Main phospholipids and their fatty acid composition in muscle and cephalothorax of the edible Mediterranean crustacean Palinurus vulgaris (spiny lobster)

The total lipids of muscle and cephalothorax of Mediterranean lobster Palinurus vulgaris were found to be 1.0% and 2.4% of the wet tissue of which the phospholipids represented 66.5% and 47.5%, respectively. The main PhL saturated, monounsaturated and polyunsaturated fatty acids in muscle and cephalothorax were C16:0, C18:0, C18:1ω-9, C18:1ω-7, C20:4ω-6, C20:5ω-3 and C22:6ω-3. 2-OH C14:0 and cyclo-17:0 fatty acids were also identified though in low percentages. The main individual PhL in muscle were found to be phosphatidylcholine (53.5%), 72.0% of which corresponded to the structure of 1,2-diacyl-glycerocholine while the rest 28.0% to 1-O-alkyl-2-acyl-glycerocholine or 1-O-(1-alkenyl)-2-acyl-glycerocholine and phosphatidylethanolamine (19.3%), 75.0% of which corresponded to the structure of 1,2-diacyl-glyceroethanolamine and 25% to 1-O-alkyl-2-acyl-glyceroethanolamine or 1-O-(1-alkenyl)-2-acyl-glyceroethanolamine. Cephalothorax main PhL were found to be PC and PE (66.4% and 18.8%, respectively). In muscle and cephalothorax PC ω-3 fatty acids amounted 7.78% and 8.60%, while in PE amounted 30.77% and 23.65% respectively. Furthermore, in both tissues PhL, cardiolipine phosphatidylserine, phosphatidylinositol, sphingomyelin and lysophosphatidylcholine, were also found.     

Optic gland implants and their effects on the gonads of Octopus.

1. Optic glands transplanted from one Octopus vulgaris into another cause enlargement of the gonads and ducts of the recipient. 2. Enlargement occurs whether or not the gland was secreting when implanted and regardless of the sex of the donor or recipient. 3. Glands derived from Eledone moschata or Octopus macropus implanted into O. vulgaris are as effective as glands derived from O. vulgaris. 4. Implants derived from Sepia officinalis or Loligo vulgaris appear to be ineffective.     

Phospholipids of Nocardia coeliaca

The lipids of Nocardia coeliaca were separated into at least 10 components by the use of thin-layer chromatography. Phosphatidylcholine was the most abundant phospholipid in this organism, accounting for 25 to 40% of the total phospholipids. The major fatty acid components of the phosphatidylcholine were 14-methyl-pentadecanoic acid (41%), the other C(15) and C(17) iso- and anteiso-fatty acids (29%), and palmitic acid (13.5%). The next most abundant phospholipid was phosphatidylethanolamine (25 to 30%), followed by phosphatidylinositol (11 to 14%) and cardiolipin (7 to 15%). Phosphatidylethanolamine and phosphatidylinositol were very similar to the phosphatidylcholine in fatty acid composition, whereas cardiolipin was characterized by a higher content of palmitic acid (30%). In all of the phospholipids examined, only trace amounts of monounsaturated fatty acids were present. When washed cells of N. coeliaca were incubated with methionine-methyl-(14)C for 1 to 3 hr, the radioactivity was mainly incorporated into the choline moiety of the phosphatidylcholine. In contrast, acetate-1-(14)C or glycerol-1-(14)C was incorporated much more slowly into the phosphatidylcholine than into the other phospholipids and neutral lipids. No phosphatidylcholine was detected in 10 other species of Nocardia examined.     

