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M. Jin, C.H. Kaschula, N.S. Desmond–Smith, S. Li, G.H. Travis; Acyl CoA:Retinol Acyltransferase (ARAT) Activity in Bovine Retinal Pigment Epithelium . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1746.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Absorption of a photon by a rhodopsin pigment induces 11–cis to all–trans isomerization of its retinaldehyde chromophore. Restoration of light sensitivity to the pigment requires chemical re–isomerization of retinaldehyde, which takes place in the retinal pigment epithelium (RPE). Fatty–acyl esters of all–trans–retinol (atROL) are substrates for the isomerase in RPE. Two retinyl–ester synthases have been described: lecithin:retinol acyltransferase (LRAT), which uses phosphatidylcholine as an acyl donor, and acyl CoA:retinol acyltransferase (ARAT), which uses palmitoyl coenzyme A (palm CoA). The presence of LRAT in RPE has been well documented. It is unclear whether ARAT, which has never been purified or cloned, is expressed in RPE. Here, we show that ARAT is present in RPE. Methods: As a negative control for ARAT, we generated 293T cells that stably express bovine LRAT (LRAT–293T cells). We assayed membranes from LRAT–293T cells, mouse liver, and bovine RPE for the conversion of atROL to all–trans–retinyl esters (atRE’s) in the presence or absence of [3H]–labeled palm CoA, using normal–phase high performance liquid chromatography (HPLC). Results: Membranes from all three cell types catalyzed palm CoA–independent synthesis of atRE’s (LRAT). We observed no stimulation of atRE synthesis with addition of [3H]–palm CoA to LRAT–293T membranes. In contrast, we observed two– to three–fold stimulation of atRE synthesis with addition of [3H]–palm CoA to membranes from mouse liver and bovine RPE. In both cases, the atRE’s incorporated significant [3H]–palmitate from the [3H]–palm CoA. We measured the rates of atRE synthesis by membranes from the three tissues at different substrate concentrations, which allowed us to calculate the kinetic parameters, KM and Vmax of LRAT and ARAT in bovine RPE for atROL. The KM of LRAT was 2.0 µM versus 10.4 µM for ARAT. The Vmax of LRAT was 7.0 nm/min/mg versus 13.0 nm/min/mg for ARAT in the same membrane sample. We confirmed our identification of ARAT by showing complete inhibition of this activity, but not LRAT, with addition of progesterone. Both activities were potently inhibited by all–trans–retinylbromoacetate. Conclusions: Greater than 50% of the total retinyl–ester synthase activity in RPE is due to ARAT. The presence of only ‘trace’ atRE’s in lrat –/– knockout tissues is because the KM of ARAT is 5–10–fold higher than the concentration of atROL in blood. Thus, LRAT functions to ‘capture’ atROL from blood, while ARAT is active at high cellular concentrations of atROL, such as following a large photobleach of rhodopsin.
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