Nervous System Membrane Proteins
Approaches to the purification of the 5-hydroxytryptamine reuptake system
from human blood platelets
Eric A. L. Biessen,* Alan S. Hornt and George T. Robillard
Department of Physical Chemistry and The Institute BIOSON and *Department of Medicinal Chemistry, University
of Groningen, Nijenborgh 16,9747 AG Groningen, The Netherlands
Introduction
Serotonin (5-HT) is one of the classical neurotransmitters. Although its physiological function is not
yet fully elucidated, it is widely accepted that the
central serotonergic system is involved in modulatory processes of affective or perceptual states and
migraine [ 1, 21. Dysfunction of the central 5-HT
system has been frequently implicated in the pathogenesis of various disorders, including chronic
depression [3-51. In the periphery, 5-HT regulates
the sensory input and motorial output, and plays a
role in pain perception [ 6 ] , gastric motility (via
S-HT,,-type receptors) and cardiovascular regulation [7]. Both the vasoconstrictive action of 5-HT
and the induction of the platelet ‘shape-change’
reaction are mediated via 5-HT2 receptors [8,9].
Physiological pathway
After synthesis, serotonin is stored in synaptic
vesicles via an ATP-dependent, low-affinity transport system. Depolarization of the presynaptic endplate membrane induces Caz+ influx, which in turn
triggers the release of serotonin, possibly by activation of intracellular microtubules [lo]. The excreted
serotonin diffuses to the postsynaptic membrane
and binds to and activates postsynaptic receptors.
Concomitantly, 5-HT is taken up into the presynaptic neuron to terminate the activation of postsynaptic receptors and to relieve 5-HT synthesis. The
relevance of reuptake for homoeostasis of 5-HT can
be derived from the high efficacy of 5-HT recycl-
Abbreviations used: 5-HT, 5-hydroxytryptamine (serotonin); NEM, N-ethylmaleimide; diamide, 1,l-azobis( N , N)-dimethylformamide; IBS, the imipramine binding site component of the 5-HT reuptake system; DTT,
dithiothreitol; h.i.c., hydrophobic interaction chromatography; GARA, y-aminobutyric acid; PAO, phenylarsine
oxide; LSL), lysergic acid diethylamide; EG, ethyleneglycol; ICs,,, concentration required to inhibit by 50%;
2A1, 2-aniinoimipramine;AMC, aminomethylcitalopram;
WGA. wheatgerm agglutinin.
*Present address: Department of Biopharmacy, Sylvius
Laboratory, Wassenaarweg 72, 2333 HI, Leiden, The
Neth,erlands.
tlleceased.
ing. Serotonin is translocated into the presynaptic
neuron via an active Na+-dependent transport system. Following reuptake it is stored in synaptic vesicles by a reserpine-sensitive, H+-dependent, active
transport system and is available for release again
[ 111. It has been postulated that the Na+-dependent
5-HT-reuptake system is functionally associated
with the hypothetical presynaptic 5-HT autoreceptor [12, 131 and, as such, is involved in feedback
processes regulating 5-HT synthesis and release.
Alternatively, accumulated 5-HT (or agonists like
LSD) can exert a regulatory effect intracellularly
without requiring the existence of an autoreceptor
~41.
Mechanism and structure of Na+dependent serotonergic reuptake
As stated, reuptake of neurotransmitters plays a
crucial role in terminating synaptic signal transduction. All of these transporters have a common absolute requirement for Na+ [ 151.
The driving force behind the transport is a
chemiosmotic ion gradient over the cell membrane.
These gradients are established by the electrogenic
N a + /K -ATPase. Serotonin transport is considered to be a two-step process. The first step
involves co-transport of 5-HT with Na+ and CI(Kin)into the cell. The second step involves K + (or
H + ) translocation from the cell interior to the
medium (Kout).K,,, appears to be rate-limiting.
