CHYOHIOLOCY

15, 222-226 ( 1978)

Influence

of Lipid Phase Transitions and Cholesterol on Protein-Lipid Interactions H. K. KIMELBERG

Dieision of Neurosurgery and Department of Biochemistry, Albany Medical College, Albany, New York 12208

Protein-lipid interactions constitute, in large part, both the structural and functional bases of biological membranes. Current concepts view the lipid component as not only forming an impermeable barrier to many compounds and a matrix within which membrane-bound proteins can be inserted, but also as having an ability to modulate the functioning of membrane proteins ( l-3). In the present work protein-lipid interactions have been studied in model phospholipid membranes, Such membranes are based, as in part are biological membranes themselves, on the physicochemical properties of phospholipids; Principally this involves their amphipathic properties, which result in their forming monolayers at polar : nonpolar interfaces or bilayers in excess aqueous media (4). A convenient type of phosphoIip?id membrane can be formed by adding phospholipids in volatile organic solvents to a clean water surface. The rapid evaporation of the solvent leaves a phosphoIipid monolayer at the air-water interface. The properties of such monolayers were worked out by Langmuir in the early part of this century (5) and a review of their properReceived October 21, 1977; accepted November 23, 1877. Synopsis paper presented at the 14th Annual Meeting of the Society for Cryobiology, August 1977, Minneapolis, Minnesota.

ties can be found in the monograph by Gaines ( 6). Liposomes are a convenient form of phospholipid bilayer membrane. They form spontaneously when dried films of most phospholipids are shaken with excess water. The initial structures consisting of numerous concentric, bilayer shells can be reduced to a limiting 250~A-diameter, single bilayer vesicle by sonication (7). Bangham et al. (8) showed in 1965 that liposomes would entrap labeled ions and release them at a steady rate with some degree of selectivity. This indicated that the phospholipid bilaycrs constituted closed membrane barriers. Such preparations and variations thereof, as well as the planar biIayer or “black lipid” membranes formed across a small hole in a Teflon cup (9), comprise a wide variety of phospholipid monolayer or bilayer preparations availabIe for investigation. These preparations can be made to resemble biological membraues more closely by incorporation of proteins, and currently there is a widespread and increasing emphasis on this approach as a tool to understanding the molecular basis of proteinlipid interactions and their functional consequences in biological membranes. Since an increasing number of purified membrane proteins are now becoming available such studies are beginning to more closely mimic the actual interactions occurring in biological membranes. The studies sum-

222 aOIl-2240/78/0152-0222$02.UO/O Copyright Q 1978 by Academic Press, Inc. Al1 righb of reprcduction in any form reserved.

LIPID

PHASE TFUSITIONS

marized in this presentation were concerned with the interactions of both soluble and membrane-bound proteins and enzymes with phospholipid monolayers and liposomes, and changes in the properties of both protein and lipid membranes were studied. The consequences of varying the “fluidity” ( 10) of the membrane lipids, by altering the fatty acyI ,groups of the phospholipids, changing the temperature, or incorporating cholesterol, are emphasized. The data in Table I show that a peripheral (1) membrane protein, cytochrome c, normally bound predominantly electrostatically to the mitochondrial inner membrane, can markedly increase the Wa+ permeability of pure phosphatidylserine (PS ) liposomes ,and expand a monolayer of the same phospholipid .at constant presTABLE

1

Effects of Cholesterol on the Interaction Cytochrome c with Phospholipid Monolayers Bilnyarsa Effect studied

of and

Composition of phospholipid membrsne Pure bovine brain phmphatidyberinc (PS)

PS/cholestar01 mixture (I/l mole &iO)

Fold increase in znNa+ permeabilil,y of liposomes

780

20

Percentage expansion of area in monolayers at constant surface pressure of 25 dynes/cm

38

9

Amount bound to lipoaomes (ma1 of PS/cytochrome c)

10

8

a Data from Ref. (11). All experiments were in 10 rnM N&l, 0:l rnM EDTA, 2 mu hbtidine, 2 mM TES solution at pH 7.4 and 2.5’ (monolayer) or 37°C (Iiposomes). Liposomes were of the Yonicated, predominanlly unilamellar type. Control permeabihty of liposomes in the absence of cytochrome c was O.OS$$$of total entrapped aNa+ diffusing out per hour. Concentrations of, cytochrome c were 10 mg/ml and 2 pg/m1 for the Iiposome and monolayer experiments, respectively.

