Phospholipid composition of dystrophic chicken erythrocyte plasmalemmae I. Isolation of a unique lipid in dystrophic erythrocyte membranes

Several structural and functional properties are characterized in nucleated erythrocyte plasmalemmae of age and sex-matched dystrophic (line 413) and normal (line 412) chickens obtained from the University of California at Davis. Plasmalemma purity is assessed through marker enzymes. Significant differences are observed in the phospholipid content between dystrophic and normal chickens. The dystrophic chicken erythrocyte plasmalemma has an increased concentration of phosphatidylserine and a decreased concentration of phosphatidylethanolamine compared with control birds. Also, a measurable and distinct polar lipid, observed only on thin-layer chromatography (TLC) plates spotted with dystrophic preparations, is visualized adjacent to phosphatidylethanolamine. These abnormalities in the dystrophic chicken erythrocyte may signal a general defect in membrane structure for chicken dystrophy.     

Phospholipid composition of dystrophic chicken erythrocyte plasmalemmae. I. Isolation of a unique lipid in dystrophic erythrocyte membranes

Several structural and functional properties are characterized in nucleated erythrocyte plasmalemmae of age- and sex-matched dystrophic (line 413) and normal (line 412) chickens obtained from the University of California at Davis. Plasmalemma purity is assessed through marker enzymes. Significant differences are observed in the phospholipid content between dystrophic and normal chickens. The dystrophic chicken erythrocyte plasmalemma has an increased concentration of phosphatidylserine and a decreased concentration of phosphatidylethanolamine compared with control birds. Also, a measurable and distinct polar lipid, observed only on thin-layer chromatography (TLC) plates spotted with dystrophic preparations, is visualized adjacent to phosphatidylethanolamine. These abnormalities in the dystrophic chicken erythrocyte may signal a general defect in membrane structure for chicken dystrophy.     

Changes in phospholipid composition during the development of Dictyostelium discoideum

The phospholipid composition of Dictyostelium discoideum cells was determined at various stages of development by two-dimensional, thin-layer chromatography and reaction thin-layer chromatography. Major phospholipids of D. discoideum which were detectable throughout all stages of development were ethanolamine phosphoglyceride and choline phosphoglyceride. Ethanolamine phosphoglyceride and choline phosphoglyceride were found as their plasmalogen forms at 45–58 and 10–24%, respectively. There were no qualitative changes in phospholipid composition during the development, but quantitative changes did occur. The relative content of ethanolamine phosphoglyceride in the total phospholipids gradually decreased from 60% at the vegetative stage to 44% at the 1-day-sorocarp stage. In contrast, choline phosphoglyceride gradually increased from 27% at the vegetative stage to 48% at the preculmination stage, and then gradually decreased to 43% during the culmination. The decrease in ethanolamine phosphoglyceride content during the middle and late development was due mainly to the decreased amount of its plasmalogen form but the increase of choline phosphoglyceride was independent of quantitative changes of its plasmalogen form. Other minor components of phospholipid did not show significant changes in their levels. The causes of these changes in contents of ethanolamine phosphoglyceride and choline phosphoglyceride were examined by label and chase experiments with [³H]ethanolamine and [¹⁴C]choline. It seems that one-third to one-half of the increased amount of choline phosphoglyceride was due to stepwise methylation of ethanolamine phosphoglyceride, and the remaining two-thirds to one-half was caused by de novo synthesis of choline phosphoglyceride from CDP-choline and diglyceride.     

An elevated content of a unique lipid in dystrophic chicken embryonic myoblast membranes

A unique lipid, ethanolamine plasmalogen, is not only present abnormally in the plasmalemmae of erythrocytes from ex ovo dystrophic chickens but is also present abnormally in the membranes from embryonic myoblasts of in ovo dystrophic chickens.     

