Biosynthetic incorporation of fatty acids with photosensitive groups into membrane lipids of cells in tissue culture.

The physical methods (13C-NMR-spectroscopy and fluorescence spectroscopy) hitherto used for the elucidation of lipid-lipid and lipid-protein interactions in artificial and simple natural membranes were extended to the application of fatty acids, phospholipids and sphingolipids with photochemical labels (azide group) in defined positions, which on photolysis generate nitrenes. These highly reactive groups react with neighbouring molecules, either lipids or polypeptide chains, with insertion or addition. Highly radioactive 12-azido[9,10-3H2]stearic acid, 12-azido[12-3H]oleic acid and 18-azido-[9,10,12,13-3H4]linoleic acid were added to the growth medium of eukaryotic cell lines in tissue culture (BHK 21 cells and Chang liver cells). They were incorporated into neutral, phosphoand sphingolipids in amounts comparable with the unsubstituted parent fatty acids. The distribution of the azido fatty acids in the phospholipids has been determined by enzymatic hydrolysis (phospholipase A2) on the basis of the distribution of their radioactivity. Radio gas chromatography and combined gas chromatography and mass spectroscopy revealed that the azide group of the radioactive fatty acids remained unaltered.     

Synthesis of fatty acids from [1-14C]acetylcoenzyme A in subcellular particles of rat epididymal adipose tissue

1. Mitochondrial and microsomal fractions of rat epididymal adipose tissue incorporated [1-(14)C]acetyl-CoA equally well into various fatty acids by a chain-elongation mechanism. C(18) and C(20) fatty acids were the two major products, and comprised about 80% of the total fatty acids synthesized in both particles. 2. When incubated in air, mitochondria synthesized stearic acid, octadecenoic acid and eicosamonoenoic acid in almost equal amounts (about 20% each), whereas in microsomal fractions, the synthesis of octadecenoic acid was more than fivefold the stearic acid formation. In both fractions, major components of synthesized monoenoic fatty acids were the Delta(11:12) isomers. Hexadecenoic acid and octadecenoic acid from whole adipose tissue contained approx. 11 and 14% of the Delta(11:12) isomer respectively. 3. When mitochondria or microsomal fractions were incubated in nitrogen, there was increased synthesis of stearic acid and palmitic acid and less of C(16) and C(18) monoenoic acids; synthesis of C(20) acids remained predominantly of the monoenoic acids. Determination of the position of the double bond in the monoenoic acids supported the view that the synthesis of hexadecenoic acid and octadecenoic acid involves a desaturase activity, whereas eicosamonoenoic acid and eicosadienoic acid are formed only by elongation of endogenous fatty acids. 4. Most of the radioactivity was found in free fatty acids (63%) and the phospholipid (26%) fraction. In phospholipids, phosphatidylcholine and phosphatidylethanolamine were the two major components. 5. Most of the fatty acids synthesized, including those not normally found in particle lipids (arachidic acid, eicosamonoenoic acid and eicosadienoic acid) were distributed fairly evenly in the phospholipid and free fatty acid fractions. However, stearic acid was found predominantly in the phospholipid fraction.     

Incorporation of spin-labeled fatty acids into bovine brain clathrin coated vesicles

Stearic acids with a nitroxide radical at selected positions have been incorporated in the phospholipid bilayers of clathrin coated vesicles, uncoated vesicles and sonicated liposomes made from the lipids extracted from the uncoated vesicles. The extent of incorporation was found minimum for stearic acids labeled on C-12 and for bilayers of uncoated vesicles. The ESR spectra of the spin-labeled fatty acids incorporated in the bilayers showed a pronounced temperature dependence (without discontinuity) and a decrease in the hyperfine splitting as the nitroxide group was inserted deeper in the hydrophobic core of the membranes. An abrupt phospholipid phase transition or a phase separation could be excluded. The presence of the external proteins (the clathrin coat) on the membranes was not found to noticeably influence the gradient of flexibility of the fatty acid chains of the phospholipids. The influence of the internal proteins embedded in the bilayers was evidenced by a detailed analysis of the ESR spectra of (7,8)SA in terms of two components: one component arising from the labels surrounded exclusively by phospholipids, the other component arising from labels of reduced mobility perturbed by the vicinity of the proteins. These results support the persistence of lipidic domains in the endocytic vesicles despite the accumulation of receptors which follows their formation.     

