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Biochim Biophys Acta. 2014 Jun;1844(6):1128-36. doi: 10.1016/j.bbapap.2014.02.022. Epub 2014 Mar 5.

Enzymatic activity of Lecithin:retinol acyltransferase: a thermostable and highly active enzyme with a likely mode of interfacial activation.

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  • 1CUO-Recherche, Hôpital du Saint-Sacrement, Centre de Recherche du CHU de Québec, Québec, Québec, Canada; Département d'Ophtalmologie, Faculté de Médecine, Université Laval, Québec, Québec, Canada; Regroupement Stratégique PROTEO, Université Laval, Québec, Québec, Canada.
  • 2CUO-Recherche, Hôpital du Saint-Sacrement, Centre de Recherche du CHU de Québec, Québec, Québec, Canada; Département d'Ophtalmologie, Faculté de Médecine, Université Laval, Québec, Québec, Canada; Regroupement Stratégique PROTEO, Université Laval, Québec, Québec, Canada. Electronic address: christian.salesse@fmed.ulaval.ca.

Abstract

Lecithin:retinol acyltransferase (LRAT) plays a major role in the vertebrate visual cycle. Indeed, it is responsible for the esterification of all-trans retinol into all-trans retinyl esters, which can then be stored in microsomes or further metabolized to produce the chromophore of rhodopsin. In the present study, a detailed characterization of the enzymatic properties of truncated LRAT (tLRAT) has been achieved using in vitro assay conditions. A much larger tLRAT activity has been obtained compared to previous reports and to an enzyme with a similar activity. In addition, tLRAT is able to hydrolyze phospholipids bearing different chain lengths with a preference for micellar aggregated substrates. It therefore presents an interfacial activation property, which is typical of classical phospholipases. Furthermore, given that stability is a very important quality of an enzyme, the influence of different parameters on the activity and stability of tLRAT has thus been studied in detail. For example, storage buffer has a strong effect on tLRAT activity and high enzyme stability has been observed at room temperature. The thermostability of tLRAT has also been investigated using circular dichroism and infrared spectroscopy. A decrease in the activity of tLRAT was observed beyond 70°C, accompanied by a modification of its secondary structure, i.e. a decrease of its α-helical content and the appearance of unordered structures and aggregated β-sheets. Nevertheless, residual activity could still be observed after heating tLRAT up to 100°C. The results of this study highly improved our understanding of this enzyme.

Copyright © 2014 Elsevier B.V. All rights reserved.

KEYWORDS:

Enzymatic activity; Lecithin:retinol acyltransferase; Retinol; Thermostability; Truncated LRAT; Visual cycle

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