509

Biochimica et Biophysica Acta, 4 0 1 ( 1 9 7 5 ) 5 0 9 - - 5 2 9 © Elsevier S c i e n t i f i c P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s BBA 77089

CHEMICALLY INDUCED LIPID PHASE SEPARATION IN MODEL MEMBRANES CONTAINING CHARGED LIPIDS: A SPIN LABEL STUDY*

H.-J. G A L L A a a n d E. S A C K M A N N b

a Max-Planck-Institut fi~r Biophysikalische Chemie, D-34 G6ttingen, and b A b tl. Experimentelle Physik III, Universit~t Ulm, D-79 Ulm/Donau, Oberer Eselsberg (G.F.R.) (Received April 10th, 1975)

Summary The lipid distribution in binary mixed membranes containing charged and uncharged lipids and the effect of Ca 2+ and polylysine on the lipid organization was studied by the spin label technique. Dipalmitoyl phosphatidic acid was the charged, and spin labelled dipalmitoyl lecithin was the uncharged (zwitterionic) component. The ESR spectra were analyzed in terms of the spin exchange frequency, Wex. By measuring Wex as a function of the molar percentage of labelled lecithin a distinction between a random and a heterogeneous lipid distribution could be made. It is established that mixed lecithinphosphatidic acid membranes exhibit lipid segregation (or a miscibility gap) in the fluid state. Comparative experiments with bilayer and monolayer membranes strongly suggest a lateral lipid segregation. At low lecithin concentration, aggregates containing between 25 % and 40 % lecithin are formed in the fluid phosphatidic acid membrane. This phase separation in membranes containing charged lipids is understandable on the basis of the Gouy-Chapman theory of electric double layers. In dipalmitoyl lecithin and in dimyristoyl phosphatidylethanolamine membranes the labelled lecithin is randomly distributed above the phase transition and has a coefficient of lateral diffusion of D = 2 . 8 . 1 0 -s cm2/s at 59 °C. Addition of Ca 2+ dramatically increases the extent of phase separation in lecithin-phosphatidic acid membranes. This chemically (and isothermally) induced phase separation is caused by the formation of crystalline patches of the Ca 2+-bound phosphatidic acid. Lecithin is squeezed out from these patches of rigid lipid. The observed dependence of Wex on the Ca 2+ concentration could be interpreted quantitatively on the basis of a two-cluster model. At low lecithin and Ca 2 + concentration clusters containing about 30 mol % lecithin are formed. At high lecithin or Ca 2 + concentrations a second type of precipitation containing 100 % lecithin starts to form in addition. A one-to-one binding of divalent ions and phosphatidic acid at pH 9 was assumed. Such a one-to*Part of this work was presented at the VIth International Conference on Magnetic Resonance in Biological Systems in Kandersteg, Switzerland, 1974.

510

one binding at pH 9 was established for the case of Mn 2 + using ESR spectroscopy. Polylysine leads to the same strong increase in the lecithin segregation as Ca ~+. The transition of the phosphatidic acid bound by the polypeptide is shifted from Tt = 47.5 ° to Tt = 62°C. This finding suggests the possibility of cooperative conformational changes in the lipid matrix and in the surface proteins in biological membranes.

Introduction Recent results in membrane research suggest that lipid bilayers and membrane b o u n d (intrinsic) proteins form a functional entity. This postulate is sustained by the finding that removal of the natural lipid environment inactivates intrinsic proteins (cf. ref. 1). Examples of intrinsic proteins are: Cytochrome oxidase, the terminal member of the mitochondrial electron transport chain [2, 3] or the cytochrome P-450/450 reductase complex, the terminal member of the hydroxylation system in liver microsomal membranes [4]. It has been established by two recent spin label studies [3, 4] that intrinsic proteins may be embedded in a cluster of rigid lipid bilayers, while this protein-lipid entity swims in the fluid (or liquid crystalline) bulk membrane. The lipid structure surrounding the P-450/450 reductase system undergoes a conformational change from a rigid to a fluid state at a "transition" temperature Tt = 32°C for rat liver microsomes [4]. This finding provides strong evidence that the lipid adjacent to the proteins differs in its molecular structure from the phospholipid forming the bulk membrane. Obviously the incorporation of intrinsic proteins in biological membranes may involve a heterogeneous lipid distribution (or lipid phase separation). The class of the so-called "extrinsic" proteins that are associated with the membrane surface are generally thought to exhibit only a very weak and negligible lipid-protein interaction. However, experiments by Kimelberg and Papahadjopoulos [5] showed that positively charged proteins such as lysozyme and cytochromes increase the Na ÷ permeability of phosphatidylserine vesicles considerably. Small changes in the lipid conformation induced by the adsorption of proteins to membrane surfaces have also been observed recently by the spin label technique [6]. A dramatic effect on the lipid distribution in binary mixed lipid bilayers containing phosphatidylcholine and phosphatidic acid is caused by adsorption of polylysine, provided the polypeptide is in its charged and therefore random coiled configuration [7]. The polylysine binds to the negatively charged phosphatidic acid which then forms rigid (crystalline) precipitations within the lipid bilayer. These experiments suggest the possibility of simultaneously and cooperatively induced conformational changes in the surface proteins and in the lipid matrix. In summarizing, the above mentioned experiments indicate that both, intrinsic and extrinsic proteins, may involve a heterogeneous lipid distribution in biological membranes. From a physical point of view two classes of conformational changes have to be considered in membranes composed of lipid mixtures:

511

(1) The crystalline-to-liquid crystalline phase transition transforming the lipid structure from a crystalline to a highly fluid state, and (2) phase separation leading to a heterogeneous lipid distribution or domain formation. Both types of conformational changes may be induced either thermally or chemically, for instance by altering the composition of the aqueous phase. Thermally induced phase separation in model membranes has been observed by several groups using calorimetry [8] or the spin label technique [9, 10, 11]. In model membranes containing charged lipids, such as phosphatidic acid, the lipid phase transition may be triggered chemically for instance by changes in the pH of the aqueous phase [12, 13]. The possibility of a chemically (and isothermally) induced phase separation has been demonstrated recently in model membranes of lecithin containing phosphatidic acid as charged lipid constituent [7]. In these experiments the conformational change has been triggered by the addition of Ca 2 +. In the present paper we performed a spin label study of these Ca 2÷ induced alterations in the lipid organization of model membranes composed of synthetic dipalmitoyl phosphatidic acid and dipalmitoyl phosphatidylcholine (lecithin). The lecithin was marked by a spin label group. Addition of Ca 2+ causes a strong increase in the broadening of the ESR-spectra. This Ca 2 +induced broadening has been reported previously by Ito and Ohnishi [14, 16]. The broadened ESR-lines were analyzed in terms of the concentration dependence of the dipolar and the spin exchange interaction. It was thus possible to distinguish whether the radical interaction is caused primarily by an increase in the lipid lateral diffusion or by lipid phase separation. In addition quantitative information on the extent of phase separation could be obtained on the basis of such an analysis. Materials and Methods

Lipids. Dipalmitoyl lecithin from Fluka and dipalmitoyl phosphatidic acid from Serdary (London, Ontario, Canada) were used w i t h o u t further purification. The dimyristoyl phosphatidylethanolamine was a gift of Dr Eibl of the Max-Planck-Institut in Gottingen. 0

~

A ~ ~ O C H 2 0 H

O ~ --"0

I01 i

•

.c:.H 3~..

