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Characterization of neuraminidases produced by various serotypes of Pasteurella haemolytica.

Straus, D C; Jolley, W L; Purdy, C W
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1993 EN
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Neuraminidases produced by 16 strains of Pasteurella haemolytica (serotypes 1 to 16) were characterized by molecular weight, antigenic identity, and substrate specificity. After growth in a chemically defined medium, stage I (lyophilized) culture supernatants were assayed for activity with N-acetylneuramin lactose, human alpha-1-acid glycoprotein, fetuin, colominic acid, and bovine submaxillary mucin. Neuraminidase produced by P. haemolytica serotype A1 (Ph A1) was purified by a combination of salt fractionation, ion-exchange chromatography on DEAE-Sephacel, and gel filtration on Sephadex G-200. Purified Ph A1 neuraminidase was used to immunize rabbits, and the resultant antiserum reduced the activity of Ph A1 neuraminidase by 46%. This antiserum also reduced the activity of neuraminidase produced by the other serotypes by between 15 and 66%. Molecular weight estimates of the neuraminidases produced by the various serotypes were obtained by gel filtration chromatography on Sephadex G-200. Fifteen of the 16 serotypes examined produced a neuraminidase with a molecular weight of approximately 150,000 to 200,000. One serotype (serotype 11) produced no material with neuraminidase activity. In addition, all 15 high-molecular-weight neuraminidases showed similar substrate specificities. That is...

Purification and reconstitution of the proton-translocating ATPase of Golgi-enriched membranes.

Young, G P; Qiao, J Z; Al-Awqati, Q
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1988 EN
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Kidney cortex microsomes enriched in Golgi markers and probably also containing endosomes were isolated by cell fractionation and found to contain a proton-translocating ATPase that was inhibited by N-ethylmaleimide (NEM). This NEM-sensitive ATPase was solubilized with n-octyl glucoside and purified using anion-exchange sievorptive chromatography on sequential DEAE-Sephadex and QAE-Sephadex columns followed by a final hydroxyapatite HPLC column. The purified enzyme, with a specific activity of 4.4 mumol.mg-1.min-1 was completely inhibited by NEM. Addition of asolectin and removal of the detergent by dialysis resulted in reconstitution of NEM-sensitive electrogenic proton transport. This vacuolar ATPase is composed of five polypeptides with apparent molecular masses of 68, 58, 40, 37, and 16 kDa.

PURIFICATION OF TYPE B STAPHYLOCOCCAL ENTEROTOXIN1

Frea, James I.; McCoy, Elizabeth; Strong, F. M.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1963 EN
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Frea, James I. (University of Wisconsin, Madison), Elizabeth McCoy, and F. M. Strong. Purification of type B staphylococcal enterotoxin. J. Bacteriol. 86:1308–1313. 1963.—A procedure for the production and isolation of type B staphylococcal enterotoxin in yields up to 4 mg per liter of toxic culture is presented. Staphylococcus aureus S6 was grown in shake flask cultures at 37 C for 24 hr in a medium consisting of 2% Hy-Case S.F., 0.3% yeast extract, calcium pantothenate, nicotinic acid, and thiamine. Cells were removed by centrifugation and the supernatant fluid was concentrated by dialysis against polyethylene glycol. Enterotoxin was isolated from the concentrate by ethanol precipitation, gel filtration on a G-100 Sephadex column, and Sephadex-column electrophoresis. The purified toxin was identical to type B reference enterotoxin on both disc electrophoresis and Ouchterlony gel diffusion. It contained no carbohydrates, lipids, or nucleic acids, gave positive tests for protein; and contained 11.6% N. The preparation showed no coagulase, alpha-hemolysin, or deoxyribonuclease activity. Emesis in monkeys resulted when the experimental toxin was injected at the rate of 0.26 μg of N per kg of body weight.

