Phases and phase transitions of the sphingolipids

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Biochimica et Biophysica Acta 1255 (1995) 213-236

etBiochi~£ BiophysicaA~ta

Review

Phases and phase transitions of the sphingolipids Rumiana Koynova 1, Martin Caffrey * Department of Chemistry, The Ohio State Unil,ersity, Columbus, OH 43210-1173, USA Received 14 April 1994; revised 22 September 1994; accepted 18 October 1994

Keywords: Cerebroside; Glycolipid; LIPIDAT; Lyotropic; Sphingomyelin; Thermotropic

Contents 1. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

214

2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

214

3. Bibliographic data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

214

4. Thermodynamic data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Sphingomyelins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Cerebrosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Sulfocerebrosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. a-Hydroxy N-acylated sulfocerebrosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Extracts from biological sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6. Dry sphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7. Sphingolipid/additive mixtures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

215 215 220 224 225 227 230 233

5. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

233

Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

233

Appendix 1: Lipid nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

234

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

234

Abbreviations: c, double bond of the cis type; d, days; DMPC, dimyristoylphosphatidylcholine, also 14:0/14:0 PC; DSC, differential scanning calorimetry; fa, fatty acid; Gal, o-galactose; GalNAc, N-acetyl-D-galactosamine; Glc, D-glucose; HI, normal hexagonal liquid crystalline phase; HII, inverted hexagonal liquid crystalline phase; IR, infra-red; l.c., liquid crystalline; L,~, lamellar liquid crystalline phase; Lac, lactose (O-/3-o-galactopyranosyl-(1 ~ 4)-ot-o-glucopyranoside); LO, lamellar gel phase; Lc, lamellar crystalline (subgel) phase; NANA, N-acetylneuraminic acid; NeuGc, N-glycoloylneuraminic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; Sph, sphingosine (4-sphingenine) base; Spn, dihydrosphingosine (sphinganine) base; Spd, generalised sphingoid base (refers to sphinganine, to its homologs and stereoisomers and to its hydroxy and unsaturated derivatives); SUV, small unilamellar vesicles; t, double bond of the trans type; T, transition temperature; XRD, X-ray diffraction; AH, transition enthalpy change * Corresponding author. i Permanent address: Central Laboratory of Biophysics, Bulgarian Academy of Science, 1113 Sofia, Bulgaria. 0005-2760/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 5 - 2 7 6 0 ( 9 4 ) 0 0 2 0 2 - 9

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R. Koynova, M. Caffrey /Biochimica et Biophysica Acta 1255 (1995) 213-236

I. Summary LIPIDAT is a computerized database providing access to the wealth of information scattered throughout the literature concerning synthetic and biologically derived polar lipid polymorphic and mesomorphic phase behavior. Herein, we present a review of the LIPIDAT data subset referring to sphingolipids together with an analysis of these data. It includes data collected over a 40-year period and consists of 867 records obtained from 112 articles in 25 different journals. An analysis of these data has allowed us to identify trends in hydrated sphingolipid phase behavior reflecting differences in fatty acyl chain length, saturation and hydroxylation, head group type, and sphingoid base identity. Information on the mesomorphism of biologically-derived and dry sphingolipids is also presented. This review includes 161 references.

2. Introduction In response to the obvious and immediate need for a centralized compendium of lipid thermodynamic data, the LIPIDAT database was established [1-3]. Its purpose is to collect, in a central location, all information on lipid mesomorphic and polymorphic transitions and miscibility. The database is considered comprehensive for glycerophospholipids, sphingolipids, glycoglycerolipids and biological membrane lipid extracts. The sphingolipids, together with the glycerophospholipids, glycoglycerolipids, and cholesterol, represent the major lipid components of biological membranes. Several sphingolipid species are found in the membranes of mammalian tissues - principally brain, but also liver, heart, and erythrocytes [10,12]. Sphingolipids are also present in the membranes of marine invertebrates [11], plants [12-14] and bacteria [9,10]. In comparison to the glycerophospholipids, the mesophase behavior of the sphingolipids has been less well studied. This, perhaps, reflects the enormous diversity in the head group constitution of these lipids which gives rise to a correspondingly diverse polymorphism and mesomorphism. Another possible reason for the paucity of data on this important lipid group arises from the fact that the chemical synthesis of the sphingolipids is considerably more complex and less far advanced than that of glycerophospholipids [18]. As a result, much of the work in this area is done with semisynthetic species which have less well-defined mesophase behavior in part because of an inherent sample heterogeneity. Nonetheless, there exists a sizeable body of literature on the thermotropic properties of the sphingolipids. The following reviews provide important background material on the sphingolipids as a class [15-20,133-135]. Here, we present an analysis of the sphingolipid data subset in LIPIDAT which serves to highlight and to summarize what is known about sphingolipid phase behavior.

It also has the purpose of identifying deficits in our knowledge of this lipid class and of providing a convenient bibliography to the sphingolipid literature. In separate publications, we have reviewed the phase behavior of hydrated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), the phosphatidylethanolamines (PE), and the glycoglycerolipids [4-6]. This series of review articles represents the next logical step to establishing the database which is to present an overview of lipid phase behavior and to set about the task of deciphering the fundamental principles which dictate lipid mesomorphism, polymorphism and metastability, based on an analysis of the data contained in the database.

3. Bibliographic data As of this writing, LIPIDAT (Version 1.0) consists of 10 687 records, each record of which contains 28 information fields [1-3]. The sphingolipid subset represents 8% of these records. Since Version 1.0 is current through June 1990, every effort has been made to include in this review all relevant sphingolipid data and literature references that have appeared since that date. The sphingolipid subset in this updated version of LIPIDAT (Version 1.1), hereafter referred to simply as LIPIDAT, consists of 867 records obtained from 112 scientific articles [21-132] published in 25 different journals. It includes data collected over a 40-year period with the first record obtained in 1953. The average data density in these articles is 7.7 records per article, which is slightly below that (8 records per article) for LIPIDAT as a whole. The annual frequency distribution for sphingolipid records in LIPIDAT is presented in Fig. 1. A growth in interest in sphingolipid phase behavior is observed in the decade of the 80's, which is when about 80% of the sphingolipid records was generated. By comparison, the

140

100 ~D

60

20

1972

1977

1982 Year

1987

1992

Fig. 1. Annual frequency of the sphingolipid records in LIPIDAT over the period 1972-1992.

R. Koynova, M. Caffrey / Biochimica et Biophysica Acta 1255 (1995) 213-236

annual frequency of sphingolipid records has decreased in the early 1990's. Twenty five different journal titles are represented in the sphingolipid record subset of which five account for 87% of the entries (Fig. 2). These include Biochimica et Biophysica Acta (BBA, 45%), Biochemistry (20%), Chemistry and Physics of Lipids (15%), Biophysical Journal (4%), and Journal of Lipid Research (3%). The distribution of sphingolipid subgroup records represented in LIPIDAT and analyzed in this review is presented in Table 1.

4. Thermodynamic data

The vast majority of the sphingolipids discussed in this article are based on ceramide or derivatives of same (Fig. 3). Ceramide consists of sphingosine (Sph, 4-sphingenine), an 18 carbon trans-monounsaturated amino diol, in amide linkage to a long chain fatty acid which is usually hydroxylated. Different types of head groups attached to the terminal hydroxyl group of ceramide give rise to the three major sphingolipid sub-classes. These include: (i) phosphorylcholine in the case of the sphingomyelins; (ii) a simple sugar in the case of the cerebrosides; and (iii) complex oligosaccharides incorporating one or more sialic acid residues in the case of the gangliosides. Sphingosine, together with dihydrosphingosine (Spn, sphinganine, a 18:0 saturated amino diol) are the most common sphingolipid bases in mammals. In higher plants and yeast, phytosphingosine (phyto-Sph, 4-hydroxysphinganine) and in marine organisms 4,8-sphingadiene, are common sphingolipid bases also [10,12]. The two asymmetric carbon atoms at positions 2 and 3 in the ceramide structure give rise to different sphingolipid diastereomers (Fig. 4). By convention, the prefixes D- and L-refer to the stereochemical configuration of the highest-numbered chiral carbon atomC3 in this case. Thus, the prefixes erythro- and threo-refer to the stereochemical configuration at C2. The naturally

J. Lipid Res. Biophys. J.

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215

Table l Distribution of sphingolipid subset records in LIPIDAT Lipid category Neutral glycosphingolipids (cerebrosides) Gal Glc Lac Sulfoglycosylsphingolipids (sulfocerebrosides) GaI-SO4 Sphingophospholipids Sphingomyelin Extracts from natural sources Cerebrosides Sulfocerebrosides Sphingomyelins Gangliosides Sphingolipid/lipid mixtures Sphingolipid/other additive mixtures Dry sphingolipids Total

No. of records (%) 87 (10%) 76 9 2 154 (18%) 154 87 (10%) 87 192 (22%) 66 19 64 43 213 (25%) 69 (8%) 62 (7%) 867 (100%)

occurring sphingolipid configuration is of the D-erythro type [20]. Of the major membrane lipid species, the sphingolipids as a class are unusual in that they have a free hydroxyl and an amide functionality in close proximity to one another and in the vicinity of the polar/apolar interface of the constituent molecules. In concert with the sugar hydroxyls when present, these functionalities facilitate the formation of extensive hydrogen-bond networks with contributions that are both intra- and intermolecular in origin. This is reflected, in part, in the characteristic thermotropism and lyotropism of the sphingolipids. Depending on the origin of the sphingolipids considered in this review, three different classes are identified as follows: (i) synthetic sphingolipids, (ii) semisynthetic sphingolipids obtained from biologically-derived sphingolipids by deacylation-reacylation procedures, and (iii) natural sphingolipids isolated from a biological source. Each class will be discussed as appropriate within the major sphingolipid categories below.

4.1. Sphingomyelins

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0

100

200

300

400

Number of records Fig. 2. Relative record contribution of the top five journals to the sphingolipid database subset (Biochimica et Biophysica Acta).

