From the Graduate School of
Science and Technology and § Department of Biology, Faculty
of Science, Kobe University, Nada, Kobe 657, Japan
Received for publication, January 22, 2001, and in revised form, April 20, 2001
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ABSTRACT |
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Detergent-resistant membrane
microdomains in the plasma membrane, known as lipid rafts, have been
implicated in various cellular processes. We report here that a
low-density Triton X-100-insoluble membrane (detergent-resistant
membrane; DRM) fraction is present in bovine rod photoreceptor outer
segments (ROS). In dark-adapted ROS, transducin and most of
cGMP-phosphodiesterase (PDE) were detergent-soluble. When ROS membranes
were exposed to light, however, a large portion of transducin localized
in the DRM fraction. Furthermore, on addition of guanosine
5'-3-O-(thio)triphosphate (GTP The phototransduction system in the photoreceptor rod outer
segments (ROS)1 of
vertebrates is a typical G protein-mediated signaling system. In the
prevailing model of phototransduction (1), light-excited rhodopsin
interacts with the GDP form of the heterotrimeric G protein transducin
and stimulates GDP-GTP exchange on its Most of the signaling proteins in ROS are membrane proteins, and they
are often modified with lipids. Rhodopsin is modified by tandem
palmitic acids at the carboxyl end of the fourth cytoplasmic loop (3).
T The lipid modification of signaling proteins has recently been
discussed in connection with its ability to lead proteins to cholesterol- and sphingolipids-enriched membrane microdomains called
lipid rafts (9). It has been proposed that lipid rafts exist in a
separate phase from the rest of the bilayer, in a state similar to the
liquid-ordered phase described in model membranes. Surrounding fluid
membrane is in a state similar to the liquid-disordered (ld) phase. Biochemically, the components of lipid rafts
are characterized by their insolubility in the detergent Triton X-100
(10). Increasing evidence suggests that cholesterol and
sphingolipid-rich lipid microdomains or rafts exist in eukaryotic cell
membranes where they have important functions (11). Trimeric G proteins
have also been implicated in signal transduction in the raft (12).
Here, we have characterized the detergent-insoluble fraction of bovine
photoreceptor ROS using sucrose density gradient ultracentrifugation. We demonstrate that important signaling proteins such as transducin and
PDE exert massive translocation between detergent-resistant membrane
(DRM) and detergent-soluble membrane domains, depending on their
activation steps. The importance of raft-like membrane domains on disc
membranes in phototransduction will be discussed.
Preparation of Triton X-100-insoluble Membrane Fraction from
Bovine ROS--
Dark-adapted bovine frozen retinas were from Lawson
Co. Ltd., Nebraska. ROS were prepared in the dark by using
an image converter (NoctovisionTM, Nippon Electric Company) as
described previously (13). ROS were suspended (12 mg protein/ml) in
Buffer A (10 mM MOPS (pH 7.2), 60 mM KCl, 30 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM BAPTA, 1 mM
phenylmethylsulfonyl fluoride, 5 µM aprotinin, 1 µM leupeptin, 1 µM pepstatin A, 1 µM E64), and stored in the dark at
100 µl of Buffer A or Buffer A containing 2 mM
GTP
To exclude the possibility that our results were artifact from freezing
and thawing of ROS membranes, we performed DRM preparation using ROS
prepared from fresh bovine retinas purchased from a local slaughter
house. Exactly the same results were obtained by using such native ROS membranes.
Immunoblotting Detection of Various Subunits of PDE and
Transducin in Fractions from Sucrose Gradient Centrifugation--
To
analyze the distribution of PDE and transducin subunits, aliquots of
each fraction were subjected to SDS-polyacrylamide gel electrophoresis
(PAGE), followed by immunoblotting with antibodies or antiserums as
described previously (14).
