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1.
F1() complexes containing equimolar ratios of the and subunits have been shown to function as active ATPases, whereas individually isolated and subunits show no real ATPase activity. These results indicate that the single-copy subunits are not required for F1-ATPase activity. The minimal F1()-core complexes exhibit, however, lower rates and some different properties from those of their parent whole F1 or 33 complexes. It is therefore concluded that for obtaining a full spectrum of the characteristic functional properties of an F1-ATPase the presence of the F1- subunit is also required. The implications of these findings on the subunit location of both catalytic and noncatalytic nucleotide binding sites is discussed.  相似文献   

2.
ATP synthase (F0F1) is driven by an electrochemical potential of H+ (H+). F0F1 is composed of an ion-conducting portion (F0) and a catalytic portion (F1). The subunit composition of F1 is 33. The active 33 oligomer, characterized by X-ray crystallography, has been obtained only from thermnophilic F1 (TF1). We proposed in 1984 that ATP is released from the catalytic site (C site) by a conformational change induced by the DELSEED sequence via -F0. In fact, cross-linking of DELSEED to stopped the ATP-driven rotation of in the center of 33. The torque of the rotation is estimated to be 420 pN·å from the H+ and H+-current through F0F1. The angular velocity () of is the rate-limiting step, because H+ increased theV max of H+ current through F0, but not theK m (ATP). The rotational unit of F0 (=ab2c10) is /5, while that in 33 is 2/3. This difference is overcome by an analog-digital conversion via elasticity around DELSEED with a threshold to release ATP. The distance at the C site is about 9.6 å (2,8-diN3-ATP), and tight Mg-ATP binding in 33 was shown by ESR. The rotational relaxation of TF1 is too rapid (=100 nsec), but the rate of AT(D)P-induced conformational change of 33 measured with a synchrotron is close to . The ATP bound between the P-loop and E188 is released by the shift of DELSEED from RGL. Considering the viscosity resistance and inertia of the free rotor (-c), there may be a stator containing OSCP (= of TF1) and F0-d to hold free rotation of 33.  相似文献   

3.
The structure of theEscherichia coli ATP synthase has been studied by electron microscopy and a model developed in which the and subunits of the F1 part are arranged hexagonally (in top view) alternating with one another and surrounding a central cavity of around 35 Å at its widest point. The and subunits are interdigitated in side view for around 60 Å of the 90 Å length of the molecule. The F1 narrows and has three-fold symmetry at the end furthest from the F0 part. The F1 is linked to F0 by a stalk approximately 45 Å long and 25–30 Å in diameter. The F0 part is mostly buried in the lipid bilayer. The subunit provides a domain that extends into the central cavity of the F1 part. The and subunits are in a different conformation when ATP+Mg2+ are present in catalytic sites than when ATP+EDTA are present. This is consistent with these two small subunits switching conformations as a function of whether or not phosphate is bound to the enzyme at the position of the phosphate of ATP. We suggest that this switching is the key to the coupling of catalytic site events with proton translocation in the F0 part of the complex.  相似文献   

4.
Exchange-out of amide tritium from labeled -subunit of 33 complex of F0F1-ATP synthase was not accelerated by ATP, suggesting that hemagglutinin-type transition of coiled-coil structure did not occur in -subunit. Local topology of nucleotide binding site and switch II region of G-protein resemble those of F1- subunit and other proteins which catalyze ATP-triggered reactions. Probably, binding of nucleotide to F0F1-ATP synthase induces conformational change of the switch II-like region with transforming subunit structure from open to closed form and this transformation results in loss of hydrogen bonds with the subunit, thus enabling the subunit to move.  相似文献   

5.
The basic structures of the catalytic portion (F1, 33) of ATP synthase are the 33 hexamer (oligomer with cooperativity) and 11 heterodimer (protomer). These were reconstituted from the and subunits of thermophilic F1 (TF1), and the 33 hexamer was crystallized. On electrophoresis, both the dimer and hexamer showed bands with ATPase activity. Using the dimer and hexamer, we studied the nucleotide-dependent rapid molecular dynamics. The formation of the hexamer required neither nucleotide nor Mg. The hexamer was dissociated into the dimer in the presence of MgADP, while the dimer was associated into the hexamer in the presence of MgATP. The hexamer, like mitochondrial F1 and TF1, showed two kinds of ATPase activity: one was cooperative and was inhibited by only one BzADP per hexamer, and the other was inhibited by three BzADP per hexamer.  相似文献   

