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Fluorometric assay for pancreatic cholesterylester hydrolase
Chemistry and Physics of Liptds, 36 (1985) 335-341
335
Elsevier Scientific Publishers Ireland Ltd.
FLUOROMETRIC ASSAY FOR PANCREATIC CHOLESTERYLESTER HYDROLASE
ANNEL1 JOUTTI, LEILA KOTAMA, JORMA A. VIRTANEN and PAAVO K.J. KINNUNEN
Departments of Basic Chemistry, Physical Chemistry, Organic Chemistry and Medical Chemistry, University of Helsinki, Slitavuorenpenger 10, Helsinki (Finland) Received September 17th, 1984 accepted November 26th, 1984
revision received November 26th, 1984
A fluorescent cholesterylester analogue, cholesteryl 6-pyrenylhexanoate (ChPH), was used as a suhstrate for pancreatic cholesterylester hydrolase (CEH, EC 3.1.1.13).'The substrate consisted of ChPH in egg phosphatidylchofinc stabilized microemuision with the aqueous phase containing deoxycholate below its critical micellat concentration. Due to the high local concentration of the pyrene moiety in the ChPH phase the fluorescence emission due to monomeric pyrene (IM) is greatly exceeded by the excimer fluorescence intensity (IF.). Upon reacting with CEH 6-pyrenylhexanoic acid and free cholesterol ate formed. The fluorescent product, 6pyrenylhexanoic acid, is transferred into the aqueous phase containing deoxycholate, thus resulting in an enhanced fluorescence due to monomeric pyrene. CEH activity can thus be assessed directly by monitoring IM vs. time without product separation. Useful assay conditions were found to be 10 ~M ChPH, 0.1 ~M egg phosphatidylcholine, 2 mM sodium deoxycholate at 250C and pH 6.5-7.0.
Keywords: cholesterylester hydrolase; fluorescence; pyrene.
Introduction Cholesterylester hydrolas¢, CEH (EC 3.1.1.13) is found in liver, pancreas [1], steroidogenic tissues [2] and arterical walls [3]. At least three types o f intracellular CEH have been detected: cytoplasmic with neutral pH optimum, microsomal with a lower pH optimum and acidic CEH found in lysosomes [4]. CEH activity is usually determined by methods involving lipid extraction such as thin-layer chromatography and liquid-liquid partition [5]. We now describe a rapid and sensitive fluorometric assay for CEH using a novel fluorescent cholesterylester analogue, cholesteryl 6-pyrenylhexanoate (ChPH). Fluorescent substrates have been employed previously for lipase/esterase assays [ 6 - 1 3 ] , and fluorescent cholesterylester analogues have been described [14] but, as far as we know, have not been used for CEH assays. The photophysics o f pyrene is well understood [9,15]. Two fluorescent relaxation pathways are available for a monomeric excited pyrene. It can return to ground 0009-3084/85/$03.30 © 1985 Elsevier Scientific Pubfishers Ireland Ltd. Published and Printed in Ireland
336 state by emission of photon with a maximum at 397 nm. The emission spectrum of monomeric pyrene exhibits vibrational structure and is designated as IM. However, if the local concentration ofpyrene is high, the excited monomeric species can collide with ground state pyrenes and give rise to an excited dimer, the excimer [15]. The excimer relaxes back to two ground state pyrenes giving rise to a broad and structureless emission band with a maximum around 470 nm and designated as IE. This property of pyrene has been used to assess the activity of phospholipase A2 [9,11 ] and to study the regulation of the catalytic expression by this enzyme by the conformation of the substrate phospholipids [12,13]. It is also applicable to the assay of cholesterylester hydrolase, described in this communication.