Serotonin reuptake can be suppressed specifically
by reuptake inhibitors like imipramine. These compounds bind to the transporter but are not translocated over the cell membrane. There are
conflicting data on the functional size of the component containing the binding site. Radiation inactivation has uncovered a component with an
apparent molecular mass of 86 kDa and 67 kDa for
the [3H]imipramine and [3H]paroxetine binding
sites, respectively [ 161. The above discrepancy
might just indicate a different type of interaction of
tricyclic and non-tricyclic reuptake inhibitors with
the transporter complex. The photo-affinity labels
2-nitroimipramine and 2-azidoimipramine have
been specifically incorporated in a 30-35 kDa protein [17, 181. Whether this protein represents the
reuptake system is questionable.
+
1991
I03
Biochemical Society Transactions
I04
Chemical modification studies have revealed
that the serotonergic [ 19-21], the GABAergic [22]
and the noradrenergic reuptake systems [23]
possess (di)thiols essential for ligand binding and
activity. Blockade of the actual binding site by the
respective ligands provides at least partial protection against thiol modification.
Although serotonin transport appears to be
more or less clarified on a macroscopic level, the
molecular basis of reuptake is poorly understood.
Isolation of this class of co-transporters has proven
to be very tedious. The GABAergic system was
solubilized and purified to homogeneity, revealing a
80 kDa glycoprotein [24,25]. However, only a sohbilization and a marginal purification of the serotonergic [26-301 transport system have been
achieved.
Coupling between carrier and
regulatory site
The imipramine binding site (IBS) is presumed to
be identical to or functionally associated with the
actual 5-HT-carrier. Three observations led to this
assumption. Firstly, the regional distribution of the
IBS closely corresponds to that of 5-HT innervation
[31, 321. Secondly, Sette etal. [33,34] demonstrated
that denervation of serotonergic neurons in the
dorsal RaphC nuclei using 5,7-dihydroxytryptamine
or electrolytic lesions eliminated both the IBS and
the 5-HT transporter to the same extent. Thirdly, a
significant correlation between the displacement
profile of [3H]imipramine binding and inhibition
profile of 5-HT reuptake has been reported by
various research grovps [35, 361. Although the
functional association of the IBS with 5-HTreuptake is commonly accepted, the nature of the
coupling between or identity of the IBS and the
5-HT-carrier is still subject to considerable uncertainty. Several lines of evidence support the hypothesis that the system consists of a mutually
exclusive regulatory and camer site. In 1983,
Barbaccia et al. showed that down-regulation of the
IBS density after chronic administration of imipramine was accompanied by an up-regulation of
5-HT reuptake in rat hippocampus [37]. Differences in the Na+ requirement of imipramine binding and 5-HT transport were reported [38]. Some
displacement, 5-HT-reuptake and chemical modification studies indicated different types of interaction
for tricyclic and non-tricyclic antidepressants or
serotonin with the 5-HT-reuptake system [ 19,
39-41].
Volume 19
Platelets as a model system for
neuronal reuptake
Platelets have proven to be a reliable and predictive
model for the pharmacological and biochemical
characterization of the 5-HT-reuptake system. The
5-HT-reuptake system in platelets and neurons
share many similarities. Both systems possess two
separate carriers for serotonin: a rather non-specific,
ATP-dependent system, which is reserpinesensitive, and a specific, imipramine-sensitive,Na coupled reuptake system. The pharmacological
profile of the latter in platelets strongly correlates
with that in brain synaptosomes [42]. Additionally,
kinetics of transport of both systems are similar.
5-HT-reuptake in platelets, on the other hand,
appears to be less selective for serotonin, in that it is
also capable of transporting noradrenaline, dopamine and tryptamine [43, 441. The IBS-receptor
distribution in the brain of Fawn-Hooded rats with
a hereditary deficiency in platelet serotonin storage
is not markedly changed, implying a different type
of genetic control [45]. In summary, decisive evidence on the identity of both systems is lacking, but
the similarities suggest that the platelet is an appropriate model system for studying neuronal 5-HT
reuptake.
