669
Intermembrane Cholesterol Transfer: Role Of Sterol Carrier Proteins and Phosphatidylserine I Friedhelm Schroeder*,a, Peter Butkoa, Ivan H a p a l a a a n d Terrence J. Scallenb aDMsion of Pharmacology and Medicinal C'hernis.~, Department of Pharmacology and Cell Biophysics, Universityof Cincinnati Medical Center, Cincinnati, Ohio 45267-0004, and the,DrDepartment of Biochemistry, School of Medicine, University of New Mexico, Albuquerque, New Mexico 87131 The effect of phosphatidylserine and sterol carrier proteins on cholesterol exchange was determined using an assay not requiring separation of donor and acceptor membrane vesicles. Sterol carrier protein-2 (SCP2, also called nonspecific lipid transfer protein), but not fatty acid binding protein (FABP, also called sterol carrier protein), enhanced the initial rate of sterol exchange between neutral zwitterionic phosphatidylcholine small unilamellar vesicles (SUV) 2.3-fold. Phosphatidylserine at 10 mol% increased the initial rate of spontaneous and of SCP2mediated (but not FABP-mediated) sterol exchange by 22% and 44-fold, respectively. The SCP2 potentiation of sterol transfer was dependent on SCP2 concentration and on phosphatidylserine concentration. The SCP2mediated sterol transfer was inhibited by a variety of cations including KCI, divalent metal ions, and neomycin. The data suggest that SCP2 increase in activity for sterol transfer may be partly ascribed to charge on the phospholipid. Lipids 25, 669-674 (1990). Rat liver sterol carrier protein-2 (SCP2) (1-3) and fatty acid binding protein (FABP) {4-7) belong to a class of intracellular proteins that bind cholesterol (1,2,4,7) and fluorescent sterol analogues (3,5-7). Because SCP2 facilitates the in vitro intermembrane transfer of many lipid classes [gangliosides (8), neutral glycosphingolipids (8), sphingomyelin (9), phospholipids (9), and cholesterol (1, 9-13)], it has been termed a nonspecific lipid transfer protein. However, some evidence indicates a more specific role for SCP2 in enhancement of sterol transfer during microsomal conversion of lanosterol to cholesterol (14,15), microsomal cholesterol esterification (16), and adrenal mitochondrial steroidogenesis (1,2,17). Since fatty acid binding protein (FABP) binds not only sterols, but also fatty acids and fatty acyl CoA, it has been termed fatty acid binding protein (7,18-20). It is important to note that in most of the investigations reporting activity for SCP2 or FABP, this activity was measured by stimulation of an acceptor membrane enzyme activity. Thus, little is actually known about the mechanism of protein carriermediated intermembrane cholesterol transfer.
MATERIALS AND METHODS Materials. 1-Palmitoyl-2-oleoyl phosphatidylcholine (PC), cholesterol, bovine brain L-a-phosphatidyl-L-serine (PS),
1A portion of this work was presented as an abstract: Nemecz, G., Butko, P., and Schroeder, F. (1989)BiophysicalJournal 5~ 137a. *To whom correspondence should be addressed. Abbreviations: DHE, dehydroergosterol; FABP, fatty acid binding protein; HPLC, high performance liquid chromatography; PC, 1-pAlmltoyl-2-oleoylphosphatidylcholine; PS, phosphatidylserine, SCP2, sterol carrier protein2; SUV, small unilamellar vesicles.
and neomycin sulfate were purchased from Avanti Polar Lipids, Inc. {Birmingham, AL), Applied Science Laboratories, Inc. (State College, PA}, Sigma Chemical Co. {St. Louis, MO}, and Sigma Chemical Co., respectively. Dehydroergosterol was synthesized as described earlier {21}. Sterols were recrystanized in alcohol, prior to use, and purity was monitored by high performance liquid chromatography {HPLC} {21}. S C P 2 w a s purified from rat liver {14). Prior to use, lyophylized SCP2 {14,22} was dissolved in distilled water to make a 100 ~ I stock solution. Unless otherwise specified, the final SCP2 or FABP concentration in the assay SUV suspension was 1.5 gM. Rat liver F A B P was generously provided by Dr. M. E. Dempsey, University of Minnesota, while recombinant rat liver F A B P was purified as described elsewhere {23). Methods. Small unilamellar vesicles (SUV) were prepared as described earlier {24}, except for the two following modifications. First, all SUV preparations were sonicated until the suspensions were clear. For control SUV {PC/sterol, 65:35, mole %} and acidic SUV (PC/PS/sterol, 55:10:35, mole %) this required 30 rnin and 4 rain sonication time, respectively. Recovery of phospholipid in SUV (24) was similar for all SUV. Second, the buffer (10 mM PIPES/0.02% NaN 3, pH 7.4) in which SUV were sonicared and redispersed was prefiltered with a 0.2 ~an filter (Millipore, Bedford, MA). In each exchange assay, total lipid concentration was near 150 ~M (donor/accepter ratio 1:10). The vesicle composition was 65 tool% phospholipid and 35 mol% sterol (dehydroergosterol in the donor, cholesterol in the acceptor vesicles). Exchange of dehydroergosterol for cholesterol between donor and acceptor membranes was monitored at 24~ in the absence and presence of S C P 2 by adaptation of the method we d~ scribed previously (25,26). The procedure was adapted to continuous measurement of polarization (3) to obtain 540 data points in 3 hr or less with a Compaq-PC computer [rather than 15-20 data points taken manually as previously done {25,26}]. The excitation source was a 450 watt xenon arc; photobleaching did not occur under the conditions used {24}. Inner-filter and light scattering artifacts were negligible due to use of dilute vesicle suspensions (absorbance at the excitation wavelength, 325 nm, was less than 0.1) and placing Janos GG-375 cut-off filters in the emission system. Use of dehydroergosterol polarization to determine initial rate of molecular sterol exchange. At high concentration, fluorescent molecules such as dehydroergosterol interact to self-quench. This interaction, resulting in radiationless energy transfer, will also decrease fluorescence polarization. Such concentration (c) dependent depolarization fits the following relationship (27): P --- po/{1 + Bc)
[1]
