Studies on the purification of Entamoeba histolytica antigens by gel filtration. I. Some physicochemical properties of the isolated fractions

1970 ◽  
Vol 16 (6) ◽  
pp. 485-492 ◽  
Author(s):  
Z. Ali Khan ◽  
E. Meerovitch
Keyword(s):  
Physicochemical Properties ◽  
Entamoeba Histolytica ◽  
Isoelectric Focusing ◽  
Phosphate Buffer ◽  
Gel Filtration ◽  
Disc Electrophoresis ◽  
Aqueous Extracts ◽  
Molecular Weights ◽  
Isoelectric Ph ◽  
Considerable Loss

Aqueous extracts, prepared from Entamoeba histolytica (DKB strain) grown at 37 °C in TTY medium with Crithidia sp., were chromatographed on Sephadex G-200 and G-100 gels equilibrated with 0.15 M phosphate buffer, pH 7.2. Seven fractions were obtained, the molecular weights of which ranged from 650 000 to [Formula: see text]. The absorption spectra of either the whole amoebic extract or the first two fractions from the G-200 column were in the region of 280 mμ. The protein to carbohydrate ratio of the whole undialyzed amoebic extract was 2.6:1, which changed to 17:1 after dialysis, indicating a considerable loss of carbohydrate. The whole amoebic extract when subjected to disc electrophoresis resolved into 26 protein bands while the fractions, in addition to having contaminants from the preceding peaks, also contained their own characteristic protein bands. The first two fractions contained, as determined by disc electrophoresis and isoelectric focusing, a variety of different anionic proteins. The isoelectric pH of the first fraction was 5.8 to 6.0 and that of the second ranged between 4.5 and 6.4.

Purification and characterization of tuberculin-active components from BCG

1975 ◽  
Vol 21 (12) ◽  
pp. 2019-2027
Author(s):  
M. Laguerre ◽  
R. Turcotte
Keyword(s):  
Physicochemical Properties ◽  
Guinea Pigs ◽  
Gel Filtration ◽  
Biologically Active ◽  
Polyacrylamide Gels ◽  
Active Components ◽  
Purification And Characterization ◽  
Molecular Weights ◽  
Antigenic Activity

The tuberculin activity of protoplasmic extracts isolated from living BCG was purified successively by gel filtration on Sephadex G-100 and G-75, and by electrophoresis on 7.5% and on gradient (6–18%) polyacrylamide gels. The tuberculin-active fractions, as determined in BCG-sensitized guinea pigs, were used as the starting material for each of the following fractionation steps.The physicochemical properties and the antigenic activity of the biologically active fractions have shown that a single component, or only a few ones with similar properties, possessed high tuberculin activity. These active components were proteins having relatively high molecular weights (about 72 000) and could behave as antigens.

Microheterogeneity and sialic acid in human plasma angiotensinogens in various physiological states

1978 ◽  
Vol 56 (9) ◽  
pp. 892-899 ◽  
Author(s):  
A. A. Faiers ◽  
A. Y. Loh ◽  
D. H. Osmond
Keyword(s):  
Sialic Acid ◽  
Rat Liver ◽  
Isoelectric Focusing ◽  
Structural Characteristics ◽  
Gel Filtration ◽  
Angiotensin I ◽  
Third Trimester ◽  
Human Renin ◽  
Isoelectric Ph ◽  
Normal Men

Pooled or individual plasmas from normal men, women, pregnant women (third trimester), anephric women, and rat liver perfusates were used as sources of angiotensinogen. The plasmas were fractionated and desalted by Sephadex gel filtration, then subjected to isoelectric focusing in a pH 4 to 6 gradient on 40 × 4 cm slabs of polyacrylamide gel. The gels were cut transversely into 0.5-cm-wide strips, the pH measured, and their angiotensinogen concentrations determined by incubation with excess human renin and radioimmunoassay of the product, angiotensin I. This revealed several peaks of angiotensinogen concentration indicative of microheterogeneity in all cases. Contrary to other claims, the isoelectric pH profiles of angiotensinogens in the various physiological states were substantially alike. Major peaks were found at pH 4.75 to 4.85 and 4.9 to 5.0 and minor peaks at pH 4.5 to 4.7 and 5.0 to 5.2; this resolution was greater than that achieved with rat liver angiotensinogens. Incubation of human angiotensinogens with neuraminidase for 3 or 16 h raised their isoelectric pH by about 0.5 U, probably due to removal of sialic acid. Since microheterogeneity persisted after desialylation, it is probably determined by structural characteristics other than sialic acid composition.

