Characterization of the Inhibitory Properties of the Monomeric and Tetrameric Forms of
Recombinant Bovine Acetylcholinesterase
Beth M. Uccellini
Comprehensive Paper
April 1, 2003
2
Abstract
Acetylcholinesterase (AChE), an essential component of cholinergic synapses,
catalyzes the hydrolysis of acetylcholine at the post-synaptic junctions, and exists in a
variety of forms in mammalian tissue. The ratio of these forms changes between healthy
individuals and those suffering from diseases such as Alzheimer’s disease (AD).
Recombinant monomeric and tetrameric forms of bovine AChE were inhibited by
edrophonium and propidium, and the former, current, and potential AD drugs, tacrine,
E2020, and (-) huperzine A, respectively. The overall range of inhibition constants was
0.00052 ± 0.00022 to 0.5862 ± 0.1720μM for the recombinant monomer and 0.00045 ±
0.0001424 to 0.5200 ± 0.0493μM for the recombinant tetramer, which are approximately
five-times smaller than the native forms.
Monoclonal anti-bodies, 25B1, 4E5, 5E8, and 6H9 that were raised against FBS
AChE were also tested as inhibitors against the recombinant forms of AChE. With 25B1,
both of the recombinant forms appeared to display higher binding affinity and more
conformational change than the native FBS AChE.
The native and recombinant
tetramers were affected in the same manner by 4E5 while the monomer was less affected
than either of the two other forms. The native tetramer appears to bind more tightly to
5E8 and 6H9 than either the recombinant monomer or tetramer.
3
Introduction
Acetylcholinesterase (AChE), a serine hydrolase, is an essential enzyme present
in the cholinergic synapses, which catalyzes the hydrolysis of the neurotransmitter,
acetylcholine (ACh). These neurons are found in the basal ganglia, the preganglionic
neurons of the autonomic nervous system, the postganglionic neurons of the
parasympathetic nervous system, and the postganglionic neurons of the sympathetic
system.1 Without AChE, ACh would build up in the postsynaptic junctions and
continuously stimulate motor neurons, which would eventually lead to paralysis. The
most serious instance of this type of paralysis comes from inhibition of enzyme upon
exposure to organophosphate chemical warfare agents.
The first X-ray crystal structure that was solved was that of AChE from Torpedo
californica.2 It showed that each subunit of the enzyme consists of 14 helices surrounded
by a 12-stranded central mixed pleated sheet in a volume of approximately 45Å by 60Å
by 65Å in an ellipsoidal shape. While AChE, like other serine hydrolases, contains a His
and a Ser residue (440 and 200 respectively) in its catalytic triad, it differs from the
others because it contains a Glu (327) instead of the normal Asp residue. The active site
of AChE is composed of several subsites, the catalytic triad, the oxyanion hole, the acyl
binding site, the choline binding site, the aromatic ring, and the external site.2 This active
site lies at the base of an approximately 20Å deep gorge that is lined with aromatic amino
acid side chains. In addition to the active site, AChE also contains one or more
peripheral binding sites where ACh or similar molecules may bind. Binding at a
peripheral site may cause conformational changes within the gorge and active site. AChE
exists in multiple molecular forms in most vertebrate tissues and predominantly in two
4
forms within mammalian brains.3 These forms are the tetrameric membrane-bound form,
a G4 globular protein, and the soluble monomeric form, a G1 protein. AChE monomers
can come together to form dimers and the tetrameric form is a dimer of dimers. AChE
may also be found but less commonly so in asymmetric forms. Figure 1 shows the
tetrameric and monomeric forms of recombinant mouse AChE.
Figure 1: (Left) Half space filled half ribbon diagram of mouse AChE tetramer showing the dimer
of dimers.4 (Right) Space filled model of mouse AChE monomer with the active site shown in yellow, the
C-terminus in red, the N-terminus in blue and peripheral binding sites in aqua. 5
AChE is one of the fastest hydrolytic enzymes with a specific activity (1.6x108 s1
M-1at 25º, pH 7.0) that comes close to the diffusion-controlled rate of encounter (108-109
s-1M-1).6 AChE catalyzes the hydrolysis of ACh into acetate and choline at the postsynaptic junctions of motor neurons. This reaction occurs through a double nucleophilic
displacement as shown in Scheme 1.7
5
The interaction of AChE with a substrate such as ACh can be described by the
general interaction of an enzyme (E) with a substrate (S) respectively. In this mechanism
S can bind to two distinct sites on the E forming two complexes, ES and SE.8 Scheme 2
outlines this mechanism.
K
E + S ⇄ ES
+
+
S
S
Kss ⇅
k2
k3
→ ROH + EA E + P
Kss⇅
SE + S ⇄ SES → SE + P
Scheme 2
In this scheme, k2 is the rate of acylation of the enzyme and k3 is the rate of deacylation
of the enzyme. Kss represents the binding of another S molecule to the ES complex, and
when the E is being inhibited, Kss describes the concentration of S where inhibition first
occurs. kcat represents the turnover rate of catalysis i.e. the number of S molecules that
are transformed into product (P) catalyzed by the E molecule when the E is completely
saturated with S molecules, and is equal to (k2*k3)/(k2+k3) derived from the steady state
6
approximation. Two other constants, Vmax and Km, are used to describe interactions of E
with S. Vmax which is represented by the equation kcat*[E] is the maximum rate of
catalysis at a specific E concentration, and Km, the Michaelis-Menten constant, which is
approximately equal to Kss+K, is the concentration of substrate at which the rate is equal
to one-half Vmax.
The inhibition of AChE is an important area of study because of the critical role
AChE plays within the body. The inhibition of an E molecule occurs when a molecule
(an inhibitor (I)) binds to the E that prevents or hinders the E from performing its
catalytic function. There are two main classes of Is, irreversible and reversible. An
irreversible I is one that dissociates very slowly from its target E. Reversible Is on the
other hand are distinguished by their rapid dissociation from the E-I complex. Within the
class of reversible Is are two subsets, competitive and noncompetitive. A competitive I
competes with the S for binding at the E’s active site while a noncompetitive I binds to
another site such as the peripheral binding site on the E. In the case of noncompetitive
inhibition, the binding of I to the peripheral site alters the conformation of the active site
thus preventing proper binding of S to E. Figure 2 shows a cartoon diagram of the
differences in the binding of S, competitive I and noncompetitive I.
7
Figure 2: Distinctions between a competitive I and a noncompetitive I. Top figure is the ES complex, the
middle figure the competitive I binding at the active site preventing S binding, the bottom the
noncompetitive I which alters E active site conformation. 9
In this study, both competitive and noncompetitive Is were used. Two inhibition
constants are used to describe the effects of these types of Is with an E. KI, the
competitive inhibition constant, represents the interaction of I with free E. αKI, the
noncompetitive inhibition constant, represents the interaction of I with the ES complex.
Scheme 3 displays the interaction of competitive and noncompetitive Is with E and ES
and shows where KI and αKI come into being.
kcat
E + S ⇄ ES → E + P
+
+
I
I
KI ⇅
αKI⇅
EI + S ⇄ ESI
Scheme 3
Five inhibitors, four monoclonal anti-bodies (MAbs), and fasciculin (FAS), a
three-fingered snake toxin, were used in this study. The inhibitors were edrophonium
8
(EDR), propidium (PROP), tacrine (TAC), E2020, and (-) huperzine A (-HupA). EDR is
a classical competitive I of AChE, and PROP is a classical noncompetitive I. TAC,
E2020, and –HupA are the former, current and potential AD treatment inhibitors,
respectively. Figure 3 displays the structures of these Is.
Figure 3: Structures of Cholinesterase Inhibitors used in this study
The MAbs used in this study were 25B1, 4E5, 6H9, and 5E8. In a 1998 study these same
MAbs were used to aid in mapping the topographical surface of the AChE and to help
discover other regions that might be involved in its catalytic functioning.10 The
production and purification of the MAbs used in this study was described by M. K.
Gentry et al.11 MAbs function as inhibitors of AChE through many possible mechanisms
9
such as, “steric occlusion of substrate entry in to the active site gorge and/or an allosteric
effect on the active site influencing substrate catalysis.”10 FAS is a snake toxin found in
the venom of the members of the mamba family, and it inhibits AChE in a similar
manner to the MAbs. MAbs and FAS partly bind at the peripheral anionic binding site
located at the rim of the active site gorge. The most advantageous feature of these
molecules is that their inhibition of AChE is reversible.
