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9
The Kinetics
of Acetylcholinesterase Inhibition and the Influence
of Fluoride and Fluoride Complexes on the
Permeability of Erythrocyte Membranes - Page 3.
4.
Carrying out the Measurements
We ran the
reactions in small 10ml measuring flasks, to which
we added 5ml of the respective buffer solution ahead
of time. Next came 0.5ml of the appropriate enzyme
solution. We used a total of three different
assays:
-
A
purified enzyme preparation from bovine
erythrocytes (produced by Serva Co of Heidelberg),
of which we dissolved 25mg ~ 50EU in 50ml of
buffer solution (either citrate-phosphate buffer
of pH 7.4, or Veronal-HCl buffer of pH 8.6).
-
A
suspension of human erythrocytes in Ringer's
solution, which was used directly
without additional treatment.
-
Human
serum, which, after dilution with an equal amount
of Ringer's solution, was
also implemented without
further treatment.
Next we
added 0.5ml of the appropriate inhibitor solution
(in the absence of inhibitor, 0.5ml of buffer), and
lastly, after setting the temperature of these
assays to 370C, 0.5ml of the
radioactively labeled ACh solution, whereby we
simultaneously started the stop watch.
When the
reaction period was completed, which in most cases
took an hour, but when determining the dependence of
the reaction rate on substrate concentration only
five minutes, we stopped the reaction by adding 2ml
of 0.1M sodium tetraphenylborate solution. After a
waiting period of one hour, which allowed for
completion of precipitate formation, we filled the
small flasks to the mark with twice distilled water.
We
transferred 1.2ml of solution into a plastic
centrifugation vessel and after centrifugation
withdrew exactly 1ml of the supernatant, which was
then measured directly in Bray's solution. The
measured impulse rate yielded a value that was
proportional to the amount of acetic acid released
and thereby to the quantity of saponified ACh. We
expressed the rate of reaction in impulses/reaction
time, or, after dividing by the specific activity of
the substrate stock solution, as µMol of released
acetic acid/reaction time.
Each series
of measurements consisted of:
-
a value to
determine the self-saponification of the ACh,
which was determined by
omitting the enzyme assay.
-
a value to
determine vo, and therefore without
addition of inhibitor, and
-
the values
with addition of inhibitor, which served to
determine the inhibition of the enzyme.
The measured
values were constantly corrected by subtracting the
self-saponification rate. The following equation
gives another overview of the derivation of the
measured values.
(equation 22)
where:
v = Rate
of reaction (in µMol acetic acid/minute)
Ib
= Total impulse rate (in impulses/minute)
IE
= impulse rate of the self-saponification (in
impulses/minute)
IU
= impulse rate of the natural radioactivity (in
impulses/minute)
A =
specific activity of the ACh stock solution (in
impulses/minute and µMol)
t =
reaction time (in minutes)
For the
measurement of the pH dependence of the inhibition,
the value of vo as well as the self-saponification
rate had to be determined separately for each pH,
since both values are pH dependent. For the
measurements that served to determine the reaction
rate as a function of substrate concentration we
used a different stock solution for each substrate
concentration, whereby the radioactivity per unit of
volume remained constant while the ACh concentration
changed. In addition, in this case we added an
excess of non-radioactively labeled ACh after the
reaction was completed, to avoid falling short of
the solubility product of the ACh-sodium
tetraphenylborate compound.
a.
AChE Inhibition by NaF
The
following measurements where to be carried out in
the concentration range equivalent to substrate
saturation, within which the reaction rate is
independent of the ACh-concentration. In order to
determine this region we carried out a measurement
in which we examined the reaction rate as a function
of the ACh concentration. The course of the
recordings is recreated in figure 7.
Figure 7
- Dependence of ACh Hydrolysis on ACh Concentration.
Phosphate-citrate buffer (following Mc.Ilvaine); pH
= 7.7 ; T = 370C ; purified AChE from
bovine erythrocytes with concentration: 0.0343mg/ml
Saturation
was reached at about 2 x 10-3M ACh. A
further increase in ACh concentration leads to a
slight reduction in the reaction rate. This
observation suggests that there is an inhibition
occurring due to excess substrate, which cannot,
however, be determined with certainty from this
measurement. Since the reaction rate barely changes,
even with significantly greater ACh concentrations,
we could still carry out the measurement at 1.4 x 10-2M
ACh. A measurement of the reaction rate as a
function of time showed that the reaction rate
remains constant over an hour. Applied to equation 2
this means that in V = k+2 • [ES],
becomes independent of [S] (reaction of zeroth
order). All of the enzyme is therefore present as
ES. Figure 8 shows a plot of the reaction rate vs.
time. 25 µMol (=5 x 10-4M) of the AChE
specific inhibitor Physostigmine were added
after 30 minutes. The curve bends off, but continues
to run linearly. The inhibition is 71.5%. The
y-intercept (at t=0) represents the share of C-14
acetic acid in the stock solution.
Figure 8
- Quantity of 14C Acetic Acid Released as
a Function of Time.
