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1.
I A Shaginian I N Ga?lonskaia V M Bondarenko 《Zhurnal mikrobiologii, epidemiologii, i immunobiologii》1980,(6):71-76
Cell-free culture filtrate boiled for 15 minutes has been found to retain its biological activity in various experimental models used for the determination of the toxicogenicity of cholera vibrio filtrates. During gel filtration of the concentrated filrate o. NAG vibrio, strain NO. 9852, through Sephadex G-75 toxic activity could be detected in the free volume of the column, which was indicative of the fact that the molecular weight of the thermostable enterotoxin was about 70,000 daltons and greater. The methods of gel diffusion and aggregated hemagglutination have been used to show that the thermostable enterotoxin of NAG vibrio No. 9852 is immunologically unrelated to cholerogen. Some data obtained in experimental models suggest that the thermostable enterotoxin probably differs from cholera enterotoxin in the mechanism of its action. 相似文献
2.
Novel method for purification of staphylococcal enterotoxin A 总被引:1,自引:0,他引:1
D Reynolds H S Tranter R Sage P Hambleton 《Applied and environmental microbiology》1988,54(7):1761-1765
A novel single-step procedure for the purification of staphylococcal enterotoxin A (SEA), namely, dye ligand affinity chromatography with the triazine dye Red A, was developed. SEA purified by this method produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The yield from 5 liters of culture supernatant was 0.113 g, corresponding to an overall yield of 55%. In some instances, purification of SEA from culture supernatants by dye ligand affinity chromatography produced two enterotoxin peaks that could be eluted from the column with 300 and 500 mM phosphate buffer (pH 6.8). Enterotoxin from these peaks produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but multiple bands were observed on isoelectric focusing gels. This method of purification represents a significant improvement in time, yields, and purity of enterotoxin over previously published purification methods. 相似文献
3.
A novel single-step procedure for the purification of staphylococcal enterotoxin A (SEA), namely, dye ligand affinity chromatography with the triazine dye Red A, was developed. SEA purified by this method produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The yield from 5 liters of culture supernatant was 0.113 g, corresponding to an overall yield of 55%. In some instances, purification of SEA from culture supernatants by dye ligand affinity chromatography produced two enterotoxin peaks that could be eluted from the column with 300 and 500 mM phosphate buffer (pH 6.8). Enterotoxin from these peaks produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but multiple bands were observed on isoelectric focusing gels. This method of purification represents a significant improvement in time, yields, and purity of enterotoxin over previously published purification methods. 相似文献
4.
Eight murine monoclonal antibodies (MAbs) against staphylococcal enterotoxin D (SED) were obtained by fusion of myeloma cells with mouse spleen cells immunized with SED only or a combination of SED and either enterotoxin A (SEA) or enterotoxin E (SEE). When only SED was used as an immunogen, six MAbs were specific for SED only, whereas one MAb was reactive with both SED and SEE when both SEs were used as immunogens. One MAb reacted with SEA, SED, and SEE when both SEA and SED were used as immunogens. A MAb with the highest reactivity to SED was used to prepare an immunosorbent for purification of SED by immunoaffinity chromatography. Approximately 70% of the partially purified SED was recovered in the eluate. The purified SED was electrophoretically and antigenically pure. Immunoaffinity chromatography proved useful in the purification of SED in terms of ease of purification, percent enterotoxin, and enterotoxin purity. 相似文献
5.
Chromatofocusing: a new method for purification of staphylococcal enterotoxins B and C1. 总被引:9,自引:2,他引:7
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A new chromatographic procedure was developed which obtained highly purified preparations of staphylococcal enterotoxins B and C1 in yields of 60% from cultures of Staphylococcus aureus and which is faster than any of the separation methods used previously. The procedure involves chromatography on carboxymethylcellulose, removal of alpha-toxin by adsorption to rabbit erythrocyte membranes, and finally, chromatofocusing as the fundamental new step. Enterotoxins were obtained in highly purified form and behaved in a homogeneous manner as determined by ultracentrifugation and electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate, with molecular weights of 34,000 for staphylococcal enterotoxin B and 30,000 for staphylococcal enterotoxin C1. Using chromatofocusing as the final purification step, we isolated three B and six C1 distinct but immunologically identical enterotoxin fractions, which were found to be devoid of any impurities and to possess a marked degree of toxicity in monkeys. 相似文献
6.
