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Inhibition of rat liver retinyl palmitate hydrolase activity by ether analogs of cholesteryl esters and acylglycerides
428
Biochimica
ef Siophysica Acta, 794 (1984) 428-434 EIsevier
BBA 51681
INHIBITION OF RAT LIVER RETINYL PALMITATE HYDROLASE ANALOGS OF CHOLESTERYL ESTERS AND A~LGLYCE~DES WILLIAM
S. BLANER
a, GIDEON
HALPERIN
b, OLGA
STEIN ‘, YECHEZKIEL
ACTIVITY BY ETHER
STEIN
b and DeWITT
S. GOODMAN
a
a Department of Medicine, College of Physicians and Surgeons of Columbia University, New York, NY (U.S.A.), b Lipid Research Luboraioty, Department of Medicine B, Hadassah University Hospital, and ’ Department of Experimental Medicine and Cancer Research, Hebrew University - Hadassah Medical School, Jerusalem (Israel) (Received December (Revised manuscript
2nd, 1983) received March
6th, 1984)
Key words: Retinyl palmitate hydroiase; acylglycerol ester hydrolase.
Cholesteryl
alkyd ether analog; Acylglyceride
ether analog; Cholesteryl
ester hydrolase;
Tri-
In previous studies, retinyl palmitate hydrolase activity in rat liver was partly characterized and was found to correlate and to partially copurify with hydrolytic activities against cholesteryl oleate and triolein. ‘Ibe present studies were designed to further explore relationships between these three lipid ester hydrolase activities, by use of non-hy~~yzable ether analogs of cholesteryl esters and a~yl~yce~~s. Cholesteryl ether analogs were potent inhibitors of all three hydrolase activities with relative potencies for the series of ethers of: linoleyl > oleyl = palmitoyl > n-butyl = n-propyl > ethyl = methyl. Retinyt paimitate hydrolase activity was most strongly inactivated by this series of analogs, with 4846% of the activity inhibited at cholesteryl ether levels of 1 PM. The acylglyceride ether analogs were much weaker inhibitors of the three hydrolase activites, with the triolein, diolein and dipalmitin analogs showing similar inhibitory potencies, greater than that of the monolein and monopalmitin analogs. The data demonstrate the potential usefuhress of ether analogs of cholesteryl esters and acylglycerides for exploring some of the ch~acte~sti~ of lipid ester hydrokse activities.
Introduction
The hydrolysis of retinyl esters is essential for the mobilization of vitamin A stores from the liver [1,2]. During hepatic mobilization, stored retinyl esters are enzymically hydrolyzed to free retinol, and the retinol is then secreted from the liver and circulated in plasma bound to its specific transport protein, retinol-binding protein [2,3]. A limited amount of information has been reported about the characteristics of retinyl ester hydrolytic enzyme activity in rat liver [4,5]. In recent studies retinyl ester hydroiase activity from rat liver was partiahy purified (approx. 200-fold) and was found to copurify with hydrolytic activities against cholesteryl oleate and triolein [6]. These three 0005-2760/84/$03.00
0 1984 Elsevier Science Publishers
B.V.
copurifying hydrolytic activities were found to have a number of similar properties (including pH optima, bile salt requirements, apparent molecular size, and hydrophobicity). However, differential solubility and ~bition studies clearly showed that the three lipid hydrolase activites are due to at least three different catalytically active centers, and at least two distinct and separable enzymes. It was suggested [6] that three separate but similar enzymes, that appear to be coordinately regulated, are involved. Non-hydroly~ble ether anaiogs of naturally occurring lipids have been used successfully in a variety of studies examining lipid metabolism and lipid biochemistry. The use of non-hydrolyzable ether analogs of cholesteryl esters has provided
429
much information about the uptake and metabolism of lipids and lipoproteins in both the intact rat [7-111 and in cells in culture in vitro [12-151. A non-hydrolyzable retinyl ether analog of retinyl palmitate has been used to study hepatic uptake from chylomicrons and its persistence in liver [16]. Ether analogs of phospholipids have been used in biochemical studies aimed at understanding the mechanism of phospholipase A, hydrolysis [17,18] and in physical studies examining model membrane systems [19,20]. In this study we report the effects of a series of cholesteryl alkyl ethers and one of acylglyceride ethers, with different alkyl chain compositions, on partially purified rat hepatic retinyl palmitate hydrolase activity. The study aimed both to explore the effects of the ether analogs on specific lipid ester hydrolase activities, and to examine the potential usefulness of such compounds for investigating some of the characteristics of such hydrolase enzymes. Materials and Methods Enzyme Preparation
The hydrolase enzyme preparation used in the work reported here was partially purified from rat liver essentially as described elsewhere [6]. The purification procedure utilized successive chromatographic fractionation of an acetone powder extract on columns of phenyl-Sepharose, DEAESepharose, DEAE-cellulose and heparin-sepharose. The partially purified enzyme preparation contained hydrolytic activities directed against cholesteryl oleate and triolein in addition to that against retinyl pahnitate. The specific activities of the three partially purified hydrolytic activities directed against retinyl palmitate, cholesteryl oleate, and triolein were respectively 61.6, 112.6 and 161.9 nmol free fatty acid formed/n& per mg. The enzyme preparation was stored at - 12 o C. Cholesteryl and acylglyceride
ether, D,L-glyceryl-1,2-dioleyl ether, L-glyceryl-3oleyl ether, D,L-glyceryl-1,Zdipalmitoyl ether and L-glyceryl-1-palmitoyl ether were the acylglyceride ethers utilized in this study. The analogs were stored at - 12” C prior to use. Stock solutions of each analog were prepared in hexane on the day of use. Determination
of enzymatic activity
Assays were carried out as previously described [5,6] in 50 mM Tris-maleate (pH 8.0) containing 0.75% (w/v) sodium cholate. Each assay mixture contained l-3 pg of partially purified enzyme protein and was incubated for 5 minutes at 37 o C. Both the protein content and incubation time of the assay were selected to ensure that approximately linear (initial) reaction rates were being measured, as determined by other studies. The substrates, retinyl [l-i4C]pahnitate, cholesteryl [l“C]oleate and tri-[1-‘4C]olein, were each used (in different assays) at final assay concentrations of 20 PM. Stock solutions of cholesteryl ether analogs and acylglyceride ether analogs were prepared in hexane and mixed with the appropriate ester substrate which was prepared in acetone. This ethersubstrate mixture was evaporated to dryness under a stream of N,, redissolved in acetone, and added to the 50 mM Tris-maleate buffer containing sodium cholate to give the desired substrate and ether analog concentrations. The reaction was started by the addition of enzyme to the assay mix and stopped by the addition of methanol/chloroform/heptane (1.41 : 1.25 : 1.OO, v/v) containing 0.1 mM palmitic acid. The free fatty acids produced as products of the reaction were extracted according to the method of Belfrage and Vaughan [23]. The enzymatic activity was calculated in terms of nmol free fatty acid formed per min, from the amount of labeled free fatty acid formed, together with the known specific activity of the substrate ester.
