Enzymes cooperating in the hydrolyzing process of benzoylcholines: Subcellular and inter-tissue comparison

Article abstract of DOI:10.1691/ph.2009.9046

Within benzoylcholine biotransformation, butyrylcholinesterase (3.1.1.8, BuChE) appears to be the key enzyme in the hydrolyzing process. Except for BuChE in the process of benzoylcholine hydrolysis, carboxylesterase (3.1.1.1, CE) could play a role in the splitting of the ester bond. The aim of this work was to clarify the interaction between BuChE and CE in the hydrolyzing process of a homologic row of benzolycholines on subcellular and inter-tissue level. Two fractions, microsomes and cytosol of rabbit lung and liver were investigated. Participation of the enzyme activities was determined on the base of kinetic inhibitory studies, using eserine as cholinesterase inhibitor. Despite the fact that in all studied fractions of both organs BuChE and CE were confirmed, only in lung microsomes exclusive BuChE activity in benzoylcholine hydrolyzing process was observed, without substrate specifity. In the other fractions studied interaction of both enzymes were recorded, whereas the benzoylcholine structure played an important role. It seems that, the portion of CE depends predominantly on substrate structure and elevates with bulk of alcoholic part of benzoylcholines. Despite the same enzyme equipment in all tissue fractions studied, the affinity of hydrolyzing enzymes interestingly differs. This might be as a result of distinct subcellular pattern of CE activity localization in lung and liver.

Full text of DOI:10.1691/ph.2009.9046

ORIGINAL ARTICLES
Department of Cell and Molecular Biology of Drugs, Faculty of Pharmacy, Comenius University, Bratislava, Slovakia
Enzymes cooperating in the hydrolyzing process of benzoylcholines:
subcellular and inter-tissue comparison
ˇ
I. Paulikova´, K. Sisˇkova´
Received February 5, 2009, accepted February 21, 2009
I. Paulikova´, Department of Cell and Molecular Biology of Drugs, Faculty of Pharmacy, Comenius
University, Kalinciakova 8, 832 32 Bratislava, Slovakia
paulikova@fpharm.uniba.sk, siskova@fpharm.uniba.sk
Pharmazie 64: 398–402 (2009)
doi: 10.1691/ph.2009.9046
Within benzoylcholine biotransformation, butyrylcholinesterase (3.1.1.8, BuChE) appears to be the key
enzyme in the hydrolyzing process. Except for BuChE in the process of benzoylcholine hydrolysis,
carboxylesterase (3.1.1.1, CE) could play a role in the splitting of the ester bond. The aim of this work
was to clarify the interaction between BuChE and CE in the hydrolyzing process of a homologic row of
benzolycholines on subcellular and inter-tissue level. Two fractions, microsomes and cytosol of rabbit
lung and liver were investigated. Participation of the enzyme activities was determined on the base of
kinetic inhibitory studies, using eserine as cholinesterase inhibitor. Despite the fact that in all studied
fractions of both organs BuChE and CE were confirmed, only in lung microsomes exclusive BuChE
activity in benzoylcholine hydrolyzing process was observed, without substrate specifity. In the other
fractions studied interaction of both enzymes were recorded, whereas the benzoylcholine structure
played an important role. It seems that, the portion of CE depends predominantly on substrate struc-
ture and elevates with bulk of alcoholic part of benzoylcholines. Despite the same enzyme equipment
in all tissue fractions studied, the affinity of hydrolyzing enzymes interestingly differs. This might be as
a result of distinct subcellular pattern of CE activity localization in lung and liver.
1. Introduction
Moreover BuChE individually varies in the rabbit livers
mainly (unpublished results). Although some observed dis-
crepancies of BuChE activity could be caused here by very
low content of BuChE, since the enzyme expression was
surprisingly low in comparison to carboxylesterases (Jbilo
et al. 1994). Not of neglectable importance are genetic var-
iations, which lead to lower or higher BuChE activity.
