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Interactions of Phosphatidylcholine Surface Monolayers with Triglyceride Cores and Enhanced ApoA-1 Binding in Lipid Emulsions Hiroyuki Saito,† Masafumi Tanaka,‡ Emiko Okamura,§ Tomohiro Kimura,§ Masaru Nakahara,§ and Tetsurou Handa*,‡ Osaka Branch, National Institute of Health Sciences, Osaka 540-0006, Japan, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan, and Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan Received November 14, 2000. In Final Form: January 29, 2001 The binding maximum of apoA-1 (N) in triolein (TO)-egg yolk phosphatidylcholine (PC) emulsions was 10-fold larger than that in PC large unilamellar vesicles (LUV) of similar size (100 nm) with no significant difference in the affinity. Replacement of the long-chain triglyceride, TO, by medium-chain triglycerides or cholesteryl oleate in emulsion cores significantly decreased the N value. The 13C NMR chemical shifts of the PC carbonyl carbon at the surface layers indicated that PC polar headgroups are more separated and exposed to water molecules in emulsions than in vesicles. The N values were satisfactorily correlated with the chemical shift, that is, the degree of separation between the carbonyl groups at the surface. Although apoA-1 binding to the PC monolayers of emulsions brings about bending of the surface layers and creates local defects in the hydrocarbon regions in a similar manner as PC LUV, the surface-core interaction seems to fill the defects with the core neutral lipids, compensates for the bending stress, and eventually increases the N value. Dependence of the core effect upon the acyl chain length of triglycerides implied important roles of the acyl chains in the surface-core interaction between PC and triglycerides.
Introduction Apolipoprotein A-1 (apoA-1) is a multifunctional exchangeable protein in animal plasma and is the major protein component of high-density lipoproteins (7-10 nm in diameter), serving as an activator of lecithin-cholesterol acyltransferase and functioning as an acceptor of cell membrane cholesterol (Chol) in the reverse Chol transport pathway.1-3 ApoA-1 molecules are also accommodated in triglyceride-rich large emulsion-like lipoproteins, chylomicrons (80-1000 nm), where the protein molecules are assumed to intercalate between the polar headgroups of surface phospholipids.4 Differences in the lipid composition of plasma lipoproteins have been presumed to be an important factor influencing the distribution of exchangeable apolipoproteins among lipoprotein particles. Several studies have shown that the surface content of Chol affects the binding of apoA-1 and other apolipoproteins to lipid emulsions as protein-free models for triglyceride-rich lipoproteins.5-8 At the triglyceride-saline interface, a large difference in apoA-1 interaction is observed between egg yolk phosphatidylcholine (PC) and Chol monolayers.9 We have reported distinct apoA-1 binding between * To whom correspondence should be addressed. Fax: +81 75 753 4601. E-mail:
[email protected]. † Osaka Branch, National Institute of Health Sciences. ‡ Graduate School of Pharmaceutical Sciences, Kyoto University. § Institute for Chemical Research, Kyoto University. (1) Fielding, C. J.; Fielding, P. E. J. Lipid Res. 1995, 36, 211-228. (2) Yokoyama, S. Biochim. Biophys. Acta 1998, 1392, 1-15. (3) Phillips, M. C.; Gillotte, K. L.; Haynes, M. P.; Johnson, W. J.; Lund-Katz, S.; Rothblat, G. H. Atherosclerosis 1998, 137, S13-17. (4) Segrest, J. P.; Garber, D. W.; Brouillette, C. G.; Harvet, S. C.; Anantharamaiah, G. M. Adv. Protein Chem. 1994, 45, 303-369. (5) Maranhao, R. C.; Tercyak, A. M.; Redgrave, T. G. Biochim. Biophys. Acta 1986, 875, 247-255. (6) Derksen, A.; Small, D. M. Biochemistry 1989, 28, 900-906. (7) Small, D. M.; Clarke, S. B.; Tercyak, A.; Steiner, J.; Gantz, D.; Derksen, A. Adv. Exp. Med. Biol. 1991, 285, 281-288. (8) Martins, I. J.; Mortimer, B.-C.; Millaer, J.; Redgrave, T. G. J. Lipid Res. 1996, 37, 2696-2705.