O CH20-P-OICH212'-N~CH3 CH3

(i)

PC* [5/10)

Spin-labelled lecithin (I) was used as one lipid constituent in the mixed membranes. The label was synthesized by acylation of 1-palmitoyl lysolecithin (from Serdary) according to the method described by Hubbell and Mc Connell [15]. The acylation was performed with the anhydride of stearic acid (Syva Associates, Palo Alto, Calif.) carrying a labelled oxazolidine ring at carbon atom number seven. The reaction p r o d u c t was purified by gel filtration on Sephadex LH-20 using an 8 : 2 chloroform/ethanol solvent.

512

Polylysine used in o u r e x p e r i m e n t s ( f r o m Miles Biochemicals) had a m o l e c u l a r weight o f a b o u t 30 000. T h e following buffers as described in ref. 16 were used: (1) pH 9.0: m i x t u r e o f 81.6 ml of 0.05 M Na2B407 and of 18.4 ml of 0.2 M H3BO3 c o n t a i n i n g 0.05 M NaC1. (2) pH 5.0: acetate b u f f e r : 70.5 ml of 1 M N a O H and 100 ml o f 1 M acetic acid was diluted with w a t e r t o a final v o l u m e o f 500 ml. (3) pH 3.0: to a solution of 0.1 M KC1 in water, 0.1 M HC1 was a d d e d until t h e r e q u i r e d pH value was reached. T h e pH values were c o n t r o l l e d with a B e c k m a n p H - m e t e r to an a c c u r a c y of 0.001 pH units. Preparation o f the vesicles Bilayer vesicles were p r e p a r e d as described earlier: the wall o f an 8 ml pear-shaped flask was c o a t e d with a thin film o f a m i x t u r e of p h o s p h a t i d i c acid and labelled lecithin in a p p r o p r i a t e a m o u n t s . T h e total a m o u n t o f lipid in the film was 0.9" 10 -5 mol. T h e n 5 ml of b u f f e r solution was a d d e d and the sample was sonicated for a b o u t 10 min at a t e m p e r a t u r e well above the lipid phase transition ( a b o u t 60°C). A f t e r a d d i t i o n of Ca 2 ÷ or p o l y l y s i n e in app r o p r i a t e a m o u n t s the vesicle dispersion was again sonicated for a b o u t 3 min. Very clear solutions were o b t a i n e d showing t h a t small vesicles were f o r m e d also in the presence of Ca 2 + or polylysine. M o n o l a y e r vesicles: p r e p a r e d as described in refs 17 and 18. Ca 2+ was added a f t e r the p r e p a r a t i o n . ESR measurements ESR spectra were t a k e n b e t w e e n 20°C and 65°C using a Varian E-line s p e c t r o m e t e r e q u i p p e d with an AEG-gaussmeter. T h e samples were sealed in capillaries with an inner d i a m e t e r of 1.1 mm. S p e c t r a were r e c o r d e d at increasing and decreasing t e m p e r a t u r e s . While r e c o r d i n g the spectra, the temperature was m o n i t o r e d to an a c c u r a c y o f 0.2°C. S p e c t r a were r e c o r d e d b y varying the m o l a r ratio, r, o f lecithin-top h o s p h a t i d i c acid b e t w e e n r = 0 and r = 0.25. T h e m o l a r ratio o f Ca2+-to p h o s p h a t i d i c acid was varied b e t w e e n R = 0 and R = 1. C o m p u t e r simulation o f E S R - s p e c t r a An a p p r o x i m a t e p r o c e d u r e for the c o m p u t e r simulation of n i t r o x i d e radical spectra m o d i f i e d b y radical i n t e r a c t i o n is described in ref. 18. This p r o c e d u r e is based on the solution o f t h e Bloch e q u a t i o n s c o n t a i n i n g additional t e r m s t h a t a c c o u n t for the spin exchange. Such a simplified m e t h o d can be applied o n l y in those cases w h e r e the anisotropies of the h y p e r f i n e interaction and the e l e c t r o n i c g-factor are largely averaged o u t b y the rapid r o t a t i o n a l m o t i o n o f the spin label in the m e m b r a n e . Such quasi-isotropic spectra m a y be simulated t o a good a p p r o x i m a t i o n b y the s u p e r p o s i t i o n of t h r e e L o r e n t z i a n lines V+I (H, A H °, ), Vo (H, A H ° ) ; V_, (H, A H °, ), c e n t e r e d at the magnetic field positions Ho - aH; Ho ; Ho + a H :

V(H) : V_, {H, AH°_,)+ Vo(H, A H °) + V+, (H, A H ° , )

513

a H is the isotropic hyperfine splitting constant which depends somewhat on the polarity of the solvent. For our computer simulation procedure we used a value of a H = 15 G. Fig. 1 exhibits the first derivative ESR spectrum of the lecithin label (I) in vesicles of phosphatidic acid at pH 9 and at a temperature (T = 59°C) above the phase transition of the lipid lamellae. The transition temperature of phosphatidic acid at pH 9 is T t = 47.5°C [7]. The line broadening at high label concentration (cf. Fig. l b ) is the result of two interaction mechanisms: the spin exchange interaction and the magnetic dipole-dipole interaction. Consequently the spectra are characterized by two independent interaction parameters, the exchange frequency Wex (in MHz) and the dipolar broadening AH d (in Gauss). The dipolar interaction may be taken into account by replacing the line widths/XH~ by AHj=AH~ + A H d In order to analyze the spectra in terms of both the exchange and the dipolar interaction, the modified Bloch equation has to be solved. Three cases had to be considered in the present work: (1) Weak radical interaction. The three ESR lines are well resolved and slightly broadened. The exchange frequency may be determined from the linewidth of the central line according to AH0 = AH0° + AH d + A H e x . The two interaction mechanisms affect the linewidth in the same way. (2) Medium interaction. The three ESR lines start to merge. An example is given in Fig. lb. The two interaction mechanisms affect the spectra in a different way. The exchange interaction tends to shift the two outer lines towards the center of the spectrum. The dipolar broadening shifts the side bands Lecithin - Label in Dipalmitoyl Phosphatidic Acid

n~

~_

10Mol%Label

F i g . 1. ( a ) F i r s t derivative E S R s p e c t r u m o f the l e c i t h i n label (I) in vesicles o f d i p a l m i t o y l p h o s p h a t i d i c acid at 5 9 ° C w i t h negligible i n t e r a c t i o n . Molar ratio l a b e l - t o - l i p i d r = 0 . 0 3 5 c o r r e s p o n d i n g t o 3 . 4 t o o l % label. - - , m e a s u r e d s p e c t r a ; . . . . , c a l c u l a t e d s p e c t r a w i t h A H ~ . 1 = 1 . 5 ; A H ~ = 1 . 0 a n d A H ° I = 2 . 5 G. ( b ) S p e c t r u m at high label c o n c e n t r a t i o n . B e s t fit b e t w e e n c a l c u l a t e d ( . . . . ) a n d e x p e r i m e n t a l s p e c t r u m ( - - ) w a s o b t a i n e d for a dipolar b r o a d e n i n g ~ / d = 0 . 7 G a n d a n e x c h a n g e f r e q u e n c y W e x = 6 . 5 M H z .

