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1.
L P van den Heuvel J van den Born J H Veerkamp T J van de Velden L Schenkels L A Monnens C H Schr?der J H Berden 《Biochimica et biophysica acta》1990,1025(1):67-76
Heparan sulfate proteoglycan (HSPG) was extracted from human tubular basement membrane (TBM) with guanidine and purified by ion-exchange chromatography and gel filtration. The glycoconjugate was sensitive to heparitinase and resistant to chondroitinase ABC, had an apparent molecular mass of 200-400 kDa and consisted of 70% protein and 30% glycosaminoglycan. The amino acid composition was characterized by its high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulfonic acid yielded core proteins of 160 and 110 kDa. The heparan sulfate (HS) chains obtained after alkaline NaBH4 treatment had a molecular mass of about 18 kDa. Results of heparitinase digestion and HNO2 treatment suggest a clustering of sulfate groups in the distal portion of the HS side chains. These chemical data are comparable to those obtained previously on glomerular basement membrane (GBM) HSPG (Van den Heuvel et al. (1989) Biochem. J. 264, 457-465). Peptide patterns obtained after trypsin, clostripain or V8 protease digestion of TBM and GBM HSPG preparations showed a large similarity. Polyclonal antisera and a panel of monoclonal antibodies raised against both HSPG preparations and directed against the core protein showed complete cross-reactivity in ELISA and on Western blots. They stained all basement membranes in an intense linear fashion in indirect immunofluorescence studies on human kidneys. Based on these biochemical and immunological data we conclude that HSPGs from human GBM and TBM are identical, or at least very closely related, proteins. 相似文献
2.
L P van den Heuvel J van den Born J H Veerkamp G H Janssen T J van de Velden L A Monnens C H Schr?der J H Berden 《The International journal of biochemistry》1990,22(8):903-914
1. Proteoglycans extracted from human and equine glomerular basement membranes (GBM) were purified by ion-exchange chromatography and gel filtration. 2. The glycoconjugates had an apparent molecular mass of 200-400 kDa and consisted of 75% protein and 25% glycosaminoglycan. Glycosidase and HNO2 treatment and the amino sugar and sulfate composition of both proteoglycan preparations identified heparan sulfate (HS) as the predominant saccharide chain. 3. Hydrolysis with trifluoromethanesulfonic acid yielded comparable core proteins with molecular masses of ca 160 and 120 kDa. 4. The HS chains had an apparent molecular mass of 18 kDa. Results of heparitinase digestion and HNO2-treatment indicated a clustering of sulfate groups in the distal part of the HS side chains. 5. Peptide mapping after trypsin, clostripain or V8 protease digestion of radiolabeled human and equine heparan sulfate proteoglycans (HSPG) preparations with three different separation techniques showed large differences. 6. Polyclonal antisera raised against the HSPGs reacted against the core proteins. Both HSPG preparations and their antisera showed ca 40% cross-reactivity. About 50% of monoclonal antisera elicited against one HSPG preparation showed reaction with both HSPG preparations. 7. Polyclonal antisera stained all basement membranes in an intense linear fashion in indirect immunofluorescence studies of kidney sections from horse, man and various mammalian species. 8. Biochemical and immunological data indicate that HSPGs from equine and human GBM have a comparable structure, but the core proteins differ considerably. 相似文献
3.
