ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 198, No. 2, December, pp. 572-579, 1979
Retinyl Esterase Activity of Purified Rat Liver Retinyl Ester Lipoprotein Complex1 CHI-CHING
CHEN AND JORAM HELLER
Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, California
90024
Received February 5, 1979; revised July 30, 1979 Retinyl ester lipoprotein complex from rat liver was shown to possess a retinyl esterase activity toward its own ligand complement. In the presence of serum albumin the retinyl esterase activity at 30°C was about fivefold larger than the activity at 4”C, while higher temperatures than 30°C led to some degradation of retinyl compounds. The pH optimum was 7.8. The esterase activity was markedly enhanced by serum albumin although the serum albumin as such had no retinyl esterase activity. In the presence of serum albumin and under optimal conditions, some ‘75to 80% of the total retinyl ester complement of the lipoprotein was hydrolyzed in 24 h. The retinyl esterase activity was totally abolished by treatment with the serine esterase inhibitor diisopropyl fluorophosphate (1.4 x 10m4M), by treatment with sulfhydryl reagents, and by detergents (0.2% of Tween 80 and sodium deoxycholate). From this series of experiments it was concluded that the retinyl ester lipoprotein complex possesses the additional physiological function of hydrolyzing its own retinyl ester complement to unesterified retinol which may then combine with serum retinol-binding protein.
In the previous paper (1) we have reported the purification and characterization of a retinyl ester lipoprotein complex from rat liver. The complex contains about 66% (by weight) of lipids, 30% protein, and some 4% of carbohydrate. Freshly isolated holoCRLP2 contains about 3% retinyl compounds, 96% of which are retinyl esters and 4% unesterified retinol. It was noted that when holo-CRLP was kept at 4”C, the relative amounts of the retinyl esters and unesterified retinol changed with time, although the total retinyl complement of the carrier complex did not change. This observation prompted the present study. The present paper describes the retinyl esterase activity of holo-CRLP. Mehadevan et al. (2) reported in 1966 the isolation from rat liver of an enzyme that hydrolyzes retinyl palmitate. The enzyme
was present in nuclear and mitochondriallysosome-rich fraction. It was dependent on the presence of bile salts in the reaction mixture and the externally added retinyl compounds used as substrate had to be dispersed in bile salts and Triton X-100. The pH optimum of the retinyl esterase reported by Mehadevan et al. was 8.6. Additions of serum albumin had no effect on the esterase activity. Yeung and Veen-Baigent (3) have subsequently reported their inability to find a retinyl palmitate hydrolyse activity in rat liver following the procedure of Mehadevan et al. (2). Nir et al. (4) reported that chick liver had minimal esterase activity toward retinyl palmitate, the predominant storage form in the liver, while it had considerable activity against retinyl acetate (which is not found naturally in the body). It is important to bear in mind that in all previous work on retinyl esterase activities of various tissues 1 This paper was supported by Grants EY 00704, externally added substrate was used. In EY 00331, and EY 00702 from the National Institutes every case the substrates that are totally of Health, USPHS. insoluble in aqueous media had to be dis* Abbreviations used: CRLP, cytosol retinyl ester persed in various detergents. In the present lipoprotein complex; serum RBP, serum retinol-binding report we have been able to dispense with protein; BSA, bovine serum albumin; DFP, diisopropyl detergents, because the retinyl esters were fluorophosphate; PSF, phenylmethylsulfonyl fluoride.
0003-9861/79/140572-08$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
572
RETINYL
ESTERASE ACTIVITY
OF RETINYL
obtained as soluble complexes with a natural carrier. The present work also provides a possible explanation for the physiological link between retinyl esters, which are the storage form in the liver, and retinol, which is the transportable form. EXPERIMENTAL
PROCEDURES
Preparation of retinyl ester carrier lipoglycoprotein The preparation of holo-CRLP from rat liver was described in detail in the preceding paper (1). In the present series of experiments holo-CRLP was normally used within 3 to 4 days after preparation. It was kept at 4°C with lo-$ M sodium azide. Since there was no difference in the esterase activity between the gel filtration CRLP and the hydroxyapatitepurified material the former was used in some experiments. Esterase assay. The assay was based on the determination of unesterified retinol in the presence of retinyl esters according to Harashima et al. (5). This method uses the conversion of unesterified retinol to anhydroretinol by dilute hydrochloric acid in alcoholic solutions. The anhydroretinol has a unique absorption spectrum with several peaks, one of which is at 399 nm, where retinyl esters have no absorption. The great advantage of this method is the lack of reactivity of retinyl ester in the dehydration reaction, Thus unesterified retinol can be accurately determined in the presence of retinyl esters. Standard reaction mixture contained 0.1 pmol retinyl esters (A,,, = 5) in 50 mM sodium phosphate buffer pH 7.8 in a final volume of 2 ml. In most cases 10 mg of bovine serum albumin (Sigma, Cohn Fraction V) was also present. The reaction was carried out at 30°C (water bath) for various times and was then stopped by adding 1 ml of 3 M NaCl (addition of salt was also essential to assure complete extraction of retinyl compounds). Three milliliters of ethanol was then added and mixed vigorously for 10 s. A further 3 ml of reagent grade benzene was added and mixed well for 30 s with a Vortex mixer. The upper benzene layer was removed after centrifuging in a clinical centrifuge for 3 min. Total retinyl compound recovery was determined by measuring the absorbance of the benzene phase at 330 nm, using a molar absorbity of E330= 49,300. To determine the amount of unesterified retinol, 2 ml of ethanolic HCl(25 ml ethanol: 2 ml of HCl) was added to 2 ml of the benzene extract and mixed well. After 10min at 22”C, 2 ml of 1 N NaOH was added and mixed well for 5 s. After centrifuging as abovetheabsorbanceofanhydroretinolinthebenzene phase was read at 399 nm. Authentic retinol was used to set up a linear standard curve. Molar absorptivity of anhydroretinol at 399 nm was 55,000, as determined by conversion of a known amount of retinol. Control experiments have shown that authentic
complex.
