scholarly journals Self-association of dermatan sulphate proteoglycans from bovine sclera

1981 ◽  
Vol 197 (2) ◽  
pp. 483-490 ◽  
Author(s):  
L Cöster ◽  
L A Fransson ◽  
J Sheehan ◽  
I A Nieduszynski ◽  
C F Phelps
Keyword(s):  
Molecular Weight ◽  
Light Scattering ◽  
Guanidine Hydrochloride ◽  
Apparent Molecular Weight ◽  
Sedimentation Equilibrium ◽  
Gel Chromatography ◽  
Side Chains ◽  
Dermatan Sulphate ◽  
Molecular Weights ◽  
High Particle

1. Two proteodermatan sulphate fractions (I and II) from bovine sclera were studied by gel chromatography, light-scattering and ultracentrifugation under various conditions. 2. Gel chromatography of proteoglycans in the absence or presence of hyaluronate was performed under associative conditions. No effect on the elution profile was noted. 3. Ultracentrifugation experiments (sedimentation-velocity and sedimentation-equilibrium) with proteoglycan I and II in 6 M-guanidine hydrochloride gave molecular weights (Mw) of 160000-220000 and 70000-100000 respectively. As the protein contents were 45% and 60% respectively, it may be calculated that proteoglycan I contained four to five side chains, whereas proteoglycan II contained one or two. Sedimentation-equilibrium runs performed in 0.15 M-NaCl gave an apparent molecular weight (Mw) of 500000-800000 for proteoglycan I and 90000-110000 for proteoglycan II. 4. In light-scattering experiments both proteoglycans I and II yielded high particle weights in 0.15 M-NaCl (3.1 × 10(6) and 3.4 × 10(6) daltons respectively). In the presence of 6 M-guanidine hydrochloride the molecular weights decreased to 410000 and 130000 respectively. The particle weights in 0.15 M-NaCl were not altered by the addition of hyaluronate or hyaluronate oligosaccharides. 5. The dermatan sulphate side chains of scleral proteoglycans (L-iduronate/D-glucuronate ratio 7:13) gave a particle weight of 100000 daltons in 0.15 M-NaCl. In 1.00 M-KCl/0.02M-EDTA the molecular weight was 24000. Addition of free scleral dermatan sulphate chains to a solution of proteoglycan II promoted further multimerization of the macromolecule.

scholarly journals Isolation and characterization of dermatan sulphate proteoglycans from bovine sclera

1981 ◽  
Vol 193 (1) ◽  
pp. 143-153 ◽  
Author(s):  
L Cöster ◽  
L A Fransson
Keyword(s):  
Sodium Acetate ◽  
Glucuronic Acid ◽  
Guanidine Hydrochloride ◽  
Sodium Dodecyl ◽  
Gel Chromatography ◽  
Side Chains ◽  
Dermatan Sulphate ◽  
Specific Chemical ◽  
Protein Core ◽  
Isolation And Characterization

1. Proteoglycans were extracted from sclera with 4 M-guanidine hydrochloride in the presence of proteinase inhibitors and purified by ion-exchange chromatography and density-gradient centrifugation. 2. The entire proteoglycan pool was characterized by compositional analyses and by specific chemical (periodate oxidation) and enzymic (chondroitinases) degradations. The glycan moieties of the molecules were exclusively galactosaminoglycans (dermatan sulphate-chondroitin sulphate co-polymers). In addition, the preparations contained small amounts of oligosaccharides. 3. The scleral proteodermatan sulphates were fractionated into one larger (I) and one smaller (II) component by gel chromatography. Proteoglycan I was eluted in a more excluded position on gel chromatography in 0.5 M-sodium acetate than in 4.0 M-guanidine hydrochloride. Reduced and alkylated proteoglycan I was eluted in the same position (in 0.5 M-sodium acetate) as was the starting material (in 4.0 M-guanidine hydrochloride). The elution position of proteoglycan II was the same in both solvents. Proteoglycans I and II had s0 20,w values of 2.8 × 10(-13) and 2.2 × 10(-13) s respectively in 6.0 M-guanidine hydrochloride. 4. The two proteoglycans differed with respect to the nature of the protein core and the co-polymeric structure of their side chains. Also proteoglycan I contained more side chains than did proteoglycan II. The dermatan sulphate side chains of proteoglycan I were D-glucuronic acid-rich (80%), whereas those of proteoglycan II contained equal amounts of D-glucuronic acid and L-iduronic acid. Furthermore, the co-polymeric features of the side chains of proteoglycans I and II were different. The protein core of proteoglycan I was of larger size than that of proteoglycan II. The latter had an apparent molecular weight of 46 000 (estimated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis), whereas the former was greater than 100 000. In addition, the amino-acid composition of the two core preparations was different. 5. As proteoglycan I altered its elution position on gel chromatography in 4 M-guanidine hydrochloride compared with 0.5 M-sodium acetate it is proposed that a change in conformation or a disaggregation took place. If the latter hypothesis is favoured, aggregation may be due to self-association or mediated by an extrinsic molecule, e.g. hyaluronic acid.

