156
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[17]
[ 17] A c y l - C o A : R e t i n o l A c y l t r a n s f e r a s e a n d Lecithin:Retinol Acyltransferase Activities of Bovine Retinal Pigment Epithelial Microsomes
By JOHN C. SAARIand D. LUCILLEBREDnERG Introduction The reactions that accompany the bleaching and subsequent regeneration of visual pigment make up the visual or regeneration cycle.~ Although visual pigment bleaching takes place in photoreceptor cells, most of the enzymatic reactions of the cycle, including the isomerization regenerating the I l-cis configuration, take place in adjacent retinal pigment epithelial (RPE) cells.2 Retinyl esters were previously thought to be solely storage forms of vitamin A in RPE; however, recent work suggests that retinyl ester is the substrate for the isomerase.3 The main fatty acids esterified to retinol are palmitate (57%), stearate (24%), and oleate (10%).4 Unusual features of retinyl ester synthesis in RPE, noted in other studies, include its occurrence in CoA-dependent and CoA-independent modes5 (Fig. 1), the presence of an endogenous acyl donor in RPE microsomes,6-s and the large amount of activity present. 5 Recent studies have shed new light on several aspects of retinyl ester synthesis in RPE. Enzymatic transfer of acyl groups from the 1-position of phosphatidylcholine (PC) to retinol has been demonstrated in RPE microsomes, suggesting that lecithin:retinol acyltransferase (LRAT) accounts for the CoA-independent retinyl esterification and that microsomal PC is the endogenous acyl donor. 9,1° Apparent acyl-CoA:retinol acyltransferase (ARAT,EC 2.3.1.76, retinol fatty-acyltransferase) activity has been shown to result, at least in part, from an initial transfer of the fatty acyl group of acyl-CoA to a lipid component of the microsomes and subsequently to G. Wald, Science 162, 230 (1968). 2 j. C. S a a r i , in " P r o g r e s s in Retinal Research" (N. N. Osborne and J. G. Chader, eels.), Vol. 9, p. 363. Pergamon, Oxford and New York, 1990. 3 p. S. Deigner, W. C. Law, F. J. Canada, and R. R. Rando, Science 244, 968 (1989). 4 S. Futterman and J. S. Andrews, J. Biol. Chem. 239, 81 (1964). s j. C. Saari and D. L. Bredberg, J. Biol. Chem. 263, 8084 (1988). 6N. I. Krinsky, J. Biol. Chem. 232, 881 (1958). E. R. Berman, J. Horowitz, N. Segal, S. Fischer, and L. Feeney-Burns, Biochim. Biophys. Acta 630, 36 (1980). s j. S. Andrews and S. Futterman, J. Biol. Chem. 239, 4073 (1964). 9 j. C. Saari and D. L. Bredberg, J. Biol. Chem. 264, 8638 (1989). to R. J. Barry, F. J. Canada, and R. R. Rando, J. Biol. Chem. 264, 9231 (1989).
METHODS IN ENZYMOLOGY, VOL. 190
Copyright © 1990 by Academic Press. Inc. All tights of reproduction In any form reserved.
[17]
A R A T AND L R A T IN RPE MICROSOMES
157
'°°t 160] d~
120" •
~er !
80 40'
0
1 '0
2'0 3'0 REACTION
4'0 TIME
5'0
60
minutes
FIG. 1. Progress curve for retinyl esterification. Addition of [3H] retinol to buffered microsomes from bovine retinal pigment epitheliumproduces an immediate burst of retinyl ester synthesis,demonstratinga CoA-independentcomponent of the reaction (El). After 30 min further addition of [3H]retinol produces little additional retinyl ester synthesis (12). However, additional retinyl ester is synthesizedin response to an addition of retinol and palmitoyl-CoA~1). (Modifiedfrom Reference 5 with permissionof the AmericanSocietyfor Biochemistryand MolecularBiology.) retinol. 5 The lipid component is likely to be a lysolecithin, although this has not been demonstrated directly. A proposed relationship of CoA-dependent and CoA-independent activities is shown in Fig. 2. L R A T has been described in rat intestinal mucosal cells it and is likely to catalyze a general reaction for retinol esterification. Methods are presented here for the determination of CoA-independent retinyl ester synthesis (lecithin:retinol acyltransferase, LRAT) with an endogenous acyl donor and with in situ synthesized PC, and of CoA-dependent retinyl ester synthesis (apparent ARAT). Assay M e t h o d s Principle. Retinyl ester synthesis is a two-substrate reaction requiring an acyl donor and retinol. An endogenous acyl donor is present in RPE microsomes, giving retinyl ester synthesis on addition of retinol alone. 5-s,12 (LRAT with endogenous acyl donor). In addition, RPE microsomes will catalyze the synthesis of [1-14C]palmitoyl-PC during an incubation with
i1 p. N. MacDonaldand D. E. Ong, J. Biol. Chem. 263, 12478 (1988). 12C. D. B. Bridges,M. S. Oka, S.-L. Fong, G. I. Liou, and R. A. Alvarcz,Neurochem. Int. 8, 527 0950).