ESTER LIPOPROTEIN
COMPLEX
573
retinyl palmitate does not react with the dehydrating agent. Also no hydrolysis of retinyl palmitate occurs under the conditions of the assay. Once the HCl had been neutralized with NaOH, anhydroretinol and retinyl ester were stable for hours. Under the experimental conditions described, the recovery of retinyl compounds in the benzene extract was 100%. Experimental points were always performed in duplicates and the agreement between the duplicates was generally 21 to 2% of total retinyl compounds. Since anhydroretinol was measured at 399 nm where retinyl esters have no absorption, as little as 0.003 pmol of retinol could be measured in the presence of 0.1 to 0.3 Fmol of retinyl esters. Retinyl esters and unesterified retinol were also determined by a column chromatography of the extract on aluminum oxide according to Futterman and Andrews (6) as described in the previous paper (1). The chromatographic method gave results that were essentially identical to the dehydration method described above. The chromatographic method is relatively slow, laborious, and potentially less reliable because of the several evaporation steps that are necessary during which retinol may be lost by oxidation. The chromatographic method was used primarily to check the specificity and reliability of the dehydration reaction. Reaction with inhibitors. Holo-CRLP or bovine serum albumin, dissolved in 50 mM sodium phosphate pH 7.4, was reacted with diisopropyl fluorophosphate (0.14 M in ethanol) at 22°C for 1 h. Final concentration of diisopropyl fluorophosphate in reaction mixture was 3 x lOA” M. Additions of diisopropyl fluorophosphate were repeated twice more at l-h intervals. Final concentration of ethanol was 1.8% (v/v). At the end of the reaction, the proteins were dialyzed against 50 mM sodium phosphate. Proteins for control experiments were subjected to the same procedure except that diisopropyl fluorophosphate was omitted. Phenylmethylsulfonyl fluoride (Sigma; 0.1 M ethanol solution) was added to holo-CRLP to final concentrations of 3 x W4 and 10m3M at 22°C. The mixture was incubated for 20 min and was then tested for esterase activity as usual. The sulfhydryl reagent mersalyl acid (Sigma; 0.1 M aqueous solution) was added to holoCRLP to final concentrations of 3 x 10e4and 10m3M, and the protein was incubated for 20 min at 22°C prior to initiating the esterase assay by the addition of serum albumin. Spectroscopy. Absorption spectra and absorbance were measured as described elsewhere (1). RESULTS
Esterase Activity of Holo-CRLP The key observation made in this study was that when holo-CRLP was kept at 4°C at pH 7.4 in 50 mM sodium phosphate, there
574
CHENANDHELLER
was a progressive increase in the proportion of unesterified retinol (Fig. 1). It should be emphasized that during this time there was no change in the total amount of retinyl compounds, only in the relative amounts of unesterified retinol and retinyl esters. The spontaneous rate of retinyl ester hydrolysis was about 1% per day at 4°C and was linear with time for the first 2 weeks (Fig. 1). Effect of Serum Albumin
When bovine serum albumin was added to low salt holo-CRLP there was a marked increase in the rate at which retinyl esters were hydrolyzed (Fig. 2). A certain minimal amount of serum albumin (about lmg under the conditions employed) was necessary in order to observe any effect by albumin (Fig. 2). The rate of retinol production was then linear with the concentration of albumin up to about 10 mg of albumin, after which it still increased with albumin concentration but at a slower rate (Fig. 2). When 50 mg of bovine serum albumin was added to 0.1 pmol of retinyl esters (as holo-CRLP), up to 75 to 80% of the total esters were hydrolyzed after 24 h at 30°C. It was most convenient to use 10 mg of serum albumin and this amount was used in all subsequent experiments unless reported otherwise. Preliminary experiments show that the enhancement of retinyl e&erase activity by serum albumin is caused by the binding of released, unesterified retinol to serum albumin. Under appropriate experimental
DAYS
-
FIG. 1. Hydrolysis of retinyl esters in holo-CRLP. Holo-CRLP after gel filtration chromatography in 50 mM sodium phosphate buffer, pH 7.4, was kept at 4°C and periodically assayed for unesterified retinol content as described under Experimental Procedures.
FIG. 2. Effect of serum albumin. Purified holoCRLP in 50 mM sodium phosphate buffer, pH 7.8, was incubated for 3 h at 30°C with various amounts of bovine serum albumin (Sigma, fraction V). At the end of the incubation period the material was assayed for unesterified retinol. Concentration of CRLP was 0.1 pmol in retinyl compounds (A,,, = 5).
conditions about 75 to 80% of the total retinyl esters in holo-CRLP were hydrolyzed and some 80 to 90% of the retinol that was produced in the reaction was transferred (bound) to serum albumin. No retinyl esters were bound to serum albumin under the same experimental condition. A detailed report on the transfer of unesterified retinol from holo-CRLP to serum albumin and other proteins will be published. Neither P-lactoglobulin nor ovalbumin led to enhancement of the retinyl e&erase activity when added to holo-CRLP in concentrations similar to serum albumin (Table I). Human serum albumin was somewhat less effective than bovine serum albumin, while removal of free fatty acids from bovine albumin increased its effectiveness to a small degree (Table I). The retinyl e&erase activity could also be detected in rat liver cytosol. The esterase activity of cytosol was identical to that of holo-CRLP that was obtained from the same preparation after purification by gel filtration. The retinyl esterase activity of cytosol was also enhanced to a similar degree by serum albumin. It was concluded from these experiments that whole rat liver cytosol does not have an inhibitor or enhancer for retinyl esterase activity. The Rate of Esterase Activity
Incubation of holo-CRLP at pH 7.8 in the presence of bovine serum albumin led to
RETINYL
ESTERASE ACTIVITY
OF RETINYL
TABLE I ENHANCEMENT OF RETINYL ESTERASE ACTIVITY OF
HOLO-CRLP BY DIFFERENT PROTEINS”
Addition None /3-Lactoglobulin Ovalbumin Human serum albumin Fraction V Bovine serum albumin Fraction V Bovine serum albumin, fatty acid free
Retinol liberated (nmol)
Relative activity (percent)
2.4 2.8 2.4
7 9 7
22.9
70
28.0
86
32.5
100
(I Holo-CRLP was incubated at pH 7.8 at 30°C for 3 h and total retinyl compounds and unesterified retinol were measured as described under Experimental Procedures. Protein additions were 10 mg. Concentration of CRLP was 0.1 wmol in retinyl compounds L%no = 5).
hydrolysis of ester at a constant rate for the first 6 h (Fig. 3). In subsequent experiments 3 h was used as standard for e&erase activity.