+
Results
Cysteine as a potential site for selective attachment of M n i t y labels
Preliminary experiments indicated that the redox
state of the reuptake receptor varied from one
membrane preparation to another and manifested
itself in an altered B,,,. value. Thiol modification
experiments indicate that dithiothreitol (DT") had a
stimulating effect on the binding of imipramine by
enhancing the B,,, by 35%, presumably by
decreasing of the fraction of oxidized IBS. Spontaneous oxidation appeared to lead to a 60% decrease in binding, whereas chemical oxidation with
1,l-azobis(N, N)-dimethylformamide
(diamide)
resulted in a 90% decrease in binding capacity. NEthylmaleimide (NEM), diamide and
(PAO)
induced a pronounced loss of [3H]imipramine
binding with a t1,2 of 22 min, 10 min and 15 min,
respectively. Saturation curves were measured and
showed that the modification by NEM, P A 0 or diamide only affected Bmm,.The affinity remained
virtually unaltered. The reversibility of P A 0 and
diamide inactivation and the ability of P A 0 to protect against alkylation by NEM was also tested.
Nervous System Membrane Proteins
After gross reduction of the IBS with 2.5 mM-DTT,
the washed membrane suspension containing 1
mM-DTT was exposed for 30 min at 20°C to 1 mMP A 0 or 5 mwdiamide. The mixture was then put
on ice. Protection against NEM alkylation was
tested by subsequent treatment of the suspension
with 7 mM-NEM for 30 min at 2o"C, followed by a
reduction step with 10 mM-DTT. Reversibility of
the oxidation was determined by reduction of the
P A 0 and diamide treated samples with excess
DTT. Diamide, P A 0 and NEM inactivate the
5-HT-reuptake system to about the same extent.
The diamide- and PAO-induced inhibition was
reversible; the [3H]imipramine binding returned to
75% of its original value. The fact that reversible
reaction with P A 0 protected against NEM-induced
alkylation indicates that NEM also reacted with the
same dithiol.
Fig. 1 examines the ability of tricyclic and
non-tricyclic compounds to protect against inactivation by P A 0 [ 191. PA0 treatment resulted in a 90%
decrease in [3H]imipramine binding. This inactivation was prevented by preincubation with tricyclics.
Even though non-tricyclics are known to be able to
displace imipramine from the IBS, they do not protect against PA0 modification. The observed inactivation after preincubation with non-tricyclics was
not due to incomplete washing, since the washing
control and the untreated control both showed the
same high value.
The rather slow NEM modification observed
in our laboratory and by Biassoni & Vaccari [21]
and Davis [ZO] suggests that NEM might inactivate
by alkylating an inert or hidden thiol group or by
reacting non-specifically. In view of the data
presented above we can exclude the latter. Either
the thiols are chemically inert or not readily accessible. The observed loss of [3H]imipraminebinding
activity after thiol modification could be explained
as an alteration in tertiary structure or as a steric
hindrance of the binding, or it could implicate the
thiol directly in the process of drug binding itself.
The selectivity with which tricyclics are able
to protect the IBS from modification by thiol
reagents affords a possible route for purification
analogous to the recently developed purification of
the calcium release channel from sarcoplasmic reticulum [46]. Cysteines outside the binding site
would be alkylated with NEM while the binding site
cysteine was protected by tricyclic compounds.
Upon removal of the tricyclics, the binding site
cysteine could be selectively labelled with
2-pyridyl-dithiopropionate-biotin conjugates and
Fig. I
Protection against PAO-induced thiol modification by
various reuptake inhibitors [ 191
Non-tricyclic inhibitors were: citalopram I0 p~ (cit);
fluoxetine 10 p~ (flu); femoxetine 10 p~ (fem); zimelidine 100 p~ (zim); norzimelidine 100 p~ (nor), and tricyclic-reuptake inhibitors were: irniprarnine I0 p~ (imi);
cyanopramine 10 ,UM (cya); clomipramine 10 p~ (clo);
amitryptiline 10 p~ (ami), and finally the endogenous
drug, 5-HT, 100 p ~After
.
reduction of the membrane
suspension with 25 mM-Dl-r (I 5 min, 20°C) the samples
were preincubated for I h (4°C) with reuptake inhibitors, followed by thiol modification with PA0 ( I mM, 30
min, 20°C). After the modification, the samples were
washed three times to remove the reuptake inhibitor
and [3H]imipraminebinding was measured. No D l 7 was
present during the binding measurements. The specific
[3H]imipraminebinding as a percentage of non-exposed,
non-preincubated control is plotted on the vertical axis.
