scholarly journals Biochemical characterization of the component parts of intestinal mucin from patients with cystic fibrosis

1984 ◽  
Vol 224 (2) ◽  
pp. 345-354 ◽  
Author(s):  
M Mantle ◽  
G G Forstner ◽  
J F Forstner
Keyword(s):  
Cystic Fibrosis ◽  
Biochemical Characterization ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Molecular Size ◽  
Equilibrium Density ◽  
Antigenic Determinants ◽  
Intestinal Mucin ◽  
Sepharose 4B

Previous studies have shown that human small-intestinal mucin consists of high-Mr glycoproteins and a smaller S-S-bonded protein of 118 kDa. The major antigenic determinants of the mucin were associated with the large glycoproteins, but depended for stability on intact disulphide bonds, and were destroyed by digestion with Pronase. In the present study we isolated and analysed the component parts of mucin from patients with cystic fibrosis with special attention being paid to the peptide constituents. After reduction with 0.2 M-beta-mercaptoethanol [5 min, 100 degrees C in 1% SDS (sodium dodecyl sulphate)], the large glycoproteins and smaller peptide with an apparent molecular size of 118 kDa were separated by equilibrium density-gradient centrifugation in CsCl, Sepharose 4B chromatography or preparative SDS/polyacrylamide-gel electrophoresis. The large glycoproteins contained about 70% of the protein of the native mucin. Digestion with Pronase resulted in a further loss of ‘naked’ protein (10% of the native mucin protein) from the C-terminal end of the glycoprotein peptide core, and left behind highly glycosylated proteins comprised mainly (70 mol%) of threonine, serine and proline. The 118 kDa component, which contained about 30% of the native mucin protein, consisted mainly of aspartic acid, serine, glutamic acid and glycine (40 mol%), plus threonine, proline, alanine, valine and leucine (35 mol%). Together with the ‘naked’ protein segment, the 118 kDa component contained most of the cysteine residues of the native mucin. Surprisingly, the peptide also contained carbohydrate (less than or equal to 5% of the native mucin carbohydrate but 50% by weight of the 118 kDa component), which included 9 mol% mannose, suggesting the presence of N-linked oligosaccharides. The peptide exhibited strong non-covalent interactions with the high-Mr glycoproteins and a tendency to self-aggregate in the absence of dissociating agents. Our findings therefore suggest that native mucin consists of large glycoproteins capable of forming disulphide bridges from their C-terminal ‘naked’ (antigenic) regions to a smaller glycopeptide having an Mr of 118 000.

scholarly journals Characterization and purification of H1TF2, a novel CCAAT-binding protein that interacts with a histone H1 subtype-specific consensus element.

1989 ◽  
Vol 9 (4) ◽  
pp. 1566-1575 ◽  
Author(s):  
P Gallinari ◽  
F La Bella ◽  
N Heintz
Keyword(s):  
Cell Cycle ◽  
Biochemical Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gene Promoter ◽  
Molecular Size ◽  
Histone H1 ◽  
Nuclear Extracts ◽  
Definition Of

Definition of mechanisms regulating human histone H1 gene transcription during the cell cycle requires the isolation and biochemical characterization of protein factors which interact with specific promoter elements. Two distinct binding activities have been identified in nuclear extracts from HeLa cells and mapped within a 180-base-pair (bp) region of a cell cycle-regulated H1 gene promoter. H1TF1 bound to an H1-specific A + C-rich sequence (AC box), 100 bp upstream of the cap site; H1TF2 interacted with the H1 subtype-specific consensus element and was dependent on the presence of an intact CCAAT box for binding. H1TF2 was purified through a combination of ion-exchange and oligonucleotide affinity chromatographies. Analysis of purified fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and UV crosslinking showed that H1TF2 was a single polypeptide of 47 kilodaltons. This factor was distinct from previously characterized CCAAT-binding proteins in both molecular size and binding properties. Fractions containing H1TF2 activity activated transcription in vitro only if programmed with an H1 DNA template carrying an intact H1TF2-binding site.

