scholarly journals Isolation and characterization of the native glycoprotein from pig small-intestinal mucus

1981 ◽  
Vol 195 (1) ◽  
pp. 267-275 ◽  
Author(s):  
M Mantle ◽  
A Allen
Keyword(s):  
Nucleic Acid ◽  
Analytical Ultracentrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Intestinal Mucus ◽  
Isolation And Characterization ◽  
Average Maximum ◽  
Small Intestinal ◽  
Covalently Bound

Glycoprotein from pig small-intestinal mucus was isolated free of non-covalently bound protein and nucleic acid with a yield of over 60%. No non-covalently bound protein could be detected by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis or by equilibrium centrifugation in a density gradient of CsCl with 4 M-guanidinium chloride. The intrinsic viscosity and reduced viscosity of the glycoprotein preparations rose with the removal of non-covalently bound protein and nucleic acid from the glycoprotein, evidence that non-covalently bound protein does not contribute to the rheological properties of the glycoprotein in the mucus. The pure glycoprotein, in contrast with impure preparations, gelled at the same concentration of glycoprotein as that present in the gel in vivo. The glycoprotein was a single component, as judged by gel filtration and analytical ultracentrifugation. The distribution of sedimentation coefficients was polydisperse but unimodal with an s025,w of 14.5S and a molecular weight of 1.72 X 10(6). The chemical composition of the glycoprotein was 77% carbohydrate and 21% protein, 52% of which was serine, threonine and proline. The glycoprotein had a strong negative charge and contained 3.1% and 18.3% by weight ester sulphate and sialic acid respectively. The molar proportion of N-acetylgalactosamine was nearly twice that of any of the other sugars present, the glycoprotein had A and H blood-group activity and the average maximum length of the carbohydrate chains was deduced to be six to eight sugar residues.

scholarly journals Isolation and Characterization of Two Proteins fromMoraxella catarrhalis That Bear a Common Epitope

1998 ◽  
Vol 66 (9) ◽  
pp. 4374-4381 ◽  
Author(s):  
John C. McMichael ◽  
Michael J. Fiske ◽  
Ross A. Fredenburg ◽  
Deb N. Chakravarti ◽  
Karl R. VanDerMeid ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Bacterial Attachment ◽  
Vaccine Candidates ◽  
Isolation And Characterization ◽  
Sds Page

ABSTRACT The UspA1 and UspA2 proteins of Moraxella catarrhalisare potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350,000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100°C. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates.

Isolation and Characterization of J-chain from Bovine Colostral Immunoglobulin M

1975 ◽  
Vol 53 (9) ◽  
pp. 943-949 ◽  
Author(s):  
R. Komar ◽  
T. K. S. Mukkur
Keyword(s):  
Gel Electrophoresis ◽  
Fast Moving ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Immunoglobulin M ◽  
J Chain ◽  
Isolation And Characterization ◽  
Moving Band ◽  
Covalently Bound

Purified bovine colostral intact immunoglobulin M (IgM) exhibited the presence of an anodal, single, fast moving band (noncovalently bound form) when subjected to analytical polyacrylamide gel electrophoresis at an alkaline pH in urea. Reduced and alkylated or sulfitolysed bovine colostral IgM (devoid of the noncovalently bound form) also showed the presence of a similar band (covalently bound form). The molecular weight of both the covalently bound and noncovalently bound forms of the fast component was determined to be 16 500 by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. In addition, the non-covalently bound form of the fast-moving component was found to be antigenically identical to the covalently bound form. The noncovalently bound form sedimented as a single peak at 1.56 S. Antiserum against the fast-moving component precipitated neither bovine colostral IgG nor μ-chains and bovine serum albumin, but precipitated native or denatured intact IgM (devoid of the non-covalently bound form) and human J-chains and vice versa, thus permitting the fast-moving components to be classified as J-chains. Radioalkylation experiments revealed the presence of 9.7 sulfhydryl groups per mole, for both the covalently and non-covalently bound forms of bovine J-chain. The stoichiometry of J-chain, determined from the densitometric tracing of the reduced and alkylated bovine colostral IgM (devoid of the noncovalently bound J-chain) in stained analytical polyacrylamide gels, revealed the presence of one J-chain per IgM molecule. On the other hand the amount of non-covalently bound form of J-chain was determined to be 1.2 per molecule of IgM.

scholarly journals Isolation and Characterization of a Novel Sialoglycopeptide Promoting Osteogenesis from Gadus morhua Eggs

