Enzymatic reduction of 11-cis-retinal bound to cellular retinal-binding protein

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266 Biochimica et Biophrsica Acta, 716 (1982) 266 272 Elsevier Biomedical Press BBA 21130 ENZYMATIC REDUCTION OF II-cis-RETINAL BOUND TO CELLULAR RETINAL-BINDING PROTEIN JOHN C. SAARI and LUCILLE BREDBERG Departnlent of Ophthalmologr RJ-IO, Unwersitr of 14'asllington School of Aledicmc,, Sc,atth,, ~l'.4 ~)81t)5 (L:.S..4.) (Rcceivcd January 12th, 1982) Ker word~: l ]-cis-Retinal reduction: Retinal binding protein," (Bocine retinal Cellular retinal-binding protein from bovine retina purifies with bound l l-cis-retinal and l l-cis-retinol as endogenous iigands. Inasmuch as these retinoids are interconvertible by a dehydrogenase reaction the accessibility of the aldehyde function of bound ll-cis-retinal to chemical and enzymatic reducing agents was determined. An ll-cis-retinol dehydrogenase from retinal pigment epithelial microsomes, first described by Lion, F., Rotmans, J.P., Daemen, F.J.M. and Bonting, S.L. (Biochim. Biophys. Acta 384, 283-292, 1975) was found to reduce complexed ll-cis-retinal at pH 5.5 and 37°C rapidly and nearly quantitatively. The product of the reduction, 1 l-cis-retinol, remained complexed with the binding protein following the reaction. Reduction proceeded 3-times more rapidly with NADH than with NADPH. No change in geometrical isomeric configuration occurred during the reaction. The dehydrogenase from retinal pigment epithelium oxidized 11-cis-retinol complexed with cellular retinal-binding protein at pH 8.5 in the presence of NAD. In spite of the ready enzymatic reduction of 1 l-cis-retinai complexed with cellular retinal-binding protein, the aldehyde function was inaccessible to several chemical reducing agents. Incubation of the complex with NaBH 4 at pH 7.5 and NaCNBH 3 or borane dimethylamine at pH 5.5 did not result in reduction of l l-cis-retinal unless the complex had been exposed to white light, a treatment known to produce all-trans- retinal which has little affinity for the binding protein. Liver alcohol dehydrogenase produced only 10% reduction of l l-cis-retinal complexed with cellular retinal-binding protein in 15 rain at 37°C when added in amounts which produced about 60% reduction of the uncomplexed retinoid. The results suggest that the interaction between the l l-cis-retinol dehydrogenase and the l l-cis-retinal complexed to cellular retinal- binding protein is a specific one and that the binding protein may function as a substrate carrier for a dehydrogenase. Introduction Of the three soluble retinoid-binding proteins which have been purified from extracts of bovine retina, only the cellular retinal-binding protein seems likely to be involved uniquely in vision. Cellular retinal-binding protein has been detected only in extracts of retina and retinal pigment epithelium [1] and binds the chromophore im- portant in vision, 11-cis-retinal [2]. An important clue to the function of cellular retinal-binding 0304-4165/82/0000-0000/$02.75 ! 1982 Elsevier Bioznedic',fl Press protein is provided by the observation that the protein, purified from bovine retina, contains two endogenous retinoids, 1 l-cis-retinol and 11-cis-ret- inal, which differ only in their state of oxidation [3]. The presence of these two bound retinoids, the existence in retinal pigment epithelium of a retinol dehydrogenase with a specificity directed towards cis-isomers of retinol [4-6] and the low solubility of retinoids in aqueous solutions [7], suggest that a potential function for cellular retinal-binding pro- tein is to carry the substrates for a dehydrogenase.

Transcript of Enzymatic reduction of 11-cis-retinal bound to cellular retinal-binding protein

Page 1: Enzymatic reduction of 11-cis-retinal bound to cellular retinal-binding protein

266 Biochimica et Biophrsica Acta, 716 (1982) 266 272 Elsevier Biomedical Press

BBA 21130

ENZYMATIC REDUCTION OF I I -c i s -RETINAL BOUND TO CELLULAR RETINAL-BINDING PROTEIN

JOHN C. SAARI and LUCILLE BREDBERG

Departnlent of Ophthalmologr RJ-IO, Unwersitr of 14'asllington School of Aledicmc,, Sc,atth,, ~l'.4 ~)81t)5 (L:.S..4.)