Characterization and purification of H1TF2, a novel CCAAT-binding protein that interacts with a histone H1 subtype-specific consensus element

1989 ◽  
Vol 9 (4) ◽  
pp. 1566-1575
Author(s):  
P Gallinari ◽  
F La Bella ◽  
N Heintz
Keyword(s):  
Cell Cycle ◽  
Biochemical Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gene Promoter ◽  
Molecular Size ◽  
Histone H1 ◽  
Nuclear Extracts ◽  
Definition Of

Definition of mechanisms regulating human histone H1 gene transcription during the cell cycle requires the isolation and biochemical characterization of protein factors which interact with specific promoter elements. Two distinct binding activities have been identified in nuclear extracts from HeLa cells and mapped within a 180-base-pair (bp) region of a cell cycle-regulated H1 gene promoter. H1TF1 bound to an H1-specific A + C-rich sequence (AC box), 100 bp upstream of the cap site; H1TF2 interacted with the H1 subtype-specific consensus element and was dependent on the presence of an intact CCAAT box for binding. H1TF2 was purified through a combination of ion-exchange and oligonucleotide affinity chromatographies. Analysis of purified fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and UV crosslinking showed that H1TF2 was a single polypeptide of 47 kilodaltons. This factor was distinct from previously characterized CCAAT-binding proteins in both molecular size and binding properties. Fractions containing H1TF2 activity activated transcription in vitro only if programmed with an H1 DNA template carrying an intact H1TF2-binding site.

scholarly journals A 70000-molecular-weight protein isolated from purified pig gastric mucus glycoprotein by reduction of disulphide bridges and its implication in the polymeric structure

1981 ◽  
Vol 197 (1) ◽  
pp. 155-162 ◽  
Author(s):  
J P Pearson ◽  
A Allen ◽  
S Parry
Keyword(s):  
Density Gradient ◽  
Protein Fraction ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Equilibrium Density ◽  
Gastric Mucus ◽  
Mucus Glycoprotein

The glycoprotein of pig gastric mucus has been isolated free of non-covalently bound protein as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and equilibrium density-gradient centrifugation. After reduction with 0.2 M-mercaptoethanol, protein was released from the glycoprotein, which consisted of a major 70000-mol.wt. component and a minor 60000-mol.wt. component. The 70000-mol.wt. protein fraction was separated from the reduced glycoprotein by either density-gradient centrifugation in CsCl or by gel filtration. Analysis of the 70000-mol.wt. protein fraction showed that, within the limits of the analysis, it was non-glycosylated, and its amino acid analysis was quite different from that of the reduced glycoprotein, which is high in serine, threonine and proline. There was a ratio of one 70000-mol.wt. protein per native glycoprotein molecule of 2 × 10(6) mol.wt. Dissociation of the native glycoprotein into glycoprotein subunits (5 × 10(5) mol.wt.) by reduction or proteolysis results in the release or hydrolysis respectively of the 70000-mol.wt. protein. A similar 70000-mol.wt. protein is demonstrated in human gastric mucus glycoprotein. A structural role for the proteins in these mucus glycoproteins is proposed.

scholarly journals Isolation and biochemical characterization of the tryptic fragments of bovine nasal-cartilage proteoglycan monomer of high buoyant density

1982 ◽  
Vol 203 (3) ◽  
pp. 683-689 ◽  
Author(s):  
H D Keiser ◽  
J B Adlersberg ◽  
H M Steinman
Keyword(s):  
Biochemical Characterization ◽  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Exchange Chromatography ◽  
Equilibrium Density ◽  
Nasal Cartilage ◽  
Cartilage Proteoglycan ◽  
Sepharose 4B

