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1.
Aspects of Subunit Interactions in the Chloroplast ATP Synthase (I. Isolation of a Chloroplast Coupling Factor 1-Subunit III Complex from Spinach Thylakoids) 总被引:1,自引:1,他引:0
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A chloroplast ATP synthase complex (CF1 [chloroplast-coupling factor 1]-CF0 [membrane-spanning portion of chloroplast ATP synthase]) depleted of all CF0 subunits except subunit III (also known as the proteolipid subunit) was purified to study the interaction between CF1 and subunit III. Subunit III has a putative role in proton translocation across the thylakoid membrane during photophosphorylation; therefore, an accurate model of subunit inter-actions involving subunit III will be valuable for elucidating the mechanism and regulation of energy coupling. Purification of the complex from a crude CF1-CF0 preparation from spinach (Spinacia oleracea) thylakoids was accomplished by detergent treatment during anion-exchange chromatography. Subunit III in the complex was positively identified by amino acid analysis and N-terminal sequencing. The association of subunit III with CF1 was verified by linear sucrose gradient centrifugation, immunoprecipitation, and incorporation of the complex into asolectin liposomes. After incorporation into liposomes, CF1 was removed from the CF1-III complex by ethylenediaminetetracetate treatment. The subunit III-proteoliposomes were competent to rebind purified CF1. These results indicate that subunit III directly interacts with CF1 in spinach thylakoids. 相似文献
2.
Aspects of Subunit Interactions in the Chloroplast ATP Synthase (II. Characterization of a Chloroplast Coupling Factor 1-Subunit III Complex from Spinach Thylakoids) 总被引:2,自引:2,他引:0
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A complex between chloroplast-coupling factor 1 (CF1) and subunit III of the membrane-spanning portion of the chloroplast ATP synthase (CF0), isolated as described in the accompanying paper (C.M. Wetzel and R.E. McCarty [1993] Plant Physiol 102: 241-249), has been further characterized. A comparison of the ATPase activities of CF1, CF1-subunit III, and the chloroplast ATP synthase (CF1-CF0) holoenzyme revealed that the properties of CF1-subunit III more closely resemble those of CF1-CF0 than those of CF1. In particular, the Ca2+-ATPase activity after reduction of the enzyme with dithiothreitol was much lower in CF1-subunit III and CF1-CF0 than in CF1, suggesting that the association of the inhibitory [epsilon] subunit is tightened by the presence of either CF0 or subunit III. Cold stability is a property of CF1-CF0 in thylakoid membranes. The ATPase activity of CF1 incubated in the cold in the presence of asolectin liposomes was lost more rapidly than that of either CF1-subunit III or CF1-CF0 incorporated into liposomes. Removal of the [epsilon] subunit from all three preparations resulted in marked stimulation of their ATPase activity. Although subunit III was also removed during depletion of the [epsilon] subunit, it is not known whether the two subunits interact directly. CF1 deficient in the [epsilon] subunit binds to liposomes containing either subunit III or CF0. Taken together, these results provide evidence that the association of CF1 and subunit III of CFo is specific and may play a role in enzyme regulation. 相似文献
3.
Subunit delta of H(+)-ATPases: at the interface between proton flow and ATP synthesis 总被引:2,自引:0,他引:2
The ATP synthases in photophosphorylation and respiration are of the F-type with a membrane-bound proton channel, F0, and an extrinsic catalytic portion, F1. The properties of one particular subunit, delta (in chloroplasts and Escherichia coli) and OSCP (in mitochondria), are reviewed and the role of this subunit at the interface between F0 and F1 is discussed. Delta and OSCP from the three sources have in common the molecular mass (approximately 20 kDa), an elongated shape (axial ratio in solution about 3:1), one high-affinity binding site to F1 (Kd approximately 100 nM) plus probably one or two further low-affinity sites. When isolated delta is added to CF1-depleted thylakoid membranes, it can block proton flow through exposed CF0 channels, as do CF1 or CF1(-delta)+ delta. This identifies delta as part of the proton conductor or, alternatively, conformational energy transducer between F0 (proton flow) and F1 (ATP). Hybrid constructs as CF1(-delta)+ E. coli delta and EF1(-delta)+ chloroplast delta diminish proton flow through CF0.CF1(-delta) + E. coli delta does the same on EF0. Impairment of proton leaks either through CF0 or through EF0 causes "structural reconstitution' of ATP synthesis by remaining intact F0F1. Functional reconstitution (ATP synthesis by fully reconstructed F0F1), however, is absolutely dependent on the presence of subunit delta and is therefore observed only with CF1 or CF1(-delta) + chloroplast delta on CF0 and EF1 or EF1(-delta) + E. coli delta on EF0. The effect of hybrid constructs on F0 channels is surprising in view of the limited sequence homology between chloroplast and E. coli delta (36% conserved residues including conservative replacements). An analysis of the distribution of the conserved residues at present does not allow us to discriminate between the postulated conformational or proton-conductive roles of subunit delta. 相似文献
4.
The chloroplast ATP-synthase catalyzes ATP synthesis coupled to transmembrane proton transport. The enzyme consists of two parts, a membrane-embedded F(0) part and an extrinsic F(1) part, which are linked by two connectors. One of these rotates during catalysis and the other remains static. Although the atomic structures of various sub-complexes and individual subunits have been reported, only limited structural information on the complex, as a whole, is available. In particular, information on the static connector is lacking. We contribute a three-dimensional map at about 20-A resolution, derived from electron cryomicroscopy of enzymes embedded in vitrified buffer followed by single particle image analysis. In the three-dimensional map both connectors, between the F(1) part and the F(0) part, are clearly visible. The static connector is tightly attached to an alpha subunit and faces the side of the neighboring beta subunit. The three-dimensional map provides a scaffold for fitting in the known atomic structures of various subunits and sub-complexes, and suggests that the oxidized, non-activated ATP-synthase from chloroplasts adopts a unique resting position. 相似文献
5.
6.
F0F1 ATP synthases synthesize ATP in their F1 portion at the expense of free energy supplied by proton flow which enters the enzyme through their channel portion F0. The smaller subunits of F1, especially subunit delta, may act as energy transducers between these rather distant functional units. We have previously shown that chloroplast delta, when added to thylakoids partially depleted of the coupling factor CF1, can reconstitute photophosphorylation by inhibiting proton leakage through exposed coupling factor CF0. In view of controversies in the literature, we reinvestigated two further aspects related to subunit delta, namely (a) its stoichiometry in CF0CF1 and (b) whether or not delta is required for photophosphorylation. By rocket immunoelectrophoresis of thylakoid membranes and calibration against purified delta, we confirmed a stoichiometry of one delta per CF0CF1. In CF1-depleted thylakoids photophosphorylation could be reconstituted not only by adding CF1 and subunit delta but, surprisingly, also by CF1 (-delta). We found that the latter was attributable to a contamination of CF1 (-delta) preparations with integral CF1. To lesser extent CF1 (-delta) acted by complementary rebinding to CF0 channels that were closed because they contained delta [CF0(+delta)]. This added catalytic capacity to proton-tight thylakoid vesicles. The ability of subunit delta to control proton flow through CF0 and the absolute requirement for delta in restoration of photophosphorylation suggest an essential role of this small subunit at the interface between the large portions of ATP synthase: delta may be part of the coupling site between electrochemical, conformational and chemical events in this enzyme. 相似文献
7.
Subunit interactions within the chloroplast ATP synthase (CF0-CF1) as deduced by specific depletion of CF0 polypeptides 总被引:1,自引:0,他引:1
The proton-linked ATP synthase (CF1-CF0) of chloroplasts consists of a catalytic component (CF1) and a membrane-embedded part (CF0) that interacts with CF1 and contains a proton channel. The subunits of CF0 which are involved in binding of CF1 were studied by examining the effect of selective depletion of subunits I, II, and IV of CF0 from the chloroplast ATP synthase on the association of the remaining CF0 subunits with CF1. Dissociated CF0 subunits were identified by sucrose density gradient centrifugation. Removal of subunit IV alone from CF0-CF1 did not cause dissociation of the other CF0 subunits from CF1. Upon removal of both subunits I and IV from CF0-CF1, subunit II also dissociated, but subunit III was still bound to CF1. Thus, at least two subunits of CF0, I and III, directly associate with CF1. Subunit II is unlikely to bind CF1 directly and may associate with subunit I. Although depletion of subunit IV does not cause dissociation of CF0 from CF1, its interaction with CF1 subunits is uncertain. 相似文献
8.
The structure of the proton-pumping vacuolar ATPase (V-ATPase) from bovine brain clathrin coated vesicles was analyzed by electron microscopy and single molecule image analysis. A three-dimensional structural model of the complex was calculated by the angular reconstitution method at a resolution of 27 A. Overall, the appearance of the V(0) and V(1) domains in the three-dimensional model of the intact bovine V-ATPase resembles the models of the isolated bovine V(0) and yeast V(1) domains determined previously. To determine the binding position of subunit H in the V-ATPase, electron microscopy and cysteine-mediated photochemical cross-linking were used. Difference maps calculated from projection images of intact bovine V-ATPase and a V-ATPase preparation in which the two H subunit isoforms were removed by treatment with cystine revealed less protein density at the bottom of the V(1) in the subunit H-depleted enzyme, suggesting that subunit H isoforms bind at the interface of the V(1) and V(0) domains. A comparison of three-dimensional models calculated for intact and subunit H-depleted enzyme indicated that at least one of the subunit H isoforms, although poorly resolved in the three-dimensional electron density, binds near the putative N-terminal domain of the a subunit of the V(0). For photochemical cross-linking, unique cysteine residues were introduced into the yeast V-ATPase B subunit at sites that were localized based on molecular modeling using the crystal structure of the mitochondrial F(1) domain. Cross-linking was performed using the photoactivatable sulfhydryl reagent 4-(N-maleimido)benzophenone. Cross-linking to subunit H was observed from two sites on subunit B (E494 and T501) predicted to be located on the outer surface of the subunit closest to the membrane. Results from both electron microscopy and cross-linking analysis thus place subunit H near the interface of the V(1) and V(0) domains and suggest a close structural similarity between the V-ATPases of yeast and mammals. 相似文献
9.
10.
Structural studies of cytochrome reductase. Subunit topography determined by electron microscopy of membrane crystals of a subcomplex 总被引:5,自引:0,他引:5
Ubiquinol: cytochrome c reductase was isolated from Neurospora mitochondria as a protein-detergent complex and dissociated by mild salt treatment. Three parts were obtained and characterized. Firstly, a complex containing the subunits III (cytochrome b), IV (cytochrome c1), VI, VII, VIII and IX; secondly, a complex containing the subunits I and II; and thirdly, the single subunit V (iron-sulphur subunit). Membrane crystals were prepared from the cytochrome bc1 subunit complex and by combining tilted electron microscopic views of the crystals, a low-resolution three-dimensional structure was calculated. This structure was compared to that of the whole cytochrome reductase (previously determined by electron microscopy of membrane crystals). Protein density absent from the structure of the subunit complex was then attributed to the missing subunits according to their size and shape and their association with the phospholipid bilayer. 相似文献
11.
The isolated H(+)-ATPase from Escherichia coli (EF(0)F(1)) was investigated by electron microscopy of samples of negatively stained monodisperse molecules, followed by single-particle image processing. The resulting three-dimensional maps showed that the F(1)-part is connected by a prominent stalk to a more peripheral part of F(0). The F(1)-part showed stain-accessible cavities inside. In three-dimensional maps from selected particles, a second stalk could be detected which was thinner than the main stalk and is thought to correspond to the stator.Three-dimensional maps of the enzyme in the absence and in the presence of the substrate analogue adenyl-beta, gamma-imidodiphosphate (AMP-PNP) were calculated. Upon binding of AMP-PNP the three-dimensional maps showed no significant changes in the F(0)-part of EF(0)F(1), whereas a major conformational change in the F(1)-part was observed. (1) The diameter of the F(1)-part decreased upon binding of AMP-PNP mainly in the upper half of F(1). (2) Enzyme particles prepared in the presence of AMP-PNP had a pointed cap at the top of the F(1)-part which was missing in its absence. (3) The stain-accessible cavity inside the F(1)-part altered its pattern significantly. 相似文献
12.