Studies on the purification of Entamoeba histolytica antigens by gel filtration. II. The antigenic properties of the isolated fractions

1970 ◽  
Vol 16 (6) ◽  
pp. 493-498 ◽  
Author(s):  
Z. Ali Khan ◽  
E. Meerovitch
Keyword(s):  
Entamoeba Histolytica ◽  
Gel Filtration ◽  
Water Soluble ◽  
The Other ◽  
Soluble Extract ◽  
Serological Tests ◽  
Molecular Weights ◽  
Antigenic Properties ◽  
Water Soluble Extract

Partially purified fractions of the water-soluble extract of Entamoeba histolytica (DKB strain) obtained by chromatography in Sephadex G-200 (F1, F2, and F4) and G-100 (F3a, F3b, F3c, and F3d) gels were used in various serological tests and their reactivities compared with the whole 1:40 antigen. The main haemagglutinating (HA) and complement-fixing (CF) activities were confined to fractions F1 and F2, which had molecular weights of 650 000 and 229 000, respectively. The 4 to 6 μg/ml of protein contained in these fractions at the optimum dilution gave antibody liters comparable to those for the whole antigen, which had about 77 μg/ml of protein. The other antigen fractions (F4, F3a, F3b, F3c, and F3d) showed very little activity. Fraction F1 had two main precipitin bands (1 and 2), which showed reaction of identity with two of the bands in fraction F2. The other fractions which showed some HA and CF reactivity had trace amounts of these antigens. It is presumed that the antigens specific for precipitin bands 1 and 2 are the main CF and HA antigens.

scholarly journals Purification and properties of α-galactosidases from Vicia faba seeds

1969 ◽  
Vol 113 (1) ◽  
pp. 49-55 ◽  
Author(s):  
P. M. Dey ◽  
J. B. Pridham
Keyword(s):  
Amino Acid ◽  
Vicia Faba ◽  
Aromatic Amino Acid ◽  
Gel Filtration ◽  
Disc Electrophoresis ◽  
Thermal Stabilities ◽  
Molecular Weights ◽  
Purification And Properties ◽  
Enzyme I ◽  
Carbohydrate Contents

Two forms of α-galactosidase, I and II, exist in Vicia faba seeds and these have been purified 3660- and 337-fold respectively. They behaved as homogeneous preparations when examined by ultracentrifugation, disc electrophoresis and gel filtration. The apparent molecular weights of enzymes I and II, as determined by gel filtration, were 209000 and 38000 respectively. The carbohydrate contents of enzymes I and II were 25% and 2·8% respectively, and the enzymes differed in their aromatic amino acid compositions. Enzyme I was split into six inactive subunits in the presence of 6m-urea. α-Galactosidases I and II showed different pH optima and Km and Vmax. values with p-nitrophenyl α-d-galactoside and raffinose as substrates, and also differed in their thermal stabilities.

ISOLATION OF LH, FSH AND TSH FROM HUMAN PITUITARIES AFTER THE REMOVAL OF HGH

1974 ◽  
Vol 77 (3) ◽  
pp. 485-497 ◽  
Author(s):  
P. A. Torjesen ◽  
T. Sand ◽  
N. Norman ◽  
O. Trygstad ◽  
I. Foss
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Disc Electrophoresis ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Molecular Weights ◽  
Pituitary Glands

ABSTRACT Highly purified human LH, FSH and TSH were isolated from batches of 300 frozen pituitary glands (200 g) by pH, acetone and ethanol fractionation, Sephadex gel filtration, ion-exchange chromatography on DEAE-cellulose and CM-Sephadex, and preparative polyacrylamide-gel electrophoresis. Sodium dodecyl-sulphate (SDS) polyacrylamide gel electrophoresis was used in order to check the purity, the identity and the molecular weight of the purified LH, FSH and TSH. This procedure showed that the hormone preparations consisted of two subunits with molecular weights of: LH: 21 300 and 17 900, FSH: 22 100 and 18 300 and TSH: 20 800 and 16 400. The purity of the hormone preparations was also evaluated by analytical disc electrophoresis at pH 8.9. The purified hormone preparations had radioimmunological activity as follows: LH: 20 000 IU/mg, FSH: 16 500 IU/mg and TSH: 5 IU/mg. All preparations had high biological potency.

scholarly journals Granulocyte/macrophage-, megakaryocyte-, eosinophil- and erythroid-colony-stimulating factors produced by mouse spleen cells

1980 ◽  
Vol 185 (2) ◽  
pp. 301-314 ◽  
Author(s):  
Antony W. Burgess ◽  
Donald Metcalf ◽  
Sue H. M. Russell ◽  
Nicos A. Nicola
Keyword(s):  
Ionic Strength ◽  
Isoelectric Focusing ◽  
Biological Activities ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Colony Stimulating Factor ◽  
Colony Stimulating Factors ◽  
Molecular Weights ◽  
Low Ionic Strength ◽  
Stimulating Factor

