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INTRODUCTION |
The continual synthesis of rhodopsin by recombination of its
apoprotein with the chromophore, 11-cis-retinal, is an
essential process that maintains visual excitation (1, 2). It has long
been known that formation of 11-cis-retinal for regeneration of rhodopsin is dependent on retinoid metabolic reactions in the retinal pigment epithelium
(RPE),1 where the majority of
enzymes of the visual cycle are located (reviewed in Ref. 3). In the
current model of the visual cycle, all-trans-retinal from
bleached rhodopsin is reduced to all-trans-retinol by an
all-trans-retinol-specific dehydrogenase (tRDH) located in
photoreceptor outer segments (4-6). The all-trans-retinol is then delivered to the RPE, where it is converted by the
lecithin:retinol acyltransferase to all-trans-retinyl ester
(7, 8). A key step in the visual cycle is performed by an
isomerohydrolase that catalyzes the formation of
11-cis-retinol from all-trans-retinyl ester
(9-10). Alternatively, the isomerization of
all-trans-retinol to 11-cis-retinol is achieved
via an intermediate with an anhydro-like carbocation structure (11). An
11-cis-retinol-specific dehydrogenase (cRDH) is able to
oxidize 11-cis-retinol to 11-cis-retinal
(12-14), which is transferred to the outer segments of photoreceptors
for recombination with opsin to form rhodopsin. Under light-adapted conditions, the rate of synthesis of 11-cis-retinal must be
sufficient for regeneration of steady-state levels of visual pigments
(15, 16).
Besides isomerohydrolase, another type of isomerase in the RPE may
include the retinochrome-like visual pigment homologues peropsin or the
RPE retinal G protein-coupled receptor (RGR) opsin (17, 18). In
contrast to the visual pigments, RGR is bound in the dark to endogenous
all-trans-retinal and is localized to intracellular
membranes in RPE and Müller cells (19, 20). Upon illumination,
all-trans-retinal bound to RGR is photoisomerized stereospecifically to the 11-cis isomer (20). These results provide evidence that RGR may function to generate
11-cis-retinal in vivo and participate in a
light-dependent photic visual cycle.
A proposed mechanism of RGR function is that 11-cis-retinal
dissociates from irradiated RGR and directly enters the pathway for
regeneration of rhodopsin under photic conditions. A central hypothesis
of the photoisomerase model is that exchange of the chromophore bound
to RGR occurs and involves distinct geometrical isomers. Retinal must
uncouple and bind anew to RGR through a stereospecific cycle that is
driven by light energy. The observation that microsomal RGR can be
labeled with 3H-labeled all-trans-retinal (21)
and that ~50% of RGR isolated from RPE is in the form of the
apoprotein (22) suggests that the binding of retinal is reversible and
that the process of binding and dissociation of the chromophore occurs
in vivo.
One of the factors that may control Schiff base hydrolysis and
dissociation of 11-cis-retinal from RGR in vivo
is specific protein interactions. We have observed that when RGR is
isolated in digitonin solution from bovine RPE microsomes by
immunoaffinity chromatography, it co-purifies consistently with a
32-34-kDa protein (p32) (22). It is possible that the co-purified
protein forms a physical complex or associates functionally with RGR.
Hence, the identification of the co-purified p32 protein may provide important insights into the mechanism of RGR function.
In this paper, we report the analysis and characterization of
co-purified p32, which has been identified as 11-cis-retinol dehydrogenase of the RPE. We demonstrate enzymatic activity of the co-purified cRDH and discuss its potential role in RGR function.
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EXPERIMENTAL PROCEDURES |
Materials--
Digitonin was obtained from
Calbiochem-Novabiochem. Hydroxylamine and all-trans-retinal
were purchased from Sigma. 11-cis-Retinal was provided by
Dr. Rosalie Crouch (Medical University of South Carolina, Charleston,
SC). An 11-cis-retinol standard was prepared by reduction of
11-cis-retinal in the presence of NaBH4 and was purified by high performance liquid chromatography (HPLC) as described previously (23). Organic solvents were HPLC grade. Dichloromethane and
hexane were obtained from Fisher. Ether and methanol were from J. T. Baker Inc. Dioxane was from Burdick & Jackson.
