scholarly journals Absence of covalently linked core protein from newly synthesized hyaluronate

1982 ◽  
Vol 207 (3) ◽  
pp. 445-457 ◽  
Author(s):  
R M Mason ◽  
C d'Arville ◽  
J H Kimura ◽  
V C Hascall
Keyword(s):  
Core Protein ◽  
Cell Layer ◽  
Gradient Centrifugation ◽  
Primary Cultures ◽  
Guanidinium Chloride ◽  
Protein Pool ◽  
Cscl Density Gradient ◽  
Chain Synthesis ◽  
Covalently Linked ◽  
Zwitterionic Detergent

1. Primary cultures of chondrocytes from the Swarm rat chondrosarcoma were labelled with either [3H]glucosamine or [14C]glucosamine, and hyaluronate synthesized by the cells was isolated from the cell layer. Parallel cultures were labelled with either [3H]serine or [3H]lysine, and identical fractions were isolated from the cell layer. Some cultures were dual-labelled. 2. In cultures labelled with [3H]serine for between 30 min and 24 h and extracted with 4.0 M-guanidine, a procedure that solubilizes predominantly extracellular macromolecules, small amounts of [3H]serine-labelled molecules were found associated with the hyaluronate fraction purified from the extract by dissociative CsCl-density-gradient centrifugation and dissociative Sepharose CL-2B chromatography. About 75% of the [3H]serine-labelled molecules in the fraction were specifically associated with hyaluronate, since they could be removed by prior treatment with proteinase-free Streptomyces hyaluronidase. The association of the [3H]serine-labelled molecules with hyaluronate was non-covalent, since they could be separated from it by further centrifugation in CsCl density gradients containing 4 M-guanidinium chloride and a zwitterionic detergent. 3. In other experiments the cultures were extracted with a sequential zwitterionic-detergent/guanidinium chloride procedure that completely solubilized the cell layer and enabled fractions containing newly synthesized cell-associated hyaluronate to be isolated. Zwitterionic detergent was present throughout. No [3H]lysine was incorporated into these fractions, irrespective of whether the cultures were pulsed concurrently with [3H]lysine and [14C]glucosamine or sequentially with [3H]lysine to prelabel the protein pool (24 h) followed by [14C]-glucosamine to label hyaluronate (1 h). 4. The results show that newly synthesized hyaluronate is not associated with covalently bound protein, and suggest that chain synthesis is initiated by a mechanism other than on to a core protein. Small amounts of [3H]serine-labelled molecules are, however, non-covalently associated with extracellular hyaluronate. Their identity is at present unknown, but they are probably of low molecular weight.

scholarly journals Characterization and N-terminal sequence of human platelet proteoglycan

1988 ◽  
Vol 255 (3) ◽  
pp. 1007-1013 ◽  
Author(s):  
J P Périn ◽  
F Bonnet ◽  
P Maillet ◽  
P Jollès
Keyword(s):  
Core Protein ◽  
Proteinase Inhibitors ◽  
Human Platelet ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Exchange Chromatography ◽  
Terminal Sequence ◽  
Guanidinium Chloride ◽  
Cscl Density Gradient ◽  
Yolk Sac Tumour

Human platelet proteoglycan (P.PG) was prepared from a 4 M-guanidinium chloride platelet extract in the presence of proteinase inhibitors. The purification procedure included CsCl-density-gradient centrifugation, DEAE-Sepharose CL-6B ion-exchange chromatography and f.p.l.c. on a Mono Q HR 5/5 column. P.PG was recovered as a polydisperse molecule, but the protein core appeared to be at least 90% homogeneous. This observation could be due to partial proteolysis of the core protein during extraction. The N-terminal sequence of the human P.PG core protein was determined up to residue 66 and was shown to be highly homologous to the propeptide of an embryonic rat yolk-sac tumour proteoglycan (PG19); the significance of this homology is discussed.

scholarly journals Rooster comb hyaluronate—protein, a non-covalently linked complex

1986 ◽  
Vol 235 (1) ◽  
pp. 117-123 ◽  
Author(s):  
C P Tsiganos ◽  
D H Vynios ◽  
D L Kalpaxis
Keyword(s):  
Protein Fraction ◽  
Core Protein ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Deae Cellulose ◽  
Solvent Mixture ◽  
Exchange Chromatography ◽  
Cscl Density Gradient ◽  
Covalently Linked