Labeling of lipids of retina subcellular fractions by [1-14C]eicosatetraenoate (20:4(n-6)) docosapentaenoate (22:5(n-3)) and docosahexaenoate (22:6(n-3))

(1-14C)-labeled (n-6) eicosatetraenoate, (n-3) docosapentaenoate and (n-3) docosahexaenoate (20:4, 22:5 and 22:6, respectively) are efficiently taken up and actively esterified into the lipids of bovine retina after 2 h incubation. Photoreceptor membranes, mitochondria, microsomes and postmicrosomal supernatants, which display significant differences in phospholipid and fatty acid compositions, are isolated after such incubations to study the labeling of lipids. The lipid classes preferentially labeled with the acids (1) largely differ among and within subcellular fractions, while (2) some common features in the treatment of the three polyenes are observed in each fraction. In all of them, the three acids are actively incorporated in phosphatidylcholine; ethanolamine glycerophospholipid, phosphatidylserine (PS) and phosphatidylinositol (PI) are highly labeled with 22:6, 22:5 and 20:4 respectively; within ethanolamine glycerophospholipid, the three label phosphatidylethanolamine in preference to plasmenylethanolamine. Most of the 14C esterified in mitochondria is in phospholipids. The endoplasmic reticulum produces in addition highly labeled triacylglycerols, also found in cytosol. High levels of 14C-labeled diacylglycerols are observed exclusively in photoreceptor membranes, where the specific radioactivity of PI is very high. The total amounts of 14C incorporated (1) are in general similar within a given fraction for the three polyenes, but (2) largely differ among fractions. The labeling of the highly unsaturated phospholipids of photoreceptor membranes is the lowest, while the postmicrosomal supernatant (whose lipids are relatively the poorest in polyenoic fatty acids) contains most of the labeled lipids isolated from retinas under these conditions. The results indicate that polyunsaturated species of retina phospholipids undergo an active synthesis and turnover, as well as an intense intracellular traffic among membranes.     

Cyclic changes in phospholipid content and composition in human endometrium during the menstrual cycle

A significant increase in total phospholipid content of the endometrium took place during the secretory phase of the human menstrual cycle (26% increase from mid-proliferative to premenstrual stage). The major phospholipid, phosphatidylcholine, was increased by 30%, whereas phosphatidylethanolamine was unchanged. Phosphatidyl-serine and -inositol underwent the largest percentage increases (40%). Phosphatidic acid levels were the only ones to decrease (-52%), a finding consistent with the role of this lipid as precursor of the increased phospholipids. The changes did not markedly affect phospholipid composition, except for a significant decrease in the proportions of phosphatidate and phosphatidylethanolamine. Arachidonate and eicosatrienoate (n-6) were the major polyunsaturated fatty acids. C22 tetra-, penta- and hexa-enoic fatty acids of the n-3 and n-4 families were also present in all major endometrial glycerophospholipids throughout the cycle. The mass changes in phospholipids during the cycle occurred without alteration of their fatty acid composition.     

Lipid composition of Trypanosoma cruzi.

1. Lipid composition of Trypanosoma cruzi epimastigote form in culture consist of 35% of phospholipids and 65% of neutral lipids. 2. Among the phospholipids, phosphatidylcholine is the more abundant (44%), followed by phosphatidylethanolamine (28%), phosphatidylinositol (12%), sphingomyelin (4%), and smaller amounts of cardiolipin, phosphatidic acid, lysolecithin, phosphatidylserine (traces), and an unidentified phospholipid (3%). 3. Pulse labeling with 32P showed highest specific incorporation in phosphatidylethanolamine, followed by phosphatidylinositol and phosphatidylcholine, suggesting a more active role for phosphatidylethanolamine in these organisms.     

Lipid composition of the electron transport membrane of Haemophilus parainfluenzae

The principal lipids associated with the electron transport membrane of Haemophilus parainfluenzae are phosphatidylethanolamine (78%), phosphatidylmonomethylethanolamine (0.4%), phosphatidylglycerol (18%), phosphatidylcholine (0.4%), phosphatidylserine (0.4%), phosphatidic acid (0.2%), and cardiolipin (3.0%). Phospholipids account for 98.4% of the extractible fatty acids. There are no glycolipids, plasmalogens, alkyl ethers, or lipo amino acid esters in the membrane lipids. Glycerol phosphate esters derived from the phospholipids by mild alkaline methanolysis were identified by their staining reactions, mobility on paper and ion-exchange column chromatography, and by the molar glycerol to phosphate ratios. Eleven diacyl phospholipids can be separated by two-dimensional thin-layer chromatography. Each lipid served as a substrate for phospholipase D, and had a fatty acid to phosphate ratio of 2:1. Each separated diacyl phospholipid was deacylated and the glycerol phosphate ester was identified by paper chromatography in four solvent systems. Of the 11 separated phospholipids, 3 were phosphatidylethanolamines, 2 were phosphatidylserines, and 2 were phosphatidylglycerols. Phosphatidylcholine, cardiolipin, and phosphatidic acid were found at a single location. Phosphatidylmonomethylethanolamine was found with the major phosphatidylethanolamine. Three distinct classes of phospholipids are separable according to their relative fatty acid compositions. (i) The trace lipids consist of two phosphatidylethanolamines, two phosphatidylserines, phosphatidylcholine, phosphatidic acid, and a phosphatidylglycerol. Each lipid represents less than 0.3% of the total lipid phosphate. These lipids are characterized by high proportions of the short (C(10) to C(14)) and long (C(19) to C(22)) fatty acids with practically no palmitoleic acid. (ii) The major phospholipids (93% of the lipid phosphate) are phosphatidylethanolamine, phosphatidylmonomethylethanolamine, and phosphatidylglycerol. These lipids contain a low proportion of the short (C(19)) fatty acids. Palmitic and palmitoleic acids represent over 80% of the total fatty acids. (iii) The fatty acid composition of the cardiolipin is intermediate between the other two classes. Both palmitoleic and the longer fatty acids represent a significant proportion of the total fatty acid.     

Seasonal changes of fatty acid composition and thermotropic behavior of polar lipids from marine macrophytes

Major glyco- and phospholipids as well as betaine lipid 1,2-diacylglycero-O-4'-(N,N,N-tri-methyl)-homoserine (DGTS) were isolated from five species of marine macrophytes harvested in the Sea of Japan in summer and winter at seawater temperatures of 20-23 and 3 degrees C, respectively. GC and DSC analysis of lipids revealed a common increase of ratio between n-3 and n-6 polyunsaturated fatty acids (PUFAs) of polar lipids from summer to winter despite their chemotaxonomically different fatty acid (FA) composition. Especially, high level of different n-3 PUFAs was observed in galactolipids in winter. However, the rise in FA unsaturation did not result in the lowering of peak maximum temperature of phase transition of photosynthetic lipids (glycolipids and phosphatidylglycerol (PG)) in contrast to non-photosynthetic ones [phosphatidylcholine (PC) and phosphatidylethanolamine (PE)]. Different thermotropic behavior of these lipid groups was accompanied by higher content of n-6 PUFAs from the sum of n-6 and n-3 PUFAs in PC and PE compared with glycolipids and PG in both seasons. Seasonal changes of DSC transitions and FA composition of DGTS studied for the first time were similar to PC and PE. Thermograms of all polar lipids were characterized by complex profiles and located in a wide temperature range between -130 and 80 degrees C, while the most evident phase separation occurred in PGs in both seasons. Polarizing microscopy combined with DSC has shown that the liquid crystal - isotropic melt transitions of polar lipids from marine macrophytes began from 10 to 30 degrees C mostly, which can cause the thermal sensitivity of plants to superoptimal temperatures in their environment.     