514 away from the center. Accordingly the two interaction parameters Wex and AH d may be determined by a trial and error m e t h o d as described in ref. 18. The spectra calculation was p e r f o r m e d with a PDP-SI c o m p u t e r equipped with a T e k t r o n i x display (model 4002 A). The latter allowed a rapid comparison of calculated and observed spectra. (3) Strong interaction. The three hype r fi ne lines have fused to a single line of Lorentzian shape. In the limiting cases of weak and strong exchange interaction, AH d and Wex may n o t be determined independently. In these cases the values of AH d have been estimated by extrapolating the values obtained for the cases of medium radical interaction. The extrapolation was based on the assumption that AH d is proportional to the radical concentration. Some values of AH d used in the present work are summarized in Table I. Experimental results

Binding of divalent ions to vesicles of phosphatidic acid Organic phosphoric acids such as phosphatidic acids may form strong complexes with divalent ions. It is therefore expected t hat such ions bind strongly to vesicles of phosphatidic acid. For paramagnetic ions the complex f o rmatio n may be directly observed via the broadening of the ESR spectra. Consider the case of Mn2+: The ESR spectrum of this ion in aqueous solutions consists of six sharp lines. Upon binding to phosphatidic acid t hat is either in solution or incorporated into lipid lamellae this hyperfine structure is completely smeared out and a broad one line spectrum is observed. It is thus possible to distinguish between free ions (in solution) and b o u n d ions. As an example the ESR spectrum of a phosphatidic acid vesicle preparation containing Mn 2+ is shown in Fig. 2a. The molar ratio, R, of Mn2+-to-lipid is R = 1.0. Obviously the spectrum consists of a superposition of a sharp sextet spectrum and a broad one line spectrum. At pH 9 the sharp sextet spectrum just starts to appear at an equimolar ratio of Mn 2+ and phosphatidic acid. By plotting the intensity I of the sharp spectrum as a function of the molar ratio, R, of Mn 2+to-lipid, the limiting value of R, at which the m em brane is saturated with ions, may be determined. Using this procedure the m axi m um binding of Mn 2÷ to phosphatidic acid vesicles was measured as a function of pH. The result is given in Fig. 2b. TABLE I V A L U E S O F D I P O L A R B R O A D E N I N G A H d I N G A U S S O F L E C I T H I N L A B E L (I) I N V E S I C L E S O F D I P A L M I T O Y L P H O S P H A T I D I C A C I D F O R 7' = 5 9 ° C ; A S A F U N C T I O N O F T H E M O L A R FRACTION OF LABEL r is t h e m o l a r r a t i o o f l e c i t h i n - t o - p h o s p h a t i d i c a c i d . (a) v a l u e s o b t a i n e d b y s i m u l a t i o n o f t h e s p e c t r a a t medium radical interaction, (b) values extrapolated. m

Molar fraction

0.01

0.034

0.05

0.1

0.12

0.16

0.20

0.0

0.0

0.2 b

0.7 a

1.oa

1.3 a

1.5a

0.0

0.7 b

1.1 a

3.5a

4.3 a

5.0b

5.5 b

r/(l+r)

AHd values

Absence of Ca 2+ Molar fraction o f C a 2+ R = 0.5

515

a}

pH9 0~60°C

b}

~o

/"

. . . .

ID

5. q

0.8

8 ®t

¢~

0.6

G .~ R_P~QH ~ J3 "5 0.4o_

R-PO4H2~- R-PO4H°+H~/~

/

± Temperature 60 C

rr O.2-

1

2

3

4

5

6

-/

8

9

10

1'1

1'2

pH Fig. 2. (a) E S R s p e c t r u m of Mn 2+ in an a q u e o u s d i s p e r s i o n of p h o s p h a t i d i c acid vesicles a t p H 9. T h e m o l a r r a t i o of M n 2 + - t o - p h o s p h a t i d i c acid was R = 1.0. T h e s h a r p 6-line s p e c t r u m is d u e t o free ions in t h e b u l k s o l u t i o n . T h e h e i g h t I of t h e line n e a r t h e c e n t r e of t h e s p e c t r u m is a m e a s u r e of t h e c o n c e n t r a t i o n of free Mn 2+. (b) p H d e p e n d e n c e of the b i n d i n g of Mn 2+ t o the s u r f a c e of p h o s p h a t i d i c acid vesicles at 6 0 ° C . T h e o r d i n a t e gives the m o l a r ratio, R *, of b o u n d Mn:+-to-lipid. A v a l u e of 1 c o r r e s p o n d s to a o n e - t o - o n e b i n d i n g of Mn 2+ to t h e lipid. T h e b i n d i n g c u r v e d o e s n o t c h a n g e a p p r e c i a b l y w i t h t e m p e r a t u r e s b e t w e e n 2 0 ° C and 6 0 ° C .

The complex formation of Mn 2 ÷ with the lipid increases sharply at pH 4 and at pH 8. Obviously the first and the second hydroxyl bond of the phosphate group dissociate at these pH values (cf. Fig. 2b). The corresponding pK values of the free phosphatidic acid are pK ~ 3.0 and pK ~ 8.0. N o w it is well known that the pK values of fatty acids and phospholipids may change by 1 - 2 pH units if these molecules are associated in a monolayer (cf. ref. 19). Fig. 2b shows that two singly charged phosphatidic acids (between pH 5 and pH 7.5) are needed to bind one Mn 2+ ion whereas one doubly charged lipid

516

forms a one-to-one complex with Mn 2+. It is reasonable to assume that the same is valid for Ca 2+. Very strong evidence for a one-to-one binding of Ca 2+ to phosphatidic acid has been obtained in our previous optical study [7].

