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1.
In order to get insight into the origin of apparent negative cooperativity observed for F1-ATPase, we compared ATPase activity and ATPMg binding of mutant subcomplexes of thermophilic F1-ATPase, α(W463F)3β(Y341W)3γ and α(K175A/T176A/W463F)3β(Y341W)3γ. For α(W463F)3β(Y341W)3γ, apparent Km's of ATPase kinetics (4.0 and 233 μM) did not agree with apparent Km's deduced from fluorescence quenching of the introduced tryptophan residue (on the order of nM, 0.016 and 13 μM). On the other hand, in case of α(K175A/T176A/W463F)3β(Y341W)3γ, which lacks noncatalytic nucleotide binding sites, the apparent Km of ATPase activity (10 μM) roughly agreed with the highest Km of fluorescence measurements (27 μM). The results indicate that in case of α(W463F)3β(Y341W)3γ, the activating effect of ATP binding to noncatalytic sites dominates overall ATPase kinetics and the highest apparent Km of ATPase activity does not represent the ATP binding to a catalytic site. In case of α(K175A/T176A/W463F)3β(Y341W)3γ, the Km of ATPase activity reflects the ATP binding to a catalytic site due to the lack of noncatalytic sites. The Eadie-Hofstee plot of ATPase reaction by α(K175A/T176A/W463F)3β(Y341W)3γ was rather linear compared with that of α(W463F)3β(Y341W)3γ, if not perfectly straight, indicating that the apparent negative cooperativity observed for wild-type F1-ATPase is due to the ATP binding to catalytic sites and noncatalytic sites. Thus, the frequently observed Km's of 100-300 μM and 1-30 μM range for wild-type F1-ATPase correspond to ATP binding to a noncatalytic site and catalytic site, respectively. 相似文献
2.
光驱动钠离子泵是近年来发现的全新的光遗传学工具蛋白,在光激发下向膜外主动运输钠离子。然而,其钠离子传递的机制仍不清楚。该家族成员NdR2的可见吸收光谱、荧光光谱和等温滴定量热的结果说明:基态下的NdR2不结合钠离子。进一步的钠离子传递动力学和功能实验发现NdR2在酸性条件下的钠离子传递发生改变。这种pH依赖性的钠离子传递有助于深入理解光驱动钠离子泵中的钠离子的传递机制。 相似文献
3.
Methods are described to classify nucleotide binding sites of the mitochondrial coupling factor F1 from yeast on the basis of their affinities and stability properties. High affinity sites or states for ATP and related adenine analogs and low affinity sites or states which bind a broad range of different nucleotide triphosphates are found. The results are discussed in terms of a two site, two cycle scheme, where binding of nucleotide at one site facilitates the release of nucleotide at a second site. 相似文献
4.
Ernst J. Wolvetang Ronald J.A. Wanders Ruud B.H. Schutgens Jan A. Berden Joseph M. Tager 《Biochimica et Biophysica Acta (BBA)/General Subjects》1990,1035(1):6-11
Highly purified peroxisomal fractions from rat liver contain ATPase activity (18.8 ± 0.1 nmol/min per mg, n = 6). This activity is about 2% of that found in purified mitochondrial fractions. Measurement of marker enzyme activities and immunoblotting of the peroxisomal fraction with an antiserum raised against the β-subunit of mitochondrial ATPase indicates that the ATPase activity in the peroxisomal fractions can not be ascribed to contamination with mitochondria or other subcellular organelles. From the sensitivity of the ATPase present in the peroxisomal fraction towards a variety of ATPase inhibitors, we conclude that it displays both V-type and F-type features and is distinguishable from both the mitochondrial F1F0-ATPase and the lysosomal V-type ATPase. 相似文献
5.
ATP synthesis by oxidative phosphorylation and photophosphorylation, catalyzed by F1F0-ATP synthase, is the fundamental means of cell energy production. Earlier mutagenesis studies had gone some way to describing the mechanism. More recently, several X-ray structures at atomic resolution have pictured the catalytic sites, and real-time video recordings of subunit rotation have left no doubt of the nature of energy coupling between the transmembrane proton gradient and the catalytic sites in this extraordinary molecular motor. Nonetheless, the molecular events that are required to accomplish the chemical synthesis of ATP remain undefined. In this review we summarize current state of knowledge and present a hypothesis for the molecular mechanism of ATP synthesis. 相似文献
6.
The ratio between the amount of oligomycin-sensitivity-conferring protein (OSCP) and the amount of the and β subunits of F1-ATPase in the mitochondria has been determined by a method combining electrophoresis, electrotransfer and immunotitration with monoclonal antibodies. The peptides separated in SDS-polyacrylamide gel electrophoresis were blotted to nitrocellulose sheets by electrotransfer. The nitrocellulose sheets were incubated with 125I-labelled purified monoclonal antibodies specific to various peptides. The 125I-labelled immune complexes were located by immunodecoration using peroxidase-conjugated second antibodies and the blotted peptides were revealed with H2O2 and -naphthol. The amount of immune complex present on the nitrocellulose was determined by counting the radioactivity present on the spots. The amount of peptide blotted is directly proportional to the amount of protein loaded on the electrophoresis. By comparing standard curves made with the isolated proteins to the values obtained in the presence of various amounts of the membrane-protein complex, one can calculate the content of this peptide in the membrane. It was found that the mitochondrial membrane contains 2 mol of OSCP per mol of F1. 相似文献
7.
Ouabain Binding, ATP Hydrolysis, and Na+ ,K+ -Pump Activity During Chemical Modification of Brain and Muscle Na+ ,K+ -ATPase 总被引:1,自引:0,他引:1
J. Teisinger H. Zemková P. Svoboda E. Amler F. Vyskoil 《Journal of neurochemistry》1992,58(3):1066-1072
The effects of 16 group-specific, amino acid-modifying agents were tested on ouabain binding, catalytical activity of membrane-bound (rat brain microsomal), sodium dodecyl sulfate-treated Na+,K(+)-ATPase, and Na+,K(+)-pump activity in intact muscle cells. With few exceptions, the potency of various tryptophan, tyrosine, histidine, amino, and carboxy group-oriented drugs to suppress ouabain binding and Na+,K(+)-ATPase activity correlated with inhibition of the Na+,K(+)-pump electrogenic effect. ATP hydrolysis was more sensitive to inhibition elicited by chemical modification than ouabain binding (membrane-bound or isolated enzyme) and than Na+,K(+)-pump activity. The efficiency of various drugs belonging to the same "specificity" group differed markedly. Tyrosine-oriented tetranitromethane was the only reagent that interfered directly with the cardiac receptor binding site as its inhibition of ouabain binding was completely protected by ouabagenin preincubation. The inhibition elicited by all other reagents was not, or only partially, protected by ouabagenin. It is surprising that agents like diethyl pyrocarbonate (histidine groups) or butanedione (arginine groups), whose action should be oriented to amino acids not involved in the putative ouabain binding site (represented by the -Glu-Tyr-Thr-Trp-Leu-Glu- sequence), are equally effective as agents acting on amino acids present directly in the ouabain binding site. These results support the proposal of long-distance regulation of Na+,K(+)-ATPase active sites. 相似文献
8.
High-level expression of soluble subunit b of F1F0 ATP synthase in Escherichia coli cell-free system
Jiazhang Lian Yi Ma Jin Cai Ming Wu Jufang Wang Xiaoning Wang Zhinan Xu 《Applied microbiology and biotechnology》2009,85(2):303-311
The overexpression of subunit b of F1F0 adenosine triphosphate (ATP) synthase from Escherichia coli is so toxic that it even prevents the transformation of plasmids encoding this protein into E. coli BL21 (DE3). In the present work, E. coli cell-free system was chosen as an alternative to express this highly toxic membrane protein. This protein was either produced
as precipitates followed by detergent resolubilization or expressed as a soluble form with detergent addition. Among several
types of tested detergents, Brij 58 could effectively solubilize approximately 85% of the target membrane protein within a
wide range of concentration (48 to 178 times critical micelle concentration [CMC]) with little effect on the expression level.
With the presence of Brij 58 at the final concentration of 96 times CMC in the E. coli cell-free system, 789 μg/mL of soluble subunit b was achieved after 4 h biosynthesis, which is the highest level for the expression of membrane proteins in a batch-mode cell-free
expression system. The present work provides a rapid and efficient procedure of expressing one membrane protein with high
cytotoxicity in the cell-free system and will be helpful to further exploration of reconstituting F1F0 ATP synthase into liposome or polymer vesicle to design a nanoelectromechanical system device. 相似文献
9.
10.
Site-directed mutagenesis was used to generate three mutations in the uncB gene encoding the a-subunit of the F0 portion of the F0F1-ATPase of Escherichia coli. These mutations directed the substitution of Arg-210 by Gln, or of His-245 by Leu, or of both Lys-167 and Lys-169 by Gln. The mutations were incorporated into plasmids carrying all the structural genes encoding the F0F1-ATPase complex and these plasmids were used to transform strain AN727 (uncB402). Strains carrying either the Arg-210 or His-245 substitutions were unable to grow on succinate as sole carbon source and had uncoupled growth yields. The substitution of Lys-167 and Lys-169 by Gln resulted in a strain with growth characteristics indistinguishable from a normal strain. The properties of the membranes from the Arg-210 or His-245 mutants were essentially identical, both being proton impermeable and both having ATPase activities resistant to the inhibitor DCCD. Furthermore, in both mutants, the F1-ATPase activities were inhibited by about 50% when bound to the membranes. The membrane activities of the mutant with the double lysine change were the same as for a normal strain. The results are discussed in relation to a previously proposed model for the F0 (Cox, G.B., Fimmel, A.L., Gibson, F. and Hatch, L. (1986) Biochim. Biophys. Acta 849, 62–69). 相似文献
11.
