Preparative method of isolating phosphoinositide fractions from brain tissue]

A simple technique for preparative isolation of chromatographically homogenous fractions of mono-, di- and triphosphoinositides from ox brain tissue is described. Podyphosphoinositides fractions were obtained after chromatography of lipid extract on DEAE cellulose, phosphomonoinositides fraction--after chromatography of polyphosphoinositide-free material on aluminium hydroxide column. Bivalent metal ions were eliminated from lipid extract using chromatography on Dowex-50 H+. Ammonium acetate was removed after precipitation of lipids in water: methanol (1:1) in the presence of 4 M this salt. The average yield of mono-, di- and triphosphates was 40, 22 and 58 mg respectively per 1 kg of brain tissue as callulated for lipid phosphorus.     

Thin-layer chromatography of the phosphoinositides

Thin-layer chromatography for the separation of mono-, di-, and triphosphoinositides (0.3-3 micro g total phosphorus) is described.     

Preparative isolation of polyphosphoinositide fractions from ox brain

A simple preparative method for chromatographic isolation of pure fractions of di- and triphosphoinositides (1-phosphatidylinositol 4-phosphate and 1-phosphatidylinositol 4,5-bisphosphate) from ox brain is described. Polyphosphoinositide fractions have been obtained by ion-exchange chromatography of the lipid extract using gradient elution with 0-0.6 M ammonium acetate in chloroform/methanol/water (20:9:1) from a DEAE-cellulose column. Before chromatography, divalent metal ions were removed from the lipid extract by passing through a Dowex-50 (H+) column and lipids were converted to the sodium salt by neutralisation with sodium hydroxide in methanol solution. After chromatography, fractions of di- and triphosphoinositides were precipitated in methanol/water mixture (1:1) by evaporation in a vacuum to a final concentration of about 4 M ammonium acetate. Necessary salts of di- and triphosphoinositides were obtained by passing the ammonium salts of the lipids through Dowex-50 (H+) and neutralising with corresponding base in methanol solution. About 0.35 mmol of diphosphoinositide and 0.63 mmol of triphosphoinositide were obtained from 1 kg of wet ox brain tissue.     

Characterization of mono-, di-, and tri-O-acetylated sialic acids on human cells

The presence of mono-, di-, and tri-O-acetylated sialic acids on human cells was demonstrated by using radiochromatographic and chemical techniques. Human melanoma cells and fresh colon tissue were biosynthetically labeled with 6- (3H) glucosamine. Radiolabeled sialic acids were hydrolytically removed from cellular glycoconjugates, purified by ion-exchange chromatography, and separated by paper chromatography on the basis of the number of O-substitutions on each sialic molecule. This analytical technique characterized radiolabeled sialic acids that migrated with the same Rf as synthetic mono-, di-, and tri-O-acetylated 14C-labeled sialic acids. The mono-O-acetylated sialic acids were characterized by their sensitivity to sodium periodate oxidation and a crude mouse liver esterase preparation. The di- and tri-O-acetylated sialic acids were characterized by their resistance to sodium periodate oxidation and sensitivity to the action of crude mouse liver esterase. Chromatographically separated di- and tri-O-acetylated sialic acids from normal human colon tissue were characterized by their respective ion molecular weights by using fast-atom bombardment-mass spectrometry. Using these methods, we chemically characterized mono, di-, and tri-O-acetylated sialic acids expressed on human cells. Aberrant expression of O-acetylated sialic acids was associated with adenocarcinoma of the colon, leading to a nearly complete loss of di- and tri-O-acetylated sialic acids.     