LIGHT-SCATTERING STUDIES OF COPOLYMERS: I. EFFECT OF HETEROGENEITY OF CHAIN COMPOSITION ON THE MOLECULAR WEIGHT

1958 ◽  
Vol 36 (12) ◽  
pp. 1616-1626 ◽  
Author(s):  
W. Bushuk ◽  
H. Benoit
Keyword(s):  
Molecular Weight ◽  
Refractive Index ◽  
Light Scattering ◽  
Average Molecular Weight ◽  
Apparent Molecular Weight ◽  
Scattering Data ◽  
Refractive Index Increment ◽  
Molecular Weights ◽  
Chain Composition ◽  
Total Molecular Weight

The classical light-scattering theory for polymer solutions is extended to solutions of copolymers which may be polydisperse in chain composition as well as in molecular weight. It is shown that much too high molecular weights will result from light-scattering data for copolymers owing to fluctuations in chain composition; the magnitude of this effect increases rapidly as the absolute value of the refractive index increment approaches zero. The methods for determining the usual weight-average molecular weight and polydispersity of composition are described.The theory was tested with experimental results on: (1) a mixture of polystyrene and poly(methylmethacrylate), considered as a copolymer with the highest possible polydispersity of composition; (2) a high conversion, statistical copolymer of styrene and methylmethacrylate (copolymer I); and (3) a block copolymer of the same monomers (copolymer II). The apparent molecular weight for copolymer I (Mw = 1.83 × 105) varied markedly with refractive index increment in much the same way as the total molecular weight for the mixture. The molecular weight of copolymer II (Mw = 1.20 × 106) remained essentially constant in the same series of solvents. The polydispersities of composition obtained were 0.72 and 0.05 for copolymer I and II respectively compared with the maximum possible value equal to 1.0.

scholarly journals Isopycnic-centrifugation studies in caesium chloride and in caesium sulphate on dermatan sulphate proteoglycans from bovine sclera

1981 ◽  
Vol 199 (3) ◽  
pp. 581-589 ◽  
Author(s):  
John K. Sheehan ◽  
Ingemar Carlstedt ◽  
Lars Cöster ◽  
Anders Malmström ◽  
Lars-Ȧke Fransson
Keyword(s):  
Molecular Weight ◽  
Buoyant Density ◽  
Broad Band ◽  
Apparent Molecular Weight ◽  
Gel Chromatography ◽  
Analytical Ultracentrifuge ◽  
Dermatan Sulphate ◽  
Caesium Chloride ◽  
Mode 2 ◽  
Isopycnic Centrifugation