Incorporation of exogenous fatty acids into phospholipids by cultured hamster fibroblasts. Effect of SV40 transformation

In situ incorporation of two saturated (palmitic, 16:0; stearic, 18:0) and three unsaturated fatty acids (oleic, 18:1; linoleic, 18:2; arachidonic, 20:4) into the four major phospholipids, sphingomyelin, PC, PI and PE, was followed. Transformed cells incorporated unsaturated fatty acids more rapidly, whereas no significant differences were found concerning saturated fatty acids. In vitro determination of phospholipid acylation showed that incorporation of coenzyme A-activated forms of two saturated fatty acids (16:0 and 18:0) and one unsaturated fatty acid (18:1) into phospholipids was increased in transformed cells. Comparison of results obtained in situ and in vitro strongly suggests that incorporation of fatty acids into phospholipids in cultured cells is not limited by acyltransferase activities.     

The fatty acid composition of the serum phospholipids of children with sickle cell disease in Nigeria

The purpose of this study was to determine the fatty acid composition of the serum phospholipids of children with sickle cell disease (SCD) in Nigeria and to compare the relative fluidity of the acyl chains of the serum phospholipids of controls versus the subjects with SCD. It is widely accepted that the fatty acid composition of an individual's serum phospholipids reflects that of their tissue phospholipids. An alteration in the fatty acid composition of membrane phospholipids could affect critical membrane-dependent enzymes and processes (e.g., ion and solute transport, hormone-receptor interactions, signal transduction pathways). We found a significant reduction in the content of polyunsaturated n-3 fatty acids in the phospholipids of subjects with SCD which could result in a reduction of the fluidity of their tissue membranes. Specifically, there was a 40-50% reduction in the proportion of total n-3 fatty acids in subjects with SCD. On the basis of calculated melting points and double bond indices of the acyl chains of the serum phospholipids, the phospholipids of the children with SCD are less fluid relative to those of their healthy counterparts. In addition, we determined that linoleic acid, arachidonic acid, and stearic acid were the major determinants of the fluidity of the acyl chains of the serum phospholipids of the healthy controls and children with SCD.     

Facile syntheses for (5Z,9Z)-5,9-hexadecadienoic acid, (5Z,9Z)-5,9-nonadecadienoic acid, and (5Z,9Z)-5,9-eicosadienoic acid through a common synthetic route

The delta 5,9 fatty acids (5Z,9Z)-5,9-hexadecadienoic acid, (5Z,9Z)-5,9-nonadecadienoic acid, and (5Z,9Z)-5,9-eicosadienoic acid were synthesized for the first time in four steps (9-12% overall yield) starting from commercially available 2-(2-bromoethyl)-1,3-dioxolane. The synthetic approach provided enough material to corroborate the structure and stereochemistry of (5Z,9Z)-5,9-nonadecadienoic acid which was recently identified in the flowers of Malvaviscus arboreus (Malvaceae). The novel phospholipids 1-hexadecanoyl-2-[(5Z,9Z)-5,9-eicosadienoyl]-sn-glycer o-3-phosphocholine and 1-octadecanoyl-2-[(5Z,9Z)-5,9-eicosadienoyl]-sn- glycero-3-phosphocholine were also synthesized from commercially available L-alpha-phosphatidylcholine (egg yolk) and characterized by positive ion electrospray mass spectrometry. These are the first examples of unsymmetrical phospholipids with saturated fatty acids at the sn-1 position and delta 5,9 fatty acids at the sn-2 position.     