Extraction and purification of the beta subunit and an active alpha beta-core complex from the spinach chloroplast CFoF1-ATP synthase. 总被引:2,自引:0,他引:2
Incubation of Rhodospirillum rubrum chromatophores with 2 M LiCl in the presence of MgATP has been shown to remove their F1 beta subunit leaving inactive but fully reconstitutable beta-less chromatophores (Gromet-Elhanan, Z., and Khanashvili, D., (1986) Methods Enzymol, 126, 528-538). A similar treatment of thoroughly washed spinach thylakoids has now been shown to release the CF1 beta subunit (CF1 beta) together with a complex containing equal amounts of CF1 alpha and CF1 beta (CF1 (alpha beta]. The purified CF1 (alpha beta) complex can reconstitute an active membrane-bound hybrid F0F1-ATPase with beta-less R. rubrum chromatophores and also catalyzes a low but very reproducible soluble MgATPase. Purified CF1 beta shows none of these activities although it can bind as efficiently as CF1 (alpha beta) to the beta-less chromatophores. By subjecting the crude spinach 2 m LiCl extract to dissociating conditions an enriched CF1 beta preparation is released. It contains traces of CF1 alpha and CF1 delta, is able to reconstitute an active hybrid F0F1-ATPase but, as the pure CF1 beta shows no soluble ATPase activity. These results indicate that trace amounts of CF1 alpha are enough for endowing CF1 beta with a reconstitutive capacity, but for exhibition of a significant soluble ATPase activity equivalent amounts of CF1 alpha and beta are required. The CF 1 (alpha beta) complex isolated and purified in this report thus represents the minimal catalytic core of the CF1-ATPase. 相似文献
13.
Recent developments on structural and functional aspects of the F1 sector of H+-linked ATPases 总被引:4,自引:0,他引:4
This review concerns the catalytic sector of F1 factor of the H+-dependent ATPases in mitochondria (MF1), bacteria (BF1) and chloroplasts (CF1). The three types of F1 have many similarities with respect to the structural parameters, subunit composition and catalytic mechanism. An alpha 3 beta 3 gamma delta epsilon stoichiometry is now accepted for MF1 and BF1; the alpha 2 beta 2 gamma 2 delta 2 epsilon 2 stoichiometry for CF1 remains as matter of debate. The major subunits alpha, beta and gamma are equivalent in MF1, BF1 and CF1; this is not the case for the minor subunits delta and epsilon. The delta subunit of MF1 corresponds to the epsilon subunit of BF1 and CF1, whereas the mitochondrial subunit equivalent to the delta subunit of BF1 and CF1 is probably the oligomycin sensitivity conferring protein (OSCP). The alpha beta gamma assembly is endowed with ATPase activity, beta being considered as the catalytic subunit and gamma as a proton gate. On the other hand, the delta and epsilon subunits of BF1 and CF1 most probably act as links between the F1 and F0 sectors of the ATPase complex. The natural mitochondrial ATPase inhibitor, which is a separate protein loosely attached to MF1, could have its counterpart in the epsilon subunit of BF1 and CF1. The generally accepted view that the catalytic subunit in the different F1 species is beta comes from a number of approaches, including chemical modification, specific photolabeling and, in the case of BF1, use of mutants. The alpha subunit also plays a central role in catalysis, since structural alteration of alpha by chemical modification or mutation results in loss of activity of the whole molecule of F1. The notion that the proton motive force generated by respiration is required for conformational changes of the F1 sector of the H+-ATPase complex has gained acceptance. During the course of ATP synthesis, conversion of bound ADP and Pi into bound ATP probably requires little energy input; only the release of the F1-bound ATP would consume energy. ADP and Pi most likely bind at one catalytic site of F1, while ATP is released at another site. This mechanism, which underlines the alternating cooperativity of subunits in F1, is supported by kinetic data and also by the demonstration of partial site reactivity in inactivation experiments performed with selective chemical modifiers. One obvious advantage of the alternating site mechanism is that the released ATP cannot bind to its original site.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
14.
