British_lournal cfHaemarology, rg79,41, 383-391.
Relation between Low Erythrocyte Acetylcholinesterase Activity and Membrane Lipids in Paroxysmal Nocturnal Haemoglobinuria* JUNJAREE S I R I W I T T A Y A K O R N A N D Y O N G Y U T H Y U r H A V O N G
Department of Biochemistry, Mahidol University, Faculty of Science, Bangkok, Thailand (Received 9 January 1978; accepted for publication 28 June 1978)
SUMMARY. Acetylcholinesterase of intact erythrocytes, their ghosts and salt soluble extracts obtained from patients with paroxysmal nocturnal haemoglobinuria (PNH) does not differ from normal with respect to K, values for acetylthiocholine, and K, values for phenyltrimethylammonium iodide. However, the enzyme from PNH sources has lower V,,, values than normal, has different thermal stability from normal, has less distinctive transition temperature in the Arrhenius plots, and is less subject to inhibition by stearic acid. These results and that from comparison of activation of deoxycholate-extracted enzyme by lipids from normal erythrocytes suggest that the low acetylcholinesterase activity in P N H erythrocytes is due, at least in part, to alteration in the lipid environment of the enzyme. A consistent feature in paroxysmal nocturnal haemoglobinuria (PNH) is the low activity of erythrocyte acetylcholinesterase (AChE) (Auditore & Hartmann, 1959; Metz et al, 1960; Kunstling & Rosse, 1969). A correlation has been shown between the decrease in enzyme activity and severity of the disease as measured by Ham’s test and cold-antibody lysis test (Metz et al, 1960), and in a heterogeneous population of cells from the patient those which are sensitive to complement lysis have little or no AChE (Kunstling & Rosse, 1969; Hartmann & Arnold, 1977). The cause for the low AChE activity of the erythrocyte is a matter of conjecture. It is unlikely that the enzyme is lost from the erythrocyte as it ages since the young cells in P N H have lower enzyme activity than the old cells (Auditore & Hartmann, 1959;Metz et al, 1960; Herz et al, 1968). The possibility of the presence of a tightly bound inhibitor has been raised (Herz et a l , 1968) but none has so far been found. Erythrocyte AChE is a membrane bound enzyme which can be extracted into solution under certain conditions (Hanahan, 1973; Mitchell & Hanahan, 1966). Deoxycholateextracted enzyme is depleted of lipids and has a low activity, which can be stimulated by addition of exogenous lipids (Sihotang, 1976).The activity of the membrane-bound enzyme is therefore probably highly influenced by its lipid environment. Since there have been several,
* This paper is dedicated to the late D r Sa-nga Pootrakul, Haematology Unit, Faculty of Medicine, Siriraj Hospital. Correspondence: Dr Yongyuth Yuthavong, Department of Biochemistry, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 4.Thailand. 0007-1048/79/030c+0383$02.00 01979 Blackwell Scientific Publications
3 83
3 84
Junjaree Siriwittayakorn and Yongyuth Yuthavong
albeit contradictory, reports on the alteration in composition and state of the membrane lipids in P N H (Formijne et al, 1957; Harris et al, 1957; Munn & Crosby, 1957; Leibetseder & Ahrens, 1959; Barry, 1959; Phillips & Roome, 1962; Bradlow et al, 1965; Meriwether & Mengel, 1966; Mengel et al, 1967; Paniker et al, 1974) it is possible that the low AChE activity is related to abnormal membrane lipids. This paper is concerned with comparison of kinetic and other characteristics of the normal and PNH enzyme. It is shown that, while the K,,, for substrate and Ki for competitive inhibitor are unchanged, other properties of the enzyme, which are likely to be dependent on its attached lipids, are different. Furthermore, lipids from normal erythrocytes have a relatively greater activating effect on the deoxycholate-extracted enzyme from P N H erythrocytes than on the enzyme from normal erythrocytes, indicating that the lower activity of the former is due, at least in part, to the lipid environment. MATERIALS AND METHODS All chemicals and reagents used were reagent grade. Phenyltrimethylammonium iodide was prepared by the reaction between stoichiometric amounts of dimethylaniline and methyl iodide in ether. Fresh blood was drawn from normal subjects and patients by venepuncture using acid citrate dextrose solution as anticoagulant (10ml whole blood per 1 . 5 ml solution). All samples were kept at 4°C until use. Erythrocyte ghosts were prepared from fresh blood by the method of Fairbanks et a1 (1971). AChE was solubilized 'from erythrocyte ghosts by 1.2 M NaCl as described by Mitchell & Hanahan (1966). The soluble extract was concentrated over Amicon PM 10Diaflo ultrafiltration membrane. All procedures were conducted at 4°C. Protein content was determined according to the method of Cowry et a1 (1951). Inhibition of AChE activity of intact erythrocytes by stearic acid was investigated following the procedures described by Aloni & Livne (1974) and Livne & Bar-Yaakov (1976). Lipids were extracted from normal erythrocytes by the method of Bligh & Dyer (1959) as modified by Kates (1972). Lipid suspension was prepared by sonicating lipid-water medium (Sihotang, 1976) for 10min (20 kcs; Branson ultransonic assembly, Branson sonic company). Deoxycholate extract of AChE was prepared by suspending erythrocyte ghosts in a solution of deoxycholate (dissolvedin IOO mM phosphate buffer, pH 8.0) to give a final concentration of 3 0 mM. The suspension was centrifuged at IOO ooog for I h at 4°C in a Beckman ultracentrifuge model Lz-65 (Wright & Plummer, 1972). The supernatant was dialysed against 5 ideal milliosmolar phosphate buffer, pH 7.4, overnight at 4OC. Acetylcholinesterase activity was assayed colorimetrically in IOO m~ phosphate buffer, pH 8.0, by the method of Ellman et a/ (1961) using t-cysteine hydrochloride as standard. Unless otherwise specified, the assay medium contained 0.75 mM acetylthiocholine bromide (ATC) 0.40 m~ 5,5-dithiobis-2-nitrobenzoicacid (DTNB) and appropriate amount of erythrocytes, ghosts or soluble extract; the enzyme reaction was followed at 25°C for at least 6 min a t 412 nm by Gilford recording photometer model 2000, attached to a Beckman D U monochromator. RESULTS
The low activity of erythrocyte AChE in P N H compared with the enzyme from normal
AChE and Membrane Lipids in P N H
38s
erythrocytes is ascribed mainly to the low V,,, values. Table I shows that the K, values are similar, while V,,, values are lower than normal for the enzyme from intact erythrocytes, ghosts and soluble sodium chloride extract of ghosts obtained from PNH blood. The difference in V,,, is more noticeable for the enzymes in ghosts and soluble extracts than in intact erythrocytes. The similarity is observed not only in K , for the substrate ATC, but also in K, for the competitive inhibitor phenyltrimethylammonium iodide, as measured with the enzyme in erythrocyte ghosts (Table 11). TABLE I. Michaelis-Menten constants (K,) and maximum velocities ( VmJ for acetylthiocholine of normal and PNH erythrocyte acetylcholinesterase
Enzyme source Intact erythrocyte Erythrocyte ghost 1 . 2M
NaCl extract
Subject*
Normal ( 5 ) PNH ( 5 ) Normal ( 5 ) PNH (10) Normal (3) PNH ( 5 )
0.143 (0.090-0.167) 0.119(0.089-0.138)
8.34 (7.633.68) 7.02 (5.89-8.61)
0.105 (0.086-0.13 I ) 0.101 (0.06&0.151)
2.28
0 . 1 2 5 (0.101-0.160)
4.61 (3.38-5.78) 1.48 (0.82-2.16)
(1.33-3.33) 1.04 (0.57-1.67)
0.102 (0.087-0.120)
The constants were determined by either following initial rates of reaction at various substrate concentrations, or by following the total progress of the reaction to completion. N o significant differences in the values from the two methods were found.