158
[17]
TISSUE AND ORGAN CULTURE PALMITATE CoA
PALMITOYL CoA
LINOLEATE - - - 4
RETINOL
LINOLEATE P-CHOLINE
RETINYL PALMITATE
CoA-INDEPENDENT ESTERIFICATION
CoA-DEPENDENT ESTERIFICATION
I~
FIo. 2. Proposed relationship between CoA-independent (LRAT) and CoA-dependent (ARAT) retinyl ester synthesis in bovine retinal pigment epithelial microsomes. A contribution arising from direct transfer of palmitate from palndtoyl-CoA to retinol has not been ruled out. [Reproduced with permission from J. C. Saari, in "Progress in Retinal Research" (N. N. Osborne and J. G. Chader, eds.), Vol. 9, p. 363. Pergamon, Oxford and New York, 1990.]
[1-14C]palmitoyllyso-PC and a fatty acyl-CoA, producing a ]4C-labeled acyl donor for retinyl ester synthesis (LRAT with exogenous acyl donor). 9 Finally, apparent ARAT activity can be measured with []4C]palmitoylCoA as an acyl donor for the reaction. 5 The assay procedure involves extraction of [aH]retinol and [3H]retinyl esters from reaction mixtures and their separation on disposable alumina columns. Quantification is obtained by liquid scintillation counting. Reagents Alumina columns: 0.4 g of 6% water-deactivated alumina; ~a dry in a Pasteur pipette loosely plugged with glass wool Assay buffer: 40 mM Tris-acetate, pH 8, containing 60/zM bovine serum albumin (BSA) and 1 mM dithiothreitol (DTT) Bovine serum albumin, fatty acid poor (Sigma, St. Louis, MO) Buffered sucrose: 0.25 M sucrose buffered with 25 mM Tris-acetate, pH 7, containing 1 mM DTT Chloroform/methanol, 2: l, v/v t4 13R. Hubbard, P. K. Brown, and D. Bownds, this series, Vol. 18 part C, p. 615. ~4j. Folch, M. Lees, and G. A. Sloane-Stanley, J.Biol. Chem. 226, 497 (1957).
[ 17]
ARAT AND LRAT IN RPE MICROSOMES
159
Fatty acyl-CoAs (Sigma), assayed and purified by high-performance liquid chromatography (HPLC) if necessary5 Thin layer chromatography (TLC) sheets, ITLC-SG silica-impregnated glass fiber sheets (Gelman Sciences, Ann Arbor, MI) TLC solvent: hexane/2-propanol/water, 6:8:1 (v/v/v), for separation of lyso-PC and PC Liquid scintillation cocktail (Ecolume, ICN Biomedicals, Inc., Costa Mesa, CA) [ 1-~4C]Palmitoyllyso-PC (Du Pont- New England Nuclear, Boston, MA), purified by TLC if necessary9 (128,000 dpm/nmol) p4C]Palmitoyl-CoA (Du Pont-NEN), purified by HPLC; 5 a working solution of I mM in assay buffer (~32,000 dpm/nmol) is prepared Petroleum ether (30-60 o boiling range) all-trans-Retinol (unlabeled) (Sigma), purified by HPLC ~5 all-trans-[3H]Retinol (Du Pont-NEN), purified by HPLC; a working solution of 2 mM in ethanol (specific activity -20,000 dpm/nmol) is prepared Ruby red bulbs, 40 W (Westinghouse) General Procedures. All operations involving retinoids are performed in darkness or under dim red illumination. Buffers are bubbled with argon before use, and tubes are flushed with argon and sealed during incubations. Butylated hydroxytoluene (BHT) is added to all solutions containing lipids (1:100, w/w, BHT/lipid). Bovine serum albumin (60 Wl¢) is included in all solutions of retinoids and fatty acyl-CoAs to minimize the known detergent effects of the latter ~6and to enhance the water solubility of the former. Isolation of Retinal Pigment Epithelial Microsomes. Bovine (calf or adult) eyes are hemisected and the anterior portion and vitreous discarded. Neural retina is removed with a blunt-tipped capsule forceps (Storz Ophthalmic Instruments, St. Louis, MO). Cold buffered sucrose (1 ml/eye) is added to the eye cup, and RPE cells are dislodged by gently stroking from center to periphery with a small brush. The dark suspension of cells is removed with a Pasteur pipette. After repeating the procedure, the cells are homogenized with 8-10 passes of a motor-driven Teflon-glass homogenizer and centrifuged at 20,000 g for 20 min at 5". The supernatant is centrifuged at 150,000 g for 1 hr at 5*. The microsomal pellet from this spin is resuspended in 10 mMTris-acetate, pH 7, 1 mM DTT (50 or 100 /zl/calf or adult eye, respectively) and stored at -80* in 50-/zl portions. Protein concentrations of the preparations are obtained using the Folin phenol procedure.~7 15j. C. Saari, L. Brcdberg, and G. G. Garwin, J. BioL Chem. 257, 13329 (1982). t6 p. p. Constantinides and J. M. Steim, J. Biol. Chem. 260, 7573 (1985). 1~G. L. Peterson, this series, Vol. 91, p. 95.
