High yield solubilization of undenatured luteinizing hormone-releasing hormone receptors from rat anterior pituitary

Ogier, Sally-Ann; Mitchell, Rory; Fink, George

doi:10.1042/bst0150138

Cited by 1 publication

(3 citation statements)

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“…Aliquots of 300 µ were used in the sub¬ sequent ligand binding assays. The PEG precipitation method of CHAPS-solubilized LHRH receptors has been shown previously (Winiger et al 1983;Ogier et al 1987) to permit recovery of fully functional LHRH-binding sites from the solubilization solution.…”

Section: Receptor Solubilizationmentioning

confidence: 99%

“…Separation of solubilized from non-solubilized specific binding sites was determined by centrifu¬ gation at 45 000 g for 1 h. In our hands, using rat tissue, this procedure gave an artifactual result of 20 + 2% of specific binding in the supernatant in the absence of any detergent (Ogier et al 1987). This was presumably due to the retention of microdispersed fragments of tissue in the dense solution.…”

Section: Solubilization Conditionsmentioning

confidence: 99%

“…For example, 8% of available sites from bovine pituitaries (Perrin, Haas, Rivier & Vale, 1983) and 20% from rat ovaries (Capponi, Aubert & Clayton, 1984) were solubilized with 3-((3-cholamidopropyl) -dimethylammonio)-1 -propanesulphonate (CHAPS; 5 mmol/1). Where higher yields have been reported (Winiger, Birabeau, Lang et al 1983) the solubilization criteria may not have been sufficiently stringent (Ogier, Mitchell & Fink, 1987).…”

Section: Introductionmentioning

confidence: 98%

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Solubilization of a large molecular weight form of the rat LHRH receptor

Ogier

Mitchell²,

Fink³

1987

Journal of Endocrinology

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The LHRH receptor has been solubilized from male rat anterior pituitary glands, using the zwitterionic detergent 3-((3-cholamidopropyl)-dimethylammonio)-1-propanesulphonate in the presence of a high concentration of sodium chloride. This method gave high yields (up to greater than 70%) of the LHRH-binding site from the membrane preparation. Ligand binding studies using LHRH analogues were carried out to determine dissociation constants for LHRH receptors both in situ in the membrane preparation and for solubilized LHRH receptors. For all the analogues the binding characteristics were similar in both preparations, suggesting that the solubilization procedure left the LHRH receptor undenatured. Gel filtration revealed an apparent molecular weight for the LHRH receptor of 100,000-160,000, with the mean value being approximately twice that found by others using sodium dodecyl sulphate-polyacrylamide gel electrophoretic techniques. The results indicate that the LHRH receptor probably exists in gonadotroph membranes as a large complex of more than one subunit.

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Section: Receptor Solubilizationmentioning

confidence: 99%

Section: Solubilization Conditionsmentioning

confidence: 99%