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Are essential fatty acids essential for membrane function?
Prog. Lipid Res. Vol. 25, pp. 41-46, 1986 Printed in Great Britain. All rights reserved
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0163-7827/86/$0.00 + 0.50 © 1986 Pergamon Journals Ltd
ESSENTIAL FATTY FOR MEMBRANE
ACIDS ESSENTIAL FUNCTION?
A. G. LEE, J. M. EAST and R. J. FROUD Department of Biochemistry, University of Southampton, Basset Crescent East, Southampton, S09 3TU, U.K.
The phospholipid composition of biological membranes is complex. A typical membrane will contain phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidylglycerols, sphingomyelin and, in neural tissue, cerebrosides. These phospholipids contain a wide variety of fatty alkyl chains, attached either by ester or (in the plasmalogens) ether linkages, chain lengths normally lying between 16 and 22 carbons with up to 6 carbon-carbon double bonds. In combination, this results in many hundreds of chemically distinct species of phospholipid. It has been argued that the observed complexity of composition demonstrates that the phospholipid requirements for a fully functional membrane are highly specific and complex. In its most extreme form, the argument would have it that each and every species of phospholipid in the membrane has its own distinct function and that a complete model for the membrane would have to include a list of all these functions. However, it is also possible to draw the exactly opposite conclusion from the same data. It can be argued that the presence of a wide variety of phospholipid species in the membrane demonstrates that the exact phospholipid composition is unimportant and that within relatively broad limits (that need to be defined) any one mixture of phospholipids will do as well as any other. I: The cell would then be free to construct its membrane using whichever fatty acyl chains and lipid head groups were currently most available. The two arguments presented above relate to two very different pictures of the membrane. The first corresponds to a highly tuned structure, but one in which whatever benefit is obtained by optimising the structure of the membrane for each of its many functions has to be offset against the problems of control. Once a certain level of complexity is reached, the cost of control will become prohibitive. The second argument pictures the membrane as a much simpler robust structure. At the cost of possible suboptimal function, little control has to be exerted over the composition of the membrane. Such a membrane will, of course, have the greater reliability. Experimentally, what has to be specified for such a membrane is the range of phospholipid structures compatible with function. Here we will consider whether there is any evidence that the essential fatty acids are essential for the proper functioning of the membrane. Fatty acids as components of phospholipids could affect membrane function in one or more of four ways: (1) effects on lipid fluidity; (2) effects on membrane thickness; (3) effects on lipid phase; (4) by specific interaction with membrane proteins. It is also necessary to consider the metabolic role of fatty acids in the cell, with equilibration between phospholipids and triglycerides, the storage roles of phospholipids in the cell, with the membranes acting as a store of arachidonic acid for eicosanoid JPLR2S-C
41
42
A . G . Lee et al.
synthesis and the role of phospholipids such as the phosphatidylinositols in signalling in the cell. Most discussions of the importance of the correct fatty acyl chain composition for membrane function have stressed effects on membrane fluidity. Phospholipids can exist in two distinct physical states depending on temperature, a gel phase (equivalent to a solid) and a liquid crystalline phase (a liquid like state). ~' In the gel phase, the phospholipids are tightly packed with the fatty acyl chains in a fully extended, all-trans form. The structure is highly ordered and rigid, with properties like those of a solid. In marked contrast, in the liquid crystalline phase, there is considerable freedom of motion for the phospholipids with rotation about C-C bonds of the fatty acyl chains and lateral diffusion of phospholipids within the plane of the membrane. In the liquid crystalline phase, the viscosity of the fatty acyl chains is comparable to that of a light machine oil so that movement of nonpolar molecules through the bilayer will be relatively free. In this sense, a membrane in the liquid crystalline phase is said to be "fluid". The concept of fluidity is familiar in normal bulk fluids, but is more complex in a two-dimensional membrane system. The chemical nature of the phospholipid molecule, with fatty acyl chains "anchored" to a relatively immobile backbone, results in a gradient of motion within the membrane, with both the rate and amplitude of motion increasing along the fatty acyl chain from the carboxyl to the methyl terminal end. j~ A proper description of the membrane requires that the rate and amplitude factors be separated. The amplitude of motion is described in terms of an order parameter which describes the time-averaged disposition in space of each group of atoms in the acyl chain. The rate of motion can be described in terms of a correlation time, a measure of the rate of motion of a group of atoms between its various possible positions in space. Formally, fluidity corresponds solely to rate of motion, but in the biological literature the word is used very informally to include both factors. Indeed, the method generally used to measure fluidity (the polarization of flourescence of the probe diphenylhexatriene) depends in a complex fashion on both factors. 23 We should first consider the effects of fatty acyl chain structure on the temperature of the transition between the gel and liquid crystalline phase (Table l). First, transition temperatures decrease with decreasing chain length. Second, the introduction of a first double bond into a saturated fatty acyl chain results in a very marked decrease in transition temperature, the introduction of a second double bond resulting in a further but smaller decrease and the introduction of still further double bonds resulting in a small increase in transition temperature. These effects can be understood in terms of the difficulty of packing unsaturated chains in the close packed gel phase. Phospholipids containing one saturated and one unsaturated chain have a phase transition temperature between the phase transition temperatures for the respective phospholipids with two identical fatty acyl chains. The transition temperatures listed in Table l show that in mammalian systems the liquid crystalline phase can be achieved without the use of polyunsaturated fatty acyl chains. Unsaturation will also affect the exact fluidity of the bilayer in the liquid crystalline phase. Seelig and Waespe-Sarcevic" have described the hydrocarbon interior of the bilayer
TABLE 1. Transition Temperatures for Phosphatidylcholines3.11
Fatty acids* 16:0/16:0 18:0/18:0 22:0/22:0 18:0/18:1 18:0/18:2 18:0/18:3 18:0/20:4 18:1/18:1
Transition temperature (°C) 42 54 75 5 - 16 - 13 - 13 -20
*The first and second fatty acids are attached to the 1- and 2-positions of the phospholipids, respectively,
Are essential fatty acids essential
43
TASLE2. Order Parameters and ATPase Activity6 Fatty acid chain ATPase aetivityt Position 1 Position 2 Orderparameter* (%) arachidonyl arachidonyl 0.52 91 linoleoyl linoleoyl 0.55 115 linoleoyl palmitoyl 0.55 89 oleoyl palmitoyl 0.57 94 oleoyl oleoyl 0.56 100 *= Order parameter for 5-doxylstearic acid at 37°C. ~fffiActivity (%) at 37°C expr-~ded relative to that for the ATPase reconstituted with dioleoylphosphatidylcholinetaken as 100%.