Isolation of RGR--
All isolation procedures were performed
under dim red light. RPE cells were isolated from fresh bovine eyes, as
described previously (22). The cells were washed in ice-cold 0.25 M sucrose, 30 mM sodium phosphate buffer, pH
6.5, and homogenized in a glass Dounce homogenizer. After a low speed
centrifugation at 300 × g, the homogenate was
centrifuged at 15,000 × g for 20 min at 4 °C. RPE
microsomes in the supernatant were sedimented by centrifugation at
150,000 × g for 1 h at 4 °C and stored at
80 °C until later use. For isolation of RGR, the microsomal
membranes were extracted three times for 1 h at 4 °C with 1.2%
digitonin solution containing 10 mM sodium phosphate, pH
6.5, 150 mM NaCl, and 0.5 mM EDTA. The extracts
were centrifuged each time at 100,000 × g for 20 min
at 4 °C. The pooled supernatants were incubated overnight at 4 °C
with Affi-Gel 10 resin (Bio-Rad) conjugated to monoclonal antibody 2F4,
which is directed to the carboxyl terminus of bovine RGR (21). The
resin was transferred to a column and washed with 25 bed volumes of
wash buffer (0.1% digitonin in 10 mM sodium phosphate, pH
6.5, 150 mM NaCl, and 0.5 mM EDTA). RGR was
eluted from the column with 10 times 0.5 bed volumes of wash buffer
containing 50 µM bovine RGR carboxyl-terminal peptide
(CLSPQRREHSREQ). The eluted fractions were pooled and concentrated
~4-fold using a Centricon 3 concentrator (Amicon, Inc., Beverly, MA).
The proteins were analyzed by SDS-polyacrylamide gel electrophoresis
and stained with Coomassie Blue. The amount of isolated protein was
determined with a densitometer by measurement of relative band
intensity and comparison with known amounts of bovine serum albumin
protein standard. The amount of RGR used in some experiments was based on a typical yield of ~64 ng/eye isolated from 15-80 eyes. In a
modification of the isolation procedure, 20% glycerol was included in
all buffers for the isolation of RGR from RPE microsomes, as indicated
in Fig. 5.
Mass Spectrometry--
Immunoaffinity-purified RGR was
electrophoresed in a 15% SDS-polyacrylamide gel and stained with
Coomassie Blue. A 32-kDa protein (p32) was found to co-purify
consistently with RGR. The p32 protein band was excised from the gel
and used for a trypsin in-gel digestion (24). A portion of the digested
peptide mixture was analyzed using a custom-built capillary liquid
chromatography system (25) interfaced directly to a Thermo-Finnigan
(San Jose, CA) LCQ ion trap mass spectrometer, as described
previously (26). Full mass range spectra, high resolution zoom scan
spectra, and fragment ion (MS/MS) spectra were collected using the
automated, data-dependent acquisition functions of the LCQ
data system. The protein identification was made by correlating the
collected MS/MS spectra to the OWL protein sequence data base using the
Sequest data base search program (27).
11-cis-Retinol Dehydrogenase Assay with Exogenous
Substrate--
The reduction activity of cRDH was measured as
described previously (12, 28). The thiobarbituric acid method
was used to monitor the colored derivative formed by retinal and
thiobarbituric acid (29). The immunoaffinity-purified RGR or RPE
microsomal proteins were used as the source of cRDH enzyme. For the
reduction of exogenous substrate, the substrate solution contained 10 nmol of exogenous retinal isomer, 12% acetone, 1.2% Tween 80, 1 mM NADH or NADPH, and 0.2 M sodium acetate
buffer, pH 5.0. Controls were performed by omitting the NADH or NADPH
cofactor or by addition of a heat-inactivated protein sample. The
reactions were initiated by the addition of 50 µl of substrate
solution to 50 µl of protein solution. After incubation at 37 °C,
the reactions were terminated by addition of 0.5 ml of ethanol.