Relatively homogeneous fractions of proteoglycan fragments were prepared from tryptic digests of the 4M-guanidinium chloride extract of bovine nasal cartilage. Glycosaminoglycan-containing fragments were separated from non-proteoglycan contaminants by ion-exchange chromatography and fractionated by equilibrium density-gradient centrifugation under dissociative conditions. The fractions of highest buoyant density were chromatographed on a column of Sepharose 4B, digested with chondroitinase ABC and chromatographed on a column of Sepharose 6B, yielding two distinct fractions: fraction B/6B-4 contained fragments from the chondroitin sulphate-bearing region of the proteoglycan monomer, and fraction B/6B-2 fragments from the keratan sulphate-rich region, most probably including a chondroitin sulphate-bearing monomer segment. By dansyl chloride analysis, fraction B/6B-2 had alanine and leucine as sole and fraction B/6B-4 had isoleucine and leucine as greatly predominant N-terminal amino acids, indicative of the relative homogeneity of these preparations of cartilage proteoglycan monomer fragments.

scholarly journals Interaction of autoantibodies to thyrotropin receptor with a hydrophilic subunit of the thyrotropin receptor

1985 ◽  
Vol 228 (1) ◽  
pp. 111-117 ◽  
Author(s):  
E Davies Jones ◽  
F A Hashim ◽  
Y Kajita ◽  
F M Creagh ◽  
P R Buckland ◽  
...  
Keyword(s):  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Water Soluble ◽  
Tsh Receptor ◽  
Human Thyroid ◽  
Thyrotropin Receptor ◽  
Freezing And Thawing ◽  
Sucrose Density Gradient Centrifugation ◽  
A Subunit

Reduction of human thyroid membranes with dithiothreitol caused the release of a water-soluble glycoprotein which neutralized the thyrotropin (TSH) receptor-binding and thyroid-stimulating activities of Graves‘ serum. Analysis of the protein by gel filtration and sucrose density gradient centrifugation allowed estimates of 3.45 nm for the Stokes’ radius, 3.6 S for the s20,w and 47 000 +/- 5000 (mean +/- S.D.; n = 4) for the Mr. The material released by dithiothreitol treatment could be crosslinked to 125I-labelled TSH coupled to N-hydroxysuccinimidyl 4-azidobenzoate (125I-HSAB-TSH), suggesting that it contained a component of the TSH receptor. Furthermore, analysis of the crosslinked material by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that it contained the TSH receptor A subunit (Mr 50 000). Several factors suggested therefore that the glycoprotein released by dithiothreitol treatment of human thyroid membranes was the TSH receptor A subunit. In particular, (a) both preparations were hydrophilic and were released from membranes by reduction, (b) they had similar Mr values and (c) both preparations crosslinked to 125I-HSAB-TSH. Material similar to the TSH receptor A subunit was released from thyroid membranes by treatment with papain, probably as a result of cleavage of the receptor A subunit at a site close to the interchain disulphide bridge. A similar mechanism, involving thyroid proteinases, was probably involved in release of material with similar properties to the TSH receptor A subunit during freezing and thawing of human thyroid homogenates.

scholarly journals Parasporal Body Formation via Overexpression of the Cry10Aa Toxin of Bacillus thuringiensis subsp. israelensis, and Cry10Aa-Cyt1Aa Synergism

2009 ◽  
Vol 75 (14) ◽  
pp. 4661-4667 ◽  
Author(s):  
Alejandro Hernández-Soto ◽  
M. Cristina Del Rincón-Castro ◽  
Ana M. Espinoza ◽  
Jorge E. Ibarra
Keyword(s):  
Bacillus Thuringiensis ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Microbial Control ◽  
Control Agent ◽  
Mass Spectrometry Analysis ◽  
Parasporal Crystal ◽  
Spectrometry Analysis ◽  
Crystal Complex

