Molecular Structures of Acetylcholinesterase from Electric Organ Tissue of the Electric Eel

Abstract

Three purified molecular forms of acetylcholinesterase (EC 3.1.1.7) with sedimentation coefficients of 18 S, 14 S, and 11 S were studied by analytical ultracentrifugation and electron microscopy. The three species have molecular weights of (1.1 ± 0.1) X 106, (7.5 4 1.5) X 106, and (3.35 4-0.25) X 106, respectively. Electron micrographs reveal that the 18S and 14S forms are asymmetric, composed of a head, containing a large number of subunits, and an elongated tail. The 11 S form of acetylcholinesterase is apparently a tetrameric structure devoid of the tail. Maleylation of 18S and 14S acetylcholinesterases abolishes their tendency to aggregate at low ionic strength.Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) in extracts of fresh electric organ tissue of electric eels consists of several components distinguished by their sedimentation coefficients (1, 2). The major component is an 18S form, which aggregates at low ionic strength (2, 3). A 14S component, which also aggregates at low ionic strength, is present in smaller amounts. Treatment of electric organ tissue with proteolytic enzymes or autolysis converts all the acetylcholinesterase to an 11S form, which does not aggregate at low ionic strength and is not present in fresh tissue (2, 3).We have purified, by affinity chromatography, the different molecular forms of acetylcholinesterase present in fresh tissue or obtained after proteolysis (3, 4). Purified 18S and 14S acetylcholinesterases retain their tendency to aggregate at low ionic strength, and the 18S, 14S, and 11S acetylcholinesterases all display similar patterns on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 2-mercaptoethanol (4). Massouli6 et al.(5) have suggested, on the basis of gel-filtration studies, that 18S and 14S acetylcholinesterases have an asymmetric structure, in contrast with 11S acetylcholinesterase, which is globular.In the following we will show, by electron microscopy and ultracentrifugation, that 18S and 14S acetylcholinesterases differ markedly in their quaternary structure from the 11S form. Similar electron microscopic observations have been recently reported by Rieger et al. (6). We will also show that the tendency of 18S and 14S acetylcholinesterases to aggregate at low ionic strength can be prevented by chemical modification. The relevance of these observations to the relationship of acetylcholinesterase with the electroplax membrane will be discussed. METHODS 18S, 14S, and llS forms of acetylcholinesterase were purified by affinity chromatography (3, 4). The 18S and -14S enzymes were separated from each other by sucrose gradient centrifugation as follows: aliquots of 1.8 ml of a mixture of purified 18S and 14S enzymes (containing about 1 mg/ml of enzyme) were layered on a 5-20% linear sucrose gradient in 1.0 M NaCl-0.01 M phosphate (pH 7.0) of total volume of 28 ml, with a 5-ml cushion of 60% sucrose in the same buffer at the bottom. Centrifugation was performed in an SW27 rotor in an L2 65-B Beckman preparative ultra...

Two Different Heparin-binding Domains in the Triple-helical Domain of ColQ, the Collagen Tail Subunit of Synaptic Acetylcholinesterase

“…For ColQ, we showed that the interaction is primarily electrostatic, since the elimination of basic charges completely abolishes heparin binding. This is consistent with chemical modification studies, in which maleylation of the ⑀-NH 2 groups of lysine residues eliminated the tendency of asymmetric AChE to form glycosaminoglycan-dependent aggregates (25), and with calorimetric measurements that indicate that heparin binding is predominantly electrostatic. For heparin-binding proteins where basic residues are involved, different consensus sequences have been described according to the structural context in which they are expressed.…”

“…It is interesting to note that the length of ColQ triple helix, measured both in electron micrographs (3,25) as in the structural model (12), is around 50 nm, which corresponds to the distance between pre-and postsynaptic membranes (36). Therefore, the distance between the two HBDs (ϳ25 nm) represents an important fraction of the space between these membranes, so that their binding to different layers of the extracellular matrix would serve to orient the enzyme for proper function.…”

The Dependence of Acetylcholinesterase Aggregation at Low Ionic Strength upon a Polyanionic Component

“…In effect, alkylation of the amine residues, either by maleic anhydride [4], which replaces positively charged by negatively charged groups, or by acetic anhydride [6], which neutralises them, completely abolished all aggregation even in the presence of added aggregating agent, without any change in the hydrodynamic properties of the isolated molecules. It is thus very likely that positive groups, such as lysine residues, participate in ionic interactions responsible for aggregation.…”

Immunochemical and Enzymic Properties of the Electric Eel Acetylcholinesterase‐Antiacetylcholinesterase System

“…Studies on the native molecular forms of acetylcholinesterase by the techniques of gel filtration [6,7] and equilibrium sedimentation [8] indicate that they are large asymmetrical structures, whereas the 1143 enzyme is of a globular form. Electron microscopy confirms the conclusions reached in the physicoEnzyme.…”

“…chemical studies. Thus the native forms of acetylcholinesterase are indeed asymmetric structures composed of a multisubunit head and an elongated tail [8,9], while the 1143 enzyme is most probably a tetrameric structure, devoid of the tail [8-lo].…”