Separation of solubilized A2 adenosine receptors of human platelets from non-receptor [3H]NECA binding sites by gel filtration |
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Authors: | Martin J Lohse Bernice Elger Jutta Lindenborn-Fotinos Karl-Norbert Klotz Ulrich Schwabe |
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Institution: | (1) Pharmakologisches Institut, Universität Heidelberg, Im Neuenheimer Feld 366, D-6900 Heidelberg, Federal Republic of Germany |
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Abstract: | Summary Human platelet membranes were solubilized with the zwitterionic detergent CHAPS (3-3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate) and the solubilized extract subjected to gel filtration. Binding of the adenosine receptor agonist 3H]NECA (5-N-ethylcarboxamidoadeno-sine) was measured to the eluted fractions. Two 3H]NECA binding peaks were eluted, the first of them with the void volume. This first peak represented between 10% and 25% of the 3H]NECA binding activity eluted from the column. It bound 3H]NECA in a reversible, saturable and GTP-dependent manner with an affinity of 46 nmol/1 and a binding capacity of 510 fmol/mg protein. Various adenosine receptor ligands competed for the binding of 3H]NECA to the first peak with a pharmacological profile characteristic for the A2 adenosine receptor as determined from adenylate cyclase experiments. In contrast, most adenosine receptor ligands did not compete for 3H]NECA binding to the second, major peak. These results suggest that a solubilized A2 receptor-GS protein complex of human platelets can be separated from other 3H]NECA binding sites by gel filtration. This allows reliable radioligand binding studies of the A2 adenosine receptor of human platelets.Abbreviations CHAPS
3-3-(cholamidopropyl)dimethylammoniol-l-propanesulfonate
- CIA
2-chloroadenosine
- CPA
N6-cyclopentyladenosine
- DPX
1,3-diethyl-8-phenylxanthine
- NECA
5-N-ethylcarboxamidoadenosine
- PAA
2-phenylaminoadenosine
- PIA
N6-phenyhsopropyladenosine
- XAC
8-{4-({(2-aminoethyl)amino}carbonyl}methyl)oxy]phenyl]-1,3-dipropylxanthine
Send offprint requests to M. J. Lohse |
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Keywords: | A2 Adenosine receptor Human platelets Radioligand binding Adenylate cyclase |
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