Heparan sulfate proteoglycan from the extracellular matrix of human lung fibroblasts. Isolation, purification, and core protein characterization 总被引:6,自引:0,他引:6
A Heremans J J Cassiman H Van den Berghe G David 《The Journal of biological chemistry》1988,263(10):4731-4739
Confluent cultured human lung fibroblasts were labeled with 35SO4(2-). After 48 h of labeling, the pericellular matrix was prepared by Triton X-100 and deoxycholate extraction of the monolayers. Heparan sulfate proteoglycan (HSPG) accounted for nearly 80% of the total matrix [35S]proteoglycans. After solubilization in 6 M guanidinium HCl and cesium chloride density gradient centrifugation, the majority (78%) of these [35S] HSPG equilibrated at an average buoyant density of 1.35 g/ml. This major HSPG fraction was purified by ion-exchange chromatography on Mono Q and by gel filtration on Sepharose CL-4B, and further characterized by gel electrophoresis and immunoblotting. Intact [35S]HSPG eluted with Kav 0.1 from Sepharose CL-4B, whereas the protein-free [35S]heparan sulfate chains, obtained by alkaline borohydride treatment of the proteoglycan fractions, eluted with Kav 0.45 (Mr approximately 72,000). When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, core (protein) preparations, obtained by heparitinase digestion of 125I-labeled HSPG fractions, yielded one major labeled band with apparent molecular mass of approximately 300 kDa. Reduction with beta-mercaptoethanol slightly increased the apparent Mr of the labeled band, suggesting a single polypeptide structure and the presence of intrachain disulfide bonds. Immunoadsorption experiments and immunostaining of electrophoretically separated heparitinase-digested core proteins with monoclonal antibodies raised against matrix and cell surface-associated HSPG suggested that the major matrix-associated HSPG of cultured human lung fibroblasts is distinct from the HSPG that are anchored in the membranes of these cells. Binding studies suggested that this matrix HSPG interacts with several matrix components, both through its glycosaminoglycan chains and through its heparitinase-resistant core. Core (protein) interactions seem to be responsible for the association of the proteoglycan with the extracellular matrix. 相似文献
4.
Multiple distinct membrane heparan sulfate proteoglycans in human lung fibroblasts 总被引:12,自引:0,他引:12
V Lories J J Cassiman H Van den Berghe G David 《The Journal of biological chemistry》1989,264(12):7009-7016
Heparitinase digestion of the hydrophobic membrane-associated heparan sulfate proteoglycans (HSPG) of fetal human lung fibroblasts yields core proteins of various sizes: i.e. monomeric core proteins of 125, 90, 64, 48, and 35 kDa and a disulfide-linked dimeric core protein composed of approximately 35-kDa subunits. By immunizing BALB/c mice with liposome-incorporated HSPG, we have obtained a total of five anti-HSPG monoclonal antibodies (Mabs, i.e. Mabs S1, 1C7, 2E9, 6G12, and 10H4) with different specificities. Polyacrylamide gel electrophoresis of 125I-labeled membrane HSPG immunoprecipitated with these Mabs revealed that Mabs 1C7 and 2E9 bind only membrane HSPG which yield a 125-kDa core protein after heparitinase digestion, whereas Mab S1-bound HSPG yield a 64-kDa core protein, and Mabs 6G12 and 10H4 retain membrane HSPG with a 48-kDa core protein. Western blotting of the heparitinase-digested proteoglycans and immunostaining with the Mabs confirmed this pattern of reactivity. However, in this assay, Mabs 6G12 and 10H4 also detected a minor approximately 90-kDa core protein in addition to the 48-kDa core protein. Except perhaps for the 10H4 epitope, the epitopes recognized by these Mabs appear to be part of the peptide moieties as they resisted complete deglycosylation of the HSPG with trifluoromethanesulfonic acid. Since these data were inconsistent with a direct relationship between the major core proteins, the 48-, 64-, and 125-kDa core proteins were immunopurified and further compared by peptide mapping with Staphylococcus aureus protease V8, trypsin, and CNBr cleavage. Clearly distinct peptide patterns were obtained for the three different core proteins. These results imply that the 48-, 64-, and the 125-kDa membrane HSPG core proteins of human lung fibroblasts are derived from distinct proteoglycans. 相似文献
5.
The structures of cell-associated heparan sulfate (HS) proteoglycans and their interaction with the plasma membrane was studied using rat ovarian granulosa cell culture. HS proteoglycans were either metabolically labeled by incubating cell cultures with [3H] leucine and [35S]sulfate or labeled in plasma membrane preparations with a photoactivatable reagent, 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (TID), a compound which has been shown to selectively label the hydrophobic membrane-binding domains of several proteins. After purification of HS proteoglycans from the labeled cell cultures or from the labeled membrane preparations by repeated Q-Sepharose ion exchange chromatography in 8 M urea, they were analyzed by Superose 6 gel filtration and octyl-Sepharose chromatography both in 4 M guanidine HCl. The results indicated that the HS proteoglycans were labeled with 125I and therefore have an intramembranous domain. Phospholipase C (Bacillus thuringiensis), which specifically cleaves phosphatidylinositol membrane anchors, released approximately 25% of the 35S-labeled HS proteoglycans from the cell surface as well as 20-30% of the 125I-label from the 125I-TID-labeled HS proteoglycans. These data indicate that a subpopulation of HS proteoglycans are intercalated into the plasma membrane through a linkage structure involving phosphatidylinositol. Phospholipase C-resistant, 125I-labeled HS proteoglycans represent those species inserted into membrane through an intercalated peptide sequence. Core protein size of phosphatidylinositol-anchored species estimated by polyacrylamide gel electrophoresis after heparitinase digestion was approximately 80 kDa, and it was significantly larger than that of the directly intercalated species (approximately 70 kDa). 相似文献
6.
Cell surface phosphatidylinositol-anchored heparan sulfate proteoglycan initiates mouse melanoma cell adhesion to a fibronectin-derived, heparin-binding synthetic peptide 总被引:4,自引:0,他引:4
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S L Drake D J Klein D J Mickelson T R Oegema L T Furcht J B McCarthy 《The Journal of cell biology》1992,117(6):1331-1341
Cell surface heparan sulfate proteoglycan (HSPG) from metastatic mouse melanoma cells initiates cell adhesion to the synthetic peptide FN-C/H II, a heparin-binding peptide from the 33-kD A chain-derived fragment of fibronectin. Mouse melanoma cell adhesion to FN-C/H II was sensitive to soluble heparin and pretreatment of mouse melanoma cells with heparitinase. In contrast, cell adhesion to the fibronectin synthetic peptide CS1 is mediated through an alpha 4 beta 1 integrin and was resistant to heparin or heparitinase treatment. Mouse melanoma cell HSPG was metabolically labeled with [35S]sulfate and extracted with detergent. After HPLC-DEAE purification, 35S-HSPG eluted from a dissociative CL-4B column with a Kav approximately 0.45, while 35S-heparan sulfate (HS) chains eluted with a Kav approximately 0.62. The HSPG contained a major 63-kD core protein after heparitinase digestion. Polyclonal antibodies generated against HSPG purified from mouse melanoma cells grown in vivo also identified a 63-kD core protein. This HSPG is an integral plasma membrane component by virtue of its binding to Octyl Sepharose affinity columns and that anti-HSPG antibody staining exhibited a cell surface localization. The HSPG is anchored to the cell surface through phosphatidylinositol (PI) linkages, as evidenced in part by the ability of PI-specific phospholipase C to eliminate binding of the detergent-extracted HSPG to Octyl Sepharose. Furthermore, the mouse melanoma HSPG core protein could be metabolically labeled with 3H-ethanolamine. The involvement of mouse melanoma cell surface HSPG in cell adhesion to fibronectin was also demonstrated by the ability of anti-HSPG antibodies and anti-HSPG IgG Fab monomers to inhibit mouse melanoma cell adhesion to FN-C/H II. 35S-HSPG and 35S-HS bind to FN-C/H II affinity columns and require 0.25 M NaCl for elution. However, heparitinase-treated 125I-labeled HSPG failed to bind FN-C/H II, suggesting that HS, and not HSPG core protein, binds FN-C/H II. These data support the hypothesis that a phosphatidylinositol-anchored HSPG on mouse melanoma cells (MPIHP-63) initiates recognition to FN-C/H II, and implicate PI-associated signal transduction pathways in mediating melanoma cell adhesion to this defined ligand. 相似文献
7.
Glucocerebrosidase was purified from human cultured dermal fibroblasts more than 2200-fold to apparent homogeneity using high performance Alkyl-Superose HR 5/5 hydrophobic interaction and Bio-Sil TSK-250 gel permeation column chromatography. Sodium dodecyl sulfate--polyacrylamide gel electrophoresis and protein staining of the catalytically active and concentrated enzyme fractions from the gel permeation columns revealed the presence of one band of Mr 64,000. The glucocerebrosidase preparation purified to homogeneity was digested with peptide N-glycosidase F that cleaves N-linked oligosaccharide structures from glycoproteins. The molecular weight of glucocerebrosidase after digestion with peptide N-glycosidase F was reduced to Mr 57,000, suggesting that the mature enzyme is a glycoprotein and that N-linked oligosaccharide constitutes a minimum of about 10% of the total molecular weight of the polypeptide. These findings are compatible with the hypothesis that glucocerebrosidase was initially synthesized as a precursor polypeptide which was subsequently glycosylated to become the mature enzyme. 相似文献
8.
P Marynen J Zhang J J Cassiman H Van den Berghe G David 《The Journal of biological chemistry》1989,264(12):7017-7024
Heparitinase treatment of cell surface-associated heparan sulfate proteoglycans (HSPG) of human lung fibroblasts reveals core proteins with apparent Mr values of 125,000, 90,000, 64,000, 48,000 and 35,000 (Lories, V., De Boeck, H., David, G., Cassiman, J.-J., and Van den Berghe, H. (1987) J. Biol. Chem. 262, 854-859). The 90- and 48-kDa core proteins share the epitope of the monoclonal antibody 6G12 which was used to screen a human lung fibroblast expression cDNA library. Rescreening of the libraries yielded clone 48K5 with an insert of 3439 base pairs. Polyclonal antibodies were raised in rabbits against a fragment of the protein encoded by the 48K5 cDNA different from the part carrying the 6G12 epitope. These antibodies specifically recognize the 90- and 48-kDa core proteins on Western blots of total cellular extracts of human lung fibroblast HSPG. The specific reactivity of the polyclonal antiserum confirms the identity of the 48K5 clone and further distinguishes the 48- and the 90-kDa core proteins, which do share the 6G12-defined epitope and at least one additional antigenic determinant with the 48K5 cDNA-encoded protein, from the 125-, 64-, and 35-kDa core proteins of cell surface HSPG of human lung fibroblasts which do not react with either antibody preparation. The protein encoded by the 48K5 clone contains a stop-transfer sequence indicative of an integral membrane protein and three potential glycosaminoglycan attachment sites. The 48K5 clone detects two major poly(A)+ RNA species in human lung fibroblasts presumably generated by the use of alternative polyadenylation signals. The 48K5 gene was mapped to chromosome 8q23 by in situ hybridization and hybridization to DNA of somatic cell hybrids. 相似文献
9.
Characterization and N-terminal sequence of a heparan sulphate proteoglycan synthesized by endothelial cells in culture. 总被引:2,自引:0,他引:2
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We have isolated from the conditioned medium of an established endothelial cell line a heparan sulphate proteoglycan whose involvement in the inhibition of the extrinsic coagulation pathway was reported in previous studies [Colburn & Buonassisi (1982) Biochem. Biophys. Res. Commun. 104, 220-227]. The proteoglycan was purified by gel filtration and ion-exchange chromatography, and appears to be free of contaminating proteins as determined by polyacrylamide-gel electrophoresis of the radioiodinated protein core before and after removal of the glycosaminoglycan chains by treatment with heparitinase. By this procedure the Mr of the protein core was estimated to be 22000. The N-terminal end was sequenced up to amino acid 25. The 21st residue is likely to be glycosylated. Analysis of the purified proteoglycan by gel-filtration chromatography yielded Kd values of 0.2 for the whole molecule and 0.35 for the glycosaminoglycan chains. The structure that emerges from these data is that of a heparan sulphate proteoglycan characterized by a relatively small protein core and few glycosaminoglycan chains. 相似文献
10.