* Numbers of determinations are given in parentheses; one or two determinations per case. t Arithmetic means are given, with ranges in parentheses. $ Expressed as pmoles ATC hydrolysed/h/Io' cells for intact erythrocyte and pmoles ATC hydrolysed/min/mg protein for erythrocyte ghosts and 1.2M NaCl extract. TABLE 11. Inhibitor constants (K,) for phenyltrimethylammonium of acetylcholinesterase of normal and P N H erythrocyte ghosts. Specijc activity Sample Normal case I Normal case 2 P N H case I P N H case 2 P N H case3
(pmoles ATC hydrolyredl min/mg protein) I .60
105
K, (M)
I .48
4.0 4.8
1.16 0.74 0.3I
4.3 4.2 4.3
K, values were determined by the method of Dixon (1953) with ATC as substrate. Substrate concentrations were 0.2 and 0.3 mM; 10 rate measurements at various inhibitor concentrations were made at each substrate concentration.
Junjaree Siriwittayakorn and Yongyuth Yuthavong
3 86
Erythrocyte AChE in YNH differs from normal in other aspects and Fig I shows differences in thermal stability of the enzyme from both sources. The PNH enzyme in ghosts is morc resistant than normal to thermal inactivation a t 55°C and 60°C (Figs I a and Ib). The sodium chloride extract of the enzyme from both sources are more stable a t 60°C than the enzymc in ghosts (Fig IC).However, the PNH enzyme is now more sensitive than normal to thermal inactivation. Herz et a1 (1968) did not find any significant differences in thermal stability between the PNH and normal enzyme. We are not certain why there is this discrepancy, but one possible explanation is the fact that the thermal stability of the enzyme both from normal and PNH erythrocytes is dependent on storage time (Siriwittayakorn, 1977). Our experiments were conducted with fresh samples, so that the effect of storage time on thermal stability was negligible. The plots of log V against I / T (Arrhenius plots) for the enzyme from normal intact erythrocytes, ghosts and sodium chloride extract are biphasic (Fig 2 ) . The results for intact erythrocytes and ghosts are similar to those of Aloni & Livne (1974). However, the biphasic 100,
I 100
20
40
80
60
100
b C
-0
1
0A
-0
-0 - 0
' A '
'' -A
-A - A -A -A -A
ZD-
O*
20
40
I0
10
110
TIME (MINI
FIG I . Thermal stability ofAChE of: (a) normal and PNH fresh ghosts a t 55°C; (b) normal and PNH fresh ghosts at 60°C; and (c) soluble extracts of normal and PNH ghosts at 60°C; the assay was made at 25°C after incubation of the enzyme for various intervals. 0, Normal; A, PNH.
AChE and Membrane Lipids in I "
387
nature of the plots is less distinctive or absent for the P N H enzyme. In the two P N H cases studied, the loss of the biphasic nature is more apparent for the enzyme with lower specific activity. The alteration in the plots of log Vagainst I/T, with consequent change in apparent activation energies, suggests that the abnormally low enzyme activity in P N H may be due to abnormal membrane lipids. This possibility is strengthened further when it is found that stearic
01
>
0.4
0
-0.4
0.4
>
b
01
0
0.4
0.4
0
C
c)
3 - 8.1
-0.4
-1.2
-01
-16
-1.1
3.1
3.2
31
1.4
3.S
3.8
3.1
FIG2.Plotsoflog Vagainst ~ / T f o AChEof:(a) r normalandPNH intact erythrocyter;(b)ghosts;and (c) sodium chloride extracts; 0.75 mM acetylthiocholine, 0.4 mM dithionitrobenzoate, IOO mM phosphate, pH 8.0. I/ is expressed as pmol/h/Ios cells for intact erythrocytes,and as pmol/min/mg protein for ghosts and soluble extracts. 0,Normal; A, PNH case I ; A, PNH case 2.
acid, which inhibits AChE through its binding with membrane lipids (Aloni & Livne, 1974; Livne & Bar-Yaakov, 1976),is less effective in inhibiting the P N H enzyme than normal (Table 111).
In order to clarify the role of membrane lipids in the abnormality of erythrocyte AChE, the enzyme from the ghosts of normal and P N H subjects was extracted with deoxycholate. After removal of deoxycholate by dialysis, the enzyme was activated by addition of total lipids extracted from normal erythrocytes. It was found that the enzyme extracted from P N H ghosts can be activated to a greater extent than the enzyme extracted from normal ghosts (Fig 3 ) .
Junjaree Siriwittayakorn and Yongyuth Yuthavong TABLE 111. Inhibition ofacetylcholinesterase of normal and P N H erythrocytes by stearic acid. The enzyme activity was assayed in IOO m~ Na,SO, buffered with 5 m~ sodium phosphate, pH 8.0, at 2s"C. The assay medium contained appropriate amount of erythrocytes, 0.4 mM DTNB, 0.75 mM ATC and 3 . 3 p g stearic acid. Mean values of four normal samples are given with ranges of activity in parentheses.
