Sulfhydryl-group modifications of Torpedo Californica acetylcholine receptor: subunit localization and effects on function

Conference · Thu May 01 00:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:5127128

McNamee, M G; Yee, A S

The effects of thiol-group modification on acetylcholine receptor (ACHR) function were measured using purified Torpedo ACHR reconstituted into soybean lipid vesicles. N-Phenyl-maleimide (NPM) was used to modify sulfhydryl groups in ACHR in the absence of any prior reduction by dithiotheitol. Modification by NPM led to the inhibition of ion channel activity without a detectable effect on ligand binding. The ion flux inhibition by NPM primarily affected channel activation, since the initial rates of activation decreased over a wide range of carbamylcholine concentrations. The /sup 3/H-NPM subunit labelling pattern of ACHR (a multisubunit membrane protein with ..cap alpha../sub 2/..beta gamma..delta stoichiometry) revealed preferential labelling of the ..gamma.. subunit. At high NPM concentration, the number of sulfhydryl groups on the ..gamma.. subunit that could be modified with NPM was two. Detergent was required during labelling for functionally relevant thiol group modifications, and most of the label was protected from protease digestion in the reconstituted membranes. These results are consistent with the presence of the NPM modification in a bilayer and/or cytoplasmic domain. Analysis of cyanogen bromide and trypsin fragments indicates that the labeled cysteines may be located in the postulated amphipathic helix region of the ..gamma.. subunit.

• Research Organization: Univ. of California, Davis
• OSTI ID: 5127128
• Report Number(s): CONF-8606151-
• Journal Information: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Vol. 45:6; ISSN FEPRA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACETYLCHOLINE
AMINES
AMMONIUM COMPOUNDS
ANIMALS
AQUATIC ORGANISMS
AUTONOMIC NERVOUS SYSTEM AGENTS
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL EFFECTS
BIOLOGICAL FUNCTIONS
DRUGS
ESTERS
FISHES
FUNCTIONS
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LIGANDS
MEMBRANE PROTEINS
NEUROREGULATORS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PARASYMPATHOMIMETICS
PROTEINS
QUATERNARY COMPOUNDS
REACTION KINETICS
RECEPTORS
THIOLS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES