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Published in final edited form as: Comp Biochem Physiol B. 1993 Jun;105(2):257–261. doi: 10.1016/0305-0491(93)90226-U

RETINOID METABOLISM AND CONVERSION OF RETINOL TO DEHYDRORETINOL IN THE CRAYFISH (PROCAMBARUS CLARKII) RETINA

Tatsuo Suzsuki *,†, Akihisa Terakita ‡,§, Andrew T C Tsin ‖
PMCID: PMC4505922  NIHMSID: NIHMS588257  PMID: 26190886

Abstract

  1. Activities of retinylester hydrolase (REHase) and retinol dehydrogenase (RDHase) were detected in the crayfish retina which has two visual pigment chromophores and three stored retinoids.

  2. RDHase and 11-cis REHase activities were localized to rhabdomal fraction and enzymes were specific to 11-cis isomers of substrates. Overall reaction from retinylester to retinal was highly specific to 11-cis isomers and this reaction was not selective between retinol and dehydroretinol.

  3. When 11-cis [3H]-retinol was incubated with retina homogenates, radioactivity was detected in 11-cis dehydroretinol.

  4. These results suggest that retinol is converted to dehydroretinol after hydrolysis of retinylesters.

INTRODUCTION

The crayfish, Procambarus clarkii is a unique invertebrate that has the rhodopsin–porphyropsin visual pigment system (Suzuki et al., 1984; Hariyama and Tsukahara, 1988; Zeiger and Goldsmith, 1989). The proportion of porphyropsin increases in response to low temperature and disappears at high temperature (Suzuki et al., 1985). 3,4-Didehydroretinal (dehydroretinal), the chromophore of porphyropsin is derived from dehydroretinol stored as esters in the retina (Suzuki et al., 1988), and this dehydroretinol may be synthesized from retinol in the retina. Additional retinoid, 3-hydroxyretinol, was found in the retina and it is probably an intermediate of the retinol–dehydroretinol conversion (Suzuki and Miyata, 1991). The proportion of dehydroretinal in the chromophores is far higher than that of dehydroretinol in stored esters (Suzuki et al., 1988, 1991). This discrepancy of retinoid composition might be explained by one of two possible mechanisms; (1) enzymes producing the chromophores favor the formation of dehydroretinoids, (2) retinol is converted to dehydroretinol after hydrolysis of retinyl esters. In order to examine these possibilities, we investigated two enzymes, retinylester hydrolase (REHase) and retinol dehydrogenase (RDHase). We also studied the conversion of retinol to dehydroretinol.

MATERIALS AND METHODS

The crayfish (Procambarus clarkii) were purchased from a local dealer and kept at 10°C in the dark until use. These conditions induced the accumulation of dehydroretinol and porphyropsin in the retina. The eyestalks were cut from animals, and the retinas were isolated from the crystalline cone layer, homogenized in distilled water and used for enzyme assay. In the experiment of enzyme localization, retinal tissue was separated into three fractions (soluble, rhabdomal and precipitate) by the centrifugation of retina homogenate on a sucrose-density gradient (10–60%) as reported previously (Suzuki et al., 1988).

Enzyme assay

All-trans and 11-cis retinyl [3H]-palmitates were synthesized by reacting retinols with the anhydride of [3H]-palmitic acid as described (Prystowsky et al., 1981) and purified by high-performance liquid chromatography (HPLC). For REHase assays the substrate concentration was adjusted to 2 nmol/10 µl ethanol (sp. act. 20.3 nCi/nmol for all-trans and 13.2 nCi/nmol for 11-cis retinyl [3H]-palmitate). The standard incubation mixtures consisted of 20–40 µg protein of retina homogenate, 10 µM substrate and 50 mM Tris–maleate buffer (pH 8.0) in a final reaction volume of 200 µl. Incubations were conducted at 25°C for 1 hr under dim light. After the incubation, [3H]-palmitic acid liberated by enzyme reaction was extracted as described by Belfrage and Vaughan (1969). One milliliter of the extract was mixed with 5 ml of Aquasol 2 (Dupont) and radioactivity was measured with a liquid scintillation counter (Packard 2500TR).