BuChE is probably not necessery for life, since individuals
with no BuChE activity do not suffer from any kind of dis-
ease relating to its lack (Li et al. 2006). Even though, this
statement could turn premature in the course of future inves-
tigations. From the toxicological point of view the participa-
tion of BuChE activity in metabolism of several xenobio-
tics, especially organophosphates is important.
CE is an enzyme widely distributed in animal cells and is,
together with BuChE, involved in the hydrolyzing process.
However a lot of isoenzymatic forms of CE occuring on
not only interspecies, but even on intertissue levels were
described.
Unexpected response of BuChE activity in the rabbit liver
has previously been the reason for the elucidation and
characterization of BuChE as well as the participating en-
zymes in the hydrolyzing process on the subcellular and
inter-tissue level using substrates of the benzoylcholine
row (Paulikova´ et al. 2006). These compounds are N-al-
kylderivates of benzoylcholines, with following general
formula: N-(2-benzoyloxyetyl)alkyldimetylammonium bro-
mides, where alkyl is 1–14 carbons long (abbreviated
BCHn, where n is the number of carbons in the alkyl side
Many drugs contain an ester bond, which is very sensitive
to esterases attack. It is a very complicated process de-
pending on many biological, toxicological and biochem-
ical aspects. It depends on the overall substrate structure
and concentration, as well as species, tissue and subcellu-
lar localization, if one or more esterases participate in the
hydrolyzing process. While the action of the majority of
drugs is terminated on this basis (local anesthetics, antide-
pressants . . . etc.), there are some releasing products,
which can also contribute to the pharmacological effects
(bambuterol, irinotecan . . . etc.).
Butyrylcholinesterase (EC 3.1.1.8, BuChE) has been in
a focus of many scientists for a long time. While this en-
zyme occures in various tissues (brain, liver, lung, kidney,
heart, blood, adipose tissue) its natural substrate has not
been found and so the exact role of BuChE remains unex-
plained. In general, it is supposed that BuChE is synthe-
sized in liver and transported to other tissues, but some
authors assume its synthesis also in some other tissues
(Lucic et al. 2005). Whereas a lot is known about BuChE
gene, sequence of amino acids, formation of molecular
forms, active site and etc., there are several contradictions
about BuChE subcellular distribution, as well as the unu-
sual behaviour of BuChE molecular forms in rabbit tissues
(Rush et al. 1980; Jbilo et al. 1994). The most significant
changes have been observed in the liver microsomal frac-
tion on the inter-species level (Paulikova´ et al. 2006).
398
Pharmazie 64 (2009) 6

ORIGINAL ARTICLES
activity. BuChE activity was confirmed with aliphatic sub-
a
2,5
2
strate – S-butyrylthiocholine. Carboxylesterase activity
was measured with p-nitrophenylacetate. Results are sho-
wen in the Table.
CYTOSOL
MICROSOMES
Figure 1a displays the whole hydrolyzing activity in cyto-
solic and microsomal fractions of rabbit lung in relation to
the lenght of alkyl side chain in benzoylcholines, when a
2 mmol/l substrate concentration was used. The course of
the hydrolyzing activity of benzoylcholines with shorter
alkyl side chain from BCH2 up to BCH8 appears to be
mutualy reverse in cytosolic and microsomal fractions in
both studied tissues. In general, the hydrolyzing process is
more intensive in microsomal fractions of liver and lung
than in the cytosol (Fig. 1a, b).
Since the affinity of BuChE to benzoylcholines differs de-
pending on the lenght of their alkyl side chain, two sub-
strates were chosen for further kinetic experiments. Fig. 2
is a review of the kinetic study of two designated sub-
strates with short and middle length chain, BCH2 and
BCH8 respectively, measured in cytosol and microsomes
of rabbit lung and liver. In lung cytosol the saturation
curve assigns two peaks (Fig. 2a), and so does not seem
to follow the typical Michaelis-Menten kinetics. In the
other studied tissue fractions this effect was not observed
(Fig. 2b, c, d). Further studies provided evidence of parti-
cipation of two enzymes in the hydrolysis of benzoylcho-
lines in all studied fractions except for the lung micro-
somes.
