Hydrolysis of 11-cis- and all-trans-retinyl palmitate by retinal pigment epithelium microsomes.
A partial characterization of the enzymatic hydrolysis of 11-cis- and all-trans-retinyl palmitate by bovine retinal pigment epithelium microsomes was carried out using a micro-radiometric method to quantitate liberated palmitic acid. Retinyl ester hydrolase (REH) activity was examined in the absence of detergent. Hydrolysis of 11-cis- and all-trans-retinyl palmitate was protein- and time-dependent. Optimal enzyme activity occurred at slightly alkaline pH (8-9). Apparent kinetic constants (Vmax and Km) for the 11-cis-REH were 2.1 nmol/min/mg protein and 66 microM, respectively. All-trans-REH demonstrated a lower maximum velocity of 0.3 nmol/min/mg protein and a slightly higher substrate affinity of 27 microM. Further characterization of 11-cis-retinyl palmitate hydrolysis involved monitoring formation of reaction products, 11-cis retinol and palmitic acid, which were found to be released in essentially a 1:1 stoichiometry. Addition of all-trans retinyl bromoacetate, a known inhibitor of lecithin:retinol acyltransferase reduced both 11-cis and all-trans-REH activities but to significantly different degrees (50 and 76%, respectively). Although the microsomal preparation exhibited LRAT activity, acyl transfer was not readily reversible as labeled palmitic acid was not transferred to added acyl acceptor compounds. These findings suggest that hydrolysis of 11-cis-retinyl palmitate by bovine retinal pigment epithelium microsomes may occur at a catalytic site distinct from that for the all-trans isomer and that this hydrolysis is not representative of a reverse transesterification reaction.
Mata
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Division of Life Sciences, University of Texas, San Antonio 78249.
Tsin
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Chambers
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JP
D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D002417:Cattle; D004224:Diterpenes; D006868:Hydrolysis; D007536:Isomerism; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism*; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D000084562:Retinyl Esters; D013379:Substrate Specificity; D014801:Vitamin A / Q000031:analogs & derivatives* / Q000378:metabolism
D004224:Diterpenes; D010169:Palmitic Acids; D000084562:Retinyl Esters; D014801:Vitamin A; C014794:retinol palmitate; D019308:Palmitic Acid; D002265:Carboxylic Ester Hydrolases; C022523:retinyl esterase