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THE JOURNAL OF EXPERIMENTAL ZOOLOGY 276:102-lll(1996)
Presence of (anAcetylcholinesterase in the
Cnidarian Actinia equina (Anthozoa:Actiniaria)
and of a Thiocholine Ester-Hydrolyzing
Esterase in the Sponge Spongia ofjCicinaZis
(Demospongiae:Keratosa)
VINCENZO TALESA, RITA ROMANI, GABRIELLA ROSI,
AND ELVIO GIOVANNINI
Department of Experimental Medicine, Division of Cell and Molecular
BLology, University of Perugia, 06100 Perugia, Italy
ABSTRACT
Cholinesterase (ChE) was studied in Cnidaria (Actinia equina) and in Porifera
(Spongia oficinalis). In A. equina a single enzyme form was detected, likely membrane-bound
through weak ionic or hydrophobic interactions. According to gel-filtration chromatography and
sedimentation analysis, it seems a GI globular monomer (78 kDa, 6.1 S) including some hydrophobic domain. This enzyme shows a good active site specificity with differently sized substrates. The
behaviour with specific ChE inhibitors and substrate inhibition is typical of the acetylcholinesterases
and makes it quite distinct from non-specific esterases also present in A. equina. In S. officinalis,
ChE-like activity is due to a smaller hydrophilic protein (50 kDa, 4.8 S). This enzyme shows a
very low substrate affinity for thiocholine esters, a poor sensitivity for positively charged ChE
inhibitors and for eserine, as well as absence of substrate inhibition with acetylthiocholine. These
results, together with those of electrophoretic analysis, suggest that in S. officinalis a particular
esterase form has also fitted for hydrolyzing choline esters with a low catalytic efficiency.
01996 Wiley-Liss, Inc.
Cholinesterases (ChEs") are a class of serine
hydrolases ubiquitous in the animal kingdom.
They likely originated from the addition of a negative charge into the active site of an ancestral
esterase, thus gaining a better specificity for choline esters as substrates. As is well known, according to substrate specificity and sensitivity t o
specific inhibitors, a ChE can be classified as acetylcholinesterase (AChE, EC 3.1.1.7) or less specific butyrylcholinesterase (BChE, EC 3.1.1.8)
(Silver, '74).
In Vertebrata, AChEs are mainly involved in the
cholinergic neurotransmission, while BChEs are
present as soluble proteins in the serum and also
occur in various tissues (Silver, '74; Toutant et al.,
'85; Massoulie et al., '93).AChEs and BChEs from
Vertebrata are encoded b,y two distinct genes. They
both are polymorphic enzymes with asymmetric
and globular forms. Asymmetric AChEs, typical
of neuromuscular junctions, consist of high molecular weight oligomers (A4, b,Alz) anchored
through a collagenous structure t o the basal
lamina within the synaptic cleft. Globular forms
(GI, G2, G4) include either amphiphilic or hydro0 1996 WILEY-LISS, INC.
philic catalytic subunits, based on the capacity of
interacting with non-ionic detergent. Detergentsoluble enzymes are membrane-linked through a
hydrophobic domain (Massoulie and Bon, '82;
Silman and Futerman, '87; Massoulik et al., '93).
In Invertebrata, a number of studies concerning ChEs from Insecta (Gnagey e t al., '87;
Toutant, %9),Nematoda (Arpagaus et al., '92, '941,
Sipunculida (Talesa et al., '93), Mollusca (Talesa
et al., '94, '95a,b), Annelida (Talesa et al., '95c)
only showed globular forms. Sometimes a dimeric
(G,) ChE, glycolipid-anchored to the cell mem-
*Abbreviations used: AChE = acetylcholinesterase; ATC = acetylthiocholine; BChE = butyrylcholinesterase; Brij 96 = 10-old ether;
BTC = butyrylthiocholine; BW284c51 = 1:5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide; ChE = cholinesterase;
DTNB = 5,5'-dithiobis-(-2 nitrobenzoic acid); HS = high-salt; HSB =
high-salt-Brij; HSDS = high-salt-detergent-soluble; HSS = high-saltsoluble; HST = high-salt-Triton; LS = low-salt; LSDS = low-salt-detergent-soluble; LSS = low-salt-soluble;LST = low-salt Triton; p-NPhB
= p-nitrophenylbutyrate; PTC = propionylthiocholine; PtdIns =
phosphatidylinositol.
Received November 8, 1995; revision accepted May 10, 1996.
