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Development of an affinity chromatography resin for the purification of carcinogen binding proteins from mouse liver
Vol.
129,
No.
May
31.
1985
1, 1985
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
155-l
62
DEVELOPMENT OF AN AFFINITY CHROMATOGRAPHY RESIN FOR THE PURIFICATION OF CARCINOGEN BINDING PROTEINS FROM MOUSE LIVER Sheila
Collins,
John D. Altman
and Michael
A. Marietta*
Department of Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received
14arch
11,
1985
Pyrene, a structural analog of benzoCa]pyrene, is an effective competing ligand for high affinity carcinogen binding proteins in mouse liver. A pyrenederivatized Sepharose gel was prepared for affinity chromatography purification of these proteins, and adsorbs all detectable C3H1BCa]P-binding activity from hepatic cytosol with the adsorption of less than 1% of total protein. Specific carcinogen binding activity is recovered from pyrene-derivatized Sepharose columns with the enrichment of a 33 kDa polypeptide. This chromatograpy resin represents a major step in the isolation, of these unusual receptor-like binding proteins for aromatic hydrocarbon carcinogens. D 1985 Academic press, h.
Recent that
bind
investigations a limited
chlorinated
interactions
cinogens
(61.
of
per
sites
cell
(1,7),
sites, potency
*
Address
are
or ability
binding
affinites the
in the
function
the
reminiscent ligands
low nanomolar
binding
microsomal
author
drug
at
range
of the for
affinities metabolizing
Room 56-229,
hormone
potent
and and drug
animal
car-
and a finite
of these
exception
proteins
(PAH)L
of
are
has been discerned
to correlate
of novel
hydrocarbons
characteristics
With
to this
existence
aromatic
features
primary
to induce
correspondence
the
Many of these
proteins.
attempts
demonstrated
polycyclic
(l-5).
no biological
although
of
with
Binding
xenobiotic-binding tor
number
pesticides
receptor
have
number
receptor-like
TCDD-binding these with
PAH-binding carcinogenic
enzymes
MIT,
Ah recep-
have been made
Cambridge,
MA 02139.
'Abbreviations used are: Ah, aryl hydrocarbon; l-AP-CB, 1-Aminopyrene Sepharose 68; BCalP, benzocalpyrene; B6, C5781/6J mice; BSA, bovine serum albumin; CL-6B, Sepharose CL-6B; Hepes, 4-(2-hydroxyethyl)l-piperazine ethanesulfonic acid; and 10% HEDG buffer, composed of 25 w Hepes, 5 mM EDTA, 1.0 n+l dithiothreitol glycerol at pH 7.6; PAH, polycyclic aromatic hydrocarbon; SDS, sodium dodecyl sulfate, SDS-PAGE, SDS-polyacrylamide gel electrophoresis; TCDD, 2,3,7,8TLC, thin-layer chromatography. tetrachlorodibenzo-p-dioxin; 0006-291X/85
155
All
$1.50
Copyright 0 1985 by Academic Press, Inc. rights of reproduction in any form reserved.
Vol. 129, No. 1, 1985
The existence
(2,3,8). bolism dence
of
substrates
significance
of
have
significance
of
the
synthesized binding previously
that
may facilitate
has been suggested
ligand
of putative
extracts
from
proteins
(9,101,
microsomal but
meta-
supporting
evi-
inconclusive.
The limitations
the
of carrier
lipophilic
remains
crude
8lOCHEMlCALAND8lOPHYSlCALRESEARCHCOMMUNlCATlONS
Ah receptor,
receptors
these
(or
described
high
their
(11).
affinity
of
proteins)
for
establishing
probing
their
Therefore,
is resin
in order
from
required. for
the
the
mouse liver
biological
structural
carcinogen-binding
purification
chromatography class
studies
and for
been discussed
an affinity protein
binding
features
to further
proteins Toward
this
isolation whose
in
study
distinct end,
we have
of a carcinogen properties
we have
(5). MATERIALS AND METHODS
ANIMALS (Barrbor, L3HIB[alP
Male
86 mice aged 6-8 weeks were obtained from Jackson Maine). Preparation of 100,000 x 1 hepatic cytosol binding assays were performed as described previously
Laboratories and standard (5).
CHEMICALS B[a]P, pyrene and 1-aminopyrene were obtained from Aldrich Chemical Company (Milwaukee, WI), TCDD from KOR Isotopes (Cambridge, MA), Hepes, dextran, Coomassie Blue 6250, fluorescamine and epoxy-activated Sepharose 68 from Sigma Chemical Company (St. Louis, MO), Norit-A charcoal from Fisher Scientific (Pittsburgh, PA). Reagents for SDS-PAGE were electrophoretic grade and obtained from either Bio-Rad Laboratories or BRL (Rockville, MD). All other chemicals were of the highest purity available. RADIOCHEMICALS [1,3,6-3H]B[a]P (specific activity 87.5 Ci/mmole) and lmethyl-l*C1 methylated BSA were obtained from New England Nuclear Corporation (Boston, MA). C3H]B[a]P was repurified before use as previously described (5). [1,6-3HlTCDD (52 Ci/mmole) was obtained from ICN Radiochemicals (Irvine, CA) with purity assessed by TLC on Silica gel in benzene:methanol (4:ll. SYNTHESIS OF l-AMINOPYRENE SEPHAROSE 6B (l-AP-6B1 l-AP-6B was prepared from epoxy-activated Sepharose 6B and 1-aminopyrene (recrystallized from cyclohexane) according to the manufacturer's instructions. The coupling reaction was carried out in p-dioxane:water (50:50), pH 9.0, at a molar ratio of 1-aminopyrene:epoxy groups of 5:l under a nitrogen atmosphere for 20-24 hours. The derivatized resin was washed extensively with methanol (141. The solvent was periodically collected, evaporated to dryness and examined for the presence of 1-aminopyrene by TLC and UV spectra. Aside from the initial washing step with coupling buffer, none was found. Extent of derivatization was determined spectrally (15) at 292 nm by suspending the desiccated gel in 80% aqueous glycerol and found to be 6.38 2 1.90 nmoles/mg gel, or 2.1 pmoles/ml packed gel. Comparable values have been obtained for steroid-modified Sepharose gels used in steroid hormone receptor purifications (14,16). Subsequent preparations of l-AP-6B routinely contained l-2 umoles ligand/ml packed gel. CHROMATOGRAPHY WITH l-AMINOPYRENE SEPHAROSE 6B l-AP-6B was equilibrated buffer overnight. Control incubations contained an equal gram amount tically treated Sepharose CL-6B. In batch experiments, the Sepharose with an equal volume of B6 mouse liver cytosol (final concentrations:
156
in HEDG of idenwas mixed 1.0 mg/ml
Vol.
