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 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 Polymorphism of Gli-D1 alleles of Kragujevac’s winter wheat cultivars (Triticum aestivum L.)

Genetika ◽  
2007 ◽  
Vol 39 (2) ◽  
pp. 273-282
Author(s):  
Desimir Knezevic ◽  
Aleksandra Novoselskaya-Dragovich
Keyword(s):  
Triticum Aestivum ◽  
Winter Wheat ◽  
Molecular Mass ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Triticum Aestivum L ◽  
Polyacrylamide Gel ◽  
Wheat Cultivars ◽  
Number Of Components ◽  
Winter Wheat Cultivars

Composition of gliadins encoded by Gli-D1 allele as well polymorphisms of Gli-D1 allele investigated in 25 wheat cultivars by using acid polyacrylamide gel electrophoresis. Electrophoregrams obtained by polyacrylamide gel electrophoresis were used for estimation variability of gliadin components and identification of gliadin blocks. Five gliadin blocks encoded by different alleles at Gli-D1 locus were apparently expressed and identified. Gliadin blocks differed according to number of components and their molecular mass. Variability of determined block components indicates that existing polymorphisms of gliadins alleles. Frequency of identified 5 alleles at Gli-D1 locus was in ratio from 4% to 52%. The highest frequency of b allele and the of g allele was found.

scholarly journals Identification of a 64 kDa heparan sulphate proteoglycan core protein from human lung fibroblast plasma membranes with a monoclonal antibody

1987 ◽  
Vol 247 (3) ◽  
pp. 765-771 ◽  
Author(s):  
H de Boeck ◽  
V Lories ◽  
G David ◽  
J J Cassiman ◽  
H van den Berghe
Keyword(s):  
Molecular Mass ◽  
Gel Electrophoresis ◽  
Human Lung ◽  
Core Protein ◽  
Heparan Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Apparent Molecular Mass ◽  
Lung Fibroblasts ◽  
Human Lung Fibroblasts ◽  
The Core

Human lung fibroblasts produce heparan sulphate proteoglycans (HSPG) that are associated with the plasma membrane. A monoclonal-antibody (Mab)-secreting hybridoma, S1, was produced by fusion of SP 2/0-AG 14 mouse myeloma cells with spleen cells from mice immunized with partially purified cellular HSPG fractions. The HSPG character of the material carrying the epitope recognized by Mab S1 was demonstrated by: (i) the co-purification of the S1 epitope with the membrane HSPG of human lung fibroblasts; (ii) the decrease in size of the material carrying the S1 epitope upon treatment with heparinase or heparitinase, and the resistance of this material to heparinase treatment after N-desulphation. The S1 epitope appears to be part of the core protein, since it was destroyed by proteinase treatment and by disulphide-bond reduction, but not by treatments that depolymerize the glycosaminoglycan chains and N-linked oligosaccharide chains. Polyacrylamide-gel electrophoresis of non-reduced heparitinase-digested membrane HSPG followed by Western blotting and immunostaining with Mab S1 revealed a single band with apparent molecular mass of 64 kDa. Membrane proteoglycans isolated from detergent extracts or from 4 M-guanidinium chloride extracts of the cells yielded similar results. Additional digestion with N-glycanase lowered the apparent molecular mass of the immunoreactive material to 56 kDa, suggesting that the core protein also carries N-linked oligosaccharides. Fractionation of 125I-labelled membrane HSPG by immuno-affinity chromatography on immobilized Mab S1, followed by heparitinase digestion and polyacrylamide-gel electrophoresis of the bound material, yielded a single labelled band with apparent molecular mass 64 kDa. Treatment with dithiothreitol caused a slight increase in apparent molecular mass, suggesting that the core protein of this membrane proteoglycan of a single subunit containing (an) intrachain disulphide bond(s).

scholarly journals Identification of four extracellular-matrix enamel proteins during embryonic-rabbit tooth-organ development

1977 ◽  
Vol 163 (3) ◽  
pp. 591-603 ◽  
Author(s):  
H L Guenther ◽  
R D Croissant ◽  
S E Schonfeld ◽  
H C Slavkin
Keyword(s):  
Molecular Weight ◽  
Extracellular Matrix ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Zealand White Rabbit ◽  
Polyacrylamide Gel ◽  
Enamel Matrix ◽  
Matrix Mineralization ◽  
Enamel Proteins

1. Investigations were designed to identify the proteins which characterize the ameloblast phenotype, and to determine to what extent these extracellular-matrix proteins were degraded as a function of enamel matrix mineralization and maturation. 2. The identification of enamel proteins was based on comparisons between the electrophoretic patterns of enamel-containing and non-enamel-containing matrix extracts isolated from specific regions within 26-day embryonic New Zealand White rabbit incisor and molar tooth organs. 3. Since enamel proteins become mineralized on secretion, matrix specimens were demineralized in cold 5% (w/v) trichloroacetic acid, extracted with buffered 6M-urea and reduced with mercaptoethanol, and then the solubilized proteins were fractionated by urea/polyacrylamide-gel electrophoresis. 4. Three enamel-specific electrophoretic components were identified in newly secreted enamel-matrix specimens and this number increased as a function of mineralization and maturation. 5. Antibodies were prepared against embryonic rabbit extracellular matrix containing enamel. Comparison etween immunoelectrophoretic patterns demonstrated that two of the three enamel components were antigenic. 6. Polyacrylamide-gel electrophoresis in sodium dodecyl sulphate was used to identify four enamel proteins of mol.wts. (1) 65 000 (2) 58000 (3) 22 000 and (4) 20 000, localized within enamel matrix. Enamel proteins (1) and (3) were phosphorylated, whereas (2) and (4) did not contain detectable phosphate. Labelled proline, leucine, tryptophan and glucosamine were incorporated into each of the four enamel proteins extracted from tooth explants incubated in the presence of radioactive precursors for 6 h. Whereas four proteins were identified in newly secreted enamel matrix, the concentrations of high-molecular-weight proteins (1) and (2) were found to decrease and the number (greater than 10) and concentration of low-molecular-weight polypeptides increased as a function of advanced enamel-matrix mineralization and maturation.

