Vol. 167, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 1383-1392
March 30, 1990
Interaction
Protein-Ligand of Nitrosamines with E-A.
Department
Received
February
Interactions: Nicotinic Acetylcholine
KAPP, S. DAYA and C.G.
WHITELEY*
of Chemistry and Biochemistry, Grahamstown, South Africa,
Rhodes 6140
20,
Receptor
University,
1990
and spectrophotometry have been used to study the binding of dipropyl, dibutyl and diphenylnitrosamine to nicotinic acetylcholine receptor isolated, and purified, from Torpedo fuscomaculata. Scatchard analysis indicates that all four ligands are true agonists of the receptor exhibiting positive cooperative binding with the existence of more than one class of binding site. The number of binding sites for the nitrosamines approximates 2. Diphenylnitrosamine binds to the receptor more tightly at low concentrations (Kd = 1.3 IIM) than the aliphatic nitrosamine (Kd, = 8-12 uM). Yet at high concenirations all nitrosamines behaved with similar Kd values Fluorimetry
dimethyl,
(27-38
PM).
~1990
Academic
Press.
1°C.
The nicotinic acetylcholine receptor is a ligand gated ion channel protein. (132) It contains, in its protein moiety, binding sites for acetylcholine and its agonists and antagonists (receptor function), the calcium channel (response function) and several types of molecular sites (molecular functions). The elucidation of its molecular mechanism of function, therefore requires an intimate understanding of its ligand binding properties. With the exception of polypeptide neurotoxins all the ligands of AChE The ligands of AChE can be subdivided may also be ligands of the receptor. into substrates and competitive inhibitors while those of the receptor into agonists and antagonists (competitive blockers). Since it had been found, in (3), that the powerful carcinogenic nitrosamines these laboratories and others act as competitive inhibitors to AChE it was reasonable to suppose that they would interact with the receptor as well. A detailed description of the interaction of these molecules with the receptor might provide insights not only into the molecular mechanism of junctional excitation and permeability change, but also into the principles on which more complex neural functions are
based.
With this in mind, and prompted by the availability in a form suitable for biochemical solubi 1i sed receptor(4) *TO whom correspondence
of highly purified studies we have
should be addressed.
1383
0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
167,
No.
undertaken binding
3, 1990
a systematic of various
Binding ligands
applied they
and the are
are
AND
investigation
nitrosamines
and klnetlc
Nitrosamines since
BIOCHEMICAL
of the
ligands source
the
the
kinetics depend
observed
to use for
of carbocations
RESEARCH
of
COMMUNICATIONS
interaction
and
receptor.
critically
properties
suitable
a rich
into
with
studies
BIOPHYSICAL
on the changes
interaction (5) (Scheme
specificity
of the
in fluorescence.
with 1).
the
receptor
Receptor "ii'
-R-NT>:
R-Receptor
RN+=N
R2N-N=O
Scheme
Careful
selection
in fluorescence information functional MATERIALS MATERIALS
of the observed
on the
basic
properties
of
1
nitrosamines
and a study
upon their
binding
principles
of
integral
to the
intracellular
membrane
of the receptor
kinetics
and changes
may provide
communication
and on the
proteins.
AND METHODS
Electric rays (Torpedo fuscomaculata) were caught in the Bushman's river Pure naja venom (a estuary off the South tast coast of South Africa. cobratoxin) was purchased from the Council of Scientific Industry and Research, Pretoria. Polybuffer exchange (PBE 94), Polybuffer 74, Sephacryl S-400 and CNBr-Sepharose 48 were purchased from Pharmacia; phenylmethylsulphonylfluoride, molecular weight standards, bovine serum albumin, electroplax of electric eel (Electrophorus electricus); carbamylcholine, acetylcholine and DEAE-Sepharose 6B were obtained from Sigma. Triton X-100 was obtained from Ethylenediaminetetraacetic acid (EDTA), sodium azide Fischer Scientific Co. and nitrosamines were brought from Aldrich Chemical Co., acrylamide, ammonium persulphate, N,N'methylene bis acrylamide, sodium dodecylsulphate (SDS) and Dialysis membranes were obtained Coomassie Brilliant Blue R-250 from Bio Rad. from Spectropor and all other inorganic and buffer materials were of reagent grade.
&?%dticm
of Receptor(4) Electric organs were excised from a freshly d Torpedo ray and stored at -8O'C until required Electric tissue (120 g) wis Eomogenised (2.0 min) in imidazole buffer (25mM, pH 7.4 150 mLs) containing sodium azide (O-01%), EDTA (lOWI) and phenylmeth~lsulphor~ylfluoride (O.lil+l). The homogenate was then centrifuged (20000 x g, 60 min, 4°C) and the supernatant discarded. The pellet was resuspended in the same buffer solution and Triton X-100 added to a final concentration of 1%. The mixture was stirred (18h, 4°C) and then centrifuged (100000 x g, 60 min), the pellet resuspended in the above buffer, centrifuged again and the supernatants pooled. 1384
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No.
3, 1990
BIOCHEMICAL
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BIOPHYSICAL
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The receptor extract (10 mBs) was added to a PBE 94 column (11 x 1.6 ems) previously equilibrated with imidazole buffer (25mM, pH 7.4, 40 mls). The column was eluted with PB 74 (pH 4) at 54 mPs h fractions (3.0 m8s) collected and monitored for absorbance at 280nm. Finall; the column was washed with sodium chloride (IM, 50 m8s) to elute the proteins not displaced by low ionic strength. Electrophoresis of fractions of receptor in 5 mm thick acrylamide gels is used to monitor the purification of the receptor. The acrylamidebisacrylamide mixture (8% and 0.2% respectively) is polymerised in 0.01M potassium phosphate (pH 7.0) containing 0.25% N,N,N,N-tetramethylenediamine. Polymerisatign is catalysed by the addition of ammonium persulphate (320 pg.cms.- ). The upper and lower buffer compartments are filled with potassium persulphate (O.OlM, pH 7.0), sample (50 ~1) is applied to the gel and a potential of 8mA/tube is applied. Protein is stained with 1% solution of Coomassie Blue in a mixture of isopropyl alcohol-water-glacial acetic acid. Destaining is achieved by extensive washings in alcohol and acetic acid. Assay of Receptor(4) A fluorescence titration assay is described which allows a rapld and reproducible means for determining receptor site concentrations without many of the difficulties associated with the use of radiochemicals. Tubocurarine, a specific competitive antagonist of nicotinic acetylcholine receptor, shows similar affinities for both membrane bound and solubil' receptor and hence may be included as a fluorescence quenching ligand. ts(td Since the intensity of excitation light decreases along the light path due to the inner filter effect and the absorption by tubocurarine, the decrease in tryptophanyl fluorescence is corrected by dividing the apparent intensity (F,) by the light intensity in the sample cell (equation 1). F, = Fa/lOECa
(1)
where F is the corrected fluorescence intensity, E is the molar extinction ntration of tubocurarine and ‘a’ is an coefficient, c is the molar cofi57 instrumental constant (0.588). An excitation wavelength of 295nm and emission wavelength of 340nm were used.
