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91.
Localization of the delta-like-1-binding site in human Notch-1 and its modulation by calcium affinity 总被引:1,自引:0,他引:1
Cordle J Redfieldz C Stacey M van der Merwe PA Willis AC Champion BR Hambleton S Handford PA 《The Journal of biological chemistry》2008,283(17):11785-11793
The Notch signaling pathway plays a key role in a myriad of cellular processes, including cell fate determination. Despite extensive study of the downstream consequences of receptor activation, very little molecular data are available for the initial binding event between the Notch receptor and its ligands. In this study, we have expressed and purified a natively folded wild-type epidermal growth factor-like domain (EGF) 11-14 construct from human Notch-1 and have used flow cytometry and surface plasmon resonance analysis to demonstrate a calcium-dependent interaction with the human ligand Delta-like-1. Site-directed mutagenesis of three of the calcium-binding sites within the Notch-(11-14) fragment indicated that only loss of calcium binding to EGF12, and not EGF11 or EGF13, abrogates ligand binding. Further mapping of the ligand-binding site within this region by limited proteolysis of Notch wild-type and mutant fragments suggested that EGF12 rather than EGF11 contains the major Delta-like-1-binding site. Analysis of an extended fragment EGF-(10-14), where EGF11 is placed in a native context, surprisingly demonstrated a reduction in ligand binding, suggesting that EGF10 modulates binding by limiting access of ligand. This inhibition could be overcome by the introduction of a calcium binding mutation in EGF11, which decouples the EGF-(10-11) module interface. This study therefore demonstrates that long range calcium-dependent structural perturbations can influence the affinity of Notch for its ligand, in the absence of any post-translational modifications. 相似文献
92.
Pollen-tube cell walls are unusual in that they are composed almost entirely of callose, a (1,3)--linked glucan with a few 6-linked branches. Regulation of callose synthesis in pollen tubes is under developmental control, and this contrasts with the deposition of callose in the walls of somatic plant cells which generally occurs only in response to wounding or stress. The callose synthase (uridine-diphosphate glucose: 1,3--d-glucan 3--d-glucosyl transferase, EC 2.4.1.34) activities of membrane preparations from cultured pollen tubes and suspension-cultured cells of Nicotiana alata Link et Otto (ornamental tobacco) exhibited different kinetic and regulatory properties. Callose synthesis by membrane preparations from pollen tubes was not stimulated by Ca2+ or other divalent cations, and exhibited Michaelis-Menten kinetics only between 0.25 mM and 6 mM uridine-diphosphate glucose (K
m 1.5–2.5 mM); it was activated by -glucosides and compatible detergents. In contrast, callose synthesis by membrane preparations from suspension-cultured cells was dependent on Ca2+, and in the presence of 2 mM Ca2+ exhibited Michaelis-Menten kinetics above 0.1 mM uridine-diphosphate glucose (K
m 0.45 mM); it also required a -glucoside and low levels of compatible detergent for full activity, but was rapidly inactivated at higher levels of detergent. Callose synthase activity in pollen-tube membranes increased ten fold after treatment of the membranes with trypsin in the presence of detergent, with no changes in cofactor requirements. No increase in callose synthase activity, however, was observed when membranes from suspension-cultured cells were treated with trypsin. The insoluble polymeric product of the pollen-tube enzyme was characterised as a linear (1,3)--d-glucan with no 6-linked glucosyl branches, and the same product was synthesised irrespective of the assay conditions employed.Abbreviations Ara
l-arabinose
- CHAPS
3-[(3-cholamidopropyl)dimethylammonia]-1-propane sulphonic acid
- DAP
diphenylamine-aniline-phosphoric acid stain
- Gal
d-galactose
- Glc
d-glucose
- Man
d-mannose
- Mes
2-(N-morpholino)ethane sulphonic acid
- Rha
d-rhamnose
- Rib
d-ribose
- TFA
trifluoroacetic acid
- UDPGlc
uridine-diphosphate glucose
- Xyl
d-xylose
This research was supported by funds from a Special Research Centre of the Australian Research Council. H.S. was funded by a Melbourne University Postgraduate Scholarship and an Overseas Postgraduate Research Studentship; S.M.R. was supported by a Queen Elizabeth II Research Fellowship. We thank Bruce McGinness and Susan Mau for greenhouse assistance, and Deborah Delmer and Adrienne Clarke for advice and encouragement throughout this project. 相似文献
93.
The temperature dependence of the partial reactions leading to turn-over of the UQH2:cyt c
2 oxidoreductase of Rhodobacter sphaeroides have been studied. The redox properties of the cytochrome components show a weak temperature dependence over the range 280–330 K, with coefficients of about 1 m V per degree; our results suggest that the other components show similar dependencies, so that no significant change in the gradient of standard free-energy between components occurs over this temperature range. The rates of the reactions of the high potential chain (the Rieske iron sulfur center, cytochromes c
1 and c
2, reaction center primary donor) show a weak temperature dependence, indicating an activation energy < 8 kJ per mole for electron transfer in this chain. The oxidation of ubiquinol at the Qz-site of the complex showed a strong temperature dependence, with an activation energy of about 32 kJ mole–1. The electron transfer from cytochrome b-566 to cytochrome b-561 was not rate determining at any temperature, and did not contribute to the energy barrier. The activation energy of 32 kJ mole–1 for quinol oxidation was the same for all states of the quinone pool (fully oxidized, partially reduced, or fully reduced before the flash). We suggest that the activation barrier is in the reaction by which ubiquinol at the catalytic site is oxidized to semiquinone. The most economical scheme for this reaction would have the semiquinone intermediate at the energy level indicated by the activation barrier. We discuss the plausibility of this simple model, and the values for rate constants, stability constant, the redox potentials of the intermediate couples, and the binding constant for the semiquinone, which are pertinent to the mechanism of the ubiquinol oxidizing site.Abbreviations (BChl)2
P870, primary donor of the photochemical reaction center
-
b/c
1 complex
ubiquinol: cytochrome c
2 oxidoreductase
- cyt b
H
cytochrome b-561 or higher potential cytochrome b
- cyt b
L
cytochrome b-566, or low potential cytochrome b
- cyt c
1, cyt c
2, cyt c
t
cytochromes c
1 and c
2, and total cytochrome c (cyt c
1 and cyt c
2)
- Fe.S
Rieske-type iron sulfur center, Q
- QH2
ubiquinone, ubiquinol
- Qz, QzH2, Qz
–
ubiquinone, ubiquinol, and semiquinone anion of ubiquinone, bound at quinol oxidizing site
- Qz-site
ubiquinol oxidizing site (also called Qo-(outside)
- Qo
(Oxidizing)
- QP
(Positive proton potential) site)
- Qc-site
uubiquinone reductase site (also called the Qi-(inside)
- QR
(Reducing), or
- QN
(Negative proton potential) site)
- UHDBT
5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazol 相似文献
94.
To date, there are no vaccines against any of the major parasitic diseases, and chemotherapy is the main weapon in our arsenal. There is an urgent need for better drugs against Leishmania. With the completion of the human genome sequence and soon that of Leishmania, for the first time we have the opportunity to identify novel chemotherapeutic treatments. This requires the exploitation of a variety of technologies. The major challenge is to take the process from discovery of drug candidates all the way along the arduous path to the marketplace. A crucial component will be the forging of partnerships between the pharmaceutical industry and publicly funded scientists to ensure that the promise of the current revolution in biology lives up to our hopes and expectations. 相似文献
95.
96.
Glucan synthesis by intact cotton fibres fed with different precursors at the stages of primary and secondary wall formation 总被引:1,自引:0,他引:1
Seed clusters of individual locules from fruit capsules of Gossypium arboreum L. with adhering intact fibres were fed with radioactive uridinediphosphoglucose (UDPG), guanosinediphosphoglucose (GDPG), glucose and sucrose. The incorporation into high molecular weight glucans of the fibres was studied. For primary wall fibres, UDPG at 1 mM was by far the best precursor, whereas sucrose was the best precursor for secondary wall fibres. No competition was observed between the incorporation of glucose from UDPG and from sucrose when the two were fed simultaneously to secondary wall fibres, indicating that their metabolic pathways are well separated when they are fed from the apoplast. Inhibitors of respiratory ATP-formation strongly inhibited incorporation of sucrose but not that of UDPG. Sucrose incorporation was studied at five different stages of development of the cotton fibres. At the stage of most intense secondary wall formation the incorporation rate was about 300 times that during primary wall formation (24 days post anthesis (DPA)). Incorporation from 1 mM UDPG or GDPG by secondary wall fibres (35 DPA) was less than twice that of primary wall fibres (22 DPA), indicating that the two sugar nucleotides are not readily used as precursors for secondary wall cellulose when they are fed to the exterior of intact cells. The high molecular weight non-cellulosic glucans formed from UDPG and sucrose at 5 and 1,000 M were solubilized in strongly alkaline solutions or dimethyl-sulfoxide (DMSO) and were partially characterized by degradation with an exo--1,3-glucanase. After feeding for one hour, at most 1/3 of the radioactivity in high molecular weight material was found in cellulose and at least 2/3 in -1,3-glucan. The proportions varied little for fibres in the age range of 30 to 48 DPA when sucrose was the precursor although the total incorporation varied by a factor of about four. The fact that at all stages of secondary wall formation -1,3-glucan is synthesized at a very high rate, but that the total amount in the cell wall does not exceed 2% in the later stages of wall formation, can be interpreted in terms of a high turnover of this polysaccharide if it is assumed that wound effects are negligible in the system under study.Abbreviations UDPG
uridinediphosphoglucose
- GDPG
guanosinediphosphoglucose
- HEPES
N-2-hydroxyethylpiperazine-N-2-ethansulphonic acid
- DMSO
dimethyl-sulfoxide
- DNP
2,4-dinitrophenol
- DPA
days post anthesis 相似文献
97.