1. Two proteodermatan sulphate species from bovine sclera (fractions PG-I and PG-II) separable by gel chromatography were studied by isopycnic centrifugations in CsCl and Cs2SO4 both in an analytical and a preparative mode. 2. In CsCl, fraction PG-I formed a broad band at a density ρ=1.75g/ml whereas fraction PG-II banded sharply at ρ=1.64g/ml. However, in Cs2SO4, fraction PG-II banded at ρ=1.51g/ml and fraction PG-I at ρ=1.40g/ml, a reversal of the relative banding positions of the two species in CsCl. 3. Preparative isopycnic centrifugations in the two caesium salts permitted further subfractionation of fractions PG-I and PG-II. In both CsCl and Cs2SO4 fraction PG-I was split into subfractions that varied greatly in uronate/protein ratios but had very similar uronate composition. In contrast, isopycnic centrifugation of fraction PG-II in Cs2SO4 gave rise to subfractions with similar uronate/protein ratios but markedly different uronate composition (iduronate content, 88–42%). 4. Subfractions of fractions PG-I and PG-II obtained in preparative centrifugations in CsCl or Cs2SO4 were examined in the analytical ultracentrifuge. These subfractions banded at discrete positions in the gradient. Estimations of apparent molecular weight for these subfractions from data from the analytical isopycnic centrifugations gave values that were much higher (around 1×106) than were those obtained previously for their ‘monomeric’ states (fraction PG-I 160000–410000, and fraction PG-II 70000–130000). 5. In CsCl, fraction PG-I may be subfractionated according to the number of side chains in the molecule. Fraction PG-I increased its net solvation (i.e. lowered its buoyant density) to a larger extent than did fraction PG-II in going from CsCl to Cs2SO4 (i.e. from lower to higher water activity). It is proposed that the presence of large amounts of iduronate in fraction PG-II makes these molecules relatively less solvated in Cs2SO4. Thus the uronate composition may be an important factor in determining the banding position of proteodermatan sulphates in density-gradient centrifugations.

Subunit components of casein micelles from bovine, ovine, caprine and equine milks

1989 ◽  
Vol 56 (1) ◽  
pp. 61-68 ◽  
Author(s):  
Tomotada Ono ◽  
Hideaki Kohno ◽  
Satoshi Odagiri ◽  
Toshio Takagi
Keyword(s):  
Molecular Weight ◽  
Light Scattering ◽  
High Performance ◽  
Laser Light ◽  
Laser Light Scattering ◽  
The Other ◽  
Gel Chromatography ◽  
Casein Micelles ◽  
Molecular Weights ◽  
Combined Use

SummarySubunit components of ovine, caprine and equine casein micelles were separated by gel chromatography using a TSK-G4000SW high-performance column and the subunit components of the fractions analysed and compared with bovine casein. Molecular weights of the casein complexes were determined by the combined use of high-performance gel chromatography and low-angle laser light scattering. The caprine and ovine caseins were separated into three peaks (F2, F3 and F4) which were similar to those of bovine casein with respect to composition and molecular weight (500, 100 and 23 K). These F2, F3 and F4 peaks consisted mainly of αs- and κ-casein, αs- and β-casein and β-casein respectively. The equine casein was separated into two components corresponding to F3 and F4 of bovine casein. These F3 and F4 peaks consisted mainly of αs- and β-casein and β-casein respectively. The molecular weight of equine F3 (850 K) was different from that of the other three species. The contents of F2 and F4 in these caseins were dependent on the contents of κ-casein and β-casein respectively.

Soluble wool Proteins. I. Light scattering and viscosity in Aqueous solution

1958 ◽  
Vol 11 (4) ◽  
pp. 581 ◽  
Author(s):  
BS Harrap ◽  
EF Woods
Keyword(s):  
Molecular Weight ◽  
Light Scattering ◽  
Intrinsic Viscosity ◽  
High Speed ◽  
Guanidine Hydrochloride ◽  
Sodium Dodecyl ◽  
Relative Viscosity ◽  
Molecular Weights ◽  
Solvent Component ◽  
Wool Proteins

