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1.
A method for the quantitative determination of protein incorporated in solubilized polyacrylamide gels 总被引:1,自引:0,他引:1
Dense and light polyacrylamide gels containing N,N′-(1,2-dihydroxyethylene)bisacrylamide (DHEBA) or N,N′-diallyltartardiamide (DATD) as the crosslinker have been tested for their solubilization properties. Both types of gel can be dissolved in 10 mm periodic acid. The time and temperature required for complete dissolution of slabs of DHEBA crosslinked gels (12 h at 50°C), however, greatly exceed those required for dissolving slabs of DATD-polyacrylamide gel (0.5 h at 22°C). Bovine serum albumin kept under the respective dissolving conditions gave a lower response in the Lowry protein assay in instances where hot periodic acid had been used. Nearly independent of the type and concentration of their constituents, the different dissolved polyacrylamide gels interfere slightly with the Lowry assay by causing some “aspecific” color development. A method is outlined enabling a reliable quantitative determination of protein incorporated in DHEBA or DATD crosslinked polyacrylamide gels. 相似文献
2.
The retention of low molecular weight proteins during electrophoresis through gradient polyacrylamide gels was improved when a gradient of N,N′,N″-triallyl citric triamide (TACT) was superimposed on the gradient of acrylamide and N,N′-methylenebisacrylamide (MBA). Gels cross-linked only with N,N′-(1,2-dihydroxyethylene)bisacrylamide (DHEBA) are soluble in dilute periodic acid or dilute aqueous solutions of bases. DHEBA cross-linked gradient gels have a smaller pore structure at high acrylamide concentrations and a more open structure at low acrylamide concentrations than gels cross-linked with MBA. Proteins labeled with tritium and carbon-14 were fractionated through DHEBA cross-linked gradient gels and the isotopes measured after solution of the gel with periodic acid. The mild solubilizing conditions enhanced isotope resolution. The characteristics of several cross-linking molecules are discussed and reasons advanced for the superiority of those with acrylamido end groups. 相似文献
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4.
Polyacrylamide gels cross-linked with N,N′-diallyltartardiamide (DATD) are, in contrast to gels cross-linked with N,N′-methylenebisacrylamide (Bis), readily solubilized by periodic acid (Anker, H. S., 1970, FEBS Lett.7, 293), thus permitting efficient analyses of electrophoretically separated, labeled biological material. The capacities of polyacrylamide gels, cross-linked with Bis and DATD, to serve as media for electrophoretic separation of proteins, were compared. As DATD-cross-linked gels were inferior to equimolar Bis-crosslinked gels with 5% cross-linking (CBis = 5%) by the criteria of more pronounced swelling, markedly softer gels and, less concentrated and bended protein zones on electrophoresis and subsequent staining, gels cross-linked with different percentage CDATD were examined. The water regain of DATD-cross-linked gels, the retardation coefficients, and free mobilities of different proteins in equimolar Bis- and DATD-cross-linked gels were determined. When the DATD concentration in gels was increased to CDATD = 27%, gels assumed physical characteristics comparable to those cross-linked with Bis at CBis = 5%. We report further the rapid, facile isolation of protein bands out of the gel matrix cross-linked with DATD. However, the isolation procedure results in an irreversible loss of biological activity. 相似文献
5.
The reaction of the monofunctional [Pt(Gly-Gly-N,N′,O)I]− complex, in which Gly-Gly is the dipeptide glycyl-glycine coordinated through two nitrogen and oxygen atoms, with the N-acetylated dipeptide l-methionyl-l-histidine (MeCOMet-His) studied by 1H NMR spectroscopy. All reactions were carried out in 50 mM phosphate buffer at pD 7.4 and at 25 °C. In the initial stage of the reaction, the platinum(II) complex forms the kinetically favored [Pt(Gly-Gly-N,N′,O)(MeCOMet-His-S)]− complex, with unidentate coordination of the MeCOMet-His dipeptide through the sulfur atom of the methionine residue. In the second stage of the reaction, complete intramolecular migration of the [Pt(Gly-Gly-N,N′,O)] unit from the sulfur to the N3 nitrogen atom of imidazole was observed and a new platinum(II)-peptide complex, [Pt(Gly-Gly-N,N′,O)(MeCOMet-His-N3)]− was formed. In comparison with previous results obtained for the reaction of [Pt(dien)Cl]+ with different methionine- and histidine-containing peptides, this migration reaction was sufficiently fast and strongly selective to the N3 atom of the imidazole ring of the histidine side chain. This study is an important step in the development of new platinum(II) complexes for selective covalent modification of peptides and proteins. 相似文献
6.
