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.
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.
Casadó
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Departament de Bioquímica i Fisiologia, Facultat de Quimica, Universitat de Barcelona, Catalunya, Spain.
Cantí
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C
Mallol
J
J
Canela
E I
EI
Lluis
C
C
Franco
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R
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002540:Cerebral Cortex / Q000378:metabolism; D002793:Cholic Acids; D019204:GTP-Binding Proteins / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000378:metabolism; D010660:Phenylisopropyladenosine; D011869:Radioligand Assay; D011983:Receptors, Purinergic / Q000737:chemistry / Q000302:isolation & purification*; D012995:Solubility; D013552:Swine; D013816:Thermodynamics
D002793:Cholic Acids; D009419:Nerve Tissue Proteins; D011983:Receptors, Purinergic; D010660:Phenylisopropyladenosine; D006165:Guanylyl Imidodiphosphate; D019204:GTP-Binding Proteins; D008274:Magnesium; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate