scholarly journals Proteoglycans of human umbilical cord arteries.

2000 ◽  
Vol 47 (4) ◽  
pp. 1081-1091 ◽  
Author(s):  
T Gogiel ◽  
S Jaworski
Keyword(s):  
Molecular Mass ◽  
Umbilical Cord ◽  
Chondroitin Sulphate ◽  
Guanidine Hydrochloride ◽  
Exchange Chromatography ◽  
Human Umbilical Cord ◽  
Chondroitinase Abc ◽  
The Core ◽  
Core Proteins ◽  
Sds Page

Proteoglycans (PGs) were dissociatively extracted from human umbilical cord arteries (UCAs) with 4 M guanidine hydrochloride containing Triton X-100 and protease inhibitors, purified by Q-Sepharose anion exchange chromatography and lyophilized. They were analysed by gel filtration, SDS/PAGE and agarose gel electrophoresis before and after treatment with chondroitinase ABC. It was found that the PG preparation was especially enriched in chondroitin/dermatan sulphate PGs. The predominant PG fraction included small PGs that emerged from Sepharose CL-2B with Kav = 0.74. Their molecular mass, estimated by SDS/PAGE, was 160-200 kDa and 90-150 kDa, i.e. it was typical for biglycan and decorin, respectively. Treatment with chondroitinase ABC yielded the core proteins of 45 and 47 kDa, characteristic for both small PGs. Remarkable amounts of the 45 kDa protein were detected in non-treated PG samples, suggesting the presence of free core proteins of biglycan and decorin. Large PGs were present in lower amounts. In intact form they were eluted from Sepharose CL-2B with Kav = 0.17 and 0.43. Digestion with chondroitinase ABC yielded the core proteins with a molecular mass within the range of 180-360 kDa but predominant were the bands of 200, 250 and 360 kDa. The large PGs probably represent various forms of versican or perlecan bearing chondroitin sulphate chains.

scholarly journals Characterization of proteoglycans isolated from associative extracts of human articular cartilage

1993 ◽  
Vol 293 (1) ◽  
pp. 165-172 ◽  
Author(s):  
V Vilím ◽  
A J Fosang
Keyword(s):  
Articular Cartilage ◽  
Molecular Mass ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Gel Chromatography ◽  
Human Articular Cartilage ◽  
Keratan Sulphate ◽  
Core Proteins ◽  
Sds Page

Approx. 10% of the total proteoglycan content of normal young human articular cartilage was extracted under associative conditions with Dulbecco's PBS. Proteoglycans isolated from the extract by Q-Sepharose chromatography were separated by gel chromatography and characterized by gradient gel SDS/PAGE and immunoblotting. Three species of small proteoglycans, two main populations of aggrecan and a population of its smaller fragments were identified. The major populations of aggrecan contained chondroitin sulphate chains, all or part of the N-terminal G1 and G2 domains and, therefore, intact keratan sulphate domains. The larger population was estimated by gradient SDS/PAGE to have a molecular mass of approx. 600 kDa or greater. The second population had an apparent molecular mass of approx. 300-600 kDa. Core proteins derived from these populations of proteoglycans separated on SDS/PAGE into several clusters of bands in the range from 120 to approx. 360 kDa. The extract further contained smaller fragments which lacked chondroitin sulphate but reacted with antibodies against keratan sulphate, and against epitopes present in the G2 domain of aggrecan. The presence of the G2 domain in a broad range of populations of decreasing size indicated extensive cleavage of the aggrecan core protein within its chondroitin sulphate domain. These findings suggest that fragmentation of aggrecan probably occurs in vivo in normal articular cartilage of young individuals. Associative extracts also contained decorin, biglycan and fibromodulin. These were resolved from aggrecan by gel chromatography and identified by immunodetection.

scholarly journals Purification and partial characterization of the major cell-associated heparan sulphate proteoglycan of rat liver

1991 ◽  
Vol 273 (2) ◽  
pp. 415-422 ◽  
Author(s):  
M Lyon ◽  
J T Gallagher
Keyword(s):  
Molecular Mass ◽  
Proteinase Inhibitors ◽  
Gradient Centrifugation ◽  
Heparan Sulphate ◽  
Exchange Chromatography ◽  
Guanidinium Chloride ◽  
Apparent Homogeneity ◽  
Core Proteins ◽  
Sds Page ◽  
Triton X