as a series of strata running parallel to the bilayer surface. Segments of the fatty acyl chains that are located in the same stratum are characterized by similar mobilities. Unsaturation complicates this picture. Since the C ffi C unit is relatively rigid, the area around the double bond will be more ordered than areas nearer the methyl end of the hydrocarbon chain. With polyunsaturated chains, such ordering effects will become even more marked. These effects have been demonstrated in a study of the motion of the flourescence probe diphenylhexatriene (DPH) in lipid bilayers. Motion can be described as 'wobbling in a cone' and characterized in terms of an orientational constraint (a cone angle) and a wobbling diffusion constant related to the rate of motion 23 The analysis shows that introduction of one double bond into a fatty acyl chain causes a marked increase in rate of motion and increase in cone angle of motion, but that further double bonds have relatively little effect.23The same pattern of change has been illustrated in studies of order parameter using spin labels where, although decreasing order is observed with increasing uusaturation, effects are small (Table 2). 6 In conclusion, double bonds, particularly after the first, have little effect on fluidity in the liquid crystalline phase. The most important effect of unsaturation, therefore, seems to be to ensure that lipids are in the liquid crystalline phase at physiological temperature. The importance of the liquid crystalline phase has been demonstrated most clearly in microorganisms such as Acholeplasma in which the fatty acyl chain composition of the membrane can be manipulated: it is found that growth requires that at least half of the lipid be in the liquid crystalline phase.16 In mammalian systems, the requirement for the liquid crystalline phase may be even more strict since in such membranes the bulk of the lipids are in the liquid crystalline phase, this being maintained against changes in environmental temperature by appropriate changes in fatty acyl chain composition. In natural membranes, the majority of the phospholipids contain one saturated and one unsaturated fatty acyl chain, the distribution being ensured by the specificity of phospholipase A2 and of choline and ethanolamine phosphotransferases: 9.u such a distribution, in combination with the range of fatty acyl chains found in natural membranes, ensures that the phase transition temperatures of the bulk of the lipids in the membrane will be below physiological temperature (Table 1). The importance of these factors is illustrated by the data in Tables 3 and 4 for the composition of muscle sarcoplasmic reticulum. In rabbit muscle sarcoplasmic reticulum, the relative content of saturated to unsaturated fatty acyl chains is about 40: 60. ~4Although the lipid composition is distinct from that in other tissues such as liver, there is no evidence that the particular composition is in any way specially suited for the function of muscle sarcoplasmic reticulum. Thus, the fatty acid composition of sarcoplasmic reticulum from rabbit, rat, chicken and human muscle all show some differences) 4'~s and that from lobster is distinctly different with a much greater proportion of polyunsaturated fatty acids (40% as compared to 9% in rabbit). ~ Changes in phospholipid composition of sarcoplasmic reticuium have been observed during development. In chicken, there is a marked decrease in palmitate and increase in linoleate with development 2 and in rabbit there is also an increase in unsaturation. 2~ The effects' of diet on total phosphatidylcholines from turkey breast have been reported (Table 4). Enriching