129,
No.
1, 1985
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Samples were incubated in a circular protein, 0,,25 - 2.5 mglml Sepharosel. shaking bath for 2.5 hours at 20-22°C. After chilling on ice, aliquots (1.0 ml) of the suspension were added to 1.5 ml Eppendorf tubes and centrifuged 1 minute in a microcentrifuge (13,000 x 1) to pellet the Sepharose. Portions of the supernatant (0.5 ml and 0.1 ml) were removed to monitor B[a]P binding activity and protein concentrations, respectively. For column chromatography, l-AP-6B (6.0 mg/mll was equilibrated overnight in HEDG buffer containing 0.1 M NaCl. Typically, 150 mg of cytosolic protein (3.5 pmol binding activity/mg) were incubated with the l-AP-6B suspension as described ,For batch chromatography at a gram ratio of 2.1 mg l-AP-6B/mg protein and a final protein concentration of 2.5 mg/ml (total volume 61.0 ml). The entire suspension was poured Subsequent operations were conducted at 4°C. into a 0.5 x 9.0 cm column (bed volume = 1.8 ml) at a maximum flow rate of 0.2 ml/min. The unbound proteins were collected, then fractions (1.0 ml) were collected (as the column was washed overnight with equilibration buffer (approximately 50 ml), which was subsequently pooled and concentrated on an Amicon YM5 membrane to 1.5 ml. A small amount of protein (1.15 mgl and only nonspecific binding activity was recovered. Elution of protein bound to l-AP-6B was performed with Hepes buffer, pH 7.6, containing 2.0 M sodium thiocyanate, Bound proteins were eluted in the first and fractions (1.0 ml) were collected. B ml. These were pooled, dialyzed against HEDG buffer (3x1 liter), and concentrated as described above to about 1 ml. This volume was further reduced with an Amicon Centriconto 400 ul (0.85 mg/ml protein). ELECTROPHORESIS SDS-PAGE was carried out using a modification method of Laemmli (17) as previously described (5). Gels were Coomassie Blue (18) or by silver staining (19).
of the stained
with
RESULTS AND DISCUSSION COMPETITION BINDING WITH PYRENE. epoxy-Sepharose
was based
protein
and the
chemical
ability
to compete
effective
with
dize,
Finally,
binding
of
a relatively
stable
be a good candidate 1-aminopyrene
and 86 liver
cytosol,
specific
derivatives
and found in affinity
data
directly
was reduced
its to be than
suggested
chromatography
was incubated binding
for
of PAHs rapidly
These
to
binding
was tested
weaker
affinity
coupling
carcinogen
assays
molecule. for
for
Pyrene
threefold
and amine
where
(not
the
compound.
shown),
hydroxy
for
in competition
in experiments
C3HlBCaIP
l-AP-68
C3H]B[alP
of a ligand
affinity
of the
of 35 nM (not
is
would
relative
stability
a number
1-aminopyrene
1-aminopyrene
levels
an I&,
While
BCalP (5).
for
on its
The choice
oxithat
development. at
1 uM with
to nonspecific
shown).
ADSORPTION OF BCalP-BINDING is shown below.
PROTEIN TO THE l-AP-CB
I4
o-cn,-Fn-cn,-o-(cn1),-O-C~-~H-CH~-NOH
GEL.
/\ -
ON \ %
157
\ \/
The structure
of
Vol.
129,
1, 1985
No.
BIOCHEMICAL
Table
I.
The results vity
mg/ml mg/ml mg/ml mg/ml rig/ml
concentration
changes
in supernatant
of
likely
not
results
phenyl-Sepharose
teins
from
effective
by comparison
with
l-AP-6B.
(40 pmoles/ml
50% of
binding
(2.3
mg/ml)
of
Samples sity
gradients
Specific the
ability
that
the
after 1.
of
majority
adsorption In Figure
conditions
than
more
by l-AP-6B.
also
bind
affinity
the
degree
better of
9S
(20)
have
3-methyl-
BCalP-binding
pro-
derivatization
on
less
than
concentration
in agreement
of
was to
by l-AP-68,
binding with
to binding
content containing
illustrate
la indicates
protein the
is
results
equilibrium
from CL-6B
den-
and to assess
Figure
the Ah receptor,
Ah receptor experiments
on 5 - 20% sucrose
receptor.
