Myosin VIIA is specifically associated with calmodulin and microtubule-associated protein-2B (MAP-2B)

2001 ◽  
Vol 354 (2) ◽  
pp. 267-274 ◽  
Author(s):  
Penio T. TODOROV ◽  
Rachel E. HARDISTY ◽  
Steve D. M. BROWN
Keyword(s):  
Binding Sites ◽  
Strong Association ◽  
Mouse Kidney ◽  
Scatchard Analysis ◽  
Tail Region ◽  
Immunoblotting Analysis ◽  
Kd Values ◽  
Myosin Viia

Myosin VIIA is a motor molecule with a conserved head domain and tail region unique to myosin VIIA, which probably defines its unique function in vivo. In an attempt to further characterize myosin VIIA function we set out to identify molecule(s) that specifically associate with it. We demonstrate that 17 and 55kDa proteins from mouse kidney and cochlea co-purify with myosin VIIA on affinity columns carrying immobilized anti-myosin VIIA antibody. N-terminal sequencing and immunoblotting analysis identified the 17kDa protein as calmodulin, whereas MS and immunoblotting analysis identified the 55kDa protein as microtubule-associated protein-2B (MAP-2B). Myosin VIIA can also be co-immunoprecipitated from kidney homogenate using anti-calmodulin or anti-MAP2 (recognizing isoforms 2A and 2B) antibodies, confirming the strong association between calmodulin and myosin VIIA and between MAP-2B and myosin VIIA. Myosin VIIA binds to calmodulin with an apparent Kd of 10-9 M. Scatchard analysis of the binding of myosin VIIA to MAP-2B provided evidence for two binding sites, with Kd values of 10-10 and 10-9 M, which have been mapped to medial and C-terminal tail domains of myosin VIIA. The characterization of the interaction of calmodulin and MAP-2B with myosin VIIA provides new insights into the function of myosin VIIA.

Characterization of Thromboxane A2/Prostaglandin H2 Receptors in Porcine Coronary Artery -The Inhibitory Effect of a Novel Dibenzoxepin Derivative, KW-3635

1992 ◽  
Vol 67 (05) ◽  
pp. 582-584 ◽  
Author(s):  
Ichiro Miki ◽  
Akio Ishii
Keyword(s):  
Coronary Artery ◽  
Binding Sites ◽  
Thromboxane A2 ◽  
Inhibitory Effect ◽  
Scatchard Analysis ◽  
Single Class ◽  
Porcine Coronary Artery ◽  
Equilibrium Binding ◽  
H2 Receptors

SummaryWe characterized the thromboxane A2/prostaglandin H2 receptors in porcine coronary artery. The binding of [3H]SQ 29,548, a thromboxane A2 antagonist, to coronary arterial membranes was saturable and displaceable. Scatchard analysis of equilibrium binding showed a single class of high affinity binding sites with a dissociation constant of 18.5 ±1.0 nM and the maximum binding of 80.7 ± 5.2 fmol/mg protein. [3H]SQ 29,548 binding was concentration-dependently inhibited by thromboxane A2 antagonists such as SQ 29,548, BM13505 and BM13177 or the thromboxane A2 agonists such as U46619 and U44069. KW-3635, a novel dibenzoxepin derivative, concentration-dependently inhibited the [3H]SQ 29,548 binding to thromboxane A2/prosta-glandin H2 receptors in coronary artery with an inhibition constant of 6.0 ± 0.69 nM (mean ± S.E.M.).

Identification and characterization of a corticotrophin-releasing hormone receptor in human placenta

1995 ◽  
Vol 133 (5) ◽  
pp. 591-597 ◽  
Author(s):  
Vicki L Clifton ◽  
Phillip C Owens ◽  
Phillip J Robinson ◽  
Roger Smith
Keyword(s):  
Human Placenta ◽  
Binding Sites ◽  
Hormone Receptor ◽  
Human Myometrium ◽  
Scatchard Analysis ◽  
Releasing Hormone ◽  
Rat Pituitary ◽  
Identification And Characterization ◽  
Crh Receptors