I A Ende G Terplan B Kickh?fen D K Hammer 《Applied and environmental microbiology》1983,46(6):1323-1330
A new chromatographic procedure was developed which obtained highly purified preparations of staphylococcal enterotoxins B and C1 in yields of 60% from cultures of Staphylococcus aureus and which is faster than any of the separation methods used previously. The procedure involves chromatography on carboxymethylcellulose, removal of alpha-toxin by adsorption to rabbit erythrocyte membranes, and finally, chromatofocusing as the fundamental new step. Enterotoxins were obtained in highly purified form and behaved in a homogeneous manner as determined by ultracentrifugation and electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate, with molecular weights of 34,000 for staphylococcal enterotoxin B and 30,000 for staphylococcal enterotoxin C1. Using chromatofocusing as the final purification step, we isolated three B and six C1 distinct but immunologically identical enterotoxin fractions, which were found to be devoid of any impurities and to possess a marked degree of toxicity in monkeys. 相似文献
7.
Abstract A chromatographic method, wheat-germ agglutinin affinity chromatography, was developed to isolate Escherichia coli heat-labile enterotoxin from human source. Isolated LT enterotoxin showed potent activity in the rabbit jejunal loop assay, and immunological and structural analogies with cholera enterotoxin in the radial immuno-hemolysis test and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. 相似文献
8.
S. typhimurium enterotoxin, partially purified in accordance with our scheme (salting out with 75% ammonium sulfate, dialysis and gel filtration in a column with Sephadex G-150, followed by electrofocusing), showed enterotoxic activity in the intestinal loop of a rabbit and yielded the positive result in the cutaneous test. S. typhimurium enterotoxin proved to be protein with a molecular weight of 140000 daltons and the isoelectric point equal to 4.4. The biological activity of S. typhimurium enterotoxin was neutralized with homologous antiserum and with antiserum to cholera enterotoxin. Heating the preparation at 75 degrees C for 30 minutes led to a considerable decrease in its enterotoxic activity. 相似文献
9.
Improved method for purification of enterotoxin from Clostridium perfringens type A. 总被引:11,自引:2,他引:9
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The purification procedure of Clostridium perfringens type A enterotoxin has been improved. The cell sonic extract was precipitated twice with ammonium sulfate, first 40% saturated to concentrate the enterotoxin and then 15% saturated. The two precipitations were followed by gel filtration on Sephadex G-100. The enterotoxin appeared to be homogeneous on 7% polyacrylamide gel electrophoresis after this three-step purification procedure, with a recovery of 56% and a 12.3-fold purification. The solubility properties at different pH values, temperatures, and ammonium sulfate concentrations are also given as basis for the purification procedure. 相似文献
10.
The purification procedure of Clostridium perfringens type A enterotoxin has been improved. The cell sonic extract was precipitated twice with ammonium sulfate, first 40% saturated to concentrate the enterotoxin and then 15% saturated. The two precipitations were followed by gel filtration on Sephadex G-100. The enterotoxin appeared to be homogeneous on 7% polyacrylamide gel electrophoresis after this three-step purification procedure, with a recovery of 56% and a 12.3-fold purification. The solubility properties at different pH values, temperatures, and ammonium sulfate concentrations are also given as basis for the purification procedure. 相似文献
11.
Purification and partial characterization of a cytotonic enterotoxin produced by Aeromonas hydrophila 总被引:2,自引:0,他引:2
This report describes the purification and partial characterization of a cytotonic enterotoxin produced by a human diarrheal isolate (SSU) of Aeromonas hydrophila. The extracellular enterotoxin was purified by (NH4)2SO4 precipitation, hydrophobic column chromatography, and chromatofocusing. The highly purified enterotoxin exhibited a molecular mass of 44 kDa and an isoelectric point in the range of 4.3 - 5.5 as determined by chromatofocusing. Western blot analysis using Aeromonas anti-enterotoxin revealed a single band at 44 kDa; however, cholera antitoxin failed to detect the enterotoxin antigen. This non-cholera toxin cross-reactive (non-CTC) enterotoxin was biologically active in vivo as determined by rabbit ligated ileal loop and rabbit skin vascular permeability assays. Biological activity also was in vitro by this toxin as measured by the elongation of Chinese hamster ovary (CHO) cells. The enterotoxic activity associated with this molecule was neutralized completely by homologous antibodies but not by cholera antitoxin. The purified toxin preparation was free of hemolytic and cytotoxic activities as determined by its inability to lyse rabbit red blood cells or damage CHO cells, respectively. Furthermore, this toxin induced the elevation of cAMP in CHO cells suggesting thereby that the mechanism of action of Aeromonas non-CTC enterotoxin may be similar to heat-labile enterotoxins of Escherichia coli and Vibrio cholerae. 相似文献
12.