The dynamic properties (storage moduli, G′ and loss moduli, G″) of tamarind gels and the influence of saccharose and polysaccharide concentrations were studied using model rings of 3 mm thickness and 20 mm diameter, prepared with three saccharose (55, 60 and 65% w/v) and three polysaccharide concentrations (1.5, 2.0 and 2.5% w/v). Small amplitude oscillatory measures were taken at 25°C in a PHYSICA LS 100 rheometer with parallel plate geometry. Results for the 9 gels showed the zone of linear viscoelasticity between 0.637 and 6.37 Pa of oscillatory shear stress. The mechanical spectra obtained after 24, 48 and 72 h evidenced the presence of syneresis with an increase in G′ as a function of time. The effects of polysaccharide concentrations on gel viscoelasticity were greater than those of saccharose. 相似文献
7.
Sabine Seeberg ne Kraft Caroline Bischof Annette Loos Sebastian Braun Nigar Jafarova Ulrich Schatzschneider 《Journal of inorganic biochemistry》2009,103(8):1126-1134
A series of five tris(2-pyridylmethyl)amine (tpa) ruthenium complexes [Ru(tpa)(N–N)](PF6)2 with N–N = bpy (2,2′-bipyridine), phen (1,10-phenanthroline), dpq (dipyrido[3,2-d:2′,3′-f]quinoxaline), dppz (dipyrido[3,2-a;2′,3′-c]phenazine), and dppn (4,5,9,16-tetraazadibenzo[a,c]naphthacene) was prepared and characterized by NMR, UV–Visible (UV/Vis), and fluorescence spectroscopy as well as cyclic voltammetry. Structures optimized with density functional theory methods (DFT, BP86, TZVP) without constraints show C1 symmetry while in solution, the 1H and 13C NMR spectra are in accordance with an average Cs symmetry. This is thought to be due to a low energy barrier for flipping of the equatorial pyridine ring from one side of the N–N plane to the other. The electronic structure of the compounds was studied with DFT and a change in the highest occupied molecular orbital (HOMO) character from Ru t2g for the bpy, phen, and dpq to N–N ligand-based for the dppz and dppn complexes was found. TDDFT calculations showed dominant N–N-based intra-ligand charge transfer (ILCT) transitions in the latter two complexes mixed with metal-to-ligand charge transfer (MLCT) bands found for all five compounds. DNA binding of the complexes was studied with UV/Vis titrations, the fluorescent ethidium bromide displacement assay, and CD spectroscopy. The affinity increases with the aromatic surface area of of the bidentate N–N ligand in the order bpy phen < dpq < dppz dppn. Viscosity measurements support an intercalative binding mode for the latter three compounds, while the others did not show a pronounced effect of the hydrodynamic properties of calf thymus (CT) DNA. 相似文献
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9.
Tetsuo Takemura Go Emoto Jun Satoh Yoshitaka Kobayashi Chihiro Yaginuma Yuta Takahashi Takamitsu Utsukihara C. Akira Horiuchi 《Journal of Molecular Catalysis .B, Enzymatic》2008,55(3-4):104-109
Both enantiomers of 2,2′-dihydroxy-4,4′,5,5′,6,6′-hexamethybiphenyl (2), a potentially useful chiral synthon, were obtained with >99% ee in high enantioselectivity by cholesterol esterase or porcine pancreas lipase (PPL)-mediated hydrolysis of the corresponding (±)-dipentanoate or (±)-dihexanoate, respectively. Absolute configuration of (S)-3-bromo-2,6′-dimethoxy-4,5,6,2′,3′,4′-hexamethyl-biphenyl (2h) was determined by X-ray analysis. 相似文献
10.