Clifton VL, Owens PC, Robinson PJ, Smith R. Identification and characterization of a corticotrophinreleasing hormone receptor in human placenta. Eur J Endocrinol 1995;133:591–7. ISSN 0804–4643 Corticotrophin-releasing hormone (CRH) causes vasodilatation in the human fetal–placental circulation and has paracrine actions in placental tissue, suggesting that CRH receptors may be present in the human placenta. We have now identified and characterized placental CRH binding sites and compared them to those described previously in human myometrium and rat pituitary. Radiolabelled ovine CRH binding to placental membranes was pH-, time-, temperature- and divalent cation-dependent and was reversible in the presence of 1 μmol/l unlabelled ovine CRH. Scatchard analysis of placentae delivered vaginally or by elective caesarean section revealed dissociation constants (Kd) of 214.5 ± 84 pmol/l (N = 8) and 45.4 ± 23.9 pmol/l (N = 9), respectively. The Kd for caesarean placental binding sites was similar to that of human myometrium (59.6 pmol/l, N = 3) and rat pituitary (82.5 pmol/l, N = 3) receptors. However, in vaginally delivered placentae the CRH binding sites had a much lower affinity (p < 0.05). The receptor densities (Bmax) of vaginally delivered and caesarean-delivered placentae were 28.6 ± 9.6 and 6.1 ± 2.8 fmol/mg, respectively (p < 0.05). Chemical cross-linking studies using disuccinimidyl suberate indicated that the molecular weight of the CRH receptor in the placenta and rat pituitary is 75 kD. We conclude that there is a high-affinity population of CRH binding sites in the human placenta that are physicochemically similar to pituitary and myometrial CRH receptors. The CRH receptor properties in the placenta change in response to labour, when CRH levels in maternal blood are highest, suggesting that placental CRH may regulate its receptor. R Smith, Endocrinology Unit, John Hunter Hospital, Locked Bag 1, Hunter Regional Mail Centre, Newcastle, NSW 2310, Australia

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.

Characterization of [3H] desmethylimipramine binding in bovine adrenal medulla: interactions with σ- and (or) phencyclidine-receptor ligands

1992 ◽  
Vol 70 (11) ◽  
pp. 1508-1514 ◽  
Author(s):  
Cheryl Rogers ◽  
Simon Lemaire
Keyword(s):  
Adrenal Medulla ◽  
Binding Sites ◽  
Scatchard Analysis ◽  
Noradrenaline Uptake ◽  
Receptor Ligands ◽  
Mk 801 ◽  
Bovine Adrenal Medulla ◽  
Sodium Dependent ◽  
Stimulation Of

High-affinity binding sites (apparent KD 2.87 nM) for [3H]desmethylimipramine ([3H]DMI), have been demonstrated and characterized in membrane preparations of bovine adrenal medulla. The binding of [3H]DMI improved upon pretreatment of the membrane with KCl and was saturable, sodium dependent, and potently inhibited by nisoxetine and imipramine. [3H]DMI binding was also inhibited by various phencyclidine (PCP)- and (or) σ-receptor ligands, with the following order of potency: haloperidol > rimcazole > (−)-butaclamol > dextromethorphan > MK-801 > (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine((+)-3-PPP) > PCP > N-(2-thienyl)cyclohexyl-3,4-piperidine (TCP) > (+)-SKF-10047 > (−)-SKF-10047. The inhibition produced by σ ligands was not attributed to stimulation of either σ1- or σ2-receptors, owing to inactivity of the selective σ-receptor ligands (+)-pentazocine and 1,3-di(2-tolyl)guanidine (DTG). The inhibition of [3H]DMI binding by σ- and PCP-receptor ligands was not attributed to PCP1- or PCP2-receptor stimulation, owing to the decreased potency (100-fold) of these ligands in [3H]DMI assays compared with the affinity for brain PCP1 sites, and the ineffectiveness of the PCP2-ligand N-(1-(2-benzo(b)thiophenyl)cyclohexyl)piperidine (BTCP). Scatchard analysis of the inhibition by the σ-ligands haloperidol and (+)-3-PPP, as well as the PCP1 receptor ligand MK-801, demonstrated noncompetitive interaction with the site bound by [3H]DMI. These studies indicate that bovine adrenomedullary membranes possess a specific receptor for the noradrenaline uptake inhibitor [3H]DMI, which is sensitive to allosteric modulation produced by PCP and σ-ligands.Key words: desmethylimipramine, σ-receptor, phencyclidine, noradrenaline uptake, adrenal medulla.

