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.

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.

SECMA 1®, a mitogenic hexapeptide from Ulva algeae modulates the production of proteoglycans and glycosaminoglycans in human foreskin fibroblast

1998 ◽  
Vol 17 (1) ◽  
pp. 18-22 ◽  
Author(s):  
R Ennamany ◽  
D Saboureau ◽  
N Mekideche ◽  
E E Creppy
Keyword(s):  
Glucuronic Acid ◽  
De Novo ◽  
Heparan Sulphate ◽  
Guanidine Hydrochloride ◽  
Sodium Deoxycholate ◽  
Effective Concentration ◽  
Skin Fibroblasts ◽  
Human Skin Fibroblasts ◽  
Dermatan Sulphate ◽  
Salt Extract

SECMA 1® is a polypeptide purified from a green algeae of the Ulva species by several gel chromatographies, showing the following sequence (Glu-Asp-Arg-Leu-Lys-Pro). In order to determine the effect of SECMA 1® on human skin fibroblasts extracellular matrix, proteoglycans (PGs) and glycosaminoglycans (GAGs) were assayed after 24 h incubation of 20 day-old foreskin fibroblasts at the 2nd passage. The results revealed that most of [35S]sulphate was associated with fibroblast membranes, which contained (67%) of the total de novo synthesized sulphated PGs, in two distinct forms: one hydrophilic (39%), and one hydrophobic (28%). The remaining `matrix' retained 5% of proteoglycans. The remaining 35S-label may represent the free label in the cytosol. After 24 h incubation of skin fibroblasts with different concentrations of SECMA 1® (2, 4 and 10 μg/ml), the [35S] sulphate incorporation into PGs of Salt-extract, sodium deoxycholate (DOC) extract and Guanidine hydrochloride (GuA-HCl)-extract was increased significantly ( P<0.005) with 4 μg/ml, as compared to untreated control. The most effective concentration (4 μg/ml) increased the different [35S]sulphate PGs extracts (NaCl, DOC and GuA-HCl) by respectively (66; 17 and 75%). The relative contents of iduronic and glucuronic acid in the GAG produced by skin fibroblasts were estimated. No effect of SECMA 1® on the incorporation of [35S]sulphate into Heparan sulphate was found. The incorporation of [35S]sulphate into (chondroïtine sulphate + heparan sulphate) and (chondroïtine sulphate + dermatan sulphate) was increased by respectively 37% and 11% by SECMA 1® (4 μg/ml).

scholarly journals Isolation and characterization of a specific enterokinase inhibitor from kidney bean (Phaseolus vulgaris)

1983 ◽  
Vol 209 (1) ◽  
pp. 91-97 ◽  
Author(s):  
R T Jacob ◽  
P G Bhat ◽  
T N Pattabiraman
Keyword(s):  
Phaseolus Vulgaris ◽  
Inhibitory Activity ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Kidney Bean ◽  
Gel Chromatography ◽  
Isolation And Characterization ◽  
Wide Range ◽  
Arginine Residues

A specific enterokinase inhibitor from kidney bean (Phaseolus vulgaris) was purified to homogeneity. It showed a single protein band on sodium dodecyl sulphate/polyacryl-amide-gel electrophoresis in the presence of mercaptoethanol, and the Mr was 31000. Aspartic acid was identified as the N-terminus of the inhibitor. The Mr by gel chromatography on Sephadex G-200 was found to be 60000, indicating the dimeric nature of the inhibitor. The inhibitor was found to be a glycoprotein. The monosaccharide moieties were glucose, mannose, glucuronic acid and glucosamine in the proportions 3.15%, 5.0%, 0.85% and 1.3% respectively. The inhibitor was most active on pig enterokinase, followed by bovine and human enterokinases. Maximal inhibitory activity was elicited by preincubation of the inhibitor with the enzyme for 15 min. Digestion with pepsin resulted in loss of inhibitory activity. The inhibitor was stable to exposure to a wide range of pH values (2-10), and exposure to pH above 10 resulted in loss of inhibitory activity. Modification of arginine residues by cyclohexane 1,2-dione and ninhydrin led to complete loss of enterokinase-inhibitory activity.

scholarly journals Isolation of 35S- and 3H-labelled proteoglycans from cultures of human embryonic skin fibroblasts