Jason P. Landrum Joseph P. Montoya 《Journal of experimental marine biology and ecology》2009,380(1-2):20-24
Aquatic crustaceans often play a major role in organic matter (OM) transformation and recycling through their feeding and excretory activities. In this study, we measured the isotopic and elemental composition of organic matter fed to Palaemonetes sp. shrimp and the fecal pellets they produced. Nitrogen (N) content of the food (8.2 ± 0.2%, mean ± SD) was significantly higher than the fecal pellets (2.0 ± 0.9%), a pattern that also applied to the carbon (C) content of food (46.7 ± 1.0%) and fecal pellets (14.3 ± 6.8%). We also found a significant decrease in the N content of undigested, macerated food (6.1 ± 0.9 %) relative to food that had been soaked in artificial seawater (ASW) and artificial seawater that had previously contained shrimp (CASW) in the absence of feeding shrimp. We found no significant difference in N or C isotopic composition between the dry food, ASW- and CASW-soaked control food, and fecal pellets. We did, however, observe a significant increase in δ15N of the undigested, macerated food (δ15N = 6.3 ± 0.6‰) relative to both the dry flake food (δ15N = 5.6 ± 0.2‰) and controls incubated in the absence of shrimp in either ASW (δ15N = 5.6 ±0.3‰) or CASW (δ15N = 5.8 ± 0.1‰). Our results differ from previous findings of isotopic alteration of OM during processing by crustaceans (copepods), suggesting that isotopic changes related to feeding might be either taxon- or food-specific. This study also provides information on the influence of grazers/shredders on both the elemental and isotopic composition of POM, suggesting that larger aquatic shredders can influence the chemical composition of particles by either physical manipulation of the POM (release of DOM) or by facilitating microbial colonization of the POM. 相似文献
11.
Takaharu Kanno Masanobu Kawanishi Takeji Takamura-Enya Volker M. Arlt David H. Phillips Takashi Yagi 《Mutation Research - Genetic Toxicology and Environmental Mutagenesis》2007,634(1-2):184-191
3-Nitrobenzanthrone (3-nitro-7H-benz[d,e]anthracen-7-one, 3-NBA) is a powerful mutagen and a suspected human carcinogen existing in diesel exhaust and airborne particulates. Recently, one of the major presumed metabolites of 3-NBA, 3-aminobenzanthrone (3-ABA), was detected in human urine samples. Here we analyzed DNA adducts formed in 3-NBA-exposed human hepatoma HepG2 cells by a 32P-postlabeling/thin layer chromatography (TLC) method and a 32P-postlabeling/polyacrylamide gel electrophoresis (PAGE) method. With HepG2 cells exposed to 3-NBA (0.36–36.4 μM) for 3 h, we obtained three spots or bands corresponding to adducted nucleotides. Two were assigned as 2-(2′-deoxyadenosin-N6-yl)-3-aminobenzanthrone-3′-phosphate (dA3′p-N6-C2-ABA) and 2-(2′-deoxyguanosin-N2-yl)-3-aminobenzanthrone-3′-phosphate (dG3′p-N2-C2-ABA), with identical mobilities to those of synthetic standards on PAGE analysis. The chemical structure of the substance corresponding to the other spot or band could not be identified. Quantitative analyses revealed that the major adduct was dA3′p-N6-C2-ABA and its relative adduct labeling (RAL) value at 36.4 μM of 3-NBA was 200.8 ± 86.1/108 nucleotide. 相似文献
12.
The present research investigates the possibility that 2 weak urea-type cytokinins, the N,N′-bis-(1-naphthyl)urea and the N,N′-bis-(2-naphthyl)urea, enhance adventitious root formation. The rooting activity was assessed using the stem slice test, the mung bean rooting test and the rooting of apple microcuttings. The two compounds influenced the adventitious rooting process differently as regards the bioassay used. In the stem slice test, in the presence of exogenous auxin, both compounds enhanced the rooted slice percentage. In mung bean shoots, the N,N′-bis-(1-naphthyl)urea enhanced the root formation at the lowest concentration used (0.01 μM) while the N,N′-bis-(2-naphthyl)urea enhanced rooting at higher concentrations. In the rooting test of apple microcuttings the N,N′-bis-(1-naphthyl)urea and the N,N′-bis-(2-naphthyl)urea slightly enhanced only the mean root number per microcutting. 相似文献
13.
Howard L. Bachrach 《Analytical biochemistry》1981,110(2)
Protein bands become visible in polyacrylamide gels containing 8
urea after chilling the gels in air for 5 to 10 min at −70°C. Urea appears to crystallize preferentially as opaque bands in regions of the gel where protein reduces the amount of free water available as solvent for the urea molecules. Thus detected, the gel sections containing protein bands from foot-and-mouth disease virus can be immediately cut out, and their proteins obtained by electrophoretic elution or extraction procedures. Analysis of the proteins for purity and concentration is then carried out by electrophoresing measured aliquots on analytical gels, staining with Coomassie brilliant blue, scanning the gels for absorbance at 600 nm, and converting peak areas to micrograms of protein using Folin phenol standard curves determined for each purified capsid protein. The most basic capsid protein and its in virion proteolytic-cleavage products stain metachromatically. 相似文献
14.