Fluorimetric Analysis Fluorimetric measurements were made with a Hitachi fluorescence spectrophotometer using an excitation light source from a Xenon 150 lamp, the fluorescence measured through the cell at an angle of 90" to the incident beam. All fluorimetric titrations were carried out at 20°C and in Increasing concentrations of ligand O.lM phosphate buffer (pH = 7.2). (O-600 PM) were titrated against a fixed concentration of receptor (100 pg.cmsm3). The mixture was excited at 295nm and emission measured at 340nm. The fluorescence data was used to determine the dissociation constant (Kd) of the ligands with the receptor. The results were analysed according to equation 2 and 3. Kd/l
-e
= (L$,/e-
Where (A) is the total total num &er of binding AF is sites by ligand; amount of ligand; AF, with ligand; (L)t IS @e
Pi
(2)
0
= A F/ A F,,,
(3)
concentration of acceptor in the system; p is the sites; e is the fractional occupancy of total acceptor the increase in fluorescence in the presence of known is the increase in fluorescence at full saturation total concentration of ligand.
The interaction of nitrosamines to the receptor, with and without the presence of d-tubocurarine and before and after-gialysis, was also investigated fluorimetrically. Purified receptor (lOCipg.cms ) in O.lM phosphate buffer (pH 7.2) (3.0 cms3) was combined with ligands and other cholinergic ligands. Excitation was measured at 295nm and emission at 340nm. Spectr-ophotom&ry
receptor,
before
The interaction and after dialysis,
of nitrosamines was studied 1385
to nicotinic acetylcholine spectrophotometrically using
Vol.
167,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
a Bausch and Lomb 1001 spectrophotomjter. Reaction mixtures (3.0 cms3) ), purified receptor material (100~1, containing gitrosamine (2.83yg.cms 100 Pg.cms- ) in imidazole buffer (O.lM, pH 7.4) were scanned between 210nm and 280nm. Dialysis was performed for 5h at 2°C with four changes of imidazole buffer (O.O25M, pH 7.4). The binding of the nitrosamines to the recepsor was also studied Reaction mixtures (3.0 ems ) contained purified spectrophotometrical!J. receptor (100 pg.cms ); 100 ~11 and ligand (2-50 PM) in imidazole buffer (O.lM, pH 7.4). The absorbance was monitored at 230nm. The determination of the number of binding sites and the dissociation constants of tt@)receptor ligand complex was determined according to the method of Scatchard The magnitude of the decrease in absorbance due'to (equations 4 and 5). the pry5ence of,ligand was used to calculate the amount of free and bound ligand .
(L), = @It where
(L)
- * A,/ *‘Axmax (Ajt
(4)
v/iLlf
= p - v/Kd
(5)
is
the total ligand concentration; (A) is the total receptor AA is the difference in absorbange observed when ligand (L) is the maximum absorbance observed when f~n~~~~a~~~~'receetor (A); AA all of the ligand molecules (L) $P$xbound to the receptor; v are the moles of bound ligand; p is the number of binding sites; (L) is the concentration of complex. free ligand and Kd is the dissociation constant of rigand-receptor
RESULTS
Fluorimetric various
Studies
ligands
In an attempt
Fluorimetry
and combinations to detect
cholinergic
ligands
relative
of dimethylnitrosamine
measured
(Fig.
receptor
the
fluorescence
1).
fluorescence
was also
fluorescence, alone
quenches
effects emitted
plot
were of binding
appeared
of the
linear
linear though
of
and d-tubocurarine
IO fold
fluorescence of the with
enhancement
presence
of 5DvM
in
d-Tubocurarine
by 20% while
fluorescence.
of ligand
The 70% quench
present.
in the
was
(5C PM)
acetylcholine
of the
The pure
ligand
DMN,
receptor
By comparison, L-tryptophan, a maximum at 348nm. Thus emission maximum
in the
receptor site
binding
receptor.
of nAChR in the
(DMN) was combined
the
of
to the
intrinsic
receptor
receptors
binding
and removal
ligand
agonist,
with
and analysis
not
the
10 fold.
was no longer
plot
nAChR underwent of
of dimethylnitrosamine, on the
nitrosamines
the
the
a maximum at 336nm.
fluorescence curve
effect
acetylcholine one class
with
at 290nm emitted
The binding the
found,
of the
the
dialysis
dialysis,
The latter
fluorescence
tryptophan
quenches
when
after
fluorescence
a 55% quench
activated
for
the
a 4% quench.
produces
for
previously
upon spectra
by approximately
observed
However,
study
acetylcholine
After
70%.
was enhanced
d-tubocurarine.
change
(DMN),
by over
to
and antagonists
fluorescence
The nitrosamine
protein
used
of agonists
a conformational
the
presence the
was also
but tended
- e versus
2 and 3).
instead
indicated
receptor. to become 1386
by 12nm. e are
compared
d-tubocurarine
The analysis the
shift
(ligand)/
diphenylnitrosamine,
(Figs.
at the
l/l
a blue
plots
existence
With d-tubocurarine, hyperbolic at higher
for
and the
of more than the analysis concentrations.
Vol.
167,
No.
3, 1990
BIOCHEMICALANDBIOPHYSICALRESEARCH
COMMUNICATIONS
16
6
01
300
320 NAVELENGTH 340 (nm) 360
0.5
360
1.0
1.5
l/(L)tM“
Fig.
1
Fluorescence3emission spectra of purified acetylcholine receptor (100 ug.cms ) excited at 295nm showing the effect of agonists, antagonists and nitrosamines. (0) nAChR + dimethylnitrosamine (DMN) (17.5mM) after dialysis; (m) nAChR after dialysis and nAChR + DMN (17.5mm) + d-tubocurarine (d-TbC) (50 PM) after dialysis; (r) nAChR and nAChR t acetylcholine (ACh) (55 uM); (x) nAChR t dTbC (50 PM); (0) nAChR + DMN (17.5mm) + ACil (55 PM); (a) nAChR t DMN (17.5mM) + d-TbC (50 PM) and nAChR + DivlN (li.SmM).
Fig.
2
Double reciprocal plots of fluorescence change on binding of dtubocurarine (d-TbC) (50 PM) (a), d-TbC (33.3 PM) (m); dimethylnitrosamine (A), acetylcholine (o), and diphenylnitrosamine (x) to the nicotinic cholinergic receptor in 1OOmM phosphate buffer (pH 7.2). The mixture was excited at 295nm and emission measured at 340nm.
This reflects
one class
and two classes contents
(Kd)
of binding sites at lower d-tubocurarine concentrations of binding sites at a higher concentration. The dissociation can then be estimated and represented (Table 1). For all 12 . 10 .
6. 4. 2
2
4
6 (Lp3
Fig.
