This chapter presents the method to measure the reaction rate of the acyl-CoA-stimulated esterification of retinol presented to microsomes in dispersed form. This enzymatic reaction is referred to as acyl-CoA: retinol acyltransferase, or ARAT. The rate of retinol esterification is determined under conditions that optimize pH and in which product formation is linear with incubation time and amount of microsomal protein (enzyme). It has been found it most convenient to utilize radiolabeled retinol of known specific radioactivity and to separate the labeled retinyl ester product from remaining labeled retinol on small columns of alumina oxide. It is also possible to separate product from reactants by high-performance liquid chromatography (HPLC) and thus to utilize either radiolabeled or unlabeled substrate or, additionally, to characterize the species of esters formed. Generally, both the retinol and fatty acyl-CoA substrates are present at or near saturating concentrations. Palmitoyl-CoA has been routinely used as the activated fatty acid because retinyl palmitate predominates among most tissue retinyl esters.
All Science Journal Classification (ASJC) codes
- Molecular Biology