Molecules ◽  
2019 ◽  
Vol 25 (1) ◽  
pp. 156
Author(s):  
Zhiliang Hei ◽  
Meihui Zhao ◽  
Yingying Tian ◽  
Hong Chang ◽  
Xuanri Shen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Gadus Morhua ◽  
Bone Growth ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Resonance Spectroscopy ◽  
Isolation And Characterization

Gadus morhua eggs contain several nutrients, including polyunsaturated fatty acids, lecithin and glycoproteins. A novel sialoglycopeptide from the eggs of G. morhua (Gm-SGPP) was extracted with 90% phenol and purified by Q Sepharose Fast Flow (QFF) ion exchange chromatography, followed by S-300 gel filtration chromatography. Gm-SGPP contained 63.7% carbohydrate, 16.2% protein and 18.6% N-acetylneuraminic acid. High-performance size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that Gm-SGPP is a 7000-Da pure sialoglycopeptide. β-elimination reaction suggested that Gm-SGPP contained N-glycan units. Amino acid N-terminal sequence analysis indicated the presence of Ala-Ser-Asn-Gly-Thr-Gln-Ala-Pro amino acid sequence. Moreover, N-glycan was connected at the third Asn location of the peptide chain through GlcNAc. Gm-SGPP was composed of D-mannose, D-glucuronic acid and D-galactose. Fourier transform-infrared spectroscopy (FT-IR), 1H-nuclear magnetic resonance spectroscopy (1H-NMR) and methylation analysis were performed to reveal the structure profile of Gm-SGPP. In vitro results showed that the proliferation activity of MC3T3-E1 cells was significantly promoted by Gm-SGPP. In vivo data revealed that Gm-SGPP increased the calcium and phosphorus content of tibias and promoted longitudinal bone growth in adolescent rats.

Glycogen phosphorylase in fish muscle: demonstration of three interconvertible forms

1990 ◽  
Vol 258 (2) ◽  
pp. C344-C351 ◽  
Author(s):  
H. Schmidt ◽  
G. Wegener
Keyword(s):  
Glycogen Phosphorylase ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Muscular Activity ◽  
Fish Muscle ◽  
Kinetic Properties ◽  
Phosphorylase Activity ◽  
Tissue Extracts

White skeletal muscle of crucian carp contains a single isoenzyme of glycogen phosphorylase, which was purified approximately 300-fold to a specific activity of approximately 13 mumol.min-1.mg protein-1 (assayed in the direction of glycogen breakdown at 25 degrees C). Tissue extracts of crucian muscle produced three distinct peaks of phosphorylase activity when separated on DEAE-Sephacel. Peaks 1 and 3 were identified, in terms of kinetic properties and by interconversion experiments, as phosphorylase b and a, respectively. Peak 2 was shown to be a phospho-dephospho hybrid. The three interconvertible forms of phosphorylase were purified and shown to be dimeric molecules at 20 degrees C. At 5 degrees C, a and the hybrid tended to form tetramers. The Mr of the subunit was estimated to be 96,400 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hybrid is kinetically homogeneous, and its kinetic properties are intermediate between those of b and a forms. The b, hybrid, and a forms of phosphorylase can be isolated from rapidly frozen muscle of crucian but in different proportions, depending on whether fish were anesthetized or forced to muscular activity for 20 s. Muscle of anesthetized crucian had 36, 36, and 28% of phosphorylase b, hybrid, and a forms, respectively, whereas the corresponding values for exercised fish were 12, 37, and 51%. Results suggest that three interconvertible forms of phosphorylase exist simultaneously in crucian muscle and that hybrid phosphorylase is active in contracting muscle in vivo.

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.

scholarly journals The interrelations between high- and low-molecular-weight forms of normal and mutant (Krabbe-disease) galactocerebrosidase

1980 ◽  
Vol 189 (1) ◽  
pp. 9-15 ◽  
Author(s):  
Yoav Ben-Yoseph ◽  
Melinda Hungerford ◽  
Henry L. Nadler
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Krabbe Disease ◽  
Low Molecular Weight ◽  
Molecular Weight Form

Galactocerebrosidase (β-d-galactosyl-N-acylsphingosine galactohydrolase; EC 3.2.1.46) activity of brain and liver preparations from normal individuals and patients with Krabbe disease (globoid-cell leukodystrophy) have been separated by gel filtration into four different molecular-weight forms. The apparent mol.wts. were 760000±34000 and 121000±10000 for the high- and low-molecular-weight forms (peaks I and IV respectively) and 499000±22000 (mean±s.d.) and 256000±12000 for the intermediate forms (peaks II and III respectively). On examination by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the high- and low-molecular-weight forms revealed a single protein band with a similar mobility corresponding to a mol.wt. of about 125000. Antigenic identity was demonstrated between the various molecular-weight forms of the normal and the mutant galactocerebrosidases by using antisera against either the high- or the low-molecular-weight enzymes. The high-molecular-weight form of galactocerebrosidase was found to possess higher specific activity toward natural substrates when compared with the low-molecular-weight form. It is suggested that the high-molecular-weight enzyme is the active form in vivo and an aggregation process that proceeds from a monomer (mol.wt. approx. 125000) to a dimer (mol.wt. approx. 250000) and from the dimer to either a tetramer (mol.wt. approx. 500000) or a hexamer (mol.wt. approx. 750000) takes place in normal as well as in Krabbe-disease tissues.

scholarly journals Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Redistribution of l-[1,5,6-3H]-fucose-labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes in vivo

1979 ◽  
Vol 182 (1) ◽  
pp. 203-212 ◽  
Author(s):  
Andrea Quaroni ◽  
Katharina Kirsch ◽  
Milton M. Weiser
Keyword(s):  
Golgi Complex ◽  
Membrane Fraction ◽  
Surface Membrane ◽  
Sodium Dodecyl ◽  
Membrane Glycoproteins ◽  
Intestinal Villus ◽  
Microvillus Membrane ◽  
Small Intestinal ◽  
Small Intestinal Villus

The biogenesis of plasmalemma glycoproteins of rat small-intestinal villus cells was studied by following the incorporation of l-[1,5,6-3H]fucose, given intraperitoneally with and without chase, into Golgi, lateral basal and microvillus membranes. Each membrane fraction showed distinct kinetics of incorporation of labelled fucose and was differently affected by the chase, which produced a much greater decrease in incorporation of label into Golgi and microvillus than into lateral basal membranes. The kinetic data suggest a redistribution of newly synthesized glycoproteins from the site of fucosylation, the Golgi complex, directly into both lateral basal and microvillus membranes. The observed biphasic pattern of label incorporation into the microvillus membrane fraction may be evidence for a second indirect route of incorporation. The selective effect of the chase suggests the presence of two different pools of radioactive fucose in the Golgi complex that differ in (1) their accessibility to dilution with non-radioactive fucose, and (2) their utilization for the biosynthesis of membrane glycoproteins subsequently destined for either the microvillus or the lateral basal parts of the plasmalemma. The radioactively labelled glycoproteins of the different membrane fractions were separated by sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis and identified by fluorography. The patterns of labelled glycoproteins in Golgi and lateral basal membranes were identical at all times. At least 14 bands could be identified shortly after radioactive-fucose injection. Most seemed to disappear at later times, although one of them, which was never observed in microvillus membranes, increased in relative intensity. All but two of the labelled glycoproteins present in the microvillus membrane corresponded to those observed in Golgi and lateral basal membranes shortly after fucose injection. The patterns of labelled glycoproteins in all membrane fractions were little affected by the chase. These data support a flow concept for the insertion of most surface-membrane glycoproteins of the intestinal villus cells.

scholarly journals Aggregation equilibria of tyrosinase of Harding-Passey mouse melanoma

1985 ◽  
Vol 228 (1) ◽  
pp. 95-101 ◽  
Author(s):  
J C Garcia-Borron ◽  
F Solano ◽  
J L Iborra ◽  
J A Lozano
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Permeation Chromatography ◽  
Aqueous Environment ◽  
Fluorescence Studies ◽  
Mouse Melanoma ◽  
Low Protein ◽  
Tryptophan Residues ◽  
Sample Dilution

The purification of two isoenzymes of tyrosinase has been carried out in Harding-Passey mouse melanoma. One is found in the cytosol and the other one bound to melanosomes. Both migrate as single bands on sodium dodecyl sulphate/polyacrylamide gels, having an apparent Mr of 58 000. Solubilized particulate tyrosinase showed an aggregation equilibrium involving a monomer, tetramer, octamer and a high-Mr micellar form with Brij 35, the solubilizing agent. H.p.l.c. studies indicated a interconversion between those species, the monomer contribution increasing with the sample dilution. The tetramer and the octamer probably represent the predominant forms in vivo. Soluble tyrosinase showed a simpler aggregation equilibrium, involving two forms, monomer and tetramer, with the same interconversion pattern. Fluorescence studies suggested that tryptophan residues were exposed to the aqueous environment when tyrosinase was dissociated by dilution. Tyrosinase shows a tendency to aggregate, at low protein concentration, and a resistance to dissociation by urea or SDS so remarkable that gel-permeation chromatography in 4M-urea does not affect the equilibrium, and the band obtained on SDS/polyacrylamide-gel electrophoresis is a dimer.

scholarly journals High-molecular weight kininogen is present in cultured human endothelial cells: localization, isolation, and characterization

