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Affinity chromatography of arterial lysosomal cholesterol ester hydrolase
273
Biochimica et Biophysics Acta, 664 (1981) 273-277 ~se~erlNortb-HoUand
Biomedical Press
BBA 57801
AFFINITY CHROMATOGR~HY OF ARTERIAL CHOL~S~ROL ESTER ~DROLASE
J.C. DOUSSET a, N. DOUSSET b, M.J. FOGLIE’ITI
LYSOSOMAL
c and 1;. DOUSTE-BLAZY
b
a Laboratoire de Biochimie (Professeur G. SouZa), Facult& de Pharmacie, 31400 Toulouse, b INSERT U 101, Biochimie des Lipides, H&pital Purpan, 31059 To~lo~e and c Labomtoire de Chimie Biologique, Facultb des Sciences Pharmace~t~q~es et Biologiq~es, 75006 Paris (France) (Received July 31st, 1980) (Revised manuscript received November 2Oth, 1980)
Key words: cholesterol ester hyd~l~e; raphy; (Arterial lysosome)
cholesterol ester synthesis; Affinity chromaiog-
summary The glycopro~ic nature of rabbit aortic lysosomal cholesterol ester hydrolase has been demonstrated by affinity chromatography on Concanavalin A-Sepharose. After chromatography, the enzyme lacks synthesizing activity. This activity is restored by addition of deactivated lysosomes containing more endogenous cholesterol. On the other hand, a hypothesis for the activation role of b~(mono~yl~yce~l) phosphate is suggested.
Introduction The presence of cholesteryl ester-synthesizing and -hydrolysing activities in aortae of various species has been demons~a~d [l--5], These activities seem to be due to the same protein, the cholesterol ester hydrolase (EC 3.1.1.13). Studies on this enzyme are of great interest because the two activities have been shown to change under conditions of atherosclerosis. The synthesizing activity is increased and the hydrolytic one is generally decreased. Generally, hydrolytic reactions are irreversible and the synthesis of cholesterol esters may depend on an effector which reverses the reaction. An inhibitor of the synthesizing activity has been described in rat and rabbit aortae [4], The enzyme purification was achieved by classical techniques for protein fractionation and it seems that the inhibitor was eluted with the enzyme. In a previous work [6] we have been able to demonstrate the lysosomd OOOS-2760/81/0000-0000/$02.50
0 Elsevier/North-Holland
Biomedical Press
274
localization of the rabbit cholesterol ester synthetase. Most of the lysosomal hydrolases are glycoproteins which possess affinity for Concanavalin A [7,8]. So, we performed the fractionation of the enzyme by affinity chromatography on Concanavalin A-Sepharose. Materials and Methods Enzymatic preparation Adult male New Zealand white Rabbits approximately 20 weeks old were given a commercial diet. During the 15 h before they were killed, the animals had free access to water but not to food. After the animals had been killed, aortae were removed from the heart to the iliac bifurcation, and cleaned of blood and periadvential tissue at 4°C. Procedures for tissue preparation and fractionation were carried out in an ice-water bath (O-4” C) and refrigerated centrifuge. The preparation was weighed and minced finely and homogenized in 20 vol. 0.01 M Tris-HCl buffer, pH 7.2, containing 9% NaCl. The homogenate was centrifuged at 750 X g for 10 min to remove the nuclear material. The supematant was then centrifuged at 5500 X g for mitochondria elimination, and at 14 000 X g for lysosome separation. The pellet containing the lysosomes was resuspended in the extraction buffer containing Mn, Mg, CaClz at 2 - 10e3 M and 0.5 M NaCl. After successive congelations and decongelations the extract was centrifuged (26 600 X g for 30 min). The supematant is the enzymatic preparation. Enzyme assays Cholesterol-esterifying activity. The incubation mixture contained 8.6 pmol citric acid, 20 pmol disodium phosphate buffer, pH 5.2,10 nmol [l-14C]cholesterol, 10 nmol oleic acid and enzymatic fraction in a final volume of 0.5 ml. Incubation was carried out by shaking for 3 h at 37’C. The reaction mixture was extracted with chloroform/methanol (2 : 1, v/v) according to Folch et al [ 91 and fractionated into cholesterol and cholesteryl esters by thin-layer chromatography on silica gel G coated plate in light petroleum/diethyl ether/acetic acid (95 : 5 : 1, v/v). After development of the chromatogram, the compounds were stained by iodine vapours and the areas containing the labelled lipids from the incubation were scraped from the plate and put into a vial containing 10 ml scintillation mixture. Esterification was determined by measuring the radioactivity of the cholesterol esters as a percentage of the total radioactivity. HydroZytic activity. The incubation mixture contained 8.6 pmol citric acid, 20 pmol disodium phosphate buffer, pH 5.2,10 nmol [ 1-14C]oleate cholesterol and enzymatic fraction. Incubation was carried out by shaking for 3 h at 37°C and the reaction mixture was treated as described for the synthesizing activity. The extract was separated into cholesteryl esters and free fatty acids. Fatty acid identification The lipids were transesterified using BF3 in methanol. The extracted methyl esters were purified on thin-layer plates with benzene as developing solvent. They were analysed by gas-liquid chromatography in an Intersmat gas chromato-
215
graph equiped with a flame ionization detector, and a column of 10% (w/w) diethyl glycol succinate on Chromosorb WAW (80-100 mesh). Bis(monoacylglycery1) phosphate identification
Bis(monoacylglycery1) phosphate phospholipase AZ treatment.