6.
A rapid procedure is described for the separation of CMP-sialic acid:lactosylceramide sialyltransferase reaction components using Sep Pak C18 cartridges. The quantitative separation of the more polar nucleotide sugar, CMP-sialic acid, and its free acid from the less polar GM3-ganglioside is simple and rapid relative to previously described methods. Recovery of GM3 is optimized by the addition of phosphatidylcholine to the reaction mixture prior to the chromatographic step. Using rat liver Golgi membranes as a source of CMP-sialic acid: lactosylceramide sialyltransferase activity (GM3 synthase; ST-1), the transfer of [14C] sialic acid from CMP-[14C] sialic acid to lactosylceramide can be quantified by this assay. The procedure is reliable and may be applicable to the isolation of ganglioside products in otherin vitro glycosyltransferase assays.Abbreviations GM3 GM3-ganglioside - II3NeuAc-LacCer NeuAc2-3Gal1-4Glc1-1Cer - GD1a GD1a-ganglioside, IV3NeuAc, II3NeuAc-GgOse4Cer, NeuAc2-3Gal1-3GalNac1-4(NeuAc2-3)Gal1-4Glc1-1Cer - GD3 GD3-ganglioside, II3(NeuAc)2LacCer, NeuAc2-8NeuAc2-3Gal1-4Glc1-1Cer - GgOse4Cer asialo-GM1 Gal1-3GalNAc1-4Gal1-4Glc1-1Cer - FucGMI fucosyl-GMI-ganglioside, Fuc1-2Gal1-3GalNAc1-4Gal1-4 Glc1-1Cer - ST-1 GM3 synthase, CMP-sialic acid:lactosylceramide sialyltransferase - LacCer lactosylceramide, Gal1-4Glc1-1Cer - CMP-NeuAc cytidine 5-monophospho-N-acetylneuraminic acid - PC phosphatidylcholine - PMSF phenylmethylsulfonyl fluoride  相似文献   

7.
The structural and functional connection between the peripheral catalytic F1 sector and theproton-translocating membrane sector F0 of the mitochondrial ATP synthase is reviewed. Theobservations examined show that the N-terminus of subunit , the carboxy-terminal and centralregion of F0I-PVP(b), OSCP, and part of subunit d constitute a continuous structure, the lateralstalk, which connects the peripheries of F1 to F0 and surrounds the central element of thestalk, constituted by subunits and . The ATPase inhibitor protein (IF1) binds at one sideof the F1F0 connection. The carboxy-terminal segment of IF1 apparently binds to OSCP. The42L-58K segment of IF1, which is per se the most active domain of the protein, binds at thesurface of one of the three / pairs of F1, thus preventing the cyclic interconversion of thecatalytic sites required for ATP hydrolysis.  相似文献   

8.
The binding of oligomycin sensitivity conferring protein (OSCP) to soluble beef-heart mitochondrial ATPase (F1) has been investigated. OSCP forms a stable complex with F1, and the F1 · OSCP complex is capable of restoring oligomycin- and DCCD-sensitive ATPase activity to F1- and OSCP-depleted submitochondrial particles. The F1 · OSCP complex retains 50% of its ATPase activity upon cold exposure while free F1 is inactivated by 90% or more. Both free F1 and the F1 · OSCP complex release upon cold exposure a part—probably 1 out of 3—of their subunits; whether subunits are also lost is uncertain. The cold-treated F1 · OSCP complex is still capable of restoring oligomycin- and DCCD-sensitive ATPase activity to F1- and OSCP-depleted particles. OSCP also protects F1 against modification of its subunit by mild trypsin treatment. This finding together with the earlier demonstration that trypsin-modified F1 cannot bind OSCP indicates that OSCP binds to the subunit of F1 and that F1 contains three binding sites for OSCP. The results are discussed in relation to the possible role of OSCP in the interaction of F1 with the membrane sector of the mitochondrial ATPase system.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - OSCP oligomycin sensitivity conferring protein - SDS sodium dodecylsulfate This paper is dedicated to the memory of David E. Green—scholar, pioneer, visionary.  相似文献   

9.
ATP synthase (FoF1) consists of F1 (ATP-driven motor) and Fo (H+-driven motor). F1 is a complex of 33 subunits, and is the rotating cam in 33. Thermophilic F1 (TF1) is exceptional in that it can be crystallized as a monomer and an 33 oligomer, and it is sufficiently stable to allow refolding and reassembly of hybrid complexes containing 1, 2, and 3 modified or . The nucleotide-dependent open–close conversion of conformation is an inherent property of an isolated and energy and signals are transferred through / interfaces. The catalytic and noncatalytic interfaces of both mitochondrial F1 (MF1) and TF1 were analyzed by an atom search within the limits of 0.40 nm across the interfaces. Seven (plus thermophilic loop in TF1) contact areas are located at both the catalytic and noncatalytic interfaces on the open form. The number of contact areas on closed increased to 11 and 9, respectively, in the catalytic and noncatalytic interfaces. The interfaces in the barrel domain are immobile. The torsional elastic strain applied through the mobile areas is concentrated in hinge residues and the P-loop in . The notion of elastic energy in FoF1 has been revised. X-ray crystallography of F1 is a static snap shot of one state and the elastic hypotheses are still inconsistent with the structure, dyamics, and kinetics of FoF1. The domain motion and elastic energy in FoF1 will be elucidated by time-resolved crystallography.  相似文献   

10.
Growth rate estimates () of phytoplankton populations that were sampled from nitrogen-limited continuous cultures and then incubated for short durations in batch culture with added14C-HCO3 were significantly different than steady-state growth rates () for 3 of 5 marine phytoplankton species. Two diatoms,Thalassiosira weissflogii andChaetoceros simplex, displayed virtually identical growth rates (=) over a wide range of, whereas for a third diatom,Phaeodactylum tricornutum, was overestimated by an average of 40% compared to. In contrast, was underestimated by the14C technique for the two remaining species: up to 40% at a steady-state of 1.0 day–1 for the chlorophyteDunaliella tertiolecta and up to 100% at of 1.4 day–1 for the haptophytePavlova lutheri. For the latter two species the divergence between and appeared to increase with increasing steady-state. A simple model of labeled and total carbon flow between the aqueous phase and cellular biomass was constructed to demonstrate that respiration was negligible when=, but was significant when>. In the cases in which<, a rapid physiological alteration presumably took place once the steady state was disturbed and cells were placed in the incubation chambers, which perhaps was related to the nutritional state of the cultures at the time of sampling. Questions thus are raised regarding our ability to measure accurately primary productivity from shipboard experiments with confined samples of phytoplankton from nutrient-impoverished waters that probably are less hardy than the laboratory cultures used in these studies.  相似文献   

11.
Summary Hordeum spontaneum C. Koch emend. Bacht. varieties have been both intercrossed and crossed with two cultivated barley varieties ofH. vulgare (L.) emend Vav. et Bacht. with a view of eliciting the nature of inheriting the spikelet-pedicel of the lateral spikelets and the shape of their apex in the said wildgrowing barley. The investigations of F1 and F2 showed the inheritance of the spikelet-pedicel to have a dominating nature and to segregate in F2 in conformity with the Mendelian monohybrid type. In the second case the forms with shorter awn-like formations, or their rudiments, were dominating.As a result ofH. spontaneum x H. vulgare hybridization along with already known forms, new formations were received, they have been conditionally named by the author:sessiliproskowetzii, proskowfertillum, ischnofertillum, and pallipodum.
Zusammenfassung Im Rahmen größerer Untersuchungen über die Abstammung und Phylogenie der Gerste wurden mehrere Varietäten vonHordeum spontaneum C. Koch emend. Bacht. sowohl untereinander als auch mit zwei Varietäten der Kulturgerste,H. vulgare (L.) emend. Vav. et Bacht., gekreuzt. Es sollte geklärt werden, wie bei den genannten Wildgersten das Stielchen (pedicel) der Seitenährchen sowie die Ausbildung des Apex der Seitenährchen (d. h. ihre Begrannung) vererbt werden. Die Untersuchung der F1 und F2 zeigte, daß das Stielchen (gegenüber ungestielten Seitenährchen) dominant und gemäß einer monohybriden Mendelspaltung vererbt wird. Bezüglich der Ausbildung des Apex der Seitenährchen ergab sich im allgemeinen Dominanz der kürzeren oder rudimentären Grannen gegenüber längeren Grannen.Im Ergebnis der Hybridisation zwischenH. spontaneum undH. vulgare wurden, neben bereits bekannten, verschiedene neue Formen gefunden, die vom Autor vorläufig wie folgt benannt werden:sessiliproskowetzii, proskowfertillum, ischnofertillum, pallipodum.Die Ergebnisse werden im Zusammenhang mit Fragen der Abstammung der Kulturgerste diskutiert.


