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Platelet Aggregation Inhibiting and Anticoagulant Effects of Oligoamines XXVInteractions of the Oligoamine RE 1492 with Biomembranes.

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21
Interactions of RE 1492 with Biomembranes
Platelet Aggregation Inhibiting and Anticoagulant Effects of Oligoamines, XXV3):
Interactions of the Oligoamine RE 1492 with Biomembranes
Annette Kesselhuta)+),Hans Ebel”, Franz v. Bruchhausenb),and Klaus Rehsea)*
Institut fUr Pharmakologieb),Institut fiir Klinische Physiologiec)and Institut fiir Pharmazie”)der Freien Universiat Berlin, Konigin-Luise-Str. 2+4,
14195 Berlin
Received April 25, 1994
The absorption of D-glucose by rat thymocytes is reduced to half of control
by 30 p o V L and decreased to 10 % by 100 pmoVL of RE 1492. This is
backed by the fact that the absorption of 2-deoxy-~-glucoseis inhibited in
the same extent. The more hydrophilic oligoamine RE 1888 had an analogous but smaller effect while spermine was ineffective. In a lipid peroxidation model RE 1492 or spermine in a concentration of 100 p o l / L nearly
completely inhibited for formation of Fe3+ions when the phospholipid was
mimicked by adenosine monophosphate. This suggests an interaction with
negatively charged membrane phospholipids. RE 1888 had an equal but
smaller effect. The effect of RE 1492 on lipid order and lipid motility was
checked on ovine lymphocyte membranes by fluorescence polarization
measurements. The steady state as well as the limiting anisotropy as an
expression for lipid order is decreased by rising concentrations of RE
1492. The use of several anthroyloxy stearic acids as fluorescent probes
also shows an increased lipid motility in several areas of the membrane
bilayer. The use of fluorescent parinaric acids suggests that areas of high
regularity, i.e. liquid crystal formation are involved, too.
The oligoamine RE 1492 (NJV’JV”-Tris-4-phenylbutyl-1,3,5-benzenetrimethanamine trihydrochloride) is a new drug which exhibits antiplatelet
and anticoagulant activities in v i f r d ) . These effects are correlated with
antithrombotic properties in v i v d ) . The common mechanism could be the
interaction of the cationic oligoamine with phospholipids which are
involved both in platelet aggregation and the fibrin formation processes.
The interaction with synthetic membranes, i.e. certain phospholipid vesicles, which we found recently3).prompted us to extend our investigations
on biological membranes and whole cells.
Antiaggregatorische und anticoagulante Eigenschaften von Oligoaminen, 25. Mitt.”:
Wechselwirkungen zwischen dem Oligoamin RE 1492 und Biomembranen
Die Aufnahme von D-(ilucose in Thymocyten von Ratten wird durch
30 pmol/L RE 1492 halbiert und durch 100
auf 10%der Kontrolle
verringert. Die Absorption von 2-Deoxy-D-glucose wird in gleichem
AusmaB gehemmt. Das hydrophilere Oligoamin RE 1888 hat eine
geringere Wirkung w h d Spermin wirkungslos ist. In einem Modell der
Lipidperoxidation wurde durch RE 1492 bzw. Spermin (ie 100 pnoVL)
die Bildung von Fe3+-Ionenfast vollst2ndig unterdriickt, wenn Adenosinmonophosphat zugegen war. Hieraus wird auf eine bevorzugte Wechselwirkung mit negativ geladenen Phospholipiden geschlossen. Der EinfluD
von RE 1888 war geringer. Der EinfluS von RE 1492 auf Lipidordnungsgrad und Lipidbeweglichkeit wurde an Membranen von Hammellymphocyten mittels Fluoreszenzpolarisation untersucht. Die limitierende
Anisotropie als MaB fur den Lipidordnungsgrad wird durch RE 1492
konzentrationsabhhgig verringert. Mit Anthroyloxyfettsauren als Fluoreszenzsonden konnte gezeigt werden, daB auch eine Erhohung der
Lipidbeweglichkeit in verschiedenen Membrantiefen gegeben ist. Durch
Octadecatriensauren konnte die Beeinflussung flussig-kristalliner
Membranareale nachgewiesen werden.