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Quantification of Retinyl Ester. Enzymatic reactions are stopped by the addition of 2 volumes of ice-cold ethanol. Following the addition of 6 volumes of petroleum ether, retinoids are extracted by forcing the lower phase into the upper phase with a Pasteur pipette about 6 times. Three volumes of water are then added and the tube centrifuged to separate the phases. The upper phase is removed, extracted with 3 volumes of water to remove traces of ethanol, and applied to an alumina column, which is washed with 1 ml of petroleum ether (bp 35- 60 o), followed by 2 ml of 3% diethyl ether in petroleum ether to elute retinyl esters and 2 ml of 50% diethyl ether in petroleum ether to elute retinol. After collection of retinoid-containing fractions in scintillation vials, the solvent is evaporated at 37 ° with a stream of argon (Reacti-Vap, Pierce, Rockford, IL), and the radioactivity is determined by liquid scintillation. Assay for LRAT, Endogenous Acyl Donor. all-trans-[aH]Retinol is added to assay buffer at 37 °, to give a final retinol concentration of 10 gal. After a 30-sec incubation at 37 °, the reaction is initiated by the addition of microsomes (10-50/tg protein/ml, final concentration) and the incubation continued. For initial rate studies an assay time of 0.5 rain is employed. The reaction is stopped with the addition of ice-cold ethanol, and retinoids are extracted as described. Assayfor LRA T, Exogenous Acyl Donor. The assay using an exogenous acyl donor involves enzymatic generation, during a preincubation, of labeled PC that serves as an acyl donor for subsequent retinyl ester synthesis. Microsomes (10-50 /tg protein/ml) are preincubated with 10 / d e [1-~4C]palmitoyllyso-PC and linoleoyl-CoA (60 #M) in assay buffer containing 150 #Air BSA. After 5 min, [aH]retinol is added (10 ~ final concentration) and the incubation continued. Following the addition of 2 volumes of cold ethanol, retinol and retinyl esters are extracted and quantiffed as described above. Retinyl ester synthesis in this assay takes place with acyl groups provided by the endogenous acyl donor (-80%) and the in situ synthesized PC (-20%). Determination of the rate of incorporation of [~4C]palmitate into retinyl esters is a measure of the latter process, whereas the rate of incorporation of [3H]retinol into retinyl esters measures the overall process. Linoleoyl-CoA generated the most active substrate of the acyl-CoAs tested. 9 Conversion of lyso-PC to PC can be determined by thin-layer chromatography on silica-impregnated glass fiber sheets following extraction of 1 volume of the reaction mixture with 4 volumes of CHC 13-methanol? Assay ofARA TActivity. Initial rates of ARAT activity can be observed following the addition of [~4C]palmitoyl-CoA and retinol to microsomes. p4C]Palmitoyl-CoA (20 #M) is added to microsomes (10-50 gg protein/ ml) and preincubated for 30-see. The reaction is initiated by the addition
[17]
ARAT
AND
LRAT IN RPE MICROSOMES
161
of [3H]retinol (10 # M final concentration). After an appropriate time (0.5- 1.5 min for routine assays), 2 volumes of cold ethanol are added and ~4C and 3H incorporation into retinyl esters determined as described above. An effect of palmitoyl-CoA on the rate of incorporation of [3H]retinol into retinyl ester is observed following a preincubation of microsomes with retinol to allow retinyl ester synthesis to apparently cease (Fig. 1). alltrans-[3H]Retinol is added to RPE microsomes in assay buffer to give a concentration of 10 pM. Portions are removed at intervals during incubation at 37 ° and the extent of incorporation of [aH]retinol into retinyl ester determined. When the reaction has apparently ceased (-20 rain, Fig. 1), palmitoyl-CoA (final concentration 20 pM) and [3H]retinol (final concentration 10/tM) are added and portions removed at intervals for approximately 20 min for analysis of retinyl esters. General Comments
Stability. Both LRAT and ARAT activities are stable for at least 6 months in microsomal fractions stored at - 8 0 ° in the presence of DTT. Storage and/or assay in the absence of DTT results in a loss of ARAT activity with retention of LRAT activity. It is likely that conflicting accounts6-s,~2 of the effect of added palmitoyl-CoA are attributable to lack of adequate sulfhydryl protection. At 37 ° in the absence of substrate, the LRAT half-life (t0 is 17 min. The t~ of palmitoyl-CoA at 37* and pH 8, measured in the presence of microsomes (60/zg/ml) but absence of retinol, is 9 min. Progress Curve of Retinyl Ester Synthesis. Addition of retinol to buffered microsomes from bovine RPE results in a burst of CoA-independent retinyl ester synthesis that appears to stop after approximately 20 rain (Fig. 1). Addition of palmitoyl-CoA and retinol at this time allows retinyl ester synthesis to resume, demonstrating a CoA-dependent component of the reaction. The CoA-independent component of the reaction appears to be catalyzed by lecithin:retinol acyltransferase (LRAT), an enzyme that transfers a fatty acid from the 1-position of phosphatidylcholine to retinol.9,t° Exogenous dipalmitoyl-PC is poorly integrated into microsomal membranes, and its addition results in tittle LRAT activity.5 However, substrate can be generated in situ as RPE microsomes catalyze synthesis of [114C]palmitoyl-2-acyl-PC from 1-[14C]palmitoyllyso-PC and fatty acyl-CoA. Addition of retinol then results in the transfer of [~4C]palmitate to retinol, demonstrating LRAT activity. Results obtained employing different fatty acylCoAs for PC synthesis suggest that the fatty acid in the 2-position is important in determining the efficiency of the substrate. 9
162
TISSUE AND ORGAN CULTURE
[ 17]
TABLE I CoA-INDEPENDENTRETINYL ESTER SYNTHESIS:SPECIFIC ACTIVITY OF MICROSOMALPREPARATIONS
Source of microsomes
Retinyl ester synthesis (nmol/min/rng)
Bovine retinal pigment epithelium a Rat intestine b Rat liver~ Rat mammary gland a Rat testes"
102 0.031 0.145 0.06 0.094
a C. Saad and D. L. Breclberg, J. Biol. Chem. 263, 8084 (1988). b D. E. Ong, B. Kakkad, and P. N. MacDonald, J. Biol. Chem. 262, 2729 (1987). c R. W. Yost, E. H. Harrison, and A. C. Ross, J. Biol. Chem. 263, 18693 (1988). aA. C. Ross, J. LipidRes. 23, 133 (1982). "L. R. Chandhary and E. C. Nelson, Biochim. Biophys. Acta 917, 24 (1987).