Retinols and dehydroretinols (11-cis and all-trans) were purified by HPLC after reducing the corresponding retinals by NaBH4. The concentration of retinols was adjusted to 2 nmol/10 µl ethanol. The RDHase activity was assayed by the method which converts aldehydes to oximes (Terakita et al., 1991). The standard incubation mixture consisted of 50 mM Tris–maleate buffer (pH 8.0), 100 mM NH2OH, 10 µM substrate and 60–70 µg protein of tissue homogenate in final volume of 200 µl. The mixture was incubated at 25°C for 1 hr under dim light and the reaction was terminated by addition of 0.5 ml cold ethanol. Retinaloximes were extracted with hexane and quantified with HPLC as previously described (Makino-Tasaka and Suzuki, 1986).

Non-radioactive retinyl (and dehydroretinyl) palmitates were synthesized from corresponding alcohols and palmitoyl chloride and purified by HPLC. Overall reaction (REHase + RDHase) was assayed in the same reaction mixture as for RDHase using retinyl and dehydroretinyl palmitates (10 µM) as substrates instead of retinols. The products, retinaloximes, were extracted and analyzed as above.

Proteins were determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard.

Conversion of retinol into dehydroretinol and 3-hydroxyretinol

11-Cis [3H]-retinol was prepared from all-trans [11,12(n)-3H]-retinol (Amersham, Bucks, UK) using the isomerase activity of retinochrome (Hara and Hara, 1984). All-trans [3H]-retinol (100 µCi/20 nmol) was dissolved in 1 ml hexane and passed through an activated MnO2 (1 g) column to oxidize to retinal. The retinal solution was evaporated and the residue was dissolved in 25µl ethanol. The retinal was dispersed in 1 ml of 0.5% digitonin solution containing ca 0.2 nmol retinochrome, 50 mM phosphate buffer (pH 6.5) and 100 mM ascorbate and irradiated with orange light. The photoproducts (main component 11-cis isomer) were reduced with NaBH4, extracted with petroleum ether, and 11-cis retinol was purified by HPLC. The concentration of 11-cis [3H]-retinol was adjusted to 10 nmol/10 µl (sp. act. 23 nCi/nmol) by addition of non-radioactive 11-cis retinol.

Incubation mixture was 100 µl of 100 mM Tris–HCl (pH 8.0, final 50 mM), 80 µl tissue homogenate (about 0.5 mg protein), 10 µl of 20 mM NADPH and 10 µl of 1 mM 11-cis [3H]-retinol in ethanol in a final volume of 200 µl. The mixture was incubated at 25°C for 3 hr under dim light and the reaction was terminated by addition of cold 0.5 ml methanol. Non-radioactive standard mixture (1.0 nmol each of all-trans retinol, 11-cis retinol, all-trans dehydroretinol, 11-cis dehydroretinol, all-trans 3-hydroxyretinol, 11-cis 3-hydroxyretinol) was added to the sample and, after addition of 1 ml distilled water, total retinoids were extracted with petroleum ether three times. The extracts were combined, evaporated under a stream of N2, and the residue dissolved in 200 µl HPLC solvent. Retinoids were separated by HPLC as reported previously (Suzuki and Miyata, 1991). A 100 µl aliquot was injected into HPLC equipped with a silica gel column (Waters) and retinols and dehydroretinols were separated with 6% dioxane/hexane at flow rate of 1.5 ml/min. Absorbance at 330 nm was monitored with a UV-detector and recorded. HPLC eluates for every 15 sec were collected into 4 ml mini-vials and mixed with 3 ml Aquasol 2, and radioactivity was determined with a liquid scintillation counter. 3-Hydroxyretinols were separated with an elution solvent of 20% dioxane/1% ethanol/hexane with the same HPLC using a residual 100 µl aliquot of the extract, and UV-absorbance and radioactivity were determined as above.

