Hydrodynamic properties of adenosine Ri receptors solubilized from rat cerebral-cortical membranes

Abstract

Adenosine Ri receptors and inhibitory guanine-nucleotide-regulatory components were solubilized from rat cerebral-cortical membranes with sodium cholate. (-)-N6-Phenylisopropyl[2,8-3H]adenosine [( 3H]PIA) binds with high affinity to the soluble receptors, which retain the pharmacological specificity of adenosine Ri receptors observed in membranes. The binding is regulated by bivalent cations and guanine nucleotides. Bivalent cations increase [3H]PIA binding by increasing both the affinity and the apparent number of receptors. Guanine nucleotides decrease agonist binding by increasing the dissociation of the ligand-receptor complex. Adenosine agonists stabilize the high-affinity form of the soluble receptor. The hydrodynamic properties of the adenosine receptor were determined with cholate extracts of membranes that were treated with [3H]PIA. Sucrose-gradient-centrifugation analysis indicates that the receptor has a sedimentation coefficient of 7.7 S. The receptor is eluted from Sepharose 6B columns with an apparent Stokes radius of 7.2 nm. Labelling of either sucrose-gradient or gel-filtration-column fractions with pertussis toxin and [32P]-NAD+ reveals that both the 41,000- and 39,000-Mr substrates overlap with the receptor activity. These studies suggest that the high-affinity adenosine-receptor-binding activity in the cholate extract represents a stable R1-N complex.

Solubilization of stable adenosine A1 receptors from rat brain

“…In contrast, PIA binding to R-N complexes is greatly increased by Mg2`and almost eliminated by GTP [2]. With adenosine-stabilized receptors some R-N complexes must persist, since the overall affinity for CPDPX was somewhat lower, and Mg2+ decreased receptor number.…”

“…The initial PIA-binding activity was low and decayed rapidly at either 40 or 20°C (Fig. 1 a) CPDPX binding was measured under control conditions, but, to maximize [3H]PIA binding, the agonist binding was measured in the presence of Mg2" to shift the receptors into the high-affinity form for agonists [2,8].…”

“…Progress on the purification of adenosine Al receptors has been hampered by the fact that the receptors tend to be occupied during detergent solubilization owing to the presence in many tissues of endogenous adenosine, which has a relatively high affinity for Al receptors. As has been well described for other receptors (and also for adenosine Al receptors) that are coupled to either the stimulation or the inhibition of adenylate cyclase, binding of agonists promotes the stabilization of R-N complexes [2,3]. For adenosine receptors, this situation, coupled with the high concentrations of endogenous adenosine, results in the exclusive extraction by detergents of adenosine-receptor-Nprotein complexes.…”

“…It appears that solubilized adenosine receptors must be reconstituted in order to bind ligands specifically. A review of the adenosine receptor solubilization literature [2,[4][5][6][7][8] reveals that, in all such reports, binding assays were performed after dilution of the detergent below its CMC, so that, in effect, a detergent-dilution reconstitution procedure [18] was carried out. {One report [5] Ci/mmol); non-specific binding was determined in the presence of 10 mM-caffeine.…”

“…In every study that has been reported describing solubilized adenosine-receptor preparations, agonists bind with high affinity [2,[4][5][6][7][8] and guanine nucleotides regulate the agonist binding [2,4,5,7,8]. Extensive pretreatment of the membranes with adenosine deaminase before solubilization results in a substantial loss of the receptors that are subsequently detected, as monitored by agonist binding [2]. The reason that this situation poses a problem in the purification of adenosine receptors is that R-N complexes are intrinsically highly unstable, having rarely been demonstrated for any receptor system to withstand fractionation procedures.…”