ESTER LIPOPROTEIN
COMPLEX
575
of detergents was observed in the absence as well as in the presence of serum albumin in the incubation medium (Table II). Effect of Various
Inhibitors
When holo-CRLP was treated with low concentrations of either diisopropyl fluorophosphate or phenylmethylsulfonyl fluoride the retinyl esterase activity was totally abolished (Fig. 6). At the concentrations used, these reagents are considered to be specific inhibitors of serine esterases; thus we tentatively conclude that the retinyl esterase activity of holo-CRLP is a serine esterase. The retinyl esterase activity is also abolished by low concentrations of mersalyl acid, a sulfhydryl reagent (Fig. 6). On the other hand the esterase activity seemed to be little affected by heparin (Fig. 6). Does Serum Albumin Esterase Activity?
Possess Retinyl
Because of the marked enhancement of the retinyl esterase activity of holo-CRLP by serum albumin we were concerned with
pH Optimum
The pH optimum of the retinyl esterase activity in 50 mM sodium phosphate was pH 7.8 (Fig. 4). Esterase activity at pH 6 or 9.5 was about 30% of the activity at pH 7.8. Effect of Temperature
The retinyl esterase activity of holo-CRLP was temperature dependent (Fig. 5), with the activity at 4°C being some 20% of the activity at 30°C. Raising the temperature to 37°C increased the rate even further but also led to loss of total retinyl compounds, presumably due to instability and loss of CRLP at the higher temperatures. The loss of retinyl compounds at 30°C was only some 5% after 24 h. Effect of Detergents
Tween 80 and sodium deoxycholate completely abolished the retinyl activity of holoCRLP while Triton X-100 reduced the activity by some 70% (Table II). The effect
FIG. 3. Time course of retinyl esterase activity. Holo-CRLP in 50 mM sodium phosphate pH 7.8 was incubated at 30°C in the presence or absence of 10 mg bovine serum albumin per 0.1 pmol retinyl esters. At the indicated times the incubation mixture was assayed for unesterified retinal.
576
CHENANDHELLER
excluding the possibility that serum albumin itself might have esterase activity. The evidence against this possibility is unambiguous and is as follows. (a) When the gel filtration purification step of CRLP (1) was performed in a buffer that contained in addition 1 M NaCl, a high molecular weight retinyl ester lipoprotein complex was still obtained.3 This complex differs from the CRLP obtained in low salt buffers by having a higher A33,,lA258ratio due to loss of an AZ5*absorbing component. Removal of the NaCl by dialysis produced a stable retinyl ester lipoprotein complex similar in many ways to that obtained by the usual purification procedure (gel filtration in low salt buffers).3 One notable difference though was the absence of spontaneous esterase activity when the dialyzed “high salt” retinol ester lipoprotein was stored at 4°C. Incubation of dialyzed “high salt” retinyl ester lipoprotein complex with serum albumin does not show any esterase activity (Fig. 7). Thus in order to observe the esterase activity one has to have a “low salt” purified holo-CRLP present (Fig. 7). (b) Serum albumin does not hydrolyze externally added retinyl palmitate (Fig. 7). (c) Treatment of serum albumin with the esterase reagent diisopropyl fluorophosphate reduces the esterase activity of holoCRLP to a slight extent (Fig. 8). On the other hand, treatment of holo-CRLP with diisopropyl fluorophosphate completely abolishes retinyl esterase activity when tested with native serum albumin (Fig. 8). In other words, there is no esterase inhibition when serum albumin is treated, but there is total inhibition of retinyl esterase activity when CRLP is treated, although the assay includes native, untreated serum albumin. (d) Essentially similar results are obtained when the enzyme is destroyed by heat denaturation. Only denaturation of holoCRLP abolishes the retinyl esterase activity (Table III). Heat treatment of serum albumin does reduce its effectiveness as enhancer of the retinyl esterase activity of native holo-CRLP (presumably due to denaturation and lack of binding of released 3 Heller and Chen, unpublished results.
PH
FIG. 4. Effect of pH on retinyl esterase activity. Holo-CRLP was incubated for 3 h at 30°C after adjusting the pH with 5 N NaOH. All incubations were in the presence of 10 mg bovine serum albumin. Unesterified retinol was assayed as described under Experimental Procedures.
unesterified retinol). The effectiveness of albumin was reduced, but the esterase activity of native holo-CRLP in the presence of denatured serum albumin was still more than twice that of the control in the absence of albumin (Table III). Denatured holoCRLP in the presence of native serum albumin completely lost the retinyl esterase activity (Table III). These experiments seem to rule out any possibility that serum albumin as such has a retinyl esterase activity in the assay system used in this work.
FIG. 5. Temperature effect. Holo-CRLP in 50 mM sodium phosphate pH 7.3 was incubated at various temperatures in the presence of 10 mg bovine serum albumin. At the indicated times the incubation mixtures were assayed for unesterified retinol.
RETINYL
ESTERASE ACTIVITY
OF RETINYL
ESTER LIPOPROTEIN
577
COMPLEX
TABLE II EFFECT OF DETERGENTSON THE RETINYL ESTERASEACTIVITY OF HOLO-CRLP” With bovine serum albumin
Without serum albumin
Detergent added
Retinol liberated (nmol)
None Triton X-100 Tween 80 Sodium deoxycholate
2.1 0.7 0.1 0.0
Relative activity (percent)
Retinol liberated (nmol) 21.5 6.0 0.5 0.0
100 33 5 0
Relative activity (percent) 100 28 2 0
‘1Holo-CRLP was incubated at pH 7.8 at 30°C for 3 h and unesterified retinol was assayed as described under Experimental Procedures. Detergents were added to a final concentration of 0.2%. Concentration of CRLP was 0.1 prnol in retinyl compounds (A,,, = 5). DISCUSSION
There have been several previous reports of retinyl esterase activity(ies) in rat liver (2, 4). The present study differs from previous studies in one fundamental aspect: No detergents were used as dispersing agents for the substrate. Previous studies had to use detergents such as Triton X-100 and/or sodium taurocholate to solubilize the retinyl ester substrate (2, 4). This was the case even when the “natural” substrate was used, i.e., a creamy lipid float obtained by centrifugation of rat liver homogenate (2, 4). In the present study the retinyl
esterase activity was part of a purified retinyl ester lipoprotein complex isolated from rat liver. The esterase activity of CRLP not only did not require detergents but was actually severely or completely inhibited by them at fairly low concentrations. Thus, while at least 0.1% (final concentration) of sodium taurocholate was essential foresterase activity described by Mahadevan et al. (2), and 1% was optimal, the retinyl esterase activity shown by holo-CRLP was totally
40/40
HOURS HOURS
FIG. 6. Effects of inhibitors. Holo-CRLP in 50 mM sodium phosphate, pH 7.8, was preincubated with diisopropyl fluorophosphate (DFP) 1.4 x 10m4M, phenylmethylsulfonyl fluoride (PSF), mersalyl acid (MA) 3 x 10e4M, or heparin for 30 min at 30°C. Bovine serum albumin was then added (10 mg/O.l prnol retinyl esters) and the material incubated at 30°C for the indicated times. The incubation mixture was then assayed for unesterified retinol.