The inhibition of AChE can either be beneficial or harmful to an organism. For
example, the use of AChE Is as treatments for neurological illnesses like Alzheimer’s
Disease (AD) is an advantageous use of inhibition. However, AChE Is are used in many
chemical warfare agents and pesticides. Here the purpose of the inhibition is to harm the
organism that comes into contact with the I. However, there is potential for the use of
reversible AChE Is (like MAbs and FAS) to provide temporary protection against the
irreversible Is found in toxic compounds such as those used in nerve gas.
AD is a debilitating illness striking senior citizens across the world. According to
the cholinergic hypothesis, memory impairments in patients with this senile dementia
disease are due to a selective and irreversible deficiency in the cholinergic functions in
the brain12. There is a selective loss of neurons containing choline acetyltransferase, the
enzyme responsible for the synthesis of ACh, resulting in decreased levels of ACh in the
cortical tissue. In a recent study, Winkler et al., demonstrated that the presence of
cerebral ACh is necessary for cognitive behavior and it can improve learning deficits and
memory loss in rats that have incurred severe damage to the nucleus basalis of Meynert.13
According to this theory then an effective treatment for AD would be to increase the
amount of ACh in the brain by giving patients AChE inhibitors.14 By inhibiting AChE
10
the decomposition of ACh by AChE is decreased, thus increasing the amount of
functioning ACh in the brain. Studies have shown that aging and AD cause a decrease in
the G4 form of AChE in the brain.15 The ratio of the monomeric form to the tetrameric
form in an AD patient differs from that of a healthy adult. Since different forms of AChE
can have different physiological functions it is important to understand their individual
inhibitory properties in regards to clinical drug development.
The purpose of this study was to characterize the monomeric and tetrameric forms
of recombinant bovine brain AChE in terms of their binding to Is, MAbs, and FAS and
also to compare their binding properties to those of the naturally occurring monomeric
and tetrameric forms of fetal bovine serum AChE. The recombinant forms of AChE
analyzed in this study were obtained from a full-length cDNA clone of 1,854 base pairs
for the mature tetrameric subunit of AChE from bovine brain. This clone was truncated
at the C-terminus to obtain a 1,745 base pair cDNA clone for the monomeric form of the
enzyme. Both of these forms were expressed in CHO-K1 cells to produce the
recombinant monomeric and tetrameric forms of AChE.
Results
The inhibition of AChE by EDR, PROP, TAC, -HupA, and E2020 was measured
via a kinetic (initial rates) method. The results were analyzed using primary and
secondary data plots. The primary data plot shows Velocity (V) vs. [acetylthiocholine
iodide (ATC)] curves for a series of [I]. Best-fit lines were plotted using an allosteric two
site design with the equation, V0 = Vmax/(1+[S]/Kss+Km/[S]), where Vmax stands for the
maximal rate of catalysis, [S] symbolizes [ATC], and V0 is the initial velocity of
catalysis. An example of this plot is shown in Figure 4.
11
V(mAbs/min)
Inhibition of Recombinant
AChE Monomer by
TacrineTrial Four
50
45
40
35
30
25
20
15
10
5
0
0.01
control
0.068 M Tacrine
0.136 M Tacrine
0.272 M Tacrine
0.4088 M Tacrine
0.5451M Tacrine
0.6813 M Tacrine
0.1
1
10
100
[ATC], mM
Figure 4: An example of a primary data plot of the inhibition of recombinant AChE monomer by TAC at
pH 8.0 and temperature 25˚.
Figure 5 displays examples of the two types of secondary data plots. The first is a plot of
Vmaxapp/Kmapp vs. [I], and this is used to determine the value of KI. Here Vmaxapp/Kmapp
represents the rate constant for the interaction of S and E in the presence of I. The second
is a plot of Vmaxapp vs. [I], and this is used to determine KI. The following equation was
used to determine these values: Vmaxapp or Vmaxapp/Kmapp = {(Vmax/Km)*KI}/{KI+[I]},
where [I] is representative of the concentration of inhibitor.