Possible structural and functional similarities between the channel part, CF0, of chloroplast ATPase (CF0CF1) and ion channels permeable to monovalent cations were investigated using high-affinity toxins mainly targeted against voltage-sensitive K+ channels. In particular, the effect of the K(+)-channel blocker alpha-dendrotoxin and the crude scorpion venom of Leiurus quinquestriatus hebraeus (LQ venom) on ATP synthesis in thylakoid membranes and in CF0CF1-containing liposomes was characterised. Alpha-dendrotoxin (K(i) approximately 5.05 microM) and the LQ venom (K(i) approximately 1.55 micrograms/ml) specifically inhibited ATP synthesis in thylakoid membranes and in CF0CF1-containing liposomes. Our results show that alpha-dendrotoxin and peptides of the LQ venom with an apparent molecular mass of about 4.0 kDa, probably isoforms of charybdotoxin, specifically bind to CF0CF1. This binding was reversible and induced a high leak conductance for H+ through CF0. The Ca(2+)-dependent ATPase activity of the isolated soluble part of CF0CF1 (CF1) was completely inhibited by 1 microM alpha-dendrotoxin, while the crude LQ venom, at concentrations up to 10 micrograms/ml, had no affect on ATPase activity. The concentration dependence of the inhibition by alpha-dendrotoxin indicates that approximately 2 mol alpha-dendrotoxin bind/mol CF0CF1 and 1 mol alpha-dendrotoxin/mol CF1. Known inhibitors of H(+)-flow-through CF0 acted in the presence of alpha-dendrotoxin synergistically. Dicyclohexylcarbodiimide and venturicidin, in contrast to their known effect of blocking H(+)-flow-through CF0, increased the leak conductance through CF0 in the presence of alpha-dendrotoxin drastically. This uncoupling effect indicates that their normal mode of blocking is a secondary effect. Binding of the inhibitors to their respective sites apparently does not affect the proton pathway in CF0, but induces a conformation which closes the channel part for H+. Protein sequence comparison between the known binding site of charybdotoxin in the shaker K+ channel from Drosophila [MacKinnon, R. & Heginbotham, L. (1990) Neuron 5, 767-771] and the choroplast ATPase showed that subunit III reveals a significant similarity (64%) in parts of its sequence (Gln28-Leu53) to the helix 5 and helix 6 (S5-S6) linker region (Ala413-Cys462; the charybdotoxin-binding site) of the shaker K+ channel. According to secondary-structure predictions, the homologous sequences in subunit III and the shaker K+ channel represent putative hydrophilic loops connecting two transmembrane alpha-helices. Apparently the shaker K+ channel and subunit III share significant topological features in these hydrophilic loops which may be part of the respective channel entrance. 相似文献
15.
Recent studies show that the epsilon subunit of bacterial and chloroplast F(1)F(0) ATPases is a component of the central stalk that links the F(1) and F(0) parts. This subunit interacts with alpha, beta and gamma subunits of F(1) and the c subunit ring of F(0). Along with the gamma subunit, epsilon is a part of the rotor that couples events at the three catalytic sites sequentially with proton translocation through the F(0) part. Structural data on the epsilon subunit when separated from the complex and in situ are reviewed, and the functioning of this polypeptide in coupling within the ATP synthase is considered. 相似文献
16.
17.
Studies reported here were undertaken to gain greater molecular insight into the complex structure of mitochondrial ATP synthase (F(0)F(1)) and its relationship to the enzyme's function and motor-related properties. Significantly, these studies, which employed N-terminal sequence, mass spectral, proteolytic, immunological, and functional analyses, led to the following novel findings. First, at the top of F(1) within F(0)F(1), all six N-terminal regions derived from alpha + beta subunits are shielded, indicating that one or more F(0) subunits forms a "cap." Second, at the bottom of F(1) within F(0)F(1), the N-terminal region of the single delta subunit and the C-terminal regions of all three alpha subunits are shielded also by F(0). Third, and in contrast, part of the gamma subunit located at the bottom of F(1) is already shielded in F(1), indicating that there is a preferential propensity for interaction with other F(1) subunits, most likely delta and epsilon. Fourth, and consistent with the first two conclusions above that specific regions at the top and bottom of F(1) are shielded by F(0), further proteolytic shaving of alpha and beta subunits at these locations eliminates the capacity of F(1) to couple a proton gradient to ATP synthesis. Finally, evidence was obtained that the F(0) subunit called "F(6)," unique to animal ATP synthases, is involved in shielding F(1). The significance of the studies reported here, in relation to current views about ATP synthase structure and function in animal mitochondria, is discussed. 相似文献
18.
