ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 288, No. 2, August 1, pp. 500-508, 1991 Fatty Acyl CoA-Dependent and -Independent Retinol Esterification by Rat Liver and Lactating Mammary Gland Microsomes’ R. Keith Randolph, K. Elise Winkler, and A. Catharine Ross2 Department of Physiology and Biochemistry, Medical College of Pennsylvania, Philadelphia, Pennsylvania 19129 Received February 1, 1991, and in revised form April 2, 1991 Retinol esterification was examined in microsomes from rat liver and lactating mammary gland as a function of the form of retinol substrate, dependence on fatty acyl CoA, and sensitivity to phenylmethylsulfonyl fluoride (PMSF). Retinol bound to cellular retinol-binding protein (CRBP) or dispersed in solvent was esterified in a fatty acyl CoA-independent, PMSF-sensitive reaction, consis- tent with 1ecithin:retinol acyltransferase (LRAT) activ- ity. LRAT activity exhibited the same K, (2 FM retinol) between tissues but a higher V,., in liver as compared to that in mammary gland (47 vs 8 pmol/min/mg micro- some protein, respectively). Solvent-dispersed retinol was also esterified in a fatty acyl CoA-dependent, PMSF- resistant reaction, consistent with acyl CoA:retinol acyl- transferase (ARAT) activity. Retinol bound to CRBP was not a good substrate for this reaction. ARAT activity dis- played a similar V,,, (300 pmol/min/mg microsome pro- tein) between tissues but K, values of 15 and 5 PM for retinol and fatty acyl CoA in mammary gland as com- pared to 30 and 25 PM, respectively, in the liver. Thus, when substrate was near or below K,,,, retinol esterifi- cation occurred predominantly by LRAT in the liver and ARAT in the mammary gland, respectively. The concen- tration of CRBP in the cytosol, determined by Western blotting, was approximately 2 PM in the liver but was almost nondetectable in the mammary gland. These data suggest that retinol esterification is regulated via different mechanisms in liver and mammary gland and support a specific role for CRBP in the liver. o ISSI Academic Press, Inc. i This work was supported by National Institutes of Health Grants HD-16484, HL-22633, HL-07443 and The Howard Heinz Endowment. * To whom correspondence and reprint requests should be addressed at: Department of Physiology and Biochemistry, Medical College of Pennsylvania, 3300 Henry Avenue, Philadelphia, PA 19129. FAX: (215) 843-8849. 500 Vitamin A is found in several body organs primarily in the form of fatty acid esters of retinol which fulfill either storage or transport functions. Following hydrolysis of stored retinyl esters in the liver, retinol is transported to other body tissues following its secretion into the circu- lation bound to retinol-binding protein (RBP)3 (1). Ret- inyl esters in the intestine and lactating mammary gland, on the other hand, are transported in their esterified form along with other neutral lipids in lymph chylomicra (2) or milk lipid droplets (3), respectively. In vivo and in vitro studies are consistent with the hy- pothesis that the synthesis of these retinyl esters occurs by different mechanisms in different tissues. For example, the composition of liver retinyl ester stores, which consist almost exclusively of retinyl palmitate and retinyl stearate (4), varies little with type of dietary fat as compared to the fatty acid composition of total liver lipids (5, 6). The retinyl esters which are transported in the milk also in- clude retinyl palmitate and stearate but also esters of long chain unsaturated and medium chain fatty acids (7). In addition, and in contrast to the liver, the retinyl esters found in milk are influenced significantly by the com- position of dietary fat (7). The physiological and bio- chemical bases for these differences are not known. To date, two retinol esterifying activities, ARAT and LRAT, have been described in the microsomes from nu- merous body organs and have been distinguished from each other by several criteria. The reaction catalyzed by ARAT was initially described in the liver (8) and has sub- sequently been reported in lactating mammary gland (9), small intestine (lo), skin (ll), lacrimal gland (12), and testis (13). Retinol esterification catalyzed by ARAT is 3 Abbreviations used: RBP, retinol-binding protein; CRBP, cellular retinol-binding protein; LRAT, 1ecithin:retinol acyltransferase; ARAT, acyl CoA:retinol acyltransferase; BSA, bovine serum albumin; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; HPLC, high per- formance liquid chromatography. 0003.9861/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.