J. Cell Sci. 12, 911-923 (1973)
Printed in Great Britain
911
ACETYLCHOLINESTERASE IN HUMAN
ERYTHROID CELLS
R. J. SKAER
Department of Medicine, University of Cambridge, Hills Road, Cambridge, England
SUMMARY
Acetylcholinesterase is present in human red cells but cannot be demonstrated by the copper
thiocholine test. The enzyme is revealed, however, in the perinuclear cisterna, endoplasmic
reticulum and Golgi apparatus of red cell precursors. It is suggested that 2 forms of the enzyme
are present, one of which can be demonstrated by the copper thiocholine test, the other cannot;
one form may be the precursor of the other. These observations may cast light on the kinetics
of red cell replacement and on the interpretation of the results from the copper thiocholine
test on other tissues such as the nervous system.
INTRODUCTION
Acetylcholinesterase (E.C. 3.1.1.7) is present in large amounts in human red cells
(Galehr & Plattner, 1928). Although the enzyme binds penicillin and is thereby inactivated (Herz, 1967), the normal functions of acetylcholinesterase in erythrocytes
are unknown. The problem of its function is particularly puzzling since serum from
most people contains isozymes of pseudocholinesterase (E.C. 3.1.1.8) which also
have the power of splitting acetylcholine (Goedde & Altland, 1968). Acetylcholinesterase does not seem to be involved with the sodium pump (Martin, 1970) or with
choline transport (Martin, 1970). Digestion with proteolytic enzymes has shown that
acetylcholinesterase is present on the outside of the erythrocyte membrane (Heller &
Hanahan, 1972). Nevertheless, the enzyme is not localized in mature red cells by the
Koelle copper thiocholine histochemical test for acetylcholinesterase with light
microscopy (Zajicek, Sylvan & Datta, 1954; Rogister & Gerebtzoff, 1958).
Even with electron microscopy, I find the copper thiocholine test and its modifications (Lewis & Shute, 1969) give a completely negative result with erythrocytes.
However, I have found that the Koelle copper thiocholine test gives a positive reaction
in red cell precursors in human bone marrow. This enzyme in the bone marrow may
well be a precursor form of the enzyme found in mature red cells and its presence
may cast light on the kinetics of red cell replacement.
MATERIALS AND METHODS
Fresh human blood was obtained from normal donors. Bone marrow was from the sternum,
opened as a result of open-heart surgery.
912
R. J. Skaer
Electron microscopy
Blood was fixed for 60 min at 20 CC in a large volume of O'$ % glutaraldehyde containing
4 niM calcium, 1-7 % sucrose and o-i M cacodylate buffer, pH 7-4. The cacodylate buffer does
not affect acetylcholinesterase activity (assayed spectrophotometrically by the method of
Ellman, Courtney, Andres & Featherstone (1961)). Fixed blood was processed by centrifugation
at 2000 rev/min, removal of die supernatant, and resuspension of the pellet in fresh medium.
Very small pieces of bone marrow were fixed, sometimes as described above but more often
for 90 min in 1-5% glutaraldehyde containing 4 mM calcium, i'7% sucrose and o-i M cacodylate buffer, pH 7-4, at 20 °C. Bone spicules were dissected away in the fixative. Fixed
tissues were washed at 4 °C for at least 12 h in o-i M cacodylate buffer, pH 7-4, containing
1 -7 % sucrose. After die cytochemical procedures die specimens were postosmicated, dehydrated,
and embedded in Araldite as described by Lewis & Shute (1969). Thin sections were examined
unstained in an AEI EM6B electron microscope operated at 60 kV.
Cytochemistry
Fixed marrow was tested for acetylcholinesterase by the mediod of Lewis & Shute (1969) but,
as suggested by Lewis (unpublished), specimens were rinsed for 30 min in an acetylcholine
wash before and after a 4-h incubation in the reaction mixture. Enzymic reaction product was
less diffuse when acetylcholine washes were used. The acetylcholine wash was prepared in the
same way as the reaction mixture but 95 mg acetylcholine iodide are substituted for 100 mg
acetylthiocholine iodide. The inhibitor for pseudocholinesterase (2XIO~*M ethopropazine
hydrochloride) that was always present in the thiocholine reaction mixture was omitted from
the acetylcholine washes. Control specimens were treated identically but 5 x IO~*M physostigmine salicylate was included in the acetylcholine washes and die reaction mixture.
Spectrophotometric assays
These were based on die mediod of Ellman et al. (1961) for acetylcholinesterase and were
performed in a Unicam SP 1800 dual-beam recording spectrophotometer at 37 °C.
Assay of the effect of fixation
Fresh red cells, prepared by Dextran sedimentation (Cutts, 1970), were washed 3 times in
sterile 0-9 % saline. The Ellman et al. test showed that diis treatment had no effect on acetylcholinesterase activity. The cells were resuspended and equal volumes of die suspension were
transferred to o-i M phosphate buffer, pH 8 (control cells) or to 0-5 % glutaraldehyde in o-i M
phosphate buffer, pH 7-2, at 20 CC. The freshly purchased glutaraldehyde (Taab Laboratories,
52 Kidmore End Road, Emmer Green, Reading, England) had an absorption peak at 230 nm that
was double the height of die peak at 280 nm. Redistilled glutaraldehyde did not absorb significandy at 230 nm. At intervals the suspension of fixed red cells was shaken, 0'4 ml removed,
the red cells spun down and resuspended in 6 ml phosphate buffer, pH 80. Acetylcholinesterase
activity was measured by die mediod of Ellman et al. (1961).
Assay of the effect of copper ions
One millilitre of a suspension of glutaraldehyde-fixed, washed, red cells, prepared as described
above, was treated for from 5 min to 5 h with 10 ml of die copper thiocholine reaction mixture
buffered widi succinate buffer at pH 5-3. The suspension was shaken, 0-4 ml removed and the
cells were rapidly spun down, washed 3 times with 0-9% saline and resuspended in 6 ml of
o-i M phosphate buffer, pH 8. Acetylcholinesterase activity was measured by the method of
Ellman et al. (1961) and was compared with the activity of die same volume of fixed control
cells treated identically but widi die 10 ml of reaction mixture replaced by 10 ml of succinate
buffer pH 5-3.
Acetylcholinesterase in red cells
913
Assay of the effect of pH
Fresh red cells prepared by dextran sedimentation (Cutts, 1970) and washed 3 times in
sterile 0-9 % saline were fixed for 30 min in 0-5 % glutaraldehyde in o-i M cacodylate buffer,
pH 7-0. The fixed cells were resuspended and washed 3 times with 4 times the packed cell
volume of sterile 0-9 % saline. The freshly stirred suspension of red cells was divided into a
control portion and a test portion. Physostigmine salicylate was added to the control portion
(to give a final concentration of 5 x IO~ 3 M). This destroyed the acetylcholinesterase activity of
the controls.
Over the pH range of 5-5-8-0, 0-067 M Sorensen phosphate buffer was used. The range was
extended to pH 5-0 and 9 0 by 0 1 M histidine/potassium hydroxide buffer to which o-6 M potassium chloride was added as an activator for the enzyme (Michel, 1949). Duplicate measurements
of enzymic activity at pH 5-5 and pH 8-o were made in both histidine buffer and in phosphate
buffer. Enzymic activity over the range pH 5-9 was also measured in 0-2 M potassium dihydrogen
phosphate/Trizma base buffer.
The test was performed as follows: 3 ml of buffer, 100 /il of a o-oi M solution of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) made up as described by Ellman et al. (1961), and 100 fi\
of a 6 x IO~'M aqueous solution of ethopropazine hydrochloride were added to each of the test
and reference cuvettes of the spectrophotometer. The suspensions of red cells were stirred
thoroughly and 10 /tl of the test red cells were added to the test cuvette; 10 fi\ of the control red
cells containing physostigmine salicylate were added to the reference cuvette. To both cuvettes
20 //I of freshly prepared 0-075 M acetylthiocholine iodide were added and the rate of production
of colour at each pH was measured at 412 nm. The final concentration of acetylthiocholine iodide
was that recommended by Ellman et al. (1961) as low enough to give optimum enzymic activity
through minimum interference by substrate inhibition.