Martials and Me~ods
Reagents The fluorescent cholesterylester analogue (PhCH) and 6-pyrenylhexanoic acid (PHA) were purchased from KSV-Chemicals Oy, Valimotie 7, Helsinki 38, Finland. Cholesterylester hydrolase (CEH, cholesterylesterase, EC 3.1.1.13) from bovine pancreas, and other chemicals were from Sigma. CEH assay The substrate for 100 assays was prepared by mixing 2.0/zrnol (1.4 mg) of ChPH and 20 nmol (14 /ag) of egg PC in chloroform. The solvent was removed under a stream of nitrogen and the dried lipid was then dissolved in 1 ml of absolute ethanol. If required this solution can be stored at -20°C. For each standard assay 10/al of this solution was rapidly injected with 20/al Hamilton syringe into 2 ml of assay buffer [16,17]. The final assay system for CEH contained, in a total volume of 2 ml, 20 nmol of ChPH, 0.2 nmol egg PC and 4.0/amol NaDOC in 50 mM Tris-HCl buffer (pH 7.0). Prior to the enzymatic reaction the assay mixture was allowed to equilibrate for approx. 5 min. The reaction was started by the addition of CEH. Fluorescence measurements were performed with a SLM 4800S spectrofluorometer using excitation wavelength of 345 nm. Assays were monitored in magnetically stirred cuvettes thermostated to 25°C and the emission intensity at 397 nm was recorded as a function of time. The assay was calibrated by adding known amounts of free PHA to the reaction mixture in the absence of enzyme and observing the monomeric pyrene emission intensity at 397 nm after each addition [ 11 ]. Results and Discussion
Characteristics of the substrate The aim of the present study was to develop a fluorometric homogeneous assay system for CEH. For this purpose a fluorescent cholesterylester analogue ChPH (Fig. 1) was employed. The pyrene moiety in the acyl chain was chosen because it
337
O
Fig. I. Chemical stxucture and CPK-spacefilling model of ChPH. allows monitoring of changes in the local concentration of 6-pyrenylhexanoate due to CEH without the separation of reaction products. Egg PC-stabilized microemulsions of cholesterylester were formed by squirting ethanol solution of these lipids into aqueous buffers [16,17]. Intensity ratios IE,/IM of pyrene were recorded for dispersions produced at varying ChPH: egg PC molar ratios (Fig. 2). Progressive increase in IE/IM was observed upon enrichment of ChPH in the emulsion droplets. The molar ratio of 100:1 of ChPH to egg PC was chosen for further experiments since it yields significant changes in IM upon CEH reaction under appropriate conditions. The substrate concentration (at constant ChPH: egg PC molar ratio) had no effect on the IE/IM ratio (data not shown). The effect of temperature on the IE/IM ratio of the substrate is shown in Fig. 3 and shows enhanced rate of excimer formation upon increase in temperature from 20°C to 45°C. Due to thermal instability of the enzyme preparation 25°C was used in the CEH assay. The rate of CEH reaction is greatly stimulated by sodium deoxycholate [18]. Accordingly, no CEH activity could be observed in the absence of NaDOC. The effect of sodium deoxycholate concentration on the IF,/IM of the substrate is shown in Fig. 4. When the concentration of NaDOC exceeds the critical micelle concentration (CMC) all of the fluorescence becomes monomeric. This apparently occurs as a consequence of solubilization of the egg PC-stabilized emulsion droplets and formation of mixed micelles of ChPH with the bile salt and phospholipid. Below the CMC of NaDOC the substrate emulsion is stable as judged by the high and unaffected IE[IM. The NaDOC concentration used for the assay (~2 mM) was below the CMC.
338
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Fig. 2. ll~/IMaS a function of [ChPHl/[egg PC] molar ratio. Excitation at 345 nm. nm and I E a t 470 nm.
IMat 397
Fig. 3. l~/IMas a function of temperature. Molar ratio of [ChPHI/[egg PC] was 100: 1. Cuvette contained 30 nmol ChPH and 0.3 nmol egg PC in 2.0 m150 mM Tris buffer, pH 7.0. Fig. 4. l~/IMas a function of NaDOC concentration. Conditions otherwise as in Fig. 3.
so
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,
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450 WAVELENGTH
,
,
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,
,
550
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Fig. 5. Hydrolysis of ChPH by CEH at 25°(: observed by pyrene fluoresoenee emisaion spectra (a) before addition of CEH, (b) 0.5 min, (c) 2.5 min, (d) 4.5 min and (e) 6.5 rain after addition of 5 ~g CEH. The reaction mixture consisted of 20 nmol ChPH, 0.2 nmol egg PC and 4.0 ~mol NaDOC in 2.0 m150 mM Tris (pH 7.0).
