Silicic Acid-impregnated Paper Research Articles

Studies on the diversity of inositol-containing yeast phospholipids: incorporation of 2-deoxyglucose into lipid.

Seven inositol-containing phospholipids in Saccharomyces cerevisiae can be resolved by two-dimensional chromatography on silicic acid-impregnated paper. Four of these lipids are stable to mild alkaline methanolysis; one of these has already been characterized as a mannosyl di(inositolphosphoryl) ceramide. Addition of labeled 2-deoxy-d-glucose to a growing culture results in label appearing in five lipids, some of which are alkali-stable and have the same R(F) values as the alkali-stable inositol-containing phospholipids. These lipids are labeled rapidly. The deoxyglucose is incorporated intact, probably in a glycosidic link, since the deoxyglucose is released by mild acid treatment.

Glycolipid biosynthesis: biosynthesis of mannose- and fucose-containing glycolipids by hela cells

1. 1. Triton X-100 extracts of HeLa cells incorporated both [ 14C]fucose and [ 14C]mannose into endogenous glycolipid acceptors using GDP-[ 14C]fucose or GDP-[ 14C]mannose as substrates. 2. 2. Both systems had an optimum temperature of 37° and required Mg 2+. 3. 3. Silicic acid-impregnated paper chromatography of the reaction products indicated that with each sugar a distinct glycolipid was synthesized. 4. 4. The [ 14C]fucose system exhibited a K m of 1.9 μM with respect to GDP-fucose, and the [ 14C]mannose system had a K m of 3.5 μM with respect to GDP-mannose. 5. 5. Using acetone precipitation it was possible to dissociate the enzyme and acceptor for each system. 6. 6. In each example the glycolipids synthesized were identified as glyco-glycerolipids and not sphingolipids or phospholipids. 7. 7. Diolein acted as an exogenous acceptor in the mannose system, while dipalmitin acted as an acceptor for the fucose system; distearin did not act as an acceptor for either sugar.

Phospholipids from Bacillus stearothermophilus.

The lipids of Bacillus stearothermophilus strain 2184 were extracted with chloroform-methanol and separated into neutral lipid and three phospholipid fractions by chromatography on silicic acid columns. The phospholipids were identified by specific staining reactions on silicic acid-impregnated paper, by chromatography of alkaline and acid hydrolysis products, and by determination of acyl ester:glycerol:nitrogen:phosphorus molar ratios. The total extractable lipid was 8% of the dry weight of whole cells and consisted of 30 to 40% neutral lipid and 60 to 70% phospholipid. The phospholipid consisted of diphosphatidyl glycerol (23 to 42%), phosphatidyl glycerol (22 to 39%), and phosphatidyl ethanolamine (21 to 32%). The concentrations of diphosphatidyl glycerol and phosphatidyl glycerol were lower in 2-hr cells than in 4- and 8-hr cells. Whole cells were fractionated by sonic treatment and differential centrifugation. The total lipid content, expressed in per cent of dry weight of each fraction was: whole protoplasts, 10%; membrane fraction, 18%; 30,000 x g particulate fraction, 22%; and 105,000 x g particulate fraction, 26%. The relative phospholipid concentrations in each fraction were about the same. As had been previously reported, none of the phospholipid was stable to alkaline hydrolysis.

The isolation and characterisation of phosphatidylglycerol and a structural isomer from pig lung

Two minor phospholipid components were isolated from a lipid extract of pig lungs. One was identified as phosphatidylglycerol. The results of alkaline hydrolysis, periodate oxidation and acetolysis showed that the other was a structural isomer of phosphatidylglycerol with the structure of a lyso- bis-phosphatidic acid. Its chromatographic characteristics on thin-layer plates of silica gel and on silicic acid-impregnated paper were quite distinct from those of phosphatidylglycerol. Both of these phospholipids were also present in lipid extracts of rat lungs and rabbit lungs.

Phospholipids of yeast: I. Improvements in analysis by two-dimensional strip-transfer paper chromatography

1. 1. A strip-transfer technique, which enables phospholipids to be eluted from one type of paper on to a second type of paper, has resulted in improved resolution of phospholipid mixtures from yeast by two-dimensional paper chromatography. The most satisfactory separations were obtained when lipid mixtures were chromatographed in the first direction on formaldehyde-treated, alumina-impregnated, or anion exchange paper, followed by elution on to silicic acid-impregnated paper and development in the second direction. Resolution of lipid mixtures was less satisfactory when chromatography was carried out in the first direction on alumina- or silicic acid-impregnated paper, followed by development on to formaldehyde paper in the second direction. The merits of the various systems are discussed and a simple apparatus is described which facilitates the strip-transfer procedure. 2. 2. The Chromatographie methods described are particularly effective for detecting minor phospholipids (phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylserine and N, N-dimethylphosphatidylethanolamine) in crude lipid extracts.

The chromatographic separation of polyphosphoinositides and studies on their turnover in various tissues

1. 1. 32P-labeled spots obtained when total lipid extracts from tissue slices are chromatographed on silicic acid-impregnated paper with phenol-ammonia as the developing solvent been characterized as di- and triphosphoinositide. This has enabled us to develop a simple, rapid, quantitative method for assaying the radioactivity incorporated into di- and triphosphoinositide in large numbers of small samples of tissue. 2. 2. Slices of salts gland, brain cortex, kidney, liver, pancreas, and heart ventricle incorporated 32P into di- and triphosphoinisitide when incubated in physiological saline containing [ 32P]orthophosphate. With the exception of liver, the incorporation into triphosphoinositide was higher. The incorporation into diphosphoinositide generally paralled that into triphosphoinositide. 3. 3. Under conditions in which 32P incorporation into phosphatidic acid and phosphatidyl inositol was stimulated by acitylcholine in salt-gland slices, there was an inhibition of incorporation into the polyphosphoinositides.

Chromatographic separation of C19-16-dehydro-steroids

The chromatography of a closely related series of 16-dehydro-steroids and their acetates on thin layer plates and silicic acid-impregnated paper is described. Fairly rapid and easy separations of most of the compounds studied can be achieved although the separation of androst-16-en-3β-ol and androsta-5, 16-dien-3β-ol is only obtained by an overrunning technique. Silicic acid-impregnated paper appears to be free from disadvantages associated with papers impregnated with other stationary phases such as phenylcellosolve, liquid paraffin and kerosene. The use of a number of colour reagents is described, including phosphomolybdic acid, phosphotungstic acid, resorcylaldehyde—sulphuric acid, the Allen reagent and uranyl nitrate in sulphuric acid. Some of the steroids give specific colours which may be useful in their identification.

Zur papierchromatographischen auftrennung von pflanzenlipiden

Crude lipid extracts may be separated rapidly by two-dimensional chromatography on a new type of silicic acid-impregnated paper. In the examination of lipid extracts of sugar beet leaves 23 different spots have been observed; 10 of these have been characterized as phospholipids and 4 as glycolipids, while the remining 9 lipid spots could not yet be classified.

Relation of structure to function in bacterial O antigens. II. Fractionation of lipids present in Boivin-type endotoxin of Serratia marcescens.

Nowotny, Alois (City of Hope Medical Center, Duarte, Calif.). Relation of structure to function in bacterial O antigens. II. Fractionation of lipids present in Boivin-type endotoxin of Serratia marcescens. J. Bacteriol. 85:427-435, 1963.-Methods for liberation of lipids from their endotoxic O antigen were investigated. In the case of endotoxin isolated from a chromogenic Serratia marcescens, best results were obtained with diluted formic acid. The crude lipid mixture consisted of at least 16 different components, which were analyzed by paper chromatography. Good separation was achieved by silicic acid-impregnated paper. Lipids were stained with metachromatic o-toluidine blue. Solubility of the crude lipid mixture was studied in different organic solvents. Preparative fractionation was developed utilizing various organic solvents on the basis of solubility differences of the lipid components. Pure fractions were obtained by silicic acid column chromatography. Since none of the lipid liberation methods so far developed can produce undegraded lipids free from split products and remnants of cell-wall polysaccharides, their usefulness is limited in comparing the chemical structure of the liberated lipid with that of the lipid intact in the cell wall. The liberation procedures cause changes within the lipid structure itself, which in turn would alter whatever potential ability it might have to elicit certain biological reactions.

Separation of lecithin and phosphatidyl ethanolamine by chromatography on paper impregnated with zinc salts.

