scholarly journals Membrane receptor for odour-binding proteins

1996 ◽  
Vol 317 (1) ◽  
pp. 23-27 ◽  
Author(s):  
Mohamed BOUDJELAL ◽  
Asipu SIVAPRASADARAO ◽  
John B. C FINDLAY
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Binding Protein ◽  
Structural Similarity ◽  
Thiol Group ◽  
The Body ◽  
Retinol Binding Protein ◽  
Proteinase K ◽  
Scatchard Analysis ◽  
Major Urinary Proteins

Specific binding of 125I-labelled bovine odour-binding protein (OBP) to isolated membranes from nasal mucosa was demonstrated. The interaction reached equilibrium within 30 min at 37 °C and was reversible. A Scatchard analysis of the equilibrium binding revealed a single population of binding sites, with the calculated equilibrium dissociation constant and maximum number of binding sites being 2.25±0.5 μM and 18.5±2 pmol/mg of membrane protein respectively (n = 2). Receptor activity was decreased on digestion by trypsin, proteinase K or endoglycosidase H, was heat labile and was sensitive to thiol-group-specific reagents. With the exception of rat and mouse major urinary proteins, which exhibit a high degree of structural similarity with OBP and bind similar ligands, other members of the lipocalin family, such as retinol-binding protein and β-lactoglobulin, failed to inhibit the binding of 125I-labelled OBP to its receptor. The receptor seems not to be restricted to olfactory tissues, as it was detected in a variety of other tissues. This suggests that OBP is unlikely to play a role only in olfactory signal transduction. It might have a much broader role within the body; possibilities include a role in detoxification or signalling.

scholarly journals Hepoxilin A3-specific binding in human neutrophils

1996 ◽  
Vol 313 (2) ◽  
pp. 537-541 ◽  
Author(s):  
Denis REYNAUD ◽  
Peter DEMIN ◽  
Cecil R. PACE-ASCIAK
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Leukotriene B4 ◽  
Proteinase K ◽  
Human Neutrophils ◽  
Scatchard Analysis ◽  
Intact Cells ◽  
Specific Binding Sites ◽  
Binding Data ◽  
Hepoxilin A3

Hepoxilins have been shown to release calcium from intracellular stores in human neutrophils [Dho, Grinstein, Corey, Su and Pace-Asciak (1990) Biochem. J. 266, 63-68; Laneuville, Reynaud, Grinstein, Nigam and Pace-Asciak (1993) Biochem. J. 295, 393-397]. In this paper we report that tritium-labelled hepoxilin A3 (8S) binds to broken neutrophil membranes in a time-, substrate- and temperature-dependent fashion. Specific binding was displaced with unlabelled hepoxilin A3. Specific binding was greatest at 37 °C. Competitive binding was best observed with unlabelled hepoxilin A3 (8S); the glutathione conjugate, HxA3-C (8S or 8R), or 12(S)-hydroxyeicosatetraenoic acid was less active. Similarly inactive in displacing the bound radiolabelled hepoxilin A3 was leukotriene B4 as well as a variety of prostaglandins and thromboxane B2. Formylmethionyl-leucylphenylalanine was similarly inactive in competing for the hepoxilin binding sites. Specific binding was inhibited by pretreatment of the broken membranes during 30 min at 37 °C with proteinase K, while specific binding of the intact cells was unaffected. Scatchard analysis of binding data revealed a single population of binding sites with apparent KD and Bmax. of 79.3±9.1 nM and 8.86±1.4 pmol/ml per 2×106 cells (±S.E.M.) respectively reflecting approx. 2.67×106 sites/cell. These results demonstrate for the first time that neutrophils contain specific binding sites to hepoxilin A3.

scholarly journals Tissue distribution of the receptor for plasma retinol-binding protein

