skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Purification and characterization of 94kDa and 80kDa forms of the muscarinic cholinergic receptor

Abstract

Two molecular forms of the muscarinic cholinergic receptor have been consistently observed in a variety of species, albeit in variable amounts. Proteins which are specifically labeled by (/sup 3/H)propylbenzilylcholine mustard ((/sup 3/H)PrBCM) were observed at 94kDa and 80kDa upon SDS-PAGE of membrane proteins prepared from brains and hearts of trout, frog, turtle, chicken, rat, and pig. They have developed a purification procedure which yields each of these proteins in a homogeneous form suitable for structural analysis. The four step procedure involves affinity chromatography on 3-(2'-aminobenzhydryloxy)tropane-sepharose, concentration on hydroxylapatite, preparative SDS-PAGE and extraction of individual bands from the gel. Limited tryptic digestion of purified (/sup 3/H)PrBCM-labeled porcine atrial muscarinic receptor yields (/sup 3/H)-labeled fragments of 75, 65, 52, 40, 35, 30, 25, and 20kDa, in close agreement with results of analogous digestions of muscarinic receptor from other species and tissues. Complete tryptic digestion and subsequent mapping by reverse-phase HPLC yields very similar profiles for (/sup 125/I)-labeled 94kDa and 80kDA receptor forms. Most peaks which elute in the hydrophobic region of the profile overlap for the two proteins while the 94kDa protein contains several additional peaks of apparent low hydrophobicity.

Authors:
; ;
Publication Date:
Research Org.:
National Institute of Health, Bethesda, MD
OSTI Identifier:
5020666
Alternate Identifier(s):
OSTI ID: 5020666
Report Number(s):
CONF-8606151-
Journal ID: CODEN: FEPRA
Resource Type:
Conference
Resource Relation:
Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States); Journal Volume: 45:6; Conference: 76. annual meeting of the Federation of American Society for Experimental Biology, Washington, DC, USA, 8 Jun 1986
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; PROTEINS; RECEPTORS; CHEMICAL COMPOSITION; PURIFICATION; ACETYLCHOLINE; BRAIN; CELL MEMBRANES; CHICKENS; CHROMATOGRAPHY; FROGS; HEART; IODINE 125; RATS; SWINE; TRACER TECHNIQUES; TRITIUM COMPOUNDS; TROUT; AMINES; AMMONIUM COMPOUNDS; AMPHIBIANS; ANIMALS; AQUATIC ORGANISMS; AUTONOMIC NERVOUS SYSTEM AGENTS; BETA DECAY RADIOISOTOPES; BIRDS; BODY; CARDIOVASCULAR SYSTEM; CELL CONSTITUENTS; CENTRAL NERVOUS SYSTEM; DAYS LIVING RADIOISOTOPES; DOMESTIC ANIMALS; DRUGS; ELECTRON CAPTURE RADIOISOTOPES; ESTERS; FISHES; FOWL; INTERMEDIATE MASS NUCLEI; IODINE ISOTOPES; ISOTOPE APPLICATIONS; ISOTOPES; LABELLED COMPOUNDS; MAMMALS; MEMBRANE PROTEINS; MEMBRANES; NERVOUS SYSTEM; NEUROREGULATORS; NUCLEI; ODD-EVEN NUCLEI; ORGANIC COMPOUNDS; ORGANS; PARASYMPATHOMIMETICS; QUATERNARY COMPOUNDS; RADIOISOTOPES; RODENTS; SEPARATION PROCESSES; VERTEBRATES 550201* -- Biochemistry-- Tracer Techniques

Citation Formats

Fracek, S.P. Jr., Venter, J.C., and Kerlavage, A.R.. Purification and characterization of 94kDa and 80kDa forms of the muscarinic cholinergic receptor. United States: N. p., 1986. Web.
Fracek, S.P. Jr., Venter, J.C., & Kerlavage, A.R.. Purification and characterization of 94kDa and 80kDa forms of the muscarinic cholinergic receptor. United States.
Fracek, S.P. Jr., Venter, J.C., and Kerlavage, A.R.. Thu . "Purification and characterization of 94kDa and 80kDa forms of the muscarinic cholinergic receptor". United States. doi:.
@article{osti_5020666,
title = {Purification and characterization of 94kDa and 80kDa forms of the muscarinic cholinergic receptor},
author = {Fracek, S.P. Jr. and Venter, J.C. and Kerlavage, A.R.},
abstractNote = {Two molecular forms of the muscarinic cholinergic receptor have been consistently observed in a variety of species, albeit in variable amounts. Proteins which are specifically labeled by (/sup 3/H)propylbenzilylcholine mustard ((/sup 3/H)PrBCM) were observed at 94kDa and 80kDa upon SDS-PAGE of membrane proteins prepared from brains and hearts of trout, frog, turtle, chicken, rat, and pig. They have developed a purification procedure which yields each of these proteins in a homogeneous form suitable for structural analysis. The four step procedure involves affinity chromatography on 3-(2'-aminobenzhydryloxy)tropane-sepharose, concentration on hydroxylapatite, preparative SDS-PAGE and extraction of individual bands from the gel. Limited tryptic digestion of purified (/sup 3/H)PrBCM-labeled porcine atrial muscarinic receptor yields (/sup 3/H)-labeled fragments of 75, 65, 52, 40, 35, 30, 25, and 20kDa, in close agreement with results of analogous digestions of muscarinic receptor from other species and tissues. Complete tryptic digestion and subsequent mapping by reverse-phase HPLC yields very similar profiles for (/sup 125/I)-labeled 94kDa and 80kDA receptor forms. Most peaks which elute in the hydrophobic region of the profile overlap for the two proteins while the 94kDa protein contains several additional peaks of apparent low hydrophobicity.},
doi = {},
journal = {Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)},
number = ,
volume = 45:6,
place = {United States},
year = {Thu May 01 00:00:00 EDT 1986},
month = {Thu May 01 00:00:00 EDT 1986}
}

Conference:
Other availability
Please see Document Availability for additional information on obtaining the full-text document. Library patrons may search WorldCat to identify libraries that hold this conference proceeding.