Specijic activity (pmollhlml packed cells)
%
Sample
No stearic acid
W i t h stearic acid
inhibition
Normal (4)
50 3 (402-581)
286 (224-3 36)
43 (41-44)
PNH Case I Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 Case 8
31
-
448 39s 3 57 287 266 23 8 I54 97
23
33 16 42 I1
21
32 18
A-A-A-A
a
A-A-A-A
A
-8 20-
0
0
(-2
0.4. 0.6 0.0 ADDED LIPIDS Img/mlJ
1d
1.2
FIG 3 . Activation of AChE in deoxycholate extract of normal and P N H erythrocytes by total lipids from normal erythrocytes. 0,Normal case I , initial activity 44 pmol/h/mg protein; 0 , normal case 2 , initial activity 21 pmol/h/mg protein; A , P N H case I , initial activity 22 pmol/h/mg protein; A. P N H case 2 , initial activity 15 pmol/h/mg protein; all samples were freshly prepared, except normal case 2 which was kept frozen for approximately 2 weeks before use.
AChE and Membraite Lipids in P N H
3 89
DISCUSSION O u r observation that AChE from PNH erythrocytes, either in intact form, ghosts or sodium chloride extract, has low V,,, but apparently normal K, for ATC (Table I), indicates that the defect is located either in the amount of the enzyme or in the catalytic step following substrate binding, but not in the binding step itself. The constancy of K , of the enzyme in PNH erythrocytes has been observed before (Ferrone et al, 1970; Herz et al, 1968).The similarity of K, values of the enzyme from normal and PNH ghosts for the competitive inhibitor phenyltrimethylammonium iodide (Table 11) of the enzyme from normal and PNH ghosts lends further support to this conclusion. While we have no data on the relative amounts of the enzyme in normal and PNH erythrocytes, studies on thermal stability (Fig I ) and other kinetic characteristics suggest that the low activity is explained, at least in part, by alteration in the enzyme present. This alteration is probably not due to a removable non-competitive inhibitor, since the low activity is found in the soluble extract and ghosts as well as in intact erythrocytes. The alteration is also apparently not due to differences in the distribution of multiple molecular forms of the enzyme, since Ott et al (1975) found the same isoelectro-focusing pattern for the PNH as the normal enzyme. The tendency of the P N H enzyme to have less prominent or no discontinuity in Arrhenius plots compared with normal, as well as differencesin apparent activation energy of the enzyme from the two sources (Fig z), suggests that in PNH erythrocytes, membrane lipids have been altered, with consequent alteration in the catalytic activity. I t is worth noting that a linear Arrhenius plot for human erythrocyte AChE can be obtained if the membrane lipids ofnormal erythrocytes are modified by linolenoyl sorbitol (Aloni & Livne, 1974). Since the AChE activity in sodium chloride extracts of both normal and PNH erythrocytes shows similar variation with temperature to the activity in the corresponding ghosts and intact erythrocytes, it is likely that only the lipids firmly bound to the enzyme influence the enzyme activity. A similar conclusion was also reached by Beauregard & Roufogalis (1977) who demonstrated that the presence of a discontinuity in the Arrhenius plot of bovine erythrocyte AChE is dependent on tightly bound phospholipid. The observation that stearic acid has less inhibitory effect on the PNH enzyme than normal (Table 111) contrasts with the unchanged affinity of the enzyme for phenyltrimethylammonium iodide (Table 11) and the unchanged K, values for ATC (Table I). Since the enzyme inhibition is apparently due to hydrophobic interaction between stearic acid and membrane lipids (Livne & Bar-Yaakov, 1976), the smaller extent of the inhibition on PNH enzyme may be due to difference in amount and/or nature of binding of stearic acid to the membrane lipids. From this and the foregoing evidence, it appears that an abnormality in membrane lipids, and not in the structure of the enzyme itself, accounts for the low activity of erythrocyte AChE in PNH. Consequently, if it is possible to replace the environment of the P N H enzyme with lipids from normal erythrocyte membrane, restoration of the enzyme activity towards normal vales might be expected. The finding (Fig 3 ) that the deoxycholate-extracted enzyme from erythrocytes can be activated by lipids from normal erythrocytes to a greater extent than the enzyme from normal erythrocytes, is consistent with this expectation. The activation of the extracted enzyme may be due to the binding and/or exchange of added lipids with the enzyme, which was solubilized together with a small amount of residual lipids attached. Since
3 90
Junjaree Siriwittayakorn and Yongyuth Yuthavong
immediate activation was observed, binding of the added lipids probably plays a greater role than exchange. The greater activation of extracted PNH enzyme may be due to difference in amount or composition of attached lipids from normal. So far, the only phospholipid shown to have an activating effect on delipidated AChE of human erythrocyte is phosphatidylserine (Sihotang, 1976).Whether this phospholipid or others is responsible for the AChE activity in PHH has not been investigated. In another instance, the catalytic activity of bovine erythrocyte AChE has been reported to be influenced by cardiolipin (Beauregard & Roufogalis, 1977). Although there is no definite explanation for the alteration it is clear that the low AChE activity of P N H erythrocytes is linked with an abnormality of the associated lipids. ACKNOWLEDGMENTS
We highly appreciate the co-operation of, and discussion with, the staff at the Haematology Unit, Department of Medicine, Faculty of Medicine, Siriraj Hospital. In particular, we would like to thank the late Dr Sa-nga Pootrakul, Dr Prawase Wasi, Dr Mongkol Kruatrachue and Dr Supa Na-Nakorn for their advice and encouragement, and for making available the DNH blood samples. Dr Prayad Komaratat and Dr Prapon Wilairat, both from the author’s department, also gave much helpful advice. This work was supported in part by a grant from the National Research Council of Thailand (toJ.S.) and a grant from the Wellcome Trust (to Y.Y.). REFERENCES ALONI,B. & LIVNE,A. (1974) Acetylcholine esterase as a probe for erythrocyte-membrane intactness. Biochimica et Biophysica Acta, 339, 359-366. AUDITORE, J.V. & HARTMANN, R.C. (1959) Paroxysmal nocturnal hemoglobinuria. 11. Erythrocyte acetylcholinesterase defect. American Journal .f Medicine, 27, 401-410. BARRY, R.M. (1959) The phospholipid distribution in the erythrocyte in paroxysmal nocturnal haemoglobinuria. Britishjournal ofHaematology, 5,2 12-2 16. BEAUREGARD, G. & ROUFOGALIS, B.D. (1977)The role of tightly bound phospholipid in the activity of erythrocyte acetylcholinesterase. Biochemical and Biophysical Research Communications, 77, 21 1-2 19. BLIGH,E.G. & DYER,W.J. (1959) A rapid method of total lipid extraction and purification. Canadian Journal ofBiochemistry and Physiology, 37,911-917. BRADLOW, B.A., LEE,J. & RUBENSTEIN, R. (1965) Erythrocyte phospholipids: quantitative thin-layer chromatography in paroxysmal nocturnal haemoglobinuria and hereditary spherocytosis. British Journal ofHaematology, 11, 3 15-322. DIXON,M. (1953) The determination of enzyme inhibitor constants. BiochemicalJournal, 55, 17-171. ELLMAN, G.L., COURTNEY, K.D., ANDRES, V., JR & FEATHERSTONE, R.M. (1961)A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology, 7, 8 8 3 5 .