ABSTRACT Bacillus thuringiensis subsp. israelensis is the most widely used microbial control agent against mosquitoes and blackflies. Its insecticidal success is based on an arsenal of toxins, such as Cry4A, Cry4B, Cry11A, and Cyt1A, harbored in the parasporal crystal of the bacterium. A fifth toxin, Cry10Aa, is synthesized at very low levels; previous attempts to clone and express Cry10Aa were limited, and no parasporal body was formed. By using a new strategy, the whole Cry10A operon was cloned in the pSTAB vector, where both open reading frames ORF1 and ORF2 (and the gap between the two) were located, under the control of the cyt1A operon and the STAB-SD stabilizer sequence characteristic of this vector. Once the acrystalliferous mutant 4Q7 of B. thuringiensis subsp. israelensis was transformed with this construct, parasporal bodies were observed by phase-contrast microscopy and transmission electron microscopy. Discrete, ca. 0.9-μm amorphous parasporal bodies were observed in the mature sporangia, which were readily purified by gradient centrifugation once autolysis had occurred. Pure parasporal bodies showed two major bands of ca. 68 and 56 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. These bands were further characterized by N-terminal sequencing of tryptic fragments using matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, which identified both bands as the products of ORF1 and ORF2, respectively. Bioassays against fourth-instar larvae of Aedes aegypti of spore-crystal complex and pure crystals of Cry10Aa gave estimated 50% lethal concentrations of 2,061 ng/ml and 239 ng/ml, respectively. Additionally, synergism was clearly detected between Cry10A and Cyt1A, as the synergistic levels (potentiation rates) were estimated at 13.3 for the mixture of Cyt1A crystals and Cry10Aa spore-crystal complex and 12.6 for the combination of Cyt1A and Cry10Aa pure crystals.

scholarly journals Affinity purification and subunit structures of β-N-acetylhexosaminidases A and B from boar epididymis

1984 ◽  
Vol 219 (3) ◽  
pp. 1009-1015 ◽  
Author(s):  
H C Parkes ◽  
J L Stirling ◽  
P Calvo
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Gradient Gel Electrophoresis ◽  
Sepharose 4B ◽  
Electrophoretic Transfer ◽  
Peptide Maps

beta-N-Acetylhexosaminidase from boar epididymis was separated into two forms, A and B, on DEAE-cellulose. Both these forms were excluded from Sepharose S-200 and had apparent Mr values of 510 000 on gradient gel electrophoresis under non-denaturing conditions. Affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylam ine coupled to CNBr-activated Sepharose 4B was used to separate and purify beta-N-acetylhexosaminidases A and B that had specific activities of 115 and 380 mumol/min per mg of protein respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of denatured beta-N-acetylhexosaminidase A gave a single major component of Mr 67 000. beta-N-Acetylhexosaminidase B also had this component, and in addition had polypeptides of Mr 29 000 and 26 000. All these polypeptides were glycosylated. Antiserum to the B form precipitated form A from solution and reacted with the 67 000-Mr component or form A after electrophoretic transfer from sodium dodecyl sulphate/polyacrylamide gels to nitrocellulose sheets. The 67 000-Mr components of forms A and B yielded identical peptide maps when digested with Staphylococcus aureus V8 proteinase, and the 29 000-Mr and 26 000-Mr components in form B may be related to the 67 000-Mr polypeptide.

scholarly journals Effects of intramammary infection on whey proteinograms of sheep during lactation

2015 ◽  
Vol 35 (3) ◽  
pp. 230-236 ◽  
Author(s):  
Vânia F. Lemos ◽  
Eduardo L.S. Guaraná ◽  
José A.B. Afonso ◽  
José J. Fagliari ◽  
Paulo C. Silva ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Biochemical Characterization ◽  
Production Systems ◽  
Polyacrylamide Gel Electrophoresis ◽  
Whey Proteins ◽  
Sodium Dodecyl ◽  
Acid Glycoprotein ◽  
Milk Samples ◽  
Lactation Stage ◽  
Potential Biomarkers

