共查询到20条相似文献,搜索用时 15 毫秒
1.
《Process Biochemistry》2007,42(5):812-819
Dual fluorescence confocal laser scanning microscopy has been used to visualise the binding of a fluorescently labelled polyclonal ovine anti-fluorescein F(ab′)2 antibody to immobilised fluorescein. The fluorescent ligand was immobilised on a Streamline quartz base agarose matrix; a resin used industrially for expanded bed chromatography, using two different fluorescein initial concentrations in order to obtain two batches of immunogen-affinity adsorbent with different immobilised ligand densities. The fluorescein specific F(ab′)2 were purified from anti-fluorescein serum pepsin digest by adsorption on immobilised antigen chromatographic resin, followed by conjugation to the fluorescent probe Alexa Fluor 660. The dual fluorescence signals from the immobilised antigen and the immuno-specific F(ab′)2 were used to map the progressive depth of the bound F(ab′)2 layer within individual adsorbent beads. In addition, the labelled anti-fluorescein F(ab′)2 was diluted to identical antigen binding activity concentrations in crude serum digest and in blank buffer and the resulting fluorescence intensity profiles were comparatively assessed for any detectable differences in binding patterns that might be caused by processing the more complex mixture of crude serum digests. It was observed that the relative immobilised ligand utilisation was higher when using the immuno-adsorbent with lower immobilised antigen density. Furthermore, the progression of the adsorbed F(ab′)2 front inside the immuno-adsorbent beads displayed closer agreement with the postulates of the shrinking core mechanism (SCM) when the immuno-adsorbent with lower immobilised antigen was used. The confocal images did not reveal any differences between the depth of the adsorption fronts of crude serum digest and pre-purified F(ab′)2 samples. 相似文献
2.
Yoav Ben-Yoseph Emmanuel Shapira David Edelman Barbara K. Burton Henry L. Nadler 《Archives of biochemistry and biophysics》1977,184(1):373-380
Two neutral β-galactosidase isozymes were purified from human liver. The initial step of purification was removal of the acidic β-galactosidases by adsorption on concanavalin A-Sepharose 4B conjugate. Subsequent purification steps included ammonium sulfate precipitation, diethylaminoethyl cellulose column chromatography, Sephadex G-100 gel filtration, and preparative polyacrylamide-gel isoelectric focusing. The final step of purification was affinity chromatography of the separated isoelectric forms on ?-aminocaproyl-β-d-galactosylamine-Sepharose 4B conjugate. The purified β-galactosidase isozymes had activity toward both β-d-galactoside and β-d-glucoside derivatives of 4-methylumbelliferone and p-nitrophenol with a pH optimum around 6.2. These enzyme forms were also found to possess lactosylceramidase II activity with a pH optimum in the range of 5.4 to 5.6, but not lactosylceramidase I activity and no activity toward galactosylceramide or GM1-ganglioside. The molecular weight was found to be in the range of 37,500–39,500 for the two neutral isozymes and they had similar Km and V values; the more acidic form (designated β-galactosidase N1) was more heat stable than the other form (designated β-galactosidase N2). Antibodies evoked against the N1 and N2 β-galactosidases gave identical precipitin lines retaining enzymatic activity. No cross-reactivity was observed between the neutral and the acidic isozymes when examined with the respective antisera. 相似文献
3.
