Isolation and characterization of dermatan sulphate proteoglycan from human uterine cervix

Proteoglycans were extracted from human uterine cervix with 4 M-guanidinium chloride in the presence of proteinase inhibitors. They were purified by density-gradient centrifugation in 4 M-guanidinium chloride/CsCl (starting density 1.32 g/ml) followed by DEAE-cellulose and Sepharose chromatography. Only one polydisperse proteoglycan was found. s020,w was 2.1S and the weight-average molecular weight was 73 000 (sedimentation-equilibrium centrifugation) to 110 500 (light-scattering). The core protein was monodisperse, with an apparent molecular weight of 47 000. The proteoglycan contained about 30% protein and probably two or three glycosaminoglycan side chains per molecule. High contents of aspartate, glutamate and leucine were found. The glycan moiety of the proteoglycan was exclusively dermatan sulphate, with a co-polymeric structure with approximately equal quantities of iduronic acid- and glucuronic acid-containing disaccharides.

Download Full-text

Isolation and characterization of human cervical-mucus glycoproteins

Biochemical Journal ◽

10.1042/bj2110013 ◽

1983 ◽

Vol 211 (1) ◽

pp. 13-22 ◽

Cited By ~ 129

Author(s):

I Carlstedt ◽

H Lindgren ◽

J K Sheehan ◽

U Ulmsten ◽

L Wingerup

Keyword(s):

Density Gradient ◽

Proteinase Inhibitors ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Disc Electrophoresis ◽

Guanidinium Chloride ◽

Molecular Weights ◽

Isolation And Characterization ◽

Mucus Glycoproteins

Mucus glycoproteins (mucins) were extracted from human cervical pregnancy mucus by 6 M-guanidinium chloride in the presence of proteinase inhibitors. Purification was subsequently achieved by isopycnic density-gradient centrifugation in CsCl/ guanidinium chloride gradients. The purified macromolecules represented approx. 85% of the total and were devoid of nucleic acids and proteins, as judged by analytical density-gradient centrifugation, disc electrophoresis and u.v. spectroscopy. Sedimentation-velocity centrifugation revealed a single unimodal peak with S20,W 50.1S in 0.2M-NaCl and 37.0S in 6 M-guanidinium chloride. Molecular weights obtained by light-scattering were 9.7 × 10(6) and 5.9 × 10(6) in 0.2M-NaCl and 6 M-guanidinium chloride respectively. The chemical analyses were typical of those of epithelial mucins. The macromolecules contained approx. 20% (w/w) of protein, and 65% (w/w) was accounted for as carbohydrate. Serine and threonine constituted 32 mol/100 mol and proline 10 mol/100 mol of the amino acids. The major sugars found were N-acetylglucosamine (12.8%), N-acetylgalactosamine (9.7%), galactose (18.7%), sialic acid (15.0%) and fucose (7.5%).

Download Full-text

Isolation and characterization of chondroitin 6-sulfate proteoglycans present in the extracellular matrix of rabbit bone marrow

Blood ◽

10.1182/blood.v72.2.745.745 ◽

1988 ◽

Vol 72 (2) ◽

pp. 745-755 ◽

Cited By ~ 6

Author(s):

E Okayama ◽

K Oguri ◽

T Kondo ◽

M Okayama

Keyword(s):

Bone Marrow ◽

Soluble Fraction ◽

Proteinase Inhibitors ◽

Gradient Centrifugation ◽

Gel Filtration ◽

Sedimentation Equilibrium ◽

Isolation And Characterization ◽

Cscl Density Gradient ◽

Core Peptide ◽

Rabbit Bone

Abstract The authors' previous studies showed that the glycosaminoglycans present in rabbit bone marrow were composed of chondroitin 6-sulfate (79%) and hyaluronic acid (16%). Immunohistochemically the chondroitin 6-sulfate was demonstrated to be in bone marrow matrix constructing hematopoietic microenvironment. In this study the authors isolated and characterized these glycosaminoglycans in their macromolecular form (i.e., proteoglycans). Bone marrow of 3-month-old rabbits was defatted with organic solvents containing proteinase inhibitors at -20 degrees C, and proteoglycans were extracted from the defatted tissue with 4 mol/L guanidine HCl containing the proteinase inhibitors. After extensive dialysis of the extract against 7 mol/L urea, more than 90% of hexuronate-containing materials was recovered in the urea-soluble fraction. The proteoglycans were purified from the urea-soluble fraction by diethyl aminoethyl (DEAE)-Sephacel chromatography, CsCl density-gradient centrifugation, and Bio-Gel A-5m gel filtration, then were rechromatographed on DEAE-Sephacel and on Bio-Gel A-5m. The proteoglycans were separated into three molecular species with different mol wts that were assessed to be 46,000, 16,000, and 8,300 by sedimentation-equilibrium centrifugation. Amino acid analyses of these proteoglycans revealed that serine and glycine accounted for approximately 60% of the total amino acids common to the three proteoglycans. The glycosaminoglycan side chains of these proteoglycans were converted stoichiometrically into unsaturated 6-sulfated disaccharide by digestion with chondroitinase AC-II, indicating that they were fully sulfated chondroitin 6-sulfate. Their apparent mol wts were estimated by gel filtration on Bio-Gel A-0.5m to be 10,900, 14,400, and 7,700. Computation of these results, taken together with their biochemical composition, revealed that the largest proteoglycan, PG-II, consisted of four chains of the chondroitin 6-sulfate and the core peptide with mol wt of approximately 4,000. The smaller two proteoglycan subpopulations, PG-I and PG-III, consisted of a single glycosaminoglycan chain linked to a small peptide with mol wts of approximately 500 and 1,000, respectively.