B[a]P
158
most
of
gel)
highest
experiments
Ah
visualization
In Control
of
but
phenyl-Sepharose
l-AP/ml
protein
brought
total
pmols
these
were
the
a class
these
analyzed
binding the
No
shown).
also
matrix,
samples
half
for
the 4S-sedimenting
lb identical allows
B[a]P
to
of
to the
C~HITCDD, which
was removed
resin
major
CL-68,
We found
at the
the
were
et al.
purify
l-2
at
recovered.
Tierney
high
while
or CL-68
capacity
vs.
of
fully
acti-
low CL-CB concentrations
cytosol.
(not
The purpose
the
at
binding
manner,
by Sepharose
was adsorbed tested
1). of
this
gel
and CL-CB were
(Figure
vast
packed
activity
l-AP-68
adsorption
liver
BCalP
l-AP-68
to partially
Despite
phenyl-Sepharose
from
is
activity
in adsorptive
phenyl-Sepharose C3H]B[a]P
from
purifcation
rat
the
activity
of 100% activity.
definition
proteins
of
dependent
concentrations
48 chromatography
binding
all
binding
significant
our
147 112 78.7 14.9 7.6
essentially
of 147% of binding
reflect
4B to be minimally
FROM WHOLE RESIN
+ 0.030 7 0.043 + 0.040 T 0.008 IO.018
tested
COMMUNICATIONS
% CONTROL 100
in a concentration
protein
from
cholanthrene
that
CL-68
The recovery does
0.790 0.599 0.421 0.080 0.041
by l-AP-68
highest
used
CL - 6B CL - 68 l-AP-6B l-AP-68 I-AP-CB
I indicate
can be removed
probably
RESEARCH
SPECIFIC C3H]-B[a]P Bound/ml 0.535 + 0.090
of Table
observed.
BIOPHYSICAL
ADSORPTION OF BCalP-BINDING ACTIVITY CYTOSOL BY l-AMINOPYRENE-SEPHAROSE-6B
CONTROL 0.25 2.50 0.25 1.25 2.50
AND
absent of Table with
Under these whole there
cytosol was no
Vol.
129,
No.
BIOCHEMICAL
1, 1985
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
n
I
24-
AND
20-
24
6
16
6
TOP
Froct~on
24
6
TOP
16
24
Number
.$p
Sucrose density gradient fractionation of carcinogen-binding proSamples were incubated with radioligands. treated with dextran-coated charcoal to remove free ligand as described (51, and centrifuged through 5-20% sucrose gradients (5.0 ml) in a SW50.1 Ti rotor at 50,000 rpm for 16 hr at 4°C. Fractions (3 drops) were collected. A: [3]B[a]P (6.5 nM) binding in cytosol before (01 and after (01 l-AP-68 incubation; 300 Pg total protein. B: [3~1~~~~ (8.5 nM) binding as in A; 1.8 mg total protein. C: C3HlBCalP (8.5 nM1 binding in salt-eluted proteins from I-AP-CB in the absence 10) or presence (01 of 1 PM unlabeled B[a]P; 75 @g total protein.
reduction
in
cytosol.
Since
species
at is
protein
which those
this
4s
4s TCDD
4s
gradient
and
either
has
(4,5),
binds
the
OF BCalP
eluted
and
with
sodium
thiocyanate
in
concentrated was
to
disrupt
significant
part
in
the
conducted
of
which
in
for
binding previously
of
ligands
bound
to and
reasons.
(5).
159
to
which this
In
protein,
addition,
region
of
the
from assay
the
4s
methods account
for
indicate
that
hepatic
of purif
interactions
binding
ts
resu
whole
BCalP
probably
been
Materials
several
hydrophobic
our
classes
have
Proteins
described
chosen
these
to
distinct
therein) These
of
ACTIVITY.
are
between
experiments.
neither
abundant
sites
references
both
as
these
relative
sedimenting
differences
and
peaks
more
lower
that
our
binding
this
this
isolate
BINDING
agent
studies
notion
(7
to
chaotropic
in
Small
proteins,
were
activity
the
TCDD
for
binding
observed potential
9s
affinity
investigators
carcinogen-binding
RECOVERY
or
polycyclics.
other
has
little
with
TCDD-binding
l-AP-68
binding
[~H]TCDD
consistent
of
B[alP
ed
the
to
date.
l-AP-6B
Methods.
column Elution
Thiocyanate
serves
we
to
presume
based Bresciani
on
as play
a
structure(21,221
a
has
Vol. 129, No. 1, 1985 shown
that
estrogen
8lOCHEMlCALAND8lOPHYSlCALRESEARCHCOMMUNlCATlONS
thiocyanate-containing receptor
in active
illustrates
the
with
a peak
profile
la).
With
recovery
binding
is
with
this
ligands
part,
allow
this
PAH-binding
this
the
the
Figure
activity
from
l-AP-6B
cytosol
(Fig.
as competing
protein
lc
is
ligand,
recovered
in a form
protein.
during
purification
However,
the
to include
from
buffers
significant
This
9s Ah receptor
of B[alP
mild
of binding
as well
as the
in
in active
containing
and
to associate
may explain,
l-AP-6B
alteration
strategies
solubility tendency
use here.
Ah receptor
elution
poor
and their
their
by affinity
Biospecific
concentrations,
without
uterine
columns.
incubation
problem.
the
calf
form.
detergents
may
properties,
more abundant
and
4s
protein.
are
samples
OF POLYPEPTIDES on l-AP-68
not
of
Proteins
adsorbed
the
proteins
as well
less
by l-AP-6B
the
protein
proteins
Tierney for
et al.
or CL-6B x 1 hepatic
column
(lanes cytosol
are displayed
weights
Most
of the
cytosolic
pro-
5 or 6) compared (lanes
to
1 and 2).
in lanes
33 kDa and 22 kDa are
Two
present.