CEH assay Pancreatic CEH acts on the substrate ChPH producing 6-pyrenylhexanoic acid and cholesterol. Subsequent changes in the emission spectra of ChPH are illustrated in Fig. 5: the monomeric pyrene fluorescence at 397 nm increases in intensity as the hydrolysis proceeds while the excirner emission decreases. An isoemissive point at 425 nm is seen. The reaction product, 6-pyrenylhexanoate was also verified using thin-layer chromatography.
u
J
pH Fig. 6. Pancreatic CEH activity as a function of pH. Emission read at 397 run as a function of time. Conditions as in Fig. 5.
340
"I"
ug 0 - -
abe-
2b
--~0
4O
[ChPH], pM Fig. 7. Pancreatic CEH activity as a function of substrate concentration. Conditions otherwise as in Fig. 5.
The pH optimum of CEH from pancreas was found to be approx. 6 . 5 - 7 . 0 (Fig. 6). The presence of NaDOC dictates the lowest attainable pH as it precipitates below pH 5.5. The enzyme reaction showed an apparent saturation at 20/alVl substrate (Fig. 7). The Lineweaver-Burk plot gives an apparent Km-value of approx. 4.7/aM ChPH. ChPH thus provides a sensitive fluorescent probe for the determination of CEH activity as the hydrolytic reaction can easily be followed by the increasing monomeric pyrene fluorescence emission without the separation of the reaction products.
References 1 V.J. Neelon and L. Lack, Biochim. Biophys. Acta, 487 (1977) 137-144. 2 R.C. Pittman and D. Steinberg, Biochim. Biophys. Acta, 487 (1977) 431 -444. 3 T. Sakurada, H. Orimo, H. Okabe, A. Noma and M. Murakami, Biochim. Biophys. Acta, 424 (1976) 204-212. 4 B. Lundberg, R. Clements and T. Ikivgren, Biochim. Biophys. Acta, 572 (1979) 492-501. 5 L.L. Gallo, R. Atasoy, G.V. Vahouny and C.R. Treadwell, J. Lipid Res., 19 (1978) 913917. 6 0 . S . Wolfbeis and E. Koller, Anal. Biochem., 129 (1983) 365-370. 7 G.T.N. Besley and S.E. Moss, Biochim. Biophys. Acta, 752 (1983) 54-64. 8 J.F. Koster, H. Vaandrager and T.J.C. van Berkel, Biochim. Biophys. Acta, 618 (1980) 98105. 9 H.S. Hendrickson and P.N. Rauk, Anal. Biochem., 116 (1981) 553-559. I0 P. Vainio, J.A. Virtanen, J.T. Sparrow, A.M. Gotto and P.K.J. Kinnunen, Chem. Phys. Lipids, 33 (1982) 21-30. 11 T. Thuren, J.A. Virtanen, P. Vainio and P.K.J. Kinnunen, Chem. Phys. Lipids, 33 (1983) 283 -292. 12 P.K.J. Kinnunen, T. Thuren, P. Vainio and J.A. Virtanen, in: V. Degiorgio and M. Corti (Eds.), Physics of Amphiphiles, Mieelles, Vesicles and Microemulsions, Elsevier,Amsterdam, 1984, in press.
341
13 T. Thuren, P. Vainio, J.A. Virtanen, K. Blomquist, P. Somerharju and P.K.J. Kinnunen,Biochemistry, (1984) in press. 14 L.A. Sklar, W.W. Mantulin and H.I. Pownall, Biochem. Biophys. Res. Commun., 105 (1982) 674--680. 15 Th. F~rster, Angew. Chem., 81 (1969) 364-370. 16 S. Bazri and E.D. Korn, Biochim. Biophys. Acta, 298 (1973) 1015-1019. 17 D.P. Via, I.F. Craig, G.W. Jacobs, L. vanWinde, A.M. Gotto and L.C. Smith, J. Lipid Res., 23 (1982) 570-576. 18 G.V. Vahouny, S. Weersing and C.R. Treadwell, Biochim. Biophys. Acta, 98 (1965)607616.
335
Elsevier Scientific Publishers Ireland Ltd.