was prepared from cardiolipin (Sigma) by
Cholesterol determination
Cholesterol was determined by the method of Rijschlau et al. [lo]. Affinity chromatography on Concanavalin A-Sepharose
The enzymatic preparation was chromatographed on a Concanavalin A-Sepharose column (10 ml) equilibrated with the extraction buffer containing Mn, Mg, CaC12at 2 - 10V3M and 0.5 M NaCl. The elution was carried out with the same buffer until no absorbance at 280 nm was observed. In a second step, the elution was performed with the buffer containing increasing concentrations of cu-methylmannoside (0.1 and 0.7 M). 3-ml fractions were collected and assayed for the enzyme activities. Results and Discussion During the chromatography of the lysosomal extract (extent of hydrolysis: 3.33 nM oleic acid liberated/h), the enzymatic activity was adsorbed on Concanavalin A-Sepharose. Its elution was performed with a buffer containing 0.1 M a-methylmannoside. However, only the hydrolytic activity was detected (total activity recovered: 2.53 nm oleic acid liberated/h, 76% of total activity) (Fig. 1). Like most of the lysosomal hydrolases, the cholesterol ester hydrolase is a glycoprotein. Its affinity for the Concanavalin A is in favour of a mannoside structure. A280
nM olelc acid
0.1 I
Fraction
No.
Fig. 1. Chromatography of the lysosomal extract on Concanavalin ASepharose. F&&ion by: (A) 0.01 M Tris-HCl buffer, pH 7.2. containing Mn, Mg and CaCl2 at 2 - 10m3 M and 0.6 M NaCl; (B) as in A + 0.1 M cy-methylmannoside: (C)as in A + 0.7 M cY-mannoside. a, Absorbance (or A) at 280 nm: a, hydrolytic activity (run01 oleic add formed/ml enzymatic fraction per h).
276 TABLE I FREE FATTY ACID COMPOSITION OF ELUTED WITH A BUFFER CONTAINING
TOTAL LYSOSOMES AND ENZYMATIC 0.1 M ff-METHYLMANNOSIDE (PERCENT
FRACTION DISTRIBU-
TION) Fatty acid
Total lysosomes
Enzymatic fraction (0.1 M)
16 : 0 16 : 1 18 : 0 18 : 1 18 : 2
43.7 8.1 15.6 29.6 2.9
44.9 1.8 17.5 27.0 2.7
The synthesizing activity is very low in the same fraction where the hydrolytic activity is detected (0.004 nmol cholesteryl oleate formed/ml enzymatic fraction per h). Furthermore, this activity is not present in any other chromatographic fractions. It seems that during the chromatography a dissociation between enzymatic protein and an activator of the synthesis occurs. Recently, Tuhackova et al. [ll] have reported that after purification by gel filtration, DEAE-cellulose and Sepharose chromatography, the cytosolic liver cholesterol e&erase was unable to esterify cholesterol. Many enzymes of lipid metabolism are stimulated by lipidic effecters. This is the case with lecithin cholesterol acyltransferase [ 12,131. The synthesizing activity of the cholesterol ester hydrolase could be stimulated by lysosomal lipoproteins, the affinity of which for the Concanavalin A is different to that of the enzyme. The endogenous cholesterol concentration of the chromatographic fraction containing the enzyme is low (0.087 nmol/ml). The synthesizing activity of arterial lysosomal cholesterol esterase could be restored by fractions containing more endogenous cholesterol than the enzymatic fraction: deactivated lysosomes (0.275 nmol/ml) and mitochondrial fractions (0.475 nmol/ml). Addition of lysosomes deactivated by heating results in the formation of cholesteryl esters with different saturated (44.6%) and unsaturated fatty acids (monoenes 40.6 and dienes 14.8). The stimulation of the synthesizing activity could result, in this case, from the presence of lysosomal lipids, the component of which could only act as substrate or activator during cholesterol esterification. These lipoproteins would have a different affinity for Concanavalin A. However, the fatty acid composition of the total lysosomal extract and. of the enzymatic fraction exhibited no major differences (Table I). Then the stimulation of synthesizing activity could be due to an enrichment in the other substrate, endogenous cholesterol. In fact, the endogenous cholesterol concentration of the chromatographic fraction containing the enzyme is lower than that in lysosomes. The fraction eluting with a higher concentration of cY-methylmannoside (0.7 M) and exhibiting no synthesizing activity but containing lipoproteins is able to restore the synthesizing activity, leading to mainly saturated (68.2%) and mono-unsaturated (31.8%) cholesteryl esters, but to a lesser extent than total deactivated lysosomes.