Characterization of melatonin receptors in the ram pars tuberalis: influence of light

1990 ◽  
Vol 123 (5) ◽  
pp. 557-562 ◽  
Author(s):  
Jean Pelletier ◽  
Bertrand Castro ◽  
Georges Roblot ◽  
Renée Wylde ◽  
Marie-Madeleine de Reviers
Keyword(s):  
Median Eminence ◽  
Binding Sites ◽  
Melatonin Receptors ◽  
Scatchard Analysis ◽  
Pars Tuberalis

Abstract. The present study was conducted to assess the binding of [125I]melatonin to frozen unfixed sections of pars tuberalis/median eminence tissue from Ile-de-France rams exposed or not exposed to light before slaughter. The specificity of [125I]melatonin binding to the pars tuberalis tissue was revealed by autoradiography and the magnitude of binding as related to the pars tuberalis area was determined after incubation and counting of pars tuberalis/median eminence sections. Subsequent studies with sections incubated with [125I]melatonin indicated that 1. the binding sites were saturable; 2. binding was stable for 24 h at 20°C, but unstable at 28 or 37°C; 3. melatonin and [12 7I]melatonin had a similar potency to compete with [125I]melatonin for binding sites, whereas other ligands such as serotonin or N-acetylserotonin were devoid of activity, and 4. by Scatchard analysis, the constant affinity Ka was found to be high in the 1010 l/mol range. Rams exposed to light throughout the night prior to slaughter presented a significant increase in the apparent number of [125I]melatonin binding sites in comparison to animals maintained under darkness (2.25±0.30 vs 1.01±0.17 fmol/mm2 pars tuberalis, p<0.01), whereas Ka values were similar in both groups. These results indicate the presence of true melatonin receptors in the pars tuberalis of the ram. Furthermore, they suggest that their apparent number is light-dependent.

scholarly journals High-resolution in vivo identification of miRNA targets by Halo-Enhanced Ago2 Pulldown

2019 ◽  
Author(s):  
Xiaoyi Li ◽  
Yuri Pritykin ◽  
Carla P. Concepcion ◽  
Yuheng Lu ◽  
Gaspare La Rocca ◽  
...  
Keyword(s):  
Binding Sites ◽  
Embryonic Stem ◽  
Genetically Engineered ◽  
Lung Cancers ◽  
Mirna Targets ◽  
Pathological Conditions ◽  
Non Coding Rnas ◽  
Mrna Binding

SUMMARYThe identification of miRNA targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily translated to an in vivo setting. To overcome these limitations and to facilitate the investigation of miRNA functions in mice, we have developed a method (HEAP: for Halo-Enhanced Ago2 Pulldown) to map miRNA-mRNA binding sites. This method is based on a novel genetically engineered mouse harboring a conditional, Cre-regulated, Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be efficiently purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, in developing embryos, in adult tissues and in autochthonous mouse models of human brain and lung cancers.The tools and the datasets we have generated will serve as a valuable resource to the scientific community and will facilitate the characterization of miRNA functions under physiological and pathological conditions.

scholarly journals Characterization of the Binding of Adenosine Diphosphate to Human Platelet Membranes

1977 ◽  
Author(s):  
C. Legrand ◽  
B. Bauvois ◽  
J. P. Caen
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Adenosine Diphosphate ◽  
Affinity Constant ◽  
Membrane Bound ◽  
High Concentrations ◽  
Kinetics Of

ADP-mediated platelet aggregation is a routinely employed test but its mechanism is poorly understood. The aim of this study was to compare the binding of ADP to plasma membranes isolated from normal platelets and thrombasthenic platelets (which do not aggregate with ADP). Binding of ADP to isolated membranes was assayed by incubation with 14C-ADP followed by Mill i pore filtration. In standard conditions, 14C-ADP was not transformed and non specific binding represented lessthan 3 % of the total binding. Using 1 μM 14C-ADP, the binding has been shown to be a rapid (t 1/2 = 2 mn 30 sec), saturable and reversible phenomenon at 37° C. The existence of a major population of binding sites, with an affinity constant Ka = 0.43 (+ 0.1) χ 106M-1, has been demonstrated. The kinetics of the binding was normal with membranes Tsolated from the platelets of 4 thrombasthenic patients and the affinity constant, when determined, was in the normal range. Dissociation of the membrane-bound 14C-ADP occurred rapidly at 37° C (t l/2c≃3mn) when samples were diluted enough (dilution 1 : 100 was currently employed) to avoid rebinding of the radioligand. Accelerated dissociation (t 1/2 ≃ 1 mn) was observed when the dilution was performed in the presence of an excess of unlabeled ADP, suggesting the existence of negatively cooperative site-site interactions among the ADP binding sites. This effect was only observed at high concentrations of ADP (> 10–5M) and its eventual role in vivo remains to be established. Two thrombasthenic membrane preparations studied in the same way dissociated as did the control membranes.