Xianhai Zhao Berit Ebert Baocai Zhang Huabin Liu Yutao Zhang Wei Zeng Carsten Rautengarten Huiling Li Xiaoyang Chen Antony Bacic Guodong Wang Shuzhen Men Yihua Zhou Joshua L. Heazlewood Ai‐Min Wu 《The Plant journal : for cell and molecular biology》2020,104(1):252-267
Rhamnogalacturonan‐II (RG‐II) is structurally the most complex glycan in higher plants, containing 13 different sugars and 21 distinct glycosidic linkages. Two monomeric RG‐II molecules can form an RG‐II‐borate diester dimer through the two apiosyl (Api) residues of side chain A to regulate cross‐linking of pectin in the cell wall. But the relationship of Api biosynthesis and RG‐II dimer is still unclear. In this study we investigated the two homologous UDP‐D‐apiose/UDP‐D‐xylose synthases (AXSs) in Arabidopsis thaliana that synthesize UDP‐D‐apiose (UDP‐Api). Both AXSs are ubiquitously expressed, while AXS2 has higher overall expression than AXS1 in the tissues analyzed. The homozygous axs double mutant is lethal, while heterozygous axs1/+ axs2 and axs1 axs2/+ mutants display intermediate phenotypes. The axs1/+ axs2 mutant plants are unable to set seed and die. By contrast, the axs1 axs2/+ mutant plants exhibit loss of shoot and root apical dominance. UDP‐Api content in axs1 axs2/+ mutants is decreased by 83%. The cell wall of axs1 axs2/+ mutant plants is thicker and contains less RG‐II‐borate complex than wild‐type Col‐0 plants. Taken together, these results provide direct evidence of the importance of AXSs for UDP‐Api and RG‐II‐borate complex formation in plant growth and development. 相似文献
98.
Redemann S Schloissnig S Ernst S Pozniakowsky A Ayloo S Hyman AA Bringmann H 《Nature methods》2011,8(3):250-252
We present a method to control protein levels under native genetic regulation in Caenorhabditis elegans by using synthetic genes with adapted codons. We found that the force acting on the spindle in C. elegans embryos was related to the amount of the G-protein regulator GPR-1/2. Codon-adapted versions of any C. elegans gene can be designed using our web tool, C. elegans codon adapter. 相似文献
99.
Health benefits of garlic and other Allium vegetables (e.g., onions), such as lipid lowering and anticancer effects, are credited to metabolic byproducts, including diallyl trisulfide (DATS). Evidence for anticancer effects of garlic derives from both population-based case-control studies, and clinical and laboratory investigations using purified garlic constituents such as DATS. Studies have shown that DATS can offer protection against chemically-induced neoplasia as well as oncogene-driven spontaneous cancer development in experimental rodents. Mechanisms underlying cancer chemopreventive effects of DATS are not completely understood, but known pharmacological responses to this natural product include alteration in carcinogen-metabolizing enzymes, cell cycle arrest, induction of apoptotic cell death, suppression of oncogenic signal transduction pathways, and inhibition of neoangiogenesis. This article reviews mechanisms and targets of cancer chemoprevention by DATS. 相似文献
100.
Ira S. Kass David A. Larsen Ching C. Wang Antony O.W. Stretton 《Experimental parasitology》1982,54(2):166-174
Avermectin B1a, a novel antiparasitic agent, paralyzes Ascaris suum without causing either flaccid paralysis or a hypercontraction. It reduces the lengthening of the acetylcholine-preconditioned A. suum muscle strip caused by γ-aminobutyric acid. It does not affect the contraction of the isolated muscle strip preparation caused by applying acetylcholine. However, preinjection with Avermectin B1a does significantly reduce the shortening caused by acetylcholine injection without affecting the paralysis of an intact ascarid worm. These results suggest that Avermectin B1a may act on the central nervous system of Ascaris sp. nematodes. 相似文献