Address reprint requests to Dr. Vincenzo Talesa, Dipartimento di
Medicina Sperimentale, Sezione di Biologia Cellulare e Molecolare,
Universita di Perugia, Via del Giochetto, 06100 Perugia, Italy.
CHOLINESTERASE IN ACTINIA AND SPONGIA
brane, coexists with another form contained in the
hemolymph (Talesa et al., '94, '95a). ChEs from
Invertebrata often display a less defined substrate
specificity than the corresponding enzymes from
Vertebrata and a wide variability in the kinetic
behaviour (Talesa et al., '90, '94).
Our previous research (Talesa et al., '92) studied soluble forms of AChE in Cnidaria (VeZeZZa
uelella, Actinia equina), a phylum in which tissue
differentiation gave the inception of the nervous
system in the animal kingdom as a primitive
nerve net. However, the presence of what appeared to be ChE has been observed in Porifera
as well (Lentz, '661, even if such a phylum did not
evolve a nervous system or sense organs and
shows only the simplest of contractile elements
(Hickman, '67; Hickman et al., '88).
Goals of the present work were further studies
of ChE in Cnidaria (Actinia equina) as well as a
more reliable identification and characterization of
possible ChE forms in Porifera (Spongia oficinalis).
In addition, bearing in mind the remote phylogenic
origin of these organisms, we tried t o detect in
them some evidence for a possible origin of ChEs
from esterases (likely carboxylesterases) also ubiquitous in the animal kingdom, based on changes
in molecular and kinetic features,
MATERIALS AND METHODS
Materials
Acetyl- (ATC), propionyl- (PTC), butyrylthiocholine (BTC) iodide as well as p-nitrophenylbutyrate (p-NPhB) used as substrates for ChE
or esterase activity measurements, respectively,
eserine sulfate, edrophonium chloride, procainamide, and 1:5-bis(4-allyldimethylammoniumphenyl)-pentan-3-onedibromide (BW284c51)used
as ChE inhibitors, diethyl-p-phenylphosphateand
bis-p-nitrophenylphosphateused as esterase inhibitors, bacitracin and aprotinin (protease inhibitors), Escherichia coli alkaline phosphatase,
marker proteins, and blue dextran for M, evaluation, fast blue BB salt, and a-naphthylacetate for
esterase staining after electrophoresis, were purchased from Sigma Chemical Co. (St. Louis,
MO). Ultrogel AcA 44 for gel-filtration chromatography was bought from LKB, Bromma, Sweden.
5,5'-dithiobis(-2-nitrobenzoicacid) (DTNB) was from
Merck (Darmstadt, FRG). Electrophoresis purity
reagents were from Bio-Rad (Melville, NY).Bacterial phosphatidylinositol (Ptd1ns)-specific phospholipase C (Bacillus cereus) and P-galactosidase
(E. coli) were bought from Boehringer (Mann-
103
heim, FRG). All other reagents used were analytical grade products from various sources and all solutions were made in twice-distilledwater.
Specimens of Actinia equina (3-5 cm in diameter, 8-10 cm in length) and Spongia officinalis
(15-20 cm in diameter) were collected during May
on the rocky bottom in shallow waters along the
coast of the Thyrrenian sea near Livorno (Italy).
Both materials were quickly frozen at -20°C and
transferred t o the laboratory in dry ice.
Composition of buffers used for
extractions, sedimentation analysis,
and gel-filtration chromatography
Low-salt (LS) buffer contained 20 mM Tris-HC1,
pH 7.4, 1mM EDTA, 5 mM MgC12,0.1 mg/ml bacitracin, and 0.008 TIU/ml aprotinin to minimize
proteolysis. Low-salt-Triton (LST) and high-salt
(HS) buffers contained LS buffer supplemented
as above plus 1%Triton X-100 or 1.0 M NaCI,
respectively. High-salt-Triton (HST) and high-saltBrij (HSB) buffer contained HS buffer plus 1%
Triton X-100 or 0.5% 10-oleil ether (Brij 961, respectively.
Assay methods
ChE activity measurements were carried out at
20°C according t o a modification of the method
early described by Ellman et al. (,61), using ATC
as substrate. In particular, as a general rule, the
assay mixture was composed of 0.89 ml of 0.1 M
Na-phosphate buffer, pH 7.2, containing 0.5 mM
DTNB, 0.1 ml of 1.0 mM substrate (final concentration), and 0.01 ml of enzyme solution
(HSDS or other extract). Esterase activity is
commonly evaluated measuring the hydrolysis
of p-nitrophenylesters as substrates (Van Lith
e t al., '89). In the present study, p-NPhB at 5 mM
final concentration was used, since it is not hydrolyzed by ChE from A. equina, as observed in a
previous report (Talesa et al., '92). Esterase activity measurements in HSDS o r other extracts
were made with the same assay mixture used for
ChE except the absence of DTNB. The product of
thiocholine reaction with DTNB (ChE) or p nitrophenol production (esterase) were spectrophotometrically determined at 412 nm (E =
13,600 M-' ern-') or 400 nm (E = 13,200 M-' cm-I),
respectively, by continuous recording. The rate
of absorbance change was linear for at least 2 min
for both activities and the slope was used to calculate the initial rate. One enzyme unit (IU) was
defined as the amount of enzyme which catalyzes
the hydrolysis of 1 pmol of substrate/min.
V. TALESA ET AL.
104
Extraction of ChE and esterase
for calculation of the sedimentation coefficients
~~.
.
(S).The gradients were emptied from the bottom
using a peristaltic pump and 40 fractions of 250
p1 each were collected. ChE or esterase activity
in each fraction was measured on the basis of the
bI2
or boo
change through 30 min in the usual
assay mixture containing 1 mM ATC or 5 mM p NPhB and 0.05 ml of each fraction. Alkaline
phosphatase and P-galactosidase activities were
determined according to Principato et al. ('84a)
or Massouli6 and Rieger ('69), respectively.
The extraction of enzymes was carried out on
the whole from 50 g of A. equina specimens and
from 100 g of S. officintrlis, operating at 5°C. The
typical procedure, whi ch was repeated several
times, started from 5 g of A. equina or 10 g of S.
officinalis. Either material, after washing with
bidistilled water and adding 20 ml of HST buffer,
was carefully minced by scissors in small pieces.
In a preliminary expeniment the resulting materials were centrifuged a t 100,OOOg for 30 min
(Beckman L 60 ultracentrifuge, SW 41 Ti rotor,
Gel-filtration chromatography and M r
24,000 rpm; Beckman, Palo Alto, CA); the superevaluation of ChEs
natants were found almost devoid of ChE as well
The M, value of ChE forms from A. equina or
as esterase activity and discarded. Therefore, as S. officinalis were estimated from the Svedberg
a general rule, either minced material in HST equation M, = 67cq NS RJ(l-p/pm), where N is the
buffer was homogenized with an Ultra-Turrax T Avogadro number, while for q (viscosity of water),
25 homogenizer and centrifuged a s above at p and pm(densities of water and enzyme protein)
100,OOOg for 1h. The supernatants thus obtained the values reported by Osterman ('86) were used.
from either source (20-22 ml) and noted as high- The Stokes radii (R,) were determined according
salt-detergent-soluble (HSDS) extracts, showed to Laurent and Killander ('64). A sample (5 ml) of
marked ATC- and p-NPhB-splitting activities that HSDS extract was applied to an Ultrogel AcA 44
were also assayed in the presence of 10-4M es- column (2.5 x 114 cm) equilibrated and eluted (0.5
erine (ChE inhibitor). Tentative experiments ml/min) with HST buffer. Fractions of ,5 ml were
with S. officinalis extracts were also carried out collected. Horse heart cytochrome c (17 A), chicken
adding to the assay mixture 10" M diethyl-p-nitro- egg 9lbumin (30 A), E. coli alkaline phosphatase
phenylphosphate or bis-p-nitrophenylphosphate, (33 A), and yeast alcohol dehydrogenase (46 A)
well-known esterase inhibitors (Van Lith et al., were used as standards. The void volume was es'89). It is also noticeable that similar amounts of timated using blue dextran. ChE activity was
solubilized enzyme activity were obtained as well evaluated as described under Density gradient
using in the extraction procedure LS, HS, or LST centrifugation. The sedimentation coefficients ( S )
buffer (LSS, HSS, LSDS extract, respectively). In to be used in the M, calculation were obtained by
addition, preliminary electrophoretic analyses (see sedimentation analysis as reported above.