Gangliosides of human, bovine, and rabbit plasma

Gangliosides were isolated from human, bovine, and rabbit plasma and were quantified by gas-liquid chromatography. Purification was achieved by sequential use of partitioning in solvents, DEAE-Sephadex chromatography, base treatment, and silicic acid chromatography. Human and bovine plasma yielded slightly more than 1 micro mole of lipid-bound sialic acid/100 ml; for rabbit plasma the value was 0.28 micro mole/100 ml. The total bovine plasma ganglioside fraction contained equal amounts of N-acetylneuraminic and N-glycolylneuraminic acids, rabbit plasma gangliosides had about 1% of the latter, and the human plasma sample contained only the former. Thin-layer chromatography revealed important differences among the plasmas from the three species, but all possessed hematosides and hexosamine-containing gangliosides. The approximate ratios of these two categories, based on sialic acid content, were (hematosides: hexosamine-type): human, 2:1; rabbit, 3:2; and bovine, 2:3. The fatty acid compositions of both categories were characteristic of extraneural gangliosides and included six major acids: palmitic, stearic, behenic, tricosanoic, lignoceric, and nervonic. The major long-chain base in each sample was sphingosine, while only a trace of the C(20) isomer was detected.     

Evidence for a Composite State of an F′his,gnd Element and a Cryptic Plasmid in a Derivative of Salmonella typhimurium LT2

The zoospores of Blastocladiella emersonii, when derived from cultures grown on solid media, contain about 11% total lipid. This lipid was separated chromatographically on silicic acid into neutral lipid (46.6%), glycolipid (15.8%), and phospholipid (37.6%). Each class was fractionated further on columns of silicic acid, Florisil, or diethylaminoethyl-cellulose, and monitored by thin-layer chromatography. Triglycerides were the major neutral lipids, mono- and diglycosyldiglycerides were the major glycolipids, and phosphatidylcholine and phosphatidylethanolamine were the major phospholipids. Other neutral lipids and phospholipids detected were: hydrocarbons, free fatty acids, free sterols, sterol esters, diglycerides, monoglycerides, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidic acid, phosphatidylserine, and phosphatidylinositol. Palmitic, palmitoleic, stearic, oleic, gamma-linolenic, and arachidonic acids were the most frequently occurring fatty acids. When B. emersonii was grown in (14)C-labeled liquid media, lipid again accounted for 11% of both mature plants and zoospores released from them. The composition of the lipid extracted from such plants and spores was also the same; however, it differed markedly from that of the lipid in spores harvested from solid media, consisting of 28.3% neutral lipid, 12.0% glycolipid, and 59.7% phospholipid. The major lipids were again triglycerides for neutral lipids, mono- and diglycosyldiglycerides for glycolipids, and phosphatidyl choline and phosphatidylethanolamine for phospholipids.     

Metabolism of fatty acids by bovine spermatozoa

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

A new chromatographic method for the preparative isolation of phosphoinositides and other anionic phospholipids

A new chromatographic procedure for preparative isolation of mono-, di- and triphosphoinositides and other anionic phospholipids with the use of adsorbents containing primary amino groups is described. Sorbents with immobilized neomycin, L-lysine and aminoalkyl groups were tested. Conditions for isolation of chromatographically pure phospholipids of separate classes on the above sorbents were developed. Isolation of polyphosphoinositides on the amino sorbents represents a new type of chromatography involving bioaffinity and ion-exchange interaction.     

Structural analysis of phosphatidylcholine of plant tissue

Pure preparations of phosphatidylcholine were isolated from spinach leaf chloroplasts, spinach leaf microsomes, and cauliflower inflorescence. The isolated phosphatidylcholine was treated with snake venom phospholipase A, and the fatty acid distribution and composition of the fatty acid methyl esters prepared from the lysophosphatidylcholine and the freed fatty acid were determined by gas-liquid chromatography. The results showed that saturated fatty acids were preferentially esterified at position 1 and unsaturated fatty acids at position 2. The phosphatidylcholine from cauliflower was also treated with phospholipase C. The resulting diglycerides were fractionated on AgNO(3)-impregnated thin-layer plates. The diglyceride fractions were transesterified and the fatty acid composition of each was determined by gas-liquid chromatography. The predominant species contained linolenic acid only (22% of the total), linolenic and oleic acids (19%), and linolenic and palmitic acids (37%). These molecular species could not be accounted for by random distribution of the fatty acids.     