RESULTS

Characterization of REHase and RDHase

The first step of retinoid metabolism for chromophore formation is the hydrolysis of stored retinyl ester to retinol. In order to eliminate the contribution of retinylester synthetase reaction, we used retinyl [3H]-palmitate as substrate and liberated [3H]-palmitic acid was determined.

The retina REHase showed interesting pH-dependencies. When all-trans retinyl palmitate was used as substrate, the enzyme activity showed a single peak and the optimum pH was 5. On the contrary, two activity peaks were found when 11-cis retinyl palmitate was used as substrate, around at pH 5 and pH 8 (Table 1). Hydrolysis was abolished by heat treatment of homogenate (90°C, 3 min). These results suggest that the preparation is the mixture of two species of REHase, one active at pH 5 may be non-specific ester hydrolase and another with activity peak at pH 8 is specific to retinoid metabolism.

Table 1.

Activities of REHase and RDHase in crayfish retinal homogenate

Substrate isomer
PH 11-cis All-trans
(pmol/mg/hr)
REHase 5.0 5447 ± 149 6098 ± 277
8.0 9155 ± 513 408 ± 144
RDHase 8.0 1239 ± 89 310 ± 19
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Enzyme activities were assayed using retinyl [3H]-palmitates for REHase and retinols for RDHase as substrate as in Materials and Methods. Mean ± SD (N = 3) are represented.

Rhabdoms and associated membranes are structures important to retinoid metabolism, because oil droplets containing retinylesters closely associated with the rhabdom structure (Suzuki et al., 1988). Three subcellular fractions were obtained from retina homogenates, and REHase was determined for 11-cis retinyl palmitate at pH 8.0. The highest specific activity was found in the rhabdomal fraction (Table 2). The isolated rhabdoms were treated with small volume of hypotonic buffer (20 mM Tris–HCl, pH 8.0) and further divided into two fractions (membranous and soluble protein fractions) by centrifugation at 100,000 g for 60 min. High REHase activity (18.5 nmol/mg/hr) was found in the precipitate but no detectable activity in the supernatant. These results indicate the REHase is a membrane-associated protein localized to rhabdom and related membranes.

Table 2.

Subcellular distribution of REHase and RDHase

Soluble Rhabdomal Heavy precipitate
(pmol/mg/hr)
REHase 1884 ± 217 11341 ± 616 1768 ± 166
RDHase 144 ± 24 3695 ± 103 566 ±117
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Three fractions were obtained by sucrose-density gradient centrifugation and specific activities were determined at pH 8.0 using 11-cis retinyl[3H]-palmitate for REHase and 11-cis retinol as substrate as in Materials and Methods. Mean ± SD (N = 3) are represented

Dehydrogenation (oxidation) of retinols is the next step of retinoid metabolism for chromophore production. Hydroxylamine was used to trap the products, retinals, to prevent backward reaction and to reduce stereoisomerization which easily occurs without NH2OH. Effects of NADP, co-factor of dehydrogenases, on the reaction were investigated. Only a slight acceleration effect was observed in the presence of 1 mM NADP under the present experimental conditions. Therefore, the dinucleotide was not added to the reaction medium.

The RDHase showed a single broad peak of activity, maximum at pH 8.0, which had nearly the same optimum pH as REHase. The activity was lost after the heat treatment of homogenate (90°C, 3 min). Substrate specificity of RDHase was studied using 11-cis and all-trans-retinyl palmitates. RDHase was specific to 11-cis isomer (Table 1).

Subcellular distribution of RDHase was studied by preparing three fractions; water soluble, rhabdomal and heavy precipitate fractions. The highest specific activity was found in rhabdomal fraction, indicating that RDHase is concentrated in rhabdom and related membranous structures (Table 2).

Overall reaction (REHase + RDHase)

Substrate selectivity of two successive reactions (REHase and RDHase) was examined by using retinyl palmitates as substrates and then determining retinaloximes as final products. The results are shown in Fig. 1. The overall reaction was highly specific to 11-cis isomers and activity for all-trans isomers was hardly detected. However, there was not a significant difference in specificity between 11-cis retinyl and 11-cis dehydroretinyl palmitates.

Fig. 1.

Fig. 1

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Substrate specificity of overall reaction (REHase + RDHase). These activities were determined at pH 8.0 using retinyl palmitates as substrates and retinaloximes as final products. Cis1, 11-cis retinyl palmitate; Cis2, 11-cis dehydroretinyl palmitate; ATI, all-trans retinyl palmitate; AT2, all-trans dehydroretinyl palmitate. Mean ± SD (N = 3) are shown.

Incorporation of radioactivity to dehydroretinol and 3-hydroxyretinol

In order to examine the conversion of retinol to 3-hydroxyretinol and/or dehydroretinol, 11-cis [3H]-retinol was incubated with retina homogenate and products were separated by HPLC. Figure 2 shows the radioactivities of HPLC eluates. During the incubation and extraction procedures, considerable amount of 11-cis retinol was isomerized to 13-cis and all-trans isomers. This stereoisomerization of retinol was observed also in the control experiments using heat-denatured preparation. Considerable radioactivity was found in the fractions of 11-cis dehydroretinol when 11-cis [3H]-retinol was incubated with retinal homogenate but this was not observed in control using heat-denatured proteins. Time course of the radioactivity of 11-cis dehydroretinol was examined in additional experiments, and it was found that the activity increased with time up to 3 hr (data not shown). These results indicate that retinol was converted to dehydroretinol by the action of an enzyme.

Fig. 2.

Fig. 2

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HPLC profiles of radioactive retinoids derived from [3H]-retinol. 11-cis[3H]-retinol was incubated with retina homogenate at pH 8.0 for 3 hr, and the products were separated by HPLC. Boiled homogenate (90°C, 3 min) was used as control (-♦-). One representative is shown. Elution positions of standard retinols are indicated by arrows: 111, 11-cis retinol; 131, 13-cis retinol; 112, 11-cis dehydroretinol;

AT1, all-trans retinol; AT2, all-trans dehydroretinol.

3-Hydroxyretinols were separated by HPLC and radioactivities were determined as above. Small amount of radioactivity was found at the position of 11-cis 3-hydroxyretinol and the peak was observed to be thermally labile. The amount of radioactivity associated with 3-hydroxyretinol remained constant during the incubation up to 3 hr. The level of radioactivity, however, was too low to establish unequivocally the formation of 3-hydroxyretinol.

DISCUSSION

In the crayfish retina, retinol and dehydroretinol are stored in the esterified form and most of them are in the 11-cis configuration (Suzuki et al., 1988). Chromophores of visual pigments, retinal and dehydroretinal, must be derived from these retinyl esters by two enzyme reactions governed by REHase and RDHase. In the present study we detected both enzyme activities which are specific to 11-cis isomers of retinoids. Little was known about these enzyme activities in invertebrate retina in contrast to many studies in retinal pigment epithelium (RPE) of vertebrate.

In bovine RPE, Berman et al. (1985) reported that REHase is localized to the lysosomal fraction and that the activity was highest at pH 4.0–4.5 with low specificity for retinyl- and fatty acyl-esters. Blaner et al. (1987) used human RPE homogenate, and reported that REHase has an activity peak at pH 8.5 and was specific to 11-cis isomer. REHase activities at pH 8 were reported also in bovine RPE and neural retina by Tsin and Malsbury (1991). The different results reported on REHase are probably due to the different preparation and different assay conditions. Mammalian RPE seems to contain two esterhydrolases, one specific to retinoid metabolism and another lysosomal, non-specific esterhydrolase. If the crayfish retina had lysosomal, non-specific esterhydrolase, besides REHase, specific to 11-cis retinyl esters, the two peaks of REHase activity at pH 8 and 5 could be reasonably explained.

