Binding of the Nucleoside Transport Inhibitor 4-Nitrobenzylthioinosine to Erythrocyte Membranes

1973 ◽  
Vol 51 (5) ◽  
pp. 666-672 ◽  
Author(s):  
M. A. Pickard ◽  
R. R. Brown ◽  
B. Paul ◽  
A. R. P. Paterson
Keyword(s):  
Binding Sites ◽  
Nucleoside Transport ◽  
Erythrocyte Membranes ◽  
Affinity Binding ◽  
Inhibitor Molecule ◽  
High Affinity Binding ◽  
High Affinity ◽  
Transport Inhibitor ◽  
Binding Data ◽  
Nucleoside Transport Inhibitor

4-Nitrobenzylthioinosine (NBMPR), a potent nucleoside transport inhibitor, was prepared in two radioactive forms and the binding of these to erythrocyte ghosts was studied. Similar binding data were obtained with inhibitor containing 14C in the purine 8-position or in the benzyl 7-position, suggesting that the entire inhibitor molecule was bound. A saturable high-affinity mode of NBMPR binding was apparent; NBMPR bound in this way was not removed by washing, but was displaced by a related inhibitor of nucleoside transport, 2-hydroxy-5-nitrobenzylthioguanosine (HNBTGR). It is postulated that the high-affinity binding sites are the nucleoside transport elements of the erythrocyte membrane. From ghosts treated with 14C-NBMPR under conditions which assured binding of the high affinity type, 14C was recovered by extractions in the form of NBMPR. Thus, this mode of NBMPR binding is reversible and covalent linkages do not appear to be involved. A low affinity mode of NBMPR binding was also demonstrated; this appeared to be a partition of NBMPR between the medium and the membrane substance. This component of bound NBMPR was not displaced by HNBTGR and was removed by washing.

scholarly journals Species differences in nucleoside transport. A study of uridine transport and nitrobenzylthioinosine binding by mammalian erythrocytes

1982 ◽  
Vol 208 (1) ◽  
pp. 83-88 ◽  
Author(s):  
S M Jarvis ◽  
J R Hammond ◽  
A R P Paterson ◽  
A S Clanachan
Keyword(s):  
Guinea Pig ◽  
Binding Sites ◽  
Species Differences ◽  
Cell Types ◽  
Nucleoside Transport ◽  
Kinetic Properties ◽  
Affinity Binding ◽  
Transport Capacity ◽  
High Affinity Binding ◽  
High Affinity

A kinetic study of the inward transport of uridine in erythrocytes of rabbit, human, mouse, rat and guinea-pig demonstrated that the apparent Km of this process was similar (about 0.2mM) in these cell types, but Vmax. values differed markedly. In this array of cell types, Vmax. values were proportional to the number of transport-inhibitory, high-affinity binding sites present per cell of each type. Transport of uridine or adenosine was not detected in dog erythrocytes, nor was saturable, high-affinity binding of nitrobenzylthioinosine demonstrable. These findings demonstrate that species differences in nucleoside transport capacity are attributable to differences in the cell-surface content of functional nucleoside transport sites, rather than to differences in the kinetic properties of these sites.

High Affinity Binding Sites for Activated Protein C and Protein C on Cultured Human Umbilical Vein Endothelial Cells

1994 ◽  
Vol 72 (03) ◽  
pp. 465-474 ◽  
Author(s):  
Neelesh Bangalore ◽  
William N Drohan ◽  
Carolyn L Orthner
Keyword(s):  
Binding Sites ◽  
Protein C ◽  
Umbilical Vein ◽  
Activated Protein C ◽  
Affinity Binding ◽  
Cell Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Gla Domain ◽  
Umbilical Vein Endothelial Cells

SummaryActivated protein C (APC) is an antithrombotic serine proteinase having anticoagulant, profibrinolytic and anti-inflammatory activities. Despite its potential clinical utility, relatively little is known about its clearance mechanisms. In the present study we have characterized the interaction of APC and its active site blocked forms with human umbilical vein endothelial cells (HUVEC). At 4° C 125I-APC bound to HUVEC in a specific, time dependent, saturable and reversible manner. Scatchard analysis of the binding isotherm demonstrated a Kd value of 6.8 nM and total number of binding sites per cell of 359,000. Similar binding isotherms were obtained using radiolabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone (PPACK) inhibited APC indicating that a functional active site was not required. Competition studies showed that the binding of APC, PPACK-APC and PC were mutually exclusive suggesting that they bound to the same site(s). Proteolytic removal of the N-terminal γ-carboxyglutamic acid (gla) domain of PC abolished its ability to compete indicating that the gla-domain was essential for cell binding. Surprisingly, APC binding to these cells appeared to be independent of protein S, a cofactor of APC generally thought to be required for its high affinity binding to cell surfaces. The identity of the cell binding site(s), for the most part, appeared to be distinct from other known APC ligands which are associated with cell membranes or extracellular matrix including phospholipid, thrombomodulin, factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment of HUVEC with antifactor VIII antibody caused partial inhibition of 125I-APC binding indicating that factor VIII or a homolog accounted for ∼30% of APC binding. Studies of the properties of surface bound 125I-APC or 125I-PC and their fate at 4°C compared to 37 °C were consistent with association of ∼25% of the initially bound radioligand with an endocytic receptor. However, most of the radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly at 37° C in an intact and functional state. These data indicate the presence of specific, high affinity binding sites for APC and PC on the surface of HUVEC. While a minor proportion of binding sites may be involved in endocytosis, the identity and function of the major proportion is presently unknown. It is speculated that this putative receptor may be a further mechanisms of localizing the PC antithrombotic system to the vascular endothelium.

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