where P0 is a constant defined as the polarization at infinite dilution (zero concentration). B = P0 X T X LIPIDS,VoL 25, No. 11(1990)
670
F. SCHROEDER E T A L constant, where Z is the fluorescence lifetime. Because dehydroergosterol fluorescence lifetime is independent of concentration in an isotropic solvent {28) or a membrane system (24}, B can be considered a constant. It is import a n t to note t h a t Po and B were calculated from a comp u t e d fit to experimentally obtained data from a series of SUV with varying dehydroergosterol but constant 35 mole % total sterol. Although E q u a t i o n [1] describes the dependency of P on dehydroergosterol concentration, it does not explain the molecular transfer of dehydroergosterol from donor to acceptor vesicles because the polarization of the mixture of donor and acceptor vesicles contains signal from both donor and acceptor vesicles simultaneously. In addition, polarization P, in contrast to anisotropy r, is not an additive p a r a m e t e r {29; also see Equation [4]). Polarization can be converted to anisotropy according to the following formula: P=
[2]
3r/(2 + r}
Insertion of Equation [2] into Equation [1] reveals t h a t anisotropy fulfills the same hyperbolic equation as polarization: [3]
r = r0/{1 + B'c)
where r 0 is the anisotropy at infinite dilution and the constant B' [B' equals {3/2) X r 0 • T X constant] relates to B from Equation [1] as B' = B {1 + r0/2). The anisotropy r of a mixture of donor and acceptor vesicles {with anisotropies rD and rA, respectively} is
[4]
r = fDrD + fArA
where fD, fA are fractional fluorescence intensities {30). Due to the 10-fold excess of acceptor vesicles over donor vesicles: CD + 10CA = CI
[5]
where CD, CA are the dehydroergosterol concentrations in donor and acceptor, respectively, and the constant c~ is the initial concentration in donor [11 times the total concentration of dehydroergosterol in the lipid phase {total donor + acceptor)].Herein, ci = 35 mol%. The relative amount XD present in the donor vesicles as a function of time is given as: XD = CD/CI
[6]
The relative amount present in the acceptor population is XA =
1 -- X D =
1
--
CD/C I
[7]
The dependence of the fluorescence intensity F on the relative concentration of dehydroergosterol in PC vesicles (containing 35 mol% sterol) can be described b y an empirical equation obtained b y polynomial fitting to the individual steady state polarization measurements: F -- 1.7 x D - - 0.7
XD 2
[8]
I t is important to note t h a t Equation [8] and subsequent mathematical t r e a t m e n t applies to the particular LIPIDS, Vol. 25, No. 11 (1990)
case of 35 mole% total sterol at 24~ and the 10-fold excess of acceptor over donor vesicles. Taking into account the latter fact and Equation [8], the fractional fluorescence intensities fD and fA of donor and acceptor vesicle populations, respectively, can be expressed as: fD = {1.7 XD - - 0.7
XD 2)
/{1.63 + 0.14 x D -- 0.77
f A = 1 - - fD
XD 2) [ 9 ]
[10]
The denominator in Equation [9] appears to be due to the normalization condition fD + fA ---- 1, which must be fulfilled at all times. Expressing r D and r A by Equation [3], in combination with Equations [4] and [5], we obtain r = r0 {fD/1 + DXDJ ~- fA/[1 + D{1 -- XD}/10]}
[11]
This formula describes anisotropy r of the donor/acceptor mixture as a function of a sole variable, the relative concentration XD of dehydroergosterol in donor vesicles. D is a new constant, D = B'ci, and fD and fA alSO are functions of x D (Eq. [8]). The number 10 in Equation [11] appears to be due to the 1:10 donor/acceptor ratio. B y applying the formula in Equation [2] on Equation [11] for m a n y values of XD, we obtain the dependency of polarization P on dehydroergosterol concentration XD. The dependency can be described with the following polynomial function: P
=
- - a X D 2 "~- b x D -F c
[121
where the parameters a = 0.185, b -- 0.028, and c --- 0.320 for the PC/sterol (65:35) vesicles used herein at 24~ Thus, the molecular transfer of dehydroergosterol during the exchange process can be calculated from steady state polarization of dehydroergosterol in the donor/acceptor SUV mixture. Importantly, P changes almost linearly with XD when x D is not much less than 1. This means that at the initial stages of exchange, the initial rate of the polarization change [dP/dt]t=0 is proportional to the initial rate of the sterol exchange --[dxD/dt]t=o. The proportionality cons t a n t can be determined from the slope of the straight line in Figure 1 {see Results and Discussion) and the experimental conditions of the measurements. The l(~fold excess of the acceptor over donor, the values of total lipid {150 ~M), total sterol {52.5 ~M) and dehydroergosterol concentration {4.77 yM) and the average [dP/dt]t=0 = 0.0018 min -1 lead to: -[dxD/dt]t= 0 ~- 30 [dP/dt]t=0
[13]
where [dxDldt]t: 0 is in nmol X rain -1 and [dP/dt]t=0 in min -1. The minus sign appears to be due to the fact that polarization P and the dehydroergosterol concentration xD in donor change in the opposite direction (while P increases, XD decreases). Initial rates of polarization change were determined from the first 1 rain for PC/PS/ sterol with SCP2 and 5 min of the record for all other SUV with or without SCP~. The accuracy of initial rate measurements was within 5%. It is important to note that initial rates primarily reflect the fast component of the biphasic kinetics of the sterol exchange. The fast component has a rate constant one order of magnitude higher
671 PHOSPHATIDYLSERINE STIMULATES STEROL CARRIER PROTEIN-2
y