Results: PAGE) displayed in the extracts from
each of the sources the Eiame patterns of ChE and Polyacrylamide gel electrophoresis (PAGE)
esterase forms. However, HSDS extracts were choElectrophoretic patterns of ChE as well as of
sen for analytical studies since they showed esterase activity from either source were studied
slightly higher specific activities (IU/ml). They by PAGE (7% acrylamide, 0.2% bis-acrylamide)
were stored at -80°C until subsequent use.
carried out at non-denaturing conditions. In particular, after a dialysis against LS buffer, 20 pl of
Density gradient centrifugation
HSDS extracuane (4 mIU ChE and 13 mIU esterase
activity, A. equina; 3 mIU ChE and 25 mIU
Sedimentation pattern of ChE and esterase acesterase
activity, S. officinalis) were run on a 16
tivity in HSDS extract from either A. equina and
x
16
x
0.15
cm slab-gel (20 mA current, 0.025 M
S. officinalis was studied by centrifugation of
Tris-0.192
M
glycine buffer, pH 8.3, 5°C). A presamples (200 pl) layered. onto 5 2 0 % sucrose denliminary
electrophoretic
analysis of LSS, HSS, and
sity gradients (10 ml in polyallomer tubes) in HS,
LSDS
extracts
was
also
carried out in the same
HST, or HSB buffer. In the last case, samples were
way.
Staining
for
ChE
activity
was obtained acpreincubated with 0.5% Brij 96 for 30 min before
cording
to
the
method
of
Juul('68)
as modified by
applying. Centrifugations were performed at 5°C
3
mM
ATC
as
substrate. In
Stenersen
('
8
0)
using
in a Beckman L60 ultracentrifuge equipped with
particular,
after
the
incubation
in
a
copper/ATC
a SW 41 Ti rotor at 36,000 rpm (222,OOOg); E.
solution,
the
gel
was
washed
2-3
hours
in twicecoli alkaline phosphataEle (6.1 S) and P-galactosidase (16 S) were included as internal standards distilled water prior to the final dithiooxamide
CHOLINESTERASE IN ACTZNIA AND SPONGZA
treatment. Staining for esterase activity was performed according to Hirose et al. ('90) with fastblue BB salt using 0.05% a-naphthyl acetate as
substrate.
Treatment of ChE with phospholipase C
and non-denaturing PAGE
Since ChE from A. equina interacts with detergents (see Results: Density gradient centrifugation), the possible presence in this enzyme of a
PtdIns anchor was studied by PtdIns-specific
phospholipase C digestion and subsequent PAGE.
Samples of HSDS extract (200 p1,40 mIU of ChE
activity) were incubated with 5 IU of phospholipase C (5 pl of pure commercial preparation) for
90 minutes at 25°C under continuous stirring.
Non-denaturing PAGE was carried out as previously detailed (Talesa et al., '93) running aliquots (20 pl? 4 mIU) of the above described
mixture, as well as an undigested control, in a
vertical slab gel (8 x 7.3 x 0.1 cm) apparatus (BioRad). Gel and running buffer contained 0.5% Triton X-100; gel staining for ChE activity was
performed following the method of Juul ('68) as
modified by Stenersen ('80). The actual activity
of phospholipase C preparation was checked by a
parallel experiment with a DS extract from the
annelid Hirudo medicinalis, containing a PtdInstailed ChE (Talesa et al., '95c). These trials were
carried out by digestion of H. medicinalis extract
as above (200 pl, 200 mIU of ChE activity) and
subsequent running of 20 pl aliquots (20 mIU) of
phospholipase C-treated or undigested enzyme.
105
Lineweaver-Burk double reciprocal plots of experimental data, using ATC, PTC, and BTC as substrates in the 0.1-1 mM concentration range (five
concentrations for each substrate).
Inhibition studies of ChE activity in HSDS extracts from both A. equina and S. officinalis were
carried out in the presence of ATC as substrate
and using well-known inhibitors of ChEs, previously employed t o study invertebrate enzymes:
procainamide (Principato et al., '89; Talesa et al.,
'90, '92), edrophonium (Talesa et al., ,931, eserine
(Silver, '74; Arpagaus et al., '92; Talesa et al.,
'95a,b) and BW284c51 (Silver, '74). Inhibitor concentrations in the 10-3-10-9M range were used in
the assay and residual ChE activities were determined (two distinct determinations for each inhibitor concentration). In particular, the reaction
was started after an extract-inhibitor incubation
of 1minute by adding the substrate solution. Substrate inhibition tests of ChE from either origin
were also performed using ATC in the 0.1-50 mM
concentration range.
RESULTS
Extraction of ChE and esterase
No significant amount of ChE or esterase activity was recovered from either A. equina or S.
officinalis in a free form by dilacerating the tissues without homogenization. The enzyme extraction, carried out by homogenization in LS, HS,
LST, or HST buffer, respectively, gave comparable
recoveries of both enzyme activities from either
source; however, the maximal yield as total
Kinetic and inhibition studies
amount and specific activity (IU/ml) was obtained
Kinetic parameters K, and V,, were deter- using HST buffer (HSDS extracts) (Table 1).