Sphingomyelin, also referred to as N-acylsphingosine1-phosphorylcholine, represents one of the simplest of the polar sphingolipids. It is a major lipid constituent of animal cell membranes. Because the chemical synthesis of sphingomyelin is considerably more complex than that of the glycerophospholipids [18] there is a paucity of studies on the fully synthetic, stereochemically pure sphingomyelins [81,87,91,107]. However, studies on semisynthetic species, obtained by deacylation-reacylation of natural sphingomyelin extracts (primarily from bovine brain), abound. The sphingoid (referring to sphinganine, to its homologs and stereoisomers and to its hydroxy and unsaturated derivatives [7]) base of bovine brain semisynthetic

216

R. Koynoua, M. Caffrey / Biochimica et Biophysica Acta 1255 (1995) 213-236

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R. KoynoL'a, M. Caffrey / Biochimica et Biophysica Acta 1255 (1995) 213-236 1C1..120H

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Fig. 4. Molecularstructureof the D-erythroand L-threostereoisomersof ceramide. sphingomyelins has a reported composition which varies from approx. 75% [113] to >90% sphingosine [126]. Small amounts of sphinganine and of C20-sphingosine are usually present also. It has been found that during the deacylation-reacylation procedure, a significant amount of the L-threo stereoisomer is formed [59,126]. Thus, the final product represents a mixture of the D-erythro and L-threo stereoiomers. The thermodynamic characteristics of the phase transitions of hydrated synthetic and semisynthetic sphingomyelins are summarized in Table 2, complete with literature references. Semisynthetic sphingomyelins with a fatty acyl chain 14 to 24 carbon atoms long exhibit a chain 'melting' transition between the lamellar gel and the lamellar liquid crystalline phases. In the case of those species with a fatty acyl chain 14 and > 20 carbon atoms long the transition, as monitored by DSC, is complex as evidenced by the presence of multiple peaks in the corresponding calorimetric traces [125,126]. The latter behavior has been ascribed to gel phase polymorphism [126]. An additional low-temperature transition is observed in the case of N24:0-Sph PC (see Appendix I) at around 40°C (entry no. 18 in Table 2; vide infra). The phase behavior of synthetic sphingomyelins of the DL-erythro configuration prepared by the method of Shapiro [137] is similar to that of the semisynthetic compounds when the chain 'melting' transition is considered (compare entry nos. 5, 12 and 19 for synthetic preparations with entry nos. 3, 9 and 18, respectively, for semisynthetic sphingomyelins in Table 2). However, close inspection of the data shows some differences with respect to low-temperature polymorphism. With the synthetic oL-erythro-N18:0-Sph PC, the gel (Lt~) phase is metastable and converts to a stable crystalline subgel phase after low-temperature incubation [69]. Upon heating, a high-enthalpy transition directly to the L,~ phase takes place at a temperature above that observed for the corresponding LI3-L,~ transition (entry no. 11 in Table 2). In contrast, for synthetic DLerythro-N-16:0-Sph PC, a transition between two lameUar gel phases of different morphology has been observed at 30 ° C, well below the Lt~-L,~ transition at 41° C [72].

217

Synthetic, stereochemically pure D-erythro- and L-threoN-18:0-Sph PC, display an L~-L,~ transition [138] similar to that of the semisynthetic compound [81]. They also exhibit a complex polymorphic behavior at low temperatures, which is different for the two diastereomers (entry nos. 13, 14 in Table 2). Further, gel phase metastability, characteristic of the stereochemically pure compounds, is not observed in the D-erythro-/L-threo-mixture [81]. A dependence on chemical and stereochemical purity of gel phase metastability and the formation of stable crystalline phases has been reported for other lipids [136,139]. The synthetic and semisynthetic sphingomyelins differ in the degree of sphingoid base homogeneity. Indeed, for the semisynthetic lipids, base composition can vary from approx. 75% [113] to > 90% sphingosine [126]. This heterogeneity with respect to sphingoid base works against the formation of lamellar crystalline phases and low-temperature forms of the gel phase. It has been proposed [16] that the sphingosine base contributes 13.5 carbon atoms to the non-polar hydrocarbon region of the lipid bilayer in the lamellar phase. The complex mesomorphism of the long-chain sphingolipids is thought to result from the packing constraints imposed by a significant hydrocarbon chain inequivalence [16]. In this respect, the phase behavior of long-chain sphingomyelins is reminiscent of the mixed-chain phosphatidylcholines [16]. When discussing mixed-chain lipids it is useful to describe them in terms of a normalized chain-length inequivalence parameter, A C / C L [16]. Here, AC is the effective chain-length difference and CL is the effective length of the longer of the two chains (both measured in number of carbon-carbon bonds). Thus, in the case of a sphingomyelin with an acyl chain consisting of n carbon atoms (n > 14), A C / C L = (n - 13.5)/(n - 1). Phase transition temperatures and enthalpies as a function of the N-acyl chain length for the sphingomyelins are presented in Fig. 5. The chain 'melting' transition temperature increases with increasing chain length from 14 to 20 carbon atoms and levels out for the longer chain compounds. As the chain inequivalence increases there is a greater tendency for the hydrocarbon chains from adjacent monolayers in a lipid bilayer to interdigitate. Thus, for chains 14, 16 and 18 carbon atoms long the sphingomyelins pack without significant interdigitation at the bilayer midplane in the lamellar gel phase. In contrast, two different types of chain interdigitation is registered in the lamellar gel phase of N-24:0-Sph PC. One is partially interdigitated, with the shorter chain from one monolayer of the bilayer arranged end to end with the longer chain from the opposite monolayer (ip..- ""|)- The other is of the mixed interdigitated type, with the shorter chains from the adjacent monolayers of the bilayer arranged end-to-end and with the longer chains essentially spanning the bilayer (r~--~l). The latter arrangement provides a surface area per head group equivalent to the cross-sectional area of three hydrocarbon chains. Thus, the transition at approx.

218

R. Koynova, M. Caffrey / Biochimica et Biophysica A cta 1255 (1995) 213-236

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R. Koynot~a, M. Caffrey / Biochimica et Biophysica Acta 1255 (1995) 213-236

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R. Koynova, M. Caffrey / Biochimica et Biophysica Acta 1255 (1995) 213-236

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hydrocarbon chains and thus the chain order-disorder transition [16]. The effect of incorporating cis-double bonds in the N-acyl chain of sphingomyelins is small compared to the effect of chain unsaturation in the glycerophospholipids. Thus, a cis-double bond at C15 in the 24 carbon atom long N-acyl chain of sphingomyelin decreases the transition temperature by approx. 16°C (cf. entry no. 20 and no. 23 in Table 2). A cis-double bond at position 9 or at positions 9 and 12 in an 18 carbon atom long N-acyl chain sphingomyelin decreases the transition temperature from 45°C to 33° C or 26° C, respectively (entry nos. 13, 21, 22 in Table 2). In contrast, the introduction of a cis-double bond at position 9 or at positions 9 and 12 in one of the acyl chains of 18:0/18:0 PC (DSPC) reduces the chain order/disorder transition temperature from 55 ° C to 6 ° C for 18:0/18:1c9 PC and to - 16° C for 18:0/18:2c9,12 PC [1].

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Acyl chain length Fig. 5. Dependence of the transition temperature (upper panel) and the enthalpy change of the lamellar gel - lamellar liquid crystalline transition (lower panel) on the chain length (in units of number of carbon atoms) of fully hydrated sphingomyelins. A key to the transition types is presented in the inset.

40°C in N-24:0-Sph PC involves the conversion from a lamellar mixed-interdigitated phase to a lamellar phase with partially interdigitated chains. The latter undergoes a transition to the lamellar liquid crystalline phase at approx. 48° C [77]. There is an apparent discontinuity in the transition enthalpy dependence on acyl chain length at a chain length value of 20 carbon atoms (Fig. 5, lower panel). This might be due to a change in the chain packing mode from non-interdigitated to interdigitated. According to the results of the analysis of the packing modes in mixed-chain glycerophospholipids [16], chain interdigitation is expected at a chain-length inequivalence value of A C / C L > 0.35. In this context, we note that A C / C L ~ 0.35 for N-20:0Spn PC. The replacement of sphingosine by its dihydro-derivatire, sphinganine, in N-16:0-Spn PC results in an increase in the lamellar gel-lamellar liquid crystalline transition temperature by approx. 7 ° C (cf. entry no. 5 and no. 26 in Table 2). This relatively small effect is ascribed to the position of the trans-double bond in sphingosine in the structurally ordered interface region where it is not expected to influence considerably the packing order of the

The cerebrosides (1-fl-glycosylceramides) are composed of a mono- or oligosaccharide head group fl-O-glycosidically linked to the terminal hydroxyl of ceramide (Fig. 3). Galactocerebrosides are a major component of the myelin sheet [10,12]. Glucocerebrosides and lactocerebrosides are the major extraneural glycosphingolipids. Cerebrosides with tri- and tetrasaccharide-containing head groups, known as globosides, are found in the erythrocyte membrane [15]. As noted for the sphingomyelins above, measurements on relatively homogeneous cerebrosides have been carried out primarily with semisynthetic preparations obtained by deacylation of natural extracts followed by reacylation with known fatty acids. The data available in LIPIDAT concerning the phase transition characteristics of the assorted cerebrosides which differ in their sugar head group, acyl chain and sphingoid base identity, are summarized in Table 3. These will be treated separately below based on sugar head group identity. 1. Galactocerebrosides

Semisynthetic galactocerebrosides with saturated fatty acyl chains 16, 18 and 24 carbon atoms long and a sphingosine base (approx. 95% sphingosine, approx. 5% sphinganine) are reported to form three different lamellar phases - two ordered low-temperature phases with different stabilities and an L,~ phase at high temperature. Their phase behavior on heating is dominated by a high-enthalpy, high-temperature lamellar crystalline-L,~ transition which takes place at 82-84 ° C and which is independent of chain length (entry nos. 1-3 in Table 3). During this transition, the extensive hydrogen bonded network, characteristic of both the galactose head group and the sphingosine moiety, falls apart simultaneously with the chain order-to-disorder transition. On cooling, an ordered

R. Koynot, a, M. Caffrey / Biochimica et Biophysica Acta 1255 (1995) 213-236

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R. Koynoca, M. Caffrey / Biochimica et Biophysica Acta 1255 (1995) 213-236