(Rcceivcd January 12th, 1982)

Ker word~: l ]-cis-Retinal reduction: Retinal binding protein," (Bocine retinal

Cellular retinal-binding protein from bovine retina purifies with bound l l-cis-retinal and l l-cis-retinol as endogenous iigands. Inasmuch as these retinoids are interconvertible by a dehydrogenase reaction the accessibility of the aldehyde function of bound ll-cis-retinal to chemical and enzymatic reducing agents was determined. An ll-cis-retinol dehydrogenase from retinal pigment epithelial microsomes, first described by Lion, F., Rotmans, J.P., Daemen, F.J.M. and Bonting, S.L. (Biochim. Biophys. Acta 384, 283-292, 1975) was found to reduce complexed ll-cis-retinal at pH 5.5 and 37°C rapidly and nearly quantitatively. The product of the reduction, 1 l-cis-retinol, remained complexed with the binding protein following the reaction. Reduction proceeded 3-times more rapidly with NADH than with NADPH. No change in geometrical isomeric configuration occurred during the reaction. The dehydrogenase from retinal pigment epithelium oxidized 11-cis-retinol complexed with cellular retinal-binding protein at pH 8.5 in the presence of NAD. In spite of the ready enzymatic reduction of 1 l-cis-retinai complexed with cellular retinal-binding protein, the aldehyde function was inaccessible to several chemical reducing agents. Incubation of the complex with NaBH 4 at pH 7.5 and NaCNBH 3 or borane dimethylamine at pH 5.5 did not result in reduction of l l-cis-retinal unless the complex had been exposed to white light, a treatment known to produce all-trans- retinal which has little affinity for the binding protein. Liver alcohol dehydrogenase produced only 10% reduction of l l-cis-retinal complexed with cellular retinal-binding protein in 15 rain at 37°C when added in amounts which produced about 60% reduction of the uncomplexed retinoid. The results suggest that the interaction between the l l-cis-retinol dehydrogenase and the l l-cis-retinal complexed to cellular retinal- binding protein is a specific one and that the binding protein may function as a substrate carrier for a dehydrogenase.

Introduction

Of the three soluble retinoid-binding proteins which have been purified from extracts of bovine retina, only the cellular retinal-binding protein seems likely to be involved uniquely in vision. Cellular retinal-binding protein has been detected only in extracts of retina and retinal pigment epithelium [1] and binds the chromophore im- portant in vision, 11-cis-retinal [2]. An important clue to the function of cellular retinal-binding

0304-4165/82/0000-0000/$02.75 ! 1982 Elsevier Bioznedic',fl Press

protein is provided by the observation that the protein, purified from bovine retina, contains two endogenous retinoids, 1 l-cis-retinol and 11-cis-ret- inal, which differ only in their state of oxidation [3]. The presence of these two bound retinoids, the existence in retinal pigment epithelium of a retinol dehydrogenase with a specificity directed towards cis-isomers of retinol [4-6] and the low solubility of retinoids in aqueous solutions [7], suggest that a potential function for cellular retinal-binding pro- tein is to carry the substrates for a dehydrogenase.

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In this report we describe studies of the interaction of bound ll-cis-retinal with dehydrogenases and aldehyde reagents and provide evidence for what appears to be a specific interaction between l l - cis-retinal bound to cellular retinal-binding protein and a dehydrogenase from retinal pigment epi- thelium.

Materials and Methods

Materials. 11-cis-[ 3 H]Retinol was prepared from 11-cis-retinal (a generous gift of Dr. Walter Scott, Hoffmann LaRoche) by reduction in ethanol with NaB3H4 (New England Nuclear, 116.5 Ci /mmol) [8]. l l-cis-[3H]-Retinal was prepared by oxidation of 11-cis-[3H]retinol with MnO 2 [9,10]. The purity of all retinoids was verified by high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC). NaBH4, NaCNBH 3 and borane dimethylamine were purchased from Alpha Division, Ventron Corporation. Horse liver al- cohol dehydrogenase, NADPH, NADH, NADP and NAD were purchased from Sigma Chemical Co.