Influence of S-ethyl Dipropylthiocarbamate (EPTC) on the Fatty Acid Composition of Wheat Leaf Galactolipids

Winter wheat (Triticum sativum L. cv. Nisu) was grown in sand which contained 0, 0.25, 0.5 and 1.0 mg S-ethyl dipropylthiocarbamate (EPTC) per kg air dry sand. The galactolipids of leaves of 21-day-old seedlings were isolated by preparative thin layer chromatography. The fatty acids of the mono- and digalactosyl diglycerides were analysed by gas liquid capillary chromatography. The major fatty acids of the wheat leaf galactolipids were palmitic, palmitoleic, stearic, oleic, vaccenic, linoleic and linolenic acids (in the monogalactosyl diglyceride fraction of untreated plants 22.5, 2.4, 3.1, 5.2, 2.5, 51.1 and 1526.6 μg and in the digalactosyl diglyceride fraction 108.8, 2.3, 10.4, 9.9, 8.2, 42.3 and 1120.7 μg/g leaf fresh weight, respectively). Total fatty acid content of the mono- and digalactosyl diglyceride fractions was decreased by 85 and 87%, respectively, at 1 mg EPTC/kg sand, while decrease in the fresh weight of the leaves was 79%. The content of linoleic and linolenic acids/g fresh weight of the leaves was decreased in the monogalactosyl diglyceride fraction by 27 and 43%, respectively, while the content of all other fatty acids was increased. In the digalactosyl diglyceride fraction the content of both linoleic and linolenic acids/g leaf fresh weight was decreased by 55%. The content of palmitic and vaccenic acids was also decreased, whereas the content of other fatty acids remained at the level of the untreated samples. The general quality of the fatty acids in the mono- and digalactosyl diglyceride fractions was altered slightly by EPTC.     

Separation of mono- and diglycerides by gas--liquid chromatography

The parameters affecting the separation and quantification of trimethylsilyl ethers of mono- and diglycerides have been investigated by gas-liquid chromatography with QF-1 and SE-30 as stationary phases and a flame ionization detector. Results have been compared with those obtained earlier for triglycerides. The isothermal characteristics of a range of trimethylsilyl ethers of mono- and diglycerides on both stationary phases showed that log retention volume was directly proportional to carbon number and inversely proportional to absolute temperature. However, glyceride derivatives with lower carbon numbers deviated from these relationships. By using various rates of programmed temperature rise, we have determined the elution temperatures (Kelvin scale) of the mono- and diglyceride trimethylsilyl ethers relative to that of glycerol trilaurate. The "carbon equivalent of a trimethylsilyl group" is defined and shown to be useful in comparing the chromatographic properties of different glyceride classes. Weight and molar correction factors have been obtained and used to analyze diglycerides derived from egg and bovine brain lecithins.     

Lipid composition of the extracellular matrix of Botrytis cinerea germlings

Six simple lipid classes (mono-, di- and tri-acylglycerols, free fatty acids, free fatty alcohols and wax esters) were identified by TLC in the extracellular matrix of Botrytis cinerea germlings and the molecular components of each class were characterized using GC-MS. The relative amounts of fatty acids and fatty alcohols within each lipid class were determined by GC-FID. Over all the lipid classes, the most abundant saturated fatty acids were palmitic (ca. 30%) and stearic acid (ca. 22%). Palmitoleic and oleic acids made up ca. 21% and 24% (respectively) of the free fatty acids, while erucic (ca. 4.1%) and linoleic (ca. 3.6%) acids were the most abundant unsaturated fatty acids in the acylglycerides. The acylglycerides also contained almost 35% long chain fatty acids (C20:0 to C28:0). Six fatty acids were identified which had odd-numbered carbon chain lengths (C15:0, C17:0, C19:0, C21:0, C23:0 and C25:0). Of these, pentacosanoic acid made up almost 14% of the fatty acids in the acylglycerides. Three methyl-branched chain fatty acids, namely isopalmitic, isoheptadecanoic and anteisopalmitic, were identified in the ECM, all in small amounts. Of the fatty alcohols identified, only palmityl and stearyl alcohols were found in the free form (ca. 57% and 43%, respectively) but arachidyl alcohol (ca. 47%) and 1-octacosanol (ca. 30%) were the most abundant fatty alcohols found in the wax ester fraction.     

