THE PROTEIN COMPOSITION OF ISOLATED MYELIN1

—Three fractions, each containing markedly different proteins, was obtained from myelin: (1) The first fraction was obtained as an insoluble residue when myelin was extracted with neutral chloroform-methanol (CM, 2:1, v/v). It was digestible with trypsin and had an amino acid composition similar to that of the acidic proteolipid protein of Wolfgkam (1966). (2) The second fraction was obtained as a precipitate by the addition of various electrolytes (KCl, NaCl, CaCl₂, MgCl₂ or HCl) to the CM (2:1 v/v) extract. This fraction consisted mainly of a basic protein which exhibited an electrophoretic mobility and amino acid composition indistinguishable from those of the basic protein obtained from white matter (Martensson and LeBaron, 1966). This procedure provided for a simple and rapid isolation of the basic protein from myelin. Depending on the conditions of precipitation, this fraction was either free of lipid or contained tri- and diphosphoinositide. The effects of different ions at differing concentrations and the yield and nature of the precipitate have been studied. (3) A third fraction remained in solution in CM (2:1, v/v) after the addition of the electrolyte. It comprised the bulk of the myelin lipids and a protein fraction which was resistant to digestion with trypsin and had an amino acid composition similar to the classical proteolipid protein of Folch-Pi and Lees (1951). The possibility of a salt-type bonding between the basic protein and the polyphosphoinositides is discussed, and values for tri- and diphosphoinositide in bovine myelin are given.     

Studies on isolation and characterization of starch from oat (Avena nuda) grains

Starch from AC Hill oat grains (Avena nuda) was isolated and some of the characteristics determined. The yield of starch was 23·4% on a whole grain basis. The shape of the granule was polyhedral to irregular, with granules 6–10 μm in diameter. Lipids were extracted by acid hydrolysis and by selective solvent extraction with chloroform-methanol 2:1 v/v (CM) at ambient temperature, followed by n-propanol-water 3:1 v/v (PW) at 90–100°C. The acid hydrolyzed extracts which represented the total starch lipids (TSL) was 1·13%. The free lipids in the CM extract (1% TSL) was 6·2%, whereas the free and bound lipids in the PW extracts was 93.0%. Neutral lipids formed the major lipid class in the CM and PW extracts. The monoacyl lipid content in both CM and PW extracts was 61·0%. The total amylose content was 19·4%, of which 13·9% was complexed by native lipids. X-ray diffraction was of the ‘A’ type. Oat starch differed from wheat starch in showing a higher swelling factor, decreased amylose leaching, coleaching of a branched starch component and amylose during the pasting process, higher peak viscosity and set-back, low gel rigidity, greater susceptibility towards acid hydrolysis, greater resistance to -amylase action and a higher freeze-thaw stability. Furthermore, in comparison to wheat starch, the amylose chains of oat starch appear to be more loosely arranged in the amorphous regions, whereas in crystalline regions, oat starch chains are more compactly packed. Lipid removal from oat and wheat starches decreased their swelling factor, peak viscosity, set-back, gelatinization temperatures, freeze-thaw stability and paste clarity (at pH > 4·0), and increased their thermal stability, amylose leaching, enthalpy of gelatinization, susceptibility towards -amylase and paste clarity (at pH < 4·0). The results also showed that the properties of AC Hill oat starch is not representative of oat starch in general.     

GLycol-EThanol-WAter (3:3:4); A SOlvent for SUdan BLack B STaining

The previously used supersaturated 60% isopropanol method (Lillie 1954, p. 303) protected the more readily soluble lipids from the solvent action of 70% alcohol but precipitation of dye particles within and around sections is sometimes a disturbing complication (cf. Vlachos 1959). The propylene glycol method of Chiffelle and Putt (Lillie, p. 304) has been found to remove much of the birefringent lipid from the sections. Glacial acetic acid dissolves most tissue lipids except those of the solvent-resistant ceroid or lipofuscin type. While solutions in 60-70% acetic acid conserve ordinary fats well (Sills and Marsh 1959) their effect on the more easily soluble lipids should be further explored.     