Heparan sulphate proteoglycans were solubilized from whole rat livers by homogenization and dissociative extraction with 4 M-guanidinium chloride containing Triton X-100 and proteinase inhibitors. The extract was subjected to trichloroacetic acid precipitation and the proteoglycan remained soluble. This was then purified to apparent homogeneity by a combination of (a) DEAE-Sephacel chromatography, (b) digestion with chondroitinase ABC followed by f.p.l.c. Mono Q ion-exchange chromatography, and (c) density-gradient centrifugation in CsCl and 4 M-guanidinium chloride. Approx. 1.5 mg of proteoglycan was obtained from 30 livers with an estimated recovery of 25%. The purified proteoglycan was eluted from Sepharose CL6B as an apparently single polydisperse population with a Kav. of 0.19 and displayed a molecular mass of greater than or equal to 200 kDa (relative to protein standards) by SDS/PAGE. Its heparan sulphate chains were eluted with a Kav. of 0.44 and have an estimated molecular mass of 25 kDa. Digestion of the proteoglycan with a combination of heparinases yielded core proteins of 77, 49 and 44 kDa. Deglycosylation using trifluoromethanesulphonic acid, though slightly decreasing the sizes, gave an identical pattern of core proteins. Electrophoretic detergent blotting demonstrated that all of the core proteins were hydrophobic and are probably integral plasma membrane molecules. The peptide maps generated by V8 proteinase digestion of the two major core proteins (77 and 49 kDa) were very similar, suggesting that these two core proteins are structurally related.

scholarly journals One of the major sulphated proteins secreted by rat hepatocytes contains low-sulphated chondroitin sulphate

1990 ◽  
Vol 272 (1) ◽  
pp. 113-118 ◽  
Author(s):  
E M Sjöberg ◽  
E Fries
Keyword(s):  
Molecular Mass ◽  
Chondroitin Sulphate ◽  
Alkali Treatment ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Exchange Chromatography ◽  
Secretory Proteins ◽  
Gel Chromatography ◽  
Chondroitinase Abc ◽  
Oligosaccharide Chain

When isolated hepatocytes are incubated with 35SO4(2-), a specific set of secretory proteins is labelled. One of these proteins is electrophoretically heterogeneous, with an apparent molecular mass of 35-45 kDa [Marcks von Würtemberg & Fries (1989) Biochemistry 28, 4088-4093]. Here we report that treatment with chondroitinase ABC converted the broad electrophoretic band of this protein, with a 50-60% loss of radioactivity, into a relatively homogeneous band with a molecular mass of 28 kDa. Size determination by gel chromatography of the protein's oligosaccharide chain (released by alkali treatment) indicated that it contained about 40 hexose units. Similar analysis of the enzyme-resistant oligosaccharide chain remaining linked to the protein after chondroitinase ABC treatment indicated a size of between six and eight hexose units. These observations suggest that the protein's oligosaccharide chain carries only three or four sulphate groups, of which one or two are located close to the polypeptide chain. Consistent with this hypothesis, the free oligosaccharide behaved like a low-sulphated glycosaminoglycan upon ion-exchange chromatography.

scholarly journals Isolation and partial characterization of heparan sulphate proteoglycans from human hepatic amyloid

1992 ◽  
Vol 288 (1) ◽  
pp. 225-231 ◽  
Author(s):  
J H Magnus ◽  
T Stenstad ◽  
G Husby ◽  
S O Kolset
Keyword(s):  
Molecular Mass ◽  
Core Protein ◽  
Alkali Treatment ◽  
Heparan Sulphate ◽  
Exchange Chromatography ◽  
Gel Chromatography ◽  
Tissue Sections ◽  
The Core ◽  
Amyloid Deposits ◽  
Sds Page

Proteoglycans were isolated from human amyloidotic liver by extraction with guanidine, followed by trichloroacetic acid precipitation, DEAE-Sephacel ion-exchange chromatography, and Sepharose CL-6B gel chromatography. A significant portion of the material was found to be free chondroitin/dermatan sulphate chains (30%), whereas the predominant part was heparan sulphate proteoglycan (HSPG) (70%). The approx. molecular mass of the HSPG was 200 kDa, as measured by gel electrophoresis and gel chromatography. The molecular mass of the core protein was shown to be 60 kDa by SDS/PAGE following de-aminative cleavage of the heparan sulphate chains. The heparan sulphate chains were liberated from the core protein by alkali treatment and found to have a molecular mass of approx. 35 kDa by Sepharose CL-6B gel chromatography. The core protein was shown, by immunoblotting, to react with a monoclonal antibody against bovine basement membrane HSPG. The presence of HSPG in amyloid deposits was further confirmed by immunohistochemistry on tissue sections from amyloidotic liver using the same antibody.