contain the same amount of dehydroergosterol, independent of whether they originally were donor or acceptor 0.3 I vesicles. What is effectively observed is a continuous dilution of dehydroergosterol in vesicles, e.g., a smooth increase in the dehydroergosterol fluorescence polarization. Changes in the initial rate of polarization change are 0.2 proportional to the number of sterol molecules transfered (Fig. 1). Theoretical justification of the method is preI I I I R sented in Materials and Methods. Polarization change is linear until up to 20% of the donor dehydroergosterol is exchanged between phosphatidylcholine/sterol (65:35) SUV (Fig. 1, A) and between phosphatidylcholine/phosphatidylserine/sterol (55:10:35) SUV (Fig. 1, B). The slopes of the two straight line segments in Figures A and 0,2 B indicate t h a t the inclusion of 10 mole % phosphatidylserine did not significantly alter the equations derived for ! ! ! exchange between phosphatidylcholine/sterol (65:35) SUV 1.0 0.8 0.6 0.4 0.2 0 DONOR (Materials and Methods). DEHYDROERGOSTEROL/TOTAL STEROI. Changes in polarization of D H E fluorescence upon mix(mole%/mole%l ing the donor and acceptor PC SUV in the presence or FIG. 1. Polarization of the donor/acceptor SUV mixture as a func- absence of S C P 2 a r e shown in Figure 2. The changes in tion of the relative concentration of dehydroergosterol in donor vesicles, The polarization of dehydroergosterol in the donorlaceep~ polarization were not due either to instability of the donor SUV (curve 1, Fig. 2) or to addition of SCP2 to donor tot mixture calculated at 24~ was plotted as a function of the ratio of donor dehydroergosterol to donor total sterol (dehydroergosterol alone (curve 2, Fig. 2). In both cases polarization was conplus cholesterol). A, PC/sterol (65:35) SUV; and B, PC/PS/stcrol stant with time. Addition of SCP2 had no effect on donor (55:10:35)SUV. The curve represents the best polynomial fit (Eq. [12]) polarization for the following reasons. First, the polariza(see Materials and Methods). The straight line indicates that the tion of d e h y d r o e r g o s t e r o l b o u n d to SCP2 is 0.143 polarization increase can be considered linear with the decrease in very similar to that in donor SUV 0.156 • 0.003; the dehydroergosterol amount in donor vesicles only at the initial • stages of the sterol exchange (until about 20% of dehydroergostcrol second, the single sterol binding site of every S C P 2 is leaves the donor). not saturated, since the ratio of SUV dehydroergosterol to SCP 2 is only 3:1 in the donor SUV/SCP2 mixture. Third, the Kd of dehydroergosterol binding to S C P 2 is than the slow component and it comprises about 10% of between 1 and 2 ~M (3). Thus, the S C P 2 sterol binding the exchangeable pool of sterol (3,25,26). site would not be expected to be completely s a t u r a t e d Turbidity determination (light scattering). Light scat- under the assay conditions used herein. Spontaneous extering at 325 n m was measured at 90 ~ in an SLM 4800 change of dehydroergosterol between PC/sterol SUV spectrofluorometer, with the cut-off filter removed from (curve 3, Fig. 2) exhibited an initial rate of polarization the emission side. change of 0.0018 + 0.0001 rain-1 (Table 1). S C P 2 increased the rate of polarization change in PC SUV (curve RESULTS A N D DISCUSSION Earlier we demonstrated t h a t dehydroergosterol (DHE) and [3H]cholesterol exchange kinetics between model membranes were very similar (25,26). The d e h y d r o e r g ~ sterol exchange process can be visualized as follows. A t time zero, all dehydroergosterol molecules are packed in the donor vesicles. They then experience a dehydroergosterol-rich environment, such t h a t dehydroergosteroldehydroergosterol interactions resulting in self-quenching and energy transfer are quite probable. This results in highly depolarized fluorescence with values near 0.156 • 0.003 (25). A t the initial stages of the exchange, the acceptor vesicles contain extremely low amounts of dehydroergosterol, so t h a t the dehydroergosterol-dehydr~ ergosterol interactions are very rare. The fluorescence signal from acceptor vesicles exhibits high polarization near 0.348 • 0,005, b u t low intensity. Under the assay conditions, donor SUV dehydroergosterol is transferred to the acceptor SUV while cholesterol from the acceptor SUV is transferred to the donor SUV in an equimolar manner. As the exchange progresses, dehydroergosterol concentration in donor vesicles decreases, while t h a t in the acceptor vesicles increases. The process continues until equilibrium is reached, which is when all the vesicles
0.3 4
! N 0
.
a.
~
~ ?
0
!
I
60
t20
180
TIME |min) FIG. 2. SCP2 stimulates dehydroergosterol exchange between palmltoyloleoyl phosphatidylcholine/sterol vesicles. All exchanges were performed at 24~ as described in Materials and Methods. Curve 1, PCfDHE (65:35) SUV, no aceeptor SUV, no SCP2; curve 2, no aceeptor SUV, SCP2; curve 3, spontaneous exchange of sterol between PC/sterol donor and aceeptor SUV, no SCP2; and curve 4, SCPTmediated exchange between PCIsterol SUV. SCP2 (1.5 pM) and/or aceeptor SUV (150 ~I) were added at the time indicated by an ~row.
LIPIDS,Vol.25,No.11(1990)
672 F. S C H R O E D E R E T AL. TABLE 1 Effect of Ionic Strength on SCP 2 Stimulated Sterol Transfera Initial rate of polarization change (rnin -1 X rain) Phospholipid
Salt
No protein
Phosphatidylcholine
0 0.6 M KCI
1.9 __.0.1 1.4
1 mM CaC12
ND
Phosphatidylserine
0 0.6 M KC1 1 mM CaC12 1 mM MgCI 2
2.2 +__ 0.1 2.2 1.8 2.0
SCP 2
4.2 + 0.1 2.5
FABP
2.0 +_ 0.1
3.2 92.3 _+ 4.3 4.0 43.3 47.6
1.5 _+ 0.1
aphosphatidylcholine and phosphatidylserine denote SUV containing 10 mole % of the respective phospholipid in addition to 55 mole % phosphatidyleholine and 35 mole % steroL Protein concentration was 1.5 I~M. Values represent the mean +- SEM (n = 3-7). KCI, CaCI2, or MgCI 2 were included in the exchange buffer when indicated.
A
4
~
"oz,,. 120
0.3
'r.e
N
E
80
n.
40
m
ee
.I 0.2
.J
tA 0
I
O:
, 60
120
180
T I M E (rain) FIG.
3. P h o s p h a t i d y l s e r i n e
potentiates
effect
of SCP 2 on
dehydroergosterol exchange between SUV. Curve 1, PC/PS/DHE (55:10:35) SUV, no acceptor SUV, n o SCP2; curve 2, no aeeeptor SUV, SCPz; curve 3, spontaneous exchange of sterol between PC/ PS/sterol donor and acceptor SUV, no SCP2; and curve 4, SCPTmediated exchange between PC/PS/sterol SUV. SCP 2 1.5 tLM) and/or aceeptor SUV (150 t4M) were added at the time indicated by arrow A (curves 3 and 4) and arrow B (curve 2).
