RPE65 from Cone-dominant Chicken Is a More Efficient Isomerohydrolase Compared with That from Rod-dominant Species

Cones recover their photosensitivity faster than rods after bleaching. It has been suggested that a higher rate regeneration of 11-cis-retinal, the chromophore for visual pigments, is required for cones to continuously function under bright light conditions. RPE65 is the isomerohydrolase catalyzing a key step in regeneration of 11-cis-retinal. The present study investigated whether RPE65 in a cone-dominant species is more efficient in its enzymatic activity than that from roddominant species. In vitro isomerohydrolase activity assay showed that isomerohydrolase activity in the chicken retinal pigment epithelium (RPE) was 11.7-fold higher than in the bovine RPE, after normalization by RPE65 protein levels. Similar to that of human and bovine, the isomerohydrolase activity in chicken RPE was blocked by two specific inhibitors of lecithin retinal acyltransferase, indicating that chicken RPE65 also uses all-trans-retinyl ester as the direct substrate. To exclude the possibility that the higher isomerohydrolase activity in the chicken RPE could arise from another unknown isomerohydrolase, we expressed chicken and human RPE65 using the adenovirus system in a stable cell line expressing lecithin retinal acyltransferase. Under the same conditions, isomerohydrolase activity of recombinant chicken RPE65 was 7.7-fold higher than that of recombinant human RPE65, after normalization by RPE65 levels. This study demonstrates that RPE65 from the cone-dominant chicken RPE possesses significantly higher specific retinol isomerohydrolase activity, when compared with RPE65 from rod-dominant species, consistent with the faster regeneration rates of visual pigments in cone-dominant retinas. Cones recover their photosensitivity faster than rods after bleaching. It has been suggested that a higher rate regeneration of 11-cis-retinal, the chromophore for visual pigments, is required for cones to continuously function under bright light conditions. RPE65 is the isomerohydrolase catalyzing a key step in regeneration of 11-cis-retinal. The present study investigated whether RPE65 in a cone-dominant species is more efficient in its enzymatic activity than that from roddominant species. In vitro isomerohydrolase activity assay showed that isomerohydrolase activity in the chicken retinal pigment epithelium (RPE) was 11.7-fold higher than in the bovine RPE, after normalization by RPE65 protein levels. Similar to that of human and bovine, the isomerohydrolase activity in chicken RPE was blocked by two specific inhibitors of lecithin retinal acyltransferase, indicating that chicken RPE65 also uses all-trans-retinyl ester as the direct substrate. To exclude the possibility that the higher isomerohydrolase activity in the chicken RPE could arise from another unknown isomerohydrolase, we expressed chicken and human RPE65 using the adenovirus system in a stable cell line expressing lecithin retinal acyltransferase. Under the same conditions, isomerohydrolase activity of recombinant chicken RPE65 was 7.7-fold higher than that of recombinant human RPE65, after normalization by RPE65 levels. This study demonstrates that RPE65 from the cone-dominant chicken RPE possesses significantly higher specific retinol isomerohydrolase activity, when compared with RPE65 from rod-dominant species, consistent with the faster regeneration rates of visual pigments in cone-dominant retinas. Visual pigments of rods and cones consist of protein opsins and the chromophore, 11-cis-retinal covalently bound to opsins via a Schiff base (1Baylor D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 560-565Crossref PubMed Scopus (270) Google Scholar). Upon light absorption, the 11-cis-retinal is photoisomerized to all-trans-retinal, which subsequently activates opsin and triggers the phototransduction cascade (1Baylor D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 560-565Crossref PubMed Scopus (270) Google Scholar, 2McBee J.K. Palczewski K. Baehr W. Pepperberg D.R. Prog. Retin. Eye Res. 2001; 20: 469-529Crossref PubMed Scopus (314) Google Scholar). The 11-cis-retinal chromophore regenerates through a series of reactions of the retinoid visual cycle (3Rando R.R. Chemical Rev. 2001; 101: 1881-1896Crossref PubMed Scopus (139) Google Scholar, 4Crouch R.K. Chader G.J. Wiggert B. Pepperberg D.R. Photochem. Photobiol. 1996; 64: 613-621Crossref PubMed Scopus (86) Google Scholar). The visual cycle has been intensively studied in rod-dominant species such as bovine and mouse. The two-cell (photoreceptor and RPE) 2The abbreviations used are:RPEretinal pigment epitheliumLRATlecithin retinol acyltransferaseAd-chRPE65adenovirus-expressing chicken RPE65Ad-hRPE65adenovirus-expressing human RPE65CHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidBTP1,3-bis[tris(hydroxymethyl)-methylamino] propaneHPLChigh-performance liquid chromatographyapo-CRBPapo-cellular retinol-binding protein Type 1AcDCMK10-N-acetamidododecyl chloromethyl ketone. 2The abbreviations used are:RPEretinal pigment epitheliumLRATlecithin retinol acyltransferaseAd-chRPE65adenovirus-expressing chicken RPE65Ad-hRPE65adenovirus-expressing human RPE65CHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidBTP1,3-bis[tris(hydroxymethyl)-methylamino] propaneHPLChigh-performance liquid chromatographyapo-CRBPapo-cellular retinol-binding protein Type 1AcDCMK10-N-acetamidododecyl chloromethyl ketone. system of 11-cis-retinal recycling has been proposed for the visual cycle. After reduction of all-trans-retinal by retinol dehydrogenase, the generated all-trans-retinol is transported from the photoreceptor to the RPE and esterified to all-trans-retinyl esters by lecithin retinol acyltransferase (LRAT). The resulting all-trans-retinyl ester can either be stored in the RPE or directly converted to 11-cis-retinol by the isomerohydrolase, which was recently identified as the microsomal protein RPE65 (5Moiseyev G. Chen Y. Takahashi Y. Wu B.X. Ma J.X. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12413-12418Crossref PubMed Scopus (399) Google Scholar, 6Jin M. Li S. Moghrabi W.N. Sun H. Travis G.H. Cell. 2005; 122: 449-459Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 7Redmond T.M. Poliakov E. Yu S. Tsai J.Y. Lu Z. Gentleman S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13658-13663Crossref PubMed Scopus (315) Google Scholar). RPE65 has been shown to be an essential enzyme in the retinoid visual cycle for 11-cis-retinal regeneration (8Redmond T.M. Yu S. Lee E. Bok D. Hamasaki D. Chen N. Goletz P. Ma J.X. Crouch R.K. Pfeifer K. Nat. Genet. 1998; 20: 344-351Crossref PubMed Scopus (773) Google Scholar). After oxidation of 11-cis-retinol by retinol dehydrogenase-5, the generated 11-cis-retinal is transported back to the photoreceptors to regenerate the visual pigments. retinal pigment epithelium lecithin retinol acyltransferase adenovirus-expressing chicken RPE65 adenovirus-expressing human RPE65 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid 1,3-bis[tris(hydroxymethyl)-methylamino] propane high-performance liquid chromatography apo-cellular retinol-binding protein Type 1 10-N-acetamidododecyl chloromethyl ketone. retinal pigment epithelium lecithin retinol acyltransferase adenovirus-expressing chicken RPE65 adenovirus-expressing human RPE65 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid 1,3-bis[tris(hydroxymethyl)-methylamino] propane high-performance liquid chromatography apo-cellular retinol-binding protein Type 1 10-N-acetamidododecyl chloromethyl ketone. It is known that cones have lower sensitivities but faster responses to light, when compared with rods (9Perry R.J. McNaughton P.A. J. Physiol. 