Substrate specificity and subcellular localization of the aldehyde-Alcohol redox-Coupling reaction in carp cones

Article abstract of DOI:10.1074/jbc.M113.521153

Our previous study suggested the presence of a novel conespecific redox reaction that generates 11-cis-retinal from 11-cisretinol in the carp retina. This reaction is unique in that 1) both 11-cis-retinol and all-trans-retinal were required to produce 11-cis-retinal; 2) together with 11-cis-retinal, all-trans-retinol was produced at a 1:1 ratio; and 3) the addition of enzyme cofactors such as NADP(H) was not necessary. This reaction is probably part of the reactions in a cone-specific retinoid cycle required for cone visual pigment regeneration with the use of 11-cis-retinol supplied from Mueller cells. In this study, using purified carp cone membrane preparations, we first confirmed that the reaction is a redox-coupling reaction between retinals and retinols. We further examined the substrate specificity, reaction mechanism, and subcellular localization of this reaction. Oxidation was specific for 11-cis-retinol and 9-cis-retinol. In contrast, reduction showed low specificity: many aldehydes, including all-trans-, 9-cis-, 11-cis-, and 13-cis-retinals and even benzaldehyde, supported the reaction. On the basis of kinetic studies of this reaction (aldehyde-alcohol redox-coupling reaction), we found that formation of a ternary complex of a retinol, an aldehyde, and a postulated enzyme seemed to be necessary, which suggested the presence of both the retinol- and aldehydebinding sites in this enzyme. A subcellular fractionation study showed that the activity is present almost exclusively in the cone inner segment. These results suggest the presence of an effective production mechanism of 11-cis-retinal in the cone inner segment to regenerate visual pigment.

Full text of DOI:10.1074/jbc.M113.521153

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 51, pp. 36589–36597, December 20, 2013
© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Substrate Specificity and Subcellular Localization of the
Aldehyde-Alcohol Redox-coupling Reaction in Carp Cones*
Received for publication, September 21, 2013, and in revised form, November 6, 2013 Published, JBC Papers in Press, November 11, 2013, DOI 10.1074/jbc.M113.521153
Shinya Sato‡, Takashi Fukagawa§, Shuji Tachibanaki‡§, Yumiko Yamano¶, Akimori Wada¶, and Satoru Kawamura‡§1
From the ‡Department of Biological Sciences, Graduate School of Science, and the §Graduate School of Frontier Biosciences, Osaka
University, Yamada-oka 1-3, Suita, Osaka 565-0871 and the ¶Department of Organic Chemistry for Life Science, Kobe
Pharmaceutical University, Motoyamakita-machi 4-19-1, Higashinada-ku, Kobe 658-8558, Japan
Background: In carp cones, 11-cis-retinal and all-trans-retinol are formed with 11-cis-retinol and all-trans-retinal present.
Results: The substrate specificity, reaction mechanism, and subcellular localization of this reaction were determined.
Conclusion: Substrate specificity is high for retinol but low for retinal, and the activity is present in the cone inner segment.
Significance: The possible contribution to efficient pigment regeneration in cones is suggested.
Our previous study suggested the presence of a novel cone-
specific redox reaction that generates 11-cis-retinal from 11-cis-
retinol in the carp retina. This reaction is unique in that 1) both
11-cis-retinol and all-trans-retinal were required to produce
11-cis-retinal; 2) together with 11-cis-retinal, all-trans-retinol
was produced at a 1:1 ratio; and 3) the addition of enzyme cofac-
tors such as NADP(H) was not necessary. This reaction is prob-
ably part of the reactions in a cone-specific retinoid cycle
required for cone visual pigment regeneration with the use of
11-cis-retinol supplied from Müller cells. In this study, using
purified carp cone membrane preparations, we first confirmed
that the reaction is a redox-coupling reaction between retinals
and retinols. We further examined the substrate specificity,
reaction mechanism, and subcellular localization of this reac-
tion. Oxidation was specific for 11-cis-retinol and 9-cis-retinol.
In contrast, reduction showed low specificity: many aldehydes,
including all-trans-, 9-cis-, 11-cis-, and 13-cis-retinals and even
benzaldehyde, supported the reaction. On the basis of kinetic
studies of this reaction (aldehyde-alcohol redox-coupling reac-
tion), we found that formation of a ternary complex of a retinol,
an aldehyde, and a postulated enzyme seemed to be necessary,
which suggested the presence of both the retinol- and aldehyde-
binding sites in this enzyme. A subcellular fractionation study
showed that the activity is present almost exclusively in the cone
inner segment. These results suggest the presence of an effective
production mechanism of 11-cis-retinal in the cone inner seg-
ment to regenerate visual pigment.
tion of the phototransduction cascade to generate an electrical
response (1, 2). The activated visual pigment then bleaches,
decomposes into opsin and all-trans-retinal, and loses its ability
to detect light. In both rods and cones, all-trans-retinal released
from bleached pigment is reduced to all-trans-retinol. To
maintain the ability to detect light in photoreceptors, visual
pigments should be regenerated. For regeneration of the pig-
ment, 11-cis-retinal is required: all-trans-retinol formed after
bleaching is converted to 11-cis-retinal through a multistep
enzymatic process called the retinoid cycle (or the visual cycle).