3
8
10
12
14
16
18
x 10-l 31
Analysis data for the binding of d-tubocurarine (d-TbC) (50 PM) (0); d-TbC (33.3 ~14) (m); dimethylnitrosamine (A); acetylcholine (a); and diphenylnitrosamine (x) to the nicotinic cholinergic receptor in 1OOmM phosphate buffer (ptl 7.2).
1387
Vol.
167,
No.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
Table
RESEARCH
COMMUNICATIONS
1
values obtained by fluorimetric titrations and (DPrN), diphenyl (DPhN) dibutyl,p,"Pt:$i%t-~t?;,oma5,'ra,nto:ad:d,:kfl (DMN) dipropyl nitrosamine (DBuN), acetylcholi;e (ACh) and d-tubocurarine (d-TbC) on nicotinic cholinergic receptor FLUORIMETRY Ligand
EC5,-,(W
d-Tbc ACh DPhN DMN DPrN DBuN
Kd,(uM)
Kd2(IrM)
.05
11.4
.12
2.3
1.45
27.3
1.96
8.4
37.2
1.84
37.0
1.87
11.7
36.8
41.1
1.91
12.1
37.9
.398 .790 56
9.13
61 77
nitrosamines
there
representing
a weak
10.60
are
SPECTROPHOTOMETRY
two equilibrium
association
cooperativity
and binds to the receptor with ~11 these results are consistent
of fluorescence
receptor-ligand samine
intensity
complex
from
site
monitored
by measurement
protein.
The protein
in the presence difference
in protein
directly is
the
(Figs.
to the
parameter
5 and 6) for
of more than
equilibrium
dissociation
least than
represented
50% more the
nitrosamines Though Only
One Class
I).
to the with
of binding
site
representation exhibits positive
-
12 x 10m2,M that the
q
to
of the
absorbance is
nitro-
DMN is
of binding for
linear
site
high
at low
Kd values
d-tubocurarine 7) it 1388
and
plot the
receptor.
The can be estimated
appeared
concentrations
to bind
(Kd
concentrations
noted
to
as the to be
indicated
q
at
1.3 LIM)
all
PM.)
and acetylcholine is
large
measured
- nAChR complex
but
at the
(27-38
The
The Scatchard
and low affinity
Yet at higher
4).
is considered
The diphenylnitrosamine receptor
(Fig.
sufficiently change,
(230nm)
not
of the
210nm and 280nm
shown
of nitrosamine
were
(Fig.
1.92 1.96 1.98
- One
wavelengths
such an interaction.
similar
seemed that
35.6
to nAChR was also
The signal
concentration
constants
2.03
of acetylcholine
ultra-violet between
at a wavelength
nitrosamines.
behaved it
data.
one class
tightly
in the
nitrosamines
(Table
aliphatic
ligands
upon binding
to follow
the
existence and are
maxima
2.0
27.0
displacement
of dimethylnitrosamine
titration
1.68
3.1
constants
addition
P
acetylcholine.
spectrum
absorbance
proportional
a reliable
physical
of changes
of the
between
by the
of nitrosamine
absorption
and absence
evaluation
difference
caused
upon
common for
The binding
Spectrophotometry
allow
are
a binding
.I8
two affinities (Kd with the interpretation
observed
M)
8
other a more tighter Acetylcholine also
cooperativity.
Kd2(v
1.30 9.6
dissociation
and the
M)
.033
2.01
to positive
changes
Kd,b
1.79
in addition and 2 PM).
P
that
interacted at high
with
concentration
Vol.
167, No. 3, 1990
BIOCHEMICALANDBIOPHYSICALRESEARCH
COMMUNICATIONS
1.5
1 Y i I s z 0.5
04
210
220
230
240 YAYELENGTH
250
260
4
Ultraviolet absorptlgn (100 ~1; 100 pg ems dimethylnitrosamine
Fig.
5
Scatchard
and Kd ) !-eceptor2as number of moles
the
the antagonist the results
one of low affinity
(Fig.
1.5
2.0
2.5
spectrum of nicotinic cholinergic receptor ) in the preSence of (m) and absence (0) of (2.83 ,.,g ems ) in imidazole buffer (IOOmM, pH 7.4).
u are
nitrosamine.
1.0
"
plot for the binding (Kd nicotinic cholinergic
the spectrophotometry.
of
0.5
280
(nn)
Fig.
(a); to
270
are consistent 7, inset).
of dimethylnitrosamine determined by of
bound
with two sites
dimethyl-
- one of high and
DISCUSSION Our studies have shown t hat the two nitrosamines - dimethylnitrosamine DPhn) interact with the agonists binding sites (DMN) and diphenylnitrosamine ( receptor. Though the results are not presented of the nicotinic acetylcholine and dibutylnitrosamine) the other nitrosamines studied (dipropylnitrosamine exhibit similar effects. The potency of a particular nitrosamine in the interaction with the receptor is determined by the dissociation constants (Kd) for the receptorThe fit into the active site of the protein, reflected by the ligand complex. values of Kd, is largely determined by the size, structure and configuration The capability of such a ligand to bind, non covalently at, or of the ligand. close to, the active site could also influence these values. Two fundamental properties of the interaction of the nitrosamines to the receptor agonist active site are implied. First, there is at least one protonated residue which combines with the ligand within the receptor-complex. Second the receptor active site must have a hydrophobic nature. Thus when the nitrosamines environment.
bind to the receptor they pass from a hydrophilic to a hydrophobic This change in environment is a prequisite for good binding of 1389
Vol.
167,
No.
3, 1990
BIOCHEMICALAND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
36 Kd2
32
1 Kdl
28
. I ‘,
24
0.5 x
3 Y 2
-ii 2. L r3 2
l
20
0
l!iiJ 12
3
4
16 Y 12
\
8 4
0.5
1.0
2.0
1.5
2.5
07
Y
Fig.
6
1.0
0.5
1.5
2.0
2.5
Y
Scatchard
plot for the binding (Kd and the nicotinic cholinergic rkeptor v are the number of spectrophotometry.
Kd ) of diphenylnitrosamine is determined by moles of bound diphenyl-
(m) to
nitrosamine. Fig.
7
Scatchard
plot
for
the
ligand.
binding (Kdp) of d-tubocurarine (A) and the nicotinic cholinergic receptor as (L)f is the concentration of by spectrophotometry. The inset v are the number of molesof bound ligand.
to the
active
acetylcholine (x) to determined
acetylcholine
site
and has already
been
invoked
free
is
as an important
factor.(") If these that
the
alkylating the
observations
ligand
are
decomposes,
species
hydrophilic
(R+)
(Scheme
and size
in the
value
of the
of the
then
as it
1).
and hydrophobic
energy
accepted
as soon
the
reaches
Obviously
environment
carbocation
reaction the
the coupled
constants
with
the
formed
(Kd)
site
suggests
into
an
ease of transition
and intermediate
dissociation
mechanism
active
for
the
between
overall
will
stability,
be reflected
receptor-nitrosamine
complex. Detection binding study
with
is
the
ligand
quenching
of the ligands
induced
fluorescence arise
is
the
containing
binding The intrinsic
nitrosamine
protein
moiety
due to the
one of the
with when
presence site
receptor
ligand the
the
ligand
fluorescence
and diphenylnitrosamine
In the present upon binding with
to the regions
in or
to
investigation the the nitrosamines
a change
protein
upon method
The decrease
supports
binding
in a protein
and most direct
interactions.
of non-polar of the
change
simplest
change.
of the
on titration
nitrosamine
fluorescence
conformational
of fluorescence
used to study
the
intrinsic
in relative
in environment
takes
place.
around
the
This
tryptophan
receptor. of the with 1390
receptor
is quenched
emission
observed
by dimethyl-
at 336nm.
d-
of may
Vol.
