TY - JOUR
T1 - Reconstitution of a Functional β‐Adrenergic Receptor using Cholate and a Novel Method for Its Functional Assay
AU - GAL, Alma
AU - BRAUN, Sergei
AU - FEDER, Debra
AU - LEVITZKI, Alexander
PY - 1983/8
Y1 - 1983/8
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0020525901&partnerID=8YFLogxK
U2 - 10.1111/j.1432-1033.1983.tb07580.x
DO - 10.1111/j.1432-1033.1983.tb07580.x
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C2 - 6307696
AN - SCOPUS:0020525901
SN - 0014-2956
VL - 134
SP - 391
EP - 396
JO - European Journal of Biochemistry
JF - European Journal of Biochemistry
IS - 2
ER -