Torpedo electric organ. Compared to normal concentration standards, this yield is
exceptionally high.
4.2.2.2 Physicochemical Properties and Subunit Structure
The physical and chemical properties of the AChR have been elucidated. Optical rota-
tory dispersion measurements indicate that the receptor consists of about 34% helix
and 28–30% β-sheet structure—a high proportion of ordered secondary structure. Some
carbohydrates are part of the molecule. The DNA encoding the receptor has been
cloned and sequenced, revealing the complete amino acid sequence of the subunits.
Thesubunit structure of the AChR varies according to its origin. There are four pep-
tide chains, referred to as α(mass ~ 40 kD),β (~ 48 kD),γ (~ 58 kD), and δ (~ 64 kD),
which can be separated by electrophoresis. The receptor of Torpedo californica has an
α 2 βγδchain composition, giving it a monomeric molecular mass of 250 kD. The α
chain is affinity-labeled specifically by [^3 H] bromoacetylcholine and by [^3 H] 4-N-
maleimidobenzyl-trimethylammonium iodide on Cys-192 and Cys-193 and therefore
must be the ACh binding subunit. The other chains are integral parts of the receptor and
do not dissociate, even in 8 M urea. The different chains have different amino acid
sequences but similar compositions. 4-N-Maleimidobenzyl-trimethylammonium
iodide, a specific affinity reagent, indicates the important fact that the quaternary-
ammonium-ion binding site (the -COO−of glutamate) and an —SH group binding to
maleimide are in close proximity. [^3 H] Bungarotoxin ([^3 H] BTX) crosslinks the αand
βchains and probably also obstructs the ion channel. The βandγchains are preferen-
tially labeled by a nitrene obtained from pyrene-sulfonylazide, a hydrophobic reagent
believed to attach itself to proteins within the core of the membrane (see figure 4.5).
208 MEDICINAL CHEMISTRY
Figure 4.5 This model of the nicotinic acetylcholine receptor shows two pentameric units cova-
lently linked through the δsubunit. One of the units is shown in cross-section, indicating the selec-
tivity gate of the ion channel in the closed state. The 43 kD protein is shown, associated with the
receptor on the cytoplasmatic side.Theligand binding sites of the AChR have been explored by a number of techniques.
The ACh binding site was first investigated using the MBTA affinity label. MBTA
binds to the native receptor with a KD= 8 × 10 −^5 M but binds much more strongly after
reduction of the membrane preparation with dithioerythritol, a mild reducing agent. In
binding to the AChR, MBTA occupies half the sites that bind [^125 I] BTX.