Subsequently, 0.2 ml of thiourea and 0.2 ml of thiobarbituric acid
reagent were added, and the samples were incubated for 30 min at room
temperature. The solutions were centrifuged at 14,000 rpm on a tabletop
centrifuge (Eppendorf Centrifuge 5415C, Germany) for 1 min. 50 µl of
distilled water was added to each supernatant to prevent precipitation.
Color development was measured at 530 nm. Ethanol was used as a reagent blank for the color development. The concentration of exogenous retinal
isomer was determined by absorbance measurements with a Hitachi
U-3000 scanning spectrophotometer. NADH and NADPH were prepared
immediately before use in 5 mM Tris-HCl, pH 7.5.
Extraction of the Chromophore of RGR by Hydroxylamine
Derivatization--
The retinal chromophore of RGR was extracted under
dim red light and analyzed by the method of hydroxylamine
derivatization, as described by Groenendijk et al. (30, 31).
In a typical extraction procedure, 100-300 µl of purified RGR were
mixed with 0.1 volume of 2 M hydroxylamine, pH 6.5, and
then 300 µl of methanol and 300 µl of dichloromethane. The sample
volume was brought to 900 µl by the addition of 10 mM
sodium phosphate buffer, pH 6.5, containing 150 mM NaCl and
0.5 mM EDTA. The extraction with dichloromethane (aqueous
buffer/methanol/dichloromethane, 1:1:1 by volume) was performed by
vortexing for 30 s. After centrifugation at 14,000 rpm for 1 min,
the lower organic phase was removed, and the upper phase was extracted
twice more with 300 µl of dichloromethane. The organic layers were
pooled and dried under a nitrogen stream. The extracted retinaloximes
were then solubilized in 600 µl of hexane, filtered through glass
wool, and dried again under nitrogen. The residue was either stored in
darkness at
80 °C or analyzed immediately by HPLC.
HPLC Analysis of Retinaloximes--
The isomers of retinaloximes
were analyzed by HPLC, as described previously (20). The extracted
retinaloximes were dissolved in hexane and applied to a LiChrosorb RT
Si60 silica column (4 × 250 mm, 5 µm) (Merck). The HPLC system
was equipped with a Beckman model 126 solvent module and model 166 UV-visible detector (Beckman Instruments). The 20-µl samples were
injected and the components were resolved in a running solvent
consisting of hexane supplemented with 8% diethyl ether and 0.33%
ethanol with flow rate of 1 ml/min. The HPLC column was calibrated
using all-trans- and 11-cis-retinaloxime standards. Identification of the retinaloxime isomers was based on the
retention times of the known retinaloxime products. Absorbance was
measured at 360 nm, and the absorbance peaks were analyzed with the
Gold Nouveau software (Beckman Instruments). The proportion of each
isomer in the loading sample was determined from the total peak areas
of both its syn- and anti-retinaloxime and was based on the following
extinction coefficients (
360, in hexane):
all-trans syn = 54,900, all-trans anti = 51,600, 11-cis syn = 35,000, 11-cis anti = 29,600, 13-cis syn = 49,000, and
13-cis anti = 52,100 (31, 32).
HPLC Analysis of 11-cis-Retinol--
11-cis-Retinol
was co-extracted with retinaloximes and analyzed by HPLC, as described
above. In some experiments, the retinoids were separated on a Resolve
silica column (3.9 × 150 mm, 5 µm) (Waters Corp., Milford, MA)
using a Waters 2690 HPLC module. The samples were resolved in a running
solvent consisting of hexane supplemented with 8% diethyl ether and
0.33% ethanol. Retinaloximes and 11-cis-retinol were
measured simultaneously with a Waters 2487 dual wavelength absorbance
detector at 360 and 320 nm, respectively; sensitivity was ~6-fold
higher than that of our Beckman system. The absorbance peaks were
analyzed with the Millennium 32 Chromatography Manager software,
version 3.20 (Waters Corp.). Identification of
11-cis-retinol was based on the retention time of a purified standard. The 11-cis-retinol standard (
max = 318 nm) was prepared from known 11-cis-retinal, as described
previously (23). The HPLC system was calibrated with 8.8-0.88 pmol of
11-cis-retinol and 9.1-0.91 pmol of
syn-all-trans-retinaloxime standards.