Affinity
labeling
studies
of whole
cytosol
purified
proteins
suggest
that
major
unfractionated
a 33 kDa species (20)
and 17 kDa are also
3 and 4.
bands.
the
23,
after
of 35,
in both is
2.
proteins
species
chromatography
chromatography
observations).
molecular
of minor
affinity
l-AP-6B
l-AP-6B
SDS-PAGE of
in Figure
100,000
abundant,
carcinogen-binding
presented
to either
from
as a number these
are
of apparent
yet
BOUND TO l-AP-6B.
unfractionated
recovered
Discrete,
binding
that
cytosolic
the
in unfractionated
in the
(14).
procedure
of both
chromatography
and of
present
of receptors
to recover
of
binding
observed
has precluded
problem
ANALYSIS
major
matrix
of
recovery
teins
that
native
difficulty
inability
Modification overcome
the
recovery
[3HlBCalP
suggesting
at micromolar
l-AP-6B
our
absent,
allow
estradiol-affinity
has been a significant
diminished
the
to
denaturation
chromatography
from
specific
BCalP
from
Irreversible
related
of
identical
indistinguishable
have
form
1 PM unlabeled
L3HIBCalP
buffers
report
3-methylcholanthrene
(Collins the
from 160
cytosol
the
and that
and Marletta,
partial rat
indicated,
purification liver
cytosol.
isolated unpublished
of multiple An
abundant
Vol. 129, No,. 1, 1985
BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMLJNlCATlONS Ml234
56
from l-AP-6B chromatography. Figure 2. SDS-PAGE of proteins M: molecular weiaht was thr&ah a 12.5% Dolvacrvlamide eel. BC"mouse liver cytosol (9.4 M, in kilodaltons inhicatedf 1: proteins bound to l-AP-CB (2.1 pg); 4: same as (15 pg); 3: unbound effluent 5: unbound effluent from l-AP-6B (5 pg); 6:
32 kDa species mouse
it
is
to l-AP-6B
effective the
their
preparation
3 (1.3
by SDS-PAGE is
analogous
fractionation
steps
as 1
Pg);
from
CL-66
to our
(5
Fg).
findings
in
liver. While
tion
in
Electrophoresis standards with Kg); 2: same
in
clear
additional
chromatography separating
carcinogen-binding
purification
that
of these
the
to achieve vast
(98-99%)
and should
activity, novel
majority
binding
purity,
greatly
proteins
over
are
this
single
of
cytosolic
enhance more
required step
procedure
proteins the
conventional
in addiis
from
selectivity
in
methods.
REFERENCES 1. 2. 3. 4. 5.
J. Biol. Chem. 251, Poland, A., Glover, E., and Kende, A.S. (1976) 4936-4946. Tierney, B., Weaver, D., Heintz, N.H., Schaeffer, W.I., and Bresnick, (1980) Arch. Biochem. BiOohVs. 200. 513-523. E. (1981) Cancer Res_,fi, Holder, G.M., , Tiernev. __ B.. _ and Esnick. 4408-4414. Zytkovicz, T.H. (19821 Cancer Res. 42, 4387-4393. M.A. (198nMol. Pharmacol. Collins, S. and Marletta, -26, 353-359.
161
E.
Vol. 129, No. 1, 1985
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
21. 22.
BIOCHEMICALAND
BIOPHYSICALRESEARCH
COMMUNICATIONS
IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Volume 32, Polynuclear Aromatic Compounds, Part I, Chemical, Environmental and Experimental Data, (1983) W.H.O., Lyon. Eisen, H.J., Hannah, R.R., Legraverand, C., Okey, A.B., and Nebert, D.W. (1983) Biochem. Actions Harm.-g, 227-257. Heintz, N.H., Tierney, B., Bresnl ck, E. and Schaeffer, W.I. (1981) Cancer Res. 41, 1794-1802. Dixit, R., Bickers, D.R., and Muk htar, H. (1,982) Biochem. Biophys. Res. Commun. 104, 1093-1101. WPdinton, O., Griffin, M.J. 3 and Tang, J. (1983) Cancer Res. 43, 4198-4206. Hollenberg, M.D. and Cuatrecasas, P., in: The Receptors (19791, O'Brien, R.D. ted). DD. 193-214. Plenum Press, New York. Bradford;-Ml' (1976) Anal. Biochem. 2, 248-254. Udenfriend, S., Stein, S., Bohlem, P. Dairman, W., Leimgruber, W., and Weigele, M. (1972) Science 178, 871-872. Sica, V., Parikh, Ix, E., Puca, G.A., and Cuatrecasas, P. (1973) 23 Biol. Chem. 248, 6543-6558. Work, T S aTWork, E. (1979) Laboratory Techniques in Biochemistry and Molecul&'Biology, Vol. 7, p.395, North Holland Publishing Co., Amsterdam. Grandics, P., Puri, R-K., and Toft, D.O. (1982) Endocrinology 110, 1055-1057. Laemmli, U.K. (1970) Nature (Lond.1 =,680-685. Fairbanks, G., Steck, T.L., and Wallach, D.F.H. (19711 Biochemistry lo, 2606-2617. Morrissey, J. (1981) Anal. Biochem. 117, 307-310. Tierney, B., Munzer, S., and Bresnick, E. (1983) Arch. Biochem. Biophys. 225. 826-835. ma, v., and Bresciani, F. (1979) Biochemistry I& 2369-2378. Bresciani, F., Sica, V., Weizs, A., Buonaguro, F.M., Bova, R., Puca, G.A., Molinari, A.M., and Endrenyi, L. in: Perspectives in Steroid Receptor Research (1980). Bresciani, F. ted.), pp. 273-298, Raven Press, New York.
