Interfaces of Molecular Recognition Sites and Biosensors; Novel Bio-Sensing Materials

2015 ◽  
Vol 1793 ◽  
pp. 1-6
Author(s):  
Kyung M. Choi
Keyword(s):  
Binding Sites ◽  
Glass Surface ◽  
High Sensitivity ◽  
Affinity Binding ◽  
Sensing Element ◽  
Practical Applications ◽  
High Affinity ◽  
High Affinity Receptor ◽  
Number Of Binding Sites ◽  
Low Sensitivity

ABSTRACTWe introduce a sensing element, “Molecularly Imprinted Polymer (MIP),” which created by “Molecular Imprinted Technique.” However, the sensitivity of MIP’s based bio-sensors limits for practical applications due to the low sensitivity. To achieve a high sensitivity of MIP’s based sensors, the synthesis of “high affinity receptor or binding sites,” such as “monoclonal particles” is a key objective. In previous studies, affinity distribution plots indicated that “high affinity binding sites” were obtained when the number of binding sites per particle decreased. It means that smaller particles are expected to have higher affinity binding sites compared to larger particles. The result motivated us to produce small-sized MIP’s particles for the achievement of higher sensitivity. Microfluidic Synthesis has taken a great attention to synthesize small particles. However, the microfluidic synthesis gave us a difficulty, especially collections of MIP’s particles from the surface of PDMS-based microchannels due to a sticking problem. Thus, we employed a new approach, which can collect MIP’s particles without any sticking problem from the surface of the reactor. It is a photopatterned MIP’s system generated on the glass surface. We prepared a photomask with micro-sized patterns and then fabricate MIP’s particles on a glass surface by photopolymerization. Uniform MIP’s patterns were printed on the glass surface. The interface between the glass surface and the MIP’s pattern was observed by SEM. Micro-sized MIP’s particles were collected from the glass surface by scratching off the photocured MIP’s patterns.

The ‘ligand-induced conformational change’ of alpha 5 beta 1 integrin. Relocation of alpha 5 subunit to uncover the beta 1 stalk region

1998 ◽  
Vol 111 (12) ◽  
pp. 1759-1766 ◽  
Author(s):  
J. Tsuchida ◽  
S. Ueki ◽  
Y. Takada ◽  
Y. Saito ◽  
J. Takagi
Keyword(s):  
Conformational Change ◽  
Binding Sites ◽  
K562 Cells ◽  
Affinity Binding ◽  
Fab Fragment ◽  
High Affinity Binding ◽  
High Affinity ◽  
Number Of Binding Sites ◽  
Beta 1 Integrin ◽  
Beta1 Subunits

Integrin heterodimers undergo a conformational change upon the binding of ligand to their extracellular domains. An anti-beta1 integrin monoclonal antibody AG89 can detect such a conformational change since it recognizes a ligand-inducible epitope in the stalk-like region of beta1 subunits. The binding of a 125I-labeled AG89 Fab fragment to alpha5 beta1 integrins on K562 cells was assessed and analyzed by the Scatchard method. High affinity binding sites for AG89 are present on cells treated with ligand peptide. In addition, results revealed that cells treated with EDTA also express AG89 binding sites with the same affinity although the number of binding sites is 4-fold lower. AG89 immunoprecipitated alpha5 beta1 complexes from surface-labeled K562 cells treated with ligand peptide. By contrast, it immunoprecipitated only beta1 chains when the ligand peptide was absent, suggesting that high affinity binding sites on EDTA-treated cells are associated with non-functional beta1 monomer. Additional studies show that the epitope for AG89 is constitutively exposed on mutant beta1 that cannot complex with alpha5. These data suggest that the AG89 epitope is masked by the alpha5 subunit. Ligand binding and integrin activation may uncover the beta1 stalk region by triggering a conformational shift of alpha5 relative to beta1.

High-affinity calcium binding sites in luminal and basolateral renal membranes

1985 ◽  
Vol 248 (4) ◽  
pp. F472-F481
Author(s):  
Z. Talor ◽  
G. Richison ◽  
J. A. Arruda
Keyword(s):  
Binding Sites ◽  
Calcium Binding ◽  
Divalent Cations ◽  
Ruthenium Red ◽  
Affinity Constant ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Ca Uptake ◽  
Number Of Binding Sites