Each value is the mean, k EM., of the number of experiments given in parentheses. Each experiment was run in
quadruplicate. Significance **P < 0.00 I compared with
control, *P < 0.0 I, tttf < 0.00 I compared with PAOexposed sample, ttP < 0.005, P
t C 0.0 I. A control of the
wash step was included.
I
Protocol:
Ligand
the protein isolated on avidin-Migel. Our initial
studies have shown that such a procedure will have
to be applied to intact platelets membranes rather
than solubilized and partially purified IBS; once
solubilized, the ability of tricyclics to protect against
non-specific NEM inactivation is sharply reduced.
This is presumably the result of exposing additional
cysteines upon solubilization which cannot be protected by the presence of tricyclic compounds.
1991
I05
Biochemical Society Transactions
I06
Hydrophobic interaction chromatography as a
possible purification step
Hydrophobic interaction chromatography (hic.) is
commonly employed in the purification of
membrane-bound proteins. Separation is based on
differences in the nature of the hydrophobic
domains of these proteins. The behaviour of the
5-HT-reuptake system on hexyl- and butyl-agarose
was assessed to establish the potential role of h.i.c.
as a prefractionating step in the purification of the
protein. Two types of experiments were performed.
The first involved direct application of crude solubilizate [platelet membranes solubilized in NaSCN
and 1% (w/v) digitonin] to the hydrophobic interaction column. The second involved application of
solubilizate, that had been previously chromatographed on hexyl-agarose, but did not bind. Application of the. crude solubilizate, previously dialysed
against buffer containing up to 500 mM-potassium
phosphate (KP,) (pH 7 4 , did not result in any
adsorption of binding activity whatsoever. When
partially purified solubilizate was used, in buffer
containing up to 1000 mM-KP,, at pH values down
to 6.0, in the presence of 100 nwimipramine, or at
20°C instead of the usual temperature of 4°C the
recovery of activity in the flow-through still
amounted to 80%. Under rather stringent conditions, using 40% (v/v) ethyleneglycol (EG) in 20
mM-KPi buffer, no specific elution of binding
activity was observed. Since the reuptake system
Fig. 2
Inhibition curves of hexyl- (0).heptylamine ( A )
(0) and
octyl-
Solubilizate was assayed in the presence of alkylamines,
at concentrations ranging from 0.1 mM to 10 mM. The
specific binding is plotted (as a percentage of the unexposed control, mean ~ s . E . M .of a determination in
triplicate) against the concentration of alkylamine.
Concentration (msi)
Volume 19
was not retained on hexyl- and butyl-agarose, more
hydrophobic resins like octyl-, phenyl- or decylagarose were tested Subjection of crude solubilizate
to these resins in 20 or 500 mM-KPi (pH 7.5)
resulted in a 6% and 26% recovery in the flowthrough, respectively. An average recovery of 4%
and 9%, respectively, was obtained upon elution
with up to 60% (v/v) EG in 20 mM-KPi or 5 mMKP, buffer. When hexyl-pretreated solubilizate was
applied to either octyl-, phenyl- or decyl-agarose, in
20 mM-KP, containing 20% (v/v) EG, 100 mMNaCI, 100 nwimipramine or 20% (v/v) EG and
100 nwclomipramine, 16%k 3% of the IBS eluted
in the flow-through and only 6%f 2% during the
elution step [using 20 mM-KP, containing up to 60
(v/v) EG]. At first glance, the observations with
butyl-/hexyl-agarose on one hand and octyl-/
decyWpheny1-agarose on the other, seem paradoxical. The former two resins did not adsorb binding
activity even under extreme conditions, while the
latter caused a complete loss of binding activity
under all conditions.