spherical PC bilayers (vesicles of 100 nm) and spherical PC monolayers (surface layers of emulsions of similar size) and also demonstrated the inverse effects of Chol on apolipoprotein binding in vesicles and emulsions.10 In these studies, we recognized crucial roles of core neutral lipids in the structural modification of emulsion surface layers and thereby in apolipoprotein binding. Indeed, a recent study using surface plasmon resonance indicated that the conformation of apoA-1 in high-density lipoproteins is influenced by core lipids.11 In the present study, triglycerides with different acyl chain lengths and cholesteryl oleate were incorporated into emulsion cores covered with surface PC monolayers, and apoA-1 binding to these lipid particles was discussed in terms of the headgroup arrangement of PC at the particle surface evaluated by a 13C NMR method. Materials and Methods ApoA-1 was isolated from pig plasma using the procedures described previously.9,10,12 The protein was further purified by affinity column chromatography (HiTrap Blue affinity column, Pharmacia Biotech) to remove trace amounts of pig serum albumin. Egg yolk phosphatidylcholine was kindly provided by Asahi Kasei Co. The purity (over 99.5%) was determined by thinlayer chromatography. Triolein (TO) obtained from NOF Co. (Tokyo) was purified using a silicate (Wakogel C-200, Wako Pure Chemicals) column to remove fatty acids, diglycerides, and monoglycerides. Cholesteryl oleate (CO) and the medium-chain triglycerides tricaprylin (C8) and tricaproin (C6) were obtained from Sigma and used without further purification. Preparation of Emulsions and Vesicles. TO-PC, C8PC, C6-PC, and CO-PC emulsions with particle diameters of (9) Handa, T.; Saito, H.; Tanaka, I.; Kakee, A.; Tanaka, K.; Miyajima, K. Biochemistry 1992, 31, 1415-1420. (10) Saito, H.; Miyako, Y.; Handa, T.; Miyajima, K. J. Lipid Res. 1997, 38, 287-294. (11) Curtiss, L. K.; Bonnet, D. J.; Rye, K.-A. Biochemistry 2000, 39, 5712-5721. (12) Handa, T.; Komatsu, H.; Kakee, A.; Miyajima, K. Chem. Pharm. Bull. 1990, 38, 2079-2082.
10.1021/la001583t CCC: $20.00 © 2001 American Chemical Society Published on Web 03/24/2001
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100-120 nm (determined from quasi-elastic light-scattering measurements, Photal LPA-3000/3100, Otsuka Electronic Co.) were prepared at 60-70 °C as described10,13,14 using a highpressure emulsifier (Nanomizer, Nanomizer Inc., Tokyo). Emulsion particles were separated from vesicles by ultracentrifugation. 31P NMR spectra of emulsion PC were recorded (Bruker AC-300 spectrometer) with a paramagnetic-shift reagent, praseodymium(III) nitrate to detect the coexistence of vesicles.14 The core (neutral) lipid/surface PC molar ratios were 85:15 for TO-PC, 88:12 for C8-PC, 93:7 for C6-PC, and 87:13 for CO-PC. A triglyceride having shorter chains and smaller molecular volume, C8 or C6, was required to be in the higher fraction to give similar emulsion size and surface area as compared to TO. PC large unilamellar vesicles (LUV) with a diameter of 100 nm and small unilamellar vesicles (SUV) with a diameter of 30 nm were prepared by extrusion10 and sonication methods, respectively. ApoA-1 Binding Studies. ApoA-1 binding assays were performed in 10 mM Tris-HCl buffer (pH 7.4), containing 150 mM NaCl, 1mM EDTA, and 0.01% NaN3. The mixtures (1.0 mL) of a constant amount of emulsions or vesicles (1-5 mM for PC) and various amounts of apoA-1 were incubated for 1 h at 25 °C and ultracentrifuged to separate the bound protein (the top fraction) from the free protein (the bottom fraction, 400 µL). The centrifugation period of 1 h was selected to give stationary values of bound protein.10 The solubilities of C8 and C6 in an aqueous medium, 0.86 and 1.17 µM, respectively, were much smaller than the emulsion triglyceride concentration, 7-13 mM.15 When the mixtures contained vesicles, 3% sucrose was added to adjust the density. After ultracentrifugation, the bottom fractions were collected, and then 100 µL of a 10% nonionic surfactant, heptaethyleneglycoldodecyl ether, was added to each fraction. These samples were left overnight at 4 °C to solubilize the small amounts of remaining lipid. The apoA-1 concentration in the fraction was determined by measuring the peak area of tryptophan fluorescence near 335 nm (excitation at 280 nm). Free apoA-1 in the same surfactant solution was used as a fluorescence standard. The lipid-bound apoA-1 amount was calculated from the difference between the protein concentrations before and after ultracentrifugation. Binding data were analyzed by a linearized plot of the equilibrium according to the following equation:16