FAIRBANKS, G., S l E C K , T.L. & WALLACH, D.F.H. (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry, 10, 2606-2617. FERRONE, S., ZANELLA, A., SIRCHIA, G. & F L O R ~A. S, (1970) Study of the acetylcholinesterase activity of AET-treated (2-aminoethylisothiouronium) normal red cells (PNH-like cells). Acta Vitaminolugica et Enzymologica, 24, 2 2 2 - 2 2 5 . FORMIJNE, P., POULIE, N.J. & RODBARD, J.A. (1937) Determination of phospholipid fractions in the human erythrocyte. Clinica Chimica Acta, 2 , 2 ~ - 3 4 . HANAHAN, D.J. (1973) The erythrocyte membrane. Variability and membrane enzyme activity. Biochimica et Biophysica Acta, 300, 3 19-340, HARRIS, I.M., PRANKERD, T.A.J. 81 WESTERMAN, M.P. (1957) Abnormality ofphospholipids in red cells of patients with paroxysmal nocturnal hacmoglobinuria. British Medical Journal, ii, 1276-1277. HARTMANN, R.C & ARNOLD, A.B. (1977) Paroxysmal nocturnal haemoglobinuria (PNH) as a clonal disorder. Annual Reviews ofMedicine, 28, 187-194. HERZ,F., KAPLAN, E. & SCHEYE, E.S. (1968) Differences between the red cell acetylcholinesterase defects of paroxysmal nocturnal hemoglobinuria and of ABO hemolytic disease. Acts Haematologica, 39.85-90. K A m , M. (1972) Techniques of Lipidology, p. 3 5 2 .
AChE and Membrane Lipids in P N H North-Holland Publishing Company, Amsterdam. KUNSTLING, T.R. & ROSSE,W.F. (1969) Erythrocyte acetylcholinesterase deficiency in paroxysmal nocturnal hemoblobinuria (F"H)-A comparison of the complement-sensitive and insensitive populations. Blood, 33,607-616. LEIAFTSEDER, F. & AHRENS, E.H., JR (1959) The fattyacid composition of red cells in paroxysmal nocturnal haemoglobinuria. British Journal ofhlaematology, 59
356364.
LIVNE,A. & BAR-YAAKOV, 0. (1976) Sensitivity of erythrocyte acetylcholinesterase to inhibition by linolenoyl sorbitol. Dependence on a transmembrane potential. Biochimica el Biophysica Acta, 419, 3 58-364. LOWRY, O.H., ROSEBROUGH, N.J., FARR,A.L. & RAND A L L , R.J. (1951) Protein measurement with the Folin phenol reagent. Journal ofBiological Chemistry, I93 9 265-275,
MENGEL, C.E., KANN,H.E., JR & MERIWETHER, W.D. ( I 967) Studies of paroxysmal nocturnal hemoglobinuria erythrocytes: increased lysis and lipid peroxide formation by hydrogen peroxide. Journal of Clinical Investigation, 46, 1715-1723. MFRIWETHER, W.D. & MENGEL, C.E. (1966)Peroxidation of lipid from normal and paroxysmal nocturnal haemoglobinuria erythrocytes. Nature, 210, 9132.
METZ, J., BRADLOW, B.A., LEWIS,S.M. & DACIE, J.V. (1960) The acetylcholinesterase activity of thc erythrocytes in paroxysmal nocturnal haemoglobinuria in relation to the severity of the disease. BritirhJournal of Haematology, 6, 372-380.
391
MITCHELL, C.D. & HANAHAN, D.J. (1966) Solubilization of certain proteins from the human erythrocyte stroma. Biochemistry, 5 , 51-57. MUNN, J.I. & CROSBY, W.H. (1957) Paroxysmal nocturnal hemoglobinuria. Evidence of defect of red cell stroma manifested by abnormalities of lipids. Proceedings of the Societyfor Experimental Biology and
Medicine, 96, 480-482. Orr, P., JENNY, B. & BRODBECK, U. (1975) Multiple molecular forms of purified human erythrocyte acetylcholinesterase. European Journal of Biorhemisfry, 573 469-480. PANIKER, N.V., ARNOLD,A.B. & HARTMANN, R.C. (1974) Studies of the role of red cell membrane peroxidation in paroxysmal nocturnal haemoglobinuria (PNH). British Journal of Haematology, 26, 39-47.
PHILLIPS, G.B. & ROOME,M.S. (1962) Quantitative chromatographic analysis of the phospholipids of abnormal human red blood cells. Proceeding3 of the Society for Experimental Biology and Medicine, 109, 360-364.
SIHOTANG, K. (1976) Acetylcholinesterase and its association with lipid. EuropeanJournal of Biochemistry, 63,519-524.
SIRIWIITAYAKORN, J. (1977) Erythrocyte acetylcholinesterase defect in paroxysmal nocturnal hemoglobinuria: studies on kinetic characteristics. Master of Science thesis, Mahidol University, Bangkok. WRIGHT, D.L.& PLUMMER,D.T. (1972) Solubilization of acetylcholinesterase from human erythrocytes by Triton X-IOOin potassium chloride solution. Biochimica et Biophysica Acta, 261, 398-401.