Retinoid-binding proteins. Cellular retinal- and cellular retinol-binding proteins were purified from extracts of bovine retina as previously described [ 11 ] through steps 2 and 3. Cellular retinal-binding protein was complexed with 11-cis-[3H]retinol by incubation of the protein with a 10-fold molar excess of the retinoid, followed by gel filtration through Sephadex G-100. Cellular retinal-binding protein was complexed with 11-cis-[3H]retinol by initially exposing the protein to white light to effect an isomerization of bound 11-cis-retinal to all-trans-retinal, followed by incubation with a 10-fold molar excess of 11-cis-[3H]retinol and gel filtration.

Analysis of retinoids. Retinoids were extracted from the proteins with 5 vol. of petroleum ether (b.p. 30-60°C) after addition of an equal volume of cold (0°C) ethanol. Analysis of the extracted retinoids was made by TLC (Silica Gel G, 5°C, cyclohexane: toluene : ethyl acetate, 50 : 30 : 20, v /v ) and by HPLC. Retinoid analysis by HPLC em- ployed a Beckman HPLC and an Altex Ultra- sphere Si column (0.46 × 25 cm) (5 ~m particle size). Geometrical isomers of retinol were resolved at room temperature with dioxane:hexane (8:92,

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v/v) . Geometrical isomers of retinal were resolved at room temperature with dioxane: hexane (3 : 97, v /v) . The flow rate was 2 ml /min in both cases. The procedures employed for both TLC and HPLC were adapted from the description given by Groenendijk et al. [12].

Dehydrogenase activity. Cellular retinal-binding protein, complexed with 11-cis-[3 H]retinal (6-15 /~M), was incubated in 0.1 M sodium acetate at pH 5.5 and 37°C with N A D H (65 /~M) and micro- somes from retinal pigment epithelium (reaction volume, 0.3 ml). After 15-20 min the reaction was terminated by the addition of 1 ml of cold ethanol (0°C) containing 2 /~g/ml each of 11-cis-retinol, 11-cis-retinal, all-trans-retinol, all-trans-retinal and all-trans-retinyl palmitate. Retinoids were then ex- tracted and determined as described above. When cellular retinal-binding protein with bound 1 l-cis- [3H]retinol was the substrate the reaction was run in 0.1 M Tris-HC1, pH 8.5, with the addition of NAD. Controls, included in all assays, lacked only the addition of pyridine nucleotide. Assays were conducted either under red illumination (Westing- house 40 W ruby red bulb) or total darkness unless specified. To effect a photoisomerization of bound ligand, cellular retinal-binding protein was exposed to a 60 W tungsten bulb (distance, 20 cm) for 15 min at 0°C.

Dehydrogenase assays employing uncomplexed retinal were performed using a fluorescence de- termination of the amount of retinol produced. After incubation with the dehydrogenase as de- scribed above, retinoids were extracted and the fluorescence of the extract measured (excitation, 335 nm; emission, 458 nm). In hexane the fluores- cence of all-trans-retinol was found to be 5-times that of l l-cis-retinol. Data obtained using this assay (shown in Fig. 1) were corrected by this factor.

ll-cis-Retinol dehydrogenase. Retinal pigment epithelial cells were obtained from fresh bovine eyes and washed as described by Berman and Feeney [13]. A microsomal fraction was obtained using a modification of the method of Zimmerman [6]. The cells were suspended in 10 vol. 0.25 M sucrose, 0.025 M Tris-acetate, pH 7.0, homoge- nized with 8-10 strokes of a Teflon-glass homo- genizer driven at 450 rpm, and centrifuged at 200 × g for 6 rain. The pellet was re-homogenized

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and centrifuged and the combined supernatants were successively centrifuged at 15000 × g for 25 min and 1 4 0 0 0 0 × g for 120 min to generate, respectively, mit0chondrial, microsomal and su- pernatant fractions. The microsomal pellet was resuspended by sonication in 0.01 M Tris-acetate, pH 7, 0.1 mM dithiothreitol and stored at - 80°C.

