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1.
Cofactors in the biosynthesis of prostaglandins F and F 总被引:1,自引:0,他引:1
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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
4.
The rotation of an asymmetric core of subunits in F0F1-ATP synthases has been proposed as a means of coupling the exergonic transport of protons through F0 to the endergonic conformational changes in F1 required for substrate binding and product release. Here we review earlier evidence both for and against subunit rotation and then discuss our most recent studies using reversible intersubunit disulfide cross-links to test for rotation. We conclude that the subunit of F1 rotates relative to the surrounding catalytic subunits during catalytic turnover by both soluble F1 and membrane-bound F0F1. Furthermore, the inhibition of this rotation by the modification of F0 with DCCD suggests that rotation in F1 is obligatorily coupled to rotation in F0 as an integral part of the coupling mechanism. 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
7.
Demonstration of peptide:N-glycosidase F activity in endo-beta-N-acetylglucosaminidase F preparations 总被引:34,自引:0,他引:34
T H Plummer J H Elder S Alexander A W Phelan A L Tarentino 《The Journal of biological chemistry》1984,259(17):10700-10704
Endo-beta-N-acetylglucosaminidase F preparations from Flavobacterium meningosepticum have been found to contain peptide:N-glycosidase activity. Only the second activity, designated as peptide:N-glycosidase F, readily cleaves the beta-aspartylglycosylamine linkage of a fetuin triantennary complex glycopeptide, as shown by the isolation of the corresponding carbohydrate-free peptide containing aspartic acid and of an intact oligosaccharide with a di-N-acetylchitobiosyl moiety at the reducing end. Both activities in the mixture will hydrolyze a high mannose octaglycopeptide from ovalbumin, with the type of product formed being influenced by pH. At pH 4.0, only the endo-beta-N-acetylglucosaminidase F activity is functional, releasing octapeptide-GlcNAc and oligosaccharide-GlcNAc. At pH 9.3, the predominant cleavage is by peptide:N-glycosidase F at the glycosylamine bond, releasing octapeptide and oligosaccharide-GlcNAc-GlcNAc. This latter oligosaccharide is then hydrolyzed by endo-beta-N-acetylglucosaminidase F to oligosaccharide-GlcNAc plus GlcNAc. 相似文献
8.
Bovine heart submitochondrial particles depleted of F1, OSCP (oligomycin sensitivity-conferring protein), and F6 require the presence of cations to rebind F1. Among the cations tested, NH4+, Cs+, and Rb+ were most efficient, followed by K+, Na+, Li+, Ca2+, and Mg2+. The extent of F1 binding approached that occurring upon supplementation with F6 and/or OSCP, and was similar to the F1 content of particles prior to depletion. In the absence of cations, F6 and/or OSCP were ineffective in promoting the binding of F1 to the depleted particles. The F1 bound to the particles in the presence of cations alone was completely insensitive to oligomycin. It remained bound to the particles after removal of the cation, and could be rendered partially (approximately 50%) or maximally (less than 80%) oligomycin-sensitive upon the subsequent addition of OSCP or of F6 and OSCP, respectively. The surface potential of the particles, as determined by microelectrophoresis, was screened by all cations tested, regardless of their ability to promote the binding of F1; this was in contrast to earlier findings with particles depleted of F1 only, where the ability of cations to promote the rebinding of F1 paralleled their efficiency to neutralize the surface charge of the particle membrane. It is concluded that the effect of cations on the binding of F1 to F1-, F6-, and OSCP-depleted particles is due to a specific interaction of the cations with certain segments or components of the membrane. The results suggest the existence of a binding site for F1 on F0 in addition to the binding site(s) provided by F6 and OSCP. 相似文献
9.
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
10.
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
11.
