Degradation of Cell Surface Heparan Sulfates Decreases the High Affinity Binding of Basic FGF to Endothelial Cells, but Not to FRTL-5 Rat Thyroid Cells

Thyroid ◽  
1995 ◽  
Vol 5 (6) ◽  
pp. 455-460 ◽  
Author(s):  
NAOYA EMOTO ◽  
OSAMU ISOZAKI ◽  
EIJI OHMURA ◽  
KAZUO SHIZUME ◽  
TOSHIO TSUSHIMA ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Affinity Binding ◽  
Thyroid Cells ◽  
High Affinity Binding ◽  
Basic Fgf ◽  
High Affinity ◽  
Heparan Sulfates ◽  
Rat Thyroid

scholarly journals Polarized fibronectin secretion and localized matrix assembly sites correlate with subendothelial matrix formation

Blood ◽  
1990 ◽  
Vol 75 (12) ◽  
pp. 2335-2342 ◽  
Author(s):  
AP Kowalczyk ◽  
RH Tulloh ◽  
PJ McKeown-Longo
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Binding Sites ◽  
Affinity Binding ◽  
Apical Surface ◽  
Matrix Assembly ◽  
High Affinity Binding ◽  
High Affinity ◽  
Fibronectin Matrix ◽  
Subendothelial Matrix

Abstract Endothelial cells in vivo form the interface between the vascular and interstitial compartments and are strategically located to mediate vascular permeability and hemostasis. One mechanism endothelial cells use to maintain a nonthrombogenic surface is to polarize basement membrane constituents to the basolateral cell surface. In the present study, we began characterization of the mechanisms used by endothelial cells in the assembly of a subcellular fibronectin matrix. Immunofluorescence microscopy was used to localize extracellular matrix fibronectin in endothelial cell cultures. In contrast to preconfluent and newly confluent cultures, post-confluent cultures assembled a fibronectin matrix that was restricted to the basolateral cell surface. To determine if endothelial cells polarize fibronectin secretion, Millicell culture inserts were used to distinguish proteins secreted from apical and basal surfaces. Preconfluent and newly confluent cultures secreted fibronectin equally into apical and basal media. In contrast, post-confluent endothelial cells secreted fibronectin preferentially into the basal chamber. The degree to which fibronectin secretion was polarized varied with time in culture and with the ability of the monolayers to act as a barrier to the movement of 125I- fibronectin from the apical to basal chamber. In addition, high affinity binding sites for exogenous 125I-fibronectin were found to be present on the basolateral, but not apical, surface of post-confluent endothelial monolayers. These results indicate that subendothelial matrix assembly correlates with polarized fibronectin secretion, culture confluence, and expression of high affinity binding sites for fibronectin on the basolateral cell surface.

scholarly journals Polarized fibronectin secretion and localized matrix assembly sites correlate with subendothelial matrix formation

Blood ◽  
1990 ◽  
Vol 75 (12) ◽  
pp. 2335-2342
Author(s):  
AP Kowalczyk ◽  
RH Tulloh ◽  
PJ McKeown-Longo
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Binding Sites ◽  
Affinity Binding ◽  
Apical Surface ◽  
Matrix Assembly ◽  
High Affinity Binding ◽  
High Affinity ◽  
Fibronectin Matrix ◽  
Subendothelial Matrix

Endothelial cells in vivo form the interface between the vascular and interstitial compartments and are strategically located to mediate vascular permeability and hemostasis. One mechanism endothelial cells use to maintain a nonthrombogenic surface is to polarize basement membrane constituents to the basolateral cell surface. In the present study, we began characterization of the mechanisms used by endothelial cells in the assembly of a subcellular fibronectin matrix. Immunofluorescence microscopy was used to localize extracellular matrix fibronectin in endothelial cell cultures. In contrast to preconfluent and newly confluent cultures, post-confluent cultures assembled a fibronectin matrix that was restricted to the basolateral cell surface. To determine if endothelial cells polarize fibronectin secretion, Millicell culture inserts were used to distinguish proteins secreted from apical and basal surfaces. Preconfluent and newly confluent cultures secreted fibronectin equally into apical and basal media. In contrast, post-confluent endothelial cells secreted fibronectin preferentially into the basal chamber. The degree to which fibronectin secretion was polarized varied with time in culture and with the ability of the monolayers to act as a barrier to the movement of 125I- fibronectin from the apical to basal chamber. In addition, high affinity binding sites for exogenous 125I-fibronectin were found to be present on the basolateral, but not apical, surface of post-confluent endothelial monolayers. These results indicate that subendothelial matrix assembly correlates with polarized fibronectin secretion, culture confluence, and expression of high affinity binding sites for fibronectin on the basolateral cell surface.

scholarly journals Mechanism of thrombin binding to endothelial cells

Blood ◽  
1983 ◽  
Vol 61 (2) ◽  
pp. 368-372 ◽  
Author(s):  
PT Bauer ◽  
R Machovich ◽  
P Aranyi ◽  
KG Buki ◽  
E Csonka ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Binding Sites ◽  
Antithrombin Iii ◽  
Affinity Binding ◽  
Heparin Binding ◽  
High Affinity Binding ◽  
Aortic Endothelial Cells ◽  
High Affinity ◽  
Lysine Residues ◽  
Two Populations

