Solubilization of A1 adenosine receptor from pig brain: characterization and evidence of the role of the cell membrane on the coexistence of high- and low-affinity states

J Neurosci Res. 1990 Aug;26(4):461-73. doi: 10.1002/jnr.490260409.


The present solubilization strategy recognizes the important role of detergent cocktails in the solubilization and subsequent stability of adenosine A1, receptors from pig brain cortical membranes. The 3-[3-(cholamidopropyl)dimethylammonio]-1-propane-sulfonate-digitonin mixture produced the extraction of up to 52% of the receptor with an enrichment of 1.2-fold with respect to crude membranes. The binding activity of the soluble extract was very stable even in the absence of glycerol. In crude membranes the existence of high- and low-affinity states was detected, but in the soluble extract and in the detergent-treated membranes only the high-affinity state was detected. Association-dissociation curves showed that in crude membranes no interconversion between high- and low-affinity sites is produced by the association of the ligand [3H]R-N6-phenylisopropyladenosine. These results suggest that the high- and low-affinity states are different conformations induced by the structure of the membrane. The modulation of the binding activity by (Gpp(NH)p) 5'-guanylylimidodiphosphate and Mg2+ was studied. In crude membranes Gpp(NH)p shifted the high-affinity state to the low-affinity state, whereas the contrary occurred when Mg2+ was used. The effect of both Mg2+ and Gpp(NH)p was also assayed with the soluble extract and with the detergent-treated membranes. In addition to a decrease of the overall binding capacity, Gpp(NH)p promoted a conversion to all low-affinity states in the detergent-treated membranes or to all very-low-affinity sites in the soluble extract. Mg2+ and Gpp(NH)p counteracted their effects in intact membranes, whereas Mg2+ could not reverse the uncoupling effect of Gpp(NH)p with solubilized or detergent-treated membranes. Thus, it is suggested that Mg2+ acts at sites other than guanine-nucleotide-sensitive sites. If high-affinity states correspond to receptor/G protein complexes and low-affinity states correspond to the uncoupled receptor, we should conclude that Mg2+, as well as the loss of membrane integrity, favours the interaction of A1 receptor molecule with G protein.

MeSH terms

  • Animals
  • Brain / metabolism*
  • Cell Membrane / metabolism*
  • Cerebral Cortex / metabolism
  • Cholic Acids
  • GTP-Binding Proteins / metabolism
  • Guanylyl Imidodiphosphate / metabolism
  • In Vitro Techniques
  • Kinetics
  • Magnesium / metabolism
  • Nerve Tissue Proteins / analysis
  • Nerve Tissue Proteins / metabolism
  • Phenylisopropyladenosine
  • Radioligand Assay
  • Receptors, Purinergic / chemistry
  • Receptors, Purinergic / isolation & purification*
  • Solubility
  • Swine
  • Thermodynamics


  • Cholic Acids
  • Nerve Tissue Proteins
  • Receptors, Purinergic
  • Phenylisopropyladenosine
  • Guanylyl Imidodiphosphate
  • GTP-Binding Proteins
  • Magnesium
  • 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate