The world's first wiki where authorship really matters (Nature Genetics, 2008). Due credit and reputation for authors. Imagine a global collaborative knowledge base for original thoughts. Search thousands of articles and collaborate with scientists around the globe.

wikigene or wiki gene protein drug chemical gene disease author authorship tracking collaborative publishing evolutionary knowledge reputation system wiki2.0 global collaboration genes proteins drugs chemicals diseases compound
Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)
 
 
 
 
 

Characterization of [35S]-ATP alpha S and [3H]-alpha, beta-MeATP binding sites in rat brain cortical synaptosomes: regulation of ligand binding by divalent cations.

1. We made a comparative analysis of the binding characteristics of the radioligands [35S]-ATP alpha S and [3H]-alpha, beta-MeATP in order to test whether these ligands can be used to analyse P2-purinoceptors in synaptosomal membranes from rat brain cortex. 2. Synaptosomes possess sites with high affinity for [35S]-ATP alpha S (Kd = 22.2 +/- 9.1 nM, Bmax = 14.8 pmol mg-1 protein). The rank order of the competition potency of the different compounds (ATP alpha S, ATP, ATP gamma S > ADP beta S, 2-MeSATP > deoxyATP, ADP > > UTP, alpha, beta-MeATP, AMP, Reactive Blue-2, suramin, isoPPADS) is consistent with pharmacological properties of P2Y-purinoceptors. 3. Under identical conditions [35S]-ATP alpha S and [3H]-alpha, beta-MeATP bind to different binding sites at synaptosomal membranes from rat brain cortex. The affinity of the [3H]-alpha, beta-MeATP binding sites (Kd = 13.7 +/- 1.8 nM, Bmax = 6.34 +/- 0.28 pmol mg-1 protein) was 38 fold higher than the potency of alpha, beta-MeATP to displace [35S]-ATP alpha S binding (Ki = 0.52 microM). ATP and ADP beta S competed at both binding sites with different affinities, 60 fold and 175 fold, respectively. The other agonists tested (2-MeSATP, UTP, GTP) did not affect specific [35H]-alpha, beta-MeATP binding at concentrations up to 100 microM. The antagonists (suramin, isoPPADS, Evan's Blue) showed completely different affinities for both binding sites. 4. Binding of [35S]-ATP alpha S on synaptosomes was regulated by GTP, which is indicative for G-protein coupled receptors. The Kd value for the high affinity binding site was reduced in the presence of GTP about 5 fold (from 1.8 nM to 8.6 nM). In the presence of Mg2+ the affinity was increased (Kd 1.8 nM versus 22 nM in the absence of Mg2+). 5. The binding of both radioligands was regulated in an opposite manner by physiological concentrations of Ca2+ and Mg2+. Binding of [3H]-alpha, beta-MeATP to synaptosomal membranes was increased 3 fold by raising the Ca2+ concentration from 10 microM to 1 mM, whereas the addition of Mg2+ in the same concentration range resulted in an 80% reduction of the binding. In contrast, [35S]-ATP alpha S binding was not influenced at the same range of Ca2+ or Mg2+ concentrations (10 microM to 1 mM). The addition of Mg2+ (5 mM) increased the affinity of [35S]-ATP alpha S for the high affinity site 10 fold. 6. Diadenosine polyphosphates had a bimodal effect on [35S]-ATP alpha S binding to synaptosomal membranes. AP5A and Ap6A enhanced binding of [35S]-ATP alpha S 1.6 fold in a concentration range between 0.1 and 50 microM. Ap3A was a weak inhibitor with a Ki value of 7.2 microM. Ap4A, AP5A and Ap6A inhibited with Ki values > 100 microM. These data support the concept that diadenosine polyphosphates do not directly interact with ATP alpha S binding sites. 7. In conclusion, on the basis of present knowledge of the interaction of P2-purinoceptor active compounds with P2x- and/or P2Y-purinoceptors, our data strongly suggest that [35S]-ATP alpha S is a useful tool to study P2Y-purinoceptors. Thus, the [35S]-ATP alpha S binding site might to a large extent represent P2Y-purinoceptors in synaptosomes from rat brain cortex. The nucleotide binding is regulated by G proteins, indicated by the effects of GTP/Mg2+ on binding.[1]

References

 
WikiGenes - Universities