Lipid composition of Limulus photoreceptor membranes.

The lipid composition has been determined for rhabdomeric photoreceptor membranes of Limulus, and these data are compared with those from photoreceptor membranes of albino rats. The comparison is of interest because the membranes of these two photoreceptor cells regulate ionic transport differently during the response to illumination. 1. Phospholipid class composition of Limulus is similar, but not identical, to that of rats. The major differences are a greater percentage of sphingomyelin in Limulus and a greater percentage of phosphatidylethanolamine in the rat. 2. Ethanolamine plasmalogens, not observed in rat photoreceptor membranes, are present in Limulus photoreceptor fractions. 3. The level of cholesterol in Limulus is higher than that usually reported for vertebrate rod outer segments. 4. The predominant polyunsaturated fatty acids of Limulus photoreceptor membrane phospholipids are 20: 4(n-6) and 20: 5(n-3) with only traces of 22: 6(n-3). This is in sharp contrast with the large percentages of 22: 6(n-3) found in rat photoreceptors. 5. The fatty acid distributions of both membrane systems are highly unsaturated, but the ratio of (n-3) to (n-6) polyunsaturates is only 1.7 for Limulus as compared to 4.6 for rat.     

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.     

Effects of increased intracellular pH-buffering capacity on the light response of Limulus ventral photoreceptor

Aspects of a possible involvement of hydrogen ions in the electrophysiological responses to light of Limulus ventral photoreceptors were investigated. A I M solution of either a zwitter-ionic pH buffer or a weakly-buffering control substance was pressure injected through a micropipette into a ventral photoreceptor cell. To estimate the amount injected, ³⁵SO₄ was included in the solution. Membrane currents induced by light flashes were measured by a voltage-clamp technique. The buffer-filled micropipette passed current and a 3 M KCl filled micropipette monitored membrane voltage. The sensitivity (peak light-induced current/stimulus energy) was measured, after dark adaptation, before and after injection. Injections of buffers, pH 6.3–7.2, to intracellular concentrations of at least 40–200 mM produced only a small mean decrease in sensitivity, approximately equal to that caused by injections of control substances. Excitation, therefore, apparently is not mediated by a change in intracellular pH. Buffers with pH values 5.4–8.4 were also injected. The time to peak of the response depended on pH, being shortened by up to 20% at pH values below 7.7 and lengthened at higher pH values. The time to peak of the response appeared to be shortened by an increase in intracellular pH-buffering capacity even when there was no change in intracellular pH.     

Lipid composition of Sporendonema epizoum

Triglycerides, free fatty acids, free and esterified ergosterol, Q₉, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, lysophosphatidylethanolamine, and three different acylglycoses were identified in the soluble lipids of Sporendonema epizoum mycelium. The same compounds as well as a sterol glycoside were also found in conidia. The mycelium is richer than the conidia in phospholipids, Q₉ and free and esterified ergosterol but contains less glycolipids. The most abundant fatty acid in all non-polar fractions is C_18:2. The prevalent fatty acid of the phospholipids is C_18:1, except for conidial phosphatidylethanolamine and mycelial lysophosphatidylethanolamine.     

Lipid composition of Daucus carota roots

Lipids extracted from Daucus carota roots were analyzed and the fatty acid composition of the triglycerides and phospholipids determined. Compariso     

Lipid composition of membranes of aminestorage organelles

1. The lipid composition of the membranes from isolated 5-hydroxytryptamine-storage organelles of blood platelets of rabbits and of those from chromaffin granules of bovine adrenal medulla was compared. 2. In contrast with the membranes of the chromaffin granules, those of the 5-hydroxytryptamine organelles did not contain lysophosphatidylcholine (lysolecithin). 3. Both the cholesterol/phospholipid ratio and the relative proportions of phosphatidylethanolamine (kephalin), phosphatidylinositol and phosphatidylserine were about the same in both membranes, whereas phosphatidylcholine (lecithin) and sphingomyelin showed somewhat higher values in the membranes of the 5-hydroxytryptamine organelles. 4. In conclusion, the release of 5-hydroxytryptamine from blood platelets is probably not correlated with the presence of lysophosphatidylcholine in the membranes of the storage organelles and may thus differ from the mechanism of catecholamine release in adrenal medulla.     

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.     

Lipid analysis of mitochondrial membranes from the yeast Pichia pastoris

Here we describe for the first time isolation and biochemical characterization of highly purified mitochondrial inner and outer membranes from Pichia pastoris and systematic lipid analysis of submitochondrial fractions. Mitochondria of this yeast are best developed during growth on glycerol or sorbitol, but also on methanol or fatty acids. To obtain organelle membranes at high quality, methods of isolation and subfractionation of mitochondria originally developed for Saccharomyces cerevisiae were adapted and employed. A characteristic feature of the outer mitochondrial membrane of P. pastoris is the higher phospholipid to protein ratio and the lower ergosterol to phospholipid ratio compared to the inner membrane. Another marked difference between the two mitochondrial membranes is the phospholipid composition. Phosphatidylcholine and phosphatidylethanolamine are major phospholipids of both membranes, but the inner membrane is enriched in cardiolipin, whereas the outer membrane contains a high amount of phosphatidylinositol. The fatty acid composition of both mitochondrial membranes is similar. Variation of the carbon source, however, leads to marked changes of the fatty acid pattern both in total and mitochondrial membranes. In summary, our data are the first step to understand the P. pastoris lipidome which will be prerequisite to manipulate membrane components of this yeast for biotechnological purposes.     

Crystal Structures of Limulus SAP-Like Pentraxin Reveal Two Molecular Aggregations

The serum-amyloid-P-component-like pentraxin from Limulus polyphemus, a recently discovered pentraxin species and important effector protein of the hemolymph immune system, displays two distinct doubly stacked cyclic molecular aggregations, heptameric and octameric. The refined three-dimensional structures determined by X-ray crystallography, both based on the same cDNA sequence, show that each aggregate is constructed from a similar dimer of protomers, which is repeated to make up the ring structure. The native octameric form has been refined at a resolution of 3 Å, the native heptameric form at 2.3 Å, and the phosphoethanolamine (PE)-bound octameric form at 2.7 Å. The existence of the hitherto undescribed heptameric form was confirmed by single-particle analysis using cryo-electron microscopy. In the native structures, the calcium-binding site is similar to that in human pentraxins, with two calcium ions bound in each subunit. Upon binding PE, however, each subunit binds a third calcium ion, with all three calcium ions contributing to the binding and orientation of the bound phosphate group within the ligand-binding pocket. While the phosphate is well-defined in the electron density, the ethanolamine group is poorly defined, suggesting structural and binding variabilities of this group. Although sequence homology with human serum amyloid P component is relatively low, structural homology is high, with very similar overall folds and a common affinity for PE. This is due, in part, to a “topological” equivalence of side-chain position. Identical side chains that are important in both function and fold, from different regions of the sequence in human and Limulus structures, occupy similar space within the overall subunit fold. Sequence and structure alignment, based on the refined three-dimensional structures presented here and the known horseshoe crab pentraxin sequences, suggest that adaptation and refinement of C-reactive-protein-mediated immune responses in these ancient creatures lacking antibody-based immunity are based on adaptation by gene duplication.     

Improved microtechnique for endotoxin assay by the Limulus amebocyte lysate test

A simple microtechnique for the endotoxin assay with Limulus amebocyte lysate is described. Mixtures of 1 μl of reagent with 1 μl of test sample are incubated in 5-μl capillaries which are subsequently dipped into a dye solution. In the absence of a firm gel the dye enters the capillaries whereas a firm gel, formed in the presence of endotoxin, prevents the stain from entering the capillaries. The technique offers a great saving of reagent; unequivocal distinction between positive and negative results, a good reproducibility, and a sensitivity equal to or higher than those in previously reported techniques could be demonstrated.     

Control of fatty acid composition of Acholeplasma laidlawii membranes

The temperature-dependent pattern of incorporation of palmitate and oleate from the growth medium into Acholeplasma laidlawii membrane lipids correlates with the physical state of the membrane defined by calorimetry. Both the pattern and the state can be changed at will by changing the fatty acid composition of the membrane lipids. The ratio of palmitate to oleate incorporated is independent of temperature when the membrane bilayer is below its transition and fully ordered, but becomes temperature dependent upon the onset of the transition and continues to be temperature dependent when the membrane is above its transition and fully fluid. This behavior is mimicked by the physical binding of palmitate and oleate to bilayers of extracted membrane lipids and to bilayers of lecithin. Selective binding by membranes may provide a means for controlling lipid fatty acid composition without invoking an enzymatic mechanism.     

Lipid phase transitions in cytoplasmic and outer membranes of Escherichia coli

The cytoplasmic and outer membranes containing either trans-Δ⁹-octadecenoate, trans-Δ⁹-hexadecenoate or cis-Δ⁹-octadecenoate as predominant unsaturated fatty acid residues in the phospholipids were prepared from a fatty acid auxotroph, Escherichia coli strain K1062. Order-disorder transitions of the phospholipids were revealed in both fractions of the cell envelope by fluorescent probing or wide angle X-ray diffraction. The mid-transition temperatures, $\text{T}\text{t}$, and the range of the transition, $\text{ΔT}$, are similar in the outer and cytoplasmic membrane. Relative to the corresponding extracted lipids, 60–80% of the hydrocarbon chains take part in the transition in the cytoplasmic membrane whereas in the outer membrane only 25–40% of the chains become ordered. The results suggest that in the outer membrane part of the lipids form fluid domains in the form of mono- and/or bilayers.     

Lipid phase transitions in fatty acid-homogeneous membranes of Acholeplasma laidlawii B

The thermotropic behaviour of fatty acid-homogeneous membranes of Acholeplasma laidlawii B was investigated by Fourier transform infrared spectroscopy. The organism was grown at 37°C in the presence of avidin, an inhibitor of fatty acid synthesis, in a medium supplemented with pentadecanoic acid-d₂₉; the enrichment of the membranes with this fatty acid was 95%. The temperature-dependent phase behaviour of the membranes was studied via the C–D stretching vibrational modes of the membrane lipids and was compared with that of the lipid extract. The high level of fatty acid homogeneity results in a sharp (for natural membranes) gel to liquid crystalline phase transition. The transition, in both the membranes and extracted lipids, is centered at about 6°C above the growth temperature. During the transition two principal liquid states are evident, one being more conformationally ordered than the other. The effect of proteins on the principal lipid phase transition is minimal. However, in the intact membranes there is evident a weaker, lower temperature transition, which is not evident in the extracted lipids.     

The unique lipid composition of gecko (Gekko Gekko) photoreceptor outer segment membranes

This study investigated the lipid and fatty acid composition of gecko photoreceptor outer segment membranes which contain the P521 cone-type pigment. The lipids of gecko photoreceptor outer segment membranes were first extracted and separated by thin layer chromatography (TLC) and then analyzed by gas chromatography (GC). Our results show that gecko photoreceptor outer segment membranes contain less phosphatidylethanolamine (PE) and more phosphatidylcholine (PC) and phosphatidylserine (PS) compared with those of bovine and frog. The content of the polyunsaturated fatty acid, docosahexaenoic acid (DHA), in PC and PS is also the highest yet reported (55 and 63%, respectively). These lipid differences may provide some insight into the specific lipid requirements of cone-type pigments.