The presence of lo4 M eserine in the assay submined for thiocholine ester-splitting enzymes
present as a single form in either studied species stantially unaffected esterase activity, while ChE
(see Results: PAGE) by computer analysis of activity was suppressed or significantly reduced
TABLE 1. Extraction of cholinesterase (ChE) and esterase from Actinia equina (5 g) and Spongia officinalis (10 g) carried
out by homogenization i n high-salt-detergent (HSDS extract), i n low-salt-detergent (LSDS extract),
high-salt (HSS extracts), or low-salt (LSS extract) buffer, as detailed in the text'
Extract
A. equina
HSDS
LSDS
HSS
S. officinalis
LSS
LSDS
LSDS
HSS
LSS
ChE (total activity [IUD
Esterase (total activity [IUI)
4.0(ND)
13.3 (13.7)
9.5
11.0
9.6
25 (23.9)
22
22
19
3.5
3.3
3.3
2.5 (1.75)
2.3
2.3
2.1
'ChE and esterase activities were evaluated using acetylthiocholine (ATC) and p-nitrophenylbutyrate (p-NPhB) as substrate, respectively.
The values in parentheses were obtained in the presence of lo4 M eserine. One enzyme unit (IU) was defined as the amount of enzyme
catalyzing the hydrolysis of 1pmol of substrateimin. ND: not detectable.
V. TALESA ET AL.
106
in the extracts from A. equina and S. officinalis,
respectively. Assays carried out (S. oficinalis) with
esterase inhibitors displayed total suppression of
both ChE and esterase activity (results not shown).
Density gradient centrifigation
Sedimentation analysis of ChE and esterase activity in the HSDS extracts from A. equina or S.
officinalis were performed with groups of four experiments. The apparent sedimentation coefficients (peaks of enzyme activity) are given as
means SD, while the sedimentation profiles result from the mean values of activity in each fraction. Sucrose gradient centrifugation of HSDS
extract from A. equina, carried out in the absence
of detergent (HS buffer), showed a single peak
of ChE activity at 10.1 2 0.4 S, shifted t o apparent 6.7 f 0.2 S and 6.1 r 0.2 S positions when
Brij 96 or Triton X-100 was, respectively, added
to the gradient (Fig. 111).The same allalysis for
esterase, with a detergent-free gradient, gave a
diffuse sedimentation pattern at high S values,
likely due to partial aggregation of enzyme, besides an activity peak at 6.7 2 0.1 S. Addition of
Triton X-100 or Brij 96 gave double peak-shaped
sedimentation profiles (9.4 2 0.3 S , 4.8 f 0.1 S,
and 8.7 2 0.2 S , 4.8 2 0.2 S, respectively), thus
suggesting the presence of at least two esterase
forms (Fig. 1B).
The same sedimentation analysis of HSDS extract from S. officinalis gave, in a detergent-devoid gradient, a single peak of ChE activity at
4.8 2 0.2 S, which remained unchanged after adding Triton X-100 (4.8 & 0.3 s) or Brij 96 (4.8 &
0.4 S) to the gradient (Fig. 10. The sedimentation profile of esterase also showed a single, although less sharply shaped, activity peak at 4.8
f 0.4 S position in a detergent-free gradient. It
was unchanged in the presence of Triton X-100
(4.8 2 0.3 S) or Brij 96 (4.8 2 0.2 S)(Fig. ID).
0.0
n
ol
0.6
4
c
c
3
0.4
3)
L
0.2
b
I
0.0
n
D
.6d
-
c
>
I
V
I
V
I
30
40
.tr
0
4
aJ
0
(I?
0.3
W
Lo
Q
L
W
c
u
0.2
0)
.tr
Lo
w
0.i
0.0
0
10
20
30
40
0
10
20
Frectlon number
Fig. 1. Sedimentation pattern in sucrose density gradient of cholinesterase (ChE) and esterase forms in high-saltdetergent-soluble (HSDS) extracts from Actinia equina (A,B)
and Spongia officinalis (C,D) prepared as detailed in the text.
Gradients (5-20% were made in HS buffer with 1%Triton X-
100 (01, 0.5% Brij 96 (O),
or no detergent (0).
ChE and esterase activities were evaluated using ATC orp-NPhB as substrate, respectively. The arrows indicate the position of E.
coli P-galactosidase (16 S) and alkaline phosphatase (6.1 S )
used as internal standards.