Binding studies
These results could signify an inactivation of binding activity rather than an 'irreversible' adsorption
upon exposure to the alkyl-agarose. One might
speculate that the phenyl, octyl and decyl moieties
interact or compete with a factor essential for the
activity of the solubilized receptor. Removal of an
essential phospholipid is the first option that one
might consider. Hexyl-pretreated solubilizate,
dialysed against 5 mM-KPi, containing 0.1% (w/v)
digitonin and 20% (v/v) EG, was applied to a 0.5 g
octyl-agarose column, equilibrated with the above
buffer with 0.03% (w/v) egg yolk L-Qphosphatidylcholine (type IX-E) or Escclerichia coZi
L-a-phosphatidylethanolamine (type IX), purified
by ether/acetone extraction as described by
Kagawa & Racker [47]. The column was washed
and eluted with 2 ml of the same buffer containing
0.1% (w/v) digitonin, 40% (v/v) EG and phospholipids. However, no significant activity was
observed either in the flow-through or in the elution
fraction.
One might argue that a specific interaction of
long-chain alkyl residues with the solubilized protein occurs, which inactivates the protein. In order
to investigate this possibility, the characteristics of
["Iimipramine binding in the presence of several
alkylamines or alcohols with varying chain lengths,
was studied. Fig. 2 illustrates the effect of hexyl-,
heptyl- and octyl-amines, at concentrations ranging
Nervous System Membrane Proteins
from 0.1 to 10 mM, on [3H]imipraminebinding. The
potency of the compounds to inhibit ['Hlimipramine binding strongly correlated with the length
of the alkyl chain. Octylamine, heptylamine and
hexylamine displayed ICs, values of 0.42 mM, 4.1
mM and > 10 mM, respectively. The same tendency
was observed for the series of alcohols, suggesting
that specific interactions of the alkyl moiety with
either the micellar structure or the solubilized protein itself underlie these phenomena. This interaction may denature the 5-HT-reuptake system
resulting in a reduction in B,,,. or gradually affect
the binding properties of the IBS, as expressed by a
change in Kd. Saturation binding studies were performed in the presence of 1 mM-hexyl- and octylamine. Hexylamine did not affect the B,,, and only
slightly increased affinity (4328 f 155 c.p.m./50 pl;
1.1 f 0.1 nM compared with 4327 f 92 c.p.m./50 pl;
0.7 f 0.1 nM for the control; P< 0.1). By contrast, the
octylamine-induced inactivation arose from a
change in af€inity (4.4f0.9 nM compared with
0.8fO.1 nM for the control; P<0.0005); the receptor density remained unchanged (4558 & 172
c.p.m./50 pl compared with 4910 c.p.m./50 pl for
the control). In general, since long-chain alkyl
groups, at concentrations matching the capacity of
the alkyl-agarose, have a detrimental effect on
['Hlimipramine binding, we may safely infer that
the same processes underlie the inactivation of the
IBS upon chromatography over octyl- or decylagarose.
The next issue to be considered involves the
reversibility of this inactivation. Fig. 3 depicts the
effect of removal of octylamine in 50 m w W i buffer.
A 60% reactivation of binding activity, relative to
the untreated control, is observed after 43 h
dialysis; it should be kept in mind that ["Iimipramine binding of the untreated dialysed control
amounted to 77% of the undialysed control. Reactivation levelled off at 7 5 4 0 % of the control value
after 100 h of dialysis. Due to the spontaneous
inactivation of both the control and the octylaminetreated sample with time, a more prolonged dialysis
of both samples was not informative.