We evaluated Ca binding by highly purified luminal (L) and basolateral (BL) tubular membranes prepared from beef kidney. Ca binding was measured by using 45Ca and a rapid-filtration technique. After Ca uptake reached equilibrium, the vesicles were lysed and the amount of 45Ca retained in the membranes was considered the bound Ca. Ca binding in both membranes accounted for approx. 80% of total Ca uptake. Analysis of binding data by Scatchard plot revealed the presence of two distinct types of binding sites in both L and BL membranes. The high-affinity binding sites showed a similar affinity constant of 10(-5)M for both L and BL membranes, but the maximum number of binding sites was 0.75 and 1.6 nmol/mg protein, respectively. In contrast, the low-affinity binding sites were similar regarding affinity constant and maximum number of binding sites in the two membranes. In L and BL membranes, high-affinity binding sites were selective for Ca, as high concentrations of divalent cations were required to inhibit Ca binding. In both membranes Ca binding was inhibited by ruthenium red, LaCl3, and detergents, and it was stimulated by calmodulin inhibitors (trifluoperazine, calmidazolium), ionophore A-23187, and ATP. These results demonstrate that L and BL membranes possess high-affinity binding sites with different capacities but similar characteristics as regards affinity constant and stimulation and inhibition of binding. The data further demonstrate that most of Ca uptake by these membranes represents binding.

scholarly journals Co-internalization of the p55 and p70 subunits of the high-affinity human interleukin 2 receptor. Evidence for a stable ternary receptor complex.

1988 ◽  
Vol 168 (5) ◽  
pp. 1923-1928 ◽  
Author(s):  
M R Fung ◽  
G Ju ◽  
W C Greene
Keyword(s):  
Binding Sites ◽  
Interleukin 2 ◽  
Affinity Binding ◽  
Receptor Complex ◽  
Homogeneous Population ◽  
High Affinity ◽  
Membrane Complex ◽  
High Affinity Receptor ◽  
Affinity Receptor ◽  
Receptor Assembly

The high-affinity IL-2-R complex is composed of at least two distinct IL-2-binding subunits, including p55 (Tac, IL-2-R alpha) and p70 (IL-2-R beta). Using a radiolabeled mAb specific for the p55 receptor subunit and cells expressing a homogeneous population of high-affinity binding sites, we demonstrate that p55 is co-internalized with p70 after IL-2 binding to the receptor complex. Endocytosis of p55 depends upon the presence of IL-2 in a form capable of effectively interacting with the p70 subunit. Whether IL-2 is required for high-affinity receptor assembly or triggering of the internalization of preassembled receptors remains unresolved. Together, these findings support the existence of a stable, high-affinity human IL-2-R membrane complex composed of at least the p55 and p70 receptor subunits and IL-2.

Demonstration of specific dopamine receptors on human pituitary adenomas

1987 ◽  
Vol 114 (4) ◽  
pp. 595-602 ◽  
Author(s):  
Masafumi Koga ◽  
Haruyoshi Nakao ◽  
Masayo Arao ◽  
Bunzo Sato ◽  
Keizo Noma ◽  
...  
Keyword(s):  
Dopamine Receptors ◽  
Binding Sites ◽  
Pituitary Adenomas ◽  
Binding Capacity ◽  
Affinity Binding ◽  
Human Pituitary ◽  
Human Pituitary Adenoma ◽  
High Affinity ◽  
Number Of Binding Sites ◽  
Dissociation Constant Kd

Abstract. Dopamine receptors on human pituitary adenoma membranes were characterized using [3H] spiperone as the radioligand. The specific [3H]spiperone binding sites on prolactin (PRL)-secreting adenoma membranes were recognized as a dopamine receptor, based upon the data showing high affinity binding, saturability, specificity, temperature dependence, and reversibility. All of 14 PRL-secreting adenomas had high affinity dopamine receptors, with a dissociation constant (Kd) of 0.85 ±0.11 nmol/l (mean ± sem) and a maximal binding capacity (Bmax) of 428 ± 48.6 fmol/mg protein. Among 14 growth hormone (GH)-secreting adenomas examined, 8 (57%) had dopamine receptors with a Kd of 1.90 ± 0.47 nmol/l and a Bmax of 131 ± 36.9 fmol/mg protein. Furthermore, 15 of 24 (58%) nonsecreting pituitary adenomas also had dopamine receptors with a Kd of 1.86 ± 0.37 nmol/l and a Bmax of 162 ± 26.0 fmol/mg protein. These results indicate that some GH-secreting adenomas as well as some nonsecreting pituitary adenomas contain dopamine receptors. But their affinity and number of binding sites are significantly lower (P < 0.05) and fewer (P < 0.001) respectively, than those in PRL-secreting adenomas.

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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