Hyaluronate from rooster comb was isolated by ion-exchange chromatography on DEAE-cellulose from tissue extracts and papain digests. The preparations were labelled with [14C]acetic anhydride and subjected to CsCl-density-gradient centrifugation in 4 M-guanidinium chloride in the presence and absence of 4% ZwittergentTM 3-12. A radioactive protein fraction was separated from the hyaluronate when the zwitterionic detergent was also present. The protein could also be separated from the glycosaminoglycan by chromatography on Sepharose CL-6B eluted with the same solvent mixture. The protein fraction contained three protein bands of Mr 15,000-17,000 as assessed by polyacrylamide-gel electrophoresis in 0.1% SDS, and seemed to lack lysozyme activity. No evidence of other protein or amino acid(s) covalently linked with the hyaluronate was obtained. The hyaluronate-protein complex may be re-formed upon mixing the components, the extent of its formation depending on the conditions used. The results show that, as in chondrosarcoma [Mason, d'Arville, Kimura & Hascall (1982) Biochem. J. 207, 445-457] and teratocarcinoma cells [Prehm (1983) Biochem. J. 211, 191-198] the rooster comb hyaluronate also is not linked covalently to a core protein.

scholarly journals Interactions between different corneal proteoglycans

1978 ◽  
Vol 173 (3) ◽  
pp. 935-939 ◽  
Author(s):  
P Speziale ◽  
M S Speziale ◽  
L Galligani ◽  
C Balduini
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Original Sample ◽  
Chemical Analyses ◽  
Guanidinium Chloride ◽  
Keratan Sulphate ◽  
Cscl Density Gradient ◽  
Two Peaks ◽  
Fraction Density

Proteoglycans were extracted from bovine cornea with 4M-guanidinium chloride and purified by CsCl-density-gradient centrifugation. Under associative conditions two fractions were found: one capable of forming assemblies of high molecular weight and another lacking this property. The heavier fraction (density 1.59 g/ml) was eluted as a single retarded peak from Sepharose 2B, but on DEAE-Sephadex chromatography, gave two peaks: the first (eluted with 0.75 M-NaCl) contained mainly proteochondroitin sulphate and the second (eluted with 1.25 M-NaCl) mainly proteokeratan sulphate. Each of these proteoglycans was more retarded on Sepharose 2B than was the original sample from density-gradient centrifugation. Re-aggregation was obtained by recombination of the two fractions. The lighter fraction (density 1.44 g/ml), containing predominantly keratan sulphate chains, was eluted from DEAE-Sephadex as a single peak with 1.25 M-NaCl and was retarded on Sepharose 2B: this fraction was not able to form aggregates with proteochondroitin sulphate. Chemical analyses of the carbohydrate and protein moieties of the proteoglycans from DEAE-Sephadex confirmed that, in the cornea, different subunits are present with characteristic aggregation properties and hydrodynamic volumes.

scholarly journals Extraction and purification of proteoglycans from mature bovine bone

1984 ◽  
Vol 224 (1) ◽  
pp. 47-58 ◽  
Author(s):  
A Franzén ◽  
D Heinegård
Keyword(s):  
Core Protein ◽  
Proteinase Inhibitors ◽  
Extraction Procedure ◽  
Deae Cellulose ◽  
Bovine Bone ◽  
Guanidinium Chloride ◽  
Extraction Solvent ◽  
Extraction And Purification ◽  
Cscl Density Gradient ◽  
Mineralized Matrix

Proteoglycans were extracted in good yields from the mineralized matrix of ground bovine bone, by using a two-step extraction procedure. Proteoglycans (8% of total), not associated with the bone mineral, were extracted at − 20 degrees C with 4M-guanidinium chloride containing proteinase inhibitors. Proteoglycans associated with the mineral, which accounted for 60% of the total, were then solubilized when EDTA was added to the extraction solvent. They were fractionated and purified in the presence of 4M-guanidinium chloride by CsCl-density-gradient centrifugations followed by chromatography on Sepharose CL-4B. Further purification was obtained by chromatography on DEAE-cellulose and hydroxyapatite in the presence of 7 M-urea. Three populations of proteoglycans and additional glycosaminoglycan peptides were obtained. The molecular dimensions of both intact molecules and of their side chains as well as their amino acid composition were different, indicating that they represent separate molecular entities. The main proteoglycan self-aggregated in the absence of 4M-guanidinium chloride or 7 M-urea, a property that was abolished when the proteoglycan core protein was fragmented.