Serine and ethanolamine incorporation into different plasmalogen pools: Subcellular analyses of phosphoglyceride synthesis in cultured glioma cells

In cultured glioma cells, plasma membrane (PM) is enriched in phosphatidylserine (PtdSer) and plasmalogens (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine). Serine can be a precursor of headgroups of both ptdSer and ethanolamine phosphoglycerides (PE) including plasmalogens and non-plasmalogen PE (NP-PE). Synthesis of phospholipids was investigated at the subcellular level using established fractionation procedures and incorporation of [³H(G)]L-serine and [1,2-¹⁴C]ethanolamine. Specific radioactivity of PtdSer from [³H]serine was 2-fold greater in PM than in microsomes, reaching maximum by 2–4 h. Labeled plasmalogen from [³H]serine appeared in PM by 4 h and increased to 48 h, whereas almost no plasmalogen accumulated in microsomes within 12 h. In contrast, labeled plasmalogen from [1,2-¹⁴C]ethanolamine appeared in both PM and microsomes at early incubation times and became enriched in PM beyond 12 h. Thus, in glioma cells: (1) greater and faster accumulation of labeled PtdSer in PM may reflect direct synthesis from serine within PM; (2) PM is a major source of PtdSer for decarboxylation and PE synthesis; (3) NP-PE in both PM and microsome provides headgroup for synthesis of plasmalogen; and, (4) plasmalogen synthesis may involve different intracellular pools depending on headgroup origin.Abbreviations NP-PE nonplasmenylethanolamine phosphoglycerides including both diacyl and alkylacyl species - PE total ethanolamine phosphoglycerides: plasmalogen-plasmenylethanolamine or alkenylacyl ethanolamine phosphoglyceride (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) - PL phospholipid - PM plasma membrane - PtdCho phosphatidylcholine - PtdSer phosphatidylserine     

Analysis of erythrocyte membrane proteins from dystrophic hamsters

To search for potentially mutant proteins, we have investigated erythrocyte ghost proteins from normal and dystrophic hamster by two-dimensional gel electrophoresis. No significant differences are observed between dystrophic and normal erythrocytes in their peptide patterns on SDS-polyacrylamide gel electrophoresis while on two-dimensional gels a protein spot of approximate Mr 20 000 with an approximate isoelectric point of 4.5 is found in erythrocytes from dystrophic animals and is consistently absent in normal erythrocytes. A large population of erythrocyte (60%) from dystrophic hamsters shows distorted shape as visualized by scanning electron microscopy. The nature of this protein and its relevance in hamster muscular dystrophy are at present not known.     

A phospholipid serine base exchange enzyme

A membrane bound L-serine exchange enzyme which catalyzes the exchange reaction between L-serine and phospholipid-base was solubilized and separated from the ethanolamine-exchange enzyme by Sepharose 4B and DEAE-cellulose column chromatography. The separated fraction was purified approximately 37-fold with a yield of 2--5%. This fraction did not possess ethanolamine or choline exchange activity. The optimal pH was approx. 8.0, the incorporation rate of L-serine into phospholipid was linear up to 20 min incubation time and the activity was maximum at 10 mM CaCl2. The calculated Km value for L-serine was 0.4 mM. Ethanolamine phospholipid was the most effective acceptor for L-serine incorporation, particularly ethanolamine plasmalogen. The Km values obtained were: 0.25 mM for ethanolamine plasmalogen, 0.25mM for pig liver phosphatidylethanolamine and 0.66 mM for egg yolk phosphatidylethanolamine. These observations suggest that the hydrophobic moiety in ethanolamine phospholipid, as well as the base moiety, is important for the affinity of the L-serine exchange enzyme. Neither ethanolamine nor choline inhibited the L-serine exchange activity. There was no detectable conversion of phosphatidylcholine or phosphatidylethanolamine to phosphatidic acid by the partially purified enzyme.     