Biochemical Heterogeneity of the Lipid Status of the Prespawn Eggs of Pink Salmon <Emphasis Type="Italic">Oncorhynchus gorbuscha</Emphasis> (Walbaum 1792) (Varzuga River,White Sea basin)

A comparative analysis of lipid and fatty acids contents in certain portions of female gonads—head, central, and caudal—of the prespawned pink salmon Oncorhynchus gorbusha has been performed. Heterogeneity of the lipid status of eggs located in certain portions of ovaries has been found: in the head portion, a high level of physiologically important eicosapentaenoic 20:5(n-3) and docosahexaenoic 22:6(n-3) fatty acids has been registered, which coincided with a higher intensity of lipid metabolism evidenced by higher ratio of 16:0/18:1(n-9); the central portion is characterized by a low level of total lipids due to phospholipids (including phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin) and cholesterol; in the caudal portion, a high amount of certain phospholipids (phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, sphingomyelin, and phosphatidylinositol) has been found. Such heterogeneity in certain portions of ovaries indicates asynchronous biochemical processes in oocytes of these portions that finally affects fertilization, growth and development of embryos, and further differentiation of the young fish.     

Effect of gamma irradiation and cystamine on kidney lipids of rats

Effect of whole body gamma irradiation (1200 r) and the effect of administration cystamine prior to irradiation has been studied on kidney total lipids, cholesterol, phospholipids (phosphatidylcholine and phosphatidylethanolamine). Irradiation significantly decreases kidney cholesterol and this decrease was not prevented by administration of cystamine prior to irradiation. Irradiation did not affect the incorporation of NaH232PO4 into kidney phosphatidylcholine and phosphatidylethanolamine but the incorporation of glucose-U-14C was significantly reduced in kidney total lipids and phosphatidylcholine. Administration of cystamine before irradiation was ineffective in modifying the incorporation of glucose-U-14C into kidney lipids and phosphatidylcholine.     

Phospholipid and fatty acid composition in mitochondria from spinach (Spinacia oleracea) leaves and petioles. A comparative study.

Essentially chlorophyll-free mitochondria from photosynthetic (leaf) and non-photosynthetic tissue (petiole) were isolated from spinach (Spinacia oleracea). Leaf mitochondria were found to contain more phosphatidylcholine than phosphatidylethanolamine compared with petiole mitochondria. Galactolipids were found in small and equal amounts (5 mol of galactolipids/100 mol of galactolipids and phospholipids) in both leaf and petiole mitochondria. Fatty acid composition showed a significant difference in the amounts of C18:2 and C18:3 acids. The C18:2/C18:3 ratio was more than twice as high in all of the phospholipids studied from petiole mitochondria compared with the ratio in leaf mitochondria. More than 50% (mol/100 mol) of the fatty acids in the major lipids (phosphatidylcholine, phosphatidylethanolamine and cardiolipin) in petiole mitochondria were C18:2. In the minor lipids (phosphatidylinositol and phosphatidylglycerol), C16:0 dominated in both leaf and petiole mitochondria.     

Incorporation into phospholipid classes and metabolism via desaturation and elongation of various 14C-labelled (n-3) and (n-6) polyunsaturated fatty acids in trout astrocytes in primary culture

The incorporation and metabolism of [1-14C]18:3(n-3), [1-14C]20:5(n-3), [1-14C]18:2(n-6), and [1-14C]20:4(n-6) were studied in primary cultures of trout brain astrocytes. There were no significant differences between the amounts of individual fatty acids incorporated into total lipid at 22 degrees C, with greater than 90% of all the fatty acids being incorporated into polar lipid classes. The distributions of 18:2(n-6), 18:3(n-3), and 20:5(n-3) in individual phospholipid classes at 22 degrees C were very similar, with 57-63 and 18-24% being incorporated into phosphatidylcholine and phosphatidylethanolamine, respectively. Approximately equal amounts of 20:4(n-6), approximately 30% of the total, were incorporated into each of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. The metabolism of the (n-3) fatty acids to longer-chain and more unsaturated species was significantly greater than that of (n-6) acids, but delta 4-desaturase activity was very low. A culture temperature of 10 degrees C increased the incorporation of all the fatty acids into total lipid and that of C20 fatty acids into polar lipid. At 10 degrees C, the incorporation of C20 fatty acids into phosphatidylethanolamine and phosphatidylinositol was increased, and the incorporation into phosphatidylcholine and phosphatidylserine was decreased. The distribution of C18 fatty acids was unchanged at the lower temperature, as was the desaturation and elongation of all the polyunsaturated fatty acids incorporated.     

Fatty acid composition of individual polar lipid classes from marine macrophytes

Major glycolipids [monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)) and phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG)] as well as betaine lipid 1,2-diacylglycero-O-4'-(N,N,N-tri-methyl)-homoserine (DGTS) were isolated from Anfeltia tobuchiensis (Rhodophyta), Laminaria japonica, Sargassum pallidum (Phaeophyta), Ulva fenestrata (Chlorophyta) and Zostera marina (Embriophyta), harvested in the Sea of Japan. GC analysis of their fatty acid (FA) composition revealed that the n-6 polyunsaturated FAs (PUFAs) shared the most part of the sum of n-6 and n-3 PUFAs in PC and PE compared with glycolipids and PG. In algae, it was related to the prevalence of 20:4n-6 over 20:5n-3 in non-photosynthetic lipids. Percentage of n-6 PUFAs as well as the sum of n-3 and n-6 PUFAs decreased in the following sequence: PC-->PE-->PG. The saturation increased in the lines of MGDG-->DGDG-->SQDG and PC-->PE-->PG. PG was close to SQDG by the level of saturation. Distribution of C(18) and C(20) PUFAs in polar lipids depended on taxonomic position of macrophytes. Balance between C(18) and C(20) PUFAs was preferably shifted to the side of C(20) PUFAs in PC and PE that was observed in contrast to glycolipids and PG from L. japonica containing both series of FAs. The set of major FAs of polar lipid classes can essentially differ from each other and from total lipids of macrophytes. For example, MGDG was found to accumulate characteristic fatty acids 16:4n-3, 16:3n-3, 18:3n-6 and 18:4n-3, 20:3n-6 in U. fenestrata, Z. marina, L. japonica and S. pallidum, respectively.     

Distribution of omega-3 and omega-6 fatty acids in the ether- and ester-linked phosphoglycerides from tissues of the rainbow trout, Salmo gairdneri

1. Data presented here demonstrate that polyunsaturated fatty acids in the phospholipids of rainbow trout tissues are compartmentalized differently than in mammalian tissues. 2. We have determined the distribution of omega-3 (n-3) and omega-6 (n-6) fatty acids in the alkyl-, alk-1-enyl-, and diacyl- subclasses of phosphatidylcholines (PC), phosphatidyl-ethanolamines (PE), phosphatidylinositols (PI), and phosphatidylserines (PS) from gill, kidney and spleen of rainbow trout. 3. Alkyl-linked PC and alk-1-enyl-linked PE were the most abundant ether-containing phospholipids, amounting to 10-15% of each class; no ether-linked PI or PS was detected. 4. C20:4 n-6 was found in high concentrations only in PI; the n-3 fatty acids were found in highest concentration in the ether-linked phospholipids as compared with the diacyl subclasses and C20:5 n-3 was especially prevalent in 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine and C22:6 n-3 was prevalent in PS.     

Lipids of the zygomycete Absidia corymbifera F-965

The cell lipids of the zygomycete Absidia corymbifera F-965 extracted with isopropanol and CHCl3-MeOH mixtures at the exponential growth phase comprise 20+/-2% of mycelium dry wt. The lipids consist of: triacylglycerols (51% of the total lipids extracted), diacylglycerols (9%), monoacylglycerols (3%), ergosterol (5%), ergosterol peroxide (5alpha,8alpha-epidioxyergosta-6,22-diene-3beta-ol) (3%), fatty-acid esters of ergosterol (less than 0.5%), free fatty acids (4%), glucocerebroside (3%), and glycerophospholipids (22%). The main phospholipids are phosphatidylethanolamine (39% of the total phospholipids), phosphatidyl-myo-inositol (17%), diphosphatidylglycerol (12%), phosphatidic acid (7%), phosphatidylcholine (6%), phosphatidylglycerol (3%), and two unusual phospholipids reported earlier, N-acetylphosphatidylethanolamine (7%) and N-ethoxycarbonyl phosphatidylethanolamine (9%). In addition, two unknown acidic phospholipids are present in traces. Saturated fatty acids of the lipids are dominated by n-hexadecanoic acid and unsaturated ones by octadecenoic acid; octadecadienoic and octadecatrienoic acids are present in lesser amounts. Ergosterol peroxide as well as the above glucocerebroside which contains 9-methylsphinga-4(E),9(E)-dienine have first been revealed in zygomycetes.     

What cephalopod remains from Xiphias gladius stomachs can imply about predator‐prey interactions in the Mediterranean Sea?

Analysis of the cephalopod beaks found in the stomachs of 69 swordfish Xiphias gladius caught in the Aegean Sea resulted in the identification of nine cephalopod taxa. Cephalopod consumption was higher in summer and autumn, and larger swordfish were more likely to feed on cephalopods. Todarodes sagittatus was the most abundant species, a finding suggesting its abundance in the Mediterranean sea.     

Two unusual glycerophospholipids from a filamentous fungus, Absidia corymbifera

The chloroform-methanol extractable lipids of the soil filamentous fungus Absidia corymbifera VKMF-965 account for about 20% by weight of dry cells and are composed of low-polarity constituents (about 75% of the total lipids), such as triacylglycerols (mainly), diacylglycerols, sterols and free fatty acids, as well as of glycolipids (about 3%) and phospholipids. The last consist largely of components common to the fungal lipids, namely, phosphatidylethanolamine (38% of the total phospholipids), phosphatidyl-myo-inositol (16%), diphosphatidylglycerol (12%), phosphatidylcholine (7%), phosphatidic acid (6%) and phosphatidylglycerol (3%), and two unusual phospholipids, PL1 (6%) and PL2 (9%). Based on the infrared (IR), (1)H-nuclear magnetic resonance (NMR), (13)C-NMR and mass spectra along with the results of degradation experiment, these two phospholipids have been established to be 1,2-diacyl-sn-glycero-3-phospho(N-acetylethanolamine), or N-acetyl phosphatidylethanolamine, and 1,2-diacyl-sn-glycero-3-phospho(N-ethoxycarbonyl-ethanolamine), respectively. These structures have been confirmed by preparing similar phospholipids from the phosphatidylethanolamine isolated from the same fungus and correlating their chromatographic behaviour, IR and (1)H-NMR spectra with those of PL1 and PL2. So far N-acetyl phosphatidylethanolamine has been detected only in cattle and human brains and a human placenta but its structure was not rigorously proved. PL2 is a novel lipid; to our knowledge no natural phospholipid with an urethane group has yet been found. The main fatty acids of both the phospholipids are n-hexadecanoic, octadecanoic and octadecadienoic ones; PL2 contains in addition a considerable amount of octadecatrienoic acid with its greater portion located at the sn-1 position.     

Reconstitution of affinity-purified dopamine D2 receptor binding activities by specific lipids