4, Fig. 2) 2.3-fold{Table I).F A B P {Table I) and recombinant F A B P {data not shown) were without effect. Inclusion of 10 mole % PS did not change dehydroergosterol polarizationin donor S U V in the absence {curve 1, Fig. 3) or presence {curve 2, Fig. 3} of SCP2. PS increased the rate (curve 3, Fig. 3 vs curve 3, Fig. 2) of the spontaneous exchange of sterolby 22% as compared to P C S U V {Table 1). More importantly, SCP2 enhanced the rate of polarizationchange (curve 4 at A and curve 2 at B, Fig. 3). The extent of this enhancement of the initialrate of sterol exchange was dependent on the concentration of phosphatidylserine in the S U V and on the SCP2 added to the vesicles {Fig. 4). SCP2 enhanced initialrate of polarization change by 40-fold between PC/PS/sterol (55:10:35) S U V and 60-fold between PC/PS/sterol {35:30:35) S U V {Fig. 4). In contrast, F A B P {Table 1) or recombinant F A B P {data not shown) did not enhance the initialrate of polarization change between PS containing SUV. LIPIDS,Vol. 25, No. 11 (1990)
0 0.5 1.5 STEROL CARRIER PROTEIN-2 [uM] FIG. 4. Effect of S C P 2 and phosphatidylserine concentration o n dehydroeegosteml excbR-ge between SUV. SCP 2 concentration was varied from 0 to 1.5 I~M as indicated in the figure. SUV were comprised of pheopholipid/sterel (65:35) with solid bars indicating PCIPSlsterol (60:5:35), czoss hatched bars indicating PC/PS/sterol (55:10:35), and open bars indicating PC/PS/sterol (35:30:35). Otherwise, all conditions were as described in legend to Figure 2.
SCP2 has a net positive charge at neutral pH, since its isoelectricpoint is 8.6 (14).PS bears a net negative charge at neutral pH. If electrostaticattractionbetween the two species accounts for the stimulatory effect of PS on the SCP2-mediated sterol exchange, then the effect should be suppressed by screening of the charges by ions in the medium. Addition of 0.6 M KCI, 1 m M CaCI2, or 1 m M MgCI2 had littleeffect on spontaneous sterol exchange between PC or PS containing S U V {Table 1).As indicated by no change in light scattering,these ions did not cause aggregation of either P C or PS containing S U V at the concentrations and under the conditions tested. In the presence of 0.6 M KCI or 1 m M CaCl2, the SCPs-mediated sterol exchange between P C S U V was inhibited by 24% and 40%, respectively {Table 1). More important, 0.6 M KCI, 1 m M CaCI2, and 1 m M MgCI~ inhibited SCP2-mediated sterol exchange between PS containing S U V by 96%, 53%, and 48%, respectively {Table 1).
673
PHOSPHATIDYLSERINE STIMULATES STEROL CARRIER PROTEIN-2
Likewise, at w M concentrations the polycation neomycin also inhibited both spontaneous {Fig. 5, insert) and SCP~-mediated {Fig. 5} sterol transfer between PS containing SUV. However, the degree of inhibition of the SCP2-mediated sterol transfer was several orders of magnitude greater than for spontaneous sterol transfer. From these data, the three following conclusions m a y be proposed. First, the ability of sterol carrier proteins to bind sterols does not necessarily allow conclusion that they enhance sterol transfer by acting as sterol carriers. SCP2 and F A B P both bind sterols {1-7} and stimulate microsomal enzymes utilizing sterol substrates {1,2,
observation is supported b y 18-fold enhancement of sterol t r a n s f e r b y SCP2 in acidic phospholipid containing monolayer m e m b r a n e s {13}. Third, SCP2 m a y facilitate t r a n s f e r of sterols b y interaction with m e m b r a n e s . This possibility is s u p p o r t e d b y the inhibition of SCP2 mediated sterol t r a n s f e r in acidic PS containing SUV b y high salt and b y neomycin. O t h e r i n v e s t i g a t o r s have shown direct interaction of SCP2 with acidic phospholipid containing monolayer membranes {13} and mitochondria {34}. H i g h salt concentration also inhibited protein mediated phosphatidylcholine t r a n s f e r between m e m b r a n e s {35}.
14-20). However, only SCP2 stimulates transfer of sterol from donor to acceptor m e m b r a n e s while F A B P does not. Thus, sterol binding and sterol t r a n s f e r e n h a n c e m e n t b y these proteins a p p e a r to be s e p a r a t e functions. Merely binding cholesterol is not sufficient to m a k e a protein a sterol t r a n s f e r protein. P e r h a p s binding of protein and cholesterol m a y not be relevant to the m e c h a n i s m of transfer. I t is certainly possible for a protein to interact with a m e m b r a n e to enhance the desorption r a t e of sterol f r o m the m e m b r a n e w i t h o u t acting as a sterol carrier p e r se. The desorption r a t e is the r a t e limiting step in spontaneous sterol transfer (31). Alternately, the binding of sterol m a y function to deliver sterol to specific enzymes without actually enhancing i n t e r m e m b r a n e sterol transfer. The latter possibility m u s t certainly be considered in view of the observation t h a t another lipid binding protein, interphotoreceptor retinol binding protein, does in fact bind retinol but, surprisingly, inhibits t r a n s f e r of retinol between m e m b r a n e s (32). Second, SCP~ m a y be m u c h more specific in its function t h a n previously believed. SCP2 enhances i n t e r m e m b r a n e t r a n s f e r of a v a r i e t y of ligands several fold (1,8-13). However, even in m e m b r a n e s contRinlng acidic phospholipids the t r a n s f e r of phospholipids was s t i m u l a t e d only 2-7-fold between model m e m b r a n e s (13,33). As shown in Table 1, SCP2 s t i m u l a t e d sterol t r a n s f e r between acidic phospholipid (PS) containing vesicles m u c h more t h a n between neutral zwitterionlc (PC) containing vesicles (2.3- vs 44-fold). This
ACKNOWLEDGMENTS
,.., A
2
~4
1
% ,-- 8 0 "7 60
o
20
ttl
n-
4O
F- 20
t 10
t
, 7 T ~, 30 50 N E O M Y C I N [uM]
,* 100
FIG. 5. Inhibition of spontaneous and SCPTmediated dehydroergc~ sterol exchange by the polycationic antibiotic, neomycin. SUV were
composed of PCIPS/sterol (55:10:35). All conditions were as described in the legend to Figure 2, except that neomycin was added at the indicated concentration. The inset refers to neomycin effects on spontaneous dehydroergosterol exchange.
This work was supported in part by grants from the USPHS DK41402 (F.S.), and AM32309 and AM10628 {T.J.S.). REFERENCES
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674 F. SCHROEDER E T A L . 26. Nemecz, G., and Schroeder, F. (1988) Biochemistry 27, 7740-7749. 27. Weber, G. (1954) Trans. Farad Soc. 50, 552-555. 28. Smutzer, G., Crawford, B.F., and Yeagle, P.L. (1986) Biochir~ Biophys. Acta 862, 361-371. 29. Weber, W.G. {1952) Biochem~ J. 51, 145-167. 30. Lakowicz, J.R. (1983) Principles of Fluorescence Spectroscopy, Plenum Press, New York, p. 145. 31. McLean, L.R., and Phillipis, M.C. (1984) Biochemistry 23, 4624-4630. 32. Ho, M.-T.P., Massey, J.B., Pownall, H.J., Anderson, R.E., and
LIPID& Vol. 25, No. 11 (1990)
Hollyfield, J.G. (1989)J. Biot Che~ 264, 928-935. 33. Altamura, N., and Landriscina, C. (1986) Int. J. Biochem. l& 513-517. 34. Megli, F.M., De Lisi, A., van Amerongen, A., Wirtz, K.W.A., and Quagliariello, E. (1986) Biochim. Biophys. Acta 861, 463-470. 35. Wirtz, K.W.A., Geurts Van Kessel, W.S.M., Kamp, H.H., and Demel, R.A. (1976) Eur. J. Biochem. 61, 515-523. [Received January 18, 1990; Revision accepted August 10, 1990]
Intermembrane Cholesterol Transfer: Role Of Sterol Carrier Proteins and Phosphatidylserine I Friedhelm Schroeder*,a, Peter Butkoa, Ivan H a p a l a a a n d Terrence J. Scallenb aDMsion of Pharmacology and Medicinal C'hernis.~, Department of Pharmacology and Cell Biophysics, Universityof Cincinnati Medical Center, Cincinnati, Ohio 45267-0004, and the,DrDepartment of Biochemistry, School of Medicine, University of New Mexico, Albuquerque, New Mexico 87131 The effect of phosphatidylserine and sterol carrier proteins on cholesterol exchange was determined using an assay not requiring separation of donor and acceptor membrane vesicles. Sterol carrier protein-2 (SCP2, also called nonspecific lipid transfer protein), but not fatty acid binding protein (FABP, also called sterol carrier protein), enhanced the initial rate of sterol exchange between neutral zwitterionic phosphatidylcholine small unilamellar vesicles (SUV) 2.3-fold. Phosphatidylserine at 10 mol% increased the initial rate of spontaneous and of SCP2mediated (but not FABP-mediated) sterol exchange by 22% and 44-fold, respectively. The SCP2 potentiation of sterol transfer was dependent on SCP2 concentration and on phosphatidylserine concentration. The SCP2mediated sterol transfer was inhibited by a variety of cations including KCI, divalent metal ions, and neomycin. The data suggest that SCP2 increase in activity for sterol transfer may be partly ascribed to charge on the phospholipid. Lipids 25, 669-674 (1990). Rat liver sterol carrier protein-2 (SCP2) (1-3) and fatty acid binding protein (FABP) {4-7) belong to a class of intracellular proteins that bind cholesterol (1,2,4,7) and fluorescent sterol analogues (3,5-7). Because SCP2 facilitates the in vitro intermembrane transfer of many lipid classes [gangliosides (8), neutral glycosphingolipids (8), sphingomyelin (9), phospholipids (9), and cholesterol (1, 9-13)], it has been termed a nonspecific lipid transfer protein. However, some evidence indicates a more specific role for SCP2 in enhancement of sterol transfer during microsomal conversion of lanosterol to cholesterol (14,15), microsomal cholesterol esterification (16), and adrenal mitochondrial steroidogenesis (1,2,17). Since fatty acid binding protein (FABP) binds not only sterols, but also fatty acids and fatty acyl CoA, it has been termed fatty acid binding protein (7,18-20). It is important to note that in most of the investigations reporting activity for SCP2 or FABP, this activity was measured by stimulation of an acceptor membrane enzyme activity. Thus, little is actually known about the mechanism of protein carriermediated intermembrane cholesterol transfer.
MATERIALS AND METHODS Materials. 1-Palmitoyl-2-oleoyl phosphatidylcholine (PC), cholesterol, bovine brain L-a-phosphatidyl-L-serine (PS),
1A portion of this work was presented as an abstract: Nemecz, G., Butko, P., and Schroeder, F. (1989)BiophysicalJournal 5~ 137a. *To whom correspondence should be addressed. Abbreviations: DHE, dehydroergosterol; FABP, fatty acid binding protein; HPLC, high performance liquid chromatography; PC, 1-pAlmltoyl-2-oleoylphosphatidylcholine; PS, phosphatidylserine, SCP2, sterol carrier protein2; SUV, small unilamellar vesicles.
and neomycin sulfate were purchased from Avanti Polar Lipids, Inc. {Birmingham, AL), Applied Science Laboratories, Inc. (State College, PA}, Sigma Chemical Co. {St. Louis, MO}, and Sigma Chemical Co., respectively. Dehydroergosterol was synthesized as described earlier {21}. Sterols were recrystanized in alcohol, prior to use, and purity was monitored by high performance liquid chromatography {HPLC} {21}. S C P 2 w a s purified from rat liver {14). Prior to use, lyophylized SCP2 {14,22} was dissolved in distilled water to make a 100 ~ I stock solution. Unless otherwise specified, the final SCP2 or FABP concentration in the assay SUV suspension was 1.5 gM. Rat liver F A B P was generously provided by Dr. M. E. Dempsey, University of Minnesota, while recombinant rat liver F A B P was purified as described elsewhere {23). Methods. Small unilamellar vesicles (SUV) were prepared as described earlier {24}, except for the two following modifications. First, all SUV preparations were sonicated until the suspensions were clear. For control SUV {PC/sterol, 65:35, mole %} and acidic SUV (PC/PS/sterol, 55:10:35, mole %) this required 30 rnin and 4 rain sonication time, respectively. Recovery of phospholipid in SUV (24) was similar for all SUV. Second, the buffer (10 mM PIPES/0.02% NaN 3, pH 7.4) in which SUV were sonicared and redispersed was prefiltered with a 0.2 ~an filter (Millipore, Bedford, MA). In each exchange assay, total lipid concentration was near 150 ~M (donor/accepter ratio 1:10). The vesicle composition was 65 tool% phospholipid and 35 mol% sterol (dehydroergosterol in the donor, cholesterol in the acceptor vesicles). Exchange of dehydroergosterol for cholesterol between donor and acceptor membranes was monitored at 24~ in the absence and presence of S C P 2 by adaptation of the method we d~ scribed previously (25,26). The procedure was adapted to continuous measurement of polarization (3) to obtain 540 data points in 3 hr or less with a Compaq-PC computer [rather than 15-20 data points taken manually as previously done {25,26}]. The excitation source was a 450 watt xenon arc; photobleaching did not occur under the conditions used {24}. Inner-filter and light scattering artifacts were negligible due to use of dilute vesicle suspensions (absorbance at the excitation wavelength, 325 nm, was less than 0.1) and placing Janos GG-375 cut-off filters in the emission system. Use of dehydroergosterol polarization to determine initial rate of molecular sterol exchange. At high concentration, fluorescent molecules such as dehydroergosterol interact to self-quench. This interaction, resulting in radiationless energy transfer, will also decrease fluorescence polarization. Such concentration (c) dependent depolarization fits the following relationship (27): P --- po/{1 + Bc)
[1]
where P0 is a constant defined as the polarization at infinite dilution (zero concentration). B = P0 X T X LIPIDS,VoL 25, No. 11(1990)
670
F. SCHROEDER E T A L constant, where Z is the fluorescence lifetime. Because dehydroergosterol fluorescence lifetime is independent of concentration in an isotropic solvent {28) or a membrane system (24}, B can be considered a constant. It is import a n t to note t h a t Po and B were calculated from a comp u t e d fit to experimentally obtained data from a series of SUV with varying dehydroergosterol but constant 35 mole % total sterol. Although E q u a t i o n [1] describes the dependency of P on dehydroergosterol concentration, it does not explain the molecular transfer of dehydroergosterol from donor to acceptor vesicles because the polarization of the mixture of donor and acceptor vesicles contains signal from both donor and acceptor vesicles simultaneously. In addition, polarization P, in contrast to anisotropy r, is not an additive p a r a m e t e r {29; also see Equation [4]). Polarization can be converted to anisotropy according to the following formula: P=
[2]
3r/(2 + r}
Insertion of Equation [2] into Equation [1] reveals t h a t anisotropy fulfills the same hyperbolic equation as polarization: [3]
r = r0/{1 + B'c)
where r 0 is the anisotropy at infinite dilution and the constant B' [B' equals {3/2) X r 0 • T X constant] relates to B from Equation [1] as B' = B {1 + r0/2). The anisotropy r of a mixture of donor and acceptor vesicles {with anisotropies rD and rA, respectively} is
[4]
r = fDrD + fArA
where fD, fA are fractional fluorescence intensities {30). Due to the 10-fold excess of acceptor vesicles over donor vesicles: CD + 10CA = CI
[5]
where CD, CA are the dehydroergosterol concentrations in donor and acceptor, respectively, and the constant c~ is the initial concentration in donor [11 times the total concentration of dehydroergosterol in the lipid phase {total donor + acceptor)].Herein, ci = 35 mol%. The relative amount XD present in the donor vesicles as a function of time is given as: XD = CD/CI
[6]
The relative amount present in the acceptor population is XA =
1 -- X D =
1
--
CD/C I
[7]
The dependence of the fluorescence intensity F on the relative concentration of dehydroergosterol in PC vesicles (containing 35 mol% sterol) can be described b y an empirical equation obtained b y polynomial fitting to the individual steady state polarization measurements: F -- 1.7 x D - - 0.7
XD 2
[8]
I t is important to note t h a t Equation [8] and subsequent mathematical t r e a t m e n t applies to the particular LIPIDS, Vol. 25, No. 11 (1990)
case of 35 mole% total sterol at 24~ and the 10-fold excess of acceptor over donor vesicles. Taking into account the latter fact and Equation [8], the fractional fluorescence intensities fD and fA of donor and acceptor vesicle populations, respectively, can be expressed as: fD = {1.7 XD - - 0.7
XD 2)
/{1.63 + 0.14 x D -- 0.77
f A = 1 - - fD
XD 2) [ 9 ]
[10]
The denominator in Equation [9] appears to be due to the normalization condition fD + fA ---- 1, which must be fulfilled at all times. Expressing r D and r A by Equation [3], in combination with Equations [4] and [5], we obtain r = r0 {fD/1 + DXDJ ~- fA/[1 + D{1 -- XD}/10]}
[11]
This formula describes anisotropy r of the donor/acceptor mixture as a function of a sole variable, the relative concentration XD of dehydroergosterol in donor vesicles. D is a new constant, D = B'ci, and fD and fA alSO are functions of x D (Eq. [8]). The number 10 in Equation [11] appears to be due to the 1:10 donor/acceptor ratio. B y applying the formula in Equation [2] on Equation [11] for m a n y values of XD, we obtain the dependency of polarization P on dehydroergosterol concentration XD. The dependency can be described with the following polynomial function: P
=
- - a X D 2 "~- b x D -F c
[121
where the parameters a = 0.185, b -- 0.028, and c --- 0.320 for the PC/sterol (65:35) vesicles used herein at 24~ Thus, the molecular transfer of dehydroergosterol during the exchange process can be calculated from steady state polarization of dehydroergosterol in the donor/acceptor SUV mixture. Importantly, P changes almost linearly with XD when x D is not much less than 1. This means that at the initial stages of exchange, the initial rate of the polarization change [dP/dt]t=0 is proportional to the initial rate of the sterol exchange --[dxD/dt]t=o. The proportionality cons t a n t can be determined from the slope of the straight line in Figure 1 {see Results and Discussion) and the experimental conditions of the measurements. The l(~fold excess of the acceptor over donor, the values of total lipid {150 ~M), total sterol {52.5 ~M) and dehydroergosterol concentration {4.77 yM) and the average [dP/dt]t=0 = 0.0018 min -1 lead to: -[dxD/dt]t= 0 ~- 30 [dP/dt]t=0
[13]
where [dxDldt]t: 0 is in nmol X rain -1 and [dP/dt]t=0 in min -1. The minus sign appears to be due to the fact that polarization P and the dehydroergosterol concentration xD in donor change in the opposite direction (while P increases, XD decreases). Initial rates of polarization change were determined from the first 1 rain for PC/PS/ sterol with SCP2 and 5 min of the record for all other SUV with or without SCP~. The accuracy of initial rate measurements was within 5%. It is important to note that initial rates primarily reflect the fast component of the biphasic kinetics of the sterol exchange. The fast component has a rate constant one order of magnitude higher
671 PHOSPHATIDYLSERINE STIMULATES STEROL CARRIER PROTEIN-2
y
contain the same amount of dehydroergosterol, independent of whether they originally were donor or acceptor 0.3 I vesicles. What is effectively observed is a continuous dilution of dehydroergosterol in vesicles, e.g., a smooth increase in the dehydroergosterol fluorescence polarization. Changes in the initial rate of polarization change are 0.2 proportional to the number of sterol molecules transfered (Fig. 1). Theoretical justification of the method is preI I I I R sented in Materials and Methods. Polarization change is linear until up to 20% of the donor dehydroergosterol is exchanged between phosphatidylcholine/sterol (65:35) SUV (Fig. 1, A) and between phosphatidylcholine/phosphatidylserine/sterol (55:10:35) SUV (Fig. 1, B). The slopes of the two straight line segments in Figures A and 0,2 B indicate t h a t the inclusion of 10 mole % phosphatidylserine did not significantly alter the equations derived for ! ! ! exchange between phosphatidylcholine/sterol (65:35) SUV 1.0 0.8 0.6 0.4 0.2 0 DONOR (Materials and Methods). DEHYDROERGOSTEROL/TOTAL STEROI. Changes in polarization of D H E fluorescence upon mix(mole%/mole%l ing the donor and acceptor PC SUV in the presence or FIG. 1. Polarization of the donor/acceptor SUV mixture as a func- absence of S C P 2 a r e shown in Figure 2. The changes in tion of the relative concentration of dehydroergosterol in donor vesicles, The polarization of dehydroergosterol in the donorlaceep~ polarization were not due either to instability of the donor SUV (curve 1, Fig. 2) or to addition of SCP2 to donor tot mixture calculated at 24~ was plotted as a function of the ratio of donor dehydroergosterol to donor total sterol (dehydroergosterol alone (curve 2, Fig. 2). In both cases polarization was conplus cholesterol). A, PC/sterol (65:35) SUV; and B, PC/PS/stcrol stant with time. Addition of SCP2 had no effect on donor (55:10:35)SUV. The curve represents the best polynomial fit (Eq. [12]) polarization for the following reasons. First, the polariza(see Materials and Methods). The straight line indicates that the tion of d e h y d r o e r g o s t e r o l b o u n d to SCP2 is 0.143 polarization increase can be considered linear with the decrease in very similar to that in donor SUV 0.156 • 0.003; the dehydroergosterol amount in donor vesicles only at the initial • stages of the sterol exchange (until about 20% of dehydroergostcrol second, the single sterol binding site of every S C P 2 is leaves the donor). not saturated, since the ratio of SUV dehydroergosterol to SCP 2 is only 3:1 in the donor SUV/SCP2 mixture. Third, the Kd of dehydroergosterol binding to S C P 2 is than the slow component and it comprises about 10% of between 1 and 2 ~M (3). Thus, the S C P 2 sterol binding the exchangeable pool of sterol (3,25,26). site would not be expected to be completely s a t u r a t e d Turbidity determination (light scattering). Light scat- under the assay conditions used herein. Spontaneous extering at 325 n m was measured at 90 ~ in an SLM 4800 change of dehydroergosterol between PC/sterol SUV spectrofluorometer, with the cut-off filter removed from (curve 3, Fig. 2) exhibited an initial rate of polarization the emission side. change of 0.0018 + 0.0001 rain-1 (Table 1). S C P 2 increased the rate of polarization change in PC SUV (curve RESULTS A N D DISCUSSION Earlier we demonstrated t h a t dehydroergosterol (DHE) and [3H]cholesterol exchange kinetics between model membranes were very similar (25,26). The d e h y d r o e r g ~ sterol exchange process can be visualized as follows. A t time zero, all dehydroergosterol molecules are packed in the donor vesicles. They then experience a dehydroergosterol-rich environment, such t h a t dehydroergosteroldehydroergosterol interactions resulting in self-quenching and energy transfer are quite probable. This results in highly depolarized fluorescence with values near 0.156 • 0.003 (25). A t the initial stages of the exchange, the acceptor vesicles contain extremely low amounts of dehydroergosterol, so t h a t the dehydroergosterol-dehydr~ ergosterol interactions are very rare. The fluorescence signal from acceptor vesicles exhibits high polarization near 0.348 • 0,005, b u t low intensity. Under the assay conditions, donor SUV dehydroergosterol is transferred to the acceptor SUV while cholesterol from the acceptor SUV is transferred to the donor SUV in an equimolar manner. As the exchange progresses, dehydroergosterol concentration in donor vesicles decreases, while t h a t in the acceptor vesicles increases. The process continues until equilibrium is reached, which is when all the vesicles
0.3 4
! N 0
.
a.
~
~ ?
0
!
I
60
t20
180
TIME |min) FIG. 2. SCP2 stimulates dehydroergosterol exchange between palmltoyloleoyl phosphatidylcholine/sterol vesicles. All exchanges were performed at 24~ as described in Materials and Methods. Curve 1, PCfDHE (65:35) SUV, no aceeptor SUV, no SCP2; curve 2, no aceeptor SUV, SCP2; curve 3, spontaneous exchange of sterol between PC/sterol donor and aceeptor SUV, no SCP2; and curve 4, SCPTmediated exchange between PCIsterol SUV. SCP2 (1.5 pM) and/or aceeptor SUV (150 ~I) were added at the time indicated by an ~row.
LIPIDS,Vol.25,No.11(1990)
672 F. S C H R O E D E R E T AL. TABLE 1 Effect of Ionic Strength on SCP 2 Stimulated Sterol Transfera Initial rate of polarization change (rnin -1 X rain) Phospholipid
Salt
No protein
Phosphatidylcholine
0 0.6 M KCI
1.9 __.0.1 1.4
1 mM CaC12
ND
Phosphatidylserine
0 0.6 M KC1 1 mM CaC12 1 mM MgCI 2
2.2 +__ 0.1 2.2 1.8 2.0
SCP 2
4.2 + 0.1 2.5
FABP
2.0 +_ 0.1
3.2 92.3 _+ 4.3 4.0 43.3 47.6
1.5 _+ 0.1
aphosphatidylcholine and phosphatidylserine denote SUV containing 10 mole % of the respective phospholipid in addition to 55 mole % phosphatidyleholine and 35 mole % steroL Protein concentration was 1.5 I~M. Values represent the mean +- SEM (n = 3-7). KCI, CaCI2, or MgCI 2 were included in the exchange buffer when indicated.
A
4
~
"oz,,. 120
0.3
'r.e
N
E
80
n.
40
m
ee
.I 0.2
.J
tA 0
I
O:
, 60
120
180
T I M E (rain) FIG.
3. P h o s p h a t i d y l s e r i n e
potentiates
effect
of SCP 2 on
dehydroergosterol exchange between SUV. Curve 1, PC/PS/DHE (55:10:35) SUV, no acceptor SUV, n o SCP2; curve 2, no aeeeptor SUV, SCPz; curve 3, spontaneous exchange of sterol between PC/ PS/sterol donor and acceptor SUV, no SCP2; and curve 4, SCPTmediated exchange between PC/PS/sterol SUV. SCP 2 1.5 tLM) and/or aceeptor SUV (150 t4M) were added at the time indicated by arrow A (curves 3 and 4) and arrow B (curve 2).