The slow kinetics of reactivation could reflect
a slow rearrangement of the protein or a retarded
removal of octylamine. T o check this, solubilizate
was exposed for 1 h at 23°C to 10 mwoctylamine,
subsequently diluted to 5, 3, 1, 0.5 and 0.3 mMoctylamine with 100 mM-Wi, incubated for 1 h at
ambient temperature and assayed for ["H]imipramine binding. An abrupt reactivation was
observed after a 20-fold dilution of the octylamine
Fig. 3
Effect of dialysis of octylamine-exposed solubilizate
on [3H]imipramine binding
Solubilizate was incubated for I h at 4°C with 10 mMoctylamine in 500 mM-KP, buffer containing 1 % (v/v)
dimethylsulphoxide, 2 mM-MgCI,, I mM-EDTA, 0.3 mMD l 7 , 0. I mM-PMSF, and subsequently dialysed against
100 mM-KP, buffer, containing 2 mM-MgCI,, I mM-EDTA,
0.3 mM-DTT and 0. I mM-PMSF (pH 7.5). At various timepoints, samples were collected and assayed, in triplicate,
for [3H]im~praminebinding at 43 h after onset of the
dialysis (0)or at I10 h after onset of the dialysis (0).
The recovery is expressed as a percentage of the
untreated dialysed control. Binding activity of this
dialysed control amounted to 77% of the undialysed
control after 43 h and 50% after I I 0 h of dialysis.
loor
8o
2
t
-1'P
60!-
X
X'
1-
I
1
1
I
1
I
20
40
60
80
100
I20
Time (h)
incubate. The combined data suggest that there are
binding sites on the reuptake protein specific for
long-chain aliphatics. Binding at these sites raises
the Ki for imipramine thus causing an apparent
inactivation when assaying at low imipramine concentrations. Whether binding at these sites was
responsible for immobilizing the carrier on octyl-,
phenyl- or decyl-agarose was examined by using
octylamine in the elution step of the chromatographic procedure, followed by prolonged dialysis
of the eluted fraction. Preliminary experiments have
demonstrated that binding activity could not be
recovered using this approach. Additional sites
appear to be involved in retaining the protein on the
resin. We have yet to find a method to selectively
remove the 'carrier in an active form.
Development and application of affinity
Chromatography resins for purification of the
5-HT-reuptake system
The Ki values for the inhibition of ["Iimipramine
binding to crude platelet membranes are in the
1991
I07
Biochemical Society Transactions
I08
5-20 nM range for imipramine, citalopram and
several derivatives of these compounds. These high
affinities make affinity chromatography an excellent
choice for purifying the reuptake system, provided
that the proper affinity resins can be developed. We
have synthesized a series of resins consisting of
immobilized citalopram, imipramine and serotonin
derivatives. Table 1 summarizes the attributes of
these resins.
Testing of the resins: retention of binding activity
The retention of binding activity to the affinity
resins is depicted in Table 2. All of the serotoninderived affinity resins show a 30% adsorption of the
IBS, regardless of the spacer type, the spacer length
and the nature of the coupling bond. This is presumably a consequence of its moderate affinity, in
the high nanomolar region, and corresponding high
dissociation rate. The desipramine resin shows an
encouraging 72% adsorption; however, the 5-HTreuptake system appeared to be irreversibly bound
or inactivated since binding activity could not be
recovered, even under stringent conditions like a
pH shock (pH 5). The 10-hydroxyimipraminederived resin does not show any adsorption as, in
fact, might be anticipated in view of the low affinity
of the parent compound, ketipramine. The
2-aminoimipramine-Afgel (2AI-Afgel), immobilized via an amide bond, shows a 65% retention,
which is considerably more than the adsorption to
the same ligand linked via an amine bond. No absolute generalizations can be made for imipramine
ligands linked at the 3-position. Of these resins,
the oxirane-coupled 3-aminomethylimipramine
Sepharose was most promising, whereas the 3-(1hexy1carboxamide)imipramine Sepharose performed badly. Apparently, the charge and chemical
nature of the functional group at the 3-position are
not the most prominent factors determining
adsorption to the ligand. Of the citalopram-derived
affinity resins, aminomethylcitalopram Sepharose
(AMC-Sepharose) and aminomethylcitalopram
Affigel (AMC-Afigel) displayed significant adsorption of binding activity. Binding is impaired when
using a C,,-spacer, possibly due to unfavourable
folding of the spacer arm.