The apparent rapid cessation of retinyl ester synthesis (Fig. 1) was initially interpreted as depletion of an endogenous acyl donor. 5 However, addition of [aH]retinol to microsomes that had been preincubated with retinol for 20 min results in rapid appearance of the label in retinyl ester, 's demonstrating that retinyl ester synthesis is continuing without a net increase in accumulation. Thus, the plateau in the progress curve (Fig. l) is likely to be due to reversal of the LRAT reaction and/or hydrolase activity balancing the rate of retinyl ester synthesis. Detailed studies of the ARAT reaction indicate that it can be divided into two steps. 5 Addition of p4C]palmitoyl-CoA to microsomes in the absence of retinol results in an initial acylation of a microsomal lipid. Incorporation of [~4C]palmitate into retinyl palmitate, in the absence of palmitoyl-CoA, can then be demonstrated following the addition of retinol, demonstrating the two-step nature of the reaction. Thus, in RPE it appears that ARAT activity can be explained, at least in part, by an initial transfer of an acyl group from acyl-CoA to a microsomal lipid followed by acyl transfer to retinol (LRAT) (Fig. 2). Direct acyl transfer from palmitoyl-CoA to retinol as an additional reaction has not been ruled out. Kinetic Properties. The specific LRAT activity of RPE microsomes with the endogenous acyl donor is the highest reported for retinyl ester ,8 j. C. Saari and D. L. Bredberg, unpublished.
[ 18]
DETERMINATION OF RETINOIDS IN HUMAN SKIN
163
synthesis, with a value approximately 103 times that reported for microsomes from liver, intestinal mucosa, testes, or rat mammary gland (Table I). Km values of approximately 2 #M for all-trans- and 11-cis-retinol are observed for LRAT activity;5 it is not known whether both substrates are processed by the same or different enzymes. The rate of the reaction is optimal between pH 7.5 and 8. Acknowledgments Supported in part by U.S. Public Health Service Grants EY02317 and EY01730, and in part by an award from Research to Prevent Blindness, Inc.
[18] H i g h - P e r f o r m a n c e L i q u i d C h r o m a t o g r a p h y o f N a t u r a l a n d S y n t h e t i c R e t i n o i d s in H u m a n S k i n S a m p l e s By ANDERSVAHLQUIST,HANS TORMX, OLA ROLLMAN, and EVA ANDERSSON
Introduction The effects of retinoids on the skin are described in detail elsewhere in this series. Typically, advanced hypovitaminosis A elicits epidermal hyperkeratinization and follicular plugging. Hypervitaminosis A, in contrast, stimulates epidermal cell turnover and retards keratinocyte differentiation. In each of these situations it may be of interest to know not only the serum concentration of vitamin A but also the skin concentration. A small, superficial skin biopsy will usually suffice for screening the endogenous concentrations of retinol and 3,4-didehydroretinol, the two predominating forms of vitamin A in human epidermis, ~provided that a sensitive analytical technique is available. The skin is also a primary target tissue for retinoid therapy. For several years, synthetic retinoids have been used topically and systemically in skin disorders such as ache, psoriasis, and keratinizing genodermatoses. In these situations, retinoid analysis of a skin biopsy may be of value for monitoring the therapeutic drug concentrations and establishing the blood-tissue concentration relationship. We describe a technique for routine analysis of picomolar amounts of both neutral (retinol, 3,4-didehydroretinol) and acidic (retinoic acid, isoI A. Vahlquist, J. Invest. Dermatol. 79, 89 (1982).
METHODS IN ENZYMOI.DGY, VOL. 190
Copyright © 1990 by Academic Press, Inc. Allrightsoft~oductioninany form reserved.