FLUOROMETRIC ASSAY FOR PANCREATIC CHOLESTERYLESTER HYDROLASE
ANNEL1 JOUTTI, LEILA KOTAMA, JORMA A. VIRTANEN and PAAVO K.J. KINNUNEN
Departments of Basic Chemistry, Physical Chemistry, Organic Chemistry and Medical Chemistry, University of Helsinki, Slitavuorenpenger 10, Helsinki (Finland) Received September 17th, 1984 accepted November 26th, 1984
revision received November 26th, 1984
A fluorescent cholesterylester analogue, cholesteryl 6-pyrenylhexanoate (ChPH), was used as a suhstrate for pancreatic cholesterylester hydrolase (CEH, EC 3.1.1.13).'The substrate consisted of ChPH in egg phosphatidylchofinc stabilized microemuision with the aqueous phase containing deoxycholate below its critical micellat concentration. Due to the high local concentration of the pyrene moiety in the ChPH phase the fluorescence emission due to monomeric pyrene (IM) is greatly exceeded by the excimer fluorescence intensity (IF.). Upon reacting with CEH 6-pyrenylhexanoic acid and free cholesterol ate formed. The fluorescent product, 6pyrenylhexanoic acid, is transferred into the aqueous phase containing deoxycholate, thus resulting in an enhanced fluorescence due to monomeric pyrene. CEH activity can thus be assessed directly by monitoring IM vs. time without product separation. Useful assay conditions were found to be 10 ~M ChPH, 0.1 ~M egg phosphatidylcholine, 2 mM sodium deoxycholate at 250C and pH 6.5-7.0.
Keywords: cholesterylester hydrolase; fluorescence; pyrene.
Introduction Cholesterylester hydrolas¢, CEH (EC 3.1.1.13) is found in liver, pancreas [1], steroidogenic tissues [2] and arterical walls [3]. At least three types o f intracellular CEH have been detected: cytoplasmic with neutral pH optimum, microsomal with a lower pH optimum and acidic CEH found in lysosomes [4]. CEH activity is usually determined by methods involving lipid extraction such as thin-layer chromatography and liquid-liquid partition [5]. We now describe a rapid and sensitive fluorometric assay for CEH using a novel fluorescent cholesterylester analogue, cholesteryl 6-pyrenylhexanoate (ChPH). Fluorescent substrates have been employed previously for lipase/esterase assays [ 6 - 1 3 ] , and fluorescent cholesterylester analogues have been described [14] but, as far as we know, have not been used for CEH assays. The photophysics o f pyrene is well understood [9,15]. Two fluorescent relaxation pathways are available for a monomeric excited pyrene. It can return to ground 0009-3084/85/$03.30 © 1985 Elsevier Scientific Pubfishers Ireland Ltd. Published and Printed in Ireland
336 state by emission of photon with a maximum at 397 nm. The emission spectrum of monomeric pyrene exhibits vibrational structure and is designated as IM. However, if the local concentration ofpyrene is high, the excited monomeric species can collide with ground state pyrenes and give rise to an excited dimer, the excimer [15]. The excimer relaxes back to two ground state pyrenes giving rise to a broad and structureless emission band with a maximum around 470 nm and designated as IE. This property of pyrene has been used to assess the activity of phospholipase A2 [9,11 ] and to study the regulation of the catalytic expression by this enzyme by the conformation of the substrate phospholipids [12,13]. It is also applicable to the assay of cholesterylester hydrolase, described in this communication.
