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1.
In Escherichia coli, the F1FO ATP synthase b subunits house a conserved arginine in the tether domain at position 36 where the subunit emerges from the membrane. Previous
experiments showed that substitution of isoleucine or glutamate result in a loss of enzyme activity. Double mutants have been
constructed in an attempt to achieve an intragenic suppressor of the b
arg36→ile and the b
arg36→glu mutations. The b
arg36→ile mutation could not be suppressed. In contrast, the phenotypic defect resulting from the b
arg36→glu mutation was largely suppressed in the b
arg36→glu,glu39→arg double mutant. E. coli expressing the b
arg36→glu,glu39→arg subunit grew well on succinate-based medium. F1FO ATP synthase complexes were more efficiently assembled and ATP driven proton pumping activity was improved. The evidence
suggests that efficient coupling in F1FO ATP synthase is dependent upon a basic amino acid located at the base of the peripheral stalk. 相似文献
2.
Bisetto E Picotti P Giorgio V Alverdi V Mavelli I Lippe G 《Journal of bioenergetics and biomembranes》2008,40(4):257-267
The role of the integral inner membrane subunit e in self-association of F0F1ATP synthase from bovine heart mitochondria was analyzed by in situ limited proteolysis, blue native PAGE/iterative SDS-PAGE, and LC-MS/MS. Selective degradation of subunit e, without disrupting
membrane integrity or ATPase capacity, altered the oligomeric distribution of F0F1ATP synthase, by eliminating oligomers and reducing dimers in favor of monomers. The stoichiometry of subunit e was determined
by a quantitative MS-based proteomics approach, using synthetic isotope-labelled reference peptides IAQL*EEVK, VYGVGSL*ALYEK,
and ELAEAQEDTIL*K to quantify the b, γ and e subunits, respectively. Accuracy of the method was demonstrated by confirming
the 1:1 stoichiometry of subunits γ and b. Altogether, the results indicate that the integrity of a unique copy of subunit
e is essential for self-association of mammalian F0F1ATP synthase.
Elena Bisetto and Paola Picotti contributed equally to this work. 相似文献
3.
A homodimer of b subunits constitutes the peripheral stalk linking the F1 and F0 sectors of the Escherichia coli ATP synthase. Each b subunit has a single-membrane domain. The constraints on the membrane domain have been studied by systematic mutagenesis. Replacement of a segment proximal to the cytoplasmic side of the membrane had minimal impact on F1F0 ATP synthase. However, multiple substitutions on the periplasmic side resulted in defects in assembly of the enzyme complex. These mutants had insufficient oxidative phosphorylation to support growth, and biochemical studies showed little F1F0 ATPase and no detectable ATP-driven proton pumping activity. Expression of the b
N2A,T6A,Q10A
subunit was also oxidative phosphorylation deficient, but the b
N2A,T6A,Q10A
protein was incorporated into an F1F0 complex. Single amino acid substitutions had minimal reductions in F1F0 ATP synthase function. The evidence suggests that the b subunit membrane domain has several sites of interaction contributing to assembly of F0, and that these interactions are strongest on the periplasmic side of the bilayer. 相似文献
4.
Giovanna Lippe Elena Bisetto Marina Comelli Stefania Contessi Francesca Di Pancrazio Irene Mavelli 《Journal of bioenergetics and biomembranes》2009,41(2):151-157
Mitochondria are central to heart function and dysfunction, and the pathways activated by different cardioprotective interventions
mostly converge on mitochondria. In a context of perspectives in innate and acquired cardioprotection, we review some recent
advances in F0F1ATPsynthase structure/function and regulation in cardiac cells. We focus on three topics regarding the mitochondrial F0F1ATPsynthase and the plasma membrane enzyme, i.e.: i) the crucial role of cardiac mitochondrial F0F1ATPsynthase regulation by the inhibitory protein IF1 in heart preconditioning strategies; ii) the structure and function of mitochondrial F0F1ATPsynthase oligomers in mammalian myocardium as possible endogenous factors of mitochondria resistance to ischemic insult;
iii) the external location and characterization of plasma membrane F0F1 ATP synthase in search for possible actors of its regulation, such as IF1 and calmodulin, at cell surface. 相似文献
5.