ether analogs
Seven cholesteryl alkyl ethers and five acylglyceride ethers were prepared and purified as described previously [21,22]. The cholesteryl alkyl ethers utilized in this study were the analogs of the methyl, ethyl, n-propyl, n-butyl, pahnitoyl, oleyl and linoleyl esters of cholesterol. Glyceryl trioleyl
Chemical and radiochemical
methodr
Protein was determined according to the method of Bradford [24] using a bovine serum albumin/ y-globulin mixture as standard. Radioactivity was determined with a liquid scintillation spectrometer (Tricarb model 3255 Packard, Lagrange, IL) utiliz-
430
ing Hydrofluor as scintillation Diagnostics, Sommerville, NJ).
solvent (National
Radioactive cho~este~l [l-““Cloleate, tri[l-r4C] olein and [l-‘4C]palmitic acid were purchased from New England Nuclear (Boston, MA). Unlabeled cholesteryl oleate, triolein, retinyl palmitate, /Icarotene, squalene, a-tocopherol, cholesterol, palmitic acid and sodium cholate were purchased from Sigma Chemical Co. (St. Louis, MO). The squalene was purified by thin-layer chromatography before use. Fig. 2. In~bition of retinyl paimitate hydrolase activity by acylglyceride ethers. All data points are the average of three individual determinations. q, glyceryl trioleyl ether; 0, D,L-
Results Prior to examining the effects of the ether analogs on enzymatic activity, the homogeneity of the substrate-ether dispersion throughout the assay mixture was examined. This dispersion was judged to be homogeneous by three criteria: (1) the labeled substrate in the presence of unlabeled ether analog was uniformly distributed throughout the mixture; (2) labeled cholesteryl linoleyl ether analog in the presence of unlabeled substrate was uniformly distributed throughout the mixture; and (3) the mixture was optically clear, similar to the assay mixture in the absence of ether analog. The uniformity of distribution was determined by radioassay of multiple samples withdrawn from various parts of
-~-_-_____-_--__-___. ._--..--__.___-‘___________,
1. Inhibition of retinyl palmitate hydrolase activity by cholesteryl alkyl ethers. Additions were made as described in Materials and Methods. All data points are the average of three indvidual determinations. 0, cholesteryl linoleyl ether; A, cholesteryl oleyl ether; 0, cholesteryl palmitoyl ether; A, cholesteryl n-butyl ether; a, cholesteryl a-propyl ether; 0, cholesteryl ethyl ether; 0, cholesteryl methyl ether; 0, cholesterol; & pahnitic acid.
glyceryl-1,2-dioleyl A, L-glyceryl-3-oleyl
ether;
A, D,L-glyceryl-1,2-dipalmitoyk
ether; 0, L-glyceryl-1-palmitoyl
ether;
ether.
the substrate solution. These observations indicate that the ether analogs and substrate were both uniformly dispersed and hence presumably accessible to enzyme protein. As can be seen in Fig. 1, the cholesteryl alkyl ethers were highly potent inhibitors of retinyl palmitate hydrolase activity. The ethers with long alkyl side-chains (C,,-C,,) were more potent inhibitors than those with short chains (C,-C,). Cholesteryl linoleyl ether was the most potent inhibitor in the series of compounds studied. Free cholesterol weakly inhibited retinyl pahnitate hydrolase activity, and free pahnitic acid was not inhibitory throughout the entire concentration range tested. Fig. 2 shows the effects of the acylglyceride ether analogs on retinyl palmitate hydrolase activity. The acylglyceride ethers were markedly less potent inhibitors of this activity than were the cholesteryl alkyl ethers. The analogs of triolein, diolein and dipal~tin were appro~mately equal in inhibitory effects, while the monoiein and monopalmitin analogs were minimally inhibitory. At concentrations of acylglyceride ether greater than 10 PM, solubility problems were encountered that prevented the testing of higher concentrations of these analogs. Figs. 3 and 4 show the effects of the cholesteryl alkyl ethers (Fig. 3) and of the acylglyceride ethers (Fig. 4) on cholesteryl oleate hydrolysis. As with
431
---..____i 0
I1
2
1
I
4
f
I
6
I,,
6
I
IO
kaleQ]t
I
I
12
I,
14
I
I
16
I,
18
I,
20
0
1,,,,,,,,,,,,,,,,,,, 2 4 6
PM
8
IO
12
14
16
I6
20
s PM
ketogl
Fig. 3. Inhibition of cholesteryl oleate hydrolase activity by cholesteryl alkyl ethers. All data points are the average of three individual dete~nations. 0, cholesteryl hnoleyl ether; A, cholesteryl oleyl ether; 0, cholesteryl pabnitoyl ether; A, cholesteryl n-butyl ether; l , cholesteryl n-propyl ether; D, cholesteryl ethyl ether; 0, cholesteryl methyl ether; l, cholesterol; n, pahnitic acid.
Fig. 5. Inhibition of triolein hydrolase activity by cholesteryl alkyl ethers. All data points are the average of three individual det~~nations. 0, cholesteryl linoleyl ether; A, cholesteryl oleyl ethers; 0, cholesteryl palmitoyl ether; A, cholesteryl n-butyl ether; 0, cholesteryl n-propyl ether; Cl, cholesteryl ethyl ether; 0, cholesteryl methyl ether; 0, cholesterol; H. palmitic acid.
retinyl palmitate hydrolase activity (Figs. 1 and 2), the cholesteryl alkyl ether analogs were much more potent inhibitors of cholesteryl oleate hydrolase activity than were the acylglyceride ether analogs. Again, the long-chain ether analogs (linoleyl, oleyl and palmitoyl side-chains) were more potent inhibitors than the short-chain analogs .(n-butyl, n-propyl, ethyl, and methyl). Free cholesterol was weakly inhibitory,, slightly less so than the methyl and ethyl cholesteryl ether analogs. Palmitic acid was without effect. The acylglyceride ether analogs were only weakly inhibitory in the concentration range examined, with the glyceryl trioleyl ether
being a slightly stronger inhibitor than the monoor diacylglycerol ether analogs. Like the retinyl palmitate and cholesteryl oleate hydrolase activities, the triolein hydrolase activity was strongly inhibited by the cholesteryl alkyl ethers (Fig. 5). Here, too, the linoleyl, oleyl and pal~toyl ether analogs were stronger inhibitors than the short-chain ethers. Neither free cholesterol nor palmitic acid was inhibitory in the concentration range examined. Triolein hydrolase activity was weakly and similarly inhibited by the triolein, diolein and dipalmitin ether analogs, and minimally or not inhibited by the monoacylglycerol analogs (Fig. 6).
