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

A C Ross

doi:10.1016/s0021-9258(18)34945-7

Abstract

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 microM) 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.

Highlights

To explore the nature of retinyl ester synthesis b y Despite interest in the relationship of vitamin A status to liver microsomes, membranesprepared from rat o r cat hepatic content, only a few studies have addressed the enzyliver were incubatuendder various conditionswith [’H] mology of vitamin A esterification in liver or in other target retinol dispersed in dimethyl sulfoxide
To determine whether the fatty acid coenzyme A [2,12] and in experiments with microsomes from used toesterify [3H]retinol under basal conditions might be derived from an endogenous poool f fatty acylCoA associated with the microsomal preparation, microsomes werepretreated at pH 7.4 with 0.5 M hydroxylamine, a reagent that reacts with coenzyme A thioesbovine retina, it was noted that addition of preformed palmitoyl-CoA did not increase retinyl ester yield [12]
Requirements for Retinyl Ester Synthesis-Liver microsomes were incubated with r3H]retinol and various additions to determine what exogenous substrates or supplements are needed for retinyl estesrynthesis

Summary

Introduction

To explore the nature of retinyl ester synthesis b y Despite interest in the relationship of vitamin A status to liver microsomes, membranesprepared from rat o r cat hepatic content, only a few studies have addressed the enzyliver were incubatuendder various conditionswith [’H] mology of vitamin A esterification in liver or in other target retinol dispersed in dimethyl sulfoxide. When micro- lactating mammary gland of the rat, I found that the rateof somes were incubated with both C3H]retinol and [“C] esterification of retinol by microsomeswas markedly enpalmitoyl-CoA of known specificradioactivities, the hanced after inclusion of ATP and reduced coenzyme A or ratio of 14Ct o ‘H in newly synthesized retinyl palmitate fatty acyl-CoA esters [34].These observations have prompted was essentiallyequal t o that of its putative substrates, a reinvestigationof retinyl ester synthesis by liver microsomes. Indicating that [14C]palmitate did not undergo signifi- I report here evidence that microsomes prepared from livers cant isotope dilution prior to acylation of [3H]retinoL of the cat and rat catalyze the esterification of retinol by a. These experiments provide direct evidence for retinol fatty acyl-CoA:retinol acyltransferase reaction. These experiments provide direct evidence for retinol fatty acyl-CoA:retinol acyltransferase reaction. esterification catalyzedby a microsomal acyl-CoA:retinol acyltransferaseand indirect evidence fora pool of MATERIALS AND METHODS

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Conclusion