167,
No.
3, 1990
Tubocurarine, specific is
a specific interaction
competition
DMN are are
BIOCHEMICAL
of DMN with
of the
for
RESEARCH
antagonist
of the
receptor.
This,
receptor
to support
COMMUNICATIONS
receptor
blocks
and the
fact
any
that
when both
acetylcholine
idea
the
the
that
there and
nitrosamines
agonists.
Scatchard
binding
BIOPHYSICAL
of the
receptor
cooperative sites
the
site evidence
nicotinic
The curves binding
active
is clear
potential
positive
competitive
at the
present,
AND
plot
and the
acetylcholine,
(Figs.
5, 6 and 7) indicate
existence
of more than
d-tubocurarine
and the
a
one class
of
nitrosamines
at the
receptor. Both reversible
the
low and high
and are displaced
tubocurarine
(Fig.
explained
over
competitive
which
well
the nitrosamine in sufficient
with
cholinergic
range takes
Furthermore
very
binding
by the
The inhibition
a 300 fold
model
nitrosamines. agree
1).
affinity
the
of tubocurarine into
account
binding
values
from
observed
in the
displacement
of nitrosamine
bound of the
the
effective
concentration
Kd values
from
It
is
the
noted
blue-shift
of
spectral
properties
sensitive
to the
suggests
agonists,
while
fluorescence (Fig.
either
of the
wavelength other
the
is
independent
emission
nature
the
where
a small
portion
of that
population
properties
of the
molecules
bound
presence
emission
ligand
sufficient
receptor spectrum,
interacts has been to the
of a blue-shift
second
with
and/or
the
displaced, class
while
of the
in acetylto
with
effector
the (d-
Nitrosamines
even
can be taken 1391
The quench
site
site.
as
therefore,
remaining bound site the emission
to the
receptor
act
shift,
a
The
blue-shift
of d-tubocurarine
sensitive at the
receptor.
ligand.
receptor
to the
the
of the
of the
bound
Nevertheless
the
occupancy
bound
the
cause
The spectral
as any nitrosamine the one receptor
or acetylcholine)
of
information
presence
and
1 that
of
character
tubocurarine
fraction
to the
(Fig.
Extension
by the
dominate
spectrum
appear
agonist
is
by fluorescence
sites
which
wavelength
Furthermore
receptor
compounds
of the
site.
bind
as antagonists.
With
competitive
nitrosamines
all
sites.
fluorimetry
bound
to the
the
agreement.
fluorescence
act
caused
receptor determined
bound.
1) can be interpreted receptor
from
for
The values of two methods used are decrease in
in excellent
compound
of the
intensity
choline
of the
others
seems characteristic
of the
by a
analysis.
agonist
are
can be
constants
is due to the
nitrosamine
ligands
that
all
estimated
to the
when the
of the nature
constants
of ligand
binding
analysis
12nm occurs
obtained(")
presence
direct
from
concentrations two binding
Scatchard
and d-
by d-tubocurarine
the
the
are
acetylcholine
constants estimated by the lead us to conclude that the
fluorescence
the
nitrosamines
agonists
of DMN binding
the
dissociation agreement to
of the
under
conditions When
protein. the of sites
spectral becomes
as evidence
for
apparent. a
Vol.
167, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
change characteristic of an agonist ligand and associated with the physiological response of the receptor protein. The fact that the nitrosamine binding functions must be characterised by both high and low affinity components explains the paradoxical observations that very high concentrations of d-tubocurarine (200. LIM) are necessary to completely displace the nitrosamine from the site binding the tubocurarine. The observed competition between the nitrosamines and tubocurarine and between acetylcholine and nitrosamines can be accounted for by a simple model that assumes that they all bind to a common site and most probably with a 1 : 1 stoichiometry. This model predicts that the total number of binding sites for dimethylnitrosamine, diphenylnitrosamine, acetylcholine and d-tubocurarine structural
and dibutylnitrosamine) would be identical. Indeed our results number of binding sites for DMN is-1.84, for DPhN is 1.96, for ACh is 2.01 and for d-tubocurarine is 1.79. In order to further unravel the mechanism of ligand interaction of the receptor we have used fluorescence titrations and spectrophotometry of various nitrosamines with the protein. The employed receptor preparations, monitoring ligands and techniques were particularly suited for this purpose. The receptor, isolated from Torpedo exhibited positive cooperativity of acetylcholine binding sites as well as with the nitrosamine ligands. Since the latter were regarded as full agonists to the frog rectus abdominis muscle and the Torpedo membrane, it must be assumed that they bind by the same mechanism. A complete analysis in terms of Kd values of binding equilibria of the receptor with a set of agonists and antagonists is presented. (and
dipropyl
conclude
that
the
We acknowledge the Medical
Research Council
(South Africa)
for financial
support. REFERENCES 1. Maelicke, Roberts, 2. Changeux,
3.
A.
(1981) in Molecular Pharmacology (A.S.V. Burgen and G.C.K. Elsevier North Holland ~~1-22. J-P., A. De Villiers - Thiery and P. Chemouilli. (1984) Science 225 : 1335-1345. Eid, P., P. Goeldner, C.G. Hirth and P. Jost. (1981) Biochemistry 20 : eds.).
2251-2256. Kapp, E.A.
and C.G. Whiteley. (1989) Biochem. Biophys. Acta (In Press). Ingelman-Sundberg, M. (1980) Pharm. Sci. 1 : 176-179. ;: Meunier, J.C., R. Sealcock, R. Olsen, and J-P. Changeux. (1974) Eur. J. Biochem. 45 : 371-394. 7. Kaneda, N., F. Tanaka, M. Kohno, K. Hayashi, and K. Vagi. (1982 ‘1 Arch. Biochem. Biophys. 218 : 376-383. 8. Scatchard, G. (1949) Proc. Natl. Acad. Sci. 51 : 660-672. 9. Khan, M., M.V.Ki Sas&y and A. Surolia. (1986) J. Biol. Chem. 2.61 : 3013-3019. 10. Jencks, W.P. (1975) Adv. Enzymol., 43 : 219-410. 11. Bennett, M.V.L., M. Wurzel and H. Grundfest. (1961) J. Gen. Physiol. 44 :
4.