The formation of mature haemopoietic cells is controlled by hormones that specifically stimulate the progenitor cells of the granulocyte/macrophage, eosinophil, megakaryocyte and erythroid pathways. PWMSC medium (pokeweed-mitogen-stimulated spleen-cell-conditioned medium) is known to contain the biological activities that control the clonal proliferation of these four progenitor cells in vitro in semi-solid agar cultures. In this study the molecular properties of these biological activities were characterized, and all four colony-stimulating factors appear to be associated with glycoproteins. These factors were precipitated between 50 and 80%-satd. (NH4)2SO4 and could be concentrated by ultrafiltration over a 10000-mol.wt.-cut-off hollow-fibre membrane. Megakaryocyte- and erythroid-colony-stimulating factors were lost when the conditioned medium was dialysed at low ionic strength (<0.03m). Neither asialo- nor sialo-erythropoietin was detectable in concentrated PWMSC medium or in the fractions purified from it by gel filtration on Sephadex G-150. The factors bound to concanavalin A–Sepharose were eluted with α-methyl-d-glucopyranoside (0.10m). Analysis by gel filtration on Sephadex G-150 indicated that the apparent molecular-weight distributions of all colony-stimulating factors were identical (37000). Treatment with neuraminidase did not alter the biological activities of any of these factors, but when the molecular weights were analysed, after neuraminidase treatment, on Sepharose CL-6B in the presence of guanidine hydrochloride (6m) all were eluted with a mol.wt. of 24000. Although the apparent molecular weights of the different factors were identical, charge differences were detectable by isoelectric focusing on thin-layer granulated gels. There appeared to be considerable charge heterogeneity associated with each factor, as all were focused over 2–4 pH units. The maximum activity of the granulocyte/macrophage-colony-stimulating factor on isoelectric focusing was at pH4.8, whereas the maximum activity for the eosinophil-colony-stimulating factor was at pH5.8. The erythroid- and megakaryocyte-colony-stimulating activities were detected in the pH ranges 4.8–5.8 and 4.6–7.1 respectively. Chromatographic differences between the granulocyte/macrophage- and eosinophil-colony-stimulating factors were also detected by hydrophobic chromatography at low ionic strength (0.15m-NaCl) on Cibacron Blue–Sepharose and at high ionic strength [2m-(NH4)2SO4] on phenyl-Sepharose. Eosinophil-colony-stimulating factor bound more strongly than the other factors to both matrices. The megakaryocyte- and erythroid-colony-stimulating activities were always associated with those for granulocytes/macrophages and eosinophils. Preparations highly enriched for eosinophil-colony-stimulating factor were also obtained by DEAE-cellulose chromatography. An overall purification of 100-fold for all of the factors was achieved with the present techniques, and, although differences were observed, only granulocyte/macrophage-stimulating factors and a small proportion of the eosinophil-stimulating factors could be completely separated from the others. Our results are consistent with the existence of separable factors for granulocyte/macrophage and eosinophil stimulation, but the megakaryocyte- and erythroid-stimulating activities were always associated with the granulocyte/macrophage- and eosinophil-stimulating activities. Thus there may be one molecule that is able to stimulate all four colony types or four very similar molecules that are difficult to separate.

scholarly journals MURINE AMYLOID FIBRIL PROTEIN: ISOLATION, PURIFICATION AND CHARACTERIZATION

1971 ◽  
Vol 19 (1) ◽  
pp. 16-28 ◽  
Author(s):  
G. G. GLENNER ◽  
D. PAGE ◽  
C. ISERSKY ◽  
M. HARADA ◽  
P. CUATRECASAS ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Amyloid Fibril ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Disc Electrophoresis ◽  
Structure Identification ◽  
Major Protein ◽  
Amyloid Protein ◽  
Molecular Weights ◽  
Sepharose 4B

Murine amyloid has been produced by four different induction methods ( Mycobacterium butyricum, casein, casein plus Freund's adjuvant and endotoxin-induced mouse amyloidosis) in several strains and obtained from mice with "spontaneous" amyloidosis. The amyloid fibrils have been concentrated from spleen and liver. Electron microscopy of all of these preparations reveals the amyloid fibril to be 100 Å in width and composed of two parallel filaments, each measuring 35-40 Å in width and having the appearance of a twisted ribbon. X-ray diffraction of all preparations reveals a "backbone" spacing at 4.75 Å and a "side chain" spacing at 11 Å indicating a "β-pleated sheet" structure. Identification of the major protein component of amyloid fibril concentrates was made by combined use of sodium dodecyl sulfate polyacrylamide disc electrophoresis, labeling of the protein with 3H-tryptophan and Sepharose 4B gel filtration. Purification of the amyloid protein from spontaneous amyloidosis liver was accomplished by sequential gel filtration with 5 M guanidine in 1 N acetic acid on Sepharose 4B and Sephadex G-100 or G-75 columns. The material is a unique glycoprotein with a molecular weight of 7200, a high content of dicarboxylic and short chain amino acids, a significant amount of tryptophan and an unreactive NH2-terminal amino acid, tentatively identified as pyrrolid-2-one-5-carboxylic acid. There are no methionine, half-cystine, hydroxylysine or hydroxyproline residues. Murine amyloid protein, therefore, has striking similarities to many human amyloid protein preparations. It differs from the human proteins in the similarity of molecular weights of different preparations.