Pf ) [PC](Pf/Pb)N - Kd
(1)
where Pf and Pb are free and bound protein concentrations (µM), respectively, [PC] is concentration of PC in emulsions or vesicles (mM), N is the binding maximum, (mmol apoA-1/mol PC) and Kd is the dissociation constant (µM). From a linear plot of Pf against [PC](Pf/Pb), Kd and N could be estimated. PC concentration was determined by phosphorus assay according to the method of Bartlett.17 The concentrations of triglycerides and CO were determined using respective enzymatic assay kits purchased from Wako Pure Chemicals. The protein concentration was determined by the method of Lowry et al.18 using bovine serum albumin (Bio Rad) as the standard. 13C NMR Spectroscopy. 13C NMR spectra were recorded with a high-resolution, multinuclear and multipurpose NMR (JEOL JNM-EX270 wide-bore type) spectrometer equipped with an Oxford superconductor magnet (6.35 T). High-quality NMR tubes with 20 mm o.d. (custom-made) were used. The concentration of emulsions or vesicles was 25-100 mM of PC. All measurements were performed at 37 °C. Free induction decays were accumulated 40 000-100 000 times. An aqueous solution of 4,4-dimethyl-4-silapentane-1-sulfonate was used as an external reference. Experimental errors of the chemical shift were 0.02 ppm. The details have been described elsewhere.19 (13) Saito, H.; Minamida, T.; Arimoto, I.; Handa, T.; Miyajima, K. J. Biol. Chem. 1996, 271, 15515-15520. (14) Saito, H.; Nishiwaki, K.; Handa, T.; Ito, S.; Miyajima, K. Langmuir 1995, 11, 3742-3747. (15) Small, D. M. In The Physical Chemistry of Lipids: Handbook of Lipid Research 4; Plenum Press: New York, 1986; pp 372-382. (16) Tajima, S.; Yokoyama, S.; Yamamoto, A. J. J. Biol. Chem. 1983, 258, 10073-10082. (17) Bartlett, G. R. J. Biol. Chem. 1959, 234, 466-468. (18) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265-275.
Figure 1. (a) Amount of bound apoA-1 represented as a function of the free protein concentration in the medium (10 mM TrisHCl, 150 mM NaCl, pH ) 7.4). n ) 8. (b) Linearized plots of apoA-1 binding data according to eq 1. The numbers represent (1) TO-PC emulsions, (2) C8-PC emulsions, (3) C6-PC emulsions, and (4) PC LUV. The solid lines in Figure 1a were calculated with Kd and N in Table 1. Standard errors for Kd and N values estimated from regression analysis are shown in Table 1.
Results ApoA-1 Binding to Emulsions and Vesicles. Figure 1a shows the binding profiles of apoA-1 to long-chain triglyceride (TO)-PC, medium-chain triglyceride (C8 or C6)-PC emulsions, and PC LUV. The amount of apoA-1 bound to the lipid particles increased with apoA-1 concentration. The linearized plots of the binding data according to eq 1 showed a reasonably good fit to straight lines, indicative of equilibrium binding of apoA-1 to the emulsion or vesicle surface (Figure 1b). From the slope and the ordinate intercept of the straight line, binding parameters, the binding maximum, N, and the dissociation constant, Kd, were obtained (Table 1). The N value in TO-PC emulsions was about 10 times larger than that in PC LUV of similar size (100 nm) with negligible change in the affinity (1/Kd). When the long-chain triglyceride TO in emulsion cores was replaced by a medium-chain triglyceride, C8 or C6, the N value was decreased significantly with no difference in the affinity (1/Kd). Table 1 also shows that reduction in PC vesicle size (SUV, 30 nm) led to a marked increase in the binding maximum N (19) Okamura, E.; Nakahara, M. J. Phys. Chem. 1999, B103, 35053509.