I-
B
0 t-
5 0 a w ii
40
20 i
1‘Oi---0
2
4
6
8
IO
CAnabl. PM Fig. 4. inhibition of cholesteryl oleate hydrolase activity by acylglyceride ethers. All data points are the average of three individual dete~nations. 0, glyceryt trioieyl ether; 0, D,LSybil-l,~-diol~l ether; A, D,L-~y~l-~,2_dipalmitoyl ether; A, L-gtyceryl-3-oleyl ether; 0, L-glyceryl-l-pahnitoyl ether.
Fig. 6. Inhibition of triolein hydrolase activity by acylglycetide ethers. All data points are the average of three individual determinations. 0, glyceryl trioleyl ether, 0, D,L-glyceryf-1,2dioleyl ether; A, D,L-~yce~l-l,2-dip~toyl ether; A, Lgtyceryl-3-oleyl ether; e, L-~y~~~-l-pa~toyl ether.
432
TABLE I RELATIVE INHIBITORY POTENCIES OF ETHER ANALOGS ON RAT LIVER HYDROLASE RETINYL PALMITATE (RPH), CHOLESTERYL OLEATE (COH) AND TRIOLEIN (TOH)
ACTIVITIES
AGAINST
The concentrations of each of the ether analogs necessary to decrease the specified hydrolase activity by 50% of control values are listed in this table. These values were obtained graphically from Figures 1 through 6 by estimating from the individual inhibition profiles the concentration of analog needed to give a 50% reduction in activity. It should be noted that the more potent an inhibitor a given compound is, the lower the concentration needed to achieve 50% reduction in activity. Ether analog
Cholesterol Cholesteryl methyl ether Cholesteryl ethyl ether Cholesteryl n-propyl ether Cholesteryl n-butyl ether Cholesteryl palmitoyl ether Cholesteryl oleyl ether Cholesteryl linoleyl ether Glyceryl trioleyl ether D,L-Glyceryl-1,2-dioleyl ether D,L-Glyceryl-1,2dipalmitoyl ether L-Glyceryl-3-oleyl ether L-Glyceryl-l-palmitoyl ether Sodium pahnitate
Concentration Inhibition
(CM) for 50%
RPH
COH
TOH
4.2 0.81 0.90 0.50 0.60 0.20 0.22 0.16 > 10 >lO >lO >lO > 10 z 20
18.0 9.6 8.4 3.1 3.0 1.4 0.90 0.80 >lO >lO 210 >lO >lO > 20
20.0 2.5 2.1 3.0 6.0 0.70 1.0 0.80 >lO > 10 > 10 > 10 210 > 20
Table I presents a summary of the effects of cholesteryl alkyl ether and acylglyceride ether analogs on each of the three hydrolase activities studied. The values listed represent the approximate concentration of each compound needed to inhibit the given hydrolase activity by 50%, as estimated graphically from the data in Figs. l-6. As already mentioned, for each of the three hydrolase activities, the cholesteryl alkyl ethers were more potent inhibitors than were the acylglyceride ether analogs. For all three hydrolase activities, the long-chain cholesteryl alkyl ethers were more potent inhibitors than were the short-chain ethers, with cholesteryl linoleyl ether being most inhibitory. Finally, the cholesteryl alkyl ethers inhibited retinyl pahnitate hydrolase activity more strongly than they did either of the other two hydrolase activites; thus, for each cholesteryl alkyl ether, 50% inhibition of retinyl palmitate hydrolase activity was achieved at a lower concentration of ether than that needed for equivalent inhibition of ether cholesteryl oleate or triolein hydrolase activity. As an additional control, three other hydrophobic, lipid-soluble compounds were examined to
ascertain whether they would inhibit retinyl palmitate, cholesteryl oleate or triolein hydrolase activites. The compounds tested included two polyisoprenoid hydrocarbons, squalene and /3carotene, and a-tocopherol. Each compound was tested against each hydrolase activity over the concentration range l-10 PM. These three compounds were found to be weakly inhibitory of the hydrolase activities, but much less potent as inhibitors than were the cholesteryl alkyl ethers. Thus, at the concentration of 4 pm, squalene showed 7-1795 inhibition, p-carotene showed 19-39% inhibition and cy-tocopherol showed 16-24% inhibition of the three hydrolase activities, compared to the activities seen without any addition. Discussion The present study was undertaken to explore the effects of ether analogs of cholesteryl esters and of acylglycerides on the enzymatic hydrolysis of retinyl palmitate, cholesteryl oleate and triolein using a partially purified enzyme preparation iso-
433
lated from rat liver. The cholesteryl alkyl ethers proved to be very potent inhibitors of all three of the hydrolase activities. A pattern of inhibitory potency for the cholesteryl alkyl ether analogs based on alkyl side-chain length was observed, with relative potencies for this series of ethers of: linoleyl > oleyl = palmitoyl > n-butyl = n-propyl > ethyl = methyl. Cholesterol was less strongly inhibitory towards the three hydrolase activities than any of the cholesteryl alkyl ethers, and palmitic acid was not inhibitory in the micromolar concentration range. Retinyl palmitate hydrolase activity was the most affected by the cholesteryl alkyl ether series, with a 48-86% reduction in hydrolytic activity caused by analog concentrations of 1 PM. The cholesteryl oleate and triolein hydrolyzing activities were less powerfully inhibited for a given concentration of ether; however, even the least potent ether analog (the methyl ether} still reduced these activities by 50% or more at a concentration of 9.6 FM. The acylglyceride ethers proved t,o be much weaker inhibitors of all three of the hydrolase activities than did the cholesteryl alkyl ethers. At concentrations of 10 PM none of the acylglyceride ether analogs was able to decrease hydrolase activity by as much as 50%. It was not possible to examine these compounds at higher concentrations due to their limited solubility in the assay mixture. The magnitude of the observed inhibition of the three hydrolase activities by the cholesteryl alkyl ether series is striking. We have estimated the apparent K, values, under the conditions employed in these studies, for the hydrolytic activities against retinyl palm&ate, cholesteryl oleate and triolein to be 11.2 FM, 6.0 FM, and 22.2 FM, respectively 161. Hence, the retinyl palmitate hydrolase activity is nearly abolished at ether concentrations which are one-tenth of the apparent K, value for the retinyl ester substrate. The extent of inhibition observed for the cholesteryl alkyl ether series on the three hydrolytic activities very significantly exceeds the potency of inhibition observed with other inhibitory reagents [6]. For instance, the hydrolase activities are sensitive to the serine esterase inhibitors diisopropylfluorophosphate and phenylmethanesulfonyl fluoride but at concentrations of these compounds between 0.1