Sialic acid was revealed in the antigens of virulent (503/830 and Schu) and vaccine (Schu-attenuated and 15-reduced) strains of F. tularensis and also in the antigens of F. novicida isolated by the treatment with trichloracetic acid, by the thiobarbiturate method of Warren. Its content depended on the culture virulence. Sialic acid was absent in the antigens of avirulent strains of the causative agent of tularemia (503-attenuated, 15-attenuated and 21/400), but, in difference from the virulent strains, there was revealed 2-desoxyribose on account of the presence of DNA. Serological activity of F. tularensis antigen decreased only 15 times in the antibody neutralization reaction after a complete release of sialic acid as a result of hydrolysis of the preparation in 0.1 N, H2SO4 at 80 degrees C for one hour, i.e. it persisted at a sufficiently high level. On the basis of investigations carried out it can be admitted that there existed a chemical association between the sialic acid and other composites in the polysaccharide of the virulent and the vaccine strains of F. tularensis, but that this acid played no significant role in the manifestation of specific properties by the antigen. 相似文献
12.
Origin of the F685 and F695 fluorescence in Photosystem II 总被引:2,自引:2,他引:0
Andrizhiyevskaya EG Chojnicka A Bautista JA Diner BA van Grondelle R Dekker JP 《Photosynthesis research》2005,84(1-3):173-180
The emission spectra of CP47-RC and core complexes of Photosystem II (PS II) were measured at different temperatures and excitation wavelengths in order to establish the origin of the emission and the role of the core antenna in the energy transfer and charge separation processes in PS II. Both types of particles reveal strong dependences of spectral shape and yield on temperature. The results indicate that the well-known F-695 emission at 77 K arises from excitations that are trapped on a red-absorbing CP47 chlorophyll, whereas the F-685 nm emission at 77 K arises from excitations that are transferred slowly from 683 nm states in CP47 and CP43 to the RC, where they are trapped by charge separation. We conclude that F-695 at 77 K originates from the low-energy part of the inhomogeneous distribution of the 690 nm absorbing chlorophyll of CP47, while at 4 K the fluorescence originates from the complete distribution of the 690 nm chlorophyll of CP47 and from the low-energy part of the inhomogeneous distribution of one or more CP43 chlorophylls. 相似文献
13.
Diez M Zimmermann B Börsch M König M Schweinberger E Steigmiller S Reuter R Felekyan S Kudryavtsev V Seidel CA Gräber P 《Nature structural & molecular biology》2004,11(2):135-141
Synthesis of ATP from ADP and phosphate, catalyzed by F(0)F(1)-ATP synthases, is the most abundant physiological reaction in almost any cell. F(0)F(1)-ATP synthases are membrane-bound enzymes that use the energy derived from an electrochemical proton gradient for ATP formation. We incorporated double-labeled F(0)F(1)-ATP synthases from Escherichia coli into liposomes and measured single-molecule fluorescence resonance energy transfer (FRET) during ATP synthesis and hydrolysis. The gamma subunit rotates stepwise during proton transport-powered ATP synthesis, showing three distinct distances to the b subunits in repeating sequences. The average durations of these steps correspond to catalytic turnover times upon ATP synthesis as well as ATP hydrolysis. The direction of rotation during ATP synthesis is opposite to that of ATP hydrolysis. 相似文献
14.
Christelle Schouppe Jacques Vaillier Renée Venard Michel Rigoulet Jean Velours Francis Haraux 《Journal of bioenergetics and biomembranes》1999,31(2):105-117
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. 相似文献
15.
Ashley E. Brammer Randy B. Stockbridge Christopher Miller 《The Journal of general physiology》2014,144(2):129-136
Many bacterial species protect themselves against environmental F− toxicity by exporting this anion from the cytoplasm via CLCF F−/H+ antiporters, a subclass of CLC superfamily anion transporters. Strong F− over Cl− selectivity is biologically essential for these membrane proteins because Cl− is orders of magnitude more abundant in the biosphere than F−. Sequence comparisons reveal differences between CLCFs and canonical Cl−-transporting CLCs within regions that, in the canonical CLCs, coordinate Cl− ion and govern anion transport. A phylogenetic split within the CLCF clade, manifested in sequence divergence in the vicinity of this ion-binding center, raises the possibility that these two CLCF subclades might exhibit differences in anion selectivity. Several CLCF homologues from each subclade were examined for F−/Cl− selectivity of anion transport and equilibrium binding. Differences in both of these anion-selectivity metrics correlate with sequence divergence among CLCFs. Chimeric constructs identify two residues in this region that largely account for the subclade differences in selectivity. In addition, these experiments serendipitously uncovered an unusually steep, Cl−-specific voltage dependence of transport that greatly enhances F− selectivity at low voltage. 相似文献
16.