Scanning isoelectric focusing has been employed for continuous monitoring of the isoelectric spectrum of highly purified cholera enterotoxin in 4% polyacrylamide gels containing 2% ampholytes pH 3–10. The resolution obtained by this technique is of high order because at no instance during focusing interruption of current occurs and thus diffusion of the isolated protein moieties is suppressed. An added aspect of scanning isoelectric focusing was that it allowed estimation of the minimal focusing time of cholera enterotoxin. Thus under the standard assay procedure, the main basic component of cholera enterotoxin was focused in 5800 sec, while the other at least 3 minor acidic and anodic components were focused in approximately 19000 sec. Focusing of cholera enterotoxin in the presence of 6m urea allowed the visualization of 5 well defined and about equal components. The proteinaceous nature of the observed peaks was verified by scanning at wavelengths other than 280 nm, staining of gels for protein, and varying the concentration of the enterotoxin. The design of scanning isoelectric focusing equipment is presented. Reproducibility, economy of sample, and ampholytes and simplicity of experimental technique were some of the features of this apparatus. The resolution of scanning isoelectric focusing was found to be superior to that of ordinary disc and SDS gel electrophoresis. 相似文献
13.
A new method for high yield purification of type beta transforming growth factor from human platelets 总被引:2,自引:0,他引:2
A J van den Eijnden-van Raaij I Koornneef E J van Zoelen 《Biochemical and biophysical research communications》1988,157(1):16-23
A new method was developed for the purification of type beta transforming growth factor from human platelets. This method is a three-step procedure including gel filtration, weak cation exchange HPLC and reverse phase HPLC. All steps are carried out at low pH using exclusively volatile acidic buffer solutions. The sterile conditions and easy removal of salt by lyophilization facilitate the quantification of the growth factor in biological assays. Based on immunological characterization the purified acid-stable, highly basic transforming growth factor beta is the beta 1 form. Using the present method pure platelet TGF beta 1 is obtained in very high yield. 40 units of outdated human platelets yield 800 micrograms pure TGF beta 1, which is about a 10-20 fold higher yield than reported for other purification procedures. 相似文献
14.
A medium containing 4% bio-trypcase and 1% yeast extract was used for the production of Staphylococcus aureus enterotoxin B. The yield obtained was estimated at 200 micrograms of enterotoxin per ml of S. aureus S-6 culture supernatant. The purification method involves chromatography on Biorex 70 resin, isoelectric focusing, and gel filtration on Sephadex G-100. The purified enterotoxin (isoionic point, pH 8.55) was shown to be homogenous protein with a molecular weight of 29,000 when tested by gel electrophoresis. 相似文献
15.
Isolation of proteins by gel filtration in 6M guanidinium chloride: application to RNA tumor viruses 总被引:16,自引:0,他引:16
Separation of RNA tumor virus proteins by gel filtration in 6m guanidinium chloride indicates that this method will effectively separate polypeptides in milligram amounts whose molecular weights differ by as little as 15 percent. Such separations are achieved because proteins in 6m guanidinium chloride have a random coil conformation with diffusion coefficients considerably smaller than the corresponding native proteins. Consequently, protein bands that emerge from a gel filtration column in 6m guanidinium chloride are remarkably sharp. A 60–70% yield of renatured RNA tumor virus proteins could be obtained following dialysis against a dilute solution of mercaptoethanol. The major proteins of avian RNA tumor viruses were obtained as pure components by gel filtration in 6m guanidinium chloride. Mammalian RNA tumor viruses appear to have a more complex protein structure, since, in some cases, additional purification was required to obtain the pure proteins. This method of protein separation might be used as the initial stop in the isolation of components from other biological macrostructures. 相似文献
16.
