490
ENZYMOLOGY AND METABOLISM
[55] Assay
[55]
of Liver Retinyl Ester Hydrolase By DALE A. COO.~R
Introduction The hydrolysis of long-chain fatty acyl esters of retinol is an important event in the release of stored hepatic vitamin A. Two widely used methods for the assay of retinyl ester hydrolase are based on the observation that liver preparations catalyze this reaction in the presence of added trihydroxy bile salts. ~ One of these methods (Method 1), developed by Prystowsky et al.,2 is based on stimulation of activity with sodium cholate and the measurement of the release of [~4C]palmitic acid from retinyl [14C]palmitate. A second method (Method 2), developed by Cooper et al., a,4 utilizes a 3-[(3-cholamidopropyl)dimethylammonio]propane sulfonate (CHAPS)/Triton X-100 detergent system and high-performance liquid chromatography (HPLC) to measure the production of unlabeled retinol. The protocols for these assays and some of the enzymatic properties of retinyl ester hydrolase are presented in this chapter. Method 1 Tissue and Substrate Preparation. Originally designed to measure rat liver retinyl ester hydrolase activity, this procedure has also been used with rat kidney, intestine, testes, and lung, 5 and chick liver. 6 Homogenates are prepared by mincing rinsed liver in 3 or more volumes (w/v) of ice-cold 50 mM Tris-maleate (pH 8.0), followed by homogenization with several strokes of a motor-driven mortar and pestle (Thomas Scientific, Philadelphia, PA) in an ice-water bath. Liver homogenates can be frozen and stored at - 2 0 ° for at least 1 year without loss of activity. Liver homogenates that contain high concentrations of retinyl esters must be diluted so that the amount of hepatic retinyl esters added to the i S. Mahadevan, N. I. Ayyoub, and O. A. Roels, J. Biol. Chem. 241, 57 (1966). J. H. Prystowsky, J. E. Smith, and D. S. Goodman, J. Biol. Chem. 256, 4498 (1981). 3 D. A. Cooper and J. A. Olson, Biochim. Biophys. Acta 884, 251 (1986). 4 D. A. Cooper, H. C. Furr, and J. A. Olson, J. Nutr. 117, 2066 (1987). 5 j. L. Napoli, A. M. McCormick, B. O'Meara, and E. A. Dratz, Arch. Biochem. Biophys. 230, 194 (1984). 6 D. Sklan and S. Donoghue, Biochim. Biophys. Acta 711, 532 (1982).
METHODS IN ENZYMOLOGY,VOL. 189
Copyright© 1990by AcademicPress, Inc. All rightsof reproductionin any formreserved.
[55]
ASSAY OF LIVER RETINYL ESTER HYDROLASE
491
assay mix does not affect the measured enzyme activity by changing the concentration and specific activity of the substrate. This condition is met if the homogenate contributes less than a few percent of the retinyl esters in the assay mix. Also, dilution may be necessary to ensure that the amount of protein added (microgram quantities) is linearly proportional to the reaction rate. Retinyl [1-14C]palmitate is prepared by the reaction of the symmetric anhydride of [1-14C]palmitic acid (New England Nuclear, Boston, MA) with all-trans-retinol (Eastman Kodak, Rochester, NY). 2 The procedures for the preparation of the anhydride 7 and of retinyl palmitate are too lengthy to be presented here, but they are identical to published procedures used in the synthesis of cholesteryl esters. 8 Assay Procedure and Activity Calculations. The reaction mixture is prepared in a screw-top glass test tube (16 × 100 mm) and consists of 100 /zl of 0.1 M Tris-maleate, pH 8.0, 40/zl of sodium cholate (1.3 mg, Sigma, St. Louis, MO), 50/zl of enzyme-containing sample, and 10/~1 of retinyl [1-14C]palmitate in ethanol (2 nmol and -0.05/zCi or 100,000 cpm). Tubes are incubated in a 37° water bath for 15-30 min. The reaction is quenched by the addition of 3.25 ml of methanol/chloroform/heptane (1.41:1.25:1.00, v : v : v ) containing 0.1 mM palmitic acid (Sigma) as a carrier (organic solvents used in Methods 1 and 2 are purchased from Fisher, Minneapolis, MN). The liquid phases are separated by centrifugation at 1000 rpm in a