Chemisto, and Physics of Lipids 14 (1975) 236-246 © North-Holland Publishing Company
PHOSPHOLIPID UNSATURATION AND PLASMA MEMBRANE ORGANIZATION P. EMMELOT and R.P. VAN HOEVEN Department of Biochemistry, Antoni van Leeuwenhoek-Laboratory, The Netherlands Cancer Institute, Amsterdam, The Netherlands
Received June 25, 1974,
accepted August 17, 1974
A comparison has been made between the unsaturation of plasma-membrane phospholipids, present in the human erythrocyte, rat liver, mouse liver and a rapidly growing rat hepatoma. Of the double bonds present in the hydrocarbon chains of the membrane phospholipids, onethird is contributed by sphingomyelin plus phosphatidyl choline and the remainder by phosphatidyl serine, ethanolamine and inositol. Assuming that the phospholipids are asymmetrically distributed in the two leaflets of the bilayer in general, the consequences of this asymmetry in combination with cholesterol content and fatty acid distribution on plasma membrane organization and function are discussed. It is suggested, that the organizational disposition of plasma membrane components other than phospholipids is at least related if not dependent upon the latters' asymmetric distribution in the bilayer.
1. Introduction Recent studies have established that the phospholipids in the bilayer of the human erythrocyte membrane are asymmetrically distributed [ 1 - 3 ] . Phosphatidyl choline and sphingomyelin appear to be located in the outer leaflet, phosphatidyl ethanolamine and phosphatidyl serine in the inner leaflet. The various phospholipid classes of the erythrocyte membrane and hepatic plasma membranes each contain more or less specific hydrocarbon chains, e.g. differing in degree of unsaturation [4--7]. The asymmetric distribution of the phospholipids could therefore imply that inner and outer leaflet markedly differ in the unsaturation of their phospholipid hydrocarbon chains. Since unsaturation is the main parameter of lipid fluidity [8, 9], this, in turn, could mean that the two leaflets differ in fluidity.* The fluidity of a lipid membrane is determined by the intra- and intermolecular motions of the lipid hydrocarbon chains at a given temperature. These motions are inversely related to the degree of packing o f the chains which may vary from tightly packed in the case of saturated * Footnote: see next page.
P. Emmelot, R.P. van Hoeven, Phospholipid unsaturation
237
to very loosely packed in that of poly-unsaturated chains. Accordingly, the hydrocarbon chain composition of the membrane phospholipids will influence the cohesiveness of the membrane in terms of osmotic stability [ 10, 11 ], mechanical breaking and sealing [ 12], and permeability [ 10, 11 ], and affect the rate of lateral displacement or mobility of the phospholipids and proteins occurring in membranes [13-15]. In the present report we present quantitative data which substantiate the aforementioned assumptions, and discuss some possible consequences.
II. Phospholipid unsaturation The number of double bonds per molecule o f phospholipid species present in outer and inner leaflets, and the total unsaturation per leaflet of the human erythrocyte membrane are illustrated in table 1. Values were computed from data reviewed by Rouser et al. [4]. Table 1 also contains the corresponding values for plasma membranes of rat and mouse livers and a rat hepatoma, isolated and analyzed by the authors [7]. It is assumed as suggested by Bretscher [16], that mammalian plasma membranes other than from the erythrocyte exhibit the same asymmetric phospholipid distribution as does the latter. This view is substantiated by the finding [17] that the sum content of sphingomyelin plus phosphatidyl choline comprises about 50% of the total phospholipids present in hepatic plasma membranes, i.e. 52, 47, 50 and 4 6 - 4 8 % for respectively, rat liver, rat hepatoma, mouse liver and two mouse hepatomas. Inspection of table 1 indicates that the two phospholipids of the outer leaflet contain the least unsaturated hydrocarbon chains, and that in general about twice as many double bonds are present in the inner as in the outer bilayer leaflet of all plasma membranes examined (those from mouse hepatomas not illustrated). Accordingly, the outer leaflet should be markedly less fluid than the inner one. However, before this conclusion is drawn the content, location and effect of cholesterol in the bilayer must be considered. * Although for a given fatty acid chain length and unsaturation phosphatidyl choline shows a lower transition temperature than does phosphatidyl ethanolamine (the transition temperature Tc for e.g. the dimyristoyl derivatives being 23°C and 48°C, respectively) [9], the contribution of head group and chain length to the fluidity is much less than that of double bonds (cf. distearoyl and dioleoyl phosphatidyl choline showing a Tc of 58°C and 22°C, respectively) [8]. Differences in lipid fluidity between inner and outer leaflets of the phospholipid bilayer are thus somewhat smaller than those derived at on the basis of the mere presence of double bonds, as illustrated in table 1. For convenience the asymmetric distribution of the phospholipids has been taken as absolute. In fact small amounts of the various phospholipid classes may have a location opposite to that of their bulk amounts in the erythrocyte membrane [2, 3,81 ]. We are at present investigating whether this might also be accompanied by difference in fatty acyl unsaturation.
