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1.
Cytokines are known to tip the balance of the coagulant-anticoagulant molecules on the endothelial cell surface toward intravascular coagulation. Their effects on endothelial cell surface-associated heparin-like compounds have not been examined yet. Incorporation of [35S]sulfate into heparan sulfate on cultured porcine aortic endothelial cells was suppressed by human recombinant interleukin-1 beta (rIL-1 beta) or tumor necrosis factor alpha (rTNF alpha) in a dose- and time-dependent manner with little effect on cell number, protein content, and [3H]leucine incorporation of cells. Maximal inhibition was achieved by incubation of cells with 100 ng/ml of rIL-1 beta or 5 ng/ml of rTNF alpha for 12-24 hours, resulting in a reduction of the synthesis of heparan sulfate on the cell surface by approximately 50%. The dose dependency was consistent with that seen in the stimulation of endothelial cell procoagulant activity by each cytokine. The suppression of heparan sulfate synthesis was sustained for at least 48 hours after pretreatment of cells with cytokines and was unchanged after the addition of indomethacin or polymyxin B. The rate of degradation of prelabeled 35S-heparan sulfate on the cell surface was not altered by cytokine treatments. Neither the size, the net negative charge, nor the proportion of the molecule with high affinity for antithrombin III of endothelial cell heparan sulfate was changed by cytokines. Furthermore, specific binding of 125I-labeled antithrombin III to the endothelial cell surface was reduced to 40-60% of control by cytokines. In parallel with reduction in binding, antithrombin III cofactor (heparin-like) activity was partially diminished in cytokine-treated endothelial cells. Thus, cytokine-mediated suppression of heparin-like substance on endothelial cells appears to be another cytokine-inducible endothelial effects affecting coagulation. 相似文献
2.
Lack of acidic fibroblast growth factor activation by heparan sulfate species from diabetic rat skin
Marie-Claude Bourin 《Glycoconjugate journal》1997,14(4):423-432
The glucosaminoglycans isolated from the skin of control and streptozotocin-diabetic rats were fractionated on ion-exchange
chromatography into a heparan sulfate (HS)-like and a heparin-like species. In addition, a low sulfated fraction was isolated
from the diabetics. The HS and heparin-like fractions isolated from the diabetics (in contrast to the low sulfated fractions)
retained high affinity for the acidic (FGF-1) and basic (FGF-2) fibroblast growth factors. In culture, the fractions purified
from the control rats and the heparin-like material isolated from the diabetics mediated the biological activity of both FGFs
in a dose-dependent manner. By contrast, the diabetic HS-like fractions promoted the biological activity of FGF-2 but not
of FGF-1. The results support the idea that the structural motives in HS required for FGF-1 and FGF-2 mediated receptor signalling
are different. They may be relevant to the impaired wound healing observed in the disease.
This revised version was published online in November 2006 with corrections to the Cover Date. 相似文献
3.
M Piepkorn P Hovingh W M Hentschel 《Biochemical and biophysical research communications》1988,151(1):327-332
The heparan sulfates synthesized in vitro by three cell lines were isolated by proteolysis and preparative anion exchange chromatography and purified free of other glycosaminoglycans by selective enzymatic degradation. The isolates from the medium of BALB/c 3T3 fibroblasts, B16.F10 melanoma cells, and a cutaneous fibrosarcoma line, along with that from the detergent-extracted cell layer of the fibroblasts, were affinity-fractionated on columns of matrix-immobilized human antithrombin III. Each heparan sulfate contained subfractions with high affinity for the proteinase inhibitor, ranging from 3-34% of the starting material. The high affinity species possessed measurable anticoagulant activities by a clotting assay (6 to 30 units/mg). Since none of the lines were derived from cell types having any known biological role in vascular homeostasis, we suggest that anticoagulant activity of the glycosaminoglycan is a random property of its primary structure. 相似文献
4.
