F1F0-ATPase from Escherichia coli with mutant F0 subunits. Partial purification and immunoprecipitation of F1F0 complexes

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.     

Sulfite inhibits the F1F0-ATP synthase and activates the F1F0-ATPase of Paracoccus denitrificans

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.     

心室内和心外膜应用腺苷对延髓PGL神经元电活动的影响

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。     

Identification of F0 subunits in the rat liver mitochondrial F0F1-ATP synthase.

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.     

Structural and functional characterization of subunits of the F0 sector of the mitochondrial F0F1-ATP synthase.

Proteolytic digestion of F1-depleted submitochondrial particles (USMP), reconstitution with isolated subunits and titration with inhibitors show that the nuclear-encoded PVP protein, previously identified as an intrinsic component of bovine heart F0 (F01) (Zanotti, F. et al. (1988) FEBS Lett. 237, 9-14), is critically involved in maintaining the proper H+ translocating configuration of this sector and its correct binding to the F1 catalytic moiety. Trypsin digestion of USMP, under conditions leading to cleavage of the carboxyl region of the PVP protein and partial inhibition of transmembrane H+ translocation, results in general loss of sensitivity of this process to F0 inhibitors. This is restored by addition of the isolated PVP protein. Trypsin digestion of USMP causes also loss of oligomycin sensitivity of the catalytic activity of membrane reconstituted soluble F1, which can be restored by the combined addition of PVP and OSCP, or PVP and F6. Amino acid sequence analysis shows that, in USMP, modification by [14C] N,N'-dicyclohexylcarbodiimide of subunit c of F0 induces the formation of a dimer of this protein, which retains the 14C-labelled group. Chemical modification of cysteine-64 of subunit c results in inhibition of H+ conduction by F0. The results indicate that proton conduction in mitochondrial F0 depends on interaction of subunit c with the PVP protein.     

The gamma subunit of F1 and the PVP protein of F0 (F0I) are components of the gate of the mitochondrial F0F1 H(+)-ATP synthase

The gamma subunit of the F1 moiety of the bovine mitochondrial H(+)-ATP synthase is shown to function as a component of the gate. Addition of purified gamma subunit to F0-liposomes inhibits transmembrane proton conduction. This inhibition can be removed by the bifunctional thiol reagent diamide. Immunoblot analysis shows that the diamide effect is likely due to disulphide bridging of the gamma subunit with the PVP protein of the F0 sector.     

北美CHIHUAHUAN荒漠啮齿动物群落动态III．变量的频次分布和预测

本文用х²方和柯尔莫哥洛夫检验分析了北美Chihuahuan荒漠啮齿动物群落6个生态学变量的频次分布,并用移动平均和指数平滑方法拟合了这些变量的动态变化。结果显示：1)结合种群密度、生物量、物种均匀性和生物量均匀性服从正态分布;2)物种数和物种多样性的频次为向右偏斜的分布,无法用常见的理论分布来近似表示;3)单移动平均和双移动平均分别较好地描述了物种多样性和物种均匀性;4)其余4个变量可用单指数平滑来较好的描述．     

人工饲养条件下高原鼠兔生长和发育的初步研究

本文研究了人工饲养条件下高原鼠兔的生长发育情况,并和其他种鼠兔的生长作了比较。室内高原鼠兔比野外的生长快,人工饲养的阿富汗鼠兔和北美鼠兔生长期短,成熟早。     

Structure of Dimeric F1F0-ATP Synthase

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.     

Evidence from immunological studies of structure-mechanism relationship of F1 and F1F0

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     

Activation and Deactivation of F0F1-ATPase in Yeast Mitochrondia

The regulation of membrane-bound proton F₀F₁ATPase by the protonmotive force and nucleotides was studied in yeastmitochondria. Activation occurred in whole mitochondria and the ATPaseactivity was measured just after disrupting the membranes with Triton X-100.Deactivation occurred either in whole mitochondria uncoupled with FCCP, or indisrupted membranes. No effect of Triton X-100 on the ATPase was observed,except a slow reactivation observed only in the absence of MgADP. BothAMPPNP and ATP increased the ATPase deactivation rate, thus indicating thatoccupancy of nucleotidic sites by ATP is more decisive than catalyticturnover for this process. ADP was found to stimulate the energy-dependentATPase activation. ATPase deactivated at the same rate in uncoupled anddisrupted mitochondria. This suggests that deactivation is not controlled byrebinding of some soluble factor, like IF1, but rather by the conversion ofthe F₁.IF1 complex into an inactive form.     

Thermal inactivation of electron-transport functions and F0F1-ATPase activities

Bovine heart submitochondrial particles in suspension were heated at a designated temperature for 3 min, then cooled for biochemical assays at 30 degrees C. By enzyme activity measurements and polarographic assay of oxygen consumption, it is shown that the thermal denaturation of the respiratory chain takes place in at least four stages and each stage is irreversible. The first stage occurs at 51.0 +/- 1.0 degrees C, with the inactivation of NADH-linked respiration, ATP-driven reverse electron transport, F0F1 catalyzed ATP/Pi exchange, NADH and succinate-driven ATP synthesis. The second stage occurs at 56.0 +/- 1.0 degrees C, with the inactivation of succinate-linked proton pumping and respiration. The third stage occurs at 59.0 +/- 1.0 degrees C, with the inactivation of electron transfer from cytochrome c to cytochrome oxidase and ATP-dependent proton pumping. The ATP hydrolysis activity of F0F1 persists to 61.0 +/- 1.0 degrees C. An additional transition, detectable by differential scanning calorimetry, occurring around 70.0 +/- 2.0 degrees C, is probably associated with thermal denaturation of cytochrome c and other stable membrane proteins. In the presence of either mitochondrial matrix fluid or 2 mM mercaptoethanol, all five stages give rise to endothermic effects, with the absorption of approx. 25 J/g protein. Under aerobic conditions, however, the first four transitions become strongly exothermic, and release a total of approx. 105 J/g protein. Solubilized and reconstituted F0F1 vesicles also exhibit different inactivation temperatures for the ATP/Pi exchange, proton pumping and ATP hydrolysis activities. The first two activities are abolished at 49.0 +/- 1.0 degrees C, but the latter at 58.0 +/- 2.0 degrees C. Differential scanning calorimetry also detects biphasic transitions of F0F1, with similar temperatures of denaturation (49.0 and 54.0 degrees C). From these and other results presented in this communication, the following is concluded. (1) A selective inactivation, by the temperature treatment, of various functions of the electron-transport chain and of the F0F1 complex can be done. (2) The ATP synthesis activity of the F0F1 complex involves either a catalytic or a regulation subunit(s) which is not essential for ATP hydrolysis and the proton translocation. This subunit is 10 degrees C less stable than the hydrolytic site. Micromolar ADP stabilizes it from thermal denaturation by 4-5 degrees C, although ADP up to millimolar concentration does not protect the hydrolytic site and the proton-translocation site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Functional and structural characterization of the talin F0F1 domain

The globular head domain of talin, a large multi-domain cytoplasmic protein, is required for inside-out activation of the integrins, a family of heterodimeric transmembrane cell adhesion molecules. Talin head contains a FERM domain that is composed of F1, F2, and F3 subdomains. A F0 subdomain is located N-terminus to F1. The F3 contains a canonical phosphotyrosine binding (PTB) fold that directly interacts with the membrane proximal NPxY/F motif in the integrin β cytoplasmic tail. This interaction is stabilized by the F2 that interacts with the lipid head-groups of the plasma membrane. In comparison to F2 and F3, the properties of the F0F1 remains poorly characterized. Here, we showed that F0F1 is essential for talin-induced activation of integrin αLβ2 (LFA-1). F0F1 has a high content of β-sheet secondary structure, and it tends to homodimerize that may provide stability against proteolysis and chaotrope induced unfolding.     

Myocardial ischemic preconditioning and mitochondrial F1F0-ATPase activity

A short period of ischemia followed by reperfusion (ischemic preconditioning) is known to trigger mechanisms that contribute to the prevention of ATP depletion. In ischemic conditions, most of the ATP hydrolysis can be attributed to mitochondrial F₁F₀-ATPase (ATP synthase). The purpose of the present study was to examine the effect of myocardial ischemic preconditioning on the kinetics of ATP hydrolysis by F₁F₀-ATPase. Preconditioning was accomplished by three 3-min periods of global ischemia separated by 3 min of reperfusion. Steady state ATP hydrolysis rates in both control and preconditioned mitochondria were not significantly different. This suggests that a large influence of the enzyme on the preconditioning mechanism may be excluded. However, the time required by the reaction to reach the steady state rate was increased in the preconditioned group before sustained ischemia, and it was even more enhanced in the first 5 min of reperfusion (101 ± 3.0 sec in preconditioned vs. 83.4 ± 4.4 sec in controls, p 0.05). These results suggest that this transient increase in activation time may contribute to the cardioprotection by slowing the ATP depletion in the very critical early phase of post-ischemic reperfusion.     

重组人肝再生增强因子在体外能刺激HTC肝癌细胞DNA合成

在成功克隆人肝再生增强因子（ｈＡＬＲ）ｃＤＮＡ的基础上,本研究将人ＡＬＲ编码区ｃＤＮＡ亚克隆于真核瞬间表达质粒ｐＣＤＮＡ Ｉ,构建了真核表达质粒ｐＣＤＮＡ Ｉ－ｈＡＬＲ,转染ｃｏｓ－７细胞后,表达产物生物活性检测表明：真核表达的ｈＡＬＲ在体外能刺激ＨＴＣ肝癌细胞增殖,这一体外活性的确定为重组人ＡＬＲ的纯化提供了简洁、可靠的检测方法。     

Mitochondrial F0F1 H+-ATP synthase. Characterization of F0 components involved in H+ translocation

The membrane F0 sector of mitochondrial ATP synthase complex was rapidly isolated by direct extraction with CHAPS from F1-depleted submitochondrial particles. The preparation thus obtained is stable and can be reconstituted in artificial phospholipid membranes to result in oligomycin-sensitive proton conduction, or recombined with purified F1 to give the oligomycin-sensitive F0F1-ATPase complex. The F0 preparation and constituent polypeptides were characterized by SDS-polyacrylamide gel electrophoresis and immunoblot analysis. The functional role of F0 polypeptides was examined by means of trypsin digestion and reconstitution studies. It is shown that, in addition to the 8 kDa DCCD-binding protein, the nuclear encoded protein [(1987) J. Mol. Biol. 197, 89-100], characterized as an intrinsic component of F0 (F0I, PVP protein [(1988) FEBS Lett. 237,9-14]) [corrected] is involved in H+ translocation and the sensitivity of this process to the F0 inhibitors, DCCD and oligomycin.     

ATP-synthetase complex (F1F0) from Escherichia coli. Purification and characterization of subunits A and B of the F0 part

An intact B890 light-harvesting pigment—protein complex has been obtained from Rhodospirillum rubrum strain S1. We show that this complex contains two types of low-M_r polypeptide. Both these polypeptides are present in the intact chromatophore membrane. Analysis of the pigment content of this complex suggests that per pair of polypeptides the complex contains 2 molecules of bacteriochlorophyll and one molecule of carotenoid.