scholarly journals Properties of fractionated chondroitin sulphate from ox nasal septa

1971 ◽  
Vol 122 (4) ◽  
pp. 477-485 ◽  
Author(s):  
Åke Wasteson
Keyword(s):  
Molecular Weight ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Random Coil ◽  
Sedimentation Equilibrium ◽  
Gel Chromatography ◽  
Equilibrium Studies ◽  
The Individual ◽  
A Charge ◽  
The Relationship

1. Chondroitin sulphate was isolated from bovine nasal septa by precipitation with cetylpyridinium chloride after digestion of the tissue with papain. 2. The material was divided into two portions, one of which was partially degraded with testicular hyaluronidase. 3. Untreated and hyaluronidase-digested material were fractionated into a total of eleven subfractions by gel chromatography on Sephadex G-200 and Sephadex G-100 respectively. 4. Chemical analyses indicated that the composition of all the fractions was similar to that of chondroitin sulphate. However, electrophoresis revealed a charge-inhomogeneity in the low-molecular-weight fractions obtained after hyaluronidase digestion. 5. The physicochemical properties of the subfractions were investigated by sedimentation-velocity, diffusion and sedimentation-equilibrium studies, osmometry, viscometry and gel chromatography. The individual fractions were essentially monodisperse and showed molecular weights ranging from 2400 to 36000. 6. The relationship between the intrinsic viscosity and the molecular weight was [η]=5.0×10−6×M1.14, indicating that the chondroitin sulphate molecules assume a shape intermediate between that of a random coil and a stiff rod. 7. The relationship between the sedimentation constant and the molecular weight (>104) was s020,w=2.3×10−2×M0.44.

Some Physical Parameters of Succinyl-Coenzyme A Synthetase of Escherichia coli

1974 ◽  
Vol 52 (7) ◽  
pp. 594-598 ◽  
Author(s):  
Anita Krebs ◽  
William A. Bridger
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Coenzyme A ◽  
Apparent Molecular Weight ◽  
Random Coil ◽  
Sedimentation Equilibrium ◽  
Physical Parameters ◽  
Equilibrium Studies ◽  
Α Helix ◽  
Succinyl Coenzyme A Synthetase

A physical study of succinyl-coenzyme A synthetase of Escherichia coli has been conducted. The extinction coefficient for the enzyme at 280 nm [Formula: see text] has been evaluated by two independent methods and found to be equal to 4.9 ± 0.2. Sedimentation equilibrium studies show that there is a marked dependence of the apparent molecular weight upon the concentration of the enzyme. At concentrations above 1 mg/ml, the enzyme exists predominantly as an α2β2 tetramer of overall molecular weight near 140 000; at lower concentrations, a significant fraction of the enzyme dissociates to an αβ dimer. The circular dichroism spectrum of the enzyme suggests a high proportion of random coil structure, with small contributions of α-helix and β-structure.

scholarly journals Degradation of heparin in mouse mastocytoma tissue

1971 ◽  
Vol 125 (4) ◽  
pp. 1119-1129 ◽  
Author(s):  
Sören Ögren ◽  
Ulf Lindahl
Keyword(s):  
Molecular Weight ◽  
Potassium Chloride ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Supernatant Fraction ◽  
Gel Chromatography ◽  
Neutral Sugar ◽  
Pulse Labelling