757-781. 1392
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 1383-1392
March 30, 1990
Interaction
Protein-Ligand of Nitrosamines with E-A.
Department
Received
February
Interactions: Nicotinic Acetylcholine
KAPP, S. DAYA and C.G.
WHITELEY*
of Chemistry and Biochemistry, Grahamstown, South Africa,
Rhodes 6140
20,
Receptor
University,
1990
and spectrophotometry have been used to study the binding of dipropyl, dibutyl and diphenylnitrosamine to nicotinic acetylcholine receptor isolated, and purified, from Torpedo fuscomaculata. Scatchard analysis indicates that all four ligands are true agonists of the receptor exhibiting positive cooperative binding with the existence of more than one class of binding site. The number of binding sites for the nitrosamines approximates 2. Diphenylnitrosamine binds to the receptor more tightly at low concentrations (Kd = 1.3 IIM) than the aliphatic nitrosamine (Kd, = 8-12 uM). Yet at high concenirations all nitrosamines behaved with similar Kd values Fluorimetry
dimethyl,
(27-38
PM).
~1990
Academic
Press.
1°C.
The nicotinic acetylcholine receptor is a ligand gated ion channel protein. (132) It contains, in its protein moiety, binding sites for acetylcholine and its agonists and antagonists (receptor function), the calcium channel (response function) and several types of molecular sites (molecular functions). The elucidation of its molecular mechanism of function, therefore requires an intimate understanding of its ligand binding properties. With the exception of polypeptide neurotoxins all the ligands of AChE The ligands of AChE can be subdivided may also be ligands of the receptor. into substrates and competitive inhibitors while those of the receptor into agonists and antagonists (competitive blockers). Since it had been found, in (3), that the powerful carcinogenic nitrosamines these laboratories and others act as competitive inhibitors to AChE it was reasonable to suppose that they would interact with the receptor as well. A detailed description of the interaction of these molecules with the receptor might provide insights not only into the molecular mechanism of junctional excitation and permeability change, but also into the principles on which more complex neural functions are
based.
With this in mind, and prompted by the availability in a form suitable for biochemical solubi 1i sed receptor(4) *TO whom correspondence
of highly purified studies we have
should be addressed.
1383
0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
167,
No.
undertaken binding
3, 1990
a systematic of various
Binding ligands
applied they
and the are
are
AND
investigation
nitrosamines
and klnetlc
Nitrosamines since
BIOCHEMICAL
of the
ligands source
the
the
kinetics depend
observed
to use for
of carbocations
RESEARCH
of
COMMUNICATIONS
interaction
and
receptor.
critically
properties
suitable
a rich
into
with
studies
BIOPHYSICAL
on the changes
interaction (5) (Scheme
specificity
of the
in fluorescence.
with 1).
the
receptor
Receptor "ii'
-R-NT>:
R-Receptor
RN+=N
R2N-N=O
Scheme
Careful
selection
in fluorescence information functional MATERIALS MATERIALS
of the observed
on the
basic
properties
of
1
nitrosamines
and a study
upon their
binding
principles
of
integral
to the
intracellular
membrane
of the receptor
kinetics
and changes
may provide
communication
and on the
proteins.
AND METHODS
Electric rays (Torpedo fuscomaculata) were caught in the Bushman's river Pure naja venom (a estuary off the South tast coast of South Africa. cobratoxin) was purchased from the Council of Scientific Industry and Research, Pretoria. Polybuffer exchange (PBE 94), Polybuffer 74, Sephacryl S-400 and CNBr-Sepharose 48 were purchased from Pharmacia; phenylmethylsulphonylfluoride, molecular weight standards, bovine serum albumin, electroplax of electric eel (Electrophorus electricus); carbamylcholine, acetylcholine and DEAE-Sepharose 6B were obtained from Sigma. Triton X-100 was obtained from Ethylenediaminetetraacetic acid (EDTA), sodium azide Fischer Scientific Co. and nitrosamines were brought from Aldrich Chemical Co., acrylamide, ammonium persulphate, N,N'methylene bis acrylamide, sodium dodecylsulphate (SDS) and Dialysis membranes were obtained Coomassie Brilliant Blue R-250 from Bio Rad. from Spectropor and all other inorganic and buffer materials were of reagent grade.
&?%dticm
of Receptor(4) Electric organs were excised from a freshly d Torpedo ray and stored at -8O'C until required Electric tissue (120 g) wis Eomogenised (2.0 min) in imidazole buffer (25mM, pH 7.4 150 mLs) containing sodium azide (O-01%), EDTA (lOWI) and phenylmeth~lsulphor~ylfluoride (O.lil+l). The homogenate was then centrifuged (20000 x g, 60 min, 4°C) and the supernatant discarded. The pellet was resuspended in the same buffer solution and Triton X-100 added to a final concentration of 1%. The mixture was stirred (18h, 4°C) and then centrifuged (100000 x g, 60 min), the pellet resuspended in the above buffer, centrifuged again and the supernatants pooled. 1384
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No.
3, 1990
BIOCHEMICAL
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The receptor extract (10 mBs) was added to a PBE 94 column (11 x 1.6 ems) previously equilibrated with imidazole buffer (25mM, pH 7.4, 40 mls). The column was eluted with PB 74 (pH 4) at 54 mPs h fractions (3.0 m8s) collected and monitored for absorbance at 280nm. Finall; the column was washed with sodium chloride (IM, 50 m8s) to elute the proteins not displaced by low ionic strength. Electrophoresis of fractions of receptor in 5 mm thick acrylamide gels is used to monitor the purification of the receptor. The acrylamidebisacrylamide mixture (8% and 0.2% respectively) is polymerised in 0.01M potassium phosphate (pH 7.0) containing 0.25% N,N,N,N-tetramethylenediamine. Polymerisatign is catalysed by the addition of ammonium persulphate (320 pg.cms.- ). The upper and lower buffer compartments are filled with potassium persulphate (O.OlM, pH 7.0), sample (50 ~1) is applied to the gel and a potential of 8mA/tube is applied. Protein is stained with 1% solution of Coomassie Blue in a mixture of isopropyl alcohol-water-glacial acetic acid. Destaining is achieved by extensive washings in alcohol and acetic acid. Assay of Receptor(4) A fluorescence titration assay is described which allows a rapld and reproducible means for determining receptor site concentrations without many of the difficulties associated with the use of radiochemicals. Tubocurarine, a specific competitive antagonist of nicotinic acetylcholine receptor, shows similar affinities for both membrane bound and solubil' receptor and hence may be included as a fluorescence quenching ligand. ts(td Since the intensity of excitation light decreases along the light path due to the inner filter effect and the absorption by tubocurarine, the decrease in tryptophanyl fluorescence is corrected by dividing the apparent intensity (F,) by the light intensity in the sample cell (equation 1). F, = Fa/lOECa
(1)
where F is the corrected fluorescence intensity, E is the molar extinction ntration of tubocurarine and ‘a’ is an coefficient, c is the molar cofi57 instrumental constant (0.588). An excitation wavelength of 295nm and emission wavelength of 340nm were used.