P. Emmelot, R.P. van Hoeven, Phospholipid unsaturation
238
Table 1 The unsaturation of plasma-membrane phospholipids. Phospholipids
Human erythrocyte a
Rat liver b
Mouse liver b
Rat hepatomab
Average number of double bonds per molecule
Outer leaflet Sphingomyelinc Phosphatidyl choline
0.26 1.97
0.24 3.12
0.27 2.38
0.13 1.98
2.95 3.16
4.66 4.24 3.64
4.90 4.78 3.68
2.36 2.98 2.88
lnner leaflet Phosphatidyl serine Phosphatidyl ethanolamine Phosphatidyl inositol
--
Number of double bonds contributed by a phospholipid to 100 molecules of total phospholipidsd
Outer leaflet Sphingomyelinc Phosphatidyl choline
7 57
6 94
6 64
3 48
64
100
70
51
38 85
70 81
64 96
26 66
22
15
123
173
175
101
187
273
245
152
Sum:
Inner leaflet Phosphatidyl serine Phosphatidyl ethanolamine Phosphatidyl inositol
--
Su m:
Outer plus inner
9
Cholesterol/PL-P(molar) e
0.81
0.65
0.80
0.89
Protein/phospholipid (w/w)
2.0
3.6
3.6
2.0
a Calculated from ref. [4]. b Refs. [7, 17]. c The double bond in the sphingosine moiety of sphingomyelin has been disregarded in the calculations since owing to its A4 position it has hardly, if any effect on lipid fluidity in the membrane. d (% composition) X (number of double bonds per molecule). e The cholesterol concentration appears to be increased if the average phospholipid molecule in the outer leaflet contains less than one double bond.
P. Ernmelot, R.P. van Hoeven, Phospholipid unsaturation
239
Ill. Cholesterol First, plasma membranes are characterized by a high content of cholesterol relative to phospholipid, as compared with intracellular membranes (table 1 ; ref. [ 17]). Secondly, various lines of evidence may suggest that cholesterol is concentrated in the outer leaflet of the plasma membrane [18, 19]. Thirdly, cholesterol has been shown to interact with the hydrocarbon chains of phospholipids and to impose upon them the "intermediate fluid condition", fluidizing gel and rigidizing liquid states of the chains at any temperature [8, 9]. By this dual effect the kinetic freedom of the methyl-terminated segments (from C10 on) of saturated and unsaturated hydrocarbon chains is differentially affected resulting in a motion of the two types of chains intermediate between that in the gel and liquid states. The conformational restriction [20] imposed by cholesterol on the hydrocarbon chains counteracts the temperature-induced transition in the physical state of the phospholipids. As a result a high cholesterol concentration relative to phospholipid (certainly at 1 : 1) abolishes the gel-to-liquid crystalline transition. The cholesterol : phospholipid molar ratio in mammalian plasma membranes (e.g. erythrocyte [21], thymocyte [22, 23], lymphocyte [24], hepatic [17] and corpus luteum [25] cells) is sufficiently high to obtain a molar ratio of 1.0 in the outer leaflet in which cholesterol is supposedly concentrated. Thus enough cholesterol is available to impose the intermediate fluid condition on at least all hydrocarbon chains present in the outer leaflet. The data of table 1 further show that cholesterol content and degree of unsaturation, especially of the phospholipids in the outer leaflet, are inversely related. The decrease in unsaturation of the rat-hepatoma in comparison with the corresponding liver membranes, is accompanied by a markedly increased cholesterol content. Also, a slight decrease was noted of the overall chain length by maximally one C atom for an individual phospholipid class [7], chain length being another, though weak parameter of fluidity [8]. Changes in fatty acyl saturation according to cholesterol content have also been observed in mycoplasma membrane [26]. These results suggest that a causal relation between degree of fatty acyl saturation and cholesterol content may exist in order to safeguard the proper fluidity of the membrane (outer leaflet). One may similarly ask if there exists a causal relation between the high unsaturation of the phospholipids in the inner leaflet and the alleged relative deficiency of cholesterol in that leaflet. Conceivably and from various published data [27, 28] it might be assumed that cholesterol would not be capable of establishing LondonVan der Waals interactions throughout very fluid, poly-unsaturated hydrocarbon chain areas at physiological temperature. If so, the asymmetric concentration of cholesterol in the two leaflets of the bilayer would be the result of the asymmetric distribution of the phospholipids. In this connexion it is of interest that recent results of Huang et al. [28] may indicate that incorporation of cholesterol (30 mole %) in egg lecithin bilayers causes an interleaflet shift of the more unsaturated molecules of this phospholipid class.
240
P. Emmelot, R.P. van Hoeven, Phospholipid unsaturation
It may also appear that cholesterol cannot to any large extent be accomodated physico-chemically in inner mitochondrial, endoplasmic reticulum (microsomal) and vertebrate rod outer segment membranes. These membranes, even the microsomal ones which are sites of cholesterol biosynthesis [29], contain very little cholesterol and sphingomyelin, whereas otherwise their phospholipids contain appreciable amounts of poly-unsaturated fatty acyls. By contrast, the myelin membrane is characterized by high cholesterol and sphingomyelin contents and a low concentration of poly-unsaturated fatty acids. (Comparative data in refs. [17, 30-32].) However, even if cholesterol would be (a) equally divided over the plasma-membrane bilayer and (b) capable of imposing intermediate fluidity on the unsaturated fatty acyls of the phospholipids in the inner leaflet, that leaflet would still be more fluid and less coherent than the outer leaflet. This may be inferred from results obtained by De Gier and Van Deenen [ 10, 11 ] but also directly from the greater fluidity of egg-yolk lecithin (poly-unsaturated)-cholesterol than of dipalmitoyl lecithincholesterol complexes [9].
