Solubilization and characterization of guanine nucleotide‐sensitive muscarinic agonist binding sites from rat myocardium

Berrie, Christopher P.; Birdsall, N. J. M.; Hulme, Edward C.; Keen, Mary; Stockton, Jane M.

doi:10.1111/j.1476-5381.1984.tb16482.x

Cited by 52 publications

(25 citation statements)

References 28 publications

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“…The preparation of EDTA-washed membranes from extraorbital lacrimal glands and myocardium was as described by Berrie et al (1984) for myocardial membranes except the KCl/sodium pyrophosphate treatment was omitted. The rapidly frozen membranes were stored at -20'C or -80'C.…”

Section: Methodsmentioning

confidence: 99%

“…The soluble receptor binding assays used a similar protocol to that for the membrane binding assays except the separation of free and bound radioligand was accomplished by gel filtration on 2 ml columns of Sephadex G50M (Berrie et al, 1984). All assays were carried out at 40C in a 1 mM Mg2I/ 20 mm Na HEPES buffer, pH 7.5 containing 1% digitonin.…”

Section: Methodsmentioning

confidence: 99%

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Modulation of the structure‐binding relationships of antagonists for muscarinic acetylcholine receptor subtypes

Pedder

Eveleigh

Poyner

et al. 1991

British J Pharmacology

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1Membranes from rat cerebral cortex, myocardium and extraorbital lacrimal gland were used as sources of M1, M2 and M3 muscarinic acetylcholine receptors respectively and the affinities of seven antagonists for the three subtypes were examined under different experimental conditions. 2 The affinities for the membrane-bound receptors were measured at different ionic strengths and temperatures and compared with those determined on the receptor solubilised in the neutral detergent digitonin or the zwitterionic detergent, CHAPSO. 3 The range of measured affinity constants of a given antagonist for a specific subtype varied from 2 (atropine at M1 receptors) to 1000 (AF-DX 116 at M2 receptors). 4 As a consequence of these changes in affinity, which were dependent on the drug, the subtype and the experimental conditions, both the structure-binding relationships of a given subtype can

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Modulation of the structure‐binding relationships of antagonists for muscarinic acetylcholine receptor subtypes

Pedder

Eveleigh

Poyner

et al. 1991

British J Pharmacology

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“…KCl-pyrophosphate extracted, EDTA-washed membranes from rat myocardium were prepared and solubilized using digitonin as described previously (Berrie et al, 1984b). Assay and analytical methods, and the sucrose density gradient centrifugation technique were as previously described (Hulme et al, 1983a,b;Berrie et al, 1984a,b;1985c digitonin, pH 7.5 at 4°C unless otherwise specified.…”

Section: Methodsmentioning

confidence: 99%

“…These conditions have previously been shown to allow the detection of stable mAChR-N protein complexes in addition to the apparently monomeric binding protein (Berrie et al, 1984b (Berrie et al, 1985c . (Hulme et al, 1981;Burgisser et al, 1982).…”

Section: Methodsmentioning

confidence: 99%

The binding of pirenzepine to digitonin‐solubilized muscarinic acetylcholine receptors from the rat myocardium

Birdsall

Hulme

Keen

1986

British J Pharmacology

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The binding of pirenzepine to digitonin‐solubilized rat myocardial muscarinic acetylcholine receptors has been examined at 4°C. Solubilization produced only small changes in the binding of N‐methylscopolamine and atropine. In contrast to the low affinity binding of pirenzepine found to be present in the membranes, high affinity binding was detected in the soluble preparation. In both preparations, pirenzepine binding was complex. High affinity pirenzepine binding (KD ∼ 3 × 10−8M) to the soluble myocardial receptors could be monitored directly using [3H]‐pirenzepine. [3H]‐pirenzepine‐labelled soluble myocardial receptors have a sedimentation coefficient of 11.1 s. This indicates that [3H]‐pirenzepine binds predominantly to the uncoupled form of the receptor. However, [3H]‐ pirenzepine‐agonist competition experiments indicated that the high affinity pirenzepine binding sites are capable of coupling with a guanosine 5′‐triphosphate (GTP)‐binding protein. Pirenzepine affinities for the soluble myocardial receptors were unaffected by their state of association with the GTP‐binding proteins found in the heart. The equilibrium binding properties of the soluble cortical and myocardial receptors were very similar. However, the binding kinetics of the myocardial receptor were much slower. It appears that the membrane environment can affect the affinity of pirenzepine for the rat myocardial muscarinic receptor. Removal of the constraint by solubilization allows the expression of high affinity pirenzepine binding.

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“…There is evidence of specific complexes from target size analysis (Uchida et al, 1982), photo affinity labelling (Avissar et al, 1983), and binding studies and references therein). In the accompanying paper we describe the solubilization and characterization of a specific muscarinic receptor-guanine nucleotide binding protein complex from the rat myocardium (Berrie et al, 1984). The evidence is, however, that the structural integrity of the muscarinic binding site is much less dependent on protein interactions than that of, for example, the nicotinic acetylcholine receptor from Torpedo.…”

Section: Solubilization Of Muscarinic Receptorsmentioning

confidence: 99%

Hydrodynamic properties of muscarinic acetylcholine receptors solubilized from rat forebrain

Berrie¹,

Birdsall²,

Haga³

et al. 1984

British J Pharmacology

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Solubilization and characterization of guanine nucleotide‐sensitive muscarinic agonist binding sites from rat myocardium

Cited by 52 publications

References 28 publications

Modulation of the structure‐binding relationships of antagonists for muscarinic acetylcholine receptor subtypes

Modulation of the structure‐binding relationships of antagonists for muscarinic acetylcholine receptor subtypes

The binding of pirenzepine to digitonin‐solubilized muscarinic acetylcholine receptors from the rat myocardium

Hydrodynamic properties of muscarinic acetylcholine receptors solubilized from rat forebrain

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