Binding of thrombin to thrombomodulin accelerates inhibition of the enzyme by antithrombin III. Evidence for a heparin-independent mechanism 总被引:4,自引:0,他引:4
The endothelial cell surface provides a receptor for thrombin-designated thrombomodulin (TM) which regulates thrombin formation and the activity of the enzyme at the vessel wall surface by serving as a potent cofactor for the activation of protein C by thrombin. Heparin-like structures of the vessel wall have been proposed as another regulatory mechanism catalyzing the inhibition of thrombin by antithrombin III. In the present study, the interaction of antithrombin III with the thrombin-TM complex and its interference with heparin and polycations were investigated by using human components and TM isolated from the microvasculature of rabbit lung. Purified TM bound thrombin and acted as a cofactor for protein C activation. The addition of heparin (0.5 unit/mL) to the reaction mixture interfered neither with the binding of thrombin to TM nor with the activation of protein C. However, the polycations protamine (1 unit/mL) as well as polybrene (0.1 mg/mL) affected the thrombin-TM interaction. This was documented by an increase in the Michaelis constant from 8.3 microM for thrombin alone to 19.5 microM for thrombin-TM with the chromogenic substrate compound S-2238 in the presence of 1 unit/mL protamine. When the inhibition of thrombin by antithrombin III was determined, the second-order rate constant k2 = 8.4 X 10(3) M-1 s-1 increased about 8-fold in the presence of TM, implying an accelerative function of TM in this reaction. Although purified TM did not bind to antithrombin III-Sepharose, suggesting the absence of heparin-like structures within the receptor molecule, protamine reversed the accelerative effect of TM in the inhibition reaction.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
5.
Anticoagulantly active heparin-like molecules from vascular tissue 总被引:28,自引:0,他引:28
Mucopolysaccharides were isolated from calf cerebral microvasculature and calf aorta. The only complex carbohydrates that exhibited anticoagulant activity were heparin-like components. The biologic potencies of calf cerebral and aortic heparin-like species were 2.92 units/mg of anti-factor Xa activity and 2.85 units/mg of anti-factor IIa activity, as well as 0.56 unit/mg of anti-factor Xa activity and 0.19 unit/mg of anti-factor IIa activity, respectively. Additional experiments revealed that the anticoagulantly active aortic components were significantly present only within the intima. The above populations of heparin-like species were affinity fractionated with antithrombin. The highly active component obtained from calf cerebral microvasculature exhibited an anti-factor Xa activity of 40.7 units/mg as well as an anti-factor IIa activity of 36.8 units/mg, constituted about 4.2% of the initial mass of the starting material, and represented about 75% of the biologic potency of the starting material. The highly active component derived from calf aorta exhibited an anti-factor Xa activity of 55.4 units/mg as well as an anti-factor IIa activity of 11.3 units/mg, constituted about 0.3% of the initial mass of the starting material, and represented about 60% of the biologic potency of the starting material. The highly active cerebral microvascular species possessed a molecular weight and charge density similar to that of heparan sulfate whereas the highly active aortic species displayed a molecular weight and charge density equivalent to that of a hexadecasaccharide fragment of heparin. 相似文献
6.
Previous studies have shown that placental protein 5 (PP5) forms complexes with heparin. In order to further elucidate the biological role of PP5 we studied the effect of plasmin and thrombin on the immunoreactivity of PP5, and the possible functional antiplasmin and antithrombin effects of purified PP5. Varying concentrations of plasmin and thrombin were added to pregnancy plasma, and the PP5 levels, measured by radioimmunoassay, were found to be elevated by 558% (plasmin and 48–87% (thrombin). Incubation of radiolabeled PP5 with plasmin resulted in the formation of radioactive fragments with smaller molecular weights. Functional studies using a chromogenic substrate confirmed that purified PP5 has an anti-plasmin activity. An average increase of 15% was observed in the antiplasmin activity when 200 ng purified PP5 was added to 150 μl of pregnancy serum. Thus, there are certain similarities between PP5 and antithrombin III. Both form complexes with heparin and have antiplasmin properties, and both were found to be heat labile. But, functional studies utilizing a chromogenic substrate failed to demonstrate any antithrombin III-like activity in the purified PP5 preparation that had antiplasmin activity. Our results show that the function of PP5 is related to the blood coagulation and fibrinolytic systems, at least through its inhibitory action on plasmin. 相似文献
7.
Kazimírová M Jancinová V Petríková M Takác P Labuda M Nosál' R 《Experimental & applied acarology》2002,28(1-4):97-105
The tropical bont tick, Amblyomma variegatum can cause intense skin irritation and inflammation and bites that often develop into septic wounds or abscess in their host.
Crude salivary gland extract (SGE) of partially engorged A. variegatum females as well as SGE protein fractions purified by three-step reverse phase HPLC procedure were tested for their anti-aggregatory
effect on isolated human blood platelets stimulated with thrombin and compared with the effect of recombinant hirudin. At
concentrations 10−3 and 5 × 10−3 μg protein/ml the following rank order of antiplatelet activity was detected: AV 16/3 (inhibitor purified from AV-III, third
purification) > SGE > AV-II (fraction from first purification) > AV-III (fraction from first purification) > hirudin. The
effect of all fractions tested was dose-dependent. For fraction AV 16/3, the inhibitory effect was 49 and 61% for 10−3 and 5 × 10−3 μg protein/ml, respectively. The results suggest that protein fractions from A. variegatum SGE possess an antithrombin effect on human blood platelets with hirudin-like activity.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
8.
Membrane Anomalies in Huntington''s Disease Fibroblasts 总被引:1,自引:0,他引:1
Friedhelm Schroeder Ingeburg E. Goetz † Eugene Roberts† 《Journal of neurochemistry》1984,43(2):526-539
Plasma membranes, microsomes, and mitochondria were isolated from paired, passage number matched, cultured human fibroblasts. The cells were obtained from skin biopsies of Huntington's disease (HD) subjects and from sex and age matched controls. All fibroblasts were cultured in identical media for three to seven passages. Enrichment of surface marker enzymes such as Na+,K+-ATPase indicated a 10-fold purification of the isolated plasma membrane. The specific activity of Na+,K+-ATPase was 62 and 82% greater in the crude homogenate and isolated plasma membrane, respectively, of HD fibroblasts than in control fibroblasts. The specific activity of plasma membrane Na+,K+-ATPase was correlated with lipid composition and with membrane structure as determined by measurement of the rotational relaxation time and limiting anisotropy of fluorescence probe molecules. Major alterations in the structure of the plasma membranes in HD fibroblasts were not noted. The rotational relaxation time and limiting anisotropy of 1,6-diphenyl-1,3,5-hexatriene and of trans-parinaric acid were not significantly different between the plasma membrane, microsomes, or mitochondria of HD versus those of control fibroblasts. trans-Parinaric acid demonstrated the coexistence of fluid and solid domains in all three subcellular membrane fractions of the normal and HD skin fibroblasts. Lastly, both trans-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene displayed characteristic breakpoints in Arrhenius plots of absorbance corrected fluorescence in plasma membranes, microsomes, and mitochondria. In all cases, similar breakpoint temperatures, indicative of phase alterations, were noted near 20 degrees and 30 degrees C. These breakpoints were unaltered in HD. In summary, the data do not support the concept of major membrane structural defects in HD. 相似文献
9.
A new form of high affinity fibroblast growth factor receptor has been purified from adult bovine brain membranes. Purification was performed by chromatography on DEAE-Trisacryl and wheat germ agglutinin-agarose followed by FGF-2 affinity chromatography. Affinity labeling of purified fractions with 125I-FGF-2 showed after cross-linking a 170-kDa complex, suggesting the existence of a 150-kDa FGF receptor. No cross-reactivity with anti-FGF receptor 1 (FGFR-1 or flg) or with anti-receptor 2 (FGFR-2 or bek) antibodies could be detected with this partially purified receptor. Heparitinase treatment of the partially purified FGF receptor abolished the formation of the ligand receptor complex. The complex was restored in the presence of heparin in a dose dependent fashion, supporting the idea that heparin-like molecules are needed for proper binding. Further purification of the receptor was achieved by heparin-Sepharose affinity chromatography and yielded a purification of over 320,000-fold. The purified receptor fraction was radiolabeled and loaded on RPLC C4 column. Eluted fractions were analysed by SDS-PAGE. A major 150-kDa band was detected. These data show for the first time a new form of FGF receptor isolated from bovine brain membranes. This purified receptor displays affinity for heparin and was therefore named heparin binding FGF receptor (HB-FGFR). It remains unclear whether the receptor is a proteo-heparin sulfate or whether heparans are strongly associated and therefore are copurified. Large scale preparations are in progress for core protein structure studies. 相似文献
10.
Rishab K. Gupta A. Marilyn Leitch Donald L. Morton 《Cancer immunology, immunotherapy : CII》1999,16(1):40-47
Summary Immune complexes (IC) were isolated from plasma of melanoma patients by absorption to staphylococcal protein A and subsequent elution with MgCl2. The isolated ICs were purified by precipitation with polyethylene glycol and sucrose density gradient ultracentrifugation after radioiodination with 125I. The purified ICs were dissociated and radiolabeled antigen/antibody components were separated by ultracentrifugation at low pH (2.6). Under these conditions, about 72% radioactivity of the purified IC remained in the light-density region as a wide band. After neutralization, 26%–60% radioactivity in the region of 5S sedimentation bound to immobilized autologous immunoglobulins, as opposed to a maximum of 23% to immobilized immunoglobulins from human normal serum. Significant levels (73%–77%) of radioactivity in 7S region bound to rabbit anti-human IgG immunobeads. Immunoprecipitation of the antigen fraction by allogeneic anti-melanoma and rabbit anti-melanoma antibodies followed by SDS-polyacrylamide gel electrophoresis revealed the presence of a fetal antigen (FA) and a melanoma tumor-associated antigen (TAA). In addition, the presence of auto-antigen(s) was indicated by using autologous antibody in immunoprecipitation. Immunoglobulins (IgG) isolated from purified IC bound to cultured melanoma, sarcoma, and normal fibroblasts, although the binding to sarcoma and normal fibroblasts could be inhibited by preincubation of isolated IgG with soluble FA but not with soluble melanoma TAA. Thus, results of this investigation provide evidence that circulating IC in melanoma patients are composed of at least IgG and different antigens, and some of these antigens are produced by their tumor. 相似文献
11.
Gupta Rishab K. Leitch A. Marilyn Morton Donald L. 《Cancer immunology, immunotherapy : CII》1983,16(1):40-47
Immune complexes (IC) were isolated from plasma of melanoma patients by absorption to staphylococcal protein A and subsequent elution with MgCl2. The isolated ICs were purified by precipitation with polyethylene glycol and sucrose density gradient ultracentrifugation after radioiodination with 125I. The purified ICs were dissociated and radiolabeled antigen/antibody components were separated by ultracentrifugation at low pH (2.6). Under these conditions, about 72% radioactivity of the purified IC remained in the light-density region as a wide band. After neutralization, 26%-60% radioactivity in the region of 5S sedimentation bound to immobilized autologous immunoglobulins, as opposed to a maximum of 23% to immobilized immunoglobulins from human normal serum. Significant levels (73%-77%) of radioactivity in 7S region bound to rabbit anti-human IgG immunobeads. Immunoprecipitation of the antigen fraction by allogeneic anti-melanoma and rabbit anti-melanoma antibodies followed by SDS-polyacrylamide gel electrophoresis revealed the presence of a fetal antigen (FA) and a melanoma tumor-associated antigen (TAA). In addition, the presence of auto-antigen(s) was indicated by using autologous antibody in immunoprecipitation. Immunoglobulins (IgG) isolated from purified IC bound to cultured melanoma, sarcoma, and normal fibroblasts, although the binding to sarcoma and normal fibroblasts could be inhibited by preincubation of isolated IgG with soluble FA but not with soluble melanoma TAA. Thus, results of this investigation provide evidence that circulating IC in melanoma patients are composed of at least IgG and different antigens, and some of these antigens are produced by their tumor. 相似文献
12.