THE separation of phospholipids by paper chromatography has generally been performed on modified papers. Marinetti and Stotz1 and Lea et al.2 report the use of silicic acid-impregnated paper, and Brown et al.3 have used silicic acid-impregnated glass-fibre ‘paper’. Rouser et al.4 investigated the possibility of using paper impregnated with mineral oil, rubber, silicone, and formamide, and paper oxidized by periodic acid, but found all these to be unsuitable. Recently, however, in this laboratory5 a method has been evolved for the separation and differential staining of phospholipids on untreated paper. During work on this method difficulty was encountered with the separation of lecithin from phosphatidyl ethanol-amine, and a procedure for separating them on paper impregnated with zinc salts is described here.

Phosphatide components of fowl blood cells

The phosphatide components of fowl blood cells were separated by chromatography on silicic acid-impregnated paper and found to consist of lecithin (42%), phosphatidyl ethanolamine (24%), sphingomyelin + lysophosphatidyl ethanolamine (21%), and small amounts of phosphatidyl serine, lysolecithin, phosphatidyl inositol and phosphatidic acid. Each component had a different fatty acid composition; in particular, phosphatidyl ethanolamine had a higher proportion of polyunsaturated fatty acids and of stearic acid, and a lower proportion of palmitic acid than lecithin. These two phosphatides were also present in their respective plasmogen forms, the aldehyde moieties of which were found to be mainly palmitaldehyde and stearaldehyde. Incubation of the total blood cells with [2− 14]acetate resulted in the incorporateion of 14C into the non-phosphatide fraction (cholesteryl esters, cholesterol and diglycerides) and to a lesser extent into the lecithin, phosphatidyl ethanolamine and sphingomyelin components.

The uptake of amino acids by lipids of pseudomonas aeruginosa

The lipids of intact Pseudomonas aeruginosa resting cells were extracted and the weights of the lipid fractions determined. The phospholipids were found to comprise a major portion (80%) of the ether-soluble lipids. Chromatograms of the phospholipid complex on silicic acid-impregnated paper revealed at least six fractions. Two-dimensional chromatograms of the aqueous acid hydrolyzate of the phospholipids indicated the presence of at least 13 ninhydrin positive compounds. There was a correlation between the uptake of dl -alanine-1-C14, dl -leucine-1-C14, and dl -phenylalanine-3-Cl4 by phospholipids of the resting cells and utilization of these amino acids by the cell as indicated by manometric studies.

Chromatographic and radioisotopic investigations of the lipid components of runner bean leaves.

The total lipids of runner bean leaves have been separated by chromatography on silicic acid-impregnated paper into at least eleven components. These have been partially identified by their Rf values, staining behaviour with various specific reagents, radioisotope incorporation data, and products of milk alkaline hydrolysis. Apart from pigments and glycerides, six phosphatide components were found, five of which have been identified as lecithin, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl inositol and phosphatidic acid; also, four distinct glycolipids, and a sulfur-containing lipid were detected. Hydrolysis studies showed that the four glycolipids contained galactose, glucose, glycerol and an unknown sugar in different molecular proportions, and that the sulfolipid contained glucose, the same unknown sugar, and an acid-stable form of sulfur.

An inositol phosphatide of peas

Inositol phosphatide has been obtained from raw peas of all stages of maturity, excluding dry seed peas, after the peas were held in frozen storage (−17.8 °C.) for several months. The liberation is believed to be enzymic in nature. Peas which had been heat treated to inactivate enzymes yielded negligible amounts of inositol phosphatide. Silicic acid-impregnated paper chromatography confirmed the presence of inositol phosphatide, and silicic acid chromatography showed its heterogeneity. The analytical data have been supplemented by analyses for inositol and fatty acids. Some discussion has been presented concerning the purity and composition of this fraction of pea lipides.

Quantitative glass paper chromatography: phosphatidyl choline and sphingomyelin

A rapid chromatographic procedure was developed for the separation of sphingomyelin, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, and free fatty acids on glass paper coated with sodium silicate. In addition, phosphatidyl choline and sphingomyelin were determined quantitatively by densitometry of the charred chromatogram, which was obtained by spraying the developed chromatogram with sulfuric acid and heating in an oven. The separation of phosphatides on sodium silicate-treated glass paper is more rapid than on silicic acid-impregnated paper, and the former is simpler to prepare. Preliminary application of this quantitative technique to human serum indicates that it may have a wide adaptability for the determination of phospholipids in natural products.