A. G. Lee et al.
44
TABLE 3. Fatty Acid Content of Phospholipids o f Rabbit Muscle Sarcoplasmic Reticulum ~4 Composition (Mole %) Phosphatidylcholine
Phosphatidylethanolamine
Fatty acid
Total
Pos 1
Pos 2
Total
14:0 16:0 18:0 18: I 18:2 20:4 22 : 3 22:5
0.2 40.0 4.0 17.0 25.0 8.0 1.0 2.0
0.4 74.0 8.0 8.0 8.0 ----
0.6 5.5 0.3 26.0 41.0 15.0 2.0 3.0
26.0 14.0 14.0 6.0 16.0 5.0 6.0
-
-
Pos 1 -
-
Pos 2 -
49.0 26.0 5.0 2.0 ----
-
5.0 2.0 21.0 I1.0 27.0 9.0 11.0
the diet in saturated fatty acids with beef has no significant effect on phospholipid composition, and the total content of saturated fatty acids remains slightly less than 50%. Addition of highly unsaturated fatty acids to the diet, however, causes a marked change in the unsaturated fatty acid content of the membrane but with little change in the saturated fatty acids; in particular, the content of the polyunsaturated fatty acids 20: 5 and 22:6 is increased at the expense of 18:1 and 18:2. In this particular membrane at least, the only important requirement appears to be that no more than 50% of the fatty acids be saturated, ensuring that the bulk of the lipid be in the liquid crystalline phase. There is no evidence that any particular pattern of unsaturation is important, and thus there is no evidence that the exact fluidity in the liquid crystalline phase has to be controlled. As well as changing fluidity, changes in fatty acid composition will result in changes in bilayer thickness. Measurements by Cornell and Separovic4 have, however, shown that such effects are smaller than might have been thought, the major changes in fact being in the area each molecule of lipid occupies in the bilayer surface. Thus, for phosphatidylcholines, changing the fatty acyl chains from 12:0 to 18:0 causes a change in thickness of the hydrocarbon region of the bilayer in the liquid crystalline phase by 4 .~,, whereas for fully extended fatty acyl chains the expected change in thickness would be 15/~,. In dilauroyl phosphatidylcholine, the fatty acyl chains are extended to 80% of their crystalline length whereas in distearoyl phosphatidylcholine the ratio is only 62%. 4 Introduction of double bonds would shorten the fatty acyl chains and thus thin the bilayer, but from the above results such effects would be expected to be quite small. An exception could be arachidonic acid, where the repeating C = C--CH2-C = C pattern forces a helical twist on the chain, so that the straightest possible structure is, in fact, somewhat shorter than the straightest conformation of oleic acid. The possible importance of bilayer thickness is illustrated by the studies on reconstituted membrane systems to be discussed later. A number of recent studies have also suggested that fatty acyl chains can affect the three-dimensional structure adopted by lipid aggregates in water and that, as well as the bilayer (iamellar) phase typical of biological membranes, a number of nonbilayer phases
TABLE 4. Effects of Diet on Fatty Acid Composition (wt %) of Phosphatidylcholines from Turkey Breast ~a Fatty acid
Standard
+ Beef fat
+ Anchovy oil
16:0 18:0 18:1 18:2 20:4 20:5 22:6 Total saturated
33.0 11.0 22.0 25.0 2.0 0.3 0.2 44.0
33.0 12.0 24.0 23.0 3.0 0.4 0.6 46.0
33.0 I 1.0 15.0 9.0 3.0 11.0 7.0 47.0
Are essential fatty acids essential TAaLE 5. Relative Activities of the (Ca2+-Mg2+)ATPase Reconstituted into Phospholipid BilayersL7 Phospholipid % activity* Dioleoylphosphatidylcholine 150 Dioleoylphosphatidylethanolamine 75 Dioleoylphosphatidylserine 50 Dipalmitoylphosphatidylcholine (45°C) 90 Dipalmitoylphosphatidylcholine (25°C) 0 *Expressed relative to that of the ATPase in the native sarcoplasmic reticulum membrane
45
TABLE 6. Relative Activities of the (Ca2+-Mg'+)ATPase Reconstituted into Bilayers of Diacylphosphatidyicholines Activity Fatty acyl chains (IU/mg) Myristoleic (14:I) 3.7 Palmitoleic (16:1) 18.7 Oleic 08:1) 24.1 Eicosoenoic (20:1) 18.2 Erucic (22:1) 11.1 Nervonic (24: 1) 3.2
are possible. 5 The phase adopted by a lipid depends on the "shape" of the lipid, the shape being defined by the relative areas occupied by the lipid head group and the fatty acyl chains. )° A cylindrical structure (in which the lipid head group and the two fatty acyl chains occupy roughly equal areas) gives a bilayer phase and other shapes the nonbilayer phases. Changes in fatty acid unsaturation will change the areas occupied by the fatty acyl chains and so affect the probability of formation of nonbilayer phases. The importance of such nonbilayer phases in real biological membranes is still very uncertain. The final factor we need to consider is specific interaction between fatty acyl chains and membrane proteins. The most common structure for a protein sequence spanning the membrane is the ~t-helix, consisting of alternate rows of "knobs and holes" spiralling around the surface of the helix. Specific binding of fatty acids, or specific recognition of carbon-carbon double bonds by such a structure, seems unlikely. In conclusion, studies with whole membranes indicate the importance of a roughly 50/50 balance