scholarly journals Four classes of cell-associated proteoglycans in suspension cultures of articular-cartilage chondrocytes

1986 ◽  
Vol 233 (3) ◽  
pp. 809-818 ◽  
Author(s):  
Y Sommarin ◽  
D Heinegård
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Cell Surface ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Suspension Cultures ◽  
Polyacrylamide Gel ◽  
Side Chains ◽  
Dermatan Sulphate ◽  
Core Proteins

The characteristics of cell-associated proteoglycans were studied and compared with those from the medium in suspension cultures of calf articular-cartilage chondrocytes. By including hyaluronic acid or proteoglycan in the medium during [35S]sulphate labelling the proportion of cell-surface-associated proteoglycans could be decreased from 34% to about 15% of all incorporated label. A pulse-chase experiment indicated that this decrease was probably due to blocking of the reassociation with the cells of proteoglycans exported to the medium. Three peaks of [35S]sulphate-labelled proteoglycans from cell extracts and two from the medium were isolated by gel chromatography on Sephacryl S-500. These were characterized by agarose/polyacrylamide-gel electrophoresis, by SDS/polyacrylamide-gel electrophoresis of core proteins, by glycosaminoglycan composition and chain size as well as by distribution of glycosaminoglycans in proteolytic fragments. The results showed that associated with the cells were (a) large proteoglycans, typical for cartilage, apparently bound to hyaluronic acid at the cell surface, (b) an intermediate-size proteoglycan with chondroitin sulphate side chains (this proteoglycan, which had a large core protein, was only found associated with the cells and is apparently not related to the large proteoglycans), (c) a small proteoglycan with dermatan sulphate side chains with a low degree of epimerization, and (d) a somewhat smaller proteoglycan containing heparan sulphate side chains. The medium contained a large aggregating proteoglycan of similar nature to the large cell-associated proteoglycan and small proteoglycans with dermatan sulphate side chains with a higher degree of epimerization than those of the cells, i.e. containing some 20% iduronic acid.

scholarly journals Dermatan sulphate proteoglycans of human articular cartilage. The properties of dermatan sulphate proteoglycans I and II

1989 ◽  
Vol 262 (3) ◽  
pp. 823-827 ◽  
Author(s):  
P J Roughley ◽  
R J White
Keyword(s):  
Articular Cartilage ◽  
Gel Electrophoresis ◽  
Core Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Human Articular Cartilage ◽  
Amino Acid Residues ◽  
Dermatan Sulphate ◽  
Sulphate Proteoglycan ◽  
Core Proteins

Dermatan sulphate proteoglycans were purified from juvenile human articular cartilage, with a yield of about 2 mg/g wet wt. of cartilage. Both dermatan sulphate proteoglycan I (DS-PGI) and dermatan sulphate proteoglycan II (DS-PGII) were identified and the former was present in greater abundance. The two proteoglycans could not be resolved by agarose/polyacrylamide-gel electrophoresis, but could be resolved by SDS/polyacrylamide-gel electrophoresis, which indicated average Mr values of 200,000 and 98,000 for DS-PGI and DS-PGII respectively. After digestion with chondroitin ABC lyase the Mr values of the core proteins were 44,000 for DS-PGI and 43,000 and 47,000 for DS-PGII, with the smaller core protein being predominant in DS-PGII. Sequence analysis of the N-terminal 20 amino acid residues reveals the presence of a single site for the potential substitution of dermatan sulphate at residue 4 of DS-PGII and two such sites at residues 5 and 10 for DS-PGI.

scholarly journals Cell-surface radioiodination with the sparingly soluble catalyst Iodogen. Difference between the dividing and non-dividing populations of rodent thymocytes

1981 ◽  
Vol 194 (1) ◽  
pp. 351-355 ◽  
Author(s):  
J G Salisbury ◽  
J M Graham
Keyword(s):  
Cell Surface ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Surface Proteins ◽  
New Method