1995 ◽  
Vol 305 (2) ◽  
pp. 419-424 ◽  
Author(s):  
S Smeland ◽  
T Bjerknes ◽  
L Malaba ◽  
W Eskild ◽  
K R Norum ◽  
...  
Keyword(s):  
Tissue Distribution ◽  
Specific Binding ◽  
Binding Protein ◽  
Cell Types ◽  
Binding Activity ◽  
Retinol Binding Protein ◽  
Scatchard Analysis ◽  
High Binding ◽  
Differentiated Cells ◽  
Protein Receptor

The tissue distribution of the retinol-binding-protein receptor has been studied by using a cell-free binding assay. High binding activity was found in placenta, retina pigment epithelial cells, bone marrow and kidneys. Specific binding activity was also found in the small intestines, spleen and liver, and to a lesser extent in lung. Scatchard analysis revealed that the difference in binding activity was due to variations in receptor level and not affinity changes. When the kidneys were separated into cortex and medulla we found that almost all the specific binding activity present in kidneys was recovered in the cortex. The choroid plexus, an important site in the delivery of nutrients to the cerebrospinal fluid, expressed very high binding activity. The pineal gland, which has been shown to store vitamin A, also showed high binding activity. Testes from immature animals showed higher binding activity than testes from mature rabbits. Cultured undifferentiated kidney keratinocytes showed about 40 times higher binding activity than differentiated cells. Skin fibroblasts demonstrated no binding activity. In conclusion, the data presented in this report show that the level of the retinol-binding-protein receptor varies considerably between cell types. The observed tissue distribution of the receptor agrees well with the present knowledge on retinol function and metabolism by various cells.

Endothelin ETA and ETB receptors in postnatal intestine

2001 ◽  
Vol 280 (4) ◽  
pp. G555-G562 ◽  
Author(s):  
Craig A. Nankervis ◽  
David J. Dunaway ◽  
Charles E. Miller
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Ischemia Reperfusion ◽  
Age Groups ◽  
Scatchard Analysis ◽  
Binding Assays ◽  
Site Model ◽  
Number Of Binding Sites ◽  
Competitive Binding Assays

We aimed to characterize endothelin (ET) receptors in the swine intestinal vasculature and to determine ischemia-reperfusion (I/R) effects on these receptors. Saturation and competitive binding assays were performed on mesenteric artery protein membranes from 1- and 40-day-old animals, both control and those subjected to 1 h of partial ischemia followed by 6 h of reperfusion in vivo. Scatchard analysis of saturation binding with 125I-labeled ET-1 in membranes from endothelium-denuded (E−) vessels revealed that the maximum number of binding sites was greater in younger animals. Competitive125I-ET-1 binding was significant for a one-site model with ET-1, ET-3, and sarafotoxin S6c (S6c) in membranes from endothelium-intact (E+) and E− vessels in both age groups. The maximum number of ET-1 binding sites was significantly greater in younger animals. In the presence of the ETAreceptor antagonist BQ-123, competitive 125I-ET-1 binding was significant for a one-site model with ET-1 and S6c in membranes from E+ vessels in both age groups. The maximum number of ET-1 binding sites was significantly greater in younger animals. After I/R, the maximum number of ET-1 binding sites was unchanged. In the presence of BQ-123, specific binding by ET-1 and S6c was eliminated in both age groups after I/R. These results suggest that both ET receptor populations are expressed to a greater degree in younger animals and I/R significantly affects the ETB receptor.

The Binding Of 125I-Von Willebrand Factor To Human Platelets In The Presence Of Ristocetin

1981 ◽  
Author(s):  
P Silber ◽  
T H Finlay
Keyword(s):  
Von Willebrand Factor ◽  
Binding Sites ◽  
Binding Constant ◽  
Specific Binding ◽  
Human Platelets ◽  
Scatchard Analysis ◽  
Binding Data ◽  
Number Of Binding Sites ◽  
Von Willebrand ◽  
Willebrand Factor

The effect of ristocetin on the binding of 125I-porcine von Willebrand factor to human platelets was studied. Previously, we had shown that 125I-porcine von Willebrand factor binds to human platelets in the absence of ristocetin. The present work demonstrates that binding is stimulated by ristocetin and this stimulation is maximal at a ristocetin concentration of 2 mg/ml. At a ristocetin concentration of 0.5 mg/ml, Scatchard analysis indicates a binding constant of 5.18 × 10-9M and the presence of 105,000 binding sites. This compares with our previous finding, in the absence of ristocetin, of a binding constant of 2.92 × 10-7M and 4760 binding sites. These binding data assume the porcine von Willebrand factor to be a tetramer with a molecular weight of 9 × 105. This study indicates that ristocetin causes tighter binding and increases the number of binding sites on human platelets for porcine von Willebrand factor. Unlabelled porcine von Willebrand factor competitively inhibits the specific binding of the labelled protein and gives a binding constant of 0.17 × 10-9M. Similar results were obtained using human von Willebrand factor.

Mineralcorticoid receptors along the nephron: [3H]aldosterone binding in rabbit tubules

1981 ◽  
Vol 241 (6) ◽  
pp. F605-F611 ◽  
Author(s):  
A. Doucet ◽  
A. I. Katz
Keyword(s):  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Rabbit Kidney ◽  
Scatchard Analysis ◽  
Thick Ascending Limb ◽  
Collecting Tubule ◽  
The Difference ◽  
Collecting Tubules ◽  
Cortical Collecting Tubule

To identify the site of mineralocorticoid action along the nephron, we measured the specific binding of [3H]aldosterone to nephron segments microdissected from aldosterone-deficient rabbits. Specific binding was defined as the difference between binding measured in the absence or in the presence of 2,000-fold excess of unlabeled hormone (in 10(-18) mol X cm tubule length-1 +/- SE). High specific binding capacity was found in the branched collecting tubule (108 +/- 4), the cortical collecting tubule (119 +/- 9), and the outer medullary collecting tubule (115 +/- 16), whereas specific binding was negligible in the proximal convoluted tubule (8 +/- 9), pars recta (2 +/- 6), medullary thick ascending limb (4 +/- 6), cortical thick ascending limb (6 +/- 2), and distal convoluted tubule (6 +/- 6). In cortical collecting tubules, Scatchard analysis of the specific [3H]aldosterone binding indicated a dissociation constant (KD) of 2.2 X 10(-9) M and a maximum number of binding sites of 157 X 10(-18) mol X cm tubule length-1. The steroid specificity was assessed from the competition of various steroids for [3H]aldosterone binding sites. Receptors from the cortical collecting tubule revealed the following sequence of affinities: aldosterone greater than DOCA greater than spironolactone greater than dexamethasone greater than 5 alpha-dihydrotestosterone = progesterone = 17 beta-estradiol, indicating that the binding sites in the collecting tubule are mineralocorticoid receptors. These results demonstrate significant [3H]aldosterone binding to receptors of high affinity and mineralocorticoid specificity only in the collecting tubule and suggest that this nephron segment is the target site of mineralocorticoid action in the rabbit kidney.

scholarly journals A yeast ARS-binding protein activates transcription synergistically in combination with other weak activating factors.

1990 ◽  
Vol 10 (3) ◽  
pp. 887-897 ◽  
Author(s):  
A R Buchman ◽  
R D Kornberg
Keyword(s):  
Dna Sequences ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Essential Gene ◽  
Specific Binding ◽  
Binding Protein ◽  
Point Mutations ◽  
Yeast Genes

ABFI (ARS-binding protein I) is a yeast protein that binds specific DNA sequences associated with several autonomously replicating sequences (ARSs). ABFI also binds sequences located in promoter regions of some yeast genes, including DED1, an essential gene of unknown function that is transcribed constitutively at a high level. ABFI was purified by specific binding to the DED1 upstream activating sequence (UAS) and was found to recognize related sequences at several other promoters, at an ARS (ARS1), and at a transcriptional silencer (HMR E). All ABFI-binding sites, regardless of origin, provided weak UAS function in vivo when examined in test plasmids. UAS function was abolished by point mutations that reduced ABFI binding in vitro. Analysis of the DED1 promoter showed that two ABFI-binding sites combine synergistically with an adjacent T-rich sequence to form a strong constitutive activator. The DED1 T-rich element acted synergistically with all other ABFI-binding sites and with binding sites for other multifunctional yeast activators. An examination of the properties of sequences surrounding ARS1 left open the possibility that ABFI enhances the initiation of DNA replication at ARS1 by transcriptional activation.

