Reconstitution of a Functional β‐Adrenergic Receptor using Cholate and a Novel Method for Its Functional Assay
Open Access
- 1 August 1983
- journal article
- research article
- Published by Wiley in European Journal of Biochemistry
- Vol. 134 (2), 391-396
- https://doi.org/10.1111/j.1432-1033.1983.tb07580.x
Abstract
Solubilization of purified turkey erythrocyte membranes at increasing cholate to protein ratios and in the presence of salt, extracts up to 20% of the β-adrenergic receptor together with the GTP stimulatory protein (Ns) of adenylate cyclase. Upon removal of the cholate, by active absorption on Bio-beads, the functional interaction between the β-receptor and the GTP regulatory protein Ns is quantitatively restored. The receptor (R) in the presence of l-isoproterenol and p[NH]ppG is able to catalyze the activation of Ns to its permanently active state, N's p[NH]ppG, with a rate constant (kon) identical to that of the native membrane. Reconstitution of the R/Ns mixture using poly(ethyleneglycol)-6000 restores the receptor binding properties as effectively as SM-2 Bio-beads. Unlike SM-2 Bio-beads, however, poly(ethyleneglycol) is not as efficient in restoring the R to Ns functional coupling. In this communication we also report on the ability to monitor quantitatively N's. p[NH]ppG, using native turkey erythrocyte membranes in the presence of Lubrol-PX as the source of the catalytic unit (c) of adenylate cyclase. The latter method is as efficient as using S49 AC- lymphoma cell membranes but much less expensive. Using this technique, we also demonstrate that when the Ns to C interaction is nullified, employing treatment with N-ethylmaleimide, the parameters which characterize R to Ns coupling remain unchanged.Keywords
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