Chemical reducing agents. Reductions with NaBH 4 were carried out in 0.1 M Tris-HC1 at pH 7.5 while those with N a C N B H 3 and borane di- methylamine were carried out in 0.1 M sodium acetate at pH 5.5, the pH used during enzymatic reductions. The concentrations employed were: 50 mM NaBH 4, 300 mM borane dimethylamine and N a C N B H 3, 6 15 /,M cellular retinal-binding and cellular retinol-binding proteins. Following in- cubation at 37°C, the reaction mixtures were di- alyzed at 5°C and the retinoids extracted and analyzed as described above. All incubations were carried out under red illumination or total dark- ness unless specified.

Results

l l-cis-Retinol dehydrogenase from retinal pig- ment epithelium. A dehydrogenase with specificity directed towards cis-retinoids was first described in retinal pigment epithelium by Lion et al. [4] and shown to be a microsomal enzyme by Zimmerman [6]. We confirm their findings concerning the specificity and distribution of this enzyme. As shown in Fig. 1, the preference of the enzyme for l l-cis-retinal over all-trans-retinal is striking and nearly absolute.

Enzymatic reduction of bound l l-cis-retinal. In- cubation of l l-cis-retinal complexed with cellular retinal-binding protein wtih N A D H and retinal pigment epithelial microsomes resulted in prompt reduction of the retinoid. As is shown in Fig. 2, approx. 85% of the bound l l-cis-retinal was re- covered as 11-cis-retinol after 20 min of reaction. In the absence of pyridine nucleotide no 11-cis- retinol was found in addition to that originally present in the substrate (usually less than 4% of the total retinoid). The extent of reduction was linearly proportional to the amount of added mi- crosomes (result not shown). N A D H was about 3-times as effective a substrate in the dehydro- genase reaction as NADPH. Incubation of l l-cis-

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Fig. I. Reduction of uncomplexed l l-cis- (© O) and all-tra.s-retinal (0 0) by a dehydrogenase from retinal pigment epithelial cells. The 125 p.M retinoid was incubated with a homogenate of retinal pigment epithelial cells and 625 FxM NADH at 37°C and pH 5.5. Extracted retinol ~vas de- termined by fluorescence.

retinal complexed with cellular retinal-binding protein with N A D H or N A D P H in the absence of microsomes produced no reduction of the retinoid showing that the binding protein itself is not a dehydrogenase. Incubation of l l-cis-retinal com- plexed with cellular retinal-binding protein at 37°C and pH 5.5 for 30 rain produced no alteration of the absorption spectrum of the protein showing that the complex was stable over the period of incubation. SDS-polyacrylamide gel electrophore- sis of cellular retinal-binding protein which had been incubated at pH 5.5 and 37°C with retinal pigment epithelial microsomes gave no indication of proteolytic degradation.

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8

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Fig. 2. Reduction of I I-c/s-retinal complexed with cellular reti- nal-binding protein by a dehydrogenase from retinal pigment epithelium. I I-cis-[3H]Retinal complexed with 12 ~M cellular retinal-binding protein was incubated at pH 5.5 and 37°C with 65 p.M NADH and pigment epithelial microsomes (100/Lg/ml, microsomal protein). At the reaction times shown retinoids were extracted from samples and analyzed by TLC.

Even though retinal pigment epithelial micro- somes are readily able to convert uncomplexed retinol to retinyl ester [ 14,15] tittle esterification of the product of reduction, 11-cis-retinol, was found in these experiments, in part, because the pH of the incubation (pH 5.5) is below the optimum for the esterification reaction. Esterification of bound 11-cis-retinol was seen in other experiments car- ried out at higher pH.

The fate of the reduced substrate, 11-cis-retinol, was examined in a further experiment, l l-cis- [3H]Retinal complexed with cellular retinal- binding protein was incubated with NADH and microsomes at pH 5.5 and 37°C for 20 rain and then chilled at 0°C to retard the reaction. Retinoid analysis of a portion of the reaction mixture indi- cated that 31% of the complexed 11-cis-[3H]retinal had been reduced. After centrifugation of the sus- pension to remove the micosomes, more than 90% of the radioactivity remained in solution showing that the retinoid had not been transferred to the microsomes during the reaction. Cellular retinal- binding protein from this experiment was then re-isolated by Sephadex G-100 gel filtration. Reti- noid analysis of a portion of the re-isolated bind- ing protein showed again that 31% of the total

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bound retinoid was 11-cis-retinol. The results dem- onstrate that 11-cis-retinol remains bound to cellu- lar retinal-binding protein following reduction.

