Pediat. Res. 7: 204-214 (1973) A Review: Human Erythrocyte Acetylcholinesterase FRITZ HERZ[I241 AND EUGENE KAPLAN Departments of Pediatrics, Sinai Hospital, and the Johns Hopkins University School of Medicine, Baltimore, Maryland, USA Introduction that this enzyme was an esterase, hence the term "choline esterase" was coined [100]. Further studies In recent years the erythrocyte membrane has received established that more than one type of cholinesterase considerable attention by many investigators. Numer- occurs in the animal body, differing in substrate ous reviews on the composition [21, 111], immunologic specificity and in other properties. Alles and Hawes [85, 116] and rheologic [65] properties, permeability [1] compared the cholinesterase of human erythro- [73], active transport [99], and molecular organiza- cytes with that of human serum and found that, tion [109, 113, 117], attest to this interest. Although although both enzymes hydrolyzed acetyl-a-methyl- many studies relating to membrane enzymes have ap- choline, only the erythrocyte cholinesterase could peared, systematic reviews of this area are limited. hydrolyze acetyl-yg-methylcholine and the two dia- More than a dozen enzymes have been recognized in stereomeric acetyl-«: /3-dimethylcholines. These dif- the membrane of the human erythrocyte, although ferences have been used to delineate the two main changes in activity associated with pathologic condi- types of cholinesterase: (1) acetylcholinesterase, or true, tions are found regularly only with acetylcholinesterase specific, E-type cholinesterase (acetylcholine acetyl- (EC. 3.1.1.7). Although the physiologic functions of hydrolase, EC. 3.1.1.7) and (2) cholinesterase or erythrocyte acetylcholinesterase remain obscure, the pseudo, nonspecific, s-type cholinesterase (acylcholine location of this enzyme at or near the cell surface gives acylhydrolase, EC. 3.1.1.8). it special significance in studies of cellular membranes and the activity alterations seen in several hemolytic Acetylcholinesterase is found in the nervous tissue disorders may be of importance in understanding cer- of all animals and in the erythrocytes of most of them, tain basic disease processes. Present knowledge con- whereas pseudocholinesterase is found in lesser cerning erythrocyte acetylcholinesterase as related to amounts in the nervous tissue of all animals and in the health and disease, age of the individual, and erythro- serum of most of them [4]. The most important differ- cyte aging is so diffuse that a review of this subject ences between acetylcholinesterase and pseudocholines- seems to be warranted. terase are indicated in Table I. Although other non- specific esterases that also hydrolyze acetylcholine are Cholinesterases present in many tissues, they can be distinguished from the cholinesterases by their insensitivity to inhibition Cholinesterases are enzymes, present almost exclu- by eserine. sively in animal tissues, that catalyze the hydrolysis of acetylcholine into acetic acid and choline. Acetylcho- Some Properties of Acetylcholinesterase line was discovered over 100 years ago, but it was not known until the 1920's that this substance was wide- Acetylcholinesterase possesses two adjacent binding spread in animal tissues and that it was important for sites for the substrate. One site is "anionic," and it the functioning of nervous tissue. After acetylcholine binds the cationic quaternary nitrogen of acetylcholine. was found in animal tissues, it was shown that the The other site is "esteratic," formed by a serine residue physiologic action of this ester was inhibited by ex- and by another nucleophilic group (imidazole of a tracts of heart tissue [66]. Later studies indicated that histidine residue) that accepts the proton released the inactivation of acetylcholine was caused by the during the enzyme reaction. While the anionic site action of an enzyme [28]. It then became apparent attracts the quaternary nitrogen of the substrate, the 204 Human erythrocyte acetylcholinesterase 205 Table I. Characteristics of cholinesterases1 such a way that in its life span of 120 days and 280 km Acetylcho- Pseudocho- of continuous travel it is an effective vehicle for the linesterase linesterase supply of oxygen to all body tissues. Although it Optimum pH 7.5-8.0 8.5 appears that the cell does not expend energy in Inhibition by excess substrate + — fulfilling its known primary functions of transporting Hydrolysis of oxygen and carbon dioxide, the maintenance of the Tributyrine — + erythrocyte in a functional state involves energy Acetyl-/3-methylcholine + — expenditure. Essential to the rapid oxygenation of Benzoylcholine — + Hydrolysis rate with butyrylcholine 10% 200% hemoglobin in the lungs and to the comparably rapid (relative