The surface proteins of dividing and non-dividing subpopulations of rat and mouse thymocytes have been labelled by using a new method of radioiodination. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and autoradiography of the labelled proteins shows distinct differences in labelling between the mouse and rat cells and also, in the case of the rat, between the dividing and non-dividing populations.

scholarly journals The small dermatan sulphate proteoglycans synthesized by fibroblasts derived from skin, synovium and gingiva show tissue-related heterogeneity

1988 ◽  
Vol 256 (1) ◽  
pp. 35-40 ◽  
Author(s):  
H Larjava ◽  
J Heino ◽  
T Krusius ◽  
E Vuorio ◽  
M Tammi
Keyword(s):  
Extracellular Matrix ◽  
Molecular Mass ◽  
Gel Electrophoresis ◽  
Core Protein ◽  
Culture Media ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Gingival Fibroblasts ◽  
Dermatan Sulphate ◽  
Different Tissues

Dermatan sulphate proteoglycans (DSPGs) synthesized in the presence of 35SO4 were characterized in culture media of fibroblast lines obtained from skin, synovium, and gingiva. The molecular mass of DSPG varied from 95-130 kDa as estimated by SDS/polyacrylamide-gel electrophoresis. Gingival fibroblasts constantly produced larger DSPGs than skin fibroblasts. This was due to the larger dermatan sulphate (DS) chains, which also showed tissue-related heterogeneity in the distribution of 4- and 6-sulphated disaccharide units. The N-glycosylated cores (44 and 47 kDa) obtained following chondroitinase ABC treatment were of identical size in all tissues. The cores from the different tissues were also of the same size (38 kDa) when addition of the N-linked oligosaccharides was inhibited by tunicamycin or when they were removed by N-glycanase treatment. No evidence for low-molecular-mass sulphated oligosaccharides was found. All tissues contained two mRNA species (1.6 and 1.9 kb) for the DSPG core protein. These data suggest that the pattern of transferase activities involved in the construction of DS chains differs from one tissue to another. This variation may modulate the functions of DSPG in the extracellular matrix.

scholarly journals Effect of tunicamycin on epidermal glycoprotein and glycosaminoglycan synthesis in vitro

1981 ◽  
Vol 198 (2) ◽  
pp. 331-338 ◽  
Author(s):  
Ian A. King ◽  
Anne Tabiowo
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Cell Adhesion ◽  
Epidermal Cell ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Membrane Glycoproteins ◽  
Glycosaminoglycan Synthesis ◽  
Core Proteins

1. When pig ear skin slices were cultured for 18h in the presence of 1mug of tunicamycin/ml the incorporation of d-[3H]glucosamine into the epidermis, solubilized with 8m-urea/5% (w/v) sodium dodecyl sulphate, was inhibited by 45–55%. This degree of inhibition was not increased by using up to 5mug of tunicamycin/ml or by treating the skin slices with tunicamycin for up to 8 days. The incorporation of (U-14C)-labelled l-amino acids under these conditions was not affected by tunicamycin. Polyacrylamide-gel electrophoresis indicated that the labelling of the major glycosaminoglycan peak with d-[3H]glucosamine was unaffected, whereas that of the faster migrating glycoprotein components was considerably decreased in the presence of tunicamycin. 2. Subcellular fractionation indicated that tunicamycin specifically inhibited the incorporation of d-[3H]glucosamine but not of (U-14C)-labelled l-amino acids into particulate (mainly plasma-membrane) glycoproteins by about 70%. The labelling of soluble glycoproteins was hardly affected. Polyacrylamide-gel electrophoresis of the plasma-membrane fraction showed decreased d-[3H]glucosamine incorporation into all glycoprotein components, indicating that the plasma-membrane glycoproteins contained mainly N-asparagine-linked oligosaccharides. 3. Cellulose acetate electrophoresis of both cellular and extracellular glycosaminoglycans showed that tunicamycin had no significant effect on the synthesis of the major component, hyaluronic acid. However, the incorporation of both d-[3H]glucosamine and 35SO42− into sulphated glycosaminoglycans was inhibited by about 50%. This inhibition was partially overcome, at least in the cellular fraction, by 2mm-p-nitrophenyl β-d-xyloside indicating that tunicamycin-treated epidermis retained the ability to synthesize sulphated glycosaminoglycan chains. Tunicamycin may affect the synthesis and/or degradation of proteoglycan core proteins or the xylosyltransferase. 4. Electron-microscopic examination of epidermis treated with tunicamycin for up to 4 days revealed no significant changes in cell-surface morphology or in epidermal-cell adhesion. Either N-asparagine-linked carbohydrates play little role in epidermal-cell adhesion or more probably there is little turnover of these components in epidermal adhesive structures such as desmosomes and hemidesmosomes during organ culture.

scholarly journals Heparin-sepharose affinity chromatography for purification of bull seminal-plasma hyaluronidase

1979 ◽  
Vol 183 (3) ◽  
pp. 531-537 ◽  
Author(s):  
P N Srivastava ◽  
A A Farooqui
Keyword(s):  
Hyaluronic Acid ◽  
Affinity Chromatography ◽  
Seminal Plasma ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Single Band ◽  
National Formulary ◽  
Turnover Number

Bull seminal-plasma hyaluronidase was purified 180-fold by chromatography on concanvalin A-Sepharose, heparin Sepharose, Sephadex G-200 and Sephacryl S-200. With hyaluronic acid as the substrate, the specific activity and turnover number of purified hyaluronidase were 3.63 mumol/min per mg (104000 National Formulary units/mg of protein) and 214 min-1 (mol of product formed/mol of enzyme per min) respectively. Polyacrylamide-gel electrophoresis indicated that the purified enzyme migrated as a single band on 7.5 and 10% (w/v) gels at pH 4.3 and 5.3. Bull seminal-plasma hyaluronidase was markedly inhibited by hydroxylamine, phenylhydrazine and semicarbazide. Purified hyaluronidase (1.25 munits; 1 unit = 1 mumol of N-acetylglucosamine liberated/min at 37 degrees C) dispersed the cumulus clot of rabbit ova in 1 h at 22 degrees C.

scholarly journals AN ELECTROPHORETIC DIFFERENCE BETWEEN SURFACE AND SECRETED IGM OF MURINE SPLENOCYTES

1973 ◽  
Vol 138 (5) ◽  
pp. 1282-1287 ◽  
Author(s):  
Ulrich Melcher ◽  
Jonathan W. Uhr
Keyword(s):  
Cell Surface ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Murine Splenocytes

µ-chains on the surface of murine splenocytes are more heterogeneous on SDS-polyacrylamide gel electrophoresis than both secreted and intracellular µ-chains labeled with [3H]tyrosine. No difference in heterogeneity among cell surface, secreted, or intracellular L chains was detected. The possible role of carbohydrate in µ-chain heterogeneity is discussed.

scholarly journals Differentiation-related changes in glycoprotein synthesis by human keratinocytes

1986 ◽  
Vol 237 (2) ◽  
pp. 519-525 ◽  
Author(s):  
G P R Roberts ◽  
J Brunt
Keyword(s):  
Cell Surface ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Keratinocytes ◽  
Polyacrylamide Gel ◽  
Galactose Oxidase ◽  
Glycoprotein Synthesis ◽  
Surface Labelling ◽  
Triton X

