Letters to the Editor

1997 ◽  
Vol 273 (4) ◽  
pp. C1437-C1439
Author(s):  
A. W. Cuthbert
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Single Channel ◽  
Letters To The Editor ◽  
High Affinity ◽  
Channel Proteins ◽  
Affinity Ligand ◽  
Ligand Binding Sites ◽  
Kinetics Of ◽  
High Affinity Ligand

The following is the abstract of the article discussed in the subsequent letter: Blazer-Yost, Bonnie L., and Sandy I. Helman.The amiloride-sensitive epithelial Na+ channel: binding sites and channel densities. Am. J. Physiol. 272 ( Cell Physiol. 41): C761–C769, 1997.—The amiloride-sensitive Na+ channel found in many transporting epithelia plays a key role in regulating salt and water homeostasis. Both biochemical and biophysical approaches have been used to identify, characterize, and quantitate this important channel. Among biophysical methods, there is agreement as to the single-channel conductance and gating kinetics of the highly selective Na+ channel found in native epithelia. Amiloride and its analogs inhibit transport through the channel by binding to high-affinity ligand-binding sites. This characteristic of high-affinity binding has been used biochemically to quantitate channel densities and to isolate presumptive channel proteins. Although the goals of biophysical and biochemical experiments are the same in elucidating mechanisms underlying regulation of Na+transport, our review highlights a major quantitative discrepancy between methods in estimation of channel densities involved in transport. Because the density of binding sites measured biochemically is three to four orders of magnitude in excess of channel densities measured biophysically, it is unlikely that high-affinity ligand binding can be used physiologically to quantitate channel densities and characterize the channel proteins.

The amiloride-sensitive epithelial Na+ channel: binding sites and channel densities

1997 ◽  
Vol 272 (3) ◽  
pp. C761-C769 ◽  
Author(s):  
B. L. Blazer-Yost ◽  
S. I. Helman
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Single Channel ◽  
Na Channel ◽  
High Affinity ◽  
Channel Proteins ◽  
Affinity Ligand ◽  
Kinetics Of ◽  
High Affinity Ligand ◽  
Epithelial Na Channel

The amiloride-sensitive Na+ channel found in many transporting epithelia plays a key role in regulating salt and water homeostasis. Both biochemical and biophysical approaches have been used to identify, characterize, and quantitate this important channel. Among biophysical methods, there is agreement as to the single-channel conductance and gating kinetics of the highly selective Na+ channel found in native epithelia. Amiloride and its analogs inhibit transport through the channel by binding to high-affinity ligand-binding sites. This characteristic of high-affinity binding has been used biochemically to quantitate channel densities and to isolate presumptive channel proteins. Although the goals of biophysical and biochemical experiments are the same in elucidating mechanisms underlying regulation of Na+ transport, our review highlights a major quantitative discrepancy between methods in estimation of channel densities involved in transport. Because the density of binding sites measured biochemically is three to four orders of magnitude in excess of channel densities measured biophysically, it is unlikely that high-affinity ligand binding can be used physiologically to quantitate channel densities and characterize the channel proteins.

scholarly journals Adenosine Receptors of Cerebral Microvessels and Choroid Plexus

1986 ◽  
Vol 6 (4) ◽  
pp. 463-470 ◽  
Author(s):  
Rajesh N. Kalaria ◽  
Sami I. Harik
Keyword(s):  
Cerebral Cortex ◽  
Ligand Binding ◽  
Choroid Plexus ◽  
Adenosine Receptors ◽  
Binding Sites ◽  
Specific Binding ◽  
Cerebral Microvessels ◽  
High Affinity ◽  
Receptor Sites ◽  
Ligand Binding Sites

We studied, by ligand binding methods, the two adenosine receptors, A, and A2, in rat and pig cerebral microvessels and pig choroid plexus. Ligand binding to cerebral microvessels was compared with that to membranes of the cerebral cortex. [3H]Cyclohexyladenosine and [3H]l-phenylisopropyladenosine were the ligands used for A1-receptors, and [3H]5'- N-ethylcarboxamide adenosine ([3H]NECA) was used to assess A2-receptors. We report that cerebral microvessels and choroid plexus exhibit specific [3H]NECA binding, but have no appreciable A1-receptor ligand binding sites. Specific binding of [3H]NECA to cerebral microvessels, choroid plexus, and cerebral cortex was saturable and suggested the existence of two classes of A2-receptor sites: high-affinity ( Kd ∼ 250 n M) and low-affinity ( Kd ∼ 1–2 μ M) sites. The Kd and Bmax of NECA binding to cerebral microvessels and cerebral cortex were similar within each species. Our results, indicating the existence of A2-receptors in cerebral microvessels, are consistent with results of increased adenylate cyclase activity by adenosine and some of its analogues in these microvessels.