Martials and Me~ods
Reagents The fluorescent cholesterylester analogue (PhCH) and 6-pyrenylhexanoic acid (PHA) were purchased from KSV-Chemicals Oy, Valimotie 7, Helsinki 38, Finland. Cholesterylester hydrolase (CEH, cholesterylesterase, EC 3.1.1.13) from bovine pancreas, and other chemicals were from Sigma. CEH assay The substrate for 100 assays was prepared by mixing 2.0/zrnol (1.4 mg) of ChPH and 20 nmol (14 /ag) of egg PC in chloroform. The solvent was removed under a stream of nitrogen and the dried lipid was then dissolved in 1 ml of absolute ethanol. If required this solution can be stored at -20°C. For each standard assay 10/al of this solution was rapidly injected with 20/al Hamilton syringe into 2 ml of assay buffer [16,17]. The final assay system for CEH contained, in a total volume of 2 ml, 20 nmol of ChPH, 0.2 nmol egg PC and 4.0/amol NaDOC in 50 mM Tris-HCl buffer (pH 7.0). Prior to the enzymatic reaction the assay mixture was allowed to equilibrate for approx. 5 min. The reaction was started by the addition of CEH. Fluorescence measurements were performed with a SLM 4800S spectrofluorometer using excitation wavelength of 345 nm. Assays were monitored in magnetically stirred cuvettes thermostated to 25°C and the emission intensity at 397 nm was recorded as a function of time. The assay was calibrated by adding known amounts of free PHA to the reaction mixture in the absence of enzyme and observing the monomeric pyrene emission intensity at 397 nm after each addition [ 11 ]. Results and Discussion
Characteristics of the substrate The aim of the present study was to develop a fluorometric homogeneous assay system for CEH. For this purpose a fluorescent cholesterylester analogue ChPH (Fig. 1) was employed. The pyrene moiety in the acyl chain was chosen because it
337
O
Fig. I. Chemical stxucture and CPK-spacefilling model of ChPH. allows monitoring of changes in the local concentration of 6-pyrenylhexanoate due to CEH without the separation of reaction products. Egg PC-stabilized microemulsions of cholesterylester were formed by squirting ethanol solution of these lipids into aqueous buffers [16,17]. Intensity ratios IE,/IM of pyrene were recorded for dispersions produced at varying ChPH: egg PC molar ratios (Fig. 2). Progressive increase in IE/IM was observed upon enrichment of ChPH in the emulsion droplets. The molar ratio of 100:1 of ChPH to egg PC was chosen for further experiments since it yields significant changes in IM upon CEH reaction under appropriate conditions. The substrate concentration (at constant ChPH: egg PC molar ratio) had no effect on the IE/IM ratio (data not shown). The effect of temperature on the IE/IM ratio of the substrate is shown in Fig. 3 and shows enhanced rate of excimer formation upon increase in temperature from 20°C to 45°C. Due to thermal instability of the enzyme preparation 25°C was used in the CEH assay. The rate of CEH reaction is greatly stimulated by sodium deoxycholate [18]. Accordingly, no CEH activity could be observed in the absence of NaDOC. The effect of sodium deoxycholate concentration on the IF,/IM of the substrate is shown in Fig. 4. When the concentration of NaDOC exceeds the critical micelle concentration (CMC) all of the fluorescence becomes monomeric. This apparently occurs as a consequence of solubilization of the egg PC-stabilized emulsion droplets and formation of mixed micelles of ChPH with the bile salt and phospholipid. Below the CMC of NaDOC the substrate emulsion is stable as judged by the high and unaffected IE[IM. The NaDOC concentration used for the assay (~2 mM) was below the CMC.
338
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®
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i
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1:2 1:1 2:1 10.1 100:1 [ChPH]: [Egg PC]
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TEM~RATURE.~
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6
8
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10
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m
12
,
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m
16
, m
18
.
-
20
[NaDOC], mM
Fig. 2. ll~/IMaS a function of [ChPHl/[egg PC] molar ratio. Excitation at 345 nm. nm and I E a t 470 nm.
IMat 397
Fig. 3. l~/IMas a function of temperature. Molar ratio of [ChPHI/[egg PC] was 100: 1. Cuvette contained 30 nmol ChPH and 0.3 nmol egg PC in 2.0 m150 mM Tris buffer, pH 7.0. Fig. 4. l~/IMas a function of NaDOC concentration. Conditions otherwise as in Fig. 3.
so
339
~ 1.C u) z uJ I - .8 z . i
(J
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> .2.J
Ilg i
360
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w
,
,
400
,
,
i
i
i
450 WAVELENGTH
,
,
500
,
,
,
,
550
nm
Fig. 5. Hydrolysis of ChPH by CEH at 25°(: observed by pyrene fluoresoenee emisaion spectra (a) before addition of CEH, (b) 0.5 min, (c) 2.5 min, (d) 4.5 min and (e) 6.5 rain after addition of 5 ~g CEH. The reaction mixture consisted of 20 nmol ChPH, 0.2 nmol egg PC and 4.0 ~mol NaDOC in 2.0 m150 mM Tris (pH 7.0).