AND INTERACTIONS TABLE

223 2

Effects of Temperature and Cholesterol on the Permeability Effects of Cytochrome c on Liposomes Prepared from Phosphatidylgiyoerols with Different Fatty Acyl Chainsa Phosphatidylglyccml

Tempersture (“C)

Fold incream in -Na+ permeability

Dipalmitoylphosphatidylglycerol (DPPC)

30 36

3 17

Doleoylphosphatidylglycerol (IIOPG)

30 36

20 14

DOPG/Cholesterol (l/l mole ratio)

30 36

2 2

(dI>ata from Ref. (11). Liposome “Na+ permeability experiments done under the same conditions as in Table 1 except t.hey were made from tbe phospholipids shown. Fold increase refers to the increase in permeability due to the addition of cytochrome c at. a final concentration of 1 mg/ml.

sure. This and other data (11-13) suggested that these effecb seen at low ionic strength may be due to penetration of a part of the protein into the bilayer interior, subsequent to its initial electrostatic binding. The marked inhibition of these effects when cholesterol is incIuded could then be due to the decreased fluidity of the membrane due to the presence of cholesterol (7), inhibiting this penetration. In support of this it can be seen from Table 1 that there is no marked difference in the binding of the protein to liposomes when cholesterol was incorporated. This effect of cholesterol varied with the protein studied. Thus, under the same conditions as in Table 1 the effects of human serum albumin (50 mg/ml) #and myelin proteclipid (0.36 mg/ml) on the Wa+ permeability of PS liposomes were reduced 60- and 3-fold, respectiveIy, by the incorporation of cholesterol ( 11). The effects of varying membrane fluidity by alterations in temperature on the effects of cytochrome c are shown in Table 2, In

II,

224

K. KPMELBERG

02 40 A -

58 O2 A -

phospholipid

+

cholesterol FIG. 1. Diagram of the .effects of temperature and cholesterol on the 3uidity of the fatty acyl chains in phospholipid membranes. In the upper two figures the effect of an increase in temperature is shown as inducing a change in the fatty acyl chains from a solid, all-truns state with close molecular packing (left hand figure) to a fluid, cis-trans state with increased molecular motion, decreased packing, and consequently an increased area per molecule and decreased membrane width. The effect of choIestero1 is to decrease the fluidity of the fluid membrane to a state intermediate between the completely solid and completely fluid membrane. The head-groups of the phospholipid molecules are represented by open circles, and the fatty acyl chains by solid lines. CholestemI is represented by a small closed circle (hydroxyl), a larger oval area (steroid skeleton), and a wavy line (aliphatic chain),

these experiments synthetic phosphatidylglycerols, which like bovine brain phosphatidylserine have a net negative charge on their polar head groups, were synthesized with different fatty diacyl groups. DPPG has two M-carbon saturated fatty acyl groups and consequently shows a transition from the solid aMruns configuration of the fatty acyl groups to a fluid cS.s-true configuration within a temperature range of 37 to 40°C (14). DOPG has two 18-carbon chains, but each has a single cis C = C double bond and consequently is fluid down to temperatures below 0°C. It can be seen that, in the case of DPPG, a marked cytochrome c-induced 22Na+ permeability increase occurred when the temperature was increased from 30°C to a temperature in the region of the phase ,transition. In

contrast a constant, high, cytochrome c-induced increase in permeability was seen for DOPG in the same temperature range. For DOPG liposomes the incorporation of cholesterol again inhibits the permeability increase. All these effects are consistent with a permeability increase due to partial penetration of the protein into the bilayer. A pictorial representation of the effects of temperature and cholesterol in decreasing the fluidity of phospholipid membranes is shown in Fig. 1. The increased motion and looser packing of the fatty acyl chains in the fluid membrane can be envisaged as allowing a protein to penetrate the bilayer more easily or undergo conformational changes and/ or translational movements required for its activity. In order to see whether similar effects

LIPID TABLE

PHASE TRASITIONS

AND INTERACTIONS

225

3

Effects of Cholesterol on the Reactivation of Lipid-Depleted (Na+ + K-t-) ATPase by Phouphatidylglycerol Liposomes with Differing Fatty Acyl Chainsa Phospholipid

sdded

(Na+ + K+) ATPW activity (Irmol of Pblg of protein/hr)

None DPPG (0.6 pmol added) DPPG/chobsterol (l/l mole ratio) DOPG (0.1 pmol added) DOPG/cholesterol (l/L.6 mole ratio)

3.132

3.3 17.8 1.4 35.2 14.9

n Determinations were made at 37-38%. R/licromoles of lipid added were optional amounti. All were sonicated liposomes. See Ref. (14) for further experimental details and Table 2 for definition of abbreviations.

3.3

34

35

36

+,$

FIG. 2. Arrhenius plots of the Activation by phosphatidylserine (PS) and phosphatidylglycerols (PC) as sonicated liposomes of a phospholipiddepleted (Na+ + K+) ATPase from rabbit kidney outer medulla. DOPG, dioleoyl PG (C18:l); DMPG, dimyristyl PG (C14); DPPG, dipaImitoy1 PG (CM); DSPG, &steamy1 PG (Cl8). See Ref. (14) for further experimental details.