Identification of a 64 kDa heparan sulphate proteoglycan core protein from human lung fibroblast plasma membranes with a monoclonal antibody. 总被引:7,自引:1,他引:6
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H de Boeck V Lories G David J J Cassiman H van den Berghe 《The Biochemical journal》1987,247(3):765-771
Human lung fibroblasts produce heparan sulphate proteoglycans (HSPG) that are associated with the plasma membrane. A monoclonal-antibody (Mab)-secreting hybridoma, S1, was produced by fusion of SP 2/0-AG 14 mouse myeloma cells with spleen cells from mice immunized with partially purified cellular HSPG fractions. The HSPG character of the material carrying the epitope recognized by Mab S1 was demonstrated by: (i) the co-purification of the S1 epitope with the membrane HSPG of human lung fibroblasts; (ii) the decrease in size of the material carrying the S1 epitope upon treatment with heparinase or heparitinase, and the resistance of this material to heparinase treatment after N-desulphation. The S1 epitope appears to be part of the core protein, since it was destroyed by proteinase treatment and by disulphide-bond reduction, but not by treatments that depolymerize the glycosaminoglycan chains and N-linked oligosaccharide chains. Polyacrylamide-gel electrophoresis of non-reduced heparitinase-digested membrane HSPG followed by Western blotting and immunostaining with Mab S1 revealed a single band with apparent molecular mass of 64 kDa. Membrane proteoglycans isolated from detergent extracts or from 4 M-guanidinium chloride extracts of the cells yielded similar results. Additional digestion with N-glycanase lowered the apparent molecular mass of the immunoreactive material to 56 kDa, suggesting that the core protein also carries N-linked oligosaccharides. Fractionation of 125I-labelled membrane HSPG by immuno-affinity chromatography on immobilized Mab S1, followed by heparitinase digestion and polyacrylamide-gel electrophoresis of the bound material, yielded a single labelled band with apparent molecular mass 64 kDa. Treatment with dithiothreitol caused a slight increase in apparent molecular mass, suggesting that the core protein of this membrane proteoglycan of a single subunit containing (an) intrachain disulphide bond(s). 相似文献
11.
Solubilization and purification of a membrane-associated 3,3'',5-tri-iodo-L-thyronine-binding protein from rat erythrocytes.
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3,3',5-Tri-iodo-L-thyronine (L-T3) binding sites from rat erythrocyte membranes were solubilized in an active form by using the zwitterionic detergent CHAPS or the anionic detergent lauroylsarcosine. The binding protein was successively purified by Sephadex G-200 and affinity chromatography. The purified material retained its binding activity and exhibited high affinity and specificity compared with those displayed in the original membrane. Yield was about 10% of the starting activity. The specific binding activity was enriched by approx. 100-fold, which represents a purity of only 0.1%. Analysis of the purified preparation on SDS/PAGE showed two major protein bands (Mr 64,000 and Mr 50,000), but these could not represent the binding protein since the purity obtained was low. However, affinity-labelling experiments with N-bromoacetyl-L-[125I]T3 in intact membranes showed that two proteins (also with Mr values of 64,000 and 50,000) bound the hormone specifically, suggesting a co-migration of hormone receptors and contaminants on gel electrophoresis. 相似文献
12.
Structural characterization of heparan sulfate proteoglycan subclasses isolated from bovine aortic endothelial cell cultures 总被引:3,自引:0,他引:3
Labeled heparan sulfate proteoglycans (HSPG) were isolated from wounded and confluent cultures of bovine aortic endothelial cells by nondegradative extraction with 4 M guanidine hydrochloride and detergent. HSPG were separated from more highly charged chondroitin or dermatan sulfate proteoglycans by ion-exchange chromatography, and subclasses of different hydrodynamic size were isolated by gel filtration. Three major subclasses of HSPG were characterized structurally with respect to the presence and relative size of protein core, the presence and amount of nonsulfated oligosaccharide, and size and structure of heparan sulfate (HS) chains. The largest (600-800-kDa) HSPG subclass (I), isolated from cell layers and media of confluent cultures, bears 38-kDa HS chains on an apparently heterogeneous class of relatively large glycoprotein cores. HSPG II (150-200 kDa), isolated from cell layer or media, has 22-kDa HS chains and smaller core glycoproteins (less than 50 kDa). HSPG III, the subclass of smallest hydrodynamic size, has 13-kDa HS chains and a glycopeptide core of less than 15 kDa. All subclasses bear varying proportions of non-sulfated oligosaccharides of similar sizes. Comparisons of HS chain structure indicated that the different subclasses have similar proportions (49-55%) of N-sulfate, with both O-sulfate and highly N-sulfated blocks of disaccharide distributed similarly along HS chains. In addition, HS chains from subclasses II and III contain sequences that are insensitive to periodate oxidation or heparitinase digestion, suggesting that they contain increased proportions of iduronate.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
13.