scholarly journals High Affinity Ligand Binding by Integrins Does Not Involve Head Separation

2003 ◽  
Vol 278 (19) ◽  
pp. 17185-17189 ◽  
Author(s):  
Bing-Hao Luo ◽  
Timothy A. Springer ◽  
Junichi Takagi
Keyword(s):  
Ligand Binding ◽  
High Affinity ◽  
Affinity Ligand ◽  
High Affinity Ligand

scholarly journals Rat and human neutrophil N-formyl-peptide chemotactic receptors. Species difference in the glycosylation of similar 35-38 kDa polypeptide cores

1991 ◽  
Vol 277 (1) ◽  
pp. 67-72 ◽  
Author(s):  
J J Remes ◽  
U E Petäjä-Repo ◽  
H J Rajaniemi
Keyword(s):  
Ligand Binding ◽  
Species Difference ◽  
Cross Linking ◽  
Complex Type ◽  
High Affinity ◽  
Affinity Ligand ◽  
Pngase F ◽  
Formyl Peptide ◽  
Membrane Bound ◽  
High Affinity Ligand

Rat and human neutrophil N-formyl-peptide chemotactic receptors were subjected to glycosidase and proteinase treatments to determine the extent and species differences of glycosylation and the carbohydrate requirement in the high-affinity ligand binding. N-Formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys was attached to rat and human neutrophils either before or after glycosidase and proteinase treatments, and the labelled receptors were solubilized after glutaraldehyde cross-linking and analysed by SDS/PAGE and autoradiography. Both the rat and human N-formyl-peptide chemotactic receptors contain only N-linked oligosaccharides, as demonstrated by their sensitivity to peptide N-glycosidase F (PNGase F) and resistance to O-glycanase treatment. The N-linked oligosaccharides seem to be of the complex type rather than the high-mannose or hybrid type and lack terminal sialic acid, as demonstrated by their resistance to endoglycosidases D and H and neuraminidase treatments. This sensitivity pattern was similar in both species, and the shift in the molecular size of the receptors to 35-38 kDa after PNGase F treatment occurred through one intermediate product, suggesting that both receptors contain a similar 35-38 kDa polypeptide core with two N-linked complex-type oligosaccharides, the heterogeneity of which is responsible for the species difference in receptor size. Papain treatment alone or followed by PNGase F produced in both species a 33-36 kDa membrane-bound fragment that was still able to bind the ligand, suggesting that the oligosaccharides are located on the approx. 2 kDa papain-cleavable polypeptide fragment of the receptors. The cleavage sites for both papain and PNGase F were hidden in occupied receptors, suggesting a conformational or topographical change in these upon ligand binding. Scatchard analyses and cross-linking experiments demonstrated that carbohydrates are not required for high-affinity ligand binding and that the 33-36 kDa membrane-bound papain fragment of both receptors contains the ligand-binding site.

scholarly journals P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin.

1995 ◽  
Vol 128 (4) ◽  
pp. 661-671 ◽  
Author(s):  
K L Moore ◽  
K D Patel ◽  
R E Bruehl ◽  
F Li ◽  
D A Johnson ◽  
...  
Keyword(s):  
Binding Sites ◽  
Fluid Phase ◽  
Human Neutrophils ◽  
Shear Stresses ◽  
High Affinity ◽  
Affinity Ligand ◽  
Activated Platelets ◽  
Static Conditions ◽  
Selectin Binding ◽  
High Affinity Ligand

Neutrophils roll on P-selectin expressed by activated platelets or endothelial cells under the shear stresses in the microcirculation. P-selectin glycoprotein ligand-1 (PSGL-1) is a high affinity ligand for P-selectin on myeloid cells. However, it has not been demonstrated that PSGL-1 contributes to the rolling of neutrophils on P-selectin. We developed two IgG mAbs, PL1 and PL2, that appear to recognize protein-dependent epitopes on human PSGL-1. The mAbs bound to PSGL-1 on all leukocytes as well as on heterologous cells transfected with PSGL-1 cDNA. PL1, but not PL2, blocked binding of 125-I-PSGL-1 to immobilized P-selectin, binding of fluid-phase P-selectin to myeloid and lymphoid leukocytes, adhesion of neutrophils to immobilized P-selectin under static conditions, and rolling of neutrophils on P-selectin-expressing CHO cells under a range of shear stresses. PSGL-1 was localized to microvilli on neutrophils, a topography that may facilitate its adhesive function. These data indicate that (a) PSGL-1 accounts for the high affinity binding sites for P-selectin on leukocytes, and (b) PSGL-1 must interact with P-selectin in order for neutrophils to roll on P-selectin at physiological shear stresses.

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