gIycerols and also by bovine brain PS. The d.ata is expressed in the form of an Arrhenius plot, and we have suggested that would indeed alter the functioning of a the ,discontinuous changes in slope seen membrane enzyme, we examined the in- represent the end (PS sand DMPG) or fluence of temperature and cholesterol on beginning (DPPG and DSPG) of the region of the solid to fluid melt of each the reactivation of a phospholipid-depIeted particular phospholipid (14). It is sig(Nat + K’) ATPase preparation (14) by the synthetic! phosphatidylglycerols dis- nificant that reactivation by DOPG, which remains fluid throughout the temperature cussed above. Table 3 shows the inhibition of this reactivation by incorporation of range studied, shows a single linear slope. Similar discontinuous Arrhenius pIots cholesterol into phosphatidylglycerol liposomes of different fatty acy1 chain cctm- have also been found to occur in. uivo for position. Figure 2, shows the effects of (Na’ + K+) ATPase activities from a temperature ton the reactivation of (Na+ variety of sources (15), In most cases a single discontinuity at around 20°C has + K+) ATPase by different phosphatidylTABLE Discontinuity Bpecieaand

4

Temperatures of (Na+ + K+) ATPase Activity Fluidity (tf) of Membrane Preparations” t.

tieaue

Lamb kidney outer medulla Sheep kidney cortex and outer medulla Gold f&h intestinal mucasa Cultured mouse SV403T3 LY% Rat brain

(&) and Membrane

YC)

20 22 21 or 12 (dependent on acclimation temperature) 24 22 (glial cells) 19 (neuron)

19 (,rynaptasomes) a Data from material reviewed in Ref. (15). See text for further details.

11 (“C)

Reference

20 18-22 -

(17) (18) (19)

-

@O) w

H. K. KIMELBERG

been found, similar to that found for Iipiddepleted (Na+ + K+) ATPase reconstituted with bovine brain PS. In those cases studied, this was also found to correspond to a fluidity transition in the lipids of the membrane preparation, as measured by a lipid-soluble probe. Also, it is of interest that the temperatures of this discontinuity can be altered in poikilotherms by variations in the acclimation temperature. Acclimation at low temperatures is known to increase the levels of unsaturated fatty acyl chains of phospholipids in poikilotherms (16). Representative data from the literature illustrating the above points are shown in Table 4. Such data suggests that the effects of membrane fluidity on proteinlipid interactions observed in model systems may also be seen in biological membranes in vivo. UndoubtedIy, the behavior in living organisms will he complicated, however, by the increased heterogeneity of lipid and protein composition seen in biological membranes. Careful comparative studies, of both model and natural systems, should clarify the molecular details of the effects of lipid fluidity on membrane function and how alterations in temperature and membrane composition can affect this. REFERENCES 1. Singer, S. J. Annu. Reo. Biochem. 43, 80% 833 (1974). 2. Bretscher, M. S. Science 181, 822-029 ( 1973). 3. Kimelberg, H. K., Mol. Cell. Biochem. 10, 171-190 (1978). 4. Bangham, A. D. In “Progress in Biophysics and Molecular Biology” (J. A. V. Butler and D. Noble, Eds.), Vol. 18, pp. 29-95. Pergamon Press, Elmsford, New York, 1968.

5. Langmuir, I. .I. Amer. Chem. Sot. 39, 184S1905 ( 1917) I 6. Gaines, G. L. “Insoluble Monolayers at Liquid-Gas Interfaces,” pp. 347-361. Interscience Publishers, New York and London, 1966. 7. Papahadjopoulos, D., and Kimelberg. H. K. In “Progress in Surface Science (S. G. Davison, Ed.), pp. 141-232. Pergamon Press, Oxford, 1974. 8. Bangham, A. D., Standish, M. M., and Watkins, J. C. J. Mol. Biol. 13, 238-252 (1965). 9. Mueller, P., Rubin, D. O., Tien, H. T., and Wescott, W. C. Nature (London) 194, 979980 (1962). 10. Marsh, D. In “Essays in Biochemistry” (P. N. Campbell and W. N. Aldridge, Eds.), Vol. 11, pp. 139-180. Academic Press, London New York/San Francisco, 1975. II. Papahadjopoulos, D., Cowden, M., and Kimelberg, H. K. Biochim. Biophys. A.&z 330, 8-26 (1973). 12. Kimelberg, H. K., and Papahadjopoulos, D.

Biochim.

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Acta

293,

805-80$1

(1971). 13. Kimelberg, H. K., and Papahadjopoulos. D., J. Bid. Chem. 246, 114%1148 (1971). 14. Kimelberg, H. K., and Papahadjopoulos, D., J. Biol. Chem. 249, 1071-1080 (1974). 15. Kimelberg, H. K., In “Cell Surface Reviews” (G. Poste and G. L. Nicolson, Eds.), Vol. 3, pp. 205-293. North-Holland Publishing Company, Amsterdam, New York, Oxford,

1977. 16. Hazel, J. R., and Presser, C. L. Physiol. Reu. 54, 620-877 (1974). 17. Grisham, C. M., and Barnett, R, E. Biochemktry 12, 2635-2637 (1973). 18. Charnook. J. S., and Bashford, C. L. Mol.

PharmucoE. 11, ‘@f&774 ( 1975). 19. Smith, M. W. Biochem, J. 105, 6.5 (1967). 20. Kimelberg, H. K., and Mayhew, E. J. Bid.

Chem. 25, 100-104 ( 1975). 21. Kin&berg, H. K., Biddlecome, S., Narumi, S., and Bourke, R. S. Brain Res. 147, in press.