Diego F. Hernández-Ramírez Elizabeth Olivares-Martínez Carlos A. Núñez-Álvarez Eneas A. Chavelas Enrique García-Hernández Gregoria Gómez-Hernández Luis Llorente Antonio R. Cabral 《Biochemical and biophysical research communications》2014
Several studies have shown that conformational changes of β2-glycoprotein I (β2GPI) when bound to negatively charged components expose cryptic epitopes and subsequent binding of anti-β2GPI from patients with antiphospholipid syndrome (APS). However, the role of the carbohydrate chains of β2GPI in this anti-β2GPI reactivity is poorly understood. We therefore studied the reactivity and inhibition of anti-β2GPI antibodies from APS patients with native, partially glycosylated β2GPI (pdβ2GPI; without sialic acid) and completely deglycosylated β2GPI (cdβ2GPI). To determine the potential biologic importance of these glycoforms and their interaction with anti-β2GPI in vitro, stimulation assays were performed with the U937 cell line. Circular dichroism (CD) and fluorescence analysis of the three β2GPI forms were also studied. We found an increased reactivity of anti-β2GPI against pdβ2GPI and cdβ2GPI compared to native β2GPI. Both deglycosylated β2GPI isoforms showed higher inhibition of the anti-β2GPI reactivity than the native protein in soluble-phase. Likewise, the antibody/β2GPI/glycoform complexes increased the synthesis of IL-6, IFNγ and TNFα and the expression of HLA-DR, CD14 and CD11c in U937 cells. CD and fluorescence studies of the glycoforms yielded considerable changes in the fluorescence signals. Our work suggests that the partial or complete removal of the carbohydrate chains uncover cryptic epitopes present in β2GPI. The differentiation and increased synthesis of pro-inflammatory cytokines by U937 cells in vitro may have pathogenetic implications. 相似文献
4.
B Eskin B Treadwell B Redfield C Spears H F Kung H Weissbach 《Archives of biochemistry and biophysics》1978,189(2):531-534
Two forms of initiation factor 2, (IF-2α, Mr, 118,000 and IF-2β, Mr 90,000) have been isolated from Escherichia coli extracts and tested for their ability to support β-galactosidase synthesis in a phage DNA-directed in vitro protein synthesis system. Although both forms are equally active in supporting the binding of fMet-tRNA to ribosomes only IF-2α functions in β-galactosidase synthesis. 相似文献
5.
Heidi Kiil Blomhoff Rune Blomhoff Terje B. Christensen 《Biochimica et Biophysica Acta (BBA)/General Subjects》1983,757(2):202-208
In order to enhance the stability of β-galactosidase, we conjugated the enzyme with dextran T-10 (Mr approx. 10 000). The conjugate contained 9–10 mol dextran/mol protein (β-galactosidase, Mr 68 000), and the specific activity retained after conjugation was 90 ± 4% (n = 3) of the initial activity. Uptake and degradation of native and conjugated β-galactosidase in isolated hepatocytes and nonparenchymal liver cells was studied. There was a marked increase in stability against degradation in both cell types when β-galactosidase was conjugated with Dextran. The degradation of dextran-conjugated enzyme was reduced by 35% in hepatocytes and by 43% in nonparenchymal cells, after 80 and 40 min, respectively, as compared with the free enzyme. However, there was insignificant difference between the uptake of native and conjugated enzyme into the liver cells. Upon intravenous infusion into rats, native and conjugated enzyme were cleared from plasma with only a slight difference in the clearance rate. The observed stability of dextran-conjugated β-galactosidase towards cellular degradation was in accordance with the in vitro experiments. The conjugate showed marked thermal stability at 50°C and enhanced resistance towards proteolysis by the broad specific protease subtilopeptidase A. This demonstrates that dextran conjugation may be used as a means of stabilizing lysosomal enzymes for therapeutic purposes. 相似文献
6.
《Journal of Molecular Catalysis .B, Enzymatic》2011,70(3-4):147-153
Bacillus macerans cyclodextrin glycosyltransferase (CGTase) (EC 2.4.1.19) was covalently immobilised on Eupergit C and used in a packed-bed reactor to investigate the continuous production of long-carbohydrate-chain alkyl glycosides from α-cyclodextrin (α-CD) and n-dodecyl-(1,4)-β-maltopyranoside (C12G2β). The effects of buffer ion strength and pH, and enzyme loading on the immobilisation yield and the enzyme activity were evaluated. Approximately 98% of the protein and 33% of the total activity were immobilised. At pH 5.15, the enzymatic half-life was 132 min at 60 °C and 18 min at 70 °C. The immobilised enzyme maintained 60% of its initial activity after 28 days storage at 4 °C. The degree of conversion was controlled by simple regulation of the flow rate through the reactor, making it possible to optimise the product distribution. It was possible to achieve a yield of the primary coupling product n-dodecyl-(1,4)-β-maltooctaoside (C12G8β) of about 50%, with a ratio between the primary and the secondary coupling product of about 10. Thermoanaerobacter sp. CGTase (Toruzyme 3.0 L) immobilised on Eupergit C had good operational stability at 60 and 70 °C thus showing the advantages of using more thermostable enzymes in biocatalysis. However, this enzyme was unsuitable for the production of C12G8β due to extensive disproportionation reactions, giving a broad product range. 相似文献
7.