scholarly journals Influence of divalent cations on the detection of somatogenic and lactogenic binding sites in mouse liver cells

1985 ◽  
Vol 228 (3) ◽  
pp. 761-764 ◽  
Author(s):  
G N Ciccia-Torres ◽  
J M Dellacha
Keyword(s):  
Binding Sites ◽  
Mouse Liver ◽  
Binding Capacity ◽  
Specific Binding ◽  
Divalent Cations ◽  
Isolated Hepatocytes ◽  
Liver Cells ◽  
Scatchard Analysis ◽  
Male Mice ◽  
Ovine Prolactin

Specific binding of 125I-labelled human somatotropin was demonstrated in isolated hepatocytes from male mice. In the presence of divalent cations (Ca2+ and Mg2+) the binding of 125I-labelled human somatotropin was competitive with ovine prolactin. Scatchard analysis of competition data indicated a KD of 1.4 +/- 0.2 nM and a binding capacity of 13 000 +/- 2000 sites/cell. In the absence of divalent cations and in the presence of EDTA, human and bovine somatotropins were found to be equally effective to displace bound 125I-labelled human somatotropin, while ovine prolactin showed a weak competition. In this case, the binding capacity was 8400 +/- 1500 sites/cell and the KD was 1.1 +/- 0.1 nM.

Comparison of growth hormone binding and metabolic response in rat adipocytes of epididymal, subcutaneous, and retroperitoneal origin

1985 ◽  
Vol 110 (1) ◽  
pp. 50-55 ◽  
Author(s):  
Stephen LaFranchi ◽  
Cheryl E. Hanna ◽  
Toni Torresani ◽  
Eugen Schoenle ◽  
Ruth Illig
Keyword(s):  
Growth Hormone ◽  
Binding Sites ◽  
Metabolic Response ◽  
Specific Binding ◽  
Human Growth ◽  
Scatchard Analysis ◽  
Rat Adipocytes ◽  
Significant Difference ◽  
Glucose Incorporation ◽  
Number Of Binding Sites

Abstract. We undertook a comparison of human growth hormone (hGH) binding and metabolic responses in rat adipocytes of epididymal, subcutaneous, and retroperitoneal origin to determine whether the site of fat depot biopsy might affect the response to hGH stimulation. The results showed highest specific binding in epididymal (3.6%), followed by subcutaneous (2.3%) and retroperitoneal adipocytes (1.5%); half-maximal binding was achieved at 14–18 ng/ml hGH for the three sites. Scatchard analysis of the binding data from each site was linear; there was no significant difference in binding affinities (2.1 to 3.3 × 109, m−1), but the number of binding sites was statiticially higher in epididymal (9.8 × 103) as compared to subcutaneous (7.5 × 103, P < 0.05) and retroperitoneal cells (3.3 × 103, P < 0.01). Stimulation with 5 to 2500 ng pituitary hGH produced a dose-related increase in glucose incorporation, with the largest increase in epididymal fat cells (31%, P <0.05) followed by subcutaneous cells (18%, P < 0.05); no significant increase was seen with retroperitoneal cells. Biosynthetic hGH produced a similar pattern of glucose incorporation in the three sites. Addition of hGH antibodies blocked the glucose incorporation in epididymal adipocytes using both pituitary-derived and biosynthetic hGH. It seems clear that this insulin-like effect is caused by hGH, not an insulin-like impurity. We conclude that the number of binding sites, perhaps related to adipose cell size, differs in adipose tissue from different locations and this influences the metabolic response to hGH stimulation.

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