1991; 433: 561-587Crossref PubMed Scopus (177) Google Scholar, 10Baylor D.A. Invest. Ophthalmol. Vis. Sci. 1987; 28: 34-49PubMed Google Scholar). There is some evidence suggesting that visual cycle reactions also proceed faster in cones than in rods (11Ala-Laurila P. Kolesnikov A.V. Crouch R.K. Tsina E. Shukolyukov S.A. Govardovskii V.I. Koutalos Y. Wiggert B. Estevez M.E. Cornwall M.C. J. Gen. Physiol. 2006; 128: 153-169Crossref PubMed Scopus (71) Google Scholar, 12Mata N.L. Radu R.A. Clemmons R.C. Travis G.H. Neuron. 2002; 36: 69-80Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). The rate of conversion of all-trans-retinal to all-trans-retinol is 10–40 times faster in isolated salamander cones than that in rods (11Ala-Laurila P. Kolesnikov A.V. Crouch R.K. Tsina E. Shukolyukov S.A. Govardovskii V.I. Koutalos Y. Wiggert B. Estevez M.E. Cornwall M.C. J. Gen. Physiol. 2006; 128: 153-169Crossref PubMed Scopus (71) Google Scholar). These observations suggest that pigment regeneration is faster in cones than in rods under light conditions. This faster pigment regeneration may require a more efficient visual cycle in cone-dominant species. Because isomerohydrolase RPE65 is a key enzyme in the visual cycle for the regeneration of 11-cis-retinal, the present study investigated whether RPE65 in the RPE of cone-dominant chicken is a more efficient isomerohydrolase than in the roddominant bovine and human. Cloning of the Chicken RPE65 cDNA and Construction of the Expression Vector—The human RPE65 cDNA was cloned as described previously (13Ma J. Zhang J. Othersen K.L. Moiseyev G. Ablonczy Z. Redmond T.M. Chen Y. Crouch R.K. Invest. Ophthalmol. Vis. Sci. 2001; 42: 1429-1435PubMed Google Scholar). For cloning of chicken RPE65, animals were purchased from a local vendor. The chicken eyes were enucleated, and the RPE carefully dissected. The total RNA was isolated from the RPE using TRIzol reagent (Invitrogen) and further purified with an RNeasy column (Qiagen, Valencia, CA). Reverse transcription was performed with the TaqMan reverse transcriptase system (Applied Biosystemes Inc., Foster City, CA) using an oligo(dT) primer. To clone the full-length chicken RPE65 cDNA, the gene-specific primers (forward primer: 5′-GCGGCCGCCACCATGTACAGCCAGGTGGAGC-3′ containing a NotI site and the Kozak sequence (14Kozak M. Nucleic Acids Res. 1984; 12: 857-872Crossref PubMed Scopus (2376) Google Scholar) and reverse primer: 5′-AAGCTTCATGCTCTTTTGAAGAGTCCATGG-3′; containing a HindIII site) were used for PCR. PCR was performed with Pfu-Turbo (Stratagene, La Jolla, CA), a high fidelity enzyme, at 94 °C for 5 min followed by 35 cycles of 94 °C for 1 min, 58 °C for 1 min, and 72 °C for 2 min. The size of the PCR product was confirmed by 0.8% of agarose gel electrophoresis, and the DNA band with expected size was isolated from the gel using the QIAquick gel extraction kit (Qiagen). The extracted RPE65 cDNA was treated with TaqDNA polymerase (Roche Applied Science) at 72 °C for 10 min. Finally, the full-length chicken RPE65 cDNA was cloned into the pGEM-T easy vector (Promega, Madison, WI) and transformed into Escherichia coli. The positive clones were sequenced by an ABI-3770 automated DNA sequencer (Applied Biosystems Inc.) from both directions to exclude any mutations. Confirmed human and chicken RPE65 cDNAs were cloned between the NotI and HindIII sites of the pShuttle-CMV vector (Qbiogene, Montreal, Canada) for construction of adenoviruses. Preparations, amplification, and titration of the recombinant adenoviruses were performed as described previously (5Moiseyev G. Chen Y. Takahashi Y. Wu B.X. Ma J.X. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12413-12418Crossref PubMed Scopus (399) Google Scholar). Purification of Native Bovine and Recombinant Chicken RPE65—Native bovine RPE65 was purified from bovine RPE microsomes as described previously (15Moiseyev G. Takahashi Y. Chen Y. Gentleman S. Redmond T.M. Crouch R.K. Ma J.X. J. Biol. Chem. 2006; 281: 2835-2840Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Recombinant chicken RPE65 was purified from 293A cells infected by the adenovirus-expressing chicken RPE65 (Ad-chRPE65) at multiplicity of infection of 100 for 24 h. Microsomes were prepared from the infected 293A cells as described previously (16Moiseyev G. Crouch R.K. Goletz P. Oatis Jr., J. Redmond T.M. Ma J.X. Biochemistry. 2003; 42: 2229-2238Crossref PubMed Scopus Google Scholar). The microsomal were in and at for 1 h. The was a column RPE65 in the was by The with the of RPE65 were and using a with The of RPE65 was by with as described previously (15Moiseyev G. Takahashi Y. Chen Y. Gentleman S. Redmond T.M. Crouch R.K. Ma J.X. J. Biol. Chem. 2006; 281: 2835-2840Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). and of RPE65 in protein in cell and microsomal were using the PubMed Scopus Google Scholar). chicken and bovine RPE65 were by both the PubMed Scopus Google Scholar) and the of and PubMed Scopus Google Scholar). protein of total microsomal from chicken and bovine RPE were the gel and with an for RPE65 (13Ma J. Zhang J. Othersen K.L. Moiseyev G. Ablonczy Z. Redmond T.M. Chen Y. Crouch R.K. Invest. Ophthalmol. Vis. Sci. 2001; 42: 1429-1435PubMed Google Scholar). The that was used in was the which is in bovine, and chicken of purified chicken and bovine RPE65 were used as in the The of the was by the The protein of RPE65 was by using the The of the purified RPE65 protein was as the function of of purified The of RPE65 in the microsomes used were the a stable cell line expressing previously Y. Moiseyev G. Chen Y. Ma J.X. 2005; PubMed Scopus Google were infected with and the adenovirus expressing human RPE65 with a multiplicity of infection of cells were used as a were for in a 100 and microsomal were prepared as described previously (16Moiseyev G. Crouch R.K. Goletz P. Oatis Jr., J. Redmond T.M. Ma J.X. Biochemistry. 2003; 42: 2229-2238Crossref PubMed Scopus Google Scholar). was to the cell to the of The was min, to and The was and the microsomal was in and stored at chicken and bovine RPE microsomes were Inc., under and in the same of was used as the for the isomerohydrolase For of microsomal protein were into of 100 containing of bovine and of The was and were extracted with of and of and at for 5 min. The generated were by as described (16Moiseyev G. Crouch R.K. Goletz P. Oatis Jr., J. Redmond T.M. Ma J.X. Biochemistry. 2003; 42: 2229-2238Crossref PubMed Scopus Google Scholar). The of retinoid was identified the of retinoid The isomerohydrolase activity was from the of the 11-cis-retinol using with as a Cloning of Chicken chicken RPE65 cDNA was cloned as described under The chicken RPE65 cDNA an a protein of with a of The acid sequence with human RPE65 of the key acid required for enzymatic activity, such as the in the T.M. Poliakov E. Yu S. Tsai J.Y. Lu Z. Gentleman S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13658-13663Crossref PubMed Scopus (315) Google Scholar, Y. Moiseyev G. Chen Y. Ma J.X. 2005; PubMed Scopus Google and the and D.R. P. R.R. Cell. Full Text Full Text PDF PubMed Scopus Google between the chicken and human RPE65 that the chicken RPE65 the which was as a site in bovine RPE65 D.R. P. R.R. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). is in RPE65 bovine, and RPE65, but is by in chicken RPE65 of RPE65 in the Chicken RPE than in the Bovine RPE65, expressed in 293A was purified by chromatography as described under The purified chicken RPE65 was as shown by The of the purified RPE65 was confirmed by as described previously (16Moiseyev G. Crouch R.K. Goletz P. Oatis Jr., J. Redmond T.M. Ma J.X. Biochemistry. 2003; 42: 2229-2238Crossref PubMed Scopus Google Scholar). The purified chicken RPE65 was used as a to RPE65 in chicken RPE by RPE65 purified from the bovine RPE was used as a for bovine protein was in chicken RPE microsomes used for the and the of chicken RPE65 was as by protein was in bovine RPE and the of bovine RPE65 was the of RPE65 was of total microsomal in chicken RPE The of RPE65 in chicken RPE was significantly lower than that of bovine RPE65, which was of total microsomal Chicken the of investigated whether the chicken isomerohydrolase all-trans-retinyl ester as to that in rod-dominant species such as the bovine and human the of chicken isomerohydrolase activity the of all-trans-retinyl ester using two specific apo-cellular retinol-binding protein Type 1 and 10-N-acetamidododecyl chloromethyl which have been previously (16Moiseyev G. Crouch R.K. Goletz P. Oatis Jr., J. Redmond T.M. Ma J.X. Biochemistry. 