Purification and Properties of Cytoplasmic and Mitochondrial Malate Dehydrogenases of Physarum polycephalum

Teague, W. Martin; Henney, Henry R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1973 EN
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Two isoenzymes of malate dehydrogenase (MDH) were demonstrated in plasmodia of Physarum polycephalum by polyacrylamide-gel electrophoresis. The more “cathodal” form was uniquely associated with mitochondria (M-MDH) and the other form was found in the soluble cytoplasm (S-MDH). The isoenzymes were separated by acetone fractionation of soluble plasmodial homogenates acidified to pH 5.0. The M-MDH was purified 201-fold by cetylpyridinium chloride treatment, fractionation with ammonium sulfate, gradient elution from sulfoethyl cellulose at pH 6.0, and Sephadex G-100 chromatography. The S-MDH was purified 155-fold by ammonium sulfate fractionation, diethylaminoethyl cellulose chromatography, gradient elution from sulfoethyl cellulose at pH 5.5, and Sephadex G-100 chromatography. The optimal cis-oxalacetate concentrations were 0.35 mM for M-MDH and 0.25 mM for S-MDH, and the optimal pH for both isoenzymes was 7.6 for oxalacetate reduction. The optimal l-malate concentrations were 5 mM for S-MDH and 6 mM for M-MDH, and both isoenzymes exhibited an optimal pH of 10.0 for L-malate oxidation. The Michaelis constants of S-MDH and M-MDH served to discriminate between the isoenzymes. The S-MDH was more heat-stable than the M-MDH. High concentrations of oxalacetate and malate inhibited S-MDH more than M-MDH. The isoenzymes were further distinguished by their utilization of analogues of nicotinamide adenine dinucleotide. Many properties of the Physarum isoenzymes were similar to those of more complex organisms...

Presence and partial characterization of internal acid protease of Aspergillus oryzae.

Tsujita, Y; Endo, A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1978 EN
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The presence and partial characterization of the internal acid protease (EC 2.4.23.6) of Aspergillus oryzae has been investigated. Although the majority of the acid protease is external and present in the culture filtrate, a significant amount of the active enzyme is firmly bound to the cells; it is not released by repeated extraction of cells with 0.9% sodium chloride but is liberated into the soluble fraction during disruption of cells. The internal acid protease, as well as the external one, was separated into two major molecular forms (F1 and F2) with molecular weights of 60,000 and 42,000, respectively, by chromatography on Sephadex G-100 and on CM-Sephadex C-50. The partially purified internal enzymes had the same catalytic and immunological properties, as did the external enzyme.

Purification and Partial Characterization of an Aminopeptidase from Lactobacillus lactis

Eggimann, Bernhard; Bachmann, Marc
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1980 EN
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A surface-bound aminopeptidase of Lactobacillus lactis cells was solubilized with lysozyme, and the extract was subjected to streptomycin sulfate precipitation, ammonium sulfate fractionation, chromatography on Sephadex G-100 and diethylaminoethyl-Sephadex A-50, and preparative polyacrylamide gel electrophoresis. The purified enzyme was homogeneous in disc electrophoretic analysis and consisted of a single polypeptide chain with a molecular weight of 78,000 to 81,000. The optimal pH and optimal temperature for enzyme activity were 6.2 to 7.2 and 47.5°C, respectively, for l-lysine-4-nitroanilide as the substrate. The enzyme was activated by Co2+ and Zn2+ ions and inhibited by Cu2+, Hg2+, and Fe3+ ions and by the metal-complexing reagents ethylenediaminetetraacetic acid, 1,10-phenanthroline, and α,α′-dipyridyl. Higher concentrations of substrate and hydrolysis products also inhibited the activity of the enzyme. The aminopeptidase had broad substrate specificity and hydrolyzed many amino acid arylamides and many peptides with unsubstituted NH2-terminal amino acids.

Lyticase: Endoglucanase and Protease Activities That Act Together in Yeast Cell Lysis

Scott, Janet H.; Schekman, Randy
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1980 EN
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Yeast lytic activity was purified from the culture supernatant of Oerskovia xanthineolytica grown on minimal medium with insoluble yeast glucan as the carbon source. The lytic activity was found to consist of two synergistic enzyme activities which copurified on carboxymethyl cellulose and Sephadex G-150, but were resolved on Bio-Gel P-150. The first component was a β-1,3-glucanase with a molecular weight of 55,000. The Km for yeast glucan was 0.4 mg/ml; that for laminarin was 5.9 mg/ml. Hydrolysis of β-1,3-glucans was endolytic, yielding a mixture of products ranging from glucose to oligomers of 10 or more. The size distribution of products was pH dependent, smaller oligomers predominating at the lower pH. The glucanase was unable to lyse yeast cells without 2-mercaptoethanol or the second lytic component, an alkaline protease. Neither of these agents had any effect on the glucanase activity on polysaccharide substrates. The protease had a molecular weight of 30,000 and hydrolyzed Azocoll and a variety of denatured proteins. The enzyme was unusual in that it had an affinity for Sephadex. Although the activity was insensitive to most protease inhibitors, it was affected by polysaccharides; yeast mannan was a potent inhibitor. The enzyme did not have any mannanase activity...