TISSUE AND ORGANCULTURE
[17]
[ 17] A c y l - C o A : R e t i n o l A c y l t r a n s f e r a s e a n d Lecithin:Retinol Acyltransferase Activities of Bovine Retinal Pigment Epithelial Microsomes
By JOHN C. SAARIand D. LUCILLEBREDnERG Introduction The reactions that accompany the bleaching and subsequent regeneration of visual pigment make up the visual or regeneration cycle.~ Although visual pigment bleaching takes place in photoreceptor cells, most of the enzymatic reactions of the cycle, including the isomerization regenerating the I l-cis configuration, take place in adjacent retinal pigment epithelial (RPE) cells.2 Retinyl esters were previously thought to be solely storage forms of vitamin A in RPE; however, recent work suggests that retinyl ester is the substrate for the isomerase.3 The main fatty acids esterified to retinol are palmitate (57%), stearate (24%), and oleate (10%).4 Unusual features of retinyl ester synthesis in RPE, noted in other studies, include its occurrence in CoA-dependent and CoA-independent modes5 (Fig. 1), the presence of an endogenous acyl donor in RPE microsomes,6-s and the large amount of activity present. 5 Recent studies have shed new light on several aspects of retinyl ester synthesis in RPE. Enzymatic transfer of acyl groups from the 1-position of phosphatidylcholine (PC) to retinol has been demonstrated in RPE microsomes, suggesting that lecithin:retinol acyltransferase (LRAT) accounts for the CoA-independent retinyl esterification and that microsomal PC is the endogenous acyl donor. 9,1° Apparent acyl-CoA:retinol acyltransferase (ARAT,EC 2.3.1.76, retinol fatty-acyltransferase) activity has been shown to result, at least in part, from an initial transfer of the fatty acyl group of acyl-CoA to a lipid component of the microsomes and subsequently to G. Wald, Science 162, 230 (1968). 2 j. C. S a a r i , in " P r o g r e s s in Retinal Research" (N. N. Osborne and J. G. Chader, eels.), Vol. 9, p. 363. Pergamon, Oxford and New York, 1990. 3 p. S. Deigner, W. C. Law, F. J. Canada, and R. R. Rando, Science 244, 968 (1989). 4 S. Futterman and J. S. Andrews, J. Biol. Chem. 239, 81 (1964). s j. C. Saari and D. L. Bredberg, J. Biol. Chem. 263, 8084 (1988). 6N. I. Krinsky, J. Biol. Chem. 232, 881 (1958). E. R. Berman, J. Horowitz, N. Segal, S. Fischer, and L. Feeney-Burns, Biochim. Biophys. Acta 630, 36 (1980). s j. S. Andrews and S. Futterman, J. Biol. Chem. 239, 4073 (1964). 9 j. C. Saari and D. L. Bredberg, J. Biol. Chem. 264, 8638 (1989). to R. J. Barry, F. J. Canada, and R. R. Rando, J. Biol. Chem. 264, 9231 (1989).
METHODS IN ENZYMOLOGY, VOL. 190
Copyright © 1990 by Academic Press. Inc. All tights of reproduction In any form reserved.
[17]
A R A T AND L R A T IN RPE MICROSOMES
157
'°°t 160] d~
120" •
~er !
80 40'
0
1 '0
2'0 3'0 REACTION
4'0 TIME
5'0
60
minutes
FIG. 1. Progress curve for retinyl esterification. Addition of [3H] retinol to buffered microsomes from bovine retinal pigment epitheliumproduces an immediate burst of retinyl ester synthesis,demonstratinga CoA-independentcomponent of the reaction (El). After 30 min further addition of [3H]retinol produces little additional retinyl ester synthesis (12). However, additional retinyl ester is synthesizedin response to an addition of retinol and palmitoyl-CoA~1). (Modifiedfrom Reference 5 with permissionof the AmericanSocietyfor Biochemistryand MolecularBiology.) retinol. 5 The lipid component is likely to be a lysolecithin, although this has not been demonstrated directly. A proposed relationship of CoA-dependent and CoA-independent activities is shown in Fig. 2. L R A T has been described in rat intestinal mucosal cells it and is likely to catalyze a general reaction for retinol esterification. Methods are presented here for the determination of CoA-independent retinyl ester synthesis (lecithin:retinol acyltransferase, LRAT) with an endogenous acyl donor and with in situ synthesized PC, and of CoA-dependent retinyl ester synthesis (apparent ARAT). Assay M e t h o d s Principle. Retinyl ester synthesis is a two-substrate reaction requiring an acyl donor and retinol. An endogenous acyl donor is present in RPE microsomes, giving retinyl ester synthesis on addition of retinol alone. 5-s,12 (LRAT with endogenous acyl donor). In addition, RPE microsomes will catalyze the synthesis of [1-14C]palmitoyl-PC during an incubation with
i1 p. N. MacDonaldand D. E. Ong, J. Biol. Chem. 263, 12478 (1988). 12C. D. B. Bridges,M. S. Oka, S.-L. Fong, G. I. Liou, and R. A. Alvarcz,Neurochem. Int. 8, 527 0950).