Fluorimetric Analysis Fluorimetric measurements were made with a Hitachi fluorescence spectrophotometer using an excitation light source from a Xenon 150 lamp, the fluorescence measured through the cell at an angle of 90" to the incident beam. All fluorimetric titrations were carried out at 20°C and in Increasing concentrations of ligand O.lM phosphate buffer (pH = 7.2). (O-600 PM) were titrated against a fixed concentration of receptor (100 pg.cmsm3). The mixture was excited at 295nm and emission measured at 340nm. The fluorescence data was used to determine the dissociation constant (Kd) of the ligands with the receptor. The results were analysed according to equation 2 and 3. Kd/l
-e
= (L$,/e-
Where (A) is the total total num &er of binding AF is sites by ligand; amount of ligand; AF, with ligand; (L)t IS @e
Pi
(2)
0
= A F/ A F,,,
(3)
concentration of acceptor in the system; p is the sites; e is the fractional occupancy of total acceptor the increase in fluorescence in the presence of known is the increase in fluorescence at full saturation total concentration of ligand.
The interaction of nitrosamines to the receptor, with and without the presence of d-tubocurarine and before and after-gialysis, was also investigated fluorimetrically. Purified receptor (lOCipg.cms ) in O.lM phosphate buffer (pH 7.2) (3.0 cms3) was combined with ligands and other cholinergic ligands. Excitation was measured at 295nm and emission at 340nm. Spectr-ophotom&ry
receptor,
before
The interaction and after dialysis,
of nitrosamines was studied 1385
to nicotinic acetylcholine spectrophotometrically using
Vol.
167,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
a Bausch and Lomb 1001 spectrophotomjter. Reaction mixtures (3.0 cms3) ), purified receptor material (100~1, containing gitrosamine (2.83yg.cms 100 Pg.cms- ) in imidazole buffer (O.lM, pH 7.4) were scanned between 210nm and 280nm. Dialysis was performed for 5h at 2°C with four changes of imidazole buffer (O.O25M, pH 7.4). The binding of the nitrosamines to the recepsor was also studied Reaction mixtures (3.0 ems ) contained purified spectrophotometrical!J. receptor (100 pg.cms ); 100 ~11 and ligand (2-50 PM) in imidazole buffer (O.lM, pH 7.4). The absorbance was monitored at 230nm. The determination of the number of binding sites and the dissociation constants of tt@)receptor ligand complex was determined according to the method of Scatchard The magnitude of the decrease in absorbance due'to (equations 4 and 5). the pry5ence of,ligand was used to calculate the amount of free and bound ligand .
(L), = @It where
(L)
- * A,/ *‘Axmax (Ajt
(4)
v/iLlf
= p - v/Kd
(5)
is
the total ligand concentration; (A) is the total receptor AA is the difference in absorbange observed when ligand (L) is the maximum absorbance observed when f~n~~~~a~~~~'receetor (A); AA all of the ligand molecules (L) $P$xbound to the receptor; v are the moles of bound ligand; p is the number of binding sites; (L) is the concentration of complex. free ligand and Kd is the dissociation constant of rigand-receptor
RESULTS
Fluorimetric various
Studies
ligands
In an attempt
Fluorimetry
and combinations to detect
cholinergic
ligands
relative
of dimethylnitrosamine
measured
(Fig.
receptor
the
fluorescence
1).
fluorescence
was also
fluorescence, alone
quenches
effects emitted
plot
were of binding
appeared
of the
linear
linear though
of
and d-tubocurarine
IO fold
fluorescence of the with
enhancement
presence
of 5DvM
in
d-Tubocurarine
by 20% while
fluorescence.
of ligand
The 70% quench
present.
in the
was
(5C PM)
acetylcholine
of the
The pure
ligand
DMN,
receptor
By comparison, L-tryptophan, a maximum at 348nm. Thus emission maximum
in the
receptor site
binding
receptor.
of nAChR in the
(DMN) was combined
the
of
to the
intrinsic
receptor
receptors
binding
and removal
ligand
agonist,
with
and analysis
not
the
10 fold.
was no longer
plot
nAChR underwent of
of dimethylnitrosamine, on the
nitrosamines
the
the
a maximum at 336nm.
fluorescence curve
effect
acetylcholine one class
with
at 290nm emitted
The binding the
found,
of the
the
dialysis
dialysis,
The latter
fluorescence
tryptophan
quenches
when
after
fluorescence
a 55% quench
activated
for
the
a 4% quench.
produces
for
previously
upon spectra
by approximately
observed
However,
study
acetylcholine
After
70%.
was enhanced
d-tubocurarine.
change
(DMN),
by over
to
and antagonists
fluorescence
The nitrosamine
protein
used
of agonists
a conformational
the
presence the
was also
but tended
- e versus
2 and 3).
instead
indicated
receptor. to become 1386
by 12nm. e are
compared
d-tubocurarine
The analysis the
shift
(ligand)/
diphenylnitrosamine,
(Figs.
at the
l/l
a blue
plots
existence
With d-tubocurarine, hyperbolic at higher
for
and the
of more than the analysis concentrations.
Vol.
167,
No.
3, 1990
BIOCHEMICALANDBIOPHYSICALRESEARCH
COMMUNICATIONS
16
6
01
300
320 NAVELENGTH 340 (nm) 360
0.5
360
1.0
1.5
l/(L)tM“
Fig.
1
Fluorescence3emission spectra of purified acetylcholine receptor (100 ug.cms ) excited at 295nm showing the effect of agonists, antagonists and nitrosamines. (0) nAChR + dimethylnitrosamine (DMN) (17.5mM) after dialysis; (m) nAChR after dialysis and nAChR + DMN (17.5mm) + d-tubocurarine (d-TbC) (50 PM) after dialysis; (r) nAChR and nAChR t acetylcholine (ACh) (55 uM); (x) nAChR t dTbC (50 PM); (0) nAChR + DMN (17.5mm) + ACil (55 PM); (a) nAChR t DMN (17.5mM) + d-TbC (50 PM) and nAChR + DivlN (li.SmM).
Fig.
2
Double reciprocal plots of fluorescence change on binding of dtubocurarine (d-TbC) (50 PM) (a), d-TbC (33.3 PM) (m); dimethylnitrosamine (A), acetylcholine (o), and diphenylnitrosamine (x) to the nicotinic cholinergic receptor in 1OOmM phosphate buffer (pH 7.2). The mixture was excited at 295nm and emission measured at 340nm.
This reflects
one class
and two classes contents
(Kd)
of binding sites at lower d-tubocurarine concentrations of binding sites at a higher concentration. The dissociation can then be estimated and represented (Table 1). For all 12 . 10 .
6. 4. 2
2
4
6 (Lp3
Fig.
