Retinol esterification by rat liver microsomes. Evidence for a fatty acyl coenzyme A: Retinol acyltransferase

To explore the nature of retinyl ester synthesis by liver microsomes, membranes prepared from rat or cat liver were incubated under various conditions with [³H] retinol dispersed in dimethyl sulfoxide. When [³H]retinol, buffer, and microsomes were incubated together (basal conditions), some [³H]retinol esterification was consistently observed. However, the rate of esterification could be increased 6- to 11-fold by addition of either palmitoyl-CoA (100 μM) or a fatty acyl CoA-generating system. To determine whether the fatty acid used to esterify [³H]retinol under basal conditions might be derived from an endogenous pool of fatty acyl-CoA associated with the microsomal preparation, microsomes were pretreated at pH 7.4 with 0.5 M hydroxylamine, a reagent that reacts with coenzyme A thioesters to form hydroxamates. This pretreatment reduced the basal reaction by 69%. However, hydroxylamine-treated microsomes still retained acyltransferase activity, as shown by a 24- to 40-fold increase in retinyl ester synthesis after addition of palmitoyl-CoA. When microsomes were incubated with both [³H]retinol and [¹⁴C]palmitoyl-CoA of known specific radioactivities, the ratio of ¹⁴C to ³H in newly synthesized retinyl palmitate was essentially equal to that of its putative substrates, indicating that [¹⁴C]palmitate did not undergo significant isotope dilution prior to acylation of [³H]retinol. These experiments provide direct evidence for retinol esterification catalyzed by a microsomal acyl-CoA:retinol acyltransferase and indirect evidence for a pool of fatty acyl-CoA in isolated liver microsomes that is available to react with [³H]retinol to form esterified retinol.