277
In another way, a mitochondrial fraction restores the capacity of ester formation (saturated and mono-unsaturated fatty acids). In this case, the activation could be due to a mitochondrial component. It is known that mitochondrial diphosphatidylglycerol can be converted in lysosomes to bis(monoacylglyceryl) phosphate [ 141. This component has been reported as an activator of some lysosomal enzymes i.e. liver lipase [ 151. According to this hypothesis, we have been able to identify by thin-layer chromatography in the lysosomal extract a component, the migration of which is identical to that of bis(monoacylglyceryl) phosphate [ 161. After Concanavalin A chromatography, lipoproteins either acting as substrate (because they are rich in exogenous cholesterol) or containing the bis(monoacylglycery1) phosphate are dissociated from the enzymatic fraction. Research is in progress to verify these hypotheses. Acknowledgements The authors wish to thank Professor R. Agid, Director of the Physiology Institute, for fruitful discussions, Thanks are also due to J. Manent for technical assistance. References 1 PateIski, J.. Bowyer. DE.. Howard, A.N.., Jennings, I.W.. Thome, C.J.R. and Gresham. G.A. (1970) Atherosclerosis 12.41-53 2 St. Chair, R.W., Lofhmd, H.B. and Clarkson, T.B. (1970) Circ. Res. 27.213-215 3 Kothari, H.V., Miller, B.F. and Kritchevski. D. (1973) Biochim. Biophys. Acta 296.446454 4 Kothari, H.V. and Kritchevski, D. (1975) Lipids 10,322-330 5 Kr&hev&i, D. and Kothari. H.V. (1978) in Advances in Lipid Research (Paoletti, R. and Kritchevski. D., eds.), Vol. 16. pp. 221-262. Academic Press, New York Dousset, J.C., Dousset. N.. El Baba, A.M., Soula. G. and Douste-Blazy. L. (1979) Artery 5,432-447 Morris, B.F., Ben Yoseph, Y. and Nadler. H.L. (1979) Biochem. J. 177.175-180 Den Tandt. W.R. (1980) Chn. Chim. Acta 102.199-205 Folch. J., Lees, M. and SloaneStanley, G.H. (1957) J. Biol. Chem. 226.497-509 R5schiau. P.. Bemt, E. and Gruber, W. (1974) Z. Khn. Chem. KIin. Biochim. 12. 226 Tuhackova, Z., Kriz. 0. and Hradec, J. (1980) Biochim. Biophys, Acta 617.439-445 Nakagawa, M. and Nishida. T. (1973) Biochim. Biophys. Acta 296,577-585 Marcel, Y.L. and Vezina. C. (1973) J. Biol. Chem. 248, 8254-8259 Poorthuis. B.J.H.M. and HostetIer, K.Y. (1978) J. Lipid Res. 19.309-316 Joutti, A., Brotherus. J. and Kimunen. P.K.J. (1980) Communication to the XXIInd International Conference on the Biochemistry of Lipids, Milan 26-28 May 16 Somerharju. P. and Renkonen. 0. (1980) Biochim. Biophys. Acta 618,407-419 6 7 8 9 10 11 12 13 14 15
Biochimica et Biophysics Acta, 664 (1981) 273-277 ~se~erlNortb-HoUand
Biomedical Press
BBA 57801
AFFINITY CHROMATOGR~HY OF ARTERIAL CHOL~S~ROL ESTER ~DROLASE
J.C. DOUSSET a, N. DOUSSET b, M.J. FOGLIE’ITI
LYSOSOMAL
c and 1;. DOUSTE-BLAZY
b
a Laboratoire de Biochimie (Professeur G. SouZa), Facult& de Pharmacie, 31400 Toulouse, b INSERT U 101, Biochimie des Lipides, H&pital Purpan, 31059 To~lo~e and c Labomtoire de Chimie Biologique, Facultb des Sciences Pharmace~t~q~es et Biologiq~es, 75006 Paris (France) (Received July 31st, 1980) (Revised manuscript received November 2Oth, 1980)
Key words: cholesterol ester hyd~l~e; raphy; (Arterial lysosome)
cholesterol ester synthesis; Affinity chromaiog-
summary The glycopro~ic nature of rabbit aortic lysosomal cholesterol ester hydrolase has been demonstrated by affinity chromatography on Concanavalin A-Sepharose. After chromatography, the enzyme lacks synthesizing activity. This activity is restored by addition of deactivated lysosomes containing more endogenous cholesterol. On the other hand, a hypothesis for the activation role of b~(mono~yl~yce~l) phosphate is suggested.