clinical centrifuge at room temperature for 15 min. Radioactivity of free fatty acids is determined by liquid scintillation spectrometry of I ml of the upper phase after addition to 10 ml of Scintiverse I (Fisher). To calculate retinyl ester hydrolase activity, the radioactivity in test assays given in counts per minute (cpm) is corrected for radioactivity in an assay mixture from which the enzyme has been omitted. The corrected counts are multiplied by 2.45 to account for the total volume of the upper phase, and the product is divided by the partition coefficient of palmitic acid. The partition coefficient is that portion of palmitic acid extracted from a tube containing an enzyme-free reaction mixture to which 10/zl of 200 /zM [1-14C]palmitic acid in ethanol has been added. The partition coefficient for palmitic acid in this solvent system is approximately 0.70. Level of Activity and Enzymatic Properties. Rat liver retinyl ester hydrolase activity ranges from 5 to 250 pmol/min/mg; this range is larger when portions of liver are assayed. 2,9 In spite of this variability, it is well 7 Z. Selinger and Y. Lapidot, J. Lipid Res. 7, 174 (1966). 8 B. R. Lentz, Y. Barenholz, and T. E. Thompson, Chem. Phys. Lipids 15, 216 (1975). 9 W. S. Blaner, J. E. Smith, R. B. Dell, and D. S. Goodman, J. Nutr. 115, 856 (1985).
492
ENZYMOLOGY AND METABOLISM
[55]
established that the assay gives consistent activity for a given enzyme preparation, zl° The apparent Km and Vmaxvalues of an approximately 200-fold purified preparation of retinyl ester hydrolase are 11.2/xM and 141 nmol/min/mg, respectively. ~1The most potent inhibitors of hydrolase activity, with concentrations giving 50% inhibition, include the following: ether analogs of cholesterol esters [especially the linoleyl ether (0.16/~M)],12 ether analogs of triglycerides (>10 txM), 12 phylloquinone (20 txM), 5 and a-tocopherol (100 izM). 5
Method 2 Tissue and Substrate Preparation. This procedure has been used with pig liver and kidney 3 and rat liver4; however, pig and rat liver hydrolases differ with regard to the substrate and CHAPS concentrations and pH giving maximal activity. The differences are noted below. Tissue homogenates are prepared as described for Method 1. Homogenates are diluted so that the amount of homogenate protein added to the reaction mixture is less than 1 mg for pig liver or 750/xg for rat liver; usually 200-300/xg is sufficient. Under these conditions, the amount of liver-derived vitamin A added to the assay mix is negligible when the liver reserves are less than 200/.~g/g (higher levels have not been tested). For each 1 ml of substrate solution, 2.63 mg (for pig assay) or 26.3 mg (for rat assay) of retinyl palmitate (Sigma) is dissolved in hexanes, and the concentration is verified by measuring the absorption at 325 nm of a known dilution. An appropriate volume of this solution is added to a screw-top test tube, and the hexanes are evaporated under a stream of argon. The residue is dissolved in 20 mg Triton X-100 (Sigma) per milliliter of assay buffer, a small stir bar is added, and the solution is stirred vigorously. The substrate solution can be used up to 2 days later when stored in the dark at 4° in an argon-flushed container. A surface layer of retinyl ester may form during storage but the solution will be homogeneous if it is warmed to room temperature and stirred prior to use. Assay Procedure and Actioity Calculations. Assays are carried out in 12 x 75 mm glass tubes with caps. For the pig enzyme, 250/zl of 200 mM CHAPS (Sigma) in 50 mM Tris-maleate, pH 8.0, is combined with 50/zl i0 E. H. Harrison, J. E. Smith, and D. S. Goodman, J. Lipid Res. 20, 760 (1979). N W. S. Blaner, J. H. Prystowsky, J. E. Smith, and D. S. Goodman, Biochirn. Biophys. Acta 794, 419 (1984). t2 W. S. Blaner, G. Halperin, O. Stein, Y. Stein, and D. S. Goodman, Biochim. Biophys. Acta 794, 428 (1984).