1979 ◽  
Vol 183 (3) ◽  
pp. 669-681 ◽  
Author(s):  
L Cöster ◽  
I Carlstedt ◽  
A Malmström
Keyword(s):  
Density Gradient Centrifugation ◽  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Guanidine Hydrochloride ◽  
Gel Chromatography ◽  
Dermatan Sulphate ◽  
Cscl Density Gradient ◽  
Embryonic Skin ◽  
Sepharose 4B ◽  
Phosphate Buffered Saline

35SO42– and [3H]-leucine-labelled proteoglycans were isolated from the medium of a fibroblast culture, from an EDTA extract of the monolayer, and from consecutive dithiothreitol and guanidine hydrochloride extracts of the cells. Proteoglycans of different sizes were isolated from the extracts by gel chromatography on Sepharose 4B. In the medium and the EDTA extract the largest proteoglycans contained only 35S-labelled galactosaminoglycan, whereas all other fractions contained in addition heparan [35S-labelled galactosaminoglycan, whereas all other fractions contained in addition heparin [35S]sulphate. The galactosaminoglycan-containing proteoglycans of the various extracts were separated into a larger component, containing chondroitin sulphate-like side chains, and a smaller component, containing dermatan sulphate. The larger proteoglycan of the medium showed reversible association-dissociation behaviour when chromatographed on Sepharose CL2B in phosphate-buffered saline and 4M-guanidine hydrochloride respectively. This property remained after removal of extraneous proteins by CsCl-density-gradient centrifugation in guanidine hydrochloride. The association was markedly increased by the addition of high-molecular-weight hyaluronic acid.

scholarly journals Biosynthesis and secretion of dermatan sulphate proteoglycans in cultures of human skin fibroblasts

1984 ◽  
Vol 220 (2) ◽  
pp. 575-582 ◽  
Author(s):  
L Cöster ◽  
I Carlstedt ◽  
A Malmström ◽  
B Särnstrand
Keyword(s):  
Glucuronic Acid ◽  
Cell Layer ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Gel Chromatography ◽  
Intracellular Accumulation ◽  
Dermatan Sulphate ◽  
Iduronic Acid ◽  
A Cell ◽  
Small Proteoglycan

Fibroblasts in culture were incubated with [3H]leucine and [35S]sulphate for 1-24 h. A large glucuronic acid-rich and a small iduronic acid-rich dermatan sulphate proteoglycan were isolated with the use of isopycnic density-gradient centrifugation, ion-exchange and gel chromatography. After 3 h the accumulation in the cell layer of the small proteoglycan reached a steady state, whereas the large one continued to increase, albeit more slowly. In the medium both proteoglycans accumulated ‘linearly’, although the large one appeared somewhat later than the small one. The composition of the polysaccharide chains and the size of the protein cores did not vary during the experiment. The two proteoglycans were synthesized at approximately similar rates, but were distributed differently in the culture. The small proteoglycan was mainly confined to the medium, whereas the large one was found in the medium as well as in a cell-associated pool. There was an intracellular accumulation of iduronic acid-rich dermatan sulphate as free polysaccharides.

scholarly journals Isolation and characterization of senile amyloid--related antigenic substance (SASSAM) from mouse serum. Apo SASSAM is a low molecular weight apoprotein of high density lipoprotein.

1983 ◽  
Vol 158 (5) ◽  
pp. 1600-1614 ◽  
Author(s):  
K Higuchi ◽  
A Matsumura ◽  
K Hashimoto ◽  
A Honma ◽  
S Takeshita ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Density Lipoprotein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Density Lipoprotein ◽  
High Density ◽  
Mouse Serum ◽  
Gel Chromatography ◽  
Precipitation Line ◽  
Isolation And Characterization