Antibodies--
Rabbit polyclonal antibodies were raised against
peptides corresponding to various bovine antigens as follows:
Asn24-Ala40 of P Cholesterol Extraction with Methyl- Other--
Proteins were analyzed by SDS-PAGE using the
following types of gradient gels: 1) 8-18%T, 0.4%C gels for
general purpose; 2) 8-18%T, 0.08%C gels for visualizing the
characteristic double bands of P Effect of Light and GTP
Protein compositions in fractions from sucrose density gradients were
analyzed by SDS-PAGE with CBB staining (Fig. 1, lower panels). Several characteristic proteins were observed in the buoyant fractions derived from all ROS (fractions 4-5). Among these
proteins, three components with molecular masses of 90,000, 88,000, and 40,000 Da seemed to vary their distribution in a
stimulus-dependent manner. In the absence of GTP
The 90,000- and 88,000-Da proteins accumulated in the DRM
fraction after simultaneous stimulation of ROS by light and GTP
We assayed the molecular activity of PDE in fractions derived from
light-bleached and GTP
Next, we examined the localization of transducin and PDE subunits in
the fractions using specific antibodies (Fig.
2). T
The most abundant 37,000-Da component observed in all DRM
fractions was identified to be rhodopsin by immunoblotting (data not
shown). About 10% of the total ROS rhodopsin was usually observed in
the DRM fractions; however, unlike PDE or transducin, rhodopsin showed
no significant translocation in any case.
Solubilization of PDE with MCD Extraction--
It is known that
the cholesterol-removing regent, methyl- Although the lateral organization of membrane lipids on the disc
membrane of ROS has been expected, there has been no concrete evidence
to support the presence of raft-like phases in ROS. However, we have
found that a low-density buoyant fraction, i.e. a
DRM, can be prepared from bovine photoreceptor disc membranes,
suggesting that there are raft-like lipid domains in ROS. Here we have
explored the effect of light and the unhydrolyzable GTP analog GTP First, we found that all transducins in the dark-adapted ROS, seemingly
GDP-T By contrast, PDE showed light- and GTP In addition to such a reduction in the negative targeting signal,
however, a component, either protein or lipid, or a physical condition
of the DRM is probably required to recruit activated PDE to the DRM. As
cholesterol depletion by MCD selectively but partially releases PDE
from the DRM, cholesterol seems to contribute, at least in part, to the
recruitment of PDE to DRM. The incomplete solubilization of PDE by MCD
suggests, however, that there is an additional component(s) or a
physical condition that holds PDE in the DRM.
A stimulus-dependent assembly of proteins, including PDE,
at the rim region of the disc membrane has been proposed (22). The
contribution of GARP2 (63,000 Da) has been implicated both in this PDE
assembly and in the inhibition of PDE. So far, however, we have not
observed a 63,000-Da protein that co-translocates with PDE to DRM,
though the existence of GARP2 in DRM has not been excluded.
It should be noted that the molecular activity of PDE in the DRM
prepared from light- and GTP On the other hand, there may be another possible mechanism
with which active PDE is anchored to the DRM. We observed major fractions of retina-specific regulator of G protein signaling (RGS9;
55,000 Da) and a novel G Conclusively, our data strongly suggested that the
raft-like phase in bovine ROS disc membranes are highly likely the
place where the signaling proteins involved in phototransduction make contact with each other in their own activation
step-dependent manners. By exploring the structure,
temporal behavior, and function of this raft-like phase on the disc
membranes, deeper insights into phototransduction system of vertebrate
photoreceptors and its adaptation mechanism would be obtained.
S) to light-bleached ROS,
transducin became detergent-soluble again. PDE was not recruited to the
DRM fraction after light stimulus alone, but simultaneous stimulation
by light and GTP
S induced a massive translocation of all PDE
subunits to the DRM. A cholesterol-removing reagent,
methyl-
-cyclodextrin, selectively but partially solubilized PDE from the DRM, suggesting that cholesterol contributes, at least in
part, to the association of PDE with the DRM. By contrast, transducin
was not extracted by the depletion of cholesterol. These data suggest
that transducin and PDE are likely to perform their functions in
phototransduction by changing their localization between two distinct
lipid phases, rafts and surrounding fluid membrane, on disc membranes
in an activation-dependent manner.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit (T
).