162
129,
No.
May
31.
1985
1, 1985
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
155-l
62
DEVELOPMENT OF AN AFFINITY CHROMATOGRAPHY RESIN FOR THE PURIFICATION OF CARCINOGEN BINDING PROTEINS FROM MOUSE LIVER Sheila
Collins,
John D. Altman
and Michael
A. Marietta*
Department of Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received
14arch
11,
1985
Pyrene, a structural analog of benzoCa]pyrene, is an effective competing ligand for high affinity carcinogen binding proteins in mouse liver. A pyrenederivatized Sepharose gel was prepared for affinity chromatography purification of these proteins, and adsorbs all detectable C3H1BCa]P-binding activity from hepatic cytosol with the adsorption of less than 1% of total protein. Specific carcinogen binding activity is recovered from pyrene-derivatized Sepharose columns with the enrichment of a 33 kDa polypeptide. This chromatograpy resin represents a major step in the isolation, of these unusual receptor-like binding proteins for aromatic hydrocarbon carcinogens. D 1985 Academic press, h.
Recent that
bind
investigations a limited
chlorinated
interactions
cinogens
(61.
of
per
sites
cell
(1,7),
sites, potency
*
Address
are
or ability
binding
affinites the
in the
function
the
reminiscent ligands
low nanomolar
binding
microsomal
author
drug
at
range
of the for
affinities metabolizing
Room 56-229,
hormone
potent
and and drug
animal
car-
and a finite
of these
exception
proteins
(PAH)L
of
are
has been discerned
to correlate
of novel
hydrocarbons
characteristics
With
to this
existence
aromatic
features
primary
to induce
correspondence
the
Many of these
proteins.
attempts
demonstrated
polycyclic
(l-5).
no biological
although
of
with
Binding
xenobiotic-binding tor
number
pesticides
receptor
have
number
receptor-like
TCDD-binding these with
PAH-binding carcinogenic
enzymes
MIT,
Ah recep-
have been made
Cambridge,
MA 02139.
'Abbreviations used are: Ah, aryl hydrocarbon; l-AP-CB, 1-Aminopyrene Sepharose 68; BCalP, benzocalpyrene; B6, C5781/6J mice; BSA, bovine serum albumin; CL-6B, Sepharose CL-6B; Hepes, 4-(2-hydroxyethyl)l-piperazine ethanesulfonic acid; and 10% HEDG buffer, composed of 25 w Hepes, 5 mM EDTA, 1.0 n+l dithiothreitol glycerol at pH 7.6; PAH, polycyclic aromatic hydrocarbon; SDS, sodium dodecyl sulfate, SDS-PAGE, SDS-polyacrylamide gel electrophoresis; TCDD, 2,3,7,8TLC, thin-layer chromatography. tetrachlorodibenzo-p-dioxin; 0006-291X/85
155
All
$1.50
Copyright 0 1985 by Academic Press, Inc. rights of reproduction in any form reserved.
Vol. 129, No. 1, 1985
The existence
(2,3,8). bolism dence
of
substrates
significance
of
have
significance
of
the
synthesized binding previously
that
may facilitate
has been suggested
ligand
of putative
extracts
from
proteins
(9,101,
microsomal but
meta-
supporting
evi-
inconclusive.
The limitations
the
of carrier
lipophilic
remains
crude
8lOCHEMlCALAND8lOPHYSlCALRESEARCHCOMMUNlCATlONS
Ah receptor,
receptors
these
(or
described
high
their
(11).
affinity
of
proteins)
for
establishing
probing
their
Therefore,
is resin
in order
from
required. for
the
the
mouse liver
biological
structural
carcinogen-binding
purification
chromatography class
studies
and for
been discussed
an affinity protein
binding
features
to further
proteins Toward
this
isolation whose
in
study
distinct end,
we have
of a carcinogen properties
we have
(5). MATERIALS AND METHODS
ANIMALS (Barrbor, L3HIB[alP
Male
86 mice aged 6-8 weeks were obtained from Jackson Maine). Preparation of 100,000 x 1 hepatic cytosol binding assays were performed as described previously
Laboratories and standard (5).
CHEMICALS B[a]P, pyrene and 1-aminopyrene were obtained from Aldrich Chemical Company (Milwaukee, WI), TCDD from KOR Isotopes (Cambridge, MA), Hepes, dextran, Coomassie Blue 6250, fluorescamine and epoxy-activated Sepharose 68 from Sigma Chemical Company (St. Louis, MO), Norit-A charcoal from Fisher Scientific (Pittsburgh, PA). Reagents for SDS-PAGE were electrophoretic grade and obtained from either Bio-Rad Laboratories or BRL (Rockville, MD). All other chemicals were of the highest purity available. RADIOCHEMICALS [1,3,6-3H]B[a]P (specific activity 87.5 Ci/mmole) and lmethyl-l*C1 methylated BSA were obtained from New England Nuclear Corporation (Boston, MA). C3H]B[a]P was repurified before use as previously described (5). [1,6-3HlTCDD (52 Ci/mmole) was obtained from ICN Radiochemicals (Irvine, CA) with purity assessed by TLC on Silica gel in benzene:methanol (4:ll. SYNTHESIS OF l-AMINOPYRENE SEPHAROSE 6B (l-AP-6B1 l-AP-6B was prepared from epoxy-activated Sepharose 6B and 1-aminopyrene (recrystallized from cyclohexane) according to the manufacturer's instructions. The coupling reaction was carried out in p-dioxane:water (50:50), pH 9.0, at a molar ratio of 1-aminopyrene:epoxy groups of 5:l under a nitrogen atmosphere for 20-24 hours. The derivatized resin was washed extensively with methanol (141. The solvent was periodically collected, evaporated to dryness and examined for the presence of 1-aminopyrene by TLC and UV spectra. Aside from the initial washing step with coupling buffer, none was found. Extent of derivatization was determined spectrally (15) at 292 nm by suspending the desiccated gel in 80% aqueous glycerol and found to be 6.38 2 1.90 nmoles/mg gel, or 2.1 pmoles/ml packed gel. Comparable values have been obtained for steroid-modified Sepharose gels used in steroid hormone receptor purifications (14,16). Subsequent preparations of l-AP-6B routinely contained l-2 umoles ligand/ml packed gel. CHROMATOGRAPHY WITH l-AMINOPYRENE SEPHAROSE 6B l-AP-6B was equilibrated buffer overnight. Control incubations contained an equal gram amount tically treated Sepharose CL-6B. In batch experiments, the Sepharose with an equal volume of B6 mouse liver cytosol (final concentrations:
156
in HEDG of idenwas mixed 1.0 mg/ml
Vol.