Vesicles of pure lecithin label The ESR-spectrum of vesicles from pure lecithin label (I) consists of a single exchange narrowed line. This line exhibits Lorentzian shape at all temperatures. A sharp decrease in the line width at T t = 44°C indicates a crystalline-to-liquid crystalline phase transition of the labelled lecithin at this temperature. This value compares well with the transition t e m p e r a t u r e of dipalmitoyl lecithin vesicles (Tt = 41 °C). Effect of Ca 2+ on the radical interaction in mixed lecithin-phosphatidic acid vesicles Typical ESR spectra of mixed vesicles composed of lecithin label (I) and dipalmitoyl phosphatidic acid are given in Fig. 3. The spectra were taken at 59°C, that is at a t em pe r at ur e well above the lipid phase transition of dipalmitoyl phosphatidic acid at pH 9. Spectra are presented as a funct i on of the lecithin content. Moreover, Fig. 3 shows how addition of increasing amounts of Ca 2÷ affects the line shape. It is found that addition of Ca 2÷ has a dramatic effect on the line shape. Consider the spectra at 10 % lecithin. Upon addition of increasing amounts of Ca 2+ the partially resolved triplet spectrum is transformed to an unstructured one line spectrum which exhibits to a good a p p ro x imatio n Lorentzian shape. Obviously the binding of Ca ~÷ to the membrane leads to a strong increase in the radical interaction. Fig. 4 shows the effect of temperature. Spectra are presented at a t e m p er atu r e below (31°C) and at two temperatures above (51°C and 59°C) the phase transition of phosphatidic acid (Tt = 47.5°C at pH 9). At molar ratios of Ca2+-to-phosphatidic acid of R = 0.50 and 1.0 Lorentzian lines are obtained at T > Tt. Their line widths decrease with increasing temperatures. Analysis of the spectra show that the exchange interaction increases with increasing temperature. By c o m p u t e r simulation of the spectra presented in Fig. 3 values of the spin exchange frequency Wex have been determined bot h as a function of lecithin c o n c e n t r a t i o n and of the Ca 2+ concentration. The results are summarized in Fig. 5. In Fig. 5a, Wex is plotted as a funct i on of the molar fraction r/(l+r), of labelled lecithin. In Fig. 5b Wex is plotted as a function ofx/(l+r)/r. Effects of pH changes Fig. 6 shows how changes in the pH of the aqueous phase affect the ESR spectra of mixed vesicles containing 16 mol % lecithin. It is seen t hat a decrease in the pH value leads to a significant decrease in the radical interaction. This effect is most p r o n o u n c e d in the presence of Ca z +. Fig. 7 summarizes the effect of pH changes on the spin exchange f r equency Wex. Between pH 7 and pH 5, where the phosphatidic acid molecules carry a single charge, Ca 2÷ addition has a much smaller effect on the label interaction than at high pH values. At pH 3, where part of the phosphatidic acid is uncharged, the influence of divalent ions on Wex is very small. Measurements could n o t be p e r f o r m e d at pH < 3 since the vesicles coagulated.

517

Oipolmitoy[ phosphatidic acid + Lecithin-Label

a

~

~o,~~oy,,oo ,o,

b

molor fraction of Ca~ 020

3.4%

20%

D i p a l m i | o y l phosphotidic acid +Lecithin-Label

mo,o%,~.~.,i°° 0.33

1%

34% 3.4%

5%

10%

12%

16%

20%

Fig. 3. First d e r i v a t i v e E S R s p e c t r a of l e c i t h i n label (I) in p h o s p h a t i d i c acid vesicles t a k e n at T = 5 9 ° C a n d p H 9. T h e n u m b e r s at t h e r i g h t side o f t h e s p e c t r a give the m o l a r p e r c e n t a g e o f l a b e l l e d l e c i t h i n w i t h r e s p e c t to t h e t o t a l lipid c o n t e n t . (a) s p e c t r a t a k e n in t h e a b s e n c e of Ca2+: (b), (c) a n d (d) s p e c t r a t a k e n in t h e p r e s e n c e o f d i f f e r e n t a m o u n t s o f Ca 2+. T h e m o l a r f r a c t i o n s of Ca 2+ w i t h r e s p e c t t o p h o s p h a t i d i c acid are in (b) 0.2; in (c) 0 . 3 3 a n d in (d) 0.5. N o t e : T h e values of t h e m o l a r f r a c t i o n are given b y R / ( I + R ) w h e r e R is t h e m o l a r r a t i o of Ca2+-to-phosphatidic acid.

518

D,palrllqtoyl PhosphatidicAcid+ 10mol 7vLecithinLabel

I ,

R=0.25

I~1, LI +

L"

L~' U

R=0.5

R=1.0

=,50

/

Fig. 4. E f f e c t o f t e m p e r a t u r e o n t h e E S R l i n e s h a p e o f m i x e d l e c i t h i n - p h o s p h a t i d i c a c i d v e s i c l e s in t h e a b s e n c e a n d i n t h e p r e s e n c e o f d i f f e r e n t a m o u n t s o f C a 2+. T h e s p e c t r a w e r e t a k e n a t p H 9. A t t h i s p H t h e t r a n s i t i o n t e m p e r a t u r e o f d i p a l m i t o y l p h o s p h a t i d i c a c i d m e m b r a n e s is T t = 4 7 . 5 ° C [ 7 ] . T h e l i n e w i d t h s o f t h e L o r c n t z i a n l i n e s a t m o l a r r a t i o s o f C a 2 + - t o - p h o s p h a t i d i c a c i d ( R = 0 . 5 a n d R = 1) d e c r e a s e with increasing temperature.

I; a

molar fraction

][

x

~) W~.;

/

~

molar fraction of Ca *+ A 033 • 0.20 x 000

w~

0:05

o'.~o

Molar Fraction

o'.~5 of L e c i t h i n

o12o r

1.r F i g . 5. C o n c e n t r a t i o n d e p e n d e n c e o f t h e e x c h a n g e f r e + q u e n c y , W e x a t p H 9 a n d a t 5 9 ° C , b o t h i n t h e a b s e n c e a n d i n t h e p r e s e n c e o f d i f f e r e n t a m o u n t s o f C a 2 . ( a ) W e x is p l o t t e d as a f u n c t i o n o f t h e m o l a r f r a c t i o n ( r / l + r ) o f l e c i t h i n , r is t h e m o l a r r a t i o o f l e c i t h i n - t o - p h o s p h a t i d i c acid. (b) Plot of Wex versus S t r a i g h t l i n e s a r e o b t a i n e d b o t h i n t h e a b s e n c e a n d i n t h e p r e s e n c e o f C a 2+ u p t o l e c i t h i n c o n c e n t r a t i o n s o f 2 0 t o o l %. A t l a r g e r l e c i t h i n c o n c e n t r a t i o n s t h e c u r v e s a r e e x p e c t e d ~o e x h i b i t a n u p w a r d d e f l e c t i o n i n d i c a t e d b y t h e c u r v e (-.-). A t a C a s+ m o l a r f r a c t i o n o f R -~ 0 . 5 i t is W e x ~ 2 7 M H z . 1+17

519

~

84Mo1%Dipalmltoyl Phosphatidicacid 16Mo1%Lecdhin-Lobei

rnolofr~ooction

, 20Gauss ,

~ / / / ~

Fig. 6. E f f e c t o f p H c h a n g e s on t h e E S R s p e c t r a o f vesicles c o n t a i n i n g 16 m o l % l e c i t h i n label. T h e n u m b e r s g i v e n at t h e r i g h t side of e a c h s p e c t r u m are t h e m o l a r f r a c t i o n , R/(I+R), o f Ca 2+ w i t h r e s p e c t t o p h o s p h a t i d i c acid.

Experiments with monolayer membranes For comparison, some experiments were also performed with vesicles formed by lipid monolayers. The interior of the vesicles contained a chloroform dibutylether mixture [17, 18]. A spectrum analysis in terms of the exchange frequency was only performed in the absence of Ca 2 +. The results are given in Fig. 8. The exchange frequencies observed in the lipid monolayers agree well with the values of Wex characterizing the corresponding bilayer system. Comparison with uncharged vesicles One aim of the present work was to compare the molecular organization of lipid lamellae composed of a mixture of charged and uncharged lipids with lipid bilayers containing only uncharged lipid mixtures. For this purpose we studied mixed vesicles of (1) lecithin label (I) in dipalmitoyl phosphatidylcholine and of (2) lecithin label in dimyristoyl phosphatidylethanolamine. Values of the exchange frequencies Wex were again determined as a function of the label concentration up to label-to-lipid molar ratios of r = 0.2 5. The re-

520

"1-

:2

molar fraction

N 1~E

o 0.50 0,33

x

0.20 20.

2.

. t., . 6.

.8

~ - ~ ---~- pH g.o pH ;" ~,. -~'''~

15.

10.

0'1

o'2

0:3

0'5

0'~

motor froction of Co÷+

Fig. 7. P l o t o f Wex as a f u n c t i o n o f m o l a x f r a c t i o n o f C a :+, R / ( I + R ) , (as m e a s u r e d w i t h r e s p e c t t o p h o s p h a t i d i c acid), f o r d i f f e r e n t p H . T h e e f f e c t o f Ca 2+ o n t h e e x c h a n g e f r e q u e n c y is g~reatly r e d u c e d a t l o w e r p H . T h i s is m o s t c l e a r l y s e e n in t h e i n s e r t o f t h e figure. B r o k e n c u r v e : v a l u e s o f Wex c a l c u l a t e d with equation A2 of the Appendix.

5 /Z x~ il l / l

o.b5

o11

°'/~I i

I

o.15

0.2 r

1.r Fig. S. C o m p a r i s o n

of exchange frequencies, Wex, of labelled lecithin (I) in membranes contaJzung

charged (dJpalzmtoy] phosphaticLic acid) ( ~ ) and uncharged ( d i p a l m i t o y l lecithin) (o) and ~ m y H s t o y l phosphatidyletbanolaznine (e) ]ipids. i values o f Wex obtained f o r phosphatidJc acid m o n o l a y e r vesicles, r is the molar ratio o f labelled lecithin-to-unlabel]ed lipid. Insert: Normalized free energy G/k T o f a b i n a r y m i x e d m o n o l a y e r c o n t a i n i n g b o t h u n c h a r g e d l i p i d a n d l i p i d c a r r y i n g o n e n e t c h a r g e . m o l a r f r a c t i o n o f c h a r g e d lipid. D e f i n i t i o n o f K cf. E q n 8.

sult is summarized in Fig. 8, together with the values of Wex obtained for the lecithin-phosphatidic acid system. A pronounced difference in the radical interaction in charged and uncharged lipid lamellae is observed. Obviously Wex is strictly proportional to the molar fraction (r/l+r) of labelled lecithin in uncharged lipid mixtures. In charged lipid lamellae, a much stronger radical inter-

521

action is observed. In addition W e x is no longer proportional to r/l+r. As will be shown below, this finding can be explained by assuming a heterogeneous distribution of the charged and uncharged lipids within the membrane.

Effects of polylysine Fig. 9 shows the effect of polylysine on the lecithin interaction in phosphatidic acid vesicles. At pH 9 the lysine groups are positively charged and the polylysine exhibits a random coil configuration. Obviously addition of polylysine induces a significant increase in the radical interaction. The temperature dependence of the ESR spectra at pH 9 and at low label concentration (r ~< 0.01) exhibits two well separated lipid phase transitions: a transition at 47.5°C is characteristic for phosphatidic acid membranes at pH 9. The second transition at 62°C is caused by regions of lipid layers to which p o l y lysine is adsorbed. This effect of polylysine has been described in more detail in our optical study with excimer probes [7]. Dipalmitoyl Phosphatidic Acid ÷ 10 mol

% Lecithin Label Addition of Poly Lys~ne

J

/

', '\

jj~

/

/

~

31 °

5g ° J

Fig. 9. E f f e c t o f p o l y l y s i n e o n E S R s p e c t r a o f m i x e d l e c i t h i n - p h o s p h a t i d i c acid m e m b r a n e s a t p H 9. S p e c t r a a t l e f t side', w i t h o u t p o l y p e p t i d e . S p e c t r a a t r i g h t side: after a d d i t i o n o f p o l y l y s i n e . N u m b e r o f p o s i t i v e l y s i n e g r o u p s e q u a l t o n u m b e r of p h o s p h a t i d i c a c i d m o l e c u l e s . A t 5 9 ° C , Wex i n c r e a s e s f r o m Wex = 6 . 7 M H z t o Wex = 9 . 5 M H z u p o n a d d i t i o n o f p o l y l y s i n e .

Discussion According to ref. 9, the radical interaction in lipid lamellae containing a labelled lipid as one constituent may be determined by two factors: (1) By the rate of lateral diffusion D of the labelled lipid molecules (2) By the distribution of the lipids within the membrane, a factor which determines the average radical distance. These two mechanisms lead to completely different concentration dependencies of the exchange frequency Wex. Provided the radical interaction is determined by the lipid lateral diffusion, Wex is proportional to the molar fraction (r/l+r) of labelled lipid: Wex

4 dcD r -3 XF l + r

= -

(5)

In this equation, dc, X and F are the critical radical interaction distance, the length of one diffusional jump and the area per lipid molecule, respectively.

522 D is the coefficient of the label lateral diffusion. This model is based on the assumption of a random lipid distribution within the lipid bilayers. If Wex is determined by the lipid distribution, that is by the formation of clusters of labelled lipid, one expects a functional dependence according to

Wex = ~/ex(J-

/~+r

where n is the n u m b e r of clusters per cm: and where Wex is the m a x i m u m exchange interaction in the interior of the cluster. 1/1+r By plotting Wex both as a function of r/l+r and as a function of \/ ,

r

respectively, one should be able to distinguish between the above two cases.

Lipid segregation in the absence o f Ca 2+ In Fig. 8, the exchange frequencies of labelled lecithin (I) in dipalmitoyl phosphatidylcholine, in dimyristoyl phosphatidylethanolamine and in dipalmitoyl phosphatidic acid are compared at a temperature (T = 59°C) well above the respective transition temperatures. Wex is plotted as a function of the label molar fraction (r/l+r). Straight lines are obtained for the ethanolamine and the lecithin membranes, clearly demonstrating that the labelled lecithin is r an d o m l y distributed and is rapidly diffusing in these lipid lamellae. From the slopes of the straight lines the lateral diffusion coefficient D may be determined according to Eqn 5. Using values of d c ~ 8 2[ and X ~ 8 A one obtains D = 2.8-10 -8 cm2/s for both the ethanolamine and the lecithin membranes at 59°C. This value of D agrees well with the diffusion coefficient for androstane in lecithin membranes (D = 3 . 1 0 .8 cm 2/s at 50°C). It is smaller by a factor of ab o u t 3 than the value of D found in the optical experiments [7] using Pyrene decanoic-acid labels. A completely different behaviour is observed for mixed lecithinphosphatidic acid vesicles. For this system Wex is not proportional to r/l+r even in the liquid crystalline state of the m e mbrane (T > Tt). Accordingly the two phospholipids segregate in these mixed systems above T t. T w o types of lipid phase separation should be considered: (1) A heterogeneity in the lipid distribution within each m o n o l a y e r of the membranes (lateral phase separation) (2) A difference in the lipid composition between the inner and the outer m o n o l a y e r of the vesicles (transversal phase separation cf. refs 20 and 21). In order to distinguish between these two possibilities we p e r f o r m e d the experiments with mixed lecithin-phosphatidic acid m o n o l a y e r vesicles. The concentration dependence of Wex for this system is also pl ot t ed in Fig. 8. Good agreement between the values of the exchange frequency in the bilayer and in the m o n o l a y e r membranes is observed. This finding provides strong evidence for the occurrence of lateral phase separation in mixed lecithinphosphatidic acid membranes. The question arises w he t he r phase separation in fluid mixed membranes containing a c o m p o n e n t with a net electric charge is physically reasonable. Considering only the repulsion forces between the charged lipids one would

523 r a t h e r e x p e c t a r a n d o m i z a t i o n in the m i x e d system. H o w e v e r , at the surface of m e m b r a n e s c o m p o s e d o f charged lipids an electric d o u b l e layer f o r m s b y the a b s o r p t i o n of c o u n t e r ions. It has b e e n e m p h a s i z e d b y O v e r b e e k [22] t h a t a very characteristic f e a t u r e of diffuse electric d o u b l e layers o f the G o u y C h a p m a n t y p e is the negative sign of its Gibbs free energy. At high surface p o t e n t i a l the free e n e r g y is p r o p o r t i o n a l to the surface charge: Gdl = - 2 F . k T

(7)

w h e r e 1" d e n o t e s the n u m b e r of charges per cm 2 . Consider a m i x e d m o n o l a y e r c o m p o s e d of charged and u n c h a r g e d lipids c o n t a i n i n g a m o l a r f r a c t i o n 7 o f charged lipids. T a k i n g into a c c o u n t the mixing e n t r o p y , the total free energy of such a m i x e d lipid layer is given b y (cf ref. 23, p. 237): G = R F(71n 3' + (1 - 3')ln(1 - 3') - K 3 ' )

(8)

w h e r e K3' = 2F is positive. In the insert o f Fig. 8, G / k T is p l o t t e d as a f u n c t i o n of the fraction, 7, of charged lipid for K = 0 and K = 1. These diagrams p r e d i c t a c o m p l e t e r a n d o m i z a t i o n o f the lipid at K = 0 ( m i n i m u m of G at 7 = 1/2). F o r K > 0 the m i n i m u m free e n e r g y is decreased c o n s i d e r a b l y and in a d d i t i o n is shifted t o higher fractions o f charged lipids ( m i n i m u m of G at 7 > 1/2). In view o f this result, lateral phase separation in m e m b r a n e s c o m p o s e d of charged and u n c h a r g e d lipids seems physically reasonable. Assume a value o f F = 60 A 2 for the area o c c u p i e d b y one p h o s p h a t i d i c acid m o l e c u l e . This w o u l d c o r r e s p o n d to a lipid d e n s i t y o f 2 . 8 . 1 0 - 1 ° m o l / c m 2 . A c c o r d i n g to E q n 7 the free e n e r g y of the electric d o u b l e layer o f a m e m b r a n e c o m p o s e d o f single charged p h o s p h a t i d i c acid m o l e c u l e s w o u l d thus be Gdl --1.8 R. F o r this value o f Gdl t h e free e n e r g y has a m i n i m u m for 7 ~ 0.8. A c c o r d i n g l y , the f o r m a t i o n o f regions enriched and d e p l e t e d in the charged lipid w o u l d lead to a l o w e r free e n e r g y of the m i x e d m e m b r a n e t h a n a r a n d o m mixture.

Enhancement of phase separation by Ca 2+ Fig. 5 shows the c o n c e n t r a t i o n d e p e n d e n c e o f Wex for d i f f e r e n t m o l a r fractions, R / I + R , o f Ca 2 + as m e a s u r e d with r e s p e c t t o the p h o s p h a t i d i c acid c o n t e n t . In Fig. 5a, Wex is p l o t t e d as a f u n c t i o n o f r/l+r. N o straight lines are o b t a i n e d b e t w e e n R = 0 and R = 1. O b v i o u s l y the radical i n t e r a c t i o n is n o t a diffusion c o n t r o l l e d process. In Fig. 5b the e x c h a n g e f r e q u e n t l y is p l o t t e d as a function of 1 ~ . Straight lines are o b t a i n e d b o t h in the p r e s e n c e and in the absence o f Ca 2 +. T h e i r slopes increase with increasing Ca 2 + c o n c e n t r a t i o n . This finding d e m o n s t r a t e s t h a t a d d i t i o n o f Ca 2 + strongly e n h a n c e s the lipid segregation in m i x e d l e c i t h i n - p h o s p h a t i d i c acid m e m b r a n e s . F r o m the slopes o f the straight lines, values o f the density, n, o f lecithin clusters m a y be e s t i m a t e d a c c o r d i n g t o E q n 6. T h e result, s u m m a r i z e d in T a b l e II, shows t h a t the cluster density, n, does n o t d e p e n d on the Ca 2 + c o n c e n t r a t i o n . H o w e v e r , since Wex increases with increasing Ca 2 + c o n c e n t r a t i o n , the size o f the lecithin clusters m u s t increase u p o n a d d i t i o n o f Ca 2 +. Such a result is e x p e c t e d f r o m the findings r e p o r t e d in o u r optical s t u d y [ 7]. T h e r e it was s h o w n t h a t the transition t e m p e r a t u r e o f Ca 2 +-bound

524

T A B L E II C H A R A O T E R I S T I C P A R A M E T E R S O F L E C I T H I N P R E C I P I T A T I O N S IN M I X E D L E C I T H I N PHOSPHATIDIC AOID MEMBRANES r a n d R m o l a r ratios of l e c i t h i n - t o - p h o s p h a t i d i c acid a n d of C a 2 + - t o - p h o s p h a t i d i c acid, r e s p e c t i v e l y . rc: v a l u e of r in t h e l e c i t h i n c o n t a i n i n g clusters of t y p e 1 (r~) or t y p e 2 (r~). n: d e n s i t y of clusters ( p e r c m 2), N: n u m b e r of m o l e c u l e s p e r cluster, ~2 r a d i u s of cluster. Molar f r a c t i o n of Ca ~+ R / ( I + R )

0.0

0.2

0.33

0.5

n \•clusters - - - ~ m ~ /~

3.4 × 10 al

3.3 x 1011

3.4 × 1011

3.2 x 10 It

Values ( W e x ( M H z ) for ~ ~2 ( A ) r = 0.25 (r c

9 ~t I = 8 0 A r~ ~ 0.5

----

----

19 ~ =45A r~--*

phosphatidic acid is shifted to very high temperatures. Consequently Ca 2+b o u n d phosphatidic acid forms rigid clusters which are e m b e d d e d in liquid crystalline lipid regions composed of non- bou nd phosphatidic acid and lecithin. As shown above, lecithin-phosphatidic acid membranes m ost probably exhibit a mosaic-like structure of lecithin enriched and lecithin depleted regions. It is therefore reasonable to assume that Ca z + attaches mainly to the lecithin depleted regions leading there to the form at i on of rigid phosphatidic acid clusters. Lecithin would be squeezed out from these crystalline regions. Judged from these considerations, the cluster density, n, is expected to remain unchanged u p o n addition of Ca 2+. Clearly the m a x i m u m exchange rate, Wex, defined in Eqn 6 depends on the molar fraction, r c / ( l + r c), of lecithin within the clusters. Depending on the Ca 2 + concentration, two limiting cases have to be considered: (1) R = 1: Nearly all phosphatidic acid is bound by Ca 2+ and is in a rigid state at pH 9. Clusters of fluid lecithin are formed which are e m b e d d e d in the rigid matrix of the Ca 2+-bound lipid. Since r c / ( l + r c ) ~ 1, Wex should be equal ~0 to the value W ex = 30 MHz characteristic for membranes of pure lecithin label. The value of Wex = 27 MHz obtained from Fig. 9 is in rather good agreement with this expectation*. For circular two-dimensional clusters the radius gt2, may be det erm i ned as a function of the lecithin c onc e nt r at i on according to (cf. ref. 9, Eqn 28a): -

~22

-

~

-

+r

(9)

As an example the value of gZ2 for r = 0.25 is given in Table II. (2) R = 0 (absence of Ca2+): T w o types of lecithin clusters have to be considered: At low label c onc e nt r at i on a first t y p e (1) of cluster containing both lecithin and phosphatidic acid will be formed. Its composition is determined by the minimum of the free energy, G m . From our above estimation of G m the clusters are expected to contain less than 50 mol % lecithin, that is 1 1 r e/(1+% ) < 0.5. These clusters would grow in size with increasing lecithin concentration, r/(l+r), up t o a limiting value: * T h e small d i s c r e p a n c y o f a ~ o u t 10 % is m o s t p r o b a b l y d u e to the fact that according to Fig. 2 the c o m p l e x f o r m a t i o n of Ca 2 w i t h p h o s p h a t i d i c acid is n o t a p e r f e c t 1 : 1 b i n d i n g at p H 9.

525 rm

1 rc

l+r m

l+r~

Up t o this c o n c e n t r a t i o n Wex is a linear f u n c t i o n of x f ( l + r ) / r as f o u n d in Fig. 5b. A t higher lecithin c o n c e n t r a t i o n s , r > rm, a second t y p e (2) o f cluster c o n t a i n i n g a p p r o x . 100 % lecithin (r2c/r2c+l ~ 1) starts to f o r m . A c c o r d i n g l y one e x p e c t s t h a t at r > > rm, Wex should increase rapidly with increasing ~0 This b e h a v i o u r lecithin c o n c e n t r a t i o n , since at r -+ ~ one e x p e c t s Wex = Wex. has been indicated in Fig. 5b b y the b r o k e n curve (- - - ). F o r circular clusters of t y p e (1), the radius ~1 is given b y 1 ~21

-

r

/1

~

+r

l+r~ 1

rc

"

(10)

This e q u a t i o n follows d i r e c t l y f r o m E q n 27 in ref. 9: T h e value of ~21 for the lecithin c o n c e n t r a t i o n r = 0.25 is given in Table II. T h e rate of spin e x c h a n g e Wex w i t h i n the t w o t y p e s of lecithin clusters is diffusion c o n t r o l l e d (at T = 59°C) and one thus e x p e c t s Wex

=

$0 rc ex l + r c

(11)

F r o m this e q u a t i o n , the m o l a r f r a c t i o n o f lecithin in the clusters of t y p e (1) m a y be estimated. Consider the case R = 0: Wex is d e t e r m i n e d b y t h e value o f the e x c h a n g e f r e q u e n c y at the lecithin c o n c e n t r a t i o n , rm, w h e r e the s e c o n d t y p e o f cluster starts to form. In principle, r m can be d e t e r m i n e d f r o m t h a t p o i n t in the p l o t o f Wex versus x / ( l + r ) / r w h e r e the straight line exhibits an upward d e f l e c t i o n (curve -.- in Fig. 5b). An u p p e r limit o f Wex is given b y the i n t e r c e p t o f the straight line with the o r d i n a t e (Wex = 12.5 MHz). A lower limit o f Wex is t h e e x c h a n g e f r e q u e n c y at r / l + r = 0.2 (I~ex = 9 MHz). T h e lecithin c o n c e n t r a t i o n in the first t y p e o f cluster thus has a value b e t w e e n l 1 1 l r c / l +r c = 0.3 and r c / l +r c = 0.4 (cf. E q n 11). Intermediate

c a s e ( 0 < R < 1)

T h e a b o v e discussion of the e x p e r i m e n t s at the limiting cases R = 0 and R = I and at 59°C lead t o the following conclusion: A t lecithin c o n c e n t r a t i o n s r < rm, p a t c h e s (clusters o f t y p e (1)) o f lipid m o n o l a y e r c o n t a i n i n g 3 0 - - 4 0 mol % lecithin (r~c = 0 . 4 3 - - 0 . 6 7 ) are f o r m e d in the fluid p h o s p h a t i d i c acid m e m b r a n e . T h e size o f these clusters increases with increasing lecithin c o n c e n t r a t i o n r (cf. E q n 10) while the cluster density, n, remains c o n s t a n t . A t r > r m (r m = r c1 = 0 . 4 3 - - 0 . 6 7 ) a s e c o n d t y p e (2) of cluster o f p u r e lecithin (r~/l+r2c = 1) starts t o f o r m . A t R = 1 clusters o f t y p e (2) are f o r m e d at all lecithin c o n c e n t r a t i o n s . A d d i t i o n o f Ca 2 + leads to the f o r m a t i o n o f rigid p h o s p h a t i d i c acid regions f r o m w h i c h lecithin is squeezed. A c c o r d i n g l y , the effective m o l a r ratio, r*, o f lecithin-to-free p h o s p h a t i d i c acid increases. A t pH 9, where a 1 : 1 b i n d i n g o f Ca 2 + and p h o s p h a t i d i c acid occurs, one obtains r r ~

=

_

_

1-R

526 Depending u p o n w he t he r r* < r m or r* > rm, addition of Ca: ÷ leads to an it:crease in the size of the clusters of t ype (1) or type (2), respectively. In bot h cases Ca 2+ addition causes an increase in the exchange frequency Wex. In the appendix a model based on the above consideration is presented which allows to calculate Wex as a function of the Ca 2+ concentration, (el. Eqn A2). As an example Wex has been calculated for an initial molar ratio, of lecithin-tophosphatidic acid of r = 0.25 and for a value rm = r~ = 0.33. The calculated values of Wex have been plotted in Fig. 7 as a function of the molar fraction, R/(I+R), o f Ca 2 + (broken curve) good agreement between the calculated and the experimental plots of Wex versus R/(I+R) is obtained for pH 9. As reported by Schreier-Muccillo et al. [23] addition of Ca" ÷ increases the apparent order of films formed from a mixture of lecithin, cholesterol and phosphatidic acid. This finding may also be explained as an effect of phase separation. Upon addition of Ca 2+ rigid patches of phosphatidic acid may form within the multilayers. As a consequence the spin probes would be restricted to the fluid memb r a ne regions f or m e d from lecithin and cholesterol and would reflect the order characteristic for this system.

pH dependence The nearly negligible effect of Ca 2+ on the exchange frequency at pH 3 (cf. Fig. 6) is expected from the finding that the binding of divalent ions to phosphatidic acid is negligibly small at this pH. An explanation for the small effect of Ca 2+ at pH 5 c a n n o t be given yet.

