Lipid composition of photoreceptor membranes from goldfish retinas

Isolation and biochemical characterization of goldfish retinal photoreceptor outer segment membranes are described. The lipid fraction is composed primarily of phospholipids (68 mol%) with substantial amounts of neutral lipids (32 mol%). Sterols account for only about 2 wt% of the membranes (about 9 mol% of the total lipids). The phospholipid class composition and fatty acid composition are similar to those of other vertebrate photoreceptor membranes. Two novel findings were the high levels of free fatty acids (21 mol% of the total lipids, primarily palmitic and docosahexaenoic acids) and the presence of relatively significant amounts of a C-32 diacylglycerol molecular species.     

Morphogenesis of the photoreceptor outer segment during postnatal development in the mouse (BALB/c) retina

Summary Disc formation of rod photoreceptor cells in developing BALB/c mice retinas was studied by rapid freeze, freeze-substitution, freeze-etching, immunocytochemistry, and myosin S-1 decoration methods. Freeze-substituted photoreceptor cells contained variously shaped vesicles in the apical swelling of the connecting cilium or the base of the outer segment during postnatal development. Rapid freezing successfully arrested pinocytosis; the fusion of small vesicles to give large ones, and the compression of certain vesicles (0.3–0.6 m) appears to lead gradually to the formation of the so-called discs. We therefore propose that membranous discs are formed by the fusion of small pinocytotic vesicles and their subsequent compression. Discs formed in this way were partially stacked, but were ordered at random during the early developmental stages. During development, a partial stack of discs was progressively rearranged to a regular form as seen in mature outer segments. Cytoskeletal actin was expected to be involved in the disc formation; it was demonstrated in the distal axoneme of the connecting cilium during development and showed no change in its distribution. However, the polarity of the actin filaments, as revealed by myosin S-1 decoration in early developmental stages, was much more variable than in the adult. Barbed ends of actin filaments were associated with the plasma membrane or the membrane of vesicles. We also found actin filaments coiled up helically on ciliary microtubules.     

The effects of high concentrations of sodium or calcium ions on the lipid composition and properties of Tetrahymena membranes

Tetrahymena pyriformis cells have been grown in media varying in NaCl concentration from 3.7 mM (normal medium) to 0.3 M and varying in CaCl₂ from 0.2 mM (normal medium) to 0.1 M. Tetrahymena grown in 0.3 M NaCl showed relatively few alterations in phospholipid composition, with significant changes being found only in the cell surface membranes (pellicle), which increased in phosphatidylethanolamine content from 39% (low Na⁺) to 48% (high Na⁺) of the total phospholipids. The small decrease in fatty acid unsaturation and increase in shorter chain fatty acids in pellicle phospholipids were not statistically significant. No significant changes in phospholipid head group composition or fatty acid distribution were observed in high Ca²⁺-grown cells. Complementary studies of membrane fluidity, as inferred from freeze-fracture electron microscopy analysis, indicated that membranes of high Na⁺-acclimated cells were similar to those of control cells, when each was measured in its respective medium. However, the outer alveolar membrane of the pellicle and the food vacuolar membrane were considerably less fluid in high-Ca²⁺ cells. The lower fluidity in vacuolar membranes may have been responsible for alterations in the cells' capacity to form food vacuoles.     

The relationship between plasma membrane lipid composition and physical-chemical properties. I. Fluorescence polarization studies of fatty acid-altered EL4 tumor cell membranes

EL4 cells were cultured with exogenous fatty acids under conditions that resulted in their incorporation into membrane phospholipids. The behavior of the fluorescent lipid probes diphenylhexatriene and perylene was monitored in intact EL4 cells and in isolated EL4 plasma membranes. In whole cells substituted with unsaturated fatty acids, there was always a marked decrease in the $\text{P}$ value of both probes compared to the $\text{P}$ value of the probes in unsubstituted cells. In whole cells substituted with saturated fatty acids, on the other hand, $\text{P}$ values for both probes were unchanged compared to unsubstituted cells. In plasma membrane isolated from EL4 cells, no difference in $\text{P}$ values for either probe was observed among membranes from unsubstituted, saturated fatty acid substituted or unsaturated fatty acid substituted cells, even when the degree of fatty acid substitution was quite substantial. Most of the fluorescent signal for both probes in whole cells appeared to come from cytoplasmic lipid droplets. The value of techniques such as fluorescent polarization for monitoring physical properties of membranes (such as ‘fluidity’) is discussed.     

Effect of age on the membrane lipid composition of Streptococcus sanguis

The cell membrane of Streptococcus sanguis contains three classes of lipid: neutral lipid, glycolipid and phospholipid. A striking difference in membrane lipid composition between cells in the exponential and in the stationary phases of growth was observed. During the exponential phase, approx. 37–45%, 14–19% and 37–45% of the lipids synthesized were found to be neutral lipid, glycolipid and phospholipid, respectively. The amount of lipid synthesized reached a maximum at the early stationary phase. The amount of phospholipid drastically declined thereafter and that of neutral lipid slightly declined. In contrast, the amount of glycolipid markedly increased and exceeded the amount of phospholipid. The phospholipid present during the exponential phase was found to be mainly phosphatidylglycerol (82–88%) and a small amount of cardiolipin (12–18%). At the stationary phase, the amount of phosphatidylglycerol greatly decreased and reached approx. 16% of that in the early stationary phase, while cardiolipin steadily increased and became the major phospholipid in the late stationary phase. The glycolipid was found to be composed of mainly mono- and diglucosyldiglycerides. At the end of the experiment (after 8 h incubation), the distribution of lipids was found to be: neutral lipid, 46%; glycolipid (monoglucosyldiglyceride, 28%; diglucosyldiglyceride, 13%) 41%; and phospholipid (phosphatidylglycerol, 3%, cardiolipin, 8%) 13%.     

Influence of environmental temperature on mitochondrial membranes

Mitochondrial phospholipids from goldfish lateral line muscle were analysed with respect to polar and apolar groups. Groups of 20 goldfish, acclimated to 5, 20 and 30°C, were used. Temperature-induced shifts of both polar and apolar groups of the mitochondrial phospholipids were observed. The fatty acid composition of mitochondrial phospholipids is characterized by a large amount of polyenoic acids, dominated by docosahexaenoic acid and by octadecadienoic acid. At the higher acclimation temperatures, a significant decrease in docosahexaenoic acid is found. However, the resultant effect of environmental temperature on the degree of unsaturation is small, in contrast to the marked effect on mean chain length. Pronounced changes in the molar ratio of phosphatidylcholine and phosphatidylethanolamine are seen; a decrease in mitochondrial phosphatidylcholine is observed at low acclimation temperature, which is compensated for by a nearly equal increase in phosphatidylethanolamine. The main phospholipids are, apparently, phosphatidylcholine, phosphatidylethanolamine and cardiolipin, comprising 90% of the total pool of 12 species. It is found that the anionic nature of the phospholipids is increased at low acclimation temperatures. We discuss this effect and its probable importance in the stabilization of the surface potential of the mitochondrial membranes.     

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.     

Isolation, characterization and protein and polar lipid compositions of Paracoccus denitrificans outer membrane

Sucrose density gradient centrifugation of Paracoccus denitrificans strains ATCC 13543 and ATCC 17741 cell envelopes plus poly-β-hydroxybutyrate, isolated from organisms broken using a French pressure cell, revealed three bands of densities: I, 1.16 g/ml; II, 1.19 g/ml; III, 1.24 g/ml. On the basis of chemical and enzymatic assays and sodium dodecyl sulfate-polyacrylamide gel electrophoresis the bands were identified as: I, cytoplasmic membrane; II, poly-β-hydroxybutyrate; III, outer membrane plus poly-β-hydroxybutyrate. Poly-β-hydroxybutyrate was removed by increased low-speed centrifugation before deposition of cell envelopes. Density gradient centrifugation of cell envelopes gave a simple pattern of two bands, cytoplasmic and outer membranes. In both strains outer membranes showed a broad protein band at M_r 70 000–83 000 upon SDS-polyacrylamide gel electrophoresis of samples solubilized at 25°C, which was not present in samples solubilized at 100°C, where a single major band was present of M_r 32 000 in strain ATCC 13543 and 35 000 in strain ATCC 17741. The major outer membrane protein stained positively for lipid in both strains, as did an M_r 70 000 protein, which was the second major protein in strain ATCC 17741. The second major outer membrane protein of stain ATCC 13543 had an M_r of 20 000 in unheated samples but 23 000 in heated samples. This protein was not present in strain ATCC 17741. Quantitative data on the polar lipid compositions of cell envelope fractions are presented.     

Lipid and protein composition and thermotropic lipid phase transitions in fatty acid-homogeneous membranes of Acholeplasma laidlawii B

The membrane composition and lipid physical properties have been systematically investigated as a function of fatty acid composition for a series of Acholeplasma laidlawii B membrane preparations made homogeneous in various fatty acids by growing cells on single fatty acids and avidin, a potent fatty acid synthetic inhibitor. The membrane protein molecular weight distribution is essentially constant as a function of fatty acid composition, but the lipid/protein ratio varies over a 2-fold range when different fatty acid growth supplements are used. The membrane lipid head-group composition varies somewhat under these conditions, particularly in the ratio of the two major neutral glycolipids. Differential thermal analytical investigations of the thermotropic phase transitions of various combinations of membrane components suggest that these compositional changes are unlikely to result in qualitative changes in the nature of lipid-protein or lipid-lipid interactions, although lesser changes of a quantitative nature probably do occur. The total lipids of membranes made homogeneous in their lipid fatty acyl chain composition exhibit sharper than normal gel-to-liquid-crystalline phase transitions of which midpoint temperatures correlate very well with the phase transition temperatures of synthetic hydrated phosphatidylcholines with like acyl chains. Our results indicate that using avidin and suitable fatty acids to grow A. laidlawii B, it is possible to manipulate the position and the sharpness of the membrane lipid phase transition widely and independently without causing major modifications in other aspects of the membrane composition. This fact makes the fatty acid-homogeneous A. laidlawii B membrane a very useful biological membrane preparation in which to study lipid physical properties and their functional consequences.     

The polar lipid composition of Mesorhizobium ciceri

The extractable lipid composition of Mesorhizobium ciceri strain HAMBI 1750 grown in a phosphate sufficient medium (79CA) is reported. Cardiolipin (CL—27% of total lipids), phosphatidylglycerol (PG—18%), phosphatidylethanolamine (PE—1%), phosphatidylcholine (PC—30%) and two methylated derivatives of PE, i.e. phosphatidyl-N, N-dimethylethanolamine (DMPE—1%) and phosphatidyl-N-monomethylethanolamine (MMPE—1%), were found to make up the phospholipids of the analysed bacteria. Nonphosphorus, ornithine-containing lipid (OL—10%) was also detected. Polar groups of phospholipids were predominantly acylated with cis-11,12-methyleneoctadecanoyl (lactobacillic) residues, whereas the ornithine lipid contained mainly 3-hexadecanoyloxy-11,12-methyleneoctadecanoic acid bound to the α-amino group.     

The relatioship between plasma membrane lipid composition and physical-chemical properties II. Effect of phospholipid fatty acid modulation on plasma membrane physical properties and enzymatic activities

The fatty acid composition of plasma membrane phospholipids of the murine T lymphocyte tumor EL4 were systematically modified in an attempt to understand the relationship between lipid bilayer composition and plasma membrane physical and biological properties. Two plasma membrane enzyme activities, adenylate cyclase and ouabain-sensitive ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase, were measured in normal and fatty acid-substituted EL4 plasma membrane fractions. The fatty acid effect on enzyme activities was similar to previously reported effects of fatty acids on cytotoxic T cell function. The activity of both enzymes was inhibited by saturated fatty acids, while unsaturated fatty acids had a moderate enhancing effect on both enzyme activities. Using two different nitroxide derivatives of stearic acid, the order parameter and approximate rotational correlation times were calculated from ESR spectra of normal and fatty acid-modified plasma membranes. No significant difference was found in either parameter in these membranes. These results, in conjunction with earlier data from our laboratory and others, suggest that caution should be exercised in inferring changes in membrane ‘fluidity’ based on lipid modulation of biological membranes.     

Phospholipid composition of dystrophic chicken erythrocyte plasmalemmae II. Characterization of a unique lipid from dystrophic erythrocyte membranes as ethanolamine plasmalogen

The phospholipid content of normal (line 412) and dystrophic (line 413) chicken erythrocyte plasmalemmae has been quantified on a developmental basis using sex matched controls. A specific minor phospholipid component, ethanolamine plasmalogen, is identified from dystrophic erythrocyte membrane preparations. To arrive at this identification, data from studies utilizing gas-liquid chromatography, thin-layer chromatography, [¹⁴C]ethanolamine incorporation, and biochemical assay for specific organic moieties were correlated. This phospholipid has the potential to alter and regulate membrane fluidity and thus membrane function. The possible presence of significant concentrations of plasmalogen in human dystrophic tissues may serve as a marker for dystrophy and thus be of clinical importance.     

Specificity of fatty acid composition of highly migratory fish. A comparison of docosahexaenoic acid content in total lipids extracted in various organs of bonito (Euthynnus pelamis)

Fatty acid composition of lipids in various organs of the bonito (Euthynnus pelamis), a typical species within the tuna species, caught in the sea off the Pacific coast of Japan, was determined by gas-liquid chromatography. The major fatty acids of all organs examined were palmitic acid (16:0), palmitoleic acid (16:1), oleic acid (18:1), icosapentaenoic acid (20:5), and docosahexaenoic acid (22:6). 22:6 (DHA) was the dominant fatty acid accounting for 25% or more of the total fatty acids in all organs of the bonito, caught in the sea off the Japanese coast.     

The effect of calcium on the bilayer stability of lipids from bovine rod outer segment disk membranes

The phase behavior of bovine rod outer segment disk lipids has been investigated using freeze-fracture and ³¹P nuclear magnetic resonance (NMR) techniques. ³¹P-NMR spectra of isolated disk membranes were taken as a function of temperature between 25°C and 45°C. The ³¹P-NMR spectrum characteristic of phospholipid bilayers was observed at all temperatures both in the absence of Ca²⁺ and in the presence of 10 mM and 50 mM Ca²⁺. A similar study was performed on lipids isolated from the disk membranes. In the absence of Ca²⁺ only lamellar phase behavior was observed. In the presence of less than 10 mM Ca²⁺, however, there was a change in morphology to non-lamellar structures. Removal of the Ca²⁺ caused the system to reassume the lamellar form.     

Phospholipid and fatty acid composition of tetrodotoxin receptor-rich membrane fragments from Electrophorus electricus

To study the interaction of voltage-sensitive Na⁺-channels with membrane lipids, the phospholipid and fatty acid composition of highly purified membrane fragments from the remarkably differentiated plasma membrane of Electrophorus electricus has been analyzed. After density gradient fractionation and carrier free electrophoresis, fractions with up to 30 pmol tetrodotoxin binding/mg protein can be obtained, which may correspond to a 50% pure preparation of the extrasynaptic part of the excitable face. Phospholipid classes and cholesterol are separated by one-dimensional thin-layer chromatography in acidic and alkaline solvent systems. The following mean molar contents are found: 40% phosphatidylcholine, 23% phosphatidylserine, 30% phosphatidylethanolamine and 7% sphingomyelin. In a series of 11 animals, significant deviations from these mean values have been observed. The fatty acid composition of the phospholipids has been determined by gas chromatography. Phosphatidylcholine contains more than 50% 16:0, and about 20% unsaturated fatty acids in the C-18 group. Compared to other plasma membrane fractions, this phospholipid is the least differentiated. By contrast, phosphatidylethanolamine and phosphatidylserine show many characteristics in different membrane fractions, especially in their unsaturated components representing more than 50%. 22:6, as the major constituent in these fractions, accounts for a quarter to a third of all fatty acids in these fractions. 18:0 is the main saturated component in these two phospholipids with abundances of typically a quarter or less of all fatty acids. Knowledge of the lipid composition of these excitable membranes may help to conserve binding and structural properties when analyzing lipid-sensitive Na⁺-channels in vitro. It is also useful as a guideline for systematic reconstitution studies.     

Modifications of membrane lipid composition following the nutritional shift-up of starved cells a comparison with membrane biogenesis in Tetrahymena

Detailed analyses of lipid composition have been made on various membrane fractions isolated at different intervals after 24 h-starved Tetrahymena cells were refed with nutrient-rich medium. During starvation there was a marked alteration in both phospholipid polar headgroup and acyl chain compositions: an increase in 2-aminoethylphospholipid and γ-linolenic acid (18 : 3) with a concurrent decrease in phosphatidylethanolamine and palmitoleic acid (16 : 1). However, following refeeding, such an altered lipid composition was rather rapidly restored to the initial level of the control cell membranes prior to starvation. This membrane lipid modification was found to occur in good accordance with the recovery of cell size and lipid synthesis. The considerable changes in the principal unsaturated fatty acids, 16 : 1 and 18 : 3, which are formed via the palmitate and stearate desaturation pathways, respectively, were suggested to be accounted for by the levels of desaturases activities. The results of the labeling experiments with radioactive precursors have demonstrated that in the refed cells, there was a more rapid and dynamic transfer or exchange between membranes as compared with that in the exponentially growing control cells. Thus, rapid ameliorative modifications of membrane lipid composition are thought to be required for the urgent growth of membrane systems in the refed cell which should be ready to initiate new division.     

Correlation between fluidity and fatty acid composition of phospholipid species in Tetrahymena pyriformis during temperature acclimation

The correlation between the fluidity of phospholipids and their fatty acid composition was studied by spin label technique and gas-liquid chromatography for three major phospholipid species in Tetrahymena pyriformis during temperature acclimation. The fluidity of 2-aminoethylphosphonolipid increased within the first 10 h of the cold-acclimation when the content of γ-linolenic acid in 2-aminoethylphosphonolipid was highest, and it then decreased up to 24 h. On the other hand, the fluidities of phosphatidylethanolamine and phosphatidylcholine showed a gradual decrease up to 24 h after the temperature shift, although γ-linolenic acid contents were highest at 10 h after the temperature shift. Thus the fluidity changes of these two phospholipids were interpreted as resulting from the altered content of other fatty acids in addition to γ-linolenic acid, since the γ-linolenic acid content was smaller than that of 2-aminoethylphosphonolipid. The results suggest that the content of γ-linolenic acid in 2-aminoethylphosphonolipid plays a role in regulating the thermal adaptation process.     

Characterization of photoreceptor cell types in the little brown bat Myotis lucifugus (Vespertilionidae)

We report the expression of three visual opsins in the retina of the little brown bat (Myotis lucifugus, Vespertilionidae). Gene sequences for a rod-specific opsin and two cone-specific opsins were cloned from cDNA derived from bat eyes. Comparative sequence analyses indicate that the two cone opsins correspond to an ultraviolet short-wavelength opsin (SWS1) and a long-wavelength opsin (LWS). Immunocytochemistry using antisera to visual opsins revealed that the little brown bat retina contains two types of cone photoreceptors within a rod-dominated background. However, unlike other mammalian photoreceptors, M. lucifugus cones and rods are morphologically indistinguishable by light microscopy. Both photoreceptor types have a thin, elongated outer segment. Using microspectrophotometry we classified the absorption spectrum for the ubiquitous rods. Similar to other mammals, bat rhodopsin has an absorption peak near 500 nm. Although we were unable to confirm a spectral range, cellular and molecular analyses indicate that M. lucifugus expresses two types of cone visual pigments located within the photoreceptor layer. This study provides important insights into the visual capacity of a nocturnal microchiropteran species.     

Sterol composition of Linneus torquatus (Nemertini) Anopla

The sterol fraction from the marine worm Linneus torquatus Coe (phylum Nemertini, class Anopla, family Lineidae) has been isolated, separated by HPLC and preparative TLC on AgNO₃-impregnated silica gel, and sterols identified using GC, GC-MS and NMR spectroscopy. It was shown that the fraction contains at least 12 sterols belonging mainly to Δ^5,22, Δ^5,24(28) and Δ⁵ series. The major sterol components were 24-methylcholesta-5,24(28)-dien-3β-ol, cholesta-5,22E-dien-3β-ol, 24-nor-cholesta-5,22-dien-3β-ol and cholesterol.