129,
No.
1, 1985
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Samples were incubated in a circular protein, 0,,25 - 2.5 mglml Sepharosel. shaking bath for 2.5 hours at 20-22°C. After chilling on ice, aliquots (1.0 ml) of the suspension were added to 1.5 ml Eppendorf tubes and centrifuged 1 minute in a microcentrifuge (13,000 x 1) to pellet the Sepharose. Portions of the supernatant (0.5 ml and 0.1 ml) were removed to monitor B[a]P binding activity and protein concentrations, respectively. For column chromatography, l-AP-6B (6.0 mg/mll was equilibrated overnight in HEDG buffer containing 0.1 M NaCl. Typically, 150 mg of cytosolic protein (3.5 pmol binding activity/mg) were incubated with the l-AP-6B suspension as described ,For batch chromatography at a gram ratio of 2.1 mg l-AP-6B/mg protein and a final protein concentration of 2.5 mg/ml (total volume 61.0 ml). The entire suspension was poured Subsequent operations were conducted at 4°C. into a 0.5 x 9.0 cm column (bed volume = 1.8 ml) at a maximum flow rate of 0.2 ml/min. The unbound proteins were collected, then fractions (1.0 ml) were collected (as the column was washed overnight with equilibration buffer (approximately 50 ml), which was subsequently pooled and concentrated on an Amicon YM5 membrane to 1.5 ml. A small amount of protein (1.15 mgl and only nonspecific binding activity was recovered. Elution of protein bound to l-AP-6B was performed with Hepes buffer, pH 7.6, containing 2.0 M sodium thiocyanate, Bound proteins were eluted in the first and fractions (1.0 ml) were collected. B ml. These were pooled, dialyzed against HEDG buffer (3x1 liter), and concentrated as described above to about 1 ml. This volume was further reduced with an Amicon Centriconto 400 ul (0.85 mg/ml protein). ELECTROPHORESIS SDS-PAGE was carried out using a modification method of Laemmli (17) as previously described (5). Gels were Coomassie Blue (18) or by silver staining (19).
of the stained
with
RESULTS AND DISCUSSION COMPETITION BINDING WITH PYRENE. epoxy-Sepharose
was based
protein
and the
chemical
ability
to compete
effective
with
dize,
Finally,
binding
of
a relatively
stable
be a good candidate 1-aminopyrene
and 86 liver
cytosol,
specific
derivatives
and found in affinity
data
directly
was reduced
its to be than
suggested
chromatography
was incubated binding
for
of PAHs rapidly
These
to
binding
was tested
weaker
affinity
coupling
carcinogen
assays
molecule. for
for
Pyrene
threefold
and amine
where
(not
the
compound.
shown),
hydroxy
for
in competition
in experiments
C3HlBCaIP
l-AP-68
C3H]B[alP
of a ligand
affinity
of the
of 35 nM (not
is
would
relative
stability
a number
1-aminopyrene
1-aminopyrene
levels
an I&,
While
BCalP (5).
for
on its
The choice
oxithat
development. at
1 uM with
to nonspecific
shown).
ADSORPTION OF BCalP-BINDING is shown below.
PROTEIN TO THE l-AP-CB
I4
o-cn,-Fn-cn,-o-(cn1),-O-C~-~H-CH~-NOH
GEL.
/\ -
ON \ %
157
\ \/
The structure
of
Vol.
129,
1, 1985
No.
BIOCHEMICAL
Table
I.
The results vity
mg/ml mg/ml mg/ml mg/ml rig/ml
concentration
changes
in supernatant
of
likely
not
results
phenyl-Sepharose
teins
from
effective
by comparison
with
l-AP-6B.
(40 pmoles/ml
50% of
binding
(2.3
mg/ml)
of
Samples sity
gradients
Specific the
ability
that
the
after 1.
of
majority
adsorption In Figure
conditions
than
more
by l-AP-6B.
also
bind
affinity
the
degree
better of
9S
(20)
have
3-methyl-
BCalP-binding
pro-
derivatization
on
less
than
concentration
in agreement
of
was to
by l-AP-68,
binding with
to binding
content containing
illustrate
la indicates
protein the
is
results
equilibrium
from CL-6B
den-
and to assess
Figure
the Ah receptor,
Ah receptor experiments
on 5 - 20% sucrose
receptor.