Purification and Some Properties of Maleylpyruvate Hydrolase and Fumarylpyruvate Hydrolase from Pseudomonas alcaligenes

Bayly, Ronald C.; Chapman, Peter J.; Dagley, Stanley; Berardino, Dario Di
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1980 EN
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Hydrolysis of the gentisate ring-cleavage product, maleylpyruvate (cis-2,4-diketohept-5-enedioic acid), was shown to be catalyzed by an enzyme, maleylpyruvate hydrolase 11, in Pseudomonas alcaligenes (P25X1) after growth with 3-hydroxybenzoate. This activity was separated from fumarylpyruvate hydrolase activity during the course of its purification which accomplished an approximately 50-fold increase in specific activity. An apparent molecular weight of 77,000 was assigned on the basis of Sephadex G-200 chromatography. Despite the presence of up to three similarly migrating bands of protein on polyacrylamide-gel electrophoresis of the purified enzyme, at least two of these bands possessed maleylpyruvate hydrolase activity. Electrophoresis on sodium dodecyl sulfate-polyacrylamide before and after reduction with mercaptoethanol gave a principal band of molecular weight of 33,000 (and a minor band of molecular weight 50,000). A number of substituted maleylpyruvates also served as substrates for maleylpyruvate hydrolase 11, but maleylacetoacetate and fumarylpyruvate were not attacked. Fumarylpyruvate hydrolase was purified approximately 40-fold to give a single band on polyacrylamide gels and with an apparent molecular weight of 73,000 by Sephadex G-200 chromatography. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis before or after reduction with mercaptoethanol...

CAP37, a human neutrophil-derived chemotactic factor with monocyte specific activity.

Pereira, H A; Shafer, W M; Pohl, J; Martin, L E; Spitznagel, J K
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1990 EN
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CAP37, an antimicrobial protein of human neutrophil granules, is a specific chemoattractant for monocytes. Purified to homogeneity by sequential chromatography over carboxymethyl Sephadex, G-75 Sephadex, and hydrophobic interaction HPLC, demonstratively endotoxin-free CAP37 was maximally chemotactic over a range of 1.3 X 10(-9)-10(-8) M. Thus it was active in the same molar concentrations as formyl-methionyl-leucyl-phenylalanine. CAP37 lacked chemotactic activity for neutrophils and lymphocytes. In checkerboard assays CAP37 had some chemokinetic activity as well. It was also chemotactic for rabbit mononuclear cells. Higher concentrations (2.7 X 10(-8) M) were required for activity with rabbit cells than with human. Sequence analysis of the first 42 NH2-terminal amino acid residues of CAP37 showed strong homologies with known serine proteases that mediate various functions in inflammation. However, a critical substitution of a serine for a histidine at position 41 suggested that CAP37 lacked serine protease action. This impression was supported by the failure of CAP37 to bind tritiated diisopropyl fluorophosphate. 89% of total CAP37 was released extracellularly from human neutrophils while they phagocytized Staphylococcus aureus. We propose that CAP37 released from neutrophils during phagocytosis and degranulation may mediate recruitment of monocytes in the second wave of inflammation.

Physicochemical and Biological Properties of Human and Canine Plasmins

Takeda, Y.; Nakabayashi, M.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1974 EN
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Three kinds of plasmin were found to be generated when plasminogen or [125I]plasminogen was incubated at 32°C for longer than 20 min in urokinase and 50% glycerol. Each plasmin was then separated by G-200 or G-75 Sephadex filtration, and its physicochemical properties were determined. The molecular weights of the three plasmins as determined by G-200 Sephadex filtration were 125,000±5,000(SD), 63,000±2,000(SD), and 31,500±1,000(SD), and those by sodium dodecyl sulfate(SDS)-polyacrylamide electrophoresis were 130,000±5,000(SD), 64,000±3,000(SD), and 32,000±1,500(SD). It was also found that during the incubation of the smallest plasmin in SDS and beta-mercaptoethanol it was further split into two smaller pieces of about 16,000 mol wt and that polymer proteins of 95,000±2,000(SD) and 48,000±1,500(SD) mol wt were formed. Despite these differences in the molecular size of the three plasmins, the specific activity of each plasmin was closely similar and in case of human plasmins averaged 29±0.9(SD) CTA units/mg plasmin and in case of canine plasmins 8.5±0.54(SD) CTA units/mg plasmin. Then, using human plasmin of the smallest size (mol wt 31,500), the total plasma antiplasmin capacity was determined in 20 normal human plasma, which averaged 7.8±2(SD) CTA units of plasmin per milliliter plasma. Studies were next made of the affinity of human [125I]-plasmin of the smallest size with albumin...