2003; 42: 2229-2238Crossref PubMed Scopus Google Scholar, Biochemistry. PubMed Scopus Google Scholar). In the of the a of 11-cis-retinol was generated from all-trans-retinol after with bovine or chicken RPE microsomes and The of of a of ester to the RPE microsomes blocked the of both esters and 11-cis-retinol and we have shown that is also a and specific of ester (16Moiseyev G. Crouch R.K. Goletz P. Oatis Jr., J. Redmond T.M. Ma J.X. Biochemistry. 2003; 42: 2229-2238Crossref PubMed Scopus Google Scholar). To all-trans-retinyl ester from bovine and chicken RPE microsomes were with of for min to the of Similar to also the of esters and 11-cis-retinol in both the chicken and bovine RPE microsomes and These suggest that the chicken isomerohydrolase also uses esters as its for the of of the of RPE65 in Chicken and Bovine RPE of the of RPE65 isomerohydrolase activity were performed using bovine RPE microsomes H. Palczewski K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. R.R. Biochemistry. 1998; PubMed Scopus Google Scholar). is known the specific activity of RPE65 is the same in cone-dominant species. have prepared bovine and chicken RPE microsomes using the same as described previously (16Moiseyev G. Crouch R.K. Goletz P. Oatis Jr., J. Redmond T.M. Ma J.X. Biochemistry. 2003; 42: 2229-2238Crossref PubMed Scopus Google Scholar). all-trans-retinyl ester has been shown to be the of the isomerohydrolase (16Moiseyev G. Crouch R.K. Goletz P. Oatis Jr., J. Redmond T.M. Ma J.X. Biochemistry. 2003; 42: 2229-2238Crossref PubMed Scopus Google Scholar, D.R. R.R. Biochemistry. 2003; 42: PubMed Scopus Google the of the ester its in was used to esters by and esters were to by To the RPE65 isomerohydrolase activity, we the of of with of chicken RPE microsomes and of bovine RPE The was at and the generated was using and by with 11-cis-retinol The of 11-cis-retinol was at its and The of isomerohydrolase the enzyme was also both for bovine and chicken the of the we the specific isomerohydrolase activity in bovine RPE microsomes to be of 11-cis-retinol of total microsomal protein min The of 11-cis-retinol in chicken RPE microsomes showed a specific activity of isomerohydrolase It has been shown that any activity in their RPE or (8Redmond T.M. Yu S. Lee E. Bok D. Hamasaki D. Chen N. Goletz P. Ma J.X. Crouch R.K. Pfeifer K. Nat. Genet. 1998; 20: 344-351Crossref PubMed Scopus (773) Google suggesting that RPE65 is the retinol in the chicken RPE has lower of RPE65 than bovine RPE, we RPE65 both in bovine and chicken RPE microsomes used for the enzymatic activity by using purified bovine and chicken RPE65 as and the isomerohydrolase activity by the of The specific isomerohydrolase as the of the of 11-cis-retinol was of 11-cis-retinol of RPE65 min for the chicken Under the same the bovine RPE65 showed a specific activity of lower than that of chicken RPE65 of the of Recombinant and Chicken is a possibility that the higher specific activity in the chicken RPE microsomes may be to the of another unknown in to RPE65, chicken RPE65 was expressed using the adenovirus system in the cell line as described previously Y. Moiseyev G. Chen Y. Ma J.X. 2005; PubMed Scopus Google Scholar). This cell line has been shown to and to isomerohydrolase activity Y. Moiseyev G. Chen Y. Ma J.X. 2005; PubMed Scopus Google Scholar). For of the activity, human RPE65 was expressed in the cells under the same as that for chicken after the with or microsomes were prepared from the infected cells as described (16Moiseyev G. Crouch R.K. Goletz P. Oatis Jr., J. Redmond T.M. Ma J.X. Biochemistry. 