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Methods in Neurosciences
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[15]
Purification of Arrestin from Bovine Retinas
Janina BuczyJko and Krzysztof Palczewski
Introduction
The binding of arrestin (also referred to as the 48K protein or S-antigen) to
freshly bleached phosphorylated rhodopsin occurs rapidly (<200 msec) and
quenches the activation of G protein (transducin) (1-4). We have shown
that arrestin also acts in the phototransduction process by blocking rapid
dephosphorylation of phosphorylated and photolyzed rhodopsin until activated rhodopsin decays (5). The dissociation of arrestin from the photoreceptor protein occurs when photolyzed rhodopsin is completely deactivated by
the removal of the photoisomerized chromophore, all-?/-a«j-retinal (6).
The amino acid sequence of arrestin was determined first for bovine arrestin (7), and the sequence has now been determined for an additional five
species. Arrestin contains a relatively high proportion of nonpolar amino
acids (55%) that are assembled in a /3-sheet conformation (7, 8). However,
very little is known about the biochemical/biophysical and structural properties of the protein.
Arrestin is also of interest because of its ability to cause an autoimmune
disease, uveoretinitis, that resembles uveitis in humans (9). Immunization
of experimental animals with arrestin results in inflammation of the uvea and
retina followed by destruction of retinal photoreceptor cells (reviewed in
Ref. 10).
Arrestin-like proteins have been found recently in nonphotosensitive cells
such as myocardium, liver, kidney, lung, and cerebellum. These proteins
may be involved in the transduction of chemical signals (11), analogous to
the function of arrestin in the transduction of light signals in the visual system.
For example, /3-arrestin has been shown to interact with the /3-adrenergic
receptor (12, 13) in a manner that is analogous to the interaction of arrestin
with rhodopsin.
A variety of methods have been developed for the purification of arrestin.
Several are based on conventional chromatographic procedures (9, 14-16)
that are time consuming and often produce a mixture of intact arrestin and
its proteolytic fragments. The method of Wilden et al. (3) utilizes the binding
of arrestin to illuminated phosphorylated rhodopsin. This procedure requires
the use of dark-adapted retina, gives low yield, and produces arrestin contaminated by vesicles of rod outer segment (ROS) membranes. We have developed an economical, highly reproducible, and simple method that overcomes
Methods in Neurosciences, Volume 15
Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.
[15] PURIFICATION OF ARRESTIN
227
the difficulties mentioned above (17). The method is based on the fact that
arrestin binds the ligands heparin and phytic acid (inositol hexakisphosphate,
InP6). Thus, after arrestin is first bound to heparin-Sepharose, it is selectively
eluted from the resin by phytic acid.
Methods
Purification of Bovine Arrestin by Affinity Chromatography on
Heparin-Sepharose
All procedures are performed at 5°C. This procedure can be applied to
bleached retinas.
Extract Preparation
Fifty frozen dark-adapted bovine retinas (obtained from Lawson, Inc., Lincoln, NE, and stored at -70°C) are thawed in a water bath at 20-25°C. The
retinas are homogenized in a glass-glass homogenizer under a red safelight
with 50 ml of ice cold 10 mM HEPES buffer, pH 7.5, containing 1 mM
benzamidine and 20 /Ag/ml leupeptin (reagents can be purchased from Fisher,
Pittsburgh, PA, or Sigma, St. Louis, MO). Soluble proteins are separated
from retinal debris by centrifugation for 25 min at 43,700 g. The supernatant
is collected and the pellet discarded.
DEAE-cellulose Chromatography
The opalescent and reddish extract (51 ml) is applied at a rate of 25 ml/hr
to a 1.6 x 17 cm column of DEAE-cellulose (Whatman, Clifton, NJ,
DE-52) that has been equilibrated with 10 mM HEPES buffer, pH 7.5. The
column is washed with 10 mM HEPES buffer, pH 7.5, containing 15 mM
NaCl, at a flow rate of 25 ml/hr until the absorbance at 280 nm drops below
0.1 and a major hemoglobin band is eluted from the column (or moves toward
the end of the column; —150 ml). The washing can be performed overnight,
if convenient. Arrestin is eluted with a gradient of NaCl (240 ml total, from
0 to 150 mM NaCl containing 10 mM HEPES buffer, pH 7.5, and 0.5 mM
benzamidine). The flow rate is 15 ml/hr, and 3-ml fractions are collected.