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R. Koynova, M. Caffrey / Biochimica et Biophysica Acta 1255 (1995) 213-236

metastable lamellar phase forms. Conversion to a stable crystalline phase depends on the cooling protocol and the acyl chain length [48,71,98]. The low-temperature metastability has been explained as arising from a decoupling of chain ordering from hydrogen bond reformation upon cooling [71]. According to X-ray diffraction data, the stable low-temperature crystalline phase of N-24:0-Sph Gal is of the partially interdigitated type. Partial chain interdigitation persists into the lamellar liquid crystalline phase of this lipid [48]. Incorporation of one or two cis-double bonds in the am±de-linked fatty acyl chain results in a decrease of the chain 'melting' transition temperature and enthalpy (cf. entry no. 2 with entry nos. 7, 9 in Table 3). As was observed with the sphingomyelins, however, the effect of chain unsaturation is not as pronounced as it is in the case of the glycerophospholipids. Cerebrosides with unsaturated acyl chains form the lamellar liquid crystalline phase at high temperature and two lamellar crystalline phases of differential stability at low temperature [98]. Replacement of lignoceric (24:0) acid with its 2-hydroxy derivative - cerebronic (24:1OH2) acid - in the corresponding galactocerebroside decreases the lamellar chain order-disorder transition temperature (cf. entry no. 3 with entry no. 10 in Table 3). It has been proposed that the hydroxy group at the 2 position in the acyl chain provides a kinetic barrier to attaining a low-temperature crystalline phase [15]. Thus, the gel phase is the only low-temperature

223

phase for this lipid. It transforms to a lamellar liquid crystalline phase at 65.7 ° C which is close to the metastable gel-liquid crystalline transition temperature in N-24:0-Sph Gal (entry no. 4 in Table 3). 2. Glucocerebrosides

The phase behavior of synthetic N-16:0-Sph Glc is similar to that of its galactose analog with respect to both phase transition temperature and enthalpy change and low temperature metastability (entry nos. 13, 14 in Table 3). The 5°C difference in the 'main' transition temperature (cf. 82°C for N-16:0-Sph Gal with 87°C for N-16:0-Sph Glc) could possibly be a result of differences in sample purity. Some major differences are apparent when the phase properties of the galacto- and glucocerebrosides are compared with those of the corresponding glycoglycerolipids. To begin with, the transition temperatures of the cerebrosides are relatively insensitive to acyl chain length (Table 3). Secondly, while the high-temperature phase for the hydrated glycoglycerolipids is of the non-bilayer type (usually the inverted hexagonal for the long-chain compounds, see Ref. [6] and references therein), the hydrated cerebrosides form lamellar phases exclusively. These differences in phase behavior perhaps reflect the more extensive intraand intermolecular hydrogen bonding prevailing in the case of the cerebrosides (vide infra). In this connection, it is interesting to note that glucose bound to ceramide

Table 5 Thermodynamic parameters of the phase transitions in hydrated sulfocerebrosides (o~-hydroxy fatty acids) Lipid

N-16:IOH2-Sph Gal-SO4

N-18:IOH2-Sph GaI-SO4

N-24:IOH2-Sph Gal-SO4

Entry no.

1 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20

* Measured from cooling scan.

Salt

0.01 M Na ÷ 0.1 M Na* 0.5 M Na + 1 M Na + 2 M Na ÷ 0.01 M K ~0.1 M K + 0.5 M K + 1 M K+ 2 M K÷ 1 M Mg 2+ 1 M Ca 2+ 2 M Li ÷ 0.01 M Na + 2 M Na + 0.1 M K + 2 M K+ 2 M Li + 2 M K+ 2 M Li +

Stable lamellar g e l Metastable lamellar g e l lamellar liquid crystal lamellar liquid crystal

Metastable-stable lamellar gel

T,°C

T,°C

AH, kcal/mol

75.6

9.9

83.4

14.3 ± 0.3

81.5

17.4 ± 2.0

T,°C

AH, kcal/mol

53.2 53.2 54.8 57.1 61.2 53.1 55.1 58.5 61.1 64.5 54-57 60.0 57.4 56.4 63.4 58.2 67.0 60.2 (67.0, 69.7) * 62.3 61.5 *

6.4 + 0.7 6.4 + 0.7 6.4 ± 0.7 6.4 + 0 . 7 6.4 5.6 ± 0.6 5.6 ± 0.6 5.6 ± 0.6 5.6 ± 0.6 5.6 3.7 3.7 6.4 6.4 ± 0.5 6.4 6.9 7.0 ± 0.3 6.5 7.6 ± 1.2 * 6.2 49.5 7.2 * 40.2 *

References

AH, kcal/mol

3.7 2.6 *

[401,[1061 [1061,11081 [1061 [1061 [1061 [1061, [1081 [106] [1061 [106] [51], [1061,[1081 [lO8] [1o8] [52], [108] [40] [1o8] [1o8] [51], [108] [52], [108] [51] [52]

224

R. KoynoL,a,M. Caffrey/ Biochimica et BiophysicaActa 1255 (1995) 213-236

extends away from the bilayer to a lesser degree and has a smaller orientational order parameter than when it is bound to diacylglycerol [159]. 3. Lactocerebrosides Data on the thermal behavior of two synthetic lactocerebrosides, specifically, N-18:0-Spn Lac and N-24:0-Spn Lac, are to be found in LIPIDAT [76]. In contrast to the sphingolipids discussed thus far, the lactocerebroside head group consists of a disaccharide. One consequence of increasing the number of monoses in the lipid is to destabilize the ordered low-temperature phase manifested as a lowering of the corresponding transition temperature (entry nos. 15, 16 in Table 3). A similar effect is observed with glycoglycerolipids [6]. 4.3. Sulfocerebrosides

The lipids in this class are acidic sulfoglycosylsphingolipids, and correspond to sulfated derivatives of the neutral glycosphingolipids (Ref. [7], Fig. 3). The major mammalian sulfocerebroside, monogalactosylceramide 13sulfate, is an important lipid in myelin representing 3.8 wt% of the total lipid [140]. The fatty acids associated with this lipid range in length from 14 to 26 carbon atoms and include the nonhydroxy and a-hydroxy forms with sizeable quantities of saturated and monounsaturated chains. The myelin sulfocerebrosides are dominated by the 24 carbon atom long fatty acids with the following distribution: 23% 24:0, 19% 24:1OH2, and 7% 24:1c15 [44]. As noted above, a long fatty acyl chain coupled with a relatively short chain at the sphingoid base site, gives rise to a pronounced chain length inequivalence in these naturally occurring sulfocerebrosides. Thermodynamic parameters of the phase transitions associated with the semisynthetic bovine brain sulfogalactocerebrosides having a uniform non-hydroxy or an o~-hydroxy fatty acid composition are summarized in Tables 4 and 5, respectively. Included in the tables are the effects of the concentration and identity of salts in solution used for dispersing the lipid. The sphingoid base composition of these semisynthetic species is as found in the original material: 94% sphingosine, 4% sphinganine, and 2% dihydroxy 18:0 and 1% dihydroxy 16:0 (hexadecasphinganine) [141]. In what follows, we will examine in turn the saturated and unsaturated non-hydroxy followed by the a-hydroxy fatty acid-containing sulfocerebrosides. 4.3.1. Saturated non-hydroxy N-acylated sulfocerebrosides As noted above for the other sphingolipids, the sulfocerebrosides within this group have been shown to access lamellar phases only. Specifically, two low-temperature gel phases have been found along with a high temperature L,~ phase (Table 4). Generally, the trend upon heating is for there to be a single transition from a stable lamellar gel

to the L,~ phase. In the cooling direction, the sequence is L~-to-metastable lamellar gel-to-stable lamellar gel. Details of the latter transitions are very much cooling rate-dependent [51,52,108]. Where measured, the transition temperature does not appear to be particularly sensitive to fatty acyl chain length. This has been interpreted as indicating that the intermolecular forces between lipids within the lamellar phase are dominated more by head group than by chain interactions [51]. The identity and concentration of cations in the dispersing aqueous medium has a pronounced effect on sulfogalactocerebroside phase behavior (Table 4). Thus, the lamellar gel-L,~ phase transition temperature increases with cation concentration. Further, monovalent cations increase the transition temperature in the order Li+< Na+< K + < Rb + which matches exactly the ionic radius sequence in this series of Group IA cations [108]. The effect has been attributed to an increased ability of the sulfocerebrosides to engage in intermolecular hydrogen bond formation upon shielding of the charge associated with the sulfate moiety in the presence of cations [108]. It has also been found that the divalent cations, Ca 2+ and Mg 2+, as well as the monovalent cation, Li +, inhibit the formation of the stable low-temperature lamellar gel phase [108]. Upon rapid cooling of N-24:0-Sph Gal-SO4 and N26:0-Sph GaI-SO4, two metastable lamellar gel phases have been found by ESR, Raman spectroscopy and X-ray diffraction measurements [52,89,142]. The high- and lowtemperature metastable phases have been identified as having partially interdigitated and mixed interdigitated type chain packing, respectively, as was reported above for the sphingomyelins (vide infra). The stable low-temperature phase is considered to be of the partially interdigitated lamellar crystalline type with orthorhombic chain packing and to have a low level of hydration and enhanced intermolecular hydrogen bonding [142]. Because of the pronounced chain length inequivalence in these sulfocerebrosides, it has been proposed that the corresponding L~ phase has chains that are partially interdigitated across the bilayer midplane ( ~ K a " ~ ) also [89,142]. 4.3.2. Unsaturated non-hydroxy N-acylated sulfocerebrosides To the best of our knowledge, there exists but a single example of an unsaturated non-hydroxy N-acylated sulfocerebroside whose phase behavior has been studied. The lipid in question is based on nervonic acid, specifically N-24:lc15-Sph GaI-SO4. The phase transition characteristics of this lipid in 2 M K + and 2 M Li are reported in Table 4 (entry nos. 31, 32). The presence of a single double bond lowers the stable lamellar gel-to-L,~ transition temperature by 19° C to 50 ° C when compared to the corresponding saturated analog (Table 4, entry no. 28). At the same time, the enthalpy change associated with the transition is almost halved. Taken together, these data suggest a less well ordered

R. Koynova, M. Caffrey / Biochimica et Biophysica Acta 1255 (1995) 213-236

packing of unsaturated chains in the bilayers of the gel phase. However, the double bond does not prevent the formation of a stable low-temperature lamellar crystalline phase [51].