Irradiation of RGR and 11-cis-Retinol Dehydrogenase Activity
toward the Endogenous Chromophore--
RGR was irradiated with 470-nm
monochromatic light using an Oriel light source (Oriel Corp.,
Stratford, CT) equipped with a 150-W xenon arc lamp. Monochromatic
light beams at 470 nm were formed by passing the light through a 470-nm
interference filter (Oriel #53845) and 455-nm long pass filter (Oriel
#51284). Nonirradiated control samples were held under dim red light,
but otherwise were treated identically. The samples contained
immunoaffinity-purified RGR in 0.1% digitonin, 10 mM
sodium phosphate, pH 6.5, 150 mM NaCl, 0.5 mM
EDTA, and 20% glycerol and were incubated in the presence of 3 mM NADH cofactor or in buffer only as indicated under
"Results." NADH was freshly prepared in 5 mM Tris-HCl
buffer, pH 7.5. The final reaction volume (100 µl) was adjusted by
the addition of 10 mM sodium phosphate, pH 6.5, 150 mM NaCl, and 0.5 mM EDTA. Incubations were
performed for the desired time at 37 °C in a quartz cuvette
positioned 60 cm from the light source. After delivery of the intended
amount of light, the mixture was taken out of the water bath, and 0.2 M hydroxylamine was added to stop the reaction. The retinal
chromophore was extracted by hydroxylamine derivatization and analyzed
by HPLC, as described previously. The incubation of RGR at 37 °C
rather than room temperature did not affect the distribution of retinal
isomers from irradiated RGR.
 |
RESULTS |
Isolation of RGR and Identification of Co-purified 32-kDa
Protein--
RPE microsomal membrane proteins were extracted in 1.2%
digitonin solution at pH 6.5, and solubilized RGR was purified by immunoaffinity chromatography under dim red light. Under these conditions, a 32-kDa protein (p32) was found to co-purify reproducibly with RGR (Fig. 1). The p32 protein band
was readily detectable in SDS-polyacrylamide gels by staining with
Coomassie Blue. It was not recognized on Western blots by the antibody
that was coupled to the immunoaffinity resin, even when the immunoblots
were overdeveloped (results not shown). The relative amount of p32 that
was co-purified with RGR was significantly increased by the inclusion
of glycerol in the isolation procedure (Fig. 1A, lane
3). When the antibody binding sites of the immunoaffinity column
were blocked by prewashing with excess peptide, neither RGR nor p32 was
eluted from the column (Fig. 1B), and p32 did not appear
isolatable as a nonspecifically adsorbed protein. The p32 band was
excised from a SDS-polyacrylamide gel and analyzed by liquid
chromatography MS/MS. When the resulting MS/MS spectra were correlated
to the data base of known protein sequences, a number of them gave
positive matches to 11-cis-retinol dehydrogenase, a known
critical enzyme of the visual cycle (Fig. 2).

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Fig. 1.
Co-purification of RGR and p32 from bovine
RPE. RGR was isolated from digitonin extracts of RPE microsomal
proteins by an immunoaffinity procedure. The isolated proteins were
electrophoresed on SDS-polyacrylamide gels and detected by Coomassie
Blue (A) or silver staining (B). A, a
10-kDa protein ladder (10-200 kDa) was used as a molecular weight
standard (lane 1). The isolation of RGR was performed in the
absence (lane 2) or in the presence (lane 3) of
20% glycerol. RGR isolated from 6.7 and 4.3 eyes was loaded in
lanes 2 and 3, respectively. B, RGR
was isolated without glycerol (lane 1). The isolation
procedure was also carried out with an immunoaffinity column that was
first prewashed with one bed volume of 600 µg/ml peptide to block all
antibody binding sites. All other purification steps were unchanged. In
this control, the 32-kDa protein was not eluted from the column
(lane 2). The arrows indicate RGR and the
co-purified ~32-kDa protein.