FIG. 7. Retinyl e&erase activity of low and high salt CRLP. Low salt holo-CRLP, 0.1 pmolketinyl compounds, in 50 mM sodium phosphate buffer, pH 7.8, was incubated at 30°C with 10 mg bovine serum albumin. High salt holo-CRLP (HS-CRLP) was similarly incubated with serum albumin. Retinyl palmitate, 0.1 Fmol, in ethanol was added to 10 mg bovine serum albumin and incubated as above. At the indicated times the incubation mixtures were assayed for unesterified retinol.
578
CHENANDHELLER
abolished by 0.2% sodium deoxycholate and 95 to 98% decreased by 0.2% Tween 80. Holo-CRLP reacts as a classical serine esterase. It is completely inhibited by low concentrations of diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. These inhibitors made it clear that the e&erase activity resides in the holo-CRLP molecule and that treatment of serum albumin does not abolish the e&erase. In the future these inhibitors should prove useful as labeling reagents for the active site of the retinyl esterase. The esterase activity shown by CRLP had other interesting properties. We found that the retinyl ester lipoprotein complex from rat liver cytosol can be purified in two forms. When the lipoprotein complex was purified in the presence of 1 M NaCl the A33,,lA258ratio increased and the esterase activity was lost.3 This gentle way of obtaining inactive CRLP proved useful in showing that the esterase activity resides in holoCRLP and that the enhancement of esterase activity by the addition of serum albumin was not due to any inherent esterase activity of albumin. It seems that serum albumin by binding the released unesterified retinol provides a “sink” for the hydrolysis products. The transfer of unesterified retinol from holo-CRLP to serum albumin and other proteins will be a subject of a forthcoming report. Possibly the most fascinating aspect of the present work is the finding of the dual nature of holo-CRLP. Thus CRLP is not only a storage form for retinyl compounds TABLE
8 $ 3 B : E y L2 h
FIG. 8. Effect of DFP on CRLP and serum albumin. Holo-CRLP or bovine serum albumin were treated with DFP to a final concentration of 2.4 x 10e3 M. After 3 h incubation the proteins were dialyzed as described under Experimental Procedures. Proteins that were not treated with DFP were subjected to the same experimental manipulations except that DFP was omitted. At the indicated times samples were taken for assay of unesterified retinol.
in the liver but in addition acts as the esterase for the predominant retinyl storage form, i.e., retinyl ester. It is well established that retinol is mobilized from the liver to the peripheral target tissues as a complex with serum RBP (‘7). Serum RBP is synthesized in the liver, and in the form isolated from the blood it is found exclusively as all-truns retinol-RBP complex (8). It is also well established that apo-serum RBP does not bind retinyl palmitate (9), the predominant III
EFFECTOFTEMPERATURE DENATURATION"
After
Holo-CRLP (0.1 firno1 retinyl compounds)
Bovine serum albumin added, 10 mg
Native Native Native Heat treated Heat treated Heat treated
None Native Heat treated None Native Heat treated
Retinol liberated, (nmol) 1.9 28.9 4.7 0.0 0.0 0.0
Relative activity (percent) 7 100 16 0 0 0
a Serum albumin or holo-CRLP were heated for 30 min at 85°C in 50 mM sodium phosphate buffer pH 7.8. cooling the esterase assay was performed as described under Experimental Procedures.
RETINYL
ESTERASE
ACTIVITY
OF RETINYL
retinyl ester in the liver (10). The small amounts of retinyl esters found in the blood are found in chylomicrons derived from the intestinal absorption of retinol(10). It seems clear that the stored retinyl ester in holoCRLP has to be hydrolyzed to free retinol before it is able to combine with apo-serum RBP to form retinol-serum RBP complex. Thus, one of the functions of holo-CRLP is to act as an esterase on its own stored retinyl ester, a reaction that produces unesterified retinol in situ. The unesterified retinol then may be transferred to aposerum RBP to form the complex retinolserum RBP which is then secreted by the liver into the bloodstream. An important aspect of this proposed scheme is that neither retinyl esters nor retinal need ever to leave their binding proteins. Since both retinyl esters and retinol are lipids that are easily damaged by the environment, besides being totally insoluble in aqueous media, this might be an important biological means of protecting these vitamins by sequestering them in storage forms that also double as enzymes. Several steps in this intracellular transfer of retinol remain to be worked out in detail. We need to know the factors that control the esterase activity in vivo, whether holo-
ESTER
LIPOPROTEIN
COMPLEX
579
CRLP also has an ester-synthesizing activity, how unesterified retinol is transferred to apo-serum RBP, and whether other proteins are involved in this transfer. We hope, now that we know some of the carriers and enzyme activities that are involved in this transfer, it will be possible to elucidate the rest of the necessary steps. REFERENCES 1. HELLER, J. (1979) Arch. Biochem. Biophys. 198, 562-571. 2. MAHADEVAN, S., AYYOUB, N. I., AND ROELS, A. 0. (1966) J. Biol. Chem. 241, 5’7-64. 3. YEUNG, D. L., AND VEEN-BAIGENT, M. J. (1971) Amer. J. Clin. Nub. 24, 172-173. 4. NIR, I., BRUCKENTAL, I., AND ASCARELLI, I. (1967) Brit. J. Nutr. 21, 557-563. 5. HARASHIMA, K., OKAZAKI, H., AND AOKI, H. (1961) J. Vitaminol. 7, 150-162. 6. FUTTERMAN, S., AND ANDREWS, J. S. (1964) J. Biol. Chem. 239, 81-84. 7. GOODMAN, DEW. S. (1974) Vitam. Harm. 32, 167-180. 8. HELLER, J., AND HORWITZ, J. (1973) J. Biol. Chm. 248, 6308-6316. 9. HORWITZ, J., AND HELLER, J. (1973) J. Biol. Chem. 249, 4712-4719. 10. GOODMAN, DEW. S., HUANG, H. S., AND SHIRATORI, T. (1965) J. Lipid Res. 6,390-396.