Arrestin is eluted as a broad peak at about 75 mM NaCl as determined
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and immunoblotting (Fig. 1).
Heparin-Sepharose Affinity Chromatography
Combined fractions containing arrestin from the DEAE-cellulose column
(fractions 26-42, total 48 ml) are applied at a flow rate of 20 ml/hr to a 1 x
228
III PHOTORECEPTOR MEMBRANES AND PROTEINS
M.W.
(x10~ 3 )
SDS-PAGE protein profile
92 - L 4
67 -'3i=
43 -'
30 -I
20-
14 _.
L 9 13 17 21 25 29 33 37 41 45 49 53
57
M.W.
Immunoblotting pattern of an
/ x 1 g-3\n monoclonal antibody
9267 43 3020 -
14-
L 9 13 17 21 25 29 33 37 41 45 49 53 57
FIG. 1 DEAE-cellulose chromatography of arrestin (see text for methods). (Top)
Coomassie blue-stained gel of 4 /JL\f the indicated fractions. (Bottom) Immunoblot
[15] PURIFICATION OF ARRESTIN
229
7.5 cm column of heparin-Sepharose that has been equilibrated with 10 mM
HEPES buffer, pH 7.5, and 100 mM NaCl. The column is washed with 10
mM HEPES buffer, pH 7.5, containing 150 mM NaCl at a flow rate of 20
ml/hr until absorbance at 280 nm drops below 0.01 (—60 ml). Arrestin is
eluted at a flow rate of 12 ml/hr by a linear gradient of InP6 (phytic acid,
Sigma, P-1916) in 10 mM HEPES buffer, pH 7.5, and 150 mM NaCl (70 ml
total, from 0 to 8 mM InP6 in the washing buffer), and 1.5-ml fractions are
collected. Arrestin is eluted as a sharp peak at about 2.5 mM InP6 as determined by the absorbance at 280 nm (InP6 does not absorb at this wavelength),
SDS-PAGE, and immunoblotting (Fig. 2). Arrestin at this stage is more than
95% pure, but the preparation contains InP6, which is a competitive inhibitor
of the interaction between arrestin and phosphorylated rhodopsin. Thus,
careful removal of InP5 is necessary. Dialysis will remove the bulk of InP6,
but complete removal can only be achieved by rechromatography on heparin-Sepharose with NaCl as the eluant.
Heparin-Sepharose Ion-Exchange Chromatography
To purify arrestin completely and to eliminate InP 5 , the protein is rechromatographed on heparin-Sepharose. Fractions containing arrestin that were
eluted from heparin-Sepharose by InP6 (fractions 7-12, 9 ml), are dialyzed
against 10 mM HEPES buffer, pH 7.5, containing 100 mM NaCl (dialyze
against 1 liter buffer for 24 hr). Next, the dialyzate containing arrestin is
applied at a flow rate of 13 ml/hr to a 1 x 4 cm column of heparin-Sepharose
that has been equilibrated with 10 mM HEPES buffer, pH 7.5, containing
100 mM NaCl. The column is washed with HEPES buffer containing 100
mM NaCl (10 ml) at a flow rate of 13 ml/hr. Arrestin is eluted with 400 mM
NaCl in the HEPES buffer as determined by absorption at 280 nm and
SDS-PAGE (Fig. 3). From 50 bovine retinas, the yield is about 4 mg of
homogeneous arrestin.