GLycol-EThanol-WAter (3:3:4); A SOlvent for SUdan BLack B STaining

The previously used supersaturated 60% isopropanol method (Lillie 1954, p. 303) protected the more readily soluble lipids from the solvent action of 70% alcohol but precipitation of dye particles within and around sections is sometimes a disturbing complication (cf. Vlachos 1959). The propylene glycol method of Chiffelle and Putt (Lillie, p. 304) has been found to remove much of the birefringent lipid from the sections. Glacial acetic acid dissolves most tissue lipids except those of the solvent-resistant ceroid or lipofuscin type. While solutions in 60–70% acetic acid conserve ordinary fats well (Sills and Marsh 1959) their effect on the more easily soluble lipids should be further explored.     

A protocol for the combined sub-fractionation and delipidation of lipid binding proteins using hydrophobic interaction chromatography

Cellular lipids frequently co-purify with lipid binding proteins isolated from tissue extracts or heterologous host systems and as such hinder in vitro ligand binding approaches for which the apo-protein is a prerequisite. Here we present a technique for the complete removal of unesterified fatty acids, phospholipids, steroids and other lipophilic ligands bound to soluble proteins, without protein denaturation. Peroxisome proliferator activated receptor gamma ligand binding domain and intracellular fatty acid binding proteins were expressed in an Escherichia coli host and completely delipidated by hydrophobic interaction chromatography using phenyl sepharose. The delipidation procedure operates at room temperature with complete removal of bound lipids in a single step, as ascertained by mass spectrometry analysis of organic solvent extracts from purified protein samples. The speed and capacity of this method makes it amenable to scale-up and high-throughput applications. The method can also easily be adapted for other lipid binding proteins that require delipidation under native conditions.     

Relationships among low MW hydrophobic proteins from wheat endosperm

Low MW proteins extractable with chloroform-methanol mixtures from wheat endosperm have been purified from different Triticum species and partially characterized. Their amino acid composition and MWs are consistent with previous genetic evidence concerning relationships among these proteins: proteins CM1 and CM2 are homoeologous (ancestral homologues); proteins CM3 and CM3′ are allelic variants; proteins 16 and 17 are homoeologous.     

Changes in the level of tightly-bound mitochondrial phospholipids induced by exogenous factors

The effects of some parameters of the incubation medium (tonicity, H+ and Pi concentration) on the phospholipid content in rat mitochondrial extracts prepared by normal extraction and by extraction with strongly acidified solvent mixtures, were studied. It was found that after normal extraction with a chloroform-methanol mixture, 8% to 25% of mitochondrial phospholipids can be additionally extracted by acidified solvent mixtures. The concentration of weakly extractable lipids depends on the tonicity and ionic composition of the incubation medium. It was suggested that there exist in mitochondria two lipid pools, a loosely and a tightly-bound ones; the equilibrium between these pools is effectively regulated by medium tonicity and other external factors.     

Influence of extraction solvent system on the extractability of lipid components from the biomass of Nannochloropsis gaditana

Microalgae oils are considered to be promising alternative sources of omega-3 LC-PUFA. The aim of this work was therefore to evaluate different solvent (mixtures), currently accepted for use in the food industry, for the extraction of lipids from Nannochloropsis gaditana, an omega-3 LC-PUFA-rich microalga. Importantly, not only the total lipid yield but also the lipid class, eicosapentaenoic acid, carotenoid, and sterol yield were investigated. It was shown that the highest yield for each of the components was obtained with dichloromethane/ethanol (1:1). All extracts except the one obtained with dichloromethane/ethanol (1:1) were enriched in neutral lipids and depleted in polar lipids, when compared to the total lipid extract (chloroform/methanol 1:1). Hexane/isopropanol (3:2) seems to be the second best option: it has the advantage of performing better at criteria such as toxicity, but has the disadvantage that almost half of the interesting oil cannot be recovered.     

