共查询到20条相似文献,搜索用时 375 毫秒
1.
I S Thakur 《Biochemistry international》1986,13(6):951-960
Prosopis juliflora pollen grain crude extract gave six different molecular weight fractions varied from 81,000 to 13,000 dalton on Sephadex G-100 gel filtration. The purity of fractions of Prosopis juliflora pollen extract were checked by polyacrylamide gel electrophoresis. The fraction had an molecular weight 20,000 dalton showed four absorption maxima whereas other fractions had single absorption maxima. Allergenic activity and nature of allergens were evaluated by in vitro Radioallergosorbent test and in vivo Passive Cutaneous Anaphylaxis test. All these tests indicated that most allergenic fractions were in the 20,000 molecular weight. 相似文献
2.
A procedure is described for the use of immunoadsorption chromatography of hydroxyindole O-methyltransferase (HIOMT). HIOMT was purified from bovine pineal extract by affinity chromatography on immunoglobulins (Ig)-Sepharose. The overall purification was about 45-fold; the yield was 84%. This enzyme constitutes about 2.0% of the soluble proteins in the pineal gland. The enzyme represented a single precipitin line on Ouchterlony double diffusion plate and immunoelectrophoresis. Ultracentrifugation analysis indicated the existence of molecular aggregates of enzyme and disc gel electrophoresis showed one main protein band and several minor bands. However sodium dodecyl sulphate (SDS) gel electrophoresis showed a single protein band with subunit molecular weight 38,000 demonstrating bovine pineal HIOMT to be polymer enzyme of a single subunit. The properties of the purified enzyme including disc gel electrophoretic pattern, the effect of pH, chemicals and substrates and immunological properties were identical with those of the crude enzyme. 相似文献
3.
Tetanus Toxin and Antigenic Derivatives I. Purification of the Biologically Active Monomer 总被引:3,自引:1,他引:2
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A procedure for the isolation of pure tetanus toxin in a lethal monomeric form was developed based on the extraction of whole cells and chromatographic techniques. A crude extract of toxin was obtained by hypertonic extraction of cells from a 72-hr culture of Clostridium tetani Massachusetts strain. The extract was precipitated with ammonium sulfate and further purified by sequential use of ion-exchange chromatography and gel filtration. The degree of purification obtained by the fractionation procedures was monitored by polyacrylamide gel electrophoresis. The pure toxin has an average specific activity of 150 x 10(6) mouse MLD per mg of N and 3,000 Lf per mg of N. Immunological purity was demonstrated by a single line on both immunoelectrophoresis and agar double diffusion. One band was obtained on polyacrylamide electrophoresis, as was a single symmetrical peak in the ultracentrifuge and on Sephadex G-100 chromatography. The pure protein has an absorbancy ratio (280/260 mmu) of 2.1 in phosphate buffer (pH 7.5). 相似文献
4.
I S Thakur 《Biochemistry international》1989,18(3):605-613
An allergenically active glycoprotein was homogeneously isolated from the aqueous extract of Prosopis juliflora pollen by ConA-Sepharose affinity chromatography. The molecular weight of this glycoprotein was 20,000 dalton, determined by gel filtration and SDS-PAGE. This fraction showed a total carbohydrate concentration of 25%. The purified glycoprotein revealed immunochemically most antigenic or allergenic and demonstrated homogeneous after reaction with P. juliflora pollen antiserum, characterized by gel diffusion, Immunoelectrophoresis and Radioallergosorbent test. 相似文献
5.
Multiple forms of human renin. Purification and characterization. 总被引:10,自引:0,他引:10
F X Galen C Devaux T Guyenne J Menard P Corvol 《The Journal of biological chemistry》1979,254(11):4848-4855
Human renin was purified from a juxtaglomerular cell tumor with a high renin content, 24.2 Goldblatt units/mg of protein. The purification procedure comprised three steps: gel filtration, DEAE-cellulose chromatography, and preparative isoelectric focusing. Five forms of renin amounting to 5.3 mg of enzyme were obtained with isoelectric points of 4.95, 5.10, 5.35, 5.55, and 5.70. They were all glycoproteins. The three major fractions had very similar specific activities, 868, 860, and 809 Goldblatt units/mg of protein. These fractions produced a single band on analytical isoelectric focusing and a single arc on immunoelectrophoresis. On polyacrylamide gel electrophoresis at pH 7.8, each fraction consisted of two renin bands with the same molecular weight, but different net charges. The molecular weight determined by gel filtration and Fergusson plot analysis on polyacrylamide gel was 38,000 to 42,000. The optimum pH determined on N-acetyltetradecapeptide substrate was 6.5, and the Km was 6.8 x 10(-6) M. These parameters were identical with those for standard human kidney renin. Antibodies raised against tumor renin completely inhibited the activity of both tumor and standard renin. Under dissociating conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel electrophoresis in the presence of 6 M urea), part of the purified enzyme dissociated into two smaller fragments (Mr = 20,000 and 25,000) containing renin activity. 相似文献
6.