A Couvineau B Amiranoff N Vauclin-Jacques M Laburthe 《Biochemical and biophysical research communications》1984,122(1):283-288
125I-GIP binds reversibly to a high affinity binding site in crude plasma membranes prepared from a hamster pancreatic beta cell tumor. The treatment of labeled membranes with the cross-linker dithiobis (succinimidylpropionate) prevents, to a greater extent, the rapid dissociation of 125I-GIP-membrane complexes which is observed when 10(-6) M native GIP is added. Polyacrylamide gel electrophoresis of membrane proteins reveals a major 125I-GIP-protein complex of Mr 64,000. This labeling decreases when increasing concentrations (10(-9) -10(-6)M) of native GIP are added but is not altered by other peptide hormones (tested at 10(-6)M) including glucagon, VIP and insulin. The Mr 64,000 complex is not observed in tissues which have no specific binding sites for GIP such as intestinal epithelium. Assuming one molecule of 125I-GIP is bound per molecule of protein, one protein with Mr 59,000 is identified as the specific GIP binding site. 相似文献
14.
Isolation and characterization of a heparin-binding domain from the amino terminus of platelet thrombospondin 总被引:31,自引:0,他引:31
V M Dixit G A Grant S A Santoro W A Frazier 《The Journal of biological chemistry》1984,259(16):10100-10105
Calcium-replete thrombospondin has been purified from outdated platelets using heparin-Sepharose affinity chromatography, gelatin-Sepharose to remove fibronectin, and gel filtration to eliminate low-molecular-weight heparin-binding proteins. Edman degradation of six different preparations revealed the amino-terminal sequence of thrombospondin (TSP) to be Asn-Arg-Ile-Pro-Glu-Ser-Gly-Gly-Asp-Asn-Ser-Val-Phe-. This sequence was obtained in initial yields as high as 85%, indicating that no blocked chains are present. Cleavage of calcium-replete TSP with thermolysin or plasmin results in the production of relatively stable fragments. Chromatography of these digests on heparin-Sepharose followed by elution with 0.6 M NaCl affords purification of an Mr 25,000 fragment from the thermolysin digest and an Mr 35,000 fragment from the plasmin digest. The binding of these fragments to heparin-Sepharose does not require divalent metal ions. Neither fragment is disulfide-bonded to other fragments present in the digests. The heparin-binding domains from both digests have similar amino acid compositions and their tryptic peptide maps on high performance liquid chromatography are identical with the exception of one peptide unique to each fragment. Automated Edman degradation in a vapor-phase sequenator of the thermolytic heparin-binding domain electroeluted from sodium dodecyl sulfate-gels indicates that the heparin-binding domain resides at the amino terminus of the Mr 180,000 TSP peptide chain. 相似文献
15.
Heparin-binding EGF-like growth factor stimulation of smooth muscle cell migration: dependence on interactions with cell surface heparan sulfate 总被引:20,自引:1,他引:19
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《The Journal of cell biology》1993,122(4):933-940
Heparin-binding EGF-like growth factor (HB-EGF), but not EGF, binds to cell surface heparan sulfate proteoglycan (HSPG). This was demonstrated in (a) the binding of 125I-HB-EGF to mutant CHO cells deficient in HS production was diminished by 70% compared to wild-type CHO cells, (b) the binding of 125I-HB-EGF to CHO cells and bovine aortic smooth muscle cells (BASMC) was diminished 80% by heparitinase or chlorate treatment, and (c) 125I-EGF did not bind to CHO cells and its binding to BASMC was not diminished at all by heparitinase and only slightly by chlorate treatment. Accordingly, the role of HB-EGF interactions with HSPG in modulating bioactivity was examined. Heparitinase or chlorate treatment of BASMC diminished the ability of HB-EGF to stimulate BASMC migration by 60-80%. A similar inhibition of migration occurred when BASMC were treated with a synthetic peptide (P21) corresponding to the sequence of the putative heparin-binding domain of HB-EGF. As a control for BASMC viability, and for specificity, it was found that heparitinase and P21 did not inhibit at all and chlorate inhibited only slightly the stimulation of BASMC migration by PDGF AB. Since heparitinase, chlorate, and P21 treatment also diminished by 70-80% the cross-linking of 125I-HB-EGF to the EGF receptor, it was concluded that the interaction of HB-EGF, via its heparin-binding domain, with cell surface HSPG was essential for its optimal binding to the EGF receptor on BASMC and hence for its optimal ability to stimulate migration. 相似文献
16.