《Enzyme and microbial technology》1986,8(11):681-685
Detachment of immobilized antibody from its support matrix in an immunosorbent system prepared by the cyanogen bromide activation route was demonstrated. The immunosorbent system, however, was stable under slightly basic conditions. Detachment of antibody from the support material occurred mainly during the elution of the antigen complexed with the immobilized antibody. The antibody was detached from the matrix by different elution buffers. The detachment pattern of antibody was independent of the number of cycles used and also independent of the support materials. A change in the molecular structure of the detached antibody occurred as revealed by an alteration in the ultraviolet absorption spectra of the released antibody. The antibody detachment from the support matrix occurred in more than one antigen-antibody system suggesting that the leakage phenomenon may be a widespread disadvantage associated with the cyanogen bromide activation procedure. Detachment of the antibody could be reduced to < 10 ng ml−1 by immobilizing antibody on the properly oxidized polysaccharide support material or on the N-hydroxysuccinimide activated ester gel. Antibody dissociation from the matrix did not occur when antibodies were immobilized by either amine or amide attachment, thus, immunosorbents prepared by such strategies are suitable for the immunochromatographic purification of proteins from complex mixtures. 相似文献
8.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1995,664(1):97-106
β2-Microglobulin (β2-M), which accumulates in the plasma of patients undergoing long-term dialysis, has been identified as the principal precursor protein of amyloid fibrils in dialysis-related amyloidosis. As no specific treatment for this affection has been yet established, an extracorporeal immunoadsorption procedure appears to be an attractive therapeutic approach to remove β2-M. Several murine monoclonal antibodies to human β2-M were developed and compared as affinity ligands. One of them was selected on the basis of its specificity and adsorption capacity. In order to achieve maximum efficiency in protein removal, different parameters of the procedure were studied and optimized: effect of antibody coupling density, determination of maximum adsorption capacity of the immunoadsorbents and influence of antigen concentration and of flow-rate on antigen capture efficiency. The conditions of regeneration of immunoaffinity sorbents were also investigated to allow their multiple use without loss of adsorption capacity. The results show the validity of the proposed technique in removing β-M from plasma of patients with chronic renal failure. 相似文献
9.
《Experimental mycology》1990,14(3):290-293
ThebrlA andabaA genes ofAspergillus nidulans regulate stages of conidiophore development and are themselves regulated during development.brlA− mutants produce conidiophore stalks devoid of vesicles, sterigmata, and spores.abaA− mutants produce most of the conidiophore structures but fail to form conidia. To assess the spatial expression of these two genes, we fused the 5′ flanking region ofbrlA orabaA to theEscherichia coli lacZ gene.A. nidulans transformants with a single copy of either fusion gene integrated at a defined heterologus locus (argB) expressedβ-galactosidase during conidiophore development, parallelingbrlA andabaA mRNA accumulation. Controls lacking the fusion genes produced little or noβ-galactosidase activity. A method forin situ detection ofβ-galactosidase was devised. Hyphae or conidiophores were permeabilized by treatment with chloroform vapors and stained with 5-bromo-4-chloroindolyl-β-d-galactoside.β-Galactosidase activity was detected in specific conidiophore cell types.brlA- andabaA-directedβ-galactosidase accumulated in vesicles, sterigmata, and immature conidia. This procedure should be applicable for determining cellular specificities of gene expression in fungi for which transformation systems exist. 相似文献
10.
