EVALUATION OF A PURIFICATION PROCEDURE FOR THE MUSCARINIC RECEPTOR FOR THE PURPOSE OF QUANTITATIVE RECEPTOR ASSAYS OF ANTICHOLINERGICS .B. THE SOLUBILIZED RECEPTOR
J. Smisterova et al., EVALUATION OF A PURIFICATION PROCEDURE FOR THE MUSCARINIC RECEPTOR FOR THE PURPOSE OF QUANTITATIVE RECEPTOR ASSAYS OF ANTICHOLINERGICS .B. THE SOLUBILIZED RECEPTOR, Preparative biochemistry, 25(4), 1995, pp. 223-251
For the purpose of quantitative receptor assays, a three-step solubili
zation procedure including three optimization sets for muscarinic rece
ptor from calf striatum was developed. The first step includes the ext
raction of the P2-pellet with n-hexane and consequently with 2 M NaCl.
By the latter, 39% of non-receptor proteins was extracted. The result
ing pellet (NaCl-pellet), enriched in muscarinic receptors by a factor
of 1.5-1.7, was solubilized with 1% digitonin. The binding parameters
of the solubilized receptor were determined for the tertiary H-3-dexe
timide (H-3-DEX) and the ary quaternary H-3-N-methylscopolamine (H-3-N
MS). The resulting receptor density measured with H-3-dexetimide was l
ower (43.3% of that for the NaCl-pellet) than that for H-3-N-methyl-sc
opolamine (56.7%). The treatment with digitonin preserved the high aff
inity for H-3-N-methylscopolamine (K-d = 0.645 nM), however the affini
ty of H-3-dexetimide decreased after solubilization (K-d = 8.526 nM).
The use of solubilized receptors in combination with hydrophilic H-3-N
MS allows to increase the ratio specific/non-specific binding, since t
he non-specific binding for this ligand to the solubilized preparation
is lower when compared with membrane-bound receptors. The above solub
ilization procedure was found preferable over directly solubilizing th
e P2-pellet since (a) the receptor density for 3H-NMS was higher for t
he solubilized NaCl-pellet by a factor of about 1.7, and (b) the treat
ment of the P2-pellet with digitonin resulted in a lowering of the K-d
to 2.422 nM. However, with respect to the plasma effect on the ligand
binding, both solubilized preparations give similar results. The use
of the solubilized NaCl-pellet or the P2-pellet can considerably impro
ve the quantitative receptor assays of plasma samples. Unlike the memb
rane-bound receptor, a high volume of plasma, such as 400 mu l, can be
added to the assay without any influence on the H-3-DEX binding when
solubilized preparation is used.