In vertebrate eyes, two pathways of the retinoid cycle are known
(3–6). The first and classic cycle includes the reactions found in
the retinal pigment epithelium (RPE).2 In this cycle (the RPE
retinoid cycle), all-trans-retinol formed after pigment bleach-
ing in rods and cones is sent to the RPE, where all-trans-retinol
is esterified, isomerohydrolyzed, and oxidized to 11-cis-retinal
(7–9). The 11-cis-retinal formed is sent back to the rods and
cones to regenerate visual pigment. The second and recently
discovered pathway is a cone-specific retinoid cycle (the cone
retinoid cycle): the reduced all-trans-retinol is sent to Müller
cells, where all-trans-retinol is isomerized to 11-cis-retinol (10,
11). The isomerized 11-cis-retinol is sent back to the cones,
where 11-cis-retinol is oxidized to 11-cis-retinal to regenerate
visual pigments (3, 5, 6).
The cone retinoid cycle has been suggested to contribute
significantly to pigment regeneration in cones. According to
Mata et al. (12), the maximum rate of the supply of 11-cis-
retinal through the RPE retinoid cycle seems to be too low to
regenerate the pigment for cones to function under high levels
of steady illumination. They estimated that the cone retinoid
cycle is 20-fold more effective than the classic RPE retinoid
cycle in regenerating the cone pigment. In fact, Kolesnikov et al.
(13) reported that, in mouse retina, the cone retinoid cycle pro-
motes M/L-cone dark adaptation 8-fold faster than the RPE
retinoid cycle.
In the vertebrate retina, light is detected by rods and cones.
Rods mediate night vision, whereas cones mediate day vision. In
both cells, a light-absorbing molecule, visual pigment, is com-
posed of a chromophore, 11-cis-retinal, and a protein moiety,
opsin. Light isomerizes 11-cis-retinal to all-trans-retinal, which
triggers a conformational change in opsin that leads to activa-
In the cone retinoid cycle, 11-cis-retinol supplied from Mül-
ler cells is oxidized to 11-cis-retinal in cones (14, 15). It has been
suggested that this oxidation is attained by retinol dehydrogen-
* This work was supported by Japan Society for the Promotion of Science
Grants 20370060 and 23227002 (to S. K.) and 22770150 and 24570085 (to
S. T.), a Human Frontier Science Program grant (to S. K.), and the Naito
Foundation (to S. T.).
1 To whom correspondence should be addressed. Tel.: 81-6-6879-4610; Fax: 2 The abbreviations used are: RPE, retinal pigment epithelium; AL-OL, alde-
81-6-6879-4614; E-mail: kawamura@fbs.osaka-u.ac.jp.
hyde-alcohol; OS, outer segment; IS, inner segment.
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ϩ
ase activities in cones with NADP as a cofactor (12). However, activity, freshly obtained purified cones were freeze-thawed
in our previous study (16), purified carp cone membranes did and then washed once by centrifugation at 104,000 ϫ g for 15
not show significant activities of 11-cis-retinol oxidation in the min at 4 °C in potassium gluconate buffer. When necessary, the
ϩ
presence of NADP . Instead, without the addition of enzyme supernatant obtained in this centrifugation was collected. The
cofactors, a novel reaction showed a high activity of 11-cis- membrane precipitates obtained were suspended in isotonic
retinal formation in the presence of both 11-cis-retinol and all- buffer (potassium gluconate buffer), hypotonic buffer (2 mM
trans-retinal. In this reaction, in addition to 11-cis-retinal, all- EDTA, 10 mM HEPES-KOH, and 1 mM dithiothreitol, pH 7.5),
trans-retinol was produced, and the stoichiometry of the hypertonic buffer (600 mM NaCl, 10 mM HEPES-KOH, and 1
formation of 11-cis-retinal and all-trans-retinol was 1:1. On the mM dithiothreitol, pH 7.5), or detergent buffer (0.48 mM dode-
basis of these findings, we postulated that, in this reaction, cyl-␤-D-maltoside and 10 mM HEPES-KOH, pH 7.5). The
11-cis-retinol was oxidized to 11-cis-retinal with the concomi- membranes suspended in detergent buffer were incubated for
tant reduction of all-trans-retinal to all-trans-retinol. This pos- 30 min at 4 °C with rotation. For other membrane samples, this
tulated retinal (AL) reduction- and retinol (OL) oxidation-cou- step was omitted. The membranes thus prepared were centri-
pling reaction (AL-OL coupling reaction) showed a maximum fuged at 104,000 ϫ g for 20 min at 4 °C to obtain a soluble
rate of supply of 11-cis-retinal estimated to be 240 times higher supernatant and a membrane precipitate fraction; these were
than that attained by the RPE retinoid cycle (16). Because such used to determine in which fraction AL-OL coupling activity
activities were not found in purified carp rods, the AL-OL cou- was present.
pling reaction could be part of the mechanisms that effectively
produce 11-cis-retinal from its alcohol in cones in the cone pared a cone outer segment membrane-rich fraction and a
retinoid cycle.
cone inner segment membrane-rich fraction3 by stepwise
To study the subcellular localization of the activity, we pre-
In this study, we first confirmed that the reaction is actually a sucrose density gradient centrifugation, a method modified
redox-coupling reaction between retinols and retinals. Then, to from the one reported previously (16). The collected mem-
further characterize the AL-OL coupling reaction, we exam- branes were suspended in potassium gluconate buffer. The
ined the substrate specificities of both oxidation and reduction, total protein content in each fraction was quantified with
the reaction mechanism, and the subcellular localization of the Coomassie Brilliant Blue staining after SDS-PAGE using BSA
activity. Oxidation was found to be specific for 11-cis-retinol as a standard, and cone visual pigment was quantified spectro-
and 9-cis-retinol, whereas reduction was observed rather photometrically (18, 19). The purity of each fraction was judged
widely with many aldehydes. On the basis of these findings, we based on the content of cone visual pigment per total proteins
now call the reaction the aldehyde-alcohol redox-coupling in each fraction.
reaction with the same abbreviation as before (AL-OL coupling
Preparation of Retinoids and 9-cis-Retinol Analogs—All-
reaction). Our results suggest that the postulated enzyme(s) trans-retinal and 9-cis-retinal were purchased from Sigma-Al-
catalyzing the AL-OL coupling has two distinct substrate-bind- drich, and 3-dehydro-all-trans-retinal (A2 all-trans-retinal)
ing sites for aldehydes and retinols. We found that the was kindly provided by Prof. T. Seki (Osaka Kyoiku University)
enzyme(s) is a tightly membrane-associated protein(s) local- or purchased from Toronto Research Chemicals. Other retinal
ized in the inner segment (most probably in the ellipsoid isomers (11-cis-retinal and 13-cis-retinal) were prepared as
region) of a cone. On the basis of these results, we discuss the described (20): ϳ10 mg of all-trans-retinal was dissolved in 4 ml
possible functional mechanisms of the reaction.
of acetonitrile at a final concentration of 10 mM and isomerized
by irradiation with a fluorescent lamp (15 watts) at a distance of
10 cm for 1 day at 4 °C. After the solvent was evaporated under
EXPERIMENTAL PROCEDURES
Preparation of Cone Membranes—Carp (Cyprinus carpio) nitrogen, a mixture of retinal isomers was dissolved in 1 ml of a
cones were purified as described (17). Animals were cared for diethyl ether/n-hexane (15:85) solvent so that the concentra-
according to institutional guidelines. Our purified cones tion of total retinal isomers was ϳ40 mM. The following manip-
retained the outer segment and the ellipsoid, but lacked the ulations were carried out under dim red light to prevent further
nucleus and the terminal region. We always counted the num- isomerization. The mixture was then applied to a JASCO LC
ber of cone cells in a small portion of our purified cone prepa- 800 HPLC system, and each isomer was separated with a pre-
ration to estimate the total number of the cells in a sample used parative normal-phase column (Develosil 60-5, Nomura
for each experiment, and in this study, the AL-OL coupling Chemical) using diethyl ether/n-hexane (15:85) as the mobile
activity is expressed as units/cone cell. Cone membranes were phase. Retinals in the eluate were detected by monitoring the
prepared as follows. Purified cones were disrupted by freeze- absorbance at 350 nm, and the eluate corresponding to each
thawing and washed three times with potassium gluconate peak on the chromatogram was collected in a test tube. To
buffer (115 mM potassium gluconate, 2.5 mM KCl, 2 mM MgCl2, identify and quantify the retinal isomer, the absorption spec-
0.2 mM EGTA, 0.1 mM CaCl2, 1 mM dithiothreitol, and 10 mM trum was measured and compared with data in the literature
HEPES-KOH, pH 7.5) by centrifugation at 104,000 ϫ g for 15 (21, 22). After purification, the solvent was evaporated under
min at 4 °C. The washed membranes were stored at Ϫ80 °C nitrogen, and each retinal isomer was dissolved in ethanol and
until used.
stored at Ϫ80 °C until used.
For the substrate specificity and kinetic analyses, stored cone
membranes were thawed, suspended in potassium gluconate
buffer, and used. To study the membrane localization of the 3 T. Fukagawa, S. Tachibanaki, and S. Kawamura, unpublished data.
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Characterization of the AL-OL Coupling Reaction in Carp Cones
hexane ϭ 1:4) to afford the aldehyde. A hexane solution of
n-butyllithium (1.1 mmol) was added to a solution of triethyl
3-methyl-4-phosphonocrotonate (1.1 mmol) and N,NЈ-di-
methyl propylene urea (3.5 mmol) in tetrahydrofuran (5.5 ml)
at 0 °C. After stirring for 30 min, the solution was cooled to
Ϫ78 °C, and a solution of the aldehyde (1 mmol) obtained as
described above and dissolved in tetrahydrofuran (3 ml) was
added. The resulting mixture was stirred for an additional 2 h.
The reaction was quenched with saturated NH4Cl, and the
products were extracted with diethyl ether. The organic layer
was dried over Na2SO4, filtered, and evaporated. The residue
was purified by column chromatography on silica gel (hexane/
AcOEt ϭ 10:1), and the alcohol was obtained by reaction with
diisobutylaluminium hydride.