Demonstration and characterization of motilin-binding sites in the rabbit cerebellum

1997 ◽  
Vol 272 (5) ◽  
pp. G994-G999 ◽  
Author(s):  
I. Depoortere ◽  
T. L. Peeters
Keyword(s):  
Binding Sites ◽  
Molecular Layer ◽  
Physiological Role ◽  
Scatchard Analysis ◽  
Affinity Binding ◽  
Lower Affinity ◽  
Erythromycin A ◽  
G Protein Coupled

This is the first report on central motilin receptors. Autoradiography on cerebellar slices revealed specific motilin-binding sites in the molecular layer of the cortex. Scatchard analysis of cold saturation studies showed the existence of a high-(pKd,hi = 9.07 +/- 0.09, where pKd is the negative logarithm of the dissociation constant) and a low-affinity binding site (pKd,lo = 6.56 +/- 0.09). Similar affinities were found with rabbit motilin and with the porcine (po) antagonist [Phe3, Leu13]po-motilin. Feline and canine motilin had a markedly lower affinity for the low-affinity site (pKd,lo = 5.29 and 4.58, respectively); chicken motilin had a lower affinity for both sites (pKd,hi = 8.36, pKd,lo = 3.97). Erythromycin A and its derivative N-trimethyl erythromycin A cnol ether also bound to cerebellar motilin receptors (pKd,hi = 7.29 and 8.91, respectively). Structure-activity studies with motilin fragments and the potency ranking of agonists suggest that a novel subtype receptor of motilin may exist in the brain. Guanosine 5'-O-(3-thiotriphosphate) (0.1 mM) reduced the number and the affinity for the high-affinity binding sites, which is evidence for G protein-coupled receptors. Our findings open new perspectives for the study of the physiological role of motilin.

scholarly journals Characterization of ligand binding and processing by bombesin receptors in an insulin-secreting cell line

1987 ◽  
Vol 247 (3) ◽  
pp. 731-738 ◽  
Author(s):  
S L Swope ◽  
A Schonbrunn
Keyword(s):  
Islet Cells ◽  
Scatchard Analysis ◽  
Dependent Manner ◽  
Temperature Dependent ◽  
Single Class ◽  
Relative Potencies ◽  
Stimulate Insulin Release ◽  
Bombesin Receptors

Bombesin is a tetradecapeptide which stimulates insulin secretion in vivo by isolated islets and by HIT-T15 cells, a clonal line of hamster pancreatic-islet cells. In the present study we have used [125I-Tyr4]bombesin to characterize bombesin receptors in HIT-T15 cells. [125I-Tyr4]Bombesin binding was time- and temperature-dependent: maximum binding occurred after 45 min, 90 min and 10 h at 37, 22 and 4 degrees C respectively. Thereafter, cell-associated radioactivity declined at 37 degrees C and 22 degrees C but not at 4 degrees C. Scatchard analysis of [125I-Tyr4]bombesin binding measured at 4 degrees C showed that HIT-T15 cells contain a single class of binding sites (approximately equal to 85000/cell) with an apparent Kd of 0.9 +/- 0.11 nM. Structurally unrelated neuropeptides did not compete for [125I-Tyr4]bombesin binding. However, the relative potencies of bombesin and four bombesin analogues in inhibiting the binding of [125I-Tyr4]bombesin correlated with their ability to stimulate insulin release. Receptor-mediated processing of [125I-Tyr4]bombesin was examined by using an acid wash (0.2 M-acetic acid/0.5 M-NaCl, pH 2.5) to dissociate surface-bound peptide from the cells. Following [125I-Tyr4]bombesin binding at 4 degrees C, more than 85% of the cell-associated radioactivity could be released by acid. When the temperature was then increased to 37 degrees C, the bound radioactivity was rapidly (t1/2 less than 3 min) converted into an acid-resistant state. These results indicate that receptor-bound [125I-Tyr4]bombesin is internalized in a temperature-dependent manner. In fact, the entire ligand-receptor complex appeared to be internalized, since pretreatment of cells with 100 nM-bombesin for 90 min at 37 degrees C decreased the subsequent binding of [125I-Tyr4]bombesin by 90%. The chemical nature of the cell-associated radioactivity was determined by reverse-phase chromatography of the material extracted from cells after a 30 min binding incubation at 37 degrees C. Although 70% of the saturably bound radioactivity was co-eluted with intact [125I-Tyr4]bombesin 90% of the radioactivity subsequently dissociated from cells chromatographed as free iodide. At least some of the degradation of receptor-bound [125I-Tyr4]bombesin appeared to occur in lysosomes, since chloroquine increased the cellular accumulation of [125I-Tyr4]bombesin at 37 degrees C and slowed the release of radioactivity.(ABSTRACT TRUNCATED AT 400 WORDS)

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