mM and 1.0 mM, well above the cholesteryl ether concentrations used in this study. At the onset of these experiments we had hoped that the different kinds of ether analogs might have differential inhibitory effects on the three copurifying activities. Thus, we wondered whether the cholesteryl alkyl ethers might particularly inhibit cholesteryl ester hydrolyzing activity, and whether the acylglyceride ethers might particularly inhibit triolein hydrolase activity. The results reported here indicate that this did not occur. In fact, retinyl palmitate hydrolase activity was found to be more sensitive to inhibition by the cholesteryl alkyl ethers than were the hydrolase activities against cholesteryl oleate or triolein. For example, at a level of 5 FM, the cholesteryl ethyl ether abolished 96% of the retinyl palmitate hydrolase activity, but only 35% and 63% respectively, of the cholesteryl oleate and triolein hydrolase activities. The quantitatively different effects of a given ether on the three hydrolase activities is consistent with our previous evidence [6] that indicated that the active centers responsible for the hydrolysis of retinyl palm&ate, cholesteryl oleate and triolein are not identical. The biochemical basis for the powerful inhibitory effects of the cholesteryl alkyl ethers on these partially purified hydrolase activities is capable of one or more of several possible explanations. One possibility is the direct binding of the ether analog to the substrate binding site, thus preventing the substrate from entering this site and undergoing catalysis. The lack of a relationship between potency of inhibition on the one hand, and structural resemblance of the ether analog to the ester substrate on the other hand, argues against this explanation. Another possibility might be the binding of the hydrophobic ethers to the hydrolase molecule in a particular way that leads to either induction of an unfavorable conformation for catalysis or to a partial obstruction of the substrate binding site. It is unlikely that a nonspecific hydrophobic interaction between the hydrolase molecule and the ether can account for the observed effects, since the highly hydrophobic hydrocarbons &carotene and squalene, as well as the acylglyceride ethers, were all much less potent inhibitors than were the cholesteryl alkyl ethers. Still another possible explanation for the present
434
observations might be the perturbation by the ether analogs of the substrate-bile salt micelle, so that the substrate may be presented to the hydrolases in a catalytically less favorable manner. If this were the case, one would need to postulate that the cholesteryl alkyl ethers’ affect the retinyl palmitate-bile salt mixed micelle more than they do the cholesteryl oleate-bile salt mixed micelle. With the information available, it is not possible to arrive at one explanation or combination of possible explanations to effectively explain the inhibitions observed. More purified preparations of the hydrolase enzymes will probably be required in order to define better the biochemical mechanisms responsible for the observed effects. This study illustrates the potential usefulness of ether analogs of cholesteryl esters and acylglycerides for exploring some of the characteristics of lipid ester hydrolase activities. The high inhibitory potency of the long-chain cholesteryl alkyl ethers suggests that they may prove of interest and value in studies of a variety of such enzymes from a number of tissue sources. Acknowledgements The excellent technical assistance of Ms. Xenia Luna is gratefully acknowledged. This investigation was supported by grants AM 05968, HL 07343 and HL 28454 from the National Institutes of Health, Bethesda, MD. References 1 Goodman, D.S. (1980) Fed. Proc. 39, 2716-2722 2 Smith, J.E. and Goodman, D.S. (1979) Fed. 2504-2509
Proc.
38,
3 Kanai, M., Raz, A. and Goodman, D.S. (1968) J. Chn. Invest. 47, 2025-2044 4 Harrison, E.H., Smith, J.E. and Goodman, D.S. (1979) J. Lipid Res. 20, X0-771 5 Prystowsky, J.H., Smith, J.E. and Goodman, D.S. (1981) J. Biol. Chem. 256, 4498-4503 6 Blaner, W.S., Prystowsky, J.H., Smith, J.E. and Goodman, D.S. (1984) Biochim. Biophys. Acta 794, 419-427 7 Stein, O., Halperin, G. and Stein, Y. (1980) B&him. Biophys. Acta 620, 247-260 8 Stein, Y., Halperin, G. and Stein, 0. (1981) Biochim. Biophys. Acta 663, 569-574 9 Stein, Y., Dabach, G., Hollander, G., Halperin, G. and Stein, 0. (1983) B&him. Biophys. Acta 752, 98-105 10 Stein, Y., Kleinman, Y., Halperin, G. and Stein, 0. (1983) B&him. Biophys. Acta 750, 300-305 11 Kleinman, Y., Halperin, G., Stein, 0. and Stein, Y. (1982). Atherosclerosis 43, l-6 12 Stein, Y., Halperin, G. and Stein, 0. (1980) FEBS Lett. 111, 104-106 13 Friedman, G., Chajek-Sham, T., Stem, O., Olivercrona, T. and Stein, Y. (1981) B&him. Biophys. Acta 666, 156-164 14 Chajek-Sham, T., Friedman, G., Halperin, G., Stein, 0. and Stein, Y. (1981) Biochim. Biophys. Acta 666, 147-155 15 Stein, O., Friedman, G., Chajek-Shaul, T., Halperin, G., Olivecrona, T. and Stein, Y. (1983) B&him. Biophys. Acta 750, 306-316 16 Goodman, D.S., Stein, O., Halperin, G. and Stein, Y. (1983) Biochim. Biophys. Acta 750, 223-230 17 Burns, R.A., Friedman, J.M. and Roberts, M.F. (1981) Biochemistry 20, 5945-5950 18 DeBose, C.D. and Roberts, M.F. (1983) J. Biol. Chem. 258, 6327-6334 19 Vaughan, D.J. and Keough, K.M. (1974) FEBS Lett. 47, 158-161 20 Lee, T.-C. and Fitzgerald, V. (1980) Biochim. Biophys. Acta 598, 189-192 21 Halperin, G. and Gatt, S. (1980) Steroids 35, 39-42 22 Paltauf, F. and Spencer, F. (1968) Chem. Phys. Lipids 2, 168-172 23 Belfrage, P. and Vaughan, M. (1969) J. Lipid Res. 10, 341-344 24 Bradford, M.M. (1976) Anal. B&hem. 72, 248-254
Biochimica
ef Siophysica Acta, 794 (1984) 428-434 EIsevier
BBA 51681
INHIBITION OF RAT LIVER RETINYL PALMITATE HYDROLASE ANALOGS OF CHOLESTERYL ESTERS AND A~LGLYCE~DES WILLIAM
S. BLANER
a, GIDEON
HALPERIN
b, OLGA
STEIN ‘, YECHEZKIEL
ACTIVITY BY ETHER
STEIN
b and DeWITT
S. GOODMAN
a
a Department of Medicine, College of Physicians and Surgeons of Columbia University, New York, NY (U.S.A.), b Lipid Research Luboraioty, Department of Medicine B, Hadassah University Hospital, and ’ Department of Experimental Medicine and Cancer Research, Hebrew University - Hadassah Medical School, Jerusalem (Israel) (Received December (Revised manuscript
2nd, 1983) received March
6th, 1984)
Key words: Retinyl palmitate hydroiase; acylglycerol ester hydrolase.