1. Heparin was prepared from mouse mastocytoma tissue by mild procedures, including extraction of mast-cell granules with 2m-potassium chloride, precipitation of the extracted polysaccharide with cetylpyridinium chloride from 0.8m-potassium chloride and finally digestion of the isolated material with testicular hyaluronidase. The resulting product (fraction GEH) represented approx. 40% of the total heparin content of the tissue. 2. Fraction GEH was fractionated by gel chromatography on Sepharose 4B into three subfractions, with average molecular weights (¯Mw) of approx. 60000–70000 (highly polydisperse material), 26000 and 9000 respectively. Treatment of each of the subfractions with alkali or with papain did not affect their behaviour on gel chromatography. Amino acid and neutral sugar analyses indicated that the two low-molecular-weight fractions consisted largely of single polysaccharide chains lacking the carbohydrate–protein linkage region. It was suggested that these heparin molecules had been degraded by an endopolysaccharidase. 3. Pulse labelling in vivo of mastocytoma heparin with [35S]sulphate showed initial labelling of large molecules followed by a progressive shift of radioactivity toward fractions of lower molecular weight. Further, heparin-depolymerizing activity was demonstrated by incubating 35S-labelled heparin in vitro with a mastocytoma 10000g-supernatant fraction. Appreciable degradation of the polysaccharide occurred, as demonstrated by gel chromatography. In contrast, no depolymerization was observed on subjecting 14C-labelled chondroitin sulphate to the same procedure.

scholarly journals Attempted isolation of a heparin proteoglycan from bovine liver capsule

1970 ◽  
Vol 116 (1) ◽  
pp. 27-34 ◽  
Author(s):  
U. Lindahl
Keyword(s):  
Potassium Chloride ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Bovine Liver ◽  
Gel Chromatography ◽  
Dermatan Sulphate ◽  
Liver Capsule ◽  
Neutral Sugars ◽  
Degraded Material ◽  
Heparin Preparation

(1) Polysaccharides were isolated from bovine liver capsule by extraction with 2m-potassium chloride followed by precipitation from 0.8m-potassium chloride with cetylpyridinium chloride. Chondroitin sulphate was eliminated by digestion with hyaluronidase. The yield of heparin was approx. 40% of that obtained after extraction of the papain-digested tissue. (2) The macromolecular properties of the hyaluronidase-digested polysaccharide were studied by gel chromatography on Sephadex G-200 of the intact, as well as of the alkali-degraded, material. The results suggested the presence of single heparin chains in addition to a dermatan sulphate proteoglycan. (3) A purified heparin preparation was analysed for amino acids and neutral sugars. Xylose, galactose and serine were found in amounts corresponding to 0.1, 0.2, and 0.4 residue/polysaccharide chain (mol.wt. 7400), respectively. It is suggested that the isolated material had been degraded by a polysaccharidase with endo-enzyme properties.

scholarly journals Purification and properties of 6-phosphogluconate dehydrogenase from sheep liver

1969 ◽  
Vol 115 (4) ◽  
pp. 639-643 ◽  
Author(s):  
R. H. Villet ◽  
K. Dalziel
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sedimentation Velocity ◽  
Sedimentation Equilibrium ◽  
Equilibrium Studies ◽  
Phosphogluconate Dehydrogenase ◽  
Sheep Liver ◽  
Equilibrium Sedimentation ◽  
Purification And Properties

A method is described for the isolation of 6-phosphogluconate dehydrogenase from sheep liver. The product appears to be homogeneous in polyacrylamide-gel electrophoresis and in sedimentation-velocity and sedimentation-equilibrium studies in the ultracentrifuge. The molecular weight is estimated as 129000 from equilibrium sedimentation.

Physicochemical Studies of Conglutin γ, a Storage Globulin From Seeds of Lupinus angustifolius

1980 ◽  
Vol 7 (1) ◽  
pp. 1 ◽  
Author(s):  
RJ Blagrove ◽  
JM Gillespie ◽  
GG Lilley ◽  
EF Woods
Keyword(s):  
Molecular Weight ◽  
Guanidine Hydrochloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Optical Rotatory Dispersion ◽  
Sedimentation Equilibrium ◽  
Native Protein ◽  
Equilibrium Studies ◽  
Physicochemical Studies ◽  
Α Helix

