Reconstitution of a functional beta-adrenergic receptor using cholate and a novel method for its functional assay.
Solubilization of purified turkey erythrocyte membranes at increasing cholate to protein ratios and in the presence of salt, extracts up to 20% of the beta-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 beta-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.
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002793:Cholic Acids / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000032:analysis; D005033:Ethylmaleimide / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D011941:Receptors, Adrenergic / Q000378:metabolism*; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D012995:Solubility; D014422:Turkeys
D002793:Cholic Acids; D011941:Receptors, Adrenergic; D011943:Receptors, Adrenergic, beta; D006165:Guanylyl Imidodiphosphate; D000262:Adenylyl Cyclases; D005033:Ethylmaleimide