It has been claimed (Andreu, J.M., Warth, R. and Muñoz, E. (1978) FEBS Lett. 86, 1–5) that the F1-ATPase of Micrococcus lysodeikticus is a glycoprotein containing mannose and glucose as the principal sugars. Even after extensive purification of M. lysodeikticus F1-ATPase by DEAE-Sephadex A25 chromatography, carbohydrate contents varying from 2.7 to 10.8% have been found. Concanavalin A-reactive components corresponding to the succinylated lipomannan have been detected and separated from the ATPase in purified F1 preparations by immunoelectrophoresis (rocket and two-dimensional) through agarose gels containing concanavalin A. Passage of the purified F1-ATPase through concanavalin A-Sepharose 4B columns removed the carbohydrate component(s) without loss of the specific activity of the ATPase. Mannose was the only sugar detectable by gas-liquid chromatography of the F1-ATPase before Con A-Sepharose 4B chromatography and it was completely eliminated after chromatography. No qualitative or quantitative changes in the subunit (, β, γ, δ and ε) profiles were detectable when the sodium dodecyl sulfate polyacrylamide gels were scanned by densitometry of F1-ATPase before and after Con A-Sepharose 4B chromatography. We conclude that there is no evidence of carbohydrate covalently linked to this F1-ATPase and that this membrane protein is not a glycoprotein. The presence of carbohydrate is attributable to contamination with lipomannan. 相似文献
12.
Tamara Azarashvili Irina Odinokova Anush Bakunts Vadim Ternovsky Olga Krestinina Jaana Tyynelä Nils-Erik Leo Saris 《Cell calcium》2014
Phosphorylated and non-phosphorylated forms of the F0F1-ATPase subunit c from rat liver mitochondria (RLM) were purified and their effect on the opening of the permeability transition pore (mPTP) was investigated. Addition of dephosphorylated subunit c to RLM induced mitochondrial swelling, decreased the membrane potential and reduced the Ca2+ uptake capacity, which was prevented by cyclosporin A. The same effect was observed in the presence of storage subunit c purified from livers of sheep affected with ceroid lipofuscinosis. In black-lipid bilayer membranes subunit c increased the conductance due to formation of single channels with fast and slow kinetics. The dephosphorylated subunit c formed channels with slow kinetics, i.e. the open state being of significantly longer duration than in the case of channels formed by the phosphorylated form that had short life spans and fast kinetics. The channels formed were cation-selective more so with the phosphorylated form. Subunit c of rat liver mitochondria was able to bind Ca2+. Collectively, the data allowed us to suppose that subunit c F0F1-ATPase might be a structural/regulatory component of mPTP exerting its role in dependence on phosphorylation status. 相似文献
13.
David B. Hicks 《BBA》2010,1797(8):1362-1377
This review focuses on the ATP synthases of alkaliphilic bacteria and, in particular, those that successfully overcome the bioenergetic challenges of achieving robust H+-coupled ATP synthesis at external pH values > 10. At such pH values the protonmotive force, which is posited to provide the energetic driving force for ATP synthesis, is too low to account for the ATP synthesis observed. The protonmotive force is lowered at a very high pH by the need to maintain a cytoplasmic pH well below the pH outside, which results in an energetically adverse pH gradient. Several anticipated solutions to this bioenergetic conundrum have been ruled out. Although the transmembrane sodium motive force is high under alkaline conditions, respiratory alkaliphilic bacteria do not use Na+- instead of H+-coupled ATP synthases. Nor do they offset the adverse pH gradient with a compensatory increase in the transmembrane electrical potential component of the protonmotive force. Moreover, studies of ATP synthase rotors indicate that alkaliphiles cannot fully resolve the energetic problem by using an ATP synthase with a large number of c-subunits in the synthase rotor ring. Increased attention now focuses on delocalized gradients near the membrane surface and H+ transfers to ATP synthases via membrane-associated microcircuits between the H+ pumping complexes and synthases. Microcircuits likely depend upon proximity of pumps and synthases, specific membrane properties and specific adaptations of the participating enzyme complexes. ATP synthesis in alkaliphiles depends upon alkaliphile-specific adaptations of the ATP synthase and there is also evidence for alkaliphile-specific adaptations of respiratory chain components. 相似文献
14.
Mohammad Delawar Hossain Shou Furuike Yasuhiro Onoue Kengo Adachi Masasuke Yoshida Kazuhiko Kinosita Jr. 《BBA》2010,1797(4):435-195
F1-ATPase is a rotary molecular motor in which the γ subunit rotates inside the cylinder made of α3β3 subunits. We have studied the effects of sodium dodecyl sulfate (SDS) on the rotational and ATP hydrolysis activities of F1-ATPase. Bulk hydrolysis activity at various SDS concentrations was examined at 2 mM ATP. Maximal stimulation was obtained at 0.003% (w/v) SDS, the initial (least inhibited) activity being about 1.4 times and the steady-state activity 3-4 times the values in the absence of SDS. Rotation rates observed with a 40-nm gold bead or a 0.29-μm bead duplex as well as the torque were unaffected by the presence of 0.003% SDS. The fraction of beads that rotated, in contrast, tended to increase in the presence of SDS. SDS seems to bring inactive F1 molecules into an active form but it does not alter or enhance the function of already active F1 molecules significantly. 相似文献
15.
F1-ATPase is a rotatory molecular motor fueled by ATP nucleotides. Different loads can be attached to the motor axis to show that it rotates in main discrete steps of 120° with substeps of ∼80° and 40°. Experimental data show the dependence on the mean rotational velocity ω with respect to the external control parameters: the nucleotide concentration [ATP] and the friction of the load γL. In this work we present a theoretical analysis of the experimental data whose main results are: 1), A derivation of a simple analytical formula for ω([ATP], γL) that compares favorably with experiments; 2), The introduction of a two-state flashing ratchet model that exhibits experimental phenomenology of a greater specificity than has been, to our knowledge, previously available; 3), The derivation of an argument to obtain the values of the substep sizes; 4), An analysis of the energy constraints of the model; and 5), The theoretical analysis of the coupling ratio between the ATP consumed and the success of a forward step. We also discuss the compatibility of our approach with recent experimental observations. 相似文献
16.
The pH dependence of the reaction of dicyclohexylcarbodiimide with the essential aspartyl-61 residue in subunit c of Escherichia coli ATP synthase was compared in membranes and in a detergent dispersed preparation of the enzyme. The rate of reaction was estimated by measuring the inactivation of ATPase activity. The reaction with the detergent dispersed form of the enzyme proved to be pH sensitive with the essential aspartyl group titrating with a pKa=8. However, when measured with E. coli membranes, the reaction proved to be pH insensitive. The results suggest that the reacting aspartyl-61 residues are shielded from the bulk aqueous solvent when in the membrane, but then become aqueous-accessible following detergent solubilization. 相似文献
17.
18.
We have measured the inhibitory potencies of several local anesthetics (procaine, lidocaine, tetracaine and dibucaine) and related compounds (chlorpromazine, procainamide and propranolol) on the ATPase activities of bovine heart submitochondrial particles and purified F1 extracted from these particles. All of these agents cause inhibition of ATPase in F1 as well as in submitochondrial particles. A linear relationship is found between the log of the octanol/water partition coefficients and the log of the concentrations required for 50% inhibition of F1. Sedimentation velocity ultracentrifugation and polyacrylamide gel electrophoresis showed that 1.0 mM tetracaine caused partial dissociation of the F1 complex. Complete reversibility of the enzyme inhibitory effects was demonstrated, however. This work shows that local anesthetics can affect protein structure and enzyme activity without the mediation of lipid. 相似文献
19.
F1-ATPase catalyzes ATP hydrolysis to drive the central γ-shaft rotating inside a hexameric cylinder composed of alternating α and β subunits. Experiments showed that the rotation of γ-shaft proceeds in steps of 120° and each 120°-rotation is composed of an 80° substep and a 40° substep. Here, based on the previously proposed models, an improved physical model for chemomechanical coupling of F1-ATPase is presented, with which the two-substep rotation is well explained. One substep is driven by the power stroke upon ATP binding, while the other one resulted from the passage of γ-shaft from previous to next adjacent β subunits via free diffusion. Using the model, the dynamics and kinetics of F1-ATPase, such as the rotating time of each substep, the dwell time at each pause and the rotation rate, are analytically studied. The theoretical results obtained with only three adjustable parameters reproduce the available experimental data well. 相似文献
20.
Lizhong Xu 《BBA》2008,1777(11):1422-1431
The enzyme F1-ATPase is a rotary nanomotor in which the central γ subunit rotates inside the cavity made of α3β3 subunits. The experiments showed that the rotation proceeds in steps of 120° and each 120° step consists of 80° and 40° substeps. Here the Author proposes a stochastic wave mechanics of the F1-ATPase motor and combines it with the structure-based kinetics of the F1-ATPase to form a chemomechanic coupled model. The model can reproduce quantitatively and explain the experimental observations about the F1 motor. Using the model, several rate-limited situations about γ subunit rotation are proposed, the effects of the friction and the load on the substeps are investigated and the chemomechanic coupled time during ATP hydrolysis cycle is determined. 相似文献