Characterization of membrane components of the flask-shaped mycoplasma Mycoplasma mobile

The cell membrane of Mycoplasma mobile was isolated by either ultrasonic or French press treatment of intact cells. The membrane fraction contained all of the cellular lipids, but only one-third of cellular proteins and had a density of 1.14 g ml-1. The soluble fraction contained the NADH dehydrogenase activity of the cells, as well as a protein with an apparent molecular mass of 55 kDa that was phosphorylated in the presence of ATP. Lipid analyses of M. mobile membranes revealed that membrane lipid could be labelled by radioactive glycerol, oleate and to a much higher extent by palmitate but not by acetic acid. The membrane lipid fraction was composed of 54% neutral and 46% polar lipid. The major constituents of the neutral lipid fraction were free fatty acid, free cholesterol and cholesterol esters (45, 25 and 20%, respectively, of total neutral lipid fraction). The free cholesterol count was 13% (w/w) of total membrane lipids with a cholesterol:phospholipid molar ratio of about 0.9. Among the polar lipids, both phospho- and glycolipids were detected. The phospholipid fraction consisted of a major de novo-synthesized phosphatidylglycerol (approximately 63% of total phospholipids), plus exogenous phosphatidylcholine and sphingomyelin incorporated in an unchanged form from the growth medium. The glycolipid fraction was dominated by a single glycolipid (approximately 90% of total glycolipids) that was preferentially labelled by palmitic acid and showed a very high saturated:unsaturated fatty acids ratio.     

Structures of minor ether lipids isolated from the aceticlastic methanogen, Methanothrix concilii GP6

Structures were determined for two phospholipids and three glycolipids purified from chloroform-methanol extracts of Methanothrix concilii GP6. Together they accounted for 14% of the total lipid and were based on a C20,20-diether core structure consisting of either 2,3-di-O-phytanyl-sn-glycerol or its 3'-hydroxy analog, namely, 2-O-[3,7,11,15-tetramethylhexadecyl]-3-O-[3'- hydroxy-3',7',11',15'-tetramethylhexadecyl]-sn-glycerol. These two core lipids formed phosphodiester bonds to ethanolamine and glycosidic bonds to beta-D-galactopyranose. A third glycolipid consisted of the triglycosyl head group beta-D-galactopyranosyl-(1----6)-[beta-D-glucopyranosyl-(1----3)]-beta-D - galactopyranose in glycosidic linkage to the 3'-hydroxydiether core lipid.     

Uncovering the unfoldome: enriching cell extracts for unstructured proteins by acid treatment

A method to enrich cell extracts in totally unfolded proteins was investigated. A literature search revealed that 14 of 29 proteins isolated by their failure to precipitate during perchloric acid (PCA) or trichloroacetic acid (TCA) treatment where also shown experimentally to be totally disordered. A near 100 000-fold reduction in yield was observed after 5% or 9% PCA treatment of total soluble E. coli protein. Despite this huge reduction, 158 and 142 spots were observed from the 5% and the 9% treated samples, respectively, on silver-stained 2-D SDS-PAGE gels loaded with 10 microg of protein. Treatment with 1% PCA was less selective with more visible spots and a greater than 3-fold higher yield. A substantial yield of unprecipitated protein was obtained after 3% TCA treatment, suggesting that the common use of TCA precipitation prior to 2-D gel analysis may result in loss of unstructured protein due to their failure to precipitate. Our preliminary analysis suggests that treating total protein extracts with 3-5% PCA and determining the identities of soluble proteins could be the starting point for uncovering unfoldomes (the complement of unstructured proteins in a given proteome). The 100 000-fold reduction in yield and concomitant reduction in number of proteins achieved by 5% PCA treatment produced a fraction suitable for analysis in its entirety using standard proteomic techniques. In this way, large numbers of totally unstructured proteins could be identified with minimal effort.     

Purification and chemical characterization of the major glycoprotein of avian myeloblastosis virus.