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Table 1. Binding Parameters of ApoA-1
TO-PC emulsions C8-PC emulsions C6-PC emulsions CO-PC emulsions PC LUV PC/Chol LUVa (PC/Chol) ) (3/2) PC SUV a
dissociation constant Kd (µM)
binding maximum N (m mol/mol PC)
0.28 ( 0.10 0.29 ( 0.06 0.36 ( 0.12 0.60 ( 0.16 0.38 ( 0.19 0.91 ( 0.18
1.89 ( 0.21 1.07 ( 0.05 0.45 ( 0.01 0.92 ( 0.09 0.17 ( 0.02 1.72 ( 0.16
0.05 ( 0.01
2.05 ( 0.04
Saito, H. et al. J. Lipid Res. 1997, 38, 287-294.
Table 2. 13C NMR Chemical Shift Differences for CdO, N(CH3)3, and NCH2CH2OP Atoms of PC Molecules from LUV Surfacea (∆δb in ppm) 13CdO
TO-PC 0.148 emulsions C8-PC 0.090 emulsions C6-PC n.d. emulsions CO-PC 0.048 emulsions PC LUV 0 (δ ) 176.048) PC/Chol LUV 0.139 (PC/Chol) ) (3/2) PC SUV 0.164c -0.216d
-N(13CH3)3
-NCH213CH2OP-
0.052
0.085
0.035
0.061
n.d.
n.d.
0.002 0 (δ ) 56.693) 0.027 -0.006
-0.014 0 (δ ) 62.059) 0.069 -0.022
a Experimental error of the chemical shifts is (0.02. b Positive value of ∆δ indicates lower field shift. c Outer leaflets (positive bending). d Inner leaflets (negative bending).
Figure 2. Effects of triglyceride in PC LUV and emulsion core on apoA-1 binding maximum. Saturating amounts of TO or C8 were added into PC LUV as surface triglycerides. n ) 8.
but a decrease in the Kd value (i.e., an increase in the binding affinity), compared with PC LUV (100 nm). Triglycerides are slightly soluble in both PC bilayers of LUV and PC monolayers of emulsions: about 3 mol % of TO in the bilayers20 and the monolayers21 and 10 mol % of C8 in the bilayers.22 The effects of the saturating amounts of TO or C8 on the N value of PC LUV were examined as shown in Figure 2. The minimal influence of the surface triglycerides indicated that the core triglycerides play major roles in the pronounced increase of the apoA-1 binding maximum at emulsion surface PC monolayers. 13C NMR of Emulsions and Vesicles. A 13C NMR method was employed to determine interfacial interactions of PC molecules at the atomic site level.19 We made use of the empirical rule that NMR signals show a significant shift to a lower field when atomic sites are in a polar environment.23,24 Typical changes in 13C NMR spectra for the carbonyl and choline methyl carbons of PC by introduction of triglyceride cores (from PC LUV to TOPC emulsions) are shown in Figure 3. Chemical shift differences from LUV, ∆δ, of the carbonyl carbon of PC were 0.148 and 0.090 ppm for TO-PC and C8-PC emulsions, respectively (Figure 3a and Table 2). Here, the positive values indicate lower field shifts. The results demonstrated that the carbonyl groups are surrounded by a more polar environment at the emulsion than at the LUV surface and that the acyl chain length of core (20) Hamiton, J. A.; Small, D. M. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 6878-6882. (21) Handa, T.; Saito, H.; Miyajima, K. Biochemistry 1990, 29, 28842890. (22) Deckelbaum, R. C.; Hamilton, J. A.; Moser, A.; BengtssonOlivecrona, G.; Butbul, E.; Carpentier, Y. A.; Gutman, A.; Olivecrona, T. Biochemistry 1990, 29, 1136-1142. (23) Rao, U. R. K.; Manohar, C.; Valaulikar, B. S.; Iyer, R. M. J. Phys. Chem. 1987, 91, 3286-3291. (24) Mishra, B. K.; Samant, S. D.; Pradhan, P.; Mishra, S. B.; Manohar, C. Langmuir 1993, 9, 894-898.
triglycerides influences the local environment around the carbonyl carbon of surface PC. Furthermore, replacement of core TO by a rigid lipid, CO, significantly decreased the ∆δ value (Table 2). The signal of the choline methyl group of PC located at the outermost region of lipid particles also showed lower field shifts in emulsions than in LUV (Figure 3b), although the magnitude of the shift difference was less than that of the carbonyl carbon. Table 2 (the third column) also shows the chemical shift difference for the R-methylene carbon of the choline group. Unfortunately, the chemical shifts for acyl chain carbons of PC in emulsions were not evaluated because of overlap with those of core lipids. When Chol was added to PC LUV bilayers, the 13C NMR signals for the carbonyl, choline methyl, and R-choline methylene groups of PC shifted toward the lower field as shown in Table 2. The ∆δ value of the carbonyl carbon was similar to the value in TO-PC emulsions. In PC SUV, the chemical shift of the carbonyl carbon split into two peaks (Table 2). The positive radius of curvature (outer leaflets) and the negative radius of curvature (inner leaflets) of the bilayers led to the lower (∆δ ) +0.164 ppm) and higher (∆δ ) -0.216 ppm) field shifts, respectively. The lower shift was ascribed to the enhanced separation of the polar headgroups and thereby to increased hydration. Outer/inner separation for the choline carbon signals was not detected under these experimental conditions, and the signals probably consisted of both contributions. As the chemical shifts for acyl chain carbons of PC were not evaluated for emulsions, we utilized the lower field shift of PC carbonyl carbon as a better index of polarity for the PC headgroup region at the emulsion or vesicle particle surface. ApoA-1 Binding and Chemical Shift of the Carbonyl Carbon. In Figure 4, the binding maximum of apoA-1 is represented as a function of the chemical shift of PC carbonyl carbon at the emulsion or vesicle surface. For PC SUV, the chemical shift of the outer leaflets of bilayers was employed. The results shown in the figure indicate a good correlation between the apoA-1 binding maximum, N, and the chemical shift, suggesting that the polarity or hydration at the PC headgroup plays crucial roles in determination of the N value of lipid particles. Effects of bound apoA-1 on the 13C NMR spectra of PC in TO-PC emulsions were evaluated as shown in Figure 5 and Table 3. The 90% maximum bound apoA-1 brought about significant higher field shifts for the carbonyl carbon but led to less pronounced effects on the choline carbons. These results demonstrated that the bound protein
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Figure 3. 13C NMR spectra of emulsions and LUV. Emulsions or LUV (25-100 mM of PC) were contained in a custom-made sample tube with 20 mm o.d. The medium was 10 mM Tris-HCl with 150 mM NaCl, pH ) 7.4. (a) 13C NMR spectra of the carbonyl group in PC LUV (1), C8-PC (2), and TO-PC (3) emulsions. (b) 13C NMR spectra of the choline methyl group in PC LUV (1), C8-PC (2), and TO-PC (3) emulsions. Table 3. Effects of ApoA-1 on the 13C NMR Chemical Shift Differences of PC between TO-PC Emulsions and PC LUV Surfaces (∆δ in ppm)a TO-PC TO-PC emulsions emulsions + apoA-1 choline methyl -N(13CH3)3 choline R-methylene -NCH213CH2OP carbonyl 13CdO
PC LUV
0.052
0.025
0 (δ ) 56.693)
0.085
0.009
0 (δ ) 62.059)
0.148
-0.016
0 (δ ) 176.048)
a
At the 90% maximum binding of apoA-1. Experimental error of the chemical shifts is (0.02.
and bound apoA-1 influences the torsion angles of the rapidly moving choline group at the outermost region. The chemical shifts of the choline carbons, therefore, seem to be affected by several complicated factors. Figure 4. ApoA-1 binding maximum, N, represented as a function of the chemical shift of carbonyl carbon of PC at the vesicle or emulsion surface. For PC SUV (30 nm), the chemical shift of the outer leaflets was employed.
Figure 5. Effects of apoA-1 binding on the 13C chemical shift of the PC carbonyl carbon at the TO-PC emulsion surface, before (1) and after (2) addition of apoA-1 (90% of the binding maximum).
resulted in a less polar environment for the headgroup of PC, especially at the carbonyl carbon site. Although the phosphorylcholine dipoles of PC are approximately parallel to the bilayer surface of LUV,25,26 it is possible that the dipole is otherwise oriented in the emulsion monolayers,27 (25) Brown, M. F.; Seelig, J. Biochemistry 1978, 17, 381-384. (26) Scherer, P. G.; Seelig, J. Biochemistry 1989, 28, 7720-7728. (27) Li, K.-L.; Tihal, C. A.; Guo, M.; Stark, R. E. Biochemistry 1993, 32, 9926-9935.
