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1.
Emil Bulatov Esther M. Martin Sneha Chatterjee Axel Knebel Satoko Shimamura Albert Konijnenberg Clare Johnson Nico Zinn Paola Grandi Frank Sobott Alessio Ciulli 《The Journal of biological chemistry》2015,290(7):4178-4191
The multisubunit cullin RING E3 ubiquitin ligases (CRLs) target post-translationally modified substrates for ubiquitination and proteasomal degradation. The suppressors of cytokine signaling (SOCS) proteins play important roles in inflammatory processes, diabetes, and cancer and therefore represent attractive targets for therapeutic intervention. The SOCS proteins, among their other functions, serve as substrate receptors of CRL5 complexes. A member of the CRL family, SOCS2-EloBC-Cul5-Rbx2 (CRL5SOCS2), binds phosphorylated growth hormone receptor as its main substrate. Here, we demonstrate that the components of CRL5SOCS2 can be specifically pulled from K562 human cell lysates using beads decorated with phosphorylated growth hormone receptor peptides. Subsequently, SOCS2-EloBC and full-length Cul5-Rbx2, recombinantly expressed in Escherichia coli and in Sf21 insect cells, respectively, were used to reconstitute neddylated and unneddylated CRL5SOCS2 complexes in vitro. Finally, diverse biophysical methods were employed to study the assembly and interactions within the complexes. Unlike other E3 ligases, CRL5SOCS2 was found to exist in a monomeric state as confirmed by size exclusion chromatography with inline multiangle static light scattering and native MS. Affinities of the protein-protein interactions within the multisubunit complex were measured by isothermal titration calorimetry. A structural model for full-size neddylated and unneddylated CRL5SOCS2 complexes is supported by traveling wave ion mobility mass spectrometry data. 相似文献
2.
Analogs of riboflavin that were altered at positions N(3), 8α, and N(10) of the 7,8-dimethylisoalloxazine ring were immobilized by covalent attachment to aminoalkylated agarose and polyacrylamide beads. These materials were used for affinity chromatographic purification of the riboflavin-carrier protein from egg white, egg yolk, and blood from laying hens, of flavokinase from rat liver, and of partially purified flavodoxin from Azotobacter vinelandii (FMN). The apo-carrier protein, which tightly complexes riboflavin (Kd ≈ 2 nm), was bound by the N(3)-, 8α-, and N(10)-flavinyl beads and was selectively displaced in moderate to high yield by 10 μm riboflavin or 1 m NaCl at pH 3.5. Flavokinase, which complexes less tightly with riboflavin (Km ≈ 12 μm), was bound by the 8α- and N(10)-flavinyl beads. Binding to the latter was sufficiently tight that the addition of riboflavin was needed to displace flavokinase from the beads. The A. vinelandii flavodoxin, which normally complexes riboflavin 5′-phosphate (K3 ≈ 5 nm) but less avidly complexes riboflavin (Kd ≈ 0.6 μm), was bound by the N(10)-flavinyl beads and eluted in low yield upon addition of FMN; most of the apoprotein denatured on the column despite the inclusion of thiol-protecting reagents. These flavin affinity materials may be generally useful for isolating a variety of other proteins that bind riboflavin. 相似文献
3.
Different biologands carrying synthetic adsorbents have been reported in the literature for protein separation. We have developed
a novel and new approach to obtain high protein adsorption capacity utilizing 2-methacrylamidohistidine (MAH) as a bioligand.
MAH was synthesized by reacting methacrylochloride and histidine. Spherical beads with an average size of 150–200 μm were
obtained by the radical suspension polymerization of MAH and 2-hydroxyethyl-methacrylate (HEMA) conducted in an aqueous dispersion
medium. p(HEMA-co-MAH) beads had a specific surface area of 17.6 m2/g. Synthesized MAH monomer was characterized by NMR. p(HEMA-co-MAH) beads were characterized by swelling test, FTIR and elemental
analysis. Then, Cu(II) ions were incorporated onto the beads and Cu(II) loading was found to be 0.96 mmol/g. These affinity
beads with a swelling ratio of 65%, and containing 1.6 mmol. MAH/g were used in the adsorption/desorption of human serum albumin
(HSA) from both aqueous solutions and human serum. The adsorption of HSA onto p(HEMA-co-MAH) was low (8.8 mg/g). Cu(II) chelation
onto the beads significantly increased the HSA adsorption (56.3 mg/g). The maximum HSA adsorption was observed at pH 3.0 Higher
HSA adsorption was observed from human plasma (94.6 mg HSA/g). Adsorption of other serum proteins were obtained as 3.7 mg/g
for fibrinogen and 8.5 mg/g for γ-globulin. The total protein adsorption was determined as 107.1 mg/g. Desorption of HSA was
obtained using 0.1 M Tris/HCl buffer containing 0.5M NaSCN. High desorption ratios (up to 98% of the adsorbed HSA) were observed.