IV. Fluidity of the plasma membrane bilayer Accordingly, a cholesterol-"buffered" outer leaflet and a more fluid inner leaflet of the phospholipid bilayer may be characteristic features of the plasma membrane and have organizational and functional significance. In the absence of cholesterol the saturated sphingomyelin would tend to attain the gel state forming immobilized patches (in the case of liver plasma membranes occupying some 40% of the surface area) that hamper translateral movement of surface proteins and also counteract the dynamic formation of membrane processes such as microvilli, and membrane flow instrumental in cell locomotion. The gel state may also confer "brittleness" to the membrane [33, 34]. Cholesterol, by imposing the intermediate fluid condition, increases the fluidity of the saturated hydrocarbon chains of sphingomyelin and decreases that of the unsaturated ones of phosphatidyl choline, and thus "harmonizes" the overall fluidity of the outer leaflet. As a consequence a well-packed yet flexible barrier counteracting random permeation of molecules results. The stabilizing effects of sphingomyelin and cholesterol on the erythrocyte membrane have also been demonstrated more directly [10, 11, 35], but it should be noted that an unwarranted increase in the cholesterol content without being matched by a change in phospholipid unsaturation, could be adverse to membrane function [34, 36]. Thus by its specific lipid composition the o u t e r leaflet of the plasma membrane bilayer forms the barrier against the environment and in this respect resembles the insulating myelin membrane. By contrast, the inner leaflet of the plasma membrane forms a more fluid and permeable structure and thus resembles intracellular membranes, such as the endoplasmic reticulum membrane which is apt to pinch off vesicles. This compatibility of structure may be important in exoand endocytotic processes. Of interest in this connexio~a is also that mitochondrial
P. Emmelot, R.P. van Hoeven, Phospholipid unsaturation
241
outer membrane phospholipids contain more saturated fatty acids than do the inner membranes ones, whereas the latter are rich in unsaturated fatty acids [37]; these conditions may be instrumental in the differences in plasticity of these two membranes [38]. The state of intermediate fluidity of the phospholipids (of the outer leaflet) of the plasma membrane could be a factor causing the lateral diffusion of lipid in the erythrocyte membrane [39] and of antigen-antibody complexes on the surface of other mammalian cells [ 15] to be at least one order of magnitude lower than that calculated for the rapid lateral diffusion of spin-labeled phospholipid in lecithin bilayer membranes and more fluid biomembranes [40, 41, 80]. Recent studies have shown that the physical state of the phospholipids may control various enzymic functions in bacterial [42-46] and mycoplasma [47] membranes, which contain little or no cholesterol and a rather simple fatty acyl spectrum. These functions show abrupt increases in rate ("break"; decrease in activation energy) at certain characteristic temperatures at which the membrane phospholipids undergo phase transition or lateral separation [45, 46] ("melt"), and they thus appear to be critically dependent on the fluid state which allows rapid lateral diffusion of the phospholipids. However, since cholesterol abolishes the gel-to-liquid phase transition [8, 9, 48] (and, by inference, lateral phase separation) [46, 47, 49] it may follow that temperature changes will only affect the viscosity of the phospholipids in the outer leaflet of mammalian plasma membranes provided that no microheterogeneity in the distribution of cholesterol and phospholipid in the plane of the membrane exists. In that case, an enzymic process (e.g. hormone-activated adenylate cyclase) [50] in a mammalian plasma membrane (liver) exhibiting a sharp discontinuity at a distinct temperature (32°C) is not likely to have its temperature-sensitive domain in the outer leaflet of the phospholipid bilayer. Since the high unsaturation of the inner leaflet phospholipids allows fluidity over a broad temperature traject encompassing 32°C, it would appear that neither the inner leaflet phospholipids could be involved in the 32°C "break". It should be noted that both cholesterol [47, 51] and poly-unsaturated phospholipids (egg phosphatidyl glycerol and phosphatidyl serine [52, 53]) have been shown to affect or abolish the break in ATPase activities of various membrane systems. Accordingly either another mechanism such as an effect of the temperature on the enzyme itself, or the location of the enzyme in a privileged lipid microdomain should be considered. The latter could be accounted for by the clustering of cholesterol molecules [54, 55] leading to cholesterol-poor regions, and the association of the enzyme with particular molecules of a phospholipid class. The former possibility has been experimentally dismissed for a (Na + K +) ATPase membrane preparation [56]. In the latter case the enzyme activity break would monitor a phase change in the lipid microdomain of the enzyme. V. Plasma membrane proteins
It may further be argued that the outer leaflet of the plasma membrane, in accor-
242
P. Emmelot, R.P. van Hoeven, Phospholipicl unsaturation
dance with its barrier function, is less penetrable by polar protein (segments) than is the inner leaflet. The higher unsaturation of the inner leaflet phospholipids will create a more polar milieu than that present in the outer leaflet and accordingly more protein may become dispersed in the inner than in the outer leaflet. Actually, the inner side of the erythrocyte membrane contains more protein than does the outer side [16]. According to the type of agent by which proteins can be released from the human erythrocyte membrane, three classes of protein have been distinguished, viz. integral (intrinsic) proteins, peripheral (extrinsic) proteins [57-59] and, most recently, a third, intermediate class of proteins [60]. Integral proteins can only be solubilized by hydrophobic-bond breaking agents such as detergents, whereas the most tenuously bound, peripheral proteins (the myosin- and actin-like spectrin molecules and glyceraldehyde-3-phosphate dehydrogenase) are released by "mild" electrostatic-bond breaking agents. The intermediate class of proteins is removed by protein "perturbants" without dissolution of the membrane core. The integral proteins principally consist of the glycoproteins, and the other two classes contain the non-glycosylated proteins located at/in the inner face of the erythrocyte membrane [60, 61] These 3 classes of proteins may grosso modo be assigned to the following locations. The integral proteins which are anchored in the hydrophobic domain are dispersed in the outer leaflet (e.g. acetylcholinesterease [62]; 5'-nucleotidase in the case of liver plasma membranes [63, 64]) or transversing the entire membrane width (glycophorin and the main erythrocyte membrane protein [65, 66]). The spectrin molecules and glyceraldehyde-3-phosphate dehydrogenase are superficially bound by electrostatic interactions at the cytoplasmic side [60, 61, 67] to, respectively, the two membrane-spanning proteins [68] and the inner face proteins which constitute the third, intermediate class of proteins. The latter proteins may penetrate the more polar milieu afforded by the highly unsaturated and loosely-packed fatty acyls of the inner leaflet of the bilayer. These proteins seem to interact both mutually and with phospholipid head groups primarily through polar bonds (hydrogen and electrostatic bonds) though 7r electron interactions between fatty acyl double bonds and aromatic and heterocyclic rings of amino acids might also be involved. Accordingly, the organizational disposition of the various membrane proteins may be related to the physico-chemical conditions existing in the phospholipid bilayer. The overall transverse distribution of protein in the membrane may be described as iceberg like, with carbohydrate antennae on the tip and cytoplasmic constituents linked to the base. If one prospects for the various membrane strata in terms of polarity one would encounter a very polar outer periphery containing carbohydrate, followed by the most hydrophobic domain in the outer phospholipid layer, a less hydrophobic and more polar inner layer, and, finally, a very polar cytoplasmic border line. Whereas the binding of micro filaments at the latter side may be genuine [69, 70] and persistent, the binding of other cytoplasmic proteins such as glyceraldehyde 3-P dehydrogenase may perhaps only be temporal or even, in the case of liver plasma membranes [71], constitute an artifact resulting from the meth-
P. Emmelot, R.P. van Hoeven, Phospholipid unsaturation
243
od used for isolating the membranes. The condition in the inner leaflet of the bilayer might also be such as to allow the transient penetration of cytoplasmic protein (enzymes?).
Vi. Sidedness of enzymes
The asymmetric disposition of membrane proteins in respect of the two membrane faces reflects their function and this is most apparent for membrane enzymes. For example, the outward faced 5'-nucleotidase may allow nucleotides to be taken up as nucleosides, and glycosyl transferases may act in intercellular contact [72] and related phenomena [73]. In addition, the particular location of an enzyme or part of an enzymic sequence or process may reflect the dependence of its function on a specific lipid environment. Thus, the vectorial [74] enzyme systems (Na + K+) ATPase and hormone-activated adenylate cyclase, which effectively span the entire membrane width, appear to be reactivated in delipidated membrane preparations by phosphatidyl serine [74, 75] or phosphatidyl inositol [75] in particular. If this reflects the native situation it may be significant that both phospholipids are anionic and that the former is, and the latter (which is lacking in human erythrocyte membranes) may for stoichiometric reasons be assumed to be an inner leaflet component (table 1). The fatty acyl profiles of these two phospholipids are highly unsaturated and resemble each other more than those of the other phospholipid classes (fig. 1). Parenthetically, the recent conclusion [76] that phosphatidyl ethanolamine may activate the (Na+-K +) ATPase equally well as phosphatidyl serine, which was based on the finding that phosphatidyl serine in a (Na ÷ - K ÷) ATPase preparation could be converted without loss of enzyme activity into phosphatidyl ethanolamine by serine decarboxylase, may not be correct if the fluidity of the hydrocarbon chains determines the activity. The catalytic units of the (Na ÷ K÷) ATPase and hormone-activated adenylate cyclase are located at the cytoplasmic side of the surface membrane [75, 77]. Accordingly, the fluid inner-leaflet phospholipids may in the case of adenylate cyclase act in the transduction step linking the hormone-receptor interaction at the outerface with the catalytic unit at the innerface of the membrane. Similarly, the fluid phosphatidyl serine may allow enzyme rotation should this be required, or conformational change [78] occurring in the course of the (Na+-K +) ATPase reaction at the cytoplasmic side. Hormone-activated adenylate cyclase and the (Na+-K +) ATPase seem to require a fluid phospholipid "halo", a lipid microdomain rich in poly-unsaturated hydrocarbon chains. In fact it has been found [79] that the interior of the bilayer immediate to the (Na+-K +) ATPase has a more fluid character than has the rest of the bilayer. In conclusion it is suggested that the organizational disposition of plasma membrane components other than phospholipids is related to, if it does not depend on, the latter's asymmetric distribution in the bilayer.
P. Emmelot, R.P. van Hoeven, Phospholipid unsaturation
244
[]
phosphatidyl choline
[]
sphingomyelin
[]
phosphatidyl ethanolamine
[]
phosphatidyl serine
t~1 phosphatidyl inositol
80 70
,,outer leaflet" phospholipids
60 50 40 30 _
•
20
E
10_
] 0
60
r-] 1
2
3
4
5
r~ 6
_
50 _
,,inner leaflet" phospholipids
40_
30 _ _~ 2 0 _ 0
E
10_
0
1
2
3
4
5
6
SATURATES
MONOENES
DIENES
TRIENES
TETRAENES
PENTAENES
HEXAENES
16:0 18:0 20:0 22:0 23:0 24:0
16:1 18:1 231 24:1
18:2
20:3
20:4
20:5 22:5
226
Fig. 1. Fatty acyl profiles in phospholipid classes derived from isolated rat-liver plasma membranes (adapted from ref. [ 7 ] ).