CHOLINESTERASE IN ACTINIA AND SPONGIA
The fairly widened shape of such a n activity peak
suggests a n adjoining sedimentation of several
molecular forms of enzyme.
ChE
0-
107
E
ChE
E
Gel-filtrationchromatography
and M revaluation of ChEs
Ultrogel AcA 44 chromatography of HSDS extract from A. equina or S. officinalis gave for both
a single ChE activity peak at Stokes radius (R,)
values of 31 and 25 A, respectively. A repetition
of such experiments displayed quite similar results. The M, values, calculated setting in the
Svedberg equation R, and S from gel-filtration and
sedimentation analysis (6.1 S and 4.8 S), were
78,000 (A. equina) and 50,000 (S. officinalis).
Polyaciylamide gel electrophoresis (PAGE)
According to the results of PAG-electrophoresis,
both ChEs from A. equina and S. officinalis migrated towards the anode as single activity bands.
On the contrary, electrophoretic patterns of esterases showed several bands of activity (two for
1 2
3
4
A. equina, more numerous for S. officinalis). In
addition, as to A. equina, a correspondence occurs
Fig. 2. Non-denaturing PAGE of cholinesterase (ChE) and
between the ChE activity band and that of the
faster migrating esterase form (Fig. 2, lanes 1, esterase (E) forms in high-salt-detergent-soluble (HSDS) extract from Actinia equina (lanes 1,2: 4 mIU ChE and 13
2), while the ChE activity band of S. officinalis mIU
E activity, respectively) and Spongia oficinalis (lanes
corresponds in position to the predominant one of 3,4: 3 mIU ChE and 25 mIU E activity, respectively) preesterase activity (Fig. 2, lanes 3,4). Such experi- pared as detailed in the text. Samples of 20 pl were run.
ments were repeated several times with quite Staining for ChE and E activity was performed according t o
the method of Juul ('68) as modified by Stenersen ('80) and
similar results.
A preliminary electrophoretic analysis of LSS, Hirose et al. ('90) respectively. 0: origin of migration.
HSS, and LSDS extracts from A. equina and S.
officinalis gave substantially the same patterns A. equina and S. oficinalis. Such kinetic constants
of ChE and esterase activity observed with HSDS were determined using different substrates. Based
on the sets of V, and Vm,/Km values, ATC is the
extract (results not shown).
best substrate for both enzymes, followed by PTC,
lkeatment of ChE with phospholipase C
while BTC is by far the less suitable one: the
and non-denaturing PAGE
Vmax(~~~)/Vmax(ATC)
ratio is 0.18 and 0.42 for the ChE
Digestion with PtdIns-specific phospholipase C from A. equina and S. officinalis, respectively. In
of HSDS extract gave no change of electrophoretic addition, while V, values for both enzymes lie
pattern of ChE from A. equina in non-denaturing roughly into the same size order, ChE from S.
conditions and in the presence of Triton X-100. The officinalis shows far higher K, values (nearly two
ChE activity band migrated towards the anode in magnitude orders). In consequence, the V,JK,
the same way as undigested control (Fig. 3, lanes values are even 100-fold lower (ATC) in compari1,2). The experiment was repeated with identical son with those of the ChE from A. equina.
results. The positive control with a ChE-containAs regards the studies with some ChE inhibiing DS extract from Hirudo medicinalis verified tors (Fig. 4), ATC-hydrolyzing activity of ChE from
the activity of phospholipase C, giving the expected A. equina showed, as a rule, a higher sensitivity
conversion of the amphiphilic slow-migrating en- than the corresponding enzyme from S. oficinalis.
zyme to a faster hydrophilic form (Fig. 3, lanes 3,4). Residual activity of the latter exceeded 50% even
with 10 M procainamide, BW284c51, or eserine,
Kinetic and inhibition studies
while Is0 was about 5 x lo4 M with edrophonium.
Table 2 gives V, and K, values for thiocholine On the contrary, ChE from A. equina showed only
ester-splitting enzymes present as single forms in traces of residual activity with
M BW284c51
V. TALESA ET AL.
108
T
C
T
C
1
2
3
4
Fig. 3. Non-denaturing PAGE of cholinesterase (ChE) in
high-salt-detergent-soluble(HSDS) extract from Actinia
equina (lanes 1,2:4 mIU of ChE activity) prior to and after
digestion with PtdIns-specific phospholipase C , carried out
as described in the text. Posritive control of phospholipase C
activity with a ChE-conta ning DS extract from Hirudo
medicinalis (lanes 3,4: 20 riIU of ChE activity). Samples of
20 pl were run. Staining for ChE activity was performed according t o the method of Juiil('68) as modified by Stenersen
('80). T,phospholipase C-treated sample; C, control undigested
sample.
or eserine, while lo3 M procainamide or edrophonium left a detectable activity. Ih0values for
this enzyme were roughly lo4 M (procainamide
and edrophonium),
M (eserine), and 5 x lo-'
M (BW284c51).