Performing the test in this way cancels out the effects of spontaneous hydrolysis of acetylthiocholine, but at pH 8-5 and above it is necessary to perform the test rapidly since spontaneous
hydrolysis of acetylthiocholine iodide is so rapid in both test and reference cuvettes that, after
a few minutes the optical density of both cuvettes is too great to be measured. Reproducible results were obtained, however, by taking the initial velocity of the reactions in
each case. The same procedure was followed for a fresh preparation of caudate nucleus from
rat brain, homogenized in 0-32 M sucrose solution at 4 °C in a Potter homogenizer and spun
down at 100 000 g in an MSE Superspeed 65 refrigerated centrifuge. The activity of the
microsomal fraction from Torpedo electric organ (kindly donated by Dr A. Boyne, Department of Biochemistry, University of Cambridge) was also measured over the same range
of pH.
The rates of enzymic hydrolysis obtained were corrected for the change of absorption of the
Ellman reaction product with pH. Fifty millilitres of Ellman reaction product were prepared
by performing the test on Torpedo acetylcholinesterase in tap water, pH 7-4; when sufficient
colour had developed the reaction was blocked by adding 5 x IO~'M physostigmine salicylate;
i-5-ml samples of the coloured solution were diluted with an equal quantity of 0-2 M potassium
dihydrogen phosphate/Trizma base buffers, pH 5-9, and the absorption was compared at
412 nm against half-strength Ellman D T N B solution at the same pH. The pH of each solution
was checked after measuring the absorption. Since the Ellman reaction product fades fairly
rapidly, especially at high pH, each measurement was interspersed with a measurement made
at pH 8. This was prepared freshly from the coloured stock solution each time. The paper
recorder was run continuously so these measurements at pH 8 could be used to follow the
fading of the stock Ellman reaction product with the passage of time. Each absorption measurement was corrected for the fading of the stock solution and the resulting figure was used to
correct for the change of absorption of reaction product with pH.
This method enables the enzymic activity of different forms of acetylcholinesterase to be
compared directly, since all determinations are carried out under identical conditions. The
particular shapes of curve obtained, however, are of relevance only to the conditions used in
this experiment. Thus the fact that enzymic activity was measured with 5,5'-dithiobis-(2-nitrobenzoic acid) may well affect the absolute values of pH optimum obtained, since this reagent
activates the hydrolysis of acetylthiocholine by human acetylcholinesterase (Brownson & Watts,
1972), and the activation might be dependent on pH. Moreover the different buffers used have
gi4
R-J- Skaer
differing effects on enzymic activity; the interactions between Tris buffer and acetylcholinesterase are complex (Pavli£, 1967) and enzymic activity in histidine buffer is slightly less than
in Sorensen's phosphate buffer.
RESULTS
Mature red cells are negative with the copper thiocholine test but do react with the
Ellman et al. (1961) spectrophotometric test for acetylcholinesterase. By applying
this test to red cells after each stage of the Koelle test, I find the lack of histochemical
reaction is due to sensitivity of the enzyme to at least 3 components of the histochemical
test: (a) fixation, (b) copper ions, and (c) pH conditions.
(a) Although acetylcholinesterase from some sources is remarkably resistant to
aldehydefixatives(Couteaux, 1955) the enzyme in human red cells is rapidly destroyed
by 3 % solutions of commercial glutaraldehyde. Large amounts of enzymic activity
survive, however, in 0-5% glutaraldehyde if fixation is not prolonged (Table 1), but
the copper thiocholine test on these fixed cells is still negative. So much enzymic
activity is left after fixation for 60 min in 0-5% glutaraldehyde at room temperature
that it is difficult to believe that, under these conditions, fixation can significantly limit
the amount of reaction product.
Table 1. Enzyme activity expressed as a percentage of that
in unfixed control cells
Period of
h
o-5
i-o
4'5
fixation,
Redistilled
0-5 % glutaraldehyde
Taab
0-5 % glutaraldehyde
85
85
73
85
85
50
(b) I have found that acetylcholinesterase activity in human red cells is very
sensitive to copper ions: 44% of the activity in controls is inhibited by the copper ions
in the thiocholine incubation mixture. Inhibition was rapid and complete within
10 min.
(c) The copper thiocholine test is performed at pH 5-3 even though the optimum
pH for most forms of acetylcholinesterase is between pH 7-0 and 9-0. The low pH is
necessary to obtain good localization of reaction product. The enzymic activity of
different forms of acetylcholinesterase expressed as a percentage of the maximum
for each form are shown for the pH range 5—9 in Fig. 1. The curves are directly comparable since all measurements were made under identical conditions. The graph
shows how varied is the response of different forms of acetylcholinesterase to pH.
The activity of the enzyme in rat brain - a form that does react with the copper thiocholine test - is much less affected by low pH than is the enzyme in red cells. Since
the copper thiocholine test is performed far from the optimum pH of acetylcholinesterase it is likely that the test will be as much affected by differences in the pH
response curve of different forms of the enzyme as by differences in absolute amounts
of the enzyme.
Acetylcholinesterase in red cells
Fig. i. Activity of acetylcholinesterase at different pH levels (Trizma base buffer).
For ease of comparison the activity at each pH is expressed as a percentage of that
at optimum pH. A, fixed red cells; O, rat brain pellet; %, Torpedo electric organ.
It is likely that these effects so reduce the level of enzyme activity that the solubility
product of copper thiocholine is not locally exceeded. The negative histochemical
reaction is probably not due to precipitated reaction product diffusing away from the
red cells, for even where diffusion is restricted by other cells as in bone marrow, and
in rouleaux, the red cells are still negative; nor is it due to loss of enzyme from the red
cells, for the enzyme is tightly bound to the membrane (Heller & Hanahan, 1972).
The lack of reaction with the copper thiocholine test is a characteristic property of
the form of acetylcholinesterase in red cells.
In red cell precursors, however, acetylcholinesterase is revealed by the copper
thiocholine test (Fig. 2). Reaction product is present in the perinuclear cisterna,
endoplasmic reticulum and Golgi apparatus of polychromatophil erythroblasts and
normoblasts. In marrow from normal adult human males most of the red cell series
is negative, but approximately 20% of the polychromatophil erythroblasts and a very
small percentage of normoblasts show a reaction product; this is in patches in the
perinuclear cisterna, and in the sparse endoplasmic reticulum. It is rare for more than
30% of the total perimeter of the nuclear membrane to react positively; the plasma
membrane is always negative. Reaction product is totally abolished in controls treated
with 5 x IO~3M physostigmine salicylate.
Much more reaction product was present in marrow from a female patient with
916
R. J. Skaer
extensive red cell replacement associated with mechanical haemolysis due to a leaky
aortic sleeve. Approximately 60 % of the polychromatophil erythroblasts were positive,
and some contained a large amount of endoplasmic reticulum that reacted positively
(Fig. 3). The Golgi apparatus was strongly positive (Fig. 4). In most cells that reacted
positively the entire profile of the perinuclear cisterna was filled with reaction product
(Fig. 5); where the perinuclear cisterna was locally enlarged, the enlargement was
filled with reaction product (Fig. 6). Thus the reaction product is localized in spaces
bounded by membranes.
