Evaluation of a purification procedure for the muscarinic receptor for the purpose of quantitative receptor assays of anticholinergics. Part A: The membrane-bound receptor.
The presented purification procedure for the muscarinic receptor from calf striatum includes the extraction of lipids with hexane in the first step and the removal of 39% of non-receptor proteins with 2 M NaCl in the second step. The simplicity of such an approach to the purification of the receptor warrants its use in the routine practice for quantitative purposes. The high affinity binding of tertiary 3H-dexetimide (3H-DEX) and quaternary 3H-N-methylscopolamine (3H-NMS) is preserved after the removal of irrelevant lipids and proteins from the P2-pellet. The overall yield of receptors--80%, when labelled with 3H-NMS, was satisfactory. Moreover, the final product, the NaCl-pellet, exerts a higher density of 3H-NMS binding sites per mg proteins by a factor of about 1.7. The overall yield of receptors and purification factor were lower, when measured with 3H-DEX. The total yield of 3H-DEX binding sites amounted to about 40% and the receptor density per mg protein decreased by a factor of 0.85. We did not succeed in the improvement of the ratio specific/non-specific binding, neither for 3H-DEX nor for 3H-NMS for the purified receptor preparations. The use of 3H-NMS is preferable to 3H-DEX in plasma sample assays because of a negligible effect of plasma on ligand binding when compared with 3H-DEX.
Smisterová
J
J
University Centre for Pharmacy, Groningen, The Netherlands.
Ensing
K
K
de Zeeuw
R A
RA
D000818:Animals; D001769:Blood; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry*; D002498:Centrifugation; D018680:Cholinergic Antagonists / Q000032:analysis* / Q000378:metabolism; D003342:Corpus Striatum / Q000737:chemistry; D003909:Dexetimide / Q000378:metabolism; D006586:Hexanes; D018727:Muscarinic Antagonists / Q000378:metabolism; D019832:N-Methylscopolamine; D011869:Radioligand Assay; D011976:Receptors, Muscarinic / Q000302:isolation & purification* / Q000378:metabolism; D012602:Scopolamine Derivatives / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D014316:Tritium
D018680:Cholinergic Antagonists; D006586:Hexanes; D018727:Muscarinic Antagonists; D011976:Receptors, Muscarinic; D012602:Scopolamine Derivatives; D014316:Tritium; D003909:Dexetimide; D012965:Sodium Chloride; D019832:N-Methylscopolamine