Measurement of AL-OL Coupling Activity—The AL-OL cou-
pling reaction was carried out under dim red light where pig-
FIGURE 1. Scheme of the synthesis of 9-cis-retinol analogs. A, tetraenol
derivative, 6Z-3,7-dimethyl-9-phenyl-2,4,6,8-nonatetraen-1-ol. B, trienol
derivative, 3,7-dimethyl-2,4,6-octatrien-1-ol. DIBAL-H, diisobutylaluminium
hydride. OTf, OSO2CF3 (O-trifluoromethanesulfonyl).
ment bleach was negligible. A cone membrane suspension (100
␮l) obtained from 1–3 ϫ 105 cones was transferred to a 5-ml
glass vial. An alcohol substrate and an aldehyde/ketone sub-
strate both dissolved in 0.5 ␮l of ethanol were added to the vial
Each retinol isomer was prepared by reduction of the corre- individually. In the case of ubiquinone, it was added as a 1,4-
sponding retinal isomer (23). Approximately 6 mg of NaBH4 dioxane solution because it was difficult to dissolve it in etha-
was added to a 100–300-␮l ethanol solution containing a reti- nol. The sample was then incubated for 5–10 min at 25 °C. As a
nal isomer, and solution was incubated for 30–60 min on ice, control, a sample containing cone membranes but devoid of
and water was added to the sample to a final volume of 500 ␮l to one of the two substrates was incubated under similar condi-
partition NaBH4 in an aqueous phase. Retinols were extracted tions. The reaction was terminated by adding 300 ␮l of ice-
twice with 450 ␮l of solvent A (10:5:0.2:85 benzene/tert-butyl chilled methanol. Retinal isomers in the sample were extracted
methyl ether/ethanol/n-hexane). After extraction, the solvent in the form of oximes as described (26) with slight modifica-
was evaporated under nitrogen, and each retinol isomer was tions: 100 ␮l of 1 M hydroxylamine, pH 7.5, was added, and the
dissolved in ethanol and stored at Ϫ80 °C until used.
sample was incubated for 40 min at 25 °C. After incubation,
Analogs of 9-cis-retinol were prepared as follows. To obtain a retinoids (i.e. both retinal oximes and retinols) were extracted
tetraenol derivative (6Z-3,7-dimethyl-9-phenyl-2,4,6,8-nona- twice with 450 ␮l of solvent A. The solvent was then evapo-
tetraen-1-ol) (Fig. 1A), a mixture of the triflate (1.0 eq), rated, and the extract was dissolved in 100 ␮l of solvent A. Each
obtained from the reaction of phenylacetaldehyde with N-phe- retinoid in the extract was separated by an analytical normal-
nyl-bis(trifluoromethanesulfonimide) (24), and tetraenylstan- phase column (Cosmosil 5SL-II, Nacalai Tesque) using the
nyl ester (1.3 eq) (25) was dissolved in dry dimethylformamide HPLC system and detected by monitoring the absorbance at
(4 ml), and CsF (2.0 eq), Pd(PPh3)4 (10 mol %), and CuI (20 mol 350 nm with solvent A as the mobile phase (26). Each retinoid
%) were added. The flask was evacuated and refilled with argon was identified from its retention time by comparison with those
five times. The mixture was stirred at 40–45 °C for 15 h, cooled of standard retinoids and quantified by comparing the peak
to room temperature, quenched with water, and extracted with area on the chromatogram with that of known amounts of each
ether. The organic layer was dried over Na2SO4, filtered, and retinoid standard that had been quantified spectrophotometri-
evaporated. The residue thus obtained was purified by column cally based on a reported extinction coefficient (27, 28). During
chromatography using neutralized SiO2/powdered KF (9:1) to incubation with cone membranes, retinoids are isomerized
give a coupling product. The coupled ester (1 eq) was dissolved thermally (16). For this reason, the activity was determined by
in CH2Cl2 (3 ml) and cooled to 0 °C. After diisobutylaluminium subtracting the amount of the isomer produced in a control
hydride (1 M solution in toluene, 2.2 eq) was added to the solu- measurement in which either aldehyde or alcohol was not
tion, the mixture was stirred at room temperature for 1 h. The added. Because an aldehyde and an alcohol are produced at a
resulting solution was quenched with aqueous silica gel (H2O/ 1:1 ratio in the AL-OL coupling reaction, the activity was mea-
SiO2 ϭ 1:4), stirred for 1 h, filtered through Celite (World Min- sured by the amount of one of the products, an aldehyde or an
erals), and dried over Na2SO4. After removal of the solvent, the alcohol. For a practical reason to avoid the complexity of the
residue was purified by column chromatography on silica gel quantification of the reaction product, in some of the measure-
(hexane/ethyl acetate ϭ 10:1) to give the alcohol.
ments, we used a non-retinoid substrate, benzaldehyde. When-
To synthesize a trienol derivative (3,7-dimethyl-2,4,6-oc- ever possible, we confirmed that the aldehyde and the alcohol
tatrien-1-ol) (Fig. 1B), a mixture of 3-methyl-2-buten-1-ol (1 were produced at a 1:1 ratio.
mmol) and MnO2 (25 mmol) in dry CH2Cl2 (20 ml) was stirred
Kinetic Analysis of the AL-OL Coupling Reaction—The
at room temperature for 6 h. After filtration through Celite, the AL-OL coupling reaction is a two-substrate reaction, and typi-
filtrate was concentrated under reduced pressure. The residue cally, there are two types of reaction mechanisms in this type of
was purified using column chromatography on silica gel (ether/ reaction: a sequential reaction and a ping-pong reaction (29).