DISCUSSION
There are, then, 2 forms of acetylcholinesterase in the red cell series. One form is
in red cell precursors; it can be demonstrated histochemically by the acetylthiocholine test and is therefore relatively insensitive to glutaraldehyde, low pH, and
copper ions, and it is present in spaces bounded by membranes. The other form is
found in erythrocytes and is membrane-bound (Heller & Hanahan, 1972). Since not
all polychromatophil erythroblasts contain the enzyme that can be demonstrated by
copper thiocholine, this isozyme is unlikely to be an essential, intrinsic component of
the perinuclear cisterna and cisternae of the endoplasmic reticulum. Both these sites
are regions around which protein synthesis occurs and the histochemical localization
of the isozyme in these regions indicates that it may have been recently synthesized.
Its occurrence in red cell precursors also suggests that this isozyme is the precursor
of the form in the red cell membrane. It might be that the incorporation of the isozyme
into the plasma membrane as a lipoprotein makes it particularly sensitive to fixation,
copper ions and acidity.
An alternative interpretation is that there is no relationship between the isozyme
revealed by the copper thiocholine test and that present in the membrane of erythrocytes. A choice between these hypotheses might be possible if the time of appearance
of the 2 isozymes were known accurately. If acetylcholinesterase activity was demonstrated histochemically in the perinuclear cisterna before acetylcholinesterase activity
was demonstrable in the plasma membrane, the first hypothesis would be more
reasonable. On the other hand, the biochemical demonstration of acetylcholinesterase
activity in the plasma membrane of early basophil erythroblasts of humans might
favour the second hypothesis.
If the isozyme revealed by the thiocholine test is a precursor of the form in red cells,
an interpretation that would predict just such a relationship between the amount of
reaction product and the replacement rate of the red cells as is suggested by the
present preliminary results is that synthesis of the precursor is discontinuous; as
differentiation stops, enzyme synthesis stops. A positive histochemical reaction might
indicate those cells that are differentiating to the next stage of erythropoiesis.
The isozyme that can be revealed histochemically, though it is enzymically active,
may have no functional significance until it is transformed into the membrane-bound
form of the enzyme. Thus there is a great need for caution in interpreting even the
positive results of the copper thiocholine test: the activity revealed may have no
Acetylcholinesterase in red cells
917
immediate functional significance, and, as in the case of mature red cells, sites that
do not react in the test may well be rich in acetylcholinesterase. Since isozymes of
acetylcholinesterase are present in the nervous system and in muscle (Koelle, Hossaini,
Akbarzadeh & Koelle, 1970) it is possible that these observations also apply there.
Masters & Holmes (1972) suggest that multiple forms of acetylcholinesterase are
merely the result of changes in aggregation of polymeric isozymes. The 2 isozymes
shown here to be present in human erythroid cells are so different in their chemical
properties it is difficult to ascribe these differences solely to the state of aggregation.
If one form is converted into the other it is likely to be the result of chemical
modification.
I am most grateful to Mr B. Milstein and Mr C. Parish of Papworth Hospital for samples of
bone marrow, and to Dr B. L. Gupta, Professor F. G. J. Hayhoe and Professor E. N. Willmer
for reading and criticizing the manuscript. I should also like to thank Dr P. R. Lewis for
helpful discussions.
REFERENCES
C. & WATTS,D. C. (1972). Interactions between 5,5'-dithiobis-(2-nitrobenzoicacid)
and cholinesterases complicating the coupled assay. Proc. Biocliem. Soc., Lancaster, September
1972. Biochem. J. (in the Press).
COUTEAUX, R. (1955). Localisation of cholinesterases at neuromuscular junctions. Int. Rev.
Cytol. 4, 335-375CUTTS, J. H. (1970). Cell Separation Methods in Haematology, pp. 49-54. New York and London:
Academic Press.
ELLMAN, G. L., COURTNEY, K. D., ANDRES, V. JR. & FEATHERSTONE, R. M. (1961). A new and
rapid colorimetric determination of acetylcholinesterase activity. Biodiem. Plutrmac. 7, 88-95.
GALEHR, O. & PLATTNER, F. (1928). t)ber das Schicksal des Acetylcholins im Blute. PflUgers
Arch. ges. Physiol. 218, 488-505.