Certain reagents utilized in the formation of polyacrylamide gels are shown to interfere in the Lowry assay for protein. Acrylamide (3–30%) and potassium ferrocyanide (0.0015–0.0105%) produced a linear response in color formation. Both compounds are capable of reducing the phenol reagent in the absence of copper and the interference can be compensated for by employing the appropriate blank. An extract of polymerized and electrophoresed gels also interferes in the Lowry assay, however, this increased color formation cannot be corrected by using a gel extract blank. Under the conditions studied, filtration, centrifugation, and dialysis did not sufficiently remove the acrylamide fines responsible for the interference. 相似文献
15.
Raymond D. Pratt John P. Johnson 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1984,805(4):405-411
Thyroid hormone (T3) has been demonstrated to inhibit the action of aldosterone on sodium transport in toad urinary bladder and rat kidney. We have exammined the effect of T3 on aldosterone action and specific nuclear binding in cultured epithelial cells derived from toad urinary bladder. In cell line TB6-C, addition of 5·10−8 M T3 to culture media for up to 3 days results in no change in short-circuit current or transepithelial resistance. This concentration of T3 completely inhibits the maximal increase in short-circuit current in response to 1·10−7 M aldosterone. The inhibition can be demonstrated with 18 h preincubation or with simultaneous addition of T3 and aldosterone. The half-maximal concentration for the inhibition of the aldosterone effect is approx. 5·10−9 M T3. T3 has no effect on cyclic AMP-stimulated short-circuit current in these cells. The effect of T3 on nuclear binding of [3H]aldosterone was examined using a filtration assay with data analysis by at least-squares curve-fitting program. Best fit was obtained with a model for two binding sites. The dissociation constants for the binding were K′d1 = (0.82 ± 0.36)·10−10 M and K′d2 = (3.2±0.60)·10−8 M.The half-maximal concentration for aldosterone-stimulated sodium transport in these cells is approx. 1·10−8 M. Analysis of nuclear aldosterone binding in cells preincubated for 18 h with 5·10−8 M T3 showed a K′d1 = (0.15 ± 0.10)·10−10 M and K′d2 = (3.5 ± 0.10)·10−8 M. We conclude that T3 i action of aldosterone on sodium transport at a site after receptor binding in the nucleus. 相似文献
16.
Viktor Stolc 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1982,721(3):236-239
The content of adenosine 3′,5′-monophosphate in human mononuclear leukocytes was enhanced 3–5-times by venoms obtained from African toad (Bufo africanus), American toad (Bufo americanus), Colorado river toad (Bufo arenarum) and Marine toad (Bufo marinus) at 25 μg/ml for 5 min of incubation at 37°C. The maximum stimulation was observed after 1–5 min of incubation. The half-maximal stimulation was observed at 0.1 μg/ml venom obtained from Colorado river toad (Bufo arenarum). The increased content of adenosine 3′,5′-monophosphate in the mononuclear leukocytes persisted without significant change for at least 30 min of incubation at 37°C. 相似文献
17.
Eleonora Winkelhausen Ruzica Jovanovic-Malinovska Slobodanka Kuzmanova Maja Cvetkovska Christo Tsvetanov 《World journal of microbiology & biotechnology》2008,24(10):2035-2043
Hydrogels based on high molecular weight poly(ethylene oxide) were synthesized by u.v.-irradiation of aqueous solutions in
presence of the photoinitiator, (4-benzoylbenzyl)trimethylammonium chloride and different crosslinkers, poly(ethylene glycol),
diacrylates and N,N′-methylenebisacrylamide. Candida boidinii cells were immobilized in these hydrogels and the gels were characterized in regards to gel fraction yield, degree of equilibrium
swelling, shear storage and loss moduli. In addition, the number average molecular weight between crosslinks and the mesh
size were estimated. The incorporated yeast cells considerably affected the viscoelastic properties of the gels. Immobilized
C. boidinii cells were used for conversion of xylose to xylitol. Of the immobilized systems tested, only the system with poly(ethylene
oxide) crosslinked with N,N′-methylenebisacrylamide exhibited xylitol production. The operational stability of this system was evaluated by seven repeated-batch
runs performed in Erlenmeyer flasks in duration of 55 days. The progressive improvement of xylose consumption, up to 73.5%,
stopped in the fifth cycle, after which it dropped to 42.7%. Although xylitol concentration never reached more than 4.2 g l−1, xylitol was produced in each of the seven cycles. The cell leakage of 1.8 g l−1 during the first 45 days, indicated very good stability of the system. 相似文献
18.