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Characterization of the AL-OL Coupling Reaction in Carp Cones
shown in Fig. 3, many aldehydes showed the activity: all-trans-
retinal, 9-cis-retinal, 13-cis-retinal, A2 all-trans-retinal, benzal-
dehyde, medium-chain aldehydes (dodecanal, nonanal, and
hexanal), citral, and a trienal derivative produced significant
amounts of 11-cis-retinal. The activities detected were similar
among the aldehydes examined, ranging from 1.03 Ϯ 0.03
amol/cone/s with citral to 5.3 Ϯ 1.6 amol/cone/s with 9-cis-
retinal. In contrast, ketones (␤-ionone and ubiquinone), pen-
tanal, 3-methyl-2-butenal, and acetaldehyde were poor sub-
strates at 250 ␮M. The compound 4-hydroxy-2-nonenal is a
lipid peroxidation product of polyunsaturated fatty acids such
as docosahexaenoic acid and is produced in the retina in light
(30). This aldehyde was not a good substrate either. It is note-
worthy that some of the aldehydes (nonanal and hexanal)
reported to be produced in mitochondria under oxidative stress
(31) supported the reaction very effectively (Fig. 3). The activity
was also measured and detected for 11-cis-retinal (Fig. 3, aster-
isk), and in this case, 9-cis-retinol instead of 11-cis-retinol was
used as the alcohol (see below).
FIGURE 2. Evidence for a redox reaction. There are potentially two possible
mechanisms that account for the production of 11-cis-retinal: by isomeriza-
tion (A) and by redox coupling (B). The products of the reaction in the pres-
ence of A2 all-trans-retinal and 11-cis-retinol were quantified (C). A2 all-trans-
retinal was converted to its alcohol (n ϭ 3).
To distinguish which was the case, the activity was measured by
varying the concentrations of both substrates.
In Fig. 3, pentanal and 3-methyl-2-butenal did not show
activities at 250 ␮M. However, when the concentration was
increased, they showed significant activities (0.44 Ϯ 0.03 amol/
RESULTS
Evidence for a Redox Reaction—In our previous study (16), cone/s with 1 mM pentanal and 0.43 Ϯ 0.12 amol/cone/s with
when 11-cis-retinol and all-trans-retinal were incubated with 2.5 mM 3-methyl-2-butenal; mean Ϯ range of variations in two
cone membranes, the corresponding aldehyde and alcohol, i.e. independent measurements). Acetaldehyde did not show sig-
11-cis-retinal and all-trans-retinol, were produced at a 1:1 ratio. nificant activities even at 8.9 mM. These results therefore indi-
Because this stoichiometry of the reaction could be easily cate that hydrophobic aldehydes are able to act as aldehyde
explained by assuming that 11-cis-retinol was oxidized to substrates, although the affinity for the possible binding site
11-cis-retinal and that all-trans-retinal was reduced to all- seems to be dependent on the carbon chain length.
trans-retinol concomitantly, we suggested that the reaction is a
For the oxidation of alcohols, all-trans-, 9-cis-, 11-cis-, and
coupling of oxidation of an alcohol and reduction of an alde- 13-cis-retinols were first examined. As shown in Fig. 3, benzal-
hyde. To confirm this idea and to exclude the possibility that dehyde can act as an aldehyde substrate. To avoid the complex-
the products were formed by isomerization, i.e. 11-cis-retinal ity of quantification of the product among various cis- and
was produced from all-trans-retinal, and all-trans-retinol was trans-isomers produced during incubation, benzaldehyde was
produced from 11-cis-retinol, we used 3-dehydro-all-trans-ret- used as the common aldehyde for measurement of the oxida-
inal (A2 all-trans-retinal) as the aldehyde and analyzed the reac- tion of retinol isomers, and the oxidized product of each retinol
tion products. If 11-cis-retinal is produced from all-trans-reti- was quantified (Fig. 4A, left panel). A retinol substrate (250 ␮M)
nal by isomerization, we should obtain A2 11-cis-retinal (Fig. and benzaldehyde (5 mM) were incubated in the presence of
2A). Instead, if 11-cis-retinal is produced by oxidation of 11-cis- cone membranes. The results show that at 250 ␮M, only 11-cis-
retinol, we should obtain A2 all-trans-retinol (Fig. 2B). Upon retinol and 9-cis-retinol supported the reaction, and all-trans-
incubation of 11-cis-retinol and A2 all-trans-retinal, we retinol and 13-cis-retinol were poor substrates (Fig. 4A). The
obtained A2 all-trans-retinol (Fig. 2C). This result clearly shows activity was slightly higher for 9-cis-retinol (8.7 Ϯ 1.2 amol/
that all-trans-retinol was formed by reduction of all-trans-ret- cone/s) compared with 11-cis-retinol (5.8 Ϯ 0.6 amol/cone/s).
inal and that 11-cis-retinal was formed by oxidation of 11-cis- To confirm that all-trans-retinol and 13-cis-retinol were poor
retinol (Fig. 2B). We therefore call this reaction the aldehyde- substrates, the activities of these alcohols were measured at the
alcohol redox-coupling reaction (AL-OL coupling reaction; see higher concentration of 2.5 mM. No activity was observed for
below). In this reaction, 11-cis-retinal and A2 all-trans-retinol all-trans-retinol, whereas a low activity (1.2 Ϯ 0.1 amol/cone/s,
were produced at a 1:1 ratio (Fig. 2C), and the addition of mean Ϯ range of variations in two independent measurements)
enzyme cofactors was not necessary, in agreement with our was observed for 13-cis-retinol at this concentration. It should
previous study (16).
be mentioned that, in general, the measured activity is depen-
Substrate Specificity of the AL-OL Coupling Reaction—To dent on the substrate and concentration used in the counter-
understand the physiological role(s) of this reaction, it would be part of the reaction. This is why the activities obtained in the
helpful to know the substrate specificity of this reaction. For the presence of 11-cis-retinol and benzaldehyde were different in
reduction of aldehydes, 11-cis-retinol was used as the alcohol Fig. 3 (250 ␮M 11-cis-retinol and 250 ␮M benzaldehyde) and Fig.