A new method developed for purification of enterotoxin C2 from Staphylococcus aureus strain 361 consisted of four steps: batchwise adsorption from culture supernatant on QAE-Sephadex; gel filtration on Sephadex G-100; chromatography on QAE-Sephadex using a buffer of constant pH and molarity; and gel filtration using a volatile buffer of constant pH and molarity; and gel filtration using a volatile buffer as the eluting solvent. The purified enterotoxin appeared homogeneous by gel immunodiffusion, gel chromatography and in the analytical ultracentrifuge, although an apparent heterogeneity was noted on QAE-Sephadex chromatography and polyacrylamide disc electrophoresis at pH 4.5. The emetic dose, ED50, by intravenous route in cynomolgus monkeys was 0.04 mug/kg of animal weight. Upon treatment with sodium dodecylsulfate, beta-mercaptoethanol and urea, enterotoxin C2 separated into 3 bands in sodium dodecylsulfate-electrophoresis. One band mol. wt 29000, and two bands of lower molecular weight were so close that they moved as a single zone. After elution from gels, the zone of lower molecular weight were so close that they moved as a single zone. After elution from gels, the zone of lower molecular weight oligopeptides emerged as a single peak at the same position as untreated enterotoxin C2 during gel filtration with buffer lacking thiol and denaturant, and gave a reaction of complete identify to enterotoxin C2 in Ouchterlony immunodiffusion. The results suggest that enterotoxin C2 is a mixture composed of intact polypeptide chains, mol. wt 29000, and two fragments cleaved in the disulfide region of molecular weight of approx. 15400 and 12800 linked by the single disulfide bond in the toxin molecule. Amino acid analysis indicates that enterotoxin C2 consists of 255 amino acid residues. 相似文献
17.
18.
Comparison of different purification procedure for extraction of staphylococcal enterotoxin A from foods. 总被引:1,自引:1,他引:0
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Different procedures commonly used for extraction, purification, and concentration of staphylococcal enterotoxins from foods were investigated with 131I- and 125I-labeled staphylococcal enterotoxin A. Loss of labeled enterotoxin A was compared with loss of total nitrogen. The results showed that in most of the common procedures, such as gel filtration, ion exchange, and heat treatment, the percentage of loss of labeled enterotoxin A was greater than the loss of total nitrogen. Chloroform extraction and acid precipitation with hydrochloric acid had nearly the same effect on the purification of both labeled enterotoxin A and total nitrogen. Ammonium sulfate precipitation proved to be practical and was successfully used for purification of enterotoxin A from sausage extract. Simultaneous use of trypsin and Pseudomonas peptidase for treatment of food extracts considerably reduced food proteins capable of interfering with serological detection of enterotoxins but did not essentailly influence the loss of enterotoxin A. 相似文献
19.
K Sato Y Miyachi N Ohsawa K Kosaka 《Biochemical and biophysical research communications》1975,62(3):696-703
Cholera enterotoxin increases C-AMP production, and stimulates testosterone secretion in the rat testis . This gonadotropic action of cholera enterotoxin is potentiated by theophylline. It is suggested that cholera enterotoxin acts on Leydig cells directly to activate their adenyl cyclase, and consequently stimulates steroidogenesis in the rat testis. However, the receptor of cholera enterotoxin seems to be located at a different site from that of human chorionic gonadotropin. 相似文献
20.
Effect of substitution of glycine for arginine at position 146 of the A1 subunit on biological activity of Escherichia coli heat-labile enterotoxin. 总被引:5,自引:0,他引:5
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K Okamoto K Okamoto A Miyama T Tsuji T Honda T Miwatani 《Journal of bacteriology》1988,170(5):2208-2211
The ADP-ribosyltransferase activity of polypeptide A1 of cholera toxin and that of Escherichia coli heat-labile enterotoxin (LT) are primarily responsible for the toxic activities of these toxins. Since the amino acid sequences of the two A1 polypeptides are very similar, their functional mechanisms are considered to be the same. Arg-146 of polypeptide A1 is thought to be involved in the active site, because this amino acid of cholera toxin has been identified as the site of self-ADP-ribosylation. However, the exact role of Arg-146 and the significance of self-ADP-ribosylation in toxicity remain unclear. We substituted Arg-146 of polypeptide A1 of LT with Gly by oligonucleotide-directed mutagenesis and examined the biological property of the resultant mutant LT. The substitution changed the mobility of subunit A on sodium dodecyl sulfate-polyacrylamide gel but did not reduce the vascular permeability activity of LT. This result indicates that Arg-146 is not absolutely required for toxic activity and that LT can express its toxic activity without self-ADP-ribosylation at Arg-146. 相似文献