The intracellular localization of exogenously supplied human platelet beta-glucuronidase in cultured skin fibroblasts derived from a beta-glucuronidase-deficient patient was studied. Four cellular fractions were obtained by differential speed centrifugation. Following two days of incubation, the exogenously supplied enzyme exhibited a distribution pattern identical to that of endogenous beta-hexosaminidase. Disruption of membranes by freezing and thawing caused a 35% increase of the enzyme activity, thus indicating a latent activity following the internalization. This indicated localization in the lysosomal fractions. Longer incubation periods led to an intracellular shift of the engulfed enzyme from the lighter lysosomal fraction to heavier particles. Once located in the heavier fraction, the enzyme was relatively stable, and participated in the catabolism of 35S-labeled mucopolysaccharides which had accumulated in the lysosomes of these fibroblasts. A marked reduction in the accumulated mucopolysaccharides of the lysosomal fraction was observed following addition of the enzyme. This was accompanied by the formation of smaller sized molecules. 相似文献
13.
Susan B. Edelstein Xandra O. Breakefield 《Biochemical and biophysical research communications》1981,98(3):836-843
The effects of several hormones known to affect monoamine oxidase activity have been studied in living human skin fibroblasts grown in culture. Of the hormones tested, the synthetic glucocorticoid dexamethasone caused the greatest increases in activity at physiologic concentrations. Increases of 10–12 fold were observed after 8–9 days of exposure to 5 × 10?8 M dexamethasone. This increase in activity was accompanied by a change in the relative proportion of the A and B types of activity in fibroblasts, from about 35% A:65% B in control cultures to 90% A:10% B in cultures exposed to dexamethasone. The increase in activity and the shift in the proportion of A and B activities could be accounted for almost exclusively by a specific increase in the number of Type A molecules. 相似文献
14.
Cultured human foreskin fibroblasts produce a factor that stimulates their growth with properties similar to basic fibroblast growth factor 总被引:2,自引:0,他引:2
Michael T. Story 《In vitro cellular & developmental biology. Plant》1989,25(5):402-408
Summary To determine if fibroblasts could be a source of fibroblast growth factor (FGF) in tissue, cells were initiated in culture
from newborn human foreskin. Cells were studied in Passages 2 to 8. Fibroblast cell lysates promoted radiolabeled thymidine
uptake by cultured quiescent fibroblasts. Seventy-nine percent of the growth-promoting activity of lysates was recovered from
heparin-Sepharose. The heparin-binding growth factor reacted on immunoblots with antiserum to human placenta-derived basic
FGF and competed with iodinated basic FGF for binding to antiserum to (1-24)bFGF synthetic peptide. To confirm that fibroblasts
were the source of the growth factor, cell lysates were prepared from cells incubated with radiolabeled methionine. Heparin
affinity purified material was immunoprecipitated with basic FGF antiserum and electrophoresed. Radiolabeled material was
detected on gel autoradiographs in the same molecular weight region as authentic iodinated basic FGF. The findings are consistant
with the notion that cultured fibroblasts express basic FGF. As these cells also respond to the mitogen, it is possible that
the regulation of their growth is under autocrine control. Fibroblasts may be an important source of the growth factor in
tissue.
Supported in part by grant DK 31063 from the National Institutes of Health, Bethesda, MD. 相似文献
15.