Characterization of the constituent fatty acids in phosphatidylinositol during early development in Rana nigromaculata

1. Qualitative and quantitative changes in phosphatidylinositol (PI) were analyzed in the eggs, embryos and tadpoles of the Japanese pond frog, Rana nigromaculata, at various stages of development. 2. The weight percentage of PI to total phospholipid and lipid was about 8.4-15.2% and 1.4-2.6%, respectively, during embryonic life. 3. At the early stages of the unfertilized egg and the two-cell embryo, the predominant fatty acids are palmitic, stearic, oleic and linoleic acid. From the dorsal lip, early gastrula stage and beyond, the percentage of linoleic acid declines and there is an increase in palmitoleic acid. A relatively large amount of arachidonic acid was noted at the unfertilized egg stage at the 1-position. 4. A large amount of arachidonic acid was also observed at the 2-position of PI in the unfertilized egg, hatching embryo and post-hatching tadpole stages, relative to palmitic and stearic acid. 5. Palmitic and stearic acid were increased at the 2-position of PI in the other embryo and the feeding tadpole stages, relative to arachidonic acid, indicating a shift in these molecular species. 6. Thus, there were marked changes in the positional distribution of the constituent fatty acids in PI during early development of R. nigromaculata.     

Molecular species of diacylphosphatidylethanolamine in rat and mouse heart given the same diet

Diacylphosphatidylethanolamine (PE) was isolated from mouse and rat heart given the same standard diet. The molecular species of PE were determined after conversion of PE into diglycerides by means of hydrolysis with phospholipase C, subsequent hydrolysis with pancreatic lipase and separation of the products by argentation TLC and capillary gaschromatography. Docosahexaenoic acid (22:6n3) containing molecular species and arachidonic acid (20:4n6) containing molecular species represented the major fractions. A preference for stearic acid to combine with poly-unsaturated fatty acids was found. Despite an abundant presence of linoleic acid (18:2n6) in the diet, molecular species containing this fatty acid represented only a minor fraction. The possible physico-chemical and physiological meaning of the presence of molecular species containing many double bonds is discussed.     

Phosphoinositides of the brain and liver of rats in ethanol poisoning]

Alcohol intoxication induced in the rat brain significant decrease in the monophosphoinositides level, but the di- and triphosphoinositides quantity did not change; the content of all the three studied fractions of the inositol-containing phospholipids in the liver decreased. The tendency to retain the normal level of di- and triphosphoinositides in the brain following alcohol intoxication may be considered as an important factor providing physiological condition of the brain metabolism.     

Lipid profiles of Aspergillus niger and its unsaturated fatty acid auxotroph, UFA2

A comparative study of the mycelial lipid composition of a wild strain (V35) and one unsaturated fatty acid auxotroph (UFA2) of Aspergillus niger has been performed. The lipid composition of both strains are qualitatively the same but quantitatively different. All the strains contain the following phospholipids: cardiolipin, phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylcholine, and phosphatidylserine; and triglycerides, diglycerides, monoglycerides, ergosterol, and sterol esters as the neutral lipids; mono- and di-galactosyl diglyceride as the major glycolipids along with small amounts of the corresponding mannose analogs. Phosphatidylethanolamine and phosphatidylcholine constitute the bulk of the phospholipids. The mutant (UFA2) contains a higher level of glycerides and lower levels of sterol (both free and esterified form), phospholipids, and glycolipids than the wild type. Aspergillus niger contains C16 to C18 saturated and unsaturated fatty acids. Small amounts of long-chain (C20 to C24) and short-chain (C10 to C14) saturated and unsaturated acids are also present. Linoleic, oleic, and palmitic are the major acids, stearic and linolenic acids being minor ones. UFA2 grows only in the presence of unsaturated fatty acid (C16 or C18) and accumulates a higher concentration of supplemented acid which influences its fatty acid profile.     