Lahcen Bichoualne Bénédicte Thiébot Monique Langris Pierre Barbey Hamid Oulhaj Jean Bocquet 《Molecular and cellular biochemistry》1994,140(1):37-48
Confluent testicular peritubular cells derived from immature rats were used to study membrane associated proteoglycans (PG) Peripheral material (heparin releasable), membrane and intracellular material (Triton X-100 releasable) were collected, purified by anion exchange chromatography then characterized by gel filtration and by hydrophobic interaction chromatography, followed by enzymatic digestion and chemical treatment. The peripheral material was constituted of two populations of PG (Kav=0 and 0.10 on Superose 6 column), each containing both heparan sulfate proteoglycans (HSPG) and chondroitin proteoglycans (CSPG) and perhaps a hybrid PG (HSCSPG). These PG being not retained on an octyl Sepharose column they were devoided of hydrophobic properties. The integral membrane proteoglycans isolated on the basis of their hydrophobic properties represented 20% of the Triton X-100 releasable material, and were exclusively constituted of proteoheparan sulfate. There were no relationships between this membrane HSPG and the peripheral HSPG as evidenced by pulse chase experiments. The mode of intercalation of the hydrophobic HSPG in the cell membrane was studied. The majority of these macromolecules (80%) were sensitive to trypsin and only a minor proportion (20%) were sensitive to phosphatidylinositol specific phospholipase C. Thus, about 80% of the hydrophobic HSPG were intercalated in the cell membrane by a hydrophobic segment of the core protein whereas about 20% were associated with the cell membrane via a phosphatidylinositol residue covalently bound to the core protein of the PG.Abbreviations PG
Proteoglycans
- CSPG
Chondroitin Sulfate Proteoglycans
- HSPG
Heparan Sulfate Proteoglycans
- HSCSPG
Heparan and Chondroitin Sulfate Proteoglycans
- DNAse I
Deoxyribonuclease I
- DMEM
Dulbeccos modified Eagle's medium
- H/D
HAM F12/DMEM
- ECM
Extracellular Matrix
- PBS
Phosphate Buffered Saline
- PI
Phosphatidylinositol
- GPI
Glycosyl Phosphatidylinositol
- PI-PLC
Phosphatidylinositol Specific Phospholipase C
- TBS
Tris Buffered Saline
- STI
Soybean Trypsin Inhibitor
- GAG
Glycosaminoglycans
- HA
Hyaluronic Acid 相似文献
17.
Brucato S Fagnen G Villers C Bonnamy PJ Langris M Bocquet J 《Biochimica et biophysica acta》2001,1510(1-2):474-487
(35)S-Radiolabeled cultured Sertoli cells from immature rat testis were extracted with detergent and the different proteoheparan sulfate (HSPG) forms of the extract were discriminated and quantified on the basis of their high anionic charge, hydrodynamic size, lipophilic properties, susceptibility to trypsin and phosphatidylinositol phospholipase C (PI-PLC). Trypsin released 50% of total cellular HSPG corresponding to 80% of total hydrophobic HSPG. Trypsin-accessible HSPG were presumed to be integral membrane species. Trypsin-resistant HSPG, probably intracellular, distributed into non-lipophilic (37.5%) and lipophilic (12.5%) populations. Biochemical analysis of PG copurified with plasma membrane confirmed the existence of hydrophobic HSPG integrated into this structure. Among hydrophobic HSPG accessible to trypsin, 35% were PI-PLC released and radiolabeled by [(3)H]inositol indicating that about one third of integral membrane HSPG were intercalated into the plasma membrane through a phosphatidylinositol anchor (glypican type). PI-PLC-resistant forms represented HSPG inserted into the membrane through a hydrophobic segment of the core protein (syndecan type). No lipophilic PG was present in other cell compartments (culture medium, cell periphery, extracellular matrix). (125)I-Iodinated hydrophobic HSPG were deglycanated and submitted to SDS-polyacrylamide gel electrophoresis. In the glypican family, a core protein (64--65 kDa) was detected, whereas in the syndecan family, bands of 60 and 68 kDa were observed which may correspond to self-association of different core proteins. In Sertoli cell, specific functional attributes of different integral membrane HSPG forms remain to be investigated. 相似文献
18.
《The International journal of biochemistry》1993,25(12):1835-1848
- 1.1. The proteoglycan peak from anion exchange chromatography of an extract of bovine aorta was digested with chondroitinase ABC. The residual heparan sulphate proteoglycans were further purified by chromatography on Sepharose CL4B and DEAE-Sephacel to yield two species, of high and low charge density.