B[a]P
158
most
of
gel)
highest
experiments
Ah
visualization
In Control
of
but
phenyl-Sepharose
l-AP/ml
protein
brought
total
pmols
these
were
the
a class
these
analyzed
binding the
No
shown).
also
matrix,
samples
half
for
the 4S-sedimenting
lb identical allows
B[a]P
to
of
to the
C~HITCDD, which
was removed
resin
major
CL-68,
We found
at the
the
were
et al.
purify
l-2
at
recovered.
Tierney
high
while
or CL-68
capacity
vs.
of
fully
acti-
low CL-CB concentrations
cytosol.
(not
The purpose
the
at
binding
manner,
by Sepharose
was adsorbed tested
1). of
this
gel
and CL-CB were
(Figure
vast
packed
activity
l-AP-68
adsorption
liver
BCalP
l-AP-68
to partially
Despite
phenyl-Sepharose
from
is
activity
in adsorptive
phenyl-Sepharose C3H]B[a]P
from
purifcation
rat
the
activity
of 100% activity.
definition
proteins
of
dependent
concentrations
48 chromatography
binding
all
binding
significant
our
147 112 78.7 14.9 7.6
essentially
of 147% of binding
reflect
4B to be minimally
FROM WHOLE RESIN
+ 0.030 7 0.043 + 0.040 T 0.008 IO.018
tested
COMMUNICATIONS
% CONTROL 100
in a concentration
protein
from
cholanthrene
that
CL-68
The recovery does
0.790 0.599 0.421 0.080 0.041
by l-AP-68
highest
used
CL - 6B CL - 68 l-AP-6B l-AP-68 I-AP-CB
I indicate
can be removed
probably
RESEARCH
SPECIFIC C3H]-B[a]P Bound/ml 0.535 + 0.090
of Table
observed.
BIOPHYSICAL
ADSORPTION OF BCalP-BINDING ACTIVITY CYTOSOL BY l-AMINOPYRENE-SEPHAROSE-6B
CONTROL 0.25 2.50 0.25 1.25 2.50
AND
absent of Table with
Under these whole there
cytosol was no
Vol.
129,
No.
BIOCHEMICAL
1, 1985
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
n
I
24-
AND
20-
24
6
16
6
TOP
Froct~on
24
6
TOP
16
24
Number
.$p
Sucrose density gradient fractionation of carcinogen-binding proSamples were incubated with radioligands. treated with dextran-coated charcoal to remove free ligand as described (51, and centrifuged through 5-20% sucrose gradients (5.0 ml) in a SW50.1 Ti rotor at 50,000 rpm for 16 hr at 4°C. Fractions (3 drops) were collected. A: [3]B[a]P (6.5 nM) binding in cytosol before (01 and after (01 l-AP-68 incubation; 300 Pg total protein. B: [3~1~~~~ (8.5 nM) binding as in A; 1.8 mg total protein. C: C3HlBCalP (8.5 nM1 binding in salt-eluted proteins from I-AP-CB in the absence 10) or presence (01 of 1 PM unlabeled B[a]P; 75 @g total protein.
reduction
in
cytosol.
Since
species
at is
protein
which those
this
4s
4s TCDD
4s
gradient
and
either
has
(4,5),
binds
the
OF BCalP
eluted
and
with
sodium
thiocyanate
in
concentrated was
to
disrupt
significant
part
in
the
conducted
of
which
in
for
binding previously
of
ligands
bound
to and
reasons.
(5).
159
to
which this
In
protein,
addition,
region
of
the
from assay
the
4s
methods account
for
indicate
that
hepatic
of purif
interactions
binding
ts
resu
whole
BCalP
probably
been
Materials
several
hydrophobic
our
classes
have
Proteins
described
chosen
these
to
distinct
therein) These
of
ACTIVITY.
are
between
experiments.
neither
abundant
sites
references
both
as
these
relative
sedimenting
differences
and
peaks
more
lower
that
our
binding
this
this
isolate
BINDING
agent
studies
notion
(7
to
chaotropic
in
Small
proteins,
were
activity
the
TCDD
for
binding
observed potential
9s
affinity
investigators
carcinogen-binding
RECOVERY
or
polycyclics.
other
has
little
with
TCDD-binding
l-AP-68
binding
[~H]TCDD
consistent
of
B[alP
ed
the
to
date.
l-AP-6B
Methods.
column Elution
Thiocyanate
serves
we
to
presume
based Bresciani
on
as play
a
structure(21,221
a
has
Vol. 129, No. 1, 1985 shown
that
estrogen
8lOCHEMlCALAND8lOPHYSlCALRESEARCHCOMMUNlCATlONS
thiocyanate-containing receptor
in active
illustrates
the
with
a peak
profile
la).
With
recovery
binding
is
with
this
ligands
part,
allow
this
PAH-binding
this
the
the
Figure
activity
from
l-AP-6B
cytosol
(Fig.
as competing
protein
lc
is
ligand,
recovered
in a form
protein.
during
purification
However,
the
to include
from
buffers
significant
This
9s Ah receptor
of B[alP
mild
of binding
as well
as the
in
in active
containing
and
to associate
may explain,
l-AP-6B
alteration
strategies
solubility tendency
use here.
Ah receptor
elution
poor
and their
their
by affinity
Biospecific
concentrations,
without
uterine
columns.
incubation
problem.
the
calf
form.
detergents
may
properties,
more abundant
and
4s
protein.
are
samples
OF POLYPEPTIDES on l-AP-68
not
of
Proteins
adsorbed
the
proteins
as well
less
by l-AP-6B
the
protein
proteins
Tierney for
et al.
or CL-6B x 1 hepatic
column
(lanes cytosol
are displayed
weights
Most
of the
cytosolic
pro-
5 or 6) compared (lanes
to
1 and 2).
in lanes
33 kDa and 22 kDa are
Two
present.