Oxidation of bound l l-cis-retinol could be demonstrated in the presence of NAD at pH 8.5. However, at this pH considerable esterification of bound 11-cis-retinol occurred leading to a compe- tition between oxidation and esterification reac- tions. In a typical experiment the following reti- noids were found after 20 min of incubation of 11-cis-[3H]retinol complexed with cellular retinal- binding protein with microsomes and NAD at 37°C: retinal, 58%; retinol, 4%; retinyl ester, 38%. The values obtained following the same incubation in the absence of NAD were: retinal, 3%; retinol, 53%; and retinyl esters, 44%.

The ratio of the extent of reduction by the microsomal dehydrogenase of 11-cis-retinal added either uncomplexed or complexed with cellular retinal-binding protein was 1.3 (uncomplexed/ complexed), showing that the dehydrogenase is about equally efficient in reducing complexed and uncomplexed l l-cis-retinal. The same ratio ob- tained with liver alcohol dehydrogenase under comparable conditions was 4.8 indicating that 11- cis-retinal bound to cellular retinal-binding protein is a relatively poor substrate for this dehydro- genase.

Retention of geometrical isomeric conformation during reduction. The TLC system employed in this study resolves all geometrical isomers of retinal from the corresponding geometrical isomer of reti- nol, the minimal requirement of the study. How- ever, not all the common geometrical isomers of retinol and retinal are well resolved from one another. All-trans and 9-cis-derivatives are not completely resolved either as alcohols or aldehydes and l l-cis and 13-cis-derivatives are not com- pletely resolved either as alcohols or aldehydes. Thus, to examine the possibility of a geometrical isomerization occurring during reduction of com- plexed l l-cis-retinal, we employed both HPLC which resolves all common geometrical isomers of retinol and retinal, and TLC analysis. The results, shown in Table I, illustrate that both methods of analysis give comparable results in determining the extent of reduction and that no geometrical isomerization occurs during enzymatic reduction of 11-cis-retinal.

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TABLE I

ENZYMATIC REDUCTION OF I I-cis-RETINAL COM- PLEXED WITH CELLULAR RETINAL-BINDING PRO- TEIN. ANALYSIS OF THE EXTRACTED RETINOIDS BY TLC AND HPLC

1 l-cis-[ 3 H]Retinal complexed with cellular retinal-binding pro- tein was incubated at pH 5.5 and 37°C with retinal pigment epithelial microsomes and NADH for 20 rain. The extracted retinoids from this reaction mixture were analyzed by TLC and HPLC. The retinoids associated with the binding protein before enzymatic reduction were: l l-cis-retinal, 96%; all-trans-retinal, 4%.

Retitmid % of total

TLC HPLC

all-trans-Retinol It 3 4 9-cis-Retinol 0 11-cis-Retinol / 48 ,42 13-cls-Retinol f 0 all-lrans-Retinal 5 9-cis-Retinal } 4 0 I l-c/s-Retinal / 43 43 13-ors-Retinal )' 0

Reaction o f bound l l -c&-ret inal with chemical

reducing agents. The reduct ion of b o u n d 1 1-cis- re t inal by a dehydrogenase f rom ret inal p igment ep i the l ium would not be r emarkab le if the al- dehyde group of the b o u n d l igand were readi ly accessible or ' exposed ' to the solvent. We therefore examined the accessibi l i ty of this funct ional group to chemical reducing agents. The results of several exper iments are presented in Table II. Borane d i m e t hy l amine and N a C N B H 3 at p H 5.5 and N a B H 4 at p H 7.5 did not reduce b o u n d 1 1-cis- re t inal complexed with cel lular r e t ina l -b ind ing p ro te in in 15 min at 37°C unless the complex had been exposed to white light p r io r to the add i t i on of the reducing agents. I r r ad ia t ion of the complex with white light previous ly has been shown to cause a pho to i somer iza t ion of b o u n d 11-cis-retinal, p roduc ing all-trans-retinal which has l i t t le aff ini ty for the b ind ing p ro te in [3]. N a B H 4 at p H 7.5 also fai led to reduce b o u n d l l-cis-retinal when the complex was incuba ted with the reducing agent and re t inal p igment epi thel ia l microsomes (no py r id ine nucleot ide) .