The major glycoprotein of avian myeloblastosis virus (AMV) has been purified to an apparent state of homogeneity by gel filtration on a Sepharose 4B column in the presence of 6 m guanidine hydrochloride followed by dialysis against distilled water and then extraction with chloroform-methanol. The AMV glycoprotein remains soluble in the aqueous phase whereas contaminating proteins precipitate, either upon dialysis against distilled water or after treatment with chloroform-methanol.Carbohydrate, represented by glucosamine, mannose, galactose, fucose, and sialic acid, constitutes 40% of the weight of AMV glycoprotein. Glucosamine is the major carbohydrate component whereas fucose and sialic acid are present in relatively low amount. Amino acid analysis indicates a relatively high content of aspartic and glutamic acid, serine, threonine, and glycine. Based on SDS-polyacrylamide gel electrophoresis, a molecular weight value of 77,500 ± 500 was determined for AMV glycoprotein.     

Purification of an Acidic Structural Protein from rat Skin Using Triton X-100

An acidic protein was extracted with neutral-salt solutions from rat skin. When precipitated by dialysis against dilute acetic acid, the structural protein was separated from contaminating soluble collagens and other soluble proteins. The precipitate was dissolved in buffers containing 1% Triton X-100 and purified to apparent size and charge homogeneity by chromatography on a DEAE Bio-Gel A column. Triton X-100 was necessary for achieving nondestructive disaggregation of the protein which could be reversed by dialyzing out the detergent against methanol-dilute acetic acid.     

Identification of novel phospholipid binding proteins in Saccharomyces cerevisiae

A proteomics approach was used to search for novel phospholipid binding proteins in Saccharomyces cerevisiae. Phospholipids were immobilized on a solid support and the lipids were probed with soluble yeast protein extracts. From this, the phosphatidic acid binding proteins were eluted and identified by mass spectrometry. Thirteen proteins were identified and 11 of these were previously unknown lipid binding proteins. The protein-lipid interactions identified would not have been predicted using bioinformatics approaches as none possessed a known lipid binding motif. A subset of the identified proteins was purified to homogeneity and determined to directly bind phospholipids immobilized on a solid support or organized into liposomes. This simple approach could be systematically applied to perform an exhaustive screen for soluble lipid binding proteins in S. cerevisiae or other organisms.     

Purification of an acidic structural protein from rat skin using Triton X-100.

An acidic protein was extracted with neutral-salt solutions from rat skin. When precipitated by dialysis against dilute acetic acid, the structural protein was separated from contaminating soluble collagens and other soluble proteins. The precipitate was dissolved in buffers containing 1% Triton X-100 and purified to apparent size and charge homogeneity by chromatography on a DEAE Bio-Gel A column. Triton X-100 was necessary for achieving nondestructive disaggregation of the protein which could be reversed by dialyzing out the detergent against methanol-dilute acetic acid.     

A novel glycosphingolipid from gram-negative aquatic bacteria.

The chloroform-methanol extractable lipids of the Gram-negative fresh-water bacteria Arcocella aquatica NO-502 and Flectobacillus major FM were found to contain an unusual ninhydrin-positive glycolipid. It was purified by two-stage silica gel-column chromatography. By the use of IR and (1)H-NMR spectroscopy, mass spectrometry and chemical-degradation experiment, the lipid was established to be 1-O-monoglycosyl ceramide, the carbohydrate moiety of which was the alpha-pyranose-ring form of 7-desoxy-7-amino-D-manno-heptulosonic acid, or 1-hydroxycarbonyl-6-deoxy-6-amino-alpha-D-mannopyranose. The ceramide portion consisted mainly (by 95% in the A. aquatica glycolipid and 80% in the F. major glycolipid) of 2-N-(2'-D-hydroxy-13'-methyltetradecanoyl)-15-methyl-4(E)-hexad ecasph ingenine. The minor molecular species differed from the major one only in fatty acid structure. The glycolipid accounted for 8 and 11% of the total lipids extracted from A. aquatica NO-502 and F. major FM cells, respectively.     