Mutagenesis was used to generate seven truncation mutants of the spinach (Spinacia oleracea) chloroplast ATP synthase delta subunit lacking 5, 11, 17, or 35 amino acid residues from the N-terminus or 3, 9, or 15 residues from the C-terminus. Interactions between these mutants and all other subunits of the chloroplast ATPase were investigated by a yeast two-hybrid system. The results indicate that the N-terminal deletions mainly affected interactions between the delta subunit and the other part of CF(1), but did not significantly affect interactions with the CF(0) sector. In contrast, C-terminal truncations of the delta subunit mainly affected its interaction with the CF(0) sector and caused little impairment in interactions with the other part of CF(1). The conformation of the delta subunit C-terminal domain seems to be more sensitive to the truncations, as shown by minimal expression driven by C-terminal deleted (nine residues) mutants. Further studies showed C-terminal truncations of the delta subunit greatly impaired its ability to restore cyclic photophosphorylation in NaBr vesicles, whereas N-terminal truncations had little effect on the ability of delta to plug the CF(0) channel. None of the mutants impaired ATP hydrolysis by CF(1). 相似文献
19.
Konno H Suzuki T Bald D Yoshida M Hisabori T 《Biochemical and biophysical research communications》2004,318(1):17-24
To understand the regulatory function of the gamma and epsilon subunits of chloroplast ATP synthase in the membrane integrated complex, we constructed a chimeric FoF1 complex of thermophilic bacteria. When a part of the chloroplast F1 gamma subunit was introduced into the bacterial FoF1 complex, the inverted membrane vesicles with this chimeric FoF1 did not exhibit the redox sensitive ATP hydrolysis activity, which is a common property of the chloroplast ATP synthase. However, when the whole part or the C-terminal alpha-helices region of the epsilon subunit was substituted with the corresponding region from CF1-epsilon together with the mutation of gamma, the redox regulation property emerged. In contrast, ATP synthesis activity did not become redox sensitive even if both the regulatory region of CF1-gamma and the entire epsilon subunit from CF1 were introduced. These results provide important features for the regulation of FoF1 by these subunits: (1) the interaction between gamma and epsilon is important for the redox regulation of FoF1 complex by the gamma subunit, and (2) a certain structural matching between these regulatory subunits and the catalytic core of the enzyme must be required to confer the complete redox regulation mechanism to the bacterial FoF1. In addition, a structural requirement for the redox regulation of ATP hydrolysis activity might be different from that for the ATP synthesis activity. 相似文献
20.
Subunit H of the vacuolar (H+) ATPase inhibits ATP hydrolysis by the free V1 domain by interaction with the rotary subunit F 总被引:1,自引:0,他引:1
The vacuolar (H+) ATPases (V-ATPases) are large, multimeric proton pumps that, like the related family of F1F0 ATP synthases, employ a rotary mechanism. ATP hydrolysis by the peripheral V1 domain drives rotation of a rotary complex (the rotor) relative to the stationary part of the enzyme (the stator), leading to proton translocation through the integral V0 domain. One mechanism of regulating V-ATPase activity in vivo involves reversible dissociation of the V1 and V0 domains. Unlike the corresponding domains in F1F0, the dissociated V1 domain does not hydrolyze ATP, and the free V0 domain does not passively conduct protons. These properties are important to avoid generation of an uncoupled ATPase activity or an unregulated proton conductance upon dissociation of the complex in vivo. Previous results (Parra, K. J., Keenan, K. L., and Kane, P. M. (2000) J. Biol. Chem. 275, 21761-21767) showed that subunit H (part of the stator) inhibits ATP hydrolysis by free V1. To test the hypothesis that subunit H accomplishes this by bridging rotor and stator in free V1, cysteine-mediated cross-linking studies were performed. Unique cysteine residues were introduced over the surface of subunit H from yeast by site-directed mutagenesis and used as the site of attachment of the photo-activated cross-linking reagent maleimido benzophenone. After UV-activated cross-linking, cross-linked products were identified by Western blot using subunit-specific antibodies. The results indicate that the subunit H mutant S381C shows cross-linking between subunit H and subunit F (a rotor subunit) in the free V1 domain but not in the intact V1V0 complex. These results indicate that subunits H and F are proximal in free V1, supporting the hypothesis that subunit H inhibits free V1 by bridging the rotary and stator domains. 相似文献