Reduction o f bound 11-cis-retinal by liver alcohol dehydrogenase. It was of interest to de te rmine if

TABLE lI

TREATMENT OF I I-cis-RETINAL COMPLEXED WITH CELLULAR RETINAL-BINDING PROTEIN WITH CHEMICAL REDUCING AGENTS

Reducing agent and l l-cis-[3H]retinal complexed with cellular retinal-binding protein were incubated for 15 min at 37°C. Retinoids wcrc then extracted and analyzed by TLC.

Reducing agent % Retinol gc Retinal

None 2 98 Borane dimethylamine

(300 raM, pH 5.5) non-irradiated (red light only) 5 95 irradiated (white light) 45 55

NaBH 4 (50 raM, pH 7.5) non-irradiated (red light only) 8 92 irradiated (white light) 96 4

NaCNBH 3 (300 raM, pH 5.5) non-irradiated (red light only) 2 98

o ther dehydrogenases were also capab le of reduc- ing 11-cis-retinal complexed with cel lular re t inal- b ind ing prote in . Horse liver a lcohol dehydro- genase was chosen for s tudy as the enzyme will reduce uncomplexed l l-cis-retinal al though the re t inoid is a re la t ively poor subs t ra te [ 16]. A m o u n t s of liver a lcohol dehydrogenase and microsomal 1 1-cis-retinol dehydrogenase that p roduced equiva- lent reduc t ion of uncomplexed 1 1-cis-retinal were c o m p a r e d in their abi l i ty to reduce bound 1 1-cis-

retinal. As shown in Table III , fiver alcohol dehy- drogenase , even when added in relat ively large amounts , is much less effective in reduct ion of b o u n d l l-cis-retinal than the p igment epi thel ial enzyme.

Discussion

The inaccess ibi l i ty to chemical agents of the a ldehyde funct ion of 11-cis-retinal complexed with cel lular re t ina l -b ind ing pro te in is quite appa ren t f rom the da ta ob ta ined in this s tudy (Table II). Rela t ive ly high concent ra t ions of reducing agents (up to 300 mM), which readi ly reduce ' free ' reti- nal, fail to reduce the bound ret inoid. This ob- servat ion is in accord with an earl ier result which de mons t r a t e d that the complex is s table in the presence of N H 2 O H unless exposed to b leaching white light [3]. I r r ad ia t ion of the complex with

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TABLE III

REDUCTION OF l l - c i s - R E T I N A L COMPLEXED WITH CELLULAR RETINAL-BINDING PROTEIN BY LIVER ALCOHOL DEHYDROGENASE AND l l - c i s - R E T I N O L DEHYDROGENASE

I I-cis-[3H]Retinal complexed with 12 /zM cellular retinal- binding protein was incubated at pH 5.5 and 37°C for 15 rain with 60 #M NADH and either 160/zg/ml liver alcohol dehy- drogenase or pigment epithelial microsomes (30/~g/ml, micro- somal protein). The amounts of the dehydrogenases added gave comparable reduction of uncomplexed 1 l-c/s-retinal under sim- ilar conditions. Retinoids were extracted and analyzed by TLC.

Dehydrogenase added % Retinol % Retinal

None (control) 1 99 Liver alcohol dehydrogenase 10 90 I 1-cis-Retinol dehydrogenase 64 36

white light is known to result in a photoisomeriza- tion of bound 11-cis-retinal producing all-trans- retinal which no longer binds specifically to the binding protein. As expected the aldehyde func- tion of the irradiated complex is readily reduced by chemical reducing agents (Table II) and reacts with NH2OH to form the oxime [3]. The picture that emerges from these studies is that of a non- covalent complex of protein and 11-cis-retinal in which the aldehyde function is either sterically or chemically protected from interaction with low molecular weight aldehyde reagents.

The facile reduction of bound 11-cis-retinal by a dehydrogenase from retinal pigment epithelial microsomes (Fig. 2) becomes all the more remarka- ble when compared to the lack of effect of the chemical reducing agents. The dehydrogenase re- duces bound l l-cis-retinal rapidly and nearly quantitatively suggesting some type of specific in- teraction between the enzyme and 11-cis-retinal or l l-cis-retinol bound to cellular retinal-binding protein. This specificity of interaction is further indicated by the observation that not all alcohol dehydrogenases which will reduce uncomplexed 11-cis-retinal are capable of reducing 11-cis-retinal bound to cellular retinal-binding protein since liver alcohol dehydrogenase acts very poorly on the bound substrate (Table III). However, the 11-cis- retinol dehydrogenase itself appears to be rela- tively nonspecific in that it reduces complexed and uncomplexed 1 l-cis-retinal equally well.