1,5
1
0,5
0
BCH2 BCH4 BCH6 BCH8 BCH10 BCH14
b
12
10
8
CYTOSOL
MICROSOMES
6
4
2
0
BCH2 BCH4 BCH6 BCH8 BCH10 BCH14
Fig. 1: The overall hydrolysis rate (v) of benzoylcholines with different
alkyl side chain length in cytosol and microsomes of rabbit lung
(a) and liver (b), when 1 mmol/l substrate concentration was used.
Values are mean ꢀ SEM from 6 separate experiments
The cooperation of two enzymes in the hydrolyzing pro-
cess was confirmed by the inhibitory kinetic studies with
the cholinesterase inhibitor eserine. Low eserine concen-
tration enabled to distinct between eserine-sensitive - true
BuChE and non-sensitive – remaining hydrolyzing activ-
ity. In cytosol of lung and liver relatively low inhibition
was recorded, when BCH8 was used as substrate (Fig. 3b,
d). When BCH2 was the substrate, in cytosol full inhibi-
tion was attained in lung and approximately 50% of re-
chain) (Csiba et al. 1986). They are quaternary ammonium
salts with antimicrobial activity.
Two fractions of lung and liver were investigated: micro-
somes and cytosol.
2. Investigations and results
Before the experiments with benzoylcholines, both tissue
fractions were tested for the presence of BuChE and CE
a
b
2,5
2
BCH2
BCH8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
1,5
1
0,5
0
0
0
0
5
10
5
10
c (S) mmol/l
c (S) mmol/l
c
d
16
14
12
10
8
2,5
2
1,5
1
6
Fig. 2:
4
Kinetic study of BCH2 and BCH8 hydrolysis
in rabbit lung (cytosol (a), microsomes (b))
and liver (cytosol (c), microsomes (d)). The
substrate (S) concentration ranges from 0.5 to
10 mmol/l. Values are mean ꢀ SEM from 6 se-
parate experiments
0,5
0
2
0
0
5
10
0
5
10
c (S)mmol/l
c (S) mmol/l
Pharmazie 64 (2009) 6
399

ORIGINAL ARTICLES
Table: Butryrylcholinesterase and carboxylesterase activity in cytosolic and microsomal fractions of rabbit lung and liver
Activity
Lung
Liver
Cytosol
Microsomes
Cytosol
Microsomes
BuChE (mmol/min ꢁ mg)
CA (mmol/min ꢁ mg)
1.26 ꢀ 0.062
34.46 ꢀ 4.223
1.46 ꢀ 0.030
88.13 ꢀ 5.852
5.32 ꢀ 0.112
125.19 ꢀ 10.936
7.37 ꢀ 0.097
107.38 ꢀ 8.293
Values are mean ꢀ SEM from 6 separate experiments
a
b
whole hydrolyzing
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
activity
remaining activity
0
5
10
0
5
10
c (BCH2) mmol/l
c (BCH8) mmol/l
c
d
1
0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
2,5
2
1,5
1
0,5
0
0
5
10
0
5
10
c (BCH2) mmol/l
c (BCH8) mmol/l
e
f
4,5
4
3,5
3
2,5
2
1,5
1
18
16
14
12
10
8
6
4
2
0
Fig. 3:
0.1 mmol/l eserine was used in the inhibition
kinetic study of BCH2 and BCH8 hydrolysis
in subcellular fractions of rabbit lung and liver.
The substrate concentration ranged from 0.5 to
10 mmol/l. For in lung microsomes full inhibi-
tion of hydrolyzing activity was achieved, the
responsible enzyme appears to be BuChE and
only the results in cytosol (a, b) are displayed.
Neither in liver cytosol (c, d), nor in micro-
somes (e, f), full BuChE inhibition was at-
tained. Values are mean ꢀ SEM from 6 sepa-
rate experiments
0,5
0
0
5
10
0
5
10
c (BCH8) mmol/l
c (BCH2) mmol/l
maining activity was observed in liver (Fig. 3a, c). The
inhibitory pattern of BCH2 and BCH8 in liver micro-
somes turned out to be reverse to that in liver cytosol
(Fig. 3e, f).