2003; 42: 2229-2238Crossref PubMed Scopus Google Scholar). To isomerohydrolase activity, of the microsomal were used in the in vitro isomerohydrolase activity assay as described under The was at from to min of the The of generated 11-cis-retinol was and using and The rate of the 11-cis-retinol was from the of the of the The rate of the was to be in the cells expressing human RPE65 and in expressing chicken RPE65, when the same of total microsomal was After normalization of the enzymatic activity by the RPE65 in the the specific isomerohydrolase activity of chicken RPE65 was higher than that of human RPE65 activity, which was suggesting that recombinant chicken RPE65 has a higher specific enzymatic activity than human RPE65 under the same conditions. To that the activity is in the of 11-cis-retinol from we in both chicken and bovine RPE microsomes under the same as that for the isomerohydrolase assay and by the total microsomal protein These were compared with isomerohydrolase in the same microsomal and by the total microsomal protein The specific activity of bovine was which was significantly higher than that of the isomerohydrolase activity of in the same chicken and isomerohydrolase were and This demonstrates that activity was higher than the isomerohydrolase activity and is in the The chicken human and bovine have cones N.L. Radu R.A. Clemmons R.C. Travis G.H. Neuron. 2002; 36: 69-80Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). have shown that cones have faster than rods (9Perry R.J. McNaughton P.A. J. Physiol. 1991; 433: 561-587Crossref PubMed Scopus (177) Google Scholar, 10Baylor D.A. Invest. Ophthalmol. Vis. Sci. 1987; 28: 34-49PubMed Google Scholar). The of cones after times faster than that of rods (9Perry R.J. McNaughton P.A. J. Physiol. 1991; 433: 561-587Crossref PubMed Scopus (177) Google Scholar, 10Baylor D.A. Invest. Ophthalmol. Vis. Sci. 1987; 28: 34-49PubMed Google Scholar). The faster in cones a faster regeneration of 11-cis-retinal after its bleaching. we to animals with cone-dominant have a more efficient isomerohydrolase in their RPE65 is the key enzyme of the visual which regenerates 11-cis-retinal, the chromophore for and visual pigments (5Moiseyev G. Chen Y. Takahashi Y. Wu B.X. Ma J.X. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12413-12418Crossref PubMed Scopus (399) Google Scholar, 6Jin M. Li S. Moghrabi W.N. Sun H. Travis G.H. Cell. 2005; 122: 449-459Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 7Redmond T.M. Poliakov E. Yu S. Tsai J.Y. Lu Z. Gentleman S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13658-13663Crossref PubMed Scopus (315) Google Scholar). In the present we have that the RPE65 in chicken eyes has significantly higher enzymatic activity, when compared with that in human and bovine This for the that cone-dominant species may have a more efficient regeneration system than that in rod-dominant species. we have that two of in and in have isomerohydrolase in the but the isomerohydrolase with protein of RPE65 in (16Moiseyev G. Crouch R.K. Goletz P. Oatis Jr., J. Redmond T.M. Ma J.X. Biochemistry. 2003; 42: 2229-2238Crossref PubMed Scopus Google Scholar). Similar with of were by Redmond T.M. Jr., Biochemistry. 2005; PubMed Scopus Google Scholar). These suggest that is in the specific of RPE65 from and The isomerohydrolase activity in purified RPE65 has been This is that RPE65 is a protein that for its but the enzymatic activity of RPE65 is to of the To the isomerohydrolase in chicken and human or bovine RPE65, we have the isomerohydrolase activity by RPE65 in To the more efficient in chicken RPE from higher of the isomerohydrolase protein or a higher activity of the enzyme, we have the of RPE65, the isomerohydrolase and compared the specific enzymatic activity of chicken RPE65 with of human and bovine To in of the to RPE65 from species, purified chicken and bovine RPE65 were