Erhard E. Pfündel 《Photosynthesis research》2009,100(3):163-177
The F
0 and F
M level fluorescence from a wild-type barley, a Chl b-less mutant barley, and a maize leaf was determined from 430 to 685 nm at 10 nm intervals using pulse amplitude-modulated
(PAM) fluorimetry. Variable wavelengths of the pulsed excitation light were achieved by passing the broadband emission of
a Xe flash lamp through a birefringent tunable optical filter. For the three leaf types, spectra of F
V/F
M (=(F
M − F
0)/F
M) have been derived: within each of the three spectra of F
V/F
M, statistically meaningful variations were detected. Also, at distinct wavelength regions, the F
V/F
M differed significantly between leaf types. From spectra of F
V/F
M, excitation spectra of PS I and PS II fluorescence were calculated using a model that considers PS I fluorescence to be constant
but variable PS II fluorescence. The photosystem spectra suggest that LHC II absorption results in high values of F
V/F
M between 470 and 490 nm in the two wild-type leaves but the absence of LHC II in the Chl b-less mutant barley leaf decreases the F
V/F
M at these wavelengths. All three leaves exhibited low values of F
V/F
M around 520 nm which was tentatively ascribed to light absorption by PS I-associated carotenoids. In the 550–650 nm region,
the F
V/F
M in the maize leaf was lower than in the barley wild-type leaf which is explained with higher light absorption by PS I in
maize, which is a NADP-ME C4 species, than in barley, a C3 species. Finally, low values of F
V/F
M at 685 in maize leaf and in the Chl b-less mutant barley leaf are in agreement with preferential PS I absorption at this wavelength. The potential use of spectra
of the F
V/F
M ratio to derive information on spectral absorption properties of PS I and PS II is discussed. 相似文献
6.
A pure, active cytochrome b
6
f was isolated from the chloroplasts of the marine green alga, Bryopsis
corticulans. To investigate and characterize this cytochrome b
6
f complex, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), absorption spectra measurement and HPLC were employed. It was shown that this purified complex contained four large subunits with apparent molecular masses of 34.8, 24, 18.7 and 16.7 kD. The ratio of Cyt b
6 to Cytf was 2.01 : 1. The cytochromeb
6
f was shown to catalyze the transfer of 73 electrons from decylplastoquinol to plastocyanin–ferricyanide per Cyt f per second. α-Carotene, one kind of carotenoid that has not been found to present in cytochrome b
6
f complex, was discovered in this preparation by reversed phase HPLC. It was different from β-carotene usually found in cytochrome b
6
f complex. The configuration of the major α-carotene component was assigned to be 9-cis by resonance Raman spectroscopy. Different from the previous reports, the configuration of this α-carotene in dissociated state was determined to be all-trans. Besides this carotene, chlorophyll a was also found in this complex. It was shown that the molecular ratios of chlorophylla, cis and all-trans-α-carotene to Cyt f in this complex were 1.2, 0.7 and 0.2, respectively. 相似文献
7.
Microcoleus chthonoplastes constitutes one of the dominant microorganisms in intertidal microbial mat communities. In the laboratory, the effects of
repeated daily exposure to ultraviolet radiation (16:8 light:dark cycle) was investigated in unicyanobacterial cultures isolated
from three different localities (Baltic Sea = WW6; North Sea = STO and Brittany = BRE). Photosynthesis and growth were measured
in time series (12–15 days) while UV-absorbing mycosporine-like amino acids (MAAs) and cellular integrity were determined
after 12 and 3 days exposure to three radiation treatments [PAR (22 μmol photon m−2 s−1) = P; PAR + UV-A (8 W m−2) = PA; PAR + UV-A + UV-B (0.4 W m−2) = PAB]. Isolate-specific responses to UVR were observed. The proximate response to radiation stress after 1-day treatment
showed that isolate WW6 was the most sensitive to UVR. However, repeated exposure to radiation stress indicated that photosynthetic
efficiency (F
v/F
m) of WW6 acclimated to UVR. Conversely, although photosynthesis in STO exhibited lower reduction in F
v/F
m during the first day, the values declined over time. The BRE isolate was the most tolerant to radiation stress with the lowest
reduction in F
v/F
m sustained over time. While photosynthetic efficiencies of different isolates were able to acclimate to UVR, growth did not.
The discrepancy seems to be due to the higher cell density used for photosynthesis compared to the growth measurement. Apparently,
the cell density used for photosynthesis was not high enough to offer self-shading protection because cellular damage was
also observed in those filaments under UVR. Most likely, the UVR acclimation of photosynthesis reflects predominantly the
performance of the surviving cells within the filaments. Different strategies were observed in MAAs synthesis. Total MAAs
content in WW6 was not significantly different between all the radiation treatments. In contrast, the additional fluence of
UV-A and UV-B significantly increased MAAs synthesis and accumulation in STO while only UV-B fluence significantly increased
MAAs content in BRE. Regardless of the dynamic photosynthetic recovery process and potential UV-protective functions of MAAs,
cellular investigation showed that UV-B significantly contributed to an increased cell mortality in single filaments. In their
natural mat habitat, M. chthonoplastes benefits from closely associated cyanobacteria which are highly UVR-tolerant due to the production of the extracellular UV-sunscreen
scytonemin. 相似文献
8.
L-Lactate cytochrome c oxidoreductase (flavocytochrome b 2, FC b 2) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b 2 producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b 2. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b 2 activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b 2 producer characterized by a sixfold increased (to 3 μmol min?1 mg?1 protein in cell-free extract) activity of the enzyme. 相似文献
9.
The FUD17 strain of Chlamydomonas reinhardtii is a photosynthesis-deficient, acetate-requiring mutant with a defect in the chloroplast atpE gene, which codes for the ε subunit of the chloroplast ATP synthase. In this work, the FUD17 mutant was examined in relation
to other known ATP synthase mutants as an initial step toward using this strain to generate altered versions of the atpE gene for site-directed mutagenesis of the ε subunit. The FUD17 strain grows well and is normally pigmented in the dark (heterotrophic
conditions), but cannot grow autotrophically in the light, even when media are supplemented with acetate. Under heterotrophic
conditions, it shows no accumulation of the ε subunit, and much lower levels of the α and β subunits of the chloroplast ATP
synthase. FUD17 shows no light-dependent oxygen evolution and shows a strong, light-dependent alteration in its chlorophyll
fluorescence. These results show that FUD17 possesses similar characteristics to other ATP synthase mutants and fails to express
an assembled ATP synthase complex on its thylakoid membrane. A preliminary attempt at site-directed mutagenesis is described
which produced a slightly truncated form of the ε subunit, which is expressed normally in the cell.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
10.
Smirnova IA Zaslavsky D Fee JA Gennis RB Brzezinski P 《Journal of bioenergetics and biomembranes》2008,40(4):281-287
The ba
3-type cytochrome c oxidase from Thermus thermophilus is phylogenetically very distant from the aa
3–type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O2 to water is catalysed at a haem a
3-CuB catalytic site. The three-dimensional structure of the ba
3 oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial,
Rhodobacter sphaeroides and Paracoccus denitrificans aa
3 oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba
3
-cytochrome c oxidase using the flow-flash method. After flash-induced dissociation of CO from the fully reduced enzyme in the presence
of oxygen we observed rapid oxidation of cytochrome b (k ≅ 6.8 × 104 s−1) and formation of the peroxy (PR) intermediate. In the next step a proton was taken up from solution with a rate constant of ~1.7 × 104 s−1, associated with formation of the ferryl (F) intermediate, simultaneous with transient reduction of haem b. Finally, the enzyme was oxidized with a rate constant of ~1,100 s−1, accompanied by additional proton uptake. The total proton uptake stoichiometry in the oxidative part of the catalytic cycle
was ~1.5 protons per enzyme molecule. The results support the earlier proposal that the PR and F intermediate spectra are similar (Siletsky et al. Biochim Biophys Acta 1767:138, 2007) and show that even though the architecture of the proton-conducting pathways is different in the ba
3 oxidases, the proton-uptake reactions occur over the same time scales as in the aa
3-type oxidases.
Smirnova and Zaslavsky contributed equally to the work described in this paper. 相似文献
11.
The structure of subunit a of the Escherichia coli ATP synthase has been probed by construction of more than one hundred monocysteine substitutions. Surface labeling with 3-N-maleimidyl-propionyl biocytin (MPB) has defined five transmembrane helices, the orientation of the protein in the membrane,
and information about the relative exposure of the loops connecting these helices. Cross-linking studies using TFPAM-3 (N-(4-azido-2,3,5,6-tetrafluorobenzyl)-3-maleimido-propionamide) and benzophenone-4-maleimide have revealed which elements of
subunit a are near subunits b and c. Use of a chemical protease reagent, 5-(-bromoacetamido)-1,10-phenanthroline-copper, has indicated that the periplasmic end
of transmembrane helix 5 is near that of transmembrane helix 2. 相似文献
12.