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How is it that a large, bulky protein, probably embedded in a membrane, is able to interact with a functional group that is not accessible to water soluble aldehyde reagents? Certainly the func- tional groups of the dehydrogenase as well as the pyridine nucleotide must be brought into proxim- ity of the aldehyde function of 11-cis-retinal. One possibility is that the interaction of 11-cis-retinal complexed with cellular retinal-binding protein with a component present in the microsomes re- suits in a lowered affinity of the binding protein for the ligand. The equilibrium between bound and free 11-cis-retinal would then be displaced towards free 11-cis-retinal allowing facile reduc- tion of the free retinoid by the dehydrogenase. This possibility, as stated, seems unlikely since NaBH 4 does not reduce 11-cis-retinal complexed with cellular retinal-binding protein even in the presence of microsomes. More subtle variations of this mechanism are possible in which interaction between 11-cis-retinal complexed with cellular reti- nal-binding protein and components of the mi- crosomes lead to conformational changes which expose bound 11-cis-retinal to functional groups of the dehydrogenase but not the bulk solvent. Further experimentation will be necessary to fully assess the mechanistic aspects of this complex reaction. However, it does seem evident that an element of specificity must exist in the interaction of the dehydrogenase or another component in the microsomes and ll-cis-retinal bound to cellular retinal-binding protein.

The l l-cis-retinol dehydrogenase used in this study was derived from retinal pigment epithelium whereas cellular retinal-binding protein was pre- pared from extracts of bovine retina. Recent stud- ies in this laboratory have verified the presence of cellular retinal-binding protein in extracts of reti- nal pigment epithelium (Saari, J.C., Garwin, G.C. and Bredberg, D.L., unpublished data). Thus, the purported substrate carrier protein and the dehy- drogenase are found in the same cell. 11-cis-Reti- nol dehydrogenase has not yet been detected in extracts of neural retina and it is not known which cell or cells of the retina contain cellular retinal-bi- nding protein.

An earlier communication from this laboratory [2] reported that liver alcohol dehydrogenase re- duced bound 11-cis-retinal, a result seemingly at

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odds with the conclusions of this communication. However the previous study was conducted before we were fully aware of the necessity of protecting cellular retinal-binding protein from white light and it is likely that the preparation of cellular retinal-binding protein used as a substrate for alcohol dehydrogenase contained substantial amounts of uncomplexed all-trans-retinal. In addi- tion, a much larger amount of liver alcohol dehy- drogenase was employed in the earlier study (1 mg) and the incubation period was much longer (1 h at 37°C). Thus we feel the results obtained in the present study are a more accurate reflection of the extent of reduction of bound 11-cis-retinal by liver alcohol dehydrogenase.

The limited solubility of retinoids in aqueous solutions would appear to demand the existence of a mechanism to allow for their orderly transport (intracellular as well as extracellular) and en- zymatic transformation. The specificity of interac- tion between the 11-cis-retinol dehydrogenase and cellular retinal-binding protein demonstrated in this study, suggests that one function of the bind- ing protein may be to act as a substrate carrier for an l l-cis-retinol dehydrogenase. However. the l l- cis-retinol dehydrogenase is clearly able to act on uncomplexed retinoid, leading one to question the physiological meaning of the enzymatic reduction of bound retinoid. The significance of this reaction should be more clear when the fraction of com- plexed and uncomplexed l l-cis-retinol and l l-cis- retinal in retina and retinal pigment epithelium is determined, a subject of current research in this laboratory.

Acknowledgements

Mr. Gregory Garwin provided assistance in preparation of the retinoid-binding proteins and retinoids, and in retinoid analysis by HPLC. Dr. Craig Wells performed initial experiments demon- strating the feasibility of this experimental ap- proach. This study was supported in part by USPHS Grants EY-02317, EY-00343, EY-01730 and by a grant from Research to Prevent Blind- ness, Inc.

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