[55]
ASSAY OF LIVER RETINYL ESTER HYDROLASE
493
of 5 mM retinyl palmitate suspension. After a 5-min preincubation at 37°, 50 /zl of homogenate is added to initiate the reaction. The mixture is vortexed gently and incubated for 30-60 rain in a Dubnoff shaking incubator at 25 strokes/min. The assay mix for the rat enzyme is composed of 350/xl of 393 mM CHAPS in 100 mM Tris-maleate, pH 7.0, 50/zl of 50 mM retinyl palmitate suspension, and 50 ~1 of liver homogenate. Incubation is for 30-45 rain. Reaction mixtures containing over 900/zM retinyl palmitate may be turbid; however, this does not affect the assay. To stop the hydrolase reaction, 500/zl absolute ethanol is added and the solution is vortexed. Next, the assay mix is extracted twice with 2 ml of hexanes containing 0.1% butylated hydroxytoluene (BHT) (Sigma). The combined hexane extracts are dried under a gentle stream of argon, and the residue is redissolved in 500/~1 2-propanol and stored in 1.5-ml Eppendorf tubes at - 2 0 °. Retinol can be analyzed by using any HPLC method that separates retinol from contaminating peaks. A proven method is based on chromatography with the following equipment: a 4.6 x 250 mm Partisil 10/25 ODS-2 column (Whatman, Clifton, N J), an Uptight guard column (Upchurch Scientific, Oak Harbor, WA) filled with pellicular octadecylsilane (Vydac, The Separation Group, Hesperia, CA), an LDC Constametric III pump (Riviera Beach, FL), an ISCO variable absorbance detector (Lincoln, NE), and a Shimadzu C-R3A integrator (Kyoto, Japan). A Waters Associates (Milford, MA) WISP Model 710 HPLC autosampler can be used to process multiple samples. After every second or third injection of 25/zl, the column should be flushed with 1 : 1 methanol/tetrahydrofuran (v/v) to elute retinyl esters, all-trans-retinol is eluted in 7.3 min with 9 : 1 (v/v) methanol/water pumped at a flow rate of 2 ml/min. The ultraviolet absorbance (325 nm) of the column effluent is monitored, and retinol is quantitated by comparison of peak area to that of a standard concentration curve. Retinyl ester hydrolase activity is calculated from the amount of retinol produced during the assay corrected for retinol formed in control incubations containing all assay components except the homogenate. If liver homogenates contain high levels of retinol, correction should be made for retinol extracted from unincubated assay mixtures (minus retinyl palmitate). This assay will detect the production of as little as 4 pmol retinol. The interassay variability is 4.7%. Level of Activity and Enzymatic Properties. Liver retinyl ester hydrolase activity in 24 pigs 3 was 13-34 pmol/min/mg. The enzyme has an apparent Km of 140 /xM. 3 Other excellent substrates and their rates of hydrolysis relative to all-trans-retinyl palmitate are as follows: the linolenic (2.1), myristic (2.1), oleic (0.8), linoleic (0.7), and stearic (0.6) esters
494
ENZYMOLOGY AND METABOLISM
[56]
of retinol as well as the 9-cis (2.4), 13-cis (5.7), and 9,13-cis (6.8) isomers of retinyl palmitate.~3 Rat liver retinyl ester hydrolase activity in 25 rats was 100-500 pmol/ min/mg and had an apparent Km of 1.3 mM. 4 In comparison to equimolar sodium cholate, CHAPS gives 9.3 times higher activity on average and reduced interanimal variability/Ethanol (0.01 to 0.5 M) stimulates hydrolase activity 20 to 86%.14 Conclusions
Although the methods described in this chapter have not been compared side by side, they have similar ease of use, sensitivity, and reproducibility. The assays differ largely with respect to composition, methodology, and the interanimal variability in activity they give. 13 D. A. Cooper and J. A. Olson, Arch. Biochem. Biophys. 2611, 705 (1988). 14 H. Friedman, S. Mobarhan, J. Hupert, D. Lucchesi, C. Henderson, P. Langenberg, and T. J. Layden, Arch. Biochem. Biophys. 269, 69 (1989).
