Lack of G protein-coupled sigma receptors in rat brain membranes: receptor-mediated high-affinity GTPase activity and [35S]GTPγS binding studies

2004 ◽  
Vol 112 (7) ◽  
pp. 873-883 ◽  
Author(s):  
Y. Odagaki ◽  
R. Toyoshima ◽  
T. Yamauchi
Keyword(s):  
Rat Brain ◽  
G Protein ◽  
Sigma Receptors ◽  
Gtpase Activity ◽  
Binding Studies ◽  
High Affinity ◽  
Brain Membranes ◽  
Rat Brain Membranes ◽  
Gtpγs Binding ◽  
G Protein Coupled

scholarly journals Characterization of tetanus toxin binding to rat brain membranes. Evidence for a high-affinity proteinase-sensitive receptor

1986 ◽  
Vol 236 (3) ◽  
pp. 845-852 ◽  
Author(s):  
E J Pierce ◽  
M D Davison ◽  
R G Parton ◽  
W H Habig ◽  
D R Critchley
Keyword(s):  
Rat Brain ◽  
Tetanus Toxin ◽  
Binding Studies ◽  
Single Class ◽  
Toxin Binding ◽  
High Affinity ◽  
Brain Membranes ◽  
Protein Receptor ◽  
Rat Brain Membranes ◽  
Competition Binding

Binding of 125I-labelled tetanus toxin to rat brain membranes in 25 mM-Tris/acetate, pH 6.0, was saturable and there was a single class of high-affinity site (KD 0.26-1.14 nM) present in high abundance (Bmax. 0.9-1.89 nmol/mg). The sites were largely resistant to proteolysis and heating but were markedly sensitive to neuraminidase. Trisialogangliosides were effective inhibitors of toxin binding (IC50 10 nM) and trisialogangliosides inserted into membranes lacking a toxin receptor were able to bind toxin with high affinity (KD 2.6 nM). The results are consistent with previous studies and the hypothesis that di- and trisialogangliosides act as the primary receptor for tetanus toxin under these conditions. In contrast, when toxin binding was assayed in Krebs-Ringer buffer, pH 7.4, binding was greatly reduced, was non-saturable and competition binding studies showed evidence for a small number of high-affinity sites (KD 0.42 nM, Bmax. 0.90 pmol/mg) and a larger number of low-affinity sites (KD 146 nM, Bmax. 179 pmol/mg). Treatment of membranes with proteinases, heat, and neuraminidase markedly reduced binding. Trisialogangliosides were poor inhibitors of toxin binding (IC50 11.0 microM), and trisialogangliosides inserted into membranes bound toxin with low affinity. The results suggest that in physiological buffers tetanus toxin binds with high affinity to a protein receptor, and that gangliosides represent only a low-affinity site.

Rat brain membranes possess two high-affinity binding sites for [3H]somatostatin

1985 ◽  
Vol 55 (2) ◽  
pp. 161-166 ◽  
Author(s):  
D.R. Weightman ◽  
C.A. Whitford ◽  
C.R. Snell ◽  
B.H. Hirst ◽  
D.E. Brundish ◽  
...  
Keyword(s):  
Rat Brain ◽  
Binding Sites ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Brain Membranes ◽  
Rat Brain Membranes

scholarly journals Purification of the G-protein G13 from rat brain membranes

1994 ◽  
Vol 303 (1) ◽  
pp. 135-140 ◽  
Author(s):  
R Harhammer ◽  
B Nürnberg ◽  
K Spicher ◽  
G Schultz
Keyword(s):  
Rat Brain ◽  
G Protein ◽  
G Proteins ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Sucrose Density Gradient Centrifugation ◽  
Brain Membranes ◽  
Rat Brain Membranes

Significant amounts of G13, a member of the recently described G12-subfamily of heterotrimeric G-proteins, have been detected in rat brain membranes by specific antisera. The alpha-subunits of G13 (G alpha 13) were purified by using a combination of conventional and subunit-exchange chromatography. Purification was facilitated by the fact that the initial anion-exchange chromatography separated G13 from most of the other G-proteins, including Gq/11. Moreover, G alpha 13-enriched fractions obtained from this chromatographic step were devoid of beta gamma-dimers, despite the absence of G-protein-activating agents. Nevertheless, the purified G alpha 13 retained its ability to interact with beta gamma-dimers under appropriate conditions, i.e. the addition of Lubrol PX instead of cholate as detergent and the omission of ethylene glycol routinely used as a protecting additive. The interaction was demonstrated by (i) the binding of G alpha 13 to immobilized beta gamma-complexes and (ii) the formation of stable heterotrimers during sucrose-density-gradient centrifugation. Furthermore, our studies on G alpha 13 provide evidence for an extremely slow basal GDP/GTP exchange rate. The purified protein showed negligible binding of guanosine 5′-[gamma-[35S]thio]triphosphate (GTP[35S]). Accordingly, dissociation of G alpha 13 from immobilized beta gamma-complexes was achieved by AlF4-/Mg2+, but not by GTP[S]. These data indicate that G13 exhibits properties highly distinct from those of other G-proteins.

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