- 2.2. Higher molecular weight material had a higher proportion of high charge density proteoglycan, while the lower molecular weight species had a higher proportion of low charge density heparan sulphate proteoglycan.
- 3.3. The two species shared epitopes as they both reacted with an antibody to heparan sulphate proteoglycan from bovine glomerular basement membrane.
- 4.4. On electron microscopy, both high and low charge density proteoglycans were visualized as ‘tadpole-like’ molecules, which showed a tendency to aggregate via their globular heads.
- 5.5. Bovine aortic smooth muscle cells were cultured in the presence of [35S]sulphate and [3H]glucosamine. Proteoglycans were isolated from medium and cell layer extract by the methods outlined above.
- 6.6. The major HSPG species isolated from medium were significantly larger than those from cell layer and displayed substantial heterogeneity in both size of HS chain after papain digestion and size of protein core after heparitinase digestion. 7. The major cell layer species yielded two HS species of widely differing mol. wt after papain digestion, and a very small protein core after heparitinase digestion. Therefore cell layer-associated HSPGs show a good deal more homogeneity than those found in the medium.
- 7.8. Further ion-exchange chromatography after digestion with chondroitinase ABC revealed HSPG species of lower charge density, possibly derived from a hybrid chondroitin sulphate-dermatan sulphate proteoglycan (CS/DSPG) after removal of the CS/DS chains.
19.
Binding and structural characteristics of a soluble lactogen-binding protein from rabbit mammary-gland cytosol.
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Specific receptors for prolactin (PRL) are known to be present on plasma membranes and intracellular membranes of mammary gland. We now report, however, the detection and characterization of a soluble lactogen-specific binding protein in high-speed (200,000 g) cytosolic preparations from pregnant- and non-pregnant-rabbit mammary gland. The binding protein was not detectable by poly(ethylene glycol) precipitation; instead, bound and free 125I-labelled human growth hormone (hGH; a potent lactogen) was separated using a mini-gel filtration technique. Specific binding of 125I-hGH reached an apparent equilibrium between 10 and 14 h at 21-23 degrees C. It was dependent on mammary-gland protein concentration and, partially, on Ca2+ or Mg2+ concentrations. Scatchard analysis revealed steep curvilinear plots, the high-affinity component having a KA of approximately 3 X 10(10) M-1. Gel filtration on calibrated Ultrogel AcA34 columns of 125I-hGH-cytosol complexes or of cytosol alone, followed by measurement of 125I-hGH binding in each eluted fraction, indicated that the binding protein had an Mr of 33,000-43,000. A specific binding protein of the same size was observed when 125I-ovine or -human PRL, but not 125I-bovine GH, was used as ligand. The apparent lactogenic specificity was confirmed by a lack of cross-reactivity of the binding protein with an anti-[GH receptor (rabbit liver)] monoclonal antibody. Polyacrylamide-gel electrophoresis of 125I-hGH covalently cross-linked to cytosol with disuccinimidyl suberate revealed binding proteins of Mr 35,000 (non-reduced) and 37,000 (reduced), results comparable with those obtained by gel filtration and indicating an absence of inter-subunit disulphide bonds. These studies have shown the presence of an apparently naturally soluble lactogen-binding protein in the cytosolic fraction of rabbit mammary gland. The relationship between this binding protein and the membrane PRL receptor is not yet known. 相似文献
20.
L Bonamusa I V Baanante 《Comparative biochemistry and physiology. B, Comparative biochemistry》1990,95(2):295-301
1. Skeletal muscle glycogen phosphorylase b has been purified from Liza ramada (mullet). 2. The Mr of the purified enzyme subunit was found to be 97,000. By gel filtration a relative Mr of 190,000 was found. 3. Proteolytic digestion of 32P-phosphorylated mullet phosphorylase gave a [32P]-labelled peptide which is observed to contain Ser, its sequence being -Gln-Ile-Ser-Val-Pro-. 4. During 'in vitro' phosphorylation of mullet phosphorylase, 32P was incorporated in different protein bands resolved by isoelectric focusing. The degree of radioactivity associated with each one changed with the incubation time. 相似文献