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1268-1276 ◽  
Author(s):  
F van Iwaarden ◽  
PG de Groot ◽  
JJ Sixma ◽  
M Berrettini ◽  
BN Bouma
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
High Molecular Weight ◽  
Light Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Endothelial Cells ◽  
Isolation And Characterization

Abstract The presence of high-molecular weight (mol wt) kininogen was demonstrated in cultured human endothelial cells derived from the umbilical cord by immunofluorescence techniques. Cultured human endothelial cells contain 58 +/- 11 ng (n = 16) high-mol wt kininogen/10(6) cells as determined by an enzyme-linked immunosorbent assay (ELISA) specific for high-mol wt kininogen. High-mol wt kininogen was isolated from cultured human endothelial cells by immunoaffinity chromatography. Nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that endothelial cell high-mol wt kininogen consisted of five protein bands with mol wts of 95,000, 85,000, 65,000, 46,000, and 30,000 daltons. Immunoblotting of the endothelial cell high-mol wt kininogen by using specific antisera against the heavy and light chain indicated that the 95,000-, 85,000-, and 65,000-dalton bands consisted of the heavy and light chain whereas the 46,000- and 30,000-dalton bands reacted only with the anti-light chain antiserum. Immunoprecipitation studies performed with lysed, metabolically labeled endothelial cells and monospecific antisera directed against high-mol wt kininogen suggested that high-mol wt kininogen is not synthesized by the endothelial cells. Endothelial cells cultured in high-mol wt kininogen-free medium did not contain high-mol wt kininogen. These studies indicate that endothelial cell high-mol wt kininogen was proteolytically cleaved in the culture medium and subsequently internalized by the endothelial cells. Binding and internalization studies performed with 125I-labeled, proteolytically cleaved, high-mol wt kininogen showed that endothelial cells can indeed bind and internalize proteolytically cleaved high-mol wt kininogen in a specific and saturable way.

scholarly journals Expression of a functional laminin receptor (alpha 6 beta 1, very late activation antigen-6) on human eosinophils

Blood ◽  
1993 ◽  
Vol 82 (9) ◽  
pp. 2872-2879 ◽  
Author(s):  
SN Georas ◽  
BW McIntyre ◽  
M Ebisawa ◽  
JL Bednarczyk ◽  
SA Sterbinsky ◽  
...  
Keyword(s):  
Divalent Cations ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Basement Membranes ◽  
Laminin Receptor ◽  
Receptor Interaction ◽  
Inflammatory Reactions ◽  
Adhesive Interactions ◽  
Activation Antigen

Abstract The mechanisms responsible for the accumulation of eosinophils at sites of allergic and other inflammatory reactions are unknown, but recent studies have implicated both eosinophil and endothelial adhesion molecules in this process. However, less well studied have been the adhesive interactions between eosinophils and the subendothelial basement membrane and interstitial connective tissues. To test the hypothesis that eosinophils might interact with extracellular matrix proteins, we analyzed purified human eosinophils for the expression and function of various beta 1 integrins. Using indirect immunofluorescence and flow cytometry, purified eosinophils from mildly allergic donors were found to consistently express the integrin subunits beta 1 (CD29), alpha 4 (CD49d, very late activation antigen [VLA]-4 alpha), and alpha 6 (CD49f, VLA-6 alpha). No significant expression of the alpha 1, alpha 2, alpha 3, alpha 5, or beta 4 subunits was detected. Platelet contamination of the eosinophil preparations was excluded by light microscopy and by the inability to detect expression of platelet glycoproteins alpha v, CD41b, and CD42b. Immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of eosinophils confirmed the expression of cell-surface beta 1, alpha 4, and alpha 6. Furthermore, eosinophils purified from allergic donors were shown to adhere to plate-bound laminin, but not to type 1 or type 4 collagen. Adhesion to laminin was concentration-dependent, required divalent cations, and was completely and specifically inhibited by the anti-alpha 6 monoclonal antibody (MoAb) GoH3 and by the anti-beta 1 MoAb 33B6. Interestingly, the anti-beta 1 MoAb 18D3 (which like 33B6 blocks eosinophil binding to VCAM-1) did not inhibit eosinophil adhesion to laminin, suggesting that there are functionally distinct epitopes on the beta 1 subunit. Eosinophils purified from 4 healthy, nonallergic donors also showed alpha 6-dependent adhesion to laminin, although these cells adhered less well. These studies establish the expression of alpha 6 beta 1 on human eosinophils and document its function as a laminin receptor. Interaction of eosinophil alpha 6 beta 1 with laminin, eg, in basement membranes, may contribute to the localization of these cells at inflammatory sites in vivo.

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