The results of substrate inhibition tests, carried
out in the presence of ATC, evidenced that such
an inhibition only concerns ChE from A. equina
and starts beyond 5 mlVI substrate (Fig. 5).
DISCUSSION
The joined results of sedimentation and electrophoretic analysis suggest the presence both in
A. equina and S. offiiciizaZis of a single thiocholine
ester-hydrolyzing enzyme and, in addition, a set
of esterases, more numerous in the latter organism. Such enzymes can be solubilized at comparable yields by homogenization in a low or high
salt buffer, with or without a detergent.
ChE from A. equina does not overlap its activity with esterase, being inactive on p-NPhB
(Talesa et al., '92) and strongly inhibited by eserine. Sedimentation pattern in a detergent-free
gradient shows t h a t such a n enzyme is a n
amphiphilic protein with some hydrophobic domain, giving aggregated oligomers (10.1 S position), fully resolved by adding detergent to the
gradient (peak shifting to 6.1-6.7 S). However,
such a hydrophobicity is not due to a PtdIns, giving the in vivo membrane-anchoring of several
amphiphilic ChEs (Silman and Futerman, '87;
Ferguson and Williams, '88; Massoulib et al., '93).
In fact, the enzyme shows no change of electrophoretic pattern in the presence of Triton X-100
after treatment with a specific phospholipase C.
The M, value (78,000) emerging from the results
of sedimentation analysis and gel-filtration chromatography approaches that (83,000) which we
found previously by SDS-PAGE of the same ChE
from A. equina (Talesa et al., '92). Therefore we
believe that the studied enzyme is a monomeric
globular form (GJ, while the M, value (330,000)
and tetrameric structure suggested in Talesa et
al. ('92) for the presumed native ChE likely concerned an aggregate.
The esterases ofA. equina are as well amphiphilic
forms with hydrophobic detergent-binding domains
giving aggregation of the enzymes, as shown by
centrifugation in a detergent-devoid gradient.
According to solubilization behaviour, both ChE
and esterase forms from A. equina could be weakly
attached t o the cell membrane through either ionic
interactions or the hydrophobic domains evidenced
by sedimentation analysis.
In S. officinalis an enzyme assay with different
substrates and inhibitors cannot state whether
ChE-like and esterase activities are mutually exclusive or overlap, since the former is only in part
inhibited by eserine and suppressed by usual esterase inhibitors. The thiocholine ester-splitting
enzyme, based on results of density gradient centrifugation, is likely a hydrophilic protein, devoid
of detergent interaction or self-aggregation.Its sedimentation coefficient (4.8 S ) approaches those of
globular monomeric (GJ ChEs from Vertebrata
(Massoulie et al., '93) or Invertebrata (Arpagaus
et al., '92), while the M, value (50,000) is rather
lower than those usually observed for monomeric
109
CHOLINESTERASE IN ACTZNZA AND SPONGZA
TABLE 2. Kinetic constants of cholinesterase (ChE) activities in high-salt-detergent-soluble (HSDS) extracts from
Actinia equina and Spongia officinalis, prepared as described in the text
(IU/ml)
0.216 k0.060
0.120 f 0.020
0.040 2 0.010
0.142 2 0.060
0.134 f: 0.028
0.060 2 0.014
ATC
PTC
BTC
ATC
PTC
BTC
A. equina
S. officinalis
K, (mM)
V,,,
Substrate
Vmax/K,
0.098 k 0.014
0.145 0.035
0.300 f: 0.086
8 * 1.09
14 k 4.20
19 4.13
2.20
0.83
0.13
1.8 x
9.6 x
3.2 x
(min-3
lo-'
lo3
lo3
'The enzyme activity was determined using actyl- (ATC), propionyl- (PTC), and butyryl-thiocholine (BTC)as substrates.,,V
and K, values
are given as M f SD of four experiments. One enzyme unit (IU)was defined as the amount of enzyme catalyzing the hydrolysis of 1 pmol of
substratelmin.