12
Vmax/Km vs. [Tacrine]
(Used to Determine KI)
Vmax vs. [Tacrine]
(Used to Determine KI)
50
40
300
Vmax
Vmax/Km
400
200
30
20
100
10
0
0
0
1
2
3
4
0
[Tacrine], M
1
2
3
Tacrine],M
Figure 5: (Left) an example of a secondary data plot of V maxapp/Km vs. [TAC], used to determine K I.
(Right) an example of secondary data plot of V maxapp vs. [TAC], used to determine KI. Both plots were
generated from data taken at pH 8.0 and 25C.
The KI and KI values (the inhibition constants) for the recombinant monomer and
tetramer as inhibited by the five inhibitors are shown in Table 1. Table 2 contains the
inhibition constants for the native monomer and tetramer.
Table 1: Inhibition constants for recombinant AChE monomer and tetramer at pH 8.0 and 25C. *αKI
values were used because PROP is a noncompetitive I.*
Inhibitor
Rec. Monomer
Rec. Tetramer
KI ± M (M)
KI ± M (M)
EDR
0.26±0.01
0.52±0.04
TAC
0.01±0.002
0.020±0.004
-HupA
0.0005±0.0002
0.0005 ± 0.00008
E2020
0.001±0.0002
0.001±0.0001
*PROP
0.586±0.2
0.39±0.1
4
13
Table 2: Inhibition constants for native FBS AChE monomer and tetramer. **These values were
determined using the Steady-State Method.**16
Native Monomer
Native Tetramer
KI ± M (M)
KI ± M (M)
EDR
0.48 ± 0.04
0.46 ± 0.03
TAC
0.04 ± 0.007
0.11 ± 0.02
**-HupA
0.008 ± 0.001
0.007 ± 0.3
E2020
0.007 ± 0.001
0.009 ± 0.001
*PROP
1.45 ± 0.01
1.4 ± 0.1
Inhibitor
The inhibition of AChE by MAbs, 25B1, 4E5, 6H9, and 5E8, and by FAS was
measured using a steady state method. The recombinant monomer and tetramer as well
as the native FBS tetramer were used to make the complexes, and residual AChE activity
was measured and plotted. Data plots were made of % AChE Activity vs. [MAb or
FAS]. These graphs were used for qualitative analysis. A quantitative anaysis of this
data could be obtained using the following equation under the specified constraints: viβ =
vi{1 + β[FAS] / (KI + vi / k)}, where [FAS] = 0, and where vi = k{([E] - KI - [FAS]) +
((KI + [FAS] + [E])2 - 4[FAS][E])1/2}/2 under the constraints that Km<<[S]<<Kss and
[FAS]≈[E]. In this system of equations [FAS] can be replaced with the concentration of
any of the MAbs, KI is the reversible inhibition constant of E by either FAS or a MAb,
and β is the fraction of residual activity. β is a measurement of how the conformational
changes induced by the binding of MAbs or FAS at the peripheral site affects AChE
activity. The quantitative analysis was not performed on this data because there were too
few data points for the computer to analyze using this complex equation. The plots of
%AChE activity vs. [MAb] or [FAS] can be seen in figures 6 through 10 for 25B1, 4E5,
5E8, 6H9, and FAS respectively. Inhibition with 25B1 shows that both of the
recombinant forms appeared to display higher binding affinity and induced more
14
conformational change compared to native FBS AChE. The binding of 4E5 to native and
recombinant tetramers was similar, whereas its binding to the monomer was less effective
than the tetrameric forms. The native tetramer appears to bind more tightly to 5E8 and
6H9 than either the recombinant monomer or tetramer. Inhibition studies with FAS
showed that there was no difference in binding between the recombinant forms and the
native tetramer.
Activity vs. [25B1]
% AChE Activity
150
FBS AChE Abs.
Tetramer Abs.
Monomer Abs.
100
50
0
0.0
0.5
1.0
1.5
2.0
[25B1], nM
Figure 6: Inhibition plot of %AChE Activity vs. [25B1] at pH 8.0,in 5mM sodium phosphate buffer with
0.01%BSA at 25˚C.
15
Activity vs. [4E5]
% AChE Activity
150
FBS AChE Abs.