With 4 figures  相似文献   

12.
The -potentials of n-alkane droplets, formed by fatty acids, were studied in model systems of the culture liquid of yeasts (Candida maltosa) capable of utilizing n-alkanes. The value of the -potential was found to depend on the droplet size. The negative -potential of submicron droplets was so high that it prevented the droplets from coagulating with cells also possessing a high negative -potential. The dominant role of submicron n-alkane droplets in the kinetics of yeast growth could be accounted for by the existence of a mechanism regulating the contact interactions of individual cells with the droplets followed by the uptake of the substrate.  相似文献   

13.
Minimal photosynthetic catalytic F1() core complexes, containing equimolar ratios of the and subunits, were isolated from membrane-bound spinach chloroplast CF1 and Rhodospirillum rubrum chromatophore RrF1. A CF1-33 hexamer and RrF1-11 dimer, which were purified from the respective F1() complexes, exhibit lower rates and different properties from their parent F1-ATPases. Most interesting is their complete resistance to inhibition by the general F1 inhibitor azide and the specific CF1 inhibitor tentoxin. These inhibitors were earlier reported to inhibit multisite, but not unisite, catalysis in all sensitive F1-ATPases and were therefore suggested to block catalytic site cooperativity. The absence of this typical property of all F1-ATPases in the 11 dimer is consistant with the view that the dimer contains only a single catalytic site. The 33 hexamer contains however all F1 catalytic sites. Therefore the observation that CF1-33 can bind tentoxin and is stimulated by it suggests that the F1 subunit, which is required for obtaining inhibition by tentoxin as well as azide, plays an important role in the cooperative interactions between the F1-catalytic sites.Abbreviations CF0F1 chloroplast F0F1 - CF1 chloroplast F1 - CF1 chloroplast F1 subunit - CF1 chloroplast F1 subunit - CF1() a complex containing equal amounts of the CF1 and subunits - MF1 mitochondrial F1 - RrF0F1 Rhodospirillum rubrum F0F1 - RrF1 R. rubrum F1 - RrF1 R. rubrum F1 subunit - RrF1 R. rubrum F1 subunit - RrF1() a complex containing equal amounts of the RrF1 and subunits - Rubisco Ribulose-1,5-bisphosphate carboxylase - TF1 thermophilic bacterium PS3 F1  相似文献   

14.
(1) Extensive studies on proton-translocating ATPase (H+-ATPase) revealed that H+-ATPase is an energy transforming device universally distributed in membranes of almost all kinds of cells. (2) Crystallization of the catalytic portion (F1) of H+-ATPase showed that F1 is a hexagonal molecule with a central hole. The diameter of F1 is about 90 Å and its molecular weight is about 380,000. (3) Use of thermophilic F1 permits the complete reconstitution of F1 from its five subunits (, , , , and ) and demonstration of the gate function of the -complex, the catalytic function of (supported by and ), and the H+-translocating functions of all five subunits. (4) Studies using purified thermostable F0 showed that F0 is an H+-channel portion of H+-ATPase. The direct measurement of H+-flux through F0, sequencing of DCCD-binding protein, and isolation of F1-binding protein are described. (5) The subunit stoichiometry of F1 may be 33. (6) Reconstitution of stable H+-ATPase-liposomes revealed that ATP is directly synthesized by the flow of H+ driven by an electrochemical potential gradient and that H+ is translocated by ATP hydrolysis. This rules out functions for all the hypothetical components that do not belong to H+-ATPase in H+-driven ATP synthesis. The roles of conformation change and other phenomena in ATP synthesis are also discussed.  相似文献   

15.
F430 is the prosthetic group of the methylcoenzyme M reductase of methanogenic bacteria. The compound isolated from Methanosarcina barkeri appears to be identical to the one obtained from the only distinctly related Methanobacterium thermoautotrophicum. F430 is thermolabile and in the presence of acetonitrile or C10 in4 sup- two epimerization products are obtained upon heating; in the absence of these compounds F430 is oxidized to 12, 13-didehydro-F430. The latter is stereoselectively reduced under H2 atmosphere to F430 by cell-free extracts of M. barkeri or M. thermoautotrophicum. H2 may be replaced by the reduced methanogenic electron carrier coenzyme F420.Abbreviations CH3S-CoM methylcoenzyme M, 2-methylthioethanesulfonic acid - HS-CoM coenzyme M, 2-mercaptoethanesulfonic acid - F430 Ni(II) tetrahydro-(12, 13)-corphin with a uroporphinoid (III) ligand skeleton - 13-epi-F430 and 12,13-di-epi-F430 the 12, 13- and 12, 13-derivatives of F430 - 12, 13-didehydro-F430 F430 oxidized at C-12 and C-13 - coenzyme F420 7,8-didemethyl-8-hydroxy-5-deazaflavin derivative - coenzyme F420H2 reduced coenzyme F420 - MV+ methylviologen semiquinone - HPLC high-performance liquid chromatography  相似文献   