Assessment of Purity
The purity of arrestin should be assessed using SDS-PAGE (18). Additionally,
an immunoblot analysis should be performed to evaluate the structural integrity of arrestin, since the N terminus of arrestin is susceptible to degradation
(19). The extent of degradation of the N terminus of the protein can be tested
of the fractions from DEAE-cellulose chromatography. The staining was performed
employing monoclonal antipeptide antibody CIOC 10 (generous gift from Dr. L.
Donoso). Molecular weight standards were from Pharmacia (Piscataway, NJ). Lane
L was loaded with 4 /xl of extract.
230
III PHOTORECEPTOR MEMBRANES AND PROTEINS
Absorption profile, 280 nm
s
.o
0.2
0.0
10
20
30
Fraction number
Immunoblotting pattern of
anti-arrestin monoclonal antibody
SDS-PAGE protein profile
M.W.
(x1(T 3 )
92674330-
20-
10
11
12
8
9
10
11
12
FIG. 2 Heparin-Sepharose/phytic acid chromatography of arrestin. (Top) The absorption profile was obtained from a heparin-Sepharose column (see text for details).
(Bottom left) Coomassie blue-stained gel of 4 /JL\f the indicated fractions. (Bottom
right) Immunoblot of the fractions from heparin-Sepharose. Staining was performed
employing monoclonal antipeptide antibody CIOC 10. Lane L was loaded with 4 ,1x1
of the pooled fractions obtained from the DEAE-cellulose chromatography step.
[15] PURIFICATION OF ARRESTIN
231
i
M.W.
(x10' 3 )
'i
-
1.2
92-67\
43 _
E
I
0.8
CM
_
03
30-
.a
o
\
S*^«_4__-
.Q
< 0.4 -
20400 mM
NaCI
14-
no
\J.\J
0
6
12
18
24
Fraction number
FIG. 3 Heparin-Sepharose/NaCl chromatography of arrestin. The absorption profile
was obtained from a heparin-Sepharose column (see text for details). (Inset) Coomassie blue-stained gel of 4 /xg of arrestin from the combined fractions (fractions 17-22).
by Edman degradation directly on the sample or on the protein blotted onto
Immobilon membranes (19).
Properties of Purified Arrestin
Stability
Arrestin prepared by this method is stable for several weeks at 0°C. It is
important that the buffer used for arrestin storage includes 0.02% NaN 3 . We
have noticed that arrestin aggregates and precipitates when exposed to very
232
III PHOTORECEPTOR MEMBRANES AND PROTEINS
low ionic strength buffer; therefore, storing in a buffer containing 100 mM
NaCl is highly recommended.
Inhibitors of Arrestin Interaction with Photolyzed and
Phosphorylated Rhodopsin
We have found that heparin and dextran sulfate compete with photoactivated
and phosphorylated rhodopsin to bind arrestin (20). Phytic acid and other
highly phosphorylated inositols inhibit this interaction (17). The dissociation
constants for arrestin and inositol phosphates are in the low micromolar
range. Several other polyanions are poor inhibitors. Neither ATP, GTP, nor
Ca2+ are ligands for arrestin, despite recent reports (discussed in Ref. 21).
Composition and Posttranslational Modification
It has been reported that bovine arrestin may contain lipids and carbohydrate
moieties, covalently attached to the protein (for review, see Ref. 22). It
was also reported that arrestin can be modified substoichiometrically by
phosphorylation, presumably by protein kinase C (23). Mass spectroscopic
analysis reveals that arrestin purified by this method does not have posttranslational modifications, other than acetylation (Fig. 4). It has been shown that
all three of the cysteines in arrestin are available as free sulfhydryl groups
(8, 24) rather than in disulfide linkage as previously suggested (7).
Molecular Weight
Based on the amino acid composition of arrestin obtained from the sequence
of the protein, the molecular weight of arrestin is 45,275 (404 amino acids).
The mass spectroscopic analysis of the native protein revealed that arrestin
is acetylated on its N-terminal methionine, and that the molecular weight is
45,317.57 ± 4.41 (Fig.4).