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P. Emmelot, R.P. van Hoeven, Phospholipid unsaturation
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PHOSPHOLIPID UNSATURATION AND PLASMA MEMBRANE ORGANIZATION P. EMMELOT and R.P. VAN HOEVEN Department of Biochemistry, Antoni van Leeuwenhoek-Laboratory, The Netherlands Cancer Institute, Amsterdam, The Netherlands
Received June 25, 1974,
accepted August 17, 1974
A comparison has been made between the unsaturation of plasma-membrane phospholipids, present in the human erythrocyte, rat liver, mouse liver and a rapidly growing rat hepatoma. Of the double bonds present in the hydrocarbon chains of the membrane phospholipids, onethird is contributed by sphingomyelin plus phosphatidyl choline and the remainder by phosphatidyl serine, ethanolamine and inositol. Assuming that the phospholipids are asymmetrically distributed in the two leaflets of the bilayer in general, the consequences of this asymmetry in combination with cholesterol content and fatty acid distribution on plasma membrane organization and function are discussed. It is suggested, that the organizational disposition of plasma membrane components other than phospholipids is at least related if not dependent upon the latters' asymmetric distribution in the bilayer.
1. Introduction Recent studies have established that the phospholipids in the bilayer of the human erythrocyte membrane are asymmetrically distributed [ 1 - 3 ] . Phosphatidyl choline and sphingomyelin appear to be located in the outer leaflet, phosphatidyl ethanolamine and phosphatidyl serine in the inner leaflet. The various phospholipid classes of the erythrocyte membrane and hepatic plasma membranes each contain more or less specific hydrocarbon chains, e.g. differing in degree of unsaturation [4--7]. The asymmetric distribution of the phospholipids could therefore imply that inner and outer leaflet markedly differ in the unsaturation of their phospholipid hydrocarbon chains. Since unsaturation is the main parameter of lipid fluidity [8, 9], this, in turn, could mean that the two leaflets differ in fluidity.* The fluidity of a lipid membrane is determined by the intra- and intermolecular motions of the lipid hydrocarbon chains at a given temperature. These motions are inversely related to the degree of packing o f the chains which may vary from tightly packed in the case of saturated * Footnote: see next page.
P. Emmelot, R.P. van Hoeven, Phospholipid unsaturation
237
to very loosely packed in that of poly-unsaturated chains. Accordingly, the hydrocarbon chain composition of the membrane phospholipids will influence the cohesiveness of the membrane in terms of osmotic stability [ 10, 11 ], mechanical breaking and sealing [ 12], and permeability [ 10, 11 ], and affect the rate of lateral displacement or mobility of the phospholipids and proteins occurring in membranes [13-15]. In the present report we present quantitative data which substantiate the aforementioned assumptions, and discuss some possible consequences.
II. Phospholipid unsaturation The number of double bonds per molecule o f phospholipid species present in outer and inner leaflets, and the total unsaturation per leaflet of the human erythrocyte membrane are illustrated in table 1. Values were computed from data reviewed by Rouser et al. [4]. Table 1 also contains the corresponding values for plasma membranes of rat and mouse livers and a rat hepatoma, isolated and analyzed by the authors [7]. It is assumed as suggested by Bretscher [16], that mammalian plasma membranes other than from the erythrocyte exhibit the same asymmetric phospholipid distribution as does the latter. This view is substantiated by the finding [17] that the sum content of sphingomyelin plus phosphatidyl choline comprises about 50% of the total phospholipids present in hepatic plasma membranes, i.e. 52, 47, 50 and 4 6 - 4 8 % for respectively, rat liver, rat hepatoma, mouse liver and two mouse hepatomas. Inspection of table 1 indicates that the two phospholipids of the outer leaflet contain the least unsaturated hydrocarbon chains, and that in general about twice as many double bonds are present in the inner as in the outer bilayer leaflet of all plasma membranes examined (those from mouse hepatomas not illustrated). Accordingly, the outer leaflet should be markedly less fluid than the inner one. However, before this conclusion is drawn the content, location and effect of cholesterol in the bilayer must be considered. * Although for a given fatty acid chain length and unsaturation phosphatidyl choline shows a lower transition temperature than does phosphatidyl ethanolamine (the transition temperature Tc for e.g. the dimyristoyl derivatives being 23°C and 48°C, respectively) [9], the contribution of head group and chain length to the fluidity is much less than that of double bonds (cf. distearoyl and dioleoyl phosphatidyl choline showing a Tc of 58°C and 22°C, respectively) [8]. Differences in lipid fluidity between inner and outer leaflets of the phospholipid bilayer are thus somewhat smaller than those derived at on the basis of the mere presence of double bonds, as illustrated in table 1. For convenience the asymmetric distribution of the phospholipids has been taken as absolute. In fact small amounts of the various phospholipid classes may have a location opposite to that of their bulk amounts in the erythrocyte membrane [2, 3,81 ]. We are at present investigating whether this might also be accompanied by difference in fatty acyl unsaturation.