4.4. a-Hydroxy N-acylated sulfocerebrosides The thermodynamic parameters of the phase transitions undergone by sulfocerebrosides N-acylated with a-hydroxy fatty acids in the presence of different cations are assembled in Table 5. One of the effects of introducing the a-hydroxyl group is to prevent the isothermal conversion of the metastable to the stable lamellar gel phase. The hydroxylated sulfocerebrosides in question transform from the metastable to a stable low-temperature lamellar phase only upon heating and in the presence of 2 M K + [51]. Another consequence of hydroxylation is an increase in the chain order-to-disorder transition temperature for both the stable and metastable lamellar gel phases (compare entry nos. 10, 19, and 28 in Table 4 and nos. 10, 17, and 19 in Table 5). This result is not unexpected since the a-hydroxyl group will stabilize the low-temperature gel phase by engaging in hydrogen bond formation. Parenthetically, we note that hydroxylation of the fatty acyl chain in ceramides has been found to effect a condensation of the lipids as monolayers at the air/water interface [143]. A persual of the data in Table 5 shows that the effect of increasing cation concentration is to raise the chain order-

225

to-disorder transition temperature. This is observed with Na +, K +, Li +, and Ca 2+ and perhaps to a lesser extent with Mg 2+. As noted previously the origin of the effect most likely relates to cation screening of the anionic sulfate group. Where comparisons are possible, the data in Table 5 indicate a small increase in the chain melting transition temperature as the fatty acyl chain is lengthened. Again, the effect is considerably less compared to that found in the glycerophospholipids. The negatively charged sulfate groups in the sulfocerebrosides introduce a repulsive interaction between adjacent lipids. One consequence of this is to destabilize the more ordered low-temperature lamellar gel phase. Thus, by comparison with the neutral cerebrosides, the anionic sulfocerebrosides generally have lower transition temperatures for comparable transitions (compare entry nos. 1-3 in Table 3 with entry nos. 1, 14, 24 in Table 4; entry no. 1 in Table 3 with entry nos. 19, 20 in Table 5). However, it appears as though the destabilizing effect of the charged sulfate can be counteracted by high salt concentration and by hydroxylating the N-fatty acyl residue. The high salt serves to screen the negative charge and the additional hydroxyl group contributes to hydrogen bonding. Thus, we find that the transition temperatures of the sulfogalactocerebrosides containing hydroxyl acyl chains at high K + concentrations are similar to those of the neutral galactocerebrosides with non-hydroxy fatty acids (compare entry nos. 17, 19 in Table 5 and entry nos. 2, 3 in Table 3).

]'able 6 Thermodynamic parameters of the phase transitions in hydrated natural sphingomyelin extracts Lipid

Entry no.

Lamellar gellamellar liquid crystal T, ° C

AH, kcal/mol

References

Bovine brain-Spd PC (fa composition: 24-50% 18:0, 12-73% 24:1c15, 2-20% 16:0, 4-10% 24:0, 1-7% 22:0)

1

35.7±5.8

6.7±1.7

[25-27], [30],[32],[33],[35],[39],[43],[53],[571, [65], [86],[100],[103],[105],[112],[119], [124], [127], [130],[132]

Bovine brain-Spd P C / I M KSCN Egg-Spd PC (fa composition: 86% 16:0, 6% 18:0, 6% 24:1c15)

2 3

21.0 37.7 _+ 1.1 *

2.2 5.4 + 1.9

[57] [27], [79], [80], [88], [105], [119], [121-123l, [125]

Bovine erythrocyte-Spd PC (fa composition: 42% 24:0, 17% 16:0, 11% 22:0, 10% 24:1c15)

4

20.0

Sheep erythrocyte-Spd PC (fa composition: 55% 24: lc15, 19% 22:0, 11% 24:0, 10% 16:0, 5% 23:0)

5

21.0, 31.0

Human erythrocyte-Spd PC (fa composition: 30% 24:1c15, 30% 16:0, 16% 24:0, 8% 18:0, 7% 22:0)

8

32 **

[23]

5.3 (total for 2 peaks)

[27]

[32]

Transition at - 34.6 ° C, AH = 0.4 kcal/mol, representing the freezing of PC head group motion has also been identified using a combination of DSC and NMR methods for this lipid extract [122]. * * Transition from gel phase MLV to liquid crystalline phase as SUV [32].

R. Koynoua, M. Caffrey /Biochimica et Biophysica Acta 1255 (1995) 213-236

226

o

,.,z

eq ~

I

eq ~.~

I

I

I

r~

?

t~

?

*

*

4-1.

{"4 4-1

~ -H

O 4-1

O 4-1

÷1

+1

-H

E

?

~

1

+1

+1

Jrl

+1

~

E

8

o

o

lb.

.=_

ID.

.o

,.

Z Z m

.o ;=

0

,-~

0 0 0

oA

e-

O0 .0 '~

~

~

~

~~ cq eq ~ ~

('~

-& & .~_ o

..~ ..~ ".~

~

o

", ; ~ ",

•o~

~

o~ O .O. .O. .O. . .

" ~

.~ ~o = zo&z~~e, :, xA~

~AeA~

~6

~

.~ ~ ,. =

~~:~q :&:~:~ ~ .°~ ~o ~~"- ~~:. ~,.~

a

~,

~v

R. Koynova, M. Caffrey / Biochimica et Biophysica Acta 1255 (1995) 213-236

4.5. Extracts from biological sources 4.5.1. Sphingomyelins Sphingomyelin extracts from several biological sources have been studied from the point of view of their thermotropic phase behavior. The corresponding transition temperatures and enthalpy values are listed in Table 6. A persual of these data shows that the natural sphingomyelins undergo mesophase transitions in the temperature range of 20 to 40°C which is close to physiological temperature for several of these species. Combining this observation with data on the interdigitated packing mode of some long-chain sphingomyelins, which are abundant constituents of the natural membrane sphingomyelins, it

227

has been speculated that these lipids may play a role in membrane transport [16]. Sphingomyelins isolated from natural sources vary with respect to the chemical structure of the constituent sphingoid base and the N-acyl chain. Although more than sixty different species of sphingoid base have been described [151], the base composition of the natural sphingomyelins is known to be relatively homogeneous. Thus, for example, bovine brain and many other biologically derived sphingomyelins consist predominantly of the sphingosine base together with a small amount of sphinganine and/or icosasphinganine (C20-sphingosine) [141]. In most mammalian tissues, palmitic (16:0) acid is the dominant fatty acid. Considerable amounts of nervonic (24:1c15), ligno-

Table 8 Structures of some major gangliosides

IUPAC-IUB Ablxevlation

Structure p

p

P

,'N=.~.,O= ~

3 GalNAc 1 - 4 4 Gal 1 .-~,4GIc 1 .-I, C e ~ ?P ?3 1 Gal

Svennerholm Notation*

Wlegandt Notation**

Gin. "

G.tl I

, .o.~-ogo. ~ r

Gm

Gt. 1

IISNeuAc-tac-Cer

GM3

Gu:I

~ SNau.~--,.SNeuAc-r¢~4-'Cer

Gin.

Gtot2a

IVSNeuAc-ASNOU~z-GOOse 4 -Cer

GTlb

G=t3b

w' ~ = - , l '

GO.I',

G.t4b

4

2NANA

p

p

P

$

3 GaiN/u: 1 - ~ 4 Gad 1 --,~4 GIC 1 -~ Cerandde 3

?

2NANA

p

P

NAHA 2 ,-.) 3 Gal 1 -~4GIc 1 ~ Ceramide

P

p

P

3 GalN/~ 1.-) 4 GoI 1 ~ 4 6 1 c 1 ~ Cerarrdde ?p 3 1" 1 Gal 2NN4A 3 1" ZNN4A

p

P

p

Gal 1 ~ 3 GaiN/k: 1-~4 Gal 1 ~ 4 G I c I ~ Cerai't,dcile 3 3 1" ? 2NN,~ 2 NANA 8

? 2NANA

p

P

P

Gal 1 ~ 3 GalNAc 1 ~ 4 Gal I -).4Gk:: 1 --z,Cer-~n'ide 3 3 I" ? 2 NANA B 2 NANA 8 't ? ZNN4A 2NANA

~u~= -e,go= -Cer

* Ref. [160]; * * Ref. [161]. * * * G-ganglioside; M-monosialo-; D-disialo-; T-trisialo-; Q-quatrosialo-; arabic numbers indicate sequence of migration in thin-layer chromatograms [160].

228

R. Koyno~,a, M. Caffrey / Biochimica et Biophysica Acta 1255 (1995) 213-236

ceric (24:0) and behenic (22:0) acids are also present [152]. In the nervous system, stearic (18:0) rather than palmitic acid is the primary fatty acid component [153]. One of the long term objectives of the LIPIDAT database analysis project is to use the results to establish the principles of lipid phase behavior as these relate to lipid chemical structure and sample composition. With these principles and rules in hand, it should be possible to predict the mesophase properties of complex mixtures, as is found in the natural sphingomyelins, from a knowledge of the identity and relative amounts of the individual lipid species that constitute them.

4.5.2. Cerebrosides The phase behavior of two types of cerebroside extracts from natural sources have been studied. These include: (1) total extracts, representing complex mixtures of individual lipid species which share a common sugar head group, but which vary in their sphingoid base and fatty acyl chain composition; and (2) specific fractions of the total extracts which possess a more homogeneous fatty acyl chain composition. The corresponding transition temperatures and enthalpy values are presented in Table 7. The bulk of the data in the table concern the monoglycosides. Data on a single lactoside and on a limited number of globoside species are also present. Low-temperature gel phase metastability, characteristic of the synthetic and semisynthetic cerebrosides, is displayed by some of the biologically-derived monoglycosylcerebrosides. Of the naturally derived cerebrosides studied thus far, the majority have unusually high lamellar orderto-disorder transition temperatures. The values in Table 7 cluster in the 50°C to 70°C range. These high transition temperatures suggest a role in membrane structural integrity [15]. Further, phase separation-induced clustering of the monoglycosyl cerebrosides in the membrane plane is

expected because of the large difference in transition temperatures between the cerebrosides and the more common phospholipids [15]. According to the data of Maggio et al. [76], an increase in the number of monoses in the glycosyl head group results in a lowering of the chain order-disorder transition temperature and enthalpy (entry nos. 12, 18, 19, 20 in Table 7) as was observed with the synthetic cerebrosides. Worthy of note is the lack of gel phase polymorphism in all of the synthetic and natural glycosphingolipids examined in this work [76]. The authors attribute this to the lower lipid concentration used in their study as compared to that used in others. It has been reported that the gross morphology of aqueous cerebroside dispersions is different from the quasi-spherical liposomes observed with the glycerophospholipids. The former includes an assortment of tubules, filaments and small liposomes (see for example, Ref. [15] and references therein). It is possible that the morphology of bulk cerebroside dispersions in water impacts on mesomorphic and polymorphic behavior. Obviously, this is an area where additional work is needed.

4.5.3. Gangliosides The gangliosides are acidic glycosphingolipids which have one or more sialic acid residues as part of their oligosaccharide head group. Sialate gives the polar head group a net negative charge at neutral pH. The most abundant form of sialic acid found in human gangliosides is /3-D-N-acetyl neuraminic acid (NANA) [8]. Gangliosides constitute 6% of the total lipids in the gray matter of the brain. They are also found in small amounts in some extraneural tissues and in erythrocyte membranes. As a result of the variety in the carbohydrate moiety, a plethora of gangliosides exist. In fact, over 100 distinct ganglioside species have been identified from different tissues [15]. These complex sphingolipids are usually classified accord-

Table 9 Thermodynamic parameters of the phase transitions in hydrated gangliosides Lipid

Entry no.

