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.     

Molecular species of mono-, di-, and triphosphoinositides of bovine brain

The mono-, di-, and triphosphoinositides of bovine brain were isolated by chromatography on columns of DEAE-cellulose, alumina, and silicic acid. The major molecular species in each phosphoinositide class were identified and quantitatively estimated by combined thin-layer and gas-liquid chromatography of the component diglycerides, which were released by hydrolysis with a specific brain phosphodiesterase. The diglycerides were treated with pancreatic lipase, and the positional distribution of the fatty acids was determined. Over 27 molecular species were identified, and these accounted for about 95% of each phosphoinositide class, but the 1-stearate 2-arachidonate derivative contributed more than 40% of the total in each class. The other molecular species also were qualitatively and quantitatively similar in the three phosphoinositide classes. All the long-chain and polyunsaturated acids were confined to the 2-position and were preferentially paired with stearic acid in the 1-position. Oleic acid in the 2-position was about equally divided between species with palmitic and stearic acids in the 1-position. These results suggest that the mono-, di-, and triphosphoinositides of the bovine brain have similar compositions and that the various molecular species may be metabolically related.     

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.     

NMR lipid profile of Agaricus bisporus

Lipids extracted from freeze dried and powdered cultivated Agaricus bisporus by the Bligh and Dwyer method, were subjected to 1D-proton and 2D-COSY NMR analysis. The diacylglycerophospholipids, mono-, di- and tri-glycerides, ether lipids, sphingolipids and steroidal lipids were studied qualitatively and quantitatively. Our findings suggested that (a) ethanolamines and cholines were the predominant diacylphospholipids, (b) sterols, mainly ergosterol, were present in relatively large quantities and (c) the phospholipid fatty acid composition consisted almost exclusively of linoleic acid. This type of detailed data on lipid composition was accurately and rapidly obtained in one step, without chemical modification of the sample. Additional information on four classes of lipid, including their fatty acid composition was obtained after separating the total lipid extract by NH₂-aminopropyl Certify II Bond Elut solid phase chromatography and analysing the NMR spectra of each class of lipids. The results demonstrated the potential of the method for the study of plant metabolism, development and taxonomy.     

Purification of rat liver mitochondrial anion carriers by triazine dye affinity chromatography

Mitochondria were prepared from rat liver, solubilized with Triton X-100 and submitted to chromatography on dry hydroxylapatite. The pass-through from the column, containing a mixture of mitochondrial carriers, was processed further by triazine dye affinity chromatography and optimal conditions for binding and elution of mitochondrial carriers were established. Activities of the mono-, di-, tricarboxylate and 2-oxoglutarate carriers were measured after reconstitution of various chromatographic fractions into asolectin vesicles, and a good resolution of different carriers was obtained. Thus the triazine dye affinity chromatography appears to be a potent new tool for purification of mitochondrial anion carriers.     

Isolation and structural characterization of twenty-one sialyloligosaccharides from galactosialidosis urine. An intact N,N'-diacetylchitobiose unit at the reducing end of a diantennary structure

Galactosialidosis urine was fractionated by gel-permeation chromatography on Bio-Gel P-6. The obtained sialic acid-containing carbohydrate fractions were purified by reversed-phase chromatography and separated according to charge by medium-pressure anion-exchange chromatography on Mono Q. The Mono Q fractions, being mixtures of sialyloligosaccharides differing mainly in sialic acid-linkage type (alpha 2-3/alpha 2-6), were subfractionated by high-performance liquid chromatography on Lichrosorb-NH2. The purified compounds were analysed by 500-MHz 1H-NMR spectroscopy. Twenty-one fully and partially sialylated N-acetyllactosamine-type compounds include mono-, di-, tri- and tetra-antennary structures. All structures have the sequence Man beta 1-4Glc-NAc at the reducing terminus in common, except one diantennary structure bearing an intact N,N'-diacetylchitobiose unit at the reducing end, which is a new feature in human glycoproteinosis urine.     