GTP-T
separates from its counterpart, the
subunit of transducin (T
), and binds the
inhibitory subunit (P
) of cGMP-phosphodiesterase (PDE),
thus releasing the constraint of P
on the catalytic
subunits (P
and P
) of PDE. The resulting
decrease in cytoplasmic cGMP leads to the closure of cGMP-gated
channels and the hyperpolarization of photoreceptor plasma membranes.
Although the signaling cascade of ROS has been intensively studied
during the past two decades, the whole mechanism has not yet been
elucidated (for review see Ref. 2).
is modified by a fatty acyl chain, and
T
is both modified by a farnesyl group and
carboxymethylated (4-6). By contrast, P
and
P
are modified by farnesyl and geranylgeranyl, respectively (7), and P
subunits are carboxymethylated (8).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
95 °C.
S was added to 400 µl of ROS suspension. The suspensions were
incubated at 0 °C for 30 min in the dark or under normal room
lighting. The following procedures were all carried out at 4 °C, and
for dark-adapted ROS all procedures were done in complete darkness. After incubation, each suspension was mixed with a 1/2 volume of Buffer
A containing 3% (w/v) Triton X-100 to a final detergent concentration
of 1% (w/v) and then homogenized by passing through a 21-gauge needle
three times. The homogenate was mixed with 2.4 M sucrose in
Buffer A to a final sucrose concentration of 0.9 M and
placed in the bottom of centrifuge tubes (SW55-Ti; Beckman). Samples
were overlaid with 0.8, 0.7, 0.6, and 0.5 M sucrose
solutions in Buffer A (900 µl each) and subjected to
ultracentrifugation (46,000 rpm for 20 h at 4 °C).
500-microliter fractions were collected from the top of the centrifuge
tube downwards and stored at 0 °C.
;
Gly24-Cys40 of P
; and
Val16-Pro28 of P
. Antibodies against
P
and P
showed high selectivity to each
antigen. An antibody against T
(Gly2-Leu15) was obtained from Calbiochem;
antibodies against T
(G
1; C-16),
T
(G
1; P-19), and heat-shock protein 90 (Hsp90) were from Santa Cruz Biotechnology, Inc.
-Cyclodextrin
(MCD)--
MCD was used to remove cholesterol from membrane fractions.
After dilution with Buffer A, the DRM (32 µg protein) was sedimented by centrifugation and resuspended in 100 µl of Buffer A containing various amounts of MCD. After incubation on ice for 1 h, fractions were centrifuged at 100,000 × g for 30 min at 4 °C.
The supernatants and pellets were separated and processed for SDS-PAGE.
Cholesterol was assayed spectrophotometrically using a diagnostic kit
(Cholesterol CII-Test; Wako).
and P
with CBB staining (15). PDE activity was assayed by a phosphate release
assay as described previously (16). Protein assay was performed
routinely by the Bradford assay (17). The content of PDE in each
fraction was assayed by densitometric scanning of immunoblotting data
on x-ray film using NIH image software. Purified PDE was used as a
standard, and protein content was measured in the linear region of
standard curves.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S on Appearance and Protein Composition
of the DRM--
First, we examined whether the DRM could be isolated
from bovine ROS. Suspensions of ROS were incubated in the dark or under room light with or without GTP
S and then homogenized in a buffer containing 1% Triton X-100. In each case, DRMs were observed as a
diffuse yellow-white band in a low-density region (1.1 ± 0.05 g/ml at 4 °C) (Fig. 1, upper
panels). Light-bleached and GTP
S-stimulated ROS gave a slightly
denser band in a slightly higher-density region (Fig. 1B,
upper panel).
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Fig. 1.
Effect of light and
GTP S on the appearance and protein composition
of the low-density Triton X-100-insoluble fraction of bovine ROS.
