Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 Sep 6;44(35):11715-21.
doi: 10.1021/bi050942m.

Chicken retinas contain a retinoid isomerase activity that catalyzes the direct conversion of all-trans-retinol to 11-cis-retinol

Nathan L Mata  1 , Alberto Ruiz, Roxana A Radu, Tam V Bui, Gabriel H Travis
Affiliations

Chicken retinas contain a retinoid isomerase activity that catalyzes the direct conversion of all-trans-retinol to 11-cis-retinol

Nathan L Mata et al. Biochemistry. .

Abstract

Vertebrate retinas contain two types of light-detecting cells. Rods subserve vision in dim light, while cones provide color vision in bright light. Both contain light-sensitive proteins called opsins. The light-absorbing chromophore in most opsins is 11-cis-retinaldehyde, which is isomerized to all-trans-retinaldehyde by absorption of a photon. Restoration of light sensitivity requires chemical re-isomerization of retinaldehyde by an enzymatic pathway called the visual cycle in the retinal pigment epithelium. The isomerase in this pathway uses all-trans-retinyl esters synthesized by lecithin retinol acyl transferase (LRAT) as the substrate. Several lines of evidence suggest that cone opsins regenerate by a different mechanism. Here we demonstrate the existence of two catalytic activities in chicken retinas. The first is an isomerase activity that effects interconversion of all-trans-retinol and 11-cis-retinol. The second is an ester synthase that effects palmitoyl coenzyme A-dependent synthesis of all-trans- and 11-cis-retinyl esters. Kinetic analysis of these two activities suggests that they act in concert to drive the formation of 11-cis-retinoids in chicken retinas. These activities may be part of a new visual cycle for the regeneration of chromophores in cones.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Visual cycle for regeneration of rhodopsin. The light-sensitive protein in rods is rhodopsin, located in the membranes of outer-segment disks. 11-cis-Retinaldehyde (11cRAL) is coupled to a lysine residue in rhodopsin through a protonated Schiff base linkage. Absorption of a photon (hv) induces 11-cis to all-trans isomerization of retinaldehyde to yield metarhodopsin, which activates the visual transduction cascade. The all-trans-retinaldehyde (atRAL) subsequently dissociates from apoopsin and is reduced to all-trans-retinol (atROL) by all-trans-retinol dehydrogenase (atRDH), which uses NADPH as a cofactor. The atROL diffuses from the outer segment and is taken up by an RPE cell, where it is esterified with a fatty acid from phosphatidylcholine (PC) in a reaction catalyzed by lecithin retinol acyl transferase (LRAT), yielding an all-trans-retinyl ester (atRE) and lysophosphatidylcholine (lyso-PC). The atRE is converted to 11-cis-retinol (11cROL) and a free fatty acid (FFA) by the isomerase (Rpe65). The 11cROL is oxidized by an 11cROL dehydrogenase (11cRDH), which uses NAD+ as a cofactor, to yield 11cRAL. The 11cRAL diffuses back to the outer segment where it combines irreversibly with aporhodopsin to form a new rhodopsin pigment molecule.
Figure 2
Figure 2
Chicken eyecup and retina following dissection. The left panel shows the RPE-containing eyecup following dissection. Patches of the pigmented RPE were rinsed away during removal of the retina. The right panel shows a chicken retina after dissection from the posterior eyecup. Note the absence of melanin pigment, indicating clean separation from the RPE.
Figure 3
Figure 3
Synthesis of retinyl esters from atROL and palm-CoA by chicken retina and bovine RPE. (A) atREs and 11cREs produced by bovine RPE microsomes at the indicated times following incubation with 10 μM atROL and 100 μM palm-CoA. (B) atREs and 11cREs produced by chicken retina microsomes under similar incubation conditions. Error bars show standard deviations (n = 4). Note the predominant synthesis of atREs by bovine RPE. Also note the synthesis of 11cREs by chicken retina after a 1 min incubation, before the appearance of atREs.
Figure 4
Figure 4
Synthesis of retinoids from [3H]atROL by chicken retina explants. (A) Time course of [3H]retinoids synthesized at the indicated incubation times from 0.1 μM [3H]atROL added to the culture medium. Error bars show standard deviations (n = 4). (B) Representative UV chromatograms at 340 nm of retinoids after 5 min (⋯) and 40 min (–) incubations. Extracts were incubated with hydroxylamine to protect the aldehydes during analysis. 11cRAL and atRAL were measured as the corresponding syn- and anti-oximes (s- or a-11cROX and s- or a-atROX). Insets show UV spectra of representative peaks from the 5 and 40 min chromatograms.
Figure 5
Figure 5
LRAT expression in RPE and retina. (A) Northern blot of RNA from chicken and bovine RPE and retinas probed with a cDNA for bovine LRAT. Each lane contained 1 μg of poly(A)+ RNA from the indicated tissue source. The mobilities of RNA size markers are shown on the left in kilobases. (B) Immunoblot of protein extracts from chicken and bovine RPE and retinas, reacted with an antibody to human LRAT. Each lane contained 10 μg of total protein from the indicated tissue source. The mobilities of protein size markers are shown at the left in kilodaltons.
Figure 6
Figure 6
Substrate kinetics of retinoid synthesis by chicken retina microsomes. (A) Retinoids synthesized following 2 min incubations at the indicated concentrations of atROL. Palm-CoA (100 μM) was added to each reaction mixture. (B) Retinoids synthesized following 2 min incubations at the indicated concentrations of 11cROL. Palm-CoA (100 μM) was added to each reaction mixture. (C) Retinoids synthesized following 2 min incubations at the indicated concentrations of palm-CoA. atROL (50 μM) was added to each reaction mixture. Data are expressed as initial reaction velocities (V0). The Vmax and KM constants derived from Eadie–Hofstee transformations of these data are presented in Table 1.
Figure 7
Figure 7
Effect of apo-CRBP1 on retinoids synthesized from atROL by chicken retinal homogenates. (A) Chicken retina homogenates were preincubated with 55 μM CRBP1 before addition of 20 μM holo-CRBP1 (atROL substrate with CRBP1). Retinoids were extracted and analyzed by HPLC at the indicated incubation times. (B) Chicken retina homogenates were incubated under conditions similar to those used for panel A without apo-CRBP1 preincubation. Results are shown for each retinoid detected as nanomoles per milligram of protein. Error bars show standard deviations (n = 4). Note the synthesis of 11cROL from atROL with no net synthesis of atREs or 11cREs in panel A.
Figure 8
Figure 8
Hypothesized isomerosynthase and alternate visual cycle in chicken retinas. Schematic drawings of the retinol isomerase and palm-CoA-dependent retinyl ester synthase in chicken retinas. The isomerase catalyzes passive interconversion of atROL and 11cROL. The ester synthase uses palm-CoA as an acyl donor to catalyze formation of an atRE or 11cRE from 11cROL or atROL, respectively. In the isomerosynthase catalytic mode (depicted here as a complex between these enzymes), the 11cROL product of isomerization is directly esterified to drive the formation of 11-cis-retinoids. This results in higher catalytic efficiency than for uncoupled atROL-dependent synthesis of atREs or 11cROL-dependent synthesis of 11cREs.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Arshavsky VY, Lamb TD, Pugh EN. G proteins and phototransduction. Annu Rev Physiol. 2002;64:153–87. - PubMed
    1. Moiseyev G, Crouch RK, Goletz P, Oatis J, Jr, Redmond TM, Ma JX. Retinyl esters are the substrate for isomerohydrolase. Biochemistry. 2003;42:2229–38. - PubMed
    1. Gollapalli DR, Rando RR. All-trans-retinyl esters are the substrates for isomerization in the vertebrate visual cycle. Biochemistry. 2003;42:5809–18. - PubMed
    1. Mata NL, Moghrabi WN, Lee JS, Bui TV, Radu RA, Horwitz J, Travis GH. Rpe65 is a retinyl ester binding protein that presents insoluble substrate to the isomerase in retinal pigment epithelial cells. J Biol Chem. 2004;279:635–43. - PubMed
    1. MacDonald PN, Ong DE. Evidence for a lecithin-retinol acyltransferase activity in the rat small intestine. J Biol Chem. 1988;263:12478–82. - PubMed

Publication types