Physicochemical studies are reported for conglutin �, the minor globulin isolated from seeds of L. angustifolius cv. Uniwhite. Isoelectric focusing of the native protein in polyacrylamide gel slabs resolved major and minor broad bands near pH 8.0 and 7.8 respectively. Following reduction of disulfide bonds with β-mercaptoethanol in 8 M urea, the smaller polypeptide chain of known sequence focused near pH 6.9 while the larger chain focused near pH 8.0. Sedimentation equilibrium studies showed that the major component in aqueous buffers at neutral pH is a hexamer of molecular weight 280 000 which dissociates to the monomer of molecular weight 47 000 at pH 4.8. The sequence molecular weight of the small subunit polypeptide is 16 517 [Elleman, T.C. (1977). Aust. J. Biol. Sci. 30, 33-45]. The molecular weights determined for the larger chain by sedimentation equilibrium or column chromatography in 6 M guanidine hydrochloride, and by dodecyl sulfate-polyacrylamide gel electrophoresis, were in the range 28 000-30 000. Optical rotatory dispersion and circular dichroism measurements have been used to establish the approximate proportions of α-helix (15%), β-structure (35%), β-turns (18%) and unordered regions (32%) in the native protein. The denaturation curve for guanidine hydrochloride and the proportions of α-helix (50%), β-turns (18%) and unordered regions (32%) in 80 % trifluoroethanol have been determined.

scholarly journals Sedimentation-equilibrium studies on the heterogeneity of two β-lactamases

1970 ◽  
Vol 118 (3) ◽  
pp. 467-474 ◽  
Author(s):  
P. H. Lloyd ◽  
A. R. Peacocke
Keyword(s):  
Molecular Weight ◽  
Diffusion Coefficients ◽  
Minor Component ◽  
Theoretical Solution ◽  
Sedimentation Equilibrium ◽  
Equilibrium Studies ◽  
Molecular Weights ◽  
A Minor

Solutions of crystalline β-lactamase I and β-lactamase II, prepared by Kuwabara (1970), were examined in the ultracentrifuge and their sedimentation coefficients, diffusion coefficients, molecular weights and heterogeneity determined. Each sample was shown to consist of a major component comprising at least 97% of the material and a minor component of much higher molecular weight. The molecular weights of the major components were 27800 for β-lactamase I and 35600 for β-lactamase II. Emphasis is placed on a straightforward practical way of analysing the sedimentation-equilibrium results on mixtures of two macromolecular components rather than on a strict theoretical solution. Appendices describe the theory of systems at both chemical and sedimentation equilibrium and the procedure for calculating the combined distribution of two components.

scholarly journals Purification and structural characterization of a cartilage matrix protein

1981 ◽  
Vol 197 (2) ◽  
pp. 367-375 ◽  
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.

IgE response in schistosomiasis japonica: characterization of a cercarial allergen in comparison with purified egg allergens

1997 ◽  
Vol 71 (3) ◽  
pp. 221-226 ◽  
Author(s):  
M. Owhashi ◽  
Y. Horii ◽  
A. Ishii
Keyword(s):  
Molecular Weight ◽  
Apparent Molecular Weight ◽  
Cross Reactivity ◽  
Schistosomiasis Japonica ◽  
Gel Chromatography ◽  
Con A ◽  
The Cross ◽  
Ige Response ◽  
The Relationship

AbstractThe relationship between the cercarial allergen and two previously isolated egg allergens (J1, J2) of Schistosoma japonicum was examined especially in terms of the cross-reactivity between them. Semi-purified cercarial allergen (JAC) was obtained from the crude extract of S. japonicum cercariae by gel chromatography on Sephadex G-200. The apparent molecular weight of JAC was estimated approximately as 60–100 kDa. JAC could bind to Con A-Sepharose, indicating its glycoprotein nature. Three groups of BALB/c mice were immunized with JAC, J1 or J2 using A1(OH)3 as adjuvant, and the cross-reactivity of each antiserum was examined by PCA. Anti-JAC, anti-Jl or anti-J2 serum was highly specific to the corresponding antigen. When IgE-ELISA of S. japonicum patient sera was performed using JAC, J1 or J2 as an antigen, the correlation between anti-J1 and anti-J2 (r = 0.78) was high, whereas the correlation between anti-JAC and anti-J1 (r = 0.27) or between anti-JAC and anti-J2 (r = 0.12) was low. These results suggest that most IgE epitopes on cercarial allergen are independent from those on egg allergens in S. japonicum.