Save / Share:
  • Isolated guinea pig chromaffin cells possess both nicotinic (nAChR) and muscarinic (mAChR) cholinergic receptors that are positively coupled to catecholamine (CA) release. Sixty to 70% of CA release is mediated by nAChRs and 30-40% by mAChRs. In the absence of added calcium, nAChR mediated CA release was reduced by 65% whereas the muscarinic response was unaffected. The addition of 100nM 12-0-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C (PKC), also resulted in an increased CA release. Temporally and quantitatively, this response resembled that of mAChR activation. Addition of optimal concentrations of nicotine (50..mu..M) and TPA (100nM) induced a synergistic increasemore » in CA release. Addition of muscarine (1mM) and TPA resulted in an additive response despite a 40-60% inhibition of mAChR mediated inositol phosphate release by TPA. Thus, in guinea pig chromaffin cells, it appears that PKC activation alone is a sufficient stimulus for CA release and that activation of both nicotinic and muscarinic receptors may further increase this enzyme's activity.« less
  • The mAChR/effector pathway for signal transduction is important in the physiology of esophagus and mAChR alterations are involved in EtOH induced changes in several organs. To see if EtOH-induced increases in lower esophageal sphincter pressure (LESP) are due to upregulation of mAChR, the authors evaluated mAChR binding and D-R curves for bethanechol (IV) induced increases in LESP, and compared these values to changes in LESP after acute and chronic EtOH. EtOH was given to cats acutely or chronically. The number of mAChR sites (Bmax) in esophagus was lowered by acute EtOH, withdrawal from chronic EtOH raised Bmax. Acute injection ofmore » EtOH to cats in withdrawal reversed this increase in mAChR density. These changes correlated with the earlier data on EtOH-induced changes in LESP. In contrast, the D-R curve for bethanechol shifted to the right. Thus, the withdrawal-associated increase in Bmax is more likely to be a compensatory response to deficits distal to the receptor recognition site than to proximal deficits and doesn't cause LESP hyperactivity. Also, receptor binding changes do not necessarily translate into physiological changes.« less
  • Reports indicate that nipecotic acid ethyl ester (NAEE) displays cholinomimetic properties in vivo. In the present study a series of physiological and biochemical tests were conducted to characterize this action. NAEE had a negative inotropic effect on the guinea pig atrium, and stimulated contraction of the guinea pig ileum and isolated mouse stomach strip at concentrations similar to bethanechol (BCH). The atrial and ilial effects were reversed by atropine. Unlike BCH, NAEE had no effect on basal acid secretion in the isolated mouse stomach at concentrations < 100 ..mu..M. NAEE was more potent than carbachol (CCH) in displacing /sup 3/H-ONBmore » binding from rat brain membranes. The potency of NAEE to inhibit antagonist binding in rat heart membranes was enhanced by Mg/sup + +/ (Hill coefficient < 1.0) and reduced by Gpp(NH)p. Like CCH, NAEE inhibited GTP-stimulated adenylate cyclase in rat brain striatal membranes. As compared to CCH, NAEE had little effect (< 5%) as a stimulator of inositol phosphate (IP) production in rat brain slices. The results indicate that NAEE is a direct-acting muscarinic receptor agonist. Moreover, its differential effects on acid secretion, IP accumulation, and adenylate cyclase suggest that it may be useful for defining cholinergic receptor subclasses.« less
  • Earlier studies with the racemic 1-azabiocyclo[2.2.2]oct-3-yl {alpha}-fluoroalkyl-{alpha}-hydroxy-{alpha}-phenylacetate (FQNPe) mixture had demonstrated high in vitro binding affinity for the muscarinic-cholinergic receptor (m-AChR). Pre-treatment of rats with this new agent significantly blocked receptor localization of subsequently injected [I-131]-Z-(-,-)-IQNP, which is an established high affinity m-AChR ligand. Syntheses and characterization of the four FQNPe stereoisomers: (-)(-) FQNPe, (-)(+) FQNPe, (+)(-) FQNPe, and (+)(+) FQNPe will be presented. The interesting NMR spectra of the diastereomeric salts formed in the resolution of racemic {alpha}-(1-chloropent-5-yl)-{alpha}-hydroxy {alpha}-phenylacetic acid will also be discussed.
  • Radioiodinated p-iodobenzyl bromide is a potentially useful alkylating agent for the introduction of radioactive iodine into biologically interesting molecules. I-125 and I-123 labeled are prepared for the first time at the no carrier added level by an electrophilic substitution reaction on p-trimethylsilylbenzyl bromide using N-chlorosuccinimide in acetic acid. After HPLC purification, isolated yields of 70-80% were obtained within 1 hour with specific activities of 600-1500 mCi/..mu..mole. The utility of radioiodinated is demonstrated by the synthesis of radioiodinated p-iododexetimide, a potent muscarinic receptor antagonist (Kd=2.7 nM). Labeled with I-125 or I-123 was reacted with norbenzyldexetimide in 55% aqueous acetonitrile at 60more » deg C for 10 mins to give, after HPLC purification, a 70-80% yield of I-125 or I-123 p-iododexetimide (>99% radiochemically pure). Specific activities of above 600 mCi/..mu..mole were obtained in 65 mins from production of radioiodinated. Currently, the authors are evaluating radioiodinated p-iododexetimide as a muscarinic cholinergic receptor imaging agent for use with single photn emission computed tomography.« less