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The active site and the phospholipid activation of rat liver lysosomal lipase are not stereospecific
Chemistry and Physics o f Lipids, 29 (1981) 235-239 Elsevier/North-Holland Scientific Publishers Ltd.
235
THE ACTIVE SITE AND THE PHOSPHOLIPID ACTIVATION OF RAT LIVER LYSOSOMAL LIPASE ARE NOT STEREOSPECIFIC
ANNELI JOUTTI, PETRI VAINIO, JAAKKO R. BROTHERUS ~ , F. PALTAUF a and PAAVO K.J. KINNUNEN Department of Medical Chemistry, University of Helsinki, Siltavuorenpenger 10 A, SF-O0170 Heisinki 17 (Finland) and alnstitute of Biochemistry, Technical University of Graz, A-8010 Graz (Austria)
Received June 15th, 1981
accepted August 17th, 1981
The stereochemical specificity of lysosomal lipase of rat liver was investigated using enantiomeric triacylglycerol analogs, sn-l-alkyl-2,3-diacylglycerol and sn-3-alkyl-l,2-diacylglycerol as substrates. Lysosomal lipase utilized both substrates with equal rates. The dependence of the activity of lysosomal lipase on the stereoconfiguration of activating acidic phospholipid was also studied. Our results showed that both sn-3-phospholipids (diphosphatidylglycerol, phosphatidylserine) and sn-l-phospholipids (bis(monoacylglycero)phosphate (BMP)) were efficient activators of this enzyme and thus the stereochernical configuration of the activating phospholipid is not important. Accordingly, the rat liver lysosomal lipase lacks stereospecificity with respect to both the triacylglycerol substrate and the acidic phospholipid activator. Keywords: lysosomal lipase; stereospecificity; acidic phospholipids; bis(monoacylglycerol) phosphate.
Introduction Liver lysosomes contain a triacylglycerol lipase which exhibits a pH optimum of approximately 4 . 0 - 4 . 5 [1,2]. The enzyme is tightly associated with the lysosomal membranes [3]. In addition to triacylglycerol hydrolase activity cholesterol esterase activity can be detected [3,4]; Deficiency of the lysosomal lipase, known as Woiman's syndrome [5,6], leads to accumulation of triacylglycerols and cholesterol esters in lysosomes. In vitro studies with a partially purified enzyme preparation have revealed that rat lysosomal lipase is strongly stimulated by acidic phospholipids [3,7]. In contrast, the lipase can be completely inhibited by heparin [3]. The stereospecificity (i.e., differentiation between position sn-1- and sn-3-acyl chains of triacylglycerols) is known for several lipases. Pancreatic lipase lacks stereospecificity [8] ; in contrast, lipoprotein lipase and hepatic endothelial lipase hydrolyze preferentially the sn-l-ester bond of the triacylglycerols [9-11 ]. In the 0009-3084/81/0000-0000/$02.50 O 1981 Elsevier/North-Holland Scientific Publishers Ltd.
236
A. Joutti et aL, Stereospecificity of rat liver lysosomal lipase
present study the stereochemical specificity of lysosomal lipase of rat liver was investigated using enantiomeric synthetic substrates [9,11]. In principle, the lysosomal lipase could also be specific for the stereochemical structure of the activating acidic phospholipids. All phospholipids tested so far were derivatives of sn-3-glycerophosphate. However, the major acidic phospholipid of lysosomes, bis(monoacylglycero)phosphate (BMP), contains only sn-l-glycerophosphate [12,13]. Therefore, we tested its efficiency as an activator of the enzyme. Our results show that the rat liver lysosomal lipase lacks stereospecificity with respect to both the triacylglycerol substrate and the phospholipid activator. Materials and Methods