D Sompolinsky J B Hertz N H?iby K Jensen B Mansa Z Samra 《Acta pathologica et microbiologica Scandinavica. Section B, Microbiology》1980,88(3):143-149
In crude water-soluble extracts of Pseudomonas aeruginosa 64 antigens can be demonstrated by crossed immunoelectrophoresis in agarose with polyvalent Pseudomonas-immunoglobulin. One of these antigens cross-reacts with antigens prepared from bacteria of a wide range of taxonomic groups. Monospecific immunoglobulins to this antigen (Common Antigen) were produced by immunization with the appropriate immunocomplex extracted from agarose. Common Antigen was purified by the combination of two fractionation methods: Precipitation of the crude extract with 18% (w/v) sodium sulfate, followed by gel filtration of the supernatant on a Sephadex G-200 column. By this method, 35% of Common Antigen from the crude extract was recovered, more than half of the fractions electrophoretically pure. Electrophoresis of reduced Common Antigen on a dodecyl sodium sulfate polyacrylamide gel revealed two protein bands with apparent molecular weights of 59-62 000 and 62-65 000, respectively. The untreated antigen, however, passed a column of Sephadex G-200 with the void volumen, indicating a substance of high molecular weight (> 4-600 000). 相似文献
7.
Partial purification and characterization of Schistosoma mansoni soluble egg antigen with Con A-Sepharose chromatography 总被引:9,自引:0,他引:9
A crude preparation of Schistosoma mansoni soluble egg antigen (SEA) was subjected to affinity chromatography with concanavalin A (Con A) bound to Sepharose 4B. The resulting Con A fractions (bound and unbound) were characterized with sodium dodecyl sulfate (SDS) gel electrophoresis, immunoelectrophoresis, immunodiffusion, and lymphocyte blastogenesis techniques. In the fraction that did not bind to Con A there were at least two distinct antigens, and there were also at least two distinct antigens in the fractions that did bind to Con A. With SDS polyacrylamide gel electrophoresis, at least 20 distinct protein bands (Coomassie blue staining) and three glycoprotein bands (PAS reactive) were present in the unbound fractions from Con A chromatography. The bound fractions separated into at least six distinct glycoproteins with SDS electrophoresis. Although both the bound and unbound fractions contained precipitating antigens, only the bound fractions were capable of eliciting lymphocyte blastogenic responses. 相似文献
8.
Molecular weight of major component proteins in crude saline extract of adult Paragonimus westermani
When the component proteins in crude saline extract of 13-week old adult Paragonimus westermani were observed by non-denaturing discontinuous-polyacrylamide gel electrophoresis (Disc-PAGE), 8 distinct bands were clearly recognized. Molecular weight (MW) of each band protein, numbered in sequence from cathodal side which appeared in 10% separating gel, was measured first by Ferguson plot utilizing different gel concentrations from 10% to 4.5%. MW of band 1 protein (known as egg protein) was 440 kDa. And MW of other band proteins were: 386 kDa in band 2, 17.4 kDa in band 3, 17 kDa in band 4, 14.3 kDa in band 5, 46 kDa in band 6, 38 kDa in band 7 and 23 kDa in band 8. When the proteins in the crude extract were separated into fractions by molecular sieve chromatography through 1.6(phi) X 70 cm sized Sephacryl S-300 Superfine column and revisualized by Disc-PAGE in 8% gel, the sequence of eluted proteins was band 1, band 2, band 6, band 7 and bands 3, 4, 5 and 8. This elution profile confirmed MW of each band protein in the crude extract as measured by Ferguson plot. 相似文献
9.