158
[17]
TISSUE AND ORGAN CULTURE PALMITATE CoA
PALMITOYL CoA
LINOLEATE - - - 4
RETINOL
LINOLEATE P-CHOLINE
RETINYL PALMITATE
CoA-INDEPENDENT ESTERIFICATION
CoA-DEPENDENT ESTERIFICATION
I~
FIo. 2. Proposed relationship between CoA-independent (LRAT) and CoA-dependent (ARAT) retinyl ester synthesis in bovine retinal pigment epithelial microsomes. A contribution arising from direct transfer of palmitate from palndtoyl-CoA to retinol has not been ruled out. [Reproduced with permission from J. C. Saari, in "Progress in Retinal Research" (N. N. Osborne and J. G. Chader, eds.), Vol. 9, p. 363. Pergamon, Oxford and New York, 1990.]
[1-14C]palmitoyllyso-PC and a fatty acyl-CoA, producing a ]4C-labeled acyl donor for retinyl ester synthesis (LRAT with exogenous acyl donor). 9 Finally, apparent ARAT activity can be measured with []4C]palmitoylCoA as an acyl donor for the reaction. 5 The assay procedure involves extraction of [aH]retinol and [3H]retinyl esters from reaction mixtures and their separation on disposable alumina columns. Quantification is obtained by liquid scintillation counting. Reagents Alumina columns: 0.4 g of 6% water-deactivated alumina; ~a dry in a Pasteur pipette loosely plugged with glass wool Assay buffer: 40 mM Tris-acetate, pH 8, containing 60/zM bovine serum albumin (BSA) and 1 mM dithiothreitol (DTT) Bovine serum albumin, fatty acid poor (Sigma, St. Louis, MO) Buffered sucrose: 0.25 M sucrose buffered with 25 mM Tris-acetate, pH 7, containing 1 mM DTT Chloroform/methanol, 2: l, v/v t4 13R. Hubbard, P. K. Brown, and D. Bownds, this series, Vol. 18 part C, p. 615. ~4j. Folch, M. Lees, and G. A. Sloane-Stanley, J.Biol. Chem. 226, 497 (1957).
[ 17]
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159
Fatty acyl-CoAs (Sigma), assayed and purified by high-performance liquid chromatography (HPLC) if necessary5 Thin layer chromatography (TLC) sheets, ITLC-SG silica-impregnated glass fiber sheets (Gelman Sciences, Ann Arbor, MI) TLC solvent: hexane/2-propanol/water, 6:8:1 (v/v/v), for separation of lyso-PC and PC Liquid scintillation cocktail (Ecolume, ICN Biomedicals, Inc., Costa Mesa, CA) [ 1-~4C]Palmitoyllyso-PC (Du Pont- New England Nuclear, Boston, MA), purified by TLC if necessary9 (128,000 dpm/nmol) p4C]Palmitoyl-CoA (Du Pont-NEN), purified by HPLC; 5 a working solution of I mM in assay buffer (~32,000 dpm/nmol) is prepared Petroleum ether (30-60 o boiling range) all-trans-Retinol (unlabeled) (Sigma), purified by HPLC ~5 all-trans-[3H]Retinol (Du Pont-NEN), purified by HPLC; a working solution of 2 mM in ethanol (specific activity -20,000 dpm/nmol) is prepared Ruby red bulbs, 40 W (Westinghouse) General Procedures. All operations involving retinoids are performed in darkness or under dim red illumination. Buffers are bubbled with argon before use, and tubes are flushed with argon and sealed during incubations. Butylated hydroxytoluene (BHT) is added to all solutions containing lipids (1:100, w/w, BHT/lipid). Bovine serum albumin (60 Wl¢) is included in all solutions of retinoids and fatty acyl-CoAs to minimize the known detergent effects of the latter ~6and to enhance the water solubility of the former. Isolation of Retinal Pigment Epithelial Microsomes. Bovine (calf or adult) eyes are hemisected and the anterior portion and vitreous discarded. Neural retina is removed with a blunt-tipped capsule forceps (Storz Ophthalmic Instruments, St. Louis, MO). Cold buffered sucrose (1 ml/eye) is added to the eye cup, and RPE cells are dislodged by gently stroking from center to periphery with a small brush. The dark suspension of cells is removed with a Pasteur pipette. After repeating the procedure, the cells are homogenized with 8-10 passes of a motor-driven Teflon-glass homogenizer and centrifuged at 20,000 g for 20 min at 5". The supernatant is centrifuged at 150,000 g for 1 hr at 5*. The microsomal pellet from this spin is resuspended in 10 mMTris-acetate, pH 7, 1 mM DTT (50 or 100 /zl/calf or adult eye, respectively) and stored at -80* in 50-/zl portions. Protein concentrations of the preparations are obtained using the Folin phenol procedure.~7 15j. C. Saari, L. Brcdberg, and G. G. Garwin, J. BioL Chem. 257, 13329 (1982). t6 p. p. Constantinides and J. M. Steim, J. Biol. Chem. 260, 7573 (1985). 1~G. L. Peterson, this series, Vol. 91, p. 95.