scholarly journals An extremely thermostable extracellular proteinase from a strain of the archaebacterium Desulfurococcus growing at 88° C

1987 ◽  
Vol 247 (1) ◽  
pp. 121-133 ◽  
Author(s):  
D A Cowan ◽  
K A Smolenski ◽  
R M Daniel ◽  
H W Morgan
Keyword(s):  
Molecular Mass ◽  
Gel Electrophoresis ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Permeation Chromatography ◽  
Polyacrylamide Gel ◽  
British Library ◽  
Extracellular Proteinase ◽  
Hydrophobic Residues ◽  
The Stability

An organism growing at 88 degrees C that closely resembles Desulfurococcus mucosus produced a single extracellular proteinase. We have purified this enzyme and carried out a preliminary characterization. The proteinase, which is a serine-type enzyme, had a molecular mass of 52,000 Da by SDS/polyacrylamide-gel electrophoresis, but only 10,000-13,000 Da by gel-permeation chromatography. Molecular mass values from sucrose-gradient centrifugation were of the same order as those from SDS/polyacrylamide-gel electrophoresis. It had an isoelectric point of 8.7, and was inhibited by di-isopropyl phosphorofluoridate, phenylmethanesulphonyl fluoride and chymostatin. Substrate-specificity studies suggested a possible preference for hydrophobic residues on the C-terminal side of the splitting point. The thermostability of this enzyme is probably greater than any other reported proteinase (t1/2 at 95 degrees C, 70-90 min; t1/2 at 105 degrees C, 8-9 min). Ca2+ chelation does not appear to be implicated in stabilization of the protein structure. The stability of the Desulfurococcus proteinase was not greatly affected by the presence of reducing reagents (e.g. dithiothreitol), some chaotropic agents (e.g. NaSCN) and some detergents, but activity was lost rapidly at 95 degrees C in the presence of the oxidizing agent NaBO3. Proteolytic activity was readily detected at temperatures up to and including 125 degrees C, although denaturation was very rapid above 115 degrees C. A number of Figures supporting some of the findings reported in this paper have been deposited in supplement SUP 50137 (14 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1987) 241, 5.

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.

Does the B820 Subcomplex of the B880 Complex Retain Carotenoids?

1996 ◽  
Vol 51 (5-6) ◽  
pp. 309-318 ◽  
Author(s):  
Andrey Moskalenko ◽  
Olga Toropygina ◽  
Nina Kuznetsova
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Molecular Mass ◽  
Gel Electrophoresis ◽  
High Performance ◽  
Rhodospirillum Rubrum ◽  
Complex Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
The Common

Abstract A study is reported on the modification of the B880-RC assembly of Chromatium minutissimum during octyl-β-D -glucopyranoside/dodecyl-β-D -maltoside/Deriphat polyacrylamide gel electrophoresis followed by electroelution with dodecyl-β-D-maltoside and high performance liquid chromatography with octyl-β-D-glucopyranoside according to the method developed by Kerfeld et al. (Biochim. Biophys. Acta 1185, 193-202 [1994a]) for isolation of the B820 subcomplexes of Chromatium purpuratum. The B880-RC assembly of Chromatium minutissimum isolated by electrophoresis was contaminated by the B 800-850 complex. It was further separated into four components, three of which were in agreement with the cited work: (i) colorless contaminations, (ii) the B880-RC assembly, (iii) the B 800-850 complex. In contrast with Kerfeld et al. (1994a), the fourth band was a band of free pigments (Bchl or Bchl-t-carotenoids) which had the same molecular mass as the B820 subcomplex of Chromatium purpuratum. For comparison, the B880-RC enriched fraction of Rhodospirillum rubrum modified by lyophilization in the presence of octyl-β-D-glucopyranoside with or w ithout carotenoids was separated by high performance liquid chromatography with octyl-β-D-glucopyranoside. The apparent molecular mass of the B820 subcomplex was 30 kD a for the sample without carotenoids and 245 kD a for that with carotenoids. The common principles of organization of the B880 complex, the interaction of the B 800- 850 complex with the B880-RC assembly, the participation of carotenoids in the stabilization of the B880 complex structure and the ability of different isolation steps to modify the structure of the B880 complex are discussed. It was concluded that there are other explanations for the presence of carotenoids in the B820 subcomplex. Hence, the question of whether the B820 subcomplex retains carotenoids remains open.

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