Lipids in fruiting bodies of the basidiomyceteOudemansiella mucida

Dried fruiting bodies of the basidiomyceteOudemansiella mucida contain 29.1% total lipids. Their qualitative analysis revealed the presence of mono-, di-, triglycerides, sterols, free fatty acids and sterolesters. Quantitatively most significant were triglycerides (37.9%) and free fatty acids (29.7%). The phospholipid fraction contained phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidic acid. Gas chromatography showed the presence of a broad spectrum of fatty acids. The ratio between the neutral and polar fractions was 6: 1, both having linoleic acid as the main component.     

Hypoxia/ischemia induces dephosphorylation of rat brain neuromodulin/GAP-43 in vivo

The in vivo state of phosphorylation and the modification of two Cys residues of neuromodulin/ GAP-43 (Nm) were analyzed by electrospray ionization-mass spectrometry (ES-MS). The protein was purified from rat brain with homogenization buffer containing 1% Nonidet P-40, protease inhibitors, protein phosphatase inhibitors, and sulfhydryl reagent, 4-vinylpyridine. Nm was purified by HPLC and ion-exchange chromatography, and the various fractions were identified by ES-MS as unphosphorylated and mono-, di-, tri-, and tetraphosphorylated species. All of these Nm species contained 2 mol of added 4-vinylpyridine per mol of Nm, suggesting that the two Cys residues are in the reduced form in the brain. In vivo, the majority of Nm is in the phosphorylated form (approximately 80%), of which the levels of the mono- and diphospho forms are higher than those of the tri- and tetraphospho species. Four in vivo phosphorylation sites, Ser41, Thr95, Ser142, and Thr172, were identified by amino acid sequencing and tandem ES-MS of the peptides derived from Lys-C endoproteinase digestion. Among these sites, only Ser41 is a known target of PKC, whereas the kinases responsible for the phosphorylation of the other three novel sites are unknown. Hypoxia/ischemia caused a preferential dephosphorylation of Ser41 and Thr172, whereas Thr95 is the least susceptible to dephosphorylation.     

Nucleosome mono, di, tri-, and tetramers from chicken embryo chromatin.

The fractionation of gram quantities of nuclease digested chromatin from chicken embryos into nucleosome mono-, di-, tri-, and tetramers is described in detail. Each of these nucleosomal species contains a fraction soluble in 0-1 M KC1 that decreases with increasing repeat number. Less histone H1 is associated with the nucleosome fractions soluble as compared to the respective fractions precipitated in 0.1 M KC1. Thermal denaturation profiles of the four nucleosomal species are monophasic. The same Tm of 78 degrees C has been determined for the KC1-soluble nucleosomes and for the KC1-insoluble monomer. The Tm of the KC1-insoluble oligomers is 79.8 degrees C. Multiphasic melting curves were recorded for nucleosomal material that was concentrated by lyophilisation or stored at 4 degrees C in 0.25 mM EDTA. Total nucleosome mono-, di-, tri-, and tetramers (consisting of both the fraction soluble and insoluble in 0.1 M KC1) have been analyzed concerning their sedimentation, diffusion, partial specific volume, and molecular weight and compared with the sedimentation and molecular weight data of KC1-soluble nucleosome mono- and tetramers.     

Lipid compounds of the umbilical cord vein and their alterations in preeclampsia

The lipid composition of vascular walls changes during development, ageing and pathological processes. Preeclampsia is the most common pregnancy-associated pathological syndrome. It is accompanied by significant remodelling of the extracellular matrix, both in the umbilical cord vessels and in the surrounding Wharton's jelly. Lipids of the umbilical cord have not been extensively studied. Here we evaluate the lipid composition of the umbilical cord vein and its alteration in preeclampsia. Thin layer chromatography and high-performance liquid chromatography were employed for these analyses. It was found that the umbilical cord vein wall, as with most human tissues, contains free fatty acids, mono-, di- and triacylglycerols, free cholesterol and its esters. The characteristic feature is the presence of high amounts of monounsaturated fatty acids, mainly myristoleic acid (C14:1) and oleic acid (C18:1), and polyunsaturated fatty acids, including eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6), which are rather minor lipid components of most human tissues. They exist both in a free form and in a form of acylglycerols and cholesterol esters. Preeclampsia is associated with an increase in the accumulation of free fatty acids, acylglycerols and cholesterol esters in the umbilical cord vein wall, with a proportional reduction in unsaturated fatty acid contents in all the investigated lipid fractions. Total amount of myristoleate was similar to control values. It is suggested that stimulation of lipolysis in maternal tissues increases supply of free fatty acids to foetal blood and promotes the accumulation fatty acids and their esters in some foetal vascular walls.     