L ysosomal lipase The lysosomal lipase of rat liver was purified to Step II according to Teng and Kaplan [3]. Rat livers were homogenized in 0.25 M sucrose containing 1 mM EDTA (pH 7.2) (3 ml/g of liver) at 4°C. The homogenate was centrifuged at 1000 × g, for 10 min. The supernatant was centrifuged at 3300 × g, for 10 min, and the resulting supernatant at 12 000 ×g, for 40 rnin. The particulate fraction from the last centrifugation was dispersed in 10 ml of 0.25 M sucrose and centrifuged at 12 000 ×g for 20 min. The pellet was homogenized in 5 ml of 2% Triton X-100 (pH 6.0). After 60 min, the suspension was centrifuged at l0 s X g in a Spinco ultracentrifuge for 60 min at 4°C. The supematant fluid was stabilized by the addition of half its volume of glycerol and stored at --20°C. Assay o f lysosomal lipase The lipase assay conditions were identical to those used by Teng and Kaplan [3] except that the volume was 200/al. The reaction mixture contained 50 mM sodium acetate (pH 4), 2% of Triton X-IO0, 1 mM tri[9,10-SH]oleoylglycerol (1.5 • lO s cpm/assay; Amersham), and indicated amounts of phospholipids. The reaction was started by the addition of enzyme. Incubations were carried out at 37°C, for 10 min, and the reactions were stopped by the addition of chloroform/methanol/ heptane (1.4 : 1.25 : 1.0, by vol.) and the phases were separated by centrifugation after addition of 0.14 M borate, whereafter radioactivity of free fatty acids was determined [14]. Phospholipids Phospholipids were isolated from male rats that had received intraperitoneaUy Triton WR 1339 which leads to accumulation of BMP in liver lysosomes [15]. The lipids from four rat livers were extracted [16]. The liver phosphatidylcholines, phosphatidylserines and BMP were obtained from the lipid extract by column chromatography on silicic acid and by thin-layer chromatography [ 12,13 ]. Diphosphatidylglycerol was purified from rat heart by the same method. Phosphorus was analyzed by the method of Bartlett [17].
A. Joutti et al., Stereospecificity of rat liver lysosomal lipase
237
Assay for stereospecificity of lysosomal lipase The enantiomeric triacylglycerol analogs, 3-O-[1-14C]octadecyl-l,2-dioleoyl-snglycerol (0.05 mCi/mmol) and 1-O-[9,10-aH]octadecyl-2,3-dioleoyl-sn-glycerol (0.11 mCi/mmol) were prepared according to Paltauf et al. [9,11 ]. The incubation mixture contained 1 /amol of the substrate, 2 mM phosphatidylserines and 2% Triton X-100 in 50 mM sodium acetate (pH 4.5). Hydrolysis of enantiomeric triacylglycerols by lysosomal lipase was carried out for 60 min at 37°C. The reaction was started by the addition of 20/ag of liver lysosomal lipase extract. At the indicated times 300-~1 aliquots were taken from the reaction mixture (2.5 ml) and extracted as described [16]. The reaction products were separated on silica gel plates using hexane/ether/acetic acid (80 : 20 : 2, by vol.) as solvent system and were counted for 14C-and 3H-radioactivity.
Results and Discussion
Stereospecificity of lysosomal lipase was determined by utilizing the enantiomeric triacylglycerol analogs, sn-l-alkyl-2,3-diacylglycerol and sn-3-alkyl-l,2diacylglycerol, as substrates (Fig. 1). It is evident that lysosomal lipase utilizes both substrates with equal rates and thus lacks stereochemical specificity. This result agrees with the postulated function of the liver lysosomal lipase in the clearance of triacylglycerols from partially degraded plasma lipoproteins [18,19]. These
100
I--
o Q <
n.
80
60
=d
o ~
4o
0 Z
,0
20
00
I
10
I
20
I
30
I
40
I
50
I
60
TIME,min
Fig. 1. Degradation of the enantiomeric triacylglycerol analogs, 1-alkyl-2,3-diacylglyceroland 3-alkyl-l,2-diacylglycerol,by liver lysosornal lipase. Detailed description of the assay mixture is given in Materials and Methods. Alkyldiacylglycerol(e, o); alkylacylglycerol (A, z~) alkylglycerol (a, a). Closed symbols, alkyl in the sn-3-position;open symbols, alkyl in the sn-l-position.
238
A. Joutti et al., Stereospeeifieity o f rat liver lysosornal lipase
ld E
6
E u 0
E
c >.
o iii
.d
0 0
I
I
I
1
2
3
[PHOSPHOLIPID],mM Pi Fig. 2. Influence of phospholipid concentration on acid lipase activity. Lipase activity of a Triton X-100 extract of lysosomal fraction (20 ~g protein) in the presence of phosphatidylcholine (o), diphosphatidylglycerol (e), phosphatidylserine (a), and BMP (o). See text for details.