Cholesteryl
alkyd ether analog; Acylglyceride
ether analog; Cholesteryl
ester hydrolase;
Tri-
In previous studies, retinyl palmitate hydrolase activity in rat liver was partly characterized and was found to correlate and to partially copurify with hydrolytic activities against cholesteryl oleate and triolein. ‘Ibe present studies were designed to further explore relationships between these three lipid ester hydrolase activities, by use of non-hy~~yzable ether analogs of cholesteryl esters and a~yl~yce~~s. Cholesteryl ether analogs were potent inhibitors of all three hydrolase activities with relative potencies for the series of ethers of: linoleyl > oleyl = palmitoyl > n-butyl = n-propyl > ethyl = methyl. Retinyt paimitate hydrolase activity was most strongly inactivated by this series of analogs, with 4846% of the activity inhibited at cholesteryl ether levels of 1 PM. The acylglyceride ether analogs were much weaker inhibitors of the three hydrolase activites, with the triolein, diolein and dipalmitin analogs showing similar inhibitory potencies, greater than that of the monolein and monopalmitin analogs. The data demonstrate the potential usefuhress of ether analogs of cholesteryl esters and acylglycerides for exploring some of the ch~acte~sti~ of lipid ester hydrokse activities.
Introduction
The hydrolysis of retinyl esters is essential for the mobilization of vitamin A stores from the liver [1,2]. During hepatic mobilization, stored retinyl esters are enzymically hydrolyzed to free retinol, and the retinol is then secreted from the liver and circulated in plasma bound to its specific transport protein, retinol-binding protein [2,3]. A limited amount of information has been reported about the characteristics of retinyl ester hydrolytic enzyme activity in rat liver [4,5]. In recent studies retinyl ester hydroiase activity from rat liver was partiahy purified (approx. 200-fold) and was found to copurify with hydrolytic activities against cholesteryl oleate and triolein [6]. These three 0005-2760/84/$03.00
0 1984 Elsevier Science Publishers
B.V.
copurifying hydrolytic activities were found to have a number of similar properties (including pH optima, bile salt requirements, apparent molecular size, and hydrophobicity). However, differential solubility and ~bition studies clearly showed that the three lipid hydrolase activites are due to at least three different catalytically active centers, and at least two distinct and separable enzymes. It was suggested [6] that three separate but similar enzymes, that appear to be coordinately regulated, are involved. Non-hydroly~ble ether anaiogs of naturally occurring lipids have been used successfully in a variety of studies examining lipid metabolism and lipid biochemistry. The use of non-hydrolyzable ether analogs of cholesteryl esters has provided
429
much information about the uptake and metabolism of lipids and lipoproteins in both the intact rat [7-111 and in cells in culture in vitro [12-151. A non-hydrolyzable retinyl ether analog of retinyl palmitate has been used to study hepatic uptake from chylomicrons and its persistence in liver [16]. Ether analogs of phospholipids have been used in biochemical studies aimed at understanding the mechanism of phospholipase A, hydrolysis [17,18] and in physical studies examining model membrane systems [19,20]. In this study we report the effects of a series of cholesteryl alkyl ethers and one of acylglyceride ethers, with different alkyl chain compositions, on partially purified rat hepatic retinyl palmitate hydrolase activity. The study aimed both to explore the effects of the ether analogs on specific lipid ester hydrolase activities, and to examine the potential usefulness of such compounds for investigating some of the characteristics of such hydrolase enzymes. Materials and Methods Enzyme Preparation
The hydrolase enzyme preparation used in the work reported here was partially purified from rat liver essentially as described elsewhere [6]. The purification procedure utilized successive chromatographic fractionation of an acetone powder extract on columns of phenyl-Sepharose, DEAESepharose, DEAE-cellulose and heparin-sepharose. The partially purified enzyme preparation contained hydrolytic activities directed against cholesteryl oleate and triolein in addition to that against retinyl pahnitate. The specific activities of the three partially purified hydrolytic activities directed against retinyl palmitate, cholesteryl oleate, and triolein were respectively 61.6, 112.6 and 161.9 nmol free fatty acid formed/n& per mg. The enzyme preparation was stored at - 12 o C. Cholesteryl and acylglyceride
ether, D,L-glyceryl-1,2-dioleyl ether, L-glyceryl-3oleyl ether, D,L-glyceryl-1,Zdipalmitoyl ether and L-glyceryl-1-palmitoyl ether were the acylglyceride ethers utilized in this study. The analogs were stored at - 12” C prior to use. Stock solutions of each analog were prepared in hexane on the day of use. Determination
of enzymatic activity
Assays were carried out as previously described [5,6] in 50 mM Tris-maleate (pH 8.0) containing 0.75% (w/v) sodium cholate. Each assay mixture contained l-3 pg of partially purified enzyme protein and was incubated for 5 minutes at 37 o C. Both the protein content and incubation time of the assay were selected to ensure that approximately linear (initial) reaction rates were being measured, as determined by other studies. The substrates, retinyl [l-i4C]pahnitate, cholesteryl [l“C]oleate and tri-[1-‘4C]olein, were each used (in different assays) at final assay concentrations of 20 PM. Stock solutions of cholesteryl ether analogs and acylglyceride ether analogs were prepared in hexane and mixed with the appropriate ester substrate which was prepared in acetone. This ethersubstrate mixture was evaporated to dryness under a stream of N,, redissolved in acetone, and added to the 50 mM Tris-maleate buffer containing sodium cholate to give the desired substrate and ether analog concentrations. The reaction was started by the addition of enzyme to the assay mix and stopped by the addition of methanol/chloroform/heptane (1.41 : 1.25 : 1.OO, v/v) containing 0.1 mM palmitic acid. The free fatty acids produced as products of the reaction were extracted according to the method of Belfrage and Vaughan [23]. The enzymatic activity was calculated in terms of nmol free fatty acid formed per min, from the amount of labeled free fatty acid formed, together with the known specific activity of the substrate ester.
