Conference paper

Spectroscopic Studies of Ligand Interactions with Acetycholinesterase

Palmer Taylor ; Division of Pharmacology, Department of Medicine, University of California, San Diego, La Jolla, California 92037
Jamson Lwebuga-Mukasa ; Division of Pharmacology, Department of Medicine, University of California, San Diego, La Jolla, California 92037
Harvey Berman ; Division of Pharmacology, Department of Medicine, University of California, San Diego, La Jolla, California 92037
Shelley Lappi ; Division of Pharmacology, Department of Medicine, University of California, San Diego, La Jolla, California 92037

Abstract

A series of ligands with suitable spectroscopic properties have
been employed for investigating the sites and specificity of ligand
association with an acetylcholinesterase purified to apparent homogeneity
from Torpedo californica. Bisquaternary ammonium .ligands
in which the nitrogens are maximally separated by 14 A, bind
with 1 : 1 stoichiometry with each 82 000 molecular weight subunit
on acetylcholinesterase. Placement of a benzoquinone moiety between
the quaternary nitrogens provides a ligand which upon
binding quenches the tryptophanyl fluorescence of the protein. The
complex exhibits 490/o of the fluorescence of the free protein and
the quenching appears to take place by radiationless energy transfer
between excited state dipoles. Since quenching appears uniform
with binding on each subunit, it is unlikely that the ligand binding
sites exist near a center or axis of symmetry between subunits.
Modification of the active s ite serine forming the respective sulfonyl
or phosphoryl esters enables one to study the orientation of the
bound bisquaternary ligand with respect to the catalytic serine and
the es.terifying group. Propidium, a second ligand, bililds to acetylcholinesterase
with an enhancement in quantum yield and a shift
in excitation and emission wavelengths. Propidium also exhibits
binding which is stoichiometric with each subunit on the enzyme
but is highly selective for a peripheral anionic site. Thus, agents
such as edrnphonium and N-methylaoridinium which bilnd preferentially
to the active center of the enzyme will not dissociate the
bound propidium. Bisquaternary ligands such as decamethonium
are mutually competitive with both the active center and the
peripheral anionic site. The long excitation and emission wavelengths
(488/632 nm) of propidium endow it with ideal acceptor
properties for energy transfer studies with various donor ligands
bound to the active center.

Keywords

Hrčak ID:

196589

URI

https://hrcak.srce.hr/196589

Publication date:

3.12.1975.

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