It was possible to reuse Cu(II) chelated-p(HEMA-co-MAH) beads without significant decreases in the adsorption capacities. 相似文献
4.
Polymer-coated magnetic beads have become widely used in biological applications because of their facile recovery and easily
modifiable surface. Herein, we report the application of magnetic beads to in vitro refolding of B. cepacia lipase. Magnetic particles (Fe3O4) prepared by co-precipitation of Fe2+ and Fe3+ ions under basic conditions were subsequently coated with carboxylic acid-containing polystyrene by emulsion polymerization.
The polymer-coated magnetic beads were then conjugated with molecular chaperone proteins to assist with refolding. The chaperone-conjugated
magnetic beads efficiently refolded B. cepacia lipase and were easily reused. The beads showed comparable refolding activity to the soluble chaperone, and retained more
than 95% of their refolding activity after five cycles of refolding B. cepacia lipase. 相似文献
5.
Jerzy Rogalski Janusz Szczodrak Andrzej Dawidowicz Zdzisław Ilczuk Andrzej Leonowicz 《Enzyme and microbial technology》1985,7(8):395-400
The major components of cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and d-xylanase (see 1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) complexes have been immobilized on glass beads activated by 3-aminopropyltriethoxysilane or 3-glycidoxypropyltrimethoxysilane. The final preparations contained over 20 mg protein g?1 glass beads. The activity retained was 71.6–98.1% for cellulase complexes and 81–100% for d-xylanase complexes. The immobilization of the enzymes spread their optimum pH range. Cellulose and d-xylan were quantitatively hydrolysed by the immobilized enzymes. The major reaction products were identified as a d-glucose and d-xylose respectively. 相似文献
6.
Summary Sera and effusion fluids of patients with breast cancer (BC) contain immune complexes (IC). Antigens present in these complexes were isolated as follows: a pool of effusions from patients with BC was fractionated with ammonium sulfate. The proteins precipitating at 40% saturation were further fractionated by filtration through a Sephadex G-200 column. The material recovered in the first peak (molecules larger than monomeric IgG) was brought to pH 3.0 to dissociate the IC, and the mixture was filtered through a column of Sephacryl S-300 at pH 3.0. Proteins smaller than monomeric IgG were collected, radioiodinated, and used as antigens (125Ag) to search for corresponding antibodies in sera of patients with BC (BCS) and of healthy individuals (NHS). 125Ag was reacted with the sera and the immune complexes obtained were precipitated with an antiserum to human Ig and analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography. Both NHS and BCS contained antibodies against two antigens; one of these appeared as a strong band of 17KD, the other as a doublet of approximately 25KD. It is concluded that some of the proteins in the IC from patients with BC are auto-antigens. No BC-specific antigens were identified. 相似文献
7.
Bruce S. Jacobson 《生物化学与生物物理学报:生物膜》1980,600(3):769-780
The plasma membrane from Dictyostelium discoideum was routinely purified 35-fold by an improved technique using beads coated with positively charged polymers. Cells were attached to the beads and bare regions between the cells were neutralized with a polyanion. The neutralization decreased contamination of the bare regions by intracellular proteins released when cells were disrupted to leave behind beads coated by plasma membrane. The neutralization increased the purification as measured by membrane-bound 125I-labeled concanavalin A. Contamination by markers for various intracellular components was markedly decreased. Various bare-site neutralization reagents were evaluated and gave different results depending upon their charge density and molecular weight. The pH of the neutralization was critical. The optimum pH for cell attachment to beads, 5.0, had little effect as regards bare-site neutralization. A new procedure is given that optimizes the essential features for the plasma membrane isolation on beads. 相似文献
8.