ether analogs
Seven cholesteryl alkyl ethers and five acylglyceride ethers were prepared and purified as described previously [21,22]. The cholesteryl alkyl ethers utilized in this study were the analogs of the methyl, ethyl, n-propyl, n-butyl, pahnitoyl, oleyl and linoleyl esters of cholesterol. Glyceryl trioleyl
Chemical and radiochemical
methodr
Protein was determined according to the method of Bradford [24] using a bovine serum albumin/ y-globulin mixture as standard. Radioactivity was determined with a liquid scintillation spectrometer (Tricarb model 3255 Packard, Lagrange, IL) utiliz-
430
ing Hydrofluor as scintillation Diagnostics, Sommerville, NJ).
solvent (National
Radioactive cho~este~l [l-““Cloleate, tri[l-r4C] olein and [l-‘4C]palmitic acid were purchased from New England Nuclear (Boston, MA). Unlabeled cholesteryl oleate, triolein, retinyl palmitate, /Icarotene, squalene, a-tocopherol, cholesterol, palmitic acid and sodium cholate were purchased from Sigma Chemical Co. (St. Louis, MO). The squalene was purified by thin-layer chromatography before use. Fig. 2. In~bition of retinyl paimitate hydrolase activity by acylglyceride ethers. All data points are the average of three individual determinations. q, glyceryl trioleyl ether; 0, D,L-
Results Prior to examining the effects of the ether analogs on enzymatic activity, the homogeneity of the substrate-ether dispersion throughout the assay mixture was examined. This dispersion was judged to be homogeneous by three criteria: (1) the labeled substrate in the presence of unlabeled ether analog was uniformly distributed throughout the mixture; (2) labeled cholesteryl linoleyl ether analog in the presence of unlabeled substrate was uniformly distributed throughout the mixture; and (3) the mixture was optically clear, similar to the assay mixture in the absence of ether analog. The uniformity of distribution was determined by radioassay of multiple samples withdrawn from various parts of
-~-_-_____-_--__-___. ._--..--__.___-‘___________,
1. Inhibition of retinyl palmitate hydrolase activity by cholesteryl alkyl ethers. Additions were made as described in Materials and Methods. All data points are the average of three indvidual determinations. 0, cholesteryl linoleyl ether; A, cholesteryl oleyl ether; 0, cholesteryl palmitoyl ether; A, cholesteryl n-butyl ether; a, cholesteryl a-propyl ether; 0, cholesteryl ethyl ether; 0, cholesteryl methyl ether; 0, cholesterol; & pahnitic acid.
glyceryl-1,2-dioleyl A, L-glyceryl-3-oleyl
ether;
A, D,L-glyceryl-1,2-dipalmitoyk
ether; 0, L-glyceryl-1-palmitoyl
ether;
ether.
the substrate solution. These observations indicate that the ether analogs and substrate were both uniformly dispersed and hence presumably accessible to enzyme protein. As can be seen in Fig. 1, the cholesteryl alkyl ethers were highly potent inhibitors of retinyl palmitate hydrolase activity. The ethers with long alkyl side-chains (C,,-C,,) were more potent inhibitors than those with short chains (C,-C,). Cholesteryl linoleyl ether was the most potent inhibitor in the series of compounds studied. Free cholesterol weakly inhibited retinyl pahnitate hydrolase activity, and free pahnitic acid was not inhibitory throughout the entire concentration range tested. Fig. 2 shows the effects of the acylglyceride ether analogs on retinyl palmitate hydrolase activity. The acylglyceride ethers were markedly less potent inhibitors of this activity than were the cholesteryl alkyl ethers. The analogs of triolein, diolein and dipal~tin were appro~mately equal in inhibitory effects, while the monoiein and monopalmitin analogs were minimally inhibitory. At concentrations of acylglyceride ether greater than 10 PM, solubility problems were encountered that prevented the testing of higher concentrations of these analogs. Figs. 3 and 4 show the effects of the cholesteryl alkyl ethers (Fig. 3) and of the acylglyceride ethers (Fig. 4) on cholesteryl oleate hydrolysis. As with
431
---..____i 0
I1
2
1
I
4
f
I
6
I,,
6
I
IO
kaleQ]t
I
I
12
I,
14
I
I
16
I,
18
I,
20
0
1,,,,,,,,,,,,,,,,,,, 2 4 6
PM
8
IO
12
14
16
I6
20
s PM
ketogl
Fig. 3. Inhibition of cholesteryl oleate hydrolase activity by cholesteryl alkyl ethers. All data points are the average of three individual dete~nations. 0, cholesteryl hnoleyl ether; A, cholesteryl oleyl ether; 0, cholesteryl pabnitoyl ether; A, cholesteryl n-butyl ether; l , cholesteryl n-propyl ether; D, cholesteryl ethyl ether; 0, cholesteryl methyl ether; l, cholesterol; n, pahnitic acid.
Fig. 5. Inhibition of triolein hydrolase activity by cholesteryl alkyl ethers. All data points are the average of three individual det~~nations. 0, cholesteryl linoleyl ether; A, cholesteryl oleyl ethers; 0, cholesteryl palmitoyl ether; A, cholesteryl n-butyl ether; 0, cholesteryl n-propyl ether; Cl, cholesteryl ethyl ether; 0, cholesteryl methyl ether; 0, cholesterol; H. palmitic acid.
retinyl palmitate hydrolase activity (Figs. 1 and 2), the cholesteryl alkyl ether analogs were much more potent inhibitors of cholesteryl oleate hydrolase activity than were the acylglyceride ether analogs. Again, the long-chain ether analogs (linoleyl, oleyl and palmitoyl side-chains) were more potent inhibitors than the short-chain analogs .(n-butyl, n-propyl, ethyl, and methyl). Free cholesterol was weakly inhibitory,, slightly less so than the methyl and ethyl cholesteryl ether analogs. Palmitic acid was without effect. The acylglyceride ether analogs were only weakly inhibitory in the concentration range examined, with the glyceryl trioleyl ether
being a slightly stronger inhibitor than the monoor diacylglycerol ether analogs. Like the retinyl palmitate and cholesteryl oleate hydrolase activities, the triolein hydrolase activity was strongly inhibited by the cholesteryl alkyl ethers (Fig. 5). Here, too, the linoleyl, oleyl and pal~toyl ether analogs were stronger inhibitors than the short-chain ethers. Neither free cholesterol nor palmitic acid was inhibitory in the concentration range examined. Triolein hydrolase activity was weakly and similarly inhibited by the triolein, diolein and dipalmitin ether analogs, and minimally or not inhibited by the monoacylglycerol analogs (Fig. 6).
