Solubilization and partial purification of retinyl ester synthetase and retinoid isomerase from bovine ocular pigment epithelium.

R J Barry, F J Cañada, R R Rando

Index: J. Biol. Chem. 264 , 9231, (1989)

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Abstract

Studies reported previously from this laboratory have demonstrated that membranes from the pigment epithelium of the vertebrate eye can transform free all-trans-retinol to 11-cis-retinol as well as 11-cis- and all trans-retinyl esters (Bernstein, P. S., Law, W. C., and Rando, R. R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1849-1853; Bernstein, P. S., Law, W. C., and Rando, R. R. (1987) J. Biol. Chem. 262, 16848-16857; Fulton, B. S., and Rando, R. R. (1987) Biochemistry 26, 7938-7945). The congeneric retinals are also formed under conditions where retinol redox activity is present. Here we report the successful solubilization of both the retinyl ester synthetase and isomerase activities from the pigment epithelium membranes of the bovine eye. The zwitterionic detergent Zwittergent 3-14(N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate; cmc 0.012%) gave optimal solubilization of both activities. Three initial criteria for successful solubilization were used. First, high speed centrifugation (greater than 150,000 x g) left the activities in the supernatant. Second, the solubilized enzymatic activities were found in the included volume upon gel filtration. Finally, the solubilized activities were quantitatively passed through a 0.22-microns filter. Employing anion exchange and gel filtration chromatography results in a partial purification of the retinyl ester synthetase (approximately 189-fold). The solubilized retinoid isomerase is also partially purified (approximately 10-14-fold) following anion exchange chromatography. It is also shown that the membrane-bound and solubilized ester synthetase catalyzes the esterification of retinol using added lecithins as exogenous acyl donors. In addition, evidence is provided indicating that there is a positional selectivity for the acyl group transfer from the lecithin to retinol. The transfer occurs largely, if not entirely, from the 1-position of the lecithin.