between saturated and unsaturated fatty acyl chains, but give no evidence for a requirement for any specific pattern of unsaturation. These conclusions can be put on a more quantitative basis using simplified, reconstituted membrane systems. One of the most studied of membrane proteins is the (Ca2+-Mg 2+ ) ATPase that can be purified from sarcoplasmic reticulum of muscle. This can be reconstituted into bilayers of defined phospholipid composition, so that the effect of phospholipid structure and phase on enzyme activity can be studied in detail. It is found that the activity of the ATPase is markedly dependent on the chemical structure of the surrounding phospholipid, both on the lipid head group (Table 5) and fatty acyl chain length (Table 6). The optimal lipid head group for activity is phosphatidylcholine, and the optimal chain length is 18, with chains shorter than 16 or longer than 22 supporting significantly lower activities. The data in Tables 5 and 6 demonstrate that the complex lipid composition of sarcoplasmic reticulum (Table 3) is not optimized for ATPase activity. Despite the importance of the chemical structure of the surrounding phospholipid for ATPase activity, it has been shown that binding constants of phospholipids to the ATPase are almost independent of structure, so that the composition of the lipid mixture around the ATPase in the membrane (the annular lipids) will parallel the bulk composition of the membrane. 7 The ATPase will not therefore be buffered from any effects of changing lipid composition in the membrane. The data in Table 5 also illustrates the importance of the liquid crystalline phase for activity; whereas the ATPase is active in bilayers of dipalmitoylphosphatidylcholine at 45°C in the liquid crystalline phase, the ATPase exhibits no activity at 25°C when the liquid will be in the gel phase? 5 The same system can be used to test for the importance of the exact fluidity in the liquid crystalline phase. Reconstitution of the ATPase into phospholipids containing a variety of polyunsaturated fatty acyl chains causes relatively small changes in activity with no correlation between activity and membrane order (Table 2). 6 Despite the presence of polyunsaturated fatty acids in native sarcoplasmic reticulum, these experiments give no evidence for their having any distinct function. Similar results have been observed in other reconstituted systems. Rat liver mlcrosomal stearoyl-CoA desaturase has been reconstituted into bilayers of egg yolk phosphatidylcholine (unsaturated, average chain length C17) and dimyristoylphosphatidylcholine
46
A . G . Lee et al.
(DMPC). At temperatures above 24°C when DMPC is in the liquid crystalline phase, the enzyme is active in both systems, the activity in egg yolk phosphatidylcholine being ca. 2-3 times that in the short chain lipid DMPC. s At lower temperatures, when DMPC will be in the gel phase, there is no activity. These results are strikingly similar to those obtained with the (Ca2+-Mg2+)ATPase. The experiments provide no evidence for the idea that desaturase activity is controlled by membrane fluidity. Rhodopsin has also been reconstituted into simple lipid bilayers. The retinal rod outer segment disk membrane (ROS) is particularly rich in unsaturated fatty acids: in cattle ROS about 50% of the fatty acid is 22:6) 7 However, when rhodopsin is reconstituted into bilayers of egg phosphatidylcholine, the kinetics of the metarhodopsin I-metarhodopsin II change are identical to those in the native ROS membrane; it was only when rhodopsin was reconstituted into a short chain lipid such as DMPC that a considerable reduction in rate was observed) 9 It has been suggested that the polyunsaturated fatty acids in ROS lead to a highly fluid membrane, and that this accounts for the fast rate of lateral diffusion observed for rhodopsin in ROS. However, the rate of diffusion of the related protein bacteriorhodopsin in bilayers of DMPC in the liquid crystalline phase has been found to be faster than for rhodopsin in ROS. 2° These reconstitution studies illustrate the importance of fatty acyl chain length for the activity of membrane proteins, and explain the restricted range of chain lengths found in natural membranes. They also demonstrate the importance of the liquid crystalline phase and explain why at least half of the fatty acyl chains should be unsaturated. They however provide no evidence suggesting that polyunsaturated fatty acids are important either because of specific interactions with membrane proteins or because of effects on lipid fluidity. If the essential fatty acids are not essential for membrane function, then their importance must be explained in terms of their other functions in the cell. Acknowledgements--We thank the ARC, SERC and Wellcome Trust for financial support.