The properties of RDHase activity in the crayfish retina are similar to those reported in vertebrate retina and RPE; specific to 11-cis isomer and pH optimum near 8. (Lionet al., 1975; Saari and Bredberg, 1982; for review, Bridges, 1984). In the present experiment, NADP had little effect on RDHase activity in contrast to the analogous reaction in vertebrate RPE. In our experiment, hydroxylamine in a high concentration was used to trap retinals produced by enzymatic reaction. This is one of the possible reasons for dinucleotide independence.

Both REHase and RDHase showed highest specific activity in rhabdomal fraction (Table 2), and further, in sub-rhabdomal membrane fraction. As reported previously, oil droplets containing retinyl esters are closely associated with rhabdom structure in the retinula cell (Suzuki et al., 1988). The structural relationship between stored sites of esters and enzyme localization seems to be reasonable for efficient formation and transport of the chromophores of the visual pigment.

An important property noticed is the substrate specificity to retinol and dehydroretinol, because the proportion of dehydroretinal in the chromophores is far higher than that of dehydroretinol in stored ester. The results of Fig. 1 show that the reactions producing chromophores from stored esters are selective for 11-cis isomers but not specific towards dehydroretinol. Therefore, the discrepancy of retinoid composition between that in chromophores and that in stored ester cannot be explained simply by the substrate specificity of the two enzymes.

A possible mechanism to explain the discrepancy of retinoid composition is the conversion of retinol to dehydroretinol after the hydrolysis of retinyl esters. The results of Fig. 2 show that retinol was converted to dehydroretinol by enzyme action of retina homogenate.

3-Hydroxyretinol found in the stores esters was considered, for in vivo studies, to be an intermediate in the retinol-dehydroretinol conversion (Suzuki and Miyata, 1991). In the present study, the radioactivity incorporated into 3-hydroxyretinol was very low and the result was not completely conclusive. However, if the 3-hydroxyretinol was indeed the intermediate, it is reasonable to expect this low activity level of hydroxyretinol. The conversion of retinol to dehydroretinol was also observed in tadpole retina (Tsin et al., 1985), but there has been no information on the involvement of 3-hydroxyretinol in the retinol–dehydroretinal conversion in the vertebrate retina.

In conclusion, the chromophores of visual pigments in the crayfish retina are produced by the actions of two enzymes, REHase and RDHase, which show no selectivity for dehydroretinol. Retinol is converted into dehydroretinol after the hydrolysis of stored retinyl esters and this conversion may be responsible for the difference in retinoid composition between that in chromophores and that in stored esters. 3-Hydroxyretinol is probably the intermediate of the retinol–dehydroretinol conversion.

Acknowledgements

We thank Dr. K. Yoshihara (Suntory Institute of Bioorganic Research) and Dr T. Seki (Osaka Kyoiku University) for their gift of 3-hydroxyretinols. We also thank Prof. Y. Kito (Osaka University) and Prof. K. Nagai (Hyogo College of Medicine) for their comments. This work was supported by the Grants-in-Aid for Scientific Research (01540610) from the Japanese Ministry of Education, Science and Culture.