Effect of cholesterol on miscibility and phase behavior in binary mixtures with synthetic ceramide 2 and octadecanoic acid. Infrared studies

The three main lipid components of the stratum corneum, namely ceramides, free fatty acids and cholesterol, play a fundamental role in the maintenance of the skin barrier. The current investigation is aimed toward understanding the miscibility and intermolecular interactions of these lipids. Toward this end, Fourier transform infrared spectroscopic studies of the three possible equimolar binary mixtures of cholesterol, a synthetic non-hydroxylated fatty acid N-acyl sphingosine with a C18 chain length (N-stearoylsphingosine, approximating human ceramide 2), and stearic acid were undertaken. The thermotropic responses of the methylene stretching and scissoring vibrations were used to evaluate chain conformation and packing respectively. Selective perdeuteration, of either the stearic acid or the ceramide acid chains, permitted separate and simultaneous evaluation of the conformational order and packing properties of the sphingosine chain, the amide linked fatty acid chains and/or the stearic acid chain. Whereas cholesterol mixed well with ceramide at physiological temperatures, the stearic acid was miscible with the cholesterol only at relatively high temperatures where the fatty acid is disordered. A complex interaction between stearic acid and ceramide was detected. A separate fatty acid-rich phase persisted until at least 50 degrees C, whereas at higher temperatures the components appear to be quite miscible. However, a preferential association of the fatty acid with the ceramide base chain is indicated. None of the binary systems studied exhibit miscibility and interactions resembling those in the ternary mixtures of these substances, which is widely used to model stratum corneum. The role of cholesterol in controlling the miscibility characteristics in the ternary system is evident.     

The influence of fatty acids on model cholesterol/phospholipid membranes

The aim of this work was to verify the influence of the saturated (SFA) (stearic acid) and the unsaturated (UFA) (oleic and alpha-linolenic) fatty acids on model cholesterol/phospholipid membranes. The experiments were based on the Langmuir monolayer technique. Cholesterol and phospholipid were mixed in the molar ratio that corresponds to the proportion of these lipids in the majority of natural human membranes. Into the binary cholesterol/phospholipid monolayers, various amounts of fatty acids were incorporated. Our investigations were based on the analysis of the interactions between molecules in ternary (cholesterol/phospholipids/fatty acid) mixtures, however, also binary (cholesterol/fatty acid and phospholipids/fatty acid) mixed system were examined. It was concluded that the influence of the fatty acids on model cholesterol/phospholipid membrane is closely connected with the shape of the fatty acid molecule, resulting from the saturation degree of the hydrocarbon chain. It was found that the saturated fatty acid makes the model membrane more rigid, while the presence of unsaturated fatty acid increases its fluidity. The increasing amount of stearic acid gradually destabilizes model membrane, however, this effect is the weakest at low content of SFA in the mixed monolayer. Unsaturated fatty acids in a small proportion make the membrane thermodynamically more stable, while higher content of UFA decreases membrane stability. This explains low proportion of the free fatty acids to other lipids in natural membrane.     

Metabolism of fatty acids by bovine spermatozoa

The incorporation of (14)C-labelled myristic, palmitic, stearic, oleic and linoleic acids in vitro into the lipids of bovine spermatozoa was measured at intervals from 2min to 2h. All acids were rapidly incorporated into diglycerides, myristic acid being metabolized to the greatest extent. Whereas the low incorporation of acids into total phospholipids reflected the relative stability of the major phospholipid fractions in sperm, the minor phospholipids, particularly phosphatidylinositol, showed comparatively high metabolic activity. Although, in general, saturated acids were incorporated more actively than unsaturated substrates, stearic acid was poorly incorporated into all lipids except phosphatidylinositol. In regard to fatty acid composition of sperm lipids it was notable that diglycerides contained myristic acid as the major component, and this acid was also a prominent moiety of phosphatidylinositol. Docosahexaenoic acid was the principal fatty acid of the major phospholipid classes. These findings have been discussed in relation to the role of lipids in the metabolism of spermatozoa.     