16.
Summary On t.l.c. plates 125I-cholera toxin binds to a disialoganglioside tentatively identified as GDlb with about 10 times less capacity than to ganglioside GM1. Binding of labeled toxin to both gangliosides was abolished in presence of excess amounts of unlabeled B subunit. Ganglioside extracts from human or pig intestinal mucosa showed toxin binding to gangliosides GM1 and GD1b. In ganglioside-containing lipid monolayers the penetration of the toxin was independent of the ganglioside binding capacity.Abbreviations GM2 Gal-NAc14Gal(3-2NeuAc)14G1c1Cer - GM1 Gal3Ga1-NAc14Gal(32NeuAc)14G1c11Cer - GD1a NeuAc23Ga113Gal-NAc14Gal(32NeuAc)14G1c11Cer - GD1b Gall3Gal-NAcl4Gal(32NeuAc82NeuAc)14Glc11Cer - GT1b NeuAc23Ga113Ga1-NAcal4Gal(3-2NeuAc82NeuAc)14G1c11Cer - dpPC 1,2-hexadecanoyl-sn-glycero-3-phosphocholine - dpPE 1,2-hexadecanoyl-sn-glycero-3-phosphoethanolamine  相似文献   

17.
The most commonly quoted mechanism of the coupling between the electrochemical proton gradient and the formation of ATP from ADP and Pi assumes that all states of the F1 portion of the ATP synthase have subunits in tight, loose, and open conformations. Models based on this assumption are inconsistent with some of the available experimental evidence. A mechanism that includes an additional subunit conformation, closed, observed in the rat liver structure overcomes these difficulties.  相似文献   

18.
F1-ATPases are large multimeric proteins that can be isolated from the membrane bound system that catalyzes the phosphorylation of ADP by inorganic phosphate in bacteria, plants, and mitochondria. They can be visualized in electron micrographs of the inner mitochondrial membranes where they appear as large protruding spheres 90 Å in diameter. The purified F1-ATPases have a molecular weight of 320,000 to 400,000 daltons and are composed of five non-identical subunits (, , , and ). The stoichiometry of these subunits in the complex is still unknown but compositions of the type 33 and 22222 were found to be consistent with some of the available experimental data. This review discusses the recent data and the experimental approaches utilized for the structural characterization of F1-ATPases.  相似文献   

19.
Neuroactive steroids and other positive modulators of GABAA receptors showed regional variation in both the efficacy and potency for modulation of [35S]TBPS binding to rat brain membrane homogenates, with biphasic concentration-dependence. GABA present in the binding assays prevented the enhancement phase of the steroid concentration-dependence plot while the antagonists bicuculline and RU5135 prevented the inhibition phase. Using recombinant GABAA receptors, expressed in insect cell line Sf9 using baculovirus, enhancement by steroids of [35S]TBPS binding was sensitive to the presence of the 2 subunit and the nature of the subunit (122S > 12, 62, 622S, and 62). As in cerebellum, addition of RU5135 reduced the inhibitory phase and revealed a small enhancement of TBPS binding by neuroactive steroids. The subunit-dependent interactions of steroid and GABA site ligands are consistent with a three-state model in which the receptor mono-liganded by GABA or steroid has a different affinity for TBPS than the resting state, and the receptor biliganded by GABA, steroid, or both has little affinity for TBPS.  相似文献   

20.
Summary A set of three 3D (1H, 13C, 15N) triple-resonance correlation experiments has been designed to provide H1-H8 intraresidue sugar-to-base correlations in purines in an unambiguous and efficient manner. Together, the HsCsNb, HsCs(N)bCb, and HbNbCb experiments correlate the H1 sugar proton to the H8 proton of the attached base by means of the {H1, C1, N9, C8, H8} heteronuclear scalar coupling network. The assignment strategy presented here allows for unambiguous H1-H8 intraresidue correlations, provided that no two purines have both the same H1 and C1 chemical shifts and the same C8 and N9 chemical shifts. These experiments have yielded H1-H8 intraresidue sugar-to-base correlations for all five guanosines in the [13C, 15N] isotopically labeled RNA duplex r(GGCGCUUGCGUC)2.  相似文献   

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