Human keratinocytes were cultured in media in which the Ca2+ concentration controlled the stage of differentiation. In media containing less than 0.1 mM-Ca2+ keratinocytes grew as a monolayer, but in the presence of 2mM-Ca2+ the cells differentiated and formed stratified colonies. Glycoproteins of both stratified and unstratified cells were radiolabelled by metabolic incorporation of radioactive precursors and by cell-surface labelling using galactose oxidase/NaB3H4. The radiolabelled keratinocytes were extracted with 0.5% Triton X-100, and the glycoproteins in both the Triton X-100-soluble and Triton X-100-insoluble fractions were analysed by polyacrylamide-gel electrophoresis in the presence of SDS. Two Triton X-100-soluble glycoproteins with high Mr values (greater than 200,000) were major glycoproteins in stratified keratinocytes, but were present in only trace amounts in unstratified keratinocytes. Characterization of these glycoproteins by examination of the effect of tunicamycin on their synthesis and the effect of neuraminidase on their migration characteristics showed that they were cell-surface sialoglycoproteins containing O-glycosidically linked oligosaccharides. Analysis of the adherent cytoskeletons left after Triton X-100 extraction of stratified and unstratified keratinocytes revealed that a glycoprotein of Mr 184,000 was decreased in stratified keratinocytes. Incubation of unstratified keratinocytes in high-Ca2+ medium resulted in a rapid modification of the glycoprotein of Mr 184,000, and it is suggested that this event may be related to desmosome formation and stratification.

scholarly journals A rapid purification of bovine testicular hyaluronidase by chromatography on dermatan sulphate-substituted 1,6-diaminohexane–sepharose 4B

1981 ◽  
Vol 199 (2) ◽  
pp. 419-426 ◽  
Author(s):  
M Lyon ◽  
C F Phelps
Keyword(s):  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
High Yield ◽  
Dermatan Sulphate ◽  
Bovine Testicular Hyaluronidase ◽  
Sepharose 4B ◽  
Testicular Hyaluronidase

The binding of bovine testicular hyaluronidase to AH-Sepharose (1,6-diaminohexane--Sepharose) gels substituted with (1) dermatan sulphate, (2) desulphated dermatan sulphate, (3) heparin and (4) de-N/O-sulphated, re-N-acetylated heparin was investigated. Hyaluronidase was found to bind to (1) and (3), but not (2) and (4). On the basis of these observations a preparative scheme for the purification of testicular hyaluronidase was developed. This consisted of two steps: (i) chromatography on dermatan sulphate-substituted AH-Sepharose 4B; (ii) chromatography on acetylated AH-Sepharose 4B. This procedure gave hyaluronidase with a specific activity of 19.1 units (mumol/min)/mg in high yield. Polyacrylamide-gel electrophoresis at pH 4.3 revealed two components, both possessing hyaluronidase activity. Sodium dodecyl sulphate polyacrylamide-gel electrophoresis likewise revealed two close bands with approximate molecular weights of 61000 and 67200.

Contractile-tailed bacteriophages adsorb toEscherichia coliO128ab lipopolysaccharide that is altered by large plasmids to provide receptors and lipopolysaccharide heterogeneity within the serogroup

1995 ◽  
Vol 41 (2) ◽  
pp. 163-169
Author(s):  
M. L. Kalmokofff ◽  
D. E. Bradley
Keyword(s):  
Escherichia Coli ◽  
Silver Staining ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Coli Strain ◽  
Polyacrylamide Gel ◽  
Clinical Isolates ◽  
Side Chains ◽  
O Antigen ◽  
Large Plasmids

The verotoxigenic Escherichia coli strain H.I.8 (originally O128:B12, now not typeable) contained a ColB+M plasmid and two morphologically identical temperate bacteriophages (HI8A and HI8B). Both phages were O128ab specific, using the lipopolysaccharide O side chains of susceptible clinical isolates as receptors. SDS polyacrylamide gel electrophoresis with silver staining of O128ab lipopolysaccharide revealed four distinct types of ladder with different interband spacings. No specificity was found between ladder type and sensitivity to either phage. One of the numerous large plasmids present in O128ab isolates was found to modify the structure of the lipopolysaccharide O side chains to provide phage receptors.Key words: bacteriophage, plasmid, lipopolysaccharide, O antigen.

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