CEH assay Pancreatic CEH acts on the substrate ChPH producing 6-pyrenylhexanoic acid and cholesterol. Subsequent changes in the emission spectra of ChPH are illustrated in Fig. 5: the monomeric pyrene fluorescence at 397 nm increases in intensity as the hydrolysis proceeds while the excirner emission decreases. An isoemissive point at 425 nm is seen. The reaction product, 6-pyrenylhexanoate was also verified using thin-layer chromatography.
u
J
pH Fig. 6. Pancreatic CEH activity as a function of pH. Emission read at 397 run as a function of time. Conditions as in Fig. 5.
340
"I"
ug 0 - -
abe-
2b
--~0
4O
[ChPH], pM Fig. 7. Pancreatic CEH activity as a function of substrate concentration. Conditions otherwise as in Fig. 5.
The pH optimum of CEH from pancreas was found to be approx. 6 . 5 - 7 . 0 (Fig. 6). The presence of NaDOC dictates the lowest attainable pH as it precipitates below pH 5.5. The enzyme reaction showed an apparent saturation at 20/alVl substrate (Fig. 7). The Lineweaver-Burk plot gives an apparent Km-value of approx. 4.7/aM ChPH. ChPH thus provides a sensitive fluorescent probe for the determination of CEH activity as the hydrolytic reaction can easily be followed by the increasing monomeric pyrene fluorescence emission without the separation of the reaction products.
References 1 V.J. Neelon and L. Lack, Biochim. Biophys. Acta, 487 (1977) 137-144. 2 R.C. Pittman and D. Steinberg, Biochim. Biophys. Acta, 487 (1977) 431 -444. 3 T. Sakurada, H. Orimo, H. Okabe, A. Noma and M. Murakami, Biochim. Biophys. Acta, 424 (1976) 204-212. 4 B. Lundberg, R. Clements and T. Ikivgren, Biochim. Biophys. Acta, 572 (1979) 492-501. 5 L.L. Gallo, R. Atasoy, G.V. Vahouny and C.R. Treadwell, J. Lipid Res., 19 (1978) 913917. 6 0 . S . Wolfbeis and E. Koller, Anal. Biochem., 129 (1983) 365-370. 7 G.T.N. Besley and S.E. Moss, Biochim. Biophys. Acta, 752 (1983) 54-64. 8 J.F. Koster, H. Vaandrager and T.J.C. van Berkel, Biochim. Biophys. Acta, 618 (1980) 98105. 9 H.S. Hendrickson and P.N. Rauk, Anal. Biochem., 116 (1981) 553-559. I0 P. Vainio, J.A. Virtanen, J.T. Sparrow, A.M. Gotto and P.K.J. Kinnunen, Chem. Phys. Lipids, 33 (1982) 21-30. 11 T. Thuren, J.A. Virtanen, P. Vainio and P.K.J. Kinnunen, Chem. Phys. Lipids, 33 (1983) 283 -292. 12 P.K.J. Kinnunen, T. Thuren, P. Vainio and J.A. Virtanen, in: V. Degiorgio and M. Corti (Eds.), Physics of Amphiphiles, Mieelles, Vesicles and Microemulsions, Elsevier,Amsterdam, 1984, in press.
341
13 T. Thuren, P. Vainio, J.A. Virtanen, K. Blomquist, P. Somerharju and P.K.J. Kinnunen,Biochemistry, (1984) in press. 14 L.A. Sklar, W.W. Mantulin and H.I. Pownall, Biochem. Biophys. Res. Commun., 105 (1982) 674--680. 15 Th. F~rster, Angew. Chem., 81 (1969) 364-370. 16 S. Bazri and E.D. Korn, Biochim. Biophys. Acta, 298 (1973) 1015-1019. 17 D.P. Via, I.F. Craig, G.W. Jacobs, L. vanWinde, A.M. Gotto and L.C. Smith, J. Lipid Res., 23 (1982) 570-576. 18 G.V. Vahouny, S. Weersing and C.R. Treadwell, Biochim. Biophys. Acta, 98 (1965)607616.