Effect of polylysine As d emo n s tr at ed in Fig. 9, positively charged polylysine has the same elfect on mixed lecithin phosphatidic acid membranes as Ca 2+. Obviously adsorption of polylysine to the mixed lipid bilayer leads to the form at i on of clusters of phosphatidic acid held together by the polypeptide. This t ype of chemically induced phase separation has been studied in more detail in ref. 7. The clusters held together by the polylysine have a chain melting t em perat ure of T t ~ 60°C which corresponds to an effective pH of a b o u t pH 2. A model of the polylysine induced lipid segregation is presented in Fig. 10. A much weaker, however, noticeable effect on the lipid distribution in mixed lecithinphosphatidic acid membranes has been observed u p o n addition of natural proteins such as c y t o c h r o m e . Smith and coworkers [25] observed an increase in the lipid order upon addition of polylysine (or lysine-leucine copolymers) to (1) brain-lipid bilayers and (2) to mixed egg lecithin-phosphatidic acid membranes. Their results (cf. Figs 2 and 3a of ref. 25) may well be interpreted in terms of the distribution of steroid labels in lysine b o u n d rigid patches of charged lipid and in n o n b o u n d fluid regions. These examples show that extrinsic proteins that are supposed to be only loosly co n n ected with the membrane may have a remarkable effect on the ilpid structure. This observation suggests that, in principle, conform at i onal changes in surface proteins may trigger in a cooperative way (structural changes) in the local lipid composition.

527

~ DPA

Polylysine /

,~

~~*~---~

2~.y.

pH 9

Fig. 10. Possible e f f e c t of r a n d o m l y coiled p o l y l y s i n e ( w i t h p o s i t i v e l y c h a r g e d N+H~) on a m i x e d m e m b r a n e of d i p a l m i t o y l p h o s p h a t i d i e acid ( D P A ) a n d d i p a l m i t o y l l e c i t h i n ( D P L ) . H y d r o c a r b o n chains t h a t axe s y m b o l i z e d b y s t r a i g h t lines are in a c r y s t a l l i n e c o n f o r m a t i o n .

K i n e t i c s o f lipid segregation T h e fluid l e c i t h i n - p h o s p h a t i d i c acid lipid lamellae m a y be c o n s i d e r e d as a t w o d i m e n s i o n a l alloy t h a t e x h i b i t s a miscibility gap. T h e rate of f o r m a t i o n of clusters o f o n e lipid c o m p o n e n t b y uphill d i f f u s i o n [26] is d e t e r m i n e d b y the rate o f t h e lipid lateral diffusion, D. T h e t i m e s r n e e d e d f o r the f o r m a t i o n of a circular cluster o f N lipid m o l e c u l e s m a y be e s t i m a t e d a c c o r d i n g to (cf. ref. 24) Eqn 13:

2NF 7T'D

F is t h e area p e r lipid m o l e c u l e (F ~ 60 X: ). With a lipid d i f f u s i o n c o e f f i c i e n t of D ~ 10 -7 c m 2/s one e s t i m a t e s r ~ 4 . 1 0 - 9. A s s u m p t i o n : t w o t y p e s of lecithin p r e c i p i t a t i o n s (clusters) m a y f o r m : a first t y p e o f cluster (1) c o n t a i n i n g a lecithin c o n c e n t r a t i o n r Ic = rm is solely p r e s e n t up to a limit of r = rm. A t r > r m a s e c o n d t y p e (2) of cluster containing 100 % lecithin starts to f o r m . g] ~ and ~22 d e n o t e the radius o f cluster o f t y p e (1) and (2), r e s p e c t i v e l y , ~212 a n d ~ 2 2 are t h e c o r r e s p o n d i n g areas o f the clusters.

528

In o r d e r t o allow for t h e f a c t t h a t t h e cluster t y p e (2) starts to f o r m at r > r m while t y p e (1) ceases growing at this limit, it is possible to i n t r o d u c e effective areas in t h e f o l l o w i n g w a y : 1 rr(~l~')2

= TRY-?.1

a2

1+0¢ 2

2

2 ~ 1+0¢ 2

w h e r e a = r * / r m . O t h e r f u n c t i o n s with a s h a r p e r step at the critical value r = r m w o u l d in principle yield the s a m e result. T h e average e x c h a n g e f r e q u e n c y m a y be c a l c u l a t e d in c o m p l e t e a n a l o g y to t h e m e t h o d d e v e l o p e d in ref. 9 (cf.Eqns 25, 26, 27 and 28a). T h e o n l y d i f f e r e n c e is t h a t in t h e p r e s e n t case t w o t y p e s ~ 0 ex and ~ e x = ~z 0 o f clusters f o r w h i c h Wex = W 1c have t o be c o n s i d e r e d . ex l+r~

T h e clusters are again divided in a core c h a r a c t e r i z e d b y an e x c h a n g e f r e q u e n c y Wex = Wex and a b e l t of w i d t h dc w h e r e Wex = 1/2 Wex. E q n 24 o f ref. 9 is t h e n valid f o r each t y p e o f cluster. By inserting t h e e f f e c t i v e areas n ~ 1*2 a n d ~22 *z, in this e q u a t i o n t h e average e x c h a n g e f r e q u e n c y is t h e n given b y Wex {(~l*)2pm

= a22Wex

+ (~2"2)}

1 + ~2

;;

= Wex~22

- ~ -

f

(a2)

In this e q u a t i o n the f o l l o w i n g a b b r e v i a t i o n s rm Pm-

l+r m

r* ; ~-

rm

r -

rm(1

-

R)

have b e e n used. T h e c o n c e n t r a t i o n d e p e n d e n c e o f FZa is given b y E q n 9. As an e x a m p l e Wex has b e e n calculated as a f u n c t i o n o f R f o r r = 0.25, rm = 0.33 and f o r a value of t h e cluster d e n s i t y n = 3.3.1011 cm-2. T h e curve has b e e n p l o t t e d in Fig. 7 ( b r o k e n line). R e a s o n a b l e a g r e e m e n t b e t w e e n the calculated and the e x p e r i m e n t a l p l o t s of Wex versus R is o b t a i n e d . Acknowledgement We are greatly i n d e b t e d t o Miss U. B o t t i n for careful p e r f o r m a n c e o f the m e a s u r e m e n t s . T h e careful writing of t h e m a n u s c r i p t b y Mrs B. S t r o b e l is gratefully a c k n o w l e d g e d . T h e w o r k was partially s u p p o r t e d b y the D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t u n d e r c o n t r a c t No. 5a 2 4 6 / 1 . References 1 S. R a z i n ( 1 9 7 1 ) B i o c h i m . B i o p h y s . A c t a 2 6 5 , 2 4 1 2 9 6 2 V a n d e r k o o i , G., S e n i o r , A . E . , C a p a l d i , R . A . a n d H a y s h i , H. ( 1 9 7 2 ) B i o c h i m . B i o p h y s . A c t a 2 7 4 , 38--48 3 G r i f f i t h , O.H., J o s t , P., C a p a l d i , R . A . a n d V a n d e r k o o i , G. ( 1 9 7 3 ) A n n . N e w Y o r k A c a d . Sci. 2 2 2 , 561--573 4 Stier, A. a n d S a c k m a n n , E. ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 3 1 1 , 4 0 0 - - 4 0 8 5 K i m e l b e r g , H . K . a n d P a p a h a d j o p o u l o s , D. ( 1 9 7 1 ) J. Biol. C h e m . 2 4 6 , 1 1 4 2 - - 1 1 5 0

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6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

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