160
TISSUE AND ORGAN CULTURE
[17]
Quantification of Retinyl Ester. Enzymatic reactions are stopped by the addition of 2 volumes of ice-cold ethanol. Following the addition of 6 volumes of petroleum ether, retinoids are extracted by forcing the lower phase into the upper phase with a Pasteur pipette about 6 times. Three volumes of water are then added and the tube centrifuged to separate the phases. The upper phase is removed, extracted with 3 volumes of water to remove traces of ethanol, and applied to an alumina column, which is washed with 1 ml of petroleum ether (bp 35- 60 o), followed by 2 ml of 3% diethyl ether in petroleum ether to elute retinyl esters and 2 ml of 50% diethyl ether in petroleum ether to elute retinol. After collection of retinoid-containing fractions in scintillation vials, the solvent is evaporated at 37 ° with a stream of argon (Reacti-Vap, Pierce, Rockford, IL), and the radioactivity is determined by liquid scintillation. Assay for LRAT, Endogenous Acyl Donor. all-trans-[aH]Retinol is added to assay buffer at 37 °, to give a final retinol concentration of 10 gal. After a 30-sec incubation at 37 °, the reaction is initiated by the addition of microsomes (10-50/tg protein/ml, final concentration) and the incubation continued. For initial rate studies an assay time of 0.5 rain is employed. The reaction is stopped with the addition of ice-cold ethanol, and retinoids are extracted as described. Assayfor LRA T, Exogenous Acyl Donor. The assay using an exogenous acyl donor involves enzymatic generation, during a preincubation, of labeled PC that serves as an acyl donor for subsequent retinyl ester synthesis. Microsomes (10-50 /tg protein/ml) are preincubated with 10 / d e [1-~4C]palmitoyllyso-PC and linoleoyl-CoA (60 #M) in assay buffer containing 150 #Air BSA. After 5 min, [aH]retinol is added (10 ~ final concentration) and the incubation continued. Following the addition of 2 volumes of cold ethanol, retinol and retinyl esters are extracted and quantiffed as described above. Retinyl ester synthesis in this assay takes place with acyl groups provided by the endogenous acyl donor (-80%) and the in situ synthesized PC (-20%). Determination of the rate of incorporation of [~4C]palmitate into retinyl esters is a measure of the latter process, whereas the rate of incorporation of [3H]retinol into retinyl esters measures the overall process. Linoleoyl-CoA generated the most active substrate of the acyl-CoAs tested. 9 Conversion of lyso-PC to PC can be determined by thin-layer chromatography on silica-impregnated glass fiber sheets following extraction of 1 volume of the reaction mixture with 4 volumes of CHC 13-methanol? Assay ofARA TActivity. Initial rates of ARAT activity can be observed following the addition of [~4C]palmitoyl-CoA and retinol to microsomes. p4C]Palmitoyl-CoA (20 #M) is added to microsomes (10-50 gg protein/ ml) and preincubated for 30-see. The reaction is initiated by the addition
[17]
ARAT
AND
LRAT IN RPE MICROSOMES
161
of [3H]retinol (10 # M final concentration). After an appropriate time (0.5- 1.5 min for routine assays), 2 volumes of cold ethanol are added and ~4C and 3H incorporation into retinyl esters determined as described above. An effect of palmitoyl-CoA on the rate of incorporation of [3H]retinol into retinyl ester is observed following a preincubation of microsomes with retinol to allow retinyl ester synthesis to apparently cease (Fig. 1). alltrans-[3H]Retinol is added to RPE microsomes in assay buffer to give a concentration of 10 pM. Portions are removed at intervals during incubation at 37 ° and the extent of incorporation of [aH]retinol into retinyl ester determined. When the reaction has apparently ceased (-20 rain, Fig. 1), palmitoyl-CoA (final concentration 20 pM) and [3H]retinol (final concentration 10/tM) are added and portions removed at intervals for approximately 20 min for analysis of retinyl esters. General Comments
Stability. Both LRAT and ARAT activities are stable for at least 6 months in microsomal fractions stored at - 8 0 ° in the presence of DTT. Storage and/or assay in the absence of DTT results in a loss of ARAT activity with retention of LRAT activity. It is likely that conflicting accounts6-s,~2 of the effect of added palmitoyl-CoA are attributable to lack of adequate sulfhydryl protection. At 37 ° in the absence of substrate, the LRAT half-life (t0 is 17 min. The t~ of palmitoyl-CoA at 37* and pH 8, measured in the presence of microsomes (60/zg/ml) but absence of retinol, is 9 min. Progress Curve of Retinyl Ester Synthesis. Addition of retinol to buffered microsomes from bovine RPE results in a burst of CoA-independent retinyl ester synthesis that appears to stop after approximately 20 rain (Fig. 1). Addition of palmitoyl-CoA and retinol at this time allows retinyl ester synthesis to resume, demonstrating a CoA-dependent component of the reaction. The CoA-independent component of the reaction appears to be catalyzed by lecithin:retinol acyltransferase (LRAT), an enzyme that transfers a fatty acid from the 1-position of phosphatidylcholine to retinol.9,t° Exogenous dipalmitoyl-PC is poorly integrated into microsomal membranes, and its addition results in tittle LRAT activity.5 However, substrate can be generated in situ as RPE microsomes catalyze synthesis of [114C]palmitoyl-2-acyl-PC from 1-[14C]palmitoyllyso-PC and fatty acyl-CoA. Addition of retinol then results in the transfer of [~4C]palmitate to retinol, demonstrating LRAT activity. Results obtained employing different fatty acylCoAs for PC synthesis suggest that the fatty acid in the 2-position is important in determining the efficiency of the substrate. 9