Relation between Low Erythrocyte Acetylcholinesterase Activity and Membrane Lipids in Paroxysmal Nocturnal Haemoglobinuria* JUNJAREE S I R I W I T T A Y A K O R N A N D Y O N G Y U T H Y U r H A V O N G
Department of Biochemistry, Mahidol University, Faculty of Science, Bangkok, Thailand (Received 9 January 1978; accepted for publication 28 June 1978)
SUMMARY. Acetylcholinesterase of intact erythrocytes, their ghosts and salt soluble extracts obtained from patients with paroxysmal nocturnal haemoglobinuria (PNH) does not differ from normal with respect to K, values for acetylthiocholine, and K, values for phenyltrimethylammonium iodide. However, the enzyme from PNH sources has lower V,,, values than normal, has different thermal stability from normal, has less distinctive transition temperature in the Arrhenius plots, and is less subject to inhibition by stearic acid. These results and that from comparison of activation of deoxycholate-extracted enzyme by lipids from normal erythrocytes suggest that the low acetylcholinesterase activity in P N H erythrocytes is due, at least in part, to alteration in the lipid environment of the enzyme. A consistent feature in paroxysmal nocturnal haemoglobinuria (PNH) is the low activity of erythrocyte acetylcholinesterase (AChE) (Auditore & Hartmann, 1959; Metz et al, 1960; Kunstling & Rosse, 1969). A correlation has been shown between the decrease in enzyme activity and severity of the disease as measured by Ham’s test and cold-antibody lysis test (Metz et al, 1960), and in a heterogeneous population of cells from the patient those which are sensitive to complement lysis have little or no AChE (Kunstling & Rosse, 1969; Hartmann & Arnold, 1977). The cause for the low AChE activity of the erythrocyte is a matter of conjecture. It is unlikely that the enzyme is lost from the erythrocyte as it ages since the young cells in P N H have lower enzyme activity than the old cells (Auditore & Hartmann, 1959;Metz et al, 1960; Herz et al, 1968). The possibility of the presence of a tightly bound inhibitor has been raised (Herz et a l , 1968) but none has so far been found. Erythrocyte AChE is a membrane bound enzyme which can be extracted into solution under certain conditions (Hanahan, 1973; Mitchell & Hanahan, 1966). Deoxycholateextracted enzyme is depleted of lipids and has a low activity, which can be stimulated by addition of exogenous lipids (Sihotang, 1976).The activity of the membrane-bound enzyme is therefore probably highly influenced by its lipid environment. Since there have been several,
* This paper is dedicated to the late D r Sa-nga Pootrakul, Haematology Unit, Faculty of Medicine, Siriraj Hospital. Correspondence: Dr Yongyuth Yuthavong, Department of Biochemistry, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 4.Thailand. 0007-1048/79/030c+0383$02.00 01979 Blackwell Scientific Publications
3 83
3 84
Junjaree Siriwittayakorn and Yongyuth Yuthavong
albeit contradictory, reports on the alteration in composition and state of the membrane lipids in P N H (Formijne et al, 1957; Harris et al, 1957; Munn & Crosby, 1957; Leibetseder & Ahrens, 1959; Barry, 1959; Phillips & Roome, 1962; Bradlow et al, 1965; Meriwether & Mengel, 1966; Mengel et al, 1967; Paniker et al, 1974) it is possible that the low AChE activity is related to abnormal membrane lipids. This paper is concerned with comparison of kinetic and other characteristics of the normal and PNH enzyme. It is shown that, while the K,,, for substrate and Ki for competitive inhibitor are unchanged, other properties of the enzyme, which are likely to be dependent on its attached lipids, are different. Furthermore, lipids from normal erythrocytes have a relatively greater activating effect on the deoxycholate-extracted enzyme from P N H erythrocytes than on the enzyme from normal erythrocytes, indicating that the lower activity of the former is due, at least in part, to the lipid environment. MATERIALS AND METHODS All chemicals and reagents used were reagent grade. Phenyltrimethylammonium iodide was prepared by the reaction between stoichiometric amounts of dimethylaniline and methyl iodide in ether. Fresh blood was drawn from normal subjects and patients by venepuncture using acid citrate dextrose solution as anticoagulant (10ml whole blood per 1 . 5 ml solution). All samples were kept at 4°C until use. Erythrocyte ghosts were prepared from fresh blood by the method of Fairbanks et a1 (1971). AChE was solubilized 'from erythrocyte ghosts by 1.2 M NaCl as described by Mitchell & Hanahan (1966). The soluble extract was concentrated over Amicon PM 10Diaflo ultrafiltration membrane. All procedures were conducted at 4°C. Protein content was determined according to the method of Cowry et a1 (1951). Inhibition of AChE activity of intact erythrocytes by stearic acid was investigated following the procedures described by Aloni & Livne (1974) and Livne & Bar-Yaakov (1976). Lipids were extracted from normal erythrocytes by the method of Bligh & Dyer (1959) as modified by Kates (1972). Lipid suspension was prepared by sonicating lipid-water medium (Sihotang, 1976) for 10min (20 kcs; Branson ultransonic assembly, Branson sonic company). Deoxycholate extract of AChE was prepared by suspending erythrocyte ghosts in a solution of deoxycholate (dissolvedin IOO mM phosphate buffer, pH 8.0) to give a final concentration of 3 0 mM. The suspension was centrifuged at IOO ooog for I h at 4°C in a Beckman ultracentrifuge model Lz-65 (Wright & Plummer, 1972). The supernatant was dialysed against 5 ideal milliosmolar phosphate buffer, pH 7.4, overnight at 4OC. Acetylcholinesterase activity was assayed colorimetrically in IOO m~ phosphate buffer, pH 8.0, by the method of Ellman et a/ (1961) using t-cysteine hydrochloride as standard. Unless otherwise specified, the assay medium contained 0.75 mM acetylthiocholine bromide (ATC) 0.40 m~ 5,5-dithiobis-2-nitrobenzoicacid (DTNB) and appropriate amount of erythrocytes, ghosts or soluble extract; the enzyme reaction was followed at 25°C for at least 6 min a t 412 nm by Gilford recording photometer model 2000, attached to a Beckman D U monochromator. RESULTS
The low activity of erythrocyte AChE in P N H compared with the enzyme from normal
AChE and Membrane Lipids in P N H
38s
erythrocytes is ascribed mainly to the low V,,, values. Table I shows that the K, values are similar, while V,,, values are lower than normal for the enzyme from intact erythrocytes, ghosts and soluble sodium chloride extract of ghosts obtained from PNH blood. The difference in V,,, is more noticeable for the enzymes in ghosts and soluble extracts than in intact erythrocytes. The similarity is observed not only in K , for the substrate ATC, but also in K, for the competitive inhibitor phenyltrimethylammonium iodide, as measured with the enzyme in erythrocyte ghosts (Table 11). TABLE I. Michaelis-Menten constants (K,) and maximum velocities ( VmJ for acetylthiocholine of normal and PNH erythrocyte acetylcholinesterase
Enzyme source Intact erythrocyte Erythrocyte ghost 1 . 2M
NaCl extract
Subject*
Normal ( 5 ) PNH ( 5 ) Normal ( 5 ) PNH (10) Normal (3) PNH ( 5 )
0.143 (0.090-0.167) 0.119(0.089-0.138)
8.34 (7.633.68) 7.02 (5.89-8.61)
0.105 (0.086-0.13 I ) 0.101 (0.06&0.151)
2.28
0 . 1 2 5 (0.101-0.160)
4.61 (3.38-5.78) 1.48 (0.82-2.16)
(1.33-3.33) 1.04 (0.57-1.67)
0.102 (0.087-0.120)
The constants were determined by either following initial rates of reaction at various substrate concentrations, or by following the total progress of the reaction to completion. N o significant differences in the values from the two methods were found.