Partial characterization and purification of a rabbit granulocyte factor that increases permeability of Escherichia coli.

Weiss, J; Franson, R C; Beckerdite, S; Schmeidler, K; Elsbach, P
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1975 EN
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Recently we reported that rapid killing of Escherichia coli by granulocytes or granulocyte fractions is accompanied by an equally rapid and discrete increase in permeability of the microbial envelope (Beckerdite, Mooney, Weiss, Franson, and Elsbach. 1974. J. Exp. Med. 140: 396-409). Most of this permeability-increasing activity (PI) is found in a crude granule preparation. PI is quantitatively recovered in a 23,000-g supernatant fraction (Sup II) after sulfuric acid extraction of granulocyte homogenates prepared in water. PI is nondialyzable, destroyed by pronase and trypsin, stable at 4degreesC for at least 2 mo, and destroyed by heating at 94degreesC. Anionic substances, such as heparin sulfate and isolated E. coli lipopolysaccharide, bind to and inhibit PI. PI has been purified up to 1,000-fold from homogenate in a yield of 50percent by acid extraction and carboxymethyl-Sephadex chromatography. Such purified fractions have bactericidal activity that equals that of disrupted granulocytes and Sup II, are similarly enriched with respect to granule-associated phospholipase, and protease activities. Whereas E. coli, sensitive to PI, binds or inactivates solubilized PI, a resistant strain of Serratia marcescens does not. Binding of PI to sensitive microorganisms seems to be necessary for expression of its biological activity since both the apparent binding to and the biological effect of PI on E. coli are completely blocked by 10-20 mM Mg2+ or Ca2+. Mg2+ or Ca2+ can reverse the effect on E. coli permeability produced by Sup II or the carboxymethyl-Sephadex fraction but not that produced by granulocyte homogenate. The close association of bactericidal...

The postnatal decline of hemoglobin F synthesis in normal full-term infants.

Bard, H
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1975 EN
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Studies were carried out during the 1st yr of life in normal infants born at term to determine the proportions of fetal hemoglobin (Hb F) and adult hemoglobin (Hb A) being synthesized, in order to describe the complete switchover from Hb F to Hb A synthesis during postnatal life. 53 blood samples from 37 infants were incubated in an amino acid mixture containing [14C]leucine and chromatographed on DEAE-Sephadex for separation of Hb F and Hb A fractions. The completeness of the CEAE-Sephadex separation of Hb A and Hb F at an age when the major portion of synthesis was of the adult type of hemoglobin was confirmed by globin chain chromatography with the use of carboxylmethyl cellulose. There was a rapid decline in Hb F synthesis postnatally until 16-20 wk of age when levels of 3.2% plus or minus SD 2.1% were reached. By combining this data with that previously published, the complete switchover from Hb F to Hb A synthesis can be described in humans in relation to postconceptional age. It follows a sigmoid curve; the steep portion, which lies between the 30th and 52nd postconceptional week, is preceded and follwoed by plateaus averaging 95% and 7% Hb F synthesis, respectively.

Uricosuric agents in uremic sera. Identification of indoxyl sulfata and hippuric acid.

Boumendil-Podevin, E F; Podevin, R A; Richet, G
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1975 EN
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Serum and urine from chronically uremic patients and normal individuals were subjected to gel filtration of Sephadex-G10. The effects of the eluted fractions on the uptake of urate and para-aminohippurate by isolated cortical tubules of rabbit kidney were investigated. According to the origin of the samples, one to three major groups of fractions inhibiting both urate and para-aminohippurate transport were disclosed. The first eluted group occurred for all the samples under study. The second one was demonstrated in both sera and urines from uremic patients but only in urines from normal individuals. The third one was exclusively detected in uremic sera and urines. Among all the compounds identified, only hippuric acid, eluted in the fractions of the second group, was capable of inhibiting the uptake of urate and para-aminohippurate in vitro. The concentration for which this inhbiitory effect of hippuric acid occurred was in the range of that existing in uremic sera. Indoxyl sulfate, which accumulates to very high concentrations in uremic serum, could not be disclosed in the above-mentioned fractions. This is explained by the strong adsorption of this indole derivative to Sephadex gel. Potassium indoxyl sulfate, when tested in vitro at the concentration existing in uremic serum...

Biologic and Immunologic Characterization and Physical Separation of ACTH and ACTH Fragments in the Ectopic ACTH Syndrome

Orth, David N.; Nicholson, Wendell E.; Mitchell, William M.; Island, Donald P.; Liddle, Grant W.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1973 EN
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Extracts of tumors from 32 patients with the ectopic ACTH syndrome were subjected to simultaneous bioassay and radioimmunoassays for ACTH. Radioimmunoassays were performed using three antisera, one of which reacts with the extreme N-terminal 1-13 amino acid sequence of ACTH, the second with the N-terminal 1-23 sequence of the ACTH molecule, and the third with the C-terminal 25-39 amino acid sequence of ACTH. There was, in general, good correlation between bioactivity and N-terminal ACTH immunoreactivity. However, there were large excesses of both extreme N-terminal and C-terminal immunoreactive materials in most tumor extracts, which were not found in extracts of three human pituitaries. Three tumor extracts were subjected to molecular sieve chromatography on Sephadex G-50 fine resin. The bioactive ACTH eluted in the same fractions as pituitary ACTH (mol wt≃4,500 daltons) and reacted equally in all three ACTH radioimmunoassay systems. The bioactive tumor ACTH was neutralized by incubation with the C-terminal antiserum, indicating it has an intact C-terminal sequence of amino acids. The next several fractions from the Sephadex column contained a material, mol wt≃3,100, which was biologically inactive and had C-terminal immunoreactivity but no N-terminal or extreme N-terminal immunoreactivity. Incubation with the N-terminal 1-23 ACTH antiserum did not adsorb these C-terminal fragments...

Postnatal Fetal and Adult Hemoglobin Synthesis in Early Preterm Newborn Infants

Bard, Harry
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1973 EN
Relevância na Pesquisa
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Studies were carried out during the postnatal period in infants born at or before the 32nd wk of gestation to determine the proportion of fetal hemoglobin (Hb F) and adult hemoglobin (Hb A) being synthesized, and to compare these studies to those previously reported on at birth from normal newborn infants 25-43 wk gestation. When the pretern infants reached the postconceptional age corresponding to term, their Hb A and Hb F synthesis was compared to a group of newborn infants at term. 53 blood samples from 25 preterm and 11 full-term infants were incubated in an amino acid mixture containing [14C]leucine, and column-chromatographed on DEAE-Sephadex for separation of Hb F and Hb A fractions. The completeness of the DEAE-Sephadex separation of Hb F and Hb A was confirmed by globin chain chromatography with the use of carboxymethylcellulose. The rate of transition from Hb F to Hb A synthesis postnatally in the preterm infants resembled that reflecting the in utero transition. At the postconceptional age corresponding to term, there was no difference in the relative amounts of Hb F and Hb A being synthesized by the preterm infants and by the term infants. The birth process did not alter the rate of transition from Hb F to Hb A.

The primary structure of the myosin head.

Maita, T; Hayashida, M; Tanioka, Y; Komine, Y; Matsuda, G
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1987 EN
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The sequence of the NH2-terminal 808 amino acid residues of chicken pectoralis muscle myosin head was determined. Three characteristic 20-, 23-, and 50-kDa fragments were isolated from a digest of myosin subfragment 1 (S1) by gel filtration on a Sephadex G-100 column in the presence of 5 M guanidine hydrochloride, followed by anion-exchange chromatography on a QAE-Sephadex A-50 column in the presence of 8 M urea. The fragments were sequenced completely by conventional methods. Peptides overlapping the 23- and 50-kDa fragments and also overlapping the 50- and 20-kDa fragments were obtained by cleaving S1 with cyanogen bromide. Comparison of the 23-kDa and 50-kDa sequences with that of the overlapping peptide indicated that no additional amino acid exists between the 23- and 50-kDa fragments and that 5 amino acids exist between the 50- and 20-kDa fragments of S1. Methylated amino acid residues were found at four positions: epsilon-N-monomethyllysine at position 35, epsilon-N-trimethyllysine residues at 130 and 550, and 3-N-methylhistidine at 754.