I-
B
0 t-
5 0 a w ii
40
20 i
1‘Oi---0
2
4
6
8
IO
CAnabl. PM Fig. 4. inhibition of cholesteryl oleate hydrolase activity by acylglyceride ethers. All data points are the average of three individual dete~nations. 0, glyceryt trioieyl ether; 0, D,LSybil-l,~-diol~l ether; A, D,L-~y~l-~,2_dipalmitoyl ether; A, L-gtyceryl-3-oleyl ether; 0, L-glyceryl-l-pahnitoyl ether.
Fig. 6. Inhibition of triolein hydrolase activity by acylglycetide ethers. All data points are the average of three individual determinations. 0, glyceryl trioleyl ether, 0, D,L-glyceryf-1,2dioleyl ether; A, D,L-~yce~l-l,2-dip~toyl ether; A, Lgtyceryl-3-oleyl ether; e, L-~y~~~-l-pa~toyl ether.
432
TABLE I RELATIVE INHIBITORY POTENCIES OF ETHER ANALOGS ON RAT LIVER HYDROLASE RETINYL PALMITATE (RPH), CHOLESTERYL OLEATE (COH) AND TRIOLEIN (TOH)
ACTIVITIES
AGAINST
The concentrations of each of the ether analogs necessary to decrease the specified hydrolase activity by 50% of control values are listed in this table. These values were obtained graphically from Figures 1 through 6 by estimating from the individual inhibition profiles the concentration of analog needed to give a 50% reduction in activity. It should be noted that the more potent an inhibitor a given compound is, the lower the concentration needed to achieve 50% reduction in activity. Ether analog
Cholesterol Cholesteryl methyl ether Cholesteryl ethyl ether Cholesteryl n-propyl ether Cholesteryl n-butyl ether Cholesteryl palmitoyl ether Cholesteryl oleyl ether Cholesteryl linoleyl ether Glyceryl trioleyl ether D,L-Glyceryl-1,2-dioleyl ether D,L-Glyceryl-1,2dipalmitoyl ether L-Glyceryl-3-oleyl ether L-Glyceryl-l-palmitoyl ether Sodium pahnitate
Concentration Inhibition
(CM) for 50%
RPH
COH
TOH
4.2 0.81 0.90 0.50 0.60 0.20 0.22 0.16 > 10 >lO >lO >lO > 10 z 20
18.0 9.6 8.4 3.1 3.0 1.4 0.90 0.80 >lO >lO 210 >lO >lO > 20
20.0 2.5 2.1 3.0 6.0 0.70 1.0 0.80 >lO > 10 > 10 > 10 210 > 20
Table I presents a summary of the effects of cholesteryl alkyl ether and acylglyceride ether analogs on each of the three hydrolase activities studied. The values listed represent the approximate concentration of each compound needed to inhibit the given hydrolase activity by 50%, as estimated graphically from the data in Figs. l-6. As already mentioned, for each of the three hydrolase activities, the cholesteryl alkyl ethers were more potent inhibitors than were the acylglyceride ether analogs. For all three hydrolase activities, the long-chain cholesteryl alkyl ethers were more potent inhibitors than were the short-chain ethers, with cholesteryl linoleyl ether being most inhibitory. Finally, the cholesteryl alkyl ethers inhibited retinyl pahnitate hydrolase activity more strongly than they did either of the other two hydrolase activites; thus, for each cholesteryl alkyl ether, 50% inhibition of retinyl palmitate hydrolase activity was achieved at a lower concentration of ether than that needed for equivalent inhibition of ether cholesteryl oleate or triolein hydrolase activity. As an additional control, three other hydrophobic, lipid-soluble compounds were examined to
ascertain whether they would inhibit retinyl palmitate, cholesteryl oleate or triolein hydrolase activites. The compounds tested included two polyisoprenoid hydrocarbons, squalene and /3carotene, and a-tocopherol. Each compound was tested against each hydrolase activity over the concentration range l-10 PM. These three compounds were found to be weakly inhibitory of the hydrolase activities, but much less potent as inhibitors than were the cholesteryl alkyl ethers. Thus, at the concentration of 4 pm, squalene showed 7-1795 inhibition, p-carotene showed 19-39% inhibition and cy-tocopherol showed 16-24% inhibition of the three hydrolase activities, compared to the activities seen without any addition. Discussion The present study was undertaken to explore the effects of ether analogs of cholesteryl esters and of acylglycerides on the enzymatic hydrolysis of retinyl palmitate, cholesteryl oleate and triolein using a partially purified enzyme preparation iso-
433
lated from rat liver. The cholesteryl alkyl ethers proved to be very potent inhibitors of all three of the hydrolase activities. A pattern of inhibitory potency for the cholesteryl alkyl ether analogs based on alkyl side-chain length was observed, with relative potencies for this series of ethers of: linoleyl > oleyl = palmitoyl > n-butyl = n-propyl > ethyl = methyl. Cholesterol was less strongly inhibitory towards the three hydrolase activities than any of the cholesteryl alkyl ethers, and palmitic acid was not inhibitory in the micromolar concentration range. Retinyl palmitate hydrolase activity was the most affected by the cholesteryl alkyl ether series, with a 48-86% reduction in hydrolytic activity caused by analog concentrations of 1 PM. The cholesteryl oleate and triolein hydrolyzing activities were less powerfully inhibited for a given concentration of ether; however, even the least potent ether analog (the methyl ether} still reduced these activities by 50% or more at a concentration of 9.6 FM. The acylglyceride ethers proved t,o be much weaker inhibitors of all three of the hydrolase activities than did the cholesteryl alkyl ethers. At concentrations of 10 PM none of the acylglyceride ether analogs was able to decrease hydrolase activity by as much as 50%. It was not possible to examine these compounds at higher concentrations due to their limited solubility in the assay mixture. The magnitude of the observed inhibition of the three hydrolase activities by the cholesteryl alkyl ether series is striking. We have estimated the apparent K, values, under the conditions employed in these studies, for the hydrolytic activities against retinyl palm&ate, cholesteryl oleate and triolein to be 11.2 FM, 6.0 FM, and 22.2 FM, respectively 161. Hence, the retinyl palmitate hydrolase activity is nearly abolished at ether concentrations which are one-tenth of the apparent K, value for the retinyl ester substrate. The extent of inhibition observed for the cholesteryl alkyl ether series on the three hydrolytic activities very significantly exceeds the potency of inhibition observed with other inhibitory reagents [6]. For instance, the hydrolase activities are sensitive to the serine esterase inhibitors diisopropylfluorophosphate and phenylmethanesulfonyl fluoride but at concentrations of these compounds between 0.1