degradation products can be used as precursors for the resynthesis of other molecules, and the stereochemical position in which the fatty acid had originally been bound is not of importance. However, the question still remains why lipoprotein lipase and hepatic endothelial lipase do possess relatively strict stereospecificity [9-11 ] and whether this distinction of lipases into two classes has any biological significance. Figure 2 illustrates the deperrdence of the lysosomal lipase activity on the concentration of added phospholipids. Lecithin did not change the activity of the enzyme. In contrast, the acidic sn-3-phospholipids, diphosphatidylglycerols and phosphatidylserines, were efficient activators of lysosomal lipase; the activity of the enzyme alone is 5-10% of that in the presence of 2 mM phosphatidylserines or diphosphatidylglycerols. These observations are in agreement with the results of Kaplan and coworkers [3,7]. The trace of lipase activity in the absence of added lipids is probably best explained by the presence of residual acidic lipid in the enzyme preparation (Step II material, Ref. 3). In addition to sn-3-glycerophospholipids, the lysosomal lipase was also strongly activated by BMP (Fig. 2). The backbone of this lipid has the uncommon stereochemical structure of sn- 1-glycerophospho-l'-glycerol [ 12,13 ]. Consequently, the stereochemical configuration of the activating phospholipid is not important, and
A. Joutti et aL, Stereospecific#y o f rat liver lysosomal lipase
239
the stimulating effect o f acidic phospholipids is likely to be mediated by a change in the charge o f the substrate [20]. BMP is specifically found in the lysosomes [ 15,21 ] ;however its function in lysosomes is not known. Since BMP is the main acidic phospholipid o f the lysosomes and an efficient activator o f liver lysosomal lipase in vitro (Fig. 2), it is possible that this lipid activates the lysosomal lipase also in vivo. The role of the uncommon sn-1stereoconfiguration o f BMP remains unknown; it may cause BMP to be resistant to the action o f lysosomal phospholipases [12]. In conclusion, we have observed that both the active site and the phospholipid activation site(s) o f the rat liver lysosomal lipase lack stereospecificity. This is in accordance with its role as a general purpose digestive enzyme, like the equally unspecific pancreatic lipase. As activators the lysosomal lipase may utilize either acidic phospholipids ingested together with the triacylglycerols, or the endogenous lysosomal BMP. In the absence of exogenous phospholipids, BMP alone is sufficient to support the degradation of triacylglycerols by the lysosomal lipase.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
K. Hayase and A.L. Tappel J. Biol. Chem., 245 (1970) 169. W. Guder, L. Weiss and O. Wieland Biochim. Biophys. Acta, 187 (1969) 183. S. Teng and A. Kaplan, J. Biol. Chem., 249 (1974) 1064. H.R. Sloan and R. Fredrickson J. Clin. Invest., 51 (1972) 1923. A.D. Patrick and B.D. Lake Nature (London), 222 (1969) 1067. B.D. Lake and A.D. Patrick J. Pediatr., 76 (1970) 262. M. Kariya and A. Kaplan J. Lipid Res., 14 (1973) 243. N.H. Tattrie, R.A. Bailey and M. Kates Arch. Biochem. Biophys., 78 (1958) 319. F. Paltauf, F. Esfaldi and A. Holasek FEBS Lett., 40 (1974) 119. B. ~kesson, S. Gronowitz and B. HersliSf FEBS Lett., 71 (1976) 241. P. Somerhaxju, T. Kuusi, F. Paltauf and P.K.J. Kinnunen FEBS Lett., 96 (1978) 170. J. Brotherus, O. Renkonen, J. Herrmann and W. Fischer Chem. Phys. Lipids, 13 (1974) 178. A. Joutti, J. Brotherus, O. Renkonen, R. Laine and W. Fischer Biochim. Biophys. Acta, 450 (1976) 206. P. Belfrage and M. Vaughan J. Lipid Res., 10 (1971) 341. J,R. Wherret and S. Huterer J. Biol. Chem., 247 (1972) 4114. J. Folch, M. Lees and G.H. Sloane-Stanley J. Biol. Chem., 266 (1957) 485. G.R. Bartlett J. Biol. Chem., 234 (1959) 459. Th. J.C. van Berkel, J.K. Kruijt and J.F. Koster Eur. J. Biochem., 58 (1975) 145. Th. J.C. van Berkel, H. Vaandrager, J.K. Kruijt and J.F. Koster Biochim. Biophys. Acta, 617 (1980) 446. A.J. Slotboom, H.M. Verheij, P.H.M. Baartmanns and L.L.M. van Deenen Chem. Phys. Lipids, 17 (1976) 128. J. Brotherus and O. Renkonen J. Lipid Res., 18 (1977) 191.
235
THE ACTIVE SITE AND THE PHOSPHOLIPID ACTIVATION OF RAT LIVER LYSOSOMAL LIPASE ARE NOT STEREOSPECIFIC
ANNELI JOUTTI, PETRI VAINIO, JAAKKO R. BROTHERUS ~ , F. PALTAUF a and PAAVO K.J. KINNUNEN Department of Medical Chemistry, University of Helsinki, Siltavuorenpenger 10 A, SF-O0170 Heisinki 17 (Finland) and alnstitute of Biochemistry, Technical University of Graz, A-8010 Graz (Austria)
Received June 15th, 1981
accepted August 17th, 1981
The stereochemical specificity of lysosomal lipase of rat liver was investigated using enantiomeric triacylglycerol analogs, sn-l-alkyl-2,3-diacylglycerol and sn-3-alkyl-l,2-diacylglycerol as substrates. Lysosomal lipase utilized both substrates with equal rates. The dependence of the activity of lysosomal lipase on the stereoconfiguration of activating acidic phospholipid was also studied. Our results showed that both sn-3-phospholipids (diphosphatidylglycerol, phosphatidylserine) and sn-l-phospholipids (bis(monoacylglycero)phosphate (BMP)) were efficient activators of this enzyme and thus the stereochernical configuration of the activating phospholipid is not important. Accordingly, the rat liver lysosomal lipase lacks stereospecificity with respect to both the triacylglycerol substrate and the acidic phospholipid activator. Keywords: lysosomal lipase; stereospecificity; acidic phospholipids; bis(monoacylglycerol) phosphate.
Introduction Liver lysosomes contain a triacylglycerol lipase which exhibits a pH optimum of approximately 4 . 0 - 4 . 5 [1,2]. The enzyme is tightly associated with the lysosomal membranes [3]. In addition to triacylglycerol hydrolase activity cholesterol esterase activity can be detected [3,4]; Deficiency of the lysosomal lipase, known as Woiman's syndrome [5,6], leads to accumulation of triacylglycerols and cholesterol esters in lysosomes. In vitro studies with a partially purified enzyme preparation have revealed that rat lysosomal lipase is strongly stimulated by acidic phospholipids [3,7]. In contrast, the lipase can be completely inhibited by heparin [3]. The stereospecificity (i.e., differentiation between position sn-1- and sn-3-acyl chains of triacylglycerols) is known for several lipases. Pancreatic lipase lacks stereospecificity [8] ; in contrast, lipoprotein lipase and hepatic endothelial lipase hydrolyze preferentially the sn-l-ester bond of the triacylglycerols [9-11 ]. In the 0009-3084/81/0000-0000/$02.50 O 1981 Elsevier/North-Holland Scientific Publishers Ltd.
236
A. Joutti et aL, Stereospecificity of rat liver lysosomal lipase
present study the stereochemical specificity of lysosomal lipase of rat liver was investigated using enantiomeric synthetic substrates [9,11]. In principle, the lysosomal lipase could also be specific for the stereochemical structure of the activating acidic phospholipids. All phospholipids tested so far were derivatives of sn-3-glycerophosphate. However, the major acidic phospholipid of lysosomes, bis(monoacylglycero)phosphate (BMP), contains only sn-l-glycerophosphate [12,13]. Therefore, we tested its efficiency as an activator of the enzyme. Our results show that the rat liver lysosomal lipase lacks stereospecificity with respect to both the triacylglycerol substrate and the phospholipid activator. Materials and Methods
L ysosomal lipase The lysosomal lipase of rat liver was purified to Step II according to Teng and Kaplan [3]. Rat livers were homogenized in 0.25 M sucrose containing 1 mM EDTA (pH 7.2) (3 ml/g of liver) at 4°C. The homogenate was centrifuged at 1000 × g, for 10 min. The supernatant was centrifuged at 3300 × g, for 10 min, and the resulting supernatant at 12 000 ×g, for 40 rnin. The particulate fraction from the last centrifugation was dispersed in 10 ml of 0.25 M sucrose and centrifuged at 12 000 ×g for 20 min. The pellet was homogenized in 5 ml of 2% Triton X-100 (pH 6.0). After 60 min, the suspension was centrifuged at l0 s X g in a Spinco ultracentrifuge for 60 min at 4°C. The supematant fluid was stabilized by the addition of half its volume of glycerol and stored at --20°C. Assay o f lysosomal lipase The lipase assay conditions were identical to those used by Teng and Kaplan [3] except that the volume was 200/al. The reaction mixture contained 50 mM sodium acetate (pH 4), 2% of Triton X-IO0, 1 mM tri[9,10-SH]oleoylglycerol (1.5 • lO s cpm/assay; Amersham), and indicated amounts of phospholipids. The reaction was started by the addition of enzyme. Incubations were carried out at 37°C, for 10 min, and the reactions were stopped by the addition of chloroform/methanol/ heptane (1.4 : 1.25 : 1.0, by vol.) and the phases were separated by centrifugation after addition of 0.14 M borate, whereafter radioactivity of free fatty acids was determined [14]. Phospholipids Phospholipids were isolated from male rats that had received intraperitoneaUy Triton WR 1339 which leads to accumulation of BMP in liver lysosomes [15]. The lipids from four rat livers were extracted [16]. The liver phosphatidylcholines, phosphatidylserines and BMP were obtained from the lipid extract by column chromatography on silicic acid and by thin-layer chromatography [ 12,13 ]. Diphosphatidylglycerol was purified from rat heart by the same method. Phosphorus was analyzed by the method of Bartlett [17].