Activation of heparin cofactor II by heparin oligosaccharides 总被引:1,自引:0,他引:1
M M Maimone D M Tollefsen 《Biochemical and biophysical research communications》1988,152(3):1056-1061
Heparin was partially depolymerized with heparinase or nitrous acid. The resulting oligosaccharides were fractionated by gel filtration chromatography and tested for the ability to stimulate inhibition of thrombin by purified heparin cofactor II or antithrombin. Oligosaccharides containing greater than or equal to 18 monosaccharide units were active with antithrombin, while larger oligosaccharides were required for activity with heparin cofactor II. Intact heparin molecules fractionated on a column of immobilized antithrombin were also tested for activity with both inhibitors. The relative specific activities of the unbound heparin molecules were 0.06 with antithrombin and 0.76 with heparin cofactor II in comparison to unfractionated heparin (specific activity = 1.00). We conclude that heparin molecules much greater than 18 monosaccharide units in length are required for activity with heparin cofactor II and that the high-affinity antithrombin-binding structure of heparin is not required. 相似文献
16.
G Mertens J J Cassiman H Van den Berghe J Vermylen G David 《The Journal of biological chemistry》1992,267(28):20435-20443
Human aortic endothelial cells (HAEC) and human umbilical vein endothelial cells (HUVEC) were labeled with 35SO(4)2- for 48 h. The membrane-associated proteoglycans were solubilized from these monolayers with detergent and purified by ion-exchange chromatography on Mono Q, incorporation in liposomes, and gel filtration. The liposome-intercalated proteoglycans were 125I-iodinated and treated with heparitinase before SDS-polyacrylamide gel electrophoresis. Radio-labeled proteins with apparent molecular masses of 130, 60, 46, 35, and 30 kDa (HAEC) and 180, 130, 62, 43, and 35 kDa (HUVEC) were detected by autoradiography. Further characterization by affinity chromatography on immobilized monoclonal antibodies and by Northern blot analysis provided evidence for the expression of syndecan, glypican, and fibroglycan in human endothelial cells. Most of the heparan sulfate which accumulated in the subendothelial matrix was implanted on a 400-kDa core protein. This protein was immunologically related to perlecan and bound to fibronectin. Binding studies on immobilized antithrombin III suggested that all membrane-associated heparan sulfate proteoglycan forms had the capacity to bind to antithrombin III but that high affinity binding was more typical for glypican. Most of the proteoglycans isolated from the extracellular matrix also bound only with low affinity to antithrombin III. These results imply that glypican may specifically contribute to the antithrombotic properties of the vascular wall. 相似文献
17.
The effects of low molecular weight (LMW) protease inhibitors of microbial origin were evaluated on the intracellular degradation of beta-galactosidase purified from Aspergillus oryzae and taken up by cultured human skin fibroblasts with beta-galactosidase deficiency. Only thiol protease inhibitors showed an effect to increase the enzyme activity. E-64, a specific inhibitor of thiol proteases, prolonged 3-fold a half life of the exogenous beta-galactosidase and when the enzyme was supplied as liposomes, the half life was prolonged 9-fold in these cells. The role of thiol proteases in the degradation of enzyme molecules was discussed. 相似文献
18.