Extinction Coefficient and Ultraviolet and Fluorescence Spectroscopy
The UV absorption maximum for arrestin is at 278 nm. An accurate extinction
coefficient was determined by amino acid analysis.These results gave an
. absorbance for a 0.1% solution at 278 nm of 0.638. On a per amide basis,
the molar extinction coefficients are e(278) = 71.5, and e(190) = 10,560
(8). The integrity of arrestin can be conveniently monitored by employing
233
[15] PURIFICATION OF ARRESTIN
100
75
w
c
CD
+~>
_c
50 -
CD
CD
DC
25 -
800
850
900
950
1000
1050
1100
1150
1200
M/Z
FIG. 4 Mass spectrum of arrestin. The predicted mass for the protein obtained from
its sequence (7) is 45,275 daltons. If 42 daltons is added for an acetyl group, the
molecular mass is then 45,317 daltons. Twenty different peaks (m/z) corresponding
to the same mass (m = 45,317 daltons) with charges varying from +56 to +37 were
identified. This verifies the molecular size of arrestin and suggests that the protein,
the N terminal of which is blocked, is acetylated on its N terminus. The analysis
was done by Drs. K. Walsh and L. Ericsson at the University of Washington.
fluorescence spectroscopy as described by Kotake et al. (25) (Fig. 5), since
the emission maximum of denatured arrestin is shifted from 306 nm for the
intact protein to 340 nm.
Binding Assay for Arrestin to Photolyzed and
Phosphorylated Rhodopsin
A convenient assay has been developed to monitor arrestin binding to photolyzed and phosphorylated rhodopsin (17). Under red light, phosphorylated
rhodopsin, which has been regenerated with 11-c/s-retinal (30 /u,M; 2 to 5
234
III PHOTORECEPTOR MEMBRANES AND PROTEINS
1.00.8
250
300
350
Wavelength (nm)
400-
£• 300'
s
™ 200 H
CD
DC
100-
300
320 340 360 380
Wavelength (nm)
400
FIG. 5 UV and fluorescence spectra of arrestin. (Top) UV spectrum of arrestin
(0.76 mg/ml) in 10 mM HEPES buffer, pH 7.5, containing 100 mM NaCl. (Bottom)
Fluorescence spectrum (excitation at 280 nm) of arrestin (1 fjiM) in 10 mM HEPES
buffer, pH 7.5, containing 100 mM NaCl at 25°C (dahsed line), along with those of
the same sample partially denatured by incubating for 10 min at 70°C (dotted line)
and buffer alone (solid line).
phosphates per molecule of rhodopsin) in 10 mM HEPES buffer, pH 7.5,
containing 100 mM NaCl and 1 mM MgCl 2 , is mixed with purified arrestin
(5 jU,M) in a total volume of 60 /id. The sample is illuminated under a 180-W
lamp for 5 min from a distance of 10 cm at 30°C. Under these conditions
45-50% of rhodopsin is bleached. The sample is centrifuged in an Airfuge
(Beckman, Fullerton, CA) at 180,000 g for 2 min. The supernatant is stored
for SDS-PAGE and/or protein content analysis. The pellet is washed with
180 /u.1 of the HEPES buffer, centrifuged under the same conditions, and
dissolved in 55 /id of 1% SDS containing 0.1% 2-mercaptoethanol for SDSPAGE analysis.
[15] PURIFICATION OF ARREST1N
235
Acknowledgments
We thank Drs. K. Walsh and L. Ericsson (University of Washington) for mass
spectroscopy analysis of arrestin, Dr. L. Donoso (Jefferson University) for a monoclonal antibody against arrestin (C10C10), and Preston Van Hooser for excellent
technical assistance. This research was supported by U.S. Public Health Service
Gant EY 09339 and by a grant from the Human Frontiers in Science Program. KP
is the recipient of a Jules and Doris Stein Research to Prevent Blindness Professorship.
References
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236
III PHOTORECEPTOR MEMBRANES AND PROTEINS
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