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Fig. 2.
Mass spectrometric identification of
p32. The co-purified p32 was excised from the SDS-polyacrylamide
gel shown in Fig. 1A, lane 2. The protein band
was subjected to trypsin in-gel digestion, and the peptide fragments
were analyzed by liquid chromatography MS/MS. The MS/MS data were
correlated to the data base of known protein sequences using Sequest.
A, peptides of p32 that matched the sequence of cRDH,
11-cis-retinol dehydrogenase (Accession no. A55429), are
indicated by lines drawn above the corresponding portion of
the sequence. Observed and calculated (in parentheses) mass
values for the doubly protonated molecular ion are given above the
line. B, high resolution (Zoom) scan used to
determine the accurate m/z value and charge state
of the ion corresponding to the peptide LLWLPASYLPAR
(Mr = 1398.8). C, MS/MS spectrum of
the peptide LLWLPASYLPAR that was matched to the protein sequence data
base. Members of the B and Y'' fragment ion series (51) are indicated
along with their observed m/z values.
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Co-purification of Retinol Dehydrogenase Activity--
An assay
was performed to verify the presence of enzymatically active cRDH in
preparations of RGR. Exogenous 11-cis-retinal was used as
the substrate. The reduction of 11-cis-retinal was followed
by its disappearance and was dependent on the amount of
immunoaffinity-purified RGR added to the reaction (Fig.
3). The activity required the NADH
cofactor. The reduction activity of cRDH was found to accompany each
step in the purification of RGR and was abolished if RGR was
heat-inactivated (results not shown). These observations indicated that
cRDH is co-purified with RGR in a detergent solution and remains an
active enzyme.

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Fig. 3.
Co-purification of RGR and
11-cis-retinol dehydrogenase activity. cRDH
activity in immunoaffinity-purified RGR was shown by enzymatic
reduction of exogenous 11-cis-retinal. Various amounts of
the eluted RGR (3.8 ng/µl) were incubated with 10 nmol of
11-cis-retinal in the presence of 1 mM NADH for
6 min at 37 °C (100 µl reaction volume). Subsequently, retinal was
determined by the thiobarbituric acid method and measured by absorbance
at 530 nm (A530). Without NADH,
A530 was 0.27 at the 25-µl RGR fraction. In
the absence of exogenous 11-cis-retinal, background
A530 was 0.01 at the 25-µl RGR fraction.
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Stereospecificity and Cofactor Dependence of Co-purified
cRDH--
Important features that distinguish cRDH from its
counterpart, the tRDH in photoreceptor cells, are substrate
stereospecificity and cofactor dependence of the enzyme. These
properties of the dehydrogenase were tested in preparations of RGR to
further characterize the co-purified cRDH. The cRDH in
immunoaffinity-purified RGR reacted with exogenous
11-cis-retinal but not with all-trans-retinal (Fig. 4A). Co-purified cRDH
preferred NADH rather than NADPH as the cofactor in the reduction
reaction (Fig. 4, A and B).

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Fig. 4.
Substrate specificity and cofactor dependence
of 11-cis-retinol dehydrogenase. The reduction
activity of co-purified cRDH and RPE microsomal membranes was analyzed
in reactions with exogenous retinal isomers. A and
B, immunoaffinity-purified RGR (90 ng) or C and
D, RPE microsomes (100 µg of protein) were incubated with
exogenous all-trans- (×) or 11-cis-retinal
( ). Each reaction contained 10 nmol of retinal isomer in the
presence of 1 mM NADH or NADPH, as shown. The reactions
were performed for the indicated length of time at 37 °C (100-µl
reaction volume). Retinal was determined by the thiobarbituric acid
method and measured by absorbance at 530 nm. A, absorbance
at the indicated time point; A0, absorbance at
time zero.