The gene (choB
b
), encoding cholesterol oxidase from Brevibacterium sp. CCTCC M201008, was cloned and sequenced by PCR (GenBank accession number: DQ345780). The gene consists of 1653 base pairs
and encodes a protein of 551 amino acids. ChoB
b
exhibited a homology of 98% with cholesterol oxidase gene from Brevibacterium
sterolicum ATCC 21387. The cholesterol oxidase gene, cloned in the vector pET-28a, was over-expressed in Escherichia
coli BL21–CodonPlus (DE3)-RP grown at 23°C in Luria-Bertani medium containing 50 μM riboflavin, the precursor of the FAD coenzyme
of the enzyme. A maximum activity of 3.7 U/mg was obtained from cell free extract of E. coli BL21-CodonPlus (DE3)-RP harboring the pET-28a-choBb. 相似文献
13.
Raif Musa-Aziz Lihong Jiang Li-Ming Chen Kevin L. Behar Walter F. Boron 《The Journal of membrane biology》2009,228(1):15-31
Others have shown that exposing oocytes to high levels of (10–20 mM) causes a paradoxical fall in intracellular pH (pHi), whereas low levels (e.g., 0.5 mM) cause little pHi change. Here we monitored pHi and extracellular surface pH (pHS) while exposing oocytes to 5 or 0.5 mM NH3/NH4
+. We confirm that 5 mM causes a paradoxical pHi fall (−ΔpHi ≅ 0.2), but also observe an abrupt pHS fall (−ΔpHS ≅ 0.2)—indicative of NH3 influx—followed by a slow decay. Reducing [NH3/NH4
+] to 0.5 mM minimizes pHi changes but maintains pHS changes at a reduced magnitude. Expressing AmtB (bacterial Rh homologue) exaggerates −ΔpHS at both levels. During removal of 0.5 or 5 mM NH3/NH4
+, failure of pHS to markedly overshoot bulk extracellular pH implies little NH3 efflux and, thus, little free cytosolic NH3/NH4
+. A new analysis of the effects of NH3 vs. NH4
+ fluxes on pHS and pHi indicates that (a) NH3 rather than NH4
+ fluxes dominate pHi and pHS changes and (b) oocytes dispose of most incoming NH3. NMR studies of oocytes exposed to 15N-labeled show no significant formation of glutamine but substantial accumulation in what is likely an acid intracellular compartment. In conclusion, parallel measurements of pHi and pHS demonstrate that NH3 flows across the plasma membrane and provide new insights into how a protein molecule in the plasma membrane—AmtB—enhances
the flux of a gas across a biological membrane.
相似文献
Walter F. Boron (Corresponding author)Email: |
14.
Many bacteria adapt to microoxic conditions by synthesizing a particular cytochrome c oxidase (cbb
3) complex with a high affinity for O2, encoded by the ccoNOQP operon. A survey of genome databases indicates that ccoNOQP sequences are widespread in all sub-branches of Proteobacteria but otherwise are found only in bacteria of the CFB group (Cytophaga, Flexibacter, Bacteroides). Our analysis of available genome sequences suggests four major strategies of regulating ccoNOQP expression in response to O2. The most widespread strategy involves direct regulation by the O2-responsive protein Fnr. The second strategy involves an O2-insensitive paralogue of Fnr, FixK, whose expression is regulated by the O2-responding FixLJ two-component system. A third strategy of mixed regulation operates in bacteria carrying both fnr and fixLJ-fixK genes. Another, not yet identified, strategy is likely to operate in the -Proteobacteria Helicobacter pylori and Campylobacter jejuni which lack fnr and fixLJ-fixK genes. The FixLJ strategy appears specific for the -subclass of Proteobacteria but is not restricted to rhizobia in which it was originally discovered. 相似文献
15.
Little information is known on what the magnitude of nitrogen (N) processed by ectomycorrhizal (ECM) fungal species in the
field. In a common garden experiment performed in a northern California oak woodland, we investigated transfer of nitrogen
applied as 15NH4 or 15NO3 from leaves to ectomycorrhizal roots of three oak species, Quercus agrifolia, Q. douglasii, and Q. garryana. Oak seedlings formed five common ectomycorrhizal morphotypes on root tips. Mycorrhizal tips were more enriched in 15N than fine roots. N transfer was greater to the less common morphotypes than to the more common types. 15N transfer from leaves to roots was greater when , not , was supplied. 15N transfer to roots was greater in seedlings of Q. agrifolia than in Q. douglasii and Q. garryana. Differential N transfer to ectomycorrhizal root tips suggests that ectomycorrhizal morphotypes can influence flows of N
from leaves to roots and that mycorrhizal diversity may influence the total N requirement of plants. 相似文献
16.
17.