Structure and dynamics of plasmalogen model membranes containing cholesterol: a deuterium NMR study

Deuterium nuclear magnetic resonance (²H-NMR) was used to investigate the structure and dynamics of the sn-2 hydrocarbon chain of semi-synthetical choline and ethanolamine plasmalogens in bilayers containing 0, 30, and 50 mol% cholesterol. The deuterium NMR spectra of the choline plasmalogen yielded well-resolved quadrupolar splittings which could be assigned to the corresponding hydrocarbon chain deuterons. The sn-2 acyl chain was found to adopt a similar conformation as observed in the corresponding diacyl phospholipid, however, the flexibility at the level of the C-2 methylene segment of the plasmalogen was increased. Deuterium NMR spectra of bilayers composed of the ethanolamine plasmalogen yielded quadrupolar splittings of the C-2 segment much larger than those of the corresponding diacyl lipids, suggesting that the sn-2 chain is oriented perpendicular to the membrane surface at all segments. Cholesterol increased the ordering of the choline plasmalogen acyl chain to the same extent as in diacyl lipid bilayers. T₁ relaxation time measurements demonstrated only minor dynamical differences between choline plasmalogen and diacyl lipids in model membranes.     

Lipoproteins secreted by cultured rat hepatocytes contain the antioxidant 1-alk-1-enyl-2-acylglycerophosphoethanolamine

Monolayer cultures of rat hepatocytes have been examined for their ability to secrete ethanolamine plasmalogen as a component of nascent lipoproteins. In culture medium from these cells, ethanolamine plasmalogen comprises approx. 20-30% of total ethanolamine glycerophospholipids when measured either as phospholipid mass or by the incorporation of [1-3H]ethanolamine. An approximately equal distribution of the plasmalogen was found throughout all lipoprotein density fractions. The content of plasmalogen in whole rat serum, was 36% of total ethanolamine glycerophospholipids. In contrast, in rat liver and cultured hepatocytes the amount of ethanolamine plasmalogen was 5-fold lower than in serum or culture medium (approx. 5% of total ethanolamine phospholipids). Normal human plasma also contains ethanolamine plasmalogen in relatively large amounts (approx. 50% of total ethanolamine phospholipids). Thus, a major function of plasmalogen biosynthetic enzymes in liver may be the provision of ethanolamine plasmalogen for secretion into lipoproteins. Previous studies (e.g., Zoeller, R.A. et al. (1988) J. Biol. Chem. 263, 11590-11596) have suggested that ethanolamine plasmalogen may function as an antioxidant for the protection of lipid and protein membrane components against oxidation. Oxidized, but not native, low-density lipoprotein is rapidly taken up by macrophages with the formation of foam cells characteristic of atherosclerotic lesions (Steinbrecher, U.P. et al. (1984) Proc. Natl. Acad. Sci. USA 81, 3883-3887). Thus, the presence of plasmalogen as part of newly secreted lipoprotein particles may prevent their oxidation and subsequent uptake by macrophages.     

LIPID COMPOSITION AND METABOLISM OF CULTURED HAMSTER BRAIN ASTROCYTES

Abstract— The lipid composition and metabolism of confluent cultures of cells derived from newborn hamster brain and having morphology characteristic of immature astrocytes or spongioblasts was investigated and compared to that of newborn hamster brain dispersions and cloned glioma cells (C6). The cells displayed stable morphology for at least 30 subcultures; thereafter spontaneous transformation occurred. No appreciable changes were observed in either composition or metabolic characteristics of any major neutral lipid or phospholipid class in successive subcultures or following transformation. The overall lipid composition of the hamster astrocyte cultures closely resembled that of newborn hamster brain, but the phospholipid composition showed substantial differences. The cells contained as a percent of lipid P relatively more ethanolamine plasmalogen, choline plasmalogen and sphingomyelin and somewhat less phosphatidylcholine and phosphatidylethanolamine. The phospholipids of the hamster astrocyte and C6 cells were similar. Of the lipid precursors examined, [U-¹⁴C]glucose was incorporated best into all preparations. C6 glioma cells incorporated both [U-¹⁴C]glucose and [1-¹⁴C]acetate most actively. From 69–88% of ³²P incorporated into hamster astrocyte phospholipids was present in choline phosphoglycerides, whereas the corresonding figure for hamster brain dispersions was 53%. The ratio of specific activities of phosphatidylcholine to phosphatidylinositol was substantially higher in the cultured cells than in the brain preparations. The small pool of choline plasmalogen in the hamster astrocytes usually achieved the highest specific activity of any phospholipid. When [U-¹⁴C]glucose and [1-¹⁴C]acetate were precursors, the bulk of label in the astrocytes appeared in choline phosphoglycerides and triacyglycerol. Our results indicate that the hamster astrocyte cell line as grown expresses distinctive features of lipid composition and metabolism which are nearly constant through many generations.     

Lipids of dystrophic and normal mouse muscle: whole tissue and particulate fractions

Myofibrillar, mitochondrial, and microsomal fractions were prepared from normal and dystrophic mouse limb muscle by differential centrifugation and analyzed for phospholipids and cholesterol. Fatty acids and aldehydes of neutral lipids and of phospholipids from whole muscle and particulate fractions were also determined. Normal microsomes contained more lecithin and less total ethanolamine phospholipids and cardiolipin than mitochondria. The myofibrils had an intermediate phospholipid composition, but their cholesterol-phospholipid ratio was smaller than that of the other two fractions. Except for an increased percentage of phosphatidalethanolamine in the dystrophic mitochondria, only the composition of the dystrophic microsomes differed from normal by containing less lecithin but more total ethanolamine phospholipid, phosphatidalethanolamine, sphingomyelin, and cholesterol. No significant differences were found in the fatty acid composition of neutral lipid extracts from normal and dystrophic preparations, but there was a significant decrease in the percentage of 22:6 in phospholipids from both dystrophic whole muscle and microsomes (-25% and -37%, respectively), whereas the 20:4 content was unaltered. By contrast, the percentages of 18:0 and total fatty aldehyde increased significantly. Phospholipid extracts from all dystrophic samples showed a significant decrease in 16:0 and an increase in 18:1 as compared with the normal.     

Identification of plasmalogen as the major phospholipid constituent of cardiac sarcoplasmic reticulum

The phospholipid molecular species of canine myocardial sarcoplasmic reticulum were identified by fast atom bombardment mass spectrometry, reverse-phase high-performance liquid chromatography, and other conventional techniques. Cardiac sarcoplasmic reticulum contains 1.4 mumol of lipid Pi/mg of protein which is comprised of 53% plasmalogen. Cardiac sarcoplasmic reticulum ethanolamine glycerophospholipid contains 73% plasmalogen that is predominantly comprised of moieties with 18-carbon vinyl ethers at the sn-1 position and arachidonic acid at the sn-2 position. In contrast, canine skeletal muscle sarcoplasmic reticulum contains only 19% plasmalogen that is predominantly comprised of ethanolamine plasmalogen (78% of skeletal muscle sarcoplasmic reticulum ethanolamine glycerophospholipid) with arachidonic and docosatetraenoic acids at the sn-2 position. The possibility that tetraenoic ethanolamine plasmalogens in both cardiac and skeletal muscle sarcoplasmic reticulum facilitate calcium translocation by their propensity for adopting a hexagonal II conformation at physiologic temperatures is discussed.     