3
8
10
12
14
16
18
x 10-l 31
Analysis data for the binding of d-tubocurarine (d-TbC) (50 PM) (0); d-TbC (33.3 ~14) (m); dimethylnitrosamine (A); acetylcholine (a); and diphenylnitrosamine (x) to the nicotinic cholinergic receptor in 1OOmM phosphate buffer (ptl 7.2).
1387
Vol.
167,
No.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
Table
RESEARCH
COMMUNICATIONS
1
values obtained by fluorimetric titrations and (DPrN), diphenyl (DPhN) dibutyl,p,"Pt:$i%t-~t?;,oma5,'ra,nto:ad:d,:kfl (DMN) dipropyl nitrosamine (DBuN), acetylcholi;e (ACh) and d-tubocurarine (d-TbC) on nicotinic cholinergic receptor FLUORIMETRY Ligand
EC5,-,(W
d-Tbc ACh DPhN DMN DPrN DBuN
Kd,(uM)
Kd2(IrM)
.05
11.4
.12
2.3
1.45
27.3
1.96
8.4
37.2
1.84
37.0
1.87
11.7
36.8
41.1
1.91
12.1
37.9
.398 .790 56
9.13
61 77
nitrosamines
there
representing
a weak
10.60
are
SPECTROPHOTOMETRY
two equilibrium
association
cooperativity
and binds to the receptor with ~11 these results are consistent
of fluorescence
receptor-ligand samine
intensity
complex
from
site
monitored
by measurement
protein.
The protein
in the presence difference
in protein
directly is
the
(Figs.
to the
parameter
5 and 6) for
of more than
equilibrium
dissociation
least than
represented
50% more the
nitrosamines Though Only
One Class
I).
to the with
of binding
site
representation exhibits positive
-
12 x 10m2,M that the
q
to
of the
absorbance is
nitro-
DMN is
of binding for
linear
site
high
at low
Kd values
d-tubocurarine 7) it 1388
and
plot the
receptor.
The can be estimated
appeared
concentrations
to bind
(Kd
concentrations
noted
to
as the to be
indicated
q
at
1.3 LIM)
all
PM.)
and acetylcholine is
large
measured
- nAChR complex
but
at the
(27-38
The
The Scatchard
and low affinity
Yet at higher
4).
is considered
The diphenylnitrosamine receptor
(Fig.
sufficiently change,
(230nm)
not
of the
210nm and 280nm
shown
of nitrosamine
were
(Fig.
1.92 1.96 1.98
- One
wavelengths
such an interaction.
similar
seemed that
35.6
to nAChR was also
The signal
concentration
constants
2.03
of acetylcholine
ultra-violet between
at a wavelength
nitrosamines.
behaved it
data.
one class
tightly
in the
nitrosamines
(Table
aliphatic
ligands
upon binding
to follow
the
existence and are
maxima
2.0
27.0
displacement
of dimethylnitrosamine
titration
1.68
3.1
constants
addition
P
acetylcholine.
spectrum
absorbance
proportional
a reliable
physical
of changes
of the
between
by the
of nitrosamine
absorption
and absence
evaluation
difference
caused
upon
common for
The binding
Spectrophotometry
allow
are
a binding
.I8
two affinities (Kd with the interpretation
observed
M)
8
other a more tighter Acetylcholine also
cooperativity.
Kd2(v
1.30 9.6
dissociation
and the
M)
.033
2.01
to positive
changes
Kd,b
1.79
in addition and 2 PM).
P
that
interacted at high
with
concentration
Vol.
167, No. 3, 1990
BIOCHEMICALANDBIOPHYSICALRESEARCH
COMMUNICATIONS
1.5
1 Y i I s z 0.5
04
210
220
230
240 YAYELENGTH
250
260
4
Ultraviolet absorptlgn (100 ~1; 100 pg ems dimethylnitrosamine
Fig.
5
Scatchard
and Kd ) !-eceptor2as number of moles
the
the antagonist the results
one of low affinity
(Fig.
1.5
2.0
2.5
spectrum of nicotinic cholinergic receptor ) in the preSence of (m) and absence (0) of (2.83 ,.,g ems ) in imidazole buffer (IOOmM, pH 7.4).
u are
nitrosamine.
1.0
"
plot for the binding (Kd nicotinic cholinergic
the spectrophotometry.
of
0.5
280
(nn)
Fig.
(a); to
270
are consistent 7, inset).
of dimethylnitrosamine determined by of
bound
with two sites
dimethyl-
- one of high and
DISCUSSION Our studies have shown t hat the two nitrosamines - dimethylnitrosamine DPhn) interact with the agonists binding sites (DMN) and diphenylnitrosamine ( receptor. Though the results are not presented of the nicotinic acetylcholine and dibutylnitrosamine) the other nitrosamines studied (dipropylnitrosamine exhibit similar effects. The potency of a particular nitrosamine in the interaction with the receptor is determined by the dissociation constants (Kd) for the receptorThe fit into the active site of the protein, reflected by the ligand complex. values of Kd, is largely determined by the size, structure and configuration The capability of such a ligand to bind, non covalently at, or of the ligand. close to, the active site could also influence these values. Two fundamental properties of the interaction of the nitrosamines to the receptor agonist active site are implied. First, there is at least one protonated residue which combines with the ligand within the receptor-complex. Second the receptor active site must have a hydrophobic nature. Thus when the nitrosamines environment.
bind to the receptor they pass from a hydrophilic to a hydrophobic This change in environment is a prequisite for good binding of 1389
Vol.
167,
No.
3, 1990
BIOCHEMICALAND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
36 Kd2
32
1 Kdl
28
. I ‘,
24
0.5 x
3 Y 2
-ii 2. L r3 2
l
20
0
l!iiJ 12
3
4
16 Y 12
\
8 4
0.5
1.0
2.0
1.5
2.5
07
Y
Fig.
6
1.0
0.5
1.5
2.0
2.5
Y
Scatchard
plot for the binding (Kd and the nicotinic cholinergic rkeptor v are the number of spectrophotometry.
Kd ) of diphenylnitrosamine is determined by moles of bound diphenyl-
(m) to
nitrosamine. Fig.
7
Scatchard
plot
for
the
ligand.
binding (Kdp) of d-tubocurarine (A) and the nicotinic cholinergic receptor as (L)f is the concentration of by spectrophotometry. The inset v are the number of molesof bound ligand.
to the
active
acetylcholine (x) to determined
acetylcholine
site
and has already
been
invoked
free
is
as an important
factor.(") If these that
the
alkylating the
observations
ligand
are
decomposes,
species
hydrophilic
(R+)
(Scheme
and size
in the
value
of the
of the
then
as it
1).
and hydrophobic
energy
accepted
as soon
the
reaches
Obviously
environment
carbocation
reaction the
the coupled
constants
with
the
formed
(Kd)
site
suggests
into
an
ease of transition
and intermediate
dissociation
mechanism
active
for
the
between
overall
will
stability,
be reflected
receptor-nitrosamine
complex. Detection binding study
with
is
the
ligand
quenching
of the ligands
induced
fluorescence arise
is
the
containing
binding The intrinsic
nitrosamine
protein
moiety
due to the
one of the
with when
presence site
receptor
ligand the
the
ligand
fluorescence
and diphenylnitrosamine
In the present upon binding with
to the regions
in or
to
investigation the the nitrosamines
a change
protein
upon method
The decrease
supports
binding
in a protein
and most direct
interactions.
of non-polar of the
change
simplest
change.
of the
on titration
nitrosamine
fluorescence
conformational
of fluorescence
used to study
the
intrinsic
in relative
in environment
takes
place.
around
the
This
tryptophan
receptor. of the with 1390
receptor
is quenched
emission
observed
by dimethyl-
at 336nm.
d-
of may
Vol.