S-Carboxymethylkerateine 2 (SCMK2) and cc-keratose, two proteins derived from wool, have been characterized by nitrogen content, ultraviolet absorption, refractive index increment, light scattering, and intrinsic viscosity. Variations in the physical properties of different batches are attributed to different degrees of aggregation during the preparation. The relative viscosity decreased with time and was generally accompanied by an increase in turbidity, indicating aggregation. The effect of heating was to accelerate the fall in viscosity and increase in turbidity. Light scattering investigations showed that dissociation occurred on dilution and in some cases this could be detected by viscosity measurements. The molecular weight of several millions for SCMK2 at pH 6.7 was reduced to less than 1 million by removal of large aggregates by high-speed centrifuging, with an increase in both dissymmetry and intrinsic viscosity. In alkaline buffers at pH 10.5 the proteins were further dissociated and gave molecular weights of the order of 450,000. The behaviour of α-keratose was similar to that of SCMK2. Measurements on SCNK2 carried out in the presence of sodium dodecyl sulphate gave a molecular weight of 142,000 for the detergent-protein complex, corresponding to 95,000 for the protein, the dissymmetry was near unity and the intrinsic viscosity 0.115 dl/g. In 10M acetic acid, 8M urea, and 641 guanidine hydrochloride the apparent molecular weights were 95,000, 140,000, and 210,000 respectively, but these values are only upper limits because of possible selective solvation of the solvent component in such three-component systems.

scholarly journals Proteoglycans of bovine periodontal ligament and skin. Occurrence of different hybrid-sulphated galactosaminoglycans in distinct proteoglycans

1982 ◽  
Vol 201 (1) ◽  
pp. 27-37 ◽  
Author(s):  
C H Pearson ◽  
G J Gibson
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Periodontal Ligament ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Sedimentation Equilibrium ◽  
Gel Chromatography ◽  
Dermatan Sulphate ◽  
Self Association

A proteoglycan purified from 4 M-guanidinium chloride extracts of bovine periodontal ligament closely resembled that of bovine skin, except for a rather lower protein content and a higher molecular weight (120 000 compared with about 90 000) by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The latter difference was explained by the molecular weights (29 000 and 16 000) of the respective dermatan sulphate components, each of which was rich in L-iduronate (about 75% of the total hexuronate). Significant amounts of other glycosaminoglycans did not occur in these proteoglycans, which were homogenous on gel chromatography and agarose/polyacrylamide-gel electrophoresis. Polydispersity was observed in sedimentation equilibrium experiments, but proteolysis or self-association of the proteodermatan sulphates may have affected these results. Ligament proteoglycans that were almost completely extracted with 0.1 M-NaCl contained less protein of a completely different amino acid composition than the proteodermatan sulphates. They were heterogeneous in size but generally smaller than cartilage proteoglycans and L-iduronate was a component, comprising about 7% of the total hexuronate of the sulphated galactosaminoglycan chains. The latter consisted of two fractions differing in molecular weight, but a dermatan sulphate with a high L-iduronate content was not present. These proteoglycans had some resemblance to D-glucuronate-rich proteoglycans of other non-cartilaginous tissues. Such compounds, however, are difficult to categorize at present.

scholarly journals Nuclear matrix of HeLa S3 cells. Polypeptide composition during adenovirus infection and in phases of the cell cycle.

1977 ◽  
Vol 72 (1) ◽  
pp. 194-208 ◽  
Author(s):  
L D Hodge ◽  
P Mancini ◽  
F M Davis ◽  
P Heywood
Keyword(s):  
Cell Cycle ◽  
Molecular Weight ◽  
Nuclear Matrix ◽  
Apparent Molecular Weight ◽  
Molecular Weight Range ◽  
Weight Range ◽  
Molecular Weights ◽  
Liver Nuclei ◽  
Hela S3 ◽  
Biochemical Analyses

A subnuclear fraction has been isolated from HeLa S3 nuclei after treatment with high salt buffer, deoxyribonuclease, and dithiothreitol. This fraction retains the approximate size and shape of nuclei and resembles the nuclear matrix recently isolated from rat liver nuclei. Ultrastructural and biochemical analyses indicate that this structure consists of nonmembranous elements as well as some membranous elements. Its chemical composition is 87% protein, 12% phospholipid, 1% DNA, and 0.1% RNA by weight. The protein constituents are resolved in SDS-polyacrylamide slab gels into 30-35 distinguishable bands in the apparent molecular weight range of 14,000 - 200,000 with major peptides at 14,000 - 18,000 and 45,000 - 75,000. Analysis of newly synthesized polypeptides by cylindrical gel electrophoresis reveals another cluster in the 90,000-130,000 molecular weight range. Infection with adenovirus results in an altered polypeptide profile. Additional polypeptides with apparent molecular weights of 21,000, 23,000, and 92,000 become major components by 22 h after infection. Concomitantly, some peptides in the 45,000-75,000 mol wt range become less prominent. In synchronized cells the relative staining capacity of the six bands in the 45,000-75,000 mol wt range changes during the cell cycle. Synthesis of at least some matrix polypeptides occures in all phases of the cell cycle, although there is decreased synthesis in late S/G2. In the absence of protein synthesis after cell division, at least some polypeptides in the 45,000-75,000 mol wt range survive nuclear dispersal and subsequent reformation during mitosis. The possible significance of this subnuclear structure with regard to structure-function relationships within the nucleus during virus replication and during the life cycle of the cell is discussed.

