COLOCALIZATION OF 11-CIS RETINYL ESTERS AND RETINYL ESTER HYDROLASE ACTIVITY IN RETINAL-PIGMENT EPITHELIUM PLASMA-MEMBRANE

PURPOSE. To identify the subcellular locale of 11-cis retinyl eaters i n bovine retinal pigment epithelium (RPE) and to characterize the enzy mic mechanism responsible for liberation of 11-cis retinoids in this c ompartment. METHODS. Endoplasmic reticulum (ER)-enriched and plasma me mbrane (PM)-enriched protein fractions were prepared from bovine RPE m icrosomes using sequential discontinuous sucrose and Percoll gradient fractionation. Enzyme markers for ER (such as carboxylesterase), and P M (such as 5'-nucleotidase [5'-ND]; alkaline phosphatase [AP]; and oua bain-sensitive Na+,K+-ATPase [ATPase]) were used to identify the subfr actions. Membrane-associated retinoids were quantified by high-perform ance liquid chromatography (HPLC) and retinyl ester hydrolase (REH) ac tivities were determined by radiometric and chromatographic (HPLC) mea ns. RESULTS. Chromatographic analyses of membrane-associated retinoids showed that 11-cis retinyl esters are localized mainly in PM-enriched fractions, whereas all-trans retinyl esters are associated predominan tly with ER-enriched membranes; profiles of the distribution of 11-cis - and all-trans REH activities were consistent with the retinyl ester distribution. Further purification of the crude PM fraction yielded a fraction (P2) that was significantly enriched with 5'-ND (fivefold), A TPase (15-fold), AP (10-fold), and 11-cis retinyl ester hydrolase (11- cis REH; threefold) activities, but was relatively devoid of carboxyle sterase and all-trans REH activities. Apparent kinetic constants (K-m( app) and V-m(app)) for 11-cis REH activity in P2 were 18 mu M and 1800 picomoles/min per mg, respectively. CONCLUSIONS. This is the first id entification of an 11-cis-specific REH activity in RPE plasma membrane . Results from these studies demonstrate the capacity of RPE plasma me mbranes to accommodate and hydrolyze 11-cis retinyl esters. Plasma mem brane storage and mobilization of 11-cis retinyl esters represents a n ovel compartmentalization of retinoid metabolism that is distinct from the sites where 11-cis retinoids are produced. The implication of the se findings for present theories of visual chromophore biosynthesis ar e discussed.