* Numbers of determinations are given in parentheses; one or two determinations per case. t Arithmetic means are given, with ranges in parentheses. $ Expressed as pmoles ATC hydrolysed/h/Io' cells for intact erythrocyte and pmoles ATC hydrolysed/min/mg protein for erythrocyte ghosts and 1.2M NaCl extract. TABLE 11. Inhibitor constants (K,) for phenyltrimethylammonium of acetylcholinesterase of normal and P N H erythrocyte ghosts. Specijc activity Sample Normal case I Normal case 2 P N H case I P N H case 2 P N H case3
(pmoles ATC hydrolyredl min/mg protein) I .60
105
K, (M)
I .48
4.0 4.8
1.16 0.74 0.3I
4.3 4.2 4.3
K, values were determined by the method of Dixon (1953) with ATC as substrate. Substrate concentrations were 0.2 and 0.3 mM; 10 rate measurements at various inhibitor concentrations were made at each substrate concentration.
Junjaree Siriwittayakorn and Yongyuth Yuthavong
3 86
Erythrocyte AChE in YNH differs from normal in other aspects and Fig I shows differences in thermal stability of the enzyme from both sources. The PNH enzyme in ghosts is morc resistant than normal to thermal inactivation a t 55°C and 60°C (Figs I a and Ib). The sodium chloride extract of the enzyme from both sources are more stable a t 60°C than the enzymc in ghosts (Fig IC).However, the PNH enzyme is now more sensitive than normal to thermal inactivation. Herz et a1 (1968) did not find any significant differences in thermal stability between the PNH and normal enzyme. We are not certain why there is this discrepancy, but one possible explanation is the fact that the thermal stability of the enzyme both from normal and PNH erythrocytes is dependent on storage time (Siriwittayakorn, 1977). Our experiments were conducted with fresh samples, so that the effect of storage time on thermal stability was negligible. The plots of log V against I / T (Arrhenius plots) for the enzyme from normal intact erythrocytes, ghosts and sodium chloride extract are biphasic (Fig 2 ) . The results for intact erythrocytes and ghosts are similar to those of Aloni & Livne (1974). However, the biphasic 100,
I 100
20
40
80
60
100
b C
-0
1
0A
-0
-0 - 0
' A '
'' -A
-A - A -A -A -A
ZD-
O*
20
40
I0
10
110
TIME (MINI
FIG I . Thermal stability ofAChE of: (a) normal and PNH fresh ghosts a t 55°C; (b) normal and PNH fresh ghosts at 60°C; and (c) soluble extracts of normal and PNH ghosts at 60°C; the assay was made at 25°C after incubation of the enzyme for various intervals. 0, Normal; A, PNH.
AChE and Membrane Lipids in I "
387
nature of the plots is less distinctive or absent for the P N H enzyme. In the two P N H cases studied, the loss of the biphasic nature is more apparent for the enzyme with lower specific activity. The alteration in the plots of log Vagainst I/T, with consequent change in apparent activation energies, suggests that the abnormally low enzyme activity in P N H may be due to abnormal membrane lipids. This possibility is strengthened further when it is found that stearic
01
>
0.4
0
-0.4
0.4
>
b
01
0
0.4
0.4
0
C
c)
3 - 8.1
-0.4
-1.2
-01
-16
-1.1
3.1
3.2
31
1.4
3.S
3.8
3.1
FIG2.Plotsoflog Vagainst ~ / T f o AChEof:(a) r normalandPNH intact erythrocyter;(b)ghosts;and (c) sodium chloride extracts; 0.75 mM acetylthiocholine, 0.4 mM dithionitrobenzoate, IOO mM phosphate, pH 8.0. I/ is expressed as pmol/h/Ios cells for intact erythrocytes,and as pmol/min/mg protein for ghosts and soluble extracts. 0,Normal; A, PNH case I ; A, PNH case 2.
acid, which inhibits AChE through its binding with membrane lipids (Aloni & Livne, 1974; Livne & Bar-Yaakov, 1976),is less effective in inhibiting the P N H enzyme than normal (Table 111).
In order to clarify the role of membrane lipids in the abnormality of erythrocyte AChE, the enzyme from the ghosts of normal and P N H subjects was extracted with deoxycholate. After removal of deoxycholate by dialysis, the enzyme was activated by addition of total lipids extracted from normal erythrocytes. It was found that the enzyme extracted from P N H ghosts can be activated to a greater extent than the enzyme extracted from normal ghosts (Fig 3 ) .
Junjaree Siriwittayakorn and Yongyuth Yuthavong TABLE 111. Inhibition ofacetylcholinesterase of normal and P N H erythrocytes by stearic acid. The enzyme activity was assayed in IOO m~ Na,SO, buffered with 5 m~ sodium phosphate, pH 8.0, at 2s"C. The assay medium contained appropriate amount of erythrocytes, 0.4 mM DTNB, 0.75 mM ATC and 3 . 3 p g stearic acid. Mean values of four normal samples are given with ranges of activity in parentheses.
