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

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:
OSTI Identifier:
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
Research Org:
National Institute of Health, Bethesda, MD
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