scholarly journals A novel low-molecular weight chondroitin sulphate proteoglycan isolated from cartilage

1981 ◽  
Vol 197 (2) ◽  
pp. 355-366 ◽  
Author(s):  
D Heinegård ◽  
M Paulsson ◽  
S Inerot ◽  
C Carlström
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Chondroitin Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sodium Dodecyl ◽  
Partial Specific Volume ◽  
Gel Chromatography ◽  
Guanidinium Chloride ◽  
Cscl Density Gradient

Proteoglycans were isolated from cartilage by extraction with 4M-guanidinium chloride followed by direct centrifugation in 4M-guanidinium chloride/CsCl at a low starting density, 1.34 g/ml. N-Ethylmaleimide was included in the extraction solvent as a precaution against contamination of proteoglycans with unrelated proteins mediated by disulphide exchange. A novel, discrete, low-buoyant-density proteoglycan (1.40-1.35 g/ml) was demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Its proteoglycan nature was revealed by the shift in the molecular size observed on gel electrophoresis after treatment with chondroitinase ABC. The core protein was monodisperse. The proteoglycan was further purified by gel chromatography with and without addition of hyaluronate. The proteoglycan constitutes less than 2% (by weight) of the total extracted proteoglycans and is not capable of interacting with hyaluronate. The same proteoglycan was purified in larger quantities by sequential associative and dissociative CsCl-density-gradient centrifugation, zonal rate sedimentation in a sucrose gradient and gel chromatography on Sepharose CL-4B. The pure proteoglycan had a molecular weight of 76 300 determined by sedimentation-equilibrium centrifugation and an apparent partial specific volume of 0.59 ml/g. It contained about 25% protein (of dry weight) and had remarkably high contents of leucine and cysteine as compared with other proteoglycans. The proteoglycan contained two to three large chondroitin sulphate chains and some oligosaccharides.

Subunit Structure and some Properties of Pyruvate Kinase of Neurospora

1975 ◽  
Vol 53 (2) ◽  
pp. 109-119 ◽  
Author(s):  
M. Kapoor
Keyword(s):  
Molecular Weight ◽  
Pyruvate Kinase ◽  
Guanidine Hydrochloride ◽  
Gel Filtration ◽  
Sedimentation Equilibrium ◽  
Phosphoryl Group ◽  
Subunit Structure ◽  
Equilibrium Studies ◽  
Variable Extent ◽  
High Concentrations

Pyruvate kinase isolated from Neurospora and purified to homogeneity has been shown to be a tetramer of molecular weight around 242 000 by gel filtration studies and 239 000 daltons by sedimentation equilibrium measurements. The monomer produced by treatment with guanidine hydrochloride is found to be 51 000–52 000 daltons by sedimentation equilibrium studies; a molecular weight of 62 000 was determined for the monomer generated by SDS treatment by electrophoresis in SDS–polyacrylamide gels. The enzyme has an isoelectric point of 6.35–6.41. Substrate saturation kinetics of PEP show a variable extent of cooperativity depending upon the buffer ions employed in the assay. ADP is the most effective phosphoryl group acceptor, GDP and IDP being poor substitutes. A divalent cation, Mg2+, is required for activity. At low concentrations, Ca2+ acts as an activator of pyruvate kinase but it is inhibitory at high concentrations. Fructose 1,6-diphosphate is the most potent allosteric activator, fructose 6-phosphate being next in order of effectiveness. Valine is a powerful inhibitor. Phenylalanine, tyrosine, and tryptophan are without any effect individually, but their simultaneous presence results in a considerable activation. Alanine does not affect this enzyme appreciably.

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