to acetylcholine) deoxygenation in the tissues is a structurally intact Inhibition by erythrocyte, i.e., the hemoglobin must be enclosed Eserine + + + + + + + + within the relatively impermeable envelope or mem- Quinidine + + + + + brane. Conditions that affect the functional and struc- Diisopropylphosphofluoridate + + + + + + tural integrity of this membrane may lead to an ineffec- 'After the data of Augustinsson [4]. Acetylcholinesterase: tive and short lived cell. erythrocytes, nervous tissue, brain, placenta. Pseudocholines- As indicated above, the erythrocyte membrane has terase: serum, pancreas, glands. been extensively studied in relation to composition, electrophilic carbon of the carbonyl group at the immunologic and rheologic properties, permeability, esteratic site forms a covalent bond with the oxygen of active transport, and molecular organization. How- serine. The alcoholic portion is split off from the ever, the possibility that membrane abnormalities of acetylcholinesterase-substrate complex, and the acety- an enzymatic nature [31, 112] may be associated with lated esteratic site reacts with water to form acetic acid several inherited or acquired hemolytic disorders has and the intact enzyme. Substances that bind at the not received much attention. The erythrocyte mem- anionic site (simple quaternary compounds) are rever- brane, composed primarily of lipid and protein, like sible acetylcholinesterase inhibitors, since they prevent the membranous structures of other cells, possesses attachment of the substrate. Other compounds, such as specific enzymes [90]. An enzyme is considered to be prostigmine, eserine, or related substances that have located on the membrane if it cannot be removed by a carbamyl ester linkage or urethane structure, com- manipulations that render the membrane hemoglobin bine with both the anionic and the esteratic sites. After free. Hence, only the enzymes indicated in Table II choline is split off, a carbamylated enzyme is left that are considered to form an integral part of the erythro- is hydrolyzed very slowly. This type of inhibition is cyte membrane. slowly reversible. Substances such as diisopropylphos- Acetylcholinesterase in the Erythrocyte phofluoridate form stable diisopropylphosphate esters with the serine residue of the esteratic site, resulting in The capacity of erythrocytes to split acetylcholine irreversibly inhibited acetylcholinesterase. This type of was first described by Galehr and Plattner [33] in inhibition characterizes insecticides and nerve gases. Compounds with a strongly cationic group at an appro- Table II. Enzymes of the erythrocyte membrane priate intramolecular distance from a nucleophilic Enzymes References group, such as 2-pyridine aldoxime methiodide, rapidly Acid proteases and peptidases [40, 92] remove the diisopropylphosphate group from the ester- Diphosphopyridinenucleotidase [35] atic site, restoring enzyme activity. 2-Pyridine aldoxime Triphosphopyridinenucleotidase [35] methiodide has been used successfully as an antidote Phosphatases of various specificities [20, 34, 41, 52, 91, 103] Cation-dependent adenosinetri- [41,75] for diisopropylphosphofluoridate poisoning. Compre- phosphatases hensive reviews on acetylcholinesterase inhibitors and 5'-Adenosinemonophosphate deam- [79] their modes of action have been published [27, 32]. inase Adenylate kinase [17, 41] The Erythrocyte and Its Membrane Diglyceride kinase [52] Phosphoglycerate kinase [72] The mature erythrocyte is a nonnucleated, bicon- Sialyltransferases [63] cave, disc-shaped cell containing a high concentration Glycolytic enzymes [16, 25, 82, 90, 105] of hemoglobin [9]. This unique cell is programmed in Acetylcholinesterase [11] 206 HERZ AND KAPLAN 1927. Alles and Hawes [1] demonstrated in 1940 that tients with paroxysmal nocturnal hemoglobinuria (see the erythrocyte enzyme resembled the brain esterase below). but differed markedly from the cholinesterase found Brauer and Root [11] in 1945 showed that after in in serum. Sabine [86] observed large increases in vitro hemolysis, acetylcholinesterase activity can be erythrocyte acetylcholinesterase activity in dogs after recovered in the erythrocyte membrane. Studies with phlebotomy and in man after blood loss. In pernicious proteolytic enzymes, which are unable to traverse the anemia a reproducible sequence of events was estab- erythrocyte membrane, have indicated that acetyl- lished: in relapse, the enzyme activity was relatively cholinesterase or its active sites are located at or near low, but it rose sharply above normal with the reticulo- the outer surface of the human erythrocyte [7, 30, 51, cyte response and then declined progressively as remis- 74]. It has been suggested on the basis of a molecular sion was established.