I S Thakur 《Biochemistry international》1991,23(3):449-459
Highly active glycoprotein allergens have been isolated from pollen of Prosopis juliflora by a combination of Sephadex G-100 gel filtration and Sodium dodecyl sulphate-Poly-acrylamide gel electrophoresis. The glycoprotein fraction was homogeneous, and had molecular weight 20,000. The purified glycoprotein allergen contained 20% carbohydrate, mainly arabinose and galactose. Enzymatic digestion of glycoprotein with protease released glycopeptides of molecular weight ranging from less than 1,000 to more than 5,000 on Sephadex G-25 gel filtration. Antigenicity or allergenicity testing of these glycopeptides by immunodiffusion, immunoelectrophoresis, and radioallergosorbent test indicated complete loss of allergenic activity after digestion with protease whereas incubation with beta-D-galactosidase and periodate oxidation had little affect on the allergenic activity of the glycoprotein fraction. But incubation with alpha-D-glucosidase did not affect the allergenic activity significantly. All these tests indicated that protein played significant role in allergenicity of P. juliflora pollen. 相似文献
10.
《Biochimica et Biophysica Acta.Protein Structure》1976,420(1):112-121
A lectin with N blood group specificity was isolated fromVicia graminea seeds. This lectin was purified from a crude extract by precipitation with ammonium sulfate, DEAE-cellulose chromatography and Sephadex G-150 gel filtration. Purification steps were followed by increase of specific activity. Its homogeneity was demonstrated by polyacrylamide gel electrophoresis, immunoelectrophoresis, electrofocusing and ultracentrifugation. This lectin is an acid glycoprotein with 7.3% carbohydrate, a high percentage of serine and contains no sialic acid. The native lectin has a molecular weight about 100 000 and dissociates into four subunits of 25 000 as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Preliminary hemagglutination inhibition has shown that the lectin was not inhibited by any of the monosaccharides contained in N blood group substances; however it was inhibited by the erythrocyte membrane major glycoprotein and the tryptic fragments obtained from erythrocytes. 相似文献
11.
A lectin with N blood group specificity was isolated from Vicia graminea seeds. This lectin was purified from a crude extract by precipitation with ammonium sulfate, DEAE-cellulose chromatography and Sephadex G-150 gel filtration. Purification steps were followed by increase of specific activity. Its homogeneity was demonstrated by polyacrylamide gel electrophoresis, immunoelectrophoresis, electrofocusing and ultracentrifugation. This lectin is an acid glycoprotein with 7.3% carbohydrate, a high percentage of serine and contains no sialic acid. The native lectin has a molecular weight about 100 000 and dissociates into four subunits of 25 000 as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Preliminary hemagglutination inhibition has shown that the lectin was not inhibited by any of the monosaccharides contained in N blood group substances; however it was inhibited by the erythrocyte membrane major glycoprotein and the tryptic fragments obtained from erythrocytes. 相似文献
12.
《Biochimica et Biophysica Acta.Protein Structure》1981,667(1):1-14
The characterization of a human fibroblast surface glycoprotein, visualized by crossed immunoelectrophoresis using rabbit antibodies against whole fibroblasts, is described. The antigen is synthesized by fibroblasts in culture and was localized both intracellularly and at the cell surface. It was highly antigenic and was detected only in human cells of mesenchymal origin.The glycoprotein occurred in two different forms with α2 and β electrophoretic mobility. The slow migrating amphiphilic β form was localized at the cell surface and showed a single protein band with an apparent molecular weight of 150 000 in SDS-polyacrylamide gel electrophoresis. By external papain treatment of intact viable cells, a water-soluble molecule was released with a reduced molecular weight (140 000) and an increased electrophoretic mobility as compared to the native membrane component. This hydrophilic form was also present intracellularly in fibroblasts not treated with exogeneous proteases. The observation that the detergent-solubilized β form was irreversibly converted to a more anodic form by incubation of whole cell extract at acidic pH, suggested that the intracellular protein represented a lysosomal degradation product of native internalized fibroblast surface glycoprotein. 相似文献
13.
Soluble extracts of Plasmodium berghei were separated into 12 fractions following preparative disc electrophoresis in polyacrylamide gel. One or two protein bands were detected in each fraction by analytical disc electrophoresis. Similarly, one or two precipitinogens were generally detected in each of Fractions 1 through 11 by immunoelectrophoresis and by double immunodiffusion in agar gel, while the unfractionated extract contained 10 precipitinogens. Antisera produced in rabbits against each fraction each contained two or three (sometimes five) antiplasmodial precipitins demonstrable by immunoelectrophoresis. Serial fractions obtained in separate runs were closely similar to each other, although some degree of overlapping sometimes occurred between neighboring fractions. Glycoproteins were detected in all the fractions, but chiefly in Fractions 4 and 12. The bulk of the RNA in the extract was located in Fraction 4, while hemoglobin was usually confined to Fraction 6. The molecular weights of the soluble components of P. berghei range between 8000 and 130,000. 相似文献
14.