Vol. 198, No. 2, December, pp. 572-579, 1979
Retinyl Esterase Activity of Purified Rat Liver Retinyl Ester Lipoprotein Complex1 CHI-CHING
CHEN AND JORAM HELLER
Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, California
90024
Received February 5, 1979; revised July 30, 1979 Retinyl ester lipoprotein complex from rat liver was shown to possess a retinyl esterase activity toward its own ligand complement. In the presence of serum albumin the retinyl esterase activity at 30°C was about fivefold larger than the activity at 4”C, while higher temperatures than 30°C led to some degradation of retinyl compounds. The pH optimum was 7.8. The esterase activity was markedly enhanced by serum albumin although the serum albumin as such had no retinyl esterase activity. In the presence of serum albumin and under optimal conditions, some ‘75to 80% of the total retinyl ester complement of the lipoprotein was hydrolyzed in 24 h. The retinyl esterase activity was totally abolished by treatment with the serine esterase inhibitor diisopropyl fluorophosphate (1.4 x 10m4M), by treatment with sulfhydryl reagents, and by detergents (0.2% of Tween 80 and sodium deoxycholate). From this series of experiments it was concluded that the retinyl ester lipoprotein complex possesses the additional physiological function of hydrolyzing its own retinyl ester complement to unesterified retinol which may then combine with serum retinol-binding protein.
In the previous paper (1) we have reported the purification and characterization of a retinyl ester lipoprotein complex from rat liver. The complex contains about 66% (by weight) of lipids, 30% protein, and some 4% of carbohydrate. Freshly isolated holoCRLP2 contains about 3% retinyl compounds, 96% of which are retinyl esters and 4% unesterified retinol. It was noted that when holo-CRLP was kept at 4”C, the relative amounts of the retinyl esters and unesterified retinol changed with time, although the total retinyl complement of the carrier complex did not change. This observation prompted the present study. The present paper describes the retinyl esterase activity of holo-CRLP. Mehadevan et al. (2) reported in 1966 the isolation from rat liver of an enzyme that hydrolyzes retinyl palmitate. The enzyme
was present in nuclear and mitochondriallysosome-rich fraction. It was dependent on the presence of bile salts in the reaction mixture and the externally added retinyl compounds used as substrate had to be dispersed in bile salts and Triton X-100. The pH optimum of the retinyl esterase reported by Mehadevan et al. was 8.6. Additions of serum albumin had no effect on the esterase activity. Yeung and Veen-Baigent (3) have subsequently reported their inability to find a retinyl palmitate hydrolyse activity in rat liver following the procedure of Mehadevan et al. (2). Nir et al. (4) reported that chick liver had minimal esterase activity toward retinyl palmitate, the predominant storage form in the liver, while it had considerable activity against retinyl acetate (which is not found naturally in the body). It is important to bear in mind that in all previous work on retinyl esterase activities of various tissues 1 This paper was supported by Grants EY 00704, externally added substrate was used. In EY 00331, and EY 00702 from the National Institutes every case the substrates that are totally of Health, USPHS. insoluble in aqueous media had to be dis* Abbreviations used: CRLP, cytosol retinyl ester persed in various detergents. In the present lipoprotein complex; serum RBP, serum retinol-binding report we have been able to dispense with protein; BSA, bovine serum albumin; DFP, diisopropyl detergents, because the retinyl esters were fluorophosphate; PSF, phenylmethylsulfonyl fluoride.
0003-9861/79/140572-08$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
572
RETINYL
ESTERASE ACTIVITY
OF RETINYL
obtained as soluble complexes with a natural carrier. The present work also provides a possible explanation for the physiological link between retinyl esters, which are the storage form in the liver, and retinol, which is the transportable form. EXPERIMENTAL
PROCEDURES
Preparation of retinyl ester carrier lipoglycoprotein The preparation of holo-CRLP from rat liver was described in detail in the preceding paper (1). In the present series of experiments holo-CRLP was normally used within 3 to 4 days after preparation. It was kept at 4°C with lo-$ M sodium azide. Since there was no difference in the esterase activity between the gel filtration CRLP and the hydroxyapatitepurified material the former was used in some experiments. Esterase assay. The assay was based on the determination of unesterified retinol in the presence of retinyl esters according to Harashima et al. (5). This method uses the conversion of unesterified retinol to anhydroretinol by dilute hydrochloric acid in alcoholic solutions. The anhydroretinol has a unique absorption spectrum with several peaks, one of which is at 399 nm, where retinyl esters have no absorption. The great advantage of this method is the lack of reactivity of retinyl ester in the dehydration reaction, Thus unesterified retinol can be accurately determined in the presence of retinyl esters. Standard reaction mixture contained 0.1 pmol retinyl esters (A,,, = 5) in 50 mM sodium phosphate buffer pH 7.8 in a final volume of 2 ml. In most cases 10 mg of bovine serum albumin (Sigma, Cohn Fraction V) was also present. The reaction was carried out at 30°C (water bath) for various times and was then stopped by adding 1 ml of 3 M NaCl (addition of salt was also essential to assure complete extraction of retinyl compounds). Three milliliters of ethanol was then added and mixed vigorously for 10 s. A further 3 ml of reagent grade benzene was added and mixed well for 30 s with a Vortex mixer. The upper benzene layer was removed after centrifuging in a clinical centrifuge for 3 min. Total retinyl compound recovery was determined by measuring the absorbance of the benzene phase at 330 nm, using a molar absorbity of E330= 49,300. To determine the amount of unesterified retinol, 2 ml of ethanolic HCl(25 ml ethanol: 2 ml of HCl) was added to 2 ml of the benzene extract and mixed well. After 10min at 22”C, 2 ml of 1 N NaOH was added and mixed well for 5 s. After centrifuging as abovetheabsorbanceofanhydroretinolinthebenzene phase was read at 399 nm. Authentic retinol was used to set up a linear standard curve. Molar absorptivity of anhydroretinol at 399 nm was 55,000, as determined by conversion of a known amount of retinol. Control experiments have shown that authentic
complex.