NATURE OF PROTEINS INTRITICALEAND ITS PARENTAL SPECIES: II. GEL FILTRATION AND DISC ELECTROPHORESIS RESULTS

1970 ◽  
Vol 50 (1) ◽  
pp. 15-24 ◽  
Author(s):  
C. H. CHEN ◽  
W. BUSHUK
Keyword(s):  
Spring Wheat ◽  
Parental Species ◽  
Acetic Acid Solution ◽  
Gel Filtration ◽  
Disc Electrophoresis ◽  
Hard Red Spring Wheat ◽  
Molecular Weights ◽  
Electrophoretic Patterns ◽  
Water Salt ◽  
Protein Components

The compositions and identity of protein components in the water, salt solution, 70% ethanol, and acetic acid solution extracts of the endosperm from one line of Triticale, its parents, and one cultivar of hard red spring wheat were investigated by gel filtration and disc electrophoresis. Albumins, globulins, and gliadins, as defined by their solubility, molecular weight, and electrophoretic mobility, were obtained by gel filtration on Bio-Gel P-150. Approximate molecular weights determined from elution volumes were 1 × 104to 3 × 1045 × 104to 9 × 104, and above 1.5 × 105for albumins and globulins, gliadins and glutenins, respectively. Each gel-filtration fraction contained several protein components as determined by disc electrophoresis. Quantitative distribution of fractions varied with species. For each fraction the amount for Triticale was intermediate between the amounts for the parental species. The gel-filtration spectrum for Triticale was similar to that for the hard red spring wheat. However, disc electrophoretic patterns for fractions obtained by gel filtration for the two species were quite different.

scholarly journals The purification and properties of the l-serine O-sulphate-degrading system of pig liver

1971 ◽  
Vol 121 (5) ◽  
pp. 747-752 ◽  
Author(s):  
N. Tudball ◽  
P. Thomas ◽  
R. Bailey-Wood
Keyword(s):  
Amino Acid ◽  
Isoelectric Focusing ◽  
Enzyme System ◽  
Gel Filtration ◽  
Pig Liver ◽  
Molecular Weights ◽  
Purification And Properties ◽  
Isoelectric Points ◽  
Degrading System ◽  
Similar Amino Acid

1. The enzyme system from pig liver responsible for the αβ-elimination of l-serine O-sulphate was purified 1000-fold. 2. Isoelectric focusing produced two enzymically active fractions with isoelectric points at pH5.6 and 5.9 respectively. 3. Osmometry and gel filtration showed both enzymes to possess molecular weights of approx. 54000. 4. The separate activities exhibited similar amino acid compositions.

scholarly journals The purification and properties of a ribonucleoenzyme, o-diphenol oxidase, from potatoes

1970 ◽  
Vol 118 (1) ◽  
pp. 15-23 ◽  
Author(s):  
K. Balasingam ◽  
W. Ferdinand
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Disc Electrophoresis ◽  
Ph Optimum ◽  
Molecular Weights ◽  
Major Form ◽  
Purification And Properties ◽  
Minimal Molecular Weight

1. o-Diphenol oxidase was isolated from potato tubers by a new approach that avoids the browning due to autoxidation. 2. There are at least three forms of the enzyme, of different molecular weights. The major form, of highest molecular weight, was separated from the others in good yield and with high specific activity by gel filtration through Bio-Gel P-300. 3. The major form is homogeneous by disc electrophoresis but regenerates small amounts of the species of lower molecular weight, as shown by rechromatography on Bio-Gel P-300. 4. There is an equal amount of RNA and protein by weight in the fully active enzyme. The RNA cannot be removed without loss of activity, and is not attacked by ribonuclease. 5. The pH optimum of the enzyme is at pH5.0 when assayed with 4-methylcatechol as substrate. It is ten times more active with this substrate than with chlorogenic acid or catechol. The enzyme is fully active in 4m-urea. 6. A minimal molecular weight of 36000 is indicated by copper content and amino acid analysis of the protein component of the enzyme. 7. The protein contains five half-cystinyl residues per 36000 daltons, a value similar to that found in o-diphenol oxidase from mushrooms. It also contains tyrosine residues although, when pure, it does not turn brown by autoxidation.

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