ChEs or catalytic subunits of these enzymes. The
set of esterases from s.officinalis shown by PAGelectrophoresis holds hydrophilic forms as well,
with a sedimentation pattern unaffected by the
presence or absence of detergent in the sucrose
gradient. Probably, both ChE-like enzyme and esterases from S. oficinalis occur in vivo as soluble
cytosolic proteins; otherwise, they could be once
...
A
80
-
60
-
40
-
20
-
n
be
W
n
4
-
.C
>
again membrane-linked by weak electrostatic interactions.
As from kinetic studies, thiocholine ester-hydrolyzing enzymes from both A. equina and S .
officinalis could be classified as AChEs according
to the higher values of V,, and Vm,Km (Fersht,
'85; Principato et al., '88) with ATC as substrate.
Moreover, both the enzymes show a good specific-
4
0
43
W
100
.c
u
r_
80
43
3
U
c
60
u)
a
Q!
40
20
0
-10
-8
-6
-4
Fig. 4. Inhibition of cholinesterase (ChE) forms in highsalt-detergent-soluble (HSDS)extracts from Actinia equina
( 0 )and Spongia officinalis (0)
assayed as detailed in the
-2
-10
-8
-6
-4
-2
text. ChE activity was evaluated using ATC as substrate. A,
procainamide; B, edrophonium; C, BW284c51; D, eserine.
V. TALESA ET AL.
110
AChEs, high sensitivity to BW284c51, and eserine
as well as substrate inhibition with ATC (Silver, '74). Moreover, the degree of inhibition by
procainamide and edrophonium is comparable
to that of some invertebrate ChEs (Talesa et al.,
.d
'95a). The AChE from A. equina also hydrolyzes
detectably a charge-devoid substrate (a-naphthyl
0.04
3
acetate),
thus giving an apparent electrophoretic
u
band
of
esterase
activity.
u 0.02
Therefore,
keeping
in mind the course of anic
u 0 .oo
mal phylogeny, it is likely that Porifera, devoid of
-1 .5
-0.5
0.5
1 .5 a nervous system and sense organs, lack enzymes
classifiable as ChEs, even if some of their esterases already gained the capacity of hydrolyzing, with a low catalytic efficiency, choline esters.
On the contrary, in Cnidaria, where the onset of
Fig. 5. Substrate inhibition curves of cholinesterase (ChE)
forms in high-salt-detergent-soluble (HSDS) extracts from tissue differentiation has given a primitive nerActinia equina (@) and Spon<giaoffcinaZis (0).
ChE activity vous system, there is a ChE showing kinetic
was evaluated using ATC as mbstrate.
and molecular features typical of such a class
of enzymes.
0.10
n
c
.C
ity level of the active site with differently sized
ATc) values approach those
substrates: Vmm(BTC)/Vma
ofAChEs from Vertebrata and Jnsecta (Silver, '74;
Gnagey et al., '87).
However, apart from such analogies, the ChElike enzyme from 5'. officinalis shows a far lower
catalytic efficiency (V,,JK, values), due t o a poor
substrate affinity for tl~iocholineesters: indeed,
Km values exceed by far those of A. equina enzyme and even those ol' well-known BChEs from
Vertebrata (Andersen and Mikalsen, '78; Principato
et al., '84b). Such findings suggest a reduced role
of electrostatic interactions in the enzyme-substrate complex formation. Considering also the
very low sensitivity for competitive positively
charged inhibitors of ChEs (procainamide,
edrophonium, BW284~51)and the absence of
substrate inhibition with ATC, it is likely that the
active site of S. ofj'icinalis enzyme is devoid of the
ChE-typical anionic moiety. Furthermore, the low
inhibition by eserine di$sagreeswith a distinctive
feature of ChEs, while it is usual for aryl- and
carboxyl-esterases (Silvw, '74). On the other hand,
the existence of an active site conformation and
catalytic mechanism dif'ferent from those of ChEs
and more suited to estsrases is in keeping with
the results of electrophoretic analysis of the enzymes from s.officinalis, where the ATC-hydrolyzing activity seems to be axnmitted to a predominant
esterase form.
On the contrary, the thiocoline ester-splitting
enzyme from A. equina seems a true AChE, based
on substrate affinity, close to that of vertebrate
ACKNOWLEDGMENTS
We are grateful t o Mr. Marco and Maurizio Rosi,
Livorno, Italy, for collecting the specimens of
Actinia equina and Spongia officinalis used in this
study. We thank Andrea Piazzoli and F'rancesco Fabi
for technical assistance. This work was supported
by a grant from the Italian CNR (94.02479.CT04).
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