Normal human serum contains two forms of acetylcholinesterase

Clinica Chimicu Acta, 158 (1986) l-6 Elsevier

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CCA 03505

Normal human serum contains two forms of acetylcholinesterase K. Sorensen a, U. Brodbeck a, A.G. Rasmussen and B. Norgaard-Pedersen b

b

a Institute of Biochemistry and Molecular Biology, University of Bern, Buhistrasse 28, CH-3012 Bern (Switzerland) and b Statens Seruminstirut, Amager Boulevard 80, DK-2300 Copenhagen S (Denmark) (Received

November 20th, 1985; revision received February accepted after revision March 25th, 1986)

8th, 1986;

Key words: Serum acetylcholinesterase; Purification; Monoclonal antibodies; Quantification; Immune-chemistry; Inhibitors; Sucrose density centrifugation

Summary

We have produced antibodies (polyclonal and monoclonal) against acetylcholinesterase, and used them for immuno-chemical demonstration and quantification of the enzyme in serum. The concentration was 1.2 IU/l. The antibodies were shown not to cross-react with human butyrylcholinesterase, using pure preparations of the enzymes. The serum acetylcholinesterase could be purified using affinity chromatography. The resulting preparation was analyzed using sucrose density gradient centrifugation. Two forms of the enzyme with sedimentation constants of 10.9s and 7.6s were observed, both reacted equally well with monoclonal antibodies towards acetylcholinesterase indicating a common epitope.

Introduction

It is well established that human serum contains abundant amounts of butyrylcholinesterase (BChE, EC 3.1.1.8) but only trace amounts of acetylcholinesterase (AChE, EC 3.1.1.7). The existence of the latter has mainly been ascertained by electrophoresis and activity staining in the absence and presence of certain inhibitors [l]. In the past it has, however, not been possible to determine reliably the amount of AChE present in serum, since there is no substrate nor inhibitor with absolute specificity for AChE or BChE and the existence in serum of a true AChE has been disputed [2,3]. Recently, we described a novel immuno-chemical method which allows the quantification of minor amounts of AChE in biological fluids even in the presence of a large excess of BChE [4]. In this assay microtiter plates are 0009-8981/86/$03.50

0 1986 Else&r

Science Publishers

B.V. (Biomedical

Division)

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coated with antibodies specific for AChE which immobilize that enzyme, whereas BChE is removed by washing. In the resuiting enzyme-antibody complex, AChE retains its activity which is then quantified by the spectrophotometric assay of Ellman et al [5]. The present study was undertaken to show the existence of AChE in human serum by immunochemical methods, to quantify it and to analyze its molecular forms. Materials and methods

Normal human serum was obtained from volunteer donors. The serum donors was pooled and used for this investigation. Pure AChE was purified from human erythrocyte membranes as elsewhere [6]. Pure human BChE was from Behringwerke (FRG, lot no. Tensilon (Edrophonium-chloride) was a gift of Hoffmann-La Roche, Basel. land.

from 40 detailed 450212. Switzer-

Antibodies The following monoclonal antibodies specific for AChE were used: (a) AE-1 obtained as a gift from Dr. Fambrough f7] and (b) HAH l-l and HAH 1-2 produced in the Hybridoma Laboratory of the Statens Seruminstitut, Copenhagen, Denmark, using standard methods [8]. Polyclonal antibodies were raised according to standard methods, as described by Harboe and Ingild [9]. Pure AChE from human erythrocytes was the antigen used for immunization for all the antibodies. Assay procedures Enzyme activity was measured either by the macro-assay of Ellman et al [5], in 3-ml polystyrene cuvettes or by the enzyme antigen immunoassay (EAIA) as described by Hangaard et al 141. Unless otherwise stated the assay solution contained 1 mmol/l acetylthiocholine-iodide, 0.25 mmol/l 2,2-bis-dithionitrobenzoate (DTNB) in 100 mmol/l sodium phosphate buffer, pH 7.4. The macro-assays were read at 412 nm on a LKB Ultrospec 4050 photometer, with on-line data-collection using a Commodore C64 micro-computer running the programs of Sorensen [lo]. The micro-titer plates used in the EAIA were read at 405 nm in a FLOW multichannel ELISA-photometer interfaced to a ~cro-computer. Sucrose density gradient centrifugation Linear sucrose gradients (5-30x w/v) were made in 10 mmol/l Tris, 144 mmol/l NaCI, containing 1% (w/v) Triton X-100, pH 7.4. Samples of 500 ~1 were carefully overlayered on the gradients contained in 14 ml centrifuge tubes. Centrifugation was carried out in a Kontron TST 41.14 Rotor on a Centrikon 2070 centrifuge at 195000 X g,, for 16 h temp. 4°C. Catalase, 11.45, was the S-value marker. The gradients were emptied from the bottom with a glass capillary using a peristaltic pump. A total of 40 fractions were collected, approximately 250 ~1 each. Individual fractions were analyzed by the EAIA method outlined above. The

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activity profiles were plotted using the build-in curve-program Graphtec MPlOOO plotter.

of the Watanabe

Results

Specificity of mono-clonal and poly-cionaf antibodies The specificity of the antibodies was tested using pure AChE from human erythrocyte membranes, and pure human BChE commercially available. Results from the solid phase assay showed that the mono-clonal antibodies EA-1, HAH l-l and HAH 1-2 as well as the polyclonal one only reacted with AChE and not BChE, even in concentrations 500 times that of BChE in serum (the highest tested). Assays carried out with butyryl-thiocholine as the substrate, gave a signal of < 1% that obtained with acetylthiochofne. Recovery In order to demonstrate that the EAIA method could quantify the AChE without interference from BChE or other serum components, recovery experiments were performed, in which a known amount of pure AChE was added to a serum sample or to a solution which contained the pure human BChE. In cases where no interference and no cross-reactivity is seen, a value of 100% recovery will be determined. Our results were: recovery of pure erythrocyte AChE in serum 100 + 6.5%; recovery of pure erythrocyte AChE in pure BChE 104 f 6.2%; recovery of serum AChE in pure BChE 100 f 6.4%. The figures are mean & SD of triplicate determinations at 5 different enzyme concentrations. *AChE in human serum

The EAIA was used to demonstrate the presence of AChE in human serum as well as to determine its concentration. Figure 1 shows a dose-response curve using mono-clonal antibodies with substrate alone as well as in presence of IO pmol/l BW284C51, an inhibitor with preference for AChE and Lysivane at 100 fimol/l which preferentially inhibits BChE. Identical curves were obtained with the polyclonal antibody. Using pure human erythrocyte membrane AChE as standard in the EAIA we could determine the activity of AChE in serum to be 1.2 IU/l serum. The existence of AChE in serum was corroborated by an experiment in which AChE was specifically removed from serum using affinity-chromatography. Serum (25 ml} was passed through a column (1.5 ml) of N,N,N-trimethylammonium-mphenylenediamine-Sepharose, an affinity resin used to purify AChE from several different sources [6,11,12]. BChE does not bind to this matrix (Gennari and Sorensen, unpublished observations) and as a consequence all BChE activity was recovered in the effluent of the column. On the other hand EAIA showed that 95% of the AChE activity in serum had been retained on this particular affinity&pharose. Activity could quantitatively and selectively be eluted with Tensilon (Edrophoni~-chlo~de), a reversible inhibitor of AChE. Further characterization of serum AChE AChE separated from BChE by the chromatography described above was analyzed by sucrose density gradient centrifugation. As shown in Fig. 2 the enzyme

DILUTION

Fig. 1. Titration curves of serum analyzed in the absence and presence of either BW284C51 or Lysivane. Different dilutions of serum were analyzed in the EAIA. Each serum dilution was performed in buffer devoid of any inhibitor (0) or in the presence of 100 gmol/l Lysivane (A) or in the presence of 10 pmol/l BW284C51 (m). The abscissa gives dilution where 0 equals undiluted serum, 1 equals serum diluted 1 : 1 with buffer, etc.

sedimented with apparent S-values of 7.6 and 10.9s. In the presence of monoclonal antibodies HAH l-2 all activity shifted towards higher S-values. The S-values now peaked at 12.3 and 14.7S, thus the increase in sedimentation constant was 3.8 and 4.6 respectively, demonstrating that the mono-clonal antibody recognized both forms of the enzyme.

FRACTION

NUttBER

Fig. 2. Sucrose density centrifugation of a purified serum described in materials and methods. Individual fractions antibodies. l, enzyme alone; l, enzyme preincubated for antibody HAH 1-2 (1.3 mg/ml). The S-values are 7.6s and 14.6s for the enzyme-antibody complex.

AChE preparation. Gradients were made up as were analyzed with the EAIA using polyclonal 4 hours at 20°C with 10 pl of the monoclonal and 10.9s for the untreated sample and 12.2s

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Discussion

We have used immuno-chemical methods to demonstrate the presence of AChE in human serum from healthy adults and our results clearly show that this enzyme is present, albeit at low concentration. If the specific activity and the subunit molecular weight of the AChE in serum are assumed similar to those of erythrocyte membrane AChE, the concentration in serum is calculated to be 3.5 pmol/l. The BChE in adult serum is given as 2.5 nmol/l [13], i.e. approximately 1000 times higher than AChE. It is, therefore, not possible to distinguish by inhibitors and substrates alone between the two enzymes although the inhibitors are > 90% specific [l]. The large difference in concentrations of the two enzymes would require inhibitors with specificities of at least 99.99%. On the other hand, after separation of AChE from BChE, BW2834C51 and Lysivane gave characteristic inhibition patterns (Fig. 1) proving that the antigen bound by the monoclonal antibodies is indeed AChE. Since our antibodies did not cross-react with BChE, the immunochemical reaction with AChE using the EAIA allowed us to quantify AChE without any of these problems. Recently, Brock et al [3] failed to detect AChE in serum added to amniotic fluid. This apparent discrepancy to our results might have been due to the suboptimal assay procedures used by those authors in combination with the low concentration of serum tested (diluted 1 : 10). The specificity of the mono-clonal antibodies for AChE as compared to BChE is of interest, since it shows that the epitopes recognized are not present (or at least not accesible) in BChE. This finding does not prove that other proteins do not react with the mono-clonal antibodies. Monoclonal antibodies have an absolute specificity for a given epitope, which may however be present on other proteins too. Recently the use of a mono-clonal antibody for the immunopurification of AChE from rabbit brain failed, since the eluate of the immuno-affinity column was only about 20% pure [14]. This indicates that the epitope which that particular antibody recognized was also present on other proteins. On the other hand AChE from human erythrocytes could successfully be purified on an immuno-affinity column

[201* The fact that two different molecular forms could be discerned by sucrose density gradient centrifugation raises the interesting question pertaining to the origin of serum AChE. The activity sedimenting at 7.6s most likely is a dimeric enzyme that could originate from the erythrocyte AChE. That enzyme is amphiphilic but can be converted by proteolytic treatment to a hydrophilic form sedimenting at 7s [5,16]. The enzyme activity sedimenting at 10.9s most likely represents a tetrameric form of AChE which might have its origin in neuronal cells which were shown to contain both amphiphilic [ll] and hydrophilic tetrameric forms [17]. Alternatively the 10.9s activity might originate from collagen tailed AChE which is known to be converted by proteolysis to tetrameric hydrophilic forms devoid of the collagen-like tail [18]. The use of mono-clonal antibodies specific for either the enzyme from the erythrocytes or the brain would solve this question. It is of interest to note that human fetal serum contains considerably more AChE, and that this extra amount is solely due to a 10.9s form of the enzyme (Sorensen unpubl.). This is also the case for the fetal bovine enzyme [19].

Acknowledgements

Part of this work was supported by The Swiss National Science Foundation; Grant number 3.300-0.82 to U.B. AGR is the recipient of a grant from the Michaelsen Foundation, Copenhagen, Denmark. References 1 Goedde HW, Doenicke A, Altland K. Pseudocholinesterasen. Phamakogenetik, Biochemie, Klinik. Berlin: Springer Verlag, 1967. 2 Norgaard-Pedersen B, Rasmussen AG, Hangaard J, Simaga S, Sorensen K, B&beck U. Acetylcholinesterase immunoassays in detection of neural-tube defects. Lancet 1985; I: 691-692. 3 Brock DJH, Barron L, Van Heyningen V. Reply to Acetylcholinesterase immunoassays in detection of neural-tube defects. Lancet 1985; I: 692. 4 Hangaard J, Sorensen K, B&beck B, Norgaard-Pedersen B. Quantitative determination of acetylcholinesterase by enzyme antigen immunoassay: methodological aspects and clinical use. Stand J Clin Lab Invest 1984; 44: 717-724. 5 Ellman GL, Courtney KD, Andres Jr V, Featherstone RM. A new and rapid calorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961; 7: 88-95. 6 Brodbeck U, Gentinetta R, Ott P. Purification by affinity chromatography of red cell membrane acetylcholinesterase. In: Azzi A, Brodbeck U, Zahler P, eds. Membrane proteins, a laboratory manual. 85-96. Berlin: Springer Verlag, 1981. 7 Fambrough DM, Engel AG, Rosenberry TL. Acetylcholinesterase of human erythrocytes and neromuscular junctions: homologies revealed by monoclonal antibodies. Proc Nat1 Acad Sci (USA) 1982; 79: 1078-1082. 8 Kohler G, Milstein C. Nature (London) 1985; 256: 495-497. 9 Harboe N, Ingild A. Immunization, isolation of immunoglobulins, estimation of antibody titre. Stand J Immunol Suppl 1. 1973; 2: 161-164. 10 Sorensen K. Computer programs for enzyme-kinetic measurements on the LKB-Ultrospec photometer. Life Sci 1984; 35: 111. 11 Sorensen K, Gentinetta R, Brodbeck U. An amphiphile amphiphile-dependent form of human brain caudate nucleus acetylcholinesterase: purification and properties. J Neurochem 1982; 39: 1050-1060. 12 Stieger S, B&beck U. Amphiphilic detergent soluble AChE from Torpedo marmorata: characterization and conversion by proteolysis to a hydrophilic form. J Neurochem 1985; 44: 48-56. 13 Whittaker M. Cholinesterases. In: Bergmeyer HU, Bergmeyer J, Grass1 M, eds. Methods of enzymatic analysis, Vol. IV, Third ed. Weinheim, FRG: Verlag Chemie, 1983: 52-74. 14 Mints KP, Brimijoin S. Two-step immunoaffinity purification of acetylcholinesterase from rabbit brain. J Neurochem 1985; 44: 225-232. 15 Weitz M, Bjerrum OJ, Brodbeck U. Characterization of an active hydrophilic erythrocyte membrane acetylcholinesterase obtained by limited proteolysis of the purified enzyme. Biochim Biophys Acta 1984; 776: 65-74. 16 Dutta-Choudhury TA, Rosenberry TL. Human erythrocyte acetylcholinesterase is an amphipathic protein whose short membrane-binding domain is removed by papain digestion. J Biol Chem 1984; 259: 5653-5660. 17 Gennari K, Brodbeck U. Molecular forms of acetylcholinesterase from human caudate nucleus: comparison of salt-soluble and detergent-soluble tetrameric species. J Neurochem 1985; 44: 697-704. 18 Massoulie J, Bon S. The molecular forms of cholinesterase and acetylcholinesterase in vertebrates. AM Rev Neurosci 1982; 5: 57-106. 19 Ralston JS, Rush RS, Doctor BP, Wolfe AD. Acetylcholinesterase from fetal bovine serum. Purification and characterization of soluble G, enzyme. J Biol Chem 1985; 260: 4312-4318. 20 Bjerrom OJ, Selmer J, Hangaard J, Larsen F. Isolation of human erythrocyte acetylcholinesterase using phase separation with Triton X-114 and monoclonal immunosorbent chromatography. J Appl Biochem 1985: 7: 356-369.