scholarly journals The degradation of cartilage proteoglycans by tissue proteinases. Proteoglycan structure and its susceptibility to proteolysis

1977 ◽  
Vol 167 (3) ◽  
pp. 629-637 ◽  
Author(s):  
P J Roughley ◽  
A J Barrett
Keyword(s):  
Ionic Strength ◽  
Cathepsin D ◽  
Core Protein ◽  
Degradation Products ◽  
Gradient Centrifugation ◽  
Limited Proteolysis ◽  
Extraction Procedure ◽  
Cathepsin G ◽  
Nasal Cartilage ◽  
Cscl Density Gradient

1. Proteoglycan was obtained from bovine nasal cartilage by a procedure involving sequential extraction with a low-ionic-strength KCl solution, then a high-ionic-strength CaCl2 solution. Purification was by CsCl-density-gradient centrifugation. 2. The CaCl2- extracted proteoglycan was subjected to proteolytic degradation by papain, trypsin, cathepsin D, cathepsin B, lysosomal elastase or cathepsin G. Degradation was allowed to proceed until no further decrease in viscosity was detectable. 3. The size and chemical composition of the final degradation products varied with the different proteinases. Cathepsin D and cathepsin G produced glycosaminoglycan-peptides of largest average size, and papain produced the smallest product. 4. The KCl-extracted proteoglycan was intermediate in molecular size and composition between the CaCl2-extracted proteoglycan and the largest final degradation products, and may have been formed by limited proteolysis during the extraction procedure. 5. It is postulated that the glycosaminoglycan chains are arranged in groups along the proteoglycan core protein. Proteolytic cleavage between the groups may be common to the majority of proteinases, whereas clevage within the groups is dependent on the specificity of each individual proteinase.

scholarly journals Physical properties of chondroitin sulphate/dermatan sulphate proteoglycans from bovine aorta

1986 ◽  
Vol 240 (2) ◽  
pp. 575-583 ◽  
Author(s):  
R Kapoor ◽  
C F Phelps ◽  
T N Wight
Keyword(s):  
Uronic Acid ◽  
Glucuronic Acid ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Polymer Concentration ◽  
Dermatan Sulphate ◽  
Guanidinium Chloride ◽  
Structural Units ◽  
The Core ◽  
Covalently Linked

Bovine aortic chondroitin sulphate/dermatan sulphate proteoglycans (PG-25, PG-35 and PG-50) were differentially precipitated with ethanol and analysed by a variety of chemical and physical techniques. The glycosaminoglycan chains of PG-25 and PG-35 contained a mixture of glucuronic acid and iduronic acid, whereas the uronic acid component of PG-50 was primarily glucuronic acid. In addition, various amounts of oligosaccharides containing small amounts of mannose, a galactose/hexosamine ratio of 1:1 and an absence of uronic acid were covalently linked to the core protein of all proteoglycans. The weight-average Mr (Mw) values of the proteoglycans determined by light-scattering in 4 M-guanidinium chloride were 1.3 × 10(6) (PG-25), 0.30 × 10(6) (PG-35) and 0.88 × 10(6) (PG-50). The s0 values of the proteoglycans were distributed between 7 and 8 S, and the reduced viscosities, eta sp./c, of all proteoglycans were dependent on the shear rate and polymer concentration. Electron microscopy of spread molecules revealed that PG-25 contained small structural units that appeared to self-associate into large aggregates, whereas PG-35 and PG-50 appeared mainly as monomers consisting of a core with various numbers of side projections. Hyaluronic acid-proteoglycan complexes occurred only with a small proportion of the molecules present in PG-35, and their formation could be inhibited by oligosaccharides. These results suggest the presence in the aorta of subspecies of chondroitin sulphate and dermatan sulphate proteoglycans, which show large variations in their physicochemical and inter- and intra-molecular association properties.

scholarly journals Purification and partial characterization of a tumour-metastasis-associated high-Mr glycoprotein from rat 13762NF mammary adenocarcinoma cells