BuChE and the participating enzymes, but individual dif-
ferences of subcellular fractions on inter-tissue level in the
hydrolyzing process using different substrates.
Two fractions of rabbit lung and liver were chosen for eluci-
dation of enzymes responsible for the hydrolysis of ben-
zoylcholines. In previous studies, BuChE was confirmed to
be the enzyme responsible for benzoylcholines hydrolysis,
whereas there was no evidence about an another enzyme,
cooperating with BuChE (Paulikova´ et al. 2006).
When S-butyrylthiocholine was used as substrate, BuChE
activity was in liver microsomal fraction significantly ele-
vated compared to cytosol. No activity changes were re-
corded between microsomes and cytosol in the lung. How-
ever, BuChE activity in the liver was severalfold higher
than in the lung. The CE activity in the liver also ex-
ceeded the CE activity in the lung. But whereas in the
lung the CE microsomal activity reached more than dou-
ble the values of those in cytosol, in liver the activity of
CE was approximately equal (Table). Interestingly, in the
3. Discussion
Despite at first sight the hydrolyzing process is metaboli-
cally simple and not energetically challenging, in reality it
is a complex of various factors and reactions which relate
to each other. On one hand there are substrates, their
structure and concentration, on the other, there are en-
zymes and their izoenzymes, as well as individual condi-
tions of this reactions. Due to this it is inevitable to inves-
tigate the metabolic processes on subcellular, inter-tissue
and inter-species levels, since izoenzymes are likely to oc-
cur. In addition, the hydrolyzing reaction is often cata-
lysed by more then one typical enzyme. From this reason
the aim of this paper was not only a general study of
400
Pharmazie 64 (2009) 6

ORIGINAL ARTICLES
4.3. Preparation of subcellular fractions
rat lung CE activity was mainly found in the cytosolic
fraction (Gaustad et al. 1991).
After decapitation, lung and liver were immediately perfunded, rinsed in
saline and stored at ꢂ20 ꢃC until analysis. Tissues were homogenized in
a medium consisting of: 0.25 mol/l saccharose solution, adjusted by
0.15 mmol/l Tris-HCl buffer to pH 7.4. Liver and lung subcellular fractions
(microsomes and cytosol) were obtained by differential centrifugation of
10–20% crude organ homogenate according to Cinti (1972). Protein con-
tent in fractions was determined by the Lowry method (Lowry et al. 1951).
For the kinetic study benzoylcholines were used due to
previous BuChE activity studies. A reverse tendency of
benzoylcholines hydrolyzing activity, depending on the al-
kyl side chain, was observed in cytosol and microsomal
fractions only in the lung. The most appropriate substrates
for hydrolyzing enzymes in lung cytosol are long-lenght
substrates in opposite to microsomes, where the conveni-
ent substrates appear to be the middle-lenght benzoylcho-
lines. In both subcellular fractions of rabbit liver middle-
lenght benzoylcholines show the highest hydrolyzing ac-
tivity. Within interspecies distinctions in rat microsomes
the hydrolyzing velocity reaches its maximal values in
short-lenght alkyl side chain substrates, which gradually
decreases towards middle-lenght chain substrates. Exactly
the opposite course of hydrolyzing activity was observed
in rabbit liver, where hydrolyzing activity increased from
short to middle-lenght side chain benzoylcholines (Olas-
zova´ et al. 1998, Paulikova´ et al. 2006). Although the lung
microsomal BuChE activity copies that in the liver, the
total hydrolyzing velocity is significantly lower.
Except BuChE carboxylesterase could be the enyzme,
playing a role in the process of benzoylcholine hydrolysis,
but never before participation of CE was considered. In
view of this, BuChE inhibitory kinetic studies were car-
ried out using eserine. Deliberately two substrates with
different affinity to BuChE were selected to elucidate the
participation of mentioned hydrolyzing enzymes in rabbit
lung and liver on subcellular level.