ROS membranes (5 mg of protein) were incubated in the dark or
under room light at 0 °C for 30 min in the presence or absence of
GTP
S (400 µM). A, light/
GTP
S;
B, light/+GTP
S; C, dark/
GTP
S;
D, dark/+GTP
S. After incubation, ROS were solubilized
with 1% Triton X-100 at 4 °C and then subjected to sucrose density
gradient centrifugation. Gradients were separated into 500-µl
fractions from top to bottom. ppt indicates the pellet
fraction resuspended in 500 µl of Buffer A. Upper panels,
low-density buoyant fraction revealed by light illumination after
centrifugation (top to bottom of fraction is
shown from left to right). Lower
panels, CBB-stained protein profiles. 5-µl aliquots of each
fraction were subjected to SDS-PAGE (8-18%T, 0.08%C) followed by CBB
staining. The positions of P
, P
,
T
, and rhodopsin are indicated.
S, light
exposure of ROS elicited massive translocation of the 40,000-Da protein
from the detergent-soluble (see Fig. 1C; fractions 9-10) to
the DRM fractions (see Fig. 1A; fraction 5). This
light-dependent accumulation of the 40,000-Da protein in
the DRM fraction was prevented by the addition of GTP
S to
light-bleached ROS (compare Fig. 1, A and B). In
contrast, no effect of GTP
S on the distribution of the 40,000-Da
protein was observed in the dark-adapted ROS (Fig. 1, C and
D). On the basis of its molecular mass and its quantity
relative to rhodopsin, we thought that the 40,000-Da protein was highly
likely to be the transducin
subunit.
S, whereas protein bands of these molecular masses diminished in the
detergent-soluble protein fractions (compare Fig. 1, A and B). Even in dark-adapted ROS, GTP
S induced indistinct but
detectable translocation of 90,000- and 88,000-Da components to the DRM
(compare fraction 5 of Fig. 1, C and D). We
thought that these bands were likely to be the
and
subunits of
PDE. In addition, it is noteworthy that the 88,000-Da protein, which
has the same apparent molecular mass as P
, is in the
soluble fraction (fraction 10). We identified this protein as Hsp90 in
terms of immunoblotting (data not shown).
S-stimulated ROS. Without exposure to GTP
S,
the molecular activity of PDE was negligible in all fractions. In
contrast, PDE in either the DRM or detergent-soluble fractions from ROS
stimulated by both light and GTP
S showed considerably higher
molecular activities. The activities in the DRM- (Fig. 1B,
fraction 5) and Triton X-100-soluble fractions (Fig. 1B,
fraction 10) were 125 ± 10 (n = 3), and 467 ± 27/(n = 3) cGMP molecule/sec/PDE molecule, respectively.
showed exactly the
same behavior as the 40,000-Da protein shown in Fig. 1. In dark-adapted
ROS, all subunits of transducin were detergent-soluble (Fig.
2B). In light-bleached ROS, 30-50% of all transducin
subunits were detergent-insoluble and located in the DRM (Fig.
2A). On addition of GTP
S to light-bleached ROS, these
subunits became detergent-soluble again. P
and
P
were detected at the same positions as the CBB-stained
90,000- and 88,000-Da bands. In light-bleached ROS, GTP
S elicited a
massive translocation of all PDE subunits from the detergent-soluble to the DRM fractions (Fig. 2C). More than 80% of all PDE
subunits were in the DRM under our experimental conditions. Even in
dark-adapted ROS, GTP
S induced small but detectable translocation of
all PDE subunits to the DRM (Fig. 2D).
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Fig. 2.
Effect of light and
GTP S on the partitioning of transducin and PDE
subunits in the DRM. Dark-adapted or light-bleached ROS was
incubated in the presence or absence of GTP
S (400 µM),
solubilized, and then subjected to sucrose density gradient
centrifugation as described in Fig. 1. Proteins in 0.2-µl aliquots of
each fraction were separated by SDS-PAGE and blotted onto
nitrocellulose membranes. Immunoblotting was done by using specific
antibodies against the subunits of PDE (P
,
P
, and P
) or transducin
(T
, T
, and T
).
A and B, subunits of transducin; C and
D, subunits of PDE; A and C, light;
B and D, dark.
-cyclodextrin, can
solubilize a certain protein component of the Triton X-100-resistant
membrane of rat brain (18). Thus, we examined the effect of MCD on the
protein composition of DRM derived from light- and GTP
S-exposed ROS.