Noemi Santiago de Weil George V. Hillyer Elaine Pacheco 《International journal for parasitology》1984,14(2):197-206
Santiago de Weil N., Hillyer G. V. and Pacheco E. 1984. Isolation of Fasciola hepatica genus-specific antigens. International Journal for Parasitology14: 197–206. The Fasciola hepatica antigens which induce antibody formation in acute fascioliasis were isolated by acid elution after reacting an F. hepatica tegument antigen extract with a CNBr-Sepharose 4B column coupled with IgG obtained from the serum of rabbits infected with fascioliasis for 6–10 weeks. These isolated antigens were further separated by gel filtration using a column packed with Sephacryl S-200. In this manner three major peaks were obtained. The best serologic antigens were found in peak 2 which had a mol. wt range of 14,000–43,000. This peak contains genus-specific F. hepatica antigens which are highly reactive with fascioliasis serum. These antigens do not cross-react with either Schistosoma mansoni or with bovine serum albumin by gel diffusion. Monitoring by ELISA and gel diffusion with heterologous and homologous antisera showed that as purification by antibody affinity chromatography proceeded, cross reactivity with S. mansoni was eliminated. The rabbit antiserum obtained against peak 2, when tested by immunoelectrophoresis with a crude F. hepatica extract shows one main band identical to the main band observed with serum from acutely infected rabbits. Up to two other minor bands can be detected using concentrated homologous antisera. Fractions obtained from preparative iso-electric focusing of the F. hepatica tegument extract were reacted with sera from rabbits with acute fascioliasis. Two main bands were observed in immunodiffusion with antigens eluting in a pH range of 7.4–8.7. When these fractions were monitored with anti peak 2 antisera, two precipitin bands appeared with antigens eluting in a pH range of 7.4–7.9. The F. hepatica genus-specific antigen pool was applied to ELISA to evaluate its ability to detect antibody in a primary F. hepatica infection in rabbits. A rise in absorbance values could be detected by 2 weeks of infection, reached high levels by 6 weeks and remained high through 28 weeks of infection. 相似文献
15.
Specific rabbit antisera against purified Hordeum vulgare seedling RNase I from two winter barley cultivars each formed a single precipitin band when reacted with the homologous crude tissue extract. RNase antigen from either cultivar was equally reactive with both antisera when evaluated by immunodiffusion and immunoelectrophoresis. A small but consistent difference in anti-RNase specificity between cultivars was shown by passive hemagglutination inhibition, suggesting that molecular differences may exist between the two RNase antigens. Immunodiffusion and rocket immunoelectrophoresis were used to qualitatively test the cross-reactivity of protein preparations from various members of the genus Hordeum and species from other related grass genera. Neither antiserum showed cross-reactivity with soluble protein preparation from species outside the genus Hordeum. A few species within the genus Hordeum were cross-reactive. A modification of rocket immunoelectrophoresis was developed to determine the amount of RNase in unpurified tissue extracts. The technique involved a template-reservoir which allowed detection of 250 ng RNase in tissue extract volumes of 50 μl. The amount of RNase in unpurified protein extracts from the two cultivars of barley was similar. 相似文献
16.
Peter F. Lens Wies van den Briel Alan Musgrave Herman van den Ende 《Archives of microbiology》1980,126(1):77-81
To identify mating type-specific glycoproteins associated with the flagellar membrane of Chlamydomonas eugametos, which could be involved in sexual agglutination, antibodies were raised in rabbits against purified gamete flagella of either mating type. The immunoglobulin (Ig) fractions exhibited partial mating-type specificity in agglutinating gametes, in the indirect immunofluorescence test and in the crossed immunoelectrophoresis test. This specificity was strongly enhanced by absorbing the fractions with flagella of the opposite mating type. Absorbed Ig fractions produced a single precipitation line with Triton extracts of gamete flagella in the crossed immunoelectrophoresis technique. On polyacrylamide gel electrophoresis this line appeared to contain two flagellar glycoprotein fractions, PAS 1 and PAS 4. Polyacrylamide gels of flagellar extracts incubated with these Ig fractions, followed by staining with peroxidase-anti-rabbit Ig resulted in the staining of only the PAS 1 and PAS 4 bands, which confirms that these components of the flagellar membrane are mating type-specific antigens.The investigations were supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO). 相似文献
17.