ESTER LIPOPROTEIN
COMPLEX
573
retinyl palmitate does not react with the dehydrating agent. Also no hydrolysis of retinyl palmitate occurs under the conditions of the assay. Once the HCl had been neutralized with NaOH, anhydroretinol and retinyl ester were stable for hours. Under the experimental conditions described, the recovery of retinyl compounds in the benzene extract was 100%. Experimental points were always performed in duplicates and the agreement between the duplicates was generally 21 to 2% of total retinyl compounds. Since anhydroretinol was measured at 399 nm where retinyl esters have no absorption, as little as 0.003 pmol of retinol could be measured in the presence of 0.1 to 0.3 Fmol of retinyl esters. Retinyl esters and unesterified retinol were also determined by a column chromatography of the extract on aluminum oxide according to Futterman and Andrews (6) as described in the previous paper (1). The chromatographic method gave results that were essentially identical to the dehydration method described above. The chromatographic method is relatively slow, laborious, and potentially less reliable because of the several evaporation steps that are necessary during which retinol may be lost by oxidation. The chromatographic method was used primarily to check the specificity and reliability of the dehydration reaction. Reaction with inhibitors. Holo-CRLP or bovine serum albumin, dissolved in 50 mM sodium phosphate pH 7.4, was reacted with diisopropyl fluorophosphate (0.14 M in ethanol) at 22°C for 1 h. Final concentration of diisopropyl fluorophosphate in reaction mixture was 3 x lOA” M. Additions of diisopropyl fluorophosphate were repeated twice more at l-h intervals. Final concentration of ethanol was 1.8% (v/v). At the end of the reaction, the proteins were dialyzed against 50 mM sodium phosphate. Proteins for control experiments were subjected to the same procedure except that diisopropyl fluorophosphate was omitted. Phenylmethylsulfonyl fluoride (Sigma; 0.1 M ethanol solution) was added to holo-CRLP to final concentrations of 3 x W4 and 10m3M at 22°C. The mixture was incubated for 20 min and was then tested for esterase activity as usual. The sulfhydryl reagent mersalyl acid (Sigma; 0.1 M aqueous solution) was added to holoCRLP to final concentrations of 3 x 10e4and 10m3M, and the protein was incubated for 20 min at 22°C prior to initiating the esterase assay by the addition of serum albumin. Spectroscopy. Absorption spectra and absorbance were measured as described elsewhere (1). RESULTS
Esterase Activity of Holo-CRLP The key observation made in this study was that when holo-CRLP was kept at 4°C at pH 7.4 in 50 mM sodium phosphate, there
574
CHENANDHELLER
was a progressive increase in the proportion of unesterified retinol (Fig. 1). It should be emphasized that during this time there was no change in the total amount of retinyl compounds, only in the relative amounts of unesterified retinol and retinyl esters. The spontaneous rate of retinyl ester hydrolysis was about 1% per day at 4°C and was linear with time for the first 2 weeks (Fig. 1). Effect of Serum Albumin
When bovine serum albumin was added to low salt holo-CRLP there was a marked increase in the rate at which retinyl esters were hydrolyzed (Fig. 2). A certain minimal amount of serum albumin (about lmg under the conditions employed) was necessary in order to observe any effect by albumin (Fig. 2). The rate of retinol production was then linear with the concentration of albumin up to about 10 mg of albumin, after which it still increased with albumin concentration but at a slower rate (Fig. 2). When 50 mg of bovine serum albumin was added to 0.1 pmol of retinyl esters (as holo-CRLP), up to 75 to 80% of the total esters were hydrolyzed after 24 h at 30°C. It was most convenient to use 10 mg of serum albumin and this amount was used in all subsequent experiments unless reported otherwise. Preliminary experiments show that the enhancement of retinyl e&erase activity by serum albumin is caused by the binding of released, unesterified retinol to serum albumin. Under appropriate experimental
DAYS
-
FIG. 1. Hydrolysis of retinyl esters in holo-CRLP. Holo-CRLP after gel filtration chromatography in 50 mM sodium phosphate buffer, pH 7.4, was kept at 4°C and periodically assayed for unesterified retinol content as described under Experimental Procedures.
FIG. 2. Effect of serum albumin. Purified holoCRLP in 50 mM sodium phosphate buffer, pH 7.8, was incubated for 3 h at 30°C with various amounts of bovine serum albumin (Sigma, fraction V). At the end of the incubation period the material was assayed for unesterified retinol. Concentration of CRLP was 0.1 pmol in retinyl compounds (A,,, = 5).
conditions about 75 to 80% of the total retinyl esters in holo-CRLP were hydrolyzed and some 80 to 90% of the retinol that was produced in the reaction was transferred (bound) to serum albumin. No retinyl esters were bound to serum albumin under the same experimental condition. A detailed report on the transfer of unesterified retinol from holo-CRLP to serum albumin and other proteins will be published. Neither P-lactoglobulin nor ovalbumin led to enhancement of the retinyl e&erase activity when added to holo-CRLP in concentrations similar to serum albumin (Table I). Human serum albumin was somewhat less effective than bovine serum albumin, while removal of free fatty acids from bovine albumin increased its effectiveness to a small degree (Table I). The retinyl e&erase activity could also be detected in rat liver cytosol. The esterase activity of cytosol was identical to that of holo-CRLP that was obtained from the same preparation after purification by gel filtration. The retinyl esterase activity of cytosol was also enhanced to a similar degree by serum albumin. It was concluded from these experiments that whole rat liver cytosol does not have an inhibitor or enhancer for retinyl esterase activity. The Rate of Esterase Activity
Incubation of holo-CRLP at pH 7.8 in the presence of bovine serum albumin led to
RETINYL
ESTERASE ACTIVITY
OF RETINYL
TABLE I ENHANCEMENT OF RETINYL ESTERASE ACTIVITY OF
HOLO-CRLP BY DIFFERENT PROTEINS”
Addition None /3-Lactoglobulin Ovalbumin Human serum albumin Fraction V Bovine serum albumin Fraction V Bovine serum albumin, fatty acid free
Retinol liberated (nmol)
Relative activity (percent)
2.4 2.8 2.4
7 9 7
22.9
70
28.0
86
32.5
100
(I Holo-CRLP was incubated at pH 7.8 at 30°C for 3 h and total retinyl compounds and unesterified retinol were measured as described under Experimental Procedures. Protein additions were 10 mg. Concentration of CRLP was 0.1 wmol in retinyl compounds L%no = 5).
hydrolysis of ester at a constant rate for the first 6 h (Fig. 3). In subsequent experiments 3 h was used as standard for e&erase activity.