P. Emmelot, R.P. van Hoeven, Phospholipid unsaturation
238
Table 1 The unsaturation of plasma-membrane phospholipids. Phospholipids
Human erythrocyte a
Rat liver b
Mouse liver b
Rat hepatomab
Average number of double bonds per molecule
Outer leaflet Sphingomyelinc Phosphatidyl choline
0.26 1.97
0.24 3.12
0.27 2.38
0.13 1.98
2.95 3.16
4.66 4.24 3.64
4.90 4.78 3.68
2.36 2.98 2.88
lnner leaflet Phosphatidyl serine Phosphatidyl ethanolamine Phosphatidyl inositol
--
Number of double bonds contributed by a phospholipid to 100 molecules of total phospholipidsd
Outer leaflet Sphingomyelinc Phosphatidyl choline
7 57
6 94
6 64
3 48
64
100
70
51
38 85
70 81
64 96
26 66
22
15
123
173
175
101
187
273
245
152
Sum:
Inner leaflet Phosphatidyl serine Phosphatidyl ethanolamine Phosphatidyl inositol
--
Su m:
Outer plus inner
9
Cholesterol/PL-P(molar) e
0.81
0.65
0.80
0.89
Protein/phospholipid (w/w)
2.0
3.6
3.6
2.0
a Calculated from ref. [4]. b Refs. [7, 17]. c The double bond in the sphingosine moiety of sphingomyelin has been disregarded in the calculations since owing to its A4 position it has hardly, if any effect on lipid fluidity in the membrane. d (% composition) X (number of double bonds per molecule). e The cholesterol concentration appears to be increased if the average phospholipid molecule in the outer leaflet contains less than one double bond.
P. Ernmelot, R.P. van Hoeven, Phospholipid unsaturation
239
Ill. Cholesterol First, plasma membranes are characterized by a high content of cholesterol relative to phospholipid, as compared with intracellular membranes (table 1 ; ref. [ 17]). Secondly, various lines of evidence may suggest that cholesterol is concentrated in the outer leaflet of the plasma membrane [18, 19]. Thirdly, cholesterol has been shown to interact with the hydrocarbon chains of phospholipids and to impose upon them the "intermediate fluid condition", fluidizing gel and rigidizing liquid states of the chains at any temperature [8, 9]. By this dual effect the kinetic freedom of the methyl-terminated segments (from C10 on) of saturated and unsaturated hydrocarbon chains is differentially affected resulting in a motion of the two types of chains intermediate between that in the gel and liquid states. The conformational restriction [20] imposed by cholesterol on the hydrocarbon chains counteracts the temperature-induced transition in the physical state of the phospholipids. As a result a high cholesterol concentration relative to phospholipid (certainly at 1 : 1) abolishes the gel-to-liquid crystalline transition. The cholesterol : phospholipid molar ratio in mammalian plasma membranes (e.g. erythrocyte [21], thymocyte [22, 23], lymphocyte [24], hepatic [17] and corpus luteum [25] cells) is sufficiently high to obtain a molar ratio of 1.0 in the outer leaflet in which cholesterol is supposedly concentrated. Thus enough cholesterol is available to impose the intermediate fluid condition on at least all hydrocarbon chains present in the outer leaflet. The data of table 1 further show that cholesterol content and degree of unsaturation, especially of the phospholipids in the outer leaflet, are inversely related. The decrease in unsaturation of the rat-hepatoma in comparison with the corresponding liver membranes, is accompanied by a markedly increased cholesterol content. Also, a slight decrease was noted of the overall chain length by maximally one C atom for an individual phospholipid class [7], chain length being another, though weak parameter of fluidity [8]. Changes in fatty acyl saturation according to cholesterol content have also been observed in mycoplasma membrane [26]. These results suggest that a causal relation between degree of fatty acyl saturation and cholesterol content may exist in order to safeguard the proper fluidity of the membrane (outer leaflet). One may similarly ask if there exists a causal relation between the high unsaturation of the phospholipids in the inner leaflet and the alleged relative deficiency of cholesterol in that leaflet. Conceivably and from various published data [27, 28] it might be assumed that cholesterol would not be capable of establishing LondonVan der Waals interactions throughout very fluid, poly-unsaturated hydrocarbon chain areas at physiological temperature. If so, the asymmetric concentration of cholesterol in the two leaflets of the bilayer would be the result of the asymmetric distribution of the phospholipids. In this connexion it is of interest that recent results of Huang et al. [28] may indicate that incorporation of cholesterol (30 mole %) in egg lecithin bilayers causes an interleaflet shift of the more unsaturated molecules of this phospholipid class.
240
P. Emmelot, R.P. van Hoeven, Phospholipid unsaturation
It may also appear that cholesterol cannot to any large extent be accomodated physico-chemically in inner mitochondrial, endoplasmic reticulum (microsomal) and vertebrate rod outer segment membranes. These membranes, even the microsomal ones which are sites of cholesterol biosynthesis [29], contain very little cholesterol and sphingomyelin, whereas otherwise their phospholipids contain appreciable amounts of poly-unsaturated fatty acyls. By contrast, the myelin membrane is characterized by high cholesterol and sphingomyelin contents and a low concentration of poly-unsaturated fatty acids. (Comparative data in refs. [17, 30-32].) However, even if cholesterol would be (a) equally divided over the plasma-membrane bilayer and (b) capable of imposing intermediate fluidity on the unsaturated fatty acyls of the phospholipids in the inner leaflet, that leaflet would still be more fluid and less coherent than the outer leaflet. This may be inferred from results obtained by De Gier and Van Deenen [ 10, 11 ] but also directly from the greater fluidity of egg-yolk lecithin (poly-unsaturated)-cholesterol than of dipalmitoyl lecithincholesterol complexes [9].