α-Mannosidase and β-N-acetylhexosaminidase, which could function in the cleavage of glycosidic linkages in the native Ricinus communis lectins, and β-galactosidase were purified some 100-fold from the endosperm tissue of castor bean seedlings. The procedure used ammonium sulphate precipitation followed by chromatography on CM-cellulose, hydroxyapatite and Sephacryl S-300 to separate the three activities. All three glycosidases were present, with the lectins, in the protein bodies of dry seed and increased in activity during the time that lectins are broken down in the vacuoles. The enzymes show optimal activity in the range pH 3–5.5. The α-mannosidase had a Km of 0.77 mM for p- nitrophenyl-α-D-mannopyranoside. The β-galactosidase showed a Km of 1.39 mM for p-nitrophenyl-β-D-galactopyranoside. The β-N-acetylhexasominidase had a Km of 0.47 mM for p-nitrophenyl-N-acetyl-β-N-glucosamide and a Km of 0.33 mM for p-nitrophenyl-N-acetyl-β-D-galactosamide. Effects of competitive inhibitors and cations were described. 相似文献
11.
《Process Biochemistry》2010,45(6):954-960
A psychrotolerant yeast Guehomyces pullulans 17-1 isolated from sea sediment in Antarctica could produce high level (17.2 U/ml) of both extracellular and cell-bound β-galactosidase. The extracellular β-galactosidase in the supernatant of the cell culture of the psychrotolerant yeast G. pullulans 17-1 was purified to homogeneity with a 2.4-fold increase in specific activity as compared to the supernatant by concentration, gel filtration chromatography (Sephadex G-200) and cation-exchange chromatography (CM-Sepharose Fast Flow cation-exchange). The molecular mass of the purified extracellular β-galactosidase was estimated to be 335 kDa. The optimal temperature and pH of the purified β-galactosidase were 50 °C and 4.0, respectively. Km and Vmax values of the purified β-galactosidase for o-nitrophenyl-β-d-galactopyranoside were 3.3 mM and 9.2 μmol/min. Lactose can be converted into glucose and galactose and a large amount of reducing sugar can be released from milk under catalysis of the purified β-galactosidase. The matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectroscopy identified a peptide ALEEYKK which is the conserved motif of the β-galactosidases from other yeasts. The results show that the enzyme may have potential applications in food industry. 相似文献
12.
β-Galactosidase (EC: 3.2.1.23), one of the glycosidases detected in Erythrina indica seeds, was purified to 135 fold. Amongst the four major glycosidases detected β-galactosidase was found to be least glycosylated, and was not retained by Con-A CL Seralose affinity matrix. A homogenous preparation of the enzyme was obtained by ion-exchange chromatography, followed by gel filtration. The enzyme was found to be a dimmer with a molecular weight of 74 kDa and 78 kDa, by gel filtration and SDS-PAGE, respectively. The optimum pH and optimum temperature for enzyme activity were 4.4 and 50 °C, respectively. The enzyme showed a Km value of 2.6 mM and Vmax of 3.86 U/mg for p-nitrophenyl-β-D-galactopyranoside as substrate and was inhibited by Zn2+ and Hg2+. The enzyme activity was regulated by feed back inhibition as it was found to be inhibited by β-D-galactose. Chemical modification studies revealed involvement of tryptophan and histidine for enzyme activity. Involvement of tryptophan was also supported by fluorescence studies and one tryptophan was found to be present in the active site of β-galactosidase. Circular dichroism studies revealed 37% α helix, 27% β sheet and 38% random coil in the secondary structure of the purified enzyme. 相似文献
13.
Rohan L. Wimalasena George S. Wilson 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1991,572(1-2)
Factors affecting the specific activity of immobilized antibodies and their biologically active fragments were studied with goat anti-mouse and goat anti-human immunoglobulin G. Antibodies were immobilized on HW 65 polymeric support matrix activated with carbonyldiimidazole, hydrazide and iodoacetic acid. The most significant factors influencing the specific activity of stochastic coupling of antibodies are multisite attachment, multiple orientations and steric hindrance imposed by crowding of antibody and the size of the antigen. In oriented immobilization the specific activity is affected only by steric hindrance. The specific activity of immunosorbents prepared by immobilization of F(ab′) fragments can be improved to almost 100% by limiting the amount of protein immobilization and the size of the antigen. The present study shows the protocols for optimizing immobilized antibody performance. 相似文献
14.