GM1 (bovine brain)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

GM2 (bovine brain) GM2 (Tay-Sachs, human origin) GM3 (bovine brain) GM3 (adrenal medulla) NeuGcGM3 (adrenal medula) GDla (bovine brain) GTlb (bovine brain) GQlb Mixed gangliosides (bovine brain) Desialated mixed gangliosides (sheep brain), SUV

References

Phase transition T, ° C

ZlH, kcal/mol

20.6 + 3.1 40.0 + 4.2 29.3 10-55 35.3 34.0 39.4 14.8 _+ 1.1 40.0 7.5 _+ 0.2

1.14 + 0.26 2.6 1.5 3 cal/g 2.5

21.0 _+ 8.5 44.0 _+ 4.3 25.0, 44.0

2.4 0.77 _+ 0.1 3.0 0.40 _+ 0.10 1 cal/g 3.9

[50], [76], [92], [96], [1201

[371,[92] [761 [29] [76] [42] [76] [421, [50], [76], [92], [120] [92] [50], [76], [120] [37] [24], [92] [24], [29], [92] [128]

R. Koynoca, M. Caffrey / Biochimica et Biophysica Acta 1255 (1995) 213-236

ing to the number and position of the sialic acid residues. The structures of and abbreviations used to represent the most common ganglioside types are shown in Table 8.

229

Gangliosides have been proposed to play a role in signal transduction across membranes [144], in the regulation of differentiation and proliferation processes [145],

Table 10 Thermodynamic parameters of the phase transitions in dry sphingolipids Lipid

Entry no.

Transition type

T, ° C

N-24:0-phyto-Sph

1

lamellar crystal F *-lamellar crystal E * (lst heating) * *

43

[ 104]

2

lamellar crystal E *-lamellar crystal C * (lst heating) * ~

98

[ 104]

3

lamellar crystal A-lamellar crystal C * (lst heating) * * *

85

[104]

4

lamellar crystal D *-lamellar crystal C * (subsequent heating)

23

[104]

5 6 7 8

lamellar crystal C *-lamellar crystal B * lamellar crystal B *-lamellar liquid crystal lamellar liquid crystal-fluid isotropic lamellar crystal B-lamellar crystal C (1 st heating) lamellar crystal B-lamellar crystal A + lamellar liquid crystal (lst heating)

106 121 150 54

[ 104] [104] [104] [102]

90

[102]

10 11

lamellar crystal A-lamellar liquid crystal lamellar liquid crystal (LC1)-lamellar liquid crystal (LC2)

120 131

[ 102] [102]

12 13

lamellar liquid crystal (LC2)-fluid isotropic lamellar crystal A-lamellar crystal B (1 st heating)

187 96

6.2 + 0.6

[102] [71]

14

lamellar crystal B'-lamellar crystal B (2nd heating)

52

2.2 + 0.1

[71]

15 16 17

lamellar crystal B-lamellar liquid crystal lamellar liquid crystal-fluid isotropic lamellar crystal A-lamellar crystal B (1 st heating)

143 180 100

11.2 + 0.3 0.8 +_ 0.1

[71] [71] [102]

18

lamellar crystal B'-lamellar crystal B (2nd heating)

50

[102]

19 20 21

lamellar crystal B-lamellar liquid crystal lamellar liquid crystal-fluid isotropic lamellar crystal D-lamellar crystal C (lst heating)

143 178 73

[ 102] [ 102] [102]

22

lamellar crystal C-lamellar crystal B (lst heating)

95

[ 102]

23 24 25

lamellar crystal B-cubic liquid crystal cubic liquid crystal-lamellar liquid crystal cubic liquid crystal-lamellar crystal A (cooling)

126 150 50

[102] [102] [102]

26 27 28 29 30 31

lamellar liquid crystal-fluid isotropic lamellar crystal-lamellar liquid crystal lamellar crystal-lamellar liquid crystal lamellar liquid crystal-fluid isotropic lamellar crystal-lamellar liquid crystal two coexisting phases (disordered + helical chain conformation) - inverted hexagonal

212 92 87.3 127.3 105 130

[102] [102] [44] [44] [77] [99]

32 33 34 35 36

lamellar gel-lamellar liquid crystal lamellar crystal-lamellar liquid crystal lamellar liquid crystal-inverted cubic inverted cubic-inverted hexagonal inverted hexagonal-fluid isotropic

75 87 144 170 208

N-2:0-Sph Gal

9

N-16:0-Sph Gal

N-18:0-Sph Gal

N-18:IOH2-Sph Gal

N-18:0-Sph GaI-SO4 (Na-salt) Bovine brain-Spd GaI-SO4 Gaucher spleen-Spd Glc Winter rye leaves-Spd GIc N-18:0-Sph PC Bovine brain-Spd PC

V-shape molecular arrangement (Ref. [104], see Fig. 6). * * Crystallized from chloroform-methanol solution by slow cooling from room temperature down to 12° C [104]. * * * Crystallized from chloroform-methanol solution at 4° C [104].

AH, kcal/mol

6.6 0.8 12.1 (3 cal/g) 3.8

References

[59] [124] [124] [ 124] [ 124]

230

R. Koynot:a, M. Caffrey/ Biochimica et BiophysicaActa 1255 (1995) 213-236

and in the modulation of membrane-linked enzymes [149]. The ability of different gangliosides to modify membrane functions is possibly related to their intrinsic tendency to aggregate. This property has been studied in simple model systems consisting of pure and mixed gangliosides in water. Thus, for example, the following members of ganglioside series: GTlb, GDla, GM1 and GM2 (Table 8), are found to form micelles of increasing molecular weight, hydrodynamic radius and asymmetry [146,147]. In contrast, the ganglioside, GM3, which has a relatively small hydrophillic head group, does not micellarize. Instead it forms unilamellar vesicles [148]. The critical micelle concentration (CMC) of the gangliosides falls in the range 10 8-10 5 M. As expected, the CMC increases with the number of sialic acid residues in the head group [150]. Mixed brain gangliosides are reported to form the normal hexagonal (H 0 mesophase at low water concentrations (18-50 wt% water). At > 60 wt% water, an isotropic, presumably micellar, phase is formed [24]. Parameters of the phase transitions observed in gangliosides from biological sources are summarized in Table 9. Double-peaked calorimetric traces have been registered for mixed brain gangliosides containing GM1, GDla, GTlb and GQlb (entry nos. 1, 2; 8, 9; 12, 13) [24,34,37,92]. The structural basis of these transitions is not yet clear. Since XRD studies indicate that the hydrocarbon chains are disordered both below and above the calorimetric transitions [24], and the enthalpies are typically low, they probably reflect interconversions between fluid mesophases. Again, a lot more work must be done before we are to fully understand the structure and thermodynamic properties of the naturally-derived gangliosides. 4.6. Dry sphingolipids

The limited data available in LIPIDAT on the polymorphic and mesomorphic behavior of dry and partially hydrated sphingolipids is summarized in Table 10. Three types of transitions typify the behavior of these lipids, as they do for dry lipids in general. These include different polymorphic interconversions between lamellar crystalline phases of different morphology, the lamellar crystal-L~ transition, and the L : f l u i d isotropic transition at high temperatures ( > 100°C). Non-bilayer liquid crystalline phases have been reported to form at temperatures between where the L,~ and the fluid isotropic phases are stable in the case of N-18:IOH2-Sph Gal [102], bovine brain sphingomyelin [124] and the glucocerebroside from winter rye leaves [99]. In addition to the references cited in Table 10, useful information concerning the molecular arrangements in dry and partially hydrated sphingolipids can be found in Refs. [155-158]. Using XRD, an anhydrous ceramide containing a phytosphingosine base and a fatty acyl chain 24 carbon atoms long has been shown to form six different lamellar crystalline phases in different temperature ranges in a manner

Fig. 6. Schematicrepresentationof the moleculararrangement in the B, C, D, E and F type lamellar crystalline phases (see Table 10) found in anhydrous tetracosanoyl-phytosphingosine(N-24:0-phyto-Sph)[104].

that depends also on sample thermal history (entry nos. 1-6 in Table 10). In only one of these phases were the ceramide molecules found to pack in a bilayer arrangement with the fatty acyl chain interdigitating between opposing monolayers (lamellar crystal A in entry no. 3, Table 10; lamellar d-spacing 51.5 A). In the other five lamellar crystalline phases, the ceramide molecules have a V-shaped conformation (Fig. 6) and pack in single layers with the sphingosine and fatty acyl chains forming separate matrices [104]. Differences between these five lamellar crystal phases refer mainly to the tilt of the hydrocarbon chains with lamellar d-spacings varying between 37.3-46.5 h,. The ceramides transform to the L= and then to the fluid isotropic phases at temperatures in excess of 120 ° C (entry nos. 6, 7 in Table 10). Dry N-acetyl-galactosylsphingosine (N-2:0-Sph Gal) and cerebrosides with fatty acyl chains 16 and 18 carbon atoms long also have been found to form only lamellar phases before melting to the fluid isotropic phase at very high temperatures (entry nos. 8-20 in Table 10). In anhydrous N-18:IOH2-Sph Gal four different lamellar crystal polymorphs as well as a high-temperature L,~ phase have been reported and the parameters of the associated phase transitions are included in Table 10 (entry nos. 21-26). In addition, an optically isotropic liquid crystalline phase with a cubic lattice was observed as an intermediate between a lamellar crystalline and the L,~ phase in the temperature range 126° C to 150°C [102] (entry nos. 23, 24 in Table 10). Typical of a cubic phase it exhibits a pronounced undercooling down to 50°C at which point it

R. Koynot a, M. Caffrey / Biochimica et Biophysica Acta 1255 (1995) 213-236

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R. Koynor'a, M. Caffrey / Biochimica et Biophysica Acta 1255 (1995) 213-236

transforms to a lamellar crystalline phase (entry no. 25 in Table 10). In contrast to the multitude of lamellar crystal phases exhibited by the previous sphingolipid, N-18:0-Sph GalSO4 has just one such polymorph with a lamellar d-spacing of 48 A. At 92 ° C, this anhydrous sulfosphingolipid transforms to the L,~ phase with a lamellar d-spacing of 62 ,~ (entry no. 27 in Table 10). Data concerning the phase behavior of sphingolipids at

different levels of hydration below full hydration are included in the few published temperature-composition phase diagrams of sphingolipid/water systems. They refer to sphingolipid extracts from biological sources and include bovine brain sphingomyelin/water [124], bovine brain galactocerebroside/water [102], human brain sulfogalactocerebroside (Na+-salt)/water [102], and bovine brain suifogalactocerebroside/water [44] systems. These phase diagrams are dominated by lamellar (crystalline, gel and