Phospholipid and nucleic acid gradients in the developing amphibian embryo

Rana pipiens embryos at the end of the blastula stage were dissociated and the cell suspension was separated into presumptive ectoderm, mesoderm, light endoderm, and heavy endoderm cells by a discontinuous density gradient centrifugation technique. The isolated germ layers were analyzed for total lipid, lipid phosphorus, plasmalogen, RNA, and DNA. Per gram dry weight, DNA showed a threefold decrease from ectoderm to heavy endoderm. On the same basis, the RNA content of the mesoderm was 34 per cent higher than that of ectoderm, and 320 and 570 per cent higher than that of light and heavy endoderm, respectively. In addition to the RNA and DNA gradients, there were at least two superimposed lipid gradients: a neutral lipid gradient decreasing from ectoderm to endoderm, and a total phospholipid gradient increasing from ectoderm to endoderm. In contrast to total phospholipid, a specific phospholipid class, ethanolamine plasmalogen, decreased from ectoderm to endoderm. The total lipid content per gram dry weight was the same in all the germ layers. Total phospholipids were analyzed quantitatively by thin layer chromatography. Phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and inositol phospholipid constituted 34, 13, 12, and 34 per cent, respectively, of the total lipid phosphorus. The phospholipid composition was different in each germ layer. The possible role of specific lipids in embryonic induction and differentiation is discussed.     

The hydrolysis of the alkenyl group from enthanolamine-plasmalogen by oligodendroglial cell-enriched fractions

The rate of hydrolysis of the 1-0-alkenyl group of sn-1-alk-1′-enyl-2-acyl-glycerylphosphorylethanolamine (alkenyl, acyl-GPE; ethanolamine plasmalogen) by plasmalogenase is higher in oligodendroglial cell-enriched fractions from bovine brain compared with fractions enriched in neuronal perikarya and astroglia. The distribution of plasmalogenase activity in membrane fractions isolated from bovine oligodendroglia has been compared with that of ‘marker’ enzymes. The highest specific activity was in a fraction enriched in plasma membranes, whilst most activity was recovered in an endoplasmic reticulum membrane fraction. In bovine oligodendroglial cell homogenates, the enzyme had a neutral pH optimum, had no requirement for divalent cations and its activity towards 1-alkenyl-GPE (lysoplasmalogen) was half that with alkenyl, acyl-GPE. C₁₆ alkenyl groups were hydrolysed more rapidly than C₁₈ alkenyl groups. With ³H-labelled alkenyl, acyl-GPE as substrate, radioactivity in released aldehydes appeared in fatty acids esterified in phospholipid while the oxidation of fatty aldehydes was blocked by the addition of NADH. An NAD-dependent aldehyde dehydrogenase was found to be present in oligodendroglia which exhibited highest activity towards C₁₄C₁₈ aldehydes (K_m, 2 μM).     

INCORPORATION OF 32P INTO THE PHOSPHOLIPIDS OF NEURONAL AND GLIAL CELL ENRICHED FRACTIONS ISOLATED FROM RABBIT CEREBRAL CORTEX: EFFECT OF NOREPINEPHRINE

Abstract— Glial cells isolated from rabbit cerebral cortex contained approximately one-third more phospholipids per unit protein than the neuronal cell bodies. The pattern of individual phospholipids was rather similar in both cell types. The incorporation of intracisternally administered ³²P into neuronal and glial phospholipid classes of rabbit brain was studied at intervals ranging from 5 to 60min. In general, for all investigated phospholipids the incorporation of the label was somewhat faster in neurons than in glial cells. Phosphatidylinositol showed the fastest and ethanolamine plasmalogen the slowest incorporation of ³²P in both neurons and glial cells. A lag phase of about 10 min could be observed before labelling of the glial phosphatidylcholine, phosphatidylethanolamine, ethanolamine plasmalogen, phosphatidylserine and sphingomyelin had occurred. Among the neuronal phospholipids a lag phase was found only for the labelling of the ethanolamine plasmalogen. Norepinephrine increased the incoropration of ³²P into phosphatidylinositol of both glia and neurons but had no effect on the specific radioactivity of ethanolamine plasmalogen and sphingomyelin. Labelling of phosphatidylcholine was slightly inhibited in both cell types by the administration of norepinephrine.     