used as protein for of chicken and bovine RPE65 in the RPE, the used for RPE65 an that is in bovine, and chicken RPE65, to by The showed that the RPE65 in chicken RPE microsomes is lower than that in the bovine In isomerohydrolase activity assay showed that the specific isomerohydrolase activity in chicken RPE microsomes is times higher than that in bovine RPE after normalization by RPE65 in the RPE These observations suggest that the higher rate of 11-cis-retinal regeneration in chicken RPE microsomes be to the higher RPE65 levels. It has been that a of the in cone-dominant species is that directly all-trans-retinol as the and directly to 11-cis-retinol of the esters N.L. Radu R.A. Clemmons R.C. Travis G.H. Neuron. 2002; 36: 69-80Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, N.L. A. Radu R.A. Travis G.H. Biochemistry. 2005; PubMed Scopus Google Scholar). the the rod-dominant retinol the of esters that a direct for the isomerohydrolase (16Moiseyev G. Crouch R.K. Goletz P. Oatis Jr., J. Redmond T.M. Ma J.X. Biochemistry. 2003; 42: 2229-2238Crossref PubMed Scopus Google Scholar, D.R. R.R. Biochemistry. 2003; 42: PubMed Scopus Google Scholar). To the of the in the by chicken RPE we used two specific inhibitors of a enzyme in the specific inhibitors of and blocked the indicating that ester is a direct of chicken RPE to that in rod-dominant species. is in line with the observations by using the that in cone-dominant species the same the of esters as that in rod-dominant species D.R. R.R. Biochemistry. 2003; 42: PubMed Scopus Google Scholar). It was also that chicken lower of all-trans-retinyl ester but higher of esters than that in eyes N.L. Radu R.A. Clemmons R.C. Travis G.H. Neuron. 2002; 36: 69-80Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, N.L. A. Radu R.A. Travis G.H. Biochemistry. 2005; PubMed Scopus Google Scholar). The higher isomerohydrolase activity in the chicken RPE could also be for the lower of all-trans-retinyl esters in the chicken RPE, all-trans-retinyl esters more converted to which is esterified to esters for N.L. Radu R.A. Clemmons R.C. Travis G.H. Neuron. 2002; 36: 69-80Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, N.L. A. Radu R.A. Travis G.H. Biochemistry. 2005; PubMed Scopus Google Scholar). It has been shown that any in the or RPE (8Redmond T.M. Yu S. Lee E. Bok D. Hamasaki D. Chen N. Goletz P. Ma J.X. Crouch R.K. Pfeifer K. Nat. Genet. 1998; 20: 344-351Crossref PubMed Scopus (773) Google suggesting that RPE65 is the in the RPE, at in It has been proposed that some cone-dominant species, chicken and may have an retinol in their eyes N.L. Radu R.A. Clemmons R.C. Travis G.H. Neuron. 2002; 36: 69-80Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, N.L. A. Radu R.A. Travis G.H. Biochemistry. 2005; PubMed Scopus Google Scholar, A. A. M. M. Palczewski K. J. J. PubMed Scopus Google Scholar). enzyme has been identified at the is a possibility that higher isomerohydrolase activity in chicken RPE may arise from another we cloned the chicken RPE65 and expressed in which but any activity Y. Moiseyev G. Chen Y. Ma J.X. 2005; PubMed Scopus Google Scholar). The isomerohydrolase assay using isolated microsomes showed that the recombinant chicken RPE65 has an isomerohydrolase activity 7.7-fold higher than that in human RPE65 after normalization by the recombinant RPE65 suggesting that recombinant chicken RPE65 is a more efficient enzyme than that in This that high activity of chicken RPE65 may to a faster regeneration rate of 11-cis-retinal in cone-dominant species. Chicken and human RPE65 sequence at the acid It to be investigated which of the acid for the higher retinol activity in chicken It is also that could to the higher enzymatic activity of chicken of the for the higher activity of chicken RPE65 could have of the for the isomerohydrolase and could also to the of a more efficient RPE65 for retinal by RPE65 mutations.

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