167,
No.
3, 1990
Tubocurarine, specific is
a specific interaction
competition
DMN are are
BIOCHEMICAL
of DMN with
of the
for
RESEARCH
antagonist
of the
receptor.
This,
receptor
to support
COMMUNICATIONS
receptor
blocks
and the
fact
any
that
when both
acetylcholine
idea
the
the
that
there and
nitrosamines
agonists.
Scatchard
binding
BIOPHYSICAL
of the
receptor
cooperative sites
the
site evidence
nicotinic
The curves binding
active
is clear
potential
positive
competitive
at the
present,
AND
plot
and the
acetylcholine,
(Figs.
5, 6 and 7) indicate
existence
of more than
d-tubocurarine
and the
a
one class
of
nitrosamines
at the
receptor. Both reversible
the
low and high
and are displaced
tubocurarine
(Fig.
explained
over
competitive
which
well
the nitrosamine in sufficient
with
cholinergic
range takes
Furthermore
very
binding
by the
The inhibition
a 300 fold
model
nitrosamines. agree
1).
affinity
the
of tubocurarine into
account
binding
values
from
observed
in the
displacement
of nitrosamine
bound of the
the
effective
concentration
Kd values
from
It
is
the
noted
blue-shift
of
spectral
properties
sensitive
to the
suggests
agonists,
while
fluorescence (Fig.
either
of the
wavelength other
the
is
independent
emission
nature
the
where
a small
portion
of that
population
properties
of the
molecules
bound
presence
emission
ligand
sufficient
receptor spectrum,
interacts has been to the
of a blue-shift
second
with
and/or
the
displaced, class
while
of the
in acetylto
with
effector
the (d-
Nitrosamines
even
can be taken 1391
The quench
site
site.
as
therefore,
remaining bound site the emission
to the
receptor
act
shift,
a
The
blue-shift
of d-tubocurarine
sensitive at the
receptor.
ligand.
receptor
to the
the
of the
of the
bound
Nevertheless
the
occupancy
bound
the
cause
The spectral
as any nitrosamine the one receptor
or acetylcholine)
of
information
presence
and
1 that
of
character
tubocurarine
fraction
to the
(Fig.
Extension
by the
dominate
spectrum
appear
agonist
is
by fluorescence
sites
which
wavelength
Furthermore
receptor
compounds
of the
site.
bind
as antagonists.
With
competitive
nitrosamines
all
sites.
fluorimetry
bound
to the
the
agreement.
fluorescence
act
caused
receptor determined
bound.
1) can be interpreted receptor
from
for
The values of two methods used are decrease in
in excellent
compound
of the
intensity
choline
of the
others
seems characteristic
of the
by a
analysis.
agonist
are
can be
constants
is due to the
nitrosamine
ligands
that
all
estimated
to the
when the
of the nature
constants
of ligand
binding
analysis
12nm occurs
obtained(")
presence
direct
from
concentrations two binding
Scatchard
and d-
by d-tubocurarine
the
the
are
acetylcholine
constants estimated by the lead us to conclude that the
fluorescence
the
nitrosamines
agonists
of DMN binding
the
dissociation agreement to
of the
under
conditions When
protein. the of sites
spectral becomes
as evidence
for
apparent. a
Vol.
167, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
change characteristic of an agonist ligand and associated with the physiological response of the receptor protein. The fact that the nitrosamine binding functions must be characterised by both high and low affinity components explains the paradoxical observations that very high concentrations of d-tubocurarine (200. LIM) are necessary to completely displace the nitrosamine from the site binding the tubocurarine. The observed competition between the nitrosamines and tubocurarine and between acetylcholine and nitrosamines can be accounted for by a simple model that assumes that they all bind to a common site and most probably with a 1 : 1 stoichiometry. This model predicts that the total number of binding sites for dimethylnitrosamine, diphenylnitrosamine, acetylcholine and d-tubocurarine structural
and dibutylnitrosamine) would be identical. Indeed our results number of binding sites for DMN is-1.84, for DPhN is 1.96, for ACh is 2.01 and for d-tubocurarine is 1.79. In order to further unravel the mechanism of ligand interaction of the receptor we have used fluorescence titrations and spectrophotometry of various nitrosamines with the protein. The employed receptor preparations, monitoring ligands and techniques were particularly suited for this purpose. The receptor, isolated from Torpedo exhibited positive cooperativity of acetylcholine binding sites as well as with the nitrosamine ligands. Since the latter were regarded as full agonists to the frog rectus abdominis muscle and the Torpedo membrane, it must be assumed that they bind by the same mechanism. A complete analysis in terms of Kd values of binding equilibria of the receptor with a set of agonists and antagonists is presented. (and
dipropyl
conclude
that
the
We acknowledge the Medical
Research Council
(South Africa)
for financial
support. REFERENCES 1. Maelicke, Roberts, 2. Changeux,
3.
A.
(1981) in Molecular Pharmacology (A.S.V. Burgen and G.C.K. Elsevier North Holland ~~1-22. J-P., A. De Villiers - Thiery and P. Chemouilli. (1984) Science 225 : 1335-1345. Eid, P., P. Goeldner, C.G. Hirth and P. Jost. (1981) Biochemistry 20 : eds.).
2251-2256. Kapp, E.A.
and C.G. Whiteley. (1989) Biochem. Biophys. Acta (In Press). Ingelman-Sundberg, M. (1980) Pharm. Sci. 1 : 176-179. ;: Meunier, J.C., R. Sealcock, R. Olsen, and J-P. Changeux. (1974) Eur. J. Biochem. 45 : 371-394. 7. Kaneda, N., F. Tanaka, M. Kohno, K. Hayashi, and K. Vagi. (1982 ‘1 Arch. Biochem. Biophys. 218 : 376-383. 8. Scatchard, G. (1949) Proc. Natl. Acad. Sci. 51 : 660-672. 9. Khan, M., M.V.Ki Sas&y and A. Surolia. (1986) J. Biol. Chem. 2.61 : 3013-3019. 10. Jencks, W.P. (1975) Adv. Enzymol., 43 : 219-410. 11. Bennett, M.V.L., M. Wurzel and H. Grundfest. (1961) J. Gen. Physiol. 44 :
4.
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