mM and 1.0 mM, well above the cholesteryl ether concentrations used in this study. At the onset of these experiments we had hoped that the different kinds of ether analogs might have differential inhibitory effects on the three copurifying activities. Thus, we wondered whether the cholesteryl alkyl ethers might particularly inhibit cholesteryl ester hydrolyzing activity, and whether the acylglyceride ethers might particularly inhibit triolein hydrolase activity. The results reported here indicate that this did not occur. In fact, retinyl palmitate hydrolase activity was found to be more sensitive to inhibition by the cholesteryl alkyl ethers than were the hydrolase activities against cholesteryl oleate or triolein. For example, at a level of 5 FM, the cholesteryl ethyl ether abolished 96% of the retinyl palmitate hydrolase activity, but only 35% and 63% respectively, of the cholesteryl oleate and triolein hydrolase activities. The quantitatively different effects of a given ether on the three hydrolase activities is consistent with our previous evidence [6] that indicated that the active centers responsible for the hydrolysis of retinyl palm&ate, cholesteryl oleate and triolein are not identical. The biochemical basis for the powerful inhibitory effects of the cholesteryl alkyl ethers on these partially purified hydrolase activities is capable of one or more of several possible explanations. One possibility is the direct binding of the ether analog to the substrate binding site, thus preventing the substrate from entering this site and undergoing catalysis. The lack of a relationship between potency of inhibition on the one hand, and structural resemblance of the ether analog to the ester substrate on the other hand, argues against this explanation. Another possibility might be the binding of the hydrophobic ethers to the hydrolase molecule in a particular way that leads to either induction of an unfavorable conformation for catalysis or to a partial obstruction of the substrate binding site. It is unlikely that a nonspecific hydrophobic interaction between the hydrolase molecule and the ether can account for the observed effects, since the highly hydrophobic hydrocarbons &carotene and squalene, as well as the acylglyceride ethers, were all much less potent inhibitors than were the cholesteryl alkyl ethers. Still another possible explanation for the present
434
observations might be the perturbation by the ether analogs of the substrate-bile salt micelle, so that the substrate may be presented to the hydrolases in a catalytically less favorable manner. If this were the case, one would need to postulate that the cholesteryl alkyl ethers’ affect the retinyl palmitate-bile salt mixed micelle more than they do the cholesteryl oleate-bile salt mixed micelle. With the information available, it is not possible to arrive at one explanation or combination of possible explanations to effectively explain the inhibitions observed. More purified preparations of the hydrolase enzymes will probably be required in order to define better the biochemical mechanisms responsible for the observed effects. This study illustrates the potential usefulness of ether analogs of cholesteryl esters and acylglycerides for exploring some of the characteristics of lipid ester hydrolase activities. The high inhibitory potency of the long-chain cholesteryl alkyl ethers suggests that they may prove of interest and value in studies of a variety of such enzymes from a number of tissue sources. Acknowledgements The excellent technical assistance of Ms. Xenia Luna is gratefully acknowledged. This investigation was supported by grants AM 05968, HL 07343 and HL 28454 from the National Institutes of Health, Bethesda, MD. References 1 Goodman, D.S. (1980) Fed. Proc. 39, 2716-2722 2 Smith, J.E. and Goodman, D.S. (1979) Fed. 2504-2509
Proc.
38,
3 Kanai, M., Raz, A. and Goodman, D.S. (1968) J. Chn. Invest. 47, 2025-2044 4 Harrison, E.H., Smith, J.E. and Goodman, D.S. (1979) J. Lipid Res. 20, X0-771 5 Prystowsky, J.H., Smith, J.E. and Goodman, D.S. (1981) J. Biol. Chem. 256, 4498-4503 6 Blaner, W.S., Prystowsky, J.H., Smith, J.E. and Goodman, D.S. (1984) Biochim. Biophys. Acta 794, 419-427 7 Stein, O., Halperin, G. and Stein, Y. (1980) B&him. Biophys. Acta 620, 247-260 8 Stein, Y., Halperin, G. and Stein, 0. (1981) Biochim. Biophys. Acta 663, 569-574 9 Stein, Y., Dabach, G., Hollander, G., Halperin, G. and Stein, 0. (1983) B&him. Biophys. Acta 752, 98-105 10 Stein, Y., Kleinman, Y., Halperin, G. and Stein, 0. (1983) B&him. Biophys. Acta 750, 300-305 11 Kleinman, Y., Halperin, G., Stein, 0. and Stein, Y. (1982). Atherosclerosis 43, l-6 12 Stein, Y., Halperin, G. and Stein, 0. (1980) FEBS Lett. 111, 104-106 13 Friedman, G., Chajek-Sham, T., Stem, O., Olivercrona, T. and Stein, Y. (1981) B&him. Biophys. Acta 666, 156-164 14 Chajek-Sham, T., Friedman, G., Halperin, G., Stein, 0. and Stein, Y. (1981) Biochim. Biophys. Acta 666, 147-155 15 Stein, O., Friedman, G., Chajek-Shaul, T., Halperin, G., Olivecrona, T. and Stein, Y. (1983) B&him. Biophys. Acta 750, 306-316 16 Goodman, D.S., Stein, O., Halperin, G. and Stein, Y. (1983) Biochim. Biophys. Acta 750, 223-230 17 Burns, R.A., Friedman, J.M. and Roberts, M.F. (1981) Biochemistry 20, 5945-5950 18 DeBose, C.D. and Roberts, M.F. (1983) J. Biol. Chem. 258, 6327-6334 19 Vaughan, D.J. and Keough, K.M. (1974) FEBS Lett. 47, 158-161 20 Lee, T.-C. and Fitzgerald, V. (1980) Biochim. Biophys. Acta 598, 189-192 21 Halperin, G. and Gatt, S. (1980) Steroids 35, 39-42 22 Paltauf, F. and Spencer, F. (1968) Chem. Phys. Lipids 2, 168-172 23 Belfrage, P. and Vaughan, M. (1969) J. Lipid Res. 10, 341-344 24 Bradford, M.M. (1976) Anal. B&hem. 72, 248-254