,NH-(CH&Ph
Cb
I
RE 1492
Interaction of RE 1492 with rat thymocytes
In order to study the influence of RE 1492 on transport
phenomena through the membranes of intact cells we have
chosen rat thymocytes and investigated the transport of
D-glucose into these cells. This is a carrier mediated,
facilitated diffusion process4) and thus indicating a fully
intact membrane including the carrier protein. When Dglucose reaches the cytosol it is metabolized partly via the
pentose phosphate pathway to ribulose-5-phosphate and
COz. In this case the latter exclusively stems from C-1 of D-
+)
Part of the PhD thesis A. Kesselhut
Arch. Phurm. (Weinheim) 328.21-27 (1995)
0-N
glucose. This metabolic transformation can therefore be
followed when 1-14C-D-glucoseis used. The amount of
14C02formed is absorbed by hyamine hydroxide.
0 VCH VerlagsgesellschaftmbH, D-69451 Weinheim, 1995 0366-6233/95/0101-0021$5.00 + .25/0
22
Kesselhut, E M , v. Bruchhausen, and Rehse
Tab. 1: Glucose transport into rat thymocytes measured as C 0 2 formation (pmol/108 cells x k
SEM, n = 3, t,) and uptake of 2-deoxyglucose (prnoV108 cells, x f SEM, n = 3, t2).
C
t’
~ m o ~ - L - l[min]
]
0
3
10
20
30
20
20
50
20
20
10
10
20
20
40
40
90
90
100
0
30
0
30
0
30
0
30
0
30
0
30
0
30
100
300
loo0
10
30
100
300
20
20
20
-
-
-
-
12
co2
[min]
[pmol]
[pmoll
15
15
15
15
15
15
15
1
1
2
2
5
5
10
10
15
15
96.03 f 1.47
96.99 f 1.15
81.04 f 0.91
56.95 f 0.70
48.04f 0.51
32.05 f 0.63
11.34 f 0.53
46.90 f 1.69
23.00 f 1.05
%.03 f 1.47
48.04 f 0.51
192.4 f 2.10
97.60 f 1.05
353.7 f 3.28
172.5 f 1.69
58.83 f 1.87
60.06f 2.81
52.48 f 1.94
35.79 f 1.53
30.35 f 1.17
20.43 f 1.22
5.58 f 0.67
3.67 f 0.38
2.08 f 0.53
7.58 f 0.94
4.62 f 0.82
19.22 f 0.63
10.15 f 0.69
36.46 f 2.15
19.23 f 1.48
58.65 f 2.01
32.61 f 1.59
77.88 f 1.94
40.55 f 1.53
82.32 f 2.17
42.01 f 1.46
60.32 f 1.84
58.14 f 1.59
59.41 f 2.04
57.96 f 1.92
52.18 f1.41
43.47 f 1.56
37.87 f 1.29
20
20
30
30
15
15
1s
15
15
15
15
The first part of Table 1 shows that the formation of C 0 2
is decreased by RE 1492 to 10% of the control values. In
order to assure that this effect is really due to the inhibition
of transport and not to the inhibition of enzymes involved
in the metabolic cascade a second method was applied. The
compound 2-deoxy-D-glucose uses the same carrier protein
in the same velocity5)as glucose but is not metabolized in
the cell. Its rise in concentration within the cell therefore
parallels the activity of the transport system. The results
obtained with UL- 14C-labelled 2-deoxy-D-glucose as a
function of various concentrations of RE 1492 are as well
shown in the first block of Table 1 data. They are corrected
for extracellular liquid by addition of 1-3H-mannitol. It is
obvious that the uptake of deoxyglucose is reduced in the
same ratio as this is the case with the I4CO2 formation.
The second part of Table 1 shows the time course of the
invasion of the two compounds at a RE 1492 concentration
of 30 pmol/L. It is striking that at any time of incubation
the transport is reduced to half in either method. For comparison a natural oligoamine i.e. spennine has been included. The third block of data shows that this compound is
unable to influence the glucose transport even in a concentration of lo00 pmol/L.