REFERENCES 1. BENNETT,J. P., SMITH, G. A., HOUSLAY, M. D., HES~rH, T. R., METCALFE,J. C. and WARREN,G. B. Biochim. Biophys. Acta S13, 310-320 (1978). 2. BOLXND,R., MAIt1"ONCm,A. and TILt.ACt, T. W. J. Biol. Chem. 249, 612-623 (1974). 3. CooLroat, K. P., ~ C. B. and KEouon, K. M. W. Biochemistry 22, 1466-1473 (1983). 4. COI~NELL,B. A. and SZPAILOVlC,F. Biochim. Biophys. Acta 733, 189-193 (1983). 5. DEKKEK,C. J., GEL~I~3VAN KESSEL,W. S. M., KLOMP,J. P. G., PmTEZS,J. and DE KRUlffF, B. Chem. Phys. Lipids 33, 93-106 (1983). 6. EAST, J. M., JONES, O. T., SIMMONDS,A. C. and LEE, A. G. J. Biol. Chem. 259, 8070-8071 (1984). 7. EmT, J. M. and LEE, A. G. Biochemistry 21, 4144--4151 (1982). 8. ENOCH, H. G., CA'rALA,A. and SaXlTTMATamR,P. J. Biol. Chem. 251, 5095-5103 (1976). 9. HOLUB, B. J. J. Biol. Chem. 253, 691--696 (1978). 10. ISRAELACHVlLI,J. N., MARtmlJ^, S. and HORN, R. G. Q. Rev. Biophys. 13, 121-200 (1980). 11. LiE, A. G. In Membrane Fluidity in Biology, Vol. 2, pp. 43-88, (ALOL~, R. C., ed.) Academic Press, New York, 1983. 12. LI~, A. G. Proc. Nutr. Soc. (in press). 13. MXDRmA,V. M. C. and AN'nJN~MADF.mA, M. C. Can. J. Biochem. 54, 516-520 (1976). 14. MARm, L. and K ~ . A. Can. J. Biochem. 51, 1248-1261 (1973). 15. MARX1,L. and KuKslS. A. Can. J. Biochem. 51, 1365-1379 (1973). 16. MCELH^NEY, R. N. Biomembranes 12, 249-269 (1984). 17. MIlJ^NICH, G. P., SKLAX,L. A., WHrrE, D. C. and DRATZ,E. A. Biochim. Biophys. Acta. 552, 294-306 (1979). 18. NEUDOERFFER,T. S. and LEx, C. H. Br. J. Nutr. 21, 691-714 (1967). 19. O'BamN, D. F., CO~A, L. F. and OTT, R. A. Biochemistry 16, 1295-1303 (1977). 20. PETERS,R. and CH~xY, R. J. Proc. Natl. Acad. Sci. U.S.A. 79, 4317-4321 (1982). 21. SXRZXLA,M. G., P I ~ , M., ZUI~.ZYCHA,E. and MICHALAK,M. Eur. J. Biochem. 57, 24-34 (1975). 22. Sm:LIG,J. and WAESl~-S~CEVlC, N. Biochemistry 17, 3310-3315 0978). 23. SI-oBas, C. D., KOIJYAMA,T., KtNOSlTA,K. and IKEGAMI,A. Biochemistry 20, 4257--4262 (1981). 24. VAN l~m~ BOSCH, H. Biochim. Biophys. Acta 604, 191-246 (1980). 25. W ~ N , (3. B., TOON,P. A., BIRD~LL, N. J. M., I.~, A. G. and MLrrCALI~,J. C. Biochemistry 13, 5501-5507 (1974).
ARE
0163-7827/86/$0.00 + 0.50 © 1986 Pergamon Journals Ltd
ESSENTIAL FATTY FOR MEMBRANE
ACIDS ESSENTIAL FUNCTION?
A. G. LEE, J. M. EAST and R. J. FROUD Department of Biochemistry, University of Southampton, Basset Crescent East, Southampton, S09 3TU, U.K.
The phospholipid composition of biological membranes is complex. A typical membrane will contain phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidylglycerols, sphingomyelin and, in neural tissue, cerebrosides. These phospholipids contain a wide variety of fatty alkyl chains, attached either by ester or (in the plasmalogens) ether linkages, chain lengths normally lying between 16 and 22 carbons with up to 6 carbon-carbon double bonds. In combination, this results in many hundreds of chemically distinct species of phospholipid. It has been argued that the observed complexity of composition demonstrates that the phospholipid requirements for a fully functional membrane are highly specific and complex. In its most extreme form, the argument would have it that each and every species of phospholipid in the membrane has its own distinct function and that a complete model for the membrane would have to include a list of all these functions. However, it is also possible to draw the exactly opposite conclusion from the same data. It can be argued that the presence of a wide variety of phospholipid species in the membrane demonstrates that the exact phospholipid composition is unimportant and that within relatively broad limits (that need to be defined) any one mixture of phospholipids will do as well as any other. I: The cell would then be free to construct its membrane using whichever fatty acyl chains and lipid head groups were currently most available. The two arguments presented above relate to two very different pictures of the membrane. The first corresponds to a highly tuned structure, but one in which whatever benefit is obtained by optimising the structure of the membrane for each of its many functions has to be offset against the problems of control. Once a certain level of complexity is reached, the cost of control will become prohibitive. The second argument pictures the membrane as a much simpler robust structure. At the cost of possible suboptimal function, little control has to be exerted over the composition of the membrane. Such a membrane will, of course, have the greater reliability. Experimentally, what has to be specified for such a membrane is the range of phospholipid structures compatible with function. Here we will consider whether there is any evidence that the essential fatty acids are essential for the proper functioning of the membrane. Fatty acids as components of phospholipids could affect membrane function in one or more of four ways: (1) effects on lipid fluidity; (2) effects on membrane thickness; (3) effects on lipid phase; (4) by specific interaction with membrane proteins. It is also necessary to consider the metabolic role of fatty acids in the cell, with equilibration between phospholipids and triglycerides, the storage roles of phospholipids in the cell, with the membranes acting as a store of arachidonic acid for eicosanoid JPLR2S-C
41
42
A . G . Lee et al.