Separation of brain phosphatidylserines according to degree of unsaturation by thin-layer chromatography

Phosphatidylserines (PS) have been prepared from bovine brain using DEAE column chromatography. A method involving AgNO₃-impragnated silica gel H thin-layer chromatography is presented for separating intact PS according to the degree of unsaturation of their fatty acids. A detailed analysis was made of the fatty acid composition of the various fractions using gas chromatography. Some data are presented on the composition of molecular species of PS in bovine brain. The two main molecular species found in cerebral cortex are tentatively assigned the structures of 1-octadecanoyl-2-docosahexaenoyl-sn-glycero-3-phosphorylserine and 1-octadecanoyl-2-octadecenoyl-sn-glycero-3-phosphorylserine.     

Regulation of the composition and positioning of saturated and monoenoic fatty acids in phosphatidylcholine

The mechanism by which polyenoic acids control the amount and positioning of monounsaturated fatty acids in choline phosphoglycerides from baby hamster kidney cells was studied. Under normal growth conditions monoenoic acids were derived from the desaturation of saturated fatty acids and comprised over 50% of the fatty acids at position 1 of the glycerol moiety. The monoene content of positions 1 and 2 decreased in response to the addition of di- and polyenoic acids to the culture medium. All the di- and polyenoic acid supplements tested inhibited the desaturation of palmitic and stearic acid and replaced monoenes at position 2. However only linoleic, linolenic, and eicosadienoic acids replaced monoenes at position 1. The results suggest that under appropriate conditions up to 25% of the choline phosphoglyceride fraction consisted of a stable molecular species containing di- or trienoic fatty acids at both the 1 and 2 positions of glycerol moiety. With eicosatrienoic or arachidonic acid supplements, on the other hand, the monoenes at position 1 were replaced with saturated fatty acids. The magnitude of these effects, particularly at position 1, was proportional to the concentration of the fatty acid supplement. The results suggest that polyenes with at least 20 carbon atoms can play a key role in determining the ultimate composition and positioning of fatty acids in baby hamster kidney choline phosphoglycerides and that this control is mediated by their ability to inhibit delta 9 desaturase and by a retailoring system specific for these polyenes.     

The lipid binding site of the phosphatidylcholine transfer protein from bovine liver

The phosphatidylcholine transfer protein (PC-TP) from bovine liver has a binding site for phosphatidylcholine (PC). Structural and molecular characteristics of this site were investigated by binding PC-analogues carrying photolabile, fluorescent and short-chain fatty acids. Analysis of the photolabeled PC/PC-TP adduct showed that the hydrophobic peptide segment Val171-Phe-Met-Tyr-Tyr-Phe-Asp177 is part of the lipid binding site for the 2-acyl chain. This site was further studied by binding PC carrying cis-parinaric acid at the sn-2-position. Time resolved fluorescence anisotropy measurements indicated that the 2-acyl chain was immobilized following the rotation of PC-TP. Similar experiments with PC carrying cis-parinaric acid at the sn-1-position demonstrated that the 1-acyl chain was immobilized as well but at a site distinctly different from that of the 2-acyl chain. Binding sites for the 1- and 2-acyl chain were then explored by use of PC-isomers carrying decanoic, lauric and myristic acid at the sn-1- (or sn-2-)-position and oleic acid at the sn-2- (or sn-1-)-position. Incubation with vesicles prepared of these PC-species indicated that binding to PC-TP diminished with decreasing acyl chain length but more so for species with short-chain fatty acids on the sn-2-position than on the sn-1-position. Transfer experiments confirmed that PC-TP discriminates between PC-isomers of apparently equal hydrophobicity favouring the transfer of these species carrying oleic acid at the sn-2-position.     

Fatty acid composition and thermal behavior of natural sphingomyelins

We found significant differences in the fatty acid composition of several bovine brain, egg yolk and sheep erythrocyte sphingomyelins. These differences in fatty acid composition influence the thermal behavior of hydrated sphingomyelin as recorded by differentail scanning calorimetry. Significant differences were also found in the temperature and complexity of the order-disorder phase transitions of bovine brain sphingomyelin obtained from different sources which, in general, correlate with the relative content of the saturated fatty acids (palmitic (C16:0) and stearic acid (C18:0) acids) and the long unsaturated nervonic acid (C24:1).