[56] A s s a y o f t h e R e t i n o i d I s o m e r a s e S y s t e m o f t h e E y e
By PAUL S. BERNSTEIN and ROBERT R. RANDO Introduction The all-trans- to 11-cis-retinoid isomerase is the key enzyme of the vertebrate visual cycle. It is responsible for the endergonic isomerization of vitamin A compounds from the all-trans isomeric form to the higher energy 11-cis configuration essential for rhodopsin regeneration in the eye. The existence of an enzyme of this type was postulated in the early 1950s by Wald and Hubbard when the vertebrate visual cycle was first discussed. ~The reproducible demonstration of the existence of an isomerase was not achieved until recently, however, requiring the use of high specific-activity radioactive retinoid substrates and high-performance liquid chromatographic (HPLC) analysis. 2 Although the isomerase has not yet been completely purified, a number of its properties are well established: (1) the isomerase is localized to R. Hubbard and G. Wald, J. Gen. Physiol. 36, 269 (1952). 2 p. S. Bernstein, W. C. Law, and R. R. Rando, Proc. Natl. Acad. Sci. U.S.A. 84, 1849 (1987).
METHODS IN ENZYMOLOGY, VOL. 189
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
ENZYMOLOGY AND METABOLISM
[55] Assay
[55]
of Liver Retinyl Ester Hydrolase By DALE A. COO.~R
Introduction The hydrolysis of long-chain fatty acyl esters of retinol is an important event in the release of stored hepatic vitamin A. Two widely used methods for the assay of retinyl ester hydrolase are based on the observation that liver preparations catalyze this reaction in the presence of added trihydroxy bile salts. ~ One of these methods (Method 1), developed by Prystowsky et al.,2 is based on stimulation of activity with sodium cholate and the measurement of the release of [~4C]palmitic acid from retinyl [14C]palmitate. A second method (Method 2), developed by Cooper et al., a,4 utilizes a 3-[(3-cholamidopropyl)dimethylammonio]propane sulfonate (CHAPS)/Triton X-100 detergent system and high-performance liquid chromatography (HPLC) to measure the production of unlabeled retinol. The protocols for these assays and some of the enzymatic properties of retinyl ester hydrolase are presented in this chapter. Method 1 Tissue and Substrate Preparation. Originally designed to measure rat liver retinyl ester hydrolase activity, this procedure has also been used with rat kidney, intestine, testes, and lung, 5 and chick liver. 6 Homogenates are prepared by mincing rinsed liver in 3 or more volumes (w/v) of ice-cold 50 mM Tris-maleate (pH 8.0), followed by homogenization with several strokes of a motor-driven mortar and pestle (Thomas Scientific, Philadelphia, PA) in an ice-water bath. Liver homogenates can be frozen and stored at - 2 0 ° for at least 1 year without loss of activity. Liver homogenates that contain high concentrations of retinyl esters must be diluted so that the amount of hepatic retinyl esters added to the i S. Mahadevan, N. I. Ayyoub, and O. A. Roels, J. Biol. Chem. 241, 57 (1966). J. H. Prystowsky, J. E. Smith, and D. S. Goodman, J. Biol. Chem. 256, 4498 (1981). 3 D. A. Cooper and J. A. Olson, Biochim. Biophys. Acta 884, 251 (1986). 4 D. A. Cooper, H. C. Furr, and J. A. Olson, J. Nutr. 117, 2066 (1987). 5 j. L. Napoli, A. M. McCormick, B. O'Meara, and E. A. Dratz, Arch. Biochem. Biophys. 230, 194 (1984). 6 D. Sklan and S. Donoghue, Biochim. Biophys. Acta 711, 532 (1982).