The study aimed to identify potential biomarkers of mammary gland infection in Santa Inês sheep. Commercial flocks of sheep provided the same hygiene, sanitary, and nutritional management under semi-intensive production systems were monitored during the lactation stage-and assessed 15, 30, 60, and 90 days after delivery (through the end of lactation and weaning). The California Mastitis Test (CMT) was performed on the mammary glands. Milk was collected for bacterial examination and protein analysis. Bacterial culture and biochemical characterization of the samples were performed. Forty-two milk samples from healthy glands (negative CMT and bacterial testing) and 43 milk samples from infected glands (positive CMT and bacterial testing) taken at the predefined time points were assessed. A rennin solution was used to obtain the whey. The proteins analysis was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which allowed for the quantification of nine whey proteins produced in healthy glands: serum albumin, lactoferrin, IgA, IgG heavy-chain (IgG HC), IgG light-chain (IgG LC), total IgG (IgG HC + IgG LC), α-lactalbumin, β-lactoglobulin, protein with MW 15.000 Da, protein with MW 29.000 Da and eleven whey proteins secreted by infected glands, including haptoglobin and α-1-acid glycoprotein. A comparison of whey proteins between healthy and infected glands showed increases (P<0.05) in the secreted and total contents of all proteins, except for IgG LC and α-lactoalbumin. The most significant changes were observed in α-1-acid glycoprotein, lactoferrin and haptoglobin, which showed three-, five-, and seven-fold increases in secretion, respectively. This study showed that haptoglobin, α-1-acid glycoprotein, lactoferrin, albumin, and the IgA and IgG immunoglobulins may serve as potential biomarkers for mammary gland infection in sheep.

scholarly journals Characterization of proteins from the cytoskeleton of Giardia lamblia

1983 ◽  
Vol 59 (1) ◽  
pp. 81-103 ◽  
Author(s):  
R. Crossley ◽  
D.V. Holberton
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Giardia Lamblia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Molecular Weights

Proteins from the axonemes and disc cytoskeleton of Giardia lamblia have been examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. In addition to tubulin and the 30 X 10(3) molecular weight disc protein, at least 18 minor components copurify with the two major proteins in Triton-insoluble structures. The most prominent minor bands have the apparent molecular weights of 110 X 10(3), 95 X 10(3) and 81 X 10(3). Protein of 30 X 10(3) molecular weight accounts for about 20% of organelle protein on gels. In continuous 25 mM-Tris-glycine buffer it migrates mostly as a close-spaced doublet of polypeptides, which are here given the name giardins. Giardia tubulin and giardin have been purified by gel filtration chromatography in the presence of sodium dodecyl sulphate. Well-separated fractions were obtained that could be further characterized. Both proteins are heterogeneous when examined by isoelectric focusing. Five tubulin chains were detected by PAGE Blue 83 dye-binding after focusing in a broad-range ampholyte gel. Giardin is slightly less acidic than tubulin. On gels it splits into four major and four minor chains with isoelectric points in the pI range from 5.8 to 6.2. The amino acid composition of the giardin fraction has been determined, and compared to Giardia tubulin and a rat brain tubulin standard. Giardins are rich in helix-forming residues, particularly leucine. They have a low content of proline and glycine; therefore they may have extensive alpha-helical regions and be rod-shaped. As integral proteins of disc microribbons, giardins in vivo associate closely with tubulin. The properties of giardins indicate that in a number of respects - molecular size, charge, stoichiometry - their structural interaction with tubulin assemblies will be different from other tubulin-accessory protein copolymers studied in vitro.

Dna Synthesis in Purified Populations of Avian Erythroid Cells

1972 ◽  
Vol 10 (1) ◽  
pp. 27-46
Author(s):  
A. F. WILLIAMS
Keyword(s):  
Biochemical Characterization ◽  
Bone Marrow Cells ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Experimental System ◽  
Equilibrium Density ◽  
Polychromatic Erythrocytes ◽  
Type Size ◽  
Haemoglobin Content

By the use of equilibrium density-gradient centrifugation erythroblasts and early polychromatic erythrocytes have been isolated from avian anaemic bone marrow. Cells from both the unfractionated and purified preparations have been characterized in terms of their histological type, size, haemoglobin content and ability to synthesize DNA. Erythroblasts were the only cells to synthesize DNA and it appeared that their progeny, the polychromatic erythrocyte, failed to enter a new S phase. The experimental system described allows biochemical characterization of earlier stages of avian erythropoiesis than has previously been possible.

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