Veronal/HCl
buffer; pH=8.6; 370C; ACh concentration
1.43 x 10-2M. Enzyme as in fig 7.
Next we
studied the inhibition of two enzyme assays as a
function of the NaF concentration at constant pH.
Human blood was extracted from a slightly blocked
arm vein, and coagulation was prevented by adding
10% isotonic citrate solution. We separated
erythrocytes and serum by centrifugation and washed
the blood cells three times with physiological NaCl
solution (0.9%), after which the cells were
suspended in an equal amount of "Ringer's solution"
(Preparation 1). The serum was diluted with an equal
amount of Ringer's solution and centrifuged away
from the precipitated fibrin (Preparation 2). - The
fibrin precipitates because the Ringer's solution
contains Ca2+ which, due to the citrate
present is no longer sufficiently complexed.– The
Ringer's solution used in the subsequent reactions
consisted of the following:
9.0 g NaCl
0.42 g KCl
0.5 g NaHCO3
0.5 g Glucose
0.24 g CaCl2
0.025 g MgCl2
--------------------------------------------------
double-distilled water –
1,000ml
This
solution, whose pH value was 7.4 and whose buffering
capacity was relatively limited, was used to offset
hemolysis of the erythrocytes, and to create the
most natural conditions possible. We proceeded as
was described in detail at the beginning of III,A,4.
Figure 9 shows the plot of the inhibitory percentage
as a function of NaF concentration.
Figure 9
– NaF Inhibition of AChE From Human Erythrocytes and
PChE From Human Serum
ACh
concentration 7.15 x 10-3M, Ringer's
solution, T=370C.
The serum-cholinesterases
are visibly more inhibited by the NaF than the AChE
from the erythrocytes. The non-monotonic course of
PChE inhibition at lower concentrations is probably
the result of differing affinities of individual
enzymes in the PChE mixture for the inhibitor.
According to equations 16,17 and 18, independent of
the type of inhibition, a straight line should arise
when vo/v-1 is plotted against the
inhibitor concentration, assuming that the number of
binding sites on the enzyme for the inhibitor is the
same as for the substrate. If several enzymes are
simultaneously involved in the reaction, a linear
dependence only develops when the affinities
(reciprocal inhibitor constants) of the
individual components for the inhibitor are equally
large, which is rather unlikely given the number of
PChEs. Figure 10 shows such a plot for the two
curves from figure 9.
Figure 10.
Dependence of (vo/v) –1 on NaF
Concentration
The course of the curves can, in
the case of the serum preparation, be approximated
by two straight lines with different slopes. This
suggests that the reaction rate is considerably
limited by just two components of the enzyme
mixture, which have different affinities for the
fluoride. The AChE of the erythrocytes yields a
linear course, which suggests that the controlled
variables of equations 16-18 are fulfilled here.
Next we determined the form of
the inhibition from a plot in accordance with
Lineweaver and Burk. Purified AChE from bovine
erythrocytes (obtained from the company Serva in
Heidelberg) again served as our enzyme specimen.
Figure 11
- Lineweaver-Burk Diagram of the Inhibition of AChE
by NaF.
Curve 1: uninhibited reaction
Curve 2: 1.43 x 10-2M
NaF measured in phosphate-citrate buffer, pH 7.7
The inhibition is competitive and
the Michaelis constant of the uninhibited reaction
is: KM = 4.2 x 10-4Mol/l. From
equation 6 one calculates the inhibitor constant to
be: KI = 6.26 x 10-3Mol/l. The
affinity of the substrate for the enzyme is
therefore, in this case, 15 times as great as that
of the inhibitor. Using equation 16, the inhibitor
constant can also be calculated from the slopes of
the lines in figure 10. The following applies:
(equation 23)
The letter “n” stands for the
slope of the lines and is graphically derived from
figure 10. We took the value for KM from
the analysis of figure 11 (4.2 x 10-4M),
and the substrate concentration had a value of 7.15
x 10-3M. By substituting these values
into equation 23 we obtain, taking the value of the
slope (n=1.08 x 102) into account, the
inhibitor constant for the AChE of the erythrocytes:
KI = 5.6 x 10-4Mol/l. This
value, however, means that the dissociation
constants of the enzyme/substrate complex (KM)
and of the enzyme/inhibitor complex (KI)
are roughly the same. A comparison with the constant
(KI = 6.26 x 10-3M) derived
from figure 11 shows that upon shifting to
physiological conditions the enzyme is more strongly
inhibited by the fluoride. Since a KM
value for the PChE of the serum is not available to
us, we can not analogously analyze the serum curve
which, due to its non-monotonic course, seems of
little purpose anyway. An inhibition of the AChE of
the erythrocytes begins at fluoride concentrations >
5 x 10-4M ~ 9.5 mg/l. The serum-cholinesterases
are already inhibited at concentrations > 7 x 10-5M
~1.3mg/l. These effects are not yet sufficient to
lead to an explanation of a vagotonic effect, as is
shown by inhibition of caries at physiological
fluoride concentrations.
Introduction |
Contents |
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3 |
4 |
5 |
6 |
7 |
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9 |