Studies on Soluble Fibrin Complexes as a Function of pH

1975 ◽  
Author(s):  
Y. Benabid ◽  
E. Concord ◽  
M. Suscillon
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Gel Electrophoresis ◽  
Gel Chromatography ◽  
Agarose Gel ◽  
Monomer Mixture ◽  
Soluble Fibrin ◽  
Molecular Weights ◽  
Fibrin Monomer

Purified fibrinogen solutions, incubated with thrombin. CNBr. Sepharose, were subjected to agarose gel chromatography and eluted at different pH (6.5; 7.5; 8.5). Among high molecular weight derivatives formed by thrombin, the major component was a dimer. Gel chromatography at pH 8.5 showed a complexes peak distinct of that from fibrinogen, whereas at pH 6.5, only the fibrinogen peak appeared: fibrin monomer was eluted with fibrinogen as demonstrated by polyacrylamid gel electrophoresis 3.75% pH 8.9. SDS urea electrophoresis after reduction indicated that complexes peak contained two α-chains (α and α′). When fibrinogen was incubated with thrombin in the presence of FSF and calcium, several derivatives with higher and higher molecular weights were formed besides the dimer, and elution profiles of chromatography were identical at pH 6.5 and 8.5, thus indicating stable complexes formation. If fibrinogen-fibrin monomer mixture was subjected to FSF action at different pH, no complexes were formed at pH 6.5. These results confirm that at pH 6.5, any association was prevented.

A PLASMINOGEN ACTIVATOR INHIBITOR (PAI-2) CIRCULATES IN TWO HIGH MOLECULAR WEIGHT FORMS IN PREGNANCY

1987 ◽  
Author(s):  
N A Booth ◽  
A Reith ◽  
B Bennett
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Plasminogen Activator ◽  
Apparent Molecular Weight ◽  
Late Pregnancy ◽  
Plasminogen Activator Inhibitor ◽  
Molecular Weights ◽  
Histiocytic Cell ◽  
Sds Page ◽  
Pai 1

Normal vascular endothelium and platelet α-granules contain an inhibitor of plasminogen activator (PAI-1) of about 48000 molecular weight, which is released by stimuli such as thrombin. An immunologically distinct inhibitor (PAI-2) of about 47000 molecular weight has been purified from placenta and from a histiocytic cell line U-937. The level of PA-inhibition in plasma is raised in late pregnancy and this may be due to increases in PAI-1 or in PAI-2 or in both.Using SDS-PAGE and zymography on fibrin/plasminogen /u-PA detector gels, we have found that normal plasma contains a band of inhibition of apparent molecular weight 40000, which can be neutralised by antiserum raised against PAI-1. Pregnancy plasma contained this band as well as additional inhibitor bands of apparent molecular weights 75000 and 130000. The novel high molecular weight PA-inhibitors were detectable by zymography at about 12 weeks gestation. They were specific for plasminogen activator and did not inhibit plasmin. They were inhibited by antiserum raised against PAI-2 from U-937 cells (a gift from Dr EKO Kruithof) and thus are immunologically related to PAI-2. They may represent circulating complexes of PAI-2 with another protein or aggregates of PAI-2, which retain inhibitory activity after SDS-PAGE. PAI-2 appears to represent a pregnancy associated protein that circulates in a number of different molecular weight forms.

Sign in / Sign up

Export Citation Format

Share Document