Table 12 A summary of the phase transitions in hydrated sphingolipids Phase transition type

Sphingolipid species

Transition temperature, ° C

Reference source

Lamellar gel (subgel)-lamellar liquid crystal

N-n:0-Sph PC, n = 14, 16, 18, 20, 22 N-18:lc9-Sph PC N-18:2c9,12-Sph PC N-24: lcl5-Sph PC N-16:0-Spn PC N-n:0-Sph Gal, n = 16, 18, 24 N-18: lc9-Sph Gal N-18:2c9,12-Sph Gal N-24:1OH2-Sph Gal N-n:0-Spn Gal, n = 18, 24 N-n:0-Sph Glc, n = 16, 18 N-n:0-Sph Lac, n = 18, 24 N-n:0-Sph Gal-SO4, n = 16, 18, 24, 26 N-24:1c15-Sph GaI-SO4 N-n:lOH2-Sph GaI-SO4, n = 16, 18, 24 bovine brain-Spd PC egg-Spd PC bovine erythrocyte-Spd PC sheep erythrocyte-Spd PC human erythrocyte-Spd PC bovine brain-Spd Gal (total extract) bovine brain-spd Gal (non-hydroxy N-fatty acids) bovine brain-Spd Gal (a-hydroxy N-fatty acids) Gaucher spleen-Spd Glc winter rye-Spd Glc bovine adrenal medula-Spd Lac bovine brain-Spd GaI-SO4 human brain-Spd GaI-SO4 human brain-Spd Gal-NAc(/31-4)Gal(/3 1-4' )Glc( /3 1-1)

25-50 33 26 28 48 82-84 45-55 28-44 66 84 85-88 74-77 50-75 36-50 53-83 36 38 20 21-31 32 66 58-76 68-71 66-84 8-56 74 52-59 40 61

Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table

bovine brain-Spd Gal(/3 1-3)GalNAc(/3 1-4)Gal(/3 1-4)Glc(/3 1-1')

54

Table 7

pig erythrocyte-Spd GalNAc(/3 1-3)Gal( a 1-4)Gal(/3 1-4)GIc(/3 l- 1')

40

Table 7

Partially interdigitated lamellar gel-lamellar liquid crystal

N-24:0-Sph PC

44-50

Table 2

Lamellar gel-lamellar gel interconversions

N-n:0-Sph PC, n = 16, 22 N-24:0-Sph PC (mixed interdigitated-partially interdigitated lamellar gel)

18-30 36-43

Table 2 Table 2

D-erythro-N-18:0-Sph PC 33-44 L-threo-N-18:0-Sph PC 35-43 N-n:0-Sph Gal, n = 16, 18, 24 43-54 N-16:0-Sph Glc 51 N-n:0-Sph GaI-SO4, n = 18, 24 42-55 N-24:10H2-Sph Gal-SO4/2 M Li + 40-50 bovine brain-Spd Gal (non-hydroxy N-fatty acids) 52 Gaucher spleen-Spd Glc 63 winter rye-Spd Glc 20

Table Table Table Table Table Table Table Table Table

2 2 2 2 2 3 3 3 3 3 3 3 4 4 5 6 6 6 6 6 7 7 7 7 7 7 7 7 7

2 2 3 3 4 5 7 7 7

R. Koynoca, M. Caffrey/ Biochimica et Biophysica Acta 1255 (1995) 213-236

liquid crystalline) phases except for the human brain sulfogalactocerebroside (Na+-salt)/water system, in which non-lamellar isotropic cubic and micellar phases are reported at high water concentrations [102]. A more detailed description of these phase diagrams is beyond the scope of this review and of LIPIDAT. It is, however, within the purview of LIPIDAG - the phase diagram database [2]. In LIPIDAG, data are presented in the form of temperaturecomposition and temperature-pressure phase diagrams. LIPIDAG encompasses lipid/water and lipid/lipid phase diagrams prepared dry and in the presence of excess aqueous phase. Binary, multi-component and theoretical phase diagrams are included in the compendium. 4. 7. Sphingolipid / additive mixtures

A search of LIPIDAT under sphingolipid-lipid mixtures/additives yields 282 records (Table 1). These include 213 records for sphingolipid/lipid mixtures and 69 records for other additives. It is the latter group that we will focus on here since the former is beyond the scope of this article. The effect of an assortment of non-lipid additives on the phase behavior of sphingolipids is summarized in Table 11. Most of the entries in the table concern myelin basic protein and its interaction with sulfogalactocerebrosides and sphingomyelin. Myelin basic protein constitutes about 30% of total myelin protein and, as its name implies, is highly cationic [154]. It is shown to increase the rate of formation of the stable iamellar gel phase in the non-hydroxy fatty acid-containing sulfocerebrosides, at least at low concentrations [40]. At high protein concentration, however, the complex (double-peaked) shape of the calorimetric heating endotherm (entry nos. 5, 6 in Table 11) has been interpreted as indicating that some of the lipid sample remains in the metastable gel state in the presence of the protein. Thus, high myelin basic protein concentrations are thought to prevent the formation of the stable low-temperature state in non-hydroxy fatty acid sulfogalactocerebrosides. For the hydroxy fatty acid-containing lipids, the formation of the stable state is probably totally suppressed in the presence of the protein. In this case, the complex transition shape observed (entry nos. 3, 10 in Table 11) is indicative of a reduced lipid-protein interaction with only part of the lipid apparently influenced by the protein. Overall, the perturbing effect of the myelin basic protein on sulfogalactocerebrosides was found to be smaller compared to that on acidic phospholipids. The difference may be related to the participation of sulfocerebrosides in extensive hydrogen bonding interactions [40]. The myelin basic protein has been shown to influence the phase transition properties of N-16:0-Sph PC. Specifically, a double-peaked transition takes place at about the same temperature where a single endotherm is observed for the pure lipid (compare entry no. 5 in Table 2 with entry no. 16 in Table 11).

233

Polylysine increases the stable lamellar gel-lamellar liquid crystalline phase transition temperature and decreases the rate of stable gel phase formation in the non-hydroxy fatty acid sulfogalactocerebrosides and especially N-24:0-Spd Gal-SO4. The latter is probably due to a more efficient shielding of the sulfocerebroside charge in the presence of the basic polylysine [40]. In contrast, no effect of polylysine is registered in the case of N-16:0-Sph PC [36]. Ethylene glycol and dimethyisulfoxide (DMSO) have been shown to change the complex polymorphic behavior of pure hydrated N-16:0-Spd Gal to that of a single reversible transition (cf. entry no. 1 in Table 3 with entry nos. 14, 15 in Table 11). A stabilization of the low-temperature gel phase is also observed. This effect can be explained by the lower hydration of the lipid in the presence of kosmotropic solutes [4l].

5. Concluding remarks The purpose of this review has been to summarize the thermotropic data available in the literature on sphingolipids. By so doing, some characteristic features of this lipid class have been identified. Generally, only three types of polymorphs/mesophases (stable and metastable lamellar gel phases and the lamellar liquid crystalline phase) and three types of phase transitions were found to occur in hydrated sphingolipids. In the long-chain sphingolipids, where chain length inequivalence is pronounced, chain interdigitation is observed in the gel phases. The relevant data are presented in summary form in Table 12 where the various sphingolipid types giving rise to particular phases or phase transitions in defined temperature ranges are identified. The data in the table are organized in such a way as to facilitate the selection of a species of sphingolipid existing in a particular phase or exhibiting a particular type of phase transition in a defined temperature range. Based on an analysis of the sphingolipid species studied to date, the sphingolipids as a group would appear to be unique in comparison to the other major membrane lipids by the paucity of polymorphs and mesophases they can access. In stark contrast, the PEs and the glycoglycerolipids can access at least eight distinct polymorphs or mesophases with a minimum of eleven transitions possible between them [5,6]. The ability of the sphingolipids to form extensive hydrogen bonded network at the polarapolar interface is likely to be the primary factor contributing to the stability of the lamellar structures.

Acknowledgements This project was funded in part through a grant for Critical Compilations of Physical, Chemical and Materials

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Data from the National Institute of Standards and Technology and grants from the National Institutes of Health (DK 36849), The Procter and Gamble Company, and Avanti Polar Lipids, Inc. to M.C.

Appendix 1

Lipid nomenclature The nomenclature used in this article and in the LIPIDAT database for the sphingolipid class is as follows. The sphingolipid molecule is considered to be composed of three identifiable parts: (i) a sphingoid (referring to sphinganine,, to its homologs and stereoisomers and to its hydroxy and unsaturated derivatives) base; (ii) the acyl chain linked to the sphingoid amide nitrogen; and (iii) the polar head group. The default structure is the D-erythro isomer with an unbranched, unmodified saturated hydrocarbon chain esterified to the amide nitrogen and the head group covalently attached at the C1 position of sphingosine. For base designation, the descriptors -Sph, -Spn, and -Spd are used for sphingosine, dihydrosphingosine (sphinganine) and a general sphingoid base, respectively. The specific stereochemical configuration with respect to the C3 and C2 positions on the base is indicated by a prefix in the lipid descriptor as O-, L- and erythro-, threo, respectively. For pure synthetic or semisynthetic lipid preparations, acyi chain residues are fully specified, using a systematic nomenclature as follows. The chain length, in units of carbon atoms (and with the first carbon of the chain defined to be the one bonded through an oxygen atom to the amide nitrogen) is given to the left of a colon (':'). Modifications to the acyl chain are indicated to the right of the colon and are listed according to number, kind, and location. First, to the right of the colon appears the number of modifications on the chain. A zero frO') indicates that the chain is in the default configuration, with no modifications. Thus, for example, N-18:0-Sph indicates Nstearoylsphingosine. Following the number of modifications, the modifications themselves are listed alphabetically. Following each modification is a number indicating the carbon atom position on the chain where the modification is located. The database nomenclature system employs a strategy to reflect the diversity of modifications to sphingolipid acyl chains by describing changes from the default on an atom-by-atom basis along the chain. Modifications include, but are not limited to, unsaturation at one or more sites along the chain, and the presence of functionalities such as hydroxyl groups or heteroatoms. For example, N-18:IOH2-Sph denotes the sphingosine base with an o~-hydroxystearyl moiety linked to the amide nitrogen. The following notation is used to denote double bonds. The

letters 'c' and 't' denote, respectively, the cis and t r a n s configuration of the double bond, followed by a number or set of numbers, identifying double bond position. Thus, the linolenoyl chain which is an 18 C acyl moiety with cis double bonds at C9, C12 and C15, is described simply by 18:3c9,12,15. In the default condition for sphingolipids, the head group is assumed to be linked at the C1 position of the ceramide base. It is indicated after the ceramide descriptor. The most common head group descriptors for sphingolipids are phosphocholine (PC) in the case of sphingomyelins, and galactose (Gal), glucose (Glc), and galactose-suifate (Gal-SO4) in the case of cerebrosides.