Addition of MCD to the DRM fraction resulted in a
dose-dependent and selective solubilization of proteins,
which gave a broad band of ~90,000 Da on SDS-PAGE gels (8-18%T,
0.4%C) (Fig. 3A).
Immunoblotting data showed that the band coincided with
P
and P
(see Fig. 3B; data not shown for P
). MCD also extracted P
in a
similar, dose-dependent manner (Fig. 3B). MCD
removed almost all of cholesterol from the DRM in a
dose-dependent manner (Fig. 3C), whereas MCD
solubilized 25 ± 4%; n = 3 of PDE from the DRM.
Notably, no other proteins in the raft, including rhodopsin, were
extracted. In contrast to PDE, no transducin was extracted by MCD from
the DRM prepared from light-bleached ROS without GTP
S (Fig.
3D).
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Fig. 3.
Specific solubilization of PDE in the DRM
with MCD. A, protein solubilized with MCD from light-
and GTP S-induced DRM. After incubating DRM (32 µg of
protein) with the indicated concentrations of MCD in 100 µl of Buffer
A for 60 min on ice, the samples were centrifuged at 100,000 × g for 30 min. Supernatants (spt) were recovered.
Pellets (ppt) were resuspended in the original volume, and
the same sample volume (10-µl) was analyzed on a CBB-stained SDS-PAGE
gel (8-16%T, 0.4%C). B, identification of extracted
protein as PDE. Proteins separated by SDS-PAGE were immunoblotted
(IB) with P
- and P
-specific
antibodies. C, extraction of cholesterol by MCD from DRM
derived from light- and GTP
S-stimulated ROS. DRM containing 1.2 pmol
of PDE was treated with varying concentration of MCD. Cholesterol
contents of extracts and residual membranes were determined. Data are
plotted as a percentage of the total cholesterol content (0.67 nmol) in
DRM. D, MCD treatment of DRM prepared from light-bleached
ROS without GTP
S. DRM was treated with 10 mM MCD, and
spt and ppt were analyzed on a CBB-stained SDS-PAGE gel.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S
on the distribution of photosignaling proteins in this raft-like fraction of ROS.
, were
detergent-soluble, whereas light exposure elicited the recruitment of a
considerable amount of transducins to DRM. Insolubility in detergents
like Triton X-100 is observed in lipid bilayers that exist in physical
states in which lipid packing is tight (10). Thus, in the dark
condition, transducins seem to be localized in detergent-soluble fluid
membrane domains of disc membranes. On the contrary, at least a portion
of transducin seems to be recruited to a raft-like tightly packed lipid
phase on disc membranes. The addition of GTP
S to light-bleached ROS
prior to solubilization inhibited this recruitment. Because
light-bleached rhodopsin (Rh*) has high affinity to
GDP-T
(19), and GTP
S
inhibited the recruitment of transducin to DRM in light-bleached ROS,
the transducin-binding site in DRM is highly likely rhodopsin. In
addition, it should be emphasized that, although the distribution
pattern of rhodopsin along the sucrose density gradient was apparently
constant in all conditions, that of transducin was drastically changed
by light exposure of ROS. Light-dependent recruitment of
transducin to DRM might be explained in two ways: 1) Rh* binds with
GDP-T
in the
detergent-soluble regions of ROS membranes, and then the complex was
recruited to the DRM; 2) some rhodopsins are originally localized on
DRM, and when bleached, they recruit
GDP-T
from
detergent-soluble membrane. In the former case,
light-dependent increase in rhodopsin should be observed
with the assembly of transducin to the DRM, though no increase in the
amount of rhodopsin in DRM was detected. Thus, this hypothesis seems to
be unlikely. However, to exclude this hypothesis, more accurate
measurement of rhodopsin, which is bound by transducin, in DRM should
be done. On the other hand, the latter explanation seems to fit well
with our data, though it may require heterogeneity of rhodopsin in the
disc membrane as a basal assumption. Our data suggest that there are
two rhodopsin pools in ROS, one in DRM and one in the detergent-soluble
membranes. Rh* on DRM seems to have a priority to make contact with
GDP-T
, in comparison to
Rh* in the detergent-soluble membranes. So far, it is difficult to
estimate the real size of the pool of rhodopsin on the raft-like phase
in native ROS. The experiment shown in Fig. 1 may lead us to
underestimate the amount of
Rh*·GDP-T
complex on the
raft-like phase in native system if it exists, because we used a
detergent to prepare the raft-like membranes from ROS. A
detergent-independent preparation method of rafts (20) may be useful to
assess the real size of such a rhodopsin pool. So far, our data
indicate that at least ~10% of rhodopsin in ROS localizes in the
raft-like phase, and a certain group of transducins selectively
interacts with them. The functional significance of the rhodopsin in
DRM and that of the transducin having affinity to such Rh* are obscure
by this time. Exploration on the localization of the raft-like phase on
disc membranes may clarify their roles in phototransduction.