substrate, and various aldehyde/ketone compounds, including 4A (250 ␮M 11-cis-retinol and 5 mM benzaldehyde).
retinal isomers, were examined at a concentration of 250 ␮M
For further analysis of alcohol specificity, 9-cis-retinol ana-
(Fig. 3). The activity was determined as the aldehyde/ketone- logs (a trienol derivative and a tetraenol derivative) were exam-
dependent formation of 11-cis-retinal (Fig. 3, left panel). As ined at 250 ␮M (Fig. 4B). In this measurement, all-trans-retinal
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Characterization of the AL-OL Coupling Reaction in Carp Cones
FIGURE 3. AL-OL coupling activities for various aldehydes and ketones. Each substrate (250 ␮M) and 11-cis-retinol (250 ␮M) were incubated in the presence
of cone membranes. The product (11-cis-retinal) was quantified, and the rate of its formation is shown by bars (mean Ϯ S.D. (n Ͼ 3) or the range of variations
(n ϭ 2); n ϭ 16 for all-trans-retinal, and n ϭ 2–3 for the others). The structure of each substrate is shown. *, in the case of the measurement of 11-cis-retinal,
9-cis-retinol was used as the alcohol substrate, and the activity was determined as the formation of 11-cis-retinol.
cate that oxidation of the AL-OL coupling reaction is highly
specific for 9-cis-retinol and 11-cis-retinol.
Reaction Mechanism of the AL-OL Coupling Reaction—The
AL-OL coupling reaction is a two-substrate reaction that uti-
lizes an alcohol substrate and an aldehyde substrate. To char-
acterize the substrate-binding mechanism of AL-OL coupling,
we performed a kinetic analysis. There are potentially two
mechanisms that account for a two-substrate reaction. One is a
sequential mechanism in which both substrates must bind to
the enzyme before the products are formed and released. The
other is a ping-pong mechanism in which one substrate first
binds to the enzyme to form one product, followed by the sec-
ond substrate binding to release the second product. These two
mechanisms could be distinguished kinetically by measuring
the enzyme activity at various concentrations of the first sub-
strate at different fixed concentrations of the second substrate.
FIGURE 4. AL-OL coupling activities for various alcohols. A, each retinol
isomer (250 ␮M) and benzaldehyde (5 mM) were incubated in the presence of
cone membranes. The aldehyde product of each isomer was quantified, and
the rate of its formation is shown by bars. B, the activities of 9-cis-retinol
analogs (250 ␮M) were measured in the presence of all-trans-retinal (250 ␮M),
and one of the products (all-trans-retinol) was quantified. The rate of its for-
mation is shown by bars. 11-cis-Retinol (250 ␮M) was used as a control. The
structure of each alcohol is shown. In both A and B, the results are shown as
When the relation between the substrate concentration and the
enzyme activity is plotted double-reciprocally, the plots give a
series of lines having a common intersection in the case of the
sequential reaction and a series of parallel lines in the case of the
ping-pong mechanism (29).
The AL-OL coupling activity was measured by varying both
the mean Ϯ S.D. (n Ͼ 3) or the range of variations (n ϭ 2).
11-cis-retinol and benzaldehyde concentrations. Fig. 5A shows
the rate of the activity as a function of the concentration of
11-cis-retinol at three fixed benzaldehyde concentrations on a
semilogarithmic scale. When the relation between the activity
and the substrate concentration was plotted double-recipro-
cally, the lines seemingly had a common intersection and were
not parallel. For this reason, the result was further analyzed on
the basis of the sequential reaction.
(250 ␮M) was used as the aldehyde substrate, and the all-trans-
retinol formed was quantified as the product of the AL-OL
coupling reaction (Fig. 4B, left panel). This way of quantifica-
tion was necessary because the oxidized products of the trienol
and tetraenol derivatives were difficult to quantify. Although
the trienal derivative was a good aldehyde substrate (Fig. 3), its
alcohol was a poor substrate (Fig. 4B). Similarly, the tetraenol
derivative was not a good substrate (Fig. 4B). (The activity of the
aldehyde of the tetraenol derivative was difficult to measure.)
These results, together with the results shown in Fig. 4A, indi-
The data in Fig. 5A were fitted to an equation formulated
for the sequential reaction (29): v ϭ (V1[A][B])/(KiAKmB
ϩ
K
mB[A] ϩ KmA[B] ϩ [A][B]), where v is the rate of the reaction;
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Characterization of the AL-OL Coupling Reaction in Carp Cones
FIGURE 5. Kinetic analysis of the reaction mechanism. The AL-OL coupling
reaction was performed in the presence of various concentrations of 11-cis-
retinol and benzaldehyde, and the activity was measured as the formation of
11-cis-retinal. A, the activity was plotted semilogarithmically as a function of
the concentration of 11-cis-retinol at three fixed benzaldehyde concentra-
tions. All of the data were fitted to the equation formulated for the sequential
reaction (solid lines; see “Results”). B, re-plot of A in a double-reciprocal plot.
The data points are shown as the mean Ϯ range of variations (n ϭ 2).
FIGURE 6. Localization of the AL-OL coupling activity in cones. A, cone
membranes were washed by centrifugation with isotonic, hypertonic, or
hypotonic buffer and buffer containing the detergent dodecyl-␤-D-maltoside
(0.48 mM). The activities of the AL-OL coupling reaction were determined in
the supernatant and precipitate fractions after centrifugation. Each activity
wasnormalizedtothatofthestartingsampleandisshownasthemean ϮS.D.