Specijic activity (pmollhlml packed cells)
%
Sample
No stearic acid
W i t h stearic acid
inhibition
Normal (4)
50 3 (402-581)
286 (224-3 36)
43 (41-44)
PNH Case I Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 Case 8
31
-
448 39s 3 57 287 266 23 8 I54 97
23
33 16 42 I1
21
32 18
A-A-A-A
a
A-A-A-A
A
-8 20-
0
0
(-2
0.4. 0.6 0.0 ADDED LIPIDS Img/mlJ
1d
1.2
FIG 3 . Activation of AChE in deoxycholate extract of normal and P N H erythrocytes by total lipids from normal erythrocytes. 0,Normal case I , initial activity 44 pmol/h/mg protein; 0 , normal case 2 , initial activity 21 pmol/h/mg protein; A , P N H case I , initial activity 22 pmol/h/mg protein; A. P N H case 2 , initial activity 15 pmol/h/mg protein; all samples were freshly prepared, except normal case 2 which was kept frozen for approximately 2 weeks before use.
AChE and Membraite Lipids in P N H
3 89
DISCUSSION O u r observation that AChE from PNH erythrocytes, either in intact form, ghosts or sodium chloride extract, has low V,,, but apparently normal K, for ATC (Table I), indicates that the defect is located either in the amount of the enzyme or in the catalytic step following substrate binding, but not in the binding step itself. The constancy of K , of the enzyme in PNH erythrocytes has been observed before (Ferrone et al, 1970; Herz et al, 1968).The similarity of K, values of the enzyme from normal and PNH ghosts for the competitive inhibitor phenyltrimethylammonium iodide (Table 11) of the enzyme from normal and PNH ghosts lends further support to this conclusion. While we have no data on the relative amounts of the enzyme in normal and PNH erythrocytes, studies on thermal stability (Fig I ) and other kinetic characteristics suggest that the low activity is explained, at least in part, by alteration in the enzyme present. This alteration is probably not due to a removable non-competitive inhibitor, since the low activity is found in the soluble extract and ghosts as well as in intact erythrocytes. The alteration is also apparently not due to differences in the distribution of multiple molecular forms of the enzyme, since Ott et al (1975) found the same isoelectro-focusing pattern for the PNH as the normal enzyme. The tendency of the P N H enzyme to have less prominent or no discontinuity in Arrhenius plots compared with normal, as well as differencesin apparent activation energy of the enzyme from the two sources (Fig z), suggests that in PNH erythrocytes, membrane lipids have been altered, with consequent alteration in the catalytic activity. I t is worth noting that a linear Arrhenius plot for human erythrocyte AChE can be obtained if the membrane lipids ofnormal erythrocytes are modified by linolenoyl sorbitol (Aloni & Livne, 1974). Since the AChE activity in sodium chloride extracts of both normal and PNH erythrocytes shows similar variation with temperature to the activity in the corresponding ghosts and intact erythrocytes, it is likely that only the lipids firmly bound to the enzyme influence the enzyme activity. A similar conclusion was also reached by Beauregard & Roufogalis (1977) who demonstrated that the presence of a discontinuity in the Arrhenius plot of bovine erythrocyte AChE is dependent on tightly bound phospholipid. The observation that stearic acid has less inhibitory effect on the PNH enzyme than normal (Table 111) contrasts with the unchanged affinity of the enzyme for phenyltrimethylammonium iodide (Table 11) and the unchanged K, values for ATC (Table I). Since the enzyme inhibition is apparently due to hydrophobic interaction between stearic acid and membrane lipids (Livne & Bar-Yaakov, 1976), the smaller extent of the inhibition on PNH enzyme may be due to difference in amount and/or nature of binding of stearic acid to the membrane lipids. From this and the foregoing evidence, it appears that an abnormality in membrane lipids, and not in the structure of the enzyme itself, accounts for the low activity of erythrocyte AChE in PNH. Consequently, if it is possible to replace the environment of the P N H enzyme with lipids from normal erythrocyte membrane, restoration of the enzyme activity towards normal vales might be expected. The finding (Fig 3 ) that the deoxycholate-extracted enzyme from erythrocytes can be activated by lipids from normal erythrocytes to a greater extent than the enzyme from normal erythrocytes, is consistent with this expectation. The activation of the extracted enzyme may be due to the binding and/or exchange of added lipids with the enzyme, which was solubilized together with a small amount of residual lipids attached. Since
3 90
Junjaree Siriwittayakorn and Yongyuth Yuthavong
immediate activation was observed, binding of the added lipids probably plays a greater role than exchange. The greater activation of extracted PNH enzyme may be due to difference in amount or composition of attached lipids from normal. So far, the only phospholipid shown to have an activating effect on delipidated AChE of human erythrocyte is phosphatidylserine (Sihotang, 1976).Whether this phospholipid or others is responsible for the AChE activity in PHH has not been investigated. In another instance, the catalytic activity of bovine erythrocyte AChE has been reported to be influenced by cardiolipin (Beauregard & Roufogalis, 1977). Although there is no definite explanation for the alteration it is clear that the low AChE activity of P N H erythrocytes is linked with an abnormality of the associated lipids. ACKNOWLEDGMENTS
We highly appreciate the co-operation of, and discussion with, the staff at the Haematology Unit, Department of Medicine, Faculty of Medicine, Siriraj Hospital. In particular, we would like to thank the late Dr Sa-nga Pootrakul, Dr Prawase Wasi, Dr Mongkol Kruatrachue and Dr Supa Na-Nakorn for their advice and encouragement, and for making available the DNH blood samples. Dr Prayad Komaratat and Dr Prapon Wilairat, both from the author’s department, also gave much helpful advice. This work was supported in part by a grant from the National Research Council of Thailand (toJ.S.) and a grant from the Wellcome Trust (to Y.Y.). REFERENCES ALONI,B. & LIVNE,A. (1974) Acetylcholine esterase as a probe for erythrocyte-membrane intactness. Biochimica et Biophysica Acta, 339, 359-366. AUDITORE, J.V. & HARTMANN, R.C. (1959) Paroxysmal nocturnal hemoglobinuria. 11. Erythrocyte acetylcholinesterase defect. American Journal .f Medicine, 27, 401-410. BARRY, R.M. (1959) The phospholipid distribution in the erythrocyte in paroxysmal nocturnal haemoglobinuria. Britishjournal ofHaematology, 5,2 12-2 16. BEAUREGARD, G. & ROUFOGALIS, B.D. (1977)The role of tightly bound phospholipid in the activity of erythrocyte acetylcholinesterase. Biochemical and Biophysical Research Communications, 77, 21 1-2 19. BLIGH,E.G. & DYER,W.J. (1959) A rapid method of total lipid extraction and purification. Canadian Journal ofBiochemistry and Physiology, 37,911-917. BRADLOW, B.A., LEE,J. & RUBENSTEIN, R. (1965) Erythrocyte phospholipids: quantitative thin-layer chromatography in paroxysmal nocturnal haemoglobinuria and hereditary spherocytosis. British Journal ofHaematology, 11, 3 15-322. DIXON,M. (1953) The determination of enzyme inhibitor constants. BiochemicalJournal, 55, 17-171. ELLMAN, G.L., COURTNEY, K.D., ANDRES, V., JR & FEATHERSTONE, R.M. (1961)A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology, 7, 8 8 3 5 .