Discussion ApoA-1 Binding to PC Surface Layers. Emulsion particles had 3- to 10-fold larger binding maximums for apoA-1, N, than PC LUV with no appreciable differences in the Kd value. The effects of core lipids on the N value were in the order TO > C8 > CO > C6 (Figure 1 and Table 1). ApoA-1 possesses tandem repetitive class A amphipathic helical domains in the sequence, which fold into the globular structure in an aqueous medium. At the lipid surface, the protein unfolds and the helices are intercalated between phospholipids.4 The class A amphipathic helices are identified as having a wide zwitterionic polar face containing clusters of positively charged residues at the polar/nonpolar interface and a cluster of negatively charged residues at the center of the polar face.4 This class of helices are postulated to act as detergents by virtue of their cross section being wedge-shaped.28,29 PC molecules with their truncated cone shape preferentially assemble into bilayers with a rather flat surface.30 Insertion of the wedge-shaped amphipathic helices of apoA-1 between PC molecules in bilayers leads to local positive bending. The unfavorable bending energy may cause a decrease in the binding maximum as seen in PC (28) Tytler, E.; Segrest, J. P.; Epand, R. M.; Nie, S.-Q.; Epand, R.F.; Mishra, V. K.; Venkatachalapathi, Y. V.; Anantharamaiah, G. M. J. Biol. Chem. 1993, 268, 22112-22118. (29) Palgunachari, M. N.; Mishra, V. K.; Lund-Katz, S.; Phillips, M. C.; Adeyeye, S. O.; Alluri, S.; Anantharamaiah, G. M.; Segrest, J. P. Arterioscler. Thromb. Vasc. Biol. 1996, 16, 328-338. (30) Ibdah, J. A.; Lund-Katz, S.; Phillips, M. C. Biochemistry 1989, 28 (8), 1126-1133.
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LUV. Although apoA-1 binding to the PC monolayers of emulsions also brings about bending of the surface layers and creates local defects in the hydrocarbon region in a similar manner as in PC LUV bilayers, interactions of surface layers with neutral lipid cores seem to fill the packing defects and increase the N value. Triglycerides induce a negative radius of curvature and exhibit a reverse hexagonal or cubic structure forming propensity in phosphatidylethanolamine bilayers.31 Insertion of a saturated amount of TO or C8 into PC LUV, however, did not give appreciable effects on the binding maximum, N (Figure 2). The results indicate a more direct interaction between surface PC and core lipids in emulsions. The acyl chain length of the triglyceride was an important factor in the surface-core interaction of emulsions: The medium-chain triglycerides, C8 and C6, lowered the interaction and diminished the binding capacity of emulsions for apoA-1, N. The replacement of TO by a rigid core lipid, CO, reduced the N value (Table 1),10 probably because of the decreased mobility and the weaker surface-core interaction (Table 2). The effect of core lipids on the PC hydration (i.e., polarity) and, therefore, the separation of the headgroups were evaluated on the basis of 13C NMR measurements (Figure 3 and Table 2). The lower field chemical shifts of carbonyl carbon of PC were satisfactorily correlated with the binding maximum of apoA-1, N, in emulsions and vesicles (Figure 4). Binding of apoA-1 to TO-PC emulsions may prevent the access of water molecules to the carbonyl groups and lead to higher field chemical shifts as shown in Figure 5 and Table 3. PC SUV are characterized by the intensely curved bilayers and asymmetric arrangement of PC.32 The outer leaflets bend positively with extra space and additional hydration between the PC headgroups, as indicated by the 13C NMR data in Table 2. The bending energy of the outer leaflets is released by the interaction with the wedgeshaped amphipathic helices of apoA-1, and the binding maximum and affinity of the protein were 12- and 7.6fold larger in PC SUV than in PC LUV: RT ln(0.05/0.38) ) -RT ln 7.6 ) -5.1 kJ/mol apoA-1 (Table 1). Chol in PC LUV. Chol causes a significant increase in the apoA-1 binding maximum of PC LUV without disturbing the membrane structure.10 However, the protein has a much lower affinity for Chol than PC at the triglyceride-saline9 and air-saline30 interfaces. On the basis