Introduction The presence of cholesteryl ester-synthesizing and -hydrolysing activities in aortae of various species has been demons~a~d [l--5], These activities seem to be due to the same protein, the cholesterol ester hydrolase (EC 3.1.1.13). Studies on this enzyme are of great interest because the two activities have been shown to change under conditions of atherosclerosis. The synthesizing activity is increased and the hydrolytic one is generally decreased. Generally, hydrolytic reactions are irreversible and the synthesis of cholesterol esters may depend on an effector which reverses the reaction. An inhibitor of the synthesizing activity has been described in rat and rabbit aortae [4], The enzyme purification was achieved by classical techniques for protein fractionation and it seems that the inhibitor was eluted with the enzyme. In a previous work [6] we have been able to demonstrate the lysosomd OOOS-2760/81/0000-0000/$02.50
0 Elsevier/North-Holland
Biomedical Press
274
localization of the rabbit cholesterol ester synthetase. Most of the lysosomal hydrolases are glycoproteins which possess affinity for Concanavalin A [7,8]. So, we performed the fractionation of the enzyme by affinity chromatography on Concanavalin A-Sepharose. Materials and Methods Enzymatic preparation Adult male New Zealand white Rabbits approximately 20 weeks old were given a commercial diet. During the 15 h before they were killed, the animals had free access to water but not to food. After the animals had been killed, aortae were removed from the heart to the iliac bifurcation, and cleaned of blood and periadvential tissue at 4°C. Procedures for tissue preparation and fractionation were carried out in an ice-water bath (O-4” C) and refrigerated centrifuge. The preparation was weighed and minced finely and homogenized in 20 vol. 0.01 M Tris-HCl buffer, pH 7.2, containing 9% NaCl. The homogenate was centrifuged at 750 X g for 10 min to remove the nuclear material. The supematant was then centrifuged at 5500 X g for mitochondria elimination, and at 14 000 X g for lysosome separation. The pellet containing the lysosomes was resuspended in the extraction buffer containing Mn, Mg, CaClz at 2 - 10e3 M and 0.5 M NaCl. After successive congelations and decongelations the extract was centrifuged (26 600 X g for 30 min). The supematant is the enzymatic preparation. Enzyme assays Cholesterol-esterifying activity. The incubation mixture contained 8.6 pmol citric acid, 20 pmol disodium phosphate buffer, pH 5.2,10 nmol [l-14C]cholesterol, 10 nmol oleic acid and enzymatic fraction in a final volume of 0.5 ml. Incubation was carried out by shaking for 3 h at 37’C. The reaction mixture was extracted with chloroform/methanol (2 : 1, v/v) according to Folch et al [ 91 and fractionated into cholesterol and cholesteryl esters by thin-layer chromatography on silica gel G coated plate in light petroleum/diethyl ether/acetic acid (95 : 5 : 1, v/v). After development of the chromatogram, the compounds were stained by iodine vapours and the areas containing the labelled lipids from the incubation were scraped from the plate and put into a vial containing 10 ml scintillation mixture. Esterification was determined by measuring the radioactivity of the cholesterol esters as a percentage of the total radioactivity. HydroZytic activity. The incubation mixture contained 8.6 pmol citric acid, 20 pmol disodium phosphate buffer, pH 5.2,10 nmol [ 1-14C]oleate cholesterol and enzymatic fraction. Incubation was carried out by shaking for 3 h at 37°C and the reaction mixture was treated as described for the synthesizing activity. The extract was separated into cholesteryl esters and free fatty acids. Fatty acid identification The lipids were transesterified using BF3 in methanol. The extracted methyl esters were purified on thin-layer plates with benzene as developing solvent. They were analysed by gas-liquid chromatography in an Intersmat gas chromato-
215
graph equiped with a flame ionization detector, and a column of 10% (w/w) diethyl glycol succinate on Chromosorb WAW (80-100 mesh). Bis(monoacylglycery1) phosphate identification
Bis(monoacylglycery1) phosphate phospholipase AZ treatment.