Tetramer Abs.
Monomer Abs.
100
50
0
0.0
0.5
1.0
1.5
2.0
[4E5], nM
Figure 7: Inhibition plot of %AChE Activity vs. [4E5] at pH 8.0,in 5mM sodium phosphate buffer with
0.01%BSA at 25˚C.
Activity vs. [5E8]
% AChE Activity
150
100
FBS AChE Abs.
Tetramer Abs.
Monomer Abs.
50
0
0.0
0.5
1.0
1.5
2.0
2.5
[5E8], nM
Figure 8: Inhibition plot of %AChE Activity vs. [5E8] at pH 8.0,in 5mM sodium phosphate buffer with
0.01%BSA at 25˚C.
16
Activity vs. [6H9]
% AChE Activity
150
Abs
Tetramer Abs.
Monomer Abs.
100
50
0
0
10
20
30
40
50
60
[6H9], nM
Figure 9: Inhibition plot of %AChE Activity vs. [6H9] at pH 8.0,in 5mM sodium phosphate buffer with
0.01%BSA at 25˚C.
Activity vs. [FAS]
%AChE Activity
150
FBS AChE
Rec. Monomer
Rec. Tetramer
100
50
0
0
5
10
15
20
25
[FAS], nM
Figure 10: Inhibition plot of %AChE Activity vs. [FAS] at pH 8.0,in 5mM sodium phosphate buffer with
0.01%BSA at 25˚C.
Discussion
Inhibitors. The results from the inhibition studies with EDR, PROP, TAC,
E2020, and –HupA show that the inhibitors were all more potent towards both of the
recombinant forms than towards the two native forms of AChE. However, there was no
17
more than a five-fold difference in KI values between any recombinant form and its
corresponding native form for any inhibitor. It is typical to see up to a five-fold
difference in KI values when AChE from different species of eukaryotes are compared.
This would be the difference between FBS AChE and mouse AChE or monomeric mouse
AChE compared to tetrameric mouse AChE. Because of this, there is no reason to
believe that the recombinants are any different from the native forms in regards to their
enzymatic inhibition properties.
Neurological function degeneracy is among the worst symptoms of AD. In terms
of AD treatment, these results show that –HupA and E2020 are equally effective against
both monomeric and tetrameric forms of AChE. The use of cholinesterase inhibitors as a
symptomatic treatment of AD remains a promising option in a world where there is no
cure for it. By binding to AChE, these inhibitors allow ACh levels to increase in the
synapsis, which allows for better nerve impulse transmission and enhances memory in
these patients. While the use of AChE inhibitors is not a cure, it is an effective short-term
symptomatic treatment.
MAbs and FAS. The inhibition studies with the MAbs and FAS demonstrate
differences between the two recombinant forms of the enzyme as well as between the
native tetramer and the recombinant tetramer. In the case of 25B1 both of the
recombinant forms of AChE appear to display higher binding affinity and induced more
conformational change compared to the native tetramer. The steeper downward slope of
the recombinant forms compared to the native tetramer. It would be expected that β for
the recombinant enzymes against 25B1 would be greater than for the native tetramer
based on the activity plot. The binding of 4E5 to the native and recombinant tetramers
18
was similar (i.e. the best-fit lines for those two forms of the enzyme nearly overlap),
whereas the binding of 4E5 to the recombinant monomer was less effective than either
tetrameric form. It would seem from these plots that the β values for the tetrameric forms
would be larger than the recombinant monomer because its slope was less negative. 4E5
appears to be more potent towards the tetrameric form and to induce more
conformational change in them. The native tetramer appears to bind more tightly to 5E8
and 6H9 than either of the recombinant forms. In both of these cases, the slope of the
native tetramer lines was much steeper than the slope of the recombinant lines. β values
for the native tetramer are anticipated to be larger than the β values for the recombinant
forms. The binding of both of the recombinant forms and the native tetramer and FAS
were identical. This implies that there is not a significant difference in how FAS binding
alters the catalytic activity given the differences between the recombinant monomer and
tetramer, and between the native and recombinant tetramer. β here is expected to be very
close for all three forms. Prior studies of these MAbs and FAS with the native FBS
AChE monomer and tetramer classify the MAbs into two groups. The first contains
25B1 and 4E5 and is classified by no interference with binding of S to the active site and
the peripheral site. The second group contains 5E8 and 6H9 where binding to AChE
caused interference in the binding of S to both the active and peripheral sites.10 Previous
studies with FAS on the native monomer and tetramer indicate that KI values represent
the binding of FAS to the aromatic residues on the rim of the active site gorge. Also, β is
representative of the allosteric effect of this binding on the active site deep down in the
gorge.10 Without performing a quantitivate analysis on this data very little can be
claimed as definite. However, these types of studies are important for future studies
19
needed to determine the effectiveness of using MAbs or even FAS as a protective
mechanism for AChE when a person is exposed to nerve gas and other chemical warfare
agents. There is great potential in this area of study for uses in the military and in
homeland security.