Structural and dynamical studies of mixed chlorophyll/phosphatidylcholine bilayers via x-ray diffraction, absorption polarization spectroscopy and nuclear magnetic resonance.

The structure and dynamics of phosphatidylcholine bilayers containing chlorophyll were studied by X-ray diffraction and absorption polarization spectroscopy in the form of hydrated orientated multilayers below the thermal phase transition of the lipid chains and by nuclear magnetic resonance in the form of single-wall vesicles above the thermal transition. Our results show that (a) chlorophyll is incorporated into the phosphatidylcholine bilayers with its porphyrin ring located anisotropically in the polar headgroup layer of the membrane and with its phytol chain penetrating in a relatively extended form between the phosphatidylcholine fatty acid chains in the hydrocarbon core of the mixed bilayer membrane and (b) the intramolecular anisotropic rotational dynamics of the host phosphatidylcholine molecules are significantly perturbed upon chlorophyll incorporation into the bilayer at all levels of the phosphatidylcholine structure. These dynamics for the host phosphatidylcholine fatty acids chains are qualitatively different from that of the incorporated chlorophyll phytol chains on a 10(-9)-10(-10)s time scale in the ideally mixed two-component bilayer.     

Fatty acid composition of total lipids and phospholipids of membrane preparations of transport ATPases

The fatty acid composition of total lipids and phospholipids of duck salt gland Na,K-ATPase (outer plasma membrane) and of rabbit skeletal muscle Ca-ATPase (intracellular membrane) was investigated. The bulk of Na,K-ATPase fatty acids is represented by palmitic (16:0), oleic (18:1), stearic (18:0) and arachidonic (20:4) acids. The duck salt gland is characterized by rather a high content of unsaturated fatty acids, especially of arachidonic acid. The unsaturation index of total-lipid fatty acids increases during purification of these preparations in the following order: homogenate greater than microsomal fraction greater than purified enzyme. The fatty acid composition of Na,K-ATPase total lipids and phospholipids reveals certain differences. Phospholipids contain more stearic and liholeic (18:2) acids than total lipids, but the level of arachidonic acid in them is twice as low. Besides, phospholipids were found to contain polyunsaturated docosohexaenic (22:6) acid. The bulk of fatty acids of rabbit skeletal muscle Ca-ATPase total lipids and phospholipids is represented by 16:0, 18:0, 18:1 and 18:2 acids. The content of polyunsaturated fatty acids in this preparation is much lower than in duck salt gland Na,K-ATPase. The fatty acid composition of total lipids and phospholipids in rabbit skeletal muscle Ca-ATPase differ insignificantly. The differences in the fatty acid composition of membrane preparations under study is conditioned mainly by the fractional composition of their lipids.     

Evolving lipid vesicles in prebiotic hydrothermal environments

We compared three different kinds of lipid vesicles made of saturated fatty acids, unsaturated fatty acids, and phospholipids for their evolutionary capabilities in a simulated hydrothermal environment.Encapsulation of the glycine monomers enhanced the oligomerization of peptides in all cases. Fatty acid vesicles remained stable at higher temperatures and efficiently utilized heat energy for this synthetic reaction. Phospholipid vesicles were destabilized by higher temperatures, and thus were found to be better suited to enhance synthetic reactions at lower temperatures     

Changes in the fatty acid profiles of red blood cell membrane phospholipids in human neonates during the first month of life