Thrombin-inhibitory activity of whale heparin oligosaccharides 总被引:1,自引:0,他引:1
Whale heparin was partially digested with a purified heparinase and the oligosaccharide fractions with 8-20 monosaccharide units were isolated from the digest by gel filtration on Sephadex G-50, followed by affinity chromatography on a column of antithrombin III immobilized on Sepharose 4B. A marked difference in the inhibitory activity for thrombin in the presence of antithrombin III was observed between the high-affinity fractions for antithrombin III of octasaccharide approximately hexadecasaccharide and those of octadecasaccharide approximately eicosasaccharide. The disaccharide compositions of these hexadeca-, octadeca-, and eicosasaccharides were analyzed by high-performance liquid chromatography after digestion with a mixture of purified heparitinases 1 and 2 and heparinase. The analytical data indicated that the proportions of trisulfated disaccharide (IdUA(2S)alpha 1----4GlcNS(6S)) and disulfated disaccharide (UA1----4GlcNS(6S)) increased with the manifestation of high thrombin-inhibitory activity, while that of monosulfated disaccharide (UA1----4GlcNS) decreased. The present observations, together with those so far reported, suggest that the presence of the former structural elements, specifically IdUA(2S)alpha 1----4GlcNS(6S), as well as the antithrombin III-binding pentasaccharide at the proper positions in the molecules of whale heparin oligosaccharides is essential for the manifestation of high inhibitory activity for thrombin in the presence of antithrombin III. The structural bases for the manifestation of the anticoagulant activity of whale and porcine heparins and their oligosaccharides are also discussed. 相似文献
19.
Structure and interactions of proteoglycans in the extracellular matrix produced by cultured human fibroblasts. 总被引:7,自引:0,他引:7
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S Johansson K Hedman L Kjelln J Christner A Vaheri M Hk 《The Biochemical journal》1985,232(1):161-168
Subconfluent cultures of human embryonic skin fibroblasts were labelled with [35S]sulphate for 3 days, after which cell-free extracellular matrix was isolated. A chondroitin sulphate proteoglycan (CSPG) and a heparan sulphate proteoglycan (HSPG) were purified from the matrix. Chromatography on Sepharose CL-2B gave peak Kav. values of 0.35 and 0.38 respectively for the CSPG and the HSPG. The polysaccharide chains released from the two PGs were of similar size (Kav. 0.50 on Sepharose CL-4B). Approx. 50% of the CSPG showed affinity for hyaluronic acid (HA). However, it differed immunologically from the HA-aggregating CSPG of human articular cartilage, and had a larger core protein (apparent molecular mass 290 kDa) than had the cartilage PG. Neither metabolically [35S]sulphate-labelled PGs, isolated from the medium of fibroblast cultures, nor chemically 3H-labelled polysaccharides (HA, CS, HS and heparin) were incorporated into the extracellular matrix when added to unlabelled cell cultures. These results indicate that the matrix PGs are not derived from the PGs present in the medium and that an interation between polysaccharide chains and matrix components is not sufficient for incorporation of PGs into the matrix. Incubation of cell-free 35S-labelled matrix with unlabelled polysaccharides did not lead to the release of any 35S-labelled material, supporting this conclusion. Furthermore, so-called 'link proteins' were not present in the fibroblast cultures, indicating that the CSPGs were anchored in the matrix in a manner different from the link-stabilized association of CSPG with HA in chondrocyte matrix. The identification of a proteinase, secreted by fibroblasts in culture, that after activation with heparin has the ability to release 35S-labelled PGs from the matrix may also indicate that the core proteins are important for the association of the PGs to the matrix. 相似文献
20.
Anticoagulantly active heparin from clam (Mercenaria mercenaria) 总被引:1,自引:0,他引:1
Heparin was isolated from Mercenaria mercenaria by ion-exchange chromatography and was fractionated into two distinct populations with immobilized antithrombin. The high-affinity glycosaminoglycan accelerated dramatically the inhibition of purified human factors IIa and Xa via purified human antithrombin. Specific anti-factor IIa and anti-factor Xa activities were 363 and 348 U.S.P. units/mg, respectively. The highly active clam heparin exhibited a molecular weight of approximately 18,000 and contained approximately 2.5 sulfate groups per disaccharide. The intrinsic fluorescence of purified human antithrombin was enhanced in the presence of the high-affinity invertebrate glycosaminoglycan to an extent comparable to the level induced by vertebrate heparin. In addition, the critical tetrasaccharides containing 3-O-sulfated glucosamine residues, which constitute part of the unique antithrombin-binding domain of mammalian heparin, were also detected in high-affinity Mercenaria heparin. 相似文献