These studies were part of a larger study on the characterization of the
recombinant monomer and tetramer. Ultimately the goal of this whole project is to
understand how the recombinant enzymes function so that they can be used for in vitro
studies instead of native forms, which have to be isolated from animals/human tissues or
plasma. The recombinant forms provide a less-expensive and more constant source of
enzyme.
Experimental Section
Materials:
The recombinant monomeric and tetrameric forms of bovine brain AChE were
engineered and expressed by Dr. Carolyn Chambers of the Walter Reed Army Institute of
Research (WRAIR). The inhibitors, EDR, PROP, TAC, -HupA, and E2020, the ATC,
5,5’-dithiobis (2-nitrobenzioc acid) (DTNB), and the sodium phosphate were obtained
from the Sigma Chemical Co. (St. Louis, MO). ATC was used as the AChE substrate,
and DTNB was used as the color indicator for the reaction between ATC and AChE.
Sodium phosphate was used as the buffer salt. The MAbs, 25B1, 4E5, 5E8, and 6H9 were
generated on site at WRAIR. FAS was obtained in its lyophilized form from Alomone
Labs (Jerusalem, Israel), and was resuspended in 50mM phosphate buffer, pH 8.0 with
0.05%BSA. All experiments were carried out at WRAIR.
20
Solutions:
All solutions were prepared using deionized (DI) water that was purified and
irradiated by a Milli-Q instrument. The 30mM ATC stock solution was prepared by
dissolving 3.470g ATC into 400mL of DI water. Similarly, the 30mM DTNB solution
was prepared by dissolving 4.7556g DTNB into 400mL of DI water. The 50mM sodium
phosphate buffer was prepared by dissolving 81.9703g of it into 10L of DI water, the pH
was then brought up to 8.0 by the addition of concentrated sodium hydroxide.
Recombinant AChE monomer and tetramer solutions were diluted with the amount of
sodium phosphate buffer (pH 8.0) to yield at 0.1unit/mL concentration.
Inhibition Studies:
Recombinant AChE monomer and tetramer were assayed with each inhibitor
(EDR, PROP, TAC, E202, and –HupA) using the same kinetic procedure. In each of
eight microcentrifuge tubes for each enzyme/inhibitor combination 270µL of enzyme and
a varying concentration of inhibitor were added. ATC solutions were prepared using a
serial dilution starting with a concentration of 30mM and decreasing by halves to
0.15mM. The dilutions were made with 50mM phosphate buffer, pH 8. 250µL of 30mM
DTNB was added to each dilution. Ten µL of the enzyme/inhibitor complexes were
plated out into 24 wells of a 96 well microtiter plate. 290µL of ATC/DTNB reaction
mixture were added to each well. The well plate was then immediately read in the
spectrophotometer plate reader for ten minutes at 412 nm and 24.5°. KI and KI values
were determined using Prism, a graphical analysis program. Non-linear least squares
methods were used in their determination using the equation already discussed. In
21
Appendix 1 an example of the inhibition protocol chart used in this experimental set-up is
displayed.
MAbs and FAS Studies:
Recombinant AChE monomer and tetramer along with the native tetramer were
complexed with the MAbs (25B1, 4E5, 5E8, and 6H9) and with FAS using the same
kinetic experimental set-up. In each of ten 15mL conical tubes a fixed amount of AChE
was added to each tube to allow for an over all concentration of 0.1 U/mL in a total
volume of 10mL. Increasing amounts of the antibody or FAS were added to tubes two
through ten. Tube One contained no antibody to serve as the experimental control.
