development. One of the important functions of these family members stems from a negative feedback loop in which E2F7 and E2F8 limit the expression of E2F1 and prevent E2F1-dependent apoptosis. 相似文献
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心室内和心外膜应用腺苷对延髓PGL神经元电活动的影响 总被引:1,自引:0,他引:1
在35只切断两侧缓冲神经和迷走神经的麻醉大鼠,观察心室内注射腺苷和心外膜涂布腺苷对延髓腹外侧头端区PGL神经元自发电活动的影响。结果如下:(1)35只大鼠共记录到121个自发放电单位,平均放电频率为22.5±1.9spikes/s。(2)心室内冲击注射腺苷(0.5μmol/kg,0.1ml)时,BP先升(△1.7±0.2kPa,P<0.001)后降(△4.6土0.5kPa,P<0.001),HR减慢(△126.5±12.3bpm,P<0.001);35个PGL神经元自发放电单位中,30个单位的放电频率由21.9士2.6增至29.2土3.4spikes/s(P<0.001),3个单位不变,2个单位减少。(3)心外膜涂布腺苷(20mmol/L),动脉血压和心率的变化不明显,22个PGL神经元自发放电频率由18.8土1.9增至26.9土2.8spikes/s(P<0.001),3个单位的放电频率无变化。(4)静脉注射选择性腺苷A1受体拮抗8-cyclopentyl-1,3-dipropylxanthine(DPCPX,500μg/kg)可完全阻断腺苷对PGL神经元的兴奋效应。(5)在左右房室沟涂布85%酚或切除双侧星状神经节后,腺苷激活PGL神经元的效应即行消失。结果提示,腺苷可通过人受体激活心交感神经传入纤维,进而兴奋PGL神经元。 相似文献