Purification and characterization of a protease produced by Vibrio cholerae non-O1 and comparison with a protease of V. cholerae O1.

Honda, T; Lertpocasombat, K; Hata, A; Miwatani, T; Finkelstein, R A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1989 EN
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A protease produced by a clinical isolate of Vibrio cholerae non-O1 was purified to apparent homogeneity by ammonium sulfate fractionation and successive column chromatography on DEAE-Sephadex A25, Sephadex G100, Mono Q, and Phenyl Superose. Like the hemagglutinin-protease of V. cholerae O1, the purified protease had both hemagglutinating and proteolytic activities. The protease was heat labile, and in contrast to crude preparations, no Arrhenius effect was observed with the purified protein. Immunological analyses indicated that the proteases (or hemagglutinins) derived from V. cholerae O1 and non-O1 are identical.

Immunochemistry of a Formamide-extracted Antigen from Clostridium perfringens Cell Walls

Johnson, Howard M.; Brenner, Kristen; Hall, Herbert E.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1969 EN
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The type-specific antigen of a strain of Clostridium perfringens involved in food poisoning was isolated from the cell wall by the use of hot formamide. The antigen appears to consist of polysaccharide or mucopeptide. The formamide extract was shown to be heterogeneous by gel filtration on Sephadex G-200. The serologically active fraction contained about 25% of the amount of protein present in the original formamide extract. Hexosamine, acetyl groups, and carbohydrate also were detected. The formamide extract showed a high degree of serological activity. The serological activity was increased twofold on Sephadex gel filtration.

Isolation and Characterization of Fractions of Mycoplasma pneumoniae I. Chemical and Chromatographic Separation

Prescott, B.; Sobeslavsky, O.; Caldes, G.; Chanock, R. M.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1966 EN
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Prescott, B. (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), O. Sobeslavsky, G. Caldes, and R. M. Chanock. Isolation and characterization of fractions of Mycoplasma pneumoniae. I. Chemical and chromatographic separation. J. Bacteriol. 91:2117–2125. 1966.—Fractionation of Mycoplasma pneumoniae, cultured on a beef heart infusion-horse serum-yeast extract medium, was carried out by chemical and chromatographic procedures. The chemical method yielded eight fractions consisting of lipid, carbohydrates, and proteins. Four protein-rich fractions were isolated by chromatographing a supernatant fluid of sonically treated organisms on Sephadex G-25. The 12 fractions were tested for serological and antigenic activity in vitro and in vivo. The lipid fraction was serologically active and the relative order of activity of the protein fractions appeared to depend on the amount of lipid present in the molecule. The highly serologically active Sephadex G-25 protein fraction 1 prepared chromatographically contained 15% lipid in the molecule, whereas the less serologically active protein fraction 2 prepared by chemical means contained 2% lipid. The acetone-extracted lipid fraction was chromatographed on thin-layer chromatography plates and found to consist of nine fractions. Serological activity was associated with only the first three spots above the origin. Lipid extracted from the protein fractions seemed to be similar to the acetone-extracted lipid from the sediment of the sonically treated organisms.

Production, Purification, and Composition of Staphylococcal α Toxin

Coulter, John R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1966 EN
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Coulter, John R. (Institute of Medical and Veterinary Science, Adelaide, Australia). Production, purification, and composition of staphyloccocal α toxin. J. Bacteriol. 92:1655–1662. 1966—Pure staphylococcal α toxin has been prepared in quantities suitable for chemical, biological, and clinical characterization. Purification was achieved by acid-methanol precipitation, chromatography on G100 Sephadex, and electrophoresis in G100 Sephadex. We recovered 25% of the crude toxin in pure form, a yield of 12 mg/liter of crude culture supernatant fluid. The pure material gave a single line on gel diffusion and on immunoelectrophoresis and gave a single symmetrical peak in the ultracentrifuge. The α toxin was highly unstable, with a half-life of 3 days at 0 C (pH 7.8); solutions of it could not be frozen, and we found no method to stabilize it. On standing, a thready precipitate appeared; it was inactive against rabbit red cells, was not lethal to rabbits, but was able to elicit specific anti-α antibody production in the rabbit. There is evidence that α toxin is an associating molecule, with a mean sedimentation coefficient of approximately 3.0 and a molecular weight of approximately 30,000. The lowest molecular weight, found by equilibrium ultracentrifugation...