A. Joutti et al., Stereospecificity of rat liver lysosomal lipase
237
Assay for stereospecificity of lysosomal lipase The enantiomeric triacylglycerol analogs, 3-O-[1-14C]octadecyl-l,2-dioleoyl-snglycerol (0.05 mCi/mmol) and 1-O-[9,10-aH]octadecyl-2,3-dioleoyl-sn-glycerol (0.11 mCi/mmol) were prepared according to Paltauf et al. [9,11 ]. The incubation mixture contained 1 /amol of the substrate, 2 mM phosphatidylserines and 2% Triton X-100 in 50 mM sodium acetate (pH 4.5). Hydrolysis of enantiomeric triacylglycerols by lysosomal lipase was carried out for 60 min at 37°C. The reaction was started by the addition of 20/ag of liver lysosomal lipase extract. At the indicated times 300-~1 aliquots were taken from the reaction mixture (2.5 ml) and extracted as described [16]. The reaction products were separated on silica gel plates using hexane/ether/acetic acid (80 : 20 : 2, by vol.) as solvent system and were counted for 14C-and 3H-radioactivity.
Results and Discussion
Stereospecificity of lysosomal lipase was determined by utilizing the enantiomeric triacylglycerol analogs, sn-l-alkyl-2,3-diacylglycerol and sn-3-alkyl-l,2diacylglycerol, as substrates (Fig. 1). It is evident that lysosomal lipase utilizes both substrates with equal rates and thus lacks stereochemical specificity. This result agrees with the postulated function of the liver lysosomal lipase in the clearance of triacylglycerols from partially degraded plasma lipoproteins [18,19]. These
100
I--
o Q <
n.
80
60
=d
o ~
4o
0 Z
,0
20
00
I
10
I
20
I
30
I
40
I
50
I
60
TIME,min
Fig. 1. Degradation of the enantiomeric triacylglycerol analogs, 1-alkyl-2,3-diacylglyceroland 3-alkyl-l,2-diacylglycerol,by liver lysosornal lipase. Detailed description of the assay mixture is given in Materials and Methods. Alkyldiacylglycerol(e, o); alkylacylglycerol (A, z~) alkylglycerol (a, a). Closed symbols, alkyl in the sn-3-position;open symbols, alkyl in the sn-l-position.
238
A. Joutti et al., Stereospeeifieity o f rat liver lysosornal lipase
ld E
6
E u 0
E
c >.
o iii
.d
0 0
I
I
I
1
2
3
[PHOSPHOLIPID],mM Pi Fig. 2. Influence of phospholipid concentration on acid lipase activity. Lipase activity of a Triton X-100 extract of lysosomal fraction (20 ~g protein) in the presence of phosphatidylcholine (o), diphosphatidylglycerol (e), phosphatidylserine (a), and BMP (o). See text for details.