-
-
-
*Deoxyglucose
We suggest that the reason for this difference is the lack
of hydrophobic interactions with the carrier protein and/or
the membrane phospholipids. This view is backed by the
results obtained with the bissydnone imine RE 1888
(4,4’-rn-phenylene-bis-3-(4-phenylbutyl)-sydnone
imine).
This compound is similar to spermine with respect to its
good solubility in water because of the polar sydnone moiety (inner salt). On the other hand it still possesses two substituents which are suitable for hydrophobic interactions.
This results in a smaller inhibition of the deoxyglucose
transport which is reduced by 25% at a concentration of
100 pmol/L of RE 1888. The inhibition of the transport is
non competitive. This can be derived from the LineweaverBurk plot which is shown in Fig. 1.
Inhibition of lipid peroxidation by RE 1492
Fe2+ions play a decisive role in lipid oxidation by molecular oxygen in that they catalyze the formation of hydroxy
radicals6).In 1988 Tadolini7)presented a relatively simple
model for the investigation of this process. In this model the
natural phospholipids are replaced by compounds which
mimic their polar head groups. These are adenosine monoArch. Pharrn. (Weinheim) 328.21-27 (1995)
23
Interactions of RE 1492 with Biomembranes
*/+
1-
-10
by addition of 1,lO-diazaphenanthrene(ferroine) by the formation of the red Fez+complex. The extinction at 5 15 nm is
recorded. Only in the presence of AMP the oxidation of the
Fez+ions could be inhibited by oligoamines. These results
are compiled in Tab. 2. When the incubation is started by
addition of 150 p m o l b FeS04 and the chelator added
immediately an extinction of 1.121 k 0.041 is measured
(exp. 1). As the oxidation of the ferrous ions proceeds, the
extinction decreases (see exp. 3, 5, 7). After 30 min only
about 10%of Fe2+ions are left (exp. 9, E = 0.135 f 0.016).
When 30 pmol/L RE 1492 are added to the incubation mixture the oxidation strongly is inhibited as a function of time
(exp. 2 , 4 , 6 , 8 , 10). This inhibition already becomes significant after 10 min (exp. 6) and gets more and more obvious
(exp. 8, 10). The same effect is observed with the natural
oligoamine spermine (exp. 11-13). The influence of the
concentration of several oligoamines on the oxidation was
investigated in the experiments 14-26. Increasing concentration of RE 1492 are correlated with increased inhibition
of the oxidation (exp. 14-18).
This already is significant with 3 pmol/L (exp. 16). With
100 pmol/L a nearly complete inhibition of Fe3+formation
is observed (exp. 18). The same effect again can be
-x
0
t/
’
/
5
10
15
20
25
30
35
40
Fig. 1: Noncompetitive inhibition of the glucose transport by RE 1492 30
F m o K ( 0 ) (+ = control values)
phosphate (AMP) (for phosphatidic acid), cytidyl choline
phosphate (for phosphatidyl choline) and glyceroinositol
phosphate (for phosphatidylinositol). The extent of “lipid
peroxidation” is measured after the incubation time stated,
Tab. 2: Inhibition of the oxidation of Fe2+-ionswith Fenron’s reagent in the presence of AMP by
means of oligoamines expressed in extinction units ( E S I ~of) the Fe2+-phenanthrolinecomplex
(cOF? = 150 FmoI/L).
!ompound
Exp.
No.