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For comparison, the activity of cRDH in RPE microsomal membranes was
also assayed. As expected, microsomal cRDH was active in the reduction
of 11-cis-retinal, and there was only little activity with
all-trans-retinal as the substrate (Fig. 4, C and D). The enzyme in the microsomal membranes displayed
substrate-specific activity with either cofactor, NADH or NADPH. These
observed properties of microsomal cRDH were consistent with the results
of previous studies (12-14).
Effect of cRDH on 11-cis-Retinal Bound to Irradiated
RGR--
Since cRDH reacts specifically with
11-cis-retinal, the role of co-purified cRDH in RGR function
may be to reduce and remove bound 11-cis-retinal from RGR
after stereospecific photoisomerization of its
all-trans-retinal chromophore. In previous experiments, cRDH
reduction activity was not observed because the required cofactor was
not added (20).
RGR was kept in the dark or irradiated with blue light. Subsequently,
each sample was incubated in the dark in the presence or absence of
NADH. The predominant chromophore extracted from nonirradiated RGR was
all-trans-retinal, as expected (Fig.
5A), and incubation with NADH
had no effect on the all-trans chromophore in the dark (Fig.
5B). The irradiation of RGR and subsequent incubation in the
dark without NADH resulted in stereospecific isomerization of ~55%
of the bound all-trans-retinal to 11-cis-retinal
(Fig. 5C); these results were in agreement with previous
studies (20). When RGR was irradiated and then incubated in the dark in
the presence of NADH, there was a significant decrease in total
retinal. The decrease in total retinal was brought about by the
selective loss of 11-cis-retinal with little effect on the
amount of the all-trans isomer (Fig. 5D). Changes
in the chromophore of RGR were most prominent when NADH was present
during irradiation of RGR. Under conditions in which the cofactor is
included and cRDH is active, irradiation of RGR resulted in significant
loss of both 11-cis- and all-trans retinal (Fig.
6).

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Fig. 5.
Interaction of co-purified
11-cis-retinol dehydrogenase with the chromophore of
RGR. Equal aliquots of RGR from the same preparation were
irradiated or not for the first 5 min without cofactor. Subsequently,
each sample (260 ng of RGR) was incubated in the dark for an additional
5 min in the presence or absence of NADH. Retinal isomers were then
extracted by the hydroxylamine derivatization method and analyzed by
HPLC. A, incubation in the dark for 10 min without NADH.
B, incubation in the dark for 5 min and then incubation in
the dark for another 5 min in the presence of 3 mM NADH.
C, irradiation for 5 min and then incubation in the dark for
5 min without NADH. D, irradiation for 5 min followed by
incubation in the dark for 5 min in the presence of 3 mM
NADH. The incubations were performed at 37 °C, and the irradiated
samples were exposed to 470-nm monochromatic light. RGR in this and all
following experiments was isolated in the presence of 20% glycerol.
11, 11-cis syn-retinaloxime; all,
all-trans syn-retinaloxime; 13, 13-cis
syn-retinaloxime. In these experiments, the anti
isomers of the retinaloximes were below the level of detection.
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Fig. 6.
Irradiation of RGR in the presence of
NADH-dependent 11-cis-retinol
dehydrogenase activity. Equal aliquots of RGR from the same
preparation were irradiated with 470-nm monochromatic light for 5 min
at 37 °C in the absence (upper trace) or in the presence
(lower trace) of 3 mM NADH (260 ng of
RGR/reaction). The retinal isomers were then extracted by the
hydroxylamine derivatization method and analyzed by HPLC.
11, 11-cis syn-retinaloxime; all,
all-trans syn-retinaloxime; 13, 13-cis
syn-retinaloxime. In these experiments, the anti
isomers of the retinaloximes were below the level of detection.