synthesis and the role of phospholipids such as the phosphatidylinositols in signalling in the cell. Most discussions of the importance of the correct fatty acyl chain composition for membrane function have stressed effects on membrane fluidity. Phospholipids can exist in two distinct physical states depending on temperature, a gel phase (equivalent to a solid) and a liquid crystalline phase (a liquid like state). ~' In the gel phase, the phospholipids are tightly packed with the fatty acyl chains in a fully extended, all-trans form. The structure is highly ordered and rigid, with properties like those of a solid. In marked contrast, in the liquid crystalline phase, there is considerable freedom of motion for the phospholipids with rotation about C-C bonds of the fatty acyl chains and lateral diffusion of phospholipids within the plane of the membrane. In the liquid crystalline phase, the viscosity of the fatty acyl chains is comparable to that of a light machine oil so that movement of nonpolar molecules through the bilayer will be relatively free. In this sense, a membrane in the liquid crystalline phase is said to be "fluid". The concept of fluidity is familiar in normal bulk fluids, but is more complex in a two-dimensional membrane system. The chemical nature of the phospholipid molecule, with fatty acyl chains "anchored" to a relatively immobile backbone, results in a gradient of motion within the membrane, with both the rate and amplitude of motion increasing along the fatty acyl chain from the carboxyl to the methyl terminal end. j~ A proper description of the membrane requires that the rate and amplitude factors be separated. The amplitude of motion is described in terms of an order parameter which describes the time-averaged disposition in space of each group of atoms in the acyl chain. The rate of motion can be described in terms of a correlation time, a measure of the rate of motion of a group of atoms between its various possible positions in space. Formally, fluidity corresponds solely to rate of motion, but in the biological literature the word is used very informally to include both factors. Indeed, the method generally used to measure fluidity (the polarization of flourescence of the probe diphenylhexatriene) depends in a complex fashion on both factors. 23 We should first consider the effects of fatty acyl chain structure on the temperature of the transition between the gel and liquid crystalline phase (Table l). First, transition temperatures decrease with decreasing chain length. Second, the introduction of a first double bond into a saturated fatty acyl chain results in a very marked decrease in transition temperature, the introduction of a second double bond resulting in a further but smaller decrease and the introduction of still further double bonds resulting in a small increase in transition temperature. These effects can be understood in terms of the difficulty of packing unsaturated chains in the close packed gel phase. Phospholipids containing one saturated and one unsaturated chain have a phase transition temperature between the phase transition temperatures for the respective phospholipids with two identical fatty acyl chains. The transition temperatures listed in Table l show that in mammalian systems the liquid crystalline phase can be achieved without the use of polyunsaturated fatty acyl chains. Unsaturation will also affect the exact fluidity of the bilayer in the liquid crystalline phase. Seelig and Waespe-Sarcevic" have described the hydrocarbon interior of the bilayer
TABLE 1. Transition Temperatures for Phosphatidylcholines3.11
Fatty acids* 16:0/16:0 18:0/18:0 22:0/22:0 18:0/18:1 18:0/18:2 18:0/18:3 18:0/20:4 18:1/18:1
Transition temperature (°C) 42 54 75 5 - 16 - 13 - 13 -20
*The first and second fatty acids are attached to the 1- and 2-positions of the phospholipids, respectively,
Are essential fatty acids essential
43
TASLE2. Order Parameters and ATPase Activity6 Fatty acid chain ATPase aetivityt Position 1 Position 2 Orderparameter* (%) arachidonyl arachidonyl 0.52 91 linoleoyl linoleoyl 0.55 115 linoleoyl palmitoyl 0.55 89 oleoyl palmitoyl 0.57 94 oleoyl oleoyl 0.56 100 *= Order parameter for 5-doxylstearic acid at 37°C. ~fffiActivity (%) at 37°C expr-~ded relative to that for the ATPase reconstituted with dioleoylphosphatidylcholinetaken as 100%.
as a series of strata running parallel to the bilayer surface. Segments of the fatty acyl chains that are located in the same stratum are characterized by similar mobilities. Unsaturation complicates this picture. Since the C ffi C unit is relatively rigid, the area around the double bond will be more ordered than areas nearer the methyl end of the hydrocarbon chain. With polyunsaturated chains, such ordering effects will become even more marked. These effects have been demonstrated in a study of the motion of the flourescence probe diphenylhexatriene (DPH) in lipid bilayers. Motion can be described as 'wobbling in a cone' and characterized in terms of an orientational constraint (a cone angle) and a wobbling diffusion constant related to the rate of motion 23 The analysis shows that introduction of one double bond into a fatty acyl chain causes a marked increase in rate of motion and increase in cone angle of motion, but that further double bonds have relatively little effect.23The same pattern of change has been illustrated in studies of order parameter using spin labels where, although decreasing order is observed with increasing uusaturation, effects are small (Table 2). 6 In conclusion, double bonds, particularly after the first, have little effect on fluidity in the liquid crystalline phase. The most important effect of unsaturation, therefore, seems to be to ensure that lipids are in the liquid crystalline phase at physiological temperature. The importance of the liquid crystalline phase has been demonstrated most clearly in microorganisms such as Acholeplasma in which the fatty acyl chain composition of the membrane can be manipulated: it is found that growth requires that at least half of the lipid be in the liquid crystalline phase.16 In mammalian systems, the requirement for the liquid crystalline phase may be even more strict since in such membranes the bulk of the lipids are in the liquid crystalline phase, this being maintained against changes in environmental temperature by appropriate changes in fatty acyl chain composition. In natural membranes, the majority of the phospholipids contain one saturated and one unsaturated fatty acyl chain, the distribution being ensured by the specificity of phospholipase A2 and of choline and ethanolamine phosphotransferases: 9.u such a distribution, in combination with the range of fatty acyl chains found in natural membranes, ensures that the phase transition temperatures of the bulk of the lipids in the membrane will be below physiological temperature (Table 1). The importance of these factors is illustrated by the data in Tables 3 and 4 for the composition of muscle sarcoplasmic reticulum. In rabbit muscle sarcoplasmic reticulum, the relative content of saturated to unsaturated fatty acyl chains is about 40: 60. ~4Although the lipid composition is distinct from that in other tissues such as liver, there is no evidence that the particular composition is in any way specially suited for the function of muscle sarcoplasmic reticulum. Thus, the fatty acid composition of sarcoplasmic reticulum from rabbit, rat, chicken and human muscle all show some differences) 4'~s and that from lobster is distinctly different with a much greater proportion of polyunsaturated fatty acids (40% as compared to 9% in rabbit). ~ Changes in phospholipid composition of sarcoplasmic reticuium have been observed during development. In chicken, there is a marked decrease in palmitate and increase in linoleate with development 2 and in rabbit there is also an increase in unsaturation. 2~ The effects' of diet on total phosphatidylcholines from turkey breast have been reported (Table 4). Enriching