Sera obtained from senescence-accelerated mouse (SAM) and normal mice contained a substance that reacted with antiserum raised against ASSAM, a novel senile amyloid fibril protein isolated from the liver of SAM. This physiological substance, termed "SASSAM" (serum ASSAM-related antigenic substance), migrated to the albumin/prealbumin region in immunoelectrophoresis and the precipitation line formed with anti-ASSAM antiserum was stained positively with both Amide Black 10 B and Oil Red O/Fat Red 7B solutions, thereby suggesting that SASSAM is an alpha lipoprotein. Using Sephadex G-200 gel chromatography, SASSAM was eluted as a high mol wt form of approximately 200,000 daltons. Fractionation of lipoprotein from normal mouse serum by preparative ultra-centrifugation disclosed that SASSAM was found mainly in high density lipoprotein, HDL (the density is between 1.063 and 1.21 g/cm3). The largest amount of SASSAM was found in the HDL2 fraction (the density is between 1.063 and 1.125) and in this fraction SAA was not detected. Furthermore, ASSAM immunoreactivity appeared in the low mol wt proteins (below 10,000 daltons) of apo HDL separated in the buffer containing 8 M urea through Sephadex G-200. In 8 M urea sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), the major components of apolipoproteins in this position, possibly corresponding to apo C proteins, have the same molecular weight, 5,200 daltons, as ASSAM and this component was labeled by anti-ASSAM antiserum after transfer to nitrocellulose paper.

scholarly journals Isolation and characterization of dermatan sulphate and heparan sulphate proteoglycans from fibroblast culture

1981 ◽  
Vol 197 (1) ◽  
pp. 217-225 ◽  
Author(s):  
I Carlstedt ◽  
L Cöster ◽  
A Malmström
Keyword(s):  
Cell Layer ◽  
Heparan Sulphate ◽  
Buoyant Density ◽  
Human Skin Fibroblast ◽  
Dermatan Sulphate ◽  
Protein Core ◽  
Sulphate Proteoglycan ◽  
Isolation And Characterization ◽  
Iduronic Acid ◽  
Heparan Sulphate Proteoglycans

35SO42(-)- and [3H]leucine-labelled proteoglycans were isolated from the medium and cell layer of human skin fibroblast cultures. Measures were taken to avoid proteolytic modifications during isolation by adding guanidinium chloride and proteolysis inhibitors immediately after harvest. The proteoglycans were purified and fractionated by density-gradient centrifugation, followed by gel and ion-exchange chromatography. Our procedure permitted the isolation of two major proteoglycan fractions from the medium, one large, containing glucuronic acid-rich dermatan sulphate chains, and one small, containing iduronic acid-rich ones. The protein core of the latter proteoglycan had an apparent molecular weight of 47000 as determined by polyacrylamide-gel electrophoresis, whereas the protein core of the former was considerably larger. The major dermatan sulphate proteoglycan of the cell layer was similar to the large proteoglycan of the medium. Only small amounts of the iduronic acid-rich dermatan sulphate proteoglycan could be isolated from the cell layer. Instead most of the iduronic acid-rich glycans appeared as free chains. The heparan sulphate proteoglycans found in the cell culture were largely confined to the cell layer. This proteoglycan was of rather low buoyant density and seemed to contain a high proportion of protein. The major part of the heparan sulphate proteoglycan from the medium had a higher buoyant density and contained a smaller amount of protein.

scholarly journals Isolation and characterization of a variant of ovomucoid

1975 ◽  
Vol 147 (1) ◽  
pp. 139-144 ◽  
Author(s):  
A Waheed ◽  
A Salhuddin
Keyword(s):  
Gel Electrophoresis ◽  
Simple Procedure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Preparative Scale ◽  
Isolation And Purification ◽  
Isolation And Characterization ◽  
Fluorescence Characteristics

A simple procedure, which can be used on a preparative scale, for the isolation and purification of a major variant of ovomucoid from egg white is described. Ovomucoid was precipitated by salt, and further fractionated by chromatography on sulphoethyl-Sephadex. It showed size homogeneity as revealed by gel chromatography and sodium dodecyl sulphate-polyacrylamide-gel electrophoresis where the mobility was consistent with a molecular weight of 28 300+/-2300. The inhibitor showed full antiryptic but no antichymotryptic activity. The u.v.-absorption and fluorescence characteristics indicated the absence of tryptophan. Polyacrylamide-gel electrophoresis in the presence of 9M-urea demonstrated absence of charge heterogeneity. The intrinsic viscosity of ovomucoid was 5.36ml/g which yielded an equivalent hydrodynamic radius (2.9nm), axial ratio (6.0) and frictional ratio (1.31) of the molecule. The Stokes radius (3.5nm), diffusion coefficient (7.8 times 10(-7 cm2/s) and frictional ratio (1.35) were calculated from gel-filtration data. These results suggest that ovomucoid exists in non-globular conformation under native conditions and that the deviation from the behaviour of a typical globular protein seems to be due both to asymmetry and hydration.