Nadia Hgarat Gildas Mouta Cardoso Filippo Rusconi Jean-Christophe Franois Danile Praseuth 《Nucleic acids research》2007,35(13):e92
Purification of specific DNA–protein complexes is a challenging task, as the involved interactions can be both electrostatic/H-bond and hydrophobic. The chromatographic stringency needed to obtain reasonable purifications uses salts and detergents. However, these components elicit the removal of proteins unspecifically bound to the chromatographic support itself, thus contaminating the purification products. In this work, a photocleavable linker connected the target oligonucleotidic sequence to the chromatographic beads so as to allow the irradiation-based release of the purified DNA–protein complexes off the beads. Our bioanalytical conditions were validated by purifying the tetracycline repressor protein onto a specific oligonucleotide. The purification factor was unprecedented, with a single contaminant. The robustness of our method was challenged by applying it to the purification of multiprotein assemblies forming onto DNA damage-mimicking oligonucleotides. The purified components were identified as well-known DNA repair proteins, and were shown to retain their enzymatic activities, as seen by monitoring DNA ligation products. Remarkably, kinase activities, also monitored, were found to be distinct on the beads and on the purified DNA–protein complexes, showing the benefits to uncouple the DNA–protein assemblies from the beads for a proper understanding of biochemical regulatory mechanisms involved in the DNA–protein assemblies. 相似文献
9.
Tomasz W. Zare¸ba Corina Pascu Waleria Hryniewicz Torkel Wadström 《Current microbiology》1997,34(1):6-11
Forty-four enterococcal strains isolated from human clinical specimens were investigated for binding of 125I-labeled fibronectin, vitronectin, thrombospondin, lactoferrin, and collagen type I and IV, and for cell surface hydrophobicity.
Most strains expressed low binding of iodine-labeled human fibronectin, collagen I and IV, and higher binding of human vitronectin,
human lactoferrin, and human thrombospondin. Bacteria grown in Todd-Hewitt broth exhibited increased binding to vitronectin
and thrombospondin. In particle agglutination assays (PAA), Enterococcus faecalis strains reacted strongly with coated latex beads in contrast to E. faecium strains, which generally did not react. The ability of enterococci to bind ECM proteins was affected by heating and proteolytic
digestion, suggesting that some protein-binding components become surface exposed after treatment with proteases. The binding
of 125I-labeled proteins to E. faecalis strain E70 was inhibited when cells were preincubated with unlabeled proteins. Preincubating cells with sulfated polymers
such as dextran sulfate (M
r
5000 and 8000), pentosan sulfate and heparin decreased binding of vitronectin, lactoferrin, and thrombospondin. The binding
of lactoferrin and thrombospondin was also decreased when bacteria were preincubated with galactose, fucose, and mannosamine,
but not with mannose. All of 30 E. faecalis strains expressed pronounced surface hydrophobicity, but 10 of 14 E. faecium strains showed hydrophilic cell surface.
Received: 22 April 1996 / Accepted: 29 June 1996 相似文献
10.
Dorothy E. Croall 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,714(2):200-208
The interactions in vitro between [3H]estradiol and liver proteins from Xenopus laevis have been examined to determine if the binding reaction meets criteria of steroid-receptors which may function in the induction of vitellogenesis. Estrogenic hormones associated with proteins in serum and liver cytosol from Xenopus laevis. However, the interactions between soluble liver proteins and estrogens apparently do not result from serum contamination of liver as specific binding was distinguishable by ligand affinity and by differential mobility on polyacrylamide gels. Steroid ligands bound by liver proteins during incubation in vitro were examined by solubility and by thin-layer chromatography. Only a small percentage (13%) of the bound radioactive ligand was recovered as the original tritium-labeled steroid, 17β-estradiol. The major ligand was recovered as a water-soluble metabolite of estradiol which was identified tentatively as an estradiol-glucoside. To investigate whether the protein-bound estradiol metabolite(s) merely masks a small amount of authentic estradiol-receptor complexes or if the metabolite could be an intermediate in estrogen function, isolated liver nuclei were incubated with liver cytosol containing 3H-labeled steroid-protein complexes or with serum protein-bound [3H]estradiol. Nuclei preferentially accumulated 3H-labelea steroids from liver cytosol protein-steroid complexes relative to [3H]estradiol from serum proteins. However, analysis of the steroids recovered in the nuclei after incubation with liver cytosol revealed that both 17β-[3H]estradiol and the 3H-labeled water-soluble metabolite were retained in vitro by nuclei. 相似文献
11.