V1 is the maximum rate; [A] and [B] are the concentrations of (n ϭ 5 for the study in detergent buffer) or the range of variations (n ϭ 2 for
the others). B–D, SDS-PAGE pattern (B), cone pigment content (C), and AL-OL
coupling activity (D) examined in the OS-rich and IS-rich preparations and in
the starting purified cones (OS-plus-IS). For SDS-PAGE (B), each sample
applied contained 2 ␮g of proteins. Cone pigment content (C) and AL-OL
couplingactivity(D)areexpressedperunitamountofproteinsandareshown
as the mean Ϯ range of variations (n ϭ 2).
substrates A and B (i.e. 11-cis-retinol and benzaldehyde, respec-
tively); KiA is the binding constant of A for the free enzyme; and
K
mA and KmB are the Michaelis constants of A and B, respec-
tively. The data fitted well to the equation with the following
parameters: V1 ϭ 4.5 amol of 11-cis-retinal formed per cone/s,
K
iA ϭ 33 ␮M, KmA ϭ 126 ␮M, and KmB ϭ 469 ␮M (Fig. 5A, solid
We then examined the subcellular localization of the activity.
lines). The results are re-plotted double-reciprocally in Fig. 5B. Purified carp cones were mechanically broken into the outer
Because the data fitted well to the equation for the sequential segment (OS) and the inner segment (IS). OS-rich and IS-rich
reaction, we concluded that the AL-OL coupling is a sequential fractions were obtained using a stepwise sucrose density gradi-
reaction.
ent. Because the AL-OL coupling activity was tightly bound to
Localization of the AL-OL Coupling Activity in Cones—It membranes, soluble proteins lost during this OS-IS separation
would be also important to determine the localization of were ignored. The SDS-PAGE pattern of the total cone mem-
the AL-OL coupling reaction in a carp cone to understand the branes containing both OS and IS membranes (Fig. 6B, OS-
physiological role(s) of the reaction. First, we examined the plus-IS) showed that, in this membrane preparation, visual pig-
membrane localization of the activity. For this purpose, freshly ment (Fig. 6B, asterisk) was a minor protein component and
obtained purified carp cones were lysed by freeze-thawing and that other proteins (presumably those expressed in the cone IS)
fractionated into a supernatant and a precipitate fraction by were the major proteins. This finding is consistent with our
centrifugation. The activity was detected exclusively in the pre- observation that the volume of the IS is much larger than that of
cipitate fraction (data not shown). For additional analyses, this the OS in carp cones (see, for example, Fig. 1A in Ref. 2). In the
precipitate was further resuspended in various kinds of buffers OS-rich fraction, cone visual pigment content was enriched by
(isotonic, hypertonic, hypotonic, and detergent) and centri- 36-fold compared with that in the starting cone membranes
fuged to obtain supernatant and precipitate fractions. In all of (Fig. 6, B and C, compare pigment contents in OS-rich and
the buffer solutions except the one containing a detergent, the OS-plus-IS), whereas in the IS-rich fraction, most of the pre-
activity was detected exclusively in the precipitate fraction (Fig. sumed IS proteins were retained, but the pigment content was
6A). In the buffer containing a detergent, the activity was reduced significantly (Fig. 6, B and C, compare pigment and
detected mostly in the supernatant fraction. These results indi- other protein content in OS-plus-IS and IS-rich). When the
cate that the protein(s) responsible for the AL-OL coupling activity of the AL-OL coupling reaction was measured, the
reaction is a tightly membrane-associated protein(s) in cone activity was almost exclusively detected in the IS-rich fraction
membranes.
(Fig. 6D). Viewing our purified cone preparations under a light
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Characterization of the AL-OL Coupling Reaction in Carp Cones
microscope showed that the majority of the cells were cone
photoreceptors, and red blood cells were the only visible con-
taminants. The AL-OL coupling activity was not detected in
purified red blood cells (data not shown). Likewise, the activity
was not detected in the RPE (16). These results strongly suggest
that the activity is present in the cone IS. Because our purified
cones lacked the nucleus and terminal regions (18), the results
shown in Fig. 6D indicate that the protein(s) responsible for the
AL-OL coupling activity is probably localized in the ellipsoid
region of a cone IS.
DISCUSSION
FIGURE 7. Model of the substrate-binding sites in a postulated enzyme(s)
catalyzing the AL-OL coupling reaction.
In this study, we first confirmed that 11-cis-retinal and all-
trans-retinol formed in the presence of cone membranes are
the products of oxidation of 11-cis-retinol and reduction of all-
trans-retinal, respectively, and not those of isomerization (Fig. nicotinoprotein alcohol dehydrogenase) contain tightly bound
2). On the basis of this result, we termed this reaction the alde- NADH and catalyze the oxidation of an alcohol with the con-
hyde-alcohol redox-coupling reaction (AL-OL coupling reac- comitant reduction of an aldehyde (32). The nicotinoprotein
tion). We further examined the substrate specificity, substrate- alcohol dehydrogenase reaction obeys a ping-pong mechanism
binding mechanism, and subcellular localization of this (32), whereas the AL-OL coupling reaction obeys a sequential
reaction. Study of the substrate specificity demonstrated that reaction (Fig. 5). For this reason, the underlying mechanisms of
reduction of aldehyde showed broad substrate specificity with a these two reactions are not the same but could be similar in the
preference for aldehydes having a long carbon chain (Fig. 3), sense that AL-OL coupling may utilize firmly bound cofactor.