Sera from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and mixed connective tissue disease (MCTD) possessed higher titer of antibody to human β2-microglobulin (β2m) than those from healthy controls and patients with Behçet's disease in the enzyme-linked immunosorbent assay. It was also confirmed by the immunoprecipitation method. Anti-β2m antibody in sera from those patients immunoprecipitated free β2m but not β2m in association with major histocompatibility complex class I antigen heavy chain. It was suggested that anti-β2m antibody in sera from either patients or healthy controls might be directed mainly against free β2m. The relationship between the anti-β2m antibody and anti-lymphocytotoxic antibody found in those patients is discussed. 相似文献
15.
Shakeel Ahmed Ansari Rukhsana Satar Fahad Alam Mohammed Husein Alqahtani Adeel Gulzar Chaudhary Muhammad Imran Naseer Sajjad Karim Ishfaq Ahmed Sheikh 《Process Biochemistry》2012,47(12):2427-2433
The present study demonstrates synthesis, characterization and surface functionalization of silver nanoparticles (AgNPs) via glutaraldehyde for high yield immobilization of Aspergillus oryzae β-galactosidase. Soluble β-galactosidase (SβG), enzyme adsorbed on unmodified AgNPs (UβG) and surface modified AgNPs (MβG) showed same pH-optima at pH 4.5. However, it was observed that MβG exhibited enhanced pH stability toward acidic and alkaline sides, and increased temperature resistance as compared to SβG and UβG. Michaelis constant, Km was increased nearly three-folds for MβG while Vmax for soluble and MβG was 0.515 mM/min and 0.495 mM/min, respectively. Furthermore, MβG showed greater resistance to product inhibition mediated by galactose as compared to it soluble counterpart and exhibited excellent catalytic activity even after its fourth successive reuse. The remarkable bioconversion rates of lactose from milk in batch reactors further revealed an attractive catalytic efficiency of β-galactosidase adsorbed on surface functionalized AgNPs thereby promoting its use in the production of lactose free dairy products. 相似文献
16.
Abdelnasser S. S. Ibrahim Ali A. Al-Salamah Ahmed Mohamed El-Toni Mohamed A. El-Tayeb Yahya B. Elbadawi Garabed Antranikian 《Biotechnology and Bioprocess Engineering》2013,18(6):1238-1249
Recently isolated Cr(VI)-reducing Amphibacillus KSUCr3 whole cells were immobilised in magnetic gels. Magnetic magnetite (Fe3O4) nanoparticles were synthesised with an average particle size of 47 nm and 80 electromagnetic unit (emu)/g saturation magnetisation. Whole cells were immobilised by entrapment in agar, agarose, alginate, or gelatin in the presence or absence of Fe3O4 nanoparticles for the preparation of both magnetic and nonmagnetic immobilised cells. Of the gels tested, alginate was selected as the best immobilisation matrix, and following optimisation of the entrapment process, the immobilisation yield reached 92.5%. In addition to the ease of separation and reuse of the magnetic cell-containing alginate beads using an external magnet, the magnetically immobilised cells showed approximately 16% higher Cr(VI) reduction activity compared with nonmagnetic immobilised cells. To improve their physical and mechanical properties, the magnetic alginate beads were successfully coated with a dense silica layer using sol-gel chemistry and Ca(OH)2, an alkaline catalyst for tetraethyl orthosilicate, to avoid leaching of Ca2+ ions. Amphibacillus KSUCr3 cells immobilised in silica-coated magnetic alginate beads showed approximately 1.4- to 3.9-fold enhancement of thermal stability compared with free cells. Furthermore, after seven batch cycles, the Cr(VI) reduction activity of free cells decreased to 48%, whereas immobilised cells still retained 81.1% of their original activity. In addition, the Cr(VI)-reduction rate of immobilised cells was higher relative to free cells, especially at higher Cr(VI) concentrations. These results supported the development of a novel, efficient biocatalysts for Cr(VI) detoxification using a combination of whole cell immobilisation, sol-gel chemistry, and nanotechnology. 相似文献
17.
