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

Abstract

A single molecular form of soluble acetylcholinesterase was isolated from a variety of mammalian tissues by use of a novel affinity matrix. This matrix was synthesised by coupling the reversible cholinesterase inhibitor, edrophonium chloride, to epoxy-activated Sepharose. This simple synthesis produced a matrix which was exceptionally stable and had the novel property of selectively binding only one molecular form of acetylcholinesterase. Soluble proteins from a variety of mammalian tissues, including brain, adrenal glands, cerebrospinal fluid, and blood, were separated by centrifugation. These contained combinations of acetylcholinesterase (EC 3.1.1.7) and cholinesterase (EC 3.1.1.8), varying from a single form of acetylcholinesterase to multiple forms of both acetylcholinesterase and cholinesterase. The edrophonium-Sepharose matrix bound only one form of acetylcholinesterase. This form of acetylcholinesterase corresponded in molecular size and electrophoretic mobility to the unique form found in cerebrospinal fluid, i.e. secretory acetylcholinesterase. Cholinesterase was not bound to the matrix.

Altered Levels of Acetylcholinesterase in Alzheimer Plasma

“…As these light species are the major species in human plasma, this low affinity may explain why previous immunoassay studies observed lower plasma AChE levels than reported in the present study. Similarly, other attempts to separate AChE from BuChE in human plasma have been unsuccessful as the affinity resin usually used to separate these enzymes (edrophonium-Sepharose), binds only soluble tetramers [54], whereas soluble light species present in plasma which have amphiphilic properties do not bind [55]. We have estimated that only ∼20% of the human plasma AChE activity bound to an edrophonium-Sepharose affinity matrix (not shown).…”

Acetylcholinesterase in Dendrobaena veneta (Oligochaeta: Opisthopora) is Present with Forms Sensitive and Insensitive to Phosphatidylinositol Phospholipase C

“…propionylthiocholine (PpSCho), butyrylthiocholine (BtSCho) used as substrates for cholinesterase activity measurements, 1 ,S-Bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide (BW 284c51), tetra(monoisopropy1)pyrophosphortetramide (iso-OMPA), eserine sulfate, edrophonium chloride, procainamide and diisopropylfluorophosphate (iPr,P-F) used as cholinesterase inhibitors, bacitracin and aprotinin (protease inhibitors), Escherichia coli alkaline phosphatase, Sephadex G-200-120, marker proteins and blue dextran for M, evaluation were purchased from Sigma Chemical Co. Ultrirgel AcA 44 was bought from LKB, while 5-S'-Dithiobis(2-nitrobenzoic acid) (Nbs,) was obtained from Merck. Affinity resin for cholinesterase purification was prepared by coupling edrophonium chloride to epoxy-activated Sepharose according to Hodgson and Chubb (1983). Hydroxyapatite and electrophoresis purity reagents were from Bio-Rad (Melville NY).…”

Dimeric forms of cholinesterase in Sipunculus nudus

“…5,5'-Dithiobis-(2-nitrobenzoic acid) (NbS,) was from Merck. Affinity resin for ChE purification was prepared coupling edrophonium chloride to epoxy-activated Sepharose (Pharmacia) according to Hodgson and Chubb [24]. Electrophoresis-purity reagents and nitrocellulose membrane were from BioRad, peroxidase-antiperoxidase complex was from Dakopatts.…”

“…Table 1 shows the purification procedure for LSS and DS ChE. Purification factors of 1700 and 1090 were, respectively, achieved with a two-step affinity chromatography performed on an edrophonium-containing gel [24]. This matrix was an efficient tool in the selective binding of both enzymes.…”