RE 1492
spamine
1
2
3
4
5
6
7
8
9
10
11
12
13
RE 1492
-s
RE 1888
Arch. Pharm. (Weinheim)328,21-27 (1995)
14
15
16
17
18
19
20
21
22
23
24
25
26
C
t
[~mol-L-1] [minl
0
30
0
30
0
30
0
30
0
30
30
30
30
0
0
0.3
3
30
100
0.3
3
30
100
0.3
3
30
100
0
0
5
5
10
10
20
20
30
30
10
20
30
0
30
30
30
30
30
30
30
30
30
30
30
30
30
1.121 f 0.041
1.103 f 0.044
0.995 f 0.032
1.049 f 0.041
0.852 f 0.053
1.025 f 0.032
0.551 f 0.028
0.912 f 0.031
0.135 f 0.016
0.813 f 0.036
1.046 f 0.043
0.857 f 0.054
0.795 f 0.035
1.121 f 0.041
0.143 f 0.035
0.184 f 0.022
0.387 f 0.034
0.813 f 0.036
1.120 f 0.038
0.206 f 0.031
0.421 f 0.045
0.789 f 0.031
1.072 f 0.044
0.183 H.027
0.227 f 0.023
0.428 f 0.021
0.7 13 f 0.036
24
Kesselhut, Ebel, v. Bruchhausen, and Rehse
achieved with spermine (exp. 19-22). The oligosydnone
imine RE 1888 as well inhibits the oxidation but to a smaller extent (see exp. 23-26). The effect of 100 pmol/L RE
1888 is comparable to 30 pmol/L RE 1492 (exp. 26 and 17)
or spermine (exp. 21).
Alteration of ovine lymphocyte membranes by oligoamines
detected by fluorescence polarization experiments
The insertion of fluorophores into biomembranes provides
an opportunity to get information about the lipid molecules
surrounding the fluorophore. This interaction most elegantly can be studied by the fluorescence polarization technique: Unlike in solution apolar fluorescent molecules in lipid
membranes show selected orientation. When irradiated with
polarized light only molecules of which the molecular axis
is parallel to the plane of the polarized light are able to
absorb this light. In consequence the emission of light
occurs with a defined angle to the plane of the light
absorbed, i.e. the fluorescent light is polarized as well. The
degree of polarization is expressed by the steady state
anisotropy rs = 111- I I [ l : (Ill+ 211)]. The parameter 111is
the part of the light emitted from plane which is parallel to
the plane of the exitation light while I, represents the light
of which the plane is perpendicular.
The limiting anisotropy r, is calculated by the formula (1)
where
r, is the maximal fluorescence anisotropy value (for DPH a
value of 0.39 was used), T~ the fluorescence lifetime of the
fluorophore, RCT the rotational correlation time, rs the
steady state anisotropy.
During the life time of the exited state T~ the fluorescent
molecules rotate in the membrane. This leads to a partial
depolarization of the light. The degree depends on the viscosity of the surrounding membrane which in turn is a function of the regularity of the membrane lipid molecules.
This is expressed by the rotation correlation time (RCT)
corresponding to lipid motility and the rotation angle 0
reflecting lipid order (for details see Experimental Part).
The influence of RE 1492 on these parameters is summarized in Tab. 3. Using the fluorescent probe 1,6-diphenylhexa- 1,3,5-triene (DPH) the steady state anisotropy rs is
decreased up to 25% by rising concentrations of RE 1492.
The same is true for the limiting anisotropy r,. Here the
decrease is even larger (I 38%). These results indicate
decreased order of the lipid molecules in the lymphocyte
membrane.
This view is supported by the widening of the rotation
angle 0 from 35" to nearly 44".A similar conclusion can be
drawn from the decrease of the life time 'tP of the exited
state from nine to seven nanoseconds. This means an
improved deactivation via facilitated molecular vibrations.
The RCT is not changed. This suggests that the cycling of
the probe itself is not altered. This might be due to the fact
that DPH is a relatively rigid probe, so that its RCT is not
sensitive to a change in lipid motility.
We, therefore, investigated the influence of RE 1492 on
the membrane with some more flexible anthroyloxy fatty
acids. These probes furthermore allow to measure changes
in lipid motility in different layers of the membrane9).Thus
2-anthroyloxystearic acid (2-AS) reflects changes in parts
of the membrane which are near the surface while 12-AS
represents alterations in deeper parts. The 16-anthroyloxypalmitic acid (16-AP) is suitable for investigating the center
of the membrane bilayer. The results obtained with these
probes are compiled in Tab. 4. The changes in lipid motility
are most dramatic in the area near the surface of the membrane. The RCT is reduced more than to 50% indicating a
strongly facilitated rotation. The limiting anisotropy r, is
decreased by 30%, thus showing decreased lipid order (exp.
