Rapid and Complete Purification of Acetylcholinesterases of Electric Eel and Erythrocyte by Affinity Chromatography

Abstract

Affinity chromatography has been used to purify acetylcholinesterase both from the electric tissue of Electrophorus electricus and from bovine erythrocyte membranes. For this purpose, several specific enzymic inhibitors of each protein were synthesized and joined covalently to an insoluble support resin. AchE is selectively retained by such inhibitor-resins when highly impure solutions are chromatographed upon them. After removal from the resin, both enzymes are electrophoretically homogeneous and they may be recovered in yields of 75% or more.In the study to be presented below, we have used affinity chromatography to purify acetylcholinesterase (AchE) from both the electric tissue of Electrophorus electricus and from bovine erythrocyte membranes; only a single purification step is required. The central feature of this technique depends upon coupling a specific inhibitor of the enzyme to an inert, insoluble support resin. Under appropriate solvent conditions, chromatography of the enzyme upon the inhibitor-resin compound selectively retains AchE and thus eliminates contaminating molecules that possess no affinity for the inhibitor ligand. Subsequently, AchE is removed from the column by elution with another specific inhibitor whose inhibition constant (Ki) is less than that of the inhibitor-resin compound.Purification by affinity chromatography therefore requires (a) synthesis of an inhibitor that can be covalently bound to a solid support; (b) attachment of the inhibitor molecule to the resin support by a chain of atoms long enough to enable the inhibitor to interact readily with the binding site(s) of the enzyme; and (c) establishing conditions under which the protein can be selectively removed from the column. (For a summary of recent studies on affinity chromatography, see refs. 1 and 2.)In earlier studies, Leuzinger and Baker (3) have used conventional procedures to purify eel AchE. Erythrocyte AchE has never been purified, although partial purification procedures have been presented (4-6). The work outlined here was initiated to facilitate complete purification of both proteins prior to comparative studies of their molecular structures and function. MATERIALS AND METHODS Measurements of enzyme activityAchE activity was measured from the amount of 0.01 NNaOH needed to maintain constant pH during hydrolysis of acetylcholine at pH 7.5 and 22°C. The incubation solvent contained Abbreviation: AchE, acetylcholinesterase. For these studies, the nonionic resin agarose (Bio-Rad) was used as the insoluble support. 2% agarose (molecular weight exclusion limit = 5 X 107 g/mol) was selected so that AchE (MW = 250,000) (3) would potentially be able to interact with inhibitor molecules bound on both the inside and the outside of the gel matrix.Cuatrecasas (2) has recently presented chemical methods for attachment of small organic molecules to agarose. The several inhibitor ligands used in this study are illustrated in Table 1 and their syntheses are described below.Trimethyl (p-aminophenyl)ammonium chloride hydro...

Studies on the localization of the cardiac glycoside receptor

“…The lack of interaction between cellular receptor or in vitro ATPase preparations and the direct conjugates of digoxin or ouabain to serum albumin has a counterpart in certain affinity chromatography systems where ligands coupled directly to a solid support matrix are unable to interact with the enzyme-binding site. In such systems, attachment of the ligand to the solid support by a long, flexible chain of atoms sometimes enables the ligand to interact readily with the enzyme-binding site (65,66,34). For this reason, the extended chain derivatives of serum albumin shown in Fig.…”

Isolation of the Secretory Form of Soluble Acetylcholinesterase by Using Affinity Chromatography on Edrophonium‐Sepharose

“…The affinity gel does not bind ChE and seems the first to be described as having this property, although most previous studies have usually concentrated on tissues in which only AChE is present, e.g., electric organ of Electrophorus electricus or bovine erythrocytes (Kalerdon et al, 1970;Berman and Young, 1971). Harris et al (1977), when analyzing extracts of guinea-pig retina, found that their column bound ChE.…”

Acetylcholinesterase and Acetylcholine Receptor.

“…The affinity ligand, N-(6-aminohexanoyi-w-aminophenyl)trimethylammonium bromide hydrobromide, was ptepared and was coupled to 6-aminohexanoi'c acid-Sepharose B (41)(42) prior to this study. Details of the ligand preparation and of the tryptic digestion protocol for the isolation of 11S AcChE are ,to be found in the 1985 Annual Report (43).…”

“…Literature procedures (21,40) for AcChE isolation were adapted, with purification by affinity chromatography (41)(42).…”