Osamu Kusano-Arai Rie Fukuda Wakana Kamiya Hiroko Iwanari Takao Hamakubo 《Analytical biochemistry》2016
The reliable assessment of monoclonal antibody (mAb) affinity against membrane proteins in vivo is a major issue in the development of cancer therapeutics. We describe here a simple and highly sensitive method for the evaluation of mAbs against membrane proteins by means of a kinetic exclusion assay (KinExA) in combination with our previously developed membrane protein display system using budded baculovirus (BV). In our BV display system, the membrane proteins are displayed on the viral surface in their native form. The BVs on which the liver cancer antigen Roundabout 1 (Robo1) was displayed were adsorbed onto magnetic beads without fixative (BV beads). The dissociation constant (Kd, ∼10−11 M) that was measured on the Robo1 expressed BV beads correlated well with the value from a whole cell assay (the coefficient of determination, R2 = 0.998) but not with the value for the soluble extracellular domains of Robo1 (R2 = 0.834). These results suggest that the BV–KinExA method described here provides a suitably accurate Kd evaluation of mAbs against proteins on the cell surface. 相似文献
12.
Abstract: Membrane-bound opiate receptors from neuroblastoma-glioma hybrid cells and from different parts of the rat brain (whole brain minus cerebellum, cortex, thalamus-hypothalamus and cerebellum) were labeled with the methionine-enkephalin analogue, D-[3H]Ala2-Met-enkephalinamide, and solubilized with the nonionic detergent Brij 36T. The protease inhibitors bacitracin, phenylmethylsulfonyl fluoride, Trasylol, and leupeptin were included in the solubilization buffer to minimize proteolysis. Two simple techniques, ammonium sulfate precipitation and activated charcoal absorbence, were adapted to separate the free and the macromolecule-bound ligands. The solubilized receptor-[3H]enkephalin complexes were partially purified by consecutive passages through Sephadex G-75 and Sepharose 6B columns. Of the three peaks of radioactivity that were observed in the effluent of the Sepharose column, two contained proteins, and one of them, with a Stokes radius of 59 Å, seemed to contain the specific opiate receptor, as evidenced by additional experiments. This peak was further purified on thiol-Sepharose or diethylaminoethanol-Sephadex columns that were eluted with a gradient of 0–50 mM dithiothreitol or with 1.0 M KCI, respectively. The receptor-[3H]enkephalin complex from neuroblastoma-glioma cells (apparent δ-type receptors) binds less to the thiol-Sepharose beads than receptor-(3H]enkephalin prepared from the hypothalamus-thalamus, which is rich in μ receptors. The [3H]enkephalin receptor complexes of the various sources also differed in their stability. The dissociation of the ligand from the neuroblastoma-glioma receptor was monophasic, with a half- life of 250 min, whereas that of two brain regions was biphasic, with half-lives of 195–330 min and 10,000 min. The methods described may be of use for further purification of soluble opiate receptors, either active or cross-linked to the ligand. 相似文献
13.
The orientation to and landing on a source of human odour by female Culex quinquefasciatus Say (Diptera: Culicidae) is observed in a wind tunnel without an airflow or with a laminar airflow of 0.2 m s?1. Odours from human feet are collected by ‘wearing’ clean glass beads inside a stocking and presenting beads in a Petri dish in a wind tunnel. Mosquitoes are activated by brief exposure to a 1 L min?1 jet of 4% CO2 positioned 10 cm from the release cage. In moving air at 0.2 m s?1, a mean ± SE of 3.45 ± 0.49 landings are observed in 10‐min trials (five mosquitoes per trial), whereas 6.50 ± 0.96 landings are recorded in still air. Furthermore, 1.45 ± 0.31 mosquitoes are recorded on beads at any one time in moving air (a measure of individuals landing versus one landing multiple times) compared with 3.10 ± 0.31 in still air. Upwind flight to beads in moving air is demonstrated by angular headings of flight immediately before landing, whereas approaches to beads in still air are oriented randomly. The mean ± SE latency until first landing is 226.7 ± 17.98 s in moving air compared with 122.5 ± 24.18 in still air. Strategies used to locate a prospective host at close range in still air are considered. 相似文献
14.