Exclusive responsibility of BuChE for studied benzoylcho-
lines hydrolysis was affirmed in the lung microsomes and
in lung cytosol explicitly for BCH2, while the middle-
length alkyl side chain substrate BCH8 required a coop-
erating CE. This is probably caused by the bulk of the
alcoholic part of benzoylcholines, that suit rather carboxyl-
esterase than BuChE. Similar results were observed in the
rat lung. In contrast to rabbit microsomes small portion of
CE activity, however without benzoylcholine specifity,
was recorded in rat lung microsomes (unpublished re-
sults). In cytosol and microsomes of rabbit liver the
BuChE activity in hydrolyzing process was supported by
CA in both the benzoylcholines. Participation of BuChE
activity decreased with elongation of the alkyl side chain
on ammonia atom. Similar results were observed in rat
liver microsomes (Paulikova´ et al. 2004).
4.4. Enzyme activity
4.4.1. Determination of BuChE activity according to Ellman
The BuChE activity was determined by a modified Ellman method (Ell-
man et al. 1961). The activity of the sister enzyme acetylcholinesterase
(3.1.1.7, AChE) was inhibited by 20 min preincubation of the tissue homo-
genate with a specific AChE inhibitor (bw284c51, 0,02 mmol/l) at 37 ꢃC.
After the following 30 min incubation of the homogenate in 0,1 mol/l
phosphate buffer, pH 7,4 with 0,5 mmol/l 5,50-dithiobis(2-nitrobenzoic)
acid at 37 ꢃC, the substrate – 1mmol/l butyrylthiocholine was added. The
absorbance was read at 436 nm for 5 min in 60 s intervals. The exclusive
participation of BuChE and AChE in butyrylthiocholine brakedown was
prooved by 5 min preincubation of the tissue homogenate with common
AChE and BuChE inhibitor – eserine (0.1 mmol/l).
4.4.2. Determination of CE activity
The carboxylesterase activity was determined with the substrate p-nitrophe-
nylacetate (Clement and Erhardt 2000). The absorbance of p-nitrophenol
was measured at 400 nm and room temperature. The 1.5 ml sample cuvette
contained 50 ml of sample, 0.3 ml of 0.55 mmol/l p-nitrophenylacetate in
methanol and 1.15 ml of 100 mmol/l potassium phosphate buffer, pH 7.4.
4.4.3. Kinetic study
4.4.3.1. Substrates
The own kinetic study of BuChE in cytosolic and microsomal fractions of
rabbit lung and liver was carried out with substrates of benzoylcholine
type. These compounds are N-alkylderivates of benzoylcholines, with fol-
lowing general formula: N-(2-benzoyloxyetyl)alkyldimetylammonium bro-
mides, where alkyl is 1–14 carbons long.
4.4.3.2. Incubation
The enzyme reaction was performed under aerobic conditions, at 37 ꢃC for
15 min (in inhibitory studies the enzyme was preincubated for 5 min with
eserine in final concentration 0.1 mmol/l). Incubation mixtures contained in
a total volume of 4 ml 0.15 mol/l potassium phosphate buffer, pH 7.4 with
0.57 mmol/l MgCl2, substrate in different concentration ranging according
to experiment and microsomal or cytosolic fractions. The reaction was in-
itiated by addition of the substrate to the mixture and finished by lowering
the pH value to 2.5 with 1 mol/l HCl.
4.4.3.3. Extraction and HPLC analysis
After the incubation, p-iodobenzoic acid in CH3OH was added to all incuba-
tion mixtures as internal standard. Incubation mixtures were extracted 3 times
into CHCl3, using 10 ml. Extracts were combined, filtered and vacuum dried.
Dried samples were re-dissolved in CH3OH and analyzed by the means of
HPLC (Helia et al. 1995). Enzyme activity was expressed in nmol of benzoic
acid formed per minute, calculated per 1 mg of protein. Obtained results were
subjected to a statistical analysis using the Student t-test.
Based on this facts, it can be concluded, that the majority
of the specific changes of benzoylcholine hydrolysis have
been limited to the microsomal fractions. No too signifi-
cant differences brought the cytosolic fraction on inter-
species and inter-tissue level. Both of enzymes BuChE
and CE cooperated with each other, whereas the portion
of CE elevated when the alkyl part of benzoylcholines
was growing.
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