Separation of solubilized A2 adenosine receptors of human platelets from non-receptor [3H]NECA binding sites by gel filtration.
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-ethylcarboxamidoadenosine) 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/l 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.
Lohse
M J
MJ
Pharmakologisches Institut, Universität Heidelberg, Federal Republic of Germany.
Elger
B
B
Lindenborn-Fotinos
J
J
Klotz
K N
KN
Schwabe
U
U
D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism; D019830:Adenosine-5'-(N-ethylcarboxamide); D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism*; D002793:Cholic Acids; D002850:Chromatography, Gel; D006801:Humans; D011869:Radioligand Assay; D011942:Receptors, Adrenergic, alpha / Q000302:isolation & purification*; D012995:Solubility
D002793:Cholic Acids; D011942:Receptors, Adrenergic, alpha; D019830:Adenosine-5'-(N-ethylcarboxamide); D000241:Adenosine; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate