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131.
Sjaak Peelen Sybren S. Wijmenga Paul J. A. Erbel Robert L. Robson Robert R. Eady Jacques Vervoort 《Journal of biomolecular NMR》1996,7(4):315-330
Summary The 1H, 15N and 13C backbone and 1H and 13C beta resonance assignments of the long-chain flavodoxin from Azotobacter chroococcum (the 20-kDa nifF product, flavodoxin-2) in its oxidized form were made at pH 6.5 and 30°C using heteronuclear multidimensional NMR spectroscopy. Analysis of the NOE connectivities, together with amide exchange rates, 3JHnH coupling constants and secondary chemical shifts, provided extensive solution secondary structure information. The secondary structure consists of a five-stranded parallel -sheet and five -helices. One of the outer regions of the -sheet shows no regular extended conformation, whereas the outer strand 4/6 is interrupted by a loop, which is typically observed in long-chain flavodoxins. Two of the five -helices are nonregular at the N-terminus of the helix. Loop regions close to the FMN are identified. Negatively charged amino acid residues are found to be mainly clustered around the FMN, whereas a cluster of positively charged residues is located in one of the -helices. Titration of the flavodoxin with the Fe protein of the A. chroococcum nitrogenase enzyme complex revealed that residues Asn11, Ser68 and Asn72 are involved in complex formation between the flavodoxin and Fe protein. The interaction between the flavodoxin and the Fe protein is influenced by MgADP and is of electrostatic nature.Abbreviations SQ
semiquinone
- FMN
riboflavin 5-monophosphate; nif, nitrogen fixation
- TSP
3-(trimethylsilyl)propionate sodium salt
- DSS
2,2-dimethyl-2-silapentane-5-sulfonate sodium salt
Supplementary Material is available on request, comprising a Materials and Methods section for the expression and purification of the A. chroococcum flavodoxin, a Table S1 containing the parameters of the titration of A. chroococcum flavodoxin with the Fe protein, and a Table S2 containing the 15N, HN, 13C, 1H, 13C, 1H and 13CO chemical shifts.To whom correspondence should be addressed. 相似文献
132.
Tiecheng Qiao Robert Witkowski Robin Henderson G. McLendon 《Journal of biological inorganic chemistry》1996,1(5):432-438
The kinetics of methemoglobin reduction by cytochrome b
5 has been studied by stopped-flow and saturation transfer NMR. A forward rate constant k
f = 2.44×104 M–1 s–1 and a reverse rate constant k
b = 540 M–1s–1 have been observed at 10 mm, pH 6.20, 25 °C. The ratio k
f/k
b = k
eq = 43.6 is in good agreement with the equilibrium constant calculated from the electrochemical potential between cyt b
5 and methemoglobin. A bimolecular collisional mechanism is proposed for the electron transfer from cyt b
5 to methemoglobin based on the kinetic data analysis. The dependence of the rate constants on ionic strengths supports such
collisional mechanism. It is also found that the reaction rate strongly depends on the conformations of methemoglobin.
Received: 20 February 1996 / Accepted: 4 June 1996 相似文献
133.
Ricardo O. Louro Teresa Catarino Carlos A. Salgueiro Jean LeGall António V. Xavier 《Journal of biological inorganic chemistry》1996,1(1):34-38
Using potentiometric titrations, two protons were found to participate in the redox-Bohr effect observed for cytochrome c
3 from Desulfovibrio vulgaris (Hildenborough). Within the framework of the thermodynamic model previously presented, this finding supports the occurrence
of a concerted proton-assisted 2e– step, ideally suited for the coupling role of cytochrome c
3 to hydrogenase. Furthermore, at physiological pH, it is shown that when sulfate-reducing bacteria use H2 as energy source, cytochrome c
3 can be used as a charge separation device, achieving energy transduction by energising protons which can be left in the acidic
periplasmic side and transferring deenergised electrons to sulfate respiration. This mechanism for energy transduction, using
a full thermodynamic data set, is compared to that put forward to explain the proton-pumping function of cytochrome c oxidase. 相似文献
134.