was prepared from cardiolipin (Sigma) by
Cholesterol determination
Cholesterol was determined by the method of Rijschlau et al. [lo]. Affinity chromatography on Concanavalin A-Sepharose
The enzymatic preparation was chromatographed on a Concanavalin A-Sepharose column (10 ml) equilibrated with the extraction buffer containing Mn, Mg, CaC12at 2 - 10V3M and 0.5 M NaCl. The elution was carried out with the same buffer until no absorbance at 280 nm was observed. In a second step, the elution was performed with the buffer containing increasing concentrations of cu-methylmannoside (0.1 and 0.7 M). 3-ml fractions were collected and assayed for the enzyme activities. Results and Discussion During the chromatography of the lysosomal extract (extent of hydrolysis: 3.33 nM oleic acid liberated/h), the enzymatic activity was adsorbed on Concanavalin A-Sepharose. Its elution was performed with a buffer containing 0.1 M a-methylmannoside. However, only the hydrolytic activity was detected (total activity recovered: 2.53 nm oleic acid liberated/h, 76% of total activity) (Fig. 1). Like most of the lysosomal hydrolases, the cholesterol ester hydrolase is a glycoprotein. Its affinity for the Concanavalin A is in favour of a mannoside structure. A280
nM olelc acid
0.1 I
Fraction
No.
Fig. 1. Chromatography of the lysosomal extract on Concanavalin ASepharose. F&&ion by: (A) 0.01 M Tris-HCl buffer, pH 7.2. containing Mn, Mg and CaCl2 at 2 - 10m3 M and 0.6 M NaCl; (B) as in A + 0.1 M cy-methylmannoside: (C)as in A + 0.7 M cY-mannoside. a, Absorbance (or A) at 280 nm: a, hydrolytic activity (run01 oleic add formed/ml enzymatic fraction per h).
276 TABLE I FREE FATTY ACID COMPOSITION OF ELUTED WITH A BUFFER CONTAINING
TOTAL LYSOSOMES AND ENZYMATIC 0.1 M ff-METHYLMANNOSIDE (PERCENT
FRACTION DISTRIBU-
TION) Fatty acid
Total lysosomes
Enzymatic fraction (0.1 M)
16 : 0 16 : 1 18 : 0 18 : 1 18 : 2
43.7 8.1 15.6 29.6 2.9
44.9 1.8 17.5 27.0 2.7
The synthesizing activity is very low in the same fraction where the hydrolytic activity is detected (0.004 nmol cholesteryl oleate formed/ml enzymatic fraction per h). Furthermore, this activity is not present in any other chromatographic fractions. It seems that during the chromatography a dissociation between enzymatic protein and an activator of the synthesis occurs. Recently, Tuhackova et al. [ll] have reported that after purification by gel filtration, DEAE-cellulose and Sepharose chromatography, the cytosolic liver cholesterol e&erase was unable to esterify cholesterol. Many enzymes of lipid metabolism are stimulated by lipidic effecters. This is the case with lecithin cholesterol acyltransferase [ 12,131. The synthesizing activity of the cholesterol ester hydrolase could be stimulated by lysosomal lipoproteins, the affinity of which for the Concanavalin A is different to that of the enzyme. The endogenous cholesterol concentration of the chromatographic fraction containing the enzyme is low (0.087 nmol/ml). The synthesizing activity of arterial lysosomal cholesterol esterase could be restored by fractions containing more endogenous cholesterol than the enzymatic fraction: deactivated lysosomes (0.275 nmol/ml) and mitochondrial fractions (0.475 nmol/ml). Addition of lysosomes deactivated by heating results in the formation of cholesteryl esters with different saturated (44.6%) and unsaturated fatty acids (monoenes 40.6 and dienes 14.8). The stimulation of the synthesizing activity could result, in this case, from the presence of lysosomal lipids, the component of which could only act as substrate or activator during cholesterol esterification. These lipoproteins would have a different affinity for Concanavalin A. However, the fatty acid composition of the total lysosomal extract and. of the enzymatic fraction exhibited no major differences (Table I). Then the stimulation of synthesizing activity could be due to an enrichment in the other substrate, endogenous cholesterol. In fact, the endogenous cholesterol concentration of the chromatographic fraction containing the enzyme is lower than that in lysosomes. The fraction eluting with a higher concentration of cY-methylmannoside (0.7 M) and exhibiting no synthesizing activity but containing lipoproteins is able to restore the synthesizing activity, leading to mainly saturated (68.2%) and mono-unsaturated (31.8%) cholesteryl esters, but to a lesser extent than total deactivated lysosomes.
