title: Colocalization of 11-cis retinyl esters and retinyl ester hydrolase activity in retinal pigment epithelium plasma membrane.
abstract: OBJECTIVE
  To identify the subcellular locale of 11-cis retinyl esters in bovine retinal pigment epithelium (RPE) and to characterize the enzymic mechanism responsible for liberation of 11-cis retinoids in this compartment.
  
  
  METHODS
  Endoplasmic reticulum (ER)- enriched and plasma membrane (PM)-enriched protein fractions were prepared from bovine RPE microsomes using sequential discontinuous sucrose and Percoll gradient fractionation. Enzyme markers for ER (such as carboxylesterase), and PM (such as 5'-nucleotidase [5'-ND]; alkaline phosphatase [AP]; and ouabain-sensitive Na+,K+-ATPase [ATPase]) were used to identify the subfractions. Membrane-associated retinoids were quantified by high-performance liquid chromatography (HPLC) and retinyl ester hydrolase (REH) activities were determined by radiometric and chromatographic (HPLC) means.
  
  
  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 predominantly 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), ATPase (15-fold), AP (10-fold), and 11-cis retinyl ester hydrolase (11-cis REH; threefold) activities, but was relatively devoid of carboxylesterase and all-trans REH activities. Apparent kinetic constants (Km(app) and Vm(app)) for 11-cis REH activity in P2 were 18 microM and 1800 picomoles/min per mg, respectively.
  
  
  CONCLUSIONS
  This is the first identification of an 11-cis-specific REH activity in RPE plasma membrane. Results from these studies demonstrate the capacity of RPE plasma membranes to accommodate and hydrolyze 11-cis retinyl esters. Plasma membrane storage and mobilization of 11-cis retinyl esters represents a novel compartmentalization of retinoid metabolism that is distinct from the sites where 11-cis retinoids are produced. The implication of these findings for present theories of visual chromophore biosynthesis are discussed.
authors.affiliation: Division of Life Sciences, The University of Texas at San Antonio, 78249-0662, USA.
authors.forename: N L
authors.initials: NL
authors.lastname: Mata
authors.forename: E T
authors.initials: ET
authors.lastname: Villazana
authors.forename: A T
authors.initials: AT
authors.lastname: Tsin
mesh_terms: D015720:5'-Nucleotidase / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015415:Biomarkers; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002851:Chromatography, High Pressure Liquid; D004224:Diterpenes; D004721:Endoplasmic Reticulum / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D012176:Retinoids / Q000378:metabolism*; D000084562:Retinyl Esters; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013347:Subcellular Fractions; D014801:Vitamin A / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D015415:Biomarkers; D004224:Diterpenes; D012176:Retinoids; D000084562:Retinyl Esters; D014801:Vitamin A; C014794:retinol palmitate; D002265:Carboxylic Ester Hydrolases; C022523:retinyl esterase; D043182:Carboxylesterase; D000469:Alkaline Phosphatase; D015720:5'-Nucleotidase; D000254:Sodium-Potassium-Exchanging ATPase