ϩ
whereas alcohol oxidation was specific for 11-cis-retinol and Actually, the addition of NADPH or NADP increased the
9-cis-retinol (Fig. 4). The binding of aldehyde and alcohol to the AL-OL coupling activity under some conditions. This line of
postulated enzyme could be explained by a sequential mecha- study is now in progress.
nism (Fig. 5). The AL-OL coupling activity was localized in the
Possible Functional Mechanisms of the AL-OL Coupling
cone IS membranes, most probably in the cone ellipsoid mem- Reaction—In our previous study (16), we postulated that, in the
branes (Fig. 6). On the basis of these findings, we discuss below AL-OL coupling reaction, the physiologically relevant aldehyde
the possible structure of the binding site in the postulated and alcohol substrates are all-trans-retinal and 11-cis-retinol,
enzyme and also possible functional mechanisms of the AL-OL respectively. The former is the product of pigment bleach, and
coupling reaction.
the latter is the retinoid transported from Müller cells to cones.
Possible Structure of the Substrate-binding Site—The double- In fact, in the presence of total cone membranes, cone visual
reciprocal plots of the relation between the substrate concen- pigment was regenerated after bleaching the pigment when
tration and the AL-OL coupling activity show a common inter- 11-cis-retinol was present during bleaching (16). This result
section (Fig. 5B). This demonstrates that the binding of an suggested that AL-OL coupling utilized all-trans-retinal
alcohol and an aldehyde to the enzyme is sequential. However, derived from the pigment as the aldehyde substrate to oxidize
it was not possible in this analysis to determine whether one of 11-cis-retinol. AL-OL coupling is advantageous in regenerating
the substrates should bind first or whether the binding order is cone visual pigment for two reasons. First, the oxidizing activity
random (29).
of 11-cis-retinol was 50 times higher than that attained by ret-
ϩ
The binding of alcohols showed high specificity for 11-cis- inol dehydrogenase activities (with NADP ) in cone mem-
retinol and 9-cis-retinol. In contrast, the binding of aldehydes branes (16), and second, the reaction was specific to 11-cis-
showed broad specificity with preference for molecules con- retinol and 9-cis-retinol (Fig. 4), both of which are effective in
taining a long carbon chain. This difference in substrate speci- regenerating the pigment.
ficity between alcohols and aldehydes, together with the kinetic
However, in this study, we found that the AL-OL coupling
analysis showing sequential binding of the substrates, suggests activity is present in the IS, most probably in the ellipsoid region
that the binding site of alcohols is different from that of alde- of a cone. This finding raises a point that needs to be explained
hydes. Because one substrate is oxidized and the other substrate in our postulation: an aldehyde, all-trans-retinal, has to be
is reduced, we speculate that the catalytic site of the redox reac- transported to the IS, and an aldehyde, 11-cis-retinal produced
tion is probably present between the binding site of alcohols from 11-cis-retinol, has to be transported to the OS. Because
and that of aldehydes (Fig. 7). However, the mechanism aldehydes are toxic, some cells contain CRALBP (cellular reti-
through which the reaction proceeds requires further study.
nal-binding protein) to transport retinals within a cell. How-
The uniqueness of the AL-OL coupling reaction is that it ever, photoreceptors do not contain such proteins (33). Despite
oxidizes an alcohol and reduces an aldehyde at a 1:1 ratio with- these facts, based on the results in previous studies, cones may
out requiring exogenous cofactors. It is a beneficial reaction be able to transport retinals within a cell.
because high energy metabolites such as NADPH are not nec-
In rods, when a large portion of pigment is bleached, the
essary. To our knowledge, this type of reaction is not known in all-trans-retinal formed leaks from the OS to the IS (34).
vertebrates, but in bacteria, some alcohol dehydrogenases (e.g. Although the cone OS contains a higher reduction activity for
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Characterization of the AL-OL Coupling Reaction in Carp Cones
7. Jin, M., Li, S., Moghrabi, W. N., Sun, H., and Travis G. H. (2005) Rpe65 is
all-trans-retinal compared with the rod OS (16, 35, 36), the
leakage of all-trans-retinal from the cone OS, if it is present,
could be a route for the supply of the aldehyde for the AL-OL
coupling reaction acting in the IS. Another potential source of
aldehydes supporting the AL-OL coupling reaction could be
the aldehydes produced in mitochondria in the ellipsoid region.
As shown in Fig. 3, medium-chain aldehydes such as hexanal
and nonanal effectively support the AL-OL coupling reaction.
These aldehydes are produced in mitochondria under oxidative
stress (31). If these aldehydes are produced under normal con-
ditions, these aldehydes can act as the aldehyde substrate to
support the AL-OL coupling reaction.
the retinoid isomerase in bovine retinal pigment epithelium. Cell 122,
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It has been suggested that cones have a specific pathway to
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retinal was applied to the cell body of a salamander cone after
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Signal Transduction:
Substrate Specificity and Subcellular
Localization of the Aldehyde-Alcohol
Redox-coupling Reaction in Carp Cones
Shinya Sato, Takashi Fukagawa, Shuji
Tachibanaki, Yumiko Yamano, Akimori
Wada and Satoru Kawamura
J. Biol. Chem. 2013, 288:36589-36597.
doi: 10.1074/jbc.M113.521153 originally published online November 11, 2013
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