Download Full-text

Isolation and characterization of chondroitin 6-sulfate proteoglycans present in the extracellular matrix of rabbit bone marrow

Blood ◽

10.1182/blood.v72.2.745.bloodjournal722745 ◽

1988 ◽

Vol 72 (2) ◽

pp. 745-755

Author(s):

E Okayama ◽

K Oguri ◽

T Kondo ◽

M Okayama

Keyword(s):

Bone Marrow ◽

Soluble Fraction ◽

Proteinase Inhibitors ◽

Gradient Centrifugation ◽

Gel Filtration ◽

Sedimentation Equilibrium ◽

Isolation And Characterization ◽

Cscl Density Gradient ◽

Core Peptide ◽

Rabbit Bone

The authors' previous studies showed that the glycosaminoglycans present in rabbit bone marrow were composed of chondroitin 6-sulfate (79%) and hyaluronic acid (16%). Immunohistochemically the chondroitin 6-sulfate was demonstrated to be in bone marrow matrix constructing hematopoietic microenvironment. In this study the authors isolated and characterized these glycosaminoglycans in their macromolecular form (i.e., proteoglycans). Bone marrow of 3-month-old rabbits was defatted with organic solvents containing proteinase inhibitors at -20 degrees C, and proteoglycans were extracted from the defatted tissue with 4 mol/L guanidine HCl containing the proteinase inhibitors. After extensive dialysis of the extract against 7 mol/L urea, more than 90% of hexuronate-containing materials was recovered in the urea-soluble fraction. The proteoglycans were purified from the urea-soluble fraction by diethyl aminoethyl (DEAE)-Sephacel chromatography, CsCl density-gradient centrifugation, and Bio-Gel A-5m gel filtration, then were rechromatographed on DEAE-Sephacel and on Bio-Gel A-5m. The proteoglycans were separated into three molecular species with different mol wts that were assessed to be 46,000, 16,000, and 8,300 by sedimentation-equilibrium centrifugation. Amino acid analyses of these proteoglycans revealed that serine and glycine accounted for approximately 60% of the total amino acids common to the three proteoglycans. The glycosaminoglycan side chains of these proteoglycans were converted stoichiometrically into unsaturated 6-sulfated disaccharide by digestion with chondroitinase AC-II, indicating that they were fully sulfated chondroitin 6-sulfate. Their apparent mol wts were estimated by gel filtration on Bio-Gel A-0.5m to be 10,900, 14,400, and 7,700. Computation of these results, taken together with their biochemical composition, revealed that the largest proteoglycan, PG-II, consisted of four chains of the chondroitin 6-sulfate and the core peptide with mol wt of approximately 4,000. The smaller two proteoglycan subpopulations, PG-I and PG-III, consisted of a single glycosaminoglycan chain linked to a small peptide with mol wts of approximately 500 and 1,000, respectively.

Download Full-text

α-Crystallin. The isolation and characterization of distinct macromolecular fractions

Biochemical Journal ◽

10.1042/bj1240337 ◽

1971 ◽

Vol 124 (2) ◽

pp. 337-343 ◽

Cited By ~ 144

Author(s):

Abraham Spector ◽

Lu-Ku Li ◽

Robert C. Augusteyn ◽

Arthur Schneider ◽

Thomas Freund

Keyword(s):

Molecular Weight ◽

Amino Acid ◽

Gel Filtration ◽

Deae Cellulose ◽

Chemical Difference ◽

Molecular Weights ◽

Isolation And Characterization ◽

Different Populations ◽

Molecular Weight Fractions

α-Crystallin was isolated from calf lens periphery by chromatography on DEAE-cellulose and gel filtration. Three distinct populations of macromolecules have been isolated with molecular weights in the ranges approx. 6×105−9×105, 0.9×106−4×106and greater than 10×106. The concentration of macromolecules at the molecular-weight limits of a population are very low. The members of the different populations do not appear to be in equilibrium with each other. Further, in those molecular-weight fractions investigated, no equilibrium between members of the same population was observed. The population of lowest molecular weight comprises 65–75% of the total material. The amino acid and subunit composition of the different-sized fractions appear very similar, if not identical. The only chemical difference observed between the fractions is the presence of significant amounts of sugar in the higher-molecular-weight fractions. Subunit molecular weights of approx. 19.5×103and 22.5×103were observed for all α-crystallin fractions.