We have studied the changes in the fatty acid profiles of red blood cell membrane phospholipids in 47 infants who were exclusively fed human milk from birth to 1 month of life. Twenty blood samples were obtained from cord, 15 at 7 days and 12 at 30 days after birth. Membrane phospholipids were obtained from erythrocyte ghosts by thin-layer chromatography and fatty acid composition was determined by gas liquid chromatography. Phosphatidylcholine showed the most important changes during early life; stearic, w6 eicosatrienoic and arachidonic acids decreased whereas oleic and linoleic acids increased. In phosphatidylethanolamine, palmitic and stearic acid declined and oleic, linoleic and docosahexenoic acids increased with advancing age. Small changes were noted for individual fatty acids in phosphatidylserine. In sphingomyelin stearic acid increased from birth to 1 month and linoleic, arachidonic and nervonic acids decreased. Total polyunsaturated fatty acids of the w6 series greater than 18 carbon atoms increased with advancing age in phosphatidylethanolamine and decreased in choline and serine phosphoglycerides and in sphingomyelin. Long chain fatty acids derived from linoleic acid decreased in phosphatidylcholine but increased in ethanolamine and serine phosphoglycerides. The different behavior in the changes observed in fatty acid patterns for each erythrocyte membrane phospholipid may be a consequence of its different location in the cell membrane bilayer and specific exchange with plasma lipid fractions.     

The metabolism of (1-14C) stearic acid in rat testicular tissue.

(1) The metabolism of stearic acid was studied in vivo following intratesticular injection of [1-14C] stearate. Soon after injection 14C activity was found mainly in the free fatty acid pool. This was followed at later time periods by transfer of label primarily to the phosphatide pool. During each time period significant amounts of label were recovered at 14CO2. (2) Analysis of 14C-labeled fatty acids from the injected testes demonstrated an initial rapid rate of oxidation and desaturation of [1-14C] stearate followed by a slower steady state rate. It was concluded that the initial rate was due to the rapid turnover of the highly labeled free fatty acid pool followed by a much slower rate as [14C] stearate was esterified to the more metabolically stable phospholipids. Elongation of the labeled stearic or its desaturated derivative was not observed. (3) The rate of desaturation in vitro of stearic acid was measured in microsomal preparations from rat testes and found to be 12.0 +/- 0.5 pmol/min/mg compared to the estimated in vivo value of 22 pmol/min/mg and the value of 390 pmol/min/mg for hepatic microsomal desaturase.     

Regiospecific effect of 1-octanol on cis-trans isomerization of unsaturated fatty acids in the solvent-tolerant strain Pseudomonas putida S12

The solvent-tolerant bacterium Pseudomonas putida S12, which adapts its membrane lipids to the presence of toxic solvents by a cis to trans isomerization of unsaturated fatty acids, was used to study possible in vivo regiospecificity of the isomerase. Cells were supplemented with linoleic acid (C18:2delta9-cis,delta12-cis), a fatty acid that cannot be synthesized by this bacterium, but which was incorporated into membrane lipids up to an amount of 15% of total fatty acids. After addition of 1-octanol, which was used as an activator of the cis-trans isomerase, the linoleic acid was converted into the delta9-trans,delta12-cis isomer, while the delta9-cis,delta12-trans and delta9-trans,epsilon12-trans isomers were not synthesized. Thus, for the first time, regiospecific in vivo formation of novel, mixed cis/trans isomers of dienoic fatty acid chains was observed. The maximal conversion (27-36% of the chains) was obtained at 0.03-0.04% (v/v) octanol, after 2 h. The observed regiospecificity of the enzyme, which is located in the periplasmic space, could be due to penetration of the enzyme to a specific depth in the membrane as well as to specific molecular recognition of the substrate molecules.     

Inositol lipids, phosphatidate and diacylglycerol share stearoylarachidonoylglycerol as a common backbone in thrombin-stimulated human platelets.