Mucus was extracted from human gastric mucosa by homogenization in distilled water. The crude extract was purified from plasma, salivary and tissue contaminants by different steps involving chromatography on Sepharose 6B, Sepharose 2B and immunosorbents. The native glycoprotein so prepared was found to be immunologically pure; it migrated as a single band in acrylamide agarose gel electrophoresis at pH 8.6 and 3.5. The molecule exhibits blood group acitivity, contains 0.71 per cent sialic acid but no sulphate. The carbohydrate moiety contains glucosamine, galactosamine, fucose, galatose and mannose in the molar proportions 3: 1.95: 2.92:4.53:0.05. 相似文献
18.
Complexes of chromogranin A and dopamine beta-hydroxylase among the chromogranins of the bovine adrenal medulla 总被引:1,自引:0,他引:1
1. The core proteins of chromaffin granules have been examined by polyacrylamide gel electrophoresis and crossed immunoelectrophoresis against monospecific antisera. 2. Dopamine beta-hydroxylase (dopamine beta-monooxygenase, EC 1.14.17.1) appeared as the major immunogen of the core proteins and accounted for 4 and 8% by weight of the crude lysate and membrane-containing fractions, respectively. 3. The non-ionic detergent, Berol, solubilized dopamine beta-hydroxylase from the membranes in a form which was immunologically identical but of lower relative mobility by crossed immunoelectrophoresis. In the absence of detergent a difference in relative mobility was also noted between the purified enzyme and that contaminated by chromogranin A. These observations suggest that several molecular forms of dopamine beta-hydroxylase may occur which differ in size and/or charge due to interactions with the contaminants under the experimental conditions. 4. The main chromogranin in the crude lysate was absent from electropherograms of the acidic chromogranins (95--96% of total protein in lysate). These were obtained free of dopamine beta-hydroxylase by concanavalin A adsorption at high ionic strength or by acidification in 2 M acetic acid. The main band reappeared upon recombination with dopamine beta-hydroxylase, indicating the presence of some dopamine beta-hydroxylase, possibly as dimers, in this main, chromogranin A band. A protein concentration-dependent aggregate of dopamine beta-hydroxylase-free chromogranin A was detected, with a relative mobility slightly faster than the main band of the crude lysate. 相似文献
19.
Immunochemically identical hydrophilic and amphiphilic forms of the bovine adrenomedullary dopamine beta-hydroxylase. 总被引:2,自引:0,他引:2
By means of a monospecific antibody, dopamine beta-hydroxylase was monitored immunoelectrophoretically in various extracts of chromaffin granules. Approximately one-third of the dopamine beta-hydroxylase present was located in the membrane fraction and could only be liberated with detergent. The dopamine beta-hydroxylases of the buffer and membrane fractions were antigenically identical, but differed in their amphiphilicity, as demonstrated by the change in precipitation patterns on removal of Triton X-100 from the gel, on charge-shift crossed immunoelectrophoresis and on crossed hydrophobic interaction immunoelectrophoresis with phenyl-Sepharose. Furthermore, immunoelectrophoretic analysis in the presence of Triton X-100 plus the cationic detergent cetyltrimethylammonium bromide indicates additional heterogeneity of the membrane-bound dopamine-beta-hydroxylase. By limited proteolysis with chymotrypsin and thermolysin the amphiphilic form could be convered into its hydrophilic counterpart. 相似文献
20.
The major protein of the pig pancreatic zymogen granule membrane is an integral glycoprotein of 92 X 10(3) daltons (Da) which amounts to 25% of the total proteins of this membrane. When zymogen granule membranes were prepared in presence of 5 mM dithiothreitol (DTT), this glycoprotein specifically vanished from the membrane preparation. During membrane purification two other fractions were produced out of the purified granules: a soluble fraction of zymogens referred to as granule content and a dense pellet. The possibility that DTT could release the 92-kDa protein from the membrane to these other fractions has been rejected. Altogether, addition of DTT during the lysis of the granules induced a total degradation of the 92-kDa protein. This hydrolysis could be inhibited by phenylmethylsulfonyl fluoride but not by N-alpha-p-tosyl-L-lysine chloromethyl ketone or L-1-tosylamide-2-phenylethylchloromethyl ketone. In the course of these experiments, using gel filtration of the granule content, it was found that the 92-kDa protein was also present in the granule content in the form of an aggregate of 300 kDa. A protease was present in this aggregate and could hydrolyse the 92-kDa protein upon addition of DTT. From immunoblotting studies and rocket immunoelectrophoresis, it was found that the soluble 92-kDa protein was antigenically similar to the membrane protein and that 44% of the immunoreactive glycoprotein of the granule was soluble in the content. A cross-reacting fragment of 65 kDa has been observed in all the fractions, yet at different levels.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献