Tristetraprolin/zinc finger protein 36 (TTP/ZFP36) binds and destabilizes some pro-inflammatory cytokine mRNAs. TTP-deficient mice develop a profound inflammatory syndrome due to excessive production of pro-inflammatory cytokines. TTP expression is induced by various factors including insulin and extracts from cinnamon and green tea. TTP is highly phosphorylated in vivo and is a substrate for several protein kinases. Multiple phosphorylation sites are identified in human TTP, but it is difficult to assign major vs. minor phosphorylation sites. This study aimed to generate additional information on TTP phosphorylation using phosphopeptide mapping and mass spectrometry (MS). Wild-type and site-directed mutant TTP proteins were expressed in transfected human cells followed by in vivo radiolabeling with [32P]-orthophosphate. Histidine-tagged TTP proteins were purified with Ni-NTA affinity beads and digested with trypsin and lysyl endopeptidase. The digested peptides were separated by C18 column with high performance liquid chromatography. Wild-type and all mutant TTP proteins were localized in the cytosol, phosphorylated extensively in vivo and capable of binding to ARE-containing RNA probes. Mutant TTP with S90 and S93 mutations resulted in the disappearance of a major phosphopeptide peak. Mutant TTP with an S197 mutation resulted in another major phosphopeptide peak being eluted earlier than the wild-type. Additional mutations at S186, S296 and T271 exhibited little effect on phosphopeptide profiles. MS analysis identified the peptide that was missing in the S90 and S93 mutant protein as LGPELSPSPTSPTATSTTPSR (corresponding to amino acid residues 83–103 of human TTP). MS also identified a major phosphopeptide associated with the first zinc-finger region. These analyses suggest that the tryptic peptide containing S90 and S93 is a major phosphopeptide in human TTP. 相似文献
15.
16.
Erythrocyte and HeLa cell plasma membranes were isolated on polylysinecoated polyacrylamide beads and the transbilayer disposition of their proteins was investigated.When membranes of intact erythrocytes were isolated on beads and then labelled by lactoperoxidase-catalysed iodination, their labelling pattern was similar to that of inside-out vesicles in solution.When the membranes of intact HeLa cells were isolated on beads and then labelled by galactose oxidase-[3H]borohydride treatment, no glycoprotein or glycolipid sugars were accessible. On the other hand, when the HeLa cell membranes were isolated on beads and then labelled by the lactoperoxidase-catalysed iodination, all of the major membrane proteins were iodinated. These experiments confirmed for HeLa cell membranes what had previously been shown for erythrocyte membranes: when the membranes of intact cells are isolated on beads, the accessibility of their surfaces to enzymatic probes is the same as would be expected of inside-out vesicles in suspension. Double-label experiments, in which the HeLa cell membranes were labelled first on the intact HeLa cells and again after isolation on beads, identified several 相似文献
17.
Erkki Ruoslahti Eva Engvall 《Biochimica et Biophysica Acta (BBA)/General Subjects》1980,631(2):350-358
Collagen-fibronectin complexes, formed by binding of fibronectin to gelatin or collagen insolubilized on Sepharose, were found to bind 20–40% of radioactivity in [35S]heparin. Fibronectin attached directly to Sepharose also bound [35S]heparin, while gelatin-Sepharose without fibronectin did not. Unlabeled heparin and highly sulfated heparan sulfate efficiently inhibited the binding of [35S]heparin, hyaluronic acid and dermatan sulfate were slightly inhibitory, while chondroitin sulfates and heparan sulfate with a low sulfate content did not inhibit.The interaction of heparin with fibronectin bound to gelatin resulted in complexes which required higher concentrations of urea to dissociate than complexes of fibronectin and gelatin alone. Heparin as well as highly sulfated heparan sulfate and hyaluronic acid brought about agglutination of plastic beads coated with gelatin when fibronectin was present. Neither fibronectin nor glycosaminoglycans alone agglutinated the beads.It is proposed that the multiple interactions of fibronectin, collagen and glycosaminoglycans revealed in these assays could play a role in the deposition of these substances as an insoluble extracellular matrix. Alterations of the quality or quantity of any one of these components could have important effects on cell surface interactions, including the lack of cell surface fibronectin in malignant cells. 相似文献
18.