A. G. Lee et al.
44
TABLE 3. Fatty Acid Content of Phospholipids o f Rabbit Muscle Sarcoplasmic Reticulum ~4 Composition (Mole %) Phosphatidylcholine
Phosphatidylethanolamine
Fatty acid
Total
Pos 1
Pos 2
Total
14:0 16:0 18:0 18: I 18:2 20:4 22 : 3 22:5
0.2 40.0 4.0 17.0 25.0 8.0 1.0 2.0
0.4 74.0 8.0 8.0 8.0 ----
0.6 5.5 0.3 26.0 41.0 15.0 2.0 3.0
26.0 14.0 14.0 6.0 16.0 5.0 6.0
-
-
Pos 1 -
-
Pos 2 -
49.0 26.0 5.0 2.0 ----
-
5.0 2.0 21.0 I1.0 27.0 9.0 11.0
the diet in saturated fatty acids with beef has no significant effect on phospholipid composition, and the total content of saturated fatty acids remains slightly less than 50%. Addition of highly unsaturated fatty acids to the diet, however, causes a marked change in the unsaturated fatty acid content of the membrane but with little change in the saturated fatty acids; in particular, the content of the polyunsaturated fatty acids 20: 5 and 22:6 is increased at the expense of 18:1 and 18:2. In this particular membrane at least, the only important requirement appears to be that no more than 50% of the fatty acids be saturated, ensuring that the bulk of the lipid be in the liquid crystalline phase. There is no evidence that any particular pattern of unsaturation is important, and thus there is no evidence that the exact fluidity in the liquid crystalline phase has to be controlled. As well as changing fluidity, changes in fatty acid composition will result in changes in bilayer thickness. Measurements by Cornell and Separovic4 have, however, shown that such effects are smaller than might have been thought, the major changes in fact being in the area each molecule of lipid occupies in the bilayer surface. Thus, for phosphatidylcholines, changing the fatty acyl chains from 12:0 to 18:0 causes a change in thickness of the hydrocarbon region of the bilayer in the liquid crystalline phase by 4 .~,, whereas for fully extended fatty acyl chains the expected change in thickness would be 15/~,. In dilauroyl phosphatidylcholine, the fatty acyl chains are extended to 80% of their crystalline length whereas in distearoyl phosphatidylcholine the ratio is only 62%. 4 Introduction of double bonds would shorten the fatty acyl chains and thus thin the bilayer, but from the above results such effects would be expected to be quite small. An exception could be arachidonic acid, where the repeating C = C--CH2-C = C pattern forces a helical twist on the chain, so that the straightest possible structure is, in fact, somewhat shorter than the straightest conformation of oleic acid. The possible importance of bilayer thickness is illustrated by the studies on reconstituted membrane systems to be discussed later. A number of recent studies have also suggested that fatty acyl chains can affect the three-dimensional structure adopted by lipid aggregates in water and that, as well as the bilayer (iamellar) phase typical of biological membranes, a number of nonbilayer phases
TABLE 4. Effects of Diet on Fatty Acid Composition (wt %) of Phosphatidylcholines from Turkey Breast ~a Fatty acid
Standard
+ Beef fat
+ Anchovy oil
16:0 18:0 18:1 18:2 20:4 20:5 22:6 Total saturated
33.0 11.0 22.0 25.0 2.0 0.3 0.2 44.0
33.0 12.0 24.0 23.0 3.0 0.4 0.6 46.0
33.0 I 1.0 15.0 9.0 3.0 11.0 7.0 47.0
Are essential fatty acids essential TAaLE 5. Relative Activities of the (Ca2+-Mg2+)ATPase Reconstituted into Phospholipid BilayersL7 Phospholipid % activity* Dioleoylphosphatidylcholine 150 Dioleoylphosphatidylethanolamine 75 Dioleoylphosphatidylserine 50 Dipalmitoylphosphatidylcholine (45°C) 90 Dipalmitoylphosphatidylcholine (25°C) 0 *Expressed relative to that of the ATPase in the native sarcoplasmic reticulum membrane
45
TABLE 6. Relative Activities of the (Ca2+-Mg'+)ATPase Reconstituted into Bilayers of Diacylphosphatidyicholines Activity Fatty acyl chains (IU/mg) Myristoleic (14:I) 3.7 Palmitoleic (16:1) 18.7 Oleic 08:1) 24.1 Eicosoenoic (20:1) 18.2 Erucic (22:1) 11.1 Nervonic (24: 1) 3.2
are possible. 5 The phase adopted by a lipid depends on the "shape" of the lipid, the shape being defined by the relative areas occupied by the lipid head group and the fatty acyl chains. )° A cylindrical structure (in which the lipid head group and the two fatty acyl chains occupy roughly equal areas) gives a bilayer phase and other shapes the nonbilayer phases. Changes in fatty acid unsaturation will change the areas occupied by the fatty acyl chains and so affect the probability of formation of nonbilayer phases. The importance of such nonbilayer phases in real biological membranes is still very uncertain. The final factor we need to consider is specific interaction between fatty acyl chains and membrane proteins. The most common structure for a protein sequence spanning the membrane is the ~t-helix, consisting of alternate rows of "knobs and holes" spiralling around the surface of the helix. Specific binding of fatty acids, or specific recognition of carbon-carbon double bonds by such a structure, seems