Separation of chloroplast polar lipids and measurement of galactolipid metabolism by high-performance liquid chromatography

Procedures are described for the separation of polar lipids from plant chloroplasts by high-performance liquid chromatography, using a polar-modified silica column. Glycolipids and phospholipids were eluted with a gradient of 2-propanol/n-hexane (80:55, v/v) and 2-propanol/n-hexane/water/methanol (80:55:15:10, v/v). The lipids were detected by uv absorbance at 202 nm. Diacylglycerol and mono-, di-, and trigalactosyldiacylglycerol and phosphatidylcholine were separated on a LiChrosorb NH2 column (7-microns particles, Merck, FRG), but acidic lipids were retained. These lipids could be quantified from their 202-nm absorbance recording. The absorption coefficients obtained depended on the mean number of double bonds in the different lipid classes. The separation was applied for a rapid monitoring of the lipid composition in thylakoids and in fractionated inner and outer envelopes. The activities of galactosyltransferases involved in galactolipid metabolism, UDPGal:diacylglycerol galactosyltransferase and galactolipid:galactolipid galactosyltransferase, could be measured quantitatively in specific assays for both enzymes.     

Separation of lipid classes by solid phase extraction

A rapid and reliable method for the separation of lipid classes is described using aminopropyl disposable columns. This method is a modification to an existing procedure that allows the separation of both neutral and acidic phospholipid fractions and a high recovery of the latter. Acidic phospholipids were eluted with a mixture of hexane-2-propanol-ethanol-0.1 M ammonium acetate-formic acid 420:350:100:50:0.5 containing 5% phosphoric acid after neutral phospholipids had been eluted with methanol. It was verified that extremely high recoveries of cholesterol (CH), triglycerides (TG), free fatty acids (FFA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidic acid (PA), sphingomyelin (SM), and cerebrosides were obtained with this method. In addition, there appeared to be no preferential losses or degradation of any particular molecular species as the fatty acid distribution of bovine brain PS and the molecular species profile of plant PI were unaltered by the procedure. Depending on the tissue, this method may yield fractions containing pure lipid classes and/or simple mixtures of lipid classes of similar polarity. These fractions may then be more easily separated by thin-layer chromatography or high performance liquid chromatography for a complete lipid class analysis.     

Determination of Fatty Acid Composition of Spore Lipids of the Moss Polytrichum commune by Glass Capillary Column Gas Chromatography

Fatty acid methyl esters of Polytrichum commune spore triglyceride and mono- and diglycosyl diglyceride fractions were analysed by glass capillary column gas chromatography provided with a precolumn system. The composition of the fatty acids in the lipid fractions differed only quantitatively: the diglycosyl diglyceride fraction was characterized by a high content of C 18: 3ω3 (67.7%), and the triglyceride and monoglycosyl diglyceride fractions by about 35%. The monoglycosyl diglyceride fraction contained a high proportion of C 14: 0 (18.4%). In all fractions the content of polyunsaturated C 20 acids was low, ranging from trace amounts to 4.9%.     

Glycosphingolipids of cultured human colon carcinoma cells and their drug-resistant sublines

Human colon carcinoma cells were analyzed for lipid phosphorus, cholesterol and glycosphingolipids. Ceramide mono-, di- and trihexosides and sulfatides were isolated by column and thin-layer chromatography and determined quantitatively on the basis of their hexose content. The complex lipid fractions so isolated were only partially resolved with the material available. Gangliosides GM2 and GM3 and globoside were major components of the fraction and were determined on the basis of their hexose, hexosamine and neuraminic acid content. The HCT 116, 116a and 116b cells contained no fucolipids. Cell lines resistant to mitomycin C, teniposide and etoposide were developed and analyzed. Over the 5 year period of the study sulfatides declined to about one-fourth of their original amounts in both parent and drug-adapted cells. HCT 116 cells adapted to mitomycin C and teniposide had 30% less ceramide monohexoside and a 45% greater cholesterol to lipid phosphorus ratio than the parent cells. Reductions in ceramide dihexoside in the drug-adapted cells were greater than those of the ceramide monohexoside. Galabiosyl ceramide was the major ceramide dihexoside in all the cells and accumulated in HCT 116a to levels 4-6-fold greater than that of the other lines as the only dihexoside.     

Novel Mono-, Di-, and Trimethylornithine Membrane Lipids in Northern Wetland Planctomycetes

Northern peatlands represent a significant global carbon store and commonly originate from Sphagnum moss-dominated wetlands. These ombrotrophic ecosystems are rain fed, resulting in nutrient-poor, acidic conditions. Members of the bacterial phylum Planctomycetes are highly abundant and appear to play an important role in the decomposition of Sphagnum-derived litter in these ecosystems. High-performance liquid chromatography coupled to high-resolution accurate-mass mass spectrometry (HPLC-HRAM/MS) analysis of lipid extracts of four isolated planctomycetes from wetlands of European north Russia revealed novel ornithine membrane lipids (OLs) that are mono-, di-, and trimethylated at the ε-nitrogen position of the ornithine head group. Nuclear magnetic resonance (NMR) analysis of the isolated trimethylornithine lipid confirmed the structural identification. Similar fatty acid distributions between mono-, di-, and trimethylornithine lipids suggest that the three lipid classes are biosynthetically linked, as in the sequential methylation of the terminal nitrogen in phosphatidylethanolamine to produce phosphatidylcholine. The mono-, di-, and trimethylornithine lipids described here represent the first report of methylation of the ornithine head groups in biological membranes. Various bacteria are known to produce OLs under phosphorus limitation or fatty-acid-hydroxylated OLs under thermal or acid stress. The sequential methylation of OLs, leading to a charged choline-like moiety in the trimethylornithine lipid head group, may be an adaptation to provide membrane stability under acidic conditions without the use of scarce phosphate in nutrient-poor ombrotrophic wetlands.     

The structures of the asparagine-linked sugar chains of bovine interphotoreceptor retinol-binding protein. Occurrence of fucosylated hybrid-type oligosaccharides

The sugar chains of interphotoreceptor retinol-binding protein purified from the interphotoreceptor matrix of bovine eyes were liberated from the polypeptide portion by hydrazinolysis followed by N-acetylation and NaB[3H]4 reduction. The oligosaccharide fraction thus obtained was separated into four acidic fractions by paper electrophoresis. The four acidic fractions were confirmed to be mixtures of mono-, di-, tri-, and tetrasialyloligosaccharides. Both N-acetyl- and N-glycolylneuraminic acids were found as sialic acids of interphotoreceptor retinol-binding protein. The monosialylated oligosaccharide fraction, which accounted for 40 molar per cent of the total oligosaccharides liberated, was a mixture of the following hybrid-type oligosaccharides: (Formula: see text) This is the first time that fucosylated hybrid-type oligosaccharides have been found in any glycoprotein. The di-, tri-, and tetrasialyloligosaccharide fractions were composed of biantennary complex-type oligosaccharides, the outer chains of which are either Sia alpha 2----(3- or 6-linked)Gal beta 1----3(Sia alpha 2----6)GlcNac or Sia alpha 2----(3- or 6-linked)Gal beta 1----4GlcNAc.     

Isolation and characterization of fractions of Mycoplasma pneumoniae. I. Chemical and chromatographic separation

Prescott, B. (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), O. Sobeslavsky, G. Caldes, and R. M. Chanock. Isolation and characterization of fractions of Mycoplasma pneumoniae. I. Chemical and chromatographic separation. J. Bacteriol. 91:2117-2125. 1966.-Fractionation of Mycoplasma pneumoniae, cultured on a beef heart infusion-horse serum-yeast extract medium, was carried out by chemical and chromatographic procedures. The chemical method yielded eight fractions consisting of lipid, carbohydrates, and proteins. Four protein-rich fractions were isolated by chromatographing a supernatant fluid of sonically treated organisms on Sephadex G-25. The 12 fractions were tested for serological and antigenic activity in vitro and in vivo. The lipid fraction was serologically active and the relative order of activity of the protein fractions appeared to depend on the amount of lipid present in the molecule. The highly serologically active Sephadex G-25 protein fraction 1 prepared chromatographically contained 15% lipid in the molecule, whereas the less serologically active protein fraction 2 prepared by chemical means contained 2% lipid. The acetone-extracted lipid fraction was chromatographed on thin-layer chromatography plates and found to consist of nine fractions. Serological activity was associated with only the first three spots above the origin. Lipid extracted from the protein fractions seemed to be similar to the acetone-extracted lipid from the sediment of the sonically treated organisms.     

PROTEIN METHYLATION BY CEREBRAL TISSUE

Abstract— Transfer of the methyl group of S -adenosyl [Me-¹⁴C]methionine into cerebral proteins, an encephalitogenic protein and histones was studied using extracts of bovine and rat brains. The brain extract contains multiple substrate proteins and their lysine and arginine residues were methylated to form N^e-mono-, -di- and -trimethyl-lysine and N ^G-mono-, N ^G, N ^G- and N^G,N^G-dimethylarginine residues respectively, at different rates. The enzyme which catalyses the methylation of arginine residues was differentiated by ammonium sulphate fractionation from that methylating lysine residues. Methylation of arginine and lysine residues of proteins was stepwise in general, from mono- to dimethyl-arginine and from mono- to di- and trimethyl-lysines. Two different enzymes which methylate histone and the encephalitogenic basic protein respectively were obtained from the cytoplasmic fraction of rat brain and their enzymic properties were examined.     

Isolation and composition of the carotenoid-containing oil droplets from cone photoreceptors.

A procedure for isolating the carotenoid-containing oil droplets of cone retinal photoreceptors of Gallus domesticus is described. The oil droplets, composed almost entirely of neutral lipids and carotenoids, have been separated into ten chromatographic components. Similar separations have been carried out on the total retinal neutral lipids for comparison. The neutral lipids represented 26.1% of the total retinal lipid. Cholesterol, cholesterol ester, mono-, di- and triacylglycerols represented 92.6% of the total neutral lipid. Each of these and other minor neutral lipid components were also present in the lipids extracted from the isolated oil droplets in correspondingly similar concentrations. However, the concentrations of carotenoids were greatly enriched in the neutral lipids of the oil droplets. Each of the major fatty acyl-containing neutral lipids from the chromatography of oil droplet lipids is greatly enriched in polyunsaturated fatty acids when compared with the corresponding component from the total neutral lipid chromatography. In the acylglycerols and free fatty acid fraction from the oil droplets, linoleic and arachidonic acid together represented 52-83% of the total polyunsaturated fatty acids present. The remainder was generally distributed about equally among six other acids. Except for the diacylglycerol fraction, linoleic acid was usually the most enriched acid in a specific oil droplet fraction when compared with any other polyunsaturated fatty acids. A similar pattern of polyunsaturated fatty acid enrichment observed in the fatty acids of the outer segment phospholipids relative to the corresponding total phospholipid fractions of this cone rich retina (Johnston, D. and Hudson, R.A. (1974) Biochim. Biophys. Acta 369, 269) suggest possible metabolic relationships between the oil droplet neutral lipids and the outer segment membrane phospholipids of the cone photoreceptors. A mechanism for the accumulation of the carotenoids in the oil droplets is also discussed.     

Biosynthetic incorporation of fluorescently labelled fatty acids in Escherichia coli

Escherichia coli cells were cultivated in a medium containing 1-pyrene butanoic acid, a fluorescent probe. Total lipids were extracted from the cells, and the extract was separated by thin-layer chromatography. The fluorescent fractions were examined using spectrofluorimetry. The starting 1-pyrene butanoic acid was shown to be biosynthetically incorporated into the bacterial lipid. Four fluorescent fractions appeared as a result; the fractions were derivatives of this compound modified in the chromophore and the fatty acid chain. The results indicate that the formation of 1-pyrene butanoic acid fluorescent metabolites can be used for studying the oxidation-reduction systems of the bacterium.