scholarly journals Identity of the core proteins of the large chondroitin sulphate proteoglycans synthesized by skeletal muscle and prechondrogenic mesenchyme

1994 ◽  
Vol 298 (1) ◽  
pp. 51-60 ◽  
Author(s):  
D A Carrino ◽  
J E Dennis ◽  
R F Drushel ◽  
S E Haynesworth ◽  
A I Caplan
Keyword(s):  
Skeletal Muscle ◽  
Molecular Mass ◽  
Cyanogen Bromide ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Linear Segment ◽  
Electron Microscopic ◽  
The Core ◽  
Core Proteins ◽  
Aggrecan Core Protein

Large, chondroitin sulphate-containing proteoglycans are synthesized by three prominent tissue in the embryonic chick limb. One of these proteoglycans is aggrecan, the phenotype-specific proteoglycan of cartilage. Another, PG-M, is produced by prechondrogenic mesenchymal cells. The third, M-CSPG, is made by developing skeletal muscle cells. While the carbohydrate components of PG-M and M-CSPG share some similarities, both of these proteoglycans clearly have different carbohydrate moieties from those of aggrecan. To compare these three proteoglycans at another level, their core protein structures were analysed in three ways: by the presence or absence of monoclonal antibody epitopes, by one-dimensional peptide display of the cyanogen bromide-cleaved core proteins and by electron microscopic imaging of the molecules. Monoclonal antibodies whose epitopes are present in aggrecan core protein were tested with core protein preparations from M-CSPG and PG-M. One of these, 7D1, recognizes both PG-M and M-CSPG, while another, 1C6, shows no reactivity for the non-cartilage proteoglycans. The absence of 1C6 reactivity is of interest, as its epitope is in a region of the aggrecan core protein known to have a functional homologue in the core proteins of PG-M and M-CSPG. The cyanogen bromide-fragmented peptide pattern of M-CSPG is the same as that of PG-M, and both are different from that of aggrecan. The aggrecan pattern has one prominent large band (molecular mass 130 kDa), some less prominent large bands (molecular mass 70-100 kDa) and several smaller bands. In contrast, the PG-M and M-CSPG patterns show no bands with molecular masses > 73 kDa, and the smaller bands (molecular mass < 40 kDa) have a different pattern to that of the smaller bands from aggrecan. The electron microscopic images of aggrecan show a core protein with one end having two globular regions separated by a short linear segment; adjacent to this is a long linear segment, which sometimes contains a third globular region at the end of the core protein opposite the end with the double-globe structure. M-CSPG and PG-M core proteins never show images with the double-globe structure. Instead, one end of the molecule has a single globular domain, and a second globular region is variably present at the opposite end of the core protein. Thus, by all three methods, the core proteins of PG-M and M-CSPG appear to be the same and both differ from the core protein of aggrecan.

CHANGES IN PROTEOGLYCAN AND SULFATED PROTEIN SYNTHESIS DURING MEGAKARYOCYTE MATURATION IN VIVO

1987 ◽  
Author(s):  
B P Schick ◽  
C J Walsh ◽  
T Jenkins-West
Keyword(s):  
Protein Synthesis ◽  
The Core ◽  
Small Peak ◽  
Core Proteins ◽  
Time Period ◽  
Sds Page ◽  
Isoelectric Points ◽  
Average Size ◽  
Triton X