GOEDDE, H. W. & ALTLAND, K. (1968). Evidence for different 'silent genes' in the human
serum pseudocholinesterase polymorphism. Ann. N.Y. Acad. Sci. 151, 540-544.
HELLER, M. & HANAHAN, D. J. (1972). Human erythrocyte membrane bound enzyme: acetylcholinesterase. Biochim. biophys. Acta 255, M 15, 251-272.
HERZ, F. (1967). Inactivation of erythrocyte acetylcholinesterase by penicillin. Nature, Loud.
214, 497-499.
KOELLE, W. A., HOSSAINI, K. S., AKBARZADEH, P. & KOELLE, G. B. (1970). Histochemical
evidence and consequences of the occurrence of isoenzymes of acetylcholinesterase. J. Histodiern. Cytochem. 18, 812-819.
LEWIS, P. R. & SHUTE, C. C. D. (1969). An electron microscopic study of cholinesterase distribution in the rat adrenal medulla. J. Microscopy 89, 181-193.
MARTIN, K. (1970). The effect of proteolytic enzymes on acetylcholinesterase activity, the
sodium pump and choline transport in human erythrocytes. Biochim. biophys. Acta 203,
BROWNSON,
182-184.
MASTERS, C. J. & HOLMES, R. S. (1972).
MICHEL, H. O. (1949). An electrometric
Isoenzymes and ontogeny. Biol. Rev. 47, 309-361.
method for the determination of red blood cell and
plasma cholinesterase activity. J. Lab. din. Med. 34, 1564-1568.
PAVLI£, M. (1967). The inhibitory effect of Tris on the activity of cholinesterases. Biochim.
biophys. Acta. 139, I33~i37ROGISTER, G. & GEREBTZOFF, M. A. (1958). Recherches histochimiques sur les acetylcholine et
choline esterases. 5. Localisation dans les elements figures du sang et dans les organes h6mopoI6tiques. Acta anat. 32, 39-50.
ZAJICEK, J., SYLVEN, B. & DATTA, M. (1954). Attempts to demonstrate acetylcholinesterase
activity in blood and bone marrow cells by a modified thiocholine technique. J. Histochem.
Cytochem. 2, 115—121.
{Received 3 August 1972)
9i 8
R.J.Skaer
Fig. 2. Unstained section of a polychromatophil erythroblast from ncrmal male bone
marrow showing a positive reaction for acetylcholinesterase in the nuclear membrane
and endoplasmic reticulum. The marrow was fixed at 20 °C in i'5 % glutaraldehyde
(Taab) for 1-5 h at pH 7-4 and processed with the copper thiocholine test, x 25000.
Acetylcholinesterase in red cells
v
920
R. J. Skaer
Fig. 3. Section of a polychromatophil erythroblast from a marrow with extensive red
cell replacement. Treated as Fig. 2 but lightly stained widi uranyl acetate. Large
amounts of endoplasmic reticulum containing reaction product are shown. The
perinuclear cisterna is also positive. The dark granule below the. nucleus is an
aggregate of ferritin. The darkness is due to the ferritin and is not enzymic reaction
product, x 12000.
Fig. 4. Unstained section of a polychromatophil erythroblast from a marrow with
extensive red cell replacement. Treated as in Fig. 2. Much reaction product is present
in the Golgi apparatus, x 60000.
Acetylcholinesterase in red cells
M. J. Skaer
v
Fig. 5. Unstained section of a polychromatophil erythroblast from a marrow with
extensive red cell replacement. Treated as Fig. 2. The amount of endoplasmic reticulum containing reaction product is more typical than that shown in Fig. 3. The dark
granule on the right of the nucleus is an aggregate of ferritin. x 25000.
Acetylcholinesterase in red cells
Fig. 6. Unstained section of a polychromatophil erythroblast from a marrow with
extensive red cell replacement. A local enlargement of the perinuclear cistema filled
with reaction product is shown. The sharpness of demarcation of the enlargement
shows that it is not produced by the obliquity of the perinuclear cistema to the plane
of the section, x 15000.
59-2