unlikely. In conclusion, studies with whole membranes indicate the importance of a roughly 50/50 balance between saturated and unsaturated fatty acyl chains, but give no evidence for a requirement for any specific pattern of unsaturation. These conclusions can be put on a more quantitative basis using simplified, reconstituted membrane systems. One of the most studied of membrane proteins is the (Ca2+-Mg 2+ ) ATPase that can be purified from sarcoplasmic reticulum of muscle. This can be reconstituted into bilayers of defined phospholipid composition, so that the effect of phospholipid structure and phase on enzyme activity can be studied in detail. It is found that the activity of the ATPase is markedly dependent on the chemical structure of the surrounding phospholipid, both on the lipid head group (Table 5) and fatty acyl chain length (Table 6). The optimal lipid head group for activity is phosphatidylcholine, and the optimal chain length is 18, with chains shorter than 16 or longer than 22 supporting significantly lower activities. The data in Tables 5 and 6 demonstrate that the complex lipid composition of sarcoplasmic reticulum (Table 3) is not optimized for ATPase activity. Despite the importance of the chemical structure of the surrounding phospholipid for ATPase activity, it has been shown that binding constants of phospholipids to the ATPase are almost independent of structure, so that the composition of the lipid mixture around the ATPase in the membrane (the annular lipids) will parallel the bulk composition of the membrane. 7 The ATPase will not therefore be buffered from any effects of changing lipid composition in the membrane. The data in Table 5 also illustrates the importance of the liquid crystalline phase for activity; whereas the ATPase is active in bilayers of dipalmitoylphosphatidylcholine at 45°C in the liquid crystalline phase, the ATPase exhibits no activity at 25°C when the liquid will be in the gel phase? 5 The same system can be used to test for the importance of the exact fluidity in the liquid crystalline phase. Reconstitution of the ATPase into phospholipids containing a variety of polyunsaturated fatty acyl chains causes relatively small changes in activity with no correlation between activity and membrane order (Table 2). 6 Despite the presence of polyunsaturated fatty acids in native sarcoplasmic reticulum, these experiments give no evidence for their having any distinct function. Similar results have been observed in other reconstituted systems. Rat liver mlcrosomal stearoyl-CoA desaturase has been reconstituted into bilayers of egg yolk phosphatidylcholine (unsaturated, average chain length C17) and dimyristoylphosphatidylcholine
46
A . G . Lee et al.
(DMPC). At temperatures above 24°C when DMPC is in the liquid crystalline phase, the enzyme is active in both systems, the activity in egg yolk phosphatidylcholine being ca. 2-3 times that in the short chain lipid DMPC. s At lower temperatures, when DMPC will be in the gel phase, there is no activity. These results are strikingly similar to those obtained with the (Ca2+-Mg2+)ATPase. The experiments provide no evidence for the idea that desaturase activity is controlled by membrane fluidity. Rhodopsin has also been reconstituted into simple lipid bilayers. The retinal rod outer segment disk membrane (ROS) is particularly rich in unsaturated fatty acids: in cattle ROS about 50% of the fatty acid is 22:6) 7 However, when rhodopsin is reconstituted into bilayers of egg phosphatidylcholine, the kinetics of the metarhodopsin I-metarhodopsin II change are identical to those in the native ROS membrane; it was only when rhodopsin was reconstituted into a short chain lipid such as DMPC that a considerable reduction in rate was observed) 9 It has been suggested that the polyunsaturated fatty acids in ROS lead to a highly fluid membrane, and that this accounts for the fast rate of lateral diffusion observed for rhodopsin in ROS. However, the rate of diffusion of the related protein bacteriorhodopsin in bilayers of DMPC in the liquid crystalline phase has been found to be faster than for rhodopsin in ROS. 2° These reconstitution studies illustrate the importance of fatty acyl chain length for the activity of membrane proteins, and explain the restricted range of chain lengths found in natural membranes. They also demonstrate the importance of the liquid crystalline phase and explain why at least half of the fatty acyl chains should be unsaturated. They however provide no evidence suggesting that polyunsaturated fatty acids are important either because of specific interactions with membrane proteins or because of effects on lipid fluidity. If the essential fatty acids are not essential for membrane function, then their importance must be explained in terms of their other functions in the cell. Acknowledgements--We thank the ARC, SERC and Wellcome Trust for financial support.
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