Abstract The interaction of human alpha-thrombin with mini-pig aortic endothelial cells was studied using 125I-labeled enzyme. Equilibrium between bound and free thrombin was attained within 1 min, and the Klotz-Hunston equations indicated two populations of binding sites. Approximately 30,000 sites/cell belonged to the high-affinity class with a Kd of about 3 x 10(-8) M. Modification of two lysine residues of thrombin with pyridoxal 5′-phosphate (PLP2-thrombin) destroyed the high- affinity binding and about three-fourths of the low-affinity bindings. When the lysine residue of thrombin involved in heparin binding was protected with heparin against chemical modification (PLP-thrombin), the modified enzyme remained similar to the native one with respect to cellular binding, with some loss of low-affinity binding only. Heparin, in a tenfold molar excess to enzyme, inhibited the binding of the native as well as the PLP-thrombin, whereas it did not influence the interaction between PLP2-thrombin and the cell. Since heparin might interfere with both the enzyme and the cell, the binding of heparin to endothelial cells was also examined. The results revealed that 3H- heparin also bound to cells. This binding was characterized by a Kd of 3 x 10(-7) M, approximately 10(6) sites/cell. Furthermore, thrombin bound to endothelial cells was released by antithrombin III. On the basis of these and other data in the literature, a model is proposed for the mechanism of the binding of thrombin to endothelial cells.

scholarly journals Interaction of the small interstitial proteoglycans biglycan, decorin and fibromodulin with transforming growth factor β

1994 ◽  
Vol 302 (2) ◽  
pp. 527-534 ◽  
Author(s):  
A Hildebrand ◽  
M Romarís ◽  
L M Rasmussen ◽  
D Heinegård ◽  
D R Twardzik ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Binding Site ◽  
Fusion Proteins ◽  
Transforming Growth Factor ◽  
Affinity Binding ◽  
Tgf Beta ◽  
High Affinity Binding ◽  
High Affinity ◽  
Core Proteins ◽  
High Affinity Binding Site

We have analysed the interactions of three proteoglycans of the decorin family, decorin, biglycan and fibromodulin, with transforming growth factor beta (TGF-beta). The proteoglycan core proteins, expressed from human cDNAs as fusion proteins with Escherichia coli maltose-binding protein, each bound TGF-beta 1. They showed only negligible binding to several other growth factors. Intact decorin, biglycan and fibromodulin isolated from bovine tissues competed with the fusion proteins for the TGF-beta binding. Affinity measurements suggest a two-site binding model with Kd values ranging from 1 to 20 nM for a high-affinity binding site and 50 to 200 nM for the lower-affinity binding site. The stoichiometry indicated that the high-affinity binding site was present in one of ten proteoglycan core molecules and that each molecule contained a low-affinity binding site. Tissue-derived biglycan and decorin were less effective competitors for TGF-beta binding than fibromodulin or the non-glycosylated fusion proteins; removal of the chondroitin/dermatan sulphate chains of decorin and biglycan (fibromodulin is a keratan sulphate proteoglycan) increased the activities of decorin and biglycan, suggesting that the glycosaminoglycan chains may hinder the interaction of the core proteins with TGF-beta. The fusion proteins competed for the binding of radiolabelled TGF-beta to Mv 1 Lu cells and endothelial cells. Affinity labelling showed that the binding of TGF-beta to betaglycan and the type-I receptors in Mv 1 Lu cells and to endoglin in endothelial cells was reduced, but the binding to the type-II receptors was unaffected. TGF-beta 2 and 3 also bound to all three fusion proteins. Latent recombinant TGF-beta 1 precursor bound slightly to fibromodulin and not at all to decorin and biglycan. The results show that the three decorin-type proteoglycans each bind TGF-beta isoforms and that slight differences exist in their binding properties. They may regulate TGF-beta activities by sequestering TGF-beta into extracellular matrix.

High-affinity binding measurements of antibodies to cell-surface-expressed antigens

2008 ◽  
Vol 373 (1) ◽  
pp. 52-60 ◽  
Author(s):  
Palaniswami Rathanaswami ◽  
John Babcook ◽  
Michael Gallo
Keyword(s):  
Cell Surface ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity

scholarly journals Mechanism of thrombin binding to endothelial cells

Blood ◽  
1983 ◽  
Vol 61 (2) ◽  
pp. 368-372
Author(s):  
PT Bauer ◽  
R Machovich ◽  
P Aranyi ◽  
KG Buki ◽  
E Csonka ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Binding Sites ◽  
Antithrombin Iii ◽  
Affinity Binding ◽  
Heparin Binding ◽  
High Affinity Binding ◽  
Aortic Endothelial Cells ◽  
High Affinity ◽  
Lysine Residues ◽  
Two Populations

The interaction of human alpha-thrombin with mini-pig aortic endothelial cells was studied using 125I-labeled enzyme. Equilibrium between bound and free thrombin was attained within 1 min, and the Klotz-Hunston equations indicated two populations of binding sites. Approximately 30,000 sites/cell belonged to the high-affinity class with a Kd of about 3 x 10(-8) M. Modification of two lysine residues of thrombin with pyridoxal 5′-phosphate (PLP2-thrombin) destroyed the high- affinity binding and about three-fourths of the low-affinity bindings. When the lysine residue of thrombin involved in heparin binding was protected with heparin against chemical modification (PLP-thrombin), the modified enzyme remained similar to the native one with respect to cellular binding, with some loss of low-affinity binding only. Heparin, in a tenfold molar excess to enzyme, inhibited the binding of the native as well as the PLP-thrombin, whereas it did not influence the interaction between PLP2-thrombin and the cell. Since heparin might interfere with both the enzyme and the cell, the binding of heparin to endothelial cells was also examined. The results revealed that 3H- heparin also bound to cells. This binding was characterized by a Kd of 3 x 10(-7) M, approximately 10(6) sites/cell. Furthermore, thrombin bound to endothelial cells was released by antithrombin III. On the basis of these and other data in the literature, a model is proposed for the mechanism of the binding of thrombin to endothelial cells.

Sign in / Sign up

Export Citation Format

Share Document