METHODS IN ENZYMOLOGY,VOL. 189
Copyright© 1990by AcademicPress, Inc. All rightsof reproductionin any formreserved.
[55]
ASSAY OF LIVER RETINYL ESTER HYDROLASE
491
assay mix does not affect the measured enzyme activity by changing the concentration and specific activity of the substrate. This condition is met if the homogenate contributes less than a few percent of the retinyl esters in the assay mix. Also, dilution may be necessary to ensure that the amount of protein added (microgram quantities) is linearly proportional to the reaction rate. Retinyl [1-14C]palmitate is prepared by the reaction of the symmetric anhydride of [1-14C]palmitic acid (New England Nuclear, Boston, MA) with all-trans-retinol (Eastman Kodak, Rochester, NY). 2 The procedures for the preparation of the anhydride 7 and of retinyl palmitate are too lengthy to be presented here, but they are identical to published procedures used in the synthesis of cholesteryl esters. 8 Assay Procedure and Activity Calculations. The reaction mixture is prepared in a screw-top glass test tube (16 × 100 mm) and consists of 100 /zl of 0.1 M Tris-maleate, pH 8.0, 40/zl of sodium cholate (1.3 mg, Sigma, St. Louis, MO), 50/zl of enzyme-containing sample, and 10/~1 of retinyl [1-14C]palmitate in ethanol (2 nmol and -0.05/zCi or 100,000 cpm). Tubes are incubated in a 37° water bath for 15-30 min. The reaction is quenched by the addition of 3.25 ml of methanol/chloroform/heptane (1.41:1.25:1.00, v : v : v ) containing 0.1 mM palmitic acid (Sigma) as a carrier (organic solvents used in Methods 1 and 2 are purchased from Fisher, Minneapolis, MN). The liquid phases are separated by centrifugation at 1000 rpm in a clinical centrifuge at room temperature for 15 min. Radioactivity of free fatty acids is determined by liquid scintillation spectrometry of I ml of the upper phase after addition to 10 ml of Scintiverse I (Fisher). To calculate retinyl ester hydrolase activity, the radioactivity in test assays given in counts per minute (cpm) is corrected for radioactivity in an assay mixture from which the enzyme has been omitted. The corrected counts are multiplied by 2.45 to account for the total volume of the upper phase, and the product is divided by the partition coefficient of palmitic acid. The partition coefficient is that portion of palmitic acid extracted from a tube containing an enzyme-free reaction mixture to which 10/zl of 200 /zM [1-14C]palmitic acid in ethanol has been added. The partition coefficient for palmitic acid in this solvent system is approximately 0.70. Level of Activity and Enzymatic Properties. Rat liver retinyl ester hydrolase activity ranges from 5 to 250 pmol/min/mg; this range is larger when portions of liver are assayed. 2,9 In spite of this variability, it is well 7 Z. Selinger and Y. Lapidot, J. Lipid Res. 7, 174 (1966). 8 B. R. Lentz, Y. Barenholz, and T. E. Thompson, Chem. Phys. Lipids 15, 216 (1975). 9 W. S. Blaner, J. E. Smith, R. B. Dell, and D. S. Goodman, J. Nutr. 115, 856 (1985).