1987 ◽  
Vol 242 (3) ◽  
pp. 779-787 ◽  
Author(s):  
P A Steck ◽  
S M North ◽  
G L Nicolson
Keyword(s):  
Cellular Localization ◽  
Proteinase Inhibitors ◽  
Cell Clone ◽  
Gradient Centrifugation ◽  
Mammary Adenocarcinoma ◽  
Metastatic Tumour ◽  
Guanidinium Chloride ◽  
Tumour Metastasis ◽  
Adenocarcinoma Cells ◽  
Cscl Density Gradient

The expression of a high-Mr sialogalactoprotein (gp580) on rat 13762NF mammary adenocarcinoma cells was identified and correlated with spontaneous metastatic potential to colonize lung [Steck & Nicolson (1983) Exp. Cell Res. 147, 255-267]. Using a highly metastatic tumour-cell clone, MTLn3, we isolated and characterized gp580 from cells growing in vitro and in vivo in the mammary fat-pads of Fischer 344 rats. The glycoprotein was extracted with 4 M-guanidinium chloride/4% Zwittergent 3-12 solution in the presence of proteinase inhibitors. The extracts were then subjected to dissociative CsCl-density-gradient centrifugation, gel filtration on Sepharose CL-2B columns and ion-exchange chromatography on DEAE-Sephacel. The isolated glycoprotein possessed low electrophoretic mobility in SDS/polyacrylamide gels, and after desialylation bound 125I-labelled peanut agglutinin. Electrophoresis of gp580 in polyacrylamide-gradient gels resulted in a diffuse but homogeneous migrating band of Mr approx. 55,000. After removal of carbohydrate, gp580 was demonstrated to have a protein core of Mr approx. 150,000. The gp580 had a high density (1.430 g/ml) on isopycnic centrifugation in 4 M-guanidinium chloride and was resistant to most proteinases and other degradative enzymes, suggesting a mucin-like structure. Amino acid and carbohydrate analyses revealed that gp580 has high contents of serine, threonine, glutamic acid, aspartic acid, glucosamine and galactosamine; several acidic and neutral oligosaccharides were obtained from alkaline-borohydride digests. Cellular localization studies suggested that gp580 is associated mainly with the cell-surface and extracellular-matrix fractions of MTLn3 cells.

scholarly journals N-Glycosylation of Haloferax volcanii Flagellins Requires Known Agl Proteins and Is Essential for Biosynthesis of Stable Flagella

2012 ◽  
Vol 194 (18) ◽  
pp. 4876-4887 ◽  
Author(s):  
Manuela Tripepi ◽  
Jason You ◽  
Sevcan Temel ◽  
Özlem Önder ◽  
Dustin Brisson ◽  
...  
Keyword(s):  
Gradient Centrifugation ◽  
Haloferax Volcanii ◽  
Content Type ◽  
Glycosylation Sites ◽  
Cscl Density Gradient ◽  
Domains Of Life ◽  
Covalently Linked ◽  
Glycosylation Pathway ◽  
A Minor ◽  
And Function

ABSTRACTN-glycosylation, a posttranslational modification required for the accurate folding and stability of many proteins, has been observed in organisms of all domains of life. Although the haloarchaeal S-layer glycoprotein was the first prokaryotic glycoprotein identified, little is known about the glycosylation of other haloarchaeal proteins. We demonstrate here that the glycosylation ofHaloferax volcaniiflagellins requires archaeal glycosylation (Agl) components involved in S-layer glycosylation and that the deletion of anyHfx. volcaniiaglgene impairs its swimming motility to various extents. A comparison of proteins in CsCl density gradient centrifugation fractions from supernatants of wild-typeHfx. volcaniiand deletion mutants lacking the oligosaccharyltransferase AglB suggests that when the Agl glycosylation pathway is disrupted, cells lack stable flagella, which purification studies indicate consist of a major flagellin, FlgA1, and a minor flagellin, FlgA2. Mass spectrometric analyses of FlgA1 confirm that its three predicted N-glycosylation sites are modified with covalently linked pentasaccharides having the same mass as that modifying its S-layer glycoprotein. Finally, the replacement of any of three predicted N-glycosylated asparagines of FlgA1 renders cells nonmotile, providing direct evidence for the first time that the N-glycosylation of archaeal flagellins is critical for motility. These results provide insight into the role that glycosylation plays in the assembly and function ofHfx. volcaniiflagella and demonstrate thatHfx. volcaniiflagellins are excellent reporter proteins for the study of haloarchaeal glycosylation processes.

scholarly journals Isolation and some structural analyses of a proteodermatan sulphate from calf skin

1983 ◽  
Vol 213 (2) ◽  
pp. 289-296 ◽  
Author(s):  
T Nakamura ◽  
E Matsunaga ◽  
H Shinkai
Keyword(s):  
Core Protein ◽  
Gradient Centrifugation ◽  
Cetylpyridinium Chloride ◽  
Periodic Acid ◽  
Molar Ratio ◽  
Gel Chromatography ◽  
Dermatan Sulphate ◽  
Periodic Acid Schiff ◽  
Cscl Density Gradient ◽  
Calf Skin

A proteodermatan sulphate was isolated from 0.15 M-NaCl and 0.45 M-NaCl extracts of newborn-calf skin. The proteoglycan was separated from collagen and hyaluronic acid by precipitation with cetylpyridinium chloride and CsCl-density-gradient centrifugation. Further purification was performed by ion-exchange, affinity and molecular-sieve chromatography. The proteoglycan bound to concanavalin A-Sepharose in 1 M-NaCl. It gave a positive reaction with periodic acid/Schiff reagent and contained 8.3% of uronic acid. The dermatan sulphate, the only glycosaminoglycan component, was composed of 74% iduronosylhexosamine units and 26% glucuronosylhexosamine units. The Mr was assessed to be 15000-20000 by gel chromatography. The core protein was found to be a sialoglycoprotein that had O-glycosidic oligosaccharides with N-acetylgalactosamine at the reducing termini. The molar ratio of oligosaccharide chains to dermatan sulphate was approx. 3:1. From these results the proposed structure of proteodermatan sulphate is: one dermatan sulphate chain (average Mr 17500), three O-glycosidic oligosaccharide chains and probably N-glycosidic oligosaccharide chain(s) bound to one core-protein molecule (Mr 55000).

scholarly journals Abnormal synthesis of cartilage-characteristic proteoglycan in azaserine-induced micromelial limbs

1989 ◽  
Vol 261 (2) ◽  
pp. 627-635 ◽  
Author(s):  
A Honda ◽  
I Tsuboi ◽  
K Kimata ◽  
Y Hirabayashi ◽  
K Yamada ◽  
...  
Keyword(s):  
Core Protein ◽  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Immunological Methods ◽  
Chick Embryos ◽  
In Ovo ◽  
Hind Limbs ◽  
Limb Morphogenesis ◽  
Indirect Immunofluorescence Technique ◽  
Chain Synthesis

Administration of azaserine (250 micrograms) to day-4 chick embryos in ovo was shown to induce micromelial limbs. In the present study, biosynthesis of cartilage-characteristic proteoglycan H (PG-H) as an index of limb chondrogenesis was examined in normal and micromelial hind limbs from day-7 chick embryos by biochemical and immunological methods. (1) Metabolic labelling of the micromelial limbs with [6-3H]-glucose and [35S]sulphate, followed by analysis of labelled proteoglycans by glycerol-density-gradient centrifugation under dissociative conditions, showed a marked reduction in PG-H synthesis. (2) PG-H synthesized by micromelial limbs differed from that synthesized by normal limbs in possessing a slower sedimenting velocity and much lower amounts of chondroitin sulphates. (3) The amount of PG-H core protein in micromelial limbs was significantly decreased to about 19% on a per limb basis and about 42% on a per DNA basis of that in normal limbs, as determined by e.l.i.s.a. (4) The transition from PG-M to PG-H during limb formation was retarded in micromelial limbs as judged by an indirect immunofluorescence technique using antibodies against PG-M and PG-H. (5) The deficiency of incorporation of labelled glucose into chondroitin sulphate chains of PG-H in micromelial limbs was partially restored by using [6-3H]-glucosamine as a precursor, suggesting that the synthesis of UDP-N-acetylhexosamine, required for chondroitin sulphate chain synthesis of PG-H in micromelial limbs, was decreased. These results suggest that the reduction in the synthesis of PG-H as well as the production of an abnormal form of PG-H during a critical period of limb morphogenesis may be important factors in explaining the micromelia induced by azaserine.

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