/ . Embryo/, exp. Morph. Vol. 51, pp. 209-215, 1979
Printed in Great Britain © Company of Biologists Limited 1979
209
The activity of cholinesterases during the
development of Xenopus laevis
By TERESA GINDI 1 AND JOHN KNOWLAND1
From the Department of Biochemistry, Oxford
SUMMARY
The activity of cholinesterases during the early development of Xenopus laevis has been
examined, and the activity of acetylcholinesterase in particular has been distinguished from
other cholinesterases. In contrast to some earlier findings, the activity of acetylcholinesterase
is low at early stages and gradually increases during development. Possible reasons for the
differences between the earlier results and those reported here are discussed.
INTRODUCTION
Acetylcholinesterase is an enzyme characteristic of neuromuscular tissue, and
for this reason measurements of its activity during embryonic development
indicate the appearance of specialized cells. In amphibia, most studies suggest
that acetylcholinesterase activity is low in early embryos (Sawyer, 1943), and
rises in later development, becoming localized at synapses (Boell & Shen, 1950;
Shen, Greenfield & Boell, 1955; Deuchar, 1966). However, one recent report
(Atherton & Lee, 1975) concludes that very early embryos of Xenopus laevis
contain high levels of acetylcholinesterase and that activity falls to a very low
level in swimming tadpoles at about stage 50, which must have functional muscles
and nerves. In view of these unexpected findings, we have re-examined the
activity of acetylcholinesterase during Xenopus development, and have also
sought to discriminate between acetylcholinesterase and other cholinesterases
of uncertain function. These include pseudocholinesterases, butyrylcholinesterases and non-specific cholinesterases, which can be distinguished from acetylcholinesterase through the use of selective inhibitors (Silver, 1974).
MATERIALS AND METHODS
Embryos of Xenopus laevis were obtained using standard methods (Gurdon,
1967), de-jellied (Dawid, 1965), thoroughly rinsed, staged (Nieuwkoop & Faber,
1967), and stored at — 70 °C. These treatments did not affect the enzyme
activity present in fresh embryos.
1
Authors' address: Department of Biochemistry, South Parks Road, Oxford 0X1 3QU,
U.K.
210
T. GINDI AND J. KNOWLAND
Five thawed embryos were homogenized in 1 ml of 0-1 M sodium phosphate
pH 8 containing 0-5 % Triton X-100. Homogenates were shaken at 37 °C for
5 min (Ho & Ellman, 1969) and centrifuged at 15000 g for 5 min. The enzyme
activity was not affected by the treatment at 37 °C. Protein concentrations of
extracts were measured using the method of Lowry, Rosebrough, Farr &
Randall, 1951.
Enzyme activities were determined colorimetrically at 25 °C using acetylthiocholine and butyrylthiocholine as substrates (Ellman, Courtney, Andres &
Featherstone, 1961), 0 - 3 3 X 1 0 ~ 3 M DTNB (dithiobisnitrobenzoate), and a
total volume of 3 ml. The absorbance change was followed for at least 10 min
using a Cecil spectrophotometer.
The inhibitors used were eserine salicylate, BW 284c51 dibromide (a gift from
Wellcome Reagents) and ethopropazinehydrochloride (May & Baker). Solutions
of these inhibitors and the substrates were made up fresh daily in 0 1 M sodium
phosphate pH 8 except for ethopropazine hydrochloride, which was dissolved
in ethanol because it is not very soluble in water. Ethopropazine was added where
required in a minimum volume of ethanol, and correction was made for the
very slight inhibition caused by the ethanol alone.
RESULTS
Measurements o/K m of acetylcholinesterase
Homogenates of tails from swimming tadpoles (stages 48-50) had high activity
with acetylthiocholine as substrate and were used to determine the Km for the
enzyme (Fig. 1). The average value obtained, using two different enzyme concentrations, was 3 x 10~4 M; and a substrate concentration of 10~3 M was used in
all subsequent assays.
Thiol groups in tissue extracts
The enzyme assay depends upon hydrolysis of the thiocholine substrates to
form thiol groups, which then react with DTNB to produce a yellow anion. In
early embryos where enzyme activity was low and large volumes of extract
(more than 100 juX) had to be used for assays, SH groups present in the extract
caused a significant absorbance change, resulting in incorrect estimates of
enzyme activity. It was necessary to pre-incubate the extracts with DTNB for
10 min before adding substrate to correct for the endogenous SH groups. Uncorrected estimates of enzyme activity could be up to ten times higher than
corrected values.
The jelly surrounding unhatched embryos consists of sulphated mucopolysaccharides (Salthe, 1963). It must be cross-linked by disulphide linkages because
cysteine at pH 8 dissolves it, but seems to contain very few free thiol groups
because the amounts of free thiol detected in homogenates made from embryos
complete with jelly, chemically de-jellied embryos, or hatched embryos differed
Activity of cholinesterase during Xenopus development
20
111
-
l/SOittf 1 )
Fig. .1. Km of acetylcholinesterase. Acetylthiocholine concentrations from 7-5 x
10~6 M to 0-5 x 10~3 M were used to measure the Km as described under Results.
by less than 10 %. Tests showed that three rinses removed all the cysteine used to
dissolve the jelly, and the reactive thiol groups in extracts were probably
contributed by glutathione and proteins.
Inhibitor studies
Three selective inhibitors were used to distinguish acetylcholinesterase from
other esterases.
Eserine should inhibit all cholinesterases, with total inhibition at about
10~5 M (Silver, 1974). Complete inactivation of the Xenopus enzyme required
treatment with 5 x 10~5 M eserine at room temperature for 1 hr. Thus a high concentration of eserine was necessary, and the rate of inactivation was slow, but
these results do indicate the presence of a cholinesterase because most nonspecific esterases are not inhibited by eserine below 10~2 M.
BW 284c51 is a powerful selective inhibitor of true acetylcholinesterases
which usually does not affect other cholinesterases (Silver, 1974). The activity
212
T. GINDI AND J. KNOWLAND
-60
-
&
20 E
10
Developmental stage
Fig. 2. Changes in acetylcholinesterase activity during development. The activity of
acetylcholinesterase at the stages indicated is expressed as n-mole of acetylthiocholine
iodide hydrolysed per min per embryo (•—•) and per mg of homogenate protein
(O-O).
in Xenopus embryos was fully inhibited by BW 284c51 at concentrations as low
as 5 x 10~6 M, and inhibition was complete within 5 min. Full inhibition at
such low concentrations is typical of a true acetylcholinesterase.
Ethopropazine is widely used to selectively inhibit butyrylcholinesterases while
studying acetylcholinesterases. At 10~4 M or higher concentrations, when most
butyrylcholinesterases should be completely inhibited, ethopropazine inhibited
the hydrolysis of acetylthiocholine by less than 10 %. Ethopropazine quite often
shows incomplete specificity, and this slight inhibition is consistent with the
Xenopus enzyme being a true acetylcholinesterase. Furthermore, direct tests
revealed no evidence for butyrylcholinesterase.
Activity with butyrylthiocholine as substrate
Butyrylthiocholine concentrations between 0-1 and 12 mM were used in
attempts to measure butyrylcholinesterase activity. The activity was low at all
stages between early gastrula and swimming tadpole, and was only slightly
inhibited by treatment for 1 h with 3 mM eserine. This indicates that the
hydrolysis of butyrylthiocholine is probably not due to a cholinesterase, but to a
non-specific esterase.
Activity of cholinesterase during Xenopus development
213
Table 1. Comparison of acetylcholinesterase activities in homogenates before and
after centrifugation
n-mole of acetylthiocholine hydrolysed per min per mg of
protein in:
A
Developmental stage
Homogenate
15000 g supernatant
49 000 g supernatant
10
33-34
50
0-40
4
56
0-51
5
63
0-65
6
72
Changes in acetylcholinesterase activity during development
To provide a convenient reference point of acetylcholinesterase activity before
development starts, measurements were made using oocytes. The activity of
acetylcholinesterase was less than 005 nmole/min per oocyte.