In wastewater treatment microbial cultures immobilised on various matrices are used to protect the microbes from confronting shock loads of organic pollutants. Because of the beneficial effect of activated carbon, it is generally used as a carrier material in comparison to other matrices. In this study mutant strain of P. pictorum (MU 174) was immobilised on ricebran based activated carbon. The effect of contact time, pH, particle size, mass of activated carbon, temperature and ionic strength on adsorption of MU 174 on activated carbon were investigated. The adsorption kinetic parameters like K
p
, K
ad
and H were also determined.Authors are grateful to Dr. K.V. Raghavan, Director, CLRI for his keen interest in publishing this work. Financial assistance by CSIR/UGC is gratefully acknowledged by Miss S. Chitra. 相似文献
18.
β-Glucosidase, β-galactosidase, and cellulase were present in the alimentary canal of Hodotermes mossambicus workers, larvae, and soldiers. Chitinase was absent. The pH and temperature optima, Michaelis constants, and energies of activation of β-glucosidase and β-galactosidase were determined. The enzymes showed different characteristics in all three castes. Enzyme content was found to differ from one developmental stage to the next. The termites lost their intestinal symbionts when ecdysing and re-inoculation took place after ecdysis. 相似文献
19.
Association of HL-A antigens andβ
2-microglobulin at the cellular and molecular level 总被引:1,自引:0,他引:1
Ralph A. Reisfeld Ernest D. Sevier Michele A. Pellegrino Soldano Ferrone Miroslav D. Poulik 《Immunogenetics》1975,2(1):183-197
Surfaces of cultured human lymphoid cells RPMI 1788, RPMI 4098, RPMI 8866, Raji, and WI-L2 were found to contain bothβ 2-microglobulin (β 2-μ) and HL-A determinants when tested by direct complement-dependent cytotoxicity andquantitative absorption with different cytotoxic antiβ 2-μ antisera and specific HL-A alloantisera. The same antigenic specificities were found in 3M KCl extracts of these cultured cells with a sensitiveβ 2-μ radioimmunoassay and an HL-A antigen blocking assay. Daudi cells provided a contrast, since noβ 2-μ or HL-A determinants were found on their surfaces or in 3 M KCl extracts prepared from them. Results from specific antibody blocking tests suggest a close association betweenβ 2-μ and HL-A determinants on plasma membranes of cultured human lymphoid cells. A solid state immunoadsorbent containing antiβ 2-μ antibodies effectively removed all detectable HL-A antigenic activity from some 3M KCl extracts of cultured human lymphoid cells as well as from some sera. Adsorption of HL-A antigens to these immunoadsorbents was specific since it was blocked only by prior addition ofβ 2-μ. Once on the antiβ 2-μ immunoadsorbents, HL-A antigens still reacted specifically with HL-A alloantibodies in quantitative absorption experiments. HL-A antigens andβ 2-μ could be eluted from antiβ 2-μ immunoadsorbents with a variety of chaotropic reagents and detergents, but thus far potassium bromide and sodium dodecyl sulfate (SDS) appear to be the most effective. SDS-PAGE of these eluates indicated that HL-A antigens were considerably purified by adsorption to antiβ 2-μ immunoadsorbents and that two major molecular size fragments were distinguishable, i.e., ∼33,000 for HL-A and ∼ 12,000 forβ 2-μ. 相似文献
20.
The method of Gaitonde and Dovey [Biochem. J.117, 907 (1970)] for the determination of tryptophan by reaction with ninhydrin in acid is adapted for the measurement of protein bound to solid support materials, including collagen. DEAE-Sephadex, DEAE-cellulose, polyacrylamide and collodion give negligible background absorbance with the reagent; collagen and activated agarose give some color, but this can be abolished by pretreating the collagen with H2O2. Collagen, Sephadex and agarose dissolve in the reagent. Levels of lactase (β-galactosidase) and glucoamylase were readily and linearly measured down to 0.2 mg in the presence of 21 mg collagen, and activity and immobilized protein content of lactase-collagen complexes were linearly related. 相似文献