Myburg AA Griffin AR Sederoff RR Whetten RW 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2003,107(6):1028-1042
Comparative genetic mapping in interspecific pedigrees presents a powerful approach to study genetic differentiation, genome evolution and reproductive isolation in diverging species. We used this approach for genetic analysis of an F1 hybrid of two Eucalyptus tree species, Eucalyptus grandis (W. Hill ex Maiden.) and Eucalyptus globulus (Labill.). This wide interspecific cross is characterized by hybrid inviability and hybrid abnormality. Approximately 20% of loci in the genome of the F1 hybrid are expected to be hemizygous due to a difference in genome size between E. grandis (640 Mbp) and E. globulus (530 Mbp). We investigated the extent of colinearity between the two genomes and the distribution of hemizygous loci in the F1 hybrid using high-throughput, semi-automated AFLP marker analysis. Two pseudo-backcross families (backcrosses of an F1 individual to non-parental individuals of the parental species) were each genotyped with more than 800 AFLP markers. This allowed construction of de novo comparative genetic linkage maps of the F1 hybrid and the two backcross parents. All shared AFLP marker loci in the three single-tree parental maps were found to be colinear and little evidence was found for gross chromosomal rearrangements. Our results suggest that hemizygous AFLP loci are dispersed throughout the E. grandis chromosomes of the F1 hybrid.Communicated by O. Savolainen 相似文献
18.
Inositol polyphosphate-5-phosphatase F (INPP5F) is one of the largest families of phosphoinositide phosphatases, 5-phosphatase. It contains a Sac domain whose amino acids
are essential for inositol polyphosphate phosphatase activities. Here, we assigned the porcine INPP5F to SSC14q29 by using SCHP and IMpRH. Sequencing of PCR products from different breeds identified an A/G polymorphism in the
last exon. The allele frequencies of this SNP showed that the Yorkshire and Duroc pigs have high G allele frequency, whereas
the local pigs have high A allele frequency. Association analysis of the genotypes with growth and carcass traits found that
different genotypes of INPP5F have significant differences in average daily gain (ADG) (P < 0.05) in Yorkshire pigs. 相似文献
19.
David J. Castillo Teresa Ballado Laura Camarena Georges Dreyfus 《Antonie van Leeuwenhoek》2009,95(1):77-90
The single subpolar flagellum of Rhodobacter sphaeroides shows an enlarged hook-filament junction. One of the two proteins that compose this section of the filament is HAP1
Rs
(FlgK
Rs
) it contains a central non-conserved region of 860 amino acids that makes this protein about three times larger than its
homologue in Salmonella enterica serovar Typhimurium. We investigated the role of this central portion of the unusually large HAP1 protein of R. sphaeroides by monitoring the effects of serial deletions in flgK
Rs
, the gene encoding HAP1
Rs
, on swimming and swarming. Two deletion mutants did not assemble functional flagella, two were paralyzed and five exhibited
reduced free-swimming speeds. Some mutants produced unusual swarming patterns on soft agar without or with Ficoll 400. A segment
of approximately 200-aa of the central region of HAP1
Rs
that aligns with the variable region of the flagellin sequence from other γ- and β-proteobacteria was also found. Therefore,
it is possible that the origin of this large central domain of HAP1
Rs
could be associated with an event of horizontal transfer and subsequent duplications and/or insertions. 相似文献
20.
Calcification, a process common to numerous marine taxa, has traditionally been considered to be a significant source of CO2 in tropical waters only. A number of relatively recent studies, however, have shown that significant amounts of CO2 are also produced in temperate waters, although none of these studies was carried out on rocky shores, which are considered
to be very productive systems. We compared the CO2 fluxes due to respiration and calcification in two temperate species, the cirripedes Chthamalus montagui and Elminius modestus. The population dynamics of both species were estimated at two sites during a 1-year experimental period in order to establish
mean organic (ash-free dry weight) and CaCO3 (dry shell weight) production. Based on these parameters, we estimated the CO2 fluxes due to respiration and calcification. CaCO3 production was estimated to be 481.0 and
at each site, representing 3.4 and respectively, of released CO2. These fluxes represent each 47% of the CO2 released as a result of respiration and calcification. The production of CaCO3 at the high-density site was: (1) among the highest values obtained for temperate organisms, and (2) comparable to the estimated
CO2 fluxes for coral reefs. As calcifying organisms are well represented in temperate ecosystems in terms of both density and
biomass, our results provide clear evidence that calcification of temperate organisms should not be underestimated. Additional
studies on other rocky shore taxa are needed before the relative importance of calcification in rocky intertidal carbon budgets
can be generalized. 相似文献