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TISSUE AND ORGAN CULTURE
[ 17]
TABLE I CoA-INDEPENDENTRETINYL ESTER SYNTHESIS:SPECIFIC ACTIVITY OF MICROSOMALPREPARATIONS
Source of microsomes
Retinyl ester synthesis (nmol/min/rng)
Bovine retinal pigment epithelium a Rat intestine b Rat liver~ Rat mammary gland a Rat testes"
102 0.031 0.145 0.06 0.094
a C. Saad and D. L. Breclberg, J. Biol. Chem. 263, 8084 (1988). b D. E. Ong, B. Kakkad, and P. N. MacDonald, J. Biol. Chem. 262, 2729 (1987). c R. W. Yost, E. H. Harrison, and A. C. Ross, J. Biol. Chem. 263, 18693 (1988). aA. C. Ross, J. LipidRes. 23, 133 (1982). "L. R. Chandhary and E. C. Nelson, Biochim. Biophys. Acta 917, 24 (1987).
The apparent rapid cessation of retinyl ester synthesis (Fig. 1) was initially interpreted as depletion of an endogenous acyl donor. 5 However, addition of [aH]retinol to microsomes that had been preincubated with retinol for 20 min results in rapid appearance of the label in retinyl ester, 's demonstrating that retinyl ester synthesis is continuing without a net increase in accumulation. Thus, the plateau in the progress curve (Fig. l) is likely to be due to reversal of the LRAT reaction and/or hydrolase activity balancing the rate of retinyl ester synthesis. Detailed studies of the ARAT reaction indicate that it can be divided into two steps. 5 Addition of p4C]palmitoyl-CoA to microsomes in the absence of retinol results in an initial acylation of a microsomal lipid. Incorporation of [~4C]palmitate into retinyl palmitate, in the absence of palmitoyl-CoA, can then be demonstrated following the addition of retinol, demonstrating the two-step nature of the reaction. Thus, in RPE it appears that ARAT activity can be explained, at least in part, by an initial transfer of an acyl group from acyl-CoA to a microsomal lipid followed by acyl transfer to retinol (LRAT) (Fig. 2). Direct acyl transfer from palmitoyl-CoA to retinol as an additional reaction has not been ruled out. Kinetic Properties. The specific LRAT activity of RPE microsomes with the endogenous acyl donor is the highest reported for retinyl ester ,8 j. C. Saari and D. L. Bredberg, unpublished.
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DETERMINATION OF RETINOIDS IN HUMAN SKIN
163
synthesis, with a value approximately 103 times that reported for microsomes from liver, intestinal mucosa, testes, or rat mammary gland (Table I). Km values of approximately 2 #M for all-trans- and 11-cis-retinol are observed for LRAT activity;5 it is not known whether both substrates are processed by the same or different enzymes. The rate of the reaction is optimal between pH 7.5 and 8. Acknowledgments Supported in part by U.S. Public Health Service Grants EY02317 and EY01730, and in part by an award from Research to Prevent Blindness, Inc.
[18] H i g h - P e r f o r m a n c e L i q u i d C h r o m a t o g r a p h y o f N a t u r a l a n d S y n t h e t i c R e t i n o i d s in H u m a n S k i n S a m p l e s By ANDERSVAHLQUIST,HANS TORMX, OLA ROLLMAN, and EVA ANDERSSON
Introduction The effects of retinoids on the skin are described in detail elsewhere in this series. Typically, advanced hypovitaminosis A elicits epidermal hyperkeratinization and follicular plugging. Hypervitaminosis A, in contrast, stimulates epidermal cell turnover and retards keratinocyte differentiation. In each of these situations it may be of interest to know not only the serum concentration of vitamin A but also the skin concentration. A small, superficial skin biopsy will usually suffice for screening the endogenous concentrations of retinol and 3,4-didehydroretinol, the two predominating forms of vitamin A in human epidermis, ~provided that a sensitive analytical technique is available. The skin is also a primary target tissue for retinoid therapy. For several years, synthetic retinoids have been used topically and systemically in skin disorders such as ache, psoriasis, and keratinizing genodermatoses. In these situations, retinoid analysis of a skin biopsy may be of value for monitoring the therapeutic drug concentrations and establishing the blood-tissue concentration relationship. We describe a technique for routine analysis of picomolar amounts of both neutral (retinol, 3,4-didehydroretinol) and acidic (retinoic acid, isoI A. Vahlquist, J. Invest. Dermatol. 79, 89 (1982).
METHODS IN ENZYMOI.DGY, VOL. 190
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