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Synthesis of 11-cis-Retinol during Irradiation of RGR--
To
confirm that 11-cis-retinal was reduced by the co-purified
cRDH with retention of the expected isomeric configuration, we
demonstrated the production of 11-cis-retinol upon
irradiation of RGR in the presence of NADH. The retinoid extracts from
irradiated preparations of RGR were analyzed after exposure to light
for different lengths of time. Absorbance was measured at 320 nm to optimize the detection of 11-cis-retinol. The irradiation of
RGR in the presence of NADH resulted in significant decline of
all-trans-retinal and a concomitant
time-dependent increase in 11-cis-retinol (Fig. 7). The amount of
11-cis-retinol formed corresponded closely to the amount of
all-trans-retinal lost at 5 min; however, the production of
11-cis-retinol lagged the more rapid decline in
all-trans-retinal in the first 1-2 min of incubation. Since
cRDH uses the 11-cis isomer specifically, we conclude that
the increase in 11-cis-retinol is due to the reaction of
cRDH on free or bound 11-cis-retinal, the photoisomerization
product of irradiated RGR.

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Fig. 7.
Production of 11-cis-retinol
by irradiation of RGR in the presence of NADH. Equal aliquots of
RGR from the same preparation were irradiated with 470-nm monochromatic
light for various lengths of time at 37 °C in the presence of 3 mM NADH (640 ng RGR/reaction). The reactions were stopped,
and the retinal isomers were extracted at the indicated times by the
hydroxylamine derivatization method. 11-cis-Retinol was
co-extracted and analyzed simultaneously by HPLC. Two representative
experiments are shown for the decline in
all-trans-retinaloxime, corresponding to the
all-trans-retinal chromophore of RGR (A), and
synthesis of 11-cis-retinol (B). The accumulation
of 11-cis-retinol did not occur without NADH. C,
HPLC chromatograms of the absorbance (AU) of
11-cis-retinol at 320 nm at various time points of a single
experiment. D, representative HPLC chromatograms of the
11-cis-retinol standard (5.5 pmol) and extracted retinoids
after 0 and 7-min incubations. The retinoids were detected by
absorbance at 320 nm using the Waters HPLC system and were
predominantly all-trans syn-retinaloxime and
11-cis-retinol (arrows) after 0 and 7-min
incubations, respectively.
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DISCUSSION |
The RGR opsin is a major all-trans-retinal-binding
protein in the RPE. Physiological evidence that supports the processing of precursor all-trans-retinol and its incorporation into
the chromophore of RGR in the dark has been obtained recently (33). Illumination of RGR results in stereospecific conversion of the bound
all-trans-retinal to 11-cis-retinal. Since
retinoid-binding proteins would be required to transfer the chromophore
to or from RGR, the mechanism by which RGR functions may depend on
specific protein interactions. In this paper, we demonstrate functional interaction in vitro between the RGR opsin and cRDH. Indeed,
the isolation of highly purified RGR from bovine RPE results in
consistent co-purification of cRDH in a manner that suggests that cRDH
binds to RGR in a protein complex.
In the presence of glycerol, co-purification of cRDH was optimal and
Coomassie Blue staining of cRDH was typically as intense and specific
as that of RGR in polyacrylamide gels. A general effect of glycerol is
to stabilize the protein complex and reduce nonspecific binding to the
affinity resin (34). The co-purified enzyme was active and held several
properties similar to those of microsomal cRDH (12-14), isolated cRDH
(28), or recombinant cRDH (35, 36). It was efficient in the reduction
of exogenous 11-cis-retinal and had little reactivity with
the all-trans isomer. NADH was preferred over NADPH as a
cofactor in the reduction reaction. In contrast, microsomal cRDH
activity was observable with both NADH and NADPH (Fig. 4, C
and D). Despite the presence in the RPE of cRDH activity
that uses NADPH (12, 37), co-purification of the
NADPH-dependent enzyme with RGR was not evident.