ATP synthase (F-ATPase) produces ATP at the expense of ion-motive force or vice versa. It is composed from two motor/generators, the ATPase (F1) and the ion translocator (F0), which both are rotary steppers. They are mechanically coupled by 360 degrees rotary motion of subunits against each other. The rotor, subunits gamma(epsilon)C10-14, moves against the stator, (alphabeta)3delta(ab2). The enzyme copes with symmetry mismatch (C3 versus C10-14) between its two motors, and it operates robustly in chimeric constructs or with drastically modified subunits. We scrutinized whether an elastic power transmission accounts for these properties. We used the curvature of fluorescent actin filaments, attached to the rotating c ring, as a spring balance (flexural rigidity of 8.10(-26) N x m2) to gauge the angular profile of the output torque at F0 during ATP hydrolysis by F1. The large average output torque (56 pN nm) proved the absence of any slip. Angular variations of the torque were small, so that the output free energy of the loaded enzyme decayed almost linearly over the angular reaction coordinate. Considering the three-fold stepping and high activation barrier (>40 kJ/mol) of the driving motor (F1) itself, the rather constant output torque seen by F0 implied a soft elastic power transmission between F1 and F0. It is considered as essential, not only for the robust operation of this ubiquitous enzyme under symmetry mismatch, but also for a high turnover rate under load of the two counteracting and stepping motors/generators. 相似文献
17.
Xiaolong L Xiaoai Z Yuanbo C Jiachang Y Zhiyong L Peidong J 《Biochemical and biophysical research communications》2006,347(3):752-757
In order to observe mechanically driven proton flux in F(0)F(1)-ATPase coupled with artificial driven rotation on F(1) simultaneously, a double channel observation system was established. An artificial delta-free F(0)F(1)-ATPase was constructed with alpha(3), beta(3), epsilon, gamma, and c(n) subunits as rotator and a, b(2) as stator. The chromatophore was immobilized on the glass surface through biotin-streptavidin-biotin system, and the magnetic bead was attached to the beta subunit of delta-free F(0)F(1)-ATPase. The mechanically driven proton flux was indicated by the fluorescence intensity change of fluorescein reference standard (F1300) and recorded by a cooled digital CCD camera. The mechanochemical coupling stoichiometry between F(0) and F(1) is about 4.15 +/- 0.2H(+)/rev when the magnetic field rotated at 0.33 Hz (rps). 相似文献
18.
Kingston N Remple JD Burnham W Stabler RM McGhee RB 《Journal of wildlife diseases》1976,12(4):562-565
One Falco rusticolus and two of 24 Falco peregrinus were positive for Plasmodium relictum. Sixteen percent of the erythrocytes (RBC) of the gyrfalcon were parasitized. Following treatment with chloroquine the parasite burden was dramatically reduced (to less than 0.01%). Infection in peregrine falcons was low (0.01-0.4% RBCs were parasitized). This is the second report of Plasmodium in the gyrfalcon and the first report in the peregrine falcon. 相似文献
19.
A stator is proposed as necessary to prevent futile rotation of the F(1) catalytic sector of mitochondrial ATP synthase (mtATPase) during periods of ATP synthesis or ATP hydrolysis. Although the second stalk of mtATPase is generally believed to fulfil the role of a stator capable of withstanding the stress produced by rotation of the central rotor, there is little evidence to directly support this view. We show that interaction between two candidate proteins of the second stalk, OSCP and subunit b, fused at their C-termini to GFP variants and assembled into functional mtATPase can be monitored in mitochondria using fluorescence resonance energy transfer (FRET). Substitution of native OSCP with a variant containing a glycine 166 to asparagine (G166N) substitution yielded a metastable complex. In contrast to the enzyme containing native OSCP, FRET could be irreversibly lowered for the enzyme containing G166N at a rate that correlated closely with the rate of enzyme activity (ATP hydrolysis). The non-hydrolysable ATP analogue, AMP-PCP did not have this effect. We conclude that two candidate proteins of the stator stalk, OSCP and b, are subject to stresses during enzyme catalytic activity commensurate with their role as a part of a stator stalk. 相似文献