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S.G. Trevino, S.T. Schuschereba, P.D. Bowman, A. Tsin; Lecithin: Retinol Acyltransferase in ARPE–19 . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1741.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The purpose of this study is to investigate the esterification of retinol and the expression of lecithin:retinol acyltransferase (LRAT) protein in ARPE–19. Methods: ARPE–19 was obtained from ATCC and grown to confluency. Cells were incubated with a 10 µM all–trans retinol, 500 µg/ml fatty acid free BSA, and 0.5% sucrose mixture for up to 24 hours. Extracted retinoids were analyzed by HPLC, quantified against authentic standard curves and identified based on relative retention, coeultion with standards, online photodiode array spectrum and saponification. LRAT activity assays were run on ARPE–19 and bovine RPE membrane protein in the presence and absence of palmitoyl–CoA and retinyl bromoacetate (RBA), a known LRAT inhibitor. Microarray analysis was performed on RNA isolated from ARPE–19. Western analysis with the truncated–LRAT (tLRAT) antibody (gift, Dr. Dean Bok, UCLA) was run on ARPE–19, bovine RPE, and bovine liver protein. Results: ARPE–19 cells did not contain any endogenous levels of retinoids detectable by HPLC. There was a rapid accumulation of intracellular all–trans retinol (∼15 pmol/ µg total protein) and gradual formation of retinyl esters which peaked at ∼6.0 pmol/ µg total protein within the 24 hour incubation period. Saponified retinyl esters showed the retinyl esters were in the all–trans configuration. Substrate saturation analysis using ARPE–19 membrane protein yielded apparent kinetic constants (Km and Vmax) for all–trans LRAT activity of 9.5 µM and 0.838 nmol/min/mg protein, respectively. In contrast to our bovine RPE control, palmitoyl–CoA did not stimulate acyl:retinol acyltransferase activity (ARAT). Addition of RBA to the assay significantly inhibited (78%) LRAT enzyme activity. Microarray expression analysis indicated that the mean fluorescence intensity level of LRAT (198.4 ± 28.3) was about one third of the house keeping gene glyceraldehydes–3–phosphate dehydrogenase (G3PDH) 663 ± 66. Western analyses with tLRAT antibody showed that LRAT protein was present in ARPE–19 at a MW of approximately 25 kD. Conclusions: This is the first study to report the internalization and esterification of retinol in ARPE–19. In addition, we have also characterized LRAT activity in ARPE–19 membrane protein. Since LRAT protein is expressed in ARPE–19 and its activity is inhibited by RBA, we conclude that the esterification of retinol is primarily due to LRAT. Furthermore, since this activity is not enhanced by the addition of CoA, ARPE–19 may not contain ARAT activity.
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