Incorporation of linoleic acid into membrane glycerophospholipids from rat brain submitted to ischemia and hypoxia

In the presence of ATP, Mg, and CoA-SH[1-¹⁴C]linoleic acid was incorporated into membrane phospholipids (P₂ fraction and synaptosomes) prepared from rat brain cortex. The relative order for linoleate incorporation was: phosphatidylcholine >phosphatidylethanolamine>phosphatidylinositol>ethanolamine plasmalogen >phosphatidylserine. The incorporation of labeled linoleate into P₂ fraction phospholipids was investigated in rats, aged 4, 16, and 90 days, after being subjected to ischemic and hypoxic conditions. With the exception of a small increase in the incorporation of the radioactivity into diacyl-GPC, little change in incorporation profile was observed with 4-day-old rats submitted to ischemic and hypoxic conditions. However, the incorporation of labeled linoleate into membrane phospholipids was decreased in 16-and 90-day-old rats after being subjected to ischemic and hypoxic conditions. Among the phospholipids, the decrease in incorporation of radioactivity was most prominent with ethanolamine plasmalogens and phosphatidylinositol although the radioactivity of phosphatidylcholine seemed to remain relatively constant. The decreased incorporation activity in these two age groups was noted along with concomitant increase in the FFA content, whereas an increase in FFA was not observed in the 4-day-old brain samples. Thus, the specific decrease in labeling of ethanolamine plasmalogens and phosphatidylinositol may be the result of increased enzymic degradation of these compounds after ischemic and hypoxic treatment. Furthermore, the decrease in incorporation of linoleate into membrane phospholipids may be due to an increase in the membrane, FFA pool which subsequently, gave a dilution of the labeled precursor.     

Differential turnover of polyunsaturated fatty acids in plasmalogen and diacyl glycerophospholipids of isolated cardiac myocytes

To investigate the relative turnover of esterified polyunsaturated fatty acids in diacylglycerophospholipids and plasmalogens in isolated cardiac myocytes, we characterized the phospholipid composition and distribution of radiolabel in different phospholipid classes and in individual molecular species of diradyl choline (CGP) and ethanolamine (EGP) glycerophospholipids after incubation of isolated cardiac myocytes with [3H]arachidonate or [14C]linoleate. Plasmalogens in CGP (55%) and EGP (42%) quantitatively accounted for the total plasmalogen content (39%) of cardiac myocyte phospholipids. Plasmalogens comprised 86% and 51% of total arachidonylated CGP and EGP mass, respectively, and [3H]arachidonate was primarily incorporated into plasmalogens in both CGP (65%) and EGP (61%) classes. The specificity activity of [3H]arachidonylated diacyl-CGP was approximately 2- to 5-fold greater than that of [3H]arachidonylated choline plasmalogen, whereas comparable specific activities were found in the [3H]arachidonate-labeled ethanolamine plasmalogen and diacyl-EGP pools. Of the total linoleate-containing CGP and EGP mass, 54% and 57%, respectively, was esterified to plasmalogen molecular species. However, [14C]linoleate was almost exclusively incorporated into diacyl-CGP (96%) and diacyl-EGP (86%). The specific activities of [14C]linoleate-labeled diacyl-CGP and diacyl-EGP were 5- to 20-fold greater than that of the [14C]linoleate-labeled plasmalogen pools. The differential incorporation of polyunsaturated fatty acids in plasmalogens and diacylglycerophospholipids demonstrates that the metabolism of the sn-2 fatty acyl moiety in these phospholipid subclasses is differentially regulated, possibly fulfilling separate and distinct physiologic roles.     