Three affinity resins, serotonin-Afgel, 2AIAffigel, AMC-Sepharose and AMC-Afgel, which
are typical examples for the various types of
ligands, were subjected to more detailed study. As
expected from the moderate affinity, the IBS is only
slightly retained by serotonin-Afigel. Binding
Table I
Synthesis and properties of the affinity resins
Bond type indicates the nature of the coupling bond between the spacer arm and the ligand. The hydrophilicity of the
spacer arm is given in the last column: ( - ) indicates hydrophobic, ( + ) mildly hydrophilic and ( + + ) strongly hydrophilic, respectively.
Spacer
Resin
Ligand
Bond type
Length (A)
Hydrophilicity
Oxirane 3-aminomethylimipramine
-2-Aminoimipramine
-Desipramine
-Aminomethylcitalopram
- IO-Hydroxyimipramine
-5-HT
3-( I -Hexylcarboxamide)imipramine
NHS-Cl-AMC
-C,,-AMC
-C,,-AMC
Affi- I0 AMC
-3-Aminomethylimipramine
-5-HT
-2-Aminoi mipramine
2-5-HT (Sigma)
I
4
7
5
3
6
2
5
5
5
5
I
6
4
Sec. amine
Sec. amine
Tert. amine
Sec. amine
Ether
Sec. amine
Amide
Amide
Amide
Amide
Amide
Amide
Amide
Amide
Amide
19
19
+
+
+
+
+
+
Volume 19
6
19
19
19
19
13
2.7
I5
27
14
14
14
14
2.7
-
+
++
+
++
++
++
++
+
Nervous System Membrane Proteins
Table 2
Testing of affinity resins
Affinity resin (0.5 g) was incubated overnight with I ml TridHCI buffer, containing 0. I % (wh) digitonin and 0.5 ml solubilizate. After packing of the gel slurry,
the column was washed with I ml of the above buffer. The flow-through was
dialysed against two changes of TridHCI buffer and assayed, in triplicate, for
[3H]imipraminebinding. Recovery is given as percentage of the control binding
in the flow-through of an analogously treated Sepharose column. The maximal
absolute error in the recovery was 10%. Ligand capacity was determined by
hydrolysis of 0.2 g resin in 2 ml 0. I M-HCIat 90°C for 2 h. The extinction of the
hydrolysate at 250 nm was determined for imipramine-derived resins, at 280
nm for serotonin-derived resins, and at 237 nm for citalopram-derived resins,
and the capacity was calculated.
Resin
Oxirane 3-aminomethylimipramine
2-Aminoimipramine
Desipramine
AMC
I0-Hydroxyimipramine
5-HT
3-( I -Hexylcarboxamide)imipramine
NHS C,,-AMC
CIB-AMC
Affi- I0 AMC
3-Aminomethylimipramine
5-HT
2-Aminoimipramine
2-5-HT
activity elutes from the column just after the flowthrough at a recovery of 76%. The weak retention
makes this resin unsuitable for purification
purposes.
Chromatography over 2AI-Affigel gave a 56%
elution immediately afier the flow-through. When
assayed at 18.5 nM instead of 3.0 nM, 89% of the
binding activity could be recovered in the flowthrough, suggesting that the affinity of the IBS is
altered during chromatography and the dialysis
step. No elution whatsoever could be observed
during both the specific and non-specific wash-step.
Both of the citalopram-derived resins demonstrated
retention of the IBS. About 38% of the applied
activity could be recovered upon specific elution of
the AMC-Afigel column with 1 pM-imipramine.
Specific elution could also be observed with AMCSepharose; the recovery strongly varied from one
experiment to another (values ranging from 0 to
68%). Prior blockade of the reuptake system with
Ligand capacity
(pmol/ml)
0.7
3.7
5 .O
4.6
2. I
4.3
3.3
3.9
3.0
5.3
3.2
6.0
8. I
10-15
I09
Recovery
(% of control)
24
85
38
47
I10
66
71
62
95
38
41
72
35
69
citalopram obstructed adsorption to the AMCAfigel. Adsorption to AMC-Sepharose could be
prevented by prior incubation of the solubilizate
with 10 pwfluoxetine, 10 pwclomipramine and 50
pM-serotonin (see Table 3). AMC-oxiraneSepharose was tested repeatedly, but due to the low
reproducibility obtained using this affinity resin,
AMC-Afigel was favoured.