degradation products can be used as precursors for the resynthesis of other molecules, and the stereochemical position in which the fatty acid had originally been bound is not of importance. However, the question still remains why lipoprotein lipase and hepatic endothelial lipase do possess relatively strict stereospecificity [9-11 ] and whether this distinction of lipases into two classes has any biological significance. Figure 2 illustrates the deperrdence of the lysosomal lipase activity on the concentration of added phospholipids. Lecithin did not change the activity of the enzyme. In contrast, the acidic sn-3-phospholipids, diphosphatidylglycerols and phosphatidylserines, were efficient activators of lysosomal lipase; the activity of the enzyme alone is 5-10% of that in the presence of 2 mM phosphatidylserines or diphosphatidylglycerols. These observations are in agreement with the results of Kaplan and coworkers [3,7]. The trace of lipase activity in the absence of added lipids is probably best explained by the presence of residual acidic lipid in the enzyme preparation (Step II material, Ref. 3). In addition to sn-3-glycerophospholipids, the lysosomal lipase was also strongly activated by BMP (Fig. 2). The backbone of this lipid has the uncommon stereochemical structure of sn- 1-glycerophospho-l'-glycerol [ 12,13 ]. Consequently, the stereochemical configuration of the activating phospholipid is not important, and
A. Joutti et aL, Stereospecific#y o f rat liver lysosomal lipase
239
the stimulating effect o f acidic phospholipids is likely to be mediated by a change in the charge o f the substrate [20]. BMP is specifically found in the lysosomes [ 15,21 ] ;however its function in lysosomes is not known. Since BMP is the main acidic phospholipid o f the lysosomes and an efficient activator o f liver lysosomal lipase in vitro (Fig. 2), it is possible that this lipid activates the lysosomal lipase also in vivo. The role of the uncommon sn-1stereoconfiguration o f BMP remains unknown; it may cause BMP to be resistant to the action o f lysosomal phospholipases [12]. In conclusion, we have observed that both the active site and the phospholipid activation site(s) o f the rat liver lysosomal lipase lack stereospecificity. This is in accordance with its role as a general purpose digestive enzyme, like the equally unspecific pancreatic lipase. As activators the lysosomal lipase may utilize either acidic phospholipids ingested together with the triacylglycerols, or the endogenous lysosomal BMP. In the absence of exogenous phospholipids, BMP alone is sufficient to support the degradation of triacylglycerols by the lysosomal lipase.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
K. Hayase and A.L. Tappel J. Biol. Chem., 245 (1970) 169. W. Guder, L. Weiss and O. Wieland Biochim. Biophys. Acta, 187 (1969) 183. S. Teng and A. Kaplan, J. Biol. Chem., 249 (1974) 1064. H.R. Sloan and R. Fredrickson J. Clin. Invest., 51 (1972) 1923. A.D. Patrick and B.D. Lake Nature (London), 222 (1969) 1067. B.D. Lake and A.D. Patrick J. Pediatr., 76 (1970) 262. M. Kariya and A. Kaplan J. Lipid Res., 14 (1973) 243. N.H. Tattrie, R.A. Bailey and M. Kates Arch. Biochem. Biophys., 78 (1958) 319. F. Paltauf, F. Esfaldi and A. Holasek FEBS Lett., 40 (1974) 119. B. ~kesson, S. Gronowitz and B. HersliSf FEBS Lett., 71 (1976) 241. P. Somerhaxju, T. Kuusi, F. Paltauf and P.K.J. Kinnunen FEBS Lett., 96 (1978) 170. J. Brotherus, O. Renkonen, J. Herrmann and W. Fischer Chem. Phys. Lipids, 13 (1974) 178. A. Joutti, J. Brotherus, O. Renkonen, R. Laine and W. Fischer Biochim. Biophys. Acta, 450 (1976) 206. P. Belfrage and M. Vaughan J. Lipid Res., 10 (1971) 341. J,R. Wherret and S. Huterer J. Biol. Chem., 247 (1972) 4114. J. Folch, M. Lees and G.H. Sloane-Stanley J. Biol. Chem., 266 (1957) 485. G.R. Bartlett J. Biol. Chem., 234 (1959) 459. Th. J.C. van Berkel, J.K. Kruijt and J.F. Koster Eur. J. Biochem., 58 (1975) 145. Th. J.C. van Berkel, H. Vaandrager, J.K. Kruijt and J.F. Koster Biochim. Biophys. Acta, 617 (1980) 446. A.J. Slotboom, H.M. Verheij, P.H.M. Baartmanns and L.L.M. van Deenen Chem. Phys. Lipids, 17 (1976) 128. J. Brotherus and O. Renkonen J. Lipid Res., 18 (1977) 191.