492
ENZYMOLOGY AND METABOLISM
[55]
established that the assay gives consistent activity for a given enzyme preparation, zl° The apparent Km and Vmaxvalues of an approximately 200-fold purified preparation of retinyl ester hydrolase are 11.2/xM and 141 nmol/min/mg, respectively. ~1The most potent inhibitors of hydrolase activity, with concentrations giving 50% inhibition, include the following: ether analogs of cholesterol esters [especially the linoleyl ether (0.16/~M)],12 ether analogs of triglycerides (>10 txM), 12 phylloquinone (20 txM), 5 and a-tocopherol (100 izM). 5
Method 2 Tissue and Substrate Preparation. This procedure has been used with pig liver and kidney 3 and rat liver4; however, pig and rat liver hydrolases differ with regard to the substrate and CHAPS concentrations and pH giving maximal activity. The differences are noted below. Tissue homogenates are prepared as described for Method 1. Homogenates are diluted so that the amount of homogenate protein added to the reaction mixture is less than 1 mg for pig liver or 750/xg for rat liver; usually 200-300/xg is sufficient. Under these conditions, the amount of liver-derived vitamin A added to the assay mix is negligible when the liver reserves are less than 200/.~g/g (higher levels have not been tested). For each 1 ml of substrate solution, 2.63 mg (for pig assay) or 26.3 mg (for rat assay) of retinyl palmitate (Sigma) is dissolved in hexanes, and the concentration is verified by measuring the absorption at 325 nm of a known dilution. An appropriate volume of this solution is added to a screw-top test tube, and the hexanes are evaporated under a stream of argon. The residue is dissolved in 20 mg Triton X-100 (Sigma) per milliliter of assay buffer, a small stir bar is added, and the solution is stirred vigorously. The substrate solution can be used up to 2 days later when stored in the dark at 4° in an argon-flushed container. A surface layer of retinyl ester may form during storage but the solution will be homogeneous if it is warmed to room temperature and stirred prior to use. Assay Procedure and Actioity Calculations. Assays are carried out in 12 x 75 mm glass tubes with caps. For the pig enzyme, 250/zl of 200 mM CHAPS (Sigma) in 50 mM Tris-maleate, pH 8.0, is combined with 50/zl i0 E. H. Harrison, J. E. Smith, and D. S. Goodman, J. Lipid Res. 20, 760 (1979). N W. S. Blaner, J. H. Prystowsky, J. E. Smith, and D. S. Goodman, Biochirn. Biophys. Acta 794, 419 (1984). t2 W. S. Blaner, G. Halperin, O. Stein, Y. Stein, and D. S. Goodman, Biochim. Biophys. Acta 794, 428 (1984).
[55]
ASSAY OF LIVER RETINYL ESTER HYDROLASE
493
of 5 mM retinyl palmitate suspension. After a 5-min preincubation at 37°, 50 /zl of homogenate is added to initiate the reaction. The mixture is vortexed gently and incubated for 30-60 rain in a Dubnoff shaking incubator at 25 strokes/min. The assay mix for the rat enzyme is composed of 350/xl of 393 mM CHAPS in 100 mM Tris-maleate, pH 7.0, 50/zl of 50 mM retinyl palmitate suspension, and 50 ~1 of liver homogenate. Incubation is for 30-45 rain. Reaction mixtures containing over 900/zM retinyl palmitate may be turbid; however, this does not affect the assay. To stop the hydrolase reaction, 500/zl absolute ethanol is added and the solution is vortexed. Next, the assay mix is extracted twice with 2 ml of hexanes containing 0.1% butylated hydroxytoluene (BHT) (Sigma). The combined hexane extracts are dried under a gentle stream of argon, and the residue is redissolved in 500/~1 2-propanol and stored in 1.5-ml Eppendorf tubes at - 2 0 °. Retinol can be analyzed by using any HPLC method that separates retinol from contaminating peaks. A proven method is based on chromatography with the following equipment: a 4.6 x 250 mm Partisil 10/25 ODS-2 column (Whatman, Clifton, N J), an