S-dependent
translocation from the detergent-soluble to the DRM fractions. As
isoprenyl moieties on peripheral membrane proteins are considered to be a negative targeting signal to lipid rafts (21), the two isoprenyl residues on P
may cause its exclusion from the raft-like domains on the disc membrane. Proteins in
ld phase membranes are soluble in Triton X-100 (10), which
is in agreement with the high detergent-solubility of unexcited PDE. Further, we speculate that the activation of PDE may bring about a
conformational change of PDE, which might reduce its targeting signal
to the ld phase presumably by rearranging the two isoprenyl moieties to an unexposed location.
S-stimulated ROS seemed not to be
suppressed. Apparent molecular activity of PDE in the DRM was
suppressed only partially, although almost the same proportions of
P
and P
were co-localized as discussed above. We have observed uni- or multilamellar vesicles (100-200 nm in diameter) in the DRM fraction by electron
microscopy.2
Therefore, the apparent suppression of the activity is highly likely because of the inaccessibility of cGMP to enzymes facing the
inner surface of the vesicles. If this is true, GARP2 would not be a
good candidate as an anchor for PDE on DRM, because it strongly
inhibits PDE (22).
subunit (G
5L; 44,000 Da) in DRM.3 Because they have a
high-affinity to the
AlF4
-GDP·T
·P
complex (23), it is highly likely that they form multiprotein complex
with
GTP
S·T
·P
2 on the raft-like membrane domains on the disc membrane. Although T
remaining in the DRM was scarcely detected by
immunoblotting (Fig. 2A), such a little portion of
T
may be sufficient for the activation of all PDE in
DRM. In any case, to elucidate the mechanism that keeps PDE in its
active form despite the co-localization of P
in DRM,
more accurate measurement of the amounts of PDE and T
and knowledge about the composition of the multiprotein complex
containing the activated PDE in DRM are essentially needed.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Shohei Maekawa and Dr. Akio Yamazaki for valuable discussions. We also express our gratitude to an anonymous reviewer for making a number of helpful comments on an earlier version of the manuscript.
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FOOTNOTES |
---|
* This work was supported in part by Grant-in-aid 12490023 from the Ministry of Education, Science and Culture of Japan (to F. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel./Fax: 81-78-803-5717; E-mail: fhayashi@kobe-u.ac.jp.
Published, JBC Papers in Press, April 23, 2001, DOI 10.1074/jbc.C100032200
2 M. Mieda, M. Kishimoto, H. Liu, K. Seno, T. Suzaki, and F. Hayashi, unpublished observation.
3 K. Seno, M. Abe, Y. Higuchi, M. Mieda, Y. Owada, H. Liu, and F. Hayashi, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are:
ROS, rod
outer segments;
PDE, cGMP-phosphodiesterase;
DRM, detergent-resistant membrane;
MCD, methyl--cyclodextrin;
ld, liquid-disordered;
MOPS, 4-morpholinpropanesulfonic acid;
BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid;
GTP
S, guanosine 5'-3-O-(thio)triphosphate;
PAGE, polyacrylamide gel electrophoresis;
Hsp, heat-shock protein;
CBB, Coomassie Brilliant Blue;
Rh*, light-bleached rhodopsin.
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