1 and 2). In the region of the 7-anthroyl group these effects
are as well obvious but smaller (exp. 3 and 4). In the deeper
layer of the 12-anthroyl group again a bisection in the RCT
and a 20% decrease of r, is observed (exp. 5 and 6).
In the center of the bilayer nearly no changes are seen.
This suggests that the cationic N-functions of RE 1492
influence the 2-AS region while the 4-phenyl group is
responsible for the changes in the 12-AS part of the membrane.
The dependence of these changes on the concentration of
RE 1492 exemplarily was investigated in the 12-AS region
of the membrane (exp. 9-13). RE 1492 in a concentration of
3 pmol/L is without effect (exp. 9). Higher concentrations
successively decrease the RCT as well as r,. The regularity
of membrane lipids is increased with decreasing temp., i.e.
more and more areas with a liquid crystalline status are
formed.
The fluorescent probes 9Z,1lE, 13E,152-octadecatetraenoic acid (CPA) and 9E,11E,13E,15E-octadecatetraenoicacid
(TPA) are most suitable for investigation of lipid motility in
these areas. The results obtained with these probes are summarized in Tab. 5. In general the steady state anisotropy is
Tab. 3: Influence of several concentrations RE 1492 on some fluorescence mimeters using
the fluorescent probe 1,6-diphenylhexa-1,3,5-triene (DPH)
0.2258M.0015 0.2238fo.0010 0.218W.0192 0.1884M.0151
O.l99Ol$.O1 0.1984fo.0188 0.1914M.025S 0.1495W.0184 0.1241fo.0341
8.89iO.06
9.10.28
8.72fo.22
7.82M.08
7.1W.14
RC"[ns]
1.43M.04
1.37fo.07
1.38M.03
1.49iO.05
1.50.04
erkg-1
~ . 9 5 f i . i 5 35.01~.19
3s.74~.26
39.9~1.18
43.m.34
Arch. Pharm. (Weinheim) 328,21-27 (1995)
25
Interactions of RE 1492 with Biomembranes
Tab. 4: Influence of RE 1492 on the rotation correlation time (RCT) and limiting anisotropy (rJ
in different layers of ovine lymphocyte membranes. (A = anthroyloxy S = stearic acid P = palmitic
acid)
Exp.
RE 1492
Probe
RCT
ro.
No.
c [umoUL1
1
0
2-AS
2.85 f 0.12
0.1 129 f 0.0019
100
2-AS
1.29 f 0.08
2
0.0801 f 0.0024
3.63 f 0.15
0
7-AS
0.0985 f 0.0030
3
100
7-AS
2.36 f 0.13
4
0.0689 f 0.0028
0
12-As
5
0.0475 f 0.0035
3.86 f 0.18
6
1.79 f 0.08
0.0410 f 0.0029
100
12-As
7
1.56 f 0.07
0
1CAP
0.0381 f 0.0010
8
1.37 f 0.08
0.0362 f 0.0013
100
1CAP
9
3.73 f 0.12
0.0462 f 0.0026
3
12AS
10
3.04 f 0.20
10
12-AS
0.0515 f 0.0030
0.0479 f 0.0029
11
20
12-As
2.80 f 0.11
12
0.0460 f 0.0027
30
1243
2.67 f 0.13
2.30 f 0.08
50
12-AS
0.0452 f 0.0029
13
1
Probe
temp. ["CI
cis-
4
15
25
37
45
4
15
25
37
45
parinaric
acid
(BA)
trans-
w
acid
CIPA)
rl
control
0.2503 f 0.0034
0.2245 f 0.0041
0.1872 f 0.0015
0.1548 f 0.0013
0.1504 f 0.0026
0.2802 f 0.0026
0.2538 f 0.0028
0.2044 f 0.0014
0.1661 f 0.0025
0.1532 f 0.0013
decreased by RE 1492 at temp. varying from 4-45°C. The
largest effects are observed at 4°C. This suggests that RE
1492 is able to increase lipid motility even in liquid cristalline membrane parts.