Photosystem (PS) II particles retaining a high rate of O2 evolution were isolated from the mesophilic filamentous cyanobacterium, Spirulina platensis. To achieve high production of PSII complexes in the cells, irradiance from halogen incandescent lamps was used. Disruption of cells by vibration of glass beads proved to be the most suitable procedure for isolation of thylakoid membranes. The selectivity of detergents for PSII particle preparation rose in the order of Triton X-100 < decyl-β-D-glucopyranoside < dodecyldimethyl-aminooxide < n-heptyl-β-D-thioglucoside < N-dodecyl-N,N-dimethylammonio-3-propane sulphonate < n-octyl-β-thioglycoside < octylglucoside < n-dodecyl-β-D-maltoside. The last four detergents yielded extracts, from which pure PSII particles not contaminated by PSI complexes could be obtained by sucrose-gradient centrifugation (20–45%) at the 43% sucrose level. We assumed both the acceptor and donor sides of the isolated n-dodecyl-β-D-maltoside (DM) particles to be intact due to high oxygen production by DM particles [1,500 meq(e?) mol?1 (Chl) s?1] achieved in the presence of all artificial acceptors tested. The PSII particle fraction from the sucrose gradient was used with immobilized metal (Cu2+) affinity chromatography (IMAC) for the preparation of the PSII core complex. By washing the column with a MES buffer containing MgCl2 and CaCl2, the phycobiliproteins were stripped off. The PSII core complex was eluted in a buffer containing 1% DM, mannitol, MgCl2, NaCl, CaCl2, and ?-aminocaproic acid. SDS-PAGE of the core complex provided pure bands of D1 and D2 proteins and PsbO protein from thylakoid membrane, which were used to raise polyclonal antibodies in rabbits. These antibodies recognized D1 and D2 not only as monomers of 31 and 32 kDa proteins, but also as heterodimers of D1, D2 corresponding to the band of 66 kDa on SDS-PAGE. This was in contrast to antibodies of synthetic determinants, which reacted only with the monomers of D1 and D2 proteins. These negative reactions against heterodimers of D1, D2 supported the hypothesis that dimeric forms of PSII reaction centre proteins have a C-terminal sequence sterically protected against a reaction with specific antibodies. 相似文献
19.
《Journal of receptor and signal transduction research》2013,33(4):529-539
AbstractA photoaffinity derivative of highly purified 125I-labelled epidermal growth factor (125I-EGF) has been synthesized. The heterobifunctional crosslinking reagent p-azidophenylglyoxal (PAPG) was bound to arginine residues in 125I-EGF. PAPG-125I-EGF bound to EGF receptors on rat fibroblasts and human A431 epidermoid carcinoma cells in culture. An apparent decreased affinity of PAPG-125I-EGF for the EGF receptor is in accord with at least one arginine being at or near the EGF receptor binding site. The PAPG-125I-EGF:EGF receptor complexes on rat cells were internalized to the same extent as control EGF:receptor complexes. A431 cells treated with PAPG-125I-EGF were irradiated with ultraviolet light and the labelled proteins were analyzed by SDS-polyacrylamide gel electrophoresis. The 3 major labelled proteins had apparent molecular weights ranging from 75,000 to 200,000. Only the labelling of the 200,000-Mr protein was prevented by the addition of excess unlabelled EGF with the PAPG-125I-EGF. This molecular weight is in agreement with the reported size of the EGF receptor plus EGF. A protein with apparent molecular weight of 100,000 was labelled by 125I-EGF by an unknown mechanism which was dependent on the dose of UV light and blocked by the addition of excess unlabelled EGF. 相似文献
20.