References [1] Caffrey, M. (1993) NIST Standard Reference Database 34 Lipid Thermotropic Phase Transition Database (LIPIDAT), National Institute of Standards and Technology, Gaithersburg, MD 208990001. E-mail: [email protected]. Fax: + 1 (301) 926 0416. [2] Caffrey, M., Moynihan, D. and Hogan, J. (1991) Chem. Phys. Lipids 57, 275-291. [3] Caffrey, M. (1993) LIPIDAT: A Database of Thermodynamic Data and Associated Information on Lipid Mesomorphic and Polymorphic Transitions. CRC Press, Boca Raton, FL 33431. [4] Caffrey, M. and Hogan, J. (1992) Chem. Phys. Lipids 61, 1-109. [5] Koynova, R. and Caffrey, M. (1994) Chem. Phys. Lipids 69, 1-34. [6] Koynova, R. and Caffrey, M. (1994) Chem. Phys. Lipids, 69, 181-207. [7] IUPAC-IUB Commision on Biochemical Nomenclature (1977) Eur. J. Biochem. 79, 11-21. [8] Lehninger, A. (1975) in Biochemistry: The Molecular Basis of Cell Structure and Function, Worth Publishers, New York [9] Murray, R., Narasimhan, R., Levine, M., Pinteric, L., Shirley, M., Lingwood, C. and Schachter, H. (1980) Cell Surface Glycolipids ACS Symposium Series 128 (Sweeley, C.C., ed.), pp. 105-125. [10] Kates. M. (1978) Prog. Chem. Fats Other Lipids 15, 301-327. [11] Hori, T., Horiguchi, M. and Hayashi, A. (Eds.) (1984) Biochemistry of Natural C-P Compounds (Maruzen, Kyoto), p. 53, 124-144. [12] Kates, M. (1970) Adv. Lipid Res. 8, 225-265. [13] Laine, R. and Renkonen, O. (1973) Biochemistry 12, 1106-1111. [14] Laine, R. and Renkonen, O. (1974) Biochemistry 13, 2837-2843. [15] Curatolo, W. (1987) Biochim. Biophys. Acta 906, 111-136. [16] Huang, C.-h. and Mason, J. (1986) Biochim. Biophys. Acta 864, 423-470. [17] Boggs, J. (1987) Biochim. Biophys. Acta 906, 353-404. [18] Barenholz, Y. and Thompson, T. (1980) Biochim. Biophys. Acta 604, 129-158. [19] Pick, L. and Bielschowsky, M. (1927) Klin. Wochenschr. 6, 16311637. [20] Shapiro, D., Flowers, H. (1962) J. Am. Chem. Soc. 84, 1047-1050. [21] Finean, J. (1953) Biochim. Biophys. Acta 10, 371-384. [22] Clowes, A., Cherry, R. and Chapman, D. (1971) Biochim. Biophys. Acta 249, 301-317. [23] Demel, R., Jansen, J., Van Dijck, P. and Van Deenen, L. (1977) Biochim. Biophys. Acta 465, 1-10. [24] Curatolo, W., Small, D. and Shipley, G. (1977) Biochim. Biophys. Acta 468, 11-20. [25] Cohen, R. and Barenholz, Y. (1978) Biochim. Biophys. Acta 509, 181-187. [26] Van Dijck, P. (1979) Biochim. Biophys. Acta 555, 89-101. [27] Calhoun, W. and Shipley, G. (1979) Biochim. Biophys. Acta 555, 436-441.

R. Koynol,a, M. Caffrey / Biochimica et Biophysica Acta 1255 (1995) 213-236

[28] Skarjune, R. and Oldfield, E. (1979) Biochim. Biophys. Acta 556, 208-218. [29] Bach, D. and Sela, B.-A. (1980) Biochim. Biophys. Acta 596, 186-(191. [30] Cullis, P. and Hope, M. (19801 Biochim. Biophys. Acta 597, 533-542. [31] Mackay, A., Wassail, S., Valic, M., Gorrissen, H. and Cushley, R. (1980) Biochim. Biophys. Acta 6111, 22-33. [32] Hui, S., Stewart, T. and Yeagle, P. (19801 Biochim. Biophys. Acta 601, 271-281. [33] Alien, T. (1981) Biochim. Biophys. Acta 640, 385-397. [34] Hinz, H.-J., Koerner, O. and Nicolau, C. 11981) Biochim. Biophys. Acta 643, 557-571. [35] Siminovitch, D. and Jeffrey, K. (1981) Biochim. Biophys. Acta 645, 270-278. [36] Epand, R.M and Moscarello, M. (1982) Biochim. Biophys. Acta 685, 230-232. [37] Bach, D., Miller, I. and Sela, B.-A. (1982) Biochim. Biophys. Acta 686, 233-239. [38] Epand, R.M., Boni, L. and Hui, S. (1982) Biochim. Biophys. Acta 692, 330-338. [39] Dasseux, J., Faucon, J., Lafleur, M., Pezolet, M. and Dufourcq, J. (1984) Biochim. Biophys. Acta 775, 37-50. [40] Boggs, J., Rangaraj, G., Moscarello, M. and Koshy, K. (1985) Biochim. Biophys. Acta 816, 208-220. [41] Curatolo, W. (1985) Biochim. Biophys. Acta 817, 134-138. [42] Maggio, B., Ariga, T., Sturtevant, J. and Yu, R. (1985) Biochim. Biophys. Acta 818, 1-12. [43] Barkai, G., Mashiach, S., Goldman, B., Kalina, M. and Shinitzky, M. (1985) Biochim. Biophys. Acta 834, 103-109. [44] Ruocco, M. and Shipley, G. (1986) Biochim. Biophys. Acta 859, 246-256. [45] Curatolo, W. (1986) Biochim. Biophys. Acta 861, 373-376. [46] Miller, I.R. and Bach, D. (1986) Biochim. Biophys. Acta 863, 121-127. [47] Mehlhorn, I., Parraga, G., Barber, K. and Grant, C. (1986) Biochim. Biophys. Acta 863, 139-155. [48] Reed, R. and Shipley, G. (1987) Biochim. Biophys. Acta 896, 153-164. [49] Tsao, Y., Freire, E. and Huang, L. (1987) Biochim. Biophys. Acta 900, 79-87. [50] Maggio, B., Sturtevant, J. and Yu, R. (1987) Biochim. Biophys. Acta 901, 173-182. [51] Boggs, J., Koshy, K. and Rangaraj, G. (1988) Biochim. Biophys. Acta 938, 361-372. [52] Boggs, J., Koshy, K. and Rangaraj, G. (19881 Biochim. Biophys. Acta 938, 373-385. [53] Johnston, D. and Chapman, D. (1988) Biochim. Biophys. Acta 939, 603 -614. [54] Mehlhorn, I., Barber, K. and Grant, C. (1988) Biochim. Biophys. Acta 943, 389-41t4. [55] Jackson, M., Johnston, D. and Chapman, D. (1988) Biochim. Biophys. Acta 944, 497-506. [56] Gardam, M. and Silvius, J. (1989) Biochim. Biophys. Acta 980, 319-325. [57] Cunningham, B., Tamura-Lis, W., Lis, L. and Collins, J. (1989) Biochim. Biophys. Acta 984, 109-112. [58] Mehlhorn, 1., Barber, K., Florio, E. and Grant, C. (19891 Biochim. Biophys. Acta 986, 281-289. [59] Maulik. P., Spirada, P. and Shipley, G. (19911 Biochim. Biophys. Acta 1062, 211-219. [611] Long, R., Hruska, F., Gesser, H. and Hsia, J. (1971) Biochem. Biophys. Res. Commun. 45, 167-173. [61] Thewalt, J., Kitson, N., Araujo, C., MacKay, A. and Bloom, M. (1992) Biochem. Biophys. Res. Commun. 88, 1247-1252. [62] Boggs, J., Mulholland, D. and Koshy, K. (1990) Biochem. Cell Biol. 68, 70-82.