Uptight guard column (Upchurch Scientific, Oak Harbor, WA) filled with pellicular octadecylsilane (Vydac, The Separation Group, Hesperia, CA), an LDC Constametric III pump (Riviera Beach, FL), an ISCO variable absorbance detector (Lincoln, NE), and a Shimadzu C-R3A integrator (Kyoto, Japan). A Waters Associates (Milford, MA) WISP Model 710 HPLC autosampler can be used to process multiple samples. After every second or third injection of 25/zl, the column should be flushed with 1 : 1 methanol/tetrahydrofuran (v/v) to elute retinyl esters, all-trans-retinol is eluted in 7.3 min with 9 : 1 (v/v) methanol/water pumped at a flow rate of 2 ml/min. The ultraviolet absorbance (325 nm) of the column effluent is monitored, and retinol is quantitated by comparison of peak area to that of a standard concentration curve. Retinyl ester hydrolase activity is calculated from the amount of retinol produced during the assay corrected for retinol formed in control incubations containing all assay components except the homogenate. If liver homogenates contain high levels of retinol, correction should be made for retinol extracted from unincubated assay mixtures (minus retinyl palmitate). This assay will detect the production of as little as 4 pmol retinol. The interassay variability is 4.7%. Level of Activity and Enzymatic Properties. Liver retinyl ester hydrolase activity in 24 pigs 3 was 13-34 pmol/min/mg. The enzyme has an apparent Km of 140 /xM. 3 Other excellent substrates and their rates of hydrolysis relative to all-trans-retinyl palmitate are as follows: the linolenic (2.1), myristic (2.1), oleic (0.8), linoleic (0.7), and stearic (0.6) esters
494
ENZYMOLOGY AND METABOLISM
[56]
of retinol as well as the 9-cis (2.4), 13-cis (5.7), and 9,13-cis (6.8) isomers of retinyl palmitate.~3 Rat liver retinyl ester hydrolase activity in 25 rats was 100-500 pmol/ min/mg and had an apparent Km of 1.3 mM. 4 In comparison to equimolar sodium cholate, CHAPS gives 9.3 times higher activity on average and reduced interanimal variability/Ethanol (0.01 to 0.5 M) stimulates hydrolase activity 20 to 86%.14 Conclusions
Although the methods described in this chapter have not been compared side by side, they have similar ease of use, sensitivity, and reproducibility. The assays differ largely with respect to composition, methodology, and the interanimal variability in activity they give. 13 D. A. Cooper and J. A. Olson, Arch. Biochem. Biophys. 2611, 705 (1988). 14 H. Friedman, S. Mobarhan, J. Hupert, D. Lucchesi, C. Henderson, P. Langenberg, and T. J. Layden, Arch. Biochem. Biophys. 269, 69 (1989).
[56] A s s a y o f t h e R e t i n o i d I s o m e r a s e S y s t e m o f t h e E y e
By PAUL S. BERNSTEIN and ROBERT R. RANDO Introduction The all-trans- to 11-cis-retinoid isomerase is the key enzyme of the vertebrate visual cycle. It is responsible for the endergonic isomerization of vitamin A compounds from the all-trans isomeric form to the higher energy 11-cis configuration essential for rhodopsin regeneration in the eye. The existence of an enzyme of this type was postulated in the early 1950s by Wald and Hubbard when the vertebrate visual cycle was first discussed. ~The reproducible demonstration of the existence of an isomerase was not achieved until recently, however, requiring the use of high specific-activity radioactive retinoid substrates and high-performance liquid chromatographic (HPLC) analysis. 2 Although the isomerase has not yet been completely purified, a number of its properties are well established: (1) the isomerase is localized to R. Hubbard and G. Wald, J. Gen. Physiol. 36, 269 (1952). 2 p. S. Bernstein, W. C. Law, and R. R. Rando, Proc. Natl. Acad. Sci. U.S.A. 84, 1849 (1987).
METHODS IN ENZYMOLOGY, VOL. 189
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