REFERENCES

  1. Belfrage P, Vaughan M. Simple liquid-liquid partition system for isolation of labeled oleic acid from mixtures with glycerides. J. Lipid Res. 1969;10:341–344. [PubMed] [Google Scholar]
  2. Berman ER, Segal N, Rothman H, Weiner A. Retinyl ester hydrolase of bovine retina and pigment epithelium: comparisons to the rat liver enzymes. Curr. Eye Res. 1985;4:867–876. doi: 10.3109/02713688509095254. [DOI] [PubMed] [Google Scholar]
  3. Blaner WS, Das SR, Gouras P, Flood MT. Hydrolysis of 11-cis and all-trans retinyl palmitate by homogenates of human retinal epithelial cells. J. biol. Chem. 1987;262:53–58. [PubMed] [Google Scholar]
  4. Bridges CDB. Retinoids in photosensitive systems. In: Sporn MB, Roberts AB, Goodman DS, editors. The Retinoids. Vol. 2. Orlando, FL: Academic Press; 1984. pp. 125–176. [Google Scholar]
  5. Hara R, Hara T. Squid m-retinochrome: two forms of metaretinochrome. Vision Res. 1984;24:1629–1640. doi: 10.1016/0042-6989(84)90321-3. [DOI] [PubMed] [Google Scholar]
  6. Hariyama T, Tsukahara Y. Seasonal variation of spectral sensitivity in crayfish retinula cells. Comp. Biochem. Physiol. 1988;91A:529–533. [Google Scholar]
  7. Lion F, Rotmans JP, Daemen FJM, Bonting SL. Stereospecificity of ocular retinol dehydrogenases and the visual cycle. Biochim. biophys. Acta. 1975;384:283–292. doi: 10.1016/0005-2744(75)90030-3. [DOI] [PubMed] [Google Scholar]
  8. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J. biol. Chem. 1951;193:265–275. [PubMed] [Google Scholar]
  9. Makino-Tasaka M, Suzuki T. Quantitative analysis of retinal and 3-dehydroretinal by high-pressure liquid chromatography. In: Chytil F, MacCormick DB, editors. Methods in Enzymology: Vitamins and Coenzymes, Part H. Vol. 123. New York; Academic Press; 1986. pp. 53–61. [DOI] [PubMed] [Google Scholar]
  10. Prystowsky JH, Smith JE, Goodman DS. Retinyl palmitate hydrolase activity in normal rat liver. J. biol. Chem. 1981;256:4498–4503. [PubMed] [Google Scholar]
  11. Saari JC, Bredberg L. Enzymatic reduction of 11-cis -retinal bound to cellular retinal-binding protein. Biochim. biophys. Acta. 1982;716:266–272. doi: 10.1016/0304-4165(82)90277-x. [DOI] [PubMed] [Google Scholar]
  12. Suzuki T, Arikawa K, Eguchi E. The effects of light and temperature on the rhodopsin–porphyropsin visual system of the crayfish, Procambarus clarkii. Zool. Sci. 1985;2:455–461. [Google Scholar]
  13. Suzuki T, Makino-Tasaka M, Eguchi E. 3-Dehydroretinal (vitamin A2 aldehyde) in crayfish eye. Vision Res. 1984;18:783–787. doi: 10.1016/0042-6989(84)90149-4. [DOI] [PubMed] [Google Scholar]
  14. Suzuki T, Miyata S. 3-Hydroxyretinol in the rhodopsin–porphyropsin retina of an invertebrate (Procambarus clarkii) Comp. Biochem. Physiol. 1991;99B:307–312. [Google Scholar]
  15. Suzuki T, Toh Y, Maeda A, Eguchi E. Retinyl and 3-dehydroretinyl esters in the crayfish retina. Vision Res. 1988;28:1061–1070. doi: 10.1016/0042-6989(88)90132-0. [DOI] [PubMed] [Google Scholar]
  16. Terakita A, Hara R, Ozaki K, Hara T. Retinol ligands of retinal-binding protein in the squid retina. Zool. Sci. 1989;6:1082. (Abstract). [Google Scholar]
  17. Tsin ATC, Alvarez RA, Fong S-L, Bridges CDB. Conversion of retinol to 3,4-didehy-droretinol in the tadpole. Comp. Biochem. Physiol. 1985;81B:415–421. doi: 10.1016/0305-0491(85)90335-9. [DOI] [PubMed] [Google Scholar]
  18. Tsin ATC, Malsbury DG. Bile salt independent retinyl ester hydrolase in the bovine eye. Brain Res. Bull. 1991;28:121–125. doi: 10.1016/0361-9230(92)90238-s. [DOI] [PubMed] [Google Scholar]
  19. Zeiger J, Goldsmith TH. Spectral properties of porphyropsin from an invertebrate. Vision Res. 1989;29:519–527. doi: 10.1016/0042-6989(89)90038-2. [DOI] [PubMed] [Google Scholar]

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