Donald Poirier Claude Labrie Yves Mrand Fernand Labrie 《The Journal of steroid biochemistry and molecular biology》1991,38(6)
Seven estradiol (E2) derivatives with an alkynylamide side chain at the 17α position were synthesized starting from ethynylestradiol (EE2). The main chemical step was the coupling reaction of the acetylide ion of EE2 with carbon dioxide, glutaric anhydride or bromoalkyl ortho ester. The synthesis of these compounds is fast (3–6 steps according to the compound) and is easily achieved with good yield. Five compounds with different side chain lenghts were evaluated for uterotrophic and antiuterotrophic activity in the CD-1 mouse. None of the tested compounds shows estrogenic activity in this sensitive in vitro system. At low doses (1 and 3 μg), a 14–57% inhibition of E2-induced uterine growth was observed while no additional inhibition was observed at the 10, 20 and 30 μg doses. In human breast carcinoma cells in culture, all compounds show estrogenic activity at high concentrations while only compound 39 (N-buty,N-methyl-8-[3′,17′β-dihydroxy estra-1′,3′,5′(10′)-trien-17′α-yl]-7-octynamide) possesses antiproliferative or antiestrogenic effects. No significant correlation could be demonstrated between alkynylamide side chain length and estrogenic or antiestrogenic activity. Among the compounds tested, the derivative of EE2 possessing a five-methylene (CH2) side chain (compound 39) possesses the best antiestrogenic activity (44 ± 7% in the CD-1 mouse uterus assay at the 3μg dose and 57 ± 4% at 0.1 nM in human ZR-75-1 cancer cells in culture). 相似文献
19.
Carla Bisi-Castellani 《Inorganica chimica acta》1984,90(3):155-159
The structures of (H2tmen)[PtCl4], (1), (H2tmen = N,N,N′,N′-tetramethylethylenediammonium), [triclinic, P
; A = 7.344(3), B = 8.345(3), C = 6.216(2) Å, α = 84.53(3), β = 109.22(3), γ = 69.43(3)°, Z = 1] and (H2tmen)[PtCl6], (2), [monoclinic, P21/a; A = 14.409(4), B = 12.736(7), C = 8.601(3) Å, β = 99.58(3)°, Z = 4] were determined from diffractometric data by Patterson and Fourier methods and refined by full-matrix least-squares to R = 0.027 and 0.039 for (1) and (2) respectively. In both cases the anions and cations are joined in polymeric chains through hydrogen bonds involving the protonated nitrogens and the co-ordinated chlorine atoms. The square-planar [PtCl4]2− and octahedral [PtCl6]2− anions are centrosymmetric; the H2tmen2+ cations are centrosymmetric in (1) with a N-C-C-N dihedral angle of exactly 180°, while in (2) the dihedral angle is 166°. The different symmetry of the organic moieties/Ci and C1 in (1) and (2) respectively] results in a different i.r. spectrum which is more complex the lower the symmetry; moreover the spectrum of a KBr pellet of (2) changes with time, finally resembling that of (1) and of the (H2tmen)Cl2 salt. 相似文献
20.
John W. Huffman Simon M. Bushell John R. A. Miller Jenny L. Wiley Billy R. Martin 《Bioorganic & medicinal chemistry》2002,10(12):7809
Three series of new cannabinoids were prepared and their affinities for the CB1 and CB2 cannabinoid recptors were determined. These are the 1-methoxy-3-(1′,1′-dimethylalkyl)-, 1-deoxy-11-hydroxy-3-(1′,1′-dimethylalkyl)- and 11-hydroxy-1-methoxy-3-(1′,1′-dimethylalkyl)-Δ8-tetrahydrocannabinols, which contain alkyl chains from dimethylethyl to dimethylheptyl appended to C-3 of the cannabinoid. All of these compounds have greater affinity for the CB2 receptor than for the CB1 receptor, however only 1-methoxy-3-(1′,1′-dimethylhexyl)-Δ8-THC (JWH-229, 6e) has effectively no affinity for the CB1 receptor (Ki=3134±110 nM) and high affinity for CB2 (Ki=18±2 nM). 相似文献