Highly polar non-glycerol containing lipids present in our mitochondrial membranes

Aqueous 80% ethanol extracts from the outer mitochondrial membranes contained a series of compounds, insoluble in chloroform-methanol (2:1) and soluble in chloroform-methanol-water (10:10:3). These compounds contain fatty acids, amino acids and hexoses. Procedures are described for the extraction, purification and cromatographic separation of these compounds. Five of them have been partially characterised. Some structural data are given. It is suggested that some relationship could exist with the glycosylation of the outer membrane proteins.     

Quantitative isolation of total glycosphingolipids from animal cells

The quantitative isolation of total glycosphingolipids from crude lipid extracts without contamination from other lipid classes is described. The method consists of (a) acetylation of total lipids with pyridine and acetic anhydride, (b) separation of acetylated glycolipids from nonglycolipids on a magnesia-silica gel (Florisil) column, and (c) deacetylation of glycolipid in chloroform-methanol-sodium methoxide. This method is useful for determination of microgram quantities of glycolipids derived from less than 1 ml of packed cells.     

The effects of hydration and divalent cations on lamellar-nonlamellar phase transitions in membranes and total lipid extracts from Acholeplasma laidlawii A-EF22 – a 2H NMR study

Acholeplasma laidlawii strain A-EF22 was grown in a medium supplemented with 75 μm α-deuterated palmitic acid (16:0-d ₂) and 75 μm α-deuterated oleic acid (18:1c-d ₂), or with 150 μm 18:1c-d ₂. The fatty acids were incorporated into the membrane lipids and ²H NMR spectra were recorded from intact membranes, total lipid extracts, and the combined glucolipid and neutral lipid fractions of a total lipid extract. The lipids in intact membranes form a bilayer structure up to at least 70 °C. The same result was obtained with membranes digested with pronase, which removes a large fraction of the membrane proteins. A reversed hexagonal liquid crystalline (H_II) phase was formed below 70 °C by the total lipid extracts hydrated with 20 and 30% (w/w) water; in the presence of 40% (w/w) water only one of the extracts formed an H_II phase below 70 °C. The H_II phase was formed at higher temperatures with an increasing water content. However, only a lamellar liquid crystalline (L _α ) phase was formed up to 70 °C by the total lipid extracts when the water concentrations were 50% (w/w) or higher. The temperature (T _LH) for the L _α to H_II phase transition in the combined glucolipid and neutral lipid fractions was only 2–3 °C lower than for the total lipids, and the phospholipids thus have a very modest influence on the T _LH value. Physiologically relevant concentrations of Ca²⁺ and Mg²⁺ ions did not affect the phase equilibria of total lipid extracts significantly. It is concluded from comparison with published data that the membrane lipids of the cell wall-less bacterium A. laidlawii have a smaller tendency to form reversed nonlamellar phases than the membrane lipids of three bacterial species surrounded by a cell wall. Received: 10 March 1997 / Accepted: 4 July 1997     

Purification and characterization of phthalanilide--lipid complexes from tissues

What appears to be a new class of phospholipids has been isolated from dog brain in the form of complexes with a substituted phthalanilide. The complexes were extracted by chloroform-methanol and purified by countercurrent distribution in solvent systems containing water, chloroform, methanol, and Freon 113. The binding of the phthalanilide congener to lipids has some ionic character. Cations such as H(+) or Ca(++) displaced the phthalanilide from its lipid complex. The pH for 50% displacement acid was about 3.8 and was independent of the purity of the complex. Thin-layer chromatography of the lipid yielded four subfractions of lipid, three of which were ninhydrin-positive and all of which yielded a group of unidentified ninhydrin-positive components on hydrolysis. Each lipid subfraction contained nitrogen, phosphorus, fatty acids, and glycerol but in different ratios. Of the known phospholipids containing nitrogen, none matches the composition and behavior of the lipids isolated as phthalanilide complexes. We have therefore concluded that the phthalanilides bind to a new class of phospholipids characterized by a high content of unidentified ninhydrin-positive components.