We investigated changes in sulfated proteoglycan (PG) and sulfated protein synthesis during megakaryocyte (MK) maturation in vivo by characterizing the (35S)-labeled molecules in MKs and platelets (PLTs) obtained daily from 3 hr to 5 days after injection of guinea pigs with (35S)sulfate. Radioactivity in macromolecules was maximal in MKs 3 hr and in PLTs 3 days after the injection. The cells were solubilized in 8M urea/50mM Tris/0.2% Triton X-100/0.1M NaCl, and PGs and sulfoproteins were separated by DEAE-Sephacel chromatography. PGs (65% of cell 35s) were eluted as two fractions, one (PG-1, 87%) with 4M Gdn HC1 and another (PG-2, 13%) with 4M Gdn HCl/2% TX-100. The Kav of PLT PG-1 on Sepharose CL-6B shifted gradually from 0.18 to 0.10 from 1-5 days after (35S) injection, and the smaller and larger PG-1 species were resolved on SDS-PAGE by fluorography. The size of PG-1 molecules was a function of glycosaminoglycan (GAG) chain length. The appearance of the different size PG-1 molecules in PLTs was accounted for by their disappearance from MKs over the same time period. Thus the size of the PG-1 synthesized by MKs decreased with MK maturation. The (35S)-PG-2 appeared in PLTs only 2-3 days after (35S) injection, had Kav 0.07 on CL-6B, but had GAGs of the same average size as those of PG-1. The hydrophobic character of PG-2 suggests that it might be the membrane PG. PG-1 and PG-2 were separated by SDS-PAGE and identified by fluorography. The core proteins of PG-1 and PG-2 were obtained by chondroitinase digestion and identified by SDS-PAGE and fluorography. The GAGs of PG-1 and PG-2 were almost entirely chondroitin-6-sulfate. The average size of PG-1 was 200,000 and its GAGs about 45,000.The sulfated proteins (20-25% of total cell 35S) eluted in the wash-through of the DEAE-Sephacel column and with 0.23M NaCl. Their isoelectric points were 4.0-6.5. They eluted as a small peak near the V0 and a major broad peak from Kav 0.3-0.6 on CL-6B columns, and could be identified as at least 8 distinct bands on SDS-PAGE by fluorography. Digestion with NaOH/NaBH4, Pronase or papain released small (35S)-labeled fragments, and the (35S) appeared to be associated with oligosaccharides. The sulfoproteins appeared in PLTs primarily 2-4 days after (35S) injection, and different proteins were labeled at different time points.

scholarly journals Glutathione S-transferases of the yeast Yarrowia lipolytica have unusually large molecular mass

1998 ◽  
Vol 333 (3) ◽  
pp. 839-845 ◽  
Author(s):  
Vivienne FOLEY ◽  
David SHEEHAN
Keyword(s):  
Molecular Mass ◽  
Yarrowia Lipolytica ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Post Translational Modification ◽  
Native Molecular Mass ◽  
Sds Page ◽  
Glutathione S Transferases ◽  
Yeast Yarrowia Lipolytica

Two similar glutathione S-transferases (GSTs), which do not bind to glutathione– or S-hexylglutathione–agarose affinity resins, have been purified from the yeast Yarrowia lipolytica. An approx. 400-fold purification was obtained by a combination of DEAE-Sephadex, phenyl-Sepharose, hydroxyapatite and Mono-Q anion-exchange chromatography. The native molecular mass of both proteins was estimated as approx. 110 kDa by both Superose-12 gel-filtration chromatography and non-denaturing electrophoresis. SDS/PAGE indicated a subunit mass of 50 kDa. Reverse-phase HPLC of purified proteins gave a single, well-resolved, peak, suggesting that the proteins are homodimers. Identical behaviour on HPLC, native electrophoresis and SDS/PAGE, N-terminal sequencing, sensitivity to a panel of inhibitors and identical specific activities with 1-chloro-2,4-dinitrobenzene as substrate suggest that the two isoenzymes are very similar. The enzymes do not immunoblot with antisera to any of the main GST classes, and N-terminal sequencing suggests no clear relationship with previously characterized enzymes, such as that of the fungus, Phanerochaete chrysosporium [Dowd, Buckley and Sheehan (1997) Biochem. J. 324, 243–248]. It is possible that the two isoenzymes arise as a result of post-translational modification of a single GST isoenzyme.

Production, purification, and characterization of an extracellular endo-β-1, 3-glucanase from a monokaryon of Schizophyllum commune ATCC 38548 defective in exo-β-1, 3-glucanase formation

1994 ◽  
Vol 40 (1) ◽  
pp. 18-23 ◽  
Author(s):  
Andreas Prokop ◽  
Peter Rapp ◽  
Fritz Wagner
Keyword(s):  
Molecular Mass ◽  
Schizophyllum Commune ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Glucanase Activity ◽  
Sds Page ◽  
S 100 ◽  
Reduced Activity