277
In another way, a mitochondrial fraction restores the capacity of ester formation (saturated and mono-unsaturated fatty acids). In this case, the activation could be due to a mitochondrial component. It is known that mitochondrial diphosphatidylglycerol can be converted in lysosomes to bis(monoacylglyceryl) phosphate [ 141. This component has been reported as an activator of some lysosomal enzymes i.e. liver lipase [ 151. According to this hypothesis, we have been able to identify by thin-layer chromatography in the lysosomal extract a component, the migration of which is identical to that of bis(monoacylglyceryl) phosphate [ 161. After Concanavalin A chromatography, lipoproteins either acting as substrate (because they are rich in exogenous cholesterol) or containing the bis(monoacylglycery1) phosphate are dissociated from the enzymatic fraction. Research is in progress to verify these hypotheses. Acknowledgements The authors wish to thank Professor R. Agid, Director of the Physiology Institute, for fruitful discussions, Thanks are also due to J. Manent for technical assistance. References 1 PateIski, J.. Bowyer. DE.. Howard, A.N.., Jennings, I.W.. Thome, C.J.R. and Gresham. G.A. (1970) Atherosclerosis 12.41-53 2 St. Chair, R.W., Lofhmd, H.B. and Clarkson, T.B. (1970) Circ. Res. 27.213-215 3 Kothari, H.V., Miller, B.F. and Kritchevski. D. (1973) Biochim. Biophys. Acta 296.446454 4 Kothari, H.V. and Kritchevski, D. (1975) Lipids 10,322-330 5 Kr&hev&i, D. and Kothari. H.V. (1978) in Advances in Lipid Research (Paoletti, R. and Kritchevski. D., eds.), Vol. 16. pp. 221-262. Academic Press, New York Dousset, J.C., Dousset. N.. El Baba, A.M., Soula. G. and Douste-Blazy. L. (1979) Artery 5,432-447 Morris, B.F., Ben Yoseph, Y. and Nadler. H.L. (1979) Biochem. J. 177.175-180 Den Tandt. W.R. (1980) Chn. Chim. Acta 102.199-205 Folch. J., Lees, M. and SloaneStanley, G.H. (1957) J. Biol. Chem. 226.497-509 R5schiau. P.. Bemt, E. and Gruber, W. (1974) Z. Khn. Chem. KIin. Biochim. 12. 226 Tuhackova, Z., Kriz. 0. and Hradec, J. (1980) Biochim. Biophys, Acta 617.439-445 Nakagawa, M. and Nishida. T. (1973) Biochim. Biophys. Acta 296,577-585 Marcel, Y.L. and Vezina. C. (1973) J. Biol. Chem. 248, 8254-8259 Poorthuis. B.J.H.M. and HostetIer, K.Y. (1978) J. Lipid Res. 19.309-316 Joutti, A., Brotherus. J. and Kimunen. P.K.J. (1980) Communication to the XXIInd International Conference on the Biochemistry of Lipids, Milan 26-28 May 16 Somerharju. P. and Renkonen. 0. (1980) Biochim. Biophys. Acta 618,407-419 6 7 8 9 10 11 12 13 14 15