0.05% BSA: 5mM Phosphate Buffer, pH 8.0 was added to each tube to bring the total
volume up to 10mL. These complexes were incubated overnight at 4˚C. Each complex
was then assayed using 30mM ATC and 30mM DTNB solutions made as described
above on a spectrophotometer. Readings of residual AChE activity were taken at 412nm
every 15sec. for a total of 3min. Plots were then made of %AChE activity vs. [MAbs] or
[FAS]. In Appendix 2 an example of the inhibition protocol chart used in this
experimental set-up is displayed.
22
Appendix 1
An example of Inhibition Protocol used for EDR, PROP, TAC, E2020, and –HupA. Upper block shows EI
complexes with 1 being the control containing E but not I, and 8 being the blank receiving neither E nor I.
Lower block shows serial ATC-DTNB, substrate, dilutions.
23
Appendix 2
An example of MAb and FAS Protocol used for 25B1, 4E5, 5E8, 6H9, and FAS. Upper block shows EMAb complexes with 1 being the control containing E but not MAb or FAS.
1
Massoulie, J., L., Doctor, B.P., Soreq, H., Velan, B., Cygler, M., Rotundo, R., Shafferman, A., Silman, I.,
and Taylor, P. (1992) Multidisciplinary Approached to Cholinesterase Functions (Shafferman, A., and
Velan, B., Eds.) Plenum Press, New York.
2
Sussman, J. L.; Harel, M.; Frolow, F.; Oefner, C.; Goldman, A.; Toker, L.; Silman, I. Science 1991, 253,
872-879.
24
3
Saxena, A.; Hur, R.S.; Doctor, B.P. The Natural Form of Fetal Bovine Serum Acetylcholinesterase is
Truncated at the C-Terminus. Unpublished.
4
(1998) Structure and Function of Cholinesterases and Related Proteins (Doctor, B.P., Taylor, P., Quinn,
D.M., Rotundo, R.L., Gentry, M.K., Eds.) Plenum Press, New York.
5
Bourne, Y.; Taylor, P.; Marchot, P. Cell, 1995, 83, 503-512.
6
Quinn, DM.; Pryor, A.N.; Selwood, T.; Lee, B.H.; Acheson, S.A.; Barlow, P.N. (1991) The Chemical
Mechanism of Acetylcholinesterase Reaction. Biological Catalysis at the Speed Limit in Cholinesterases:
Structure, Function, Mechanism, Genetics and Cell Biology (Massoulie, J., Bacou, F., Barnard, E.,
Chatonnet, A.; Doctor, B.P.; Quinn, M.D., eds.), pp 252-257 Am. Chem. Soc., Washington DC.
7
Enyedy, I.J.; Kovach, I.M.; Brooks, B.R. J. Am. Chem. Soc. 1998, 120, 8043-8050.
8
Radic, Z.; Pickering, N.; Vellom, D.C.; Camp, S.; Taylor, P. Biochem 1993, 32, 12074-12084.
9
Berg, J.M.; Tymoczko, J.L.; Stryer, L. (2002) Biochemistry (5th Ed). W.H. Freeman, New York.
10
Saxena, A., Hur, R., and Doctor, B. P. Biochemistry 1998 37 (1), 145-154.
11
Gentry, M.K.; Moorad, D. R.; Hur, R. S.; Saxena, A.; Ashani, Y.; Doctor, B. P. J. Neurochem. 1995 64,
842-849.
12
Perry, E. K. Br. Med. Bull. 1986 42, 63-69.
13
Winkler, J.; Suhr, S. T.; Gage, F. H.; Thal, L. J.; Fisher, L. J. Nature 1995 375, 484-487.
14
Becker, R. E., and Giacobini, E. Drug Dev. Res. 1988 12, 163-195.
15
Meneguz, A.; Bisso, G.M.; and Michalek, H. Neurochem. Res. 1992, 17, 785-790.
16
Saxena, A.; Hur, R.S.; Doctor, B.P. Subunit Association Affects Peripheral Anionic Site Function in
Acetylcholinesterase. Unpublished.