scholarly journals Isolation and characterization of high-buoyant-density proteoglycans from bovine femoral-head cartilage

1983 ◽  
Vol 213 (2) ◽  
pp. 355-362 ◽  
Author(s):  
M Lyon ◽  
J Greenwood ◽  
J K Sheehan ◽  
I A Nieduszynski
Keyword(s):  
Light Scattering ◽  
Femoral Head ◽  
Chondroitin Sulphate ◽  
Guanidine Hydrochloride ◽  
Buoyant Density ◽  
Radius Of Gyration ◽  
Gel Chromatography ◽  
Isolation And Characterization ◽  
Femoral Head Cartilage

Proteoglycans were extracted from bovine (15-18 months old) femoral-head cartilage. The heterogeneity of the A1D1 proteoglycan fraction was examined by gel chromatography, sedimentation velocity, sucrose rate-zonal centrifugation and CS2SO4 isopycnic centrifugation. In all cases polydisperse but unimodal distributions were obtained. Chemical analysis of the preparation yielded a galactosamine/glucosamine molar ratio of 7:1, and 13C n.m.r. spectroscopy showed that the chondroitin sulphate comprised equal proportions of the 4- and 6-sulphate isomers. Gel chromatography of a papain and Pronase digest of the proteoglycan indicated that the chondroitin sulphate chains had a Mn of approx. 10500. The mean buoyant density of the proteoglycan in pure CS2SO4 was 1.46 g/ml. Physical characterization of the proteoglycan preparation in 4M-guanidine hydrochloride, pH 7.4, by using conventional light-scattering gave a radius of gyration of 42 nm and a Mw of 0.96 × 10(6). Quasi-elastic light-scattering in the same solvent yielded a translational diffusion coefficient, D020, of 5.41 × 10(-8) cm2 × S-1, and ultracentrifugation gave a sedimentation coefficient, S020, of 12.0S. Thus from sedimentation-diffusion studies a Mw of 1.36 × 10(6) was calculated. The possible origins for the differences in the two molecular-weight estimates are discussed. It is concluded that the high-buoyant-density proteoglycans from bovine articular cartilage are significantly smaller than those from bovine nasal septum, and that this is largely due to the smaller size of their chondroitin sulphate chains.

scholarly journals Isolation and characterization of tryase, a serine proteinase from rat liver

1981 ◽  
Vol 193 (1) ◽  
pp. 251-259 ◽  
Author(s):  
J Saklatvala ◽  
J S Bond ◽  
A J Barrett
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Sodium Dodecyl ◽  
Liver Homogenate ◽  
Deae Cellulose ◽  
Serine Proteinase ◽  
Apparent Molecular Weight ◽  
Soya Bean ◽  
Gel Chromatography ◽  
Isolation And Characterization

1. A new serine proteinase, tryase, was isolated from the membrane fraction of a post-nuclear supernatant of rat liver homogenate. The enzyme was solubilized with 1 M-MgCl2 and purified to homogeneity by DEAE-cellulose chromatography and affinity chromatography with soya-bean trypsin inhibitor linked to Sepharose 4B. 2. The enzyme was identified on sodium dodecyl sulphate/polyacrylamide gels by reaction with radiolabelled di-isopropyl phosphorofluoridate. Unreduced its molecular weight was 32 500, reduced it was 28 000. 3. The enzyme readily hydrolysed azocasein and tripeptide nitroanilide substrates with an arginine or lysine residue adjacent to the leaving group. D-Pro-Phe-Arg-NPhNO2 was used routinely (Km = 0.25 mM). Tryase showed little activity on blocked arginine esters or amides. 4. It was inhibited by di-isopropyl phosphorofluoridate, benzamidine, aprotinin, soya-bean and lima-bean trypsin inhibitors, Ile-Leu-Arg-CH2Cl and Phe-Ala-Arg-CH2Cl. It was not inhibited by Tos-Lys-CH2Cl. 5. Subcellular-fractionation studies showed that tryase was associated with particles similar in their sedimentation properties to lysosomes, but, since it was not present in tritosomes, it was not in the classical lysosome. 6. Rat liver contained other neutral proteinases; one of these was a serine proteinase with an apparent molecular weight of 90 000 on gel chromatography.

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