Eur. J. Biochem. 61, 515-523 (1976)
The Protein-Mediated Transfer of Phosphatidylcholine between Membranes The Effect of Membrane Lipid Composition and Ionic Composition of the Medium Karel W. A. WIRTZ. Wouter S. M . GEURTS VAN KESSEL, Hein H. KAMP, and Rudolf A. DEMEL Biochemisch Laboratorium, Rijksuniversiteit te Utrecht (Rcceived August 4/0ctober 3, 1975)
The phosphatidylcholine exchange protein from bovine liver catalyzes the transfer of phosphatidylcholine between rat liver mitochondria and sonicated liposomes. The effect of changes in the liposomal lipid composition and ionic composition of the medium on the transfer have been determined. In addition, it has been determined how these changes affected the electrophoretic mobility i.e. the surface charge of the membrane particles involved. Transfer was inhibited by the incorporation of negatively charged phosphatidic acid, phosphatidylserine, phosphatidylglycerol and phosphatidylinositol into the phosphatidylcholine-containing vesicles ;zwitterionic phosphatidylethanolamine had much less of an inhibitory effect while positively charged stearylamine stimulated. The cation Mg2+ and, to a lesser extent, K + overcame the inhibitory effect exerted by phosphatidic acid, in that concentration range where these ions neutralized the negative surface charge most effectively. Under conditions where Mg2+ and K + affected the membrane surface charge relatively little inhibition was observed. In measuring the protein-mediated transfer between a monolayer and vesicles consisting of only phosphatidylcholine, cations inhibited the transfer in the order La3+ > M$+ 2 Ca2+ > K + = Na+. Inhibition was not related to the ionic strength, and very likely reflects an interference of these cations with an electrostatic interaction between the exchange protein and the polar head group of phosphatidylcholine. The phosphatidylcholine exchange protein from bovine liver functions as a carrier of phosphatidylcholine between membrane interfaces [l]. Recently it was demonstrated that the protein-mediated transfer of phosphatidylcholine between phosphatidylcholine liposomes was inhibited by incorporation of phosphatidic acid or phosphatidylinositol into these liposomes [2]. Steady state analysis of the kinetic data indicated that the apparent dissociation constant of the exchange protein-liposome complex decreased with an increasing phosphatidic acid content of the liposomes [3]. This was interpreted to mean that an increase of the negative surface charge facilitated the interaction of the exchange protein with the membrane interface resulting in less protein free in the medium to function as a carrier i.e. inhibition of transfer. The former studies implied that physical chemical properties of the interface may have an effect on the activity of the exchange protein. In the present study this concept has been elaborated by correlating the protein-mediated transfer of phosphatidylcholine between rat liver mitochondria and liposomes with the
surface charge of these membrane structures. Comparable studies on the relationship between surface charge and phospholipase action have indicated that the surface charge of a phospholipid interface may control the formation of the proper enzyme-phospholipid complex [4]. The surface charge of a membrane is the resultant of two counteracting factors, namely, the surface charge density of the membrane and the counter ion concentration in the medium [ 5 ] . In this study we have altered the surface charge of the liposome independently of that of the mitochondrion by introducing charged amphipathic molecules into the liposomal bilayer. In addition, the surface charge of both liposome and mitochondrion have been affected simultaneously by changing the ionic composition of the medium. In order to investigate whether the activity of the exchange protein is dependent on the ionic strength of the medium, the transfer of phosphatidylcholine has been measured between a monolayer and liposomes both of which consisting only of phosphatidylcholine
Transfer of Phosphatidylcholine between Membranes
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[l]. Under these conditions the cation concentration at the membrane surface should ideally equal the bulk ion concentration since the membranes involved lack an overall surface charge. While this study was in progress it was reported that the activity of an exchange protein from bovine heart was inhibited by increasing the ionic strength of the medium [6]. Part of this investigation has been reported at the Eigth Meeting of the Federation of European Biochemical Societies, Amsterdam, August 1972.
the procedure of Ames and Dubin [20]. The irradiated liposomes were used in the assays for measuring the transfer of ['4C]phosphatidylcholine.The hand-shaken liposomal suspension was used for determination of the electrophoretic mobility of the liposomes (see below). Assay A
Determination of the transfer of ['4C]phosphatidylcholine from 14C-labelled mitochondria to 3Hlabelled liposomes was performed according to a MATERIALS AND METHODS modification of the assay of phosphatidylcholine exchange activity as previously described [14]. MitoPhosphatidylcholine was isolated from egg yolk chondria containing ['4C]phosphatidylcholine (10 mg and used for the preparation of phosphatidic acid of protein) were incubated with 3H-labelled liposomes [7,8]. Phosphatidylethanolamine and phosphatidyl(1 pmol of phosphatidylcholine) and phosphatidylinositol were isolated from rat liver, and phosphatidylcholine exchange protein (0.01 mg) in a total volume serine from bovine brain by applying the lipid ex2.5 ml of buffer (pH 7.4) for 20 min at 25 "C. In of tracts to a TEAE-cellulose column 191. Phosphatidyladdition to the 1 pmol of phosphatidylcholine the glycerol prepared from egg yolk phosphatidylcholine liposomes contained various amounts of other amwas kindly donated by Dr B. Verheij. After equilibraphipathic molecules. See the legends to the figures for tion with 0.1 M NaCl in the chloroform/methanol/ composition of liposomes and buffer. At the end of water system of Folch et al. [lo] the phospholipids incubation the mitochondria were sedimented at were stored in chloroform at - 20 "C. l-Palmitoyl-29000rev./min for 5min in the SS-34 rotor of the oleoyl-sn-glycero-3-phosphorylcholine was syntheSorvall centrifuge in Sorvall tubes (no. 250) with sized according to established procedures [ll]. A adapters. The pellets were discarded. The pH of the ['4C]methyl group was introduced into this compound according to the method of Stoffel et al. [12]. [7~r-~H]- supernatant obtained was adjusted to 5.1 by addition of 0.5 ml of 0.2 M sodium acetate in 0.25 M sucrose Cholesteryloleate was prepared from TH]cholesterol (pH 5.0) followed by a centrifugation at 9000 rev./min (The Radiochemical Centre, Amersham, England) for 5 min. This pH adjustment caused an aggregation and oleoyl chloride by the method of Swell and of those mitochondria which had remained in the Treadwell [ 131. Phosphatidylcholine exchange protein supernatant after the first centrifugation. Controls was isolated from bovine liver [14]. Protein was without exchange protein were carried through the determined by the method of Lowry et al. [15]. entire procedure. The mitochondrial supernatant Stearylamine was obtained from E. Merck AG containing the liposomes was collected, the lipids were (Darmstadt) and used without further purification. extracted, and the 14C/3Hratio of the liposomal lipid Male rats (200- 300 g) of the Wistar albino strain extract corrected for a control incubation, was deterwere fasted overnight and injected intraperitoneally mined as described [14]. The percentage of [l4C]phoswith 20 pCi [Me-'4C]choline (The Radiochemical phatidylcholine transferred from mitochondria to Centre, Amersham, England). After 45 min livers a/h x X x 100 where a and h are the liposomes, is were excised and the mitochondrkd containing ['"CIradioactivity in the 3H-labelled liposomes and 14Cphosphatidylcholine were isolated as described [ 161. labelled mitochondria, respectively, before incubation The final mitochondrial pellet was resuspended in the and X the corrected 14C/3H ratio of the liposomal incubation buffer (see legends to figures) and used the lipids after incubation. same day. Protein was determined by the biuret method [17]. Hand-shaken liposomes containing a trace of Assay B [3H]cholesteryloleate (0.01 7; w/w) were prepared in The rate of transfer of ['4C]phosphatidylcholine the incubation buffer following the procedure of from a monolayer consisting of 16:0/18: 1 [Me-14C]de Gier et a f . [18]. The liposomal suspension (2phosphatidylcholine to phosphatidylcholine liposomes 10 ~ m o l phospholipid/ml) was irradiated ultrawas performed as described [l]. For experimental desonically in a Branson sonifier for 5 min at 80 W outtails, see legends to Fig. 7. put. Liposomes consisting of only phosphatidylcholine were sonicated for 30 min. The liposomes were Measurements of Electrophoretic Mobility stored in the refrigerator and used the next day. Lipid phosphorus was determined by the method of Chen Electrophoretic mobilities of hand-shaken liposomes were determined at 25 "C in a microelectrophoet af. [19] after destruction of the sample according to
K. W. A. Wirtz, W. S . M. Geurts van Kessel, H. H. Kamp, and R. A. Demel
resis apparatus of the type described by Bangham et al. [21,22]. Mobilities of the mitochondria were determined under similar conditions by Dr S. Nordling (Department of Pathology, University of Helsinki, Helsinki). The velocities of the particles are expressed in terms of ps-' V-' cm. The media in which the mobilities were determined, were similar to those in which the transfer of ['4C]phosphatidylcholine was measured. In the present study the electrophoretic mobility determined for handshaken liposomes may be similar to that of the irradiated single bilayer liposomes. This will be correct if the radius of both types of particles is large compared with the reciprocal thickness of the double layer [23]. Single bilayer liposomes, however, may not meet this criterion as their radius is in the order of 15-25pm [24,25]. Then the determined electrophoretic mobilities have relative significance [23]. RESULTS Transfrr of (I4 C]Phosphatidylcholine (Assay A )
The conditions of incubation as given in the Methods section, were chosen such that the exchange protein was limiting in the transfer of [14C]phosphatidylcholine from the ''C-labelled mitochondria to the liposomes. The liposomes used were ultrasonically dispersed for 5 min because sonication increases the liposomal phosphatidylcholine pool that participates in the transfer process. This is shown in Fig. 1 where transfer reached a maximum with 0.5 pmol of phosphatidylcholine if the liposomes were irradiated for 10 min. In the instance of a 1-min irradiation, 1 pmol of phosphatidylcholine was necessary to attain the same level of transfer. However, up to 4 pmol of phosphatidylcholine of hand-shaken liposomes were not sufficient to reach this maximal level. The liposomes used in the preceding experiments contained 2 mol- phosphatidic acid. In general, an increase of the content of charged amphipathic molecules facilitates the ultrasonic dispersion of the liposomal phospholipid [25].
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517
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Liposomal PtdCho (Krnol)
Fig. 1. Percent transjkr oj' mitochondrial ['4CJpho.spliatidylcholine as a function of amount of liposomal phosphatidylcholine. Mitochondria containing ['4C]phosphatidylcholine (10 mg of protein) were incubated with liposornes (1 prnol of phosphatidylcholine, 0.02 prnol phosphatidic acid, 0.01 by weight, [7a-3H]cholesteryloleate) in 2.5 rnl 0.25 M sucrose/0.001 M EDTA/0.010 M KCl/O.OlO M Tris (pH 7.4) as described in Methods. Liposomes used were sonicated for different periods of time. (M Hand) shaken liposomes; ( x - x ) liposomes sonicated for 1 min; (-0) liposomes sonicated for 10 rnin. PtdCho, phosphatidylcholine
x,
Transjer of ['4C]Phosphatidylcholine to Mi-xed Liposomes
Under the standard conditions of incubation a transfer of approximately 10% was observed with liposomes containing 2 mol- % phosphatidic acid (Fig. 1). In order to determine whether the composition of the liposomes affected the transfer, liposomes were prepared with various amounts of negatively charged phosphatidic acid and positively charged stearylamine (Fig. 2A). Relative to the transfer with liposomes containing 2 mol- % phosphatidic acid, transfer decreased by increasing the liposomal phos-
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Fig. 2. Electrophoretic mobility o/' mixrd liposomes conlaining phosphatidylcholine (PidCho) and various amounts of phosphalidic acid and stearylamine, and the transfer of mitochondrial [I4C]phosphatidylcholine to these liposomes. Hand-shaken liposornes in 0.25 M sucrose/0.001M EDTA/0.010 M KCl/O.OlO M Tris (pH 7.4) were used for the determination of the electrophoretic mobility (B). Sonicated liposomes were used in the transfer experiments as described in Methods (A)
518
Transfer of Phosphatidylcholine bctween Membranes
phatidic acid content, and increased by incorporation of stearylamine. However, stearylamine became inhibitory at a concentration above 10 mol-"/,. In summary, a transfer of 15% with liposomes containing 10 mol- % stearylamine declined to 4.5 % with liposomes containing 20 mol- "/, phosphatidic acid. Concomitantly, the electrophoretic mobility of the liposomes changed from a cathodic mobility of 2 ps -'V-' cm for 10 mol-% stearylamine into V - l cm for 20 molan anodic mobility of - 4 ps phosphatidic acid (Fig. 2B). These results suggest that the transfer of phosphatidylcholine between mitochondria and liposomes may be inhibited by increasing the negative surface charge of the liposomes. The latter relationship has been corroborated with liposomes which contained various amounts of phosphatidylserine, phosphatidylglycerol and phosphatidylinositol. The presence of these negatively charged phospholipids had a very similar effect on the electrophoretic mobility of the liposomes (Fig. 3 B). Mobility increased from - 1.5 ps-' V-' cm in the absence of these phospholipids to - 5.5 ps-' V P 1cm in the presence of 20 molIn general it was observed that the mobility of liposomes containing only phosphatidylcholine varied between - 1.O and - 1.5 ps-' V-' cm at the low ionic strength of the medium (I = 0.013, see legend to Fig. 3). This anodic mobility may be due to traces of impurity such as free fatty acids, in the electrically neutral phosphatidylcholine. In agreement with what was observed for phosphatidic acid, negatively charged phosphatidylserine, phosphatidylglycerol and phosphatidylinositol had a pronounced inhibitory effect on the transfer (Fig. 3A). Up to 8 mol-%, these phospholipids inhibited to the same extent; above 8 molit was phosphatidylinositol that exerted the strongest inhibition. For example, 20 mol- % phosphatidylinositol inhibited the transfer by 70 "/,. Studies with phosphatidylethanolamine, however, indicated that the surface charge is not the only parameter of importance in the protein-mediated transfer. Incorporation of phosphatidylethanolamine had very little effect on the electrophoretic mobility of the liposomes (Fig.3B). In spite of this lack it was inhibitory in the transfer although the inhibition was much less extensive than that exerted by the negatively charged phospholipids (Fig. 3 A).
+
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Effect of Mono-, Di- and Trivalent Cations The transfer of ['4C]phosphatidylcholine has been measured between I4C-labelled mitochondria and liposomes (assay A) and between 14C-labelled monolayers and liposomes (assay B) at various cation concentrations. The effect of these ions on the electrophoretic mobility i.e. surface charge of the membranes involved have been determined.
5 10 15 20 25 Negatively charged phospholipids (rnol %)
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Fig. 3. Electrvphoretic mobility of mixed liposomes mid the transjkr mitochondrial ['4C]pho.vpluztidylcholine to these liposomes. For experimental details, see lcgend to Fig. 2. The liposomes contained phosphatidylcholine and various amounts of phosphatidylethanolphosphatidylserine ), (M phosphatidyl), amine (M glycerol ( x x ), and phosphatidylinositol M ()
of
~
It is shown in Fig.4B that in 0.25 M sucrose/ 0.01 M Tris-HC1 (pH 7.4) liposomes containing 12 mol- "/, phosphatidic acid, and mitochondria have mobilities of - 4.1 and - 2.0 ps-l V-' cm, rcspectively. Increase of the K concentration under conditions where isotonicity of the medium was maintained, resulted in a decrease of the anodic mobilities of both membrane particles. In 0.15 M KC1/0.01 M Tris-HC1 (pH 7.4) liposomes and mitochondria have mobilities of - 1.6 and - 0.8 ps-' V-' cm, respectively. Effect of K + on the transfer was little (Fig.4A). Transfer passed through a maximum at 0.015-0.030 M KCl at which concentration transfer had increased from 6 to 8 Similar experiments were performed with Mg2+ (Fig. 5). Liposomes with a mobility of - 4 ps-' V-' cm in 0.25 M sucrose/O.Ol M Tris-HC1 (pH 7.4) did not move in an electric field upon addition of 0.01 M MgC1,. In the presence of 0.002 M MgC12 mobilities of both liposome and mitochondrion had decreased to - 1.0 and -0.7 ps-' V-' cm, respectively. M$+ had an equally pronounced effect on the transfer +
x.
K . W. A. Wirtz, W. S . M. Ceurts van Kessel, H. H. Kamp, and R. A. Demel
519
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Fig. 4. Ejfect of Kt on the electrophoretic mobility qfliposomes and mitochondria, and on the transfer of mitochondrial [i4C]phosphatidylcholine. Hand-shaken liposomes consisting of phosphatidylcholine and phosphatidic acid (12 mol-%) were prepared in 0.25 M sucrose/O.Ol M Tris-HCI (pH 7.4). in 0.15 M KCI/O.Ol M TrisHC1 (pH 7.41, and in mixtures of these buffers. (B) Electrophoretic mobilities of liposomes ( x -x ) and mitochondria ( 0 4 ) were determined in these media. (A) Transfcr of mitochondrial ['4C]phosphatidylcholine was determined in the same media as described in Methods
Fig. 5 . effect of M g z + on the electrophoretic mobility of' liposomes and mitochondria, and on the fransjer of mitochondrial ['4C]phosphatidylcholine. Hand-shaken liposomes consisting of phosphatidylcholine and phosphatidic acid (14 mol- %) were prepared in 0.25 M sucrose/O.Ol M Tris-HCI (pH 7.4). MgCI, was added to the sucrose/Tris medium and the electrophoretic mobilities of liposomes ( x -x ) and mitochondria (-0) were determined (B). Transfer of mitochondrial [14C]phosphatidylcholine was determined in the same media as described in Methods (A)
(Fig. 5A). A maximum transfer was obtained in the presence of 0.002 M MgCl, at which concentration transfer had increased from 6 % to 13.5%; higher concentrations of MgCI, became inhibitory. It appears from Fig. 5 that M$+ stimulated transfer in that concentration range where this cation was most effective in reducing the surface charge of liposome and mitochondrion. In addition the results suggest that Mg2+ became inhibitory at these concentrations where this ion changed the surface charge of the membranes more gradually. In support of this conclusion an experiment was carried out with liposomes containing 5 mol- % stearylamine. Because of positively charged stearylamine, the liposomes had a cathodic mobility of 1.7 pspl V-' cm that was barely affected by Mg2+ (Fig. 6B). Under these conditions M$+ inhibited the transfer of [14C]phosphatidylcholine over the whole concentration range
(Fig. 6A). This suggests that Mg2+ is inhibitory in the transfer except under these conditions where the inhibition is overcome by stimulation due to a reduction of the surface charge of the negatively charged membranes. In order to provide additional evidence for the inhibition exerted by cations, transfer was measured between a ''C-labelled monolayer and liposomes both of which consisting of only phosphatidylcholine. Exchange protein was limiting under the incubation conditions (see legend to Fig. 7). The rate of transfer was expressed as a percentage of [14C]phosphatidylcholine transferred to the subphase per min and was calculated from the decrease of the surface radioactivity after addition of the exchange protein as outlined previously [l]. It is seen from Fig.7 that an increase of the ionic concentration of the medium decreases the rate of transfer no significant difference
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Transfer of Phosphatidylcholine between Membranes
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Fig. 7. Ej'ect of cations on lhe exchange of' phosj3hatidvlcholine between a ['4C]phosphatidylcholine-containing monoluyer and liposomes. The monolayer consisted of 20.6 nmol 16:0/18: 1 [Me-'4C]phosphatidylcholine and covered a surface area of 74.1 cmz (a surface pressure of 30 dynes cm-'). Liposomes were prepared from egg yolk phosphatidylcholine as described in Methods section. The subphase contained 1.25 pmol liposomal phosphatidylcholine in a total volume of 75 ml 0.01 M Tris/O.Ol M sodium acetate/HCl (pH 7.4). The exchange reaction was initiated by addition of 5.7nmol exchange protein (125 pg) to the subphase. The rate of transfer of [14C]phosphatidylcholineto the subphase was calculatcd from the initial slope which represented the dilution of the surface radioactivity. For additional experimcntal details see [l ]
MgCIz (mM)
Fig. 6. Eflect of M?+ on the electrophoretic mobility of liposomes and mitochondria, and on the transfer of mitochondria1 ['4C]phosphatidylcholine. See legend to Fig. 5. The liposomes contained phosphatidylcholine and 5 mol- "/, stearylamine
being observed between different cations of the same class of valency. However, Ca2+ or Mg2+ inhibited about 40 times as effectively as Na' or K + . With La3 the same inhibition occurred at concentrations about another factor of 40 lower than for the divalent cations. This indicates that inhibition is not just related to the ionic strength but becomes more effective with increasing valency. +
DISCUSSION The protein-mediated transfer of phosphatidylcholine between mitochondria and liposomes will be a function of the phosphatidylcholine pools available in these two membrane particles [3]. Recently evidence was provided that only phosphatidylcholine present in the outer liposomal monolayer participated in the transfer process [26]. Prolonged irradiation of a liposomal suspension results in single bilayer liposomes with approximately 65 % of the phosphatidylcholine in the outer monolayer [27,28]. Since about 9 % is present in the outer monolayer of hand-shaken
liposomes, irradiation will result in a 7-fold increase of the amount of phosphatidylcholine available for transfer [29], Thls would explain as to why irradiation of a liposomal suspension enhanced the transfer (Fig.1). It is of interest to note, however, that e.g. 0.2 pmol phosphatidylcholine of a 10-min irradiated liposomal suspension gives the same transfer as 3.6 pmol of a hand-shaken liposomal suspension. In view of the above this factor of 18 cannot be explained by just an increase of phosphatidylcholine in the outer monolayer. Instead the results suggest that in addition to a concentration Factor the transfer per unit area of outer monolayer is faster for irradiated than for hand-shaken liposomes. From nuclear magnetic resonance results it has been inferred that ultrasonic irradiation introduces subtle changes in the orientation of phosphatidylcholine molecules in the bilayer [30,31]. This may be one of the reasons for the irradiated liposomes to be more effective in the transfer process. Changes in molecular organization have also been inferred in explaining the faster rate of hydrolysis of phosphatidylcholine by phospholipase B from Penicillium notatum after ultrasonic irradiation [32]. The exchange protein from bovine liver contains one molecule of non-covalently bound phosphatidylcholine [I, 14,331. Upon interaction with a membrane the protein can exchange its endogenous phosphatidyl-
K. W. A. Wirtz, W. S. M. Geurts van Kessel, H. H. Kamp, and R. A. Demel
choline molecule for one present in the membrane [ l , 331. It is presumably by this mechanism that in the present study the protein transfers ['4C]phosphatidylcholine from the 14C-labelled mitochondria to the liposomes. Previously in studying the transfer of phosphatidylcholine between single bilayer liposomes, it was demonstrated that the transfer decreased with an increase of negatively charged phosphatidic acid in the liposomes [2,3]. An analysis of the kinetic data indicated that the apparent dissociation constant of the exchange protein-liposome complex decreased in a parallel fashion resulting in an inhibition of transfer [3]. In the present study it has been confirmed that the incorporation of phosphatidic acid into phosphatidylcholine liposomes inhibits transfer (Fig. 2). The inhibition is not specifically related to this phospholipid as other negatively charged phospholipids like phosphatidylserine, phosphatidylglycerol and phosphatidylinositol inhibited also (Fig. 3). This argues in favor of the activity of the protein being controlled by the negative surface charge of the interface. Studies on the action of human pancreatic phospholipase A2 indicated that phosphatidylcholine liposomes were only susceptible to enzymatic attack provided acidic phospholipids were present [34,35]. This suggests that in accordance with what we have seen with the exchange protein, the acidic phospholipids lower the dissociation constant of the phospholipase-liposome complex; however, in contrast to an inhibitory effect on the activity of the exchange protein, a stimulation of phospholipase activity is observed. The decrease of the dissociation constant discussed above, may be related to a loosening of the polar head region of the bilayer by the mutually repulsive acidic phospholipids [3]. An optimal transfer was obtained with slightly positively charged liposomes containing 5 - 10 molstearylamine (Fig. 2). Since the isoelectric point of the exchange protein is 5.8, the overall charge of the protein will be negative at the pH of incubation (pH 7.4). This may facilitate the interaction of the protein with phosphatidylcholine present at a positively charged interface. Similar arguments have been used to explain why the activity of phospholipase B from Penicillium notatum and phospholipase C from Clostridium perfringens were sharply delineated by the surface charge of the substrate [4]. It is of interest to note that phosphatidylethanolamine which does not contribute to the liposomal surface charge, exerted also an inhibitory effect, be it to a much lesser extent than the acidic phospholipids (Fig. 3). A similar observation has been made for sphingomyelin [2]. This demonstrates that in addition to the membrane surface charge other factors may regulate the activity of the exchange protein. Since the surface charge is related to the ion concentration of the medium, the effect of K + and Mg2+
521
on the transfer activity and surface charge have been determined (Fig. 4- 6). It appears that these cations stimulated the transfer of phosphatidylcholine in that concentration range where these ions were most effective in neutralizing the contribution of the acidic phospholipids to the surface charge. In dealing with negatively charged mitochondria and liposomes, both surface charge and activity were to a much lesser extent affected by K + than M g + . This confirms the general notion that monovalent cations interact only weakly with negatively charged phospholipids [36- 381 ; this in contrast to the strong interaction of divalent cations [36,37,39-421. The above stimulation suggests that cations counteract the effect of phosphatidic acid on the interaction of the exchange protein with the liposome [3]. It is currently under investigation whether the stimulatory effect of particularly Mg2+ may be understood in terms of divalent cations altering the dissociation constant of the exchange protein-liposome complex. The specificity of the exchange protein as regards the transfer of phosphatidylcholine alludes to an electrostatic interaction between the protein and the polar head group of phosphatidylcholine [14]. This may explain why M$+ and, to a lesser extent, K f inhibited the transfer under conditions where these ions had no or relatively little effect on the surface charge of the liposomes (Fig.4-6). Effect of cations on the transfer was also measured under conditions where surface charge would play a negligible role i.e. between a monolayer and liposomes consisting of only phosphatidylcholine (Fig. 7). An increase of the ion concentration inhibited the transfer in the following order of effectiveness: La3+ > M 2 + 2 CaZ+> K' = Na'. This inhibition was not related to the ionic strength as, for example, a 50% reduction of the transfer required 0.4 mM LaCl, as compared to 15 mM CaCl,; 300 mM KC1 was not sufficient to attain this extent of inhibition. In agreement with the present study, 0.1 mM LaC1, did not inhibit the transfer of phospholipids between mitochondria and microsomes in the presence of the 105000 x g supernatant from rat liver [43]. It is thought that the order of inhibition mentioned above could reflect the tendency of the various cations used to concentrate near the negatively charged phosphate group of the zwitterionic phosphatidylcholine. This may disturb the presumed electrostatic interaction between the protein and the polar headgroup of phosphatidylcholine [14]. On the other hand, it cannot be excluded that the inhibition is due to an interaction of the cations with the protein. Direct evidence for cations interacting with phosphatidylcholine is still controversial [40,44]. However, cation-dipole interactions should be considered to understand why cations increase the fluorescence of 8-anilino-1-napthalene sulfonate in suspensions con-
522
taining phosphatidylcholine [45 -471. The cations increase the fluorescence in the order La3+ > Mg2+ = Ca2+ > K + = N a + ; the increase is independent of the ionic strength. It is of interest to note that there may be a parallel between the cations inhibiting the transfer and increasing the fluorescence. Recently, Na', Ca2+ and Mgz+ were found to inhibit also the transfer of phosphatidylcholine as catalysed by the exchange protein from bovine heart, between mitochondria and liposomes [6]. In contrast to the present study, the extent of inhibition was governed by the ionic strength. In addition, the latter inhibition required relatively high ion concentrations ; e.g. 350 mM CaCl, gave a 50 % reduction of transfer. Apart from this the exchange proteins from bovine heart differ from the phosphatidylcholine exchange protein from bovine liver in molecular weight and isoelectric point [14,48]. From the preceding results it appears that in v i m phospholipids other than phosphatidylcholine control the activity of the phosphatidylcholine exchange protein from bovine liver. Recently similar observations have been made with regard to the exchange proteins from bovine brain which stimulate preferentially the transfer of phosphatidylinositol [49,50]. If the activity of the various exchange proteins present in the cell are affected by the phospholipids already present in the membrane, then this has to be envisaged as a control mechanism. In vivo, this mechanism may be important in determining the phospholipid composition of the membrane [51]. Thc authors thank Mrs A. Snel-Niemeyer for expert technical assistance. They are grateful to Professor Dr L. L. M. van Deenen for his continuous interest and advice.
REFERENCES 1. Demel, R. A,, Wirtz, K. W. A., Kamp, H. H., Geurts van Kessel, W. S. M. & van Deenen, L. L. M. (1973) Nut. New Biol. 246, 102-105. 2. Hellings, J. A., Kamp, H. H., Wirtz, K. W. A. & van Deenen, L. L. M. (1974) Eur. J . Biochem. 47, 601-605. 3 . Van den Bessekaar, A. M. H. P., Helmkamp, G . M. & Wirtz, K . W. A. (1975) Biochemistry, 14, 1852- 1858. 4. Dawson, R. M. C. (1968) Biological Membranes, Physical FactandFunction (Chapman, D., ed.) pp. 203 - 232, Academic Press, New York. 5. Overbeek, J. T. G. (1952) . , Colloid Science (Kruyt, . - H. R., ed.) vol. 1, pp. 78 - 79, Elsevier Publishing Company, Amsterdam. 6. Johnson, L. W. & Zilversmit, D. B. (1975) Biochim. Biophys. Acta, 375, 165- 175. 7. Papahadjopoulos, D. & Miller, N. (1967) Biochim. Biophys. Acta, 135, 624-638. 8. Davidson, F. M . & Long, C. (1958) Biochem. J . 69,458-466. 9. Rouser, G., Kritchevsky, G., Yamamoto, A., Simon, G., Galli, C. & Bauman, A. J. (1969) Methods Enzymol. 14, 272-317. 10. Folch, J., Lees, M. & Sloane-Stanley, G. (1957) J . B i d . Chem. 226,497 - 509.
Transfer of Phosphatidylcholine between Membranes 11. Van Deenen, L. L. M. & de Haas, G. H. (1964) Ade. Lipid Res. 2, 167-234. 12. Stoffel, W., Lekim, D. & Tschung, T. S. (1971) HoppeSeyler's Z . Physiol. Chem. 352, 1058- 1064. 13. Swell, L. & Treadwell, C. R. (1955) J . Biol. Chem. 212, 141150. 14. Kamp, H. H., Wirtz, K. W. A. & van Deenen, L. L. M. (1973) Biochim. Biophys. Acfa,318, 313- 325. 15. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J . Biol. Chem. 193, 265-275. 16. Wirtz, K. W. A. & Zilversmit, D. B. (1969) Biochim. Biophys. Acts, 193, 105-116. 17. Gornall, A. G., Bardawill, C. J . & David, M. M. (1949) J . Biol. Chem. 177,751-766. 18. De Gier, J., Mandersloot. J . G. & van Deenen, L. L. M. (1968) Biochim. Biophys. Acta, 150, 666- 675. 19. Chen, A. S., Toribara, T. Y. & Warner, H. (1956) Anal. Chem. 28,1756- 1758. 20. Ames, R. N. & Dubin, D. T. (1960) J . Biol. Chem. 235, 755769. 21. Bangham, A. D., Flemans, R., Heard, D. H. & Seaman, G. V. F. (1958) Nuture (Lond.) 182, 642-644. 22. Seaman, G. V. F. & Heard, D. H. (1960) J . Gen. Physiol. 44,251 -268. 23. Overbeek, J. T. G. (1952) Colloid Science (Kruyt, H. R., ed.) vol. 1, pp. 207- 210, Elsevier Publishing Company, Amster-
dam. 24 Hauser, H. & Irons, L. (1972) Hoppe-Seyler's Z . Physiol. Chem. 353,1579- 1590. 25 Johnson, S. M. (1973) Biochim. Biophys. Acta, 307,27-41. 26 Johnson, L. W., Hughes, M. E. & Zilversmit, D. B. (1975) Biochim. Biophys. Acta, 375, 176- 185. 27 Kornberg, R. D. & McConnell, H. M. (1971) Biochemistry, 10,1111-1120. 28 Finer, E. G., Flook, A. G. & Hauser, H. (1972) Biochim. Biophys. Acta, 260, 49- 58. 29 Bangham, A. D., de Gier, J. & Greville, G. D. (1967) Chem. Phys. Lipids, I , 225 - 246. 30 Sheetz, M. P. & Chan, S. I. (1972) Biochemistry, 11,4573-4581. 31 Lichtenberg, D., Petersen, N. O., Cirardet, J. L., Kainosho, M., Kroon, P. A,, Seiter, C. H. A,, Feigenson, G. W. & Chan, S. I. (1975) Biochim. Biophys. Acta, 382, 10-21. 32. Dawson, R. M. C. & Hauser, H. (1967) Biochim. Biophys. Acfa, 137, 518-524. 33. Kamp, H. H., Wirtz, K. W. A. & van Deenen, L. L. M. (1975) Biochim. Biophys. Acta, 398,401 -414. 34. De Haas, G . H., Heemskerk, C. H. T., van Deenen, L. L. M., Baker, R. W. R., Gallai-Hatchard, J., Magee, W. L. & Thompson, R. H. S. (1963) Biochemical Problems of Lipids (Frazer, A. C., ed.) vol. 1, pp. 244-250, Elsevier Publishing Company, Amsterdam. 35. Pieterson, W. A. (1973) Ph. D. Thesis, University of Utrecht, p. 9. 36. Abramson, M. B., Katzman, R., Wilson, C. E. & Gregor, H. P. (1964) J . Biol. Chem. 23Y, 4066-4072. 37. Hauser, H. & Dawson, R. M. C. (1967) Eur. J . Biochem. I , 61 -69. 38. McLaughlin, S. G. A., Szabo, G. & Eisenmann, G . (1971). J . Gen. Physiol. 58, 667 -687. 39. Papahadjopoulos, D. (1968) Biochim. Biophy~y. Acta, 163, 240- 254. 40. Seimiya, T. & Ohki, S. (1973) Biochim. Biophy.r Acfa, 298, 546- 561. 41. Trauble, H. & Eibl, H. (1974) Proc. Nail Acad. Sci. U.S.A. 71, 214-219. 42. Tocanne, J . F., Ververgaert, P. H. J. Th., Verkleij, A. J. & van Deenen, L. L. M. (1974) Chem. Phys. Lipids 12,201-219. 43. Kamath, S. A. & Rubin, E. (1973) Arch. Biochem. Biophys. 158, 312- 322.
K. W. A. Wirtz, W. S. M. Geurts van Kessel, H. H. Kamp, and K. A. Demel 44. Colacicco, G. (1971) Biochim. Biophys. Arm, 266, 313-319. 45. Vanderkooi, J. & Martonosi, A. (1969) Arch. Biochem. Bio~ C I , V S . 133, 1.53-163. 46. Gomperts, B., Lantelme, F. & Stock, R. (1970) J . Memhrune Biol. 3. 241 -266. 47. Trluble, H. (1971) Nurur~~i.s.senscha~ieti, 58, 277 -284. 48. Ehnholm, C. & Zilversmit, D. B. (1973) J . B i d . Chem. 24X, 1719-1724.
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49. Harvey, M. S., Helmkamp, G . M., Wirtz, K . W. A,, & van Deenen, L. L. M. (1974) FEBS Lett. 46, 260-262. 50. Helmkamp, G. M., Harvey, M. S., Wirtz, K. W. A. & van Deenen, L. L. M. (1974) J . Biol. Chem. 249, 6382-6389. 51. Harvey, M. S., Helmkamp, G. M., Kamp, H. H. & Wirtz, K . W . A. (1974) in Biornrmhrunes: Lipkf.7, Pioteim and Recepfptors (Burton, R . M . & Packer, L., cds) pp. 191 -205. BI-Sciencc Publications Division, Webstcr Groves, Missouri.
K. W. A. Wirlz, W. S. M. Geurts van Kcssel, H. H. Kamp, and R. A. Demel, Riochemisch Laboratorium, Rijksuniversiteit te Utrecht, Transitorium 3, Universiteit’s Centrum “De Uithof’, Padualaan 8, Utrecht, The Netherlands
The Protein-Mediated Transfer of Phosphatidylcholine between Membranes The Effect of Membrane Lipid Composition and Ionic Composition of the Medium Karel W. A. WIRTZ. Wouter S. M . GEURTS VAN KESSEL, Hein H. KAMP, and Rudolf A. DEMEL Biochemisch Laboratorium, Rijksuniversiteit te Utrecht (Rcceived August 4/0ctober 3, 1975)
The phosphatidylcholine exchange protein from bovine liver catalyzes the transfer of phosphatidylcholine between rat liver mitochondria and sonicated liposomes. The effect of changes in the liposomal lipid composition and ionic composition of the medium on the transfer have been determined. In addition, it has been determined how these changes affected the electrophoretic mobility i.e. the surface charge of the membrane particles involved. Transfer was inhibited by the incorporation of negatively charged phosphatidic acid, phosphatidylserine, phosphatidylglycerol and phosphatidylinositol into the phosphatidylcholine-containing vesicles ;zwitterionic phosphatidylethanolamine had much less of an inhibitory effect while positively charged stearylamine stimulated. The cation Mg2+ and, to a lesser extent, K + overcame the inhibitory effect exerted by phosphatidic acid, in that concentration range where these ions neutralized the negative surface charge most effectively. Under conditions where Mg2+ and K + affected the membrane surface charge relatively little inhibition was observed. In measuring the protein-mediated transfer between a monolayer and vesicles consisting of only phosphatidylcholine, cations inhibited the transfer in the order La3+ > M$+ 2 Ca2+ > K + = Na+. Inhibition was not related to the ionic strength, and very likely reflects an interference of these cations with an electrostatic interaction between the exchange protein and the polar head group of phosphatidylcholine. The phosphatidylcholine exchange protein from bovine liver functions as a carrier of phosphatidylcholine between membrane interfaces [l]. Recently it was demonstrated that the protein-mediated transfer of phosphatidylcholine between phosphatidylcholine liposomes was inhibited by incorporation of phosphatidic acid or phosphatidylinositol into these liposomes [2]. Steady state analysis of the kinetic data indicated that the apparent dissociation constant of the exchange protein-liposome complex decreased with an increasing phosphatidic acid content of the liposomes [3]. This was interpreted to mean that an increase of the negative surface charge facilitated the interaction of the exchange protein with the membrane interface resulting in less protein free in the medium to function as a carrier i.e. inhibition of transfer. The former studies implied that physical chemical properties of the interface may have an effect on the activity of the exchange protein. In the present study this concept has been elaborated by correlating the protein-mediated transfer of phosphatidylcholine between rat liver mitochondria and liposomes with the
surface charge of these membrane structures. Comparable studies on the relationship between surface charge and phospholipase action have indicated that the surface charge of a phospholipid interface may control the formation of the proper enzyme-phospholipid complex [4]. The surface charge of a membrane is the resultant of two counteracting factors, namely, the surface charge density of the membrane and the counter ion concentration in the medium [ 5 ] . In this study we have altered the surface charge of the liposome independently of that of the mitochondrion by introducing charged amphipathic molecules into the liposomal bilayer. In addition, the surface charge of both liposome and mitochondrion have been affected simultaneously by changing the ionic composition of the medium. In order to investigate whether the activity of the exchange protein is dependent on the ionic strength of the medium, the transfer of phosphatidylcholine has been measured between a monolayer and liposomes both of which consisting only of phosphatidylcholine
Transfer of Phosphatidylcholine between Membranes
516
[l]. Under these conditions the cation concentration at the membrane surface should ideally equal the bulk ion concentration since the membranes involved lack an overall surface charge. While this study was in progress it was reported that the activity of an exchange protein from bovine heart was inhibited by increasing the ionic strength of the medium [6]. Part of this investigation has been reported at the Eigth Meeting of the Federation of European Biochemical Societies, Amsterdam, August 1972.
the procedure of Ames and Dubin [20]. The irradiated liposomes were used in the assays for measuring the transfer of ['4C]phosphatidylcholine.The hand-shaken liposomal suspension was used for determination of the electrophoretic mobility of the liposomes (see below). Assay A
Determination of the transfer of ['4C]phosphatidylcholine from 14C-labelled mitochondria to 3Hlabelled liposomes was performed according to a MATERIALS AND METHODS modification of the assay of phosphatidylcholine exchange activity as previously described [14]. MitoPhosphatidylcholine was isolated from egg yolk chondria containing ['4C]phosphatidylcholine (10 mg and used for the preparation of phosphatidic acid of protein) were incubated with 3H-labelled liposomes [7,8]. Phosphatidylethanolamine and phosphatidyl(1 pmol of phosphatidylcholine) and phosphatidylinositol were isolated from rat liver, and phosphatidylcholine exchange protein (0.01 mg) in a total volume serine from bovine brain by applying the lipid ex2.5 ml of buffer (pH 7.4) for 20 min at 25 "C. In of tracts to a TEAE-cellulose column 191. Phosphatidyladdition to the 1 pmol of phosphatidylcholine the glycerol prepared from egg yolk phosphatidylcholine liposomes contained various amounts of other amwas kindly donated by Dr B. Verheij. After equilibraphipathic molecules. See the legends to the figures for tion with 0.1 M NaCl in the chloroform/methanol/ composition of liposomes and buffer. At the end of water system of Folch et al. [lo] the phospholipids incubation the mitochondria were sedimented at were stored in chloroform at - 20 "C. l-Palmitoyl-29000rev./min for 5min in the SS-34 rotor of the oleoyl-sn-glycero-3-phosphorylcholine was syntheSorvall centrifuge in Sorvall tubes (no. 250) with sized according to established procedures [ll]. A adapters. The pellets were discarded. The pH of the ['4C]methyl group was introduced into this compound according to the method of Stoffel et al. [12]. [7~r-~H]- supernatant obtained was adjusted to 5.1 by addition of 0.5 ml of 0.2 M sodium acetate in 0.25 M sucrose Cholesteryloleate was prepared from TH]cholesterol (pH 5.0) followed by a centrifugation at 9000 rev./min (The Radiochemical Centre, Amersham, England) for 5 min. This pH adjustment caused an aggregation and oleoyl chloride by the method of Swell and of those mitochondria which had remained in the Treadwell [ 131. Phosphatidylcholine exchange protein supernatant after the first centrifugation. Controls was isolated from bovine liver [14]. Protein was without exchange protein were carried through the determined by the method of Lowry et al. [15]. entire procedure. The mitochondrial supernatant Stearylamine was obtained from E. Merck AG containing the liposomes was collected, the lipids were (Darmstadt) and used without further purification. extracted, and the 14C/3Hratio of the liposomal lipid Male rats (200- 300 g) of the Wistar albino strain extract corrected for a control incubation, was deterwere fasted overnight and injected intraperitoneally mined as described [14]. The percentage of [l4C]phoswith 20 pCi [Me-'4C]choline (The Radiochemical phatidylcholine transferred from mitochondria to Centre, Amersham, England). After 45 min livers a/h x X x 100 where a and h are the liposomes, is were excised and the mitochondrkd containing ['"CIradioactivity in the 3H-labelled liposomes and 14Cphosphatidylcholine were isolated as described [ 161. labelled mitochondria, respectively, before incubation The final mitochondrial pellet was resuspended in the and X the corrected 14C/3H ratio of the liposomal incubation buffer (see legends to figures) and used the lipids after incubation. same day. Protein was determined by the biuret method [17]. Hand-shaken liposomes containing a trace of Assay B [3H]cholesteryloleate (0.01 7; w/w) were prepared in The rate of transfer of ['4C]phosphatidylcholine the incubation buffer following the procedure of from a monolayer consisting of 16:0/18: 1 [Me-14C]de Gier et a f . [18]. The liposomal suspension (2phosphatidylcholine to phosphatidylcholine liposomes 10 ~ m o l phospholipid/ml) was irradiated ultrawas performed as described [l]. For experimental desonically in a Branson sonifier for 5 min at 80 W outtails, see legends to Fig. 7. put. Liposomes consisting of only phosphatidylcholine were sonicated for 30 min. The liposomes were Measurements of Electrophoretic Mobility stored in the refrigerator and used the next day. Lipid phosphorus was determined by the method of Chen Electrophoretic mobilities of hand-shaken liposomes were determined at 25 "C in a microelectrophoet af. [19] after destruction of the sample according to
K. W. A. Wirtz, W. S . M. Geurts van Kessel, H. H. Kamp, and R. A. Demel
resis apparatus of the type described by Bangham et al. [21,22]. Mobilities of the mitochondria were determined under similar conditions by Dr S. Nordling (Department of Pathology, University of Helsinki, Helsinki). The velocities of the particles are expressed in terms of ps-' V-' cm. The media in which the mobilities were determined, were similar to those in which the transfer of ['4C]phosphatidylcholine was measured. In the present study the electrophoretic mobility determined for handshaken liposomes may be similar to that of the irradiated single bilayer liposomes. This will be correct if the radius of both types of particles is large compared with the reciprocal thickness of the double layer [23]. Single bilayer liposomes, however, may not meet this criterion as their radius is in the order of 15-25pm [24,25]. Then the determined electrophoretic mobilities have relative significance [23]. RESULTS Transfrr of (I4 C]Phosphatidylcholine (Assay A )
The conditions of incubation as given in the Methods section, were chosen such that the exchange protein was limiting in the transfer of [14C]phosphatidylcholine from the ''C-labelled mitochondria to the liposomes. The liposomes used were ultrasonically dispersed for 5 min because sonication increases the liposomal phosphatidylcholine pool that participates in the transfer process. This is shown in Fig. 1 where transfer reached a maximum with 0.5 pmol of phosphatidylcholine if the liposomes were irradiated for 10 min. In the instance of a 1-min irradiation, 1 pmol of phosphatidylcholine was necessary to attain the same level of transfer. However, up to 4 pmol of phosphatidylcholine of hand-shaken liposomes were not sufficient to reach this maximal level. The liposomes used in the preceding experiments contained 2 mol- phosphatidic acid. In general, an increase of the content of charged amphipathic molecules facilitates the ultrasonic dispersion of the liposomal phospholipid [25].
-
517
I
I
Liposomal PtdCho (Krnol)
Fig. 1. Percent transjkr oj' mitochondrial ['4CJpho.spliatidylcholine as a function of amount of liposomal phosphatidylcholine. Mitochondria containing ['4C]phosphatidylcholine (10 mg of protein) were incubated with liposornes (1 prnol of phosphatidylcholine, 0.02 prnol phosphatidic acid, 0.01 by weight, [7a-3H]cholesteryloleate) in 2.5 rnl 0.25 M sucrose/0.001 M EDTA/0.010 M KCl/O.OlO M Tris (pH 7.4) as described in Methods. Liposomes used were sonicated for different periods of time. (M Hand) shaken liposomes; ( x - x ) liposomes sonicated for 1 min; (-0) liposomes sonicated for 10 rnin. PtdCho, phosphatidylcholine
x,
Transjer of ['4C]Phosphatidylcholine to Mi-xed Liposomes
Under the standard conditions of incubation a transfer of approximately 10% was observed with liposomes containing 2 mol- % phosphatidic acid (Fig. 1). In order to determine whether the composition of the liposomes affected the transfer, liposomes were prepared with various amounts of negatively charged phosphatidic acid and positively charged stearylamine (Fig. 2A). Relative to the transfer with liposomes containing 2 mol- % phosphatidic acid, transfer decreased by increasing the liposomal phos-
I
30
I
I
10 Phosphatid i t acid
20
(mol%)
I 0
I
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10 20 30 Stearylarnine
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Fig. 2. Electrophoretic mobility o/' mixrd liposomes conlaining phosphatidylcholine (PidCho) and various amounts of phosphalidic acid and stearylamine, and the transfer of mitochondrial [I4C]phosphatidylcholine to these liposomes. Hand-shaken liposornes in 0.25 M sucrose/0.001M EDTA/0.010 M KCl/O.OlO M Tris (pH 7.4) were used for the determination of the electrophoretic mobility (B). Sonicated liposomes were used in the transfer experiments as described in Methods (A)
518
Transfer of Phosphatidylcholine bctween Membranes
phatidic acid content, and increased by incorporation of stearylamine. However, stearylamine became inhibitory at a concentration above 10 mol-"/,. In summary, a transfer of 15% with liposomes containing 10 mol- % stearylamine declined to 4.5 % with liposomes containing 20 mol- "/, phosphatidic acid. Concomitantly, the electrophoretic mobility of the liposomes changed from a cathodic mobility of 2 ps -'V-' cm for 10 mol-% stearylamine into V - l cm for 20 molan anodic mobility of - 4 ps phosphatidic acid (Fig. 2B). These results suggest that the transfer of phosphatidylcholine between mitochondria and liposomes may be inhibited by increasing the negative surface charge of the liposomes. The latter relationship has been corroborated with liposomes which contained various amounts of phosphatidylserine, phosphatidylglycerol and phosphatidylinositol. The presence of these negatively charged phospholipids had a very similar effect on the electrophoretic mobility of the liposomes (Fig. 3 B). Mobility increased from - 1.5 ps-' V-' cm in the absence of these phospholipids to - 5.5 ps-' V P 1cm in the presence of 20 molIn general it was observed that the mobility of liposomes containing only phosphatidylcholine varied between - 1.O and - 1.5 ps-' V-' cm at the low ionic strength of the medium (I = 0.013, see legend to Fig. 3). This anodic mobility may be due to traces of impurity such as free fatty acids, in the electrically neutral phosphatidylcholine. In agreement with what was observed for phosphatidic acid, negatively charged phosphatidylserine, phosphatidylglycerol and phosphatidylinositol had a pronounced inhibitory effect on the transfer (Fig. 3A). Up to 8 mol-%, these phospholipids inhibited to the same extent; above 8 molit was phosphatidylinositol that exerted the strongest inhibition. For example, 20 mol- % phosphatidylinositol inhibited the transfer by 70 "/,. Studies with phosphatidylethanolamine, however, indicated that the surface charge is not the only parameter of importance in the protein-mediated transfer. Incorporation of phosphatidylethanolamine had very little effect on the electrophoretic mobility of the liposomes (Fig.3B). In spite of this lack it was inhibitory in the transfer although the inhibition was much less extensive than that exerted by the negatively charged phospholipids (Fig. 3 A).
+
I 12t-A
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1
' - 1
-'
3
OO
5
15
10
20
25
30
x.
Effect of Mono-, Di- and Trivalent Cations The transfer of ['4C]phosphatidylcholine has been measured between I4C-labelled mitochondria and liposomes (assay A) and between 14C-labelled monolayers and liposomes (assay B) at various cation concentrations. The effect of these ions on the electrophoretic mobility i.e. surface charge of the membranes involved have been determined.
5 10 15 20 25 Negatively charged phospholipids (rnol %)
0
30
Fig. 3. Electrvphoretic mobility of mixed liposomes mid the transjkr mitochondrial ['4C]pho.vpluztidylcholine to these liposomes. For experimental details, see lcgend to Fig. 2. The liposomes contained phosphatidylcholine and various amounts of phosphatidylethanolphosphatidylserine ), (M phosphatidyl), amine (M glycerol ( x x ), and phosphatidylinositol M ()
of
~
It is shown in Fig.4B that in 0.25 M sucrose/ 0.01 M Tris-HC1 (pH 7.4) liposomes containing 12 mol- "/, phosphatidic acid, and mitochondria have mobilities of - 4.1 and - 2.0 ps-l V-' cm, rcspectively. Increase of the K concentration under conditions where isotonicity of the medium was maintained, resulted in a decrease of the anodic mobilities of both membrane particles. In 0.15 M KC1/0.01 M Tris-HC1 (pH 7.4) liposomes and mitochondria have mobilities of - 1.6 and - 0.8 ps-' V-' cm, respectively. Effect of K + on the transfer was little (Fig.4A). Transfer passed through a maximum at 0.015-0.030 M KCl at which concentration transfer had increased from 6 to 8 Similar experiments were performed with Mg2+ (Fig. 5). Liposomes with a mobility of - 4 ps-' V-' cm in 0.25 M sucrose/O.Ol M Tris-HC1 (pH 7.4) did not move in an electric field upon addition of 0.01 M MgC1,. In the presence of 0.002 M MgC12 mobilities of both liposome and mitochondrion had decreased to - 1.0 and -0.7 ps-' V-' cm, respectively. M$+ had an equally pronounced effect on the transfer +
x.
K . W. A. Wirtz, W. S . M. Ceurts van Kessel, H. H. Kamp, and R. A. Demel
519
15
-
-A
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-a E
i 0.250 0
0.125 Sucrose ( M )
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0.150
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2
Y
v-
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6
L (Y vv)
g 3
KCI ( M )
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-'I
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0 - 4 r 0.250
3 .0.125
v-
I
0
Sucrose ( M )
0
0.075
0.150
KCI ( M )
M g C b (mM)
Fig. 4. Ejfect of Kt on the electrophoretic mobility qfliposomes and mitochondria, and on the transfer of mitochondrial [i4C]phosphatidylcholine. Hand-shaken liposomes consisting of phosphatidylcholine and phosphatidic acid (12 mol-%) were prepared in 0.25 M sucrose/O.Ol M Tris-HCI (pH 7.4). in 0.15 M KCI/O.Ol M TrisHC1 (pH 7.41, and in mixtures of these buffers. (B) Electrophoretic mobilities of liposomes ( x -x ) and mitochondria ( 0 4 ) were determined in these media. (A) Transfcr of mitochondrial ['4C]phosphatidylcholine was determined in the same media as described in Methods
Fig. 5 . effect of M g z + on the electrophoretic mobility of' liposomes and mitochondria, and on the fransjer of mitochondrial ['4C]phosphatidylcholine. Hand-shaken liposomes consisting of phosphatidylcholine and phosphatidic acid (14 mol- %) were prepared in 0.25 M sucrose/O.Ol M Tris-HCI (pH 7.4). MgCI, was added to the sucrose/Tris medium and the electrophoretic mobilities of liposomes ( x -x ) and mitochondria (-0) were determined (B). Transfer of mitochondrial [14C]phosphatidylcholine was determined in the same media as described in Methods (A)
(Fig. 5A). A maximum transfer was obtained in the presence of 0.002 M MgCl, at which concentration transfer had increased from 6 % to 13.5%; higher concentrations of MgCI, became inhibitory. It appears from Fig. 5 that M$+ stimulated transfer in that concentration range where this cation was most effective in reducing the surface charge of liposome and mitochondrion. In addition the results suggest that Mg2+ became inhibitory at these concentrations where this ion changed the surface charge of the membranes more gradually. In support of this conclusion an experiment was carried out with liposomes containing 5 mol- % stearylamine. Because of positively charged stearylamine, the liposomes had a cathodic mobility of 1.7 pspl V-' cm that was barely affected by Mg2+ (Fig. 6B). Under these conditions M$+ inhibited the transfer of [14C]phosphatidylcholine over the whole concentration range
(Fig. 6A). This suggests that Mg2+ is inhibitory in the transfer except under these conditions where the inhibition is overcome by stimulation due to a reduction of the surface charge of the negatively charged membranes. In order to provide additional evidence for the inhibition exerted by cations, transfer was measured between a ''C-labelled monolayer and liposomes both of which consisting of only phosphatidylcholine. Exchange protein was limiting under the incubation conditions (see legend to Fig. 7). The rate of transfer was expressed as a percentage of [14C]phosphatidylcholine transferred to the subphase per min and was calculated from the decrease of the surface radioactivity after addition of the exchange protein as outlined previously [l]. It is seen from Fig.7 that an increase of the ionic concentration of the medium decreases the rate of transfer no significant difference
+
Transfer of Phosphatidylcholine between Membranes
520
15
-
12
." 0
5
E
u
9 I
I
"0
0.05
Ion
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I
0.10 I l concentration ( M )
0.30
Fig. 7. Ej'ect of cations on lhe exchange of' phosj3hatidvlcholine between a ['4C]phosphatidylcholine-containing monoluyer and liposomes. The monolayer consisted of 20.6 nmol 16:0/18: 1 [Me-'4C]phosphatidylcholine and covered a surface area of 74.1 cmz (a surface pressure of 30 dynes cm-'). Liposomes were prepared from egg yolk phosphatidylcholine as described in Methods section. The subphase contained 1.25 pmol liposomal phosphatidylcholine in a total volume of 75 ml 0.01 M Tris/O.Ol M sodium acetate/HCl (pH 7.4). The exchange reaction was initiated by addition of 5.7nmol exchange protein (125 pg) to the subphase. The rate of transfer of [14C]phosphatidylcholineto the subphase was calculatcd from the initial slope which represented the dilution of the surface radioactivity. For additional experimcntal details see [l ]
MgCIz (mM)
Fig. 6. Eflect of M?+ on the electrophoretic mobility of liposomes and mitochondria, and on the transfer of mitochondria1 ['4C]phosphatidylcholine. See legend to Fig. 5. The liposomes contained phosphatidylcholine and 5 mol- "/, stearylamine
being observed between different cations of the same class of valency. However, Ca2+ or Mg2+ inhibited about 40 times as effectively as Na' or K + . With La3 the same inhibition occurred at concentrations about another factor of 40 lower than for the divalent cations. This indicates that inhibition is not just related to the ionic strength but becomes more effective with increasing valency. +
DISCUSSION The protein-mediated transfer of phosphatidylcholine between mitochondria and liposomes will be a function of the phosphatidylcholine pools available in these two membrane particles [3]. Recently evidence was provided that only phosphatidylcholine present in the outer liposomal monolayer participated in the transfer process [26]. Prolonged irradiation of a liposomal suspension results in single bilayer liposomes with approximately 65 % of the phosphatidylcholine in the outer monolayer [27,28]. Since about 9 % is present in the outer monolayer of hand-shaken
liposomes, irradiation will result in a 7-fold increase of the amount of phosphatidylcholine available for transfer [29], Thls would explain as to why irradiation of a liposomal suspension enhanced the transfer (Fig.1). It is of interest to note, however, that e.g. 0.2 pmol phosphatidylcholine of a 10-min irradiated liposomal suspension gives the same transfer as 3.6 pmol of a hand-shaken liposomal suspension. In view of the above this factor of 18 cannot be explained by just an increase of phosphatidylcholine in the outer monolayer. Instead the results suggest that in addition to a concentration Factor the transfer per unit area of outer monolayer is faster for irradiated than for hand-shaken liposomes. From nuclear magnetic resonance results it has been inferred that ultrasonic irradiation introduces subtle changes in the orientation of phosphatidylcholine molecules in the bilayer [30,31]. This may be one of the reasons for the irradiated liposomes to be more effective in the transfer process. Changes in molecular organization have also been inferred in explaining the faster rate of hydrolysis of phosphatidylcholine by phospholipase B from Penicillium notatum after ultrasonic irradiation [32]. The exchange protein from bovine liver contains one molecule of non-covalently bound phosphatidylcholine [I, 14,331. Upon interaction with a membrane the protein can exchange its endogenous phosphatidyl-
K. W. A. Wirtz, W. S. M. Geurts van Kessel, H. H. Kamp, and R. A. Demel
choline molecule for one present in the membrane [ l , 331. It is presumably by this mechanism that in the present study the protein transfers ['4C]phosphatidylcholine from the 14C-labelled mitochondria to the liposomes. Previously in studying the transfer of phosphatidylcholine between single bilayer liposomes, it was demonstrated that the transfer decreased with an increase of negatively charged phosphatidic acid in the liposomes [2,3]. An analysis of the kinetic data indicated that the apparent dissociation constant of the exchange protein-liposome complex decreased in a parallel fashion resulting in an inhibition of transfer [3]. In the present study it has been confirmed that the incorporation of phosphatidic acid into phosphatidylcholine liposomes inhibits transfer (Fig. 2). The inhibition is not specifically related to this phospholipid as other negatively charged phospholipids like phosphatidylserine, phosphatidylglycerol and phosphatidylinositol inhibited also (Fig. 3). This argues in favor of the activity of the protein being controlled by the negative surface charge of the interface. Studies on the action of human pancreatic phospholipase A2 indicated that phosphatidylcholine liposomes were only susceptible to enzymatic attack provided acidic phospholipids were present [34,35]. This suggests that in accordance with what we have seen with the exchange protein, the acidic phospholipids lower the dissociation constant of the phospholipase-liposome complex; however, in contrast to an inhibitory effect on the activity of the exchange protein, a stimulation of phospholipase activity is observed. The decrease of the dissociation constant discussed above, may be related to a loosening of the polar head region of the bilayer by the mutually repulsive acidic phospholipids [3]. An optimal transfer was obtained with slightly positively charged liposomes containing 5 - 10 molstearylamine (Fig. 2). Since the isoelectric point of the exchange protein is 5.8, the overall charge of the protein will be negative at the pH of incubation (pH 7.4). This may facilitate the interaction of the protein with phosphatidylcholine present at a positively charged interface. Similar arguments have been used to explain why the activity of phospholipase B from Penicillium notatum and phospholipase C from Clostridium perfringens were sharply delineated by the surface charge of the substrate [4]. It is of interest to note that phosphatidylethanolamine which does not contribute to the liposomal surface charge, exerted also an inhibitory effect, be it to a much lesser extent than the acidic phospholipids (Fig. 3). A similar observation has been made for sphingomyelin [2]. This demonstrates that in addition to the membrane surface charge other factors may regulate the activity of the exchange protein. Since the surface charge is related to the ion concentration of the medium, the effect of K + and Mg2+
521
on the transfer activity and surface charge have been determined (Fig. 4- 6). It appears that these cations stimulated the transfer of phosphatidylcholine in that concentration range where these ions were most effective in neutralizing the contribution of the acidic phospholipids to the surface charge. In dealing with negatively charged mitochondria and liposomes, both surface charge and activity were to a much lesser extent affected by K + than M g + . This confirms the general notion that monovalent cations interact only weakly with negatively charged phospholipids [36- 381 ; this in contrast to the strong interaction of divalent cations [36,37,39-421. The above stimulation suggests that cations counteract the effect of phosphatidic acid on the interaction of the exchange protein with the liposome [3]. It is currently under investigation whether the stimulatory effect of particularly Mg2+ may be understood in terms of divalent cations altering the dissociation constant of the exchange protein-liposome complex. The specificity of the exchange protein as regards the transfer of phosphatidylcholine alludes to an electrostatic interaction between the protein and the polar head group of phosphatidylcholine [14]. This may explain why M$+ and, to a lesser extent, K f inhibited the transfer under conditions where these ions had no or relatively little effect on the surface charge of the liposomes (Fig.4-6). Effect of cations on the transfer was also measured under conditions where surface charge would play a negligible role i.e. between a monolayer and liposomes consisting of only phosphatidylcholine (Fig. 7). An increase of the ion concentration inhibited the transfer in the following order of effectiveness: La3+ > M 2 + 2 CaZ+> K' = Na'. This inhibition was not related to the ionic strength as, for example, a 50% reduction of the transfer required 0.4 mM LaCl, as compared to 15 mM CaCl,; 300 mM KC1 was not sufficient to attain this extent of inhibition. In agreement with the present study, 0.1 mM LaC1, did not inhibit the transfer of phospholipids between mitochondria and microsomes in the presence of the 105000 x g supernatant from rat liver [43]. It is thought that the order of inhibition mentioned above could reflect the tendency of the various cations used to concentrate near the negatively charged phosphate group of the zwitterionic phosphatidylcholine. This may disturb the presumed electrostatic interaction between the protein and the polar headgroup of phosphatidylcholine [14]. On the other hand, it cannot be excluded that the inhibition is due to an interaction of the cations with the protein. Direct evidence for cations interacting with phosphatidylcholine is still controversial [40,44]. However, cation-dipole interactions should be considered to understand why cations increase the fluorescence of 8-anilino-1-napthalene sulfonate in suspensions con-
522
taining phosphatidylcholine [45 -471. The cations increase the fluorescence in the order La3+ > Mg2+ = Ca2+ > K + = N a + ; the increase is independent of the ionic strength. It is of interest to note that there may be a parallel between the cations inhibiting the transfer and increasing the fluorescence. Recently, Na', Ca2+ and Mgz+ were found to inhibit also the transfer of phosphatidylcholine as catalysed by the exchange protein from bovine heart, between mitochondria and liposomes [6]. In contrast to the present study, the extent of inhibition was governed by the ionic strength. In addition, the latter inhibition required relatively high ion concentrations ; e.g. 350 mM CaCl, gave a 50 % reduction of transfer. Apart from this the exchange proteins from bovine heart differ from the phosphatidylcholine exchange protein from bovine liver in molecular weight and isoelectric point [14,48]. From the preceding results it appears that in v i m phospholipids other than phosphatidylcholine control the activity of the phosphatidylcholine exchange protein from bovine liver. Recently similar observations have been made with regard to the exchange proteins from bovine brain which stimulate preferentially the transfer of phosphatidylinositol [49,50]. If the activity of the various exchange proteins present in the cell are affected by the phospholipids already present in the membrane, then this has to be envisaged as a control mechanism. In vivo, this mechanism may be important in determining the phospholipid composition of the membrane [51]. Thc authors thank Mrs A. Snel-Niemeyer for expert technical assistance. They are grateful to Professor Dr L. L. M. van Deenen for his continuous interest and advice.
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K. W. A. Wirlz, W. S. M. Geurts van Kcssel, H. H. Kamp, and R. A. Demel, Riochemisch Laboratorium, Rijksuniversiteit te Utrecht, Transitorium 3, Universiteit’s Centrum “De Uithof’, Padualaan 8, Utrecht, The Netherlands