Embryos from gastrula to premetamorphic tadpoles were assayed for acetylcholinesterase activity. The 15000 g supernatant from at least 30 whole embryos
was used at each stage and every determination was repeated three times or
more. Corrections for non-enzymic hydrolysis of substrate and thiol groups
present in extracts were applied, and activities were expressed per embryo and
per mg of protein in the homogenates. The results are plotted in Fig. 2.
Because previous studies (Atherton & Lee, 1975) used a 49000g supernatant
for enzyme assays, several measurements were made at different developmental
stages using supernatants obtained by centrifuging homogenates at 49000 g
for 1 h. The total activity in the 49000 g supernatant was the same as that in the
15000# supernatant, and expressing enzyme activity in terms of protein in
either the whole homogenate or the supernatant only does not affect the changes
in activity during development; there is ten times as much activity at stage 33 as
there is at stage 10 (Table 1).
DISCUSSION
Homogenates of Xenopus laevis embryos were found to contain an enzyme
which hydrolyses acetylthiocholine. It is inhibited by eserine and BW 284c51
but not by ethopropazine, and is therefore a true acetylcholinesterase. It is more
resistant to eserine than most mammalian acetylcholinesterases, but some other
amphibian enzymes are unusually resistant to eserine (Hawkins & Mendel,
1946).
Homogenates of Xenopus embryos also hydrolysed butyrylthiocholine to a
small extent, but this activity was not inhibited by eserine and was probably due
to non-specific esterases. In a previous report of a high butyrylcholinesterase
activity in early Xenopus embryos (Atherton & Lee, 1975), the effect of eserine
was not tested, and thiol groups present in the extract may have reacted with
214
T. GINDI AND J. KNOWLAND
DTNB in the assay mixture, so that the significance of the observations may be
uncertain.
The enzyme activities were measured relative to protein concentration in the
homogenate or supernatant and also per embryo. The protein content of the
embryos used changed very little during the stages of development studied and
so the changes in acetylcholinesterase activity observed are similar whether the
number of embryos or their protein content is used to standardize the measurements. We prefer to use the number of embryos, because most of the protein in
early Xenopus embryos is insoluble yolk protein.
The results standardized using homogenate protein differ from those expressed
per embryo mainly in the point of inflexion on the graph around stage 40. This
reflects the slight drop in protein content as the residual yolk is consumed just
before feeding starts, and the total content of enzyme per embryo does not rise.
The results found are similar to those in the salamander (Sawyer, 1942, 1943).
Acetylcholinesterase activity begins to rise at stage 22, when the spinal cord and
brain are beginning to develop. By stage 26, when the embryo can first move
spontaneously, the activity has doubled, and it progressively increases as the
embryo becomes more active, hatches and starts to swim. The increase in
acetylcholinesterase activity therefore correlates well with the development of
the nervous and muscular system. Activity at early stages is very low, and small
changes are difficult to detect, due mainly to non-enzymic hydrolysis of substrate
and thiols present in tissue components, which may partly explain the high
values obtained previously for gastrula and earlier stages (Atherton & Lee,
1975). In contrast with the earlier report, there was no indication of a fall in
acetylcholinesterase activity between stages 44 and 50, and since the tadpoles
become increasingly active at this time, a fall in acetylcholinesterase activity
seems unlikely.
We are grateful to Drs E. D. Adamson and M. G. Ord for helpful comments on the manuscript. T.G. also thanks the Principal and Fellows of Lady Margaret Hall, Oxford, for
financial assistance from the E. P. Abraham Cephalosporin Fund. This work was supported
by the Medical Research Council.
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{Received 13 November 1978, revised 9 January 1979)