Gel-filtered human platelets were stimulated with 5i.u. of thrombin/ml for times up to 1 min. The fatty acid composition of inositol-containing phospholipids, phosphatidic acid and diacylglycerol was determined by g.l.c. in control and thrombin-stimulated platelet suspensions. Inositol phospholipids were found to have similar proportions of stearic and arachidonic acids, the sum of these representing 86.6% of the total fatty acids in phosphatidylinositol (PtdIns), 76.9% in phosphatidylinositol 4-phosphate (PtdIns4P) and 85.4% in phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. However, arachidonic and stearic acids were less abundant in phosphatidic acid (PtdA) and diacylglycerols in non-stimulated platelets. A transient decrease in the mass of PtdIns(4,5)P2 was observed after 5-10s of thrombin stimulation, followed by an increase after 30s. The amounts of PtdIns4P and PtdIns decreased throughout the experiment. A transient accumulation of stearoylarachidonoylglycerol was observed at 5s, whereas stearoylarachidonoylglycerol 3-phosphate (PtdA) was produced in increasing amounts throughout the experiment. The decrease in inositol-containing phospholipids was not fully compensated for by the production of diacylglycerol or PtdA [or PtdIns(4,5)P2] at 1 min. All the changes in inositol phospholipids, as well as those observed in diacylglycerols and PtdA, were due to a parallel reduction or increase in the contents of stearic and arachidonic acids, with a stoichiometry equal to 1. Taken together, this suggests an interconversion of all these lipids with the utilization of a common backbone, stearoylarachidonoylglycerol. The deacylation of this diacylglycerol could account for up to 4-5nmol of arachidonate/10(9) platelets after 1 min stimulation by thrombin.     

Patterns of fatty acid release from endogenous substrates by human platelet homogenates and membranes.

We describe a method for measuring the release of fatty acids from endogenous substrates of human platelet homogenates and membranes. The method depends on the availability of lipids whose fatty acids are odd-chained and therefore suitable as internal reference compounds that, at the time of lipid extraction, can be added to an incubation to permit subsequent quantification of the content of free fatty acids or fatty acids esterified to specific lipids. We found four types of lipolytic activities in human platelets. In homogenates at pH 4.0 a triglyceride lipase operated as shown by the synchrony of triglyceride degradation and release of glycerol and those fatty acids that are the predominant constituents of triglycerides. However, enough arachidonic acid was released at this pH level to suggest some phospholipid breakdown, since triglycerides hold relatively small amounts of this acid. With membranous preparations, in the alkaline pH range there were two peaks of fatty acid release with accompanying degradation of phospholipids. At pH 8.5, where release of the saturated acids, palmitic and stearic, predominated, their sum was 3.5 times that of arachidonic acid. At pH 9.5 the release of palmitic and stearic acids was only slightly below their peak values; however, the release of arachidonic acid nearly equaled the sum of the saturated acids. Linoleic acid was not released in representative amounts by those reactions that released arachidonic acid, despite the overwhelming propensity of both to be esterified at the 2-position of phospholipids. Pertinently, the choline phospholipids are linoleic-rich and the non-choline phospholipids linoleic-poor, while both have a generous endowment of arachidonic acid. With this in mind, we raise the possibility that the phospholipase A2 of human platelets is an endoenzyme because of its tendency to act on those phospholipids that are thought to comprise the inner layer of the cell membrane.     

Modification of membrane lipid: physical properties in relation to fatty acid structure.