Purification
A two-step purification procedure has been
developed for partial purification based on wheatgerm agglutinin (WGA) afinity and citalopram
affinity chromatography [48].In a typical purification procedure, solubilizate was subjected to a prepurification step on WGA-Sepharose to reduce the
digitonin level and to eliminate some proteins or
phospholipids which might interfere with the
affinity chromatography step. Although most of the
protein eluted from the column in the flow-through,
1991
Biochemical Society Transactions
graphic steps (e.g. h.p.1.c. on Pharmacia Mono Q
columns), when applied earlier in the purification,
Effect of blockade of the 5-HT-reuptake system with
resulted in specific binding and elution of activity.
fluoxetine. clomipramine and serotonin on the retenThe pharmacological profile of the partially
tion of binding activity to amino methylcitalopram
purified
transporter was examined using displaceSepharose
ment binding studies with six typical reuptake inhiResin (0.4 g) was incubated overnight with 0.5 ml
bitors, namely 5-HT, imipramine, citalopram,
solubilizate, which was preincubated vyith 10 p ~ - zimelidine, femoxeline and clomipramine. Ki values
fluoxetine or I0 p > -clomipramine or 50 p-serotonin or
of these compounds were comparable with values
buffer for 60 min at 4°C. After packing, the column was
obtained using crude platelet membranes
washed with 0.5 ml TridHCI buffer containing 50 mM( r = 0.982) or solubilizate ( r = 0.927), suggesting a
TridHCI (pH 7.5) 100 mM-NaCI, 5 mM-KCI, 2 mM-MgCI,,
purification of the intact 5-HT-reuptake system.
I mM-EDTA, 0.3 mM-Dm, 0. I mM-PMSF and 0. I% (wh)
digitonin. The flow-through was collected, dialysed for 6
1. Tricklebank, M. D. (1985) Trends Pharmacol. Sci. 5,
h against I I and 18 h against 5 I assay buffer and assayed
403-407
for [3H]imipramine binding. Values are expressed as a
2. Fozzard, J. R. (1990) Proceedings of an International
percentage of total applied [3H]imipramine bindSymposium on Serotonin, pp. 81-90, Kluwer Ac.
ing f S.E.M.
Publ., Dordrecht
3. Van Praag, H. M. & Medlewicsz. J. (1983) Management of Depressions with Monoamine Precursors,
Preincu bation
Retention
Table 3
I10
50 pM-serotonin
I0 pM-clomipramine
10 pM-fluoxetine
67f21%
25f I I%
- 13f I I%
8f 13%
more than 88 f 4% (n = 8) of the imipramine binding capacity was retained. Elution of the column
using an N-acetylglucosamine gradient resulted in
70 f6% ( n= 8) recovery of binding activity. SDSPAGE of the eluted fractions confirmed the large
purification factor realized by this step. The purification factor was estimated to be 50-70-fold, both
on the basis of the EZs4
profile and scanning of the
polyacrylamide gels. The WGA pool was immediately incubated with AMC-Afigel. A column was
packed and washed. Directly following the washing
steps, binding activity was eluted using 10 PMcitalopram. Almost all the binding activity of the
prepurified solubilizate was retained by the affinity
column (91 f 2%; n=8). Binding activity could be
recovered in part (22f4W)upon elution with 10
,uM-citalopram. Analysis of the eluted fractions on
SDS-PAGE after labelling of the proteins with
Na1251revealed the appearance of a 78 kDa and a
55 kDa protein concomitantly with recovery of
binding activity. The purity was estimated to be
5 1 0 % on the basis of PAGE analysis, resulting in
an overall purification of 10000-fold. Complete
purification has not yet been achieved due to the
instability of the partially pure protein. All attempts
at additional chromatography resulted in loss of virtually all activity even though the same chromato-
Volume 19
Karger, New York
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Received 30 August 1990
1991