Experimental Part
Studies with rat thymocytes
The thymocytes were isolated according to Goldfinelo)from male wistar
rats weighing 100-150 g. The animals were killed by decapitation. Thymus
glands were removed quickly and washed in ice cold Krebs-Ringer buffer
pH 7.4 (120 mmol/L NaCI, 5 mmol/L KCI, 1 mmol/L CaCI2, 2.5 mmo&
MgC12, 1.5 mmol/L NaH2PO4, 25 mmol/L Tris, 15 mmoW Hepes). The
tissue was freed from blood vessels and gently minced with scissors. The
thymocytes thereby liberated were filtered through gauze. Larger tissue
fragments that were removed, were resuspended in buffer, minced and
filtered again. This process was repeated three times. The cell suspension
so obtained was washed by centrifugation at 2.200 rpm (800 x g) for 5
min. The supernatant was decanted and the cell pellet resuspended in
buffer again. The thymocytes were counted in a Neubauer chamber.
Additional buffer was added to the cells to yield a final cell concentration
of loRcells/ml. Cell viability, which was determined by trypan blue
exclusion, was > 95% throughout this period as well as after realization of
the following experiments.
Arch. Pharm. (Weinheim) 328.21-27 (1995)
rs RE 1492
1100 umoVLl
0.1828 f 0.0022
0.1712 f 0.0024
0.1639 f 0.0031
0.1619 f 0.0013
0.1573 fO.OO1l
0.2105 f 0.0019
0.1889 f O.O(l23
0.1767 f 0.0012
0.1643 f 0.0023
0.1571 f 0.m
For the glucose transport experiments (measurement of the C02 formation) 10' cells were pipetted into coulter counter vessels and equilibrated
for 15 min at 37OC. Thereafter the various compounds were added and the
suspension was incubated again for 15 min at 37OC. To start the uptake, I L4C-~-glucose
was added to the medium to a final concentration of 37
KBq/ml. Immediately after this procedure, sieve inserts were placed into
the coulter counter vessels out of the medium, carrying a little glass vial.
Then the vessels were closed thightly with a rubber top. Further incubation
was carried out at 37°C for the time stated. By injection through the rubber
top hyamine hydroxide was placed into the little glass vials to absorb the
C0 2 formed during 30 min of incubation at room temp. To stop the glucose transport, 8 N H2S04was added to the cell suspension. Finally the
glass vials, including the absorbed C02, were transferred to counting vials
with the aid of scintillation fluid ready protein plus. The samples were
counted in a p-scintillation counter with an external standard for quench
correction.
Studies on 2-deoxyglucose uptake were conducted basing on a method
described by Segal") in a modified manner. Cells were prepared as
described above. The cell suspension (10' cells) was pipetted in polyethylene microtubes. Pre-equilibration and incubation with the compounds
were similar to the C02-fonnation experiments. The uptake was initiated
by adding 3,7 KBq/ml 2-deo~y-D-U-~~C-glucose
and ~-l-~H-mannitol
as
an extracellular fluid marker. After the proper time the samples were centrifuged for 30 sec at 4°C with 5 OOO rpm and then placed on crushed ice.
An aliquote of the supernatant was transferred quickly to counting vials
with scintillation fluid. The rest of the supematant was removed by aspira-
26
tion and the cell pellet resuspended in 5% trichloroacetic acid. The entire
suspension was transferred quantitatively to counting vials containing scintillation fluid. All samples (supernatant and cell pellets) were counted in a
0-scintillation counter with automatic quench correction.
Because the final cell pellets inevitably contained occluded extracellular
fluid and to determine only the absorbed 2-deoxy-D-glucose, ~ - l - ~ H - m a n nitol was added as an extracellular fluid marker. The p-scintillation counter counted the pulses of 3H and I4C in different channels, so that the ratio
of 3H and I4C in the extracellular fluid with the samples containing the
supernatant could be calculated. Therefore, it is possible to subtract the
part of extracellular 2-deoxyglucose in the samples that contained the cell
pellet. So corrections for occluded extracellular fluid in the cell pellet
could be made.