The role of lipids in maintaining ligand binding properties of affinity-purified bovine striatal dopamine D2 receptor was investigated in detail. The receptor, purified on a haloperidol-linked Sepharose CL6B affinity column, exhibited low [3H]spiroperidol binding unless reconstituted with soybean phospholipids. In order to understand the role of individual phospholipids in maintaining the receptor binding activity, the purified preparation was reconstituted separately with individual phospholipids and assayed for [3H]spiroperidol binding. Except for phosphatidylcholine and phosphatidylethanolamine, that respectively restored 30 and 20% binding as compared to that obtained with soybean lipids, reconstitution with other lipids had very little effect. When various combinations of phospholipids were used for reconstitution, a phosphatidylcholine and phosphatidylserine mixture seemed to almost fully restore the receptor binding. A mixture of phosphatidylcholine and phosphatidylethanolamine was as effective as phosphatidylcholine alone in reconstituting ligand binding; however, when phosphatidylserine was also included in the mixture, there was a pronounced increase in binding (about 2-fold compared to the soybean lipids and about 6-fold compared to the phosphatidylcholine-phosphatidylethanolamine mixture). Substitution of other phospholipids or cholesterol for phosphatidylserine in phosphatidylcholine and phosphatidylethanolamine mixture had little effect. Maximal reconstitution of [3H]spiroperidol binding was obtained with phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine mixture (2:2:1, w/w) at a concentration of 0.5 mg/ml. The reconstituted receptor exhibited high affinity binding for [3H]spiroperidol which was comparable to that obtained with membrane or solubilized preparations. Various dopaminergic antagonists and agonists showed appropriate order of potency for the reconstituted receptor. The presently described reconstitution data suggest a role of specific phospholipids in preserving the binding properties of dopamine D2 receptor and should prove useful in studies on functional reconstitution of the receptor.     

Modulation by phospholipids of the activity of monoamine oxidase purified from pig liver

Monoamine oxidase was purified from pig liver mitochondria to homogeneity. The enzyme sample contained a large amount of phospholipids. Depletion of lipids from the enzyme sample resulted in a decrease in its activity, while activity was restored by the binding of the lipid-depleted enzyme to phosphatidylcholine, phosphatidylethanolamine, or mitochondrial lipids. Upon binding the lipid-depleted enzyme to the mixture of phosphatidylcholine and phosphatidylethanolamine (molar ratio, 1 : 1), the enzymatic activity toward serotonin was elevated over that of the purified enzyme, but not toward benzylamine, suggesting a change in substrate specificity. Upon lipid depletion, inhibition by deprenyl became weaker, while that by clorgyline became stronger. This alteration was reversed by the binding to lipids. By the binding of the lipid-depleted enzyme to some lipids such as the mixture of phosphatidylcholine and phosphatidylethanolamine (molar ratio, 1 : 1), inhibition by clorgyline became even weaker than for the original enzyme sample.     

Asymmetry of lipid organization in cholinergic synaptic vesicle membranes.

The lipid composition of purified Torpedo cholinergic synaptic vesicles was determined and their distribution between the inner and outer leaflets of the vesicular membrane was investigated. The vesicles contain cholesterol and phospholipids at a molar ratio of 0.63. The vesicular phospholipids are (mol% of total phospholipids): phosphatidylcholine (40.9); phosphatidylethanolamine (24.6); plasmenylethanolamine (11.5); sphingomyelin (12); phosphatidylserine (7.3); phosphatidylinositol (3.7). The asymmetry of the synaptic vesicle membranes was investigated by two independent approaches: (a) determining accessibility of the amino lipids to the chemical label trinitrobenzenesulphonic acid (TNBS); (b) determining accessibility of the vesicular glycerophospholipids to phospholipase C (Bacillus cereus). TNBS was found to render the vesicles leaky and thus cannot be used reliably to determine the asymmetry of Torpedo synaptic vesicle membranes. Incubation of the vesicles with phospholipase C (Bacillus cereus) results in biphasic hydrolysis of the vesicular glycerophospholipids. About 45% of the phospholipids are hydrolysed in less than 1 min, during which no vesicular acetylcholine is released. In the second phase, the hydrolysis of the phospholipids slows down markedly and is accompanied by loss of all the vesicular acetylcholine. These findings suggest that the lipids hydrolysed during the first phase are those comprising the outer leaflet. Analysis of the results thus obtained indicate that the vesicular membrane is asymmetric: all the phosphatidylinositol, 77% of the phosphatidylethanolamine, 47% of the plasmenylethanolamine and 58% of the phosphatidylcholine were found to reside in the outer leaflet. Since phosphatidylserine is a poor substrate for phospholipase C (B. cereus), its distribution between the two leaflets of the synaptic vesicle membrane is only suggestive.