Isolation of cholinergic receptor protein(s) from Torpedo nobiliana by affinity chromatography

Biochemistry ◽  
1974 ◽  
Vol 13 (14) ◽  
pp. 2822-2827 ◽  
Author(s):  
David E. Ong ◽  
Robert N. Brady
Blood ◽  
1996 ◽  
Vol 87 (3) ◽  
pp. 926-937 ◽  
Author(s):  
JL Spivak ◽  
LS Avedissian ◽  
JH Pierce ◽  
D Williams ◽  
WD Hankins ◽  
...  

The full-length murine erythropoietin receptor was expressed in Spodoptera frugiperda (Sf9) cells using a recombinant baculovirus vector. Erythropoietin receptor protein production was maximal 48 hours after infection, as determined by metabolic labeling and immunoblotting; receptor protein varied in molecular mass from 62 to 76 kD. Erythropoietin receptors produced in Sf9 cells could be solubilized using CHAPS in a form capable of binding erythropoietin, and the solubilized receptor bound to immobilized Concanavalin A (Con A) and wheat germ agglutinin, as well as to immobilized recombinant human erythropoietin. Analysis of the distribution of erythropoietin receptors in Sf9 plasma membrane and cytosol fractions using lectin affinity chromatography revealed that membrane-bound receptor had a higher apparent molecular mass and contained the bulk of receptors that bound to wheat germ agglutinin. The receptor was purified by sequential affinity chromatography on Con A-Sepharose and immobilized erythropoietin. Erythropoietin receptors expressed in Sf9 cells were inserted into the plasma membrane in the correct orientation, bound 125I-erythropoietin with a single affinity (kD, 330 pmol/L), and were internalized after ligand binding. However, kD varied inversely with the number of cell surface receptors. Solubilized erythropoietin receptors in whole-cell lysates and isolated plasma membranes exhibited high-affinity binding, with kD values of 92 and 57 pmol/L, respectively. Erythropoietin bound to the surface of infected Sf9 cells could be cross-linked to two proteins with molecular masses of 90 and 65 kD using the homobifunctional cross-linker, disuccinimidyl suberate (DSS). Similar results were obtained with solubilized receptors in whole-cell lysates, and both proteins could be immunoprecipitated by an antiserum to the erythropoietin receptor carboxyl-terminal domain.


FEBS Letters ◽  
1971 ◽  
Vol 16 (2) ◽  
pp. 92-94 ◽  
Author(s):  
J.P. Bourgeois ◽  
S. Tsuji ◽  
P. Boquet ◽  
J. Pillot ◽  
A. Ryter ◽  
...  

1981 ◽  
Vol 77 (4) ◽  
pp. 373-385 ◽  
Author(s):  
K Yoshii ◽  
Y Kobatake ◽  
K Kurihara

Properties of the receptor sites for L-amino acids in taste cells of the bullfrog (Rana catesbeiana) were examined by measuring the neural activities of the glossopharyngeal nerve under various conditions. (a) The frogs responded to 12 amino acids, but the responses to the amino acids varied with individual frogs under natural conditions. The frog tongues, however, exhibited similar responses after an alkaline treatment that removes Ca2+ from the tissue. The variation in the responses under natural conditions was apparently due to the variation in the amount of Ca2+ bound to the receptor membrane. (b) The responses to hydrophilic L-amino acids (glycine, L-alanine, L-serine, L-threonine, L-cysteine, and L-proline) were of a tonic type, but those to hydrophobic L-amino acids (L-valine, L-leucine, L-isoleucine, L-methionine, L-phenylalanine, and L-tyrptophan) were usually composed of both phasic and tonic components. (c) The properties of the tonic component were quite different from those of the phasic component: the tonic component was largely enhanced by the alkaline treatment and suppressed by the acidic treatment that increases binding of Ca2+ to the tissue. Also, the tonic component was suppressed by the presence of low concentrations of salts, or the action of pronase E, whereas the phasic component was unchanged under these conditions. These properties of the phasic component were quite similar to those of the response to hydrophobic substances such as quinine. These results suggest that the hydrophilic L-amino acids stimulate receptor protein(s) and that the hydrophobic L-amino acids stimulate both the receptor protein and a receptor site similar to that for quinine. (d) On the basis of the suppression of the responses to amino acids by salts, the mechanism of generation of the receptor potential is discussed.


2011 ◽  
Vol 6 (4) ◽  
pp. 518-523
Author(s):  
Negrin Negrev ◽  
Yuri Nyagolov ◽  
Margarita Stefanova ◽  
Emiliya Stancheva

AbstractEffects of the hormones of the hypothalamic-pituitary-thyroid axis on some basic parameters of the activity of protein C anticoagulation pathway in rats are studied. Thyrotropin-releasing hormone (0.06 mg/kg body mass), thyrotropin (1 IU/kg), triiodothyronine (T3) (0.08 mg/kg), thyroxine (T4) (0.08 mg/kg), administered subcutaneously for three consecutive days on four different groups of rats increased significantly activated protein C, free protein S and protein S activity, and reduced the soluble endothelial protein C receptor. Protein C antigen and total protein S were significantly elevated only by thyrotropin-releasing hormone and thyroid-stimulating hormone, but they were not affected by T3 and T4 treatment. The data indicate the hypothalamic-pituitary-thyroid axis is involved in the regulation of the protein C anticoagulation pathway in rats by activation of this system, suggesting a tendency of hypocoagulability.


2020 ◽  
Vol 10 (1) ◽  
Author(s):  
S. D. Lam ◽  
N. Bordin ◽  
V. P. Waman ◽  
H. M. Scholes ◽  
P. Ashford ◽  
...  

Abstract SARS-CoV-2 has a zoonotic origin and was transmitted to humans via an undetermined intermediate host, leading to infections in humans and other mammals. To enter host cells, the viral spike protein (S-protein) binds to its receptor, ACE2, and is then processed by TMPRSS2. Whilst receptor binding contributes to the viral host range, S-protein:ACE2 complexes from other animals have not been investigated widely. To predict infection risks, we modelled S-protein:ACE2 complexes from 215 vertebrate species, calculated changes in the energy of the complex caused by mutations in each species, relative to human ACE2, and correlated these changes with COVID-19 infection data. We also analysed structural interactions to better understand the key residues contributing to affinity. We predict that mutations are more detrimental in ACE2 than TMPRSS2. Finally, we demonstrate phylogenetically that human SARS-CoV-2 strains have been isolated in animals. Our results suggest that SARS-CoV-2 can infect a broad range of mammals, but few fish, birds or reptiles. Susceptible animals could serve as reservoirs of the virus, necessitating careful ongoing animal management and surveillance.


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