Most noteworthy was the substrate stereospecificity of cRDH in
reactions with the chromophore of RGR. The co-purified enzyme was inert
to all-trans-retinal bound to RGR in the dark but active toward endogenous 11-cis-retinal that was generated by
irradiation of RGR. The cRDH was remarkably stable during
co-purification with RGR over a period of 28 h, and its activity
did not require purification of the enzyme in the presence of NADH, as
reported previously (28).
The functional significance of RGR and cRDH interaction may lie in the
mechanism of chromophore dissociation from RGR. Like squid rhodopsin
and other invertebrate visual pigments (38, 39), the chromophore of RGR
opsin does not readily dissociate after photoisomerization in
vitro, and irradiation does not lead to bleaching of RGR, as
determined by difference absorption spectra before and after light
exposure (22). If RGR operates in vivo as a stereospecific
photoisomerase to directly generate the 11-cis chromophore
in the visual cycle, then all-trans-retinal should be
photoisomerized and 11-cis-retinal should be released
from RGR catalytically. The previous experiments on irradiation of RGR
were run without NADH and achieved at most ~50% net conversion of
all-trans- to 11-cis-retinal (20). We now can
demonstrate more complete photoisomerization of the bound
all-trans-retinal to the 11-cis isomer. When RGR
is irradiated in the presence of NADH, isomerization of the chromophore
is coupled to efficient reduction by cRDH. These results strongly
suggest that 11-cis-retinol is generated in a photic visual
cycle. Possible steps in further processing of
11-cis-retinol include its conversion to
11-cis-retinyl esters or binding to cellular
retinaldehyde-binding protein. The results do not exclude an additional
mechanism of dissociation and direct transfer of
11-cis-retinal from RGR to cellular retinaldehyde-binding protein or any effect of cellular retinaldehyde-binding protein on the
interaction between RGR and cRDH.
The functional coupling of RGR with cRDH extends the evidence that RGR
and rhodopsin have evolved with distinct, yet parallel, features. The
chromophores of RGR and rhodopsin are photoisomerized in opposite
directions; hence, each is bound to 11-cis- and
all-trans-retinal at a specific photochemical state of the
opsin. After photoisomerization of the chromophore,
11-cis-retinal from RGR is reduced by cRDH, and
all-trans-retinal from rhodopsin is reduced by tRDH to the respective alcohols. The cRDH and tRDH are highly homologous retinol dehydrogenases (6, 35, 36). The reduction of
all-trans-retinal participates in the inactivation of
rhodopsin and is a rate-limiting step of the visual cycle at high light
levels (5). The tRDH is tightly associated with the rod outer segments
(40-43); however, its orientation in the rod outer segment membrane
and possible binding to rhodopsin are unknown. In comparison, there is
evidence that the catalytic domain of cRDH in the RPE is situated
toward the lumen of the smooth endoplasmic reticulum (44). This
membrane topology of cRDH suggests that 11-cis-retinal from
irradiated RGR is reduced by cRDH and presented at the lumenal surface
of the smooth endoplasmic reticulum for transport or further processing of the alcohol.
The physiological significance of the 11-cis-retinal
reduction reaction involving cRDH and RGR needs to be corroborated by in vivo studies. Another reductase role for cRDH was first
proposed by Lion et al. (12). Most models of the visual
cycle since then have emphasized the oxidation reaction of cRDH
instead, because no experiment to regenerate rhodopsin with
11-cis-retinol in excised mammalian retina has succeeded
(45-47). On the other hand, cone pigments in excised retina can be
regenerated with 11-cis-retinol (48). In this case, the
reduction of 11-cis-retinal from RGR to
11-cis-retinol might be sufficient for chromophore synthesis by the RPE in a cone visual cycle. Alternatively, the
11-cis-retinol from RGR may be converted to the retinyl
ester for storage or usage as a requisite intermediate in the synthesis
of 11-cis-retinal. Our results provide a new argument for a
reductase role of cRDH in addition to its role in oxidation of
11-cis-retinol. The placement of RGR and cRDH in a common
pathway may generate additional insights into the molecular defect in
cases of fundus albipunctatus that involve mutations in the human cRDH
gene (49, 50).