of the deuterium quadrupole splitting of choline methylenes and the 31P chemical shielding anisotropy of PC, Brown and Seelig reported that Chol in PC bilayers acts as a spacer molecule, increasing the separation between the headgroups.25 The reduction of 31P NMR signal width observed by addition of Chol to PC bilayers is explained in terms of enhanced motional freedom of the phosphate group, consistent with the spacing out of or increased separation between the headgroups.33 Furthermore, Chol-induced red shifts of the asymmetric stretching band of the PC phosphate are attributed to enhanced hydration of the PC headgroups in bilayers.34 Chol, acting as a spacer in the polar group region of PC bilayers and decreasing the spontaneous curvature of mixed bilayers,35
induces inverted cubic or hexagonal structures of lipids.36 The lower field shifts of 13C NMR signals observed in PC/ Chol LUV (Table 2) supported the spacing effect of Chol in the polar group region of LUV. The inversed wedgeshaped lipid Chol in bilayers releases the bending stress brought about by the apoA-1 interaction and increases the number of binding sites, but slightly decreases the affinity for the protein as shown in Table 1: RT ln(0.91/ 0.38) ) 2.2 kJ/mol apoA-1. On the basis of polarographic measurements, Lecompte et al. suggested that Chol diminishes the interactions between the nonpolar face of amphipathic helices of apoA-1 and the hydrocarbon chains of PC membranes.37 Surface-Core Interaction in Emulsions. Our previous studies of steady-state and time-resolved fluorescence anisotropy on the effects of core lipids on the surface PC monolayers of emulsions indicated that the interaction of surface PC with core TO restricts the acyl chain motion of the surface monolayers.13,14 Lateral diffusion of fluorescent PC was evaluated in TO-PC, C8-PC, and C6PC emulsions, showing the reduced rigidifying effect of the medium-chain triglycerides on PC monolayers (unpublished data). Combination of the fluorescence anisotropy with the13C NMR results indicated that the core neutral lipids have two distinct interfacial effects on the surface layers: (1) spacing out or separation of the polar headgroups of PC in surface monolayers and (2) condensation or increase in rigidity of the acyl chain region of surface PC monolayers. “Interdigitation” of acyl chains between PC and core neutral lipids in emulsions is suggested as in very low-density lipoproteins.38,39 The dependency of the surface-core interaction on the acyl chain length of triglycerides can be explained by interdigitation interactions. Further studies on the effect of the acyl chain length of core triglycerides are currently in progress and will be published soon.
(31) Epand, R. M.; Epand, R. F.; Lancaster, C. R. D. Biochim. Biophys. Acta 1988, 945, 161-166. (32) Huang, C.; Mason, J. T. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 308-310. (33) Guo, W.; Hamilton, J. A. Biochemistry 1995, 34, 14174-14184. (34) Casal, H. L.; Mantsch, H. H.; Hauser, H. Biochemistry 1987, 26, 4408-4416. (35) Saito, H.; Kawagishi, H.; Tanaka, M.; Tanimoto, T.; Okada, S.; Komatsu, H.; Handa, T. J. Colloid Interface Sci. 1999, 219, 129-134.
(36) Israelachivili, J. In Intermolecular & Surface Forces, 2nd ed.; Academic Press: London, 1992; pp 366-394. (37) Lecompte, M.-F.; Bras, A.-C.; Dousset, N.; Portas, I.; Salvayre, R.; Ayrault-Jarrier, M. Biochemistry 1998, 37, 16165-16171. (38) Morrisett, J. D.; Gaubatz, J. W.; Tarver, A. P.; Allen, J. K.; Pownall, H. J.; Laggner, P.; Hamilton, J. A. Biochemistry 1984, 23, 5343-5352. (39) Mims, M. P.; Guyton, J. R.; Morrisett, J. D. Biochemistry 1992, 25, 474-483.
Abbreviations apoA-1 Chol PC NMR TO CO C8 C6 LUV SUV Tris
apolipoprotein A-1 cholesterol egg yolk phosphatidylcholine nuclear magnetic resonance triolein cholesteryl oleate tricaprylin tricaproin large unilamellar vesicles small unilamellar vesicles tris (hydroxymethyl)aminomethane
Acknowledgment. This work was supported in part by Grants-in-Aid for Scientific Research 10771274, 12771387 (to H.S.) and 12470488, 12470489 (to T.H.) from JSPS and grants from the Human Science Foundation of Japan and from the Naito Foundation. LA001583T