Affinity
labeling
studies
of whole
cytosol
purified
proteins
suggest
that
major
unfractionated
a 33 kDa species (20)
and 17 kDa are also
3 and 4.
bands.
the
23,
after
of 35,
in both is
2.
proteins
species
chromatography
chromatography
observations).
molecular
of minor
affinity
l-AP-6B
l-AP-6B
SDS-PAGE of
in Figure
100,000
abundant,
carcinogen-binding
presented
to either
from
as a number these
are
of apparent
yet
BOUND TO l-AP-6B.
unfractionated
recovered
Discrete,
binding
that
cytosolic
the
in unfractionated
in the
(14).
procedure
of both
chromatography
and of
present
of receptors
to recover
of
binding
observed
has precluded
problem
ANALYSIS
major
matrix
of
recovery
teins
that
native
difficulty
inability
Modification overcome
the
recovery
[3HlBCalP
suggesting
at micromolar
l-AP-6B
our
absent,
allow
estradiol-affinity
has been a significant
diminished
the
to
denaturation
chromatography
from
specific
BCalP
from
Irreversible
related
of
identical
indistinguishable
have
form
1 PM unlabeled
L3HIBCalP
buffers
report
3-methylcholanthrene
(Collins the
from 160
cytosol
the
and that
and Marletta,
partial rat
indicated,
purification liver
cytosol.
isolated unpublished
of multiple An
abundant
Vol. 129, No,. 1, 1985
BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMLJNlCATlONS Ml234
56
from l-AP-6B chromatography. Figure 2. SDS-PAGE of proteins M: molecular weiaht was thr&ah a 12.5% Dolvacrvlamide eel. BC"mouse liver cytosol (9.4 M, in kilodaltons inhicatedf 1: proteins bound to l-AP-CB (2.1 pg); 4: same as (15 pg); 3: unbound effluent 5: unbound effluent from l-AP-6B (5 pg); 6:
32 kDa species mouse
it
is
to l-AP-6B
effective the
their
preparation
3 (1.3
by SDS-PAGE is
analogous
fractionation
steps
as 1
Pg);
from
CL-66
to our
(5
Fg).
findings
in
liver. While
tion
in
Electrophoresis standards with Kg); 2: same
in
clear
additional
chromatography separating
carcinogen-binding
purification
that
of these
the
to achieve vast
(98-99%)
and should
activity, novel
majority
binding
purity,
greatly
proteins
over
are
this
single
of
cytosolic
enhance more
required step
procedure
proteins the
conventional
in addiis
from
selectivity
in
methods.
REFERENCES 1. 2. 3. 4. 5.
J. Biol. Chem. 251, Poland, A., Glover, E., and Kende, A.S. (1976) 4936-4946. Tierney, B., Weaver, D., Heintz, N.H., Schaeffer, W.I., and Bresnick, (1980) Arch. Biochem. BiOohVs. 200. 513-523. E. (1981) Cancer Res_,fi, Holder, G.M., , Tiernev. __ B.. _ and Esnick. 4408-4414. Zytkovicz, T.H. (19821 Cancer Res. 42, 4387-4393. M.A. (198nMol. Pharmacol. Collins, S. and Marletta, -26, 353-359.
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6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
21. 22.
BIOCHEMICALAND
BIOPHYSICALRESEARCH
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IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Volume 32, Polynuclear Aromatic Compounds, Part I, Chemical, Environmental and Experimental Data, (1983) W.H.O., Lyon. Eisen, H.J., Hannah, R.R., Legraverand, C., Okey, A.B., and Nebert, D.W. (1983) Biochem. Actions Harm.-g, 227-257. Heintz, N.H., Tierney, B., Bresnl ck, E. and Schaeffer, W.I. (1981) Cancer Res. 41, 1794-1802. Dixit, R., Bickers, D.R., and Muk htar, H. (1,982) Biochem. Biophys. Res. Commun. 104, 1093-1101. WPdinton, O., Griffin, M.J. 3 and Tang, J. (1983) Cancer Res. 43, 4198-4206. Hollenberg, M.D. and Cuatrecasas, P., in: The Receptors (19791, O'Brien, R.D. ted). DD. 193-214. Plenum Press, New York. Bradford;-Ml' (1976) Anal. Biochem. 2, 248-254. Udenfriend, S., Stein, S., Bohlem, P. Dairman, W., Leimgruber, W., and Weigele, M. (1972) Science 178, 871-872. Sica, V., Parikh, Ix, E., Puca, G.A., and Cuatrecasas, P. (1973) 23 Biol. Chem. 248, 6543-6558. Work, T S aTWork, E. (1979) Laboratory Techniques in Biochemistry and Molecul&'Biology, Vol. 7, p.395, North Holland Publishing Co., Amsterdam. Grandics, P., Puri, R-K., and Toft, D.O. (1982) Endocrinology 110, 1055-1057. Laemmli, U.K. (1970) Nature (Lond.1 =,680-685. Fairbanks, G., Steck, T.L., and Wallach, D.F.H. (19711 Biochemistry lo, 2606-2617. Morrissey, J. (1981) Anal. Biochem. 117, 307-310. Tierney, B., Munzer, S., and Bresnick, E. (1983) Arch. Biochem. Biophys. 225. 826-835. ma, v., and Bresciani, F. (1979) Biochemistry I& 2369-2378. Bresciani, F., Sica, V., Weizs, A., Buonaguro, F.M., Bova, R., Puca, G.A., Molinari, A.M., and Endrenyi, L. in: Perspectives in Steroid Receptor Research (1980). Bresciani, F. ted.), pp. 273-298, Raven Press, New York.
162