The intramolecular electron-transfer rate constant for the Cu(II)–topaNH2⇌ Cu(I)–topaSQ equilibrium in methylamine oxidase has been measured by temperature-jump relaxation techniques. At pH 7.0 the estimated kobs = 150±30 s–1 for both methylamine and benzylamine; assuming the equilibrium constant is ≈0.7–1 at pH 7.0 and 296 K, this would correspond
to a forward electron-transfer rate constant kET≈ 60–75 s–1. Although substantially slower than the previously determined kET≈ 20 000 s–1 for pea seedling amine oxidase [5] steady-state kinetics measurements established that kET > kcat≈ 4–10 s–1. Thus the Cu(I)-semiquinone state is a viable intermediate in methylamine oxidase turnover.
Received: 16 August 1995 / Accepted: 21 December 1995 相似文献
135.
Cytoplasmic determinants that specify the fate of endoderm, muscle and epidermis cells are known to be localized in specific
areas of fertilized eggs of ascidians. The presence of such cytoplasmic determinants in unfertilized eggs was demonstrated
in previous studies, but no information has yet been proved about their distribution. To investigate the distribution of cytoplasmic
determinants in unfertilized eggs, we devised a method for distinguishing the polarity of unfertilized eggs using vital staining
and we performed cytoplasmic-transfer experiments by fusing blastomeres and cytoplasmic fragments from various identified
regions of unfertilized eggs. Cytoplasmic fragments, that contained cortical and subcortical material, from five different
positions along the animal-vegetal axis were prepared, and they were fused with a4.2 (presumptive-epidermis) or A4.1 (non-epidermis)
blastomeres. The ectopic development of endoderm, muscle and epidermis cells that was promoted by the transplanted cytoplasm
was assessed by examining the expression of alkaline phosphatase (ALP), myosin and epidermis-specific antigen, respectively.
Differentiation of endoderm and muscle was observed at higher frequencies as cytoplasmic fragments closer to the vegetal pole
were transplanted. Conversely, formation of epidermis was observed at higher frequencies as cytoplasmic fragments closer to
the animal pole were transplanted. The results suggest that, in cortical and subcortical regions of unfertilized ascidian
eggs, endoderm and muscle determinants are widely distributed along a gradient, with maximum activity at the vegetal pole,
whilst epidermis determinants are also distributed along a gradient but with maximum activity at the animal pole.
Recieved: 10 June 1996 / Accepted: 12 September 1996 相似文献
136.
研究了层理鞭枝藻藻胆体在不同浓度磷酸缓冲溶液中解离过程中荧光发射光谱的变化和光能传递。完整藻胆体的77K荧光光谱中只有一个峰,位于685nm它是末端发射体(核心-膜连接多肽和别藻蓝蛋白-B)的荧光峰。部分解离藻胆体的荧光光谱的主峰位移至652nm:次峰位于685nm;660nm为一弱荧光发射肩。它们依次为C-藻蓝蛋白,末端发射体和别藻蓝蛋白的荧光。严重解离藻胆体的荧光主峰移644nm;次峰由685nm移至682nm;660nm荧光发射肩消失。这表明C-藻蓝蛋白所捕获的光能已不能传递给别藻蓝蛋白,但可传递给末端发射体洞时又表明C-藻蓝蛋白不仅与别藻蓝蛋白相连接而且还与末端发射体相连接。提出该藻胆体光能传递链如下:核心-膜连接多肽藻红蓝蛋白→C-藻蓝蛋白→别藻蓝蛋白别藻蓝蛋白-B 相似文献
137.
The cellular pathway of sugar uptake in developing cotyledons of Vicia faba L. and Phaseolus vulgaris L. seed was evaluated using a physiological approach. The cotyledon interface with the seed coat is characterised by a specialised dermal cell complex. In the case of Vicia faba cotyledons, the epidermal component of the dermal cell complex is composed of transfer cells. Sucrose is the major sugar presented to the outer surface of both cotyledons and it is taken up from the apoplasm unaltered. Estimated sucrose concentrations within the apparent free space of Vicia and Phaseolus cotyledons were 105 and 113 mM respectively. Rates of in-vitro uptake of [14C]sucrose by cotyledon segments or by whole cotyledons following physical removal or porter inactivation of the outer cells demonstrated that, for both Vicia and Phaseolus cotyledons, the dermal cell complexes are the most intense sites of sucrose uptake. Accumulation of [14C]sucrose in the storage parenchyma of whole cotyledons was directly affected by experimental manipulation of uptake by the outer cell layers and plasmolytic disruption of the interconnecting plasmodesmata. These findings indicated that sucrose accumulated by the dermal cell complexes is transported symplasmically to the storage parenchyma. Overall, it is concluded that the dermal cell complexes of the developing legume embryo, irrespective of the presence or absence of wall ingrowths, are the major sites for the uptake of sucrose released from the maternal tissues to the seed apoplasm. Thereafter, the accumulated sucrose is transported radially inward through the symplast to the storage parenchyma.Abbreviations AFS
apparent free space
- CF
5-(6)-carboxyfluorescein
- CFDA
5-(6)-carboxyfluorescein diacetate
- Mes
2-(N-morpholino)ethanesulfonic acid
- PCMBS
p-chloromercuribenzenesulfonic acid
- SRG
sulphorhodamine G
The investigation was supported by funds from the Research Management Committee, The University of Newcastle and the Australian Research Council. One of us, R. McDonald, gratefully acknowledges the support of an Australian Postgraduate Research Award. We are grateful to Stella Savoury for preparing the photomicrographs. 相似文献
138.