Lipid composition and metabolism in testicular and ejaculated ram spermatozoa

1. Spermatozoa collected directly from the testis of the conscious ram contain 25% more phospholipid than ejaculated spermatozoa. The concentration of lecithin, phosphatidylethanolamine and ethanolamine plasmalogen was greater in testicular spermatozoa; little difference was observed in choline plasmalogen. Both types of spermatozoa had significant amounts of cardiolipin and alkyl ether phospholipid. 2. The fatty acids in the phospholipid extracted from testicular spermatozoa have a very high content of palmitic acid. The phospholipids of ejaculated spermatozoa contained less palmitic acid, but more myristic acid. 3. Ejaculated spermatozoa contained less acyl ester and cholesterol. It is suggested that lipids are a source of substrate for spermatozoa during their passage through the epididymis. 4. Testicular spermatozoa when incubated with [U-¹⁴C]glucose incorporated more radioactivity into the glycerol part of the phospholipid and neutral lipid fractions than did ejaculated cells. The distribution of radioactivity in the individual phospholipids and neutral lipids was similar for both cell types. No radioactivity was detected in choline plasmalogen, which accounted for approx. 40% of the total phospholipid. 5. Testicular spermatozoa incorporated more radioactivity from glucose into formate than into acetate, whereas a higher proportion of radioactivity was found in acetate in ejaculated cells. 6. The implications of these lipid changes in the process of spermatozoal maturation are discussed.     

Structural characterization of the glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase by fast atom bombardment mass spectrometry

The glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase (EC 3.1.1.7) is composed of a glycan linked through a glucosamine residue to an inositol phospholipid that is resistant to the action of phosphatidylinositol-specific phospholipase C. Deamination cleavage of the glucosamine with nitrous acid released the inositol phospholipid which was purified by high performance liquid chromatography. Analysis by fast atom bombardment mass spectrometry with negative ion monitoring and by the complementary technique of collision-induced dissociation revealed molecular and daughter ions that indicated a plasmanylinositol with a palmitoyl group on an inositol hydroxyl. The intact membrane anchor was released from reductively methylated human erythrocyte acetylcholinesterase by proteolysis with papain or Pronase, deacylated by base hydrolysis, and purified by high performance liquid chromatography. Positive and negative ion fast atom bombardment mass spectrometry of the major products isolated by high performance liquid chromatography indicated the following structure for the complete glycoinositol phospholipid anchor. (formula; see text) Methylation of free amino groups by reduction with deuterium instead of hydrogen permitted determination of the number of free amino groups in individual fragment ions as further confirmation of structural assignments. The structure of the glycan portion of the human erythrocyte acetylcholinesterase membrane anchor appears to be similar to that described for Trypanosome brucei variant surface glycoprotein MITat 1.4 (variant 117) (Ferguson, M.A.J., Homans, S.W., Dwek, R.A., and Rademacher, T.W. (1988) Science 239, 753-759) except for the absence of a galactose antenna and the presence of a phosphorylethanolamine on the hexose adjacent to glucosamine.     

Structure and dynamics of plasmalogen model membranes containing cholesterol: a deuterium NMR study

Deuterium nuclear magnetic resonance (2H-NMR) was used to investigate the structure and dynamics of the sn-2 hydrocarbon chain of semi-synthetical choline and ethanolamine plasmalogen in bilayers containing 0, 30, and 50 mol% cholesterol. The deuterium NMR spectra of the choline plasmalogen yielded well-resolved quadrupolar splittings which could be assigned to the corresponding hydrocarbon chain deuterons. The sn-2 acyl chain was found to adopt a similar conformation as observed in the corresponding diacyl phospholipid, however, the flexibility at the level of the C-2 methylene segment of the plasmalogen was increased. Deuterium NMR spectra of bilayers composed of the ethanolamine plasmalogen yielded quadrupolar splittings of the C-2 segment much larger than those of the corresponding diacyl lipids, suggesting that the sn-2 chain is oriented perpendicular to the membrane surface at all segments. Cholesterol increased the ordering of the choline plasmalogen acyl chain to the same extent as in diacyl lipid bilayers. T1 relaxation time measurements demonstrated only minor dynamical differences between choline plasmalogen and diacyl lipids in model membranes.