FAIRBANKS, G., S l E C K , T.L. & WALLACH, D.F.H. (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry, 10, 2606-2617. FERRONE, S., ZANELLA, A., SIRCHIA, G. & F L O R ~A. S, (1970) Study of the acetylcholinesterase activity of AET-treated (2-aminoethylisothiouronium) normal red cells (PNH-like cells). Acta Vitaminolugica et Enzymologica, 24, 2 2 2 - 2 2 5 . FORMIJNE, P., POULIE, N.J. & RODBARD, J.A. (1937) Determination of phospholipid fractions in the human erythrocyte. Clinica Chimica Acta, 2 , 2 ~ - 3 4 . HANAHAN, D.J. (1973) The erythrocyte membrane. Variability and membrane enzyme activity. Biochimica et Biophysica Acta, 300, 3 19-340, HARRIS, I.M., PRANKERD, T.A.J. 81 WESTERMAN, M.P. (1957) Abnormality ofphospholipids in red cells of patients with paroxysmal nocturnal hacmoglobinuria. British Medical Journal, ii, 1276-1277. HARTMANN, R.C & ARNOLD, A.B. (1977) Paroxysmal nocturnal haemoglobinuria (PNH) as a clonal disorder. Annual Reviews ofMedicine, 28, 187-194. HERZ,F., KAPLAN, E. & SCHEYE, E.S. (1968) Differences between the red cell acetylcholinesterase defects of paroxysmal nocturnal hemoglobinuria and of ABO hemolytic disease. Acts Haematologica, 39.85-90. K A m , M. (1972) Techniques of Lipidology, p. 3 5 2 .
AChE and Membrane Lipids in P N H North-Holland Publishing Company, Amsterdam. KUNSTLING, T.R. & ROSSE,W.F. (1969) Erythrocyte acetylcholinesterase deficiency in paroxysmal nocturnal hemoblobinuria (F"H)-A comparison of the complement-sensitive and insensitive populations. Blood, 33,607-616. LEIAFTSEDER, F. & AHRENS, E.H., JR (1959) The fattyacid composition of red cells in paroxysmal nocturnal haemoglobinuria. British Journal ofhlaematology, 59
356364.
LIVNE,A. & BAR-YAAKOV, 0. (1976) Sensitivity of erythrocyte acetylcholinesterase to inhibition by linolenoyl sorbitol. Dependence on a transmembrane potential. Biochimica el Biophysica Acta, 419, 3 58-364. LOWRY, O.H., ROSEBROUGH, N.J., FARR,A.L. & RAND A L L , R.J. (1951) Protein measurement with the Folin phenol reagent. Journal ofBiological Chemistry, I93 9 265-275,
MENGEL, C.E., KANN,H.E., JR & MERIWETHER, W.D. ( I 967) Studies of paroxysmal nocturnal hemoglobinuria erythrocytes: increased lysis and lipid peroxide formation by hydrogen peroxide. Journal of Clinical Investigation, 46, 1715-1723. MFRIWETHER, W.D. & MENGEL, C.E. (1966)Peroxidation of lipid from normal and paroxysmal nocturnal haemoglobinuria erythrocytes. Nature, 210, 9132.
METZ, J., BRADLOW, B.A., LEWIS,S.M. & DACIE, J.V. (1960) The acetylcholinesterase activity of thc erythrocytes in paroxysmal nocturnal haemoglobinuria in relation to the severity of the disease. BritirhJournal of Haematology, 6, 372-380.
391
MITCHELL, C.D. & HANAHAN, D.J. (1966) Solubilization of certain proteins from the human erythrocyte stroma. Biochemistry, 5 , 51-57. MUNN, J.I. & CROSBY, W.H. (1957) Paroxysmal nocturnal hemoglobinuria. Evidence of defect of red cell stroma manifested by abnormalities of lipids. Proceedings of the Societyfor Experimental Biology and
Medicine, 96, 480-482. Orr, P., JENNY, B. & BRODBECK, U. (1975) Multiple molecular forms of purified human erythrocyte acetylcholinesterase. European Journal of Biorhemisfry, 573 469-480. PANIKER, N.V., ARNOLD,A.B. & HARTMANN, R.C. (1974) Studies of the role of red cell membrane peroxidation in paroxysmal nocturnal haemoglobinuria (PNH). British Journal of Haematology, 26, 39-47.
PHILLIPS, G.B. & ROOME,M.S. (1962) Quantitative chromatographic analysis of the phospholipids of abnormal human red blood cells. Proceeding3 of the Society for Experimental Biology and Medicine, 109, 360-364.
SIHOTANG, K. (1976) Acetylcholinesterase and its association with lipid. EuropeanJournal of Biochemistry, 63,519-524.
SIRIWIITAYAKORN, J. (1977) Erythrocyte acetylcholinesterase defect in paroxysmal nocturnal hemoglobinuria: studies on kinetic characteristics. Master of Science thesis, Mahidol University, Bangkok. WRIGHT, D.L.& PLUMMER,D.T. (1972) Solubilization of acetylcholinesterase from human erythrocytes by Triton X-IOOin potassium chloride solution. Biochimica et Biophysica Acta, 261, 398-401.