IV. Fluidity of the plasma membrane bilayer Accordingly, a cholesterol-"buffered" outer leaflet and a more fluid inner leaflet of the phospholipid bilayer may be characteristic features of the plasma membrane and have organizational and functional significance. In the absence of cholesterol the saturated sphingomyelin would tend to attain the gel state forming immobilized patches (in the case of liver plasma membranes occupying some 40% of the surface area) that hamper translateral movement of surface proteins and also counteract the dynamic formation of membrane processes such as microvilli, and membrane flow instrumental in cell locomotion. The gel state may also confer "brittleness" to the membrane [33, 34]. Cholesterol, by imposing the intermediate fluid condition, increases the fluidity of the saturated hydrocarbon chains of sphingomyelin and decreases that of the unsaturated ones of phosphatidyl choline, and thus "harmonizes" the overall fluidity of the outer leaflet. As a consequence a well-packed yet flexible barrier counteracting random permeation of molecules results. The stabilizing effects of sphingomyelin and cholesterol on the erythrocyte membrane have also been demonstrated more directly [10, 11, 35], but it should be noted that an unwarranted increase in the cholesterol content without being matched by a change in phospholipid unsaturation, could be adverse to membrane function [34, 36]. Thus by its specific lipid composition the o u t e r leaflet of the plasma membrane bilayer forms the barrier against the environment and in this respect resembles the insulating myelin membrane. By contrast, the inner leaflet of the plasma membrane forms a more fluid and permeable structure and thus resembles intracellular membranes, such as the endoplasmic reticulum membrane which is apt to pinch off vesicles. This compatibility of structure may be important in exoand endocytotic processes. Of interest in this connexio~a is also that mitochondrial
P. Emmelot, R.P. van Hoeven, Phospholipid unsaturation
241
outer membrane phospholipids contain more saturated fatty acids than do the inner membranes ones, whereas the latter are rich in unsaturated fatty acids [37]; these conditions may be instrumental in the differences in plasticity of these two membranes [38]. The state of intermediate fluidity of the phospholipids (of the outer leaflet) of the plasma membrane could be a factor causing the lateral diffusion of lipid in the erythrocyte membrane [39] and of antigen-antibody complexes on the surface of other mammalian cells [ 15] to be at least one order of magnitude lower than that calculated for the rapid lateral diffusion of spin-labeled phospholipid in lecithin bilayer membranes and more fluid biomembranes [40, 41, 80]. Recent studies have shown that the physical state of the phospholipids may control various enzymic functions in bacterial [42-46] and mycoplasma [47] membranes, which contain little or no cholesterol and a rather simple fatty acyl spectrum. These functions show abrupt increases in rate ("break"; decrease in activation energy) at certain characteristic temperatures at which the membrane phospholipids undergo phase transition or lateral separation [45, 46] ("melt"), and they thus appear to be critically dependent on the fluid state which allows rapid lateral diffusion of the phospholipids. However, since cholesterol abolishes the gel-to-liquid phase transition [8, 9, 48] (and, by inference, lateral phase separation) [46, 47, 49] it may follow that temperature changes will only affect the viscosity of the phospholipids in the outer leaflet of mammalian plasma membranes provided that no microheterogeneity in the distribution of cholesterol and phospholipid in the plane of the membrane exists. In that case, an enzymic process (e.g. hormone-activated adenylate cyclase) [50] in a mammalian plasma membrane (liver) exhibiting a sharp discontinuity at a distinct temperature (32°C) is not likely to have its temperature-sensitive domain in the outer leaflet of the phospholipid bilayer. Since the high unsaturation of the inner leaflet phospholipids allows fluidity over a broad temperature traject encompassing 32°C, it would appear that neither the inner leaflet phospholipids could be involved in the 32°C "break". It should be noted that both cholesterol [47, 51] and poly-unsaturated phospholipids (egg phosphatidyl glycerol and phosphatidyl serine [52, 53]) have been shown to affect or abolish the break in ATPase activities of various membrane systems. Accordingly either another mechanism such as an effect of the temperature on the enzyme itself, or the location of the enzyme in a privileged lipid microdomain should be considered. The latter could be accounted for by the clustering of cholesterol molecules [54, 55] leading to cholesterol-poor regions, and the association of the enzyme with particular molecules of a phospholipid class. The former possibility has been experimentally dismissed for a (Na + K +) ATPase membrane preparation [56]. In the latter case the enzyme activity break would monitor a phase change in the lipid microdomain of the enzyme. V. Plasma membrane proteins
It may further be argued that the outer leaflet of the plasma membrane, in accor-
242
P. Emmelot, R.P. van Hoeven, Phospholipicl unsaturation