235

[63] Yagi, K., Uchiyama, F., Ohki, K., Kojima, N. and Nozawa, Y. (1984) Biochem. Int. 9, 791-797. [64] Viani, P., Marchesini, S., Cervato, G. and Cestaro, B. (1986) Biochem. Int. 12, 125-135. [65] Barenholz, Y., Suurkuusk, J., Mountcastle, D., Thompson, T. and Biltonen, R. (1976) Biochemistry 15, 2441-2447. [66] Correa-Freire, M., Freire, E., Barenholz, Y., Biltonen, R. and Thompson, T. (19791 Biochemistry 18, 442-445. [67] Calhoun, W. and Shipley, G. (19771 Biochemistry 18, 1717-1722. [68] Estcp, T., Mountcastle, D., Barenholz, Y., Biltonen, R. and Thompson, T. (1979) Biochemistry 18, 2112-2117. [69] Estep, T., Calhoun, W., Barenholz, Y., Biltonen, R., Shipley, G. and Thompson, T. (19801 Biochemistry 19, 20-24. [70] Freire, E., Bach, D., Correa-Freirc, M., Miller, 1.R. and Barenholz, Y. (1980) Biochemistry 19, 3662 3665. [71] Ruocco, M., Atkinson, D., Small, D., Skarjune, R., Oldfield, E. and Shipley, G. 119811 Biochemistry 20, 5957-5966. [72] Lentz, B., Hoechli, M. and Barenholz, Y. (19811 Biochemistry 20, 6803-6809. [73] Estep, T., Freire, E., Anthony, F., Barenholz, Y., Biltonen, R. and Thompson, T. (1981) Biochemistry 20, 7115-7118. [74] Curatolo, W. (1982) Biochemistry 21, 1761-1764. [75] Barenholz, Y., Freire, E., Thompson, T., Correa-Freire, M., Bach, D. and Miller, I.R. (1983) Biochemistry 22, 3497-3501. [76] Maggio, B., Ariga, T., Sturtevant. J. and Yu, R. 11985) Biochemistry 24, 1084-1092. [77] Levin, 1., Thompson, T., Barenholz, Y. and Huang, C. (1985) Biochemistry 24, 6282-6286. [78] Curatolo, W. and Jungalwala, F. (1985) Biochemistry 24, 66086613. [79] Anderle, G. and Mendelsohn, R. 119861 Biochemistry 25, 21742179. [80] McKeone, B., Pownall, H. and Masscy, J. 11986) Biochemistry 25, 7711-7716. [81] Bruzik, K. and Tsai, M. 11987) Biochemistry 26, 5364-5368. [82] Ollmann, M., Scharzmann, G., Sandhoff, K. and Galla, H. (1987) Biochemistry 26, 5943-5952. [83] Masserini, M., Palestini, P., Venerando, B., Fiorilli, A., Acquotti, D. and Tcttamani, G. (19881 Biochemistry 27, 7973-7978. [84] Rintoul, D. and Welti, R. (19891 Biochemistry 28, 26-31. [85] Masserini, M., Palestini, P. and Frcirc, E. (19891 Biochemistry 28, 5029-5034. [86] Schrocdcr, F. and Nemecz, G. (1989) Biochemistry 28, 5992-6000. [87] Bruzik, K., Sobon, B. and Salamonczyk, G. (19911) Biochemistry 29, 4(117-4021. [88] Kan, C.-C., Ruan, Z.-s. and Bittman, R. (1991) Biochemistry 30, 7759-7766. [89] Stevenson, C., Rich, N. and Boggs, J. (19921 Biochemistry 31, 1875-1881. [90] Molotkovsky, J., Gromova, I. and Bergelson, L. (1991) Biol. Membr. 8, 934-942. [91] Tsai, M., Bruzik, K., Wisner, D. and Liu, S. 11987) Biophos. Analog., 561-570. [92] Bunow, M. and Bunow, B. (19791 Biophys. J. 27, 325-337. [93] Ruocco, M., Shipley, G. and Oldfield, E. 11983) Biophys. J. 43, 91-101. [94] Ruocco, M. and Shipley, G. (1984) Biophys. J. 46, 695-707. [95] Hresko, R., Sugar, I., Barenholz, Y. and Thompson, T. (19871 Biophys. J. 51, 725-733. [96] Reed, R. and Shipley, G. (1988) Biophys. J. 53, 124a-n. [97] Reed, R. (1988) Biophys. J. 53, 253a-n. [98] Reed, R. and Shipley, G. 119891 Biophys. J. 55, 281-292. [99] Lynch, D., Caffrey, M., Hogan, J. and Steponkus, P. (1992) Biophys. J. 61, 1289-1300. [100] Luscher-Mattli, M. (1989) Biopolymers 28, 799-817. [101] Margolis, L., Tikhonov, A. and Vasilieva, E. (1980) Cell 19, 189 195.

236

R. Koynol~a, M. Caffrey / Biochimica et Biophysica Acta 1255 (1995) 213-236

[102] Abrahamsson, S., Pascher, I., Larsson, K. and Karlsson, K.-A. (1972) Chem. Phys. Lipids 8, 152-179. [103] Faiman, R. (1979) Chem. Phys. Lipids 23, 77-84. [104] Dahlen, B. and Pascher, I., (1979) Chem. Phys. Lipids 24, 119-133. [105] Epand, R.M. and Epand, R.F. (1980) Chem. Phys. Lipids 27, 139-150. [106] Koshy, K. and Boggs, J. (1983) Chem. Phys. Lipids 34, 41-53. [107] Cohen, R., Barenholz, Y., Gatt, S. and Dagan, A. 11984) Chem. Phys. Lipids 35, 371-384. [108] Boggs, J., Koshy, K. and Rangaraj, G. (1984) Chem. Phys. Lipids 36, 65-89. [109] Pink, D., Macdonald, A. and Quinn, B. (1988) Chem. Phys. Lipids 47, 83-95. [110] Molotkovsky, J., Mikhalyov, 1., lmbs, A. and Bergelson, L. (199l) Chem. Phys. Lipids 58, 199-212. [111] Nurul Amin, M., Collins, J., Tamura-Lis, W., Lis, L. and Quinn, P. (1989) Colloids Surfaces 36, 459-467. [112] Oldfield, E. and Chapman, D. (1972) FEBS Lett. 21,303 306. [113] Neuringer, L., Sears, B., Jungalwala, F. and Shriver, E. (1979) FEBS Lett. 104, 173-175. [114] Cestaro, B., Cervato, G., Di Silvestro, G., Sozzani, P. and Berra, B. (1981) ltal. J. Biochem. 30, 429-436. [115] Cestaro, B., Marchesini, S., Cervato, G., Viani, P. and Vesely, S. (1984) Ital. J. Biochem. 33, 381-391. [116] Utsumi, H., Suzuki, T., Inoue, K. and Nojima, S. (1984) J. Biochem. 96, 97-11)5. [117] Kojima, H., Hanada-Yoshikawa, K., Katagiri, A. and Tamai, T. (1988) J. Biochem. 103, 126-131. [118] Untracht, S. and Shipley, G. (1977)J. Biol. Chem. 252, 4449-4457. [119] Rujanavech, C., Henderson, P. and Silbert, D. (1986) J. Biol. Chem. 261, 7204-7214. [120] Maggio, B., Sturtevant, J. and Yu, R. (1987) J. Biol. Chem. 262, 2652-2659. [121] Chien, K., Huang, W., Jean, J. and Wu, W. (1991) J. Biol. Chem. 266, 3252-3259. [122] Wu, W., Chi, L., Yang, Y. and Fang, S. (1991) J. Biol. Chem. 266, 13602-13606. [123] Chi, L., Hsieh, C. and Wu, W. (1992) J. Chin. Chem. Soc. 39, 35-42. [124] Shipley, G., Avecilla, L. and Small, D. (1974) J. Lipid Res. 15, 124-131. [125] Ahmad, T., Sparrow, J. and Morrisett, J. (1985) J. Lipid Res. 26, 11611-1165.

[126] Spirada, P., Maulik, P., Hamilton, J. and Shipley, G. 11987) J. Lipid Res. 28, 710-718. [127] Lamba, O., Borchman, D., Sinha, S., Lal, S., Yappert, M. and Lou, M. 11991) J. Mol. Struct. 248, 1-24. [128] Bhattacharyya, M., Sarkar, M. and Nandy, P. (1991) J. Surface Sci. Technol. 7, 323-330. [129] Merajver, S. and Sheridan, J. (1981) J. Theor. Biol. 93, 737-755. [130] Borchman, D., Lamba, O., Salmassi, S., Lou, M. and Yappert, M. (1992) Lipids 27, 261-265. [131] Harris, R., Groh, G., Baxter, D. and Hitzemann, R. (1984) Mol.

Pharmacol. 25, 410-417. [132] Brandenburg, K. and Seydel, U. (1986) Z. Naturforsch. 41c, 453467. [133] Barenholz, Y. and Gatt, S. 11982) Phospholipids, New Comprehensive Biochemistry, Vol. 4 (Hawthorne, J. and Ansell, G., eds.) Elsevier, Amsterdam. [134] Sundaralingam, M. (1974) Ann. N.Y. Acad. Sci. 195, 324-355. [135] Tilcock, C. (1986) Chem. Phys. Lipids 40, 109-125. [136] Boyanov, A., Koynova, R. and Tenchov, B. (1986) Chcm. Phys. Lipids 39, 155-163 [137] Shapiro, D. (1969) Chemistry of Sphingolipids, Herman, Paris. [138] Bruzik, K. (1986) J. Chem. Soc., Chem. Commun., 329-331. [139] Tenchnv, B., Boyanov, A. and Koynova, R. (1984) Biochemistry 23, 3553-3558. [140] Norton, W. and Cammer, W. (1984) in Myelin (Morell, P., ed.), pp. 147 195, Plenum Press, New York. [141] Karlsson, K.-A. (1970) Chem. Phys. Lipids 5, 6-43. [142] Stinson, R. and Boggs, J. (1989) Biochim. Biophys. Acta 986, 234-240. [143] Abramson, M., Katzman, R., Wilson, C. and Gregor, H. (1964)J. Biol. Chem. 239, 4066-4072. [144] Fishman, P. (1988) Fidia Res. Set. 14, 183-202. [145] Hakomori, S. (1981) Annu. Rev. Biochem. 50, 733-764. [146] Corti, M., Cantu, L., Sonnino, S. and Tettamanti, G. (1988) Fidia Res. Set. 14, 79-91. [147] Corti, M., Cantu, E., Sonnino, S. and Tettamanti, G. (1990) Chem. Phys. Eipids 55, 223-229. [148] Sonnino, S., Cantu, L., Acquotti, D., Corti, M. and Tettamanti, G. (1990) Chem. Phys. Lipids, 52, 231-241. [149] Leon, A., Facci, E., Toffano, G., Sonnino, S. and Tettamanti, G. (1981) J. Neurochem. 37, 350-357. [150] Ulrich-Bott, B. and Wiegandt, H. (1984) J. Lipid Res. 25, 12331245. [151] Karlsson, K. 11970) Lipids 5, 878-891. [152] White, D. (1973) in Form and Function of Phospholipids (Ansell, G., Dawson, R. and Hawthorne, J., eds.), pp. 441-482, Elsevier, Amsterdam. [153] Rouser, G., Nelson, C., Fleischer, S. and Simon, G. (1968) in Biological membrane (Chapman, D., ed.), p. 5, Academic Press, New York. [154] Boggs, J., Wood, D. and Moscarello, M. (1981) Biochemistry 20, 1065-1/)73. [155] Pascher, I. and Sundell, S. (1977) Chem. Phys. Lipids 20, 175-191. [156] Pascher, I. (1976) Biochim. Biophys. Acta 455, 433-451. [157] Loefgren, H. and Pascher, I. (1977) Chem. Phys. Lipids 20, 273-284. [158] Nyholm, P.-G., Pascher, 1. and Sundell, S. (1990) Chem. Phys. Lipids 52, 1-10. [159] Jarrell, H., Giziewicz, J. and Smith, 1. 11986) Biochemistry 25, 3950-3957. [160] Svennerholm, L. (1963) J. Neurochem. 10, 613-623. [161] Wiegandt, H. (1971) Adv. Lipid Res. 9, 249-289.