4, Fig. 2) 2.3-fold{Table I).F A B P {Table I) and recombinant F A B P {data not shown) were without effect. Inclusion of 10 mole % PS did not change dehydroergosterol polarizationin donor S U V in the absence {curve 1, Fig. 3) or presence {curve 2, Fig. 3} of SCP2. PS increased the rate (curve 3, Fig. 3 vs curve 3, Fig. 2) of the spontaneous exchange of sterolby 22% as compared to P C S U V {Table 1). More importantly, SCP2 enhanced the rate of polarizationchange (curve 4 at A and curve 2 at B, Fig. 3). The extent of this enhancement of the initialrate of sterol exchange was dependent on the concentration of phosphatidylserine in the S U V and on the SCP2 added to the vesicles {Fig. 4). SCP2 enhanced initialrate of polarization change by 40-fold between PC/PS/sterol (55:10:35) S U V and 60-fold between PC/PS/sterol {35:30:35) S U V {Fig. 4). In contrast, F A B P {Table 1) or recombinant F A B P {data not shown) did not enhance the initialrate of polarization change between PS containing SUV. LIPIDS,Vol. 25, No. 11 (1990)
0 0.5 1.5 STEROL CARRIER PROTEIN-2 [uM] FIG. 4. Effect of S C P 2 and phosphatidylserine concentration o n dehydroeegosteml excbR-ge between SUV. SCP 2 concentration was varied from 0 to 1.5 I~M as indicated in the figure. SUV were comprised of pheopholipid/sterel (65:35) with solid bars indicating PCIPSlsterol (60:5:35), czoss hatched bars indicating PC/PS/sterol (55:10:35), and open bars indicating PC/PS/sterol (35:30:35). Otherwise, all conditions were as described in legend to Figure 2.
SCP2 has a net positive charge at neutral pH, since its isoelectricpoint is 8.6 (14).PS bears a net negative charge at neutral pH. If electrostaticattractionbetween the two species accounts for the stimulatory effect of PS on the SCP2-mediated sterol exchange, then the effect should be suppressed by screening of the charges by ions in the medium. Addition of 0.6 M KCI, 1 m M CaCI2, or 1 m M MgCI2 had littleeffect on spontaneous sterol exchange between PC or PS containing S U V {Table 1).As indicated by no change in light scattering,these ions did not cause aggregation of either P C or PS containing S U V at the concentrations and under the conditions tested. In the presence of 0.6 M KCI or 1 m M CaCl2, the SCPs-mediated sterol exchange between P C S U V was inhibited by 24% and 40%, respectively {Table 1). More important, 0.6 M KCI, 1 m M CaCI2, and 1 m M MgCI~ inhibited SCP2-mediated sterol exchange between PS containing S U V by 96%, 53%, and 48%, respectively {Table 1).
673
PHOSPHATIDYLSERINE STIMULATES STEROL CARRIER PROTEIN-2
Likewise, at w M concentrations the polycation neomycin also inhibited both spontaneous {Fig. 5, insert) and SCP~-mediated {Fig. 5} sterol transfer between PS containing SUV. However, the degree of inhibition of the SCP2-mediated sterol transfer was several orders of magnitude greater than for spontaneous sterol transfer. From these data, the three following conclusions m a y be proposed. First, the ability of sterol carrier proteins to bind sterols does not necessarily allow conclusion that they enhance sterol transfer by acting as sterol carriers. SCP2 and F A B P both bind sterols {1-7} and stimulate microsomal enzymes utilizing sterol substrates {1,2,
observation is supported b y 18-fold enhancement of sterol t r a n s f e r b y SCP2 in acidic phospholipid containing monolayer m e m b r a n e s {13}. Third, SCP2 m a y facilitate t r a n s f e r of sterols b y interaction with m e m b r a n e s . This possibility is s u p p o r t e d b y the inhibition of SCP2 mediated sterol t r a n s f e r in acidic PS containing SUV b y high salt and b y neomycin. O t h e r i n v e s t i g a t o r s have shown direct interaction of SCP2 with acidic phospholipid containing monolayer membranes {13} and mitochondria {34}. H i g h salt concentration also inhibited protein mediated phosphatidylcholine t r a n s f e r between m e m b r a n e s {35}.
14-20). However, only SCP2 stimulates transfer of sterol from donor to acceptor m e m b r a n e s while F A B P does not. Thus, sterol binding and sterol t r a n s f e r e n h a n c e m e n t b y these proteins a p p e a r to be s e p a r a t e functions. Merely binding cholesterol is not sufficient to m a k e a protein a sterol t r a n s f e r protein. P e r h a p s binding of protein and cholesterol m a y not be relevant to the m e c h a n i s m of transfer. I t is certainly possible for a protein to interact with a m e m b r a n e to enhance the desorption r a t e of sterol f r o m the m e m b r a n e w i t h o u t acting as a sterol carrier p e r se. The desorption r a t e is the r a t e limiting step in spontaneous sterol transfer (31). Alternately, the binding of sterol m a y function to deliver sterol to specific enzymes without actually enhancing i n t e r m e m b r a n e sterol transfer. The latter possibility m u s t certainly be considered in view of the observation t h a t another lipid binding protein, interphotoreceptor retinol binding protein, does in fact bind retinol but, surprisingly, inhibits t r a n s f e r of retinol between m e m b r a n e s (32). Second, SCP~ m a y be m u c h more specific in its function t h a n previously believed. SCP2 enhances i n t e r m e m b r a n e t r a n s f e r of a v a r i e t y of ligands several fold (1,8-13). However, even in m e m b r a n e s contRinlng acidic phospholipids the t r a n s f e r of phospholipids was s t i m u l a t e d only 2-7-fold between model m e m b r a n e s (13,33). As shown in Table 1, SCP2 s t i m u l a t e d sterol t r a n s f e r between acidic phospholipid (PS) containing vesicles m u c h more t h a n between neutral zwitterionlc (PC) containing vesicles (2.3- vs 44-fold). This
ACKNOWLEDGMENTS
,.., A
2
~4
1
% ,-- 8 0 "7 60
o
20
ttl
n-
4O
F- 20
t 10
t
, 7 T ~, 30 50 N E O M Y C I N [uM]
,* 100
FIG. 5. Inhibition of spontaneous and SCPTmediated dehydroergc~ sterol exchange by the polycationic antibiotic, neomycin. SUV were
composed of PCIPS/sterol (55:10:35). All conditions were as described in the legend to Figure 2, except that neomycin was added at the indicated concentration. The inset refers to neomycin effects on spontaneous dehydroergosterol exchange.
This work was supported in part by grants from the USPHS DK41402 (F.S.), and AM32309 and AM10628 {T.J.S.). REFERENCES
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