Differential scanning calorimetry (DSC) and electron spin resonance (ESR) measurements were made to characterize how modifications in the fatty acid composition of Escherichia coli affected the thermotropic phase transition(s) of the membrane lipd. When the fatty acid composition contained between 20 and 60% saturated fatty acids, the DSC curves for isolated phospholipids and cytoplasmic membranes showed a broad (15-25 degree C) gel to liquid-crystalline phase transition, the position of which depended on the particular fatty acid composition. Utilizing multiple lipid mutants, enrichment of the membrane phospholipids with a single long-chain cis-monoenoic fatty acid in excess of that possible in a fatty acid levels less than 20% and gradually replaced the broad peak as the cis-monoenoic fatty acid content increased. These results were obtained with phospholipids, cytoplasmic membranes, and whole cells. With these same phopholipids, plots of 2,2,6,6-tetramethylpiperidinyl-1-oxy partitioning and ESR order parameters vs. 1/T revealed discontinuities at temperatures 40-60 degrees C above the calorimetrica-ly measured gel to liquid-crystalline phase transitions. Moreover, when the membrane phospholipids were enriched with certain combinations of cis-monenoic fatty acids (e.g., cis-delta 9-16:1 plus cis-delta 11-18:1) the DSC curve showed a broad gel to liquid crystalline phase change below 0 degrees C but the ESR studies revealed no discontinuities at temperatures above those of the gel to liquid-crystalline transition. These results demonstrated that enrichment of the membrane lipids with molecules in which both fatty acyl chains are identical cis-monoenoic residues led to a distinct type of liquid-crystalline phase. Furthermore, a general conclusion from this study is that Escherichia coli normally maintains a heterogeneous mixture of lipid molecules and, by so doing, prevents strong lipid-lipid associations that lead to the formation of lipid domains in the membrane.     

Phospholipids from the hepatopancreas of Indian horseshoe crab Carcinoscropius rotundicauda

Total phospholipids were extracted from the heart, hepatopancreas, and hemolymph of the Indian horseshoe crab Carcinoscorpius rotundicauda by the conventional method. Characteristic group reaction and 2-dimensional thin-layer chromatography on silica gel were used for identification of different phospholipids. The phospholipid profile obtained from hemolymph and 2 major organs are comparable and show phosphatidyl choline (PC) and phosphatidyl ethanolamine to be the major phospholipids. A phospholipid has been consistently detected migrating immediately below the PC in the thin-layer chromatogram of lipids extracted from the hepatopancreas. When mixed methyl esters of this slower moving PC are resolved on a silica gel plate ran in hexane ether:acetic acid 80:20:1, with appropriate controls, an additional spot is seen just below the normal methyl ester, indicating a difference between the fatty acid compositions of 2 PC (e.g., regular and slower). The slower mixed methyl esters were found to comprise mainly the 4 saturated fatty acids: lauric, myristic, palmitic, and stearic. The slow moving PC seems to consist mainly of molecular species with the above-mentioned saturated fatty acids at both Sn 1 and Sn 2 positions.     

Evidence that trans-bilayer interdigitation of glycosphingolipid long chain fatty acids may be a general phenomenon

'Interdigitation' is a term coined to describe the phenomenon whereby pure phosphatidylcholines with intramolecular fatty acid chain length heterogeneity when hydrated to form bilayers may insert the methyl ends of long fatty acids from one side across more than half of the membrane thickness to protrude amongst the acyl chains of the opposite side of the bilayer (Keough, K.M.W. and Davis, P.J. (1979) Biochemistry 18, 1453-1459; Huang, C. and Mason, J.T. (1986) Biochim. Biophys. Acta 864, 423-470). In this article we address the fate of long fatty acid chains of glycosphingolipids present as minor components in membranes of non-interdigitating phosphatidylcholines. In this pursuit, derivatives of galactosyl ceramide, lactosyl ceramide, globoside and GM1 were synthesized having either 18-carbon or 24-carbon fatty acid with a spin label covalently attached at C-16. Labelled glycolipids were incorporated at 1-2 mol% into bilayers of synthetic phosphatidylcholines, their mixtures with cholesterol, or natural egg phosphatidylcholine. In each case the C-16 carbon of the glycolipid long chain fatty acid showed considerably greater 'order' and immobility than did C-16 of the fatty acid which was similar in length to the host matrix phospholipids. We interpret this as strong evidence that the long chain fatty acid interdigitates across the mid point of the bilayer in the systems studied. Clearly this phenomenon did not require that the phospholipid host matrix have mixed chain lengths. Furthermore it was totally independent of glycolipid family: for a given host matrix and (glycolipid) fatty acid chain length the order parameter values found were the same amongst all four glycolipid families tested.