“Lipid” pero.xi&tion studies
The influence of the oligoamines on iron oxidation in the presence of
compounds mimicking phospholipid polar heads was determined as
described by Tadolini’). Briefly, the samples contained 10 pnoW adenosine monophosphate or 30 pnolb cytidinyl choline phosphate respectively 30 p,movL glyceroinositol phosphate in 5 m m o K Mops buffer pH 7.2.
The oligoamines were added, and the reaction was started with 150
pnom FeS04. At the time stated the reactions were stopped by addition
of 4 mmol/L 1.10-diazaphenanthrene and the extinction at 515 nm was
read immediately.
Fluorescence polarization experiments with sheep
lymphocytes
Preparation of sheep lymphocytes
The cells were prepared based on the method described by MehdiI2).
First intestinal lymphatic ganglia were removed from freshly slaughtered
sheep at the local slaughter house and packed in crushed ice. Adherent
tissue (fat and fascia) was removed and the lymphatic ganglia were given
in a meat mincer. Lymphocytes were liberated by filtering the minced lymphatic tissue through a sieve and rinsing with ice cold Krebs-Ringer buffer
pH 7.4. Then the cells were washed in the buffer three times by centrifugation for 15 min at 2.800 rpm at 4°C. The pellet was resuspended in hypotonic medium (1 mmol/L Tris-HCI buffer pH 7.4 containing 1 mg/L
DNAse to prevent cell aggregation), and the cells were disrupted by
homogenization. The cell suspension was homogenized - after stirring at
4OC for 60 min - in a Dounce homogenizer with ten strokes of a loose fitting teflon pestle. Sucrose (0.25 mom) was added to the homogenate, and
this mixture was centrifuged for 15 min at 11 000 rpm (4OC). The resulting
supernatant was adjusted to 1.23 m o m sucrose (= crude “membrane” suspension). In the Beckman rotor tubes the following sucrose gradient was
piled up:
6 ml of 2 mom sucrose, 6 ml of 1.31 mol/L sucrose, 17 ml of the crude
“membrane” suspension (1.23 mol/L sucrose) and 6 ml of 0.8 mol/L
sucrose, all in 1 m m o m Tris-HCI buffer pH 7.4. The sucrose gradient was
centrifuged for 16 h at 25 OOO rpm at 4°C. The top portion of the 2 mol/L
sucrose layer was aspirated and washed three times in 1 mmol/L Tris-HCI
buffer pH 7.4 for 1 h at 50 OOO rpm (4OC). At last the pellet was resuspended in 20 mmol/L Hepes-Tris buffer pH 7.4 containing 300 mmoUL mannitol, carefully homogenized and portionized frozen in liquid N1.Stored like
this, these membrane vesicles can be used for six weeks for fluorescence
polarization measurements. The enzyme 5’-nucleotidase which is a typical
membrane specific enzyme, was enriched 8-times in comparison to the cell
homogenate.
Kesselhut, Ebel, v. Bruchhausen, and Rehse
Instrumentation for fluorescence polarization measurements
A model of the spectrofluorometer (SLM 4800) used in the fluorescence
polarization experiments was shown by Lokowiczr3’.The measurement of
the steady state anisotropy was conducted by using the T-formate or two
channel method. Thereby the intensities of the parallel and the
perpendicular components of the emitted light were measured
simultaneously using two separate detection systemsr3).The determination
of the steady state anisotropy as well as the phase and modulation
measurements of fluorescence lifetimes were carried out to calculate the
fluorescence lifetime. The sample was excited with light, the intensity of
which was modulated in a sinusoidal manner by using an ultrasonic
modulator. Because of the time lag between absorption and emission, the
emission was delayed in phase and demodulated relative to the exciting
light. The phase delay and the demodulation factor could be used to
calculate the fluorescence lifetime. The third kind of measurement was the
differential polarized phase fluorometry. This is a method to investigate
the time dependent decays of anisotropy. The samples were excited with
polarized sinusoidally modulated light and the phase difference between
the perpendicular and the parallel components of the emission was
measured. The degree of phase difference depends on the rate of
fluorophore rotation and phase difference is indirect proportional to
limiting anisotropy r, corresponding to the lipid order of the membrane.