Production of extracellular β-1, 3-glucanase activity by a monokaryotic Schizophyllum commune strain was monitored and results indicated that the β-glucanase activity consisted of an endo- β-1, 3-glucanase activity, besides a negligible amount of β-1, 6-glucanase and β-glucosidase activity. Unlike the β-1, 3-glucanase production of the dikaryotic parent strain S. commune ATCC 38548, the β-1, 3-glucanase formation of the monokaryon was not regulated by catabolite repression. The endo- β-1, 3-glucanase of the monokaryon was purified from the culture filtrate by lyophilization, anion exchange chromatography on Mono Q, and gel filtration on Sephacryl S-100. It appeared homogeneous on SDS-PAGE with a molecular mass of 35.5 kDa and the isoelectric point was 3.95. The enzyme was only active toward glucans containing β-1, 3-linkages, including lichenan, a β-1, 3-1, 4-D-glucan. It attacked laminarin in an endo-like fashion to form laminaribiose, laminaritriose, and high oligosaccharides. While the extracellular β-glucanases from the dikaryotic S. commune ATCC 38548 degraded significant amounts of schizophyllan, the endo- β-1, 3-glucanase from the monokaryon showed greatly reduced activity toward this high molecular mass β-1, 3-/β-1, 6-glucan. The Km of the endoglucanase, using laminarin as substrate, was 0.28 mg/mL. Optimal pH and temperature were 5.5 and 50 °C, respectively. The enzyme was stable between pH 5.5 and 7.0 and at temperatures below 50 °C. The enzyme was completely inhibited by 1 mM Hg2+. Growth of the monokaryotic S. commune strain was not affected by its constitutive endo- β-1, 3-glucanase formation.Key words: endo- β-1, 3-glucanase, Schizophyllum commune, monokaryon, constitutive endo- β-1, 3-glucanase formation.

scholarly journals Identification of cytosolic protein tyrosine kinases of human prostate by renaturation after SDS/PAGE

1992 ◽  
Vol 284 (3) ◽  
pp. 653-658 ◽  
Author(s):  
Y Durocher ◽  
A Chapdelaine ◽  
S Chevalier
Keyword(s):  
Tyrosine Kinases ◽  
Cell Function ◽  
Guanidine Hydrochloride ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Protein Tyrosine Kinases ◽  
Partial Purification ◽  
Major Band ◽  
Protein Tyrosine ◽  
Sds Page

The identification of protein tyrosine kinases (PTKs) was successfully achieved by renaturation in gels after SDS/PAGE. To this effect, samples were mixed with a PTK substrate, namely the polydispersed co-polymer of glutamic acid and tyrosine [poly(Glu, Tyr), M(r) from 30,000 to 94,000], and were simultaneously submitted to electrophoresis. Following guanidine hydrochloride denaturation, renaturation and phosphorylation with [gamma-32P]ATP, kinase activity was detected by autoradiography. When applied to cytosol from human hyperplastic prostate, eleven protein kinases were detected, among which one major (M(r) 50,000) and two minor proteins (M(r) 40,000 and 38,000) were identified as PTKs by the presence of phosphotyrosine. Incubation of the gel in hot alkali after glutaraldehyde cross-linking almost completely eliminated the detection of non-PTK enzymes. On the other hand, in the absence of poly(Glu,Tyr), no PTK activity was detected. Partial purification of cytosolic PTKs indicates that the native M(r) of the major phosphotransferase was 44,000, as estimated by gel filtration following ammonium sulphate precipitation and anion-exchange chromatography. Upon renaturation after electrophoresis, this fraction showed only one major band active on poly(Glu,Tyr) which was associated with the polypeptide of M(r) 50,000. This enzyme was also identified following two-dimensional electrophoresis and renaturation in the presence of poly(Glu,Tyr), allowing the determination of a pI in the range 7.5-7.8. Thus PTKs can be easily renatured following electrophoresis and rapidly identified on the basis of their M(r) and pI in both crude or partially purified preparations. With the crucial role played by PTKs in the activation of cell function and carcinogenesis, this procedure could be useful in the identification of such enzymes and in distinguishing them from their substrates in gels.

scholarly journals Production of the HBc Protein from Different HBV Genotypes in E. coli. Use of Reassociated HBc VLPs for Packaging of ss- and dsRNA

2021 ◽  
Vol 9 (2) ◽  
pp. 283
Author(s):  
Ivars Petrovskis ◽  
Ilva Lieknina ◽  
Andris Dislers ◽  
Juris Jansons ◽  
Janis Bogans ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ammonium Sulfate Precipitation ◽  
E Coli ◽  
The Core ◽  
Hbv Genotypes ◽  
Core Proteins ◽  
B Virus

The core proteins (HBc) of the hepatitis B virus (HBV) genotypes A, B, C, D, E, F, and G were cloned and expressed in Escherichia coli (E. coli), and HBc-formed virus-like particles (VLPs) were purified with ammonium sulfate precipitation, gel filtration, and ion exchange chromatography (IEX). The best VLP yield was found for the HBc of the HBV genotypes D and G. For the HBc of the HBV genotypes D, F, and G, the possibility of dissociation and reassociation maintaining the native HBc structure was demonstrated. Single-stranded (ss) and double-stranded (ds) ribonucleic acid (RNA) was successfully packed into HBc VLPs for the HBV genotypes D and G.

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