dance with its barrier function, is less penetrable by polar protein (segments) than is the inner leaflet. The higher unsaturation of the inner leaflet phospholipids will create a more polar milieu than that present in the outer leaflet and accordingly more protein may become dispersed in the inner than in the outer leaflet. Actually, the inner side of the erythrocyte membrane contains more protein than does the outer side [16]. According to the type of agent by which proteins can be released from the human erythrocyte membrane, three classes of protein have been distinguished, viz. integral (intrinsic) proteins, peripheral (extrinsic) proteins [57-59] and, most recently, a third, intermediate class of proteins [60]. Integral proteins can only be solubilized by hydrophobic-bond breaking agents such as detergents, whereas the most tenuously bound, peripheral proteins (the myosin- and actin-like spectrin molecules and glyceraldehyde-3-phosphate dehydrogenase) are released by "mild" electrostatic-bond breaking agents. The intermediate class of proteins is removed by protein "perturbants" without dissolution of the membrane core. The integral proteins principally consist of the glycoproteins, and the other two classes contain the non-glycosylated proteins located at/in the inner face of the erythrocyte membrane [60, 61] These 3 classes of proteins may grosso modo be assigned to the following locations. The integral proteins which are anchored in the hydrophobic domain are dispersed in the outer leaflet (e.g. acetylcholinesterease [62]; 5'-nucleotidase in the case of liver plasma membranes [63, 64]) or transversing the entire membrane width (glycophorin and the main erythrocyte membrane protein [65, 66]). The spectrin molecules and glyceraldehyde-3-phosphate dehydrogenase are superficially bound by electrostatic interactions at the cytoplasmic side [60, 61, 67] to, respectively, the two membrane-spanning proteins [68] and the inner face proteins which constitute the third, intermediate class of proteins. The latter proteins may penetrate the more polar milieu afforded by the highly unsaturated and loosely-packed fatty acyls of the inner leaflet of the bilayer. These proteins seem to interact both mutually and with phospholipid head groups primarily through polar bonds (hydrogen and electrostatic bonds) though 7r electron interactions between fatty acyl double bonds and aromatic and heterocyclic rings of amino acids might also be involved. Accordingly, the organizational disposition of the various membrane proteins may be related to the physico-chemical conditions existing in the phospholipid bilayer. The overall transverse distribution of protein in the membrane may be described as iceberg like, with carbohydrate antennae on the tip and cytoplasmic constituents linked to the base. If one prospects for the various membrane strata in terms of polarity one would encounter a very polar outer periphery containing carbohydrate, followed by the most hydrophobic domain in the outer phospholipid layer, a less hydrophobic and more polar inner layer, and, finally, a very polar cytoplasmic border line. Whereas the binding of micro filaments at the latter side may be genuine [69, 70] and persistent, the binding of other cytoplasmic proteins such as glyceraldehyde 3-P dehydrogenase may perhaps only be temporal or even, in the case of liver plasma membranes [71], constitute an artifact resulting from the meth-
P. Emmelot, R.P. van Hoeven, Phospholipid unsaturation
243
od used for isolating the membranes. The condition in the inner leaflet of the bilayer might also be such as to allow the transient penetration of cytoplasmic protein (enzymes?).
Vi. Sidedness of enzymes
The asymmetric disposition of membrane proteins in respect of the two membrane faces reflects their function and this is most apparent for membrane enzymes. For example, the outward faced 5'-nucleotidase may allow nucleotides to be taken up as nucleosides, and glycosyl transferases may act in intercellular contact [72] and related phenomena [73]. In addition, the particular location of an enzyme or part of an enzymic sequence or process may reflect the dependence of its function on a specific lipid environment. Thus, the vectorial [74] enzyme systems (Na + K+) ATPase and hormone-activated adenylate cyclase, which effectively span the entire membrane width, appear to be reactivated in delipidated membrane preparations by phosphatidyl serine [74, 75] or phosphatidyl inositol [75] in particular. If this reflects the native situation it may be significant that both phospholipids are anionic and that the former is, and the latter (which is lacking in human erythrocyte membranes) may for stoichiometric reasons be assumed to be an inner leaflet component (table 1). The fatty acyl profiles of these two phospholipids are highly unsaturated and resemble each other more than those of the other phospholipid classes (fig. 1). Parenthetically, the recent conclusion [76] that phosphatidyl ethanolamine may activate the (Na+-K +) ATPase equally well as phosphatidyl serine, which was based on the finding that phosphatidyl serine in a (Na ÷ - K ÷) ATPase preparation could be converted without loss of enzyme activity into phosphatidyl ethanolamine by serine decarboxylase, may not be correct if the fluidity of the hydrocarbon chains determines the activity. The catalytic units of the (Na ÷ K÷) ATPase and hormone-activated adenylate cyclase are located at the cytoplasmic side of the surface membrane [75, 77]. Accordingly, the fluid inner-leaflet phospholipids may in the case of adenylate cyclase act in the transduction step linking the hormone-receptor interaction at the outerface with the catalytic unit at the innerface of the membrane. Similarly, the fluid phosphatidyl serine may allow enzyme rotation should this be required, or conformational change [78] occurring in the course of the (Na+-K +) ATPase reaction at the cytoplasmic side. Hormone-activated adenylate cyclase and the (Na+-K +) ATPase seem to require a fluid phospholipid "halo", a lipid microdomain rich in poly-unsaturated hydrocarbon chains. In fact it has been found [79] that the interior of the bilayer immediate to the (Na+-K +) ATPase has a more fluid character than has the rest of the bilayer. In conclusion it is suggested that the organizational disposition of plasma membrane components other than phospholipids is related to, if it does not depend on, the latter's asymmetric distribution in the bilayer.
P. Emmelot, R.P. van Hoeven, Phospholipid unsaturation
244
[]
phosphatidyl choline
[]
sphingomyelin
[]
phosphatidyl ethanolamine
[]
phosphatidyl serine
t~1 phosphatidyl inositol
80 70
,,outer leaflet" phospholipids
60 50 40 30 _
•
20
E
10_
] 0
60
r-] 1
2
3
4
5
r~ 6
_
50 _
,,inner leaflet" phospholipids
40_
30 _ _~ 2 0 _ 0
E
10_
0
1
2
3
4
5
6
SATURATES
MONOENES
DIENES
TRIENES
TETRAENES
PENTAENES
HEXAENES
16:0 18:0 20:0 22:0 23:0 24:0
16:1 18:1 231 24:1
18:2
20:3
20:4
20:5 22:5
226
Fig. 1. Fatty acyl profiles in phospholipid classes derived from isolated rat-liver plasma membranes (adapted from ref. [ 7 ] ).
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