ESTER LIPOPROTEIN
COMPLEX
575
of detergents was observed in the absence as well as in the presence of serum albumin in the incubation medium (Table II). Effect of Various
Inhibitors
When holo-CRLP was treated with low concentrations of either diisopropyl fluorophosphate or phenylmethylsulfonyl fluoride the retinyl esterase activity was totally abolished (Fig. 6). At the concentrations used, these reagents are considered to be specific inhibitors of serine esterases; thus we tentatively conclude that the retinyl esterase activity of holo-CRLP is a serine esterase. The retinyl esterase activity is also abolished by low concentrations of mersalyl acid, a sulfhydryl reagent (Fig. 6). On the other hand the esterase activity seemed to be little affected by heparin (Fig. 6). Does Serum Albumin Esterase Activity?
Possess Retinyl
Because of the marked enhancement of the retinyl esterase activity of holo-CRLP by serum albumin we were concerned with
pH Optimum
The pH optimum of the retinyl esterase activity in 50 mM sodium phosphate was pH 7.8 (Fig. 4). Esterase activity at pH 6 or 9.5 was about 30% of the activity at pH 7.8. Effect of Temperature
The retinyl esterase activity of holo-CRLP was temperature dependent (Fig. 5), with the activity at 4°C being some 20% of the activity at 30°C. Raising the temperature to 37°C increased the rate even further but also led to loss of total retinyl compounds, presumably due to instability and loss of CRLP at the higher temperatures. The loss of retinyl compounds at 30°C was only some 5% after 24 h. Effect of Detergents
Tween 80 and sodium deoxycholate completely abolished the retinyl activity of holoCRLP while Triton X-100 reduced the activity by some 70% (Table II). The effect
FIG. 3. Time course of retinyl esterase activity. Holo-CRLP in 50 mM sodium phosphate pH 7.8 was incubated at 30°C in the presence or absence of 10 mg bovine serum albumin per 0.1 pmol retinyl esters. At the indicated times the incubation mixture was assayed for unesterified retinal.
576
CHENANDHELLER
excluding the possibility that serum albumin itself might have esterase activity. The evidence against this possibility is unambiguous and is as follows. (a) When the gel filtration purification step of CRLP (1) was performed in a buffer that contained in addition 1 M NaCl, a high molecular weight retinyl ester lipoprotein complex was still obtained.3 This complex differs from the CRLP obtained in low salt buffers by having a higher A33,,lA258ratio due to loss of an AZ5*absorbing component. Removal of the NaCl by dialysis produced a stable retinyl ester lipoprotein complex similar in many ways to that obtained by the usual purification procedure (gel filtration in low salt buffers).3 One notable difference though was the absence of spontaneous esterase activity when the dialyzed “high salt” retinol ester lipoprotein was stored at 4°C. Incubation of dialyzed “high salt” retinyl ester lipoprotein complex with serum albumin does not show any esterase activity (Fig. 7). Thus in order to observe the esterase activity one has to have a “low salt” purified holo-CRLP present (Fig. 7). (b) Serum albumin does not hydrolyze externally added retinyl palmitate (Fig. 7). (c) Treatment of serum albumin with the esterase reagent diisopropyl fluorophosphate reduces the esterase activity of holoCRLP to a slight extent (Fig. 8). On the other hand, treatment of holo-CRLP with diisopropyl fluorophosphate completely abolishes retinyl esterase activity when tested with native serum albumin (Fig. 8). In other words, there is no esterase inhibition when serum albumin is treated, but there is total inhibition of retinyl esterase activity when CRLP is treated, although the assay includes native, untreated serum albumin. (d) Essentially similar results are obtained when the enzyme is destroyed by heat denaturation. Only denaturation of holoCRLP abolishes the retinyl esterase activity (Table III). Heat treatment of serum albumin does reduce its effectiveness as enhancer of the retinyl esterase activity of native holo-CRLP (presumably due to denaturation and lack of binding of released 3 Heller and Chen, unpublished results.
PH
FIG. 4. Effect of pH on retinyl esterase activity. Holo-CRLP was incubated for 3 h at 30°C after adjusting the pH with 5 N NaOH. All incubations were in the presence of 10 mg bovine serum albumin. Unesterified retinol was assayed as described under Experimental Procedures.
unesterified retinol). The effectiveness of albumin was reduced, but the esterase activity of native holo-CRLP in the presence of denatured serum albumin was still more than twice that of the control in the absence of albumin (Table III). Denatured holoCRLP in the presence of native serum albumin completely lost the retinyl esterase activity (Table III). These experiments seem to rule out any possibility that serum albumin as such has a retinyl esterase activity in the assay system used in this work.
FIG. 5. Temperature effect. Holo-CRLP in 50 mM sodium phosphate pH 7.3 was incubated at various temperatures in the presence of 10 mg bovine serum albumin. At the indicated times the incubation mixtures were assayed for unesterified retinol.
RETINYL
ESTERASE ACTIVITY
OF RETINYL
ESTER LIPOPROTEIN
577
COMPLEX
TABLE II EFFECT OF DETERGENTSON THE RETINYL ESTERASEACTIVITY OF HOLO-CRLP” With bovine serum albumin
Without serum albumin
Detergent added
Retinol liberated (nmol)
None Triton X-100 Tween 80 Sodium deoxycholate
2.1 0.7 0.1 0.0
Relative activity (percent)
Retinol liberated (nmol) 21.5 6.0 0.5 0.0
100 33 5 0
Relative activity (percent) 100 28 2 0
‘1Holo-CRLP was incubated at pH 7.8 at 30°C for 3 h and unesterified retinol was assayed as described under Experimental Procedures. Detergents were added to a final concentration of 0.2%. Concentration of CRLP was 0.1 prnol in retinyl compounds (A,,, = 5). DISCUSSION
There have been several previous reports of retinyl esterase activity(ies) in rat liver (2, 4). The present study differs from previous studies in one fundamental aspect: No detergents were used as dispersing agents for the substrate. Previous studies had to use detergents such as Triton X-100 and/or sodium taurocholate to solubilize the retinyl ester substrate (2, 4). This was the case even when the “natural” substrate was used, i.e., a creamy lipid float obtained by centrifugation of rat liver homogenate (2, 4). In the present study the retinyl
esterase activity was part of a purified retinyl ester lipoprotein complex isolated from rat liver. The esterase activity of CRLP not only did not require detergents but was actually severely or completely inhibited by them at fairly low concentrations. Thus, while at least 0.1% (final concentration) of sodium taurocholate was essential foresterase activity described by Mahadevan et al. (2), and 1% was optimal, the retinyl esterase activity shown by holo-CRLP was totally
40/40
HOURS HOURS
FIG. 6. Effects of inhibitors. Holo-CRLP in 50 mM sodium phosphate, pH 7.8, was preincubated with diisopropyl fluorophosphate (DFP) 1.4 x 10m4M, phenylmethylsulfonyl fluoride (PSF), mersalyl acid (MA) 3 x 10e4M, or heparin for 30 min at 30°C. Bovine serum albumin was then added (10 mg/O.l prnol retinyl esters) and the material incubated at 30°C for the indicated times. The incubation mixture was then assayed for unesterified retinol.
