Title : Expression and renaturation of the N-terminal extracellular domain of torpedo nicotinic acetylcholine receptor alpha-subunit - Schrattenholz_1998_J.Biol.Chem_273_32393 |
Author(s) : Schrattenholz A , Pfeiffer S , Pejovic V , Rudolph R , Godovac-Zimmermann J , Maelicke A |
Ref : Journal of Biological Chemistry , 273 :32393 , 1998 |
Abstract :
The N-terminal extracellular region (amino acids 1-209) of the alpha-subunit of the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue was expressed as inclusion bodies in Escherichia coli using the pET 3a vector. Employing a novel protocol of unfolding and refolding, in the absence of detergent, a water-soluble globular protein of 25 kDa was obtained displaying approximately 15% alpha-helical and 45% beta-structure. The fragment bound alpha-[3H]bungarotoxin in 1:1 stoichiometry with a KD value of 0.5 nM as determined from kinetic measurements (4 nM from equilibrium binding). The kinetics of association of toxin and fragment were of second order, with a similar rate constant (8.2 x 10(5) M-1 s-1) as observed previously for the membrane-bound heteropentameric nAChR. Binding of small ligands was demonstrated by competition with alpha-[3H]bungarotoxin yielding the following KI values: acetylcholine, 69 microM; nicotine, 0.42 microM; anatoxin-a, 3 miroM; tubocurarine, 400 microM; and methyllycaconitine, 0.12 microM. The results demonstrate that the N-terminal extracellular region of the nAChR alpha-subunit forms a self-assembling domain that functionally expresses major elements of the ligand binding sites of the receptor. |
PubMedSearch : Schrattenholz_1998_J.Biol.Chem_273_32393 |
PubMedID: 9829968 |
Schrattenholz A, Pfeiffer S, Pejovic V, Rudolph R, Godovac-Zimmermann J, Maelicke A (1998)
Expression and renaturation of the N-terminal extracellular domain of torpedo nicotinic acetylcholine receptor alpha-subunit
Journal of Biological Chemistry
273 :32393
Schrattenholz A, Pfeiffer S, Pejovic V, Rudolph R, Godovac-Zimmermann J, Maelicke A (1998)
Journal of Biological Chemistry
273 :32393