Regarding the steady state anisotropy rs, lifetime of the fluorophore T~ and
phase difference A-phase, the following parameters could be calculated:
- r, the limiting anisotropy which corresponds to the order of membrane
lipids
- RCT, the rotation correlation time, which reflects the “microviscosity”
resp. the lipid motility and is the time, a fluorophore needs to rotate
around its own axis,
- 0,
the rotation angle of the fluorophor, that is directly derived from r,
and depends on lipid order.
All values were recorded and analysed by an online computer program.
Preparation of membrane-fluorophor samples and fluorescence
polarization measurements
The samples contain the thawed membrane vesicles (adjusted to a
protein amount of 300 l g ) in 10 mmol/L Hepes-Tris buffer pH 7.4, to
which 100 mmol/L mannitol had been added (MHT buffer). This
suspension was carefully homogenized in glass potters by a few strokes,
the compound and the fluorescent probe, e.g. 1 pmoW DPH were added
and incubated with the exclusion of light for 30 min at 3 7 T . Than the
incorporation of DPH into the lipid phase of the membranes was finished
and did not change anymore. The samples were centrifuged at 20 OOO rpm
at 4°C for 20 min and the pellet was resuspended in MHT buffer. So
excess DPH and compound, which were not bound to the membrane
vesicles, were removed and could not influence the further measurements.
The fluorescence polarization measurements with DPH as fluorescent
probe were carried out at 25°C. The wavelength of the excitation
monochromator was set to 360 nm. The emission light was measured
through a Schott KV 418 filter. In the experiments each parameter (e.g. rs.
T ~ was
) determined ten times.
Different to the experiments with DPH, the concentration of the added
anthroyloxy fatty acids in the samples was 2 Fmol/L, the incubation temp.
25OC, the wavelength of excitation light 365 nm, the emitting light measured also at 418 nm. The concentration of the parinaric acids was 1
pmom. TPA was incubated for 30 min at 37OC. CPA for 2 h at 37°C.
Excitation wavelength was 321 nm (CPA) or 323 nm (TPA), respectively.
The emission was determined at 408 nm for both probes.
Arch. Pharm. (Weinheim) 328.21-27 (1995)
27
Interactions of RE 1492 with Biomembranes
References
8
1
K. Rehse, U. Liikens, G. Claus, Arch. Pharm. (Weinheim) 1987,320,
9
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1233- 1238.
K. Rehse, A. Kesselhut, V. Schein, M. K b p f e , B. Rose, E. Unsold,
Arch. Pharm. (Weinheim) 1991,324,301-305.
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D.L. Baly, R. Horuk, Eiochim. Eiophys. Acta 1988,947,571-590.
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B. Halliwell, J.M.C. Gutteridge, Eiochem. J. 1984,2/9, 1-14.
B. Tadolini, Eiochem. J. 1988,249.33-36.
Arch. Pharm. (Weinheim)328,21-27 (1995)
11
12
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W.J. van Blitterswyk, R.P. van Hoeven, B.W. van der Meer, Eiochim.
Biophys. Acta 1981,644,323-332.
K.R. Thulbom, L.M. Tilley, W.H. Sawyer, F.E. Treloar, Biochim. Biophys. Acta 1979,558, 166-178.
I.D. Goldfine, G.J. Smith, C.G. Sirnons, S.H. Ingbar, E.C. Jorgensen,
J . Eiol. Chem. 1976,251,4233-4238.
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S.Q. Mehdi, S.S. Nussey, Eiochem. J . 1975,145, 105-1 11.
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[Ph246]
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effect, platelet, 1492, oligoamines, biomembranes, inhibition, xxvinteractions, anticoagulant, aggregation, oligoamide
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