FIG. 7. Retinyl e&erase activity of low and high salt CRLP. Low salt holo-CRLP, 0.1 pmolketinyl compounds, in 50 mM sodium phosphate buffer, pH 7.8, was incubated at 30°C with 10 mg bovine serum albumin. High salt holo-CRLP (HS-CRLP) was similarly incubated with serum albumin. Retinyl palmitate, 0.1 Fmol, in ethanol was added to 10 mg bovine serum albumin and incubated as above. At the indicated times the incubation mixtures were assayed for unesterified retinol.
578
CHENANDHELLER
abolished by 0.2% sodium deoxycholate and 95 to 98% decreased by 0.2% Tween 80. Holo-CRLP reacts as a classical serine esterase. It is completely inhibited by low concentrations of diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. These inhibitors made it clear that the e&erase activity resides in the holo-CRLP molecule and that treatment of serum albumin does not abolish the e&erase. In the future these inhibitors should prove useful as labeling reagents for the active site of the retinyl esterase. The esterase activity shown by CRLP had other interesting properties. We found that the retinyl ester lipoprotein complex from rat liver cytosol can be purified in two forms. When the lipoprotein complex was purified in the presence of 1 M NaCl the A33,,lA258ratio increased and the esterase activity was lost.3 This gentle way of obtaining inactive CRLP proved useful in showing that the esterase activity resides in holoCRLP and that the enhancement of esterase activity by the addition of serum albumin was not due to any inherent esterase activity of albumin. It seems that serum albumin by binding the released unesterified retinol provides a “sink” for the hydrolysis products. The transfer of unesterified retinol from holo-CRLP to serum albumin and other proteins will be a subject of a forthcoming report. Possibly the most fascinating aspect of the present work is the finding of the dual nature of holo-CRLP. Thus CRLP is not only a storage form for retinyl compounds TABLE
8 $ 3 B : E y L2 h
FIG. 8. Effect of DFP on CRLP and serum albumin. Holo-CRLP or bovine serum albumin were treated with DFP to a final concentration of 2.4 x 10e3 M. After 3 h incubation the proteins were dialyzed as described under Experimental Procedures. Proteins that were not treated with DFP were subjected to the same experimental manipulations except that DFP was omitted. At the indicated times samples were taken for assay of unesterified retinol.
in the liver but in addition acts as the esterase for the predominant retinyl storage form, i.e., retinyl ester. It is well established that retinol is mobilized from the liver to the peripheral target tissues as a complex with serum RBP (‘7). Serum RBP is synthesized in the liver, and in the form isolated from the blood it is found exclusively as all-truns retinol-RBP complex (8). It is also well established that apo-serum RBP does not bind retinyl palmitate (9), the predominant III
EFFECTOFTEMPERATURE DENATURATION"
After
Holo-CRLP (0.1 firno1 retinyl compounds)
Bovine serum albumin added, 10 mg
Native Native Native Heat treated Heat treated Heat treated
None Native Heat treated None Native Heat treated
Retinol liberated, (nmol) 1.9 28.9 4.7 0.0 0.0 0.0
Relative activity (percent) 7 100 16 0 0 0
a Serum albumin or holo-CRLP were heated for 30 min at 85°C in 50 mM sodium phosphate buffer pH 7.8. cooling the esterase assay was performed as described under Experimental Procedures.
RETINYL
ESTERASE
ACTIVITY
OF RETINYL
retinyl ester in the liver (10). The small amounts of retinyl esters found in the blood are found in chylomicrons derived from the intestinal absorption of retinol(10). It seems clear that the stored retinyl ester in holoCRLP has to be hydrolyzed to free retinol before it is able to combine with apo-serum RBP to form retinol-serum RBP complex. Thus, one of the functions of holo-CRLP is to act as an esterase on its own stored retinyl ester, a reaction that produces unesterified retinol in situ. The unesterified retinol then may be transferred to aposerum RBP to form the complex retinolserum RBP which is then secreted by the liver into the bloodstream. An important aspect of this proposed scheme is that neither retinyl esters nor retinal need ever to leave their binding proteins. Since both retinyl esters and retinol are lipids that are easily damaged by the environment, besides being totally insoluble in aqueous media, this might be an important biological means of protecting these vitamins by sequestering them in storage forms that also double as enzymes. Several steps in this intracellular transfer of retinol remain to be worked out in detail. We need to know the factors that control the esterase activity in vivo, whether holo-
ESTER
LIPOPROTEIN
COMPLEX
579
CRLP also has an ester-synthesizing activity, how unesterified retinol is transferred to apo-serum RBP, and whether other proteins are involved in this transfer. We hope, now that we know some of the carriers and enzyme activities that are involved in this transfer, it will be possible to elucidate the rest of the necessary steps. REFERENCES 1. HELLER, J. (1979) Arch. Biochem. Biophys. 198, 562-571. 2. MAHADEVAN, S., AYYOUB, N. I., AND ROELS, A. 0. (1966) J. Biol. Chem. 241, 5’7-64. 3. YEUNG, D. L., AND VEEN-BAIGENT, M. J. (1971) Amer. J. Clin. Nub. 24, 172-173. 4. NIR, I., BRUCKENTAL, I., AND ASCARELLI, I. (1967) Brit. J. Nutr. 21, 557-563. 5. HARASHIMA, K., OKAZAKI, H., AND AOKI, H. (1961) J. Vitaminol. 7, 150-162. 6. FUTTERMAN, S., AND ANDREWS, J. S. (1964) J. Biol. Chem. 239, 81-84. 7. GOODMAN, DEW. S. (1974) Vitam. Harm. 32, 167-180. 8. HELLER, J., AND HORWITZ, J. (1973) J. Biol. Chm. 248, 6308-6316. 9. HORWITZ, J., AND HELLER, J. (1973) J. Biol. Chem. 249, 4712-4719. 10. GOODMAN, DEW. S., HUANG, H. S., AND SHIRATORI, T. (1965) J. Lipid Res. 6,390-396.