P. P. Jauhar 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1995,90(6):865-871
As the first step in the transfer of barely yellow dwarf virus resistance and salt tolerance from decaploid tall wheatgrass (Thinopyrum ponticum) into hexaploid bread wheat (Triticum aestivum L.), octoploid intergeneric hybrids (2n = 8x = 56) were synthesized by crossing the tall wheatgrass cultivar Alkar with wheat cvs. Fukuhokomugi (Fuko) and Chinese Spring. (Fuko x Alkar) F1 hybrids were studied in detail. The F1 hybrids were perennial and generally resembled the male wheatgrass parent with regard to morphological features and gliadin profile. Most hybrids were euploid with 56 chromosomes and showed high chromosome pairing. On an average, in 6 hybrids 83.6% of the complement showed chiasmatic association, some between wheat and wheatgrass chromosomes. Such a high homoeologous pairing would be obtained if Ph1, the major homoeologous pairing suppressor in wheat, was somehow inactivated. Some of the Fuko x Alkar hybrids had high pollen fertility (18.5–42.0% with a mean of 31.5%) and high seed fertility (3–29 seeds wtih a mean of 12.3 seeds per spike), offering excellent opportunities for their direct backcrossing onto the wheat parent. 相似文献
139.
W. L. Li P. D. Chen L. L. Qi D. J. Liu 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1995,90(3-4):526-533
A species-specific repeated sequence, pHvNAU62, was cloned from Haynaldia villosa, a wheat relative of great importance. It strongly hybridized to H. villosa, but not to wheat. In situ hybridization localized this sequence to six of seven H. villosa chromosome pairs in telomeric or sub-telomeric regions. Southern hybridization to whea-H. villosa addition lines showed that chromosomes 1V through 6V gave strong signals in ladders while chromosome 7V escaped detection. In addition to H. villosa, several Triticeae species were identified for a high abundance of the pHvNAU62 repeated sequence, among which Thinopyrum bassarabicum and Leymus racemosus produced the strongest signals. Sequence analysis indicated that the cloned fragment was 292 bp long, being AT rich (61%), and showed 67% homology of pSc7235, a rye repeated sequence. Isochizomer analysis suggested that the present repeated sequence was heavily methylated at the cytosine of the CpG dimer in the genome of H. villosa.It was also demonstrated that pHvNAU62 is useful in tagging the introduced 6VS chromosome arm, which confers a resistance gene to wheat powdery mildew, in the segregating generations. 相似文献
140.
W. B. Curry M. D. Grabe I. V. Kurnikov S. S. Skourtis D. N. Beratan J. J. Regan A. J. A. Aquino P. Beroza J. N. Onuchic 《Journal of bioenergetics and biomembranes》1995,27(3):285-293
The simplest views of long-range electron transfer utilize flat one-dimensional barrier tunneling models, neglecting structural details of the protein medium. The pathway model of protein electron transfer reintroduces structure by distinguishing between covalent bonds, hydrogen bonds, and van der Waals contacts. These three kinds of interactions in a tunneling pathway each have distinctive decay factors associated with them. The distribution and arrangement of these bonded and nonbonded contacts in a folded protein varies tremendously between structures, adding a richness to the tunneling problem that is absent in simpler views. We review the pathway model and the predictions that it makes for protein electron transfer rates in small proteins, docked proteins, and the photosynthetic reactions center. We also review the formulation of the protein electron transfer problem as an effective two-level system. New multi-pathway approaches and improved electronic Hamiltonians are described briefly as well. 相似文献