Download Full-text

Extraction and purification of proteoglycans from mature bovine bone

Biochemical Journal ◽

10.1042/bj2240047 ◽

1984 ◽

Vol 224 (1) ◽

pp. 47-58 ◽

Cited By ~ 49

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.

Download Full-text

The macromolecular structure of human cervical-mucus glycoproteins. Studies on fragments obtained after reduction of disulphide bridges and after subsequent trypsin digestion

Biochemical Journal ◽

10.1042/bj2130427 ◽

1983 ◽

Vol 213 (2) ◽

pp. 427-435 ◽

Cited By ~ 77

Author(s):

I Carlstedt ◽

H Lindgren ◽

J K Sheehan

Keyword(s):

Proteinase Inhibitors ◽

Gradient Centrifugation ◽

Cervical Mucus ◽

Trypsin Digestion ◽

Radius Of Gyration ◽

Guanidinium Chloride ◽

Disulphide Bonds ◽

Tentative Model ◽

The Relationship ◽

Mucus Glycoproteins

Human cervical-mucus glycoproteins (mucins) were extracted with 6 M-guanidinium chloride in the presence of proteinase inhibitors and purified by isopycnic density-gradient centrifugation. The whole mucins (Mr approx. 10 × 10(6] were degraded into ‘subunits’ (Mr approx. 2 × 10(6] by reduction of disulphide bonds. Trypsin digestion of the ‘subunits’ produced glycopeptides with Mr approx. 380000, which appear to be rod-like with a length of approx. 105 nm. The relationship between the radius of gyration and the Mr value obtained by light-scattering for whole mucins, ‘subunits’ and ‘domains’ suggest that cervical-mucus glycoproteins are linear flexible macromolecules composed of, on the average, four or five ‘domains’/subunit and four subunits/whole mucin macromolecule. The shape-dependent particle scattering function for the whole mucins and the ‘subunits’ are in accordance with that of a linear flexible chain. No evidence for a branched or a star-like structure was found. A tentative model for cervical mucins is proposed.

Download Full-text

Purification and structural characterization of a cartilage matrix protein

Biochemical Journal ◽

10.1042/bj1970367 ◽

1981 ◽

Vol 197 (2) ◽

pp. 367-375 ◽

Cited By ~ 93

Author(s):

M Paulsson ◽

D Heinegård

Keyword(s):

Molecular Weight ◽

Matrix Protein ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Cartilage Matrix ◽

Sedimentation Equilibrium ◽

Intact Protein ◽

Gel Chromatography ◽

Guanidinium Chloride ◽

Cscl Density Gradient

The cartilage matrix protein is a major non-collagenous protein in bovine cartilage. It was purified from a 5 M-guanidinium chloride extract of bovine tracheal cartilage by sequential CsCl-density-gradient centrifugation, gel chromatography in guanidinium chloride and differential precipitation. The molecular weight of the intact protein is 148 000, determined by sedimentation-equilibrium centrifugation. It was dissociated to three subunits of molecular weight 52 000 by reduction of disulphide bonds. The cartilage matrix protein was insoluble in low-salt solutions and behaved abnormally on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The content of cysteine was high, whereas the contents of aromatic amino acids were low. The carbohydrate content was 3.9% (w/w). Glycopeptides obtained after papain digestion were heterogenous on gel chromatography. Asparagine/aspartic acid was enriched in the purified glycopeptides, indicating the presence of N-glycosidic linkages to protein.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj2420779 ◽

1987 ◽

Vol 242 (3) ◽

pp. 779-787 ◽

Cited By ~ 11

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.

Download Full-text

The Isolation and Characterization of the ATPase Inhibitory Protein (TN-I) from Bovine Cardiac Muscle

Canadian Journal of Biochemistry ◽

10.1139/o75-165 ◽

1975 ◽

Vol 53 (11) ◽

pp. 1207-1213 ◽

Cited By ~ 17

Author(s):

L. D. Burtnick ◽

W. D. McCubbin ◽

C. M. Kay

Keyword(s):

Molecular Weight ◽

Cardiac Muscle ◽

Calcium Binding ◽

Sodium Dodecyl ◽

Basic Amino Acid ◽

Sedimentation Equilibrium ◽

Amino Acid Residues ◽

Inhibitory Protein ◽

Isolation And Characterization

The inhibitory component of the troponin complex (TN-I) was purified from bovine cardiac muscle, using a combination of ion exchange and molecular exclusion chromatographies in the presence of urea. It has the ability to inhibit the Mg2+-activated ATPase (EC 3.6.1.3) of a synthetic cardiac actomyosin preparation and this inhibition is reversed by the addition of cardiac calcium binding component of troponin (TN-C). Conventional sedimentation equilibrium experiments suggest a molecular weight for cardiac TN-I of 22 900 ± 500. However, sodium dodecyl sulfate (SDS) gels indicate a molecular weight of 27 000 ± 1000. The mobility of TN-I on SDS gels may be anomalous due to the high proportion of basic amino acid residues in the protein. Cardiac TN-I and TN-C interact to form a tight complex, even in the presence of 6 M urea. The results of this study invite direct comparison with results published for rabbit skeletal TN-I.

Download Full-text